Protective effects of VEGF treatment on focal cerebral ischemia in rats

Introduction

Ischemic cerebrovascular diseases are a common type of aggressive disease with high rates of incidence, mortality and morbidity. Therefore, studies on the pathophysiology of these diseases and new treatment strategies are necessary, since there are currently no effective clinical treatments. For example, the factors that promote angiogenesis might be helpful for the recovery of patients with cerebral ischemia.

VEGF plays a significant role in the increase of vascular permeability (1,2). In the brain, VEGF is considered the main medium of peritumoral vasogenic brain edema (3,4). Animal and clinical experiments have shown that shortly after focal cerebral ischemia, leakage in the vascular permeability barrier occurs, leading to cerebral edema. VEGF expression peaks when vasogenic brain edema following ischemia is most severe, so the high VEGF expression may be associated with blood-brain barrier leakage. It has already been suggested in previous studies that in patients with cerebral ischemia, the microvascular density of the ischemic hemisphere increased significantly when compared with contralateral lesions, and the number of microvessels in the penumbra area may be correlated with the survival time of the stroke patients (5–7).

Results showed that following persistent cerebral ischemia in rats, local injection of VEGF in the cerebral cortex promoted angiogenesis 7 days following ischemia. The treatment group of 30 μg/ml VEGF showed the most significant effect and the group of 10 μg/ml showed the least significant effect, which indicated that the effect of angiogenesis may be related to the concentration of VEGF. Within a certain range, the higher the dose, the greater the effect.

In this study, the effects of exogenous VEGF on the infarct volume of cerebral ischemia, neurological deficits, cerebral edema, microvascular density and apoptosis were investigated. Electron microscopy of vascular endothelial cells and apoptotic cell ultrastructure were performed to study whether excessive proliferation causes formation of the vascular tumor-like structures. The protective roles of VEGF suggest that exogenous VEGF may be appropriate for clinical use.

Materials and methods

Animals

Forty healthy male Wistar rats 3–4 months old, each weighing 250–300 g, were provided by the Experimental Animal Center of Shandong Medical University (China). The rats were randomly divided into 4 groups, administered PBS and 10, 20 or 30 μg/ml of VEGF. The PBS group served as the control. The infarct volume ratio and the neurological deficits were determined by MRI. Microvessel density, apoptosis in brain slices and brain edema were determined. The focal cerebral ischemia rat model was made using the middle cerebral artery occlusion (MCAO) method described by Longa et al(8). All animal experiments were conducted according to the animal experimental guidelines of Jining Medical College (Shandong, China). The study was approved by the ethics committee of Jining Medical College.

Drug treatments

VEGF was diluted to concentrations of 10, 20 or 30 μg/ml using sterile 0.1 M PBS containing 0.1% fetal calf serum. VEGF solutions were slowly injected into the left cerebral cortex of rats. For the control group, sterile 0.1 M PBS containing 0.1% fetal calf serum was injected into the left cerebral cortex of the rats. Each rat was injected three times at 1, 24 and 48 h after infarct. Observation was performed 7 days after infarction.

Neurological deficit score

The neurological deficit scores were calculated according to the 0–5 grade scoring criteria described by Longa et al(8), with the greater number indicating more severe function deficits.

Infarct volume ratio

The 1.5T MRI machine was used to determine the anatomy structure of the rat brains, with the following parameters: 3 inches of surface coils, a large matrix (256×256), small view field (FOV, 8×4 cm), 3 mm of thickness and 0.3 mm of intervals. According to the location and extent of the abnormal signal area on each layer of the scanned image, the ratio of the infarct volume to the contralateral cerebral hemisphere was calculated.

Microvessel density

The immunohistochemical SP method was performed to detect microvessel density, using antibody against laminin concentration (1:200; Santa Cruz Biotechnology, Inc., USA). Ten high power microscopic fields (40×10) were randomly selected in the infarct cortex and basal ganglia in each slice. The number of positive vessels per field was calculated.

Apoptosis

The oligonucleotide terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling method (TUNEL) was used to detect in situ apoptosis. At 40×10 high power, 10 fields were randomly selected around the infarct cortex and basal ganglia in each slice. The number of total cells and positive cells were counted to calculate the positive cell rates.

Electron microscopy

Rat tissues were fixed in 4% glutaraldehyde and examined by electron microscopy. The brain tissue was rinsed with 2% glutaraldehyde and then fixed with 1% S4O4. Following acetone dehydration and the EPON-821 embedment, 1-μm ultrathin sections were cut. With sodium acetate and citrate double staining, the ultrastructure of apoptotic cells, endothelial cell proliferation and deterioration tendency were observed by transmission electron microscopy.

Statistical analysis

Data were analyzed by SPSS software and given as the mean ± SD. Single-factor analysis of variance, q-test and t-test were performed. P<0.05 was considered to indicate statistically significant differences.

Results

VEGF reduces infarct volume and improves neurological functions

To determine whether VEGF treatment affects focal cerebral ischemia in rats, rats were administered PBS or VEGF at concentrations of 10, 20 or 30 μg/ml. The effects of VEGF on rat infarct volume and neurological deficits were investigated as described in Materials and methods and the results are presented in Table I.

Table I Treatment of rats with different concentrations of VEGF.

Table I

Treatment of rats with different concentrations of VEGF.

	PBS	10 μg/ml	20 μg/ml	30 μg/ml
Nos. of rats	7	8	8	9
Infarct volume (%)	43.21±7.12	37.26±5.81a	33.62±8.24b	27.64±6.77c
Neurological deficit scores	2.86±0.69	2.50±0.76a	2.00±0.54b	1.78±0.67c

a P>0.05,

b P<0.05,

c P<0.01.

