Establishment and Evaluation of a Diabetic Rat Model with Lower Limb Arteriosclerosis Obliterans

Summary

Lower extremity arteriosclerosis obliterans is a common complication of diabetes mellitus. Here, we present a novel animal modeling method that simulates the progression of atherosclerotic occlusive disease by injecting ethanol into blood vessels to disrupt vascular structure, thereby providing a new platform for the treatment of vascular diseases.

Abstract

This study aimed to establish and validate a novel rat model of lower extremity arteriosclerosis obliterans (ASO) in diabetic rats. Unlike traditional femoral artery ligation, we injected 95% ethanol (0.1 mL) into the external iliac artery. Postoperatively, arterial occlusion and hindlimb ischemia were assessed on days 1, 3, 7, and 14 using Doppler ultrasound, thermal imaging, and wet muscle weight measurement. Histological analysis of blood vessels and muscle tissues was performed to evaluate the model's success rate and stability. High-concentration ethanol (95%) induces vascular endothelial cell damage, leading to femoral artery occlusion. Compared to the control group, Doppler ultrasound revealed a progressive reduction in arterial diameter and increased flow velocity in the affected limb. Thermal imaging demonstrated decreased limb temperature, while muscle weight measurement showed a significant reduction in the ipsilateral muscle mass. Histopathological analysis confirmed arterial lumen occlusion, decreased microvascular density, and progressive lesion exacerbation over time. In conclusion, iliac artery injection of 95% ethanol effectively replicates the pathophysiological characteristics of lower limb arterial occlusion, providing a novel model for studying arteriosclerosis obliterans in diabetic rats.

Introduction

Diabetic lower extremity arterial occlusion (DLEAO) is a leading cause of disability and mortality in diabetic patients, severely impairing quality of life and imposing a substantial socioeconomic burden¹. The pathogenesis of diabetic arterial disease involves complex interactions among hyperglycemia, dyslipidemia, chronic inflammation, and vascular dysfunction². Animal models have been instrumental in elucidating these mechanisms, providing critical insights for therapeutic development. Animal models serve as valuable tools for simulating the pathophysiology of peripheral arterial disease (PAD) in human diabetic patients. These models enable high-throughput, reproducible studies to investigate disease progression and elucidate underlying mechanisms. Furthermore, preclinical trials utilizing animal models provide essential data for novel therapeutics, including pharmacological agents, gene therapy, and regenerative medicine approaches (e.g., stem cell therapy)³. Such foundational research is critical for informing subsequent clinical trials and advancing translational medicine.

Currently, the femoral artery ligation (FAL) method is widely used in laboratories to establish rat models of hindlimb ischemia⁴. This approach rapidly induces ischemic manifestations in the lower limbs⁵; however, the resulting arterial thrombosis differs from the pathophysiological progression observed in diabetic patients with lower extremity arterial disease (LEAD), which involves endothelial injury, inflammatory factor accumulation, and smooth muscle cell proliferation^2,6. Additionally, FAL triggers rapid neovascularization and collateral vessel formation, restoring low-flow perfusion shortly after modeling^5,7. This contrasts with the chronic progression of diabetic LEAD. Therefore, there is a need to develop an animal model that more accurately replicates the pathophysiological mechanisms of human arteriosclerosis.

