Egypt. J. Histol. Vol. 32, No. 2, Dec. 2009: 358 - 367
(ISSN: 1110 - 0559)
Original Article
Effect of Sodium Fluoride on the Cerebellar Cortex of Adult Albino Rats
and the Possible Protective Role of Vitamin B6: A Light and Electron
Microscopic Study
Omayma Kamel Afifi
Department of Histology, Faculty of Medicine, Tanta University
ABSTRACT
Introduction: Sodium fluoride (NaF) toxicity has been a subject of extensive studies over the last years because of their
environmental persistence and world widespread distribution. Its toxicity on many organs has received considerable
attention, but its neurotoxicity has not been entirely studied.
Aim of the Work: Studying the histological changes occurring in the cerebellar cortex of albino rats following chronic
exposure to NaF and the possible protective role of vitamin B6.
Materials and Methods: Twenty adult male albino rats were divided into group I (control), group II (each animal was
daily injected I P with 3.6 mg vitamin B6), group III (each animal received 10 mg/kg NaF orally once daily) and group
IV (each rat was given vitamin B6 concomitantly with NaF at the same dose, period and route of group II and III). After
three months the cerebellum was dissected and prepared for light and electron microscopic study.
Results: The cerebellum of NaF treated animals exhibited severe degenerative changes especially in Purkinje cells.
There was multilayer disposition of these cells associated with structural changes in the form of dilated rER and Golgi
complex, swollen mitochondria in addition to marginated nuclear chromatin. The surrounding neuropil appeared
vacuolated with accumulation of neuroglial cells. Many myelinated nerve fibers displayed disruption in myelination and
irregular neurofilaments. Silver stained sections showed accumulation of cytoskeletal elements in Purkinje cells. On
the other side, these changes were ameliorated in rats of group IV which received vitamin B6 concomitantly with NaF.
Conclusion: Chronic administration of NaF induced degenerative changes in the cerebellar cortex which could be
ameliorated by vitamin B6.
Key Words: Sodium fluoride, vitamin B6, cerebellar
Corresponding Author: Omayma Kamel Afifi
cortex, rats.
Tel.: 012157980
E-mail: omaymakamel_afifi@ hotmail. com
INTRODUCTION
Fluorine is a chemical electronegative element in the
periodic table and a member of the halogen family, which
is widely distributed in the environment. It is a natural
component of the earth’s crust and occurs in varying
concentration in rocks, soil, water and air. It is present in
several chemical forms either organic or inorganic, one
of the commonest inorganic fluorides is sodium fluoride1.
Like many elements, it is beneficial to human health in
trace amounts, but can be toxic in excess2.
commonly used as pesticide, rodenticides, fungicide, and
a constituent in glass, enamel and dental laboratories3,4.
It is present in high level in some sea foods and in
bone products as gelatin. The dark green vegetables are
considered a major source of fluoride, as it accumulates
it in their leaf which is obtained from the soil and water5.
Cooking in Teflon (fluorinated ethylene) lined cookware
also increase the concentration of fluoride in our daily
foods prepared inside them6.
Since the mid of 1940s, fluoride has been added to tap
water to reduce dental caries in American communities.
Then, it is added to drinking water for the purpose
of clarification in many countries. The water based
beverages such as beers, colas, carbonated soft drinks,
sport drinks and reconstituted juices contain variable
amount of fluoride. Sodium fluoride was the first fluoride
compound used in fluorination of drinking water and
it is still commonly used for that purpose. It is also
Commercially, it is used to etch glass, ceramics and
computer chips, refine metals and petroleum products,
make ceramic materials more porous, inhibit fermentation
in breweries and wineries, polish aluminum and is used as
a refrigerant and as a rust remover. As a result, exposure
to fluoride is greater than had been anticipated and
because of its multiple sources, it is difficult to determine
the daily fluoride consumption3.
34 (1159-2009)
358
Effect of Sodium Fluoride on the Cerebellar Cortex of Adult Albino Rats and the Possible Protective Role of Vitamin B6
Fluoride is toxic by all routes especially when
consumed in excessive amount. Long-term intake of
high levels of fluoride in human causes neurological
complications such as paralysis of limbs, vertigo,
spasticity in extremities and impaired mental acuity. In
experimental animals, chronic fluoride toxicity causes
altered neuronal and cerebrovascular integrity7.
sodium phosphate buffer at pH 7.4 containing 2.5%
gluteraldehyde solution14. The cerebellum from each
animal was dissected out and specimens were taken
from the lateral lobe and processed for light and electron
microscopic study.
For light microscopic study, the specimens were
fixed in 10% neutral- buffered formalin, processed for
preparation of paraffin section. Sections were cut and
stained with hematoxylin and eosin (H&E) for general
examination and Glees and Marsland,s silver stain for
cytoskeleton demonstration15.
Vitamin B6 is a water soluble vitamin and is a part
of vitamin B complex group. It could be helpful in some
neurological condition as peripheral neuropathy and its
deficiency has been linked to several neuropsychiatric
disorders8. It is a collective term comprising pyridoxine,
pyridoxol and pyridoxamine. It is involved in several
key biological processes, where it acts as a coenzyme
for more than one hundred enzymes participating in
protein metabolism9. It is also an essential cofactor
for homocysteine re-methylation, and its deficiency
is associated with an increase in blood homocysteine
levels which is a risk factor for cerbrovascular diseases,
Alzheimer,s and dementia10.
