Toxigenesis and Treatment of Scorpion Envenomation in
Man and Experimental Animals in Sudan
Submitted by
Nuzha Abdellatief Sidahmed Abdoon
PhD Candidate
College of Pharmacy
Khartoum University
Khartoum, Sudan
2004
This thesis was defended on 30- November- 2005 and approved.
Supervisors
1- Dr. Abdelrahim Elsayed Ali
2- Dr. Elamin Ibrahim Elnima
3- Dr. Mohammed Haj-Ali
Examination Committee
1- Dr. Mosa Mohammad Mosa
2- Dr. Edres
3- Dr. Elamin Ibrahim Elnima
To
My Mother and Father
To My Husband
Table of Contents
Title
Examination Committee
Dedication
Table of Contents
Titles of Tables
Titles of Figures
Acknowledgment
Summary
1. CHAPTER ONE
LITERATURE REVIEW
1.1. Nomenclature and distribution of scorpions
1.2. Component of scorpion venoms
1.3. Mechanism of action of scorpion venom toxins
1.4. General effects of scorpion venom
1.5. Effects of scorpion venoms on skeletal muscles
1.6. Effect of scorpion venoms on smooth muscles
1.7. Effect of scorpion venoms on the cardiovascular system
1.8. Effect of scorpion venoms on the respiratory system
1.9. Scorpion envenomation and inflammation
1.10. Cytokines and inflammation
1.11. Nitric oxide and inflammation
1.12. Treatment of scorpion envenomation
2. CHAPTER TWO
INTRODUCTION
3. CHAPTER THREE
MATERIALS AND METHODS
3.1. Materials
3.1.1. Materials for clinical characteristics of scorpion-envenomed
patients admitted to Dongla Hospital in Sudan
3.1.2. Materials for Cardiovascular experiments
3.1.3. Materials for effect of selected anti-inflammatory drugs on the
lethal actions of Leiurus quinquestriatus scorpion venom
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3.2. Methods
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3.2.1. Methods of clinical characteristics of scorpion-envenomed
patients admitted to Dongla Hospital in Sudan
3.2.1.1. Phase I: Reserch design
3.2.1.2. Phase II: Patient selection
3.2.1.3. Phase III: Preparation of Patient information sheets
3.2.1.4. Phase IV: Methods used for laboratory investigations
3.2.1.4.1. Determination of blood Glucose
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3.2.1.4.2. Determination of blood urea nitrogen
3.2.1.4.3. Determination of blood creatinine
3.2.1.4.4. Determination of blood total protein
3.2.1.4.5. Determination of blood white blood cell count
3.2.1.5. Phase V: Distribution and collection of patient information sheets
3.2.1.6. Phase VI: Data Analysis
3.2.1.7. Study Limitations
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3.2.2. Methods for Cardiovascular experiments
3.2.2.1 Animal preparation and protocol
3.2.2.2. Methods used for biochemical determinations in rabbit serum
3.2.2.2.1 Determination of serum cytokine IL 8
3.2.2.2.2. Determination of serumTNFα
3.2.2.2.3. Determination of serum total nitrites and endogenous nitrites
3.2.2.2.4. Determination of serum albumin
3.2.2.2.5. Determination of serum total protein
3.2.2.2.6. Determination of serum alanine aminotransferase (ALT)
3.2.2.2.7. Determination of serum aspartate aminotransferase (AST)
3.2.2.2.8. Determination of serum creatinine
3.2.2.2.9. Determination of blood urea nitrogen (BUN)
3.2.2.2.10. Determination of serum lactate dehydrogenase (LDH)
3.2.2.2.11. Determination of serum creatine kinase (CK)
3.2.2.2.12. Determination of serum glucose
3.2.2.2.13. Determination of serum carbon dioxide
3.2.2.2.14. Determination of serum chloride
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3.2.2.3. Methods used for Hematological determinations in rabbit serum
3.2.2.3.1. Determination of serum total and deferential white blood cells
A: Peroxidase method for determination of total WBC
B: Determination of Basophils
3.2.2.3.2. Determination of red blood cells (RBC)
3.2.2.4. Statistical analysis
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3.2.3. Methods for effect of selected anti-inflammatory drugs on the lethal
actions of Leiurus quinquestriatus scorpion venom
3.2.3.1 Lethality studies in mice
3.2.3.2. Statistical analysis
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4. CHAPTER FOUR
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RESULTS
4.1. Results for Clinical Characteristics of scorpion-envenomed Patients
admitted to Dongla Hospital in Sudan
4.1.1. General information about envenomed patients
4.1.2. General signs and symptoms of envenomed patients
4.1.3. Cardiovascular and respiratory symptoms of envenomed patients
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4.1.4. General laboratory investigations of envenomed patients
3.1.5. Treatment given to envenomed patients
4.2. Result for Correlation between blood pressure and selected biochemical
parameters, including cytokines and NO, in conscious rabbits injected
subcutaneously with Leiurus quinquestriatus scorpion venom
4.2.1. Cardiovascular experiments
4.2.1.1. Effect of venom on MABP
4.2.1.1.1. Effect of normal saline on MABP of conscious rabbits
4.2.1.1.2. Effect of LQQ venom on MABP of conscious rabbits
4.2.2. Effect of venom on lung body weight index of conscious rabbits
4.2.2.1. Effect of LQQ scorpion venom on lung body weight index
of conscious rabbits
4.2.3. Effect of LQQ venom on selected biochemical parameters
4.2.3.1. Effect of LQQ venom on serum IL 8 and TNFα
4.2.3.1.1. Effect of s.c. injection of normal saline on serum IL 8
and TNFα in conscious rabbits
4.2.3.1.2. Effect of s.c. injection of LQQ venom on serum IL 8
and TNFα in conscious rabbits
4.2.3.2. Effect of LQQ venom on serum nitrate and nitrite levels
4.2.3.2.1. Effect of s.c. injection of normal saline on serum nitrate
and nitrite in conscious rabbits
4.2.3.2.2. Effect of s.c. injection of LQQ venom on serum nitrate
and nitrite in conscious rabbits
4.2.3.3. Effect of LQQ venom on serum glucose
4.2.3.3.1. Effect of s.c. injection of normal saline on serum
glucose concentration in conscious rabbits
4.2.3.3.2. Effect of s.c. injection of LQQ scorpion venom on serum
glucose concentration in conscious rabbits
4.2.3.4. Effect of LQQ venom on ALT, AST, LDH and CK serum levels
4.2.3.4.1. Effect of s.c. injection of normal saline on CK, LDH, ALT
and AST serum levels in conscious rabbits
4.2.3.4.2. Effect of s.c. injection of LQQ venom on CK, LDH, ALT
and AST serum levels in conscious rabbits
4.2.3.5. Effect of LQQ venom on serum albumin and total protein levels
4.2.3.5.1. Effect of s.c. injection of normal saline on serum total protein
and albumin levels in conscious rabbits
4.2.3.5.2 Effect of s.c. injection of LQQ venom on serum total protein
and albumin levels in conscious rabbits
4.2.3.6. Effect of LQQ venom on BUN and creatinine serum levels
4.2.3.6.1. Effect of s.c. injection of normal saline on BUN and creatinine
serum levels in conscious rabbits
4.2.3.6.2. Effect of s.c. injection of LQQ venom on BUN and creatinine
serum levels in conscious rabbits
4.2.3.7. Effect of LQQ venom on serum carbon dioxide and chloride levels
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4.2.3.7.1 Effect of s.c. injection of normal saline on serum carbon
dioxide and chloride levels in conscious rabbits
4.2.3.7.2. Effect of s.c. injection of LQQ venom on serum carbon dioxide
and chloride levels in conscious rabbits
4.2.4. Effect of LQQ venom on selected hematological parameters
4.2.4.1 Effect of LQQ venom on serum RBC,WBC, basophils and
neutrophils
4.2.4.1.1. Effect of s.c. injection of normal saline on serum RBC,
WBC, basophils and neutrophils in conscious rabbits
4.2.4.1.2. Effect of s.c. injection of LQQ venom on serum RBC,WBC,
basophils and neutrophils in conscious rabbits
4.3. Result for effect of Selected Anti-inflammatory Drugs on the Lethal
actions of Leiurus quinquestriatus Scorpion Venom
4.3.1. Effect of i.v. and s.c. injection of 0.9 % NaCl into mice
4.3.2. Effect of i.v. and s.c. injection of LQQ venom on survival of mice
4.3.3. Effect of montelukast, hydrocortisone, or indomethacin
administration on survival of mice
4.3.4. Effect of oral administration of Montelukast on the survival of mice
injected subcutaneously with LQQ scorpion venom
4.3.5. Effect of intravenous injection of hydrocortisone on survival of mice
injected subcutaneously with LQQ scorpion venom
4.3.6. Effect of intravenous injection of indomethacin on survival of mice
injected subcutaneously with LQQ scorpion venom
5. CHAPTER FIVE
GENERAL DISCUSSION
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6. CHAPTER SIX
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REFERENCES
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Titles of Tables
Table (1a, 1b, 1c): General Clinical Data of Patients Stung By Scorpions In Dongla
Hospital in Sudan
Table (2): Percentage of surviving and non-surviving adults and children
admitted to Dongla Hospital in Sudan following scorpion envenomation
Table (3): Comparison of personal details of survivors and non-survivors admitted
to Dongla Hospital in Sudan following scorpion envenomation
Table (4): Comparison of cardiovascular and respiratory signs and symptoms in
survivors and non-survivors admitted to Dongla hospital in Sudan
following scorpion envenomation
Table (5): Comparison of signs and symptoms in survivors and non-survivors admitted
to Dongla Hospital following scorpion envenomation
Table(6): Comparison of blood laboratory parameters of survivors and non-survivors
admitted to Dongla Hospital in Sudan following scorpion envenomation
Table (7): Comparison of treatment protocol used for survivors and non-survivors
admitted to Dongla Hospital in Sudan following scorpion envenomation
Table(8): % of control mean arterial blood pressure measured at different time
intervals following the injection of Leiurus quinquestriatus venom into
conscious rabbits.
Table (9): Lung / body weight index of control and Leiurus quinqestriatus venom–
injected rabbits
Table (10): Serum IL8 levels in control and Leiurus quinquesrriatus venom-treated
rabbits
Table (11): Serum TNF α concentration of control and Leiurus quinquesrriatus venomtreated rabbits
Table (12): Total nitrite concentration in serum of control and Leiurus quinquestriatus
venom-treated rabbits.
Table (13): Serum endogenous nitrate concentration in control and Leiurus
quinquestriatus venom-treated rabbits
Table (14): Serum nitrate concentration in control and Leiurus quinquestriatus
venom-treated rabbits.
Table(15 ): Serum glucose concentration in control and Leiurus quinquesrriatus
venom-treated rabbits
Table (16): Serum CK and LDH levels in control and Leiurus quinquestriatus
venom-treated rabbits
Table (17): Serum AST and ALT concentration in control and Leiurus quinquestriatus
venom-treated rabbits
Table (18): Serum total protein and albumin levels in control and Leiurus
quinquestriatus venom-treated rabbits
Table (19): Serum BUN and creatinine levels in control and Leiurus quinquestriatus
venom-treated rabbits
Table (20): Serum CO2 and chloride levels in control and Leiurus quinquestriatus
venom-treated rabbits
Table (21):
RBC and WBC concentration in control and Leiurus quinquestriatus
venom-treated rabbits
Table (22):
Serum basophils and neutrophils levels in control and Leiurus
quinquestriatus venom-treated rabbits
Table (23): Determination of intravenous (A) and subcu/taneous (B) LD50 of Egyptian
Leiurus quinquestriatus scorpion venom
Table (24): Determination of intravenous (A) and subcutaneous (B) LD50 of Sudanese
Leiurus quinquestriatus scorpion venom
Table (25): The effect of s.c. injection of Leiurus quinquestriatus venom alone or after
montelukast on the lethality of mice.
Table (26): The effect of s.c. injection of Leiurus quinquestriatus venom alone or after
hydrocortisone on the lethality of mice
Table (27): The effect of s.c. injection of Leiurus quinquestriatus venom alone or after
indomethacin on the lethality of mice
Table (28): Percentage of survival and average survival time of mice injected with
Leiurus quinquestriatusscorpion venom alone or after montelukast,
hydrocortisone or indomethacin.
Titles of Figures
Fig (1): Percent of mean arterial blood pressure (MABP) measured at different times in
conscious rabbits injected with leiurus quinquestriatus (LQQ) venom.
Fig (2): Serum IL 8 (A) and TNF α concentration of control (0.9%NaCl, 0.05ml kg-1)
and LQQ venom (0.4 mgkg-1) rabbits.
Fig (3): Serum total nitrite (A) and endogenous nitrite (B) concentration of control
(0.9%NaCl, 0.05ml kg-1) and LQQ venom (0.4 mgkg-1) rabbits.
Fig (4): Serum Nitrate concentration of control (0.9%NaCl, 0.05ml kg-1) and LQQ
venom (0.4 mgkg-1) rabbits.
Fig (5): Serum glucose concentration of control (0.9%NaCl, 0.05ml kg-1) and LQQ
venom (0.4 mgkg-1) rabbits.
Fig (6): Serum CK (A) and LDH (B) concentration of control (0.9%NaCl, 0.05ml kg-1)
and LQQ venom (0.4 mgkg-1) rabbits.
Fig (7): Serum AST (A) and ALT (B) concentration of control (0.9%NaCl, 0.05ml kg-1)
and LQQ venom (0.4 mgkg-1) rabbits.
Fig (8): Serum total protein (A) and Albumine (B) concentration of control (0.9%NaCl,
0.05ml kg-1) and LQQ venom (0.4 mgkg-1) rabbits.
Fig (9): Serum BUN (A) and creatinine (B) concentration of control (0.9%NaCl, 0.05ml
kg-1) and LQQ venom (0.4 mgkg-1) rabbits.
Fig (10): Serum CO2 (A) and CL (B) concentration of control (0.9%NaCl, 0.05ml kg-1)
and LQQ venom (0.4 mgkg-1) rabbits.
Fig (11): Serum RBC (A) and WBC (B) concentration of control (0.9%NaCl, 0.05ml kg1
) and LQQ venom (0.4 mgkg-1) rabbits.
Fig (12): Serum basophil (A) and neutrophil (B) concentration of control (0.9%NaCl,
0.05ml kg-1) and LQQ venom (0.4 mgkg-1) rabbits.
Fig (13): Survival distribution function curve of mice injected with L. quinquestriatus
(250 µg kg-1) alone or 2 hours after montelukasr (MK, 20 mg kg--1).
Fig (14): Survival distribution function curve of mice injected with L. quinquestriatus
(300µg kg-1) alone or 2 hours after montelukasr (MK, 20 mg kg-1).
Fig (15): Survival distribution function curve of mice injected with L. quinquestriatus
(250 µg kg-1) alone or 30 min after hydrocortizone (HCTZ, 10 mg kg-1).
Fig (16): Survival distribution function curve of mice injected with L. quinquestriatus
(300 µg kg-1) alone or 30 min after hydrocortizone (HCTZ, 10 mg kg-1).
Fig (17): Survival distribution function curve of mice injected with L. quinquestriatus
(250 µg kg-1) alone or 30 min after indomethacin (IND, 20 mg kg-1).
Fig (18): Survival distribution function curve of mice injected with L. quinquestriatus
(300 µg kg-1) alone or 30 min after indomethacin (IND, 20 mg kg-1).
ACKNOWLEDGEMENT
I must extend my profound appreciation to Prof. Abdelrahim Elsayed Ali,
Prof. Elamin Ibrahim Elnima and Dr. Mohammed Haj-Ali, for their excellent
supervision, invaluable advice and constructive criticism, that helped me get through
may intricate phases during the different stages of preparation of this thesis.
I would like to express my sincere gratitude and cordial thanks to Dr. Amal Fatani,
professor of pharmacology, College of Pharmacy, King Saud University, Kingdom of
Saudi Arabia. My extreme thanks to her, for her encouragement, guidance, enthusiasm
and beneficial advice in the course of writing this thesis.
Very special thanks must go to Dr. Mohammad Ismail Hamed, Professor of
Pharmacology, 6 October University, Egypt, for his superb helpfulness, kind advice and
support which are appreciable and unforgettable.
I which to express my deepest thanks and appreciation to members of National
Antivenom and Vaccine Production Center, King Fahad National Guard Hospital,
Kingdom of Saudi Arabia for their keen help and valuable cooperation during this
work, especially the director Dr Mohammed Al-Eheideb and Production manager Dr.
Mohammed Atef Abuzaid. Their understanding, advice and beneficial aid helped me
tremendously.
I must extend my gratitude to several people in Dongla hospital, Dongla, Sudan (Dr.
Ibrahim, Dr. Abdullah, Dr. Kokwag, biochemist Amany and Dr. Zohir) who have
willingly helped in various ways during the task of collecting the data for clinical part of
this thesis.
It is my pleasure to thank Dr. Abdulkarim Hashim, for his keen assistance and
cooperation and enormous help.
My final and most sincere thanks go to my family. I owe particular thanks to my father
and mother for always encouraging me to follow intellectual pursuits. My appreciation
and great thanks to my husband, Osman for his unfailing support and limitless
encouragement. My thanks to my brothers (Saif, Mubarak and Abdullah) for making
everything worthwhile, and my sisters Nazera and Nujood, for always being there for
me. A special thanks goes to my daughter Azza who had created a lively environment to
which I could devote the many hours required to bring forth this thesis.
Summary
The cumulative actions of scorpion neurotoxins are complex and may be traced to
activation of different ion channels with subsequent release of various transmitters and
modulators including inflammatory mediators. This would lead to various pathological
manifestations including acute respiratory distress syndrome (ARDS), systemic
inflammatory responses syndrome (SIRS) and multiple organ dysfunction or failure
(MOD or MOF), especially in the later stages when hypotension and bradycardia are
dominant.
This study attempted to examine the deleterious effects that occur following scorpion
envenomation, whether in humans or experimental animals. This would shed some light
on the role of the mediators released by the venom toxins and how best to combat their
actions, thus ultimately saving lives. Up to now treatment of scorpion envenomation is
either by antivenoms (not agreed upon by all) and management of signs and symptoms
as they occur. Although some investigators have examined what are the changes,
whether biochemical or hematological, that occur during scorpion envenomation, in
both humans and experimental animals, few have utilized this information to provide
therapeutic tools that would actually combat venom-induced effects.
In this study a preliminary clinical investigation was undertaken to examine scorpion
envenomed victims admitted to the main hospital in Dongla, Sudan. Their signs,
symptoms, laboratory tests, therapeutic intervention and outcome were all recorded in
a six-month period. The study indicated that more than two thirds of those admitted
were male and one-third children, of the latter more than 60% died. Approximately
three quarters of the stung patients seeked medical help within an hour with the
percentage of survivors decreasing as the time of admittance was delayed, probably due
to non-reversible damages occurring from the cascade of transmitters and modulators
released by the venom via its action on ionic channels. It was obvious from the study
that the more severe the signs and symptoms upon admittance, the greater the chance of
a sordid prognosis and death. A higher percentage died of patients admitted with
certain symptoms, more notably presence of convulsions, restlessness, excessive
vomiting, hyperglycemia, acidosis, hypo- or hypertension, brady- or tachycardia,
presence of arrhythmia and/-or signs of respiratory distress. Similarly, the present work
pointed towards a significant correlation between the level of certain biochemical and
hematological parameters and the morbidity following scorpion envenomation, even
when measured immediately after admittance in to the hospital. For instance, a poor
prognosis usually occurred in patients with evidence of altered kidney, liver and cardiac
functions. In addition envenomed patients with high WBC counts had greater morbid
outcomes. This study unfortunately showed a high mortality rate among admitted
envenomed victims, suggesting the inadequacy of the treatment protocol utilized. This
depended on inadequate dose and route of a scorpion antivenom and symptomatic
therapy including hydrocortisone and an antihistamine.
The unacceptable high death rate of patients admitted to Dongla hospital in Dongla
prompted the necessity of performing additional experiments on conscious New
Zealand white rabbits injected with the venom of the scorpion mostly identified by the
stung patients, Leiurus quinquestriatus. This was done to pinpoint exactly what occurs
following scorpion envenomation and how best to combat it. Blood pressure was
measured intermittently up to 12 hours and correlated with selected serum biochemical
and hematological parameters collected simultaneously, including the measurement of
inflammatory modulators such as nitric oxide and the cytokines, IL-8 and TNF.
In the present study, subcutaneous injection of a sub-lethal dose of LQQ venom into
conscious rabbits, caused a triphasic effect on blood pressure consisting of an initial
insignificant reduction, a prolonged increase, followed by a gradual terminal
hypotensive and bradycardic phase that culminated in death. Biochemical and
hematological changes observed in rabbits injected with LQQ venom was comparable to
that seen in stung patients admitted to Dongla hospital. Moreover, the present study
also confirmed the ability of the scorpion venom to enhance release of the potent
vasodilator nitric oxide and the inflammatory mediators cytokines IL-8 and TNF. These
are known to play a role in the venom-evoked terminal hypotension and multiple organ
dysfunctions; the latter was evidenced by the altered biochemical and hematological
parameters.
It is known that cytokines and the activation of inflammatory processes ultimately
lead to several detrimental effects on different systems following scorpion
envenomation. Therefore it was thought necessary to preform a series of protection
experiments, whereas agents that block different steps in the inflammatory process
were administered to mice prior to the injection of LQQ venom, in order to assess their
ability to prolong survival.
Animals were divided into groups and given montelukast (10 or 20 mg kg–1, po.),
hydrocortisone (5 or 10 mg kg–1, iv.), or indomethacin (10 or 20 mg kg–1, iv.). All
animals were then injected subcutaneously with either 0.25 or 0.3mg kg–1 of LQQ
venom. Signs and symptoms of envenomation were recorded and percent survival after
24 hours as well as the time of death of the diseased animals were determined in each
group.
Montelukast, hydrocortisone and indomethacin significantly prolonged the
death time of non-surviving animals as well as the percentage of survived animals per
group, with montelukast having greater protecting power. This study showed that antiinflammatory drugs may play an important role in protection against the lethal effects
of scorpion venoms and might be of great value in ameliorating the detrimental effects
observed with scorpion envenomation and prolonging survival. Moreover, most of the
pathological effects are concentrated during the late terminal hypotensive phase
indicating that early treatment of envenomed victims is essential.
CHAPTER - Ι
LITERATURE REVIEW
LITERATURE REVIEW
1.1 Nomenclature and distribution of scorpions:
Scorpions belong to a relatively small order of the subspecies Arachnoida, class
Arachnomorpha. Over 1400 species of scorpions have been described, however, few are
dangerous to humans, and these belong to the Buthidae family: genera Leiurus,
Androctonus, Parabuthus, Pandinus, Buthus, Buthotus (Old World scorpions) or
Centruroides and Tityus (New World scorpions) (Balozet, 1971; Bϋcherl, 1971; Vachon,
1979). The classification from Africa and the Middle East has been summarized by
Vachon (1966), while the distribution of scorpions in the Northern and Central parts of
Africa and the countries of the Middle East was described by Balozet (1971). According
to Balozet, in Northwest Africa, five species are dangerous; Androctonus australis,
Buthus occitanus, Buthacus arenicola, Androctonus amoreuxi and Androctonus aeneas.
In Libya, A. australis and Leiurus quinquestriatus are found. This formidable last
species is common in Egypt, where it is associated with A. amoreuxi and Androctonus
crassiacuda, an association that also occurs in the Northern and Central parts of Sudan.
Scorpions are mostly confined to tropical and subtropical climates with dispersions
into the temperate zone of both hemispheres (Bϋcherl, 1971), however, some prefer
colder and moister climates. Many species live under stones or sometimes in stone
fences and rock crevices; some occasionally inhabit empty borrows of rodents or
isopods, or even occasionally enter houses and tents. The period of activity of scorpions
extends throughout the warm season, and they may wander on warm nights in winter
(Shulov and Levy, 1978). Moreover, scorpions are nocturnal creatures; during the day
they seek shelter under stones, debris or inside houses.
1.2. Components of scorpion venoms
Scorpion venoms are complex mixtures of mucous, low molecular weight components
(salts and organic compounds), and many basic neurotoxic proteins (Watt and Simard,
1984). They may also contain autacoids such as serotonin (Adam and Weiss, 1958) and
histamine (Ismail et al., 1975). Unlike snake venoms, scorpion venoms are poor in
enzymes (Balozet, 1971), these enzymes and non-protein materials do not seem to play
any role in scorpion envenomation. Because venoms from the family Buthidae are toxic
to humans, they have been better-characterized biochemically and pharmacologically
(Goyffon and Kovoor, 1978). The main toxic component of venoms from this family are
the neurotoxins which are low molecular weight basic polypeptides composed of a single
polypeptide chain cross-linked by four disulfide bridges (Miranda et al., 1970; Rochat et
al., 1979). The specificity of these toxins varies considerably (Zlotkin et al., 1978), and
scorpion venom toxicity in humans has been mainly attributed to the pharmacological
properties of these neurotoxins specially those active in mammals.
