Ablation of neurogenesis attenuates recovery of motor function after focal cerebral ischemia in middle-aged mice.

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

Conditional depletion of neurogenesis inhibits long-term recovery after experimental stroke in mice.

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

Infarct volume or volume loss in middle-aged DCX-TK⁽⁻⁾ and DCX-TK⁽⁺⁾ transgenic mice after dMCAO.

<p>(<b>A</b>) Middle-aged DCX-TK⁽⁻⁾ and DCX-TK⁽⁺⁾ transgenic mice were treated with vehicle or GCV for 14 days, underwent MCAO, and were killed 24 hr later. Top panel: representative images of infarct area in H&E-stained coronal brain sections. Bottom panel: infarct volumes, expressed as percentage hemispheric volume. *<i>P</i><0.05. (<b>B</b>) Middle-aged DCX-TK⁽⁻⁾ and DCX-TK⁽⁺⁾ transgenic mice were treated with vehicle or GCV for 14 days, underwent dMCAO, and were killed 12 weeks later. Top panel: representative images of atrophy area in H&E-stained coronal brain sections. Bottom panel: volume loss, expressed as percentage hemispheric volume. *<i>P</i><0.05.</p

EBST, limb-placing and rotarod testing in middle-aged DCX-TK⁽⁻⁾ and DCX-TK⁽⁺⁾ mice after dMCAO.

<p>Middle-aged DCX-TK⁽⁻⁾ and DCX-TK⁽⁺⁾ mice underwent dMCAO after treated with vehicle or GCV for 14 days, then behavioral tests were conducted at intervals over the next 12 wks. (<b>A</b>) Scores of elevated body swing test (EBST; lower scores correspond to more severe deficits). (<b>B</b>) Scores of limb-placing test (lower scores represent more severe deficits). (<b>C</b>) Scores of rotarod test (lower scores represent more severe deficits). *<i>P</i><0.05.</p

Conditional depletion of DCX- and BrdU-immunopositive cells in SVZ and dentate SGZ of middle-aged DCX-TK⁽⁺⁾ transgenic mice.

<p>(<b>A</b>) Middle-aged DCX-TK⁽⁺⁾ and DCX-TK⁽⁻⁾ mice were treated for 14 days with vehicle or GCV, received behavioral training, and then underwent MCAO or dMACO. Behavioral testing was conducted for 12 weeks after dMCAO, following which some mice were given BrdU on the last day after dMCAO. Mice were then euthanized for measurement of damaged volume and immunocytochemistry. In addition, BrdU was injected at 24 hr prior to termination of GCV administration to test whether neurogenesis was inhibited. (<b>B</b>) Representative images of DCX- and BrdU-immunopositive cells in dentate SGZ of middle-aged DCX-TK⁽⁺⁾ transgenic mice treated with vehicle or GCV (left panel). DCX- and BrdU-immunopositive cells in SVZ of middle-aged DCX-TK⁽⁺⁾ transgenic mice treated with vehicle or GCV (right panel). (<b>C</b>) Representative images of DCX (green)/BrdU-immunopositive cells (red) in SVZ of middle-aged DCX-TK⁽⁺⁾ transgenic mice treated with vehicle (left pane). High magnification view (right panel) shows that DCX (green) and BrdU (red) were colocalized in single cells.</p

DCX-immunopositive cells in SVZ and dentate SGZ of vehicle- and GCV-treated, wild-type and DCX-TK transgenic mice, 12 weeks after MCAO.

<p>(A) Representative images of DCX-immunoreactive cells in SVZ (top) and dentate SGZ (bottom) from vehicle (left)- and GCV (right)-treated DCX-TK(+) transgenic mice. (B) Quantification of DCX-immunoreactive cells in SVZ (left panel) and dentate SGZ (right panel) from GCV (red bars)- and vehicle (black bars)-treated DCX-TK(+) and DCX-TK(-) mice. There were no significant differences between vehicle- and GCV-treated groups.</p

Neurobehavioral deficits in vehicle- and GCV-treated, wild-type and DCX-TK transgenic mice, 12 weeks after MCAO.

<p>Transgenic (DCX-TK(+)) and wild type (DCX-TK(-)) mice were treated for 14 days with vehicle (PBS) or GCV, then underwent MCAO. Behavioral testing was performed at the indicated times after MCAO. (<b>A</b>) Beam-walking test scores, expressed as the mean numbers of forelimb (left panel) or hindlimb (right panel) slip steps when traversing an elevated narrow beam; higher scores represent more severe deficits. (<b>B</b>) Corner test scores, expressed as a percentage of rearing to the contralesional (impaired) side; lower scores represent more severe deficits. (<b>C</b>) Elevated body swing test scores, expressed as a percentage of turns to the contralesional (impaired) side; lower scores represent more severe deficits. (<b>D</b>) Limb-placing test scores, expressed as a score derived from the number of correct limb placements; lower scores represent more severe deficits. *, <i>P</i><0.05 compared to vehicle-treated DCX-TK(+) mice.</p

Transplantation of human neural precursor cells in Matrigel scaffolding improves outcome from focal cerebral ischemia after delayed postischemic treatment in rats

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