For electron microscopy, very small pieces were
processed and embedded in Epon. Semithin sections
were cut, stained with toulidine blue and examined by
light microscope to choose the selected areas. Ultrathin
sections were cut with LKB ultramicrotome using
diamond knife on copper grids and stained with uranyl
acetate followed by lead citrate for examination with
JEOL transmission electron microscope.
RESULTS
The effects of fluoride compounds on human health
have received worldwide attention because of their
environmental persistence, industrial exposure and
widespread distribution. Its neurotoxicity is well proven
clinically but, the detailed histological changes have not
been fully studied and it is not well recognized if these
neurological symptoms could be prevented or minimized
by protective agents. So, the present work aimed to study
the structural changes which might result from sodium
fluoride on the cerebellar cortex of adult male albino rats
and to evaluate whether vitamin B6 was able to reduce or
improve these changes.
General examination:
The water and vitamin B6 received rats appeared
normal and did not exhibit any signs of neurological
abnormality. On the other hand, three rats from NaF
received animals exhibited some neurological signs
in the form of reduction in activity, muscle weakness,
tremors with movement, loss of equilibrium and walked
like to be drunk and at the end of the experiment their
hind limbs were paralysed. Animals of group IV which
received vitamin B6 concomitantly with NaF showed
only reduction of activity up to the end of the study.
MATERIALS AND METHODS
Microscopic results:
The present study was carried out on twenty adult
albino rats, weighing 180-200 gms each. All animals
were kept in clean properly ventilated cages and received
balanced diet and water. The animals were divided into
four groups (five rats each): Group I (control), Group II
(vitamin B6 group): Each animal was injected I P with
3.6 mg vitamin B6 daily. The dose was calculated after
applying the interspecies dosage conversion scheme,
on daily human high therapeutic dose of vitamin B6 as
200 mg/day11,12. Group III (NaF received group): Each
animal received 10 mg/kg sodium fluoride orally by
gastric tube once daily13. Sodium fluoride was obtained
from El-Gomhuria Company for Chemicals and Medical
Trading, Tanta, Egypt in the form of white powder. Group
IV (protective group): Each rat was given vitamin B6 at
the same dose and route as in group II concomitantly
with the NaF at the same dose and route of group III.
Control Group:
H & E sections of the cerebellar cortex of this group
(I) as well as vitamin B6-treated group (II) showed the
well known normal structure. They showed three distinct
layers from outside inwards; the molecular layer, the
Purkinje cell layer and the granular cell layer. The outer
molecular layer was formed of nerve fibers with few
scattered stellate and basket cells. The Purkinje cells
were arranged in one row between the molecular and
granular layers. They showed large pyriform or flask
shaped cell bodies, centrally located vesicular nuclei
with apparent nucleoli and basophilic Nissel,s granules
in their cytoplasm. The granular layer was composed of
closely packed numerous small granular cells with dark
spherical nuclei (Fig. 1). By Glees and Marsland,s silver
stain, the molecular layer showed many nerve fibers
including regularly arranged axons of granular cells and
dendrites of Purkinje cells. The cytoplasm of Purkinje
cells showed homogenous brown stained cytoplasmic
The animals were examined daily for neurological
signs by simple observational methods. Three months
after the experiment, all rats were anaesthetized
and perfused with 4% paraformaldehyde in 0.1M
359
Omayma Kamel Afifi
cytoskeletal elements (Fig. 2). By transmission electron
microscope, the cerebellar cortex did not demonstrate
substantial differences among rats in control group and
vitamin B6-treated group and it was similar to the well
known normal ultrstructral picture. The Purkinje cells
showed large spherical central euochromatic nuclei and
cytoplasm containing free ribosomes, rough endoplasmic
reticulum, Golgi complexes and mitochondria (Fig. 3).
The surrounding neuropil showed some neuroglial cells
and many nerve fibers either unmyelinated or myelinated
with regular compact myelin sheath (Figs. 4,5). The
axoplasm of these nerve fibers contained regularly
arranged microtubules and neurofilaments (Fig. 6).
inbetween many apparently normal cells with central
open face nuclei. Many cells still surrounded with small
vacuolated neuropil. The molecular and granular layers
were more or less similar to control (Fig. 16). As regards
silver stained sections, the cerebellar cortex appeared
more or less as those of control sections (Fig. 17).
Utrastructurally, the animals that received NaF and
vit B6 simultaneously showed partial improvement and
preservation of the normal structure of cerebellar cortex.
Most of the Purkinje cells appeared nearly similar to the
control and few of them displayed irregular outline of
their nuclei (Fig. 18). Most of nerve fibers were normal
with regular compact myelin sheath around normal axons
while few scattered fibers were mildly affected (Fig. 19).
Sodium fluoride received group:
Examination of the H&E stained sections of this group
(III) revealed multifocal neuronal affection especially
on the Purkinje cell layer. The monolayer arrangement
of Purkinje cells was disrupted in many areas, as they
disappeared completely in several areas while they
revealed multilayer deposition with loss of the normal
pyriform shape in others (Figs. 7,8). Most of Purkinje
cells either in multilayer or monolayer areas, were
shrunken having irregular outlines with deep homogenous
cytoplasm and absence of Nissel,s granules. They were
surrounded with vacuolated neuropil (perineural spaces)
with accumulation of neuroglial cells around some of
them. As regard to nuclei, few cells showed vesicular
nuclei, others showed pyknotic nuclei or complete
karyolysis (Figs. 7,9,10). The molecular and granular
layers were less affected; multiple vacuolated areas
associated with increased microglial nuclei are seen in
molecular layer while the granular layer showed pyknosis
of their nuclei (Figs. 8-10). Glees and Marsland,s silver
stained sections showed irregular arranged nerve fibers
in the molecular layer while the Purkinje cells showed
a massive accumulation of the cytoplasmic cytoskeletal
elements that displayed deep argyrophilia (Fig. 11).
