Spatial Probe Test Research Articles

Effect of Nattokinase in D-galactose- and Aluminum Chloride-induced Alzheimer's Disease Model of Rat.

Alzheimer's disease (AD) is the most common form of dementia worldwide. Nattokinase is a serine protease extracellularly produced by natto, a fermented product of Bacillus subtilis var. natto. In this study, we investigated the therapeutic effects of nattokinase in a rat model of AD induced by aluminum and D-galactose. Forty Wistar rats were randomly divided into four groups: normal, vehicle, and orally administered nattokinase (NK65 and NK130 groups). Except for the normal group, all groups were treated with AlCl3 and D-galactose for 10 weeks. The NK65 and NK130 groups additionally received 65 mg/kg/day and 130 mg/kg/day nattokinase, respectively. We analyzed β-amyloid levels in the cerebrospinal fluid (CSF), and the spatial reference test was evaluated using the Morris water maze test. After the Morris water maze test, rats of all groups were subjected to micro-computed tomography (μCT) to assess constructional changes in the brain. Aluminum concentration and β-amyloid levels were analyzed by histochemical staining in all brain tissues. Oral administration of nattokinase in the AD rat model increased free-form β-amyloid levels in the CSF and improved aluminum and amyloid plaque accumulation in the brain. Brain μCT images showed enhanced brain volume with fewer constructional changes after treatment with nattokinase. In the behavioral tests, both the escape latency time in the spatial reference test and the time taken to cross the platform area in the spatial probe test improved partially. The results suggest that nattokinase has potential therapeutic applications in the treatment of AD.

Using Deep Ocean Water in the Fermentation of Antrodia cinnamomea to Boost Magnesium Ion Bioabsorption and Anti-Inflammatory Effects in the Brain of an Alzheimer’s Disease Rat Model

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by the deposition of amyloid β-peptide (Aβ) and subsequent oxidative inflammatory response, leading to brain damage and memory loss. This study explores the potential of Antrodia cinnamomea (AC), a Taiwan-native fungus known for its anti-inflammatory and antioxidant properties. The metabolites of AC, including dehydroeburicoic acid (DEA), 4-acetylantroquinonol B (4-AAQB), dehydrosulphurenic acid (DSA), and polysaccharides, were of particular interest. In the experiment, deep ocean water (DOW) was used to facilitate the solid-state fermentation of Antrodia cinnamomea NTTU 206 (D-AC), aiming to enhance its functional components. The impact of D-AC on the modulation of AD-related risk factors and the augmentation of cognitive abilities was subsequently evaluated in an AD rat model. This model was established via consecutive infusions of Aβ40 into the brain over a 28-day period. The administration of D-AC resulted in remarkable improvements in the rats’ reference memory, spatial probe test, and working memory. Notably, it restored the hippocampal magnesium levels by upregulating the expression of the magnesium transporter MAGT1. Concurrently, D-AC significantly downregulated the expressions of β-secretase 1 (BACE1) and the phosphorylated tau protein (p-tau), which were both implicated in AD progression. Additionally, it mitigated inflammatory responses, as suggested by the decreased levels of tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in the hippocampus and cerebral cortex. Ultimately, the ability of D-AC to restore the brain magnesium levels, attenuate inflammatory responses, and reduce hippocampal Aβ40 deposition led to significant improvements in the cognitive decline of AD rats. D-AC demonstrated a comparable efficacy with its counterpart, AC fruiting bodies (F-AC group), despite their componential differences. This study underscores the potential of D-AC, enriched through fermentation, as a novel dietary strategy for Alzheimer’s disease prevention.

Electroacupuncture at "Baihui"（GV20） and "Yongquan"（KI1） improves learning-memory ability of APP/PS1 double transgenic mice by regulating endosomal-lysosomal system

To explore the mechanism of electroacupuncture（EA） in improving learning-memory ability in Alzheimer's disease （AD） mice from the perspective of endosomal-lysosomal system. Male APP/PS1 transgenic mice were randomly divided into model group and EA group （n=10 in each group） and 10 male C57BL/6 wild mice were taken as the normal group. EA （1 Hz/50 Hz, 1 mA） was applied at bilateral "Yongquan"（KI1） and acupuncture was applied at "Baihui" （GV20） for 15 min. The mice of the model and normal groups were subjected to restriction with the same method as those of the EA group for 15 min. The treatment was conducted once every other day for 6 weeks. The spatial learning-memory ability （shown by escape latency of place navigation test and the time of crossing the target platform and total swimming distance in the target quadrant in 1 min of spatial probe test ） was detected by Morris water maze test. The immunoactivity of senile plaques （SP） in the hippocampus tissue was detected by immunohistochemistry. The ultrastructural characters of hippocampal neurons were observed by transmission electron microscope, and the expression levels of Ras-related protein 5 （Rab5）, Ras-related protein 7 （Rab7） and cathepsin D （CTSD） in the hippocampus were detected by Western blot, separately. Compared with the normal group, the escape latency, SP immunoactivity, and protein expression levels of Rab5, Rab7 and CTSD were significantly increased （P<0.05, P<0.01）, while the number of crossing the original platform and the total swimming distance in the platform quadrant were considerably reduced （P<0.05） in the model group. In contrast to the model group, the EA group had a marked decrease in the escape latency, SP immunoactivity, and protein expression levels of Rab5, Rab7 and CTSD （P<0.05, P<0.01）, and a striking increase in the number of crossing the original platform and the swimming distance in the platform quadrant （P<0.05）. Results of transmission electron microscope showed an accumulation of endosome, lysosome, and endolysosomes in the hippocampal neurons in the model group, which was evidently milder in the EA group. EA of GV20 and KI1 can improve the learning-memory ability of AD mice, which may be related to its function in reducing hippocampal Aβ deposition and down-regulating endosomal-lysosomal system activity.

