A new method of microglia sorting and functional characteristics of spinal microglia in aged rats / 中国应用生理学杂志

Objective: To establish an improved method of separating microglia from aged rats and to observe the biological characteristics of spinal microglia of aged rats. Methods: Young SD rats (2 months) were used as control group. Single cell suspension of rat microglia were prepared by trypsin, trypsin substitutes or mechanical net rubbing method. Then, by assessing the purity and survival rate of cells, and observing the morphological characteristics and analyzing the inflammatory functional characteristics, we optimized the isolation and purification method of microglia from aged rats (20 months old) , and observed the functional characteristics of spinal microglia in aged rats. Results: The survival rate of cells digested by pancreatic enzyme was low(young rats 83%, aged rats 60%). Although the survival rate of mechanical net rubbing method was higher than that of pancreatic enzyme digest methods (95%), the cell acquisition rate was lower(young rats(0.207±0.020)×106, aged rats(0.243±0.023)×106). Trypsin substitute dissociation combining density gradient centrifugation method was the best way to get abundant, active and higher survival microglia, and the purity reached more than 85%. We used this method to separate microglia from spinal cord of rats. Compared with the young rats, the spinal cord tissue of old rats was larger, the digestive fluid volume was higher, but the digestion time was shorter. Compared with the young rats, the aged rat spinal microglia had larger and rounder cell body, fewer and shorter protrusions, it tended to be activated morphologically, the level of proinflammatory cytokine IL-1β of microglia in aged rats was lower, and the level of antiinflammatory factor IL-10 was higher. Conclusion: The method of trypsin substitute dissociation combined with density gradient centrifugation was successfully established to isolate and purify microglia from spinal cord of rats, the spinal microglia of old rats showed anti-inflammatory phenotype.

Protective Effects of Tongmaitang Yanming Capsules on Retinal Damage of Aged Rats / 中国药房

OBJECTIVE:To explore the protective effects of Tongmaitang yanming capsules on retinal damage of aged rats. METHODS:Aged rats(aged 24 months)were randomly divided into Tongmaitang yanming capsule high-dose,medium-dose and low-dose groups (1.750,0.875,0.378 g/kg),positive control drug group (Calcium dobesilate capsules,0.175 g/kg) and model group,with 10 rats in each group. They were given relevant medicine intragastrically once a day for consecutive 90 days. At the end of the 90th day,another 10 rats aged 1.5 months were included into blank control group. After medication,60 rats were provid-ed with water but no food for 12 hours. Blood sample was collected from femoral,and the serum levels of GSH-Px,GSH,SOD and MDA was determined,respectively;right eyes were sampled,pathological change of retina was observed by HE staining and the number of retinal cell layer (RCL) neurons was counted. RESULTS:Compared with blank control group,serum levels of GSH-Px,GSH and SOD decreased in model group,while MDA level increased;the number of RCL neurons was (59 ± 4)% of blank control group (P<0.05 or P<0.01),and retina damage was found. Compared with model group,the serum levels of GSH-Px,GSH and SOD increased in positive control drug group and Tongmaitang yanming capsule groups,while MDA level de-creased;the number of RCL neurons in those groups were (98 ± 7)%,(69 ± 5)%,(78 ± 7)% and (95 ± 6)% of blank control group,respectively (P<0.05 or P<0.01);retina damage was relieved. CONCLUSIONS:Tongmaitang yanming capsules can in-hibit aging-induced lipid peroxidation of rat retina,prevent the progressive loss of retina neurons and protect aging-induced retina damage of rats.

Establishment and identification of type 2 diabetes mellitus-induced Alzheimer’s disease rat model / 西安交通大学学报(医学版)

Objective To establish the rat model of type 2 diabetes mellitus (T2DM)-induced Alzheimer’s disease (AD).Methods The rat model of T2DM was established by continuous high fat and high glucose diet in aged rats. Then the rat model was screened and identified by using Morris water maze, cerebrospinal fluid microdialysis technique,ELISA,electrophysiological technique and pathologic method,respectively.Results Compared with the normal group and the T2DM group,the rats in T2DM+DM group had obviously learning and memory impairment;the level ofβ-AP in the hippocampus was significantly higher and the frequency of the spikes induced by Achin the hippocampus was notably lower.Conclusion The rat model of T2DM-induced AD can be successfully established by continuous high fat and high glucose diet in rats.

