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Erythropoietin attenuates neuronal injury and potentiates the expression of pCREB in anterior horn after transient spinal cord ischemia in rats
Surgical Neurology 68 (2007) 297 – 303 www.surgicalneurology-online.com
Ischemia
Erythropoietin attenuates neuronal injury and potentiates the expression of pCREB in anterior horn after transient spinal cord ischemia in rats Atac¸ Sfnmez, MD, PhDa,4,1, Birol Kabakc¸V, MDb,1, Enver Vardar, MDf, Duygu Gqrel, MDc, ¨ lker Sfnmez, MD, PhDd, Yahya T. Orhanc, U ¨ nal Ac¸ikel, MDb, Necati Gfkmen, MDe U Departments of aLearning Resources Center Research Laboratory, bCardiovascular Surgery, cPathology, dHistology and Embryology, and e Anesthesiology and Reanimation, School of Medicine, Dokuz Eylul University Inciralti, TR-35340 Izmir, Turkey f Department of Pathology, Social Security Educational Hospital, TR-35350 Izmir, Turkey Received 30 June 2006; accepted 3 November 2006
Abstract
Background: Recent studies have suggested that EPO activates the CREB transcription pathway and increases BDNF expression and production, which contributes to EPO-mediated neuroprotection. We investigated whether EPO has a neuroprotective effect against ISCI in rats and examined the involvement of CREB protein phosphorylation in this process. Methods: Spinal cord ischemia was produced by balloon occlusion of the abdominal aorta below the branching point of the left subclavian artery for 5 minutes, and rHu-EPO (1000 U/kg BW) was administered intravenously after the onset of the reperfusion. Neurologic status was assessed at 1, 24, and, 48 hours. After the end of 48 hours, spinal cords were harvested for histopathologic analysis and immunohistochemistry for pCREB. Results: All sham-operated rats had a normal neurologic outcome, whereas all ischemic rats suffered severe neurologic deficits after ISCI. Erythropoietin treatment was found to accelerate recovery of motor deficits and prevent the loss of motoneurons in the spinal cord after transient ischemia. Ischemic spinal cord injury induced the phosphorylation of pCREB at the anterior horn of the spinal cord, and EPO treatment significantly potentiated expression of pCREB increase at the anterior horn of the spinal cord. Conclusions: These results demonstrate that a single dose of EPO given before ISCI provides significant neuroprotection and potentiates the expression of pCREB in this region. D 2007 Elsevier Inc. All rights reserved.
Keywords:
Aorta; Spinal cord ischemia; Erythropoietin; pCREB; Neuroprotection; Rat
1. Introduction Abbreviations: ANOVA, analysis of variance; BDNF, brain-derived neurotrophic factor; BW, body weight; cAMP, cyclic adenosine monophosphate; CREB, cyclic adenosine monophosphate responsive element binding protein; EPO, erythropoietin; ISCI, ischemic spinal cord injury; MAP, mitogen-activated protein; MSDI, motor sensory deficit index; NGF, nerve growth factor; PBS, phosphate-buffered saline; pCREB, phosphorylated CREB; pCREB-IR, pCREB immunoreactivity; rHu-EPO, recombinant human erythropoietin; VEGF, vascular endothelial growth factor. 4 Corresponding author. Tel.: +90 232 412 46 79; fax: +90 232 259 05 41. E-mail address: [email protected] (A. Sfnmez). 1 These authors contributed equally to this work. 0090-3019/$ – see front matter D 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.surneu.2006.11.045
Ischemic spinal cord injury remains the most dreaded complication of operations for treatment of descending thoracic and thoracoabdominal aortic aneurysms [11,13]. The necessity for aortic cross-clamping carries a risk of distal organ ischemia including kidneys, liver, intestines, and spinal cord. The incidence of spinal cord complications ranges from 8% to 30% and depends on the nature and extent of the disease and the duration of aortic crossclamping [4]. Neuronal injury arising from spinal cord
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ischemia is believed to result from diverse but interrelated processes such as glutamate-mediated excitotoxicity, nitric oxide overproduction, inflammation, apoptosis, and freeradical generation [30]. These cellular stimuli are known to activate members of the MAP kinase family, which participate in numerous intracellular signaling pathways that can initiate reparative processes or cell death. Various agents and techniques that ameliorate these different processes have been tested in experimental model of spinal cord ischemia [11,13,25]. Erythropoietin is a hematopoietic cytokine hormone that has recently been shown to exert neuroprotection against various insults such as in vitro and in vivo. Erythropoietin and EPO receptors have both been reported in the brain cortex, cerebellum, hippocampus, pituitary gland, and spinal cord [24]. Erythropoietin has been reported to activate specific receptors in the central nervous system and was found to be neurotrophic and neuroprotective against to excitotoxicity, ischemia, hypoxia, trauma, and inflammation, and serum growth factor deprivation in both in vitro and in vivo models [18,24,40]. Acute and delayed beneficial action of systemically administered EPO has also been reported in rabbit ISCI [3]. The mechanisms of EPO to produce neuroprotective effects are reduction in glutamate toxicity, increased production of neuronal anti-apoptotic factors, reduced nitric oxide–mediated injury, anti-inflammatory effects, and antioxidant properties [18]. These findings have greatly enhanced the value of EPO as possible therapeutic strategy and focused attention on the molecular mechanisms underlying its neuroprotective effect. CREB is a transcription factor with multiple functions [26,33] and is believed to play a key role in cell survival [2,7,39]. Reportedly, oxidant injury regulates the activity of CREB and thus survival [5,15,17,29]. CREB is constitutively expressed in neuronal nuclei, and its activation occurs via phosphorylation at serine 133 (Ser133) by various kinases, including MAP kinases [26,33]. Extracellular signal-regulated kinase phosphorylates CREB through p90rsk. Accordingly, various signaling cascades converge for CREB phosphorylation, such that CREB is now known to play a pivotal role in many physiologic activities including neuronal development, regeneration, memory function, synaptic plasticity, and cell repair [1,6,14,16,28,31,32,38]. For instance, CREB mediates neuronal responses to various neurotrophins including BDNF and NGF, which regulate neuronal survival, differentiation, neurogenesis, and synaptic function [8]. Conversely, CREB itself controls BDNF transcription [36]. CREB activation is an important event in neural plasticity and neuroprotection against injury in the developing brain, and drugs that activate cAMP-CREB signaling would provide novel therapeutic approaches for the treatment of hypoxic-ischemic brain injury [34,39]. Recently, it has been reported that EPO activates the CREB transcription pathway and increases BDNF expression and production, and this increase contributes to EPO-mediated neuroprotection in primary hippocampal neurons [37].
