2.1.

Slaughterhouse Kidneys

Six slaughterhouse-retrieved porcine kidneys were obtained from a local abattoir. Pigs (female Dutch Landrace pigs, approximately 5 months old with an average weight of 130 kg) were slaughtered for consumption purposes and were handled according to standardized legal procedures. The pigs were stunned by electricity and died from exsanguination. Approximately 2 liters of blood were collected in a beaker with 25,000 IU of heparin (LEO Pharma A/S, Ballerup, Denmark) during exsanguination. Kidneys were removed from the cadaver en bloc, the renal artery was dissected free, and surrounding tissue was removed. The left kidney was used for all the experiments since this side displayed a better visible arterial bifurcation. After 30 min of warm ischemia (i.e., the time between the stop of the circulation of blood and the start of the cold flush), the kidneys were flushed with 500 ml cold 4°C saline. Next, the kidneys were installed in a kidney holder and placed on the hypothermic machine perfusion (HMP) (Kidney Assist Transporter, Organ assist^®, Groningen, The Netherlands) at 4°C and perfused for three and a half hours at a mean pressure of 25 mmHg. HMP was oxygenated (100% ${\mathrm{O}}_2$) at a rate of $100\mathrm{ml}/\min$.

2.2.

Normothermic Machine Perfusion

The setup of the normothermic machine perfusion (NMP) was described in detail elsewhere,¹³ using a centrifugal pump head (Deltastream DP3^®, MEDOS Medizintechnik AG, Heilbronn, Germany) controlled by in-house developed software (Sophistikate^®, Labview, National Instruments, Austin, United States).¹⁴ The software not only enables both flow and pressure directed perfusion but also allows to switch between pulsatile sinusoidal and constant flow. The temperature was regulated using a Jubalo water heating system and set at 37°C. An integrated heat exchanger (HILITE 1000^®, MEDOS Medizintechnik AG, Heilbronn, Germany) was built in the oxygenator. The flow sensor is a clamp-on flow sensor (ME7PXL clamp^®, Transonic Systems Inc., Ithaca, United States). The pressure sensor is a Truewave^® disposable pressure transducer (Edwards Lifesciences, Irvine, Unites States). As perfusion medium, 500 ml of autologous leukocyte depleted blood was used. The blood was diluted with 300 ml Ringers lactate (Baxter, Utrecht, The Netherlands) and supplemented with 10 ml 8.4% bicarbonate (B. Braun Melsungen AG, Melsungen, Germany), 10 ml 5% glucose (Baxter, Utrecht, The Netherlands), 6 mg mannitol (Baxter, Utrecht, The Netherlands), 0.33 ml dexamethasone (Centrafarm, Etten-Leur, The Netherlands), $100\mathrm{mg}/200\mathrm{mg}$ amoxicilline/clavulanic acid (Sandoz B.V. Almere, The Netherlands), 90 mg creatinine (Sigma-Aldrich, St. Louis), and 0.1 ml sodium nitroprusside (Sigma-Aldrich, St. Louis). Plasma was added to reach a hematocrit of 24%. A constant infusion ($20\mathrm{ml}/\mathrm{h}$) of a mixture of 90 ml Aminosol (Aminoplasmal, B. Braun Melsungen AG, Melsungen, Germany), 1 ml insulin (NovoRapid^®, Novo Nordisk, Bagsværd, Denmark), and 3 ml bicarbonate was maintained. Carbogen (95% ${\mathrm{O}}_2$ and 5% ${\mathrm{CO}}_2$) was supplied via the oxygenator at a flow rate of $500\mathrm{ml}/\min$. The complete NMP setup is shown in Fig. 1.

Fig. 1

Schematic overview of the experimental setup which allows for simultaneous blood flow measurements using LSCI and total RBF. The LSCI setup consists of a grayscale camera with optics, a red 638-nm laser with a diffuser, and a computer to run the LSCI algorithm. The laser light homogenously illuminates the kidney, which allows for full-field, real-time perfusion imaging of the cortical blood flow. The kidney is connected to a computer that measures and regulates the total RBF, pressure, and temperature. The autologous blood is oxygenated using carbogen and warmed using a warm water bath. The syringe pump is used to add compounds to keep the blood composition stable. The urine is collected to monitor urine production.

