Bharathi Hattiangady

Stem/progenitor cell proliferation factors FGF‐2, IGF‐1, and VEGF exhibit early decline during the course of aging in the hippocampus: Role of astrocytes

Dentate neurogenesis, important for learning and memory, declines dramatically by middle age. Although studies have shown that this age‐related decrease can be reversed to some extent by exogenous applications of mitogenic factors, it is unclear whether one or more of these factors exhibits decline by middle age. We hypothesize that multiple stem/progenitor cell proliferation factors exhibit early decline during the course of aging in the hippocampus, and some of these declines are linked to age‐related alterations in hippocampal astrocytes. We measured the concentrations of fibroblast growth factor‐2 (FGF‐2), insulin‐like growth factor‐1 (IGF‐1), and vascular endothelial growth factor (VEGF) in the hippocampus of young, middle‐aged, and aged F344 rats, using enzyme‐linked immunosorbent assay (ELISA). In addition, we quantified the total number of FGF‐2 immunopositive (FGF‐2+) and glial fibrillary acidic protein immunopositive (GFAP+) cells in the dentate gyrus and the entire hippocampus. Our results provide new evidence that the concentrations of FGF‐2, IGF‐1, and VEGF decline considerably by middle age but remain steady between middle age and old age. Further, decreased concentrations of FGF‐2 during aging are associated with decreased numbers of FGF‐2+ astrocytes. Quantification of GFAP+ cells, and GFAP and FGF‐2 dual immunostaining analyses, reveal that aging does not decrease the total number of astrocytes but fractions of astrocytes that express FGF‐2 decline considerably by middle age. Thus, dramatically decreased dentate neurogenesis by middle age is likely linked to reduced concentrations of FGF‐2, IGF‐1, and VEGF in the hippocampus, as each of these factors can individually influence the proliferation of stem/progenitor cells in the dentate gyrus. Additionally, the results demonstrate that decreased FGF‐2 concentration during aging is a consequence of age‐related impairment in FGF‐2 synthesis by astrocytes.

Aging does not alter the number or phenotype of putative stem/progenitor cells in the neurogenic region of the hippocampus.

To investigate whether dramatically waned dentate neurogenesis during aging is linked to diminution in neural stem/progenitor cell (NSC) number, we counted cells immunopositive for Sox-2 (a putative marker of NSCs) in the subgranular zone (SGZ) of young, middle-aged and aged F344 rats. The young SGZ comprised approximately 50,000 Sox-2+ cells and this amount did not diminish with aging. Quantity of GFAP+ cells and vimentin+ radial glia also remained stable during aging in this region. Besides, in all age groups, analogous fractions of Sox-2+ cells expressed GFAP (astrocytes/NSCs), NG-2 (oligodendrocyte-progenitors/NSCs), vimentin (radial glia), S-100beta (astrocytes) and doublecortin (new neurons). Nevertheless, analyses of Sox-2+ cells with proliferative markers insinuated an increased quiescence of NSCs with aging. Moreover, the volume of rat-endothelial-cell-antigen-1+ capillaries (vascular-niches) within the SGZ exhibited an age-related decline, resulting in an increased expanse between NSCs and capillaries. Thus, decreased dentate neurogenesis during aging is not attributable to altered number or phenotype of NSCs. Instead, it appears to be an outcome of increased quiescence of NSCs due to changes in NSC milieu.

The window and mechanisms of major age-related decline in the production of new neurons within the dentate gyrus of the hippocampus

