Optical coherence tomography and computer-aided diagnosis of a murine model of chronic kidney disease.

Chronic kidney disease (CKD) is characterized by a progressive loss of renal function over time. Histopathological analysis of the condition of glomeruli and the proximal convolutional tubules over time can provide valuable insights into the progression of CKD. Optical coherence tomography (OCT) is a technology that can analyze the microscopic structures of a kidney in a nondestructive manner. Recently, we have shown that OCT can provide real-time imaging of kidney microstructures in vivo without administering exogenous contrast agents. A murine model of CKD induced by intravenous Adriamycin (ADR) injection is evaluated by OCT. OCT images of the rat kidneys have been captured every week up to eight weeks. Tubular diameter and hypertrophic tubule population of the kidneys at multiple time points after ADR injection have been evaluated through a fully automated computer-vision system. Results revealed that mean tubular diameter and hypertrophic tubule population increase with time in post-ADR injection period. The results suggest that OCT images of the kidney contain abundant information about kidney histopathology. Fully automated computer-aided diagnosis based on OCT has the potential for clinical evaluation of CKD conditions.

Multi-modality Optical Imaging of Rat Kidney Dysfunction: In Vivo Response to Various Ischemia Times.

We observed in vivo kidney dysfunction with various ischemia times at 30, 75, 90, and 120 min using multi-modality optical imaging: optical coherence tomography (OCT), Doppler OCT (DOCT), and two-photon microscopy (TPM). We imaged the renal tubule lumens and glomerulus at several areas of each kidney before, during, and after ischemia of 5-month-old female Munich-Wistar rats. For animals with 30 and 75 min ischemia times, we observed that all areas were recovered after ischemia, that tubule lumens were re-opened and the blood flow of the glomerulus was re-established. For animals with 90 and 120 min ischemia times, we observed unrecovered areas, and that tubule lumens remained close after ischemia. TPM imaging verified the results of OCT and provided higher resolution images than OCT to visualize renal tubule lumens and glomerulus blood flow at the cellular level.

Optical Coherence Tomography of the Aging Kidney.

OBJECTIVES: The aging kidney exhibits a progressive decline in renal function with characteristic histopathologic changes and is a risk factor for renal transplant. However, the degree to which the kidney exhibits this decline depends on several factors that vary from one individual to the next. Optical coherence tomography is an evolving noninvasive imaging technology that has recently been used to evaluate acute tubular necrosis of living-human donor kidneys before their transplant. With the increasing use of kidneys from older individuals, it is important to determine whether optical coherence tomography also can distinguish the histopathology associated with aging. MATERIALS AND METHODS: In this investigation, we used Munich-Wistar rats to evaluate the ability of optical coherence tomography to detect histopathologic changes associated with aging. Optical coherence tomography observations were correlated with renal function and conventional light microscopic evaluation of these same kidneys. RESULTS: With the onset of severe proteinuria at 10 to 12 months of age, optical coherence tomography revealed tubular necrosis/atrophy, interstitial fibrosis, tubular dilation, and glomerulosclerosis. With a further deterioration in kidney function at 16 to 18 months of age (as indicated by rising creatinine levels), optical coherence tomography revealed more extensive interstitial fibrosis and tubular atrophy, increased tubular dilation with cyst formation and more sclerotic glomeruli. CONCLUSIONS: The foregoing observations suggest that optical coherence tomography can be used to detect the histopathology of progressive nephropathy associated with aging.

Low-fluence rate, long duration photodynamic therapy in glioma mouse model using organic light emitting diode (OLED).

