Post-Operational Photodynamic Therapy of the Tumor Bed: Comparative Analysis for Cold Knife and Laser Scalpel Resection.

In this paper, we report on a study regarding the efficiency of the post-operational phototherapy of the tumor bed after resection with both a cold knife and a laser scalpel in laboratory mice with CT-26 tumors. Post-operational processing included photodynamic therapy (PDT) with a topically applied chlorin-based photosensitizer (PS), performed at wavelengths of 405 or 660 nm, with a total dose of 150 J/cm2. The selected design of the tumor model yielded zero recurrence in the laser scalpel group and 92% recurrence in the cold knife group without post-processing, confirming the efficiency of the laser scalpel in oncology against the cold knife. The application of PDT after the cold knife resection decreased the recurrence rate to 70% and 42% for the 405 nm and 660 nm procedures, respectively. On the other hand, the application of PDT after the laser scalpel resection induced recurrence rates of 18% and 30%, respectively, for the considered PDT performance wavelengths. The control of the penetration of PS into the tumor bed by fluorescence confocal microscopy indicated the deeper penetration of PS in the case of the cold knife, which presumably provided deeper PDT action, while the low-dose light exposure of deeper tissues without PS, presumably, stimulated tumor recurrence, which was also confirmed by the differences in the recurrence rate in the 405 and 660 nm groups. Irradiation-only light exposures, in all cases, demonstrated higher recurrence rates compared to the corresponding PDT cases. Thus, the PDT processing of the tumor bed after resection could only be recommended for the cold knife treatment and not for the laser scalpel resection, where it could induce tumor recurrence.

The Effect of Diabetes Mellitus Type 1 on the Energy Metabolism of Hepatocytes: Multiphoton Microscopy and Fluorescence Lifetime Imaging.

A decrease in the regenerative potential of the liver during the development of non-alcoholic fatty liver disease (NAFLD), which is observed in the vast majority of patients with diabetes mellitus type 1, significantly increases the risk of postoperative liver failure. In this regard, it is necessary to develop new approaches for the rapid intraoperative assessment of the condition of liver tissue in the presence of concomitant liver pathology. A modern label-free approach based on multiphoton microscopy, second harmonic generation (SHG), and fluorescence lifetime imaging microscopy (FLIM) allow for the evaluation of the structure of liver tissue as well as the assessment of the metabolic state of hepatocytes, even at the cellular level. We obtained optical criteria and identified specific changes in the metabolic state of hepatocytes for a reduced liver regenerative potential in the presence of induced diabetes mellitus type 1. The obtained criteria will expand the possibilities for the express assessment of the structural and functional state of liver tissue in clinical practice.

Preclinical Studies on the Safety and Toxicity of Photoditazine in the Antibacterial Photodynamic Therapy of Uropathogenic Bacteria.

The 'dusting' technique of lithotripsy for the removal of infected urinary calculi and the wide use of drainage after endoscopic surgery may stimulate spreading of multidrug-resistant bacterial strains. Antibacterial photodynamic therapy (PDT) is one promising method for the elimination these strains. The purpose of our study was to evaluate alterations of renal pelvis morphology and renal function in laboratory animals after bactericidal regimens of PDT. Renal pelvises of pigs were filled with Photoditazine and then assessed either by examining the accumulation of Photoditazine in the urothelium or by illumination with a laser at a wavelength of 662 nm. A renal test and a complete blood count was performed to assess a negative effect of the treatment on health. Structural alterations of the kidney tissues were analyzed by histological examination. No photosensitizer fluorescence was detected in the urothelium of the pelvis. Histological study showed that PDT caused minor changes to the urothelium of the renal pelvis but did not affect the underlying connective tissue. No renal function abnormalities were found after PDT. Thus, the study indicates that antibacterial PDT is a safety technique that can complement common antibiotic therapy in the surgical treatment of urolithiasis.

Effect of Hepatic Pathology on Liver Regeneration: The Main Metabolic Mechanisms Causing Impaired Hepatic Regeneration.

