| Background and objectivesVascular dementia is caused by cerebrovascular disease, with impaired learning and memory as the main clinical manifestations of chronic acquired severe cognitive dysfunction syndrome, common sequelae of cerebrovascular disease. Apoptosis is currently considered the pathogenesis of vascular dementia is one.Hippocampus with learning and memory function on the major brain areas is susceptible to ischemic injury. Studies suggest that chronic cerebral ischemia can lead to apoptosis, degeneration of hippocampal cells, and atrophy damage which often lead to the emergence of learning and memory impairment, and is the pathological basis for vascular dementia.P38MAPK plays an important role in p38mapk signaling pathway. iNOS is inducible nitric oxide synthase. Recently, studies have found that iNOS and p38MAPK in acute cerebral ischemia tissue play a role in the process of brain damage through apoptosis.Butylphthalide, which is independently developed in China, is a generation new drug for resistance of non-hemorrhagic cerebral ischemic stroke. It can recover the brain functions, and reduce the clinical symptoms not only by re-constructing the blood of ischemic tissue but also by blocking apoptosis by protecting the mitochondria and reduction of calcium store release of calcium ions et al. Although butylphthalide can inhibit apoptosis, the process of apoptosis is extremely complex. Now, there are few reports on it being used to deal with learning and memory impairment of vascular dementia. So, by vascular dementia rat model, we intend to observe apoptosis, the expression of iNOS and p38MAPK in the hippocampus, and to study the changes of learning and memory after being interfered by butylphthalide, which expects to look for new drugs for the clinical treatment of vascular dementia and to explore for vascular dementia specific molecular mechanismsMaterials and methods1. Groups of experimental animals42 healthy male adult Wistar rats were randomly divided into sham-operation group, model group, butylphthalide group. Each group consisted of 14 animals.2. Animal modelBilateral common carotid artery with a permanent two-side ligation was used in the experiment. Three groups of rats were treated with 10% chloral hydrate (3.5 mg/ kg) intraperitoneal injection of anesthesia. After the success of anesthesia, each rat was given skin incision along the neck, and separated with blunt way from the subcutaneous fascia and muscle tissue to bilateral common carotid arteries. In the rats assigned to model group and butylphthalide group, each common carotid artery was double ligated with No 0 silk suture, and cut from the middle of double ligation. After surgery, each rat was given warm. When they woke, were backed into the clean cage to continue to be raise. Sham-operation group of rats received the same surgical operation without ligation.3. Test dose, method and durationTwo weeks after surgery, the rat received butylphthalide (65 mg/kg) diluted to 2ml with vegetable oil to the stomach in butylphthalide treatment group; but, in the model group and in sham-operation, was only given 2ml vegetable oil. In three groups, each rat was once a day for 2 months.4. Determination of learning and memory in ratsTwo months after the drug oral administration, we test learning and memory ability of rats with a Y-type maze. The correct response was that the electric shock rai could go directly to safety zone, when it was shocked by electric. The total numbers of shock, which were before occurring 9 correct responses times in consecutive 10 responses, were learning ability of rats.24 hours later, each rat was measured 10 times shock, correct response times were representative of memory ability.5. The production of hippocampal tissue sectionsFollowing the Maze testing, the hippocampus drained blood and separated from the brain was put into 4% formaldehyde and kept 24h. Then, it was given conventional dehydration, transparent, being dipped into wax, embedding, coronal orientation slice which thick was 5μm.6. Immunohistochemistry staining and Tunel stainingThese sections were stained by iNOS and p38MAPK immunohistochemistry and TUNEL staining kit requirements before looking at them under the optical microscope at 4007. Image acquisition and analysisMicroscope imaging system made in Germany and Lecia Biosens Digital Imaging System v1.6 were used in the study. Three horizons about the same size were randomly selected and measured gray value in each slice. Then, the value averaged o all the slices were selected as a result. The result was expressed as mean±standard deviation(x±s).8. Statistical analysisData were analyzed using one-way ANOVA and statistical software with SPSS17. LSD method was used to detect the differences between groups. Statistically significant was accepted at p<0.05.Results1. The learning and memory conditions of three groups'ratsIn three groups of rats, the fastest learning and forgetting the least belonged to the sham-operation groups'rats. Their numbers of shocks that were 34.07±3.09 times were at the least before occurring 9 correct responses times in consecutive 10 ones, while the responses of correct times of the rat kept were 8.07±1.07 in consecutive 10 times after 24 hours. In the butylphthalide group, the rats required for the 60.78±4.26 shocks times before occurring 9 correct responses times in consecutive 10 responses. After 24 hours,6.42±1.45 times were kept by the rat. In the model group, they required for the 74.21±3.01times. Comparison among the three groups was statistically significant difference; P value was less than 0.05.2. Hippocampus immunohistochemical staining of iNOSINOS immunohistochemistry appeared in the cytoplasm for different degrees of brown. In the sham-operation group, there were only light brown very small cells to be seen under the light microscope. But, a larger number of dark brown and yellow ones could be seen in the model group. A brown-yellow fewer cells were seen in butylphthalide group, which average gray of positive cells was larger than in the sham-group, but was lower than in the model group. Comparison of the butylphthalide group and the sham-operation group, p value was less than 0.05. Meanwhile, the same state occurred by comparison of the butylphthalide group and the model group.3 Hippocampus immunohistochemical staining of p38MAPKP38MAPK staining should appear in the nucleus for different degrees of brown. In the sham-operation group, a small amount of yellow light brown cells were only seen under the light microscope. In the model group, large amounts dark brown yellow cells were seen. Brown yellow positive cells were seen in the butylphthalide group, which average gray value of positive cells was larger than in the sham-group, but was lower than in the model group. Comparison of the butylphthalide group and the sham-operation group, p value was less than 0.05. Meanwhile, the same state occurred by comparison of the butylphthalide group and the model group.4. Hippocampus cells Tunel stainingIn the sham-operation group, a few cells occurred, which nuclei were stained brown and were fragmentation. Their volume of cytoplasm became smaller than normal volume. In the model group, quantities of such cells increased. In the butylphthalide, such cells were also seen by us, which quantities were less than in the model group but more than in the sham-operation group, their numbers were 61.92±4.19%. In the butylphthalide group, the numbers of apoptotic cells were 35.00±3.32%. The apoptotic numbers of the sham-group were 5.07±2.40%, P<0.05.Conclusions1 Butylphthalide can play the role of preventing and delaying the development for the learning and memory disorders of vascular dementia 2. In the development of vascular dementia, there is a certain relationship with INOS and p38MAPK3. Butylphthalide can block the apoptosis of hippocampal cells to obtain improvement of the memory and learning capacity of vascular dementia by probably reducing the expression of iNOS and p38MAPK. |
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