Vascular endothelial growth factor increases the function of calcium-impermeable AMPA receptor GluA2 subunit in astrocytes via activation of protein kinase C signaling pathway.

Astrocytic calcium signaling plays pivotal roles in the maintenance of neural functions and neurovascular coupling in the brain. Vascular endothelial growth factor (VEGF), an original biological substance of vessels, regulates the movement of calcium and potassium ions across neuronal membrane. In this study, we investigated whether and how VEGF regulates glutamate-induced calcium influx in astrocytes. We used cultured astrocytes combined with living cell imaging to detect the calcium influx induced by glutamate. We found that VEGF quickly inhibited the glutamate/hypoxia-induced calcium influx, which was blocked by an AMPA receptor antagonist CNQX, but not D-AP5 or UBP310, NMDA and kainate receptor antagonist, respectively. VEGF increased phosphorylation of PKCα and AMPA receptor subunit GluA2 in astrocytes, and these effects were diminished by SU1498 or calphostin C, a PKC inhibitor. With the pHluorin assay, we observed that VEGF significantly increased membrane insertion and expression of GluA2, but not GluA1, in astrocytes. Moreover, siRNA-produced knockdown of GluA2 expression in astrocytes reversed the inhibitory effect of VEGF on glutamate-induced calcium influx. Together, our results suggest that VEGF reduces glutamate-induced calcium influx in astrocytes via enhancing PKCα-mediated GluA2 phosphorylation, which in turn promotes the membrane insertion and expression of GluA2 and causes AMPA receptors to switch from calcium-permeable to calcium-impermeable receptors, thereby inhibiting astrocytic calcium influx. The present study reveals that excitatory neurotransmitter glutamate-mediated astrocytic calcium influx can be regulated by vascular biological factor via activation of AMPA receptor GluA2 subunit and uncovers a novel coupling mechanism between astrocytes and endothelial cells within the neurovascular unit.

Nigrostriatal Dopamine Acting on Globus Pallidus Regulates Sleep.

Lesions of the globus pallidus externa (GPe) produce a profound sleep loss (∼45%) in rats, suggesting that GPe neurons promote sleep. As GPe neuronal activity is enhanced by dopamine (DA) from the substantia nigra pars compacta (SNc), we hypothesized that SNc DA via the GPe promotes sleep. To test this hypothesis, we selectively destroyed the DA afferents to the caudoputamen (CPu) using 6-hydroxydopamine and examined changes in sleep-wake profiles in rats. Rats with 80-90% loss of SNc neurons displayed a significant 33.7% increase in wakefulness (or sleep reduction). This increase significantly correlated with the extent of SNc DA neuron loss. Furthermore, these animals exhibited sleep-wake fragmentation and reduced diurnal variability of sleep. We then optogenetic-stimulated SNc DA terminals in the CPu and found that 20-Hz stimulation from 9 to 10 PM increased total sleep by 69% with high electroencephalograph (EEG) delta power. We finally directly optogenetic-stimulated GPe neurons and found that 20-Hz stimulation of the GPe from 9 to 10 PM increased total sleep by 66% and significantly increased EEG delta power. These findings elucidate a novel circuit for DA control of sleep and the mechanisms of abnormal sleep in BG disorders such as Parkinson's disease and Huntington's disease.

Identification of a direct GABAergic pallidocortical pathway in rodents.

Interaction between the basal ganglia and the cortex plays a critical role in a range of behaviors. Output from the basal ganglia to the cortex is thought to be relayed through the thalamus, but an intriguing alternative is that the basal ganglia may directly project to and communicate with the cortex. We explored an efferent projection from the globus pallidus externa (GPe), a key hub in the basal ganglia system, to the cortex of rats and mice. Anterograde and retrograde tracing revealed projections to the frontal premotor cortex, especially the deep projecting layers, originating from GPe neurons that receive axonal inputs from the dorsal striatum. Cre-dependent anterograde tracing in Vgat-ires-cre mice confirmed that the pallidocortical projection is GABAergic, and in vitro optogenetic stimulation in the cortex of these projections produced a fast inhibitory postsynaptic current in targeted cells that was abolished by bicuculline. The pallidocortical projections targeted GABAergic interneurons and, to a lesser extent, pyramidal neurons. This GABAergic pallidocortical pathway directly links the basal ganglia and cortex, and may play a key role in behavior and cognition in normal and disease states.

