Experimental & Translational Stroke Medicine - Latest Articles

An experimental Protocol for mimicking Pathomechanisms of Traumatic Brain Injury in Mice

Christiane Albert-Weissenberger — 2012-02-02T00:00:00Z

Traumatic brain injury (TBI) is a result of an outside force causing immediate mechanical disruption of brain tissue and delayed pathogenic events. In order to examine injury processes associated with TBI, a number of rodent models to induce brain trauma have been described. However, none of these models covers the entire spectrum of events that might occur in TBI. Here we provide a thorough methodological description of a straightforward closed head weight drop mouse model to assess brain injuries close to the clinical conditions of human TBI.

Sex-Independent Neuroprotection with Minocycline after Experimental Thromboembolic Stroke

Md Nasrul Hoda — 2011-12-16T00:00:00Z

Background: Minocycline provides neurovascular protection reducing acute cerebral injury. However, it is unclear whether minocycline is effective in females. We tested minocycline in both sexes and aged animals using a novel embolic stroke model in mice that closely mimics acute thromboembolic stroke in humans. Methods: Five groups of mice were subjected to thromboembolic stroke: adult males, aged males, adult females, aged females, and adult ovariectomized females. They were treated with phosphate saline (vehicle) or minocycline (6 mg/kg) immediately after stroke onset. Behavioral outcomes, infarct volumes and cerebral blood flow were assessed. The effect of minocycline on expression and activity of MMP-9 was analyzed. Results: The model resulted in reproducible infarct in the experimental groups. As expected, adult females were significantly more resistant to cerebral ischemic injury than males. This advantage was abolished by aging and ovariectomy. Minocycline significantly reduced the infarct volume (P<0.0001) and also improved neurologic score (P<0.0001) in all groups. Moreover, minocycline treatment significantly reduced mortality at 24 hours post stroke (P=0.037) for aged mice (25% versus 54%). Stroke up-regulated MMP-9 level in the brain, and acute minocycline treatment reduced its expression in both genders (P<0.0001). Conclusion: In a thromboembolic stroke model minocycline is neuroprotective irrespective of mouse sex and age.

Role of blood cell-associated angiotensin II type 1 receptors in the cerebral microvascular response to ischemic stroke during angiotensin-induced hypertension.

Mutsumi Nagai — 2011-11-16T00:00:00Z

Background: Angiotensin II type 1 receptor (AT1R) blockers lower the incidence of ischemic stroke in hypertensive patients and attenuate brain inflammation and injury in animal models. Although AT1R on both blood cells (BC) and vascular endothelial cells (EC) can be activated by angiotensin II (Ang II) to elicit inflammation, little is known about the relative contributions of AT1R expressed on BC and EC to the brain injury responses to ischemia and reperfusion (I/R) in the setting of angiotensin-induced hypertension. Methods: The contributions of BC- and EC-associated AT1R to I/R-induced brain inflammation and injury were evaluated using wild type (WT), AT1aR-/-, and bone marrow chimera mice with either a BC+/EC+ (WT→WT) or BC-/EC+ (AT1aR-/-→WT) distribution of AT1aR. The adhesion of leukocytes and platelets in venules, blood brain barrier (BBB) permeability and infarct volume were monitored in postischemic brain of normotensive and Ang II-induced hypertensive mice. Results: The inflammatory (blood cell adhesion) and injury (BBB permeability, infarct volume) responses were greatly exaggerated in the presence of Ang II-induced hypertension. The Ang II-enhanced responses were significantly blunted in AT1aR-/- mice. A similar level of protection was noted in AT1aR-/- →WT mice for BBB permeability and infarct volume, while less or no protection was evident for leukocyte and platelet adhesion, respectively. Conclusions: BC- and EC-associated AT1aR are both involved in the brain injury responses to ischemic stroke during Ang II-hypertension, with EC AT1aR contributing more to the blood cell recruitment response and BC AT1aR exerting a significant influence on the BBB disruption and tissue necrosis elicited by I/R.

Translational research in sepsis - an ultimate challenge?

Tim Kampmeier — 2011-11-14T00:00:00Z

In the era of evidence-based medicine, large, randomized, controlled, multicenter studies represent the "summit of evidence". In contrast to specialties like cardiology, the majority of randomized, controlled trials in critical care medicine, however, have failed to demonstrate a survival benefit; notably, despite encouraging results from experimental and phase-II clinical studies. The difficulty in translating our theoretical knowledge into successful multicenter randomized, controlled trials and subsequent treatment recommendations may represent one reason, why the mortality of septic shock still averages between 40-60%, although our knowledge about the underlying pathophysiology has considerably increased and international guidelines have widely been implemented. The present article elucidates some of the difficulties in translating research from bench to bedside.

