The effects of chronic alcoholization on the expression of brain-derived neurotrophic factor and its receptors in the brains of mice genetically predisposed to depressive-like behavior

Brain-derived neurotropic factor (BDNF) plays an important role in mechanisms of depression. Precursor protein of this factor (proBDNF) can initiate apoptosis in the brain, while the mature form of BDNF is involved in neurogenesis. It is known that chronic alcoholization leads to the activation of apoptotic processes, neurodegeneration, brain injury, and cognitive dysfunction. In this work, we have studied the influence of long-term ethanol exposure on the proBDNF and BDNF protein levels, as well as on the expression of genes that encode these proteins in the brain structures of ASC mice with genetic predisposition to depressive-like behavior and in mice from parental nondepressive CBA strain. It was shown that chronic alcoholization results in a reduction of the BDNF level in the hippocampus and an increase in the amount of TrkB and p75 receptors in the frontal cortex of nondepressive CBA mice. At the same time, the long-term alcoholization of depressive ASC mice results in an increase of the proBDNF level in the frontal cortex and a reduction in the p75 protein level in the hippocampus. It has also been shown that, in depressive ASC mice, proBDNF and BDNF levels are significantly lower in the hippocampus and the frontal cortex compared with nondepressive CBA strain. However, no significant differences in the expression of genes encoding the studied proteins were observed. Thus, changes in the expression patterns of proBDNF, BDNF, and their receptors under the influence of alcoholization in the depressive ASC strain and nondepressive CBA strain mice are different.     

Transient focal cerebral ischemia down-regulates glutamate transporters GLT-1 and EAAC1 expression in rat brain

Transient focal cerebral ischemia leads to extensive excitotoxic neuronal damage in rat cerebral cortex. Efficient reuptake of the released glutamate is essential for preventing glutamate receptor over-stimulation and neuronal death. Present study evaluated the expression of the glial (GLT-1 and GLAST) and neuronal (EAAC1) subtypes of glutamate transporters after transient middle cerebral artery occlusion (MCAO) induced focal cerebral ischemia in rats. Between 24h to 72h of reperfusion after transient MCAO, GLT-1 and EAAC1 protein levels decreased significantly (by 36% to 56%, p < 0.05) in the ipsilateral cortex compared with the contralateral cortex or sham control. GLT-1 and EAAC1 mRNA expression also decreased in the ipsilateral cortex of ischemic rats at both 24h and 72h of reperfusion, compared with the contralateral cortex or sham control. Glutamate transporter down-regulation may disrupt the normal clearance of the synaptically-released glutamate and may contribute to the ischemic neuronal death.     

Comparative Effect of Treadmill Exercise on Mature BDNF Production in Control versus Stroke Rats

Physical exercise constitutes an innovative strategy to treat deficits associated with stroke through the promotion of BDNF-dependent neuroplasticity. However, there is no consensus on the optimal intensity/duration of exercise. In addition, whether previous stroke changes the effect of exercise on the brain is not known. Therefore, the present study compared the effects of a clinically-relevant form of exercise on cerebral BDNF levels and localization in control versus stroke rats. For this purpose, treadmill exercise (0.3 m/s, 30 min/day, for 7 consecutive days) was started in rats with a cortical ischemic stroke after complete maturation of the lesion or in control rats. Sedentary rats were run in parallel. Mature and proBDNF levels were measured on the day following the last boot of exercise using Western blotting analysis. Total BDNF levels were simultaneously measured using ELISA tests. As compared to the striatum and the hippocampus, the cortex was the most responsive region to exercise. In this region, exercise resulted in a comparable increase in the production of mature BDNF in intact and stroke rats but increased proBDNF levels only in intact rats. Importantly, levels of mature BDNF and synaptophysin were strongly correlated. These changes in BDNF metabolism coincided with the appearance of intense BDNF labeling in the endothelium of cortical vessels. Notably, ELISA tests failed to detect changes in BDNF forms. Our results suggest that control beings can be used to find conditions of exercise that will result in increased mBDNF levels in stroke beings. They also suggest cerebral endothelium as a potential source of BDNF after exercise and highlight the importance to specifically measure the mature form of BDNF to assess BDNF-dependent plasticity in relation with exercise.     

