Monitoring Renal Hemodynamics and Oxygenation by Invasive Probes: Experimental Protocol

Renal tissue hypoperfusion and hypoxia are early key elements in the pathophysiology of acute kidney injury of various origins, and may also promote progression from acute injury to chronic kidney disease. Here we describe methods to study control of renal hemodynamics and tissue oxygenation by means of invasive probes in anesthetized rats. Step-by-step protocols are provided for two setups, one for experiments in laboratories for integrative physiology and the other for experiments within small-animal magnetic reso-

The majority of the preclinical studies that generated this concept utilized a set of invasive probes to measure renal hemodynamics and oxygenation in anaesthetized rats [12,[14][15][16][19][20][21][22]. These probes typically include (1) a perivascular flow probe for measurement of total renal blood flow, (2) laser-Doppler-probes for assessment of local tissue perfusion, (3) Clark-type electrodes or fluorescencequenching optodes for measurements of local tissue partial pressure of oxygen (pO 2 ), and (4) devices for invasive measurement of arterial blood pressure. These methods are considered the gold standard for the study of renal hemodynamics and oxygenation because the methods-with the exception of the laser-Doppler-provide calibrated quantitative data [23][24][25]. The methodological principles of these techniques are detailed in the chapter by Cantow K et al. "Quantitative Assessment of Renal Perfusion and Oxygenation by Invasive Probes: Basic Concepts." Besides the study of the pathophysiology of AKI and CKD, these methods have also been used to study (1) mechanisms of control of renal hemodynamics and oxygenation in healthy rats, (2) the effects of various substances on this control, and (3) several putative preventive or therapeutic approaches for AKI and CKD [20][21][22][26][27][28][29][30].
It is well known that, due to the considerable capacity of the organism's homeostatic control systems to-at least partiallycompensate for disturbances of, or injury to, certain control elements, these alterations are often not easily detectable when studied by measuring baseline data only. Therefore, the control systems must be "challenged" in order to unmask such alterations. This is done by dedicated test interventions (see the chapter by Cantow K et al. "Reversible (Patho)Physiologically Relevant Test Interventions: Rationale and Examples") [19,27,28,31].
As all established modalities available in today's experimental and translational research, these techniques have shortcomings and methodological restraints, in particular, the invasiveness that preclude the survival of the animals and therefor the implementation in long-term studies, and, of course, their use in humans. Magnetic resonance imaging (MRI) offers noninvasive techniques to obtain insight into renal perfusion and oxygenation under (patho)physiological conditions. MRI affords full kidney coverage, soft tissue contrast that helps to differentiate the renal layers, seconds to minutes temporal resolution, support of longitudinal studies, and high anatomical detail [24,25,[32][33][34].
However, the validity and efficacy of functional MRI techniques for quantitative characterization of renal tissue perfusion and oxygenation and its changes in various (patho)physiological scenarios remains to be established [24,33,[35][36][37]. In particular, the weakness of MRI, its qualitative nature, needs to be addressed by calibration with quantitative methods, that is, the gold standard physiological techniques. Realizing the need of tracking invasive physiological parameters and MR parameters simultaneously for the same kidney, an integrated multimodality approach designated as MR-PHYSIOL was developed by our group [24,33,38]. It combines the measurements by the invasive probes described above (hereafter called PHYSIOL) with renal functional MRI data acquired by an ultrahigh field small animal scanner. By means of this hybrid setup, the first steps toward calibration of the blood oxygenation-sensitive parameter T 2 * (so-called blood oxygenation level-dependent MRI, BOLD-MRI) were done. Dedicated (patho)physiologically relevant test intervention including short periods of suprarenal aortic occlusion, hypoxia, and hyperoxia were applied to modulate renal perfusion and oxygenation, in order to detail the relationship between renal T 2 * and tissue oxygenation [24,33,39]. Of course, the MR-PHYSIOL setup can be used for calibration of functional MR parameters other than oxygenation-sensitive T 2 * as well.
In the following, step-by-step protocols are provided for two setups, one for stand-alone experiments in laboratories for integrative physiology (PHYSIOL) and the other for experiments within dedicated small-animal magnetic resonance scanners by use of the hybrid setup (MR-PHYSIOL).
This experimental protocol chapter is complemented by a separate chapter describing the basic concepts, which is part of this book.
This chapter is part of the book Pohlmann A, Niendorf T (eds) (2020) Preclinical MRI of the Kidney-Methods and Protocols. Springer, New York.

