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Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
Alpha-2 agonist attenuates ischemic injury in spinal cord neurons Kirsten A. Freeman, MD,a,* Ferenc Puskas, MD, PhD,b Marshall T. Bell, MD,a Joshua M. Mares, BS,a Lisa S. Foley, MD,a Michael J. Weyant, MD,a Joseph C. Cleveland Jr., MD,a David A. Fullerton, MD,a Xianzhong Meng, MD, PhD,a Paco S. Herson, PhD,b and T. Brett Reece, MDa a b
Department of Surgery, University of Colorado, Denver, Colorado Department of Anesthesiology, University of Colorado, Denver, Colorado
article info
abstract
Article history:
Background: Paraplegia secondary to spinal cord ischemiaereperfusion injury remains a
Received 6 May 2014
devastating complication of thoracoabdominal aortic intervention. The complex in-
Received in revised form
teractions between injured neurons and activated leukocytes have limited the understand-
4 December 2014
ing of neuron-specific injury. We hypothesize that spinal cord neuron cell cultures subjected
Accepted 17 December 2014
to oxygen-glucose deprivation (OGD) would simulate ischemiaereperfusion injury, which
Available online 23 December 2014
could be attenuated by specific alpha-2a agonism in an Akt-dependent fashion. Materials and methods: Spinal cords from perinatal mice were harvested, and neurons cultured
Keywords:
in vitro for 7e10 d. Cells were pretreated with 1 mM dexmedetomidine (Dex) and subjected to OGD
Aortic surgery
in an anoxic chamber. Viability was determined by MTT assay. Deoxyuridine-triphosphate
Ischemiaereperfusion injury
nick-end labeling staining and lactate dehydrogenase (LDH) assay were used for apoptosis
Paraplegia
and necrosis identification, respectively. Western blot was used for protein analysis.
Dexmedetomidine
Results: Vehicle control cells were only 59% viable after 1 h of OGD. Pretreatment with Dex significantly preserves neuronal viability with 88% viable (P < 0.05). Dex significantly decreased apoptotic cells compared with that of vehicle control cells by 50% (P < 0.05). Necrosis was not significantly different between treatment groups. Mechanistically, Dex treatment significantly increased phosphorylated Akt (P < 0.05), but protective effects of Dex were eliminated by an alpha-2a antagonist or Akt inhibitor (P < 0.05). Conclusions: Using a novel spinal cord neuron cell culture, OGD mimics neuronal metabolic derangement responsible for paraplegia after aortic surgery. Dex preserves neuronal viability and decreases apoptosis in an Akt-dependent fashion. Dex demonstrates clinical promise for reducing the risk of paraplegia after high-risk aortic surgery. ª 2015 Elsevier Inc. All rights reserved.
1.
Introduction
Paraplegia remains a devastating complication in thoracoabdominal aortic intervention resulting in significant
burdens on the patients, their families, and the medical system. Despite advances in surgical practice, paraplegia continues to be an obstacle in complex thoracoabdominal aortic surgery [1]. Spinal cord injury still occurs in up to 20% of
* Corresponding author. Department of Surgery, University of Colorado, 12631 E 17th Ave, Mail Stop C310, Aurora, CO 80045. Tel.: þ1 303 724 4682; fax: þ1 303 724 2806. E-mail address: [email protected] (K.A. Freeman). 0022-4804/$ e see front matter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2014.12.033
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high-risk patients [2]. Ischemia to the spinal cord leads to complex metabolic derangement and inflammatory responses that further contribute to neuronal degeneration, known as ischemiaereperfusion (IR) injury [3]. IR injury has been an area of intensive investigation with alpha-2 agonists being widely studied. Alpha-2 agonists have been shown to attenuate IR injury in cerebral [4e7], cardiac [8], lung [9], and renal [10] models. Dexmedetomidine (Dex), a highly selective alpha-2a agonist, has been shown in in vivo murine models to improve neuronal viability and provide functional attenuation of spinal cord IR injury [11]. Dex is commonly used in the intensive care unit as a sedative, so translation into a neuroprotective agent would not be challenging. However, the mechanism by which Dex provides neuroprotection remains to be elucidated, especially within specific populations of cells within the spinal cord. IR produces cytokine release [12] and induces apoptotic events, which initiate a cascade of processes that lead to cell injury and death. Promoting the phosphatidylinositol 3kinase (PI3K-Akt) survival pathway reduces apoptotic cell death [13]. The Akt pathway is a prosurvival pathway, which has been implicated as having a role in the protection by Dex [8,14e16]. Our previously published murine model showed the protective effects of Dex in spinal cord IR injury [11]; however, the neurons could not be separated from the other cellular milieu for specific investigation. We hypothesize that isolated spinal cord neurons subjected to oxygen-glucose deprivation (OGD) would simulate IR injury seen in aortic surgery and that Dex preserves neuronal viability through an Akt-dependent mechanism.
then decapitated. The vertebral column was dissected out, and the spinal cord was removed en bloc via injection of phosphate-buffered saline (PBS, pH 7.4) through the spinal canal. Spinal cord tissue was minced and then digested in Hibernate-A (Invitrogen, Carlsbad, CA) with Papain (Worthington, Lakewood, NJ). Neurons were isolated using an OptiPrep (SigmaeAldrich, St Louis, MO) density gradient adapted from Brewer [17] and plated on Poly-D-Lysine (SigmaeAldrich) coated plates at 300,000 cells per well on a 24-well plate in Neurobasal-A (Invitrogen), B27 (Invitrogen), Glutamax (Invitrogen), and Penicillin/Streptomycin (Gibco, New York, NY). On in vitro day three, AraC (SigmaeAldrich) was added to prevent astrocyte replication. Cell cultures were maintained in a humidified atmosphere containing 5% CO2 at 37 C and underwent half media change every 3 d. The cultures were >90% neurons as seen by morphology on light microscope and confirmed with microtubule-associated protein 2 (MAP2) positive staining. Cultures of neurons from perinatal mice are considered mature on in vitro day 7e10, and thus were used for experimentation after in vitro day 7.
2.4.
Immunofluorescence
2.