Up-regulation of hexokinase1 in the right ventricle of monocrotaline induced pulmonary hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a proliferative arteriopathy associated with a glycolytic shift during heart metabolism. An increase in glycolytic metabolism can be detected in the right ventricle during PAH. Expression levels of glycolysis genes in the right ventricle during glycolysis that occur in monocrotaline (MCT)-induced pulmonary hypertension (PH) remain unknown. METHODS: PH was induced by a single subcutaneous injection of MCT (50 mg/kg) into rats, eventually causing right heart failure. Concurrently, a control group was injected with normal saline. The MCT-PH rats were randomly divided into three groups according to MCT treatment: MCT-2 week, 3 week, and 4 week groups (MCT-2w, 3w, 4w). At the end of the study, hemodynamics and right ventricular hypertrophy were compared among experimental groups. Expression of key glycolytic candidate genes was screened in the right ventricle. RESULTS: We observed an increase in mean pulmonary arterial pressure, right ventricular systolic pressure and right ventricular hypertrophy index three weeks following MCT injection. Alterations in the morphology and structure of right ventricular myocardial cells, as well as the pulmonary vasculature were observed. Expression of hexokinase 1 (HK1) mRNA began to increase in the right ventricle of the MCT-3w group and MCT-4w group, while the expression of lactate dehydrogenase A (LDHA) was elevated in the right ventricle of the MCT-4w group. Hexokinase 2(HK2), pyruvate dehydrogenase complex α1 (PDHα1), and LDHA mRNA expression showed no changes in the right ventricle. HK1 mRNA expression was further confirmed by HK1 protein expression and immunohistochemical analyses. All findings underlie the glycolytic phenotype in the right ventricle. CONCLUSIONS: There was an increase in the protein and mRNA expression of hexokinase-1 (HK1) three and four weeks after the injection of monocrotaline in the right ventricle, intervention of HK1 may be amenable to therapeutic intervention.

Alleviating sleep disturbances and modulating neuronal activity after ischemia: Evidence for the benefits of zolpidem in stroke recovery.

AIMS: Sleep disorders are prevalent among stroke survivors and impede stroke recovery, yet they are still insufficiently considered in the management of stroke patients, and the mechanisms by which they occur remain unclear. There is evidence that boosting phasic GABA signaling with zolpidem during the repair phase improves stroke recovery by enhancing neural plasticity; however, as a non-benzodiazepine hypnotic, the effects of zolpidem on post-stroke sleep disorders remain unclear. METHOD: Transient ischemic stroke in male rats was induced with a 30-minute middle cerebral artery occlusion. Zolpidem or vehicle was intraperitoneally delivered once daily from 2 to 7 days after the stroke, and the electroencephalogram and electromyogram were recorded simultaneously. At 24 h after ischemia, c-Fos immunostaining was used to assess the effect of transient ischemic stroke and acute zolpidem treatment on neuronal activity. RESULTS: In addition to the effects on reducing brain damage and mitigating behavioral deficits, repeated zolpidem treatment during the subacute phase of stroke quickly ameliorated circadian rhythm disruption, alleviated sleep fragmentation, and increased sleep depth in ischemic rats. Immunohistochemical staining showed that in contrast to robust activation in para-infarct and some remote areas by 24 h after the onset of focal ischemia, the activity of the ipsilateral suprachiasmatic nucleus, the biological rhythm center, was strongly suppressed. A single dose of zolpidem significantly upregulated c-Fos expression in the ipsilateral suprachiasmatic nucleus to levels comparable to the contralateral side. CONCLUSION: Stroke leads to suprachiasmatic nucleus dysfunction. Zolpidem restores suprachiasmatic nucleus activity and effectively alleviates post-stroke sleep disturbances, indicating its potential to promote stroke recovery.

Pontine control of rapid eye movement sleep and fear memory.

