RESUMEN
We hypothesize that the injection of JP4-039, a mitochondria-targeted nitroxide, prior to irradiation of the mouse retina may decrease apoptosis and reduce neutrophil and macrophage migration into the retina. In our study, we aimed to examine the effects of JP4-039 in the mouse retina using fluorescent microscopy, a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and flow cytometry. Forty-five mice and one eye per mouse were used. In Group 1, fluorescent microscopy was used to determine retinal uptake of 10 µL (0.004 mg/µL) of intravitreally injected BODIPY-labeled JP4-039 at 0, 15, and 60 min after injection. In Group 2, the TUNEL assay was performed to investigate the rate of apoptosis after irradiation in addition to JP4-039 injection, compared to controls. In Group 3, flow cytometry was used to determine the extent of inflammatory cell migration into the retina after irradiation in addition to JP4-039 injection, compared to controls. Maximal retinal uptake of JP4-039 was 15 min after intravitreal injection (p < 0.0001). JP4-039-treated eyes had lower levels of retinal apoptosis (35.8 ± 2.5%) than irradiated controls (49.0 ± 2.7%; p = 0.0066) and demonstrated reduced migration of N1 cells (30.7 ± 11.7% vs. 77.7 ± 5.3% controls; p = 0.004) and M1 cells (76.6 ± 4.2 vs. 88.1 ± 3.7% controls, p = 0.04). Pretreatment with intravitreally injected JP4-039 reduced apoptosis and inflammatory cell migration in the irradiated mouse retina, marking the first confirmed effect of this molecule in retinal tissue. Further studies may allow for safety profiling and potential use for patients with radiation retinopathy.
Asunto(s)
Apoptosis , Movimiento Celular , Mitocondrias , Retina , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Ratones , Retina/efectos de los fármacos , Retina/metabolismo , Retina/efectos de la radiación , Retina/patología , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/efectos de la radiación , Movimiento Celular/efectos de los fármacos , Movimiento Celular/efectos de la radiación , Ratones Endogámicos C57BL , Masculino , Óxidos de Nitrógeno/farmacología , Inflamación/patologíaRESUMEN
PURPOSE: Pseudomonas aeruginosa keratitis is a severe ocular infection that can lead to perforation of the cornea. In this study we evaluated the role of bacterial quorum sensing in generating corneal perforation and bacterial proliferation and tested whether co-injection of the predatory bacteria Bdellovibrio bacteriovorus could alter the clinical outcome. P. aeruginosa with lasR mutations were observed among keratitis isolates from a study collecting samples from India, so an isogenic lasR mutant strain of P. aeruginosa was included. METHODS: Rabbit corneas were intracorneally infected with P. aeruginosa strain PA14 or an isogenic ΔlasR mutant and co-injected with PBS or B. bacteriovorus. After 24 h, eyes were evaluated for clinical signs of infection. Samples were analyzed by scanning electron microscopy, optical coherence tomography, sectioned for histology, and corneas were homogenized for CFU enumeration and for inflammatory cytokines. RESULTS: We observed that 54% of corneas infected by wild-type PA14 presented with a corneal perforation (n = 24), whereas only 4% of PA14 infected corneas that were co-infected with B. bacteriovorus perforate (n = 25). Wild-type P. aeruginosa proliferation was reduced 7-fold in the predatory bacteria treated eyes. The ΔlasR mutant was less able to proliferate compared to the wild-type, but was largely unaffected by B. bacteriovorus. CONCLUSION: These studies indicate a role for bacterial quorum sensing in the ability of P. aeruginosa to proliferate and cause perforation of the rabbit cornea. Additionally, this study suggests that predatory bacteria can reduce the virulence of P. aeruginosa in an ocular prophylaxis model.