Methods
On in vitro day 8, the cells were fixed with 4% paraformaldehyde on their culture slides, then blocked with 5% normal goat serum in 0.1% Triton X-100 (Sigma-Aldrich, St Louis, MO). Cells were incubated with primary antibody MAP2 (Cell Signaling Technology) at 1:50 overnight at 4 C, followed by secondary antibody Anti-Rabbit IgG Alexa Fluor 488 Conjugate (Cell Signaling Technology) at 1:1000 for 1 h. Prolong Gold Anti-Fade Reagent with 40 , 6-diamidino-2-phenylindole (Cell Signaling Technology) was added to slides and coverslips applied. Images were acquired using Intelligent Imaging Innovations Slidebook (Denver, CO) software.
2.1.
Materials
2.5.
Dex, atipamezole, and LY294002 were purchased from Tocris (Ellisville, MO). The dose of Dex was determined by prior dose response curves. All antibodies including pAkt, Akt, and secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA).
2.2.
Animals
All experiments were approved by the Animal Care and Use Committee at the University of Colorado at Denver Health Sciences Center, and conformed to the Guide for the Care and Use of Laboratory Animals (US National Institutes of Health publication No. 85-23, National Academy Press, Washington DC, revised 1996). Postnatal day 2e3 old C57BL/6 mice from Jackson Laboratories (Bar Harbor, ME) were used for all experiments. Each litter was considered n ¼ 1 (all pups from 1 litter are considered as one group, and then a second litter would be a second n).
2.3.
Cell culture
Primary spinal cord neuron cultures were obtained from 2e3day-old mice. Briefly, mice were euthanized with isoflurane
Oxygen-glucose deprivation
On the day of the experiment, the experimental medium Dulbecco Modified Eagle Medium without glucose (Gibco) was placed in the Ruskin Bug Box Plus humidified airtight hypoxic chamber for 2 h. The Ruskin Bug Box Plus was used as per manufacturer’s protocol to maintain an environment of 95% N2/5%CO2 at 37 C and verified with Anaerobic Indicator (Oxoid Ltd, Basingstoke, Hants, United Kingdom). The maintenance culture medium was removed, cells washed with PBS, and experimental hypoxic medium was added to the cell culture wells. OGD was induced by placing the plates in the hypoxic chamber. Dex at 1 mM or vehicle control (VC) was added 30 min before OGD and added to the OGD media during anoxia (Fig. 1). The concentration of Dex used was determined by a literature review. Antagonists and/or inhibitors were added before Dex at a concentration of 10 mM.
2.6.
Viability studies
Cell viability was determined by MTT Cell Proliferation Kit (Roche, Indianapolis, IN) according to the procedure provided by the manufacturer. Briefly, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) was added to each well at a concentration of 0.5 mg/mL and incubated for 4 h at 37 C,
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Fig. 1 e Experimental model. This figure shows our experimental model including OGD to simulate ischemia and return to incubator to simulate reperfusion.
then dimethyl sulfoxide solution was added. The absorbance at 630 nm was measured on a BioTek Synergy H1 Hybrid microplate reader (Winooski, VT). Cell viability is the percentage absorbance relative to nonischemic control standard error of the mean (SEM).
2.7.
Apoptosis assay
Apoptosis was determined by deoxyuridine-triphosphate nick-end labeling (TUNEL) with use of the NeuroTACS II In Situ Apoptosis Detection Kit (Trevigen, Gaithersburg, MD) according to the procedure provided by the manufacturer. Briefly, cells were first fixed and permeabilized on their culture slides. The cells were incubated with TUNEL reaction mix and counterstained. Staining was quantified as TUNEL positive cells per high power field averaged from 10 random fields SEM.
2.8.
Necrosis assay
After experimentation, the cell culture supernatant was collected. Lactate dehydrogenase (LDH) Cytotoxicity Assay Kit was purchased from Cayman Chemical Company (Ann Arbor, MI). The assay was performed according to instructions provided by the manufacturer. Briefly, prepared LDH standard and experimental samples were placed on a 96-well plate. LDH reaction solution was added to the standard and samples. After incubation, the absorbance at 490 nm was measured. Data are presented as average LDH concentration in mU/mL SEM.
Protein concentration was analyzed with NanoDrop Spectrophotometer (NanoDrop Technologies, Wilmington, DE) then placed in 4 Laemmli sample buffer with b-mercaptoethanol and boiled at 100 C. Each sample was loaded onto 4%e20% gradient gels (Bio-Rad, Hercules, CA) with equivalent protein per lane, run at 160 V for 50 min, and transferred to nitrocellulose membranes. Membranes were blocked with 5% bovine serum albumin-tris-buffered saline with tween, incubated overnight with primary antibody at 4 C, and then incubated with secondary antibody. Immunoblots were detected with Pierce ECL Western Blotting Substrate (Thermo Scientific) and visualized after exposure to x-ray film. ImageJ (National Institutes of Health, Bethesda, MD) software was used for quantification.
2.11.
Statistics
Results are presented as mean standard error. Data were analyzed with StatView software (SAS Institute Inc, Cary, NC). Statistical analysis was performed using Student t-test and one-way analysis of variance. A P value of 90% of the cells in culture. Perinatal isolated neurons are considered mature in culture on day 7e10 in vitro.
2.10.
3.2.
2.9.
Enzyme-linked immunosorbant assay
Western blots
Cells were rinsed with PBS and collected with Mammalian Protein Extraction Reagent (Thermo Scientific, Rockford, IL).
Spinal cord neuronal viability with OGD
Spinal cord neurons were subjected to 1, 2, and 4 h of OGD in a humidified hypoxic chamber with glucose-free media.
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Fig. 2 e Neuronal culture images. (Left) Spinal cord neurons on day 7 in vitro by light microscopy at 320 magnification. (Right) MAP2 immunofluorescence staining of neurons and nuclei under oil immersion 3100 magnification.
Viability was assessed with MTT reduction assay. OGD has a significant effect on the viability of spinal cord neuronal cells (Fig. 3). After 1 h of OGD, cell viability was significantly reduced to 59% of nonischemic control (P < 0.05). After 2 h, the viability was further significantly reduced to 39% of nonischemic control (P < 0.05), and then after 4 h further significantly reduced to 14% of nonischemic control (P < 0.05). Results are expressed as percentage of nonischemic control values. We chose 1 h of OGD for further studies given the amount of viable neurons still remaining in culture to elucidate a mechanism by which these neurons survive.
the MTT assay, an assay where living cells are detected by their ability to reduce MTT. Dex treatment of the cells without OGD did not have any significant effects on the viability of the cells suggesting that Dex does not have any adverse effects on the neurons.
3.4.
