title: Comparison of different quantification methods to determine hippocampal damage after cerebral ischemia.
abstract: BACKGROUND
  Experimental stroke studies use multiple techniques to evaluate histopathological damage. Unfortunately, sensitivity and reproducibility of these techniques are poorly characterized despite pivotal influence on results.
  
  
  METHODS
  The present study compared several quantification methods to differentiate between two severities of global cerebral ischemia and reperfusion. Male Sprague-Dawley rats were randomized to moderate (10min) or severe (14min) ischemia by bilateral carotid occlusion (BCAO) with hemorrhagic hypotension. Neuronal cell count was determined in hippocampus at bregma -3.14mm and -3.8mm on day 3 and 28 post insult by counting neurons in the whole CA1 or in one to three defined regions of interest (ROI) placed in NeuN and Fluoro-Jade B stained sections.
  
  
  RESULTS
  In healthy rats hippocampal neurons were arranged uniformly, while distribution became inhomogeneous after ischemia. The number of NeuN and Fluoro-Jade B positive cells was dependent on localization. Differences between ischemia severities became more prominent at 28 days compared to 3 days. Fluoro-Jade B positive cell count increased at 28 days, staining rather injured not dying neurons.
  
  
  METHODS
  Placement of counting windows has a major influence on extent of differences between degree of neuronal injury and variations within groups.
  
  
  CONCLUSIONS
  The investigated quantification methods result in inconsistent information on the degree of damage. To obtain consistent and reliable results observation period should be extended beyond 3 days. Due to inhomogeneous distribution of viable neurons in CA1 after ischemia neuronal counting should not be performed in a single ROI window, but should be performed in multiple ROIs or the whole CA1 band.
authors.affiliation: Department of Anesthesiology, Medical Center of Johannes Gutenberg-University, Mainz, Germany; Department of Anesthesiology, Union Hospital, Huazhong University of Science and Technology, Wuhan, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Wang
authors.affiliation: Institute of Medical Biostatistics, Epidemiology and Informatics, Medical Center of the Johannes Gutenberg-University, Mainz, Germany.
authors.forename: Antje
authors.initials: A
authors.lastname: Jahn-Eimermacher
authors.affiliation: Department of Anesthesiology, Medical Center of Johannes Gutenberg-University, Mainz, Germany.
authors.forename: Melanie
authors.initials: M
authors.lastname: Brückner
authors.affiliation: Department of Anesthesiology, Medical Center of Johannes Gutenberg-University, Mainz, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Werner
authors.affiliation: Department of Anesthesiology, Medical Center of Johannes Gutenberg-University, Mainz, Germany.
authors.forename: Kristin
authors.initials: K
authors.lastname: Engelhard
authors.affiliation: Department of Anesthesiology, Medical Center of Johannes Gutenberg-University, Mainz, Germany. Electronic address: thal@uni-mainz.de.
authors.forename: Serge C
authors.initials: SC
authors.lastname: Thal
mesh_terms: D000818:Animals; D034961:Antigens, Nuclear / Q000378:metabolism; D048588:Benzoxazines; D002545:Brain Ischemia / Q000473:pathology*; D056547:CA1 Region, Hippocampal / Q000473:pathology*; D002452:Cell Count; D004396:Coloring Agents; D004195:Disease Models, Animal; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006651:Histocytochemistry / Q000379:methods*; D020300:Intracranial Hemorrhages / Q000473:pathology; D019585:Intracranial Hypotension / Q000473:pathology; D008297:Male; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000473:pathology; D011897:Random Allocation; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D012720:Severity of Illness Index; D013997:Time Factors
chemical_list: D034961:Antigens, Nuclear; D048588:Benzoxazines; D004396:Coloring Agents; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009419:Nerve Tissue Proteins; C546339:Rbfox3 protein, rat; C435731:fluoro jade; C028911:cresyl violet
keywords: Brain damage; Brain ischemia; Hippocampus; Histology; Morphometry; Quantification