QiShenYiQi Inhibits Tissue Plasminogen Activator–Induced Brain Edema and Hemorrhage after Ischemic Stroke in Mice

Background: Thrombolysis with tissue plasminogen activator (tPA) remains the only approved drug therapy for acute ischemic stroke. However, delayed tPA treatment is associated with an increased risk of brain hemorrhage. In this study, we assessed whether QiShenYiQi (QSYQ), a compound Chinese medicine, can attenuate tPA-induced brain edema and hemorrhage in an experimental stroke model.Methods: Male mice were subjected to ferric chloride-induced carotid artery thrombosis followed by mechanical detachment of thrombi. Then mice were treated with QSYQ at 2.5 h followed by administration of tPA (10 mg/kg) at 4.5 h. Hemorrhage, infarct size, neurological score, cerebral blood flow, Evans blue extravasation, FITC-labeled albumin leakage, tight and adherens junction proteins expression, basement membrane proteins expression, matrix metalloproteinases (MMPs) expression, leukocyte adhesion, and leukocyte infiltration were assessed 24 h after tPA administration.Results: Compared with tPA alone treatments, the combination therapy of QSYQ and tPA significantly reduced hemorrhage, infarction, brain edema, Evans blue extravasation, albumin leakage, leukocyte adhesion, MMP-9 expression, and leukocyte infiltration at 28.5 h after stroke. The combination also significantly improved the survival rate, cerebral blood flow, tight and adherens junction proteins (occludin, claudin-5, junctional adhesion molecule-1, zonula occludens-1, VE-cadherin, α-catenin, β-catenin) expression, and basement membrane proteins (collagen IV, laminin) expression. Addition of QSYQ protected the downregulated ATP 5D and upregulated p-Src and Caveolin-1 after tPA treatment.Conclusion: Our results show that QSYQ inhibits tPA-induced brain edema and hemorrhage by protecting the blood-brain barrier integrity, which was partly attributable to restoration of energy metabolism, protection of inflammation and Src/Caveolin signaling activation. The present study supports QSYQ as an effective adjunctive therapy to increase the safety of delayed tPA thrombolysis for ischemic stroke.

Dimethyl fumarate prevents acute lung injury related cognitive impairment potentially via reducing inflammation

Objective Dimethyl fumarate (DMF) has been reported to exert a protective role against diverse lung diseases and cognitive impairment-related diseases. Thus this study aimed to investigate its role on acute lung injury (ALI) and related cognitive impairment in animal model. Methods C57BL/6 mice were divided into four groups: control group, DMF group, ALI group, and ALI + DMF group. For ALI group, the ALI mice model was created by airway injection of LPS (50 μL, 1 μg/μL); for ALI + DMF group, DMF (dissolved in 0.08% methylcellulose) was treated twice a day for 2 days, and on the third day, mice were injected with LPS for ALI modeling. Mice pre-administered with methylcellulose or DMF without LPS injection (PBS instead) were used as the control group and DMF group, respectively. Morris water maze test was performed before any treatment (0 h) and 6 h after LPS-induction (54 h) to evaluate the cognitive impairment of mice. Next, the brain edema and blood brain barrier (BBB) permeability of ALI mice were assessed by brain water content, Evans blue extravasation and FITC-Dextran uptake assays. In addition, the effect of DMF on the numbers of total cells and neutrophils, protein content in BALF were quantified; the inflammatory factors in BALF, serum, and brain tissues were examined by ELISA, qRT-PCR, and Western blot assays. The effect of DMF on the cognitive impairment-related factor HIF-1α level in lung and brain tissues was also examined by Western blot. Results DMF reduced the numbers of total cells, neutrophils and protein content in BALF of ALI mice, inhibited the levels of IL-6, TNF-α and IL-1β in BALF, serum and brain tissues of ALI mice. The protein expressions of p-NF-κB/NF-κB and p-IKBα/IKBα was also suppressed by DMF in ALI mice. Morris water maze test showed that DMF alleviated the cognitive impairment in ALI mice by reducing the escape latency and path length. Moreover, DMF lessened the BBB permeability by decreasing cerebral water content, Evans blue extravasation and FITC-Dextran uptake in ALI mice. The HIF-1α levels in lung and brain tissues of ALI mice were also lessened by DMF. Conclusion In conclusion, DME had the ability to alleviate the lung injury and cerebral cognitive impairment in ALI model mice. This protective effect partly associated with the suppression of inflammation by DMF.