Irisin Peptide Protects Brain Against Ischemic Injury Through Reducing Apoptosis and Enhancing BDNF in a Rodent Model of Stroke

Evidence has shown therapeutic potential of irisin in cerebral stroke. The present study aimed to assess the effects of recombinant irisin on the infarct size, neurological outcomes, blood–brain barrier (BBB) permeability, apoptosis and brain-derived neurotrophic factor (BDNF) expression in a mouse model of stroke. Transient focal cerebral ischemia was established by middle cerebral artery occlusion (MCAO) for 45 min and followed reperfusion for 23 h in mice. Recombinant irisin was administrated at doses of 0.1, 0.5, 2.5, 7.5, and 15 µg/kg, intracerebroventricularly (ICV), on the MCAO beginning. Neurological outcomes, infarct size, brain edema and BBB permeability were evaluated by modified neurological severity score (mNSS), 2,3,5-triphenyltetrazolium chloride (TTC) staining and Evans blue (EB) extravasation methods, respectively, at 24 h after ischemia. Apoptotic cells and BDNF protein were detected by TUNEL assay and immunohistochemistry techniques. The levels of Bcl-2, Bax and caspase-3 proteins were measured by immunoblotting technique. ICV irisin administration at doses of 0.5, 2.5, 7.5 and 15 µg/kg, significantly reduced infarct size, whereas only in 7.5 and 15 µg/kg improved neurological outcome (P?<?0.001). Treatment with irisin (7.5 µg/kg) reduced brain edema (P?<?0.001) without changing BBB permeability (P?>?0.05). Additionally, irisin (7.5 µg/kg) significantly diminished apoptotic cells and increased BDNF immunoreactivity in the ischemic brain cortex (P?<?0.004). Irisin administration significantly downregulated the Bax and caspase-3 expression and upregulated the Bcl-2 protein. The present study indicated that irisin attenuates brain damage via reducing apoptosis and increasing BDNF protein of brain cortex in the experimental model of stroke in mice.     

Development of mature BDNF‐specific sandwich ELISA

Mature brain‐derived neurotrophic factor (mBDNF) plays a vital role in the nervous system, whereas proBDNF elicits neurodegeneration and neuronal apoptosis. Although current enzyme‐linked immunosorbent assay (ELISA) has been widely used to measure BDNF levels, it cannot differentiate mBDNF from proBDNF. As the function of proBDNF differs from mBDNF, it is necessary to establish an ELISA assay specific for the detection of mBDNF. Therefore, we aimed to establish a new mBDNF‐specific sandwich ELISA. In this study, we have screened and found a combination of antibodies for a sandwich ELISA. A monoclonal antibody and sheep anti‐BDNF were chosen as capture and detection antibody for sandwich ELISA respectively. The new ELISA showed no cross‐reactivity to human recombinant NT‐3, NT‐4, nerve growth factor and negligible cross‐reactivity (0.99–4.99%) for proBDNF compared to commercial ELISA kits (33.18–91.09%). The application of the new mBDNF ELISA was shown through the measurement of mBDNF levels in different brain regions of rats and in the brain of β‐site amyloid precursor protein cleaving enzyme 1 (BACE1)^?/? and WT mice and compared to western blot. Overall, this new ELISA will be useful for the measurement of mBDNF levels with high specificity.

     

Effects of prenatal stress exposure on soluble Aβ and brain-derived neurotrophic factor signaling in male and female APPswe/PS1dE9 mice

Chronic stress and stress-related disorders, such as major depression (MD), have been shown to increase the risk for developing Alzheimer's disease (AD). Brain-derived neurotrophic factor (BDNF) has been postulated as a neurophysiological link between these illnesses. Our previous research has indicated that exposing the APPswe/PS1dE9 mouse model of AD to prenatal maternal stress (PS) induced a depressive-like phenotype, specifically in female mice. Considering the role of BDNF in depressive-like behavior and its interactions with amyloid-β (Aβ), our aim was to explore whether these mice would also exhibit alterations in soluble Aβ, mature BDNF (mBDNF), proBDNF, and the receptors TrkB and p75(NTR) in comparison to non-stressed animals. Our results demonstrate that female APPswe/PS1dE9 mice have higher levels of hippocampal proBDNF and soluble Aβ as compared to their male littermates. Additionally, a tendency was observed for PS to lower mBDNF protein levels in the hippocampus, but only in female mice, while receptor levels remained unaltered by sex or PS exposure. Given that female mice both have higher proBDNF and Aβ levels, these findings suggest an underlying role for BDNF signaling and Aβ production in the selective vulnerability of women for MD and AD development.     

