Cerebral angiogenesis and expression of VEGF after subarachnoid hemorrhage (SAH) in rats

Brain Research 981 (2003) 58–69 www.elsevier.com / locate / brainres

Research report

Cerebral angiogenesis and expression of VEGF after subarachnoid hemorrhage (SAH) in rats ´ * J. Josko Silesian Medical School, Chair and Department of Environmental Medicine and Epidemiology, 19 H. Jordan Str., 41 -808 Zabrze, Poland Accepted 25 April 2003

Abstract Subarachnoid hemorrhage (SAH) leads to the development of vasospasm in which endothelin-1 plays a very important role. The effect of its vasoconstricting action is hypoxia of the nervous tissue, which stimulates the release of growth factors. Vascular endothelial growth factor (VEGF) released in excessive amounts from hypoxically altered cerebrovascular endothelial cells is the most potent angiogenic factor and may enhance angiogenesis after SAH. If endothelin-1 is mainly responsible for vasospasm after SAH, it is possible that early administration of endothelin converting enzyme inhibitor or endothelin receptor antagonist may protect neurons against. The aim of the study was to establish whether prolonged vasospasm and endothelial cell hypoxia stimulate VEGF expression and, in consequence, promote angiogenesis in the central nervous system after subarachnoid hemorrhage. Investigations were also performed to determine whether the administration of phosphoramidon, an endothelin-converting enzyme (ECE) inhibitor, and BQ-123, an endothelin receptor ETA antagonist, suppresses angiogenesis and VEGF expression. Experiments were carried out in male Wistar rats injected with phosphoramidon or BQ-123 into the cisterna magna following the induction of subarachnoid hemorrhage. The brains were removed 48 h after the hemorrhage for histopathological and immunohistochemical examinations of VEGF expression and angiogenesis in the cerebral hemispheres, brainstem, and cerebellum. Statistical analysis was performed using nonparametric Wilcoxon test (P,0.05). The results obtained have shown for the first time a close correlation between endothelial hypoxia after SAH in cerebral microvessels and enhanced angiogenesis. There is also an increase in VEGF expression in cerebral vessels and neurons within the cerebral hemispheres, brainstem, and cerebellum. The administration of phosphoramidon or BQ-123 has been found to inhibit angiogenesis. Angiogenesis in the chronic phase of SAH-induced vasospasm is the result of prolonged narrowing of vessels due to excessive secretion of endothelin by damaged endothelial cells. Present results obtained indicate that it is possible to reduce or prevent the late effects of SAH, i.e., neuronal hypoxia and cerebral edema, through the inhibition of endothelin-1 induced vasospasm.  2003 Elsevier B.V. All rights reserved. Keywords: Angiogenesis; Vascular endothelial growth factor; Subarachnoid hemorrhage; Endothelin-1; BQ-123; Phosphoramidon.

1. Introduction Subarachnoid hemorrhage (SAH) frequently leads to prolonged cerebral vasospasm resulting in vascular pathology due to endothelial cell ischemia and neuronal hypoxia [54]. Endothelial damage is manifested by changes such as cellular edema, capillary occlusion and thickening of the muscle layer [52]. It has been demonstrated that endothelin-1 (ET-1) plays a key role in the development of cerebral vasospasm, and that hypoxic cells, regardless of the cause of hypoxia, produce increased amounts of vascular endothelial growth *Tel.: 148-32-272-2847; fax: 148-32-272-2847. ´ E-mail address: [email protected] (J. Josko). 0006-8993 / 03 / $ – see front matter  2003 Elsevier B.V. All rights reserved. doi:10.1016 / S0006-8993(03)02920-2

factor (VEGF), the main factor responsible for angiogenesis and enhanced vascular permeability. Our previous investigations showed a reversal of SAHinduced constriction of the cerebral basilar artery following the administration of BQ-123, a selective ETA blocker [27]. Other investigators also reported an increase in ET-1 concentrations in the cerebrospinal fluid and blood of patients after SAH [50,56]. In many cases, plasma ET-1 levels closely correlated with the thrombus size and the magnitude of circulatory disturbances [59]. Both in hypoxia, regardless of its cause, and brain tumor, the affected cells were found to produce increased amounts of VEGF, a major angiogenic factor and enhancer of vascular permeability [7,9,20,44,49]. Angiogenesis has so far been demonstrated in embryo-

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genesis [48], diabetic retinopathy [1,2], post-infarction myocardium [38], psoriasis [13], lower limb ischemia [57], endometriosis [41,53], rheumatoid arthritis [19,35] and, most of all, neoplasms [45,51]. There are no reports in literature concerning angiogenesis after SAH, except for my previous investigations which together with the recent findings have formed the basis for present understanding of this interesting problem [31–33]. The aim of the study was to establish whether prolonged cerebral vasospasm and endothelial cell hypoxia stimulate VEGF expression and, in consequence, promote angiogenesis in the central nervous system after subarachnoid hemorrhage. Investigations were also performed to determine whether the administration of phosphoramidon, an endothelinconverting enzyme (ECE) inhibitor, or BQ-123, an endothelin receptor ETA antagonist, suppresses angiogenesis and VEGF expression.

