Correlation of CD34+ cells with tissue angiogenesis after traumatic brain injury in a rat model

Abstract

Increasing evidence suggests that circulating endothelial progenitor cells, which are a subpopulation of hematopoietic progenitor CD34(+) cells, play a critical role in neovascularization and tissue repair. We have tested the hypothesis that traumatic brain injury (TBI) could mobilize CD34(+) cells to peripheral blood and brain tissue, a process critical for vascular repair, in a rat model of TBI. Male Wistar rats were subjected to controlled fluid percussion. Blood and brain tissue were collected before and after TBI to measure the levels of CD34(+) cells in peripheral blood and to detect their accumulation in the damaged cerebral tissue. Compared with surgery controls, CD34(+) cells significantly increased in the peripheral blood and accumulated in the brain tissue of TBI rats. Immunohistochemistry detected new vessels with incomplete CD34(+) endothelial-like cell lining and an increased number of microvessels in the injured and surrounding tissue. The results demonstrate a close correlation between an increase in circulating CD34(+) cells in response to traumatic injury and angiogenesis in TBI rat brain. They also suggest that transplantation of CD34(+) cells or augmentation of endogenous CD34(+) cells may be a novel therapeutic approach for patients with TBI.

Figures

FIG. 1.

Flow cytometry detection of CD34+ cells. (A) Isotype control antibody and (B) CD34-PE antibody: representative images of flow cytometrical detection of CD34+ cells (expressed as 1 × 104 mononuclear cells; mean ± SEM) in peripheral blood. (C) Number of circulating CD34+ cells at 0, 24, 48, 72, 120, and 168 h after TBI. Repeated measures ANOVA showed significant effects of injury (F = 27.34, p < 0.001), time (F = 39.58, p < 0.001) on the number of circulating CD34+ cells, and interaction between injury and time (F = 6.253, p < 0.001). The post hoc test (LSD) further indicated that CD34+ cell numbers in rats from TBI group were higher than those in controls except at baseline (0 h) and 168 h after TBI. *p < 0.01, **p < 0.05 compared with control at the same time point (Student t test; n = 14).

FIG. 2.

Detection of CD34+ cells in the injured brain tissue: CD34+ cells that were spindle-shape cells and stained brown were counted in brain tissue from the injury zone (A) and ispilateral hippocampus (B) 24 h after TBI. (C and D) Representative images from the contralateral and ipsilateral hippocampus, respectively, showing significantly more CD34+ cells accumulated in ipsilateral hippocampus compared to that in the contralateral hippocampus. (E and F) Representative images from the injured tissue and tissue from the areas remote from the injury zone, showing fewer CD34+ cells in the remote tissue. Marker G indicated injury zone. The images were representative of CD34-stained tissue sections. Bars, 50 μm (A, B, E, F) or 100 μm (C and D).

FIG. 3.

Quantification of CD34+ cells in brain tissue. (A) Changes in the number of CD34+ cells in brain tissue from TBI and control rats (per 100x). By two-way ANOVA, a significant difference was found in the effects of injury (F = 306.80, p < 0.001), time (F = 101.08, p < 0.001), and interaction (F = 88.46, p < 0.001) between injury and time. Post hoc test (LSD) further indicated that CD34+ cells number in TBI rats were higher than that in controls except at baseline (*p < 0.01, **p < 0.05 compared with control at the same time point; Student t test; n = 14). (B) Patterns of change in CD34+ cells in peripheral blood and brain tissue of TBI rats.

FIG. 4.

Detection of CD34+ microvasculature: brain tissues from indicated regions (small images, left) were immunochemically stained for CD34. (A and B) Representative of CD34 stained tissue from the boundary of the injured cerebral tissue, showing CD34+ endothelial-like lumen structures on day 3 after TBI. (C and D) Representative OF CD34 staining of tissue from the injured tissue, also showing CD34+ endothelial-like lumen structures, but only at approximately day 14 after TBI. Arrows indicated the vascular lumen-like structure. The images were representative from CD34-stained tissue sections. Bar, 30 μm (A and B) or 50 μm (C and D).

FIG. 5.

Changes in CD34+ cells and MVD after TBI. (A) Dynamic changes of post-traumatic MVD of TBI group and control group. By two-way ANOVA, significant difference was shown in the effects of injury (F = 116.94, p < 0.001) and time (F = 25.23, p < 0.001) on MVD number, and interaction (F = 19.20, p < 0.001) between injury and time. Post hoc test (LSD) further indicated that MVD number in TBI rats were significantly higher than that in controls except at baseline and 24 h after TBI (*p < 0.01 compared with control at the same time point; Student t test; n = 14). (B) Common pattern of changes in CD34+ cells and CD34+ MVD in the injured tissue.