British Journal of Neurosurgery, December 2014; 28(6): 739–745 © 2014 The Neurosurgical Foundation ISSN: 0268-8697 print / ISSN 1360-046X online DOI: 10.3109/02688697.2014.915007
ORIGINAL ARTICLE
A pre-injury high ethanol intake in rats promotes brain edema following traumatic brain injury Weichuan Wu1, Runfa Tian1, Shuyu Hao1, Feifan Xu1, Xiang Mao2 & Baiyun Liu1 1Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Beijing, P. R. China,
and 2Department of Clinical Medicine, Anhui Medical University, Hefei, P. R. China endothelial growth factor (VEGF) are downstream regulatory factors of HIF-1α,5–8 and both of these proteins are highly associated with brain edema after TBI. Aquaporins (AQPs) are a family of glial membrane water channel proteins that transport water in or out of glial cells.
Abstract Drinking is a risk factor for traumatic brain injury (TBI), and ethanol can aggravate the outcome by promoting brain edema. The mechanism involved is not fully understood. It has been confirmed that aquaporin-4 (AQP4) and vascular endothelial growth factor (VEGF) play pivotal roles in cytotoxic/vasogenic brain edema individually, and both of these proteins are downstream regulatory factors of hypoxiainducible factor-1a (HIF-1a). In this study, we used a fluid percussion injury (FPI) model in rats to determine the effects of acute ethanol intake on the expression levels of HIF-1a, AQP4, and VEGF prior to FPI. The animals were sacrificed 1, 2, 3, and 4 days post-injury. We found that the expression levels of HIF-1a and AQP4 were significantly upregulated in the ethanol-pretreated groups, whereas the VEGF expression level was not. In addition, there was a positive correlation between HIF-1a and AQP4. The results of this study indicate that cytotoxic brain edema may play an important role in the early stage of FPI in ethanol-pre-treated animals and that HIF-1a and AQP4 might be involved. Keywords: brain edema; ethanol intake; fluid percussion injury; traumatic brain injury
Introduction Traumatic brain injury (TBI) is a critical disease due to its high morbidity and mortality. As a major complication of TBI, brain edema is responsible for an increase in intracranial pressure, which if not treated promptly and effectively may eventually lead to brain herniation. Post-trauma brain edema consists of two different types: cytotoxic and vasogenic brain edema, both of which are involved in the pathophysiological development of TBI.1 Hypoxia-inducible factor-1α (HIF-1α), a transcription factor induced under hypoxic conditions, controls the downstream expression of some types of proteins. This protein is expressed at very low levels in cells but is prominently upregulated in TBI rats.2–4 Aquaporin-4 (AQP4) and vascular
Fig. 1. Expression of HIF-1α protein determined by Western blot analysis after FPI. ( ) Representative Western blot illustrating the different expression levels of HIF-1α at different time points. ( ) Densitometric analysis of HIF-1α expression. Values are the mean standard error of eight animals for each treatment and post-injury time point studied.
Correspondence: Baiyun Liu, Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Beijing 100050, P. R. China. Tel: 86010-67059175. E-mail: [email protected] Received for publication 4 April 2013; accepted 6 April 2014
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Fig. 2. Expression of HIF-1α protein determined by immunofluorescence after FPI. HIF-1α was hardly found in group A and peaked on post-injury day 2 in groups B and C.
The discovery of AQPs subverted the traditional opinion that simple diffusion is the prominent mode by which water enters cells.9 AQP4 is a key factor responsible for cytotoxic edema. This protein is primarily found in astrocyte foot processes that are attached to capillary walls, accounting for 85–90% of the surface area of these capillaries.10 Because of the proportion of astrocytes in the brain, astrocyte swelling plays an important role in cytotoxic edema.11 VEGF is a key factor in the regulation of angiogenesis, and expression of this protein is regulated by HIF-1α.5,12 HIF1α expression is up-regulated under hypoxic conditions, and this protein controls the expression of VEGF, which is responsible for the increase in vascular permeability.13,14 Recently, it has been reported that VEGF impaired the integrity of the blood-retina barrier, and estrogen, an inhibitor of VEGF, attenuates VEGF-initiated blood-retina barrier breakdown in rats.15 As a result of the increasing permeability of the blood-brain barrier, macromolecules and water move out of the vasculature.
