Metab Brain Dis (2014) 29:483–493 DOI 10.1007/s11011-013-9446-7

ORIGINAL PAPER

Glia activation and its role in oxidative stress Olalekan Michael Ogundele & Adams Olalekan Omoaghe & Duyilemi Chris Ajonijebu & Abiodun Ayodele Ojo & Temitope Deborah Fabiyi & Olayemi Joseph Olajide & Deborah Tolulope Falode & Philip Adeyemi Adeniyi

Received: 13 July 2013 / Accepted: 21 October 2013 / Published online: 13 November 2013 # Springer Science+Business Media New York 2013

Abstract Glia activation and neuroinflamation are major factors implicated in the aetiology of most neurodegenerative diseases (NDDs). Several agents and toxins have been known to be capable of inducing glia activation an inflammatory response; most of which are active substances that can cause oxidative stress by inducing production of reactive oxygen species (ROS). Neurogenesis on the other hand involves metabolic and structural interaction between neurogenic and

Highlights 1. Structural evidence reveals neuronal damage cytoplasmic fragmentation and loss of axonal projections in neuronal cells for the treatment group 2. Glia activation increased with cyanide treatment and oxidative stress 3. Neuronal metabolism increased both in oxidative stress and neurogenesis as shown by NSE immunopositivity and was more prominent in neurogenic cells of the PVZ both in the control and treatment category 4. Cell proliferation increased in PVZ and parietal cortex with cyanide treatment. The increase was much more prominent in neurogenic cells of the PVZ. The cortical cell increase was found to be glia (GFAP immunopositive) rather than neuronal O. M. Ogundele (*) : P. A. Adeniyi Department of Anatomy, College of Medicine and Health Sciences, Afe Babalola University, Ado-Ekiti, Nigeria e-mail: [email protected] A. O. Omoaghe : D. C. Ajonijebu : T. D. Fabiyi Department of Physiology, College of Medicine and Health Sciences, Afe Babalola University, Ado-Ekiti, Nigeria A. A. Ojo Department of Chemical Sciences, College of Sciences, Afe Babalola University, Ado-Ekiti, Nigeria D. T. Falode Department of Oncology, American Hospitals and Resorts, Lekki, Lagos State, Nigeria O. J. Olajide Department of Anatomy, College of Health Sciences, University of Ilorin, Ilorin, Nigeria

glia cells of the periventricular zone (PVZ); a region around the third ventricle. This study investigates glia activation (GFAP ), cell proliferation (Ki-67) and neuronal metabolism (NSE) during neurogenesis and oxidative stress by comparing protein expression in the PVZ against that of the parietal cortex. Adult Wistar Rats were treated with normal saline and 20 mg/Kg KCN for 7 days. The tissue sections were processed for immunohistochemistry to demonstrate glia cells (anti Rat-GFAP), cell proliferation (anti Rat-Ki-67) and neuronal metabolism (anti Rat-NSE) using the antigen retrieval method. The sections from Rats treated with cyanide showed evidence of neurodegeneration both in the PVZ and cortex. The distribution of glia cells (GFAP), Neuron specific Enolase (NSE) and Ki-67 increased with cyanide treatment, although the increases were more pronounced in the neurogenic cell area (PVZ) when compared to the cortex. This suggests the close link between neuronal metabolism and glia activation both in neurogenesis and oxidative stress. Keywords Neuron . Glia . Neurogenesis . Proliferation . Oxidative Stress . Cortex . PVZ Abbreviations PVZ Periventricular zone GFAP Glia fibrillary acidic protein NSE Neuron specific enolase Ki 67- Antigen Ki-67 KCN Potassium Cyanide ROS Reactive oxygen Species RNS Reactive nitrogen species NO Nitric oxide ACSF Accessory cerebrospinal fluid BSA Bovine serum albumin DNA Deoxyribonucleic acid DAB 3′3′ Diaminobenzidine Tetrachloride