Non-clinical evidence supports anti-inflammatories as more effective medication than antihistamines against tarantula local effects envenomation

BackgroundTarantulas are the most common invertebrates pets, especially in North America and Europe. The most commercialized genera are from Southern Asia and tropical Americas, represented by Vitalius, found in Southeastern Brazil, and Brachypelma, common in Mexican desert. Bites by these spiders in humans occurs during manipulation and generally result in clinical manifestations such local pain, erythema and oedema, with the possibility of secondary local infection. Hence, the cases are usually treated with prescription free drugs such as antihistamines and anti-inflammatories.MethodsIn this work, we investigated the post treatment with commercial nonsteroidal and steroidal anti-inflammatories and anti-histamines administered by oral and intraperitoneal routes on rat paw oedema induced by venoms of V. dubius and B. smithi. Hydroplethysmometer standard oedema measurement and Evans blue extravasation were performed. Dose standardization experiments showed that the V. dubius is more potent than B. smithi, and doses were established at 30 μg/paw and 60 μg/paw respectively.ResultsThe oral post-administration of ketoprofen (non-selective cyclooxygenase inhibitor) and prednisolone (steroidal anti-inflammatory) markedly reduced a paw oedema evoked by only V. dubius venom, but loratadine (H1-antihistamine) had negligible effect on rat paw oedema induced by both venoms. Intraperitoneal administration, ketoprofen (20 mg Kg−1) and loratadine (5 mg Kg−1) reduced the rat paw oedema induced by V. dubius and B. smithi while methylprednisolone (10 mg Kg−1) only inhibited the oedema induced by V. dubius.ConclusionThese results suggest that the pos-treatment with nonsteroidal and steroidal anti-inflammatory drugs are more potent than antihistamines in attenuating the local effect induced by V. dubius and B. smithi venoms.

Cutaneous Hypersensitivity as an Indicator of Visceral Inflammation via C-Nociceptor Axon Bifurcation

Pain on the body surface can accompany disorders in the deep tissue or internal organs. However, the anatomical and physiological mechanisms are obscure. Here, we provided direct evidence of axon bifurcation in primary C-nociceptive neurons that innervate both the skin and a visceral organ. Double-labeled dorsal root ganglion (DRG) neurons and Evans blue extravasation were observed in 3 types of chemically-induced visceral inflammation (colitis, urocystitis, and acute gastritis) rat models. In the colitis model, mechanical hypersensitivity and spontaneous activity were recorded in vivo from double-labeled C-nociceptive neurons in S1 or L6 DRGs. These neurons showed significantly enhanced responses to both somatic stimulation and colorectal distension. Our findings suggest that the branching of C-nociceptor axons contribute to cutaneous hypersensitivity in visceral inflammation. Cutaneous hypersensitivity on certain locations of the body surface might serve as an indicator of pathological conditions in the corresponding visceral organ.

Abstract TP350: Escin Attenuates the Impairment of Neurological Function by Ameliorating Systemic Inflammation in Intracerebral Hemorrhagic Mice

Background and Purpose: Escin could attenuate cerebral ischemia-induced brain injury. The present study aimed to investigate whether Escin may attenuate the impairment of neurological function by ameliorating systemic inflammation in ICH mice. Methods: The ICH model was prepared by intrastriatal injection of collagenase. At 0.5 h, 2 h, 6 h and 12 h after Escin injection, the concentration of Escin in the serum and brain were detected. The effects of Escin on the Garcia test were evaluated. Brain water content was observed via a wet/dry weight method. Evans blue extravasation in the cerebrums were also assayed. The serum IL-1β level was detected with the ELISA kit. The ICH mice were further treated with Escin plus IL-1β. At 24 h of the ICH, Garcia test, and brain water content, the Evans blue extravasation and serum IL-1β levels were evaluated. Additionally, the expression of RhoA, ROCK1, IκBα, nuclear NF-κB, cytosolic NF-κB, Occludin and Claudin-5 in mice brain tissue were investigated with the Western blot. Results: Escin was detected in the serum of the Control group and ICH group at 0.5 h, 2 h, 6 h and 12 h after Escin administration. Escin was not detected in brain tissues at any time points in the animals. At 24 h and 72 h of ICH, the Garcia test scores in the Escin groups were significantly increased ( P < 0.05 or P < 0.01). Brain water contents and Evans blue extravasation of the right basal ganglia in the Escin groups were significantly reduced ( P < 0.05 or P < 0.01). Escin abated the increase of IL-1β induced by ICH ( P < 0.05 or P < 0.01). IL-1β administration reversed the effect of Escin on Garcia test scores, the brain water contents, and the Evans blue extravasation ( P < 0.01). In the ICH group, the levels of RhoA, ROCK1, nuclear NF-κB were increased while the expression of IκBα, cytosolic NF-κB, Occludin, Claudin-5 were reduced ( P < 0.05 or P < 0.01). However, Escin abated RhoA, ROCK1, nuclear NF-κB and augmented IκBα, cytosolic NF-κB, Occludin, and Claudin-5. IL-1β administration partially blocked the Escin-mediated regulation of IL-1β/RhoA/NF-κB signaling pathway. Conclusion: Escin cannot penetrate the blood brain barrier (BBB). Escin improves neurological function and by inhibiting systemic inflammation, and then regulating IL-1β/RhoA/NF-κB signaling pathway in BBB.

