Quantifying Acute Myocardial Injury Using Ratiometric Fluorometry

Early reperfusion is the best therapy for myocardial infarction (MI). Effectiveness, however, varies significantly between patients and has implications for long-term prognosis and treatment. A technique to assess the extent of myocardial salvage after reperfusion therapy would allow for high-risk patients to be identified in the early post-MI period. Mitochondrial dysfunction is associated with cell death following myocardial reperfusion and can be quantified by fluorometry. Therefore, we hypothesized that variations in the fluorescence of mitochondrial nicotinamide adenine dinucleotide (NADH) and flavoprotein (FP) can be used acutely to predict the degree of myocardial injury. Thirteen rabbits had coronary occlusion for 30 min followed by 3 h of reperfusion. To produce a spectrum of infarct sizes, six animals were infused cyclosporine A prior to ischemia. Using a specially designed fluorometric probe, NADH and FP fluorescence were measured in the ischemic area. Changes in NADH and FP fluorescence, as early as 15 min after reperfusion, correlated with postmortem assessment infarct size (r=0.695, p\u3c0.01). This correlation strengthened with time (r=0.827, p\u3c0.01 after 180 min). Clinical application of catheter-based myocardial fluorometry may provide a minimally invasive technique for assessing the early response to reperfusion therapy

Surface Fluorescence Studies of Tissue Mitochondrial Redox State in Isolated Perfused Rat Lungs

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

Optical Imaging of Lipopolysaccharide-induced Oxidative Stress in Acute Lung Injury from Hyperoxia and Sepsis

Reactive oxygen species (ROS) have been implicated in the pathogenesis of many acute and chronic pulmonary disorders such as acute lung injury (ALI) in adults and bronchopulmonary dysplasia (BPD) in premature infants. Bacterial infection and oxygen toxicity, which result in pulmonary vascular endothelial injury, contribute to impaired vascular growth and alveolar simplification seen in the lungs of premature infants with BPD. Hyperoxia induces ALI, reduces cell proliferation, causes DNA damage and promotes cell death by causing mitochondrial dysfunction. The objective of this study was to use an optical imaging technique to evaluate the variations in fluorescence intensities of the auto-fluorescent mitochondrial metabolic coenzymes, NADH and FAD in four different groups of rats. The ratio of these fluorescence signals (NADH/FAD), referred to as NADH redox ratio (NADH RR) has been used as an indicator of tissue metabolism in injuries. Here, we investigated whether the changes in metabolic state can be used as a marker of oxidative stress caused by hyperoxia and bacterial lipopolysaccharide (LPS) exposure in neonatal rat lungs. We examined the tissue redox states of lungs from four groups of rat pups: normoxic (21% O2) pups, hyperoxic (90% O2) pups, pups treated with LPS (normoxic + LPS), and pups treated with LPS and hyperoxia (hyperoxic + LPS). Our results show that hyperoxia oxidized the respiratory chain as reflected by a ~ 31% decrease in lung tissue NADH RR as compared to that for normoxic lungs. LPS treatment alone or with hyperoxia had no significant effect on lung tissue NADH RR as compared to that for normoxic or hyperoxic lungs, respectively. Thus, NADH RR serves as a quantitative marker of oxidative stress level in lung injury caused by two clinically important conditions: hyperoxia and LPS exposure

