Recombinant adeno-associated viral (rAAV) vectors are potentially powerful tools for gene therapy of CNS diseases, but their penetration into brain parenchyma is severely limited by the blood-brain barrier (BBB) and current delivery relies on invasive stereotactic injection. Here we evaluate the local, targeted delivery of rAAV vectors into the brains of mice by noninvasive, reversible, microbubble-facilitated focused ultrasound (FUS), resulting in BBB opening that can be monitored and controlled by magnetic resonance imaging (MRI). Using this method, we found that IV-administered AAV2-GFP (green fluorescence protein) with a low viral vector titer (1×109 vg/g) can successfully penetrate the BBB-opened brain regions to express GFP. We show that MRI monitoring of BBB-opening could serve as an indicator of the scale and distribution of AAV transduction. Transduction peaked at 3 weeks and neurons and astrocytes were affected. This novel, noninvasive delivery approach could significantly broaden the application of AAV-viral-vector-based genes for treatment of CNS diseases.
BackgroundInflammation or nerve injury-induced upregulation and release of chemokine CC chemokine ligand 2 (CCL2) within the dorsal root ganglion (DRG) is believed to enhance the activity of DRG nociceptive neurons and cause hyperalgesia. Transient receptor potential vanilloid receptor 1 (TRPV1) and tetrodotoxin (TTX)-resistant Nav1.8 sodium channels play an essential role in regulating the excitability and pain transmission of DRG nociceptive neurons. We therefore tested the hypothesis that CCL2 causes peripheral sensitization of nociceptive DRG neurons by upregulating the function and expression of TRPV1 and Nav1.8 channels.MethodsDRG neuronal culture was prepared from 3-week-old Sprague–Dawley rats and incubated with various concentrations of CCL2 for 24 to 36 hours. Whole-cell voltage-clamp recordings were performed to record TRPV1 agonist capsaicin-evoked inward currents or TTX-insensitive Na+ currents from control or CCL2-treated small DRG sensory neurons. The CCL2 effect on the mRNA expression of TRPV1 or Nav1.8 was measured by real-time quantitative RT-PCR assay.ResultsPretreatment of CCL2 for 24 to 36 hours dose-dependently (EC50 value = 0.6 ± 0.05 nM) increased the density of capsaicin-induced currents in small putative DRG nociceptive neurons. TRPV1 mRNA expression was greatly upregulated in DRG neurons preincubated with 5 nM CCL2. Pretreating small DRG sensory neurons with CCL2 also increased the density of TTX-resistant Na+ currents with a concentration-dependent manner (EC50 value = 0.7 ± 0.06 nM). The Nav1.8 mRNA level was significantly increased in DRG neurons pretreated with CCL2. In contrast, CCL2 preincubation failed to affect the mRNA level of TTX-resistant Nav1.9. In the presence of the specific phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002 or Akt inhibitor IV, CCL2 pretreatment failed to increase the current density of capsaicin-evoked inward currents or TTX-insensitive Na+ currents and the mRNA level of TRPV1 or Nav1.8.ConclusionsOur results showed that CCL2 increased the function and mRNA level of TRPV1 channels and Nav1.8 sodium channels in small DRG sensory neurons via activating the PI3K/Akt signaling pathway. These findings suggest that following tissue inflammation or peripheral nerve injury, upregulation and release of CCL2 within the DRG could facilitate pain transmission mediated by nociceptive DRG neurons and could induce hyperalgesia by upregulating the expression and function of TRPV1 and Nav1.8 channels in DRG nociceptive neurons.
Objective-Alternating shear stress, which resembles the flow condition in stenotic arteries, induces platelet aggregation.This study investigated how exercise training and deconditioning influence alternating shear-induced platelet aggregation (ASIPA) and clarify the mechanisms underlying ASIPA. Methods and Results-Thirty healthy male sedentary subjects were randomly divided into control and trained groups. The trained men were trained on a bicycle ergometer at Ϸ60% of maximal oxygen consumption for 30 minutes per day, 5 days per week for 8 weeks, and then were deconditioned for 8 weeks. The experimental results indicate the following:(1) short-term strenuous exercise increases the extent of ASIPA and is accompanied by increased the von Willebrand factor (vWF) binding and P-selectin expression on platelets in both the control and trained groups, whereas the enhancement of platelet function decreases after exercise training in trained subjects; (2) at rest and immediately after exercise, ASIPA and the vWF binding and P-selectin expression on platelets are reduced by training, but remain unchanged in the control group; and (3) Key Words: training Ⅲ detraining Ⅲ shear stress Ⅲ platelets Ⅲ adhesion molecules L ifestyle habits such as exercise may have significant influence on the risk of major vascular thrombotic events. 1,2 Previous studies have suggested that the risk of primary cardiac arrest transiently increases during vigorous exercise, 3,4 whereas regular exercise is associated with an overall decreased risk of cardiovascular diseases. 5,6 Shear-induced platelet aggregation (SIPA) is important in arterial thrombosis, which is a major contributing factor for atherothrombotic occlusion of blood vessels. 7 Therefore, it is important to distinguish the shearmediated thrombotic events that occur between short-term bouts of exercise and physical conditioning. According to our earlier study of male patients with stable angina, strenuous acute exercise can increase the capacity of adhered platelets to withstand physiological flow shear stress. 8 A previous investigation also demonstrated that SIPA increased after strenuous treadmill exercise in patients with effort angina. 9 However, the effects of exercise training and detraining on shearmediated platelet activation and its underlying mechanisms have not yet been studied. See page 265Pathological, high shear stress induces binding of von Willebrand factor (vWF) to the platelet glycoprotein (GP) Ib complex on platelets. This interaction transduces signals in platelets, subsequently activating GP IIb/IIIa complex. The activated GP IIb/IIIa complex then binds to fibrinogen, stabilizing the aggregated platelets. 10,11 Our recent investigation showed that short-term intense exercise promoted SIPA, possibly by improving the ability of vWF to bind to platelets and the subsequent activation of GPIIb/IIIa complexes to sustained high shear stress. 12 In fact, blood flow in stenotic vessel follows a complex pattern of hydrodynamics in that flow rate first increases dramatical...
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