Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits

Podsumowanie

We describe a surgical procedure used to induce peripheral ischemia in rabbits with hyperlipidemia and diabetes. This surgery acts as a preclinical model for conditions experienced in peripheral artery disease in patients. Angiography is also described as a means to measure the extent of introduced ischemia and recovery of perfusion.

Streszczenie

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular disease is the development of ischemia. In severe cases, patients can develop critical limb ischemia in which they experience constant pain and an increased risk of limb amputation. Current therapies for peripheral ischemia include bypass surgery or percutaneous interventions such as angioplasty with stenting or atherectomy to restore blood flow. However, these treatments often fail to the continued progression of vascular disease or restenosis or are contraindicated due to the overall poor health of the patient. A promising potential approach to treat peripheral ischemia involves the induction of therapeutic neovascularization to allow the patient to develop collateral vasculature. This newly formed network alleviates peripheral ischemia by restoring perfusion to the affected area. The most frequently employed preclinical model for peripheral ischemia utilizes the creation of hind limb ischemia in healthy rabbits through femoral artery ligation. In the past, however, there has been a strong disconnect between the success of preclinical studies and the failure of clinical trials regarding treatments for peripheral ischemia. Healthy animals typically have robust vascular regeneration in response to surgically induced ischemia, in contrast to the reduced vascularity and regeneration in patients with chronic peripheral ischemia. Here, we describe an optimized animal model for peripheral ischemia in rabbits that includes hyperlipidemia and diabetes. This model has reduced collateral formation and blood pressure recovery in comparison to a model with a higher cholesterol diet. Thus, the model may provide better correlation with human patients with compromised angiogenesis from the common co-morbidities that accompany peripheral vascular disease.

Wprowadzenie

Peripheral arterial disease (PAD) is a common circulatory disorder in which the progression of atherosclerotic plaque formation leads to a narrowing of blood vessels in the limbs of the body. The recent increase in risk factors for atherosclerosis, including diabetes, obesity, and inactivity, has led to increasing prevalence of vascular disease¹. Currently, it is estimated that 12%–20% of the general population over 60 years old has peripheral arterial disease². A major consequence of peripheral arterial disease is the development of peripheral ischemia, most commonly found in the lower limbs. In severe cases, patients can develop critical limb ischemia, a state in which there is constant pain due to a lack of blood flow. Patients with critical limb ischemia have a 50% likelihood of having one limb amputated within one year of diagnosis. Furthermore, patients with diabetes have a higher incidence of peripheral arterial disease and poorer outcomes following interventions for revascularization^3,4. Current therapies for peripheral ischemia include percutaneous interventions such as atherectomy and stenting or surgical bypass. However, for many patients these treatments only provide short-term benefits and many are not healthy enough for major surgical procedures. In this work, we describe a preclinical animal model for testing new treatments targeting peripheral vascular disease that incorporates the generation of peripheral ischemia in rabbits through surgical ligation in the context of the diabetic disease state.

The hind limb ischemia model in rabbits has been used as a physiological model for obstructive vascular disease and preclinical precursor to human studies for over half a century^5,6. Rabbits are often a preferred species for studies on peripheral ischemia due to the developed musculature of the ankle and calf muscle, in contrast to common large animal models that are ungulates (animals with hooves). Several recent reviews have addressed the use of this model and others in modeling peripheral vascular disease in humans^7,8. Similar models using hind limb ischemia in rabbits were used in preclinical studies of growth factors^{9,10,11,12,13,14,15,16,17,18,19,20}, gene therapy^{21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44}, and stem cells^{45,46,47,48,49,50,51}for therapeutic neovascularization in the limbs. Unfortunately, the clinical trials that followed these successful animal studies did not show significant benefits for patients⁵².

One suggested explanation of the reason for this translational failure is that the condition of peripheral ischemia in human patients is one that includes resistance to angiogenic signals^{53,54,55,56,57,58,59}. Several studies have shown defects in angiogenic signaling pathways in diabetes and hyperglycemia. Diabetes and hyperlipidemia lead to a loss of heparan sulfate proteoglycans and an increase in enzymes that cut heparan sulfate, presenting a potential mechanism for resistance to therapeutic angiogenesis/arteriogenesis with growth factors^60,61. Thus, a key feature of a model for peripheral ischemia should include an aspect of therapeutic resistance so that therapies may be evaluated in the context of the disease state present in human patients.

