Neuroprotection and neuroregeneration of retinal ganglion cells after intravitreal carbon monoxide release

PLoS One. 2017 Nov 27;12(11):e0188444. doi: 10.1371/journal.pone.0188444. eCollection 2017.


Purpose: Retinal ischemia induces apoptosis leading to neurodegeneration and vision impairment. Carbon monoxide (CO) in gaseous form showed cell-protective and anti-inflammatory effects after retinal ischemia-reperfusion-injury (IRI). These effects were also demonstrated for the intravenously administered CO-releasing molecule (CORM) ALF-186. This article summarizes the results of intravitreally released CO to assess its suitability as a neuroprotective and neuroregenerative agent.

Methods: Water-soluble CORM ALF-186 (25 μg), PBS, or inactivated ALF (iALF) (all 5 μl) were intravitreally applied into the left eyes of rats directly after retinal IRI for 1 h. Their right eyes remained unaffected and were used for comparison. Retinal tissue was harvested 24 h after intervention to analyze mRNA or protein expression of Caspase-3, pERK1/2, p38, HSP70/90, NF-kappaB, AIF-1 (allograft inflammatory factor), TNF-α, and GAP-43. Densities of fluorogold-prelabeled retinal ganglion cells (RGC) were examined in flat-mounted retinae seven days after IRI and were expressed as mean/mm2. The ability of RGC to regenerate their axon was evaluated two and seven days after IRI using retinal explants in laminin-1-coated cultures. Immunohistochemistry was used to analyze the different cell types growing out of the retinal explants.

Results: Compared to the RGC-density in the contralateral right eyes (2804±214 RGC/mm2; data are mean±SD), IRI+PBS injection resulted in a remarkable loss of RGC (1554±159 RGC/mm2), p<0.001. Intravitreally injected ALF-186 immediately after IRI provided RGC protection and reduced the extent of RGC-damage (IRI+PBS 1554±159 vs. IRI+ALF 2179±286, p<0.001). ALF-186 increased the IRI-mediated phosphorylation of MAP-kinase p38. Anti-apoptotic and anti-inflammatory effects were detectable as Caspase-3, NF-kappaB, TNF-α, and AIF-1 expression were significantly reduced after IRI+ALF in comparison to IRI+PBS or IRI+iALF. Gap-43 expression was significantly increased after IRI+ALF. iALF showed effects similar to PBS. The intrinsic regenerative potential of RGC-axons was induced to nearly identical levels after IRI and ALF or iALF-treatment under growth-permissive conditions, although RGC viability differed significantly in both groups. Intravitreal CO further increased the IRI-induced migration of GFAP-positive cells out of retinal explants and their transdifferentiation, which was detected by re-expression of beta-III tubulin and nestin.

Conclusion: Intravitreal CORM ALF-186 protected RGC after IRI and stimulated their axons to regenerate in vitro. ALF conveyed anti-apoptotic, anti-inflammatory, and growth-associated signaling after IRI. CO's role in neuroregeneration and its effect on retinal glial cells needs further investigation.

MeSH terms

  • Animals
  • Axons / drug effects
  • Axons / metabolism
  • Calcium-Binding Proteins / genetics
  • Calcium-Binding Proteins / metabolism
  • Carbon Monoxide / metabolism*
  • Caspase 3 / genetics
  • Caspase 3 / metabolism
  • Cell Movement / drug effects
  • Cells, Cultured
  • Coordination Complexes / administration & dosage
  • Coordination Complexes / pharmacology
  • Coordination Complexes / therapeutic use
  • Female
  • GAP-43 Protein / genetics
  • GAP-43 Protein / metabolism
  • Glial Fibrillary Acidic Protein / metabolism
  • HSP70 Heat-Shock Proteins / metabolism
  • HSP90 Heat-Shock Proteins / metabolism
  • Intravitreal Injections
  • Male
  • Microfilament Proteins / genetics
  • Microfilament Proteins / metabolism
  • Mitogen-Activated Protein Kinases / metabolism
  • NF-kappa B / genetics
  • NF-kappa B / metabolism
  • Nerve Regeneration* / drug effects
  • Neuroglia / drug effects
  • Neuroglia / metabolism
  • Neuroprotection* / drug effects
  • Neuroprotective Agents / pharmacology
  • Neuroprotective Agents / therapeutic use
  • Phosphorylation / drug effects
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Rats, Sprague-Dawley
  • Reperfusion Injury / drug therapy
  • Reperfusion Injury / genetics
  • Reperfusion Injury / pathology
  • Retinal Ganglion Cells / drug effects
  • Retinal Ganglion Cells / metabolism*
  • Retinal Ganglion Cells / pathology
  • Tubulin / metabolism
  • Tumor Necrosis Factor-alpha / genetics
  • Tumor Necrosis Factor-alpha / metabolism


  • Aif1 protein, rat
  • Calcium-Binding Proteins
  • Coordination Complexes
  • GAP-43 Protein
  • Glial Fibrillary Acidic Protein
  • HSP70 Heat-Shock Proteins
  • HSP90 Heat-Shock Proteins
  • Microfilament Proteins
  • NF-kappa B
  • Neuroprotective Agents
  • RNA, Messenger
  • Tubb3 protein, rat
  • Tubulin
  • Tumor Necrosis Factor-alpha
  • triscarbonyl(histidinato)molybdate(III)
  • Carbon Monoxide
  • Mitogen-Activated Protein Kinases
  • Caspase 3

Grant support

This work was conducted at the Eye Center, Medical Center – University of Freiburg, Faculty of Medicine, Freiburg, Germany, and granted by The German Research Foundation (DFG, Grants: BI 1567/2-1 and GO 2158/3-1). The funder (DFG) had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. The article processing charge was funded by the German Research Foundation (DFG) and the Albert Ludwigs University Freiburg in the funding programme Open Access Publishing.