Intranasally delivered bFGF enhances neurogenesis in adult rats following cerebral ischemia

Basic fibroblast growth factor (bFGF) is a very important mitogenic factor with proved neurogenesis effects in the central nervous system. Intranasal administration can bypass blood-brain barrier and deliver drugs into the brain directly. We investigated whether intranasal administration of bFGF at later time points after ischemia could promote adult neurogenesis and improve neurologic functions. Rats received bFGF or saline intranasally once daily for 6 consecutive days, starting at 1 day after transient middle cerebral artery occlusion (MCAO). Bromodeoxyuridine (BrdU) was injected at 5 and 6 days after MCAO. Rats were killed at 7 or 28 days after MCAO. Neurogenesis was assessed by immunostaining for BrdU and cell type-specific markers. Neurological functions were evaluated by the modified Neurological Severity Scores. Compared with the control animals, intranasal administration of bFGF improved behavioral recovery without affecting infarct size, and enhanced proliferation of progenitor cells in the subventricular zone and the subgranular zone of the dentate gyrus (DG). Furthermore, the new proliferated cells could differentiate into neurons (BrdU+NeuN+ cells) in the striatum and DG at 28 days after MCAO. Intranasal administration of bFGF offers a non-invasive alternative for the treatment of stroke.     

Neurogenesis in the striatum of the quinolinic acid lesion model of Huntington's disease

The presence of ongoing neurogenesis in the adult mammalian brain raises the exciting possibility that endogenous progenitor cells may be able to generate new neurons to replace cells lost through brain injury or neurodegenerative disease. We have recently demonstrated increased cell proliferation and the generation of new neurons in the Huntington's disease human brain. In order to better understand the potential role of endogenous neuronal replacement in neurodegenerative disorders and extend our initial observations in the human Huntington's disease brain, we examined the effect of striatal cell loss on neurogenesis in the subventricular zone (SVZ) of the adult rodent forebrain using the quinolinic acid (QA) lesion rat model of Huntington's disease. Cell proliferation and neurogenesis were assessed with bromodeoxyuridine (BrdU) labeling and immunocytochemistry for cell type-specific markers. BrdU labeling demonstrated increased cell proliferation in the SVZ ipsilateral to the QA-lesioned striatum, resulting in expansion of the SVZ in the lesioned hemisphere. Quantification revealed that QA lesion-induced striatal cell loss produced a significant increase in the area of BrdU-immunoreactivity in the SVZ ipsilateral to the lesioned hemisphere between 1 and 14 days post-lesion compared with sham-lesioned animals, with the greatest increase observed at 7 days post-lesion. These changes were associated with an increase in cells in the anterior SVZ ipsilateral to the lesioned striatum expressing the antigenic marker for SVZ neuroblasts, doublecortin (Dcx). Importantly, we observed Dcx-positive cells extending from the SVZ into the QA-lesioned striatum where a subpopulation of newly generated cells expressed markers for immature and mature neurons. This study demonstrates that loss of GABAergic medium spiny projection neurons following QA striatal lesioning of the adult rat brain increases SVZ neurogenesis, leading to the putative migration of neuroblasts to damaged areas of the striatum and the formation of new neurons.     

Platelet-derived growth factor (PDGF-BB) and brain-derived neurotrophic factor (BDNF) induce striatal neurogenesis in adult rats with 6-hydroxydopamine lesions

The effects of i.c.v. infused platelet-derived growth factor and brain-derived neurotrophic factor on cell genesis, as assessed with bromodeoxyuridine (BrdU) incorporation, were studied in adult rats with unilateral 6-hydroxydopamine lesions. Both growth factors increased the numbers of newly formed cells in the striatum and substantia nigra to an equal extent following 10 days of treatment. At 3 weeks after termination of growth factor treatment, immunostaining of BrdU-labeled cells with the neuronal marker NeuN revealed a significant increase in newly generated neurons in the striatum. In correspondence, many doublecortin-labeled neuroblasts were also observed in the denervated striatum following growth factor treatment. Further evaluation suggested that a subset of these new neurons expresses the early marker for striatal neurons Pbx. However, no BrdU-positive cells were co-labeled with DARPP-32, a protein expressed by mature striatal projection neurons. Both in the striatum and in the substantia nigra there were no indications of any newly born cells differentiating into dopaminergic neurons following growth factor treatment, such that BrdU-labeled cells never co-expressed tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis. In conclusion, our results suggest that administration of these growth factors is capable of recruiting new neurons into the striatum of hemiparkinsonian rats.     

