| Objective: Amyotrophic lateral sclerosis (ALS) is a progressiveneurodegenerative disorder which selectively affects upper and lower motorneurons. ALS is characterized by a combination of upper and lower neurondegeneration except the sensory system and autonomic nervous system. Thepathogenesis is not clear.5-10%of ALS cases are familial (fALS), themajority of cases (90-95%) are sporadic (sALS). The clinical manifestation ofsporadic and familial ALS are very similar. Approximately20%of fALS and4%of sALS are linked to mutations in the gene encoding for the ubiquitouscopper/zinc superoxide dismutase (SOD1). Among this the G93A (glycine toalanine substitution at the93rd codon) site is the most common mutant.SOD1-G93A transgenic mice display progressive motor neuron degenerationand similar to the cases of human ALS. So the mutant SOD1-G93A mice arethe most common animal model to investigate ALS in the world.The diagnosis is based on clinical aspects, electrophysiological andneuropathological changes associated with clinically proven impairment ofupper motor neuron, with chronic and progressive development. Although Thediagnostic criteria in El Escorial reformulated in1998does not clear thatsensory impairments isn't necessary, almost all clinicians diagnose ALSaccording no sensory impairments when they diagnose ALS. There was reportabout sensory impairments of ALS in1990s last century. Although dominatedby motor dysfunction there is increasing evidence that ALS is a multisystemdisorder in which the autonomic system, spinocerebellar tracts, dorsal columns,basal ganglia and extra-motor cortex may also be affected. These findingshave challenged the current understandings of this disease.In the present study we have investigated the potential alterations oflumbar spinal cord motor and sensory roots in transgenic mice expressing a hSOD1mutant. We tested the mutant SOD1protein, P62, LC3Ⅱ, GCLC andHO-1using western blot aimed at proving sensory impairments andinvestigating the pathogenesis. Our work will provide experimental evidencefor diagnosis and differential diagnosis of ALS.Methods:1Breeding of transgenic miceAll animals were kept at constant temperature and humidity and sterileconditions (specific pathogen free, SPF), fed with sterilized SPF rodents feedparticles. Experiments were carried out according to the regulations oflaboratory animal management of HeBei province. Animals were maintainedwith ad libitum access to water and diet, three or five mice per cage.Transgenic SOD1-G93A mice and their non-transgenic littermates weregenerated by breeding hemizygous male B6SJL-Tg (SOD1-G93A)1Gur/Jmice to female B6SJLF1hybrids, both of which were purchased from theJackson Laboratory (Bar Harbor, ME, USA) and originally produced byGurney et al. The genetic offspring mice were identified at three weeks of ageby genotyping for human SOD1with tissue from tail.2Motor function score and experimental groups2.1Motor function ScoreThe mice were evaluated for signs of motor deficit daily, beginning at50days of age, with the following4point scoring system.(1).4point, no sign of motor dysfunction, no weight loss.(2).3point, Hind limb tremors are evident when suspended by the tail.(3).2point, Gait abnormalities are present.(4).1point, Dragging of at least one hind limb.(5).0point, Inability to right itself within30s when placed on either side orits back.2.2Experimental groupsMice were divided into three groups including pre-symptomatic (60days),clinical onset (about100-120days), and end stage (about130-150days),based on the motor function score of SOD1-G93A mice. SOD1-G93A transgenic mice were model groups, non-transgenic littermate mice werecontrol groups. Each group had6mice at each time point. Pre-symptomaticgroup was60-day-old mice, without clinical symptoms and no weight loss.Clinical onset was defined when the mice began to lose weight, and show gaitabnormalities. When the mice lost>20%of their body weight, and could notupright itself in30seconds after being placed on either side or its back, it wasscored as end stage.3Methods：3.1SamplingAfter anesthesia with10%chloral hydrate, lumbar spinal cord werepreserved in the fixative after transcardially perfusion with phosphate-bufferedsaline (PBS), followed by4%paraformaldehyde in PBS, ventral root (VR)and dorsal root (DR) were freshly dissected, put into1.5ml centrifuge tubes,froze in liquid nitrogen, and then stored in-80℃refrigerator.3.2Hematoxylin-eosin stainVR and DR tissues fixed in4%paraformaldehyde wereparaffin-embedded and cut into5μm sections. The sections were thendeparaffinized, hydrated and impregnated in hematoxylin solution for5minutes. The sections were thoroughly washed in water for1-3seconds, thentreated in1%hydrogen ethanol for1-3seconds, followed by water rinsingagain. The sections were then impregnated in0.5%eosin Y solution for1-3minutes. Sections were then watered, dehydrated and mounted.3.3Toluidine blue stainVentral root (VR) and dorsal root (DR) of the lumbar enlargements werefixed in4%glutaraldehyde for24h and then in1%osmium tetroxide for1h.Tissues were dehydrated through graded acetones and embedded in epoxyresin Epon812. Semithin sections (1μm) were cut, placed on the slides, andoven dried. The slides were stained with1%toluidine blue solution for10min,rinsed with water, dehydrated and mounted.3.4Naumenko-Feigin silver stainVR and DR tissues fixed in4%paraformaldehyde were paraffin-embedded and cut into12μm sections. The sections were thendeparaffinized, hydrated and impregnated in0.14%silver nitrate at56-60℃for24h. After further treatment in2%sodium sulfate and1%hydroquinonefor6min, the sections were thoroughly washed in water for15min and thentoned