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 JoVE Biology

Murine Model of Hindlimb Ischemia

1, 1, 2, 1

1Division of Cardiovascular Medicine, Stanford University, 2Department of Anesthesiology, University of California, San Francisco

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    Summary

    The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.

    Date Published: 1/21/2009, Issue 23; doi: 10.3791/1035

    Cite this Article

    Niiyama, H., Huang, N. F., Rollins, M. D., Cooke, J. P. Murine Model of Hindlimb Ischemia . J. Vis. Exp. (23), e1035, doi:10.3791/1035 (2009).

    Abstract

    In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide. Medical therapies for symptomatic relief are limited. Surgical or endovascular interventions are useful for some individuals, but long-term results are often disappointing. As a result, there is a need for developing new therapies to treat PAD. The murine hindlimb ischemia preparation is a model of PAD, and is useful for testing new therapies. When compared to other models of tissue ischemia such as coronary or cerebral artery ligation, femoral artery ligation provides for a simpler model of ischemic tissue. Other advantages of this model are the ease of access to the femoral artery and low mortality rate.

    In this video, we demonstrate the methodology for the murine model of unilateral hindimb ischemia. The specific materials and procedures for creating and evaluating the model will be described, including the assessment of limb perfusion by laser Doppler imaging. This protocol can also be utilized for the transplantation and non-invasive tracking of cells, which is demonstrated by Huang et al.1.

    Protocol

    1. Induction of Unilateral Hindlimb Ischemia

    1. The surgical tools needed for this operation include: fine pointed forceps, pointed forceps, spring scissors, surgical scissors, needle holder, and retractor. We make our own retractor using a paperclip because it is smaller than commercially available retractors. Sterilize these tools prior to surgery by an autoclave or a hot-bead sterilizer. A cautery tool and sterile fine pointed cotton swabs will also be needed for this surgery. It is recommended that the tools be re-sterilized at the tips as needed during the procedure.
    2. When the tools are ready, place the mouse into the anesthesia induction chamber containing 1–3% isoflurane in 100% oxygen at a flow rate of 1L/min.
    3. Leave the mouse in the induction chamber until it is unresponsive to external stimuli. Then remove the animal from the induction chamber. It is recommended to flush the anesthetic from the box prior to opening the lid, to decrease operator exposure to isoflurane.
    4. Then place the animal in the supine position onto the pre-operating table and connect it to a continuous flow of isoflurane. Using an electric shaver, remove the hair from the hindlimb. Apply hair removal cream to thoroughly remove hair.
    5. Place the mouse in the supine position over a draped heated pad on the operating table, and connect it to a continuous flow of isoflurane. Extend and secure the hindlimb with a piece of tape. Once the hindlimb is secure, wipe the skin with three alternating betadine and alcohol scrubs. For the remainder of the surgical procedure, use a dissection microscope at 10X or 20X magnification to obtain an enlarged view of the hindlimb region.
    6. Using fine forceps and surgical scissors, make an incision of the skin, approximately 1 cm long, from the knee towards the medial thigh.
    7. Using phosphate buffered saline (PBS)-moistened fine pointed cotton swabs, gently brush away subcutaneous fat tissue surrounding the thigh muscle.
    8. Next, apply the cautery transversely to incise and dissect through the subcutaneous fat tissue to reveal the underlying femoral artery.
    9. Use a retractor to open the wound and to obtain a better view of the lower extremity vasculature.
    10. Using fine forceps and a fine pointed cotton swab, gently pierce through the membranous femoral sheath to expose the neurovascular bundle.
    11. Then, using a clean set of fine forceps and cotton swab, dissect and separate the femoral artery from the femoral vein and nerve at the proximal location near the groin. After the dissection, pass a strand of 7-0 silk suture underneath the proximal end of the femoral artery. Occlude the proximal femoral artery using double knots. Place the tie on the vessel as proximal in the wound as possible in order to leave length for the second tie and an intervening segment that will be transected.
    12. Separate the femoral artery from the femoral vein at the distal location close to the knee. Pass a strand of 7-0 suture underneath the distal end of the femoral artery proximal to the popliteal artery. Occlude the vessel using double knots.
    13. Occlude the distal femoral artery with a second set of double knots just proximal to the first set of knots. This second set of sutures will be used for gripping the artery during the transaction procedure.
    14. Similarly, for gripping purposes, occlude the proximal femoral artery with a second set of double knots just distal to the first set of knots.
    15. Transect the segment of femoral artery between the distal and proximal knots with a fine pointed cotton swab and a pair of spring scissors. Use caution not to pierce the femoral vein wall.
    16. Remove the retractor and close the incision using 5-0 Vicryl sutures. These sutures do not need to be removed at a later time, as they dissolve on their own.
    17. Once the incision is closed, place the animal on top of a draped heated pad in the recovery cage and monitor continuously until awake.
    18. After animal has recovered for 1 hour, proceed with the laser Doppler blood perfusion step in order to confirm the ischemia induction.