1.3. Mechanism of action of scorpion venom toxins
The cumulative actions of scorpion neurotoxins are complex and many in vivo and in
vitro effects may be traced to activation of different ion channels. Scorpion neurotoxins
act mainly on voltage-activated Na+ channels (Watt and Simard, 1984). There are two
types of scorpion toxins known to act on these channels, α- and β-toxins, both types
increase the excitability of neurons, leading to repetitive action potentials and an
increase in transmitters release (Wang and Strichartz, 1983; Borneman and Hahin,
1993). Such transmitters include noradrenaline (Langer et al., 1975), adrenaline (Jacobs
et al., 1978), glutamate, aspartate, gamma butyric acid (Coutinho-Netto et al., 1980),
acetylcholine (Gomez and Farell, 1985), and dopamine (Sirinathsinghji et al., 1989).
Other mediators have also been postulated to be released such as serotonin (CunhaMelo et al., 1973), permeability increasing factor (Leme et al., 1978), kinins (Lahiri and
Chaudhuri, 1983; Fatani et al., 1998), platelet activating factor (Freire-Maia and De
Matos, 1993), and- /- or cytokines (Sofer et al., 1996; Barraviera et al., 1995, Fukuhara
et al., 2003). Although scorpion venoms are classic sources of Na+ channel toxins, these
venoms contain, in addition, minor components that act selectively on voltage-gated or
Ca++ activated K+ channels (Miller et al., 1985; Moczydlwiski et al., 1988; Dreyer, 1990;
Strong, 1990; Blaustein et al., 1991; Crest et al., 1992), chloride channels ( DeBin and
Strichartz, 1991; DeBin et al., 1993; Lippens et al., 1995), and Ca++-gated Ca++-selective
channels in skeletal and-/-or cardiac sarcoplasmic reticulum (Valdivia et al., 1991; El-
Hayek et al., 1995). Thus, certain effects of the venom may be attributed to the action of
neurotoxins on these channels.
1.4. General effects of scorpion venoms
In humans, scorpion venom causes extremely severe local pain and paresthesias, signs
of central nervous system stimulation such as agitation, restlessness, hyperexitability,
vomiting and hypo- or hyperthermia. Evidence for muscular involvement, such as
shivering, increased muscle tone, twitching and convulsion have been observed. Also,
signs of parasympathetic stimulation, such as increased salivation, lachrymation,
perfuse perspiration, gastric hyper distention, diarrhea, and involuntary micturtion,
have been reported. Hyperglycemia, hyponatremia, hyperkalemia, leucocytosis, and
elevated cardiac enzymes levels have also been reported. Respiratory abnormalities may
manifest as irregular respiratory movement, hyperpnea alternating with hypopnea,
bronchial hypersecretion, expiratory wheezing, pulmonary edema, and respiratory
paralysis. Cardiovascular manifestation of envenomation includes brady- or
tachycardia, arrhythmias, hypertension, hypotension, myocardial infarction, heart
failure, shock, and cardiovascular collapse. (Compos et al., 1980; Goyffon et al., 1982;
Dittrich et al., 1995; Ismail, 1995). In experimental animals, scorpion venom cause
vocalization, great agitation, accelerated respiration, hypersalivation, lachrymation,
shivering, muscle fasciculation, periodic spastic contractions, spastic paralysis, slow and
irregular breathing patterns, signs of hypertension and-/- or hypotension, cardiac
arrhythmias, gasping convulsions, cardiac and respiratory arrest and extreme rigidity
of the body after death (Balozet, 1971; Zlotkin et al., 1978)
1.5. Effects of scorpion venoms on skeletal muscles
The stimulatory effects of scorpion venoms on skeletal musculature are among the
most typical manifestations observed following scorpion poisoning in both humans and
experimental animals (Bücheral, 1971; Freire-Maia and Campos, 1989). Many
mechanisms have been postulated to explain the action of scorpion venoms on skeletal
muscles. The vascular contractions evoked by scorpion venom on striated muscle was
attributed to the central action of the venom on spinal motor neurons (Del Pozo and
Anguiano, 1974), while others attributed muscle contracture of the denervated skeletal
muscles to the direct action of venom on the muscle (Yarom and Meiri., 1972; Walther
et al., 1976). In addition, venom-elicited muscle twitches and decurarizing activity was
related to the release of acetylcholine-like substance (Vital Brazil et al., 1973).
Furthermore, an increase in pre-and postjunctional action at the neuromuscular
junction was also suggested for scorpion venoms-/-toxins including LQ venom (Parnas et
al., 1970; Warnick et al., 1976).
1.6. Effect of scorpion venoms on smooth muscles
Scorpion venoms enhance the activity of the autonomic nervous system leading to
release of acetylcholine that cause muscle contracture (Ismail et al., 1975; Zlotkin et al.,
1978; Fujimoto et al., 1979) and exaggerated rhythmic spasmodic activities (CunhaMelo et al., 1973; Tintpulver et al., 1976). In experimental animals, the stimulatory
effects evoked by the venom on smooth muscle was ascribed to prejunctional
mechanisms in which venom enhanced nor epinephrine release (Gwee et al., 1994).
Venom actions on smooth muscle were also attributed to venom-evoked stimulation of
non-adrenergic non-cholinergic nerves such as the release of substance P (Cunha-Melo
et al., 1973; Freire-Maia et al., 1976b) and nitric oxide (Gwee et al., 1996).
1.7. Effects of scorpion venoms on the cardiovascular system
Scorpion venoms in most instances cause a vagal-mediated initial short-lasting
hypotension followed by a pronounced and long-lasting hypertension that is considered
a major etiological factor in the development of cardiac failure and pulmonary edema
(Gueron and Yarom, 1970; Gueron et al., 1980, 1992; Gueron and Ovsyshcher, 1989;
Freire-Maia and Campos, 1987, 1989; Bawaskar and Bawaskar, 1987; Ismail, 1995).
Venom-induced hypertension has been attributed to increased sympathetic stimulation
and neurotransmitter release as a result of activation of the fast Na+ channels and
blocking the Ca++ activated K+ channels (Ismail, 1995). Venom-released catecholamines
would act on α-and β- adrenoceptors (Friera-Maia and Campos, 1989; Sofer and
Gueron, 1992) and possibly activate the renin-angiotensin system (La Grange, 1977;
Radha Krishna Murthy and Vakil, 1990). Other mechanisms implicated in the venomevoked hypertension include aldosterone hypersecretion (Gueron et al., 1992) and a
direct action of the venom on vascular smooth muscle cells ( Savino and Catanzaro,
1979; Wang et al., 1994).
Following the venom-elicited hypertension, there is usually a prolonged late
hypotension resistant to treatment by autonomic blockers and conventional treatment,
and may ultimately lead to shock and death. Postulated mechanisms include a
cholinergic effect, hypovolemia secondary to excessive fluid loss, and possible presence
of large amounts of potent vasodilator substances, such as kinins and-/-or
prostaglandins (Gajalakshmi, 1978; Gueron et al., 1980; Ismail et al., 1992; Sofer and
Gueron, 1992; Ismail, 1995).
Several investigators have described different types of cardiac arrhythmias
following scorpion envenomation. These include sinus tachycardia and bradycardia,
atrial fibrillation; first and second-degree heart block, atrioventricular(AV) dissociation
with accelerated functional rhythm, premature atrial or ventricular beats, ventricular
tachycardia and ventricular fibrillation (Gueron et al., 1967; Gueron and Yarom, 1970;
Bawaskar and Bawaskar, 1989). According to Freire-Maia et al. (1974) and Gueron et
al. (1980), venom-induced bradycardia and conduction defects appear to be due to
venom-evoked release of acetylcholine acting on vagal ganglia and postganglionic nerve
endings. On the other hand, tachycardia and ventricular ectopic beats are arrhythmias
resulting possibly from venom-released catecholamines acting on cardiac βadrenoceptors. Myocardial ischemia and infarction have been observed after scorpion
envenomation and have been explained by relative and/ -or temporary myocardial
hypoxia due to decrease in coronary blood flow during tachycardia and positive
inotropic effects (Campos et al., 1980). An increase in oxygen consumption induced by
venom-released catecholamines and the subsequent electrolyte changes, could also play
a role in the relative myocardial hypoxia and infarction (Freire-Maia and Campos,
1989;
Ismail,
1995).
Scorpion
venoms
appear
to
cause
some
of
their
electrocardiographic abnormalities through the resultant electrolyte changes (Ismail,
1995).
Several authors have investigated heart failure due to scorpion envenomation.
Gueron et al. (1980) ascribed the increased filling pressure of the left ventricle (LV)
principally to an ischemic impairment of LV compliance in association with an increase
in LV after- load. Both phenomena were attributed to the outpouring of catecholamines
usually evident in scorpion envenomation. It is also quite possible that the
catecholamine-induced increase in myocardial contractility is of short duration and is
followed by “stunning” of the myocardium with decreased systolic performance as a
result of increased myocardial oxygen demand and decrease in supply (Gueron and
Sofer, 1990). The experimentally proven effects of scorpion venom on Na+, K+ or Ca++
channels of cardiac cells may also play a role in the pathogenesis of cardiac dysfunction
(Gueron and Sofer, 1990; Abroug et al., 1991).
1.8. Effects of scorpion venoms on the respiratory system
Clinical reports of victims stung by scorpions described different types of abnormal
respiratory movements that may terminate in death (Ismail, 1995). Venom-elicited
respiratory abnormalities have been additionally reported in experimental animals (Del
Pozo, 1968; Freire-Maia et al., 1970, 1973, 1976a; Ismail et al., 1972, 1973; Stahnke,
1978). These include, tachypnea, hyperpnea, abnormal periodic breathing patterns with
gasping and apneic episodes, stridor with expiratory wheezing, and-/-or respiratory
failure (Stahnke, 1950; Gueron et al., 1967; Zlotkin et al., 1978; Campos et al., 1980;
Gueron and Ovsyshcher, 1987; Freire-Maia and Campos, 1987, 1989; Sofer and
Gueron, 1988). Various mechanisms were proposed to explain these effects, including
an action on the central nervous system (Magalhaes, 1938; Del Pozo, 1968), the carotid
bodies (Patterson and Wooly, 1971; Ismail et al., 1972, 1973) and the afferent vagal
fibers (Freire-Maia et al., 1976b).
Several investigators have observed that many patients stung by scorpions die with
acute pulmonary edema (Gueron and Yarom, 1970; Gueron et al., 1967; Campos et al.,
1980; Freire-Maia and Campose, 1989; Ismail, 1995, De-Matos et al., 2001). The
pathogenesis of lung edema induced by scorpion venom is very complex, and a number
of postulations have been proposed. As the scorpion venom induces acute arterial
hypertension, it seems likely that the hypertension may also have an effect on the
venom-evoked edema formation (Freire-Maia et al., 1978; Freire-Maia and Campos,
1989). In addition, Rossi et al. (1974) indicated that pulmonary edema might stem from
a modification of the alveolus-capillary barrier induced by pulmonary toxins.
Moreover, acute left ventricular failure resulting from massive catecholamine release or
myocardial damage induced by the venom has been suggested as a possible pathogenic
mechanism (Gueron and Yarom, 1970; Freire-Maia et al., 1978; Gueron et al., 1980). A
non-cardiogenic origin of pulmonary edema has likewise been proposed by other
authors (Rahav and Weiss, 1990; Mathur et al., 1993; Amaral, 1994). It has been
postulated that scorpion venom-evoked pulmonary edema may be due, at least in part,
to release of mediators such as permeability increasing factors, platelet activating
factors, leukotrienes, kinins, prostaglandins, and/ -or cytokines (Leme et al., 1978;
Freire-Maia and De Matos, 1993; De Matos et al., 1997; Fatani et al., 1998).
1.9. Scorpion envenomation and inflammation
The release of cytokines and other mediators, related to the inflammatory process
may additionally account for the several manifestations observed following scorpion
envenomation. For example, acute respiratory distress syndrome (ARDS) has been
reported in children stung by scorpion venoms (Sofer et al., 1996; Meki and Mohey ElDean, 1998; Magalhaes et al., 1999), a syndrome that may encompass scorpion venomelicited non-cardiogenic pulmonary edema. Acute respiratory distress syndrome is a
well-defined clinical entity that is invariably associated with increased liquid in the lung.
(Ronaldo and Ingram, 1991). This syndrome is related to the uncontrolled liberation
and production of cytokines and other products of activated macrophages, lymphocytes
and tissue resident cells. The major offenders appear to be monocytic phagocytes and
polymorphonuclear leukocytes that adhere to endothelial surfaces and undergo a
respiratory burst to inflict oxidant injury and release mediators of inflammation such
as leukotrienes, thromboxanes, and prostaglandins that can lead to acute lung injury.
Initially, the injury to the alveolocapillary membrane result in leakage of liquid,
macromolecules, and cellular components from the blood vessels into the interstitial
space and, with increasing severity, into the alveoli. Alveolar collapse occurs secondary
to the effect of the alveolar liquid. The regional dysfunction is nonhomogeneous; it leads
to severe ventilation perfusion inbalance and the shunting of blood through regions in
which alveoli are collapsed or filled with liquid. The lungs are heavy, edematous, and
nearly airless with regions of hemorrhage, atellectasis, and consolidation engorgement
of vessels with red blood cells, and aggregates of platelets and polymorphonuclear
leukocytes along with interstitial and alveolar hemorrhage is common (Dietch, 1992).
Several of these effects have been observed following venom -induced pulmonary edema
(Gueron et al., 1990; Sofer and Gueron, 1990; Freire-Maia and De-Matos, 1993).
However, the mechanisms underlying the ability of scorpion venom to initiate the
inflammatory cascade and the consequent increase in vascular permeability of the lung
are not clear.
Further evidence for the ability of scorpion venoms to trigger inflammatory
responses comes from reports of severely envenomed victims presenting with clinical
signs of systemic inflammatory response syndrome (SIRS), a condition that may be
involved in the pathogenesis of shock, cardiac dysfunction and pulmonary edema, all of
which are present after scorpion envenomation (Amaral et al., 1993, 1994). Systemic
inflammatory response syndrome may be caused by different microorganisms, noninfectious processes such as release of cytokines, tissue injury and-/-or pancreatitis, are
effects also present in scorpion envenomation, all these can result in SIRS and may lead
to organ dysfunction and ultimately to multiple organ failure (MOF) (Sofer et al., 1996).
Moreover, Scorpion venoms can stimulate the neuro-endocrine immunological axis
by its ability to release catecholamines, corticosteroids, bradykinins and prostaglandins;
agents proven to induce the release of immunological mediators (Chaudry et al., 1989).
Mekki and Mohey-El-Dean (1998) stated that cytokines, in conjunction with the neuroendocrine axis, play a major role in the responses to venom-evoked tissue injury
eventually leading to MOF. In addition, the direct effect of the venom on different
organs may also play a role in the venom-induced MOF (See reviews by Freire-Maia
and Campos, 1989; Ismail, 1995). Multiple organ failure has been documented to occur
after a number of diverse clinical conditions including intoxication and tissue injury
(Bone, 1996).
Thus, it appears from the different signs and symptoms in both experimental and
clinical studies following scorpion envenomation, that scorpion venom stimulates the
neuroendocrinal-immunological axis and lead to release of several inflammatory and
immunological mediators as bradykinins, prostaglandins and cytokines.
1.10. Cytokines and inflammation
Cytokines, which are released in response to inflammation, are a diverse group of
proteins, usually glycoproteins, of relatively low-molecular weight. They regulate
important biological processes, such as cell growth and differentiation, cell activation,
inflammation, immunity, tissue repair, fibrosis, and morphogenesis (Akira et al., 1990;
Paul and Seder, 1994).
Initially, cytokines were thought to be produced only by
immune cells upon various insults, such as microorganisms and their products,
antigens, cell-to-cell interactions, cytokines, and other environmental changes.
Nowadays, it is well accepted that other cells produce cytokines when challenged with
various environmental or inflammatory insults, and these molecules are the soluble
messages of cell communications (Voronov et al., 1999).
Cytokines usually act in
picomolar concentrations through specific high -affinity cell surface receptors, which
transmit cytokine signals to the nucleus. They act mainly in a paracrine and autocrine
manner on neighboring cells and themselves, but they can also act in an endocrine
fashion on distant cells (Andus, 1991).
Cytokines mediate all phases of the inflammatory responses, both local and systemic,
and their network determines the outcome of these responses. (Dinarello, 1996; Lijne,
1996). Activation of macrophages during inflamation release a broad spectrum of
cytokines and inflammatory mediators; IL-1, IL-6 and TNF-α are of the utmost
importance as pro-inflammatory cytokines, since they produce a wide spectrum of
biological activities that help coordinate the body responses to infections (Cybulsky et
al., 1988; Dinarello, 1994, 1996). At the inflammation site other cellular events, such as
mast cell degradation in addition to platelet aggregation and activation, can also result
in the release of mediators, which are chemotactic for macrophages plus monocytes and
activate their functions (Radermecker et al., 1994; Razin et al., 1995).
There are 2 types of T-helper cells (Th) that secret cytokines in response to antigenic
stimulation; Th1 cells secrete mainly IFN-γ, IL-2 and TNF-β whereas Th2 cells secrete
cytokines that mediate humeral immunity mainly IL-4, IL-5, IL-10 and IL-13. (Gollob
et al., 1996; Wang et al., 1996). Th2 responses are anti-inflammatory, because they
inhibit the generation of pro-inflammatory cytokines, especially IL-1 and TNF by
macrophages (Paul and Seder, 1994). IL-12 is the major cytokine that stimulates Th
cells differentiation towards Th1 cells (Powrie and Coffman, 1993; Paul and Seder,
1994). While IL-4 stimulate Th cells differentiation towards TH2 (Pene et al., 1988;
Scott, 1993; Romagnani, 1994). Other factors, such as nature of the antigen, the dose,
the protocol and route of immunization, and the genetic responsiveness of the host may
affect the polarization of Th cell responses (Varney et al., 1993; Jutel et al., 1995;
Mchugh et al., 1995).
The net amount of pro-inflammatory cytokines especially the “alarm cytokines” (IL-1
and TNF) and the duration of their secretion determine the nature of the inflammatory
responses, both local and systemic. Low amounts usually result in local inflammation,
while high quantities may result in septic shock (Moldawer, 1994; Tracey and Cerami,
1994; Bemelmans et al., 1996). The local effects include the activation of vascular
endothelium, increase in vascular permeability, and access of leucocytes into the
affected tissue and their activation, and local tissue destruction. The systemic
manifestations include fever, the acute-phase responses, and inductions of systemic
shock in severe inflammatory processes (Voronov et al., 1999). As the circulation
becomes flooded with inflammatory mediators, the integrity of the capillary walls is
destroyed. Cytokines spill out into end organs, producing additional sites of damage and
this can result in multiple organ dysfunctions and death may ensure (Bone, 1996).
1.11. Nitric oxide and inflammation
Nitric oxide (NO) is a major secretory product of mammalian cells that initiates
host defense, homeostatic and development functions by either direct effect or
intercellular signaling. NO is the product of a five-electron oxidation of the amino acid
L-argenine mediated by one of three isoforms of nitric oxide synthase (Moncada et al.,
1989). As a direct effector, NO is thought to activate regulatory proteins, Kinases, and
proteases that are directed by reactive oxygen intermediates (Schreck and Baeuerle,
1991). As a messenger molecule, NO covalently interacts with target molecule based on
redox potential (Nathan, 1992). Activation of the immune system is associated with an
increase in macrophage NO production (Stuehr and Marlwtta, 1985). NO exerts a
variety of homeostatic influences as an activator of soluble guanylyl cyclase (Ignarro,
1990), a neuronal potentiator, a peripheral nervous system neurotransmitter
(Garthwaite, 1991), and a contraction regulator of both smooth muscle and vascular
tissue (Conner and Grisham, 1995). In addition, NO has been linked to the formation of
olfactory (Kendrich, et al., 1997) and synaptic memories and remodeling (Nowad, 1992)
Several evidences have immerged for the possible role of nitric oxide (NO) release in
several effects observed during scorpion envenomation. Meki and Mohey El-Dean
(1998), demonstrated that serum NO levels were significantly increased in scorpion
envenomed children and that these levels correlated well with the severity of the case,
the degree of persistent hypotension, and showed a direct relationship with organ
failure score. The authors attributed the increase in NO to venom-evoked increased
release of acetylcholine, bradykinins and-/-or cytokines. NO is a mediator, modulator
and pathological entity that plays a role in several diseases including asthma and
neurodegenerative disorders (Moncada, 1992), in addition, to its significant role during
host defense and immunological reactions (Anggard, 1994).
1.12. Treatment of scorpion envenomation
Several protocols have been advocated to treat the multitude of the scorpion venom
elicited pathological changes in different systems. Freire-Maia and Campos (1987, 1989)
recommended treatment of mild cases of envenomation with symptomatic measures
and/ -or antivenom, and severe cases with symptomatic measures, support of vital
functions and i.v. injection of antivenom. Sofer and Gueron (1988), however,
recommended close monitoring of patients, for pulmonary or central nervous system
complications, if hypertension is present i.v. hydralazine can be used. The majority of
investigators considered neutralizing the venom itself by the use of antivenom the only
specific treatment for scorpion sting (Freire Maia and Campos, 1989; Rahdakrishna
Murthy et al., 1992; Ismail et al; 1994). However, some investigators are not convinced
of the value of the antivenom in the prevention and abolition of the cardiovascular
manifestations (Bawaskar and Bawaskar., 1994; Gueron et al; 1992). Ismail (1994)
recommended the use of antivenom and appropriate initial treatment of fever, vomiting
with dehydration and convulsions to be given. Few have tried to combat the venomevoked effect on the inflammatory process, which manifested as ARDS, SIRS and MOF.
Thus, it seems that the ability of scorpion venom toxins to act on specific channels, the
resultant cascade of substances reported to be released by the venom, and the
consequent activation of several systems including the inflammatory responses, may
explain the pathological complications observed in different systems following scorpion
envenomation. Much work is needed to verify the involvement of relevant mediators of
the inflammatory process in scorpion envenoming and whether blockage of these
mediators would prove to be of any value in its management.
CHAPTER - ΙΙ
INTRODUCTION
INTRODUCTION
Scorpion envenomation is a health hazard that must be reckoned with, especially in
countries where scorpions are abundant and lifestyle necessitates coming in close
encounter with them (Ismail, 1995). Scorpion stings may cause more deaths than snake
bites in several parts of the world (Warrell, 1993). According to this author the most
dangerous genera are Leiurus and Androctonus (North Africa and Middle East),
Parabothus (Southern Africa), Tityus (Latin America), Centruroides (Mexico and North
America) and Mesobothus (India).
Several investigators have studied envenomed patients in various countries, for
example Egypt (Meki et al., 2003 a, b), Tunisia (Abroug et al., 1995), Algeria (Alamir et
al., 1992), Morocco (Ghalim, 2000), Zimbabwe (Bergman, 1997), South Africa (Muller,
1993), Saudi Arabia (El-Amin, 1992; Dittrich et al., 1995; Mahaba, 1996; Al-Asamari
and Al-Seif, 2004), Jerusalem (Dudin et al., 1991); Iran (Radmanesh, 1990), North
America (Curry et al., 1984; Russel and Madon, 1984; Bond, 1999), Mexico (Calderon et
al., 1996, Osnaya-Romero, 2001), Argentine (De Roodt et al., 2003), Brazil (Corrado et
al., 1968., Freire-Maia and Campos, 1989; Fukuhara et al., 2003), India (Bawaskar and
Bawaskar, 1991; Das et al., 1995) and Australia (Isbister et al., 2003). According to these
investigators regardless of the different species and geographical location, signs and
symptoms following scorpion envenomation are more or less similar. These include:
intense local pain, vomiting, diarrhia, hypersalivation, sweating, involuntary
micturition, hyponatremia, hyperkalemia, leucocytosis, elevated cardiac enzymes,
hypertension or hypotension, congestive heart failure, myocardial ischemia and
infarction, arrhythmias, pulmonary edema, acute respiratory distress syndrome
(ARDS), systemic inflammatory responses syndrome (SIRS), acute pancreatitis and
multiple organ failure (MOF) (Campos et al., 1980; Freire-Maia and Campos 1989;
Dittrich et al., 1995; Meki and Mohey El-Dean 1998; Gueron et al., 1992; Meki et al.,
2003 b)
Treatment of scorpion envenomation is generally symptomatic, with no clear
consensus between the different practitioners on the ideal treatment protocol.