Another astonishing finding about the neuroprotective effect of EPO is that treatment of stroke with EPO enhances neurogenesis and angiogenesis, and improves neurologic function in rats by increasing VEGF and BDNF [40]. In this study, we investigated whether EPO has a neuroprotective effect against ISCI in rats and examined the possible involvement of CREB protein phosphorylation in this process. 2. Material and methods 2.1. Animal care Seventeen Wistar albino rats (4-5 months old, male) weighing 300 to 375 g were used in this study. Animals were housed under standard conditions in the Animal Research Laboratory at Dokuz Eylql University. The study protocol was approved by the Animal Research Ethical Committee of Dokuz Eylql University. 2.2. Study groups Animals were randomly divided into 3 groups. 1. 2.
3.
Sham operation (n = 3): underwent the surgical procedure but the aorta was not occluded. Control group (ischemia + saline) (n = 7): received normal saline intravenously immediately after the onset of the reperfusion. EPO group (ischemia + EPO) (n = 7): received rHuEPO (Eprex, Cilag, Zug, Switzerland) administered intravenously immediately after the onset of the reperfusion at a dose of 1000 U/kg BW.
2.3. Surgical procedure Anesthesia was induced with 2% to 3% halothane in oxygen and with 1.5% halothane (in 100% O2) delivered through a facemask during the surgery. The end-tidal carbon dioxide and halothane concentrations were monitored with a capnograph (Anesthesia Gas Monitoring 1304, Bruel and Kjaer, Naerum, Denmark) during surgery. The left femoral artery was also cannulated for monitoring the arterial blood pressure and arterial blood gases. Temperature was measured with a rectal probe and controlled by feedback to the heating lamp. The rats were kept at 37.08C F 0.58C during the ischemic period. Spinal cord ischemia was produced as described by Gilad and Gilad [12]. In brief, a balloon catheter was inserted (F2 Fogarty) through the abdominal aorta, below the kidneys, and the balloon (0.25 mL air) was inflated below the branching point of the left subclavian artery for 5 minutes, thereby blocking blood flow completely. Thereafter, the catheter was deflated and removed, and the aorta was closed, using a piece of the catheter as a bridging cannula. It was reported that longer periods of ischemia in this model may result in pulmonary edema and death within 24 hours after reperfusion. Heparin (200 U) was administrated as an intravenous bolus before aortic occlusion.
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2.4. Monitoring of physiologic parameters The cardiac pulse rate, mean arterial pressure, and rectal temperature were recorded from the monitor (Biopac MP 30 and Biopac BSL prov. 3.6.5, Biopac Systems, Santa Barbara, Calif). Partial carbon dioxide tension (Paco2), partial oxygen tension (Pao2), pH, hematocrit, and glucose were analyzed in the arterial blood samples. Arterial blood gas measurements were performed with the Radiometer ABL 700 Series (Radiometer ABL, Copenhagen, Denmark). These physiologic parameters were examined separately before starting the surgical procedure, before the aortic occlusion, and after the restoration of aortic perfusion. 2.5. Evaluation of behavioral outcome The neurologic evaluations were done in a blinded fashion. Motor performance was scored beginning at the first hour after spinal cord ischemia and then at 24 and 48 hours postoperatively. Four tests were used: (1)
(2)
(3)
(4)
Righting reflex—the ability to turn over when placed on back—was scored as: 1, normal righting; 0, weak or no righting. Placing reflex—the ability to grasp a ledge with hind limbs when suspended in air—was scored as: 1, normal placing; 0, weak or no placing. An arbitrary rating scale [12]: 4, normal walking and posture; 3, limping or mildly impaired walking with slight hind limb spasticity; 2, unilateral hind limb flaccid/spastic paralysis; 1, partial paralysis where movement occurs by bending the legs at the knees (toes are bent); 0, complete paralysis of the lower back and extremities (where movement is by dragging the lower torso). Pain response [22]: 2, withdraws from toe pinch; 1, squeals but does not withdraw; 0, no reaction.
The data from the four tests were combined to give an MSDI ranging from 0 (complete paralysis) to 8 (normal). Motor sensory deficit index was calculated for each animal at each time point.
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5 lmol/L paraffin cross sections stained with hematoxylin and eosin, by counting the motoneurons (only profiles containing nucleoli) in the ventral horn area of the representative levels of the spinal cord. Motoneurons were identified by their size ( N 25 lmol/L in diameter) and location as conveniently demonstrated in lumbar spinal cord sections taken through the enlarged ventral horn of this region. Counts of motoneurons are presented as average numbers of cells per section. 2.8. Immunohistochemistry for pCREB Five-micrometer-thick sections from the tissue blocks were placed onto poly l-lysine–coated slides. Tissue sections were deparaffinized and rehydrated before immunostaining. Antigen retrieval was performed by heating the tissue sections in a microwave oven before immunostaining. Immunohistochemistry was performed using an avidinbiotinylated enzyme complex system (Vectastain ABC Elite Kit, Vector Laboratories, Burlingame, Calif). In brief, the sections were washed in PBS (pH 7.4) for 5 minutes. They were then quenched with 0.3% H2O2 for 10 minutes to block endogenous peroxidase and washed three times for 5 minutes each in PBS. The sections were subsequently preincubated for 30 minutes in blocking serum to block nonspecific binding of antibody. Sections for detection of pCREB were incubated for 30 minutes at room temperature with the affinity-purified rabbit polyclonal antirat pCREB antibody (Upstate Biotechnology, Lake Placid, NY) at a 1:1000 dilution. This antibody is specific for pCREB in which the Ser133 residue is phosphorylated. After the primary incubation, the slides were rinsed in PBS and subsequently incubated with the biotinylated secondary antibody (antirabbit goat antibody) and 1% normal goat serum for 90 minutes. The sections were then rinsed and incubated with avidin-biotinylated peroxidase conjugate. They were finally developed in 0.02% diaminobenzidine with 0.02% H2O2. Sections were examined under
2.6. Euthanasia At 48 hours after spinal cord ischemia, the animals were deeply anesthetized (120 mg/kg BW sodium pentobarbital IP), transcardially perfused/fixed with a solution of 0.1 mol/L phosphate buffer (pH 7.4) containing 10% formalin, and the spinal cord removed and postfixed in the same solution for at least 24 hours. Thereafter, the spinal cord was cut into blocks corresponding to lumbar levels L1 and L5, and embedded in paraffin blocks. 2.7. Histopathology Five-micrometer-thick transverse sections were obtained on a microtome (Leica, Wetzlar, Germany) through the lumbar spinal cord, and sections were stained with hematoxylin and eosin. Delayed nerve cell death was measured in
Fig. 1. Therapeutic effects of EPO (1000 U/kg BW intravenous) or saline treatments on neurologic functions (combined motor and sensory score) after spinal cord ischemia. Values are mean F SD (*P b .05 when compared to control and EPO groups, **P b .05 compared to sham and control groups, ***P b .05 compared to control group).