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Introduction Neurogenesis is the process whereby neurons are being formed (Perederiy and Westbrook 2013); the process occurs mainly in the developing brain and is restricted to certain parts of the adult brain; the hippocampus and lateral ventricles, where neuronal cell division and migration have been observed (Wang et al. 2013; Perederiy and Westbrook 2013). From developmental point of view, the developing neurons possess anaerobic metabolic machinery which is changed to entirely aerobic metabolic system in the adult brain (Morte et al. 2013). This otherwise makes the adult neurons more susceptible to injury and stress related cell death. This is also evident in neural or nervous system injuries; such as spinal cord injury, ischemia perfusion in cardiovascular accidents and other forms of oxidative stress based neurodegenerative diseases (Terazawa et al. 2013). In recent years, scientists have tried to graft neural stem cells to such injury sites to facilitate replacement of degenerating neurons, but this has recorded only little success (Knuckles et al. 2012). For the purpose of this study, the region around the third ventricle (Periventricular zone; PVZ) is of interest as the studies of Xu et al. 2013 showed that cell division and neuronal proliferation occur in this region. In addition to having neurogenic cells, the PVZ/region around the third ventricle is also rich in sub-ventricular astrocytes and parenchymal glia cells (Robins et al. 2013), thus it is a preferred site for the investigation of neurogenesis and glia cell activation. The cerebrospinal fluid secreted in the choroid plexus of the lateral ventricles will normally drain into the third ventricle via the interventricular foramen. The third ventricle is connected inferiorly to the fourth ventricle by the cerebral aqueduct (Devos and Miller 2013). This region is characterized by abundant neurogenic cells and glia cells; detecting glia cells via immunohistochemistry of GFAP (a class III intermediate filament) can give an insight to the crucial link between glia activation and neurogenesis (Nazarenko et al. 2011) if mapped with in this region, that is the PVZ. A major event in mammalian neurogenesis is the migration of cells (neuronal and glial cells), proliferation of cells which in turn induces a fluctuation in energy metabolism of the proliferating cells during the developmental process (Jungenitz et al. 2013). The shift and fluctuation in metabolic pattern is evident from the fact that metabolism of the neuronal cell is anaerobic in utero as against the entirely aerobic metabolic machinery observed in most adult neurons (Murphy et al. 2012). Neuron specific enolase (NSE) have been found to be increased in proliferating neonatal neurons and degenerating adult neurons (Zhou et al. 2012), as it serves as a major indicator of glucose metabolism, this protein is peculiar to the neuronal cells of the central and peripheral nervous system and in certain cells of the adrenal gland (Knolhoff et al. 2013). Thus, NSE immunohistochemistry can be used in tracking the distribution of metabolic activities within the layers of the

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PVZ. The PVZ is believed to contain cells that are proliferating and otherwise metabolically hyperactive compared to the cortical neurons (Knolhoff et al. 2013; Mutso et al. 2012). In neuronal degeneration, the role of glial cells involves regulation of neuronal metabolism to support the stressed neurons (Zabel and Kirsch 2013). Up-regulation of glia activities is thus an indicator of stress and most times denotes the onset of degeneration and chemical imbalance (Béraud et al. 2012). However, since cortical neurons are aplastic, glia activation is restricted to neurodegeneration only. To study glia activation in neurogenesis, it is important to map the distribution of activated glia cells (GFAP) against regions of NSE and Ki-67 activity in the PVZ to understand the metabolic relationship between astrocytes and proliferating neurons around the third ventricle (neurogenesis) (Nazarenko et al. 2011). The link between the metabolic pattern (NSE), glia activation (GFAP) and neuronal proliferation (Ki-67) can also be compared between the normal, and cyanide treated rats where oxidative stress and neurodegeneration would have been induced. Uncontrolled glia activation and neuroinflamation contributes greatly to brain damage such as those observed in cyanide induced toxicity (Skaper et al. 2013). These factors, that is, reactive oxygen species (ROS) generated and activated glia cells may lead to transient brain damage such as movement disorders and cognitive dysfunctions. Studies have shown that substances with antioxidant potential reduce the damaging effect of ROS, oxidative stress and the extent of glia cell proliferation in the brain (Hui et al. 2013). We have therefore investigated the relationship between glia activation/neurogenesis in the PVZ and glia activation/ oxidative stress in the PVZ and parietal cortex by immunodetection of GFAP, NSE and Ki-67 in these regions.

