Selective degeneration of central photoreceptors after hyperbaric oxygen in normal and metallothionein-knockout mice

Abstract

Purpose: Metallothioneins (MTs) in the brain and retina are believed to bind metals and reduce free radicals, thereby protecting neurons from oxidative damage. This study was undertaken to investigate whether retinal photoreceptor (PR) cells lacking MTs are more susceptible to hyperbaric oxygen (HBO)-induced cell death in vivo.

Methods: Wild-type (WT) and MT-knockout (MT-KO) mice lacking metallothionein (MT)-1 and MT-2 were exposed to three atmospheres of 100% oxygen for 3 hours, 3 times per week for 1, 3, or 5 weeks. The control animals were not exposed. Histologic analysis of PR viability was performed by counting rows of nuclei in the outer nuclear layer (ONL). Ultrastructure studies verified PR damage.

Results: HBO exposure produced a major loss of PR cells in the central retinas of WT and MT-KO mice, with no effect on the peripheral retina even at the longest (5 weeks) exposures. The degree of PR damage and cell death increased with duration of HBO exposure. One week of HBO exposure was insufficient to cause PR death, but tissue damage was observed in the inner and outer segments. At 3 weeks, the rows of PR nuclei in the central retina were significantly reduced by 38% in WT and 28% in MT-KO animals. At 5 weeks, PR loss was identical in WT (34%) and MT-KO (34%) animals and was comparable to that in WT at 3 weeks.

Conclusions: The data suggest that MT-1 and -2 alone are not sufficient for protecting PRs against HBO-induced cell death. The selective degeneration of central PRs may provide clues to mechanisms of oxidative damage in retinal disease.

Figure 1

Sample retinal section: WT animal, 0 weeks of HBO. A low-powered light photomicrograph shows the entire retinal section, spanning the superior (s) and inferior (i) retinal hemispheres, with the optic nerve head (ONH) at its center. Beginning at the ONH, every 200 μm in either direction marked a single bin, as shown in the inset. The bins closest to the ONH (i1–i6 and s1–s6) were considered to be the central retina. Inset: a 50-μm wide sampling area was selected at the center of each bin for counting the number of PR nuclei and rows of nuclei in the ONL of the retina.

Figure 2

Changes in ONL thickness after HBO exposure. The mean number of rows of PR nuclei in the ONL (ordinate) varied as a function of retinal eccentricity (abscissa, when inferior and superior values were combined), that is, the distance from the optic nerve in unit bins. Each curve plotted the changes in ONL thickness after different durations of HBO treatment (0, 1, 3, and 5 weeks). Each value (±SD) was the average for four sections (n = 3 animals). (A) Data from WT retina; (B) data from MT-KO retina.

Figure 3

PR cell loss with increased duration of HBO exposure: MT-KO versus WT retina. The histograms give the number (mean ± SD) of rows of PR nuclei for the central retinas (Fig. 2, bins 1–6) at each HBO exposure time. The bars are 1 SD from the mean (n = 3). *Significant difference between the two values at 3 weeks (t-test, P = 0.002).

Figure 4

HBO-induced structural abnormalities in the PR IS and OS of WT and MT-KO mice preceded the loss of PR nuclei. (A, B) MT-KO retina, central region. (A) Control, no HBO. OS and IS were long and well aligned, with no signs of IS swelling, and the ONL was of normal thickness. (B) 1-week HBO. The number of rows of PR nuclei in the ONL was similar to that in the untreated retina (A), however, signs of IS swelling and OS shortening were already observed. (C, D) WT retina, central region, showing a higher magnification of the OS and IS layers at 0 and 5 weeks of HBO. (C) At 0 weeks of HBO, OS and IS were long and well aligned. (D) At 5 weeks of HBO, OS and IS became shortened, fragmented, swollen, and darkened. In addition, significant loss of PR nuclei had occurred by this stage (also see Figs. 5B, 6B). All findings were the same for both WT and MT-KO retinas. Scale bars, 25 μm.

Figure 5

Regional variation in HBO-induced PR damage. Central retinas (A, B) and peripheral retinas (C, D) of WT mice; similar findings were obtained in MT-KO mice (see Figs. 6A, 6B). (A) Central retina of WT mouse that was not exposed to HBO. The ONL, which contains the PR nuclei, was approximately 6 to 10 rows thick, and the PR IS and OS appeared healthy and vertically aligned. (B) Central retina of WT mouse exposed to 5 weeks of HBO. The ONL thickness has been reduced to only three to five rows of nuclei, indicating a significant loss of PRs. In (B), thinning of PR IS and OS layers, as well as swelling and disorganization of the remaining IS and OS also accompanied PR cell loss (compare to control retina in A). (C) Peripheral retina of WT mouse, not exposed to HBO. The ONL contained six to nine rows of PR nuclei, the IS appeared intact, and the OS were well organized. (D) Peripheral retina of WT mouse, after HBO exposure. Even after 5 weeks of HBO, the peripheral retina appeared virtually identical with the control (C), showing normal PR nuclei, IS, and OS. RPE, retinal pigment epithelium; OPL, outer plexiform layer; INL, inner nuclear layer. Scale bar, 25 μm.

Figure 6

Variability of cell damage in MT-KO central retinas at 3 weeks of HBO. (A) Untreated MT-KO retina, 0 weeks of HBO. Similar to untreated WT retina, PRs appeared healthy, with approximately 10 rows of nuclei in the ONL and intact, well-aligned IS and OS. (B) MT-KO retina, 5 weeks of HBO. As in the WT, HBO has caused a remarkable thinning of the ONL to only three to five rows of nuclei, reflecting a dramatic loss of PRs. (C, D) Various degrees of damage were observed at 3 weeks, ranging from minimal damage without any cell loss (C), similar to untreated (A) or 1-week treated (Fig. 4B) retinas, to extensive damage with considerable cell loss (D), comparable to 5-week exposed retinas (B). Scale bar, 25 μm.

Figure 7

Ultrastructural analysis of PR IS and OS of WT and MT-KO mice after different HBO exposures. (A–C) WT retina at 0, 3, and 5 weeks of HBO. (D–F) MT-KO retina at 0, 3, and 5 weeks of HBO. Overall, the MT-KO retinas looked similar to the wild-type retinas at each successive stage of HBO exposure. Well-aligned PR OS at 0 weeks of HBO (A, D) became shortened (B, E) and fragmented (C, F). (Note in D that despite the slightly cross-sectional cut through the OS, the thickness of the OS layer is well illustrated.) IS became swollen (C, E, F), and there was a progressive thinning of the OS and IS layers, as evidenced by the repositioning of the ONL more proximate to the RPE, with longer HBO exposures (compare A and D, in which the ONL was not visible in the micrograph, to C and F, in which the ONL was relatively closer to the RPE). Evidence of degenerating PRs is represented by darkened, pyknotic tissue (B, C, E: *, double arrows). At 3 weeks of HBO, the transition from normal morphology to pathologic was evident at various stages. The micrograph in (B) highlights the shortening of OS and IS, whereas (E) shows more prominent swelling of the IS. Scale bars, 5 μm.