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Embryonic stem cells (ESCs) derived from outbred mice which share several genetic characteristics similar to humans have been requested for developing stem cell-based bioengineering techniques directly applicable to humans. Here, we report the generation of ESCs derived from the inner cell mass of blastocysts retrieved from 9-week-old female outbred ICR mice mated with 9-week-old male outbred ICR mice (ICRESCs). Similar to those from 129/Ola mouse blastocysts (E14ESCs), the established ICRESCs showed inherent characteristics of ESCs except for partial and weak protein expression and activity of alkaline phosphatase. Moreover, ICRESCs were not originated from embryonic germ cells or pluripotent cells that may co-exist in outbred ICR strain-derived mouse embryonic fibroblasts (ICRMEFs) used for deriving colonies from inner cell mass of outbred ICR mouse blastocysts. Furthermore, instead of outbred ICRMEFs, hybrid B6CBAF1MEFs as feeder cells could sufficiently support in vitro maintenance of ICRESC self-renewal. Additionally, ICRESC-specific characteristics (self-renewal, pluripotency, and chromosomal normality) were observed in ICRESCs cultured for 40th subpassages (164 days) on B6CBAF1MEFs without any alterations. These results confirmed the successful establishment of ESCs derived from outbred ICR mice, and indicated that self-renewal and pluripotency of the established ICRESCs could be maintained on B6CBAF1MEFs in culture.

Various trials have been conducted to develop therapies for serious untreatable diseases. Among these, those using stem cells have shown great promise, and adipose-derived mesenchymal stem cells (ADMSCs) are easier to obtain than other types of stem cells. Prior to clinical trials, characterization of ADMSCs with monoclonal antibodies should be performed. However, it is difficult to use species-specific antibodies for veterinarians. This study was conducted to confirm the panel of human antibodies applicable for use in immunophenotypic characterization of canine adipose-derived stem cells and feline ADMSCs extracted from subcutaneous adipose tissue collected during ovariohysterectomy. For flow cytometric immunophenotyping, the third passages of canine ADMSC and feline ADMSC and human CD31, CD34, CD42, CD44, CD62 and CD133 antibodies were used. Of these, CD133 reacted with canine cells (3.74%) and feline cells (1.34%). CD133 is known as a marker related with more primitive stem cell phenotype than other CD series. Because this human CD133 was not a species-specific antibody, accurate percentages of immunoreactivity were not confirmed. Nevertheless, the results of this study confirmed human CD133 as a meaningful marker in canine and feline ADMSCs.

In the present study, we examined the effects of Dendropanax morbifera Léveille leaf extract (DML) on dgalactose-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.

Cyclooxygenase-2 (COX-2) is a known inducible inflammatory mediator. COX-2 is constitutively expressed in the hippocampus and may regulate synaptic plasticity. The present study investigated the age‑associated alterations in white blood cell counts and hippocampal COX‑2 expression in healthy mice using immunohistochemical and western blot analyses at 1 month postnatal (PM1), PM3, PM6, PM12 and PM24. White blood cell counts were significantly decreased in the PM24 group when compared with the PM1 group. In addition, lymphocyte counts were decreased in the PM24 group when compared with all other groups. By contrast, monocyte, neutrophil and eosinophil counts were increased in the PM24 group; however, this did not reach statistical significance. COX‑2 expression was identified in the granule cells of the dentate gyrus and in the pyramidal cells of the hippocampal CA2/3 region. COX‑2 immunoreactivity was maintained until PM18, however, the levels significantly decreased by PM24. These results suggest that, despite alterations in the differential white blood cell counts, the significant decrease in constitutive COX‑2 expression in the hippocampus may be associated with degenerative age-associated alterations in synaptic plasticity in the hippocampus.

