Assessing phagocytic clearance of cell death in experimental stroke by ligatable fluorescent probes

J Vis Exp. 2014 May 27;(87):51261. doi: 10.3791/51261.

Abstract

We describe a new histochemical approach for visualization of phagocytic clearance in focal brain ischemia. The approach permits the study of elimination of dead cells in stroke by waste-management phagocytes of any cellular lineage. Although numerous cells of different origins that are capable of phagocytosis are present in ischemic brain, only part of them actively engulf and digest cell corpses. The selective visualization, quantification and analysis of such active phagocytic waste-management are helpful in assessing brain response to ischemia. Efficient cell death clearance is important for brain recovery from ischemic injury, as it opens the way for the subsequent regenerative processes. The failure to clean the corpses would result in a toxic reaction caused by non-degraded DNA and proteins. The described procedure uses fluorescent probes selectively ligated by a viral topoisomerase to characteristic DNA breaks produced in all phagocytes during engulfment and digestion of cells irreversibly damaged by ischemia. The method is a new tool for the investigation of brain reaction to ischemic injury.

Publication types

  • Research Support, American Recovery and Reinvestment Act
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Video-Audio Media

MeSH terms

  • Animals
  • Brain / blood supply
  • Brain / pathology
  • Brain Ischemia / genetics
  • Brain Ischemia / pathology
  • Cell Death
  • DNA Breaks*
  • Disease Models, Animal
  • Fluorescent Dyes / chemistry*
  • Histocytochemistry / methods
  • Oligonucleotide Probes / chemistry*
  • Phagocytosis
  • Rats
  • Stroke / genetics
  • Stroke / pathology*

Substances

  • Fluorescent Dyes
  • Oligonucleotide Probes