BDNF-TrkB signaling pathway-mediated microglial activation induces neuronal KCC2 downregulation contributing to dynamic allodynia following spared nerve injury.

Mechanical allodynia can be evoked by punctate pressure contact with the skin (punctate mechanical allodynia) and dynamic contact stimulation induced by gentle touching of the skin (dynamic mechanical allodynia). Dynamic allodynia is insensitive to morphine treatment and is transmitted through the spinal dorsal horn by a specific neuronal pathway, which is different from that for punctate allodynia, leading to difficulties in clinical treatment. K+-Cl- cotransporter-2 (KCC2) is one of the major determinants of inhibitory efficiency, and the inhibitory system in the spinal cord is important in the regulation of neuropathic pain. The aim of the current study was to determine whether neuronal KCC2 is involved in the induction of dynamic allodynia and to identify underlying spinal mechanisms involved in this process. Dynamic and punctate allodynia were assessed using either von Frey filaments or a paint brush in a spared nerve injury (SNI) mouse model. Our study discovered that the downregulated neuronal membrane KCC2 (mKCC2) in the spinal dorsal horn of SNI mice is closely associated with SNI-induced dynamic allodynia, as the prevention of KCC2 downregulation significantly suppressed the induction of dynamic allodynia. The over activation of microglia in the spinal dorsal horn after SNI was at least one of the triggers in SNI-induced mKCC2 reduction and dynamic allodynia, as these effects were blocked by the inhibition of microglial activation. Finally, the BDNF-TrkB pathway mediated by activated microglial affected SNI-induced dynamic allodynia through neuronal KCC2 downregulation. Overall, our findings revealed that activation of microglia through the BDNF-TrkB pathway affected neuronal KCC2 downregulation, contributing to dynamic allodynia induction in an SNI mouse model.

An Anterior Cingulate Cortex-to-Midbrain Projection Controls Chronic Itch in Mice.

Itch is an unpleasant sensation that provokes the desire to scratch. While acute itch serves as a protective system to warn the body of external irritating agents, chronic itch is a debilitating but poorly-treated clinical disease leading to repetitive scratching and skin lesions. However, the neural mechanisms underlying the pathophysiology of chronic itch remain mysterious. Here, we identified a cell type-dependent role of the anterior cingulate cortex (ACC) in controlling chronic itch-related excessive scratching behaviors in mice. Moreover, we delineated a neural circuit originating from excitatory neurons of the ACC to the ventral tegmental area (VTA) that was critically involved in chronic itch. Furthermore, we demonstrate that the ACC→VTA circuit also selectively modulated histaminergic acute itch. Finally, the ACC neurons were shown to predominantly innervate the non-dopaminergic neurons of the VTA. Taken together, our findings uncover a cortex-midbrain circuit for chronic itch-evoked scratching behaviors and shed novel insights on therapeutic intervention.

Characterisation and application of recombinant FVIII-neutralising antibodies from haemophilia A inhibitor patients.

The development of anti-drug antibodies (ADAs) is a serious outcome of treatment strategies involving biological medicines. Coagulation factor VIII (FVIII) is used to treat haemophilia A patients, but its immunogenicity precludes a third of severe haemophiliac patients from receiving this treatment. The availability of patient-derived anti-drug antibodies can help us better understand drug immunogenicity and identify ways to overcome it. Thus, there were two aims to this work: (i) to develop and characterise a panel of recombinant, patient-derived, monoclonal antibodies covering a range of FVIII epitopes with varying potencies, kinetics and mechanism of action, and (ii) to demonstrate their applicability to assay development, evaluation of FVIII molecules and basic research. For the first objective we used recombinant antibodies to develop a rapid, sensitive, flexible and reproducible ex vivo assay that recapitulates inhibitor patient blood using blood from healthy volunteers. We also demonstrate how the panel can provide important information about the efficacy of FVIII products and reagents without the need for patient or animal material. These materials can be used as experimental exemplars or controls, as well as tools for rational, hypothesis-driven research and assay development in relation to FVIII immunogenicity and FVIII-related products.

Cangrelor PK/PD analysis in post-operative neonatal cardiac patients at risk for thrombosis.

