Neuroprotective effect of the RNS60 in a mouse model of transient focal cerebral ischemia.

BACKGROUND: Stroke is a major cause of death, disability, and public health problems. Its intervention is limited to early treatment with thrombolytics and/or endovascular clot removal with mechanical thrombectomy without any available subacute or chronic neuroprotective treatments. RNS60 has reduced neuroinflammation and increased neuronal survival in several animal models of neurodegeneration and trauma. The aim here was to evaluate whether RNS60 protects the brain and cognitive function in a mouse stroke model. METHODS: Male C57BL/6J mice were subjected to sham or ischemic stroke surgery using 60-minute transient middle cerebral artery occlusion (tMCAo). In each group, mice received blinded daily administrations of RNS60 or control fluids (PNS60 or normal saline [NS]), beginning 2 hours after surgery over 13 days. Multiple neurobehavioral tests were conducted (Neurological Severity Score [mNSS], Novel Object Recognition [NOR], Active Place Avoidance [APA], and the Conflict Variant of APA [APAc]). On day 14, cortical microvascular perfusion (MVP) was measured, then brains were removed and infarct volume, immunofluorescence of amyloid beta (Aß), neuronal density, microglial activation, and white matter damage/myelination were measured. SPSS was used for analysis (e.g., ANOVA for parametric data; Kruskal Wallis for non-parametric data; with post-hoc analysis). RESULTS: Thirteen days of treatment with RNS60 reduced brain infarction, amyloid pathology, neuronal death, microglial activation, white matter damage, and increased MVP. RNS60 reduced brain pathology and resulted in behavioral improvements in stroke compared to sham surgery mice (increased memory-learning in NOR and APA, improved cognitive flexibility in APAc). CONCLUSION: RNS60-treated mice exhibit significant protection of brain tissue and improved neurobehavioral functioning after tMCAo-stroke. Additional work is required to determine mechanisms, time-window of dosing, and multiple dosing volumes durations to support clinical stroke research.

Expanding the horizon of research into the pathogenesis of the white matter diseases: Proceedings of the 2021 Annual Workshop of the Albert Research Institute for White Matter and Cognition.

White matter pathologies are critically involved in the etiology of vascular cognitive impairment-dementia (VCID), Alzheimer's disease (AD), and Alzheimer's disease and related diseases (ADRD), and therefore need to be considered a treatable target ( Roseborough A, Hachinski V, Whitehead S. White matter degeneration - a treatable target? Roseborough et al. JAMA Neurol [Internet]. 2020 Apr 27;77(7):793-4, [1] . To help address this often-missed area of research, several workshops have been sponsored by the Leo and Anne Albert Charitable Trust since 2015, resulting in the incorporation of "The Albert Research Institute for White Matter and Cognition" in 2020. The first annual "Institute" meeting was held virtually on March 3-4, 2021. The Institute provides a forum and workspace for communication and support of the advancement of white matter science and research to better understand the evolution and prevention of dementia. It serves as a platform for young investigator development, to introduce new data and debate biology mechanisms and new ideas, and to encourage and support new research collaborations and directions to clarify how white matter changes, with other genetic and health risk factors, contribute to cognitive impairment. Similar to previous Albert Trust-sponsored workshops (Barone et al. in J Transl Med 14:1-14, [2]; Sorond et al. in GeroScience 42:81-96, [3]), established expert investigators were identified and invited to present. Opportunities to attend and present were also extended by invitation to talented research fellows and younger scientists. Also, updates on institute-funded research collaborations were provided and discussed. The summary that follows is a synopsis of topics and discussion covered in the workshop.

Cerebrovascular disease promotes tau pathology in Alzheimer's disease.