Results Histology The general morphology of the PVZ and the neocortex was demonstrated via staining in Hematoxylin and Eosin to outline the cell membrane, cytoplasm, nucleus and relative cell distribution (count). Cyanide treatment led to neurodegeneration both in the neurogenic cells of the PVZ and the pyramidal cells of the parietal cortex (Fig. 2g and h) compared with the control (Fig. 1g and h); the control neurons showed prominent axonal projections and pronounced cell bodies. The cytoplasm was darkly stained and the fibres arranged as lamellated structures. In the PVZ, the neurogenic cells were seen with pronounced rounded darkly stained nuclei organized into 3 to 4 layers (Fig. 1g). Cell movement occurred in 2 directions (considered in 2D; x-axis and y-axis) (arrows in Fig. 5g). In the treatment category, the appearance of the cells showed thin membranes, clear cytoplasm with a single fragmented centrally placed nuclei giving the cells a ghost-like appearance (Fig. 2g). Some of the cells were ruptured, thus creating vacuolar spaces in the cortex (arrows in

Metab Brain Dis (2014) 29:483–493 Fig. 1 Comparative histology of the PVZ (a–c) and the parietal cortex (d–f). Histology of the PVZ (a–c) of control animals treated with normal saline in vivo, stained in Hematoxylin and Eosin to show the general outline of neuronal cells in this region. a The appearance of the cells of the PVZ at ×100 showed the relative distributions of neuronal cells around the third ventricle. b At higher magnification ×400 (the cells are observed as round clusters with prominent nuclei forming about 3 to 4 layers (arrows)). c Increase in cell number was observed close to the hypothalamus, suggesting a close relationship between this region and the hypothalamus as a potential site for adult neurogenesis (arrow head) (×1,000). d The 6-layered neocortex is shown at ×100 for the control. e and f: Higher magnification photomicrographs for the parietal cortex at ×400 and ×1,000 respectively. Arrow shows the multipolar morphology of cortical neurons. g and i Comparative and quantitative morphology of cells in the PVZ and the pyramidal cells in the parietal cortex. g The outline of the cells can be described as rounded with prominent condensed neuclei; the cells were observed as clusters in 2 directions (arrows; Magnification ×1,000). h The axonal projections of cortical pyramidal neurons (arrow head) are seen as unidirectional projections to the adjacent layer (Magnification ×1,000). i Bar chart showing quantitaive measurement (cell count per unit area) for the PVZ and parietal cortex of the control group

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Fig. 2h). The number of cell layers of the PVZ was reduced and the cell count for both regions showed reduction in cell number (Fig. 2i) when compared to the control (Fig. 1i). Immunohistochemistry Glia activation (GFAP) This study showed that glia activation is a major factor both in neurogenesis and oxidative

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stress. The studies by Magistretti and Co-Workers (1996) have greatly described the intimate interdependent metabolic and structural relationship between the neurons and glia cells (astrocytes); the role of the glia family is often supportive as well as regulatory. This is responsible, in part, for the changes the distribution of glia cells during neurogenesis and oxidative stress. The distribution of GFAP immunopositive cells (Fig. 3a–c) showed

486 Fig. 2 Histology of the PVZ and parietal cortex of rats treated with cyanide in vivo. a–c The number of layers of neurogenic cells observed in the PVZ have been reduced to 2 layers with an increase in density of the fibrous layer above this region (Magnification ×100, ×400 and ×1,000). d–f Histology of the parietal cortex at ×100 and ×400. f: At ×1,000, the general outline of the pyramidal cells shows certain level of degeneration; the cytoplasm is fragmented with a single small centrally placed nuclei, the cell body stained very lightly with H&E. Arrows indicate sites of degeneration in the PVZ; (arrows) indicates degenerating neuronal cells in the paraietal cortex. g, h and i Comparative histology of the neurogenic cells and pyramidal cells post cyanide treatment in vivo. Reduction in cell number is a prominent feature of the PVZ with features of neuronal degeneration characterized by an increased cell size with fragmented cytoplams and no observable axons (h). Small cell bodies around the neruons represents glia cells around the degenrating neurons (arrow head in g; Magnification ×1,000). i Quantitative measurement of cell population per unit area represented with a Bar chart

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uniform distribution of glia cells both in the parietal cortex (Fig. 3d–f) and the PVZ (Fig. 3a–c, g) while in cyanide treated Rats, oxidative stress induced by ROS over production triggered glia proliferation around the distressed neurons in the cortex and the PVZ. This activation of glia cells was more pronounced in the PVZ (Fig. 4a–c) than in the cortex (Fig. 4d–f).