The protective effects of plasma gelsolin on stroke outcome in rats

Huong Le — 2011-11-02T00:00:00Z

Background: To date, recombinant tissue plasminogen activator (rtPA) is the only approved drug for ischemic stroke. It is intravenously administered functioning as a thrombolytic agent and is used to obtain reperfusion of the affected area of the brain. Excitotoxicity, inflammation and apoptosis are all involved in delayed neuronal death following stroke and offer multiple opportunities to intervene with neuroprotective agents. Gelsolin (GSN) is an actin- and calcium-binding protein mediating the disassembly of actin filaments and activity of calcium channels. It also functions as a regulator of apoptosis and inflammatory responses. This study tests the hypothesis that increasing the concentration of the form of GSN known as plasma GSN (pGSN) near an infarct will provide neuroprotection following ischemic stroke. Methods: We induced middle cerebral artery occlusion (MCAO) in male rats via intracranial injection of endothelin-1 (ET-1), a potent vasoconstrictor, and then treated with local delivery of pGSN. Whole brain laser Doppler perfusion imaging was performed through the skull to assess MCAO effectiveness. Cylinder and vibrissae tests evaluated sensorimotor function before and 72 h after MCAO. Infarct volumes were examined 72 h after MCAO via 2, 3, 5-triphenyltetrazolium chloride (TTC) assay. Results: Estimates of relative cerebral perfusion were significantly decreased in all groups receiving MCAO with no differences detected between treatments. Despite equivalent initial strokes, the infarct volume of the pGSN treatment group was significantly reduced compared with the untreated MCAO rats at 72 h. ET-1 induced significant deficits in both cylinder and vibrissae tests while pGSN significantly limited these deficits. Conclusion: Gelsolin could be a promising drug for protection against neurodegeneration following ischemic stroke.

The ATP-binding cassette transporters ABCB1 and ABCC1 are not regulated by hypoxia in immortalised human brain microvascular endothelial cells

Pauline Patak — 2011-10-26T00:00:00Z

Background: ATP-binding cassette transporters at the blood-brain barrier are actively regulated upon ischemic stroke in a way that impedes the access of pharmacological compounds to the brain tissue. The luminal endothelial transporter ABCB1 was recently shown to be increased, whereas the abluminal transporter ABCC1 was decreased on ischemic brain capillaries. In vitro studies using epithelial cells suggested that ABCB1 is regulated during hypoxia in a hypoxia-inducible factor (HIF)-1α-dependent way. Methods: In order to investigate whether hypoxia might be responsible for the expression changes of ABCB1 and ABCC1 in the ischemic brain, the immortalised human brain microvascular endothelial cell line hCMEC/D3 was exposed to hypoxia (1%) or anoxia (0%). Cell lysates were analysed by Western blot to detect the protein expression of ABCB1, ABCC1, HIF-1α and HIF-2α. Results: During hypoxia, an accumulation of HIF-1α and HIF-2α was noticed in hCMEC/D3 cells that followed different time kinetics. Both HIF-1α and HIF-2α abundance increased within 4 h of hypoxia. HIF-1α levels decreased to below detection levels within 16 h of hypoxia, whereas HIF-2α remained elevated even after 48 h. No changes of ABCB1 and ABCC1 expression were detected, neither on the mRNA nor protein level. Conclusion: Our data suggests that other factors than hypoxia may be responsible for the expression changes of ATP-binding cassette transporters in the ischemic brain.

Comparison of humoral neuroinflammation and adhesion molecule expression in two models of experimental intracerebral hemorrhage

Arthur Liesz — 2011-10-03T00:00:00Z

Background: Inflammatory cascades contribute to secondary injury after intracerebral hemorrhage (ICH) via humoral factors and cell-mediated cytotoxicity. Several experimental models were previously developed to analyze post-hemorrhagic neuroinflammation. However, neuroinflammatory markers have not been compared face-to-face between these models so far, and therefore, pathophysiological conclusions drawn from only one individual model may not be valid. Methods: We compared neuroinflammatory pathways in the two most common murine models: striatal injection of autologous blood or collagenase. Expression of pro- and anti-inflammatory cytokines (IL-1, TNF-α, IFN-γ, IL-6, TGF-β and IL-10) as well adhesion molecule expression (VCAM-1, ICAM-1) was analyzed by RT-PCR at several time points after ICH induction. Outcome and physiological parameters were compared between the models. Results: Both models induced a profound and dynamic increase in the expression of pro-inflammatory cytokines and adhesion molecules. However, blood injection resulted in significantly more pronounced alteration of these markers than collagenase injection. This difference was associated with worse outcome after blood injection compared to the collagenase model despite equal ICH volumes. Conclusions: This is the first study performing a face-to-face comparison of neuroinflammatory pathways in the two most widely used murine ICH models, revealing substantial differences between the models. This discrepancies need to be taken into account in designing future studies employing experimental ICH models, especially when analyzing neuroinflammatory pathways and therapies.

