Number Of BrdU-immunoreactive Cells Research Articles

NDP-α-MSH induces intense neurogenesis and cognitive recovery in Alzheimer transgenic mice through activation of melanocortin MC4 receptors.

Melanocortins exert neuroprotection in a variety of experimental neurodegenerative disorders, including Alzheimer's disease (AD). Further, in previous research we showed that these endogenous peptides stimulate neurogenesis in an acute neurodegenerative disorder such as ischemic stroke. In the present research, we investigated the potential neurogenic effect of melanocortins in AD using APPSwe transgenic mice (Tg2576). To this purpose, 24week-old animals were prepared for 5-bromo-2'-deoxyuridine (BrdU) labeling of proliferating cells on days 1-11 of the study. Treatment of Tg2576 mice with nanomolar doses of the melanocortin analog [Nle(4),D-Phe(7)]α-melanocyte-stimulating hormone (NDP-α-MSH), administered once daily from day 1 to 50, improved brain histology and cognitive functions relative to saline-treated Tg2576 animals. No signs of toxicity were observed. Immunohistochemical examination of the hippocampus at the end of the study (day 50) showed that NDP-α-MSH-treated Tg2576 mice had a greater number of BrdU immunoreactive cells colocalized with NeuN (an indicator of mature neurons) and Zif268 (an indicator of functionally integrated neurons) in the dentate gyrus, relative to saline-treated Tg2576 animals; no newly formed astrocytes were found. Animal pretreatment with the selective melanocortin MC4 receptor antagonist HS024 before each NDP-α-MSH administration prevented all the beneficial effects of the peptide. The present data indicate that MC4 receptor stimulation by a melanocortin prevents cognitive decline in experimental AD, this effect being associated not only with neuroprotection but also with an intense neurogenesis. MC4 receptor agonists could be innovative and safe candidates to counteract AD progression in humans.

Up-regulation of the canonical Wnt-3A and Sonic hedgehog signaling underlies melanocortin-induced neurogenesis after cerebral ischemia

In experimental cerebral ischemia, melanocortin MC4 receptor agonists induce neuroprotection and neurogenesis with subsequent long-lasting functional recovery. Here we investigated the molecular mechanisms underlying melanocortin-induced neurogenesis. Gerbils were subjected to transient global cerebral ischemia, then they were treated every 12h, and until sacrifice, with 5-bromo-2′-deoxyuridine (BrdU; to label proliferating cells), and the melanocortin analog [Nle4,d-Phe7]α-melanocyte-stimulating hormone (NDP-α-MSH) or saline. NDP-α-MSH increased hippocampus dentate gyrus (DG) expression of Wnt-3A, β-catenin, Sonic hedgehog (Shh), Zif268, interleukin-10 (IL-10) and doublecortin (DCX), as detected at days 3, 6 and 10 after the ischemic insult. Further, an elevated number of BrdU immunoreactive cells was found at days 3 and 10, and an improved histological picture with reduced neuronal loss at day 10, associated with learning and memory recovery. Pharmacological blockade of the Wnt-3A/β-catenin and Shh pathways, as well as of melanocortin MC4 receptors, prevented all effects of NDP-α-MSH. These data indicate that, in experimental brain ischemia, treatment with melanocortins acting at MC4 receptors induces neural stem/progenitor cell proliferation in the DG by promptly and effectively triggering the canonical Wnt-3A/β-catenin and Shh signaling pathways. Activation of these pathways is associated with up-regulation of the repair factor Zif268 and the neurogenesis facilitating factor IL-10, and it seems to address mainly toward a neuronal fate, as indicated by the increase in DCX positive cells.

