Rajesh N. Raman

Real-time assessment of in vivo renal ischemia using laser autofluorescence imaging

Potentially transplantable kidneys experience warm ischemia, and this injury is difficult to quantify. We investigate optical spectroscopic methods for evaluating, in real time, warm ischemic kidney injury and reperfusion. Vascular pedicles of rat kidneys are clamped unilaterally for 18 or 85 min, followed by 18 or 35 min of reperfusion, respectively. Contralateral, uninjured kidneys serve as controls. Autofluorescence and cross-polarized light scattering images are acquired every 15 s using 335-nm laser excitation (autofluorescence) and 650+/-20-nm linearly polarized illumination (light scattering). We analyze changes of injured-to-normal kidney autofluorescence intensity ratios during ischemia and reperfusion phases. The effect of excitation with 260 nm is also explored. Average injured-to-normal intensity ratios under 335-nm excitation decrease from 1.0 to 0.78 at 18 min of ischemia, with a return to baseline during 18 min of reperfusion. However, during 85 min of warm ischemia, average intensity ratios level off at 0.65 after 50 min, with no significant change during 35 min of reperfusion. 260-nm excitation results in no autofluorescence changes with ischemia. Cross-polarized light scattering images at 650 nm suggest that changes in hemoglobin absorption are not related to observed temporal behavior of the autofluorescence signal. Real-time detection of kidney tissue changes associated with warm ischemia and reperfusion using laser spectroscopy is feasible. Normalizing autofluorescence changes under 335 nm using the autofluorescence measured under 260-nm excitation may eliminate the need for a control kidney.

A non-contact method and instrumentation to monitor renal ischemia and reperfusion with optical spectroscopy

The potential of NADH autofluorescence as an in vivo intrinsic optical signature to monitor tissue metabolism is well recognized and supported by experimental results mainly in animal models. In this work, we propose a non-contact implementation of this method using large area excitation and employing a normalization method to account for non-metabolic signal changes. Proof of principle in vivo experiments were carried out using an autofluorescence imaging experimental system and a rat renal ischemia model. A hand-held fiber-optic probe was utilized to test the ability of the signal normalization method to address operational conditions associated with the translation of this method to a clinical setting. Preliminary pre-clinical in vivo test of the probe system was carried out using the same rat model.

Quantification of in vivo autofluorescence dynamics during renal ischemia and reperfusion under 355 nm excitation

We explore a method to quantitatively assess the ability of in vivo autofluorescence as a means to quantify the progression of longer periods of renal warm ischemia and reperfusion in a rat model. The method employs in vivo monitoring of tissue autofluorescence arising mainly from NADH as a means to probe the organs function and response to reperfusion. Clinically relevant conditions are employed that include exposure of the kidney to ischemia on the order of tens of minutes to hours. The temporal profile during the reperfusion phase of the autofluorescence intensity averaged over an area as large as possible was modeled as the product of two independent exponential functions. Time constants were extracted from fits to the experimental data and their average values were found to increase with injury time.

Factors influencing rat survival in a warm renal ischemia model: time to adapt the protocols.

INTRODUCTION Survival in warm renal ischemia models is not only dependent on the treatment or surgical technique being evaluated, but also on factors inherent to the model itself. Use of rats of various strains in previous studies makes interstudy comparison difficult when trying to design an appropriate model control that would yield intermediate survival. In this study, impact of rat strain on survival after prolonged warm renal ischemia in the setting of delivery-controlled inhalational anesthesia was evaluated. MATERIALS AND METHODS Under general delivery-controlled inhalation anesthesia with isoflurane, Dahl salt-sensitive, Wistar-Furth, Sprague-Dawley, and spontaneously hypertensive rats (n = 66 rats) were subjected to 150 minutes of unilateral renal warm ischemia time, subsequent reperfusion, and contralateral nephrectomy. Animals were followed up for 1 month, after which survivors were euthanized and morphologic changes in kidneys were scored. RESULTS Thirty-day survival was: Dahl salt sensitive, 78%; Wistar-Furth, 67%; Sprague-Dawley, 55%; and spontaneously hypertensive rats, 0% (P < .0001). Histologic acute injury scores were higher for non-survivors versus 30-day survivors (P < .0001). CONCLUSION Our data strongly suggest that rat strain is a major factor influencing survival and that strain and warm ischemia time selections must be considered together when designing a model control yielding intermediate survival. Further study is warranted in order to compare the effect of delivery-controlled inhalational versus historical anesthesia methods on animal survival.

Evaluation of the contribution of the renal capsule and cortex to kidney autofluorescence intensity under ultraviolet excitation

The use of reduced nicotinamide adenine dinucleotide (NADH) fluorescence to gain metabolic information on kidneys in response to an alteration in oxygen availability has previously been experimentally demonstrated, but signal quantification has not, to date, been addressed. In this work the relative contribution to rat kidney autofluorescence of the capsule versus cortex under ultraviolet excitation is determined from experimental results obtained using autofluorescence microscopy and a suitable mathematical model. The results allow for a quantitative assessment of the relative contribution of the signal originating in the metabolically active cortex as a function of capsule thickness for different wavelengths.