Electroacupuncture at "Baihui"（GV20） and "Yongquan"（KI1） improves learning-memory ability of APP/PS1 double transgenic mice by regulating endosomal-lysosomal system

To explore the mechanism of electroacupuncture（EA） in improving learning-memory ability in Alzheimer's disease （AD） mice from the perspective of endosomal-lysosomal system. Male APP/PS1 transgenic mice were randomly divided into model group and EA group （n=10 in each group） and 10 male C57BL/6 wild mice were taken as the normal group. EA （1 Hz/50 Hz, 1 mA） was applied at bilateral "Yongquan"（KI1） and acupuncture was applied at "Baihui" （GV20） for 15 min. The mice of the model and normal groups were subjected to restriction with the same method as those of the EA group for 15 min. The treatment was conducted once every other day for 6 weeks. The spatial learning-memory ability （shown by escape latency of place navigation test and the time of crossing the target platform and total swimming distance in the target quadrant in 1 min of spatial probe test ） was detected by Morris water maze test. The immunoactivity of senile plaques （SP） in the hippocampus tissue was detected by immunohistochemistry. The ultrastructural characters of hippocampal neurons were observed by transmission electron microscope, and the expression levels of Ras-related protein 5 （Rab5）, Ras-related protein 7 （Rab7） and cathepsin D （CTSD） in the hippocampus were detected by Western blot, separately. Compared with the normal group, the escape latency, SP immunoactivity, and protein expression levels of Rab5, Rab7 and CTSD were significantly increased （P<0.05, P<0.01）, while the number of crossing the original platform and the total swimming distance in the platform quadrant were considerably reduced （P<0.05） in the model group. In contrast to the model group, the EA group had a marked decrease in the escape latency, SP immunoactivity, and protein expression levels of Rab5, Rab7 and CTSD （P<0.05, P<0.01）, and a striking increase in the number of crossing the original platform and the swimming distance in the platform quadrant （P<0.05）. Results of transmission electron microscope showed an accumulation of endosome, lysosome, and endolysosomes in the hippocampal neurons in the model group, which was evidently milder in the EA group. EA of GV20 and KI1 can improve the learning-memory ability of AD mice, which may be related to its function in reducing hippocampal Aβ deposition and down-regulating endosomal-lysosomal system activity.

247-LB: Link between Hyperglycemia and Cognitive Deficits in T2DM Animal Model

The Goto-Kakizaki (GK) rat is a model of spontaneously developing non-obese type 2 diabetes mellitus (T2DM). With age, GK rats exhibit insulin resistance, decreased pancreatic function, and hyperglycemia. Studies have shown that GK rats experience cognitive impairment concurrent with hyperglycemia, but the mechanisms is unclear. In this study, we aimed to elucidate the potential link between hyperglycemia and cognitive impairment in this unique T2DM model. Longitudinal assessments of fasting blood glucose (FBG) were performed in male GK rats and age-matched Wistar rats. Our results showed that FBG in GK group was significantly higher than that in the Wistar group starting from 6 weeks of age, and there was progressive fasting hyperglycemia in GK group. In the spatial probe test of Morris water maze (MWM) at 33 weeks old, the GK rats spent less time in the target quadrant (12.16 ± 2.18s vs. 22.17 ± 1.21, P&amp;lt;0.001) and took more time to reach the location of the formerly hidden platform (36.78 ± 6.141 vs. 6.422 ± 0.8783s, P&amp;lt;0.001) than Wistar rats, suggesting that GK rats showed spatial memory deficits. In mechanistic studies, insulin receptors (IR) and synaptic proteins were down-regulated and impaired insulin signaling pathways in the hippocampus of GK rats (Figure), suggesting impaired neuroplasticity and neuropathological processes may lead to cognitive dysfunction. Disclosure L. Chen: Board Member; Hua Medicine. J. Ni: None. L. Feng: Employee; Hua Medicine. X. Zhang: None. Q. Wang: None. Y. Chen: Stock/Shareholder; Silaiputai (Shanghai) Biopharmaceutical Company. Funding Hua Medicine Co., Ltd.