Small vessel cerebrovascular disease, visualized as white matter hyperintensities on T2-weighted magnetic resonance imaging, contributes to the clinical presentation of Alzheimer's disease. However, the extent to which cerebrovascular disease represents an independent pathognomonic feature of Alzheimer's disease or directly promotes Alzheimer's pathology is unclear. The purpose of this study was to examine the association between white matter hyperintensities and plasma levels of tau and to determine if white matter hyperintensities and tau levels interact to predict Alzheimer's disease diagnosis. To confirm that cerebrovascular disease promotes tau pathology, we examined tau fluid biomarker concentrations and pathology in a mouse model of ischaemic injury. Three hundred ninety-one participants from the Alzheimer's Disease Neuroimaging Initiative (74.5 ± 7.1 years of age) were included in this cross-sectional analysis. Participants had measurements of plasma total-tau, cerebrospinal fluid beta-amyloid, and white matter hyperintensities, and were diagnosed clinically as Alzheimer's disease (n = 97), mild cognitive impairment (n = 186) or cognitively normal control (n = 108). We tested the relationship between plasma tau concentration and white matter hyperintensity volume across diagnostic groups. We also examined the extent to which white matter hyperintensity volume, plasma tau, amyloid positivity status and the interaction between white matter hyperintensities and plasma tau correctly classifies diagnostic category. Increased white matter hyperintensity volume was associated with higher plasma tau concentration, particularly among those diagnosed clinically with Alzheimer's disease. Presence of brain amyloid and the interaction between plasma tau and white matter hyperintensity volume distinguished Alzheimer's disease and mild cognitive impairment participants from controls with 77.6% and 63.3% accuracy, respectively. In 63 Alzheimer's Disease Neuroimaging Initiative participants who came to autopsy (82.33 ± 7.18 age at death), we found that higher degrees of arteriosclerosis were associated with higher Braak staging, indicating a positive relationship between cerebrovascular disease and neurofibrillary pathology. In a transient middle cerebral artery occlusion mouse model, aged mice that received transient middle cerebral artery occlusion, but not sham surgery, had increased plasma and cerebrospinal fluid tau concentrations, induced myelin loss, and hyperphosphorylated tau pathology in the ipsilateral hippocampus and cerebral hemisphere. These findings demonstrate a relationship between cerebrovascular disease, operationalized as white matter hyperintensities, and tau levels, indexed in the plasma, suggesting that hypoperfusive injury promotes tau pathology. This potential causal association is supported by the demonstration that transient cerebral artery occlusion induces white matter damage, increases biofluidic markers of tau, and promotes cerebral tau hyperphosphorylation in older-adult mice.

Biocompatibility studies of fluorescent diamond particles-(NV)~800nm (part V): in vitro kinetics and in vivo localization in rat liver following long-term exposure.

BACKGROUND: We recently reported on long-term comprehensive biocompatibility and biodistribution study of fluorescent nanodiamond particles (NV)-Z-average 800nm (FNDP-(NV)) in rats. FNDP-(NV) primary deposition was found in the liver, yet liver function tests remained normal. PURPOSE: The present study aimed to gain preliminary insights on discrete localization of FNDP-(NV) in liver cells of the hepatic lobule unit and venous micro-vasculature. Kinetics of FDNP-(NV) uptake into liver cells surrogates in culture was conducted along with cell cytokinesis as markers of cells' viability. METHODS: Preserved liver specimens from a pilot consisting of two animals which were stained for cytoskeletal elements (fluorescein-isothiocyanate-phalloidin) were examined for distribution of FNDP-(NV) by fluorescent microscopy (FM) and Confocal-FM (CFM) using near infra-red fluorescence (NIR). Hepatocellular carcinoma cells (HepG-2) and human umbilical vein endothelial cells (HUVEC) were cultured with FNDP-(NV) and assayed for particle uptake and location using spectrophotometric technology and microscopy. RESULTS: HepG-2 and HUVEC displayed rapid (<30 mins) onset and concentration-dependent FNDP-(NV) internalization and formation of peri-nuclear corona. FM/CFM of liver sections revealed FNDP-(NV) presence throughout the hepatic lobules structures marked by spatial distribution, venous microvascular spaces and parenchyma and non-parenchyma cells. CONCLUSION: The robust presence of FNDP-(NV) throughout the hepatic lobules including those internalized within parenchyma cells and agglomerates in the liver venous micro-circulation were not associated with macro or micro histopathological signs nor vascular lesions. Cells cultures indicated normal cytokinesis in cells containing FNDP-(NV) agglomerates. Liver parenchyma cells and the liver microcirculation remain agnostic to presence of FNDP-(NV) in the sinusoids or internalized in the hepatic cells.

Long-term biocompatibility of fluorescent diamonds-(NV)-Z~800 nm in rats: survival, morbidity, histopathology, particle distribution and excretion studies (part IV).