Asunto(s)
Perforación Corneal , Infecciones Bacterianas del Ojo , Queratitis , Infecciones por Pseudomonas , Animales , Conejos , Pseudomonas aeruginosa , Infecciones por Pseudomonas/microbiología , Queratitis/tratamiento farmacológico , Córnea/patología , Bacterias , Proliferación Celular , Infecciones Bacterianas del Ojo/microbiologíaRESUMEN
Purpose: Pseudomonas aeruginosa keratitis is a severe ocular infection that can lead to perforation of the cornea. In this study we evaluated the role of bacterial quorum sensing in generating corneal perforation and bacterial proliferation and tested whether co-injection of the predatory bacteria Bdellovibrio bacteriovorus could alter the clinical outcome. P. aeruginosa with lasR mutations were observed among keratitis isolates from a study collecting samples from India, so an isogenic lasR mutant strain of P. aeruginosa was included. Methods: Rabbit corneas were intracorneally infected with P. aeruginosa strain PA14 or an isogenic Δ lasR mutant and co-injected with PBS or B. bacteriovorus . After 24 h, eyes were evaluated for clinical signs of infection. Samples were analyzed by scanning electron microscopy, optical coherence tomography, sectioned for histology, and corneas were homogenized for CFU enumeration and for inflammatory cytokines. Results: We observed that 54% of corneas infected by wild-type PA14 presented with a corneal perforation (n=24), whereas only 4% of PA14 infected corneas that were co-infected with B. bacteriovorus perforate (n=25). Wild-type P. aeruginosa proliferation was reduced 7-fold in the predatory bacteria treated eyes. The Δ lasR mutant was less able to proliferate compared to the wild-type, but was largely unaffected by B. bacteriovorus . Conclusion: These studies indicate a role for bacterial quorum sensing in the ability of P. aeruginosa to proliferate and cause perforation of the rabbit cornea. Additionally, this study suggests that predatory bacteria can reduce the virulence of P. aeruginosa in an ocular prophylaxis model.
RESUMEN
In dry age-related macular degeneration (AMD), LCN2 (lipocalin 2) is upregulated. Whereas LCN2 has been implicated in AMD pathogenesis, the mechanism remains unknown. Here, we report that in retinal pigmented epithelial (RPE) cells, LCN2 regulates macroautophagy/autophagy, in addition to maintaining iron homeostasis. LCN2 binds to ATG4B to form an LCN2-ATG4B-LC3-II complex, thereby regulating ATG4B activity and LC3-II lipidation. Thus, increased LCN2 reduced autophagy flux. Moreover, RPE cells from cryba1 KO, as well as sting1 KO and Sting1Gt mutant mice (models with abnormal iron chelation), showed decreased autophagy flux and increased LCN2, indicative of CGAS- and STING1-mediated inflammasome activation. Live cell imaging of RPE cells with elevated LCN2 also showed a correlation between inflammasome activation and increased fluorescence intensity of the Liperfluo dye, indicative of oxidative stress-induced ferroptosis. Interestingly, both in human AMD patients and in mouse models with a dry AMD-like phenotype (cryba1 cKO and KO), the LCN2 homodimer variant is increased significantly compared to the monomer. Sub-retinal injection of the LCN2 homodimer secreted by RPE cells into NOD-SCID mice leads to retinal degeneration. In addition, we generated an LCN2 monoclonal antibody that neutralizes both the monomer and homodimer variants and rescued autophagy and ferroptosis activities in cryba1 cKO mice. Furthermore, the antibody rescued retinal function in cryba1 cKO mice as assessed by electroretinography. Here, we identify a molecular pathway whereby increased LCN2 elicits pathophysiology in the RPE, cells known to drive dry AMD pathology, thus providing a possible therapeutic strategy for a disease with no current treatment options.Abbreviations: ACTB: actin, beta; Ad-GFP: adenovirus-green fluorescent protein; Ad-LCN2: adenovirus-lipocalin 2; Ad-LCN2-GFP: adenovirus-LCN2-green fluorescent protein; LCN2AKT2: AKT serine/threonine kinase 2; AMBRA1: autophagy and beclin 1 regulator 1; AMD: age-related macular degeneration; ARPE19: adult retinal pigment epithelial cell line-19; Asp278: aspartate 278; ATG4B: autophagy related 4B cysteine peptidase; ATG4C: autophagy related 4C cysteine peptidase; ATG7: autophagy related 7; ATG9B: autophagy related 9B; BLOC-1: biogenesis of lysosomal organelles complex 1; BLOC1S1: biogenesis of lysosomal organelles complex 1 subunit 1; C57BL/6J: C57 black 6J; CGAS: cyclic GMP-AMP synthase; ChQ: chloroquine; cKO: conditional knockout; Cys74: cysteine 74; Dab2: DAB adaptor protein 2; Def: deferoxamine; DHE: dihydroethidium; DMSO: dimethyl sulfoxide; ERG: electroretinography; FAC: ferric ammonium citrate; Fe2+: ferrous; FTH1: ferritin heavy chain 1; GPX: glutathione peroxidase; GST: glutathione S-transferase; H2O2: hydrogen peroxide; His280: histidine 280; IFNL/IFNλ: interferon lambda; IL1B/IL-1ß: interleukin 1 beta; IS: Inner segment; ITGB1/integrin ß1: integrin subunit beta 1; KO: knockout; LC3-GST: microtubule associated protein 1 light chain 3-GST; C-terminal fusion; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LCN2: lipocalin 2; mAb: monoclonal antibody; MDA: malondialdehyde; MMP9: matrix metallopeptidase 9; NLRP3: NLR family pyrin domain containing 3; NOD-SCID: nonobese diabetic-severe combined immunodeficiency; OS: outer segment; PBS: phosphate-buffered saline; PMEL/PMEL17: premelanosome protein; RFP: red fluorescent protein; rLCN2: recombinant LCN2; ROS: reactive oxygen species; RPE SM: retinal pigmented epithelium spent medium; RPE: retinal pigment epithelium; RSL3: RAS-selective lethal; scRNAseq: single-cell ribonucleic acid sequencing; SD-OCT: spectral domain optical coherence tomography; shRNA: small hairpin ribonucleic acid; SM: spent medium; SOD1: superoxide dismutase 1; SQSTM1/p62: sequestosome 1; STAT1: signal transducer and activator of transcription 1; STING1: stimulator of interferon response cGAMP interactor 1; TYR: tyrosinase; VCL: vinculin; WT: wild type.
