VEGF antagonism reduces edema formation and tissue damage after ischemia/reperfusion injury in the mouse brain

VEGF is mitogenic, angiogenic, and a potent mediator of vascular permeability. VEGF causes extravasation of plasma protein in skin bioassays and increases hydraulic conductivity in isolated perfused microvessels. Reduced tissue oxygen tension triggers VEGF expression, and increased protein and mRNA levels for VEGF and its receptors (Flt-1, Flk-1/KDR) occur in the ischemic rat brain. Brain edema, provoked in part by enhanced cerebrovascular permeability, is a major complication in central nervous system pathologies, including head trauma and stroke. The role of VEGF in this pathology has remained elusive because of the lack of a suitable experimental antagonist. We used a novel fusion protein, mFlt(1-3)-IgG, which sequesters murine VEGF, to treat mice exposed to transient cortical ischemia followed by reperfusion. Using high-resolution magnetic resonance imaging, we found a significant reduction in volume of the edematous tissue 1 day after onset of ischemia in mice that received mFlt(1-3)-IgG. 8–12 weeks after treatment, measurements of the resultant infarct size revealed a significant sparing of cortical tissue. Regional cerebral blood flow was unaffected by the administration of mFlt(1-3)-IgG. These results demonstrate that antagonism of VEGF reduces ischemia/reperfusion–related brain edema and injury, implicating VEGF in the pathogenesis of stroke and related disorders.

J. Clin. Invest.104:1613–1620 (1999).

Brain edema is a major and often life-threatening complication of a variety of brain injuries. Clinically, late stage edema, as defined by the disruption of the blood-brain barrier, is the most common form of brain edema (1). A number of physiological conditions need to be present for the formation of cerebral edema, including altered cerebrovascular permeability, hydrostatic pressure, and appropriate osmotic and colloidal gradients. Increased cerebrovascular permeability is, however, the single most important factor for the development of brain edema.

VEGF is a potent mediator of vascular permeability (2–4). VEGF is responsible for the plasma extravasation associated with tumor microvasculature, and its effect on dermal microvessels is much greater than any other known mediators of vessel permeability (e.g., histamine) (5). Diffuse VEGF mRNA expression has been observed in adult rat brain, but at relatively low abundance (6). However, reduced oxygen tension is a known trigger for VEGF expression (7), and enhanced levels of VEGF and its receptors, Flt-1 and Flk-1/KDR, were shown to be induced in the rat brain after focal cerebral ischemia (8–10). Similar expression has recently been shown to occur in the human brain after ischemic stroke (11). The role of VEGF in the pathogenesis of stroke, and the formation of cerebral edema is unclear with contradictory experimental observations cited in the literature. For example, Nag et al. (12) used a cortical cold-injury rat model to demonstrate the presence of murine VEGF in permeable pial vessels and arterioles within the damaged tissue, and, from this observation, they inferred that VEGF is 1 of several factors that may mediate edema formation. On the other hand, Hayashi et al. (13) reported that VEGF itself, when applied topically to the surface of a reperfused rat brain after transient cerebral artery occlusion, reduced ischemic brain damage, infarct volume, and edema formation.

To date, the role of VEGF in the pathophysiology of cerebral infarction remains unknown. Because the majority of research studies are performed on rodents, the lack of a suitable pharmacological antagonist effective in either rat or mouse prevents a clear understanding of the contribution of VEGF in the pathogenesis of stroke and related disorders. Recently, however, Ferrara et al. (4) took advantage of the high affinity and cross-reactivity of VEGF binding to the tyrosine kinase receptor, Flt-1 (14, 15). They demonstrated that the truncated receptor, consisting of the first 3 immunoglobulin-like domains of the murine Flt-1 fused to a Fc-IgG from the mouse, mFlt(1–3)-IgG, is an effective inhibitor of rat and mouse VEGF. Furthermore, this fusion protein has an adequate systemic half-life to be suitable for in vivo efficacy studies. Using this fusion protein to sequester VEGF, we have been able to show that VEGF is partly responsible for the cortical edema formation in a model of focal ischemic injury with reperfusion.