In diabetic animal models, mechanical injury to the femoral artery intima via balloon catheter or chemical induction (e.g., ferric chloride stimulation) effectively triggers pathological changes, including endothelial denudation, inflammatory cell infiltration, and smooth muscle cell migration/proliferation⁸. This approach better replicates the hallmark pathological features of human atherosclerosis. Compared to the traditional FAL model, the core advantage of the ethanol-induced method lies in its ability to more accurately simulate the complex pathophysiological process of diabetic LEAD. Ethanol directly disrupts the vascular endothelium, activating platelet aggregation and thrombosis, thereby mimicking the initial event in diabetic LEAD⁹. More importantly, by gradually destroying the vascular microstructure, ethanol effectively avoids the rapid collateral circulation compensation commonly seen in the FAL model, thus successfully replicating the typical chronic hypoperfusion state of diabetic LEAD¹⁰. In contrast, the femoral artery ligation-induced ischemia model has notable limitations: rodents (e.g., mice, rats) rapidly compensate for acute ischemia due to their well-developed collateral circulation, leading to inconsistent ischemic severity and poor recapitulation of human critical limb ischemia (CLI)^11,12. Moreover, human peripheral artery disease (PAD) primarily stems from the chronic progression of atherosclerosis, whereas the femoral artery ligation model represents an acute ischemic event-fundamentally distinct pathophysiological mechanisms¹³. The primary objective of this study was to establish and characterize a stable, long-term experimental model of diabetic lower extremity arteriosclerosis obliterans (LEAOD) that accurately mimics disease progression in patients. Ethanol-induced occlusion is operationally straightforward, requiring only minimally invasive injection, which reduces the technical barrier and animal mortality. Its key parameters are easily standardized, ensuring high reproducibility. Moreover, the method is cost-effective, eliminates the need for specialized equipment, and is readily transferable across laboratories. Currently, there remains a lack of durable animal models for validating novel therapeutic interventions.

Protocol

This study was conducted under a protocol approved by the Animal Ethics Committee of Yantai Yuhuangding Hospital. This study utilized 40 male Sprague-Dawley rats (weight: 250-300 g, age: 6 weeks) housed under a 12 h light/dark cycle. Starting at 7 weeks of age, they were fed a high-fat, high-sucrose diet¹⁴^,¹⁵ ad libitum, with free access to water for 8 weeks. At 4 weeks into the diet, diabetes was induced via streptozotocin (STZ) injection¹⁶. After 8 weeks, successful establishment of the diabetic model was confirmed prior to subsequent experiments. Hindlimb arterial occlusion surgery was performed on 14-week-old rats as described below.

1. Surgical induction of hindlimb arteriosclerosis obliterans in diabetic rats

1. Prepare all surgical instruments and materials as listed in the Table of Materials and illustrated in Figure 1.
2. Anesthetize the rat using an anesthesia machine delivering isoflurane (1.5-2.5%) and oxygen (O₂, 1-2 L/min). Maintain spontaneous respiration throughout. Assess the depth of anesthesia via the toe-pinch reflex to ensure a surgical plane of anesthesia.
3. Place the rat in a supine position on a restraining platform. Remove hair from the abdomen to the lower limbs using depilatory cream and a razor blade.
4. Secure the limbs. Disinfect the hindlimb skin with alcohol. Make a 2-3 cm incision on the medial right thigh using curved forceps and scissors.
   NOTE: All surgical procedures were performed by an experienced surgeon to ensure consistency and reproducibility of the animal models.
5. Under a microscope, dissect the surrounding tissues to expose the external iliac artery/vein and femoral nerve. Isolate the vessels and nerve. Trace the external iliac artery to its bifurcation into superficial and deep femoral arteries.
6. Clamp the femoral artery midsegment and deep femoral artery with vascular clips. Slowly inject 0.1 mL of 95% ethanol into the distal external iliac artery. After 60 s, remove the clips; achieve hemostasis by compression. Close the wound layer by layer.
   NOTE: During the injection, immediate blanching of the artery and transient tremor of the ipsilateral foot were observed, confirming successful intravascular delivery of ethanol.
7. Do not immobilize the affected limb postoperatively. Administer penicillin sodium injection (400,000 IU) intramuscularly to prevent infection.
   NOTE: In this unilateral lower limb arterial occlusion model, the contralateral limb serves as the control. The occlusion time is calculated starting at 24 h post procedure.