During the last decades, death rates following scorpion envenomation were variable
and ranged from 0.36% in Mexico, 1.86% in Brazil, 2% in Algeria, 7% in southern
Libya, (Warrell 1993; Mahab 1996; Garcia et al., 2003; Fukuhara et al., 2003) and 4-8%
in Saudi Arabia (Ismail et al., 1990; Mahaba 1996). Factors cited for the high death
rate, especially in less developed countries included presence of dangerous species,
inadequate treatment facilities, illiteracy of patients, late arrival of patients,
inappropriate medication (Fatani, 1990). Thankfully, in recent years there has been a
surge of interest in scorpion envenomation and several mechanisms of actions have
emerged both on ion channels and different systems of the body (Harvey et al., 1994;
Ismail, 1995; Fatani et al., 1998, 2000; Magalhaes et al., 1999; Trasiuk, 1994; Meki et al.,
2003a; Fukuhara et al., 2003) . This has led to the upgrade in treatment modalities and
a lessening in mortality rates in several countries. For example in Saudi Arabia death
rates have been reported to have dropped from 4-6 to <0.05 during the last decade
(Ismail, 1994) and in Mexico, after the use of appropriate antivenom the rate dropped
from 0.36% to < 0.01 (Garcia et al., 2003). The severity of scorpion envenoming and the
high incidence of mortality especially among children, attributed mainly to cardiorespiratory pathology, have stimulated the investigation of the scorpion-envenoming
syndrome. The cardiovascular effects have been described as occurring in two phases,
an initial inotropic phase characterized by hypertension, tachycardia, and increased
myocardial contractility, followed by a hypokinetic stage with hypotension and
impairment of left ventricular systolic functions (Freire-Maia and Campose, 1989;
Gueron et al, 1992; Fatani, et al., 2000; Fukuhara, et al, 2003). The increased blood
pressure, left ventricular pressure and contractility have been related to the release of
catecholamines by the sympathetic nervous system and adrenal glands (Freire-Maia
and Campose, 1989). On the contrary, the prolonged late hypotension observed in
scorpion envenomation, that may ultimately lead to death, was postulated to be due to
the venom-evoked cholinergic effects, hypovolemia secondary to excessive fluid loss,
and/-or the possible presence of large amount of potent vasodilator substances, such as
nitric oxide, kinins, prostaglandins and/ -or cytokines (Gajalakshim, 1978; Gueron et
al., 1980; Ismail et al., 1992; Sofer and Gueron, 1992; Ismail, 1995; Meki and Mohey ElDeen, 1998; Magalhaes et al 1999; Meki et al., 2003a).
In addition, scorpion venoms may lead in both human and experimental animals to
tachypnea, hyperpnea, gasping and apneac episodes and / -or cardiogenic and noncardiogenic pulmonary edema [PE](Gueron et al., 1967; Campose et al., 1980; Gueron
and Ovsyshcher, 1987; Sofer and Gueron, 1988; Freire- Maia and De Matos, 1993; De
Matos et al., 1997; D’ Suze et al., 1999; Andrade et al., 2002, Bertazzi et al., 2003).
Venom-elicited tachypnea and abnormal periodic breathing patterns were explained by
the action of scorpion venom on the central nervous system, the carotid bodies and / -or
the afferent vagal fibers (Del Pozo, 1968; Ismail et al., 1973; Freire-Maia et al., 1976 a,
b) On the other hand, cardiogenic PE was related to acute left ventricular failure
resulting from massive release of catecholamine or myocardial damage induced by the
venom (Gueron and Yarom, 1970; Gueron et al., 1980). Conversely, non-cardiogenic PE
was probably related to an increased vascular permeability produced by histamine,
bradykinins, prostaglandins, platelet activating factors, cytokines, and kinins (Campose
et al., 1980; Gueron and Ovsyshcher, 1987; Sofer and Gueron, 1988; De Matos, 1997;
Fatani et al., 1998; Meki and Mohey El-Dean, 1998; Andrade et al., 2002; Meki et al.,
2003a). In addition to the cardiac and respiratory system, others are affected by
scorpion venom encompassing liver and kidney function, inflammatory response,
metabolism in addition to circulation and blood cells (Shnkaran et al., 1987; Correa et
al., 1997), due to its action on Na+ channels (Riccioppo Neto, 1983; Watt and Simard,
1984; Matos et al, 1999), and the resultant cascade of neurotransmitters and mediators
that are released (Sampanio et al., 1996; Barraviera, 1996; Fukuhara et al., 2003).
Human envenoming by scorpions is a life-threatening hazard and fatal accident
commonly reported in many regions of the world, especially in children (Freire-Maia et
al., 1994). Neuro- and cardio-toxins are present in the majority of scorpion venoms
(Freire-Maia and Campos, 1989), and from a pharmacological point of view, these two
toxins are the most important components of scorpion venoms. The cumulative actions of
scorpion neurotoxins may be traced to activation of different ion channels. The venom
action on sodium channel of neuronal terminals may lead to depolarization of axonal
membrane, releasing several neurotransmitters, which affect various systems, including
gastrointestinal tract, respiratory, cardiovascular and nervous systems (Freire-Maia et
al., 1974; Gueron et al., 1992; Freire-Maia and Campos, 1989).
Despite venom variability among different species of scorpions, human envenomation
produces closely similar clinical manifestations terminating, in severe cases, in pulmonary
edema, seizures, cardiac failure and shock (Gueron and Yarom; 1970, Ismail, 1994). In
addition to neurotransmitters, other mediators such as those affecting the inflammatory
processes, have been released following scorpion envenomation including kinins,
ecosanoids, cytokines, platelet activating factors and nitric oxide (Ismail et al., 1992; De
Matos et al., 1997, 1999; Fatani et al., 1998; Meki and Mohey El-Dean, 1998; Fukuhara et
al., 2003; Meki et al., 2003a). following scorpion envenomation, the release of cytokines
and other mediators, may account for several of the inflammatory manifestations
observed such as acute respiratory distress syndrome (ARDS), systemic inflammatory
responses syndrome (SIRS) and multiple organ failure (MOF) (Freir-Maia and Campos,
1989; Meki and Mohey El-Dean, 1998; Meki et al., 2003b). ARDS, a syndrome that is
related to the uncontrolled production and liberation of cytokines and other products of
activated macrophage, lymphocytes and tissue resident cells, has been reported in
children stung by scorpions (Sofer et al., 1996; D’Suze et al, 1999) and may encompass the
venom elicited non-cardiogenic pulmonary edema observed by several investigators
(Deitch, 1992; Sofer et al., 1996; Meki and Mohey El-Dean, 1998; Magalhaes et al., 1999).
Furthermore, clinical signs and symptoms of SIRS, a condition that may be caused by
the massive release of cytokines and may be involved in the pathogenesis of shock,
cardiac dysfunction and pulmonary edema (Bone, 1996) has been documented in severely
envenomed victims (Amaral et al. 1994; Sofer et al., 1996). Moreover, it was stated that
cytokines, in conjunction with the neuro-endocrine axis, might play a major role in the
response to venom-evoked tissue injury eventually leading to MOF (Sofer et al., 1996).
Further studies are needed to examine these venom- induced effects.
In general, treatment of the scorpion-stung victims usually includes an antivenom,
although its use remains controversial, and symptomatic therapies including
vasodilators, autonomic blockers, Glucose-insulin infusion and inotropic drugs
(Campos et al., 1980; Murthy and Vakil, 1988; Karnad et al., 1989; Gueron and Sofer,
1990; El-Amin, 1992). Although several investigators have mentioned the increased
levels of selected cytokines following scorpion envenomation, few have tried to combat
its effect and thus attempt to ameliorate venom induced ARDS, SIRS, and MOF.
Ismail et al. (1992) demonstrated the effectiveness of indomethacin and hydrocorisone,
anti-inflammatory agents, in prolonging the survival of LQQ envenomed rats. In
addition, Fatani et al. (1998) demonstrated that administration of aprotinin, a kallikren
kinin inhibitor, or icatibant, a B2 bradykinin antagonist, significantly attenuated the
venom-elicited cardio-respiratory abnormalities and increased the survival of
envenomed rabbits. This would suggest a role for inflammatory mediators,
prostaglandins or kinins in venom-evoked effects such as cardio-respiratory changes,
ARDS, SIRS, and MOF.
Unfortunately in many developing countries where facilities and up to date medical
advancements are not totally adhered to, high death incidence due to scorpion
envenomation remains a problem. Sudan, my country, is a developing nation that is
trying its best despite its political problems. Although Sudan is inhabited by a number
of different scorpion species, official data on the number of envenomations that occur in
Sudan, especially in cities other than the capital such as my hometown Dongla, are
scarce and incomplete. However, from personal observations and communications with
health professionals in a major hospital in Dongla, the death rate following scorpion
envenomation was thought to be unacceptably high. Thus, it was thought necessary to
attempt to study envenomation cases and their outcome during a certain length of time
in Dongla hospital, a representative of the only hospital in the third major city in Sudan,
Dongla City.
Thus in this study it was thought necessary to take samples of the scorpions in that
region and perform an experimental study to see what exactly occurs in animals
injected with crude venom isolated from Leiurus quinquestriatus collected from that
region. More importantly, it was thought essential to examine the different stages of
scorpion envenomation and the damages that take place wither early or late, to be able
to pinpoint what occurs in each stage and to understand why therapy was not effective
in the case of deceased patients.
Consequently, the aim of this study is to discern whether blockers of the different
steps in inflammatory sequence of events of scorpion envenoming could reduce the
venom-elicited inflammatory responses and prolong survival. This may lead to the
immergence of new treatment modalities that would help save lives.
CHAPTER - Ι Ι Ι
MATERIAL AND METHODS
MATERIAL AND METHODS
3.1. Materials
3.1.1.
Materials for Preliminary clinical study, Clinical Characteristics of scorpionenvenomed Patients admitted to Dongla Hospital in Sudan
The following drugs were utilized in Dongla Hospital in Sudan for
treatment of scorpion envenomed patients: Anti-scorpion serum, polyvalent
(Vacsera, Egypt, 1 ampoule, 40 LD50 / ml, i.m.), hydrocortisone (10 mg kg1
, i.v.), chlorpheniramine (0.09 mgKg-1, i.v.), xylocaine 1% (0.5 ml, im),
3.1.2. Materials for Correlation between blood pressure and selected biochemical and
hematological parameters, in conscious rabbits injected with L. quinquestriatus
venom
Leiurus quinquestriatus quinquestriatus (LQQ) venom was obtained from
mature scorpions, collected from Aswan in Egypt, by electrical stimulation of
the telson as described by Ismail et al., (1973). The venom was then extracted
by distilled water, centrifuged and the supernatant freeze-dried using HTRO
SICC lyophilizer (Germany). The dried venoms were kept at – 200C, when
required, the venom was reconstituted by addition of 0.9 % NaCl solution
Interleukin 8 (IL8) and Tumor necrosis factor-alpha (TNF-α) Immunoassay
Kits were purchased from Biosource international (Europe S. A.). Nitric oxide
assay kit was purchased from R $ D systems (USA), and Heparin from BBreun (Germany), whilst all other chemical used (analytical grade) were
purchased from Sigma (USA).
3.1.3. Materials for effect of Selected Anti-inflammatory Drugs on the Lethal
actions of Leiurus quinquestriatus Scorpion Venom
Leiurus quinquestriatus quinquestriatus (LQQ) venom was obtained from
mature scorpions by the same method described in (3. 2.1)
Montelukast and indomethacin were purchased from Merck Sharp and Dohm
(UK), hydrocortisone from Upjohn (USA).
3.2. Programming and methodology
3.2.1. Clinical Characteristics of scorpion-envenomed Patients:
This study was the first step of a larger intended project that would be one of many in the long journey towards the improvement of
health care in my hometown Dongla in Sudan, a country known for its flourishing fauna and abundance of snakes and scorpions. It was
decided that the first stage of this clinical project, which is represented in this study, would constitute approaching the main hospital in
the city and recording information about patients admitted in a certain period of time following scorpion envenomation. The second
stage, which would be undertaken after this project’s completion, would comprise a more comparative and comprehensive study of
envenomed patients in other hospitals and regions of Sudan. Thus, this current project covering the first stage entailed the division of
the work into five phases
• Phase I:
Project design
• Phase II: Patient selection
• Phase III: Preparation of Patient information sheets
• Phase IV: Methods used for laboratory investigations
• Phase VI: Distribution and collection of sheets
• Phase VII: Data Analysis
3.2.1.1. Phase 1: Research Design
A search of publications related to clinical scorpion envenomation in both
developed and developing countries was performed and the articles were reviewed,
concentrating on signs, symptoms, management and outcomes. In addition, to the
search, several telephone calls and personal meetings were conducted with the
health professionals in Dongla Hospital to assess the scope of the problem. Lists of
names are available upon request.
The first logical step in the long journey towards the improved health care
management in Dongla city in general and scorpion envenomed patients, in
particular, was to assess the present situation of the latter-mentioned patients in a
major hospital in Dongla. The project would gauge the number of admitted patients,
the handling of their condition and outcome. This would shed some light on benefits
and pitfalls of existing practices and methods for improving institutional efficiency.
3.2.1.2. Phase 2: Patient Selection
Scorpion envenomed patients admitted to Dongla Hospital with moderate to
severe signs and symptoms during a 6 month period (beginning of January 2002
to end of July 2002) were the target population. It would have been ideal to
survey cases of scorpion envenomation in different hospitals all around Sudan.
However, due to the difficulty of undertaking such a large venture while I am at
the present spending a lot of time in Saudi Arabia, therefore, a hospital in my
hometown Dongla was chosen in this preliminary study. The rest would be
undertaken in later stages. The patients were chosen for this study if they met
the inclusion criteria.
Inclusion criteria included: All patients admitted to Dongla Hospital within the
specified time, were stung by a scorpion and exhibited moderate to severe signs
and symptoms of envenomation.
3.2.1.3. Phase 3: Preparation of patient information sheets
A patient information sheet similar to standard forms utilized in hospitals was
prepared, with each sheet comprising of 3 sections. The first section asked
patients about their demographic personal profiles, such as name, age, weight
and sex, in addition to time lapse between sting and entry to hospital, site of sting
and type of scorpion. The second section concentrated on the physicians
assessment of their signs and symptoms, in addition to treatment provided and
outcome. The third section contained all laboratory tests performed.
3.2.1.4. Phase 4: Methods used for laboratory investigation:
3.2.1.4.1. Determination of blood Glucose:
This was performed according to the method of Teller (1956). The test
based on the dehydrogenation of beta-d- glucose to gluconic acid and
hydrogen peroxides by glucose oxidase enzyme. The hydrogen peroxides
is split by peroxidase in the presence of di-ammino 2, 2-azino-bis )3-ethylbenzthiazoline-6-sulfonate). The absorbance was measured at 436 nm.
3.2.1.4.2. Determination of blood urea nitrogen
This was carried out using an adaptation of the Berthelot’s reaction as
described by Fawcett and Scott (1960). Urea is hydrolyzed by urease to
ammonium carbonate, the ammonium ions react with hypochlorite and
phenol to give a blue endophenol, the concentration of which is
proportional to the amount of urea present in the sample.
3.2.1.4.3. Determination of blood creatinine:
This was carried out according to the method described by Hare (1950)
creatinine reacts with picric acid in the presence of sodium hydroxide to
form a red alkaline creatinine picrate complex. The absorbance is
measured at 546 nm. In a spectrophotometer.
3.2.1.4.4. Determination of blood total protein:
This was done according to the method described by Henry et al., (1974).
Pyrogallol red complexes with proteins in an acid environment containing
molybdate ions. The resulting blue-colored complex absorbance is measured
spectophotometrically.
3.2.1.4.5. Determination of blood white blood cell count:
Peroxidase method was used for determination of total WBC, The cells was
stained by metaloperoxidase and the cell size was determined and the number of
white blood cells and their population were counted.
3.2.1.5. Phase 5: Distribution and Collection of patient information sheets
A covering letter was initially faxed to director of Dongla Hospital asking for
permission and explaining the intent of the project. It also requested the help of
selected physicians and laboratory personnel that would participate in admitting
patients, recording signs and symptoms and following up laboratory investigations.
Upon the hospital’s approval, a number of meetings were conducted by the main
researcher and participants explaining how to complete the sheet. At the end of the
designated 6-month period, information sheets about all envenomed patients
admitted to the hospital during that period would be collected by myself. The initial
meetings with the participants were pursued by additional visits, telephone calls or
faxes to verify or further explain specific aspects to complement the work.
Unfortunately after 6 month only 30 clinical sheets were handed in, and due to
shortage of time and the need to continue the rest of the proposed protocol in the
thesis, it was decided to consider it a preliminary clinical investigation to be
expanded at a later time. Reasons for the low turnover, despite abundance of
scorpions in the region, was explained by the hospital personnel as the preference of
the population to home remedies and herbal practitioners, in addition to their
natural fear of hospitals. Moreover, only patients that were admitted to the hospital
with moderate to severe signs and symptoms were included. This excluded all
patients stung by scorpions and seen in outpatient clinics.
3.2.1.6. Phase 6: Data Analysis
The data in the present study was intended to be as a preliminary clinical
investigation. Percentages of patients exhibiting a specific sign or symptom or
laboratory level were calculated and results were analyzed utilizing the Fishers
Exact CHI square test and the Statistical Program Instat, version 2.0.
3.2.1.7. Study Limitations
The limitations of this study are consistent with that observed with similar
preliminary clinical investigations. Moreover, inclusion of only moderate to
severely envenomed victims may have inadvertently excluded others seen in the
outpatient clinics of the same hospital or even other hospitals outside the city in
the surrounding regions. Furthermore, it did not address victims seen by other
types of practitioners and take into account the diverse and medically suspicious
nature of Dongla inhabitants. Adversely, physicians and technicians who chose
to participate in the study may have neglected to include patients, probably due
to factors outside of their control and due to the lack of day-to-day supervision
by main investigator. This was not possible due to prior commitments in Saudi
Arabia. Additional studies are required to determine if the number of cases in
this study reflect true numbers of severely envenomed victims. Unfortunately,
the limited number of patients narrowed the amount of analysis that could be
performed and entailed categorizing it as a preliminary clinical investigation.
3.2.2. Animal preparation and protocol
During all experiments, the institution's guide for the care and use of laboratory
animals was followed. Conscious Buskat rabbits (2.82 Kg ± 0.09, New Zealand
white) were heparinized (1000 IU kg
–1
via marginal ear vein) and prepared for
blood pressure recording as described by Fatani (1990). Arterial blood pressure was
measured from the left central ear artery via a force displacement pressure
transducer connected to physiograph (Narco Biosystems, USA). In addition, for the
biochemical and hematological determinations a cannula (Gauge 18) filled with
heparinized saline was inserted into the right central ear artery and samples
collected at 0, 1, 3, 6 and 12 hours after venom injection. Only animals that survived
up to the 12 hr time limit of the experiment were included in this study and at the
end of the experiment, the animals were killed by an overdose of diethyl ether. In
time-matched control experiments, rabbits were injected with 0.9% NaCl (0.05 ml
kg-1). Lung / body weight index, indicative of lung water content was calculated by
dividing the lung weight/ total body weight and multiplying by 100 (Almeida et al.,
1976).
3.2.2.1. Methods used for biochemical determinations in rabbit serum
3.2.2.1.1 Determination of serum cytokine IL 8:
The assay was based on an oligoclonal system (Baggiolini et al., 1989), in which a
blend of monoclonal antibodies (Mabs) directed against distinct epitopes of IL8 were
used. Standards or samples containing IL8 reacted with capture monoclonal
antibodies coated on the microtiter well and with a monoclonal antibody(Mab2)
labeled with horseradish peroxides (HRP). After an incubation period allowing the
formation of a sandwich: coated Mabs1- IL8- Mabs2-HRP, the microtiter plate was
washed to remove unbound enzyme labled antibodies. Bound enzyme-labeled
antibodies were measured through a chromogenic reaction. Chromogenic solution
(TMB+H2O2) was added and incubated. The reaction was stopped with the addition
of stop solution (H2SO4). The amount of IL8 turnover was determined
colorimetrically by measuring the absorbance, which is proportional to the IL8
concentration, and the microtiter plate was then read at 450 nm using microtiter
plate reader (Linearity up to 3 OD). A standard curve was plotted and IL8
concentration in the sample was determined by interpolation from the standard
curve
3.2.2.1.2. Determination of serumTNFα:
This assay was based on a polyclonal antibody specific for TNFα (Piguet et al.,
1987). The polyclonal antibody had been coated onto the wells of the microtiter
strips provided. Samples, including standards of known TNFα content were pipetted
into these wells, followed by the addition of a biotinylated monoclonal second
antibody. During the first incubation, the TNFα antigen binds simultaneously to the
immobilized (capture) antibody on one site, and to the solution phase biotinylated
antibody on a second site. After removal of excess second antibody, streptavidineperoxidase enzyme was added. This binds to the biotinylated antibody to complete
the four-member sandwich. After a second incubation and washing to remove all the
unbound enzyme, a substrate solution was added, which is acted upon by the bound
enzyme to produce a color which was measured using an ELISA reader. The
intensity of this colored product is directly proportional to the concentration of
TNFα present in the original specimen. A standard curve was plotted and TNFα
concentration in the sample was determined by interpolation from the standard
curve.
3.2.2.1.3. Determination of serum total nitrites and endogenous nitrites:
Serum nitric oxide was determined utilizing R & D nitric oxide assay system
(R&D, USA) according to the method of Ding et al. (1988)
The transient and volatile nature of NO makes it unsuitable for most conventional
detection methods. However, most of the NO is oxidized to nitrite (NO2-) and Nitrate
(NO3-), whereas the concentration of these anions have been used as quantitative
measure of NO production. After the conversion of (NO3-), to (NO2-) , the
spectrophotometric measurement of (NO2-) is accomplished by using the Griess
reaction:
H+
NO + O2 -
Æ ONO2-
→
NO3- +
H+
H2O
2NO + O2
→ N2O4
→
NO3- +
2H+
H2O
NO + NO2- → N2O3
→
2NO2- + 2H+
A standard curve was created for endogenous and total nitrite concentration by
reducing the data using computer system capable of generating a linear curve fit
(Instat statistical program and graph pad, UK). The nitrate concentration was
determined by subtracting the endogenous nitrite concentration from the total
nitrite concentration.
3.2.2.1.4. Determination of serum albumin:
This and all following biochemical parameters were determined using Hitachi 917
machine, Boehringer Mannheim (Germany).
Albumin binds to Bromocresol-Green at PH 4.1 to form a blue-green complex
(Doumas and Biggs, 1972).The color of the complex intensity was measured as an
endpoint reaction at 596 nm, and was directly proportional to the albumin
concentration
3.2.2.1.5. Determination of serum total protein:
Pyrogallol red complexes with proteins in an acid environment containing
molbdate ions (Teitz, 1970). The resulting blue colored complex absorbs
maximally at 600 nm. The optical density is directly proportional to the protein
concentration within the sample and was measured spectrophotometrically.
3.2.2.1.6. Determination of serum alanine aminotransferase (ALT):
The method used depends on the addition of α- ketoglutarate as a second reagent. The concentration of NADH was measured by
its absorbance at 340 nm, and the rate of absorbance decrease is proportional to the ALT activity (Titz, 1995).
3.2.2.1.7. Determination of serum aspartate aminotransferase (AST):
The method involves the oxidation of NADH to NAD using MDH (malin
dehydrogenase) without pyroxal phosphate as described by International Federation
of Clinical Chemistry, committee on standards (1980). The rate of NADH decreases
was photometrically determined in the UV range and is directly proportional to
AST activity.
3.2.2.1.8. Determination of serum creatinine:
Creatinine and picric acid react in an alkaline solution to form a creatinine-picric
acid complex (modified Jaffe reaction) (Tietz, 1995). The reaction is rate-blanked
and compensated for non-specific proteins. The color intensity is directly
proportional to the creatinine concentration and was measured photometrically at
505 nm.
3.2.2.1.9. Determination of blood urea nitrogen (BUN):
Urea is hydrolyzed by urease to form CO2 and NH3. The NH3 reacts with αketoglutarate and NADH in the presence of glutamate dehydrogenase to form
glutamate and NAD (Roch-Ramel, 1967). The decrease in absorbance due to NADH
consumption was measured kinetically in the UV range at 340 nm
3.2.2.1.10. Determination of serum lactate dehydrogenase (LD):
Pyruvate is catalyzed by lactate dehydrogenase to lactate (Wahlefeled, 1983). At the
same time NADH is oxidized to NAD. The rate of decrease in NADH is directly
proportional to the LDH activity and was determined photometrically in the UV
range.