    2. Laser Doppler Blood Perfusion

    1. To begin the laser Doppler perfusion step, place the mouse into the anesthesia induction chamber containing 1–3% isoflurane in 100% oxygen at a flow rate of 1L/min.
    2. Leave the mouse in the induction chamber until it is unresponsive to external stimuli. Then remove the animal from the induction chamber. It is recommended to flush the anesthetic from the box prior to opening the lid, to decrease operator exposure to isoflurane.
    3. Place the animal on the pre-operating table connected to continuous flow of isoflurane. Then remove the hair from the hindlimb using an electric shaver followed by hair removal cream as necessary.
    4. After removing the hair, place the animal on a 37°C heated surface for 5 minutes under continuous flow of isoflurane. Monitor the core temperature to ensure euthermia, as temperature changes will markedly affect perfusion.
    5. After 5 minutes, place the animal in the supine position on a non- reflective light- absorbing surface such as green-colored cloth, connected to a continuous flow of isoflurane. Extend the hindlimbs.
    6. Next, turn on the laser Doppler imager and the acquisition software and initialize the software. Specify the size of the field of view and resolution. It is best to keep the field of view and pixel density consistent between animals in order to make future analysis easier.
    7. Open a new file. Press start to begin acquiring the image data. Generally, the imager automatically detects the distance of the mouse, but if prompted, specify the distance of the animal to the imager. The laser will show the borders of the field of view and then start acquiring data.
    8. After acquisition is complete, the image will begin to show a range of colors that are indicative of the level of blood perfusion to the legs. The colors can be set to a specific perfusion range to better compare data between animals.
    9. When the data acquisition is complete, save the file.
    10. Then return the animal to the recovery cage and monitor the animal continuously until awake.
    11. To analyze the data, use the threshold adjustment in the analysis software to substract any background noise. A threshold of 0.2 is usually a reasonable value. Select 2 regions of interest, or ROIs, that cover each hindlimb area. A variety of landmarks can be used to standardize the ROI between limbs and animals. Then determine the mean perfusion and variability in the ROI. The perfusion difference and perfusion ratio between the ischemic limb and control limb can then be easily determined.
    12. This procedure can be repeated to follow changes in hindlimb perfusion over time. At a desired time point, the animal can be euthanized for assessment of tissue function and comparison to perfusion image data.

    3. Representative Results:

    The anatomy of the hindlimb vasculature is shown in Figure 12. A representative diagram of the hindlimb after femoral artery explantation is shown in Figure 2. To confirm the induction of ischemia to the hindlimb, laser Doppler perfusion image analysis demonstrates a dramatic reduction in blood flow to the ischemic limb, in comparison to the control limb, as shown in Figure 3.

    Figure 1

    Figure 1. Anatomy of the hindlimb vasculature.  Asterisks indicate the locations of ligation for the induction of hindlimb ischemia.

    Image 2

    Figure 2. Representative diagram showing the anatomy of the hindlimb after ligation of the femoral artery at the proximal and distal sites after the removal of the femoral artery.

    Figure 3

    Figure 3. Laser Doppler images showing blood flow before and after the induction of ischemia to the left hindlimb (indicated by arrow).

