Ligation In Rats Research Articles

The hydroalcoholic extract of watercress attenuates protein oxidation, oxidative stress, and liver damage after bile duct ligation in rats.

Cholestatic liver disease is recognized by extreme collagen formation and deposition, which is mediated by free radicals. The aim of the current study was to investigate the probable hepatoprotective effects of hydroalcoholic extract of watercress (WC) against oxidative stress and liver injury in bile duct ligation (BDL)- induced cholestatic rats. A total of 32 male Wistar rats were divided into four groups; sham control (SC), BDL, SC + hydroalcoholic extract of WC and BDL + hydroalcoholic extract of WC. WC-treated rats received daily WC 500 mg/kg/day for 10 days. Biochemical tests, hepatic oxidative stress markers, and antioxidant enzymes activity were estimated. Further, liver hydroxyproline content was assayed and histological analysis was made. The BDL model markedly elevated the protein carbonyl (PCO) and hydroxyproline contents and decreased the glutathione peroxidase (GPx) activity. Hydroalcoholic extract of WC significantly decreased the surge in liver PCO and hydroxyproline levels and increased the reduced GPx enzyme activity contents in the hepatic tissue. As determined by hematoxylin and eosin staining, BDL considerably induced hepatocyte necrosis. Moreover, these changes were significantly attenuated by the hydroalcoholic extract of WC treatment. Our data indicate that the hydroalcoholic extract of WC extract attenuated liver damage in BDL rats by decreasing the hydroxyproline content and histopathological indexes. Also, it reduced oxidative stress by preventing the hepatic protein oxidation and enhancing the activity of the GPx enzyme via antioxidative effect and free-radical scavenging. Our findings suggest that hydroalcoholic extract of WC could be a beneficial new curative agent for cholestatic liver damage.

A Net Mold-Based Method of Biomaterial-Free Three-Dimensional Cardiac Tissue Creation.

Ischemic cardiomyopathy poses a significant public health burden due to the irreversible loss of functional cardiac tissue. Alternative treatment strategies include creation of three-dimensional (3D) cardiac tissues to both replace and augment injured native tissue. In this study, we utilize a net mold-based method to create a biomaterial-free 3D cardiac tissue and compare it to current methods using biomaterials. Cardiomyocytes, fibroblasts, and endothelial cells were combined using a hanging drop method to create spheroids. For the net mold patch method, spheroids were seeded into a net mold-based system to create biomaterial-free 3D cardiac patches. For the gel patch, spheroids were embedded in a collagen gel. Immunohistochemistry revealed increased alignment, vascularization, collagen I expression, cell viability, and higher density of cells in the net mold patch compared with the gel patch. Furthermore, in vivo testing in a left anterior descending artery ligation rat model found increased ejection fraction and smaller scar area following implantation of the net mold patch. We present a novel and simple reproducible method to create biomaterial-free 3D net mold patches that may potentially improve the treatment of heart failure in the future.

Foxp3 plasmid-encapsulated PLGA nanoparticles attenuate pain behavior in rats with spinal nerve ligation

Microglia play a critical role in neuropathic pain. Since upregulated Foxp3 in microglia enhances tissue repair by resolving neuroinflammation in excitotoxin-induced neuronal death, it may attenuate neuropathic pain in a similar manner. Therefore, this study tests whether Foxp3 introduced with poly (D, L-lactic-co-glycolic acid) (PLGA) nanoparticles (Foxp3 NPs) can alleviate neuropathic pain by inhibiting microglia activity. The prepared Foxp3 NPs had an anti-inflammatory effect on lipopolysaccharide-stimulated BV2 cells in vitro, and localized to spinal microglia in vivo. Further, the Foxp3 NPs significantly attenuated pain behavior induced by spinal nerve ligation in rats for 7 days by suppressing microglial activity, followed by the downregulation of pro-nociceptive genes and the upregulation of anti-nociceptive genes in the spinal dorsal horn. Collectively, these data suggest that Foxp3 NPs effectively relieve neuropathic pain in animals by reducing microglia activity and subsequent modulation of neuroinflammation, and may be of therapeutic value in the treatment of neuropathic pain.

