Neuroprotective Effects Of Dl-3-n-butylphthalide In A Transgenic Mouse Model Of Familial Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease(MND) or Lou Gehrig's disease, is a devastating neurodegenerative disordercharacterized by progressive muscular dystrophy, paralysis, bulbar symptomsand resulting from the progressive and selective loss of upper motor neuronsin the motor cortex and lower motor neurons in the brain stem as well asspinal cord, finally died of respiratory failure. Approximately90%of ALScases are sporadic, the remaining10%of patients are familial. Approximately20%of familial cases are caused by mutations in the superoxide dismutase(SOD1) gene. Nevertheless, there is no cure for this lethal disease, withmortality usually occurring within3–5years after symptom onset. Althoughmany compounds targeting have been evaluated clinically, Riluzole is the onlytherapeutic drug that has been approved by the Food and Drug Administrationfor ALS treatment, however, it only extends the life span of an ALS patient bya few months. Thus, there is an urgent need for an efficacious therapeutictreatment for ALS patients.Although the disease pathogenesis is still unknown at present, Aberrantphosphorylation of the high-molecular-weight neurofilament(NFH) proteinhas been postulated to be a primary pathogenic mechanism in the genesis ofneurofilamentous aggregates in spinal cord motor neurons in ALS.Neurofilaments belong to a family of intermediate cytoskeletal proteins. Thethree NF subunit proteins, defined on the basis of molecular mass include a200-kDa (NFH, high molecular weight NF), a160-kDa (NFM, intermediatemolecular weight NF) and a68-kDa (NFL, low molecular weight NF) protein.The homopolymerization of NFL is critical to NF assembly, NF-M and NF-His thought to facilitate NF cross-linking, stabilize the axonal cytoskeleton,enhance axonal growth and determine axonal caliber and axoplasm transport. Usually, Non-phosphorylated neurofiaments were detected in the cytoplasm ofneurons. The distribution of phosphorylated neurofiaments were in axonal anddetermined axonal caliber. However, the highly phosphorylated tail domain ofNFH is most resistant to proteolysis of NFs, which will lead to the abnormalaccumulation of phosphorylated Neurofilaments(p-NF) within perikarya.Eventually lead to neuronal degeneration and death.Dl-3n-butylphthalide(DL-NBP) was extracted from the seeds of Apiumgraveolens Linn, Chinese celery. Afterward, DL-NBP was synthesized andapproved by the State Food and Drug Administration of China for clinical usein stroke patients in2002. Previous studies showed that DL-NBP reducedoxidative stress, improved mitochondrial function, blocked inflammatoryreactions, and reduced neuronal apoptosis. Rencenly, DL-NBP improvedcognitive performance in an animal model of Alzheimer's disease. However,DL-NBP has not been tested as a therapeutic treatment in ALS.Based on these studies, we decided to examine the neuroprotective effectof DL-NBP in preventing diease progression in SOD1-G93A mice. In addition,we also investigated potential neuroprotective effect mechanism of DL-NBP.PartⅠNeuroprotective effects of different dosage ofDl-3-n-Butylphthalide when treatment started at pre-symptomatic stagein a transgenic mouse model of familial amyotrophic lateral sclerosisObjective: To examine the neuroprotective effect of DL-NBP inpreventing diease progression in SOD1-G93A mice. in addition, we alsodetected the distribution and expression of p-NF in the spinal cord ofSOD1-G93A mice.Methods:1Treatment groups and drug administrationThe forty-eight SOD1-G93A mice were randomly divided into fourexperimental groups (n=12,six males and six females): three Dl-NBP treatedgroups (60,120, and180mg/kg/d) and one vehicle control group.DL-NBP (purity>98%, was synthesized by the ShijiazhuangPharmaceutical Group NBP Pharmaceutical co.,Ltd) was orally gavage administered; DL-NBP was dissolved in corn oil at three concentrations (6,12and18mg/ml), and fresh corn oil were prepared daily. Vehicle control groupof SOD1-G93A mice were treated with corn oil. Drugs and corn oil wereadministered by oral gavage to the mice in a volume of10ml/kg body weightonce per day. The twelve non-transgenic mices (n=12, six males and sixfemales) were not received any treatment. Treatment was started at42days ofage of the SOD1-G93A mice.2Evaluation of motor function2.1Determination of Symptom Onset and Survival end points of miceAssessment of general condition starting at83days of age. We used abehavioural score system based on the score developed by Vercelli et al from1to5defined as follows: Onset of symptoms was defined as score is4; Survivalend points of mice was defined as score is1.5: healthy without any symptoms of paralysis4: slight signs of destabilized gait and paralysis of the hind limbs3: obvious paralysis and destabilized gait2: fully developed paralysis of the hind limbs, animals only crawl on theforelimbs1: fully developed paralysis of the hind limbs, the ALS mice were unableto right themselves after being placed on their back for20s2.2RotarodBeginning at10weeks of age, After an adaptation period of5days ofdaily testing. the rotarod analysis was performed weekly. The rotation startedat1rpm. Within180s the rotation speed increased up to30rpm. Each animalwas given three trials and the longest latency without falling was recorded.3ImmunohistochemistryMice were anesthetized with10%chloral hydrate and perfusedtranscardially with4%paraformaldehyde in0.1M phosphate buffer