2. Determination of lower limb temperature

1. Anesthetize the rat and place it in a supine position on the surgical platform.
2. Shave both hind limbs and position them symmetrically.
3. Measure the temperature of both hind limbs using an infrared thermography system.

3. Arterial diameter and blood flow velocity

1. Anesthetize the rat.
2. Prepare the ultrasound system and probe.
   1. Turn on the ultrasound machine by pressing the power button on the front panel.
   2. Select the high-frequency linear array probe (e.g., 40 MHz) and attach it to the system.
   3. Apply a generous amount of acoustic coupling gel to the probe surface.
3. Locate the superficial femoral artery.
   1. Place the rat in a supine position on a warming pad to maintain body temperature.
   2. Shave the fur from the medial thigh area bilaterally, approximately 1-2 cm above the knee joint, using electric clippers.
   3. Gently place the probe perpendicular to the skin surface over the shaved area.
   4. Adjust the probe position in B-mode to visualize the femoral artery as a pulsatile, anechoic tubular structure.
4. Measure arterial diameter.
   1. Freeze the B-mode image by pressing the Freeze button on the machine or software interface.
   2. Select the Measurement tool from the on-screen menu.
   3. Use the caliper function to measure the internal diameter from the near to the far wall of the artery.
   4. Repeat the measurement three times at the same location and record the average value.
5. Measure blood flow velocity.
   1. Switch to pulsed-wave Doppler mode by clicking on the PW Doppler icon.
   2. Position the sample gate over the center of the artery lumen.
   3. Adjust the Doppler angle to ≤ 60° using the angle correction tool.
   4. Press Update or Capture to record the velocity waveform over at least three cardiac cycles.
   5. Use the software's automated tracing function to calculate peak systolic velocity (PSV) and end-diastolic velocity (EDV).
   6. Save the data by clicking Save and exporting the results to a spreadsheet (e.g., CSV, Excel) file.

4. Muscle weighing

1. Following deep anesthesia, euthanize the rat by cervical dislocation.
2. Dissect the gastrocnemius muscles from both hindlimbs, preserving the Achilles tendons (Figure 2).
3. Immediately place the muscles on an electronic balance to measure wet weight.
4. Use the contralateral muscles as controls.
5. Compare measurements from four time points and present them in a curve graph.

5. Histopathological analysis

1. At each of the four time points, euthanize one group of rats rapidly.
2. Harvest the bilateral superficial femoral arteries immediately.
3. Fix the arterial tissues in 4% paraformaldehyde.
4. Embed the tissues in paraffin and section them at 5 µm thickness.
5. Perform hematoxylin and eosin (H&E) staining to examine histopathological changes.

6. Statistical analysis

1. Express all data as mean ± standard deviation (SD). Consider differences statistically significant at a p-value of <0.05.

Results

This study utilized intra-arterial ethanol injection to establish a model in diabetic SD rats (n = 40). The model was characterized by an 85% success rate (33/40), based on ultrasonic confirmation of reduced contralateral arterial diameter and blood flow velocity within 14 days post-injection. A model-related mortality rate of 8% (5/40) was recorded, predominantly during the initial period. While ethanol's cytotoxicity poses a risk of non-specific tissue damage such as gangrene, careful monitoring confirmed that the obse...

Discussion

In summary, we have successfully established a rat model of progressive arterial occlusion that closely mimics the clinical manifestations observed in diabetic patients. Consistent evaluations of hindlimb hypoperfusion were achieved through ultrasonographic measurements and muscle wet weight assessments for periods exceeding two weeks. Ligation of the femoral artery induced superficial vascular occlusion, while allowing blood recirculation through the intact deep venous system¹⁷. When ligation is ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
GraphPad version 8.0.1			RRID:SCR_002798
Phone-based Infrared Thermometer	HIKMICRO		HM-TJP10B-3AMF
Sprague-Dawley rat	Jinan Pengyue Experimental Animal Breeding Co., Ltd.		250 g
Ultrasound machine	Vevo 2100