Neuroprotection and neuroregeneration of retinal ganglion cells after intravitreal carbon monoxide release

Abstract

Purpose: Retinal ischemia induces apoptosis leading to neurodegeneration and vision impairment. Carbon monoxide (CO) in gaseous form showed cell-protective and anti-inflammatory effects after retinal ischemia-reperfusion-injury (IRI). These effects were also demonstrated for the intravenously administered CO-releasing molecule (CORM) ALF-186. This article summarizes the results of intravitreally released CO to assess its suitability as a neuroprotective and neuroregenerative agent.

Methods: Water-soluble CORM ALF-186 (25 μg), PBS, or inactivated ALF (iALF) (all 5 μl) were intravitreally applied into the left eyes of rats directly after retinal IRI for 1 h. Their right eyes remained unaffected and were used for comparison. Retinal tissue was harvested 24 h after intervention to analyze mRNA or protein expression of Caspase-3, pERK1/2, p38, HSP70/90, NF-kappaB, AIF-1 (allograft inflammatory factor), TNF-α, and GAP-43. Densities of fluorogold-prelabeled retinal ganglion cells (RGC) were examined in flat-mounted retinae seven days after IRI and were expressed as mean/mm2. The ability of RGC to regenerate their axon was evaluated two and seven days after IRI using retinal explants in laminin-1-coated cultures. Immunohistochemistry was used to analyze the different cell types growing out of the retinal explants.

Results: Compared to the RGC-density in the contralateral right eyes (2804±214 RGC/mm2; data are mean±SD), IRI+PBS injection resulted in a remarkable loss of RGC (1554±159 RGC/mm2), p<0.001. Intravitreally injected ALF-186 immediately after IRI provided RGC protection and reduced the extent of RGC-damage (IRI+PBS 1554±159 vs. IRI+ALF 2179±286, p<0.001). ALF-186 increased the IRI-mediated phosphorylation of MAP-kinase p38. Anti-apoptotic and anti-inflammatory effects were detectable as Caspase-3, NF-kappaB, TNF-α, and AIF-1 expression were significantly reduced after IRI+ALF in comparison to IRI+PBS or IRI+iALF. Gap-43 expression was significantly increased after IRI+ALF. iALF showed effects similar to PBS. The intrinsic regenerative potential of RGC-axons was induced to nearly identical levels after IRI and ALF or iALF-treatment under growth-permissive conditions, although RGC viability differed significantly in both groups. Intravitreal CO further increased the IRI-induced migration of GFAP-positive cells out of retinal explants and their transdifferentiation, which was detected by re-expression of beta-III tubulin and nestin.

Conclusion: Intravitreal CORM ALF-186 protected RGC after IRI and stimulated their axons to regenerate in vitro. ALF conveyed anti-apoptotic, anti-inflammatory, and growth-associated signaling after IRI. CO's role in neuroregeneration and its effect on retinal glial cells needs further investigation.

Fig 1. Protection of retinal ganglion cells (RGC) by intravitreal-administered ALF-186 after ischemia-reperfusion-injury (IRI).

(A) Representative images of flat mounted retinae seven days after IRI to the left eye (left picture: untreated right eye without any treatment, picture 2–4: IRI eye after intravitreal injection of PBS, ALF-186, or iALF-186). RGC had been retrogradely labeled with fluorogold one week prior to intervention. A significant reduction of RGC-density was detectable after IRI and PBS or iALF treatment, while ALF protected RGC after IRI. Scale bar indicates 200 μm. (B) Quantification of RGC-density (cells/mm²; mean±SD; n = 6) revealed a marked protection of RGC after ischemia in the ALF-treated eyes in comparison to iALF or PBS. *** = p<0.001.

Fig 2. Effect of ALF-186 on mitogen-activated protein (MAP) kinases ERK1/2 and p38.

(A) Representative western blot image showing the suppression of phosphorylated ERK1/2 compared to total ERK1/2 after IRI+ALF-186 treatment. The contralateral eye to each treated eye was indicated with (-) to demonstrate, that no MAP-kinase alterations were detectable in and between the right eyes. (B) Densitometric analysis of n = 6 western blots for phosphorylated ERK1/2 after IRI+treatment given in box plots. ALF-186 not affected the IRI-induced upregulation of ERK1/2, which was equally present after PBS, ALF or iALF-treatment, p>0.97. (C) Representative western blot image showing the increase of phosphorylated p38 compared to total p38 after intravitreal ALF-186 treatment. (D) Densitometric analysis of n = 7 western blots for phosphorylated p38 after IRI+treatment given in box plots. IRI+PBS vs. IRI+ALF-186, p = 0.267; IRI+ALF-186 vs. IRI+iALF-186, p = 0.032.

Fig 3. Intravitreal ALF-186 decreased the IRI-induced capsase-3 and NF-κB mRNA expression and affected the heat-shock response differently.