Valproic acid reduces brain damage induced by transient focal cerebral ischemia in rats: potential roles of histone deacetylase inhibition and heat shock protein induction

Growing evidence from in vitro studies supports that valproic acid (VPA), an anti-convulsant and mood-stabilizing drug, has neuroprotective effects. The present study investigated whether VPA reduces brain damage and improves functional outcome in a transient focal cerebral ischemia model of rats. Subcutaneous injection of VPA (300 mg/kg) immediately after ischemia followed by repeated injections every 12 h, was found to markedly decrease infarct size and reduce ischemia-induced neurological deficit scores measured at 24 and 48 h after ischemic onset. VPA treatment also suppressed ischemia-induced neuronal caspase-3 activation in the cerebral cortex. VPA treatments resulted in a time-dependent increase in acetylated histone H3 levels in the cortex and striatum of both ipsilateral and contralateral brain hemispheres of middle cerebral artery occlusion (MCAO) rats, as well as in these brain areas of normal, non-surgical rats, supporting the in vitro finding that VPA is a histone deacetylase (HDAC) inhibitor. Similarly, heat shock protein 70 (HSP70) levels were time-dependently up-regulated by VPA in the cortex and striatum of both ipsilateral and contralateral sides of MCAO rats and in these brain areas of normal rats. Altogether, our results demonstrate that VPA is neuroprotective in the cerebral ischemia model and suggest that the protection mechanisms may involve HDAC inhibition and HSP induction.     

δ-Opioid Receptor Activation Rescues the Functional TrkB Receptor and Protects the Brain from Ischemia-Reperfusion Injury in the Rat

Objectives

δ-opioid receptor (DOR) activation reduced brain ischemic infarction and attenuated neurological deficits, while DOR inhibition aggravated the ischemic damage. The underlying mechanisms are, however, not well understood yet. In this work, we asked if DOR activation protects the brain against ischemic injury through a brain-derived neurotrophic factor (BDNF) -TrkB pathway.

Methods

We exposed adult male Sprague-Dawley rats to focal cerebral ischemia, which was induced by middle cerebral artery occlusion (MCAO). DOR agonist TAN-67 (60 nmol), antagonist Naltrindole (100 nmol) or artificial cerebral spinal fluid was injected into the lateral cerebroventricle 30 min before MCAO. Besides the detection of ischemic injury, the expression of BDNF, full-length and truncated TrkB, total CREB, p-CREB, p-ATF and CD11b was detected by Western blot and fluorescence immunostaining.

Results

DOR activation with TAN-67 significantly reduced the ischemic volume and largely reversed the decrease in full-length TrkB protein expression in the ischemic cortex and striatum without any appreciable change in cerebral blood flow, while the DOR antagonist Naltrindole aggregated the ischemic injury. However, the level of BDNF remained unchanged in the cortex, striatum and hippocampus at 24 hours after MCAO and did not change in response to DOR activation or inhibition. MCAO decreased both total CREB and pCREB in the striatum, but not in the cortex, while DOR inhibition promoted a further decrease in total and phosphorylated CREB in the striatum and decreased pATF-1 expression in the cortex. In addition, MCAO increased C11b expression in the cortex, striatum and hippocampus, and DOR activation specifically attenuated the ischemic increase in the cortex but not in the striatum and hippocampus.

Conclusions

DOR activation rescues TrkB signaling by reversing ischemia/reperfusion induced decrease in the full-length TrkB receptor and reduces brain injury in ischemia/reperfusion     

Acute connexin43 temporal and spatial expression in response to ischemic stroke

Connexin43 (Cx43) gap junctions expressed in astrocytes can significantly impact neuronal survival in stroke. However, little is known regarding Cx43 spatial and temporal expression during the initial stages of brain ischemia. Using immunohistochemistry and Western blot analysis, we examined Cx43 spatial and temporal expression as a function of neuronal injury within the first 24 h after permanent middle cerebral artery occlusion (pMCAO). Western blot analysis showed a significant increase in Cx43 protein expression in the core ischemic area at 2 and 3 h after pMCAO. However, after 6 h of pMCAO Cx43 levels were significantly reduced. This reduction was due to cell death and concomitant Cx43 degradation in the expanding focal ischemic region, while the peri-infarct zone revealed intense Cx43 staining. The neuronal cell-death marker Fluoro-Jade C labeled injured neurons faintly at 1 h post-pMCAO with a time-dependent increase in both intensity and size of punctate staining. In addition, decreased microtubule-associated protein 2 (MAP2) immunoreactivity and thionin staining similarly indicated cell damage beginning at 1 h after pMCAO. Taken together, Cx43 expression is sensitive to neuronal injury and can be detected as early as 2 h post-pMCAO. These findings underscore Cx43 gap junction as a potential early target for therapeutic intervention in ischemic stroke.     

