Expression Of ET-1 And Notch1 Signaling Related Proteins In The Spinal Cord Of Amyostrophic Lateral Sclerosis Transgenic Mice

Objective:Amyotrophic lateral sclerosis(ALS)is a neurodegenerative disorder characterized by the death of motor cortex,brain stem,and spinal cord motoneurons.The main clinical symptoms include progressive muscle weakness and atrophy,motor deficit and respiratory failure.The average life expectancy is 3-5 years.Based on clincial statistics,ALS morbidity is about 0.15-0.25 /million.5%-10% of all ALS is familial and the remaining 90% is sporadic.The possible pathogenesis include immflammation,oxidative stress,protein aggregates,mitochondrial dysfunction,defective axonal transportion,excitotoxicity and deficiency of neurotrophic factors,however any s ingle hypothesis can not fully explain the cause of ALS pathology.Notch signal pathway plays an active role in cell fate decis ions,migration,growth,symaptic plasticity and neuronal survival.It contains four Notch receptors(Notch1-4),two types of ligands(Jagged1-2,Delta-like1,3,4)and its target genes(Hairy famility and Hes familiy).Mature oligodendrocytes play important roles in the central nervous system and many diseases.It generates compact multilayered myelin sheaths around axons and provides neurons with energy support which is essential to maintain their functional intergrity.Studies indicate that in active MS les iions lacking remyelination,Endothelin-1(ET-1)is highly expressed by reactive astrocytes,which activates Notch1 in oligodendrocytes and suppresses oligodendrocytic differentiation and remyelination.Since similar pathologic changes of oligodendrocytes including degeneration and dysmaturity exist in ALS,wether similar ET-1 and Notch1 signaling changes contribute to ALS pathogenesis is rarely reported.Therefore,this paper mainly studies the oligodendrocytic changes,and expression of ET-1,Notch1 intra-cellular domain(NICD),Jagged1,Delta-like 4(DLL4)and Hes-1 protein and their cellular localization in lumbar spinal cord of transgenic mouse model of amyotrophic lateral sclerosis,and disscusses the possible role of ET-1 and Notch1 signaling in ALS pathogenes is.The results of this study will helps to explore new possible therapeutic targets in ALS.Methods: 1 Transgenic miceAll mice were fed at constant temperature,constant temperature and humidity in sterile environment,kept with sterilized SPF rodent particles and water.Animal experiment process was in accordance with the regulations of laboratory animal managent of Hebei Province.Transgenic SOD1-G93 A mice are bred from male hemizygous carriers(B6SJL-Tg(SOD1-G93A)1Gur /J)and female B6SJL/F1,both of which were obtained from Jackson Laboratory(Bar Harbor,ME,USA).Tail tissues(2 mm)were taken from the offsprings at 30 days of age and DNA was extracted for identification of the mutant SOD1 gene using PCR.2 Motor function scoring and grouping 2.1 Motor function scoringFrom 60 days of age,mice were weighed and tested for rotarod experiment,2 times / week,and evaluated for motor function scoring.Motor deficit were evaluated following 4 point scoring system.1)4 points if normal(no sign of motor dysfunction,no loss of body weight)2)3 points if hind limb tremors are evident when suspended by the tail3)2 points if gait abnormalities are present4)1 point for dragging of at least one hind limb5)0 point for inability to right itself within 30 seconds when the mice are placed on either side or its back 2.2 Experimental groupsAccording to the clinical characteristics of SOD1-G93 A mice,5 groups are included: 30-day group,60-day group,90-day group,clinical onset group,and end stage group.Non-transgenic littermates are used for control.Each group has 6 mice.Disease onset was determined when the following criteria were both met: a,two continuous weight losses were observed after the animals reached their peak body weight.b,earliest gait abnormalities were shown.End stage was defined when the animals can no longer right themselves 30 s after being placed on their backs or side,scoring 0 points.3 Experimental methods 3.1 SamplingEach group of mice was collected for tissues at according time points.After intraperitoneal injection of 10% chloral hydrate(350mg/Kg),mice were intracardially perfused with PBS and following with 4% paraformaldehyde.Lumbar spinal cord were dissected and preserved in fixative at 4?.3.2 Immunohistochemical stainingFixed lumbar spinal cord was cryoprotected in 30% sucrose at 4? overnight,imbedded in OCT compound,and fast freezed in liquid nitrogen.The spinal cord was cut into 25?m sections using LEICA CM1850.The sections were rinsed in 0.01 M PBS for three times,then treated with 1% H202 in 0.01 M PBS for 10 minites to inactivate endogenous peroxidase.After washing in 0.01 M PBS for three times,10 min for each;the sections were blocked in 5%normal serum,0.3%Triton X-100 in PBS for 1h at room temperature.The sections were then incubated overnight at 4? with primary antibodies.After risning 3 times in TPBS,and subsequently incubatio n in biotin-conjugated secondary antibody in PBS for 1h at room temperature,the sections were washed three times in TPBS,followed by incubation in horseradish peroxidase conjugated streptavidin for 40 min at room temperature.After rins ing,DAB reagent was used for visualization.The sections were then spread onto the slides,dehydrated in ethanol,cleared in xylene,sealed with permount and analyzed by Olympus BX51.3.3 ImmunofluorescenceSimilar with immunohistochemical staining,25?m lumbar spinal cord sections were rinsed for 2 times,each for 5 min,perforated with 1%Triton X-100(in 0.01 M PBS solution)for 30 min at room temperature.After rinsing for 2 minutes,the sections were then blocked in 5% normal serum,0.3% Triton X-100,2% milk(0.01 M PBS)and incubated in double primary antibodies,4? overnight.The sections were rinsed for 3 times,each for 10 min,and incubated in the appropriate fluorophore-conjugated secondary antibodies for 2 hours at room temperature,kept dark.After rins ing,the slices were mounted with mounting medium containing DAPI.Olympus FV1000 was used for image observation.Results:1 There is degeneration of oligodendrocytes and oligodendrocyte porgenitors increase in the lumbar spinal cord of ALS mice.With the development of disease,APC positive oligodendrocytes red uced and NG2 positive oligodendrocyte progenitors increased in the lumbar anterior horn,which is most obvious in the end stage of ALS mice.2 With the progression of the disease,ET-1 positive cells increase in the lumbar spinal cord of ALS mice and reactive astrocytes express ET-1.3 With the progression of the disease,NICD positive neurons decrease,while NICD positive oligodendrocyte porgenitors increase and NICD is frequently found in the nucleus,especially at the end stage of ALS mice.4 With the progression of the disease,Jagged1 positive cells increase in the lumbar spinal cord and Jagged1 protein is expressed in activated microglia.5 With the progression of the disease,DLL4 positive neurons decrease,while DLL4 positive astrocytes increase in t he lumbar spinal cord of ALS mice.6 Hes-1 is expressed mainly in the nucleus and Hes-1 positive cells increase with disease progression.Conclusions:1 Degeneration of oligodendrocytes and increase of oligodendrocyte progenitors exist in the lumbar spinal cord of SOD1-G93 A transgenic mice.2 Reactive astrocytes express ET-1 protein in the lumbar spinal cord of SOD1-G93 A transgenic mice.3 Notch1 activation and increased expression of its ligands and target proteins exist in the lumbar spinal cord of SOD1-G93 A transgenic mice.