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.

Scutellarin enhances anti-tumor immune responses by reducing TNFR2-expressing CD4+Foxp3+ regulatory T cells.

One characteristic of tumor-associated CD4+Foxp3+ regulatory T cells (Tregs) is the high expression of tumor necrosis factor receptor type II (TNFR2), a receptor that mediates the decisive effect of tumor necrosis factor (TNF) in the activation and expansion of Tregs. There is increasing evidence that inhibition of TNFR2 can enhance anti-tumor immune responses. Therefore, we screened Chinese herbal extracts for their capacity to block TNF-TNFR2 interaction. The results showed that the treatment with a Chinese herb extract could inhibit TNFR2-induced biological responses in vitro, including the proliferation of TNFR2+ Tregs. Our subsequent study led to the identification of flavonoid compound scutellarin was responsible for the activity. Our results showed that scutellarin is able to disrupt the interaction of TNF-TNFR2 and inhibited the phosphorylation of p38 MAPK, a down-stream signaling component of TNFR2. Importantly, in vivo scutellarin treatment markedly enhanced the efficacy of tumor immunotherapy with CpG oligodeoxynucleotide in mouse CT26 colon cancer model. This effect of scutellarin was associated with the reduction of the number of tumor-infiltrating TNFR2-expressing Tregs and increased tumor infiltration of interferon-γ-producing CD8+ T cells. Our result also suggests that scutellarin or its analogs may be used as an adjuvant to enhance the anti-tumor effect of immunotherapeutic agent by eliminating TNFR2+ Treg activity.

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.

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.