Limits...
Cyclic GMP in the pig vitreous and retina after experimental retinal detachment.

Diederen RM, La Heij EC, Lemmens MA, Kijlstra A, de Vente J, Hendrikse F - Mol. Vis. (2008)

Bottom Line: To further investigate this phenomenon, we developed an experimental retinal detachment model in pigs.Experimental unilateral retinal detachments were induced in pig eyes by subretinal injection of 0.25% sodium hyaluronate.Experimental retinal detachment in the pig eye leads to a decrease of cGMP levels in vitreous similar to that observed in clinical studies.

View Article: PubMed Central - PubMed

Affiliation: Eye Research Institute Maastricht, Department of Ophthalmology, University Hospital Maastricht, The Netherlands.

ABSTRACT

Purpose: Earlier studies have revealed a decreased level of cGMP in vitreous fluid obtained from patients with a retinal detachment. To further investigate this phenomenon, we developed an experimental retinal detachment model in pigs.

Methods: Experimental unilateral retinal detachments were induced in pig eyes by subretinal injection of 0.25% sodium hyaluronate. Fourteen days later the vitreous and retinas were analyzed for cGMP expression. Following enucleation, the retinas were incubated in the presence of a nonselective phosphodiesterase inhibitor (IBMX), and the particulate guanylyl cyclase stimulator atrial natriuretic peptide (ANP) or the soluble guanylyl cyclase stimulator sodium nitroprusside (SNP). cGMP was visualized in retinal wholemounts by immunochemistry combined with a computer based stereology system. cGMP levels in vitreous were determined by ELISA.

Results: The mean vitreous cGMP level in pig eyes with a retinal detachment (1.45 pmol/ml) was significantly lower compared to the mean level of cGMP in healthy pig eyes (4.61 pmol/ml; p=0.028 was considered significant). In the inner retina, ANP as well as SNP induced cGMP immunoreactivity in both detached and healthy retinas. After incubation with ANP, cGMP could also be detected in the outer nuclear layer of the detached retina, whereas this was not the case in the normal retina.

Conclusions: Experimental retinal detachment in the pig eye leads to a decrease of cGMP levels in vitreous similar to that observed in clinical studies. This model may be helpful to analyze the mechanisms involved in cGMP dynamics following retinal detachment.

Show MeSH

Related in: MedlinePlus

In vitro expression of cyclic guanosine monophosphate (cGMP) in the photoreceptor layer of a normal or detached pig retina. Small pieces of retina were incubated with IBMX (a non-specific PDE inhibitor) and the particulate guanylyl cyclase (pGC) stimulator atrial natriuretic peptide (ANP). The expression of cGMP (cGMP: green color) in the retina was then analyzed by immunohistochemistry and nuclei were counterstained with Hoechst 33342 (red). Under these conditions no cGMP signal was visible in the photoreceptor layer of the healthy retina (Panel A). The detached retina on the other hand displayed strong cGMP expression in the photoreceptor layer (Panels B and C). Panel B is a double staining exposure for the nuclei and cGMP, showing the presence of cGMP in the nuclei of the cells (yellow) and in the cytoplasma (green). In Panel C we only show the green signal, showing strong cGMP immunoreactivity in the same piece of retina. Scale bar represents 25 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2254957&req=5

f2: In vitro expression of cyclic guanosine monophosphate (cGMP) in the photoreceptor layer of a normal or detached pig retina. Small pieces of retina were incubated with IBMX (a non-specific PDE inhibitor) and the particulate guanylyl cyclase (pGC) stimulator atrial natriuretic peptide (ANP). The expression of cGMP (cGMP: green color) in the retina was then analyzed by immunohistochemistry and nuclei were counterstained with Hoechst 33342 (red). Under these conditions no cGMP signal was visible in the photoreceptor layer of the healthy retina (Panel A). The detached retina on the other hand displayed strong cGMP expression in the photoreceptor layer (Panels B and C). Panel B is a double staining exposure for the nuclei and cGMP, showing the presence of cGMP in the nuclei of the cells (yellow) and in the cytoplasma (green). In Panel C we only show the green signal, showing strong cGMP immunoreactivity in the same piece of retina. Scale bar represents 25 μm.