Fig. 1: A photomicrograph of cerebellar cortex of control group
showing molecular cell layer (Mo), Purkinje cell layer (P), and granular
cell layer (Gr) (H&E X 400). Inset showing Purkinje cell (P) with pale
open face nucleus and prominent Nissl,s granules.
H&E X 1000.
The ultrathin sections of the Purkinje cells of this
group showed dilatation of rER and Golgi, in addition
to swollen mitochondria with destroyed cristae while
the nuclei were irregular in outline but exhibited
euochromatin (Fig. 12). The neuropil of the cerebellar
islands showed loss of structural details and accumulation
of many neuroglial cells inbetween the affected axons.
The myelinated axons revealed areas of degenerative
changes in the form of disruption, splitting and loss of the
lamellar compact structure of myelin layers. Many axons
appeared swollen with irregular outline and revealed
disorganization of their neurofilaments and many swollen
mitochondria with destroyed cristae (Figs. 13-15).
Sodium Fluoride and Vitamin B6 Received Group:
Fig. 2: A photomicrograph of a section in the cerebellar cortex of a
control rat showing regular nerve fibers in molecular layer (→) and
Purkinje cells with homogenous cytoplasmic cytoskeletal elements (P).
Glees & Marsland,s silver stain X250.
H&E stained sections of animals of this group (IV)
showed monolayer arrangement of Purkinje cells with
mild disorganization. Few Purkinje cells still affected
360
Effect of Sodium Fluoride on the Cerebellar Cortex of Adult Albino Rats and the Possible Protective Role of Vitamin B6
Fig. 3: An electron micrograph of a section in the cerebellar cortex
of a control rat showing Purkinje cell containing euchromatic nucleus
(N) with fine dispersed chromatin and prominent nucleolus (Nu). The
cytoplasm contains many free ribosomes (→), rER, Golgi apparatus (G)
and mitochondria (M).
X 7500.
Fig. 4: An electron micrograph of a section in the cerebellar cortex of a
control rat showing neuropil (perineural spaces) containing neuroglial
cells (Ng) around blood capillary (Bc) and transverse section of many
myelinated and unmyelinated nerve fibers (→).
X 5000.
Fig. 5: An electron micrograph of a transverse section in the cerebellar
cortex of a control rat showing many myelinated nerve fibers containing
many mitochondria (M).
X 7500.
361
Fig. 6: An electron micrograph of a section in the cerebellar cortex of a
control rat showing an axon (→) with regularly arranged microtubules
and neurofilaments (F).
X 7500.
Fig. 7: A photomicrograph of a section in the cerebellar cortex of a NaF
treated rat showing severely affected area with disappearance of many
Purkinje cells. Notice, few shrunken irregular and scattered Purkinje
cells (P) surrounded with vacuolated neuropil.
H&E X 400.
Fig. 8: A photomicrograph of a section in the cerebellar cortex of a NaF
treated rat showing many layers of irregular, distorted Purkinje cells (P)
inbetween outer molecular (Mo) and inner granular (Gr) layers.
H&E X 400.
Omayma Kamel Afifi
Fig. 9: Higher magnification of the previous figure showing Purkinje
cells with absence of Nissel,s granules. The surrounding neuropil is
vacuolated and many neuroglial cells (→) accumulate around Pukinje
cells.
H&E X 1000.
Fig. 12: An electron micrograph of a section in the cerebellar cortex of
a NaF treated rat showing cytoplasm of Purkinje cell containing dilated
Golgi (G) and vacuolated mitochondria (M). Notice nucleus with normal
euochromatin (N).
X 75000.
Fig. 10: A photomicrograph of a section in the cerebellar cortex of a
NaF treated rat showing a monolayer of Purkinje cells (P) with deep
homogenous esinophilic cytoplasm surrounded with vacuolated neuropil.
H&E X 400.
Fig. 13: An electron micrograph of a section in the cerebellar cortex of a
NaF treated rat showing many neuroglial cells (Ng) inbetween axons with
irregular outline (→) adjacent to a blood capillary (Bc).
X 5000.
Fig. 11: A photomicrograph of a section in the cerebellar cortex of a NaF
treated rat showing many layers of distorted Purkinje cells with massive
accumulation of cytoskeletal elements (P). The molecular layer showed
irregularly arranged nerve fibers (→).
Glees & Marsland,s silver stain X 250.
Fig. 14: An electron micrograph of a section in the molecular layer of
cerebellar cortex of NaF treated group showing many nerve fibers with
vacuolated mitochondria (M) and focal areas of splitting of myelin sheath
(→) in the vacuolated neuropil (V).
X 10000.
362
Effect of Sodium Fluoride on the Cerebellar Cortex of Adult Albino Rats and the Possible Protective Role of Vitamin B6
Fig. 15: An electron micrograph in a section in the cerebellar island of
a NaF treated rat showing an axon with few swollen mitochondria with
destroyed cristae (M) and irregularly arranged neurofilaments (F). Notice
focal degenerated area (→) and vacuolated neuopil (V).
X 7500.
Fig. 18: An electron micrograph of a section in the cerebellar cortex
of a group (IV) showing an apparently normal Purkinje cell with
regular outline and normal rER, ribosomes (→) and Golgi (G). Notice
euochromatic nucleus (N) with irregular outline.