Although the expression of cyclooxygenase-2 (COX-2) is closely associated with inflammation in the brain, it is constitutively expressed in the brain, and its expression is regulated by synaptic activity. The present study investigated postnatal expression of COX‑2 in the hippocampus in C57BL/6 mice at postnatal days (P) 1, 7, 14, 28, and 56. In addition, the presented study examined the effects of COX‑2 on synaptic plasticity through Arc, phosphorylated cAMP response element‑binding protein (pCREB), N‑methyl‑d‑aspartate receptor 1 (GluN1), and GluN2A/2B immunohistochemistry, which was performed on COX‑2 knockout (KO) and wild‑type (WT) mice. Extremely weak COX‑2 immunoreactivity was detected in the hippocampal CA1‑3 areas in addition to the dentate gyrus at P1. Conversely, COX‑2 immunoreactivity was observed in the stratum pyramidale of the CA1‑3 regions and in the outer granule cell layer of the dentate gyrus at P7. Additionally, although peak COX‑2 immunoreactivity was observed in all hippocampal sub‑regions, including the dentate gyrus at P14, it was significantly decreased at P14. Finally, COX‑2 immunoreactivity and the distribution pattern seen at P56 in the hippocampal CA1‑3 regions were similar to those observed at P28, whereas, they were identified in the inner half of the granule cell layer of the dentate gyrus. The western blot analysis revealed that the COX‑2 protein levels peaked at P14 and were decreased at P28 and P56. Additionally, the number of Arc and pCREB immunoreactive cells as well as GluN1 and GluN2A/2B immunoreactivity of COX‑2 KO mice were significantly decreased in the dentate gyrus when compared with that in WT mice. Taken together, the results of the present study suggest that COX‑2 serves an important role in synaptic plasticity in the dentate gyrus and changes in the levels of its constitutive expression are associated with the hippocampal dentate gyrus postnatal development.

Transient forebrain ischemia promotes a robust increase in neuroblast differentiation in the hippocampal dentate gyrus that peaks 7–15 days after the surgery. In this study, we compared the glucose transporter 3 (GLUT3)-dependent glucose utilization and the dynamin-1 (DNM1)-dependent neurite growth in the hippocampus of Mongolian gerbils 15 days after the induction of transient forebrain ischemia. The animals were subjected to a 5 min transient ischemia protocol and sacrificed 15 days after the surgery. Both doublecortin (DCX) immunoreactive neuroblasts and DCX total protein levels were abundantly increased in the ischemic group compared to the levels observed in the control group. In addition, animals in the ischemic group showed elevated GLUT3 immunoreactivity in the subgranular zone of the dentate gyrus compared to animals in the control group. Based on the double immunofluorescent study, increased DCX-immunoreactive neuroblasts were co-localized with GLUT3-immunoreactive components in the dent...

Recent evidence exists that glucose transporter 3 (GLUT3) plays an important role in the energy metabolism in the brain. Most previous studies have been conducted using focal or hypoxic ischemia models and have focused on changes in GLUT3 expression based on protein and mRNA levels rather than tissue levels. In the present study, we observed change in GLUT3 immunoreactivity in the adult gerbil hippocampus at various time points after 5 minutes of transient forebrain ischemia. In the sham-operated group, GLUT3 immunoreactivity in the hippocampal CA1 region was weak, in the pyramidal cells of the CA1 region increased in a time-dependent fashion 24 hours after ischemia, and in the hippocampal CA1 region decreased significantly between 2 and 5 days after ischemia, with high level of GLUT3 immunoreactivity observed in the CA1 region 10 days after ischemia. In a double immunofluorescence study using GLUT3 and glial-fibrillary acidic protein (GFAP), we observed strong GLUT3 immunoreactivity in the astrocytes. GLUT3 immunoreactivity increased after ischemia and peaked 7 days in the dentate gyrus after ischemia/reperfusion. In a double immunofluorescence study using GLUT3 and doublecortin (DCX), we observed low level of GLUT3 immunoreactivity in the differentiated neuroblasts of the subgranular zone of the dentate gyrus after ischemia. GLUT3 immunoreactivity in the sham-operated group was mainly detected in the subgranular zone of the dentate gyrus. These results suggest that the increase in GLUT3 immunoreactivity may be a compensatory mechanism to modulate glucose level in the hippocampal CA1 region and to promote adult neurogenesis in the dentate gyrus.

