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Spinal cord protection via alpha-2 agonist-mediated increase in glial cell-line–derived neurotrophic factor

Open ArchivePublished:October 14, 2014DOI:https://doi.org/10.1016/j.jtcvs.2014.10.037

      Abstract

      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.

      CTSNet classification

      Abbreviations and Acronyms:

      ELISA (enzyme-linked immunosorbent assay), GDNF (glial cell line–derived neurotrophic factor), IR (ischemia–reperfusion), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), OGD (oxygen glucose deprivation), SEM (standard error of the mean)
      Alpha-2 agonism with dexmedetomidine protected the spinal cord from injury by increasing glial cell–derived neurotrophic factor in astrocytes.
      These in vitro studies propose a novel strategy to protect the spinal cord from injury. Dexmedetomidine, a clinically available sedative, is an agonist of neuronal α2-adrenergic receptors. This agent stimulated spinal cord astrocytes to increase glial cell-line--derived neurotrophic factor, a potent neuroprotective factor for motor neurons. This new approach may prevent paraplegia during aortic surgery.
      See Editorial Commentary pages 586-7.
      Delayed paraplegia secondary to ischemia–reperfusion (IR) injury remains a devastating complication of thoracoabdominal aortic surgery. Advances have been made in adjuvant protective techniques, including hypothermic circulatory arrest and other adjuncts, but paraplegia continues to be a postoperative setback.
      • Conrad M.F.
      • Ye J.Y.
      • Chung T.K.
      • Davison J.K.
      • Cambria R.P.
      Spinal cord complications after thoracic aortic surgery: long-term survival and functional status varies with deficit severity.
      Ischemia and the inflammatory responses of reperfusion are both known to contribute to neuronal degeneration resulting in spinal cord dysfunction.
      • Stirling D.P.
      • Yong V.W.
      Dynamics of the inflammatory response after murine spinal cord injury revealed by flow cytometry.
      Alpha-2 agonists have been shown to attenuate IR injury, but the mechanism for protection has yet to be elucidated. Cerebral,
      • Zhang Y.
      • Kimelberg H.
      Neuroprotection by Alpha-2 adrenergic agonists in cerebral ischemia.
      • Ma D.
      • Hossain M.
      • Rajakumaraswamy N.
      • Arshad M.
      • Sanders R.D.
      • Franks N.P.
      • et al.
      Dexmedetomidine produces its neuroprotective effect via the alpha 2A-adrenoceptor subtype.
      cardiac,
      • Ibacache M.
      • Sanchez G.
      • Pedrozo Z.
      • Galvez F.
      • Humeres C.
      • Echevarria G.
      • et al.
      Dexmedetomidine preconditioning activates pro-survival kinases and attenuates regional ischemia/reperfusion injury in rat heart.
      lung,
      • Gu J.
      • Chen J.
      • Xia P.
      • Tao G.
      • Zhao H.
      • Ma D.
      Dexmedetomidine attenuates remote lung injury induced by renal ischemia-reperfusion in mice.
      and renal
      • Gu J.
      • Sun P.
      • Zhao H.
      • Watts H.R.
      • Sanders R.D.
      • Terrando N.
      • et al.
      Dexmedetomidine provides renoprotection against ischemia-reperfusion injury in mice.
      models have all shown attenuation of IR injury by alpha-2 agonists, and the highly selective alpha-2a agonist dexmedetomidine has been shown to provide functional attenuation of spinal cord IR injury in our in vivo murine model.
      • Bell M.T.
      • Puskas F.
      • Smith P.D.
      • Agoston V.A.
      • Fullerton D.A.
      • Meng X.
      • et al.
      Attenuation of spinal cord ischemia-reperfusion injury by specific α-2a receptor activation with dexmedetomidine.
      The method by which dexmedetomidine protected the mice in our model has not been elucidated.
      Astrocytes are the support cells of the central nervous system. Research has demonstrated that astrocytes play a critical role in neuroprotection.
      • Barreto G.E.
      • Gonzalez J.
