Authors thank E. Ferrantelli and G. Liappas for their support setting the 5/6 nephrectomy protocol, R. S&#225;nchez-D&#237;az and P. Mart&#237;n for the assistance with ureic nitrogen assessments, and E. Hevia and F. N&#250;&#241;ez for the assistance with mice care. This work was supported by grants SAF2016-80648R from the &#34;Ministerio de Econom&#237;a y Competitividad&#34;/Fondo Europeo de Desarrollo Regional (MINECO/FEDER) to Manuel L&#243;pez-Cabrera and PI 15/00598 from&#160;Fondo de Investigaciones Sanitarias (FIS)-FEDER funds, to Abelardo Aguilera.

All methods described here have been approved by the Institutional Animal Care and Use Committee of the Molecular Biology Center Severo Ochoa (Madrid, Spain).
Note: C57BL/6J female mice aged 12 to 14 weeks and weighing approximately 20 g at the start of the study were used. All the animals were housed under standard conditions and were given food and water ad libitum. Health conditions were checked daily.&#160;The material required, such as gloves, drape, catheter, suture and needles,...

The authors have nothing to disclose.

The first published data analyzing PD alterations by using a &#34;close system&#34; technique was performed in 200929 . This close system means that the entire device is located inside the body and fluid is injected with a needle through an access port. The most important technical problem in long-term animal models of fluid infusion through a catheter is the occurrence of obstruction. Possible options are to perform omentectomy or add heparin to the PDFs to reduce peritoneal adhesions. Neverthele...

