Red Cell Microparticles Suppress Hematoma Growth Following Intracerebral Hemorrhage in Chronic Nicotine-Exposed Rats.

Spontaneous intracerebral hemorrhage (sICH) is a disabling stroke sub-type, and tobacco use is a prominent risk factor for sICH. We showed that chronic nicotine exposure enhances bleeding post-sICH. Reduction of hematoma growth is a promising effective therapy for sICH in smoking subjects. Red-blood-cell-derived microparticles (RMPs) are hemostatic agents that limit hematoma expansion following sICH in naïve rats. Considering the importance of testing the efficacy of experimental drugs in animal models with a risk factor for a disease, we tested RMP efficacy and the therapeutic time window in limiting hematoma growth post-sICH in rats exposed to nicotine. Young rats were chronically treated with nicotine using osmotic pumps. sICH was induced in rats using an injection of collagenase in the right striatum. Vehicle/RMPs were administered intravenously. Hematoma volume and neurological impairment were quantified ≈24 h after sICH. Hematoma volumes in male and female nicotine-exposed rats that were treated with RMPs at 2 h post-sICH were significantly lower by 26 and 31% when compared to their respective control groups. RMP therapy was able to limit hematoma volume when administered up to 4.5 h post-sICH in animals of both sexes. Therefore, RMPs may limit hematoma growth in sICH patients exposed to tobacco use.

Red Blood Cell Microparticles Limit Hematoma Growth in Intracerebral Hemorrhage.

BACKGROUND: Spontaneous intracerebral hemorrhage (sICH) is the deadliest stroke subtype with no effective therapies. Limiting hematoma expansion is a promising therapeutic approach. Red blood cell-derived microparticles (RMPs) are novel hemostatic agents. Therefore, we studied the potential of RMPs in limiting hematoma growth and improving outcomes post-sICH. METHODS: sICH was induced in rats by intrastriatal injection of collagenase. RMPs were prepared from human RBCs by high-pressure extrusion. Behavioral and hematoma/lesion volume assessment were done post-sICH. The optimal dose, dosing regimen, and therapeutic time window of RMP therapy required to limit hematoma growth post-sICH were determined. We also evaluated the effect of RMPs on long-term behavioral and histopathologic outcomes post-sICH. RESULTS: RMP treatment limited hematoma growth following sICH. Hematoma volume (mm3) for vehicle- and RMP- (2.66×1010 particles/kg) treated group was 143±8 and 86±4, respectively. The optimal RMP dosing regimen that limits hematoma expansion was identified. RMPs limit hematoma volume when administered up to 4.5-hour post-sICH. Hematoma volume in the 4.5-hour post-sICH RMP treatment group was lower by 24% when compared with the control group. RMP treatment also improved long-term histopathologic and behavioral outcomes post-sICH. CONCLUSIONS: Our results demonstrate that RMP therapy limits hematoma growth and improves outcomes post-sICH in a rodent model. Therefore, RMPs have the potential to limit hematoma growth in sICH patients.

Chronic Nicotine Exposure Increases Hematoma Expansion following Collagenase-Induced Intracerebral Hemorrhage in Rats.

Spontaneous intracerebral hemorrhage (sICH) is a deadly stroke subtype, and tobacco use increases sICH risk. However epidemiological studies show that, there are no confirmatory studies showing the effect of tobacco use on sICH outcome. Therefore, we evaluated the effect of chronic nicotine exposure (as a surrogate for tobacco use) on outcomes following sICH. Young male and female rats were randomly assigned to either nicotine (4.5 mg/kg b.w. per day) or vehicle (saline) treatment (2-3 weeks) groups. sICH was induced by injecting collagenase into the right striatum. Neurological score and hematoma volume were determined 24 h post-sICH. The hematoma volumes in nicotine-treated male and female rats were significantly higher by 42% and 48% when compared to vehicle-treated male and female rats, respectively. Neurological deficits measured in terms of neurological score for the nicotine-treated male and female groups were significantly higher when compared to the respective vehicle-treated male and female groups. Our results show that chronic nicotine exposure increases hematoma volume post-sICH in rats of both sexes. Identifying the mechanism of nicotine-dependent increase in hematoma growth post-sICH will be crucial to understanding the detrimental effect of tobacco use on the severity of bleeding following intracerebral hemorrhage.