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Neuronal metabolism (NSE) and glia activation (GFAP) Mapping glia distribution against proliferative and (or) stress induced neurometabolism was done by demonstrating Neuron specific enolase (Gamma enolase). This is to distinguish the rapid metabolism in this set of neurons from the initial alpha enolase characteristic of regular metabolism. Increased neuronal metabolism was observed in the neurogenic cells of the

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Fig. 3 a–c and d–f anti-Glia fibrillary acidic protein (GFAP: Type III intermediate filament) immunohistochemistry to demonstrate glia cells around the neurogenic cells of the PVZ and the parietal cortex of the control group. a The distribution of glia cells in the neurogenic area can be observed at low magnification (×100) as brownish reaction deposits of 3′3′-DAB. At ×400, the projections of the glia cells are prominent and are seen as star shaped brown structures interspersed in between the neuronal cells for both the PVZ (b) and the neocortex (e). Arrows in c and f shows the glia cells at ×1,000 for the PVZ and parietal cortex respectively. Astrocytes are prominent at ×400 (e and arrow in b). g Equal glia cell count per unit area was observed for both brain regions in the control (score of 5)

PVZ (Fig. 5a–c); higher than in the cortical cells (Fig. 5d–f). NSE immunopositivity also outlined the structure of metabolically active dividing spindle shaped neurons joined by two slender cellular processes but directed in opposite directions (Fig. 5g). In the cyanide treated groups NSE expression level increased in the parietal cortex (Fig. 6d–f) and the PVZ (Fig. 6a–c) and the spindle shaped outline was lost for the dividing cells (Fig. 6g). It can be deduced that for the PVZ, NSE level increased both in neurogenesis (Fig. 5a–c; Control) and in oxidative stress (Fig. 6a–c; KCN treatment) although the expression level was seen to be higher in oxidative stress. In the parietal cortex, NSE immunopositive cells were restricted to certain areas with both pyramidal cells and surrounding small cells expressing the protein both for the control and treatment groups (Figs. 5d–f and 6d–f). Ki-67 and cell proliferation The PVZ, an area around the third ventricle have long been described as a potential area of active neurogenesis while the parietal cortex is a less active neurogenic axis. Ki-67 positive cells were greatly distributed around the PVZ (Fig. 7a and b) and the number increased greatly with KCN treatment (Fig. 8a and b). This increase corresponds with the observed increase in glia expression (Fig. 4a–c; GFAP). In the parietal cortex, the increased Ki67 immunopositivity might be as a result of glia proliferation rather than neuronal proliferation. The count of Ki-67 immunopositive cells were also higher in the neurogenic cell area (PVZ) both in the control (Fig. 7a and b) and treatment (Fig. 8a and b) when compared against the cortical Ki-67 expression for control (Fig. 7c and d) and treatment category (Fig. 8c and d).

Discussion Energy metabolism is an encompassing phenomenon describing the process of day to day neuronal survival, feeding and neuronal cell death (Kim et al. 2012). The intimate relationship between the neurons and the glia cells is more than just ordinary metabolic and structural interaction but also regulatory (Oliva et al. 2013). In the neonatal brain, the developing neurons are plastic and are capable of cell division and proliferation; they are also endowed with anaerobic metabolic machinery making them less susceptible to injury (Oliva et al. 2013). The adult neuron is almost entirely dependent on aerobic metabolism, thus making them vulnerable to oxidative stress and metabolic injuries (Zhang et al. 2013). The glycolytic process is completed in the neurons and lactate is exported to the astrocytes coupled with glutamate release where the Kreb’s cycle is completed and ATP is exported back to the neurons coupled with glutamine (Eid et al. 2013; Magistretti and Pellerin 1996). During oxidative stress such a process experiences a paradigm shift due to alteration in the

