- Email: [email protected]
Neuroprotective effect of erythropoietin after experimental cold injury–induced vasogenic brain edema in rats
Available online at www.sciencedirect.com
Surgical Neurology 70 (2008) 498 – 502 www.surgicalneurology-online.com
Trauma
Neuroprotective effect of erythropoietin after experimental cold injury–induced vasogenic brain edema in rats Ozerk Okutan, MD a,⁎, Omer Faruk Turkoglu, MD b , Hayriye Beril Gok, MD a , Etem Beskonakli, MD, PhD a a
Department of Neurosurgery, Ankara Ataturk Research and Education Hospital, Ankara, Turkey Department of Neurosurgery, Ankara Numune Research and Education Hospital, Ankara, Turkey Received 5 February 2007; accepted 18 July 2007
b
Abstract
Background: The aims of this study were to evaluate the efficiency of EPO in the treatment of cold injury–induced brain edema, apoptosis, and inflammation and to compare its effectiveness with DSP. Methods: One hundred fifteen adult male Sprague-Dawley rats weighing between 280 and 300 g were used for the study. Rats were divided into 5 groups. Controls received craniotomy only. The injury group underwent cold injury and had no medication. In the EPO group, a single dose of 1000 IU/kg body weight of EPO was administered. The DSP group received 0.2 mg/kg body weight of DSP. The vehicle group received a vehicle solution containing human serum albumin, which is the solvent for EPO. Brain edema was formed by cold injury using metal sterile rods with a diameter of 4 mm that were previously cooled at −80°C. Twenty-four hours after the injury, animals were decapitated and brain tissues were investigated for brain edema, tissue MPO and caspase-3 levels, and ultrastructure. Results: A significant increase in brain water content was revealed in injury group of rats at 24 hours after cold injury. Injury significantly increased tissue MPO and caspase-3 levels and resulted in ultrastructural damage. Both EPO and DSP markedly decreased tissue MPO and caspase-3 levels and preserved ultrastructure of the injured brain cortex. Conclusions: Erythropoietin and DSP were found to be neuroprotective in cold injury–induced brain edema model in rats via anti-apoptotic and anti-inflammatory actions. © 2008 Elsevier Inc. All rights reserved.
Keywords:
Apoptosis; Blood-brain barrier; Caspase; Erythropoietin; Inflammation; Myeloperoxidase; Neuroprotection
1. Introduction Erythropoietin, a member of the hematopoietic growth factor family, mainly produced by the kidney stimulates erythropoiesis in response to hypoxia. However, there is a growing body of evidence from experimental studies suggesting that EPO also has important nonhematopoietic functions in the CNS. Systemic administration of EPO as a pretreatment or posttreatment has been reported to be Abbreviations: BBB, blood-brain barrier; CNS, central nervous system; EM, electron microscopy; EPO, erythropoietin; DSP, dexamethasone sodium phosphate; MPO, myeloperoxidase; OsO4, osmium tetroxide; TMB, tetramethylbenzidine. ⁎ Corresponding author. Tel.: +90 505 5657239. E-mail address: [email protected] (O. Okutan). 0090-3019/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.surneu.2007.07.061
neuroprotective in different forms of CNS injury, including traumatic brain injury [1], subarachnoid hemorrhage [4], intrauterine hypoxic/ischemic brain injury [12], and spinal cord contusion injury [5,9]. Neuroprotective properties of EPO mainly attributed to its antioxidative [5,12], antiapoptotic [9], anti-inflammatory actions [9], and stimulation of neurogenesis and angiogenesis [17]. Increased BBB permeability plays a role in the pathogenesis of various CNS injuries [14,18]. Vasogenic brain edema due to increased BBB permeability is associated with a wide spectrum of neurologic disorders and also is a common problem in neurosurgical practice [10]. A potential protective role for EPO in experimental vasogenic brain edema model has not been addressed before. Therefore, the present study was designed to study whether EPO decreases
O. Okutan et al. / Surgical Neurology 70 (2008) 498–502
vasogenic brain edema, and has anti-apoptotic, anti-inflammatory properties in a rat model of vasogenic brain edema. We also compared the effect of EPO with DSP, the latter of which has been used widely as a therapeutic agent for the treatment of vasogenic brain edema in clinical practice. 2. Materials and methods 2.1. Experimental animals The experimental protocol was approved by the Local Animal Care and Use Committee of Ankara Numune Research and Education Hospital (Ankara, Turkey). One hundred fifteen adult male Sprague-Dawley rats weighing between 280 and 300 g were divided randomly into 5 groups: control (sham), injury, EPO, DSP, and vehicle groups. 2.2. Cold injury model Rats were subjected to cold injury as described previously [8,15]. General anesthesia was induced with ketamine (80 mg/ kg IP) and xylazine hydrochloride (8 mg/kg IP). All of the animals in each group were fixed using a stereotaxic frame before craniectomy. The scalp was incised on the midline, and the skull was exposed. A craniectomy of 5 × 5 mm was performed in the left parietal region between coronary and lambdoid sutures, and the dura was kept intact. A metal sterile rod with a diameter of 4 mm cooled at −80°C was applied to the surface of the parietal cortex. The metal probes weighing 10 g were kept in contact with parietal cortex for 1 minute. The rats were allowed to recover after incision closure and housed individually until euthanization. All animals had free access to food and water. Twenty-four hours after the injury, the animals were killed and the brains were rapidly removed. 2.3. Administration of drugs The EPO group received a single dose of 1000 IU/kg IP of EPO (CILAG AG, Zug, Switzerland) immediately after cold injury. The vehicle group received the same volume of human albumin. The DSP group received 0.2 mg/kg IP of DSP (Deva Drug Company, Istanbul, Turkey) immediately after cold injury. 2.4. Brain water content Twenty-four hours after the injury, the brains (n = 7 for each group) were removed and immediately separated into right and left hemispheres. The hemispheres were weighed after removal (wet weight) and again after drying in an oven at 105°C for 24 hours as described by Xi et al [18]. The percentage of water content was calculated as [(wet weight − dry weight)/wet weight] ×100%. 2.5. Determination of MPO activity Rats (n = 7 for each group) were deeply anesthetized with ketamine 24 hours after cold injury and then decapitated. The brains were rapidly removed, and the ipsilateral brain cortices were separated and flash frozen in liquid nitrogen.
