EVALUATION OF PROTECTIVE EFFECT OF TEST SAMPLE IN SURGICALLY INDUCED NEUROPATHY PAIN USING CHRONIC CONSTRUCTIVE INJURY (CCI) MODEL IN EXPERIMENTAL RAT

 

EVALUATION OF PROTECTIVE EFFECT OF TEST SAMPLE IN SURGICALLY INDUCED NEUROPATHY PAIN USING CHRONIC CONSTRUCTIVE INJURY (CCI) MODEL IN EXPERIMENTAL RAT

1.0  INTRODUCTION:

The chronic constriction injury (CCI) is a partial nerve injury, mostly used in rodents, which is produced by tying several ligatures around a nerve, such that these ligatures slightly constrict the nerve. This induces an incomplete nerve injury, which entails behavioral signs of hyperalgesia in the animals. Neuropathic pain is a common syndrome, which may result from many disorders including diabetic polyneuropathy, radicular back pain, postherpetic neuralgia, stroke, and spinal cord injury. It is characterized by hyperalgesia, allodynia and spontaneous pain. The CCI of the sciatic nerve is a widely used model of neuropathic pain which evokes a series of molecular, biochemical and cytoarchitec- tural changes in primary sensory neurons and produces neuro- pathic pain sensations similar to those observed in humans. Ligation of the sciatic nerve also leads to the cardinal symptoms of neuropathic pain, namely apparent spontaneous pain, allodynia, and hyperalgesia.

 

2.0  TEST SYSTEM DETAILS:

Species             : Rattus norvegicus (Rat)

Strain                : Wistar or Sprague Dawley

Age                   : 8-10 weeks

Body Wight        : 160-180 g

Sex                   : Male or Female

No. of animals    : 8 /Group

 

3.0  ALLOCATION OF GROUPS:

Groups	Treatment	Dose; ROA	No. of Animals
G1	Normal Control	Normal saline or 0.25% Na-CMC	8
G2	Sham Control	Normal saline or 0.25% Na-CMC	8
G3	Disease Control	Normal saline or 0.25% Na-CMC	8
G4	Reference Drug- Gabapentin	75 mpk; i.p.	8
G5	Plant Extract-1	X mpk; p.o.	8
G6	Plant Extract-2	XX mpk; p.o.	8
G7	Plant Extract-3	XXX mpk; p.o.	8

*The dose and ROA (Routes of administration) will be decided based on the type of reference drug

 

4.0 METHODOLOGY:

· The study protocol (Form B) shall be approved from the IAEC before commencing the experiment.

· Animals shall be procured from the CPCSEA authorized vendor.

· Animals shall be quarantined for 1 week as per the in house SOP.

· Healthy animals will be selected, randomized based on body weight and divided into five different groups consisting of 8 animals each.

· Induction of Neuropathy Pain

· Each rat will be anaesthetized with an intraperitoneal injection of a mixture of xylazine (10 mg/kg) and ketamine (100 mg/kg) and then placed in a prone position on a heat-regulated pad with an application of lubricating ophthalmic ointment to the eyes.

· Left thigh will be elevated and the fur on the posterior skin will be shaved using electric clippers.

· Exposed skin will be swabbed with a povidone iodine topical 10% w/v solution and a 3–4 cm incision will be made parallel to the long axis of the femur and down the centre of the biceps femoris muscle.

· The connective tissue between the gluteus superficialis and the biceps femoris muscles will be cut under a magnifying glass.

· Using ribbon retractors, the gap between the two muscles will be widened and approximately 10 mm of the sciatic nerve (proximal to the sciatic trifurcation) will be freed from the surrounding connective tissue with a micro-scissor.

· Four ligatures (suture 4-0) will be tied with a double knot, 1 mm apart and proximal to the trifurcation of the sciatic nerve.

· The constriction of the nerve will be minimal and immediately stopped when a brief twitch will be observed. The muscle layer will be then closed with sutures and the skin will be fastened using a surgical stapler.

· Identical operation will be performed, except that the sciatic nerve will not be ligated in sham-operated rats.

· Treatment and assessment of neuropathy pain:

· Group G1 animal will be treated as normal control and treated with normal saline or Na- CMC.

· Animals of group G2 & G3 will be treated as sham control (without nerve ligation) and disease control (nerve ligation) or administered with normal saline or Na- CMC.

· Group G4, G5 and G7 will be treated with test compound at different dose levels.

· All the animals will be administered with vehicle or test sample or reference drug post surgery and continued throughout the experiment.

· The behavioral tests will be consisted of mechanic allodynia and thermal hyperalgesia before surgery (day −1) and 1, 3, 7 and 14 days after surgery.

5.0 END POINT PARAMETER(S):

·       Body Weight

·       Feed water consumption

·       Mechanical allodynia

·       Thermal Hyperalgesia

·       Serum cytokines - TNF-α and IL-1β 

6.0  REFERENCE(S):

6.1 M. Shahid, F. Subhan, N. Ahmad, G. Ali, S. Akbar, K. Fawad, R.D.E. Sewell. Topical gabapentin gel alleviates allodynia and hyperalgesia in the chronic sciatic nerve constriction injury neuropathic pain model. Eur J Pain (2016).

6.2 Szu-Han Chen, Chia-Ching Wu, Sheng-Che Lin, Wan-Ling Tseng, Tzu-Chieh Huang, Anjali Yadav, Fu-I Lu, Ya-Hsin Liu, Shau-Ping Lin and Yuan-Yu Hsueh. Investigation of Neuropathology after Nerve Release in Chronic Constriction Injury of Rat Sciatic Nerve. Int. J. Mol. Sci. 2021, 22, 4746

6.3 Yasuyuki Suzuki,1 Hiromasa Mitsuhata,2 Mitsutoshi Yuzurihara,1 and Yoshio Kase. Antiallodynic Effect of Herbal Medicine Yokukansan on Peripheral Neuropathy in Rats with Chronic Constriction Injury. Evidence-Based Complementary and Alternative Medicine Volume 2012, Article ID 953459, 8 pages doi:10.1155/2012/953459.

6.4 Paul J. Austin, Ann Wu, Gila Moalem-Taylor. Chronic Constriction of the Sciatic Nerve and Pain Hypersensitivity Testing in Rats. Journal of Visualized Experiments. March 2012 |  61  | e3393 | Page 1 of 6

6.5 Risa Takemoto*, Seiwa Michihara, Li-Kun Han, Nina Fujita and Ryuji Takahashi. Ninjin'yoeito Alleviates Neuropathic Pain Induced by Chronic Constriction Injury in Rats. Front. Nutr., 04 February 2021. https://doi.org/10.3389/fnut.2021.525629

6.6 Feng Liang, Miao Liu, Xin Fu, Xueying Zhou, Peng Chen, Fanglei Han. Dexmedetomidine attenuates neuropathic pain in chronic constriction injury by suppressing NR2B, NF-jB, and iNOS activation. Saudi Pharmaceutical Journal 25 (2017) 649–654

6.7 Jun-YihChen, Li-Wen Chu, Kuang-ICheng, Su-Ling Hsieh, Yung-Shun Juan & Bin-NanWu. Valproate reduces neuroinfammation and neuronal death in a rat chronic constriction injury model. Sci. reports. (2018) 8:16457 | DOI:10.1038/s41598-018-34915-5

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