      • Torres Y.
      • Morales L.
      Astrocytic-neuronal crosstalk: implications for neuroprotection from brain injury.
      • Takano T.
      • Oberheim N.A.
      • Cotrina M.L.
      • Nedergaard M.
      Astrocytes and ischemic injury.
      • Panickar K.S.
      • Norenberg M.D.
      Astrocytes in cerebral ischemic injury: morphological and general considerations.
      • Bekker A.
      • Sturaitis M.K.
      Dexmedetomidine for neurological surgery.
      In particular, they are critical in IR injury of neurons for a variety of reasons. Astrocytes are the most abundant cell type in the nervous system
      • Barreto G.
      • White R.E.
      • Ouyang Y.
      • Xu L.
      • Giffard R.G.
      Astrocytes: targets for neuroprotection in stroke.
      with significant interconnections through gap junction channels.
      • Giaume C.
      • Koulakoff A.
      • Roux L.
      • Holcman D.
      • Rouach N.
      Astroglial networks: a step further in neuroglial and gliovascular interactions.
      They play a vital role in coupling neuronal activity blood flow that underlies the hemodynamic responses.
      • Arai K.
      • Lo E.H.
      Astrocytes protect oligodendrocyte precursor cells via MEK/ERK and PI3K/Akt signaling.
      Astrocytes are also central to neuroprotection by release of neurotrophins.
      • Migliore M.M.
      • Ortiz R.
      • Dye S.
      • Campbell R.B.
      • Amiji M.M.
      • Waszczak B.L.
      Neurotrophic and neuroprotective efficacy of intranasal GDNF in a rat model of Parkinson’s disease.
      • Nam J.H.
      • Leem E.
      • Jeon M.T.
      • Jeong K.H.
      • Park J.W.
      • Jung U.J.
      • et al.
      Induction of GDNF and BDNF by hRheb(S16H) transduction of SNpc neurons: neuroprotective mechanisms of hRheb(S16H) in a model of Parkinson’s disease.
      • Akerud P.
      • Canals J.M.
      • Snyder E.Y.
      • Arenas E.
      Neuroprotection through delivery of glial cell line-derived neurotrophic factor by neural stem cells in a mouse model of Parkinson's disease.
      Specifically, astrocytes are the major source of a glial cell line–derived neurotrophic factor (GDNF).
      • Trendelenburg G.
      • Dirnagl U.
      Neuroprotective role of astrocytes in cerebral ischemia: focus on ischemic preconditioning.
      GDNF has been shown to be a potent neurotrophic factor for motor neurons and to modulate neuronal death,
      • Nicole O.
      • Ali C.
      • Docagne F.
      • Plawinski L.
      • MacKenzie E.T.
      • Vivien D.
      • et al.
      Neuroprotection mediated by glial cell line-derived neurotrophic factor: involvement of a reduction of NMDA-induced calcium influx by the mitogen-activated protein kinase pathway.
      as well as to support neuronal survival and regeneration.
      • Pajenda G.
      • Hercher D.
      • Marton G.
      • Pajer K.
      • Feichtinger G.
      • Maleth J.
      • et al.
      Spatiotemporally limited BDNF and GDNF overexpression rescues motoneurons destined to die and induces elongative axon growth.
      GDNF has yet to be examined in spinal cord IR injury; however, GDNF has shown functional recovery in a model of traumatic spinal cord injury.
      • Ansorena E.
      • De Berdt P.
      • Ucakar B.
      • Simon-Yarza T.
      • Jacobs D.
      • Schakman O.
      • et al.
      Injectable alginate hydrogel loaded with GDNF promotes functional recovery in a hemisection model of spinal cord injury.
      An ongoing question is how to enhance the astrocyte production of GDNF. Significantly, the alpha-2 agonist dexmedetomidine has been linked to increased production of GDNF.
      • Yan M.
      • Dai H.
      • Ding T.
      • Dai A.
      • Zhang F.
      • Yu L.
      • et al.
      Effects of dexmedetomidine on the release of glial cell line-derived neurotrophic factor from rat astrocyte cells.
      We hypothesize that alpha-2 agonism with dexmedetomidine increases GDNF production in spinal cord astrocytes to provide spinal cord protection after IR injury.