488 Fig. 4 Comparative immunohistochemistry using anti-GFAP (polyclonal„ antibody) to map the distribution of glia cells in the PVZ and parietal cortex post treatment with orally administered cyanide at 20 mg/Kg for 7 days. a and d The glia cells are much more prominent as dense brownish cells with numerous projections at ×100 (arrow). b and e At ×400, the general morphology of the immunopositive glia cells are prominent with increased cell number especially around the neurogenic cells of the PVZ (b). Also increased glia presence was seen around degenerating pyramidal cells of the parietal cortex (e). The count at ×400 shows increased glia presence in the PVZ with a score of 10, higher than what was observed in the cortex at the same magnification with a score of 7 (g). The size of these glia cells were also increased in the PVZ. c and f Arrows indicates multiple glia projections and glia cell bodies around degenrating neurons as a form of response to oxidative stress (Magnification ×1,000)

neuronal glycolytic metabolic pattern, thus signalling stress, followed by glia (astrocytic) proliferation as shown in this study by GFAP immunopositivity. Since each phase the of astrocytic-neuronal glutamate-glucose cycle produces specific amounts of ATP, inhibition of the metabolic process will reduce the ability of the neuron to complete the glycolytic process- to generate its own ATP to facilitate glutamateglucose transport. A defective glutamate-glucose transport will lead to a reduction in glutamate-glutamine cycling between the neuron and the astrocytes; thus the number of astrocytes increases rapidly to facilitate and counter the metabolic shift in order to make energy substrates available to the neuron under stress. In this study, a defective glycolytic process is indicated by the increase in NSE expression post treatment with cyanide. The enolase being an enzyme of the glycolytic pathway exists in a specific isoform as NSE, it has also been previously used a marker to identify neurons versus glia (Zhou et al. 2012). The increase observed in this study can be accounted for as NSE increases to stabilize the glycolytic pathway during high energy demands, thus a defective glycolysis will lead to an increase in NSE expression. The use of cyanide in this study describes a mitochondria signalling pathway for oxidative stress. This mitochondria based pathway utilizes the ability of cyanide to inhibit the Heme a3-Cuβ binuclear centre of Cytochrome C Oxidase (CcOX) (Contestabile et al. 2012). The high tendency of cyanide to bind to this centre reduces the oxygen conversion capacity at Complex V of the electron transport chain (Complex V facilitates the conversion of molecular oxygen into water). The result is accumulation of oxygen and electrons from reduced food molecules in the mitochondria matrix, thus forming activated oxygen radicals, otherwise called reactive oxygen species (ROS) (Contestabile et al. 2012; Geissler et al. 2013) . ROS formation is a major contributor to the molecular trigger for glia activation, keeping in mind that activation of glia involves rapid proliferation, division and migration (May et al. 2013). What is the link between glia activation, cell death in oxidative stress and neurogenesis in general? There is no straight answer to this question as it requires a multidirectional approach of understanding the

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mechanism of oxidative stress induction, oxidative stress induced cell death, mechanism of glia-neuron metabolism in stress, cell death and neurogenesis. Mitochondria signalling and cell death The ROS formed from the mitochondria signalling pathway can drive cell death through apoptosis and necrosis. The ROS is a free radical

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Fig. 5 Immunohistochemical demonstration of NSE; also known as„ Neuron Specific Enolase (NSE) Immunocytochemistry Gamma enolase or enolase 2 to demonstrate a switch from alpha enolase a b to NSE in neurogenesis. a and b The prominent NSE immuno positivity in the PVZ (a) and less positivity in the control parietal cortex (b) at a magnification of ×100. The immunopositive cells were mostly dividing cells characterized by a spindle shape and a clear space in between showing the axis of seperation of both daughter cells during neurogenesis (Arrows in b and c). e and f Certain enlarged cells show extreme positivity in the PVZ and cortex. g Axis of seperation of a dividing neurogenic cell demonstrated by NSE immunocytochemistry and rendered in gray scale (×1,000)