X 7500
Fig. 16: A photomicrograph of a section in the cerebellar cortex of a
group (IV) showing monolayer of Purkinje cells inbetween the molecular
(Mo) and granular layer (Gr). Notice some affected Purkinje cells (P1) in
between normal ones with open face nuclei (P2).
H&E X 400.
Fig. 19: An electron micrograph of a section in the cerebellar cortex of
a group (IV) showing normal nerve fibers with regular compact myelin
sheath (→) and the surrounding neuropil as in control group. X 7500.
DISCUSSION
Fluoride is a two-edged sword for human health,
because fluoridation of drinking water seems to be
effective for its clarification and treatment of dental
caries, whereas undesirable adverse effects of fluoride
have been reported3. Fluoride is completely and quickly
absorbed from the gastrointestinal tract and affects
many organs16. A few studies related to the effect of oral
ingestion of NaF for long period on cerebellum have been
reported so, the current study was designed to evaluate
the effect of oral intake of NaF, which is one of the major
routes of fluoride on the structure of the cerebellar cortex
with or without vitamin B6.
Fig. 17: A photomicrograph of a section in the cerebellar cortex of a
group (IV) showing regular arranged nerve fibers in molecular layer (→)
and homogenous argyrophilia of Purkinje cells (P).
Glees & Marsland,s silver stain X 250.
363
In this work, sodium flouride induced some
neurological abnormality and selective structural changes
in the Purkinje cells while the molecular and granular
Omayma Kamel Afifi
layers were less affected. The neurological signs noticed
in this group could be attributed to cerebellar affection
with subsequent cerebellar ataxia. The cerebellum is
responsible for smooth and accurate movements and
any lesions in it manifest many signs of cerebellar
ataxia which were noticed in some animals of this group
as muscle weakness, tremors with movement, loss of
equilibrium, gait disturbance and hind limbs paralysis17.
Another previous study confirmed that ingested fluoride
was retained by the cerebellum, interfering with its
physiology and inducing neurotoxicity, cell damage and
even cell death18.
and accumulation of glial cells in the NaF treated group.
As regards the vacuolation, it might be attributed to
the shrinkage of Purkinje cells and withdrawal of their
processes secondary to cytoskeletal affection and leaving
pericellular spaces21. Concerning, the increased neuroglial
cells (gliosis), this might be a common rapid response to
CNS injury and is regulated by neurons through soluble
mediators and cell–cell contact. In the healthy and resting
condition, these cells function as supportive glial cells.
Their activation is a key factor in the defense of the neural
parenchyma against inflammation, trauma, ischaemia,
brain tumours and neurodegeneration. They transform
into potentially cytotoxic cells in response to neuronal
or terminal degeneration, or both. Activated microglia
are mainly scavenger cells but also perform various
other functions in tissue repair, neural regeneration and
induction of immune responses. The microglia activation
may sustain a chronic inflammation leading to neuronal
dysfunction and cell death. This might be mediated by
the microglial release of extracellular toxic reactive
oxygen and nitrogen species23.
This study revealed that NaF induced selective
structural changes and disorganization in Purkinje cells.
Their arrangement was disrupted; some disappeared
completely while in other areas they exhibited multilayer
accumulation. This might be caused by displacement
from some areas to be accumulated in others. Most of
them lost the pyriform shape and showed irregular
outline. These findings could be correlated with the
increased argyrophilia which was shown in silver-stained
sections and cytoskeletal disorganization noticed by
electron microscope in this study. It was suggested that
the neurofilaments accumulation is an essential factor
in the development of distal axonal degeneration19.
Similar findings were observed by Shivarajashankara
et al.7 who found that chronic fluoride intoxication caused
marked neurodegenerative changes in rats especially in
the early stages of life. In addition, these changes may
form the neural basis for impaired learning and memory,
abnormal behaviour patterns and disturbed overall body
physiology.
The major ultrastructural changes in the form of
dilated Golgi, mitochondrial swelling with disturbance
in their cristae and nuclear irregularity were seen in NaF
treated group. Many investigators mentioned that these
disorders were considered to be secondary to direct
toxicity on neuronal cells that induced profound disorder
of intercellular biochemical events, such as inhibition
of oxidative phosphorylation, abnormal pro¬duction of
proteins, and dysfunction in the detoxication21.
There are many major pathophysiological
mechanisms through which fluoride ingestion might
cause intoxication in many organs. Formerly, it was
reported that fluoride inhibits Na+/K+-ATPase, leading
to extracellular release of potassium with subsequent
hyperkalamia. In addition, fluoride ions chelate calcium
leading to hypocalcaemia and interference with calcium
function24. Subsequently, it had been known that fluoride
induced inflammatory reaction, cell cycle arrest and
apoptosis in different systems. Also, it showed diverse
actions on a variety of cellular and physiological
functions including inhibition of a variety of enzymes as
metalloenzymes that were proved to be involved in many
essential metabolic processes as glycolysis, oxidative
phosphorylation and neuro-transmission25.
In this work the appearance of distorted shrunken
Purkinje cells seemed to be a result of damage to
the system associated with structural and functional
biosynthesis of cell proteins. Some authors suggested
that the appearance of dark neurons might reflect a
certain phase of apoptosis as they displayed markedly
condensed cytoplasm and nucleoplasm20, while others
believed that dark small neurons is usually ischemic
due to possible substantial abnormalities in the capillary
wall of the cerebellar cortex with subsequent disorders
in the structural elements of the blood-brain barrier to
neurons21.
