The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two mice can reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

Degeneration of retinal pigment epithelium (RPE) is one of the most critical phenotypic changes of age-related macular degeneration (AMD), the leading cause of vision loss in the elderly. While cultured polarized RPE cells with original properties are valuable in in vitro models to study RPE biology and the consequences of genetic and/or pharmacological manipulations, the procedure to establish mouse primary PRE cell culture or pluripotent stem cell-derived RPE cells is time-consuming and yields a limited number of cells. Thus, establishing a mouse in situ RPE culture system is highly desirable. Here we describe a novel and efficient method for RPE explant culture that allows for obtaining biologically relevant RPE cells in situ. These RPE explants (herein referred to as RPE flatmounts) are viable in culture for at least 7 days, can be efficiently transduced with adenoviral constructs, and/or treated with a variety of drugs/chemicals followed by downstream analysis of the signaling pathways/biological processes of interest, such as assessment of the autophagy flux, inflammatory response, and receptor tyrosine kinases stimulation. This method of RPE explant culture is highly beneficial for pharmacological and mechanistic studies in the field of RPE biology and AMD research.

Vascular cognitive impairment and dementia (VCID) results from vascular brain injury. Given VCID's high incidence, which is expected to continue rising as the population ages, it is critical to establish a robust animal model for the disease. This paper presents a novel method of creating a mouse model of VCID that is based on asymmetric bilateral common carotid artery stenosis, which mimics human chronic cerebral hypoperfusion caused by carotid atherosclerosis. Briefly, common carotid arteries (CCAs) are ligated to different gauge needles (32 G for the right CCA and 34 G for the left CCA) using 7-0 silk sutures followed by immediate needle removal. The remaining suture rings cause persistent blood flow reduction and long-term cognitive impairment associated with white matter injury, microinfarcts, and reactive gliosis, thus closely mimicking the pathogenesis of VCID. Importantly, in this needle model, the clinical representations do not revert with time, providing reliable long-term cognitive impairment. Moreover, the survival rate 24 weeks post surgery was 81.6%, which is higher compared to the other established models of VCID with a similar level of blood flow reduction. Additional advantages include low material cost and compatibility with MRI to monitor brain injury in live animals since no metal is implanted. The main challenge in employing the needle model of VCID is the requirement for developing advanced surgical skills since mouse CCAs are less than 0.6 mm in diameter and are very fragile. High-quality visual representation of the surgery will thus help researchers to master this technique and advance our understanding of VCID, potentially leading to the development of novel therapeutic modalities to decrease the devastating cognitive decline associated with VCID.