J Korean Med Sci.  2012 Mar;27(3):291-299. 10.3346/jkms.2012.27.3.291.

Increased Expression of EMMPRIN and VEGF in the Rat Brain after Gamma Irradiation

• Wei M
• Li H
• Huang H
• Xu D
• Zhi D
• Liu D
• Zhang Y

• KMID: 1793001
• DOI: http://doi.org/10.3346/jkms.2012.27.3.291

Abstract

The extracellular matrix metalloproteinase inducer (EMMPRIN) has been known to play a key regulatory role in pathological angiogenesis. A elevated activation of vascular endothelial growth factor (VEGF) following radiation injury has been shown to mediate blood-brain barrier (BBB) breakdown. However, the roles of EMMPRIN and VEGF in radiation-induced brain injury after gamma knife surgery (GKS) are not clearly understood. In this study, we investigated EMMPRIN changes in a rat model of radiation injury following GKS and examined potential associations between EMMPRIN and VEGF expression. Adult male rats were subjected to cerebral radiation injury by GKS under anesthesia. We found that EMMPRIN and VEGF expression were markedly upregulated in the target area at 8-12 weeks after GKS compared with the control group by western blot, immunohistochemistry, and RT-PCR analysis. Immunofluorescent double staining demonstrated that EMMPRIN signals colocalized with caspase-3 and VEGF-positive cells. Our data also demonstrated that increased EMMPRIN expression was correlated with increased VEGF levels in a temporal manner. This is the first study to show that EMMPRIN and VEGF may play a role in radiation injuries of the central nervous system after GKS.

Keyword

EMMPRIN; Immunohistochemistry; Radiosurgery; Radiation Injuries, Experimental; Vascular Endothelial Growth Factor

MeSH Terms

Animals
Antigens, CD147/*metabolism
Brain/blood supply/metabolism/pathology/*radiation effects
Brain Injuries/metabolism/pathology
Caspase 3/metabolism
Gamma Rays/*adverse effects
Immunohistochemistry
Male
Microscopy, Electron, Transmission
Parietal Lobe/metabolism/pathology/radiation effects
Radiation Injuries, Experimental/metabolism
Radiosurgery/adverse effects
Rats
Rats, Wistar
Time Factors
Vascular Endothelial Growth Factor A/*metabolism

Figure

• 

  Fig. 1 Radiation damage observed 12 weeks after irradiation. Upper left, drawing of a coronal section of the rat brain showing the isodose curve with a maximal center dose of 75 Gy. (A and B) Photomicrographs of coronal sections stained with hematoxylin/eosin are shown from the irradiated (A) and nonirradiated (B) cortex. Enlarged vessels were observed in GKS-irradiated regions. Scale bar = 250 µm. (C, D, and E) EB extravasation in the target region. The significant EB fluorescence (red) was observed in the target region (C). There was no EB fluorescence detected in the nonirradiated cortex (D). Scale bar = 50 µm. The EB concentration in the irradiated tissue was significantly higher than in the sham-operated group (F). (E) Ultrastructural observations of brain capillary endothelial cells. Changes included the enlargement of endothelial nuclei, pykno-chromatin (short arrow), process of the plasma membrane (short arrow) and astroglial edema (long arrow). A, astrocyte; EC, endothelial cell; N, nuclei; L, capillary lumen; BM, basement membrane. (F) The brain water content in the parietal cortex increased in GKS groups compared with the controls. The data are presented as the mean ± SD. *P < 0.001 compared with the sham-operated group.

• 

  Fig. 2 Immunodetection of EMMPRIN after GKS. (A-F) Representative photomicrographs of EMMPRIN immunostaining in GKS-irradiated regions. Low baseline levels of EMMPRIN are observed in the sham-operated rat cortex (A), whereas EMMPRIN staining increases compared with the sham control at 8 and 12 weeks after GKS (B, C). (D-F) Higher magnification image of EMMPRIN signal. Arrows indicate positive EMMPRIN signal. Scale bar = 250 µm (A-C), or 50 µm (D-F). (G) Immunoblot of EMMPRIN protein expression in the damaged cortex after GKS. Bar graph shows the densitometric analysis of EMMPRIN-immunoreactive bands (H) and the temporal change of EMMPRIN-expressing cells after GKS (I). The data are presented as the mean ± SD. *P < 0.001 compared with the sham group.

• 

  Fig. 3 Quantitative PCR analysis for EMMPRIN in the cortex ipsilateral to the irradiation at 1, 4, 8, and 12 weeks after GKS. The average mRNA expression level in the sham brains was set to 1. Histogram and bars represent the mean ± SD of three replicas. *P < 0.001 compared with the sham group.

• 

  Fig. 4 Immunodetection of VEGF after GKS. (A-F) Representative photomicrographs of VEGF immunostaining after GKS. A few cells in the cortex of sham-operated rats are VEGF-positive (A). However, significant increases in expression of the VEGF occur in the irradiated cortex at 8 and 12 weeks after GKS (B, C). (D-F) Higher magnification image of the VEGF signal. Arrows indicate positive VEGF signal. Scale bar = 250 µm (A-C), or 50 µm (D-F). (G) Immunoblot analysis of VEGF protein expression in the damaged brain tissues 1 to 12 weeks after GKS. Bar graph shows the densitometric analysis of VEGF-immunoreactive bands (H) and the temporal change of VEGF (+) cells (I) after GKS. The data are presented as the mean ± SD. *P < 0.001 compared with the sham group.

• 

  Fig. 5 Quantitative PCR analysis for VEGF in the cortex ipsilateral to the irradiation at 1, 4, 8, and 12 weeks after GKS. The average mRNA expression level in the sham brains was set to 1. Histogram and bars represent the mean ± SD of three replicas. *P < 0.001 compared with the sham group.

• 

  Fig. 6 Representative microphotographs of immunofluorescence double staining for EMMPRIN (red) and caspase-3 immunoreactivity (green) in the target region. Colocalization of EMMPRIN (A) and caspase-3 (B) was observed in the irradiated cortex following GKS. C is a merged image of A and B. No double-labeling was found in the control groups (D). Scale bar = 50 µm.

• 

  Fig. 7 Relationship between EMMPRIN levels and VEGF expression. (A-F) Immunofluorescence double staining for EMMPRIN (red) and VEGF immunoreactivity (green) in the target region at 12 weeks after radiosurgery. Double-labeling of EMMPRIN-immunopositive cells (A) with VEGF (B) was not observed in the sham-operated control cortex (C), whereas the colocalization of EMMPRIN (D) with VEGF (E) was observed in the irradiated cortex (F, yellow). Scale bar = 50 µm. (G) Correlation between EMMPRIN-positive cells and VEGF expression in the irradiated cortex after GKS.

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