Necrostatin-1 attenuates delayed paraplegia after transient spinal cord ischemia in rabbits by inhibiting the upregulation of receptor-interacting protein kinase 1 and 3

by Selleckchem | Aug 17 2023

Background: Delayed-onset paraplegia is a disastrous complication after thoracoabdominal aortic open surgery and thoracic endovascular aortic repair. Studies have revealed that transient spinal cord ischemia caused by temporary occlusion of the aorta induces delayed motor neuron death owing to apoptosis and necroptosis. Recently, necrostatin-1 (Nec-1), a necroptosis inhibitor, has been reported to reduce cerebral and myocardial infarction in rats or pigs. In this study, we investigated the efficacy of Nec-1 in delayed paraplegia after transient spinal cord ischemia in rabbits and assessed the expression of necroptosis- and apoptosis-related proteins in motor neurons.

Methods: This study used rabbit transient spinal cord ischemia models using a balloon catheter. They were divided into a vehicle-treated group (n = 24), Nec-1-treated group (n = 24), and sham-controls (n = 6). In the Nec-1-treated group, 1 mg/kg Nec-1 was intravascularly administered immediately before ischemia induction. Neurological function was assessed using the modified Tarlov score, and the spinal cord was removed 8 h and 1, 2, and 7 days after reperfusion. Morphological changes were examined using hematoxylin and eosin staining. The expression levels of necroptosis-related proteins (receptor-interacting protein kinase (RIP) 1 and 3) and apoptosis-related proteins (Bax and caspase-8) were assessed using western blotting and histochemical analysis. We also performed double-fluorescence immunohistochemical studies of RIP1, RIP3, Bax and caspase-8.

Results: Neurological function significantly improved in the Nec-1-treated group compared with that in the vehicle-treated group 7 days after reperfusion (median 3 and 0, p = 0.025). Motor neurons observed 7 days after reperfusion were significantly decreased in both groups compared with in the sham group (vehicle-treated, p < 0.001; Nec-1-treated, p < 0.001). However, significantly more motor neurons survived in the Nec-1-treated group than in the vehicle-treated group (p < 0.001). Western blot analysis revealed RIP1, RIP3, Bax, and caspase-8 upregulation 8 h after reperfusion in the vehicle-treated group (RIP1, p = 0.001; RIP3, p = 0.045; Bax, p = 0.042; caspase-8, p = 0.047). In the Nec-1-treated group, the upregulation of RIP1 and RIP3 was not observed in any time point, whereas that of Bax and caspase-8 was observed 8 h after reperfusion (Bax, p = 0.029; caspase-8, p = 0.021). Immunohistochemical study revealed the immunoreactivity of these proteins in motor neurons. Double-fluorescence immunohistochemistry revealed the induction of RIP1 and RIP3, and that of Bax and caspase-8 in the same motor neurons.

Conclusions: These data suggest that Nec-1 reduces delayed motor neuron death and attenuates delayed paraplegia after transient spinal cord ischemia in rabbits by selectively inhibiting necroptosis of motor neurons, with minimal effect on their apoptosis.

Comments:

The study investigated the efficacy of necrostatin-1 (Nec-1), a necroptosis inhibitor, in reducing delayed paraplegia after transient spinal cord ischemia in rabbits. Delayed-onset paraplegia is a severe complication that can occur after thoracoabdominal aortic open surgery and thoracic endovascular aortic repair. The condition is characterized by delayed motor neuron death due to apoptosis and necroptosis resulting from transient spinal cord ischemia caused by temporary occlusion of the aorta.

The researchers used rabbit models of transient spinal cord ischemia induced by a balloon catheter. The rabbits were divided into three groups: a vehicle-treated group, a Nec-1-treated group, and sham-controls. The Nec-1-treated group received an intravascular administration of 1 mg/kg Nec-1 immediately before inducing ischemia. The researchers assessed neurological function using the modified Tarlov score and examined the spinal cord morphological changes using hematoxylin and eosin staining. They also analyzed the expression levels of necroptosis-related proteins (receptor-interacting protein kinase 1 and 3, RIP1 and RIP3) and apoptosis-related proteins (Bax and caspase-8) through western blotting and histochemical analysis. Additionally, double-fluorescence immunohistochemical studies were performed to examine the presence of these proteins in motor neurons.

The results showed that neurological function significantly improved in the Nec-1-treated group compared to the vehicle-treated group seven days after reperfusion. The number of motor neurons observed seven days after reperfusion was significantly reduced in both groups compared to the sham group. However, the Nec-1-treated group had significantly more surviving motor neurons compared to the vehicle-treated group. Western blot analysis revealed upregulation of RIP1, RIP3, Bax, and caspase-8 eight hours after reperfusion in the vehicle-treated group. In contrast, the Nec-1-treated group did not show upregulation of RIP1 and RIP3 at any time point, but the upregulation of Bax and caspase-8 was observed eight hours after reperfusion. Immunohistochemical studies confirmed the presence of these proteins in motor neurons, and double-fluorescence immunohistochemistry showed the induction of RIP1 and RIP3, as well as Bax and caspase-8, in the same motor neurons.

Based on these findings, the researchers concluded that Nec-1 reduces delayed motor neuron death and attenuates delayed paraplegia after transient spinal cord ischemia in rabbits by selectively inhibiting necroptosis of motor neurons, with minimal effect on their apoptosis. This study suggests that Nec-1 may hold promise as a therapeutic agent for preventing or reducing the severity of delayed paraplegia in patients undergoing surgeries involving temporary occlusion of the aorta.