Advanced technique of myocardial no-reflow quantification using indocyanine green.

The post-ischemic no-reflow phenomenon after primary percutaneous coronary intervention (PCI) is observed in more than half of subjects and is defined as the absence or marked slowing of distal coronary blood flow despite removal of the arterial occlusion. To visualize no-reflow in experimental studies, the fluorescent dye thioflavin S (ThS) is often used, which allows for the estimation of the size of microvascular obstruction by staining the endothelial lining of vessels. Based on the ability of indocyanine green (ICG) to be retained in tissues with increased vascular permeability, we proposed the possibility of using it to assess not only the severity of microvascular obstruction but also the degree of vascular permeability in the zone of myocardial infarction. The aim of our study was to investigate the possibility of using ICG to visualize no-reflow zones after ischemia-reperfusion injury of rat myocardium. Using dual ICG and ThS staining and the FLUM multispectral fluorescence organoscope, we recorded ICG and ThS fluorescence within the zone of myocardial necrosis, identifying ICG-negative zones whose size correlated with the size of the no-reflow zones detected by ThS. It is also shown that the contrast change between the no-reflow zone and nonischemic myocardium reflects the severity of blood stasis, indicating that ICG-negative zones are no-reflow zones. The described method can be an addition or alternative to the traditional method of measuring the size of no-reflow zones in the experiment.

Autofluorescence spectroscopy in photodynamic therapy for skin rejuvenation: A theranostic approach in aesthetic medicine.

BACKGROUND: The method of photodynamic therapy for skin rejuvenation (PDT-SR) provides an improvement in appearance with a safe and painless effect. The quality of treatment is most often assessed subjectively. The most informative morphological control methods are rarely used due to the invasiveness of the sampling procedure. AIM: This study aimed to find out the possibility of using skin autofluorescence spectroscopy (SAF) for an objective assessment of changes occurring in the skin during PDT-SR. METHODS: This study included 12 volunteers (10 women, 2 men) aged 32 to 79 years. Two (n = 6) or three (n = 6) PDT sessions were performed at intervals of 13-30 days. Photosensitizer chlorin e6, exposure 20 min, energy density 18-24 J/cm2 were used. SAF spectra were recorded using a two-wavelength fiber optic spectrometer under excitation at wavelengths (λex) of 365 nm and 440 nm. Measurements were made both before and after each PDT session and up to 25-238 days from the start of treatment. For the evaluation, we used the spectra AF365(λ) and AF440(λ) averaged over 40 points corrected for diffuse reflection at λex=440 nm in the range λem= 460-700 nm, as well as the spectra of the ratios AFN365(λ) and AFN440(λ), which were obtained by dividing the intensities of the current spectra by the intensities collected before PDT-SR. RESULTS: PDT-SR led to changes in both the intensity and shape of the spectra. Analysis of the spectra using numerical fitting of the spectra showed that the main changes can be explained by changes in the content of advanced glycation end products (AGEs), as well as lipofuscin-like lipopigments (LPs) and porphyrins (PPs). The spectra of AGEs upon excitation at wavelengths of 365 and 440 nm differ, which may be due to the formation of two types of bonds, with collagen and elastin. By the end of the study, the vast majority of the examined volunteers showed a significant decrease of the parameters characterizing both of these types of AGEs, AGE365 (0.56-1.2) and AGE440 (0.58-1.01), relative to the beginning of the study. In most cases, a decrease was also noted for LPs and PPs. AGE365 and AGE440 were positively correlated with the age of the volunteers (r2 = 0.26-0.46 %). A steady decrease in the content of AGEs occurred approximately on the 40th day. CONCLUSION: SAF spectroscopy makes it possible to assess changes in the content of AGEs, LPs, and PPs in the skin during PDT-SR. The method has great potential for non-invasive monitoring of the treatment process, as well as its improvement, including through its personalization. In addition, the method can be used to study the mechanisms of age-related skin changes at the molecular level and to study the processes of rejuvenation.

Effects of three types of bariatric interventions on myocardial infarct size and vascular function in rats with type 2 diabetes mellitus.

