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Journal of The Analgesics, 2013, 1, 26-32
Original Article: Effects of Acetaminophen on Motor and Sensory
Functions in Adult Rats after Sciatic Nerve Crush
D. Kapoukranidou1,*, T-V. Kalamara1 and D. Hadjipavlou-Litina2
1
Department of Physiology, School of Medicine, Faculty of Health Sciences, Aristotle University of
Thessaloniki, Greece
2
Department of Pharmaceutical Chemistry, School of Pharmacy, Faculty of Health Sciences, Aristotle
University of Thessaloniki, Greece
Abstract: Introduction: Acetaminophen, also known as paracetamol, is one of the most widely used non opioid
treatments for pain. Anti-inflammatory, remyelinating and neuroprotective effects have been documented for
acetaminophen. The most accepted theory is that of paracetamol’s positive effects on the serotonergic descending
inhibitory pathways, as well as its interactions with opioidergic systems, eicosanoid systems and/or nitric oxide
pathways. Various injuries of the central and peripheral nervous systems induce neuropathic pain, a chronic debilitating
condition. The peripheral axons, in contrast to the central nervous system axons have the noticeable ability to regenerate
after injury. The purpose of this study was to determine if the administration of paracetamol in a single-dose, immediately
after the crush of sciatic nerve would result in earlier nerve regeneration and motor and sensory rehabilitation.
Material and Methods: We evaluated a left sciatic nerve crush injury model. Adult Wistar albino rats were divided into two
groups. The control group (n=7) did not receive any drug after the surgery. The treated group (n=7) received
intraperitoneally a single dose of paracetamol (0.01 mmol/ml/kg), 8 hours after the surgery. The locomotor recovery was
tested by the wide flat runway, the inclined plane runway and the grid walking test. The sensory function recovery was
assessed by the nerve pinch test. The performance of the rats was tested one day before the surgery and on days 1st,
8th, 15th and 22nd after the sciatic nerve crush injury.
Results: The runway measurements showed a remarkable improvement of the treated group on the 22nd day, close to
the preoperative level. Moreover, on days 15th and 22nd the treated group presented a better performance with a lower
number of footfalls on the grid. The pinch test indicated earlier signs (8th day) of sensory recovery of the treated group.
Conclusion: The present study suggests that acetaminophen may offer new strategies for the treatment of peripheral
nerve injury. Further investigation is in progress to explore the possible effects of the single-dose treatment in a longer
observation period, as well as the possible effects of repeated acetaminophen administration pre- and/or postoperatively.
Keywords: Acetaminophen, Sciatic nerve crush, Behavioral tests.
1. INTRODUCTION
Peripheral nerve injuries vary from simple nerve
compression lesions, mainly through mechanical
deformation, to the most common stretch-related
injuries and severe laceration of the nerve trunks [1].
Seddon and Sunderland have developed peripheral
nerve injury grading systems. According to their
severity, these injuries have been divided by Seddon
into three wide categories: neurapraxia, axonotmesis
and neurotmesis [2]. Sunderland classifies further the
three injury types described by Seddon into five
categories by severity [3]. Peripheral nerves respond to
injury in a unique way.
In contrast to the central nervous system, which
cannot usually regenerate, the axons in the periphery
appear to have a noticeable ability to regenerate after
injury, since the neuronal environment allows the
*Address correspondence to this author at the Department of Physiology,
School of Medicine, Faculty of Health Sciences, Aristotle University of
Thessaloniki, Greece; Tel: +30 2310999328; E-mail: [email protected]
E-ISSN: 2311-0317/13
intrinsic growth capacity of neurons to activate, as well
as permits the regrowth of axons and their
remyelination, in which the Schwann cells play a
dominant role [4]. Despite this outstanding ability,
functional recovery is often poor, particularly when the
nerves are damaged close to the spinal cord and far
from their targets [5]. Therefore, peripheral nerve
injuries constitute a major source of chronic disability
with wide-ranging consequences for the patient and
high costs to the society [1]. Not only professional life
problems but also severely impaired leisure activities
[1] and posttraumatic psychological stress [6] are likely
to follow a peripheral nerve injury. In addition,
neuropathic pain, a chronic, neurodegenerative and
debilitating condition generally resistant to presently
available pharmacological and surgical interventions is
frequently an incidence of peripheral nerve injury [7].