      Materials and Methods

       Materials

      Dexmedetomidine and atipamezole were purchased from Tocris Bioscience (Ellisville, Mo). Anti-GDNF neutralizing antibody was purchased from Abcam (Cambridge, Mass).

       Animals

      The Animal Care and Use Committee at the University of Colorado at Denver Health Sciences Center approved all experiments. This investigation 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). C57BL/6 mice aged postnatal day 2 to 3 from Jackson Laboratories (Bar Harbor, Maine) were used for all experiments. Each litter in total was considered n = 1.

       Cell Culture

      Primary spinal cord neuron cultures and primary astrocyte cultures were obtained from mice aged 2 to 3 days. Briefly, the mice were euthanized with isoflurane and then decapitated. The vertebral column was dissected out, and the spinal cord was removed en bloc via injection of cold phosphate-buffered saline (pH 7.4) through the spinal canal. The spinal cord tissue was minced and then digested in a solution of Hibernate-A (Invitrogen, Carlsbad, Calif) with Papain (Worthington, Lakewood, NJ). Neurons and astrocytes were isolated using an OptiPrep (Sigma-Aldrich, St Louis, Mo) density gradient adapted from Brewer and Torricelli.
      • Brewer G.
      • Torricelli J.
      Isolation and culture of adult neurons and neurospheres.
      Neurons were plated on plates coated with Poly-D-Lysine (Sigma-Aldrich) at approximately 300,000 cells/well on a 24-well plate in 1 mL culture media of Neurobasal-A (Invitrogen), B27 (Invitrogen), GlutaMAX (Invitrogen), and penicillin/streptomycin (Gibco, New York, NY). On in vitro day 3, AraC (Sigma-Aldrich) 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 days. The cultures were greater than 90% neurons as seen by morphology on light microscope and confirmed with microtubule-associated protein 2–positive neuronal staining. Cells were used at in vitro days 7 to 10 for experimentation, which is considered mature for neuronal cultures.
      Astrocytes were placed in culture flasks or plated on 12-well plates in culture media of Neurobasal-A (Invitrogen), B27 (Invitrogen), GlutaMAX (Invitrogen), fetal bovine serum (Gibco), and penicillin/streptomycin (Gibco). Astrocytes were maintained in culture and allowed to grow to confluence before experimentation. Astrocyte medium used for experimentation was collected and stored in a −80°C refrigerator until use.

       Oxygen Glucose Deprivation

      On the day of the experiment, the experimental medium of Dulbecco's Modified Eagle's Medium without glucose (Gibco) was placed in the Ruskinn Bug Box Plus (Ruskinn Technology Ltd, Bridgend, South Wales, UK) humidified airtight hypoxic chamber for 2 hours. The Ruskinn Bug Box Plus was used per the manufacturer's protocol to maintain an environment of 95%N2/5%CO2 at 37°C. A hypoxic environment was verified with Anaerobic Indicator Strips (Oxoid Ltd, Basingstoke, Hants, UK) before placement of media in the chamber, and hypoxia of the media after 2 hours was tested with an indicator strip. The neuronal maintenance culture medium was then removed, the cells were washed with phosphate-buffered saline, and 1 mL of glucose-deprived medium previously treated in the hypoxic chamber was added to each of the cell culture wells on a 24-well plate. Oxygen glucose deprivation (OGD) was induced by placing the plates in the hypoxic chamber for the experimental time periods. A hypoxic environment in the chamber was tested with an indicator strip before placement of cells into the chamber and on the completion of experimentation time to confirm a hypoxic environment. After OGD was completed, neurons were returned to a normal incubator for reperfusion and OGD media were replaced with normal maintenance neuronal media versus experimental astrocyte media.

       Viability Studies

      For viability studies, neuronal cells were cultured on 24-well plates. Cell viability was determined with the MTT Cell Proliferation Kit (Roche Diagnostics, Indianapolis, Ind) according to the procedure provided by the manufacturer. Briefly, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to each well at a concentration of 0.5 mg/mL and incubated for 4 hours at 37°C, and then dimethyl sulfoxide solution was added to the wells. The absorbance at 630 nm was measured on a BioTek Synergy H1 Hybrid microplate reader (BioTek Instruments Inc, Winooski, Vt). Cell viability is presented as the percentage of absorbance relative to nonischemic control.

       Enzyme-Linked Immunosorbent Assay

      After the experiments were conducted, cell culture supernatants were collected. A standard enzyme-linked immunosorbent assay (ELISA) was performed using these supernatants. A GDNF (Abcam) ELISA was performed according to the procedure provided by the manufacturer, and the absorbance at 450 nm was measured on a BioTek microplate reader. Data are presented as concentration in mean picograms/milliliter ± standard error of the mean.

       Statistics

      Results are presented as mean ± standard error. Data were analyzed with StatView software (SAS Institute Inc, Cary, NC). Statistical analysis was performed using 1-way analysis of variance and Student t test.