Propofol Anesthesia Impairs the Maturation and Survival of Adult-born Hippocampal Neurons

Adult neurogenesis occurs in the hippocampus of most mammals, including humans, and plays an important role in hippocampal-dependent learning. This process is highly regulated by neuronal activity and might therefore be vulnerable to anesthesia. In this article, the authors investigated this possibility by evaluating the impact of propofol anesthesia on mouse hippocampal neurons generated during adulthood, at two functionally distinct maturational stages of their development. Adult-born hippocampal neurons were identified using the cell proliferation marker bromodeoxyuridine or a retroviral vector expressing the green fluorescent protein in dividing cells and their progenies. Eleven or 17 days after the labeling procedure, animals (n = 3-5 animals per group) underwent a 6-h-long propofol anesthesia. Twenty-one days after labeling, the authors analyzed the survival, differentiation, and morphologic maturation of adult-born neurons using confocal microscopy. Propofol impaired the survival and maturation of adult-born neurons in an age-dependent manner. Anesthesia induced a significant decrease in the survival of neurons that were 17 days old at the time of anesthesia, but not of neurons that were 11 days old. Similarly, propofol anesthesia significantly reduced the dendritic maturation of neurons generated 17 days before anesthesia, without interfering with the maturation of neurons generated 11 days before anesthesia. These results reveal that propofol impairs the survival and maturation of adult-born hippocampal neurons in a developmental stage-dependent manner in mice.

Posttraumatic hypothermia increases doublecortin expressing neurons in the dentate gyrus after traumatic brain injury in the rat

Previous studies have demonstrated that moderate hypothermia reduces histopathological damage and improves behavioral outcome after experimental traumatic brain injury (TBI). Further investigations have clarified the mechanisms underlying the beneficial effects of hypothermia by showing that cooling reduces multiple cell injury cascades. The purpose of this study was to determine whether hypothermia could also enhance endogenous reparative processes following TBI such as neurogenesis and the replacement of lost neurons. Male Sprague–Dawley rats underwent moderate fluid-percussion brain injury and then were randomized into normothermia (37°C) or hypothermia (33°C) treatment. Animals received injections of 5-bromo-2′-deoxyuridine (BrdU) to detect mitotic cells after brain injury. After 3 or 7days, animals were perfusion-fixed and processed for immunocytochemistry and confocal analysis. Sections were stained for markers selective for cell proliferation (BrdU), neuroblasts and immature neurons (doublecortin), and mature neurons (NeuN) and then analyzed using non-biased stereology to quantify neurogenesis in the dentate gyrus (DG). At 7days after TBI, both normothermic and hypothermic TBI animals demonstrated a significant increase in the number of BrdU-immunoreactive cells in the DG as compared to sham-operated controls. At 7days post-injury, hypothermia animals had a greater number of BrdU (ipsilateral cortex) and doublecortin (ipsilateral and contralateral cortex) immunoreactive cells in the DG as compared to normothermia animals. Because adult neurogenesis following injury may be associated with enhanced functional recovery, these data demonstrate that therapeutic hypothermia sustains the increase in neurogenesis induced by TBI and this may be one of the mechanisms by which hypothermia promotes reparative strategies in the injured nervous system.

Treatment of cerebral ischemia with melanocortins acting at MC4 receptors induces marked neurogenesis and long-lasting functional recovery

Melanocortins produce neuroprotection against ischemic stroke with subsequent long-lasting functional recovery, through melanocortin MC(4) receptor activation. Here we investigated whether the long-lasting beneficial effect of melanocortins in stroke conditions is associated with a stimulation of neurogenesis. Gerbils were subjected to transient global cerebral ischemia by occluding both common carotid arteries for 10 min; then, they were prepared for 5-bromo-2'-deoxyuridine (BrdU) labeling of proliferating cells. Delayed treatment (up to 9 h after the ischemic injury) for 11 days with the melanocortin analog [Nle(4),D-Phe(7)]α-melanocyte-stimulating hormone (NDP-α-MSH) improved learning and memory throughout the 50-day observation period. Immunohistochemical examination of the hippocampus on day 50 showed, in the dentate gyrus, an elevated number of BrdU immunoreactive cells colocalized with NeuN (used as indicator of mature neurons) and Zif268 (used as indicator of functionally integrated neurons). Retrospective analysis during the early stage of neural stem/progenitor cell development (days 3 and 4 after stroke) showed, in NDP-α-MSH-treated gerbils, a high degree of daily cell proliferation and revealed that NDP-α-MSH favorably affects Wnt-3A signaling pathways and doublecortin expression. Pharmacologic blockade of MC(4) receptors prevented all effects of NDP-α-MSH. These data indicate that treatment of cerebral ischemia with MC(4) receptor agonists induces, with a broad window of therapeutic opportunity, long-lasting functional recovery associated with a large number of mature and likely functional newborn neurons in brain injured areas. Our findings reveal previously undescribed effects of melanocortins which might have major clinical implications.

