This manuscript describes a method for targeted delivery to a single kidney via a catheter placed in the infrarenal abdominal aorta in the mouse.
There is a need for directed injections to enable increased and specific renal exposure for efficient evaluation of drug targets in the renal research field. Accumulation of drugs in certain organs may give rise to adverse and unwanted effects, depending on the nature of injectate. To minimize spillover and/or accumulation in other tissues, the herein described method directs the formulation into the renal artery bloodstream by inserting a catheter in the infra renal aorta, just below where it branches into the renal artery, resulting in the kidney as first reached organ and distributing of formulation throughout the kidney.
This manuscript provides a detailed description of the method, as well as its challenges and difficulties. It guides the experimenter to become skillful with this type of microsurgery that requires accuracy under sterile conditions. Speed is crucial for minimizing the ischemia and practicing the procedure will increase the chance of successful injections without adverse effects. By modulating the time between injection and reperfusion as well as the injected volume, the risk of spillover to other organs is mitigated.
Note that this technique is suitable for single dosing strategies.
In the preclinical research field within the pharmaceutical industry, model- and method- development is part of the daily work. There is an increasing interest for the ability to direct drugs to specific organs, or even separate compartments of an organ, without major spillover/entrapment, via bloodstream, into other tissues. This to enable increased and specific exposure for efficient evaluation of drug targets in models of different disease areas 1, 2, 3, 4.
A common way of delivering substances is by systemic routes (e.g., via the tail vein) since it is less invasive than the method described in this manuscript. However, systemic administration increases the risk of increased metabolism or accumulation of the compound in other tissues than the intended target organ, when passing through filtering organs such as the lung, the liver and the spleen 2,3,5. Aside from not reaching the destined tissue, this could potentially give rise to adverse and/or unwanted effects, depending on the nature of injectate. Only very small molecules pass the capillaries of filtering organs and therefore targeted delivery is especially important if working with larger molecules 6.
To minimize spillover and/or accumulation of injected formulation, in other tissues, the herein described method directs the formulation into the renal artery bloodstream through a catheter inserted in the abdominal aorta just below where it branches into the renal artery, resulting in the kidney as first reached organ. Another advantage with this guided administration is that a lower dose/volume can be used in order to reach the same level of exposure as achieved via systemic administration 3.
Other routes of administration have been explored, for example, injections via a catheter directly into the renal artery. In our hands, this was found to present a higher risk of failure to restore the circulation to the kidney. The very small diameter of the renal artery (approximately 0.35-0.55 mm in diameter) makes the incision relatively large and pose a risk of obstruction and/or embolism when closing the entry hole. According to our experience ischemic-related damage to the kidney often occurred when using this method, and we therefore developed this new way of successfully target the kidney by injecting substances through incision of the larger aorta to target the kidney.
There are similar techniques being developed in rats that also manifests the challenges and risk of stenosis/thrombosis working with injections directly into the renal artery 5. This supports our findings since the vessels in mice are even smaller.
This manuscript and video describe, in detail, how injections can be directed into the renal artery in mice through a catheter inserted in the infrarenal aorta, as well as guidance in how to overcome common difficulties in the procedure, to work in the safest way possible and thereby increase reproducibility.
Experimental procedures were approved by the Regional Laboratory Animal Ethics Committee of Gothenburg, Sweden.
1. Preoperative care
2. Surgical procedure
3. Postoperative care
The film is a mix of videos from different surgeries to present the ones with the best video quality. Some sequences have been taken from practice sessions where the mouse is not supposed to wake up. Therefore, the mouse is not always properly draped. When an animal is supposed to wake up, we always work in an aseptic way.
Histological analyses revealed that performing the injections directly into the renal artery gave rise to kidney injury, likely due to insufficient recirculation of blood flow with irreversible ischemia as a result (Figure 2). Injection through the abdominal aorta, on the other hand, did not cause any ischemic injury (Figure 3).
Evans blue dye was used to visualize that perfusion via the aorta will result in the kidney being the first reached organ (Figure 4). No other organs turned blue until after the passage out through the renal vein.
We have also showed that using this method we can increase engraftment of injected formulations to the perfused kidney using mesenchymal stem cells (Figure 5).
Figure 1: Placement of sutures and catheter. Please click here to view a larger version of this figure.
Top picture: Describing where to place the first ligature, cranially of the left kidney. Middle picture: Overview over the surgery area right before placement of catheter (2.11 in protocol). Bottom picture: Overview over the surgery area right before injection (2.10 in protocol)
Figure 2: Injections directly into renal artery. Design of, and IHC results from, a study with injection of NaCl directly into the renal artery Please click here to view a larger version of this figure.
Figure 3: IHC results at day 7 after aorta administration. IHC results from a study with injection of NaCl into the renal artery via abdominal aorta, 7 days after injection Please click here to view a larger version of this figure.