Animals
For PHYSIOL, male Wistar rats with body mass of 300-400 g are used. For MR-PHYSIOL, the spatial constraints dictated by the MR environment require the use of relatively small rats ( 350 g). The rats are allowed ad libitum food (standard maintenance diet) and water and must be housed at standardized conditions (e.g., group housed in Makrolon type IV cages with elevated lids under conventional SPF conditions; for cage enrichment paper towels as nesting material and pieces of wood for gnawing should be provided).   for MR-PHYSIOL: due to the long extension leads necessary to meet MR safety requirements, this probe has a larger body size. The reflector is made of ceramics (it does not induce MR artifacts), is L-shaped and offers no mechanism to lock the vessel. Therefore, a gauze is attached to the probe's cable, which will be fixed to the retroperitoneal muscles by means of sutures to avoid probe displacement depicted in Fig. 2. The probes are attached to an OxyLite/ OxyFlo-apparatus (Oxford Optronics, Oxford, UK). Safety information: For MR-PHYSIOL make sure that the positioning of the OxyLite/OxyFlo-apparatus meets the safety requirements of the MR environment.

Surgical Preparation
8. For continuous logging of the signals from the probes for arterial blood pressure, renal blood flow, cortical and medullary pO 2 and flux, their analog outputs must be digitized and recorded. An analog-digital converter (e.g., DT 9800-16SE-BNC, Data Translation GmbH, Bietigheim-Bissingen, Germany) permits connection to the USB port of a PC. A dedicated data acquisition software (HAEMODYN, Hugo Sachs Elektronik-Harvard Apparatus, March, Germany) allows for calibration of the probe signals and their continuous recording. 9. A vascular occluder (see Fig. 3): the remotely controlled hydraulic occluder is custom-made; it was designed and manufactured in our laboratory. The occluder consists of an inflatable tube ("head" of the occluder) connected by a catheter to a syringe. The "head" of the occluder is made of a high-grade silicone elastomer tube (Silikonkautschuk, Detakta Isolier-und Messtechnik GmbH & Co KG, Germany). For the connection between the "head" of the occluder and a regulative syringe an inextensible extension catheter (Portex Polythene Tubing; Ref 10. Vascular catheter(s) for administration of fluids (e.g., isotonic saline) and/or solutions of substances used for selective test interventions (e.g., drugs), and for repeated blood sampling (e.g., for measurements of blood gases and hemoximetry). Catheters for venous and arterial insertion are made in our laboratory using Portex Tubing (polythene).
11. For fixation and stabilization of the probes for PHYSIOL: two micromanipulators (e.g., type M-44 and MN-153, Narishige group, Tokyo, Japan) mounted on a rotatable magnetic pedestal (e.g., type M9, World Precision Instruments, Sarasota, FL, USA) as depicted in Fig. 4. The operation table needs a steel surface (e.g., type Micro-g, Technical Manufacturing Corporation, Peabody, MA, USA) to fixate the magnetic pedestals in positions required by the individual placements of the probes within the rat. For MR-PHYSIOL: to achieve stabile positions of the probes and to ensure safe transfer of the animal equipped with the probes to the scanner, a custom-made portable animal holder must be used (see Fig. 5). It was designed and built in our laboratory using 3D CAD (Autodesk Inventor 2012; Autodesk, San Rafael, CA, USA) and rapid prototyping (BST 1200es; Alphacam GmbH, Schorndorf, Germany). The holder must meet the geometry of the MR setup; it has a half-pipe shape with a section of reduced diameter to allow for the 4-element surface RF coil to be placed beneath. A mark on the holder indicates the center of the RF coil. A bridge-like construction, positioned at the end of the hind paws of the rat, enables fixation of all leads that connect the physiological 12. A respiratory (anesthetic) mask through which the spontaneously breathing rat is provided with air or other gas mixtures at   Due to the small size of rats in comparison with humans a much higher spatial resolution is required to depict the kidney with adequate detail. This, in turn, demands a high signal-to-noise ratio (SNR), which must be achieved by use of tailored MR equipment.