      Results

       Spinal Cord Cell Cultures

      By using our spinal cord isolation protocol, we were successfully able to culture and maintain primary cultures of spinal cord astrocytes and neurons as seen by the light microscopy image in Figure 1. Astrocytes are identified by light microscope by their star-shaped morphology. Astrocytes are robust cells in culture and grow to confluence easily, whereas neurons are more fragile cells in culture. Neurons are likewise identifiable by morphology on light microscope by their cell bodies and projections. Mature neurons in culture will develop a network of projections as shown in Figure 1.
      Figure thumbnail gr1
      Figure 1Light microscopy image of primary spinal cord astrocytes (left) and primary spinal cord neurons (right) at 100× magnification.

       Glial Cell Line–Derived Neurotrophic Factor Production

      When primary spinal cord astrocytes were treated with dexmedetomidine, we found that GDNF concentration increased in a dose-dependent and time-dependent fashion (Figure 2). GDNF was measured with a GDNF ELISA kit at varying time points after treatment with dexmedetomidine. The concentration of dexmedetomidine used was 0.1 μmol/L, 1 μmol/L, and 10 μmol/L. Although not significant, there was an increase in GDNF concentration at 0.1 μmol/L at 16 hours, 24 hours, and 36 hours. However, there was a significant increase in GDNF production at 24 hours with dexmedetomidine at 1 μmol/L and 10 μmol/L compared with vehicle control (P < .05). The GDNF level appears to peak at 24 hours, given the levels at 36 hours were lower, indicating the GDNF is degraded or used by the cells between the 24- and 36-hour timepoints.
      Figure thumbnail gr2
      Figure 2Astrocyte production of GDNF with dexmedetomidine treatment. A, There was a significant increase at 24 hours with 1 μmol/L and 10 μmol/L of dexmedetomidine compared with vehicle control (*P < .05). Data are presented as mean GDNF concentration in pg/mL ± standard error of the mean (SEM), n = 4. B, There is a significant increase in GDNF production at 24 hours compared with other time points with dexmedetomidine treatment of 1 μmol/L (*P < .05). Data are presented as mean GDNF concentration in pg/mL ± SEM, n = 4. Dex, Dexmedetomidine; GDNF, glial cell line–derived neurotrophic factor.
      There was not a significant difference between 1 μmol/L and 10 μmol/L treatment when compared with each other. We also found an increase in GDNF production with 1 μmol/L and 10 μmol/L treatment at 4 hours, 8 hours, 16 hours, and 36 hours, but these increases were not statistically significant. On the basis of these data, we decided to use astrocyte-conditioned medium at 24 hours post-treatment with 1 μmol/L of dexmedetomidine for further studies to determine whether GDNF preserves the viability of neurons injured by IR.

       Neuronal Viability With Oxygen Glucose Deprivation

      We then wanted to determine whether GDNF produced in the astrocyte medium could salvage neurons injured by IR injury. OGD was used as our model for IR injury. We used 30 minutes, 1 hour, 2 hours, and 4 hours of ischemia time followed by 24 hours of reperfusion to determine an ischemic dose-response curve for neuronal viability as assessed by MTT assay (Figure 3). We found that there was not a significant decrease in neuronal viability at 30 minutes of OGD (90% viable), but that there was a significant decrease in neuronal viability at 1 hour of OGD to only 61% viable (P < .05). In addition, there was a further significant decrease at 2 hours (P < .05) and 4 hours of OGD (P < .05) with 33% and 20% viable, respectively. One hour of OGD was chosen for further studies given that there was a significant decrease in neuronal viability, but enough neurons remained viable for further experimentation.
      Figure thumbnail gr3
      Figure 3Ischemic dose-response curve of neurons subjected to OGD. At 1 hour, there is a significant decrease in viability (*P < .05) and a further significant decrease at 2 hours (#P < .05) and 4 hours (++P < .05). There was a decrease, but not statistically significant, at 30 minutes. Viability determined by MTT assay and presented as mean percent control ± SEM, N = 5. OGD, Oxygen glucose deprivation.

