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Photobiomodulation (PBM) by far-red (FR) to near-infrared (NIR) light has been demonstrated to restore the function of damaged mitochondria, increase the production of cytoprotective factors and prevent cell death. Our laboratory has shown that FR PBM improves functional and structural outcomes in animal models of retinal injury and retinal degenerative disease. The current study tested the hypothesis that a brief course of NIR (830 nm) PBM would preserve mitochondrial metabolic state and attenuate photoreceptor loss in a model of retinitis pigmentosa, the P23H transgenic rat. P23H rat pups were treated with 830 nm light (180 s; 25 mW/cm 2 ; 4.5 J/cm 2 ) using a light-emitting diode array (Quantum Devices, Barneveld, WI) from postnatal day (p) 10 to p25. Sham-treated rats were restrained, but not treated with 830 nm light. Retinal metabolic state, function and morphology were assessed at p30 by measurement of mitochondrial redox (NADH/FAD) state by 3D optical cryo-imaging, electroretinography (ERG), spectral-domain optical coherence tomography (SD-OCT), and histomorphometry. PBM preserved retinal metabolic state, retinal function, and retinal morphology in PBM-treated animals compared to the sham-treated group. PBM protected against the disruption of the oxidation state of the mitochondrial respiratory chain observed in sham-treated animals. Scotopic ERG responses over a range of flash intensities were significantly greater in PBM-treated rats compared to sham controls. SD-OCT studies and histological assessment showed that PBM preserved the structural integrity of the retina. These findings demonstrate for the first time a direct effect of NIR PBM on retinal mitochondrial redox status in a well-established model of retinal disease. They show that chronic proteotoxic stress disrupts retinal bioenergetics resulting in mitochondrial dysfunction, and retinal degeneration and that therapies normalizing mitochondrial metabolism have considerable potential for the treatment of retinal degenerative disease.

Tissues with high-energy demand including the heart are rich in the energy-producing organelles, mitochondria, and sensitive to mitochondrial dysfunction. While alterations in mitochondrial function are increasingly recognized in cardiovascular diseases, the molecular mechanisms through which changes in mitochondria lead to heart abnormalities have not been fully elucidated. Here, we report that transgenic mice overexpressing a novel regulator of mitochondrial dynamics, transmembrane protein 135 (Tmem135), exhibit increased fragmentation of mitochondria and disease phenotypes in the heart including collagen accumulation and hypertrophy. The gene expression analysis showed that genes associated with ER stress and unfolded protein response, and especially the pathway involving activating transcription factor 4, are upregulated in the heart of Tmem135 transgenic mice. It also showed that gene expression changes in the heart of Tmem135 transgenic mice significantly overlap with those of aged mice in addition to the similarity in cardiac phenotypes, suggesting that changes in mitochondrial dynamics may be involved in the development of heart abnormalities associated with aging. Our study revealed the pathological consequence of overexpression of Tmem135, and suggested downstream molecular changes that may underlie those disease pathologies.

Branching morphogenesis is a key developmental process during organogenesis, such that its disruption frequently leads to long-term consequences. The kidney and eye share many etiologies, perhaps, due to similar use of developmental branching morphogenesis and signaling pathways including cell death. Tipping the apoptotic balance towards apoptosis imparts a ureteric bud and retinal vascular branching phenotype similar to one that occurs in papillorenal syndrome. Here, to compare ureteric bud and retinal vascular branching in the context of decreased apoptosis, we investigated the impact of Bim, Bcl-2’s rival force. In the metanephros, lack of Bim expression enhanced ureteric bud branching with increases in ureteric bud length, branch points, and branch end points. Unfortunately, enhanced ureteric bud branching also came with increased branching defects and other undesirable consequences. Although we did see increased nephron number and renal mass, we observed glomeruli collapse. Retinal vascular branching in the absence of Bim expression had similarities with the ureteric bud including increased vascular length, branching length, segment length, and branching interval. Thus, our studies emphasize the impact appropriate Bim expression has on the overall length and branching in both the ureteric bud and retinal vasculature.

Introduction: Obesity is a major risk factor associated with multiple pathological conditions including diabetes and cardiovascular disease. Endothelial dysfunction is an early predictor of obesity. However, little is known regarding how early endothelial changes trigger obesity. In the present work we report a novel endothelial-mediated mechanism essential for regulation of metabolic homeostasis, driven by c-Myc. Methods: We used conditional knockout (EC-Myc KO) and overexpression (EC-Myc OE) mouse models to investigate the endothelial-specific role of c-Myc in metabolic homeostasis during aging and high-fat diet exposure. Body weight and metabolic parameters were collected over time and tissue samples collected at endpoint for biochemical, pathology and RNA-sequencing analysis. Animals exposed to high-fat diet were also evaluated for cardiac dysfunction. Results: In the present study we demonstrate that EC-Myc KO triggers endothelial dysfunction, which precedes progressive increase in body weight during aging, under normal dietary conditions. At endpoint, EC-Myc KO animals showed significant increase in white adipose tissue mass relative to control littermates, which was associated with sex-specific changes in whole body metabolism and increase in systemic leptin. Overexpression of endothelial c-Myc attenuated diet-induced obesity and visceral fat accumulation and prevented the development of glucose intolerance and cardiac dysfunction. Transcriptome analysis of skeletal muscle suggests that the protective effects promoted by endothelial c-Myc overexpression are associated with the expression of genes known to increase weight loss, energy expenditure and glucose tolerance. Conclusion: Our results show a novel important role for endothelial c-Myc in regulating metabolic homeostasis and suggests its potential targeting in preventing obesity and associated complications such as diabetes type-2 and cardiovascular dysfunction.

