Loss of vision in glaucoma results from the selective death of retinal ganglion cells (RGCs). Tum... more Loss of vision in glaucoma results from the selective death of retinal ganglion cells (RGCs). Tumor necrosis factor α (TNFα) signaling has been linked to RGC damage, however, the mechanism by which TNFα promotes neuronal death remains poorly defined. Using anin vivorat glaucoma model, we show that TNFα is upregulated by Müller cells and microglia/macrophages soon after induction of ocular hypertension. Administration of XPro1595, a selective inhibitor of soluble TNFα, effectively protects RGC soma and axons. Using cobalt permeability assays, we further demonstrate that endogenous soluble TNFα triggers the upregulation of Ca2+-permeable AMPA receptor (CP-AMPAR) expression in RGCs of glaucomatous eyes. CP-AMPAR activation is not caused by defects in GluA2 subunit mRNA editing, but rather reflects selective downregulation of GluA2 in neurons exposed to elevated eye pressure. Intraocular administration of selective CP-AMPAR blockers promotes robust RGC survival supporting a critical rol...
Macrophages are key regulators of inflammation and repair, but their heterogeneity and multiple r... more Macrophages are key regulators of inflammation and repair, but their heterogeneity and multiple roles in the liver are not fully understood. We aimed herein to map the intrahepatic macrophage populations and their function(s) during acute liver injury. We used flow cytometry, gene expression analysis, multiplex-immunofluorescence, 3D-reconstruction, and spatial image analysis to characterize the intrahepatic immune landscape in mice post-CCl4-induced acute liver injury during three distinct phases: necroinflammation, and early and late repair. We observed hepatocellular necrosis and a reduction in liver resident lymphocytes during necroinflammation accompanied by the infiltration of circulating myeloid cells and upregulation of inflammatory cytokines. These parameters returned to baseline levels during the repair phase while pro-repair chemokines were upregulated. We identified resident CLEC4F+ Kupffer cells (KCs) and infiltrating IBA1+CLEC4F- monocyte-derived macrophages (MoMFs) as...
Animal models of the Boston keratoprosthesis type 1 (KPro) are needed to study glaucoma damage af... more Animal models of the Boston keratoprosthesis type 1 (KPro) are needed to study glaucoma damage after KPro implantation to control confounding comorbidities common in human KPro recipients. The purpose of this study was to determine the feasibility of establishing a reproducible mouse model of glaucoma after KPro surgery, specifically that of a miniaturized mouse model of KPro (mKPro). In the present study, a total of 20 corneas of donor C57BL/6 mice (n = 10) were implanted in one eye of each recipient BALB/C mice (n = 20), assembled as part of the mKPro, either with or without intraoperative lensectomy. Main feasibility outcomes consisted in incidence rates of loss of tone, capsule nicking, and lens extrusion, as well as acquisition of posterior segment OCT images. With lensectomy (n = 10), loss of ocular tone and retinal detachment occurred in 100% of mice. Without lensectomy (n = 10), capsule nicking and opening, as well as lens extrusion, occurred in 80% of mice. Causes of these complications included the large proportion of intraocular volume occupied by the lens, the shallow anterior chamber, and thus the lack of available intraocular volume to implant the KPro if the lens remains present. Successful mouse KPro surgery may require a great deal of practice to be useful as a reproducible model. Animal models ought to be pursued further by research teams in future studies.
Additional file 2: Supplementary Figure 1. (A) The number of YFP-positive RGC does not change at ... more Additional file 2: Supplementary Figure 1. (A) The number of YFP-positive RGC does not change at 2 weeks of ocular hypertension (OHT) relative to non-injured controls (Student's t-test, n.s.: not significant, N = 5 mice/group). (B) Real-time qPCR analysis confirms that retinal YFP gene expression does not change within 2 weeks of OHT damage (Student's t-test, n.s.: not significant, N = 5 mice/group). (C-E) RGC co-expressing YFP and SMI-32 are selected for dendritic arbor imaging and 3D reconstruction. Supplementary Figure 2. Analysis of whole-mounted retinas immunolabeled with an antibody recognizing pAMPKThr172 revealed a substantial increase of AMPK activity in RGC axons, visualized with SMI-32, at two weeks after glaucoma induction. Scale bars = 25 μm. Supplementary Figure 3. (A, B) Retinal imaging showed no fluorescein extravasation in eyes injected with compound C or vehicle. (C) In contrast, marked fluorescein extravasation was found in ischemic retinas subjected to ce...