    Discussion

    There are some sources of variability to consider while planning and performing hindlimb ischemia models. First, the level of ischemia may vary according to the location of the ligation with respect to that of side branches. Therefore, for consistency of the model, the animals should undergo the same level of arterial ligation. Another source of variability in ischemic recovery is related to the age of the animals, with young animals (6–8 weeks old) having faster and more complete recovery rates than older animals (8–10 months old), as assessed by hemodynamic (i.e. laser Doppler perfusion) or functional (i.e. treadmill testing) measures. For studies in which one is assessing an angiogenic agent, it may be preferable to use older animals, because a greater difference between groups may be observed with a therapeutic intervention. Conversely, for studies in which one is assessing an anti-angiogenic factor, it may be preferable to use younger animals to maximize effect size3.

    If performed correctly, there should be minimal complications such as bleeding, infection, or mortality. If bleeding occurs by accidental disruption of the femoral vein or other vessels, moderate pressure with a sterile cotton tipped applicator or gauze should be applied to the site of hemorrhage until the bleeding stops. Animals showing signs of infection should be treated with anti-infective agents. Animals exhibiting significant gangrene may need to beeuthanized. This complication is more common in older animals, and in some strains, such as BALB c mice4. In addition, the hindlimb ischemia model may cause mild to moderate pain. Therefore, analgesics such as buprenorphine or carprofen should be administered as needed for treatment of pain, according to recommendations of the IACUC.

    In conclusion, we have demonstrated a simple and reproducible method for studying PAD in a murine model of hindlimb ischemia. The hindlimb ischemia model with laser Doppler imaging analysis is an excellent system for studying vascular pathologies and for assessing therapeutic candidates.

    Disclosures

    Acknowledgements

    The authors thank Andrea Axtell, Satoshi Itoh, MD, Jeff Velotta, MD, Grant Hoyt, Robert C. Robbins, MD, Jin Yu, MD, Tim Doyle, PhD, and the Stanford Small Animal Imaging Core for technical assistance. The authors also thank A.M. Bickford, Inc. for support of veterinary equipment. This research was supported by research grants from the National Institutes of Health (R01 HL-75774, R01 CA098303, R21 HL085743, 1K12 HL087746), the California Tobacco Related Disease Research Program of the University of California (15IT-0257 and 1514RT-0169), and the California Institute for Regenerative Medicine (RS1-00183). N.H. is supported by a fellowship from the American Heart Association.

    Materials

    Name Type Company Catalog Number Comments
    Surgical Tools Tool Fine Science Tools
    Constix Cotton Swabs Tool Contec SC-4
    Betadine (Povidone-iodine) Reagent PDI
    70% alcohol Reagent Kendall
    Phosphate Buffered Saline Reagent Invitrogen
    7-0 silk suture Tool Genzyme
    5-0 vicryl suture Tool Ethicon Inc.
    Electric shaver Tool GE Healthcare
    Cautery Tool Baxter Internationl Inc.
    Laser Doppler and PeriScan PIM 3 System Equipment Perimed

    References

    1. Huang, N.F., Niiyama, H., De, A., & Cooke, J.P. Transplantation and non-invasive tracking of embryonic stem cell-derived endothelial cells for treatment of hindlimb ischemia. JoVE (2008).
    2. Cook, M.J. The anatomy of the laboratory mouse. Academic Press, New York (1976).
    3. Niiyama, H., Kai, H., Yamamoto, T., Shimada, T., Sasaki, K., Murohara, T., Egashira, K., & Imaizumi, T. Roles of endogenous monocyte chemoattractant protein-1 in ischemia-induced neovascularization. J. Am. Coll. Cardiol. 44, 661-6 (2004).
    4. Dokun, A.O, Keum, S., Hazarika, S., Li, Y., Lamonte, G.M., Wheeler, F., Marchuk, D.A., & Annex, B.H. A quantitative trait locus (LSq-1) on mouse chromosome 7 is linked to the absence of tissue loss after surgical hindlimb ischemia. Circulation. 117, 1207-15 (2008).