Neuropathic pain is associated with neuroinflammation occurring as the organism responds to tissue
injury by recruiting immune cells and releasing
mediators [8]. Anti-inflammatory substances present
neuroprotective properties in an experimental model of
© 2013 Pharma Professional Services
Effects of Acetaminophen on Motor and Sensory Functions in Adult
autoimmune neuritis [9] and pain suppression after
peripheral nerve injury [10]. It has been shown that
anti-inflammatory substances, such as peripheral
prostaglandins, play a significant role in the
pathogenesis of neuropathic pain and hyperalgesia that
follow a peripheral nerve injury [11].
Nonsteroidal anti-inflammatory drugs (NSAIDs) are
a group of drugs widely used as cyclooxygenase
(COX) selective and non-selective inhibitors. Recent
studies indicate that the NSAIDs ibuprofen and
indomethacin suppress the increased RhoA activity of
neurons exposed to axonal growth inhibitors or after
central nervous system trauma [12]. Moreover, acetyl
salicylic acid [13], at anti-inflammatory dose, and
celecoxib [14] significantly accelerate functional
recovery following peripheral nerve crush in the rat.
These studies suggest the potential utility of NSAIDs in
the development of new therapeutic approaches to
treat peripheral nerve injury. Acetaminophen, also
known as paracetamol, is generally considered as an
NSAID which demonstrates potent anti-inflammatory,
antipyretic and analgesic activity, but lacks other typical
NSAID actions, such as antiplatelet activity and
gastrotoxicity [15].
In spite of its wide use, the mechanism of action of
acetaminophen is largely unknown. It seems likely that
at least one mechanism may contribute to
acetaminophen’s analgesic
activity, while the
eicosanoid, opioidergic, serotonergic and cannaboid
system might be responsible for the drug’s activity too
[16]. The rationale for the present study is that,
because acetaminophen has been previously
demonstrated neuroprotective effects [17], it might be
effective in earlier recovery of motor and sensory
functions, when administered immediately after the
compression of sciatic nerve in rat.
Journal of The Analgesics, 2013, Vol. 1, No. 2
27
2. MATERIALS AND METHODS
2.1. Animals
Fourteen adult Wistar albino rats of both sexes were
randomly divided into two groups. They were housed in
groups of 3-4 and maintained in a temperature and
humidity controlled room on a 12-hour light cycle, with
food and water available ad libitum. Animals were kept
under the accordance of protocols approved by the
institutional animal care and use committee. One day
prior to the surgery and the behavioral testing, rats
were handled by the investigators and introduced to the
testing environment for a brief period of time.
2.2. Induction of Sciatic Nerve Injury
Rats were anesthetized with chloral hydrate 4.5%
(1ml/100g). The sciatic nerve of the left hind paw was
revealed after blunt dissection through biceps femoris
and freed from the surrounding connective tissue. After
the exposure, the nerve was crushed once for 30
seconds at standardized force (Figure 1). The muscle
was reapproximated and the skin incision was closed
using 6-0 sutures. The examination of the loss of
sensory and motor function in the operated limb
established the crush completeness. Digits in the
operated limb were pinched with a blunt forceps.
Absence of foot withdrawal and vocalization was
recorded as loss of sensory and motor function.
Animals were placed under a heating lamp until they
recovered from the anesthesia and then they returned
to their home cages.
2.3. Drug and Time of Administration
The control group (n=7) did not receive any drug
after the surgery. A single dose of paracetamol (0.01
mmol/ml/kg) was administered to the treated group
(n=7) intraperitoneally, 8 hours after the surgery. The
Figure 1: Photograph illustrating the crush injury induced in the exposed left sciatic nerve using the artery forceps.