The PGC-1α Activator ZLN005 Ameliorates Ischemia-Induced Neuronal Injury In Vitro and In Vivo

Oxidative stress is a great challenge to neurons following cerebral ischemia. PGC-1α has been shown to act as a potent modulator of oxidative metabolism. In this study, the effects of ZLN005, a small molecule that activate PGC-1α, against oxygen–glucose deprivation (OGD)- or ischemia-induced neuronal injury in vitro and in vivo were investigated. Transient middle cerebral artery occlusion (tMCAO) was performed in rats and ZLN005 was administered intravenously at 2 h, 4 h, or 6 h after ischemia onset. Infarct volume and neurological deficit score were detected to evaluate the neuroprotective effects of ZLN005. Well-differentiated PC12 cells, which were subjected to OGD for 2 h followed by reoxygenation for 22 h, were used as an in vitro ischemic model. Changes in expression of PGC-1α, its related genes, and antioxidant genes were determined by real-time quantitative PCR. The results showed that ZLN005 reduced cerebral infarct volume and improved the neurological deficit in rat with tMCAO, and significantly protected OGD-induced neuronal injury in PC12 cells. Furthermore, ZLN005 enhanced expression of PGC-1α in PC12 cells and in the ipsilateral hemisphere of rats with tMCAO. Additionally, ZLN005 increased antioxidant genes, including SOD1 and HO-1, and significantly prevented the ischemia-induced decrease in SOD activity. Taking together, the PGC-1α activator ZLN005 exhibits neuroprotective effects under ischemic conditions and molecular mechanisms possibly involve activation of PGC-1α signaling pathway and cellular antioxidant systems.     

Precursor form of brain-derived neurotrophic factor and mature brain-derived neurotrophic factor are decreased in the pre-clinical stages of Alzheimer's disease

Brain-derived neurotrophic factor (BDNF) is critical for the function and survival of neurons that degenerate in the late stage of Alzheimer's disease (AD). There are two forms of BDNF, the BDNF precursor (proBDNF) and mature BDNF, in human brain. Previous studies have shown that BDNF mRNA and protein, including proBDNF, are dramatically decreased in end-stage AD brain. To determine whether this BDNF decrease is an early or late event during the progression of cognitive decline, we used western blotting to measure the relative amounts of BDNF proteins in the parietal cortex of subjects clinically classified with no cognitive impairment (NCI), mild cognitive impairment (MCI) or mild to moderate AD. We found that the amount of proBDNF decreased 21 and 30% in MCI and AD groups, respectively, as compared with NCI, consistent with our previous results of a 40% decrease in end-stage AD. Mature BDNF was reduced 34 and 62% in MCI and AD groups, respectively. Thus, the decrease in mature BDNF and proBDNF precedes the decline in choline acetyltransferase activity which occurs later in AD. Both proBDNF and mature BDNF levels were positively correlated with cognitive measures such as the Global Cognitive Score and the Mini Mental State Examination score. These results demonstrate that the reduction of both forms of BDNF occurs early in the course of AD and correlates with loss of cognitive function, suggesting that proBDNF and BDNF play a role in synaptic loss and cellular dysfunction underlying cognitive impairment in AD.     

