Depletion of neurogenesis worsens functional outcome in young-adult mice after focal cerebral ischemia, but whether a similar effect occurs in older mice is unknown. Using middle-aged (12-month-old) transgenic (DCX-TK(+)) mice that express herpes simplex virus thymidine kinase (HSV-TK) under control of the doublecortin (DCX) promoter, we conditionally depleted DCX-positive cells in the subventricular zone (SVZ) and hippocampus by treatment with ganciclovir (GCV) for 14 days. Focal cerebral ischemia was induced by permanent occlusion of the middle cerebral artery (MCAO) or occlusion of the distal segment of middle cerebral artery (dMCAO) on day 14 of vehicle or GCV treatment and mice were killed 24 hr or 12 weeks later. Increased infarct volume or brain atrophy was found in GCV- compared to vehicle-treated middle-aged DCX-TK(+) mice, both 24 hr after MCAO and 12 weeks after dMCAO. More severe motor deficits were also observed in GCV-treated, middle-aged DCX-TK(+) transgenic mice at both time points. Our results indicate that ischemia-induced newborn neurons contribute to anatomical and functional outcome after experimental stroke in middle-aged mice.
Background and Purpose
Interhemispheric inhibition via the corpus callosum has been proposed as an exacerbating factor in outcome from stroke.
We measured infarct volume and behavioral outcome following middle cerebral artery occlusion in callosotomized rats and acallosal mice.
Neither callosotomy in rats nor callosal agenesis in mice improved infarct volume or behavioral outcome after middle cerebral artery occlusion.
These findings argue against a role for transcallosal projections in exacerbating focal cerebral ischemia.
corpus callosum; stroke; ischemia; callosotomy; callosal agenesis
Animal studies indicate that adult renal stem/progenitor cells can undergo rapid proliferation in response to renal injury, but whether the same is true in humans is largely unknown. To examine the profile of renal stem/progenitor cells responsible for acute tubular necrosis in human kidney, double- and triple-immunostaining was performed using proliferative marker and stem/progenitor protein markers on sections from ten kidneys with acute tubular necrosis and four normal adult kidneys. The immunopositive cells were recorded using two-photon confocal laser scanning microscopy. We found that dividing cells were present in the tubules of the cortex and medulla, as well as the glomerulus in normal human kidney. Proliferative cells in the parietal layer of Bowman’s capsule expressed CD133, and dividing cells in the tubules expressed immature cell protein markers paired box gene 2, vimentin and nestin. After acute tubular necrosis, Ki67-positive cells in the cortex tubules significantly increased compared to normal adult kidney. These Ki67-positive cells expressed CD133 and paired box gene 2, but not the cell death marker, activated caspase-3. In addition, the number of dividing cells increased significantly in patients with acute tubular necrosis, who subsequently recovered, compared to patients with acute tubular necrosis, who consequently developed protracted acute tubular necrosis or died. Our data suggest that renal stem/progenitor cells may reside not only in the parietal layer of Bowman’s capsule, but also in the cortex and medulla in normal human kidney, and the proliferative capacity of renal stem/progenitor cells after acute tubular necrosis may be an important determinant of a patient’s outcome.
ATN; kidney; progenitor cells; stem cells; outcome; proliferation
Little is known about the relationship between neuronal cell transplantation and endogenous neurogenesis after experimental stroke. We found previously that transplantation of neuronal precursors derived from BG01 human embryonic stem cells reduced infarct volume and improved behavioral outcome after distal middle cerebral artery occlusion (MCAO) in rats. In this study, transplantation was performed 14 d after distal MCAO and doublecortin (Dcx)-expressing cells in the subventricular zone (SVZ) and subgranular zone of dentate gyrus (SGZ) were counted 60 d post-transplant. Transplantation increased neurogenesis (Dcx expression) in ipsilateral SVZ, but not in contralateral SVZ or either SGZ, in both young adult (3 mo-old) and aged (24-mo-old) rats. These findings suggest that cell-based therapy for stroke may be associated with changes in endogenous adaptive processes, including neurogenesis.