Asunto(s)
Ferroptosis , Degeneración Macular , Animales , Humanos , Ratones , Anticuerpos Monoclonales , Autofagia/fisiología , Inflamasomas/metabolismo , Lipocalina 2/genética , Degeneración Macular/genética , Degeneración Macular/metabolismo , Degeneración Macular/patología , Ratones Endogámicos NOD , Ratones SCID , Nucleotidiltransferasas/metabolismoRESUMEN
Rhodopsin-associated (RHO-associated) retinitis pigmentosa (RP) is a progressive retinal disease that currently has no cure. RHO protein misfolding leads to disturbed proteostasis and the death of rod photoreceptors, resulting in decreased vision. We previously identified nonretinoid chaperones of RHO, including YC-001 and F5257-0462, by small-molecule high-throughput screening. Here, we profile the chaperone activities of these molecules toward the cell-surface level of 27 RP-causing human RHO mutants in NIH3T3 cells. Furthermore, using retinal explant culture, we show that YC-001 improves retinal proteostasis by supporting RHO homeostasis in RhoP23H/+ mouse retinae, which results in thicker outer nuclear layers (ONL), indicating delayed photoreceptor degeneration. Interestingly, YC-001 ameliorated retinal immune responses and reduced the number of microglia/macrophages in the RhoP23H/+ retinal explants. Similarly, F5257-0462 also protects photoreceptors in RhoP23H/+ retinal explants. In vivo, intravitreal injection of YC-001 or F5257-0462 microparticles in PBS shows that F5257-0462 has a higher efficacy in preserving photoreceptor function and delaying photoreceptor death in RhoP23H/+ mice. Collectively, we provide proof of principle that nonretinoid chaperones are promising drug candidates in treating RHO-associated RP.
Asunto(s)
Retinitis Pigmentosa , Rodopsina , Animales , Modelos Animales de Enfermedad , Homeostasis , Ratones , Chaperonas Moleculares , Células 3T3 NIH , Células Fotorreceptoras Retinianas Bastones/metabolismo , Rodopsina/genética , Rodopsina/metabolismoRESUMEN
Visual information is transmitted from the eye to the brain along the optic nerve, a structure composed of retinal ganglion cell (RGC) axons. The optic nerve is highly vulnerable to damage in neurodegenerative diseases, such as glaucoma, and there are currently no FDA-approved drugs or therapies to protect RGCs from death. Zebrafish possess remarkable neuroprotective and regenerative abilities. Here, utilizing an optic nerve transection (ONT) injury and an RNA-seq-based approach, we identify genes and pathways active in RGCs that may modulate their survival. Through pharmacological perturbation, we demonstrate that Jak/Stat pathway activity is required for RGC survival after ONT. Furthermore, we show that immune responses directly contribute to RGC death after ONT; macrophages/microglia are recruited to the retina and blocking neuroinflammation or depleting these cells after ONT rescues survival of RGCs. Taken together, these data support a model in which crosstalk between macrophages/microglia and RGCs, mediated by Jak/Stat pathway activity, regulates RGC survival after optic nerve injury.
