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Selected Abstracts

Mitochondrial protection by the JNK inhibitor leflunomide rescues mice from acetaminophen-induced liver injury,

HEPATOLOGY, Issue 2 2007
Calivarathan Latchoumycandane
Acetaminophen (APAP) is a widely used analgesic and antipyretic drug that is safe at therapeutic doses but which can precipitate liver injury at high doses. We have previously found that the antirheumatic drug leflunomide is a potent inhibitor of APAP toxicity in cultured human hepatocytes, protecting them from mitochondria-mediated cell death by inhibiting the mitochondrial permeability transition. The purpose of this study was to explore whether leflunomide protects against APAP hepatotoxicity in vivo and to define the molecular pathways of cytoprotection. Male C57BL/6 mice were treated with a hepatotoxic dose of APAP (750 mg/kg, ip) followed by a single injection of leflunomide (30 mg/kg, ip). Leflunomide (4 hours after APAP dose) afforded significant protection from liver necrosis as assessed by serum ALT activity and histopathology after 8 and 24 hours. The mechanism of protection by leflunomide was not through inhibition of cytochrome P450 (CYP),catalyzed APAP bioactivation or an apparent suppression of the innate immune system. Instead, leflunomide inhibited APAP-induced activation (phosphorylation) of c-jun NH2 -terminal protein kinase (JNK), thus preventing downstream Bcl-2 and Bcl-XL inactivation and protecting from mitochondrial permeabilization and cytochrome c release. Furthermore, leflunomide inhibited the APAP-mediated increased expression of inducible nitric oxide synthase and prevented the formation of peroxynitrite, as judged from the absence of hepatic nitrotyrosine adducts. Even when given 8 hours after APAP dose, leflunomide still protected from massive liver necrosis. Conclusion: Leflunomide afforded protection against APAP-induced hepatotoxicity in mice through inhibition of JNK-mediated activation of mitochondrial permeabilization. (HEPATOLOGY 2007.) [source]

Pretreatment with the ciclosporin derivative NIM811 reduces delayed neuronal death in the hippocampus after transient forebrain ischaemia

JOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 4 2010
Masaaki Hokari Dr
Abstract Objectives There have been several previous studies showing that ciclosporin, a ligand for cyclophilin D (CypD), reduces mitochondrial permeability transition (mPT) and ameliorates delayed neuronal death. NIM811 is a non-immunosuppressive ciclosporin derivative that also inhibits mPT, but has significantly less cytotoxicity than ciclosporin. Actually, in animal experiments, several investigators have reported that NIM811 ameliorates central nervous system disorders, such as traumatic brain injury, transient focal cerebral ischaemia and spinal cord injury. Therefore, we evaluated whether the ciclosporin derivative, NIM811 reduces mPT and ameliorates delayed neuronal death in the hippocampal CA1 sectors in mice when subjected to transient forebrain ischaemia. Methods Male C57BL/6 mice were treated with 50 mg/kg ciclosporin, 10, 50 or 100 mg/kg NIM811 or phosphate-buffered saline. At 30 min post-injection, all mice were subjected to 20 min bilateral common carotid artery occlusion (BCCAO). To estimate delayed neuronal death, the sections were prepared for HE staining and terminal deoxynucleotidyl transferase-mediated dUTP end-labelling (TUNEL) staining at 72 h after 20 min BCCAO. Furthermore, using 5,5,,6,6,-tetrachloro-1,1,,3,3,-tetraethylbenzimidazolocarbocyanine iodide (JC-1) staining technique, we evaluated whether NIM811 (1, 10, 100 or 1000 ,m) inhibited mPT in the neurons exposed to 100 ,m glutamate. Results Both delayed neuronal injury and apoptosis in the hippocampal CA1 sectors were significantly ameliorated at 72 h after transient forebrain ischaemia in the mice treated with 100 mg/kg NIM811 or 50 mg/kg ciclosporin. The treatments with 100 ,m and 1000 ,m NIM811 significantly inhibited the reduction of mitochondrial membrane potential in the neurons exposed to 100 ,m glutamate. Conclusions These findings strongly suggest that NIM811 inhibits mPT and ameliorates delayed neuronal death in mice subjected to transient forebrain ischaemia. [source]

Ischemic preconditioning and intermittent clamping improve murine hepatic microcirculation and Kupffer cell function after ischemic injury

