| Bu Yang Huan Wu Decoction from Qing dynasty Wang Qing-ren's "Medical forest error correction", is composing by seven kinds of traditional Chinese drugs, such as raw astragalus, radix angelica sinensis, radix paeoniae rubra, ligusticum chuanxiong hort, semen persicae, carthamus tinctorius 1. and lumbricus. It has the effectiveness of invigorating and promoting vital energy, activating blood circulation to dissipate blood stasis, dredging the meridian passage. Now the party is widely used in ischemic brain injury, spinal cord and peripheral nerve injury. In recent years, many researches show that BYHWD can improve ischemia-reperfusion injury, by many targets, many link. In order to explore the BYHWD on its targets which reduce the ability of learning and memoring of ischemic brain injury and its mechanism of prevention.And to clarify that BYHWD for promoting nerve damage repair, improve the ability of learning and memory the clinical value of traditional herbal, for offering new studies basis to the clinical application of the traditional Chinese herbal medicine. We have started the following topics about BYHWD for research as follow:1. Made a model of chronic cerebral ischemia in rats, devide in groups, and give drugs Using permanent ligation rats bilateral common carotid artery method (2-VO) for modeling chronic cerebral ischemia, This method is essentially caused by cerebral ischemia reperfusion chronic low oxygen, which eventually led to the nerve cell function decline, learning and memory capacity obstacles, simulate human for atherosclerosis, arterial lumen stenosis, cerebral ischemia infarction. This experiment chooses SPF, male, weight 180-220g SD rats as 2-VO mode. Before the operation we have to forbid the rats eat anything(not contain water), intraperitoneal injection water chloric aldehyde (0.35 g.k g-1) to anesthesia, then tied the rates on the surgery shelf, cut the hair on the neck and show the rates'skin, disinfect by the iodine and 75% ethanol, cut the skin and subcutaneous tissue, about 1.5 cm, separate the bilateral carotid artery and the vagal between the anterior cervical muscles and the bilateral cervical muscles, make a double ligation through bilateral carotid artery,,and then suture, put some sulfonamides sterilization powder on the wound to prevent infection. 10 rats in the sham operation group do the same procedures with the the mode group beside suture. After 3 weeks, the rats begin the chronic ischemic attacks, at the forth week begin the morris water maze test..Base on the result of the morris water maze test, the rats as the mode successful are devided into four guoups randomly:,model control group, BYHWD low-dose group, BYHWD high-dose group, positive control group.5 animals groups:sham-operated control group (given an equal volume of physiological saline), model control group (given an equal volume of physiological saline), BYHWD low-dose group (20g/kg), BYHWD high-dose group (40g/kg), positive control group (Ginaton tablet,0.045g/kg), n=10 animals, according to 1ml/100g intragastric administration, continuous administration of four weeks.2. The effectiveness of BYHWD on the ability of learning and memory in chronic cerebral ischemia rats This topic apply to the morris water maze test and the shuttle box test to explore the effectiveness of BYHWD on the ability of learning and memory in chronic cerebral ischemia rats. The Morris water maze test contain Place navigation test(PNT) and Spatial probe test(SPT). PNT spend 5 days, a circulation each day,4 times each ciculation. Choose a position to make the rats face to water and put into the water, observing and recording how long does the rats climbed platform. Results showed that all groups of animals progressively shorten latency of finding the platform, thereinto, comparing the model control group and sham operation control group, in the training of the first, second and a three-day incubation period of time, it has a significant difference (P<0.05); with the model control group, BYHWD high-dose group and the positive drug control group on the first day of BYHWD low-dose group in the first, second and third days were significantly different (P <0.05), prompted all drug groups after modeling can improve learning and memory in rats. Spatial probetest(SPT):platform used to measure the spatial position memory capacity of rats after learning to find the platform. At the sixth day, remove platform, then select the second point to make the rats face to water and put them into the water, write down how many times do the rats swim through the the original platform in 120 seconds. The results showed that the model control group, BYHWD high-and low-dose group and positive drug control group, the number of platforms were reduced through the sham-operated control group,42.46%,56.16%,64.38%,76.71%, of which model control group and the sham-operated control group were significant difference. (P<0.05). with the model control group, the ability of platform spatial location memory for drug-treated rats was significantly different in which the positive control group markedly enhanced. (P<0.05).2.2 The two-way avoidance test (shuttle box test) using experimental box automatic recording each rat the reaction and and cubation period on electrical stimulation (light or and buzzer). Recording the rat actively avoid time and recording how long do the rat actively avoid and passive avoidance.Result showed in the four tests, each group animals, the number of active avoidance and active avoidance response latency (AARL) was found to first increase and then slightly decreasing trend. Each dose group of BYHWD and the positive drug control group, the frequency and duration compared with model control group increased, passive avoidance response latency was firstly decrease, and then turn into the increasing trend. But there was no significant difference. Base on the result of the Morris water maze test, it suggests that drug-treated rats after modeling can be enhanced the action learning ability and stressed the memmory.3.The reseach mechanism of improving the ability of