2. Materials and methods

2.1. Animals Male Wistar rats weighing 220–250 g were used. The animals were housed two per cage under controlled conditions of lighting (light on from 06:00 to 18:00 h), temperature (20–22 8C) and humidity (50–60%), with free access to water and food (Murigran, Motycz). All experiments were performed between 14:00 and 15:00 h on animals anesthetized with i.p. ketamine (100 mg kg 21 ). The animals were divided into 10 groups of 10 rats each:

(1) control2no surgical intervention (2) nonSAH2cannulation only (3) aSAH2sham SAH by artificial cerebrospinal fluid (4) SAH–subarachnoid hemorrhage (5) nonSAH1BQ-123 (6) aSAH1BQ-123 (7) SAH1BQ-123 (8) nonSAH1phosphoramidon) (9) aSAH1phosphoramidon (10) SAH1phosphoramidon The experimental protocol was reviewed and approved by the institutional care and use committee.

2.2. Cannulation of cisterna magna The cisterna magna (CM) was cannulated 7 days before SAH or aSAH to allow time for disappearance of any effects of the procedure [28,29]. A technique by Solomon et al., with slight modification, was used [55]. The procedure was carried out using an operating microscope and a stereotaxic apparatus. A midline parietooccipital and

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nape incision was made to expose the parietal and occipital bones, the atlas arch and the apicooccipital membrane. A 0.8-mm hole was drilled at the parietooccipital suture level, through which a cannula (Venocath-18, Abbot) was inserted into the cisterna magna (CM). Proper positioning of the end of the cannula in CM and free outflow of the cerebrospinal fluid through the cannula were checked.

2.3. Subarachnoid hemorrhage ( SAH) The hemorrhage was induced by intracisternal administration of 100 ml nonheparinized blood drawn through a 0.6-mm Neoflon catheter from the axillary artery prepared in the operating microscope. Sham SAH (aSAH) was induced by intracisternal administration of 100 ml artificial CSF. The BQ-123 antagonist and phosphoramidon (40 nmol in 50 ml CSF) was administered three times: 20 min. before SAH and aSAH, 60 min after SAH and aSAH, and 24 h after SAH and aSAH [11]. The same pattern of BQ-123 and phosphoramidon administration was used in the aSAH group. The results given are those from measurements made at 48 h after SAH and aSAH. Upon completion of this phase of the experiment, the animals were anesthetized with ketamine (100 mg kg 21 i.p.) and the brains were removed for histological examination.

2.4. Brain removal After thoracotomy and heart exposure, a catheter was inserted into the left ventricle and 100 ml phosphatebuffered saline (PBS), pH 7.4, was administered under 120 cm H 2 O pressure, followed by 200 ml fixative fluid (1% glutaraldehyde and 4% formaldehyde in PBS). The vessels were thus washed and fixed, and the excess fluid was allowed to escape through an incision in the right atrium. Next, the skin in the cerebrocranial region was incised and the brain was removed and immersed in the fixative for 24 h at 4 8C. After that time it was placed in cold PBS and subjected to histological examination.

2.5. Histological examination Each brain was fixed in the fixative fluid (4% formaldehyde and 1% glutaraldehyde) for 12 h at 4 8C and washed in cold PBS, pH 7.4. The brainstem and cerebellum were then dissected and the former was cut transversely at 1 / 2 pons level. The cerebrum was processed through alcohols and xylens to paraffin (Paraplast X-tra, Sigma) in a histoprocessor. Paraffin sections, 5-mm thick, were cut and stained with cresyl violet. Vascular density measurements were made using a computerized system of microscope image analysis, equipped with Kontron KS 400 software and connected to a Zeiss Axioplan 2 microscope. A 3400 magnification was used. In each preparation 25 visual fields from the dorsal

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hippocampal level of the cerebral hemispheres and from the 1 / 2 pons level of the brainstem (ventral part) were evaluated. For the procedure of capillary counting a macro, working in KS 400 system was designed. The macro was based on the interactive algorithm, discriminating clear spaces, corresponding to cross-sections of capillaries (blood cells were washed out during perfusion fixation), seen on the dark background of stained tissue. Spaces smaller than 2.25 mm were excluded from analysis, as they did not have a visible endothelial cell nucleus and thus could not be a capillary lumena. Surface vascular densities (vessels / mm 2 ), means and standard deviations were calculated.

