David C. Hess

Minocycline to Improve Neurologic Outcome in Stroke (MINOS): A dose-finding study

Background and Purpose— Minocycline is a promising anti-inflammatory and protease inhibitor that is effective in multiple preclinical stroke models. We conducted an early phase trial of intravenous minocycline in acute ischemic stroke. Methods— Following an open-label, dose-escalation design, minocycline was administered intravenously within 6 hours of stroke symptom onset in preset dose tiers of 3, 4.5, 6, or 10 mg/kg daily over 72 hours. Minocycline concentrations for pharmacokinetic analysis were measured in a subset of patients. Subjects were followed for 90 days. Results— Sixty patients were enrolled, 41 at the highest dose tier of 10 mg/kg. Overall age (65±13.7 years), race (83% white), and sex (47% female) were consistent across the doses. The mean baseline National Institutes of Health Stroke Scale score was 8.5±5.8 and 60% received tissue plasminogen activator. Minocycline infusion was well tolerated with only 1 dose limiting toxicity at the 10-mg/kg dose. No severe hemorrhages occurred in tissue plasminogen activator-treated patients. Pharmacokinetic analysis (n=22) revealed a half-life of approximately 24 hours and linearity of parameters over doses. Conclusions— Minocycline is safe and well tolerated up to doses of 10 mg/kg intravenously alone and in combination with tissue plasminogen activator. The half-life of minocycline is approximately 24 hours, allowing every 24-hour dosing. Minocycline may be an ideal agent to use with tissue plasminogen activator.

Delayed minocycline inhibits ischemia-activated matrix metalloproteinases 2 and 9 after experimental stroke

BackgroundMatrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) are increased in the brain after experimental ischemic stroke in rats. These two proteases are involved with the degradation of the basal lamina and loss of stability of the blood brain barrier that occurs after ischemia and that is associated with thrombolytic therapy in ischemic stroke. Minocycline is a lipophilic tetracycline and is neuroprotective in several models of brain injury. Minocycline inhibits inflammation, apoptosis and extracellular matrix degradation. In this study we investigated whether delayed minocycline inhibits brain MMPs activated by ischemia in a model of temporary occlusion in Wistar rats.ResultsBoth MMP-2 and MMP-9 were elevated in the ischemic tissue as compared to the contra-lateral hemisphere after 3 hours occlusion and 21 hours survival (p < 0.0001 for MMP-9). Intraperitoneal minocycline at 45 mg/kg concentration twice a day (first dose immediately after the onset of reperfusion) significantly reduced gelatinolytic activity of ischemia-elevated MMP-2 and MMP-9 (p < 0.0003). Treatment also reduced protein concentration of both enzymes (p < 0.038 for MMP-9 and p < 0.018 for MMP-2). In vitro incubation of minocycline in concentrations as low as 0.1 μg/ml with recombinant MMP-2 and MMP-9 impaired enzymatic activity and MMP-9 was more sensitive at lower minocycline concentrations (p < 0.05).ConclusionMinocycline inhibits enzymatic activity of gelatin proteases activated by ischemia after experimental stroke and is likely to be selective for MMP-9 at low doses. Minocycline is a potential new therapeutic agent to acute treatment of ischemic stroke.

Optimal delivery of minocycline to the brain: implication for human studies of acute neuroprotection

Minocycline is currently under development as a neuroprotective agent in many different brain diseases. In more than a dozen experimental investigations in various models of brain injury, high doses of minocycline have been administered intraperitoneally. This report details new concerns with this route of administration and makes a case for intravenous dosing in experimental animals, particularly for acute neuroprotection, to optimize delivery to the brain and facilitate translation to human studies.

Minocycline for Short-Term Neuroprotection

Minocycline is a widely used tetracycline antibiotic. For decades, it has been used to treat various gram‐positive and gram‐negative infections. Minocycline was recently shown to have neuroprotective properties in animal models of acute neurologic injury. As a neuroprotective agent, the drug appears more effective than other treatment options. In addition to its high penetration of the blood‐brain barrier, minocycline is a safe compound commonly used to treat chronic infections. Its several mechanisms of action in neuroprotection—antiinflammatory and antiapoptotic effects, and protease inhibition—make it a desirable candidate as therapy for acute neurologic injury, such as ischemic stroke. Minocycline is ready for clinical trials of acute neurologic injury.

