| ObjectiveTo discuss the variation rule of apoptosis and its related genes of the brain tissue around the hematoma in cerebral hemorrhage rats at the acute stage and to study the affection on the apoptosis and its related genes treated by the combination methods of both the acupuncture and traditional Chinese medicineMethodsWe totally used 192 Sprague-Dawley, which was to abbreviate SD, 3 month old male rats. The body weight was 250 to 300 gram. We used random digits table to divided them into 6 big groups, that was the control group, the sham operated group, the model group, the simple acupuncture group, the simple traditional Chinese medicine and the combination of both acupuncture and the traditional Chinese medicine group. As to the control group, it took 8 rats. As to the model group, it took 56 rats and divided into 8 groups, which was the 6 hours group, the 24 hour group, the 72 hours group, the 7 days group, 2 weeks group, 3 weeks group and the 4 weeks group. As to the other four groups, each group took 32 rats and divided into the 6 hours group, the 24 hour group, the 72 hours group and 7 days group. As to these small groups, each group took 8 rats.We used stereo taxis instrument to inject 0.6U collagenaseⅦ, its concentration was 0.2 U per microlitre, into the right caudate nucleus of rats to establish the model of cerebral hemorrhage. As to the sham operated group, we injected 3 microlitre of physiological saline. No operation for the control group. No treatment for the control group, the sham operated group and model group. The acupuncture-point was Dazhui, Fengfu and Shuigou. The treatment method of acupuncture was undertaken once a day. The method of the traditional Chinese medicine was intragastric administration of musk, borneolum syntheticum and Stephania tetrandra S.Moore extract, twice a day.As to the combination of both acupuncture and the traditional Chinese medicine group, the juncture of acupuncture, the acupoint selection, the maneuver of acupuncture, was similar to that of the simple acupuncture group. And the composition, the concentration the intragastric administration time was similar to that of the simple apoptosis and its related genes group.The methods of ethnology, immunohistochemistry and pathology were used. We observed the variation rule of the neurological impairment score, apoptosis and its related genes in different point of time. We studied the effects on those indexes above by the treatment method of simple acupuncture, the simple apoptosis and its related genes and the combination of both the acupuncture and the traditional Chinese medicine.ResultsAs to the neurological impairment score, results were as follows:Firstly, the treatment method of the combination of both the acupuncture and the traditional Chinese medicine could improve the nerve function of the rats with intracerebral hemorrhage.In the model groups, the neurological impairment could be seen in the 6 hours group. Then the neurological impairment lasted for 48 hours. The neurological function began to recover after one week. In the simple acupuncture group, the simple traditional Chinese medicine and the combination of both acupuncture and the traditional Chinese medicine group, the neurological impairment could be seen in the 6 hours group. Then the neurological impairment score attended its peak in the 24 hours group and began to recover after 48 hours, this time point was earlier than that in the model group. To compare with the model groups, the neurological impairment score of the 48 hours of the model group was more serious than that in the 48 hours of the simple acupuncture group, the simple traditional Chinese medicine and the combination of both acupuncture and the traditional Chinese medicine group (P <0.05) . There showed no significantly difference of the neurological impairment score in all time points of the simple acupuncture group, the simple traditional Chinese medicine and the combination of both acupuncture and the traditional Chinese medicine group (P>0.05) .As to the case fatality, the model group took 11 dead rats. But the simple acupuncture group took 5 and the simple traditional Chinese medicine and the combination of both acupuncture and the traditional Chinese medicine group took 4 respectively. Most of the death cases were occurred within 48 hours. The case fatality in 3 big groups above was 23.21 %, 15.60%, 12.50% and 12.50% respectively. There was no significantly difference in the case fatality within these 4 big groups (P>0.05) . Our study showed that the nerve function in the simple acupuncture group, the simple traditional Chinese medicine and the combination of both acupuncture and the traditional Chinese medicine group rehabilitated earlier than that in the model group. This proved that those three methods of treatment could improve the nerve function of intracerebral hemorrhage rats. But all those three methods of treatment could not decrease the case fatality.Secondly, the pathomorphologyical changes of the brain tissue in rats of the model group were as follows:Under the light microscope, widespread bleeding, and histolysis could be seen in the 6 hours model group. Red cell lysis in the hematoma and generous neutrophilic granulocyte infiltration could be seen in the 24 hours model group. In the 72 hours model group, the red blood cell began to absorb in the centre of hematoma and the neutrophilic granulocyte infiltration could be seen. The lymphocyte and mononuclear cell could be seen in this group. In the 7 days model group, the neutrophilic granulocyte was few and the lymphocytosis and desmoplasia could be seen in the centre of hematoma. As to the 2 weeks model group, the dropsy was obviously resolved and there was chronic inflammation and desmoplasia around the hematoma. This proved that there did have inflammatory reaction around the hematoma.Thirdly, the treatment method of both acupuncture and the traditional Chinese medicine could relief the apoptosis of the brain tissue around the hematoma in cerebral hemorrhage rats of the model group at the acute stage.This study used the method