    Comments

    43 Comments

    Is there really "8–10 months old" mice,in your discussion section ? Or it should be 8–10 weeks old?   Thank you.
    Reply

    Posted by: AnonymousApril 16, 2009, 4:58 AM

    Dear Chris, As indicated in the discussion, we prefer using old mice (8-10 months old) to mimic the population of patients who tend to suffer from peripheral arterial disease (>60 years). Young mice (<8 weeks old) have a faster recovery rate than old mice (8-10 months old) and more easily recover without any therapeutic intervention.  Therefore, the fast recovery rate of young mice  may interfere with studies to assess the therapeutic effect of new treatments. Thank you for your inquiry. Sincerely, Ngan Huang, PhD
    Reply

    Posted by: AnonymousApril 16, 2009, 6:34 PM

    Dear Sir or Madam,
    I study in University of Thessaly (Greece), department of Biochemistry & Biotechnology. I have to make a presentation about hindlimb Ischemia and your video above would help me a lot. So, I would like to know if there is any possibility to obtain this video. Thank you in advance for your time.
    Yours faithfully,
    Dimitriou Tilemachos
    Reply

    Posted by: AnonymousMay 31, 2009, 9:55 AM

    Dear Dimitriou Tilemachos,
    You have the permission of the authors to use the video for your presentation as long as you cite this publication appropriately. However, you should contact the publisher for their permission as well. You can also contact the publisher for your request to obtain a copy of the video., as we do not have a copy of it.

    Sincerely,
    Ngan Huang, PhD
    Reply

    Posted by: AnonymousMay 31, 2009, 6:41 PM

    Do you believe that using laser doppler is really necessary to assure perfusion deficits and ischemia? Why not just traditional echo/doppler. We have echo, but not Laser Doppler and were hoping to do PAD studies in mice. Laser Doppler is great , but very expensive, and am not sure if we want to make this type of investment to study PAD in mice. What are your thoughts?
    Reply

    Posted by: AnonymousAugust 24, 2009, 1:42 PM

    Standard echo/doppler dŒs not have the resolution to accurately assess conduit artery diameter and flow in the mouse.
    Furthermore, after induction of hindlimb ischemia, flow is via collaterals that are too small to be imaged by standard echo/doppler.
    Reply

    Posted by: AnonymousAugust 24, 2009, 4:56 PM

    It is a excellent video and will help me a lot.
    I want to know how long the ischemial will last in these age mice.
    Reply

    Posted by: xiaohui z.August 29, 2009, 7:10 AM

    Thanks for your nice comments. The ischemic deficit persists for at least 4 weeks in the older C57Bl mice. The ischemic deficit is even worse in Balb/c mice. See the paper by Dokun et al in Circulation ²008
    Reply

    Posted by: AnonymousAugust 29, 2009, 3:41 PM

    Thank you for this article. It is very helpful. A few questions:
    1. Why is it necessary to dissect out the artery? Why not just ligate the vein and artery together?
    ². Why is it necessary to resect a section of vessel? Why not just place one ligature? Or if you want to place two, why not just cut the part in the middle without bothering to remove that segment? Thank you.
    Reply

    Posted by: AnonymousJanuary 6, 2010, 8:53 PM

    Regarding ligating the artery and vein together, what clinical condition are you trying to mimic? You might consider the paper by JT Shepherd published in Circulation in 195². This is available at http://circ.ahajournals.org/cgi/reprint/6/²/²81. "The calf blood flow has been studied in the human subject before and during acute occlusion of the common femoral artery and vein. The results suggest that the concomitant venous occlusion has a detrimental effect on the development of the collateral circulation."
    Reply

    Posted by: AnonymousJanuary 9, 2010, 11:25 AM

    This is an excellent publication. I would like to know if the authors can comment on the differences between the PeriMed PIM3 imaging system compared to the Moor LDI² system, namely whether you achieve the same image resolution and whether quantitation of ROI is the similar. This will aid us in deciding on which system to use. Thanks
    Reply

    Posted by: AnonymousDecember 2, 2010, 12:49 PM

    I would be happy to provide this information, and further instrument comparison data.
    Please feel free to contact me with any instrument-related queries.

    Sam Pakvis
    Perimed Inc
    spakvis@perimed-instruments.com
    70²-987-4655
    Reply

    Posted by: AnonymousAugust 24, 2011, 12:11 PM

    We would also be pleased to offer any comparison data, along with an on-site demonstration of our application specific system if desired. Please let us know if you have any further questions and we'll be happy to help.

    Moor Instruments, Inc.
    sales@moorinc.com
    30² 798 7470
    Reply

    Posted by: AnonymousFebruary 14, 2012, 9:19 AM

    First of all I would like to thank you. I learned so much thing from this paper and video as resulted me a Ph.D degree. I watched many times the video how to do ischemic surgery.