Ischemia; stroke; transplantation; neurogenesis; subventricular zone
We reported previously that Notch signaling is activated in human arteriovenous malformations (AVMs) and that intracerebral hemorrhage (ICH) in humans is accompanied by increased neurogenesis. The former phenomenon may be involved in AVM pathogenesis and the latter in the brain’s response to ICH-induced injury. Here we describe increased expression of the hypoxia-inducible neuroprotective protein, neuroglobin (Ngb), in neurons surrounding unruptured AVMs and in the perihematomal region adjacent to ICH. In these disorders, as in other clinical settings, such as ischemic stroke, AVM- and ICHinduced overexpression of Ngb may be stimulated by ischemic hypoxia and may help to constrain brain injury.
Neuroglobin; Arteriovenous malformation; Intracerebral hemorrhage; Hypoxia; Ischemia
Neural precursor cell (NPC) transplantation may have a role in restoring brain function after stroke, but how aging might affect the brain’s receptivity to such transplants is unknown. We reported previously that transplantation of human embryonic stem cell (hESC)-derived NPCs together with biomaterial (Matrigel) scaffolding into the brains of young adult Sprague-Dawley rats 3 wks after distal middle cerebral artery occlusion (MCAO) reduced infarct volume, and improved neurobehavioral performance. In this study we compared the effect of NPC and Matrigel transplants in young adult (3-mo-old) and aged (24-mo-old) Fisher 344 rats from the National Institute on Aging’s aged rodent colony. Distal MCAO was induced by electrocoagulation and hESC-derived NPCs were transplanted into the infarct cavity 3 wks later. Aged rats developed larger infarcts, but infarct volume and performance on the cylinder and elevated body swing tests, measured 6–8 wks post-transplant, were improved by transplantation. We conclude that advanced age does not preclude a beneficial response to NPC and Matrigel transplantation following experimental stroke.
transplant; neural precursor; ischemia; stroke; brain
Transplantation of neural cells is a potential approach for stroke treatment, but disruption of tissue architecture may limit transplant efficacy. One strategy for enhancing the ability of transplants to restore brain structure and function is to administer cells together with biomaterial scaffolding. We electrocoagulated the distal middle cerebral artery in adult rats and, 3 weeks later, injected one of the following into the infarct cavity: artificial cerebrospinal fluid, Matrigel scaffolding, human embryonic stem cell-derived neuronal precursor cells, scaffolding plus cells, or cells cultured in and administered together with scaffolding. Five weeks after transplantation, the latter two groups showed ∼50% and ∼60% reductions, respectively, in infarct cavity volume. Rats given cells cultured in and administered together with scaffolding also showed (1) survival and neuronal differentiation of transplanted cells shown by immunostaining for neuronal marker proteins and cleaved caspase-3, and by patch-clamp recording, 8 weeks after transplantation and (2) improved outcome on tests of sensorimotor and cognitive functions, 4 to 9 weeks after transplantation. These results indicate that transplantation of human neural cells together with biomaterial scaffolding has the potential to improve the outcome from stroke, even when treatment is delayed for several weeks after the ischemic event.
ischemia; neurogenesis; neuronal progenitor cell; stem cells; stroke; transplantation
A role for the Notch signalling pathway in the formation of arteriovenous malformations during development has been suggested. However, whether Notch signalling is involved in brain arteriovenous malformations in humans remains unclear. Here, we performed immunohistochemistry on surgically resected brain arteriovenous malformations and found that, compared with control brain vascular tissue, Notch-1 signalling was activated in smooth muscle and endothelial cells of the lesional tissue. Western blotting showed an activated form of Notch-1 in brain arteriovenous malformations, irrespective of clinical presentation and with or without preoperative embolization, but not in normal cerebral vessels from controls. In addition, the Notch-1 ligands Jagged-1 and Delta-like-4 and the downstream Notch-1 target Hes-1 were increased in abundance and activated in human brain arteriovenous malformations. Finally, increased angiogenesis was found in adult rats treated with a Notch-1 activator. Our findings suggest that activation of Notch-1 signalling is a phenotypic feature of brain arteriovenous malformations, and that activation of Notch-1 in normal vasculature induces a pro-angiogenic state, which may contribute to the development of vascular malformations.
