We developed a simplified mouse model of spinal cord ischemia using the aortic cross-clamping technique used in larger species. By lowering the level of aortic cross-clamping to the middle segment of the thoracic aorta, mice were able to survive long-term. In addition, by using a left thoracotomy, immediate access to the thoracic aorta was provided and did not require disruption of bony structures or interruption of flow in the inferior vena cava. Thus, it is less invasive and facilitates recovery from surgery necessary to create the ischemic insult. Mice in the sham group did not have any motor functional deficit even at 1 h after surgery when compared with baseline values, indicating that the thoracotomy procedure, itself, was well tolerated. The severity of neurological deficit was closely associated with ischemia duration and loss of ventral horn motor neurons. During aortic occlusion, blood flow velocity in the dorsal lumbar spinal cord fell to <10 % of baseline flow, confirming severe lumbar spinal cord ischemia. The results also demonstrated that 10 min of aorta cross-clamping is the optimal ischemia duration to study the pathological mechanism and therapy development in the C57Bl/6J mouse should long-term outcome measurements be desired. This is based on a mortality rate of 20%, but persistent functional deficits over a 28-day recovery interval. Strain differences have been reported for sensitivity to cerebral ischemia (Wellons et al. 2000). It is plausible that other mouse strains will respond differently to this form of spinal cord ischemia.
With 12 min ischemia, 4 of 7 mice died at post-injury 2–7 days for undefined reasons, but plausibly due to organ (kidney, gut) failure. Surviving mice showed a persistent neurological deficit. Lang-Lazdunski et al. (Lang-Lazdunski et al., 2000
) gave a subcutaneous injection of heparin (400 IU/kg) in their model. We did not use heparin prior to ischemia. The use of heparin might provide a different post-ischemic recovery. In order to determine if the lack of heparin in this model caused thrombosis and further to spinal cord ischemia, we examined the spinal cord histology at 24 h following 12 min of aortic clamping. Dead neurons were found in the ventral horn. However, no thrombosis was in adjacent vessels (data not shown). We examined the distal aorta during aortic clamping and no abnormal signs were found. Finally, toe color rapidly returned to normal after removal of the aneurysm clip. Thus, we could not find evidence that is that 10 min of aortic clamping led to aortic thrombosis, complicating ischemia caused by aortic cross-clamping.
The vertebral artery arises from the subclavian artery and gives off branches to supply the spinal cord. To obtain more reliable ischemia, Lang Lazdunski et al. (Lang-Lazdunski et al., 2000
) clamped the subclavian artery to further decrease spinal cord blood flow. In our model, spinal cord blood flow was decreased by an average of >90% from of baseline after thoracic aortic clamping only. This was similar to the post-ischemic blood flow change of Tiara and Marsala (Taira and Marsala, 1996
) who subjected rats to mid-thoracic endo-aortic balloon occlusion. To our knowledge a method of intra-aortic balloon occlusion has not been developed in the mouse. Thoracic aortic occlusion was sufficiently severe to induce spinal cord ischemia in our model, and it appears that it is not necessary to add subclavian artery occlusion, plausibly decreasing post-operative surgical morbidity. There were no deaths in mice subjected to sham surgery or ischemia intervals of 10 min or less during one week post-ischemia. This indicates that the surgical procedure allows full recovery by avoiding surgical manipulation of vital structures in the mediastinum. The total anesthesia/surgery time required to perform the procedure was less than 30 min. Ten min of ischemia induced a reliable injury and injured mice had 100% motor deficit in the first 24 hours and recovered about 30–50% of normal motor function by 7 days post-ischemia.
Several groups have reported that spontaneous recovery of sensory motor function occurs after spinal cord injury in rodents (Farooque et al., 2006
; Gulino et al., 2007
; Lapointe et al., 2006
; Lee et al., 2009
; Weidner et al., 2001
; Wolpaw and Tennissen, 2001
). This recovery is strain-dependent (Lapointe et al., 2006
), gender-related (Farooque et al., 2006
) and use-dependent (Wolpaw and Tennissen, 2001
). A small proportion of nerve fibers spared by the original injury were found to grow new connections to other cells. Called sprouting, this re-growth occurs spontaneously without therapeutic intervention. When sprouting is prevented, functional recovery does not occur (Weidner et al., 2001
). It was also found that synaptic plasticity modulates the spontaneous recovery of locomotion after spinal cord hemisection (Gulino et al., 2007
). Enhanced H-reflex response promotes functional recovery (Lee et al., 2009
). Our study also showed that motor deficit severity is associated with ischemia duration and that this also affects rate of spontaneous functional recovery. Ung et al. (Ung et al., 2007
) reported that post-injury BBB scores reached a spontaneous recovery plateau by 28 days. Guertin et al. (Guertin, 2005
) also chose post-injury 28 days as a period for quantitatively assessing hind limb movement recovery without intervention in adult paraplegic mice. Thereby, we decided to use a 28 day recovery interval in the long-term survival experiment.
Body temperature and blood glucose are two important factors affecting outcome in ischemic injury. Therefore, body temperature and glucose control are likely to be crucial to obtain a stable injury in this model besides fully cross-clamping the aorta.
There were few necrotic neurons remaining in the ventral horn at 7 days after either 10 or 12 min ischemia. We assume that most degenerated or dead neurons had undergone phagocytosis by this time. We stained some slides from one mouse subjected to 12 min ischemia and euthanasia at 24 hours post-injury. Numerous neurons in the ventral horn exhibited cytoplasmic eosinophilia and pyknotic homogenous nuclei. Infarct was mainly localized in the central part of the intermediate zone and the dorsomedial aspect of the dorsal horn and was characterized by destruction of normal tissue, infiltrated neutrophils and mononuclear phagocytes, and gliosis. This pattern is consistent with previous experimental studies of aortic occlusion-induced spinal cord ischemia in other animal models (Kanellopoulos et al., 1997
; Marsala and Yaksh, 1994
; Taira and Marsala, 1996
; Yamamoto et al., 1994
Because less surgical invasiveness is required in this model, the injured mice were able to quickly recover and did not have surgical complications confounding spinal cord ischemia. In the experiment evaluating post-ischemia long-term survival, 80% (4 out of 5) mice survived for the full 28 day observation interval, all of them having had severe motor functional deficits immediately after injury.