with1%gold chloride for5min. After a brief rinse with water, thesections were treated with freshly prepared2%oxalic acid for10min,followed by water rinsing again. The sections were then incubated in5%sodium thiosulfate for3min and after rinsing with water placed in0.1%LuxolFast Blue solution at56-60℃overnight. The sections were rinsed with95%ethanol to remove excess stain and further treated alternating between0.5%lithium carbonate and70%ethanol until individual fibers were distinguishable.Sections were then dehydrated and mounted.3.5Western blottingThe DR and VR were prepared using a total protein extraction kitfollowing the manufacturer's instruction and quantified using the BCA method.Thirty micrograms of protein from each sample was run on10%SDS-PAGEgels, and blotted onto PVDF membranes. After blocking in5%skim milk for1hour, primary antibodies including goat anti-SOD1(1:500), rabbitanti-ACTIN (1:500), rabbit anti-P62(1:1500), rabbit anti-LC3Ⅱ(1:1500),rabbit anti-GCLC(1:500) or rabbit anti-HO-1(1:500) was added respectivelyand the membranes were incubated overnight at4℃. Next day, the membraneswere rinsed with TPBS for3times,5minutes every time. Then anti-rabbit oranti-goat fluorescence-labeled secondary antibody (1:5000) was added. Afterincubation for1h at room temperature, the membranes were rinsed for3timeswith TPBS, and1time with PBS,5minutes every time. The bands of interestwere detected using an Odyssey Infrared Imaging System (LI-COR, Lincoln,NE). Band intensity was quantified, normalized by the GAPDH band, andstatistically analyzed.3.6Statistical analysisResults were expressed as mean±S.D. Statistical analyses were performedusing one-way ANOVA with SPSS13.0.4statistical software. Differences were considered significant at P <0.05.Results:1Identification of transgenic miceThe PCR amplification products of genome DNA from offspring micedemonstrates: a324bp band, which is the PCR product of IL-2, and a236bpband, which is the PCR product of hSOD1gene.2Clinical manifestationsSOD1-G93A transgenic mice did not show any clinical changes at60days of age and scored4. At about80-90days of age, the transgenic miceshowed tremor of their hind limbs when suspended by tail and scored3. Atabout100-120days of age, one or two hind limbs of SOD1-G93A mice beganto weak and showed muscle atrophy, and gait abnormality was observed. Atthe beginning of gait abnormality, the mice were scored2. Then, muscleweakness progressed and hind limbs couldn't support the body. Subsequently,at least one hind limb paralyzed and was dragged when walking. At this time,the mice were scored1. At about130-150days of age, the SOD1-G93A micecan't upright itself within30sec and lost weight>20%. The mice were scored0and considered as end stage. The above manifestations were not observedwith non-transgenic littermates that were scored4all the time.3The morphological changes of nerve root in lumbar spinal cordWhen stained by hematoxylin-eosin, we observed that nerve fibers of VRand DR of transgenic mice were irregular in shape, and cellular componentsincreased. In the semithin sections stained by toluidine blue, axonaldegeneration could be observed in the transverse sections of both VR and DRat the end stage. The density of myelinated nerve fibers was greatly reduced.Displayed by the silver staining, the axons in the VR and DR of SOD1-G93Amice were swollen and disintegrated.4The expression of target proteins4.1Compared with non-transgenic littermates, the expression of SOD1in theVR and DR of SOD1-G93A mice increased at60days (P﹤0.05), and itsexpression increased dramatically at onset stage, especially at end stage (P﹤ 0.05).The expression of SOD1in the VR of SOD1-G93A mice is more thanthat in the DR at every stage (p﹤0.05).4.2LC3Ⅱa nd P62expression in the VR and DR of SOD1-G93A transgenicmice and non-transgenic littermates. Compared with non-transgeniclittermates, the expression of LC3Ⅱ i n the VR and DR of SOD1-G93A miceincreased at pre-symptom, onset stage and end stage (p＜0.05). It is obviousthat the expression of P62in the VR is more than that in the DR at the threestages (p＜0.05). Compared with non-transgenic littermates, the expression ofP62in the VR of SOD1-G93A mice increased at presymptomatic stage, and itsexpression increased dramatically at onset and end stages (P＜0.05). Theexpression of P62in the VR is more than that in the DR at the three stages (p＜0.05).The expression of P62in DR of SOD1-G93A mice began to increaseat onset stage and increased dramatically at the end stage (P＜0.05).4.3The expression of GCLC and HO-1in VR and DR of SOD1-G93A miceincreased at different disease stages compared with their littermates, and theirexpression in VR is more than in DR (P﹤0.05).Conclusions:There were obvious nerve fiber degeneration in both ventral root anddorsal root of lumbar spinal cord in SOD1-G93A transgenic mice, and thedegeneration in the former was more severe than in the later. The expressionof mutant SOD1protein in DR and VR of lumbar spinal cord in G93Atransgenic mice increased with the progress of the disease. At every stage, theexpression of SOD1in VR was more than that in DR. There was the samechange about autophagy marker LC3Ⅱ and oxidative stress marker GCLC,HO-1. The expression of autophagic substrate P62in VR began to increase atthe presymptomatic stage and its expression increased dramatically at onsetand end stages. The expression of P62in DR of SOD1-G93A mice began toincrease dramatically at the end stage (P＜0.05). Our results support there aresensory injuries in SOD-G93A mice, which are related to the aggregates ofmutant SOD1protein, oxidative stress and autophagic changes. |
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