High Field MR Demonstrates Effect of Glucocorticoids in a Novel Murine Model of VEGF-Induced Vasogenic Brain Edema

Roger Murayi¹, Martin Piazza¹, Jeeva Munasinghe¹, Nancy Edwards¹, Stuart Walbridge¹, Marsha Merrill¹, and Prashant Chittiboina¹

¹Surgical Neurology Branch/NINDS, National Institutes of Health, Bethesda, MD, United States

Synopsis

The molecular mechanisms mediating the formation of peritumoral vasogenic brain edema (VBE) and its abrogation from glucocorticoid treatment is poorly understood. In order to study the molecular underpinnings and temporal evolution of these processes we successfully developed a murine model of VBE confirmed by high field MRI and histopathologic studies. Furthermore, we demonstrate a differential effect of systemic glucocorticoids on blood-brain-barrier breakdown and edema formation. This in-vivo model will allow for further investigation into the molecular mechanisms of VBE formation and potentially provide additional targets for its treatment.

Purpose

1) To develop an accurate murine model of vasogenic brain edema that demonstrates temporal evolution with high field MRI.

2) To study the temporal effects of glucocorticoids on vasogenic brain edema and blood-brain-barrier breakdown.

Introduction

Vasogenic brain edema (VBE) associated with brain tumors is predominantly a result of vascular endothelial growth factor (VEGF) secreted by the tumor^1,2. If left untreated VBE causes swelling and increased intracranial pressure which leads to neurologic deficits, herniation, and ultimately death. The molecular basis of VBE formation and its abrogation by glucocorticoids (GC) is poorly understood. A model would allow for the study of the molecular mechanisms underlying vasogenic edema formation, the effects of glucocorticoids, and potentially reveal novel targets for its treatment. Current models of VBE (e.g. cold injury model) are associated with significant necrosis and inflammation unlike VBE^3,4. Additionally, serial imaging with high field MRI helps understand the underlying pathogenesis of blood brain barrier (BBB) dysfunction that is not captured by terminal histopathologic evaluation ⁵. Herein, we describe a novel, accurate in-vivo model of VBE and the effect of systemic GC on evolution of VBE and BBB breakdown.

Methods

Rats were implanted with a stereotactically placed cannula connected to an osmotic pump which infused solution into the rat striatum. There were three conditions: 1) 0.1% w/v rat serum albumin (RSA) in phosphate-buffered saline (PBS) infusion as a control, 2) VEGF (2-5ug/ml) in 0.1% w/v RSA in PBS, or 3) VEGF infusion as in 2 with intraperitoneal dexamethasone injections (0.45mg/kg, twice daily from days 2 - 6). Infusions occurred at 1ul/hour and were continued for 6 days following 4-5 day preinfusion with normal saline at 0.5ul/hour. High field (9.4T) MRI T2W and T1W with/without gadolinium contrast images were obtained to evaluate VBE and BBB breakdown at day 2 and day 6 of infusion. Histopathological characterization of inflammation, gliosis and necrosis was performed after final imaging.

Results

Edema Formation
With VEGF infusion, MR on day 6 of infusion revealed extensive T2 hyperattenuation in the juxtacanalicular region and tracking along the external capsule of the ipsilateral side. Animals treated with VEGF demonstrated a significantly larger volume (42.90 ± 3.842 mm3) of T2 hyperattenuation at 144 hours when compared with the volume (8.585 ± 1.664 mm3) in control animals (p<0.0001). Quantitative T2 maps of entire rodent brains confirmed that local T2 values were significantly elevated in animals receiving VEGF infusions (ANOVA, F ratio =360.4, p<0.0001) (Figure 1).

BBB breakdown
Post-contrast T1 hyperattenuation in the juxtacanalicular region indicating BBB breakdown was observed in rats being infused with VEGF. The hyperintensity was not noted reliably in early (2d) phases following VEGF infusion. At the later time periods (6d) the volume of T1 hyperintesity (34.97 ± 8.99 mm3) was significantly less compared with the region of edema (p<0.0001) (Figure 2).

Histopathology
Hematoxylin and eosin staining demonstrated no evidence of tissue necrosis in either control or VEGF group. In animals receiving VEGF, an impressive amount if juxtacanalicular neuropil separation and a lack of inflammation was noted. Immunoflourescence confirmed astrocyte activation with increased GFAP staining in animals receiving VEGF when compared with uninfused brain or with control infusion.

Glucocorticoid Effect
Rats receiving 3 days of IP dexamethasone injections showed resolution of VBE, but continued to show BBB breakdown in the juxtacanalicular region (Figure 3).