In this work, we describe a rabbit model of peripheral ischemia through surgical ligation of the femoral arteries. A lead-in period with the induction of diabetes and hyperlipidemia is incorporated into the model. We compared this model to another model that incorporates a higher fat diet without diabetes and found that the model with diabetes and lower level of hyperlipidemia was more effective in reducing blood vessel growth. Our model combines advancements that have been used by separate groups, with the goal of providing a practical and standardized method to achieve consistent results in peripheral vascular disease research.

Protokół

Studies involving animals were performed with the approval of the University of Texas at Austin and the UTHealth Science Center at Houston Institutional Animal Care and Use Committee (IACUC), the Animal Care and Use Review Office (ACURO) of The United States Army Medical Research and Materiel Command Office of Research Protections, and in accordance with NIH guidelines for animal care.

1. Induction of diabetes and hyperlipidemia

1. Transition the New Zealand rabbits (4–6 months old) from one cup of standard alfalfa chow to 0.1% cholesterol chow over the course of four days. For days 1–5, use standard chow to cholesterol chow ratios of 1:0, 3:1, 1:1, 1:3, and 0:1, respectively. After two weeks on 0.1% cholesterol chow, induce rabbits to have diabetes using alloxan injection as described in the following steps
2. Sedate the rabbits using 35–75 mg/mL ketamine and 1–2 mg/mL acepromazine via subcutaneous injection and prep for an IV injection by introducing a catheter into the marginal left ear vein using a 22 g catheter.
3. Collect a drop of blood from the rabbits via hub of the ear vein catheter for the baseline blood glucose level (BGL) measurement. Any standard glucometer can be used. Normal glucose levels for a rabbit are typically in the range of 80 to 150 mg/dL.
4. Inject alloxan at 100 mg/kg reconstituted in saline to a volume of 8 mL through the ear catheter slowly over an 8-minute period using a syringe pump.
5. Check the BGL every hour for the next 12 h using a standard glucometer to monitor for hypoglycemia.
   1. Place the rabbit in a restrainer.
   2. Anesthetize the ear with 2.5% lidocaine/2.5% prilocaine cream.
   3. Take blood from the lateral ear vein using a 27 G needle and measure BGL using a standard meter.
6. Measure the BGL two times a day for the first 7 days. Give the rabbits an injection of insulin if the BGL reaches or exceeds 350 mg/dL.
7. Prepare a 3-mm stainless steel ball for implantation as a size marker during angiograms prior to the day of surgery.
   1. Cut a 10-mm circular piece of silastic sheeting out of a larger sheet using a biopsy punch.
   2. Mount the ball in the center of the sheet using clear silicone sealant.
   3. Completely cover the ball with the sealant. Allow the sealant to cure for a minimum of 24 h.
   4. Place the ball in an open 2 inch x 3 inch low density polyethylene bag and place it into a sterilization bag to be sterilized with ethylene oxide gas.

2. Preparation of rabbit for surgery

1. Anesthetize the rabbit using 20–40 mg/kg ketamine and 2 mg/kg midazolam via subcutaneous injection. Place the rabbit on 1.5%–3% isoflurane (typically 2%) throughout the initial sedation using a mask. Give an injection of alfaxalone to maintain anesthesia via an intramuscular injection of 3 mg/kg.
2. Once anesthetized, remove the mask and insert a cuffed endotracheal tube, into the airway and connect to a ventilator. Continue to administer isoflurane at 1.5%–3%.
3. Collect blood from the central artery from either ear for a baseline chemistry panel.
4. Place a 22 G ear vein catheter in the lateral ear vein for Lactated Ringer's Solution drip throughout the surgical procedure. Alternatively, normal saline (0.9% sodium chloride) can be used.
5. Using the lateral vein in the opposite ear, place a catheter in the vein and deliver alfaxalone at 6 mg/kg/h. Gradually increase the alfaxalone to 8 mg/kg/h while decreasing isoflurane to 0.6% during the prep period.
6. To limit pain and risk of infection, administer buprenorphine (0.01 mg/kg) and enrofloxacin (5 mg/kg) using a subcutaneous injection with a 25 G needle.
7. Trim the hair on the neck, right and left inner thighs, and back using clippers (#40 blade). Hair is removed from the back to maintain contact with the grounding pad.
8. Place a blood pressure cuff on each of the hind limbs and measure initial blood pressure. Place the cuff just below the knee with the probe just above the hock on the lateral surface.
9. Position the rabbit on the surgery table on its back and scrub and drape the surgery sites. This includes the neck for carotid artery access and inner right thigh for femoral artery access. Perform the sterilization scrub with alternating scrubs of 2% chlorhexidine and 70% ethyl alcohol. Repeat this three times, then apply a final spray with 2% chlorhexidine solution.
10. Place a 3-mm stainless steel ball that has been sterilized inside a low density polyethyene bag on top of the right (scrubbed) leg near the upper part of the thigh to serve as a size reference during angiogram measurements. Place a sterile drape over the leg until the time of surgery. Leave the ball inside the sterile plastic bag during the first angiogram.