learning and memory in chronic cerebral ischemia rats.3.1 Determination of cortex blood flow To explore the effectiveness of BYHWD on the blood flow in chronic cerebral ischemia rats, this topic using the rat brain solid positioner on each group to measure cortenx blood flow and compare the result. The results, in addition to sham-operated group outside each dose group were significantly decreased cerebral blood flow. In the model control group, BYHWD high-and low-dose group, the positive drug control group, cortical blood flow values were decreased to sham-operated group 50.22%(P<0.05),73.90%,67.30%,75.65%. But cerebral blood flow of BYHWD each dose group and the positive drug control group was significantly less than the model group. Compare with the model group and the control group, BYHWD high-dose group and the positive drug control group were significantly different with it (P<0.05), and the BYHWD degree of improvement of cerebral blood flow was enhanced with increasing dose. It suggests that BYHWD can obviously reduce the blood flow in chronic cerebral ischemia rats, and improve ischemia.3.2 Determination of blood flow changesChronic cerebral ischemia, change with blood flow, it's performance contain viscosity of whole blood and plasma, blood platelet aggregation rate increases, HCT increase, plasma fibrinogen increased, etc. This experiment use the blood instrument to measure the blood high, medium and low shear viscosity in each group of rats.The results showed that compared with the sham operation group, model control group significantly increased blood viscosity, which in the shear rate 150,30,5 mPas.s significant difference (P<0.05); compare with the model group, BYHWD each dose group and the positive drug control group was able to reduce the blood viscosity, in order to reduce cerebral ischemic may result in abnormalities of blood rheology on the brain tissue damage. Thereinto BYHWD high dose in the shear rate 1mPas.s and BYHWD low-dose in the shear rate 150,30,5,1 mPas.s were significant difference (P <0.05). It suggests that BYHWD can reduce viscosity of blood and improve ischemia.3.3 The effect of changing the chronic cerebral ischemia organization formTo compare the variant of each group rats ischemia organization form, and observe the effect of changing the chronic cerebral ischemia organization form.According to the procedures and finishing the tissue samples hematoxylin-eosin staining and tar violet staining. The result as follow: Hematoxylin-eosin staining:(1) Sham operation group:the cells surrounding space is normal, form is regular, the cell in hippocampal CA1 area line orderly. (2) Model group:the brain tissue is osteoporotic, the parietal lobe cortex mild ischemia change, neuron decrease, the cell line loosely, the cell surrounding space increase, capillary surrounding appear gap obviously, the nerve cell in hippocampal CA1 area decrease, line disorderly, not hierarchical, and some cells are pyknotic and deep stained.(3) Medicine group:the positive drug control group and BYHWD low does group and high does group's rats Cortex neuron have a little increase.,the form is not regular, the cell in hippocampal CA1 area line disorderly, the Volume of the nerve cell become a little small.Tar violet staining:(1) Sham operation group:the cells'form are regular, cytoplasm containing rich tar purple, stain is well-distributed, surrounding Nissl's body is blue, the nucleus without any color, nucleoli is stained, the cell in hippocampal CA1 area line orderly. About 4 to 5 floor, the cells surrounding space is normal.(2) Model group:the Volume of the cortex pyramid cells become small, the form is not regular, pyknotic is deep violet, cells and vascular surrounding space become big obviously, the cell in hippocampal CA1 area is loose, and line disorderly, some cytoplasm Nissl's body become small obviously, rather more disappear, some cells pyknotic is violet.(3) Medicine group:the cells of the positive drug control group and BYHWD low does group and high does group's rats in hippocampal CA1 area are about 2 to 3 floor, line disorderly, the volume of the nerve cells become a little small, the form is not regular, and stain is not well-distributed, Nissl's body reduce.It suggests that BYHWD can protect chronic cerebral ischemia rats'nerve cell in hippocampal CA1 area, and reduce nerve cell dying. 3.4 The effectiveness of PAF content in brain tissueAdopt enzyme-linked immunosorbent assay (ELISA assay) to measure PAF content in brain tissue. The results showed that compared with the sham operation group, model control group, PAF content in brain tissue of each BYHWD dose and the positive drug control group was significantly increased and different (P<0.01). Comparing the model control group, the PAF content in brain tissue of BYHWD high-and low-dose, and positive drug control group was significantly reduced and different (P<0.01). Results show that can reduce inflammation through decrease the brain release PAF, improve the chronic cerebral ischemia.4. Conclusion4.1 BYHWD can obviously enhance action learning ability and learning memory ability, it is relevant to drugs does.4.2 The mechanism that BYHWD can obviously enhance action learning ability and learning memory ability is relevant to these factors:obviously improve mode rats blood flow decreasing, improve ischemia, reduce mode rats blood viscosity, improve brain tissue blood perfusion, protect mode rats nerve cells in hippocampal CA1 area, reduce nerve cells dying, decrease mode rats brain tissue release PAF, reduce mode rats brain tissue inflammation., prevent PAF leading inflammation amplification effect is relevant to chronic ischemia mode rats brain tissue secondary damage.4.3This project suggest us that through the role of many links and target point,can obviously improve chronic ischemia, reduce viscosity of blood, protect nerve cells, prevent PAF leading secondary damage, thus improve learning and memory ability decline induced by the chronic cerebral ischemia, and it afford BYHWD can improve learning memory ability decrease by ischemia a new experiment basis. |
