Effects of EGb761 on synaptophysin expression in hippocampus of vascular dementia rats

AIM: To investigate the effects of Ginkgo biloba extract 761 (Egb761) on synaptophysin (SYN) expression in hippocampus of vascular dementia (VD) rats.METHODS: VD rat models, established by repeatedly cerebral ischemia/reperfusion, were randomly divided into two groups: model group and EGb761 treated group (both n=30), and another 30 condition-matched rats were selected as the sham-operated controls. Spatial learning and memory abilities of rats were assessed by Morris water maze (MWM) task, and SYN expression in hippocampal formation of rats in different groups was detected by immunohistochemical technique and image analysis.RESULTS: The MWM escape latency (EL) in model group was highly longer than that in the sham-operated group (P<0.01), while the EL of EGb761-treated group was significantly shorter than that in model group, but still longer than that in the sham-operated group at 1 m, 2 m and 4 m after VD modeling operation (P<0.05 or P<0.01). Immunohistochemical analysis showed that the SYN immunoreactive expression in hippocampal formation in model group greatly decreased and mean optical density of SYN expression highly increased compared with both sham-operated group and EGb761-treated group at three time points (P<0.01).CONCLUSION: EGb761 can increase the expression of SYN in hippocampus, which may be one of important mechanisms of EGb761 in improving learning and memory dysfunction of VD rats.     

Histopathological observation of cerebral cortex and hippocampus in the mouse with synthetic vascular dementia

AIM:To observe pathomorphological changes in cerebral cortex and hippocampus in the mouse with synthetic vascular dementia.METHODS:The synthetic vascular dementia model was produced in the mouse. Animals were killed 7 d, 15 d, and 30 d after the operation, brain tissues were removed and embedded in paraffin. Section of 8μm thickness were stained with hematoxylin-eosin(HE)and Nissl methods, and observed with light microscope.RESULTS:The cerebral cortex in the mouse became thinner on the seventh day, karyopyknosis in partial nervous cells was formed, the number of local neurons was reduced, sieve structure was observed, and glial cells pro liferated, with the similar results 15 d and 30 d afteroperation.Model mouseπs hippocampal cells in CA₁ area were reduced and almost disappeared 30 d after operation.At the same time, glial cells were abundantly proliferated, tu bercles were formed.Cells in CA₂, CA₃ area were also reduced and hippocampal sclerosis occurred.CONCLUSION:Delayed necrosis of hippocampal pyramidal cells may be the pathological basis of ischemia cerebral vascular dementia.     

Effect of concentrated decoction of Chinese herbal compound Buyanghuanwutang on cAMP-PKA-CREB signaling pathway in hippocampus of rats with vascular dementia

AIM: To evaluate the role of concentrated decoction of Chinese herbal compound Buyanghuanwutang (BYHWT) in cyclic adenosine monophosphate(cAMP)-protein kinase A(PKA)-cAMP response element-binding protein(CREB) signaling pathway in hippocampus of rats with vascular dementia (VD). METHODS: The rats were randomly divided into sham operation group (sham-operated rats treated with normal saline), VD model group (VD rats treated with normal saline), BYHWT treatment group (VD rats treated with BYHWT) and nimodipment treating group (VD rats treated with nimodipine). The rat model of VD was build by the method of four-vessel occlusion. The rats in all 4 groups were administered with the corresponding reagents for successive 30 days. The content of cAMP was measured by radioimmunoassay. The expression of PKA catalytic subunit (PKAc) was observed by Western blotting. The changes of DNA-binding activity of CREB in rat hippocampus were detected by electrophoretic mobility shift assay. RESULTS: The content of cAMP, the expression of PKAc and the DNA-bingding activity of CREB in the hippocampus of VD rats were lower than those in the hippocampus of sham-operated rats (P<0.01). The above indexes in both nimodipine treatment group and BYHWT treatment group were definitely higher than those in VD model group (P<0.01). CONCLUSION: BYHWT increases the content of cAMP, the expression of PKAc and the DNA-binding activity of CREB in VD rat hippocampus, thus strengthening the cAMP-PKA-CREB signaling pathway.     