3. Angiography

1. Expose the right common carotid artery
   1. Make a 4–5 cm long incision just lateral to the trachea using a scalpel with a #15 blade.
   2. Use blunt dissection to expose the carotid artery and open the incision using small Weitlaner retractors. Carefully isolate the carotid artery from the jugular vein and vagus nerve. Typically, a curved Metzenbaum scissors and a curved mosquito hemostat are used for the blunt dissection. Be sure to get full separation of the carotid artery from the nerve and jugular vein to make the ligatures only ligate the artery.
2. Place a ligature using a 4-0 silk suture at the proximal and distal ends of the exposed artery. Tie off the distal end of carotid with a surgeon’s knot followed by four square knots. On the proximal end, use a ligaloop to allow it to be tightened or loosened as needed. The use of a ligaloop placed at the proximal end of the exposed artery can help secure the introducer and catheter.
3. Administer 500 IU of heparin through the IV. Use approximately 0.5 mL of 1% lidocaine applied along the exposed carotid to dilate the vessel. One treatment is usually sufficient, but it can be repeated as needed. Cut approximately half way through the carotid artery using a scalpel or iris scissors, then place the 4-inch wire insertion tool into the artery.
4. Feed a 0.014 inch x 185 cm guidewire through the insertion tool to the aortic bifurcation at the iliac crest in the descending aorta. Remove the insertion tool and insert a 3F pigtail angiographic catheter over the wire.
5. Advance the pigtail catheter to be 2 cm proximal to the aortic bifurcation at the iliac crest in the descending aorta.
6. Position the tip of the catheter between the seventh lumbar and first sacral vertebrae. Test the location of the catheter by manually injecting a 2–4 mL of contrast agent.
7. Administer an intra-arterial injection of 100 μg nitroglycerin through the catheter to increase vasodilation.
8. Administer 0.8 mL of 1% lidocaine to the rabbit through the catheter to assist with vasodilation during the angiogram. Attach the tubing for the injector to the catheter and remove any air bubbles in the line. Inject 8-9 mL of contrast media using automated angiographic injector through the catheter.
9. Record serial images of the hind limbs using angiography.
   1. Set the power injector to inject contrast at 3 mL/sec for a total of 8-9 mL. Perform digital subtraction angiography at 6 frames per second.
   2. Select the serial images created and alter a photo of each angiogram using approximately -40% setting to minimize appearance of bone and capture a complete picture of the vessel perfusion with contrast. An example angiogram of the vascular flow after femoral artery ligation/excision is shown in Figure 1.

4. Isolation of the femoral artery

1. Make a longitudinal incision in the skin over the right femoral artery using a scalpel (#15 blade). Ensure that the incision extends inferiorly from the inguinal ligament ending at the area just proximal to the patella (approximately 6 cm).
2. Use blunt dissection with curved Metzenbaum scissors or a curved mosquito hemostat to expose the femoral artery.
3. Use Weitlaner retractors to hold the incision open.
4. Add 0.5 mL of 1% lidocaine locally to reduce nerve irritation and promote vasodilation.
5. Continue blunt dissection of the tissues to free the entire length of the femoral artery along with all branches of the femoral artery, including the inferior epigastric, deep femoral, lateral circumflex, and superficial epigastric arteries (Figure 2A).
6. Dissect further along the popliteal and saphenous arteries as well as the external iliac artery (Figure 2A). Periodically moisten the area with saline to protect from tissue damage. If the blunt dissection is performed along the femoral groove (between the muscles) there is no need to cut the muscle.
7. Carefully separate the artery from the vein and nerve as shown in Figure 2B,C. Ligate the arteries indicated by the diagram with 4.0 silk sutures by placing two ties with enough space between them to cut the artery. These ties are performed with a Surgeon’s knot followed by four square knots.
8. Cut between the two ties on the ligated arteries using the small Metzenbaum scissors. Excise the femoral artery from its proximal origin as a branch of the external iliac artery to the point distally where it bifurcates to form the saphenous and popliteal arteries.