    My question is regarding for ischemia and angiogenesis. If the surgery done like ligation and excision of both femoral artery and vein. DŒs it have same effect with only artery ligation and excision? Which one should be done for ischemic condition and angiogenic development?

    Thank you very much again
    Mehmet Zeynel Cilek, Ph.D
    Okayama University, Japan
    Reply

    Posted by: Mehmet Zeynel C.April 24, 2011, 9:36 PM

    Dear Mehmet,

    I am the animal technician at the Cooke lab and Dr. Cooke has directed me to answer some of your questions. We would expect the excision and ligation of both the femoral artery and vein to have a similar effect to ligating and excising the femoral artery only, except that it would create more ischemia and necrosis. We find that ligating/excising the artery alone is sufficient to cause ischemia and actually preferable to ligating both, since there is a higher chance of the animal losing the entire limb or dying if the vein was excised as well. Ligating only the artery can also cause necrosis, but it is usually localized to the tŒs and sometimes foot.
    Reply

    Posted by: AnonymousApril 25, 2011, 2:02 PM

    Dear Mehmet,

    I am the animal technician at the Cooke lab and Dr. Cooke has directed me to answer some of your questions. We would expect the excision and ligation of both the femoral artery and vein to have a similar effect to ligating and excising the femoral artery only, except that it would create more ischemia and necrosis. We find that ligating/excising the artery alone is sufficient to cause ischemia and actually preferable to ligating both, since there is a higher chance of the animal losing the entire limb or dying if the vein was excised as well. Ligating only the artery can also cause necrosis, but it is usually localized to the tŒs and sometimes foot.
    Reply

    Posted by: AnonymousApril 25, 2011, 2:02 PM

    Thank you for an excellent, clear demonstration of this method. I have read through the comments and questions and could not find the response to a question that interested me. The question was: Why is it necessary to ressect a section of vessel? Why not just place one ligature? Or if you place two, why not just cut the part in the middle without bothering to remove that segment? It'd be great to have your response to these points. Thanks
    Thanks.
    Reply

    Posted by: AnonymousMay 30, 2011, 7:11 PM

    Hi MJC,
    We have studied blood flow recovery in mice after excision of the femoral artery as well as when the proximal and distal ends were ligated without excision. We find that ligation without excision leads to a much faster recovery in limb perfusion, sometimes reaching baseline within ² weeks, presumably due to collateral blood flow. However, when the femoral artery is excised after ligation, the blood flow recovery after ² weeks is generally only about 50-60% of baseline.

    Regards,
    Ngan Huang, PhD
    Reply

    Posted by: AnonymousMay 31, 2011, 12:54 PM

    Hi MJC,
    To provide additional clarification, we have found this observation in blood flow recovery to be true of the C57BL6 and Nod SCID strains. We have not used BalbC mice, but they are purportedly more susceptible to limb loss after induction of hindlimb ischemia, and do not recover as well.

    Regards,
    Ngan Huang, PhD
    Reply

    Posted by: AnonymousMay 31, 2011, 1:55 PM

    Thank you for your fast response, that is really useful information. We have just started to use this model and were curious as to whether excision was necessary, but now we will continue to excise the vessel. Thanks again.
    Reply

    Posted by: AnonymousMay 31, 2011, 6:46 PM

    Dear authors,
    This video is the most important and useful among all the resources a new phd student could find to do his research on PAD. Now coming to my question,
    Q1. Among Balb/c and C57BL/6 mice which one do you think has a higher inflammation in ischemic limb?
    Q². On top of it which one do you think should give higher inflammatory response; increased collateral flow and shear stress (in case of artery ligation only) OR elevation of venous pressure and reduced blood flow (when ligate both artery and vein).
    Sir/Ma'am, I am a new PhD student and appreciate your kind suggestion.
    Warm regards
    Vivek
    Reply

    Posted by: vivek m.June 10, 2011, 1:08 AM

    Dear Vivek,
    We apologize for the delay in replying. We have not studied the inflammatory response between various strains. However, according to the literature, the collateral vasculature dŒs vary between the Balb/c and C57 strains such that perfusion recovery is slower in Balb/c mice.