As shown in Table I, there were no significant differences in infarct volume and neurological deficit scores between the PBS and VEGF (10 μg/ml) group (P>0.05). However, when rats were treated with 20 μg/ml VEGF, infarct volume ratios and neurological deficit scores were both decreased when compared with the PBS group. When rats were treated with 30 μg/ml VEGF, the mean infarct volume ratio decreased to 27.64±6.77%, while the mean infarct volume ratio of the PBS group was 43.21±7.12%. The mean neurological deficit score of the 30 μg/ml VEGF group was 1.78±0.67, whereas the mean score of the PBS group was 2.86±0.69. These results suggest that treatments with VEGF reduce the infarct volume and improve neurological functions.

VEGF increases microvessel generation

To determine the effects of VEGF treatment on microvessel generation, the microvessel numbers in the cerebral cortex and basal ganglia of rats administered PBS or VEGF were detected. As shown in Fig. 1, treatments with 20 and 30 μg/ml VEGF significantly improved microvessel numbers compared with the PBS group. Treatment with 30 μg/ml VEGF increased microvessel numbers ~10-fold when compared with the PBS group, suggesting that VEGF increases microvessel generation.

Figure 1 VEGF increases microvessel generation. The microvessel numbers in the cerebral cortex and basal ganglia were detected on Day 7 post-treatment. Rats were administered PBS or VEGF (10, 20 or 30 μg/ml VEGF).

VEGF inhibits apoptosis in the cerebral cortex and basal ganglia

To determine the effects of VEGF treatment on apoptosis, the apoptotic cell numbers in the cerebral cortex and basal ganglia from rats administered PBS or VEGF (10, 20 or 30 μg/ml) were determined using the TUNEL method. As shown in Fig. 2A, VEGF treatments with 20 and 30 μg/ml VEGF significantly decreased apoptotic cell numbers when compared with the PBS group. The ratio of the apoptotic cell numbers to the total cell numbers from rats in each group was calculated and is shown in Fig. 2B. These results suggest that VEGF inhibits apoptosis in the cerebral cortex and basal ganglia.

Figure 2 VEGF inhibits apoptosis in the cerebral cortex and basal ganglia. (A) The apoptotic cell numbers in the cerebral cortex and basal ganglia from rats administered PBS or VEGF (10, 20 or 30 μg/ml) were determined using the TUNEL method. Apoptotic cells are indicated with arrows. (B) The ratio of the apoptotic cell numbers to the total cell numbers from rats in each group was calculated and is presented here.

VEGF improves growth of vascular endothelial cells

To determine the effects of VEGF on vascular endothelial cells, electron microscopy was performed to detect vascular endothelial cell changes in the ischemic peripheral brain zone of rats administered PBS or VEGF (10, 20 or 30 μg/ml). As shown in Fig. 3, for the rats in the PBS group, vascular endothelium was thin with partial necrosis and necrosis was observed in axons and dendrites. In rats administered 10 μg/ml VEGF, similar results were observed when compared with the PBS group. However, when the rats were administered VEGF at a concentration of 20 μg/ml, more proliferative endothelial cells were observed and the rats in the 30 μg/ml VEGF group presented the most proliferative endothelial cells.

Figure 3 Electron microscopy of the vascular endothelium in the ischemic peripheral brain zone of rats treated with PBS or VEGF. The rats were injected with (A) PBS, (B) 10 μg/ml VEGF (C) 20 μg/ml and (D) 30 μg/ml VEGF.

Discussion

In the present study we have shown that application of VEGF reduced apoptosis 7 days after ischemia. Infarct volume was reduced and neurological function was improved, which may be due to the effects of VEGF. Thus, VEGF may play a protective role. This effect is consistent with findings from previous studies. For example, it has been reported that VEGF stimulates axon growth and improves neuronal survival of mice on the neck and dorsal root ganglion (9). Also, VEGF has been found to promote the growth of cultured midbrain neurons (10,11). Bilateral hippocampal ischemia test of rats indicated a direct protective effect of VEGF on neurons (12). Jin et al(13) found the neurotrophic effect of VGEF in studies of the mouse hippocampal HN33 cells (14).

The concentration and dosage of VEGF used in this experiment were based on previous VEGF experiments (15,16). For example, Hayashi et al(15) applied VEGF on the surface of the cerebral cortex with similar concentrations. Zhang et al(16) applied intravenous infusion of 1 mg/kg VEGF165. In this study, slow and repeated injections were used due to the short half-life of the VEGF protein.

Previous studies focused on the effects of VEGF on angiogenesis following cerebral ischemia (15,17), and in ventricle and brain parenchyma (18,19). It has been demonstrated that the application of VEGF in the ventricle of non-ischemic adult rats would also promote angiogenesis (20), but it is generally accepted that VEGF would have a pro-angiogenic effect only after it binds to its specific receptor on endothelial cells (21,22). The effect of VEGF on brain angiogenesis should be explored in future studies.

Cell death caused by ischemic brain damage may be caused by the same pathway (23,24). Neuronal death following ischemia presents with forms of necrosis and apoptosis, related to active and passive cell death mechanisms, respectively (25–27). Activation of the apoptotic response may be an important step related to cell death, thus apoptosis may affect the final infarct volume (28). The inhibition of apoptosis and the reduction of ischemic penumbra would be effective in treating ischemic vascular diseases.

References