| OBJECTIVE:Alzheimer’s disease (AD) is the most common form of dementiaut in elderly population, associated with progressive memory loss and cognitive dysfunction. It is characterized by extensive cortical neuropathology, including extracellular amyloid-containing neural plaques, intracellular neurofibrillary tangles, and cell and synapse loss in the cortical and subcortical regions of human brain. It is an international difficult problem to find a way to cure it. Acupuncture treatment of alzheimer’s disease, have certain curative effect。Investigate its mechanism, Gui believed that acupuncture can be affected by medical nerve cell apoptosis. And Bao thought that acupuncture may have to affect the neurotransmitter dopamine. A newest research indicated that acupuncture can do some effect to the isoprostane, and so on. All of these studies gave some certain explanations about how acupuncture cure alzheimer’s disease.This paper basing on the PET, from the angle of brain functional imaging, to explore the mechanism of acupuncture curing AD. PET is a non-invasive imaging technique and can observe the activity of cellular metabolism, and it has been applied to explore acupoint specificity and relationship between needling specificity and the response in cerebral regions. Such as JIA S W et al、Dong J C et al、Yin L et al all had done the similar researches basing on PET to explore how needling work. Their results showed that needling in different acupoints could stimulate different brain regions. Our literature review indicated that:(1) All o f these studies were on human bodies, and no PET study needling on rats. (2) No brain functional imaging researches on AD. This paper may firstly use rats, base on PET, chose ST-36(Zhusanli) as the researching point, aim to explore how it work when needling in ST-36.METHODS:This experiment was divided into three parts, part one was to analysis the Brain glucose metabolism of AD modle rats basing on PET; part two and part three was to research the mechanism of needling ST36 curing AD modle rats basing on PET scanning. And the detail methods was as bellow:Time and settingThe study was performed at the Experimental Animals Center of China academy of Chinese medical sciences, PET Experimental Animals Center of General Hospital of PLA, And Inst. of High Energy Physics of Chinese Academy of Science from September 2009 to January 2010.Experimental animals and groupingA total of 75 healthy Wistar rats with 38 male and 37 female,8 weeks old and weighing 200-250g, were provided by the animal center of China Academy of Chinese Medical Sciences. The entire experimental procedure was in accordance with the Guidance suggestions for the care and Use of Laboratory Animals, the Ministry of Science and Technology of the People’s Republic of China. The 75 animals were housed in separate cages under conditions of controlled illumination (12:12 h light/dark cycle), humidity, and temperature(18-22℃). After one-week acclimatization to the home cage, rats were randomly assigned to the healthy control(n=15,8male,7female) and the pre-model(n=60,30male,30female) groups. The HC was not subjected to any treatment. While the pre-model group received D-gal (20 mg in 2 ml 0.9% saline, intraperitoneal injection every day) for 6 weeks. After the six weeks’D-gal intraperitoneal injection, the pre-model group rats were anesthetized with pentobarbital (50 mg/kg). The head was placed in a stereotaxic frame, and a needle with a syringe (Hamilton) was placed in the right NBM:0.9 mm posterior and 2.8 mm lateral to the bregma and 6.9 mm vertically from the skull surface. A stereotaxic injection of 6 Ag of ibotenic acid in 0.3 Al of phosphate-buffered saline (pH 7.4) was performed at a rate of 0.1 Al/min. The syringe was left in place for more than 3 min at the site of injection. The position of the lesion was confirmed using Nissle stain. Tow weeks after the IBO injection, all of the animals(including the HC and the pre-model group) received Y-maze test, compared to HC, rats in the pre-model group fitted the stander of AD model rat were included into the next step. That was a total of 45(23male,22female) AD model succeed rats(10,4male,6female, died during the modeling treat,5,3male,2female, unfitted the stander of AD model rat) and 15healthy rats were included. The total of 60 rats in the Pre-model group,10(4male, and 6female) died during modeling; 5(3male, 2female) was unfit to the criterion of AD model after being tested by Y-maze, were excluded the following study. A total of 45 AD model rats were divided into 3 groups according to the random number table. The details were as follows:the model group(n=15,7male,8female); the sham point group(n=15,7male,8female); the ST36 group(n=15,8male,7female). All of the above 4groups received PET scan.Y-mazeThe apparatus was made of gray plastic with three identical arms (manufactured by Zhangjiagang biomedical instrument plant of Jiangsu province, China). It was regarded as a correct response only if the rat directly moved into the secure arm of Y-maze when it received a footshock (0.6 mA). The rats were trained until they had reached the learning criterion; namely, having 9 correct responses within 10 