The rat carotid balloon injury model described below allows researchers to evaluate drugs or therapeutics that negate injury-induced arterial hyperplasia. Detailed pre-surgical preparation, surgical procedure, and post-surgical cares of the animal are described.
The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is also a valuable model system to test, and to evaluate therapeutics and drugs that negate maladaptive remodeling in the vessel. The injury, or barotrauma, in the vessel lumen caused by an inflated balloon via an inserted catheter induces subsequent neointimal growth, often leading to hyperplasia or thickening of the vessel wall that narrows, or obstructs the lumen. The method described here is sufficiently sensitive, and the results can be obtained in relatively short time (2 weeks after the surgery). The efficacy of the drug or therapeutic against the induced-remodeling can be evaluated either by the post-mortem pathological and histomorphological analysis, or by ultrasound sonography in live animals. In addition, this model system has also been used to determine the therapeutic window or the time course of the administered drug. These studies can leadto the development of a better administrative strategy and a better therapeutic outcome. The procedure described here provides a tool for translational studies that bring drug and therapeutic candidates from bench research to clinical applications.
Angioplasty is an endovascular procedure used to widen the narrowed or obstructed arteries resultant from pathological conditions such as atherosclerosis. One common complication of angioplasty is the post-operational neointimal hyperplasia, or restenosis, which occurs owing to surgical injuries and the subsequent inflammation-induced vascular remodeling. These conditions lead to proliferation of vascular smooth cells, and multiple pathophysiological consequences1-3. Neointimal hyperplasia re-thickens the vessel, and occurs in up to 60% post-angioplasty patients within the first year. Therefore, restenosis is a major setback of the widely used angioplasty procedure4. Although the implantation of the drug-elution stent may help to prevent restenosis, only selected candidates can undergo this costly procedure5.
Both animal and clinical studies have established that chronic inflammation generated by vascular injury and /or surgical wound serves as the main stimulus for post-angioplasty neointimal growth2,4. The rat carotid balloon injury model mimics the clinical situation and therefore serves as a valuable model system to identify cellular factors that involve in the vascular remodeling and vascular cell proliferation6-9. This model system is also a highly useful tool to evaluate and/or screen for drugs and therapeutic reagents that suppress neointimal growth in pre-clinical translational studies10-14.
Compared to the murine carotid wire injury model15 and the murine femoral artery wire injury model16, the rat carotid balloon injury model has the advantages of being sufficiently large in size for the ease of surgical procedure that facilitates reproducibility of the inflicted injury. It can provide a larger number of primary cells (e.g. vascular smooth muscle cells, endothelial cells) for additional in vitro studies to delineate the molecular mechanism governing vascular remodeling. Importantly, compared to mice, rats are also known to be a better model for physiological and toxicological studies17. Although a disadvantage or limitation of the rat model is the lack of genetic modified and gene knockout models, this disadvantage can be overcome by the availability of the rat genomic sequence and the recent development of powerful genomic editing tools such as CRISPR-CAS technology that renders possible manipulation of vast ranges of genomic sequences in different model systems18,19.
Although the rat balloon injury model has been used by multiple labs and various comprehensive protocols have been published20,21, this protocol aims at providing more details at pre-surgery preparations and may guide researchers new to this procedure to set up this surgical practice. We also emphasize the post-surgery care of the animals that allows not only post-mortem pathological and histomorphological analyses of the therapeutic effects on arterial remodeling, but also ultrasound sonographic studies in live animals13,22.
NOTE: the use of rat balloon injury model and associated procedures including injection of recombinant sRAGE and ultrasound sonographic studies have been approved by the Animal Care and Use Committee (ACUC) of National Institute on Aging, NIH.
1. Pre-surgery Preparations
2. Surgical Procedures
3. Administration of Therapeutics, Analgesics, Post-surgery Care, and Euthanasia
Two weeks after the balloon injury, the rat is euthanized and the carotid arteries are isolated for histo-morphological analysis. Both operated left carotid artery and un-operated right artery are cross-sectioned, processed, and paraffin-embedded. The paraffin samples are then further thin-sectioned, and stained with hematoxylin-eosin (H & E). Histo-morphological analyses are performed using a digital imaging analysis system. The details of artery harvest and histo-morphological analysis have been described13,23. The balloon injury-elicited neointimal growth, or the thickening of the vessel wall, if left untreated (or treated with saline as the placebo), is evident as shown in Figure 1B, compared to non-operated vessel section from the same rat (Figure 1A). The vessel section from the rat received therapeutic reagent (in this case, sRAGE) that blocks neointimal growth showed significantly reduced thickening of the vessel wall (Figure 1C). The effects of the balloon injury as well as the therapeutic treatment can also be evaluated in vivo using ultrasound sonography22, which collaborate well with the results from histo-morphological analysis (Figure 2). The details for the evaluation of the efficacy and the administration window of therapeutic sRAGE in the rat balloon injury model have been described in previous publications13,22. To evaluate therapeutic reagents that reduce or block the injury-elicited vessel wall remodeling, a group of rats (n = 8-15) are to be operated in order to obtain statistically meaningful conclusions.
Figure 1: Representative results from the rat carotid balloon injury model. H & E staining of the carotid artery cross-sections (A) the non-operated right carotid artery section; (B) the balloon injured and placebo-treated left carotid artery section (arrows indicate the neotimal areas); (C) the balloon injured and sRAGE-treated left carotid artery section. Please click here to view a larger version of this figure.