2.6. Immunohistochemical study The immunohistochemical study was performed on 5mm thick paraffin sections. For identification of VEGF protein a monoclonal anti-VEGF (C-1) antibody (Santa Cruz Biotechnology, Santa Cruz, USA, sc-7269) was used. The antibody identifies a basic form of VEGFA , namely an epitope corresponding to amino acids 1–140 of VEGF, and is not cross-reactive with other VEGF / PIGF growth factors family members. After deparaffination of tissue sections microwave retrieval of target antigen was performed (235 min, temperature 95 8C, in citric acid buffer, pH 6.0). Then, endogenous peroxidase was blocked with 3% H 2 O 2 solution for 5 min. The following stages of procedure were performed at room temperature. The sections were incubated with blocking serum for 20 min, with primary antibody for 3 h and with secondary antibody for 30 min. After this, sections were incubated with horseradish peroxidase–streptavidin complex for 30 min and with peroxidase substrate– diaminobenzidine (DAB) for 5 min. Finally, sections were rinsed, counterstained with Meyer hematoxylin and mounted. Expression of VEGF was assessed semi-quantitatively in five cell types of brain hemispheres: ependymal cells of the lateral ventricle, choroid plexus cells of the lateral ventricle, hippocampal neurons, thalamic neurons, smooth muscle cells of subarachnoid vessels. Within the brainstem and cerebellum the following cells were investigated in ependymal cells of the fourth ventricle, choroid plexus cells of the fourth ventricle, cerebellar cortex cells (including Purkinje cells), brainstem neurons, smooth muscle cells of subarachnoid vessels. For estimation of VEGF expression the following scale was used: 0, no reaction; 1, weak (diffuse-type) reaction; 2, strong reaction in less than 50% of cells; 3, strong reaction in most cells. Evaluation of the immunohistochemical staining was carried out independently by two observers. Sections were initially placed into ‘no reaction’, ‘weak reaction’ and ‘strong reaction’ categories by agreement between the two observers over a conference microscope. For further

quantification of ‘strong reaction’, the cells under studies were counted as follows. In small areas, i.e., ventricular ependyma, choroid plexus and subarachnoid vessels all the discernible cells were assessed and the percentage of positive-stained was calculated. In large areas (hippocampal, thalamic, brainstem and cerebellar neurons) at least 1000 of the cells were assessed with the employment of an eyepiece grid at a magnification of 3400 on randomly selected 0.1-mm 2 fields. For each specimen the number of points in five studied cell types was added, so the maximal score was 15 points.

2.7. Statistical analysis For the statistical analysis the nonparametric Wilcoxon test was used. Statistical significance was as P,0.05.

3. Results

3.1. Angiogenesis in cerebral hemispheres ( Fig. 1) In control animals, mean vascular density (the number of vessels in 1 mm 2 ) was 441.26177.6 (Fig. 2). Similar densities were observed in the nonSAH and aSAH groups, the values being 370.0665.9 (P50.4) and 400.46265.0 (P50.7).After SAH induction, the density increased in relation to the control value, and was 840.26233.9 (P, 0.001). Administration of BQ-123 had significant influence on angiogenesis after SAH. In the nonSAH1BQ-123 group the vascular density was 576.46191.1 (P50.5) while in the aSAH1BQ-123 group it was 600.26279.0 (P50.4). In the SAH1BQ-123 group the density was 517.06199.4, which was statistically significant compared to the SAH group (P,0.05). Administration of phosphoramidon also had significant influence on angiogenesis after SAH. In the nonSAH1phosphoramidon group the vascular density was 370.0665.9 (P50.9), while in the aSAH1 phosphoramidon group it was 414.26239.7 (P50.7). The differences were not statistically significant. In the SAH1 phosphoramidon group the density was 458.96208.4, which was statistically significant compared to the SAH group (P,0.001). An example of a histological picture of angiogenesis in cerebral hemispheres in the SAH group is presented in Fig. 3.