Beta-nerve growth factor (β-NGF) is known to be a major leading cause of neuronal plasticity. To identify the possible action mechanisms of β-NGF gene therapy for sciatic nerve recovery, experimental dogs were randomly divided into control, pyridoxine, and pyridoxine + β-NGF groups. We observed chronological changes of morphology in the dorsal root ganglia in response to pyridoxine toxicity based on cresyl violet staining. The number of large neurons positive for cresyl violet was dramatically decreased after pyridoxine intoxication for 7 days in the dorsal root ganglia and the neuron number was gradually increased after pyridoxine withdrawal. In addition, we also investigated the effects of β-NGF gene therapy on neuronal plasticity in pyridoxine-induced neuropathic dogs. To accomplish this, tyrosine kinase receptor A (TrkA), βIII-tubulin and doublecortin (DCX) immunohistochemical staining was performed at 3 days after the last pyridoxine treatment. TrkA-immunoreactive neurons were dramatically decreased in the pyridoxine group compared to the control group, but strong TrkA immunoreactivity was observed in the small-sized dorsal root ganglia in this group. TrkA immunoreactivity in the dorsal root ganglia was similar between β-NGF and control groups. The numbers of βIII-tubulin- and DCX-immunoreactive cells decreased significantly in the pyridoxine group compared to the control group. However, the reduction of βIII-tubulin- and DCX-immunoreactive cells in the dorsal root ganglia in the β-NGF group was significantly ameliorated than that in the pyridoxine group. These results indicate that β-NGF gene therapy is a powerful treatment of pyridoxine-induced neuropathic damage by increasing the TrkA and DCX levels in the dorsal root ganglia. The experimental protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University, South Korea (approval No. SNU-060623-1, SNU-091009-1) on June 23, 2006 and October 9, 2009, respectively.

Ischemia causes oxidative stress in the endoplasmic reticulum (ER), accelerates the accumulation of unfolded and misfolded proteins, and may ultimately lead to neuronal cell apoptosis. In the present study, we investigated the effects of protein disulfide-isomerase A3 (PDIA3), an ER-resident chaperone that catalyzes disulfide-bond formation in a subset of glycoproteins, against oxidative damage in the hypoxic HT22 cell line and against ischemic damage in the gerbil hippocampus. We also confirmed the neuroprotective effects of PDIA3 by using PDIA3-knockout HAP1 cells. The HT22 and HAP1 cell lines showed effective (dose-dependent and time-dependent) penetration and stable expression of the Tat-PDIA3 fusion protein 24 h after Tat-PDIA3 treatment compared to that in the control-PDIA3-treated group. We observed that the fluorescence for both 2′,7′-dichlorofluorescein diacetate (DCF-DA) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL), which are markers for the formation of hydrogen peroxide (H2O2)-induced reactive oxygen species and apoptosis, respectively, was higher in HAP1 cells than in HT22 cells. The administration of Tat-PDIA3 significantly reduced the (1) DCF-DA and TUNEL fluorescence in HT22 and HAP1 cells, (2) ischemia-induced hyperactivity that was observed 1 day after ischemia/reperfusion, (3) ischemia-induced neuronal damage and glial (astrocytes and microglia) activation that was observed in the hippocampal CA1 region 4 days after ischemia/reperfusion, and (4) lipid peroxidation and nitric oxide generation in the hippocampal homogenates 3–12 h after ischemia/reperfusion. Transient forebrain ischemia significantly elevated the immunoglobulin-binding protein (BiP) and C/EBP-homologous protein (CHOP) mRNA levels in the hippocampus at 12 h and 4 days after ischemia, relative to those in the time-matched sham-operated group. Administration of Tat-PDIA3 ameliorated the ischemia-induced upregulation of BiP mRNA levels versus the Tat peptide- or control-PDIA3-treated groups, and significantly reduced the induction of CHOP mRNA levels, at 12 h or 4 days after ischemia. Collectively, these results suggest that Tat-PDIA3 acts as a neuroprotective agent against ischemia by attenuating oxidative damage and blocking the apoptotic pathway that is related to the unfolded protein response in the ER.

An 11-year-old, intact male Pekingese was brought to the Veterinary Teaching Hospital of Kangwon National University with a 10-day history of seizures. Fifteen days before coming to Kangwon National University, the dog had visited a local animal hospital for lameness, and non-steroidal anti-inflammatory drugs were prescribed to treat this symptom. However, 10 days before coming to our hospital, the dog experienced generalized seizures. Two days before his arrival, generalized ataxia and mental dullness also occurred. Our examinations revealed no remarkable findings on a routine blood test or X-ray. However, the neurological examinations confirmed mental dullness, generalized ataxia, and a lack of menace response and pupillary light reflexes. Nine hours later, dyspnea occurred, and 12 hours after that, the patient was euthanized per the client’s request. A necropsy of transverse sections confirmed the presence of a prominent midline shift due to extended tumor growth. On histopathological analyses, pseudopalisading necrosis of the glial cells and microvascular proliferation were observed. In immunohistochemical analysis, glial fibrillary acidic protein, proliferating cell nuclear antigens, and ionized calcium binding adaptor molecule 1 immunoreactive cells were observed in the tumor area. Based on the results, the tumor was confirmed to be a glioblastoma. Primary intracranial tumors are rare in the veterinary field. This case report describes the clinical and histopathological findings of glioblastoma in a Pekingese.

