doi: 10.1161/hs0202.103399
(Stroke. 2002;33:586.)
© 2002 American Heart Association, Inc.
Original Contributions |
Flow Cytometric Analysis of Inflammatory Cells in Ischemic Rat Brain
From the Schering-Plough Research Institute, Milan, Italy.
Correspondence to Massimiliano Beltramo, PhD, Schering-Plough Research Institute, Via Olgettina 58, 20132 Milan, Italy. E-mail massimiliano.beltramo{at}spcorp.com
Background and Purpose— Inflammation plays a key role in cerebral ischemia through activation of microglia and infiltration by leukocytes. Flow cytometry is a well-established method for quantitative and qualitative analysis of inflammatory cells. However, this technique has not been applied to the study of cerebral ischemia inflammation. The aim of this study was to establish a flow cytometric method to measure inflammatory cells in ischemic brain.
Methods— To perform flow cytometry on brain tissue, we developed 2 cell-isolation methods based on different mechanical dissociation and Percoll gradient separation techniques. The methods were tested on a rat model of permanent middle cerebral artery occlusion. Morphological and immunophenotypic analyses, with the use of anti-CD11b, anti-CD45, and 
ß T-cell receptor antibodies, were employed to identify and quantify inflammatory cells.
Results— Both methods gave consistent results in terms of yield and reproducibility. The cell suspension contained granulocytes, macrophages, lymphocytes, and neural cells. Morphological and immunophenotypic analyses enabled the identification of a cell-scatter gate (R1a) enriched in inflammatory cells. With both methods, a higher number of events in R1a were recorded in the ischemic hemisphere than in the nonischemic hemisphere (P
0.001). CD11b, CD45, and ß T-cell receptor staining confirmed that this augmentation was a reflection of the increase in the number of granulocytes, cells of the monocytic lineage, and lymphocytes.
Conclusions— Quantitative flow cytometric analysis of ischemic rat brain is feasible and provides a reliable and rapid assay to assess neuroinflammation in experimental models of brain ischemia.
Key Words: cerebral ischemia • flow cytometry • granulocytes • inflammation • microglia • rats
This article has been cited by other articles:
 |
|
 |
|
  L. E. Gralinski, S. L. Ashley, S. D. Dixon, and K. R. Spindler Mouse Adenovirus Type 1-Induced Breakdown of the Blood-Brain Barrier J. Virol., September 15, 2009; 83(18): 9398 - 9410. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Gelderblom, F. Leypoldt, K. Steinbach, D. Behrens, C.-U. Choe, D. A. Siler, T. V. Arumugam, E. Orthey, C. Gerloff, E. Tolosa, et al. Temporal and Spatial Dynamics of Cerebral Immune Cell Accumulation in Stroke Stroke, May 1, 2009; 40(5): 1849 - 1857. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. D'Mello, T. Le, and M. G. Swain Cerebral Microglia Recruit Monocytes into the Brain in Response to Tumor Necrosis Factor{alpha} Signaling during Peripheral Organ Inflammation J. Neurosci., February 18, 2009; 29(7): 2089 - 2102. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Muhammad, W. Barakat, S. Stoyanov, S. Murikinati, H. Yang, K. J. Tracey, M. Bendszus, G. Rossetti, P. P. Nawroth, A. Bierhaus, et al. The HMGB1 Receptor RAGE Mediates Ischemic Brain Damage J. Neurosci., November 12, 2008; 28(46): 12023 - 12031. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. S. Chauhan, D. G. Sterka Jr., D. L. Gray, K. L. Bost, and I. Marriott Neurogenic Exacerbation of Microglial and Astrocyte Responses to Neisseria meningitidis and Borrelia burgdorferi J. Immunol., June 15, 2008; 180(12): 8241 - 8249. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. S. Shaftel, T. J. Carlson, J. A. Olschowka, S. Kyrkanides, S. B. Matousek, and M. K. O'Banion Chronic Interleukin-1{beta} Expression in Mouse Brain Leads to Leukocyte Infiltration and Neutrophil-Independent Blood Brain Barrier Permeability without Overt Neurodegeneration J. Neurosci., August 29, 2007; 27(35): 9301 - 9309. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. C. Daginakatte and D. H. Gutmann Neurofibromatosis-1 (Nf1) heterozygous brain microglia elaborate paracrine factors that promote Nf1-deficient astrocyte and glioma growth Hum. Mol. Genet., May 1, 2007; 16(9): 1098 - 1112. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Zhou, R. A. Gault, T. R. Kozel, and W. J. Murphy Protection from Direct Cerebral Cryptococcus Infection by Interferon-{gamma}-Dependent Activation of Microglial Cells J. Immunol., May 1, 2007; 178(9): 5753 - 5761. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. W. Newcomb, S. Demaria, Y. Lukyanov, Y. Shao, T. Schnee, N. Kawashima, L. Lan, J. K. Dewyngaert, D. Zagzag, W. H. McBride, et al. The Combination of Ionizing Radiation and Peripheral Vaccination Produces Long-term Survival of Mice Bearing Established Invasive GL261 Gliomas Clin. Cancer Res., August 1, 2006; 12(15): 4730 - 4737. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. D. Huber, C. R. Campos, K. S. Mark, and T. P. Davis Alterations in blood-brain barrier ICAM-1 expression and brain microglial activation after {lambda}-carrageenan-induced inflammatory pain Am J Physiol Heart Circ Physiol, February 1, 2006; 290(2): H732 - H740. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. V. Arumugam, J. W. Salter, J. H. Chidlow, C. M. Ballantyne, C. G. Kevil, and D. N. Granger Contributions of LFA-1 and Mac-1 to brain injury and microvascular dysfunction induced by transient middle cerebral artery occlusion Am J Physiol Heart Circ Physiol, December 1, 2004; 287(6): H2555 - H2560. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Nakase, G. Sohl, M. Theis, K. Willecke, and C. C.G. Naus Increased Apoptosis and Inflammation after Focal Brain Ischemia in Mice Lacking Connexin43 in Astrocytes Am. J. Pathol., June 1, 2004; 164(6): 2067 - 2075. [Abstract] [Full Text] [PDF]
|
|