3.2. Angiogenesis in brainstem and cerebellum ( Fig. 4) No statistically significant differences in vascular density were observed between the control and experimental groups. The values obtained in particular groups were: control, 443.06170.4; nonSAH, 539.56373.4 (P50.4); aSAH, 611.46141.7 (P50.3); and SAH, 624.66211.8 (P50.1). After BQ-123 administration, the densities in particular groups did not change significantly and were: nonSAH1BQ-123, 521.26179.6 (P50.9); aSAH1BQ-

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Fig. 1. Angiogenesis in cerebral hemispheres (number of vessels / mm 2 ). *P,0.001, as compared with controls, **P,0.05, as compared with SAH, ***P,0.001, as compared with SAH. Results of groups nonSAH and aSAH not presented on any figures, because as an additional control groups they did not reveal any statistical significance compared to the control group.

123, 471.16209.9 (P50.5); SAH1BQ-123, 503.76252.2 (P50.2). After phosphoramidon administration, the densities in particular groups did not change significantly and

were: nonSAH1phosphoramidon, 517.76145.6 (P50.9); aSAH1phosphoramidon, 451.26149.9 (P50.5); SAH1 phosphoramidon, 550.76379.1 (P50.5). An example of a

Fig. 2. Angiogenesis in cerebral hemispheres: control group. Rat hippocampal cortex. Scarcely distributed capillaries, including larger ones are visible (arrow). The mean vessel density in this specimen was 441.2 vessels / mm 2 . Cresyl violet stain. Magnification, 3400.

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Fig. 3. Angiogenesis in cerebral hemispheres: SAH group. Rat hippocampal cortex. Numerous vessels predominantly small capillaries are visible (arrows). The mean vessel density in this specimen was 860 vessels / mm 2 . Note empty vessel lumena, resulting from perfusion fixation. Cresyl violet stain. Magnification, 3400.

Fig. 4. Angiogenesis in brainstem and cerebellum (number of vessels / mm 2 ). Results of groups nonSAH and aSAH not presented on any figures, because as an additional control groups they did not reveal any statistical significance compared to the control group.

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histological picture of angiogenesis in brainstem in the SAH group is presented in Fig. 5.

3.3. Expression of VEGF in cerebral hemispheres ( Fig. 6) In the control group expression of VEGF was 7.261.2. Cannulation of cisterna magna alone (nonSAH group) had no significant influence on VEGF expression (7.561.3, P50.9). Administration of an artificial cerebrospinal fluid (aSAH group) also did not change significantly VEGF expression (6.861.5, P50.2). In the SAH group VEGF expression was significantly increased (10.361.6, P,0.05) as compared with all reference groups, i.e., control, nonSAH and aSAH. Intracisternal administration of BQ-123 in nonSAH animals had no significant influence on VEGF expression (8.060.9, P50.1). Similarly, administration of BQ-123 did not change significantly VEGF expression in the aSAH group (7.361.2, P50.2). Administration of BQ-123 in the SAH group also had no significant influence on VEGF expres-

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sion (9.661.9, P50.7). Intracisternal administration of phosphoramidon in nonSAH animals had no significant influence on VEGF expression (8.561.6, P50.1). Similarly, administration of phosphoramidon did not change significantly VEGF expression in the aSAH group (7.261.7, P50.6). Administration of phosphoramidon in the SAH group also had no significant influence on VEGF expression (10.761.2, P50.7). An example of a histological picture of expression of VEGF in cerebral hemispheres in the SAH group is presented in Fig. 7.

3.4. Expression of VEGF in the brainstem and cerebellum ( Fig. 8) In the control animals expression of VEGF was 6.661.3; in nonSAH 6.561.5; and in aSAH 7.560.5. The differences were not significant statistically (P50.8 and P50.1). A significant increase of VEGF expression was found in rats after SAH (10.061.4) P,0.05 as compared with controls, nonSAH and aSAH. Intracisternal administration of BQ-123 in nonSAH and aSAH animals had no

Fig. 5. Angiogenesis in brainstem: SAH group. Ventral surface of the pons. Scarcely distributed capillaries, including larger ones are visible (arrow). The mean vessel density in this specimen was 410 vessels / mm 2 . HE stain. Magnification, 3400.

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Fig. 6. Expression of VEGF in cerebral hemispheres. *P,0.05, as compared with controls. Results of SAH1BQ-123 and SAH1phosphoramidon groups did not reveal any statistical significance compared to the SAH group. Results of groups nonSAH and aSAH not presented on any figures, because as an additional control groups they did not reveal any statistical significance compared to the control group.

