| Spinal cord injury (SCI) is the spinal cord function obstacle below level ofpost-injury, which due to various causes of spinal cord structure and function damage.The function obstacle including motor, sensory, sphincter function and so on. It isreported that China has had a ten-fold increase in SCIs caused by car crashes,construction and mining accidents in the past decade. In addition to the debilitatingeffects and permanently reduced quality of life, an enormous social, financial andemotional cost for the victims, their relations and the government is brought about bySCI.Although the great progress in treatment of spinal cord injury has been maderecently, there are few effect drugs to treat patient who suffered SCI because of itspathogenesis is not entirely clear. Therefore, There is an urgent need to focus on theproblem of further understand the mechanisms related to the development of diseaseabout spinal cord injury and to seek better and more efficient treatment to improve thequality of life of the patients.There are a lot of mechanism about motor function deficits after spinal cordinjury and also made a lot of progress. In recent years, the study found thatpost-traumatic systemic stress response is significantly, and autophagy is a cellbiological process that is a central component of the integrated stress response, whichplay a pivotal role in maintaining homeostasis in the body. Autophagy is anintracellular degradation system that delivers protein and organelle to the lysosome,which plays a wide variety of physiological and pathophysiological roles. Autophgyare involved in the development of many diseases, but too much or too littleautophagy is linked to pathophysiological roles. Recently studies confirmed thatautophagy is activated in traumatic spinal cord injury, and which promoted secondarycell death, affected functional recovery. Therefore, the characteristics of autophagicexpression after SCI need to be investigated, and we hypothesized that secondaryinjury will be controlled and function outcome will be improved if we can interruptcell autophagy in some extent after SCI, which will represent a novel therapeutic strategy for SCI.For the treatment of autophagy, we need to find a drug of inhibiting autophagy,and at the same time does not cause damage to the body. Recently, In manyexperimental animals models previously described, HDAC inhibitors(HDACi)exhibit neuroprotective effects for various neurological conditions. Valproic acid(VPA) is a histone deacetylases (HDAC) inhibitor, and plays a key role in manyaspects of fundamental cellular activities such as gene transcription, study haveshown that autophagy can be induced by VPA in glioma cell lines, which shows thatVPA can influce the level of autophagy. Therefore, the level of autophagy in spinalcord tissue may be influced and the motor function may be improved by VPA afterSCI.In this study involved in both animal and cell research. The study includes thefollowing three sections:1. To investigate the characteristics of autophagic expression after spinal cord injury.2. To investigate the change of autophagic expression and the neuroprotective effectof treatment of VPA in acute SCI.3. To establish the cell injury models by H2O2and overexpress the Beclin-1gene,and to observe the role of autophagy in the treatment of VPA in actue spinal cordinjury.Section one The characteristics of autophagic expressionin spina cord tissue after spinal cord injury.ObjectiveTo establish the spinal core injury contusion and observe the expression of bothprotein and mRNA levels of Beclin-1and LC3at at different time points after spinalcord injury, make clear the characteristics of autophagic expression after spinal cordinjury. So as to reveal the possible mechanism of secondary spinal cord injuryresearch and lay the research foundation for further study. Research methods1AnimalsAdult femaleWistar rats,220~250g, were purchased from the Animal Center ofShanxi Medical University, P. R. China.2Spinal cord injury modelAnimals were anesthetized with10%chloral hydrate (0.3ml/100g, I.P.), SCI wasperformed using a modified New York University Impactor. In brief, The T10cordsegmental level was exposed by performing laminectomy. In the present study,contusion injuries were induced by dropping a10g weight rod (tip diameter:2.5mm)from a height of25mm onto the exposed spinal cord. The sham-operated animalsreceived only a laminectomy to expose the spinal cord, but were not subjected to animpact