Expression of antioxidant enzymes in rat retinal ischemia followed by reperfusion.
To evaluate the expression and protein levels of antioxidant enzymes in the rat retina exposed to oxidative stress induced by ischemia-reperfusion injury. Retinal ischemia was induced in female Wistar rats by ligation of the optic nerve and vessels behind the left eye bulb, and was followed by reperfusion for 0, 3, 6, or 24 hours. The right eye served as control. RNA and protein were extracted simultaneously from each retina. Expressions of the endogenous antioxidant enzymes glutathione peroxidase (GPx1), catalase (CAT), copper/zinc superoxide dismutase, manganese superoxide dismutase, and the catalytic subunit of glutamylcysteine ligase (GCLc) were analyzed with real-time reverse transcription polymerase chain reaction and related to the endogenous control cyclophilin B. Protein levels were measured with Western blot analysis. During the early phase (0 or 3 hours) of reperfusion, no changes were seen in enzyme expression. After 6 hours, GCLc expression increased by a factor of 1.14 (P = .034), followed by a decline of 0.80 after 24 hours (P = .00004), according to the comparative Ct method. After 24 hours of reperfusion, GPx1 expression increased by a factor of 1.14 (P = .028), and CAT had decreased by 0.82 (P = .022). Expressions of copper/zinc superoxide dismutase and manganese superoxide dismutase showed a tendency toward a decrease by factors of 0.86 (P = .055) and 0.88 (P = .053), respectively, after 24 hours. Protein levels did not differ for any of the antioxidants, regardless of reperfusion time. The slightly increased messenger RNA expression of GPx1 after 24 hours of reperfusion with a concomitant very modest decrease in CAT and GCLc expression and no change in protein levels indicate a very modest, if any, response to oxidative stress generated by ischemia followed by reperfusion in rat retina.
Agardh
Carl-David
CD
Unit on Vascular Diabetic Complications, Department of Clinical Sciences, Malmö University Hospital, 205 02 Malmö, Sweden. carl-david.agardh@med.lu.se
Gustavsson
Carin
C
Hagert
Per
P
Nilsson
Marie
M
Agardh
Elisabet
E
D000818:Animals; D002374:Catalase / Q000032:analysis / Q000235:genetics; D021983:Cyclophilins / Q000032:analysis / Q000235:genetics; D005260:Female; D005721:Glutamate-Cysteine Ligase / Q000032:analysis / Q000235:genetics; D005979:Glutathione Peroxidase / Q000032:analysis / Q000235:genetics; D007511:Ischemia / Q000378:metabolism*; D019696:Peptidylprolyl Isomerase / Q000032:analysis / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D015427:Reperfusion Injury / Q000378:metabolism*; D012171:Retinal Vessels / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000032:analysis / Q000235:genetics
D012333:RNA, Messenger; C081786:cyclophilin B; D002374:Catalase; D005979:Glutathione Peroxidase; D013482:Superoxide Dismutase; D021983:Cyclophilins; D019696:Peptidylprolyl Isomerase; D005721:Glutamate-Cysteine Ligase