Minocycline and Tissue-Type Plasminogen Activator for Stroke Assessment of Interaction Potential

Background and Purpose— New treatment strategies for acute ischemic stroke must be evaluated in the context of effective reperfusion. Minocycline is a neuroprotective agent that inhibits proteolytic enzymes and therefore could potentially both inactivate the clot lysis effect and decrease the damaging effects of tissue-type plasminogen activator (t-PA). This study aimed to determine the effect of minocycline on t-PA clot lysis and t-PA–induced hemorrhage formation after ischemia. Methods— Fibrinolytic and amidolytic activities of t-PA were investigated in vitro over a range of clinically relevant minocycline concentrations. A suture occlusion model of 3-hour temporary cerebral ischemia in rats treated with t-PA and 2 different minocycline regimens was used. Blood–brain barrier basal lamina components, matrix metalloproteinases (MMPs), hemorrhage formation, infarct size, edema, and behavior outcome were assessed. Results— Minocycline did not affect t-PA fibrinolysis. However, minocycline treatment at 3 mg/kg IV decreased total protein expression of both MMP-2 (P=0.0034) and MMP-9 (P=0.001 for 92 kDa and P=0.0084 for 87 kDa). It also decreased the incidence of hemorrhage (P=0.019), improved neurologic outcome (P=0.0001 for Bederson score and P=0.0391 for paw grasp test), and appeared to decrease mortality. MMP inhibition was associated with decreased degradation in collagen IV and laminin-α1 (P=0.0001). Conclusions— Combination treatment with minocycline is beneficial in t-PA–treated animals and does not compromise clot lysis. These results also suggest that neurovascular protection by minocycline after stroke may involve direct protection of the blood–brain barrier during thrombolysis with t-PA.

Minocycline Development for Acute Ischemic Stroke

Minocycline, a tetracycline antibiotic, has shown anti-inflammatory, anti-apoptotic, and neuroprotective effects in many models of cerebral ischemia and neurodegenerative disease. Its high penetration of the blood–brain barrier, good safety profile, and delayed therapeutic window make it an ideal candidate for use in stroke. In animal models, minocycline reduced infarct size and improved neurologic outcome when administered acutely, with similar neuroprotective benefits seen following delayed administration. To date, two early phase clinical trials have shown minocycline to be safe and potentially effective in acute ischemic stroke, alone or in combination with tissue plasminogen activator. A large efficacy clinical trial is now needed to confirm previous studies, allow for subgroup analysis, and pinpoint the potential place for minocycline in acute stroke therapy.

Intracerebral xenotransplantation of GFP mouse bone marrow stromal cells in intact and stroke rat brain: graft survival and immunologic response.

The present study characterized survival and immunologic response of bone marrow stromal cells (BMSCs) following transplantation into intact and stroke brains. In the first study, intrastriatal transplantation of BMSC (60,000 in 3 μl) or vehicle was performed in normal adult Sprague-Dawley male rats that subsequently received daily cyclosporin A (CsA, 10 mg/kg, IP in 3 ml) or vehicle (olive oil, similar volume) starting on day of surgery up to 3 days posttransplantation. Animals were euthanized at 3 or 30 days posttransplantation and brains were processed either for green fluorescent protein (GFP) microscopy or flow cytometry (FACS). Both GFP epifluorescence and FACS scanning revealed GFP+ BMSCs in both groups of transplanted rats with or without CsA, although significantly increased (1.6- to 3-fold more) survival of GFP+ BMSCs was observed in the immunosuppressed animals. Further histologic examination revealed widespread dispersal of BMSCs away from the graft core accompanied by many long outgrowth processes in non-CsA-transplanted animals, whereas a very dense graft core, with cells expressing only sporadic short outgrowth processes, was observed in CsA-transplanted animals. There were no detectable GFP+ BMSCs in nontrans-planted rats that received CsA or vehicle. Immunologic response via FACS analysis revealed a decreased presence of cytotoxic cells, characterized by near complete absence of CD8+ cells, and lack of activation depicted by low CD69 expression in CsA-treated transplanted animals. In contrast, elevated levels of CD8+ cells and increased activation of CD69 expression were observed in transplanted animals that received vehicle alone. CD4+ helper cells were almost nondetectable in transplanted rats that received CsA, but also only minimally elevated in transplanted rats that received vehicle. Nontransplanted rats that received either CsA or vehicle displayed very minimal detectable levels of all three lymphocyte markers. In the second study, a new set of male Sprague-Dawley rats initially received bilateral stereotaxic intrastriatal transplantation of BMSCs and 3 days after were subjected to unilateral transient occlusion of middle cerebral artery. The animals were allowed to survive for 3 days after stroke without CsA immunosuppression. Epifluorescence microscopy revealed significantly higher (5-fold more) survival of transplanted GFP+ BMSCs in the stroke striatum compared with the intact striatum. The majority of the grafts remained within the original dorsal striatal transplant site, characterized by no obvious migration in intact striatum, but with long-distance migration along the ischemic penumbra in the stroke striatum. Moreover, FACS scanning analyses revealed low levels of immunologic response of grafted BMSCs in both stroke and intact striata. These results, taken together, suggest that xenotransplantation of mouse BMSCs into adult rats is feasible. Immunosuppression therapy can enhance xenograft survival and reduce graft-induced immunologic response; however, in the acute phase posttransplantation, BMSCs can survive in intact and stroke brain, and may even exhibit long-distance migration and increased outgrowth processes without immunosuppression.