5. Repeat angiography

1. Use 4-0 silk suture to attach the silastic sheet, with 3-mm stainless steel ball to the upper part of the quadriceps muscle. Pull the skin over the ball after it is in place.
2. Administer an intra-arterial injection of 100 μg nitroglycerin through the catheter to increase vasodilation.
3. If needed, administer another 0.8 mL of 1% lidocaine to the rabbit through the catheter to assist with vasodilation during the angiogram.
4. Inject 8-9 mL of contrast media using an automated angiographic injector.
5. Perform angiography as described in step 3.9.

6. Wound closure and recovery

1. Remove the catheter from the right artery. Tie off the artery using the 4-0 silk suture that is already in place around the artery.
2. Suture both wounds closed. Close muscle and subcuticular layers using 4-0 polydioxanone or 3-0 polyglactin 910 on a taper needle (see Table of Materials) in a continuous suture pattern. Close the skin using 4-0 polydioxanone or 4-0 polyglactin 910 on a reverse cutting needle (see Table of Materials) in a buried continuous subcuticular suture pattern.
   NOTE: If available, polydioxanone is preferred for both.
3. Administer intradermal injections of 0.25% bupivacaine near the incisions using a syringe with a 25 G needle. Insert the needle and inject 0.5 mL while the needle is pulled back. Give one injection per side of the wound for the incision on the neck (two injections on the neck) and two injections per side of the wound for the incision on the leg (four injections on the leg; six injections in total). The total volume injected is 3 mL (0.5 mL x 6 injections).
4. Administer subcutaneous injections of 0.5 mg/kg meloxicam and sustained release buprenorphine at 0.12 mg/kg.
5. Monitor the rabbit as it recovers from anesthesia. The rabbit will automatically start to swallow as it wakes up from anesthesia. Once the swallowing response occurs, remove the endotracheal tube. Provide close monitoring and thermal support until the rabbit is able to maintain cardiovascular function and body temperature. Return the rabbit to its enclosure once it is able to ambulate.
6. Employ fresh vegetables and/or syringe feeding of a critical care diet along with subcutaneous saline injections if the rabbit does not tolerate chow after the surgery. Cabbage, broccoli, cauliflower, carrots, or other in seasonal vegetables can be used. Shred the vegetables and mix them together to aid in the rabbit returning to eating.

7. Monitoring

1. Anesthetize the rabbits every two weeks to acquire blood pressure on both legs as described in step 2.8. Harvest blood from the central artery of the ear for use in blood chemistry assays. Alternatively, take blood from the saphenous vein or cephalic vein. Take approximately 2 mL at each time point. Use a standard blood chemistry panel for analysis. If needed, add tests for low density lipoprotein (LDL), high density lipoprotein (HDL), or hemoglobin A1c (HbA1c).
2. Take a very small amount of blood for BGL measurements.

8. Treatment

1. Prepare ten syringes with treatment, carrier, and crosslinker. Fill each syringe just prior to use with 100 μL of calcium sulfate slurry and then 100 μL of 2% sodium alginate with growth factors or other treatments such that the alginate is nearest the tip of the syringe.
2. Administer one prepared injection into the muscle before preparing the next. This reduces the time that the alginate interacts with the calcium sulfate in the syringe. Space the injections evenly along both sides of the femoral artery on the thigh. To achieve uniform injections, create a silicone sheet with holes to guide the injection, as described in other studies¹⁹. This can be easily prepared by using a biopsy punch to create holes in commercially available silicone sheeting.