injury. The contact surface of the impactor is round and slightly chamfered atthe edge to prevent tearing the dura. The diameter is chosen to be slightly less thanthat of the cord to clear the edges of the vertebral canal as the impactor compressesthe cord. Manual bladder expression was performed on each animal twice dailybefore the recovery of sphincter control.3. Spinal cord tissue dissected out for protein and RNA extractionAt different time points (1h,2h,6h,24h,48h,72h) after SCI and immediatelyafter the sham operation, the animals were anesthetized and the spinal cord tissuecontaining the injury epicenter were dissected out for protein and total RNAextraction.4. The determination of LC3and Beclin-1expression by Western blot analysisIn brief, after the spinal cord tissues were homogenized, centrifuged, thesupernatants were collected and protein concentrations were determined by the BCAmethod. Equal amounts of protein (LC3:40μg protein/lane, Beclin-1:40μg protein/lane) were electrophoresed on the15%SDS-polyacrylamide gel and thenelectrophoretically transferred to a polyvinylidene difuoride membrane. Afterblocking with blocking solution, the membrane was incubated with monoclonalantibody against either LC3or Beclin-1. Following incubation with secondaryantibody, the blot was developed with an ECL chemiluminescent detection kit and visualized with a Kodak Image Station400. The density of the scanned protein bandswere measured by ImageJ1.44p software and the results were presented as apercentage change of the loading control.5. The determination of LC3and Beclin-1expression by Real-time PCRmRNA expressions of Beclin-1and LC3were detected by Real-time PCR withSYBR Green (Sigma) detection in the Mx3005Real-time PCR system. Total RNAwas extracted from the spinal tissue at different time points and was reverselytranscribed into cDNA in right conditions, and then the cDNA was amplified in theMx3005P Real-time PCR instrument. The results, expressed as the fold difference inthe number of LC3or Beclin-1copies relative to the number of GAPDH gene copies,were determined by the relative quantitative2-ΔΔCtmethod. ΔΔCt=ΔCt (target gene)-ΔCt (GAPDH) and ΔCt (target gene)=Ct (experimental-target)-Ct (control-target)andΔCt (GAPDH)=Ct (experimental-GAPDH)-Ct (control-GAPDH).6. Immunofluorescence stainingFrozen section at the epicenter collected at2h following the injury were washedin0.1M PBS (PH7.35) containing0.3%Triton X-100for10min, and blocked with3%milk and5%FBS in0.1M PBS for1h at room temperature, by incubation with arabbit anti-LC3antibody (1:400; Cell Signaling) and mouse anti-Beclin-1(1:200;Santa Cruz Biotechnology) overnight at4°C. After rinsing with PBS, the sectionswere then incubated with anti-rabbit or goat anti-mouse fluorescently conjugatedsecondary antibodies for40min at room temperature.2-(4-Amidino-phenyl)-6-Indolecarba-midine dihydrochloride (DAPI, Beyotime Biotech, China) was used tostain the cell nucleoli.7. Statistical analysisAll calculations and the statistical analysis were performed using the statisticalsoftware program SPSS15.0. All values are presented as means±SD. Differencesbetween two means were compared by Student’s t test. All of the other values wereanalyzed by ANOVA followed by a Fisher least significant difference (post hoc) testfor multiple comparisons. Probabilities of*P <0.05were considered statisticallysignificant. Results1. The model of spinal cord injury was established with successAll the rats survived the surgery, Physiological parameters, including meanarterial blood pressure (MABP), pH values, body temperatures, blood gasses, andserumglucose were not significantly different between the sham and SCI groups,either at2h before or1day after SCI (Table1). In the present study, contusioninjuries were induced by dropping a10g weight rod (tip diameter:2.5mm) from aheight of25mm onto the exposed spinal cord. It is succeed if the reflection of tail andhind limbs was appeared. The sham-operated animals received only a laminectomy toexpose the spinal cord, but were not subjected to an impact injury.2. The expressions of LC3and Beclin-1were increased in SCI ratsTo investigate changes of autophagy at different time points after SCI, the mRNAexpressions of Beclin-1and LC3were measured by Real-time PCR and the proteinlevels were assessed by Western blot analysis. The level of autophagy wassignificantly increased (*p <0.05) at1h,2h, and6h post injury compared with thesham group. Both protein and mRNA levels