BACKGROUND: The treatment of gliomas poses significant clinical challenges due to resistance to chemo and radiation therapy, and treatment side effects. Metronomic photodynamic therapy (mPDT), which involves long treatment time with low fluence rate and multiple or continuous photosensitizer administrations, has potential in treating gliomas without threatening the quality of life and has been demonstrated in rats and rabbits. mPDT in small animals such as mouse is not yet shown due to lack of lightweight illumination device for long periods of time. METHODS: We presented low fluence rate (3mW/cm(2)) and long duration (3.7h) PDT treatment in a nude mouse model of human glioblastoma by using organic light emitting diode (OLED) with single dose of 5-aminolevulinic acid (ALA) administration as photosensitizer. Tumor volume was measured using bioluminescent imaging and the animal survival time was recorded. Additionally, we have performed limited PDT dosimetric measurements of PpIX fluorescence, tumor oxygenation and hemoglobin concentration in 3 PDT mice. RESULTS: For animals with similar pre- and immediate post-light tumor volume, the averaged total survival time of PDT mice is 40.5±9.2 days that are significantly longer than the control mice (26.0±2.0 days). The post-light survival time of PDT mice is 14.3±5.9 days that are marginally longer than the control group (8.0±0.0 days). In the dosimetric measurement, good maintenance of PpIX fluorescence in one PDT mouse has relatively improved survival time, compared with the other two PDT mice (i.e., 24 days versus 16 and 17 days). CONCLUSIONS: This pilot study demonstrated the feasibility of low-fluence rate and long treatment time of ALA-PDT using OLED without anesthetization of animals. The response of PDT treated animals with similar pre- and post-light tumor volume is encouraging to show a longer survival time than the controls. The dosimetric indices such as photosensitizer fluorescence and tissue oxygenation would help understand the possible treatment barriers for further improvement of treatment plans.

Correlation of NADH fluorescence lifetime and oxidative phosphorylation metabolism in the osteogenic differentiation of human mesenchymal stem cell.

Reduced nicotinamide adenine dinucleotide (NADH) fluorescence lifetime has been broadly used as a metabolic indicator for stem cell imaging. However, the direct relationship between NADH fluorescence lifetime and metabolic pathway and activity remains to be clarified. In this study, we measured the NADH fluorescence lifetime of human mesenchymal stem cells (hMSCs) as well as the metabolic indictors, such as adenosine triphosphate (ATP) level, oxygen consumption, and lactate release, up to 4 weeks under normal osteogenic differentiation and oxidative phosphorylation-attenuated/inhibited differentiation by oligomycin A (OA) treatment. NADH fluorescence lifetime was positively correlated with oxygen consumption and ATP level during energy transformation from glycolysis to oxidative phosphorylation. Under OA treatment, oxidative phosphorylation was attenuated/inhibited (i.e., oxygen consumption remained the same as controls or lower), cells showed attenuated differentiation under glycolysis, and NADH fluorescence lifetime change was not detected. Increased expression of the overall complex proteins was observed in addition to Complex I. We suggested special caution needs to be exercised while interpreting NADH fluorescence lifetime signal in terms of stemcell differentiation.

Mannitol infusion within 15 min of cross-clamp improves living donor kidney preservation.

BACKGROUND: Optical coherence tomography (OCT) revealed that cells lining proximal convoluted tubules of living donor kidneys (LDKs) procured by laparoscopic procedures were very swollen in response to the brief period of ischemia experienced between the time of arterial vessel clamping and flushing the excised kidney with cold preservation solution. Damage to the tubules as a result of this cell swelling resulted in varying degrees of acute tubular necrosis (ATN) that slowed the recovery of the donor kidneys during the first 2 weeks after their transplantation. METHODS: To prevent this cell damage during LDK procurement, we changed the protocol for intravenous administration of mannitol (i.e., 12.5 or 25 g) to the donor. Specifically, we reduced the time of mannitol administration from 30 to 15 min or less before clamping the renal artery. RESULT: OCT revealed that this change in the timing of mannitol administration protected the human donor proximal tubules from normothermic-induced cell swelling. An evaluation of posttransplant recovery of renal function showed that patients treated with this modified protocol returned to normal renal function significantly faster than those treated with mannitol 30 min or more before clamping the renal artery. CONCLUSION: Because slow graft recovery in the first weeks after transplantation represents a risk factor for long-term graft function and survival, we believe that this change in pretreatment protocol will improve renal transplants in patients receiving LDK.

Clinical applications of optical coherence tomography in urology.