Ischemic brain injury in diabetes and endoplasmic reticulum stress.

Diabetes is a widespread disease characterized by high blood glucose levels due to abnormal insulin activity, production, or both. Chronic diabetes causes many secondary complications including cardiovascular disease: a life-threatening complication. Cerebral ischemia-related mortality, morbidity, and the extent of brain injury are high in diabetes. However, the mechanism of increase in ischemic brain injury during diabetes is not well understood. Multiple mechanisms mediate diabetic hyperglycemia and hypoglycemia-induced increase in ischemic brain injury. Endoplasmic reticulum (ER) stress mediates both brain injury as well as brain protection after ischemia-reperfusion injury. The pathways of ER stress are modulated during diabetes. Free radical generation and mitochondrial dysfunction, two of the prominent mechanisms that mediate diabetic increase in ischemic brain injury, are known to stimulate the pathways of ER stress. Increased ischemic brain injury in diabetes is accompanied by a further increase in the activation of ER stress. As there are many metabolic changes associated with diabetes, differential activation of the pathways of ER stress may mediate pronounced ischemic brain injury in subjects suffering from diabetes. We presently discuss the literature on the significance of ER stress in mediating increased ischemia-reperfusion injury in diabetes.

Tobacco Use: A Major Risk Factor of Intracerebral Hemorrhage.

Spontaneous intracerebral hemorrhage (sICH) is one of the deadliest subtypes of stroke, and no treatment is currently available. One of the major risk factors is tobacco use. In this article, we review literature on how tobacco use affects the risk of sICH and also summarize the known effects of tobacco use on outcomes following sICH. Several studies demonstrate that the risk of sICH is higher in current cigarette smokers compared to non-smokers. The literature also establishes that cigarette smoking not only increases the risk of sICH but also increases hematoma growth, results in worse outcomes, and increases the risk of death from sICH. This review also discusses potential mechanisms activated by tobacco use which result in an increase in risk and severity of sICH. Exploring the underlying mechanisms may help alleviate the risk of sICH in tobacco users as well as may help better manage tobacco user sICH patients.

Exposure to recurrent hypoglycemia alters hippocampal metabolism in treated streptozotocin-induced diabetic rats.

AIMS: Exposure to recurrent hypoglycemia (RH) is common in diabetic patients receiving glucose-lowering therapies and is implicated in causing cognitive impairments. Despite the significant effect of RH on hippocampal function, the underlying mechanisms are currently unknown. Our goal was to determine the effect of RH exposure on hippocampal metabolism in treated streptozotocin-diabetic rats. METHODS: Hyperglycemia was corrected by insulin pellet implantation. Insulin-treated diabetic (ITD) rats were exposed to mild/moderate RH once a day for 5 consecutive days. RESULTS: The effect of RH on hippocampal metabolism revealed 65 significantly altered metabolites in the RH group compared with controls. Several significant differences in metabolite levels belonging to major pathways (eg, Krebs cycle, gluconeogenesis, and amino acid metabolism) were discovered in RH-exposed ITD rats when compared to a control group. Key glycolytic enzymes including hexokinase, phosphofructokinase, and pyruvate kinase were affected by RH exposure. CONCLUSION: Our results demonstrate that the exposure to RH leads to metabolomics alterations in the hippocampus of insulin-treated streptozotocin-diabetic rats. Understanding how RH affects hippocampal metabolism may help attenuate the adverse effects of RH on hippocampal functions.

Automated Assessment of Hematoma Volume of Rodents Subjected to Experimental Intracerebral Hemorrhagic Stroke by Bayes Segmentation Approach.