28
Journal of The Analgesics, 2013, Vol. 1, No. 2
Kapoukranidou et al.
paracetamol solution was freshly prepared on surgery
day (day 0).
were walking on the grid and the number of errors
(hindlimb slips) was determined.
2.4. Locomotor Testing
2.4.4. Sciatic Functional Index (SFI)
The animals were subjected to locomotor testing
st
th
th
one day before the surgery and on the 1 , 8 , 15 and
nd
22 day after the sciatic nerve injury. The day of the
surgery is considered as day 0. Locomotor behavioral
tests have been previously used for the study of the
recovery of motor functions after spinal injury in rats
[18-20]. In a recent survey, various simple and complex
runways were used for the assessment of the
locomotor performance of rats after compression of the
sciatic nerve [21]. In the present study, the locomotor
function recovery was examined through the flat
runway test, the inclined plane runway test, the grid
walking test and the Sciatic Functional Index (SFI)
calculation. The two runway tests conducted were
evaluated with a modified score technique (Table 1).
Table 1: Evaluation of Locomotor Behavior of Rats on
Different Runways
Score
Behavior
0 points
No movement in hind limb
1 point
Mild movement in hindlimb
2 points
Walking with mild deficit
3 points
Normal walking (near normal)
2.4.1. Flat Runway
Rats were trained to walk on a wooden board (1m x
15cm) for 120 seconds. The hindlimb movement was
assessed with the scoring mentioned before.
2.4.2. Inclined Plane Runway
A wooden board (1m x 15cm) fitted at a 45 degree
angle to the ground was used in this test. Rats were
trained to walk over the runway for 120 seconds and
their ability to balance their body position was
evaluated with the above scoring.
2.4.3. Grid Runway
A wooden apparatus (50x40x40cm) was used in
this test. A metallic horizontal grid with square holes
(3x3cm) was placed 10cm above apparatus’s base.
The roof of the apparatus was open. The 50cm side
was also open from the base until the grid, so that the
lower surface of the grid was visible to the
investigators. The animals were evaluated as they
SFI is the current standard for measuring functional
recovery following rat sciatic nerve crush. It was first
described in 1982 by DeMedinacely et al. [22] and was
subsequently modified by Bain et al. [23]. Its calculation
involves the measurement of wide-ranging relationships between feet and toes of the hindlimb of
recovering animals. In the present study, the plantar
surfaces of the hindlimbs of the rats were impregnated
with ink and then the animals were allowed to walk on
a paper along a corridor, leaving footprints during
walking. A wooden corridor (1m x 8cm) with raised
sidewalls was used. Two and a half white paper strips
(40 x 7.5cm) were placed along the corridor and were
fixed with adhesive tape closely together and at the
base and the sidewalls of the corridor. Each rat was
trained to walk steadily along the corridor without stops
until it made at least three successful efforts of
continuous walking.
The footprints were allowed to dry (Figure 2) and
were then converted into digital images using a
computer and a high resolution scanner. The image
processing was done by using the ImageJ. This is an
open-source image analysis software created in 1997
by Rayne Rasband (National Institute of Health), widely
used in medical applications, especially neuroscience,
for measuring distances and surface and plotting the
data [24]. The footprints were analyzed as described by
Bain et al. [23] Measurements included:
•
print length (PL): distance from the heel to the
third toe,
•
toe spread (TS): distance from the first to the fifth
toe on both sides,
•
intermediate toe spread (ITS): distance between
the middle of the second and fourth toe.
All measurements were taken both from the
experimental (EPL, ETS, EIT) and the normal (NPL,
NTS, NIT) side. The formula used to calculate SFI was
as follows: SFI= -38.3 (EPL-NPL)/NPL+109.5 (ETSNTS)/NTS+ 13.3 (EIT-NIT)/NIT-8.8. SFI values close to
-100 indicate severe damage, while SFI values close to
0 indicate normal function.