for20minutes.The lumbar enlargements were carefully dissected and fixed in4%pareformaldehyde for48hours. The tissues were then cut into30-um sectionsusing a vibratome. We observed the distribution of SMI-31and SMI-32in lumbar anterior horn by Immunohistochemistry.4Immunoblotting analysisMice were anesthetized with10%chloral hydrate by350mg/kg, thenextract the lumbar spinal cord of mice immediately, put it into liquid nitrogenfreezing,-80℃storage. total protein extracts were obtained from L1-L5spinal cord sections.Whole tissue extracts were prepared using a total proteinextraction kit. Then we can detected the expression level of SMI-31byWestern-blotting.5Statistical AnalysisWe used SPSS version13.0for Windows (SPSS) for allstatisticalanalyses. The data are depicted as mean values and standard errors of themean (SEM). Kaplane-Meier survival analysis was used for diease onset andsurvival comparisons,and repeated measures ANOVA test was used forassessment of rotarod performance comparisons. Biochemical andpathological results were analyzed using ANOVA followed by Newman-Keulspost hoc test and the Mann-Whitney U test.p <0.05was considered asstatistically significant.Results:1Disease onset and lifespan1.1Disease onsetTreatment with DL-NBP at dose of60,120and180mg/kg significantlydelayed disease onset of SOD1-G93A transgenic mice by about11,6,13days,respectively, compared with vehicle control. there was statistical significance.1.2LifespanTreatment with DL-NBP at dose of60,120and180mg/kg significantlyprolonged lifespan of SOD1-G93A transgenic mice by about6,2,12days,respectively, compared with vehicle control. However,180mg/kg treatmentgroup showed significantly extended the lifespan of SOD1-G93A transgenicmice.2Evaluation of motor functionPerformance of rotarod was significantly improved from13weeks to16 weeks at the dose of180mg/kg treatment group. there was statisticalsignificance. Although60mg/kg and120mg/kg treatment group could slightlyimprove performance of rotarod compared with vehicle control group, therewas no statistical significance (P>0.05).3ImmunohistochemistryThe result of immunohistochemistry for SMI-32indicated that the totalnumber of SMI-32positive in lumbar anterior horn were increased in180mg/kg treatment group, compared with vehicle group. there was remarkablestatistical significance (P=0.000), and ventron horn motor neurons were large,dark, and the processes were longer.The result of immunohistochemistry for SMI-31indicated that thenumber of SMI-31positive spina cord ventron horn motor neuron wasdecreased in180mg/kg treatment group, and a weak staining and inclusionbody for SMI-31was observed in perikarya of motor neurons, compared withvehicle group. there was remarkable statistical significance (P=0.000).4Immunoblotting analysisThe result indicates that the level of SMI-31also decreased at DL-NBPtreatment group compared with vehicle control group, there was statisticalsignificance (P=0.006).Conclusion: DL-NBP delays disease onset and extends the lifespan ofSOD1-G93A transgenic mice, improves motor performance, and restraintsthe abnormal accumulation of p-NF within perikarya of SOD1-G93Atransgenic mice.Part Ⅱ Behavioural effects of Dl-3-n-Butylphthalide when treatmentstarted at late-symptomatic stage in a transgenic mouse model of familialamyotrophic lateral sclerosisObjective: To examine the neuroprotective effect of DL-NBP inextending lifespan and improving motor function in male SOD1-G93A mice.Method:1Treatment groups and drug administrationTwenty male SOD1-G93A transgenic mice were randomly divided into the following two groups (n=10,male).(1) DL-NBP treatment group, orallygavage administration with180/kg DL-NBP from90days;(2) Vehicle controlgroup, orally gavage administration with corn oil from90days.DL-NBP (purity>98%, was synthesized by the ShiJiaZhuangPharmaceutical Group NBP Pharmaceutical co.,Ltd) was dissolved in corn oilat three concentrations18mg/ml, and fresh corn oil were prepared daily.Drugs or vehicle(corn oil) were orally gavage administered to the mice in avolume of10ml/kg body weight once per day.2Evaluation of motor functionBeginning at13weeks of age. After an adaptation period of5days ofdaily testing. the rotarod analysis was performed weekly. The rotation startedat1rpm.Within180s the rotation speed increased up to30rpm. Each animalwas given three trials and the longest latency without falling was recorded.3Determination of Survival End Points of MiceWe used a behavioural score system based on the score developed byVercelli et al from1to5defined as follows: Onset of symptoms was definedas score is4. Survival end points of mice was defined as score is1.4Statistical AnalysisWe used SPSS version13.0for Windows (SPSS) for all statisticalanalyses. The data are depicted as mean values and standard errors of themean (SEM). Kaplane-Meier survival analysis was used for survivalcomparisons, and repeated measures ANOVA test was used for assessment ofrotarod performance comparisons. P<0.05was considered as statisticallysignificant.Result:1lifespanLifespan of DL-NBP treatment group was prolonged by8days,compared with vehicle group, there was statistical difference(P=0.046).2Evaluation of motor function (rotarod test)From13weeks to18weeks, DL-NBP treatment group could slightlyimproved motor performance compared with vehicle group, although there was no statistical difference (P>0.05).Conclusion: Treament with DL-NBP from late-symptomatic stagesignificantly delayed increased the lifespan of male SOD1-G93A mice.