continuous training trials. The shorter the time required for training, the greater the capacity for learning. If the number of training times exceeded 70, the test was stopped and a learning time score of 70 wasassigned.Needling methodNeedling applications were as follows:after the overlying skin was cleaned with tincture iodine and alcohol, the sterile acupuncture needles (0.10mm in diameter and 0.9mm in length, manufactured by the Shuzhou Medical Appliance Factory, China)were then inserted perpendicularly into the points for 0.5-0.6 mm, and gently twisted, lifted and thrusted in an evenly reinforcing and reducing method. The twisting was within a range of 90-180°and at a rate of 60-90 times/min. The lifting and thrusting were within a range of approximately 0.1-0.2mm and at a rate of 60-90 times/min.[18F]FDG-PET imagingThe first part:All rats were sent to the PET-CT center of the Experimental Animals Center of General Hospital of PLA at 8:00 am after 24 h fasting, and then went through the following sequential procedure:(1) a 20min rest in a darkroom. (2) a tracer injection (18F-FDG, synthesized with Mini Tracer accelerator. O.llmci/kg dosage) via tail vein. (3) a 40min rest:After tracer injection, the model group stayed freely in a small box for 30min.30min later, the model group received gas(Isoflurane) anesthesia, the total time of gas anesthesia was almost 10min. While the sham-point group rats and the ST36 group rats were got needling application in the exact point 2 min before [18F]FDG injection. Then [18F]FDG was injected via tail vein with needle steady in the acupoint(or sham-point). After finishing injection(almost 1 min), needling manipulation continued to carry out as before. And the after injection’s needling manipulation lasted 7 min. Total time of doing this was almost 10min. Then let the rats stayed freely in a small box just as the model group have done.20 min later, sent the rats to the gas anesthesia box to anesthesia, 10min later when the rat were being fully anesthesia, rats were sent to receive PET scanning. (4) a PET scan: PET scans were performed on a Biograph Duo BGO scanner (Siemens, Germany). The images covered the whole brain and were paralleled to the AC-PC line. Image acquisition was started after a 40min uptake period (bed:1; collection mode:3D; slice thickness:3mm; slice interval:2mm; matrix size:256×256; total counts:3×109). Upon the completion of data acquisition, the images were reconstructed using ordered subset expectation maximization (OSEM) with 6 iterations and 16 subsets.The second part:All rats were sent to the PET-CT center of the Experimental Animals Center of General Hospital of PLA at 8:00 am after 24 h fasting, and then went through the following sequential procedure:(1) a 20min rest in a darkroom. (2) a tracer injection (18F-FDG, synthesized with Mini Tracer accelerator. 0.11mci/kg dosage) via tail vein. (3) a 40min rest:After tracer injection, all rats stayed freely in a small box for 35min.35min later, all rats received gas(Isoflurane) anesthesia, the total time of gas anesthesia was almost 40min. (4) a PET scan:PET scans were performed on a Biograph Duo BGO scanner (Siemens, Germany). The images covered the whole brain and were paralleled to the AC-PC line. Image acquisition was started after a 40min uptake period (bed:1; collection mode:3D; slice thickness:3mm; slice interval:1.5mm; matrix size:256×256; total counts:3×109). Upon the completion of data acquisition, the images were reconstructed using ordered subset expectation maximization (OSEM) with 6 iterations and 16 subsets.Statistical analysisThe PET images were processed with the Statistical Parametric Mapping 2.0 (SPM 2.0, the Welcome Department of Cognitive Neurology, University College London, UK). After realignment and normalization, the images were smoothed spatially using a 15mm×15mm×15mm Gaussian kernel. The data of HC, the model group, the sham-point group and the ST36 group were analyzed with a two-sample t test, (extend threshold:k=100 voxels, p<0.005).RESULTS:The first PET scan results:compare to the healthy control group, the modle group showed lower brain glucose metabolism in right parietal lobe and a higer point in the limbic system. compared to the model group, the activated brain areas in the sham-point group were mainly centered on bilateral limbic system, right frontal lobe, striatum; while they were mainly centered on bilateral limbic system(piriform cortex), bilateral temporal lobe(olfactory cortex), right amygdale and right hippocampus in the ST36 group. Compared to the sham-point group, the ST36 group showed activated brain regions in bilateral amygdale and left temporal lobe.The second PET scan results:(1) results between different groups:Compared to the normal group, the model group shown a higher glycol metabolism in the R Poaterior Lobe, R Pons:tegmentum, R Medulla Oblongata, L Anterior Lobe, L Poaterior Lobe, L Mesencephalon: tegmentum, L Medulla Oblongata; Compared to the model group, the sham point group shown a higher glycol metabolism in the L Occipital Lobe:Visual Cortex; compared to the model group, the ST36 group shown a higher glycol metabolism in the brain areas of R Dorsal Thalamus:lateral nucleus group, R Mesencephalon: tegmentum; compared to the sham-point group, the ST36 group shown a higher glycol metabolism in the brain areas of R Anterior Lobe.