LIVER TRANSPLANTATION, Issue 4 2004
Katarína Vajdová
The aim of this study was to evaluate whether the protective effect of intermittent clamping and ischemic preconditioning is related to an improved hepatic microcirculation after ischemia/reperfusion injury. Male C57BL/6 mice were subjected to 75 or 120 min of hepatic ischemia and 1 or 3 hours of reperfusion. The effects of continuous ischemia, intermittent clamping, and ischemic preconditioning before prolonged ischemia on sinusoidal perfusion, leukocyte-endothelial interactions, and Kupffer cell phagocytic activity were analyzed by intravital fluorescence microscopy. Kupffer cell activation was measured by tissue levels of tumor necrosis factor (TNF)-,, and the integrity of sinusoidal endothelial cells and Kupffer cells were evaluated by electron microscopy. Continuous ischemia resulted in decreased sinusoidal perfusion rate and phagocytic activity of Kupffer cell, increased leukocyte-endothelial interactions and TNF-, levels. Both protective strategies improved sinusoidal perfusion, leukocyte-endothelial interactions and phagocytic activity of Kupffer cells after 75-minutes of ischemia, and intermittent clamping also after 120 minutes ischemia. TNF-, release was significantly reduced and sinusoidal wall integrity was preserved by both protective procedures. In conclusion, both strategies are protective against ischemia/reperfusion injury by maintaining hepatic microcirculation and decreasing Kupffer cell activation for clinically relevant ischemic periods, and intermittent clamping appears superior for prolonged ischemia. (Liver Transpl 2004;10:520,528.) [source]

Dietary Steatotic Liver Attenuates Acetaminophen Hepatotoxicity in Mice

MICROCIRCULATION, Issue 1 2006
YOSHIYA ITO
ABSTRACT Objective: To determine whether hepatic steatosis is susceptible to acetaminophen (APAP) hepatotoxicity. Methods: Male C57Bl/6 mice were fed a "Western-style" diet (high fat and high carbohydrate) for 4 months to develop severe hepatic steatosis with mild increases in alanine aminotransferase (ALT) levels. These were compared to mice fed a standard chow diet. Results: Treatment with APAP (300 mg/kg, orally) to mice fed a regular chow increased ALT levels (519-fold) and caused hepatic centrilobular injury at 6 h. APAP increased hepatic cytochrome-P (CYP)-2E1 mRNA levels (17-fold). In vivo microscopic studies showed that APAP caused a 30% decrease in sinusoidal perfusion and the infiltration of red blood cells into the space of Disse. Electron microscopy demonstrated that numerous gaps were formed in sinusoidal endothelial cells. Mice fed the "Western-style" diet were protected from APAP hepatotoxicity as evidenced by 89% decrease in ALT levels and less centrilobular injury, which was associated with 42% decrease in CYP2E1 mRNA levels. The APAP-induced liver microcirculatory dysfunction was minimized in mice fed the "Western-style" diet. Conclusions: These results suggest that hepatic steatosis elicited by the "Western-style" diet attenuated APAP-induced hepatotoxicity by inhibiting CYP2E1 induction and by minimizing sinusoidal endothelial cell injury, leading to protection of liver microcirculation. [source]

Early Hepatic Microvascular Injury in Response to Acetaminophen Toxicity

MICROCIRCULATION, Issue 5 2003
YOSHIYA ITO
ABSTRACT Objective: The hepatic toxic response to acetaminophen (APAP) is characterized by centrilobular (CL) necrosis preceded by hepatic microvascular injury and congestion. The present study was conducted to examine changes in liver microcirculation after APAP dosing. Methods: Male C57Bl/6 mice were treated with APAP (600 mg/kg body weight) by oral gavage. The livers of anesthetized mice were examined using established in vivo microscopic methods at 0, 0.5, 1, 2, 4, 6, 12 hours after APAP. Results: The levels of hepatic transaminases (i.e., alanine aminotransferase [ALT] and aspartate transaminase) increased minimally for up to 2 hours. Thereafter, their levels were significantly and progressively increased. The numbers of swollen sinusoidal endothelial cells (SECs) in periportal regions were increased (3.5-fold) from 0.5 to 6 hours, and those in CL regions were increased (4.0-fold) at 0.5 and 1 hour. The intensity of in vivo staining for formaldehyde-treated serum albumin, which is a specific ligand for SECs, was reduced from 2 to 12 hours. Erythrocytes infiltrated into the space of Disse as early as 2 hours, and the area occupied by these cells was markedly increased at 6 hours. Sinusoidal perfusion was reduced from 1 through 12 hours, with a nadir (35% decrease) at 4 and 6 hours. Phagocytic Kupffer cell activity was significantly elevated from 0.5 through 12 hours. Although gadolinium chloride minimized the changes in sinusoidal blood flow and reduced ALT levels 6 hours after APAP, it failed to inhibit endothelial swelling, extravasation of erythrocytes, and CL parenchymal necrosis. Conclusions: These results confirm that APAP-induced SEC injury precedes hepatocellular injury, supporting the hypothesis that SECs are an early and direct target for APAP toxicity. These findings also suggest that reduced sinusoidal perfusion and increased Kupffer cell activity contribute to the development of APAP-induced liver injury. [source]