Since optical coherence tomography (OCT) was first demonstrated in 1991, it has advanced significantly in technical aspects such as imaging speed and resolution, and has been clinically demonstrated in a diverse set of medical and surgical applications, including ophthalmology, cardiology, gastroenterology, dermatology, oncology, among others. This work reviews current clinical applications in urology, particularly in bladder, urether, and kidney. Clinical applications in bladder and urether mainly focus on cancer detection and staging based on tissue morphology, image contrast, and OCT backscattering. The application in kidney includes kidney cancer detection based on OCT backscattering attenuation and non-destructive evaluation of transplant kidney viability or acute tubular necrosis based on both tissue morphology from OCT images and function from Doppler OCT (DOCT) images. OCT holds the promise to positively impact the future clinical practices in urology.

Quantitative physiology and immunohistochemistry of oral lesions.

Angiogenesis and hypoxia are reported to correlate with tumor aggressiveness. In this study, we investigated the potential of optically measured total hemoglobin concentration (THC) and blood oxygen saturation (StO2) as a quantitative measure of angiogenesis and hypoxia in oral lesions with an immunohistochemical comparison. 12 normal subjects and 40 oral patients (22 oral squamous cell carcinoma (SCC), 18 benign/premalignant lesions including 11 verrucous hyperplasia (VH) and 7 hyperkeratosis/parakeratosis (HK)) were studied. The results showed that the THC measurement was consistent with vascular endothelial growth factor (VEGF) and microvessel staining in the stromal area, but StO2 was not associated with HIF-1α. We observed inflammation induced neovascular formation in the stromal area of VH and HK that were likely attributed to higher-than-control THC and StO2 and resulted in no difference in optical measurements between all lesions. However, we found that in majority of SCC, the ratio of THC and StO2 levels between lesions and the surrounding tissues provide potential distinguishing characteristics from VH, which are not visually differentiable from SCC, with a sensitivity, specificity, and accuracy of 91%, 68%, and 76%, respectively.

NADH fluorescence as a photobiological metric in 5-aminolevlinic acid (ALA)-photodynamic therapy.

Photodynamic therapy (PDT) dosimetry is complex as many factors are involved and varied interdependently. Monitoring the biological consequence of PDT such as cell death is the most direct approach to assess treatment efficacy. In this study, we performed 5-aminolevlinic acid (ALA)-PDT in vitro to induce different cell death modes (i.e., slight cell cytotoxicity, apoptosis, and necrosis) by a fixed fluence rate of 10 mW/cm(2) and varied fluences (1, 2, and 6 J/cm(2)). Time course measurements of cell viability, caspase-3 activity, and DNA fragmentation were conducted to determine the mode of cell death. We demonstrated that NADH fluorescence lifetime together with NADH fluorescence intensity permit us to detect apoptosis and differentiate it from necrosis. This feature will be unique in the use of optimizing apoptosis-favored treatments such as metronomic PDT.

Reduced nicotinamide adenine dinucleotide fluorescence lifetime detected poly(adenosine-5'-diphosphate-ribose) polymerase-1-mediated cell death and therapeutic effect of pyruvate.

Noninvasive detection of cell death has the potential for definitive diagnosis and monitoring treatment outcomes in real time. Reduced nicotinamide adenine dinucleotide (NADH) fluorescence intensity has long been used as a noninvasive optical probe of metabolic states. NADH fluorescence lifetime has recently been studied for its potential as an alternative optical probe of cellular metabolic states and cell death. In this study, we investigated the potential using NADH fluorescence intensity and/or lifetime to detect poly(adenosine-5'-diphosphate-ribose) polymerase-1 (PARP-1)-mediated cell death in HeLa cells. We also examined if NADH signals respond to treatment by pyruvate. The mechanism of PARP-1-mediated cell death has been well studied that extensive PARP-1 activation leads to cytosolic nicotinamide adenine dinucleotide depletion resulting in glycolytic inhibition, mitochondrial failure, and death. Pyruvate could restore electron transport chain to prevent energy failure and death. Our results show that NADH fluorescence lifetime, not intensity, responded to PARP-1-mediated cell death and the rescue effect of pyruvate. This lifetime change of NADH fluorescence happened before the collapse of mitochondrial membrane potential and mitochondrial uncoupling. Together with our previous findings in staurosporine-induced cell death, we suggest that NADH fluorescence lifetime increase during cell death is mainly due to increased protein-protein interactions but not the intracellular NADH content.

Increase of reduced nicotinamide adenine dinucleotide fluorescence lifetime precedes mitochondrial dysfunction in staurosporine-induced apoptosis of HeLa cells.