AIMS: We often experience dreams of strong irrational and negative emotional contents with postural muscle paralysis during rapid eye movement (REM) sleep, but how REM sleep is generated and its function remain unclear. In this study, we investigate whether the dorsal pontine sub-laterodorsal tegmental nucleus (SLD) is necessary and sufficient for REM sleep and whether REM sleep elimination alters fear memory. METHODS: To investigate whether activation of SLD neurons is sufficient for REM sleep induction, we expressed channelrhodopsin-2 (ChR2) in SLD neurons by bilaterally injecting AAV1-hSyn-ChR2-YFP in rats. We next selectively ablated either glutamatergic or GABAergic neurons from the SLD in mice in order to identify the neuronal subset crucial for REM sleep. We finally  investigated the role of REM sleep in consolidation of fear memory using rat model with complete SLD lesions. RESULTS: We demonstrate the sufficiency of the SLD for REM sleep by showing that photo-activation of ChR2 transfected SLD neurons selectively promotes transitions from non-REM (NREM) sleep to REM sleep in rats. Diphtheria toxin-A (DTA) induced lesions of the SLD in rats or specific deletion of SLD glutamatergic neurons but not GABAergic neurons in mice completely abolish REM sleep, demonstrating the necessity of SLD glutamatergic neurons for REM sleep. We then show that REM sleep elimination by SLD lesions in rats significantly enhances contextual and cued fear memory consolidation by 2.5 and 1.0 folds, respectively, for at least 9 months. Conversely, fear conditioning and fear memory trigger doubled amounts of REM sleep in the following night, and chemo-activation of SLD neurons projecting to the medial septum (MS) selectively enhances hippocampal theta activity in REM sleep; this stimulation immediately after fear acquisition reduces contextual and cued fear memory consolidation by 60% and 30%, respectively. CONCLUSION: SLD glutamatergic neurons generate REM sleep and REM sleep and SLD via the hippocampus particularly down-regulate contextual fear memory.

Melatonin supplementation in the subacute phase after ischemia alleviates postischemic sleep disturbances in rats.

BACKGROUND: Sleep disorders are highly prevalent among stroke survivors and impede stroke recovery. It is well established that melatonin has neuroprotective effects in animal models of ischemic stroke. However, as a modulator of endogenous physiological circadian rhythms, the effects of melatonin on poststroke sleep disorders remain unclear. In the present study, we investigated how melatonin delivered intraperitoneally once daily in the subacute phase after stroke onset, influencing neuronal survival, motor recovery, and sleep-wake profiles in rats. METHODS: Transient ischemic stroke in male Sprague-Dawley rats was induced with 30 min occlusion of the middle cerebral artery. Melatonin or vehicle was delivered intraperitoneally once daily in the subacute phase, from 2 to 7 days after stroke. Electroencephalogram and electromyogram recordings were obtained simultaneously. RESULTS: Compared to the effects observed in the vehicle-treated ischemic group, after 6 daily consecutive treatment of melatonin at 10 mg/kg starting at ischemic/reperfusion day 2, the infarct volume was significantly decreased (from 39.6 to 26.2%), and the degeneration of axons in the ipsilateral striatum and the contralateral corpus callosum were significantly alleviated. Sensorimotor performances were obviously improved as evidenced by significant increases in the latency to falling off the wire and in the use of the impaired forelimb. In addition to those predictable results of reducing brain tissue damage and mitigating behavioral deficits, repeated melatonin treatment during the subacute phase of stroke also alleviated sleep fragmentation through reducing sleep-wake stage transitions and stage bouts, together with increasing stage durations. Furthermore, daily administration of melatonin at 9 a.m. significantly increased the nonrapid eye movement sleep delta power during both the light and dark periods and decreased the degree of reduction of the circadian index. CONCLUSIONS: Melatonin promptly reversed ischemia-induced sleep disturbances. The neuroprotective effects of melatonin on ischemic injury may be partially associated with its role in sleep modulation.

Basal ganglia control of sleep-wake behavior and cortical activation.

The basal ganglia (BG) are involved in numerous neurobiological processes that operate on the basis of wakefulness, including motor function, learning, emotion and addictive behaviors. We hypothesized that the BG might play an important role in the regulation of wakefulness. To test this prediction, we made cell body-specific lesions in the striatum and globus pallidus (GP) using ibotenic acid. We found that rats with striatal (caudoputamen) lesions exhibited a 14.95% reduction in wakefulness and robust fragmentation of sleep-wake behavior, i.e. an increased number of state transitions and loss of ultra-long wake bouts (> 120 min). These lesions also resulted in a reduction in the diurnal variation of sleep-wakefulness. On the other hand, lesions of the accumbens core resulted in a 26.72% increase in wakefulness and a reduction in non-rapid eye movement (NREM) sleep bout duration. In addition, rats with accumbens core lesions exhibited excessive digging and scratching. GP lesions also produced a robust increase in wakefulness (45.52%), and frequent sleep-wake transitions and a concomitant decrease in NREM sleep bout duration. Lesions of the subthalamic nucleus or the substantia nigra reticular nucleus produced only minor changes in the amount of sleep-wakefulness and did not alter sleep architecture. Finally, power spectral analysis revealed that lesions of the striatum, accumbens and GP slowed down the cortical electroencephalogram. Collectively, our results suggest that the BG, via a cortico-striato-pallidal loop, are important neural circuitry regulating sleep-wake behaviors and cortical activation.

