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. 1991 Dec;11(6):639–659. doi: 10.1007/BF00741451

Induction of proliferating cell nuclear antigen (PCNA)-immunoreactive cells in goldfish retina following intravitreal injection with 6-hydroxydopamine

K Negishi 1,2,✉, W K Stell 2, T Teranishi 1, A Karkhanis 2, V Owusu-Yaw 2, Y Takasaki 4
PMCID: PMC11567366  PMID: 1685943

Abstract

  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 (2µg in 2µl 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 theouter 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 ⩽7 days with respect to the injected eye.

  5. 6-OHDA also increased the density of PCNA-ir cells in theinner nuclear layer (INL) to >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 (Negishiet 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.

  10. The proliferative response of rod neuroblasts in the injected eye could be due in part to impairment of dopaminergic regulation of the neuroblasts and also in part to mitogen(s) released from damaged cells. The proliferative response of rod neuroblasts in theuninjected control eye, however, is likely to be caused by diffusible mitogen(s) released from the treated retina.

Key words: fish retina, immunohistochemistry, rod precursor cells, proliferating cell nuclear antigen (PCNA), 6-hydroxydopamine, regeneration

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