Background Mammary gland tumors are the most common tumors in sexually intact female dogs; however, they are rare in male dogs. This study was conducted to investigate the relationship between sexual hormones and mammary gland tumors in a male dog. Case presentation A 13-year-old, intact male Cocker Spaniel presented to the Veterinary Teaching Hospital of Kangwon National University, Republic of Korea, with an acute right ruptured caudal abdominal mass. Physical examination revealed a 14 × 14 cm ruptured mass in the right caudal abdomen, as well as a 1.5 × 1.5 cm mass in the first right mammary gland. The estrogen and progesterone concentrations in serum were within normal levels. Total mastectomy was done on the right side mammary glands. Following surgery, the site was fully recovered; however, a mass that had grown to 2 × 2 cm was found in the left fifth mammary gland and a testis tumor was also found over the period of 4 months. Mastectomy was performed on the left caudal mammary gland and castration was also performed. After the final surgery, the dog fully recovered. Histopathological examination of all three masses revealed high grade mammary adenocarcinoma in the mammary gland and the testis was diagnosed as Leydig cell adenoma. Immunohistochemical analysis revealed that the estrogen and progesterone receptors were expressed on limited cells in mammary and testis tumors. Conclusion The results of this study suggest that mammary tumors and testes tumors can occur in male dogs without relationship to female sexual hormone.

In the present study, we examined change of ionized calcium-binding adapter molecule 1 (Iba-1) in the adult and aged gerbil spinal cords. Significant change of morphological feature and neuronal cell loss were not observed in both adult and aged spinal cords of gerbil after NeuN immunohistochemistry and Fluoro-Jade B histofluoresce staining. Iba-1-immunoreactive microglia broadly distributed in the spinal cord. Most of Iba-1-immunoreactive microglia showed ramified forms in the adult gerbil cervical and lumbar spinal cords. However, morphological changes of Iba-1-immunoreactive microglia were observed in the cervical and lumbar regions of the aged gerbil spinal cord. These microglia were showed a hypertrophied body with shortened swollen processes which was characteristic of activated microglia. In addition, Iba-1 protein level significantly higher in aged cervical and lumbar spinal cords than those in the adult gerbil. The present study showed an increase of activated forms of Iba-1-immunoreactive microglia and its protein level without marked changes in morphological features and neuronal loss in the aged spinal cord compared to those in the adult gerbil spinal cord. This result suggests that the increase of Iba-1 expression in the aged spinal cord may be closely associated with age-related changes in aged gerbil spinal cord.

Despite that porcine spermatogonial stem cells (pSSCs) have been regarded as a practical tool for preserving eternally genetic backgrounds derived from pigs with high performance in the economic traits or phenotypes of specific human diseases, there were no reports about precise definition of niche conditions promoting proliferation and maintenance of pSSCs. Accordingly, we tried to determine niche conditions supporting proliferation and maintenance of undifferentiated pSSCs for short-term. For these, undifferentiated pSSCs were progressively cultured in different composition of culture medium, seeding density of pSSCs, type of feeder cells and concentration of growth factors, and then total number of and alkaline phosphatase (AP) activity of pSSCs were investigated at post-6 day culture. As the results, the culture of 4 × 105 pSSCs on mitotically in activated 2 × 105 STO cells in the mouse embryonic stem cell culture medium (mESCCM) supplemented with 30 ng/ml glial cell line-derived neurotrophic factor (GDNF) was identified as the best niche condition supporting effectively the short-term maintenance of undifferentiated pSSCs. Moreover, the optimized short-term culture system will be a basis for developing long-term culture system of pSSCs in the following researches