Nigrostriatal and mesolimbic control of sleep-wake behavior in rat.

The midbrain dopamine system via the dorsal and ventral striatum regulates a wide range of behaviors. To dissect the role of dopaminergic projections to the dorsal striatum (nigrostriatal projection) and ventral striatum (mesolimbic projection) in sleep-wake behavior, we selectively chemogenetically stimulated nigrostriatal or mesolimbic projections and examined the resulting effects on sleep in rats. Stimulation of nigrostriatal pathways increased sleep and EEG delta power, while stimulation of mesolimbic pathways decreased sleep and reduced cortical EEG power. These results indicate that midbrain dopamine signaling in the dorsal or ventral striatum promotes sleep or wake, respectively.

Altered Behaviors and Impaired Synaptic Function in a Novel Rat Model With a Complete Shank3 Deletion.

Mutations within the Shank3 gene, which encodes a key postsynaptic density (PSD) protein at glutamatergic synapses, contribute to the genetic etiology of defined autism spectrum disorders (ASDs), including Phelan-McDermid syndrome (PMS) and intellectual disabilities (ID). Although there are a series of genetic mouse models to study Shank3 gene in ASDs, there are few rat models with species-specific advantages. In this study, we established and characterized a novel rat model with a deletion spanning exons 11-21 of Shank3, leading to a complete loss of the major SHANK3 isoforms. Synaptic function and plasticity of Shank3-deficient rats were impaired detected by biochemical and electrophysiological analyses. Shank3-depleted rats showed impaired social memory but not impaired social interaction behaviors. In addition, impaired learning and memory, increased anxiety-like behavior, increased mechanical pain threshold and decreased thermal sensation were observed in Shank3-deficient rats. It is worth to note that Shank3-deficient rats had nearly normal levels of the endogenous social neurohormones oxytocin (OXT) and arginine-vasopressin (AVP). This new rat model will help to further investigate the etiology and assess potential therapeutic target and strategy for Shank3-related neurodevelopmental disorders.

Stimulation of the Pontine Parabrachial Nucleus Promotes Wakefulness via Extra-thalamic Forebrain Circuit Nodes.

Human and animal studies have identified an especially critical role for the brainstem parabrachial (PB) complex in regulating electrocortical (electroencephalogram [EEG]) and behavioral arousal: lesions of the PB complex produce a monotonous high-voltage, slow-wave EEG and eliminate spontaneous behaviors. We report here that targeted chemogenetic activation of the PB complex produces sustained EEG and behavioral arousal in the rat. We further establish, using viral-mediated retrograde activation, that PB projections to the preoptic-basal forebrain and lateral hypothalamus, but not to the thalamus, mediate PB-driven wakefulness. We exploited this novel and noninvasive model of induced wakefulness to explore the EEG and metabolic consequences of extended wakefulness. Repeated (daily) chemogenetic activation of the PB was highly effective in extending wakefulness over 4 days, although subsequent PB activation produced progressively lesser wake amounts. Curiously, no EEG or behavioral sleep rebound was observed, even after 4 days of induced wakefulness. Following the last of the four daily induced wake bouts, we examined the brains and observed a chimeric pattern of c-Fos expression, with c-Fos expressed in subsets of both arousal- and sleep-promoting nuclei. From a metabolic standpoint, induced extended wakefulness significantly reduced body weight and leptin but was without significant effect on cholesterol, triglyceride, or insulin levels, suggesting that high sleep pressure or sleep debt per se does not, as previously implicated, result in a deleterious metabolic phenotype.

Ventromedial prefrontal cortex regulates depressive-like behavior and rapid eye movement sleep in the rat.