responsible for peroxidation of lipids in biomembranes. Extensive membrane damage is characteristic of necrotic cell death (Fig. 2a). ROS also reacts with nitrogen and nitrogen containing compounds to form reactive nitrogen species (RNS) and Nitric oxide (NO). Although NO is a naturally occurring endogenous modulator of cellular activity, when produced in excess, it can trigger DNA cleavage, hence apoptosis. Thus a major significance of glia activation induced by oxidative stress is the rapid onset of neuronal degeneration triggered by ROS over production similar to the findings of Campanini et al. (2013). The PVZ comparing the general architecture of the control (Fig. 1a–c, g) and the treatment (Fig. 2a–c, g), reduction in cell number was seen in the treatment group as cell count showed a decline from 16 in the control to 9 in the test category (Figs. 1i and 2i). This indicates that oxidative stress induced by cyanide toxicity reduced the progression of neurogenesis in this region similar to the findings of Sykora et al. (2013) and Zou et al. (2012). The product of such stress includes glia activation (Madeira et al. 2013); in this context, the glia cells considered includes the glia cells forming within the neurogenic zone and those around the PVZ. Glia activation and glia in neurogenesis are inseparable as it is a function of metabolic and structural interdependence between the neurons and glia cells both in the developing and the developed brain (Episcopo et al. 2013). From literature, the rate of neuronal cell process formation indirectly affects the rate of glia cell differentiation (Shi et al. 2013), while in neurogenesis, glia activation also implies increased glia cell population by rapid division (Nikolakopoulou et al. 2013). The rate of such glia formation was found to be higher in the PVZ of the treatment group such that a score of 10 was recorded compared to the control with a score of 5, similar to the findings of Khozhaĭ and Otellin (2013). This indicates that glia differentiation increased under stress when compared to normal neurogenesis observed in the control; this is evident in the Ki-67 immunostaining where the positivity increased in the PVZ for the treatment (Fig. 8a and b) when compared to the control (Fig. 7a and b). To demonstrate neurogenesis in this study, we employed NSE immunohistochemistry to distinguish neurons from the glia (Fig. 5a–c, g) -dividing neuronal cells were

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also seen in the control while cyanide treatment disoriented the division process in the treatment group (Fig. 6a–c, g). Parietal cortex The parietal cortex is one of the sites in the brain where neurogenesis have not been described before

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(Poluch and Juliano 2013). It thus serves as a better candidate to compare glia activation in neurogenic sites versus non neurogenic sites in oxidative stress induced by cyanide toxicity. The mechanism of cyanide induced toxicity and stress in the cortex have been closely linked to ROS formation and NO activation which can either induce apoptosis or necrosis; depending on the dosage of treatment (with changes in the

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Immunohistochemistry of NSE (Gmma enolase) in the cortex and PVZ of adult wistar rats treated with 20 mg/Kg of KCN for 7 days. a and d Higher immunopositivity was observed in the KCN treated group when compared to the control (Fig. 5a and d) for the PVZ and parietal cortex respectively. c and f The neuronal cells in both the parietal cortex and PVZ expressing NSE are enlarged and are characterized by structural features of onset neuronal degeneration (arrows). b The layered outline observed in the PVZ of control rats (Fig. 5b) is absent in the treatment, the dividing cells are not prominent rather each neuron is seen as a single, large immunopositive cell and the orientation of the dividing cells is distorted (g ). This strongly differentiates NSE expression in neurogenesis from NSE expression in oxidative stress (Magnification ×100, ×400 and ×1,000)

membrane, cytoplasm and nucleus; as seen in Fig. 2h). The extent of such cell death is seen as a reduction in cell count per unit area. This count is important to deduce the probable toxicity response in plastic cells of the PVZ and non-plastic cells of the cortex, in order to build a premise around the independent cytotoxic pathways adopted by different parts of the brain. The cellular changes differ between these two categories of cells, although a reduction in cell number was seen in the PVZ, the cytoarchitecture of the cells did not