The α2,3-selective potentiator of GABAA receptors, KRM-II-81, reduces nociceptive-associated behaviors induced by formalin and spinal nerve ligation in rats

Clinical evidence indicates that positive allosteric modulators (PAMs) of GABAA receptors have analgesic benefit in addition to efficacy in anxiety disorders. However, the utility of GABAA receptor PAMs as analgesics is compromised by the central nervous system side effects of non-selective potentiators. A selective potentiator of GABAA receptors associated with α2/3 subunits, KRM-II-81(5-(8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazo[1,5-a][1,4]diazepin-3-yl)oxazole), has demonstrated anxiolytic, anticonvulsant, and antinociceptive effects in rodents with reduced motoric side effects. The present study evaluated the potential of KRM-II-81 as a novel analgesic. Oral administration of KRM-II-81 attenuated formalin-induced flinching; in contrast, diazepam was not active. KRM-II-81 attenuated nociceptive-associated behaviors engendered by chronic spinal nerve ligation (L5/L6). Diazepam decreased locomotion of rats at the dose tested in the formalin assay (10 mg/kg) whereas KRM-II-81 produced small decreases that were not dose-dependent (10–100 mg/kg). Plasma and brain levels of KRM-II-81 were used to demonstrate selectivity for α2/3- over α1-associated GABAA receptors and to define the degree of engagement of these receptors. Plasma and brain concentrations of KRM-II-81 were positively-associated with analgesic efficacy. GABA currents from isolated rat dorsal-root ganglion cultures were potentiated by KRM-II-81 with an ED50 of 32 nM. Measures of respiratory depression were reduced by alprazolam whereas KRM-II-81 was either inactive or produced effects with lower potency and efficacy. These findings add to the growing body of data supporting the idea that α2/3-selective GABAA receptor PAMs will have efficacy and tolerability as pain medications including those for neuropathic pain. Given their predicted anxiolytic effects, α2/3-selective GABAA receptor PAMs offer an additional inroad into the management of pain.

Biomarker Analysis of Orally Dosed, Dual Active, Matrix Metalloproteinase (MMP)-2 and MMP-9 Inhibitor, AQU-118, in the Spinal Nerve Ligation (SNL) Rat Model of Neuropathic Pain.

There is an unmet medical need for the development of non-addicting pain therapeutics with enhanced efficacy and tolerability. The current study examined the effects of AQU-118, an orally active inhibitor of metalloproteinase-2 (MMP-2) and MMP-9, in the spinal nerve ligation (SNL) rat model of neuropathic pain. Mechanical allodynia and the levels of various biomarkers were examined within the dorsal root ganglion (DRG) before and after oral dosing with AQU-118. The rats that received the SNL surgery exhibited significant mechanical allodynia as compared to sham controls. Animals received either vehicle, positive control (gabapentin), or AQU-118. After SNL surgery, the dorsal root ganglion (DRG) of those rats dosed with vehicle had elevated messenger RNA (mRNA) expression levels for MMP-2, IL1-β & IL-6 and elevated protein levels for caspase-3 while exhibiting decreased protein levels for myelin basic protein (MBP) & active IL-β as compared to sham controls. Rats orally dosed with AQU-118 exhibited significantly reduced mechanical allodynia and decreased levels of caspase-3 in the DRG as compared to vehicle controls. Results demonstrate that oral dosing with the dual active, MMP-2/-9 inhibitor, AQU-118, attenuated mechanical allodynia while at the same time significantly reduced the levels of caspase-3 in the DRG.

AMPK agonist AICAR ameliorates portal hypertension and liver cirrhosis via NO pathway in the\xa0BDL rat model