Optical Studies of Oxidative Stress in Lung Tissue: Rodent Models

Objectives: There currently exists a need for reliable measurements of tissue metabolic state at cellular levels. The objective of this research was to study tools capable of evaluating cellular redox states in intact tissue. To meet this goal, three different instruments (cryoimager, fluorometer, and fluorescent microscope) were used to study the metabolism and functions of the mitochondria at different levels and regimes (cryo, ex vivo, in vivo and in vitro). Introduction: Through optical monitoring of autofluorescent mitochondrial metabolic coenzymes, as well as exogenous fluorophores, the state of mitochondria can be probed in real time in many intact organs and in vitro. Autofluorescent mitochondrial metabolic coenzymes, studied here, include NADH (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide), and the ratio of these fluorophores, referred to as the mitochondrial redox ratio (RR), can be used as a quantitative metabolic marker of the tissue. Exogenous fluorophores include but are not limited to tetramethylrhodamine (TMRM) and Mito-SOX, which are used to evaluate the mitochondrial membrane potential and level of reactive oxygen species (ROS) in the mitochondria, respectively. Methods: Different optical imaging and acquisition techniques were studied to evaluate oxidative stress in lung tissue and cells in cryogenic temperatures, in vivo, ex vivo, and in vitro. Though in essence the underlying technological and biological principles appear to be the same, imaging in each of these regimes imposed unique challenges requiring significantly different approaches to system design, data acquisition, and processing. A brief description of each technique is provided here and each is described in detail in the following chapters. The first device utilized is a cryoimager, which sequentially slices tissue and acquires fluorescence images of up to five fluorophores in cryogenic temperatures (-40oC). Rapid freezing of organs preserves the tissue\u27s metabolic state and subsequent low temperature fluorescence imaging (cryoimaging) provides high fluorescence quantum yield as compared with room temperature. Sequential slicing of the tissue provides 3D spatial distribution of NADH and FAD fluorescence intensities throughout the tissue. These studies were conducted using the cryoimager in the Biophotonics Lab on different models of lung injuries including ischemia, hyperoxia, and BPD (bronchopulmonary dysplasia). The second device is a fluorometer, which was designed and implemented in the Biophotonics Lab. It is capable of monitoring the dynamics of the metabolism of the tissue through the use of optical surface fluorescence measurements of NADH and FAD. The ratio of these fluorophores, referred to as the mitochondrial redox ratio (RR), can be used as a quantitative metabolic marker of tissue. Surface fluorescence signals from NADH and FAD were acquired in the absence (baseline) and presence of metabolic perturbers (e.g. pentachlorophenol, rotenone, or potassium cyanide), in the presence of blood, and eventually in vivo. The third instrument, a fluorescent microscope, is used to image slides and dishes containing stained cells (e.g. endothelial cells, perycites, or fibroblasts) from lungs, hearts, and retinas to study their structure and dynamics at cellular level. Images of retinas were classified as normal or injured using developed cytometry tools and morphologic parameters. For heart and lung, the dynamics of concentration of reactive oxygen species (mainly superoxide) and calcium is monitored over time in cultured live cells. Results: In the cryogenic temperatures, lung treatment with KCN (inhibitor of Complex IV), resulted in an increase in RR and sets the upper limit of the NADH signal level while injured lungs (BPD model, hyperoxia and IR) showed a more oxidized chain compared with control lungs, and as a result more oxidative stress. In ex vivo fluorometric studies, an increase in RR from chain inhibitors (including KCN and rotenone), and a decrease in the same due to an uncoupler (PCP), all from baseline was observed which was consistent with the cryoimaging results. The same experiments in isolated perfused lungs previously treated with hyperoxia showed the same direction but different levels indicating the impairment in different complexes due to hyperoxia. Segmentation algorithm developed here showed 90% accuracy comparing to manual counting, and studying the cells in retina slides confirms apoptosis and oxidative stress in retinas from injured mice. In live cells, studying the dynamics of calcium concentration in the presence of different perturbations enabled us to study the behavior of mitochondrial regulated calcium channels. Also, changes in the Mito-SOX channel gave us the dynamics of mitochondrial ROS in the presence of chain perturbers (chemicals and gas). Conclusion: Optical instrumentation combined with signal and image processing tools provide quantitative physiological and structural information of diseased tissue due to oxidative stress

Mitochondrial redox studies of oxidative stress in kidneys from diabetic mice

Chronic hyperglycemia during diabetes leads to increased production of reactive oxygen species (ROS) and increased oxidative stress (OS). Here we investigated whether changes in the metabolic state can be used as a marker of OS progression in kidneys. We examined redox states of kidneys from diabetic mice, Akita/+ and Akita/+;TSP1–/– mice (Akita mice lacking thrombospondin-1, TSP1) with increasing duration of diabetes. OS as measured by mitochondrial redox ratio (NADH/FAD) was detectable shortly after the onset of diabetes and further increased with the duration of diabetes. Thus, cryo fluorescence redox imaging was used as a quantitative marker of OS progression in kidneys from diabetic mice and demonstrated that alterations in the oxidative state of kidneys occur during the early stages of diabetes

