Changes of fat-mass and obesity-associated protein expression in the hippocampus in animal models of high-fat diet-induced obesity and D-galactose-induced aging / 한국실험동물학회지

Fat-mass and obesity-associated protein (Fto) is highly expressed in the brain including, the hippocampus, and its expression is significantly decreased in the brain of Alzheimer’s disease patients. In the present study, we measured Fto immunoreactivity and protein levels in the hippocampus of obese and aged mice, which were induced by high-fat diet for 12 weeks and D-galactose treatment for 10 weeks, respectively. The obesity and aging phenotypes were assessed by physiological parameters and Morris water maze test, respectively. High fat diet fed mice showed significant increases in body weight and blood glucose levels compared to that in the control or D-galactose-induced aged mice. In addition, treatment with D-galactose significantly decreased the spatial memory. Fto immunoreactivity in the control group was mainly detected in the pyramidal cells of the CA1 and CA3 regions and in the granule cells of the dentate gyrus. In the hippocampus of high-fat diet-fed mice, Fto immunoreactive structures were similarly found in the hippocampus compared to that in the control group, but Fto immunoreactivity in high-fat diet-fed mice was also found in the stratum oriens and radiatum of the CA1 and CA3 regions and the polymorphic layer of the dentate gyrus. In the hippocampus of D-galactose-induced aged mice, fewer Fto immunoreactive structures were detected in the granule cell layer of the dentate gyrus compared to the control group. Fto mRNA and protein levels based on quantitative real-time polymerase chain reaction and western blot assays were slightly increased in the hippocampus of high-fat diet-fed mice compared to that in control mice. In addition, Fto mRNA and protein levels were significantly decreased in the aged hippocampus compared to that in the control group. Fto protein levels are susceptible to the aging process, but not in the hippocampus of high-fat diet-induced obesity. The reduction of Fto in aged mice may be associated with reduced memory impairment in mice.

Heat shock protein 70 increases cell proliferation, neuroblast differentiation, and the phosphorylation of CREB in the hippocampus / 한국실험동물학회지

In the present study, we investigated the effects of heat shock protein 70 (HSP70) on novel object recognition, cell proliferation, and neuroblast differentiation in the hippocampus. To facilitate penetration into the blood–brain barrier and neuronal plasma membrane, we created a Tat-HSP70 fusion protein. Eight-week-old mice received intraperitoneal injections of vehicle (10% glycerol), control-HSP70, or Tat-HSP70 protein once a day for 21 days. To elucidate the delivery efficiency of HSP70 into the hippocampus, western blot analysis for polyhistidine was conducted. Polyhistidine protein levels were significantly increased in control-HSP70- and Tat-HSP70-treated groups compared to the control or vehicle-treated group. However, polyhistidine protein levels were significantly higher in the Tat-HSP70-treated group compared to that in the control-HSP70-treated group. In addition, immunohistochemical study for HSP70 showed direct evidences for induction of HSP70 immunoreactivity in the control-HSP70- and Tat-HSP70-treated groups. Administration of Tat-HSP70 increased the novel object recognition memory compared to untreated mice or mice treated with the vehicle. In addition, the administration of Tat-HSP70 significantly increased the populations of proliferating cells and differentiated neuroblasts in the dentate gyrus compared to those in the control or vehicle-treated group based on the Ki67 and doublecortin (DCX) immunostaining. Furthermore, the phosphorylation of cAMP response element-binding protein (pCREB) was significantly enhanced in the dentate gyrus of the Tat-HSP70-treated group compared to that in the control or vehicle-treated group. Western blot study also demonstrated the increases of DCX and pCREB protein levels in the Tat-HSP70-treated group compared to that in the control or vehicle-treated group. In contrast, administration of control-HSP70 moderately increased the novel object recognition memory, cell proliferation, and neuroblast differentiation in the dentate gyrus compared to that in the control or vehicle-treated group. These results suggest that Tat-HSP70 promoted hippocampal functions by increasing the pCREB in the hippocampus.