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each group. All the rats in the groups survived the entire observation period. 3.1. Physiologic parameters The physiologic parameters (Paco2, Pao2, pH, hematocrit, glucose, and end-tidal CO2 volumes) were not significantly different between groups (data are not given here). 3.2. Neurobehavioral outcome All rats in the sham operation group had a normal postoperative neurologic outcome throughout the observation period. All EPO-treated rats had some form of flaccid paraplegia after recovery from anesthesia but began to recover motor function between 1 and 48 hours postoperatively, whereas all rats in the control group remained paraplegic throughout the observation period, with no improvement in either sensory or motor function (Fig. 1). 3.3. Histopathologic examinations Viable cell count in the ventral horn area of the spinal cord showed that the extent of ischemic damage was grossly proportional to the neurologic score. Viable cell number in the control group was significantly lower than in both sham and EPO-treated groups (20 F 2, 28 F 1, and 26 F 2, respectively). However, there was no significant difference between the viable cell number of sham and EPO-treated groups (Fig. 2). 3.4. pCREB Immunohistochemistry Fig. 2. A: Representative photomicrograph of the ventral horn region in cross sections through the spinal cord lumbar segments illustrating apoptotic motoneuron (bar = 150 lm), and quantitative data indicating the motor neuron number per section (*P b .05 vs sham and EPO; ANOVA followed by Tukey’s test) (B).
a light microscope. In the absence of primary antibody, no positive immunostaining for pCREB was observed. The number of cells exhibiting pCREB-IR was estimated from the anterior horn of spinal cord sections from L1 and L5 segmental levels for each animal. Counts of pCREB-IR cells are presented as average numbers of cells per section. All pCREB-IR–positive neurons at the anterior horn of the spinal cord were counted without considering the intensity of the staining. The pathologist who performed the examinations was blinded to the experimental conditions. 2.9. Statistical analysis All data are expressed as mean F SD. One-way ANOVA was used for data analysis, followed by the Tukey test for post hoc analysis. A P value b .005 was considered to be statistically significant. 3. Results Two rats in the EPO group and 2 rats in the control group died either during or after the operation (3 with cardiac tamponade and 1 with pulmonary edema). Those 4 rats were replaced by other rats to reach the appropriate number in
Fig. 3C summarizes the numbers of immunopositive cells in the anterior horn of spinal cord sections. The number of pCREB-IR neurons in the control and EPO-treated group was significantly increased compared to that in the shamoperated group (313 F 1, 365 F 16, and 147 F 5.6, respectively) in the anterior horn of the spinal cord. In this region, the number of immunoreactive cells in the EPOtreated group was also significantly higher than in the control group (Fig. 3A and B). 4. Discussion The major findings in the present study are as follows: (1) exogenous administration of EPO has a neuroprotective potential against ISCI in rats; (2) there are significant increases in the expression of pCREB-IR in the anterior horn of the spinal cord after ISCI in rats; (3) CREB protein phosphorylation response to ISCI was potentiated by a single dose of EPO treatment in the anterior horn of the spinal cord. Multiple models of nervous system injury (mouse, rat, gerbil, and rabbit) have been used to demonstrate the effectiveness of EPO as a neuroprotective agent, including focal and global cerebral ischemia, experimental autoimmune encephalomyelitis, kainic acid–induced seizures, experimental traumatic brain injury, neurotoxin-induced experimental parkinsonism, neonatal hypoxic-ischemic brain injury,
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Fig. 3. When compared to sham group (A), ISCI markedly increased pCREB expression in the rat spinal ventral horn (B) and intravenous administration of EPO potentiated (C) this increase in the same region. D: Quantitative data indicating the number of pCREB immunoreactive neurons per section (magnification, 200). *P b .05 vs sham; **P b .05 vs control; ANOVA followed by Tukey’s test.