Degenerative structural changes in axons with
splitting and disruption of their myelination in cerebellar
cortex of NaF treated rats were recorded as a component of
a dying – back process of neuronal injury. The disruption
in myelination was attributed to the changes in myelin
basic protein secondary to membrane damage and axonal
degeneration after exposure to toxic substance. Defect
in myelination was also attributed to increased water
content in degenerating nerve causing intramyelinic
edema with separation of myelin lamellae22.
However, the mechanisms underlying the
neurotoxicity of endemic fluorosis still remain unknown.
It was found that the administration of fluoride resulted
in decreased cerebellar and cerebral protein levels13.
Fluorine was able to pass through the blood-brain barrier
and accumulates in the central nervous system of exposed
animals. Chronic fluoride intoxication was accompanied
by behavioral disorders, degenerative changes and
abnormalities of aerobic metabolism of the neurons26.
Recently, it was found that fluoride induced significant
increase in lactate dehydrogenase release, intracellular
During this work, the neuropil revealed vacuolation
364
Effect of Sodium Fluoride on the Cerebellar Cortex of Adult Albino Rats and the Possible Protective Role of Vitamin B6
reactive oxygen species (ROS), malondialdehyde levels
and the percentage of apoptosis. On the other hand,
fluoride induced decrease in the glutathione levels and
glutathione peroxidase activities, accompanied by
the markedly reduced superoxide dismutase activity.
So, fluoride collectively could cause oxidative stress,
apoptosis, and decreased mRNA and protein expression
levels of neural cell adhesion molecules in rat neurons,
contributing to the neuronal dysfunction and synaptic
injury27,28.
4.
5.
6.
In the present work, the concomitant administration
of vitamin B6 and NaF reduced the signs of neurotoxicity
caused by NaF and revealed mild neurotoxic effect on
the Purkinje cells of adult male albino rats. This was in
parallel with the report that vitamin B6 may be able to
increase the amount of energy available to the neuron
via alternative pathways and it is important for proper
neuronal connections and myelination. In addition,
vitamin B6 acts as cofactor in production of essential fatty
acids and have a role in the synthesis of neurotransmitters
including serotonin, dopamine, norepinephrine and
gamma-aminobutyric acid; all of which have a role in the
proper function of nervous system29,30.
7.
8.
9.
10.
There is growing evidence in the literatures that there
is a relationship between low serum B-vitamins, elevated
homocysteine, and cognitive impairment. The high-dose
of B vitamin supplementation even in individuals with
normal levels of B vitamins was effective in reducing
the level of homocysteine which is sulfur amino acid
involved in essential metabolic pathways. Homocysteine
elevation induces neuron loss which might be via
impaired DNA repair and induction of apoptotic cell
death. There is intriguing evidence that reduction of
homocysteine levels can be readily achieved with high
doses of folic acid, vitamin B12 and vitamin B6 31,32.
11.
12.
13.
14.
From this current work, it could be concluded that
NaF induced severe toxic effect on the cerebellar cortex.
These changes could be partially reduced by concomitant
administration of vitamin B6. Considering the mild
degenerative changes still observed in some cells it might
be due to low dose of vitamin B6 used in the present work.
The dose of vitamin B6 might be needed to be adjusted to
cope with the toxicity of NaF or its pretreatment would
be earlier before drug intake.
15.
16.
17.
18.
19.
REFERENCES
1.
2.
3.
Pessan JP, Silva SM, Lauris JRP, Sampaio FC, Whitford GM
and Buzalaf MAR. (2008): Fluoride uptake by plaque from
water and from dentifrice. J.Dent.Res.; 87(5):461-465.
Fordyce FM, Vrana K, Zhovinsky E, Povoroznuk V, Toth
G, Hope BC, Iljinsky U and Baker J. (2007): A health risk
assessment for fluoride in Central Europe. Environ.Geochem.
Health Apr; 29(2):83-102.
Lund K, Refsnes M, Sandstrom T, Sostrand P, Schwarze P,
Boe J and Kongerud J. (1999): Increased CD3 positive cells in
20.
21.
365
bronchoalveolar lavage fluid after hydrogen fluoride inhalation.
Scand.J.Work Environ.Health Aug; 25(4):326-334.
O’Neil MJ, Smith A, Heckelman PE, Budavari S. (2001):
The Merck Index: An encyclopedia of chemicals, drugs and
biologicals.13th ed. John Wiley & Sons.
Fung KF, Zhang ZQ, Wong JWC and Wong MH. (1999):
Fluoride contents in tea and soil from tea plantations and the
release of fluoride into tea liquor during infusion. Environ.Pollut.;
104(2):197-205.
Horowitz HS. (1999): Proper use of fluoride products in
fluoridated communities. Lancet May 1; 353(9163):1462.
Shivarajashankara YM, Shivashankara AR, Gopalakrishna
BP, Muddanna Rao S and Hanumanth Rao S. (2002):
Histological changes in the brain of young fluoride-intoxicated
rats. Fluoride ; 35(1):12-21.
Okada H, Moriwaki K, Kanno Y, Sugahara S, Nakamoto H,
Yoshizawa M and Suzuki H. (2000): Vitamin B6 supplementation
can improve peripheral polyneuropathy in patients with chronic
renal failure on high-flux haemodialysis and human recombinant
erythropoietin. Nephrol.Dial.Transplant. Sep; 15(9):1410-1413.