Cortical Bilateral Adaptations in Rats Submitted to Focal Cerebral Ischemia: Emphasis on Glial Metabolism

This study was performed to evaluate the bilateral effects of focal permanent ischemia (FPI) on glial metabolism in the cerebral cortex. Two and 9 days after FPI induction, we analyze [¹⁸F]FDG metabolism by micro-PET, astrocyte morphology and reactivity by immunohistochemistry, cytokines and trophic factors by ELISA, glutamate transporters by RT-PCR, monocarboxylate transporters (MCTs) by western blot, and substrate uptake and oxidation by ex vivo slices model. The FPI was induced surgically by thermocoagulation of the blood in the pial vessels of the motor and sensorimotor cortices in adult (90 days old) male Wistar rats. Neurochemical analyses were performed separately on both ipsilateral and contralateral cortical hemispheres. In both cortical hemispheres, we observed an increase in tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), and glutamate transporter 1 (GLT-1) mRNA levels; lactate oxidation; and glutamate uptake and a decrease in brain-derived neurotrophic factor (BDNF) after 2 days of FPI. Nine days after FPI, we observed an increase in TNF-α levels and a decrease in BDNF, GLT-1, and glutamate aspartate transporter (GLAST) mRNA levels in both hemispheres. Additionally, most of the unilateral alterations were found only in the ipsilateral hemisphere and persisted until 9 days post-FPI. They include diminished in vivo glucose uptake and GLAST expression, followed by increased glial fibrillary acidic protein (GFAP) gray values, astrocyte reactivity, and glutamate oxidation. Astrocytes presented signs of long-lasting reactivity, showing a radial morphology. In the intact hemisphere, there was a decrease in MCT2 levels, which did not persist. Our study shows the bilateralism of glial modifications following FPI, highlighting the role of energy metabolism adaptations on brain recovery post-ischemia.     

Paradoxical Sleep Deprivation Aggravates and Prolongs Incision-Induced Pain Hypersensitivity via BDNF Signaling-Mediated Descending Facilitation in Rats

The mechanisms underlying the pronociceptive effect of paradoxical sleep deprivation (PSD) are not fully established. The modulation of BDNF signaling-mediated descending facilitation from the rostral ventromedial medulla (RVM) of brain stem has been demonstrated in persistent pain models of inflammatory pain, but not in incisional pain model. Recent study has shown that PSD increases the expression of brain-derived neurotrophic factor (BDNF) in the brainstem structure. Therefore, in the current study, we asked whether the BDNF signaling-mediated descending facilitation was involved in the PSD-induced pronociceptive effect on incisional pain and delay the recovery period of postoperative pain in rats. Our results found that a preoperative 24 h PSD significantly aggravated the pain hypersensitivity after incision and prolonged the duration of postoperative pain. The lesions of ipsilateral dorsolateral funiculus partly reversed the PSD-induced pronociceptive effect on incisional pain. Interestingly, the 24 h PSD, but not incision significantly enhanced the levels of BDNF protein expression in the RVM areas of rats. Furthermore, at 1 day or 4 days after incision, intra-RVM microinjection of a BDNF antibody partly reversed the PSD-induced pronociceptive effects in incisional rats, while it did not change the cumulative pain scores and paw withdrawal thresholds in rats receiving only plantar incision. These findings suggest that the preoperative PSD may aggravate and prolong the incision-induced pain hypersensitivity via BDNF signaling-mediated descending facilitation.     

Decreased expression of vesicular GABA transporter, but not vesicular glutamate, acetylcholine and monoamine transporters in rat brain following focal ischemia

In nerve terminals, vesicular transporters pack neurotransmitters into synaptic vesicles, which is an essential prerequisite for transmitter release. To date, three distinct families of vesicular transporters have been identified which are specific for (a) excitatory amino acids (glutamate and aspartate), (b) inhibitory amino acids (GABA and glycine) and (c) acetylcholine and monoamines. The present study evaluated the effect of transient focal cerebral ischemia on the expression of these vesicular transporters in adult rat brain. Ischemia was induced by a 1 h transient middle cerebral artery occlusion (MCAO) in spontaneously hypertensive rats. At various reperfusion periods (3-72 h), mRNA levels of the vesicular transporters were estimated in the contralateral and the ipsilateral cerebral cortex by real-time PCR analysis. Following transient focal ischemia, mRNA expression of the vesicular GABA transporter (VGAT) decreased significantly by 3 h of reperfusion and remained at a significantly lower level than sham until at least 72 h of reperfusion. Western blotting showed a significant decrease in the VGAT immunoreactive protein levels in the ipsilateral cortex of rats subjected to focal ischemia and 24 h reperfusion. Immunohistochemistry demonstrated many VGAT immunopositive puncta in the contralateral cortex, which were significantly decreased in the ipsilateral cortex at 24 h reperfusion. Focal ischemia had no effect on the mRNA levels of the vesicular transporters specific for glutamate/aspartate, acetylcholine and monoamines at either 6 h or 24 h of reperfusion.     