Chemokine (C-X-C motif) receptor 4 regulates lung endothelial barrier permeability during resuscitation from hemorrhagic shock

Chemokine (C-X-C motif) receptor 4 (CXCR4) agonists have been shown to protect lung endothelial barrier function in vitro. In vivo effects of CXCR4 modulation on lung endothelial permeability are unknown. Here we tested the effects of the CXCR4 agonist ubiquitin and the antagonist AMD3100 on lung vascular permeability and cytokine concentrations in a rat hemorrhage model. Animals were hemorrhaged (mean arterial blood pressure 30 mmHg for 30 min), treated with vehicle, ubiquitin (0.7 and 3.5 μmol/kg) or AMD3100 (3.5 μmol/kg), and resuscitated with crystalloids. Evans blue extravasation was employed to quantify lung vascular permeability. Ubiquitin dose-dependently reduced Evans blue extravasation into the lung. AMD3100 increased Evans blue extravasation. With AMD3100, TNFα levels in lung homogenates were increased; while TNFα levels were lower with ubiquitin, these differences did not reach statistical significance. Our findings suggest that CXCR4 regulates lung vascular permeability and further point towards CXCR4 as a drug target to confer lung protection during resuscitation from traumatic-hemorrhagic shock.

Sheng-Di-Da-Huang Decoction Inhibited Inflammation Expressed in Microglia after Intracerebral Hemorrhage in Rats

Objects. Sheng-Di-Da-Huang Decoction was used as an effective hemostatic agent in ancient China. However, its therapeutic mechanism is still not clear. Inflammatory injury plays a critical role in ICH-induced secondary brain injury. After hemolysis, hematoma components are released, inducing microglial activation via TLR4, which initiates the activation of transcription factors (such as NF-κB) to regulate expression of proinflammatory cytokine genes. This study aimed to verify the anti-inflammatory effects of Sheng-Di-Da-Huang Decoction on ICH rats. Materials and Methods. Intracerebral hemorrhage was induced by injection of bacterial collagenase (0.2 U) in rats. Neurological deficits, brain water content, Evans blue extravasation, expression of TLR4, NF-κB, Iba-1 positive cells (activated microglia), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) were examined 1, 3, 7, and 14 days after collagenase injection. MR images were also studied. Results. Sheng-Di-Da-Huang Decoction remarkably improved neurological function, reduced brain water content as well as Evans blue extravasation, downregulated expression of TLR4, NF-κB, TNF-α, and IL-1β, and inhibited microglial activation. Conclusions. Sheng-Di-Da-Huang Decoction reduced inflammation reaction after ICH through inhibited inflammation expressed in microglia.