While it is well known that production of new neurons from neural stem/progenitor cells (NSC) in the dentate gyrus (DG) diminishes greatly by middle age, the phases and mechanisms of major age‐related decline in DG neurogenesis are largely unknown. To address these issues, we first assessed DG neurogenesis in multiple age groups of Fischer 344 rats via quantification of doublecortin‐immunopositive (DCX+) neurons and then measured the production, neuronal differentiation and initial survival of new cells in the subgranular zone (SGZ) of 4‐, 12‐ and 24‐month‐old rats using four injections (one every sixth hour) of 5′‐bromodeoxyuridine (BrdU), and BrdU–DCX dual immunostaining. Furthermore, we quantified the numbers of proliferating cells in the SGZ of these rats using Ki67 immunostaining. Numbers of DCX+ neurons were stable at 4–7.5 months of age but decreased progressively at 7.5–9 months (41% decline), 9–10.5 months (39% decline), and 10.5–12 months (34% decline) of age. Analyses of BrdU+ cells at 6 h after the last BrdU injection revealed a 71–78% decline in the production of new cells per day between 4‐month‐old rats and 12‐ or 24‐month‐old rats. Numbers of proliferating Ki67+ cells (putative NSCs) in the SGZ also exhibited similar (72–85%) decline during this period. However, the extent of both neuronal differentiation (75–81%) and initial 12‐day survival (67–74%) of newly born cells was similar in all age groups. Additional analyses of dendritic growth of 12‐day‐old neurons revealed that newly born neurons in the aging DG exhibit diminished dendritic growth compared with their age‐matched counterparts in the young DG. Thus, major decreases in DG neurogenesis occur at 7.5–12 months of age in Fischer 344 rats. Decreased production of new cells due to proliferation of far fewer NSCs in the SGZ mainly underlies this decline.

Newly born cells in the ageing dentate gyrus display normal migration, survival and neuronal fate choice but endure retarded early maturation

Addition of new granule cells to the dentate gyrus (DG) from stem or progenitor cells declines considerably during ageing. However, potential age‐related alterations in migration, enduring survival and neuronal fate choice of newly born cells, and rate of maturation and dendritic growth of newly differentiated neurons are mostly unknown. We addressed these issues by analysing cells that are positive for 5′‐bromodeoxyuridine (BrdU), doublecortin (DCX), BrdU and DCX, and BrdU and neuron‐specific nuclear antigen (NeuN) in the DG of young adult, middle‐aged and aged F344 rats treated with daily injections of BrdU for 12 consecutive days. Analyses performed at 24 h, 10 days and 5 months after BrdU injections reveal that the extent of new cell production decreases dramatically by middle age but exhibits no change thereafter. Interestingly, fractions of newly formed cells that exhibit appropriate migration and prolonged survival, and fractions of newly born cells that differentiate into neurons, remain stable during ageing. However, in newly formed neurons of the middle‐aged and aged DG, the expression of mature neuronal marker NeuN is delayed and early dendritic growth is retarded. Thus, the presence of far fewer new granule cells in the aged DG is not due to alterations in the long term survival and phenotypic differentiation of newly generated cells but solely owing to diminished production of new cells. The results also underscore that the capability of the DG milieu to support neuronal fate choice, migration and enduring survival of newly born cells remains stable even during senescence but its ability to promote rapid neuronal maturation and dendritic growth is diminished as early as middle age.

Brain-derived neurotrophic factor, phosphorylated cyclic AMP response element binding protein and neuropeptide Y decline as early as middle age in the dentate gyrus and CA1 and CA3 subfields of the hippocampus

The hippocampus is very susceptible to aging. Severely diminished dentate neurogenesis at middle age is one of the most conspicuous early changes in the aging hippocampus, which is likely linked to an early decline in the concentration of neurotrophic factors and signaling proteins that influence neurogenesis. We analyzed three proteins that are well-known to promote dentate neurogenesis and learning and memory function in the dentate gyrus and the hippocampal CA1 and CA3 subfields of young, middle-aged and aged F344 rats. These include the brain-derived neurotrophic factor (BDNF), the transcription factor phosphorylated cyclic AMP response element binding protein (p-CREB) and the neuropeptide neuropeptide Y (NPY). The BDNF was analyzed via ELISA and BDNF immunohistochemistry, the p-CREB through densitometric analysis of p-CREB immunopositive cells, and the NPY via stereological counting of NPY-immunopositive interneurons. We provide new evidence that the BDNF concentration, the p-CREB immunoreactivity and the number of NPY immunopositive interneurons decline considerably by middle age in both dentate gyrus and CA1 and CA3 subfields of the hippocampus. However, both BDNF concentration and NPY immunopositive interneuron numbers exhibit no significant decrease between middle age and old age. In contrast, the p-CREB immunoreactivity diminishes further during this period, which is also associated with reduced BDNF immunoreaction within the soma of dentate granule cells and hippocampal pyramidal neurons. Collectively, these results suggest that severely dampened dentate neurogenesis observed at middle age is linked at least partially to reduced concentrations of BDNF, p-CREB and NPY, as each of these proteins is a positive regulator of dentate neurogenesis. Dramatically diminished CREB phosphorylation (and persistently reduced levels of BDNF and NPY) at old age may underlie the learning and memory impairments observed during senescence.