Expression of phosphorylated cell cycle related proteins in developing and aging rat hippocampus

AIM: To observe the expression of phosphorylated cell cycle related proteins cyclin-dependent kinase (CDK) and the retinoblastoma protein (Rb) in the developing and aging rat hippocampus. METHODS: The immunofluorescent staining for anti-phospho- CDK2, anti-phospho- CDC2, anti-phospho-Rb and anti-NeuN antibodies was used to determine their expressions and distributions in Wistar rat hippocampus at different ages. The expressions of phosphorylated cell cycle related proteins were also assessed by Western blotting analysis. RESULTS: (1) The number of neurons of CA1 area in hippocampus decreased during the period from 1 day to 15 months. (2) The positive expressions of phospho- CDK2 and phospho-Rb increased with aging. (3) No apparent change in the expression of phospho- CDC2 in the hippocampus of all the five groups was observed. CONCLUSION: The expressions of phospho-CDK2 and phospho-Rb suggest that differentiated neuron can re-enter the cell cycle and more neurons enter the cell cycle in the aged rat brain. Re-entering the cell cycle of differential neurons probably results in apoptosis.     

The effect of buyanghuanwu decoction on expression of AMPA receptor GluR1 subunit in mRNA and protein levels in rat hippocampus with vascular dementia

AIM: To observe the effect of buyanghuanwu decoction, a Chinese medicine, on the expression of AMPA receptor GluR1 subunit in mRNA and protein levels in rat hippocampus with vascular dementia (VD). METHODS: One hundred and forty-four rats were randomly divided into 4 groups: sham-operation group, VD model group, nimodipine group and buyanghuanwu decoction treatment group. The rat model of VD was built up by the method of 4 vessel occlusion. The VD rats were intragastrically treated with buyanghuanwu decoction suspension (pharmacognostic 50 g·kg-1·d-1) and nimodipine suspension (20 mg·kg-1·d-1) for 30 d. The learning and memory abilities were evaluated by Morris water maze testing. The change of GluR1 protein in hippocampal neurons in each group of rats was measured with immunohistochemistry and Western blotting techniques. The expression of GluR1 mRNA in hippocampus was determined by real-time fluorescence quantitative PCR. RESULTS: Compared to sham-operation group, the average escaping latency period (s) of Water maze tests in VD rats prolonged significantly and cross-platform time (numbers/min) shortened distinctly (P<0.05). The VD rats treated with buyanghuanwu decoction significantly improved the above-mentioned learning and memory performances (P<0.05); no significant difference of above-mentioned learning and memory performances among the rats in sham-operation group, nimodipine group and buyanghuanwu decoction treatment group was observed (P>0.05). Compared to the rats in sham-operation group, the mRNA and protein levels of GluR1 were apparently decreased in VD rats (P<0.05). The mRNA and protein levels of GluR1 in the neurons of hippocampus in buyanghuanwu decoction treated VD rats were higher than those in the model animals (P<0.05), and no difference was discovered in the rats among sham operation group, buyanghuanwu decoction treatment group and nimodipine group (P>0.05). CONCLUSION: Buyanghuanwu decoction improves the learning and memory abilities in VD rats. The therapeutic mechanism is associated with lessening the neuron injury on CA1 field in hippocampus and restoring the mRNA and protein expression of GluR1.     

Tubulin expression of basal cells in patients with Alzheimer's disease and vascular dementia

AIM: To investigate quantitatively the tubulin expression of basal cells in patients with Alzheimer's disease (AD) and vascular dementia (VD) and evaluate its significance in diagnosing Alzheimer's disease. METHODS: α-tubulin of basal cells was examined using LSAB immunohistochemical technique, and analyzed qualitatively and quantitatively by image analyzer. RESULTS: The integrated absorbance (IA, 261.43±25.21) and the average absorbance (AA, 1.89±0.14) in the AD group were significantly higher than those (IA, 120.55±19.71 and AA, 0.85±0.14, respectively) in the VD group (P<0.01). CONCLUSION: These data suggest that the α-tubulin of basal cells could be served as a extraneuronal biological marker in diagnosing Alzheimer's disease early.     

