Sönke Schwarting

Hemostatic Therapy in Experimental Intracerebral Hemorrhage Associated With the Direct Thrombin Inhibitor Dabigatran

Background and Purpose— Dabigatran-etexilate (DE) recently has been approved for stroke prevention in atrial fibrillation. However, lack of effective antagonists represents a major concern in the event of intracerebral hemorrhage (ICH). The aims of the present study were to establish a murine model of ICH associated with dabigatran, and to test the efficacy of different hemostatic factors in preventing hematoma growth. Methods— In C57BL/6 mice receiving DE (4.5 or 9.0 mg/kg), in vivo and in vitro coagulation assays and dabigatran plasma levels were measured repeatedly. Thirty minutes after inducing ICH by striatal collagenase injection, mice received an intravenous injection of saline, prothrombin complex concentrate (PCC; 100 U/kg), murine fresh-frozen plasma (200 &mgr;L), or recombinant human factor VIIa (8.0 mg/kg). ICH volume was quantified on brain cryosections 24 hours later. Results— DE substantially prolonged tail vein bleeding time and ecarin clotting time for 4 hours corresponding to dabigatran plasma levels. Intracerebral hematoma expansion was observed mainly during the first 3 hours on serial T2* MRI. Anticoagulation with high doses of DE increased the hematoma volume significantly. PCC and, less consistently, fresh-frozen plasma prevented excess hematoma expansion caused by DE, whereas recombinant human factor VIIa was ineffective. Prevention of hematoma growth and reversal of tail vein bleeding time by PCC were dose-dependent. Conclusions— The study provides strong evidence that PCC and, less consistently, fresh-frozen plasma prevent excess intracerebral hematoma expansion in a murine ICH model associated with dabigatran. The efficacy and safety of this strategy must be further evaluated in clinical studies.

Inhibition of lymphocyte trafficking shields the brain against deleterious neuroinflammation after stroke

T lymphocytes are increasingly recognized as key modulators of detrimental inflammatory cascades in acute ischaemic stroke, but the potential of T cell-targeted therapy in brain ischaemia is largely unexplored. Here, we characterize the effect of inhibiting leukocyte very late antigen-4 and endothelial vascular cell adhesion molecule-1-mediated brain invasion-currently the most effective strategy in primary neuroinflammatory brain disease in murine ischaemic stroke models. Very late antigen-4 blockade by monoclonal antibodies improved outcome in models of moderate stroke lesions by inhibiting cerebral leukocyte invasion and neurotoxic cytokine production without increasing the susceptibility to bacterial infections. Gene silencing of the endothelial very late antigen-4 counterpart vascular cell adhesion molecule-1 by in vivo small interfering RNA injection resulted in an equally potent reduction of infarct volume and post-ischaemic neuroinflammation. Furthermore, very late antigen-4-inhibition effectively reduced the post-ischaemic vascular cell adhesion molecule-1 upregulation, suggesting an additional cross-signalling between invading leukocytes and the cerebral endothelium. Dissecting the specific impact of leukocyte subpopulations showed that invading T cells, via their humoral secretion (interferon-γ) and immediate cytotoxic mechanisms (perforin), were the principal pathways for delayed post-ischaemic tissue injury. Thus, targeting T lymphocyte-migration represents a promising therapeutic approach for ischaemic stroke.

Deletion of Cellular Prion Protein Results in Reduced Akt Activation, Enhanced Postischemic Caspase-3 Activation, and Exacerbation of Ischemic Brain Injury