The effect of gastric bypass surgery on cognitive function of Alzheimer's disease and the role of GLP1-SGLT1 pathway

ObjectiveGastric bypass surgery has been shown to improve metabolic profiles via GLP1, which may also have cognitive benefits for Alzheimer's disease (AD) patients. However, the exact mechanism requires further investigation. MethodsRoux-en-Y gastric bypass or sham surgery was performed on APP/PS1/Tau triple transgenic mice (an AD mice model) or wild type C57BL/6 mice. Morris Water Maze (MWM) test was used to evaluate the cognitive function of mice and animal tissue samples were obtained for measurements two months after the surgery. Additionally, STC-1 intestine cells were treated with siTAS1R2 and siSGLT1, and HT22 nerve cells were treated with Aβ, siGLP1R, GLP1 and siSGLT1 in vitro to explore the role of GLP1-SGLT1 related signaling pathway in cognitive function. ResultsThe MWM test showed that bypass surgery significantly improved cognitive function in AD mice as measured by navigation and spatial probe tests. Moreover, bypass surgery reversed neurodegeneration, down-regulated hyperphosphorylation of Tau protein and Aβ deposition, improved glucose metabolism, and up-regulated the expression of GLP1, SGLT1, and TAS1R2/3 in the hippocampus. Furthermore, GLP1R silencing down-regulated SGLT1 expression, whereas SGLT1 silencing increased Tau protein deposition and exacerbated dysregulated of glucose metabolism in HT22 cells. However, RYGB did not alter the level of GLP1 secretion in the brainstem (where central GLP1 is mainly produced). Additionally, GLP1 expression was upregulated by RYGB via TAS1R2/3-SGLT1 activation sequentially in the small intestine. ConclusionRYGB surgery could improve cognition function in AD mice through facilitating glucose metabolism and reducing Tau phosphorylation and Aβ deposition in the hippocampus, mediated by peripheral serum GLP1 activation of SGLT1 in the brain. Furthermore, RYGB increased GLP1 expression through sequential activation of TAS1R2/TAS1R3 and SGLT1 in the small intestine.

Knockdown of CBX7 inhibits ferroptosis in rats with cerebral ischemia and improves cognitive dysfunction by activating the Nrf2/HO-1 pathway

Iron-dependent lipid peroxidation-induced ferroptosis has been implicated in the pathogenesis of cerebral ischemia. Ischemia-reperfusion (I/R) contributes to cognitive dysfunction. Chromobox7 (CBX7) was upregulated in neural progenitor cells, followed by hypoxia and ischemia, and the suppression of CBX7 protected against renal and cerebral I/R injuries. However, the role of CBX7 in ferroptosis and cognitive dysfunction in rats with cerebral ischemia has not yet been reported. In our study, first, rats with cerebral I/R were established through middle cerebral artery occlusion (MCAO). Intracerebroventricular injection of shRNA targeting CBX7 (sh-CBX7) enhanced brain water content, and reduced neurological deficit scores and infarct size in rats post MCAO. Second, rats were subjected to 4 trials per day for 4 consecutive days in a Morris water maze. The time to find the platform in with 1 min was recorded as escape latency. On day 5, the platform was removed to conduct a spatial probe test. Rats were given 1 min to find the position of the platform, and the times for crossing the target within 1 min were recorded. Crossing the platform time is the time spent in each quadrant. Data from the Morris water maze showed that cognitive dysfunction in ischemic rats was ameliorated by the injection of sh-CBX7, which decreased the swimming distance and escape latency of ischemic rats, increased the times for crossing the target and platform in the maze. Moreover, injection of sh-CBX7 down-regulated levels of MDA and ROS, while it up-regulated SOD and GSH-Px to repress the oxidative stress. Third, knockdown of CBX7 in ischemic rats decreased the concentration of the ferric ions and the protein expression of transferrin receptor 1 (TFR1), but enhanced the solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4), resulting in the inhibition of ferroptosis. Lastly, expressions of Nrf2, HO-1 and NAD(P)H:quinone oxidoreductase 1 (NQO1) were up-regulated in ischemic rats post sh-CBX7 injection. In conclusion, suppression of CBX7 attenuated I/R injury, improved cognitive dysfunction and inhibited ferroptosis through the activation of the Nrf2/HO-1 signaling pathway.

Subclinical hypothyroidism in pregnancy rats impaired offspring's spatial learning and memory and the cerebellar development