Changes of myelin basic protein in the hippocampus of an animal model of type 2 diabetes / 한국실험동물학회지

In this study, we observed chronological changes in the immunoreactivity and expression level of myelin basic protein (MBP), one of the most abundant proteins in the central nervous system, in the hippocampus of Zucker diabetic fatty (ZDF) rats and their control littermates (Zucker lean control; ZLC). In the ZLC group, body weight steadily increased with age; the body weight of the ZDF group, however, peaked at 30 weeks of age, and subsequently decreased. Based on the changes of body weight, animals were divided into the following six groups: early (12-week), middle (30-week), and chronic (52-week) diabetic groups and their controls. MBP immunoreactivity was found in the alveus, strata pyramidale, and lacunosum-moleculare of the CA1 region, strata pyramidale and radiatum of the CA3 region, and subgranular zone, polymorphic layer, and molecular layer of the dentate gyrus. MBP immunoreactivity was lowest in the hippocampus of 12-week-old rats in the ZLC group, and highest in 12-week-old rats in the ZDF group. Diabetes increased MBP levels in the 12-week-old group, while MBP immunoreactivity decreased in the 30-week-old group. In the 52-week-old ZLC and ZDF groups, MBP immunoreactivity was detected in the hippocampus, similar to the 30-week-old ZDF group. Western blot results corroborated with immunohistochemical results. These results suggested that changes in the immunoreactivity and expression of MBP in the hippocampus might be a compensatory response to aging, while the sustained levels of MBP in diabetic animals could be attributed to a loss of compensatory responses in oligodendrocytes.

Bacopa monnieri extract improves novel object recognition, cell proliferation, neuroblast differentiation, brain-derived neurotrophic factor, and phosphorylation of cAMP response element-binding protein in the dentate gyrus / 한국실험동물학회지

Bacopa monnieri is a medicinal plant with a long history of use in Ayurveda, especially in the treatment of poor memory and cognitive deficits. In the present study, we hypothesized that Bacopa monnieri extract (BME) can improve memory via increased cell proliferation and neuroblast differentiation in the dentate gyrus. BME was administered to 7-week-old mice once a day for 4 weeks and a novel object recognition memory test was performed. Thereafter, the mice were euthanized followed by immunohistochemistry analysis for Ki67, doublecortin (DCX), and phosphorylated cAMP response element-binding protein (CREB), and western blot analysis of brain-derived neurotrophic factor (BDNF). BME-treated mice showed moderate increases in the exploration of new objects when compared with that of familiar objects, leading to a significant higher discrimination index compared with vehicle-treated mice. Ki67 and DCX immunohistochemistry showed a facilitation of cell proliferation and neuroblast differentiation following the administration of BME in the dentate gyrus. In addition, administration of BME significantly elevated the BDNF protein expression in the hippocampal dentate gyrus, and increased CREB phosphorylation in the dentate gyrus. These data suggest that BME improves novel object recognition by increasing the cell proliferation and neuroblast differentiation in the dentate gyrus, and this may be closely related to elevated levels of BDNF and CREB phosphorylation in the dentate gyrus.

Decrease in glucose transporter 1 levels and translocation of glucose transporter 3 in the dentate gyrus of C57BL/6 mice and gerbils with aging / 한국실험동물학회지

In the present study, we compared the cell-specific expression and changes protein levels in the glucose transporters (GLUTs) 1 and 3, the major GLUTs in the mouse and gerbil brains using immunohistochemistry and Western blot analysis. In both mouse and gerbils, GLUT1 immunoreactivity was mainly found in the blood vessels in the dentate gyrus, while GLUT3 immunoreactivity was detected in the subgranular zone and the molecular layer of the dentate gyrus. GLUT1-immunoreactivity in blood vessels and GLUT1 protein levels were significantly decreased with age in the mice and gerbils, respectively. In addition, few GLUT3-immunoreactive cells were found in the subgranular zone in aged mice and gerbils, but GLUT3-immunoreactivity was abundantly found in the polymorphic layer of dentate gyrus in mice and gerbils with a dot-like pattern. Based on the double immunofluorescence study, GLUT3-immunoreactive structures in gerbils were localized in the glial fibrillary acidic protein-immunoreactive astrocytes in the dentate gyrus. Western blot analysis showed that GLUT3 expression in the hippocampal homogenates was slightly, although not significantly, decreased with age in mice and gerbils, respectively. These results indicate that the reduction in GLUT1 in the blood vessels of dentate gyrus and GLUT3 in the subgranular zone of dentate gyrus may be associated with the decrease in uptake of glucose into brain and neuroblasts in the dentate gyrus. In addition, the expression of GLUT3 in the astrocytes in polymorphic layer of dentate gyrus may be associated with metabolic changes in glucose in aged hippocampus.