Acidic fibroblast growth factor delivered intranasally induces neurogenesis and angiogenesis in rats after ischemic stroke

<title/>Background: Enhancing angiogenesis and neurogenesis is a novel therapeutic strategy for stroke treatment. Acidic fibroblast growth factor (aFGF) has been shown to have both angiogenesis and neurogenesis effects in animals with cerebral ischemia. But aFGF can not enter the brain freely after system administration due to the filtration of the blood-brain barrier (BBB). Intranasal administration of aFGF as a noninvasive method can bypass the BBB and enter the central nervous system directly without systemic adverse effects.Methods: To investigate the therapeutic effects of intranasally delivered aFGF, adult male Sprague Dawley rats were subjected to middle cerebral artery occlusion (MCAO) and intranasally administrated with aFGF or saline starting at 24 hours and once daily for the subsequent 6 days. BrdU (50mg/kg) was intraperitoneally injected daily for 13 days. A modified neurological severity scores test was performed before and at 1, 7, 14 days after MCAO. Infarct volumes were evaluated after hematoxylin and eosin staining. Immunohistochemistry was performed to detect BrdU immunoreactive cells and BrdU / DCX double labeled cells. Microvessels were labeled by FITC-dextran and the numbers, length and diameters of vessels were also measured.Results: Intranasal aFGF did not significantly reduce the lesion size, but did improve neurological functional recovery. In the subventricular zone and the striatum, numbers of BrdU immunoreactive cells were significantly increased in aFGF group at day 14, and the majority of BrdU positive cells were co-labeled with DCX. At 14 days after ischemia, the percentage of BrdU positive endothelial cells around the ischemic lesions were significantly increased in aFGF group, compared with control. Quantitative analysis of FITC-dextran perfusing vessels revealed a significant increase of vessels in the boundary regions of ischemia in the rats treated with aFGF. But there were no significant differences concerning the length and the diameter of the vessels between groups.Conclusion: In summary, aFGF may enhance neurogenesis and angiogenesis after focal cerebral ischemia. Intranasal administration of aFGF may be a feasible approach for ischemic stroke treatment.

Combination of stem cell mobilized by granulocyte-colony stimulating factor and human umbilical cord matrix stem cell: therapy of traumatic brain injury in rats.

Clinical studies of treating traumatic brain injury (TBI) with autologous adult stem cells led us to examine the impression of a combination therapy. This was performed by intravenous injection of human umbilical cord matrix stem cell (hUCMSC-Wharton(,)s jelly stem cell) with bone marrow cell mobilized by granulocytecolony stimulating factor (G-CSF) in rats injured with cortical compact device. Adult male Wistar rats (n= 50) were injured with controlled cortical impact device and divided into five groups. All injections were performed 1 day after injury into the tail veins of rats. Neurological functional evaluation of animals was performed before and after injury using modified neurological severity scores (mNSS). Animals were sacrificed 42 days after TBI and brain sections were stained by Brdu immunohistochemistry. Statistically significant improvement in functional outcome was observed in treatment groups when compared with control (P< 0.01). mNSS showed no significant differences among the hUCMSC and G-CSF treated groups at any time point (end of trial). Rats with hUCMSC + G-CSF treatment had a significant improvement on mNSS at 5 and 6 week compared to other treatment group (P< 0.01). Histological analysis in G-CSF+ hUCMSC treated traumatic rats exhibited significant increase in numbers of Brdu immunoreactive cells in their traumatic core compared with other labeled group.