Cardiac and coronary function in the Langendorff-perfused mouse heart model

EXPERIMENTAL PHYSIOLOGY, Issue 1 2009
Melissa E. Reichelt
The Langendorff mouse heart model is widely employed in studies of myocardial function and responses to injury (e.g. ischaemia). Nonetheless, marked variability exists in its preparation and functional properties. We examined the impact of early growth (8, 16, 20 and 24 weeks), sex, perfusion fluid [Ca2+] and pacing rate on contractile function and responses to 20 min ischaemia followed by 45 min reperfusion. We also assessed the impact of strain, and tested the utility of the model in studying coronary function. Under normoxic conditions, hearts from 8-week-old male C57BL/6 mice (2 mm free perfusate [Ca2+], 420 beats min,1) exhibited 145 ± 2 mmHg left ventricular developed pressure (LVDP). Force development declined by ,15% (126 ± 5 mmHg) with a reduction in free [Ca2+] to 1.35 mm, and by 25% (108 ± 3 mmHg) with increased pacing to 600 beats min,1. While elevated heart rate failed to modify ischaemic outcome, the lower [Ca2+] significantly improved contractile recovery (by >30%). We detected minimal sex-dependent differences in normoxic function between 8 and 24 weeks, although age modified contractile function in males (increased LVDP at 24 versus 8 weeks) but not females. Both male and female hearts exhibited age-related reductions in ischaemic tolerance, with a significant decline in recovery evident at 16 weeks in males and later, at 20,24 weeks, in females (versus recoveries in hearts at 8 weeks). Strain also modified tolerance to ischaemia, with similar responses in hearts from C57BL/6, 129/sv, Quackenbush Swiss and FVBN mice, but substantially greater tolerance in BALB/c hearts. In terms of vascular function, baseline coronary flow (20,25 ml min,1 g,1) was 50,60% of maximally dilated flows, and coronary reactive and functional hyperaemic responses were pronounced (up to 4-fold elevations in flow in hearts lacking ventricular balloons). These data indicate that attention to age (and sex) of mice will reduce variability in contractile function and ischaemic responses. Even small differences in perfusion fluid [Ca2+] also significantly modify tolerance to ischaemia (whereas modest shifts in heart rate do not impact). Ischaemic responses are additionally strain dependent, with BALB/c hearts displaying greatest intrinsic tolerance. Finally, the model is applicable to the study of vascular reactivity, providing large responses and excellent reproducibility. [source]

Expression of Endothelial Cell Adhesion Molecules in Neovascularized Tissue

MICROCIRCULATION, Issue 4 2000
GINA VALLIEN
ABSTRACT Objective: Recent studies indicate that endothelial cells of newly formed blood vessels are activated and exhibit a distinct phenotype that may influence the responses of these microvessels to an inflammatory stimulus. The objective of this study was to compare the basal and cytokine-stimulated expression of endothelial cell adhesion molecules in neovascularized tissue to normal (nonproliferating) vascular beds. Methods: The expression of P- and E-selectin, VCAM-1, ICAM-1, ICAM-2, and PECAM-1 was measured, using the dual radiolabeled mAb technique, in subcutaneously implanted (for 10,15 days) polyurethane sponges, skin, heart, lung, and intestine of male C57BL/6 mice (background). Results: Basal values of PECAM-1 and ICAM-2 revealed a low vascular density in the implanted sponge matrices that is comparable to skin. When normalized for vascular surface area (PECAM-1 or ICAM-1 expression), the basal level of E- and P-selectin expression was highest in neovascularized sponge and skin. TNF-, elicited an increased expression of all endothelial CAMs, except PECAM-1 and ICAM-2, but the responses were blunted in sponge and skin, relative to other vascular beds. Conclusions: These findings indicate that endothelial cells in newly formed blood vessels exhibit a pattern of basal and cytokine-induced expression of certain adhesion glycoproteins that is similar to nonproliferating cutaneous vessels. [source]

Morphometric characterization of murine articular cartilage,Novel application of confocal laser scanning microscopy

MICROSCOPY RESEARCH AND TECHNIQUE, Issue 9 2009
Kathryn S. Stok
Abstract A new technique for characterization of the three-dimensional morphology of murine articular cartilage is proposed. The technique consists of a novel application of confocal laser scanning microscopy (CLSM), where the objective was to develop and validate it for cartilage measurements in murine joints. Murine models are used in arthritis research, because they are well-described for manipulating the disease pathophysiology, facilitating our understanding of the disease, and identifying new targets for therapy. A calibration and reproducibility study was carried out to provide a consistent testing methodology for quantification of murine joints. The proximal tibial condyles from male C57BL/6 mice were scanned using a CLS microscope with an isotropic voxel size of 5.8 ,m. Measurements and analyses were repeated three times on different days, and in a second step the analysis was repeated three times for a single measurement. Calculation of precision errors (coefficient of variation) for cartilage thickness and volume was made. The bias of the system was estimated through comparison with histology. This technique showed good precision, with errors in the repeated analysis ranging from 0.63% (lateral thickness) to 3.48% (medial volume). The repeated analysis alone was robust, with intraclass correlations for the different compartments between 0.918 and 0.991. Measurement bias was corrected by scaling the confocal images to 32% of their width to match histology. CLSM provided a fast and reproducible technique for gathering 3D image data of murine cartilage and will be a valuable tool in understanding the efficacy of arthritis treatments in murine models. Microsc. Res. Tech. 2009. © 2009 Wiley-Liss, Inc. [source]

