| Intracerebral hemorrhage (ICH) is a common and often fatal stroke subtype that produces severe neurological deficits in survivors with no effective treatment. Intracerebral hemorrhage causes brain damage through multiple mechanisms. An understanding of evolution of brain injury after intracerebral hemorrhage is important to determine the strategy of treatment. Hemorrhagic stroke is accompanied by intense inflammation, including an initial infiltration by neutrophils followed by macrophage accumulation. Both cell types express inflammatory molecules capable of damaging the central nervous system through a complex cascade of events. Erythrocyte lysis is a potential cause of delayed edema formation, and complement activation and membrane attack complex (MAC) may play a role in clot lysis. Macrophages and resident microglia are activated and produce elevated levels of cytokines. Experimental and clinical cerebral hemorrhages induce a rapid production of proinflammatory cytokines in both brain parenchyma and cerebrospinal fluid. The purpose of this study was to investigate the expression of cytokines in tissue surrounding hematoma. Male Wistar rats were anesthetized , and ICH was induced by intrastiatal administration of auto arterial blood, killed at time of 6,12,24 hour and 3,5,7,day. Brains were dissected in the coronal plane through the needle entry site. The anterior portion, which contained half of the hematoma, was weighted to obtain the wet weight. Brain samples were then dried in a gravity oven weight (Blue M. Electric Co) at 100°C for 24 hours to obtain the dry weight. The water content was determined as (Wet Weight-Dry Weight)/Wet Weight. The posterior portion was immediatedly frozen in liquid nitrogen for analyze of IL-1β,IL-6 and TNF-α protein levels. To define expression and location after ICH, two animals at different time points were anesthetized and killed by perfusion with 4% paraformaldehyde in 0.1mmol/L PBS, fixed brains were cut coronally through the needle entry site, and slices including the hematoma site were embedded in paraffin. The area of brain damage on the hematoxylin and eosin–stained coronal slice at the level of the needle injection site was defined by the presence of blood collections, tissue rarefaction caused by edema, and dying cells around the injection sites.Sections (6 mm) were dewaxed and rehydrated in 0.1 mol/L PBS. After blocking steps, they were incubated with rabbit polyclonals anti–TNFα,anti-IL-6(0.1μg/ml,0.005μg/ml,respectively, provided by PEPROTECH )and mouse monoclonal anti-IL-1β(10μg/ml, provided by SEROTEC)overnight at 4°C followed by biotinylated goat anti-rabbit IgG and goat anti-mouse IgG, then streptavidin- peroxidase, then diaminobenzidine. TNFα,IL-6 and IL-1βimmunoreactivity were assessed in a positive manner in tissues surrounding the hematoma site. For Western blot analysis, tissues of frozen brain dissections were processed cell lysis.2μg protein was run on 7.5% polyacrylamide gels with a 4% stacking gel (SDS-PAGE). And then hybridized with either primary rabbit polyclonal antibodies reactive with TNFαand IL-6, or with mouse monoclonal antibody to IL-1β, and subsequently incubated with secondary anti-mouse or anti-rabbit antibodies conjugated with horseradish peroxidase. Finally, membranes were processed for proteins by using the color analysis system of picture.Results reveals that ICH induction elevated IL-6 and TNF-α protein levels after the onset of injury compared with animals injected with saline by immunoblot(P<0.001). The protein level of TNF-α reached peak at day 5. The protein level of IL-6 reached its peak at day 1. And then have an obvious decrease. Its curve is coincident with the curve of water content. And seemly the peak of water content was delayed the peak of IL-6 level. The study did not detected the expression of IL-1β, maybe the tissues used in immunoblot analysis were taken from cerebral white substance. Immunohistochemical labeling indicated that ICH was accompanied by elevate...
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