Redox-responsive inorganic fluorescent nanoprobes for serodiagnosis and bioimaging

Redox reactions play fundamental roles in life and are at the core of metabolism. Thus, observing and quantifying these reactions is crucial for diagnostics and therapy. Recent advances in inorganic fluorescent nanoprobes have revolutionized the field, enabling in vitro diagnostics by providing reliable tools for real-time, quantitative determination of redox biomolecule levels in biological samples and cells. Due to their high stability, these probes are also widely used in bioimaging, providing real-time information for in vivo diagnostics and guiding treatment of diseases associated with redox biomolecules. This review explores the diverse landscape of inorganic fluorescent nanoprobes designed for the detection of biologically relevant reactive oxygen and nitrogen species. The discussion is divided into several sections, each focusing on nanoprobes tailored for specific oxidative species. The impact of tailored nanoprobes in diagnostics and imaging-guided treatment depends on their chemical composition, surface property, and fluorescence mechanism. The discussions highlight the current strengths and weaknesses, which will help to design more efficient redox-responsive inorganic fluorescent nanoprobes in the future

Photobiomodulation in Inherited Retinal Degeneration

The retinal degenerative disease, retinitis pigmentosa (RP), is the most common cause of inherited blindness in the developed world and is caused by the progressive degeneration of rod photoreceptor cells preceding cone degeneration. Mitochondrial dysfunction and oxidative stress have been shown to play a significant role in the pathogenesis of RP and other retinal degenerative diseases. A growing body of evidence indicates that exposure of tissue to low energy photon irradiation in the far-red to near-infrared (NIR) range of the spectrum, (photobiomodulation or PBM) acts on mitochondria-mediated signaling pathways to attenuate oxidative stress and prevent cell death. These studies tested the hypothesis that PBM acts in the retina to promote mitochondrial integrity and function, prevent photoreceptor cell death and preserve retinal function in an established rodent model of retinitis pigmentosa, the P23H rhodopsin transgenic rat. Retinal function, structural integrity, surviving photoreceptors and the mitochondrial redox state were assessed using electroretinography, spectral domain optical coherence tomography, histomorphometry and cryofluorescence redox imaging. PBM did not alter the structural and functional characteristics of retina in a non-dystrophic animal strongly supporting the safety of PBM. Establishing the safety of PBM is essential to advance the therapy to clinical use. 830 nm PBM exerted a robust retinoprotective effect compared to 670 nm PBM in the P23H transgenic rat model. 830 nm PBM during the critical period of photoreceptor degeneration in P23H transgenic rat profoundly attenuated retinal degeneration resulting in the preservation of retinal function; retinal morphology and retinal metabolic state in comparison to the sham-treated group. An in vivo longitudinal study corroborated the structural preservation observed in the cross-sectional study. These findings provide evidence supporting the therapeutic utility of PBM in the treatment of retinal degenerative disease. They also further our understanding of the mechanism of action of PBM by showing that it improves mitochondrial function in the retinae of RP animals. By exploiting, the cells own mechanism of self-repair, PBM has the potential for translating into clinical practice as an innovative, non-invasive stand-alone or adjunct therapy for the prevention and treatment of retinal diseases