To investigate the physiological role of VEGF in the pathogenesis of stroke we chose a murine model of focal cerebral ischemia with reperfusion. Occlusion of the right MCA together with the right CCA with reperfusion produces a reproducible and discrete focal infarct in the cerebral cortex (16). In this model, delayed tissue damage secondary to the transient arterial occlusion is found with profound tissue reactivity and edema occurring 1–2 days after the ischemia insult, and its severity depends upon the duration of occlusion (18). The extent of the edematous tissue can be visualized using T2-weighted, fast-spin echo MRI sequence obtained 24 hours after the onset of ischemia. As illustrated in Figure 1, a pronounced hyperintensity on the T2-weighted scan was evident at 24 hours. It covered a large volume of the right cortex, extending from the primary motor cortex in the forebrain and as far as the interaural line and included the primary visual cortex. The edematous changes were confined to the cortex and did not extend beyond the corpus callosum. The lesion volume, as defined by the region of high signal intensity within the ischemic cortex, was greater after 45 minutes of ischemia than it was after 30 minutes (30.5 ± 1.6 mm³ and 22.5 ± 3.4-mm³, mean lesion volume ± SEM, for 45- and 30-minute occlusion times, respectively; P < 0.05). This is consistent with the notion that early reperfusion is beneficial despite the occurrence of secondary injury associated with reflow. Representative T2-weighted MRI (i.e., the animal closest to the mean for each group) recorded 24 hours after 45 minutes of focal ischemia are shown in Figure 1. There is a clear reduction in the extent of the cerebral edema associated with mFlt(1–3)-IgG treatment compared with the control group. The volume of the edematous tissue was quantified, and the results are shown in Figure 1b. Administration of mFlt(1–3)-IgG caused a significant reduction in the volume of cerebral edema as defined by the region of hyperintensity on the T2-weighted MRI scan acquired 1 day after onset of ischemia (∼30% reduction; P < 0.05, Student’s t-test; n = 15 and 16 in control and treatment groups, respectively).

Figure 1

mFlt(1–3)-IgG treatment reduction of cortical edema volume is demonstrated by T2-weighted MRI. (a) Representative MRI data for the 2 mice closest to the mean for each group are shown. A series of 1-mm contiguous coronal slices were made covering the entire mouse brain. The cortical edema, as defined by the region of T2 hyperintensity, is reduced with mFlt(1–3)-IgG treatment (bottom row) compared with control group (top row). The area of high signal intensity above the skull is due to inflammation associated with the surgical procedure. The volume of edematous tissue is clearly delineated by the region of high signal intensity within the cerebral cortex, and, in the control animal, extends from the frontal cortex caudally to almost the full extent of the cerebral cortex. (b) mFlt(1–3)-IgG produced a significant reduction in the lesion volume, as measured from the T2-weighted MRI (data from the individual animals are shown together with the mean lesion volume ± SEM; *P < 0.05).

As evident from the histology, this lesion continues to evolve for many days, or possibly weeks, after its onset and eventually produces a core of pan-necrosis and cavitation, with little evidence of selective neuronal death in the surrounding tissue (Figure 2). To establish whether mFlt(1–3)-IgG–mediated reduction of acute cerebral edema conferred long-term salvage of cortical tissue in this model, the surviving animals (25 of 31; 4 from the control group and 2 from the treated group died) were re-examined between 8 and 12 weeks later. At this time, the final size of the infarct can be deduced from the degree of remaining cortical tissue. This can be determined using high-resolution anatomical MRI that accurately depicts the lesion volume. Again, a fast-spin echo technique was used, but with more signal averaging and reduced T2-weighting to allow for sufficient signal-to-noise for higher resolution studies. Using this imaging sequence, we were able to obtain in-plane resolution of about 125 μm with 0.5-mm-thick slices. The imaging time for this study was about 30 minutes per mouse.

Figure 2

Histology revealed a discreet cortical lesion with cavitation and gliosis several months after ischemia/reperfusion injury. Temporary ligation of the MCA in this model induced a consistent focal lesion. In each animal, evaluated 8–12 weeks after injury, focal infarction induced discrete cavitation and loss of a unilateral focal area of tissue in the cerebral cortex involving the somatosensory cortex, predominantly the S1 forelimb and barrel fields with extension into the secondary somatosensory cortex. (a) The extent of tissue damage is seen from the low-magnification H&E sections located approximately 0.5 mm anterior and 2 mm posterior to Bregma. The motor cortex was unaffected. This cortical lesion was typically full thickness and extended from the external capsule with corpus callosum as the inner limit and the meninges as the outer limit. (b) Higher magnification H&E section. (c) The Cresyl violet and Luxol fast blue section. In each animal, the infarcted area was a cavitary lesion that contained a dense population of foamy macrophages (gitter cells), rare neutrophils, and few stromal elements that supported small vessels. The small vessels within the lesion commonly had a mild perivascular infiltrate of lymphocytes. With the exception of a mild increase in glial cells and occasional necrotic neurons in the immediately adjacent neuropile, the discrete infarct was bordered by histologically normal tissue, including the adjacent cerebral cortex and the underlying caudate putamen.