       Neuronal Viability With Dexmedetomidine-Treated Astrocyte Media

      Astrocyte-conditioned media, dexmedetomidine-treated astrocyte media, are defined here as astrocyte media that are 24 hours post-treatment with 1 μmol/L of dexmedetomidine, which has the maximal amount of GDNF (Figure 2). The dexmedetomidine-treated astrocyte media were then placed on neurons that had been subjected to OGD in replacement of the normal neuron media that would be used during reperfusion time. Viability was assessed with the use of an MTT assay. As anticipated, the results show that at 1 hour of OGD, the viability of the neurons is significantly decreased (P < .05) to approximately 62%. We also found that with 1 hour of OGD there was a significant preservation in viability to 77% (P < .05) with the dexmedetomidine-treated astrocyte media compared with standard neuron reperfusion media (Figure 4). The alpha-2 inhibitor atipamezole was given to all the neuronal cells to prevent direct effects of residual dexmedetomidine in the astrocyte-conditioned media.
      Figure thumbnail gr4
      Figure 4Effects of astrocyte-conditioned media on neuronal viability. There is a significant decrease in neuronal viability down to 62% at 1 hour OGD with control media compared with no OGD (*P < .05). With 1 hour of OGD, there is a significant increase in neuronal viability to 77% with dexmedetomidine-treated astrocyte media (#P < .05) compared with control neuron media. Results presented as mean percent nonischemic control ± SEM, N = 8. OGD, Oxygen glucose deprivation.

       Glial Cell Line–Derived Neurotrophic Factor Preservation of Neuronal Viability

      To determine whether the preservation in viability that was seen from the dexmedetomidine-treated astrocyte media was due to GDNF, we used a neutralizing GDNF antibody to block the effects of GDNF on the neurons. By using the GDNF neutralizing antibody, we found that neurons had a significant loss in viability down to 66% compared with the 77% viable neurons given dexmedetomidine-treated astrocyte media without the neutralizing antibody (P < .05) (Figure 5). This illustrates that GDNF is an important factor in neuronal survival after IR injury.
      Figure thumbnail gr5
      Figure 5GDNF preserves neuronal viability. With 1 hour of OGD, there is an increase in neuronal viability with astrocyte-conditioned media to 77% compared with 62% with control reperfusion media (*P < .05). The GDNF neutralizing antibody produced a significant loss in viability down to 66% compared with those given astrocyte-conditioned medium without the neutralizing antibody (#P < .05). There was no statistical significance between the astrocyte-conditioned media with neutralizing antibody and the control media. Results presented as mean percent nonischemic control ± SEM, N = 8. Ab, Antibody; GDNF, glial cell line–derived neurotrophic factor.
      There was no statistical significance between the control reperfusion media and the dexmedetomidine-treated astrocyte media with GDNF neutralizing antibody; however, there was an increased viability with the dexmedetomidine-treated astrocyte media with neutralizing antibody to 66% compared with the control media at 62%. This means that neurons did not lose all the preservation in viability that the astrocyte-conditioned medium provided when the GDNF neutralizing antibody was used, suggesting that there may be other attributes in the dexmedetomidine-treated astrocyte media that is also providing protection independently of GDNF.