Asunto(s)
Inmunidad Innata , Quinasas Janus/inmunología , Traumatismos del Nervio Óptico/inmunología , Células Ganglionares de la Retina/inmunología , Factores de Transcripción STAT/inmunología , Transducción de Señal/inmunología , Proteínas de Pez Cebra/inmunología , Pez Cebra/inmunología , Animales , Animales Modificados Genéticamente , Femenino , Quinasas Janus/genética , Masculino , Traumatismos del Nervio Óptico/genética , Factores de Transcripción STAT/genética , Transducción de Señal/genética , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
A cornea is innervated by sensory nerves, which branch into thick trunks, subbasal plexuses, and sensory endings. Appropriate assessment of nerve structure in a tissue provides a more complete understanding of the role of nerves in health and disease. Here, we present a whole-mount immunohistochemistry protocol that facilitates evaluation of nerve architecture throughout the mouse cornea. The fixation step in this protocol allows for reliable detection of nerve structures within the cornea and likely other tissues. For complete details on the use and execution of this protocol, please refer to Yun et al, (2020).
Asunto(s)
Córnea , Inmunohistoquímica/métodos , Nervio Oftálmico , Animales , Córnea/anatomía & histología , Córnea/inervación , Disección , Femenino , Masculino , Ratones , Nervio Oftálmico/anatomía & histología , Nervio Oftálmico/química , Nervio Oftálmico/citologíaRESUMEN
Herpes simplex virus type 1 (HSV-1)-infected corneas can develop a blinding immunoinflammatory condition called herpes stromal keratitis (HSK), which involves the loss of corneal sensitivity due to retraction of sensory nerves and subsequent hyperinnervation with sympathetic nerves. Increased concentrations of the cytokine VEGF-A in the cornea are associated with HSK severity. Here, we examined the impact of VEGF-A on neurologic changes that underly HSK using a mouse model of HSV-1 corneal infection. Both CD4+ T cells and myeloid cells produced pathogenic levels of VEGF-A within HSV-1-infected corneas, and CD4+ cell depletion promoted reinnervation of HSK corneas with sensory nerves. In vitro, VEGF-A from infected corneas repressed sensory nerve growth and promoted sympathetic nerve growth. Neutralizing VEGF-A in vivo using bevacizumab inhibited sympathetic innervation, promoted sensory nerve regeneration, and alleviated disease. Thus, VEGF-A can shape the sensory and sympathetic nerve landscape within the cornea, with implications for the treatment of blinding corneal disease.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Córnea/inervación , Córnea/metabolismo , Queratitis Herpética/etiología , Células Mieloides/inmunología , Células Mieloides/metabolismo , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Fibras Adrenérgicas , Animales , Córnea/inmunología , Córnea/virología , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Técnica del Anticuerpo Fluorescente , Herpesvirus Humano 1 , Humanos , Inmunofenotipificación , Queratitis Herpética/metabolismo , Queratitis Herpética/patología , Leucocitos/inmunología , Leucocitos/metabolismo , Leucocitos/patología , Depleción Linfocítica , Ratones , Neuritis , Índice de Severidad de la EnfermedadRESUMEN
PURPOSE: Neurotrophic keratopathy (NK) produces persistent epithelial erosion which is hard to treat effectively. Recently, corneal neurotization surgery has produced reinnervation of the cornea with resolving neurotrophic keratopathy. We hypothesized that the generation of corneal epithelial nerves after neurotization surgery would not only restore the integrity of corneal epithelium but also produce a change in the configuration of the palisades of Vogt (POV), which houses the corneal epithelial stem cells. METHODS: We assessed a patient with unilateral congenital corneal anesthesia with optical coherence tomography pre-neurotization and post-neurotization. RESULTS: Over the course of 2 years, the patient gained corneal epithelial sensation and corneal and limbal epithelium was restored to normal thickness with corresponding changes in the POV. CONCLUSIONS: The intimate relationship between epithelium and sensory nerves of the cornea has been well documented; however, changes in the corneal epithelial stem cell niche in conjunction with development of innervation have not previously been reported. Considering the architecture of the corneal nerves in conjunction with the architecture of the POV may assist in developing treatments that can support the regeneration and maintenance of epithelium during nerve regeneration.
