Increased Expression of Vascular Endothelial Growth Factor Attenuates Contusion Necrosis without Influencing Contusion Edema after Traumatic Brain Injury in Rats

Abstract

To clarify the role of vascular endothelial growth factor (VEGF) in the formation of contusion edema and necrosis after traumatic brain injury, we examined the time course of changes in the VEGF expression (enzyme-linked immunosorbent assay), cerebrovascular permeability (extravasation of Evans blue), and water content (dry-wet weight method) of the contused brain tissue in a cortical impact injury model using rats. In addition, we tested the effects of administration of bevacizumab (VEGF monoclonal antibody) on changes in the cerebrovascular permeability and water content of the contused brain tissue, as well as the neurological deficits (rota rod test) and volume of contusion necrosis. Increased VEGF expression was maximal at 72 h after injury (p<0.003). Increases in cerebrovascular permeability and water content, however, became maximal within 24 h (p<0.001) after injury (p<0.01), respectively. Administration of bevacizumab did not influence these changes in cerebrovascular permeability and water content, but led to a significant rise in the neurological deficits at 72 h–14 days (p<0.05 or 0.01) and the volume of contusion necrosis at 21 days (p<0.001) after injury. These findings suggest that increased expression of VEGF after injury does not contribute to the formation of contusion edema, but attenuates the formation of contusion necrosis. This is probably because of an increased angiogenesis and improved microcirculation in the areas surrounding the core of contusion.

Introduction

Vascular endothelial growth factor (VEGF) is a protein that exerts pronounced effects on both vascular permeability and angiogenesis.^1
–3 Five phenotypes of VEGF (VEGF-A, -B, -C, -D, and -E)^4,5 and three VEGF receptors (VEGFR-1, -2 and -3) have been identified.^6,7 Among the three VEGFRs, VEGFR-2 (Flk-1) is thought to play the most important role in facilitating the proliferation, migration, lumen formation, and survival of endothelial cells, and thereby affects vascular permeability and angiogenesis.^8,9

An increase in VEGF expression has been demonstrated after ischemic brain injury.^10
–12 Several lines of evidence have indicated that increased VEGF expression contributes to edema formation through increased cerebrovascular permeability, or improves the microcirculation through angiogenesis after ischemic brain injury.^{10,13

–17} It has been suggested that the increased VEGF expression after ischemic brain injury may be beneficial for protecting the brain tissue if increased cerebrovascular permeability or edema formation is controlled by other means.^18,19 Little is yet known, however, regarding the changes in VEGF expression and their role after traumatic brain injury (TBI).^20
–22

Cerebral contusion after after TBI is characterized by contusion edema,²³ which is often believed to be caused at least in part by an increased cerebrovascular permeability, in addition to cell swelling by microthrombosis surrounding the core of contusion.^24,25 It is possible, therefore, that an increase in VEGF expression, if it occurs after TBI, could either worsen the contusion edema through an increase in cerebrovascular permeability or reduce the volume of contusion necrosis by an increase in angiogenesis.

In the present study, to clarify the role of VEGF in the formation of contusion edema and contusion necrosis after TBI, we examined the time course of changes in the VEGF expression, cerebrovascular permeability, and water content of the contused brain tissue in a cortical impact injury model using rats. Further, we tested the effects of administration of bevacizumab, VEGF monoclonal antibody, on changes in the cerebrovascular permeability and water content of the contused brain tissue.

An increase in cerebrovascular permeability may accelerate contusion edema. In contrast, an increase in angiogenesis may protect neuronal cells from the ischemia induced by microthrombosis. These two events would have opposite effects on the neurological deficits and volume of contusion necrosis after injury. We therefore also examined the effects of administration of bevacizumab on neurological deficits and volume of contusion necrosis, seeking to clarify the overall consequences of changes in VEGF expression and the potential role of therapeutic modification of VEGF activity in TBI.

Methods

Seven- to 9-week–old adult male Sprague-Dawley rats, weighing 200–300 g each, were used. The experiments were performed in compliance with the Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research (National Research Council, National Academy Press, Washington, DC, 2003) and the Guidelines for Animal Experiments, Nihon University School of Medicine, and in accordance with the Laboratory Animal Handling Guidelines, Nihon University School of Medicine.

Induction of cerebral contusion

The cephalic region was fixed on a stereotaxic apparatus under halothane anesthesia, and the rectal temperature was maintained at 37.0±0.5°C using a heating pad. A cerebral contusion was induced using a controlled cortical impact device (Virginia Commonwealth University Medical Center, Richmond, VA) by exposing the cranial bone and by forming a hole with a diameter of 6 mm in the left parietal cortex between the bregma and lambda. A 5-mm flat-tipped impactor was placed on the dural surface at a 20-degree angle, and injury was induced using a 6-m/s impact velocity, 3-mm impact depth, and 150-millisecond impact dwell time.²⁶ In a separate group of animals (control group), only the cranial hole was formed.

