Jin-Chung Chen

The opioid receptor binding of dezocine, morphine, fentanyl, butorphanol and nalbuphine.

The ability of morphine, fentanyl, butorphanol, nalbuphine, and dezocine to compete with radiolabeled ligands for binding at the mu1, mu2, kappa1, and delta opioid receptors and the sigma receptor was characterized. In the absence of sodium, the potency of opioid receptor competition at each receptor site was found to be: mu1-fentanyl > butorphanol > morphine > or = dezocine = nalbuphine; mu2-butorphanol > fentanyl > nalbuphine > morphine = dezocine; kappa1-butorphanol > nalbuphine >> morphine > or = dezocine > fentanyl; and delta-butorphanol > nalbuphine > or = dezocine > morphine > fentanyl. For all five compounds, competition at the sigma receptor was weak, with nalbuphine and dezocine having Kis of approximately 0.5 microM and the other opioids having Kis of greater than 1 microM. Since the presence of 100 mM NaCl during the competitive binding decreased the K(i), to varying degrees, of all five opioids at the mu1 and delta receptors and of some of the opioids at the mu2 and kappa1 receptors, the five compounds studied appear to differ in efficacy at the five receptor sites.

Hemorrhage detection during focused-ultrasound induced blood-brain-barrier opening by using susceptibility-weighted magnetic resonance imaging.

High-intensity focused ultrasound has been discovered to be able to locally and reversibly increase the permeability of the blood-brain barrier (BBB), which can be detected using magnetic resonance imaging (MRI). However, side effects such as microhemorrhage, erythrocyte extravasations or even extensive hemorrhage may also occur. Although current contrast-enhanced T1-weighted MRI can be used to detect the changes in BBB permeability, its efficacy in detecting tissue hemorrhage after focused-ultrasound sonication remains limited. The purpose of this study is to investigate the feasibility of magnetic resonance susceptibility-weighted imaging (MR-SWI) for identifying possible tissue hemorrhage associated with disruption of the BBB induced by focused ultrasound in a rat model. The brains of 42 Sprague-Dawley rats were subjected to 107 sonications, either unilaterally or bilaterally. Localized BBB opening was achieved by delivering burst-mode focused ultrasound energy into brain tissue in the presence of microbubbles. Rats were studied by T2-weighted and contrast-enhanced T1-weighted MRI techniques, as well as by SWI. Tissue changes were analyzed histologically and the extent of apoptosis was investigated with the terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling method. The results demonstrated that SWI is more sensitive than standard T2-weighted and contrast-enhanced T1-weighted MRI techniques in detecting hemorrhages after brain sonication. Longitudinal study showed that SWI is sensitive to the recovery process of the damage and, therefore, could provide important and complementary information to the conventional MR images. Potential applications such as drug delivery in the brain might be benefited.

Up-regulation of dorsal root ganglia BDNF and trkB receptor in inflammatory pain: an in vivo and in vitro study.