of LC3and Beclin-1peaked at2hpost-surgery versus sham group [Fig.1-4]3. Spinal cord injury increased the fluorescence stainingInjury increased the staining of LC3and Beclin-1at2h following spinal cordinjury by fluorescence staining at the epicenter sections.Summary:The expression of autophagy is significantly increased in the spinal cord tissuefollowing spinal cord injury. The expression of both mRNA and protein levels ofBeclin-1and LC3were significantly increased at1h,2h, and6h after SCI andpeaked at2h after SCI by Real-time PCR and Western blot analysis. In addition,Injury increased the staining of LC3and Beclin-1at2h following spinal cord injuryby fluorescence staining at the epicenter sections. Section two Valproic acid reduces autophagy in spinal core tissueand promotes functional recovery after spinal cord injury in ratsObjectiveTo investigate the change of autophagic expression and the neuroprotectiveeffect of treatment of VPA in acute SCI.Research methods1Drug TreatmentRats randomly received VPA (300mg/kg, ip, twice daily) or saline vehicle ofequal volume for two weeks, starting immediately after the injury. A group of shamrats was included as additional control.2The determination of LC3and Beclin-1expression by Western blot and Realtime PCR analysisTo investigate changes of autophagy at2h after SCI, the mRNA expressions ofBeclin-1and LC3bwteen the vehicle groups and VPA treatment groups weremeasured by Real-time PCR and the protein levels were assessed by Western blotanalysis.3Histological Assessments of the neuroprotection of VPA3.1Luxol Fast Blue (LFB) staining for the myelin sheathTo evaluate the extent of spared myelin in white matter, the cross sections werestained using Luxol Fast Blue (LFB) and examined at42d after SCI. In brief, thesections were placed in95%ethanol for5min and stained in0.1%solvent blue38(Sigma) in acidifed95%ethanol at57°C oven overnight. After rinsing excess stainwith95%ethanol and distilled water, sections were then differentiated with0.05%lithium carbonate solution and70%ethanol several times until the gray matter waslargely unstained and white matter sharply defined. Sections were rinsed in distilledwater and then counterstained in cresyl violet solution for30–40sec. Then thesections were differentiated in95%ethanol for1min after rinsing in distilled water，dehydrated in100%ethanol for2×2min, cleared in xylene for2×2min and coveredby using a resinous medium. The images (at×40magnification) were captured usingan Olympus digital camera. LFB-positive area (blue) of spared white matter were analyzed by ImageJ1.44p software program, and the ratio of LFB staining of residualwhite matter to normal white matter was calculated.3.2Nissl’s staining for the motoneuronsNine sections collected at42d after the injury was stained with Nissl’s stainingto inspect ventral horn motoneurons. In brief, the sections were placed into1:1alcohol/chloroform overnight and then rehydrated with95%ethanol to distilled water,stained in0.1%cresyl violet solution for10minutes at37℃. After rinsing withdistilled water, sections were then differentiated with95%ethanol for1min,dehydrated in100%ethanol for2×2min, cleared in xylene for2×2min and coveredby using a resinous medium. The area for counting motoneurons was defined bydrawing a horizontal line from one side of the transverse section to the other passingthrough the central canal and the numbers of ipsilateral ventral horn motoneuronswere calculated by ImageJ1.44p software4Behavioral TestingMotor function of the hind limbs were evaluated by the BBB scalefor6weeksfollowing SCI. The BBB locomotor rating scale is widely used to test behavioralconsequences of spinal cord injury to the rat. The BBB scale was determined by threetrained observers who were unawared of the experimental procedures, and averaged..5Statistical analysisAll calculations and the statistical analysis were performed using the statisticalsoftware program SPSS15.0. All values are presented as means±SD. Differencesbetween two means were compared by Student’s t test. All of the other values wereanalyzed by ANOVA followed by a Fisher least significant difference (post hoc) testfor multiple comparisons. BBB scores was analyzed with a repeated measuresANOVA followed by the Tukey-Kramer test. Probabilities of P<0.05were consideredstatistically significant. Results1. Valproic acid promotes functional recovery and neuroprotective effect afterspinal cord injury in rats1.1VPA promoted significant neuroprotection following SCIVPA reduced myelin sheath damage, as reflected by Luxol fast blue (LFB)staining compared with the vehicle control. The area of total tissue preserved ininjured rats receiving VPA or vehicle was apparently smaller than in the shamcontrol，but VPA increased the LFB-positive spared myelin sheath ratio (30.96±0.58%vs.19.67±0.83%in vehicle control,*P <0.05) at1mm rostral to the epicenterat42d after SCI.