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This article summarizes the anatomically driven pathophysiology of spinal cord injury, the impact of ischemia reperfusion on the cellular level, current research in developing neuronal ischemic tolerance, and promotion of collateralization. It addresses neuroprotective strategies in modern clinical practice, current pharmacologic interventions, and continued challenges in the management of complex aortic disease. The pathophysiology of spinal cord injury includes disruption and recovery of collateral blood flow and the effects of malperfusion on the spinal cord. The optimal approach to spinal cord protection is to employ an integrated and protocolized set of strategies to simultaneously maximize spinal cord blood flow, improve ischemic tolerance, and promote collateralization. In the laboratory setting, investigation should continue to pursue a more granular understanding of ischemic injury and seek to translate protective therapies to clinical practice., (Copyright © 2019. Published by Elsevier Inc.)

Background: Paraplegia remains the most feared complication of complex thoracoabdominal aortic intervention. Although erythropoietin (EPO) has demonstrated neuroprotective effects in spinal cord ischemia, it does not work until expression of the beta common receptor subunit of the EPO receptor (βcR) is induced by ischemia. We hypothesized that the βcR can be induced by diazoxide (DZ), amplifying the neuroprotective effects of EPO in spinal cord ischemia-reperfusion injury., Methods: For the DZ time trial, adult male C57/BL6 mice received DZ (20 mg/kg) by oral gavage. Spinal cords were harvested after 0, 12, 24, 36, and 48 hours of administration. To evaluate optimal dosing, DZ was administered at 0, 5, 10, 20, and 40 mg/kg. The expression of βcR was assessed by Western blot analysis. Five groups were studied: PBS (pretreatment)+PBS (immediately before), PBS+EPO, DZ+PBS, DZ+EPO, and sham (without cross-clamping). Spinal cord ischemia was induced by 4 minutes of thoracic aortic cross-clamping. Functional scoring (Basso Mouse Score) was done at 12-hour intervals for 48 hours, and spinal cords were harvested for histological analysis., Results: Western blot analysis demonstrated that optimal βcR up-regulation occurred at 36 hours after DZ administration, and the optimal DZ dosage for βcR induction was 20 mg/kg. Motor function at 48 hours after treatment was significantly better preserved in the DZ+EPO group compared with all other groups, and was significantly better preserved in the DZ only and EPO only groups compared with control (PBS+PBS)., Conclusions: Pharmacologic up-regulation of βcR with DZ can increase the efficacy of EPO in preventing spinal cord ischemia and reperfusion injury. Improved understanding of this synergetic mechanism may serve to further prevent ischemic complications for high-risk aortic intervention., (Copyright © 2018 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.)

Background: Paraplegia from spinal cord ischemia-reperfusion (SCIR) remains an elusive and devastating complication of complex aortic operations. Erythropoietin (EPO) attenuates this injury in models of SCIR. Upregulation of the EPO beta common receptor (βcR) is associated with reduced damage in models of neural injury. The purpose of this study was to examine whether EPO-mediated neuroprotection was dependent on βcR expression. We hypothesized that spinal cord neurons subjected to oxygen-glucose deprivation would mimic SCIR injury in aortic surgery and EPO treatment attenuates this injury in a βcR-dependent fashion., Methods: Lentiviral vectors with βcR knockdown sequences were tested on neuron cell cultures. The virus with greatest βcR knockdown was selected. Spinal cord neurons from perinatal wild-type mice were harvested and cultured to maturity. They were treated with knockdown or nonsense virus and transduced cells were selected. Three groups (βcR knockdown virus, nonsense control virus, no virus control; n = 8 each) were subjected to 1 hour of oxygen-glucose deprivation. Viability was assessed. βcR expression was quantified by immunoblot., Results: EPO preserved neuronal viability after oxygen-glucose deprivation (0.82 ± 0.04 versus 0.61 ± 0.01; p < 0.01). Additionally, EPO-mediated neuron preservation was similar in the nonsense virus and control mice (0.82 ± 0.04 versus 0.80 ± 0.05; p = 0.77). EPO neuron preservation was lost in βcR knockdown mice compared with nonsense control mice (0.46 ± 0.03 versus 0.80 ± 0.05; p < 0.01)., Conclusions: EPO attenuates neuronal loss after oxygen-glucose deprivation in a βcR-dependent fashion. This receptor holds immense clinical promise as a target for pharmacotherapies treating spinal cord ischemic injury., (Copyright © 2017 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 10 6 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