Liver regeneration has been studied for many decades, and the mechanisms underlying regeneration of normal liver following resection are well described. However, no less relevant is the study of mechanisms that disrupt the process of liver regeneration. First of all, a violation of liver regeneration can occur in the presence of concomitant hepatic pathology, which is a key factor reducing the liver's regenerative potential. Understanding these mechanisms could enable the rational targeting of specific therapies to either reduce the factors inhibiting regeneration or to directly stimulate liver regeneration. This review describes the known mechanisms of normal liver regeneration and factors that reduce its regenerative potential, primarily at the level of hepatocyte metabolism, in the presence of concomitant hepatic pathology. We also briefly discuss promising strategies for stimulating liver regeneration and those concerning methods for assessing the regenerative potential of the liver, especially intraoperatively.

Label-Free Imaging Techniques to Evaluate Metabolic Changes Caused by Toxic Liver Injury in PCLS.

Abuse with hepatotoxic agents is a major cause of acute liver failure. The search for new criteria indicating the acute or chronic pathological processes is still a challenging issue that requires the selection of effective tools and research models. Multiphoton microscopy with second harmonic generation (SHG) and fluorescence lifetime imaging microscopy (FLIM) are modern label-free methods of optical biomedical imaging for assessing the metabolic state of hepatocytes, therefore reflecting the functional state of the liver tissue. The aim of this work was to identify characteristic changes in the metabolic state of hepatocytes in precision-cut liver slices (PCLSs) under toxic damage by some of the most common toxins: ethanol, carbon tetrachloride (CCl4) and acetaminophen (APAP), commonly known as paracetamol. We have determined characteristic optical criteria for toxic liver damage, and these turn out to be specific for each toxic agent, reflecting the underlying pathological mechanisms of toxicity. The results obtained are consistent with standard methods of molecular and morphological analysis. Thus, our approach, based on optical biomedical imaging, is effective for intravital monitoring of the state of liver tissue in the case of toxic damage or even in cases of acute liver injury.

Effect of pH, Norepinephrine and Glucose on Metabolic and Biofilm Activity of Uropathogenic Microorganisms.

Urinary tract infection (UTIs) aremainly caused by a number of anatomical and physiological dysfunctions, but there are also some iatrogenic factors, including the use of certain medications, that contribute to the development of UTIs. The virulence of bacteria that colonize the urinary tract may be modified by pH and by the presence of soluble substances in urine, such as norepinephrine (NE) and glucose. In this work, we studied the influence of NE and glucose across a range of pHs (5, 7, 8) on the biomass, matrix production and metabolism of uropathogenic strains of Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus and Enterococcus faecalis. We used Congo red and gentian violet to stain the extracellular matrix and biomass, respectively, of biofilms. The optical density of staining of the biofilms was measured using a multichannel spectrophotometer. The metabolic activity was analyzed by MTT assay. It was shown that NE and glucose stimulate biomass production both in the Gram-negative and Gram-positive uropathogens. The metabolic activity in the presence of glucose was higher at pH 5 for E. coli (in 4.0 ± 0.1 times), Ps. aeruginosa (in 8.2 ± 0.2 times) and Kl. pneumoniae (in 4.1 ± 0.2 times). Matrix production of Kl. pneumoniae increased under NE (in 8.2 ± 0.2 times) and in the presence of glucose (in 1.5 ± 0.3 times). Thus, NE and glucose in urine may lead to persistent UTI under patient stress and in the case of metabolic glucose disorders.

FLIM imaging revealed spontaneous osteogenic differentiation of stem cells on gradient pore size tissue-engineered constructs.