In vivo noninvasive detection of apoptosis represents a new tool that may yield a more definite diagnosis, a more accurate prognosis, and help improve therapies for human diseases. The intrinsic fluorescence of reduced nicotinamide adenine dinucleotide (NADH) may be a potential optical biomarker for the apoptosis detection because NADH is involved in the respiration for the mitochondrial membrane potential (ΔΨ) formation and adenosine-5'-triphosphate (ATP) synthesis, and the depletion of ΔΨ and ATP level is the hallmark of apoptosis. We have previously observed the NADH fluorescence lifetime change is associated with staurosporine (STS)-induced mitochondria-mediated apoptosis. However, its relationship with mitochondrial functions such as ΔΨ, ATP, and oxygen consumption rate is not clear. In this study, we investigated this relationship. Our results indicate that the NADH fluorescence lifetime increased when ΔΨ and ATP levels were equal to or higher than their values of controls and decreased before the depletion of ΔΨ and ATP, and the oxygen consumption rate did not change. These findings suggest that the increased NADH fluorescence lifetime in STS-induced cell death occurred before the depletion of ΔΨ and ATP and activation of caspase 3, and was not simply caused by cellular metabolic change. Furthermore, the NADH fluorescence lifetime change is associated with the pace of apoptosis.

Increasing damage to tumor blood vessels during motexafin lutetium-PDT through use of low fluence rate.

Photodynamic therapy (PDT) with low light fluence rate has rarely been studied in protocols that use short drug-light intervals and thus deliver illumination while plasma concentrations of photosensitizer are high, creating a prominent vascular response. In this study, the effects of light fluence rate on PDT response were investigated using motexafin lutetium (10 mg/kg) in combination with 730 nm light and a 180-min drug-light interval. At 180 min, the plasma level of photosensitizer was 5.7 ng/microl compared to 3.1 ng/mg in RIF tumor, and PDT-mediated vascular effects were confirmed by a spasmodic decrease in blood flow during illumination. Light delivery at 25 mW/cm(2) significantly improved long-term tumor responses over that at 75 mW/cm(2). This effect could not be attributed to oxygen conservation at low fluence rate, because 25 mW/cm(2) PDT provided little benefit to tumor hemoglobin oxygen saturation. However, 25 mW/cm(2) PDT did prolong the duration of ischemic insult during illumination and was correspondingly associated with greater decreases in perfusion immediately after PDT, followed by smaller increases in total hemoglobin concentration in the hours after PDT. Increases in blood volume suggest blood pooling from suboptimal vascular damage; thus the smaller increases after 25 mW/cm(2) PDT provide evidence of more widespread vascular damage, which was accompanied by greater decreases in clonogenic survival. Further study of low fluence rate as a means to improve responses to PDT under conditions designed to predominantly damage vasculature is warranted.

Fluence rate-dependent intratumor heterogeneity in physiologic and cytotoxic responses to Photofrin photodynamic therapy.

Photodynamic therapy (PDT) can lead to the creation of heterogeneous, response-limiting hypoxia during illumination, which may be controlled in part through illumination fluence rate. In the present report we consider (1) regional differences in hypoxia, vascular response, and cell kill as a function of tumor depth and (2) the role of fluence rate as a mediator of depth-dependent regional intratumor heterogeneity. Intradermal RIF murine tumors were treated with Photofrin PDT using surface illumination at an irradiance of 75 or 38 mW cm(-2). Regional heterogeneity in tumor response was examined through comparison of effects in the surface vs. base of tumors, i.e. along a plane parallel to the skin surface and perpendicular to the incident illumination. 75 mW cm(-2) PDT created significantly greater hypoxia in tumor bases relative to their surfaces. Increased hypoxia in the tumor base could not be attributed to regional differences in Photofrin concentration nor effects of fluence rate distribution on photochemical oxygen consumption, but significant depth-dependent heterogeneity in vascular responses and cytotoxic response were detected. At a lower fluence rate of 38 mW cm(-2), no detectable regional differences in hypoxia or cytotoxic responses were apparent, and heterogeneity in vascular response was significantly less than that during 75 mW cm(-2) PDT. This research suggests that the benefits of low-fluence-rate PDT are mediated in part by a reduction in intratumor heterogeneity in hypoxic, vascular and cytotoxic responses.

