| PrefaceSpinal cord injury can cause primary damage, a series of secondary cascade damages and a series of protective plerosis to avoid spinal cord injury and promote rehabilitation of it. Now researchers have found that NO/NOS plays a role in spinal injury and spinal protection after spinal cord injury. The damage on spinal cord due to NO/NOS is mainly through neurobiochemical mechanism, whereas reduction in spinal cord injury and promotion of its rehabilitation are mainly through the mechanism of blood vessels regulation consequently. Researchers also found that in the two subtypes of cNOS , nNOS plays an important role in aggravating SCI through neurobiochemical mechanism , whereas eNOS plays an important role in alleviating SCI and speeding its rehabilitation through blood vessel regulation . Further researches are still required to reveal the chang between the two subtypes of cNOS after acute spinal cord injury ( ASCI ) . Our study aims to investigate the change between the two subtype of cNOS after acute spinal cord injury and the effect of L - Arg injected into abdominal cavity on it . Consequently, it establish an basement for the treatment of SCI by restraining the injury mechanism and promoting protective role of NO/NOS after spinal cord injury.Materials1. Experiment animals and its groupFifty Healthy pure Wister rats (female or male) , with weight from 250 to 300 g , are grouped into four groups randomly .Group A: Normal control (five rats)Group B: false surgical group, laminectomy merely . 30min, Ih and 2h postoperatively. Five rats each timepoint .Group C : SCI group , underwent spinal cord injury . 30min, 1 h and 2 h after injury . five rats each timepoint .Group D ; SCI and L - Arg injection group , underwent SCI and were injected L - Arg into abdominal cavity . 30min, 1 h and 2 h after the treatment, five rats each timepoint.2. Main reagent1) Rabbit anti - rat nNOS monoclonal antibody and eNOS monoclonal antibody.2) SABC Kit and DAB Kit3 ) Parenteral solution L - Arg 5g/20ml3. Experiment instruments1) Modified Aliens beater (self made)2)Paraffin - microtome; Olympus B+ 60 light microscope; + 10 operative microscope3) Olympus /Dp10 Digital microcamera4) Olympus C -W95 software; Meta -Morph automatic true color image analyzerExperiment method1.Establishment of animal modeWe made rat spinal injury model in accordance with modified Allen ?beating method. The injured segment was T10. The beating force was 80g. cm. The Omni - laminectomy was performed in T10 segment in false operative group and the spinal cord wasn' t injured.2.Collection of specimen; Fabrication of paraffin section and stain of histochemistry and HEThe specimen of spinal cord was taken out of directly with X 10 operative microscope after laminectomy in control group.The false operative group; The specimen of spinal cord was taken out of at 30 min, 1h 2h after simple laminectomy.SCI group; The specimen of spinal cord was taken out of at 30minlh, 2h after SCI.SCI + L - Arg group: The specimen was taken out of at 30min, 1h, 2h.These specimens were made into paraffin section and stained with HE and immunochemical method ( nNOS and eNOS) , using SABC method and DAB stain.3.Results and statistical analysis1. Observe the degeneration and necrosis of spinal tissue in HE staining of every group under microscope..2. Observe the deposit and distribution of positive reaction products of eNOS and nNOS under microscope3. Image analyzing : 5 pieces of immunohistochemical stained sections were taken from each group. The five images were collectedfrom anterior horn to posterior horn in each section under microscope and analyzed. The AG ( average gray value) and OD ( optical density ) of the nNOS and eNOS are measured. All the data were marked in (x+s) style. SPSS 10.0 was used for statistical analysis.Result1.HE and immunohistochemical staining1. Control group and false operation groupThe form and structure of s... |
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