Adhesion of Leukocytes to Cerebral Venules Precedes Neuronal Cell Death and Is Sufficient to Trigger Tissue Damage After Cerebral Ischemia

So you described a problem, hinted at a solution, but didn't tell us where we can find that solution so we can get our doctors to use it. Useless.

Adhesion of Leukocytes to Cerebral Venules Precedes Neuronal Cell Death and Is Sufficient to Trigger Tissue Damage After Cerebral Ischemia

Rebecca Isabella Sienel^1,2†, Hiroharu Kataoka^3†, Seong-Woong Kim⁴, Fatma Burcu Seker^1,2 and Nikolaus Plesnila^1,2*

• ¹Laboratory of Experimental Stroke Research, Institute for Stroke and Dementia Research (ISD), University of Munich Medical Center, Munich, Germany
• ²Munich Cluster of Systems Neurology (Synergy), Munich, Germany
• ³Department of Neurosurgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
• ⁴Department of Neurosurgery, University of Giessen, Giessen, Germany

Background: Leukocytes contribute to tissue damage after cerebral ischemia; however, the mechanisms underlying this process are still unclear. This study investigates the temporal and spatial relationship between vascular leukocyte recruitment and tissue damage and aims to uncover which step of the leukocyte recruitment cascade is involved in ischemic brain injury.

Methods: Male wild-type, ICAM-1-deficient, anti-CD18 antibody treated, or selectin-deficient [fucusyltransferase (FucT IV/VII^−/−)] mice were subjected to 60 min of middle cerebral artery occlusion (MCAo). The interaction between leukocytes and the cerebrovascular endothelium was quantified by in vivo fluorescence microscopy up to 15 h thereafter. Temporal dynamics of neuronal cell death and leukocyte migration were assessed at the same time points and in the same tissue volume by histology.

Results: In wild-type mice, leukocytes started to firmly adhere to the wall of pial postcapillary venules two hours after reperfusion. Three hours later, neuronal loss started and 13 h later, leukocytes transmigrated into brain tissue. Loss of selectin function did not influence this process. Application of an anti-CD18 antibody or genetic deletion of ICAM-1, however, significantly reduced tight adhesion of leukocytes to the cerebrovascular endothelium (-60%; p < 0.01) and increased the number of viable neurons in the ischemic penumbra by 5-fold (p < 0.01); the number of intraparenchymal leukocytes was not affected.

Conclusions: Our findings suggest that ischemia triggers only a transient adhesion of leukocytes to the venous endothelium and that inhibition of this process is sufficient to partly prevent ischemic tissue damage.

Introduction

Ischemic stroke is one of the most frequent causes of death and disability worldwide (1, 2). Current therapies include thrombolysis with rtPA or rtPA in combination with mechanical thrombectomy (3–5); however, only up to 30% of stroke patients are eligible for these interventions (5). Hence, there is an ongoing and urgent need for the development of novel therapeutic options for the 70% of stroke patients who do not receive any causal treatment.

For more than four decades, inflammation has been recognized as a major pathomechanism, which is responsible for brain injury following ischemic stroke (6–11). A plethora of elegant experimental and clinical studies discovered that ischemia triggers an acute innate immune response within the brain parenchyma which results in the production of inflammatory cytokines, the upregulation of adhesion molecules on endothelial cells, and the subsequent recruitment of granulocytes and monocytes into ischemic tissue within the first few hours and days after vessel occlusion (12–19). Later on, T-lymphocytes invade the infarcted tissue and may cause further damage (20–22). Despite these impressive steps forward in our understanding of postischemic inflammation, none of the above-mentioned mechanisms translated into a viable therapeutic approach for stroke patients (14). Hence, reevaluation of previous experimental findings and identification of significant knowledge gaps may help identify so far unexplored or neglected therapeutic principles related to postischemic leukocyte recruitment.

Histopathological studies in human tissue, nonhuman primates, and rodents and investigations using radioactively labeled leukocytes in humans and experimental animal models univocally demonstrate that cerebral ischemia is associated with accumulation of polymorphonuclear leukocytes (PMNs) or granulocytes in the brain (10, 11, 23–31). According to experimental studies using radioactively labeled leukocytes or direct visualization of leukocytes by intravital microscopy, recruitment of leukocytes to the ischemic brain starts within the first 2 h after the onset of ischemia (9, 32–35), a time course also supported by investigations in stroke patients (30, 36). The nature of this accumulation seems to follow two different mechanisms: leukocytes may plug capillaries and arterioles during ischemia, thus participating in the so-called no reflow phenomenon (33, 37, 38), and/or they may adhere to the endothelium of postcapillary venules due to upregulation of adhesion molecules (27, 32, 34, 35). No matter which concept of accumulation is followed, most laboratories report that depletion of granulocytes or inhibition or deletion of adhesion molecules reduces ischemic brain damage and improves outcome following experimental stroke (27, 33, 37, 39–50). Hence, there is general agreement that leukocytes accumulate in the brain within the first few hours after cerebral ischemia and inhibition or deletion of adhesion molecules reduce ischemic brain damage. Beyond this generally accepted concept, however, many crucial issues on the role of leukocytes for ischemic tissue damage are still unsolved or highly debated (17, 27, 28). One of the main reasons for this discussion is that most of the above-cited studies used static, histopathological techniques to investigate leukocytes after stroke. Therefore, our knowledge about the dynamics of adhesion, transmigration, and accumulation of leukocytes in the brain after a stroke and how these processes are related to tissue damage is still surprisingly limited.

Technically, leukocyte dynamics after stroke can be addressed by longitudinal in vivo imaging; however, the few studies using this approach either focused on the very first hours after cerebral ischemia, a time when the neuronal injury was not yet present, or on time points later than 24 h after stroke, when the ischemic injury had already occurred (27, 32–35, 51). Consequently, we still do not know whether leukocytes are present in the brain when ischemic damage occurs or whether leukocytes are present at the site of injury (6, 7, 17, 19–21, 52–57). To answer these two important issues, we investigated the full-time course and sequence of leukocyte accumulation to the ischemic brain in parallel with neuronal cell death and tried to decipher which part of the leukocyte adhesion cascade may be involved in ischemic tissue damage.

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