(A) Fold induction of caspase-3 mRNA expression after PBS, ALF-186, or iALF injection after IRI compared to GAPDH in relation to the corresponding non-ischemic contralateral retina analyzed by RT-PCR (n = 6; mean±SD; *** = p<0.001; *** = p<0.001). ALF decreased the IRI-induced caspase-3 expression significantly. (B) Fold induction of NF-κB mRNA expression after PBS, ALF-186, or iALF injection+IRI (n = 6; mean±SD; *** = p<0.001; ** = p<0.01). Intravitreal CO suppressed NF-κB mRNA expression after IRI. (C+D) Fold induction of HSP-70 (C) and HSP-90 (D) mRNA expression 24 h after IRI (n = 6; mean±SD; * = p<0.05). While HSP-70 expression was decreased, HSP-90 was induced by ALF.

Fig 4. Effect of ALF-186 on AIF-1, TNF-α, and Gap-43 mRNA expression.

(A) Fold induction of AIF-1 mRNA expression after IRI (n = 6; data are mean±SD; ** = p<0.05; * = p<0.05). (B) Fold induction of TNF-α mRNA expression after ALF-186 (n = 6; data are mean±SD; *** = p<0.001). (C) Fold induction of Gap-43 mRNA expression after ALF-186 (n = 6; data are mean±SD; both *** = p<0.001).

Fig 5. Immunohistochemical analysis of cells migrating out of ischemic retinal explants.

Retinal explants were transferred onto laminine-1 coated cultures for six days to allow RGC regeneration. Next, explants were analyzed by immunohistochemistry. Different cell types migrated out of the retinal explants during the time in culture. (A+E) Two types of DAPI-positive nuclei were apparent: smaller (circle) and bigger (asterisk). (B+D) The smaller nuclei belonged to iba-1 positive microglia cells (circle). (F+H) The bigger nuclei belonged to GFAP-positive Müller cells and astrocytes (asterisk). (C+G) Both glial cell types did not compromise the growth behavior of the beta-III tubulin positive RGC axons. Scale bar upper row 300 μm, lower row 600 μm.

Fig 6. ALF-186 augmented the IRI-induced regenerative response of RGC-axons in vitro.

(A+B) Box-Whisker-Plots showing the amount of regenerated RGC axons per explant for all treatment groups (n = 7 retinae, averaged value of 5–8 adhered explants per retina) explanted two (A) or seven (B) days after IRI. The growth of RGC axons was more pronounced in the ischemic eyes compared to contralateral eyes and stronger after seven than two days post IRI. Furthermore, axonal resprouting was tendentiously strongest after IRI and ALF-injection, without reaching statistical significance compared to IRI+iALF. (C) Representative pictures of regenerated RGC axons of all groups seven days after IRI. Quantification was performed using fluorescence microscopy (Zeiss ApoTome) to count the beta-III tubulin positive axons (red) after six days in culture which passed a defined distance of 200 μm to the explant margin. Cell nuclei were stained with DAPI (blue). GFAP-positive processes (green) were discriminated from RGC axons by immunolabeling. The RGC axons and GFAP-processes used each other as a guidance system, showing high affinity (asterisks).

Fig 7. ALF-186 induced the extent of GFAP-positive glial cell migration out of the retinal explants.

(A) The amount of GFAP-positive migrating glial cells was highest in the IRI+ALF treated group, followed by the IRI+iALF treated group at day six in vitro (p = 0.06). A slight GFAP-reactivity could also be found in the contralateral retinae after ALF-injection into the left eye (contralateral^ALF) compared to contralateral^iALF (p = 0.15). (B-E) Representative explants in lower and higher magnification of all treatment groups on day seven post IRI. The massive migration of GFAP-positive cells was obvious in the IRI+ALF group (B) and descended from IRI+iALF (C) to contralateral^ALF (D) to contralateral^iALF (E). The asterisk beside the explant-mosaic marked the origin of the pictures in the lower row, showing this location in higher magnification. Two types of GFAP-positive cells were distinguishable beside the explant margin: 1) longitudinal-shaped cells remaining firmly anchored to the explant, frequently without a visible cell nucleus but a dense filamentous appearance; 2) star-shaped cells with an expansive cytoplasm and prominent centralized nucleus, unconnected and farther from the explant with several processes. Scale bars in both rows 600 μm. Upper row: original tile 581.25 x 581.25, identifiable in the mosaic-reconstruction of the explants; explant-diameter trepanned was 1.5 mm, the circumference of the explant was marked (white dashed line).

Fig 8. ALF-and IRI-induced beta-III tubulin immunoreactivity in GFAP-positive cells in culture.

(A) Box-Whisker-Plots showing the amount of GFAP and beta-III tubulin coexpressing macroglial cells that migrated out of the retinal explants. The median amount of GFAP+ and beta-III tubulin+ cells per explant was significantly higher in both IRI groups and can be particularly found in the IRI+ALF group. (B-D) Retinal explant after IRI+ALF. GFAP (green, C) was expressed in all the migrating Müller cells and astrocytes. Some of these cells and the RGC-axons were beta-III tubulin immunopositive (red, D). (E-G) Immunoreactivity was located in different cytoskeletal areas. While GFAP-positive structures showed filamentous patterns and were concentrated perinuclearly and at the margin of cells (F), the beta-III tubulin was associated with microtubules and predominantly found in the perinuclear region (G). Scale bars B-D 600μm.