2.5. Sensory Testing
Recovery of sensory function was analyzed using
the pinch test. At midtigh, the sciatic nerve is
Effects of Acetaminophen on Motor and Sensory Functions in Adult
Journal of The Analgesics, 2013, Vol. 1, No. 2
29
Figure 2: (A). Collection of footprints, (B). Measurements with ImageJ.
composed of about 27000 axons, of which 71% are
myelinated and unmyelinated sensory axons (23% and
48% respectively), while only 6% are myelinated motor
axons and 23% are sympathetic unmyelinated axons
[25]. Therefore, control of the sensation of pain
provides information about the degree of sciatic nerve
regeneration.
pinching were recorded. Painful stimuli should be
applied on the outer lateral side of the plantar surface,
since the medial side of the sensory function is
originated from the saphenous nerve (Figure 3). The
tests were performed one day before the operation, on
the day following the operation and were repeated at 1week intervals for three weeks postoperatively.
2.5.1. Pinch Test
All the in vivo experiments were repeated at least in
triplicate and were subjected to statistic analysis using
Microsoft Office Excel 2010 and ImageJ 1.47.
The lateral side of the rat’s paw was pinched with a
clip. Animals demonstrating a withdrawal response to
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Journal of The Analgesics, 2013, Vol. 1, No. 2
Kapoukranidou et al.
3. RESULTS
3.3. Sciatic Functional Index
3.1. Flat Runway and Inclined Plane Runway
The two groups were comparable, as there was no
statistically significant difference in the SFI scores
before the operation. The variance of the SFI on the
same day is attributed to differences of the weight and
sex of the rats. After the nerve suppression, rats were
unable to lean on the left hindlimb and displayed
clockwise rotation. The sciatic nerve innervates the
soleus and gastrocnemius muscle. The operation
resulted in paralysis of these muscles, foot drop,
inability of toe support and characteristic dragging
st
th
movement of the fingers. On both the 1 and the 8
day, the left plantar footprint was unclear and the SFI
was -93.3. The NPL and EPL are increased and the
th
ETS and EITS are decreased on the 15 day
nd
compared to the preoperative results. On the 22 day,
the NPL and EPL were decreased as well as the ETS
and EITS in comparison to the last measurement. On
nd
the 22 day the SFI value for the treated group
Preoperatively, all rats had normal gait score of 3.
The treated group appeared to have a faster recovery,
nd
with a statistically significant improvement on the 22
day (p=0.015<0.05) (Figures 4A, B).
3.2. Grid Walking Test
Preoperatively, a small number of falls in the holes
of the grid was observed. The number of falls
significantly increased in both groups after the sciatic
nerve crush, due to motor function impairment.
Although no significant differences were observed,
paracetamol improved the performance of the rats
th
th
especially on the 8 day. On the 15 day the scores
nd
were close to normal. The scores on the 22 day were
even better than the observed preoperatively (Figure
4C).
A
B
C
Figure 4: Illustrations of the time course for recovery of locomotor function of sciatic nerve crushed rats of both groups on
different runways (A) Flat runway, (B) Inclined plane runway, (C) Grid runway.
Effects of Acetaminophen on Motor and Sensory Functions in Adult
appeared to be closer to the normal than to the control
group, with a statistically significant difference
(p=0.044<0.05) (Figure 5).
Journal of The Analgesics, 2013, Vol. 1, No. 2
31
could be a limitation to our study. Therefore, further
investigations are in progress to show the involvement
of other systems/factors in the regulation of motor and
sensory functions induced by paracetamol.
DISCLOSURE
The authors report no conflicts of interest in this
work.
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Figure 5: Effect of intraperitoneal administration of
acetaminophen on Sciatic Functional Index in animals
subjected to sciatic nerve compression. The values represent
mean scores (n=7 rats per group).
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4. DISCUSSION
In the current study paracetamol was shown to be
effective recovering motor and sensory functions in
adult rats after sciatic nerve crush, when administered
intraperitoneally immediately after the compression of
the nerve. Sciatic nerve crush in rats represents a
predictive model to study molecules with a therapeutic
potential in human neuropathic pain syndromes. In
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nd
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Accepted on 16-12-2013
Published on 31-12-2013
DOI: http://dx.doi.org/10.14205/2311-0317.2013.01.02.1
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