Name: Technical Aspects of the Mouse Aortocaval Fistula (Video) | JoVE Journal
Uploaded: 2013-07-11T13:00:00.000+00:00
Duration: 6 min 12 s
Description: Learn a suture-free technique for creating mouse aortocaval fistulas using needle puncture and Doppler ultrasound validation. This streamlined method improves reproducibility and reduces surgical time for cardiovascular research.

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This work was supported in part by the National Institute of Health grant R01-HL095498, as well as with the resources and the use of facilities at the VA Connecticut Healthcare System, West Haven, CT.

1. Anesthesia and Pre-operative Procedures
<ol>
 <li>Induction of general anesthesia 
 Approval by the appropriate Institutional Animal Care and Use Committee is obtained. Male C57/black mice, aged 10 weeks, are anesthetized. Deliver 4% isoflurane with 0.8 L/min oxygen to a Plexiglas box via an isoflurane vaporizer and place the mouse inside. Alternatively, if preferred by the surgeon, intraperitoneal anesthesia can be used.</li>
 <li>Preserving anesthesia 
 After confirmation of anesthesia by lack of reaction to toe pinch, the mouse is put on the operation table in a supine position with a silicone mask. Isoflurane is decreased to 2% to maintain appropriate anesthesia level.</li>
 <li>Hair removal 
 Ventral hair is removed from the neck to lower abdomen using a hair remover. Hair remover is preferred because the hair that is left using a shaver may interfere with the Doppler ultrasound.</li>
</ol>
2. Preoperative Ultrasound
<ol>
 <li>Objectives of the ultrasound 
 Doppler ultrasound examination is performed prior to AVF surgery so as to record the base level findings that can be compared with post-operative findings to see anatomical and physiological changes.</li>
 <li>Points of observation 
 The jugular vein, supra- and infra-renal inferior vena cava (IVC), and abdominal aorta are examined using both pulse Doppler and B-mode.</li>
 <li>Measurements 
 Using the B-mode, diameter of each vasculature is measured. In addition, maximum velocity (peak systolic velocity in arteries) and waveforms are recorded in each of the measuring points described above.</li>
</ol>
3. Operative Procedures
<ol>
 <li>Mouse Preparation 
Prepare the incision site with a topical antiseptic and continue proper aseptic technique during the surgery including using a surgical drape, sterile gloves and instruments, etc.</li>
 <li>Laparotomy 
 Midline abdominal incision is made from lower edge of the liver to just above the pubis.</li>
 <li>Exposure of the aorta and IVC. 
 A retractor is inserted and all of the bowels are pulled out of the abdominal cavity to the right side and kept wrapped in gauze soaked with saline. Dissect the membrane that connects the retroperitoneum and lower colon to get full view of the aorta and IVC. Apply pressure on the bladder if it is dilated and gets in way.</li>
 <li>Exposure of the puncture site 
 Puncture site will be between half to three quarters of the distance from left renal vein to aortic bifurcation. (Figure 1) Dissect the left margin of the aorta so that it can be punctured. Keep in mind that the aorta is positioned about 45 degrees behind the IVC and it must be dissected posteriorly in addition to the left. (Figure 2) The most important point of this surgery is not to dissect between the aorta and IVC. Keep far away from them.</li>
 <li>Exposure of the clamp site of the aorta 
 Dissect the infrarenal aorta from surrounding tissues. Be careful not to injure the IVC on the left and left renal veins with its tributaries on the right.</li>
 <li>Clamping of the aorta 
 Clamp the proximal aorta by applying microsurgery clips.</li>
 <li>Rotation of the aorta 
 By grasping the connective tissue surrounding the aorta, rotate the aorta so that the puncture can be made from slightly dorsal side of the aorta (Figure 2).</li>
 <li>Puncture of the aorta through to IVC 
 Keeping the aorta in a rotated position, puncture the aorta through to IVC using a 25 G needle. (Figure 2) If successful, the needle can be seen through the thin IVC wall.</li>
 <li>Hemostasis 
 By pulling the retroperitoneal tissues, including the muscles, wrap the puncture site of the aorta. Do not compress the aorta against the IVC, as it may clot the AVF (Figure 3).</li>
 <li>De-clamping the aorta 
 After confirmation of the primary hemostasis, de-clamp the aorta. Additional bleeding may occur but it can be stopped with further compression described above. Upon de-clamping, arterial blood can be seen flowing into the IVC instead of dark venous blood flow.</li>
 <li>Closure of the abdomen 
 Return the bowels into its natural position and close the abdomen.</li>
</ol>
4. Post-operative Procedures
<ol>
 <li>Post-operative care 
 After closure of the abdomen, isoflurane is stopped. Post-operative care including analgesia and wound care is applied in accordance with the instructions recommended by Institutional Animal Care and Use Committee. For analgesia we use buprenorphine at 0.1 mg/kg intrasmuscularly every 12 hr for 24 hr following the surgical procedures.</li>
 <li>Post-operative ultrasound 
 On the first day after operation, Doppler ultrasound is performed to confirm the AVF. In addition, other variables are measured to check the changes from pre-operative values.</li>
</ol>