Fig. 7. Expression of VEGF in cerebral hemispheres: hippocampal cortex, SAH group. A strong reaction in neurons is seen (arrows). Immunostaining for VEGF. Magnification 3200.

significant influence on VEGF expression (6.561.5, P5 0.2; and 8.361.2, P50.1; respectively). Administration of BQ-123 in the SAH group also had no significant influence on VEGF expression (10.062.9, P50.7). Intracisternal

administration of phosphoramidon in nonSAH and aSAH animals had no significant influence on VEGF expression (8.161.3, P50.2; and 8.261.2, P50.1; respectively). Administration of phosphoramidon in the SAH group also

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Fig. 8. Expression of VEGF in the brainstem and cerebellum. *P,0.05, as compared with controls. Results of SAH1BQ-123 and SAH1phosphoramidon groups did not reveal any statistical significance compared to the SAH group. Results of groups nonSAH and aSAH not presented on any figures, because as an additional control groups they did not reveal any statistical significance compared to the control group.

had no significant influence on VEGF expression (12.261.0, P50.1). An example of a histological picture of expression of VEGF in brainstem in the SAH group is presented in Fig. 9.

4. Discussion The present study focuses on the chronic phase of vasospasm after SAH and hence the determination of angiogenesis and VEGF expression after 48 h. The increase in vascular density at 48 h was nearly 2-fold. The administration of BQ-123, a specific endothelin ETA receptor antagonist or phosphoramidon, an ECE inhibitor, reduced the angiogenesis after SAH in cerebral hemispheres. No significant increase in the density of brainstem capillaries was observed. These differences regarding reaction to hypoxia could be explained by special features of cerebral circulation, namely its spatial distribution. Cerebral blood flow (CBF) is near by two times higher in the cortex in comparison with subcortical areas and it is still subjected to redistribution, depending on actual physiological requirements [6,8]. The cerebral cortex is more susceptible to hypoxia than the brainstem. Furthermore, the ratio neuronal cell body / neuronal processes (dendrites and neurites) number is much higher in the cortex than in the brainstem, thus explaining its vulnerability during hypoxia [8,14]. Studies by other authors have shown that cerebral circulation adapts to hypoxia in two ways, through angiogenesis and dilatation of microvessels. The reaction to hypoxia depends on the brain area and is not homogeneous. In the cerebral cortex, striatum and hippocampus the number of vessels per 1 mm 2 tissue rises significantly,

whereas in the cerebellum and medulla oblongata there is only a tendency to an increase [47]. The potential mechanism of prolonged SAH-induced vasospasm involves many neurogenic, local biochemical and, most of all, endothelial factors. Among the latter, endothelin-1 which causes a significant, long-lasting vasospasm with resultant damage of ischemic endothelial cells plays a major role. Vascular endothelial cells are the main source of ET-1 after SAH, other sites of its production being ventricular choroid plexus, infiltrating macrophages [16], astrocytes [51], neurons [37], and glia cells [39]. Endothelin-induced vasospasm involves not only microvasculature but also vessels with large diameter. In an earlier study in rats, the cerebral basilar artery was found to be constricted due primarily to the action of endothelin -1, as shown by the reversal of vasospasm following the administration of BQ-123, an ETA receptor antagonist [27], or phosphoramidon, an ECE inhibitor [30]. One can assume that endothelial cells hypoxia after SAH is caused mainly by an increase of ET-1 concentration in these cells and in the blood. This increase is counterbalanced by the synthesis of vasodilatatory substances in endothelial cells, especially the nitric oxide (NO), stimulated by endothelin itself via ET B1 receptor and by activating constitutive nitric oxide synthase (eNOS) [24]. It is suggested that in the vasospasm after SAH the relation between vasoconstrictive and vasodilatatory substances is disturbed in favour of constrictors. Furthermore, the hemoglobin released from extravasated blood after SAH directly causes constriction of vascular smooth muscle cells and simultaneously indirectly induces vasospasm by activating ET-1 production and inhibiting synthesis of NO [21,40]. Free oxygen radicals, reducing the action of NO, are

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Fig. 9. Expression of VEGF in brainstem and cerebellum: SAH group. Cerebellar cortex. A diffuse-type staining in Purkinje cells (arrows). Immunostaining for VEGF. Magnification, 3200.