(2) Brain activated ereas after treatment compared to before treatment in the same group:compared to before treatment, activation brain areas after treatment in the normal group were as bellow:parietal lobe:sensory cortex; and compared to before treatment, activation brain areas after treatment in the model group were as bellow:R Frontal Lobe:Prelimbic Cortex, R Frontal Lobe:Motor Cortex, R Occipital Lobe:Visual Cortex, R Limbic System:Retrosplenial Cortex, R Corpus Callosum R Parietal Lobe:Sensory Cortex, R Limbic System:Cingulate Gyrus, R Dorsal, Thalamus:lateral nucleus group, R Hippocampus, R Hippocampus:Dentate Gyrus, R Anterior Lobe, R Poaterior Lobe, R Mesencephalon:superior colliculus, R Mesencephalon:inferior colliculus, R Mesencephalon:tegmentum, R Mesencephalon: pretectal area, L Frontal Lobe:Motor Cortex, L Occipital Lobe:Visual Cortex, L Limbic System:Retrosplenial Cortex, L Corpus Callosum, L Parietal Lobe:Sensory Cortex, L Limbic System:Cingulate Gyrus, L Limbic System:Septal Area, L Striatum, L Dorsal Thalamus:lateral nucleus group, L Hippocampus, L Hippocampus: Dentate Gyrus, L Anterior Lobe,L Poaterior Lobe, L Mesencephalon:superior colliculus, L Mesencephalon:inferior colliculus, L Mesencephalon:pretectal area, L Parietal Lobe:Parietal Association Cortex; compared to before treatment, activation brain areas after treatment in the sham point group were as bellow:R Frontal Lobe: Prelimbic Cortex, R Frontal Lobe:Motor Cortex, R Occipital Lobe:Visual Cortex, R Limbic System:Retrosplenial Cortex, R Corpus Callosum, R Parietal Lobe:Sensory Cortex, R Limbic System:Cingulate Gyrus, R Hippocampus, R Mesencephalon: superior colliculus, R Mesencephalon:inferior colliculus, R Mesencephalon: tegmentum, L Frontal Lobe:Motor Cortex, L Occipital Lobe:Visual Cortex, L Limbic System:Retrosplenial Cortex, L Corpus Callosum, L Parietal Lobe:Sensory Cortex, L Limbic System:Cingulate Gyrus, L Hippocampus, L Anterior Lobe, L Mesencephalon:superior colliculus, L Mesencephalon:inferior colliculus, L Parietal Lobe:Parietal Association Cortex; then, compared to before treatment, activation brain areas after treatment in the ST36 group were as bellow:R Olfactory Bulb, R Frontal Lobe:Prelimbic Cortex, R Frontal Lobe:Motor Cortex, R Temporal Lobe: Olfactory Cortex, R Occipital Lobe:Visual Cortex, R Limbic System:Retrosplenial Cortex, R Corpus Callosum, R Parietal Lobe:Sensory Cortex, R Limbic System: Cingulate Gyrus, R Hippocampus, R Anterior Lobe, R Cerebellum Nucleus, R Mesencephalon.superior colliculus, R Mesencephalon:inferior colliculus, R Mesencephalon:tegmenturn, R Pons:tegmentum, L Frontal Lobe:Prelimbic Cortex, L Frontal Lobe:Motor Cortex, L Temporal Lobe:Auditory Cortex, L Temporal Lobe: Olfactory Cortex, L Occipital Lobe:Visual Cortex, L Limbic System:Retrosplenial Cortex, L Claustral layer, L Corpus Callosum, L Parietal. Lobe:Sensory Cortex, L Accumbens Nucleus, L Limbic System:Cingulate Gyrus, L Limbic System:Septal Area, L Striatum, L Dorsal Thalamus:lateral nucleus group, L Hippocampus, L Hippocampus:Dentate Gyrus, L Anterior Lobe, I, Mesencephalon:superior colliculus, L Mesencephalon:inferior colliculus, L Mesencephalon:tegmentum, L Pons: tegmentum, L Medulla Oblongata, L Third Ventricle.Y-maze results:The learning and memory skill in different days existed significant difference. In the normal group, there were no significant difference between the first day and the second day, but bo th of the tow days were much higher than the value of the third day, the forth day and the thirty-one day.In the model group, the value of the first day was much higher than that of the other next four days. And there was no significant different between the thirty one day and the second, the third and the forth day. In the sham point group, the value of the first day was much higher than the next four days and the value of the thirty one day was much lower than that of the second, the third and the forth day. In the ST36 group, the value of the first day was much higher than the next four days and the value of the thirty one day was much lower than that of the second, the third and the forth day.Results according to multiple comparison analysis shown that, the value of the five days in the normal group were much lower than that in the other three groups. Besides, the value of the thirty one day in the model group was much higher than that in the other three groups.CONCLUSIONS:intraperitoneal injection D-gal combined with Nuclei basales injection IBO is a feasible way to make AD modle. PET imaging indicated that PET imaging may be a good way the help earlier diagnosis AD. we assumed that needling in sham-point or ST36 can increase the blood perfusion and glycol metabolism in certain brain areas to improve the cognitive of AD patients. after 20 days therapy, it actived some particulary brain earas that may express cuvative effection. And the Y-maze results indicated that, needling ST36, can improve the memory level of the AD modle rats. |
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