      Discussion

      Delayed paraplegia is a devastating complication of thoracoabdominal aortic surgery, which is secondary to IR injury. Alpha-2 agonists, including dexmedetomidine, have been shown to attenuate IR injury in many organ systems; however, the mechanism continues to not be well understood. Our laboratory has primarily focused on spinal cord IR injury and delayed paraplegia.
      • Bell M.T.
      • Puskas F.
      • Smith P.D.
      • Agoston V.A.
      • Fullerton D.A.
      • Meng X.
      • et al.
      Attenuation of spinal cord ischemia-reperfusion injury by specific α-2a receptor activation with dexmedetomidine.
      • Smith P.D.
      • Puskas F.
      • Meng X.
      • Lee J.H.
      • Cleveland Jr., J.C.
      • Weyant M.J.
      • et al.
      The evolution of chemokine release supports a bimodal mechanism of spinal cord ischemia and reperfusion.
      • Smith P.D.
      • Puskas F.
      • Meng X.
      • Cho D.
      • Cleveland Jr., J.C.
      • Weyant M.J.
      • et al.
      Ischemic dose-response in the spinal cord: both immediate and delayed paraplegia.
      Through our in vivo murine model, we have further explored the treatment benefits and mechanisms of dexmedetomidine, although there are limitations to the in vivo model.
      The limitations demonstrated in the in vivo murine model are multiple. First, the spinal cord can be treated only as a single entity. More clearly, the injection of dexmedetomidine affects every cell in the spinal cord. Once treatment has been completed and the mice are sacrificed, the entire spinal cord homogenate is studied with all cellular components included. With the in vitro model developed for this study, primary spinal cord neurons and primary spinal cord astrocytes can be cultured separately. Therefore, the effects of treatment to individual cell lines can be studied in isolation. We then are able to study the subsequent effects on each cell type independently in addition to the effects of substance released by those cell lines on one another.
      Astrocytes were formerly thought to play minimal role in neuroprotection; however, an ever increasing body of literature suggests that they may be more critical to neuronal preservation.
      • Barreto G.E.
      • Gonzalez J.
      • Torres Y.
      • Morales L.
      Astrocytic-neuronal crosstalk: implications for neuroprotection from brain injury.
      • Panickar K.S.
      • Norenberg M.D.
      Astrocytes in cerebral ischemic injury: morphological and general considerations.
      • Bekker A.
      • Sturaitis M.K.
      Dexmedetomidine for neurological surgery.
      Astrocytes are the most abundant cell type and have been shown to play key roles in both normal and pathologic central nervous functioning. Structurally, astrocytes organize the architecture of the central nervous system and help organize communication pathways.
      • Barreto G.
      • White R.E.
      • Ouyang Y.
      • Xu L.
      • Giffard R.G.
      Astrocytes: targets for neuroprotection in stroke.
      Astrocytes are also the principal housekeeping cells of the nervous system, functioning in supportive tasks to control ion and water homeostasis, release neurotrophic factors, shuttle metabolite and waste products, and participate in the formation of the blood–brain barrier.
      • Takano T.
      • Oberheim N.A.
      • Cotrina M.L.
      • Nedergaard M.
      Astrocytes and ischemic injury.
      Failure of these support mechanisms can be detrimental to neuronal survival, particularly in an injured state.
      Astrocytes are known to be a major source of GDNF.
      • Trendelenburg G.
      • Dirnagl U.
      Neuroprotective role of astrocytes in cerebral ischemia: focus on ischemic preconditioning.
      GDNF is a potent neurotrophic factor for motor neurons.
      • Nicole O.
      • Ali C.
      • Docagne F.
      • Plawinski L.
      • MacKenzie E.T.
      • Vivien D.
      • et al.
      Neuroprotection mediated by glial cell line-derived neurotrophic factor: involvement of a reduction of NMDA-induced calcium influx by the mitogen-activated protein kinase pathway.
      It has also been shown to support neuronal survival and regeneration,
      • Pajenda G.
      • Hercher D.
      • Marton G.
      • Pajer K.
      • Feichtinger G.
      • Maleth J.
      • et al.
      Spatiotemporally limited BDNF and GDNF overexpression rescues motoneurons destined to die and induces elongative axon growth.
      and has been a major source of research in the Parkinson's literature for motor neuron recovery.
      • Migliore M.M.
      • Ortiz R.
      • Dye S.
      • Campbell R.B.
      • Amiji M.M.
      • Waszczak B.L.
      Neurotrophic and neuroprotective efficacy of intranasal GDNF in a rat model of Parkinson’s disease.
      • Nam J.H.
      • Leem E.
      • Jeon M.T.
      • Jeong K.H.
      • Park J.W.
      • Jung U.J.
      • et al.
      Induction of GDNF and BDNF by hRheb(S16H) transduction of SNpc neurons: neuroprotective mechanisms of hRheb(S16H) in a model of Parkinson’s disease.
      • Akerud P.
      • Canals J.M.
      • Snyder E.Y.
      • Arenas E.
      Neuroprotection through delivery of glial cell line-derived neurotrophic factor by neural stem cells in a mouse model of Parkinson's disease.
      Specifically crucial to our model is that GDNF production has been linked to alpha-2 agonism in cerebral models.
      • Yan M.
      • Dai H.
      • Ding T.
      • Dai A.
      • Zhang F.
      • Yu L.
      • et al.
      Effects of dexmedetomidine on the release of glial cell line-derived neurotrophic factor from rat astrocyte cells.
      We hypothesized that alpha-2 agonism with dexmedetomidine increases GDNF in spinal cord astrocytes to provide spinal cord protection against neurons subjected to IR injury. The results of our study indicate that primary spinal cord astrocytes grown in vitro significantly produce GDNF with alpha-2 stimulation with 1 μmol/L and 10 μmol/L of dexmedetomidine. Our data suggest that GDNF is in its highest concentration after 24 hours of treatment. GDNF appears to be degraded or used by the cells between 24 and 36 hours given the decline in GDNF seen at 36 hours. There are significant clinical implications to the GDNF peak that we have observed in our experiments. It suggests that administration of dexmedetomidine in the clinical setting would likely need to be 1 day before the planned surgery to maximize GDNF concentration in the spinal cord at the time of IR injury.
      The data show that dexmedetomidine-treated astrocyte media with high levels of GDNF are neuroprotective for neurons subjected to OGD. OGD is our in vitro model for IR injury. We accomplish OGD by treating the cell with glucose-free media and placing the cells in a hypoxic chamber to mimic ischemia followed by replacement with maintenance media with glucose and placement back into a normal incubator to simulate reperfusion. This model uses an experimental media free of glucose and oxygen media, which is crucial to our ischemic model given that clinically ischemia deprives cells of both their source of oxygen and glucose, thereby separating it from a simple hypoxia-reoxygenation model.
      After 1 hour of OGD, only approximately 60% of neurons remain viable after 24 hours of reperfusion. With dexmedetomidine-treated astrocyte media during reperfusion, the neuronal viability is significantly increased to 77% viability. This study also found that use of a GDNF neutralizing antibody abolishes the majority of the protective effects provided by the dexmedetomidine-treated astrocyte media, suggesting that the majority of the effect on viability is due to neuroprotection by GDNF.

