Development of a novel knee contracture mouse model by immobilization using external fixation.

AIMS: Several studies have used animal models to examine knee joint contracture; however, few reports detail the construction process of a knee joint contracture model in a mouse. The use of mouse models is beneficial, as genetically modified mice can be used to investigate the pathogenesis of joint contracture. Compared to others, mouse models are associated with a lower cost to evaluate therapeutic effects. Here, we describe a novel knee contracture mouse model by immobilization using external fixation. METHODS: The knee joints of mice were immobilized by external fixation using a splint and tape. The passive extension range of motion (ROM), histological and immunohistochemical changes, and expression levels of fibrosis-related genes at 2 and 4 weeks were compared between the immobilized (Im group) and non-immobilized (Non-Im group) groups. RESULTS: The extension ROM at 4 weeks was significantly lower in the Im group than in the Non-Im group (p < 0.01). At 2 and 4 weeks, the thickness and area of the joint capsule were significantly greater in the Im group than in the Non-Im group (p < 0.01 in all cases). At 2 weeks, the mRNA expression levels of the fibrosis-related genes, except for the transforming growth factor-ß1, and the protein levels of cellular communication network factor 2 and vimentin in the joint capsule were significantly higher in the Im group (p < 0.01 in all cases). CONCLUSION: This mouse model may serve as a useful tool to investigate the etiology of joint contracture and establish new treatment methods.

Efficacy of isoproterenol in the evaluation of dormant conduction and arrhythmogenic foci identification in atrial fibrillation ablation.

BACKGROUND: Catheter ablation for atrial fibrillation (AF) is an established therapy. However, postoperative recurrence is a serious issue caused by the reconduction of the isolated pulmonary veins (PV) and the onset of non-PV foci. The objectives of this study were to elucidate dormant conduction, confirm PV arrhythmia substrate, induce non-PV foci after PV isolation, and assess the acute efficacy of high dose isoproterenol (ISP) when administered in addition to adenosine. METHODS: The study consisted of 100 patients with drug-refractory AF (paroxysmal and persistent) who underwent ablation therapy (either radio-frequency or cryoballoon ablation) as the first-line of therapy at our hospital. All patients first underwent PV isolation (PVI) and were administered adenosine followed by ISP (6 µg × 5 min). The effects were observed, and the therapeutic strategy was evaluated. RESULTS: Persistent dormant conduction due to ISP administration was observed in 13 patients. In over half of the patients, arrhythmia substrates were identified in the PV. Ten patients presented with persistent PV firing. The ablation of non-PV foci was additionally performed in 23 patients. CONCLUSIONS: We found that dormant conduction, as a result of ISP administration, is persistent and ISP is useful when performing an ablation. In addition, ISP administration is useful for the identification of PV arrhythmia substrates and induction of non-PV foci. However, the effectiveness of ISP may be partially due to the complementary effect of adenosine, and, therefore, a combination of the two drugs seems preferable.

Effects of metformin on knee joint capsule fibrosis in a diabetic mouse model.

Aims: The antidiabetic agent metformin inhibits fibrosis in various organs. This study aims to elucidate the effects of hyperglycaemia and metformin on knee joint capsule fibrosis in mice. Methods: Eight-week-old wild-type (WT) and type 2 diabetic (db/db) mice were divided into four groups without or with metformin treatment (WT met(-/+), Db met(-/+)). Mice received daily intraperitoneal administration of metformin and were killed at 12 and 14 weeks of age. Fibrosis morphology and its related genes and proteins were evaluated. Fibroblasts were extracted from the capsules of 14-week-old mice, and the expression of fibrosis-related genes in response to glucose and metformin was evaluated in vitro. Results: The expression of all fibrosis-related genes was higher in Db met(-) than in WT met(-) and was suppressed by metformin. Increased levels of fibrosis-related genes, posterior capsule thickness, and collagen density were observed in the capsules of db/db mice compared with those in WT mice; these effects were suppressed by metformin. Glucose addition increased fibrosis-related gene expression in both groups of mice in vitro. When glucose was added, metformin inhibited the expression of fibrosis-related genes other than cellular communication network factor 2 (Ccn2) in WT mouse cells. Conclusion: Hyperglycaemia promotes fibrosis in the mouse knee joint capsule, which is inhibited by metformin. These findings can help inform the development of novel strategies for treating knee joint capsule fibrosis.

Impact of C-Reactive Protein on Long-Term Cardiac Events in Stable Coronary Artery Disease Patients with Chronic Kidney Disease.