Sustained sleep fragmentation results in delayed changes in hippocampal-dependent cognitive function associated with reduced dentate gyrus neurogenesis

Sleep fragmentation (SF) is prevalent in human sleep-related disorders. In rats, sustained SF has a potent suppressive effect on adult hippocampal dentate gyrus (DG) neurogenesis. Adult-generated DG neurons progressively mature over several weeks, and participate in certain hippocampal-dependent cognitive functions. We predicted that suppression of neurogenesis by sustained SF would affect hippocampal-dependent cognitive functions in the time window when new neurons would reach functional maturity. Sprague–Dawley rats were surgically-prepared with electroencephalogram (EEG) and electromyogram (EMG) electrodes for sleep state detection. We induced sleep-dependent SF for 12 days, and compared SF animals to yoked sleep fragmentation controls (SFC), treadmill controls (TC) and cage controls (CC). Rats were injected with bromodeoxyuridine on treatment days 4 and 5. Rats were returned to home cages for 14 days. Cognitive performance was assessed in a Barnes maze with 5 days at a constant escape position followed by 2 days at a rotated position. After Barnes maze testing rats were perfused and DG sections were immunolabeled for BrdU and neuronal nuclear antigen (NeuN), a marker of mature neurons.SF reduced BrdU-labeled cell counts by 32% compared to SFC and TC groups. SF reduced sleep epoch duration, but amounts of rapid eye movement (REM) sleep did not differ between SF and SFC rats, and non-rapid eye movement (NREM) was reduced only transiently. In the Barnes maze, SF rats exhibited a progressive decrease in escape time, but were slower than controls. SF animals used different search strategies. The use of a random, non-spatial search strategy was significantly elevated in SF compared to the SFC, TC and CC groups. The use of random search strategies was negatively correlated with NREM sleep bout length during SF. Sustained sleep fragmentation reduced DG neurogenesis and induced use of a non-spatial search strategy, which could be seen 2 weeks after terminating the SF treatment. The reduction in neurogenesis induced by sleep fragmentation is likely to underlie the delayed changes in cognitive function.     

Induction of striatal neurogenesis enhances functional recovery in an adult animal model of neonatal hypoxic-ischemic brain injury

While intraventricular administration of epidermal growth factor (EGF) expands the proliferation of neural stem/progenitor cells in the subventricular zone (SVZ), overexpression of brain-derived neurotrophic factor (BDNF) is particularly effective in enhancing striatal neurogenesis. We assessed the induction of striatal neurogenesis and consequent functional recovery after chronic infusion of BDNF and EGF in an adult animal model of neonatal hypoxic-ischemic (HI) brain injury. Permanent brain damage was induced in CD-1® (ICR) mice (P7) by applying the ligation of unilateral carotid artery and hypoxic condition. At 6 weeks of age, the mice were randomly assigned to groups receiving a continuous 2-week infusion of one of the following treatments into the ventricle: BDNF, EGF, BDNF/EGF, or phosphate buffered saline (PBS). Two weeks after treatment, immunohistochemical analysis revealed an increase in the number of BrdU⁺ cells in the SVZ and striata of BDNF/EGF-treated mice. The number of new neurons co-stained with BrdU and βIII-tubulin was also significantly increased in the neostriata of BDNF/EGF-treated mice, compared with PBS group. In addition, the newly generated cells were expressed as migrating neuroblasts labeled with PSA-NCAM or doublecortin in the SVZ and the ventricular side of neostriata. The new striatal neurons were also differentiated as mature neurons co-labeled with BrdU⁺/NeuN⁺. When evaluated post-surgical 8 weeks, BDNF/EGF-treated mice exhibited significantly longer rotarod latencies at constant speed (48 rpm) and under accelerating condition (4–80 rpm), relative to PBS and untreated controls. In the forelimb-use asymmetry test, BDNF/EGF-treated mice showed significant improvement in the use of the contralateral forelimb. In contrast, this BDNF/EGF-associated functional recovery was abolished in mice receiving a co-infusion of 2% cytosine-b-d-arabinofuranoside (Ara-C), a mitotic inhibitor. Induction of striatal neurogenesis by the intraventricular administration of BDNF and EGF promoted functional recovery in an adult animal model of neonatal HI brain injury. The effect of Ara-C to completely block functional recovery indicates that the effect may be the result of newly generated neurons. Therefore, this treatment may offer a promising strategy for the restoration of motor function for adults with cerebral palsy (CP).     

