The Characteristic Of Neurons In Lateral Horn Of Spinal Cord Of Family Amyotrophic Lateral Sclerosis Animal Model

Objective: Amyotrophic lateral sclerosis(ALS) is a severe neurodegener-ative disease with unknown etiology, affecting motor cortex neurons,brainstem motor nuclei neurons, and spinal anterior horn a-motorneuron.mostly result in The main characteristic is progressive loss of upperand lower motor neurons. Recent years, studies have found partly ALS patienthave the symptom of autonomic nervous system, extrapyramidalnon-movement structure and sensory systems which beyond motor neuronarea, suggesting that ALS may be a multi-system disease. Murata Y havefound that a smaller change in nocturnal blood pressure (BP) and loss ofcorrelation between BP and heart rate (HR) in ALS, which may resulting incirculatory collapse, cardiac arrest or even sudden death[1]. otherDysautonomias in ALS, such as increased or reduced sweating[2],reducedsaliva flow rate[3],delayed gastric emptying[4]et al.Takahashi H and Kennedy PG have repotrted that loss of neurons inintermediolateral nucleus (IML)[5-6]and Itoh T have found increasedimmunoreactivity of sympathetic neurons in the intermediolateral nucleus(IML)[7], and Kihira T intracytoplasmic inclusions in arophic neurons[8], suchas Bunina bodies in Onuf's nucleus. A patient with ALS and an SOD1(V118L)mutation who has developed autonomic failure exhibited marked neuronal lossin autonomic nuclei in the medulla and in the IML column[9]. We also foundrest heart rate increased in the ALS mouse model of pre-symtomaticstage[10],which imply that autonomic functin have been injured in the earlystage.The preganglionic neurons of the sympathetic nervous system lies at T1to L2or L3, The grey matter in that area of the spinal cord institute theintermediolateral column which have an important role in autonomic function. This experiment aims to observe the expression and disturbution featureof acetylcholinesterase in lateral horn of the spinal cord at different stage ofALS,to better discuss the feature of lateral horn of spinal cord in ALS theand thus provide a theoretical basis for understanding the mechanisms of thediease.Methods:1Animal modelBreeding familial amyotrophic lateral sclerosis transgenic mice animalmodels (SOD1^G93A) Reference Vercelli A description,4score as diease onsetstage,1score as end stage. SOD1^G93Amice of60days as pre-symptomaticstage Control groups for the experiment were non-transgenic mice in the samenest and have same duration with experimental one,3mice in each groups.2MaterialAfter10%chloral hydrate350mg/kg intraperitoneal injection ofanesthesia, animal tissues were fixated via heart perfusion by4%paraformaldehyde for20min, the thoracic spinal cord of the mice were cut intothe upper level (T1-T3), middle level (T4-T6), lower level(T7-T9), all theblocks were fixated in4%paraformaldehyde for48hour.3Toluidine blue stainingThen they were dehydrated by gradient ethanol, transparentized byxylene, embedded by paraffin and made into20serial conventionalhistological sections (5μm thick).5μm thick paraffin sections were stainedwith toluidine blue (Nissl staining) to display the structure and morphologyfeacture of the intermediolateral column neurons. According to a previouslydescribed method referred by Oppenheimer[12].4Immunohistochemical stainingAfter10%chloral hydrate350mg/kg intraperitoneal injection ofanesthesia, animal tissues were fixated via heart perfusion by4%paraformaldehyde for20min, the thoracic spinal cord of the mice were cut intothe upper level (T1-T3), middle level (T4-T6), lower level(T7-T9), all thesegment were fixated in4%paraformaldehyde for12hour and then directly made into25μm thick sections, then they were used to immunohistostain ofAChE.5Statistical analysisThe mean numbers of neurons and standard deviations were calculatedfor sum of two sides in the lateral horn in the upper (T1-T3), middle (T4-T6)and lower (T7-T9) the thoracic spinal cords (Table1-2). Statistical analysiswere performed using one-way ANOVA followed by Student's t-test withSPSS13.0statistical software. Differences were considered significant at P <0.05.Results:1Animal modelB6SJL-BTg (SOD1^G93A)1Gur/J hemizygous male andB6SJLF1/J+/+female1:1hybrid,at