3.2.2.1.11. Determination of serum creatine kinase (CK):
Creatine phosphate and ADP is catalyzed by CK to creatine and ATP. Hexokinase
catalyses glucose and ATP to G6P and ADP, which in the presence of G6P-DH
forms gluconate-6-p and NADPH and hydrogen (Szasz et al., 1976). The rate of
NADPH
formation
is
proportional
photometrically in the UV range.
3.2.2.1.12. Determination of serum glucose:
to
CK
activity
and
was
measured
Glucose and ATP are catalyzed by hexokinase to G-6-P and ADP. The G-6-P is
then catalyzed by G6PDH in the presence of NADP to gluconate-6-phosphate to
form NADPH (Slein, 1974). The rate of NADPH formation is directly proportional
to the glucose concentration and was measured photometrically in the UV range at
340 nm.
3.2.2.1.13. Determination of serum carbon dioxide
This was based on the automated procedure developed by Skeggs and
Hochstrasser (1964) wherein, carbon dioxide released by acid is absorbed by an
alkaline solution containing phenolphethalin. The decrease in red color is
proportional to the carbon dioxide content of the sample and was measured
spectrophotometrically.
3.2.2.1.14. Determination of serum chloride
This determination was carried according to method described by Zall et al.,
(1956). The sample was added to an air-segmented stream of chloride sample diluent.
The stream is then dialyzed against an air-segmented stream of chloride recipient
solution to remove the chloride sample from protein and serum pigment
interferences. The chloride color reagent was added and the absorbance was
measured at 480 nm.
3.2.2.2. Methods used for Hematological determinations in rabbit serum
Hematological parameters were performed using ADVIA 120 Bayer Diagnostic
(UK) according to the program embedded in the machine (Provan and Henson,
1997)
3.2.2.2.1. Determination of serum total and deferential white blood cells (WBC):
A: Peroxidase method for determination of total WBC:
WBC and neutrophils. Method used 3 reagents to stain intra-cellular
meloperoxidase and then passed the cell through a flow cell where light scatter
and absorption was used to determine each cell’s size and level of staining. The
information was plotted on a cytogram; cluster analysis was then employed to
determine the thresholds to separate the three cell populations.
B: Determination of Basophils:
This method made use of the resistance of basophils to acid lysis and
differentiates them from the rest of the WBC population. This method is a twostage reaction utilizing one reagent.
1-Twelve microlitres of blood were mixed with the Baso Reagent (Phthalic
acid and surfactants), which together with the increased temperature in the
reaction chamber (33 Co) initially lysed the RBC and platelets.
2-The cytoplasm was then stripped from all WBC except the basophils.
3-The WBC was then passed through the laser flow cell where the cells size
and complexity were detected and recorded on the Baso cytogram.
4-Cluster analysis was employed to identify and enumerate each cell
population.
3.2.2.2.2. Determination of red blood cells (RBC):
The RBC method makes use of Mie’s theory of light scatter of spheres.
Utilizing the laser optics low angle and high angle scatter to determine the
size and hemoglobin content of each RBC.
1-Two microlitres of whole blood were diluted in the RBC reagent (Sodium
dodecyl sulphate and gluteraldehyde, which spheres the RBC and fixes it.
Sphering the cells eliminates the problems experienced with the variability
in RBC shape.
2-The cells were then passed through the flowcell in the laser optics bench
where the forward light scatters.
3-The non-linear Mie maps are then replotted using linear translation.
3.2.2.3. Statistical analysis
Statistical analysis was performed using analysis of variance (ANOVA) and TukeyKramer Multiple Comparisons Post Test, with P values of <0.05 considered significant.
3.2.3 Lethality studies in mice
Swiss Albino white male mice (20 + 0.1 g) were obtained from the Experimental
Animal Unit, National Antivenom and Vaccine Production Center. The mice were kept
at an ambient temperature and given free access to food and water. During the
experiment, the institution’s guide for the care and use of laboratory animals was
followed.
Mice were divided into different sets to determine both the intravenous and
subcutaneous doses that would kill 50 % of the animals (LD 50) and the minimum lethal
dose (MLD) for LQQ venom collected from Egypt and Sudan, utilizing the method
described by Miller and Tainter (1944). Briefly, each set comprised of 10 animals with
groups in the first and second sets injected subcutaneously with one of the following
doses (0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325 or 0.35 mg kg-1) of the Egyptian or
Sudanese LQQ scorpion venoms, respectively. The experiment was then repeated in a
similar way, with the venom-injected i.v. via the tail vein (0.15, 0.175, 0.2, 0.225, 0.25,
0.275, 0.3, or 0.325 mg kg-1) for both types of venom. The signs and symptoms of
envenomation as well as the number of dead animals after 24 hours were recorded. The
LD50 and MLD were calculated using a special logarithmic / graph paper.
In another set of experiments, animals were divided into groups (each n =20), and
given montelukast (10 or 20 mg kg–1, p.o.), hydrocortisone (5 or 10 mg kg
–1
, i.v.), or
indomethacin (10 or 20 mg kg
–1
, i.v.). Doses according to Eum, et al., 2003;
Burkovskaia, 1986; Pakulska et al., 2003 respectively. All animals were then injected
subcutaneously with either 0.25 or 0.3mg kg–1 of LQQ venom. In control groups mice
were treated with 0.2 ml/ mouse 0.9 % NaCl, montelukast, hydrocortisone or
indomethacin. Moreover, two groups of animals were given LQQ venom alone, 0.25 or
0.3mg kg–1 as a positive control. Signs and symptoms of envenomation were recorded
and mortality was determined in each group.
3.2.3.1. Statistical analysis
Statistical analysis was performed utilizing Wilcoxon cavarience survival analysis, with
P values of <0.05 considered significant.
CHAPTER - ΙV
RESULTS
RESULTS
4.1. Clinical Characteristics of scorpion-envenomed Patients admitted to
Dongla Hospital in Sudan:
A total of 30 cases of scorpion stings were reported by Dongla Hospital during a six
month period between the beginning of January and the end of July 2002. This number
does not include patients seen in the outpatients clinic and only represents those
admitted, usually with moderate to severe signs and symptoms of envenomation. In
addition, the small number of cases was explained by some of the health professionals in
the hospital as fear of the mostly rural inhabitants from hospitals and their greater
belief in local herbal practices. This study was considered as a preliminary clinical
investigation that would point towards which aspects should be taken into consideration
and emphasized in the next subsequent major investigation planned.
Tables (1 a, b, and c) record all information obtained from each of the 30 patients
including general information, signs and symptoms, laboratory investigations,
treatment protocol and outcome. In the remaining tables (Tables 2-7), an attempt was
made to analyze the data, despite the small number of participants, to gain insight into
the trends of the results. All patients were compared for prognosis in term of mortality
and afterwards divided into those who died and those who survived. All other data were
studied and risk factors that might have affected the prognosis were considered between
both groups. These included demographic factors such as age, sex and factors relating
to the sting such as clinical condition and treatment.
4.1.1 General information about envenomed patients:
As seen in Table 2, of the 30 patients 11 (36.7%) were children (from 4-16 years old)
while the remainder (19, 63.3%) were adults (18-82). Seven of the children (63.6%) and
6 of the adults (31.6%) died in the hospital despite medical intervention, with the total
percentage of non-survivors being 43% (13 out of 30).
The number of patients
reaching the hospital within 1 hr of envenomation were 22 (73%) victims and those
admitted after that time (65-180 min) were 8 (27%). Of the patients admitted early
(<1hr) the percentage of children versus adults were similar (45% vs 55%,
respectively), with 6 out of 10 children (60%) and 3 out of 12 adults (25%) dying from
the severity of their symptoms. On the other hand, only one child was admitted late (90
min) and 7 adults, three of whom died in the hospital (P>0.05, children vs adults) (Table
3). There was also not much difference in the site of sting with the lower limbs
mentioned by 60% of all victims and 40% the upper limbs. A significant number of
patients (P<0.01) identified the yellow scorpion (Leiurus quinquestriatus) as the culprit
(21 out of 30, 70%) versus 9 (30%) either did not know or mentioned another type
(Table 3).
4.1.2. General signs and symptoms of envenomed patients:
Table 5 depicts signs and symptoms exhibited by envenomed patients, survivors and
non-survivors, where it appears that severity of the symptoms correlated with
morbidity. Upon admittance, 60% of the patients (18 of the 30) were very restless; 40%
of these eventually died (12 of the 18), while most of the remaining non-restless patients
(11 out of 30, 37%) survived (P<0.01 vs non-survivors). In addition, it appears that
presence of convulsions was an important indicator of morbidity since none of the
patients that had convulsions (8 out of 30, 27%) survived, whilst from the 22 patients
who had no convulsions (73% of all victims) only 5 died (17% of total, P<0.001 vs
survivors). Furthermore, there was a significant difference in patients presenting with
either fever (11, 37% of total) or dehydration (13, 43% of total), between the number of
survivors (3 [10%] and 4 [13%], with the 2 symptoms, respectively) and non-survivors
(8 [27%] and 9 [30%], with the 2 symptoms, respectively, P<0.05). Moreover, half of the
victims (2/3 of which were adults) presented with emesis, 40% of them did not survive;
in the half where this symptom was not recorded only 1 child (3%) died (P<0.001
survivors vs non-survivors). Similarly, 12 (40%) out of 21 patients with excessive
sweating (70% of total, 2/3 of which were adults) ended up dying, while again 1 (3%) of
the patients that did not have this symptom (30% of total) eventually died (P<0.05)
(Table 5).
4.1.3. Cardiovascular and respiratory symptoms of envenomed patients:
Selected cardiovascular and respiratory parameters were measured in all envenomed
patients including blood pressure, heart rate, presence of arrhythmia or abnormal
breathing patterns (Table 4). Eighteen of the 30 victims when admitted were
hypotensive (60%) and 7 of them eventually died (23% of total). On the other hand, 7 of
the patients were hypertensive (23%), with 5 of them dying later on, while of the 5 (17%
of total) who were normotensive upon entry, only one died (3% of total). In relation to
the time of entry, the number of hypo-, normo- and hypertensive patients that were
admitted within 1 hr after envenomation were 11 (37%), 5 (17%) and 6 (20%),
respectively. On the other hand all entering the hospital after 1 hr were hypotensive (8,
27%) and none were normo- or hypertensive. On a similar note, 16 of the 30 patients
(53%) exhibited tachcardia upon admittance, many of them were also hypotensive and
7 of them eventually died (23% of total). Conversely, 10 victims entered hospital with
bradycardia (33%), again many were also hypertensive, with 9 of them dying later on
(P<0.05 bradycardic non-survivors vs survivors). Of the 8 (27% of total) who had
normal heart rate values upon entry, only one died (3% of total) (Table 4).
Presence of arrythmia upon admittance of envenomed victims appeared to be a major
determinant of morbidity, since of the 50% who entered with arrhythmia (15 patients)
40% died (12). On the other hand only 1 patient (3%) of the remaining patients that did
not exhibit arrhythmia deceased (P<0.0001 arrhythmic non-survivors vs survivors).
Another important indicator of morbidity was presence of respiratory distress upon
entry into hospital, including gasping, wheezing or abnormal respiratory patterns. Of
the 16 patients (53% of total) that had signs of respiratory distress 11 died (37%), whilst
from the 14 who had normal respiratory patterns (47% of all victims) only 2 died (7%
of total, P<0.01 vs. survivors) (Table 4).
4.1.4. General laboratory investigations of envenomed patients:
There appeared to be a significant correlation between the severity of certain
biochemical indicators and the morbidity following scorpion envenomation. For
instance, the majority of patients (23 out of 30, 77%) admitted were hyperglycemic (>6
mmol/L), of these more than half did not survive (13, 43% of total). On the contrary,
none of the patients that had normal glucose levels (7, 23% of total) died (P<0.01 nonsurvivors vs survivors).
To investigate the kidney function, an organ known to be quickly affected in acute
cases of intoxication (Susan, 1998), blood urea nitrogen (BUN), blood creatinine levels
and total protein were measured. Although only 11 of the 30 patients (37%) had high
BUN levels (>7.3 mmol/L), however, all of them died, in addition to 2 (7% of total) of
the 19 (63% of total) with normal BUN levels (P<0.0001 non-survivors vs survivors).
Similarly, 14 of the admitted patients (47% of total) had elevated blood creatinine levels
(>120 µmol/L), with 12 of them (40% of total) eventually dying. Only 1 (3% of total) of
the remaining patients with normal creatinine levels (16, 53% of total) did not survive
(Table 6). On the other hand, blood total protein was not a major determinant since
only 10% of the admitted patients (3) had low levels (<85 g/L), and 2 of these eventually
died (7% of total). Eleven of the remaining 27 patients (37%) with normal levels also
did not survive.
Anther important indicator of morbidity following scorpion envenomation appeared
to be leucocytosis. Of the 11 patients that had high WBC counts in blood (37% of total)
only one survived (3% of total) versus 16 survivors (53% of total) from the 19 (63% of
total) who had normal WBC levels (P<0.0001 non-survivors vs survivors).
4.1.5. Treatment given to envenomed patients:
Use of polyvalent anti-scorpion serum, according to the method followed in this
hospital did not appear to be beneficial, whether given with or without other drugs
including hydrocortisone. From the 16 patients (53% of total) receiving anti-scorpion
serum 7 (23%) died. On the other hand, from the 14 (47%) of the admitted patients that
did not receive antivenom 6 (20%) victims deceased while 8 (27%) survived (Table 7).
Treatment
Laboratory
investigations
cardiovascular &
Respiratory
systems
General information
Table (1a): General Clinical Data of Patients Stung By Scorpions In Dongla Hospital in Sudan
Outcome
Item
Pt.1
Pt 2
Pt. 3
Pt. 4
Pt. 5
Pt. 6
Pt. 7
Pt. 8
Pt 9
Pt10
Age yr
Sex
20
M
10
M
35
F
16
M
7
F
10
F
30
F
25
F
4
F
32
M
Weight kg
45
54
56
34
25
16
62
62
13
62
Time min
30
10
30
30
30
60
120
50
20
50
Site
LL
L. L
U. L
L. L
U. L
L. L
L. L
U. L
L.L
RL
Type
-
-
LQ
LQ
-
LQ
-
-
LQ
LQ
No
No
Yes
No
No
No
Yes
No
No
No
No
No
No
No
No
No
Yes
yes
yes
No
Fever
No
No
No
No
No
No
Yes
No
Yes
No
Hydration
Mild
N
N
N
N
N
N
N
N
N
Emesis
No
No
No
No
No
No
Yes
No
Yes
yes
Sweating
Yes
Yes
Yes
No
No
No
Yes
No
Yes
yes
HR, b/m
90
85
82
75
88
80
92
85
120
60
90/40
85/50
90/60
68/50
50/30
140/95
R
R
R
N
N
Restless
Convulsion
BP, mmHg 80/60
Rhythm
R
R
R. Distress
N
N
N
100/70 80/60 100/70
R
N
R
R
IR
IR
N
N
W
G
S.time, min
45
20
40
30
30
60
140
60
30
60
glucose
126
98
123
88
75
130
170
180
200
190
BUN
2.3l
4
6.2
4.3
7
4.2
6.1
4.7
9.3
13
creatinine
99
113
118
100
114
127
112
115
142
154
T. protein
80
67
72
82
76
65
74
84
96
62
Hb%
70
79
82
82
66
73
82
75
65
85
WBC x10-9
5
4.9
13
4.8
7.8
4.3
5.5
7
17
15
AV
L.A
No
Yes
Yes
Yes
No
Yes
No Yes
No
No
No
Yes
Yes
No
Yes
No
Yes
No
Yes
No
HCTZ
No
Yes
No
Yes
Yes
Yes
Yes
No
No
No
Chloro
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
No -
Yes
C
C
C
C
C
C
C
C
D
D
-
Data is of scorpion envenomed patients (total n=30) admitted to Dongla Hospital, Sudan, from January – July
2002. Refer to Methods and Results for further details and dosages of drugs.
-
Age (years), Sex (M, male; F, female), Time of admission (min after injection), site of injection (L.L, lower
limb; U.L., upper limb), type of scorpion (Lq, Leiurus Quinquestriatus, or -, Unknown), and hydration status (N,
normal; Dehyd, dehydration). HR b/m, heart rate in beats /min; BP mmHg, blood pressure; R, regular; IR,
irregular rhythm; R. Dist-ress, respiratory distress; G, gasping;.S Time, min (sample time after sting in min);
glucose (mg dL-1); BUN (blood urea nitrogen, mMol L-1); Creatinine (µMol L-1); T. protein (g L-1); WBC (white
blood cells, cells L-1).
-
AV, anti-scorpion serum; L.A., local anaesthetic; HCTZ; hydrocortisone; Chloro, chloropheniramine; C,
cured; D, died
Treatment
Laboratory
investigations
cardiovascular &
Respiratory systems
General information
Table (1b): General Clinical Data of Patients Stung By Scorpions In Dongla Hospital in Sudan
Outcome
Item
Pt.11
Pt 12
Pt. 13
Pt. 14
Age yr
Sex
39
M
32
M
35
F
28
F
Weight kg
84
72
75
64
Pt. 15
Pt. 16
Pt. 17
Pt. 18
Pt 19
Pt20
45
M
62
M
71
M
65
M
18
M
39
M
57
82
83
78
46
85
Time min
60
100
60
45
120
50
45
60
180
40
Site
LL
L. L
L.L
U.L
U. L
L. L
U.L
U. L
L.L
L.L
LQ
LQ
LQ
LQ
-
-
-
-
Yes
No
No
No
Yes
No
Yes
No
Yes
No
No
No
No
No
Yes
No
No
No
Type
LQ
Restless
Yes
Convulsion No
LQ
Fever
Yes
No
No
No
No
No
No
No
Yes
No
Hydration
N
N
N
DH
DH
N
N
N
DH
DH
Emesis
Yes
No
No
Yes
No
Yes
No
No
Yes
No
Sweating
Yes
Yes
Yes
Yes
Yes
Yes
No
No
Yes
No
HR, b/m
67
88
80
62
62
60
80
90
100
80
80/60
120/75
R
BP, mmHg 150/90 80/60
Rhythm
IR
R
R. Distress
G
W
116/80 180/100 70/50
R
N
175/95 162/90 89/60
IR
IR
IR
R
R
IR
W
W
G
N
N
G
N
S.time, min
60
120
70
80
130
70
30
60
190
60
glucose
170
200
130
260
123
270
145
138
200
140
BUN
7.3
6.5
3.6
11
6.5
10.2
7.1
5.7
5.8
6.4
creatinine
110
98
85
170
116
150
114
110
109
98
T. protein
83
67
56
80
64
60
82
85
68
74
Hb%
90%
86%
70%
79%
82%
82%
66%
73%
82%
75%
WBC x10-9
5.7
4.2
8.2
18
4.7
19
4.8
9
6.5
7
AV
Yes
Yes
Yes
Yes
No
No
Yes
No
Yes
Yes
L.A
Yes
No
Yes
Yes
Yes
Yes
No
Yes
No
No
HCTZ
No
No
Yes
Yes
No
Yes
No
Yes
Yes
No
Chloro
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
C
C
C
D
C
D
C
C
C
C
-
Data is of scorpion envenomed patients (total n=30) admitted to Dongla Hospital, Sudan, from January – July
2002. Refer to Methods and Results for further details and dosages of drugs.
-
Age (years), Sex (M, male; F, female), Time of admission (min after injection), site of injection (L.L, lower
limb; U.L., upper limb), type of scorpion (Lq, Leiurus Quinquestriatus, or -, Unknown), and hydration status (N,
normal; Dehyd, dehydration). HR b/m, heart rate in beats /min; BP mmHg, blood pressure; R, regular; IR,
irregular rhythm; R. Dist-ress, respiratory distress; G, gasping;.S Time, min (sample time after sting in min);
glucose (mg dL-1); BUN (blood urea nitrogen, mMol L-1); Creatinine (µMol L-1); T. protein (g L-1); WBC (white
blood cells, cells L-1).
-
AV, anti-scorpion serum; L.A., local anaesthetic; HCTZ; hydrocortisone; Chloro, chloropheniramine; C,
cured; D, died
cardiovascular &
Respiratory systems
General information
Table (1c): General Clinical Data of Patients Stung By Scorpions In Dongla Hospital in Sudan
Item
Pt.21
Pt 22
Age yr
Sex
82
F
Weight kg
71
Laboratory
investigations
Treatment
Pt. 24
13
F
35
F
6
M
42
56
35
Pt. 25
Pt. 26
Pt. 27
Pt. 28
Pt 29
Pt30
7
M
10
M
45
F
11
M
7
F
23
M
20
33
57
32
26
55
Time min
120
60
30
45
60
30
130
50
70
90
Site
U. L
U. L
U. L
L. L
L..L
U. L
U. L
L..L
L..L
L..L
Type
LQ
LQ
LQ
LQ
LQ
LQ
LQ
LQ
LQ
LQ
Yes
No
No
No
Yes
yes
Yes
yes
No
No
Yes
yes
Yes
No
Yes
yes
yes
yes
Restless
Yes
Convulsion yes
Fever
yes
No
No
yes
yes
No
Yes
yes
Yes
yes
Hydration
DH
N
N
DH
N
DH
N
N
N
DH
Emesis
Yes
Yes
No
Yes
Yes
No
Yes
Yes
Yes
yes
Sweating
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
yes
HR, b/m
100
100
110
100
70
115
100
100
110
85
80/60
92/65
130/95
50/30
60/40
50/30
60/40
90/60
IR
R
R
IR
IR
IR
IR
IR
N
N
W
G
W
G
BP, mmHg 60/50 100/80
Rhythm
IR
IR
R. Distress
N
W
S.time, min 150
Outcome
Pt. 23
glucose
250
W
N
70
50
60
60
35
140
160
120
150
270
190
130
140
150
270
160
140
170
BUN
13
12
13
5.7
10.3
11
6.5
10.2
6.3
12.2
creatinine
122
118
120
112
115
130
160
118
160
118
T. protein
Hb%
76
65
82
85
65
80
87
62
56
75
81
82
64
80
62
78
68
80
92
87
WBC x10-9
15
14
21
6.4
9.3
20
8.6
20
8.6
20
No
No
No
No
No
No
AV
Yes
No
Yes
No
L.A
No
No
No
No
HCTZ
No
No
No
Yes
Yes
Chloro
No
Yes
Yes
Yes
Yes
D
D
D
C
D
Yes
Yes
Yes
Yes
Yes
No
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
D
D
D
D
D
No
Yes
-
Data is of scorpion envenomed patients (total n=30) admitted to Dongla Hospital, Sudan, from January – July
2002. Refer to Methods and Results for further details and dosages of drugs.
-
Age (years), Sex (M, male; F, female), Time of admission (min after injection), site of injection (L.L, lower
limb; U.L., upper limb), type of scorpion (Lq, Leiurus Quinquestriatus, or -, Unknown), and hydration status (N,
normal; Dehyd, dehydration). HR b/m, heart rate in beats /min; BP mmHg, blood pressure; R, regular; IR,
irregular rhythm; R. Dist-ress, respiratory distress; G, gasping;.S Time, min (sample time after sting in min);
glucose (mg dL-1); BUN (blood urea nitrogen, mMol L-1); Creatinine (µMol L-1); T. protein (g L-1); WBC (white
blood cells, cells L-1).
-
AV, anti-scorpion serum; L.A., local anaesthetic; HCTZ; hydrocortisone; Chloro, chloropheniramine; C,
cured; D, died
Table (2): Percentage of surviving and non-surviving adults and children admitted to
Dongla Hospital in Sudan following scorpion envenomation
Group
Children 36.7% (11)
Adults 63.3% (19)
Dead
23 % (7)
20 % (6)
Alive
13 % (4)
43 % (13)
P value
>0.05
>0.05
-Data are from; patients admitted to Dongla Hospital between January-July 2002, refer to
Methods for further details.
-Total patients in study were 30; numbers between brackets are actual number of patients and percentages are
percent of total number of patients.
