Executioner Caspase Activity and Photoreceptor Apoptosis After Blunt Ocular Trauma

Abstract

 

Purpose

 

Blunt ocular trauma causes commotio retinae (CR), a condition in which macular photoreceptor degeneration permanently reduces vision in 26% of patients. After experimental CR in rats, photoreceptors die by both necrosis and apoptosis, initiated by caspase-9.<br /> The executioner caspases in CR have not been described and no treatments to prevent photoreceptor loss are available. Accordingly, we assessed the role of caspases-3, -6 and -7 as mediators of photoreceptor apoptosis in a rat model of CR, to identify a mechanism which might highlight potential therapeutic targets.

 

Methods

 

Bilateral CR was induced in rats using ballistic ocular trauma. Caspase activity was assessed by immunohistochemistry and in western blots. Caspase-6 was inhibited using unilateral intravitreal injection of penetratin (Pen1)-linked caspase-6 dominant negative (C6DN) protein after ballistic injury compared with control eyes treated with Pen1 injection alone and retinal function assessed by ERG a-wave amplitude and photoreceptor survival by outer nuclear layer (ONL) thickness on retinal sections. n=6-8 /analysis.

 

Results

 

Active caspase-3 and -7 were not found. Cleaved caspase-6 immunolocalised to photoreceptor somata and retinal levels of cleaved caspase-6 increased 48 hours after injury (Fig 1A-B). Caspase-6 inhibition by intravitreal Pen1-XBIR3 injection reduced a-wave amplitude but did not affect ONL thickness 14 days after ballistic injury compared to control eyes (Fig 2).

 

Conclusions

 

After experimental CR, caspase-6 was the only executioner caspase activated, but its inhibition did not increase photoreceptor survival or function, suggesting that either classical executioner caspases are not implicated, or that pathways alternative to apoptosis become active when caspase-6 is inhibited.  

 

Fig 1. A-B. On western blots, retinal levels of full length (p33-35) caspase-6 were unchanged, the cleaved (p18) form increased up to 48 hours. (*p=0.009). C-D. Levels of cleaved (p17) and full length (p32) caspase-3 were unchanged. E-F. Cleaved caspase-7 was not detected and full length caspase-7 (P35) was unchanged.

 

 

Fig 2. A. Caspase-6 inhibition caused a small, but statistically significant reduction in retinal function, an effect that was more apparent at higher stimulus intensities (p<0.001). B. ONL thickness after experimental CR in Pen1-C6DN-treated eyes was unchanged compared to control eyes.