Progress in Neuroprotective Strategies for Preventing Epilepsy

Neuroprotection is increasingly considered as a promising therapy for preventing and treating temporal lobe epilepsy (TLE). The development of chronic TLE, also termed as epileptogenesis, is a dynamic process. An initial precipitating injury (IPI) such as the status epilepticus (SE) leads to neurodegeneration, abnormal reorganization of the brain circuitry and a significant loss of functional inhibition. All of these changes likely contribute to the development of chronic epilepsy, characterized by spontaneous recurrent motor seizures (SRMS) and learning and memory deficits. The purpose of this review is to discuss the current state of knowledge pertaining to neuroprotection in epileptic conditions, and to highlight the efficacy of distinct neuroprotective strategies for preventing or treating chronic TLE. Although the administration of certain conventional and new generation anti-epileptic drugs is effective for primary neuroprotection such as reduced neurodegeneration after acute seizures or the SE, their competence for preventing the development of chronic epilepsy after an IPI is either unknown or not promising. On the other hand, alternative strategies such as the ketogenic diet therapy, administration of distinct neurotrophic factors, hormones or antioxidants seem useful for preventing and treating chronic TLE. However, long-term studies on the efficacy of these approaches introduced at different time-points after the SE or an IPI are lacking. Additionally, grafting of fetal hippocampal cells at early time-points after an IPI holds considerable promise for preventing TLE, though issues regarding availability of donor cells, ethical concerns, timing of grafting after SE, and durability of graft-mediated seizure suppression need to be resolved for further advances with this approach. Overall, from the studies performed so far, there is consensus that neuroprotective strategies need to be employed as quickly as possible after the onset of the SE or an IPI for considerable beneficial effects. Nevertheless, ideal strategies that are capable of facilitating repair and functional recovery of the brain after an IPI and preventing the evolution of IPI into chronic epilepsy are still hard to pin down.

Predictable Chronic Mild Stress Improves Mood, Hippocampal Neurogenesis and Memory

Maintenance of neurogenesis in adult hippocampus is important for functions such as mood and memory. As exposure to unpredictable chronic stress (UCS) results in decreased hippocampal neurogenesis, enhanced depressive- and anxiety-like behaviors, and memory dysfunction, it is believed that declined hippocampal neurogenesis mainly underlies the behavioral and cognitive abnormalities after UCS. However, the effects of predictable chronic mild stress (PCMS) such as the routine stress experienced in day-to-day life on functions such as mood, memory and hippocampal neurogenesis are unknown. Using FST and EPM tests on a prototype of adult rats, we demonstrate that PCMS (comprising 5 min of daily restraint stress for 28 days) decreases depressive- and anxiety-like behaviors for prolonged periods. Moreover, we illustrate that decreased depression and anxiety scores after PCMS are associated with ∼1.8-fold increase in the production and growth of new neurons in the hippocampus. Additionally, we found that PCMS leads to enhanced memory function in WMT as well as NORT. Collectively, these findings reveal that PCMS is beneficial to adult brain function, which is exemplified by increased hippocampal neurogenesis and improved mood and cognitive function.

Hippocampal neurogenesis and neural stem cells in temporal lobe epilepsy

Virtually all mammals, including humans, exhibit neurogenesis throughout life in the hippocampus, a learning and memory center in the brain. Numerous studies in animal models imply that hippocampal neurogenesis is important for functions such as learning, memory, and mood. Interestingly, hippocampal neurogenesis is very sensitive to physiological and pathological stimuli. Certain pathological stimuli such as seizures alter both the amount and the pattern of neurogenesis, though the overall effect depends on the type of seizures. Acute seizures are classically associated with augmentation of neurogenesis and migration of newly born neurons into ectopic regions such as the hilus and the molecular layer of the dentate gyrus. Additional studies suggest that abnormally migrated newly born neurons play a role in the occurrence of epileptogenic hippocampal circuitry characteristically seen after acute seizures, status epilepticus, or head injury. Recurrent spontaneous seizures such as those typically observed in chronic temporal lobe epilepsy are associated with substantially reduced neurogenesis, which, interestingly, coexists with learning and memory impairments and depression. In this review, we discuss both the extent and the potential implications of abnormal hippocampal neurogenesis induced by acute seizures as well as recurrent spontaneous seizures. We also discuss the consequences of chronic spontaneous seizures on differentiation of neural stem cell progeny in the hippocampus and strategies that are potentially useful for normalizing neurogenesis in chronic temporal lobe epilepsy.