Background and Purpose— The physiological function of cellular prion protein (PrPc) is not yet understood. Recent findings suggest that PrPc may have neuroprotective properties, and its absence increases susceptibility to neuronal injury. The purpose of this study was to elucidate the role of PrPc in ischemic brain injury in vivo. Methods— PrP knockout (Prnp0/0) and Prnp+/+ wild-type (WT) mice were subjected to 60-minute transient or permanent focal cerebral ischemia followed by infarct volume analysis 24 hours after lesion. To identify effects of PrPc deletion on mechanisms regulating ischemic cell death, expression analysis of several proapoptotic and antiapoptotic proteins was performed at 6 and 24 hours after transient ischemia and in nonischemic controls using Western blot or immunohistochemistry. Results— Prnp0/0 mice displayed significantly increased infarct volumes after both transient or permanent ischemia when compared with WT animals (70.2±23 versus 13.3±4 mm3; 119.8±24 versus 86.4±25 mm3). Expression of phospho-Akt (Ser473) was significantly reduced in Prnp0/0 compared with WT animals both early after ischemia and in sham controls. Furthermore, postischemic caspase-3 activation was significantly enhanced in the basal ganglia and the parietal cortex of Prnp0/0 mice. In contrast, expression of total Akt, Bax, and Bcl-2 did not differ between both groups. Conclusions— These results demonstrate that PrPc deletion impairs the antiapoptotic phosphatidylinositol 3-kinase/Akt pathway by resulting in reduced postischemic phospho-Akt expression, followed by enhanced postischemic caspase-3 activation, and aggravated neuronal injury after transient and permanent cerebral ischemia.

Hematopoietic Stem Cells Reduce Postischemic Inflammation and Ameliorate Ischemic Brain Injury

Background and Purpose— Systemic injection of hematopoietic stem cells after ischemic cardiac or neural lesions is one approach to promote tissue repair. However, mechanisms of possible protective or reparative effects are poorly understood. In this study we analyzed the effect of lineage-negative bone marrow-derived hematopoietic stem and precursor cells (Lin−-HSCs) on ischemic brain injury in mice. Methods— Lin−-HSCs were injected intravenously at 24 hours after onset of a 45-minute transient cerebral ischemia. Effects of Lin−-HSCs injection on infarct size, apoptotic cell death, postischemic inflammation and cytokine gene transcription were analyzed. Results— Green fluorescent protein (GFP)-marked Lin−-HSCs were detected at 24 hours after injection in the spleen and later in ischemic brain parenchyma, expressing microglial but no neural marker proteins. Tissue injury assessment showed significantly smaller infarct volumes and less apoptotic neuronal cell death in peri-infarct areas of Lin−-HSC–treated animals. Analysis of immune cell infiltration in ischemic hemispheres revealed a reduction of invading T cells and macrophages in treated mice. Moreover, Lin−-HSC therapy counter-regulated proinflammatory cytokine and chemokine receptor gene transcription within the spleen. Conclusions— Our data demonstrate that systemically applied Lin−-HSCs reduce cerebral postischemic inflammation, attenuate peripheral immune activation and mediate neuroprotection after ischemic stroke.

FTY720 Reduces Post-Ischemic Brain Lymphocyte Influx but Does Not Improve Outcome in Permanent Murine Cerebral Ischemia

Background The contribution of neuroinflammation and specifically brain lymphocyte invasion is increasingly recognised as a substantial pathophysiological mechanism after stroke. FTY720 is a potent treatment for primary neuroinflammatory diseases by inhibiting lymphocyte circulation and brain immigration. Previous studies using transient focal ischemia models showed a protective effect of FTY720 but did only partially characterize the involved pathways. We tested the neuroprotective properties of FTY720 in permanent and transient cortical ischemia and analyzed the underlying neuroimmunological mechanisms. Methodology/Principal Findings FTY720 treatment resulted in substantial reduction of circulating lymphocytes while blood monocyte counts were significantly increased. The number of histologically and flow cytometrically analyzed brain invading T- and B lymphocytes was significantly reduced in FTY720 treated mice. However, despite testing a variety of treatment protocols, infarct volume and behavioural dysfunction were not reduced 7d after permanent occlusion of the distal middle cerebral artery (MCAO). Additionally, we did not measure a significant reduction in infarct volume at 24h after 60 min filament-induced MCAO, and did not see differences in brain edema between PBS and FTY720 treatment. Analysis of brain cytokine expression revealed complex effects of FTY720 on postischemic neuroinflammation comprising a substantial reduction of delayed proinflammatory cytokine expression at 3d but an early increase of IL-1β and IFN-γ at 24 h after MCAO. Also, serum cytokine levels of IL-6 and TNF-α were increased in FTY720 treated animals compared to controls. Conclusions/Significance In the present study we were able to detect a reduction of lymphocyte brain invasion by FTY720 but could not achieve a significant reduction of infarct volumes and behavioural dysfunction. This lack of neuroprotection despite effective lymphopenia might be attributed to a divergent impact of FTY720 on cytokine expression and possible activation of innate immune cells after brain ischemia.

