Induction of proliferating cell nuclear antigen (PCNA)-immunoreactive cells in goldfish retina following intravitreal injection with 6-hydroxydopamine.
1. The dopaminergic neurotoxin, 6-hydroxydopamine (6-OHDA), was injected intravitreally into the eyes of juvenile (5- to 6-cm) goldfish. 2. Proliferation of rod neuroblasts caused by 6-OHDA (2 micrograms in 2 microliters saline) was detected in retinal wholemounts by immunofluorescence for proliferating cell nuclear antigen (PCNA) 3, 7, 14, 20, or 30 days after injection. 3. The injected dose of 6-OHDA was sufficient to cause permanent loss of dopaminergic interplexiform and serotonergic amacrine cells in the injected eye but not in the contralateral control eye. 4. 6-OHDA increased the density (mm-2) of PCNA-ir cells in the outer nuclear layer (ONL) of the injected eye to 2.65 times the initial density 20-30 days after injection, and it increased the density of PCNA-ir cells in the ONL of the contralateral, untreated eye, equally but after a delay of less than or equal to 7 days with respect to the injected eye. 5. 6-OHDA also increased the density of PCNA-ir cells in the inner nuclear layer (INL) to greater than 20 times the initial density 7 days after injection, followed by a rapid decline almost to control levels by 14 days after injection. 6. The sequence of responses to 6-OHDA, with PCNA-ir cells first scattered in the ONL and then clustered in the INL, suggests that neuroblasts from the ONL migrate to the INL to compensate for toxin-induced cell loss. 7. Double staining for 5-bromodeoxyuridine (BrUdR; a thymidine analogue) and PCNA, carried out on 7 days after intravitreal injection with 6-OHDA, showed that 77% of all PCNA-ir cells in the outer nuclear layer had been in S phase during the previous 24 hr. 8. Immunoreactivity for PCNA was found to be a valid marker for rod neuroblasts which have entered S phase within 1-2 days before sampling and was shown to be especially convenient for investigating the distribution of proliferating cells in whole mounts. 9. In controls injected unilaterally with saline or saline plus 1% dimethyl sulfoxide (DMSO), the differences in densities of PCNA-ir rod precursor nuclei 2-30 days after injection vs. day 0 (uninjected) were statistically insignificant in both injected and uninjected eyes (Negishi et al., 1991). Therefore the local effect of injecting 6-OHDA was due to 6-OHDA itself, not to mechanical damage or nonspecific actions of foreign substances.(ABSTRACT TRUNCATED AT 400 WORDS)
Department of Neurophysiology, University of Kanazawa School of Medicine, Japan.
K
K
Negishi
W K
WK
Stell
T
T
Teranishi
A
A
Karkhanis
V
V
Owusu-Yaw
Y
Y
Takasaki
D000818:Animals; D001324:Autoantigens / Q000378:metabolism; D001973:Bromodeoxyuridine / Q000378:metabolism; D002452:Cell Count; D002455:Cell Division; D005455:Fluorescent Antibody Technique; D006054:Goldfish; D007267:Injections; D009687:Nuclear Proteins / Q000378:metabolism*; D016627:Oxidopamine / Q000494:pharmacology*; D018809:Proliferating Cell Nuclear Antigen; D012160:Retina / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D014822:Vitreous Body / Q000187:drug effects
D001324:Autoantigens; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; D016627:Oxidopamine; D001973:Bromodeoxyuridine