Notch-1; AVM; human; brain; signalling; angiogenesis
To explore the expression of Notch1 signaling pathway in nasopharyngeal carcinoma (NPC).
We performed immunocytochemistry on surgically resected NPC using antibodies against embryonic stem (ES) cell proteins and against Notch1 signaling components.
We found that ES cell protein markers SOX2 and OCT4 were expressed in a subpopulation of cells for all three subtypes of NPC but barely in the normal control. Double immunostaining shows that SOX2- and OCT4-positive cells coexpressed proliferative markers, suggesting that human NPC may contain cancer stem–like cells. In addition, we found that Notch1 signaling was activated in NPC. Confocal images show that the Notch1 signaling activated form and Hes1, a downstream target of Notch1 signaling, was predominantly found in SOX2- and OCT4-positive cells.
Our findings suggest that the Notch1 signaling pathway might be a regulator of cancer stem–like cells in NPC.
cancer stem cells; nasopharyngeal carcinoma; Notch1 signaling; OCT4; proliferation; SOX2
Transplantation of neural cells is a potential approach for the treatment of stroke, but the disruption of tissue architecture that accompanies stroke may limit the efficacy of transplantation. One strategy for enhancing the ability of transplants to restore brain structure and, thereby, function is to administer cells together with biomaterial scaffolding. We occluded the middle cerebral artery in adult rats and, 3 wks later, injected one of the following into the infarct cavity: (a) artificial cerebrospinal fluid, (b) Matrigel scaffolding, (c) human neuronal precursor cells, (d) scaffolding plus cells, or (e) cells cultured in and administered together with scaffolding. When tested up to 9 wks later, the latter group showed reduced infarct size, survival and neuronal differentiation of transplanted cells, and improved outcome on behavioral tests of sensorimotor and cognitive function. These results indicate that transplantation of human neural cells together with the scaffolding in which they are cultured has the potential to improve outcome from stroke, even when treatment is delayed for several weeks after the ischemic event.
stroke; ischemia; transplantation; neurogenesis; stem cells; neuronal progenitor cell
The observed age-related decline in neurogenesis may result from reduced proliferation or increased death rate of neuronal precursor cells (NPCs). We found that caspase-3, but not caspase-6, -7, or -9, was activated in NPCs in neurogenic regions of young, young-adult, middle-aged and aged rat brains. The number of capase-3-immunoreactive cells was highest in young and lowest in aged rats. Surprisingly, intraventricular administration of a caspase-3 inhibitor failed to restore the number of BrdU-positive cells in the aged dentate gyrus, suggesting that the age-related decline in neurogenesis may be attributable primarily to reduced proliferation. Additionally, we also found that NPCs in the subventricular zone of young-adult and aged rat brain were increased after focal cerebral ischemia, suggesting that the increase in neurogenesis induced by ischemia may result from an increase in the rate of NPC proliferation, but not from a decrease in NPC death. Thus, our results suggest that age-related and injury-induced changes in the rate of neurogenesis are controlled at the level of NPC proliferation. Furthermore, our results may imply that the mechanisms that maintain a stable population of NPCs in the normal adult and in the ischemic brain, which account for the observed age-dependent reduction or injury-induced increases in neurogenesis, impinge on the regulation of cell division at the NPC level.
Neurogenesis; aging; cell death; ischemia; dentate gyrus; subventricular zone
The Notch1 signaling pathway is regarded as one of the main regulators of neural stem cell behavior during development, but its role in the adult brain is less well understood. We found that Notch1 was mainly expressed in doublecortin (DCX)-positive cells corresponding to newborn neurons, whereas the Notch1 ligand, Jagged1, was predominantly expressed in glial fibrillary acidic protein (GFAP)-positive astrocytic cells in the subventricular zone (SVZ) of the normal adult brain. These findings were confirmed by conditional depletion of DCX-positive cells in transgenic mice carrying herpes simplex virus thymidine kinase (HSV-TK) under the control of the DCX promoter. In addition, the activated form of Notch1 (Notch intracellular domain, NICD) and its downstream transcriptional targets, Hes1 and sonic hedgehog (Shh), were also expressed in SVZ cells. Increased activation of Notch1 signaling increased SVZ cell proliferation, whereas inhibiting Notch1 signaling resulted in a reduction of proliferating cells in the SVZ. Levels of NICD, Hes1, and Shh were increased in the SVZ at 4 and 24 h after focal cerebral ischemia. Finally, ischemia-induced cell proliferation in the SVZ was blocked by inhibition of the Notch1 signaling pathway, suggesting that Notch1 signaling may have a key role in normal adult and ischemia-induced neurogenesis.
