ROS overproduction has been implicated as a key mediator of photoreceptor death in many ocular diseases, including RD,27
age-related macular degeneration,33
and macular dystrophy.36
We thus wanted to investigate whether ROS scavengers can have an effect on RD-induced photoreceptor cell loss.
In our rodent model of experimental RD, we found evidence of a substantial increase in oxidative stress. Elevated levels of oxidized lipids and proteins in the form of 4-HNE and PCC, respectively, were found in the retina after RD. Treatment with the free radical scavenger edaravone, significantly reduced the expression of both markers (), along with a decrease in TUNEL-positive cells in the photoreceptor layer (). This finding indicates that ROS may be an important therapeutic target in preventing photoreceptor cell death after RD. We should note that the dose of edaravone used in our study was significantly higher than the doses used in patients that suffer stroke, but is similar to that used in other animal studies. The discordance in the doses needed between animals and humans presumably can be attributed to the different pharmacokinetics and route of administration (intravenous in humans versus intraperitoneal in rats).
Along with ROS expression, RD induces proinflammatory cytokine and chemokine secretion. Vitreous samples from patients with RD exhibited significantly higher levels of TNF-α37,38
compared with samples from patients with a macular hole or idiopathic premacular fibrosis. In vivo experimental RD studies also showed that RD is strongly associated with the production of MCP-1 and TNF-α.23,27
In the present study, edaravone treatment significantly reduced the expression of the inflammatory cytokines TNF-α and MCP-1 and the number of infiltrating macrophages after RD (, ).
In experimental RD, the apoptosis of photoreceptor cells has been related to the activation of both intrinsic and extrinsic apoptotic pathways.24,41,42
The present study also confirmed that RD increased the activation of caspase-8, -3, and -9. However, their activity was significantly decreased after edaravone treatment (). This result is in line with the ischemia–reperfusion injury model study which showed that edaravone functions as a neuroprotective agent by blocking cytosolic release of cytochrome c
and caspase-3 activation43,44
and by suppressing the Fas-signaling pathway.45
Moreover, these data indicate that reduction of ROS also has an effect in modulating the intrinsic apoptotic pathway. In fact, ROS are known triggers of the intrinsic apoptotic cascade via interactions with proteins of the mitochondrial permeability transition complex.46
Previous studies demonstrated that upregulation in expression of Bcl-2, the antiapoptotic protein located in the outer membrane of the mitochondria, inhibits the opening of the permeability transition complex, thus decreasing apoptotic cell death.47
Furthermore, Bcl-2 overexpression has been found to inhibit photoreceptor degeneration,48
and retinal neurons overexpressing Bcl-2 are protected against axotomy-induced cell death.49
With induction of RD, a trend in decrease of Bcl-2 along with a statistically significant upregulation of Bax (the proapoptotic member of the Bcl-2 family) was noted (). Edaravone treatment lead to increased levels of Bcl-2 protein, whereas the Bax levels were unaffected, thus tilting the balance in favor of the antiapoptotic member of the Bcl-2 family of proteins.
MAP kinase (ERK, JNK, and p38) is a family of stress-related kinases that have been implicated in various neural injuries and diseases.50
Among the members of the family, ERK1/2 has been implicated by some studies as a death-promoting kinase and could be activated by oxidative stress and ROS.51,52
reported that RD is associated with ERK1/2 activation, but not with that of JNK or p38. In the present study, ERK1/2 was activated after RD, and its activation was significantly reduced with edaravone treatment (). This finding suggests that ERK activation may be related to the participation of ROS in neuronal cell death, in concordance with an in vitro study showing that oxidative stress can activate ERK and that complete inhibition of the first phase of ERK1/2 activation can protect cells from oxidative stress–induced cell death.53
In summary, in our rodent model of RD, cell death, as determined by TUNEL positivity, was localized in the photoreceptor layer. After systemic treatment with edaravone, concomitant reduction of ROS, macrophage infiltration, inflammatory cytokines, caspase activation, and photoreceptor cell death were noted in the rat retina. This result leads to the conclusion that edaravone may decrease cell death by inhibiting macrophage function and infiltration. We previously reported that MCP-1 is a key mediator of early infiltration of macrophage/microglia after RD.27
In accordance with this result, MCP-1 upregulation after RD was substantially decreased with edaravone treatment. However, upregulation in the antiapoptotic protein Bcl-2 and downregulation of ERK1/2 with edaravone treatment suggests that edaravone may have a direct effect in preventing photoreceptor cell death.
Edaravone has been studied, not only in in vivo experimental settings, but also in clinical practice. Most of the studies included patients with acute ischemic stroke (AIS). A clinical trial has shown that the administration of edaravone alone within 72 hours of the onset of AIS significantly reduced the infarct volume and produced sustained benefits during a 3-month follow-up period.54
Recently, a study showed that improved visual acuity was observed when edaravone was administered in conjunction with vitrectomy in patients with branch retinal vein occlusion.55
Taken all together, these findings suggest that edaravone could be used in retinal diseases related to oxidative damage characterized by separation of the neurosensory retina from the RPE, including rhegmatogenous RD, AMD, retinal vein occlusion, and diabetic retinopathy. However, there is a report suggesting possible toxicity resulting in renal failure in stroke patients treated with edaravone.56
We observed photoreceptor toxicity with edaravone treatment at higher doses, detecting increased cell death in the photoreceptor layer (). Nevertheless, we did not see any toxic effect with a lower dose when given for 5 days. Therefore, although there is a need for additional studies to determine the most appropriate dosing, these results suggest that reducing oxidative stress may offer a new therapeutic target in treating RD.
In conclusion, this study provides substantial evidence of the role of ROS in photoreceptor cell death after RD. After RD, the ROS level is upregulated, along with increased production of proinflammatory cytokines and subsequent caspase activation resulting in the cell death of photoreceptor cells. We were able to reduce photoreceptor cell loss by reducing ROS with edaravone. It is likely that edaravone absorbed free radicals generated by RD and/or inhibited their functions, thereby preventing free radical–mediated photoreceptor death after RD.