Figure 2: Correlation of ultrasound sonography and histology. sRAGE (0.5 ng/g) treated carotid vessels shown in scatter plot of data from (A) lumen diameter and (B) vessel wall thickness at 2 weeks post-injury (the non-operated, injured with sRAGE, and placebo-treated, n = 12 of each group). (C) Representative sonographic and histologic (100X) images. (Figure from reference22, with permission from the publisher). Please click here to view a larger version of this figure.
There have been two methods used to inflate the balloon in order to generate the injury that removes the mural endothelium in the carotid arterial lumen. One is to fill the attached syringe with liquid20, and the other is to use air pressure21. We prefer to use liquid-filled syringe because the exact liquid volume (0.02 ml) will be used for each procedure. This renders a precise and reproducible inflation of the balloon, leading to a similar level of injury in each animal undergoing the procedure. Using liquid in the syringe indeed requires removal of air bubbles in the liquid. This can be achieved by pushing the bubbles out from the opening of the catheter. After removing a majority of air bubbles, any residual, tiny air bubbles will dissipate in 1-2 hr. We suggest that researchers make pre-surgery materials ready 1-2 hr prior to the procedure, and check the degree of the balloon inflation before each individual surgery to ensure the appropriate inflation.
For the surgery, the surgeon and his/her assistant(s) must wear all necessary protection gears including face masks, sterile gloves, and surgical gowns to protect both animals and researchers from infection. It is important to use fully grown, male rats with similar bodyweights (e.g. between 380-450 g) to ensure the proper size of carotid artery for the introduction of standard 2F Fogarty balloon embolectomy catheter used for this procedure. Before the surgery, check the level of isoflurane in the vaporizer to assure sufficient amount of the anesthetic that covers the entire surgery, and refill the vaporizer if necessary before each surgery starts. Check the pressure gauge of the oxygen tank to ensure sufficient oxygen for the surgery. To ensure a proper depth of anesthesia is reached, one can also monitor the respiratory rate of the rat. A normal rat has a respiratory rate of ~85 breath/min; an increase in respiratory depth and regular rhythm, and a decrease in respiratory rate signifies surgical anesthesia.
During the surgery, anesthesia must be continuously monitored and maintained by checking respiration, pedal reflex, and the response of the animal to the surgical stimulus. Of note, the surgeon should change gloves if his/her hands touch non-sterile parts of the animal during the surgery such as checking pedal reflex to prevent potential contamination and infection. In addition, the surgeon and the assistant should also carefully monitor the vital signs of the animal during the operation to prevent anesthetic overdose and take quick corrective actions to avert the situation if it occurs by turning off the isoflurane and increase oxygen level to 100%. The signs of anesthetic overdose include slow and shallow respirations, and weak and irregular pulse. We also suggest a good preparation of arteriotomy before the balloon injury including a complete separation of carotid artery sections from their adjacent tissues. This will help to avoid unexpected bleedings during the operation.
The animal under anesthesia, including during the post-surgical recovery phase, should never be left unattended. As an alternative to the regular buprenorphine (0.03 mg/kg b.i.d.) during the 48 hr post-surgery period, sustained-release buprenorphine (buprenorphine SR) can also be used. Injection of buprenorphine SR (1.0-1.2 mg/kg) immediately after the surgery will cover the post-surgical period up to 72 hr. In general, postsurgical complications are not anticipated; but a careful monitory practice ensures the welfare of the animals and is a policy by the institutional ACUC.
Because it is most important to reproduce the same level of the balloon injury in the study group of animals (8-15), adequate surgical skills and familiarity with the procedure are highly recommended. For researchers new to this procedure, we suggest first to practice the entire procedure including the preparation of arteriotomy, the balloon injury and the wound closure on rat carcasses that may be available in the institutional animal facility. Practicing the surgical procedure with veterinarians or researchers familiar with the procedure also ensures to obtain reproducible and consistent balloon injury results.
In our view, drugs and/or therapeutics that negate neointimal growth are best administered immediately after balloon injury in order to offset the inflammation at early stage. Some reagents may need to be administered continually during the subsequent days of recovery to achieve the effect. An early intervention helps to block or reduce the ensuing vascular remodeling13,22,24.
The authors have nothing to disclose.
The work was supported by the intramural research program of the NIH, National Institute on Aging, and by a Priority Research Centers Program grant from the National Research Foundation (NRF-2009-0093812) funded by the Ministry of Science, Information and Communication Technology, & Future Planning, the Republic of Korea (H.T.). We thank Dr. Han-sol Park for putting “material” part together for the manuscript.
2 F Fogarty balloon embolectomy catheter | Edwards Lifesciences |
Standard scalpel | Fine Science Tools |
Small curved forceps (Large radius Dumont#7shanks curved) | Fine Science Tools |
Large, medium and small micro-scissors | Roboz |
Needles (20 G) | TycoHealthcare |
Micro-surgery forceps with micro-blunted atraumatic tips | Fine Science Tools |
Atraumatic straight small arterial clamps | Fine Science Tools |
Retractor with maximum spread 5.5 cm long blunt teeth | Fine Science Tools |
Silk suture (4.0 and 6.0 ) | Fine Science Tools |
Syringe (1.0 ml) | BD |
Curity gauze sponges | AllegroMedical |
Cotton tip applicators sterile and non-sterile | Puritan Medical Products |
Compact hot bead sterilizer | Fine Science Tools |
Self-regulating heating pad | Fine Science Tools |
ADS200 anesthesia system/ventilator | Paragon Medical |
Isoflurane (forane), liquid form | Baxter |
Sodium chloride 0.9% (Saline) | Hospira |
Buprenex (buprenorphine) | Reckitt Benckiser Healthcare (UK) Ltd. |
70% alcohol | Fisher |
1: 10 Betadine | Fisher |