Quantification of VEGF expression

The VEGF expression was quantified using an enzyme-linked immunosorbent assay kit.²² The rats were beheaded under pentobarbital anesthesia (50 mg/kg, intraperitoneally [i.p.]), and 50 mg of the cerebral cortex was collected from the area surrounding the cerebral contusion. After the samples had been homogenized, the supernatant fluid was measured with a fluorescence photometer by centrifugation. The amount of VEGF was determined at 3, 6, 12, 24, and 72 h, and 5 and 7 days after formation of the cerebral contusion (n=5 for each). The control group of animals was subjected to VEGF quantification by an identical procedure (n=5).

Immunohistochemistry of VEGF, Flk-1 and von Willebrand factor (vWf )

Using a cryostat and coronal frozen sections with a thickness of 14 μm, immunohistochemical staining was performed based on the following procedure²¹: a hydrophilic section was fixed with phosphate-buffered saline (PBS) containing 1% of formalin. After a thorough washing, removal of endogenous peroxidase was performed for 20 min with ethanol containing 0.3% H₂O₂. As the primary antibody, VEGF rabbit-polyclonal antibody (Santa Cruz-152, Sweden; dilution 1:50), Flk-1 rabbit-polyclonal antibody (Santa Cruz-315, Sweden; dilution 1:50), and vWf mouse-polyclonal antibody (Serotec Antibodies, UK; dilution 1:20) were used. The vWf staining was performed as an indicator of new blood vessels. The respective antibodies were diluted with 0.01 M triethenolamine-buffer (TB; pH: 8.5 Na⁺ free) and a sensitization of the primary antibodies was performed at 4°C for 24 h. In response to the respective primary antibodies, sensitization of the secondary antibodies was performed for 3 h at room temperature, using 1% of anti-rabbit or mouse IgG, 3% of goat or horse serum, and 0.01 M TB (pH: 8.5 Na⁺ free). An Elite Kit (Vectastain, Vector Laboratories, Burlingame, Canada) was used for the coloring. As regards the coloring substance, 3, 3' -diaminobenzidine was used, and it was sensitized with nickel chloride.

Measurement of cerebrovascular permeability and water content

The extent of extravasation of Evans blue (EB) dye into the brain parenchyma was measured with a fluorescence photometer. Based on a method described by Uyama and associates,²⁷ at 1 h before sample extraction, 2% EB liquid solution was administered (4 mL/kg) via the caudal vein. The animals were transcardially perfused with PBS through the left ventricle until colorless perfusion was attained under anesthesia with pentobarbital (50 mg/kg, i.p.), and the brain parenchyma was removed. Centrifugal separation was performed after 50% trichloroacetic acid had been added for homogenization. The supernatant fluid was extracted, and 100% ethanol was added to measure the quantity of EB with a fluoro-plate reader at 3, 6, 12, 24, 48, and 72 h after injury (n=6 for each). The control group of animals was subjected to EB measurement by an identical procedure (n=6).

The contusion edema was evaluated from the water content of the brain tissue at 3, 6, 12, 24, 48, and 72 h after formation of the cerebral contusion (n=5 for each), using the dry-weight method. After the rats had been beheaded under anesthesia with pentobarbital (50 mg/kg, i.p.) the brain parenchyma was removed, the cerebrum was divided into the right and left cerebral hemispheres, and their weights were measured. Each sample was dried for 24 h at 110°C using a drying oven, the weight was measured, and its water content was calculated based on the following formula: water content=[(wet weight - dry weight)/wet weight] ×100.²⁸ The control group of animals was subjected to water content quantification by an identical procedure (n=5).

Evaluation of neurological deficits and contusion necrosis

Neurological deficits were evaluated with a rota rod device.^29
–31 The time taken for the rates to fall from the rotating rotor was measured. The baseline value was determined in each rat immediately before the induction of cerebral contusion, and the values measured at 24 and 72 h, and 7, 14, and 21 days after formation of the cerebral contusion were expressed as the ratio to baseline.

