P. Yogeeswari et al. / European Journal of Medicinal Chemistry 46 (2011) 2964e2970
2969
5.1.8. N0-(5-nitrofuran-2-yl)methylene))-5-(4-nitrophenyl)furan-2-
carbohydrazide (10)
degree of spontaneous (ongoing) pain and tests of hind limb
withdrawal to cold and mechanical stimuli (dynamic mechanical
allodynia, cold allodynia and mechanical hyperalgesia). A mini-
mum of 10 min separated the testing procedures to reduce the
influence of prior nociceptive testing. The order of testing was as
follows: spontaneous pain, dynamic allodynia, cold allodynia and
lastly mechanical hyperalgesia. All of the behavioral responses
were timed with a stopwatch.
IR (KBr): 3300, 3035,1680,1660,1550,1400,1360,1350, 835 cmꢂ1
;
1H NMR (DMSO-d6)
d (ppm): 7.10 (d, 1H, AreH), 7.22 (d, 1H, AreH),
7.38e7.42 (m, 2H, AreH), 7.79 (d, 2H, AreH), 7.93 (d, 2H, AreH), 8.13
(s,1H, NH), 8.3 (s,1H). Calculated for C16H10N4O7: C, 51.90; H, 2.72; N,
15.13, O, 30.25; found: C, 52.01; H, 2.61; N, 15.09, O, 30.29.
5.2. Pharmacology
5.2.3.1. Spontaneous pain. Spontaneous pain was assessed for
a total time period of 5 min as described previously by Choi et al.
[17]. The operated rat was placed inside an observation cage that
was kept 5 cm from the ground level. An initial acclimatization
period of 10 min was given to each of the rats. A total number of
four rats (n ¼ 4) were assigned to this group. The test consisted of
noting the cumulative duration that the rat holds its ipsilateral paw
off the floor. The paw lifts associated with locomotion or body
repositioning was not counted. It’s been suggested that those paw
lifts in the absence of any overt external stimuli are associated with
spontaneous pain, and are correlative of ongoing pain [13].
Albino mice (Swiss strain, 20e25 g) and albino rats (Wistar,
200e320 g) of either sex were used as experimental animals. All
experiments were approved by the Institutional Animal Ethics
Committee. Animals were housed six (mice) and four (rats) per
cage at constant temperature under a 12 h light/dark cycle (lights
on at 7:00 AM), with food and water ad libitum. The synthesized
compounds 1e10 were suspended in 30% v/v polyethylene glycol
(PEG) 400. Lamotrigine and carbamazepine were obtained as gift
samples from M/s IPCA Laboratories, India. Gabapentin was
obtained as a generous gift sample from M/s Wockhardt Labora-
tories, India. Aspirin used for the study was commercially available
from Central Drug House, India.
5.2.3.2. Dynamic component of mechanical allodynia. All of the
operated rats were assessed for dynamic allodynic response
according to the procedure described by Field et al. [18,19]. The
operated rat was placed inside an observation cage that was kept
5 cm from the ground level. An initial acclimatization period of
10 min was given to each of the rats. A total number of four rats
(n ¼ 4) were assigned to this group. A positive dynamic allodynic
response consisted of lifting the affected paw for a finite period of
time in response to mild stroking on the plantar surface using
a cotton-bud. This stimulus is non-noxious to a normal-behaving
rat. The latency to paw withdrawal was then noted down. If no
paw withdrawal was shown within 15 s, the test was terminated
and animals were assigned this withdrawal time. Hence, 15 s
effectively represented no withdrawal.
5.2.1. Induction of peripheral mononeuropathy-CCI model
Unilateral mononeuropathy was produced in rats using the CCI
model performed essentially as described by Bennett and Xie [15].
The rats were anesthetized with an intraperitoneal dose of pento-
barbital sodium (65 mg/kg) with additional doses of the anesthetic
given as needed. Under aseptic conditions, a 3-cm incision was
made on the lateral aspect of the left hindlimb (ipsilateral) at the
mid-thigh level with the right hindlimb serving as the control
(contralateral). The left paraspinal muscles were then separated
from the spinous processes and the common left sciatic nerve was
exposed just above the trifurcation point. Four loose ligatures were
then made with a 4-0 braided silk suture around the sciatic nerve
with about 1-mm spacing as reported elsewhere [16]. The wound
was then closed by suturing the muscle using chromic catgut with
a continuous suture pattern. Finally, the skin was closed using silk
thread with horizontal-mattress suture pattern. A sham surgery
(n ¼ 4) was performed by exposing the sciatic nerve as described
above, but not damaging it. Povidone iodine ointment was applied
topically on the wound and gentamicin antibiotic (4 mg/kg) was
given intramuscularly for five days after surgery. The animals were
then transferred to their home-cages and left for recovery.
5.2.3.3. Cold allodynia. The rats demonstrating unilateral mono-
neuropathy were assessed for acute cold allodynia sensitivity using
the acetone drop application technique as described by Caudle et al.
[20]. The operated rat was placed inside an observation cage that
was kept 5 cm from the ground level and was allowed to acclima-
tize for 10 min or until exploratory behavior ceased. A total number
of four rats (n ¼ 4) were assigned to this group. Few drops
(100e200
mL) of freshly dispensed acetone were squirted as a fine
mist onto the midplantar region of the affected paw. A cold allo-
dynic response was assessed by noting down the duration of paw
withdrawal response. For each measurement, the paw was sampled
three times and a mean calculated. At least 3 min elapsed between
each test.
5.2.2. Pharmacological interventions
Baseline sensory response values were measured for each group
of animals (n ¼ 4) pre-operatively and 9 days post-operatively.
Animals displaying allodynic and hyperalgesic responses in CCI rat
models, were then administered the relevant drug according to
a
pre-determined randomization table and testing was re-
5.2.3.4. Mechanical hyperalgesia. Mononeuropathic rats were
assessed for mechanical hyperalgesia sensitivity according to the
procedure described by Gonzalez et al. [21]. The operated rat was
placed inside an observation cage that was kept 5 cm from the
ground level. An initial acclimatization period of 10 min was given
to each of the rats. A total number of four rats (n ¼ 4) were assigned
to this group. Hindpaw withdrawal duration was measured after
a mild pinprick stimulus to the midplantar surface of the ipsilateral
(left) hindpaw. A withdrawal was defined as being abnormally
prolonged if it lasted at least 2 s. The mean withdrawal duration
was taken from a set of three responses.
performed at 0.5, 1, 1.5, 2 and 2.5 h post-drug administration. Each
group of animals was used for only one drug administrationprotocol
to ensure no ‘carry-over’ effects. Compounds 1e10 (100 mg/kg, i.p.)
were administered at t ¼ 0, in 30% v/v PEG 400. The vehicle control
group of rats received only the solvent (30% v/v PEG 400). Three
positive control groups were run alongside drug treatment groups
using lamotrigine, carbamazepine and gabapentin (100 mg/kg, i.p.).
The treatment protocol remained the same for these three drugs. No
drug testing was performed for sham-operated rats.
5.2.3. Sensory testing (nociceptive assays)
Four nociceptive assays aimed at determining the severity of
behavioral neuropathic responses namely allodynia and hyper-
algesia were performed. The assays involved measurement of the
5.2.4. Statistical analysis
All data are expressed as means ꢀ standard error of mean (SEM).
The data were analyzed using one-way ANOVA, and Bonferroni’s