Immunohistochemical Study on the Distribution of Estrogen Receptor-alpha in the Hippocampus of the Normal Aged Rat

BACKGROUND: In recent years, estrogen has also been shown to modulate the development and function of the brain, bur not exclusively in areas involved with sexual behavior. Among the most novel and fascinating effects of estrogen are those on cognitive function and memory process and their alterations during aging and neurodegenarative disease like Alzheimer. Estrogen receptors distributed not only in the hypothalamus but many different areas, like cerebral cortex, hippocampus, basal forebrain, midbrain, spinal cord, and the diverse action of estrogen is supported by this fact. Numerous studies suggest thai estrogen may be beneficial in preserving cognitive function, but it is not clear yet. PURPOSE: In this study, we perform the immunohistochemical staining in the hippocampus of normal aged rat, and show the distribution of estrogen receptor compared with the neonatal rat. METHODS: we have used antibodies against a estrogen receptor(ER)-alpha to determine their distribution in neonatal and aged SD rat hippocampus. RESULTS: In neonatal rat hippocampus, ER-alpha immunoreactivity was observed in the nucleus of Purkinje cells, whereas in aged rat hippocampus, ER-a immunoreactivity was found mainly in the cytoplasm of Purkinje cells. CONCLUSION: We showed the age related intracellular differential distribution of ER-alpha immunoreactivity in the rat hippocampus. But, further investigations are required to establish whether functional relations like cognitive ability exist with this different intracellular expression of ER-alpha immunoreactivity.

In situ hybridization study on the distribution of nNOS mRNA-positive neurons in the cerebral cortex and cerebellum of aged rats / 대한해부학회지

Nitric oxide (NO) is a diffusible cellular mediator generated by the enzyme nitric oxide synthase (NOS). NO involvement has been demonstrated in mechanisms of synaptic plasticity, particularly in hippocampal long-term potentiation, a mechanism that underlies certain forms of learning and memory. Several data have shown that NO production is decreased in the aged rat. Changes in the nNOS mRNA-containing neurons with aging were demonstrat-ed by in situ hybridization. nNOS mRNA-positive cells in aged rats were present in all cortical areas, hippocampus, and cerebellum and the distribution was similar to that of the young adult group. The number of nNOS mRNA-positive cells was significantly decreased in the occipital (86.2%), parietal (81.2%), and temporal cortices (79.4%), also in hippocampal CA1 (86.2%), dentate gyrus (92.3%), and cerebellar Purkinje cells (73.9%). The most severe decrease was found in hippocampal area. These results are consistent with the former studies showing NO decrease in aging brain and nNOS mRNA decrease indicates the involvement of neuronal system containing NOS in the aging brain, especially for learning and memory.

Immunohistochemical localization of heme oxygenase isozymes in the aged rat retina / 대한해부학회지

Heme oxygenase (HO)-1 and -2 isozymes are important markers for the oxidative stress. We localized HO-1 and HO-2 immunoreactivities and assessed the effect of aging on distribution of HO in the young and aged rat retina by immunohistochemical method. HO-1 and HO-2 showed partly different patterns of localization, indicating possibilities of different regulation of these two isozymes in the retina. HO-2 immunoreactivity was localized to some retinal neurons in ganglion cell layer and inner nuclear layer involving both of plexiform layers and pigment epithelium. The distribution of HO-2 positive cells did not show any difference between aged and young rat. In the case of HO-1, the immunoreactivity is found in retinal layers except outer nuclear layer and layer of rods and cones of young rat retina. The distribution of HO-1 immunoreactivity in old rat retina does not show remarkable difference compared with that in young rat retina, except in inner nuclear layer, outer plexiform layer and pigment epithelium. HO-1 positive components increased in inner nuclear layer, and decreased in outer plexiform layer and pigment epithelium with aging. This may suggest that the possibility of decrease in HO-1 mediated protective function against oxidative stress in outer retinal region of old rat. Another isozyme, HO-2, may not be influenced by normal aging process in rat retina. However, the localization of HO-1 and HO-2 in retina suggests that these two isozymes contribute to visual impairment in normal aging process.

Immunocytochemical Study on the Changes of Cell-Death Controlling Factors in the Hippocampal Formation and Entorhinal Cortex of Aged Rats / 신경정신의학

OBJECTIVES: Hippocampal formation and entorhinal cortex play a part in learning and memory. This study sought to investigate the change of cell-death controlling factors in the hippocampal formation and entorhinal cortex of aged rats. METHODS: Ten aged rats and ten controls were studied. We performed immunocytochemical method using antibodies against NOS, VIP, c-fos , bcl-2, bax and p53 and in situ hybridization. RESULTS: 1) The number of nNOS-immunoreactive(IR) neurons in the entorhinal cortex was significantly decreased in the aged rats(>30%). Morphologically, the number of dendritic branches seemed to be decreased and the length of dendrites showed a tendency to by shortened in the aged group. A major loss of nNOS mRNA positive neurons was observed in the hippocampal formation of the aged rats(>30%). 2) VIP-IR neurons were predominantly bipolar cell. VIP-IR cells were mildly decreased in the hippocampus and subiculum(30%). 4) Bcl-2 mRNA positive neurons were moderately decreased in the hippocampus, subiculum and entorhinal cortex(15-30%), and severely decreased in dentate gyrus of the aged rats(>30%). 5) Bax-IR neurons were similarly distributed between the control and the aged rats, but bax-IR neurons of the aged group, as compared to the control group, were weakly immunostained. 6) P53-IR neurons were only observed in hippocampal CA1 region of the aged rats. CONCLUSION: These results indicate the involvement of neuronal system containing NOS, VIP, c-fos, bcl-2 and p53 in the brain aging process, and provide the morphological evidence for the changes in immunoreactivity of cell-death controlling factors in the hippocampal formation and entorhinal cortex of aged rats.

