When is a control not a control? Reactive microglia occur throughout the control contralateral visual pathway in experimental glaucoma

Purpose Animal models show retinal ganglion cell injuries that replicate features of glaucoma and the contralateral eye is commonly used as an internal control. There is significant cross-over of retinal ganglion cell axons from the ipsilateral to the contralateral side at the level of the optic chiasm which may confound findings when damage is restricted to one eye. The effect of unilateral glaucoma on neuroinflammatory damage to the contralateral visual pathway has largely been unexplored. Methods Ocular hypertensive glaucoma was induced unilaterally or bilaterally in the rat and retinal ganglion cell neurodegenerative events were assessed. Neuroinflammation was quantified in the retina, optic nerve head, optic nerve, lateral geniculate nucleus, and superior colliculus by high resolution imaging, and in the retina by flow cytometry and protein arrays. Results Following ocular hypertensive stress, peripheral monocytes enter the retina, and microglia become reactive. This effect is more marked in animals with bilateral ocular hypertensive glaucoma. In rats where glaucoma was induced unilaterally there was significant microglia activation in the contralateral (control) eye. Microglial activation extended into the optic nerve and terminal visual thalami, where it was similar across hemispheres irrespective of whether ocular hypertension was unilateral or bilateral. Conclusions These data suggest that caution is warranted when using the contralateral eye as control in unilateral models of glaucoma. Translational Relevance Use of a contralateral eye as a control may confound discovery of human relevant mechanism and treatments in animal models. We also identify neuroinflammatory protein responses that warrant further investigation as potential disease modifiable targets. ### Competing Interest Statement The authors have declared no competing interest. * AUC : (area under the curve), FC : (flow cytometry), GCL : (ganglion cell layer), H&E : (haematoxylin and eosin), HC : (hierarchical clustering), IF : (immunofluorescence), IHC : (immunohistochemistry), IOP : (intraocular pressure), IPL : (inner plexiform layer), MDC : (myeloid-derived cell), NND : (nearest neighbor distance), NT : (normotensive), NFL : (nerve fiber layer), OHT : (ocular hypertension).