Enhanced production and dendritic growth of new dentate granule cells in the middle-aged hippocampus following intracerebroventricular FGF-2 infusions

Declined production and diminished dendritic growth of new dentate granule cells in the middle‐aged and aged hippocampus are correlated with diminished concentration of fibroblast growth factor‐2 (FGF‐2). This study examined whether increased FGF‐2 concentration in the milieu boosts both production and dendritic growth of new dentate granule cells in the middle‐aged hippocampus. The FGF‐2 or vehicle was infused into the posterior lateral ventricle of middle‐aged Fischer (F)344 rats for 2 weeks using osmotic minipumps. New cells born during the first 12 days of infusions were labeled via daily intraperitoneal injections of 5′‐bromodeoxyuridine (BrdU) and analysed at 10 days after the last BrdU injection. Measurement of BrdU+ cells revealed a considerably enhanced number of new cells in the subgranular zone (SGZ) and granule cell layer (GCL) of the dentate gyrus (DG) ipsilateral to FGF‐2 infusions. Characterization of β‐III tubulin+ neurons among newly born cells suggested an increased addition of new neurons to the SGZ/GCL ipsilateral to FGF‐2 infusions. Quantification of DG neurogenesis at 8 days post‐infusions via doublecortin (DCX) immunostaining also revealed the presence of an enhanced DG neurogenesis ipsilateral to FGF‐2 infusions. Furthermore, DCX+ neurons in FGF‐2‐infused rats exhibited enhanced dendritic growth compared with their counterparts in vehicle‐infused rats. Thus, subchronic infusion of FGF‐2 is efficacious for stimulating an enhanced DG neurogenesis from neural stem/progenitor cells in the middle‐aged hippocampus. As dentate neurogenesis is important for hippocampal‐dependent learning and memory and DG long‐term potentiation, strategies that maintain increased FGF‐2 concentration during ageing may be beneficial for thwarting some of the age‐related cognitive impairments.

Implications of decreased hippocampal neurogenesis in chronic temporal lobe epilepsy.

Temporal lobe epilepsy (TLE), characterized by spontaneous recurrent motor seizures (SRMS), learning and memory impairments, and depression, is associated with neurodegeneration, abnormal reorganization of the circuitry, and loss of functional inhibition in the hippocampal and extrahippocampal regions. Over the last decade, abnormal neurogenesis in the dentate gyrus (DG) has emerged as another hallmark of TLE. Increased DG neurogenesis and recruitment of newly born neurons into the epileptogenic hippocampal circuitry is a characteristic phenomenon occurring during the early phase after the initial precipitating injury such as status epilepticus. However, the chronic phase of the disease displays substantially declined DG neurogenesis, which is associated with SRMS, learning and memory impairments, and depression. This review focuses on DG neurogenesis in the chronic phase of TLE and first confers the extent and mechanisms of declined DG neurogenesis in chronic TLE. The available data on production, survival and neuronal fate choice decision of newly born cells, stability of hippocampal stem cell numbers, and changes in the hippocampal microenvironment in chronic TLE are considered. The next section discusses the possible contribution of declined DG neurogenesis to the pathophysiology of chronic TLE, which includes its potential effects on spontaneous recurrent seizures, cognitive dysfunction, and depression. The subsequent section considers strategies that may be useful for augmenting DG neurogenesis in chronic TLE, which encompass stem cell grafting, administration of distinct neurotrophic factors, physical exercise, exposure to enriched environment, and antidepressant therapy. The final section suggests possible ramifications of increasing the DG neurogenesis in chronic epilepsy.