Elsevier

Neuroscience

Volume 279, 24 October 2014, Pages 139-154
Neuroscience

Inflammatory cell recruitment after experimental thromboembolic stroke in rats

https://doi.org/10.1016/j.neuroscience.2014.08.023Get rights and content

Highlights

  • Ischemic stroke leads to significant neutrophil and monocyte/macrophage recruitment.

  • Microglia remain numerically unchanged but shifted to an immunoreactive phenotype.

  • Newly characterized T and B cell subpopulations accumulate in ischemia-affected brain.

  • Precisely because mimicking human stroke, embolic models qualify for future research.

Abstract

Inflammatory mechanisms were recently identified as contributors to delayed neuronal damage after ischemic stroke. However, therapeutic strategies are still lacking, probably related to the outstanding standardization on inflammatory cell recruitment emerging from predominantly artificial stroke models, and the uncertainty on functional properties of distinct subpopulations. Using a rodent model of stroke that closely reflects human embolic ischemia, this study was focused on the local recruitment of immunoreactive cells as well as their functional and regional characterization. Wistar rats underwent thromboembolic middle cerebral artery occlusion, followed by intravenous injection of the blood–brain barrier permeability marker fluorescein-conjugated albumin at 24 h. One hour later, brain tissue was subjected to multi-parameter flow cytometry and Pappenheim staining to characterize cells invaded into the ischemia-affected hemisphere, compared to the contralateral side. Immunofluorescence labeling was applied to explore the distribution patterns of recruited cells and their spatial relationships with the vasculature. One day after ischemia onset, a 6.12-fold increase of neutrophils and a 5.43-fold increase of monocytes/macrophages was found in affected hemispheres, while these cells exhibited enhanced major histocompatibility complex class II expression and allocation with vessels exhibiting impaired blood–brain barrier integrity. Microglia remained numerically unaltered in ischemic hemispheres, but shifted to an activated phenotype indicated by CD45/CD86 expression and morphological changes toward an ameboid appearance in the bordering zone. Ischemia caused an increase of lymphoid cells in close vicinity to the affected vasculature, while further analyses allowed separation into natural killer cells, natural killer T cells, T cells (added by an unconventional CD11b+/CD3+ population) and two subpopulations of B cells. Taken together, our study provides novel data on the local inflammatory response to experimental thromboembolic stroke. As concomitantly present neutrophils, monocytes/macrophages and lymphoid cells in the early stage after ischemia induction correspond to changes seen in human stroke, future stroke research should preferably use animal models with relevance for clinical translation.

Introduction

Despite intensified experimental and clinical research, stroke remains a major cause of death worldwide and contributes to an enormous financial burden (Donnan et al., 2008). On the cellular level, the gleaned pathophysiological knowledge on ischemic stroke enabled a more comprehensive perspective of tissue damage considering time-dependent mechanisms of neuronal affection (Dirnagl et al., 1999). While excitotoxicity causes cell death mainly within minutes to hours after ischemia onset, inflammation became increasingly important since this mechanism peaks later, appropriating a clinically relevant target for interventions (Dirnagl et al., 1999, Endres et al., 2008).

Based on studies using predominantly histological techniques, it is generally accepted that the inflammatory response includes infiltration of myeloid cells and lymphocytes into the ischemia-affected area, complemented by local glial activation (Kochanek and Hallenbeck, 1992, Tomita and Fukuuchi, 1996, Stoll et al., 1998, Danton and Dietrich, 2003, Wang et al., 2007, Jin et al., 2010, Denes et al., 2010). Using flow cytometry and fluorescence-activated cell sorting in rodent brains subjected to experimental ischemia, Campanella et al., 2002, Stevens et al., 2002 and Gelderblom et al. (2009) have investigated involved cellular populations and their temporal characteristics in more detail. Thereby, rising numbers of neutrophils and macrophages in the ischemic hemisphere were found to occur within hours continuing until day 2 and 3 after ischemia onset, while lymphoid cells appeared delayed in time and in a much lower amount. After a first attempt to explore the functional relevance of cellular infiltration by Weston et al. (2007), demonstrating an association of numerically increased myeloid cells (i.e. neutrophils) with larger infarct volumes after experimental ischemic stroke in rats, two recent studies addressed the role of T lymphocytes in transient and permanent cerebral ischemia in mice, yielding contradicting neuroprotective (Liesz et al., 2009) and detrimental (Kleinschnitz et al., 2010) properties (Magnus et al., 2012).