Recent studies have indicated that the Adenosine 5‘-monophosphate (AMP)-activated protein kinase (AMPK) pathway is closely involved in liver fibrosis and other fibrotic diseases. However, whether targeting the AMPK pathway can rescue liver fibrosis and its complications, such as portal hypertension, is unknown. This study aimed to explore the therapeutic value of AICAR (5-aminoimidazole-4-carboxyamide ribonucleoside), an agonist of the AMPK pathway, on liver fibrosis and portal hypertension in bile duct ligation (BDL) rats. In vitro experiments showed that the gene expression levels of TGF-b, a-SMA, and collagen 1 in primary rat hepatic stellate cells (HSCs) were significantly decreased after AICAR treatment. The p-eNOS expression and nitric oxide (NO) production were increased by AICAR administration in sinusoidal endothelial cells (SECs). For in vivo animal studies, AICAR acutely decreased portal pressure in the BDL and CCL4 fibrotic rats, but not in the partial portal vein ligation (PVL) rats, without changes in systemic hemodynamics. It was also observed by using intravital fluorescence microscopy that AICAR led to sinusoidal vasodilation in situ experiment. We propose that the relevant mechanisms may be related to the activation of the AMPK/NO pathway in SECs and that this activation promoted NO production in the liver, thereby promoting hepatic sinusoid microcirculation and decreased intrahepatic resistance. The results were verified using the NO inhibitor L-NAME. Chronic AICAR treatment also showed profound beneficial effects on the BDL model rats. The hemodynamic condition was greatly improved, but the positive effect could be partially blocked by L-NAME. Moreover, AICAR also decreased hepatic fibrogenesis in the BDL rats.Key messagesAcute and chronic use of AICAR could alleviate portal pressure without changing systemic hemodynamics.AICAR induced sinusoidal vasodilation by improving NO bioavailability and ameliorating endothelial dysfunction in vivo and in vitro.AICAR could alleviate liver cirrhosis in the BDL model rats.

The Impact of Electrical Charge Delivery on Inhibition of Mechanical Hypersensitivity in Nerve-Injured Rats by Sub-Sensory Threshold Spinal Cord Stimulation

Spinal cord stimulation (SCS) represents an important neurostimulation therapy for pain. A new ultra-high frequency (10,000 Hz) SCS paradigm has shown improved pain relief without eliciting paresthesia. We aim to determine whether sub-sensory threshold SCS of lower frequencies also can inhibit mechanical hypersensitivity in nerve-injured rats and examine how electric charge delivery of stimulation may affect pain inhibition by different patterns of subthreshold SCS. We used a custom-made quadripolar electrode (Medtronic Inc., Minneapolis, MN, USA) to provide bipolar SCS epidurally at the T10 to T12 vertebral level. According to previous findings, SCS was tested at 40% of the motor threshold, which is considered to be a sub-sensory threshold intensity in rats. Paw withdrawal thresholds to punctate mechanical stimulation were measured before and after SCS in rats that received an L5 spinal nerve ligation. Both 10,000 Hz (10 kHz, 0.024 msec) and lower frequencies (200 Hz, 1 msec; 500 Hz, 0.5 msec; 1200 Hz; 0.2 msec) of subthreshold SCS (120 min) attenuated mechanical hypersensitivity, as indicated by increased paw withdrawal thresholds after stimulation in spinal nerve ligation rats. Pain inhibition from different patterns of subthreshold SCS was not governed by individual stimulation parameters. However, correlation analysis suggests that pain inhibition from 10 kHz subthreshold SCS in individual animals was positively correlated with the electric charges delivered per second (electrical dose). Inhibition of neuropathic mechanical hypersensitivity can be achieved with low-frequency subthreshold SCS by optimizing the electric charge delivery, which may affect the effect of SCS in individual animals.

Spinal glial cell line-derived neurotrophic factor infusion reverses reduction of Kv4.1-mediated A-type potassium currents of injured myelinated primary afferent neurons in a neuropathic pain model.

High frequency spontaneous activity in injured primary afferents has been proposed as a pathological mechanism of neuropathic pain following nerve injury. Although spinal infusion of glial cell line-derived neurotrophic factor reduces the activity of injured myelinated A-fiber neurons after fifth lumbar (L5) spinal nerve ligation in rats, the implicated molecular mechanism remains undetermined. The fast-inactivating transient A-type potassium current (IA) is an important determinant of neuronal excitability, and five voltage-gated potassium channel (Kv) alpha-subunits, Kv1.4, Kv3.4, Kv4.1, Kv4.2, and Kv4.3, display IA in heterologous expression systems. Here, we examined the effect of spinal glial cell line-derived neurotrophic factor infusion on IA and the expression of these five Kv mRNAs in injured A-fiber neurons using the in vitro patch clamp technique and in situ hybridization histochemistry. Glial cell line-derived neurotrophic factor infusion reversed axotomy-induced reduction of the rheobase, elongation of first spike duration, and depolarization of the resting membrane potential. L5 spinal nerve ligation significantly reduced the current density of IA and glial cell line-derived neurotrophic factor treatment reversed the reduction. Among the examined Kv mRNAs, only the change in Kv4.1-expression was parallel with the change in IA after spinal nerve ligation and glial cell line-derived neurotrophic factor treatment. These findings suggest that glial cell line-derived neurotrophic factor should reduce the hyperexcitability of injured A-fiber primary afferents by IA recurrence. Among the five IA-related Kv channels, Kv4.1 should be a key channel, which account for this IA recurrence.

