| PrefaceThe complement system is a multimolecular system composed by protein in liquid phase or cell membrane. The complement system mediated tissue injury directly or indirectly after it was activated by classical route or shunt replacement route. Czurko had proved by immuno-histological method that Ig G infiltration and increase of C3 immunoac-tivity were present in central ischemia area 3 days after brain ischemia in mice. In 1999 Watanabe observed in mice 3 - 24 hrs after acute spinal cord injury ( ASCI) the microglia with unsaturated C3R decreased apparently with indicate C3R were saturated by iC3b in short term and complement system participate with secondary injury in spinal. As to complement system in injured spinal tissue the distribution condition , change by time and acting mechanism require further research. Finding of the role mechanism of complement system in spinal injury will afford theoretical basis for anticomplement treatment of ASCI.Materials1. Experiment animals and GroupingEighty healthy SD rats weighted from 250 - 300g were afforded by experiment animal department of CMU. They were divided into teams randomly. ( A ) Normal control team, n = 5; ( B ) operation team; There are 5 chronophase point corresponding to 12h,ld,3d,5d,7d after re-section of Lamina of vertebra As for each chronophase point, n =5. ( C) Saline team( NS) :There are 5 chronophase point corresponding to 12h,1d,3d,5d,7d after spinal injury. As for each chronophase point, n =5. 0.5ml NS was injected through caudal vein in 1 hour after the rat spinal was injured. (D)sCRl team:There are 5 chronophase point corresponding to 12h, ld,3d,5d,7d after spinal injury. As for each chronophase point, n = 5. sCR1 was injected through caudal vein in dose of 6mg/kg 1 hour after the rat spinal was injured. 2. Main reagent2.1 rabbit antirat C9,mice antirat CD59 antibody2. 2 recombination soluble receptor I of complement (sCR1)2. 3 SABC kit3. Main instrument3. 1 Modified Allen ?beater (self made)3.2 Cryo - microtome3.3 Olympus BX60 light microscope3.4 Coolsnapfx digital graphic collection card, PIV computer3.5 Meta Morph automatic true color image analyzerMethod1. Establishment of animal modelWe made dorsum injury model of rat spinal in accordance with modified Allen's beating method. The injured segment was T10 . The beating force was 50gcmf. Total laminar resection was performed in T10 segment in false operation team and the spinal was intact.2. Take material , section preparatio and stain of histocyte. Anesthesia was applied to all the rats again 12h ,ld, 3d, 5d and7 days after injury. In the middle of the lesion area we took 1cm spinal tissue, we take the part of injury for serial section . The thickness of slice was 12um. HE staining , Nissl's staining and immunohisto-chemical staining of C9NCD59 were applied to all the sections.3. Observation marker and statistics analyse:3. 1 Observe the degeneration and necrosis condition of spinal tissue in every team under microscope.3. 2 Observe the deposit and distributing condition of positive reaction product of C9 and CD59 which was brown particle under microscope.3. 3 Count the average number of neutrophil in 5 high power field of 6 section taken the part of injury in each team.3.4 Image analyzing; We took 6 pieces of C9 stained section from the part of injury in each team and measured the threshold area(TA) and average gray value ( AG). All the data was marked in ( X ?S ) style. SPSS10. 0 was used for statistic analyzing.Result1. Staining of histocyte.1. 1 Normal control team and false operation team.The form and structure of spinal tissue was normal in HE staining under microscope . Nissl's body was shown as stained patch in cyto-plasma of neuron after Nissls staining. No C9 and CD59 positive staining was observed in each chronopoint.1.2 NS team and sCRl team1.2. 1 Twelve hours after spinal injury: There was patch like hemorrhage in gray matter of NS team. Neutrophil infiltration... |
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