Even prior to these reports, a discussion emerged about the role of ischemia-induced inflammatory processes, while both deleterious consequences (such as cytokine-mediated cell death) and beneficial effects (i.e. the initiation of neuronal recovery and plasticity) were discussed (Stoll et al., 1998, Chamorro and Hallenbeck, 2006, Endres et al., 2008, Kriz and Lalancette-Hébert, 2009, Lakhan et al., 2009). The remaining uncertainty might hamper the development of drugs influencing the inflammatory response following ischemic stroke. The situation is further complicated by recent data indicating that the course of cellular recruitment critically depends on the applied animal model: Comparing filament-based models in mice with varying times of ischemia, Zhou et al. (2013) found an increasing number of neutrophils during the first 5 days after permanent ischemia, but a decreasing number following transient ischemia. Analyses of microglia activation at 24 h revealed that permanent ischemia led to a numerical increase of cells while transient ischemia caused cell counts not different from sham-operated animals. A very recent report by Möller et al. (2014) focused on ischemic consequences in spontaneous hypertensive rats, which became attractive due to the naturally existing arterial hypertension as a shared risk factor for stroke in humans. In these rats, permanent cerebral ischemia caused a neutrophil accumulation at day 1 followed by a decline at day 4, which was also found for monocytes – quite different from earlier studies.

These observations together with the ongoing attempts to improve preclinical stroke research with emphasis on translational issues (Fisher et al., 2009, Young et al., 2007) lead to the question on details about the local inflammatory response in a rodent model of stroke that is comparable to the pathophysiology of stroke in man. The present study applied for the first time flow cytometric analyses combined with in situ immunohistochemical staining to characterize the early inflammatory cell recruitment after thromboembolic stroke in rats. This model is currently believed to best mimic the human condition due to shared mechanisms of stroke formation (Durukan and Tatlisumak, 2007, Bräuninger and Kleinschnitz, 2009): a blood clot running inside of the internal carotid artery that finally occludes the middle cerebral artery.

Section snippets

Experimental design

All experiments involving animals had been conducted according to the European Communities Council Directive (86/609/EEC) and were approved by local authorities (Landesdirektion Sachsen, Leipzig, Germany, reference number TVV-34/11). Generally, efforts were made to minimize the number of animals in total and suffering of animals, which were housed in a temperature and humidity controlled room with 12 h of light/dark cycle and free access to food and water. The here presented data originated from

Results

The applied thromboembolic stroke model led to neurobehavioral alterations as evaluated by a mean Menzies score of 2.7 ± 0.7 at 4 h as well as 2.8 ± 0.9 at 24 h after ischemia induction (p = 0.602 between the time points). Therefore, animals fulfilled the pre-defined study inclusion criterion of a sufficient neurobehavioral deficit indicating right-hemispheric focal cerebral ischemia in the middle cerebral artery territory as a pre-condition for the intended cellular characterization at day 1.

Discussion

The present study aimed on the cellular inflammatory response during the first day after cerebral ischemia using a rat model of thromboembolic stroke, which has been chosen to closely mimic the human pathophysiology (Durukan and Tatlisumak, 2007, Young et al., 2007). With respect to earlier studies which applied flow cytometry for characterization of invading cells into the ischemic brain as induced by filament insertion or electrocoagulation (Campanella et al., 2002, Stevens et al., 2002,

Conclusions

Our study provides for the first time details on the inflammatory cell recruitment within ischemia-affected brain regions after thromboembolic stroke in rats, a model that has become attractive concerning translational aspects. While microglia failed to increase numerically in the ischemia-affected hemisphere, flow cytometry indicated a clear shift to an immunoreactive state, which was supported by histochemically visualized morphological alterations toward an ameboid configuration in the

Conflict of interest

The authors declare that there is no conflict of interest.

Acknowledgments

The authors would like to thank Dr. Petra Fink-Sterba and Ms. Sigrid Weisheit (Medical Experimental Centre, University of Leipzig, Leipzig) for animal care. Ms. Ulrike Scholz (Fraunhofer Institute for Cell Therapy and Immunology, Dept. Cell Engineering/GLP, Leipzig) is acknowledged for excellent technical assistance.

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