BACKGROUND: There is an urgent clinical need for targeted strategies aimed at the treatment of bone defects resulting from fractures, infections or tumors. 3D scaffolds represent an alternative to allogeneic MSC transplantation, due to their mimicry of the cell niche and the preservation of tissue structure. The actual structure of the scaffold itself can affect both effective cell adhesion and its osteoinductive properties. Currently, the effects of the structural heterogeneity of scaffolds on the behavior of cells and tissues at the site of damage have not been extensively studied. METHODS: Both homogeneous and heterogeneous scaffolds were generated from poly(L-lactic acid) methacrylated in supercritical carbon dioxide medium and were fabricated by two-photon polymerization. The homogeneous scaffolds consist of three layers of cylinders of the same diameter, whereas the heterogeneous (gradient pore sizes) scaffolds contain the middle layer of cylinders of increased diameter, imitating the native structure of spongy bone. To evaluate the osteoinductive properties of both types of scaffold, we performed in vitro and in vivo experiments. Multiphoton microscopy with fluorescence lifetime imaging microscopy was used for determining the metabolic states of MSCs, as a sensitive marker of cell differentiation. The results obtained from this approach were verified using standard markers of osteogenic differentiation and based on data from morphological analysis. RESULTS: The heterogeneous scaffolds showed improved osteoinductive properties, accelerated the metabolic rearrangements associated with osteogenic differentiation, and enhanced the efficiency of bone tissue recovery, thereby providing for both the development of appropriate morphology and mineralization. CONCLUSIONS: The authors suggest that the heterogeneous tissue constructs are a promising tool for the restoration of bone defects. And, furthermore, that our results demonstrate that the use of label-free bioimaging methods can be considered as an effective approach for intravital assessment of the efficiency of differentiation of MSCs on scaffolds.

Optical Biomedical Imaging Reveals Criteria for Violated Liver Regenerative Potential.

To reduce the risk of post-hepatectomy liver failure in patients with hepatic pathologies, it is necessary to develop an approach to express the intraoperative assessment of the liver's regenerative potential. Traditional clinical methods do not enable the prediction of the function of the liver remnant. Modern label-free bioimaging, using multiphoton microscopy in combination with second harmonic generation (SHG) and fluorescence lifetime imaging microscopy (FLIM), can both expand the possibilities for diagnosing liver pathologies and for assessing the regenerative potential of the liver. Using multiphoton and SHG microscopy, we assessed the structural state of liver tissue at different stages of induced steatosis and fibrosis before and after 70% partial hepatectomy in rats. Using FLIM, we also performed a detailed analysis of the metabolic state of the hepatocytes. We were able to determine criteria that can reveal a lack of regenerative potential in violated liver, such as the presence of zones with reduced NAD(P)H autofluorescence signals. Furthermore, for a liver with pathology, there was an absence of the jump in the fluorescence lifetime contributions of the bound form of NADH and NADPH the 3rd day after hepatectomy that is characteristic of normal liver regeneration. Such results are associated with decreased intensity of oxidative phosphorylation and of biosynthetic processes in pathological liver, which is the reason for the impaired liver recovery. This modern approach offers an effective tool that can be successfully translated into the clinic for express, intraoperative assessment of the regenerative potential of the pathological liver of a patient.

Norepinephrine Effects on Uropathogenic Strains Virulence.

The degree of virulence correlates with adhesion, biofilm formation, motility and the capacity to quickly colonize biological surfaces. The virulence of the bacteria that have colonized the urinary tract may be modified by substances dissolved in urine. One such substance is the norepinephrine (NE) hormone, which may be present in human urine, especially in times of stress and under changes in the activity of the renin-angiotensin-aldesterone system. In this work, we study the influence of NE on the biomass, biofilm formation, matrix production, adhesion, motility and metabolism of uropathogenic strains of E. coli and S. aureus. We used Congo red and gentian violet staining for detection of matrix and biomass formation, respectively. The optical density was measured by a multichannel spectrophotometer. The motility of bacterial cells was measured on semi-solid agar at 24 h and 48 h. The metabolic activity was analyzed by MTT assay. It was shown that the metabolic activity of E. coli was stimulated by NE, which led to the increasing synthesis of virulence factors such as biofilm production, adhesion, and motility. At the same time, NE did not activate the S. aureus strain's metabolism and did not change its adhesion and motility. Thus, the virulence activity of uropathogenic E. coli may be modified by NE in urine.

Multiphoton microscopy assessment of the structure and variability changes of dermal connective tissue in vulvar lichen sclerosus: A pilot study.

