Effects of proBDNF on cell proliferation and differentiation in hippocampal dentate gyrus in Alzheimer' disease rat model

To discuss the value of 11C-PIB PET and 18F-FDG micro PET imaging in Alzheimer's disease (AD) rat model. The AD rat model was established by injection of Abeta1-40 into rat's hippocampus. Learning and memory function were estimated by the Morris water maze. Amyloid deposit and neuron loss were observed by Congo red staining and HE staining respectively. 11C-PIB PET and 18F-FDG micro PET scan were performed. Meanwhile the findings of PET imaging were compared with the results of behavior test and histology. The uptake of 18F-FDG in hippocampus of AD rat model group was lower than that of control group (P < 0.01). The Morris water maze showed that the escape latent period of rat model group was longer than that of control group (P < 0.01). Neuron loss was found in rat model brain. Thus the result of micro-PET imaging was matched with those of the Morris water maze and HE staining. 11C-PIB was specifically bound to beta-amyloid in rat model brain, the uptake of 11C-PIB in AD rat model was higher than that of control (P < 0.05). Abeta deposit was observed in Congo red staining so that the result of 11C-PIB Micro-PET imaging was matched with that of beta-amyloid deposition. 11C-PIB Micro-PET imaging shows the plaque deposition while 18F-FDG PET imaging reflects the change of glucose metabolism in hippocampus in AD rat model. And PET scan can be used to verify the successful establishment of AD model rat model.

p-Coumaric acid mitigates passive avoidance memory and hippocampal synaptic plasticity impairments in aluminum chloride-induced Alzheimer's disease rat model

Baicalin has been shown to promote spatial learning and neural regeneration, which might increase the differentiation of neural stem cells in Alzheimer's disease (AD) rat models. We aimed to study the role of baicalin on neuronal pentraxin-1 (NPTX-1), neuronal pentraxin-2 (NPTX-2), and C-reactive protein (CRP) in AD model rats. The 30 male Sprague Dawley rats were divided into three groups: the control group, the AD model group, and the AD + baicalin group. Then, the Morris water maze was used to verify the effect of baicalin on the memory and spatial learning of rats. Immunohistochemistry and immunofluorescence were used to observe the expression of NPTX-1, NPTX-2, and CRP in brain tissue. Compared with the AD model group, the AD rats treated with baicalin spent significantly less time finding escape latencies (P = 0.008) and had longer cross-platform times in the target quadrant (P = 0.015). In addition, the AD + baicalin group had significantly higher numbers of hippocampal neurons compared with the AD model group (P < 0.05). Baicalin also obviously decreased the apoptosis of neurons. Moreover, compared with the AD model group, the NPTX-1 and CRP expression in the AD + baicalin group was significantly reduced (P = 0.000) while the expression of NPTX-2 in the brain tissue of AD rats was significantly increased (P = 0.000). Baicalin can play a therapeutic role by downregulating NPTX-1, upregulating NPTX-2, and downregulating CPR in AD model rats.

To determine the influence of tanshinone on the levels of nitric oxide synthase (NOS) and acetylcholinesterase (AChE) in the brain of an Alzheimer's Disease (AD) rat model and on its potential therapeutic mechanism. 100 Male Sprague Dawley rats were divided into three groups: control group, model group and tanshinone treatment group. 10 microg A beta 1-42 was injected bilaterally into the dorsal lateral region of the dentate gyrus in the hippocampus of rats in the model and tanshinone treatment groups to prepare the AD models. 24h after modeling, tanshinone, 50mg/kg, was administered by gastric perfusion to rats in the tanshinone treatment group. Later, immunohistochemical assay and Western blot analysis were used to detect expression of neuronal NOS (nNOS) and inducible NOS (iNOS) in the rat hippocampus. Activity of AChE in each subregion (CA1 approximately CA4) of rats' hippocampus was determined by a histochemical technique. Expression of nNOS in the model group was down-regulated whereas iNOS was up-regulated. After A beta 1-42 injection, the number of AChE positive fibers in each subregion (CA1 approximately CA4) of the hippocampus was decreased compared with controls. With tanshinone administration, the changes were improved to varying degrees. Tanshinone modulates AChE and NOS proteins concentrations in the hippocampus of AD rats. This may have therapeutic potential in AD rats.

