Neuroinflammatory Mechanism Of Cognitive Dysfunction Mediated By Neuropathic Pain

Part I Effect of neuropathic pain on cognitive dysfunction and its neuroinflammatory mechanismObjectives:Neuropathic pain(NPP)is always accompanied by cognitive dysfunction in various degrees,and the underlying mechanism is not elucidated.In this study,a rat NPP model was established by creating chronic constrictive injury(CCI)of the rat sciatic nerve.In the perspective of the regulation of central inflammation that occurred in hippocampal neurons,methods of behavior,molecular biology,and morphology were utilized to explore the effect of neuropathic pain on cognitive function.Methods:Sixty adult male SD rats were randomly divided into 3 groups:namely control group(CONTROL),sham operation group(SHAM),and NPP model group(CCI),with 20 rats in each group.The sciatic nerve of the rats in the CCI group was ligated,while it was only exposed without ligation in the SHAM group.The rats in the CONTROL group did not receive any surgery.Mechanical pain and thermal pain thresholds were measured as the basic threshold one day before modeling,which were measured 3 days,7 days,10 days,and 14 days after the operation,respectively.Moreover,the Morris water maze test was carried out 15-19 days after the operation.Subsequently,all the rats were sacrificed and hippocampus specimens were collected after full perfusion.Inflammatory factors including tumor necrosis factor-alpha(TNF-α)and interleukin-1β(IL-1β)in rat hippocampus were determined by enzyme-linked immunosorbent assay(ELISA).Ionized calcium binding adapter molecule 1(Iba 1,a marker of microglia),glial fibrillary acidic protein(GFAP,a marker of astrocyte),and Nestin(a marker of neural stem cell)were measured by western blotting.Furthermore,expression of Ibal,GFAP,NeuN(a marker of mature neurons),DCX(a marker of neural stem cell),and Caspase-8(a marker of apoptosis)in the rat hippocampus were detected by immunofluorescence.And the morphological changes were observed through an electron microscope.Results:1.Compared with the rats in the CONTROL group,the mechanical pain threshold of the rats in the CCI group on the 3rd,7th,10th,and 14th days after operation decreased significantly(P<0.01),and the result continued until the end of the experiment.Likewise,the thermal pain threshold of the rats in the CCI group decreased obviously(P<0.01).The indicators above of the rats were not altered in the SHAM group(P>0.05).There was no significance in the change of thermal pain behavior in control side limbs of the rats in each group,indicating the success of modeling.2.In the Morris water maze experiment,the average swimming speed of the rats in each group did not change significantly(P<0.05).In the positioning navigation experiment,the difference in the escape latency of the rats in each group was of no significance on the first day(P>0.05).On the second day,the average escape latency of the rats in the CCI group trended to prolong,however,with no statistical significance(P<0.05).On days 3,7,10,and 14,the average escape latency of CCI rats was longer compared with the rats in the CONTROL group(P<0.001),with shorter stay time in the target quadrant(P<0.05)and reduced times in crossings within 60 s(P<0.001),suggesting severe spatial memory ability impairment of the CCI rats.However,there was no statistical difference in the above indicators in the SHAM group compared with the CONTROL group(P>0.05).3.Protein expression of TNF-α and IL-1β in the hippocampus of the rats in the CCI group(P<0.001)and the SHAM group(P<0.05)increased significantly compared with the rats in the CONTROL group.4.Compared with the rats in the CONTROL group,there was no significant difference in protein expression of Iba-1 and GFAP(detected by western blotting)in the hippocampus of the SHAM rats(P>0.05).However,the protein expression of Iba-1 and GFAP increased notably in the hippocampus of the CCI rats(P<0.01).Under the electron microscope,microglia and astrocytes in the dentate gyrus(DG)area of the rats’hippocampus in the CCI group were activated.5.Compared with the rats in the CONTROL group,protein expression of Nestin was elevated obviously in the hippocampus of CCI rats(P<0.01),which was not altered in the hippocampus of SHAM rats(P>0.05).6.Immunofluorescence was used to detect the expression of NeuN and Caspase-8 in the hippocampal DG area of the three groups of rats.There was no difference in the number of NeuN and Caspase-8 positive cells in the hippocampus of the CONTROL rats and the SHAM rats(P>0.05).Compared with the SHAM group,the number of NeuN positive cells significantly decreased(P<0.001),and the number of Caspase-8 positive cells significantly increased(P<0.001)in the hippocampus of the rats in the CCI group.Conclusions:In the CCI model,neuropathic pain can lead to defects in learning ability and spatial memory in rats,The mechanism may be associated with the activation of microglia and astrocytes and the mass release of inflammatory factors(IL-1β and TNF-α)in the hippocampus caused by pain.Subsequently,leading to the damage of mature and new neurons and weakening the cognitive functions of the rats.Part Ⅱ Effect of Infliximab on cognitive dysfunction mediated by neuropathic pain and the underlying mechanismObjectives:Infliximab(IFX),a TNF-α monoclonal antibody,is widely used in various diseases due to its inhibitory effect on inflammation,oxidative stress,and apoptosis.In this study,IFX was administered to the lateral ventricle to observe the effect of EFX on the central inflammatory response,mature neuron apoptosis,and occurrence of neural stem cells in CCI rats to explore the mechanism of IFX in the treatment of neuropathic pain-mediated cognitive dysfunction.Methods:Eighty adult male SD rats were randomly divided into 4 groups:namely sham operation group(SHAM),sham operation group treated with infliximab(SHAM+IFX),NPP model group(CCI),and NPP model group treated with infliximab(CCI+IFX),with 20 rats in each group.The sciatic nerve of the rats in the CCI group was ligated,while it was only exposed without ligation in the SHAM group.On