Spinal cord neuronal apoptosis with OGD
Spinal cord neurons were then pretreated with alpha-2a agonist Dex before the OGD. These data show that cells treated with Dex before OGD have significant (P < 0.05) preservation of neuronal viability compared with the reduction in viability seen in VC þ OGD cells (Fig. 4). Preservation of neuron viability with Dex þ OGD was 88% of nonischemic control values compared with that of the 59% viability in the VC þ OGD cells. The viability was determined with the use of
The MTT assay mentioned previously showed us that some cells subjected to OGD die. We were interested in the mode of death the neuron undergoes with OGD. Using a TUNEL stain, which indicates the programmed cell death of apoptosis (Fig. 5), we found that OGD compared with nonischemic control cells have a significantly higher cells and/ or high power field that were TUNEL positive (P < 0.05), and the Dex þ OGD cells have significantly fewer TUNEL positive cells (P < 0.05). These data indicate that neurons subjected to OGD die by apoptosis. These data also show that treatment with Dex before OGD reduces apoptotic cell death. Overall, this suggests that OGD leads to apoptosis as the mode of cell death and that Dex provides protection by decreasing apoptosis.
Fig. 3 e Effects of OGD on viability of neurons. Cells were placed in a hypoxic chamber for 1, 2, or 4 h and viability determined by MTT assay. One hour of OGD significantly reduced viability versus control (*P < 0.05) and further reduced at 2 h (#P < 0.05) and 4 h (AP < 0.05). Results expressed as percentage of control values ± SEM, n [ 3.
Fig. 4 e Effects of pretreatment with Dex and OGD on viability of cells. VC D OGD cells had significantly reduced viability compared with that of nonischemic control (*P < 0.05). Neuronal viability with Dex D OGD was significantly preserved compared with that of VC D OGD cells (#P < 0.05). Cell viability was determined by MTT Assay. Results expressed as percentage of control values ± SEM, n [ 3.
3.3.
Spinal cord neuronal viability with Dex
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Fig. 5 e Neuronal apoptosis after OGD. (Left) Light microscope images of dark TUNEL-stained apoptotic cells under 310 magnification. (Right) TUNEL positive cells/high power field are significantly increased (*P < 0.05) in VC D OGD cells compared with those in nonischemic control and significantly decreased (#P < 0.05) in Dex D OGD compared with VC D OGD. n [ 3.
3.5.
Spinal cord neuronal necrosis with OGD
The apoptosis assays suggest that OGD results in apoptotic cell death of injured neurons, and that Dex provides protection by reducing apoptosis. However, we also wanted to determine if any cells subjected to OGD died by mode of necrosis and whether Dex had any effect on a necrotic cell death. An LDH assay was conducted to assess for necrosis (Fig. 6), and we found that OGD did not cause a significant change in necrosis compared with nonischemic controls. We also found that treatment with Dex did not cause a significant difference in necrosis. Because there was no difference detected in the necrosis as shown via the LDH assay, in conjunction with the previous findings that apoptosis is significantly increased in the cells subjected to OGD, these data suggest again that cells subjected to OGD die by apoptosis not necrosis.
3.6.
Cytokine production
Inflammation is one of the hallmarks of IR injury. We were interested in determining the changes that Dex has on
Fig. 6 e Neuronal necrosis after OGD. Necrosis is not significantly different between treatment groups. Necrosis is identified by LDH concentration in the supernatant. Results are expressed as average LDH concentration in mU/ mL ± SEM, n [ 4.
inflammatory cytokines produced by neurons. Production of tumor necrosis factor-alpha was not significantly altered by Dex treatment; however, production of IL-6 was significantly changed with Dex pretreatment (Fig. 7). After 1 h of OGD, the IL-6 production in VC þ OGD were significantly increased compared with nonischemic control (P < 0.05), with an increase that was nearly a three-fold. IL-6 production in Dex þ OGD was attenuated (P < 0.05) with a decrease of 42% compared with VC þ OGD cells.
3.7.
Phosphorylation of Akt
Western blot analysis was used to determine the phosphorylation of Akt. The data indicate that phosphorylation of Akt is significantly increased by Dex treatment (Fig. 8). Without OGD, Dex-treated cells have a significant increase in phosphorylated Akt compared with VC-treated cells (P < 0.05). Similarly, after 1 h of OGD, Dex-treated cells have a significant increase in phosphorylated Akt compared with VC-treated cells
Fig. 7 e Effects of OGD and Dex on production of inflammatory cytokine IL-6. The production of IL-6 after OGD is significantly increased (*P < 0.05) versus nonischemic control. IL-6 production with OGD was attenuated by Dex (#P < 0.05) compared with VC. Results are presented as pg/mL ± SEM. n [ 3.
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PI3K-Akt inhibitor, to further characterize the survival mechanism provided by Dex. The data in Figure 9 show that the addition of an alpha-2a antagonist abolishes the protective effect of Dex (P < 0.05), indicating Dex provides protection via the alpha-2a receptor. Similarly, with the addition of a PI3K-Akt inhibitor, the protective effects of Dex are also eliminated (P < 0.05). These data indicate that Dex proceeds to provide protection through the activation of the PI3K-Akt pathway.
4.
Fig. 8 e Western blot showing the effects of Dex on phosphorylation of Akt compared with total Akt. Dex treatment has a significant increase in phosphorylated Akt compared with that in VC (*P < 0.05). Similarly, after 1 h of OGD, Dex D OGD cells have a significant increase in phosphorylated Akt compared with that of VC D OGD cells (#P < 0.05), n [ 3. The immunoblot image n [ 2. (Color version of the figure is available online.)
(P < 0.05). Additionally, the data suggest that OGD does not change the phosphorylation of Akt, which suggests that OGD alone simply may not change pAkt or potentially that OGD may need a much longer time frame to change the level of pAkt.
3.8.