Essentials An optimal therapeutic strategy has yet to be established to prevent early shunt thrombosis. A phase 1 study of cangrelor was performed in neonates after palliation of congenital heart disease. PD endpoint of >90% platelet inhibition in 60% of patients was achieved at 0.5 µg/kg/min dosing. No serious adverse events related to drug administration were observed, including bleeding. ABSTRACT: Background Systemic-to-pulmonary artery shunt thrombosis is a significant cause of early postoperative mortality in neonates after palliation of congenital heart disease. In the context of thromboprophylaxis, an optimal therapeutic strategy has yet to be established before aspirin administration. Cangrelor, a fast-acting, reversible P2Y12 inhibitor, may fill this unmet need. Objectives To evaluate the pharmacokinetics (PK), pharmacodynamics (PD), and safety of cangrelor in neonates undergoing stage 1 palliation. Methods This prospective, open-label, single-arm study evaluated two cangrelor dosing cohorts following placement of a systemic-to-pulmonary artery shunt, right ventricle-to-pulmonary artery shunt, or ductal stent. Drug concentrations and platelet reactivity, assessed by light transmission aggregometry and in microfluidic assays (MF), were measured. Results Twenty-two patients were consented and 15 received a 1-hour infusion of cangrelor at either 0.5 µg/kg/min (cohort 1) or 0.25 µg/kg/min (cohort 2). Whereas the primary PD endpoint was achieved at the higher dose (ie, reduction in maximal platelet aggregation by ≥90% in 60% of participants), only 29% of those in cohort 2 attained this goal. Comparable and statistically significant results were obtained in MF assays (P < .0001 vs. baseline). Drug levels during infusion were 3-fold higher in cohort 1 vs. cohort 2 (P < .001). Most participants (70%) had undetectable drug levels by 10 minutes postinfusion with full recovery in platelet function at 1 hour. No drug-related bleeding events occurred. Conclusions Favorable PK/PD properties of cangrelor 0.5 µg/kg/min dosing and safety profile warrant further evaluation in neonates following palliative cardiac procedures.

Microfluidic hemophilia models using blood from healthy donors.

BACKGROUND: Microfluidic clotting assays permit drug action studies for hemophilia therapeutics under flow. However, limited availability of patient samples and Inter-donor variability limit the application of such assays, especially with many patients on prophylaxis. OBJECTIVE: To develop approaches to phenocopy hemophilia using modified healthy blood in microfluidic assays. METHODS: Corn trypsin inhibitor (4 µg/mL)-treated healthy blood was dosed with either anti-factor VIII (FVIII; hemophilia A model) or a recombinant factor IX (FIX) missense variant (FIX-V181T; hemophilia B model). Treated blood was perfused at 100 s-1 wall shear rate over collagen/tissue factor (TF) or collagen/factor XIa (FXIa). RESULTS: Anti-FVIII partially blocked fibrin production on collagen/TF, but completely blocked fibrin production on collagen/FXIa, a phenotype reversed with 1 µmol/L bispecific antibody (emicizumab), which binds FIXa and factor X. As expected, emicizumab had no significant effect on healthy blood (no anti-FVIII present) perfused over collagen/FXIa. The efficacy of emicizumab in anti-FVIII-treated healthy blood phenocopied the action of emicizumab in the blood of a patient with hemophilia A perfused over collagen/FXIa. Interestingly, a patient-derived FVIII-neutralizing antibody reduced fibrin production when added to healthy blood perfused over collagen/FXIa. For low TF surfaces, reFIX-V181T (50 µg/mL) fully blocked platelet and fibrin deposition, a phenotype fully reversed with anti-TFPI. CONCLUSION: Two new microfluidic hemophilia A and B models demonstrate the potency of anti-TF pathway inhibitor, emicizumab, and a patient-derived inhibitory antibody. Using collagen/FXIa-coated surfaces resulted in reliable and highly sensitive hemophilia models.

Fibrin Modulates Shear-Induced NETosis in Sterile Occlusive Thrombi Formed under Haemodynamic Flow.

