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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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Effects of 3-MA treatment in I/R after 24 h.In 3-MA-treated I/R retinas treated, LC3-positivity is markedly reduced (A), whereas lysosomal activity (LAMP1) is unchanged (B) compared to I/R retinas. (C) Cleaved caspase-3-positive cells are absent in the untreated retinas (upper panel), whereas a strong increase in cleaved caspase 3-positivity in the GCL is seen at 24 h (middle panel), significantly prevented by 3-MA treatment (bottom panel). (D) TUNEL-positive neurons are found occasionally in the control retina, whereas they increase dramatically after I/R in the GCL (arrowheads) and in INL (arrow). This increase in TUNEL-positive cells is prevented by 3-MA treatment (bottom panel). (E) I/R increase GFAP immunoreactivity (red) in the retina: in the untreated retina, only the end feet of the Müller cells (arrowheads) are GFAP-positive. GFAP expression strongly increase 24 h after I/R, and is prevented by 3-MA administration. In the I/R and I/R + 3-MA retinas, GFAP positivity is detectable in the end feet (arrowheads) and radial processes (arrows) of Müller cells. Following 3-MA treatment, the immunoreactivity is decreased versus I/R retina especially in the end feet of Müller cells. ILM: inner limiting membrane. Abbreviations as in Figure 1. (*): vitreous. Scale bar: 50 µm (100 µm in D).
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pone-0022514-g005: Effects of 3-MA treatment in I/R after 24 h.In 3-MA-treated I/R retinas treated, LC3-positivity is markedly reduced (A), whereas lysosomal activity (LAMP1) is unchanged (B) compared to I/R retinas. (C) Cleaved caspase-3-positive cells are absent in the untreated retinas (upper panel), whereas a strong increase in cleaved caspase 3-positivity in the GCL is seen at 24 h (middle panel), significantly prevented by 3-MA treatment (bottom panel). (D) TUNEL-positive neurons are found occasionally in the control retina, whereas they increase dramatically after I/R in the GCL (arrowheads) and in INL (arrow). This increase in TUNEL-positive cells is prevented by 3-MA treatment (bottom panel). (E) I/R increase GFAP immunoreactivity (red) in the retina: in the untreated retina, only the end feet of the Müller cells (arrowheads) are GFAP-positive. GFAP expression strongly increase 24 h after I/R, and is prevented by 3-MA administration. In the I/R and I/R + 3-MA retinas, GFAP positivity is detectable in the end feet (arrowheads) and radial processes (arrows) of Müller cells. Following 3-MA treatment, the immunoreactivity is decreased versus I/R retina especially in the end feet of Müller cells. ILM: inner limiting membrane. Abbreviations as in Figure 1. (*): vitreous. Scale bar: 50 µm (100 µm in D).

Mentions: To evaluate whether 3-Methyladenine (3-MA) inhibited autophagic and lysosomal activity, we studied the LC3 and LAMP1 expression in GCL-neurons 24 h after increase in IOP and treatment with 3-MA: rare LC3-positive vacuoles were observed (Figure 5 A), while spread LAMP1 vesicles were diffused in the cytoplasm (Figure 5 B). Therefore we demonstrated that 3-MA inhibits autophagy but not lysosomal activity. Cleaved caspase-3 (Figure 5 C) and TUNEL positive (D) in the retina following I/R (middle panel) were reduced following 3-MA treatment (lower panel) compared to untreated (upper panel).


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)

Effects of 3-MA treatment in I/R after 24 h.In 3-MA-treated I/R retinas treated, LC3-positivity is markedly reduced (A), whereas lysosomal activity (LAMP1) is unchanged (B) compared to I/R retinas. (C) Cleaved caspase-3-positive cells are absent in the untreated retinas (upper panel), whereas a strong increase in cleaved caspase 3-positivity in the GCL is seen at 24 h (middle panel), significantly prevented by 3-MA treatment (bottom panel). (D) TUNEL-positive neurons are found occasionally in the control retina, whereas they increase dramatically after I/R in the GCL (arrowheads) and in INL (arrow). This increase in TUNEL-positive cells is prevented by 3-MA treatment (bottom panel). (E) I/R increase GFAP immunoreactivity (red) in the retina: in the untreated retina, only the end feet of the Müller cells (arrowheads) are GFAP-positive. GFAP expression strongly increase 24 h after I/R, and is prevented by 3-MA administration. In the I/R and I/R + 3-MA retinas, GFAP positivity is detectable in the end feet (arrowheads) and radial processes (arrows) of Müller cells. Following 3-MA treatment, the immunoreactivity is decreased versus I/R retina especially in the end feet of Müller cells. ILM: inner limiting membrane. Abbreviations as in Figure 1. (*): vitreous. Scale bar: 50 µm (100 µm in D).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3142183&req=5

pone-0022514-g005: Effects of 3-MA treatment in I/R after 24 h.In 3-MA-treated I/R retinas treated, LC3-positivity is markedly reduced (A), whereas lysosomal activity (LAMP1) is unchanged (B) compared to I/R retinas. (C) Cleaved caspase-3-positive cells are absent in the untreated retinas (upper panel), whereas a strong increase in cleaved caspase 3-positivity in the GCL is seen at 24 h (middle panel), significantly prevented by 3-MA treatment (bottom panel). (D) TUNEL-positive neurons are found occasionally in the control retina, whereas they increase dramatically after I/R in the GCL (arrowheads) and in INL (arrow). This increase in TUNEL-positive cells is prevented by 3-MA treatment (bottom panel). (E) I/R increase GFAP immunoreactivity (red) in the retina: in the untreated retina, only the end feet of the Müller cells (arrowheads) are GFAP-positive. GFAP expression strongly increase 24 h after I/R, and is prevented by 3-MA administration. In the I/R and I/R + 3-MA retinas, GFAP positivity is detectable in the end feet (arrowheads) and radial processes (arrows) of Müller cells. Following 3-MA treatment, the immunoreactivity is decreased versus I/R retina especially in the end feet of Müller cells. ILM: inner limiting membrane. Abbreviations as in Figure 1. (*): vitreous. Scale bar: 50 µm (100 µm in D).
Mentions: To evaluate whether 3-Methyladenine (3-MA) inhibited autophagic and lysosomal activity, we studied the LC3 and LAMP1 expression in GCL-neurons 24 h after increase in IOP and treatment with 3-MA: rare LC3-positive vacuoles were observed (Figure 5 A), while spread LAMP1 vesicles were diffused in the cytoplasm (Figure 5 B). Therefore we demonstrated that 3-MA inhibits autophagy but not lysosomal activity. Cleaved caspase-3 (Figure 5 C) and TUNEL positive (D) in the retina following I/R (middle panel) were reduced following 3-MA treatment (lower panel) compared to untreated (upper panel).

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