days 1,5,and 9 after surgery,rats in groups of SHAM+IFX and CCI+IFX were administered with IFX to the ventricle bilaterally once a day(10 mg/ml,10 μl).Mechanical pain and thermal pain thresholds were measured as the basic threshold one day before modeling,which were measured 3 days,7 days,10 days,and 14 days after the operation,respectively.Moreover,the Morris water maze test was carried out 15-19 days after the operation.Subsequently,all the rats were sacrificed and hippocampus specimens were collected after full perfusion.Inflammatory factors including TNF-α and EL-1β in rat hippocampus were determined by ELISA.Iba 1,GFAP,and Nestin were measured by western blotting.Furthermore,expression of Ibal,GFAP,NeuN,DCX,and Caspase-8 in the rat hippocampus were detected by immunofluorescence.And the morphological changes were observed through an electron microscope.Results:1.Compared with the rats in the SHAM group,the mechanical pain threshold of the rats in the CCI+IFX group decreased on the operation side(P<0.01)on the 3rd day after modeling.Meanwhile,the mechanical pain and thermal pain thresholds decreased on the 7th,10th,and 14th days after surgery,among which the indicators on the 7th day after surgery reached the lowest levels(P<0.01).The mechanical pain and thermal pain thresholds of the SHAM rats did not change after administered with IFX(P<0.01).However,the mechanical pain and thermal pain thresholds of the rats in the CCI+IFX group increased on the 3rd,7th,10th,and 14th days after modeling compared with the rats in the CCI group(P<0.01).2.In the Morris water maze experiment,there was no difference in the average swimming speed of rats in each group(P>0.05).In the positioning navigation experiment,the average escape latency of the rats in the SHAM+IFX group at each time point did not alter significantly compared with the rats in the SHAM group(P>0.05).On the first and second days of the positioning navigation experiment,there was no difference in the escape latency in the CCI and CCI+IFX groups compared with the SHAM group(P>0.05),while the average level of escape latency in these two groups was prolonged on the 3rd day(P<0.01).On the 4th day,the average escape latency of the rats in the CCI group was still prolonged(P<0.05),which was recovered in the CCI+IFX group(P>0.05).In the experiment of space exploration,there was no significant change in target quadrant residence time ratio and crossing times within 60 s between the SHAM+IFX group and the SHAM group(P>0.05).The target quadrant residence time ratio of the rats decreased in the CCI group(P<0.01)and was not changed in the CCI+IFX group(P>0.05)compared with the rats in the SHAM group.While the crossing times within 60 s of the rats in the CCI and CCI+IFX groups were both decreased(P<0.01).When compared with the rats in the CCI group,treatment with IFX significantly increased the target quadrant residence time ratio and the crossing times within 60 s of the rats(P<0.01).3.IFX treatment did not change protein expression of TNF-α and IL-1β in the hippocampus of the SHAM rats(P>0.05).Protein expression of TNF-α and IL-1β in the hippocampus of the rats in the CCI and CCI+IFX groups increased significantly compared with the rats in the SHAM group(P<0.01).However,IFX treatment significantly decreased the expression of TNF-α and IL-1β in the hippocampus of the CCI rats(P<0.01).4.Compared with the rats in the SHAM group,there was no significant difference in protein expression of Iba-1 and GFAP(detected by western blotting)in the hippocampus of the rats in the SHAM+IFX group(P>0.05),while the protein expression of Iba-1 and GFAP increased in both groups of CCI and CCI+IFX(P<0.01).Treatment with IFX in CCI rats down-regulated the expression of these two proteins(P<0.01).Immunofluorescence results showed that microglia and astrocytes in the DG area of rats’hippocampus are activated in the CCI and CCI+IFX groups.However,the number of microglia and astrocytes reduced significantly after IFX treatment in CCI rats,with cell shrinkage and increased branch retraction.Taken together,morphology results showed that the microglia and astrocytes in the hippocampus were presented as a low activation state in the CCI rats treated with IFX.5.Compared with the rats in the SHAM group,the protein expression of Nestin(detected by western blotting)in the hippocampus of the rats in the CCI group decreased significantly(P<0.01).The protein expression of Nestin in the hippocampus of the CCI rats treated with IFX did not alter compared with the SHAM rats(P>0.05).However,it elevated when compared with the CCI rats(P<0.01).The results of immunofluorescence showed that the DCX positive cells were mainly distributed in the subgranular zone(SGZ)of the hippocampal dentate gyrus.The number of DCX positive cells in the rats reduced in the CCI group compared with the SHAM group(P<0.001),while the number of DCX positive cells in the CCI rats treated with IFX recovered to the same level as the SHAM rats(P>0.05).IFX treatment increased the number of DCX positive cells in the CCI rats(P<0.01).6.Immunofluorescence was used to detect the expression of NeuN and Caspase-8 in the hippocampal DG area of the four groups of rats.The results showed that the number of NeuN positive cells significantly decreased(P<0.001),and the number of Caspase-8 positive cells significantly increase(P<0.001)in the hippocampus of the CCI rats compared with the SHAM rats.While the number of NeuN and Caspase-8 positive cells in the CCI rats treated with INX recovered to the same level as the SHAM rats(P>0.05).Compared with the rats in the CCI group,IFX treatment increased the number of NeuN positive cells(P<0.001)and decreased the number of Caspase-8 cells(P<0.001)in the hippocampus of the CCI rats.Conclusions:Infliximab was able to treat cognitive dysfunction in NPP rats and achieved exciting efficacy.The mechanism may be associated with the anti-inflammation and anti-oxidative stress effect of infliximab,thereby reducing the effect of the central inflammatory response on mature neurons and nerve cell development in the DG region of the hippocampus.