Simulating a clinical condition of intracerebral hemorrhage (ICH) in animals is key to research on the development and testing of diagnostic or treatment strategies for this high-mortality disease. In order to study the mechanism, pathology, and treatment for hemorrhagic stroke, various animal models have been developed. Measurement of hematoma volume is an important assessment parameter to evaluate post-ICH outcomes. However, due to tissue preservation conditions and variables in digitization, quantification of hematoma volume is usually labor intensive and sometimes even subjective. The objective of this study is to develop an automated method that can accurately and efficiently obtain unbiased cerebral hematoma volume. We developed an application (MATLAB program) that can delineate the brain slice from the background and use the Hue information in the Hue/Saturation/Value (HSV) color space to segment the hematoma region. The segmentation threshold of Hue is calculated based on the Bayes classifier theorem so that the minimum error is mathematically ensured and automated processing is enabled. To validate the developed method, we compared the outcomes from the developed method with the hemoglobin content by the spectrophotometric assay method. The results were linearly correlated with statistical significance. The method was also validated by digital phantoms with an error less than 5% compared with the ground truth from the phantoms. Hematoma volumes yielded by the automated processing and those obtained by the operator's manual operation are highly correlated. This automated segmentation approach can be potentially used to quantify hemorrhagic outcomes in rodent stroke models in an unbiased and efficient way.

Preclinical Evaluation of Safety and Biodistribution of Red Cell Microparticles: A Novel Hemostatic Agent.

BACKGROUND: Uncontrollable bleeding is a major cause of mortality and morbidity worldwide. Effective hemostatic agents are urgently needed. Red cell microparticles (RMPs) are a highly promising hemostatic agent. This study evaluated the safety profile of RMPs preliminary to clinical trial. METHODS AND RESULTS: RMPs were prepared from type O+ human red blood cell by high-pressure extrusion. Male rats were treated with RMPs either a 1 × bolus, or 4 × or 20 × administered over 60 minutes. The vehicle-treated group was used as a control. Effects on physiological parameters were evaluated; namely, blood pressure, body and head temperature, hematocrit, and blood gases. We did not observe any adverse effects of RMPs on these physiological parameters. In addition, brain, heart, and lungs of rats treated with 4 × dose (bolus followed by infusion over 60 minutes) or vehicle were examined histologically for signs of thrombosis or other indications of toxicity. No thrombosis or indications of toxicity in brain, heart, or lungs were observed. Studies revealed that RMPs were distributed mainly in liver, spleen, and lymph nodes, and were potentially excreted through the kidneys. CONCLUSIONS: Our study indicates that RMP administration appears not to have any negative impact on the parameters studied and did not produce thrombosis in heart, brain, and lungs. However, more detailed long-term studies confirming the safety of RMP as a hemostatic agent are warranted.

Blockade of Acid-Sensing Ion Channels Attenuates Recurrent Hypoglycemia-Induced Potentiation of Ischemic Brain Damage in Treated Diabetic Rats.

Diabetes is a chronic metabolic disease and cerebral ischemia is a serious complication of diabetes. Anti-diabetic therapy mitigates this complication but increases the risk of exposure to recurrent hypoglycemia (RH). We showed previously that RH exposure increases ischemic brain damage in insulin-treated diabetic (ITD) rats. The present study evaluated the hypothesis that increased intra-ischemic acidosis in RH-exposed ITD rats leads to pronounced post-ischemic hypoperfusion via activation of acid-sensing (proton-gated) ion channels (ASICs). Streptozotocin-diabetic rats treated with insulin were considered ITD rats. ITD rats were exposed to RH for 5 days and were randomized into Psalmotoxin1 (PcTx1, ASIC1a inhibitor), APETx2 (ASIC3 inhibitor), or vehicle groups. Transient global cerebral ischemia was induced overnight after RH. Cerebral blood flow was measured using laser Doppler flowmetry. Ischemic brain injury in hippocampus was evaluated using histopathology. Post-ischemic hypoperfusion in RH-exposed rats was of greater extent than that in control rats. Inhibition of ASICs prevented RH-induced increase in the extent of post-ischemic hypoperfusion and ischemic brain injury. Since ASIC activation-induced store-operated calcium entry (SOCE) plays a role in vascular tone, next we tested if acidosis activates SOCE via activating ASICs in vascular smooth muscle cells (VSMCs). We observed that SOCE in VSMCs at lower pH is ASIC3 dependent. The results show the role of ASIC in post-ischemic hypoperfusion and increased ischemic damage in RH-exposed ITD rats. Understanding the pathways mediating exacerbated ischemic brain injury in RH-exposed ITD rats may help lower diabetic aggravation of ischemic brain damage.

