Inflammation as well as angiogenesis may participate in the pathophysiology of brain radiation necrosis

Abstract

Radiation necrosis (RN) after intensive radiation therapy is a serious problem. Using human RN specimens, we recently proved that leaky angiogenesis is a major cause of brain edema in RN. In the present study, we investigated the same specimens to speculate on inflammation's effect on the pathophysiology of RN. Surgical specimens of symptomatic RN in the brain were retrospectively reviewed by histological and immunohistochemical analyses using hematoxylin and eosin (H&E) staining as well as immunohistochemical staining for VEGF, HIF-1α, CXCL12, CXCR4, GFAP, CD68, hGLUT5, CD45, IL-1α, IL-6 TNF-α and NF-kB. H&E staining demonstrated marked angiogenesis and cell infiltration in the perinecrotic area. The most prominent vasculature was identified as thin-walled leaky angiogenesis, i.e. telangiectasis surrounded by prominent interstitial edema. Two major cell phenotypes infiltrated the perinecrotic area: GFAP-positive reactive astrocytes and CD68/hGLUT5-positive cells (mainly microglias). Immunohistochemistry revealed that CD68/hGLUT5-positive cells and GFAP-positive cells expressed HIF-1α and VEGF, respectively. GFAP-positive cells expressed chemokine CXCL12, and CD68/hGLUT5-positive cells expressed receptor CXCR4. The CD68/hGLUT5-positive cells expressed pro-inflammatory cytokines IL-1α, IL-6 and TNF-α in the perinecrotic area. VEGF caused leaky angiogenesis followed by perilesional edema in RN. GFAP-positive cells expressing CXCL12 might attract CXCR4-expressing CD68/hGLUT5-positive cells into the perinecrotic area. These accumulated CD68/hGLUT5-positive cells expressing pro-inflammatory cytokines seemed to aggravate the RN edema. Both angiogenesis and inflammation might be caused by the regulation of HIF-1α, which is well known as a transactivator of VEGF and of the CXCL12/CXCR4 chemokine axis.

Keywords: CXCL12/CXCR4 chemokine axis; brain radiation necrosis; inflammation; microglia; pro-inflammatory cytokine.

Figure 1.

A hematoxylin and eosin (H&E)-stained specimen from Case 5. Thin-walled enlarged capillaries indicating telangiectasis (arrow) and the proliferation of arterioles can be seen in the area between the necrotic core and normal brain tissue. These blood vessels were accompanied by interstitial edema (arrowhead) due to plasma leakage. The original objective magnification was ×100.

Figure 2.

H&E staining and immunohistochemistry for GFAP, CD68, CXCL12 and CXCR4 of the surgical specimen from Case 2 in the left column and for Case 3 in the right column. H&E staining showed the necrotic core and the surrounding tissue (perinecrotic area). Immunohistochemical staining for GFAP, CD68, CXCL12 and CXCR4 revealed GFAP- and CXCL12-positive astrocytic cells, and CD68- and CXCR4-positive oval cells in the perinecrotic area. CD68- and CXCR4-positive cells were also observed in small numbers inside the necrotic core. On the other hand, no or scarce immunoreactivity of GFAP and CXCL12 was observed in the necrotic core. The original objective magnifications were ×40 and ×200.

Figure 3.

Double immunofluorescence staining of the specimen from Case 2. The expression of HIF-1α was not detected in reactive astrocytes, as revealed by GFAP (a), but was detected in CD68-positive cells (b). VEGF was expressed in GFAP-positive cells (c) but was hardly expressed in CD68-positive cells (d). Similarly, CXCL12 was expressed in GFAP-positive reactive astrocytes (e) but not in CD68-positive cells (f). In contrast, CXCR4 was not expressed in GFAP-positive cells (g) but was expressed in CD68-positive cells (h). The original objective magnification was ×400.

Figure 4.

Double immunofluorescence staining of Case 2. IL-1α, IL-6 and TNF-α were not expressed in reactive astrocytes, as revealed by GFAP-positive cells (a, c, e), but were expressed in CD68-positive cells (b, d, f). The original objective magnification was ×400.

Figure 5.

Hypothesis of the pathophysiology of brain radiation necrosis. (a) Vascular damage around the irradiated tumor tissue caused tissue ischemia. This hypoxia induced hGLUT5-positive microglias to express HIF-1α around the necrotic core. (b) Under HIF-1α regulation, VEGF was expressed in reactive astrocytes, causing leaky and fragile angiogenesis. (c) CXCL12/CXCR4 signaling is also regulated by HIF-1α. (d) CXCL12-expressing reactive astrocytes might draw CXCR4-expressing macrophages and lymphocytes by chemotaxis into the perinecrotic area. (e) These accumulated hGLUT5-positive microglias producing NF-κβ and pro-inflammatory cytokines seemed to aggravate radiation necrosis.