of terminal- deoxynucleotidyl transferase mediated d-UTP nick end labeling, which was abbreviated for TUNEL. Results showed that the TUNEL positive cells had hyacinthine or black nucleus, which was irregular and different size. In the model group, the black TUNEL positive cells started to occur and increased and were with deepen staining in the 24 hours and 72 hours model groups. Statistics showed that the TUNEL positive cells began to increase in the 6 hours model group, rapidly increased in the 24 hours model group and was at its peak in the 72 hours model group. It was still higher than the normal value in the 3 weeks model group. The amount of the TUNEL positive cells in the 72 hours model group was significantly higher than that of the other time points of model groups (P <0.01) . And the amount of the TUNEL positive cells in both the 2 week and 4 week model groups was significantly lower than that of the other time points of model groups (P <0.01) .The odds and ends of UNEL positive cells could be seen in the cerebral cortex of the control group. There were sporadic UNEL positive cells around the pin hole in the sham operated group. This proved that the apoptosis attended the course of damaging of the nerve cell after the intracerebral hemorrhage. The UNEL positive cells most were neuron and small part of them were astrocyte and vascular endothelial cell. This showed that the neuron was easier to be damaged in the hemorrhagic brain injure. At the same time, the astrocyte and vascular endothelial cell both was affected. Thus we thought the apoptosis was a mark of the brain tissue damage around the hematoma. It reflected the damage degrees to some extent. The peak of the apoptosis was correspondent with that of the inflammatory reaction after intracerebral hemorrhage, which was from 24 hours to 72 hours. We could consider that the inflammatory reaction after intracerebral hemorrhage was concerned with apoptosis. The more serious the inflammatory reaction, the more serious the apoptosis, the more serious the brain tissue damage.The variation tendency of the UNEL positive cells in the simple acupuncture group, the simple traditional Chinese medicine and the combination of both acupuncture and the traditional Chinese medicine group was similar to that in the model group. But the amount of the apoptosis cell in the 24 hours, 72 hours and 7 days simple acupuncture group, the simple traditional Chinese medicine and the combination of both acupuncture and the traditional Chinese medicine group was significantly lower than that in the corresponding time points of the model groups (P<0.05 or P<0.01 ).The amount of the apoptosis cell in the 6 hours, 72 hours and 7 days simple acupuncture group and the simple traditional Chinese medicine and the combination group was significantly lower than that in the corresponding time points of the model groups (P<0.05 or P<0.01) . Comparison among the three treatment group showed that the amount of the apoptosis cell in the 24 hours and 72 hours simple acupuncture group and 72 hours simple traditional Chinese medicine group was significantly higher than that of the corresponding time points in the combination of both acupuncture and the traditional Chinese medicine group (P<0.05 or P<0.01) . This showed that both the simple acupuncture and the simple traditional Chinese medicine could relief apoptosis. But the effect of the combination of both the acupuncture and the traditional Chinese medicine was better than that in the simple acupuncture and the simple traditional Chinese medicine.Fourthly, the method of the combination of both acupuncture and the traditional Chinese medicine could increase the expression of bcl-2 around hematoma in cerebral hemorrhage rats at the acute stage.Our study used immunohistochemical method to mark bcl-2/bax. Results showed that there was buffy fine grain in the cytoplasm or the cell membrane of the bcl-2/bax positive cell. The nucleus was light blue. Most bcl-2/bax positive cells were neuron. There was slight expression of the amber catachromasis both bcl-2 and bax positive cell in the control group. There was much expression of the yellow trachychromatic bax positive cell and small quantity of amber catachromasis bcl-2 positive cell in the 24 hours and 72 hours model groups. There was much expression of the yellow trachychromatic bcl-2 positive cell in the 7 days model groups. As to the three treatment groups, there was much expression of the yellow trachychromatic bcl-2 positive cell in the 72 hours groups.Statistics showed that the expression of bax had regularity in the model group. It began in the 24 hours group, reached its peak in the 72 hours group and began to decrease in the 7 days group. It kept the normal level of expression in the 2 weeks and 4 weeks groups. As to the expression of bcl-2, there was a little increase in the 6 hours model group. Then it decreased in the 24 hours and 72 hours groups. It began to increase in the 7 days group and lasted for 4 weeks. There was slight expression of bcl-2 and bax in both the control group and the sham operated group. Most of the bcl-2 and bax positive cells, just as the TUNEL positives cells, were neuron. Analysis of the linear correlation showed there was negative correlation between bcl-2 and bax (r=-0.568, P<0.01) . The correlation between the amount of the TUNEL positives cells and the expression of bax was positive (r=0.415和0.662, P <0.01). And the correlation between the amount of the TUNEL positives cells and the expression of bcl-2 was negative (r=-0.709, P<0.01) .The peak of the bax was corresponded with that of the TUNEL.The expression of bcl-2 positive cells in the simple acupuncture group, the simple traditional Chinese medicine and the combination of both acupuncture and the traditional Chinese medicine group was increased and at its peak in the 72 hours. The expression of bcl-2 positive cells in the 72 hours simple acupuncture group, the 72 hours simple traditional Chinese medicine and the 24 hours and 72 hours combination of both acupuncture and the traditional Chinese medicine group was