Dendropanax morbifera Léveille extract ameliorates D-galactose-induced memory deficits by decreasing inflammatory responses in the hippocampus / 한국실험동물학회지

In the present study, we examined the effects of Dendropanax morbifera Léveille leaf extract (DML) on D-galactose-induced morphological changes in microglia and cytokines, including pro-inflammatory cytokines (interleukin [IL]-1β, IL-6, and tumor necrosis factor [TNF]-α) and anti-inflammatory cytokines (IL-4 and IL-10) in the hippocampus. Administration of DML to D-galactose-treated mice significantly improved D-galactose-induced reduction in escape latency, swimming speed, and spatial preference for the target quadrant. In addition, administration of DML to D-galactose-treated mice significantly ameliorated the microglial activation and increases of IL-1β, IL-6, and TNF-α levels in the hippocampus. Administration of D-galactose significantly reduced IL-4 levels in the hippocampus, while administration of DML to D-galactose-treated mice significantly increased IL-4 level. However, we did not observe any significant changes in IL-10 levels in hippocampal homogenates. These results suggest that DML reduces D-galactose-induced mouse senescence by reducing pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α, as well as increasing anti-inflammatory cytokine IL-4.

Immunohistochemical localization of glucose transporter 1 and 3 in the scrotal and abdominal testes of a dog / 한국실험동물학회지

Glucose is essential for testicular function; the uptake of carbohydrate-derived glucose by cells is mediated by glucose transporters (GLUTs). In the present study, we investigated the activity of GLUT1 and GLUT3, the two main isoforms of GLUTs found in testes, in the left scrotal and right abdominal testes of a German Shepherd dog. Immunohistochemical analysis showed that GLUT1 immunoreactivity was absent in the scrotal and abdominal testes. In contrast, weak to moderate GLUT3 immunoreactivity was observed in mature spermatocytes as well as spermatids in the scrotal testis. In the abdominal testis, relatively strong GLUT3 immunoreactivity was detected in Leydig cells only and was absent in mature spermatocytes and spermatids. GLUT3 immunoreactivity was significantly decreased in the tubular region of abdominal testis and significantly increased in the extra-tubular (interstitial) region of abdominal testis compared to observations in the each region of scrotal testis, respectively. These results suggest that GLUT3 is the major glucose transporter in the testes and that abdominal testes may increase the uptake of glucose into interstitial areas, leading to an increased risk of developing cancer.

Effects of aluminum on the reduction of neural stem cells, proliferating cells, and differentiating neuroblasts in the dentate gyrus of D-galactose-treated mice via increasing oxidative stress

Aluminum (Al) accumulation increases with aging, and long-term exposure to Al is regarded as a risk factor for Alzheimer's disease. In this study, we investigated the effects of Al and/or D-galactose on neural stem cells, proliferating cells, differentiating neuroblasts, and mature neurons in the hippocampal dentate gyrus. AlCl3 (40 mg/kg/day) was intraperitoneally administered to C57BL/6J mice for 4 weeks. In addition, vehicle (physiological saline) or D-galactose (100 mg/kg) was subcutaneously injected to these mice immediately after AlCl3 treatment. Neural stem cells, proliferating cells, differentiating neuroblasts, and mature neurons were detected using the relevant marker for each cell type, including nestin, Ki67, doublecortin, and NeuN, respectively, via immunohistochemistry. Subchronic (4 weeks) exposure to Al in mice reduced neural stem cells, proliferating cells, and differentiating neuroblasts without causing any changes to mature neurons. This Al-induced reduction effect was exacerbated in D-galactose-treated mice compared to vehicle-treated adult mice. Moreover, exposure to Al enhanced lipid peroxidation in the hippocampus and expression of antioxidants such as Cu, Zn- and Mn-superoxide dismutase in D-galactose-treated mice. These results suggest that Al accelerates the reduction of neural stem cells, proliferating cells, and differentiating neuroblasts in D-galactose-treated mice via oxidative stress, without inducing loss in mature neurons.