Major depressive disorder (MDD) is a debilitating disease with symptoms like persistent depressed mood and sleep disturbances. The prefrontal cortex (PFC) has been implicated as an important structure in the neural circuitry of MDD, with pronounced abnormalities in blood flow and metabolic activity in PFC subregions, including the subgenual cingulate cortex (sgACC, or Brodmann area 25). In addition, deep brain stimulation in the sgACC has recently been shown to alleviate treatment-resistant depression. Depressed patients also show characteristic changes in sleep: insomnia, increased rapid-eye-movement (REM) sleep and shortened REM sleep latency. We hypothesized that sleep changes and depressive behavior may be a consequence of the abnormal PFC activity in MDD. The rat ventromedial PFC (vmPFC, prelimbic and infralimbic cortices) is considered to be the homolog of the human sgACC, so we examined the effect of excitotic lesions in the vmPFC on sleep-wake and depressive behavior. We also made lesions in the adjacent dorsal region (dmPFC) to compare the effect of this similar but distinct mPFC region. We found that both dmPFC and vmPFC lesions led to increased REM sleep, but only vmPFC-lesioned animals displayed increased sleep fragmentation, shortened REM latency and increased immobility in the forced swim test. Anatomic tracing suggests that the mPFC projects to the pontine REM-off neurons that interact with REM-on neurons in the dorsal pons. These results support our hypothesis that neuronal loss in the rat vmPFC resembles several characteristics of MDD and may be a critical area for modulating both mood and sleep.

Neuronal activity (c-Fos) delineating interactions of the cerebral cortex and basal ganglia.

The cerebral cortex and basal ganglia (BG) form a neural circuit that is disrupted in disorders such as Parkinson's disease. We found that neuronal activity (c-Fos) in the BG followed cortical activity, i.e., high in arousal state and low in sleep state. To determine if cortical activity is necessary for BG activity, we administered atropine to rats to induce a dissociative state resulting in slow-wave electroencephalography but hyperactive motor behaviors. Atropine blocked c-Fos expression in the cortex and BG, despite high c-Fos expression in the sub-cortical arousal neuronal groups and thalamus, indicating that cortical activity is required for BG activation. To identify which glutamate receptors in the BG that mediate cortical inputs, we injected ketamine [N-methyl-d-aspartate (NMDA) receptor antagonist] and 6-cyano-nitroquinoxaline-2, 3-dione (CNQX, a non-NMDA receptor antagonist). Systemic ketamine and CNQX administration revealed that NMDA receptors mediated subthalamic nucleus (STN) input to internal globus pallidus (GPi) and substantia nigra pars reticulata (SNr), while non-NMDA receptor mediated cortical input to the STN. Both types of glutamate receptors were involved in mediating cortical input to the striatum. Dorsal striatal (caudoputamen, CPu) dopamine depletion by 6-hydroxydopamine resulted in reduced activity of the CPu, globus pallidus externa (GPe), and STN but increased activity of the GPi, SNr, and putative layer V neurons in the motor cortex. Our results reveal that the cortical activity is necessary for BG activity and clarifies the pathways and properties of the BG-cortical network and their putative role in the pathophysiology of BG disorders.

GABA transporter-1 inhibitor NO-711 alters the EEG power spectra and enhances non-rapid eye movement sleep during the active phase in mice.

GABA transporter subtype 1 (GAT1) constructs high affinity reuptake sites for GABA in the CNS and regulates GABAergic transmission. Compounds that inhibit GAT1 are targets often used for the treatment of epilepsy; however sedation has been reported as a side effect of these agents, indicating potential sedative and/or hypnotic uses for these compounds. In the current study, we observed the sleep behaviors of mice treated with NO-711, a selective GAT1 inhibitor, in order to elucidate the role of GAT1 in sleep-wake regulation during the active phase. The data revealed that NO-711 at a high dose of 10 mg/kg caused a marked enhancement of EEG activity in the frequency ranges of 3-25 Hz during wakefulness as well as rapid eye movement (REM) sleep. During the non-REM (NREM) sleep, NO-711 (10 mg/kg) elevated EEG activity in the frequency ranges of 1.5-6.75 Hz. Similar changes were found in mice treated with a low dose of 3 mg/kg. NO-711 administered i.p. at a dose of 1, 3 or 10 mg/kg significantly shortened the sleep latency of NREM sleep, increased the amount of NREM sleep and the number of NREM sleep episodes. NO-711 did not affect the sleep latency and the amount of REM sleep. NO-711 dose-dependently increased c-Fos expression in sleep-promoting nucleus of the ventrolateral preoptic area and median preoptic area. However, c-Fos expression was decreased in the wake-promoting nuclei, tuberomammillary nucleus and lateral hypothalamus. These results indicate that NO-711 can increase NREM sleep in mice.