Artificial Calculus Bovis inhibits neuron loss in hippocampus and hilus and protects the GAD positive cells in hippocampus of epileptic rats

AIM:To probe into the anti-epilepsy action of artificial Calculus Bovis, by observing its effect on the behavioral of the experimental epileptic rats, neuron loss in the hippocampus and hilus, and GAD positive cell alteration in the hippocampus. METHODS:SD rats were divided into three groups:group A (artificial Calculus Bovis treatment group);group B (acute epilepsy group) and group C (control group). A model of acute epilepsy rats was established by PTZ. The rat’s behavioral alteration was observed by the Racine’ scale. The neurons in the hippocampus and hilus were calculated by Nissl staining. The GAD positive cells were observed by immunohistochemical staining. RESULTS:The latency of the first seizure in group A was longer than that in group B, while the seizure times in group A was less than that in group B. Besides, in group A, both the neuron loss amount in the hippocampus and hilus and the GAD positive cell loss amount in the hippocampus were less than those in group B. CONCLUSION:The artificial Calculus Bovis prolonged the latency of the first seizure time, decreased the frequency of seizure, and prevented the neuron loss and protected the GAD positive cells.     

Effects of taurine-zinc on learning and memory abilities of vascular dementia mice

AIM:To observe the effects of taurine-zinc (TZC) on the learning and memory abilities of vascular dementia (VD) mice and to investigate the related mechanism. METHODS:The mice were randomly divided into model group, sham group, and TZC at 50 mg/kg, 100 mg/kg and 200 mg/kg groups. The mice in drug groups were given TZC by gavage at 10 mL/kg once daily. The mice in sham group and model group were given equal volume of distilled water. VD mice were established by intercepting both common carotid arteries and bleeding at caudal vein after 14 d of gavage. The levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were detected by ELISA. The levels of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) were measured via spectrophotometer. Step-down test and Morris water maze test were used to examine the abilities of learning and memory in the mice. RESULTS:TZC at 50 mg/kg, 100 mg/kg and 200 mg/kg reduced the levels of TNF-α, IL-1β, iNOS and NO in the brain tissues. In the water maze test, TZC at 100 mg/kg and 200 mg/kg significantly decreased the error times and latency compared with model group. In the step-down test, the escape latency was prolonged and error times were lowered significantly by treatment with TZC at 50 mg/kg, 100 mg/kg and 200 mg/kg as compared with model group. CONCLUSION:TZC improves the abilities of learning and memory, which might be related to the reduction of TNF-α, IL-1β, iNOS and NO levels in VD mice.     

Gene and protein alterations in the hippocampus of rats with temporal lobe epilepsy

AIM:To examine the expression profiles of both genes and proteins in hippocampus of rats with temporal lobe epilepsy (TLE) for revealing the molecular mechanisms of TLE and looking for the candidate targets and new therapeutic approaches in clinical practice.METHODS:Rat temporal lobe epilepsy was induced by administration of lithium chloride and pilocarpine (LiCl-PILO).The expression spectra of genes and proteins were constructed through the techniques of cDNA microarray,two-dimensional (2D) electrophoresis and Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).Subsequently,the differentially expressed genes and proteins were identified and analyzed.RESULTS:There were 192 genes of differential expression observed in hippocampal tissues of LiCl-PILO-induced temporal lobe epilepsy,and 159 genes have been registered in Genbank database,in which 84 genes were up-regulated while 75 genes were down-regulated.78 protein spots of differential display were screened out,in which 31 proteins were detected to be down-regulated and 47 were up-regulated.Finally,5 proteins were identified.CONCLUSION:These genes and proteins found in our study may play pivotal roles in the pathogenic mechanisms of epilepsy and may promise new therapeutic targets for refractory epilepsy in the future.     