499
Brain-associated myeloperoxidase (MPO) activity was determined as described previously [9]. Frozen tissue samples were weighed and homogenized in 1:10 (wt/vol) ice-cold 10 mmol TRIS buffer (pH 7.4) by the use of a dounce homogenizer. The homogenate (1 mL) was centrifuged at 5000g for 5 minutes, and the pellet was resuspended in equal volumes (1 mL) of 50 mmol phosphate buffer (pH 6.0) containing 0.5% hexadecyltrimethyl ammonium bromide and 5 mmol EDTA. The resulting suspension was centrifuged at 5000g for 2 minutes, and the supernatant was used for the activity measurement. Myeloperoxidase activity was measured in a final volume of 1 mL containing 80 mmol phosphate buffer (pH 5.4), 0.5% hexadecyltrimethyl ammonium bromide, 1.6 mmol synthetic substrate TMB initially dissolved in dimethylformamide, 2 mmol H2O2, and the sample. The reaction was started at 37°C by the addition of H2O2. The initial rate of MPO-catalyzed TMB oxidation was followed by recording the increase of absorbance at 655 nm. Myeloperoxidase activity was expressed as the amount of the enzyme producing one absorbance change per minute under assay conditions. Tissue-associated MPO activity was calculated as units per gram of wet tissue. 2.6. Determination of caspase-3 activity Caspase-3 activity of tissue samples was measured by the use of EnzChek caspase-3 kit no. 1 produced by Molecular Probes (Invitrogen, Carlsbad, Calif). Tissues were homogenized in 10 mmol ice-cold TRIS buffer (pH 7.4) containing 100 mmol NaCl, 2 mmol EDTA, and 0.02% Triton X-100. Tissue homogenates were centrifuged at 5000 rpm for 5 minutes in a microcentrifuge, and the supernatant was used for the measurement of caspase-3 activity. In a final volume of 1 mL, 200 μL of supernatant was incubated for 30 minutes at room temperature in the working solution containing synthetic caspase-3 substrate Z-DEVD-AMC. The fluorescence of samples was measured using excitation at 342 nm and emission at 441 nm in a standard fluorometer. Because the procedure is designed for use with a fluorescence microplate reader, we increased volumes accordingly as recommended by the manufacturer. 7-Amino-4-methylcoumarine supplied in the kit was used as a reference standard. Caspase-3 activity was calculated as micromoles per gram of wet tissue. 2.7. Sample preparation for EM Rats (n = 2 for each group) were deeply anesthetized with ketamine 24 hours after cold injury and then decapitated for the EM studies. The tissues used for transmission electron microscopy were obtained from the injury site. They were kept in the phosphate-buffered 2.5% glutaraldehyde and 2% paraformaldehyde solution for 24 hours, postfixed with phosphate-buffered 2% OsO4 for 1 hour, and dehydrated in a graded series of alcohol. After araldite embedding, 1- to 2-μm semithin sections were obtained with an LKB NOVA ultratome, stained with toluidine blue, and pictured with a Nikon Ophtipod light microscope (Tokyo, Japan). The same
500
O. Okutan et al. / Surgical Neurology 70 (2008) 498–502
ultratome was used to obtain 60- to 90-nm-thick sections that were contrasted with uranyl acetate and lead citrate and pictured with a JOEL JEM 1200 electron microscope (Tokyo, Japan). A single physician performed all the evaluations in a blinded manner. 2.8. Statistical analysis All the data collected from the experiment were coded, recorded, and analyzed using SPSS 10.0.1 for Windows (SPSS Inc, Chicago, Ill). The 1-way analysis of variance for parametric data and Mann-Whitney U tests were used for comparing differences among the groups. When analysis of variance showed a significant difference, the post hoc multiple comparison test was applied to demonstrate the differences. In each test, the data were expressed as the mean ± SD, and P b .05 was accepted as statistically significant. 3. Results 3.1. Blood-brain barrier disruption and brain edema A significant increase in brain water content was revealed in the injury group of rats at 24 hours after cold injury compared with the control group of rats in the left hemisphere. The mean water content of the left hemisphere was 78% ± 0.8% in the control group and 82.5% ± 1.2% in the injury group (Fig. 1; *P b .05 vs control). The mean water content of the left hemisphere was 80.5% ± 0.7%, 79.9% ± 1%, and 81.9% ± 0.6% for the EPO, DSP, and vehicle groups, respectively (Fig. 1). Both EPO and DSP treatments significantly reduced brain water content after injury (†P b .05 vs injury). The vehicle group did not show a difference from the injury group (P N .05 vs injury). No statistical difference among all groups in water content of right hemisphere was observed (data not shown). 3.2. Tissue MPO activity A significant increase in MPO activity in the left brain cortex was revealed in the injury group 24 hours after the cold injury when compared with the control group in the left
Fig. 1. Quantification of brain water content in the ipsilateral brain hemisphere 24 hours after cold injury. Compared with control animals, brain water content was markedly increased in the injury group. Both EPO and DSP groups had significantly decreased ipsilateral hemisphere water content compared to the injury group (*P b .05 vs control and †P b .05 vs injury). There was no statistical difference among the groups for contralateral hemisphere water content (data not shown) (n = 7 for each group).