The expression and meaning of Hippo signaling pathway in carotid artery ligation induced arterial remodeling model of rats

To establish an arterial remodeling model of rats and to investigate the expression and role of Hippo signaling pathway in this model. In the model group (n=40), the left common carotid artery was removed through the median incision of the neck. The 6-0 non-absorbable line was used to ligate the carotid artery near the proximal end as far as possible, completely blocking the blood flow. The common carotid artery of rats in control group (n=20) was not ligated using the operative line. After 14 days, the animals were sacrificed and the common carotid arteries were separated through the original surgical pathway and the arteries from the ligature to the distal end were collected. Arterial morphology and fibrosis were observed by HE and MASSON staining. Immunohistochemical staining was used to detect the expressions of anti-α smooth muscle actin (α-MSA) and proliferating cell nuclear antigen (PCNA) in the carotid artery. Western blot was used to detect the expressions of yes associated protein (YAP), transcriptional coactivator with PDZ-binding motif (TAZ), TEAD1, Bcl-2-like protein 4 (Bax), and B-cell lymphoma-2 (Bcl-2). Compared with the control group, the HE staining showed that the vascular remodeling was obvious, the ratio of the neointima/middle membrane was increased significantly, and the MASSON staining indicated that the fibrosis was significantly increased in model group. The immunohistochemical staining suggested that the expressions of α-SMA and PCNA were increased significantly; Western blot suggested that the expressions of YAP, TAZ, TEAD1, and Bcl-2 were increased in carotid artery of the model group. While the expression of Bax and the ratio of Bax/Bcl-2 were decreased. A rat model of arterial remodeling mediated by carotid artery ligation was established successfully in this study. Hippo signaling pathway was proved to be activated in the arterial remodeling model induced by carotid artery ligation in rats, and might regulate the change of Bax/Bcl-2 ratio related to proliferation and apoptosis, and subsequently involved in the proliferation of smooth muscle cells to promote vascular remodeling.

Effect of taxifolin on oxidative gastric injury induced by celiac artery ligation in rats

Purpose: To examine the effect of taxifolin on I/R induced gastric injury in rats using biochemical and histopatholohical methods. Methods: Eighteen albino Wistar male rats equally grouped as; gastric I/R (I/R), 50 mg/kg taxifolin + gastric I/R (TAX+ I/R) and sham operation applied (SHAM). Ischemia induced for 1 hour, and reperfusion induced for 3 hours. Results: Oxidant parameters like, Malondialdehyde (MDA) and Hydroxyguanine (8-OHdG) were higher, whereas total glutathione (tGSH) was lower in the I/R group according to SHAM group, histopathological findings such as marked destruction, edema, and proliferated dilated congested blood vessels were observed severely in the I/R group, whereas there was not any pathological finding except mild dilated congested blood vessels in the TAX+ I/R group. Conclusion: The taxifolin can be clinically beneficial in the treatment of gastric injury due to I/R procedure.

Metformin attenuates oxidative stress and liver damage after bile duct ligation in rats.