significantly higher that in the corresponding time points in the model group (P<0.05) . The expression of bcl-2 positive cells in the 24 hours, 72 hours and 7 days combination of both acupuncture and the traditional Chinese medicine group was significantly higher that in the corresponding time points in the single acupuncture group (P<0.05 or P<0.01) .The expression of bcl-2 positive cells in all time points of the combination of both acupuncture and the traditional Chinese medicine group was significantly higher that in the corresponding time points in the single traditional Chinese medicine group (P<0.05 or P<0.01). The expression of bcl-2 positive cells in the 6 hours single acupuncture group was significantly higher than that in the 6 hours single traditional Chinese medicine group (P<0.05) . This proved that all the three methods of treatment could increase the expression of bcl-2 positive cells, and the method of the combination of both acupuncture and the traditional Chinese medicine showed better effect in this aspect.The peak of the expression of bax positive cells in the control group could not be seen in the three treatment groups. There was no significant difference between all time point of these three treatment groups and the control group (P>0.05) . And there was no significant difference among all time points of these three treatment groups (P>0.05) .The expression of bax positive cells in the 72 hours single acupuncture group and 72 hours the combination of both acupuncture and the traditional Chinese medicine group was significantly lower than that in the 72 hours model group (P<0.05) . It hints there was no significant affection on the expression of bax positive cells by the single method of traditional Chinese medicine. But the methods of the single acupuncture and the combination of both acupuncture and the traditional Chinese medicine, they could restrain the expression of bax positive cells to some extent.Fifthly, the treatment method of the combination of both acupuncture and the traditional Chinese medicine could restrain the expression of caspase-3 around the hematoma in cerebral hemorrhage rats at the acute stage.Our study used immunohistochemical method to mark caspase-3. Results showed that there was slight amber expression of the caspase-3 positive cells in the control group. Most of the caspase-3 positive cells were neuron. There was much yellow trachychromatic expression of the caspase-3 positive cells in the 6 hours, 24 hours model groups.Statistics showed that the expression of caspase-3 positive cells around the hematoma was significantly increased in the 6 hours model group. It lasted to 72 hours and went down to the normal level in the 7 days group. It went down under the normal in the 3 weeks group but went up to the normal level in the 4 weeks group. That was, the expression of caspase-3 positive cells was earlier than the occurrence of apoptosis. Just as the TUNEL positive cells, most of the caspase-3 positive cells were neuron. The peak of the expression of the TUNEL positive cells was 24 hours to 72hours, while the peak of the expression of the caspase-3 positive cells was 6 hours to 72 hours. Correlation analysis showed there was positive correlation between the number of TUNEL positive cells and that of caspase-3 positive cells (r=0.662, P<0.01).The variation rules of the expression of the caspase-3 positive cells in the three treatment groups were identical to that in the control group. But it began to go down after 72 hours. There was no significant difference among all time points of the three treatment groups in the expression of the caspase-3 positive cells (P>0.05). The expression of the caspase-3 positive cells in the 6 hours, 24 hours and 72 hours the combination of both acupuncture and the traditional Chinese medicine group was significantly lower than that of the corresponding time points of the model groups (P<0.05 or P<0.01) . The expression of the caspase-3 positive cells in the24 hours combination of both acupuncture and the traditional Chinese medicine group was significantly lower than that of the corresponding time point of the simple traditional Chinese medicine group (P<0.05) .ConclusionResults of pathobiology and immunohistochemistry showed that there did have apoptosis around the hematoma of the intracerebral hemorrhage in the acute stage and the peak of apoptosis was from 24 hours to 72 hours.The expression of bax and caspase-3, which were apoptosis promotion genes related to apoptosis, appeared high. And its peak of expression was 24 hours to 72 hours and 6 hours to 72 hours respectively. But the he expression of bcl-2, which were apoptosis suppressor gene, appeared high in the 6 hours model group and then began to decrease. Its high expression was after 7 days.The correlation analysis showed there was positive correlation between the apoptosis and the expression of both bax and caspase-3 positive cells. There was negative correlation between the apoptosis and the expression of bcl-2 positive cells (P <0.01) .The beginning time point and the peak time point of the expression of the caspase-3 positive cells was earlier than that of apoptosis. The peak of the expression of the bax positive cells was corresponding with that of the TUNEL positive cells.The treatment method of simple acupuncture could increase the expression of the bcl-2 positive cells and restrain that of the caspase-3. The treatment method of simple traditional Chinese medicine could only increase the expression of the bcl-2 positive cells and had no depressant effect on that of the bax and caspase-3. The combination of both acupuncture and the traditional Chinese medicine group could increase the expression of the bcl-2 positive cells and restrain that of the caspase-3 and bax, so as to restrain apoptosis. This showed that the method of the combination of both acupuncture and the traditional Chinese medicine had better effect in the aspect of apoptosis inhibition than the method of the simple acupuncture and the simple traditional Chinese medicine.
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