Pharmacokinetics of Human Red Blood Cell Microparticles Prepared Using High-Pressure Extrusion Method.

Red blood cell microparticles (RMPs) is a high potency hemostatic agent, which may serve as a viable therapeutic approach. They generate thrombin in vitro and effective in arresting bleeding in animal bleeding models. However, prior to ascertaining the clinical efficacy of RMPs, detailed preclinical evaluation is necessary. Therefore, we aimed to characterize RMPs, ascertain their stability, and determine their pharmacokinetics in rats. RMPs were prepared from human RBCs by a high-pressure extrusion method. Pharmacokinetic parameters were computed from groups receiving various RMPs dosing regimens. Volume of distribution, elimination rate constant, and clearance for RMPs were also assessed. Major portion of prepared microparticles were RMPs and a very small portion of particles were from platelets and leukocytes. RMPs were stable when stored at 5 and -20°C for at least 12 months. In vivo half-life was found to vary for each paradigm, but in general, was less than 2 min for most of the paradigms evaluated. Our results demonstrate that RMPs are stable during prolonged storage and have a short half-life. Therefore, the clinical use of RMPs as a hemostatic agent, within a tailored treatment paradigm, may be advantageous in achieving prolonged systemic therapeutic benefit without provoking any thrombotic complications.

Ameliorative potential of conditioning on ischemia-reperfusion injury in diabetes.

Diabetes is a serious metabolic disease characterized by hyperglycemia. Diabetes also leads to several long-term secondary complications. Cardiovascular disease is an important complication of diabetes and is a major contributor to morbidity and mortality in diabetic subjects. The discovery of conditioning-induced ischemic or anoxic tolerance has led to the demonstration of the protective potential of conditioning as a treatment strategy to mitigate ischemia-reperfusion injury. Diabetes modulates multiple metabolic pathways and signal transduction cascades. Some of these pathways may overlap with mechanisms that mediate the beneficial effects of conditioning from the body's reaction to a sublethal insult, indicating the possibility of a potential interaction between diabetes and conditioning. Studies demonstrate that diabetes abrogates the ameliorative effect of various forms of conditioning, such as ischemic preconditioning, ischemic postconditioning, remote ischemic conditioning and pharmacological conditioning, on ischemia-reperfusion injury in various animal models. Moreover, drugs used to treat diabetes may have a potential impact on protection afforded by conditioning from ischemic injury. Studies also indicate a potential impact of various anti-diabetic drugs on conditioning-induced protection. Overall, the literature suggests that a better understanding of the overlap among pathways activated by diabetes and those involved in induction of ischemia tolerance may help identify ideal conditioning paradigms to protect diabetic subjects from ischemic injury.

Impact of Hypoglycemia on Brain Metabolism During Diabetes.

Diabetes is a metabolic disease afflicting millions of people worldwide. A substantial fraction of world's total healthcare expenditure is spent on treating diabetes. Hypoglycemia is a serious consequence of anti-diabetic drug therapy, because it induces metabolic alterations in the brain. Metabolic alterations are one of the central mechanisms mediating hypoglycemia-related functional changes in the brain. Acute, chronic, and/or recurrent hypoglycemia modulate multiple metabolic pathways, and exposure to hypoglycemia increases consumption of alternate respiratory substrates such as ketone bodies, glycogen, and monocarboxylates in the brain. The aim of this review is to discuss hypoglycemia-induced metabolic alterations in the brain in glucose counterregulation, uptake, utilization and metabolism, cellular respiration, amino acid and lipid metabolism, and the significance of other sources of energy. The present review summarizes information on hypoglycemia-induced metabolic changes in the brain of diabetic and non-diabetic subjects and the manner in which they may affect brain function.

Acidosis mediates recurrent hypoglycemia-induced increase in ischemic brain injury in treated diabetic rats.

