| Background and objectiveIn recent years, alcoholism with a slight external force, causing traumatic subarachnoid hemorrhage (TSAH) to death shows a rising trend. TSAH was often reported alcohol-related, but its mechanism of occurrence and death has not yet been entirely clear. Our team has established a stable TSAH model after alcoholism in rats, with the incidence and mortality rate of TSAH 81% and 76%, in the chronic group, much higher than 27% and 4.8% in the acute group (P<0.01). Therefore, we proposed that the long-term cumulative toxicity of alcoholism on the central nervous system (CNS), especially that in the brain stem central life on the metabolism, function and morphology, commonly cause death with the synergistic effect of TSAH and concussion injury. This study is based on previous work, by observing neuronal axonal transport, signal transduction that is closely related to the Tau protein,β-APP expression and distribution, to further discuss the correlation of Tau Protein,β-APP and the mechanism of death associated with TSAH in cases of chronic alcoholism in rat brains.Materials and Methods1 Animals model and groupsCL-class male SD rats weighing 300±30 g, sub-conventional breeding were randomly divided: chronic water group(n=10), chronic water group with strike(n=10), chronic alcoholism group(n=10), chronic alcoholism group with strike(n=29), chronic alcoholism group stop drinking 1 month(n=6). Intragastric administration with drinking water or white wine (Beijing Red Star Erguotou, 52% v / v) for 4 weeks, dose: 2 weeks before 8 ml / kg / time, 2 weeks after 12 ml / kg / time, 9:00 and 16: 00 interval 7 hours twice one day ig.2 MethodsDisposal of the rats after 4 w: chronic water group and chronic alcoholism group were killed by femoral artery bleeding after intraperitoneal injection of pentobarbital 2 h after the last gavage, chronic water group with strike and chronic alcoholism group with strike were given a concussion blow by a home-made device with pendulum, with a simultaneous monitor of ECG for 15 min. Two hours after strike the rats were killed using the above method. The dead rats in chronic alcoholism group with strike were classified as dead group. Immediately after death, the rats were fixed by perfusion of fixative (2.5% glutaraldehyde, 2% paraformaldehyde) for 30 min, using aortic cannulation. The whole brains were extracted and fixed in 4% neutral paraformaldehyde for 6 h. Sagittal sections of the brain were prepared for routine paraffin serial sections, HE, Bielschowsky's silver staining and Tau protein,β-APP immunohistochemical staining. The pathological changes were observed with light microscopy, image acquisition system, mining plans, Image-Pro Plus 6.0 image analysis software, cumulative integral optical density (IOD sum) and other quantitative pathological measurements. Brain tissues for electron microscopy were immediately placed in 2.5% glutaraldehyde, washed, fixed, embedded, sectioned and stained by conventional methods, and were observed using the JEM-1400 transmission electron microscope.3 Statistical AnalysisAll experimental data analysis was performed using the softs of SPSS 17.0 and Excel 2003, including analysis of variance (ANOVA) test, independent sample T test, chi-square test. P<0.05 or P<0.01is regarded as significant.Results1 General observationFor rats in chronic water group, diet and spirit were kept in good condition, and the weight increased continuously, especially in the second week, it had a significant increase (P<0.01). For rats in chronic alcoholism group, the hair was rough and dull, the mental state was poor. With the increase of alcoholism, gradually, the rats showed the loss of body weight, malnutrition, decrease of food intake and activity, body limp and other changes of chronic alcoholism. Chronic alcoholism rats decreased gradually in weight, with the weight after 1w, 3w, 4w was significantly lower than that before alcoholism (P<0.05).There were no significant changes in the heart rate before and after strike for rats in chronic water group and chronic alcoholism group (P>0.05). For rats in chronic water group with strike, the electrocardiogram transiently showed slow heart rate, increased QRS amplitude waves for 0.3±0.16 s; for rats in chronic alcoholism group, heart rate slowed down, QRS wave amplitude significantly increased and recoveried after 1.7±0.36 s. The recovery time was significantly longer than that in the chronic water group with strike (P<0.05); dead rats significantly increased QRS amplitude, the heart rate slowed down after 10 s-1 min,then became a straight line.TSAH morbidity and mortality in alcoholism group with strike were 79.3%, 51.7%, significantly higher than that in the water with strike group (P<0.05). According to Fisher grading, the grade of TSAH alcoholism with strike group was higher than that in water with strike group.2 General observation and histopathological observationIn chronic water group, the brain surface was smooth without vascular texture. By HE staining, the neurons and glial cell were found to be normal in morphology and distribution. Nerve fibers arranged regularly and closely with uniform size and staining. Bielschowsky's silver staining showed that brain stem nerve fibers were straight and arranged closely. The brains of chronic water group with strike showed mild cerebral congestion and scattered and limited hemorrhage. Neurons and glial cells had mild edema, and Nissl bodies were stained more lightly with the number reduced. Nerve fibers were irregularly thicken, broken, had expanded perivitelline and arranged loosely, with no TSAH, contusion or other lesions.Chronic alcoholism rats showed mild cerebral congestion on brain surface with vascular texture. Neurons arranged in disorder, reduced in number. Some of them sporadically had type 1 (pyknotic darkly stained) or type 2 (swelling lightly stained) changes. The cells were swollen and round, the nucleus increased in size and displaced. Central nissl bodies dissolved and disappeared; glial cells proliferated; cerebellar Purkinje cell shrinked, reduced in number, with uneven spacing. Nerve fibers were arranged slightly loosely, with increased gap, as well as mild irregular thickening and deformation, but there were no TSAH, contusion and other changes found. In chronic alcoholism