The role of nucleus accumbens core/shell in sleep-wake regulation and their involvement in modafinil-induced arousal.

BACKGROUND: We have previously shown that modafinil promotes wakefulness via dopamine receptor D(1) and D(2) receptors; however, the locus where dopamine acts has not been identified. We proposed that the nucleus accumbens (NAc) that receives the ventral tegmental area dopamine inputs play an important role not only in reward and addiction but also in sleep-wake cycle and in mediating modafinil-induced arousal. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we further explored the role of NAc in sleep-wake cycle and sleep homeostasis by ablating the NAc core and shell, respectively, and examined arousal response following modafinil administration. We found that discrete NAc core and shell lesions produced 26.5% and 17.4% increase in total wakefulness per day, respectively, with sleep fragmentation and a reduced sleep rebound after a 6-hr sleep deprivation compared to control. Finally, NAc core but not shell lesions eliminated arousal effects of modafinil. CONCLUSIONS/SIGNIFICANCE: These results indicate that the NAc regulates sleep-wake behavior and mediates arousal effects of the midbrain dopamine system and stimulant modafinil.

Role of Basal Ganglia in sleep-wake regulation: neural circuitry and clinical significance.

Researchers over the last decade have made substantial progress toward understanding the roles of dopamine and the basal ganglia (BG) in the control of sleep-wake behavior. In this review, we outline recent advancements regarding dopaminergic modulation of sleep through the BG and extra-BG sites. Our main hypothesis is that dopamine promotes sleep by its action on the D2 receptors in the BG and promotes wakefulness by its action on D1 and D2 receptors in the extra-BG sites. This hypothesis implicates dopamine depletion in the BG (such as in Parkinson's disease) in causing frequent nighttime arousal and overall insomnia. Furthermore, the arousal effects of psychostimulants (methamphetamine, cocaine, and modafinil) may be linked to the ventral periaquductal gray (vPAG) dopaminergic circuitry targeting the extra-BG sleep-wake network.

D(1)/D(2) receptor-targeting L-stepholidine, an active ingredient of the Chinese herb Stephonia, induces non-rapid eye movement sleep in mice.

L-stepholidine, an active ingredient of the Chinese herb Stephonia, is the first compound known to have mixed dopamine D(1) receptor agonist/D(2) antagonist properties and to be a potential treatment medication for schizophrenia. In schizophrenic patients insomnia is a common symptom and could be partly related to the presumed over-activity of the dopaminergic system. To elucidate whether stepholidine modulates sleep behaviors, we observed its effects on sleep-wake profiles in mice. The results showed that stepholidine administered i.p. at doses of 20, 40 or 80 mg/kg significantly shortened the sleep latency to non-rapid eye movement (non-REM, NREM) sleep, increased the amount of NREM sleep, and prolonged the duration of NREM sleep episodes, with a concomitant reduction in the amount of wakefulness. Stepholidine at doses of 40 and 80 mg/kg increased the number of state transitions from wakefulness to NREM sleep and subsequently from NREM sleep to wakefulness. However, stepholidine had no effect on either the amount of REM sleep or electroencephalogram power density of either NREM or REM sleep. Immunohistochemistry study showed that stepholidine dose-dependently increased c-Fos expression in neurons of the ventrolateral preoptic area, a sleep center in the anterior hypothalamus, as compared with the vehicle control. These results indicate that stepholidine initiates and maintains NREM sleep with activation of the sleep center in mice, suggesting its potential application for the treatment of insomnia.

VEGF enhance cortical newborn neurons and their neurite development in adult rat brain after cerebral ischemia.