The oxygenation level of a tissue is an important marker of the health of the tissue and has a direct effect on performance. It has been shown that the blood flow to the paretic muscles of hemiparetic post-stroke patients is significantly reduced compared to non-paretic muscles. It is hypothesized that hemodynamic activity in paretic muscles is suppressed as compared to non-paretic muscles, and that oximetry can be used to measure this disparity in real-time. In order to test this hypothesis, a custom-made oximetry device was used to measure hemodynamic activity in the forearm extensor muscles in post-stroke patients’ paretic and non-paretic sides and in a control population during three exercise levels calibrated to the subject’s maximum effort. The change in oxygenation (ΔOxy) and blood volume (ΔBV) were calculated and displayed in real-time. Results show no apparent difference in either ΔOxy or ΔBV between control subjects’ dominant and non-dominant muscles. However, the results show a significant difference in ΔOxy between paretic and non-paretic muscles, as well as a significant difference between normalized post-stroke and control data. Further work will be necessary to determine if the observed difference between the paretic and non-paretic muscles changes over the course of physical therapy and can be correlated with functional improvements.

Significance: Three-dimensional (3D) vascular and metabolic imaging (VMI) of whole organs in rodents provides critical and important (patho)physiological information in studying animal models of vascular network. Aim: Autofluorescence metabolic imaging has been used to evaluate mitochondrial metabolites such as nicotinamide adenine dinucleotide (NADH) and flavine adenine dinucleotide (FAD). Leveraging these autofluorescence images of whole organs of rodents, we have developed a 3D vascular segmentation technique to delineate the anatomy of the vasculature as well as mitochondrial metabolic distribution. Approach: By measuring fluorescence from naturally occurring mitochondrial metabolites combined with light-absorbing properties of hemoglobin, we detected the 3D structure of the vascular tree of rodent lungs, kidneys, hearts, and livers using VMI. For lung VMI, an exogenous fluorescent dye was injected into the trachea for inflation and to separate the airways, confirming no overlap between the segmented vessels and airways. Results: The kidney vasculature from genetically engineered rats expressing endothelial-specific red fluorescent protein TdTomato confirmed a significant overlap with VMI. This approach abided by the “minimum work” hypothesis of the vascular network fitting to Murray’s law. Finally, the vascular segmentation approach confirmed the vascular regression in rats, induced by ionizing radiation. Conclusions: Simultaneous vascular and metabolic information extracted from the VMI provides quantitative diagnostic markers without the confounding effects of vascular stains, fillers, or contrast agents.

The kidney is one of the most radiosensitive organs; it is the primary dose-limiting organ in radiotherapies for upper abdominal cancers. The role of mitochondrial redox state in the development and treatment of renal radiation injury, however, remains ill-defined. This study utilizes 3D optical cryo-imaging to quantify renal mitochondrial bioenergetics dysfunction after 13 Gy leg-out partial body irradiation (PBI). Furthermore, the mitigating effects of lisinopril (lisino), an anti-hypertensive angiotensin converting enzyme inhibitor, is assessed in renal radiation-induced injuries. Around day 150 post-irradiation, kidneys are harvested for cryo-imaging. The 3D images of the metabolic indices (NADH, nicotinamide adenine dinucleotide, and FAD, flavin adenine dinucleotide) are acquired, and the mitochondrial redox states of the irradiated and irradiated + lisino kidneys are quantified by calculating the volumetric mean redox ratio (NADH/FAD). PBI oxidized renal mitochondrial redox state by 78%. The kidneys from the irradiated + lisino rats showed mitigation of mitochondrial redox state by 93% compared to the PBI group. The study provides evidence for an altered bioenergetics and energy metabolism in the rat model of irradiation-induced kidney damage. In addition, the results suggest that lisinopril mitigates irradiation damage by attenuating the oxidation of mitochondria leading to increase redox ratio.