Treatment of fever after stroke: Conflicting evidence

Approximately 50% of patients hospitalized for stroke develop fever. In fact, experimental evidence suggests that high body temperature is significantly correlated to initial stroke severity, lesion size, mortality, and neurologic outcome. Fever occurring after stroke is associated with poor outcomes. We investigated the etiology of fever after stroke and present evidence evaluating the efficacy and safety of interventions used to treat stroke‐associated fever. Oral antipyretics are only marginally effective in lowering elevated body temperature in this population and may have unintended adverse consequences. Nonpharmacologic approaches to cooling have been more effective in achieving normothermia, but whether stroke outcomes can be improved remains unclear. We recommend using body temperature as a biomarker and a catalyst for aggressive investigation for an infectious etiology. Care must be taken not to exceed the new standard of a maximum acetaminophen dose of 3 g/day to avoid patient harm.

Human Neural Stem Cell Extracellular Vesicles Improve Recovery in a Porcine Model of Ischemic Stroke

Background and Purpose— Recent work from our group suggests that human neural stem cell–derived extracellular vesicle (NSC EV) treatment improves both tissue and sensorimotor function in a preclinical thromboembolic mouse model of stroke. In this study, NSC EVs were evaluated in a pig ischemic stroke model, where clinically relevant end points were used to assess recovery in a more translational large animal model. Methods— Ischemic stroke was induced by permanent middle cerebral artery occlusion (MCAO), and either NSC EV or PBS treatment was administered intravenously at 2, 14, and 24 hours post-MCAO. NSC EV effects on tissue level recovery were evaluated via magnetic resonance imaging at 1 and 84 days post-MCAO. Effects on functional recovery were also assessed through longitudinal behavior and gait analysis testing. Results— NSC EV treatment was neuroprotective and led to significant improvements at the tissue and functional levels in stroked pigs. NSC EV treatment eliminated intracranial hemorrhage in ischemic lesions in NSC EV pigs (0 of 7) versus control pigs (7 of 8). NSC EV–treated pigs exhibited a significant decrease in cerebral lesion volume and decreased brain swelling relative to control pigs 1-day post-MCAO. NSC EVs significantly reduced edema in treated pigs relative to control pigs, as assessed by improved diffusivity through apparent diffusion coefficient maps. NSC EVs preserved white matter integrity with increased corpus callosum fractional anisotropy values 84 days post-MCAO. Behavior and mobility improvements paralleled structural changes as NSC EV–treated pigs exhibited improved outcomes, including increased exploratory behavior and faster restoration of spatiotemporal gait parameters. Conclusions— This study demonstrated for the first time that in a large animal model novel NSC EVs significantly improved neural tissue preservation and functional levels post-MCAO, suggesting NSC EVs may be a paradigm changing stroke therapeutic.

Limb and hemispatial hypometria.

In a previous study, we demonstrated that unilateral cerebral lesions produce hypometric limb movements of the contralateral arm and hemispatial (i.e., directional) hypometria for movements towards contralateral hemispace. In the present study, we investigated 10 patients with right cerebral lesions and 25 healthy controls using a task to uncouple deficits in sensory perceptual systems and motor-action output systems on directional hypometria. This task required participants, with their eyes closed, to reproduce lateral and medial horizontal displacements (15-27 cm) with each arm. Each participant was seated at a waist high table and had their hand placed at an origin point aligned with the axillary fold on the same side. Their hand was moved by the investigator from the origin point to a target point and brought back to the point of origin (input displacement). The participant was then asked to return their hand to either the same target point or to an equidistant target point in the opposite direction. Healthy dextral participants were significantly more hypometric with their right arm, but patients with right cerebral lesions exhibited an opposite pattern with overall left arm hypometria. In addition, patients were significantly more hypometric for movements when output displacements were toward left hemispace. No effect was found for direction of sensory input. The results suggest that the directional hypometria is predominantly produced by hemispatial output deficits.