Drinking is a risk factor for TBI. It has been observed that high acute ethanol intake exacerbates the prognosis of individuals with TBI by promoting post-trauma brain edema.16–18 Cultured rat primary astrocytes and astroglia swell when exposed to ethanol in an acute manner.19 Recent studies have been conducted from the perspectives of oxidative stress,20–22 inflammatory responses,23 and cell survival.18 In this study, we examined the expression of HIF-1α, VEGF, and AQP4, which are highly correlated to either cytotoxic or vasogenic brain edema, to determine the mechanism by which the acute intake of a high dose of ethanol exacerbates post-trauma brain edema.
Materials and methods Materials HIF-1α and VEGF antibodies were purchased from Santa Cruz Biotechnology. Rabbit anti-beta-actin was purchased from Cell Signaling Technology. Mouse anti-beta-tubulin
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Fluid percussion injury Rats were anesthetized (10% chloral hydrate, 0.3 ml/100 g, i.p.) and were placed in a stereotaxic frame in the prone position. A median incision was made, and the right temporal muscle was reflected. A 4-mm hole was made in the parietal bone 1 mm from the sagittal line and between the bregma and lambda. A Luer-Lok cap was cemented over the hole to allow the attachment of the FP device via a plastic tube that was filled with saline in advance. The rats were subjected to injury of moderate severity (2.6–2.8 atm) by releasing the pendulum hammer to produce an impact that was conducted by the saline in the plastic tube to the brain surface, as previously described.27 The Luer-Lok cap was not removed and skin was not sutured until the rats had recovered spontaneous respiration for a few seconds. A heating pad was used to maintain the body temperature throughout all procedures. Sham-operated rats underwent all procedures except fluid percussion injury (FPI).
Histopathology After brain removal, they were cut from the contusion core into two parts. The anterior parts were used for histopathological analysis, and the posterior parts were used for biochemical analysis. The former were sectioned into 10-μm slices on a cryostat and were stored at 80°C for immunofluorescence staining.
Immunofluorescence staining Fig. 3. Expression of VEGF protein determined by Western blot analysis after FPI. ( ) Representative Western blot illustrating the different expression levels of VEGF at different time points. ( ) Densitometric analysis of VEGF expression. Values are the mean standard error of eight animals for each treatment and post-injury time point studied.
and anti-AQP4 antibodies were purchased from Abcam. All other chemicals were purchased from Zhongshan Goldenbridge Biotechnology Co., Ltd.
Animals Male Sprague–Dawley rats (270–300 g) were purchased from Vital River Laboratory Animal Technology Co. Ltd. and caged in a room with a 12-hour dark/light cycle. The rats had free access to food and water until 24 h before the experiment. All procedures that involved animals were approved by the local ethics committee for the use of experimental animals and were conducted in accordance with institutional guidelines. Rats were divided into three groups: A, sham operated; B, 0.9% saline at 10 ml/kg; and C, 3 g/kg ethanol at 10 ml/ kg. Saline and ethanol were administered intraperitoneally to rats in groups B and C 1 h prior to injury, as previously described.18 Animals in group B maintained a normal serum sodium and normal pathophysiological status (B, P, R, SPO2) before injury. Groups B and C both consisted of 32 animals. Group A consisted of 20 animals. Rats were decapitated, and their brains were harvested and rinsed in ice-cold artificial cerebrospinal fluid (CSF) at 1, 2, 3, or 4 days after injury. These time points were chosen based on previous studies that showed peak expression of HIF-1α and VEGF at 2 and 4 days post-injury, respectively.24–26
Coronal sections from the anterior parts of the contusion core were used for immunofluorescence staining. These sections were fixed with 4% paraformaldehyde for 20 min and washed with PBS three times. Sections that were used to detected HIF1α expression were treated with 0.3% Triton X-100 and then washed with PBS. Hydrogen peroxide (1%) was used to eliminate endogenous peroxidase activity. Then, the sections were blocked with goat serum at room temperature for 10 min. After that, the sections were incubated with primary antibodies against HIF-1α (sc-53546, 1:100; Santa Cruz Biotechnology), VEGF (sc-152, 1:200; Santa Cruz Biotechnology), or AQP4 (ab46182,1:100; Abcam) overnight at 4°C. After these sections were washed with PBS three times, they were incubated with the secondary antibodies that were diluted in 1 ml of reagentquality water (Cat. no. 03-18-06 and Cat. no. 02-15-06; KPL; 1:10) for 2 h at 37°C. Unconjugated secondary antibodies were washed away with PBS. Cells were exposed to DAPI (0.5 mg/ ml, Sigma) at room temperature for 5 min to stain the nuclei. Then, the sections were observed under a fluorescence microscope (DM14000B, Leica, Germany).