AIM: Chronic inflammation is associated with atherosclerosis development. Chronic kidney disease (CKD) is an independent risk factor for cardiovascular events and is associated with chronic inflammation. We aimed to investigate the influence of C-reactive protein (CRP), an important marker of inflammation, on the clinical outcomes of patients with CKD and stable coronary artery disease (CAD) undergoing percutaneous coronary intervention (PCI). METHODS: Among patients with stable CAD and CKD who underwent PCI, 516 patients whose CRP levels were available before the PCI procedure were identified. The patients were divided into two groups according to the CRP levels: those with CRP ≥ 2.0 mg/L (high-CRP group) and those with CRP ＜2.0 mg/L (low-CRP group). The primary endpoint of this study was the occurrence of major adverse cardiac events (MACE), defined as a composite of cardiac death, myocardial infarction, and unplanned revascularization. RESULTS: Overall, the mean age of the patients was 72.5±9.7 years, and 20.7% were female. The median CRP level was 1.43 mg/L (0.6-4.9 mg/L). The median follow-up period was 3.6 years. The occurrence of MACE was significantly higher in the high-CRP group than in the low-CRP group (log-rank p＜0.001). Notably, the incidence rate of cardiac death was significantly higher in the high-CRP group (log-rank p＜0.001). According to the multivariable analysis, CRP level ≥ 2.0 mg/L was found to be a significant predictor of MACE (hazard ratio [HR]: 1.54, 95% confidence interval [CI]: 1.04-2.28, p=0.003), as well as estimated glomerular filtration rate (HR: 0.98, 95% CI: 0.97-0.99, p＜0.01). CONCLUSION: High-CRP levels adversely affect long-term cardiac events in patients with stable CAD and CKD.

A case of accessory pathway mapped with ultra-high-resolution mapping led to a coronary sinus diverticulum.

Ultra-high-resolution mapping is useful in the ablation of accessory pathways. However, in patients with accessory pathways in the coronary sinus (CS) diverticulum, treatment with endocardial ablation may be challenging. Patients suspected of having subepicardial accessory pathways may require the examination of the venous anomaly using CS angiography.

Effect of metformin treatment and its time of administration on joint capsular fibrosis induced by mouse knee immobilization.

Joint contracture leads to major patient discomfort. Metformin, one of the most extensively used oral drugs against type 2 diabetes has recently been found to suppress tissue fibrosis as well. However, its role in suppressing tissue fibrosis in joint contractures remains unknown. In this study, we examined the role of metformin treatment in suppressing joint capsular fibrosis and the most effective time of its administration. Joint capsular fibrosis was induced by immobilizing the knee joints of mice using splints and tapes. Metformin was administered intraperitoneally every alternate day after immobilization. Histological and immunohistochemical changes and expression of fibrosis-related genes were evaluated. Metformin treatment significantly suppressed fibrosis in joint capsules based on histological and immunohistochemical evaluation. Joint capsular tissue from metformin-treated mice also showed decreased expression of fibrosis-related genes. Early, but not late, metformin administration showed the same effect on fibrosis suppression in joint capsule as the whole treatment period. The expression of fibrosis-related genes was most suppressed in mice administered with metformin early. These studies demonstrated that metformin treatment can suppress joint capsular fibrosis and the most effective time to administer it is early after joint immobilization; a delay of more than 2 weeks of administration is less effective.

Heat hypersensitivity is attenuated with altered expression level of spinal astrocytes after sciatic nerve injury in TRPV1 knockout mice.

Transient receptor potential vanilloid 1 (TRPV1) modulates pain. Studies have indicated that TRPV1 is upregulated in the spinal dorsal horn in the neuropathic pain model, but its mechanism is unknown. Here, we examined the mechanism by which TRPV1 modulates neuropathic pain by employing partial sciatic nerve ligation (pSNL) in adult male C57BL/6 J (wild-type: WT) and TRPV1 knockout (Trpv1-/-) mice. We analyzed mechanical/heat sensitivities (von Frey test/hot plate test) and glial/neuronal activities (Iba-1/GFAP/FosB by immunofluorescence) in laminae I and II in the L5 ipsilateral dorsal horn of the spinal cord. Mechanical/heat sensitivities, expression levels of microglial Iba-1 and astrocytic GFAP, and the number of FosB-positive neurons were significantly increased on days 7 and 14 in the pSNL group compared with the sham-operated and non-operated groups of both WT and Trpv1-/- mice. While mechanical sensitivity was comparable between WT and Trpv1-/- mice, the threshold against heat sensitivity was markedly prolonged in Trpv1-/- than WT mice on day 14 after pSNL. Conversely, the increment of FosB positive neurons was significantly attenuated in Trpv1-/- than WT mice on days 7 and 14 after pSNL. These results suggest that TRPV1 may modulate thermal perception via increased astrocytes in the dorsal horn of the spinal cord.

The appearance of a single left atrial tachycardia as two different types on preoperative surface and intracardiac ECG of pacemaker due to progressive cardiac conduction disturbance.

We treated a patient with PCCD whose single left AT appeared as two different types on preoperative surface and intracardiac ECG from a pacemaker. The diagnosis was hindered by the fact that the conduction block encompassed interatrial block and the pacemaker A-wave was captured at the right atrial appendage.

Disruption of the aldehyde dehydrogenase 2 gene increases the bone anabolic response to intermittent PTH treatment in an ovariectomized mouse model.