Perineuronal germinal cells in the rat cerebral cortex

The generation and renewal of cells in the adult mammalian central nervous system maintains brain functions, including plasticity. Even in the cerebral cortex of adult mammals, glial cells are thought to be replaced with newly generated cells every 100 days. Recently, we demonstrated that this proliferation is stimulated by neural activity. However, whether any germinal areas exist in the cortical parenchyma is unknown. Here, we examined the proliferating cell dynamics in the cerebral cortex of adult rats using BrdU labeling and immunohistochemistry for NeuN and lamin B1. At 2 h after a single injection of BrdU, more than 80% of BrdU-labeled cells were observed in the perineuronal territory in which the BrdU-labeled nuclei were located within 5 μm from neuronal nuclei. The ratio of perineuronal cells to nonperineuronal cells in BrdU-labeled cells gradually decreased over the 2 weeks following BrdU injection. These observations indicate that numerous cortical cells proliferate in the perineuronal territory, the germinal soil, and that part of these newly generated cells migrate from the perineuronal territory into the surrounding areas during the 2 weeks following mitosis.     

Fibroblast growth factor 2 enhances striatal and nigral neurogenesis in the acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson's disease

In response to injury, endogenous precursors in the adult brain can proliferate and generate new neurons, which may have the capacity to replace dysfunctional or dead cells. Although injury-induced neurogenesis has been demonstrated in animal models of stroke, Alzheimer's disease (AD) and Huntington's disease (HD), studies of Parkinson's disease (PD) have produced conflicting results. In this study, we investigated the ability of adult mice to generate new neurons in response to the parkinsonian toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which causes selective degeneration of nigrostriatal dopamine neurons. MPTP lesions increased the incorporation of 5-bromo-2′-deoxyuridine-5′-monophosphate (BrdU), as well as the number of cells that co-expressed BrdU and the immature neuronal marker doublecortin (DCX), in two neuroproliferative regions—the subgranular zone of the dentate gyrus (DG) and the rostral subventricular zone (SVZ). BrdU-labeled, DCX-expressing cells were not found in the substantia nigra (SN) of MPTP-treated mice, where neuronal cell bodies are destroyed, but were present in increased numbers in the striatum, where SN neurons lost in PD normally project. Fibroblast growth factor-2 (FGF-2), which enhances neurogenesis in a mouse model of HD, also increased the number of BrdU/DCX-immunopositive cells in the SN of MPTP-treated mice. Thus, MPTP-induced brain injury increases striatal neurogenesis and, in combination with FGF-2 treatment, also stimulates neurogenesis in SN.     

Intracranial self-stimulation enhances neurogenesis in hippocampus of adult mice and rats

Running is known to promote neurogenesis. Besides being exercise, it results in a reward, and both of these factors might contribute to running-induced neurogenesis. However, little attention has been paid to how reward and exercise relate to neurogenesis. The present study is an attempt to determine whether a reward, in the form of intracranial self-stimulation (ICSS), influences neurogenesis in the hippocampus of adult rodents. We used bromodeoxyuridine labeling to quantify newly generated cells in mice and rats that experienced ICSS for 1 h per day for 3 days. ICSS increased the number of 5-bromodeoxyuridine (Brdu)-labeled cells in the hippocampal dentate gyrus (DG) of both species. The effect, when examined at 1 day, 1 week, and 4 weeks post-ICSS, was predominantly present in the side ipsilateral to the stimulation, although it was distributed to the contralateral side. We also found in rats that, 4 weeks after Brdu injection, surviving newborn cells in the hippocampal DG of the ICSS animals co-localized with a mature neuron marker, neuronal nuclei (NeuN), and these surviving cells in rats were double-labeled with Fos, a marker of neuronal activation, after the rats had been trained to perform a spatial task. The results demonstrate that ICSS can increase newborn neurons in the hippocampal DG that endure into maturity.     

