Carbon monoxide treatment reduces microglial activation in the ischemic rat retina

Graefes Arch Clin Exp Ophthalmol. 2016 Oct;254(10):1967-1976. doi: 10.1007/s00417-016-3435-6. Epub 2016 Jul 21.


Purpose: Ischemia and reperfusion (I/R) injury damages retinal neurons. Retinal injury is accompanied by activation of microglia, which scavenge the dead or dying neurons, but increasing evidence now indicates that amoeboid-shaped microglia cells activated in the brain after ischemia have neurotoxic and damaging properties in their own right. A previous study showed that postconditioning with carbon monoxide (CO) protects retinal ganglion cells (RGCs) after I/R through anti-apoptotic and anti-inflammatory mechanisms. The present study was designed to investigate and quantify the activation of retinal microglia after I/R with and without CO postconditioning.

Methods: Adult Sprague-Dawley rats underwent retinal ischemia by increasing the ocular pressure to 120 mmHg for 1 h through a needle inserted into the anterior chamber. Reperfusion was induced by removing the needle. After I/R, one group of animals was kept in a CO (250 ppm) atmosphere for 1 h; the other group was kept in room air (Air). At 1, 2, 3, and 7 days after I/R, the eyes were enucleated and fixed. Intracardiac blood was analyzed for systemic effects of CO or I/R. Retinal cross sections were taken from the middle third of the eye and were stained with anti-Iba-1. Microglia cells were graded as amoeboid or ramified phenotypes according to morphologic criteria. Retinal thicknesses were determined.

Results: Evaluation of retinal tissue revealed a significant reduction of amoeboid microglia cells after I/R + CO when compared to the I/R + Air group. The peak number of amoeboid microglia was observed at day 2 post-I/R + Air. This rise was attenuated by CO postconditioning (815 versus 572 cells/mm2 for I/R + Air versus I/R + CO, respectively; p = 0.005). CO reduced and further postponed the peak in the numbers of amoeboid and ramified microglia cells in ischemic eyes and prevented microglial activation in the contralateral eyes. I/R-induced leucocytosis was inhibited by CO inhalation. The reduction of retinal thickness after I/R was more serious after Air inhalation when compared to the CO group.

Conclusions: Numerous activated microglia cells appear in the inner retina after I/R, and CO-treatment significantly attenuates this glial response. Antagonism of microglial activation may be a further neuroprotective effect of CO, apart from its direct anti-apoptotic capacity.

Keywords: Ischemia; Microglia; Neuroinflammation; Neuroprotection; Retina; Retinal ganglion cell.

MeSH terms

  • Animals
  • Blood Cells
  • Calcium-Binding Proteins / metabolism
  • Carbon Monoxide / administration & dosage*
  • Disease Models, Animal
  • Female
  • Fluorescent Antibody Technique, Indirect
  • Male
  • Microfilament Proteins / metabolism
  • Microglia / metabolism*
  • Microglia / pathology
  • Neuroprotective Agents
  • Rats
  • Rats, Sprague-Dawley
  • Reperfusion Injury / metabolism
  • Reperfusion Injury / pathology
  • Reperfusion Injury / prevention & control*
  • Retinal Degeneration / metabolism
  • Retinal Degeneration / pathology
  • Retinal Degeneration / prevention & control*
  • Retinal Ganglion Cells / metabolism*
  • Retinal Ganglion Cells / pathology
  • Retinal Vessels / pathology


  • Aif1 protein, rat
  • Calcium-Binding Proteins
  • Microfilament Proteins
  • Neuroprotective Agents
  • Carbon Monoxide