subarachnoid hemorrhage, spinal cord ischemia and injury, retinal ischemia, and peripheral nerve injury [10]. Although it was recently reported that exogenous administration of EPO provides neuroprotection after spinal cord ischemic injury in rabbits, we tested the neuroprotective effects of EPO in a rat spinal ischemia model. It has been concluded that ISCI model in rats is much more convenient than spinal cord injury model in rabbits because of two reasons: (1) rabbit spinal arterial vascularization differs from that in humans, whereas arterial vascularization is almost similar in human and rat; (2) evaluation of the model in rabbits has been found compromising owing to the limited behavioral repertoire of rabbits, which precludes sophisticated scoring and neurologic assessment [1,3,19,20]. However, results of our spinal ischemia model in rats to evaluate the effectiveness of EPO against ischemic insult support the findings of a previous study on rabbit. The discovery of the production of EPO by neuronal cells in man was made only 15 years ago, and attention has been focused on the neurotrophic and neuroprotective function of EPO in in vivo and in vitro conditions of neuronal damage. Although the mechanisms of EPO that produce neuroprotective effects are not identified yet, it has been concluded that EPO may act in a coordinated fashion at multiple levels, including limiting the production of tissue-
injuring molecules such as reactive oxygen species and glutamate, reversal of vasospasm, stimulation of angiogenesis, attenuation of apoptosis, modulation of inflammation, and recruitment of stem cells, and, thus, may protect neurons by a combination of these mechanisms [10]. CREB is a basic leucine zipper family transcription factor that plays a key role in neuronal development, function, and survival, and its transcriptional activity is upregulated in response to many cellular stimuli, including cAMP, Ca2+, hypoxia, ultraviolet light, and growth factors such as BDNF and IGF-1 [9,17,28,29,34,42]. The canonical CREB activation pathway involves stimulus-induced phosphorylation of CREB on Ser133 by several kinds of protein kinases, such as protein kinase A, Ca2+/calmodulin-dependent kinase IV, and ribosomal S6 kinase 2 to facilitate the transcription of genes with the CRE motif [39,41]. CREB is a bona fide neuron survival factor, as Ser133 pCREB can activate the relative gene transcription to further promote protein expression such as c-Fos, Jun-B, Bcl-2, and neurotrophins. These gene products regulate neuronal regeneration, survival, repair, and protection during the case of nervous system stress injury, such as hypoxia [23,34,35,39]. Not only has CREB phosphorylation been implicated in the resistance of cells to various insults, but a number of well-established neuroprotective agents exert their actions
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via pathways that converge on the CREB protein. In the present study, the neuroprotective effects of EPO treatment have also been studied in terms of changes in CREB protein phosphorylation after ischemic injury. It was previously reported that intracellular Ca2+ increase is one of the early events triggered by EPO to be critically involved in neuroprotection [21,27]. In addition, Viviani et al [37] demonstrated that EPO activates the CREB transcription pathway and increases BDNF expression and production, which contributes to EPO-mediated neuroprotection in primary hippocampal neurons. Our findings suggest that CREB protein phosphorylation response to ISCI was potentiated by EPO treatment in the anterior horn of the spinal cord. Potentiation of CREB phosphorylation observed in this region appears to be compatible with the in vitro findings in primary hippocampal neurons. Taken together, all these results support the idea that EPO acts in the nervous system as a direct protective factor against ischemic insult in neurons via activation of CREB protein phosphorylation. And CREB-protein targeting by using EPO might be an ideal way to achieve neuroprotective effects, a strategy that will have major implications for preventing ISCI. References [1] Ahn S, Ginty DD, Linden DJ. A late phase of cerebellar long-term depression requires activation of CaMKIV and CREB. Neuron 1999; 23(3):414 - 5. [2] Bonni A, Brunet A, West AE, Datta SR, Takasu MA, Greenberg ME. Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and independent mechanisms. Science 1999; 12(286):1358 - 62. [3] Celik M, Gokmen N, Erbayraktar S, Akhisaroglu M, Konakc S, Ulukus C, Genc S, Genc K, Sagiroglu E, Cerami A, Brines M. Erythropoietin prevents motor neuron apoptosis and neurologic disability in experimental spinal cord ischemic injury. Proc Natl Acad Sci U S A 2002;99(4):2258 - 63. [4] Coselli JS, LeMaire SA, de Figueiredo LP, Kirby RP. Paraplegia after thoracoabdominal aortic aneurysm repair: is dissection a risk factor? Ann Thorac Surg 1997;63(1):28 - 36. [5] Crossthwaite AJ, Hasan S, Williams RJ. Hydrogen peroxide mediated phosphorylation of ERK1/2, Akt/PKB and JNK in cortical neurones: dependence on Ca (2+) and PI3-kinase. J Neurochem 2002;80:24 - 35. [6] De Cesare D, Fimia GM, Sassone-Corsi P. Signaling routes to CREM and CREB: plasticity in transcriptional activation. Trends Biochem Sci 1999;24:281 - 5. [7] Du K, Montminy M. CREB is a regulatory target for the protein kinase Akt/PKB. J Biol Chem 1998;273:32377 - 9. [8] Finkbeiner S, Tavazoie SF, Maloratsky A, Jacobs KM, Harris KM, Greenberg ME. CREB: a major mediator of neuronal neurotrophin responses. Neuron 1997;19:1031 - 47. [9] Gao Y, Gao G, Long C, Han S, Zu P, Fang L, Li J. Enhanced phosphorylation of cyclic AMP response element binding protein in the brain of mice following repetitive hypoxic exposure. Biochem Biophys Res Commun 2006;340(2):661 - 7. [10] Genc S, Koroglu TF, Genc K. Erythropoietin and the nervous system. Brain Res 2004;1000(1-2):19 - 31. [11] Gharagozloo F, Larson J, Dausmann MJ, Neville RF, Gomes MN. Spinal cord protection during surgical procedures on the descending thoracic and thoracoabdominal aorta: review of current techniques. Chest 1996;109:799 - 809.
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effects of EPO in a rat model of spinal cord ischemia. This was caused by occluding the aorta for 5 minutes. Subsequently, EPO was administered intravenously. Forty-eight hours later, the animals were sacrificed. Erythropoietin improved the clinical outcome, and this result corresponded with a significantly higher number of motoneurons and pCREB cells in the spinal cord. CREB should be of crucial importance in cell survival and repair. Erythropoietin enhances the activation of CREB by means of phosphorylation. These results add another brick to the building of EPO, a very interesting and important hematopoietic cytokine hormone. This molecule is involved in several beneficial mechanisms in the body. In the central nervous system, EPO promotes the expression of several growth factors and would be involved in neurogenesis and angiogenesis. The role of EPO in neuroprotection is further confirmed by the results of this study. Therefore, EPO could be useful, at least for attenuating the eventual consequences on the spinal cord of aorta clamping during aortic aneurysm surgery. Eduardo Fernandez, MD Istituto di Neurochirurgia Universita` Cattolica del Sacro Cuore 00168 Rome, Italy
This is a relatively straightforward study, demonstrating a neuroprotective effect of EPO in a model of spinal cord ischemia in the rat. It joins a growing number of papers demonstrating this effect. The authors wanted to shed light on the mechanism of action of EPO, by staining for the phosphorylated form of the transcription factor CREB. The study is small, using a small number of animals and a limited number of parameters—only pCREB immunohistochemistry is performed. This limits the ability to connect the dots between the administration of EPO and the change in pCREB. Nevertheless, this is a welcome contribution, encouraging further study into the mechanism of action of EPO.