group with strike, congestion was found in brain surface, with thick patchy TSAH found mainly in ventral brain stem, and no cerebral contusion found. Neurons and glial cells were in a high degree of hydropic degeneration, with the perivitelline space expanded, and more nucleus pyknosis, dissolved, disappeared. Central Nissl bodies displaced peripherally in the cell, dissolved, disappeared at different levels, neuronophagia and satellitosis were commonly found; neurons sporadically disappeared, gliosis proliferated; cerebellar Purkinje cells were unregularly reduced in number with uneven staining, density and pyknosis; red and dark neurons were found more commonly in medulla; nerve fibers were loose and swollen, cell gap was widened with irregular thickening, distort, break, and local swelling. Chronic alcoholism group with a stop of 1 month had milder pathological changs in neurons and nerve fibers than chronic alcoholism group.3 TEM changesIn chronic water group, the brain stem was dense, nerve fiber axonal had uniform thickness, regular shape and high electron density. The myelin was dark with uniform thickness, and was tightly shafted with the membrane. Axoplasm and myelin of nerve fiber were dense and uniform. Nerve skeleton fiber had uniform thickness. Neuronal nuclei had rich mitochondria, Golgi complex, endoplasmic reticulum, which were regular in size, shape and arrangement, with ribosome commonly found. For chronic water group with strike, the brain tissue was loose, loose and swollen and lower electron density. Thickness and coloration of axonal nerve fiber and thickness of myelin were uneven. Skeleton of nerve fibers were loose and swollen. The myelin sporadically had layered fracture. Mitochondria of neurons were loose and swelled with its cristae expanded. Endoplasmic reticulum expanded mildly, and synaptic cleft widened mildly, and density of dense plaque was decreased.For chronic alcoholism rats, the brain stem was loose and swollen, axonal nerve fibers were widespreadly uneven in thickness and shape, disorganized with the myelin layer irregular and uneven in thickness and density. Nerve fiber skeleton had uneven thickness with some of them dissolved. The myelin sporadically had layered fracture. Mitochondria of neurons was obviously swelling and loose, with various crest length and thickness, and the outer membrane disintegrated. Neurons with the pyknosis of cell body and nuclear were found, with the nuclear membrane folded, heterochromatin increased and clustered, nucleolar condensated; Glial cells increased in number and the interstitial was loose and swollen.In chronic alcoholism with strike group, the myelin sheath of nerve fibers was highly loose with serious edema in the brain stem. Lamellar separation, folding, and retraction were found in the myelin sheath with floc attachment. Vesicle-like protrusions formed in part of the region and broke into the surrounding gap. The microtubules and microfilaments collapsed with some of them replaced by membranous structures and some membranous organelles (including cavitation mitochondria) clustering around them. The number of synapses reduced, synaptic vesicles accumulated; glial cells increased, interstitial occurred edema. Axoplasm and organelles accumulated gradually in the changed membranes and myelin-axis and resulted in reactive axonal swelling.Size of neurons decreased, electron density of cytoplasm and nucleus increased, nuclear membrane folded, heterochromatin clustered, nucleolus disappeared or became smaller, the nuclear cytoplasm ratio increased, the extracellular gap was widened, the membrane was disintegrated; loose and swollen, mitochondria and endoplasmic reticulum loose swelling.4 The expression of Tau,β-APP4.1 The IOD sum of TauIn normal rats, Tau protein was expressed mainly in the axons and the cytoplasm. In the chronic alcoholism group, the Tau IOD sum of brainstem, cerebellum, hippocampus, frontal lobe and other brain regions reduced significantly (P<0.05); In the chronic water group with strike, Tau IOD sum of the frontal lobe and other brain regions was significantly higher (P<0.05), but that of brain stem, cerebellum, hippocampus and other parts had no significant change (P>0.05); In the chronic alcoholism and strike group, the brainstem, cerebellum, hippocampus, frontal lobe and other parts of Tau IOD sum value was significantly higher (P<0.05). In the chronic alcoholism group with strike, Tau IOD sum of the brain stem, hippocampus, frontal lobe and other parts were significantly higher in the death rats than in the survival rats (P<0.05).4.2 The IOD sum ofβ-APPIn normal rats,β-APP was mainly expressed in the neurons cytoplasm. In chronic alcoholism rats, the brain stem, cerebellum, hippocampus and other brain regions had significantly higher IOD sum (P<0.05), but that of frontal lobe not (P>0.05). In the chronic water group with strike,β-APP IOD sum was significantly higher in the brain stem, hippocampus and other brain regions (P<0.05), but not in cerebellum, frontal lobe and other parts (P>0.05). In the chronic alcoholism and strike group,β-APP IOD sum was significantly reduced in the rat brain stem, cerebellum, hippocampus, frontal lobe and other parts (P<0.05), but that in the dead rats were significantly higher than that in the survival (P<0.05).Conclusions1. Chronic alcoholism damaged the cytoskeleton structure of nerve fibers in CNS, might degrade the compensation of conduction, and lead to the increase of vulnerability, suggesting that it may be the pathological basis of the high mortality in concussion after chronic alcoholism.2. Expression and distribution of Tau protein,β-APP in rat brain stem, cerebellum, hippocampus, frontal lobe suggested that chronic alcoholism significantly degraded the expression of Tau protein and caused overexpression ofβ-APP in CNS, as might be one of the mechanisms of white matter atrophy and reduction of neurite number, contribute the pathological basis in death of TSAH in chronic alcoholism.3. Combined with the results of previous work in our team, consider that the toxicological cumulative effects of chronic alcoholism in CNS constitute an important pathological basis in nerve injury in TSAH of the alcoholism-related, possibly, one of the mechanisms to promote the death with mild concussion.
|
PDF Full Text Request