Inhibitors
We then assessed the viability of the neurons after OGD with atipamezole, an alpha-2a antagonist, and with LY294002, a
Discussion
Paraplegic complications of aortic surgery remain devastating in aortic intervention. Surgical adjuncts have reduced the frequency of this complication [1], but the incidence remains up to 20% in extensive thoracoabdominal aortic aneurysms [2]. Spinal cord ischemic injury is a complex entity resulting from the combined insult of ischemia followed by reperfusion injury. IR injury is characterized by disruption of cellular homeostasis during the ischemic event and inflammatory damage with reperfusion [13]. Patients exhibit a bimodal distribution of paralysis including immediate and delayed paraplegia [18,19] where ischemia is implicated as the cause of immediate paraplegia [20]. Apoptosis of neurons has been suggested as the leading cause of delayed paraplegia [21]. Our first aim was the development of a model of neuronal IR injury by growing neurons in vitro and subjecting them to OGD. OGD is our model for ischemia, and return to normal media afterward mimics reperfusion. We found specifically that increasing OGD time resulted in decreasing viability. We chose 1 h of OGD for further studies because with a loss of nearly 40% there was significant injury, but there would be substantial cells still remaining to undergo further studies. With a successful model of IR injury in neuronal cell cultures, we were able to then move forward with our second aim. The next objective was to determine the effects of Dex on neuronal cell cultures in our model of IR. Previous publications have demonstrated that Dex produces its
Fig. 9 e Protective effects of Dex are abolished with alpha-2a inhibition and Akt inhibition. Alpha-2a antagonist atipamezole and PI3K-Akt inhibitor LY294002 abolished the protective effects of Dex with a significant reduction in viability compared with Dex D OGD cells (*P < 0.05). Neuronal cell viability assessed with MTT assay, expressed as percentage of nonischemic control cells ± SEM, n [ 3.
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neuroprotective effect via the alpha-2a receptor [7]. The data from our in vitro OGD of neurons corroborate our previous findings of preservation of neuronal viability and functional outcomes with Dex treatment seen in our murine in vivo model [11,22]. Before this model, spinal cord neurons were studied along with the other cells of spinal cord tissue in a spinal cord homogenate, therefore were unable to study the isolated metabolic injury to the spinal cord neurons as an individual cell line. Our study found that pretreatment with Dex before OGD preserves neuronal viability, decreases subsequent apoptosis, and attenuates inflammatory cytokine IL-6 production. Although the pathway by which Dex protects the spinal cord has not been previously well established, the PI3K-Akt pathway has been implicated in other organ systems. IR injuries in the kidney, the brain, and the heart have been attenuated by Dex and have shown an increase in phosphorylation of Akt [8,14e16]. Our data support these findings that Dex in spinal cord neurons activates the PI3K-Akt pathway and shows increase in phosphorylation of Akt. Specifically, our data suggest that Dex acts via the alpha-2a receptor given that antagonism of this receptor removed the neuroprotection. Furthermore, Dex exerts its protection through an Akt-dependent pathway as the protection was eliminated by a PI3K-Akt inhibitor. Further investigation into the effect of Dex on spinal cord neurons particularly into downstream effects of the activated PI3K-Akt pathway still remains to be explored. Specifically, we would like to broaden our understanding by investigating the downstream cellular transcription factor cyclic adenosine monophosphate response element-binding protein and other neurotrophic factors such as B-cell lymphoma-2 and brain derived neurotrophic factor. The model of our study allows for specific investigation into neuron-specific effects of Dex; however, there are limitations to our model. First, growth of neurons in culture requires use of perinatal neurons, as adult neurons are unable to survive in culture. Additionally, an in vitro model does not permit us to determine the complete functionality of the neurons as an in vivo model allows. Second, the neurons are unable to interact in their normal microenvironment. Spinal cord neurons in vivo are able to interact with supporting cells such as astrocytes and microglia. These cells have a variety of roles during IR injury, whereby sometimes they contribute to further neuronal degeneration by release of inflammatory cytokines and changes in ion channel permeabilities [23]. Conversely, astrocytes have been implicated as playing an important role in the neuroprotection induced by alpha-2a agonists via modulation of ionic homeostasis and production of neurotrophins [4,24].
5.
Conclusions
Isolated neuronal cell cultures demonstrate that Dex preserves viability through the alpha-2a receptor and Akt in the setting of OGD. Dex reduces the ischemic injury to spinal cord neurons, demonstrating the effect on metabolic tolerance specific to the ischemic injury. Dex demonstrates promise in modifying the effects of both ischemic and metabolic injury
27
from ischemic insults, which may further reduce the incidence of paraplegia after aortic intervention.
Acknowledgment The authors acknowledge and thank the University of Colorado Department of Surgery, Division of Cardiothoracic Surgery for their financial support. Authors’ contributions: M.T.B., J.M.M., and L.S.F. assisted with the conduction of experimentations. F.P., M.J.W., J.C.C., D.A.F., X.M., P.S.H., and T.B.R. provided research direction, assisted with analysis of results, and provided assistance with writing/editing of the article.
Disclosure The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in the article.
references
[1] Conrad MF, Ye JY, Chung TK, Davison JK, Cambria RP. Spinal cord complications after thoracic aortic surgery: long-term survival and functional status varies with deficit severity. J Vasc Surg 2008;48:47. [2] Greenberg RK, Lu Q, Roselli EE, et al. Contemporary analysis of descending thoracic and thoracoabdominal aneurysm repair: a comparison of endovascular and open techniques. Circulation 2008;118:808. [3] Stirling DP, Yong VW. Dynamics of the inflammatory response after murine spinal cord injury revealed by flow cytometry. J Neurosci Res 2008;86:1944. [4] Zhang Y, Kimelberg H. Neuroprotection by alpha-2 adrenergic agonists in cerebral ischemia. Curr Neuropharmacology 2005;3:317. [5] Engelhard K, Werner C, Eberspa E, et al. The effect of the alpha 2-agonist dexmedetomidine and the N-methyl-D aspartate antagonist S(þ)-ketamine on the expression of apoptosis regulating proteins after incomplete cerebral ischemia and reperfusion in rats. Neurosurg Anesth 2003;96:524. [6] Cai J, Li J, Han Y, Wang H. Alpha2-adrenergic receptors in spiral ganglion neurons may mediate protective effects of brimonidine and yohimbine against glutamate and hydrogen peroxide toxicity. Neuroscience 2013;228:23. [7] Ma D, Hossain M, Rajakumaraswamy N, et al. Dexmedetomidine produces its neuroprotective effect via the alpha 2A-adrenoceptor subtype. Eur J Pharmacol 2004; 502:87. [8] Ibacache M, Sanchez G, Pedrozo Z, et al. Dexmedetomidine preconditioning activates pro-survival kinases and attenuates regional ischemia/reperfusion injury in rat heart. Biochim Biophys Acta 2012;1822:537. [9] Gu J, Chen J, Xia P, Tao G, Zhao H, Ma D. Dexmedetomidine attenuates remote lung injury induced by renal ischemiareperfusion in mice. Acta Anaesthesiol Scand 2011;55:1272. [10] Gu J, Sun P, Zhao H, et al. Dexmedetomidine provides renoprotection against ischemia-reperfusion injury in mice. Crit Care 2011;15:R153. [11] Bell MT, Puskas F, Smith PD, et al. Attenuation of spinal cord ischemia-reperfusion injury by specific a-2a receptor activation with dexmedetomidine. J Vasc Surg 2012;56:1398. [12] Smith PD, Puskas F, Meng X, et al. The evolution of chemokine release supports a bimodal mechanism of