Discussion

In this report, we demonstrate that the chronic infusion of VEGF via mini-osmotic pump into rat striata recreates VBE without necrosis/inflammation. Additionally, high field in-vivo imaging allows monitoring of temporal evolution of VBE and BBB breakdown. This is a significant refinement in the creation of a temporally accurate model of VBE compared with earlier attempts.^2,6,7 We then demonstrate that dexamethasone exposure following initial BBB breakdown prevents VBE, but does not induce reversal of BBB breakdown in the juxtacanalicular region. Dexamethasone has multiple effects in the setting of tumor related VBE.⁸ MRI imaging provides powerful tools to understand the mechanisms of VBE.⁹ Using serial MRI imaging, we have demonstrated the differential effects of GC on VBE and BBB breakdown. Further study needs to be continued to understand the molecular underpinnings of these effects.

Conclusion

Chronic VEGF infusion in a rat model creates an accurate model of VBE, and our results suggest differential effects of GC on VBE and BBB breakdown.

Acknowledgements

This research was made possible through the National Institutes of Health (NIH) Medical Research Scholars Program, a public-private partnership supported jointly by the NIH and generous contributions to the Foundation for the NIH from the Doris Duke Charitable Foundation, The American Association for Dental Research, The Howard Hughes Medical Institute, and the Colgate-Palmolive Company, as well as other private donors.

For a complete list, please visit the Foundation website at: http://fnih.org/work/education-training-0/medical-research-scholars-program

References

1. Berkman, R. a et al. Expression of the vascular permeability factor/vascular endothelial growth factor gene in central nervous system neoplasms. J. Clin. Invest. 91, 153–9 (1993).

2. Proescholdt, M. A. et al. Vascular endothelial growth factor (VEGF) modulates vascular permeability and inflammation in rat brain. J. Neuropathol. Exp. Neurol. 58, 613–27 (1999).

3. Klatzo, I., Chui, E., Fujiwara, K. & Spatz, M. Resolution of vasogenic brain edema. Adv. Neurol. 28, 359–73 (1980).

4. Nag, S. Cold-injury of the cerebral cortex: immunolocalization of cellular proteins and blood-brain barrier permeability studies. J Neuropathol Exp Neurol 55, 880–888 (1996).

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6. Rite, I., Machado, A., Cano, J. & Venero, J. L. Intracerebral VEGF injection highly upregulates AQP4 mRNA and protein in the perivascular space and glia limitans externa. Neurochem. Int. 52, 897–903 (2008).

7. Jiang, S., Xia, R., Jiang, Y., Wang, L. & Gao, F. Vascular endothelial growth factors enhance the permeability of the mouse blood-brain barrier. PLoS One 9, e86407 (2014).

8. Fan, Z. et al. Dexamethasone alleviates tumor-associated brain damage and angiogenesis. PLoS One 9, e93264 (2014).

9. Pishko, G. L., Muldoon, L. L., Pagel, M. a, Schwartz, D. L. & Neuwelt, E. a. Vascular endothelial growth factor blockade alters magnetic resonance imaging biomarkers of vascular function and decreases barrier permeability in a rat model of lung cancer brain metastasis. Fluids Barriers CNS 12, 5 (2015).

Figures

Figure 1: VEGF infusion results in increased volume and intensity of T2W signal. 3 dimensional segmentation analysis reveals a significantly increased region of T2 hyper intensity at 144 h with VEGF infusion (A). Quantitative analysis of T2 signal in the juxtacanalicular regions reveal significant increase in T2 signal due to VEGF infusion at 36 h and beyond (B).

Figure 2: Volume of blood brain barrier breakdown is less than vasogenic edema. The volume of post contrast T1 hyper intensity is significantly lower than the volume of T2 hyper intensity at 144 h (A). Normalized T1 values in the juxtacanalicular region are elevated at 36 h and beyond with VEGF infusion (B). Representative images from one animal demonstrate the vasogenic edema (asterixes) on T2W images extend beyond the junta-canalicular regions (C, D and E).

Figure3: VEGF infused rat with glucocorticoid treatment shows absence of edema with and presence of BBB breakdown. The volume of edema present in T2W images for GC injected rats is comparable to that of controls at 144hrs and significantly less than VEGF rats without GC treatment(A). Edema is absent from the external capsule (C). T1W post-contrast scans in GC treated condition show contrast enhancement comparable to that of VEGF rats without GC treatment (B) located juxtacannicularly (E).

Proc. Intl. Soc. Mag. Reson. Med. 24 (2016)

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