In this article, we offer a novel classification of progressive changes in the connective tissue of dermis in vulvar lichen sclerosus (VLS) relying on quantitative assessment of the second harmonic generation (SHG) signal received from formalin fixed and deparaffinized tissue sections. We formulate criteria for distinguishing four degrees of VLS development: Initial-Mild-Moderate-Severe. Five quantitative characteristics (length and thickness type I Collagen fibers, Mean SHG signal intensity, Skewness and Coherence SHG signal) are used to describe the sequential degradation of connective tissue (changes in the structure, orientation, shape and density of collagen fibers) up to the formation of specific homogeneous masses. Each of the degrees has a characteristic set of quantitatively expressed features. We focus on the identification and description of early, initial changes of the dermis as the least specific. The results obtained by us and the proposed classification of the degrees of the disease can be used to objectify the dynamics of tissue changes during treatment.

A Collagen Basketweave from the Giant Squid Mantle as a Robust Scaffold for Tissue Engineering.

The growing applications of tissue engineering technologies warrant the search and development of biocompatible materials with an appropriate strength and elastic moduli. Here, we have extensively studied a collagenous membrane (GSCM) separated from the mantle of the Giant squid Dosidicus Gigas in order to test its potential applicability in regenerative medicine. To establish the composition and structure of the studied material, we analyzed the GSCM by a variety of techniques, including amino acid analysis, SDS-PAGE, and FTIR. It has been shown that collagen is a main component of the GSCM. The morphology study by different microscopic techniques from nano- to microscale revealed a peculiar packing of collagen fibers forming laminae oriented at 60-90 degrees in respect to each other, which, in turn, formed layers with the thickness of several microns (a basketweave motif). The macro- and micromechanical studies showed high values of the Young's modulus and tensile strength. No significant cytotoxicity of the studied material was found by the cytotoxicity assay. Thus, the GSCM consists of a reinforced collagen network, has high mechanical characteristics, and is non-toxic, which makes it a good candidate for the creation of a scaffold material for tissue engineering.

Toxicological Analysis of Hepatocytes Using FLIM Technique: In Vitro versus Ex Vivo Models.

The search for new criteria indicating acute or chronic pathological processes resulting from exposure to toxic agents, testing of drugs for potential hepatotoxicity, and fundamental study of the mechanisms of hepatotoxicity at a molecular level still represents a challenging issue that requires the selection of adequate research models and tools. Microfluidic chips (MFCs) offer a promising in vitro model for express analysis and are easy to implement. However, to obtain comprehensive information, more complex models are needed. A fundamentally new label-free approach for studying liver pathology is fluorescence-lifetime imaging microscopy (FLIM). We obtained FLIM data on both the free and bound forms of NAD(P)H, which is associated with different metabolic pathways. In clinical cases, liver pathology resulting from overdoses is most often as a result of acetaminophen (APAP) or alcohol (ethanol). Therefore, we have studied and compared the metabolic state of hepatocytes in various experimental models of APAP and ethanol hepatotoxicity. We have determined the potential diagnostic criteria including the pathologically altered metabolism of the hepatocytes in the early stages of toxic damage, including pronounced changes in the contribution from the bound form of NAD(P)H. In contrast to the MFCs, the changes in the metabolic state of hepatocytes in the ex vivo models are, to a greater extent, associated with compensatory processes. Thus, MFCs in combination with FLIM can be applied as an effective tool set for the express modeling and diagnosis of hepatotoxicity in clinics.

Energy Metabolism and Intracellular pH Alteration in Neural Spheroids Carrying Down Syndrome.

Brain diseases including Down syndrome (DS/TS21) are known to be characterized by changes in cellular metabolism. To adequately assess such metabolic changes during pathological processes and to test drugs, methods are needed that allow monitoring of these changes in real time with minimally invasive effects. Thus, the aim of our work was to study the metabolic status and intracellular pH of spheroids carrying DS using fluorescence microscopy and FLIM. For metabolic analysis we measured the fluorescence intensities, fluorescence lifetimes and the contributions of the free and bound forms of NAD(P)H. For intracellular pH assay we measured the fluorescence intensities of SypHer-2 and BCECF. Data were processed with SPCImage and Fiji-ImageJ. We demonstrated the predominance of glycolysis in TS21 spheroids compared with normal karyotype (NK) spheroids. Assessment of the intracellular pH indicated a more alkaline intracellular pH in the TS21 spheroids compared to NK spheroids. Using fluorescence imaging, we performed a comprehensive comparative analysis of the metabolism and intracellular pH of TS21 spheroids and showed that fluorescence microscopy and FLIM make it possible to study living cells in 3D models in real time with minimally invasive effects.