Sub-chronic administration of rimonabant causes loss of antidepressive activity and decreases doublecortin immunoreactivity in the mouse hippocampus

Rimonabant is a cannabinoid receptor 1 antagonist, and is used to treat anorexia and obesity. However, it has been suggested that rimonabant may act as a depressant. In the present study, we investigated the depressive effects of rimonabant using behavioral and biochemical methods. A single treatment with rimonabant (10 mg/kg, p.o.) reduced immobility duration in the forced swimming test (FST) to a level similar to that observed for the tricyclic antidepressant, imipramine (15 mg/kg, i.p.). However, mice treated with rimonabant for 2 weeks did not show any significant reductions in immobility duration versus vehicle-treated controls. To investigate why the antidepressant effect of rimonabant disappeared after extended treatment, we carried out 5-bromo-2-deoxyuridine (BrdU) and doublecortin (DCX) immunohistochemistry assay. Numbers of BrdU-immunoreactive cells were not significantly changed after administering rimonabant (10 mg/kg, p.o.) for 2 weeks in the hippocampal dentate gyrus (DG), but interestingly, numbers of DCX-immunopositive cells in the DG were significantly reduced after 2 weeks of rimonabant treatment at doses of 1 or 10 mg/(kg day) compared with vehicle-treated controls ( P < 0.05). These results suggest that sub-chronic treatments with rimonabant inhibit cell proliferation in DG, and that a lack of antidepressive activity may be related to a reduction in cell proliferation in this region.

Gender Differences in Febrile Seizure–induced Proliferation and Survival in the Rat Dentate Gyrus

Febrile seizures are fever-associated early-life seizures that are thought play a role in the development of epilepsy. Seizure-induced proliferation of dentate granule cells has been demonstrated in several adult animal models and is thought to be an integral part of epileptogenesis. The aim of the present study was to investigate proliferation and survival of dentate gyrus (DG) cells born after early-life hyperthermia (HT)-induced seizures in male and female rats. At postnatal day (PN) 10, male and female rats were exposed to heated air to induce seizures. Littermates were used as normothermia controls. Convulsive behavior was observed by two researchers. From PN11 to PN16, rats were injected with bromodeoxyuridine (BrdU) to label dividing cells. The number of BrdU-immunoreactive cells in the DG was counted at PN17 and PN66. At PN17, male as well as female HT rats had the same amount of BrdU-positive cells compared with controls. At PN66, significantly more BrdU-positive cells were left in HT females (53%) than in controls (44%, percentage of BrdU-positive cells at PN17), whereas no difference was found between HT males and male controls. The net result of proliferation and survival at PN66 was that female HT rats had the same number of BrdU-immunoreactive cells as controls, whereas male HT rats had 25% more BrdU-immunoreactive cells than did controls (p < 0.05). Early-life seizures cause a sexually dimorphic cytogenic response that results in an increased population of newborn DG cells in young adult males, while leaving that of young adult females unaltered.

Low-dose photodynamic therapy increases endothelial cell proliferation and VEGF expression in nude mice brain

We tested whether low-dose photodynamic therapy (PDT) induces an angiogenic response in the normal brain of nude mice (n=20). Normal brains of nude mice were subjected to PDT at low doses (Photofrin: 2 mg/kg; optical: 2 J/cm(2) and 4 J/cm(2)). BrdU (50 mg/kg) was injected (intraperitoneally, i.p.) daily from PDT treatment to sacrifice (1 and 2 weeks after PDT). Laser scanning confocal microscopy, immunohistochemistry, and immunofluorescence staining were performed to assay angiogenic response. Morphological results show no significant tissue damage induced by PDT and two- and three-dimensional image analyses revealed no significant difference in vascular structure between the areas of exposure to PDT and contralateral areas in all mice. However, the number of BrdU immunoreactive cells were significantly increased in the areas of PDT treatment compared with contralateral hemisphere in both groups, and the number of BrdU-positive cells increased in a PDT-dose-dependent manner. Furthermore, immunohistochemical data indicate that PDT at these low doses significantly induces the expression of the vascular endothelial growth factor (VEGF) in PDT-treated regions in the 1-week group, but not in the 2-week group. These data indicate that low-dose PDT results in increased VEGF expression and endothelial cell proliferation in normal brains.

