IGF-1 Alleviates Sciatic Nerve Inflammation In ALS-SOD1^G93A Transgenic Mice

Amyotrophic lateral sclerosis?ALS?is a common degenerative motor neuron disease,mainly characterized by the gradual degeneration of motor neurons in the brain stem and spinal cord,causing muscle atrophy and ultimately muscle atrophy.Most of the patients die of respiratory muscle paralysis within 3-5 years.ALS can be divided into two species:sporadic?sALS?and familial?fALS?,of which s ALS accounts for about 90%,fALS about 10%,and 1/5 of f ALS is caused by the mutation of the superoxide dismutase 1?SOD1?gene.Human mutant SOD1^G93A transgenic mice are widely applied to study the etiology,pathogenesis and treatment of ALS.At present,the pathogenesis of ALS is not clear.Many molecular and cellular mechanisms are involved in the degeneration and apoptosis of motor neurons.Although ALS is not an inflammatory disease,more and more evidence has been shown in recent years that inflammatory mechanisms play an important role in the pathogenesis of ALS,and there are inflammatory reactions in both ALS patients and animal models.Moreover,during the process of ALS disease,the activated microglia and inflammatory factors,such as tumor necrosis factor??TNF??,can directly lead to neuro-degeneration.Selective knockout of the mutant SOD1 gene in microglia can prolong the course of ALS disease and selectively inhibit NF-kappa B,an important factor in the regulation of microglia,can slow down the death of motor neurons and the disease progression.However,there are few studies focus on peripheral nerve inflammation in ALS.Gene therapy is to transfer genes into target cells through a certain way,so as to correct gene defects or play therapeutic roles.Adeno-associated virus?AAVs?has the advantages of long term high expression,low immunogenicity and low pathogenicity,and is one of the most commonly used carriers.The insulin-like growth factor 1?IGF1?is a nutrient factor similar to the insulin molecule,which is produced by endocrine,paracrine and autocrine ways.In addition to regulating cell proliferation,differentiation and maturation,IGF1can also repair nerve damages.SOD1^G93A transgenic mice injected with AAV2-h IGF1 delayed onset and extended survival.Injecting the IGF1 gene AAV2 or AAV4 into the central nervous system of SOD1^G93A93A mice can relieve their motor dysfunction and prolong their survival.Our laboratory used AAV9virus vector,with higher efficiency of spinal cord transmission,obtained similar results.The mechanism of IGF1 protects ALS mice includes IGF1activates motoneuron autophagy pathway and reduces the level of the pathogenic SOD1 protein.In addition,IGF1 increases DAO and inhibits the apoptosis of motor neurons.IGF1 can play an anti-inflammatory role by inhibiting the NF-kappa B activity in macrophage.Previous studies focused on the protective effects of IGF1 on neurons in the spinal cord of SOD1^G93A93A transgenic mice and reducing the activation of glial cells in the anterior horn of the lumbar spinal cord of ALS mice,and little attention was paid to the effect of IGF-1 on the sciatic nerve inflammation in ALS mice.In this study,we used toluidine blue,laser confocal and western immunoblotting to observe the pathological changes and neuroinflammation of the sciatic nerve in the ALS mice during disease progression.The myelin sheath of the sciatic nerve in the presymptomatic and control mice did not have obvious degeneration,no macrophage infiltration and inflammatory factor TNF?;with the progression of the disease,the myelin degeneration increased during the onset of the disease.Macrophage infiltration and activation and TNF?secretion increased;at end-stage myelin degeneration and inflammation reached the peak.We intramusclarly injected the AAV9coded double strands human IGF-1 gene?scAAV9-hIGF1?to ALS transgenic mice.We observed that the myelin degeneration of the sciatic nerve was reduced,the activation of macrophage was decreased,and the secretion of inflammatory factor TNF?alpha decreased.We found that IGF1 alleviated sciatic nerve inflammation in ALS mice.To confirm this finding,we further applied the CRISPR-Cas9 system to knock out the IGF1 gene in ALS transgenic mice.We found that inflammation in sciatic nerve of ALS mice aggravated after knockdown of IGF1 gene.These findings indicate that IGF1not only alleviates lumbar spinal inflammatory response,we firstly observed that IGF1 also reduces sciatic nerve inflammation in ALS mice.Part one Inflammatory changes of sciatic nerve in ALS transgenic miceObjective:To observe pathological changes,the activation of macrophage and the expression of inflammatory factors in the sciatic nerve of normal mice and the ALS transgenic mice during disease progression?presymptom,onset and endstage?.Methods:We selected SOD1G93A mice as animal models,fed them in the animal houses of constant temperature,constant humidity and no special pathogenic bacteria?SPF?.The primer sequences and identification method provided by the American Jackson laboratory were used to test the female mice,and mice were grouped into SOD1 positive transgenic SODl^G93A93A mice and SOD1^G93A negative ones according to the results of DNA identification.We selected 9 SOD1^G93A negative mice?littermate,130-150 days?and 27SOD1^G93A positive mice.The positive mice were divided into 3goups according to the course of disease:9 presymptom mice?60 days?,9 onset ones?100-120 days?,9 endstage ones?130-150 days?.3 mice in Each group was perfused with 5%glutaraldehyde and 3 ones for 4%polyformaldehyde infusion or 3/group freshly decected.The pathological changes of sciatic nerve in mice were observed by toluidine blue staining,and the infiltration and activation