BackgroundDuring inflammation, immune cells accumulate in damaged areas and release pro-inflammatory cytokines and neurotrophins. Brain-derived neurotrophic factor (BDNF) plays a neuromodulatory role in spinal cord dorsal horn via the post-synaptic tyrosine protein kinase B (trkB) receptor to facilitate pain transmission. However, the precise role of BDNF and trkB receptor in the primary sensory neurons of dorsal root ganglia (DRG) during inflammation remains to be clarified. The aim of this study was to investigate whether and how BDNF-trkB signaling in the DRG is involved in the process of inflammatory pain.MethodsWe used complete Freunds adjuvant- (CFA-) induced and tumor necrosis factor-α- (TNF-α-) induced inflammation in rat hindpaw as animal models of inflammatory pain. Quantification of protein and/or mRNA levels of pain mediators was performed in separate lumbar L3-L5 DRGs. The cellular mechanism of TNF-α-induced BDNF and/or trkB receptor expression was examined in primary DRG cultures collected from pooled L1-L6 DRGs. Calcitonin gene-related peptide (CGRP), BDNF and substance P release were also evaluated by enzyme immunoassay.ResultsCFA injection into rat hindpaw resulted in mechanical hyperalgesia and significant increases in levels of TNF-α in the inflamed tissues, along with enhancement of BDNF and trkB receptor as well as the pain mediators CGRP and transient receptor potential vanilloid receptor subtype 1 (TRPV1) in DRG. Direct injection of TNF-α into rat hindpaw resulted in similar effects with retrograde transport of TNF-α along the saphenous nerve to DRG during CFA-induced inflammation. Primary DRG cultures chronically treated with TNF-α showed significant enhancement of mRNA and protein levels of BDNF and trkB receptor, BDNF release and trkB-induced phospho-ERK1/2 signal. Moreover, CGRP and substance P release were enhanced in DRG cultures after chronic TNF-α treatment or acute BDNF stimulation. In addition, we found that BDNF up-regulated trkB expression in DRG cultures.ConclusionsBased on our current experimental results, we conclude that inflammation and TNF-α up-regulate the BDNF-trkB system in DRG. This phenomenon suggests that up-regulation of BDNF in DRG may, in addition to its post-synaptic effect in spinal dorsal horn, act as an autocrine and/or paracrine signal to activate the pre-synaptic trkB receptor and regulate synaptic excitability in pain transmission, thereby contributing to the development of hyperalgesia.

Postnatal changes of brain monoamine levels in prenatally malnourished and control rats

The effects of age and prenatal protein malnutrition (6% casein diet) on the concentration of monoamine neurotransmitters and their metabolites and precursors in the hippocampal formation, striatum, brain stem and cerebral cortex were investigated in 1‐, 15‐, 30‐, 45‐, 90‐ and 220‐day‐old rats. Concentrations of all neurotransmitters, i.e. dopamine, norepinephrine and serotonin, changed significantly during the development. However, two main patterns were recognized. Serotonin in all areas, and dopamine in the striatum, increased from birth to day 45, and declined significantly in 90‐day‐old rats. In contrast, norepinephrine in all areas, and dopamine in areas other than the striatum, showed the lowest levels in 30‐day‐old rats, with levels increasing gradually after this age. Concentrations of metabolites paralleled changes in corresponding neurotransmitter levels. Prenatal protein malnutrition did not significantly affect any neurotransmitter concentrations with the exception of increased tryptophan levels (181%) in the hippocampal formation of newborn rats and decreased tyrosine levels (59%) in the striatum of day 30 rats. The results indicate that the monoamine transmitter content varied dynamically throughout postnatal life; however, they seem to counteract the insult from prenatal protein malnutrition after postnatal nutritional rehabilitation.

Effects of Exercise Training and Deconditioning on Platelet Aggregation Induced by Alternating Shear Stress in Men

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 ≈60% 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) deconditioning reverses the effects of training on resting and postexercise state. Conclusions— Exercise training suppresses the extent of ASIPA, probably by reducing vWF binding to platelets and P-selectin expression on platelets. However, deconditioning reverses the training effects.

Down-regulation of the glial glutamate transporter GLT-1 in rat hippocampus and striatum and its modulation by a group III metabotropic glutamate receptor antagonist following transient global forebrain ischemia.

Our goals were to identify biochemical markers for transient global ischemia-induced delayed neuronal death and test possible drug therapies against this neuronal damage. Four-vessel occlusion (4-VO) for 20 min was used as a rat model. The temporal expression profiles of three glutamate transporters (GLT-1, GLAST and EAAC1) were evaluated in the CA1 region of the hippocampus and the striatum. The protein levels of the GLT-1 were significantly down-regulated between 3 and 6 h after ischemia-reperfusion in the CA1 region and striatum, returned to the control (2-VO) levels 24 h after reperfusion and remained unchanged for up to 7 days. The levels of GLAST in the CA1 region and striatum, and EAAC1 in the CA1 region did not change after ischemia from 1 h to 7 days. Pretreatment with group III metabotropic glutamate receptor antagonist s-alpha-MCPA (20 microg/5 microl) 30 min prior to 4-VO significantly restored the GLT-1 levels in the CA1 region caused by global ischemia at both 3 and 6 h after reperfusion. The loss of pyramidal neurons in the CA1 region due to ischemia-reperfusion could also be prevented by intraventricular pretreatment with s-alpha-MCPA. The current findings pinpoint the significance of GLT-1 during ischemia/reperfusion and suggest a potential application of group III metabotropic glutamate receptor antagonist against ischemic/hypoxic neuronal damage.