Objective To investigate the effects of tenuigenin (TEN) on expression of brain-derived neurotrophic factor (BDNF), and its receptor tyrosine protein kinase B (TrkB) in the hippocampal CA1 region of Alzheimer's disease (AD) model rats. Methods Sixty male Wistar rats were divided randomly into 4 groups: the control group, the model group, 12.50 mg/ml TEN group and 37.50 mg/ml TEN group. AD model rats were made by injecting ibotenic acid into Meynert basal nuclei of aging rats induced by D-galactose. The expressions of BDNF and its receptor TrkB in the hippocampal CA1 region were measured by immunohistochemistry method. Results The positive expressions of BDNF and TrkB were pale brown and mainly in neuronal cell membrane of the hippocampal CA1 region measured by immunohistochemistry method. The average absorbance values of BDNF and its receptor TrkB in the control group were 0.47 ± 0.02 and 0.46 ± 0.05, while in the model group were 0.30 ± 0.02 and 0.21 ± 0.07 which were significantly lower than that of the control group (P = 0.000, for all). The average absorbance values of BDNF and its receptor TrkB in 12.50 mg/ml TEN group were 0.35 ± 0.05 and 0.32 ± 0.07, which were significantly higher than that of the model group ( P = 0.000, for all) and 37.50 mg/ml TEN group were 0.43 ± 0.05 and 0.37 ± 0.03, which were significantly higher than that of the model group ( P = 0.000, for all). The average absorbance values of BDNF and its receptor TrkB in 37.50 mg/ml TEN group increased significantly than that in 12.50 mg/ml TEN group ( P = 0.000). Conclusions TEN can dose-dependently increase BDNF and its receptor TrkB expression in the hippocampal CA1 region of Alzheimer's disease model rats, which may partly explain the beneficial effects of TEN on cognitive function. doi: 10.3969/j.issn.1672-6731.2014.05.011

Central and peripheral administrations of levothyroxine improved memory performance and amplified brain electrical activity in the rat model of Alzheimer's disease

Objective To investigate neurogenesis in the dentate gyrus of adult rat following conditioned fear stimulus.Methods 24 adult male rats were randomly divided into conditioned fear stimulus group and control group,and all rats were intraperitoneally injected bromodeoxyuridine(BrdU)3 days before conditioned fear stimuli.Animals in conditioned fear stimulus group were given monotone matching foot shock and made conditioned fear stimulus model,animals in control group only give monotone.Freezing time was detected before stimuli and 7th,14th days after stimuli,then rats were sacrificed 7th or 14th day after fear conditioned stimulus,and immunohistochemistry for Brdu was detected.Results Behavioral assessment showed that the percents of freezing time in conditioned fear stimulus group 7th,14th days after conditioned fear stimulus were significantly high(88.68%,91.33%)than those in control group(11.18%,14.23%,P<0.01)and before stimulus(24.11%,16.11%,P<0.01).Compared to control group(1567±28,580±5),Brdu positive cells in dentate gyrus of rat in conditioned fear stimulus group(1364±21,296±22)were decreased,and it reached to a statistical significance on 14th day (P<0.05).Conclusion The decreased neurogenesis in the dentate gyrus of adult rat following conditioned fear stimuli may have relation to fear and other emotion memory. Key words: Conditioned fear stimulus; Neurogenesis; Hippocampus; Rat

The study of therapeutic efficacy and mechanisms of Schisandra chinensis and Evodia rutaecarpa combined treatment in a rat model of Alzheimer's disease

Nitric oxide impairs spatial learning and memory in a rat model of Alzheimer’s disease via disturbance of glutamate response in the hippocampal dentate gyrus during spatial learning

Cognitive dysfunction in Alzheimer's disease is strongly associated with a reduction in synaptic plasticity, which may be induced by oxidative stress. Testosterone is beneficial in learning and memory, although the underlying protective mechanism of testosterone on cognitive performance remains unclear. This study explored the protective mechanism of a subcutaneous injection of 0.75 mg testosterone on cognitive dysfunction induced by bilateral injections of amyloid beta 1-42 oligomers into the lateral ventricles of male rats. Morris water maze test results demonstrated that testosterone treatment remarkably reduced escape latency and path length in Alzheimer's disease rat models. During probe trials, testosterone administration significantly elevated the percentage of time spent in the target quadrant and the number of platform crossings. However, flutamide, an androgen receptor antagonist, inhibited the protective effect of testosterone on cognitive performance in Alzheimer's disease rat models. Nissl staining, immunohistochemistry, western blot assay, and enzyme-linked immunosorbent assay results showed that the number of intact hippocampal pyramidal cells, the dendritic spine density in the hippocampal CA1 region, the immune response and expression level of postsynaptic density protein 95 in the hippocampus, and the activities of superoxide dismutase and glutathione peroxidase were increased with testosterone treatment. In contrast, testosterone treatment reduced malondialdehyde levels. Flutamide inhibited the effects of testosterone on all of these indicators. Our data showed that the protective effect of testosterone on cognitive dysfunction in Alzheimer's disease is mediated via androgen receptors to scavenge free radicals, thereby enhancing synaptic plasticity.