BACKGROUND: Thromboembolic events are a major cause of heart attacks and strokes. However, diagnosis of the location of high risk vascular clots is hampered by lack of proper technologies for their detection. We recently reported on bio-engineered fluorescent diamond-(NV)-Z~800nm (FNDP-(NV)) conjugated with bitistatin (Bit) and proven its ability to identify iatrogenic blood clots in the rat carotid artery in vivo by Near Infra-Red (NIR) monitored by In Vivo Imaging System (IVIS). PURPOSE: The objective of the present research was to assess the in vivo biocompatibility of FNDP-(NV)-Z~800nm infused intravenously to rats. Multiple biological variables were assessed along this 12 week study commissioned in anticipation of regulatory requirements for a long-term safety assessment. METHODS: Rats were infused under anesthesia with aforementioned dose of the FNDP-(NV), while equal number of animals served as control (vehicle treated). Over the 12 week observation period rats were tested for thriving, motor, sensory and cognitive functions. At the termination of study, blood samples were obtained under anesthesia for comprehensive hematology and biochemical assays. Furthermore, 6 whole organs (liver, spleen, brain, heart, lung and kidney) were collected and examined ex vivo for FNDP-NV) via NIR monitored by IVIS and histochemical inspection. RESULTS: All animals survived, thrived (no change in body and organ growth). Neuro-behavioral functions remain intact. Hematology and biochemistry (including liver and kidney functions) were normal. Preferential FNDP-(NV) distribution identified the liver as the main long-term repository. Certified pathology reports indicated no outstanding of finding in all organs. CONCLUSION: The present study suggests outstanding biocompatibility of FNDP-(NV)-Z~800nm after long-term exposure in the rat.

Pilot study on biocompatibility of fluorescent nanodiamond-(NV)-Z~800 particles in rats: safety, pharmacokinetics, and bio-distribution (part III).

INTRODUCTION: We hereby report on studies aimed to characterize safety, pharmacokinetics, and bio-distribution of fluorescent nanodiamond particles (NV)-Z~800 (FNDP-(NV)) administered to rats by intravenous infusion in a single high dose. METHODS: Broad scale biological variables were monitored following acute (90 minutes) and subacute (5 or 14 days) exposure to FNDP-(NV). Primary endpoints included morbidity and mortality, while secondary endpoints focused on hematology and clinical biochemistry biomarkers. Particle distribution (liver, spleen, lung, heart, and kidney) was assessed by whole organ near infrared imaging using an in vivo imaging system. This was validated by the quantification of particles extracted from the same organs and visualized by fluorescent and scanning electron microscopy. FNDP-(NV)-treated rats showed no change in morbidity or mortality and preserved normal motor and sensory function, as assessed by six different tests. RESULTS: Blood cell counts and plasma biochemistry remained normal. The particles were principally distributed in the liver and spleen. The liver particle load accounted for 51%, 24%, and 18% at 90 minutes, 5 days, and 14 days, respectively. A pilot study of particle clearance from blood indicated 50% clearance 33 minutes following the end of particle infusion. CONCLUSION: We concluded that systemic exposure of rats to a single high dose of FDNP-(NV)-Z~800 (60 mg/kg) appeared to be safe and well tolerated over at least 2 weeks. These data suggest that FNDP-(NV) should proceed to preclinical development in the near future.

Vascular thrombus imaging in vivo via near-infrared fluorescent nanodiamond particles bioengineered with the disintegrin bitistatin (Part II).

The aim of this feasibility study was to test the ability of fluorescent nanodiamond particles (F-NDP) covalently conjugated with bitistatin (F-NDP-Bit) to detect vascular blood clots in vivo using extracorporeal near-infrared (NIR) imaging. Specifically, we compared NIR fluorescence properties of F-NDP with N-V (F-NDPNV) and N-V-N color centers and sizes (100-10,000 nm). Optimal NIR fluorescence and tissue penetration across biological tissues (rat skin, porcine axillary veins, and skin) was obtained for F-NDPNV with a mean diameter of 700 nm. Intravital imaging (using in vivo imaging system [IVIS]) in vitro revealed that F-NDPNV-loaded glass capillaries could be detected across 6 mm of rat red-muscle barrier and 12 mm of porcine skin, which equals the average vertical distance of a human carotid artery bifurcation from the surface of the adjacent skin (14 mm). In vivo, feasibility was demonstrated in a rat model of occlusive blood clots generated using FeCl3 in the carotid artery bifurcation. Following systemic infusions of F-NDPNV-Bit (3 or 15 mg/kg) via the external carotid artery or femoral vein (N=3), presence of the particles in the thrombi was confirmed both in situ via IVIS, and ex vivo via confocal imaging. The presence of F-NDPNV in the vascular clots was further confirmed by direct counting of fluorescent particles extracted from clots following tissue solubilization. Our data suggest that F-NDPNV-Bit associate with vascular blood clots, presumably by binding of F-NDPNV-Bit to activated platelets within the blood clot. We posit that F-NDPNV-Bit could serve as a noninvasive platform for identification of vascular thrombi using NIR energy monitored by an extracorporeal device.