Fig. 2. Tissue MPO activity levels in the ipsilateral brain cortex at 24 hours after cold injury. A significant increase in MPO activity in the left brain cortex was revealed in the injury group 24 hours after the cold injury when compared with the control group (*P b .05 vs control). Both EPO and DSP treatments prevented an increase in MPO levels (†P b .05 vs injury), although the MPO levels after both treatments were significantly higher than those in the control samples. The vehicle group did not show a difference from the injury group (P N .05 vs injury) (n = 7 for each group).
brain cortex (*P b .05 vs control), indicating neutrophil activation in the injured brain cortex (Fig. 2). Both EPO and DSP treatments prevented increase in MPO levels (†P b .05 vs injury), although the MPO levels after both treatments were significantly higher than control samples. The vehicle group did not show a difference from the injury group (P N .05 vs injury). 3.3. Tissue caspase-3 activity The injury rats showed a massive activation of caspase-3 in the injured cortex 24 hours after cold injury (*P b .05 vs control) (Fig. 3). Both EPO and DSP treatments reduced caspase-3 activation drastically at 24 hours (†P b .05 vs injury). The vehicle group did not show a difference from the injury group (P N .05 vs injury). 3.4. Ultrastructural findings The cold injury produced obvious ultrastructural damage as shown in Fig. 4. The injury group showed severe injury with extensive edematous spaces. The myelin sheaths of the
Fig. 3. Tissue caspase-3 activity levels in the ipsilateral brain cortex at 24 hours after cold injury. The injury rats showed a massive activation of caspase-3 in the injured cortex (*P b .05 vs control). Both EPO and DSP treatments reduced caspase-3 activation drastically at 24 hours (†P b .05 vs injury). The vehicle group did not show a difference from the injury group (P N .05 vs injury) (n = 7 for each group).
O. Okutan et al. / Surgical Neurology 70 (2008) 498–502
501
Fig. 4. Electron microscopic evaluation of cortical ultrastructure at 24 hours after cold injury. The injury group showed severe injury with extensive edematous spaces at 24 hours after cold injury. The myelin sheaths of the axons were destroyed. In the EPO (1000 IU/kg) treatment group, the ultrastructure was very well protected. The ultrastructure was normal except for some limited edematous spaces. Treatment with 0.2 mg/kg DSP also showed obvious ultrastructural protection. However, the EPO group showed better results than the DSP group (original magnification ×7500).
axons were destroyed. In the EPO (1000 IU/kg) treatment group, the ultrastructure was very well protected. The neuronal bodies and axonal structures were normal except for some limited edematous spaces. Treatment with 0.2 mg/kg DSP also showed obvious ultrastructural protection. However, the EPO group showed better results compared to the DSP group. 4. Discussion It is well established that an increase in the permeability of BBB due to endothelial cell dysfunction results in brain edema in a wide spectrum of neurologic disorders [8,10,14]. Severe brain edema causes not only brain herniation but also increases intracranial pressure and causes a decrease of cerebral blood perfusion, resulting in secondary ischemia and cell death [8]. Hence, development of new treatment strategies for brain edema is important to decrease mortality and morbidity in clinical practice. Cold injury has been reported to be a useful model for investigating the mechanism of CNS injury followed by vasogenic brain edema. It is has been demonstrated that both apoptosis and necrosis contribute to cellular damage after cold injury [8]. Apoptosis is regulated by an intracellular proteolytic cascade, mediated by the caspase family. Results from the present study clearly showed that caspase-3 activation occurs in the ipsilateral brain cortex at 24 hours after cold injury, indicating apoptotic cell death that was consistent with previous observations [8]. However, administration of EPO inhibited caspase-3 activation and significantly reduced BBB permeability, which is followed by decreased brain water content (Fig. 1). These results suggest that EPO protects brain against cold injury via preserving endothelial integrity and attenuating apoptotic cell death. Because BBB maintains the homeostasis of the brain microenvironment, preserving endothelial integrity is also crucial for neuronal activity and function [13]. Previous studies showed that neurovascular protection is an important target to prevent or reduce neuronal cell death [14]. Both in vivo and in vitro experimental models have demonstrated neuronal and vascular protection in the CNS by EPO [6]. It has been shown that EPO exerts vascular protection by
preserving endothelial cell integrity and maintains the establishment of cell-to-cell junctions [2,7]. Apoptosis, implicated in endothelial cell death after cerebral ischemia, may lead not only to increased BBB permeability but also to increased infiltration of proinflammatory cells into the injured tissue [3,19]. In the present study, there was a significant increase in MPO activity and brain edema, both of which indicate increased permeability of BBB and infiltration of proinflammatory cells into the injured tissue after cold injury. However, exogenous administration of EPO decreased tissue MPO activity significantly. It has been shown that mononuclear cell infiltration is reduced in EPO-treated animals with traumatic brain injury [1]. Erythropoietin may reduce the inflammatory response by decreasing cell death and/or by preventing the release of proinflammatory cytokines such as tumor necrosis factor and interleukin 6 [11,16]. Despite recent arguments on the effectiveness and complications of DSP, many clinics still use it for human vasogenic brain edema. However, despite its cytoprotective attributes, EPO therapy may also have clinical toxicity [6]. In the present study, we compared the effectiveness of EPO against DSP. Dexamethasone sodium phosphate treatment prevented the increase in caspase-3 and MPO activity to a similar degree as EPO; however, better ultrastructural preservation was observed in the EPO-treated group.
