The Effect Of Acupuncture On Learning And Memory Impairment In Hypoxic-ischemic Brain Damage

How to deal with the asphyxia of new baby is the important problem in Perinatal stage. Asphyxia leads to hypoxemia and acidosis, which injure many organs. And Hypoxic-Ischemic Brain Damage is the most constant reason of brain death and impariment. Now the succeed rate of asphyxia rescue increase along with the use of multi function inspection, so there are more HIBD patient. 25 percent of the survivors suffer the sequela such as paralysis, falling sickness, imbecility, deafness and so on. In developing countries the incidence of HIBD is 3-12 percent, nearly 30 percent of these babies die or be deformity. 15-20 percent of dead fetus in Bengal are because of HIBD;7-10 percent of new babies suffer HIBD in China, about one third of these babies died, three hundred thousand of these babies are deformity in some degree. The expert think there are no unanimous, efficient, integrated treatment to cure it. And the drug for long term curative effect is very few.Researches show that acupuncture can meliorate learning and memory impairment, which is caused by AD, VD, DM and so on. Someone reports that in HIBD early stage point injection can accelerate the sicken babies brain growth, reduce the happening of paralysis. To HIBD rats in early stage stimulating "baihui" point by electroacupuncture may affect the expression of fos protein in hippocampi, so as to improve the learning and memory ability of the new born rats. Now, the integration therapy included acupuncture and other methods is the most constant therapy in clinical practice. And there is good curative effect for acupuncture to deal with HIBD, it can improve the learning and memory ability of sicken babies. But there are few deep research to expatiate the mechanism, so we will use behavior experiment, pathology method, electrophysiology method, etc. expatiate the mechanism of improving learning and memory ability of the sicken babies who suffer HIBD by acupuncture, in search of theoretical basis for clinical practice.The aim of present study was to determine the effects of acupuncture on the late-onset behavioral, morphological and electrophysiological changes in the brain afterasphyxia in perinatal stage of rats and analyze the underlying mechanism by means of behavioral, histochemical and electrophysiological techniques. The study includes two parts of experiments as follows:Part I: Preparation of Animal Model of Learning and Memory Impairment Induced by Asphyxia in Perinatal Stage of rats and the Effect of Acupuncture on Memory DeclineThe aim of this part of experiment was to prepare the model of learning and memory impairment induced by asphyxia in perinatal and determine the effect of acupuncture by use of behaviorak pathological staining (H-E staining) and electrophysiological techniques.Methods: Preparation of Animal Model of Learning and Memory Impairment Induced by Simulating Asphyxia in Perinatal Stage of rats and acupuncture treatment.A total of 102 neonate Wistar rats were used in the present study. The animals of postnatal 1 hour were put into closed container for 1 hour to induce brain damage. Acupuncture therapy was administered at the postnatal 8 days of animal models. The animals were divided into 3 groups: a normal control group (Con n=22), an HIBD group (HIBD n=26) and HIBD with acupuncture group (HIBD+ACU n=20). Learning and memorial behaviors were investigated using the Morris water maze test. The situation of neuron loss in the area of hippocampus was determined by the pathological H-E staining method.LTP was measured in the CA1 area in hippocampal slices. Briefly, rats were decapitated, and the brains were rapidly removed from the skull. The medial part of the hippocampus was cut in 450 jum thick transverse slices. Slices were transferred into a recording chamber containing oxygenated (95%O2, 5%CO2) ASCF. Bipolar stainless-steel electrodes were placed on the afferent fibers of Schaffer collateral commissural fibers of the hippocampus. Glass microelectrodes were used to record population spike (PS) in the CA1 area. A stimulus-response curve to the stimulative strength of 1.5 times of the threshould was recorded for 15 mins to obtain a baseline recording of the PS. After baseline recordings, a high-frequency train of stimuli (lOOHz for Is) was applied, and PS was recorded for another 60min. The average slope and amplitude of the PS at baseline was considered as 100%, and changes in slope and amplitude were expressed as percentages of the values of baseline. Data are presented as Mean±SD. Stimulus-responserelations of the f-EPSP and the expression of LTP among the groups were compared by use of one-way AVOVAR for repeated measurements. Result:1. Overview of HIBD animal modelThe rats will be restless after 10 minutes hypoxia, will be cyanosis, and breath faster and deeper after 15-20 minutes, will move restless acutely, stand on the back legs, lean forward to the bottle opening, breath heavily after 20-30minutes;will be cyanosis more seriously, seemed dark, move slowly and little. There is no difference among all