The prolyl 4-hydroxylase inhibitor GSK360A decreases post-stroke brain injury and sensory, motor, and cognitive behavioral deficits.

There is interest in pharmacologic preconditioning for end-organ protection by targeting the HIF system. This can be accomplished by inhibition of prolyl 4-hydroxylase (PHD). GSK360A is an orally active PHD inhibitor that has been previously shown to protect the failing heart. We hypothesized that PHD inhibition can also protect the brain from injuries and resulting behavioral deficits that can occur as a result of surgery. Thus, our goal was to investigate the effect of pre-stroke surgery brain protection using a verified GSK360A PHD inhibition paradigm on post-stroke surgery outcomes. Vehicle or an established protective dose (30 mg/kg, p.o.) of GSK360A was administered to male Sprague-Dawley rats. Initially, GSK360A pharmacokinetics and organ distribution were determined, and then PHD-HIF pharmacodynamic markers were measured (i.e., to validate the pharmacological effects of the GSK360A administration regimen). Results obtained using this validated PHD dose-regimen indicated significant improvement by GSK360A (30mg/kg); administered at 18 and 5 hours prior to transient middle cerebral artery occlusion (stroke). GSK360A exposure and plasma, kidney and brain HIF-PHD pharmacodynamics endpoints (e.g., erythropoietin; EPO and Vascular Endothelial Growth Factor; VEGF) were measured. GSK360A provided rapid exposure in plasma (7734 ng/ml), kidney (45-52% of plasma level) and brain (1-4% of plasma level), and increased kidney EPO mRNA (80-fold) and brain VEGF mRNA (2-fold). We also observed that GSK360A increased plasma EPO (300-fold) and VEGF (2-fold). Further assessments indicated that GSK360A reduced post-stroke surgery neurological deficits (47-64%), cognitive dysfunction (60-75%) and brain infarction (30%) 4 weeks later. Thus, PHD inhibition using GSK360A pretreatment produced long-term post-stroke brain protection and improved behavioral functioning. These data support PHD inhibition, specifically by GSK360A, as a potential strategy for pre-surgical use to reduce brain injury and functional decline due to surgery-related cerebral injury.

First translational 'Think Tank' on cerebrovascular disease, cognitive impairment and dementia.

As the human population continues to age, an increasing number of people will exhibit significant deficits in cognitive function and dementia. It is now recognized that cerebrovascular, metabolic and neurodegenerative diseases all play major roles in the evolution of cognitive impairment and dementia. Thus with our more recent recognition of these relationships and our need to understand and more positively impact on this world health problem, "The Leo and Anne Albert Charitable Trust" (Gene Pranzo, Trustee with significant support from Susan Brogan, Meeting Planner) provided generous support for this inaugural international workshop that was held from April 13-16, 2015 at the beautiful Ritz Carlton Golf Resort in North Naples, Florida. Researchers from SUNY Downstate Medical Center, Brooklyn, NY organized the event by selecting the present group of translationally inclined preclinical, clinical and population scientists focused on cerebrovascular disease (CVD) risk and its progression to vascular cognitive impairment (VCI) and dementia. Participants at the workshop addressed important issues related to aging, cognition and dementia by: (1) sharing new data, information and perspectives that intersect vascular, metabolic and neurodegenerative diseases, (2) discussing gaps in translating population risk, clinical and preclinical information to the progression of cognitive loss, and (3) debating new approaches and methods to fill these gaps that can translate into future therapeutic interventions. Participants agreed on topics for group discussion prior to the meeting and focused on specific translational goals that included promoting better understanding of dementia mechanisms, the identification of potential therapeutic targets for intervention, and discussed/debated the potential utility of diagnostic/prognostic markers. Below summarizes the new data-presentations, concepts, novel directions and specific discussion topics addressed by this international translational team at our "First Leo and Anne Albert Charitable Trust 'Think Tank' VCI workshop".