DJ-927, a novel oral taxane, overcomes P-glycoprotein-mediated multidrug resistance in vitro and in vivo

CANCER SCIENCE, Issue 5 2003
Motoko Shionoya
DJ-927 is a novel taxane, which was selected for high solubility, non-neurotoxicity, oral bioavailability, and potent antitumor activity. In this study, we compared the in vitro and in vivo efficacy of DJ-927 with those of paclitaxel and docetaxel. DJ-927 exhibited stronger cytotoxicity than paclitaxel and docetaxel in various tumor cell lines, especially against P-glycoprotein (P-gp)-expressing cells. The cytotoxicity of DJ-927, unlike those of other taxanes, was not affected by the P-gp expression level in tumor cells, or by the co-presence of a P-gp modulator. When intracellular accumulation of the three compounds was compared, intracellular amounts of DJ-927 were much higher than those of paclitaxel or docetaxel, particularly in P-gp-positive cells. In vivo, DJ-927 showed potent antitumor effects against two human solid tumors in male BALB/c- nu/nu mice, and yielded significant life-prolongation in a murine liver metastasis model with male C57BL/6 mice, in which neither paclitaxel nor docetaxel was effective. The results demonstrate the superior efficacy of orally administered DJ-927 over intravenously administered paclitaxel or docetaxel against P-gp-expressing tumors, probably due to higher intracellular accumulation. A phase I clinical trials of DJ-927 is currently ongoing in the US. (Cancer Sci 2003; 94: 459,466) [source]

Enhanced Chondrogenesis and Wnt Signaling in PTH-Treated Fractures,

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 12 2007
Sanjeev Kakar
Abstract Studies have shown that systemic PTH treatment enhanced the rate of bone repair in rodent models. However, the mechanisms through which PTH affects bone repair have not been elucidated. In these studies we show that PTH primarily enhanced the earliest stages of endochondral bone repair by increasing chondrocyte recruitment and rate of differentiation. In coordination with these cellular events, we observed an increased level of canonical Wnt-signaling in PTH-treated bones at multiple time-points across the time-course of fracture repair, supporting the conclusion that PTH responses are at least in part mediated through Wnt signaling. Introduction: Since FDA approval of PTH [PTH(1,34); Forteo] as a treatment for osteoporosis, there has been interest in its use in other musculoskeletal conditions. Fracture repair is one area in which PTH may have a significant clinical impact. Multiple animal studies have shown that systemic PTH treatment of healing fractures increased both callus volume and return of mechanical competence in models of fracture healing. Whereas the potential for PTH has been established, the mechanism(s) by which PTH produces these effects remain elusive. Materials and Methods: Closed femoral fractures were generated in 8-wk-old male C57Bl/6 mice followed by daily systemic injections of either saline (control) or 30 ,g/kg PTH(1,34) for 14 days after fracture. Bones were harvested at days 2, 3, 5, 7, 10, 14, 21, and 28 after fracture and analyzed at the tissue level by radiography and histomorphometry and at the molecular and biochemical levels level by RNase protection assay (RPA), real-time PCR, and Western blot analysis. Results: Quantitative ,CT analysis showed that PTH treatment induced a larger callus cross-sectional area, length, and total volume compared with controls. Molecular analysis of the expression of extracellular matrix genes associated with chondrogenesis and osteogenesis showed that PTH treated fractures displayed a 3-fold greater increase in chondrogenesis relative to osteogenesis over the course of the repair process. In addition, chondrocyte hypertrophy occurred earlier in the PTH-treated callus tissues. Analysis of the expression of potential mediators of PTH actions showed that PTH treatment significantly induced the expression of Wnts 4, 5a, 5b, and 10b and increased levels of unphosphorylated, nuclear localized ,-catenin protein, a central feature of canonical Wnt signaling. Conclusions: These results showed that the PTH-mediated enhancement of fracture repair is primarily associated with an amplification of chondrocyte recruitment and maturation in the early fracture callus. Associated with these cellular effects, we observed an increase in canonical Wnt signaling supporting the conclusion that PTH effects on bone repair are mediated at least in part through the activation of Wnt-signaling pathways. [source]