| 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.Former studies show the mechanisms of DAI include ischemia-reperfusion injury, calcium overload, abnormal expression of cytokines and neurotransmitters etc.Andre Wennersen has observed apoptosis in brain tissue in TBI (traumic brain injury) by using TUNEL stain. They also found a increasing of the expression of Bax and Bcl-2 in nerve cells after TBI. These findings confirm that trauma can induce apoptosis in CNS.The Janus kinase-signal transducer and activator of transcription pathway functions as downstream effectors of cytokine and growth factor receptor signaling. This pathway plays an important role in regulating immune responses, cell proliferation, apoptosis and cellular homeostasis in human health and disease. Stat1 is the first STAT fators discoveried as key mediators of IFN signaling. It has been shown to be activated by the IFNs, cytokines such as INF-α, INF-γ, IL-6 and IL-10, and noncytokine signals such as GM-CSF, FGF and PDGF. Stat1 plays a role in enhancing apoptotic cell death by inducing the expression of the pro-apoptotic caspase-1, Bax, Fas and FasL genes, as well as down-regulate the expression of anti-apoptotic genes such as Bcl-2 and Bcl-x.Neuroepithelial cells play an important role in nervous system development, repair and regeneration of nerve tissue, neuronimmunity and synaptic transmission etc. Using some special approaches, investigators have shown that trauma injuces astrocytic and other types of neuroepithelial cells hyperplasia, hypertrophy.The changes after TBI can induce apoptosis in brain tissue, and Stat1 takes part in apoptotic signal. If there was a stimulation of JAK-STAT pathway in DAI, and taken part in apoptosis has not been known. We induced DAI in SD rats using an injury model adapted from Marmarou et al. in 1994. To observe the activation of Stat1, and the relationship between Ser pStat1 and the apoptosis of nerve cells in DAI animal model.Methods: Induction of head trauma: Sprague-Dawley rats, each weighing 240 to 280gm, were randomly divided into nine groups: control group, sham group, 6h, 12h, 24h, 48h, 72h, 5d and 10d 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 (1.75m) 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 Bielschowsky and Esterification-silver stain to verify the axonal changes. Flow cytometry was used to assay the ratio of apoptosis of the nerve cells. RT-PCR was applied to examine the expression of bax and bcl-2. Immunocytochemical technique was used to examine the expression ofβ-APP and Phospho-Stat1 (Ser727).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: There was no alteration in brain tissue in control group. In strike group slight congestion and edema were observed in cortex and cerebellum at 12h after injury. And waving and enlargement of axons were observed in cerebellum and brain stem at this time point. The findings were severe in the 24h and 48h injured rats.Immunochemistry in brain tissue: In control groupβ-APP expressed little and did not accumulate in axons.β-APP expressed higher at 6h after injury, and accumulated in the axonal segment at 12h. The accumulation was severe in the 48h injured rats. Than the expression ofβ-APP began to decrease, and disappeard from the axons little by little.2 The ratio of apoptosis of nerve cells was very low in control group and sham group. Compared with control group, the ratio of apoptosis was increased a little at 6h and 12h, and showed no significant difference (P>0.05). The ratio of apoptosis of nerve cells peaked at 24h after injury, and decreased at 48h. The ratio of apoptosis decreased further at 72h, 5d and 10d, but was still higher than that of control group (P <0.05).3 The ratio of bax/bcl-2 mRNA was upregulated at 6h after injury, and showed no significant difference compared with the control group (P>0.05). The ratio increased further at 12h, and higher than that of control group (P<0.05). The ratio of bax/bcl-2 mRNA was increased to the top at 24h. After 48h the ratio of bax/bcl-2 mRNA began to decrease compared with 24h group, and decreased further at 72h and 5d, but was still higher than that of control group (P<0.05). The ratio of bax/bcl-2 mRNA showed no significant difference compared with the control group (P>0.05).4 In control group Ser p-Stat1 express little in brain tissue. The expression of Ser p-Stat1 increased evidently compared with that of control group (P<0.01), which was mainly located in nuclei. The Ser p-Stat1 expression increased further at 12h, and get to the top at 24h. After 48h the protein expression began to decrease compared with 24h group, and decreased further at 72h, 5d and 10d, but was still higher than that of control group (P<0.01). And the expression of Ser p-Stat1 increased to the top at 12h in hippocampus.Ser p-Stat1 expressed mainly in neurons, and in some neuroepithelial cells.5 The changes of the expression of Phospho-Stat1 (Ser727) in brain tissue after injury have positive correlation with the changes of the ratio of apoptosis. And the correlation coefficient is 0.761 (P<0.05).Conclusions: DAI was induced in Sprague-Dawley rats using an injury model adapted from Marmarou et al. in 1994. The injury of DAI can induce the apoptosis of nerve cells. The expression of Ser p-Stat1 increased evidently after DAI, and peaked at 24h after injury. Ser p-Stat1 not only expressed in neurons, but also in some neuroepithelial cells. The changes of the expression of Phospho-Stat1 (Ser727) in brain tissue after injury have positive correlation with the changes of the ratio of apoptosis.
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