(Fig.7)1.2VPA increased the number of motoneurons of the spinal cord tissueAt42d after the injury, there was a trend of increasing motoneurons near theinjury epicenter in VPA-treated rats (relative to vehicle control). VPA significantlyincreased the number of motoneurons at2mm both rostral and caudal to the injuryepicenter. The difference was statistically significant.(*p<0.05vs. vehicle control)(Fig.8)1.3VPA promoted significant functional recovery following SCITo examine the effect of VPA treatment on hind limb recovery following SCI,the BBB score was measured once a week for6weeks. During the behavior testing,the BBB scores in the VPA-treated animals were consistently higher than in thevehicle-treated animals from4weeks to6weeks post injury. At42d, the BBB scoresin the sham control was normal, but it declined apparently in VPA-treated andvehicle-treated groups, however, the BBB scores were significantly higher in theVPA-treated animals compared with the vehicle-treated animals. The difference wasstatistically significant.(*P <0.05, Fig.9).2. VPA reduced the levels of autophagy2.1VPA led to reduction in the levels of Beclin-1Treatment with VPA resulted in a decrease in Beclin-1protein and mRNA levelsat2h following SCI compared with vehicle-treated rats by Western blot andReal-time PCR analysis. Analysis of the band, Beclin-1and LC3II protein levelswere markedly decreased in the VPA-treated rats versus the vehicle-treated rats, thedifference was statistically significant.(*p <0.05,**p <0.01Fig.10,11).2.2VPA led to reduction in the levels of LC3Treatment with VPA resulted in a decrease in LC3II protein and mRNA levels at 2h following SCI compared with vehicle-treated rats by Western blot and Real-timePCR analysis. Analysis of the band, Beclin-1and LC3II protein levels were markedlydecreased in the VPA-treated rats versus the vehicle-treated rats, the difference wasstatistically significant.(*p <0.05,**p <0.01Fig.12,13).2.3Spinal cord injury increased the staining of LC3in neuronThe LC3expression was confirmed in neurons definitely by doubleimmunofluorescent staining, LC3immunofluorescence staining in the epicentersections displayed a punctate pattern around the cell soma at2h after spinal cordinjury. The vehicle-treated rats exhibited increased the staining of LC3comparedwith sham control, but VPA decreased the staining of LC3, as revealed by decreasedamount of punctate LC3dots in confocal microscopy.(Fig.14)Summary:Post-SCI treatment with VPA improved Basso-Beattie-Bresnahan (BBB) scale,increased the number of ventral horn motoneurons and reduced myelin sheathdamage compared with vehicle treated animals at42d following SCI. In addition, theautophagy level was decreased at2h following spinal cord injury by Western blot,Real time PCR and double immunofluorescent staining of LC3and NeuN analysis. Section three The role of autophagy in the process of valproic acidaffect the cell viability and axonal growth of SH-SY5Y cellsObjectiveTo observe the changes in neurons by establishing the damaged SH-SY5Y modelto imitate neurons in the spinal cord tisse after SCI, and to investigate the role ofautophagy in the damaged SH-SY5Y cell treated with VPA after the autophagy levelwas upregulated by overexpression of the Beclin1protein Research methodsCell linesSH-SY5Y cell lines: purchased from the Shanghai Cell Bank, Chinese Academyof Sciences.1. The estabalishment of hydrogen peroxide damaged SH-SYSY cell modelThe SH-SYSY cell was cultured in the96-well plates for24hours, then addeddifferent concentration of H2O2(0,50,200,300,400μmol/L)，after12hours, theculture was termed and the cells were collected, the cell viability of SH-SYSY cellswere determined by the method of CCK8kit.2. The detection of cell viability, axonal growth and the level of autophagy ofSH-SY5Y cells(1) The cell viability of SH-SY5Y cells was determind by CCK8method after thedamaged cells were treated by VPA.(2) The images of the cells were captured by inverted microscope and relativeneurite length was measured using NIH Image1.55software. Cells werecultured in6-well plates, the neurite length was measure of50neurons perwell, and the average value was calculated, the present experiment was repeatedthree times.