David A. Greenberg

Vascular endothelial growth factor (VEGF) stimulates neurogenesis in vitro and in vivo

Vascular endothelial growth factor (VEGF) is an angiogenic protein with neurotrophic and neuroprotective effects. Because VEGF promotes the proliferation of vascular endothelial cells, we examined the possibility that it also stimulates the proliferation of neuronal precursors in murine cerebral cortical cultures and in adult rat brain in vivo. VEGF (>10 ng/ml) stimulated 5-bromo-2′-deoxyuridine (BrdUrd) incorporation into cells that expressed immature neuronal marker proteins and increased cell number in cultures by 20–30%. Cultured cells labeled by BrdUrd expressed VEGFR2/Flk-1, but not VEGFR1/Flt-1 receptors, and the effect of VEGF was blocked by the VEGFR2/Flk-1 receptor tyrosine kinase inhibitor SU1498. Intracerebroventricular administration of VEGF into rat brain increased BrdUrd labeling of cells in the subventricular zone (SVZ) and the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG), where VEGFR2/Flk-1 was colocalized with the immature neuronal marker, doublecortin (Dcx). The increase in BrdUrd labeling after the administration of VEGF was caused by an increase in cell proliferation, rather than a decrease in cell death, because VEGF did not reduce caspase-3 cleavage in SVZ or SGZ. Cells labeled with BrdUrd after VEGF treatment in vivo include immature and mature neurons, astroglia, and endothelial cells. These findings implicate the angiogenesis factor VEGF in neurogenesis as well.

Neurogenesis in dentate subgranular zone and rostral subventricular zone after focal cerebral ischemia in the rat

Because neurogenesis persists in the adult mammalian brain and can be regulated by physiological and pathological events, we investigated its possible involvement in the brains response to focal cerebral ischemia. Ischemia was induced by occlusion of the middle cerebral artery in the rat for 90 min, and proliferating cells were labeled with 5-bromo-2′-deoxyuridine-5′-monophosphate (BrdUrd) over 2-day periods before sacrificing animals 1, 2 or 3 weeks after ischemia. Ischemia increased the incorporation of BrdUrd into cells in two neuroproliferative regions—the subgranular zone of the dentate gyrus and the rostral subventricular zone. Both effects were bilateral, but that in the subgranular zone was more prominent on the ischemic side. Cells labeled with BrdUrd coexpressed the immature neuronal markers doublecortin and proliferating cell nuclear antigen but did not express the more mature cell markers NeuN and Hu, suggesting that they were nascent neurons. These results support a role for ischemia-induced neurogenesis in what may be adaptive processes that contribute to recovery after stroke.

Evidence for stroke-induced neurogenesis in the human brain

Experimental stroke in rodents stimulates neurogenesis and migration of newborn neurons from their sites of origin into ischemic brain regions. We report that in patients with stroke, cells that express markers associated with newborn neurons are present in the ischemic penumbra surrounding cerebral cortical infarcts, where these cells are preferentially localized in the vicinity of blood vessels. These findings suggest that stroke-induced compensatory neurogenesis may occur in the human brain, where it could contribute to postischemic recovery and represent a target for stroke therapy.

Neuroglobin protects the brain from experimental stroke in vivo.

Neuroglobin (Ngb) is an O2-binding protein localized to cerebral neurons of vertebrates, including humans. Its physiological role is unknown but, like hemoglobin, myoglobin, and cytoglobin/histoglobin, it may transport O2, detoxify reactive oxygen species, or serve as a hypoxia sensor. We reported recently that hypoxia stimulates transcriptional activation of Ngb in cultured cortical neurons and that antisense inhibition of Ngb expression increases hypoxic neuronal injury, whereas overexpression of Ngb confers resistance to hypoxia. These findings are consistent with a role for Ngb in promoting neuronal survival after hypoxic insults in vitro. Here we report that in rats, intracerebroventricular administration of an Ngb antisense, but not sense, oligodeoxynucleotide increases infarct volume and worsens functional neurological outcome, whereas intracerebral administration of a Ngb-expressing adeno-associated virus vector reduces infarct size and improves functional outcome, after focal cerebral ischemia induced by occlusion of the middle cerebral artery. We conclude that Ngb acts as an endogenous neuroprotective factor in focal cerebral ischemia and may therefore represent a target for the development of new treatments for stroke.

Neuroglobin-overexpressing transgenic mice are resistant to cerebral and myocardial ischemia.