5. Conclusions Administration of EPO in the cold injury–induced edema model was found to be neuroprotective by anti-inflammatory and anti-apoptotic mechanisms. However, different time points, different doses, and application routes should be tested to confirm results and to show longer-term outcome. Further research will explore the potential use of EPO for vasogenic brain edema in humans. Acknowledgments We would like to thank Hakan Eroglu, MSc, for his kind help with the statistical analysis. We also thank Dr Ihsan Solaroglu for his valuable comments and careful editing of the manuscript.
502
O. Okutan et al. / Surgical Neurology 70 (2008) 498–502
References [1] Brines ML, Ghezzi P, Keenan S, et al. Erythropoietin crosses the blood-brain barrier to protect against experimental brain injury. Proc Natl Acad Sci U S A 2000;97:10526-31. [2] Chong ZZ, Kang JQ, Maiese K. Apaf-1, Bcl-xL, cytochrome c, and caspase-9 form the critical elements for cerebral vascular protection by erythropoietin. J Cereb Blood Flow Metab 2003;23:320-30. [3] del Zoppo GJ, Mabuchi T. Cerebral microvessel responses to focal ischemia. J Cereb Blood Flow Metab 2003;23:879-94. [4] Grasso G. Neuroprotective effect of recombinant human erythropoietin in experimental subarachnoid hemorrhage. J Neurosurg Sci 2001;45: 7-14. [5] Kaptanoglu E, Solaroglu I, Okutan O, et al. Erythropoietin exerts neuroprotection after acute spinal cord injury in rats: effect on lipid peroxidation and early ultrastructural findings. Neurosurg Rev 2004; 27:113-20. [6] Maiese K, Li F, Chong ZZ. New avenues of exploration for erythropoietin. JAMA 2005;293:90-5. [7] Martinez-Estrada OM, Rodriguez-Millan E, Gonzalez-De Vicente E, et al. Erythropoietin protects the in vitro blood-brain barrier against VEGF-induced permeability. Eur J Neurosci 2003;18:2538-44. [8] Murakami K, Kondo T, Yang G, et al. Cold injury in mice: a model to study mechanisms of brain edema and neuronal apoptosis. Prog Neurobiol 1999;57:289-99. [9] Okutan O, Solaroglu I, Beskonakli E, et al. Recombinant human erythropoietin decreases myeloperoxidase and caspase-3 activity and improves early functional results after spinal cord injury in rats. J Clin Neurosci 2007; doi:10.1016/j.jocn.2006.01.022.
[10] Rabinstein AA. Treatment of cerebral edema. Neurologist 2006;12: 59-73. [11] Siren AL, Fratelli M, Brines M, et al. Erythropoietin prevents neuronal apoptosis after cerebral ischemia and metabolic stress. Proc Natl Acad Sci U S A 2001;98:4044-9. [12] Solaroglu I, Solaroglu A, Kaptanoglu E, et al. Erythropoietin prevents ischemia-reperfusion from inducing oxidative damage in fetal rat brain. Childs Nerv Syst 2003;19:19-22. [13] Stanness KA, Westrum LE, Fornaciari E, et al. Morphological and functional characterization of an in vitro blood-brain barrier model. Brain Res 1997;771:329-42. [14] Tsubokawa T, Yamaguchi-Okada M, Calvert JW, et al. Neurovascular and neuronal protection by E64d after focal cerebral ischemia in rats. J Neurosci Res 2006;84:832-40. [15] Turkoglu OF, Eroglu H, Okutan O, et al. The efficiency of dexamethasone sodium phosphate–encapsulated chitosan microspheres after cold injury. Surg Neurol 2005;64(Suppl 2):S11-6. [16] Villa P, Bigini P, Mennini T, et al. Erythropoietin selectively attenuates cytokine production and inflammation in cerebral ischemia by targeting neuronal apoptosis. J Exp Med 2003;198:971-5. [17] Wang L, Zhang Z, Wang Y, et al. Treatment of stroke with erythropoietin enhances neurogenesis and angiogenesis and improves neurological function in rats. Stroke 2004;35:1732-7. [18] Xi G, Hua Y, Keep RF, et al. Brain edema after intracerebral hemorrhage: the effects of systemic complement depletion. Acta Neurochir Suppl 2002;81:253-6. [19] Zhang Y, Zhang X, Park TS, et al. Cerebral endothelial cell apoptosis after ischemia-reperfusion: role of PARP activation and AIF translocation. J Cereb Blood Flow Metab 2005;25:868-77.