Despite many researches related with in-vitro culture of porcine spematogonial stem cells (SSCs), adherent culture system widely used has shown a limitation in the maintenance of porcine SSC self-renewal. Therefore, in order to overcome this obstacle, suspension culture, which is known to have numerous advantage over adherent culture, was applied to the culture of porcine SSCs. Porcine SSCs retrieved from neonatal testes were suspension-cultured for 5 days or 20 days, and characteristics of suspension-cultured porcine SSCs including proliferation, alkaline phosphatase (AP) activity, and self-renewal-specific gene expression were investigated and compared with those of adherent-cultured porcine SSCs. As the results, the suspension-cultured porcine SSCs showed entirely non- proliferative and significantly higher rate of AP-positive cells and expression of self-renewal-specific genes than the adherent-cultured porcine SSCs. In addition, long-term culture of porcine SSCs in suspension condition induced significant decrease in the yield of AP staining-positive cells on post-day 10 of culture. These results showed that suspension culture was inappropriate to culture porcine SSCs, because the culture of porcine SSCs in suspension condition didn’t stimulate proliferation and maintain AP activity of porcine SSCs, regardless of culture periods.

Background: In the present study, we investigated the effects of oil products from two Allium species: Allium sativum (garlic) and Allium hookeri (Chinese chives) on cell proliferation and neuroblast differentiation in the mouse dentate gyrus. Methods: Using corn oil as a vehicle, the essential oil from garlic (10 ml/kg), or Chinese chives (10 ml/kg) was administered orally to 9-week-old mice once a day for 3 weeks. One hour following the last treatment, a novel object recognition test was conducted and the animals were killed 2 h after the test. Results: In comparison to the vehicle-treated group, garlic essential oil (GO) treatment resulted in significantly increased exploration time and discrimination index during the novel object recognition test, while Chinese chives essential oil (CO) reduced the exploration time and discrimination index in the same test. In addition, the number of Ki67-immunoreactive proliferating cells and doublecortin-immunoreactive neuroblasts significantly increased in the dentate gyrus of GO-treated animals. However, administration of CO significantly decreased cell proliferation and neuroblast differentiation. Administration of GO significantly increased brain-derived neurotrophic factor (BDNF) levels and decreased acetylcholinesterase (AChE) activity in the hippocampal homogenates. In contrast, administration of CO decreased BDNF protein levels and had no significant effect on AChE activity, compared to that in the vehicle-treated group. Conclusions: These results suggest that GO significantly improves novel object recognition as well as increases cell proliferation and neuroblast differentiation, by modulating hippocampal BDNF protein levels and AChE activity, while CO impairs novel object recognition and decreases cell proliferation and neuroblast differentiation, by reducing BDNF protein levels in the hippocampus.

Transient forebrain ischemia depletes glucose and oxygen levels in the brain. In this pathological condition, lactate serves an important role in cellular metabolism as the end product of glycolysis. The present study investigated the expression of monocarboxylate transporter 4 (MCT4) in lactate metabolism in the hippocampal CA1 region following induction of transient forebrain ischemia. MCT4 immunoreactivity was detected in CA1 pyramidal cells of the sham-operated group. Animals from the ischemic group exhibited a transient decrease in MCT4 immunoreactivity in the CA1 region between 30 min and 3 h following ischemia compared with the sham‑operated group. The initial decrease in immunoreactivity observed between 30 min and 3 h following ischemia was followed by an increase at 2 days after the treatment. A significant increase in MCT4 immunoreactivity levels was observed 2 days after ischemia compared with the sham‑operated group. Limited MCT4 immunoreactivity was observed in the pyramidal neurons 3 days after ischemia. At 4‑10 days after ischemia, MCT4 immunoreactivity was detected in the strata radiatum, oriens and pyramidale. Furthermore, MCT4 immunoreactivity levels in the CA1 region exhibited a time‑dependent increase following ischemia. The results indicated that there were transient alterations observed in the localization of MCT4 following the induction of ischemia, and further studies are required to investigate the association between MCT4 expression and lactate metabolism in providing energy to the post‑ischemic brain.