Subclinical hypothyroidism (SCH) is a very common preclinical condition during pregnancy. The adverse effect of maternal clinical hypothyroidism (CH) on the nervous system development of offspring is beyond doubt, but it is still controversial in SCH. The aim of this study was to investigate whether spatial learning and memory ability of offspring is inhibited in SCH rat model and its possible mechanism. 45 Wistar female rats were randomly divided into SCH, CH and control (CON) groups, which were induced by semi-thyroid electrocauterization, total thyroidectomy and sham operation, respectively. Rat pups were sacrificed at embryonic day 14 (E14), E18, postnatal day 1 (P1), P3, and P10, and pups' cerebellar tissues were collected. The proliferation, differentiation and migration of cerebellar cells were observed, and RNA level of the thyroid hormone receptor α (TRα) and TRβ in the cerebellum was detected by real-time PCR, respectively. Morris Water Maze (MWM) test was performed to detect the spatial learning and memory ability of pups at P40. Our data indicated that maternal SCH will significantly extend the offspring's escape latency time, and pups perform worse in the spatial probe test compared with the CON group. Except for E14, the proliferation of pups' cerebellar granule cells (GCs), and the migration of pups' Purkinje cells (PCs) in the SCH group was significantly inhibited compared with that in the CON group at other time points (P < 0.05 or P < 0.01), and the differentiation of cerebellar astrocytes (As) in SCH group was higher than that in CON group at P3 and P10. Except for E14, the expression of TRα mRNA in SCH group was significantly lower than that in CON group (P < 0.05 or P < 0.01). And the difference of the differentiation of As and the spatial learning and memory between SCH and CH groups was not statistically significant. Our findings suggested that SCH during pregnancy nuisances the offspring's spatial learning and memory. It may be related to the decrease of the expression of TRα in cerebellum, which may further inhibit the proliferation of GCs and the migration of PCs, and increase the differentiation of As.

Electroacupuncture improves learning and memory functions in a rat cerebral ischemia/reperfusion injury model through PI3K/Akt signaling pathway activation.

Electroacupuncture has been widely used to treat cognitive impairment after cerebral ischemia, but the underlying mechanism has not yet been fully elucidated. Studies have shown that autophagy plays an important role in the formation and development of cognitive impairment, and the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway plays an important role in autophagy regulation. To investigate the role played by the PI3K/Akt signaling pathway in the electroacupuncture treatment of cerebral ischemia/reperfusion rat models, we first established a rat model of cerebral ischemia/reperfusion through the occlusion of the middle cerebral artery using the suture method. Starting at 2 hours after modeling, electroacupuncture was delivered at the Shenting (GV24) and Baihui (GV20) acupoints, with a dilatational wave (1-20 Hz frequency, 2 mA intensity, 6 V peak voltage), for 30 minutes/day over 8 consecutive days. Our results showed that electroacupuncture reduced the infarct volume in a rat model of cerebral ischemia/reperfusion injury, increased the mRNA expression levels of the PI3K/Akt signaling pathway-related factors Beclin-1, mammalian target of rapamycin (mTOR), and PI3K, increased the protein expression levels of phosphorylated Akt, Beclin-1, PI3K, and mTOR in the ischemic cerebral cortex, and simultaneously reduced p53 mRNA and protein expression levels. In the Morris water maze test, the latency to find the hidden platform was significantly shortened among rats subjected to electroacupuncture stimulation compared with rats without electroacupuncture stimulation. In the spatial probe test, the number of times that a rat crossed the target quadrant was increased in rats subjected to electroacupuncture stimulation compared with rats without electroacupuncture stimulation. Electroacupuncture stimulation applied to the Shenting (GV24) and Baihui (GV20) acupoints activated the PI3K/Akt signaling pathway and improved rat learning and memory impairment. This study was approved by the Animal Ethics Committee of the First Affiliated Hospital of Henan University of Traditional Chinese Medicine, China (approval No. 8150150901) on March 10, 2016.

Effects of post-training administration of LY341495, as an mGluR2/3 antagonist on spatial memory deficit in rats fed with high-fat diet

High-fat diets (HFDs) adversely influence glutamate metabolism and neurotransmission. The precise role of the group II metabotropic glutamate receptors (mGluR2/3) antagonist on spatial memory deficit following consumption of HFD has not yet been clarified. Therefore, in this study, we examined the effects of post-training administration of mGluR2/3 antagonism; LY341495 on spatial memory in rats fed with HFD (for 10 weeks) by using Morris Water Maze (MWM) task. The training session for testing memory acquisition in MWM consisted of 4 trials per day for 4 consecutive days. Twenty-four hours after the last training session the spatial probe test (retention) was given. Intraperitoneal injection (i.p) injection of LY341495 was done 30min before probe test. Our results showed that 10 weeks consumption of HFD had no significant effect on escape latency and swimming distance in memory acquisition. Our finding showed that consumption of a HFD leads to reference memory impairment in the probe test. HFD animals spent less time in the target zone in compare with control animals. Also, LY341495 improved HFD-induced reference memory (retention) impairment. HFD animals treated with LY341495 spent more time in the target zone in compare with HFD animals. Escape latencies to find the visible platform during visual task were same in all experimental groups, indicating no visual impairment in the animals. We propose that a HFD may act through mGluR2/3 within the brain to reduce synaptic plasticity, which impairs memory retrieval, and post-training administration of LY341495 can reduce HFD-induced reference memory impairment.