Surgical Neurology 70 (2008) 498 – 502 www.surgicalneurology-online.com
Trauma
Neuroprotective effect of erythropoietin after experimental cold injury–induced vasogenic brain edema in rats Ozerk Okutan, MD a,⁎, Omer Faruk Turkoglu, MD b , Hayriye Beril Gok, MD a , Etem Beskonakli, MD, PhD a a
Department of Neurosurgery, Ankara Ataturk Research and Education Hospital, Ankara, Turkey Department of Neurosurgery, Ankara Numune Research and Education Hospital, Ankara, Turkey Received 5 February 2007; accepted 18 July 2007
b
Abstract
Background: The aims of this study were to evaluate the efficiency of EPO in the treatment of cold injury–induced brain edema, apoptosis, and inflammation and to compare its effectiveness with DSP. Methods: One hundred fifteen adult male Sprague-Dawley rats weighing between 280 and 300 g were used for the study. Rats were divided into 5 groups. Controls received craniotomy only. The injury group underwent cold injury and had no medication. In the EPO group, a single dose of 1000 IU/kg body weight of EPO was administered. The DSP group received 0.2 mg/kg body weight of DSP. The vehicle group received a vehicle solution containing human serum albumin, which is the solvent for EPO. Brain edema was formed by cold injury using metal sterile rods with a diameter of 4 mm that were previously cooled at −80°C. Twenty-four hours after the injury, animals were decapitated and brain tissues were investigated for brain edema, tissue MPO and caspase-3 levels, and ultrastructure. Results: A significant increase in brain water content was revealed in injury group of rats at 24 hours after cold injury. Injury significantly increased tissue MPO and caspase-3 levels and resulted in ultrastructural damage. Both EPO and DSP markedly decreased tissue MPO and caspase-3 levels and preserved ultrastructure of the injured brain cortex. Conclusions: Erythropoietin and DSP were found to be neuroprotective in cold injury–induced brain edema model in rats via anti-apoptotic and anti-inflammatory actions. © 2008 Elsevier Inc. All rights reserved.
Keywords:
Apoptosis; Blood-brain barrier; Caspase; Erythropoietin; Inflammation; Myeloperoxidase; Neuroprotection
1. Introduction Erythropoietin, a member of the hematopoietic growth factor family, mainly produced by the kidney stimulates erythropoiesis in response to hypoxia. However, there is a growing body of evidence from experimental studies suggesting that EPO also has important nonhematopoietic functions in the CNS. Systemic administration of EPO as a pretreatment or posttreatment has been reported to be Abbreviations: BBB, blood-brain barrier; CNS, central nervous system; EM, electron microscopy; EPO, erythropoietin; DSP, dexamethasone sodium phosphate; MPO, myeloperoxidase; OsO4, osmium tetroxide; TMB, tetramethylbenzidine. ⁎ Corresponding author. Tel.: +90 505 5657239. E-mail address: [email protected] (O. Okutan). 0090-3019/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.surneu.2007.07.061
neuroprotective in different forms of CNS injury, including traumatic brain injury [1], subarachnoid hemorrhage [4], intrauterine hypoxic/ischemic brain injury [12], and spinal cord contusion injury [5,9]. Neuroprotective properties of EPO mainly attributed to its antioxidative [5,12], antiapoptotic [9], anti-inflammatory actions [9], and stimulation of neurogenesis and angiogenesis [17]. Increased BBB permeability plays a role in the pathogenesis of various CNS injuries [14,18]. Vasogenic brain edema due to increased BBB permeability is associated with a wide spectrum of neurologic disorders and also is a common problem in neurosurgical practice [10]. A potential protective role for EPO in experimental vasogenic brain edema model has not been addressed before. Therefore, the present study was designed to study whether EPO decreases
O. Okutan et al. / Surgical Neurology 70 (2008) 498–502
vasogenic brain edema, and has anti-apoptotic, anti-inflammatory properties in a rat model of vasogenic brain edema. We also compared the effect of EPO with DSP, the latter of which has been used widely as a therapeutic agent for the treatment of vasogenic brain edema in clinical practice. 2. Materials and methods 2.1. Experimental animals The experimental protocol was approved by the Local Animal Care and Use Committee of Ankara Numune Research and Education Hospital (Ankara, Turkey). One hundred fifteen adult male Sprague-Dawley rats weighing between 280 and 300 g were divided randomly into 5 groups: control (sham), injury, EPO, DSP, and vehicle groups. 2.2. Cold injury model Rats were subjected to cold injury as described previously [8,15]. General anesthesia was induced with ketamine (80 mg/ kg IP) and xylazine hydrochloride (8 mg/kg IP). All of the animals in each group were fixed using a stereotaxic frame before craniectomy. The scalp was incised on the midline, and the skull was exposed. A craniectomy of 5 × 5 mm was performed in the left parietal region between coronary and lambdoid sutures, and the dura was kept intact. A metal sterile rod with a diameter of 4 mm cooled at −80°C was applied to the surface of the parietal cortex. The metal probes weighing 10 g were kept in contact with parietal cortex for 1 minute. The rats were allowed to recover after incision closure and housed individually until euthanization. All animals had free access to food and water. Twenty-four hours after the injury, the animals were killed and the brains were rapidly removed. 2.3. Administration of drugs The EPO group received a single dose of 1000 IU/kg IP of EPO (CILAG AG, Zug, Switzerland) immediately after cold injury. The vehicle group received the same volume of human albumin. The DSP group received 0.2 mg/kg IP of DSP (Deva Drug Company, Istanbul, Turkey) immediately after cold injury. 2.4. Brain water content Twenty-four hours after the injury, the brains (n = 7 for each group) were removed and immediately separated into right and left hemispheres. The hemispheres were weighed after removal (wet weight) and again after drying in an oven at 105°C for 24 hours as described by Xi et al [18]. The percentage of water content was calculated as [(wet weight − dry weight)/wet weight] ×100%. 2.5. Determination of MPO activity Rats (n = 7 for each group) were deeply anesthetized with ketamine 24 hours after cold injury and then decapitated. The brains were rapidly removed, and the ipsilateral brain cortices were separated and flash frozen in liquid nitrogen.
