Elevated MMP Expression in the MRL Mouse Retina Creates a Permissive Environment for Retinal Regeneration

Abstract

Purpose: The MRL/MpJ (healer) mouse is an established model for autoimmune studies and was recently identified as having a profound ability to undergo scarless regeneration of the tissue in the ear and heart. This regenerative capacity has been linked to elevated matrix metalloproteinase (MMP)-2 and -9 expression, giving this mouse the ability to degrade and remove inhibitory basement membrane molecules. Although elevated MMP expression has been reported in somatic tissues in this strain, little is known about MMP expression and the response to injury in the MRL/MpJ mouse retina. The purpose of this study was to investigate whether increased MMP expression and subsequent decreased inhibitory extracellular matrix molecule deposition in the MRL/MpJ mouse retina produces a permissive regenerative environment.

Methods: Experiments were performed using 3- to 4-week-old MRL/MpJ, retinal degenerative (rd1), and C57BL/6 (wild-type) mice. Western blotting, oligo-microarray, and immunohistochemical analyses were used to determine the level and location of MMP and extracellular matrix (ECM) protein expression. Retinal responses to injury were modeled by retinal detachment in vivo and in retinal explantation in vitro. The capacity of the retinal environment to support photoreceptor cell migration, integration, or regeneration was analyzed using hematoxylin-eosin, immunohistochemical staining, and cell counting.

Results: Compared with C57BL/6J animals, MRL/MpJ mice exhibit elevated levels of MMP-2, -9, and -14 and decreased levels of the inhibitory proteins neurocan and CD44 within the retina. Although similar increases in MMP-2, -9, and CD44s (CD44 degradation product) were observed in the rd1 retina, elevated levels of the inhibitory ECM molecules (neurocan and CD44) remained. Thus, the MRL retinal environment, which expresses lower levels of inhibitory ECM molecules after injury, was more conducive to regeneration and enhanced photoreceptor integration in vitro than C57BL/6J or rd1 controls.

Conclusions: The MRL mouse retina shows elevated MMP expression and decreased levels of scar-related inhibitory molecules, which leads to a retinal environment that is more permissive for neural regeneration and cell integration after in vitro retinal explantation.

FIGURE 1

ECM array gene expression analysis from BL6, rd1, and MRL mouse retinas. (A-E) Representative microarray gene chips for BL6 (A1), rd1 (A2), and MRL (A3) mice, with corresponding densitometric analysis of basal MMP-9 (AB, B), MMP-2 (AC, C), MMP-14 (AD, D), and MMP-13 (AE, E) RNA expression. Retinal expression of MMP-9 (AB, B), MMP-2 (AC, C), and MMP-14 (AD, D) RNA was significantly higher in rd1 and MRL mice than in BL6 wild-type controls. Retinal expression of MMP-13 (AE, E) was significantly lower in rd1 and MRL mice than in BL6 controls (n = 12). Significance was tested using one-way ANOVA, with Tukey testing for post hoc comparisons. *P < 0.05; **P < 0.001.

FIGURE 2

Western blot analysis of MMP-2, -9, and -14 in adult BL6, rd1, and MRL mouse retina. (A-C) Representative Western blots and corresponding densitometric analyses of MMP-2 (A), MMP-9 (B), and MMP-14 (C) normalized against β-actin (loading control) in BL6, rd1, and MRL mouse retina explants. MMP-2 (A) and MMP-9 (B) expression was significantly higher in MRL and rd1 retinas than in BL6 wild-type controls. Similarly, a significantly higher level of MMP-14 (C) expression was detected in the MRL mouse retina than in the retina of rd1 injury and BL6 wild-type controls (n = 12). Significance was tested using one-way ANOVA with Tukey testing for post hoc comparisons. *P < 0.05; **P < 0.001.

FIGURE 3

Analysis of CD44 and neurocan expression in adult BL6, rd1, and MRL mouse retina explants. (A-C) Representative Western blots and corresponding densitometric analyses of CD44s (A, noninhibitory CD44 degradation product), neurocan (B), and neurocan-CT (C, noninhibitory c-terminal neurocan degradation product) normalized to β-actin (loading control) in BL6, rd1, and MRL mouse retina explants. CD44s (A) and neurocan-CT (C) expression was significantly higher, whereas full-length neurocan (B) expression was significantly lower in MRL mouse retina than in BL6 controls. CD44s (A) and full-length neurocan (B) expression were significantly higher in rd1 mouse retina than in B6 controls (n = 12). (D-I) Retinal explants were plated in serum-free media for 7 days and subsequently fixed, cryoprotected, sectioned, and immunolabeled for full-length CD44 (D-F) and neurocan (G-I). Immunocytochemical staining revealed decreased CD44 (F) and neurocan (I) expression in MRL mouse retinas compared with B6 (D, G) and degenerative rd1 (E, H) controls (n = 12). Significance was tested using one-way ANOVA with Tukey testing for post hoc comparisons. *P < 0.05; **P < 0.001. Scale bar = 40 μm.

FIGURE 4

Decreased CD44 and neurocan deposition in MRL mice after 14-day retinal detachment. Retinal detachments were induced by inserting a micropipette through the nasal retina and injecting 1 to 2 μL of 1.4% sodium hyaluronate into the subretinal space of age-matched BL6 and MRL mice. Eyes were enucleated 14 days after surgery and subsequently fixed, cryoprotected, sectioned, and immunostained. (A-L) Representative micrographs illustrating CD44 (red) and neurocan (blue) expression at injection and retinal detachment sites in BL6 (A, C, E) and MRL (B, D, F) mice (n = 6). Scale bar = 40 μm.

FIGURE 5

Elevated MMP and subsequent decreased expression of CD44 and neurocan correlate with enhanced photoreceptor integration in the MRL mouse retina. Abutting retinal explants were performed in which GFP-positive photoreceptor sheets isolated from adult mice were transplanted on top of BL6, rd1, or MRL mouse retinas. Explants were cultured in serum-free neurobasal media for 7 days and subsequently fixed, cryoprotected, sectioned, and analyzed using confocal microscopy for GFP expression and subsequent cell counting. Although minimal photoreceptor integration was detected in abutting Bl6 (A, D) and rd1 (B, D) cultures, indicated by a lack of GFP-positive cells in the host tissue, increased integration of GFP-positive photoreceptor cells was detected in the MRL mouse retinas (C, D), indicated by numerous GFP-positive cells that have migrated to the host tissue (n = 12). Significance was tested using one-way ANOVA with Tukey testing for post hoc comparisons. **P < 0.001. Scale bar = 20 μm.

FIGURE 6

Schematic diagram based predominantly on in vitro explant studies, summarizing the environmental differences among BL6, rd1, and MRL mouse retinas. Unlike the BL6, injury to the MRL mouse retina stimulated enhanced MMP-2, -9, and -14 expression, which led to enhanced enzymatic activity, glial barrier degradation, and photoreceptor integration. Although similar elevations in MMP-2 and -9 were observed in the rd1 mouse, inhibitory ECM deposition, poor glial barrier formation, and poor photoreceptor integration persisted.