In experimental animal research body temperature (BT) is measured for the objective determination of an animals' physiological condition. Invasive, probe-based measurements are stressful and can influence experimental outcome. Alternatively BT can be determined touch-free from the emitted heat of the organism at a single spot using infrared thermometers [1]. To get visual confirmation and find more appropriate surfaces for measurement a hand-held thermal imager was equipped with a self-made, cheap, 3D-printable close-up lens system that reproducibly creates eight-time magnified thermal images and improves sensitivity. This setup was used to establish ocular surface temperature (OST), representing the temperature of the brain-heart axis, as a touch-free alternative for measurement of BT in mice, rats, rabbits and humans. OST measurement after isoflurane exposure and myocardial infarction (MI) experiments in mice revealed high physiological relevance and sensitivity, the possibility to discriminate between MI and sham operations in one hour and even long-term outcome-predictive capabilities of OST after MI. Summarized here we present:Self-made close-up lens for thermal imaging cameras for eight-time magnificationEstablishment of OST for touch-free determination of BT in rodents and humansShort-and long-term predictive capabilities of OST in experimental MI in mice. (C) 2016 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Myocardial infarction (MI) leads to necrosis and uncontrolled release of cellular content. Binucleated and polyploid cardiomyocytes contain high amounts of chromatin, a DNA polymer of histones which are cytotoxic. We hypothesized that chromatin from necrotic cells accumulates in the non-perfused, ischemic infarct region, causing local high concentrations of cytotoxic histones, thereby potentiating damage to the heart after MI. The endonuclease DNase1 is capable of dispersing extracellular chromatin through linker DNA digestion which could lead to a decrease in local histone concentrations and cytotoxicity. It was confirmed that after permanent coronary artery ligation in mice, extracellular histones accumulated within the infarcted myocardium. In vitro, histones caused myocyte cytotoxicity. For protection against histone-mediated cytotoxicity after MI in vivo, DNase1 was administered within the first 6 h after induction. Indeed, DNase1 accumulation in the infarcted region of the heart was observed, as well as effective disruption of extracellular cytotoxic chromatin and subsequent reduction of high local histone concentrations. Functionally, acute DNase1 treatment resulted in significantly improved left ventricular remodeling in mice as measured by serial echocardiography, while mortality, infarct size and inflammatory parameters were unaffected. Notably, improved cardiomyocyte survival within the infarct region was observed and might account for the protective effects in acutely DNase1-treated animals. Disruption of extracellular cytotoxic chromatin within the infarcted heart by acute DNase1 treatment is a promising approach to protect myocytes from histone-induced cell death and subsequent left ventricular dysfunction after MI.

Picrosirius red (PSR) staining is a commonly used histological technique to visualize collagen in paraffin-embedded tissue sections. PSR stained collagen appears red in light microscopy. However it is largely unknown that PSR stained collagen also shows a red fluorescence, whereas live cells have a distinct green autofluorescence. Both emission patterns can be detected using standard filter sets as found in conventional fluorescence microscopes. Here we used digital image addition and subtraction to determine the relative area of the pure collagen and live cell content in heart tissue in a semi-automated process using standard software. This procedure, which considers empty spaces (holes) within the section, can be easily adapted to quantify the collagen and live cell areas in healthy or fibrotic tissues as aorta, lung, kidney or liver by semi-automated planimetry exemplified herein for infarcted heart tissue obtained from the mouse myocardial infarction model. •Use of conventional PSR stained paraffin-embedded tissue sections for fluorescence analysis.•PSR and autofluorescence images are used to calculate area of collagen and area of live cells in the tissue; empty spaces (holes) in tissue are considered.•High throughput analysis of collagen and live cell content in tissue for statistical purposes.