-Fishers Exact test was used, P<0.05 considered significant
Table (3): Comparison of personal details of survivors and non-survivors admitted
to Dongla Hospital in Sudan following scorpion envenomation
Time of hospitalization
following envenomation
Sex
Site of sting
Group
Type of scorpion
0-60 min
65-180 min
Male
Female
Lower
Limb
Upper
Limb
LQQ
Other/
Unknown
NonSurvivors
(13)
30% (9)
13% (4)
20% (6)
23% (7)
23% (7)
20% (6)
43%(13)
0 % (0)
Survivors
(17)
43% (13)
13% (4)
37%(11)
20% (6)
37%(11)
20% (6)
27 % (8)
30% (9)
P
Value
>0.05
>0.05
>0.05
<0.01*
-Data are from patients admitted to Dongla Hospital between January-July 2002, refer to Methods for further
details.
-Total patients in study were 30, numbers between brackets are actual number of patients
and percentages are percent of total number of patients.
- L.Limb: lower limb; U.Limb: upper limb; LQQ: the yellow scorpion Leiurus quinquestriatus
quinquestriatus.
-Fishers Exact test was used, with P values <0.05 considered significant (*, P<0.01).
Table (4): Comparison of cardiovascular and respiratory signs and symptoms in survivors and nonsurvivors admitted to Dongla hospital in Sudan following scorpion envenomation
Sign and symptoms
Criteria
Non-survivors
Survivors
P value
Tachycardia
(85-140 bpm)
23 %
(7)
30 %
(9)
>0.05
Bradycardia
(50-67 bpm)
17 %
(9)
3%
(1)
<0.05*
Normal heart rate
(70-80 bpm)
3%
(1)
23 %
(7)
Hypertension
(130/95 mmHg)
17 %
(5)
7%
(2)
>0.05
Hypotension
(80/60 mmHg)
23 %
(7)
37 %
(11)
>0.05
Normal blood pressure
(120/80 mmHg)
3%
(1)
13 %
(4)
Arrhythmias
(Abnormal waves)
40 %
(12)
10 %
(3)
<0.001***
Respiratory distress
(Abnormal breathing)
37 %
(11)
17 %
(5)
<0.01**
- Data are from patients admitted to Dongla Hospital between January-July 2002, refer to methods for further
details.
-Total patients in study were 30; numbers between brackets are actual number of patients
patients and percentages are percent of total number of patients.
- Significance indicated by P value utilizing Fishers Exact test, values < 0.05 considered significant (*, P<0.05;
**, P<0.01; ***, P<0.001).
Table (5): Comparison of signs and symptoms in survivors and non-survivors admitted to
Dongla Hospital following scorpion envenomation
Sign and symptoms
Non-survivors
Survivors
P value
Restlessness Present
40 %
(12)
20 %
(6)
Restlessness absent
3%
(1)
37 %
(11)
Convulsion present
27 %
(8)
0%
(0)
Convulsion absent
17 %
(5)
57 %
(17)
Fever present
27 %
(8)
10 %
(3)
Fever absent
17%
(5)
47 %
(14)
Dehydration present
30 %
(9)
13 %
(4)
Dehydration absent
13 %
(4)
43 %
(13)
Emesis present
40 %
(12)
10 %
(3)
Emesis absent
3%
(1)
47 %
(14)
Sweating present
40 %
(12)
30 %
(9)
Sweating absent
3%
(1)
27 %
(8)
<0.01**
<0.001***
<0.05*
<0.05*
<0.001***
<0.05*
- Data are from patients admitted to Dongla Hospital between January-July 2002, refer to methods for
further details.
-Total patients in study were 30; numbers between brackets are actual number of patients
and percentages are percent of total number of patients.
-Significance indicated by P value utilizing Fishers Exact test (*, P<0.05; **, P<0.01; ***, P<0.001).
.
Table(6): comparison of blood laboratory parameters of survivors and non-survivors
admitted to Dongla Hospital in Sudan following scorpion envenomation
Laboratory parameters
Criteria
Glucose high
>6 mmol/l
43%
(13)
33%
(10)
Glucose normal
4.1-5 mmol/l
0%
(0)
23%
(7)
Total protein low
<80 g/.L
7%
(2)
3%
(1)
Total protein normal or high
64-83 g/.L
37%
(11)
53%
(16)
Uera nitrogen high
>8 mmol/L
37%
(11)
0%
(0)
2.1-7.1 mmol/L
07%
(2)
57%
(17)
Creatinine high
>116 umol/L
40%
(12)
7%
(2)
Creatinine normal
80-115 umol/L
3%
(1)
50%
(15)
WBC high
>12x10-9 cell/L
33%
(10)
3%
(1)
WBC normal
4-11x10-9 cell/L
10%
(3)
53%
(16)
Uera nitrogen normal
Non-survivors
survivors
P value
<0.01*
>0.05
<0.0001**
<0.0001**
<0.0001**
- Data are from patients admitted to Dongla Hospital between January-July 2002, refer to methods for further
details.
-Total patients in study were 30; numbers between brackets are actual number of patients
and percentages are percent of total number of patients.
- Significance indicated by P value utilizing Fishers Exact test test (*, P<0.01; **, P<0.0001).
Table (7): Comparison of treatment protocol used for survivors and nonsurvivors admitted to Dongla Hospital in Sudan following scorpion envenomation
Group
Antivenom with or without drugs
Dead
Alive
P value
Drugs only No antivenom
23 % (7)
20 % (6)
30 % (9)
27 % (8)
P>0.05
P>0.05
- Data are from patients admitted to Dongla Hospital between January-July 2002, refer to Methods for further
details.
-Total patients in study were 30; numbers between brackets are actual number of patients
and percentages are percent of total number of patients.
-Antivenom: Antiscorpion serum polyvalent (Vacsera, Egypt, 1 ampoule i.m.).
-Drugs: hydrocortisone (10 mg/kg, iv), chlorpheniramine (0.09 mg/Kg, iv), xylocaine 1% (0.5 ml, im)
- significance indicated by P value and calculated utilizing Fishers Exact test, considered
significant if P<0.05
4.2.Correlation between blood pressure and
selected biochemical
parameters, including cytokines and NO, in conscious rabbits injected
subcutaneously with Leiurus quinquestriatus
scorpion venom
4.2.1. Cardiovascular experiments:
4.2.1.1 Effect of venom on mean arterial blood pressure (MABP)
4.2.1.1.1. Effect of normal saline on MABP of conscious rabbits
In time-matched control experiments, conscious rabbits injected with 0.9%
NaCl (0.05 ml kg-1, s.c.) showed no significant difference in the baseline
values of MABP (103 ± 3.9 mmHg) and all animals were alive and well at
the end of the 12 hr experiment with no signs of abnormal toxicity. In this
and subsequent experiments undertaken, unless otherwise specified, eight
rabbits were used in each group.
4.2.1.1.2. Effect of LQQ scorpion venom on MABP of conscious rabbits
LQQ venom was administered s.c. in doses ranging from 0.3 up to 0.5 mg
kg-1. Although animals treated with the lower dose of venom showed typical
signs and symptoms of envenomation, they quickly recovered. On the other
hand, rabbits injected with the higher dose died early before the end of the
time limit of the experiment thus a decision was made to utilize 0.4 mg kg-1
in the subsequent experiments.
In general, injection of LQQ venom caused a triphasic effect consisting of
an initial transient reduction in MABP at 1hr (from 100% + 0.02) of control
to 47.5 + 2.8, P<0.05 vs 0 time), afterwards MABP increased peaking at 2 hr
(max 206 + 11.8, P< 0.05 vs 0 hr), then returning to almost normal values
approximately 3hrs after venom injection. This was usually followed by a
gradual terminal hypotensive phase that started to be significantly different
than pre-venom injection values at 4 hrs and onwards (P< 0.05 vs 0 hr and
P< 0.01 vs 1and3 hrs, P< 0.001 vs 2hr, (Table 8, Figure 1).
4.2.2. Effect of venom on lung body weight index of conscious rabbits
4.2.2.1. Effect of LQQ scorpion venom on lung body weight index of conscious rabbits
When LQQ venom was administered s.c. into rabbits in dose of 0.4 mg kg-1,it
produced a significant increase in lung body weight index of the rabbits,
indicative of increased lung water content, (0.58 + 0.03 vs 0.34 + 0.01 of control
saline –treated group, P<0.01). (Table 9).
4.2.3. Effect of LQQ venom on selected biochemical parameters
4.2.3.1. Effect of LQQ venom on serum IL 8 and TNFα
4.2.3.1.1. Effect of subcutaneous injection of normal saline on serum IL 8 and
TNFα in conscious rabbits
Subcutaneous injection of 0.9% Nacl 0.05 ml kg-1 into conscious rabbits
did not produce any significant change from baseline concentration of
either IL 8 (11.89 + 2.2 pgml-1) or TNFα (49.65 + 3.6 pgml-1) throughout the
time limit of the experiment (Fig. 2, Tables 10 and 11).
4.2.3.1.2. Effect of subcutaneous injection of LQQ venom on serum IL 8 and
TNFα in conscious rabbits
Subcutaneous administration of LQQ (0.4 mg kg-1) venom into rabbits caused
a significant increase in serum IL 8 and TNFα concentrations (P<0.0001, each
vs all values from saline and venom-treated groups, Two way ANOVA). Post
ANOVA testing in venom-treated group, showed that both serum IL 8 and
TNFα gave a similar pattern, whereas their values became significantly higher
than pre-venom injection levels 3 hr after venom administration (P<0.001 for
both IL8 and TNFα). Both continued to increase at 6 and 12 hours becoming
significantly different than values from time 0-6 hr after venom injection
(P<0.001).
When values from the venom-treated groups were compared to that obtained
from the saline time–matched control animals, it was found that both IL8 and
TNFα levels were significantly higher than values seen at all times in salinetreated rabbits (P<0.001). (Tables 10 and 11, Fig. 2)
4.2.3.2. Effect of LQQ venom on serum nitrate and nitrite levels
4.2.3.2.1. Effect of subcutaneous injection of normal saline on serum nitrate and
nitrite in conscious rabbits
Control saline-treated (0.9% NaCl, 0.05 ml kg-1, s.c.) conscious restrained
rabbits did not exhibit any significant changes from baseline values whether in
serum total nitrite (76.8 + 0.54 µmol L-1), endogenous nitrite (74.3 + 0. 6 µmol
L-1) or nitrate levels (2.6 + 0.26 µmol L-1), throughout the time limit of the
experiment (12 hours). Both nitrates and nitrites are indicative of increase in
NO levels, with total nitrites being a combination of endogenous nitrites and
nitrates (Fig. 3 and 4, Tables 12-14)
4.2.3.2.2. Effect of subcutaneous injection of LQQ venom on serum nitrate and
nitrite in conscious rabbits:
In general, during the 12 hr of the experiment, subcutaneous administration
of LQQ venom into rabbits provoked a significant increase in serum total
nitrites (P<0.0001), endogenous nitrites (P<0.0001) and nitrates concentrations
(P<0.0001, each vs all values from saline and venom-treated groups, Two way
ANOVA). Post ANOVA testing in venom-treated group, showed that both
serum total nitrite and nitrate gave a similar pattern, whereas their values
became significantly higher than pre-venom injection levels 3 hr after venom
administration (P<0.05 and P<0.01 for nitrate and total nitrite, respectively).
Both continued to increase at 6 and 12 hours becoming significantly different
than values from time 0-6 hr after venom injection (P<0.001 for nitrate and
total nitrite, respectively). On the other hand, serum endogenous nitrites were
only significantly different than pre-venom injection values at 6 and 12 hr after
the venom (P<0.01).
When values from the venom-treated groups were compared to that obtained from
the saline time–matched control animals, both serum endogenous and total nitrites
levels were significantly higher than values seen at all times in saline-treated rabbits.
This was especially obvious in the later stages of envenomation 3 hr (P<0.05 and
P<0.01 for both endogenous and total nitrites, respectively), 6 hr (P<0.05 and
P<0.001) and 12 hr (P<0.0001) after LQQ venom injection. Conversely, in venominjected rabbits serum nitrate concentration was found to be statistically significant
than all values in the saline-treated animals at 6 and 12 hours post-venom
administration (P<0.001). (Tables 12-14, Fig. 3 and 4) )
4.2.3.3. Effect of LQQ venom on serum glucose
4.2.3.3.1. Effect of normal saline on serum glucose concentration in conscious
rabbits
Conscious rabbits injected with 0.9% NaCl (0.05 ml kg-1, s.c.) showed no
significant difference from baseline values of serum glucose concentration (206
+ 0.9 mmol L-1), during the time limit of the experiment (Fig. 5, Table 15)
4.2.3.3.2. Effect of LQQ scorpion venom on serum glucose concentration in conscious
rabbits
When LQQ venom was injected subcutaneously into the rabbits in a dose
equal to 0.4 mg kg
-1
, serum glucose concentration reached its maximum
increase 6 hours after venom injection (712.9 + 48.5 vs. 205.5 + 0.9 mmol L-1 ) at
zero time, P<0.001). Although values decreased afterwards, it remained
significantly higher than base line level at 12 hours (515 + 38.4 mmol L-1,
P<0.001). Additionally, at these two times glucose levels were significantly
higher than that observed in all times in corresponding time-matched control
group (P<0.001). (Fig. 5, Table 15)
4.2.3.4. Effect of LQQ venom on CK, LDH, ALT and AST serum levels
4.2.3.4.1. Effect of normal saline on CK, LDH, ALT and AST serum levels in
conscious rabbits
When conscious rabbits were injected with 0.9% NaCl (0.05 ml kg-1, s.c.), for
up to 12 hours they showed no significant difference from baseline values of
serum CK (14.7+ 0.9 x 102 U L-1), or LDH (7.9+ 0.01 U L-1), AST (40.3 + 1.2 U
L-1), or ALT (54.3 + 1.1 U L-1) (Fig. 6 and 7, Tables 16 and 17)
.
4.2.3.4.2. Effect of LQQ venom on CK, LDH, ALT and AST serum levels in
conscious rabbits
Injection of LQQ venom (0.4 mg kg -1, s.c.), caused a significant increase in
serum CK, LDH (indicative of myocardial function), AST and ALT (indicative
of liver function) levels 6 and 12 hours after venom injection (All, P<0.001 vs 0,
1 and 3 hr after the venom). Additionally, in envenomed animals the values
seen 6 and12 hr after venom administration in these parameters were
significantly higher than those observed at all times in their corresponding
time-matched control groups (P<0.001) (Fig. 6 and 7, Tables 16 and 17).
4.2.3.5. Effect of LQQ venom on serum albumin and total protein levels
4.2.3.5.1. Effect of normal saline on serum total protein and albumin levels in
conscious rabbits
Subcutaneous administration of 0.05 ml kg-1 of 0.9% NaCl into conscious
rabbits, did not significantly affect baseline values of serum total protein (68+
0.42 g L-1), or albumin levels (51.5+ 1.2 g L-1) during the whole period of the
experiment (12 hr) (Fig. 8, Table 18).
4.2.3.5.2. Effect of LQQ venom on serum total protein and albumin levels in
conscious rabbits
Although subcutaneous injection of LQQ venom decreased serum total
protein, indicative of liver function, however the values were only
significant at 6 (P<0.001 vs 0 and 1 hr after venom) and 12 hr after the
venom (P<0.001 vs 0, 1, 3 and 6 hr). Moreover, when the values observed in
envenomed animals were compared to all those in the corresponding timematched control group, they were found to be significantly lower at 3 hr
(P<0.01), 6 hr and 12 hr (P<0.001). Although serum albumin demonstrated
a similar trend following LQQ injection, however the changes were not
significant. (Table 18, Fig. 8).
4.2.3.6. Effect of LQQ venom on BUN and creatinine serum levels
4.2.3.6.1. Effect of normal saline on BUN and creatinine serum levels in
conscious rabbits
Injection of 0.9% NaCl (0.05 ml kg-1, s.c.) into conscious rabbits did not
result in any significant difference from baseline values in serum BUN (9.1+
0.08 mmol L-1), or creatinine levels (101.1+ 1.17 mmol L-1) for upto 12
hours (Fig. 9, Table 19).
4.2.3.6.2. Effect of LQQ venom on BUN and creatinine serum levels in conscious
rabbits
Once again, 6 and 12 hrs after the injection of LQQ venom (0.4 mg kg -1,
s.c.), the venom significantly elevated both serum BUN and creatinine,
indicative of kidney function, levels 6 and 12 hours after venom injection
(All, P<0.001 vs 0, 1 and 3 hr after the venom). Additionally, in envenomed
animals the values seen 6 and 12 hr after venom administration in these
parameters were significantly higher than those observed at all times in
their corresponding time-matched control groups (P<0.001). (Table 19, Fig.
9).
4.2.3.7. Effect of LQQ venom on serum carbon dioxide and chloride levels
4.2.3.7.1. Effect of LQQ venom on serum carbon dioxide and chloride levels
In time matched control experiments, 0.9% NaCl did not significantly
affect baseline values of serum carbon dioxide (18.1+ 0.44 mmol L-1), or
chloride levels (104.5+ 0.26 mmol L-1) during the 12 hr time limit of the
experiment (Fig. 10, Table 20).
4.2.3.7.2. Effect of LQQ venom on serum carbon dioxide and chloride levels in
conscious rabbits
Following the subcutaneous injection of LQQ venom serum carbon
dioxide gradually decreased, becoming significantly different at 6 hr
(P<0.01 vs 0 and 1 hr after venom) and 12 hr (P<0.001 vs 0, 1, 3 and 6 hr).
Furthermore, at these two times the values were also significantly lower
than those seen at all times in the corresponding time-matched control
group (P<0.001). Conversely, serum chloride levels peaked 6 hr after LQQ
venom injection (P<0.001 vs. pre venom values). Afterwards the levels
decreased becoming at 12 hr significantly lower than values seen at 0, 1 and
3 hr after venom injection (P<0.001). Moreover, in envenomed animals
values observed at 6 hr were also significantly higher than those seen at all
times in time matched controls (P<0.001) (Fig. 10, Table 20).
4.2.4. Effect of LQQ venom on selected hematological parameters
4.2.4.1. Effect of LQQ venom on serum RBC, WBC, Basophils and Neutrophils
4.2.4.1.1. Effect of subcutaneous injection of normal saline on serum RBC, WBC,
Basophils and Neutrophils in conscious rabbits
In time-matched control experiments, conscious rabbits injected with
0.9% NaCl (0.05 ml kg-1, s.c.) showed no significant difference from baseline
values of red blood cells (RBC, 6.16 + 0.1x108 cell L-1), white blood cells
(WBC, 13.18 + 0.08 x 108 cell L-1), basophils (2.9 + 0.13 x 108 cell L-1) and
neutrophils (4.8 + 0.29 x 108 cell L-1) during the time course of the
experiment (Fig. 11 and 12, Tables 21 and 22).
4.2.4.1.2. Effect of subcutaneous injection of LQQ venom on serum RBC, WBC,
basophils and neutrophils in conscious rabbits
Injection of LQQ venom in a dose equal to 0.4 mg kg
-1
, caused a
significant decrease in RBC and increase in WBC values 12 hours after
venom injection (reached 5.5 + 0.14 and 22.2 + 0.82, respectively, both
P<0.001 vs 0 time in control and venom treated groups). Moreover, LQQ
venom led to a gradual reduction in basophil levels that was significant at
both 6 hr (P<0.01 vs and 1 hr in venom treated groups) and 12 hr after
venom injection (P<0.001 vs 0, 1 and 3 hr). Additionally, in envenomed
animals the values seen 3, 6 and 12 hr after the venom were significantly
different than those observed at all times in corresponding time-matched
control group (P<0.01, P<0.01 and P<0.001, respectively). Conversely, the
venom produced a significant increase in neutrophil values at 6 hr (P<0.001
vs 0 and 1 hr in venom treated groups) and 12 hr after venom injection
(P<0.001 vs 0, 1 and 3 hr). Furthermore, in venom-treated rabbits the
values at 3, 6 and 12 hr after venom injection were significantly different
than all values in the time-matched control group (P<0.01, P<0.001 and
P<0.001, respectively) (Fig. 11 and 12, Tables 21and 22).
300
Rabbit 1
Rabbit 2
Rabbit 3
Rabbit 4
Rabbit 5
Rabbit 6
Rabbit 7
Rabbit 8
MEAN
Effect of LQQ venom on MABP
MABP (% of control)
250
200
150
100
50
0
0
3
Time 6
(hr)
9
Fig. ( 1 ) Percent of mean arterial blood pressure (MABP) measured at
different times in conscious rabbits injected with Leiurus quinquestriatus
(LQQ) venom
Conscious New Zealand white rabbits were injected s.c. with LQQ scorpion
venom (0.4 mg/kg) and MABP (% of control pre-venom injection levels)
monitered intermittantly for 12 hours. Points are individual values of the rabbits
(n=8) and mean of all values. Refer to methods and results for further details
12
Table (8): % of control mean arterial blood pressure measured at different time
intervals following the injection of Leiurus quinquestriatus venom
into conscious rabbits
Rabbit
1
2
3
4
5
6
7
8
0hr 0.5hr
100
35
100
40
100
45
100
50
100
55
100
45
100
60
100
50
Mean
S.E.M
100
0.03
47.5
2.8
1hr
90
100
140
120
150
130
155
130
2hr
240
180
180
200
170
230
260
185
3hr
110
120
100
130
110
100
140
90
4hr
60
70
50
60
40
50
40
60
5hr
60
60
55
40
40
40
50
50
6hr
50
50
30
40
45
45
40
35
7hr 8hr 9hr 10hr 11hr 12hr
30 40 45
35
35
35
45 40 40
30
30
30
30 30 40
30
30
30
40 35 30
35
30
30
40 35 35
35
35
30
40 35 35
30
35
35
35 35 40
30
30
30
30 30 35
40
30
30
126.9 205.6 112.5 53.8 49.4 41.9 36.3
8.06 11.8
5.9
3.7 3.1 2.5 2.1
35
1.3
37.5
1.6
33.1
1.3
-
Percent of control mean arterial blood pressure (MABP) was measured in conscious New
Zealand male rabbits (n=8) prior to and up to 12 hours following s.c. injection of Leiurus
quinquestriatus quinquestriatus (LQQ) scorpion venom. Refer to Methods and Results for
further details.
-
Values are of individual animals in addition to mean and S.E.M of all rabbits.
Table (9): Lung / body weight index of control and Leiurus quinqestriatus
venom– injected rabbits
Rabbit No.
0.9% Nacl
Leiurus quinqestriatus venom
1
2
3
4
5
6
7
8
0.32
0.36
0.37
0.31
0.32
0.35
0.30
0.38
0.57
0.49
0.51
0.62
0.54
0.66
0.53
0.72
Mean + SEM
0.34 + 0.01
0.58 + 0.03 *
-Values are for individual male New Zealand rabbits (n=8) with values representing those
obtained from individual rabbits in addition to mean and standard error of the mean
(S.E.M).
31.9
0.9
31.3
0.8
-Animals were treated with either 0.9% Nacl (0.05 ml kg –1, sc.) or LQQ venom (0.4 mg kg -1, sc.).
After end of experiment animals were killed by overdose of diethyl ether and lung/ body weight
index calculated. Refer to methods and results sections for further details.
-
Values are significantly different from control group at * (P <0.01).
&
*
Serum IL 8 Concentration
A
700
IL 8 conc.(pg/ml)
600
Cont.
Venom
+
*
500
400
#
*
300
200
100
0
0
2
4
6
8
10
12
14
Tim e (hr)
Seum TNF alpha concentration
B
1000
TNF alpha con. (pg/ml)
900
&
*
Cont.
Venom
800
700
+
*
600
#
*
500
400
300
200
100
0
0
2
4
6
8
Tim e (hr)
10
12
14
Fig ( 2): Serum IL8 (A) and TNF alpha (B) concentrations in serum of control
(0.9% Nacl, 0.05ml/kg) and LQQ venom (0.4 mg/kg) treated rabbits. Points
represent the mean and vertical lines SEM of 8 rabbits at each time point. At *
values are significantly different from each groug respective control at all times
(* p< 0.001). At # values are significantly different from each corresponding
venom treated groups at zeroand 1 hour (# p<0.001) and at + values are
significantly different from each respective venom treated group at zero, 1and 3
hours (+ p<0.001) while at & values are significantly differnt from their
corresponding venom treated groups at zero, 1, 3 and 6 hours (& p<0.001).
Table (10): Serum IL8 levels in control and L. quinquesrriatus venom-treated rabbits
Time/ Rabbit No.
