The lack of mechanistic understanding of spinal cord ischemia-reperfusion injury has hindered further adjuncts to prevent paraplegia following high risk aortic operations. Thus, the development of animal models is imperative. This manuscript demonstrates reproducible lower extremity paralysis following thoracic aortic occlusion in a murine model.
Background
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered advancements to decrease the occurrence this injury. The current model demonstrates reproducible lower extremity paralysis following thoracic aortic occlusion.
Methods
Adult male C57BL6 mice were anesthetized with isoflurane. Through a cervicosternal incision the aorta was exposed. The descending thoracic aorta and left subclavian arteries were identified without entrance into pleural space. Skeletonization of these arteries was followed by immediate closure (Sham) or occlusion for 4 min (moderate ischemia) or 8 min (prolonged ischemia). The sternotomy and skin were closed and the mouse was transferred to warming bed for recovery. Following recovery, functional analysis was obtained at 12 hr intervals until 48 hr.
Results
Mice that underwent sham surgery showed no observable hind limb deficit. Mice subjected to moderate ischemia for 4 min had minimal functional deficit at 12 hr followed by progression to complete paralysis at 48 hr. Mice subjected to prolonged ischemia had an immediate paralysis with no observable hind-limb movement at any point in the postoperative period. There was no observed intraoperative or post operative mortality.
Conclusion
Reproducible lower extremity paralysis whether immediate or delayed can be achieved in a murine model. Additionally, by using a median sternotomy and careful dissection, high survival rates, and reproducibility can be achieved.
Lower extremity paralysis continues to complicate thoracoabdominal interventions. The injury, known as spinal cord ischemia-reperfusion injury (SCIR), results in paralysis in up to 20% of high risk patients1. Surgical adjuncts such as left heart bypass, lumbar cerbrospinal fluid drains, hypothermic circulatory arrest and intercostal artery reimplantation have reduced the incidence of this complication2; however, far too many patients continue to be affected.
Clinically, spinal cord ischemia and reperfusion injury is seen as either immediate or delayed paralysis following intervention3. However, our understanding of this injury has been stifled by a lack of mechanistic detail. As a result, few options are available to attenuate the injury once it has occurred.
We have thus enlisted a small animal, murine, model of spinal cord ischemia, and reperfusion injury to better characterize its pathogenesis. The majority of studies to date have used larger animal models to characterize this injury, namely rat4, rabbit5, and pig6 models. However, these are limited by their cost, complexity, variable reproducibility, and, most importantly, lack of available techniques for genetic manipulation. The most reliable of these published animal models involves infrarenal cross clamping of the abdominal aorta in rabbits. However, human anterior spinal neurons most often derive their vascular supply from more proximal branches7. Variable vascular anatomy of the spinal cord in these models adds to difficulty in transitioning their results into clinical use.
This manuscript presents a model for immediate or delayed paraplegia following thoracic aortic occlusion that is clinically relevant and easy to employ. Exposure of the aortic arch via mini sternotomy is less invasive and can elicit highly reproducible results with minimal morbidity and mortality. While this model in not without challenges and technical nuances, these can be overcome with careful dissection and tissue handling to produce a model of hind limb paralysis that can be easily implemented.
1. Preoperative Preparation and Anesthesia
2. Rectal Probe Laser Doppler Placement
3. Dissection of Aortic Arch/Subclavian Artery
4. Closure of Sternotomy and Skin
5. Recovery and Postoperative Assessment
Mice underwent sham surgery (n=3) or aortic occlusion for 4 (n=3) to 8 min (n=3). Postoperatively mice were graded by the Basso Mouse Score (Figure 1). Mice that underwent sham surgery had no observable functional deficits at any point postoperatively. Mice subjected to moderate ischemia (4 min) had near normal hind-limb function at 12 hr with progressive functional decline to complete paralysis by 48 hr. Mice in prolonged ischemia group (8min) had complete paralysis following surgery without any recoverable function (Figure 2).
Figure 1. Basso Score for Hind Limb Motor Function8. Scoring system for hind limb neurologic dysfunction graded from 0 (no function) to 9 (normal function). Click here to view larger image.
Figure 2. Postoperative Hind-Limb Function. The severity of neurologic deficit was graded using the Basso Mouse Scale at 12 hr intervals postoperatively until 48 hr. Click here to view larger image.
Paraplegia secondary to spinal cord ischemia reperfusion is the result of a complex of poorly understood pathologies9. While this is most commonly seen after thoracoabdominal aortic surgery, a variety of other insults such as aortic dissections, trauma, embolic phenomena, vasculitis, and systemic hypotension10 can result in paraplegia. To gain further understanding of this injury and provide future targets to eliminate this injury, animal models have become a necessity.
Patients afflicted with this complication exhibit either an immediate or delayed paralysis. This model and others adequately parallel11 the bimodal distribution of paralysis seen clinically. While moderate ischemia (4 min) produced a delayed paralysis, prolonged ischemia (8 min) resulted in an immediate and permanent paralysis.
The presented model has advantages compared to those requiring a lateral thoracotomy12,13. The median sternotomy exposure leaves the pleural cavity intact and eliminates the need for tracheal intubations or reestablishing negative pressure in the pleural space. Clinically, the sternotomy is associated with much less postoperative pain in than the lateral thoracotomy and might reduce postoperative analgesic requirements.
This model is not without limitations. As with other models, murine surgery has a learning curve and good surgical technique is imperative. Mice can quickly succumb if dissection is not done with care. The most common causes of intraoperative mortality are hemorrhage or pneumothorax. To avoid potentially fatal hemorrhage the sternotomy must be made midline. If the sternotomy is made too laterally a transaction of internal mammary arteries and subsequent hemorrhage may occur. Additionally, rough dissection of descending aorta and subclavian artery can result in hemorrhage the will be difficult or impossible to control. Pneumothoraces are another potentially fatal complication. These typically occur during the placement of retraction sutures or dissection of the aorta. If retraction sutures are place too laterally or the aortic arch is not dissected with care a pneumothorax can occur and are universally fatal.
Further steps need to be employed to ensure reproducibility. Normothermia is critical. During surgery mice are kept as close to 36.5 °C as possible. Even mild hypothermia has been shown in to have significant neuroprotective effects in spinal cord and stroke models14,15. Furthermore, laser Doppler measurements should be closely monitored and mice with only 90% reduction in distal flow should be included for comparison. Incomplete occlusion of the aorta or subclavian arteries can results in higher distal flows and results that are highly variable.
In conclusion, the murine model of lower extremity paralysis presented can be easily adopted, yielding highly reproducible results. Applications of this model can provided the investigator with a means to study the immediate and delayed paralysis. Moreover, adoption of this model might prove beneficial in combating this devastating complication.
The authors have nothing to disclose.
We would like to thank the Thoracic Surgery Foundation for Research and Education for their Financial Support of this project.
VMS Anesthesia Machine | MDS Matrx | ||
Isoflurane | Vet One | 13985-528-60 | 2.0% through nose cone |
Induction Chamber | Vet Equip | 941444 | |
Heating Bed | Vestavia Scientific | ||
Laser Doppler Monitor | Moor Instruments | VMS-LDF1 | |
5-0 Suture, Polyester | Surgidac | VD-551 | Taper Needel |
Microdissecting Clips | Biomedical Research Insturments | 14-1030, 14-1060 | |
Surgical Instruments | Fine Surgical Instruments | Forceps, needle holder |