Changes of cortisol content in plasma and hippocampus after focal cerebral ischemia-reperfusion in rats

AIM: To investigate the relationship between glucocorticoid (Gc) and injury of hippocampus neurons and the effect of Gc on dementia episode after cerebral ischemia-reperfusion. METHODS: The rat model of middle cerebral artery occlusion (MACO) was established. Cortisol contents in hippocampus and plasma of the model rats were examined by means of the radioimmunoassay at 2 h, 6 h, 12 h, 24 h after reperfusion. RESULTS: The levels of cortisol content in model group were significantly higher than those in sham group and normal group both in hippocampus and plasma. The highest cortisol content was observed at 6 hours after reperfusion. HE staining showed that the impairment of hippocampus neurons was aggravated progressively with reperfusion interval elongating. CONCLUSION: The increased cortisol in hippocampus and plasma, after 2 h cerebral ischemia and 24 h reperfusion, could aggravate the injury of hippocampus neurons and lead to dementia post stroke.     

Effect of ovariectomization on Alzheimer-like phosphorylation of Tau in hippocampus of rats

AIM: To explore the effect of ovariectomization on Alzheimer-like phosphorylation of Tau in hippocampus of Sorague-Dawlery rats. METHODS: An animal model was developed using ovariectimized (OVX) rats, and the phosphorylation of Tau protein was measured by Western blot. RESULTS: The levels of phosphorylated Tau at PHF-1epitope were elevated in ovariectimized rat brain hippocampus 4 weeks and 8 weeks after ovariectomization, when compared with sham-OVX rats (P<0.05). On the other hand, the levels of non-phosphorylated Tau at Tau-1site were decreased in same brain regions at the same time point examined (P<0.05). No significant difference of phosphorylated Tau at those epitops was seen 1 week, 2 weeks, 3 weeks after ovariectomization between sham-OVX group and OVX group (P>0.05). CONCLUSION: Ovariectomization may induce Alzheimer-like hyperphosphorylation of Tau protein in brain hippocampus of rats.     

Effects of prenatal stress on neurons and neuronal ultrastructure of developing hippocampus in rats

AIM:To investigate the effects of prenatal stress (PS) on neurons and neuronal ultrastructure of hippocampus in offspring rats, and to explore the role of the overproduction of oxidants. METHODS:One month male offspring rats were obtained to observe the neuronal number, neuronal ultrastructure and the number of nNOS -positive cell in hippocampus. RESULTS:The neuronal number of CA1 and CA4 subregions in late gestation stress (LS) offspring decreases significantly. The neuronal ultrastructure of CA1 subregion in MS (stress in 7-13 days of gestation) and LS offspring appeared bulgy mitochondria, unclear membrane and irregular electron density. Lipofuscin pigments increased; The number of nNOS-positive cell in CA1, CA2, CA3 subregions and DG of MS group and the whole hippocampus of LS group increased significantly. CONCLUSION:PS damaged the neurons and neuronal ultrastructure of hippocampus of offspring rats. The damages were associated with the overproduction of oxidants.     

Expression of CRF and PKC proteins in hippocampus of rats with cerebral ischemia and reperfusion

AIM: To observe the expression of CRF and PKC in rats with cerebral ischemia.METHODS: Using immunohistochemistry technique we measured the expression quantitatively of CRF and PKC proteins in the hippocampus in rats induced by MCAO at 2 h,6 h and 24 h after reperfusion,contrast to CRF antagonist.RESULTS: (1) CRF: there were lots of positive and deeper dyeing neurons in hippocampus in model group and normal saline group rats,while there were a few positive and lighter dyeing neurons in sham group and CRF antagonist group.The positive expression areas of CRF protein in hippocampus in model group and normal saline group were significantly bigger than those in sham group and CRF-antagonist group(P<0.01),respectively.(2) PKC：there were a great number of denser positive granules in hippocampus in model group and normal saline group rats,while there were a few of scattered positive granules in sham group and CRF antagonist group.The positive expression areas of CRF protein in hippocampus in model group and normal saline group were significantly bigger than that in sham group and CRF-antagonist group (P<0.01),respectively.CONCLUSION: The high expression of CRF and PKC induced by cerebral ischemia may be one important factors that resulted in the delayed neuronal death in hippocampus.The CRF protein activated PKC expression,indicating an important pathology mechanism of nerve tissue damage induced by CRF.     