Kidney Function and Renal Disease
Kidneys are essential organs involved in homeostasis, blood filtration and hormone production. There are various conditions that lead to kidney failure and to the subsequent onset of uremia, which has been defined as the group of systemic symptoms due to the accumulation of waste products in the blood retained due to kidney function disorders1. Moreover, since homeostatic capability is also affected when there is a renal failure, hypertension due to volume overload may occur, which is also dangerous as it can lead to heart failure1. When the functional capability of kidneys is less than 10%-15%, the patient must undergo one of the following therapeutic options: hemodialysis, peritoneal dialysis (PD) or renal transplantation.
PD is an interesting option that allows patients to continue treatment from the comfort of their home or practically anywhere, thus avoiding the need for frequent hospital visits and stays. The PD technique eliminates small toxic molecules and excess water generated by the body2 through the instillation of an osmotic fluid (peritoneal dialysis fluid, PDF) into the peritoneal cavity. This instillation generates the osmotic gradient necessary for the exchange of solutes and water between the peritoneal capillary and PDF, a process known as ultrafiltration (UF).
Peritoneal Injury Induced by Peritoneal Dialysis
The peritoneal cavity is covered by a membrane (PM) composed of a monolayer of mesothelial cells resting on a matrix, which also houses few blood vessels, fibroblasts, macrophages and other cell populations. Unfortunately, the peritoneal membrane always suffers some alterations during PD treatment, such as apoptosis and loss of mesothelial cells, mesenchymal transition of mesothelial (MMT) and endothelial (end-MT) cells, recruitment of inflammatory cells and fibrocytes, vascular alterations, angiogenesis, lymphangiogenesis and/or fibrosis3,4,5,6,7,8,9. These alterations are responsible for the development of an UF capacity failure10, which precludes the continuation of the therapy, requiring that the patient must receive an alternative treatment to survive (hemodialysis or renal transplantation). Therefore, for these patients, it is essential to delay or control the development of these peritoneal alterations.
It has been speculated that uremia alone may cause inflammation11, but the most important local factor is PDF bioincompatibility. Most PDFs use glucose as the osmotic agent, which causes inflammation. Due to PDF storage times and sterilization, glucose suffers a process of degradation, and new products from this reaction appear, generating more inflammation, MMT and apoptosis12,13. Moreover, there is also the possibility of mechanical damage due to the instillation method. All these factors, acting continuously, may generate a persistent and recurrent inflammatory state, leading to chronic inflammation, which drives to membrane deterioration and, conclusively, UF failure. How this damage could be reduced or avoided is still a matter of study.
Analyzing the Development of Lesions: From Human Samples to Animal Models
Working with human biopsies is a limiting factor due to the difficulty in obtaining tissue samples. These samples can only be obtained from surgeries performed due to catheter malfunction or transplantation, usually after years of PD treatment. This approach is useful for the analysis of pathological changes suffered by a peritoneal membrane exposed to PDF, but is not sufficient to study the development of the process. Another possibility is to analyze cells drained from dialysis effluent, but this still fails to provide a complete scenario. Merging both techniques is only possible with animal models. The peritoneal structure is similar among mammals, and therefore there are models with different animal species. There are a few studies based on sheep (Rodela et al.14 and Barrell et al.15) and rabbit16,17 models; however, smaller animals are preferable as they are easier to house and maintain, and are also more economical. The use of rats18,19,20,21,22,23,24 offers a shorter treatment time needed to observe morpho-functional alterations. It has represented a very useful model to explore different issues such as the effect of anti-fibrotic drugs as for example BMP-7 (bone morphogenic protein-7)25 and RAS (renin-angiotensin system) targeting26,27,28.
However, the murine model has emerged as an ideal model with many benefits over others. The most interesting advantage is the possibility of using genetically modified mice to study the molecular and cellular basis of peritoneal damage. In fact, mice are often employed for the analysis of numerous diseases, as there are many different strains with various well-known genetic backgrounds. Other advantages include the reduced space required for housing, reduced cost of experiments (due to the animals&#39; smaller size), ease of handling, the availability of reagents and the increasing amount of available information on the different strains of mice since they have been most commonly used animals in research.
A mice-based model employing an implanted device has been the most recently established model for PD29,30, and has been shown to mimic peritoneal deterioration suffered by PD patients due to exposure to PDFs. This model has collaborated to understand the pathological processes implicated31,32,33. Moreover, it has been used to validate various potential treatments for ameliorating this deterioration using immune modulators and anti-inflammatory drugs and other anti-fibrotic and anti-angiogenic agents, such as COX-2 (cyclooxygenase-2) inhibitors34, PPAR-&#947; (peroxisome proliferator-activated receptor-&#947;) agonists35, Tamoxifen36, Paricalcitol (a vitamin D receptor activator that modulates the immune reaction)37, Rapamycin38 and Nebivolol39.
Developing the Mouse Model with an Implanted Catheter
The goal of this model is to resemble, as much as possible, the technique used in human PD patients, allowing to perform extended treatments of PD in small animals. So far, three techniques for instillation of dialysis fluid into the peritoneum have been tested in mice. The first one, blind puncture of the front abdominal wall, is controversial due to the multiple risks that it may incur, such as peritoneal damage, bleeding and, as is blindly performed, visceral puncture. The second technique is the so-called &#34;open permanent system&#34;, in which the device for injecting the fluid is placed outside the body. This procedure is most similar to that performed in humans. However, it does not allow the development of long-term experiments, as it may increase the chances of infection, and generally requires the use of anesthesia to instill PDF, which may interfere with the results. The third technique is the &#34;closed system&#34;. With this approach, the entire device used for fluid instillation is located inside the animal&#39;s body. Fluid is injected with a needle through an access port, which is placed subcutaneously. This procedure reduces the risk of peritoneal infection and bleeding as well as the need for anesthesia.
To study the effect of uremia in PD, a recent murine model has also been stablished40 based on the PDF infusion model with catheter. This model brings in a novel technique to perform a nephrectomy in mice, thus reducing renal function. In the present article, a modification of the protocol employed by Ferrantelli et al. in 201540 has been developed. This new protocol allows catheter implantation while nephrectomy, reduces the length of the wound inflicted during surgery and facilitates access to the kidneys.