Noninvasive and targeted gene delivery into the brain using microbubble-facilitated focused ultrasound.

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.

Enhanced Cdk5 Activity and p35 Translocation in the Ventral Striatum of Acute and Chronic Methamphetamine-Treated Rats

The cyclin-dependent kinase Cdk5 and DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of Mr 32 kDa)-dependent signaling have been implicated in the regulation of dopaminergic neurotransmission after chronic cocaine treatment. In this study, we examined if Cdk5 signaling participates in the behavioral and biochemical effect of acute and chronic methamphetamine (METH) treatment. We found that Cdk5 activity and the membrane fraction of p35 protein, a Cdk5 activator, in the ventral striatum increased transiently after an injection of 4 mg/kg METH, while intra-accumbens treatment with a Cdk5 inhibitor, roscovitine, prevented the acute METH-induced locomotor activation. The phosphorylation of DARPP-32 at both Thr75 and Thr34 was differentially regulated after acute METH treatment, but the levels of total Cdk5, p35, and DARPP-32 remained the same. To determine if Cdk5 signaling was associated with behavior sensitization to METH, rats that received repetitive injections of METH (4 mg/kg) for 14 consecutive days were analyzed at withdrawal day 7. The results indicate that Cdk5 activity and p35 translocation in the ventral striatum were upregulated in METH-sensitized rats; treatment with roscovitine in the nucleus accumbens effectively suppressed the 1 mg/kg METH-induced behavioral sensitization. Concomitantly, a decrease in the amount of PP-2A and DARPP-32 phosphorylation at Thr34, but an increase in phosphorylation of DARPP-32/Thr75, was observed in the ventral striatum of sensitized rats. The overall results demonstrate that Cdk5/p35 and downstream signaling in the ventral striatum play a critical role in the effects of acute METH treatment as well as the development of behavioral METH sensitization.

CC chemokine ligand 2 upregulates the current density and expression of TRPV1 channels and Nav1.8 sodium channels in dorsal root ganglion neurons

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.

D3 dopamine receptors are down-regulated in amphetamine sensitized rats and their putative antagonists modulate the locomotor sensitization to amphetamine

D(3) dopamine receptor agonists inhibit locomotor activity in rodents and modulate the reinforcing effect of psychostimulants; however, their functional role during behavioral sensitization remains unclear. In the present study, we intend to investigate if D(3) dopamine receptors alter during the amphetamine sensitization and test if manipulation of D(3) receptors would affect the development of locomotor sensitization to amphetamine. We have found that D(3) dopamine receptors are down-regulated in the limbic forebrain in chronic amphetamine-treated (5 mg/kg x 7 days) animals. The levels of both D(3) receptor protein (B(max) value) and mRNA decreased significantly in the behaviorally sensitized rats compared to the saline-treated controls. When animals were co-administered a putative D(3) receptor antagonist (U99194A or GR103691; 20 microg x 7 days; intracerebroventricle) and amphetamine (5 mg/kg x 7 days, i.p.), the locomotor sensitization to amphetamine was significantly inhibited. However, when the putative D(3) receptor antagonist U99194A was administered during the amphetamine withdrawal period at day 10, it did not affect the development of locomotor sensitization. Furthermore, pretreatment with the preferential D(3) agonist 7-hydroxydipropylaminotetralin partially blocked the inhibitory effect of U99194A on locomotor sensitization. These data prove the participation of D(3) dopamine receptors in the development of amphetamine sensitization and, in addition, suggest a potential application for D(3) antagonists in the prevention of amphetamine addiction.