| Objective: Diffuse axonal injury (DAI) is seen as wide-spread damage in the white matter of brain characterized by morphological changes to axons throughout the brain and brain stem. Severe DAI is characterized by immediate onset of coma at the time of injury, followed by persistent coma and vegetative state or severe persistent disability. DAI may largely account for the clinical manifestations of brain trauma. However, DAI is extremely difficult to detect noninvasively and is poorly defined as clinical syndrome. Maxwell summarized that in human DAI the majority of axons do not undergo immediate disruption at the time of injury (primary axotomy), but rather are exposed to non-disruptive axonal injury leading, after several hours, to disruption (secondary axotomy).Microglia plays an important role in nervous system development, repair and regeneration of nerve tissue, neuronimmunity and synaptic transmission etc. Microglia responded most early to ischemic brain injury. And microglia with increased expression of the macrophage colony-stimulating factor receptor are found surrounding plaques in Alzheimer's disease (AD) and in mouse models for AD and after ischemic or traumatic brain injury(TBI). Increased expression of macrophage colony-stimulating factor receptor causes microglia to adopt an activated state that results in proliferation, release of cytokines, and enhanced phagocytosis. As already mentioned, certain cell surface markers of importance in immune regulation, such as CD11b and MHC class II molecules, are constitutively expressed on ramified microglia in the normal adult brain. However, in response to a variety of CNS insults such as TBI, ramified microglia have the capacity not only to dramatically change their morphology to reactive or amoeboid forms but also to rapidly up-regulate a large number of receptor types and produce a myriad of secretory products that are thought to contribute to the defense of and, potentially, damage to the insulted brain. Activated microglia can express receptors and the cognate ligands for interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α) and so on. It is also important to note that although microglia possess receptors for and can be activated by IL-6 or alpha, beta, and gamma interferons (IFN-α, IFN-β, and IFN-γ), and it appears that microglia are incapable of generating appreciable quantities of these critical activating cytokines. IL-6 level in brain tissues from the impact site may represents the severity of traumatic brain injury. And IL-6 conveyed from the glial cells may contribute to the pathogenic mechanism of DAI formation. Microglia plays an important role in nervous system development, repair and regeneration of nerve tissue, neuronimmunity and synaptic transmission etc. The reaction of microglia and the expression of IL-6, in DAI rats, are not clear.We induced DAI in Sprague-Dawley (SD) rats using an injury model adapted from Marmarou et al. in 1994. We want to observe the activation of microglia and the expression of IL-6 of DAI animal. So, we can explain the relationship between microglia and the second brain injury in DAI animal model better.Methods: Induction of head trauma: SD rats, each weighing 280 to 320g, were randomly divided into nine groups: control group, sham group, 0h, 1h, 3h, 6h, 12h, 24h, 48h, 72h, 5d and 7d group. Amidline scalp incision was performed followed by periosteal elevation to expose the central area of the skull vault between the coronal and lambdoid sutures. A stainless-steel disc 1 cm in diameter was firmly fixed to this central portion of the skull vault. When the trauma device was ready, the rat was placed in the prone position on a foam bed with the disc centered immediately under the lower end of the Plexiglas tube of the trauma device. The weight (450g) was allowed to drop freely from the designated height (2m) through the Plexiglas tube onto the disc; the foam bed together with the rat was moved away from underneath the tube immediately after impact to ensure a single hit. The rat was then transferred back to the operating table and observed for a couple of minutes. The skull vault was inspected for the presence of any fracture. The scalp was sutured. Rats that died on impact and those with skull fractures were excluded from the study. Animals in the control groups were surgically prepared for impact in the same way as above, but were not subjected to the head trauma.The brain was removed, sections 5μm thick were cut and stained with HE, Bielschowsky, Cresyl violet and Weil's myelin staining to verify the axonal changes. Immunocytochemical technique was used to examine the expression of CD11b and IL-6.The data were presented as Mean±SD and analyzed with ANOVA and LSD using SPSS statistical program. A level of P<0.05 was considered as statistical significance.Results: 1 Change of pathological section:HE staining in brain tissue: There was no alteration in brain tissue in control group and sham group. In strike group slight congestion and edema were observed in cortex, cerebellum and brain stem at 6h after injury. The findings were severe in the 24h and 48h injured rats.Bielschowsky staining in brain tissue: There was no alteration in brain tissue in control group and sham group. In strike group, waving and enlargement of axons were observed in cerebellum and brain stem in the 12h injured rats. The findings were severe in the 24h and 48h injured rats.Weil's myelin staining in brain tissue: In control group and sham group, the structure of rat brain tissue was clear. Transverse section of myelin showed ring-like, and longitudinal section of the myelin looked like fishbone. Myelin gap widened significantly, and myelin seemed edema and disintegration in the 24h and 48h injured rats and decreased at 5d.Cresyl violet nissl bodies staining in brain tissue: There was no alteration in brain tissue in control group and sham group. Nissl bodies of neurons, round the damaged brain tissue, decreased significantly, and came into large coarse granulars in the 24h and 48h injured rats.2 Immunochemistry in brain tissue:The activation of microglia at different time points: In control group and sham group CD11b, the surface marker of microglia, expressed little. CD11b expressed higher at 3h after injury compared with that of control group (P<0.05), and accumulated in the axonal segment at 24h. And ramified microglias changed their morphology to reactive or amoeboid forms. The findings were severe in the 12h and 24h injured rats, and decreased at 72h, but was still higher than that of control group (P<0.05).The changes of the expression of IL-6 at different time points: In control group and sham group IL-6 expresses little in brain tissue. The expression of IL-6 increased evidently at 3h ,compared with that of control group (P<0.05). The IL-6 expression increased further at 6h, and get to the top at 12h. After 48h the protein expression began to decrease compared with 12h group, and decreased further at 72h, 5d and 7d, but was still higher than that of control group (P<0.05).Conclusions: DAI was induced in SD rats using an injury model adapted from Marmarou et al. in 1994. The injury of DAI can induce the activation of microglia. The expression of IL-6 increased evidently after DAI, and peaked at 12h after injury. IL-6 not only expressed in neuroepithelial cells, but also in some neurons. Relationship between microglia and the second brain injury in DAI animal model was approved.
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