The size of the infarct was determined by delineating the amount of remaining cortex and comparing it with the contralateral hemisphere. The size of the cortical infarction is significantly reduced by the administration of mFlt(1–3)-IgG measured 8–12 weeks later (30% reduction in infarct size (P < 0.01, Student’s t-test; n = 11 and 14 in control and treatment groups, respectively) (Figure 3). There was good agreement between the infarct volume measured by MRI and that determined using conventional histology (r = 0.80, n = 12) (Figure 4). Further, from the MRI studies, the severity of tissue edema as measured at 24 hours correlated with size of the infarct measured 8–12 weeks later (r = 0.62, n = 12) (Figure 4). Accordingly, the treated animals exhibited a reduction in the development of cerebral edema, which may provide further preservation of cortical tissue.

Figure 3

mFlt-(1–3)-IgG treatment affords long-term tissue salvage as demonstrated by high-resolution anatomical MRI. The size of the infarct can be readily delineated from the coronal MRI sections by measuring the area of the remaining cortex and comparing it with the contralateral hemisphere. Using this approach, the size of the cortical damage was found to be significantly reduced in the mFlt(1–3)-IgG–treated group compared with control group (*P < 0.01).

Figure 4

The histological measure of the infarct size correlates to MRI predictors of infarction. (a) The appearance of the cortical infarction by histology (bottom row), together with the equivalent high resolution MRI (top row) at 8–12 weeks after onset of ischemia. (b) The infarction volume determined by histology was compared with the infarct volume measured with high-resolution, anatomical MRI and the extent of edema as seen from the early T2-weighted MRI. Good correlation between the histology and MRI measurement made at both 8–12 weeks and 24 hours after ischemia/reperfusion was seen.

It should be noted that although a reduction in the area of cortical edema, as defined on the T2-weighted images, correlates with long-term preservation of cortical tissue, the extent of tissue affected is much greater than the ultimate size of the infarction. This observation supports the notion that although spontaneous resolution of tissue edema can occur, edema itself may contribute to the pathogenesis of infarction in this model. The discrepancy between the high-resolution anatomical MRI measurement of cortical damage and that measured by histology presumably reflects the difference in the accuracy of each approach and is greater in cases with smaller lesion volumes. MRI lacks the resolution offered by the histological analysis, and errors from inaccuracies in slice thickness and partial volume can confound the measurement.

In a separate study designed to determine the effects of mFlt(1–3)-IgG on cerebral blood flow, laser Doppler was used to record the changes in relative flow during occlusion of the MCA. A reduction in relative blood flow to 5–10% of the baseline value was seen in all animals examined. There was no significant difference in the mean relative tissue perfusion between the control and mFlt(1–3)-IgG and treated animals in any of the 3 areas examined (Figure 5). To determine the extent of the edematous pathology MRI was performed on these animals 24 hours after surgery. A significant reduction in the lesion volume, as defined by the region of T2 hyperintensity, was seen in this study (22.5 ± 3.4 mm³ and 5.5 ± 3.3 mm³ mean lesion volume for control and mFlt(1–3)-IgG–treated groups, respectively; P < 0.01) (Figure 6). These data show that the reduction in the cortical edema, as assessed by T2-weighted MRI, afforded by pretreatment with the fusion protein is not mediated by a change in the CBF during ischemia. Further, a single dose of mFlt(1–3)-IgG in this model of mild transient ischemia is efficacious.

Figure 6

A single dose of mFlt(1–3)-IgG given 30 minutes before onset of ischemia affords a significant reduction in lesion volume. The lesion volume is defined as the region of single hyperintensity on the T2-weighted MRI recorded 1 day after 30 minutes of ischemia with reperfusion. Values for individual animals are shown together with their means ± SEM (*P < 0.01).

The formation of cerebral edema and central nervous system (CNS) inflammation are often a late stages in the pathogenesis of infarction resulting from cerebral ischemia. The beneficial effects of prompt and adequate restoration of blood flow after an acute ischemic event may be undermined by reperfusion injury to the cerebral microvasculature comprising the cerebral microcirculation, and thus contribute to the formation of cerebral edema and the potentially fatal developments that follow (19, 20). Numerous biochemical mediators of vascular permeability have been proposed, but, as of yet, no therapies specifically aimed at modulating cerebrovascular permeability have been used. Corticosteroids are used for acute therapy of cerebral edema, and, although the mode of action is not certain, it has been hypothesized that they may act by directly altering vascular permeability at the capillary bed level or by the inhibition of the production of permeability factors by tumor cells. Beneficial effects of corticosteroids have been demonstrated in patients with primary or metastatic brain tumors, but most clinical studies of craniocerebral trauma have failed to demonstrate efficacy.