0hr/Rabbit
1
2
3
4
5
6
7
8
Mean +SEM
1hr/Rabbit
1
2
3
4
5
6
7
8
Mean +SEM
3hr/Rabbit
1
2
3
4
5
6
7
8
Mean +SEM
6hr/Rabbit
1
2
3
4
5
6
7
8
Mean +SEM
12hr/Rabbit 1
2
3
4
5
6
7
8
Mean +SEM
Control IL 8
7.36
9.09
8.92
21.92
10.65
8.05
7.53
21.58
11.89 + 2.2
5.8
8.91
8.75
22.62
10.48
8.75
3.54
18.98
10.98 + 2.3
12.21
8.75
8.92
22.79
10.83
8.75
7.53
15.85
11.95 + 1.8
12.39
8.92
8.75
21.06
10.48
8.92
7.01
16.03
11.70 + 1.7
12.39
8.92
8.92
26.26
10.31
8.92
7.01
15.85
12.32 + 2.2
LQQ IL 8
7.53
10.65
29.90
10.31
7.36
14.29
8.92
4.06
11.63 +2.8
30.07
34.93
36.31
51.40
31.63
47.06
24.00
32.33
35.97 + 3.2
149.19
150.92
152.66
149.19
142.26
154.39
149.19
147.46
149.41 + 1.3
411.01
463.03
447.42
371.13
395.40
404.07
459.56
464.76
427.05 + 12.7
664.16
634.68
636.42
598.27
679.76
660.69
641.62
664.16
647.47 + 8.9
-Values (from 1-8) are for individual male new Zeland rabbits (n=8) followed by their corresponding
mean + SEM.
- IL 8 concentration (pg/ml) was measured in serum obtained from blood collected from central
ear artery of animal treated with either saline( 0.05 ml)kg-1) or LQQ (0.4 mg kg-1). Refer to
methods for further details.
Table (11): Serum TNF α concentration of control and Leiurus quinquesrriatus venom-treated rabbits
Time/ Rabbi No.
0hr/Rabbit
1
2
3
4
5
6
7
8
Mean +SE
1hr/Rabbit
1
2
3
4
5
6
7
8
Mean +SE
3hr/Rabbit
1
2
3
4
5
6
7
8
Mean +SE
6hr/Rabbit
1
2
3
4
5
6
7
8
Mean +SE
12hr/Rabbit
1
2
3
4
5
6
7
8
Mean +SE
Control TNF α
42.95
42.44
43.47
46.04
41.41
51.20
69.22
60.47
49.65 + 3.6
43.98
42.95
42.43
69.74
43.47
60.47
41.41
41.92
48.3 + 3.8
44.50
42.44
45.53
69.74
60.47
41.92
42.44
69.22
52.03 + 4.4
43.98
43.47
46.04
70.77
59.44
42.44
41.92
69.74
52.2 + 4.4
43.98
42.95
45.01
64.59
60.98
38.83
41.41
65.10
50.4 + 3.9
LQQ TNF α
43.98
45.53
60.47
52.74
30.59
43.98
66.65
59.44
50.42 +4.1
110.43
104.25
98.07
143.91
109.40
116.10
121.76
125.88
116.22 + 5.1
270.62
251.56
246.93
227.87
259.81
265.99
291.22
291.23
263.16 +7.7
450.39
437.51
468.93
456.05
479.75
470.23
439.06
487.47
462.30 + 6.8
671.36
671.36
1170.99
995.87
707.42
944.36
825.89
995.87
872.89 + 64.8
-Values (from 1-8) are for individual male new Zeland rabbits (n=8) followed by their corresponding
Mean + SEM.
- TNF α concentration (pg/ml) was measured in serum obtained from blood collected from central
ear artery of animal treated with either saline( 0.05 ml)kg-1) or LQQ (0.4 mg kg-1). Refer to
methods for further details.
300
Total nitrite
A
250
##
**
Control
Venom
##
**
Total nitrite con
200
#
**
150
100
50
0
0
1
3
6
12
Time (hour )
180
160
140
Endogenous nitrite concentration
B
cont
Venom
#
*
#
*
#
*
Nitrite conc
120
100
80
60
40
20
0
0
1
Time 3(hour)
6
12
Fig (3): Total nitrite(A) and endogenous nitrite(B) concentration in serum of
control (0.9% Nacl.0.05 ml/kg, sc.) and LQQ venom (0.4 mg/kg, sc.) treated
rabbits. Values represent means and vertical lines SEM of8 rabbits at each
time point. A t * values are significantly different from each group respective
control at all times (*p<0.01,**p<0.001).At # values are significantly different
from their corresponding venom treated group at zero time (#p<0.001,
##p<0.001).
Table (12): Total nitrite concentration in serum of control and Leiurus
quinquestriatus venom-treated rabbits.
Rabbit
C1
C2
C3
C4
C5
C6
C7
C8
Mean +SEM
V1
V2
V3
V4
V5
V6
V7
V8
Mean +SEM
0hr
1hr
3hr
6hr
12hr
78.0
77.8
79.9
78.3
77.8
74.8
66.4
67.4
74.6
74.6
78.1
78.4
67.5
74.6
78.0
78.1
72.5
62.3
78.3
78.0
78.3
78.4
78.4
78.5
78.3
75.2
75.1
75.4
75.5
75.4
76.6
76.5
76.3
76.7
76.4
75.2
75.3
75.2
75.4
75.3
76.8 + 0.54
75.0+1.42
72.8+2.21
76.5+0.59
76.7+0.52
77.9
79.1
91.0
125.8
129.5
77.7
82.2
91.0
102.9
139.0
77.9
131.1
167.1
218.5
297.5
73.3
79.7
91.4
201.2
258.4
75.3
132.0
165.0
200.7
214.1
81.0
143.2
155.8
219.5
272.9
76.4
141.6
152.0
247.9
263.4
77.6
101.2
148.2
180.7
238.6
77.1+0.79
111.3+10.13
132.7+12.36
182.2+15.55
226.7+21.93
-Values (from 1-8) are for individual male New Zealand rabbits (n=8) followed by their corresponding Mean + SEM
-Serum total nitrite concentration (µmol L-1) was measured in serum obtained from central ear artery of animal
treated with either 0.9 % Nacl (0.05 ml kg-1) or LQQ. venom (0.4 mg kg-1). Refer to methods and results section
for further details.
Table (13): Serum endogenous nitrate concentration in control and Leiurus
quinquestriatus venom-treated rabbits.
Rabbit
0hr
1hr
3hr
6hr
12hr
C1
76.7
74.9
78.6
77.0
74.9
C2
71.7
62.6
63.6
70.5
70.8
C3
75.6
75.8
65.3
71.4
74.9
C4
75.0
69.3
59.1
74.5
74.5
C5
75.4
72.2
75.2
74.7
75.1
C6
72.6
72.5
72.9
72.4
72.9
C7
73.4
73.3
73.1
72.9
72.9
C8
73.6
73.7
73.0
73.5
73.4
74.3+0.60
71.8+1.48
70.1+2.35
73.4+0.72
73.7+0.52
75.0
75.9
86.3
82.4
70.1
75.8
77.4
85.0
49.8
76.4
74.7
125.1
145.1
162.2
234.7
72.0
73.7
69.4
138.5
189.3
72.8
126.6
144.9
137.9
147.5
78.1
136.9
135.6
150.4
201.0
74.1
137.1
140.6
172.8
187.7
76.0
99.6
137.8
86.5
129.0
74.8+0.68
106.5+9.93
118.1+11.28
122.6+15.57
154.5+21.06
Mean+SEM
V1
V2
V3
V4
V5
V6
V7
V8
Mean+SEM
-Values (from 1-8) are for individual male new Zealand rabbits (n=8) followed by their corresponding
Mean + SEM
-Serum endogenous nitrite concentration (µmol L-1) was measured in serum obtained from central ear
artery of animal treated with either 0.9 % Nacl (0.05 ml kg-1) or LQQ. Venom (0.4 mg kg-1). Refer to
methods and results section for further details.
Nitrate concentration
Control
Venom
#
90
#
**
80
**
Nitrate conc.(umol/L)
70
60
50
40
&
30
*
20
10
0
0
1
3
6
12
Time (hour)
Fig (4) Nitrate concentration determined by subtracting endoenous nitrite oncentration from total nitrite.
Points represents the mean and vertical lines SEM of 8 rabbits. At * values are significantly different from
control at all times(*p<0.05,**p<0.001). At $ values are significantly different from venom treted gp at
zero hr (& p<0.05) and at# values are significantly different from venom treated go. at zero, 1and 3 hr.(#
p<0.001)
Table (14): Serum nitrate concentration in control and Leiurus
quinquestriatus venom-treated rabbits.
Rabbit
C1
C2
C3
C4
C5
C6
C7
C8
Mean +SEM
V1
V2
V3
V4
V5
V6
V7
V8
Mean +SEM
0hr
1hr
3hr
6hr
12hr
1.3
2.9
1.3
1.3
2.9
3.2
3.8
3.8
4.1
3.8
2.5
2.5
2.2
3.2
3.2
3.2
3.2
3.2
3.8
3.5
2.9
3.2
3.2
3.8
3.2
2.5
2.5
2.5
3.2
2.5
3.2
3.2
3.2
3.8
3.5
1.6
1.6
2.2
1.9
1.9
2.6+0.26
2.9+0.23
2.7+0.28
3.1+0.36
3.1+0.22
2.9
3.2
4.7
43.4
59.4
1.9
4.7
6.0
53.1
62.5
3.2
6.0
22.0
56.2
62.8
1.3
6.0
22.0
62.5
69.1
2.5
5.4
20.1
62.8
66.6
2.9
6.3
22.0
69.1
71.9
2.2
4.4
11.3
75.1
75.7
1.6
1.6
10.4
94.2
109.6
2.3+0.24
4.7+0.57
14.8+2.66
64.6+5.46
72.2+5.67
-Values (from 1-8) are for individual male new Zealand rabbits (n=8) followed by their corresponding
Mean + SEM
-Serum nitrate concentration (µmol/L) was calculated by subtracting the endogenous nitrite from
total nitrite concentration. Refer to methods and results section for further details.
#
*
800
700
Glucose concentration
cont.
Venom
+
*
Glucose conc.(mmol/L)
600
500
400
300
200
100
0
0
1
3
6
12
Time (hour)
Fig (5): Serum glucose concentration of control (saline 0.05 ml/kg) and LQQ (0.4 mg/kg ) treated
rabbits. Values represents the mean + SEM of 8 rabbits at each time point. At * values are
significantly different from control group at all times (*p<0.001). At # values are significantly
different from venom treated group at zero, 1, and 3 hr (#p<0.001), while at + values are
significantly diff-erent from venom treated group at zero, 1, 3, and 6 hr (+P<0.001).
Table(15 ): Serum glucose concentration in control and Leiurus quinquesrriatus venom-treated rabbits
Time/Rabbit No.
0hr/Rabbit
1
2
3
4
5
6
7
8
Mean+SEM
1hr/Rabbit
1
2
3
4
5
6
7
8
Mean+SEM
3hr/Rabbit
1
2
3
4
5
6
7
8
Mean+SEM
6hr/Rabbit
1
2
3
4
5
6
7
8
Mean+SEM
12hr/Rabbit 1
2
3
4
5
Cont Glucose mmol/L
LQQ Glucose mmol/L
209
208
207
204
202
205
205
202
202
208
205
210
206
210
208
204
205.5+0.9
206.4+1.1
209
233
207
236
207
240
206
237
203
252
207
270
206
260
207
210
206.5+0.6
242.3+6.5
211
210
209
207
208
211
207
203
203
200
210
213
209
212
208
207
208.1+0.9
207.9+1.6
211
621
211
568
210
912
210
940
212
626
211
684
210
670
211
682
210.8+0.3
712.9+48.5
209
512
207
420
203
612
207
621
208
413
205
480
7
206
672
8
208
680
Mean + SEM
206.6+0.7
551.3+38.4
-Values(from 1-8) are for individual conscious male New Zealand rabbits (n=8) Followed by their
corresponding Mean + SEM .
- Serum glucose concentrations (mmol L-1) were measured in blood collected from central ear
artery of animal treated with either 0.9% Nacl (0.05 ml kg-1) or LQQ (0.4 mg kg-1 ) . Refer to
methods and results sections for further details.
#
90
A
*
CK. concentration
#
*
Cont
Venom
80
CK.conc.X 103 (U/L)
70
60
50
40
30
20
10
0
0
1
3
6
Time (hour)
25
LDH conc.(u/L)
20
B
#
LDH concentration
#
*
*
Cont
Venom
15
10
5
0
0
1
3
6
12
Time (hour)
Fig (6) :Serum CK (A) and LDH (B) concentrations of control (saline 0.05ml/kg)
and LQQ (0.4 mg/kg) treated rabbits. Values represent the mean +SEM of 8
rabbits at each time point. At * values are significantly different from each groug
respective control at all times (* P<0.001). At # values are significantly different
from each group respective venom treated at zero, 1, and 3 hours (# P<0.001).
Table (16): Serum CK and LDH levels in control and Leiurus quinquestriatus venom-treated rabbits
Time / Rabbit No.
0hr/Rabbit
1
2
3
4
5
6
7
8
Mean + SEM
1hr/Rabbit
1
2
3
4
5
6
7
8
Mean + SEM
3hr/Rabbit
1
2
3
4
5
6
7
8
Mean + SEM
6hr/ Rabbit 1
2
3
4
5
6
7
8
Mean + SEM
12hr/ Rabbit 1
2
3
4
5
6
7
8
Mean + SEM
-Values(from 1-8) are for
Mean + SEM
Control CK
LQQ CK
Control LDH
LQQ LDH
17.8
16.4
8.0
8.6
10.0
17.8
7.9
8.2
16.2
15.3
8.1
8.1
15.4
16.7
8.1
8.1
12.1
14.9
7.7
7.6
14.6
16.4
7.9
7.9
15.4
15.2
7.8
8.5
16.2
16.1
8.0
8.1
14.7 + 0.9
16.1+ 0.33
7.9 + 0.01
8.1+ 0.11
17.8
18.6
8.0
10.4
11.2
18.7
7.8
10.3
16.4
21.2
8.0
9.9
15.4
32.8
7.9
9.3
12.7
19.7
7.7
9.8
14.8
21.2
7.9
8.9
15.3
16.3
7.8
10.2
16.4
17.3
7.9
9.3
15.0 + 0.75
20.7 + 1.8
7.9 + 0.04
9.8 + 0.19
18.0
23.5
8.1
10.0
16.3
21.2
7.9
9.8
16.4
25.3
8.0
9.3
15.5
36.5
8.0
8.8
12.9
26.3
7.8
8.9
15.0
24.3
7.8
8.7
15.3
21.0
8.0
10.0
16.4
23.2
7.9
9.1
15.7 + 0.52
25.2 + 1.7
7.94 + 0.04
9.3 + 0.19
18.3
75.1
7.8
15.0
16.4
77.6
8.1
14.5
17.0
87.3
7.9
31.6
16.2
91.1
7.6
20.2
13.1
63.5
7.5
15.4
15.2
97.3
7.9
30.0
15.6
61.2
7.8
12.0
16.5
76.5
7.6
10.0
16.0 + 0.53
78.7 + 4.5
7.8 + 0.07
18.6 + 2.86
18.4
72.1
8.2
19.2
17.0
67.5
7.9
18.3
17.2
77.2
7.9
20.6
16.4
81.1
8.2
19.5
14.5
53.5
8.2
21.1
15.2
72.3
8.1
18.3
15.6
52.3
7.9
18.2
16.5
62.1
8.0
19.3
16.4 + 0.44
67.3 + 3.73
8.1 + 0.05
19.3 + 0.38
individual male New Zealand rabbits (n=8) Followed by their corresponding
- Serum CK and LDH concentrations (u L-1 ) were measured in blood collected from central ear artery
of animal treated with either 0.9% Nacl (0.05 ml kg-1) or LQQ (0.4 mg kg-1 ) . Refer to methods and
results sections for further details.
160
140
A
#
*
#
AST concentration
Cont
Venom
*
100
80
60
40
20
0
0
1
3
6
12
Tim e (hour)
90
B
80
Cont
Venom
ALT concentration
#
*
#
*
70
ALT conc. (u/L)
AST conc. u/L
120
60
50
40
30
20
10
0
0
1
3
6
12
Tim e (hour)
Fig(7) :Serum AST (A) and ALT (B) concentrations of control (saline 0.05
ml/kg) and LQQ (0.4 mg/kg) treated rabbits. Values represent the mean +
SEM of 8 rabbits at each time point. At * values are significantly different from
each group respective control at all times (*p<0.001), while at # values are
significantly different from each respective venom treated group at zero, 1, and
3 hours (#p<0.001).
Table (17): Serum AST and ALT concentration in control and Leiurus quinquestriatus venom-treated rabbits
Time /Rabbit No
0hr/Rabbit
1
2
3
4
5
6
7
8
Cont AST
LQQ AST
Cont ALT
40
41
38
37
40
40
39
38
40
41
37
38
40
39
48
38
Mean + SEM
40.3+1.2
39.0+0.5
1hr/Rabbit
1
41
48
2
39
39
3
40
50
4
39
60
5
41
43
6
37
40
7
41
41
8
48
40
Mean + SEM
40.9+1.2
45.1+2.6
3hr/Rabbit
1
40
47
2
38
47
3
41
42
4
39
48
5
48
47
6
38
47
7
39
46
8
46
47
Mean + SEM
41.1+1.3
46.37+0.65
6hr/ Rabbit 1
42
110
2
41
99
3
43
140
4
39
117
5
46
142
6
36
120
7
40
132
8
48
142
Mean + SEM
41.9+1.4
125.3+5.7
12hr/ Rabbit 1
41
122
2
40
117
3
43
152
4
40
130
5
45
149
6
40
143
7
40
140
8
47
153
Mean + SEM
42.0+1
138.3+4.9
-Values(from 1-8) are for individual male New Zealand rabbits (n=8)
Mean + SEM
LQQ ALT
58
58
57
57
52
60
49
56
56
49
52
51
56
57
54
54
54.3+1.1
55.3+1.3
55
57
55
55
50
60
46
56
52
50
50
52
51
54
54
50
51.6+1.1
54.3+1.2
55
57
54
56
50
65
47
56
50
51
51
50
52
54
52
52
51.4+0.9
55.1+1.7
56
77
53
67
52
79
48
80
51
79
50
62
50
62
47
74
50.9+1.0
72.5+2.7
57
79
54
70
52
83
49
86
54
85
51
66
50
63
51
76
52.3+0.9
76.0+3.11
Followed by their corresponding
-Serum AST and ALT concentrations (u L-1) were measured in blood collected from central ear artery
of animal treated with either 0.9% Nacl (0.05 ml kg-1) or LQQ (0.4 mg kg-1 ) . Refer to methods and
results sections for further details.
A
80
Total Protein concentration
C0nt
Venom
70
+
**
*
Total protein conc.(g /L)
60
#
**
50
40
30
20
10
0
0
1
3
6
12
Tim e (hour )
60
B
Cont
Venom
Albumin concentration
Albumin conc. (g /L)
50
&
&
40
30
20
10
0
0
1
3
6
Tim e (hour)
Fig (8) : Serum total protein (A) and albumin (B) concentrations of control (0.9 % Nacl 0.05 ml/kg, sc.) and
LQQ venom (0.4 mg/Kg, sc) treated rabbits. Bars represent the mean and vertical lines SEM of 8 rabbits at each
time point. At *, values are significantly different from their respective control groups at all times (* p <0.01,
** p< 0.001), while at &, values are significantly different from their respective control groups at zero and 1
hour (& p< 0.01). At +, values are significantly different from theircorresponding venom treated group at zero
and 1 hour (+ p< 0.001), while at #, values are significantly different from their respective venom treated group
at zero, 1, 3, and 6 hours (# p<0.001).
Table (18) Serum total protein and albumin levels in control and Leiurus quinquestriatus venom-treated rabbits
Time / Rabbit No.
Control (TP)
LQQ (TP)
Control (Alb)
LQQ (Alb)
0hr/Rabbit
1
96
67
55
53
2
68
68
50
48
3
66
61
49
44
4
69
66
47
49
5
67
65
48
50
6
69
60
55
50
7
69
64
53
51
8
67
66
55
46
68.0 + 0.42
Mean + SEM
51.5 + 1.20
48.4 + 2.20
64.6 + 1.00
1hr/Rabbit
1
69
65
56
52
2
67
67
47
46
3
66
61
48
44
4
69
64
47
46
5
66
65
48
46
6
69
59
55
49
7
66
62
55
50
8
67
63
56
44
Mean + SEM
67.4 + 0.50
63.3 + 0.90
51.5 + 1.52
47.4 + 2.08
3hr/Rabbit
1
68
63
55
50
2
67
66
46
46
3
66
56
48
41
4
68
62
47
42
5
66
59
47
45
6
68
58
52
48
7
65
60
53
50
8
60
59
49
40
Mean + SEM
66.0 + 0.94
60.4 + 1.10
49.6 + 1.16
45.3 + 1.40
6hr/ Rabbit 1
68
61
54
48
2
66
61
46
43
3
67
51
48
40
4
67
56
46
40
5
65
58
47
40
6
67
52
53
42
7
62
61
53
58
8
58
52
50
38
Mean + SEM
65.0 + 1.20
56.5 + 1.55
49.6 + 1.18
43.4 + 1.35
12hr/ Rabbit 1
69
60
53
46
2
67
56
46
40
3
67
50
47
42
4
67
48
46
41
5
62
47
48
40
6
68
42
53
43
7
66
54
52
57
8
61
50
49
36
Mean + SEM
65.9 + 1.00
50.9 + 2.00
49.3 + 1.07
43.63 + 1.35
-Values(from 1-8) are for individual male New Zealand rabbits (n=8) Followed by their corresponding
Mean + SEM
- Serum total protein (TP) and albumin (Alb) concentrations (g L-1) were measured in blood collected
from central ear artery of animal treated with either 0.9% Nacl (0.05 ml kg-1) or LQQ (0.4 mg kg-1) .
Refer to methods and results sections for further details.
BUN concentration
16
BUN conc (mmol/L)
14
A
#
cont
Venom
#
*
*
12
10
8
6
4
2
0
0
1
3
6
12
Time (hour)
200
Creatinine conc (mmol/L)
180
160
#
*
Creatinine concentration
B
#
*
Cont
Venom
140
120
100
80
60
40
20
0
0
1
3
6
12
Time (hour)
Fig (9): Serum BUN (A) and creatinine (B) concentrations of control (0.9% Nacl
0.05ml/kg,sc.)and LQQ (0.4 mg/Kg, sc.) treated rabbits. Bars represent the mean
and vertical line SEM of 8 rabbits at each time point. At *, values are significantly
different from their respective controls at all times (*p<0.001). At #, values are
significantly different from their corresponding venom treated group at zero, 1 and
3 hours(#p<0.001).
Table (19): Serum BUN and creatinine levels in control and Leiurus quinquestriatus venom-treated rabbits
Time / Rabbit No. Control (BUN)
LQQ (BUN)
Control (Cr.)
LQQ (Cr.)
0hr/Rabbit
1
9.1
9.1
100
109
2
9.1
8.9
99
100
3
8.9
9.1
99
101
4
9.3
9.3
101
102
5
8.7
8.8
101
99
6
9.2
9.2
109
99
7
9.1
10
99
103
8
9.2
8.5
101
101
Mean + SEM
9.1+ 0.08
9.1+ 0.16
101.1 + 1.17
101.8 + 1.15
1hr/Rabbit
1
9.1
9.2
100
92
2
9.2
9.6
99
104
3
9.0
9.7
99
128
4
9.4
9.9
100
108
5
8.8
9.4
100
110
6
9.2
9.5
109
107
7
9.1
10
99
97
8
9.2
8.6
100
93
Mean + SEM
9.1+ 0.07
9.5 + 0.16
100.8 + 1.19
104.9 + 4.11
3hr/Rabbit
1
9.1
9.9
100
102
2
9.3
10.1
98
120
3
8.9
10.6
97
128
4
9.5
11.0
100
121
5
8.9
10.7
99
117
6
9.0
9.5
109
120
7
9.0
10.2
98
99
8
9.2
8.7
99
100
Mean + SEM
9.1+ 0.07
10.1+ 0.26
100 + 1.33
113.4 + 3.98
6hr/ Rabbit 1
9.2
13.3
99
182
2
9.1
13.0
99
200
3
8.9
14.6
98
199
4
9.5
14.5
102
134
5
8.9
13.7
99
149
6
9.0
9.7
109
139
7
9.2
13.1
98
170
8
9.1
10.2
100
163
Mean + SEM
9.1+ 0.07
12.8 + 0.65
100.5 + 1.30
166.4 + 9.32
12hr/ Rabbit 1
9.5
13.2
102
181
2
9.5
13.0
99
199
3
9.0
13.0
99
199
4
9.9
13.2
101
130
5
8.9
12.6
100
149
6
9.1
9.2
110
130
7
9.6
13.4
100
160
8
9.3
10.6
103
153
Mean + SEM
9.4 + 0.12
12.3 + 0.55
101.8 + 1.28
162.6 + 9.82
-Values (from 1-8) are for individual male New Zealand rabbits (n=8) Followed by their corresponding
Mean + SEM .