Surgical Preparation
For PHYSIOL, surgery is performed at the same operation table at which the subsequent experiment is executed. For MR-PHYSIOL, surgery must be performed outside the MR scanner room (in a neighboring preparation room) for safety reasons.
2. After reaching the required depth of anesthesia, that is, the state of surgical tolerance (determined by specific physiological signs such as muscle relaxation degree, absence of the paw withdrawal and eye lid reflexes, absence of the swallowing reflex, and whisker movement), carefully shave the coat in the abdominal, inguinal, and ventral neck areas of the rat (hair clipper Elektra II GH2, Aesculap AG, Tuttlingen, Germany).  . Bluntly dissect the connective tissue until the femoral vein, artery, and nerve are exposed.
6. Gently separate the nerve. Do not cut or damage the nerval tissue.
7. Separate the vein from the artery by using fine tip forceps, while trying to release an approximately 7-8 mm length vein fragment from the surrounding tissue.
8. Place three pieces of 4.0 threads under the femoral artery: situate the first thread distal (i.e., toward the leg), the second thread proximal (i.e., toward the body) and the third one between them.
9. Pull the first thread toward the leg and tie a ligature to the distal artery by using a triple knot.
10. Prepare loops with loose surgical knots on the remaining two threads.
11. Pull the second thread slightly toward the body so that the blood flow from femoral artery is inhibited.
12. Make a tiny incision in the exposed segment of the femoral artery behind the third thread using fine tip scissors. Fill a catheter with tapered tip with saline.
13. Grasp the catheter with the forceps and gently insert through the incision into the lumen of the femoral artery.
14. Tie the third knot slightly, fix catheter and arterial wall with a forceps, relax the tensed second thread, and push the catheter with a second forceps slowly deeper (%10 mm) into the artery in direction of the abdomen. 15. Rinse the catheter carefully with saline, and make sure that it is patent.
16. Tie the prepared loose knot of the third and second threads. 24. Place the Transonic flow probe around the left renal artery and start monitoring RBF. It is important that the head of the flow probe is filled with fluid. For PHYSIOL, attach the cable of the probe on the "clutch" of a micromanipulator (see Fig. 4a). Position and fixate the probe (Type 1RB) by means of the micromanipulator in the appropriate position of the artery (see Fig. 6b). For MR-PHYSIOL, the placement of the probe upon the artery must be done without the benefit of a micromanipulator. A gauze is attached to the probe cable (Type MV2PSB-MRI) as shown in Fig. 1b. To avoid displacements of the probe, the gauze is fixed to the retroperitoneal muscles by sutures (see Fig. 6c) (see Note 5).
25. For PHYSIOL, attach the cortical and medullary laser-flux-pO 2 probes, respectively, at one of the two "clutches" (see Fig. 4b) of a micromanipulator. With the help of the micromanipulator, the probes are placed into the renal cortex (depth about 1-1.5 mm below the capsule) and the medulla (3-4 mm below the capsule), respectively, as shown in Fig. 6b (see Note 6).
26. For MR-PHYSIOL, remove the customary Luer-Lock connectors of the laser-flux-pO2 probes and carefully fix the fiber glass cores to the sheathing using a clamp. Provide the probe with a customary silicone tubing; its length must be adjusted to the distance between the caudal and the cranial extremities of the left kidney. Attach tailored patches of gauze to the end of silicone tubing (see Fig. 2b). Measure the distance between the caudal and the cranial extremities of the left kidney by a caliper gauge. Based on this measurement, the cortical laserflux-pO2-probe must be carefully prepared so that the distance between insertion point and the tip exactly matches the individual kidney's diameter minus 1.5 mm.
27. For MR-PHYSIOL, advance the cortical laser-flux-pO 2 -probe meticulously from the caudal extremity of the kidney along the caudocranial axis (see Fig. 6c). To prevent craniocaudal displacement, the patch of gauze fixed to the silicone tubing of the probe (see Fig. 2b) must be stuck to the capsule of kidney's ventral surface by a thin layer of Histoacryl glue.
28. For MR-PHYSIOL, prepare the medullary laser-flux-pO 2probe so that the distance between insertion point and the tip exactly matches the distance 3-4 mm. Carefully advance the medullary laser-flux-pO 2 -probe from the caudal extremity of the kidney along the caudocranial axis. Stick the patch of gauze fixed to the silicone tubing of the probe to the capsule of kidney's ventral surface by Histoacryl glue (see Fig. 6c).
29. For MR-PHYSIOL, carefully fix the two probes' tubing to the retroperitoneal muscles by sutures in order to prevent displacement (see Fig. 6c).
30. Fill the abdominal cavity with warm saline (37 C). For PHYSIOL, intermittent exchange of this fluid throughout the experiment is done via the open abdominal wall. As the abdomen will be closed for MR-PHYSIOL (see below), for replenishment of abdominal saline a catheter must be placed into the abdominal cavity. Furthermore, for MR-PHYSIOL, a fiber-optical temperature probe is placed in close proximity to the left kidney, in order to monitor the temperature of the kidney throughout the experiment. 31. Connect the probes with OxyLite/OxyFlo-apparatus and start the monitoring of tissue pO 2 and laser-Doppler-flux. 32. For MR-PHYSIOL, mark the localization of the investigated kidney's upper and lower pole from the outside on the abdominal skin using a pen. Check that the kidney (pen markings on skin) is still aligned with the mark on the portable animal holder that indicates the center of the RF coil-if necessary carefully correct the animal's position. This is essential for optimal positioning of the rat in the MRI scanner (i.e., optimal position of the rat's kidney relative to the MR coil).
33. For MR-PHYSIOL, place the special bridge of the portable animal holder right behind the rat's hind paws and fix all the technical extensions (temperature probe, Transonic probe, laser-flux-pO 2 -probes, aortic occluder line and abdominal flushing catheter) to the bridge (see Fig. 5). Close the abdominal cavity of the rat by continuous suture while passing all extensions through the caudal cutting edge of the median abdominal incision. The extensions of the laser-flux-pO 2probes must be led through the abdominal wall using a small incision in the left inguinal region. 34. Place a respiratory mask loosely around the muzzle of the spontaneously breathing rat. Open air supply to a rate of 1000 mL/min. 35. Restart the HAEMODYN software (to start a new data file) and check the quality of all physiological signals, that is, arterial pressure (measured caudal the aortic occlude and therefore equivalent to renal artery pressure, RAP), renal blood flow (RBF), cortical and medullary laser-Doppler-flux, and cortical and medullary pO 2 .