Effect of electroacupuncture of different acupoint groups on learning-memory ability and expression of IL-1β and TNF-α in hippocampus and prefrontal cortex in rats with Alzheimer's disease

To compare the effect of electroacupuncture (EA) of acupoint group for "reinforcing the kidney and regulating Governor Vessel" and acopoint group for "reinforcing the kidney and lung and regulating Governor Vessel" on lear-ning-memory ability and expression of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) proteins in the hippocampus and prefrontal cortex (PFC) in Alzheimer's disease （AD） rats, so as to explore the efficacy of the two acupoint groups and mechanisms underlying improvement of AD. Forty male SD rats were randomly divided into control, sham operation, model, "Baihui" + "Shenshu" (GV20+BL23, for "reinforcing the kidney and regulating Governor Vessel") EA and GV20+BL23+ "Feishu" (BL13, GV20+BL23+BL13, for "reinforcing the kidney and lung and regulating Governor Vessel") EA groups (n=8 rats in each group). The AD model was established by bilateral injection of amyloid β peptide （Aβ25-35，10 μL) into bilateral hippocampus, and rats of the sham operation group received injection of normal saline. After successful establishment of the model，EA (2 Hz, 2 mA) was applied to these acupoints for 15 min, once daily for 10 days. Then, the learning-memory ability was assessed by using Morris water maze tests, and the expression levels of TNF-α and IL-1β proteins in the PFC and hippocampus tissues were detected by using Western blot. Following modeling, the average escape latency of place navigation test were significantly increased (P<0.05) and the platform crossing times of spatial probe test was significantly decreased in the model group than in the control and sham operation groups (P<0.05). The expression levels of IL-1β and TNF-α proteins in the PFC and hippocampus were apparently up-regulated in the model group than in the control and the sham operation groups (P<0.000 1, P<0.001, P<0.01). After the intervention, the increase of the average escape latency and expression of IL-1β and TNF-α in the PFC and hippocampus, and the decrease of space exploration test were revised in both GV20+BL23 EA and GV20+BL23+BL13 EA groups (P<0.05,P<0.01). No significant differences were found between the GV20+BL23 and GV20+BL23+BL13 EA groups in the above mentioned indexes (P>0.05). EA of both GV20+BL23 and GV20+BL23+BL13 acupoint can improve learning-memory ability of AD rats, which is associated with their effects in down-regulating the expression of IL-1β and TNF-α in the PFC and hippocampus to reduce inflammatory reaction. There were no significant differences between the two acupoint groups in the therapeutic effects.

Hippocampal low-frequency stimulation improves cognitive function in pharmacoresistant epileptic rats

ObjectiveThe aims of the present study were to observe the changes of cognitive function in a pilocarpine-induced rat model of epilepsy, and to investigate the effects of hippocampal low-frequency stimulation (Hip-LFS) on cognitive function in rats with pharmacoresistant epilepsy. MethodsA total of 100 male Sprague Dawley rats were randomly selected to establish an epilepsy model. Rats with successfully induced epilepsy were injected intraperitoneally with phenobarbital and phenytoin for pharmacoresistance selection. The selected pharmacoresistant epileptic (PRE) rats were assigned to a pharmacoresistant control group (PRC group), or a group that received Hip-LFS (LFS group). The same number of rats with pharmacosensitive epilepsy formed the PSC group, and a normal control (NCR) group was included. A novel object recognition (NOR) test, and a Morris water maze (MWM) task were used to assess cognitive function in all groups. ResultsThe epileptic rats showed decreased abilities of learning and memory compared with normal control. The rats in the LFS group displayed significantly shorter escape latency in place navigation, spent longer times in the target quadrant, and traveled longer distances on the platform in the spatial probe test than PRC group. In the NOR test, compared with the PRC group, the discrimination index of the LFS group was significantly increased. Compared with the PRC group, the average frequency and duration of seizures were also decreased in the LFS group. ConclusionsThe present pilocarpine-induced rat model of epilepsy showed impaired cognitive function, especially in rats with PRE. The Hip-LFS treatment could effectively improve the cognitive function of rats with PRE.

Leaf extracts from Dendropanax morbifera L\xe9veille mitigate mercury-induced reduction of spatial memory, as well as cell proliferation, and neuroblast differentiation in rat dentate gyrus

BackgroundThe brain is susceptible to methylmercury toxicity, which causes irreversible damage to neurons and glia and the leaf extract Dendropanax morbifera Léveille (DML) has various biological functions in the nervous system. In this study, we examined the effects of DML on mercury-induced proliferating cells and differentiated neuroblasts.MethodsDimethylmercury (5 μg/kg) and galantamine (5 mg/kg) was administered intraperitoneally and/or DML (100 mg/kg) was orally to 7-week-old rats every day for 36 days. One hour after the treatment, novel object recognition test was examined. In addition, spatial probe tests were conducted on the 6th day after 5 days of continuous training in the Morris swim maze. Thereafter, the rats were euthanized for immunohistochemical staining analysis with Ki67 and doublecortin and measurement for acetylcholinesterase (AChE) activity.ResultsDimethylmercury-treated rats showed reduced discrimination index in novel object recognition test and took longer to find the platform than did control group. Compared with dimethylmercury treatment alone, supplementation with DML or galatamine significantly ameliorated the reduction of discrimination index and reduced the time spent to find the platform. In addition, the number of platform crossings was lower in the dimethylmercury-treated group than in controls, while the administration of DML or galantamine significantly increased the number of crossings than did dimethylmercury treatment alone. Proliferating cells and differentiated neuroblasts, assessed by Ki67 and doublecortin immunohistochemical staining was significantly decreased in the dimethylmercury treated group versus controls. Supplementation with DML or galantamine significantly increased the number of proliferating cells and differentiated neuroblasts in the dentate gyrus. In addition, treatment with dimethylmercury significantly increased AChE activity in hippocampal homogenates, while treatment with dimethylmercury+DML or dimethylmercury+galantamine significantly ameliorated this increase.ConclusionsThese results suggest that DML may be a functional food that improves dimethylmercury-induced memory impairment and ameliorates dimethylmercury-induced reduction in proliferating cells and differentiated neuroblasts, and demonstrates corresponding activation of AChE activity in the dentate gyrus.