    Regards,
    Ngan Huang, PhD
    Reply

    Posted by: AnonymousJune 26, 2011, 7:05 PM

    First of all I would like to thank you. .

    My question is regarding analgesia for transplantation of embryonic stem cell in a unilateral hindimb ischemia. I was suprised to fined so little reffrences regarding the appropriat analgesia. Can you recomemed wich analgesia (buprenorphine or carprofen ) will not interfere with the study.
    Thank you very much

    Tal Klein, D.V/M
    Pharmaseedltd, Israel
    Reply

    Posted by: AnonymousJuly 31, 2011, 5:50 AM

    Hi Tal. Since carprofen is a non-steroidal anti-inflammatory drug that can affect angiogenesis, we do not recommend using it for ischemia studies. Instead, we typically use lidocaine for analgesia.

    Ngan Huang, PhD

    Ngan Huang, PhD
    Reply

    Posted by: AnonymousAugust 1, 2011, 8:05 PM

    Hi Tal. Since carprofen is a non-steroidal anti-inflammatory drug that can affect angiogenesis, we do not recommend using it for ischemia studies. Instead, we typically use lidocaine for analgesia.

    Ngan Huang, PhD

    Ngan Huang, PhD
    Reply

    Posted by: AnonymousAugust 1, 2011, 8:05 PM

    Hi Tal. Since carprofen is a non-steroidal anti-inflammatory drug that can affect angiogenesis, we do not recommend using it for ischemia studies. Instead, we typically use lidocaine for analgesia.

    Ngan Huang, PhD

    Ngan Huang, PhD
    Reply

    Posted by: AnonymousAugust 1, 2011, 8:05 PM

    Hi Tal. Since carprofen is a non-steroidal anti-inflammatory drug that can affect angiogenesis, we do not recommend using it for ischemia studies. Instead, we typically use lidocaine for analgesia.

    Ngan Huang, PhD

    Ngan Huang, PhD
    Reply

    Posted by: AnonymousAugust 1, 2011, 8:05 PM

    Hi Tal. Since carprofen is a non-steroidal anti-inflammatory drug that can affect angiogenesis, we do not recommend using it for ischemia studies. Instead, we typically use lidocaine for analgesia.

    Ngan Huang, PhD

    Ngan Huang, PhD
    Reply

    Posted by: AnonymousAugust 1, 2011, 8:05 PM

    Hi Tal. Since carprofen is a non-steroidal anti-inflammatory drug that can affect angiogenesis, we do not recommend using it for ischemia studies. Instead, we typically use lidocaine for analgesia.

    Ngan Huang, PhD

    Ngan Huang, PhD
    Reply

    Posted by: AnonymousAugust 1, 2011, 8:05 PM

    Hi Tal. Since carprofen is a non-steroidal anti-inflammatory drug that can affect angiogenesis, we do not recommend using it for ischemia studies. Instead, we typically use lidocaine for analgesia.

    Ngan Huang, PhD

    Ngan Huang, PhD
    Reply

    Posted by: AnonymousAugust 1, 2011, 8:05 PM

    Hi Tal. Since carprofen is a non-steroidal anti-inflammatory drug that can affect angiogenesis, we do not recommend using it for ischemia studies. Instead, we typically use lidocaine for analgesia.

    Ngan Huang, PhD

    Ngan Huang, PhD
    Reply

    Posted by: AnonymousAugust 1, 2011, 8:05 PM

    Dear Tal Klein,

    Do you have any published literature where carprophen affecting the angeogenesis. As I am going to replace carprophen with buprenorphine.

    Regards,

    DC
    Reply

    Posted by: DC S.June 26, 2013, 8:33 PM

    Hi Dr. Huang,

    I really appreciate you and your colleagues doing this video. I have a question about the analgesia. How often do you administer lidocaine? I'm trying to get an idea of if "as needed means" once every few days or once every few hours.

