Effects Of Probiotics On Sciatic Nerve Structure And Macrophage Infiltration In Mouse Model Of Amyotrophic Lateral Sclerosis

Objective:1.To study the changes in sciatic nerve structure and macrophage infiltration in a mouse model of amyotrophic lateral sclerosis（amyotrophic lateral sclerosis,ALS）during presymptomatic,onset and terminal stages.2.To study whether probiotics can improve the sciatic nerve structural abnormalities and reduce macrophage infiltration in the end-stage of ALS mouse model.Methods:The ALS mouse model is a SOD1^G93Atransgenic mouse from Jackson laboratory in the United States.We raise and breed mice in an animal room with constant temperature,humidity and no specific pathogenic bacteria（SPF）.40-day-old female mice were genotyped by PCR identification,which were divided into control SOD1^WTmice and experimental SOD1^G93Amice.Nine SOD1^WTmice were selected and treated with normal saline(CK-SOD1^WT)at 60 days.Nine SOD1^G93Amice were selected and treated with normal saline(CK-SOD1^G93A)at 60 days.Six SOD1^G93Amice were selected and treated with probiotic suspension(Pro-SOD1^G93A)at 60 days.The mice in CK-SOD1^WTgroup and CK-SOD1^G93Agroup were randomly divided into 3groups:pre-symptom group（about 60 days）,onset group（about 100 days）and end-stage group（about 120 days）.The Pro-SOD1^G93Agroup was randomly divided into two groups:onset group（about 100d）and end stage group（about120d）.After scoring the neurological function of CK-SOD1^WTgroup,CK-SOD1^G93Agroup and Pro-SOD1^G93Agroup,the sciatic nerve was taken to observe the structure of sciatic nerve under light microscope and transmission electron microscope,and the macrophages in sciatic nerve were labeled with CD68 antibody immunofluorescence staining.Results:1.Neurological function score.At 60 days,100 days and 120 days,there was no dyskinesia,hindlimb muscle atrophy or muscle tremor in CK-SOD1^WTgroup,and the neurological function score was 5.Compared with the CK-SOD1^WTgroup in the same period,there was no obvious motor disturbance,hindlimb muscle atrophy or muscle tremor in the CK-SOD1^G93Agroup at 60 days,and the neurological function score was 5（nasty 9）.When suspended,adduction and tremor appeared in the hindlimb of the CK-SOD1^G93Agroup at 100 days,and the muscle of the hindlimb atrophied compared with that of the CK-SOD1^WTgroup,and the neurological function score was 4 points（P<0.001）.At 120 days,in the CK-SOD1^G93Agroup,the hind limbs were completely paralyzed,the muscles of the hind limbs were obviously atrophied,and the mice could not turn over within 30seconds after lying on their back,and the neurological function score was 1（P<0.001）.The neurological function score of Pro-SOD1^G93Agroup treated with probiotics was 4.67±0.51 at 100d（P<0.01）,which was significantly better than that of CK-SOD1^G93Agroup at 100d.At 120 days,the neurological function score was 1.67±0.33（P>0.05）.Compared with the CK-SOD1^G93Agroup at 120 days,the neurological function showed a tendency to improve,but the difference was not statistically significant.2.Cross section of sciatic nerve was observed under light microscope after toluidine blue staining.On the 60th,100th and 120th day,the number of myelinated nerve fibers in the CK-SOD1^WTgroup was large and closely arranged,the myelin sheath shape was regular,no myelin ball and no obvious axonal degeneration were found.With the extension of survival time,there was no significant change in the number of myelinated nerve fibers in the sciatic nerve,but the myelin sheath area increased.Compared with the CK-SOD1^WTgroup at the same time,the number of myelinated nerve fibers and the area of myelin sheath were not significantly decreased in the CK-SOD1^G93Agroup at 60 days.The myelinated fibers were closely arranged,no myelin ball and no obvious axonal degeneration were found.On the 100th day,in CK-SOD1^G93Agroup,the number of myelinated nerve fibers and the area of myelin sheath decreased significantly（P<0.001）.The arrangement of myelin sheath was loose,the gap was enlarged,part of myelin ball and axonal degeneration could be seen.On the 120th day,in CK-SOD1^G93Agroup,the number of myelinated nerve fibers and the area of myelin sheath decreased significantly（P<0.001）,the arrangement of myelinated fibers loosened and the gap enlarged more obviously,the medulla increased and the axonal degeneration was obvious.With the extension of survival time,the number of myelinated nerve fibers of sciatic nerve decreased,the area of myelin sheath decreased,and the structural destruction of sciatic nerve gradually