The Roles Of Spinal Cord Cdk5/p35 And Astrocytes In Rats Neuropathic Pain

ObjectivesIn the present study,to investigate the roles of Cdk5/p35 and astrocytes in rats neuropathic pain.Firstly rat CCI neuropathic pain model was established,then neuropathic pain-like behavior was determined,the expression levels of Cdk5,p35,PSD95 and GFAP in the spinal cord dorsal horns from the lumbar enlargement of rats were subsequently detected.Secondly,for further investigate the role of Cdk5/p35 and astrocytes in the mechanism for the development and maintenance of neuropathic pain.A chronic constriction injury(CCI)model was established and the Cdk5 inhibitor,roscovitine,was intrathecally administrated.Methods1 The effects of spinal cord Cdk5/p35 and astrocytes on the development and maintenance of neuropathic painSixty adult male Sprague-Dawley rats(220~250g)were used.animals were randomly divided into surgery group(CCI group)(n=48)and sham operation group(Sham group)(n=12).The changes of the mechanical paw withdrawal threshold and the thermal paw withdrawal latency in the right hind paw of rats were measured on 1 day before CCI surgery,3 day,7day,14 day and 21 day post-CCI surgery.The ratswere euthanized on the corresponding time,and the spinal cord from the lumbar enlargement of rats were obtained.The expression of Cdk5,p35,PSD95 and GFAP proteins in the spinal cord dorsal horn were detected by Western blot in each group(n=6).The expression of Cdk5,p35 and GFAP proteins were detected and the morphology of astrocytes was observed in the spinal cord dorsal horn in each group(n=6)on day 7 and day 14 post-CCI surgery by immunofluorescence.2 Roscovitine alleviated neuropathic pain by Cdk5/p35 and astrocytes mechanismThirty-two adult male SD rats(220~250g)were used.Animals were randomly divided into 4 groups: Sham+Veh group(sham operation,intrathecal administration of 5% DMSO in saline),Sham+Ros group(sham operation,intrathecal administration of roscovitine),CCI+Veh group(CCI surgery,intrathecal administration of 5% DMSO in saline),CCI+Ros group(CCI surgery,intrathecal administration of roscovitine),8ratsin each group.A chronic constriction injury(CCI)of the common sciatic nerve was performed in rat following intrathecal catheterization was succeed for 7-day.The injection volume was 20μl of Vehicle or drug solution.Roscovitine was intrathecally administrated 100μg,once a day on postoperative days 3-14.The mechanical paw withdrawal threshold and the thermal paw withdrawal latency of rats were measured on the 1day before CCI surgery,day 3,day 7 and day 14 post-CCI surgery.Rats were sacrificed on day 14 post-CCI surgery and the spinal cord from the lumbar enlargement of rats were obtained.The level expression of Cdk5,p35,PSD95 and GFAP proteins in the lumbar spinal cord dorsal horn were detected in each group by Western blotanalysis.1 Behavioral measurement of ratsThere was no significant difference in normal baseline values of the mechanical paw withdrawal threshold and thermal paw withdrawal latency between the two groups before CCI surgery(P>0.05).Compared with the Sham group,the mechanical paw withdrawal threshold and the thermal paw withdrawal latency of the CCI group were significantly decreased(P<0.05),and lasted until the day 21 post-CCI surgery.2 Western blot analysis examined the protein expression of Cdk5,p35,PSD95 and GFAP in the spinal cord dorsal hornCompared with Sham group,the expression levels of p35,PSD95 and GFAP in CCI group significantly increased and lasted until day 21 post-CCI surgery(P<0.05).There was no significant difference in the expression of Cdk5(P>0.05).3 Immunofluorescence stainingexamined protein expression of Cdk5,p35 and GFAP in the spinal cord dorsal hornCompared with sham group,the immunoreactivitystainingof p35 and GFAP were significantly increased in the superficial spinal cord dorsal horn,the expression of p35 and GFAP were significantly increased in the lumbar spinal cord dorsal horn of CCI rats on day 7 and day 14 post-CCI surgery(P<0.05);astrocytes were activated;while there was no significantdifference in thestaining intensity of Cdk5(P>0.05).4 Behavioral measurement of ratsCompared with the Sham+Veh group,thepaw withdrawal threshold to mechanical stimuli and paw withdrawal latency to thermal stimuli in the ipsilateral hind paw of the CCI+Veh group were decreased(P<0.05),and lasted until the day 14 post-CCI surgery.Compared with the CCI+Veh group,the paw Resultswithdrawal threshold to mechanical stimuli and paw withdrawal latency to thermal stimuli in the ipsilateral hind paw of the CCI+Ros group were significantly increased(P<0.05).5 Western blot analysis examined protein expression of Cdk5,p35,PSD95 and GFAP in spinal cord dorsal hornCompared with the Sham+Veh group,the expression levels of p35,PSD95 and GFAP in the CCI+Veh group were significantly increased(P<0.05),but there was no statistically significant in Cdk5 expression(P>0.05);Compared with CCI+Veh group,the expression levels of p35,PSD95 and GFAP in CCI+Ros group were significantly decreased(P<0.05),but there was no statistically significant in Cdk5 expression(P>0.05).Conclusions1 CCI-induced sciatic nerve injury in rats induced neuropathic pain and increased activity of Cdk5/p35,changed synaptic plasticity,activated astrocytes in spinal cord dorsal horn.Cdk5/p35 activity is involved in developement and maintenance of neuropathic pain;astrocytes activation contribute toNP.2 Intrathecal administration of roscovitine,a Cdk5 inhibitor,has a significant analgesic effect on CCI-induced neuropathic pain by inhibiting Cdk5 activity and activation of astrocytes.The increase of Cdk5 activity and astrocyteactivation play an important role in the development and maintenance of neuropathic pain.