Administration of CD34+ cells after stroke enhances neurogenesis via angiogenesis in a mouse model

Abstract

Thrombo-occlusive cerebrovascular disease resulting in stroke and permanent neuronal loss is an important cause of morbidity and mortality. Because of the unique properties of cerebral vasculature and the limited reparative capability of neuronal tissue, it has been difficult to devise effective neuroprotective therapies in cerebral ischemia. Our results demonstrate that systemic administration of human cord blood-derived CD34(+) cells to immunocompromised mice subjected to stroke 48 hours earlier induces neovascularization in the ischemic zone and provides a favorable environment for neuronal regeneration. Endogenous neurogenesis, suppressed by an antiangiogenic agent, is accelerated as a result of enhanced migration of neuronal progenitor cells to the damaged area, followed by their maturation and functional recovery. Our data suggest an essential role for CD34(+) cells in promoting directly or indirectly an environment conducive to neovascularization of ischemic brain so that neuronal regeneration can proceed.

Figure 1

Endothelial proliferation in situ after stroke. On days 1, 3, 7, and 14 after stroke, the number of proliferating cells (BrdU⁺) and proliferating endothelial cells (co-staining for BrdU and CD31) was determined in the left cortical area of 1–1.5 mm distal from the midline. (A) Immunohistological analysis of proliferating cells labeled with BrdU (green), anti-mouse CD31 IgG (red), and both (yellow). The number of cells visualized with BrdU (B) and the subpopulation BrdU⁺ cells also displaying mouse CD31 (i.e., double positives) (C) are shown. Ten HPFs were evaluated for each animal (n = 6 per group) by two investigators blinded to the experimental protocol. Note in C, cells displaying mouse CD31 are termed endothelial cells (ECs). Black bars, ipsilateral; white bars, contralateral. *P < 0.05 versus control. Scale bar: 30 μm.

Figure 2

Transplantation of CD34⁺ cells after stroke accelerates neovascularization. (A–D) Mice subjected to stroke received CD34⁺ cells (A and B), CD34^– cells (C), or PBS alone (D) on day 2. Animals were infused with carbon black ink and killed at 24 hours after cell transplantations. Sections were stained with TTC. Neovascularization was noted at the border zone between the ACA and MCA areas (arrowheads show microvessels), especially in animals treated with CD34⁺ cells compared with those receiving CD34^– cells or PBS alone. (E) An angiographic score for each experimental condition based on analysis of 6 mice per group. (F–H) Activated endothelial cells were observed with antibody specific for mouse CD13 in the ACA area. F: CD34⁺ cells; G: CD34^– cells; H: PBS. (I) CBF was measured in the MCA area just outside of the penumbra, and values in animals treated with CD34⁺ cells (black bars), CD34^– cells (gray bars), or PBS (white bars) were compared with values before cell transplantation at times corresponding to days 1, 7, and 14 after cell transplantation (n = 6 per group). Data shown are relative CBF versus day the measurement was performed. (J and K) Labeling vasculature by infusion of carbon black ink demonstrated neovasculature at the border of the MCA and ACA cortex in animals treated with CD34⁺ cells (J) and CD34⁺/Flk-1^– cells (K) on day 7 after cell transplantation. Scale bars: 0.5 mm (A) and 0.1 mm (B, F, and J). *P < 0.05 versus PBS.

Figure 3

CD34⁺ cell transplantation induces cortical expansion after stroke. (A–F) On day 14 (A–C), and day 90 (D–F) after cell transplantation, the brains of mice were evaluated grossly. Compared with poststroke mice treated with PBS (A and D) or infused with CD34^– cells (B and E), animals transplanted with CD34⁺ cells (C and F) showed an increase in area occupied by the left cortex. (G) Cortical regeneration was induced by CD34⁺ cells transplantation: triangles, CD34⁺ cells; squares, CD34^– cells; diamonds, PBS. In each case, there were 6 animals per group. Scale bar: 2 mm (A). *P < 0.05 versus PBS.

Figure 4

CD34⁺ cell transplantation accelerates neuronal regeneration after stroke. (A and B) On day 14, animals receiving CD34⁺ cells after stroke displayed migration of NPCs toward the ischemic area by PSA-NCAM immunostaining. (C–E) Analysis of serial sections displayed expression of neuronal stem cell markers, Musashi-1 (C) and DCX (D). Note that expression of DCX was limited to the area proximal to the SVZ. PSA-NCAM⁺ NPCs also expressed NeuN (E). Small NeuN⁺ nuclei were observed in PSA-NCAM⁺ NPCs, whereas more intensely staining and larger nuclei represent mature neurons. (F) On day 14 on the contralateral side, PSA-NCAM⁺ NPCs were limited to the SVZ; that is, no migration of NPCs was observed. (G) Migration of NPCs with small NeuN⁺ nuclei toward cortex was observed in poststroke mice treated with PBS on day 14 after cell transplantation. (H) The average number of NPCs in the white matter at the lower left of the left cortex per HPF from 5 animals under each condition. Three sections were evaluated in each animal, and n = 5 per group. Arrowheads delineate individual NPCs or demarcate areas rich in NPCs. Scale bars: 1 mm (A), 0.2 mm (B and F), and 0.4 mm (G). *P < 0.05 versus PBS.