Commentary It is well known that clamping of the aorta during surgery for aortic aneurysms can cause spinal cord ischemia. The authors of the present study demonstrated some positive
Ben Roitberg, MD Department of Neurosurgery University of Illinois at Chicago Chicago, IL 60612, USA
Ischemia
Erythropoietin attenuates neuronal injury and potentiates the expression of pCREB in anterior horn after transient spinal cord ischemia in rats Atac¸ Sfnmez, MD, PhDa,4,1, Birol Kabakc¸V, MDb,1, Enver Vardar, MDf, Duygu Gqrel, MDc, ¨ lker Sfnmez, MD, PhDd, Yahya T. Orhanc, U ¨ nal Ac¸ikel, MDb, Necati Gfkmen, MDe U Departments of aLearning Resources Center Research Laboratory, bCardiovascular Surgery, cPathology, dHistology and Embryology, and e Anesthesiology and Reanimation, School of Medicine, Dokuz Eylul University Inciralti, TR-35340 Izmir, Turkey f Department of Pathology, Social Security Educational Hospital, TR-35350 Izmir, Turkey Received 30 June 2006; accepted 3 November 2006
Abstract
Background: Recent studies have suggested that EPO activates the CREB transcription pathway and increases BDNF expression and production, which contributes to EPO-mediated neuroprotection. We investigated whether EPO has a neuroprotective effect against ISCI in rats and examined the involvement of CREB protein phosphorylation in this process. Methods: Spinal cord ischemia was produced by balloon occlusion of the abdominal aorta below the branching point of the left subclavian artery for 5 minutes, and rHu-EPO (1000 U/kg BW) was administered intravenously after the onset of the reperfusion. Neurologic status was assessed at 1, 24, and, 48 hours. After the end of 48 hours, spinal cords were harvested for histopathologic analysis and immunohistochemistry for pCREB. Results: All sham-operated rats had a normal neurologic outcome, whereas all ischemic rats suffered severe neurologic deficits after ISCI. Erythropoietin treatment was found to accelerate recovery of motor deficits and prevent the loss of motoneurons in the spinal cord after transient ischemia. Ischemic spinal cord injury induced the phosphorylation of pCREB at the anterior horn of the spinal cord, and EPO treatment significantly potentiated expression of pCREB increase at the anterior horn of the spinal cord. Conclusions: These results demonstrate that a single dose of EPO given before ISCI provides significant neuroprotection and potentiates the expression of pCREB in this region. D 2007 Elsevier Inc. All rights reserved.
Keywords:
Aorta; Spinal cord ischemia; Erythropoietin; pCREB; Neuroprotection; Rat
1. Introduction Abbreviations: ANOVA, analysis of variance; BDNF, brain-derived neurotrophic factor; BW, body weight; cAMP, cyclic adenosine monophosphate; CREB, cyclic adenosine monophosphate responsive element binding protein; EPO, erythropoietin; ISCI, ischemic spinal cord injury; MAP, mitogen-activated protein; MSDI, motor sensory deficit index; NGF, nerve growth factor; PBS, phosphate-buffered saline; pCREB, phosphorylated CREB; pCREB-IR, pCREB immunoreactivity; rHu-EPO, recombinant human erythropoietin; VEGF, vascular endothelial growth factor. 4 Corresponding author. Tel.: +90 232 412 46 79; fax: +90 232 259 05 41. E-mail address: [email protected] (A. Sfnmez). 1 These authors contributed equally to this work. 0090-3019/$ – see front matter D 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.surneu.2006.11.045
Ischemic spinal cord injury remains the most dreaded complication of operations for treatment of descending thoracic and thoracoabdominal aortic aneurysms [11,13]. The necessity for aortic cross-clamping carries a risk of distal organ ischemia including kidneys, liver, intestines, and spinal cord. The incidence of spinal cord complications ranges from 8% to 30% and depends on the nature and extent of the disease and the duration of aortic crossclamping [4]. Neuronal injury arising from spinal cord
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ischemia is believed to result from diverse but interrelated processes such as glutamate-mediated excitotoxicity, nitric oxide overproduction, inflammation, apoptosis, and freeradical generation [30]. These cellular stimuli are known to activate members of the MAP kinase family, which participate in numerous intracellular signaling pathways that can initiate reparative processes or cell death. Various agents and techniques that ameliorate these different processes have been tested in experimental model of spinal cord ischemia [11,13,25]. Erythropoietin is a hematopoietic cytokine hormone that has recently been shown to exert neuroprotection against various insults such as in vitro and in vivo. Erythropoietin and EPO receptors have both been reported in the brain cortex, cerebellum, hippocampus, pituitary gland, and spinal cord [24]. Erythropoietin has been reported to activate specific receptors in the central nervous system and was found to be neurotrophic and neuroprotective against to excitotoxicity, ischemia, hypoxia, trauma, and inflammation, and serum growth factor deprivation in both in vitro and in vivo models [18,24,40]. Acute and delayed beneficial action of systemically administered EPO has also been reported in rabbit ISCI [3]. The mechanisms of EPO to produce neuroprotective effects are reduction in glutamate toxicity, increased production of neuronal anti-apoptotic factors, reduced nitric oxide–mediated injury, anti-inflammatory effects, and antioxidant properties [18]. These findings have greatly enhanced the value of EPO as possible therapeutic strategy and focused attention on the molecular mechanisms underlying its neuroprotective effect. CREB is a transcription factor with multiple functions [26,33] and is believed to play a key role in cell survival [2,7,39]. Reportedly, oxidant injury regulates the activity of CREB and thus survival [5,15,17,29]. CREB is constitutively expressed in neuronal nuclei, and its activation occurs via phosphorylation at serine 133 (Ser133) by various kinases, including MAP kinases [26,33]. Extracellular signal-regulated kinase phosphorylates CREB through p90rsk. Accordingly, various signaling cascades converge for CREB phosphorylation, such that CREB is now known to play a pivotal role in many physiologic activities including neuronal development, regeneration, memory function, synaptic plasticity, and cell repair [1,6,14,16,28,31,32,38]. For instance, CREB mediates neuronal responses to various neurotrophins including BDNF and NGF, which regulate neuronal survival, differentiation, neurogenesis, and synaptic function [8]. Conversely, CREB itself controls BDNF transcription [36]. CREB activation is an important event in neural plasticity and neuroprotection against injury in the developing brain, and drugs that activate cAMP-CREB signaling would provide novel therapeutic approaches for the treatment of hypoxic-ischemic brain injury [34,39]. Recently, it has been reported that EPO activates the CREB transcription pathway and increases BDNF expression and production, and this increase contributes to EPO-mediated neuroprotection in primary hippocampal neurons [37].