Ginkgo biloba extracts (GBE) have been shown to effectively improve cognitive function in patients with Alzheimer's disease (AD). One potential therapeutic strategy for AD is to prevent loss of adult hippocampal neurons. While recent studies have reported that GBE protects against oxidative stress in neurons, the underlying mechanisms remain unclear. In this study, an AD-like rat model was established via bidirectional injection of amyloid beta 25-35 (Aβ25-35; 20 μg) in the hippocampal CA1 region. Learning and memory abilities of experimental rats were AD assessed in response to oral administration of 7.5 g/L or 15 g/L Ginkgo biloba extract 50 (GBE50) solution and the peroxidation phenomenon of hippocampal mitochondria determined via analysis of mitochondrial H2O2 and several related enzymes. Levels of the oxidative stress-related signaling factor cytochrome C (Cyto C), apoptosis-related proteins (Bax, Bcl-2 and caspase-3) and caspase-activated DNase (CAD) were further detected via western blot. 8-Hydroxydeoxyguanosine (8-OHdG), the major product of DNA oxidative stress, was evaluated to analyze DNA status. Our results showed elevated H2O2 levels and monoamine oxidase (MAO) activity, and conversely, a decrease in the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the hippocampus of AD rats. Administration of GBE50 regulated the activities of these three enzymes and induced a decrease in H2O2. GBE50 exerted regulatory effects on abnormally expressed apoptotic proteins in the AD rat hippocampus, enhancing the expression of Bcl-2, inhibiting release of Cyto C from mitochondria, and suppressing the level of caspase-3 (excluding cleaved caspase-3). Furthermore, GBE50 inhibited DNA damage by lowering the generation of 8-OHdG rather than influencing expression of CAD. The collective findings support a protective role of GBE50 in hippocampal neurons of AD-like animals against mitochondrial oxidative stress.

Objective To observe neural stem cells proliferation, migration and differentiation in hippocampus in developing rats with status epileptictus. Methods 320 healthy SD rats at age 7, 14, 21, 28 d (P7, P14, P21, P28) were randomly divided into status epilepticus (SE) and normal control group. In each group those rats at the same age were further randomly divided into 1, 7, 14, 21, 28 d five time points after PTZ-induced SE (n=8). New cell proliferation and migration were observed by immunohistochemistry studies in the dentate gyrus. Double labeling with Brdu/NeuN and Brdu/GFAP was performed in the P14 rats. Results Nestin positive cells appeared in the dentate gyrus on 1 d after SE in P7, P14, P21, P28 rats. The number of nestin positive cells gradually increased on 7 d and reached a peak on 14 d, then gradually reduced on 21 d, finally fell to a minimum on 28 d after SE. The numbers of nestin positive cells on 7 d（177.00±3.22，t=16.033）and 14 d （195.00±3.41，t=28.840） were significantly higher in the SE group than the NS group (147.50±2.08，136.50±2.65，both P＜0.05). The smaller age of rats with SE onset, the greater the nestin intensity. But the number of nestin positive cells in the dentate gyrus of normal rats were gradually decreased with increasing age. Nestin positive cells were distributed in subgranular zone of dentate gyrus on 1 d and 7 d after SE, then gradually migrated to the granule cell layer on 14 d with morphological changes. Small part of nestin positive cells were ectopically migrated to the hilus of dentate gyrus in P14, P21, P28 age rats, and were also seen in the CA1，CA3 of hippocampus and cortex with various cell morphology. For differentiation of newly generated cells, most of Brdu positive cells co-expressed NeuN and about 4%—5% cells co-expressed GFAP. Conclusions SE could induce neurogenesis in the hippocampal dentate gyrus area in developing rats which has age-related characteristics. Most new cells migrate from the subgranular zone to the granule cell layer of the dentate gyrus, and a small number of newly generated cells ectopically migrated to the hilus of dentate. The majority of newly generated cells differentiate into neurons, and the others differentiate into glial. Key words: Status epilepticus; Hippocampus; Neurogenesis; Neural stem cells