Western blot analysis Protein extracts were obtained from the posterior part as described above. Homogenates were centrifuged at 13 000 rpm for 15 min at 4°C. Supernatants were diluted in loading buffer and heated for 5 min at 95°C. Then, 50 μg/10 μl of protein was loaded onto a 10% (HIF-1α) or 12% (VEGF, AQP4) SDS-PAGE gel. After separation, the proteins were transferred to a nitrocellulose membrane at room temperature for 90 min or 50 min at 7 V, respectively. After being blocked with 5% skim milk, the membranes
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Fig. 4. Expression of VEGF protein determined by immunofluorescence after FPI. VEGF was hardly found in group A and peaked on post-injury day 4 in groups B and C.
were incubated overnight with a primary antibody against HIF-1α (1:2000, sc-53546, lot# 10910), VEGF (1:4000, sc-152, lot# E2411, Santa Cruz Biotechnology), AQP4, or rabbit beta-actin (1:2000, lot:6, #4967S, Cell Signaling) at 4°C. After an 1-hour incubation with the secondary antibody at room temperature, the bands were visualized by the addition of HRP Substrate Peroxide Solution using a FluorChem Imaging System (FC2, Cell Biosciences). The relative band intensities were determined using Odyssey software, version 2.0 (LI-COR).
Statistical analysis Values are expressed as the mean standard error and were analyzed by a one-way analysis of variance, followed by the Student Newman–Keuls test. A value of p 0.05 was considered significant.
Results Consistent with previous studies, this study showed that the expression of HIF-1α is significantly up-regulated in
both FPI rats and ethanol-pre-treated injured rats (Fig. 1). HIF-1α expression was significantly up-regulated after FPI on post-injury day 1 in both groups (p 0.01) and peaked on post-injury day 2 (p 0.001). HIF-1α expression was then down-regulated on post-injury day 3 and day 4. HIF-1α expression was significantly higher in ethanolpretreated injured rats than in the FPI rats on post-injury days 1 (p 0.01), 2 (p 0.01), and 3 (p 0.01). However, no significant difference was observed between the ethanolpretreated injured rats and the FPI rats on post-injury day 4 (p 0.069). Immunofluorescence staining for HIF-1α indicated that this protein was located in the cytoplasm in most HIF-1α-positive cells (Fig. 2). VEGF, responsible for vasogenic brain edema after FPI, is a major target regulated by HIF-1α. After FPI, VEGF expression was significantly up-regulated on post-injury days 2 and 3 and peaked on post-injury day 4 in both groups (p 0.05) (data not shown). No difference was observed in the expression of VEGF between FPI rats and ethanol-pre-treated injured rats on post-injury day 1 (p 0.68) or 2 (p 0.523). However, VEGF expression was significantly reduced in the
Ethanol promotes edema after TBI
Fig. 5. Expression of AQP4 protein determined by Western blot analysis after FPI. ( ) Representative Western blot illustrating the different expression levels of AQP4 at different time points. ( ) Densitometric analysis of AQP4 expression. Values are the mean standard error of eight animals for each treatment and post-injury time point studied.