groups in weight while their birth (f>0.05). A month later, all rats grow more weight, but the HIBD group grows more slowly (P<0.05, compared with Con group).2. Morris-water maze testMorris-water maze test is divided into hidden-platform acquisition training and probe trial testing.In hidden-platform acquisition training, the escape latency of HIBD group is longer than the Con groups (P<0.05),but after acupuncture treatment, the escape latency of rats became shorter.After training for seven consecutive days the rats were tested in the water maze without the platform for probe trial testing. Both the swimming time and distance in the target quadrant were measured. The swimming time of the rats in model group (29.44±4.05%) was significantly shorter than that in other groups (P<0.05 as compared with Con group and HIBD+ACU group). The swimming distance of rats in model group(21.86±4.67%) was the shortest among all five groups (P<0.05 as compared with other groups) .The above results indicate that the memory in the rats of model group was been deficient. However, after acupuncture application, the learning and memory capability of the rats has been improved (P<0.05 as compared with HIBD group).3. Pathological changes in the CA1 area of hippocampus of the ratsNeuron loss in the CA1 area of the rats in the model group and attenuation of the neuron loss in the CA1 area were investigated after acupuncture application.4. Long-term potentiation (LTP) in the hippocampusIn addition to behavioral learning, we examined long-term potentiation(LTP) in the hippocampus slice of the rats. The average slope and amplitude of the PS at baseline was considered as 100%, and changes in slope and amplitude were expressed as percentages ofthe values of baseline.After high frequency stimulation the latency of PS became shorter during 60min. High-frequency stimulation of afferent fibers led to an initial increase in the PS slope and amplitude in all groups. But this initial increase was markedly impaired in HIBD animals as compared to controls (P<0.05). In acupuncture treated group, LTP expression was significantly improved as compared to HIBD rats (P<0.05). The duration of LTP in the control group was longer than that in the HIBD group. The stimulus-response relation of the PS slope and amplitude 60 min after high-frequency stimulation in the HIBD group appeared to be reduced than that of control rats, this difference was statistically significant(P<0.05). And the difference between the HIBD+ACU group and the HIBD group was statistically significant (P<0.05).Part II: Mechanism Underlying the Effect of Acupuncture on Learning and Memory Impairment in Hypoxic-ischemic Brain DamageEmpolying immunohistochemical staining method and patch-clamp technique, the role of Glu and GABA in the learning and memory impairment induced by HIBD was explored and the effect of acupuncture on activity of inhibitory and excitatory neurons in the areas of CA1 and CA3 of hippocampus was also determined.Methods:1. The animal model, the experimental groups and acupuncture treatment were the same as described in Part I.2. Immunohistochemistry processAfter Morris-water maze test, the brain samples were fixed and collected for histochemical studies. The rats were deeply anesthetized with sodium pentobarbital (40mg/kg body weight) and perfused through heart with the fixatives for 2h. The brain was dissected out and postfixed in the same fixative for 0.5h. Then the tissue were cryoprotected in 30%concentions of sucrose in 0.1M sodium phosphate buffer at 4 °C and sectioned at 40 u m by frozen section instrument.The immunohistochemistry staining method was used to detect the changes of Glu and GABA expression in the regions of CA1 in the hippocampus. Glu or GABA-reactive sections were analyzed under MIS2000 image analyzer with 20 x objective. White light was used for illumination, and the lighting conditions were held constantly for all themeasurements. The gray densitometrical assay was then performed. The measured area was identical for each slice. About thirty areas were randomly selected for measurement in each group. Statistical analysis among the groups was performed by one way ANOVA.3. Electrophysiological recording by patch clampElectrical signals were recorded in inhibitory and excitatory neurons by whole-cell clamp under infrared differential interference contrast optics. Briefly, rats were decapitated, and the brains were rapidly removed from the skull. The medial part of the hippocampus was cut in 400 ju,m thick transverse slices. Slices were transferred into a recording chamber containing oxygenated (95%O2, 5%CO2) ASCF. Action potentials were recorded in the two types of neurons by whole-cell clamp with AxonClamp-200B amplifiers. Neurons were selected based on their morphology under IR-DIC optics and on their electrophysiological properties. Compared to excitatory neurons, inhibitory neurons display round or irregular soma. Inhibitory neurons possess high input resistance and small decay time constant in response to hyperpolarization pulses. Depolarization pulses evoke high frequency action potentials that typically show