2.3.

Laser Speckle Contrast Imaging Setup

LSCI is based on the principle of coherent laser light that is backscattered from tissue, which forms a speckle pattern at the detector. The phase shift of this backscattered light results in a random interference pattern, the so-called speckles. Due to motion in the tissue, i.e., movement of the RBCs, the interference pattern starts fluctuating, causing a dynamic speckle pattern, which is blurred by the finite exposure time of the detector. The speckle contrast $K$ is calculated using Eq. (1).

2.4.

Hemodynamic Experiments

We have designed a set of four hemodynamic experiments to study the LSCI as a tool for organ perfusion measurement (Fig. 2). During all experiments, the temperature, pressure, the (arterial) RBF, and renal resistance were measured. Subsequently, we measured the cortical perfusion using LSCI. These experiments were designed to examine the power to differentiate between well and poorly perfused tissues over time (Secs. 2.4.1 to 2.4.3) and to analyze the techniques’ ability to distinguish between local perfusion deficits, i.e., spatial resolution, and the speed at which this is detected, i.e., temporal resolution (Secs. 2.4.3 and 2.4.4). These experiments were repeated on five different kidneys. In a separate extra kidney, we have performed RBF, LSCI, and sidestream dark-field (SDF) imaging simultaneously while performing a repeated local ischemia and three gas bolus injection (Sec. 2.5).

Fig. 2

A time-scaled overview of the total experiment. The experiment was 4 h in total and can globally be split up into four different experiments; the reperfusion of the kidney, a stepwise modulation of arterial renal flow, the induction of a local ischemic area, and the gas bubble infusion of ${\mathrm{O}}_2$, carbogen (95% ${\mathrm{O}}_2$ and 5% ${\mathrm{CO}}_2$), room air, and ${\mathrm{N}}_2$. The table indicates the techniques that have been used to monitor the hemodynamic changes (RBF, renal blood flow; LSCI, laser speckle contrast imaging; and SDF, sidestream dark-field imaging). The graph represents the hemodynamic changes as measured by the arterial RBF probe [RBF (ml/min)].

2.5.

Sidestream Dark-Field Imaging Experiments

SDF imaging can track the movement of individual RBCs enabling quantitative blood flow measurements and the detection of subtle microvascular changes.^17–19 SDF imaging is a contact method with a relatively small FOV of $\sim 1{\mathrm{mm}}^2$. The device has a total magnification of $750\times$. The penetration depth is around $750\mu \mathrm{m}$. The small FOV in combination with a shallow penetration depth and the fact that it is a contact method makes SDF imaging less than ideal for the visualization or the RCM. The green light emitted by the system scatters through the tissue and is absorbed by the hemoglobin in the RBCs resulting in dark RBCs in contrast to background tissue. Using SDF imaging as a quantitative measure of blood flow allows us to compare the RBF and LSCI with SDF. However, it must be noted that RBF measures both cortical and medullar blood flow, whereas LSCI and SDF measure only the RCM with the difference of being full field (LSCI) versus a small FOV (SDF). The SDF microscope (MicroScan^® Video Microscope System, MicroScan BV, Amsterdam, The Netherlands) was held in place using a tripod connected to a laboratory table to minimize movement artifacts. The tripod has $X$- and $Y$-axis precision adjustment screws to place the microscope perpendicular to the renal cortex without inducing pressure artifacts. The tip of the SDF microscope was covered with a plastic cap. Images were recorded at $10\text{frames}/\mathrm{s}$ with a $720\times 576\text{pixel}$ resolution. The video signal was digitalized using an S-VHS to USB frame grabber and stored on a computer for further offline processing. The SDF microscope uses pulsating green light-emitting diodes that are placed around the charge-coupled device at the tip of the microscope. The data were analyzed using custom software (Matlab, Mathworks, Natick, Massachusetts) that calculated the mean pixel intensity (MPI) within the whole frame by taking the average pixel intensity. The MPI is a measure of the number of RBCs. As the number of RBCs within the frame increase, the images darken hence the MPI is a relative measure for the number of RBCs (Video 3). The region of interest for the laser speckle perfusion units (LSPU) was placed 1 cm away from the SDF camera.

