Histological evidence of delayed ischemic brain tissue damage in the rat double-hemorrhage model

The rat double-SAH model is one of the standard models to simulate delayed cerebral vasospasm (CVS) in humans. However, the proof of delayed ischemic brain damage is missing so far. Our objective was, therefore, to determine histological changes in correlation with the development of symptomatic and perfusion weighted imaging (PWI) proven CVS in this animal model. CVS was induced by injection of autologous blood in the cisterna magna of 22 Sprague-Dawley rats. Histological changes were analyzed on day 3 and day 5. Cerebral blood flow (CBF) was assessed by PWI at 3 tesla magnetic resonance (MR) tomography. Neuronal cell count did not differ between sham operated and SAH rats in the hippocampus and the cerebral cortex on day 3. In contrast, on day 5 after SAH the neuronal cell count was significantly reduced in the hippocampus (p<0.001) and the inner cortical layer (p=0.03). The present investigation provides quantitative data on brain tissue damage in association with delayed CVS for the first time in a rat SAH model. Accordingly, our data suggest that the rat double-SAH model may be suitable to mimic delayed ischemic brain damage due to CVS and to investigate the neuroprotective effects of drugs.

Delayed gadolinium-enhanced magnetic resonance imaging of hip joint cartilage: pearls and pitfalls

With the increasing advances in hip joint preservation surgery, accurate diagnosis and assessment of femoral head and acetabular cartilage status is becoming increasingly important. Magnetic resonance imaging (MRI) of the hip does present technical difficulties. The fairly thin cartilage lining necessitates high image resolution and high contrast-to-noise ratio (CNR). With MR arthrography (MRA) using intraarticular injected gadolinium, labral tears and cartilage clefts may be better identified through the contrast medium filling into the clefts. However, the ability of MRA to detect varying grades of cartilage damage is fairly limited and early histological and biochemical changes in the beginning of osteoarthritis (OA) cannot be accurately delineated. Traditional MRI thus lacks the ability to analyze the biological status of cartilage degeneration. The technique of delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) is sensitive to the charge density of cartilage contributed by glycosaminoglycans (GAGs), which are lost early in the process of OA. Therefore, the dGEMRIC technique has a potential to detect early cartilage damage that is obviously critical for decision-making regarding time and extent of intervention for joint-preservation. In the last decade, cartilage imaging with dGEMRIC has been established as an accurate and reliable tool for assessment of cartilage status in the knee and hip joint.This review outlines the current status of dGEMRIC for assessment of hip joint cartilage. Practical modifications of the standard technique including three-dimensional (3D) dGEMRIC and dGEMRIC after intra-articular gadolinium instead of iv-dGEMRIC will also be addressed.

DSS induced colitis increases portal LPS levels and enhances hepatic inflammation and fibrogenesis in experimental NASH

Intestinal bacterial overgrowth and increased permeability are features of non alcoholic steatohepatitis (NASH). Bacterial endotoxin has been shown to promote NASH progression. Application of dextran sulfate sodium (DSS) is a colitis model in mice characterized by damage of the intestinal barrier. This study was designed to investigate if application of DSS aggravates experimental NASH.

The proapoptotic Bcl-2 family member Bim plays a central role during the development of virus-induced hepatitis

The proapoptotic Bcl-2 homolog Bim was shown to control the apoptosis of both T cells and hepatocytes. This dual role of Bim might be particularly relevant for the development of viral hepatitis, in which both the sensitivity of hepatocytes to apoptosis stimuli and the persistence of cytotoxic T cells are essential factors for the outcome of the disease. The relevance of Bim in regulating survival of cytotoxic T cells or induction of hepatocyte death has only been investigated in separate systems, and their relative contributions to the pathogenesis of T cell-mediated hepatitis remain unclear. Using the highly dynamic model system of lymphocytic choriomeningitis virus-mediated hepatitis and bone marrow chimeras, we found that Bim has a dual role in the development of lymphocytic choriomeningitis virus-induced, T cell-mediated hepatitis. Although the absence of Bim in parenchymal cells led to markedly attenuated liver damage, loss of Bim in the lymphoid compartment moderately enhanced hepatitis. However, when both effects were combined in Bim(-/-) mice, the effect of Bim deficiency in the lymphoid compartment was overcompensated for by the reduced sensitivity of Bim(-/-) hepatocytes to T cell-induced apoptosis, resulting in the protection of Bim(-/-) mice from hepatitis.

