Study The Relation Between Pathomorphologic Changes And Expressions Of M-Calpain, Calpastatin In Chronic Alcoholism Brains And The Death Mechanism Of TSAH

Background and objectiveWith more and more persons reported to die with traumatic subarachnoid haemorrhage (TSAH) after drinking, forensic workers pay more attention to these cases at home and abroad. Using the TSAH model after drinking established by our research group,we found significantly higher morbility and mortality (81% and 76%) in the chronic alcoholism rats, compared with that in the acute alcoholism rats (27% and 4.8%). According to the domestic and international related reports and the results of our previous research, we assume that alcohol accumulative damage to central nervous system (CNS) result in TSAH deaths of in the chronic group. However , up to now, the rapid death in the chronic alcoholism rats with TSAH cannot be well accounted for with previous studies resulting from alcohol damages on morphology or metabolism in vital center of the brain stem. Therefore, we tried to explore the mechanisms on rapidly deaths of alcoholism TSAH rats from the relationship between patho-morphologic changes and m-Calpain / Calpastatin expression in brain stems.Materials and Methods1 Animals and groupSixty-five healthy male SD (Sprague-Dawley) rats (Clean animal), each weighting 300±30 g, were bred in several cages and randomly divided into control-water group (W), control-water with hit group (WH), alcoholism group (A), alcoholism with hit group (AH) and alcoholism group with stop administration one month (AS). The rats were administered intragastrically with 8 ml/kg/time liquor (clean water or Hongxing Erguotou wine) for the first two weeks and with 12 ml/kg/time liquor for the first two weeks, two times each day.2 MethodsFour weeks later, after administration, the experiment was performed according to detailed group. The animals were killed by exsanguination respectly in these group without hit, and the rats were given a concussion hit in these hit group with a simple self-pendulum, monitoring of electrocardiogram. About 15 minutes after hit or immediately after death, these animals were fixed via the way of infusing formalin into aorta with the help of aortic cannlation. Then the brain tissues were collected, post-fixed with 4% paraformaldehyde for 6 h, made paraffin embedded section, and stained with HE, Toluidine Blue, immunohistochemistry (IHC) of m-Calpain and Calpastatin. The patho-morphologic changes were observed and taken picture by patho-true-color image analytical system (CMIASWIN), and SUM IOD and MENA IOD were got by Image-Pro Plus 6.0.3 Statistical analysisAll data were represented as mean±standard deviation and analyzed by SPSS 17.0 (ANONA , Independent-samples T-test and chi square test) and Excel 2003 software. The significant differents marked P<0.05 or P<0.01.Results1 General Observation and Incidence rate and Mortality rate of TSAHWeight of Chronic alcoholism rats decreased after one week (P<0.05). After two weeks, with the hair roughened, yellowed and shed, the alcoholism rats showed fatigue, inactivity, and apathy, decrease of food intake, emaciated and diarrhea and so on. And the weight decreased sharply after three weeks (P<0.01). The rats of control-water rats kept in good condition and their body weight showed a slow growth (P>0.05), until the 2th week, it got a significant difference (P<0.05). Chi square test showed that the rats in WH group had significant higer incidence (79.3%) and mortality (51.7%) of TSAH compared with other group (P<0.01).2 ECG changes before and after hitThere were no significant differences in the Heart Rate Changes before and after hit between WH group and AH group (P>0.05) besides instantaneous interference of myoelectricity. ECG of WH group showed transient decreased heart rate and increased wave amplitude of QRS wave group, and recovery time was 0.3±0.16s. ECG of AH group showed more decreased heart rate and increased wave amplitude of QRS wave group, and recovery time was 1.7±0.36s , longer than WH group (P<0.05). ECG of alcoholism death group with hit showed serious abnormality with anomalous heart rate and wave amplitude.3 Macroscopic and Morphologic ObservationRat brains in W group showed smooth surfaces, normal structure and distribution of neurons and gliocytes, regularity of neurofibra arrangement, clear construction of Nissl bodies. In addition, with toluidine blue staining, neurons were sharply demarcated, with hyacinthine endochylema, large round nuclei, clearly nucelus, even chromatin, and Nissl bodies around perinuclear or dendrites showed deep blue color, with small block or granular size. Rat brains in WH group showed slight cellular swelling of neurons and gliocytes, light-colored Nissl bodies and loose neurofibras,and TSAH and cerebral contusion were no found. Rat brains in A group showed irregular form, scattered typeⅠcell (shrunken