Omi/HtrA2 Regulates a Mitochondria-Dependent Apoptotic Pathway in a Murine Model of Septic Encephalopathy

Background/Aims: the pathogenesis of sepsis-associated encephalopathy (SAE) is multifactorial, involving neurotransmitter alterations, inflammatory cytokines, oxidative damage, mitochondrial dysfunction, apoptosis, and other factors. Mitochondria are major producers of reactive oxygen species, resulting in cellular injury. Omi/HtrA2 is a proapoptotic mitochondrial serine protease involved in caspase-dependent cell death; it is translocated from mitochondria to the cytosol after an apoptotic insult. We previously found that UCF-101, a specific inhibitor of Omi/HtrA2, has neuroprotective effects on cerebral oxidative injury and cognitive impairment in septic rats. In this study, the mechanisms and molecular pathways underlying these effects were investigated. Methods: Male Sprague–Dawley rats were subjected to cecal ligation and puncture (CLP) or sham-operated laparotomy and were administered vehicle or UCF-101 (10 µmol/kg). The hippocampus was isolated for subsequent analysis. Omi/HtrA2 expression in the mitochondria or cytosol was evaluated by immunofluorescence or western blotting. Terminal deoxynucleotidyl transferase dUTP nick end labeling staining was utilized to evaluate levels of apoptosis, and western blotting was used to evaluate apoptosis-related proteins, such as cleaved caspase-3, caspase-9, and poly (ADP-ribose) polymerase (PARP). Tight junction expression was assessed by immunofluorescence and western blotting. Mitochondrial function, inflammatory cytokines, and oxidative stress were also assayed. In addition, a wet/dry method was used to evaluate brain edema and Evans blue extravasation was used to evaluate blood–brain barrier (BBB) integrity. Results: After CLP treatment, the hippocampus exhibited a mild increase in Omi/HtrA2 expression; cytosolic Omi/HtrA2 expression increased significantly, whereas mitochondrial Omi/HtrA2 expression was reduced, indicating that CLP-induced oxidative stress resulted in the translocation of Omi/HtrA2 from mitochondria to the cytosol. Hippocampal cleaved caspase-3, caspase-9, and PARP levels were significantly higher in animals treated with CLP than in sham-operated animals, while XIAP expression was lower. Treatment with UCF-101 prevented the mobilization of Omi/HtrA2 from mitochondria to the cytosol, attenuated XIAP degradation, and decreased cleaved caspase-3, caspase-9, and PARP expression as well as apoptosis. UCF-101 also reversed the decreased mitochondrial complex I, II, and III respiration and the reduced ATP caused by CLP. In addition, UCF-101 treatment resulted in a significant improvement in BBB integrity, as demonstrated by increased occludin, claudin-5, and zonula occludens 1 levels and reduced Evans blue extravasation. No significant effects of UCF-101 on brain edema were found. Inflammatory cytokines and oxidative stress were significantly higher in the CLP-treated group than in the sham-operated group. However, the inhibition of Omi/HtrA2 by UCF-101 significantly alleviated these responses. Conclusion: Our data indicated that Omi/ HtrA2 regulates a mitochondria-dependent apoptotic pathway in a murine model of septic encephalopathy. Inhibition of Omi/HtrA2 by UCF-101 leads to neuroprotection by inhibiting the cytosolic translocation of Omi/HtrA2 and antagonizing the caspase-dependent apoptosis pathway. Therapeutic interventions that inhibit Omi/HtrA2 translocation or protease activity may provide a novel method to treat SAE.

Unilateral Opening of Rat Blood-Brain Barrier Assisted by Diagnostic Ultrasound Targeted Microbubbles Destruction

Objective. Blood-brain barrier (BBB) is a key obstacle that prevents the medication from blood to the brain. Microbubble-enhanced cavitation by focused ultrasound can open the BBB and proves to be valuable in the brain drug delivery. The study aimed to explore the feasibility, efficacy, and safety of unilateral opening of BBB using diagnostic ultrasound targeted microbubbles destruction in rats.Methods. A transtemporal bone irradiation of diagnostic ultrasound and intravenous injection of lipid-coated microbubbles were performed at unilateral hemisphere. Pathological changes were monitored. Evans Blue extravasation grades, extraction from brain tissue, and fluorescence optical density were quantified. Lanthanum nitrate was traced by transmission electron microscopy.Results. After diagnostic ultrasound mediated microbubbles destruction, Evans Blue extravasation and fluorescence integrated optical density were significantly higher in the irradiated hemisphere than the contralateral side (allp<0.01). Erythrocytes extravasations were demonstrated in the ultrasound-exposed hemisphere (4±1, grade 2) while being invisible in the control side. Lanthanum nitrate tracers leaked through interendothelial cleft and spread to the nerve fiber existed in the irradiation side.Conclusions. Transtemporal bone irradiation under DUS mediated microbubble destruction provides us with a more accessible, safer, and higher selective BBB opening approach in rats, which is advantageous in brain targeted drugs delivery.