Postnatal changes in glucose transporter 3 expression in the dentate gyrus of the C57BL/6 mouse model / 한국실험동물학회지

In this study, we observed the ontogenetic changes in glucose transporter 3 (GLUT3) immunoreactivity, a major neuronal GLUT, in the dentate gyrus of mouse brains at various ages: postnatal day (P) 1, 7, 14, 28, and 56. At P1, cresyl violet staining showed abundant neurons in the dentate gyrus, whereas the granule cell layer was ill-defined. At P7, the granule cell layer was observed, and cresyl violet-positive cells were dispersed throughout the polymorphic layer. At P14, the granule cell layer was well-defined, and cresyl violet positive cells were detected abundantly in the polymorphic layer. At P28 and P56, cresyl violet-positive cells were observed in the granule cell layer, as well as in the polymorphic layer. At P1, GLUT3 immunoreactivity was detected in the dentate gyrus. At P7, GLUT3 immunoreactive cells were scattered in the polymorphic and molecular layer. However, at P14, GLUT3 immunoreactivity was observed in the polymorphic layer as well as subgranular zone of the dentate gyrus. At P28, GLUT3 immunoreactivity was detected in the polymorphic layer of the dentate gyrus. At P56, GLUT3 immunoreactivity was observed predominantly in the subgranular zone of the dentate gyrus. GLUT3 immunoreactive cells were mainly colocalized with doublecortin, which is a marker for differentiated neuroblasts, in the polymorphic layer and subgranular zone of dentate gyrus at P14 and P56. These results suggest that the expression of GLUT3 is closely associated with postnatal development of the dentate gyrus and adult neurogenesis.

Differential expression of estrogen receptor α and progesterone receptor in the normal and cryptorchid testis of a dog / 한국실험동물학회지

Descending of the testes is an important process for spermatogenesis and cryptorchidism is one of the most relevant genital defects in dogs. In a previous study, we observed abnormal morphology and proliferation of Sertoli cells in a cryptorchid testis. In the present study, we investigated the expression of estrogen and progesterone receptors in the normal and cryptorchid testis of a dog. Elective orchidectomy was performed on the dog's abdominal right testis (undescended, cryptorchid) and scrotal left testis (descended, normal). In the normal testis, estrogen receptor α immunoreactivity was detected in Leydig cells alone, while estrogen receptor α immunoreactivity in the cryptorchid testis was significantly prominent in the Sertoli cells as well. In addition, progesterone receptor immunoreactivity in the control testis was detected in the spermatids, but was not detected in the cryptorchid testis. This result suggests that unilateral cryptorchidism causes increases of estrogen receptor α expression in Sertoli cells.

Reduction of adult hippocampal neurogenesis is amplified by aluminum exposure in a model of type 2 diabetes

In this study, we investigated the effects of chronic aluminum (Al) exposure for 10 weeks on cell proliferation and neuroblast differentiation in the hippocampus of type 2 diabetic rats. Six-week-old Zucker diabetic fatty (ZDF) and Zucker lean control (ZLC) rats were selected and randomly divided into Al- and non-Al-groups. Al was administered via drinking water for 10 weeks, after which the animals were sacrificed at 16 weeks of age. ZDF rats in both Al- and non-Al-groups showed increases in body weight and blood glucose levels compared to ZLC rats. Al exposure did not significantly affect body weight, blood glucose levels or pancreatic β-cells and morphology of the pancreas in either ZLC or ZDF rats. However, exposure to Al reduced cell proliferation and neuroblast differentiation in both ZLC and ZDF rats. Exposure to Al resulted in poor development of the dendritic processes of neuroblasts in both ZLC and ZDF rats. Furthermore, onset and continuation of diabetes reduced cell proliferation and neuroblast differentiation, and Al exposure amplified reduction of these parameters. These results suggest that Al exposure via drinking water aggravates the impairment in hippocampal neurogenesis that is typically observed in type 2 diabetic animals.