Bailey AL, Wright AJ and Southon S. (1999): High performance
liquid chromatography method for the determination of pyridoxal5-phosphate in human plasma: How appropriate are cut-off values
for vitamin B6 deficiency? Eur.J.Clin.Nutr. Jun; 53(6):448-455.
Malouf R and Grimley EJ. (2003): Vitamin B6 for cognition.
Cochrane Database Syst.Rev. ;(4):CD004393.
Paget GE and Barnes JM. (1964): Toxicity tests In:
Laurence DR and bacharach AL. Evaluation of drug activities:
Pharmacometrics: Academic Press,London. p.135-166.
Gaby AR. (2003): “Safe upper levels” for nutritional supplements:
One giant step backward. J.Orthomol.Med. ; 18(3-4):126-130.
Błaszczyk I, Grucka Mamczar E, Kasperczyk S and Birkner
E. (2008): Influence of fluoride on rat kidney antioxidant
system: Effects of methionine and vitamin E. Biol.Trace Elem.
Res.; 121(1):51-59.
Bozzola JJ and Russell LD. (1998): Electron microscopy:
Principles and techniques for biologists.2nd ed.Jones & Bartlett
Publishers.
Drury RA and Wallington EA. (1980): Carleton’s histological
technique.5th ed.Oxford University Press: London.
Amira S, Soufane S and Gharzouli K. (2005): Effect of sodium
fluoride on gastric emptying and intestinal transit in mice. Exp.
Toxicol.Pathol. Aug; 57(1):59-64.
FitzGerald MJT and Folan Curran J. (2002): Clinical
neuroanatomy and related neuroscience.5th ed. W.B. Saunders.
Trabelsi M, Guermazi F and Zeghalc N. (2001): Effect of
fluoride on thyroid function. Fluoride ; 34(3):165-173.
Hirai T, Mizutani M, Ochiai K, Umemura T and Itakura C.
(1999): Distal axonopathy does not occur without neurofilament
accumulation in gamma-diketone neuropathy: Comparative
studies of normal and neurofilament-deficient quail. Acta
Neuropathol. Jun; 97(6):552-556.
Ratan RR, Murphy TH and Baraban JM. (1994): Oxidative
stress induces apoptosis in embryonic cortical neurons.
J.Neurochem. Jan; 62(1):376-379.
Sobaniec Lotowska ME. (2001): Ultrastructure of Purkinje cell
perikarya and their dendritic processes in the rat cerebellar cortex
in experimental encephalopathy induced by chronic application
of valproate. Int.J.Exp.Pathol. Dec; 82(6):337-348.
Omayma Kamel Afifi
22.
23.
24.
25.
26.
27.
Manzo L, Artigas F, Martinez E, Mutti A, Bergamaschi E,
Nicotera P, Tonini M, Candura SM, Ray DE and Costa LG.
(1996): Biochemical markers of neurotoxicity. A review of
mechanistic studies and applications. Hum.Exp.Toxicol. Mar; 15
Suppl 1:S20-S35.
Vilhardt F. (2005): Microglia: Phagocyte and glia cell. Int. J.
Biochem.Cell Biol. Jan; 37(1):17-21.
Boink AB, Wemer J, Meulenbelt J, Vaessen HA and de Wildt
DJ. (1994): The mechanism of fluoride-induced hypocalcaemia.
Hum.Exp.Toxicol. Mar; 13(3):149-155.
Refsnes M, Thrane EV, Lag M, Hege Thoresen G and
Schwarze PE. (2001): Mechanisms in fluoride-induced
interleukin-8 synthesis in human lung epithelial cells. Toxicology
Oct 15; 167(2):145-158.
Lubkowska A, Chlubek D, Machoy Mokrzyńska A, Noceń
I, Zyluk B and Nowacki P. (2004): Concentrations of fluorine,
aluminum and magnesium in some structures of the central
nervous system of rats exposed to aluminum and fluorine in
drinking water [Stezenie fluoru, glinu i magnezu w wybranych
strukturach ośrodkowego układu nerwowego szczurów
eksponowanych na glin i fluor w wodzie pitnej.]. Ann.Acad.Med.
Stetin. ; 50(S1):73-76.
Trivedi MH, Verma RJ, Chinoy NJ, Patel RS and Sathawara
NG. (2007): Effect of high fluoride water on intelligence of
28.
29.
30.
31.
32.
366
school children in India. Fluoride ; 40(3):178-183.
Zhang M, Wang A, Xia T and He P. (2008): Effects of fluoride
on DNA damage, S-phase cell-cycle arrest and the expression of
NF-κB in primary cultured rat hippocampal neurons. Toxicol.
Lett. ; 179(1):1-5.
Amadio S, D’Ambrosi N, Trincavelli ML, Tuscano D,
Sancesario G, Bernadi G, Martini C and Volonte C. (2005):
Differences in the neurotoxicity profile induced by ATP and
ATPgammaS in cultured cerebellar granule neurons. Neurochem.
Int. Oct; 47(5):334-342.
Araujo JA, Landsberg GM, Milgram NW and Miolo A.
(2008): Improvement of short-term memory performance
in aged beagles by a nutraceutical supplement containing
phosphatidylserine, Ginkgo biloba, vitamin E and pyridoxine.
Can.Vet.J. Apr; 49(4):379-385.
Balk EM, Raman G, Tatsioni A, Chung M, Lau J and
Rosenberg IH. (2007): Vitamin B6, B12 and folic acid
supplementation and cognitive function: A systematic review of
randomized trials. Arch.Intern.Med. ; 167(1):21-30.