The aim of the current study was to investigate the antioxidative effect of metformin (MTF) on bile duct ligation (BDL)-induced hepatic disorder and histological damage in rats. The rats were divided into 4 groups including sham control (SC), BDL alone (BDL surgery), MTF1 (BDL surgery and administration of 250 mg/kg of MFM) and MTF2 (BDL surgery and administration of 500 mg/kg of MTF). After BDL, the animals treated with MTF by gavage for 10 days. Hematoxylin and eosin staining, biochemical analysis and oxidative stress markers were assayed to determine histological alterations, liver functions, and oxidant/antioxidant status. Hepatotoxicity was verified by remarkable increase in plasma levels of aminotransferases and alkaline phosphatase activity and liver histology 10 days after the BDL surgery. Our finding showed that treatment with MTF markedly reduced plasma alkaline phosphatase and alleviated liver injury indices (P ≤ 0.05). Furthermore, BDL caused a considerable increase in the protein carbonyl and malondialdehyde content (P ≤ 0.05). However, MTF reduces oxidative stress by constraining the protein oxidation and lipid peroxidation, and increases antioxidant reserve by increasing the ferric reducing ability of plasma and reducing glutathione levels. MTF exerts antioxidative effects in the liver fibrosis and may represent a hepato-protective effect when given to rats with BDL-induced hepatic injury.

MicroRNA-7a ameliorates neuropathic pain in a rat model of spinal nerve ligation via the neurofilament light polypeptide-dependent signal transducer and activator of transcription signaling pathway.

Neuropathic pain is a type of chronic pain induced by either central or peripheral nerve injury. MicroRNAs have been recently linked to many diseases, including neuropathic pain. However, the role of miR-7a in neuropathic pain still remains elusive. Thus, we aim to investigate the effects of miR-7a on neuropathic pain based on the spinal nerve ligation rat model. After establishment of spinal nerve ligation rat models, rats were infected with adeno-associated virus-neurofilament light polypeptide, adeno-associated virus-miR-7a or treated with metformin. The paw withdrawal threshold and paw withdrawal latency were assessed afterward, and the expression of miR-7a and neurofilament light polypeptide as well as their interaction was determined. Subsequently, miR-7a was overexpressed or silenced in dorsal root ganglion cells to investigate the role of miR-7a in neuropathic pain. Furthermore, the regulatory effect of neurofilament light polypeptide on neuropathic pain was detected using plasmid overexpressing neurofilament light polypeptide. Spinal nerve ligation rat model exhibited upregulation of neurofilament light polypeptide but downregulation of miR-7a. In addition, neurofilament light polypeptide accumulation or miR-7a inhibition decreased paw withdrawal threshold and paw withdrawal latency. Then, neurofilament light polypeptide accumulation or miR-7a inhibition was observed to increase the phosphorylation level of signal transducer and activator of transcription. miR-7a was found to directly target neurofilament light polypeptide and downregulate neurofilament light polypeptide. In addition, inhibiting the signal transducer and activator of transcription signaling pathway was also revealed to increase paw withdrawal threshold and paw withdrawal latency. Collectively, our study demonstrated that miR-7a ameliorated neuropathic pain via blocking the signal transducer and activator of transcription signaling pathway by repressing neurofilament light polypeptide. These findings, if taken further, can be of important clinical significance in treating patients with neuropathic pain.

AICAR-Induced AMPK Activation Inhibits the Noncanonical NF-κB Pathway to Attenuate Liver Injury and Fibrosis in BDL Rats.

Background To evaluate the AMP-activated protein kinase- (AMPK-) mediated signaling and NF-κB-related inflammatory pathways that contribute to cholestatic diseases in the bile duct ligation (BDL) rat model of chronic cholestasis and verify the protective role of 5-Aminoimidazole-4-carboxamide1-β-D-ribofuranoside (AICAR) against hepatic injury and fibrosis triggered by cholestasis-related inflammation. Methods Animals were randomly divided into three groups: sham-operated group, BDL group, and BDL+ AICAR group. Cholestatic liver injury was induced by common BDL. Two weeks later, rats in BDL+AICAR group started receiving AICAR treatment. Hepatic pathology was examined by haematoxylin and eosin (H&E) and sirius red staining and hydroxyproline assay was performed in evaluating the severity of hepatic cirrhosis. Real-time PCR and Western blot were performed for RNA gene expression of RNA and protein levels, respectively. Results The BDL group showed liver injury as evidenced by histological changes and elevation in serum biochemicals, ductular reaction, fibrosis, and inflammation. The mRNA expression of canonical NF-κB inflammatory cytokines such as TNF-α, IL-1β, TGF-β, and the protein of noncanonical NF-κB, P100, and P52 was upregulated in the livers of BDL rats. The BDL rats with the administration of AICAR could induce AMPK activation inhibiting the noncanonical NF-κB pathway to attenuate liver injury and fibrosis in BDL rats. Conclusion The BDL model of hepatic cholestatic injury resulting in activation of Kupffer cells and recruitment of immune cells might initiate an inflammatory response through activation of the NF-κB pathway. The AMPK activator AICAR significantly alleviated BDL-induced inflammation in rats by mainly inhibiting the noncanonical NF-κB pathway and thus protecting against hepatic injury and fibrosis triggered by BDL.