Efficient copackaging and cotransport yields postsynaptic colocalization of neuromodulators associated with synaptic plasticity

Recent data suggest that tissue plasminogen activator (tPA) influences long-term plasticity at hippocampal synapses by converting plasminogen into plasmin, which then generates mature brain-derived neurotrophic factor (mBDNF) from its precursor, proBDNF. Motivated by this hypothesis, we used fluorescent chimeras, expressed in hippocampal neurons, to elucidate (1) mechanisms underlying plasminogen secretion from hippocampal neurons, (2) if tPA, plasminogen, and proBDNF are copackaged and cotransported in hippocampal neurons, especially within dendritic spines, and (3) mechanisms mediating the transport of these neuromodulators to sites of release. We find that plasminogen chimeras traffic through the regulated secretory pathway of hippocampal neurons in dense-core granules (DCGs) and that tPA, plasminogen, and proBDNF chimeras are extensively copackaged in DCGs throughout hippocampal neurons. We also find that 80% of spines that contain DCGs contain chimeras of these neuromodulators in the same DCG. Finally, we demonstrate, for the first time, that neuromodulators undergo cotransport along dendrites in rapidly mobile DCGs, indicating that neuromodulators can be efficiently recruited into active spines. These results support the hypothesis that tPA mediates synaptic activation of BDNF by demonstrating that tPA, plasminogen, and proBDNF colocalize in DCGs in spines, where these neuromodulators can undergo activity-dependent release and then interact and/or mediate changes that influence synaptic efficacy. The results also raise the possibility that frequency-dependent changes in extents of neuromodulator release from DCGs influence the direction of plasticity at hippocampal synapses by altering the relative proportions of two proteins, mBDNF and proBDNF, that exert opposing effects on synaptic efficacy.     

ProBDNF collapses neurite outgrowth of primary neurons by activating RhoA

Background

Neurons extend their dendrites and axons to build functional neural circuits, which are regulated by both positive and negative signals during development. Brain-derived neurotrophic factor (BDNF) is a positive regulator for neurite outgrowth and neuronal survival but the functions of its precursor (proBDNF) are less characterized.

Methodology/Principal Findings

Here we show that proBDNF collapses neurite outgrowth in murine dorsal root ganglion (DRG) neurons and cortical neurons by activating RhoA via the p75 neurotrophin receptor (p75NTR). We demonstrated that the receptor proteins for proBDNF, p75NTR and sortilin, were highly expressed in cultured DRG or cortical neurons. ProBDNF caused a dramatic neurite collapse in a dose-dependent manner and this effect was about 500 fold more potent than myelin-associated glycoprotein. Neutralization of endogenous proBDNF by using antibodies enhanced neurite outgrowth in vitro and in vivo, but this effect was lost in p75NTR^−/− mice. The neurite outgrowth of cortical neurons from p75NTR deficient (p75NTR^−/−) mice was insensitive to proBDNF. There was a time-dependent reduction of length and number of filopodia in response to proBDNF which was accompanied with a polarized RhoA activation in growth cones. Moreover, proBDNF treatment of cortical neurons resulted in a time-dependent activation of RhoA but not Cdc42 and the effect was absent in p75NTR^−/− neurons. Rho kinase (ROCK) and the collapsin response mediator protein-2 (CRMP-2) were also involved in the proBDNF action.

Conclusions

proBDNF has an opposing role in neurite outgrowth to that of mature BDNF. Our observations suggest that proBDNF collapses neurites outgrowth and filopodial growth cones by activating RhoA through the p75NTR signaling pathway.     

Changes in the level of glial fibrillary acidic protein (GFAP) after mild and severe focal cerebral ischemia