Intra-arterial administration of recombinant tissue-type plasminogen activator (rt-PA) causes more intracranial bleeding than does intravenous rt-PA in a transient rat middle cerebral artery occlusion model

R. Christian Crumrine — 2011-09-20T00:00:00Z

Background: Intra-arterial (IA) administration of rt-PA for ischemic stroke has the potential for greater thrombolytic efficacy, especially for a large thrombus in the M1 or M2 segment of the middle cerebral artery (MCA). Intracranial hemorrhage (ICH) is a concern with IA or intravenous (IV) administration especially as the therapeutic window is extended. However, because IA administration delivers a higher local concentration of agent, the incidence and severity of ICH may be greater than with similar doses IV. We investigated the safety of rt-PA administration by IA compared to IV infusion following 6 hours of MCA occlusion (MCAo) with reflow in the spontaneously hypertensive rat (SHR). Methods: Male SHRs were subjected to 6 hours MCAo with 18 hours reflow using a snare ligature model. They were treated with IA saline, IA rt-PA (1, 5, 10, 30 mg/kg), or IV rt-PA (10 and 30 mg/kg) by a 10 to 60 minute infusion beginning approximately 1 minute before reflow. The rats were recovered for 24 hours after MCAo onset at which time Bleeding Score, infarct volume, and Modified Bederson Score were measured. Results: Greater hemorrhagic transformation occurred with 10 and 30 mg/kg rt-PA administered IA than IV. The IV 10 mg/kg rt-PA dosage induced significantly less bleeding than did the 1 or 5 mg/kg IA groups. No significant increase in infarct volume was observed after IA or IV treatment. Rats treated with 30 mg/kg rt-PA by either the IA or IV route had greater neurological dysfunction compared to all other groups. Conclusions: Administration of rt-PA by the IA route following 6 hours of MCAo results in greater ICH and worse functional recovery than comparable dosages IV. Significantly greater bleeding was observed when the IA dose was a tenth of the IV dose. The increased bleeding did not translate in larger infarct volumes.

Platelet glycoprotein Ibalpha is an important mediator of ischemic stroke in mice

Simon De Meyer — 2011-09-13T00:00:00Z

Background: Platelets play an important role in ischemic stroke. GPIbα is a major platelet receptor that is critical for platelet adhesion to exposed subendothelial matrix components at sites of vascular damage. Methods: In this study, we used transgenic mice in which the extracellular part of GPIbα is replaced by human interleukin 4-receptor (GPIbα/IL4Rα). We observed normal brain vasculature in these mice. We compared infarct size in GPIbα/IL4Rα and wild-type (WT) mice 23 hours after 1-hour transient middle cerebral artery occlusion (tMCAO). In addition, the functional outcome was evaluated using a modified Bederson score. Results: We found a significantly smaller infarct size in GPIbα/IL4Rα mice compared to WT mice (38.0 ± 6.5 mm3 vs. 74.2 ± 8.6 mm3, p < 0.001). The decrease in infarct size was functionally relevant as indicated by a significantly better functional Bederson score in GPIbα/IL4Rα mice compared to WT animals (1.3 ± 0.4 vs. 2.7 ± 0.3, p < 0.05). Conclusions: Our data illustrate and further confirm the important role of platelet GPIbα in ischemic stroke, suggesting that targeted inhibition of this receptor may open new avenues in stroke treatment.

Calorie Restriction and Stroke

Silvia Manzanero — 2011-09-12T00:00:00Z

Stroke, a major cause of disability and mortality in the elderly, occurs when a cerebral blood vessel is occluded or ruptured, resulting in ischemic damage and death of brain cells. The injury mechanism involves metabolic and oxidative stress, excitotoxicity, apoptosis and inflammatory processes, including activation of glial cells and infiltration of leukocytes. In animal models, dietary energy restriction, by daily calorie reduction (CR) or intermittent fasting (IF), extends lifespan and decreases the development of age-related diseases. Dietary energy restriction may also benefit neurons, as suggested by experimental evidence showing that CR and IF protect neurons against degeneration in animal models. Recent findings by our group and others suggest the possibility that dietary energy restriction may protect against stroke induced brain injury, in part by inducing the expression of neurotrophic factors, such as brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor (bFGF); protein chaperones, including heat shock protein 70 (Hsp70) and glucose regulated protein 78 (GRP78); antioxidant enzymes, such as superoxide dismutases (SOD) and heme oxygenase-1 (HO-1), silent information regulator T1 (SIRT1), uncoupling proteins and anti-inflammatory cytokines. This article discusses the protective mechanisms activated by dietary energy restriction in ischemic stroke.