       Study Limitations

      No study is without limitations, especially an in vitro study meant to model a clinical scenario of IR injury seen in delayed paraplegia. In vitro dosing is difficult to translate to human subjects, but our previous in vivo model indicates that dosing can be appropriately increased to see treatment effects in a murine model and likely could be translated into human subjects without difficulty. Further, in vitro studies are crucial for studying individual cells but do not allow for the study of the cells within their normal microenvironment, and thus may limit understanding of treatment effects. Additional studies of astrocyte-conditioned medium are also needed to determine whether there are other properties that may provide neuroprotection in addition to GDNF. In particular, another future direction for our studies should involve co-culturing neurons and astrocytes to further mimic the true microenvironment the motor neurons are surrounded by in the spinal cord.
      Another specific limitation of our in vitro study is the need to use perinatal mice for culturing neurons, as well as the inability to separate motor from sensory neurons in the spinal cord. A hallmark of in vitro cell culture is that cells need to appropriately survive to allow experimentations to be done. Surviving in culture can even be a struggle for neurons isolated from perinatal mice, but adult neuron cultures are so unpredictable in their ability to survive, studies of them are difficult to interpret, particularly the experimentations designed to detect changes in viability that we were studying. Therefore, we use a perinatal model to have predictable cell survival in culture. Also, there is an inability to separate motor from sensory neurons in our culture. Therefore, we do not know precisely which cell populations we are salvaging with dexmedetomidine; however, we do know that GDNF has been shown to specifically rescue motor neurons.
      • Nam J.H.
      • Leem E.
      • Jeon M.T.
      • Jeong K.H.
      • Park J.W.
      • Jung U.J.
      • et al.
      Induction of GDNF and BDNF by hRheb(S16H) transduction of SNpc neurons: neuroprotective mechanisms of hRheb(S16H) in a model of Parkinson’s disease.
      • Akerud P.
      • Canals J.M.
      • Snyder E.Y.
      • Arenas E.
      Neuroprotection through delivery of glial cell line-derived neurotrophic factor by neural stem cells in a mouse model of Parkinson's disease.
      • Pajenda G.
      • Hercher D.
      • Marton G.
      • Pajer K.
      • Feichtinger G.
      • Maleth J.
      • et al.
      Spatiotemporally limited BDNF and GDNF overexpression rescues motoneurons destined to die and induces elongative axon growth.
      • Ansorena E.
      • De Berdt P.
      • Ucakar B.
      • Simon-Yarza T.
      • Jacobs D.
      • Schakman O.
      • et al.
      Injectable alginate hydrogel loaded with GDNF promotes functional recovery in a hemisection model of spinal cord injury.
      Therefore, there is a possibility that one cell type is being preferentially saved over the other; however, our perinatal neuronal culture results mirror the functional outcomes found in our in vivo adult mouse model, and thus we believe our in vitro model is a good representation of spinal cord neuronal cell death of motor neurons that account for paraplegia seen in our adult murine in vivo studies.

      Conclusions

      Our data suggest that astrocytes and GDNF play an important role in neuroprotection from IR injury and may provide a strategy for protection of the spinal cord during aortic surgery.
      The authors thank the Department of Surgery, University of Colorado Denver , for financial support for this project.

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