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spinal cord ischemia and reperfusion. Circulation 2012;126: S110. Lopez-Neblina F, Toledo AH, Toledo-Pereyra LH. Molecular biology of apoptosis in ischemia and reperfusion. J Invest Surg 2005;18:335. Yuan Y, Guo Q, Pingping X, Wang N, Song Z. Ischemic postconditioning protects brain from ischemia/reperfusion injury by attenuating endoplasmic reticulum stress-induced apoptosis through PI3K-Akt pathway. Brain Res 2011;1367:85. Zhu YM, Wang CC, Chen L, et al. Both PI3K/Akt and ERK1/2 pathways participate in the protection by dexmedetomidine against transient focal cerebral ischemia/reperfusion injury. Brain Res 2013;1494:1. Zhang F, Ding T, Yu L, Zhong Y, Dai H, Yan M. Dexmedetomidine protects against oxygen-glucose deprivation-induced injury through the I2 imidazoline receptor-PI3K/AKT pathway in rat C6 glioma cells. J Pharm Pharmacol 2012;64:120. Brewer G, Torricelli J. Isolation and culture of adult neurons and neurospheres. Nat Protoc 2007;2:1490. Fang B, Li X, Sun X, et al. Ischemic preconditioning protects against spinal cord ischemia-reperfusion injury in rabbits by
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Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
Alpha-2 agonist attenuates ischemic injury in spinal cord neurons Kirsten A. Freeman, MD,a,* Ferenc Puskas, MD, PhD,b Marshall T. Bell, MD,a Joshua M. Mares, BS,a Lisa S. Foley, MD,a Michael J. Weyant, MD,a Joseph C. Cleveland Jr., MD,a David A. Fullerton, MD,a Xianzhong Meng, MD, PhD,a Paco S. Herson, PhD,b and T. Brett Reece, MDa a b
Department of Surgery, University of Colorado, Denver, Colorado Department of Anesthesiology, University of Colorado, Denver, Colorado
article info
abstract
Article history:
Background: Paraplegia secondary to spinal cord ischemiaereperfusion injury remains a
Received 6 May 2014
devastating complication of thoracoabdominal aortic intervention. The complex in-
Received in revised form
teractions between injured neurons and activated leukocytes have limited the understand-
4 December 2014
ing of neuron-specific injury. We hypothesize that spinal cord neuron cell cultures subjected
Accepted 17 December 2014
to oxygen-glucose deprivation (OGD) would simulate ischemiaereperfusion injury, which
Available online 23 December 2014
could be attenuated by specific alpha-2a agonism in an Akt-dependent fashion. Materials and methods: Spinal cords from perinatal mice were harvested, and neurons cultured
Keywords:
in vitro for 7e10 d. Cells were pretreated with 1 mM dexmedetomidine (Dex) and subjected to OGD
Aortic surgery
in an anoxic chamber. Viability was determined by MTT assay. Deoxyuridine-triphosphate
Ischemiaereperfusion injury
nick-end labeling staining and lactate dehydrogenase (LDH) assay were used for apoptosis
Paraplegia
and necrosis identification, respectively. Western blot was used for protein analysis.
Dexmedetomidine
Results: Vehicle control cells were only 59% viable after 1 h of OGD. Pretreatment with Dex significantly preserves neuronal viability with 88% viable (P < 0.05). Dex significantly decreased apoptotic cells compared with that of vehicle control cells by 50% (P < 0.05). Necrosis was not significantly different between treatment groups. Mechanistically, Dex treatment significantly increased phosphorylated Akt (P < 0.05), but protective effects of Dex were eliminated by an alpha-2a antagonist or Akt inhibitor (P < 0.05). Conclusions: Using a novel spinal cord neuron cell culture, OGD mimics neuronal metabolic derangement responsible for paraplegia after aortic surgery. Dex preserves neuronal viability and decreases apoptosis in an Akt-dependent fashion. Dex demonstrates clinical promise for reducing the risk of paraplegia after high-risk aortic surgery. ª 2015 Elsevier Inc. All rights reserved.
1.
Introduction
Paraplegia remains a devastating complication in thoracoabdominal aortic intervention resulting in significant
burdens on the patients, their families, and the medical system. Despite advances in surgical practice, paraplegia continues to be an obstacle in complex thoracoabdominal aortic surgery [1]. Spinal cord injury still occurs in up to 20% of
* Corresponding author. Department of Surgery, University of Colorado, 12631 E 17th Ave, Mail Stop C310, Aurora, CO 80045. Tel.: þ1 303 724 4682; fax: þ1 303 724 2806. E-mail address: [email protected] (K.A. Freeman). 0022-4804/$ e see front matter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2014.12.033
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high-risk patients [2]. Ischemia to the spinal cord leads to complex metabolic derangement and inflammatory responses that further contribute to neuronal degeneration, known as ischemiaereperfusion (IR) injury [3]. IR injury has been an area of intensive investigation with alpha-2 agonists being widely studied. Alpha-2 agonists have been shown to attenuate IR injury in cerebral [4e7], cardiac [8], lung [9], and renal [10] models. Dexmedetomidine (Dex), a highly selective alpha-2a agonist, has been shown in in vivo murine models to improve neuronal viability and provide functional attenuation of spinal cord IR injury [11]. Dex is commonly used in the intensive care unit as a sedative, so translation into a neuroprotective agent would not be challenging. However, the mechanism by which Dex provides neuroprotection remains to be elucidated, especially within specific populations of cells within the spinal cord. IR produces cytokine release [12] and induces apoptotic events, which initiate a cascade of processes that lead to cell injury and death. Promoting the phosphatidylinositol 3kinase (PI3K-Akt) survival pathway reduces apoptotic cell death [13]. The Akt pathway is a prosurvival pathway, which has been implicated as having a role in the protection by Dex [8,14e16]. Our previously published murine model showed the protective effects of Dex in spinal cord IR injury [11]; however, the neurons could not be separated from the other cellular milieu for specific investigation. We hypothesize that isolated spinal cord neurons subjected to oxygen-glucose deprivation (OGD) would simulate IR injury seen in aortic surgery and that Dex preserves neuronal viability through an Akt-dependent mechanism.
