| Objective: Diffuse axonal injury (DAI) is also called diffuse alba injury, with the feature of wide-spread degeneration in the white matter of brain evoked by either the shear or tensile forces generated by the injury. DAI can occur itself or concomitantly with other head injury. It is considerded to be the most common cause of coma without haematoma, nervous functional impairment or vegetative state after traumatic brain injury. Therefore, DAI usually results in high handicap rate and high mortality rate.Since the macroscopic abnormalities in brain tissues of DAI are usually obsolete, it is difficult to diagnose DAI in forensic postmortem examinations by macroscopic observation. Therefore, the identification of DAI has been a tough problem in the forensic medicine. It is reported that the morphologic changes of axons in the alba, particularly in corpus callosum and brain stem is the characteristic changes of DAI, which can be used as a marker to diagnosis DAI. In the present study, DAI in Sprague-Dawley rats was induced by using an injury model according to Marmarou A et al. reported in 1994. The pathological alterations of relevant neurons and axons in central nervous system (CNS) after DAI were observed, in order to further explore the pathological mechanisms of DAI and provide experimental evidence for forensic pathology and clinical forensic medcine.Methods: Sprague-Dawley rats (weight: 383±26g) were randomly divided into twelve groups: normal control group(N), sham group(S), injured group including different phase after injury: 0h, 1h, 3h, 6h, 12h, 24h, 48h, 72h, 5d and 7d. DAI model in Sprague-Dawley rats was induced by an injury model established by Marmarou A et al. Sprague-Dawley rats were weighted and anesthetized by Chloral Hydrate, after achieving a proper anesthesia level, the scalp of the rat was shaved and a midline incision was performed, decoherenced hypoderma and periosteum to expose the central area of the skull vault between the coronal and lambdoid sutures. A 10-mm-diameter stainless steel disc with a thickness of 3 mm was affixed to the skull at the midline between the bregma and lambdoid sutures, then sutured epactal incision. On the next day, rats with etherization were placed prone on a sponge bed(12×12×43cm) contained in a Plexiglas box. The rats were placed in the prone position with the disc centered under the lower end of the Plexiglas tube of the head-injury device. An impact was made against the vertex of the skull at the center of the stainless steel disc by dropping a weight (copper, 450g) from a predermined height (2m), immediately after the impact, the sponge bed in Plexiglas box with rat was quickly removed to avoid a second impact. Rats were then transferred back to the operating table and observed for a couple of minutes. After the impact the animals were allowed to recover and monitored for signs of distress and skull fractures. Normal control group rats gave no disposal; Sham group rats were subjected to the same conditions including placing the animals under the impactor but without batting. After general anaesthesia, rats were transcardially perfused with paraform, brain was carefully removed and paraffin imbedded, the morphological changes of neural and axonal injury were observed under microscopy by HE staining, thionine staining, silver impregnation technique and immunohistochemical staining, DNA injury was observed by TUNEL.The data were presented as Mean±SD and analyzed with ANOVA and LSD by SPSS statistical program. A level of P<0.05 was considered to be significant.Results:1 Coma and apnea occurred immediately after hitting (≤20s). The average time of coma is 6.90±2.48min. According to the standard of ethological score in rats, all injured rats got a lower score than control group.2 There was no obvious alteration in sham group compared with normal control group. In the early time of injured group, the volume of neurons increased, the karyotheca was unclear, nucleolus was deeply stained or disappeared, pink shrunken neurons were observed in hippocamp, cerebral cortex and brain stem. The number of pink shrunken neurons increased at 24h postinjury, nuclear pyknosis, cytoplasm and nucleus adhesion (called dark neurons) were observed. There were still some dark neurons at 7d postinjury.3 The results of thionine staining: the Nissl's body disaggregated from nuclear perim and expanded, then dissolved or disappeared. The number of positive cells increased at 3h after injury in cerebral cortex and hippocamp, peaked at 48h after injury, and still higher at 7d post injury than control group(P<0.05).4 The results of Gless'silver staining: The structure of brain tissue was clear, neurofibra appeared slick and lined up in order in normal and sham control groups. Varicose axons, waving axons and retraction balls were demonstrated when more than 12h after injury, which were much more severe at 24h and 48h postinjury and decreased at 72h postinjury. But it failed to detect injured axons in groups of shorter survival periods. Injury presents recovery at 72h postinjury.5 The result of immunostaining forβ-APP: There was no expression ofβ-APP in normal and sham control group. Varicose axons, waving axons and retraction balls were detected byβ-APP at 1h postinjury in corpus callosum and brain stem. The number and diameter of positive axons increased and achieved peak at 24h and 48h postinjury. The number of positive axons decreased at 72h post injury, and there was no conspicuous difference between 7d postinjury and control group (P>0.05). 6 The result of TUNEL for apoptosis: There were only a few positive cells at 0h, 1h, 3h postinjury in injured group and control group. The number of positive cells increased at 6h post injury, achieved peak at 24h postinjury in hippocampus and 48h postinjury in cerebral cortex, decreased at 72h postinjury, and were still higher at 7d postinjury in cerebral cortex than control group(P<0.05).Conclusions:1 DAI model in Sprague-Dawley rats was successfully established with high reproducibility, which could be used for the further study of DAI.2 With the extension of injury time, the injured axonal amounts and diameter were gradually changed during DAI.3 DAI can lead to neuronal degeneration, death, especially apoptosis, which exists for a long time. |
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