(3) Both the protein and mRNA level of Beclin1and LC3were detected by Westernblot and Real-time PCR analysis after the damaged cells were treated by VPA.3. Construction and identification of plasmid vectors of the pcDNA3.l(+)-Beclin-1Construction of plasmid vectors of the pcDNA3.l (+)-Beclin-1was performed bythe Biological Science and Technology Co., Ltd. Guangzhou Saiye. The extraction ofrecombinant plasmid was performed on the steps according to the specificationEndoFree Plasmid Mini Kit II. The concentration gradient test of G418.has been doneto determine the optimal concentration for the selection of positive clones. Cellstransfected operations was performed by the lipofectamine2000transfection manualof invivogen Company. The identification of transfected cell was performed by thedetermination the LC3and Beclin1in the mRNA content by real-time PCR and in theprotein level by Western blot. 4. The detection of cell viability, axonal growth and the level of autophagy ofSH-SY5Y cells after the overexpression of the autophagic beclin-1proteinTogether up5. Statistical analysisAll calculations and the statistical analysis were performed using the statisticalsoftware program SPSS15.0. All values are presented as means±SD. Differencesbetween two means were compared by Student’s t test. All of the other values wereanalyzed by ANOVA followed by a Fisher least significant difference (post hoc) testfor multiple comparisons.. Probabilities of P<0.05were considered statisticallysignificant.Results1. The estabalishment of H2O2damaged SH-SYSY cell model byhydrogen peroxide.The results showed that with the increase concentration of H2O2, OD valuesshowed a downward trend. According to the results, we selected a concentrationof400μmol/L L H2O2to estabalish the cell model for the further experiments.（Fig.15）2. The role of VPA on damaged SH-SY5Y cell2.1The cells survival rate increased by treatment with VPACompared with control group (54±9), the cells survival rate was significantlyincreased after the SH-SY5Y cell treated with VPA.(77±7, P <0.05).2.2The neurite length of SH-SY5Y cells were increased by treatment with VPABoth the protein and mRNA level of Beclin1and LC3were detected by Westernblot and Real-time PCR analysis after the damaged cells were treated by VPA.Compared with control group, the level of autophagy was significantly increased inH2O2group, but the level of autophagy was inhibited significantly after the cellstreated with VPA.(Fig18-21)2.3VPA led to reduction in the levels of autophagyCompared with control group (22±9), the neurite length of SH-SY5Y cellswere significantly increased after the SH-SY5Y cell treated with VPA.(49±8, P<0.05). 3. The effect of valproic acid to promote axonal growth and cell survival wasinhibited after the overexpression of Beclin13.1The level of autophagy were determined after the pcDNA3.l(+)-Beclin-1transfected to the SH-SY5Y cellBoth the protein and mRNA level of Beclin1and LC3were detected by Westernblot and Real-time PCR analysis after the pcDNA3.l(+)-Beclin-1transfected to thecells.Compared with control group, the level of autophagy was significantly increasedin pcDNA3.l(+)-Beclin-1group, but the level of autophagy was not changed in thepcDNA3.l(+) group compared with the pcDNA3.l(+)-Beclin-1group.(Fig18-21)3.2The effect of VPA improving the cells survival rate was inhibited after theoverexpression of Beclin1protein.The cells survival rate was determined after the overexpression of Beclin1protein by CCK8Kit. Compared with pcDNA3.l(+) group (72±15), the cellssurvival rate was significantly decreased in the pcDNA3.l(+)-Beclin-1group.（55±11， P <0.05）[Fig27]3.3The effect of VPA improving the neurite length was inhibited after theoverexpression of Beclin1protein.The neurite length was mearsured after the overexpression of Beclin1protein byusing NIH Image1.55software. Compared with pcDNA3.l(+) group (49±12), thecells survival rate was significantly decreased in the pcDNA3.l(+)-Beclin-1group.（527±10P <0.05）[Fig27]SummeryVPA improved the cell viability，axonal growth and decreased the autophagylevel of damaged SH-SY5Y cells. The neuroprotection of VPA was inhibited whenthe autophagy was upregulated. This indicates that cell autophagy may be play a rolein the process of VPA treating spinal cord injury. |
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