of macrophages in the sciatic nerve were observed by immunofluorescence staining,and the changes of CD11b,CD68 and TNF alpha protein expression in the sciatic nerve of mice were detected by Western blotting.Results:1.In the control group,all the myelin sheaths of the sciatic nerve were normal,without myelin degeneration and phagocytes infiltration.There was no significant difference between the sciatic nerve of the presymptomatic mice and the control mice;with disease progression,the myelin sheaths of the sciatic nerve in onset mice was partially changed with the phagocytes swallowed degenerated myelin sheath,and the sciatic nerves in the endstage mice were seriously damaged.Myelin disintegration and demyelination occurred and a large number of phagocytes distributed along sciatic nerves.2.There was no obvious infiltration and activation of macrophages in the sciatic nerve of the control group.Compared with the control mice,the infiltration and activation of macrophages in the sciatic nerve were not obvious in the presymptomatic mice;the infiltration and activation of the sciatic macrophages in onset mice were increased and reached the peak in the endstage mice.3.The mice in the control group expressed a low level of TNF?,and the level of TNF?in the sciatic nerve in the presymptomatic mice was not different from that of the control mice.Whereas with disease progression,they were up-regulated in the onset mice,and reached the peak a in the sciatic nerve of the endstage mice.Part two Intramuscular injection of scAAV9-hIGF1 reduces sciatic nerve inflammation in ALS transgenic miceObjective:to observe sciatic nerve pathological changes,macrophage activation and inflammatory factor TNF alpha in ALS transgenic mice after intramuscular injection of scAAV9-hIGF1.Methods:We selected SOD1^G93A93A transgenic mice as animal model of the ALS,the animals were bred at constant temperature,constant humidity,without special pathogenic bacteria.90-day-old ALS transgenic mice were randomly assigned to scAAV9-hIGF1 and scAAV9-GFP group.mice were perfused with 5%glutaraldehyde?3/group?,4%polyformaldehyde?3/group?or removed freshly?3/group?.The pathological changes of sciatic nerve were observed after toluidine blue staining,and the infiltration and activation of macrophages in the sciatic nerve were observed by immunofluorescence staining,and the changes of CD11b,CD68 and TNF alpha protein in the sciatic nerve were detected by Western blotting.Results:1.Compareing with the GFP control group,the myelin degeneration of the sciatic nerve and the infiltration of phagocytic cells in the AAV9-IGF1group were significantly reduced;2.Compareing with the GFP control group,macrophage infiltration and activation in the AAV9-IGF1 group decreased;3.Compareing with the GFP control group,the level of inflammatory factor TNF alpha in the AAV9-IGF1 group reduced;Part three Intrathecal injection of scAAV9-sgRNA-IGF1-Cas9aggravates sciatic nerve inflammation in ALS transgenic female mice.Objective:To observe the changes of sciatic nerve pathology,macrophage infiltration activation and inflammatory factor TNF alpha in ALS mice after intrathecal injection of scAAV9-sgRNA-IGF1-Cas9.Methods:We selected SOD1^G93A transgenic mice as animal model of the ALS,the animals were bred at constant temperature,constant humidity,without special pathogenic bacteria.30-day-old ALS transgenic female mice wererandomlyassignedtoscAAV9-sgRNA-LacZ-Cas9and scAAV9-sgRNA-IGF1-Cas9 group.Mice were perfused with 5%glutaraldehyde?3/group?,4%polyformaldehyde?3/group?or removed freshly?3/group?.The pathological changes of sciatic nerve were observed after toluidine blue staining,and the infiltration and activation of macrophages in the sciatic nerve were observed by immunofluorescence staining,and the changes of CD11b,CD68 and TNF alpha protein in the sciatic nerve were detected by Western blotting.Results:1.Compareing with the scAAV9-sgRNA-LacZ-Cas9 control group,the myelin degeneration of the sciatic nerve and the infiltration of phagocytic cells in the scAAV9-sgRNA-IGF1-Cas9 group were significantly increased;2.Compareing with the scAAV9-sgRNA-LacZ-Cas9 control group,macrophage infiltration and activation in the scAAV9-sgRNA-IGF1-Cas9group were increased;3.Compareing with the scAAV9-sgRNA-LacZ-Cas9 control group,the level of inflammatory factor TNF alpha in the scAAV9-sgRNA-IGF1-Cas9group was increased;Conclusions:1.In this study,an internationally recognized ALS mouse model,SOD1^G93A93A transgenic mice,was studied.We found that during disease progression the sciatic nerve pathology and macrophage infiltration in the sciatic nerve of SOD1^G93A transgenic mice increased gradually and macrophage marker CD11b,macrophage activated marker CD68 and inflammatory factor TNF?were gradually increased with the disease progresses.It proves besides the inflammation in spinal cord of SOD1^G93A93A transgenic mice,the sciatic nerve also has neuroinflammation,which may provide new targets for the treatment of ALS.2 Consistant with the previous study that IGF1 reduces the activation of microglia in the lumbar spinal cord of SOD1^G93A mice.We find that intramuscular injection of scAAV9-IGF1 alleviates myelin degeneration and reduces macrophages activation and infiltration into the sciatic nerve and decreased the level of inflammatory factor TNF?,suggesting the therapeutic effect of IGF1 on the ALS sciatic nerve inflammation.3.IGF1 knockdown in SOD1^G93A transgenic mice by intrathecal injection of scAAV9-sgRNA-IGF1-Cas9 lesds to worse sciatic nerve pathology,more macrophage invasion and higher the inflammatory factor TNF alpha.All of these results further proved the anti-inflammation in the sciatic nerves.