generated in excess [4,21]. Consequent deficiency of NO is strongly connected with vasospasm [17]. In 1998, Chua et al. [10] demonstrated that lysophosphatidic acid (LPA) markedly elevated endothelin-1 expression in endothelial cells. mRNA levels for endothelin1 increased within an hour after addition of LPA and subsequently declined. LPA stimulated AP-1 transcription factor binding as a result of activation of ET-1 gene activity. Thus, the authors concluded that increasing ET-1 by LPA could augment and prolong the vasoconstrictive actions of this lipid mediator. The prolonged vasospasm after SAH induces hypoxia, which is the direct cause of increased expression of VEGF, and as we found in our previous study, also stimulates brain angiogenesis [31]. The above findings suggest that endothelial hypoxia is also responsible for disturbed angiogenesis, which is manifested by an increase in the production of VEGF, a potent angiogenic factor, released in abundance from ischemic areas [22,36]. Endothelin-1 is also known to induce production of VEGF in human vessels [46].

Hypoxia, endothelin-1, VEGF, FGF-b, TNFa, hemoglobin, NO, and many other substances induce also heme oxygenase (HO-1). HO-1 induces expression hypoxia-inducible factor 1a (HIF 1a), including the gene encoding VEGF [42]. The expression of VEGF closely correlates with the expression of its receptors in the same hypoxic endothelial cells. Moreover, VEGF modulates the activity of its receptors, particularly Flk-1, which have high binding affinity for VEGF [43]. The cerebral ischemia in animals is usually studied on the model of middle cerebral artery occlusion. These studies revealed an increase of VEGF expression in ischemic areas of the brain [23,25,26]. Hypoxia strongly induces VEGF expression in vivo and in vitro models. Animal studies revealed an increase of VEGF expression as early as 3 h after induction of hypoxia, with a peak intensity after 48 h [5]. It is suggested that sphingosine 1-phosphate (SPP) and phosphatidic acid (PA) induce many important endothelial cell responses associated with angiogenesis, including

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liberation of endothelial cells from established monolayers, chemotactic, migration, proliferation, adherens junction assembly and morphogenesis into capillary-like structures [18]. That bioactive lipids enhanced the ability of VEGF and FGF-b to induce angiogenic responses [18]. VEGF expression can be induced by exposure to hypoxia and this expression is mediated by hypoxia-inducible factor 1 (HIF-1) [12]. HIF-1 activates the transcription of genes whose products are required angiogenesis (plasminogen activator inhibitor, VEGF, endothelin-1, inducible nitric oxide synthase, and insulin-like growth factor) [12]. VEGF is more potent as a permeability factor than histamine or bradykinin [15]. In local as well as in diffuse brain ischemia angiogenesis is accompanied by increased permeability of endothelium contributing to the brain edema [3,34]. This increased permeability may last for several days or even months. In a mouse model, administration of VEGF antagonist—mFlt (1–3)—IgG after chronic hypoxia reduced brain edema, confirming the role of VEGF in its pathogenesis [58]. VEGF increases permeability of the blood–brain barrier (BBB) by inducing the growth and secretion of NO and stimulating cGMP activity [20] Dysfunction of BBB enables the penetration of plasma compounds to the nervous tissue, inducing the proliferation of microglia. The microglial cells release NO and superoxide free radicals, which are highly toxic for neurons. It is intriguing to ask whether induction of angiogenesis in brain hemispheres after SAH is a favourable phenomenon, taking into account simultaneous induction of brain edema by VEGF? To answer this question further studies are needed, with examination of cerebral blood flow and functional status of neurons in the presence of extravasated plasma. To date, cerebral angiogenesis is thought to be disadvantageous and undesirable effect, accompanying brain edema and neoplasms. So, it seems reasonable to prevent vasospasm after SAH by applying endothelin receptor blockers, thus eliminating the action of a strong vasospasm inducer. In our previous studies we have demonstrated that blockage of ETA receptors with a pentapeptide BQ-123 or blockage of ECE with phosphoramidon not only inhibits vasospasm of basilar artery after SAH [27] but also restrains angiogenesis [30,31]. In the presented study it was demonstrated that administration of BQ-123 or phosphoramidon does not influence the expression of VEGF. The significant increase of VEGF expression in cerebral hemispheres and different areas of the brainstem is merely an indicator of the brain ischemia caused by vasospasm. Induction of ischemia-induced angiogenesis in the brain is a delayed phenomenon, most probably compensating effects of long-term hypoxia of endothelial cells. The role of VEGF, besides its angiogenic and mitogenic action, especially after SAH, remains unknown and justifies further studies on VEGF in brain ischemia.

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Acknowledgements The present study was supported by grants from Silesian Medical School.

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