Effect of Jieyuwan on dendritic spines in prefrontal cortex and hippocampus in WKY depression-like rats

AIM To observe the changes of dendritic spines in prefrontal cortex and hippocampus of Wistar-Kyoto (WKY) depression-like rats, and to explore the effects of Jieyuwan (JYW) on them. METHODS The male WKY rats were selected as the experimental group, and the same strain of Wistar rats were selected as the control group. Firstly, sucrose preference test, open-field experiment and forced swimming test were used to detect the behavior changes in the rats as their baseline. Then, all WKY rats were randomly divided into model (WKY+NaCl) group, WKY+JYW group and WKY+citalopram group. All WKY rats and Wistar rats (Wistar+NaCl group) were administered intragastrically for 21 d, and the changes of behavior after administration were detected by the same behavioral methods. Golgi staining was used to observe the pathological characteristics of dendritic spines in the prefrontal cortex and hippocampus, and Western blot was used to detect the protein expression level of postsynaptic density protein-95 (PSD-95) in the prefrontal cortex and hippocampus. RESULTS Before administration, WKY rats clearly showed depression-like behavior, the density of dendritic spines in the prefrontal cortex and hippocampus decreased significantly (P<0.01), and the protein expression level of PSD-95 was significantly reduced (P<0.01). After treatment with the drugs, the depression-like behavior of WKY rats was significantly attenuated, the density of dendritic spines in the prefrontal cortex and hippocampus increased (P<0.01), and the protein expression level of PSD-95 also increased (P<0.01). CONCLUSION Jieyuwan significantly attenuates the depression-like behavior of WKY rats, and affects the structural changes of dendritic spines and the expression of PSD-95 protein, which further proves that dendritic spines may be one of the importantearly structural changes in depression.     

Effects of dexmedetomidine on behaviors and expression of BDNF and mTOR in hippocampus of depressive rats

AIM: To investigate the effects of dexmedetomidine (DEX) on the behaviors and the expression of brain-derived neurotrophic factor (BDNF) and mammalian target of rapamycin (mTOR) in the hippocampus of depressive rats. METHODS: Sprague-Dawley (SD) rats were randomly divided into 5 groups: sham operation group, model group, and DEX (2.5, 5 and 10 μg/kg) groups. The rats were randomly selected in each group (n=12). The rat depression model was established by chronic unpredictable mild stress and ovariectomy. The rats in DEX groups received daily DEX treatment via intraperitoneal injection for 21 d. The forced swimming immobility time (FSIT) and open-field test were used to evaluate the antidepressant effect of DEX. Escape latency and times of crossing the flat were evaluated by Morris water maze. The histological changes of hippocampal neurons were determined by Nissl staining. The mRNA levels of interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) were detected by RT-qPCR. The protein expression of IL-1β, IL-6, TNF-α and BDNF, and the phosphorylation levels of protein kinase A (PKA), cAMP response element-binding protein (CREB), tropomyosin-related kinase B (TrkB), phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt) and mTOR in hippocampus were evaluated by Western blot. RESULTS: Compared with model group, the FSIT was significantly reduced and the spontaneous activity was markedly increased in DEX groups. The damage of the hippocampal neurons was obviously attenuated, the escape latency was obviously decreased, and times of crossing the flat were markedly increased (P<0.05 or P<0.01). The levels of IL-1β, IL-6 and TNF-α were obviously decreased, and the protein levels of p-PKA, p-CREB, BDNF, p-TrkB and p-PI3K, p-Akt, p-mTOR in hippocampal tissues were obviously increased (P<0.05 or P<0.01). CONCLUSION: Dexmedetomidine improves the behaviors and the spatial learning and memory ability of depressive model rats, which may be related to its anti-inflammatory effects, as well as up-regulating the protein levels of BDNF and p-TrkB, and activating PI3K/Akt/mTOR signaling pathway in the hippocampus.     