Asunto(s)
Córnea/inervación , Enfermedades de la Córnea/cirugía , Regeneración Nerviosa/fisiología , Transferencia de Nervios/métodos , Sensación/fisiología , Adulto , Córnea/fisiopatología , Enfermedades de la Córnea/fisiopatología , Epitelio Corneal/patología , Humanos , Masculino , Microscopía Confocal , Tomografía de Coherencia ÓpticaRESUMEN
Optical Coherence Tomography (OCT) is an imaging modality that has been widely adopted for visualizing corneal, retinal and limbal tissue structure with micron resolution. It can be used to diagnose pathological conditions of the eye, and for developing pre-operative surgical plans. In contrast to the posterior retina, imaging the anterior tissue structures, such as the limbus and cornea, results in B-scans that exhibit increased speckle noise patterns and imaging artifacts. These artifacts, such as shadowing and specularity, pose a challenge during the analysis of the acquired volumes as they substantially obfuscate the location of tissue interfaces. To deal with the artifacts and speckle noise patterns and accurately segment the shallowest tissue interface, we propose a cascaded neural network framework, which comprises of a conditional Generative Adversarial Network (cGAN) and a Tissue Interface Segmentation Network (TISN). The cGAN pre-segments OCT B-scans by removing undesired specular artifacts and speckle noise patterns just above the shallowest tissue interface, and the TISN combines the original OCT image with the pre-segmentation to segment the shallowest interface. We show the applicability of the cascaded framework to corneal datasets, demonstrate that it precisely segments the shallowest corneal interface, and also show its generalization capacity to limbal datasets. We also propose a hybrid framework, wherein the cGAN pre-segmentation is passed to a traditional image analysis-based segmentation algorithm, and describe the improved segmentation performance. To the best of our knowledge, this is the first approach to remove severe specular artifacts and speckle noise patterns (prior to the shallowest interface) that affects the interpretation of anterior segment OCT datasets, thereby resulting in the accurate segmentation of the shallowest tissue interface. To the best of our knowledge, this is the first work to show the potential of incorporating a cGAN into larger deep learning frameworks for improved corneal and limbal OCT image segmentation. Our cGAN design directly improves the visualization of corneal and limbal OCT images from OCT scanners, and improves the performance of current OCT segmentation algorithms.
RESUMEN
Medical devices, such as contact lenses, bring bacteria in direct contact with human cells. Consequences of these host-pathogen interactions include the alteration of mammalian cell surface architecture and induction of cellular death that renders tissues more susceptible to infection. Gram-negative bacteria known to induce cellular blebbing by mammalian cells, Pseudomonas and Vibrio species, do so through a type III secretion system-dependent mechanism. This study demonstrates that a subset of bacteria from the Enterobacteriaceae bacterial family induce cellular death and membrane blebs in a variety of cell types via a type V secretion-system dependent mechanism. Here, we report that ShlA-family cytolysins from Proteus mirabilis and Serratia marcescens were required to induce membrane blebbling and cell death. Blebbing and cellular death were blocked by an antioxidant and RIP-1 and MLKL inhibitors, implicating necroptosis in the observed phenotypes. Additional genetic studies determined that an IgaA family stress-response protein, GumB, was necessary to induce blebs. Data supported a model where GumB and shlBA are in a regulatory circuit through the Rcs stress response phosphorelay system required for bleb formation and pathogenesis in an invertebrate model of infection and proliferation in a phagocytic cell line. This study introduces GumB as a regulator of S. marcescens host-pathogen interactions and demonstrates a common type V secretion system-dependent mechanism by which bacteria elicit surface morphological changes on mammalian cells. This type V secretion-system mechanism likely contributes bacterial damage to the corneal epithelial layer, and enables access to deeper parts of the tissue that are more susceptible to infection.