Local glucocorticoid production in the mouse lung is induced by immune cell stimulation

Glucocorticoids (GC) are potent anti-inflammatory and immunosuppressive steroid hormones, mainly produced by the adrenal glands. However, increasing evidence supports the idea of additional extra-adrenal sources of bioactive GC. The lung epithelium is constantly exposed to a plethora of antigenic stimuli, and local GC synthesis could contribute to limit uncontrolled immune reactions and tissue damage.

ENU-induced phenovariance in mice: inferences from 587 mutations

Background We present a compendium of N-ethyl-N-nitrosourea (ENU)-induced mouse mutations, identified in our laboratory over a period of 10 years either on the basis of phenotype or whole genome and/or whole exome sequencing, and archived in the Mutagenetix database. Our purpose is threefold: 1) to formally describe many point mutations, including those that were not previously disclosed in peer-reviewed publications; 2) to assess the characteristics of these mutations; and 3) to estimate the likelihood that a missense mutation induced by ENU will create a detectable phenotype. Findings In the context of an ENU mutagenesis program for C57BL/6J mice, a total of 185 phenotypes were tracked to mutations in 129 genes. In addition, 402 incidental mutations were identified and predicted to affect 390 genes. As previously reported, ENU shows strand asymmetry in its induction of mutations, particularly favoring T to A rather than A to T in the sense strand of coding regions and splice junctions. Some amino acid substitutions are far more likely to be damaging than others, and some are far more likely to be observed. Indeed, from among a total of 494 non-synonymous coding mutations, ENU was observed to create only 114 of the 182 possible amino acid substitutions that single base changes can achieve. Based on differences in overt null allele frequencies observed in phenotypic vs. non-phenotypic mutation sets, we infer that ENU-induced missense mutations create detectable phenotype only about 1 in 4.7 times. While the remaining mutations may not be functionally neutral, they are, on average, beneath the limits of detection of the phenotypic assays we applied. Conclusions Collectively, these mutations add to our understanding of the chemical specificity of ENU, the types of amino acid substitutions it creates, and its efficiency in causing phenovariance. Our data support the validity of computational algorithms for the prediction of damage caused by amino acid substitutions, and may lead to refined predictions as to whether specific amino acid changes are responsible for observed phenotypes. These data form the basis for closer in silico estimations of the number of genes mutated to a state of phenovariance by ENU within a population of G3 mice.

Analgesic and anti-inflammatory actions of robenacoxib in acute joint inflammation in dog

The objectives of this study were to establish dose-response and blood concentration-response relationships for robenacoxib, a novel nonsteroidal anti-inflammatory drug with selectivity for inhibition of the cyclooxygenase (COX)-2 isoenzyme, in a canine model of synovitis. Acute synovitis of the stifle joint was induced by intra-articular injection of sodium urate crystals. Robenacoxib (0.25, 0.5, 1.0, 2.0 and 4.0 mg/kg), placebo and meloxicam (0.2 mg/kg) were administered subcutaneously (s.c.) 3 h after the urate crystals. Pharmacodynamic endpoints included data from forceplate analyses, clinical orthopaedic examinations and time course of inhibition of COX-1 and COX-2 in ex vivo whole blood assays. Blood was collected for pharmacokinetics. Robenacoxib produced dose-related improvement in weight-bearing, pain and swelling as assessed objectively by forceplate analysis (estimated ED(50) was 1.23 mg/kg for z peak force) and subjectively by clinical orthopaedic assessments. The analgesic and anti-inflammatory effects of robenacoxib were significantly superior to placebo (0.25-4 mg/kg robenacoxib) and were non-inferior to meloxicam (0.5-4 mg/kg robenacoxib). All dosages of robenacoxib produced significant dose-related inhibition of COX-2 (estimated ED(50) was 0.52 mg/kg) but no inhibition of COX-1. At a dosage of 1-2 mg/kg administered s.c., robenacoxib should be at least as effective as 0.2 mg/kg of meloxicam in suppressing acute joint pain and inflammation in dogs.

Inhibition of damage-regulated autophagy modulator-1 (DRAM-1) impairs neutrophil differentiation of NB4 APL cells

The damage-regulator autophagy modulator 1 (DRAM-1) is a lysosomal protein that positively regulates autophagy in a p53-dependent manner. We aimed at analyzing the role of DRAM-1 in granulocytic differentiation of APL cells. We observed a significant increase of DRAM-1 expression during all-trans retinoic acid (ATRA)-induced neutrophil differentiation of NB4 APL cells but not in ATRA-resistant NB4-R2 cells. Next, knocking down DRAM-1 in NB4 APL cells was sufficient to impair neutrophil differentiation. Given that DRAM-1 is a transcriptional target of p53, we tested if DRAM-1 is regulated by the p53 relative p73. Indeed, inhibiting p73 prevented neutrophil differentiation and DRAM-1 induction of NB4 cells. In conclusion, we show for the first time that p73-regulated DRAM-1 is functionally involved in neutrophil differentiation of APL cells.

