Activated complement classical pathway in a murine model of oxygen-induced retinopathy

Abstract

Aim: To investigate whether the complement system is involved in a murine model of oxygen-induced retinopathy (OIR).

Methods: Forty C57BL/6J newborn mice were divided randomly into OIR group and control group. OIR was induced by exposing mice to 75%±2% oxygen from postnatal 7d (P7) to P12 and then recovered in room air. For the control group, the litters were raised in room air. At the postnatal 17d (P17), gene expressions of the complement components of the classical pathway (CP), the mannose-binding lectin (MBL) pathway and the alternative pathway (AP) in the retina were determined by quantitative real-time polymerase chain reaction (RT-PCR). Retinal protein expressions of the key components in the CP were examined by Western blotting.

Results: Whole mounted retina in the OIR mice showed area of central hypoperfusion in both superficial and deep layers and neovascular tufts in the periphery. The expressions of C1qb and C4b genes in the OIR retina were significantly higher than those of the controls. The expression of retinal complement factor B (CFB) gene in OIR mice was significantly lower than those of the controls. However, the expressions of C3 and complement factor H (CFH) genes were higher. The protein synthesis of the key components involved in the CP (C1q, C4 and C3) were also significantly higher in OIR mouse retina. Although MBL-associated serine protease 1 (MASP1) and MASP2 were detected in both the OIR and the control groups, the expressions were weak and the difference between the two groups was not significant.

Conclusion: Our data suggest that the complement system CP is activated during the pathogenesis of murine model of OIR.

Keywords: classical pathway; complement activation; mouse; oxygen-induced retinopathy; retina.

Figure 1. Representative retinal flat mounts of an OIR and a control mouse eye perfused with high-molecular-weight fluorescein-dextran. The photographs were taken with a fluorescence microscope at 5 times magnification at P17

A: Room air raised mouse exhibited normal retinal vascular architecture. The vessels formed a radial branching pattern in the superficial layer and a polygonal reticular pattern in the deep layer. B: Mouse exposed to hyperoxia showed reduced vascular development, which leads to the development of neovascularization. Arrow indicates area of central hypoperfusion in both the superficial and the deep layers. Arrow head indicates peripheral neovascular tufts.

Figure 2. The expressions of the major complement system components mRNA in the OIR mouse retina determined by quantitative RT-PCR. Gene expressions of selected complement components of CP, MBL pathway and AP in the retina of the OIR and the control mice were shown

A&B: mRNA levels for complement components C1qb and C4b mainly in the CP were significantly higher in the OIR mouse retina (n=3); C&D: mRNA levels for complement components MASP1 and MASP2 specifically in the MBL pathway were comparable between the two groups (n=3); E&F: mRNA levels for complement components CFB and CFH in the AP. The expression of CFB was significantly lower, while the expression of CFH was significantly higher than those of the controls (n=3); G: In the terminal pathway, the expression of C3 in the OIR mouse retina was significantly higher than those of the controls (n=3; Bars represent means±SEM; ^aP<0.001, ^bP<0.01, ^cP<0.05).

Figure 3. Retinal protein expressions of the key components of complement system mainly involved in the CP in the OIR and the control groups

A: Retinal C1q, C4 and C3 protein levels in the OIR and the control groups were determined by Western blotting (n=6). B: Densitometry analysis of the C1q, C4 and C3 protein levels in the OIR mouse retina were significantly higher than those of the control mice (Bars represent means±SEM; ^bP<0.01).