OBJECTIVES: Cerebral ischemia is a serious possible manifestation of diabetic vascular disease. Recurrent hypoglycemia (RH) enhances ischemic brain injury in insulin-treated diabetic (ITD) rats. In the present study, we determined the role of ischemic acidosis in enhanced ischemic brain damage in RH-exposed ITD rats. METHODS: Diabetic rats were treated with insulin and mild/moderate RH was induced for 5 days. Three sets of experiments were performed. The first set evaluated the effects of RH exposure on global cerebral ischemia-induced acidosis in ITD rats. The second set evaluated the effect of an alkalizing agent (Tris-(hydroxymethyl)-aminomethane: THAM) on ischemic acidosis-induced brain injury in RH-exposed ITD rats. The third experiment evaluated the effect of the glucose transporter (GLUT) inhibitor on ischemic acidosis-induced brain injury in RH-exposed ITD rats. Hippocampal pH and lactate were measured during ischemia and early reperfusion for all three experiments. Neuronal survival in Cornu Ammonis 1 (CA1) hippocampus served as a measure of ischemic brain injury. FINDINGS: Prior RH exposure increases lactate concentration and decreases pH during ischemia and early reperfusion when compared to controls. THAM and GLUT inhibitor treatments attenuated RH-induced increase in ischemic acidosis. GLUT inhibitor treatment reduced the RH-induced increase in lactate levels. Both THAM and GLUT inhibitor treatments significantly decreased ischemic damage in RH-exposed ITD rats. CONCLUSIONS: Ischemia causes increased acidosis in RH-exposed ITD rats via a GLUT-sensitive mechanism. Exploring downstream pathways may help understand mechanisms by which prior exposure to RH increases cerebral ischemic damage.

Diabetic aggravation of stroke and animal models.

Cerebral ischemia in diabetics results in severe brain damage. Different animal models of cerebral ischemia have been used to study the aggravation of ischemic brain damage in the diabetic condition. Since different disease conditions such as diabetes differently affect outcome following cerebral ischemia, the Stroke Therapy Academic Industry Roundtable (STAIR) guidelines recommends use of diseased animals for evaluating neuroprotective therapies targeted to reduce cerebral ischemic damage. The goal of this review is to discuss the technicalities and pros/cons of various animal models of cerebral ischemia currently being employed to study diabetes-related ischemic brain damage. The rational use of such animal systems in studying the disease condition may better help evaluate novel therapeutic approaches for diabetes related exacerbation of ischemic brain damage.

Hyperglycemia / hypoglycemia-induced mitochondrial dysfunction and cerebral ischemic damage in diabetics.

Enhancement of ischemic brain damage is one of the most serious complications of diabetes. Studies from various in vivo and in vitro models of cerebral ischemia have led to an understanding of the role of mitochondria and complex interrelated mitochondrial biochemical pathways leading to the aggravation of ischemic neuronal damage. Advancements in the elucidation of the mechanisms of ischemic brain damage in diabetic subjects have revealed a number of key mitochondrial targets that have been hypothesized to participate in enhancement of brain damage. The present review initially discusses the neurobiology of ischemic neuronal injury, with special emphasis on the central role of mitochondria in mediating its pathogenesis and therapeutic targets. Later it further details the potential role of various biochemical mediators and second messengers causing widespread ischemic brain damage among diabetics via mitochondrial pathways. The present review discusses preclinical data which validates the significance of mitochondrial mechanisms in mediating the aggravation of ischemic cerebral injury in diabetes. Exploitation of these targets may provide effective therapeutic agents for the management of diabetes-related aggravation of ischemic neuronal damage.

Pharmacogenomics and pharmacogenetics of thiazolidinediones: role in diabetes and cardiovascular risk factors.

The most important goal in the treatment of patients with diabetes is to prevent the risk of cardiovascular disease (CVD), the first cause of mortality in these subjects. Thiazolidinediones (TZDs), a class of antidiabetic drugs, act as insulin sensitizers increasing insulin-dependent glucose disposal and reducing hepatic glucose output. TZDs including pioglitazone, rosiglitazone and troglitazone, by activating PPAR-γ have shown pleiotropic effects in reducing vascular risk factors and atherosclerosis. However, troglitazone was removed from the market due to its hepatoxicity, and rosiglitazone and pioglitazone both have particular warnings due to being associated with heart diseases. Specific genetic variations in genes involved in the pathways regulated by TDZs have demonstrated to modify the variability in treatment with these drugs, especially in their side effects. Therefore, pharmacogenomics and pharmacogenetics are an important tool in further understand intersubject variability per se but also to assess the therapeutic potential of such variability in drug individualization and therapeutic optimization.