Cocaine selectively increases proliferation in the adult murine hippocampus

Cocaine abuse continues to be a significant problem in the USA and elsewhere. Cocaine is an indirect agonist for dopamine, norepinephrine and serotonin with numerous potential downstream effects, including processes and signals associated with adult neurogenesis. Since drug addiction is associated with brain plasticity, we hypothesized that cocaine exposure would alter cellular proliferation in two adult neurogenic regions (the subventricular and subgranular zones). We used bromodeoxyuridine (BrdU) to track newly generated cells in the brains of adult mice after chronic cocaine or saline exposures. No differences were found in the number or migration patterns of BrdU-labeled cells in the forebrain neurogenic areas. However, cocaine produced a significant increase in the number of hippocampal BrdU-labeled cells.     

Postischemic exercise decreases neurogenesis in the adult rat dentate gyrus

Running exercise enhances neurogenesis in the normal adult and aged hippocampus. However, the effect of exercise on neurogenesis in the ischemic hippocampus is unclear. Here, we show that running exercise has different effects on ischemic and non-ischemic brain. Young (3-4-month-old) normotensive Wistar rats were used for this study. We administered bromodeoxyuridine (BrdU) to rats 7 days after the induction of transient forebrain ischemia or sham operation. BrdU-labeled cells were increased in the ischemic subgranular zone (SGZ) and granule cell layer (GCL) and double immunofluoresence showed approximately 80% of BrdU-labeled cells expressed neuronal markers. To assess the effect of running exercise on neurogenesis, BrdU-labeled cells in these regions were quantified after 1 day and 14 days. In sham-operated rats, the numbers of BrdU-labeled cells were significantly increased (2.2-fold) in the SGZ and GCL in response to running exercise. The numbers of BrdU-labeled cells were increased in response to ischemia, however, they were decreased 14 days after BrdU administration and running exercise accelerated the reduction in BrdU-labeled cells in ischemic rats. These findings suggest that running exercise has a negative effect on neurogenesis in the ischemic hippocampus. This may be important with respect to assessment of therapeutic approaches for functional recovery after stroke.     

Fate mapping and lineage analyses demonstrate the production of a large number of striatal neuroblasts after transforming growth factor alpha and noggin striatal infusions into the dopamine-depleted striatum

Infusion of transforming growth factor alpha (TGFalpha) into the adult dopamine (DA)-depleted striatum generates a local population of nestin(+)/proliferating cell nuclear antigen (PCNA)(+) newborn cells. The precise origin and fate of these new striatal cells are unknown, making it difficult to direct them for neural repair in Parkinson's disease. Experiments in rats using 5-bromo-2'-deoxyuridine (BrdU) to label neural progenitor cells showed that during TGFalpha infusion in the DA-depleted striatum, newborn striatal cells formed a homogeneous population of precursors, with the majority coexpressing nestin, Mash1, Olig2, and epidermal growth factor receptor, consistent with the phenotype of multipotent C cells. Upon TGFalpha pump withdrawal, the subventricular zone (SVZ) was repopulated by neuroblasts. Strikingly, during this period, numerous clusters of doublecortin(+)/polysialylated neuronal cell adhesion molecule(+) neuroblasts were also produced in the ipsilateral medial striatum. In parallel, striatal BrdU(+)/glial fibrillary acidic protein(+) astrocytes were generated, but no BrdU(+)/O4(+)/CNPase(+) oligodendrocytes were generated. Infusion of the neuralizing bone morphogenetic protein antagonist noggin after TGFalpha pump withdrawal increased the neuroblast-to-astrocyte ratio among new striatal cells by blocking glial differentiation but did not alter striatal neurogenesis. At no time or treatment condition were differentiated neurons generated, including DA neurons. Using 6-hydroxydopamine-lesioned nestin-CreER(T2)/R26R-YFP mice that allow genetic fate-mapping of SVZ nestin(+) cells, we show that TGFalpha-generated striatal cells originate from SVZ nestin(+) precursors that confirmed data from the rats on the phenotype and fate of striatal nestin(+)/PCNA(+) cells upon TGFalpha withdrawal. This work demonstrates that a large population of multipotent striatal C-like cells can be generated in the DA-depleted striatum that do not spontaneously differentiate into DA neurons.     