Asunto(s)
Toxinas Bacterianas/metabolismo , Células Epiteliales/metabolismo , Epitelio Corneal/metabolismo , Infecciones por Proteus/metabolismo , Proteus/metabolismo , Infecciones por Serratia/metabolismo , Serratia marcescens/metabolismo , Animales , Toxinas Bacterianas/genética , Muerte Celular , Células Epiteliales/microbiología , Células Epiteliales/patología , Epitelio Corneal/microbiología , Epitelio Corneal/patología , Humanos , Ratones , Perforina/genética , Perforina/metabolismo , Proteus/genética , Infecciones por Proteus/genética , Infecciones por Proteus/microbiología , Infecciones por Proteus/patología , Células RAW 264.7 , Infecciones por Serratia/genética , Infecciones por Serratia/microbiología , Infecciones por Serratia/patología , Serratia marcescens/genética , Porcinos , Sistemas de Secreción Tipo V/genética , Sistemas de Secreción Tipo V/metabolismoRESUMEN
The retinal pigment epithelium (RPE) is a specialized monolayer of pigmented cells within the eye that is critical for maintaining visual system function. Diseases affecting the RPE have dire consequences for vision, and the most prevalent of these is atrophic (dry) age-related macular degeneration (AMD), which is thought to result from RPE dysfunction and degeneration. An intriguing possibility for treating RPE degenerative diseases like atrophic AMD is the stimulation of endogenous RPE regeneration; however, very little is known about the mechanisms driving successful RPE regeneration in vivo. Here, we developed a zebrafish transgenic model (rpe65a:nfsB-eGFP) that enabled ablation of large swathes of mature RPE. RPE ablation resulted in rapid RPE degeneration, as well as degeneration of Bruch's membrane and underlying photoreceptors. Using this model, we demonstrate for the first time that zebrafish are capable of regenerating a functional RPE monolayer after RPE ablation. Regenerated RPE cells first appear at the periphery of the RPE, and regeneration proceeds in a peripheral-to-central fashion. RPE ablation elicits a robust proliferative response in the remaining RPE. Subsequently, proliferative cells move into the injury site and differentiate into RPE. BrdU incorporation assays demonstrate that the regenerated RPE is likely derived from remaining peripheral RPE cells. Pharmacological disruption using IWR-1, a Wnt signaling antagonist, significantly reduces cell proliferation in the RPE and impairs overall RPE recovery. These data demonstrate that the zebrafish RPE possesses a robust capacity for regeneration and highlight a potential mechanism through which endogenous RPE regenerate in vivo.
Asunto(s)
Degeneración Macular/genética , Regeneración/genética , Epitelio Pigmentado de la Retina/crecimiento & desarrollo , cis-trans-Isomerasas/genética , Animales , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/crecimiento & desarrollo , Apoptosis/genética , Lámina Basal de la Coroides/crecimiento & desarrollo , Lámina Basal de la Coroides/metabolismo , Diferenciación Celular/genética , Modelos Animales de Enfermedad , Proteínas Fluorescentes Verdes/genética , Humanos , Imidas/administración & dosificación , Larva/genética , Larva/crecimiento & desarrollo , Degeneración Macular/patología , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/patología , Quinolinas/administración & dosificación , Retina/crecimiento & desarrollo , Retina/patología , Epitelio Pigmentado de la Retina/metabolismo , Vía de Señalización Wnt/efectos de los fármacos , Pez Cebra/genética , Pez Cebra/crecimiento & desarrolloRESUMEN
Purpose: To correlate outflow function and outflow tract vessel diameter changes induced by nitric oxide (NO). Methods: In a porcine anterior segment perfusion model, the effects of a nitric oxide donor (100 µM DETA-NO) on outflow facility were compared with controls (n = 8 per group) with trabecular meshwork (TM) and after circumferential ab interno trabeculectomy (AIT). Outflow structures were assessed with spectral-domain optical coherence tomography (SD-OCT) before and after NO, or an NO synthase inhibitor (100 µM L-NAME) and the vasoconstrictor, endothelin-1 (100 pg/mL ET-1). Scans were processed with a custom macroscript and aligned for automated reslicing and quantification of cross-sectional outflow tract areas (CSA). Results: The facility increased after DETA-NO (Δ of 0.189 ± 0.081 µL/min·mm Hg, P = 0.034) and AIT (Δ of 0.251 ± 0.094 µL/min·mm Hg, P = 0.009), respectively. Even after AIT, DETA-NO increased the facility by 61.5% (Δ of 0.190 ± 0.074 µL/min·mm Hg, P = 0.023) and CSA by 13.9% (P < 0.001). L-NAME + ET-1 decreased CSA by -8.6% (P < 0.001). NO increased the diameter of focal constrictions 5.0 ± 3.8-fold. Conclusions: NO can dilate vessels of the distal outflow tract and increase outflow facility in a TM-independent fashion. There are short, focally constricting vessel sections that display large diameter changes and may have a substantial impact on outflow.
