title: Feet on the ground: Physical support of the inner retina is a strong determinant for cell survival and structural preservation in vitro.
abstract: OBJECTIVE
  The purpose of this study was to explore the importance of local physical tissue support for homeostasis in the isolated retina.
  
  
  METHODS
  Full-thickness retinal sheets were isolated from adult porcine eyes. Retinas were cultured for 5 or 10 days using the previously established explant protocol with photoreceptors positioned against the culture membrane (porous polycarbonate) or the Müller cell endfeet and inner limiting membrane (ILM) apposed against the membrane. The explants were analyzed morphologically using hematoxylin and eosin staining, immunohistochemistry, TUNEL labeling, and transmission electron microscopy (TEM).
  
  
  RESULTS
  Standard cultures displayed a progressive loss of retinal lamination and extensive cell death, with activated, hypertrophic Müller cells. In contrast, explants cultured with the ILM facing the membrane displayed a maintenance of the retinal laminar architecture, and a statistically significant attenuation of photoreceptor and ganglion cell death. Transmission electron microscopy revealed intact synapses as well as preservation of normal cellular membrane structures. Immunohistochemistry showed no signs of Müller cell activation (glial fibrillary acidic protein [GFAP]), with maintained expression of important metabolic markers (glutamine synthetae [GS], bFGF).
  
  
  CONCLUSIONS
  Providing physical support to the inner but not the outer retina appears to prevent the tissue collapse resulting from perturbation of the normal biomechanical milieu in the isolated retinal sheet. Using this novel paradigm, gliotic reactions are attenuated and metabolic processes vital for tissue health are preserved, which significantly increases neuronal cell survival. This finding opens up new avenues of adult retinal tissue culture research and increases our understanding of pathological reactions in biomechanically related conditions in vivo.
authors.affiliation: Department of Ophthalmology, Lund University Hospital, Lund, Sweden.
authors.forename: Linnéa
authors.initials: L
authors.lastname: Taylor
authors.forename: Karin
authors.initials: K
authors.lastname: Arnér
authors.forename: Ingrid Holmgren
authors.initials: IH
authors.lastname: Taylor
authors.forename: Fredrik
authors.initials: F
authors.lastname: Ghosh
mesh_terms: D000818:Animals; D017209:Apoptosis; D002454:Cell Differentiation; D002470:Cell Survival; D002478:Cells, Cultured; D063928:Ependymoglial Cells / Q000378:metabolism / Q000648:ultrastructure*; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D046529:Microscopy, Electron, Transmission; D012160:Retina / Q000378:metabolism / Q000648:ultrastructure*; D013552:Swine
chemical_list: D005904:Glial Fibrillary Acidic Protein
keywords: Müller cell; neuron-glia interactions; photoreceptors; retinal biomechanics; tissue culture