Endovascular external carotid artery occlusion for brain selective targeting: a cerebrovascular swine model.

BACKGROUND: The choice of an animal model for cerebrovascular research is often determined by the disease subtype to be studied (e.g. ischemic stroke, hemorrhage, trauma), as well as the nature of the intervention to be tested (i.e. medical device or pharmaceutical). Many initial studies are performed in smaller animals, as they are cost-effective and their encephalic vasculature closely models that of humans. Non-human primates are also utilized when confirmation or validation is required on higher levels and to test larger devices. However, working with primates is complex and expensive. Intermediate sized animal models, such as swine and sheep, may represent a valuable compromise. Their cerebrovascular anatomy, however, comes with challenges because of the natural higher external carotid artery perfusion and the existence of a rete mirabile. We describe a modification to the traditional swine cerebrovascular model that significantly enhances selective brain hemispheric perfusion, limiting external carotid perfusion and dilution. RESULTS: We investigated whether unilateral endovascular coil-embolization of external carotid artery branches in swine would lead to increased brain perfusion, altering cerebral circulation so that it more closely models human cerebral circulation. Equal amounts of approximately 4 °C cold saline were injected in 6 Yorkshire pigs into the ipsilateral common carotid artery before and after embolization. Hemispheric temperature changes from pre- and post-embolization were obtained as a measure of brain perfusion and averaged and compared using non-parametric statistical tests (Wilcoxon signed rank test, Mann-Whitney U Test). Graphs were plotted with absolute changes in hemispheric temperature over time to determine peak temperature drop (PTD) and corresponding time to peak (TTP) following the cold bolus injection. There was a 288 ± 90% increase in ipsilateral brain cooling after embolization indicating improved selective blood flow to the brain due to this vascular modification. CONCLUSION: We have developed an effective, selective vascular brain model in swine that may be useful as a practical and cost-reducing intermediate step for evaluating target dose-responses for central nervous system drugs and brain selective interventions, such as local hypothermia.

Feasibility of near-infrared spectroscopic tomography for intraoperative functional cerebral monitoring: a primate study.

OBJECTIVE: The wide-ranging manipulations to the cardiovascular system that frequently occur during cardiac surgery can expose the brain to variations in its blood supply that could prove deleterious. As a first step to developing a resource suitable for monitoring such changes, we detected the hemodynamic events induced in the brain of a primate model, using high-density near-infrared spectroscopy combined with tomographic reconstruction methods and validated the findings using established radiologic and histologic techniques. METHODS: Continuous monitoring of the relative changes in the components of the cerebral hemoglobin signal was performed using high-density near-infrared spectroscopy (270 source-detector channel array) in anesthetized bonnet macaques with the brain exposed to induced ischemia and other acute events. A comparative analysis (exact binomial test) applied to reconstructed 3-dimensional images before and after the events and between cerebral hemispheres, combined with postprocedure magnetic resonance imaging, and postmortem histopathologic examination of the macaques' brains was performed to document and validate the spatial features revealed by the optical findings. RESULTS: Relative changes in the measured and calculated components of the hemoglobin signal, in response to the performed manipulations, revealed substantial concurrence among the reconstructed 3-dimensional images, magnetic resonance imaging of the macaques' brains, and postmortem histopathologic examination findings. Concurrence was seen when the manipulated hemoglobin concentration and associated oxygenation levels were either increased or decreased, and whether they were bilateral or restricted to a specified hemisphere. CONCLUSIONS: Continuous near-infrared spectroscopy tomography has been shown to accurately capture and localize cerebral ischemia, vasodilatation, and hemorrhage in primates in real time. These findings are directly applicable to clinical intraoperative functional cerebral monitoring.

Long-term post-stroke changes include myelin loss, specific deficits in sensory and motor behaviors and complex cognitive impairment detected using active place avoidance.

