Method for three-dimensional visualization of neurodegeneration in cupric-silver stained serial rat brain slices.

The spatial distribution of neurodegeneration in brains is difficult to visualize when working from 2-D serial slices. In studies where repetitive operant behavior measurements are made over several weeks following organic solvent exposure, definitive evidence of degeneration in brain structures may have been significantly cleared by the time the tissue is prepared histologically. The only remaining evidence that injury has occurred may be nothing more than neuronal and cellular debris. By choosing stains that are specific for this type of residual and/or indicative of specific pathology, a 3-D representation of the spatial distribution of the neuronal and cellular debris fields within the organ can be highlighted and displayed. We present a method for visualizing the spatial distribution of neuronal degeneration that can result from low-level organic solvent exposure scenarios. A cupric-silver stain highly specific for neuronal degeneration is used to identify neuronal debris fields in 73 serial slices of brains of rodents that were exposed to toluene vapors. Serial brain sections stained with cupric-silver are scanned at 600 dpi using a gray-scale protocol. Using commercially available software, scans are assembled into 3-D images showing both topographical and internal anatomical details. The reassembled images are further processed into stereo pairs. Gray-scale scans are compared to the original sections to establish gray-scale ranges for healthy and damaged tissue and artifact staining.

Perfusion preservation of rodent kidneys in a portable preservation device based on fluidics technology.

BACKGROUND: Technology that can implement the basic requirements for successful organ preservation in a portable configuration has yet to be realized. METHODS: This work evaluates kidney preservation in a new class of portable organ preservation technology based on fluidics principles. During hypothermic pulsatile perfusion preservation (HPPP), oxygen consumption, renal vascular resistance (RVR), pH, pCO2, and perfusion pressure were measured. After 24 hr of preservation, perfusate distribution was assessed, and oxygen consumption, RVR, and glomerular filtration rate (GFR) were compared in perfused, statically stored, and freshly harvested kidneys. RESULTS: During HPPP, perfusion pressure was 5.8+/-3.3 mmHg with oxygen delivery to the organs in excess of 3.5 times the organ metabolic requirement. During function measurements, RVR was not statistically different in the three groups; however, both oxygen consumption and GFR in the statically stored organs were significantly lower than in HPPP stored or freshly harvested kidneys. CONCLUSIONS: Our findings suggest that full portability in a hypothermic perfusion preservation device seems feasible utilizing fluidics-based technology.

The application of fluidics technology for organ preservation.

A prototype design of a portable, pulsatile, perfusion preservation device based on a novel application of fluidics technology was tested to evaluate its ability to oxygenate preservation solution and to examine the relationship between organ resistance, perfusion pressure, and perfusion flow characteristics. The effects of organ resistance on pulse rate, perfusion pressure, and perfusion flow were modeled. Interstitial PO2 in canine hearts stored at 4 degrees C for 12 hours in the fluidics device (n = 5) and in static hypothermic storage (n = 5) was also compared. Increasing outflow resistance did not have an effect on operating frequency of the fluidics actuator. Perfusion pressure rose as outflow resistance was increased, and the flow of preservation solution decreased proportionately. The PO2 of the preservation solution increased to 300 mm Hg in two hours and reached a plateau that exceeded 400 mm Hg within six hours. The aortic flow profile during pulsatile perfusion resembled a square wave function with a mean pulse duration of 0.30 +/- 0.05 seconds. Oxygen delivery by the fluidics perfusion device exceeded the oxygen requirements of the hypothermically preserved organs at all resistance levels. Initial interstitial PO2 in the hearts of both groups was greater than 150 mm Hg. In perfused hearts, PO2 declined 30% by the 12th hour, whereas complete depletion of oxygen was noted in the static storage group within six hours. The fluidics organ perfusion/transport apparatus weighs less than 18 kg, uses no electrical power, and can operate continuously for 10 to 12 hours expending 780 L of oxygen.

Oxygen consumption during oxygenated hypothermic perfusion as a measure of donor organ viability.

Hypothermic machine perfusion (HMP) for the preservation of kidneys, recovered from extended criteria organ donors (ECDs), presents the opportunity for assessing ex vivo parameters that may have value in predicting postimplantation organ viability. Organ perfusion and vascular resistance are the parameters most frequently cited as the basis for the decision to use or discard a donor kidney. The limitation of these measures is emphasized by the observation that a significant percentage of ECD kidneys with poor perfusion parameters can provide life-sustaining function after transplantation. It has been suggested that whole organ oxygen consumption (OC) during oxygenated HMP may better reflect the proportion of viable tissue in the organ and more reliably predict posttransplant organ function. Our study correlates renal OC and renal vascular resistance (RVR) during oxygenated HMP with postpreservation glomerular filtration rates (GFRs) in rodent kidneys after 24 hours of oxygenated HMP. Kidneys from adult rodents were preserved for 24 hours using oxygenated HMP and static cold storage (SCS). During oxygenated HMP preservation, organ OC, renal organ flow rates, and RVR were serially measured. After the preservation period, organs were mounted onto a Langendorff device for warming to normal body temperature and measurement of GFR. Oxygen consumption and RVR during HMP were correlated with postpreservation GFR. Oxygen consumption during oxygenated HMP was significantly correlated (r2 = 0.871; p < 0.05) with postpreservation GFR, suggesting that higher OC predicts better postpreservation GFR. In contrast, RVR was poorly correlated with postpreservation GFR (r2 = 0.258; p = 0.199). Glomerular filtration rate in SCS kidneys was 0.002 ± 0.003 ml/min/g. We demonstrate that measurement of organ OC during oxygenated HMP may have significant value in predicting postpreservation organ function.

Novel portable hypothermic pulsatile perfusion preservation technology: Improved viability and function of rodent and canine kidneys.

BACKGROUND: A reliable, portable, cost effective device for perfusion preservation of donor organs remains elusive. A portable, organ perfusion device design for hypothermic, machine perfusion (HMP) that successfully supported rodent kidneys for 24 hours was evaluated in canine kidneys. MATERIAL/METHODS: Freshly recovered rodent and canine kidneys were subjected to 24 hours of HMP or static storage (SS). Organ perfusion and cell membrane integrity were examined in HMP and SS rodent kidneys. Canine kidney function was evaluated in an isolated organ preparation. Oxygen consumption (OC), renal vascular resistance (RVR), and glomerular filtration rates (GFR) were compared. RESULTS: Perfusion pressure during HMP averaged 16 mmHg with oxygen delivery roughly 4 fold greater than the canine kidney's metabolic requirements. Following 24 hours of preservation, RVR was significantly elevated while OC and GFR were significantly lower in the SS organs compared to the HMP stored or freshly recovered kidneys. CONCLUSIONS: This organ preservation technology appears to provide an excellent preservation environment for kidneys such that post-transplant delayed graft function is minimized. Additionally, compared to current machine perfusion systems, the preservation system described in this work is significantly reduced in size, weight, and complexity, such that total portability may be possible.