Mentions: The expression of cGMP in the retina was analyzed by immunohistochemistry. No cGMP expression could be observed in any of the retinal cells (either detached or healthy) if phosphodieasterase activity had not been blocked with IBMX. This indicates that under normal conditions, cGMP has an extremely short half life. In the absence of cyclase stimulation but in the presence of PDE blockers, we observed cGMP immunoreactivity in the inner nuclear layer of the detached as well as the healthy retinas. Addition of 0.1 μM ANP as a stimulator of pGC, combined with PDE inhibition, resulted in the appearance of a strong cGMP signal in both the ganglion cells and the inner nuclear layer of the healthy retina. No cGMP signal was visible in the outer nuclear layer of the healthy retina (Figure 2A). The detached retina on the other hand displayed strong cGMP expression not only in the ganglion cells and the inner nuclear layer but also in the outer nuclear layer (Figure 2B,C). Figure 2B shows both the nuclei of the cells (Hoechst staining; red signal) and cGMP (green signal) staining of the outer nuclear layer of a detached retina. Figure 2C only presents the green signal, showing strong cGMP immunoreactivity in the same piece of retina. Following stimulation with ANP in the presence of IBMX, both the control eye and eye with the detached retina showed cGMP-immunolabeling in the inner and outer plexiform layer. Figure 3 presents the labeling of the outer plexiform layer of a detached retina with Hoechst (Figure 3A) and cGMP (Figure 3B) separately and together (Figure 3C) in the same piece of retina. Incubating the retinas in the presence of 100 μM SNP, as a stimulator of sGC in combination with PDE inhibition (with IBMX) resulted in cGMP-immunoreactivity in the ganglion cells, the inner nuclear cell layer (Figure 4 C,D) and the nerve fiber layer of the detached- and healthy retinas (Figure 4A,B). Stimulation with SNP (+ IBMX) did not result in the appearance of cGMP immunoreactivity in the photoreceptor cells of the detached or healthy retina. Incubation of retinas with ANP or SNP (+ IBMX) both resulted in cGMP-immunoreactivity in the inner nuclear layer, although not in all cell types (Figure 4C,D). The aforedescribed experiments thus only showed a difference in cGMP expression between the retinas with and without experimental detachment following in vitro stimulation of pGC. In vitro, we demonstrated that ANP induced synthesis of cGMP in the outer nuclear layer of the detached retina, whereas this was not the case in the attached retina.


Cyclic GMP in the pig vitreous and retina after experimental retinal detachment.

Diederen RM, La Heij EC, Lemmens MA, Kijlstra A, de Vente J, Hendrikse F - Mol. Vis. (2008)

In vitro expression of cyclic guanosine monophosphate (cGMP) in the photoreceptor layer of a normal or detached pig retina. Small pieces of retina were incubated with IBMX (a non-specific PDE inhibitor) and the particulate guanylyl cyclase (pGC) stimulator atrial natriuretic peptide (ANP). The expression of cGMP (cGMP: green color) in the retina was then analyzed by immunohistochemistry and nuclei were counterstained with Hoechst 33342 (red). Under these conditions no cGMP signal was visible in the photoreceptor layer of the healthy retina (Panel A). The detached retina on the other hand displayed strong cGMP expression in the photoreceptor layer (Panels B and C). Panel B is a double staining exposure for the nuclei and cGMP, showing the presence of cGMP in the nuclei of the cells (yellow) and in the cytoplasma (green). In Panel C we only show the green signal, showing strong cGMP immunoreactivity in the same piece of retina. Scale bar represents 25 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2254957&req=5