<table border="0" cellpadding="0" cellspacing="0" style="width:1207px;" width="1207">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:592px;">Minute Mouse Port 4French with retention beads and cross holes</td>
			<td style="width:180px;">Access technologies</td>
			<td style="width:244px;">MMP-4S-061108A</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:592px;">Posi-Grip Huber point needles 25 ga. X 1/2&#180;&#180;&#160;</td>
			<td style="width:180px;">Access technologies</td>
			<td style="width:244px;">PG25-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:592px;">High Temperature Cautery Kit</td>
			<td style="width:180px;">Bovie</td>
			<td style="width:244px;">18010-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:592px;">Forane</td>
			<td style="width:180px;">abbVie</td>
			<td style="width:244px;">880393.4 HO</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:592px;">non absorbable suture 6/0</td>
			<td style="width:180px;">Laboratorio Agar&#243;</td>
			<td style="width:244px;">6121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:592px;">Scissors&#160;</td>
			<td style="width:180px;">Fine Science Tools</td>
			<td style="width:244px;">14079-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:592px;">forceps</td>
			<td style="width:180px;">Fine Science Tools</td>
			<td style="width:244px;">11002-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:592px;">clamp</td>
			<td style="width:180px;">Fine Science Tools</td>
			<td style="width:244px;">13002-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:592px;">Buprenorphine 0,3 mg/ml</td>
			<td style="width:180px;"></td>
			<td style="width:244px;"></td>
			<td>pharmaceutical product</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:592px;">cotton swabs</td>
			<td style="width:180px;"></td>
			<td style="width:244px;"></td>
			<td>pharmaceutical product</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:592px;">Dalsy (Ibuprofen) 20mg/mL oral suspension</td>
			<td style="width:180px;">AbbVie&#160;S.R.L.&#160;</td>
			<td style="width:244px;"></td>
			<td>pharmaceutical product</td>
		</tr>
	</tbody>
</table>

surgical techniques for catheter placement and 5/6 nephrectomy in murine models of peritoneal dialysis

Peritoneal dialysis (PD) is a renal replacement therapy consistent on the administration and posterior recovery of a hyperosmotic fluid in the peritoneal cavity to drain water and toxic metabolites that functionally-insufficient kidneys are not able to eliminate. Unfortunately, this procedure deteriorates the peritoneum. Tissue damage triggers the onset of inflammation to heal the injury. If the injury persists and inflammation becomes chronic, it may lead to fibrosis, which is a common occurrence in many diseases. In PD, chronic inflammation and fibrosis, along with other specific processes related to these ones, lead to ultrafiltration capacity deterioration, which means the failure and subsequent cessation of the technique. Working with human samples provides information about this deterioration but presents technical and ethical limitations to obtain biopsies. Animal models are essential to study this deterioration since they overcome these shortcomings.
A chronic mouse infusion model was developed in 2008, which benefits from the wide range of genetically modified mice, opening up the possibility of studying the mechanisms involved. This model employs a customized device designed for mice, consisting of a catheter attached to an access port that is placed subcutaneously at the back of the animal. This procedure avoids continuous puncture of the peritoneum during long-term experiments, reducing infections and inflammation due to injections. Thanks to this model, peritoneal damage induced by chronic PD fluid exposure has been characterized and modulated. This technique allows the infusion of large volumes of fluids and could be used for the study of other diseases in which inoculation of drugs or other substances over extended periods of time is necessary.
This article shows the method for the surgical placement of the catheter in mice. Moreover, it explains the procedure for a 5/6 nephrectomy to mimic the state of renal insufficiency present in PD patients.

Figure 1 shows all the materials required to follow the procedures described in protocol section. For this example, mice submitted or not to nephrectomy (8 animals per group) (Figure 2) were exposed during 40 days (two injections per day, waiting at least 2 hours between both) to a mixture of two different PDFs, commonly used in the clinical practice: Extraneal (icodextrin-based PDF) and Dianeal (glucose-based PDF). A group with ...

Watch this Scientific Journal Video about Surgical Techniques for Catheter Placement and 5/6 Nephrectomy in Murine Models of Peritoneal Dialysis at JoVE.com

Surgical Techniques for Catheter Placement and 5/6 Nephrectomy in Murine Models of Peritoneal Dialysis

This article shows the method for the surgical placement in mice of an intraperitoneal catheter attached to an access port that is positioned at the back of the animal. Moreover, it explains the procedure for a 5/6 nephrectomy to resemble the uremic state of PD patients.

Peritoneal dialysis (PD) is a renal replacement therapy consistent on the administration and posterior recovery of a hyperosmotic fluid in the peritoneal ...