a constant temperature, humidity,12hourlight/dark cycles, no special pathogens (the Specific pathogen free,SPF)environment. Sterilization of SPF particles rodent feed and sterile water,cut end of the tail to extract DNA, PCR, agarose gel electrophoresis results areshown (Fig.1): at200-300bp of between the bands (236bp) for mSOD1thePCR products, such mice for SOD1^G93Apositive transgenic mice. This entrywith a non-mSOD1PCR products is non-SOD1^G93A transgenic mice.2Duantitative study of toluidine blue2.1upper level(T1-T3)Cell counts of in upper level of spinal cord of SOD1^G93Atransgenic miceof differernt stage were7.4±3.0,7.0±2.0,3.7±1.5; the control groups were5.3±1.9,4.1±1.2;6.1±1.9. Compared with end stage, both presymptomaticand diease onset stage are apparent different(p<0.05); cell counts decreasedsignificantly(p<0.05) in diease onset stage and end stage compared withcontrol group. The neurons in the pre-symptomatic stage and disease onsetstage are nomal, but neurons in the end stage were found degeneration.2.2middle level(T4-T6)Cell counts of in middle level of spinal cord of SOD1^G93Atransgenic miceof differernt stage were4.5±1.6,4.3±1.5,2.5±1.2, the overall mean have no difference (P <0.05); the control groups were4.1±2.0,4.5±2.0,5.5±1.6.Compared with the control group, the cell counts decreasedsignificantly(p<0.05) in end stage. The neurons in the pre-symptomatic stageand disease onset stage are nomal, but neurons in the end stage were founddegeneration.2.3lower level(T7-T9)Cell counts of in lower level of spinal cord of SOD1^G93Atransgenic miceof differernt stage were4.4±1.6,5.0±1.8,2.4±0.8; the control groups were5.0±2.0,4.4±1.2,5.5±1.6. Compared with end stage, both presymptomaticand diease onset stage are apparent different (p<0.05); Compared with thecontrol group, the cell counts decreased significantly (p<0.05) in end stage.The neurons in the pre-symptomatic stage and disease onset stage are nomal,but neurons in the end stage were found degeneration.3Cell counts of AchE-positive neuron3.1Cell counts of AchE-positive neuron in upper level of spinal cord ofSOD1^G93Atransgenic mice of differernt stage were6.5±1.2,6.0±2.0,3.7±1.2;the control groups were5.8±1.9,7.8±1.5,5.0±1.0; compared withend stage, both presymptomatic and diease onset stage are apparent different(p<0.05); Compared with the control group, the cell counts decreasedsignificantly(p<0.05) in diease onset stage. The AchE-positive neurons ofpre-symptomatic stage and diease onset stage SOD1^G93Atransgenic mice haveintegrity of the structure, with nucleus colorless and dark brown cytoplasm,while of the end stage, the neurons deformation, pale brown cytoplasm andNissl bidy is not clear.3.2middle level(T4-T6)Cell counts of in middle level of spinal cord of SOD1^G93Atransgenic miceof differernt stage were6.6±2.6,4.9±2.0,3.0±1.4; compared with end stage,both presymptomatic and diease onset stage are apparent different (p<0.05);the control groups were4.8±1.0,5.5±0.6,5.8±1.0. Compared with thecontrol group, cell counts decreased significantly(p<0.05) in end stage. TheAchE-positive neurons of pre-symptomatic stage and diease onset stage SOD1^G93Atransgenic mice have integrity of the structure, with nucleuscolorless and dark brown cytoplasm, while of the end stage, the neuronsdeformation, pale brown cytoplasm and Nissl bidy is not clear.3.3lower level(T7-T9)Cell counts of AchE-positive neuron in the lower level of spinal cord ofSOD1^G93Atransgenic mice of differernt stage were6.7±1.8,6.3±2.6,3.0±1.0,the overall mean have no difference (P <0.05); the control groupswere5.3±1.2,5.3±1.5,5.0±0.0. Compared with the control group, cellcounts decreased significantly(p<0.05) in end stage.The AchE-positiveneurons of pre-symptomatic stage and diease onset stage SOD1^G93Atransgenicmice have integrity of the structure, with nucleus colorless and dark browncytoplasm, while of the end stage, the neurons deformation, pale browncytoplasm and Nissl bidy is not clear.(Fig.6)Conclusion: The degeneration and loss of the intermediaolateral nucleusof spinal cord exisit in ALS, specially in the end stage of the disease, inadditon, it have no apparent difference between upper, middle and lower levelof the thoracic spinal cord.