Figure 4: Injection of Evans blue dye. Top picture showing a serial view of an injection via the aorta of Evans blue dye. Bottom picture showing a cross-section of a kidney after injection via aorta of Evans blue dye. Please click here to view a larger version of this figure.
Figure 5: Abdominal arterial infusion significantly increased engraftment of mesenchymal stem cells in the left kidney compared to the right kidney. Human bone marrow derived mesenchymal stem cells were labeled with CM-Dil (red fluorescence) in suspension. One million labeled MSCs were infused through abdominal artery in three mice. Three hours after MSD infusion, mice were terminated. Left and right kidneys were harvested and embedded in OCT for cryosectioning. Please click here to view a larger version of this figure.
This method has been successful in delivering formulations to the kidney without causing renal damage. It can be used for delivery of any kind of formulations (e.g., small molecules, stem/progenitor cells, or microvesicles). The method can be applied in healthy animals or in renal disease models.
In Figure 2 and Figure 3, the histology is presented. Figure 2 shows histology 180 minutes after perfusion while Figure 3 shows history 7 days after infusion. The reason for this is that the experiments with renal artery was only 180 minutes long. To be sure we had no chronic damage with this new method, we purposely waited 7 days to evaluate them. The n-number is small but this is just an example.
Note that this newly developed method of aorta administration to target the kidney is an invasive method with relatively low throughput and is suitable for single dosing strategies only. Repeated dosing is not possible with this method.
With minor adjustments of the ligature placements this method could possibly be used to infuse both kidneys simultaniously5,7. With the increased risk of spillover through aortic branching we decided to keep the occluding ligature as close to the kidney as possible, leading the injection to the left kidney only. It can also be used in addition to other models or techniques like uninephrectomy or perhaps even simultaneous renal ischemia reperfusion injury (IRI). In our opinion, doing the injection only 24 hours after IRI3 surgery is too harsh and will affect the animal's recovery substantially.
When using new strains of mice, one should always perform a pilot experiment on some animals from that strain to ensure that the method does not need to be adjusted in some way. For example, in some strains the vessel-branching from the aorta can be positioned a bit different. The position of the ligatures may change somewhat to ensure that the formulation ends up at the right location.
Dehydration
Opening the abdominal cavity poses a great risk for dehydration. It is therefore imperative to add fluids (around 37 °C) both during and after surgery to ensure good basis for recovery post operatively. We usually give 1 mL/mice s.c. post-surgery.
Ischemia
Prolonged surgery time or if failing to reach full recirculation will result in ischemia. It is important to record the actual ischemia time and make sure that circulation to the kidney (and back legs) are restored. Constant training and refreshing of skills are, as always, important. Performing the procedure faster reduces the time the kidney is exposed to ischemia. Keeping the ischemia time (ligation of aorta) to around 5 min (10 min max) has been shown not to harm the kidney.
Spillover
Considering the risk of spillover to other organs when the injected formulation is re-entering the circulation through the renal vein, the injected volume should be kept to a minimum. We recommend a maximum volume of 50 µL. Dosage has also been found, by others, to be a critical factor for efficiency3.
Adverse effects (thrombosis and paralysis)
Initially when setting up the model we experienced non-consistent problems with paralysis in the back legs. After relevant dose of anticoagulant (10 IU of heparin in tail vein) preoperatively, this adverse effect was significantly decreased. After consulting experts in the field, we also started to be even more careful in working with atraumatic surgery, meaning no pinching in any tissues and touching as little areas as possible. These actions taken together reduced the frequency of paralysis from around 50% to virtually none.
Initially during method development, we applied anticoagulant tissue on the renal artery to close the incision hole. This was not possible when doing injections into the aorta since the pressure is so much higher in the aorta. Anticoagulant also increases the risk of thrombosis if it enters the vessel lumen.
Make sure not to have adventitia exposed in the lumen of the aorta when suturing since adventitia is very thrombogenic. Also avoid any inverting wound edges or narrowing the aorta by suturing to thigh.
We now have an established method for delivering formulations directly to the kidney.
The authors have nothing to disclose.
Thanks to René Remie at RRSSC for helping me finetune the technique and Instech labs for collaboration in producing a special designed catheter. A big thanks also to all colleagues at AstraZeneca R&D for productive discussions with tips and tricks as well as Xerox for completing the video with sound.
Blunt hook 5mm 8/pack | Cooper surgical | 3316-8G | |
ETHILON Nylon Suture 11/0 | Ethicon | W2881 | For vessel |
Microsurgery forceps curved | Karo Pharma | FRC-15 RM-8 | |
Microsurgery forceps straight | Karo Pharma | FRS-15 RM-8 | |
Mouse renal artery cannula, 3mm 32ga stainless steel, 10cm 2Fr PU, fits 25ga | Instech | C07SS-MRA1813 | |
Vicryl, 6-0, BV-1 needle | Angthos | W9575 | For abdominal cavity |