<ol><li>Allon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Current+management+of+vascular+access%5BTitle%5D)+AND+%22Clinical+Journal+of+the+American+Society+of+Nephrology%22%5BJournal%5D)">Current management of vascular access.</a> Clinical Journal of the American Society of Nephrology. 2 (4), 786-800 (2007).</li>
<li>Dixon, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Why+don%27t+fistulas+mature%3F%5BTitle%5D)+AND+%22Kidney+International%22%5BJournal%5D)">Why don't fistulas mature?</a> Kidney International. 70 (8), 1413-1422 (2006).</li>
<li>Roy-Chaudhury, P., Spergel, L. M., Besarab, A., Asif, A., Ravani, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biology+of+arteriovenous+fistula+failure%5BTitle%5D)+AND+%22Journal+of+Nephrology%22%5BJournal%5D)">Biology of arteriovenous fistula failure.</a> Journal of Nephrology. 20 (2), 150-163 (2007).</li>
<li>Kudo, F. A., Muto, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Venous+identity+is+lost+but+arterial+identity+is+not+gained+during+vein+graft+adaptation%5BTitle%5D)+AND+%22Arteriosclerosis%2C+Thrombosis%2C+and+Vascular+Biology%22%5BJournal%5D)">Venous identity is lost but arterial identity is not gained during vein graft adaptation.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 27 (7), 1562-1571 (2007).</li>
<li>Muto, A., Yi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Eph-B4+prevents+venous+adaptive+remodeling+in+the+adult+arterial+environment%5BTitle%5D)+AND+%22The+Journal+of+Experimental+Medicine%22%5BJournal%5D)">Eph-B4 prevents venous adaptive remodeling in the adult arterial environment.</a> The Journal of Experimental Medicine. 208 (3), 561-575 (2011).</li>
<li>Croatt, A. J., Grande, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+a+model+of+an+arteriovenous+fistula+in+the+rat%3A+the+effect+of+L-NAME%5BTitle%5D)+AND+%22The+American+Journal+of+Pathology%22%5BJournal%5D)">Characterization of a model of an arteriovenous fistula in the rat: the effect of L-NAME.</a> The American Journal of Pathology. 176 (5), 2530-2541 (2010).</li>
<li>Misra, S., Fu, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+rat+femoral+arteriovenous+fistula+model%3A+increased+expression+of+matrix+metalloproteinase-2+and+-9+at+the+venous+stenosis%5BTitle%5D)+AND+%22Journal+of+Vascular+and+Interventional+Radiology%22%5BJournal%5D)">The rat femoral arteriovenous fistula model: increased expression of matrix metalloproteinase-2 and -9 at the venous stenosis.</a> Journal of Vascular and Interventional Radiology. 19 (4), 587-594 (2008).</li>
<li>Kojima, T., Miyachi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+relationship+between+venous+hypertension+and+expression+of+vascular+endothelial+growth+factor%3A+hemodynamic+and+immunohistochemical+examinations+in+a+rat+venous+hypertension+model%5BTitle%5D)+AND+%22Surgical+Neurology%22%5BJournal%5D)">The relationship between venous hypertension and expression of vascular endothelial growth factor: hemodynamic and immunohistochemical examinations in a rat venous hypertension model.</a> Surgical Neurology. 68 (3), 277-284 (2007).</li>
<li>Caplice, N. M., Wang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neoangiogenesis+and+the+presence+of+progenitor+cells+in+the+venous+limb+of+an+arteriovenous+fistula+in+the+rat%5BTitle%5D)+AND+%22American+Journal+of+Physiology+Renal+Physiology%22%5BJournal%5D)">Neoangiogenesis and the presence of progenitor cells in the venous limb of an arteriovenous fistula in the rat.</a> American Journal of Physiology Renal Physiology. 293 (2), F470-F475 (2007).</li>
<li>Nath, K. A., Kanakiriya, S. K., Grande, J. P., Croatt, A. J., Katusic, Z. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increased+venous+proinflammatory+gene+expression+and+intimal+hyperplasia+in+an+aorto-caval+fistula+model+in+the+rat%5BTitle%5D)+AND+%22The+American+Journal+of+Pathology%22%5BJournal%5D)">Increased venous proinflammatory gene expression and intimal hyperplasia in an aorto-caval fistula model in the rat.</a> The American Journal of Pathology. 162 (6), 2079-2090 (2003).</li>
<li>Karram, T., Hoffman, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+of+cardiac+hypertrophy+by+a+controlled+reproducible+sutureless+aortocaval+shunt+in+the+mouse%5BTitle%5D)+AND+%22Journal+of+Investigative+Surgery%22%5BJournal%5D)">Induction of cardiac hypertrophy by a controlled reproducible sutureless aortocaval shunt in the mouse.</a> Journal of Investigative Surgery. 18 (6), 325-334 (2005).</li>
<li>Perry, G. J., Mori, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+variation+in+angiotensin-converting+enzyme+does+not+prevent+development+of+cardiac+hypertrophy+or+upregulation+of+angiotensin+II+in+response+to+aortocaval+fistula%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Genetic variation in angiotensin-converting enzyme does not prevent development of cardiac hypertrophy or upregulation of angiotensin II in response to aortocaval fistula.</a> Circulation. 103 (7), 1012-1016 (2001).</li>
<li>Guzman, R. J., Krystkowiak, A., Zarins, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+and+sustained+medial+cell+activation+after+aortocaval+fistula+creation+in+mice%5BTitle%5D)+AND+%22The+Journal+of+Surgical+Research%22%5BJournal%5D)">Early and sustained medial cell activation after aortocaval fistula creation in mice.</a> The Journal of Surgical Research. 108 (1), 112-121 (2002).</li>
<li>Castier, Y., Lehoux, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+neointima+lesions+associated+with+arteriovenous+fistulas+in+a+mouse+model%5BTitle%5D)+AND+%22Kidney+International%22%5BJournal%5D)">Characterization of neointima lesions associated with arteriovenous fistulas in a mouse model.</a> Kidney International. 70 (2), 315-320 (2006).</li>
</ol>