TAT-Hsp70-mediated neuroprotection and increased survival of neuronal precursor cells after focal cerebral ischemia in mice

Cerebral ischemia stimulates endogenous neurogenesis within the subventricular zone and the hippocampal dentate gyrus of the adult rodent brain. However, such newly generated cells soon die after cerebral ischemia. To enhance postischemic survival of neural precursor cells (NPC) and long-lasting neural regeneration, we applied the antiapoptotic chaperone heat shock protein 70 (Hsp70) fused to a cell-penetrating peptide derived from the HIV TAT to ensure delivery across the blood-brain barrier and the cell membrane. After transient focal cerebral ischemia in mice, TAT-Hsp70 was intravenously injected concomitant with reperfusion and additionally on day 14 after stroke. TAT-Hsp70 treatment resulted in smaller infarct size (27.1 ± 9.0 versus 109.0 ± 14.0 and 88.5 ± 26.0 mm3 in controls) and in functional improvement as assessed by the rota rod, tight rope, and water maze tests when compared with saline- and TAT-hemagglutinin-treated controls. In addition, postischemic survival of endogenous doublecortin (Dcx)-positive NPC was improved within the lesioned striatum of TAT-Hsp70-treated animals for up to 4 weeks after stroke without changing overall cell proliferation of BrdU+ cells. Thus, TAT-Hsp70 treatment after stroke may be a promising tool to act neuroprotective and improve postischemic functional outcome, and also to increase survival of endogenous NPC after stroke.

Comparative Effectiveness of Hemostatic Therapy in Experimental Warfarin-Associated Intracerebral Hemorrhage

Background and Purpose— Intracerebral hemorrhage associated with oral anticoagulants has a poor prognosis. Current treatment guidelines are based on case series and plausibility only, and a common consensus on effective hemostatic therapy is missing. We compared the effectiveness of diverse hemostatic approaches in a mouse model of warfarin-associated intracerebral hemorrhage. Methods— Male C57BL/6 mice received anticoagulant treatment with warfarin (0.4 mg/kg for 3 days). Intracerebral hemorrhage was induced by striatal injection of collagenase, and 30 minutes later, mice received an intravenous injection of saline (200 &mgr;L n=15), prothrombin complex concentrate (100 U/kg, n=10), fresh-frozen plasma (200 &mgr;L, n=13), recombinant human Factor VII activated (3.5 mg/kg, n=8 and 10 mg/kg, n=8), or tranhexamic acid (400 mg/kg, n=12). Intracerebral hemorrhage volume was quantified on T2-weighted images after 24 hours. Results— Mean hematoma volumes were 7.4±1.8 mm3 in the nonwarfarin controls and 21.9±5.0 mm3 in the warfarin group receiving saline. Prothrombin complex concentrate (7.5±2.3 mm3) and fresh-frozen plasma (8.7±2.1) treatment resulted in significantly smaller hematoma volume compared with saline. Recombinant human Factor VII activated (10 mg/kg: 14.7±3.4; 3.5 mg/kg: 15.0±6.8 mm3) and tranexamic acid (16.2±4.1 mm3) were less effective. Water content in the hemorrhagic hemisphere was similar in all groups except for tranexamic acid in which it was significantly increased. Conclusions— Prothrombin complex concentrate and fresh-frozen plasma effectively prevent hematoma growth in murine warfarin-associated intracerebral hemorrhage, whereas Factor VIIa was less effective. Tranexamic acid exacerbates perihematoma edema in this mouse warfarin-associated intracerebral hemorrhage model.