Asunto(s)
Humor Acuoso/fisiología , Factores Relajantes Endotelio-Dependientes/farmacología , Óxido Nítrico/farmacología , Malla Trabecular/efectos de los fármacos , Animales , Endotelina-1/farmacología , Inhibidores Enzimáticos/farmacología , Presión Intraocular/fisiología , NG-Nitroarginina Metil Éster/farmacología , Porcinos , Tomografía de Coherencia Óptica , Malla Trabecular/diagnóstico por imagen , Malla Trabecular/fisiopatología , TrabeculectomíaRESUMEN
Purpose: To test the hypothesis that human, monkey, pig, sheep, cow, and goat eyes exhibit circumferential, radial, and interweaving collagen architecture in the posterior sclera. Methods: We analyzed 1,327 cryosections from the posterior poles of 4 human, 4 monkey, 5 pig, 8 sheep, 1 goat, and 2 cow eyes. Images were acquired using polarized light microscopy and processed to obtain polar fiber orientations relative to the center of the canal. Circumferential, radial, and interweaving regions were identified and analyzed for mean fiber orientation and anisotropy and region width and thickness. Results: Every eye exhibited circumferential, radial, and interweaving fibers in consistent locations. Radial fibers extended out from near the canal into the peripapillary and peripheral sclera in the innermost sclera. Circumferential fibers were directly adjacent to the canal and most prevalent in the outermost, posterior sclera. Interweaving fibers were found throughout the sclera thickness. Across all species, median anisotropy in the radial, circumferential, and interweaving regions were 0.95, 0.96, and 0.28, respectively. Conclusions: Regions of radial, circumferential, and interweaving fibers occur in the posterior pole sclera of human, monkey, pig, sheep, cow, and goat eyes. The consistency across species in scleral architecture suggests that they are primary organizational components whose functions should be better understood.
Asunto(s)
Colágeno/metabolismo , Disco Óptico/metabolismo , Esclerótica/metabolismo , Animales , Anisotropía , Bovinos , Cabras , Humanos , Macaca , Microscopía de Polarización , Ovinos , PorcinosRESUMEN
Purpose: Collagen is the main load-bearing component of the eye, and collagen crimp is a critical determinant of tissue mechanical behavior. We test the hypothesis that collagen crimp morphology varies over the human cornea and sclera and with age. Methods: We analyzed 42 axial whole-globe sections from 20 normal eyes of 20 human donors, ranging in age from 0.08 (1 month) to 97 years. The sections were imaged using polarized light microscopy to obtain µm-scale fiber bundle/lamellae orientation from two corneal and six scleral regions. Crimp morphology was quantified through waviness, tortuosity, and amplitude. Results: Whole-globe median waviness, tortuosity, and amplitude were 0.127 radians, 1.002, and 0.273 µm, respectively. These parameters, however, were not uniform over the globe, instead exhibiting distinct, consistent patterns. All crimp parameters decreased significantly with age, with significantly different age-related decreases between regions. The crimp morphology of the limbus changed the most drastically with age, such that it had the largest crimp in neonates, and among the smallest in the elderly. Conclusions: Age-related decreases in crimp parameters are likely one of the mechanisms underlying age-related stiffening of the sclera and cornea, potentially influencing sensitivity to IOP. Further work is needed to determine the biomechanical implications of the crimp patterns observed. The comparatively large changes in the crimp morphology of the limbus, especially in the early years of life, suggest that crimp in this region may play a role in eye development, although the exact nature of this is unclear.
Asunto(s)
Envejecimiento/fisiología , Colágeno/metabolismo , Córnea/metabolismo , Esclerótica/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Femenino , Voluntarios Sanos , Humanos , Lactante , Recién Nacido , Masculino , Microscopía de Polarización , Persona de Mediana Edad , Donantes de TejidosRESUMEN
Collagen fibers play a central role in normal eye mechanics and pathology. In ocular tissues, collagen fibers exhibit a complex 3-dimensional (3D) fiber orientation, with both in-plane (IP) and out-of-plane (OP) orientations. Imaging techniques traditionally applied to the study of ocular tissues only quantify IP fiber orientation, providing little information on OP fiber orientation. Accurate description of the complex 3D fiber microstructures of the eye requires quantifying full 3D fiber orientation. Herein, we present 3dPLM, a technique based on polarized light microscopy developed to quantify both IP and OP collagen fiber orientations of ocular tissues. The performance of 3dPLM was examined by simulation and experimental verification and validation. The experiments demonstrated an excellent agreement between extracted and true 3D fiber orientation. Both IP and OP fiber orientations can be extracted from the sclera and the cornea, providing previously unavailable quantitative 3D measures and insight into the tissue microarchitecture. Together, the results demonstrate that 3dPLM is a powerful imaging technique for the analysis of ocular tissues.