Proceedings of the National Academy of Sciences, 2022
Significance The current lack of understanding of the mechanisms leading to neurovascular deficit... more Significance The current lack of understanding of the mechanisms leading to neurovascular deficits in glaucoma is a major knowledge gap in the field. Retinal pericytes regulate microcirculatory blood flow and coordinate neurovascular coupling through interpericyte tunneling nanotubes (IP-TNTs). We demonstrate that pericytes constrict capillaries in a calcium-dependent manner during glaucomatous stress, decreasing blood supply and compromising neuronal function. Moreover, ocular hypertension damages IP-TNTs and impairs light-evoked neurovascular responses. The reestablishment of calcium homeostasis in pericytes restores vascular and neuronal function, and prevents retinal ganglion cell death in glaucomatous eyes. This study provides important insights into the therapeutic potential of pericytes to counter vascular dysregulation in glaucoma.
Figure S2. Connexin-43 blocking peptide had no effect on intracellular calcium in control non-isc... more Figure S2. Connexin-43 blocking peptide had no effect on intracellular calcium in control non-ischemic retinas. (A, B) Fluo-4 indicator reported no effect on intracellular calcium in control non-ischemic retinas after intravitreal injection of connexin-43 blocking peptide (peptide 5). (B) Vessels were labeled with lectin (red) and nuclei with DAPI (blue). Scale bar in A-B = 5 μm. (TIF 7894 kb)
Background The maintenance of complex dendritic arbors and synaptic transmission are processes th... more Background The maintenance of complex dendritic arbors and synaptic transmission are processes that require a substantial amount of energy. Bioenergetic decline is a prominent feature of chronic neurodegenerative diseases, yet the signaling mechanisms that link energy stress with neuronal dysfunction are poorly understood. Recent work has implicated energy deficits in glaucoma, and retinal ganglion cell (RGC) dendritic pathology and synapse disassembly are key features of ocular hypertension damage. Results We show that adenosine monophosphate-activated protein kinase (AMPK), a conserved energy biosensor, is strongly activated in RGC from mice with ocular hypertension and patients with primary open angle glaucoma. Our data demonstrate that AMPK triggers RGC dendrite retraction and synapse elimination. We show that the harmful effect of AMPK is exerted through inhibition of the mammalian target of rapamycin complex 1 (mTORC1). Attenuation of AMPK activity restores mTORC1 function and...
Loss of vision in glaucoma results from the selective death of retinal ganglion cells (RGCs). Tum... more Loss of vision in glaucoma results from the selective death of retinal ganglion cells (RGCs). Tumor necrosis factor α (TNFα) signaling has been linked to RGC damage, however, the mechanism by which TNFα promotes neuronal death remains poorly defined. Using anin vivorat glaucoma model, we show that TNFα is upregulated by Müller cells and microglia/macrophages soon after induction of ocular hypertension. Administration of XPro1595, a selective inhibitor of soluble TNFα, effectively protects RGC soma and axons. Using cobalt permeability assays, we further demonstrate that endogenous soluble TNFα triggers the upregulation of Ca2+-permeable AMPA receptor (CP-AMPAR) expression in RGCs of glaucomatous eyes. CP-AMPAR activation is not caused by defects in GluA2 subunit mRNA editing, but rather reflects selective downregulation of GluA2 in neurons exposed to elevated eye pressure. Intraocular administration of selective CP-AMPAR blockers promotes robust RGC survival supporting a critical rol...
Macrophages are key regulators of inflammation and repair, but their heterogeneity and multiple r... more Macrophages are key regulators of inflammation and repair, but their heterogeneity and multiple roles in the liver are not fully understood. We aimed herein to map the intrahepatic macrophage populations and their function(s) during acute liver injury. We used flow cytometry, gene expression analysis, multiplex-immunofluorescence, 3D-reconstruction, and spatial image analysis to characterize the intrahepatic immune landscape in mice post-CCl4-induced acute liver injury during three distinct phases: necroinflammation, and early and late repair. We observed hepatocellular necrosis and a reduction in liver resident lymphocytes during necroinflammation accompanied by the infiltration of circulating myeloid cells and upregulation of inflammatory cytokines. These parameters returned to baseline levels during the repair phase while pro-repair chemokines were upregulated. We identified resident CLEC4F+ Kupffer cells (KCs) and infiltrating IBA1+CLEC4F- monocyte-derived macrophages (MoMFs) as...
