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Activation of autophagy in a rat model of retinal ischemia following high intraocular pressure.

Piras A, Gianetto D, Conte D, Bosone A, Vercelli A - PLoS ONE (2011)

Bottom Line: Inhibition of autophagy by 3-methyladenine partially prevented death of neurons and reduces apoptotic markers, 24 h post-lesion.The number of neurons in the GCL decreased significantly following I/R (I/R 12.21±1.13 vs controls 19.23±1.12 cells/500 µm); this decrease was partially prevented by 3-methyladenine (17.08±1.42 cells/500 µm), which potently inhibits maturation of autophagosomes.Therefore, targeting autophagy could represent a novel and promising treatment for glaucoma and retinal ischemia.

View Article: PubMed Central - PubMed

Affiliation: Neuroscience Institute of the Cavalieri Ottolenghi Foundation, Orbassano, Torino, Italy. antonio.piras@unito.it

ABSTRACT
Acute primary open angle glaucoma is an optic neuropathy characterized by the elevation of intraocular pressure, which causes retinal ischemia and neuronal death. Rat ischemia/reperfusion enhances endocytosis of both horseradish peroxidase (HRP) or fluorescent dextran into ganglion cell layer (GCL) neurons 24 h after the insult. We investigated the activation of autophagy in GCL-neurons following ischemia/reperfusion, using acid phosphatase (AP) histochemistry and immunofluorescence against LC3 and LAMP1. Retinal I/R lead to the appearance of AP-positive granules and LAMP1-positive vesicles 12 and 24 h after the insult, and LC3 labelling at 24 h, and induced a consistent retinal neuron death. At 48 h the retina was negative for autophagic markers. In addition, Western Blot analysis revealed an increase of LC3 levels after damage: the increase in the conjugated, LC3-II isoform is suggestive of autophagic activity. Inhibition of autophagy by 3-methyladenine partially prevented death of neurons and reduces apoptotic markers, 24 h post-lesion. The number of neurons in the GCL decreased significantly following I/R (I/R 12.21±1.13 vs controls 19.23±1.12 cells/500 µm); this decrease was partially prevented by 3-methyladenine (17.08±1.42 cells/500 µm), which potently inhibits maturation of autophagosomes. Treatment also prevented the increase in glial fibrillary acid protein immunoreactivity elicited by I/R. Therefore, targeting autophagy could represent a novel and promising treatment for glaucoma and retinal ischemia.

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AP and endocytic activities in the retina.A–F: acid phosphatase (AP) activity is visualized with Gomori's (A, C, E) and Barka & Anderson (B and D) staining, counterstained with methyl green. 12 h after I/R, positive cells are located only in the ganglion cell layer (GCL), and are identified by their content of typical brown cytoplasmic granules (arrowheads in A) and by the red reaction (B, arrows) in the GCL and INL. 24 h after I/R, AP-positive cells are visible in both the GCL (arrows) and INL (arrowheads) (C–D). Only weak staining in the GCL is seen with the Gomori technique 48 h after I/R (E). No labeling is found in control retinas (F). Markedly positive cytoplasmic granules are visible in GCL-cells at higher magnification (G). H–I: 24h after I/R and intravitreal injection of HRP (H) or 4.4 kDa FITC-labelled dextran (I, arrows), corresponding granules are visible in neurons. GCL  =  ganglion cell layer; IPL  =  inner plexiform layer; INL  =  inner nuclear layer; OPL  =  outer plexiform layer; ONL  =  outer nuclear layer. Scale bars  =  100 µm and 10 µm (G and H).
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pone-0022514-g001: AP and endocytic activities in the retina.A–F: acid phosphatase (AP) activity is visualized with Gomori's (A, C, E) and Barka & Anderson (B and D) staining, counterstained with methyl green. 12 h after I/R, positive cells are located only in the ganglion cell layer (GCL), and are identified by their content of typical brown cytoplasmic granules (arrowheads in A) and by the red reaction (B, arrows) in the GCL and INL. 24 h after I/R, AP-positive cells are visible in both the GCL (arrows) and INL (arrowheads) (C–D). Only weak staining in the GCL is seen with the Gomori technique 48 h after I/R (E). No labeling is found in control retinas (F). Markedly positive cytoplasmic granules are visible in GCL-cells at higher magnification (G). H–I: 24h after I/R and intravitreal injection of HRP (H) or 4.4 kDa FITC-labelled dextran (I, arrows), corresponding granules are visible in neurons. GCL  =  ganglion cell layer; IPL  =  inner plexiform layer; INL  =  inner nuclear layer; OPL  =  outer plexiform layer; ONL  =  outer nuclear layer. Scale bars  =  100 µm and 10 µm (G and H).

Mentions: Overall, retinal morphology was conserved following I/R (Figure 1). In I/R retinas, AP activity was detected at 12 h following I/R (Figure 1 A–B), was maximal by 24 h (Figure 1 C–D) and declined at 48 h (Figure 1 E). Both methods used to visualize enzyme activity showed robust staining at 24 h post-insult, although staining was darker with the Barka and Anderson technique. Most intense staining was localized to GCL; sporadic positively stained cells were also visible in the inner nuclear layer (Figure 1 C–D, arrowheads). At high magnification, Gomori staining revealed clusters of small, intensely stained granules, preferentially located in the periphery of the cytoplasm (Figure 1 G), as is characteristic of lysosomal systems. Almost all GCL-neurons were stained, but to different degrees: the larger the cell the more intensely reactive it was. The use of NaF in the incubation medium resulted in a complete inhibition of enzymatic activity. In control sections non-specific reactivity for AP was detectable in retinal neurons (Figure 1 F).