Neuroglobin (Ngb), a protein related to myoglobin and hemoglobin but expressed predominantly in the brain, is induced by neuronal hypoxia and cerebral ischemia and protects against hypoxic or ischemic neuronal injury. We engineered transgenic mice that overexpress murine Ngb under the control of a chicken β-actin promoter, resulting in enhanced Ngb expression in multiple cell types and multiple tissues, including brain and heart. In Ngb-overexpressing transgenic mice compared with wild-type littermates, the volume of cerebral infarcts after occlusion of the middle cerebral artery was reduced by ≈30%, and the volume of myocardial infarcts produced by occlusion of the left anterior descending coronary artery was reduced by ≈25%. Ngb overexpression was associated with enhanced expression of endothelial nitric oxide synthase in vascular endothelial cells. These findings extend prior evidence for cytoprotection by Ngb and suggest both direct (parenchymatous) and indirect (vasomotor) protective mechanisms.

Endocannabinoids protect cerebral cortical neurons from in vitro ischemia in rats.

The endogenous cannabinoids (endocannabinoids) anandamide and 2-arachidonylglycerol increased cell viability in cerebral cortical neuron cultures subjected to 8 h of hypoxia and glucose deprivation. This effect was observed at nanomolar concentrations, was reproduced by a non-hydrolyzable analog of anandamide, and was unaltered by CB1 or CB2 cannabinoid receptor antagonists. Like synthetic cannabinoids, endocannabinoids can protect neurons from hypoxic injury, and may represent endogenous neuroprotective molecules in cerebral ischemia.

Vascular Endothelial Growth Factor Overexpression Delays Neurodegeneration and Prolongs Survival in Amyotrophic Lateral Sclerosis Mice

We sought genetic evidence for the involvement of neuronal vascular endothelial growth factor (VEGF) in amyotrophic lateral sclerosis (ALS). Mice expressing human ALS mutant superoxide dismutase-1 (SOD1) were crossed with mice that overexpress VEGF in neurons (VEGF+/+). We report that SOD1G93A/VEGF+/+ double-transgenic mice show delayed motor neuron loss, delayed motor impairment, and prolonged survival compared with SOD1G93A single transgenics. These findings indicate that neuronal VEGF protects against motor neuron degeneration, and may have therapeutic implications for ALS.

Transgenic ablation of doublecortin-expressing cells suppresses adult neurogenesis and worsens stroke outcome in mice

Injury stimulates neurogenesis in the adult brain, but the role of injury-induced neurogenesis in brain repair and recovery is uncertain. One strategy for investigating this issue is to ablate neuronal precursors and thereby prevent neurogenesis, but this is difficult to achieve in a specific fashion. We produced transgenic mice that express herpes simplex virus thymidine kinase (TK) under control of the promoter for doublecortin (Dcx), a microtubule-associated protein expressed in newborn and migrating neurons. Treatment for 14 days with the antiviral drug ganciclovir (GCV) depleted Dcx-expressing and BrdU-labeled cells from the rostral subventricular zone and dentate gyrus, and abolished neurogenesis and associated neuromigration induced by focal cerebral ischemia. GCV treatment of Dcx-TK transgenic, but not WT, mice also increased infarct size and exacerbated postischemic sensorimotor behavioral deficits measured by rotarod, limb placing, and elevated body swing tests. These findings provide evidence that injury-induced neurogenesis contributes to stroke outcome and might therefore be a target for stroke therapy.

Erythropoietin protects cultured cortical neurons, but not astroglia, from hypoxia and AMPA toxicity

In addition to its better-known hemopoietic action, erythropoietin (Epo) has neurotrophic properties and neuroprotective effects in some models of hypoxic-ischemic injury. To define further the cellular mechanisms underlying neuroprotection by Epo, we studied the effects of Epo on hypoxia with glucose deprivation in cultured rat cortical neurons and astroglia and on exposure to excitotoxins in cultured rat cortical neurons. Epo (30 pM) reduced neuronal, but not astroglial, cell death from hypoxia with glucose deprivation, and also attenuated the neurotoxic effect of (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), but not other excitotoxins. Epo appears to protect against cerebral ischemia through a direct effect on neurons that may be mediated in part by AMPA receptors.

CB1 cannabinoid receptor induction in experimental stroke

Cannabinoids protect cortical neurons from ischemic injury by interacting with CB1 receptors. Because a variety of neuroprotective genes are induced in cerebral ischemia, we examined the effect of experimental stroke, produced by 20 minutes of middle cerebral artery occlusion in rats, on CB1 receptor expression. Western blotting and immunohistochemistry showed that CB1 expression on neurons was increased in the arterial boundary zone of the cortical mantle, beginning by 2 hours and persisting for 72 hours or more after ischemia. These findings are consistent with a neuroprotective role for endogenous cannabinoid signaling pathways and with a potential therapeutic role in stroke for drugs that activate CB1 receptors. Ann Neurol 2000;48:257–261