Transplantation of TAT-Bcl-xL-transduced neural precursor cells: long-term neuroprotection after stroke.

Neural precursor cells (NPC) are an interesting tool in experimental stroke research, but their therapeutic potential is limited due to poor long-term survival. We therefore in vitro transduced subventricular zone-(SVZ)-derived NPC with the anti-apoptotic fusion protein TAT-Bcl-x(L) and analyzed NPC survival, differentiation, and post-stroke functional deficits after experimental ischemia in mice. Survival of TAT-Bcl-x(L)-transduced NPC, which were injected at day 7 post-stroke into the ischemic striatum, was significantly increased at 4 weeks after stroke. Increased survival of NPC was associated with reduced infarct injury and decreased post-stroke functional deficits. Animals grafted with TAT-Bcl-x(L)-transduced NPC showed an increased number of immature cells expressing the neuronal marker doublecortin. Since mature neuronal differentiation of NPC was not observed, reduced post-stroke injury cannot be attributed to enhanced neuronal regeneration, but rather to indirect by-stander effects of grafted NPC. In line with this, NPC-mediated neuroprotection of cortical neurons in vitro was associated with increased secretion of growth factors. Thus, in vitro transduction of cultivated NPC with TAT-Bcl-x(L) results in enhanced resistance of transplanted NPC followed by long-term neuroprotection and ameliorated functional deficits after transient focal cerebral ischemia in mice.

Enhanced poly(ADP‐ribose) polymerase‐1 activation contributes to recombinant tissue plasminogen activator‐induced aggravation of ischemic brain injury in vivo

Recombinant tissue plasminogen activator (rt‐PA) treatment improves functional outcome after acute ischemic stroke, inducing reperfusion by its thrombolytic activity. Conversely, there is evidence that rt‐PA can mediate neuronal damage after ischemic brain injury in vivo. In addition to other mechanisms, enhancement of N‐methyl‐D‐aspartate (NMDA) receptor signalling has been proposed to underlie rt‐PA‐mediated neurotoxicity. However, the role of poly(ADP‐ribose) polymerase‐1 (PARP‐1) activation, which mediates postischemic excitotoxic cell death, in rt‐PA‐mediated aggravation of ischemic brain injury has not been established and was therefore addressed in this study. After permanent focal cerebral ischemia, intravenous rt‐PA application significantly increased early postischemic PARP‐1 activation within ischemic hemispheres and infarct volumes compared with control mice without affecting cerebral blood flow. Rt‐PA induced increase in PARP‐1 activation, and infarct volumes could be blocked by the PARP inhibitor 3‐aminobenzamide. Moreover, the rt‐PA‐induced increase in PARP‐1 activation was also prevented by the NMDA antagonist MK‐801. In summary, we demonstrate that rt‐PA treatment enhances postischemic PARP‐1 activation, which contributes to rt‐PA induced aggravation of ischemic brain injury in vivo. Furthermore, we provide evidence that NMDA receptor activation is required for rt‐PA‐mediated effects on postischemic PARP‐1 activation.

Response to Letter by Moll Regarding Article, “Hemostatic Therapy in Experimental Intracerebral Hemorrhage Associated With the Direct Thrombin Inhibitor Dabigatran”

Response: We appreciate the interest of Dr Moll1 in our experimental study in which we examined the effect of different hemostatic approaches on early hematoma growth in mice treated with dabigatran etexilate. In contrast to another recently published article2 using a similar experimental model, treatment with high doses of dabigatran in our study induced an excess early hematoma growth compared with nonanticoagulated mice, which we documented by serial MRI. Moreover, we showed that prothrombin complex (PCC) was the most consistently effective agent preventing the excess hematoma growth in mice treated with either 4.5 mg/kg or 9 mg/kg of dabigatran etexilate intraperitoneally. Finally, we tested the effect of 3 different doses of PCC on …