Tau hyperphosphorylation is a main cause of neuronal loss in Alzheimer's disease, which can be caused by many factors, including oxidative stress. The multifunctional protein p62, which exists in neurofibrillary tangles and causes aggregation of hyperphosphorylated tau, not only serves as a receptor in selective autophagy, but also regulates oxidative stress. However, whether p62 participates in oxidative stress-induced tau hyperphosphorylation remains unclear. In this study, we produced an Alzheimer's disease rat model by injecting β-amyloid protein into the hippocampus and β-galactose intraperitoneally. Hematoxylin-eosin staining was used for morphological analysis of brain tissue, and western blotting, immunohistochemistry and reverse transcription-PCR were employed to study p62 and autophagy related proteins, antioxidant defense system kelch-like ECH-associated protein 1-NF-E2-related factor 2 related proteins and hyperphosphorylated tau, respectively. The number of neurons in the brain decreased in Alzheimer's disease rats, and the autophagy related proteins Atg12-Atg5, microtubule-associated protein 1 light chain 3-phosphatidylethanolamine and Beclin1 increased significantly, while p62 expression reduced. Expression of kelch-like ECH-associated protein 1 increased, NF-E2-related factor 2 protein and the downstream gene products of glutamate cysteine ligase catalytic subunit and glutamate cysteine ligase modulatory subunit decreased, and hyperphosphorylated tau increased. These findings demonstrate that autophagy levels increased and p62 levels decreased in the brains of Alzheimer's disease rats. Moreover, the anti-oxidative capability of the NF-E2-related factor 2-antioxidant response element pathway was decreased, which may be the cause of tau hyperphosphorylation in Alzheimer's disease brain tissue and the subsequent structural and functional damage to neurons.

Objective To investigate the effect of combined auricular needling and nerve growth factor (NGF) treatment on learning and memory abilities of Alzheimer's disease (AD) model rats, as well as their possible mechanisms. Methods Forty male SD rats were randomly assigned into four groups: normal control group, AD model group, auricular needling group, and combined auricular needling and NGF treatment group. Okadaic acid was injected into the hippocampal CA1 region of rats to establish AD models, simultaneously, 5 μL NGF (0.1 g/L) was injected into the lateral ventricle of cerebrum for 5 times every other day while acupuncture at ear-kidney-brain auricular points was implemented as well. Morris Water Maze behavioral test was performed to assess the learning and memory abilities of the rats. The neuron apoptosis of the hippocampus was detected by flow cytometer. The expressions of glial fibrillary acidic protein (GFAP) and choline acetyl-transferase (ChAT) were examined by immunohistochemistry. The neurofibrillary tangle (NFT) was observed through Bielschowsky staining of the hippocampus, and Western blotting was used to observe the Tau protein hyperphosphorylation at Ser396 locus. Results As compared with auricular needling group, the combined auricular needling and NGF treatment group had significantly shortened average escape latency in place navigation test, prolonged active time in the third quadrant in spatial probe test, increased number of platform-crossing, decreased apoptosis ratio, decreased GFAP expression and NFT in the hippocampal CA1 region, increased ChAT expression in hippocampal CA1 region, and decreased phosphorylated Tau expression at the site of Ser396 (P<0.05). Conclusion (1) Acupuncture at Ear-Kidney-Brain auricular points combined with NGF obviously improves the learning and memory abilities of the model rats; and it is more effective than auricular needling alone. (2) The mechanisms might be related with reduced neurons apoptosis, decreased abnormal activation and hyperplasia of astrocyte, improved central cholinergic system functions, and inhibited hyperphosphorylated Tau expressions. Key words: Alzheimer's disease; Auricular needling; Nerve growth factor; Tau protein

Both oxidative stress and inflammation are elevated in brains of Alzheimer's disease patients, but their pathogenic significance still remains unclear. Current evidence support the hypothesis that non-steroidal anti-inflammatory drugs (NSAIDs) and antioxidant therapy might protect against the development of Alzheimer's disease, and ibuprofen has the strongest epidemiological support. In the present work our attention was focused on (R)-alpha-lipoic acid considered as a potential neuroprotective agent in Alzheimer's disease therapy. In particular, we investigated a new co-drug (1) obtained by joining (R)-alpha-lipoic acid and ibuprofen via a diamide bond, for evaluating its potential to antagonize the deleterious structural and cognitive effects of beta-amyloid (1-40) in an infused Alzheimer's disease rat model. Our results indicated that infusion of beta-amyloid (1-40) impairs memory performance through a progressive cognitive deterioration; however, ibuprofen and co-drug 1 seemed to protect against behavioural detriment induced by simultaneous administration of beta-amyloid (1-40) protein. The obtained data were supported by the histochemical findings of the present study: beta-amyloid protein was less expressed in 1-treated than in ibuprofen and (R)-alpha-lipoic acid alone-treated cerebral cortex. Taken together, the present findings suggest that co-drug 1 treatment may protect against the cognitive dysfunction induced by intracerebroventricular infusion of beta-amyloid (1-40) in rats. Thus, co-drug 1 could prove useful as a tool for controlling Alzheimer's disease-induced cerebral amyloid deposits and behavioural deterioration.