The renal capsule had to be locally removed at the tip of the SDF microscope to be able to image the RCM during this part of the experiments. SDF imaging, RBF measurements, and LSCI were performed simultaneously in only one kidney due to the complex nature of imaging the RCM using SDF imaging. Five consecutive repetitions of the short local ischemia experiments were executed followed three gas injections with oxygen, one with room air, and one with nitrogen, respectively.

2.6.

Data Analysis

Data are presented as $\text{mean}\pm \mathrm{SD}$, unless stated otherwise. The correlations between LSCI in LSPU (A.U.) and RBF (ml/min), and SDF in MPI (A.U.) were calculated using a coefficient of determination, $R^2$. Applicable parametrical paired tests were used. A $p$-value of $<0.05$ was considered statistically significant. The experiments described in Sec. 2.4 were repeated five times to rule out unique events.

3.1.

Reperfusion Experiment

The reperfusion experiment was performed five times in five kidneys. During the hour, all kidneys warmed-up to 37°C, which led to an increase in RCM (Fig. 3.). This experiment showed resemblance with the reperfusion during a kidney transplantation. The correlation between the normalized LSPU (A.U.) and the RBF (ml/min) was $R^2=0.94\pm 0.02$ ($p<0.0001$). The good correlation might be explained by the tendency of a kidney to allocate blood to the cortex first, which is the flow that we measure using LSCI.

Fig. 3

Graph of average laser speckle units perfusion (LSPU) (A.U.) and average renal arterial blood flow (RBF) (ml/min) of five kidneys during the first hour of NMP after the HMP is captured in the reperfusion experiment. The kidney slowly warms-up from 4°C to 37°C with a resulting increase RBF.

3.2.

Flow Experiment

The flow experiment was performed 10 times in five kidneys. The $R^2$ of the normalized LSPU (A.U.) and RBF (ml/min) was $0.59\pm 0.31$ ($p>0.05$). A change in total RBF was not followed by a similar change in the cortex (i.e., RCM) resulting in a moderate correlation. As seen in Fig. 4, there seems to be a hemodynamic response, redirecting flow to the cortex. There was an apparent hemodynamic response when the RBF was decreased in a low flow state ($\sim 100\mathrm{ml}/\min$). When the RBF was increased, a classical short-term reperfusion overshoot was observed.

Fig. 4

A typical flow experiment trace with normalized LSPU in A.U. and RBF in ml/min. The flow was decreased by steps of $50\mathrm{ml}/\min$ every 4 min and subsequently increased with steps of $50\mathrm{ml}/\min$ to the starting level when a flow of $50\mathrm{ml}/\min$ was reached.

3.3.

Local Ischemia

The local ischemia experiment was performed on five kidneys. The experiment failed for two kidneys; one due to malfunctioning of the balloon catheter and the other one due to the appearance of the ischemic area on the posterior side of the kidney. Typical images are shown in Figs. 5(a) and 5(b). The data are depicted in bar graphs in Fig 5(c) and a typical trace of an ischemic region of interest is displayed in Fig. 5(d). The results of the short (5 min) and long (15 min) ischemic period are found in Table 1. The short and long ischemic period did not show any significant differences. The baseline is the average of the time period before the induction of the local ischemia. The ischemia is the average of the ischemic period. The reperfusion is the maximum value directly after the release of the local ischemia and the postocclusion is the average of the time after the reperfusion. The LSPU values are normalized compared to the baseline.