Activation of autophagy in a rat model of retinal ischemia following high intraocular pressure.

Piras A, Gianetto D, Conte D, Bosone A, Vercelli A - PLoS ONE (2011)

AP and endocytic activities in the retina.A–F: acid phosphatase (AP) activity is visualized with Gomori's (A, C, E) and Barka & Anderson (B and D) staining, counterstained with methyl green. 12 h after I/R, positive cells are located only in the ganglion cell layer (GCL), and are identified by their content of typical brown cytoplasmic granules (arrowheads in A) and by the red reaction (B, arrows) in the GCL and INL. 24 h after I/R, AP-positive cells are visible in both the GCL (arrows) and INL (arrowheads) (C–D). Only weak staining in the GCL is seen with the Gomori technique 48 h after I/R (E). No labeling is found in control retinas (F). Markedly positive cytoplasmic granules are visible in GCL-cells at higher magnification (G). H–I: 24h after I/R and intravitreal injection of HRP (H) or 4.4 kDa FITC-labelled dextran (I, arrows), corresponding granules are visible in neurons. GCL  =  ganglion cell layer; IPL  =  inner plexiform layer; INL  =  inner nuclear layer; OPL  =  outer plexiform layer; ONL  =  outer nuclear layer. Scale bars  =  100 µm and 10 µm (G and H).
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Related In: Results  -  Collection

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pone-0022514-g001: AP and endocytic activities in the retina.A–F: acid phosphatase (AP) activity is visualized with Gomori's (A, C, E) and Barka & Anderson (B and D) staining, counterstained with methyl green. 12 h after I/R, positive cells are located only in the ganglion cell layer (GCL), and are identified by their content of typical brown cytoplasmic granules (arrowheads in A) and by the red reaction (B, arrows) in the GCL and INL. 24 h after I/R, AP-positive cells are visible in both the GCL (arrows) and INL (arrowheads) (C–D). Only weak staining in the GCL is seen with the Gomori technique 48 h after I/R (E). No labeling is found in control retinas (F). Markedly positive cytoplasmic granules are visible in GCL-cells at higher magnification (G). H–I: 24h after I/R and intravitreal injection of HRP (H) or 4.4 kDa FITC-labelled dextran (I, arrows), corresponding granules are visible in neurons. GCL  =  ganglion cell layer; IPL  =  inner plexiform layer; INL  =  inner nuclear layer; OPL  =  outer plexiform layer; ONL  =  outer nuclear layer. Scale bars  =  100 µm and 10 µm (G and H).
Mentions: Overall, retinal morphology was conserved following I/R (Figure 1). In I/R retinas, AP activity was detected at 12 h following I/R (Figure 1 A–B), was maximal by 24 h (Figure 1 C–D) and declined at 48 h (Figure 1 E). Both methods used to visualize enzyme activity showed robust staining at 24 h post-insult, although staining was darker with the Barka and Anderson technique. Most intense staining was localized to GCL; sporadic positively stained cells were also visible in the inner nuclear layer (Figure 1 C–D, arrowheads). At high magnification, Gomori staining revealed clusters of small, intensely stained granules, preferentially located in the periphery of the cytoplasm (Figure 1 G), as is characteristic of lysosomal systems. Almost all GCL-neurons were stained, but to different degrees: the larger the cell the more intensely reactive it was. The use of NaF in the incubation medium resulted in a complete inhibition of enzymatic activity. In control sections non-specific reactivity for AP was detectable in retinal neurons (Figure 1 F).

Bottom Line: Inhibition of autophagy by 3-methyladenine partially prevented death of neurons and reduces apoptotic markers, 24 h post-lesion.The number of neurons in the GCL decreased significantly following I/R (I/R 12.21±1.13 vs controls 19.23±1.12 cells/500 µm); this decrease was partially prevented by 3-methyladenine (17.08±1.42 cells/500 µm), which potently inhibits maturation of autophagosomes.Therefore, targeting autophagy could represent a novel and promising treatment for glaucoma and retinal ischemia.

View Article: PubMed Central - PubMed

Affiliation: Neuroscience Institute of the Cavalieri Ottolenghi Foundation, Orbassano, Torino, Italy. antonio.piras@unito.it

ABSTRACT
Acute primary open angle glaucoma is an optic neuropathy characterized by the elevation of intraocular pressure, which causes retinal ischemia and neuronal death. Rat ischemia/reperfusion enhances endocytosis of both horseradish peroxidase (HRP) or fluorescent dextran into ganglion cell layer (GCL) neurons 24 h after the insult. We investigated the activation of autophagy in GCL-neurons following ischemia/reperfusion, using acid phosphatase (AP) histochemistry and immunofluorescence against LC3 and LAMP1. Retinal I/R lead to the appearance of AP-positive granules and LAMP1-positive vesicles 12 and 24 h after the insult, and LC3 labelling at 24 h, and induced a consistent retinal neuron death. At 48 h the retina was negative for autophagic markers. In addition, Western Blot analysis revealed an increase of LC3 levels after damage: the increase in the conjugated, LC3-II isoform is suggestive of autophagic activity. Inhibition of autophagy by 3-methyladenine partially prevented death of neurons and reduces apoptotic markers, 24 h post-lesion. The number of neurons in the GCL decreased significantly following I/R (I/R 12.21±1.13 vs controls 19.23±1.12 cells/500 µm); this decrease was partially prevented by 3-methyladenine (17.08±1.42 cells/500 µm), which potently inhibits maturation of autophagosomes. Treatment also prevented the increase in glial fibrillary acid protein immunoreactivity elicited by I/R. Therefore, targeting autophagy could represent a novel and promising treatment for glaucoma and retinal ischemia.

Show MeSH
Related in: MedlinePlus