Effects of baicalin on diabetic neuropathic pain involving transient receptor potential vanilloid 1 in the dorsal root ganglia of rats.

Diabetic peripheral neuropathy is the most common complication of diabetes mellitus and leads to sensory symptoms, including diabetic neuropathic pain (DNP). DNP is a major public health problem because it has a considerable impact on life quality of diabetes mellitus patients. Therefore, development of novel effective analgesics for DNP relief and treatment is warranted. Transient receptor potential vanilloid 1 (TRPV1) has a crucial role in nociceptive transmission under pathological forms of pain. Baicalin is a flavonoid compound extracted from a medicinal herb, Huang Qin, it possesses antioxidant properties and has an analgesic effect on nitroglycerin-induced migraine in rats and neuropathic pain in spinal nerve ligation rats. However, the effects of baicalin on DNP are unclear. Therefore, the aim of this study is to examine the effects of baicalin on DNP. Our data show that a single dose of baicalin (40 µg/kg) had a transient analgesic effect on streptozotocin (STZ)-induced DNP rats. Moreover, cumulative injection of baicalin prevented the development of STZ-induced DNP in rats in a dose-dependent manner. In addition, baicalin dose-dependently suppressed the expression of TRPV1 in dorsal root ganglia of STZ-induced DNP rats. Therefore, the analgesic role of baicalin in DNP probably occurred through TRPV1. Baicalin may play an important analgesic role in DNP and might serve as a potential compound in clinical treatment and prevention of DNP.

Insulin reverses major portal hypertension-related derangements in rats with liver cirrhosis and diabetes.

Liver cirrhosis is accompanied by increased intrahepatic resistance and angiogenesis-related portosystemic collaterals formation. Diabetic patients suffer from abnormal vasoresponsiveness and angiogenesis that can be ameliorated by glucose control. However, the relevant presentation is not clear in those with cirrhosis and diabetes, in whom insulin is the treatment of choice. Liver cirrhosis was induced in Sprague-Dawley rats with common bile duct ligation (BDL) and sham rats were used as controls. Streptozotocin 60 mg/kg (STZ, i.p., to induce diabetes) or vehicle was injected. The rats received BDL and STZ injections were injected with insulin or vehicle. On the 29th day after the procedure, the groups were surveyed for (1) systemic and portal hemodynamics; (2) mesenteric vascular density; (3) severity of portosystemic collaterals; (4) hepatic resistance using in situ liver perfusion; (5) histology survey of mesentery and liver; and (6) mesentery angiogenesis- and liver fibrogenesis-related protein expressions. Compared with the cirrhotic rats, the cirrhotic diabetic rats had lower body weight, cardiac output, superior mesenteric arterial (SMA) resistance and portal venous (PV) resistance, and higher SMA and PV flow, which were mostly reversed by insulin. The cirrhotic diabetic rats also had increased mesenteric vascular density, and enhanced pERK, pAkt, VEGF, VEGFR2 protein expressions that were reversed by insulin. Insulin decreased the degree of shunting in the diabetic cirrhotic rats. Hepatic perfusion pressure and severity of liver fibrosis were not significantly influenced by diabetes and insulin treatment in the cirrhotic rats. In conclusion, diabetes aggravated hemodynamic derangements, mesenteric angiogenesis and collaterals in the cirrhotic rats, which were mostly ameliorated by insulin. Further clinical investigations are warranted.

