Background: Continuous and primary in vitro cultures are largely used to study cellular mechanisms occurring in several pathologic-like or pathological conditions. Continuous cell lines allow to perform long-lasting experiments since they do not undergo senescence.
Methods: The immortalized Moorfields/Institute of Ophtalmology-Müller 1 (MIO-M1) cell type represents a valuable model to analyze the mechanistic pathways characterizing Müller glial cells, both in health and in disease. MIO-M1 can be used to dissect the response of these glial cells following treatments which mimic pathological condition. For instance, MIO-M1 are useful to study the response of this cell type to stress condition as the case of oxidative stress (OS) (cultured with hydrogen peroxide), pathological neovascularization (cultured with VEGF), hypoxic or hyperoxic condition (cultured in low or high oxygen chamber). On the other hand, primary cultures allow to specifically analyze cellular responses without the interference of the whole organ, although the experimental treatment is performed in vivo. Primary Müller cells can be used to perform electrophysiological analyses of different cell sites.
Discussion: We describe how to manage MIO-M1 cells and how to analyze their response to different stress conditions; moreover, we report how to isolate and identify primary Müller cells and how to perform patch clamp and single cell recordings on them.
Background: Sodium iodate (SI) is a chemical widely applied to induce retina degeneration in animal models. SI treatment caused formation of rosettes/folds in the outer nuclear layer (ONL) of the rat retina, but it was previously unclear whether SI also forms rosettes in mice. In addition, SI induced retina degeneration was never addressed in non-separate sclerochoroid/retina pigment epithelium/retina whole mount. Here we displayed features of retina degeneration including rosette formation in mice and developed a morphological analytic assessment using sclerochoroid/retina pigment epithelium/retina whole mounts.
Methods: SI was intraperitoneally injected in Sprague-Dawley (SD) rats and C57BL/6J mice using a single dose (50 mg/kg) or with a dose range (10 to 50 mg/kg) in BALB/C mice. Rat retinas were investigated up to 2-week post-injection by histology and whole mounts, and mouse retinas were investigated up to 3-week post-injection by histology, fluorescent staining of sections and/or sclerochoroid/retina pigment epithelium/retina whole mounts for the morphological evaluations of the SI-induced retina damage.
Results: SI-induced retina damage caused photoreceptor (PR) degeneration and rosettes/folds formation, as well as retina pigment epithelium degeneration and inward migration. It displayed mixed nuclei from choroid to PRs, due to layer disorganization, as shown by single horizontal images in the sclerochoroid/retina pigment epithelium/retina whole mounts. Measurement of the PR rosette area induced by SI provided a quantitative, morphological evaluation of retina degeneration.
Conclusions: The method of non-separate sclerochoroid/retina pigment epithelium/retina whole staining and mount allows us to observe the integral horizontal view of damage from sclera to PR layers, which cannot be addressed by using sectioned and separate whole mount methods. This method is applicable for morphological evaluation of retina damage, especially in the subretinal layer.
Background: Retinal degeneration is a common feature of several retinal diseases, such as retinitis pigmentosa and age-related macular degeneration (AMD). In this respect, experimental models of photo-oxidative damage reproduce faithfully photoreceptor loss and many pathophysiological events involved in the activation of retinal cell degeneration. Therefore, such models represent a useful tool to study the mechanisms related to cell death. Their advantage consists in the possibility of modulating the severity of damage according to the needs of the experimenter. Indeed, bright light exposure could be regulated in both time and intensity to trigger a burst of apoptosis in photoreceptors, allowing the study of degenerative mechanisms in a controlled fashion, compared to the progressive and slower rate of death in other genetic models of photoreceptor degeneration.
Methods: Here, an exemplificative protocol of bright light exposure in albino rat is described, as well as the main outcomes in retinal function, photoreceptor death, oxidative stress, and inflammation, which characterize this model and reproduce the main features of retinal degeneration diseases.
Discussion: Models of photo-oxidative damage represent a useful tool to study the mechanisms responsible for photoreceptor degeneration. In this respect, it is important to adapt the exposure paradigm to the experimental needs, and the wide range of variables and limitations influencing the final outcomes should be considered to achieve proper results.
Trial Registration: None.