Combination antiretroviral therapy (cART)-induced hippocampal disorders: Highlights on therapeutic potential of Naringenin and Quercetin

IntroductionIn spite of the multiple benefits of combination antiretroviral therapy (cART) on HIV positive patients, prolonged usage has been reported to exacerbate oxidative stress, and induce neurological and cognitive dysfunction, thus, the need to search for an adjuvant therapy to ameliorate the oxidative and improve treatment adherence with better virological outcome. This study aimed at determining the potential therapeutic effects of Quercetin and Naringenin on cART-induced cyto-architectural, neuro-behavioral and immunohistochemical changes in the hippocampus of the adult Wister rats. Materials and MethodsThe animals were grouped as follows: Control, DMSO, 24 mg/kg cART (Tenovovir 300 mg, Lamivudine 300 mg and Efavirenz 600 mg), 50 mg/kg Naringenin, 50 mg/kg Quercetin, cART + Naringenin, cART + Quercetin were administered orally for 8 weeks. At the end of administration, neurobehavioural test was conducted, animals were euthanized and hippocampus was processed for oxidative stress markers, histology, TNF-α, and Monoamine oxidase-B expression. ResultsAt the end of 8 weeks of administration, 24 mg/kg cART decreased superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH) and increased Malondialdehyde (MDA). Whereas, 50 mg/kg quercetin, and 50 mg/kg Naringenin decreased the oxidative stress (increased SOD, CAT, GSH, and reduced MDA) induced by cART (reduced SOD, CAT, GSH, and increased MDA). In addition, hematoxylin and eosin stained hippocampus showed that quercetin and naringenin prevented neurodegenerative changes (marked cytoplasmic shrinkage and several pyknotic nuclei in the dentate gyrus and cornus ammonis regions) in cART-treated rats. Furthermore, immunohistochemical studies revealed that quercetin and naringenin attenuates cART-induced upregulation of monoamine oxidase-B (MAO-B) expression. Likewise, from the Morris water maze neurobehavioral studies, naringenin and quercetin also ameliorated cART-induced memory impairments (initial spatial memory, reversal spatial memory and probe tests). ConclusionThis study shows that Naringenin and Quercetin have a good potential in reversing cART-induced hippocampal disorders in Wistar rats.

Potential contribution of microRNA-125b targeting p38MAPK to relieving intermittent hypoxia-induced dementia of rat models

Allowing for the correlation between intermittent hypoxia and impaired cognition, we intended to figure out whether and how dementia-relevant biomolecules (e.g. miR-125b and p38MAPK) might participate in weakening of intelligence in the context of intermittent hypoxia. Within this investigation, four groups of rat models were established, including rats treated with none, 10% continuous hypoxic (CH), 10% chronic intermittent hypoxia (CIH) and 5% chronic intermittent hypoxia (CIH). Moreover, the learning, memorizing and cognitive abilities of rats were evaluated through performing morris water maze, spatial navigation test and spatial probe test, respectively. Furthermore, expressions of miR-125b and p38MAPK were determined based on reverse transcription-polymerase chain reaction (RT-PCR) or western blotting. And the luciferase reporter gene assay was prepared to validate the targeted relationship between miR-125b and p38MAPK. Consequently, our study results showed that intermittent hypoxia generated more serious cognitive impairment to rats than persistent hypoxia (P < 0.05), and the rats treated with intermittent hypoxia were observed with down-regulated miR-125b expression and up-regulated phospho-p38MAPK expression (P < 0.05). Furthermore, the in-vitro tests exhibited that down-regulated expression of miR-125b and phosphorylation of p38MAPK could promote apoptosis of SH-SY5Y cells, and also depress their viability and proliferation (P < 0.05). The luciferase reporter gene assay also validated a targeted relationship between miR-125b and p38MAPK in SH-SY5Y cells. In conclusion, miR-125b targeting p38MAPK might be involved with modifying learning, memorizing and cognitive abilities of rats that were managed with intermittent hypoxia, which might provide theoretical basis for treating intermittent hypoxia-related dementia.

