Pimonidazole binding in C6 rat brain glioma: relation with lipid droplet detection

Abstract

In C6 rat brain glioma, we have investigated the relation between hypoxia and the presence of lipid droplets in the cytoplasm of viable cells adjacent to necrosis. For this purpose, rats were stereotaxically implanted with C6 cells. Experiments were carried out by the end of the tumour development. A multifluorescence staining protocol combined with digital image analysis was used to quantitatively study the spatial distribution of hypoxic cells (pimonidazole), blood perfusion (Hoechst 33342), total vascular bed (collagen type IV) and lipid droplets (Red Oil) in single frozen sections. All tumours (n=6) showed necrosis, pimonidazole binding and lipid droplets. Pimonidazole binding occurred at a mean distance of 114 microm from perfused vessels mainly around necrosis. Lipid droplets were principally located in the necrotic tissue. Some smaller droplets were also observed in part of the pimonidazole-binding cells surrounding necrosis. Hence, lipid droplets appeared only in hypoxic cells adjacent to necrosis, at an approximate distance of 181 microm from perfused vessels. In conclusion, our results show that severe hypoxic cells accumulated small lipid droplets. However, a 100% colocalisation of hypoxia and lipid droplets does not exist. Thus, lipid droplets cannot be considered as a surrogate marker of hypoxia, but rather of severe, prenecrotic hypoxia.

Figure 1

Haematoxylin and eosin (A) and Red Oil (B) staining of a rat brain section observed 25 days postimplantation. The tumour exhibited an extensive area of necrosis limited by cells arranged into pseudopalisade (A). Lipid droplets were observed mainly in the necrotic zone and also in the cytoplasm of viable tumoral cells surrounding necrosis (B). Enlargements of B show the presence of small lipid droplets (indicated by arrows) in viable cells at the periphery of necrosis (1) and large lipid droplets within the necrosis (2). T=tumour, NE=necrosis, PP=cells arranged into pseudopalisade, NT=nontumoral tissue, LD=lipid droplets.

Figure 2

Pseudocoloured composite binary image obtained after sequential scanning for total vascularisation (red), perfusion (blue) and hypoxia (green), respectively, in a rat brain section. NE=necrosis, PP=cells arranged into pseudopalisade.

Figure 3

Pseudocoloured 12-bit grey-level image obtained in a selected ROI including at least one perfused vessel near a hypoxic region adjoining tissue necrosis. The black rectangle represents the area in which distances were measured (A). The intensity of pimonidazole binding increases from a perfused vessel to the edge of necrosis (A). This hypoxic gradient is illustrated in (B); the green arrow shows the mean distance where hypoxia (pO₂<10 mmHg) starts to occur: approximately 114 μm. The pink arrow shows the mean distance where lipid droplets start to occur: approximately 181 μm. Thus, small lipid droplets are present in hypoxic regions, but only in cells adjacent to necrosis (A). Ves=perfused vessel, Hyp=hypoxic region, LD=lipid droplets, NE=necrosis.