Neurogenesis inhibition in the dorsal vagal complex by chronic immobilization stress in the adult rat

The dorsal vagal complex (DVC) is the brainstem integrative center that mediates the satiety reflex and relays autonomic neural responses to stress. The DVC displays adult neurogenesis, intrinsic neural stem cells and a high brain-derived neurotrophic factor (BDNF) content, effectors of plasticity that are modulated by stress in the hippocampus. In this study we asked whether neurogenesis and BDNF expression in the DVC are altered by stress, in parallel with food intake reduction. To this end, neurogenesis was assessed in adult rats in vivo by repetitive 5-bromo-2′-deoxyuridine (BrdU) administration without (controls) or with daily sessions of immobilization stress (1 h/day), and were allowed to survive for 2 weeks after the end of BrdU treatment. Neurogenic proliferation in the brainstem was detected by immunohistochemistry and confocal microscopy mainly in the area postrema and the nucleus tractus solitarius; newly formed neurons amounted to about 35% of all BrdU-labeled cells in the DVC of control rats. Chronic immobilization stress induced a significant decrease in neurogenic proliferation in the DVC which reached 50% in the area postrema. The number of newly-formed neurons was also decreased by chronic immobilization stress in the DVC, and this effect was again maximal in the area postrema; the proportion of BrdU-labeled cells that were neurons was unchanged. In vitro neurosphere assay was then performed on microdissected DVC tissue from another cohort of chronically stressed and control rats. Chronic immobilization stress induced a significant decrease of the total neurosphere number per rat DVC in both primary and secondary cultures, indicating that intrinsic neural stem cell frequency was decreased by chronic stress in DVC tissue. Tissue BDNF concentration in the DVC, as assessed by enzyme-linked immunosorbent assay, was not significantly altered when compared with controls after 3, 6, 9 or 13 days of chronic immobilization stress. These results further characterize neurogenesis in the DVC and suggest its involvement in the long-term regulation of food intake.     

Developmental exposure to 3,4-methylenedioxymethamphetamine results in downregulation of neurogenesis in the adult mouse hippocampus

3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) is a powerful releaser of 5-HT and chronic use of this drug can cause depletion of monoamines. Recently, concerns about the risk of adult brain damage due to fetal exposure to MDMA have been raised. We investigated whether developmental MDMA exposure affected adult neurogenesis in C57 black/6 mice. MDMA (1.25 or 20 mg/kg, p.o.) or vehicle was administered daily to the mother from prenatal 6th day to postnatal 21st day. When the offspring were 11 weeks old, they were injected with 5-bromo-2'-deoxyuridine (BrdU) (120 mg/kg, i.p.) once a day for 4 days. After 24 h or 28 days, the animals were killed to count the BrdU-positive cells in the dentate gyrus. At 24 h after the last BrdU injection, the number of BrdU-positive cells in the offspring developmentally exposed to MDMA was significantly lower than that of the control group. At 28 days post-BrdU labeling, BrdU-positive cells in the dentate gyrus of female offspring with developmental exposure to high dose MDMA were significantly fewer compared with the control group. In addition, most BrdU-positive cells were co-labeled with the mature neuronal marker, neuronal nuclei, while a few BrdU-labeled cells were merged with an astrocyte marker. Our results suggest that developmental exposure to MDMA can result in decreases in the proliferation and survival of mature newborn cells in the adult dentate gyrus.     

Molarless condition suppresses proliferation but not differentiation rates into neurons in the rat dentate gyrus

The dentate gyrus (DG) of the hippocampal complex is one of the few areas of the rodent brain where neurogenesis continues throughout adulthood. We investigated the effects of the molarless condition on cell proliferation, rate of differentiation into neurons in the subgranular zone of the DG, and plasma corticosterone levels. The molarless condition decreased cell proliferation in the DG and increased plasma corticosterone levels. Approximately 80% of newly generated cells differentiated into neurons and the remaining 20% of the cells differentiated into astrocytes. These ratios were not significantly different between control and molarless rats. In conclusion, the rates of neurogenesis and gliogenesis in the DG are suppressed by the molarless condition, and this suppression might be associated with the increased corticosteroid levels in molarless subjects.     