This method can help answer key questions in the Peritoneal Dialysis field about Peritoneal or Systemic alterations in response to fluid exposure either under uremic or not uremic conditions. The main advantages of this technique are that large amounts of fluid can be injected intraperitoneally over a long treatment period and that it allows the use of genetically modified animals. The implication of this technique extend toward the therapy and diagnostics of structural and functional peritoneal deterioration, as it facilitates potential, therapeutic strategic testing and biomarker evaluation.We first had the idea for this method when we anticipated the need for using genetically modified mice to starting the molecular cues involved in peritoneal damage. For catheter placement, after shaving the right flanks and backs of each animal, confirm a lack of response to toe pinch and apply ointment to the animal's eyes. Next, place the first animal on its left flank on the surgical table and disinfect the exposed skin with 1%chlorhexidine gluconate solution.Use blunt scissors to make a 0.5 centimeter cut skin incision on the right flank of the body. Then, carefully separate the skin from the adjacent muscle layer until the skin covering the entire back of the animal is well separated. Make a one millimeter diameter incision in the muscle layer through the skin incision and insert the tip of the catheter and the first plastic ring into the muscle incision.Suture the peritoneal wall tightly around the catheter with the 5-0 or 6-0 non-absorbable suture, with one plastic ring inside the peritoneal cavity and the other between the muscle and the skin. The sutures should be prepared before the incision is made and the muscle should not be released until the catheter is inserted. Otherwise, since the incision is very small, it could be difficult to find it again.Insert the access port into the subcutaneous space towards the tail of the mouse and close the wound with a second suture. Then, return the animal to its cage after full sternal recumbency. Four to seven days later, to initiate the peritoneal dialysis treatment, first load a syring with two milliliters of peritoneal dialysis fluid, and disinfect the skin surrounding the port with 1%chlorhexidine gluconate solution.Then use one hand to restrain the animal by the tail and access port, and inject the fluid into the port with the other hand using a beveled needle. For a 5/6 Nephrectomy and catheter placement, after preparing the animals for surgery, as demonstrated, make a 0.5 centimeter skin incision on the left side of the animal close to the bottom of the ribs. Open a small incision in the muscle to extract the left kidney from the peritoneum, and remove the capsule and the adrenal gland.Use a cauterizer to burn and cut the extremities of the kidney and return the kidney to the peritoneal cavity. Then, close the muscle and the skin wounds with insoluble 5-0 or 6-0 sutures and return the animal to its cage when it has fully recovered. The next day, make a 0.5 centimeter skin incision on the right side of the animal and separate the skin at the back of the animal from the muscle, as demonstrated.Make a 0.3 to 0.4 centimeter incision in the muscle and extract the right kidney from the peritoneal cavity. Remove the capsule and the adrenal gland and use a non-absorbable 5-0 or 6-0 suture to ligate the kidney vein, artery, and ureter. Now remove the kidney completely and insert the catheter so that the muscle is maintained between the two plastic rings, as demonstrated, before closing the muscle wound.Introduce the access port into the sub-cutaneous space and close the skin, as demonstrated. Then return the animal to its cage with monitoring until full recovery, and allow the animal to recover for at least 10 days before administering the hyperosmotic fluid, as demonstrated. Mouse perial peritoneal tissues obtained from the most distant area of the catheter, demonstrate an increased thickness and cell recruitment during dialysis that is further aggravated in the nephrectomized animals.Thymus epithelial cells also exhibit an altered morphology due to inter-cellular union degradation induced by the peritoneal dialysis. Serum samples from nephrectomized mice, indicate that a 5/6 Nephrectomy induces a uremic state, increasing the Ureic Nitrogen levels during the course of the experiment, compared to baseline. More over, when the kidneys are fully functional, the Ureic Nitrogen levels remain similar to the basal state, even in mice exposed to peritoneal dialysis fluid.These techniques can be completed in five to seven minutes. Without the nephrectomy, 15 to 20 minutes. For the left kidney cauterization, 20 to 25 minutes.For the right kidney removal and catheter placement, if its methodology is performed properly. After its development, this technique paved the way for researchers in the field of peritoneal dialysis to explore therapeutic options for peritoneal preservation in renal patients subjected to peritoneal dialysis treatment.

surgical-techniques-for-catheter-placement-56-nephrectomy-murine

Research

JoVE Journal

Engineering

14.7K 조회수.  Spanish National Research Council. This method can help answer key questions in the Peritoneal Dialysis field about Peritoneal or Systemic alterations in response to fluid exposure either under uremic or not uremic conditions. The main advantages of this technique are that large amounts of fluid can be injected intraperitoneally over a long treatment period and that it allows the use of genetically modified animals. The implication of this technique extend toward the therapy and diagnostics of structural and functional periton...