then decapitated. The vertebral column was dissected out, and the spinal cord was removed en bloc via injection of phosphate-buffered saline (PBS, pH 7.4) through the spinal canal. Spinal cord tissue was minced and then digested in Hibernate-A (Invitrogen, Carlsbad, CA) with Papain (Worthington, Lakewood, NJ). Neurons were isolated using an OptiPrep (SigmaeAldrich, St Louis, MO) density gradient adapted from Brewer [17] and plated on Poly-D-Lysine (SigmaeAldrich) coated plates at 300,000 cells per well on a 24-well plate in Neurobasal-A (Invitrogen), B27 (Invitrogen), Glutamax (Invitrogen), and Penicillin/Streptomycin (Gibco, New York, NY). On in vitro day three, AraC (SigmaeAldrich) was added to prevent astrocyte replication. Cell cultures were maintained in a humidified atmosphere containing 5% CO2 at 37 C and underwent half media change every 3 d. The cultures were >90% neurons as seen by morphology on light microscope and confirmed with microtubule-associated protein 2 (MAP2) positive staining. Cultures of neurons from perinatal mice are considered mature on in vitro day 7e10, and thus were used for experimentation after in vitro day 7.
2.4.
Immunofluorescence
2.
Methods
On in vitro day 8, the cells were fixed with 4% paraformaldehyde on their culture slides, then blocked with 5% normal goat serum in 0.1% Triton X-100 (Sigma-Aldrich, St Louis, MO). Cells were incubated with primary antibody MAP2 (Cell Signaling Technology) at 1:50 overnight at 4 C, followed by secondary antibody Anti-Rabbit IgG Alexa Fluor 488 Conjugate (Cell Signaling Technology) at 1:1000 for 1 h. Prolong Gold Anti-Fade Reagent with 40 , 6-diamidino-2-phenylindole (Cell Signaling Technology) was added to slides and coverslips applied. Images were acquired using Intelligent Imaging Innovations Slidebook (Denver, CO) software.
2.1.
Materials
2.5.
Dex, atipamezole, and LY294002 were purchased from Tocris (Ellisville, MO). The dose of Dex was determined by prior dose response curves. All antibodies including pAkt, Akt, and secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA).
2.2.
Animals
All experiments were approved by the Animal Care and Use Committee at the University of Colorado at Denver Health Sciences Center, and conformed to the Guide for the Care and Use of Laboratory Animals (US National Institutes of Health publication No. 85-23, National Academy Press, Washington DC, revised 1996). Postnatal day 2e3 old C57BL/6 mice from Jackson Laboratories (Bar Harbor, ME) were used for all experiments. Each litter was considered n ¼ 1 (all pups from 1 litter are considered as one group, and then a second litter would be a second n).
2.3.
Cell culture
Primary spinal cord neuron cultures were obtained from 2e3day-old mice. Briefly, mice were euthanized with isoflurane
Oxygen-glucose deprivation
On the day of the experiment, the experimental medium Dulbecco Modified Eagle Medium without glucose (Gibco) was placed in the Ruskin Bug Box Plus humidified airtight hypoxic chamber for 2 h. The Ruskin Bug Box Plus was used as per manufacturer’s protocol to maintain an environment of 95% N2/5%CO2 at 37 C and verified with Anaerobic Indicator (Oxoid Ltd, Basingstoke, Hants, United Kingdom). The maintenance culture medium was removed, cells washed with PBS, and experimental hypoxic medium was added to the cell culture wells. OGD was induced by placing the plates in the hypoxic chamber. Dex at 1 mM or vehicle control (VC) was added 30 min before OGD and added to the OGD media during anoxia (Fig. 1). The concentration of Dex used was determined by a literature review. Antagonists and/or inhibitors were added before Dex at a concentration of 10 mM.
2.6.
Viability studies
Cell viability was determined by MTT Cell Proliferation Kit (Roche, Indianapolis, IN) according to the procedure provided by the manufacturer. Briefly, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) was added to each well at a concentration of 0.5 mg/mL and incubated for 4 h at 37 C,
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Fig. 1 e Experimental model. This figure shows our experimental model including OGD to simulate ischemia and return to incubator to simulate reperfusion.
then dimethyl sulfoxide solution was added. The absorbance at 630 nm was measured on a BioTek Synergy H1 Hybrid microplate reader (Winooski, VT). Cell viability is the percentage absorbance relative to nonischemic control standard error of the mean (SEM).
2.7.
Apoptosis assay
Apoptosis was determined by deoxyuridine-triphosphate nick-end labeling (TUNEL) with use of the NeuroTACS II In Situ Apoptosis Detection Kit (Trevigen, Gaithersburg, MD) according to the procedure provided by the manufacturer. Briefly, cells were first fixed and permeabilized on their culture slides. The cells were incubated with TUNEL reaction mix and counterstained. Staining was quantified as TUNEL positive cells per high power field averaged from 10 random fields SEM.
2.8.
Necrosis assay
After experimentation, the cell culture supernatant was collected. Lactate dehydrogenase (LDH) Cytotoxicity Assay Kit was purchased from Cayman Chemical Company (Ann Arbor, MI). The assay was performed according to instructions provided by the manufacturer. Briefly, prepared LDH standard and experimental samples were placed on a 96-well plate. LDH reaction solution was added to the standard and samples. After incubation, the absorbance at 490 nm was measured. Data are presented as average LDH concentration in mU/mL SEM.
Protein concentration was analyzed with NanoDrop Spectrophotometer (NanoDrop Technologies, Wilmington, DE) then placed in 4 Laemmli sample buffer with b-mercaptoethanol and boiled at 100 C. Each sample was loaded onto 4%e20% gradient gels (Bio-Rad, Hercules, CA) with equivalent protein per lane, run at 160 V for 50 min, and transferred to nitrocellulose membranes. Membranes were blocked with 5% bovine serum albumin-tris-buffered saline with tween, incubated overnight with primary antibody at 4 C, and then incubated with secondary antibody. Immunoblots were detected with Pierce ECL Western Blotting Substrate (Thermo Scientific) and visualized after exposure to x-ray film. ImageJ (National Institutes of Health, Bethesda, MD) software was used for quantification.