Another astonishing finding about the neuroprotective effect of EPO is that treatment of stroke with EPO enhances neurogenesis and angiogenesis, and improves neurologic function in rats by increasing VEGF and BDNF [40]. In this study, we investigated whether EPO has a neuroprotective effect against ISCI in rats and examined the possible involvement of CREB protein phosphorylation in this process. 2. Material and methods 2.1. Animal care Seventeen Wistar albino rats (4-5 months old, male) weighing 300 to 375 g were used in this study. Animals were housed under standard conditions in the Animal Research Laboratory at Dokuz Eylql University. The study protocol was approved by the Animal Research Ethical Committee of Dokuz Eylql University. 2.2. Study groups Animals were randomly divided into 3 groups. 1. 2.
3.
Sham operation (n = 3): underwent the surgical procedure but the aorta was not occluded. Control group (ischemia + saline) (n = 7): received normal saline intravenously immediately after the onset of the reperfusion. EPO group (ischemia + EPO) (n = 7): received rHuEPO (Eprex, Cilag, Zug, Switzerland) administered intravenously immediately after the onset of the reperfusion at a dose of 1000 U/kg BW.
2.3. Surgical procedure Anesthesia was induced with 2% to 3% halothane in oxygen and with 1.5% halothane (in 100% O2) delivered through a facemask during the surgery. The end-tidal carbon dioxide and halothane concentrations were monitored with a capnograph (Anesthesia Gas Monitoring 1304, Bruel and Kjaer, Naerum, Denmark) during surgery. The left femoral artery was also cannulated for monitoring the arterial blood pressure and arterial blood gases. Temperature was measured with a rectal probe and controlled by feedback to the heating lamp. The rats were kept at 37.08C F 0.58C during the ischemic period. Spinal cord ischemia was produced as described by Gilad and Gilad [12]. In brief, a balloon catheter was inserted (F2 Fogarty) through the abdominal aorta, below the kidneys, and the balloon (0.25 mL air) was inflated below the branching point of the left subclavian artery for 5 minutes, thereby blocking blood flow completely. Thereafter, the catheter was deflated and removed, and the aorta was closed, using a piece of the catheter as a bridging cannula. It was reported that longer periods of ischemia in this model may result in pulmonary edema and death within 24 hours after reperfusion. Heparin (200 U) was administrated as an intravenous bolus before aortic occlusion.
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2.4. Monitoring of physiologic parameters The cardiac pulse rate, mean arterial pressure, and rectal temperature were recorded from the monitor (Biopac MP 30 and Biopac BSL prov. 3.6.5, Biopac Systems, Santa Barbara, Calif). Partial carbon dioxide tension (Paco2), partial oxygen tension (Pao2), pH, hematocrit, and glucose were analyzed in the arterial blood samples. Arterial blood gas measurements were performed with the Radiometer ABL 700 Series (Radiometer ABL, Copenhagen, Denmark). These physiologic parameters were examined separately before starting the surgical procedure, before the aortic occlusion, and after the restoration of aortic perfusion. 2.5. Evaluation of behavioral outcome The neurologic evaluations were done in a blinded fashion. Motor performance was scored beginning at the first hour after spinal cord ischemia and then at 24 and 48 hours postoperatively. Four tests were used: (1)
(2)
(3)
(4)
Righting reflex—the ability to turn over when placed on back—was scored as: 1, normal righting; 0, weak or no righting. Placing reflex—the ability to grasp a ledge with hind limbs when suspended in air—was scored as: 1, normal placing; 0, weak or no placing. An arbitrary rating scale [12]: 4, normal walking and posture; 3, limping or mildly impaired walking with slight hind limb spasticity; 2, unilateral hind limb flaccid/spastic paralysis; 1, partial paralysis where movement occurs by bending the legs at the knees (toes are bent); 0, complete paralysis of the lower back and extremities (where movement is by dragging the lower torso). Pain response [22]: 2, withdraws from toe pinch; 1, squeals but does not withdraw; 0, no reaction.
The data from the four tests were combined to give an MSDI ranging from 0 (complete paralysis) to 8 (normal). Motor sensory deficit index was calculated for each animal at each time point.
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5 lmol/L paraffin cross sections stained with hematoxylin and eosin, by counting the motoneurons (only profiles containing nucleoli) in the ventral horn area of the representative levels of the spinal cord. Motoneurons were identified by their size ( N 25 lmol/L in diameter) and location as conveniently demonstrated in lumbar spinal cord sections taken through the enlarged ventral horn of this region. Counts of motoneurons are presented as average numbers of cells per section. 2.8. Immunohistochemistry for pCREB Five-micrometer-thick sections from the tissue blocks were placed onto poly l-lysine–coated slides. Tissue sections were deparaffinized and rehydrated before immunostaining. Antigen retrieval was performed by heating the tissue sections in a microwave oven before immunostaining. Immunohistochemistry was performed using an avidinbiotinylated enzyme complex system (Vectastain ABC Elite Kit, Vector Laboratories, Burlingame, Calif). In brief, the sections were washed in PBS (pH 7.4) for 5 minutes. They were then quenched with 0.3% H2O2 for 10 minutes to block endogenous peroxidase and washed three times for 5 minutes each in PBS. The sections were subsequently preincubated for 30 minutes in blocking serum to block nonspecific binding of antibody. Sections for detection of pCREB were incubated for 30 minutes at room temperature with the affinity-purified rabbit polyclonal antirat pCREB antibody (Upstate Biotechnology, Lake Placid, NY) at a 1:1000 dilution. This antibody is specific for pCREB in which the Ser133 residue is phosphorylated. After the primary incubation, the slides were rinsed in PBS and subsequently incubated with the biotinylated secondary antibody (antirabbit goat antibody) and 1% normal goat serum for 90 minutes. The sections were then rinsed and incubated with avidin-biotinylated peroxidase conjugate. They were finally developed in 0.02% diaminobenzidine with 0.02% H2O2. Sections were examined under
2.6. Euthanasia At 48 hours after spinal cord ischemia, the animals were deeply anesthetized (120 mg/kg BW sodium pentobarbital IP), transcardially perfused/fixed with a solution of 0.1 mol/L phosphate buffer (pH 7.4) containing 10% formalin, and the spinal cord removed and postfixed in the same solution for at least 24 hours. Thereafter, the spinal cord was cut into blocks corresponding to lumbar levels L1 and L5, and embedded in paraffin blocks. 2.7. Histopathology Five-micrometer-thick transverse sections were obtained on a microtome (Leica, Wetzlar, Germany) through the lumbar spinal cord, and sections were stained with hematoxylin and eosin. Delayed nerve cell death was measured in
Fig. 1. Therapeutic effects of EPO (1000 U/kg BW intravenous) or saline treatments on neurologic functions (combined motor and sensory score) after spinal cord ischemia. Values are mean F SD (*P b .05 when compared to control and EPO groups, **P b .05 compared to sham and control groups, ***P b .05 compared to control group).
