The Expression Of Nestin And HSP70 Following Traumatic Brain Injury In Rats

Objective Brain contusion is a kind of idiopathic brain injury with a serious consequence to the central nervous system. Even individuals with only a moderate injury often have a long-lasting disruption of their way of life or die from brain trauma. Recently, neural stem cells (NSCs) have been identified in adult mammals that, have the potential to self-renew and as well as the potential to differentiate into the neurons or neuroglia. Neuroepithelial stem cell protein (nestin) is an intermediate filament protein, transiently and abundantly expressed early in embryogenesis, e.g. Many animal studies demonstrate that nestin has also been detected in a number of pathological conditions including traumatic, ischemic and excitotoxic cerebral injury. Nestin is obviously up regulated and the nestin expression is also dependent upon a spatio-temporal pattern. Heat shock proteins (HSPs) play an important role in cellular function. HSPs can protect cells from injury induced by environmental challenges, such as hypoxia, ischemia, high temperature, endotoxin, infection, et al. HSP 70 proteins are usually constitutively present at low concentrations in cells, but are actively synthesized to reach much higher concentrations as cells react to aggressive situations. Many studies have demonstrated HSP 70 is a sensitive data to estate the early-post injury time in forensic medicine aspect.On the base of immunohistochemistry method, we observe the alteration of nestin/HSP70 at various intervals in the experimental traumatic brain injury (TBI) and study the relationship between the alteration and the post-injury interval. To investigate whether or not the changes of nestin can be used as a sensitive data for estimating the early-post injury time in forensic medicine aspect.Methods Experiments were performed on 46 male Sprague-Dawley rats weighting 250～350g. The rats were divided into the normal control, the sham operation control, the operation control on the dead rats and the TBI groups. The animal models of brain contusion were made according to the method described by Edward. The test group rats were killed after various survival interval (0.5h,6h,12h,1d,3d,7d,14d and 28d). The animals were sacrificed at various times after injury. Rat brains were fixed with 4% paraformaldehyde in 0.1M phosphate buffer, pH 7.3. The specimens were then embedded in paraffin. Immunohistochemical and HE staining were used to evaluate the expression of nestin/HSP 70 in the cortex, hippocampal dentate gyrus and the corpus callosum of injury side. SPSS software was used for statistical analyses. A one-way ANOVA was used to compare injured and control groups at each time point for immunohistochemical analyses. Signifiance was at P<0.05.Results A HE staining showed that the injury of the motor-sensory cortex was restricted to within the gray matter without any involvement of either the white matter or the hippocampal tissue. Immunohistochemical staining of nestin showed that nestin was absent from astrocytes, microglia and neurons in control rats. Only endothelial, hippocampal dentate gyrus and select subventricular cells expressed nestin in both hemispheres of control rats. This expression, however, was slight and scattered. HSP 70 was expressed at a low level in neurons and micrangium endothelial cells. After induction of brain contusion, the expression of nestin/HSP 70 was up regulated and dependent upon a spatio-temporal pattern.(1) Nestin positive cells increased at 0.5h and reached the maximum level 7d after brain contusion, and then the expression decreased gradually to 14d. The intensity of nestin staining expression decreased to normal at 28d in the cortex and the hippocampal dentate gyrus. But nestin positive cells still remain to weak expression at 28d in the corpus callosum of injury side.(2) Expression of HSP 70 positive cells increased at 0.5h and reached the maximum level 12h after brain contusion, and then the expression decreased gradually to 24h. The intensity of HSP 70 staining expression increased to a high level at 3d and decreased to normal at 28d.Conclusion In this animal study, we used the ameliorative controlled cortical impact model created by Edward originally, which is a more precise model of brain injury than the other animal brain injury models. Our experiments clearly demonstrate that reactive astrocytes express nestin immunoreactivity. Nestin-positive immunolabeling was only find in endothelial and select subventricular cells of both hemispheres of control rats, whereas after trauma, nestin immunoreactive astrocytes were localized to the boundary of the ipsilateral cortical lesion and also present far from the cortical lesion of the injury rats. The nestin expression was dependent upon a spatio-temporal pattern.In this immunohistochemical study on the nestin and HSP 70, the expression of nestin/HSP 70 demonstrate nestin-postive and HSP 70-postive cells appear, increase to the peak, decrease gradually to the control rats. Forthermore, both nestin and HSP 70 expression are obviously related to the injury time.Our results with nestin suggest that the changes of nestin immunohistochemical staining can be used as a sensitive data for estimating the early-post injury time in forensic medicine aspect. The expression of nestin also can be used as an important identification to diagnose brain injury and to distinguish antemortem and postmortem brain injury.