Setting Up
Animal for MR-PHYSIOL Examination 1. Transfer the animal into the MR scanner room using the portable animal holder (see Note 7). Position portable animal holder with the rat on the MR scanner's animal bed.
2. Switch on the small animal monitoring system. Place the pneumatic pillow on the abdomen, and cover the animal with the warming blanket. Watch the respiration trace on the monitor of the small animal monitoring system and adjust pillow position until the respiratory motion is captured well (see Note 8). Set the trigger options of the small animal monitoring system such that the trigger gate opens for the duration of the expiratory phase.
3. Position the animal bed in the MR scanner such that the kidney of interest is located at the isocenter of the magnet. Because MRI can interfere with parameters acquired by invasive probes (as has been observed for laser-Doppler flux), short intervals without MR measurements must be implemented. In the following, an exemplary protocol is given for an MR-PHYSIOL experiment. It must be noted, that for these experiments, the closest coordination among the operator(s) of the MR scanner, the person(s) who perform the test interventions and those who run the electronic data storage (including setting markers for events such as start and end of an intervention) is of the essence.
1. On the PC that acquires the physiological data set a "START" marker in the HAEMODYN software to monitor and store a first set of baseline data.
2. On the MR system run the protocol(s) of your choice (T 1 -, T 2 -, T 2 *-mapping, DWI, etc.) to acquire a set of baseline data. Duplicate these scans for repeated measurements during/ between/after the test interventions and ensure that all parameters remain identical, including the shim and receiver gain.
3. Start of hypoxia. Change the gas flowing through the respiratory mask to 10% O 2 -90% N 2 .
4. Set markers and acquire both physiological and MR data during the hypoxic challenge.

5.
End of hypoxia. Change the gas flowing through the respiratory mask back to air (21% O 2 ).
6. Set markers and acquire both physiological and MR data during the recovery.
7. Start of hyperoxia. Change the gas flowing through the respiratory mask to 100% O 2 .
8. Set markers and acquire both physiological and MR data during the hyperoxic challenge.
9. End of hyperoxia. Change the gas flowing through the respiratory mask back to air (21% O 2 ).
10. Set markers and acquire both physiological and MR data during the recovery.
12. Set markers and acquire both physiological and MR data during the occlusion (see Note 9).
14. Set markers and acquire both physiological and MR data during the recovery.

End of Experiment
1. Carefully remove the respiratory mask from the animal's muzzle.
2. Cut all sutures; remove the fluid from the abdominal cavity using a pipette.
3. Control and note the overall condition of the kidney after experiment (e.g., surface coloring and its homogeneity). 5. Exsanguinate the animal by cutting the abdominal aorta.