There are several reports that have described AVF in rats, including femoral AVF,6, 7 carotid to jugular AVF8 and aorto-caval AVF.9, 10 The rat is several times larger than the mouse, allowing easier surgery in the rat compared to the mouse. Nevertheless, in order to take advantage of mouse genetic strains and variants the procedure must be extended to include mouse models. Several previously published reports introduced the AVF model in mice, but these reports have generally been in the ...

The most common option chosen for renal replacement therapy in patients with end stage renal disease is hemodialysis. Access for hemodialysis is performed by arteriovenous fistula (AVF), arteriovenous graft, or temporary catheter placement. Although AVF are the preferred mode of access, AVF are still far from perfect. One-year primary AVF patency rates are only 60-65%, with many failures due to neointimal hyperplasia. The molecular biology and physiology of normal AVF formation and maladaptive formation remains poorly characterized. As such, much is to be gained in improving AVF outcomes with the accompanying gain in understanding of the regulation of fistula formation.1-3
Exposure of a vein to arterial flow is commonly studied in the context of vein grafts that are direct models of surgical bypass.4, 5 Although vein grafts and AVF similarly expose a vein to an arterial environment, there are several critical differences between them. First, the resistance of the distal runoff bed is much lower in AVF, as the AVF drains to the right atrium whereas the vein graft drains to the often diseased distal small arteries, resulting in increased blood flow and shear stress in the AVF. Second, the blood in the venous limb of the AVF can be a mixture of venous and arterial blood leading to lower oxygenation compared to the purely arterial content in the vein graft. Third, the vein in the AVF is not removed from its bed, leading to preservation of its vaso venorum and nervous innervations, compared to the complete transposition of the vein graft into the arterial bed. Lastly, the number of anastomoses is usually two in vein grafts whereas an AVF typically has only one. In this study, we introduce a mouse model of aortocaval fistula that can be used for the study of AVF.

<table><tbody><tr><td>Vevo 770 ultrasound machine</td><td>Visualsonics</td><td>20-60 Mhz scan head; RMV-704</td><td></td></tr><tr><td>Vascular clamp</td><td>Roboz Surgical Instrument Co.</td><td>RS-5424</td><td></td></tr><tr><td>Clamp applying forceps</td><td>Roboz Surgical Instrument Co.</td><td>RS-5410</td><td></td></tr><tr><td>25 Gauge Needle</td><td>Becton Dickinson</td><td>BD PrecisionGlide Needle 25G x 7/8 #305124</td><td></td></tr></tbody></table>

technical aspects of the mouse aortocaval fistula

Technical Aspects of the Mouse Aortocaval Fistula

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta and inferior vena cava (IVC) are exposed. The proximal infrarenal aorta and the distal aorta are dissected for clamp placement and needle puncture, respectively. Special attention is paid to avoid dissection between the aorta and the IVC. After clamping the aorta, a 25 G needle is used to puncture both walls of the aorta into the IVC. The surrounding connective tissue is used for hemostatic compression. Successful creation of the AVF will show pulsatile arterial blood flow in the IVC. Further confirmation of successful AVF can be achieved by post-operative Doppler ultrasound.

