| ?Background?Traumatic brain injury(TBI) occurs when a mechanical force imposed to the head results in one or more secondary changes, such as disturbance of consciousness, neurological or neuropsychological damage, skull fractures, cerebral injury or even death. TBI is one of the most challenging diseases for human, especially severe brain injury with the mortality between 30% and 50%. There are more than 100,000,000(one hundred million) suffering from various types and different degrees brain injury. TBI including many different types resulting from the complex structure of the brain, the difference of anatomy and physiological function. Diffuse brain injury is one of a serious brain trauma, which plays an important role in TBI. Brain injury has become an important disease in our country with the increasing traffic accident and falling injury in recent years. The case about DBI is rapidly increased in forensic medicine. Meantime, DBI and its complications often lead to make the false forensic identification or even conceal the truth of a case. The diagnostic techniques and methods have increased with the progress of science and technology and the research of DBI. Presently, the diagnosis, disease evaluation and injury time estimation of DBI are based on clinical material, an autopsy, conventional histopathological finding and immunohistochemical staining in forensic medicine. However, neuroimaging with CAT scans and MRI is normal in most cases. On the other hand, the autopsy could not be performed timely resulting from all kinds of reasons when medical disputes about DBI caused by traffic accidents. Moreover, it is difficult to make accurate diagnosis according to conventional histopathology when tissue autolysis caused by different preservation methods and poorly fixed specimen. The present research has proved that direct impact, hypoxia-ischemia brain injury, inflammatory mediators, cytokines and oxygen free radicals may lead to neuronal death. Therefore, an intensive study of the change regularity of biomarkers after DBI is of important significance for medicolegal expertise and clinical therapy.The biomarker of screening research is an important aspect in forensic identification. The existing study indicate that there are endogenous brain protective and damage factors after DBI, such as neuroglobin(NGB), amyloid-? protein and their relevant oxygen free radicals. NGB referring as globin is mainly expressed in the rodent brain, which play an important role for oxygen storage and transport in the central nervous system. NGB overexpression protects neuronal cells from ?-amyloid induced cell injury. Amyloid ? protein(A?) accumulation and aggregation in brain parenchyma and perivascular region are thought to be the main cause of Alzheimer's disease(AD), which are the cleavage products of the transmembrane ?-amyloid precursor protein(?-APP). Neuronal injury are often contributed to oxidative stress and intracellular calcium overload caused by A?. Presently, the current study have confirmed that NGB is used to eliminate free radicals prevent neuron from injury. And it can protect neuron from the damage of A?. Therefore, we use the improved Marmarou free-fall impact model to copy DBI, and the pathological changes in brain tissue was observed. Meantime, detecting the expression of NGB and A?, and to study the change rules of A? and NBG in the brainstem after DBI may provide an important foundation for forensic diagnosis, damage assessment and injury time reference.?Objectives?We explore the feasibility of A? and NGB as molecular markers in brainstem after DBI via studying the expression and variation of A? and NGB at different time points.?Materials and Methods?1. Adult healthy female SD rats were randomly divided into the control group and DBI groups. The rats in DBI groups were sacrificed at 1h, 3h, 6h, 12 h, 24 h, 48 h and 72 h respectively. Each group owned 10 rats.2. DBI model was DBI group by referring to Marmarou method. Observe the behavioral changes in rats with DBI and the rat's brainstem extraction was prepared for hematoxylin-eosin staining, Luxol fast blue staining and immunohistochemistry of ?-APP to confirm the model is successful. The changes in morphology and expression are observed using A? and NGB immunohistochemistry. The concentration of A? and NGB were detected by Western blotting. Finally, the co-localization of ?-APP and A?, A? and NGB were revealed by double immunofluorescence labeling method. 3. Data are shown as mean ± SEM, and a one-way ANOVA was used for comparative analysis between groups. A value of p<0.05 was considered as the threshold for statistical significance. The changes and differences of each group in the brainstem were made through statistical analysis. All statistical data was analyzed using IBM SPSS 18.0. Office excel(Office 2010) was used for figures and related charts.?Results?1. Pathological changes in rat brainstem injury of each group are consistent with DBI morphology. Significant axonal swelling and tortuous axons are observed, and axonal retraction balls are be seen at 3h group. Moreover, typical RBs are increasing with time. 2. Immunohistochemistry showed that the expression of A? and NGB were increased at 1h post-injury. The expression of NGB were firstly decreased at 3h and 6h post-injury, and the expression of A? was still increased and reached the first peak at 6h post-injury. At 12h~72h post-injury, the NGB was fluctuated. Meanwhile, A? was reached the second peak at 24 h post-injury, and followed by a significant drop. Compared with the control group, a significant increase of the expression of A? in brainstem was observed at 1h, 3h, 6h, 12 h and 72 h post-trauma. There were significant difference of the expression of NGB at 1h, 3h, 12 h, 24 h and 48 h post-injury compared with the control. 3. Western blotting revealed that the expression of NGB was increased and reached the peak at 1h post-injury, and A? were significantly increased. The level of NGB started to fall,and there was no significant change in the content of A? at 3h post-trauma. The expression of NGB was still decreased,A? was significantly increased and reached the first peak at 6h post-trauma. NGB rose again,and A? fell down at 12 h post-injury. NGB still fall and A? reached the second peak at 24 h. NGB expression showed small fluctuations and A? decreased again at 48 h and 72 h, but still higher than normal. 4. ?-APP and A? were expressed consistently in cytoplasm and axon of brainstem. A? and NGB were observed in cytoplasm, and A? also occurred around small blood vessels. The study indicated that NGB was also specifically expressed in nervous cells of brainstem.?Conclusion?The changes of the A? and NGB in rats' brainstem play important roles for the early diagnosis of DBI. Especially 1h, 6h and 24 h are important time for diagnosis and treatment of DBI. |
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