This study demonstrates the technique to make a minimally invasive and easily reproducible model of spinal cord ischemia in rats. Various degrees of hind limb motor deficit can be produced by controlling the aortic occlusion time.
Spinal cord ischemia is a fatal complication following thoracoabdominal aortic aneurysm surgery. Researchers can investigate the strategies for preventing and treating this complication using experimental models of spinal cord ischemia. The model described here demonstrates varying degrees of paraplegia that relate to the length of occlusion following thoracic aortic occlusion in a rat spinal cord ischemia model.
A 2-Fr. balloon-tipped catheter was advanced through the femoral artery into the descending thoracic aorta until the catheter tip was placed at the left subclavian artery in anesthetized male Sprague-Dawley rats. Spinal cord ischemia was induced by inflating the catheter balloon. After a set period of occlusion (9, 10, or 11 min), the balloon was deflated. Neurologic assessment was performed using the motor deficit index at 24 h after surgery, and the spinal cord was harvested for histopathological examination.
Rats that underwent 9 min of aortic occlusion showed mild and reversible motor impairment in the hind limb. Rats subjected to 10 min of aortic occlusion presented with moderate but reversible motor impairment. Rats subjected to 11 min of aortic occlusion displayed complete and persistent paralysis. The motor neurons in the spinal cord sections were more preserved in rats subjected to shorter duration of aortic occlusion.
Researchers can achieve a reproducible hind limb motor deficit following thoracic aortic occlusion using this spinal cord ischemia model.
Paraplegia is a fatal complication of thoracoabdominal aortic aneurysm surgery. It results from spinal cord ischemia-reperfusion injury that occurs during cross-clamping and unclamping of the aorta.1 Several strategies including systemic hypothermia and cerebrospinal drainage have been introduced to protect the spinal cord,2,3,4 but many patients remain affected by the injury.
Several animal spinal cord ischemia models have been introduced to investigate its pathogenesis and devise protective strategies against the injury. In the current study, we outline a rat model of spinal cord ischemia based on Taira and Marsala's method.5 The spinal circulation system in rats is very similar to the spinal cord vascular and collateral system in humans, although there are some differences in the size and location.6,7 Thus, a rat is an anatomically suitable animal to utilize for an experimental model investigating the pathogenesis, complications, and treatment of spinal cord ischemia. Moreover, this spinal cord ischemia model produces reliable aortic occlusion with minimal intervention by utilizing an intravascular balloon occlusion of the thoracic aorta.
In this study, we demonstrated that this rat model of spinal cord ischemia induces reproducible motor deficits in the hind limbs that vary in severity depending on the aortic occlusion time.
This protocol was approved by the Institutional Animal Care and Use Committee of Seoul National University Bundang Hospital. Animal care and experiments were conducted according to the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals.
1. Surgical Preparation
2. Femoral Artery Catheterization
3. Carotid Artery Catheterization
4. Tail Artery Catheterization
5. Induction of Spinal Cord Ischemia
6. Post-surgical Care and Neurologic Assessment
7. Histopathologic Evaluation
8. Statistical Analysis
During a period of spinal cord ischemia, aortic occlusion was performed for 9 min (n=3), 10 min (n=3), or 11 min (n=3). The motor deficit index in rats is presented in Table 2. Rats that underwent 9 min of aortic occlusion showed a mild and reversible motor impairment in the hind-limb. Rats subjected to 10 min of aortic occlusion presented with moderate motor deficit, but not complete paralysis. Rats that underwent 11 min of occlusion time displayed complete and persistent paralysis.
Representative photographs of spinal cord sections stained with H & E are shown in Figure 1. The motor neurons in the spinal cord sections were more preserved in rats subjected to a shorter duration of aortic occlusion.
Figure 1. Histologic Examination of Spinal Cord Sections
The motor neurons were more preserved in rats subjected to a shorter duration of aortic occlusion (original magnification, 200X). Scale bar in all images = 50 µm. Please click here to view a larger version of this figure.
Ambulation (walking with lower extremities) | Placing/stepping reflex |
0: normal (symmetrical and coordinated ambulation) | 0: normal |
1: toes flat under body when walking but ataxia present | 1: weak |
2: knuckle-walking | 2: no stepping |
3: movement in lower extremities but unable to knuckle-walk | |
4: no movement, drags lower extremities |
Table 1. Evaluation of Ambulation and the Placing/stepping Reflex.
Aortic occlusion time | 24 h after surgery |
9 min (n = 3) | 2 (2 − 3) |
10 min (n = 3) | 4 (4 − 4) |
11 min (n = 3) | 5 (5 − 6) |
Table 2. Motor Deficit Index. Values are presented as median (interquartile range).