9. Endpoint angiography, euthanasia, perfusion fixation and tissue harvest

1. At the endpoint date, perform angiography as described in step 3 but use the left carotid artery for access.
2. After angiography, move the animal to the necropsy table and perform perfusion fixation to preserve the hind limb tissues:
   1. Increase the isoflurane to 3%–4% and perform a toe pinch to confirm the anesthesia is sufficiently deep.
   2. Administer 1000-2000 IU of heparin intravenously.
   3. Create an incision along the midline of the ribcage and spanning the length of the diaphragm using a scalpel with a #20 blade.
   4. With the rib cage exposed, cut the ribs just left of the midline using rib cutters. Use Weitlaner retractors to expose the heart.
   5. Set up the pump with output tubing with an inner diameter of 1/8 inch and a 18G needle at the end. Preload the line with saline and have at least 600 mL of saline and formalin prepared in separate containers for the perfusion.
   6. Insert the 18 G needle connected to the pump into the left ventricle via the apex of the heart. Insert another 18 G needle (unattached to anything) into the right atrium and allow blood to flow out into the downdraft of the necropsy table.
   7. Use a perfusion pump to control the flow of approximately 500 mL of saline into the heart. Use a pump setting to flow 110 mL/min.
   8. Once the fluid coming from the heart is clear, move the tubing from the saline reservoir to one filled with a 10% formalin solution. Twitching will occur in all four limbs if the perfusion is working properly. Pump approximately 500 mL of formalin solution into the left ventricle.
   9. Turn the pump off and remove the needles from the heart.
3. Remove both hind limbs at the hip by cutting around the hip joint with a scalpel with #20 blade. Use a small rib cutter to remove the limbs. Use the non-ischemic limb as a control.
4. Store the limbs in formalin for 24 h at 4 °C and then stored in 70% ethanol at 4 °C.
5. For histological analysis, take multiple biopsies from the limbs. We have used eight 6-mm biopsies taken at regions across the thigh and calf in both limbs.
   NOTE: While ankle blood pressure measurement and angiography are the most commonly used methods for measuring recovery of blood flow, other methods can be used to track recovery of the animals including Doppler ultrasound, laser Doppler imaging, infrared thermography⁶², microsphere determined perfusion^63,64, computed tomography (CT) imaging, and magnetic resonance imaging (MRI)⁶⁵.

Wyniki

Following induction of diabetes and initiation of the 0.1% cholesterol diet, the total cholesterol for the rabbits with diabetes and cholesterol diet was 123.3 ± 35.1 mg/dL (n = 6 male rabbits) averaged overall time points and rabbits. The BGL level for these rabbits was 248.3 ± 50.4 mg/dL (n = 6 male rabbits). A time course for blood chemistries and leg blood pressure ratios in a typical rabbit is shown in Figure 3 in comparison to rabbits under a higher choles...

Dyskusje

We have presented a preclinical model for inducing hind limb ischemia in rabbits with diabetes and hyperlipidemia. In many studies, there is ambiguity to the technique used to create hind limb ischemia in rabbits. In mice, the severity and recovery from hind limb ischemia is highly dependent on the location the ligation and technique used to induce ischemia. The significance of the technique presented in this work is that it allows for the consistent induction of ischemia that does not fully recover after 8 weeks in diab...