Effect of ascorbic acid on chaperone-mediated autophagy in hippocampus of rats with seizures

AIM: To investigate the role of chaperone-mediated autophagy (CMA) and oxidative stress in hippocampal injury induced by seizures and the neuroprotective effect of antioxidant ascorbic acid (AA) on epileptic rats. METHODS: The rats were randomly divided into control group, group with 24 h seizures induced by pilocarpine, group with 24 h seizures and AA pretreatment, and group of AA treatment alone. Both mRNA and protein levels of lysosome-associated membrane protein type 2a (LAMP2a) were tested by RT-PCR and Western blotting to evaluate the activity of CMA. The levels of malondialdehyde (MDA) and superoxide dismutase (SOD) were also measured by chemical methods. RESULTS: The significant increases in mRNA and protein levels of LAMP2a were found in hippocampus of the rats with 24 h seizures. The level of MDA highly increased and the level of SOD obviously decreased in the rats with seizures. AA pretreatment significantly decreased the levels of LAMP2a and MDA induced by seizures, but increased the activity of SOD. CONCLUSION: Both CMA and oxidative stress are activated in hippocampal injury induced by seizures. AA inhibits CMA and reduces oxidative stress activated by seizures by reducing LAMP2a and MDA, and has a potential antiepileptic function.     

Effect of EGB on pituitary-testicular axis and expressions of LHR and StAR in type II diabetic rats

AIM: To observe the effect of the extract of Ginkgo biloba(EGB) on pituitary-testicular axis and the mRNA expressions of luteinizing hormone receptor (LHR) and steroidogenic acute regulatory protein (StAR). METHODS: Thirty male Sprague-Dauley rats were divided into three groups randomly: normal control group, type II diabetic group and EGB treatment group. After fed with high-fat diet for 4 weeks, the later two groups were injected with strepozotocin intraperitoneally to induce type II diabetes mellitus. The EGB treatment group was given EGB at the dose of 50 mg/kg once a day for 12 weeks by intragastric administration. The normal control and diabetic group were given normal saline of equal volume per day for 12 weeks. The indices of blood glucose, insulin and low-density lipoprotein-cholesterol (LDL-c) were measured. The morphologic change of testicular tissue was observed under light microscopy (LM) and transmission electron microscopy (TEM) respectively. The concentrations of blood luteinizing hormone (LH) and testosterone (T) were assayed by the technique of enzyme linked immunosorbent assay (ELISA). The mRNA expressions of LHR and StAR from Leydig cells were detected by RT-PCR. RESULTS: The concentrations of blood glucose, insulin and LDL-c increased obviously, and the testis weights lessened obviously in type II diabetic groups compared to those in normal control groups. Rare spermatogenic cells of seminiferous tubule and germinal arrest were observed in diabetic group under LM. Ultrastructural analysis of testicular tissue by TEM showed dilation of the endoplasmic reticulum and mitochondrial swelling in Leydig cell and sertoli cell in diabetic group. The level of blood LH and T decreased in type II diabetic groups in comparison with that in the normal control group. Compared to normal groups, the mRNA expression of StAR in type II diabetic groups decreased, while the mRNA expression of LHR increased. After the treatment of EGB, the pathological change of testis was relieved, the concentrations of blood glucose, insulin and LDL-c were decreased, the level of blood LH and T, and the mRNA expression of StAR were increased, and the mRNA expression of LHR descended compared to type II diabetic groups. CONCLUSION: EGB may increase the LH-induced testosterone production by correcting metabolic disorder of glucose and lipid, improving the function of pituitary-testicular axis and regulating the expression of LHR and StAR mRNA.