Probing Metabolism and Viscosity of Cancer Cells using Fluorescence Lifetime Imaging Microscopy.

Viscosity is an important physical property of a biological membrane, as it is one of the key parameters for the regulation of morphological and physiological state of living cells. Plasma membranes of tumor cells are known to have significant alterations in their composition, structure, and functional characteristics. Along with dysregulated metabolism of glucose and lipids, these specific membrane properties help tumor cells to adapt to the hostile microenvironment and develop resistance to drug therapies. Here, we demonstrate the use of fluorescence lifetime imaging microscopy (FLIM) to sequentially image cellular metabolism and plasma membrane viscosity in live cancer cell culture. Metabolic assessments are performed by detecting fluorescence of endogenous metabolic cofactors, such as reduced nicotinamide adenine dinucleotide NAD(P)H and oxidized flavins. Viscosity is measured using a fluorescent molecular rotor, a synthetic viscosity-sensitive dye, with a strong fluorescence lifetime dependence on the viscosity of the immediate environment. In combination, these techniques enable us to better understand the links between membrane state and metabolic profile of cancer cells and to visualize the changes induced by chemotherapy.

Multiphoton tomography in differentiation of morphological and molecular subtypes of breast cancer: A quantitative analysis.

In this study multiphoton tomography, based on second harmonic generation (SHG), and two-photon-excited fluorescence (TPEF) was used to visualize both the extracellular matrix and tumor cells in different morphological and molecular subtypes of human breast cancer. It was shown, that quantified assessment of the SHG based imaging data has great potential to reveal differences of collagen quantity, organization and uniformity in both low- and highly- aggressive invasive breast cancers. The values of quantity and uniformity of the collagen fibers distribution were significantly higher in low-aggressive breast cancer compared to the highly-aggressive subtypes, while the value representing collagen organization was lower in the former type. Additionally, it was shown, that TPEF detection of elastin fibers and amyloid protein may be used as a biomarker of detection the low-aggressive breast cancer subtype. Thus, TPEF/SHG imaging offers the possibility of becoming a useful tool for the rapid diagnosis of various subtypes of breast cancer during biopsy as well as for the intraoperative determinination of tumor-positive resection margins.

Mapping metabolism of liver tissue using two-photon FLIM.

Although fluorescence lifetime imaging microscopy (FLIM) has been extensively applied to study cellular metabolism in the liver, there is neither an established approach to analyze the data, nor have appropriate protocols been developed to maintain the optical metabolic characteristics in the ex vivo liver tissue sample. Here, we show that a tri-exponential decay fitting model for the fluorescence signal from nicotinamide adenine dinucleotide (NAD(P)H) and the use of ex vivo samples allows the most appropriate processing of the FLIM data. Moreover, we determine the medium that maintains the initial metabolic state of hepatocytes (liver cells), most effectively. Our results should be particularly relevant for the interrogation of liver samples, not only in laboratory research, but also in clinical settings in the future.

In vivo assessment of functional and morphological alterations in tumors under treatment using OCT-angiography combined with OCT-elastography.

Emerging methods of anti-tumor therapies require new approaches to tumor response evaluation, especially enabling label-free diagnostics and in vivo utilization. Here, to assess the tumor early reaction and predict its long-term response, for the first time we apply in combination the recently developed OCT extensions - optical coherence angiography (OCA) and compressional optical coherence elastography (OCE), thus enabling complementary functional/microstructural tumor characterization. We study two vascular-targeted therapies of different types, (1) anti-angiogenic chemotherapy (ChT) and (2) photodynamic therapy (PDT), aimed to indirectly kill tumor cells through blood supply injury. Despite different mechanisms of anti-angiogenic action for ChT and PDT, in both cases OCA demonstrated high sensitivity to blood perfusion cessation. The new method of OCE-based morphological segmentation revealed very similar histological structure alterations. The OCE results showed high correlation with conventional histology in evaluating percentages of necrotic and viable tumor zones. Such possibilities make OCE an attractive tool enabling previously inaccessible in vivo monitoring of individual tumor response to therapies without taking multiple biopsies.