Sirtuin-2 inhibition affects hippocampal functions and sodium butyrate ameliorates the reduction in novel object memory, cell proliferation, and neuroblast differentiation / 한국실험동물학회지

We investigated the effects of the sirtuin-2 (SIRT2) inhibitor AK-7 on novel object memory, cell proliferation, and neuroblast differentiation in the dentate gyrus. In addition, we also observed the relationships with sodium butyrate, a histone deacetylase inhibitor, on the hippocampal functions. To investigate the effects of AK-7 on hippocampal functions, 10-week-old C57BL/6 mice were daily injected intraperitoneally with 20 mg/kg AK-7 alone or in combination with subcutaneous administration of 300 mg/kg sodium butyrate, a histone deacetylase inhibitor, for 21 days. A novel object recognition test was conducted on days 20 (training) and 21 (testing) of treatment. Thereafter, the animals were sacrificed for immunohistochemistry for Ki67 (cell proliferation) and doublecortin (DCX, neuroblast differentiation). AK-7 administration significantly reduced the time spent exploring new objects, while treatment in combination with sodium butyrate significantly alleviated this reduction. Additionally, AK-7 administration significantly reduced the number of Ki67-positive cells and DCX-immunoreactive neuroblasts in the dentate gyrus, while the treatment in combination with sodium butyrate ameliorated these changes. This result suggests that the reduction of SIRT2 may be closely related to age-related phenotypes including novel object memory, as well as cell proliferation and neuroblast differentiation in the dentate gyrus. In addition, sodium butyrate reverses SIRT2-related age phenotypes.

Comparison of pharmacological and genetic inhibition of cyclooxygenase-2: effects on adult neurogenesis in the hippocampal dentate gyrus

Inducible cyclooxygenase-2 (COX-2) has received much attention because of its role in neuro-inflammation and synaptic plasticity. Even though COX-2 levels are high in healthy animals, the function of this factor in adult neurogenesis has not been clearly demonstrated. Therefore, we performed the present study to compare the effects of pharmacological and genetic inhibition of COX-2 on adult hippocampal neurogenesis. Physiological saline or the same volume containing celecoxib was administered perorally every day for 5 weeks using a feeding needle. Compared to the control, pharmacological and genetic inhibition of COX-2 reduced the appearance of nestin-immunoreactive neural stem cells, Ki67-positive nuclei, and doublecortin-immunoreactive neuroblasts in the dentate gyrus. In addition, a decrease in phosphorylated cAMP response element binding protein (pCREB) at Ser133 was observed. Compared to pharmacological inhibition, genetic inhibition of COX-2 resulted in significant reduction of neural stem cells, cell proliferation, and neuroblast differentiation as well as pCREB levels. These results suggest that COX-2 is part of the molecular machinery that regulates neural stem cells, cell proliferation, and neuroblast differentiation during adult hippocampal neurogenesis via pCREB. Additionally, genetic inhibition of COX-2 strongly reduced neural stem cell populations, cell proliferation, and neuroblast differentiation in the dentate gyrus compared to pharmacological inhibition.

Treadmill exercise prevents diabetes-induced increases in lipid peroxidation and decreases in Cu,Zn-superoxide dismutase levels in the hippocampus of Zucker diabetic fatty rats

In the present study, we investigated the effects of treadmill exercise on lipid peroxidation and Cu,Zn-superoxide dismutase (SOD1) levels in the hippocampus of Zucker diabetic fatty (ZDF) rats and lean control rats (ZLC) during the onset of diabetes. At 7 weeks of age, ZLC and ZDF rats were either placed on a stationary treadmill or made to run for 1 h/day for 5 consecutive days at 16~22 m/min for 5 weeks. At 12 weeks of age, the ZDF rats had significantly higher blood glucose levels and body weight than the ZLC rats. In addition, malondialdehyde (MDA) levels in the hippocampus of the ZDF rats were significantly higher than those of the ZLC rats whereas SOD1 levels in the hippocampus of the ZDF rats were moderately decreased. Notably, treadmill exercise prevented the increase of blood glucose levels in ZDF rats. In addition, treadmill exercise significantly ameliorated changes in MDA and SOD1 levels in the hippocampus although SOD activity was not altered. These findings suggest that diabetes increases lipid peroxidation and decreases SOD1 levels, and treadmill exercise can mitigate diabetes-induced oxidative damage in the hippocampus.