In vivo quantification of autofluorescence dynamics during renal ischemia and reperfusion under dual UV excitation

We explore an optical spectroscopy approach to monitor the progression of ischemia and reperfusion in situ using a rat model. The system utilizes the sensitivity of NADH emission to changes in cell metabolism during ischemia and reperfusion. In addition, the emission from tryptophan is employed as a normalization against changes in other optical properties of the tissue. Ischemia was induced in one kidney followed by at least 60 minutes of reperfusion. During both phases, autofluorescence images of the exposed surfaces of both the ischemic kidney and the normal (control) kidney were acquired and the respective average emission intensities were determined. Preliminary results indicate that the kinetics of the ratio of the emissions under these two excitations is related to the injury time.

Predictive assessment of kidney functional recovery following ischemic injury using optical spectroscopy

Abstract. Functional changes in rat kidneys during the induced ischemic injury and recovery phases were explored using multimodal autofluorescence and light scattering imaging. The aim is to evaluate the use of noncontact optical signatures for rapid assessment of tissue function and viability. Specifically, autofluorescence images were acquired in vivo under 355, 325, and 266 nm illumination while light scattering images were collected at the excitation wavelengths as well as using relatively narrowband light centered at 500 nm. The images were simultaneously recorded using a multimodal optical imaging system. The signals were analyzed to obtain time constants, which were correlated to kidney dysfunction as determined by a subsequent survival study and histopathological analysis. Analysis of both the light scattering and autofluorescence images suggests that changes in tissue microstructure, fluorophore emission, and blood absorption spectral characteristics, coupled with vascular response, contribute to the behavior of the observed signal, which may be used to obtain tissue functional information and offer the ability to predict posttransplant kidney function.

Real-time imaging of tissue microstructures using intrinsic optical signatures

We explore imaging of tissue microstructures using autofluorescence and light scattering methods implemented through a hyperspectral microscope design. This system utilizes long working distance objectives that enable off-axis illumination of tissue thereby allowing for excitation at any optical wavelength without requiring change of optical elements within the microscope. Spectral and polarization elements are easily and rapidly incorporated to take advantage of spectral variations of spectroscopic optical signatures for enhanced contrast. The collection efficiency has been maximized such that image acquisition may be acquired within very short exposure times, a key feature for transferring this technology to a clinical setting. Preliminary studies using human and animal tissues demonstrate the feasibility of this approach for real-time imaging of intact tissues as they would appear in the operating room.

Multi-Color Autofluorescence and Scattering Spectroscopy Provides Rapid Assessment of Kidney Function Following Ischemic Injury

A major source of kidneys for transplant comes from deceased donors whose tissues have suffered an unknown amount of warm ischemia prior to retrieval, with no quantitative means to assess function before transplant. Toward addressing this need, non-contact monitoring of optical signatures in rat kidneys was performed in vivo during ischemia and reperfusion. Kidney autofluorescence images were captured under ultraviolet illumination (355 nm, 325 nm, and 266 nm) in order to provide information on related metabolic and non-metabolic response. In addition, light scattering images under 355 nm, 325 nm, and 266 nm, 500 nm illumination were monitored to report on changes in kidney optical properties giving rise to the observed autofluorescence signals during these processes. During reperfusion, various signal ratios were generated from the recorded signals and then parametrized. Time-dependent parameters derived from the ratio of autofluorescence under 355 nm excitation to that under 266 nm excitation, as well as from 500 nm scattered signal, were found capable of discriminating dysfunctional kidneys from those that were functional (p < 0.01) within hours of reperfusion. Kidney dysfunction was confirmed by subsequent survival study and histology following autopsy up to a week later. Physiologic changes potentially giving rise to the observed signals, including those in cellular metabolism, vascular response, tissue microstructure, and microenvironment chemistry, are discussed.

Autofluorescence dynamics during reperfusion following long-term renal ischemia in a rat model

Optical properties of near-surface kidney tissue were monitored in order to assess response during reperfusion to long (20 minutes) versus prolonged (150 minutes) ischemia in an in vivo rat model. Specifically, autofluorescence images of the exposed surfaces of both the normal and the ischemic kidneys were acquired during both injury and reperfusion alternately under 355 nm and 266 nm excitations. The temporal profile of the emission of the injured kidney during the reperfusion phase under 355 nm excitation was normalized to that under 266 nm as a means to account for changes in tissue optical properties independent of ischemia as well as changes in the illumination/collection geometrical parameters in future clinical implementation of this technique using a hand-held probe. The scattered excitation light signal was also evaluated as a reference signal and found to be inadequate. Characteristic time constants were extracted using a fit to a relaxation model and found to have larger mean values following 150 minutes of injury. The mean values were then compared with the outcome of a chronic survival study where the control kidney had been removed. Rat kidneys exhibiting longer time constants were much more likely to fail. This may lead to a method to assess kidney viability and predict its ability to recover in the initial period following transplantation or resuscitation.