Animal models of the Boston keratoprosthesis type 1 (KPro) are needed to study glaucoma damage af... more Animal models of the Boston keratoprosthesis type 1 (KPro) are needed to study glaucoma damage after KPro implantation to control confounding comorbidities common in human KPro recipients. The purpose of this study was to determine the feasibility of establishing a reproducible mouse model of glaucoma after KPro surgery, specifically that of a miniaturized mouse model of KPro (mKPro). In the present study, a total of 20 corneas of donor C57BL/6 mice (n = 10) were implanted in one eye of each recipient BALB/C mice (n = 20), assembled as part of the mKPro, either with or without intraoperative lensectomy. Main feasibility outcomes consisted in incidence rates of loss of tone, capsule nicking, and lens extrusion, as well as acquisition of posterior segment OCT images. With lensectomy (n = 10), loss of ocular tone and retinal detachment occurred in 100% of mice. Without lensectomy (n = 10), capsule nicking and opening, as well as lens extrusion, occurred in 80% of mice. Causes of these complications included the large proportion of intraocular volume occupied by the lens, the shallow anterior chamber, and thus the lack of available intraocular volume to implant the KPro if the lens remains present. Successful mouse KPro surgery may require a great deal of practice to be useful as a reproducible model. Animal models ought to be pursued further by research teams in future studies.
Additional file 2: Supplementary Figure 1. (A) The number of YFP-positive RGC does not change at ... more Additional file 2: Supplementary Figure 1. (A) The number of YFP-positive RGC does not change at 2 weeks of ocular hypertension (OHT) relative to non-injured controls (Student's t-test, n.s.: not significant, N = 5 mice/group). (B) Real-time qPCR analysis confirms that retinal YFP gene expression does not change within 2 weeks of OHT damage (Student's t-test, n.s.: not significant, N = 5 mice/group). (C-E) RGC co-expressing YFP and SMI-32 are selected for dendritic arbor imaging and 3D reconstruction. Supplementary Figure 2. Analysis of whole-mounted retinas immunolabeled with an antibody recognizing pAMPKThr172 revealed a substantial increase of AMPK activity in RGC axons, visualized with SMI-32, at two weeks after glaucoma induction. Scale bars = 25 μm. Supplementary Figure 3. (A, B) Retinal imaging showed no fluorescein extravasation in eyes injected with compound C or vehicle. (C) In contrast, marked fluorescein extravasation was found in ischemic retinas subjected to ce...
Proceedings of the National Academy of Sciences, 2022
Significance The current lack of understanding of the mechanisms leading to neurovascular deficit... more Significance The current lack of understanding of the mechanisms leading to neurovascular deficits in glaucoma is a major knowledge gap in the field. Retinal pericytes regulate microcirculatory blood flow and coordinate neurovascular coupling through interpericyte tunneling nanotubes (IP-TNTs). We demonstrate that pericytes constrict capillaries in a calcium-dependent manner during glaucomatous stress, decreasing blood supply and compromising neuronal function. Moreover, ocular hypertension damages IP-TNTs and impairs light-evoked neurovascular responses. The reestablishment of calcium homeostasis in pericytes restores vascular and neuronal function, and prevents retinal ganglion cell death in glaucomatous eyes. This study provides important insights into the therapeutic potential of pericytes to counter vascular dysregulation in glaucoma.
Figure S2. Connexin-43 blocking peptide had no effect on intracellular calcium in control non-isc... more Figure S2. Connexin-43 blocking peptide had no effect on intracellular calcium in control non-ischemic retinas. (A, B) Fluo-4 indicator reported no effect on intracellular calcium in control non-ischemic retinas after intravitreal injection of connexin-43 blocking peptide (peptide 5). (B) Vessels were labeled with lectin (red) and nuclei with DAPI (blue). Scale bar in A-B = 5 μm. (TIF 7894 kb)
Background The maintenance of complex dendritic arbors and synaptic transmission are processes th... more Background The maintenance of complex dendritic arbors and synaptic transmission are processes that require a substantial amount of energy. Bioenergetic decline is a prominent feature of chronic neurodegenerative diseases, yet the signaling mechanisms that link energy stress with neuronal dysfunction are poorly understood. Recent work has implicated energy deficits in glaucoma, and retinal ganglion cell (RGC) dendritic pathology and synapse disassembly are key features of ocular hypertension damage. Results We show that adenosine monophosphate-activated protein kinase (AMPK), a conserved energy biosensor, is strongly activated in RGC from mice with ocular hypertension and patients with primary open angle glaucoma. Our data demonstrate that AMPK triggers RGC dendrite retraction and synapse elimination. We show that the harmful effect of AMPK is exerted through inhibition of the mammalian target of rapamycin complex 1 (mTORC1). Attenuation of AMPK activity restores mTORC1 function and...
Uploads
Papers by Adriana Di Polo