Toll-like receptors in ischaemia and its potential role in the pathophysiology of muscle damage in critical limb ischaemia

Toll-like receptors (TLRs) are key receptors of the innate immune system which are expressed on immune and nonimmune cells. They are activated by both pathogen-associated molecular patterns and endogenous ligands. Activation of TLRs culminates in the release of proinflammatory cytokines, chemokines, and apoptosis. Ischaemia and ischaemia/reperfusion (I/R) injury are associated with significant inflammation and tissue damage. There is emerging evidence to suggest that TLRs are involved in mediating ischaemia-induced damage in several organs. Critical limb ischaemia (CLI) is the most severe form of peripheral arterial disease (PAD) and is associated with skeletal muscle damage and tissue loss; however its pathophysiology is poorly understood. This paper will underline the evidence implicating TLRs in the pathophysiology of cerebral, renal, hepatic, myocardial, and skeletal muscle ischaemia and I/R injury and discuss preliminary data that alludes to the potential role of TLRs in the pathophysiology of skeletal muscle damage in CLI.

β-Caryophyllene ameliorates cisplatin-induced nephrotoxicity in a cannabinoid 2 receptor-dependent manner

(E)-β-caryophyllene (BCP) is a natural sesquiterpene found in many essential oils of spice (best known for contributing to the spiciness of black pepper) and food plants with recognized anti-inflammatory properties. Recently it was shown that BCP is a natural agonist of endogenous cannabinoid 2 (CB(2)) receptors, which are expressed in immune cells and mediate anti-inflammatory effects. In this study we aimed to test the effects of BCP in a clinically relevant murine model of nephropathy (induced by the widely used antineoplastic drug cisplatin) in which the tubular injury is largely dependent on inflammation and oxidative/nitrative stress. β-caryophyllene dose-dependently ameliorated cisplatin-induced kidney dysfunction, morphological damage, and renal inflammatory response (chemokines MCP-1 and MIP-2, cytokines TNF-α and IL-1β, adhesion molecule ICAM-1, and neutrophil and macrophage infiltration). It also markedly mitigated oxidative/nitrative stress (NOX-2 and NOX-4 expression, 4-HNE and 3-NT content) and cell death. The protective effects of BCP against biochemical and histological markers of nephropathy were absent in CB(2) knockout mice. Thus, BCP may be an excellent therapeutic agent to prevent cisplatin-induced nephrotoxicity through a CB(2) receptor-dependent pathway. Given the excellent safety profile of BCP in humans it has tremendous therapeutic potential in a multitude of diseases associated with inflammation and oxidative stress.

Mechanisms of nanoparticle-mediated photomechanical cell damage

Laser-assisted killing of gold nanoparticle targeted macrophages was investigated. Using pressure transient detection, flash photography and transmission electron microscopy (TEM) imaging, we studied the mechanism of single cell damage by vapor bubble formation around gold nanospheres induced by nanosecond laser pulses. The influence of the number of irradiating laser pulses and of particle size and concentration on the threshold for acute cell damage was determined. While the single pulse damage threshold is independent of the particle size, the threshold decreases with increasing particle size when using trains of pulses. The dependence of the cell damage threshold on the nanoparticle concentration during incubation reveals that particle accumulation and distribution inside the cell plays a key role in tissue imaging or cell damaging.

Involvement of drug-specific T cells in acute drug-induced interstitial nephritis

Drug-induced interstitial nephritis can be caused by a plethora of drugs and is characterized by a sudden impairment of renal function, mild proteinuria, and sterile pyuria. For investigation of the possible pathomechanism of this disease, drug-specific T cells were analyzed, their function was characterized, and these in vitro findings were correlated to histopathologic changes that were observed in kidney biopsy specimens. Peripheral blood mononuclear cells from three patients showed a proliferative response to only one of the administered drugs, namely flucloxacillin, penicillin G, and disulfiram, respectively. The in vitro analysis of the flucloxacillin-reactive cells showed an oligoclonal immune response with an outgrowth of T cells bearing the T cell receptor Vbeta9 and Vbeta21.3. Moreover, flucloxacillin-specific T cell clones could be generated from peripheral blood, they expressed CD4 and the alphabeta-T cell receptor, and showed a heterogeneous cytokine secretion pattern with no clear commitment to either a Th1- or Th2-type response. The immunohistochemistry of kidney biopsies of these patients revealed cell infiltrations that consisted mostly of T cells (CD4+ and/or CD8+). An augmented presence of IL-5, eosinophils, neutrophils, CD68+ cells, and IL-12 was observed. In agreement with negative cytotoxicity assays, no cytotoxicity-related molecules such as Fas and perforin were detected by immunohistochemistry. The data indicate that drug-specific T cells are activated locally and orchestrate a local inflammation via secretion of various cytokines, the type of which depends on the cytokine pattern secreted and which probably is responsible for the renal damage.