Bile acids elicited endothelium-dependent vasoconstrictor hypo-activity through TRPV4 channels in the thoracic aorta of bile duct ligation rats

Numerous studies have demonstrated the impaired cardiovascular reactivity in cholestasis patients and bile duct ligated animals. However, the underlying mechanism remains uncertain. Transient receptor potential cation V4 (TRPV4) channels are reported to be naturally expressed in the cardiovascular system, especially on endothelial cells. However, the role of TRPV4 (transient receptor potential vanilloid 4) in regulating vascular reactivity is poorly established. In this study, we first determined that bile acids elicited endothelium-dependent vasoconstrictor hypo-activity via TRPV4 channels, which further activated cyclooxygenase 2 (COX2). Myography results demonstrated that the vascular contractile response was attenuated in BDL rats when exposed to 60 mmol/L KCl. Real time PCR and western blotting results showed that bile duct ligation (BDL) induced a time-dependent increase in TRPV4 expression levels. In addition, bile acids upregulated the expression of TRPV4 protein, which proved to be located on the cell surface of endothelial cells, and induced intracellular Ca2+ events. The relaxation response was increased while the contractile response was decreased in BDL rats, and those effects were reversed by a TRPV4 inhibitor (HC067047). Contractions induced by norepinephrine were primarily inhibited by the COX2 inhibitor, but not the NOS inhibitor, and the expression of COX2 was downregulated after TRPV4 inhibition. These data indicated that TRPV4/COX2 pathways in the endothelium are involved in vasoconstrictor hypo-activity. Our current results suggested that the TRPV4 pathway is involved in the regulation of bile acids in vasoconstrictor hypo-activity in bile duct ligation rats.

Stimulating muscarinic M1 receptors in the anterior cingulate cortex reduces mechanical hypersensitivity via GABAergic transmission in nerve injury rats

Cholinergic systems modulate synaptic transmission across the neuraxis and play an important role in higher brain function including cognition, arousal and nociception. The anterior cingulate cortex (ACC) is a fundamental brain region for nociception and chronic pain, and receives cholinergic projections mainly from basal forebrain. Recently, we found that the activation of muscarinic M1 receptors in the ACC produced antinociceptive behavior in response to mechanical stimulation. However, it has not been tested whether stimulating muscarinic receptors in the ACC can reduce mechanical hypersensitivity in animal models of chronic pain.Here, we tested whether the activation of muscarinic M1 receptors in the ACC can alleviate mechanical hypersensitivity in a nerve injury model. The activation of muscarinic M1/M4 receptors by McN-A-343 injected into the contralateral side of the ACC, but not into the ventral posterolateral nucleus, was found to dose-dependently reduce mechanical hypersensitivity 7 days following partial sciatic nerve ligation in rats. The reduction of mechanical hypersensitivity by McN-A-343, was blocked by a selective muscarinic M1 antagonist, but not a M4 receptor antagonist. Importantly, the nerve injury model did not change the protein expression of muscarinic M1 receptors in the ACC. Additionally, a type A γ-aminobutyric acid (GABAA) receptor agonist injected into the ACC reduced the mechanical hypersensitivity in this injury model. Finally, a GABAA receptor antagonist blocked the reduction of mechanical hypersensitivity by McN-A-343 in the injury model. Collectively, these results suggest that activations of muscarinic M1 receptors in the ACC reduce nerve injury-induced mechanical hypersensitivity through GABAergic transmission via GABAA receptors.

Enzyme scaffolding for metabolic engineering endeavors

Spinal cord stimulation (SCS) represents an important neurostimulation therapy for pain. A new ultra-high frequency (10,000 Hz) SCS paradigm has shown improved pain relief without eliciting paresthesia. We aim to determine whether sub-sensory threshold SCS of lower frequencies also can inhibit mechanical hypersensitivity in nerve-injured rats and examine how electric charge delivery of stimulation may affect pain inhibition by different patterns of subthreshold SCS.Materials and Methods: We used a custom-made quadripolar electrode (Medtronic Inc., Minneapolis, MN, USA) to provide bipolar SCS epidurally at the T10 to T12 vertebral level. According to previous findings, SCS was tested at 40% of the motor threshold, which is considered to be a sub-sensory threshold intensity in rats. Paw withdrawal thresholds to punctate mechanical stimulation were measured before and after SCS in rats that received an L5 spinal nerve ligation.Results: Both 10,000 Hz (10 kHz, 0.024 msec) and lower frequencies (200 Hz, 1 msec; 500 Hz, 0.5 msec; 1200 Hz; 0.2 msec) of subthreshold SCS (120 min) attenuated mechanical hypersensitivity, as indicated by increased paw withdrawal thresholds after stimulation in spinal nerve ligation rats. Pain inhibition from different patterns of subthreshold SCS was not governed by individual stimulation parameters. However, correlation analysis suggests that pain inhibition from 10 kHz subthreshold SCS in individual animals was positively correlated with the electric charges delivered per second (electrical dose).Conclusions: Inhibition of neuropathic mechanical hypersensitivity can be achieved with low-frequency subthreshold SCS by optimizing the electric charge delivery, which may affect the effect of SCS in individual animals.

