The Effects Of Different Interferences On Chronic Neuropathic Pain In Rats

Part 1 Changes of c-jun, NGF and sodium channel expression in dorsal rootganglion in the different models of chronic neuropathic pain Objective To investigate the changes of c-jun expression and three types of sodium channel transcript in dorsal root ganglion(DRG) neurons and NGF expression both in DRG and bilateral sciatic nerves in different models of chronic neuropathic pain. Methods Thirty-six male Sprague-Dawley rats were equally divided into 3 groups. The CCI model was made by loose ligation of sciatic nerve by chromic gut and the SNI model was established by tight ligation and section of tibial and the common peroneal nerve and leaving the sural nerve intact. The mechanical and thermal pain threshold were measured before operation and 1, 3, 5, 7, 9, 11, 13 days after operation. Three animals in each group were deeply anesthetized and rapidly decapitated 1, 7 and 14 days after surgery, respectively. The bilateral L_4-6 DRGs and sciatic nerves were removed. The changes of c-jun and NGF expression was determined by immunochemistry. The other 3 animals in each group were deeply anesthetized and rapidly decapitated 14 days after surgery. The L_4-6 DRG of the operated side was removed and crushed and total RNA was extracted with trizol reagent. The contralateral side was used as control. The changes of sodium channel Nav1.8,Nav1.9 and Nav1.3 expression was determined by semi-reverse transcriptase-PCR. Results The mechanical and thermal pain threshold was significantly lowered 3 and 5 days after CCI respectively and reached the lowest level on day 13 as compared with that in control group (P<0.01). The change of mechanical allodynia in SNI model occurred 1 day after injury. No thermal hyperalgesia in SNI model. The immediate early gene (IEGs) c-jun in DRG was elevated early 1 days after CCI and SNI as compared with that in control group(no expression was detected) (P<0.05)and maintained expression afterwards. The NGF expression in CCI sciatic nerve was elevated after CCI as compared with that in control group, while in dorsal root ganglion(DRG) neurons NGF-IR was downregulated 1 day after CCI and returned to the control level 7 days after operation (P<0.05)and maintained expression afterwards. There was no change of NGF expression in SNI groups. The expressions of sensory neuron specific Nav1.8 and Nav1.9 transcript was reduced 14 days after CCI as compared with that in control group(P<0.05). TTX-S sodium channel Nav1.3 transcript was elevated after CCI as compared with that in control group(no expression was detected) (P<0.05). In contrast, there was no changes of all three sodium channel transcripts in dorsal root ganglion(DRG) neurons in SNI model.Conclusion In CCI group, the rats mainly developed thermal hyperalgesia after operation, while in SNI group they only exhibited mechanical allodynia. The IEGs c-jun is considered as the marker of nerve injury and it can induce the expression of NGF. The changes of NGF after CCI is due to interuption of retrograde convey. Sodium channel is involved in the hyperexcitability of the primary sensory neurons after CCI but not in SNI model, partly induced by the changes of NGF expression. Part 2 Effects of intrathecally administerd Nav1.8 antisense oligonucleotide onthe expression of sodium channel mRNA in dorsal root ganglion Objective: To investigate the effects of Nav1.8 antisense oligonucleotide(ASODN) on the expression of sodium channel mRNA in dorsal root ganglion(DRG) neurons in chronic neuropathic pain.Methods: The CCI model was made by loose ligation of sciatic nerve trunk in 24 male SD rats under general anesthesia. The mechanical and thermal pain threshold were measured before operation and 1, 3, 5, 7, 9, 11, 13 days after operation. On the 7th postoperative day the animals were randomly divided into 4 groups of 3 animals in each: (1) group 1(CCI+NS);(2) group 2(CCI+45μg Nav1.8 mismatch oligonucleotide(MSODN)); (3) group 3(CCI+45μg Nav1.8 ASODN);(4) group 4(CCI+90μg Nav1.8 ASODN). The drugs was injected intrathecally twice a day for 5 consecutive days in all groups. The animals were deeply anesthetized and rapidly decapitated 14 days after surgery. The bilateral L_4-6 DRGs was removed and crushed and total RNA was extracted. The change of Nav1.8 sodium channel expression was determined by RT-PCR and also the c-jun expression by immunochemistry. Results: Pain threshold on ipsilateral side of nerve injury was significantly lowered after CCI as compared with that on contralateral side and elevated 3 days after Nav1.8 ASODN injection, even completely reversed the threshold with 90μg. Sensory neuron sodium channel Nav1.8 transcript was downregulted after Nav1.8 ASODN injection with 45μg dosage(group 3) as compared with that in group 1 and 2 (P<0.01) and even greater with 90μg dosage(group 4). The c-jun expression was elevated after operation and maintained high expression. Conlcusion: The intrathecally injection Nav1.8 ASODN can reverse the mechanical allodynia and thermal hyperalgesia partially by downregulation of the Nav1.8 transcript expression and needs further investigation. The effects of 90μg dosage is greater than that of 45μg one. The Nav1.8 ASODN has no effect on nerve repair. Part 3 Effects of exogenous NGF on the expression of sodium channel mRNAin dorsal root ganglionObjective To investigate the effects of exogenous NGF on the expression of sodium channel mRNA in dorsal root ganglion(DRG) neurons in chronic neuropathic pain. Methods Forty-eight male Sprague-Dawley rats were anesthetized with the intraperitoneal injection of 300mg ? kg^-1 choral hydrate. The CCI model was made by loose ligation of sciatic nerve trunk by 4-0 chromic cutgut. The mechanical and thermal pain threshold were measured before operation and 1, 3, 5, 7, 9, 11, 13 days after operation. The animals were randomly divided into 8 groups of 5 animals in each according to the drugs given or not: (1) group 1, CCI;(2) group 2 (CCI +NS); (3) group 3 to 5, CCI plus different dosages of NGF at 4, 8 and 16μg/kg given immediately after CCI; (4)group 6 to 8, CCI plus the same dosages of NGF given 4 days after CCI. The injection of NGF to the target muscle was administered immediately once a day after the nerve ligation for 14 consecutive days in group 3-5 and injected 4 days later in group 6-7 with the same dosages corresponding to group 6-8. The animals were deeply anesthetized and rapidly decapitated 14 days after surgery. The bilateral L_4-6 DRGs were removed and crushed and total RNA was extracted with trizol reagent. The change of Nav1.8 and Nav1.3 sodium channel expression was determined by RT-PCR and NGF expression on bilateral sciatic nerves by immunochemistry.Results Pain threshold was significantly lowered after CCI in group 1 and 2 as compared with that in contralateral side and elevated 5 days after NGF injection in group 3 to 5 but still maintained at low level in group 6-8. The sodium channel Nav1.8 transcript was elevated significantly after NGF injection with 4μg/kg dosage as compared with that in CCI group(P＜0.01) and even greater with 8μg/kg and 16μg/kg dosage, while Nav1.3 transcript was downregulated. NGF expression was elevated in all NGF-administration groups. Conclusion: The exogenous NGF can administered by target muscle injection. It can relieved the hyperalgesia immediate after CCI partly by reversing the changes of Nav1.8 and Nav1.3 channel expression.