Immunohistochemical studies on the distribution of microcirculation-controlling factors in the cerebral cortex and hippocampal formation of the aged rat / 대한해부학회지

Recent studies have explored certain changes of neurons containing neuropeptides that are involved in the cerebral microcirculation with aging. However, the degree of loss of vasoactive intestinal polypeptide (VIP)- and neuropeptide Y (NPY)-containing neurons in the aged CNS has not yet been established with certainty. Nitric oxide (NO) from the neuronal NO synthase (nNOS) appears to play the principal role in the cerebral flow response to functional activation. Several findings suggest that NO production may be decreased in the aged rat. Therefore, changes with aging of VIP-, NPY- and NOS-containing neurons were demonstrated by immunohistochemistry in this study. A major loss of VIP-immunoreactive (IR) neurons in the aged rat brain was observed in the frontal cortex area 3, parietal cortex area 1, hindlimb area, temporal cortex area 1 & 2, monocular part of occipital cortex area 1, occipital cortex area 2, and retrosplenial cortex. The axis of VIP neurons in the aged group showed an irregular orientation tendency, especially in layers II and III. Major loss of NPY-IR neurons in the aged rat brain were observed in the retrosplenial cortex, frontal cortex areas 1 and 2, parietal cortex areas 1 and 2, occipital cortex areas 1 and 2, the temporal cortex, hippocampus proper and cingulate cortex. Loss of NPY-IR neurons was observed mostly in layers V and VI. The number of NOS-IR cells was significantly decreased in the aged rat, but the extent of changes was variable in each area.Morphologically, the number of dendritic branches seemed to be decreased in the aged group and the length of dendrites of VIP-, NPY- and NOS-IR neurons showed a tendency to shorten. These results indicate the involvement of VIP-, NPY- and NOS-IR neurons in the aging process in relation to the increased incidence to cerebrovascular disorders in the elderly, and provide the first morphological evidence for the loss of VIP-, NPY- and NOS-IR neurons in each area of cerebral cortex and the hippocampus of the aged rat by immunohistochemistry.

Immunocytochemical study on the distribution of NOS-immunoreactive neurons in the cerebral cortex of aged rats / 대한해부학회지

Nitric oxide (NO) involvement has been demonstrated in mechanisms of synaptic plasticity, particularly in hippocampal long-term potentiation, a mechanism that underlies certain forms of learning and memory. Further, NO has been shown to regulate various neurotransmitters which play an important role in learning and memory. Several findings suggest that NO production may be decreased in the aged rat. Changes in the nNOS-containing neurons with aging were demonstrated by immunocytochemistry and in situ hybridization. NOS-immunoreactive cells in aged rats were present in all cortical areas and the hippocampus, and the pattern of distribution was similar to that of the control group. The number of NOS-immunoreactive cells in the cerebral cortex was significantly decreased in the aged rats, but the extent of changes was variable in each area, and ranged from mild decrease (50%). Severely decreased areas were the cingulate cortex, parietal cortex area 1, temporal cortex area 1, 2, 3, medial part of occipital cortex area 2, monocular and binocular part of occipital cortex area 1, entorhinal cortex, hippocampus proper, dentate gyrus and subiculum. Moderately decreased areas (30~50%) were frontal cortex area 1, 2, 3, parietal cortex area 2, forelimb, hindlimb, lateral part of occipital cortex area 2. Slightly decreased area was insular cortex. Morphologically, the number of dendritic branches seemed to be decreased in aged group and the length of dendrites of NOS-IR neurons showed a tendency to shorten. These results indicate the involvement of neuronal system containing NOS in the aging brain, and provide the first morphological evidence for the loss of NOS neurons in the cerebral cortex of the aged rats by immunocytochemistry. Further multidisciplinary investigations involving normal aging and neurodegenerative disease such as Alzheimer's disease are needed to clarify the importance of nitric oxide changes in the cerebral cortex with aging.

Effects of sapogenin from zhimu (ZMS) and its isomer on learning and memory ability and muscarinic subtype M_1 receptor density in aged rats / 中国药理学通报

AIM To observe the effects of ZMS and ZMR(isomer of ZMS),two active components of Zhimu on learning and memory ability and muscarinic subtype M 1 receptor density in aged rats. METHODS 24 month-old SD rats were randomly divided into aged control group, ZMS and ZMR treatment group. Young rats were used as normal control group. The learning and memory ability was detected by Y-maze method. The muscarinic subtype M 1 receptor density in the brain was detected by 3H-QNB binding tests. RESULTS It was found that daily oral administration of ZMS and ZMR for 40 d significantly enhanced the learning and memory ability and the muscarinic subtype M 1 receptor density in the brain of the aged rats. CONCLUSION These results suggested that ZMS and ZMR probably have potential preventive and curative action for the progressive deterioration of the cholinergic system in Alzheimers disease (AD).