Persistent neurobehavioral deficits and brain changes need validation for brain restoration. Two hours middle cerebral artery occlusion (tMCAO) or sham surgery was performed in male Sprague-Dawley rats. Neurobehavioral and cognitive deficits were measured over 10 weeks included: (1) sensory, motor, beam balance, reflex/abnormal responses, hindlimb placement, forepaw foot fault and cylinder placement tests, and (2) complex active place avoidance learning (APA) and simple passive avoidance retention (PA). Electroretinogram (ERG), hemispheric loss (infarction), hippocampus CA1 neuronal loss and myelin (Luxol Fast Blue) staining in several fiber tracts were also measured. In comparison to Sham surgery, tMCAO surgery produced significant deficits in all behavioral tests except reflex/abnormal responses. Acute, short lived deficits following tMCAO were observed for forelimb foot fault and forelimb cylinder placement. Persistent, sustained deficits for the whole 10 weeks were exhibited for motor (p<0.001), sensory (p<0.001), beam balance performance (p<0.01) and hindlimb placement behavior (p<0.01). tMCAO produced much greater and prolonged cognitive deficits in APA learning (maximum on last trial of 604±83% change, p<0.05) but only a small, comparative effect on PA retention. Hemispheric loss/atrophy was measured 10 weeks after tMCAO and cross-validated by two methods (e.g., almost identical % ischemic hemispheric loss of 33.4±3.5% for H&E and of 34.2±3.5% for TTC staining). No visual dysfunction by ERG and no hippocampus neuronal loss were detected after tMCAO. Fiber tract damage measured by Luxol Fast Blue myelin staining intensity was significant (p<0.01) in the external capsule and striatum but not in corpus callosum and anterior commissure. In summary, persistent neurobehavioral deficits were validated as important endpoints for stroke restorative research in the future. Fiber myelin loss appears to contribute to these long term behavioral dysfunctions and can be important for cognitive behavioral control necessary for complex APA learning.

Comparison of passive leg raising and hyperemia on macrovascular and microvascular responses.

Passive leg raising is a simple diagnostic maneuver that has been proposed as a measure of arterial vasodilator reserve and possibly endothelial function. While passive leg raising has previously been shown to lower blood pressure, increase flow velocity and cause brachial artery dilation, its effects on microvascular flow has not been well studied. Also, passive leg raising has been directly compared previously to upper arm but never to lower arm occlusion of blood flow induced hyperemia responses. We compared changes in macrovascular indices measured by brachial artery ultrasound and microvascular perfusion measured by Laser Doppler Flowmetry induced by passive leg raising to those provoked by upper arm and lower arm induced hyperemia in healthy subjects. Upper arm induced hyperemia increased mean flow velocity by 398%, induced brachial artery dilatation by 16.3%, and increased microvascular perfusion by 246% (p<.05 for all). Lower arm induced hyperemia increased flow velocity by 227%, induced brachial artery dilatation by 10.8%, and increased microvascular perfusion by 281%. Passive leg raising increased flow velocity by 29% and brachial artery dilatation by 5.6% (p<.05 for all), but did not change microvascular perfusion (-5%, p=ns). In conclusion, passive leg raising increases flow velocity orders of magnitude less than does upper arm or lower arm induced hyperemia. Passive leg raising-induced brachial artery dilatation is less robust than either of these hyperemic techniques. Finally, although upper arm and lower arm hyperemia elicits macrovascular and microvascular responses, passive leg raising elicits only macrovascular responses.

A novel calpain inhibitor for treatment of transient retinal ischemia in the rat.

After an acute ischemia/reperfusion of the rat retina, the activation of cytotoxic proteases, including calpain, results in necrosis and apoptosis of retinal ganglion cells resulting in their degeneration. Using a systemically administered calpain inhibitor that crosses the blood-retinal barrier would provide for novel systemic intervention that protects the retina from acute injury and loss of function. Herein, we study a novel calpain peptide inhibitor, cysteic-leucyl-argininal (CYLA), in an in-vivo rat model of retinal ischemia to determine functional protection using electroretinography. The CYLA prodrug was administered intraperitoneally before and/or after ischemia-reperfusion at concentrations of 20-40 mg/kg. We found that administering 20 mg/kg of CYLA only after ischemia provides significant preservation of retinal function.

Thrombopoietin protects the brain and improves sensorimotor functions: reduction of stroke-induced MMP-9 upregulation and blood-brain barrier injury.

