RESUMEN
Age and elevated intraocular pressure (IOP) are the two primary risk factors for glaucoma, an optic neuropathy that is the leading cause of irreversible blindness. In most people, IOP is tightly regulated over a lifetime by the conventional outflow tissues. However, the mechanistic contributions of age to conventional outflow dysregulation, elevated IOP and glaucoma are unknown. To address this gap in knowledge, we studied how age affects the morphology, biomechanical properties and function of conventional outflow tissues in C57BL/6 mice, which have an outflow system similar to humans. As reported in humans, we observed that IOP in mice was maintained within a tight range over their lifespan. Remarkably, despite a constellation of age-related changes to the conventional outflow tissues that would be expected to hinder aqueous drainage and impair homeostatic function (decreased cellularity, increased pigment accumulation, increased cellular senescence and increased stiffness), outflow facility, a measure of conventional outflow tissue fluid conductivity, was stable with age. We conclude that the murine conventional outflow system has significant functional reserve in healthy eyes. However, these age-related changes, when combined with other underlying factors, such as genetic susceptibility, are expected to increase risk for ocular hypertension and glaucoma.
Asunto(s)
Envejecimiento , Homeostasis , Presión Intraocular , Ratones Endogámicos C57BL , Animales , Presión Intraocular/fisiología , Envejecimiento/fisiología , Ratones , Glaucoma/fisiopatología , Glaucoma/patología , Glaucoma/metabolismo , Masculino , HumanosRESUMEN
Atomic force microscopy (AFM) is a valuable tool for assessing mechanical properties of biological samples, but interpretations of measurements on whole tissues can be difficult due to the tissue's highly heterogeneous nature. To overcome such difficulties and obtain more robust estimates of tissue mechanical properties, we describe an AFM force mapping and data analysis pipeline to characterize the mechanical properties of cryosectioned soft tissues. We assessed this approach on mouse optic nerve head and rat trabecular meshwork, cornea, and sclera. Our data show that the use of repeated measurements, outlier exclusion, and log-normal data transformation increases confidence in AFM mechanical measurements, and we propose that this methodology can be broadly applied to measuring soft tissue properties from cryosections.
Asunto(s)
Microscopía de Fuerza Atómica , Animales , Microscopía de Fuerza Atómica/métodos , Ratones , Ratas , Esclerótica/fisiología , Esclerótica/diagnóstico por imagen , Córnea/fisiología , Córnea/diagnóstico por imagen , Malla Trabecular/fisiología , Malla Trabecular/diagnóstico por imagen , Crioultramicrotomía/métodos , Disco Óptico/diagnóstico por imagen , Disco Óptico/fisiología , Fenómenos BiomecánicosRESUMEN
Murine models are commonly used to study glaucoma, the leading cause of irreversible blindness. Glaucoma is associated with elevated intra-ocular pressure (IOP), which is regulated by the tissues of the aqueous outflow pathway. In particular, pectinate ligaments (PLs) connect the iris and trabecular meshwork (TM) at the anterior chamber angle, with an unknown role in maintenance of the biomechanical stability of the aqueous outflow pathway, thus motivating this study. We conducted histomorphometric analysis and optical coherence tomography-based finite element (FE) modeling on three cohorts of C57BL/6 mice: "young" (2-6 months), "middle-aged" (11-16 months), and "elderly" (25-32 months). We evaluated the age-specific morphology of the outflow pathway tissues. Further, because of the known pressure-dependent Schlemm's canal (SC) narrowing, we assessed the dependence of the SC lumen area on varying IOPs in age-specific FE models over a physiological range of TM/PL stiffness values. We found age-dependent changes in morphology of outflow tissues; notably, the PLs were more developed in older mice compared to younger ones. In addition, FE modeling demonstrated that murine SC patency is highly dependent on the presence of PLs and that increased IOP caused SC collapse only with sufficiently low TM/PL stiffness values. Moreover, the elderly model showed more susceptibility to SC collapse compared to the younger models. In conclusion, our study elucidated the previously unexplored role of PLs in the aqueous outflow pathway, indicating their function in supporting TM and SC under elevated IOP.
Asunto(s)
Humor Acuoso , Glaucoma , Humanos , Anciano , Ratones , Animales , Humor Acuoso/metabolismo , Presión Intraocular , Ratones Endogámicos C57BL , Malla Trabecular/metabolismoRESUMEN
Age and elevated intraocular pressure (IOP) are the two primary risk factors for glaucoma, an optic neuropathy that is the leading cause of irreversible blindness. In most people, IOP is tightly regulated over a lifetime by the conventional outflow tissues. However, the mechanistic contributions of age to conventional outflow dysregulation, elevated IOP and glaucoma are unknown. To address this gap in knowledge, we studied how age affects the morphology, biomechanical properties and function of conventional outflow tissues in C57BL/6 mice, which have an outflow system similar to humans. As reported in humans, we observed that IOP in mice was maintained within a tight range over their lifespan. Remarkably, despite a constellation of age-related changes to the conventional outflow tissues that would be expected to hinder aqueous drainage and impair homeostatic function (decreased cellularity, increased pigment accumulation, increased cellular senescence and increased stiffness), outflow facility, a measure of conventional outflow tissue fluid conductivity, was stable with age. We conclude that the murine conventional outflow system has significant functional reserve in healthy eyes. However, these age-related changes, when combined with other underlying factors, such as genetic susceptibility, are expected to increase risk for ocular hypertension and glaucoma.
RESUMEN
Atomic force microscopy (AFM) is a valuable tool for assessing mechanical properties of biological samples, but interpretations of measurements on whole tissues can be difficult due to the tissue's highly heterogeneous nature. To overcome such difficulties and obtain more robust estimates of tissue mechanical properties, we describe an AFM force mapping and data analysis pipeline to characterize the mechanical properties of cryosectioned soft tissues. We assessed this approach on mouse optic nerve head and rat trabecular meshwork, cornea, and sclera. Our data show that the use of repeated measurements, outlier exclusion, and log-normal data transformation increases confidence in AFM mechanical measurements, and we propose that this methodology can be broadly applied to measuring soft tissue properties from cryosections.
RESUMEN
Automated methods for rapidly purifying and concentrating bacteria from environmental interferents are needed in next-generation applications for anything from water purification to biological weapons detection. Though previous work has been performed by other researchers in this area, there is still a need to create an automated system that can both purify and concentrate target pathogens in a timely manner with readily available and replaceable components that could be easily integrated with a detection mechanism. Thus, the objective of this work was to design, build, and demonstrate the effectiveness of an automated system, the Automated Dual-filter method for Applied Recovery, or aDARE. aDARE uses a custom LABVIEW program that guides the flow of bacterial samples through a pair of size-based separation membranes to capture and elute the target bacteria. Using aDARE, we eliminated 95% of the interfering beads of a 5 mL-sample volume containing 107 CFU/mL of E. coli contaminated with 2 µm and 10 µm polystyrene beads at 106 beads/mL concentration., The target bacteria were concentrated to more than twice the initial concentration in 900 µL of eluent, resulting in an enrichment ratio for the target bacteria of 42 ± 13 in 5.5 min. These results show the feasibility and effectiveness of using size-based filtration membranes to purify and concentrate a target bacterium, in this case E. coli, in an automated system.