Effect of acute exposure to hypobaric hypoxia on learning and memory in adult Sprague-Dawley rats

The present study aimed to observe the effects of acute exposure to hypobaric hypoxia on learning and memory in adult Sprague-Dawley (SD) rats as well as the changes in N-methyl-D-aspartate receptor (NMDAR) subunits. Learning and memory abilities were evaluated with Morris water maze following simulated hypoxia at 5000 m or 7000 m for 2, 4 or 6 days. The number of red blood cells, the level of hemoglobin, oxidative stress markers, the extent of neuronal damage in the hippocampal CA1, and the expression of NMDAR subunits were all measured. In place navigation test, the escape latency significantly increased only in the 5000 M 6Day (P < 0.05) group than the latency in the plain group on the fourth day. In the spatial probe test, the times of platform passing showed no significant difference between the hypoxia and plain groups. Polycythemia, an increased ratio of superoxide dismutase/malondialdehyde and degeneration of neurons appeared in an elevation-dependent and duration-dependent manner in those hypoxia groups. Furthermore, the protein expression of NR1 increased and the protein expression levels of NR2A, NR2B, and NR3A were all decreased in the 5000M 6Day and 7000M 6Day groups. In summary, the feeble effect of acute exposure to simulated hypobaric hypoxia on learning and memory in adult SD rats may be attributed to increased antioxidative capacities and decreased expression of NR3A. And moreover, differences in the expressions of NMDAR subunits were closely related to the altitude and duration.

Augmented improvement of cognition and memory by aripiprazole add-on for cilostazol treatment in the chronic cerebral hypoperfusion mouse model

Cerebrovascular dysfunction is associated with cognitive impairment in vascular dementia patients. This study aimed to explore augmented improvement of cognition and memory by aripiprazole add-on for cilostazol treatment in vascular dementia model. Male C57BL/6 mice were subjected to BCAS, and spatial probe and memory retention were examined using the Morris water maze (MWM) test. In the present study, the escape latency on the first day after 3rd week was 21.4 ± 4.0 s in sham-operated mice, and 76.3 ± 4.2 s in the vehicle-treated BCAS mice. In the spatial probe tests in the 3rd week, aripiprazole (1 mg/kg/day) showed time-dependently amelioration in spatial learning and memory impairments in contrast to 0.5 mg/kg/day. After treatment with 20 mg/kg/day of cilostazol for 3 weeks, the escape latency significantly decreased to 26.6 ± 5.8 s on the first day and further shortened to 21.6 ± 6.8 s on the fourth day. When the BCAS mice were concurrently treated with 0.5 mg/kg/day aripiprazole plus 20 mg/kg/day of cilostazol for 3 weeks, the escape latency was more shortened from 20.4 ± 1.2 s (1st day) to 14.9 ± 1.7 s on the 4th day of the 3-week trials. Furthermore, decreased spatial memory retention in BCAS mice was significantly alleviated by aripiprazole plus cilostazol cotreatment, indicating the benefit of aripiprazole add-on therapy. In line with these, significantly increased mBDNF and P-CREB levels and reduced apoptosis were identified in the BCAS mouse brain dentate gyrus by cotreatment as contrasted to each monotherapy. These results may provide the synergistic therapeutic avenues for augmented improvement of cognition and memory by cotreatment with aripiprazole plus cilostazol in cases of vascular dementia.

The MEK/ERK/CREB signaling pathway is involved in atrazine induced hippocampal neurotoxicity in Sprague Dawley rats

Atrazine (ATR) is a commonly used artificial synthetic herbicide world-wide, which has been implicated as a potential threat to human health. Previous studies have demonstrated that exposure to ATR affects hippocampus-dependent learning and memory in rodents, but the exact molecular mechanism remains to be elucidated. In this study, we investigated the effect of ATR on the hippocampus of postnatal day 35 male Sprague Dawley (SD) rats administered doses of either 10 or 100 mg/kg body weight (BW)/day of ATR for a period of 30 days. A Morris water maze (MWM) test revealed that ATR treatment impaired memory performance in the spatial probe test, especially amongst the high-dose group. Moreover, analysis by electron microscopy showed that hippocampal neuron ultrastructure in the dentate gyrus (DG) and cornu ammonis 1 (CA1) sub-regions was impaired in the ATR-treated groups. Finally, a downregulation in the mRNA and protein expression levels of members of the MEK/ERK/CREB pathway and downstream factors brain-derived neurotrophic factor (BDNF) and Zif268 was observed in hippocampal tissue following ATR treatment. Taken together, these results suggest that developmental exposure to ATR is able to induce functional and morphological lesions in the hippocampus of SD rats, and that the MEK/ERK/CREB signaling pathway may be involved in this process.

