title: Automatic Counting of Microglial Cells in Healthy and Glaucomatous Mouse Retinas.
abstract: Proliferation of microglial cells has been considered a sign of glial activation and a hallmark of ongoing neurodegenerative diseases. Microglia activation is analyzed in animal models of different eye diseases. Numerous retinal samples are required for each of these studies to obtain relevant data of statistical significance. Because manual quantification of microglial cells is time consuming, the aim of this study was develop an algorithm for automatic identification of retinal microglia. Two groups of adult male Swiss mice were used: age-matched controls (naïve, n = 6) and mice subjected to unilateral laser-induced ocular hypertension (lasered; n = 9). In the latter group, both hypertensive eyes and contralateral untreated retinas were analyzed. Retinal whole mounts were immunostained with anti Iba-1 for detecting microglial cell populations. A new algorithm was developed in MATLAB for microglial quantification; it enabled the quantification of microglial cells in the inner and outer plexiform layers and evaluates the area of the retina occupied by Iba-1+ microglia in the nerve fiber-ganglion cell layer. The automatic method was applied to a set of 6,000 images. To validate the algorithm, mouse retinas were evaluated both manually and computationally; the program correctly assessed the number of cells (Pearson correlation R = 0.94 and R = 0.98 for the inner and outer plexiform layers respectively). Statistically significant differences in glial cell number were found between naïve, lasered eyes and contralateral eyes (P<0.05, naïve versus contralateral eyes; P<0.001, naïve versus lasered eyes and contralateral versus lasered eyes). The algorithm developed is a reliable and fast tool that can evaluate the number of microglial cells in naïve mouse retinas and in retinas exhibiting proliferation. The implementation of this new automatic method can enable faster quantification of microglial cells in retinal pathologies.
authors.affiliation: Department of Neurobiology, Barrow Neurological Institute, St. Joseph's Hospital and Medical Center, Phoenix, Arizona, United States of America.
authors.forename: Pablo
authors.initials: P
authors.lastname: de Gracia
authors.affiliation: Instituto de Investigaciones Oftalmológicas Ramón Castroviejo, Universidad Complutense de Madrid, Madrid, Spain.|Facultad de Óptica y Optometría, Universidad Complutense de Madrid, Madrid, Spain.
authors.forename: Beatriz I
authors.initials: BI
authors.lastname: Gallego
authors.affiliation: Instituto de Investigaciones Oftalmológicas Ramón Castroviejo, Universidad Complutense de Madrid, Madrid, Spain.|Facultad de Medicina, Universidad Complutense de Madrid, Madrid, Spain.
authors.forename: Blanca
authors.initials: B
authors.lastname: Rojas
authors.affiliation: Instituto de Investigaciones Oftalmológicas Ramón Castroviejo, Universidad Complutense de Madrid, Madrid, Spain.|Facultad de Óptica y Optometría, Universidad Complutense de Madrid, Madrid, Spain.
authors.forename: Ana I
authors.initials: AI
authors.lastname: Ramírez
authors.affiliation: Instituto de Investigaciones Oftalmológicas Ramón Castroviejo, Universidad Complutense de Madrid, Madrid, Spain.|Facultad de Óptica y Optometría, Universidad Complutense de Madrid, Madrid, Spain.
authors.forename: Rosa
authors.initials: R
authors.lastname: de Hoz
authors.affiliation: Instituto de Investigaciones Oftalmológicas Ramón Castroviejo, Universidad Complutense de Madrid, Madrid, Spain.|Facultad de Óptica y Optometría, Universidad Complutense de Madrid, Madrid, Spain.
authors.forename: Juan J
authors.initials: JJ
authors.lastname: Salazar
authors.affiliation: Instituto de Investigaciones Oftalmológicas Ramón Castroviejo, Universidad Complutense de Madrid, Madrid, Spain.|Facultad de Medicina, Universidad Complutense de Madrid, Madrid, Spain.
authors.forename: Alberto
authors.initials: A
authors.lastname: Triviño
authors.affiliation: Instituto de Investigaciones Oftalmológicas Ramón Castroviejo, Universidad Complutense de Madrid, Madrid, Spain.|Facultad de Medicina, Universidad Complutense de Madrid, Madrid, Spain.
authors.forename: José M
authors.initials: JM
authors.lastname: Ramírez
mesh_terms: D000465:Algorithms; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D002452:Cell Count / Q000295:instrumentation / Q000379:methods*; D049109:Cell Proliferation; D004195:Disease Models, Animal; D015870:Gene Expression; D005901:Glaucoma / Q000209:etiology / Q000235:genetics / Q000473:pathology*; D007429:Intraocular Pressure; D007834:Lasers / Q000009:adverse effects; D008297:Male; D051379:Mice; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D017628:Microglia / Q000378:metabolism / Q000473:pathology*; D009798:Ocular Hypertension / Q000209:etiology / Q000235:genetics / Q000473:pathology*; D012160:Retina / Q000378:metabolism / Q000473:pathology*
chemical_list: C491049:Aif1 protein, mouse; D015415:Biomarkers; D002135:Calcium-Binding Proteins; D008840:Microfilament Proteins