increased.After intervention with probiotics,compared with the CK-SOD1^G93Agroup in the same period,the number of myelinated nerve fibers in the sciatic nerve of the Pro-SOD1^G93Agroup increased at 100d and 120d,but there was no statistical significance.The myelin sheath area increased significantly at100d and 120d（P<0.01and 120d,respectively）.The myelinated fibers arranged more tightly,the space decreased,the myelin ball decreased and the axonal degeneration was alleviated.3.Observation of cross section of sciatic nerve under transmission electron microscope.On the 60th,100th and 120th day in CK-SOD1^WTgroup,most of the myelinated nerve fibers in the sciatic nerve were normal,neurofilaments,microtubules and mitochondria could be seen in the axons,and there were no edema of mitochondria,no vacuoles under the axial membrane,and the myelin sheath lamellar concentric circle.Compared with the CK-SOD1^WTgroup at the same time,the myelinated nerve fibers in the sciatic nerve in the CK-SOD1^G93Agroup had varying degrees of degeneration:axon degeneration or even disappearance,mitochondrial edema in the axon,subaxial edema,myelin bending and invagination,myelin lamellar folding,release,delamination or even complete disintegration,myelin ball formation,phagocytosis of denatured nerve fibers.Compared with the CK-SOD1^WTgroup in the same period,there were myeloid balls in the sciatic nerve of the CK-SOD1^G93Agroup at 60 days,but there was no significant difference（P>0.001）,and the number of medullary balls increased significantly at 100 days and 120 days（P<0.001）.With the prolongation of survival time,the number of medulla in sciatic nerve in CK-SOD1^G93Agroup increased,and the destruction of sciatic nerve structure was aggravated.After intervention with probiotics,compared with the CK-SOD1^G93Agroup in the same period,the degeneration degree of myelinated nerve fibers in Pro-SOD1^G93Agroup decreased at 100d and 120d:although the distribution of myelinated nerve fibers was sparse,the degree of axonal degeneration,subaxial edema,myelinolysis and even disintegration of myelinated nerve fibers decreased,and the number of myelin balls decreased,but the difference was not statistically significant（P>0.05）.4.The infiltration of macrophages in the longitudinal section of sciatic nerve was observed after immunofluorescence staining.There was no obvious infiltration of CD68⁺macrophages in the sciatic nerve of CK-SOD1^WTgroup at 60 d,100 d and 120 d.Compared with the CK-SOD1^WTgroup at the same time,several CD68⁺macrophages were found in the sciatic nerve of the CK-SOD1^G93Agroup on the 60th day,but there was no obvious swelling of the sciatic nerve.On the100th day,a large number of CD68⁺macrophages could be seen in the sciatic nerve of CK-SOD1^G93Agroup,and the sciatic nerve was obviously swollen.At120 days,there was still CD68⁺macrophage infiltration and sciatic nerve atrophy in CK-SOD1^G93Agroup.With the prolongation of survival time,macrophage infiltration was the most serious at 100 days,suggesting that the increase of inflammatory reaction may be closely related to the occurrence of ALS symptoms.After intervention with probiotics,compared with the CK-SOD1^G93Agroup in the same period,the infiltration of CD68⁺macrophages in the sciatic nerve of the Pro-SOD1^G93Agroup decreased at 100 and 120 days,suggesting that probiotics can alleviate the infiltration of macrophages in the sciatic nerve and reduce the inflammatory reaction.Conclusion:1.With the progress of ALS,ALS mouse model showed muscle weakness,atrophy and tremor in the hindlimb,and gradually aggravated.After the intervention of probiotics,the muscle weakness,atrophy and tremor of the hind limbs of ALS mice were improved.2.In the early stage of symptoms,ALS mouse model of sciatic nerve has been myelinated nerve fiber degeneration changes,with the progress of the disease,myelinated nerve fiber degeneration aggravated,visible phagocytic infiltration.After the intervention of probiotics,the degeneration of myelinated nerve fibers and phagocyte infiltration in the sciatic nerve of ALS mice at the onset and end stage were alleviated.3.Macrophage infiltration could be seen in the sciatic nerve of ALS mice in pre-symptom,onset and end-stage.After the intervention of probiotics,the infiltration of macrophages in the sciatic nerve of ALS mice decreased,suggesting that probiotics can alleviate the infiltration of macrophages in the sciatic nerve of ALS mice,reduce the inflammatory reaction,alleviate the degeneration of sciatic nerve,and then improve the symptoms of mice.