- Serum BUN and creatinine (Cr) concentrations (mmolL-1) were measured in blood collected from
central ear artery of animal treated with either 0.9% Nacl (0.05 ml kg-1) or LQQ (0.4 mg kg-1 ) . Refer
to methods and results sections for further details.
25
A
CO2 concentration
CO2 conc.(mmol/L)
20
Cont
Venom
#
*
+
*
15
10
5
0
0
1
3
6
12
Time (hour)
120
CL concentration
B
CL conc.(mmol/L)
115
Cont
Venom
*
110
105
$
100
95
90
0
1
3
6
12
Time (hour)
Fig (10) : Serum CO2 (A) and CL (B) concentrations of control (0.9 % Nacl 0.05 ml/kg sc.) and
LQQ (0.4 mg/KG, sc.) treated rabbits. Bars represent the mean and vertical lines SEM of 8
rabbits at each time point. At *, values are significantly different from their respective control
groups at all times (* p< 0.001). At +, values are significantly different from their corresponding
venom treated group at zero, 1, 3, and 6 hour (+,p< 0.001), while at #, values are significantly
different from their respective venom treated groups at zero and 1 hour only (# p< 0.01). At $,
values are significantly different fromtheir respective venom treated group at 1, 3, and 6 hour ($
p< 0.001)
Table (20): Serum CO2 and chloride levels in control and Leiurus quinquestriatus venom-treated rabbits
LQQ (CO2)
LQQ (CL)
Time / Rabbit No.
Control (CO2)
Control (CL)
0hr/Rabbit
1
17
18
104
107
2
18
17
104
104
3
18
18
106
104
4
18
19
104
104
5
19
20
104
104
6
20
19
105
105
7
16
20
104
105
8
19
17
104
106
Mean + SEM
18.1+ 0.44
18.5 + 0.42
104.4 + 0.26
104.9 + 0.4
1hr/Rabbit
1
18
21
103
110
2
18
16
104
110
3
19
15
107
102
4
18
22
104
109
5
19
20
104
108
6
22
18
104
109
7
18
20
101
109
8
19
18
102
109
Mean + SEM
18.6 + 0.26
18.8 + 0.86
103.6 + 0.62
108.3 + 0.92
3hr/Rabbit
1
20
19
102
112
2
18
13
105
103
3
18
14
107
99
4
18
21
104
110
5
20
18
105
111
6
22
16
104
114
7
20
18
102
112
8
20
17
102
103
Mean + SEM
19.5 + 0.50
17.0+ 0.93
103.9 + 0.64
108.0 + 1.94
6hr/ Rabbit 1
20
17
102
114
2
16
12
104
113
3
18
12
107
104
4
17
16
103
115
5
20
16
105
114
6
22
12
104
114
7
20
18
102
114
8
21
17
105
114
Mean + SEM
19.3 + 0.73
15.0 + 0.91
104.0 + 0.6
112.8 + 1.26
12hr/ Rabbit 1
19
13
102
100
2
17
10
103
93
3
18
11
107
102
4
18
15
103
99
5
20
12
105
101
6
19
10
105
112
7
19
13
104
101
8
20
10
106
100
Mean + SEM
18.8 + 0.37
11.8 + 0.65
104.4 + 0.6
101.0 + 1.85
-Values(from 1-8) are for individual male New Zealand rabbits (n=8) Followed by their corresponding
Mean + SEM .
-Serum carbon dioxide( CO2) and Chloride (CL) concentrations (mmol L-1) were measured in
blood collected from central ear artery of animal treated with either 0.9% Nacl (0.05 ml kg-1) or
LQQ (0.4 mg kg-1 ) . Refer to methods and results sections for further details.
7
cont
venom
A
RBC concentration
+
*
RBC conc.x109 (cell/L)
6
5
4
3
2
1
0
0
1
3
6
12
Time (hour)
25
B
+
*
cont
venom
WBC concentration
WBC conc.x109 (cell/L)
20
15
10
5
0
0
1
3
6
12
Time (hour)
Fig (11) :Serum RBC (A) and WBC (B) concentrations of control (saline 0.05
ml/kg) and LQQ (0.4 mg/kg) treated rabbits. Values represent the mean + SEM
of 8 rabbits at each time point. At * values are significantly different from each
group respective control at all times (*p < 0.001). At + values are significantly
different from each group respective venom treated group at zero, 1, 3and 6
hr.(+ p < 0.001).
Table (21): RBC and WBC concentration in control and Leiurus quinquestriatus venom-treated rabbits
LQQ (RBC)
Time / Rabbit No.
Control (RBC)
Control (WBC)
0hr/Rabbit
1
6.3
6.4
13.2
2
6.0
6.3
13.0
3
6.6
6.6
13.1
4
6.6
6.5
13.2
5
6.3
6.5
13.1
6
6.3
6.0
12.9
7
6.0
5.8
12.6
8
6.2
6.3
13.0
Mean + SEM
6.3 + 0.01
6.3 + 0.01
13.2 + 0.05
1hr/Rabbit
1
6.3
6.3
13.2
2
5.8
6.4
13.0
3
6.6
6.6
12.9
4
6.6
6.9
13.1
5
6.2
7.0
12.9
6
6.2
5.8
12.7
7
5.9
5.8
12.5
8
6.1
6.2
13.0
Mean + SEM
6.2 + 0.10
6.4 + 0.15
12.9 + 0.08
3hr/Rabbit
1
6.2
6.2
13.4
2
6.0
6.0
13.6
3
6.5
6.5
13.2
4
6.5
6.5
13.4
5
6.1
6.9
13.0
6
6.2
5.9
13.1
7
5.9
5.9
13.0
8
6.0
6.0
13.2
Mean + SEM
6.2 + 0.01
6.2 + 0.12
13.2 + 0.08
6hr/ Rabbit 1
6.0
6.1
13.4
2
5.9
6.0
13.5
3
6.4
6.4
13.1
4
6.3
6.5
13.6
5
6.0
6.8
13.2
6
6.1
5.2
13.0
7
5.9
5.7
13.1
8
6.0
5.8
13.0
Mean + SEM
6.1+ 0.01
6.1+ 0.18
13.2 + 0.08
12hr/ Rabbit 1
6.0
5.6
13.8
2
6.0
5.2
13.5
3
6.3
5.8
13.2
4
6.1
6.0
13.7
5
6.0
6.1
13.4
6
5.9
5.1
13.5
7
5.9
5.3
13.4
8
5.9
5.2
13.2
Mean + SEM
6.0 + 0.01
5.5 + 0.14
13.4 + 0.08
-Values(from 1-8) are for individual male New Zealand rabbits (n=8) Followed by their
Mean + SEM .
LQQ (WBC)
13.3
13.4
13.1
13.0
13.1
12.9
12.8
13.2
13.2 + 0.07
13.6
10.4
13.1
13.0
12.9
13.0
13.1
13.4
12.8 + 0.04
12.9
10.0
12.9
12.0
12.0
13.2
12.9
13.0
12.3 + 0.37
12.6
13.2
14.0
12.9
14.0
12.3
13.1
13.4
13.2 + 0.46
25.7
25.2
19.2
22.6
20.2
21.6
20.8
23.0
22.2 + 0.82
corresponding
- Serum RBC and WBC concentrations (cell/L) were measured in blood collected from central ear
artery of animal treated with either 0.9% Nacl (0.05 ml kg-1) or LQQ (0.4 mg kg-1 ) . Refer to
methods and results sections for further details.
3.5
Basophil concentration
A
Basophil conc.x 108 (cell/L)
Cont
Venom
3
2.5
*
2
+
**
1.5
#
**
1
0.5
0
0
1
3
6
12
Time (hr)
Neutrophil conc.x 108 (cell/L)
18
16
B
Cont
Venom
#
**
Neutrophil concentration
+
14
**
12
*
10
8
6
4
2
0
0
1
3
6
Time (hour)
Fig (12) :Serum basophil (A) and neutrophil (B) concentration of contro
l(saline 0.05 ml/kg) and LQQ (0.4 mg /kg) treated rabbits. Values represent
the mean + SEM of 8 rabbits at each time point. At * values are significantly
different from respective control group at all times (*p < 0.01 .* *p< 0.001 ) .At
+ values are significantly different from respective venom treated group at
zero and 1 hr (+p<0.001),while at # values are significantly different from
respective venom treated group at zero, 1& 3 hr (#P<0.001) .
Table (22): Serum basophils and neutrophils levels in control and Leiurus quinquestriatus venom-treated rabbits
Time /Rabbit No.
Control (Baso.)
LQQ (Baso.)
Control (Neut)
LQQ (Neut)
0hr/Rabbit
1
3
3
4
5
2
3
3
5
5
3
3
3
5
5
4
3
3
4
5
5
3
3
5
4
6
2
2
4
6
7
3
3
6
4
8
3
2
6
6
Mean + SEM
2.9 + 0.13
2.8 + 0.16
4.8 + 0.29
5.0 + 0.27
1hr/Rabbit
1
3
2
4
6
2
3
3
6
9
3
3
3
6
9
4
3
2
4
9
5
3
3
5
4
6
2
3
4
6
7
3
3
6
4
8
3
2
6
6
Mean + SEM
2.9 + 0.13
2.6 + 0.18
5.1+ 0.34
6.6 + 0.75
3hr/Rabbit
1
3
1
5
9
2
3
3
6
9
3
3
2
4
10
4
2
2
6
11
5
2
2
5
10
6
2
2
6
9
7
4
1
6
10
8
3
1
6
10
Mean + SEM
2.8 + 0.25
1.8 + 0.25
5.5 + 0.26
9.75 + 0.25
6hr/ Rabbit 1
3
1
5
9
2
3
2
6
9
3
4
2
4
21
4
3
1
7
13
5
3
1
6
8
6
2
1
5
12
7
4
1
4
9
8
2
1
6
8
Mean + SEM
3 +0.27
1.2 + 0.16
5.4 + 0.37
11.1+ 1.56
12hr/ Rabbit 1
3
1
4
13
2
3
1
6
13
3
4
1
5
25
4
2
1
8
16
5
3
1
7
15
6
2
0
3
12
7
4
1
5
12
8
2
0
6
13
Mean + SEM
2.9 + 0.3
0.75 + 0.16
5.5 + 0.57
14.9 + 1.53
-Values(from 1-8) are for individual male New Zealand rabbits (n=8) Followed by their corresponding
Mean + SEM .
- Serum basophilsand neutrophils concentrations (cell/L) were measured in
blood collected from central ear artery of animal treated with either 0.9% Nacl
(0.05 ml kg-1) or LQQ (0.4 mg kg-1 ) . Refer to methods and results sections for
further details.
4.3. Effect of Selected Anti-inflammatory Drugs on the Lethal Actions of
Leiurus quinquestriatus Scorpion Venom:
4.3.1. Effect of intravenous and subcutaneous injection of 0.9 % NaCl into mice:
Injection of 0.2 ml/mouse of 0.9 % NaCl (n =10) caused no signs of abnormal
toxicity. All mice were alive at the end of the time limit of the experiment (300 min).
4.3.2. Effect of intravenous and subcutaneous injection of LQQ venom on the survival of
mice:
When the Egyptian LQQ venom was injected into mice in gradually increasing
doses ranging between 0.15 and 0.325 mg kg–1 i.v. or ranging between 0.175 and
0.325 mg kg–1 s.c., the LD50 of both routes were more or less similar (0.23 + 0.02 and
0.255 + 0.018 mg kg–1 , respectively). In addition, the MLD were calculated to be
0.325 and 0.35 mg kg–1 for the i.v. and s.c., respectively(Tables 25 A and B). On the
other hand, the Sudanese scorpion venom when injected in doses ranging from 0.15
and 0.325 mg kg–1 i.v. or ranging between 0.175 and 0.35 mg kg–1 sc., the LD50 were
0.225 + 0.018 and 0.245 + 0.02 mg kg–1 for iv. and s.c., respectively and MLD were
0.325 and 0.35 mg kg–1 respectively (Table 26 A and B).
All animals injected with the venom showed signs of scorpion envenomation which
included, fighting behavior, lachrymation, hypersalivation, micturition, defecation,
increased respiration, tremors and occasional convulsions. Before death, the animals
exhibited decreased motor activity, depressed respiration, gasping and convulsions.
Post mortum macroscopical examination of the hearts and lungs showed cardiac arrest
in systole with congestion of the heart plus hemorrhagic patches in the lung.
In all subsequent experiments, the Egyptian LQQ venom was utilized in doses
comparable to i.v. and s.c. LD50 (0.25 mg kg–1) and in a dose slightly less than the
MLD (0.3 mg kg–1). The percentage survival of animals / group was found to be 50 %
and the average time of death of non surviving animals was 42.45+1.6 min when the
lower dose was injected (0.25 mg kg–1), while the percentage of surviving animal /
group was 10% and the average time of death was 28.85+1.1 min when the higher
dose was used (0.3 mg kg–1)
4.3.3. Effect of montelukast, hydrocortisone, and indomethacin administration on
survival
of mice:
Oral administration of montelukast (10 or 20 mg kg–1) to mice caused no signs of
abnormal toxicity. All mice were alive at the end of the experiment. Likewise,
intravenous injection of hydrocortisone (5 or 10 mg kg–1) or indomethacin (10 or 20
mg kg–1) caused no outward signs of abnormal toxicity and at the end of the
experiment (300 min) all mice were alive and well.
4.3.4. Effect of oral administration of Montelukast on the survival of mice injected
subcutaneously with LQQ scorpion venom:
Montelukast (MK) in doses of either 10 or 20 mg kg–1, administered orally two
hours before injection of LQQ venom (0.25 mg kg–1 s.c.) significantly increased the
percentage of surviving animals from 50% with venom alone to 85% and 90% with
the low and high doses of montelukast, respectively . Also, the average time of death
of non-surviving animals was increased from 42.45+1.6 min to 193.75+1.4 and
217.6+0.6 min, respectively (P < 0.001). Likewise, the low and high doses of MK
increased the percentage of surviving animals from 10% to 50% and 70% respectively
in animals injected after 2 hours with 0.3 mg kg–1 LQQ. Moreover, the average time
of death of non-surviving mice was also increased from 28.85+1.1 min to 107.8+1.3
and 115.1+1.5 min versus the low and high dose of the drug, respectively (p < 0.0001)
(Tables 27 and 30, also Fig. 13 and 14 representative of the higher dose of MK with
both doses of venom).
4.3.5. Effect of intravenous injection of hydrocortisone on the survival of mice injected
subcutenously with LQQ scorpion venom:
When hydrocortisone (HCTZ) in doses of 5 and 10 mg kg–1 was injected
intravenously into mice half an hour before LQQ venom (0.25 mg kg–1 , s.c.), it
increased the percentage of surviving mice from 50% with venom alone to 70 % and
75%, with the low and high doses of the drug, respectively . Additionally, the average
time of death of non-surviving animals were prolonged from 42.45+1.6 min to
108.9+1.4 and 101.1+0.5 min, respectively (p < 0.05). When the higher dose of LQQ
venom was utilized (0.3 mg kg–1 s.c.), the percentage of surviving mice increased
from 10% to 45%, with the tow doses of HCTZ, while the average time of death of
non-surviving mice were increased from 28.85+1.1 min to 80.45+1.4 and 84.5+1.7
min, respectively (p < 0.0001) (Tables 28 and 30, also Fig. 15 and 16 representative
of the higher dose of HCTZ with both doses of venom).
4.3.6. Effect of intravenous injection of indomethacin on the survival of mice injected
subcutenously with LQQ scorpion venom:
Intravenous injection of indomethacin (IND) 10 and 20 mg kg–1 into mice half an
hour before injection of LQQ venom (0.25 mg kg–1, s.c.) significantly increased the
percentage of surviving animals from 50% with venom alone to 70% (with either the
low or high dose of the drug). The average time of death of non-surviving animals
was increased from 42.45+1.6 min to 100.05+0.4 and 99+0.4 min, with the 1st and 2nd
doses of the drug, respectively (p<0.05). Using the same 2 doses of the drug with a
higher dose of the venom (0.3 mg kg–1), the percentage of surviving animals was
increased from 10% with LQQ venom alone to 40% and 35%, with the drug’s 2
concentrations, respectively, and the average time of death of non-surviving animals
was increased from 28.85+1.1 min to 80.2+2.3 and 75.96+1.7 min, respectively (p<
0.001) (Tables 29 and 30, also Fig. 17 and 18 representative of the higher dose of IND
with both doses of venom).
Table (25): Determination of intravenous (A) and subcu/taneous (B) LD50 of
Egyptian Leiurus quinquestriatus scorpion venom
A:
Group
LQQ Dose (mg kg-1)
Number of dead mice
Dead %
1
0.150
0
0
2
0.175
1
10
3
0.200
2
20
4
0.225
4
40
5
0.250
5
50
6
0.275
7
70
7
0.300
9
90
8
0.325
10
100
Group
LQQ Dose (mg kg-1)
B:
1
2
3
4
5
6
7
8
Number of dead mice
Dead %
0.175
0
0
0.200
1
10
0.225
3
30
0.250
4
40
0.275
5
50
0.300
8
80
0.325
9
90
0.350
10
100
Correction formula used for the 0.0 % dead: 100 (0.25/n); for 100 % dead, 100 (n-25/n). n: no. of mice/group.
Refer to methods for further details. the LD50 of both routes were more or less similar (0.23 + 0.02 and 0.255 +
0.018 mg kg–1 , respectively).
Table (26): Determination of intravenous (A) and subcutaneous (B) LD50 of
Sudanese Leiurus quinquestriatus scorpion venom
A:
Group
1
2
3
4
5
6
7
8
LQQ Dose (mg kg-1)
Number of dead mice
Dead %
0.150
0
0
0.175
1
10
0.200
3
30
0.225
4
40
0.250
6
60
0.275
8
80
0.300
9
90
0.325
10
100
B:
Group
LQQ Dose (mg kg-1)
1
0.175
0
0
2
0.200
2
20
3
0.225
3
30
4
0.250
5
50
5
0.275
7
70
6
0.300
8
80
7
0.325
9
90
8
0.350
10
100
Number of dead mice
Dead %
Correction formula used for the 0.0 % dead: 100 (0.25/n); for 100 % dead, 100 (n-25/n). n: no. of mice/group.
Refer to methods for further details. The Sudanese scorpion venom LD50 were 0.225 + 0.018 and 0.245 + 0.02 mg kg–1
for iv. and sc., respectively.
Table (27): The effect of s.c. injection of LQQ venom alone or after montelukast
on the lethality of mice.
Animal
Number
LQQ alone (µg kg -1)
250
300
LQQ (250 µg kg -1) and MK
MK 10mgkg-1
MK20mg kg-1
LQQ (300 µg kg -1) and MK
MK10mg kg-1
MK 20mg kg-1
Survival time (min)
1
Surv.
32
Surv.
Surv.
106
Surv.
2
Surv.
28
Surv.
Surv.
108
Surv.
3
36
17
Surv.
Surv.
95
Surv.
4
Surv.
29
172
Surv.
Surv.
108
5
26
Surv.
Surv.
210
Surv.
Surv.
6
28
23
Surv.
Surv.
112
Surv.
7
45
26
Surv.
Surv.
106
Surv.
8
47
28
195
Surv.
Surv.
115
9
Surv.
36
Surv.
Surv.
Surv.
110
10
Surv.
31
Surv.
Surv.
Surv.
Surv.
11
37
35
Surv.
Surv.
105
Surv.
12
35
25
Surv.
218
Surv.
Surv.
13
Surv.
Surv.
Surv.
Surv.
97
100
14
42
26
Surv.
Surv.
98
Surv.
15
Surv.
28
193
Surv.
Surv.
98
16
Surv.
30
Surv.
Surv.
102
Surv.
17
45
21
Surv.
Surv.
Surv.
118
18
Surv.
30
Surv.
Surv.
107
Surv.
19
38
31
Surv.
Surv.
Surv.
Surv.
20
Surv.
29
Surv.
Surv.
Surv.
Surv.
mean Wt of
mice + SE
% of survival
Mice / gp.
20 + 0.1
20 + 0.12
20 + 0.1
21 + 0.11
20 + 0.1
20 + 0.12
50 %
10 %
85 %
90 %
50 %
70 %
mean death
time +S.E.M
42.45+1.6
28.85+ 1.1
193.75+1.4**
217.6+0.6**
107.8+1.3***
115.1+1.5***
-LQQ : leiurus quinquestriatus scorpion venom; MK: Montelukast; *: Surv: animal survived for more than 72 hours; Wt: weight; gp:
group. Refer to methods for further details.
-Wilcoxon
survival
statistics
(*P<0.05,
**P<0.001,
***P<0.0001)
Fig (13). Survival distribution function curve of mice injected with L. quinquestriatus
venom (250 µg kg –1, s. c, solid line) alone or 2 hours after montelukast (MK, 20 mg
kg –1, p. o, dotted line). Y-axis stands for survival with (0) indicating death and (1)
survival. X-axis represents time in minutes. Refer to methods and results for further
details.
Fig. (14). Survival distribution function curve of mice injected with L.
quinquestriatus venom (300 µ g kg -1, s.c. solid line) alone or 2 hours after
montelukast (MK, 20 mg kg –1, p.o, dotted line). Y-axis stands for survival
with (0) indicating death and (1) survival. X-axis represents time in minutes.
Refer to methods and results for further details.
Table (28): The effect of s.c. injection of LQQ venom alone or after hydrocortisone
on the lethality of mice
Animal
Number
LQQ alone (µg kg -1)
250
300
LQQ (250 µg kg -1) and HCT
HCT 5mgkg-1
HCT10mgkg-1
LQQ (300 µg kg -1) and HCT
HCT 5mgkg-1
HCT10mgkg-1
Survival time ( min )
1
Surv.
32
Surv.
Surv.
Surv.
Surv.
2
Surv.
28
Surv.
Surv.
Surv.
Surv.
3
36
17
Surv.
98
85
85
4
Surv.
29
112
Surv.
68
Surv.
5
26
Surv.
Surv.
Surv.
Surv.
87
6
28
23
Surv.
Surv.
78
Surv.
7
45
26
98
Surv.
Surv.
72
8
47
28
Surv.
95
Surv.
Surv.
9
Surv.
36
Surv.
Surv.
77
75
10
Surv.
31
92
Surv.
75
82
11
37
35
Surv.
Surv.
Surv.
80
12
35
25
101
Surv.
64
79
13
Surv.
Surv.
Surv.
Surv.
82
Surv.
42
26
100
101
Surv.
Surv.
15
Surv.
28
Surv.
Surv.
Surv.
90
16
Surv.
30
107
96
Surv.
64
17
45
21
Surv.
Surv.
78
81
18
Surv.
30
Surv.
102
77
Surv.
19
38
31
Surv.
Surv.
80
Surv.
20
Surv.
29
Surv.
Surv.
80
86
Mean Wt+SE
19 + 0.1
20 + 0.1
20 + 0.12
21 + 0.1
20 + 0.1
20 + 0.11
% of survival
Mice / gp.
50 %
10 %
70%
75 %
45 %
45 %
14
Mean time of
42.45 + 1.6 28.85+ 1.1 108.9+1.4*
101.1+0.5*
80.45+1.4***
84.5+1.7***
death of non
survivors
-LQQ : leiurus quinquestriatus scorpion venom; HCT: Hydrocortisone; Surv.: animal survived for more than 72 hours, Wt:
weight; gp: group; Av: average. Refer to methods for further details.
-Wilcoxon survival statistics (*P<0.05, **P<0.001, ***P<0.0001)
Fig. (15). Survival distribution function curve of mice injected with L. quinqestriatus
venom (250 µ kg –1, s.c, solid line) alone or 30 minute after hydrocortisone (HCTZ, 10
mg kg –1, i.v, dotted line). Y-axis stands for survival with (0) indicating death and (1)
survival. X-axis represents time in minutes. Refer to methods and results for further
details.
Fig. (16). Survival distribution function curve of mice injected with
L.quinqestriatus venom (300 µg kg –1, s.c.dotted line) alone or 30 minutes after
hydrocortisone (HCTZ, 10 mg kg –1, i.v, solid line). Y-axis stands for survival
with (0) indicating death and (1) survival. X-axis represents time in minutes.
Refer to methods and results for further details.