AIMS: The effects of three types of bariatric interventions on myocardial infarct size were tested in the rat model of type 2 diabetes mellitus (T2DM). We also evaluated the effects of bariatric surgery on no-reflow phenomenon and vascular dysfunction caused by T2DM. MAIN METHODS: Rats with T2DM were assigned into groups: without surgery, sham-operated, ileal transposition, Roux-en-Y gastric bypass, and sleeve gastrectomy. Oral glucose tolerance, glucagon-like peptide-1, and insulin levels were measured. Six weeks after surgery, the animals were subjected to myocardial ischemia-reperfusion followed by histochemical determination of infarct size (IS), no-reflow zone, and blood stasis area size. Vascular dysfunction was characterized using wire myography. KEY FINDINGS: All bariatric surgery types caused significant reductions in animal body weight and resulted in T2DM compensation. All bariatric interventions partially normalized glucagon-like peptide-1 responses attenuated by T2DM. IS was significantly smaller in animals with T2DM. Bariatric surgery provided no additional IS limitation compared with T2DM alone. Bariatric surgeries reversed T2DM-induced enhanced contractile responses of the mesenteric artery to 5-hydroxytryptamine. Sleeve gastrectomy normalized decreased nitric oxide synthase contribution to the endothelium-dependent vasodilatation in T2DM. SIGNIFICANCE: T2DM resulted in a reduction of infarct size and no-reflow zone size. Bariatric surgery provided no additional infarct-limiting effect, but it normalized T2DM-induced augmented vascular contractility and reversed decreased contribution of nitric oxide to endothelium-dependent vasodilatation typical of T2DM. All taken together, we suggest that this type of surgery may have a beneficial effect on T2DM-induced cardiovascular diseases.

Photodynamic theranostics of central lung cancer: Present state and future prospects.

Endobronchial photodynamic therapy (PDT) in central lung cancer (CLC) shows feasibility even in late stage disease. Our experience with chlorin e6 based photosensitizers (PS), including in combination with medical cancer treatment, demonstrated regression of tumor lesions of the trachea and bronchi in 94 % of patients with central NSCLC. It is possible to increase the efficiency of the treatment and achieve its personalization by using fluorescent bronchoscopes, which provide fluorescence guided PDT - photodynamic theranostics (PT). PT allows to clarify localization the area of treatment due to visualization of tumor foci which are invisible in white light, to carry out targeted irradiation and at the same time to monitor its effectiveness using the effects of bleaching/flare-up of PS. PT prospects are associated with the transition to the near-infrared (NIR) region, which makes possible to increase the depth of light penetration. The first experiments using the combined NIR/visible PT system showed the possibility of detecting tumor sites using the OS-BPT method (On-Site Bronchoscopic Photodynamic Theranostics), which consists in NIR visualization of tumor when indocyanine green (ICG) is injected directly during examination in a minimal dose. This allows the technology to be used for CLC screening in the future. Further progress of endobronchial PT will be determined by the development of clinically available devices and new NIR PSs with targeted properties, high singlet oxygen yield and fluorescence.

Potential of indocyanine green near-infrared fluorescence imaging in experimental and clinical practice.

BACKGROUND: Visualization of Indocyanine Green (ICG) near-infrared (NIR) fluorescence is a promising technique for biomedical applications because of its deep tissue penetration, low autofluorescence, weak dependence on ambient light, safety for patients and medical staff. METHODS: To apply this technique to animal studies and clinical practice, we developed multispectral NIR fluorescence imaging system FLUM-808, which can be used in experimental studies and clinical practice, in open and endoscopic surgery (fluorescence-guided surgery). The object is illuminated either directly or through an illumination channel depending on what optical instrument is used (camera lens, fiber endoscope, rigid endoscope, surgical microscope, etc.). The system is able to simultaneously display NIR images and images captured under white light. RESULTS: This article provides examples of imaging techniques used during open and endoscopic surgery with ICG as an NIR fluorescent dye and photosensitizer, demonstrates applications of the technique for experimental and clinical purposes: for intraoperative imaging of blood and lymphatic vessels; for detection of tumors and sentinel lymph nodes; for assessment of tissue, organ, or anastomotic blood supply. CONCLUSIONS: This method significantly helps to accurately assign the areas of increased fluorescence to certain anatomical structures during surgery. NIR fluorescence diagnosis can be used in important experimental studies and in clinical practice. The described results of ICG studies can help in developing new fluorescence-based diagnostic and theranostic approaches employing more effective exogenous fluorophores and photosensitizers.

Endobronchial photodynamic therapy under fluorescence control: Photodynamic theranostics.

BACKGROUND: Photodynamic therapy (PDT) has several advantages. However, one of the disadvantages is its inability to be individualized according to biological characteristics of malignant tumors. The objective of this study was to investigate a strategy for individualized endobronchial PDT in the treatment of centrally located non-small cell lung cancer. METHODS: New approach suggests taking fluorescence-based measurements of chlorine E6 photosensitizer (PS) accumulation in the malignant tumor tissue, and assess PS consumption rate during PDT. Two randomized groups of 45 patients took part in the comparative study of standard PDT procedure, 662nm, pulse-periodic mode, therapeutic light (reference group - RG) versus the investigated individualized approach under fluorescence control after irradiation with violet light, 408nm, diagnostic light (study group - SG). The PDT-treatment parameters and results of follow-up bronchoscopy were compared between the groups. RESULTS: 43 (96%) of 45 patients in SG demonstrated intense fluorescence in the area of the tracheal/bronchial tumor stenosis. 4 (9%) of 45 patients (SG) demonstrated fluorescence of mucosa areas distant from the main tumor lesion after violet light irradiation. Mean fluence during the whole PDT procedure was 95±20J/cm2 (range 60-130J/cm2), which was significantly lower than in RG (p=0.01). Total exposure time was significantly lower in SG (365±65s), compared with RG (690±65s), P=0.001. According to the follow-up bronchoscopy the difference in the PDT-treatment results between the groups is statistically insignificant. CONCLUSIONS: The investigated strategy suggests using fluorescence control of the efficacy of PDT-treatment (photodynamic theranostics) to optimize and individualize the PDT procedure.