doublecortin; ischemia; Jagged1; neurogenesis; Notch1; subventricular zone
Neurogenesis occurs in discrete regions of normal brains of adult mammals including humans, and is induced in response to brain injury and neurodegenerative disease. Whether intracerebral hemorrhage can also induce neurogenesis in human brain is unknown. Specimens were obtained from patients with primary intracerebral hemorrhage undergoing surgical evacuation of an intracerebral hematoma, and evaluated by two-photon laser scanning confocal microscopy. We found that neural stem/progenitor cell-specific protein markers were expressed in cells located in the perihematomal regions of the basal ganglia and parietal lobe of the adult human brain after primary intracerebral hemorrhage (n = 5). Cells in this region also expressed cell proliferation markers, which colocalized to the same cells that expressed neural stem/progenitor cell-specific proteins. Our data suggest that intracerebral hemorrhage induces neurogenesis in the adult human brain.
brain; human; intracerebral hemorrhage; neural stem cells; neurogenesis
Basic fibroblast growth factor (FGF-2) has been reported to protect against ischemic injury in the brains of young adult rodents. However, little is known about whether FGF-2 retains this capability in the aged ischemic brain. Since stroke in human is much more common in older people than among younger adults, to address this question is clinically important. In this study, aged (24-month-old) rats were treated with intracerebroventricular infusion of FGF-2 or vehicle for 3 days, beginning 48 hr before (pre-ischemia), 24 hr after (early post-ischemia), or 96 hr after (late post-ischemia) 60 min of middle cerebral artery occlusion, and were killed 10 days after ischemia. Aged rats given FGF-2 pre-ischemia showed better symmetry of movement and forepaw outstretching, and reduced infarct volumes, compared to rats treated with vehicle, but no significant improvement was found in aged rats given FGF-2 after focal ischemia. In contrast, young adult (3-month-old) rats treated with FGF-2 for 3 days beginning 24 hr post-ischemia showed significant neurobehavioral improvement and better histological outcome. In addition, we also found that newborn neurons in the rostral subventricular zone (SVZ) were increased in aged rats treated with FGF-2 prior to ischemia. However, unlike in young adult ischemic rats, only a few of newly generated cells migrated into the damaged region in aged brain after focal ischemia. These findings point to differences in the response of aged versus young adult rats to FGF-2 in cerebral ischemia, and suggest that such differences need to be considered in the development of neuroprotective agents for stroke.
aging; growth factor; ischemia; brain; neurogenesis; neuroprotection
We reported previously that ablation of doublecortin (DCX)-immunopositive newborn neurons in mice worsens anatomical and functional outcome measured 1 day after experimental stroke, but whether this effect persists is unknown. We generated transgenic mice that express herpes simplex virus thymidine kinase under control of the DCX promoter (DCX-TK transgenic mice). DCX-expressing and recently divided cells in the rostral subventricular zone (SVZ) and hippocampus of DCX-TK transgenic mice, but not wild-type mice, were specifically depleted after ganciclovir (GCV) treatment for 14 days. Focal cerebral ischemia was induced by permanent distal middle cerebral artery occlusion (MCAO) on day 14 of vehicle or GCV treatment, and mice were killed 12 weeks after MCAO. Infarct volume was significantly increased and neurologic deficits were more severe in GCV- compared to vehicle-treated DCX-TK transgenic mice at first 8 weeks, after depletion of DCX- and bromodeoxyuridine-immunoreactive cells in the SVZ and dentate gyrus following focal ischemia. Our results indicate that endogenous neurogenesis in a critical period following experimental stroke influences the course of long-term recovery.