Intra-articular calcium phosphate cement: Its fate and impact on joint tissues in a rabbit model

Clinical application of injectable ceramic cement in comminuted fractures revealed penetration of the viscous paste into the joint space. Not much is known on the fate of this cement and its influence on articular tissues. The purpose of this experimental study was to assess these unknown alterations of joint tissues after intra-articular injection of cement in a rabbit knee. Observation periods were from 1 week up to 24 months, with three rabbits per group. Norian SRS cement was injected into one knee joint, the contralateral side receiving the same volume of Ringers' solution. Light microscopic evaluation of histologic sections was performed, investigating the appearance of the cement, inflammatory reactions, and degenerative changes of the articular surface. No signs of pronounced acute or chronic inflammation were visible. The injected cement was mainly found as a single particle, anterior to the cruciate ligaments. It became surrounded by synovial tissues within 4 weeks and showed signs of superficial resorption. In some specimens, bone formation was seen around the cement. Degeneration of the articular surface showed no differences between experimental and control side, and no changes over time became apparent. No major degenerative changes were induced by the injected cement. The prolonged presence of cement still seems to make it advisable to remove radiologically visible amounts from the joint space.

Statin therapy induces ultrastructural damage in skeletal muscle in patients without myalgia

Muscle pain and weakness are frequent complaints in patients receiving 3-hydroxymethylglutaryl coenzymeA (HMG CoA) reductase inhibitors (statins). Many patients with myalgia have creatine kinase levels that are either normal or only marginally elevated, and no obvious structural defects have been reported in patients with myalgia only. To investigate further the mechanism that mediates statin-induced skeletal muscle damage, skeletal muscle biopsies from statin-treated and non-statin-treated patients were examined using both electron microscopy and biochemical approaches. The present paper reports clear evidence of skeletal muscle damage in statin-treated patients, despite their being asymptomatic. Though the degree of overall damage is slight, it has a characteristic pattern that includes breakdown of the T-tubular system and subsarcolemmal rupture. These characteristic structural abnormalities observed in the statin-treated patients were reproduced by extraction of cholesterol from skeletal muscle fibres in vitro. These findings support the hypothesis that statin-induced cholesterol lowering per se contributes to myocyte damage and suggest further that it is the specific lipid/protein organization of the skeletal muscle cell itself that renders it particularly vulnerable.

Neuregulin-1 beta attenuates doxorubicin-induced alterations of excitation-contraction coupling and reduces oxidative stress in adult rat cardiomyocytes

Treatment of metastatic breast cancer with doxorubicin (Doxo) in combination with trastuzumab, an antibody targeting the ErbB2 receptor, results in an increased incidence of heart failure. Doxo therapy induces reactive oxygen species (ROS) and alterations of calcium homeostasis. Therefore, we hypothesized that neuregulin-1 beta (NRG), a ligand of the cardiac ErbB receptors, reduces Doxo-induced alterations of EC coupling by triggering antioxidant mechanisms. Adult rat ventricular cardiomyocytes (ARVM) were isolated and treated for 18-48 h. SERCA protein was analyzed by Western blot, EC coupling parameters by fura-2 and video edge detection, gene expression by RT-PCR, and ROS by DCF-fluorescence microscopy. At clinically relevant doses Doxo reduced cardiomyocytes contractility, SERCA protein and SR calcium content. NRG, similarly as the antioxidant N-acetylcystein (NAC), did not affect EC coupling alone, but protected against Doxo-induced damage. NRG and Doxo showed an opposite modulation of glutathione reductase gene expression. NRG, similarly as NAC, reduced peroxide- or Doxo-induced oxidative stress. Specific inhibitors showed, that the antioxidant action of NRG depended on signaling via the ErbB2 receptor and on the Akt- and not on the MAPK-pathway. Therefore, NRG attenuates Doxo-induced alterations of EC coupling and reduces oxidative stress in ARVM. Inhibition of the ErbB2/NRG signaling pathway by trastuzumab in patients concomitantly treated with Doxo might prevent beneficial effects of NRG in the myocardium.