BACKGROUND:Activation of innate immunity, especially infiltration of monocytes, is critical for proper wound healing and scar formation after myocardial infarction (MI). Therefore, we tested the hypothesis that interleukin-13 (IL-13), which influences the differentiation of monocytes/macrophages and has profibrotic properties, modulates wound healing and remodeling after MI. METHODS AND RESULTS:MI was induced by permanent ligation of the left coronary artery in both male and female wild-type (WT)/IL-13(-/-) mice. Real-time polymerase chain reaction demonstrated that expression of IL-13 was induced in left and right ventricular myocardium of WT mice within days in response to MI. Fifty-six-day survival was significantly impaired (65% in WT versus 34% in IL-13(-/-)) in male but not female IL-13(-/-) (55% in WT versus 54% in IL-13(-/-)) mice. Serial echocardiography showed significantly increased left ventricular dilation in male IL-13(-/-) compared with WT mice starting from day 1 after MI, despite comparable infarct size. Fluorescence-activated cell sorter analysis revealed less leukocyte infiltration in male IL-13(-/-) mice on day 3. Real-time polymerase chain reaction analysis demonstrated reduced expression of marker genes of alternative activation in monocytes sorted from the infarct zone of male IL-13(-/-) in comparison with WT mice on day 3 after MI. CONCLUSIONS:Genetic deficiency of IL-13 worsens outcome after MI in male mice. Our data indicate that IL-13 regulates leukocyte recruitment and induces M2-like monocyte/macrophage differentiation, which modifies wound healing within the infarct zone.

The long known toxicity of free chromatin mediated by histones regained attention after discovery of neutrophil extracellular traps (NETs). Free histones from necrotic cells or NETs can damage prokaryotic and eukaryotic cells and are responsible for the aggravation of a growing list of diseases. DNases degrade the toxic chromatin polymer to nucleosomes and efficiently reduce local high histone concentrations. Therefore, DNase activity as a biomarker is of growing interest in basic and clinical research. Here a detailed one-step protocol is presented that allows rapid and sensitive detection of DNases down to 400 fg/μl per reaction based on the detection of fluorescent ethidium bromide/DNA complexes in a 96-well plate reader. The flexible protocol uses an internal standard for background correction and allows convenient and reliable data analysis using common laboratory equipment and chemicals without elaborate preparations. The DNase activity of a sample is clearly defined by substrate amount, incubation time, and (if appropriate) a DNase standard for absolute quantification in Kunitz units per milligram sample protein. Quantitative kinetic determination is possible within less than 1 h down to 5 pg DNases/μl per reaction.

Rationale: An exaggerated or persistent inflammatory activation after myocardial infarction (MI) leads to maladaptive healing and subsequent remodeling of the left ventricle. Foxp3(+) CD4(+) regulatory T cells (T-reg cells) contribute to inflammation resolution. Therefore, T-reg cells might influence cardiac healing post-MI.Objective: Our aim was to study the functional role of T-reg cells in wound healing post-MI in a mouse model of permanent left coronary artery ligation.Methods and Results: Using a model of genetic T-reg-cell ablation (Foxp3(DTR) mice), we depleted the T-reg-cell compartment before MI induction, resulting in aggravated cardiac inflammation and deteriorated clinical outcome. Mechanistically, T-reg-cell depletion was associated with M1-like macrophage polarization, characterized by decreased expression of inflammation-resolving and healing-promoting factors. The phenotype of exacerbated cardiac inflammation and outcome in T-reg-cell-ablated mice could be confirmed in a mouse model of anti-CD25 monoclonal antibody-mediated depletion. In contrast, therapeutic T-reg-cell activation by superagonistic anti-CD28 monoclonal antibody administration 2 days after MI led to improved healing and survival. Compared with control animals, CD28-SA-treated mice showed increased collagen de novo expression within the scar, correlating with decreased rates of left ventricular ruptures. Therapeutic T-reg-cell activation induced an M2-like macrophage differentiation within the healing myocardium, associated with myofibroblast activation and increased expression of monocyte/macrophage-derived proteins fostering wound healing.Conclusions: Our data indicate that T-reg cells beneficially influence wound healing after MI by modulating monocyte/macrophage differentiation. Moreover, therapeutic activation of T-reg cells constitutes a novel approach to improve healing post-MI.

