Catalpol increases brain angiogenesis and up-regulates VEGF and EPO in the rat after permanent middle cerebral artery occlusion

Abstract

To investigate the role and mechanism of catalpol in brain angiogenesis in a rat model of stroke, the effect of catalpol (5 mg/kg; i.p) or vehicle administered 24 hours after permanent middle cerebral artery occlusion (pMCAO) on behavior, angiogenesis, ultra-structural integrity of brain capillary endothelial cells, and expression of EPO and VEGF were assessed. Repeated treatments with Catalpol reduced neurological deficits and significantly improved angiogenesis, while significantly increasing brain levels of EPO and VEGF without worsening BBB edema. These results suggested that catalpol might contribute to infarcted-brain angiogenesis and ameliorate the edema of brain capillary endothelial cells (BCECs) by upregulating VEGF and EPO coordinately.

Keywords: Angiogenesis; Catalpol; EPO; Permanent occlusion of middle cerebral artery; VEGF.

Figure 1

Effects of intraperitoneal injection with catalpol on sensorimotor performance in post-surgical rats at days 1, 4, 7 and 15. (A) Post-stroke treatment with catalpol reduced Bederson's score in stroke rats and (B) decreased beam working score in stroke rats. The data are presented as mean ± SE. * p<0.05 compared with vehicle group, ^#p<0.01 compared with sham operation group.

Figure 2

The vascular pattern in cerebral cortical surface in rats 15 days after pMCAO. (A) In the vehicle-treated group, the pale brain surface had few vessel branch points, infarct areas were characterized by liquefactive necrosis, cortical surface vessels were scarce and rearranged, several discontinued vascular structures were observed, and the radial patterns were lost. (B) In the catalpol-treated group, brain surface vessel branch points increased obviously, vascular structures continued, focus on the infarct area was present, and the vessel radial patterns and brain tissue infarct area were close to normal. Arrow points to vascular structures around the ischemia area.

Figure 3

Angiogenesis surrounding ischemic cortical area as demonstrated by immunocytochemistry and laser scanning confocal microscopy. The effects of Catalpol on angiogenesis were indicated by double-staining for VWF, a marker of endothelial cells and for proliferating cell nuclear antigen (PCNA), a marker of cell proliferation. Co-labeling of PCNA (green) and VWF (red) demonstrates angiogenesis, i.e., endothelial proliferation in the capillaries, in the peri-infarcted area at 15 days after pMCAO. Co-localization of PCNA and VWF is yellow. (A) Sham operation group, (B) Vehicle-treated group, (C) Catalpol-treated group. Bars = 150 μm in A, B, and C. This analysis demonstrated that few vessels were double-stained by VWF and PCNA in sham-operated rats (A), but significant remodeling of the microvessel network occurred in the infarcted hemisphere and the number of vessels with small diameter and short segment increased at 15 days after the stroke. More vessels were double-stained for VWF and PCNA in catalpol-treated group (C). Statistical analyses are shown in the graph of the number of vessels co- labeled for PCNA and VWF (D). The results above agreed with the results of IOD analyses in the co-localization area (E). (^*P < 0.01).

Figure 4

Ultrastructural observations of brain capillary endothelial cells (BCECs). In the vehicle-treated group (A), BCEC edema, chromatin rarefaction (short arrow), and chondriosome swelling (long arrow) were observed. (B) In the catalpol group, BCECs, pykno-chromatin (short arrow), and chondriosome number and shape (long arrow) were close to normal or near normal. Bars = 1μm.

Figure 5

Catalpol upregulated EPO and VEGF expression in pMCAO rat brains. Immunohistochemistry results showing neurons with EPO (A, B, C, 200×) and VEGF (D, E, F, 200×) in the peri-infarcted area of a pMCAO rat, a sham-operated rat (A & D), a vehicle-treated rat (B & E), and a catalpol-treated rat (C & F). EPO and VEGF expression detected by western blot showed in (G). The internal control was β-actin. Vs vehicle group ^*p < 0.05. The experiments repeated three times and 6 rats used in each group. Statistical bars shown as H and I respectively.