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each group. All the rats in the groups survived the entire observation period. 3.1. Physiologic parameters The physiologic parameters (Paco2, Pao2, pH, hematocrit, glucose, and end-tidal CO2 volumes) were not significantly different between groups (data are not given here). 3.2. Neurobehavioral outcome All rats in the sham operation group had a normal postoperative neurologic outcome throughout the observation period. All EPO-treated rats had some form of flaccid paraplegia after recovery from anesthesia but began to recover motor function between 1 and 48 hours postoperatively, whereas all rats in the control group remained paraplegic throughout the observation period, with no improvement in either sensory or motor function (Fig. 1). 3.3. Histopathologic examinations Viable cell count in the ventral horn area of the spinal cord showed that the extent of ischemic damage was grossly proportional to the neurologic score. Viable cell number in the control group was significantly lower than in both sham and EPO-treated groups (20 F 2, 28 F 1, and 26 F 2, respectively). However, there was no significant difference between the viable cell number of sham and EPO-treated groups (Fig. 2). 3.4. pCREB Immunohistochemistry Fig. 2. A: Representative photomicrograph of the ventral horn region in cross sections through the spinal cord lumbar segments illustrating apoptotic motoneuron (bar = 150 lm), and quantitative data indicating the motor neuron number per section (*P b .05 vs sham and EPO; ANOVA followed by Tukey’s test) (B).
a light microscope. In the absence of primary antibody, no positive immunostaining for pCREB was observed. The number of cells exhibiting pCREB-IR was estimated from the anterior horn of spinal cord sections from L1 and L5 segmental levels for each animal. Counts of pCREB-IR cells are presented as average numbers of cells per section. All pCREB-IR–positive neurons at the anterior horn of the spinal cord were counted without considering the intensity of the staining. The pathologist who performed the examinations was blinded to the experimental conditions. 2.9. Statistical analysis All data are expressed as mean F SD. One-way ANOVA was used for data analysis, followed by the Tukey test for post hoc analysis. A P value b .005 was considered to be statistically significant. 3. Results Two rats in the EPO group and 2 rats in the control group died either during or after the operation (3 with cardiac tamponade and 1 with pulmonary edema). Those 4 rats were replaced by other rats to reach the appropriate number in
Fig. 3C summarizes the numbers of immunopositive cells in the anterior horn of spinal cord sections. The number of pCREB-IR neurons in the control and EPO-treated group was significantly increased compared to that in the shamoperated group (313 F 1, 365 F 16, and 147 F 5.6, respectively) in the anterior horn of the spinal cord. In this region, the number of immunoreactive cells in the EPOtreated group was also significantly higher than in the control group (Fig. 3A and B). 4. Discussion The major findings in the present study are as follows: (1) exogenous administration of EPO has a neuroprotective potential against ISCI in rats; (2) there are significant increases in the expression of pCREB-IR in the anterior horn of the spinal cord after ISCI in rats; (3) CREB protein phosphorylation response to ISCI was potentiated by a single dose of EPO treatment in the anterior horn of the spinal cord. Multiple models of nervous system injury (mouse, rat, gerbil, and rabbit) have been used to demonstrate the effectiveness of EPO as a neuroprotective agent, including focal and global cerebral ischemia, experimental autoimmune encephalomyelitis, kainic acid–induced seizures, experimental traumatic brain injury, neurotoxin-induced experimental parkinsonism, neonatal hypoxic-ischemic brain injury,
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Fig. 3. When compared to sham group (A), ISCI markedly increased pCREB expression in the rat spinal ventral horn (B) and intravenous administration of EPO potentiated (C) this increase in the same region. D: Quantitative data indicating the number of pCREB immunoreactive neurons per section (magnification, 200). *P b .05 vs sham; **P b .05 vs control; ANOVA followed by Tukey’s test.