Background: Paraplegia remains a devastating complication of aortic surgery, occurring in up to 20% of complex thoracoabdominal repairs. Erythropoietin (EPO) attenuates this injury in models of spinal cord ischemia. Upregulation of the beta-common receptor (βcR) subunit of the EPO receptor is associated with reduced damage in murine models of neural injury. This receptor activates anti-apoptotic pathways including signaling transducer and activator of transcription 3 (STAT3). We hypothesized that spinal cord ischemia-reperfusion injury upregulates the βcR subunit with a subsequent increase in activated STAT3., Methods: Adult male C57/BL6 mice received an intraperitoneal injection of 0.5 mL of EPO (10 U/kg) or 0.9% saline after induction of anesthesia. Spinal cord ischemia was induced through sternotomy and 4-minute thoracic aortic cross-clamp. Sham mice underwent sternotomy without cross-clamp placement. Four groups were studied: ischemic and sham groups, each with and without EPO treatment. After 4 hours of reperfusion, spinal cords were harvested and homogenized. The βcR subunit expression and STAT3 activation were evaluated by immunoblot., Results: Ischemia reperfusion increased βcR subunit expression in spinal cords of ischemia + saline and ischemia + EPO mice compared with shams (3.4 ± 1.39 vs 1.31 ± 0.3, p = 0.01 and 3.80 ± 0.58 vs 1.56 ± 0.32, p = 0.01). Additionally, both ischemic groups demonstrated increased STAT3 activation compared with shams (1.35 ± 0.14 vs 1.09 ± 0.07, p = 0.01 and 1.66 ± 0.35 vs 1.08 ± 0.17, p = 0.02)., Conclusions: Ischemia-reperfusion injury induces EPO receptor βcR subunit expression and early downstream anti-apoptotic signaling through STAT3 activation. Further investigation into the role of the βcR subunit is warranted to determine tissue protective functions of EPO. Elucidation of mechanisms involved in spinal cord protection is essential for reducing delayed paraplegia., (Copyright © 2015 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Objectives: Delayed paraplegia secondary to ischemia-reperfusion injury is a devastating complication of thoracoabdominal aortic surgery. Alpha-2 agonists have been shown to attenuate ischemia-reperfusion injury, but the mechanism for protection has yet to be elucidated. A growing body of evidence suggests that astrocytes play a critical role in neuroprotection by release of neurotrophins. We hypothesize that alpha-2 agonism with dexmedetomidine increases glial cell-line-derived neurotrophic factor in spinal cord astrocytes to provide spinal cord protection., Methods: Spinal cords were isolated en bloc from C57BL/6 mice, and primary spinal cord astrocytes and neurons were selected for and grown separately in culture. Astrocytes were treated with dexmedetomidine, and glial cell-line-derived neurotrophic factor was tested for by enzyme-linked immunosorbent assay. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to assess neuronal viability., Results: Spinal cord primary astrocytes treated with dexmedetomidine at 1 μmol/L and 10 μmol/L had significantly increased glial cell-line-derived neurotrophic factor production compared with control (P < .05). Neurons subjected to oxygen glucose deprivation had significant preservation (P < .05) of viability with use of dexmedetomidine-treated astrocyte media. Glial cell-line-derived neurotrophic factor neutralizing antibody eliminated the protective effects of the dexmedetomidine-treated astrocyte media (P < .05)., Conclusions: Astrocytes have been shown to preserve neuronal viability via release of neurotrophic factors. Dexmedetomidine increases glial cell-derived neurotrophic factor from spinal cord astrocytes via the alpha-2 receptor. Treatment with alpha-2 agonist dexmedetomidine may be a clinical tool for use in spinal cord protection in aortic surgery., (Copyright © 2015 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.)