Figure 5

Therapeutic neovascularization, due to CD34⁺ cell transplantation after stroke, enhances neurogenesis. (A–F) On day 14 after CD34^– cell transplantation, mature cortical neurons were observed up to the edge of the ischemic region displaying neuronal markers, NeuN (A) and MAP-2 (B), whereas only a thin layer of migrating PSA-NCAM⁺ NPCs was observed at the ischemic edge (C). In contrast, after transplantation of CD34⁺ cells, expanded cortical areas displaying a low density of NeuN⁺ (D) and MAP-2⁺ cells (E) were observed beyond the boundary demarcating mature neurons. Migration of NPCs into this expanded area was also observed by PSA-NCAM staining (F). (G–I) On day 14, TUNEL⁺ cells were visualized around the lower part of the expanded cortical area. Whereas massive cell death was observed in animals receiving CD34^– cell transplantation (G), the number of TUNEL⁺ profiles was strongly reduced in mice transplanted with CD34⁺ cells (H). (I) The average number of TUNEL⁺ cells per HPF. Three sections were evaluated in each animal; n = 5 per group. Arrowheads indicate the expanded cortical areas displaying a low density of indicated marker. Scale bars: 100 μm (A) and 50 μm (G). *P < 0.05 for CD34⁺ versus CD34^– cell transplantation.

Figure 6

Therapeutic neovascularization supports survival of regenerating neurons. (A–C) Animals treated with PBS alone (A), CD34^– cells (B), or CD34⁺ cells (C) were infused with BrdU, killed, and studied immunohistochemically with antibody to BrdU (red), NeuN (green), or both (yellow). (D) The average number of double-positive cells (stained with antibody to BrdU and NeuN) per HPF on day 90 after cell transplantation. Ten fields were evaluated in each animal, and n = 6 per group. *P < 0.05 versus PBS. (E) On day 90 after cell transplantation, brain sections from animals treated with PBS alone, CD34^– cells, or CD34⁺ cells were stained with antibody to NeuN, and the number of total neurons in the left cortex was counted. (E) The average number of total NeuN⁺ cells in the left cortex. n = 6 per group; *P < 0.05 versus PBS. (F and G) On day 90 after cell transplantation, mouse CD31 was visualized immuno-histologically in forebrain sections from poststroke animals. Newly formed vascular networks were observed in the expanded cortex. The vascular pattern displayed by these neovessels on the ipsilateral (F, ischemic side) was different from that observed on the contralateral side (G). (H) On day 90, human CD31 was visualized immunohistologically. Human endothelial cells were observed in the regenerating cortex of animals treated with CD34⁺ cells after stroke. The arrowhead shows a human CD31⁺ endothelial cell. Scale bars: 50 μm (A), 100 μm (F and G), and 20 μm (H).

Figure 7

CD34⁺ cell transplantation in 24-week-old animals. (A–C) On day 35 after cell transplantation, the brains were evaluated. Compared with poststroke mice treated with CD34^– cells (A), animals transplanted with CD34⁺ cells (B) showed an increase in area occupied by the left cortex. Significant cortical regeneration was induced by CD34⁺ cells transplantation (C). (D and E) Compared with CD34^– cell transplantation (D), increased evidence of activated vasculature was observed in animals receiving CD34⁺ cells (E), as detected with mouse specific anti-CD13 antibody. (F–H) Migration of NPCs (small NeuN⁺ nuclei migrating toward the cortex) was observed in poststroke mice treated with CD34^– cells (F) and with CD34⁺ cells (G). However, a significant increase in migrating NPCs was induced by CD34⁺ cell transplantation (H). (I and J) A thin layer of migrating PSA-NCAM⁺ NPCs was observed at the ischemic edge of the cortex in animals treated with CD34^– cells (I), compared with a much thicker layer in those receiving CD34⁺ cells (J). Scale bars: 2 mm (A), 0.1 mm (D and I), and 0.4 mm (F). n = 4 in each group; *P < 0.05 versus CD34^– cells.