Materiały

Name	Company	Catalog Number	Comments
0.9% Sodium Chloride	Henry Schein Medical	1537468 / 1531434	250 mL bag / 1000 mL irrigation btl
1 mL Syringe	VWR	BD309628
10 mL Syringe	VWR	BD309695
10% Formalin	Fisher-Scientific	23-245684
18G Needle	VWR	89219-294
20G Needle	VWR	89219-340
25G Needle	VWR	89219-290
27G Needle	VWR	89219-288
5 mL Syringe	VWR	BD309646
5% Dextrose	Patterson Veterinary	07-800-9689
Acepromazine	Patterson Veterinary	VEDC207
Alfaxalone	Patterson Veterinary	07-891-6051
Alginate	Sigma-Aldrich	PHR1471-1G
Alloxan Monohydrate	Sigma-Aldrich	A7413
Angiography Equipment	Toshiba	Infinix-i
Angiography Injector	Medrad
Anti-Mouse Ab Alexa 594	Thermo Fisher Scientific	A-11032	Secondary Antibody for IHC
Anti-Rabbit Ab Alexa 488	Thermo Fisher Scientific	A-11008	Secondary Antibody for IHC
a-SMA Antibody	Abcam	ab5694	Primary Antibody for IHC
Baytril	Bayer Animal Health	724089904201	Enrofloxacin
Blood Chemistry Panel	IDEXX	2616	Rabbit Panel
Blood Pressure Cuff	WelchAllyn		Flexiport Disposable BP Cuff-infant size 7
Blood Pressure Monitor	Vmed Technology		Vmed Vet-Dop2
Bupivacaine	Henry Schein Medical	6023287
Buprenorphine	Patterson Veterinary	42023017905
Buprenorphine SR	ZooPharm
Calcium Sulfate	CB Minerals		Food and Pharmaceutical Grade USP and FCC
Chlorhexidine Scrub	Patterson Veterinary	07-888-4598
Chloroform	Fisher-Scientific	C298-4
Cholesterol	Sigma-Aldrich	C8503
DAPI	Thermo Fisher Scientific	62248
Ear Vein Catheter	Patterson Veterinary	SR-OX165	Surflo IV catheters
Endotracheal tube	Patterson Veterinary		Sheridan Brand, Depends on Rabbit Size
Glucometer	Amazon	B001A67WH2	Accu-Chek Aviva
Glucometer Test Strips	McKesson Medical-Surgical	788222	Accu-Chek Aviva Plus
Guidewire	Boston Scientific	39122-01
Hair Clippers	Amazon	B000CQZI3Q	Oster #40 blade
Heating Pad	Cincinnati Subzero	273
Heating Pad Pump	Gaymar		Gaymar T/Pump
Hemostat	Fine Science Tools	13009-12	Curved Mosquito Hemostat
Heparin	Patterson Veterinary
Insertion Tool	Merit Medical Systems	MAP550	metal wire insertion tool
Insulin	HPB Pharmacy		Novalin R & Novalin N
Insulin Syringes	McKesson Medical-Surgical	942674
Introducer	Cook Medical	G28954	3F Check Flo Performer Introducer
Isoflurane	Henry Schein Medical	1100734
Ketamine	Patterson Veterinary	856440301
Lactated Ringers	McKesson Medical-Surgical	186662
Lidocaine	McKesson Medical-Surgical	239936
Lidocaine/Prilocaine cream	McKesson Medical-Surgical	761240
Ligaloop	V. Mueller	CH117 / CH116	White Mini / Yellow Mini
Mazola Corn Oil	Amazon	B0049IIVCI
Medrad Syringe	McKesson Medical-Surgical	346920	150 mL
Meloxicam	Patterson Veterinary
Metal ball sutures	Ethicon-Johnson & Johnson	K891H	4-0 silk C-1 30"
Metzenbaum Scissors	Fine Science Tools	14019-13
Midazolam	Henry Schein Medical	1215470
Nitroglycerin	McKesson Medical-Surgical	927528
PECAM Antibody	Novus Biologicals	NB600-562	Primary Antibody for IHC
Perfusion Pump	Masterflex
Pigtail Catheter	Merit Medical Systems	1310-21-0053	3F pigtail
Polydioxanone (PDS II) suture	McKesson Medical-Surgical	129271	4-0 taper RB-1 (needle comes on suture)
Polydioxanone (PDS II) suture	McKesson Medical-Surgical	129031	4-0 reverse cutting FS-2
Polyglactin 910 (Vicryl) suture	Butler	7233-41	3-0 taper RB-1
Polyglactin 910 (Vicryl) suture	McKesson	104373	4-0 reverse cutting FS-2
Rabbit Chow (Alfalfa)	LabDiet	5321
Rabbit Restrainer	VWR	10718-000
Rib Cutters	V. Mueller
Scalpel	Fine Science Tools	10003-12
Scalpel Blade	Fine Science Tools	10015-00	#15 blade
Silk Sutures	Ethicon-Johnson & Johnson	A183H	4-0 silk ties 18"
Stainless Steel Ball	McMaster-Carr	1598K23	3-mm diameter
Surgical Drapes	Gepco	8204S
Syringe Pump	DRE Veterinary		Versaflow VF-300
Visipaque contrast media	McKesson Medical-Surgical	509055
Weitlaner Retractor	Fine Science Tools	17012-13