Table (29): The effect of s.c. injection of LQQ venom alone or after indomethacin
on the lethality of mice
Animal
Number
LQQ alone (µg kg -1)
250
300
LQQ (250 µg kg -1) and IND
IND 10mgkg-1
IND 20mgkg-1
LQQ (300 µg kg -1) and IND
IND 10mgkg-1
IND 20mgkg-1
Survival time ( min )
1
Surv.
32
Surv.
Surv.
60
Surv.
2
Surv.
28
Surv.
Surv.
77
Surv.
3
36
17
99
95
75
Surv.
4
Surv.
29
101
96
Surv.
79
5
26
Surv.
Surv.
Surv.
Surv.
82
6
28
23
98
Surv.
65
63
7
45
26
Surv.
Surv.
Surv.
77
8
47
28
95
100
78
72
9
Surv.
36
Surv.
Surv.
Surv.
75
10
Surv.
31
Surv.
Surv.
90
62
11
37
35
Surv.
98
Surv.
64
12
35
25
98
Surv.
72
Surv.
13
Surv.
Surv.
Surv.
Surv.
Surv.
Surv.
14
42
26
96
95
Surv.
78
15
Surv.
28
Surv.
Surv.
Surv.
60
16
Surv.
30
Surv.
96
79
76
17
45
21
Surv.
Surv.
73
78
18
Surv.
30
Surv.
Surv.
68
79
19
38
31
Surv.
Surv.
72
Surv.
20
Surv.
29
Surv.
Surv.
75
Surv.
Mean Wt+SE
20 + 0.1
21 + 0.1
20 + 0.12
20 + 0.1
20 + 0.11
21 + 0.1
% of survival
50 %
10 %
70%
70 %
40 %
35 %
Mice / gp.
mean time of
42.45+1.6 28.85+1.1 100.05+0.4*
99+0.4*
80.2+2.3***
75.96+1.7**
death of non
survivors
-LQQ : leiurus quinquestriatus scorpion venom;IND: Indomethacin, Surv.: animal survived for more than 72 hours, Wt:
weight, gp: group; Av: average. Refer to methods for further details.
-Wilcoxon survival statistics (*P<0.05, **P<0.001, ***P<0.0001)
Fig.(17). Survival distribution function curve of mice injected with L.quinqestriatus
venom (250 µg kg –1, s.c. solid line) alone or 30 minutes after indomethacin (IND,
20 mg kg –1, i.v, dotted line). Y-axis stands for survival with (0) indicating death
and (1) survival. X-axis represents time in minutes. Refer to methods and results
for further details.
Fig.(18). Survival distribution function curve of mice injected with
L.quinqestriatus venom (300 µg kg –1, s.c, dotted line) alone or 30 minutes after
indomethacin (IND, 20 mg kg –1, i.v, solid line). Y-axis stands for survival with
(0) indicating death and (1) survival. X-axis represents time in minutes. Refer to
methods and results for further details.
Table (30): Percentage of survival and average survival time of mice injected with LQQ
scorpion venom alone or after montelukast, hydrocortisone or indomethacin.
LQQ venom (250 µg kg –1)
Group
% of surviving
mice / group.
mean survival
time (min)
LQQ Venom
alone
50%
MK 10 mgkg-1
and LQQ
LQQ venom (300 µg kg –1)
% of surviving
mice / group.
mean survival
time (min)
42.45+1.6
10 %
28.85+1.1
85%
193.75+1.4**
50 %
107.8+1.3***
MK 20 mgkg-1
and LQQ
90%
217.6+0.6**
70 %
115.1+1.5***
HCT 5 mgkg-1
and LQQ
70%
108.9+1.4*
45%
80.45+1.4***
HCT 10 mgkg-1
and LQQ
75 %
101.1+0.5*
45 %
84.5+1.7***
IND 10 mgkg-1
and LQQ
70%
100.05+0.4*
40 %
80.2+2.3***
IND 20 mgkg-1
and LQQ
70%
99+0.4*
35 %
75.96+1.7**
LQQ: Leiurus quinquestriatus scorpion venom; MK: Montelukast; HCT: Hydrocortisone; IND: Indomethacin. Refer
to methods for further details.
-Wilcoxon survival statistics (*P<0.05, **P<0.001, ***P<0.0001)
CHAPTER - V
GENERAL DISCUSSION
5. GENERAL DISCUSSION
This project consisted of three portions all undertaken to gain greater insight into the actions
of scorpion venoms. The first section comprised a preliminary clinical investigation on the
characteristics and outcome of patients admitted to a major hospital following scorpion
envenomation during a selected period of time. The second phase of the project entailed the
injection of LQQ venom, the culprit behind the majority of the stings in the patients studied,
into conscious rabbits to further understand the actions of the venom on different systems.
Lastly, the project attempted to check the effectiveness of selected treatment modalities
thought to be able to prolong survival and improve the outcome of mice injected with LQQ
venom.
The preliminary clinical investigation was performed to gain insight into the trends and
characteristics of scorpion envenomed patients, treatment modalities utilized and prognosis,
following their admittance to Dongla Hospital in Dongla, Sudan, during a six-month period
(January-July 2002). This study indicated that more than two thirds of those admitted were
male, which is not surprising due to their adventurous nature and the necessity of being
outdoors more often than females (Gordillo et al., 2000; De Roodet et al., 2003; Al-Asmari
and Al-saif, 2004). The study also showed that one third of the victims were children with
more than 60% of them eventually dying versus more than 70% of the adults improving and
eventually being discharged. Several studies have demonstrated that children are more
affected by scorpion toxins, possibly due to their smaller body mass and immaturity of some
of their systems (Meki and Mohey El-Dean, 1998; Freire-Maia et al, 1994; Magalhaes et al.,
1999; Isbister et al., 2003; Al-Asmari and Al-Saif, 2004).
It was apparent from the clinical portion of the study that approximately three quarters of
the stung patients seeked medical help within an hour with the percentage of survivors
decreasing as the time of admittance was delayed. It is known that during the late stages of
envenomation, several neurotransmitters and mediators are excessively released as a result of
the venom-induced action on ionic channels, resulting in deleterious effects on different
systems of the body (Watt and Simard, 1984; Freire-Maia and Campos, 1989; Ismail, 1995;
Meki et al., 2003a; Meki et al, 2003b; Fukuhara et al., 2003). It would seem that the later the
arrival of the envenomed patient to the hospital, the greater the chance of non-reversible
damages occurring.
In an attempt to map the time course of LQQ scorpion envenomation and what occurs in the
early and the late stages, an experimental portion was designed whereas blood pressure (BP),
biochemical and hematological changes were recorded simultaneously at different intervals
following the injection of LQQ venom into conscious white rabbits. The results showed that
LQQ venom caused a triphasic effect on BP consisting of an initial insignificant transient fall,
a gradual increase and a gradual terminal hypotension that ended in death of the rabbits.
The hypertension seen in the rabbits during the early stages of envenomation has been
explained by the well-known direct effect of the venom on ion channels, especially
sodium, resulting in a massive release of catecholamines from synaptic nerve ending and
the adrenal medulla (Harvey et al., 1994: Ismail, 1995; Fatani et al., 1998, 2000; Tarasiuk
et al, 1994; Meki et al., 2003a; Fukuhara et al., 2003). These catecholamines would act on
α adrenoceptors raising the blood pressure, in addition to the possible role of circulating
potent vasoconstrictors such as angiotensin, renin and/-or endothelins. In the clinical
portion of this study, one quarter of the scorpion envenomed patients admitted to Dongla
Hospital were hypertensive and bradycardic, unfortunately 70-80% of these victims
eventually did not survive. Scorpion venom evoked hypertension is considered a major
etiological factor in the development of subsequent pulmonary edema, excessive cardiac
stimulation, arrhythmias and left ventricular heart failure, usually seen following
scorpion envenomation (Abroug et al, 1991; Gueron et al., 1992b; Bawaskar and
Bawaskar, 1992 a, b; Ismail, 1995; De Matos et al., 1999; De Matos et al., 2001; Andrade,
2002; Wang et al., 1994;
Meki et al., 2003a). Moreover, although the experimental
portion of this study showed that not much biochemical changes occurred during early
hypertensive stage, markers showing damages in the cardiovascular system, such as
creatine kinase (CK) and lactate dehydrogenase (LD) were elevated in the later stages (6
hours and onwards).
Moreover, in the experimental part of this study, injecting conscious rabbits with LQQ
venom led to a prominent long lasting hypotension, following the venom-evoked
hypertension phase that ended in death. Similarly in the preliminary clinical investigation,
patients admitted early to the hospital were hyper-, normo- or slightly hypotensive, while
those who entered after 1 hour and onwards were all hypotensive. Much controversy remains
regarding this late venom-elicited hypotensive phase and the accompanying deterioration of
the general condition, both of which culminate in death. Postulated mechanisms include
venom-evoked enhanced release of the neurotransmitter acetylcholine, exaggerated B2vasodilator effect from venom-released catecholamine, a catecholamine depletion syndrome,
hypovolemia secondary to excessive fluid loss and/-or presence of large amounts of
inflammatory mediators and potent vasodilator substances, such as kinins, cytokines, nitric
oxide and /-or prostaglandins (Freire Maia et al., 1979; Sofer and Gueron, 1992; Ismail,
1995; Fatani et al., 1998; Magalhaes et al., 1999; Meki et al., 2003a).
In this study, the biochemical analysis of the serial samples of blood collected from
conscious rabbits following s.c. Injection of LQQ venom, confirmed the role of the several
inflammatory agents such as the potent vasodilator nitric oxide, TNFα, IL-8, in addition
to leucocytosis. This is in accordance with Meki and Mohy- Eldeen (1998) Meki et al
(2003a) and Fukuhara et al., (2003) who demonstrated the elevation of the cytokines
IL1β, IL6, IL8, IL10, TNFα, white blood cells and/-or NO in envenomed victims. It was
postulated that the severity of envenoming was affected by the amplification of
inflammatory cascade response followed by release of chemical mediators such as
cytokines (Meki and Mohey- Eldeen, 1998; Magalhaes et al., 1999). Additionally, the
augmented synthesis of NO, observed in rabbits injected with LQQ venom could explain
the venom-induced terminal hypotension and is probably caused by the enhancement of
the constitutive isoform of NO synthases by cytokines, acetylcholine and bradykinin, all
of which have been proven to be released in large amounts after scorpion stings (Fatani et
al., 1998; Meki and Mohey El-Deen, 1998; Meki et al 2003a). Furthermore, cytokines
such as TNF-α, IL-1β, or IL-6 have also been implicated in stimulating the release of
leukotrienes and chemokines, which in turn, by their hematopoietic stimulatory effects,
activate leukocyte formation.
(Dietch, 1992; Meki et al., 2003a; Fukuhara et al.,
2003). Leucocytosis was not only observed in the experimental portion of this study, but
also in the preliminary clinical investigation. Thirty seven percent (11) of the 30
envenomed patients admitted to the hospital-exhibited leucocytosis, one only survived,
demonstrating its role in affecting morbidity following envenomation.
Neutrophils were also increased in the serum of conscious rabbits injected with the
venom in the experimental portion of this study. This could be due to the venom-induced
enhanced cytokine release, where cytokines such as IL-8 are known neutophil chemoattractants/activators (Boujoukos et al., 1993). These neutrophils might, in addition to
leucocytosis, contribute to occurrence of injury, ischemia and oedema in multi-mediated
processes in different organs. (Boujoukos et al., 1993; Fukuhara et al., 2003; Meki et al.,
2003a) Multiple organ dysfunction has been reported following scorpion envenomation
by several authors and played a role in increasing morbidity (Freire-Maia and Campos,
1989; Meki and Mohey El-Dean, 1998; Meki et al., 2003 b). Dysfunction of several organs
was also observed during the late stages, 6 hours and onwards after injecting the venom
into conscious rabbits. The dysfunction was evidenced by the elevation of markers
showing damages in the cardiovascular system, in addition to the liver and kidney
functions. These markers included: elevation of serum creatine kinase (CK), lactate
dehydrogenase (LD), aspartate aminotransferase (AST), alanine transferase (ALT),
blood urea nitrogen (BUN) and creatinine (Cr). The latter-mentioned two parameters
were also elevated in several of the envenomed patients admitted to Dongla hospital in the
clinical portion of this study. Similar changes were described by several investigators
following scorpion envenomation (El-Asmar, 1984; Ismail and Abd- Elsalam, 1988; Sofer
and Gueron, 1988; Fatani, 1990; Hering et al., 1993; Correa et al., 1997; Magalhaes et al.,
1999; Bertazzi et al., 2003).
As evidenced by the multitude of effects taking place during the late hypotensive phase
in envenomed rabbits, this terminal stage appears to be multi-factorial. The majority of
the deleterious effects are probably the resultant of the cascade of events induced by the
venom’s ability to act on sodium channels and thus enhance the release of several
neurotransmitters and neuro-modulators in different systems of the body. Not only the
cardiovascular, hepatic and renal systems are altered, but others as well. For example,
this study confirmed that scorpion venoms affect the respiratory system, as evidenced by
the noticeable pulmonary edema shown by the increased lung/body weight index
calculated after death of rabbits injected with venom. Furthermore, several forms of
respiratory distress were observed in many of the patients admitted to Dongla hospital
after being stung. Both clinical reports and experimental studies following scorpion
envenomation described different types of abnormal respiratory conditions such as
tachypnea, hyperpnea, gasping, wheezing, acute pulmonary edema, acute respiratory
distress syndrome and /-or respiratory failure (Freire-Maia and Campos, 1989; Ismail,
1995; Sofer et al., 1996; Meki and Mohey El-Dean, 1998; Magalhaes et al., 1999; Matos et
al., 1999; D’Suze et al., 1999; Andrade et al., 2002; Bertazzi et al., 2003).
Moreover, many of the envenomed victims admitted to the hospital in this study
exhibited signs of central nervous system stimulation such as restless, excessively
vomiting, feverish and several had convulsions. These symptoms were also mentioned by
several investigators following scorpion stings in both humans and experimental animals
(Goyffon et al., 1982; Gueron and Ovsyshcher, 1987; Amaral et al., 1993; Matos et al.,
1999). More importantly, the present clinical investigation insinuated that severity of
these symptoms seen in the envenomed victims correlated with morbidity. For example
none of the patients that had convulsions (8 out of 30) survived, whilst only 5 died of the
remaining 22 patients who had no convulsions.
Moreover upon admittance, 60% of the patients were very restless; 40% of these
eventually died, while most of the remaining non-restless patients survived. A similar
pattern was detected in patients presenting with fever or emesis. Metabolic changes are
also evident following scorpion envenomation. For instance, the majority of envenomed
patients admitted to Dongla hospital in this study were hyperglycemic; of these more
than half did not survive. On the contrary, none of the patients that had normal glucose
levels died. Hyperglycemia was also evident from 6 hours and onwards in conscious
rabbits injected with LQQ venom in this study. The hyperglycemia seen in this study
could be explained by the venom-elicited β2 adrenoceptor mediated metabolic effects,
release of tissue and medullary catecholamines, serotonin content of the venom,
inhibition of insulin release and/-or pancreatitis that have been observed in human and
experimental animals (Corrado et al., 1968; El- Asmar et al., 1974, Ismail et al., 1977;
Shnkaran et al., 1987; Ismail and Abd –Elsalam, 1988; Frier-Maia and Campose, 1989).
Moreover, the significant decrease in serum carbon dioxide level and increase in serum
chloride level also observed in the present study in the conscious rabbits are in
accordance with that reported by some investigators who related this effect to the
occurrence of acidemia. The latter effect could be explained as being due to hypoxia
leading to accumulation of lactic acid. Alternatively metabolic acidosis could have
occurred as a resultant of the depressant effect of venom on respiration (Hodhod et al.,
1977; El-Asmar et al., 1977; Tash et al., 1982).
The clinical portion of the present study showed that the treatment protocol in Dongla
Hospital included a local anesthetic at site of injection, antivenom (1 ampoule, i.m.), an
antihistamine, and hydrocortisone. Unfortunately, they did not appear to be effective in the
dose and routes utilized, since a very high percent (43%) of admitted patients ended up
dying! It is apparent that if not properly treated the outcome following scorpion
envenomation may be quite morbid!
The appropriate treatment of scorpion envenoming remains controversial. Several authors
consider that symptomatic management is sufficient (Gueron and Ovsyshcher, 1987; Sofer
and Gueron, 1988; Gueron, and Sofer, 1990; Abrough et al, 1999), others on the other hand
strongly recommend the use of proper doses of specific scorpion antivenoms to neutralize
circulating venom and prevent further damages from occurring (Freire-maia and Campos,
1989; Ismail, 1994; Ghalim et al, 2000; Possani, 2000; Osnaya-Romero et al., 2001, de
Roodet et al, 2003). It was stated by Ismail (1994) that the ineffectiveness of antivenom,
reported by some investigators, may be due to the low potency of antivenom, different
scorpion species utilized in its preparation, use of inadequate doses, inappropriate route and/or late administration. This study showed that there was no difference in mortality between
those given antivenom and others who were not. However, it must be kept in mind that only 1
ampoule (able to neutralize 40 LD50 of venom) was injected intramuscularly, a dose, potency
and route proven to be ineffective (Ismail, 1992). Significant decreases in fatal cases were
recorded in scorpion-envenomed victims injected intravenously with higher quantities of
specific antivenom (2-5 ampoules, each neutralizing 60 LD50 of venom) (Ismail et al 1992,
Ismail, 1994; Calderon-Aranda et al., 1996; Osnaya-Romero et al, 2001). This would further
indicate the importance of administration of specific antivenom in the proper dose and route.
The clinical part of the work demonstrated that in 70% of the cases victims identified
Leiurus quinquestriatus as the culprit, which is a highly venomous scorpion. LQQ scorpions
are abundantly present in Middle Eastern countries such as Saudi Arabia, Egypt and Sudan.
The present study confirmed its lethality with the LD50 calculated to approximately 0.25 mg
kg-1 when injected s.c. or iv. whether obtained from scorpions collected from Southern Egypt
or North of Sudan. This is in agreement with Yahel-Niv and Zlotkin (1979) and Fatani (1990)
who found the LD50 of LQQ to be approximately 0.25 mg kg-1. Thus potent antivenom would
be essential to combat the toxins of this venomous scorpion.
Unfortunately,
No
consensus
appears
present
between
different
therapeutic
recommendations in cases of scorpion envenomation, and to the best of our knowledge
comprehensive Meta analyses comparing different treatment modalities are lacking.
Recommended therapies included the use of diuretics and oxygen, mannitol, steroids and
benzodiazepine (Freire-Maia and Campos, 1987, 1989; Gueron and Ovsyshcher, 1987; Dudin
et al., 1991), vasodilators such as prazosine and /-or nifedipine (Bawaskar and Bawaskar,
1992b; Osnaya-Romero et al, 2001), calcium gluconate, antihistamine (Muller, 1993),
acetaminophen (Osnaya-Romero et al, 2001) and hydrocortisone (Fatani, 1990; OsnayaRomero et al, 2001).
During the author’s work in the National Antivenom and Vaccine Production Center in
Riyadh, Saudi Arabia, a comprehensive treatment protocol was set forth by Ismail (1994) and
applied by the ministry of Health in different hospitals in Saudi Arabia. From that time up to
the present, the results of the protocol’s implementation led to the reduction of death rate of
scorpion-envenomed patients from 4-8% to 0.05% (Ismail, unpublished data). This protocol
included a specific antivenom (5 ampoules injected slowly i.v.). Afterwards, patients are
given according to their symptoms: a local anesthetic for pain, an antihistaminic for
inflammatory reactions, diazepam for convulsions, a diuretic such as furosemide and fluid
restriction for pulmonary edema, an antipyretic (acetaminophen) for fever, a vasodilator for
hypertension and/or ventilatory and cardiac support when needed (Ismail 1994). It is essential
that Dongla Hospital review its treatment in light of the high morbidity rate of admitted
envenomed victims.
The unacceptable high death rate of patients admitted to Dongla hospital in Dongla Sudan
forced us to look more thoroughly into the occurrences that take place during scorpion
envenomation, in an attempt to determine the best way to prevent these actions and thus save
lives. The experimental portion of the study shed some light on some of these occurrences
and demonstrated the venom-enhanced release of potent inflammatory agents such as
cytokines. Since cytokines appear to play a role in venom-evoked terminal hypotension,
ARDS, SIRS and MOD (Freir-Maia and Campos, 1989; Meki and Mohy- Eldeen, 1998;
Meki et al., 2003a and b; Fukuhara et al 2003), agents which block the inflammatory
processes and/-or cytokines may probably prevent these actions. Thus a decision was taken
to pretreat mice injected with LQQ venom with several anti-inflammatory agents including
hydrocortisone, indomathacin and a leukotriene/-cytokine inhibotor, montelukast.
This study demonstrated the effectiveness of montelukast in greatly protecting LQQ
envenomed mice and significantly prolonging their survival time. Montelukast is known
to block the action of cysteinyl leukotrienes (LT) C4, D4 and E4, mediated by cysteinyl
leukotriene 1 receptors. These leukotrienes are products of the 5-lipo-oxygenase
pathway of the arachidonic pathway metabolism (Henderson et al., 2002, Nayak, 2004).
In addition, cysteinyl leukotriene receptor 1 antagonists excert part of their antiinflammatory effects through the suppression of T helper type 2 (Th2) cytokines and
thus the subsequent inhibition of production of allergen-specific IgE, mast cell
degranulation, leukocyte trafficking, eosinophilia, T cell activation, release of
histamines, leukotrienes, and pro-inflammatory cytokines such as interleukins 4, 5 and
13 (Henderson et al., 2002; Eum et al., 2003; Wu et al., 2003). Moreover, there are now
accumulating evidence to suggest a causal relationship between the overproduction of
certain cytokines and both morbidity and mortality (Roumen et al., 1993). The efficacy
of montelukast further points to the possibility that scorpion venoms release various
cytokines and that they play an important role in the venoms-induced pathological
organ damages and the subsequent lethality.
The experimental part of this study also showed that administration of
hydrocortisone significantly increased both the percentage of surviving mice and the
average time of death of non-surviving animals. This result is in agreement with that
obtained by Ismail et al. (1992) who demonstrated the ability of hydrocortisone to
significantly prolong survival of LQQ-envenomed mice. It is well documented that
glucocorticoids, including hydrocortisone, inhibit the production of factors that are
critical in generating the inflammatory response. As a result there is decreased release
of vasoactive and chemoatractive factors, diminished secretion of lipolytic and
proteolytic enzymes, decreased extravasation of leukocytes to areas of inflammation,
and ultimately-decreased fibrosis (Chrousos, 1995). The postulated beneficial effects of
steroids in scorpion envenomation include the modulation of inflammatory mediators
and correction of putative acute adrenal insufficiency and consecutive hypovolemic
shock induced by scorpion envenomation (Mohamad et al., 1954; Ismail et al., 1974a,
Ismail et al., 1992).
Moreover, in this project it was noticed that injection of indomethacin into mice, half
an hour before injection of LQQ venom, significantly prolonged both the percentage
survival and the average time of death of non-surviving animals. It is well known that
indomethacin, a non-steriodal anti-inflammatory drug (NSAI), inhibits the enzymatic
production of prostaglandins, which are known to participate in the pathogenesis of
inflammation and fever (Moncada et al., 1978, Samuelsson, 1983). In addition, these
drugs can inhibit cell migration, activation and function of neutrophils, and leukocytes
adhesions, conditions that are known to occrue during the course of inflammation and
in scorpion envenomation, perhaps by inhibition of membrane-associated processes
independent of their ability to inhibit prostaglandins synthesis (Abramson and
Weissmann, 1989). Several studies have emphasized the relevance of pro-inflammatory
mediators including prostaglandins in the pathophysiological manifestations of human
and experimental scorpion envenomation (Freire-Maia et al., 1978; Fatani, et al., 1998;
De Matos et al., 1997). Freire-Maia et al. (1978) reported that injection of indomethacin
was capable of decreasing the severity of lung edema induced by scorpion toxins in rats.
Moreover, Ismail et al. (1992) related the terminal hypotension, which was resistant to
hypertensive agents that were shown in the majority of children who died from scorpion
stings, to the possibility of the involvement of either prostaglandins or kinins.
In conclusion, it is apparent from this study the multitude of effects that occur following scorpion
envenomation, especially in the late stages. If not properly treated the outcome may be morbid as was seen in
envenomed patients admitted to the main Hospital in Dongla, Sudan. Moreover, it was evident in the present
study that inhibitors of different steps in the inflammatory process may play an important role in prolonging
the survival following scorpion envenomation. Further studies are, however, needed to prove the value of
such agents in clinical cases. The end goal should be the ability to save peoples lives and improve their
chances of survival following scorpion envenomation.
CHAPTER - VΙ
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