Paclitaxel alleviates liver fibrosis induced by bile duct ligation in rats: Role of TGF-β1, IL-10 and c-Myc

Liver fibrosis is a global health issue that causes morbidity and mortality with no currently available treatment. It has been shown that low dose paclitaxel (PTX) can stabilize microtubules and inhibit the profibrotic transforming growth factor-beta 1 (TGF-β1) signaling pathway. In this study the effect of treatment with low dose PTX was examined using a model of cholestatic liver fibrosis. Bile-duct ligation (BDL) was induced in rats for 2 weeks then PTX (0.3 mg/kg/ip) was administered three times a week for 2 weeks. Administration of PTX ameliorated BDL-induced elevation in biomarkers of hepatocellular damage (alanine transaminase; ALT and aspartate transaminase; AST) and obstructive cholestatic injury (total bilirubin and gamma glutamyl transferase; γ-GT). PTX was able to correct the increase in liver weight to body weight ratio and the bile duct proliferation induced by BDL. Additionally, PTX treatment corrected the BDL-induced fibrosis of portal tracts, elevation of hydroxyproline content and increased alpha smooth muscle actin (α-SMA) mRNA and protein expression. This antifibrotic effect of PTX was further examined through its inhibitory effect on TGF-β1 mRNA and protein expression in addition to c-Myc mRNA expression. Furthermore, PTX rectified the BDL-induced decrease in interleukin-10 (IL-10) mRNA and protein expression. In conclusion, this study suggests that PTX at low dose has the potential to treat BDL-induced liver fibrosis in rats possibly through suppression of TGF-β1 and c-Myc and activation of IL-10 pathways.

Analgesic effects of ASP3662, a novel 11β-hydroxysteroid dehydrogenase 1 inhibitor, in rat models of neuropathic and dysfunctional pain.

Glucocorticoids are a major class of stress hormones known to participate in stress-induced hyperalgesia. Although 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) is a key enzyme in the intracellular regeneration of glucocorticoids in the CNS, its role in pain perception has not been assessed. Here, we examined the effects of ASP3662, a novel 11β-HSD1 inhibitor, on neuropathic and dysfunctional pain. The enzyme inhibitory activities and pharmacokinetics of ASP3662 were examined, and its antinociceptive effects were evaluated in models of neuropathic pain, fibromyalgia and inflammatory pain in Sprague-Dawley rats. ASP3662 inhibited human, mouse and rat 11β-HSD1 but not human 11β-HSD2, in vitro. ASP3662 had no significant effect on 87 other possible targets (enzymes, transporters and receptors). ASP3662 inhibited in vitro conversion of glucocorticoid from its inactive to active form in extracts of rat brain and spinal cord. Pharmacokinetic analysis in Sprague-Dawley rats showed that ASP3662 has CNS-penetrability and long-lasting pharmacokinetic properties. Single oral administration of ASP3662 ameliorated mechanical allodynia in spinal nerve ligation (SNL) and streptozotocin-induced diabetic rats and thermal hyperalgesia in chronic constriction nerve injury rats. ASP3662 also restored muscle pressure thresholds in reserpine-induced myalgia rats. Intrathecal administration of ASP3662 was also effective in SNL rats. However, ASP3662 had no analgesic effects in adjuvant-induced arthritis rats. ASP3662 is a potent, selective and CNS-penetrable inhibitor of 11β-HSD1. The effects of ASP3662 suggest that selective inhibition of 11β-HSD1 may be an attractive approach for the treatment of neuropathic and dysfunctional pain, as observed in fibromyalgia.