499
Brain-associated myeloperoxidase (MPO) activity was determined as described previously [9]. Frozen tissue samples were weighed and homogenized in 1:10 (wt/vol) ice-cold 10 mmol TRIS buffer (pH 7.4) by the use of a dounce homogenizer. The homogenate (1 mL) was centrifuged at 5000g for 5 minutes, and the pellet was resuspended in equal volumes (1 mL) of 50 mmol phosphate buffer (pH 6.0) containing 0.5% hexadecyltrimethyl ammonium bromide and 5 mmol EDTA. The resulting suspension was centrifuged at 5000g for 2 minutes, and the supernatant was used for the activity measurement. Myeloperoxidase activity was measured in a final volume of 1 mL containing 80 mmol phosphate buffer (pH 5.4), 0.5% hexadecyltrimethyl ammonium bromide, 1.6 mmol synthetic substrate TMB initially dissolved in dimethylformamide, 2 mmol H2O2, and the sample. The reaction was started at 37°C by the addition of H2O2. The initial rate of MPO-catalyzed TMB oxidation was followed by recording the increase of absorbance at 655 nm. Myeloperoxidase activity was expressed as the amount of the enzyme producing one absorbance change per minute under assay conditions. Tissue-associated MPO activity was calculated as units per gram of wet tissue. 2.6. Determination of caspase-3 activity Caspase-3 activity of tissue samples was measured by the use of EnzChek caspase-3 kit no. 1 produced by Molecular Probes (Invitrogen, Carlsbad, Calif). Tissues were homogenized in 10 mmol ice-cold TRIS buffer (pH 7.4) containing 100 mmol NaCl, 2 mmol EDTA, and 0.02% Triton X-100. Tissue homogenates were centrifuged at 5000 rpm for 5 minutes in a microcentrifuge, and the supernatant was used for the measurement of caspase-3 activity. In a final volume of 1 mL, 200 μL of supernatant was incubated for 30 minutes at room temperature in the working solution containing synthetic caspase-3 substrate Z-DEVD-AMC. The fluorescence of samples was measured using excitation at 342 nm and emission at 441 nm in a standard fluorometer. Because the procedure is designed for use with a fluorescence microplate reader, we increased volumes accordingly as recommended by the manufacturer. 7-Amino-4-methylcoumarine supplied in the kit was used as a reference standard. Caspase-3 activity was calculated as micromoles per gram of wet tissue. 2.7. Sample preparation for EM Rats (n = 2 for each group) were deeply anesthetized with ketamine 24 hours after cold injury and then decapitated for the EM studies. The tissues used for transmission electron microscopy were obtained from the injury site. They were kept in the phosphate-buffered 2.5% glutaraldehyde and 2% paraformaldehyde solution for 24 hours, postfixed with phosphate-buffered 2% OsO4 for 1 hour, and dehydrated in a graded series of alcohol. After araldite embedding, 1- to 2-μm semithin sections were obtained with an LKB NOVA ultratome, stained with toluidine blue, and pictured with a Nikon Ophtipod light microscope (Tokyo, Japan). The same
500
O. Okutan et al. / Surgical Neurology 70 (2008) 498–502
ultratome was used to obtain 60- to 90-nm-thick sections that were contrasted with uranyl acetate and lead citrate and pictured with a JOEL JEM 1200 electron microscope (Tokyo, Japan). A single physician performed all the evaluations in a blinded manner. 2.8. Statistical analysis All the data collected from the experiment were coded, recorded, and analyzed using SPSS 10.0.1 for Windows (SPSS Inc, Chicago, Ill). The 1-way analysis of variance for parametric data and Mann-Whitney U tests were used for comparing differences among the groups. When analysis of variance showed a significant difference, the post hoc multiple comparison test was applied to demonstrate the differences. In each test, the data were expressed as the mean ± SD, and P b .05 was accepted as statistically significant. 3. Results 3.1. Blood-brain barrier disruption and brain edema A significant increase in brain water content was revealed in the injury group of rats at 24 hours after cold injury compared with the control group of rats in the left hemisphere. The mean water content of the left hemisphere was 78% ± 0.8% in the control group and 82.5% ± 1.2% in the injury group (Fig. 1; *P b .05 vs control). The mean water content of the left hemisphere was 80.5% ± 0.7%, 79.9% ± 1%, and 81.9% ± 0.6% for the EPO, DSP, and vehicle groups, respectively (Fig. 1). Both EPO and DSP treatments significantly reduced brain water content after injury (†P b .05 vs injury). The vehicle group did not show a difference from the injury group (P N .05 vs injury). No statistical difference among all groups in water content of right hemisphere was observed (data not shown). 3.2. Tissue MPO activity A significant increase in MPO activity in the left brain cortex was revealed in the injury group 24 hours after the cold injury when compared with the control group in the left
Fig. 1. Quantification of brain water content in the ipsilateral brain hemisphere 24 hours after cold injury. Compared with control animals, brain water content was markedly increased in the injury group. Both EPO and DSP groups had significantly decreased ipsilateral hemisphere water content compared to the injury group (*P b .05 vs control and †P b .05 vs injury). There was no statistical difference among the groups for contralateral hemisphere water content (data not shown) (n = 7 for each group).