In the current study, we demonstrated a rat model of spinal cord ischemia based on Taira and Marsala's method5 that induces variable degrees of motor deficit in the hind limb depending on the aortic occlusion time.
The length of aortic occlusion can affect the degree of motor deficit. If the aortic occlusion time is longer, the motor deficit becomes more severe. Thus, researchers can achieve a certain degree of motor deficit by controlling the aortic occlusion time in this model.
Our model involves the ligation of the common carotid artery and the femoral artery, and the possibility of neurologic deficits resulting from the ligation of these arteries is a potential concern. However, rats have efficient collateral network systems. Thus, when the carotid artery or femoral artery is ligated, sufficient blood flow can be provided by the extensive collateral network. Unilateral carotid artery occlusion is reported to produce only minor effects on cerebral blood flow.10,11 Although unilateral carotid artery occlusion can produce stroke in rats, this occurs only when in combination with severe systemic hypoxia.12 During our experiment, no systemic hypoxia occurred and none of the rats presented with neurological deficits suggestive of cerebral infarction. Furthermore, when the femoral artery of rats is ligated, the collateral circulation provides sufficient blood flow to the hind limb muscles.13 This collateral circulation provides sufficient blood flow to the hind limb muscles when they are at rest, but does not provide sufficient flow during exercise.14
Furthermore, the proximal arterial pressure during spinal cord ischemia affects the development of motor deficits. According to a previous study,5 the collateral blood supply almost disappeared at the proximal arterial pressure of 40 mmHg during aortic clamping in a rat spinal ischemia model. Thus, subsequent studies maintained the proximal arterial pressure at 40 mmHg during spinal cord ischemia in their models of rat spinal cord ischemia.9,15,16 However, in this protocol, we maintained the proximal arterial pressure at 80 mmHg during aortic occlusion because it is recommended to maintain the mean arterial pressure at 80 mmHg or greater for preserving adequate spinal cord perfusion during spinal cord ischemia in the clinical practice,17 although there might be a difference in what constitutes an adequate proximal arterial pressure between humans and rodents.
The spinal cord vasculature and collateral system of rats and humans are similar,6,7 which makes rats an appropriate choice for an experimental spinal cord ischemia model. However, it should not be discounted that the results may be different according to the species in which spinal cord ischemia occurs.
In conclusion, researchers can easily adopt this rat model of spinal cord ischemia and achieve highly reproducible findings. Furthermore, they can modify the aortic occlusion time to vary the degree of motor deficit produced. As such, this model can facilitate further studies examining the underlying pathophysiology of neurologic complications following thoracoabdominal aortic aneurysm, and allow the development of neuroprotective strategies against these complications.
The authors have nothing to disclose.
The authors have no acknowledgements.
Fogarty Arterial Embolectomy catheter | Edward Life Sciences | 120602F | a balloon-tipped catheter inserted into the femoral artery |
BD Insyte-N Autoguard Shielded IV catheter | BD | 381411 | 24-gauge intravenous catheter |
50mL syringe | KOREA VACCINE | KOVAX-SYRINGE 50mL | Facial mask |
1mL syringe | KOREA VACCINE | KOVAX-SYRINGE 1ml | |
Recal probe | HARVARD APPARATUS | 50-7221F | Rectal probe for temperature monitoring |
Micro dissecting spring scissor | Jeung do bio & Plant co.LTD. | JD-S-10 | Micro-scissor |
SCISSOR (SHARP-SHARP) | Jeung do bio & Plant co.LTD. | S-51-12-S | Scissors |
Retractor | Jeung do bio & Plant co.LTD. | JD-S-74A | Retractor |
Micro forcep | Jeung do bio & Plant co.LTD. | JD-S-29 | Micro-forceps |
MOSQUITO FORCEP (Curved) | Jeung do bio & Plant co.LTD. | S-44-CPK | Curved forceps |
DRESSING FORCEP | Jeung do bio & Plant co.LTD. | S-37-16S | Blunted forceps |
4/0 black silk | Woori Medical | S431 | 4.0 black silk suture |
3-WAY STOCK | Seonwon Medcal | D-98-01 | 3-way stopcock |
Patient monitor | PHILIPS | MP20 | The arterial pressure monitoring device. |
Heating blanket | Self production | Heating blanket | |
Microtube and external reservoir | Self production | Microtube and external reservoir | |
Heparin | JW Pharmaceutical | Heparin | |
0.9% NS 1000ml | JW Pharmaceutical | Normal saline | |
Isoflurane | Hana Med | Isoflurane |