Aisen PS, Schneider LS, Sano M, Diaz-Arrastia R, van Dyck
CH, Weiner MF, Bottiglieri T, Jin S, Stokes KT, Thomas RG
and Thal LJ. (2005): High-dose B vitamin supplementation and
cognitive decline in Alzheimer disease: A randomized controlled
trial. JAMA Oct; 300(15):1774-83.
‫ﺗﺄﺛﻴﺮ ﻓﻠﻮرﻳﺪ اﻟﺼﻮدﻳﻮم ﻋﻠﻰ ﻗﺸﺮة ﻣﺨﻴﺦ اﻟﺠﺮد اﻷﺑﻴﺾ اﻟﺒﺎﻟﻎ واﻟﺪور اﻟﻮﻗﺎﺋﻲ اﻟﻤﺤﺘﻤﻞ ﻟﻔﻴﺘﺎﻣﻴﻦ ب‪ :6‬دراﺳﺔ‬
‫ﺑﺎﻟﻤﺠﻬﺮ اﻟﻀﻮﺋﻲ واﻹﻟﻜﺘﺮوﻧﻲ‬
‫أﻣﻴﻤﺔ آﺎﻣﻞ ﻋﻔﻴﻔﻲ‬
‫ﻗﺴﻢ اﻟﻬﺴﺘﻮﻟﻮﺟﻴﺎ‪ ،‬آﻠﻴﺔ اﻟﻄﺐ‪ ،‬ﺟﺎﻣﻌﺔ ﻃﻨﻄﺎ‬
‫ﻣﻠﺨﺺ اﻟﺒﺤﺚ‬
‫إن ﺳﻤﻴﺔ ﻓﻠﻮرﻳﺪ اﻟﺼﻮدﻳﻮم أﺻﺒﺤﺖ ﻣﺤﻞ دراﺳﺎت ﻋﺪﻳﺪة ﻓﻲ اﻟﺴﻨﻮات اﻟﻘﻠﻴﻠﺔ اﻷﺧﻴﺮة وذﻟﻚ ﻟﻮﺟﻮدﻩ اﻟﻤﺴﺘﻤﺮ ﺑﺎﻟﺒﻴﺌﺔ واﻧﺘﺸﺎرﻩ ﻋﻠﻲ ﻣﺴﺘﻮي‬
‫ﻋﺎﻟﻤﻲ‪ .‬و ﻗﺪ ﺣﻈﻲ ﺗﺄﺛﻴﺮﻩ اﻟﺴﻤﻲ ﻋﻠﻰ أﻋﻀﺎء آﺜﻴﺮة ﺑﻜﺜﻴﺮ ﻣﻦ اﻻهﺘﻤﺎم وﻟﻜﻦ ﺗﺄﺛﻴﺮﻩ اﻟﺴﻤﻲ ﻋﻠﻰ اﻟﺠﻬﺎز اﻟﻌﺼﺒﻲ ﻟﻢ ﻳﺪرس آﺜﻴﺮا‪ .‬وﻟﺬﻟﻚ ﻓﻘﺪ‬
‫آﺎن اﻟﻬﺪف ﻣﻦ هﺬا اﻟﺒﺤﺚ هﻮ دراﺳﺔ اﻟﺘﻐﻴﺮات اﻟﻬﺴﺘﻮﻟﻮﺟﻴﺔ اﻟﺤﺎدﺛﺔ ﻓﻲ ﻗﺸﺮة ﻣﺨﻴﺦ اﻟﺠﺮذ اﻷﺑﻴﺾ ﺑﻌﺪ ﺗﻌﺮﺿﻪ اﻟﻤﺰﻣﻦ ﻟﻔﻠﻮرﻳﺪ اﻟﺼﻮدﻳﻮم‬
‫وآﺬﻟﻚ ﺗﻘﻴﻴﻢ اﻟﺪور اﻟﻮﻗﺎﺋﻲ اﻟﻤﺤﺘﻤﻞ ﻟﻔﻴﺘﺎﻣﻴﻦ ب ‪ .6‬وﻗﺪ اﺳﺘﺨﺪم ‪ 20‬ﺟﺮذا أﺑﻴﻀﺎ ﺑﺎﻟﻐﺎ ﺗﻢ ﺗﻘﺴﻴﻤﻬﺎ اﻟﻲ‪ :‬اﻟﻤﺠﻤﻮﻋﺔ اﻷوﻟﻲ ﺿﺎﺑﻄﺔ‪ ،‬واﻟﻤﺠﻤﻮﻋﺔ‬