Data Analysis
The analyses of MRI data acquired for MR-PHYSIOL experiments depend on the respective MR protocol and are detailed in dedicated analysis chapters for each MR method, which are part of the book Pohlmann A, Niendorf T (eds) (2020) Preclinical MRI of the Kidney-Methods and Protocols, Springer, New York. Temporal alignment of MR and PHYSIOL data is achieved by identifying the starting point of the experiment within both data sets. For a direct comparison of the MR-PHYSIOL parameters only PHYSIOL data acquired during the relevant MRI acquisitions can be used. While the PHYSIOL parameters are measured with subsecond temporal resolution the acquisition of the MR parameters (derived from a MR image) requires much more time (e.g., about 60 s for a typical T 2 * mapping). PHYSIOL and MR parameters can only be compared based on the (low) temporal resolution of the MRI. For this purpose the average value of each physiological parameter over the acquisition time of each MRI scan is calculated. For PHYSIOL data that are influenced by MR acquisition (such as Laser flux signals) the averages over the times without MR acquisition must be taken. Finally, group analyses of the results (e.g., relative changes of MR and PHYSIOL data; see Note 10) are done, as shown by exemplary data obtained by MR-PHYSIOL during a short suprarenal aortic occlusion and recovery in Fig. 7.

Notes
1. Instead of a respiratory (anesthetic) mask, a tracheal cannula can be used, as it is custom-made in our lab using designed and made in our laboratory using polythene tubing. The ventral region of the throat and the trachea are opened surgically, the cannula is inserted and fixated by suture.
2. As another dedicated test of control of hemodynamics and oxygenation, hypercapnia can be used with an inspiratory fraction of CO 2 of 5% in air.
ä Fig. 7 (continued) medullary tissue partial pressure of oxygen (Tissue pO 2 ) as monitored by invasive methods (PHYSIOL) simultaneously with cortical and medullary T 2 * data (MR) acquired by a 9.4 T small animal MR scanner during suprarenal aortic occlusion and recovery. Date are given as relative changes (mean AE SEM) versus baseline (immediately before the occlusion) 3. Urethane supports anesthesia throughout the surgical preparation and the MRI examination (for several hours) and leaves cardiovascular and respiratory reflexes largely undisturbed.

(a)
In order to prevent additional pressure on the aorta (and therefore development of an unintended kidney ischemia) the positioning and fixation of the aortic occluder must be performed under careful monitoring of the overall condition of the kidney (e.g., surface coloring and its homogeneity); (b) since the occluder consists of silicone and polyethylene and is positioned about 15 mm away from the kidney, it does not cause artefacts in MRI that affect the kidney.

(a)
The signal of the probe used for PHYSIOL is too weak due to the long extension leads. For MR-PHYSIOL a probe with a larger body size and a reflector made of ceramics instead of metal must be used (see Fig. 1). Its reflector does not induce MR artifacts, is L-shaped and offers no mechanism to lock the vessel, which presents a significant challenge for the implantation if the probe. (b) The bulk of the intestine bears the risk to cause additional pressure that can dislocate the probe and cause probe pressure on the aorta, the renal artery and vein, or the kidney itself. (c) Since Transonic measurements rely on ultrasound an appropriate coupling into tissue is of high relevance. To this end the abdominal cavity must be filled with saline solution (37 C) and no air bubbles must remain between probe and vessel. An additional catheter was placed in the abdominal cavity to replenish saline leakage in time course of the experiment.
6. Before inserting the tip of the respective probe, a small incision about the diameter of the probe is made into the renal capsule by means of the tip of a hypodermic needle to facilitate insertion of the rather dull tip probe.
7. Special attention during transfer must be paid to the tube/ cable extensions (aortic occluder, abdominal flushing, and all probes). Make sure to keep tubes or cable close to the animal bed so they cannot get caught anywhere on the way into the magnet! 8. The peak-to-peak amplitude of the respiratory trace should span about 2/3 of the vertical axis on the display. Any gross movement (for instance during repositioning the pillow) will lead to large peaks and force the monitoring system to adapt the signal amplification, so that temporarily the signal will become much smaller on the display. Keep an eye on the magnification, which is given left next to the display, this will drop to a low value such as 15Â-wait until it recovered back to a value around 100Â before further adjusting it.
9. If unsuccessful, repeat the occlusion and inflate the occluder with higher hydraulic pressure and make sure that the fluid reservoir for the inflation is sufficiently filled. Always check that the inflation is sufficient to bring total renal blood flow (RBF) rapidly toward zero. 10. It is usually more practical and useful to compare relative changes in the parameters rather than absolute changes. To do this divide all parameter values by that of the last baseline value (e.g., see Fig. 7).