Background: Despite investigation into preventable pharmacologic adjuncts, paraplegia continues to complicate thoracoabdominal aortic interventions. The alpha 2a adrenergic receptor agonist, dexmedetomidine, has been shown to preserve neurologic function and neuronal viability in a murine model of spinal cord ischemia reperfusion, although the mechanism remains elusive. We hypothesize that dexmedetomidine will blunt postischemic inflammation in vivo following thoracic aortic occlusion with in vitro demonstration of microglial inhibition following lipopolysaccharide (LPS) stimulation., Methods: Adult male C57BL/6 mice underwent 4 minutes of aortic occlusion. Mice received 25 μg/kg intraperitoneal dexmedetomidine (n = 8) or 0.9% normal saline (n = 7) at reperfusion and 12-hour intervals postoperatively until 48 hours. Additionally, sham mice (n = 3), which had aortic arch exposed with no occlusion, were included for comparison. Functional scoring was done at 6 hours following surgery and 12-hour intervals until 60 hours when spinal cords were removed and examined for neuronal viability and cytokine production. Additional analysis of microglia activation was done in 12 hours following surgery. Age- and sex-matched mice had spinal cord removed for microglial isolation culture. Cells were grown to confluence and stimulated with toll-like receptor-4 agonist LPS 100 ng/mL in presence of dexmedetomidine or vehicle control for 24 hours. Microglia and media were then removed for analysis of protein expression., Results: Dexmedetomidine treatment at reperfusion significantly preserved neurologic function with mice in treatment group having a Basso Score of 6.3 in comparison to 2.3 in ischemic control group. Treatment was associated with a significant reduction in microglia activation and in interleukin-6 production. Microglial cells in isolation when stimulated with LPS had an increased production of proinflammatory cytokines and markers of activation. Treatment with dexmedetomidine significantly attenuated microglial activation and proinflammatory cytokine production in vitro with a greater than twofold reduction in tumor necrosis factor-α., Conclusions: Alpha 2a agonist, dexmedetomidine treatment at reperfusion preserved neurologic function and neuronal viability. Furthermore, dexmedetomidine treatment resulted in an attenuation of microglial activation and proinflammatory cytokine production both in vivo and in vitro following LPS stimulation. This finding lends insight into the mechanism of paralysis following thoracic aortic interventions and may guide future pharmacologic targets for attenuating spinal cord ischemia and reperfusion., (Copyright © 2014 Society for Vascular Surgery. Published by Mosby, Inc. All rights reserved.)

Objective: Dexmedetomidine, an α-2a adrenergic agonist, given pre- and postoperatively was previously shown to attenuate neuronal injury in a murine model of spinal cord ischemia-reperfusion. In the brain, α-2 agonists have been shown to induce the phosphorylation of cyclic AMP response-element binding protein (CREB), a transcription factor necessary for neuron survival. We hypothesized that the α-2a adrenergic agonist given preoperatively increases CREB-mediated neuroprotective proteins, attenuating neuronal injury and cytoarchitectural decay., Methods: Mice (ie, C57BL/6 mice) underwent 5 minutes of aortic occlusion via median sternotomy. Mice received 25 μg/kg dexmedetomidine or equivalent normal saline at 24 hours, 12 hours, and 30 minutes preoperatively. Functional outcomes were recorded at 6 to 48 hours postoperatively when spinal cords were removed for histologic analysis. Spinal cords were examined for protein kinase B, CREB, B-cell lymphoma 2, and brain-derived neurotrophic factor following treatment alone or ischemia-reperfusion surgery., Results: Following aortic occlusion, mice in the treatment group had preserved neurologic function at all time points (P < .05). Histologic analysis showed preserved cytoarchitecture and decreased neuronal injury in the treatment group when compared with ischemic controls. Additionally, analysis of spinal cord homogenate following surgery and pretreatment revealed a significant (P < .05) increase in B-cell lymphoma 2 and brain-derived neurotrophic factor expression and protein kinase B and CREB phosphorylation with α-2a adrenergic agonist pretreatment., Conclusions: Pretreatment with the α-2a agonist dexmedetomidine preserved neurologic function and attenuated neuronal injury following thoracic aortic occlusion in mice. This relationship was associated with an increased phosphorylation of protein kinase B and CREB and subsequent up-regulation of antiapoptotic factor B-cell lymphoma 2 and brain-derived neurotrophic factor. Thus, α-2a receptor agonism-induced CREB phosphorylation and contributes to dexmedetomidine's protective mechanism in the spinal cord following ischemia., (Copyright © 2014. Published by Mosby, Inc.)