Rapid establishment of mild cognitive impairment models in rats

Objective To explore method of rapidly establishing mild cognitive impairment models in rats by D-galactose (D-gal) and the appropriate dose. Methods Totally 100 male SD rats, the model groups A, B, C, D were given hypodermic injection of D-gal with the concentration of 100, 300, 1 000, 2 000 mg/(kg·day) respectively for 1 week, and the control group O received the same volume of saline. After injection, the Morris water maze (MWM) began, then the levels of malondialdehyde (MDA), superoxide dismutase (SOD) and acetylcholinesterase (AChE) in the brain homogenate were determined, and the pathomorphologic changes of the hippocampus were observed by HE staining. Results (1) The survival rate of each group was 100%. The weight of group D gained less and the swimming speed was slower than in group O. (2) At day 2-5 of the place navigation test of MWM, the escape latencies in group C and D were prolonged as compared withthose in group O. For in the spatial probe test, the number of crossing the platform in 60 s of groups C and D was (3.05±0.76) and (2.05±0.69) respectively, significantly less than in group O (4.40±1.05, P=0.000, 0.000). (3) In groups C and D, the levels of MDA were (29.19±2.35) and (32.57±2.05) respectively, significantly higher than in group O [(20.52±2.08) ng/ml, P=0.000, 0.000]. The levels of SOD were (1.46±0.12) and (1.34±0.13) IU/ml respectively, significantly lower than in group O [(1.74±0.12) IU/ml, P=0.001, 0.000]. The levels of AChE were (3.52±0.70) and (4.02±0.39) ng/ml respectively, significantly higher than in group O [(1.72±0.33) ng/ml, P=0.000, 0.000]. (4) The pathomorphologic changes of the hippocampus showed: in group C, several pyramidal cells lost, density of cell was slightly decreased, pyknotic nucleis increased and some pyramidal cells were dark-stained and a small amount of apoptotic cells were seen in the CA1 region of hippocampus, while cerebral white matter was normal; in group D, density of pyramidal cells was significantly decreased, arrange was obviously loose and in a mess, and large number of cells were condensed, dark-stained and apoptosis occurred in the CA1 region. Conclusion Rats given hypodermic injection of D-gal with 1 000 mg/(kg·day) conformed to the pathological process and its characteristics of mild cognitive impairment. Key words: D-galactose; Mild cognitive impairment; Model, animal

The Effect of Toll‐Like Receptor‐4 on the Behavioral Impairment in Aβ1–42 Induced AD Mouse Model

ObjectiveTo investigate the role of toll like receptor 4 (TLR4) in the cognition impairment in β‐amyloid fragment 1–42 (Aβ1–42) induced Alzheimer's disease (AD) mouse model.MethodsTwenty four TLR4 gene knockout mice (TLR4KO, male, body weight: 25–30g) and 24 wild type mice (WT, male, body weight: 25–30g) were randomly assigned into 4 groups: Normal saline control (WT.NS, wild type mice were injected 1ul normal saline into bilateral hippocampus), AD model group (WT. Aβ, wild type mice were injected with 1ul Aβ1–42 (5ug/ul) into bilateral hippocampus), TLR4KO‐normal saline control (TLR4KO.NS, TLR4KO mice were injected with 1ul normal saline into bilateral hippocampus), and TLR4KO Aβ group (TLR4KO.Aβ, TLR4KO mice were injected with 1ul Aβ1–42 (5ug/ul) into bilateral hippocampus). Seven days after injection, the learning and memorial functions were tested using Morris Water Maze (MWM). The repeated measures analysis of variance was used to analyze navigation test, the one‐way ANOVA and LSD post‐hoc comparisons were used for probe trial analyses. HE staining was performed.; and the expression of Bcl‐2 and Caspase‐3 was assayed by Western blot, the results was analyzed by one‐way ANOVA.Results1. Compared with normal saline control groups(WT.NS), the AD groups (WT. Aβ) showed a longer escape latency (P&lt;0.05), increased percentage of time in outer annulus (P&lt;0.05) in the place navigation test, reduced percentage of time in the platform quadrant and decreasing times of traversing platform (P&lt;0.05) in the spatial probe test. Compared with AD model groups(WT.Aβ), the escape latency and the percentage of time in outer annulus were decreased in TLR4KOAβ groups (P&lt;0.05), and the percentage of time in the platform quadrant and the times of traversing platform were increased (P&lt;0.05). There was no significant difference of swimming speed amonge each group (P&gt;0.05). 2. Hematoxylin eosin staining showed that compared with normal saline control groups, the neurons in AD groups distribute loosely and irregularly, and more dead neurons were observed. TLR4KOAβ groups showed a attenuated damage in hippocamus compared with the AD groups. 3. The results of western blots suggested that compared with control groups, the expression of Bcl‐2 in AD groups were significant reduced (P&lt;0.05) and the expression of Caspase‐3 were increased (P&lt;0.05); the expression of Bcl‐2 in the TLR4KO Aβ groups were increased (P&lt;0.05) and the level of Caspase‐3 was reduced (P&lt;0.05) when compared with AD groups.Conclusion1. Injection of 5ug Aβ1–42 into bilateral hippocampus can obvious impaire the learning and memorial functions and induced histological damage in hippocampus of mice, which provides an effective method for establishing mouse model of AD; 2. TLR4 knockout can attenuate cognitive impairment and tissue damage induced by hippocampal injection of Aβ1–42 in the AD model, which may be partly achieved by reducing apoptosis.Support or Funding InformationThis work was supported by China National Nature Scientific Foundation (81571469 and 81271268)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.