The effects of bevacizumab treatment in a rat model of retinal ischemia and perfusion injury

Abstract

Purpose: To create a model of an ischemic retina with temporary ischemia and reperfusion (IR) and to examine the possible antiapoptotic and neurodegenerative effects of a vascular endothelial growth factor (VEGF) antagonist.

Methods: Three groups were formed. Rats were subjected to continued ischemia for 45 min, and then reperfusion was allowed for 2 days. For the first group, ischemia was induced, but an anti-VEGF agent was not administered. For the second group, 2 days before ischemia, 0.005 ml (0.125 mg) of bevacizumab was administered intravitreally, and then the ischemic model was created. The last group's intraocular pressure was not increased as in the control group, and only a cannula was introduced into the anterior chamber through the cornea. Six animals from each group were subjected to histomorphometry, and four were subjected to immunohistochemical and histopathologic examinations. For a histomorphometric examination, the number of cells in the retinal ganglion cell (RGC) layer was counted using the optical dissector method. For immunohistochemistry, the vascular endothelial growth factor receptor-2 (VEGFR-2) levels and apoptosis were examined in the retinal and choroidal tissue.

Results: It was observed that in an IR injury, bevacizumab reduces the death and apoptosis of cells in the RGC layer. It was also identified that although bevacizumab is a large molecule, the agent affects the choroid and reduces the amount of VEGFR-2 in this tissue.

Conclusions: IR may be used as a model of ischemic retinopathy that includes VEGF-dependent vascular permeability and neurodegeneration. Although VEGF is a neurotrophic molecule, in IR injury, treatment with bevacizumab, which is an anti-VEGF agent, decreases apoptosis, showing that excess function of this molecule can be hazardous.

Figure 1

Stereological analysis of the rat retina demonstrates surviving cells in the ganglion cell layer. The control group has the highest number of living cells as expected since they did not receive any manipulation. The important data in this graph is that the bevacizumab (BEV) group has more living cells than the ischemia and reperfusion (IR) group. This is an important finding that shows bevacizumab is neuroprotective in the event of ischemia and reperfusion. *p<0.01, **p<0.001 and ***p<0.0001. Error bars are median ± standard error of the mean (SEM), where n=6.

Figure 2

Comparison of histological cross sections of the control, IR, and BEV groups. White arrows indicate the cells at the retinal ganglion cell layer. Staining: Hematoxylin and eosin. Scale bar: 50 µm. C = control; IR = ischemia and reperfusion; BEV = bevacizumab.

Figure 3

The apoptotic indices of the three groups. As expected, the lowest apoptosis is in the control group since they did not receive any insult. The important finding in this graph is the significantly lower apoptosis in the bevacizumab (BEV) group compared with the ischemia-reperfusion (IR) group. **p<0.01 and ***p<0.001. Error bars are median ± standard error of the mean (SEM), where n=6.

Figure 4

Immunohistochemical evaluation of TUNEL-stained tissue samples: control, IR, and BEV. The black arrows point out the apoptotic ganglion cells in the retina layers. Scale bar: 50 µm. C = control; IR = ischemia and reperfusion; BEV = bevacizumab.

Figure 5

VEGFR-2 staining intensity of the RGC layer. There are two important points in this graph. The first one is the high VEGF response to the ischemia and reperfusion and its significant suppression by bevacizumab (** p<0.01 and *** p<0.001). Data expressed as median ± standard error of the mean (SEM; n=6). BEV = bevacizumab; IR = ischemia-reperfusion.

Figure 6

Immunohistochemical evaluation of VEGFR-2-stained tissue samples: C, IR, and BEV. VEGFR-2 staining intensity increased in the retina and choroid layers of the ischemia and reperfusion (IR) group, but this result was decreased in the bevacizumab (BEV) group. Stars indicate the choroid layer. C = control. Scale bar: 100 µm.

Figure 7

VEGFR-2 intensity of the choroid layer of the eye. Data expressed as median ± standard error of the mean (SEM; n=6). The response is the same as in the ganglion cell layer (GCL) as VEGFR-2 is suppressed by bevacizumab (BEV) statistically significantly (** p<0.01 and *** p<0.001). IR = ischemia and reperfusion.