Fig. 2. Tissue MPO activity levels in the ipsilateral brain cortex at 24 hours after cold injury. A significant increase in MPO activity in the left brain cortex was revealed in the injury group 24 hours after the cold injury when compared with the control group (*P b .05 vs control). Both EPO and DSP treatments prevented an increase in MPO levels (†P b .05 vs injury), although the MPO levels after both treatments were significantly higher than those in the control samples. The vehicle group did not show a difference from the injury group (P N .05 vs injury) (n = 7 for each group).
brain cortex (*P b .05 vs control), indicating neutrophil activation in the injured brain cortex (Fig. 2). Both EPO and DSP treatments prevented increase in MPO levels (†P b .05 vs injury), although the MPO levels after both treatments were significantly higher than control samples. The vehicle group did not show a difference from the injury group (P N .05 vs injury). 3.3. Tissue caspase-3 activity The injury rats showed a massive activation of caspase-3 in the injured cortex 24 hours after cold injury (*P b .05 vs control) (Fig. 3). Both EPO and DSP treatments reduced caspase-3 activation drastically at 24 hours (†P b .05 vs injury). The vehicle group did not show a difference from the injury group (P N .05 vs injury). 3.4. Ultrastructural findings The cold injury produced obvious ultrastructural damage as shown in Fig. 4. The injury group showed severe injury with extensive edematous spaces. The myelin sheaths of the
Fig. 3. Tissue caspase-3 activity levels in the ipsilateral brain cortex at 24 hours after cold injury. The injury rats showed a massive activation of caspase-3 in the injured cortex (*P b .05 vs control). Both EPO and DSP treatments reduced caspase-3 activation drastically at 24 hours (†P b .05 vs injury). The vehicle group did not show a difference from the injury group (P N .05 vs injury) (n = 7 for each group).
O. Okutan et al. / Surgical Neurology 70 (2008) 498–502
501
Fig. 4. Electron microscopic evaluation of cortical ultrastructure at 24 hours after cold injury. The injury group showed severe injury with extensive edematous spaces at 24 hours after cold injury. The myelin sheaths of the axons were destroyed. In the EPO (1000 IU/kg) treatment group, the ultrastructure was very well protected. The ultrastructure was normal except for some limited edematous spaces. Treatment with 0.2 mg/kg DSP also showed obvious ultrastructural protection. However, the EPO group showed better results than the DSP group (original magnification ×7500).
axons were destroyed. In the EPO (1000 IU/kg) treatment group, the ultrastructure was very well protected. The neuronal bodies and axonal structures were normal except for some limited edematous spaces. Treatment with 0.2 mg/kg DSP also showed obvious ultrastructural protection. However, the EPO group showed better results compared to the DSP group. 4. Discussion It is well established that an increase in the permeability of BBB due to endothelial cell dysfunction results in brain edema in a wide spectrum of neurologic disorders [8,10,14]. Severe brain edema causes not only brain herniation but also increases intracranial pressure and causes a decrease of cerebral blood perfusion, resulting in secondary ischemia and cell death [8]. Hence, development of new treatment strategies for brain edema is important to decrease mortality and morbidity in clinical practice. Cold injury has been reported to be a useful model for investigating the mechanism of CNS injury followed by vasogenic brain edema. It is has been demonstrated that both apoptosis and necrosis contribute to cellular damage after cold injury [8]. Apoptosis is regulated by an intracellular proteolytic cascade, mediated by the caspase family. Results from the present study clearly showed that caspase-3 activation occurs in the ipsilateral brain cortex at 24 hours after cold injury, indicating apoptotic cell death that was consistent with previous observations [8]. However, administration of EPO inhibited caspase-3 activation and significantly reduced BBB permeability, which is followed by decreased brain water content (Fig. 1). These results suggest that EPO protects brain against cold injury via preserving endothelial integrity and attenuating apoptotic cell death. Because BBB maintains the homeostasis of the brain microenvironment, preserving endothelial integrity is also crucial for neuronal activity and function [13]. Previous studies showed that neurovascular protection is an important target to prevent or reduce neuronal cell death [14]. Both in vivo and in vitro experimental models have demonstrated neuronal and vascular protection in the CNS by EPO [6]. It has been shown that EPO exerts vascular protection by
preserving endothelial cell integrity and maintains the establishment of cell-to-cell junctions [2,7]. Apoptosis, implicated in endothelial cell death after cerebral ischemia, may lead not only to increased BBB permeability but also to increased infiltration of proinflammatory cells into the injured tissue [3,19]. In the present study, there was a significant increase in MPO activity and brain edema, both of which indicate increased permeability of BBB and infiltration of proinflammatory cells into the injured tissue after cold injury. However, exogenous administration of EPO decreased tissue MPO activity significantly. It has been shown that mononuclear cell infiltration is reduced in EPO-treated animals with traumatic brain injury [1]. Erythropoietin may reduce the inflammatory response by decreasing cell death and/or by preventing the release of proinflammatory cytokines such as tumor necrosis factor and interleukin 6 [11,16]. Despite recent arguments on the effectiveness and complications of DSP, many clinics still use it for human vasogenic brain edema. However, despite its cytoprotective attributes, EPO therapy may also have clinical toxicity [6]. In the present study, we compared the effectiveness of EPO against DSP. Dexamethasone sodium phosphate treatment prevented the increase in caspase-3 and MPO activity to a similar degree as EPO; however, better ultrastructural preservation was observed in the EPO-treated group.
