Angiogenesis and improved cerebral blood flow in the ischemic boundary area detected by MRI after administration of sildenafil to rats with embolic stroke

Abstract

To dynamically investigate the long-term response of an ischemic lesion in rat brain to the administration of sildenafil, male Wistar rats subjected to embolic stroke were treated with sildenafil (n=11) or saline (n=10) at a dose of 10 mg/kg administered subcutaneously 24-h after stroke and daily for an additional 6 days. Magnetic resonance images were acquired and functional performance was measured in all animals at 1 day, 2 days and weekly for 6 weeks post-stroke. All rats were sacrificed 6 weeks after stroke and endothelial barrier antigen immunostaining was employed for morphological analysis and quantification of cerebral vessels. Map-ISODATA was computed from T(1), T(2) and T(1sat) maps. ISODATA derived tissue signatures characterize the degree of ischemic injury. Based on the map-ISODATA calculated at 6 weeks, the ischemic lesion for each animal was divided into two specific regions, the ischemic boundary and ischemic core. The temporal profiles of cerebral blood flow (CBF) and tissue signature were retrospectively tracked in these two regions and were compared with histological evaluation and functional outcome. After 1 week of sildenafil treatment, the ischemic lesion exhibited two significantly different regions, with higher CBF level and correspondingly, lower tissue signature value in the boundary region than in the core region. Sildenafil treatment did not significantly reduce the lesion size, but did enhance angiogenesis. Functional performance was significantly increased after sildenafil treatment compared with the control group. Administration of sildenafil to rats with embolic stroke enhances angiogenesis and selectively increases the CBF level in the ischemic boundary, and improves neurological functional recovery compared to saline-treated rats.

Fig. 1

ISODATA (a) and coregistered EBA-immunostained tissue slice (b) of a rat brain (6-weeks after stroke) showing the two specific regions in the ischemic lesion area and the relationship between angiogenesis area and lesion boundary. The ischemic boundary region (a, transparent ROI) was created by shrinking the rim of ischemic lesion 4 pixels to the ischemic center. The remaining lesion area located inside the boundary region was identified as the ischemic core region (a, solid ROI). The angiogenesis area (b, yellow track) identified by immunohistological evaluation was partially covered by the ischemic lesion area characterized by ISODATA (b, red track). This overlapping area (b, green area) within the lesion was encompassed by the perimeter boundary ROI (comparing a with b).

Fig. 2

Temporal profiles of the ischemic lesion size for the treated and control groups (a) and blood pressure measurements (b). Although the mean values of lesion size were lower in the treated group than in the control group (average from 1 to 6 weeks: 41% vs. 49% of the ipsilateral hemisphere), there was no significant difference between the two groups throughout the experimental period. Sildenafil administered subcutaneously to rats at a dose of 10mg/Kg resulted in a transient but not a significant reduction of blood pressure compared with the pre-treatment level, with the lowest mean blood pressure exhibited 40 minutes after administration and the value gradually returning to the pre-treatment level 120 minutes after administration.

Fig. 3

Immunoreactive cerebral vessels from a treated animal (a, ipsilateral side; b, contralateral side. Bar = 100μm), vessel density (c) and relative CBF (d) in lesion boundary area 6-weeks after stroke. Treatment with sildenafil significantly increased cerebral vessel number in the lesion boundary area compared with the control group (c). Relative CBF in the lesion boundary area was also significantly improved (d) compared with the control group. Significance of difference: * = p < 0.05, comparing treated and control groups in the ischemic boundary region; + = p < 0.05, comparing ischemic boundary region with the homologous area in the contralateral (normal) side of rat brain in treated group and control group, respectively.

Fig. 4

Evolution of CBF (a–b) and tissue signature (c–d) in different regions. Ischemic boundary and ischemic core areas were identified as two significantly different regions by both CBF (a) and tissue signature values (c) for the treated group after the 1-week time point. These two regions, however, for the control group did not differ in both CBF (b) and tissue signature values (d) until 6-weeks. Significance of difference: * = p < 0.05, comparing boundary and core regions at the same time points.

Fig. 5

Neurological functional outcome (a–b) and correlation analysis (c). Treatment with sildenafil significantly improved performance on NSS (a) and on foot-fault test (b) from 2-weeks and 3-weeks after stroke, respectively, until the end of observation compared with control group. Significance of difference: * = p < 0.05, comparing treated and control groups at the same time points. Higher CBF level in the boundary region correlates to lower percentage of foot-faults (c).