Reduced nicotinamide adenine dinucleotide (NADH) fluorescence for the detection of cell death.

NADH/FAD fluorescence spectroscopy/imaging is an extremely useful tool to probe cellular metabolism and has been applied in the clinic such as early cancer detection. Recently, the potential of using NADH/FAD fluorescence as a biomarker to detect cell death has been investigated for development of cancer treatments with higher efficacy. This review aims to provide the updated information in cell death detection using the NADH/FAD fluorescence spectroscopy and imaging based on measurement of the intensity or lifetime of NADH or FAD fluorescence. The response of NADH fluorescence lifetime to metabolic perturbation, hypoxic environment, and anaerobic glycolysis (e.g., in precancerous tissues and stem cells) is also reviewed to discuss the nature and implications of the lifetime change of NADH fluorescence. Further studies are required to understand the actual site and mechanism of NADH binding of a specific death pathway for future successful in vivo detection of cell death using the NADH fluorescence lifetime.

Diffuse reflectance spectroscopy detects increased hemoglobin concentration and decreased oxygenation during colon carcinogenesis from normal to malignant tumors.

Blood content and tumor oxygen level are important biomarkers and prognostic indicators in patients with colorectal cancer (CRC). However, noninvasive measurements of both quantities in human colon are limited. In this study, we extracted the total hemoglobin concentration (THC) and oxygen saturation (StO(2)) of normal, premalignant, and malignant colonic tissues in 27 patients using a diffuse reflectance instrument and algorithms based on the diffusion equation. The mean+/-standard error of THC and StO(2) from all normal sites (n=26) is 93.4+/-17.1microM and 67.2+/-3.7%, respectively. THC increased to 136.9+/-23.8microM and 153.8+/-38.6microM and StO(2) decreased to 51.3+/-7.0% and 26.4+/-6.1% for premalignant and malignant tissues, respectively. The disease-to-normal THC ratios are 3.2+/-1.1 and 4.4+/-1.9 and the disease-to-normal StO(2) ratios are 0.7+/-0.1 and 0.5+/-0.1 for pr alignant and malignant tissues, respectively. These results demonstrate the feasibility of a robust optical method to assess colon THC and StO2 at all stages of carcinogenesis in vivo so that the angiogenesis and hypoxia of the disease and the therapeutic role can be studied in CRC patients.

Reduced nicotinamide adenine dinucleotide fluorescence lifetime separates human mesenchymal stem cells from differentiated progenies.

The metabolic changes of human mesenchymal stem cells (hMSCs) during osteogenic differentiation were accessed by reduced nicotinamide adenine dinucleotide (NADH) fluorescence lifetime. An increase in mean fluorescence lifetime and decrease in the ratio between free NADH and protein-bound NADH correlated with our previously reported increase in the adenosine triphosphate (ATP) level of hMSCs during differentiation. These findings suggest that NADH fluorescence lifetime may serve as a new optical biomarker for noninvasive selection of stem cells from differentiated progenies.

Differentiation of apoptosis from necrosis by dynamic changes of reduced nicotinamide adenine dinucleotide fluorescence lifetime in live cells.

Direct monitoring of cell death (i.e., apoptosis and necrosis) during or shortly after treatment is desirable in all cancer therapies to determine the outcome. Further differentiation of apoptosis from necrosis is crucial to optimize apoptosis-favored treatment protocols. We investigated the potential modality of using tissue intrinsic fluorescence chromophore, reduced nicotinamide adenine dinucleotide (NADH), for cell death detection. We imaged the fluorescence lifetime changes of NADH before and after staurosporine (STS)-induced mitochondria-mediated apoptosis and hydrogen peroxide (H2O2)-induced necrosis, respectively, using two-photon fluorescence lifetime imaging in live HeLa cells and 143B osteosarcoma. Time-lapsed lifetime images were acquired at the same site of cells. In untreated cells, the average lifetime of NADH fluorescence was approximately 1.3 ns. The NADH average fluorescence lifetime increased to approximately 3.5 ns within 15 min after 1 microM STS treatment and gradually decreased thereafter. The NADH fluorescence intensity increased within 15 min. In contrast, no significant dynamic lifetime change was found in cells treated with 1 mM H2O2. Our findings suggest that monitoring the NADH fluorescence lifetime may be a valuable noninvasive tool to detect apoptosis and distinguish apoptosis from necrosis for the optimization of apoptosis-favored treatment protocols and other clinical applications.