Pharmacological modulation of farnesyltransferase subtype I attenuates mecamylamine-precipitated nicotine withdrawal syndrome in mice.

This study was designed to investigate the effect of FTI-276 trifluoroacetate, a selective inhibitor of subtype I, on the development of the mecamylamine-induced nicotine withdrawal syndrome. Mice were administered nicotine (2.5 mg/kg, subcutaneously) four times daily for 7 days. To precipitate nicotine withdrawal, mice were administered one injection of mecamylamine (3 mg/kg, intraperitoneally) 1 h after the last nicotine injection on the test day (day 8). Behavioral observations were made for a period of 30 min immediately after mecamylamine treatment. FTI-276 trifluoroacetate treatment markedly and dose-dependently attenuated the precipitated nicotine withdrawal syndrome, measured by a composite withdrawal severity score, jumping frequency, hyperalgesia in the tail flick test, and anxiety-like behavior in the elevated plus maze test. The results suggest that FTI-276 trifluoroacetate can inhibit the development of a precipitated nicotine withdrawal syndrome, and thus that farnesyltransferase subtype I may be a viable pharmacological target to tackle the problem of nicotine addiction.

Pharmacological modulation of geranylgeranyltransferase and farnesyltransferase attenuates opioid withdrawal in vivo and in vitro.

Geranylgeranyltransferase and farnesyltransferase I, are noted to mediate a number of signal transduction cascades which are known to be involved in the causation of opioid withdrawal syndrome. GGTI-2133 and FTI-276 are selective modulators of geranylgeranyltransferase and farnesyltransferase subtype 1 respectively. Therefore, the present study investigated the effect of GGTI-2133 and FTI-276 on propagation of morphine dependence and resultant withdrawal signs in vivo, in sub-chronic morphine mouse model, and in vitro, in isolated rat ileum. Morphine was administered twice daily for 5 days following which a single day 6 injection of naloxone (8 mg/kg, i.p.) precipitated opioid withdrawal syndrome in mice. Withdrawal syndrome was quantitatively assessed in terms of withdrawal severity score and the frequency of jumping, rearing, fore paw licking & circling. Naloxone induced contraction in morphine withdrawn isolated rat ileum was employed as an in vitro model of opioid withdrawal syndrome. An isobolographic study design was employed to assess a potential synergistic activity between GGTI-2133 and FTI-276. GGTI-2133 and FTI-276 dose dependently attenuated naloxone induced morphine withdrawal syndrome both in vivo and in vitro. GGTI-2133 was also observed to exert a synergistic interaction with FTI-276. It is concluded that GGTI-2133 and FTI-276 attenuate the propagation of morphine dependence and reduce withdrawal signs possibly by a geranylgeranyl transferase; farnesyltransferase activation pathway linked mechanisms potentially in an interdependent manner.

Ro 32-0432 attenuates mecamylamine-precipitated nicotine withdrawal syndrome in mice.

G protein-coupled receptor kinase 5 is noted to mediate a number of signal transduction cascades involved in the causation of nicotine withdrawal syndrome. Therefore, the present study investigated the effect of Ro 32-0432, a G protein-coupled receptor kinase 5 inhibitor, on propagation of nicotine dependence and resultant withdrawal signs in subchronic nicotine mouse model. Our experimental protocol consisted of administration of nicotine, (2.5 mg/kg, subcutaneously), four times daily for 7 days. In order to precipitate nicotine withdrawal, mice were given one injection of mecamylamine (3 mg/kg, intraperitoneally) 1 h after the last nicotine injection on the test day (day 8). Behavioral observations were made for a period of 30 min immediately after mecamylamine treatment. Withdrawal syndrome was quantitated in terms of a composite withdrawal severity score, jumping frequency, nicotine-induced hyperalgesia by tail flick method, and withdrawal syndrome-related anxiety was assessed by elevated plus maze test results. Ro 32-0432 dose dependently attenuated mecamylamine-induced nicotine withdrawal syndrome in mice. It is concluded that Ro 32-0432 attenuates the propagation of nicotine dependence and reduce withdrawal signs possibly by G protein-coupled receptor kinase 5 activation-linked mechanisms.