f2: In vitro expression of cyclic guanosine monophosphate (cGMP) in the photoreceptor layer of a normal or detached pig retina. Small pieces of retina were incubated with IBMX (a non-specific PDE inhibitor) and the particulate guanylyl cyclase (pGC) stimulator atrial natriuretic peptide (ANP). The expression of cGMP (cGMP: green color) in the retina was then analyzed by immunohistochemistry and nuclei were counterstained with Hoechst 33342 (red). Under these conditions no cGMP signal was visible in the photoreceptor layer of the healthy retina (Panel A). The detached retina on the other hand displayed strong cGMP expression in the photoreceptor layer (Panels B and C). Panel B is a double staining exposure for the nuclei and cGMP, showing the presence of cGMP in the nuclei of the cells (yellow) and in the cytoplasma (green). In Panel C we only show the green signal, showing strong cGMP immunoreactivity in the same piece of retina. Scale bar represents 25 μm.
Mentions: The expression of cGMP in the retina was analyzed by immunohistochemistry. No cGMP expression could be observed in any of the retinal cells (either detached or healthy) if phosphodieasterase activity had not been blocked with IBMX. This indicates that under normal conditions, cGMP has an extremely short half life. In the absence of cyclase stimulation but in the presence of PDE blockers, we observed cGMP immunoreactivity in the inner nuclear layer of the detached as well as the healthy retinas. Addition of 0.1 μM ANP as a stimulator of pGC, combined with PDE inhibition, resulted in the appearance of a strong cGMP signal in both the ganglion cells and the inner nuclear layer of the healthy retina. No cGMP signal was visible in the outer nuclear layer of the healthy retina (Figure 2A). The detached retina on the other hand displayed strong cGMP expression not only in the ganglion cells and the inner nuclear layer but also in the outer nuclear layer (Figure 2B,C). Figure 2B shows both the nuclei of the cells (Hoechst staining; red signal) and cGMP (green signal) staining of the outer nuclear layer of a detached retina. Figure 2C only presents the green signal, showing strong cGMP immunoreactivity in the same piece of retina. Following stimulation with ANP in the presence of IBMX, both the control eye and eye with the detached retina showed cGMP-immunolabeling in the inner and outer plexiform layer. Figure 3 presents the labeling of the outer plexiform layer of a detached retina with Hoechst (Figure 3A) and cGMP (Figure 3B) separately and together (Figure 3C) in the same piece of retina. Incubating the retinas in the presence of 100 μM SNP, as a stimulator of sGC in combination with PDE inhibition (with IBMX) resulted in cGMP-immunoreactivity in the ganglion cells, the inner nuclear cell layer (Figure 4 C,D) and the nerve fiber layer of the detached- and healthy retinas (Figure 4A,B). Stimulation with SNP (+ IBMX) did not result in the appearance of cGMP immunoreactivity in the photoreceptor cells of the detached or healthy retina. Incubation of retinas with ANP or SNP (+ IBMX) both resulted in cGMP-immunoreactivity in the inner nuclear layer, although not in all cell types (Figure 4C,D). The aforedescribed experiments thus only showed a difference in cGMP expression between the retinas with and without experimental detachment following in vitro stimulation of pGC. In vitro, we demonstrated that ANP induced synthesis of cGMP in the outer nuclear layer of the detached retina, whereas this was not the case in the attached retina.

Bottom Line: To further investigate this phenomenon, we developed an experimental retinal detachment model in pigs.Experimental unilateral retinal detachments were induced in pig eyes by subretinal injection of 0.25% sodium hyaluronate.Experimental retinal detachment in the pig eye leads to a decrease of cGMP levels in vitreous similar to that observed in clinical studies.

View Article: PubMed Central - PubMed

Affiliation: Eye Research Institute Maastricht, Department of Ophthalmology, University Hospital Maastricht, The Netherlands.

ABSTRACT

Purpose: Earlier studies have revealed a decreased level of cGMP in vitreous fluid obtained from patients with a retinal detachment. To further investigate this phenomenon, we developed an experimental retinal detachment model in pigs.

Methods: Experimental unilateral retinal detachments were induced in pig eyes by subretinal injection of 0.25% sodium hyaluronate. Fourteen days later the vitreous and retinas were analyzed for cGMP expression. Following enucleation, the retinas were incubated in the presence of a nonselective phosphodiesterase inhibitor (IBMX), and the particulate guanylyl cyclase stimulator atrial natriuretic peptide (ANP) or the soluble guanylyl cyclase stimulator sodium nitroprusside (SNP). cGMP was visualized in retinal wholemounts by immunochemistry combined with a computer based stereology system. cGMP levels in vitreous were determined by ELISA.

Results: The mean vitreous cGMP level in pig eyes with a retinal detachment (1.45 pmol/ml) was significantly lower compared to the mean level of cGMP in healthy pig eyes (4.61 pmol/ml; p=0.028 was considered significant). In the inner retina, ANP as well as SNP induced cGMP immunoreactivity in both detached and healthy retinas. After incubation with ANP, cGMP could also be detected in the outer nuclear layer of the detached retina, whereas this was not the case in the normal retina.

Conclusions: Experimental retinal detachment in the pig eye leads to a decrease of cGMP levels in vitreous similar to that observed in clinical studies. This model may be helpful to analyze the mechanisms involved in cGMP dynamics following retinal detachment.

Show MeSH
Related in: MedlinePlus