Estradiol and soy extract increase the production of new cells in the dentate gyrus of old rats

In young rodents, estradiol increases cell proliferation in the dentate gyrus of the hippocampus. However, it is unknown if the old brain retains this response to estradiol. Here we assessed the generation of new cells in the dentate gyrus of old rats after administration of estradiol or a soy extract, since soy is used as an alternative to hormonal replacement therapy in postmenopausal women. In a first experiment, 12-month-old animals were ovariectomized and studied at 14, 18 or 22 months of age. The production of new cells, assessed by the incorporation of bromodeoxyuridine (BrdU), was similar in 14- and 18-month-old rats. However, there was a significant reduction in the number of BrdU-immunoreactive cells at 22 months of age. In a second experiment, 22-month-old ovariectomized animals were treated for 10 weeks with a weekly s.c. injection of 150 μg estradiol valerianate or with 60 mg/kg per day soy extract added to the drinking water. Both treatments increased significantly the production of new cells in the dentate gyrus. These findings indicate that the brains of old rats retain the ability to increase the production of new cells in response to estradiol and soy extracts.

The multi-herbal formula Guibi-tang enhances memory and increases cell proliferation in the rat hippocampus

Guibi-tang (GBT) is a multi-herbal medicinal formula that has been used to treat amnesia in the traditional Korean medicine system for hundreds of years. In this study, we investigated the effects of GBT on learning and memory function in Wistar rats by using the foot shock passive avoidance test. In addition, cell proliferation was examined using 5-bromo-2-deoxyuridine (BrdU) uptake and immunostaining in the dentate gyrus of the rat hippocampus after oral administration of GBT. In the passive avoidance test, the retention time of the GBT-treated group was significantly longer (almost 2.5-fold) than that of the control group. The number of BrdU-immunoreactive cells in the dentate gyrus was significantly higher in the GBT-treated group (almost twofold) than in the control group. These results suggest that administration of GBT to rats not only improves learning and memory but also increases the proliferation of cells in the dentate gyrus of the hippocampus.

Decreased cell proliferation in the dentate gyrus of α7 nicotinic acetylcholine receptor heterozygous mice

It is suggested that deficits of α7 nicotinic acetylcholine receptors (nAChRs) might be associated with the cognitive impairments of Alzheimer's disease and schizophrenia, and that agonists for α7 nAChR could be useful for treatment of cognitive dysfunction in these diseases. This study was undertaken to examine the role of α7 nAChRs on hippocampal cellular proliferation of adult mice. The number of 5-bromo-2′-deoxyuridine (BrdU)-immunoreactive cells in the granule cell layer (GCL) and hilus of α7 nAChR heterozygous (+/−) mice was significantly decreased as compared with that of wild-type (+/+) mice. In contrast, the number of BrdU-immunoreactive cells in GCL and hilus of α7 nAChR homozygous (−/−) mice was not different from that of wild-type mice. The results suggest that reduced levels of α7 nAChRs may decrease cell proliferation of adult hippocampus.

5-HT 1A receptor antagonist administration decreases cell proliferation in the dentate gyrus

This study investigated the action of 5-HT 1A receptor antagonists on cell proliferation in the dentate gyrus of adult rats. Three antagonists (NAN-190, p-MPPI and WAY-100635) all produced a statistically significant ∼30% reduction in the number of BrdU-immunoreactive cells in the dentate gyrus. This suggests that 5-HT 1A receptor activity is important during naturally occurring cell proliferation in the dentate gyrus, and perhaps neurogenesis, and is one of the many factors involved in its regulation.