Increased neurogenesis in brains of scrapie-infected mice

Persistent neurogenesis occurs in the adult brain throughout the life of all mammals. Recent studies have shown that neurogenesis was increased in adult gerbil and rat brains after ischemia. Neurogenesis has not been examined during neurodegenerative diseases such as scrapie. To investigate the regeneration of neurons after scrapie-infection, we infused 5-bromo-2'-deoxyuridine (BrdU), a DNA replication indicator, into both control and scrapie-infected mice. Mice were sacrificed at 150 days post-infection, i.e., at the start of clinical disease and a time when PrP(Sc) was readily detected in brain by both immunostaining and Western blot. We investigated expression of BrdU in each region of brain and observed cellular localization of BrdU using various cell markers such as neuronal nuclear (NeuN), microtubule-associated protein 2 (MAP2) and glial fibrillary acidic protein (GFAP). Immunohistochemically, BrdU-labeled cells were observed in the striatum, hippocampus, and brain stem of scrapie-infected brains. BrdU-labeled cells were much more prevalent in the hippocampus of scrapie-infected mice compared to hippocampus of control brains. In scrapie mice, there was more staining in hippocampus than in other brain regions. We also found that BrdU-positive cells colocalized with the neuronal markers NeuN and MAP2, whereas BrdU staining was not merged with GFAP, an astrocytic marker. Taken together, our results suggest that scrapie-infection induces region-specific increases in neuron regeneration.     

局灶性脑缺血后侧脑室室下区神经发生及其与血管内皮生长因子关系的研究

为了研究成年大鼠局灶性脑缺血后侧脑室室下区(SVZ)神经发生的情况及其与血管内皮生长因子(VEGF)的关系,探讨脑缺血后神经发生及其调控机制,本研究通过大脑中动脉阻断法(MCAO)建立大鼠局灶性脑缺血模型,5-溴-2-脱氧尿核苷(BrdU)标记增殖的神经前体细胞,用免疫荧光双标记法动态检测BrdU、TuJ1、MAP-2、GFAP的表达,同时观察增殖细胞表达VEGF及其受体情况。结果显示:与对照组相比,大鼠SVZ的BrdU阳性细胞数在脑缺血后4 d组明显增加,14 d组达到高峰;Br-dU/TuJ1、BrdU/MAP-2阳性双标细胞数在脑缺血后14 d组开始增加,28 d组达到高峰;但BrdU/GFAP阳性双标细胞数则无明显变化;增殖的BrdU阳性细胞同时表达VEGF及其受体FLK-1。以上结果提示:大鼠局灶性脑缺血可激活SVZ自体神经前体细胞原位增殖、分化,且增殖的细胞同时表达VEGF及其受体可能是脑缺血后神经发生增强的调节机制之一。     

Serotonin and neurogenesis in the hippocampal dentate gyrus of adult mammals

It is well documented that in mammals new neurons are generated in the dentate gyrus (DG) and integrated into hippocampal circuits throughout their life. However, functions of these newly generated cells are still hotly debated. One of the important factors that may influence the rate of DG neurogenesis is serotonin. Apart from being a neurotransmitter and neuromodulator it plays many other roles in the central nervous system, including the role of a trophic factor influencing functional state of neurons. In this review I discuss the changing views on adult hippocampal neurogenesis then briefly describe the anatomy and function of the hippocampus, focusing on its serotonergic innervation and receptors. Further, the possible role of serotonin and the newly generated DG neurons in hippocampus-dependent memory is discussed. Finally mechanisms by which serotonin and its receptors influence neurogenesis in the adult DG are summarized and hypotheses linking the decreased rate of DG neurogenesis with mechanisms of depression are discussed.     