Multiparametric Optical Bioimaging Reveals the Fate of Epoxy Crosslinked Biomeshes in the Mouse Subcutaneous Implantation Model.

Biomeshes based on decellularized bovine pericardium (DBP) are widely used in reconstructive surgery due to their wide availability and the attractive biomechanical properties. However, their efficacy in clinical applications is often affected by the uncontrolled immunogenicity and proteolytic degradation. To address this issue, we present here in vivo multiparametric imaging analysis of epoxy crosslinked DBPs to reveal their fate after implantation. We first analyzed the structure of the crosslinked DBP using scanning electron microscopy and evaluated proteolytic stability and cytotoxicity. Next, using combination of fluorescence and hypoxia imaging, X-ray computed microtomography and histology techniques we studied the fate of DBPs after subcutaneous implantation in animals. Our approach revealed high resistance to biodegradation, gradual remodeling of a surrounding tissue forming the connective tissue capsule and calcification of crosslinked DBPs. These changes were concomitant to the development of hypoxia in the samples within 3 weeks after implantation and subsequent induction of angiogenesis and vascularization. Collectively, presented approach provides new insights on the transplantation of the epoxy crosslinked biomeshes, the risks associated with its applications in soft-tissue reconstruction and can be transferred to studies of other types of implants.

Raster-scan optoacoustic angiography of blood vessel development in colon cancer models.

Raster-scan optoacoustic angiography at 532 nm wavelength with 50 µm lateral resolution at 2 mm diagnostic depth was used for quantitative characterization of neoangiogenesis in colon cancer models. Two tumor models of human colon adenocarcinoma (HT-29) and murine colon carcinoma (CT26) different in their histology and vascularization were compared. Tumors of both origins showed an inhomogeneous distribution of areas with high and low vascularization. Rapidly growing CT26 tumor demonstrated a higher rate of vessel growth from the periphery to the center. Peculiarities of the vascularity of tumor models revealed by optoacoustic imaging were confirmed by fluorescent microscopy with FITC-dextran and morphological analysis. The obtained results may be important for the investigation of tumor development and for improvement of colon cancer treatment strategies.

Can "Indirect" Contact Laser Surgery be Used for Fluorescence-Image Guided Tumor Resections? Preliminary Results.

Ensuring the complete removal of tumor tissue is the main challenge during resection operations. Recently, a technique of "indirect" contact laser surgery has been developed. In this study we assess the possibility of using such surgery for fluorescence image-guided tumor resection. Mouse colon adenocarcinoma CT-26 cells stably expressing the fluorescent protein mKate-2 was used as the tumor model. Resections of the tumor nodes were performed with either a scalpel blade, a laser scalpel with a bare tip, or a laser scalpel with a strongly absorbing coating on the fiber tip. Tumor-positive resection margins were detected using an IVIS Spectrum fluorescence imaging system. After tumor resection with the scalpel blade over half of the animals needed one additional resection to remove residual tumor cells. Animals in this group showed tumor recurrence within 7 days. Fluorescence imaging of the tumor bed, performed after resection to assess the presence of tumor cell clusters, was sufficiently effective only with a bloodless resection. The laser scalpels both with the bare tip and with the strongly absorbing coating on the tip provided such bloodless tumor resection in contact mode. Fewer animals required additional resections when the bare tipped scalpel was used and this also resulted in a reduction in tumor recurrence. After resections were carried out with the laser scalpel with the strongly absorbing coating on the tip, fluorescence was detected in the operative field and this led to undertaking additional resections, although subsequent investigation suggested that this was "false" fluorescence, resulting from the effects of the scalpel rather than the presence of residual tumor cells. The method of laser resection with a strongly absorbing coating on the tip therefore did not appear to demonstrate definite advantages over laser resection with a bare tip when removing tumors.