5. Conclusions Administration of EPO in the cold injury–induced edema model was found to be neuroprotective by anti-inflammatory and anti-apoptotic mechanisms. However, different time points, different doses, and application routes should be tested to confirm results and to show longer-term outcome. Further research will explore the potential use of EPO for vasogenic brain edema in humans. Acknowledgments We would like to thank Hakan Eroglu, MSc, for his kind help with the statistical analysis. We also thank Dr Ihsan Solaroglu for his valuable comments and careful editing of the manuscript.
502
O. Okutan et al. / Surgical Neurology 70 (2008) 498–502
References [1] Brines ML, Ghezzi P, Keenan S, et al. Erythropoietin crosses the blood-brain barrier to protect against experimental brain injury. Proc Natl Acad Sci U S A 2000;97:10526-31. [2] Chong ZZ, Kang JQ, Maiese K. Apaf-1, Bcl-xL, cytochrome c, and caspase-9 form the critical elements for cerebral vascular protection by erythropoietin. J Cereb Blood Flow Metab 2003;23:320-30. [3] del Zoppo GJ, Mabuchi T. Cerebral microvessel responses to focal ischemia. J Cereb Blood Flow Metab 2003;23:879-94. [4] Grasso G. Neuroprotective effect of recombinant human erythropoietin in experimental subarachnoid hemorrhage. J Neurosurg Sci 2001;45: 7-14. [5] Kaptanoglu E, Solaroglu I, Okutan O, et al. Erythropoietin exerts neuroprotection after acute spinal cord injury in rats: effect on lipid peroxidation and early ultrastructural findings. Neurosurg Rev 2004; 27:113-20. [6] Maiese K, Li F, Chong ZZ. New avenues of exploration for erythropoietin. JAMA 2005;293:90-5. [7] Martinez-Estrada OM, Rodriguez-Millan E, Gonzalez-De Vicente E, et al. Erythropoietin protects the in vitro blood-brain barrier against VEGF-induced permeability. Eur J Neurosci 2003;18:2538-44. [8] Murakami K, Kondo T, Yang G, et al. Cold injury in mice: a model to study mechanisms of brain edema and neuronal apoptosis. Prog Neurobiol 1999;57:289-99. [9] Okutan O, Solaroglu I, Beskonakli E, et al. Recombinant human erythropoietin decreases myeloperoxidase and caspase-3 activity and improves early functional results after spinal cord injury in rats. J Clin Neurosci 2007; doi:10.1016/j.jocn.2006.01.022.
[10] Rabinstein AA. Treatment of cerebral edema. Neurologist 2006;12: 59-73. [11] Siren AL, Fratelli M, Brines M, et al. Erythropoietin prevents neuronal apoptosis after cerebral ischemia and metabolic stress. Proc Natl Acad Sci U S A 2001;98:4044-9. [12] Solaroglu I, Solaroglu A, Kaptanoglu E, et al. Erythropoietin prevents ischemia-reperfusion from inducing oxidative damage in fetal rat brain. Childs Nerv Syst 2003;19:19-22. [13] Stanness KA, Westrum LE, Fornaciari E, et al. Morphological and functional characterization of an in vitro blood-brain barrier model. Brain Res 1997;771:329-42. [14] Tsubokawa T, Yamaguchi-Okada M, Calvert JW, et al. Neurovascular and neuronal protection by E64d after focal cerebral ischemia in rats. J Neurosci Res 2006;84:832-40. [15] Turkoglu OF, Eroglu H, Okutan O, et al. The efficiency of dexamethasone sodium phosphate–encapsulated chitosan microspheres after cold injury. Surg Neurol 2005;64(Suppl 2):S11-6. [16] Villa P, Bigini P, Mennini T, et al. Erythropoietin selectively attenuates cytokine production and inflammation in cerebral ischemia by targeting neuronal apoptosis. J Exp Med 2003;198:971-5. [17] Wang L, Zhang Z, Wang Y, et al. Treatment of stroke with erythropoietin enhances neurogenesis and angiogenesis and improves neurological function in rats. Stroke 2004;35:1732-7. [18] Xi G, Hua Y, Keep RF, et al. Brain edema after intracerebral hemorrhage: the effects of systemic complement depletion. Acta Neurochir Suppl 2002;81:253-6. [19] Zhang Y, Zhang X, Park TS, et al. Cerebral endothelial cell apoptosis after ischemia-reperfusion: role of PARP activation and AIF translocation. J Cereb Blood Flow Metab 2005;25:868-77.