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Fig. 7 Anti Rat-Ki-67 immuno staining (polyclonal) in the control Rats. a Ki-67 immunopositive cells of the PVZ at ×100. b Immunohistochemistry of Ki-67 in proliferating cells of the parietal cortex at ×100; nuclear inclusions of Ki-67 are denoted by arrows. d Few Ki-67 immunopositive cells were observed in the cortex, while the region around the PVZ (b) showed numerous immunopositive cells. Cell proliferation is higher around the PVZ (b) when compared with the parietal cortex (d). Arrows indicate Ki-67 immunopositive cells (Magnification ×100 and 400)

Metab Brain Dis (2014) 29:483–493 Fig. 8 Immuno labelling of proliferative protein marker Ki-67 in cells of the PVZ (a and b) and the parietal cortex (c and d) of Wistar rats treated with 20 mg/Kg KCN for 7 days. High expression of Ki-67 was observed in the PVZ (a and b) compared to the cortex. The extent of immunopositivity also increased around pyramidal cells of the parietal cortex (c and d) but not as much as that observed in the PVZ. Arrows indicate Ki-67 immunopositive cells (Magnification ×100 and ×400)

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change much compared to the control (Fig. 1g) while prominent changes were seen in the cortex (Fig. 1h)-the cell number reduced from 9 in the control, to 4 in the test category (Bar chart; Figs. 1i and 2i). Cortical glia activation was observed to have increased from a score of 5 to 7 per unit area following treatment with cyanide, although not as prominent as that seen in the PVZ. This is possible and expected as the PVZ also contains neurogenic glia cells that stained positive to the anti-GFAP. NSE showed immunopositivity only in the neurons under stress (Fig. 5c and d) and no evidence of division was seen when compared to the PVZ.

dividing cells showing that oxidative stress can increase NSE expression in cells even though there was no observable cell division in such cells (Fig. 6d–f; Parietal cortex). Cell proliferation (Ki-67) increased in the PVZ and parietal cortex, in a similar manner as glia activation post treatment with cyanide; although in the control, Ki-67 distribution was prominent in the PVZ and reduced in the cortex. Cell proliferation increased under stress, GFAP and NSE immunostaining showed that the proliferation was more of glia rather than neuronal in cortex, and also proliferation was higher in the PVZ (Figs. 5a–c and 6a–c) when compared to the cortical region (Figs. 5d–f and 6d–f).

Glia activation, cell proliferation and metabolism The neurons under stress or dividing neurogenic cells both require energy and support to ensure survival. This is evident from the increased GFAP positive cells around PVZ (Fig. 3a). In the control group, the glia presence was relatively low in the cortex with more of astrocytes seen (Fig. 3a). Following treatment with cyanide, the population of glia cells increased around degenerating cortical neurons (Fig. 4b). It was very interesting to observe the doubling glia activation effect in the neurogenic area (PVZ) under stress (KCN treatment); where for we have neurogenesis and oxidative stress as a combined state in this region post cyanide treatment. We deduced at this point that both neurogenesis and stress can trigger glia activation (Figs. 3 and 4). Furthermore, the relative count of glia cells increased in the neurogenic area more than in the cortex. The metabolic pattern mapped by NSE distribution showed that dividing neurogenic cells expressed NSE levels higher than non-dividing ones, thus NSE expression in neurogenesis is phasic and probably restricted to dividing and migrating neuronal cells (Fig. 5a–c). Post cyanide treatment, NSE levels increased in all cells, that is, both the dividing and non-

Conclusion This study shows that an intimate relationship exists between glia activation observed in neurogenesis and that seen in oxidative stress. The distribution of glia cells (GFAP immunopositivity) is similar to the pattern of NSE immunopositivity showing that the primary requirement for glia activation is a metabolic shift which is obtainable in neurogenesis resulting from rapid cell proliferation and migration; and oxidative stress to ensure neuronal survival.

