A New and Reliable Guide for Studies of Neuronal Loss Based on Focal Lesions and Combinations of In Vivo and In Vitro Approaches

Abstract

In this study, we describe a simple and reliable method to study neuroprotective effects in living and organized neural tissue. This method, which was based on retinal explants for in vivo focal lesions, was conceived as a collection of modular procedures, which can be customized for particular demands. With this model, it is possible to combine immunohistochemistry with image data analysis to track the two- or three-dimensional redistribution of proteins as a time/space function of primary cell loss. At the same time, it is possible to finely control the exposure of the tissue to specific drugs and molecules. In order to illustrate the use of the proposed method, we tested the effects of two different nanotube compounds on retinal explant viability. Transcriptome analyses can be separately performed in the lesion focus and penumbra with laser capture microdissection followed by polymerase chain reaction analyses. In addition, other common experimental drawbacks, such as high individual variance, are eliminated. With intraocular injections, treatments can be verified in vivo, with one eye serving as the experimental tissue and the other serving as the control tissue. In summary, we describe a flexible and easy method, which can be useful in combination with a broad variety of recently developed neuroprotective strategies, to study neurodegeneration.

VDAC1 regulates neuronal cell loss after retinal trauma injury by a mitochondria-independent pathway

“…The procedures for the mechanical trauma experiments were previously described [ 53 ]. Briefly, animals were anesthetized by intraperitoneal injection of a mixture of ketamine (75 mg/kg) and xylazine (10 mg/kg) and submitted to six local mechanical lesions in the retina, performed using a thin needle (28-gauge, 12.7 mm).…”

Complementary research models and methods to study axonal regeneration in the vertebrate retinofugal system

“…Protocols for culturing retinal explants from chicken embryos have been described by different research groups (Hoff et al 1999;Thangaraj et al 2011;Paschon et al 2013;Shirazi Fard et al 2015), however, until now, chick explants have only rarely been employed to study axonal regeneration (Thanos et al 1992). Likewise, porcine retinal explants are mostly used to analyze neuroprotection and cell survival, rather than neurite outgrowth MartinezFernandez;de la Camara et al 2014;Taylor et al 2014;Bell et al 2016).…”