    Thanks!
    Maggie Kuo
    Reply

    Posted by: AnonymousOctober 27, 2011, 4:50 PM

    Hey Maggie,

    I'm the animal technician in charge of the hindlimb ischemia surgeries for the Cooke lab. We use lidocaine to temporarily relieve post-operative pain as a local anesthetic. These animals should be monitored constantly after surgery (approx. every ten minutes for an hour and hourly after that), and if you see the standard signs of distress like rough hair coat, curling in a hunched position, failure to eat/drink, etc., you should administer more lidocaine to the surgery site. But generally lidocaine will not be enough to reduce pain, and typically IACUC suggests the administration of an analgesic like buprenorphine daily for a week after surgery. You should check with the IACUC at your institution to see what analgesic they recommend and how often it should be administered.

    Best,
    Jerry
    Reply

    Posted by: AnonymousOctober 27, 2011, 9:45 PM

    Thanks for the very informative video. My question was also about analgesia. Do the animals generally experience much tissue necrosis, or limb loss as a result of this procedure? If a limb is heavily necrotic should it be amputated? Is it only necessary to closely monitor the animals and give extra analgesia in the week following surgery or is this sometimes necessary at later timepoints as the limb becomes progressively more necrotic? Thanks, John
    Reply

    Posted by: AnonymousMarch 7, 2012, 12:38 PM

    Dear John,

    Necrosis and limb loss is more dependent on age and strain of animal; in C57s, there is typically no limb loss with some tŒ necrosis, but older animals (²0 weeks+) can exhibit limb loss. Balb/c mice are known to have worse recovery than other strains. If the limb is heavily necrotic, your institution's veterinary services would probably suggest early euthanasia; I would check with them first. Extra analgesia can usually be given as needed, so if the limb becomes heavily necrotic you can consider giving more. But keep in mind that certain analgesics can affect angiogenesis -- again, I would check with your institution's veterinary services/IACUC.

    Best,
    Jerry
    Reply

    Posted by: AnonymousMarch 7, 2012, 11:27 PM

    Dear Dr. Huang:
    Thank you very much for sharing this excellent video on HLI. Do you usually see linear correlation between the increase of blood flow, as measured by laser Doppler, and the functional recovery and/or ischemic clinical scores of the ischemic limb?
    Thanks,
    Moses Chen
    Reply

    Posted by: AnonymousMarch 8, 2012, 3:49 PM

    Dear Moses,

    The ischemic clinical scores are a very rough approach to assessing ischemia-induced disability. The perfusion can become quite low before any gangrene is observed. We have not correlated perfusion to gangrene, but it is likely that the more severe gangrene would be associated with more severe blood flow reduction.

    Best,
    Jerry
    Reply

    Posted by: AnonymousMarch 26, 2012, 2:06 PM

    Hi - Thanks for this incredibly informative publication. It can be difficult to access such specialised knowledge easily and this video has made it possible to see the whole procedure in a clear manner. I had a question with regard to choice of mouse strain for such a study. I have read the previous questions and comments on this page about BalBC vs C57s in terms of severity of ischemia. We need to use nude animals for our study. I have found it difficult to source nude C57s. Our current options seem to be Beige Nude, Athymic nude, BALB/c nude, NMRI nude and MF1 nude. Apparently the athymic nude is derived from a BALB/c so it may suffer the same harsh ischemia as the original strain. I have no idea about the other strains - most papers don't go into much detail about the strain they use (they just mention nude mice) so I am finding it hard to make a decision. If you had any suggestions or advice it would be most appreciated. Many thanks, John
    Reply

    Posted by: John S.May 3, 2012, 6:41 AM

    Hi - Thank you for such an informative publication. It can be difficult to easily access such specialised knowledge and this video makes the whole procedure very clear. I had a question about mouse strains for use with this model. I have read the previous comments about C57 vs BALB/c in terms of severity of ischemia. We have to use nude mice for our study. We are having a hard time sourcing nude C57s. The current options available to us are Beige nude, athymic nude, BALB/c nude, NMRI nude and MF1 nude. The athymic nude is derived from a BALB/c so might display the same poor recovery as this strain. I noticed that you had used NOD SCID mice before but we don't want to use a diabetic animal. Most papers I've read don't usually go into much detail of the strain of nude mouse they have used for their hindlimb ischemia experiments, but merely mention that they used nude mice. I was hoping you might have some input or suggestions with regard to the most appropriate strain to use to test a pro-angiogenic therapeutic - ie. a mouse that will display a suitable level of ischemia for the duration of a four week study. I think we only have access to relatively young animals also, so would their ability to recover rapidly also be an issue? Many thanks, John Smith
    Reply