2.11.
Statistics
Results are presented as mean standard error. Data were analyzed with StatView software (SAS Institute Inc, Cary, NC). Statistical analysis was performed using Student t-test and one-way analysis of variance. A P value of 90% of the cells in culture. Perinatal isolated neurons are considered mature in culture on day 7e10 in vitro.
2.10.
3.2.
2.9.
Enzyme-linked immunosorbant assay
Western blots
Cells were rinsed with PBS and collected with Mammalian Protein Extraction Reagent (Thermo Scientific, Rockford, IL).
Spinal cord neuronal viability with OGD
Spinal cord neurons were subjected to 1, 2, and 4 h of OGD in a humidified hypoxic chamber with glucose-free media.
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Fig. 2 e Neuronal culture images. (Left) Spinal cord neurons on day 7 in vitro by light microscopy at 320 magnification. (Right) MAP2 immunofluorescence staining of neurons and nuclei under oil immersion 3100 magnification.
Viability was assessed with MTT reduction assay. OGD has a significant effect on the viability of spinal cord neuronal cells (Fig. 3). After 1 h of OGD, cell viability was significantly reduced to 59% of nonischemic control (P < 0.05). After 2 h, the viability was further significantly reduced to 39% of nonischemic control (P < 0.05), and then after 4 h further significantly reduced to 14% of nonischemic control (P < 0.05). Results are expressed as percentage of nonischemic control values. We chose 1 h of OGD for further studies given the amount of viable neurons still remaining in culture to elucidate a mechanism by which these neurons survive.
the MTT assay, an assay where living cells are detected by their ability to reduce MTT. Dex treatment of the cells without OGD did not have any significant effects on the viability of the cells suggesting that Dex does not have any adverse effects on the neurons.
3.4.
Spinal cord neuronal apoptosis with OGD
Spinal cord neurons were then pretreated with alpha-2a agonist Dex before the OGD. These data show that cells treated with Dex before OGD have significant (P < 0.05) preservation of neuronal viability compared with the reduction in viability seen in VC þ OGD cells (Fig. 4). Preservation of neuron viability with Dex þ OGD was 88% of nonischemic control values compared with that of the 59% viability in the VC þ OGD cells. The viability was determined with the use of
The MTT assay mentioned previously showed us that some cells subjected to OGD die. We were interested in the mode of death the neuron undergoes with OGD. Using a TUNEL stain, which indicates the programmed cell death of apoptosis (Fig. 5), we found that OGD compared with nonischemic control cells have a significantly higher cells and/ or high power field that were TUNEL positive (P < 0.05), and the Dex þ OGD cells have significantly fewer TUNEL positive cells (P < 0.05). These data indicate that neurons subjected to OGD die by apoptosis. These data also show that treatment with Dex before OGD reduces apoptotic cell death. Overall, this suggests that OGD leads to apoptosis as the mode of cell death and that Dex provides protection by decreasing apoptosis.
Fig. 3 e Effects of OGD on viability of neurons. Cells were placed in a hypoxic chamber for 1, 2, or 4 h and viability determined by MTT assay. One hour of OGD significantly reduced viability versus control (*P < 0.05) and further reduced at 2 h (#P < 0.05) and 4 h (AP < 0.05). Results expressed as percentage of control values ± SEM, n [ 3.
Fig. 4 e Effects of pretreatment with Dex and OGD on viability of cells. VC D OGD cells had significantly reduced viability compared with that of nonischemic control (*P < 0.05). Neuronal viability with Dex D OGD was significantly preserved compared with that of VC D OGD cells (#P < 0.05). Cell viability was determined by MTT Assay. Results expressed as percentage of control values ± SEM, n [ 3.
3.3.
Spinal cord neuronal viability with Dex
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Fig. 5 e Neuronal apoptosis after OGD. (Left) Light microscope images of dark TUNEL-stained apoptotic cells under 310 magnification. (Right) TUNEL positive cells/high power field are significantly increased (*P < 0.05) in VC D OGD cells compared with those in nonischemic control and significantly decreased (#P < 0.05) in Dex D OGD compared with VC D OGD. n [ 3.
3.5.
Spinal cord neuronal necrosis with OGD
The apoptosis assays suggest that OGD results in apoptotic cell death of injured neurons, and that Dex provides protection by reducing apoptosis. However, we also wanted to determine if any cells subjected to OGD died by mode of necrosis and whether Dex had any effect on a necrotic cell death. An LDH assay was conducted to assess for necrosis (Fig. 6), and we found that OGD did not cause a significant change in necrosis compared with nonischemic controls. We also found that treatment with Dex did not cause a significant difference in necrosis. Because there was no difference detected in the necrosis as shown via the LDH assay, in conjunction with the previous findings that apoptosis is significantly increased in the cells subjected to OGD, these data suggest again that cells subjected to OGD die by apoptosis not necrosis.
3.6.
Cytokine production
Inflammation is one of the hallmarks of IR injury. We were interested in determining the changes that Dex has on
Fig. 6 e Neuronal necrosis after OGD. Necrosis is not significantly different between treatment groups. Necrosis is identified by LDH concentration in the supernatant. Results are expressed as average LDH concentration in mU/ mL ± SEM, n [ 4.
inflammatory cytokines produced by neurons. Production of tumor necrosis factor-alpha was not significantly altered by Dex treatment; however, production of IL-6 was significantly changed with Dex pretreatment (Fig. 7). After 1 h of OGD, the IL-6 production in VC þ OGD were significantly increased compared with nonischemic control (P < 0.05), with an increase that was nearly a three-fold. IL-6 production in Dex þ OGD was attenuated (P < 0.05) with a decrease of 42% compared with VC þ OGD cells.
3.7.
Phosphorylation of Akt
Western blot analysis was used to determine the phosphorylation of Akt. The data indicate that phosphorylation of Akt is significantly increased by Dex treatment (Fig. 8). Without OGD, Dex-treated cells have a significant increase in phosphorylated Akt compared with VC-treated cells (P < 0.05). Similarly, after 1 h of OGD, Dex-treated cells have a significant increase in phosphorylated Akt compared with VC-treated cells
Fig. 7 e Effects of OGD and Dex on production of inflammatory cytokine IL-6. The production of IL-6 after OGD is significantly increased (*P < 0.05) versus nonischemic control. IL-6 production with OGD was attenuated by Dex (#P < 0.05) compared with VC. Results are presented as pg/mL ± SEM. n [ 3.
