Ashok K. Shetty

Efficacy of doublecortin as a marker to analyse the absolute number and dendritic growth of newly generated neurons in the adult dentate gyrus.

Doublecortin (DCX), a microtubule‐associated phosphoprotein, has been recently utilized as a marker of newly born neurons in the adult dentate gyrus (DG). Nonetheless, it is unknown whether DCX exclusively labels newly formed neurons, as certain granule cells with the phenotype of differentiated neurons express DCX. We addressed the authenticity of DCX as a marker of new neurons in the adult DG by quantifying cells that are positive for 5′‐bromodeoxyuridine (BrdU), DCX and both BrdU and DCX in hippocampal tissues of adult rats treated with daily injections of BrdU for 12 consecutive days. We provide new evidence that neurons visualized with DCX immunostaining in the adult rat DG are new neurons that are predominantly born during the 12 days before euthanasia. This is confirmed by the robust expression of BrdU in 90% of DCX‐positive neurons in the DG of animals injected with BrdU for 12 days. Furthermore, DCX expression is specific to newly generated healthy neurons, as virtually all DCX‐positive cells express early neuronal antigens but lack antigens specific to glia, undifferentiated cells or apoptotic cells. As DCX expression is also robust in the dendrites, DCX immunocytochemistry of thicker sections facilitates quantification of the dendritic growth in newly born neurons. Thus, both absolute number and dendritic growth of new neurons that are generated in the adult DG over a 12‐day period can be quantified reliably with DCX immunostaining. This could be particularly useful for analysing changes in dentate neurogenesis in human hippocampal tissues as a function of ageing or neurodegenerative diseases.

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.

Characterization of neuronal/glial differentiation of murine adipose-derived adult stromal cells

Neural tissue has limited capacity for intrinsic repair after injury, and the identification of alternate sources of neuronal stem cells has broad clinical potential. Preliminary studies have demonstrated that adipose-derived adult stromal (ADAS) cells are capable of differentiating into mesenchymal and non-mesenchymal cells in vitro, including cells with select characteristics of neuronal/glial tissue. In this study, we extended these observations to test the hypothesis that murine (mu) ADAS cells can be induced to exhibit characteristics of neuronal and glial tissue by exposure to a cocktail of induction agents. We characterized the differentiation of muADAS cells in vitro using immunohistochemistry and immunoblotting, and examined whether these cells respond to the glutamate agonist N-methyl-D-aspartate (NMDA). We found that induced muADAS cells express proteins indicative of neuronal/glial cells, including nestin, GFAP, S-100, NeuN, MAP2, tau, and beta-III tubulin. Induced muADAS cells express gamma-aminobutyric acid (GABA), the NR-1 and NR-2 subunits of the glutamate receptor, GAP-43, synapsin I, and voltage-gated calcium channels. Finally, induced muADAS cells demonstrate decreased viability in response to NMDA. These findings suggest that muADAS cells can be induced to exhibit several phenotypic, morphologic, and excitotoxic characteristics consistent with developing neuronal and glial tissue.

Reactive astrocytes express the embryonic intermediate neurofilament nestin.

Nestin is a neurofilament protein expressed by the immediate precursors to neurons and glia in rats and humans. Nestin immunoreactivity in the rat CNS was studied following kainic acid (KA) hippocampal lesions. Numerous nestin positive cells within the KA lesion were confirmed to be reactive astrocytes by their immunoreactivity for glial fibrillary acidic protein (GFAP). The number of these cells decreased with time after the KA lesion and no astrocyte immunostaining for nestin was observed in control animals. A subset of nestin-positive cells in the ventricular subependymal region appeared to be radial glial cells, extending to cell body layers. Nestin is one of several embryonic markers expressed by reactive astrocytes, suggesting an embryonic reversion induced by the KA lesion, possibly to enhance functional recovery.

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.

Chronic alcohol exposure reduces hippocampal neurogenesis and dendritic growth of newborn neurons

Hippocampal neurogenesis is known as the formation of new neurons from the proliferating neural progenitor cells (NPC) at the dentate gyrus. Cell proliferation, survival, differentiation and maturation are critical stages leading to the generation of healthy neurons. As all of these stages can be influenced by alcohol exposure, we studied the effects of chronic alcohol on each process by immunocytochemistry for stage‐specific antigens and prelabelling newborn cells with bromodeoxyuridine (BrdU). Rats were administered alcohol liquid diet or control diet for one, two or four weeks. We found that cell proliferation was inhibited as proliferating cell nuclear antigen (PCNA) expression was reduced by approximately 50% in alcohol‐treated animals at all time points. Doublecortin (DCX), a microtubule protein expressed early in differentiating neurons, was progressively decreased over the duration of exposure and significantly reduced after two and four weeks of drinking. Morphological analyses of DCX‐positive cells revealed that four weeks of alcohol treatment reduced the size of the dendritic tree including the total length of apical dendrites, number of nodes and endings. Furthermore, BrdU labelling demonstrated a dramatic decrease in cell survival after four weeks of drinking, while cell death was increased by such treatment. Confocal analysis indicated that over 80% of BrdU+ cells colabelled with NeuN suggesting that alcohol reduced neurogenesis. In conclusion, chronic alcohol exposure disrupts neurogenesis by decreasing NPC proliferation, inhibiting cell survival and altering morphological maturation of newborn neurons. These data implicate impaired hippocampal neurogenesis with the cognitive and affective dysfunction associated with chronic alcoholism.