Upconverting nanoparticles in ultrasensitive detection of cardiac troponin I

Development of high-sensitivity immunoassays is a continuous interest in medical diagnostics, especially in the case of such diseases where higher sensitivity analyte measurements improve the prognosis of treatment. One such analyte is cardiac troponin I, used for detection of cardiac events. One of the key factors determining immunoassay sensitivity is the reporter, which labels the analytes in the assays and produces the measurable signal. Upconverting nanoparticles (UCNPs) are promising candidates for reporters in sensitive immunoassays. Their unique ability to convert near-infrared light into higher energy visible light by stacking photons, producing emission exhibiting anti-Stokes shift. As no other natural materials are capable of the process, measurement of UCNPs can be designed to completely dismiss any background signal originating from autofluorescence. However, reaching the maximal sensitivity they theoretically enable has been hindered by their tendency to non-specifically bind to solid surfaces in assays and to each other, forming nanoparticle clusters of varying sizes. The extent of these tendencies has been linked to the surface chemistry of the UCNPs. The aim of this thesis was to study the surface chemistry of UCNPs and apply them as reporters in different immunoassay technologies for detection of cTnI. During the research, surface coating of UCNPs with poly(acrylic acid) was studied and successfully improved, leading to reduced non-specific binding and cluster formation tendency. The performance of the UCNPs with the novel surface was compared to other surface chemistry approaches in microtiter plate assays utilizing either analog or digital readout method, and a lateral flow assay. Another aim was to develop the used assay technologies utilizing upconversion to reach extreme sensitivities. Reagents and conditions in analog microtiter plate assay for cTnI were thoroughly investigated to fine-tune the performance, and the limit of detection (LoD) reached an unprecedented low value of 0.13 ng/L for an analog assay. In addition, a mechanical actuator for automation of a lateral flow assay for cTnI was fabricated via 3D-printing, and when combined with the improved UCNPs, an LoD of 1.5 ng/L was reached, bringing high-sensitivity pointof- care detection of cTnI a step closer to reality. KEYWORDS: cardiac biomarker, luminescence, nanoparticle monodispersityHerkkien immunomääritysmenetelmien kehitys on jatkuvan kiinnostuksen aiheena diagnostiikan tutkimuksessa. Herkät biomerkkiainetestit ovat haluttuja etenkin sydänkohtauksen kaltaisissa tiloissa, jossa merkkiaineen pitoisuus verenkierrossa kasvaa samalla, kun hoitoennuste heikkenee. Yksi merkittävimmistä immunomääritysten herkkyyteen vaikuttavista tekijöistä on määritysleima, joka sitoutuu analyyttiin ja tuottaa mitattavan signaalin. Käänteisviritteisten nanopartikkelien (engl. upconverting nanoparticles, UCNP) kyky muuntaa matalaenergistä viritysvaloa korkeaenergiseksi emissioksi tekee niistä lupaavan määritysleiman herkkiin immunomäärityksiin, koska UCNP:iden signaalin mittaus voidaan tehdä niin, ettei autofluoresenssista koituvaa taustasignaalia havaita, mahdollistaen äärimmäisen herkät immunomääritykset. UCNP:iden taipumus sitoutua epäspesifisesti erilaisiin pintoihin ja muodostaa erikokoisia kasaumia on hidastanut niiden käyttöönottoa herkkinä immunomääritysleimoina. Tämän ominaisuuden on osoitettu riippuvan voimakkaasti partikkelien pintakemiasta. Tämän tutkimuksen tavoite oli tutkia UCNP:iden pintakemiaa ja hyödyntää niitä määritysleimoina erilaisissa cTnI:tä havaitsevissa immunomäärityksissä. Tutkimuksessa selvitettiin UCNP:iden poly(akryylihappo) (PAA) -pinnoituksen onnistumiseen vaikuttavia tekijöitä ja onnistuttiin merkittävästi vähentämään PAA-pintaisten UCNPeiden epäspesifistä sitoutumista ja kasautumistaipumusta. Näitä UCNPleimoja verrattiin muulla tavoin pinnoitettuihin UCNP-leimoihin mikrotiitterilevypohjaisessa immunomäärityksessä käyttäen joko analogista tai digitaalista mittausmenetelmää, sekä lateraalivirtausmäärityksessä. Toinen tavoite oli kehittää käytettyjä määritysmenetelmiä äärimmäisten herkkyyksien saavuttamiseksi. Mikrotiitterilevymäärityksen reagenssit ja toteutusmenetelmä tutkittiin tarkoin ja hienosäädettiin. Tällä tekniikalla saavutettiin 0,13 ng/L havaitsemisherkkyys cTnI:lle, mikä on ennätyksellistä analogisissa mikrotiitterilevymäärityksissä. Lisäksi suunniteltiin mekaaninen 3D-tulostettu laite automatisoimaan cTnI:tä havaitseva lateraalivirtausmääritys. Yhdistettynä paranneltuihin PAA-UCNP:ihin, saavutettiin 1,5 ng/L havaitsemisherkkyys, mikä tuo äärimmäisen herkät cTnI:n vieritestausmenetelmät askeleen lähemmäs todellisuutta. ASIASANAT: sydänmerkkiaine, luminesenssi, nanopartikkelien yksittäisyy