We designed a fiber-optic-based optoelectronic fluorometer to measure emitted fluorescence from the auto-fluorescent electron carriers NADH and FAD of the mitochondrial electron transport chain (ETC). The ratio of NADH to FAD is called the redox ratio (RR = NADH/FAD) and is an indicator of the oxidoreductive state of tissue. We evaluated the fluorometer by measuring the fluorescence intensities of NADH and FAD at the surface of isolated, perfused rat lungs. Alterations of lung mitochondrial metabolic state were achieved by the addition of rotenone (complex I inhibitor), potassium cyanide (KCN, complex IV inhibitor) and/or pentachlorophenol (PCP, uncoupler) into the perfusate recirculating through the lung. Rotenone- or KCN-containing perfusate increased RR by 21 and 30%, respectively. In contrast, PCP-containing perfusate decreased RR by 27%. These changes are consistent with the established effects of rotenone, KCN, and PCP on the redox status of the ETC. Addition of blood to perfusate quenched NADH and FAD signal, but had no effect on RR. This study demonstrates the capacity of fluorometry to detect a change in mitochondrial redox state in isolated perfused lungs, and suggests the potential of fluorometry for use in in vivo experiments to extract a sensitive measure of lung tissue health in real-time.

Fluorescence optical techniques use light in the UV-blue spectral range to measure tissue metabolism non-invasively. In this research, fluorescence optical techniques were utilized for ex vivo and in vivo myocardium spectroscopy and imaging in small animal models. For in vivo heart studies optical methods are used to assess tissue structure and biochemistry associated with heart attack (myocardial infarction) that is the leading cause of death in United States. An experimental endeavor of this complexity was made possible through the collaboration of the ESE Department and the School of Medicine. Fluorescence-based techniques demonstrate the potential of online, in vivo and noninvasive diagnosis of tissue metabolism. Based upon small animal models, we have discovered an intrinsic tissue signal to open up a field of optical spectroscopy called in vivo \"optical biopsy\". At present, the gold standard for detecting tissue abnormality is histopathalogy of excisional biopsies. This procedure requires physical removal of tissue, staining for better resolution and specimen handling. Although this gold standard provides spatial resolution, it suffers from sampling error and offline data analysis. We have investigated the tissue metabolic states in normal and abnormal cases by fluorescence spectroscopy and imaging of tissue intrinsic probes. We utilized fluorometery as the optical technique to acquire fluorescence signals of these fluorophores. We have also used fluorescence imaging to image the biochemical changes in the whole tissue surface and subsurface. One abnormal tissue state occurs when cells undergo programmed cell death or so-called \"apoptosis\". Apoptosis is an orchestrated process in which cells commit suicide. For instance, the therapeutic method of photodynamic therapy (PDT) of tumor cells can trigger the initiation of apoptosis. Another example is myocardial infarction that causes myocytes (heart cells) to become apoptotic. It is known that myocardial infarction produces a wave of apoptotic myocytes that will propagate around the infarction area and has been hypothesized to eventually cause heart failure. Fluorescence spectroscopy and imaging as diagnostic tools are able to monitor the biochemical changes during this disease process. Keywords. Optical diagnostic tools, fluorescence spectroscopy, fluorescence imaging, apoptosis, ischemia, hypoxia, redox state, mitochondria, perfused heart, infracted heart, phosphorescence, Nicotinamide adenine dinucleotide (NADH), flavoprotein (FP), cryo-imaging.

Abstract It has been shown that body mass index (BMI) and obesity may affect the mineral density of bones, regionally on weight-bearing bones or systemically through hormones and cytokines. The objective of this study was to evaluate the effect of BMI on bone mineral density (BMD) of the radius. In this cross-sectional study, 260 patients, 233 postmenopausal women and 27 men over 50, were included who underwent a bone densitometry scanning using dual-energy X-ray absorptiometry after obtaining an informed consent. The scanning was performed in three areas (i.e., spine, proximal femur, and radius), then densitometric data (BMD, T- and Z-score) were extracted. Regression analysis was performed to evaluate the effect of independent variables of age, gender, and BMI on the BMD of the above regions. By grouping the patients in two categories (BMI <25 as normal or underweight and BMI >25 as overweight and obese), the discordance in the diagnosis following the inclusion of radius into interpretation (diagnosis based on 2 vs. 3 areas), was assessed by an agreement test. The study is approved by the ethics committee of the university. Of 260 participants in the present study, mean and standard deviation for age were 61.48 ± 8.95 for all patients, 65.81 ± 10.59 for male and 60.98 ± 8.62 for women. An increasing effect of BMI was found to be statistically significant in weight-bearing areas (total femur and femoral neck) and BMI increase was not associated with increased BMD of radius. An agreement test between two diagnoses is used that showed a discordance of 28.5% in diagnosis (diagnosis based on 2 vs. 3 areas) with a kappa coefficient of 0.547 ( P = 0.001). In total, 25.4% was minor discordance and 3.1% was major discordance. Based on the results of this study, it is concluded that the BMI is not associated with increased BMD in bones that are not weight bearing, such as radius. Therefore, it may be preferred to include the densitometric data of radius into the diagnosis.