Proliferation and differentiation of progenitor cells in the cortex and the subventricular zone in the adult rat after focal cerebral ischemia.

Progenitor cells in the subventricular zone of the lateral ventricle and in the dentate gyrus of the hippocampus can proliferate throughout the life of the animal. To examine the proliferation and fate of progenitor cells in the subventricular zone and dentate gyrus after focal cerebral ischemia, we measured the temporal and spatial profiles of proliferation of cells and the phenotypic fate of proliferating cells in ischemic brain in a model of embolic middle cerebral artery occlusion in the adult rat. Proliferating cells were labeled by injection of bromodeoxyuridine (BrdU) in a pulse or a cumulative protocol. To determine the temporal profile of proliferating cells, ischemic rats were injected with BrdU every 4 h for 12 h on the day preceding death. Rats were killed 2-14 days after ischemia. We observed significant increases in numbers of proliferating cells in the ipsilateral cortex and subventricular zone 2-14 days with a peak at 7 days after ischemia compared with the control group. To maximize labeling of proliferating cells, a single daily injection of BrdU was administered over a 14-day period starting the day after ischemia. Rats were killed either 2 h or 28 days after the last injection of BrdU. A significant increase in numbers of BrdU immunoreactive cells in the subventricular zone was coincident with a significant increase in numbers of BrdU immunoreactive cells in the olfactory bulb 14 days after ischemia and numbers of BrdU immunoreactive cells did not significantly increase in the dentate gyrus. However, 28 days after the last labeling, the number of BrdU labeled cells decreased by 90% compared with number at 14 days. Clusters of BrdU labeled cells were present in the cortex distal to the infarction. Numerous cells immunostained for the polysialylated form of the neuronal cell adhesion molecule were detected in the ipsilateral subventricular zone. Only 6% of BrdU labeled cells exhibited glial fibrillary acidic protein immunoreactivity in the cortex and subcortex and no BrdU labeled cells expressed neuronal protein markers (neural nuclear protein and microtubule associated protein-2). From these data we suggest that focal cerebral ischemia induces transient and regional specific increases in cell proliferation in the ipsilateral hemisphere and that proliferating progenitor cells may exist in the adult cortex.

Functional and morphometric study of cryopreserved human parathyroid tissue transplanted into nude mice.

As part of an ongoing study of the characteristics of transplanted fresh normal and pathologic parathyroid tissues, we transplanted cryopreserved human parathyroid tissue into nude mice. Hyperplastic glands cryopreserved for various lengths of time (<2 weeks and >1 year) were transplanted into the gluteus muscle of nude mice. Specimens grafted were of two sizes: 10 mg and 30 mg. Serum human parathyroid hormone (hPTH) concentration was estimated by a double-antibody immunoradiometric assay prior to transplantation and 2, 4, and 8 weeks after transplantation. A low-calcium diet was given to some mice to evaluate any effects on the grafts of low serum concentrations of calcium. All mice were killed 4 hours after injection of bromodeoxyuridine (Brd U) for assessment of the cell proliferation in grafted parathyroid tissue. Although hPTH secretion of cryopreserved tissue was only half that of fresh tissue, the cryopreserved tissue released hPTH, as did fresh tissue. hPTH secretion was accelerated by stimulation of a low-calcium diet, and PTH secretion was positively correlated with the volume of transplanted tissue. The number of Brd U-immunoreactive cells was correlated with the serum concentration of hPTH (r = 0.95), which indicates that cell proliferation is closely related to PTH secretion under the condition of successful transplantation. The experimental transplantation of cryopreserved tissue into nude mice, coupled with a concise immunohistochemical study of the grafts conducted prior to their transplantation into humans, can contribute to the evaluation of PTH secretion and cell proliferation.