The animals were sacrificed at 21 days after injury under anesthesia with pentobarbital (50 mg/kg, i.p.) for estimation of the volume of the contusion necrosis. The brain parenchyma was removed and eight coronal frozen sections were cut every 1 mm including the contusion necrosis at a thickness of 14 μm and processed for hematoxylin-eosin staining (n=5). Sections were digitized, and the areas of contusion necrosis were outlined using National Institutes of Health Image software. The volume of contusion necrosis was calculated by integration of the necrotic area and the thickness of each section.³²

Administration of bevacizumab

The role of changes in VEGF activity was evaluated from the effects of the VEGF monoclonal antibody, bevacizumab (Avastin, 100 mg/4 mL; Roche, Switzerland), on the neurological deficits and volume of contusion necrosis. Animals were divided into two groups: an administration group (n=5) and a non-administration group (n=5), both of which were subjected to injury. The neurological deficits and volume of contusion necrosis were compared between these two groups. A separate group of animals (sham group) was subjected to an identical surgical procedure except for the actual induction of cerebral contusion (n=5).

Bevacizumab (10 mg/kg) was administered to the administration group via the caudal vein of the rats immediately after injury.^33,34 The period that bevacizumab remains inside the body has been reported to be 17.1±4.7 days.³⁵ Because the osmotic pressure of bevacizumab is approximately equivalent to that of physiological saline, the same amount of saline was administered intravenously to the sham group and the non-administration group.

Statistical analysis

Mean values were compared using two-way analysis of variance followed by the Dunnett test post hoc correction. A p value of<0.05 was considered significant for all comparisons. Data are presented as the means±standard deviation.

Results

Changes in VEGF expression, cerebrovascular permeability, and water content

Compared with the control group, a significant increase in VEGF expression began to be detected at 24 h after injury (p<0.01), and became maximal at 72 h (p<0.003) after injury (Fig. 1). The distribution of the expression of VEGF, Flk-1, and vWf was investigated at 72 h after injury when the VEGF expression became maximal. The immunoreactivity of VEGF was distributed abundantly in the cytoplasm of the vascular endothelial cells around the cerebral contusion. It was found that the expression of Flk-1 and vWf appeared to be increased together with the expression of VEGF in the areas surrounding the core of contusion and the ipsilateral hippocampus at 72 h after injury. Such increases in VEGF, Flk-1, and vWf expression were not observed in the contralateral brain tissue (Fig. 2 –4).

FIG. 1.

Time course of vascular endothelial growth factor (VEGF) expression. Enzyme-linked immunosorbent assay revealed that VEGF was elevated as early as 24 h after injury. A significant difference was observed at 24 and 72 h and 5 days after injury. *p<0.01; **p<0.003 (n=5 for each).

FIG. 2.

Immunohistochemical staining of vascular endothelial growth factor (VEGF) at 3 days after injury. (A) contusion area, ×20; (B) vascular endothelial cells; (C) vascular endothelial cells on the contralateral side; *core of contusion. Color image is available online at www.liebertpub.com/neu

FIG. 3.

Immunohistochemical staining of vascular endothelial growth factor receptor-2 (Flk-1) at 3 days after injury. (A) contusion area, ×20; (B) vascular endothelial cells; (C) vascular endothelial cells on the contralateral side; *core of contusion. Color image is available online at www.liebertpub.com/neu

FIG. 4.

Immunohistochemical staining of von Willebrand factor at 3 days after injury. (A) contusion area, ×40; (B) vascular endothelial cells; (C) vascular endothelial cells on the contralateral side; *core of contusion. Color image is available online at www.liebertpub.com/neu

Compared with the control group, the cerebrovascular permeability increased and became maximal between 3 and 24 h (p<0.001) after injury (Fig. 5). An increase in water content of the brain tissue was detected at 6 h (p<0.03) after injury and became maximal between 6 and 24 h (p<0.001) after injury, and then decreased (Fig. 6). No increase in either the cerebrovascular permeability or the water content of the contralateral brain tissue was observed.

FIG. 5.

Evans blue (EB) extravasation. The EB extravasation was maximal at 6 h after injury. *p<0.03, **p<0.001 compared with the control (n=6 for each).

FIG. 6.

Water content. The water content became maximal at 24 h after injury. *p<0.03, **p<0.001 compared with the control (n=5 for each).

Effects of bevacizumab administration

Based on the above findings, we examined the effects of administration of bevacizumab on the cerebrovascular permeability and water content at 6 and 24 h after injury, respectively. Compared with the control group, no significant effects on the increased cerebrovascular permeability and water content were noted in the group that underwent bevacizumab administration (Fig. 7).

FIG. 7.

Effect of bevacizumab. Left: Effect of bevacizumab on extravasation of Evans blue (EB) (n=4 for each). Right: Effect of bevacizumab on water content (n=4 for each). No significant changes were observed.

