Continuous illumination of the retina: ultrastructural and immunocytochemical study of apoptosis in control and adrenalectomized rats.

J.J. LOPEZ-COSTA, E.M. LÓPEZ, M. ACKERMAN, M.B. PIAGGIO, J. SÁNCHEZ DE BUSTAMANTE AND J. COIRINI.

Buenos Aires

Congreso; XVII International Congress of Eye Research.; 2006

Experimental Eye Research Society

Continuous illumination of the retina: Ultrastructural and immunocytochemical study of apoptosis in control and adrenalectomized rats. JJ. López-Costa, EM. López, M. Ackerman,.MB. Piaggio, J. Sánchez de Butamante and H. Coirini.             1IBCyN ¨Prof. E. De Robertis¨; 2Hospital de Clínicas, UBA; 3Hospital Universitario Austral, 4IBYME, 4Dept Bioquímica Humana, Facultad de Medicina, UBA-CONICET, Buenos Aires, Argentina. Our previous studies have shown that adrenalectomy partially prevents light-induced retinal degeneration (LIRD) at light microscopy. In the present work, the ultrastructural alterations of the retina and the immunocytochemical characterization of apoptotic nuclei was performed in both sham operated (CTL) and adrenalectomized (ADX) rats. Animal experiments were conducted in compliance with the ARVO Guidelines of Animal Use Statement. Sprague Dawley rats were adrenalectomized under Ketamine anesthesia (50 mg/kg, IP). After 18 hours of recovery; adrenalectomized (ADX) and CTL rats were submitted to continuous illumination (CI) (12000 lux) up to 7 days. Animals were offered water (CTL) or saline solution (NaCl, 0.9%) (ADX) and food ad libitum. Rats were anesthetized with chloral hydrate (350 mg/kg, IP) and sacrificed either before or after 1, 2, 5 and 7 days of CI. The eyes were fixed by immersion in a solution containing 2.5% glutaraldehyde and were dehydrated and embedded in Durcupan. Ultrathin sections were stained with lead citrate and were observed with a Zeiss 110 electron microscope. Another group of CTL and ADX animals were fixed using a 4 % paraformaldehyde solution. The eyes were removed, postfixed and cryoprotected. Cryostat sections were processed by immunocytochemistry using a Caspase-3 antibody. After 24 hs of CI, alterations of outer and inner photoreceptor segments were observed. Retinal degeneration increased along days 2 and 5, and was maximal after 7 days of CI, when cell debris were observed between the swollen retinal pigment epithelium and the thin outer nuclear layer (ONL). RPE showed an important phagocytic activity after 48 hs of illumination in CTL rats while maximal alterations in ADX rats were observed after 5 days of CI. An important quantity of pycnotic nuclei and nuclear fragmentation were observed in ONL in CTL rats but these changes were less prominent in ADX rats. Mitochondrial alterations were detected in inner nuclear layer of CTL rats after 1 and 2 days of CI but in ADX animals after 5 days of CI. Degenerated cells were observed in ganglion cell layer in both groups after 5 days of CI but thick electron dense processes of Muller cells were predominatly observed in CTL retinas. Immunocytochemical studies revealed labeled nuclei in ONL in both conditions confirming that apoptosis is involved in light induced retinal degeneration. As changes were more pronounced in CTL than in ADX rats, these evidences further support the idea that adrenal hormones could facilitate LIRD. (UBACYT-M020).