    Posted by: John S.May 3, 2012, 6:55 AM

    Hi John,
    In regards to immunodeficient mouse strains, we have not done extensive comparisons of the nude mice strains. We have found that Beige nude mice immediately after HLI reduces blood flow to about ²5% of normal, but haven't fully characterized their perfusion recovery over time. We have not tried the other nude strains for comparison. We do have alot of experience working with NOD.SCID mice that are IL²receptor gamma deficient. Among SCID mice, this strain is supposed to be the most immunocompromised, so we typically use this strain for our studies in which human cells are delivered. If you're studies allow for the use of SCID mice, our recent publication (Rufaihah J et al. ATVB ²011) describes the temporal kinetics of perfusion recovery using this strain. Ultimately, the level of ischemia induction is somewhat dependent on the operator, so it would be worthwhile to characterize the level of flow reperfusion in your own hands.
    Regards,
    Ngan Huang
    Reply

    Posted by: Ngan H.May 5, 2012, 1:35 PM

    Hi, Thanks for the very nice work!
    My question is: Will all the muscles of hind limb have ischemia after hind limb ligation?
    Some papers used "adductor muscle". "Gastrocnemius muscle" was used in your paper: Rufaihah J et al. ATVB ²011. Is there big difference between these different muscles?
    Thanks a lot!
    Dong Wang
    Reply

    Posted by: Dong W.October 15, 2012, 9:24 PM

    Hello Dong,

    Thank you for your comment. Not all of the muscles of the hind limb will have ischemia post ligation of the femoral artery. The leg is most ischemic below the knee and we do see some perfusion in the thigh region, when analyzed by the laser doppler.


    For cell transplantation applications, a logistical consideration is the maximum allowable volume to be injected. This is when the different muscles become important. The gastrocnemius muscle is relatively small and can only accept a single injection of 30ul at most. In contrast, the muscles in the thigh can be injected multiple times for up to ²00ul in total volume.

    Thanks,
    Arshi
    Reply

    Posted by: Arshi J.November 7, 2012, 6:19 PM

    Dear Authors,
    Thank you for valuable and informative publication.

    Two surgical methods i.e. Superficial femoral artery (SFA) ligation and SFA excision surgical methods was performed in BALB/c mice (10 -1² wks). The mice from both the surgical methods showed limb salvage, tŒ necrosis and foot necrosis. At 7th and 14 th day, mice were sacrificed respectively. Gastronomicus muscle (GM) was seperated, weighed and histopathology (H&E sataining) was performed. The results showed significant change in GM weight and histopathology when compared to SHAM control on both 7th and 14th day samples.

    Based upon the results obtained in BALB/c mice , the above mentioned experiment (SFA ligation and SFA excision )was performed in SCID mice. The animals from both surgical methods didn't show limb salvage, tΠnecrosis and foot necrosis. Results showed no change in GM weight and histopathology when compared to SHAM control on both 7th and 14th day samples.

    My Question: Why ischemia is not inducing in SCID mice when compared BALB/c mice?

    Thank you,
    Ranjith Kumar
    Reply

    Posted by: ranjith kumar c.June 5, 2013, 3:05 PM

    Hi Authors,
    Thanks for this informative publication. We recently have started a study where we produce what we had planned to be a severe hindlimb ischemia model in athymic nudes, and administered cells and/or drugs in an injectable hydrogel into the thigh muscle as a pro-angiogenic therapeutic. We are encountering a lot of variability in the ischemic response to the surgery. One week in, all our controls (no treatment) appear fine, with regard to necrosis etc and still have full use of the limb, yet in our doppler readings there is still up to a 90% reduction in prefusion. Some of the other animals have necrotic toes and a couple have autoamputated, yet some appear completely fine, at least visually. Is it common to encounter such variability, and how many animals per group do you find is necessary to achieve statistically robust data? We have used an n=8 but are starting to suspect that this may not be enough, at least in our hands. Is there often a disconnect between doppler readings and necrotic response in some animals? Many thanks
    Reply

    Posted by: John S.July 1, 2013, 12:58 PM

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