Rationale: Myocardial infarction (MI) leads to necrosis of multinucleated and polyploid myocytes. This causes uncontrolled release of cellular content like chromatin to the infarct area. Chromatin is mainly comprised of histones which are essential for controlling and packing of DNA but paradoxically are also known to be cytotoxic. This makes free chromatin a toxic DNA polymer creating local high concentrations of hazardous histones. Objective: We hypothesized that chromatin from necrotic cells accumulates in ischemic myocardium, creates local high concentrations of cytotoxic histones, and thereby potentiates ischemic damage to the heart after MI. The endonuclease DNase1 is capable of dispersing extracellular chromatin through linker DNA digestion and could decrease local histone concentrations and cytotoxicity. Methods and Results: After permanent coronary artery ligation in mice we found extracellular histones accumulated within the infarcted myocardium. Histone cytotoxicity towards isolated myocytes was confirmed in vitro. To reduce histone related cytotoxicity in vivo DNase1 was injected within the first 6 hours after induction of MI. DNase1 accumulated in the infarcted region of the heart, effectively disrupted extracellular cytotoxic chromatin and thereby reduced high local histone concentration. Animals acutely treated with DNase1 revealed significantly improved left ventricular remodeling as measured by serial echocardiography up to 28 days after MI (e.g. NaCl vs DNase1, papillary end diastolic area [mm 2 ]: 23.26 ± 2.06 vs 18.90 ± 1.24, n=9 vs 10, p<0,05). Treatment did not influence mortality, infarct size or inflammatory parameters as determined by neutrophil infiltration and RTQ-PCR analysis of characteristic cytokines. However improved myocyte survival was discovered within the infarct region which might account for the protective effects in DNase1 treated animals (NaCl vs DNase1: 3.0 ± 0.7% vs 8.3 ± 2.3%; p<0.05; n=7 vs 8). Conclusions: Targeting extracellular cytotoxic chromatin within the infarcted heart by DNase1 is a promising approach to preserve myocytes from histone induced cell death and to conserve left ventricular function after MI. The efficacy of other chromatin degrading agents is now under investigation.

Background: The proinflammatory activation of innate immunity by myocardial ischemic injury has been recognized for long time. Our recent data have indicated that activation of CD4+ T cells, presumably by auto-antigen recognition, is a prerequisite for formation of a stable scar and prevention from left ventricular dilation after experimental myocardial infarction in mice. We here hypothesized that regulatory CD4+CD25+Foxp3+CD4+ T cells might improve left ventricular wound healing and prevent from adverse remodeling after myocardial infarction. Results: Experimental myocardial infarction in mice induced the proliferation and activation of CD4+CD25+Foxp3+ regulatory T cells, as demonstrated by intracellular expression of the Ikaros family transcription factor Helios, in heart draining lymph nodes. Pretreatment of mice with an anti-CD25 antibody before myocardial infarction efficiently depleted CD4+CD25+Foxp3+ regulatory T cells and increased mortality after myocardial infarction as compared to mice treated with an isotype-matched control antibody of irrelevant specificity, i.e., 25% survival in anti-CD25 treated mice vs. 55,9% survival in control antibody treated animals. Therapeutic activation of regulatory CD4+CD25+Foxp3+ T cells by a superagonistic anti-CD28 antibody (CD28-SA) applied at day 2 after myocardial infarction prevented, compared to mice treated with an isotype-matched control antibody, from left ventricular rupture and resulted in improved survival (47.1% survival in the control group vs. 76.6% survival in the CD28-SA treated group). CD28-SA treatment lead to expansion of CD4+CD25+Foxp3+ T-cells in the peripheral blood and increased their frequency in the infarcted myocardium. This was associated with increased expression of several molecules known to facilitate wound healing by promoting the formation of a stable scar such as osteopontin and coagulation factor XIII. Conclusion: CD4+CD25+Foxp3+ regulatory T-cells are a prerequisite for proper myocardial wound healing and can be therapeutically activated to improve outcome after experimental myocardial infarction.

Background: Leukocytes, especially neutrophils, contribute to myocardial ischemia-reperfusion injury. We could recently demonstrate that myocardial wound healing is facilitated by CD4+ T-cells. We therefore studied the role of T-cells during ischemia-reperfusion of the myocardium. Methods and Results: Leukocyte infiltration was determined by FACS analysis and immunohistology after experimental ischemia-reperfusion in mice. Significantly more CD3+ T-cells infiltrated the mouse myocardium after 30 minutes of ischemia and 24 hours of reperfusion in comparison to sham operated mice. CD4+ T-cells (47%) and CD8+ T-cells (30%) were the predominant T-cell subsets among all CD45+CD3+ leukocytes recruited to the myocardium during reperfusion. NK T-cells and γδ T-cells could also be detected in reperfused myocardium. Evans Blue/ TTC-staining demonstrated that CD4 KO (which lack CD4+ T-cells), OTII T-cell receptor transgenic mice (which have a high specificity for ovalbumin derived peptides) but not CD8 KO (which lac...