Experimental procedure Animal preparation and treatment 10 F1 Generation adult Wistar rats were divided into two groups of 5 animals each (designated group 1 and group 2). The first group were treated with 20 mg/Kg BW of KCN for 7 days (Short term administration) to induce oxidative stress and neurodegeneration in the Parietal cortex and PVZ. The second group were given normal saline and were fed with normal rat chow and water for 7 days. The animals were sacrificed by cervical dislocation following the approval of the Animal use in Research

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Committee of the Afe Babalola University. The whole brain was exposed via dissection and placed in cold freshly prepared artificial cerebrospinal fluid ACSF [ACSF composition was as follows (mM): KCl, 2.5; MgSO4, 1.0; NaH2PO4, 1.25; NaHCO3, 26; d-glucose, 20; ascorbic acid, 0.45; CaCl2, 2.0; and NaCl, 125 (osmolality, 290–310 mosmol kg−1; pH 7.3– 7.4)] at 4 °C in a petri dish (prepared according to the method of Chunyan et al. 2007) to remove blood materials from the tissue. The whole brain was fixed in formolcalcium and processed to obtain paraffin wax embedded tissue blocks. Sectioning was done along the corona plane where the third ventricle and the cortex were prominent. Histology Tissue sections were processed for routine Hematoxylin and Eosin staining using the methods of Zhang et al. (2012). Immunohistochemistry The following proteins were labelled in the PVZ and cortical tissue sections (adopted and modified protocol from Xiong et al. 2013); Ki-67 and NSE were mapped in the layers of the cortex to differentiate between neurogenesis active regions and inactive regions of the brain both for the control and the oxidative stress induced treatment groups. GFAP was mapped in the PVZ and cortical tissue to demonstrate glia activation in neurogenesis and neuronal degeneration. Rat anti-GFAP was prepared in PBS 8.0 and desalted in G25 sephadex column (protein G column); the anti-GFAP was diluted at 1:100. Anti-NSE was desalted and diluted at 1:100 in Tris Buffer Saline (TBS). Rat Anti-Ki-67 (polyclonal) was diluted to 1:500 and re-suspended in PBS at PH 8.0. Procedure The paraffin wax embedded sections were mounted on a glass slide in preparation for antigen retrieval where the slides were immersed in urea overnight and then placed in a microwave for 45 min to re-activate the antigens and proteins in the tissue sections. The sections were treated with 1 % Bovine serum albumin (BSA; Sigma) to block nonspecific protein reactions following which the primary antibody treatment was done using diluted anti-NSE (1:50), antiGFAP (1:50) and anti-Ki-67 (1:50) on the pre-treated sections for one hour. Secondary antibody treatment was done using Biotinylated goat serum (1:100) for one hour [Biotinylated secondary antibody, anti-NSE, anti-Ki-67 and anti-GFAP were procured from Novocastra; Leica Biosystems]. The immunopositive reactions were developed using a polymer 3′3′ Diaminobenzidine Tetrachloride (DAB; Sigma) with colour intensification involving the use of mathenamine silver kit (Sigma). The sections were counterstained in Hematoxylin and treated in 1 % acid alcohol (freshly prepared). Cell count The images were captured using a MV550 digital Cameroscope connected to a computer interface and an

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Olympus Binocular research microscope. The resolution of the Cameroscope is 5.1 MP and the images were obtained at three different magnifications of ×100, ×400 and ×1, 000. In the micrographs we differentiated the general structure of the neurons in the cortex (pyramidal cell layer) and the rounded dividing neurogenic cells of the PVZ. Cell count was done at ×400 using the Open Office Draw Suite. The cells were counted per unit area first by identification followed by marking of each of the identified cells. Each of the identified and marked cells were counted and represented with bar charts. At the magnification of ×400, the individual immunopositive glia cells were scored by a unit of 1 per cell. A score of 10 was recorded in the PVZ post treatment while a score of 7 was recorded in the parietal cortex. Both regions were scored 5 in the control category (Fig. 3g); also shown in the bar chart. Acknowledgments We acknowledge the contributions of Mr. Madukwe Jonathan of the Department of Histopathology, National Hospital Abuja. We will also like to appreciate Mr. Oso of the Department of Chemical Sciences, Mrs. Olaiya of the Physiology Laboratory, Afe Babalola University for their assistance in regent preparation and treatment of the animals throughout the duration of the experiment. Conflict of interest The Authors hereby declare there is no conflict of interest associated with this study or any of the procedures and materials used for the purpose of the study.

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