Objectives: Adenosine receptor activation at reperfusion has been shown to ameliorate ischemia-reperfusion injury of the spinal cord, but the effects of therapy given in response to ischemic injury are unknown. We hypothesized that adenosine receptor activation with ATL-146e would produce similar protection from ischemic spinal cord injury, whether given at reperfusion or in a delayed fashion., Methods: Twenty-two New Zealand white rabbits were divided into three groups. All three groups, including the ischemia-reperfusion group (IR, n = 8), underwent 45 min of infrarenal aortic occlusion. The early treatment group (early, n = 8) received 0.06 mug/kg/min of ATL-146e for 3 h beginning 10 min prior to reperfusion. The delayed treatment group (delayed, n = 6) received ATL-146e starting 1 h after reperfusion. After 48 h, hind limb function was graded using the Tarlov score. Finally, lumbar spinal cord neuronal cytoarchitecture was evaluated., Results: Hemodynamic parameters were similar among the groups. Hind limb function at 48 h was significantly better in the early group (3.5 +/- 1.0) compared to the IR group (0.625 +/- 0.5, P < or = 0.01). There was a trend towards better hind limb function in the early group compared to the delayed group (2.4 +/- 1.1, P = 0.08). Hind limb function was similar between delayed and IR groups. Hematoxylin-eosin spinal cord sections demonstrated preservation of viable motor neurons in the early group compared to the delayed and IR groups., Conclusions: Early therapy with ATL-146e provided better protection in this study; therefore, therapy should not be delayed until there is evidence of ischemic neurological deficit. This study suggests that adenosine receptor activation is most effective as a preventive strategy at reperfusion for optimal protection in spinal cord ischemia-reperfusion injury.

Background: ATL-146e protects the spinal cord from ischemia/reperfusion injury, presumably via adenosine A(2A) receptor activation, but this relationship remains unproven. We hypothesized that spinal cord functional and cytoarchitectural preservation from ATL-146e would be lost with simultaneous administration of the specific adenosine A(2A) antagonist ZM241385 (ZM), thus proving that adenosine A(2A) receptor activation is responsible for the protective effects of this compound., Methods: New Zealand White rabbits underwent 45 minutes of infrarenal aortic cross-clamping. Groups (n = 10) included sham, ischemia, ischemia plus ATL-146e (ATL-146E), ischemia plus ZM, or ischemia with both compounds (agonist-antagonist). Tarlov scores were recorded every 12 hours. After 48 hours, the spinal cord was fixed for histology and microtubule-associated protein 2 immunohistochemistry., Results: Tarlov scores at 48 hours were significantly better in the sham and ATL-146E groups (5.0 and 3.9, respectively) compared with the other three groups (all < or =1.3; P < .001). On hematoxylin and eosin, neuronal viability was higher in the sham, ATL-146E, and agonist-antagonist groups compared with the control and ZM groups (P < .05). Microtubule-associated protein 2 expression was preserved in the sham and ATL-146E groups but was lost in the ATL + ZM, ZM241385, and control groups., Conclusions: ATL-146e preserves the spinal cord in terms of both cytoarchitecture and function after reperfusion of the ischemic spinal cord, but this preservation is not completely blocked by competitive adenosine A(2A) receptor antagonism. Although ATL-146e does seem to partially function through activation of the adenosine A(2A) receptor, the neuroprotective mechanism may not be limited to this particular receptor.