In the present study, we examined the temporal and spatial expression profiles of GFAP mRNA and protein in a focal cerebral ischemia model with ischemic injury confined to the cerebral cortex in the right middle cerebral artery (MCA) territory. Northern blot analysis showed a respective 5.5-fold and 7.2-fold increase in the GFAP mRNA in the ischemic right MCA cortex in rats subjected to 30-min (mild) or 60-min (severe) ischemia followed by 72-hr reperfusion. The GFAP mRNA signal remained elevated up to 2-week reperfusion. Interestingly, increased GFAP mRNA signal was clearly demonstrated for the first time in the left MCA cortex. A significant 1.5-fold and 5-fold increase was observed after 72-hr reperfusion following mild and severe ischemia, respectively. However, unlike the ischemic right MCA cortex, this induction was transient in the non-ischemic left MCA counterpart. In situ hybridization studies further revealed characteristic spatial induction profile following mild vs. severe ischemia. In mild ischemia, following 24-hr reperfusion, increase in GFAP mRNA was observed mainly within the ischemic right MCA cortex. Following 72-hr reperfusion, GFAP mRNA signal was observed in virtually the entire ischemic cortex, particularly the amygdala region, then gradually reduced and restricted to right MCA territory and subcortical thalamic nucleus following 2-week reperfusion. On the other hand, in severe ischemia, following 24-hr reperfusion increased GFAP mRNA signal was observed in area surrounding right MCA territory (infarct region) and outer cortical layers within the right MCA territory. Following 72-hr reperfusion, no signal was detected within right MCA cortex; however, increased GFAP signal was detected throughout the remaining ipsilateral cortex and subcortical region, as well as the contralateral cerebral cortex. GFAP mRNA signals then gradually reduced its intensity and was restricted to area surrounding necrosis and ipsilateral thalamic nucleus following 2-week reperfusion. GFAP-like immunoreactivity was also detected in area expressing GFAP mRNA. It is very likely that de novo synthesis was responsible for this increase. In summary, increased GFAP signal was noted in both ipsilateral and contralateral cerebral following mild and severe ischemia. Although the temporal induction profile for mild vs. severe ischemia was similar, the spatial induction profile was different. The mechanism leading to this differential induction and their physiological and functional significance are not clear at present. It is very likely that some local factors may involve, nevertheless, the detailed mechanisms remain to be fully explored.     

The HDAC inhibitor, sodium butyrate, stimulates neurogenesis in the ischemic brain

In the healthy adult brain, neurogenesis normally occurs in the subventricular zone (SVZ) and hippocampal dentate gyrus (DG). Cerebral ischemia enhances neurogenesis in neurogenic and non-neurogenic regions of the ischemic brain of adult rodents. This study demonstrated that post-insult treatment with a histone deacetylase inhibitor, sodium butyrate (SB), stimulated the incorporation of bromo-2'-deoxyuridine (BrdU) in the SVZ, DG, striatum, and frontal cortex in the ischemic brain of rats subjected to permanent cerebral ischemia. SB treatment also increased the number of cells expressing polysialic acid–neural cell adhesion molecule, nestin, glial fibrillary acidic protein, phospho-cAMP response element-binding protein (CREB), and brain-derived neurotrophic factor (BDNF) in various brain regions after cerebral ischemia. Furthermore, extensive co-localization of BrdU and polysialic acid–neural cell adhesion molecule was observed in multiple regions after ischemia, and SB treatment up-regulated protein levels of BDNF, phospho-CREB, and glial fibrillary acidic protein. Intraventricular injection of K252a, a tyrosine kinase B receptor antagonist, markedly reduced SB-induced cell proliferation detected by BrdU and Ki67 in the ipsilateral SVZ, DG, and other brain regions, blocked SB-induced nestin expression and CREB activation, and attenuated the long-lasting behavioral benefits of SB. Together, these results suggest that histone deacetylase inhibitor-induced cell proliferation, migration and differentiation require BDNF–tyrosine kinase B signaling and may contribute to long-term beneficial effects of SB after ischemic injury.     

Galectin-1 Enhances Astrocytic BDNF Production and Improves Functional Outcome in Rats Following Ischemia

Galectin-1, an endogenous mammalian lectin, has been implicated in a variety of CNS disorders. However, its role in cerebral ischemia is still elusive. In the present study, we investigated the effect of recombinant galectin-1 on production of astrocytic brain-derived neurotrophic factor (BDNF) and functional recovery following ischemia. Endogenous galectin-1 was found to be markedly upregulated, paralleled with increased astrocytic BDNF production under ischemic conditions both in vitro and in vivo. Administration of galectin-1significantly enhanced the expression and secretion of astrocytic BDNF in dose dependent manner. Moreover, rats subjected to photochemical cerebral ischemia showed reduced neuronal apoptosis in ischemic boundary zone and improved functional recovery after brain infusion of galectin-1 (1 μg/days, 7 days). These results suggest that induction of BDNF in astrocytes by galectin-1 may be a promising intervention to attenuate brain damage after stroke.