Currently, nearly all forms of therapy for brain edema remain nonspecific and are directed at reducing intracranial pressure — the principal complication of brain edema — rather than reducing edema formation specifically. It is interesting to note that dexamethasone, used for the treatment of cerebral edema associated with neoplasia, has been shown to inhibit the biological activity of VEGF (21–23). Because dexamethasone is known to be efficacious in reducing the formation of brain edema in animal models of stroke, it is interesting to speculate that the observed protective effect of dexamethasone may be mediated through its action on VEGF.

With the exception of specialized cell types, such as epithelial cells in the choroid plexus, only diffuse expression of VEGF has been observed in adult brain (24). However, transient hypoxia/ischemia results in marked increase in VEGF and its receptors at both the mRNA and protein level. Under systemic hypoxia, for example, VEGF expression was seen in glial cells as well neurons in the hippocampus and dentate gyrus (24). Expression of VEGF and its receptors after occlusion of the MCA in the rat brain has been studied by a number of investigators (8–10). Enhanced VEGF expression was seen within the ischemic territory and was found to be localized in most cell types, including neurons and glia, as well as infiltrating macrophages. VEGF expression has been identified with cells associated with CNS inflammation, including microglial cells/macrophages (25) and reactive astrocytes, suggesting a central role in CNS inflammation (26). VEGF expression does not appear to be localized to endothelial cells in vivo after a hypoxic insult, although marked receptor expression is noted, suggesting a paracrine action under these conditions (24). In stroke studies, although the pattern and temporal profile of expression is dependent on the model as well as the techniques used for biochemical analysis, in general, expression of VEGF and its receptors occurs hours after onset of hypoxic-ischemia insult and persists for several days (8, 10). The time course for VEGF expression is consistent with the formation of cerebral edema and the inflammation associated with these models of stroke. Further, it has been shown that prolonged exposure to hypoxia results in a transient increase in VEGF that is maximal 1–2 days after onset of the hypoxic insult, and does not persist despite continued hypoxia (27).

VEGF is also known as vascular permeability factor, because of its ability to induce vascular leak (28). Some of the original assays used for the characterization of VEGF were based on vascular permeability enhancement to plasma protein, as demonstrated using the Miles assay. More recently, it has been shown that VEGF has a dramatic increase in hydraulic conductivity in isolated, perfused frog mesentery (2), confirming that VEGF has a specific action on microvessel permeability that is independent of any hemodynamic alterations that may be induced by VEGF. Although the relationship between the endothelial cell permeability enhancement and its mitogenic and angiogenic properties remain uncertain (28), VEGF and its endothelial cell receptors are overexpressed in a number of non-neoplastic pathologies. These are characterized by hyperpermeability to circulatory plasma proteins, including retinopathies, rheumatoid arthritis, psoriasis, and other inflammatory conditions. Numerous studies exist that provide corroborative evidence linking VEGF to the formation of edema. The high abundance of VEGF in brain tumors, together with the prevalence of peritumor edema has lead a number of groups to investigate the correlation between the extent of cerebral edema, often as determined from the MRI exam, with the levels of VEGF from tissues samples (29–32). All of these studies demonstrate an association between VEGF expression and vasogenic edema, suggesting a role for enhanced microvascular permeability effects of VEGF in the formation of cerebral edema. These studies cannot, however, distinguish cause from effect. Without the appropriate pharmacological antagonist, the contribution of VEGF to the formation of cerebral edema could not be verified.

The underlying mechanisms responsible for the reduction in cortical tissue damage seen in the present study remain unclear. Although it is possible that the observed effects could be mediated via a direct consequence of changes in vascular water flux across the blood-brain barrier, we have not measured vascular permeability directly. It should be stated that measurements of the extravasation of a particular solute (for example, albumin using Evans blue staining) are dependent upon a number of different variables and are not a direct measure of permeability. Furthermore, although a number of investigators have demonstrated extravasation of Evans blue dye after focal ischemia, it has also been shown that albumin plays essentially no role in edema formation (33). Because the primary objective of the present study was to assess the pathophysiological consequences of VEGF antagonism, we chose to monitor brain edema directly using T2-weighted MRI techniques. Other physiological actions of VEGF, for example vasodilation (34) and VEGF-induced hypotension (35), could confound interpretation of our experimental observations. Although they cannot be excluded as possible contributory mechanisms, hemodynamic perturbations during the occlusion period was not detected in our study.

In summary, we have shown that VEGF expression after transient ischemia in the brain contributes to the formation of cerebral edema. We have established that mFlt(1–3)-IgG treatment significantly reduces the acute appearance of cortical edema, and this is likely to offer long-term salvage of cortical tissue. Further work is required to identify the exact mechanisms responsible for the protection seen in this pathology, nevertheless, these results suggest a novel strategy for the management and prevention of cerebral edema associated with acute neurological disorders such as stroke and closed-head injury.

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