Ischemia induces cell proliferation and neurogenesis in the gerbil hippocampus in response to neuronal death

We studied hippocampal cellular proliferation and neurogenesis processes in a model of transient global cerebral ischemia in gerbils by labelling dividing cells with 5'-Bromo-2'-deoxyuridine (BrdU). Surrounding the region of selective neuronal death (CA1 pyramidal layer of the hippocampus), an important increase in reactive astrocytes and BrdU-labelled cells was detected 5 days after ischemia. A similar result was found in the dentate gyrus (DG) 12 days after ischemia. The differentiation of the BrdU+ cells was investigated 28 days after BrdU administration by analyzing the morphology, anatomic localization and cell phenotype by triple fluorescent labelling (BrdU, adult neural marker NeuN and DNA marker TOPRO-3) using confocal laser-scanning microscopy. This analysis showed increased neurogenesis in the DG in case of ischemia and triple positive labelling in some newborn cells in CA1. Seven brain hemispheres from gerbils subjected to ischemia did not develop CA1 neuronal death; hippocampus from these hemispheres did not show any of the above mentioned findings. Our results indicate that ischemia triggers proliferation in CA1 and neurogenesis in the DG in response to CA1 pyramidal neuronal death, independently of the reduced cerebral blood flow or the cell migration from subventricular zone (SVZ).     

Imipramine enhances cell proliferation and decreases neurodegeneration in the hippocampus after transient global cerebral ischemia in rats

This study was aimed to determine whether imipramine chronic treatment promotes neurogenesis in the dentate gyrus (DG) and interferes with neuronal death in the CA1 subfield of the hippocampus after transient global cerebral ischemia (TGCI) in rats. After TGCI, animals were treated with imipramine (20 mg/kg, i.p.) or saline during 14 days. 5-Bromo-2′-deoxyuridine-5′-monophosphate (BrdU) was injected 24 h after the last imipramine or saline injection to label proliferating cells. In order to confirm the effect of TGCI on neuronal death and cell proliferation, a group of animals was sacrificed 7 days after TGCI. Neurogenesis and neurodegeneration were evaluated by doublecortin (DCX)-immunohistochemistry and Fluoro-Jade C (FJC)-staining, respectively. The rate of cell proliferation increases 7 days but returns to basal levels 14 days after TGCI. There was a significant increase in the number of FJC-positive neurons in the CA1 of animals 7 and 14 days after TGCI. Chronic imipramine treatment increased cell proliferation in the SGZ of DG and reduced the neurodegeneration in the CA1 of the hippocampus 14 days after TGCI. Immunohistochemistry for DCX detected an increased number of newly generated neurons in the hippocampal DG 14 days after TGCI, which was not affected by imipramine treatment. Further studies are needed to evaluate whether imipramine treatment for longer time would be able to promote survival of newly generated neurons as well as to improve functional recovery after TGCI.     

Enhanced neurogenesis after transient global ischemia in the dentate gyrus of the rat

The dentate gyrus is one of the few areas of the mammalian brain where new neurons are continuously produced in adulthood. Certain insults such as epileptic seizures and ischemia are known to enhance the rate of neuronal production. We analyzed this phenomenon using the temporary occlusion of the two carotid arteries combined with arterial hypotension as a method to induce ischemia in rats. We measured the rate of cell production and their state of differentiation with a mitotic indicator, bromodeoxyuridine (BrdU), in combination with the immunohistochemical detection of neuronal markers. One week after the ischemic episode, the cell production in dentate gyrus was increased two- to threefold more than the basal level seen in control animals. Two weeks after ischemia, over 60% of these cells became young neurons as determined by colabeling with BrdU and a cytoplasmic protein (CRMP-4) involved in axonal guidance during development. Five weeks after the ischemia, over 60% of new neurons expressed calbindin, a calcium-binding protein normally expressed in mature granule neurons. In addition to more cells being generated, a greater proportion of all new cells remained in the differentiated but not fully mature state during the 2- to 5-week period after ischemia. The maturation rate of neurons as determined by the calbindin labeling and by the rate of migration from a proliferative zone into the granule cell layer was not changed when examined 5 weeks after ischemia. The results support the hypothesis that survival of dentate gyrus after ischemia is linked with enhanced neurogenesis. Additional physiological stimulation after ischemia may be exploited to stimulate maturation of new neurons and to offer new therapeutic strategies for promoting recovery of neuronal circuitry in the injured brain.