short duration, deep fast afterhyperpolarization and little accommodation in frequency/amplitude. These properties match the criteria for inhibitory neurons.Standard pipette solution for recording action potentials contained(mmol/L): KC1 140> HEPES 10> EGTA 10 (pH 7.4). Fresh pipette solutions were filtered with centrifuge filters(0.1ytim pores), and their osmolarity was 295-305 mOsm. The resistance of pipettes was 5-10 Mil. We measured the threshold of action potentials and the number of action potentials. Data are presented as Mean±SD. Comparisions between all groups were examined by Mest.Results:1. Immunohistochemistry resultsThe positive products of Glu and GABA all were seen in the CA1 areas in the hippocampus. The gray density of the Glu reactive products in rats with HIBD were markedly higher than that in the control group (P<0.05). However, acupuncture decreased the Glu expression in the CA1 area of hippocampus (P<0.001). The gray density of the GABA reactive products in rats with HIBD were markedly lower than that in the control group (P<0.05). However, acupuncture raised the GABA expression in the CA1 area of hippocampus (P<0.001).2. Patch clamp resultWe compared the vulnerability of inhibitory and excitatory neurons to HIBD and the effects of acupuncture in slices. The neurons were distinguished based on electrophysiological properties. Compared to excitatory neurons, depolarization pulses evoke fast spiking at inhibitory neurons, where action potential show short duration, deep fast afterhyperpolarization and little accommodation in frequency and amplitude. These criteria are used to identify inhibitory neurons. In addition to increasing the number of action potential, the HIBD lowered the threshold of action potential of excitatory neurons. But for inhibitory neurons, the HIBD reduced the number of action potential, but raised the threshold of action potential. Quantitative measurement shows that acupuncture improved the activity of two types of neurons. The difference between the three groups was statistically significant (P<0.05).DiscussionIn mammals, the hippocampus appears to be involved in some specific forms of behavioral learning. The hippocampus is thought to serve a temporary function in the storage of memory because when the hippocampus is damaged, recent but not remote memories are parallely impaired. On a cellular level, learning is thought to involve activity-dependent plastic modifications of synaptic strength of specific neuronal pathways. One such activity activity-dependent modification is long-term potentiation(LTP) in the hippocampus, which has attracted considerable attention in the search for the mechanisms of learning and memory. LTP can be defined as a prolonged enchancement of the efficacy of excitatory synaptic transmission, which can be elicited in vivo and in vitro by brief high-frequency stimulation of excitatory afferent. Hippocampal LTP has been linked to water maze performance and this form of synaptic plasticity is widely used as a model to study the cellular mechanisms of learning and memory. In our experiment, in addition to behavioral learning by Morris water maze test, we examined synaptic plasticity in the hippocampus of the rats in vitro. In HIBD rats disturbances in water maze learning and hippocampal LTP consistently occur in parallel, in agreement with findings in the control group. The results showed that acupuncture can improve synaptic plasticity by improving the strength of LTP and prolonging the period of LTP. Both declines in the behavioral performance and in hippocampal LTP could be partially prevented by acupuncture.Neural excitotoxicity in the HIBD is believed to be related to an increase in glutamate release, intracellular calcium and free radicals, as well as a deficit of GABA neurotransmission. Clinical attempts to correct these deficiencies are less promising. Morphological studies showed a progressive loss of inhibitory neurons after in vivo ischemia. By simultaneously recording the activities of inhibitory and excitatory neurons in cortical slices with patch-clamp technique, in vitro ischemia primarily impairs the excitability and GABAergic synaptic transmission of inhibitory neurons. The present immunohistochemistry results showed acupuncture decreased the Glu expression and raised the GABA expression in the CA1 area of hippocampus. We discovered that HIBD impaired the activity of inhibitory and excitatory neurons, but acupuncture treatment decreased the excitability of excitatory neurons and rescuing the function of inhibitory neurons.ConclusionIn conclusion, experimental simulating asphyxia in perinatal stage of rats induces learning and memorial impairment and alteration of hippocampal LTP, which are harmful to the process of memory and learning. Acupuncture therapy partially prevents these impairments by improving function of inhibitory and excitatory neurons through decreased the expression of GABA and upgraded the expression of Glu in the hippocampus, thus improve the synaptic plasticity and efficiency of excitatory of the synaptic transmission so as to improve the learning and memory function.