In vivo visualization and ex vivo quantification of experimental myocardial infarction by indocyanine green fluorescence imaging.

The fluorophore indocyanine green accumulates in areas of ischemia-reperfusion injury due to an increase in vascular permeability and extravasation of the dye. The aim of the study was to validate an indocyanine green-based technique of in vivo visualization of myocardial infarction. A further aim was to quantify infarct size ex vivo and compare this technique with the standard triphenyltetrazolium chloride staining. Wistar rats were subjected to regional myocardial ischemia (30 minutes) followed by reperfusion (n = 7). Indocyanine green (0.25 mg/mL in 1 mL of normal saline) was infused intravenously for 10 minutes starting from the 25th minute of ischemia. Video registration in the near-infrared fluorescence was performed. Epicardial fluorescence of indocyanine green corresponded to the injured area after 30 minutes of reperfusion. Infarct size was similar when determined ex vivo using traditional triphenyltetrazolium chloride assay and indocyanine green fluorescent labeling. Intravital visualization of irreversible injury can be done directly by fluorescence on the surface of the heart. This technique may also be an alternative for ex vivo measurements of infarct size.

Autofluorescence spectroscopy for NADH and flavoproteins redox state monitoring in the isolated rat heart subjected to ischemia-reperfusion.

BACKGROUND: Reduction of myocardial ischemia-reperfusion injury in the patients undergoing cardiac surgery under cardiopulmonary bypass represents an important goal. Intraoperative monitoring of myocardial metabolic state using continuous registration of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) fluorescence might contribute to the solution of the problem. The successful application of fluorescent spectroscopy in the clinical field requires additional refinement of the technique, particularly using excitation of both NADH and FAD with different wavelengths. METHODS: The experiments were performed on the isolated Langendorff-perfused rat hearts (n=28) subjected to either regional or global ischemia-reperfusion. Two principles of NADH and FAD autofluorescence (AF) measurement were used for ischemia monitoring: (1) analysis of photographs and videos obtained with multispectral organoscopy technique allowing the assessment of both spatial and temporal characteristics of the process (n=16); (2) continuous registration of tissue redox state in a representative area of the heart by application of local spectroscopy, assisted by fiber optic spectrometer (n=12). RESULTS: It was found that regional myocardial ischemia resulted in a rapid, substantial increase in the intensity of NADH AF excited at 360nm in the ischemic versus non-ischemic area of the heart. The same result was obtained when the heart was made globally ischemic, while the restoration of perfusate flow completely reversed the increase in NADH AF. During the transition from ischemia to reperfusion, the spatial heterogeneity of myocardial AF was noted on video recordings, probably reflecting the microheterogeneity of myocardial blood flow. Local spectroscopy studies demonstrated opposite changes in the NADH and FAD AF during ischemia. Using both methodological approaches, we found that repetitive brief episodes of global myocardial ischemia resulted in progressive decrease in the magnitude of AF elevation, which might point to preconditioning effect. CONCLUSIONS: The application of multispectral fluorescent organoscopy offers the advantage of monitoring myocardial redox state at the level of the entire heart. Local spectroscopy is characterized by better precision and, in addition, provides the unique opportunity to measure AF in different parts of the spectrum. AF measurements are non-invasive, rapid, and technically easy to perform. For future clinical applications, it might be recommended to combine the measurement of redox state of both NADH and FAD, using excitation wavelength and emission filter optimal for each fluorophore.

Targeted drug delivery into reversibly injured myocardium with silica nanoparticles: surface functionalization, natural biodistribution, and acute toxicity.

The clinical outcome of patients with ischemic heart disease can be significantly improved with the implementation of targeted drug delivery into the ischemic myocardium. In this paper, we present our original findings relevant to the problem of therapeutic heart targeting with use of nanoparticles. Experimental approaches included fabrication of carbon and silica nanoparticles, their characterization and surface modification. The acute hemodynamic effects of nanoparticle formulation as well as nanoparticle biodistribution were studied in male Wistar rats. Carbon and silica nanoparticles are nontoxic materials that can be used as carriers for heart-targeted drug delivery. Concepts of passive and active targeting can be applied to the development of targeted drug delivery to the ischemic myocardial cells. Provided that ischemic heart-targeted drug delivery can be proved to be safe and efficient, the results of this research may contribute to the development of new technologies in the pharmaceutical industry.