After practice, survival on the first post-operative day is typically over 95%; at post-operative day 7, survival was 91.3%. Figures 1 and 2 show the ideal puncture site in coronal and cross-sectional views; placement of the puncture site is critical to obtaining good survival without excessive hemorrhage. Intra-operative deaths and early post-operative deaths are typically due to hemorrhage. This tends to occur with excessive dissection between the aorta and the IVC, or from inadequate hemostatic pressu...

The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis.

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta ...

The overall goal of this procedure is to create successful aorta, cable fistulas, and this is accomplished by first carefully dissecting the aorta near the puncture site. Next, the aorta is clamped proximal to the desired position of the aorta caval fistula. Then a 25 gauge needle is used to make a puncture from the left aorta to the inferior vena cava.Finally, hemostasis is accomplished without occluding the aorta caval fistula. Ultimately, Doppler ultrasound is used to show an increase in caval blood flow. The main advantage of this technique over existing methods like aortic caval, an osmosis, is that it is a straightforward method that can be completed in short time and does not use sutures that require time and training Prior to the operation.Use pulse doppler to take baseline recordings of the supra and infra renal inferior vena cava or IVC and abdominal aorta using B mode, measure the diameter of each vasculature as well as the maximum velocity and waveform in each of the measuring points. Begin the procedure by autoclaving all surgical instruments and vascular clamps. After anesthetizing a male C 57 black, 10 week old mouse in 4%isof fluorine with 0.8 liters per minute.Oxygen, use a toe pinch to check level of sedation. Place the mouse in a supine position. Attach a silicone mask and administer 2%isof fluorine.Use a chemical hair remover to remove the hair from the neck to the lower abdomen. First, disinfect the surgical area with 70%ethanol. Under a surgical scope, make a midline incision from the lower edge of the liver to just above the pubis.Insert a retractor and remove the entire bowel from the abdominal cavity. Then wrap it in saline soaked gauze and place it on the right side. Dissect the membrane that connects the retroperitoneum and lower colon to get a full view of the aorta and IVC.Apply pressure on the bladder if it is dilated and gets in the way. Next, add a site between half to three quarters of the distance from the left renal vein to the aortic bifurcation. Dissect the left margin of the aorta so that it can be punctured.The aorta is positioned about 45 degrees behind the IVC, and therefore it must be dissected posteriorly and to the left. Taking care not to dissect between the aorta and IVC, then carefully dissect the infrarenal aorta from the surrounding tissues, so as not to injure the IVC on the left and left renal vein with its tributaries on the right. Using microsurgery clips, clamp the proximal aorta, grasp the connective tissue surrounding the aorta and IVC and rotate it so that the puncture can be made from a slightly dorsal side of the aorta.While in this rotated position, use a 25 gauge needle to puncture the aorta through to the IVC. The needle should be seen through the thin IVC wall. After confirming a successful hemostasis dec clamp, the aorta rather than dark venous blood flow arterial blood can now be seen flowing into the IVC.Finally, return the bowels to their natural position and use six oh sutures to close the abdomen. Discontinue the isof fluorine and administer 0.1 milligrams per kilogram of buprenorphine intramuscularly every 12 hours for 24 hours postoperatively, house the animal in a solitary cage during recovery and administer wound care in accordance with your institution's animal care and use guidelines. The following day performed Doppler ultrasound to confirm the A VF and to check for changes in measurements taken prior to surgery.As shown in these coronal and cross-sectional views of the ideal puncture site placement is critical to obtaining good survival without excessive hemorrhaging, which typically occurs with too much dissection between the aorta and IVC or from inadequate hemostatic. Pressure after practice survival on the first postoperative day is typically over 95%and approximately 91%at postoperative day seven. This schematic demonstrates the importance of wrapping the puncture site with connective tissue prior to applying pressure for hemostasis, allowing good hemostasis without compression of the A VF.In our experience, a successful A VF can be confirmed with ultrasound by the third day.Once the A VF is confirmed by duplex to be present, it will usually remain stable until day 28. This figure shows typical doppler ultrasound findings in a successful A VF formation. Changes in waveforms are observed at the level of the A VF in the IVC and towards the heart.The overall A VF confirmation rate on postoperative day three is 71.4%Once mastered, this technique can be completed in 15 to 20 minutes if performed correctly.

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Technical Aspects of the Mouse Aortocaval Fistula

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