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

Abstract

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.

Figures

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).

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.

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.

Figure 5

The relative change in CBF after MCA occlusion was unaffected by pretreatment with mFlt(1–3)-IgG. Relative CBF was measured using laser Doppler flowmetry with a 1.0-mm straight steel probe positioned close to the right parietal bone above the MCA territory. Relative blood flow was monitored before and 10–15 minutes after occlusion of the MCA at the 3 positions shown in b. Relative flow values represent the mean percent change in flow normalized to the pre-ischemic level recorded from position B (mean ± SEM). A reduction in CBF was seen upon occlusion for all mice examined, but was unaffected by pretreatment with mFlt(1–3)-IgG.

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).