Aldehyde dehydrogenase 2 (ALDH2) is the enzyme that oxidizes the acetaldehyde produced by alcohol metabolism. This variant not only affects the response to alcohol but is also associated with several diseases, such as esophageal cancer, myocardial infarction, and particularly osteoporosis. In our previous study, we reported that compared to wild-type (WT) mice, Aldh2 knockout (KO) mice naturally have a strong bone formation ability, and high expression of parathyroid hormone receptor (PTHR1) in osteocytes. The effect of the Aldh2 gene on bone metabolism in response to intermittent PTH treatment is unknown. The purpose of this study was to clarify the effect of the Aldh2 gene on the bone anabolic response to intermittent PTH treatment in ovariectomized mice. Female KO and WT mice were ovariectomized at 8 weeks of age. At 14 weeks of age, the KO and WT mice were divided into vehicle-treated (Veh) and PTH-treated (PTH) groups (i.e., the WT-Veh, WT-PTH, KO-Veh and KO-PTH groups). PTH (1-34) and vehicle were subcutaneously administered to each group at a dose of 40 µg/kg body weight (BW) five times per week for 4 weeks. Micro-CT showed that the bone volume (BV), trabecular number (Tb.N), connectivity density (Conn.D), and cortical thickness (Ct.Th) values in the KO-PTH mice were significantly higher than those in the KO-Veh mice. Histomorphometric analysis showed that the BV, Tb.N, and mineral apposition rate (MAR) values in the KO-PTH group were significantly higher than those in the KO-Veh group. The mRNA expression level of PTHR1 in the KO-PTH group was significantly increased and that of p21 in the KO-PTH group was significantly decreased compared with the levels in the KO-Veh group. The expression of PTHR in osteocytes from the KO-PTH group was also significantly increased compared with that in osteocytes from the KO-Veh group. Furthermore, cell cultures revealed that the ALP+CFU-f/total CFU-f percentage was significantly higher in the KO-PTH group than in the KO-Veh group. We concluded that in ovariectomized Aldh2 KO mice, the bone anabolic response to intermittent PTH treatment was significantly enhanced compared to that in WT mice, which may be mediated by the high expression level of PTHR1.

Transient receptor potential vanilloid 1 and 4 double knockout leads to increased bone mass in mice.

Calcium balance is important in bone homeostasis. The transient receptor potential vanilloid (TRPV) channel is a nonselective cation channel permeable to calcium and is activated by various physiological and pharmacological stimuli. TRPV1 and TRPV4, in particular, have important roles in intracellular Ca2+ signaling and extracellular calcium homeostasis in bone cells. TRPV1 and TRPV4 separately mediate osteoclast and osteoblast differentiation, and deficiency in any of these channels leads to increased bone mass. However, it remains unknown whether bone mass increases in the absence of both TRPV1 and TRPV4. In this study, we used TRPV1 and TRPV4 double knockout (DKO) mice to evaluate their bone mass in vivo, and osteoclast and osteoblast differentiation in vitro. Our results showed that DKO mice and wild type (WT) mice had no significant difference in body weight and femur length. However, the results of dual-energy X-ray absorption, microcomputed tomography, and bone histomorphometry clearly showed that DKO mice had higher bone mass than WT mice. Furthermore, DKO mice had less multinucleated osteoclasts and had lower bone resorption. In addition, the results of cell culture using flushed bone marrow from mouse femurs and tibias showed that osteoclast differentiation was suppressed, whereas osteoblast differentiation was promoted in DKO mice. In conclusion, our results suggest that the increase in bone mass in DKO mice was induced not only by the suppression of osteoclast differentiation and activity but also by the augmentation of osteoblast differentiation and activity. Our findings reveal that both the single deficiency of TRPVs and the concurrent deficiency of TRPVs result in an increase in bone mass. Furthermore, our data showed that DKO mice and single KO mice had varying approaches to osteoclast and osteoblast differentiation in vitro, and therefore, it is important to conduct further studies on TRPVs regarding the increase in bone mass to explore not only individual but also a combination of TRPVs.

Influence of proton pump inhibitors on blood dabigatran concentrations in Japanese patients with non-valvular atrial fibrillation.

BACKGROUND: Dabigatran is a direct thrombin inhibitor used to decrease the risk of ischemic stroke in patients with non-valvular atrial fibrillation (NVAF). Its prodrug, dabigatran etexilate (DE) is often co-administrated with a proton pump inhibitor (PPI) because of its adverse effects on the gastrointestinal tract. Drug-drug interactions between DE and PPIs in daily clinical practice have not been fully elucidated. METHODS: Changes in blood dabigatran concentration (DC) were investigated using the dilute thrombin time test in a randomized, open-label, two-period crossover study including 34 Japanese patients with NVAF receiving dabigatran therapy with or without PPI. RESULTS: The average trough DC was significantly higher without PPI than with PPI (83 ± 42.3 vs. 55.5 ± 24.6 ng/mL, respectively; P < 0.001). Similarly, the average peak DC was significantly higher without PPI than with PPI (184.1 ± 107.7 vs. 124 ± 59.2 ng/mL, respectively; P = 0.0029). The average ratio of DC change at the trough and peak levels did not differ significantly among the three PPI types. CONCLUSIONS: PPI administration significantly decreased the trough and peak DCs in patients with NVAF. Therefore, when prescribing PPIs for patients with NVAF in a clinical setting, the possibility that the bioavailability of dabigatran may decrease should be considered.