subarachnoid hemorrhage, spinal cord ischemia and injury, retinal ischemia, and peripheral nerve injury [10]. Although it was recently reported that exogenous administration of EPO provides neuroprotection after spinal cord ischemic injury in rabbits, we tested the neuroprotective effects of EPO in a rat spinal ischemia model. It has been concluded that ISCI model in rats is much more convenient than spinal cord injury model in rabbits because of two reasons: (1) rabbit spinal arterial vascularization differs from that in humans, whereas arterial vascularization is almost similar in human and rat; (2) evaluation of the model in rabbits has been found compromising owing to the limited behavioral repertoire of rabbits, which precludes sophisticated scoring and neurologic assessment [1,3,19,20]. However, results of our spinal ischemia model in rats to evaluate the effectiveness of EPO against ischemic insult support the findings of a previous study on rabbit. The discovery of the production of EPO by neuronal cells in man was made only 15 years ago, and attention has been focused on the neurotrophic and neuroprotective function of EPO in in vivo and in vitro conditions of neuronal damage. Although the mechanisms of EPO that produce neuroprotective effects are not identified yet, it has been concluded that EPO may act in a coordinated fashion at multiple levels, including limiting the production of tissue-
injuring molecules such as reactive oxygen species and glutamate, reversal of vasospasm, stimulation of angiogenesis, attenuation of apoptosis, modulation of inflammation, and recruitment of stem cells, and, thus, may protect neurons by a combination of these mechanisms [10]. CREB is a basic leucine zipper family transcription factor that plays a key role in neuronal development, function, and survival, and its transcriptional activity is upregulated in response to many cellular stimuli, including cAMP, Ca2+, hypoxia, ultraviolet light, and growth factors such as BDNF and IGF-1 [9,17,28,29,34,42]. The canonical CREB activation pathway involves stimulus-induced phosphorylation of CREB on Ser133 by several kinds of protein kinases, such as protein kinase A, Ca2+/calmodulin-dependent kinase IV, and ribosomal S6 kinase 2 to facilitate the transcription of genes with the CRE motif [39,41]. CREB is a bona fide neuron survival factor, as Ser133 pCREB can activate the relative gene transcription to further promote protein expression such as c-Fos, Jun-B, Bcl-2, and neurotrophins. These gene products regulate neuronal regeneration, survival, repair, and protection during the case of nervous system stress injury, such as hypoxia [23,34,35,39]. Not only has CREB phosphorylation been implicated in the resistance of cells to various insults, but a number of well-established neuroprotective agents exert their actions
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via pathways that converge on the CREB protein. In the present study, the neuroprotective effects of EPO treatment have also been studied in terms of changes in CREB protein phosphorylation after ischemic injury. It was previously reported that intracellular Ca2+ increase is one of the early events triggered by EPO to be critically involved in neuroprotection [21,27]. In addition, Viviani et al [37] demonstrated that EPO activates the CREB transcription pathway and increases BDNF expression and production, which contributes to EPO-mediated neuroprotection in primary hippocampal neurons. Our findings suggest that CREB protein phosphorylation response to ISCI was potentiated by EPO treatment in the anterior horn of the spinal cord. Potentiation of CREB phosphorylation observed in this region appears to be compatible with the in vitro findings in primary hippocampal neurons. Taken together, all these results support the idea that EPO acts in the nervous system as a direct protective factor against ischemic insult in neurons via activation of CREB protein phosphorylation. And CREB-protein targeting by using EPO might be an ideal way to achieve neuroprotective effects, a strategy that will have major implications for preventing ISCI. References [1] Ahn S, Ginty DD, Linden DJ. A late phase of cerebellar long-term depression requires activation of CaMKIV and CREB. Neuron 1999; 23(3):414 - 5. [2] Bonni A, Brunet A, West AE, Datta SR, Takasu MA, Greenberg ME. Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and independent mechanisms. Science 1999; 12(286):1358 - 62. [3] Celik M, Gokmen N, Erbayraktar S, Akhisaroglu M, Konakc S, Ulukus C, Genc S, Genc K, Sagiroglu E, Cerami A, Brines M. Erythropoietin prevents motor neuron apoptosis and neurologic disability in experimental spinal cord ischemic injury. Proc Natl Acad Sci U S A 2002;99(4):2258 - 63. [4] Coselli JS, LeMaire SA, de Figueiredo LP, Kirby RP. Paraplegia after thoracoabdominal aortic aneurysm repair: is dissection a risk factor? Ann Thorac Surg 1997;63(1):28 - 36. [5] Crossthwaite AJ, Hasan S, Williams RJ. Hydrogen peroxide mediated phosphorylation of ERK1/2, Akt/PKB and JNK in cortical neurones: dependence on Ca (2+) and PI3-kinase. J Neurochem 2002;80:24 - 35. [6] De Cesare D, Fimia GM, Sassone-Corsi P. Signaling routes to CREM and CREB: plasticity in transcriptional activation. Trends Biochem Sci 1999;24:281 - 5. [7] Du K, Montminy M. CREB is a regulatory target for the protein kinase Akt/PKB. J Biol Chem 1998;273:32377 - 9. [8] Finkbeiner S, Tavazoie SF, Maloratsky A, Jacobs KM, Harris KM, Greenberg ME. CREB: a major mediator of neuronal neurotrophin responses. Neuron 1997;19:1031 - 47. [9] Gao Y, Gao G, Long C, Han S, Zu P, Fang L, Li J. Enhanced phosphorylation of cyclic AMP response element binding protein in the brain of mice following repetitive hypoxic exposure. Biochem Biophys Res Commun 2006;340(2):661 - 7. [10] Genc S, Koroglu TF, Genc K. Erythropoietin and the nervous system. Brain Res 2004;1000(1-2):19 - 31. [11] Gharagozloo F, Larson J, Dausmann MJ, Neville RF, Gomes MN. Spinal cord protection during surgical procedures on the descending thoracic and thoracoabdominal aorta: review of current techniques. Chest 1996;109:799 - 809.
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effects of EPO in a rat model of spinal cord ischemia. This was caused by occluding the aorta for 5 minutes. Subsequently, EPO was administered intravenously. Forty-eight hours later, the animals were sacrificed. Erythropoietin improved the clinical outcome, and this result corresponded with a significantly higher number of motoneurons and pCREB cells in the spinal cord. CREB should be of crucial importance in cell survival and repair. Erythropoietin enhances the activation of CREB by means of phosphorylation. These results add another brick to the building of EPO, a very interesting and important hematopoietic cytokine hormone. This molecule is involved in several beneficial mechanisms in the body. In the central nervous system, EPO promotes the expression of several growth factors and would be involved in neurogenesis and angiogenesis. The role of EPO in neuroprotection is further confirmed by the results of this study. Therefore, EPO could be useful, at least for attenuating the eventual consequences on the spinal cord of aorta clamping during aortic aneurysm surgery. Eduardo Fernandez, MD Istituto di Neurochirurgia Universita` Cattolica del Sacro Cuore 00168 Rome, Italy
This is a relatively straightforward study, demonstrating a neuroprotective effect of EPO in a model of spinal cord ischemia in the rat. It joins a growing number of papers demonstrating this effect. The authors wanted to shed light on the mechanism of action of EPO, by staining for the phosphorylated form of the transcription factor CREB. The study is small, using a small number of animals and a limited number of parameters—only pCREB immunohistochemistry is performed. This limits the ability to connect the dots between the administration of EPO and the change in pCREB. Nevertheless, this is a welcome contribution, encouraging further study into the mechanism of action of EPO.
Commentary It is well known that clamping of the aorta during surgery for aortic aneurysms can cause spinal cord ischemia. The authors of the present study demonstrated some positive
Ben Roitberg, MD Department of Neurosurgery University of Illinois at Chicago Chicago, IL 60612, USA