Binding of proteins to DNA is usually considered 1D with one protein bound to one DNA molecule. In principle, proteins with multiple DNA binding domains could also bind to and thereby cross-link different DNA molecules. We have investigated this possibility using high-mobility group A1 (HMGA1) proteins, which are architectural elements of chromatin and are involved in the regulation of multiple DNA-dependent processes. Using direct stochastic optical reconstruction microscopy ( d STORM), we could show that overexpression of HMGA1a-eGFP in Cos-7 cells leads to chromatin aggregation. To investigate if HMGA1a is directly responsible for this chromatin compaction we developed a DNA cross-linking assay. We were able to show for the first time that HMGA1a can cross-link DNA directly. Detailed analysis using point mutated proteins revealed a novel DNA cross-linking domain. Electron microscopy indicates that HMGA1 proteins are able to create DNA loops and supercoils in linearized DNA confirming the cross-linking ability of HMGA1a. This capacity has profound implications for the spatial organization of DNA in the cell nucleus and suggests cross-linking activities for additional nuclear proteins.

Early healing after myocardial infarction (MI) is characterized by a strong inflammatory reaction. Most leukotrienes are pro-inflammatory and are therefore potential mediators of healing and remodeling after myocardial ischemia. The enzyme 5-lipoxygenase (5-LOX) has a key role in the transformation of arachidonic acid in leukotrienes. Thus, we tested the effect of 5-LOX on healing after MI. After chronic coronary artery ligation, early mortality was significantly increased in 5-LOX−/− when compared to matching wildtype (WT) mice due to left ventricular rupture. This effect could be reproduced in mice treated with the 5-LOX inhibitor Zileuton. A perfusion mismatch due to the vasoactive potential of leukotrienes is not responsible for left ventricular rupture since local blood flow assessed by magnetic resonance perfusion measurements was not different. However, after MI, there was an accentuation of the inflammatory reaction with an increase of pro-inflammatory macrophages. Yet, mortality was not changed in chimeric mice (WT vs. 5-LOX−/− bone marrow in 5-LOX−/− animals), indicating that an altered function of 5-LOX−/− inflammatory cells is not responsible for the phenotype. Collagen production and accumulation of fibroblasts were significantly reduced in 5-LOX−/− mice in vivo after MI. This might be due to an impaired migration of 5-LOX−/− fibroblasts, as shown in vitro to serum. In conclusion, a lack or inhibition of 5-LOX increases mortality after MI because of healing defects. This is not mediated by a change in local blood flow, but through an altered inflammation and/or fibroblast function.

Background High mobility group A (HMGA) proteins regulate gene transcription through architectural modulation of chromatin and the formation of multi-protein complexes on promoter/enhancer regions. Differential expression of HMGA variants has been found to be important for distinct differentiation processes and deregulated expression was linked to several disorders. Here we used mouse C2C12 myoblasts and C2C12 cells stably over-expressing HMGA1a-eGFP to study the impact of deregulated HMGA1 expression levels on cellular differentiation. Results We found that induction of the myogenic or osteogenic program of C2C12 cells caused an immediate down-regulation of HMGA1. In contrast to wild type C2C12 cells, an engineered cell line with stable over-expression of HMGA1a-eGFP failed to differentiate into myotubes. Immunolocalization studies demonstrated that sustained HMGA1a-eGFP expression prevented myotube formation and chromatin reorganization that normally accompanies differentiation. Western Blot analyses showed that elevated HMGA1a-eGFP levels affected chromatin composition through either down-regulation of histone H1 or premature expression of MeCP2. RT-PCR analyses further revealed that sustained HMGA1a expression also affected myogenic gene expression and caused either down-regulation of genes such as MyoD, myogenin, Igf1 , Igf2 , Igfbp1-3 or up-regulation of the transcriptional repressor Msx1 . Interestingly, siRNA experiments demonstrated that knock-down of HMGA1a was required and sufficient to reactivate the myogenic program in induced HMGA1a over-expressing cells. Conclusions Our data demonstrate that HMGA1 down-regulation after induction is required to initiate the myogenic program in C2C12 cells. Sustained HMGA1a expression after induction prevents expression of key myogenic factors. This may be due to specific gene regulation and/or global effects on chromatin. Our data further corroborate that altered HMGA1 levels influence the expression of other chromatin proteins. Thus, HMGA1 is able to establish a specific chromatin composition. This work contributes to the understanding of how differential HMGA1 expression is involved in chromatin organization during cellular differentiation processes and it may help to comprehend effects of HMGA1 over-expression occurring in malign or benign tumours.