Intragastric gavage with denatonium benzoate acutely induces neuronal activation in the solitary tract nucleus via the vagal afferent pathway

Natural toxic substances have a bitter taste and their ingestion sends signals to the brain leading to aversive oral sensations. In the present study, we investigated chronological changes in c-Fos immunoreactivity in the nucleus tractus solitarius (NTS) to study the bitter taste reaction time of neurons in the NTS. Equal volumes (0.5 mL) of denatonium benzoate (DB), a bitter tastant, or its vehicle (distilled water) were administered to rats intragastrically. The rats were sacrificed at 0, 0.5, 1, 2, 4, 8, or 16 h after treatment. In the vehicle-treated group, the number of c-Fos-positive nuclei started to increase 0.5 h after treatment and peaked 2 h after gavage. In contrast, the number of c-Fos-positive nuclei in the DB-treated group significantly increased 1 h after gavage. Thereafter, the number of c-Fos immunoreactive nuclei decreased over time. The number of c-Fos immunoreactive nuclei in the NTS was also increased in a dose-dependent manner 1 h after gavage. Subdiaphragmatic vagotomy significantly decreased DB-induced neuronal activation in the NTS. These results suggest that intragastric DB increases neuronal c-Fos expression in the NTS 1 h after gavage and this effect is mediated by vagal afferent fibers.

Unilateral cryptorchidism induces morphological changes of testes and hyperplasia of Sertoli cells in a dog / 한국실험동물학회지

Cryptorchidism is one of the most common genital defects in dogs. This study investigated the effects of abdominal cryptorchidism on morphology, cell proliferation, and Sertoli cell condition in a dog with spontaneous unilateral cryptorchidism. Elective orchidectomy was performed on the abdominal right testis and the scrotal left testis. Significant reductions in numbers of spermatogonia, spermatocytes, and spermatids were observed in hematoxylin and eosin stained sections of the cryptorchid testis. The size of the epididymal duct was smaller than that of the control testis. Based on Ki67 immunohistochemistry, the proliferative activity of spermatogonia and spermatocytes was significantly decreased in the cryptorchid testis. However, proliferative activity was increased in the epididymal duct. Based on GATA-4 immunohistochemistry, Sertoli cells were relatively resistant to cryptorchidism, and the proliferative activity of Sertoli cells was markedly increased in the cryptorchid testis than in the control testis. These results suggest that spontaneous unilateral cryptorchidism causes morphological defects in spermatogonia and spermatocytes in the testis and changes the size of the efferent ductule of the epididymis. In addition, spontaneous unilateral cryptorchidism increases proliferative activity of Sertoli cells, which may be a predisposing factor for Sertoli cell cancer in cryptorchid testes.

Cell proliferation and neuroblast differentiation in the dentate gyrus of high-fat diet-fed mice are increased after rosiglitazone treatment

In this study, we determined how rosiglitazone (RSG) differentially affected hippocampal neurogenesis in mice fed a low-fat diet (LFD) or high-fat diet (HFD; 60% fat). LFD and HFD were given to the mice for 8 weeks. Four weeks after initiating the LFD and HFD feeding, vehicle or RSG was administered orally once a day to both groups of mice. We measured cell proliferation and neuroblast differentiation in the subgranular zone of the dentate gyrus using Ki67 and doublecortin (DCX), respectively, as markers. In addition, we monitored the effects of RSG on the levels of DCX and brain-derived neurotrophic factor (BDNF) in hippocampal homogenates. At 8 weeks after the LFD feeding, the numbers of Ki67- and DCX-positive cells as well as hippocampal levels of DCX and BDNF were significantly decreased in the RSG-treated group compared to the vehicle-treated animals. In contrast, the numbers of Ki67- and DCX-positive cells along with hippocampal levels of DCX and BDNF in the HFD fed mice were significantly increased in the RSG-treated mice compared to the vehicle-treated group. Our data demonstrate that RSG can modulate the levels of BDNF, which could play a pivotal role in cell proliferation and neuroblast differentiation in the hippocampal dentate gyrus.