‫اﻟﺜﺎﻧﻴﺔ ﺗﻢ ﺣﻘﻨﻬﺎ ﻓﻲ اﻟﺒﺮﻳﺘﻮن ب ‪ 10‬ﻣﺠﻢ ﻓﻴﺘﺎﻣﻴﻦ ب‪ 6‬واﻟﻤﺠﻤﻮﻋﺔ اﻟﺜﺎﻟﺜﺔ وﻗﺪ أﻋﻄﻲ آﻞ ﻣﻨﻬﺎ ‪10‬ﻣﺠﻢ ﻓﻠﻮرﻳﺪ اﻟﺼﻮدﻳﻮم ‪/‬آﺠﻢ ﺑﺎﻟﻔﻢ ﻳﻮﻣﻴﺎ أﻣﺎ‬
‫اﻟﻤﺠﻤﻮﻋﺔ اﻟﺮاﺑﻌﺔ ﻓﻘﺪ أﻋﻄﻰ آﻞ ﻣﻨﻬﺎ ﻓﻠﻮرﻳﺪ اﻟﺼﻮدﻳﻮم ﻣﺘﻼزﻣﺎ ﻣﻊ ﻓﻴﺘﺎﻣﻴﻦ ب ‪ 6‬ﺑﻨﻔﺲ اﻟﺠﺮﻋﺔ واﻟﻄﺮﻳﻘﺔ واﻟﻤﺪة ﻟﻠﻤﺠﻤﻮﻋﺔ اﻟﺜﺎﻧﻴﺔ واﻟﺜﺎﻟﺜﺔ‪.‬‬
‫وﺑﻌﺪ ﺛﻼﺛﺔ أﺷﻬﺮ ﺷﺮﺣﺖ ﻗﺸﺮة اﻟﻤﺨﻴﺦ وﺗﻢ ﺗﺤﻀﻴﺮهﺎ ﻟﻠﻔﺤﺺ ﺑﺎﻟﻤﻴﻜﺮوﺳﻜﻮب اﻟﻀﻮﺋﻲ واﻻﻟﻜﺘﺮوﻧﻲ‪ .‬وﻗﺪ أﻇﻬﺮت ﻗﺸﺮة اﻟﻤﺨﻴﺦ ﻟﻠﺠﺮذان‬
‫اﻟﻤﻌﺎﻟﺠﺔ ﺑﻔﻠﻮرﻳﺪ اﻟﺼﻮدﻳﻮم ﺗﺄﺛﻴﺮات هﺎدﻣﺔ ﺧﺎﺻﺔ ﻓﻰ ﺧﻼﻳﺎ ﺑﻴﺮآﻨﺠﻲ اﻟﺘﻲ ﻇﻬﺮت ﻓﻰ ﻃﺒﻘﺎت ﻋﺪﻳﺪة وآﺬﻟﻚ ﺗﻐﻴﺮات ﺗﺮآﻴﺒﻴﺔ آﺎﻻﺗﺴﺎع ﻓﻲ‬
‫اﻟﺸﺒﻜﺔ اﻻﻧﺪوﺑﻼزﻣﻴﺔ اﻟﺨﺸﻨﺔ وﺟﻬﺎز ﺟﻮﻟﺠﻲ واﻧﺘﻔﺎخ ﻓﻲ اﻟﻤﻴﺘﻮآﻮﻧﺪرﻳﺎ ﺑﺎﻹﺿﺎﻓﺔ إﻟﻲ ﺗﻄﺮف ﻓﻲ اﻟﻜﺮوﻣﺎﺗﻴﻦ‪ .‬وﻗﺪ ﻇﻬﺮت ﺑﻌﺾ اﻟﻔﺠﻮات ﻓﻰ‬
‫اﻟﺤﻴﺰ اﻟﻤﺤﻴﻂ ﺑﺎﻟﺨﻼﻳﺎ ﻣﻊ زﻳﺎدة ﻓﻲ ﺧﻼﻳﺎ اﻟﺪﺑﻖ اﻟﻌﺼﺒﻲ‪ .‬إﻣﺎ اﻷﻟﻴﺎف اﻟﻌﺼﺒﻴﺔ اﻟﻤﺤﺎﻃﺔ ﺑﺎﻟﻤﻴﻠﻴﻦ ﻓﻘﺪ ﺣﺪث اﺧﺘﻼل ﻓﻲ ﻋﻤﻠﻴﺔ ﺗﻜﻮن ﻏﻼف‬
‫اﻟﻤﻴﻠﻴﻦ وﻋﺪم اﻧﺘﻈﺎم اﻷﻟﻴﺎف اﻟﻌﺼﺒﻴﺔ‪ .‬وﻗﺪ أﻇﻬﺮت ﺻﺒﻐﺔ اﻟﻔﻀﺔ ﺗﺠﻤﻊ ﻓﻲ اﻟﻌﻨﺎﺻﺮ اﻟﻤﻜﻮﻧﺔ ﻟﺪﻋﺎﻣﺔ اﻟﺨﻠﻴﺔ‪ .‬وﻋﻠﻲ اﻟﻮﺟﻪ اﻷﺧﺮ ﻓﺎن هﺬﻩ‬
‫اﻟﺘﻐﻴﺮات ﻗﻠﺖ ﻓﻰ ﺟﺮذان اﻟﻤﺠﻤﻮﻋﺔ اﻟﺮاﺑﻌﺔ اﻟﺘﻲ ﻋﻮﻟﺠﺖ ﺑﻔﻴﺘﺎﻣﻴﻦ ب‪ 6‬ﻣﻊ ﻓﻠﻮرﻳﺪ اﻟﺼﻮدﻳﻮم‪ .‬وﻣﻦ هﺬﻩ اﻟﺪراﺳﺔ ﻧﺴﺘﻨﺘﺞ أن ﺗﻨﺎول ﻓﻠﻮرﻳﺪ‬
‫اﻟﺼﻮدﻳﻮم اﻟﻤﺰﻣﻦ ﻳﺆدي اﻟﻲ ﺗﻐﻴﺮات هﺎدﻣﺔ ﻓﻲ ﻗﺸﺮة اﻟﻤﺨﻴﺦ اﻟﺘﻲ ﻳﻤﻜﻦ ﺗﻘﻠﻴﻠﻬﺎ ﺑﻮاﺳﻄﺔ ﻓﻴﺘﺎﻣﻴﻦ ب‪.6‬‬
‫‪367‬‬