and dark) and typeⅡcell (swollen and pale) , lessen quantity of neurons and hyperplasia of gliocytes. Neurons showed an appearance of atrophy and degeneration, or being swollen and full, with chromatolysis and nucelus larger and deflected. Also, Purkinje cells arranged in loose, and some condensed even dissolved away. There were congestion and edema in rat brains of AH with thick patchy hemorrhage of TSAH, involving the whole brain and its more common saw at the ventra brain stem. And cerebral contusion were also no found. Neurons and gliocytes arranged loosely, wider pericellular space, and more neuronophagia, karyolysis (dissolution). Nissl's bodies showed progressive dissolving, lost and chromatin condensation and aggregation to various degrees; Purkinje cells of uneven arranges showed uneven cytoplasm staining, nuclei condensed and dark. Oblongata showed many nuclear condensed and neurons acidophilia metachromatic named dark neurons. Interstitial edema with wider space occurred in neurofibras, and there were lots of neurofibras with irregular thickening, twisting, breaking in brain stems. In AS group, the pathological changes of neurons and gliocytes were slightly similar to that in brains of AH group, but had less dark neurons and receded swelling.4 TEM Ultrastructural ObservationIn result of electron microscope, neurons in W grou showed normal shape, plenty organelles (chondriosome, Golgi's complex, endoplasmic reticulum) in the cytoplasm, regular shape of nuclear, compact auxiliary fibers with symmetrical diameter. In WH group, TEM showed that swelling mitochondria and eurysma cristae, dilated endoplasmic reticulum, and swelling auxiliary fibers, decreasing intensity in macula densa. TEM ultrastructural observation of rat brains in A group, revealed that obvious hydropic mitochondria, eurysma cristae and unintegrated cytolemma. There also were some pyknosis neurons with wrinkle caryotheca, pyknosis chromatospherite and assemble heterochromatin. And interstitial oedema, raritas auxiliary fibers with unsymmetrical diameter and dictyo-demyelinating were also found. The neurons in AH group with the decreased synapses had two significant features: pyknosis (typeⅠ) and swelling (typeⅡ). The former include shrinkage, high electronic dense of cytoplasm and nuclei, irregular crease of nuclear membrane, gathered mass of heterochromatin under the nuclear membrane, and disappearance or shrinkage of nucleole. The latter then include broken cell membranes, severe swelling and higher dense groundplasm of organelles in the cytoplasm, such as endoplasmic reticulum and mitochondrion. In addition, multiplication of glial cells with interstitial edema and neurofibers with irregular thickening were present. Lossen myelin and their distortion, disassociation, rupture from axon and collapse and disappearance of microtubules and microfilaments, synaptic vesicle accumulation were also found. The severe patho-ultrastructural changes of neurons and gliocytes in AS group has been relieved to some degree.5 Immunohistochemistry expression observationIn the primate brains, m-Calpain and Calpastatin were widely distributed in cell membrane, perikarya and their proximal processes and were detected in the brain stem neurons, the cerebellar granular layer and Purkinje cells. The two factorial ANOVA showed that not only a hit or alcoholism had significant effect on m-Calpain/Calpastatin IOD of rat brains (P<0.01), but also the interaction between hit and alcoholism also had (P<0.05, P<0.01). M-Calpain was expressed the least in W group, and Calpastatin the most. On the contrary, m-Calpain was excpressed the most in AH group, and Calpastatin the least. There was not significant difference between A group and AH group in m-Calpain/Calpastatin IOD of rat brains (P>0.05). And m-Calpain IOD-mean of death rats in AH group were higher than that of survival rats on the posterior ventri-brain stem (P<0.05), Calpastatin IOD-mean were lower.Conclusions1. Marked pathohistological and ultrastructural changes were found in the central nervous system of chronic alcoholism rats. The dark neurons, seeming to be a reversible pathologic injury, were found more in AH group, which suggested that the dark neurons might contribute to the patho-morphological basis of the high mortality in rats with TSAH and chronic alcoholism.2. The hit, alcoholism and their interaction had result in strong expression of m-Calpain and weak expression of Calpastatin in brain stem, which suggested that alcoholism or hit might independently or synergisticly activate Calpains'signalling and participate in the neural injury procedure. That might be the patho-metabolism basis of the high mortality rate of chronic alcoholism rats with TSAH.3. Combined with the previous research of our team, alcoholic toxic effects might play a key role in death with TSAH and chronic alcoholism, alcoholism and the hit should exert their effect synergisticly.