Neutrophils can release extracellular traps (NETs) in infectious, inflammatory and thrombotic diseases. NETs have been detected in deep vein thrombosis, atherothrombosis, stroke, disseminated intravascular coagulation and trauma. We have previously shown that haemodynamic forces trigger rapid NETosis within sterile occlusive thrombi in vitro. Here, we tested the effects of thrombin, fibrin and fibrinolysis on shear-induced NETosis by imaging NETs with Sytox Green during microfluidic perfusion of factor XIIa-inhibited or thrombin-inhibited human whole blood over fibrillar collagen (±tissue factor). For perfusions under venous pressure drops (19 mm Hg/mm-clot), thrombin generation did not alter the near-zero level of NET generation. In contrast, production of thrombin/fibrin led to a twofold reduction in neutrophil accumulation and a sixfold reduction in NET generation after 30 minutes of arterial perfusion (163 mm Hg/mm-clot). Exogenously added tissue type plasminogen activator (tPA) drove robust fibrinolysis; however, tPA did not trigger NETosis under venous flow. In contrast, tPA did enhance NET generation in clots subjected to arterial pressure drops. After 45 minutes of arterial perfusion, clots treated with 30 nM tPA had a threefold increase in total NET production and a twofold increase in normalized NET generation (measured as deoxyribonucleic acid:neutrophil) compared with fibrin-rich clots. Blocking fibrin polymerization resulted in similar level of NET release seen in tPA-treated clots, whereas ε-aminocaproic acid abolished the NET-enhancing effect of tPA. Therefore, fibrin suppresses NET generation and the absence of fibrin promotes NETs. We demonstrated that shear-induced NETosis was strongly inversely correlated with fibrin in sterile occlusive clots.

Contact Pathway Function During Human Whole Blood Clotting on Procoagulant Surfaces.

Microfluidic thrombosis assays allow the control of anticoagulation, hemodynamics, pharmacology, and procoagulant surfaces containing collagen ± tissue factor (TF). With corn trypsin inhibitor (CTI) ranging from low (1-4 µg/mL) to high levels (40-60 µg/mL), the function of Factor XIIa (FXIIa) can be modulated in the presence of low or high surface TF. With high CTI and no collagen/TF in the assay, no thrombin is generated during 15-min microfluidic perfusion. At low CTI (no TF), the generation of FXIa leads to fibrin polymerization at ~300 s after the initiation of flow over collagen, an onset time shortened at zero CTI and prolonged at high CTI. The engagement of FXIa was difficult to observe for clotting on high TF surfaces due to the dominance of the extrinsic pathway. Low TF surfaces allowed observable crosstalk between extrinsic pathway generation of thrombin and thrombin-mediated activation of FXIa, a feedback detected at >5 min and attenuated with polyphosphate inhibitor. From thrombin-antithrombin immunoassay of the effluent of blood flowing over collagen/TF, the majority of thrombin was found captured on intrathrombus fibrin. Additionally, extreme shear rates (>10,000 s-1) can generate massive von Willebrand Factor fibers that capture FXIIa and FXIa to drive fibrin generation, an event that facilitates VWF fiber dissolution under fibrinolytic conditions. Finally, we found that occlusive sterile thrombi subjected to pressure drops >70 mm-Hg/mm-clots have interstitial stresses sufficient to drive NETosis. These microfluidic studies highlight the interaction of contact pathway factors with the extrinsic pathway, platelet polyphosphate, VWF fibers, and potentially shear-induced NETs.

Using microfluidic devices to study thrombosis in pathological blood flows.

Extreme flows can exist within pathological vessel geometries or mechanical assist devices which create complex forces and lead to thrombogenic problems associated with disease. Turbulence and boundary layer separation are difficult to obtain in microfluidics due to the low Reynolds number flow in small channels. However, elongational flows, extreme shear rates and stresses, and stagnation point flows are possible using microfluidics and small perfusion volumes. In this review, a series of microfluidic devices used to study pathological blood flows are described. In an extreme stenosis channel pre-coated with fibrillar collagen that rapidly narrows from 500 µm to 15 µm, the plasma von Willebrand Factor (VWF) will elongate and assemble into thick fiber bundles on the collagen. Using a micropost-impingement device, plasma flow impinging on the micropost generates strong elongational and wall shear stresses that trigger the growth of a VWF bundle around the post (no collagen required). Using a stagnation-point device to mimic the zone near flow reattachment, blood can be directly impinged upon a procoagulant surface of collagen and the tissue factor. Clots formed at the stagnation point of flow impingement have a classic core-shell architecture where the core is highly activated (P-selectin positive platelets and fibrin rich). Finally, within occlusive clots that fill a microchannel, the Darcy flow driven by ΔP/L > 70 mm-Hg/mm-clot is sufficient to drive NETosis of entrapped neutrophils, an event not requiring either thrombin or fibrin. Novel microfluidic devices are powerful tools to access physical environments that exist in human disease.