This study was conducted to determine the protective efficacy and mechanisms of thrombopoietin (TPO) intervention in experimental focal stroke. Male rats underwent 2 hours of left middle cerebral artery occlusion (MCAO) followed by 22 hours of reperfusion. Vehicle or TPO (0.03 to 1.00 µg/kg) was administered intravenously immediately after reperfusion. Brain infarct and swelling, neurologic deficits, matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), TPO and c-Mpl (TPO receptor) mRNA, MMP-9 enzyme activity and protein expression, and the integrity of the blood-brain barrier (BBB) were subsequently measured. MCAO reperfusion produced a large infarct and swelling after stroke. Thrombopoietin significantly reduced these in a dose-dependent manner. The most effective TPO dose, 0.1 µg/kg, when administrated immediately or 2 hours after reperfusion, significantly reduced infarct and swelling and ameliorated neurologic deficits after stroke. Stroke-induced increases in cortical MMP-9 mRNA, enzyme activity and protein expression, TIMP-1 mRNA, and Evans blue extravasation were reduced by TPO intervention. Thrombopoietin did not alter cortical TPO or c-Mpl mRNA expression, blood pressure, heart rate, blood hematocrit, or platelets. This is the first demonstration of TPO's efficacy in reducing ischemic brain injury and improving functional outcome, partly by inhibiting the stroke-induced increase in MMP-9 and the early, negative effects on the BBB.

Selective CETP inhibition and PPARalpha agonism increase HDL cholesterol and reduce LDL cholesterol in human ApoB100/human CETP transgenic mice.

Cholesteryl ester transfer protein (CETP) plays a key role in high-density lipoprotein (HDL) cholesterol metabolism, but normal mice are deficient in CETP. In this study, transgenic mice expressing both human apolipoprotein B 100 (ApoB-100) and human CETP (hApoB100/hCETP) were used to characterize the effects of CETP inhibition and peroxisome proliferator-activated receptor alpha (PPARalpha) agonism on lipid profiles. Torcetrapib (3, 10, and 30 mg/kg), a CETP inhibitor, fenofibrate (30 mg/kg), a weak PPARalpha agonist, and GW590735 (3 and 10 mg/kg), a potent and selective PPARalpha agonist were given orally for 14 days to hApoB100/hCETP mice and lipid profiles were assessed. The average percentages of HDL, low-density lipoprotein (LDL), and very-low-density lipoprotein (VLDL) cholesterol fractions in hApoB100/hCETP mice were 34.8%, 61.6%, and 3.6%, respectively, which is similar to those of normolipidemic humans. Both torcetrapib and fenofibrate significantly increased HDL cholesterol and reduced LDL cholesterol, and there was a tendency for torcetrapib to reduce VLDL cholesterol and triglycerides. GW590735 significantly increased HDL cholesterol, decreased LDL and VLDL cholesterol, and significantly reduced triglycerides. Maximal increases in HDL cholesterol were 37%, 53%, and 84% with fenofibrate, torcetrapib, and GW590735, respectively. These results, in mice that exhibit a more human-like lipid profile, demonstrate an improved lipid profile with torcetrapib, fenofibrate, and GW590735, and support the use of selective PPARalpha agonism for the treatment of lipid disorders. In addition, these data demonstrate the use of hApoB100/hCETP transgenic mice to identify, characterize, and screen compounds that increase HDL cholesterol.

Pharmacologic interventions for stroke: looking beyond the thrombolysis time window into the penumbra with biomarkers, not a stopwatch.

BACKGROUND AND PURPOSE: The majority of pharmacological agents for stroke were developed based on the assumption that neurological deficits will be reduced upon the successful interruption of biochemical mechanisms leading to neuronal death. Despite significant evidence of preclinical efficacy, none of these agents succeeded. They either failed to demonstrate efficacy in the clinic or their development was halted for safety, strategic, or commercial reasons. SUMMARY OF REVIEW: This "neuroprotection strategy" has focused primarily on targets in the neurotoxic environment that occurs under ischemic conditions. In many cases, these agents were designed to tackle events that are known to start almost immediately after onset of ischemia, which is far before a realistic therapeutic time window opens for most, if not all, patients with stroke. In other instances, they were evaluated beyond a realistic timeframe in which one could expect significant salvageable tissue or penumbra to exist. Surprisingly, most of these agents were not evaluated in conjunction with strategies for improving perfusion to the affected tissue, indicating an overoptimistic assumption that neuroprotection alone could be sufficient to halt injury caused by an abrupt interruption of brain blood flow. CONCLUSIONS: We provide a constructive translational medicine perspective about how one could improve the drug development process with the hope that the probability for success can increase in our quest to establish a novel therapy for stroke.