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Ischemic Wound in Rat Model Development for Therapeutic Angiogenesis

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Dymon Morgan

July 28 2011

11 minutes


Mentors:    Amish Raval, M.D., Nehal Shah MBBS, Kimberly Maciolek

Department: Medicine

Background: Critical limb ischemia (CLI) is a widely prevalent disease affecting approximately 500 to 1,000 people per million persons per year in North America. CLI is characterized by rest pain, ulcers, and gangrene. Current treatment methods include surgical bypass and angioplasty. A large proportion of patients are not eligible for these treatments and amputation and mortality rates in treated patients remain high. In addition, a number of patients have chronic wounds whose healing is exacerbated by tissue ischemia in critical limb ischemia. Therapeutic angiogenesis has emerged as a very promising therapy to create new blood vessels that may accelerate wound healing. There is a need to develop a reliable ischemic wound animal model to eventually evaluate therapeutic angiogenic agents. I hypothesize that i) bilateral wounds created in non-ischemic rats will heal at the same rate ii) ischemia will reduce the rate of healing in the ischemic limb compared to the control limb.

Methods: Female Sprague Dawley Rats (100-250gm) were divided into 2 groups i) control rats (n=3) where bilateral excisional wounds were created ii) ischemic rats (n=3) where unilateral hindlimb ischemia was created in addition to bilateral excisional wounds. Hindlimb ischemia was created by ligating and excising the left common iliac artery (at the origin) and the femoral artery at the inguinal ligament. Blood vessels of all control limbs were dissected but not excised or ligated. Wounds were created using a 4mm punch biopsy. Serial photographs were taken for 5 days post-surgery and wound size area was calculated using the software Image J. The rats hind limbs were imaged with laser Doppler perfusion imaging (LDPI).Scans were done under resting conditions. Rats were anesthetized with 5 percent inhalation isoflurane induction in a pre-filled chamber for 4 minutes. Constant inhalation of 2 percent isoflurane via nose cone was required for maintenance. Ten minutes of acclimatization was allowed between anesthesia induction and imaging. To prevent movement during scans, both hind limbs were abducted and fixed in a symmetric position with tape. Five consecutive resting scans were taken. 

Results: For the control group the rate of wound healing was 53% in the left limb and 59% percent in the right limb. Furthermore, in the ischemic group the Laser Doppler Perfusion ratio showed a reduction of blood flow by 46% and after 5 days, the blood perfusion ratio showed a reduction of blood flow by 54%. The rate of wound healing for the ischemic group was 46% in the ischemic limb and 50% in the non-ischemic limb of the ischemic rat.

Conclusions: Overall, there is large variability in wound size at the onset and the rate of wound healing over time that was not anticipated. From the information obtained, it is evident that some limitations remain with the model. Thus, it would be necessary to re-evaluate the creation of wounds and examine the model more. Once a solid model has been created, pro-angiogenic agents can be injected in the model to study therapeutic angiogenesis.      


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