Fig. 5

The inflation of a balloon catheter in an arterial bifurcation induces a local ischemic area. The local ischemia was induced consecutively for a short (5 min) and long (15 min) period in each kidney. (a) Pseudo-color image of the RCM before induction of local ischemia made using LSCI. (b) Pseudo-color images of the RCM while local ischemia is induced made using LSCI. (c) Relative drop of the RCM measured in LSPU in A.U. for the short and long ischemic period. The results are normalized on the average of the baseline period. (d) A typical local ischemia trace measured by RBF (ml/min) and the RCM in normalized LSPU (A.U.) where the LSPU is normalized on the min/max of the whole local ischemia experiment (Video 1, MP4, 8.2 MB [URL: https://doi.org/10.1117/1.JBO.26.5.056004.1]).

Table 1

Relative change in LSPU normalized compared to the baseline for the ischemia, reperfusion, and post-occlusion period for both the short and long ischemic period, respectively. The R2 value is calculated between the RBF in ml/min and the LSPU in A.U. for the short and long ischemic period.

3.4.

Gas-Bubble Infusion

The gas-bubble infusion was performed once on five kidneys and characterized by a slow, local return of RCM, as depicted in Fig. 6. The data can be found in Table 2 where the relative drop in LSPU is calculated in relation to baseline level. The rise time is defined as the time it takes for the LSPU to return to the baseline level. This was longer than 600 s for nitrogen and thus the exact rise time could not be measured. The $R^2$ was calculated with the LSPU (A.U.) and the RBF (ml/min). The data are visualized in bar graphs in Figs. 7(a) and 7(b) for the average drop (%) and rise time (s), respectively. Figure 7(c) displays typical LSCI traces for this experiment in one kidney.

Table 2

The relative drop in LSPU in A.U. compared to baseline after the injection of oxygen, carbogen, room air, and nitrogen. The time to baseline is the total time required for the LSPU to reach baseline. This was longer than 600 s for nitrogen and thus was not measured. The R2 value is calculated between the RBF in ml/min and the LSPU in A.U.

Fig. 6

Pseudo-color images of the RCM during a gas-bubble (oxygen) injection made using LSCI. The injected oxygen forms a gas embolism in the blood vessels. These emboli prevent blood from passing through and thereby hamper the perfusion. When the gas dissolves in the perfusion medium, the embolism disappears and perfusion reappears. (a) Before oxygen injection, (b) directly after oxygen injection, (c)–(f) slow, local return of the RCM (Video 2, MP4, 9.2 MB [URL: https://doi.org/10.1117/1.JBO.26.5.056004.2]).

Fig. 7

The infusion of a gas bolus induces a short complete ischemia that is followed by growing areas of local reperfusion. (a) The average percentage drop in LSPU in A.U. after injection of the respective gas. (b) The recovery time of the LSPU to pre-injection perfusion level in seconds (c) Typical LSPU traces of the respective gasses of one kidney.

3.5.

Sidestream Dark-Field Imaging Comparison

The results of the local ischemia, oxygen, room air, and nitrogen injection are shown in Table 3. All experiments were performed on the same kidney. A representative example of the LSCI pseudo-color images and the corresponding SDF images are shown in Fig. 8 and Video 3. SDF-LSPU showed the best overall correlation compared to the LSPU-RBF and SDF-RBF $R^2$-values.

Table 3

The R2 values of the SDF, the LSPU, and the total RBF during the SDF imaging experiment. The local ischemia, oxygen, room air, and nitrogen injection were performed n times on the same kidney. The experiment consisted of five short consecutive local ischemic periods followed by three oxygen, one room air, and one nitrogen injection.

Fig. 8

Pseudo-color LSCI images and SDF images of a local ischemia induced by inflation of an arterial balloon catheter. The LSCI and SDF images were recorded simultaneously during the experiment.– Pseudo-color LSCI images of (a) pre-local ischemia, (b) during local ischemia, and (c) reperfusion after local ischemia. (d)–(f) SDF images of the corresponding LSCI images, darker SFD images indicate more RBCs present in the FOV, i.e., better perfusion (Video 3, MP4, 11.6 MB [URL: https://doi.org/10.1117/1.JBO.26.5.056004.3]).