To study the effect of VEGF overexpression on development of cortical newborn neurons in the brains after stroke, we injected human VEGF(165)-expressive plasmids (phVEGF) into the lateral ventricle of rat brains with a transient middle cerebral artery occlusion (MCAO). An injection of phVEGF significantly promoted angiogenesis (BrdU(+)-von Willebrand's factor(+)) and reduced infarct volume in the rat brain after MCAO. Single labeling of 5'-bromodeoxyuridine (BrdU) and double staining of BrdU with lineage-specific neuronal markers were used to indicate the proliferated cells and maturation of newborn neurons in the brain section of rats at 2, 4, and 8 weeks after MCAO. The results showed that BrdU positive (BrdU(+)) cells existed in ipsilateral frontal cortex within 8 weeks after MCAO and reached the maximum at 2 weeks of reperfusion. The phVEGF treatment significantly increased BrdU(+) cells compared with the control plasmid (pEGFP) injection. Cortical neurogenesis was indicated by the presence of newborn immature (BrdU(+)-Tuj1(+)), newborn mature (BrdU(+)-MAP-2(+)), and newborn GABAergic (BrdU(+)-GAD67(+)) neurons. All these neurons declined within 8 weeks after MCAO in the controls. Injection of phVEGF significantly increased BrdU(+)-Tuj1(+) neurons at 2 weeks, and BrdU(+)-MAP-2(+) neurons and BrdU(+)-GAD67(+) neurons at 4 and 8 weeks, respectively after MCAO. Moreover, phVEGF treatment significantly increased neurite length and branch numbers of BrdU(+)-MAP-2(+) newborn neurons compared with pEGFP treatment. These results demonstrate that VEGF enhances maturation of stroke-induced cortical neurogenesis and dendritic formation of newborn neurons in adult mammalian brains.

VEGF overexpression enhances striatal neurogenesis in brain of adult rat after a transient middle cerebral artery occlusion.

To elucidate whether vascular endothelial growth factor (VEGF) improves stroke-induced striatal neurogenesis, we intraventricularly injected human VEGF(165)-expressive plasmid (phVEGF) mixed with liposome into adult rats after a transient middle cerebral artery occlusion (MCAO). The results showed that EGFP, a reporter protein, positive cells appeared at 2 hr, further enhanced at 4 hr, reached the maximum at 3 days and still remained at 14 days after a single injection. Treatment with phVEGF increased angiogenesis, as indicated by double staining of vWF, a marker of endothelial cells, and 5'-bromodeoxyuridine (BrdU), a marker of cell proliferation. The phVEGF treatment dose-dependently reduced infarct volume of brain at 2 weeks after MCAO. The neuroprotection by VEGF could be obtained when the plasmid was injected within 2 hr after stroke. Moreover, VEGF overexpression significantly increased cell proliferation in the ipsilateral SVZ and the numbers of BrdU(+)-CRMP-4(+) and BrdU(+)-Tuj1(+), two markers of immature newborn neurons, and BrdU(+)-MAP-2(+), a marker of mature newborn neurons, cells in the ipsilateral striatum to MCAO. Present results show that VEGF plasmid treatment after stroke can significantly reduce infarct volume and enhance striatal neurogenesis in adult rat brain. This suggests that VEGF overexpression acquires significant functions of neuronal protection and repair in the injured brain, which provides a possibility to develop a novel therapeutic strategy for the patients with stroke.

Enhancement of ischemia-induced tyrosine phosphorylation of Kv1.2 by vascular endothelial growth factor via activation of phosphatidylinositol 3-kinase.

Our studies observed that, consistent with the literature, ischemic/hypoxic insults increased the expression of voltage-gated potassium channel (Kv) 1.2 potassium channel as well as elevating the endogenous level of vascular endothelial growth factor (VEGF) in neurons of adult rat brain following middle cerebral artery occlusion and in SH-SY5Y cells after hypoxia and glucose deprivation. Concomitantly, we also observed that ischemic injury increased the tyrosine phosphorylation of Kv 1.2 in in vivo and in vitro; the introduction of exogenous VEGF could attenuate cell death in in vitro models. Furthermore, we found that the protective effect of VEGF is mediated through its up-regulative actions on the tyrosine phosphorylation of Kv 1.2, which in turn has a direct influence on cell viability after ischemic insult. In substantiation of this result, we used anti-sense methodology to suppress the expression of endogenous VEGF, which significantly inhibited the tyrosine phosphorylation of Kv 1.2 and increased cell death elicited by ischemic/hypoxic injury. Finally, the enhancement of the tyrosine phosphorylation of the channel by VEGF in neuronal cells was significantly attenuated in the presence of wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-K), or genestin, an inhibitor of tyrosine kinase, thus suggesting that the phosphorylation of Kv 1.2 induced by VEGF is mechanistically linked to the PI3-K pathway.