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PI3K-Akt inhibitor, to further characterize the survival mechanism provided by Dex. The data in Figure 9 show that the addition of an alpha-2a antagonist abolishes the protective effect of Dex (P < 0.05), indicating Dex provides protection via the alpha-2a receptor. Similarly, with the addition of a PI3K-Akt inhibitor, the protective effects of Dex are also eliminated (P < 0.05). These data indicate that Dex proceeds to provide protection through the activation of the PI3K-Akt pathway.
4.
Fig. 8 e Western blot showing the effects of Dex on phosphorylation of Akt compared with total Akt. Dex treatment has a significant increase in phosphorylated Akt compared with that in VC (*P < 0.05). Similarly, after 1 h of OGD, Dex D OGD cells have a significant increase in phosphorylated Akt compared with that of VC D OGD cells (#P < 0.05), n [ 3. The immunoblot image n [ 2. (Color version of the figure is available online.)
(P < 0.05). Additionally, the data suggest that OGD does not change the phosphorylation of Akt, which suggests that OGD alone simply may not change pAkt or potentially that OGD may need a much longer time frame to change the level of pAkt.
3.8.
Inhibitors
We then assessed the viability of the neurons after OGD with atipamezole, an alpha-2a antagonist, and with LY294002, a
Discussion
Paraplegic complications of aortic surgery remain devastating in aortic intervention. Surgical adjuncts have reduced the frequency of this complication [1], but the incidence remains up to 20% in extensive thoracoabdominal aortic aneurysms [2]. Spinal cord ischemic injury is a complex entity resulting from the combined insult of ischemia followed by reperfusion injury. IR injury is characterized by disruption of cellular homeostasis during the ischemic event and inflammatory damage with reperfusion [13]. Patients exhibit a bimodal distribution of paralysis including immediate and delayed paraplegia [18,19] where ischemia is implicated as the cause of immediate paraplegia [20]. Apoptosis of neurons has been suggested as the leading cause of delayed paraplegia [21]. Our first aim was the development of a model of neuronal IR injury by growing neurons in vitro and subjecting them to OGD. OGD is our model for ischemia, and return to normal media afterward mimics reperfusion. We found specifically that increasing OGD time resulted in decreasing viability. We chose 1 h of OGD for further studies because with a loss of nearly 40% there was significant injury, but there would be substantial cells still remaining to undergo further studies. With a successful model of IR injury in neuronal cell cultures, we were able to then move forward with our second aim. The next objective was to determine the effects of Dex on neuronal cell cultures in our model of IR. Previous publications have demonstrated that Dex produces its
Fig. 9 e Protective effects of Dex are abolished with alpha-2a inhibition and Akt inhibition. Alpha-2a antagonist atipamezole and PI3K-Akt inhibitor LY294002 abolished the protective effects of Dex with a significant reduction in viability compared with Dex D OGD cells (*P < 0.05). Neuronal cell viability assessed with MTT assay, expressed as percentage of nonischemic control cells ± SEM, n [ 3.
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neuroprotective effect via the alpha-2a receptor [7]. The data from our in vitro OGD of neurons corroborate our previous findings of preservation of neuronal viability and functional outcomes with Dex treatment seen in our murine in vivo model [11,22]. Before this model, spinal cord neurons were studied along with the other cells of spinal cord tissue in a spinal cord homogenate, therefore were unable to study the isolated metabolic injury to the spinal cord neurons as an individual cell line. Our study found that pretreatment with Dex before OGD preserves neuronal viability, decreases subsequent apoptosis, and attenuates inflammatory cytokine IL-6 production. Although the pathway by which Dex protects the spinal cord has not been previously well established, the PI3K-Akt pathway has been implicated in other organ systems. IR injuries in the kidney, the brain, and the heart have been attenuated by Dex and have shown an increase in phosphorylation of Akt [8,14e16]. Our data support these findings that Dex in spinal cord neurons activates the PI3K-Akt pathway and shows increase in phosphorylation of Akt. Specifically, our data suggest that Dex acts via the alpha-2a receptor given that antagonism of this receptor removed the neuroprotection. Furthermore, Dex exerts its protection through an Akt-dependent pathway as the protection was eliminated by a PI3K-Akt inhibitor. Further investigation into the effect of Dex on spinal cord neurons particularly into downstream effects of the activated PI3K-Akt pathway still remains to be explored. Specifically, we would like to broaden our understanding by investigating the downstream cellular transcription factor cyclic adenosine monophosphate response element-binding protein and other neurotrophic factors such as B-cell lymphoma-2 and brain derived neurotrophic factor. The model of our study allows for specific investigation into neuron-specific effects of Dex; however, there are limitations to our model. First, growth of neurons in culture requires use of perinatal neurons, as adult neurons are unable to survive in culture. Additionally, an in vitro model does not permit us to determine the complete functionality of the neurons as an in vivo model allows. Second, the neurons are unable to interact in their normal microenvironment. Spinal cord neurons in vivo are able to interact with supporting cells such as astrocytes and microglia. These cells have a variety of roles during IR injury, whereby sometimes they contribute to further neuronal degeneration by release of inflammatory cytokines and changes in ion channel permeabilities [23]. Conversely, astrocytes have been implicated as playing an important role in the neuroprotection induced by alpha-2a agonists via modulation of ionic homeostasis and production of neurotrophins [4,24].
5.
Conclusions
Isolated neuronal cell cultures demonstrate that Dex preserves viability through the alpha-2a receptor and Akt in the setting of OGD. Dex reduces the ischemic injury to spinal cord neurons, demonstrating the effect on metabolic tolerance specific to the ischemic injury. Dex demonstrates promise in modifying the effects of both ischemic and metabolic injury
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from ischemic insults, which may further reduce the incidence of paraplegia after aortic intervention.
Acknowledgment The authors acknowledge and thank the University of Colorado Department of Surgery, Division of Cardiothoracic Surgery for their financial support. Authors’ contributions: M.T.B., J.M.M., and L.S.F. assisted with the conduction of experimentations. F.P., M.J.W., J.C.C., D.A.F., X.M., P.S.H., and T.B.R. provided research direction, assisted with analysis of results, and provided assistance with writing/editing of the article.
Disclosure The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in the article.
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