Background Nerve growth factor (NGF) is known not only as a major factor for neuronal plasticity but also as a pain stimulator. Although there have been several trials with NGF for its application in the regeneration or protection of the nervous system, the pain induced by NGF remains a challenge to be overcome. In this study, the pain induced by NGF gene therapy was evaluated. Results Vehicle or recombinant dog NGF plasmid was administered into the intrathecal space of dogs. Twenty-four hours after the vehicle or NGF plasmid inoculation, dogs were subcutaneously treated with 150 mg/kg pyridoxine every day for 7 days. For pain assessment, physical examination and electrophysiological recording were performed. Only in the vehicle-treated group, weight loss occurred, while NGF plasmid inoculation significantly improved this physical abnormalities. In the vehicle-treated group, electrophysiological recordings showed that H-reflex disappeared at 24 h after the last pyridoxine treatment. However, in the NGF plasmid inoculated group, the H-reflex were normal. In the results of immunohistochemistry, the NGF plasmid administration efficiently expressed in the dorsal root ganglia and significantly increased the pyridoxine-induced reduction of calcitonin gene-related peptide (CGRP) immunoreactive neurons, but not in substance P immunoreactive neurons, in the dorsal root ganglia. Conclusions Given these results, we reason that NGF gene therapy in pyridoxine induced neuropathic dogs does not induce neuropathic pain with this dosage, even with increasing the expression of CGRP. Electronic supplementary material The online version of this article (doi:10.1186/s12868-015-0236-5) contains supplementary material, which is available to authorized users.

In general, the seminiferous tubule basement membrane (STBM), comprising laminin, collagen IV, perlecan, and entactin, plays an important role in self-renewal and spermatogenesis of spermatogonial stem cells (SSCs) in the testis. However, among the diverse extracellular matrix (ECM) proteins constituting the STBM, the mechanism by which each regulates SSC fate has yet to be revealed. Accordingly, we investigated the effects of various ECM proteins on the maintenance of the undifferentiated state of SSCs in pigs. First, an extracellular signaling-free culture system was optimized, and alkaline phosphatase (AP) activity and transcriptional regulation of SSC-specific genes were analyzed in porcine SSCs (pSSCs) cultured for 1, 3, and 5 days on non-, laminin- and collagen IV-coated Petri dishes in the optimized culture system. The microenvironment consisting of glial cell-derived neurotrophic factor (GDNF)-supplemented mouse embryonic stem cell culture medium (mESCCM) (GDNF-mESCCM) demonstrated the highest efficiency in the maintenance of AP activity. Moreover, under the established extracellular signaling-free microenvironment, effective maintenance of AP activity and SSC-specific gene expression was detected in pSSCs experiencing laminin-derived signaling. From these results, we believe that laminin can serve as an extracellular niche factor required for the in vitro maintenance of undifferentiated pSSCs in the establishment of the pSSC culture system.

This study aims to track the longitudinal alteration pattern on the trabecular bone microarchitecture at tibial epiphysis induced by T-OA over time using in vivo micro computed tomography (). Ten SD rats were divided into control (n = 5) and T-OA (n = 5) groups. Anterior cruciate ligament transaction was performed for the T-OA group. The results showed that the alteration pattern on the trabecular bone microarchitecture at tibial epiphysis in the T.OA group was definitely different compared with that in the CON group from 0 to 8 weeks (approximately 4-16%, P > 0.05). In particular, a difference was observed in the bone formation and density distributions over time (from 0 or 4 to 8 weeks; approximately 5.15%, P

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 immuno-histochemistry 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.

To date, the methods available for isolating spermatogonial stem cells (SSCs) from porcine testicular cells have a low efficiency of cell separating. Therefore, we tried to develop a novel isolation technique with a high-yield cell separating ability to isolate SSCs from porcine testes.We confirmed the presence of SSCs by measuring alkaline phosphatase (AP) activity and SSC-specific gene expression in neonatal porcine testis-derived testicular cells. Subsequently, the isolation of SSCs from testicular cells was performed using different techniques as follows: differential plating (DP), double DP, Petri dish plating post-DP, magnetic-activated cell sorting (MACS), and MACS post-DP. Positive AP staining was used to assess and compare the isolation efficiency of each method.Petri dish plating post-DP resulted in the highest isolation efficiency. The putative SSCs isolated using this method was then further characterized by analyzing the expression of SSC-specific genes and -related proteins, and germ cell-specific genes. OCT4, NANOG, EPCAM, THY1, and UCHL1 were expressed transcriptionally, and OCT4, NANOG, SOX2, TRA-1-60, TRA-1-81, and PLZF were expressed translationally in 86 % of the isolated SSCs. In contrast, no difference was observed in the percentage of cells expressing luteinizing hormone receptor (LHR), a Leydig cell-specific protein, or GATA4, a Sertoli cell-specific protein, between SSCs and negative control cells. In addition, transcriptional expression of VASA, a primordial germ cell-specific marker, and DAZL, a premeiotic germ cell-specific marker, wasn't and was detected, respectively.We successfully developed a novel high-yield technique to isolate SSCs from porcine testes to facilitate future porcine SSC-related research.