Background: Paraplegia remains a devastating complication of thoracoabdominal aortic procedures resulting from spinal cord ischemia and reperfusion injury (SCIR). Pharmacologic interventions have not proven efficacious in attenuating this injury, with poor understanding of the underlying mechanisms. The resident macrophages, or microglia in the spinal cord, may play a significant role in SCIR. The macrolide antibiotic, minocycline, has been shown in stroke models to inhibit microglial activation. This study hypothesized that microglial inhibition by minocycline after SCIR will attenuate injury with preservation of motor function. Methods: Mature male C57Bl/6 mice underwent 4 minutes of thoracic aortic occlusion with reperfusion. Mice receiving minocycline 30 minutes before ischemia and daily thereafter (90 mg/kg and 45 mg/kg, respectively) were compared with mice receiving vehicle controls. Hind-limb motor function was measured at 12-hour intervals, with spinal cord harvest for histologic and immunologic comparison at 60 hours. Results: Minocycline treatment significantly preserved hind limb motor function in all mice (n = 7) compared with complete paralysis in all untreated mice (n = 8), reaching significance from 24 hours of reperfusion through 60 hours. Immunofluorescent staining for Iba-1 revealed significant inhibition of microglial activation by minocycline treatment. Vehicle control sections demonstrated a greater degree of apoptosis compared with minocycline-treated spinal cord sections. Conclusions: Minocycline limits microglial activation, paralleling functional preservation after aortic cross-clamping. These data suggest functional microglia contribute to reperfusion injury after spinal cord ischemia. The effects of minocycline demonstrate a potential pharmacological therapy as well as demonstrating a potential cellular target in preventing paraplegia after aortic intervention. [ABSTRACT FROM AUTHOR]

Background--Paraplegia continues to complicate thoracoabdominal aortic interventions. The elusive mechanism of spinal cord ischemia-reperfusion injury has delayed the development of pharmacological adjuncts. Microglia, the resident macrophages of the central nervous system, can have pathological responses after a variety of insults. This can occur through toll-like receptor 4 (TLR-4) in stroke models. We hypothesize that spinal cord ischemia-reperfusion injury after aortic occlusion results from TLR-4-mediated microglial activation in mice. Methods and Results--TLR-4 mutant and wild-type mice underwent aortic occlusion for 5 minutes, followed by 60 hours of reperfusion when spinal cords were removed for analysis. Spinal cord cytokine production and microglial activation were assessed at 6 and 36 hours after surgery. Isolated microglia from mutant and wild-type mice were subjected to oxygen and glucose deprivation for 24 hours, after which the expression of TLR-4 and proinflammatory cytokines was analyzed. Mice without functional TLR-4 demonstrated decreased microglial activation and cytokine production and had preserved functional outcomes and neuronal viability after thoracic aortic occlusion. After oxygen and glucose deprivation, wild-type microglia had increased TLR-4 expression and production of proinflammatory cytokines. Conclusions--The absence of functional TLR-4 attenuated neuronal injury and microglial activation after thoracic aortic occlusion in mice. Furthermore, microglial upregulation of TLR-4 occurred after oxygen and glucose deprivation, and the absence of functional TLR-4 significantly attenuated the production of proinflammatory cytokines. In conclusion, TLR-4- mediated microglia activation in the spinal cord after aortic occlusion is critical in the mechanism of paraplegia after aortic cross-clamping and may provide targets for pharmacological intervention. [ABSTRACT FROM AUTHOR]

Background: Paraplegia remains a significant complication of thoracoabdominal aortic surgery. The study of this spinal cord ischemia and reperfusion injury (SCIR) has been stifled by the constraints of current models. Large animal models are costly and restricting with regard to mechanisms of both injury and protection. While few studies of a murine model of SCIR have been published, the dose-response curve of the ischemic injury has not yet been published. We hypothesized that a viable dose-response curve of the neurological injury relative to the ischemic time could be developed. Materials and Methods: Through a cervicothoracic approach, the aortic arches of C57bl/6 mice were exposed to the origins of the left common carotid and the left subclavian arteries. The aorta was clamped between these two vessels with additional occlusion of the left subclavian artery. Ischemic times ranged from 3 to 12 min. Hindlimb motor function was observed for 48 h. Survival and motor function were evaluated. Spinal cord tissue was preserved for histologic analysis. Results: Survival curves showed increasing mortality with increasing spinal cord ischemia. This model reproduced spinal cord injury ranging from immediate paraplegia to progressive paraparesis depending on the ischemic time. Spinal cord histology mirrored the extent of functional deficits. Conclusions: Reproducible spinal cord ischemic injury is possible in a murine model and is directly translatable to thoracoabdominal surgery. The dose-response curve demonstrates that different degrees of injury can be studied from dense paraplegia to delayed onset injury depending on the ischemic insult. [Copyright &y& Elsevier]