Effect of photosensitizer dose on fluence rate responses to photodynamic therapy.

Photodynamic therapy (PDT) regimens that conserve tumor oxygenation are typically more efficacious, but require longer treatment times. This makes them clinically unfavorable. In this report, the inverse pairing of fluence rate and photosensitizer dose is investigated as a means of controlling oxygen depletion and benefiting therapeutic response to PDT under conditions of constant treatment time. Studies were performed for Photofrin-PDT of radiation-induced fibrosarcoma tumors over fluence rate and drug dose ranges of 25-225 mW cm(-2) and 2.5-10 mg kg(-1), respectively, for 30 min of treatment. Tumor response was similar among all inverse regimens tested, and, in general, tumor hemoglobin oxygen saturation (SO2) was well conserved during PDT, although the highest fluence rate regimen (225 mWx2.5 mg) did lead to a modest but significant reduction in SO2. Regardless, significant direct tumor cell kill (>1 log) was detected during 225 mWx2.5 mg PDT, and minimal normal tissue toxicity was found. PDT effect on tumor oxygenation was highly associated with tumor response at 225 mWx2.5 mg, as well as in all other regimens tested. These data suggest that high fluence rate PDT can be carried out under oxygen-conserving, efficacious conditions at low photosensitizer dose. Clinical confirmation and application of these results will be possible through use of minimally invasive oxygen and photosensitizer monitoring technologies, which are currently under development.

Quantitative comparison of tissue oxygen and motexafin lutetium uptake by ex vivo and noninvasive in vivo techniques in patients with intraperitoneal carcinomatosis.

Near-infrared diffuse reflectance spectroscopy (DRS) has been used to noninvasively monitor optical properties during photodynamic therapy (PDT). This technique has been extensively validated in tissue phantoms; however, validation in patients has been limited. This pilot study compares blood oxygenation and photosensitizer tissue uptake measured by multiwavelength DRS with ex vivo assays of the hypoxia marker, 2-(2-nitroimida-zol-1[H]-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide (EF5), and the photosensitizer (motexafin lutetium, MLu) from tissues at the same tumor site of three tumors in two patients with intra-abdominal cancers. Similar in vivo and ex vivo measurements of MLu concentration are carried out in murine radiation-induced fibrosarcoma (RIF) tumors (n=9). The selection of optimal DRS wavelength range and source-detector separations is discussed and implemented, and the association between in vivo and ex vivo measurements is examined. The results demonstrate a negative correlation between blood oxygen saturation (StO(2)) and EF5 binding, consistent with published relationships between EF5 binding and electrode measured pO(2), and between electrode measured pO(2) and StO(2). A tight correspondence is observed between in vivo DRS and ex vivo measured MLu concentration in the RIF tumors; similar data are positively correlated in the human intraperitoneal tumors. These results further demonstrate the potential of in vivo DRS measurements in clinical PDT.

Hemodynamic responses to antivascular therapy and ionizing radiation assessed by diffuse optical spectroscopies.

Diffuse optical methods were used to monitor two different therapies in K1735 malignant mouse melanoma tumor models: anti-vascular therapy and radiation therapy. Anti-vascular therapy induced acute variation in hemodynamic parameters within an hour, and radiation therapy induced longitudinal changes within 2 weeks. During anti-vascular therapy, the drug Combretastatin A-4 3-O-Phosphate (CA4P, 2.5 mg/200 mul PBS/mouse) significantly decreased tissue blood flow (65%) and blood oxygenation (38%) one hour after injection. In the longitudinal study, single-fraction ionizing radiation (12 Gy x 1) induced significant reduction of tissue blood flow (36%) and blood oxygenation (24%) 14 days after radiation. The results correlated well with contrast enhanced ultrasound, tumor histology, and a nitroimidazole hypoxia marker (EF5). The research provides further evidence that noninvasive diffuse optical spectroscopies can be useful tools for monitoring cancer therapy in vivo.