Asunto(s)
Colágeno/metabolismo , Córnea/diagnóstico por imagen , Córnea/metabolismo , Imagenología Tridimensional , Microscopía de Polarización , Esclerótica/diagnóstico por imagen , Esclerótica/metabolismo , Animales , Pollos , Humanos , Fenómenos Ópticos , Ovinos , Tendones/diagnóstico por imagen , Tendones/metabolismoRESUMEN
Although elevated intraocular pressure (IOP) and age are major risk factors for glaucoma, their effects on glaucoma pathogenesis remain unclear. This study examined the onset and progression of glaucomatous changes to ocular anatomy and physiology, structural and physiological brain integrity, and visuomotor behavior in the DBA/2J mice via non-invasive tonometry, multi-parametric magnetic resonance imaging (MRI) and optokinetic assessments from 5 to 12 months of age. Using T2-weighted MRI, diffusion tensor MRI, and manganese-enhanced MRI, increasing IOP elevation at 9 and 12 months old coincided with anterior chamber deepening, altered fractional anisotropy and radial diffusivity of the optic nerve and optic tract, as well as reduced anterograde manganese transport along the visual pathway respectively in the DBA/2J mice. Vitreous body elongation and visuomotor function deterioration were observed until 9 months old, whereas axial diffusivity only decreased at 12 months old in diffusion tensor MRI. Under the same experimental settings, C57BL/6J mice only showed modest age-related changes. Taken together, these results indicate that the anterior and posterior visual pathways of the DBA/2J mice exhibit differential susceptibility to glaucomatous neurodegeneration observable by in vivo multi-modal examinations.
Asunto(s)
Envejecimiento , Encéfalo/fisiopatología , Modelos Animales de Enfermedad , Ojo/fisiopatología , Glaucoma/fisiopatología , Presión Intraocular , Vías Visuales/fisiopatología , Animales , Femenino , Imagen por Resonancia Magnética/métodos , Masculino , Ratones , Ratones Endogámicos DBA , Nervio Óptico/fisiopatologíaRESUMEN
The underground environment imposes unique demands on life that have led subterranean species to evolve specialized traits, many of which evolved convergently. We studied convergence in evolutionary rate in subterranean mammals in order to associate phenotypic evolution with specific genetic regions. We identified a strong excess of vision- and skin-related genes that changed at accelerated rates in the subterranean environment due to relaxed constraint and adaptive evolution. We also demonstrate that ocular-specific transcriptional enhancers were convergently accelerated, whereas enhancers active outside the eye were not. Furthermore, several uncharacterized genes and regulatory sequences demonstrated convergence and thus constitute novel candidate sequences for congenital ocular disorders. The strong evidence of convergence in these species indicates that evolution in this environment is recurrent and predictable and can be used to gain insights into phenotype-genotype relationships.
Asunto(s)
Adaptación Biológica , Evolución Biológica , Ecosistema , Ojo , Mamíferos , Fenómenos Fisiológicos Oculares , AnimalesRESUMEN
Plasma-mediated ab interno trabeculectomy with the trabectome was first approved by the US Food and Drug Administration in 2004 for use in adult and pediatric glaucomas. Since then, increased clinical experience and updated outcome data have led to its expanded use, including a range of glaucomas and angle presentations, previously deemed to be relatively contraindicated. The main benefits are a high degree of safety, ease, and speed compared to traditional filtering surgery and tube shunts. The increasing burden of glaucoma and expanding life expectancy has resulted in demand for well-trained surgeons. In this article, we discuss the results of trabectome surgery in standard and nonstandard indications. We present training strategies of the surgical technique that include a pig eye model, and visualization exercises that can be performed before and at the conclusion of standard cataract surgery in patients who do not have glaucoma. We detail the mechanism of enhancing the conventional outflow pathway and describe methods of visualization and function testing.
RESUMEN
Increasing prevalence and cost of glaucoma have increased the demand for surgeons well trained in newer, microincisional surgery. These procedures occur in a highly confined space, making them difficult to learn by observation or assistance alone as is currently done. We hypothesized that our ex vivo outflow model is sensitive enough to allow computing individual learning curves to quantify progress and refine techniques. Seven trainees performed nine trabectome-mediated ab interno trabeculectomies in pig eyes (n = 63). An expert surgeon rated the procedure using an Operating Room Score (ORS). The extent of outflow beds accessed was measured with canalograms. Data was fitted using mixed effect models. ORS reached a half-maximum on an asymptote after only 2.5 eyes. Surgical time decreased by 1.4 minutes per eye in a linear fashion. The ablation arc followed an asymptotic function with a half-maximum inflection point after 5.3 eyes. Canalograms revealed that this progress did not correlate well with improvement in outflow, suggesting instead that about 30 eyes are needed for true mastery. This inexpensive pig eye model provides a safe and effective microsurgical training model and allows objective quantification of outcomes for the first time.