Compared with the sham group, significant neurological deficits were detected in the non-administration group by the rota rod test at 24 and 72 h after injury (p<0.05; Fig. 8). Such deficits were no longer observed at 7 days after injury. In the group that underwent bevacizumab administration, significant neurological deficits were detected at 24 and 72 h, and 7 and 14 days after injury (p<0.03 or 0.01), although neurological performance tended to recover gradually from 7 days and thereafter (Fig. 8).

FIG. 8.

Motor performances. Motor performance was measured by the use of a rota rod device. Significant differences were noted between the non-administered group and the bevacizumab administered group at 72 h and 7 and 14 days after injury. *p<0.05, **p<0.01 as compared to the non-administration group (n=5 for each).

Compared with the non-administration group, the neurological deficits were significantly greater in the group that underwent bevacizumab administration at 72 h and 7 and 14 days after injury (p<0.05 or 0.01; Fig. 8), indicating that recovery was delayed when bevacizumab was administered. Compared with the control group, the volume of contusion necrosis at 21 days after injury was significantly greater in the group to which bevacizumab was administered (p<0.001; Fig. 9).

FIG. 9.

Volume of contusion necrosis at 21 days after injury. The necrosis volume of the bevacizumab administered group was greater than that of the non-administered group. *p<0.001 (n=5 for each).

Discussion

Role of VEGF in the formation of contusion edema

Our experiments demonstrated an increase in VEGF expression after TBI. The increase in VEGF expression displayed an entirely different time course from the increased cerebrovascular permeability and the development of contusion edema, however.

The increased VEGF expression was maximal at 72 h after injury. In contrast (Fig. 1, 5), an increase in cerebrovascular permeability was observed at 3 h after injury and became maximal between 3 and 24 h after injury. The increase in water content of the brain tissue was detected at 6 h after injury and became maximal between 6 and 24 h after injury. These time courses for the increase in cerebrovascular permeability and the development of contusion edema are consistent with previously published data.³⁶

In addition, inhibition of VEGF activity by the administration of bevacizumab did not alter either the increase in cerebrovascular permeability or the increase in water content of the brain tissue. These findings clearly suggest that the increase in VEGF expression is not involved in the formation of contusion edema.

Role of VEGF in the formation of contusion necrosis

The increase in VEGF expression was observed in the area surrounding the core of contusion. Because blood flow is disrupted by microthrombosis in this area,^24,25 the increase in VEGF expression appears to be a response to ischemia.³⁷

The increase in VEGF expression was maximal at 72 h after injury. Lennmyr and associates¹² have demonstrated that in a rat model of middle cerebral artery occlusion, the expression of VEGF and VEGFR (Flt-1 and Flk-1) was most strongly observed around the peripheral region of the cerebral infarct at 72 h after induction of ischemia. This similarity in time course supports the inference that the increased VEGF expression represents a response to ischemia.

The increase in VEGF expression at 72 h after injury was associated with an increased vWf expression, suggesting that VEGF activates angiogenesis. Zhang and colleagues³⁸ reported that administration of VEGF in a rat model of cerebral embolism increased angiogenesis and improved the microcirculation in the peripheral region of the cerebral infarct at 9 days after induction of ischemia. It appears that the increased VEGF and vWf expression may be associated with an improved microcirculation in the area surrounding the core of contusion necrosis.

When bevacizumab was administered, the neurological deficits at 72 h–14 days after injury and the volume of contusion necrosis at 21 days after injury became clearly greater. These findings support the inference that the increased VEGF expression promoted angiogenesis and improved the microcirculation in the area surrounding the core of contusion. This would help to protect the functions of neuronal cells from the ischemia in this area and eventually reduce the volume of contusion necrosis.

Role of VEGF in development of neurological outcome

The results of the present study suggest that increased VEGF expression may inhibit the development of neurological deficits. It seems reasonable to assume that this effect may be caused by a reduced volume of contusion necrosis. In addition, the increased VEGF expression may attenuate the neurological deficits through the repair of neural circuits, because VEGF has been reported to increase neurogenesis after TBI.³⁹

The present findings suggest that increased VEGF expression plays a beneficial role in reducing the volume of contusion necrosis and attenuating the neurological deficits after TBI. A better understanding of the mechanisms of action of VEGF may help to facilitate the development of new therapeutic approaches to TBI.

Footnotes

Acknowledgments

We gratefully acknowledge Dr. Shinichiro Kokubun, M.D., Ph.D., and Dr. Chiaki Hidai, M.D., Ph.D., Division of Physiology, Department of Biomedical Sciences, Nihon University School of Medicine, for their expert technical assistance.

Author Disclosure Statement

This study was supported by a Grant-in-Aid for Scientific Research of Japan (C) to Tatsuro Mori, M.D., Ph.D. (no. 16591463). For the remaining authors, no competing financial interests exist.

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