title: Evaluation of TgH(CX3CR1-EGFP) mice implanted with mCherry-GL261 cells as an in vivo model for morphometrical analysis of glioma-microglia interaction.
abstract: BACKGROUND
  Glioblastoma multiforme is the most aggressive brain tumor. Microglia are prominent cells within glioma tissue and play important roles in tumor biology. This work presents an animal model designed for the study of microglial cell morphology in situ during gliomagenesis. It also allows a quantitative morphometrical analysis of microglial cells during their activation by glioma cells.
  
  
  METHODS
  The animal model associates the following cell types: 1- mCherry red fluorescent GL261 glioma cells and; 2- EGFP fluorescent microglia, present in the TgH(CX3CR1-EGFP) mouse line. First, mCherry-GL261 glioma cells were implanted in the brain cortex of TgH(CX3CR1-EGFP) mice. Epifluorescence - and confocal laser-scanning microscopy were employed for analysis of fixed tissue sections, whereas two-photon laser-scanning microscopy (2P-LSM) was used to track tumor cells and microglia in the brain of living animals.
  
  
  RESULTS
  Implanted mCherry-GL261 cells successfully developed brain tumors. They mimic the aggressive behavior found in human disease, with a rapid increase in size and the presence of secondary tumors apart from the injection site. As tumor grows, mCherry-GL261 cells progressively lost their original shape, adopting a heterogeneous and diffuse morphology at 14-18 d. Soma size increased from 10-52 μm. At this point, we focused on the kinetics of microglial access to glioma tissues. 2P-LSM revealed an intense microgliosis in brain areas already shortly after tumor implantation, i.e. at 30 min. By confocal microscopy, we found clusters of microglial cells around the tumor mass in the first 3 days. Then cells infiltrated the tumor area, where they remained during all the time points studied, from 6-18 days. Microglia in contact with glioma cells also present changes in cell morphology, from a ramified to an amoeboid shape. Cell bodies enlarged from 366 ± 0.0 μm(2), in quiescent microglia, to 1310 ± 146.0 μm(2), and the cell processes became shortened.
  
  
  CONCLUSIONS
  The GL261/CX3CR1 mouse model reported here is a valuable tool for imaging of microglial cells during glioma growth, either in fixed tissue sections or living animals. Remarkable advantages are the use of immunocompetent animals and the simplified imaging method without the need of immunohistochemical procedures.
authors: Laboratório de Tecnologias para Terapia Gênica, ASS 128, ICC Sul, Universidade de Brasília-UnB, Campus Darcy Ribeiro, FAV., Brasília, DF, Brasil, 70910-970. fernando@veterinario.med.br.|Molecular Physiology, Center for Integrative Physiology and Molecular Medicine (CIPMM), University of Saarland, Homburg, Germany. fernando@veterinario.med.br.
authors: Fernando F B
authors: FF
authors: Resende
authors: Molecular Physiology, Center for Integrative Physiology and Molecular Medicine (CIPMM), University of Saarland, Homburg, Germany. Xianshu.Bai@uniklinikum-saarland.de.
authors: Xianshu
authors: X
authors: Bai
authors: Laboratório de Neurofisiologia e Biologia Molecular, Department Morfologia Fisiologia e Patologia Básica, FORP, Universidade de São Paulo - USP, Ribeirão Preto, SP, Brasil, 14040-904. eadelbel@forp.usp.br.
authors: Elaine Aparecida
authors: EA
authors: Del Bel
authors: Molecular Physiology, Center for Integrative Physiology and Molecular Medicine (CIPMM), University of Saarland, Homburg, Germany. frank.kirchhoff@uks.eu.
authors: Frank
authors: F
authors: Kirchhoff
authors: Molecular Physiology, Center for Integrative Physiology and Molecular Medicine (CIPMM), University of Saarland, Homburg, Germany. Anja.Scheller@uniklinikum-saarland.de.
authors: Anja
authors: A
authors: Scheller
authors: Laboratório de Tecnologias para Terapia Gênica, ASS 128, ICC Sul, Universidade de Brasília-UnB, Campus Darcy Ribeiro, FAV., Brasília, DF, Brasil, 70910-970. ricardo.titze@pq.cnpq.br.
authors: Ricardo
authors: R
authors: Titze-de-Almeida
mesh_terms: D000818:Animals; D000073883:CX3C Chemokine Receptor 1; D045744:Cell Line, Tumor; D002540:Cerebral Cortex / Q000378:metabolism / Q000473:pathology / Q000648:ultrastructure*; D004195:Disease Models, Animal; D066246:ErbB Receptors / Q000235:genetics; D005909:Glioblastoma / Q000235:genetics / Q000473:pathology / Q000648:ultrastructure*; D005910:Glioma / Q000235:genetics / Q000473:pathology / Q000648:ultrastructure*; D006801:Humans; D051379:Mice; D017628:Microglia / Q000378:metabolism / Q000473:pathology / Q000648:ultrastructure; D018613:Microscopy, Confocal; D019707:Receptors, Chemokine / Q000235:genetics
chemical_list: D000073883:CX3C Chemokine Receptor 1; C471885:Cx3cr1 protein, mouse; D019707:Receptors, Chemokine; C541485:EGFR protein, mouse; D066246:ErbB Receptors