ethanol-pretreated injured rats compared with the FPI rats on post-injury days 3 and 4 (p 0.05) (Fig. 3). Immunofluorescence staining for VEGF indicated that this protein was located in the cytoplasm in most VEGF-positive vascular endothelial cells and glial cells (Fig. 4). AQP4, responsible for cytotoxic brain edema after FPI, is also a major target regulated by HIF-1α. After FPI, AQP4 expression was significantly up-regulated on postinjury day 1 (p 0.001) in both groups and then gradually returned to the basal level on post-injury day 4 (group A vs. group C, p 0.055) (Fig. 5). On post-injury days 1 and 2, AQP4 expression was significantly higher in the ethanol-pretreated injured group than in the FPI group (p 0.03, p 0.001). No significant differences were observed on post-injury day 3 between the ethanolpretreated injured group and the FPI group (p 0.097). Immunofluorescence staining for AQP4 indicated that this protein was located in the cytoplasm in most AQP4positive cells (Fig. 6).
Discussion This study showed that the expression levels of HIF-1α and AQP4 are significantly up-regulated in FPI rats after the acute intake of a high dose of ethanol, whereas the expression of
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VEGF was down-regulated. We also found there is a positive correlation between HIF-1α and AQP4. A previous study showed that both the density and diameter of the microvasculature are significantly decreased in fluid-percussion-injured cortical hemispheres of rats.28 As a result of microvascular impairment, cerebral blood flow is decreased, and tissue oxygen delivery is reduced. Under hypoxic conditions, a series of molecular biological changes occur in astrocytes. HIF1α, the expression of which is one of the most prominent cellular responses to hypoxia,29 regulates the expression of a broad range of genes that participate in angiogenesis, vasomotor control, iron metabolism, and cell death.30 It has been observed that hypoxia could induce cortical laminar necrosis in infants with severe TBI.31 We found that HIF-1α expression is significantly upregulated in FPI rats compared with sham-operated rats and is significantly higher in ethanol-pretreated injured rats than in saline-pretreated injured rats. Consistent with our study, a previous study demonstrated that the expression of AQP4 is significantly upregulated in FPI rats.32 In addition, that study found that the pharmacological inhibition of HIF-1α with 2-methoxyestradiol, which can down-regulate HIF-1α expression, significantly reduced the up-regulated levels of AQP4. These results suggest that AQP4 expression is regulated by HIF-1α. It also has been observed that AQP4 expression was significantly activated in cultured astrocytes in the early stage after FPI.33,34 In our study, we found that AQP4 expression was detected in both groups B and C and that this expression is significantly higher in ethanol-pretreated rats than in saline-pretreated rats after FPI. This result suggests that AQP4 is responsible for ethanol’s aggravating effect on brain edema after FPI. In addition, AQP4 expression was positively correlated with HIF-1α expression. The expression of both proteins peaked at 24 h after FPI. It has been observed that ethanol activates the expression of HIF-1α in mast cells35 and induces oxidative stress in the brain cells of FPI rats18,36,37 Oxidative stress is an oxygenconsuming process that will contribute to hypoxia. Therefore, we hypothesize that ethanol augments the expression of HIF-1α in FPI rats by inducing oxidative stress. The detailed mechanism needs to be investigated further. The up-regulated expression of VEGF has been considered to be the predominant cause of post-trauma brain edema for many years due to the blood-brain barrier destruction in and around the contusion.38,39 Previous research detected cerebral microbleeds in mild TBI.40 Lenzlinger et al. found that inhibiting VEGF significantly reduces regional cerebral edema in TBI rats.41 Kimura et al. also found that a VEGF antagonist reduces the development of brain edema and venous infarction in a 2-vein occlusion model in rats.42 We also found that VEGF expression is significantly upregulated in FPI rats, which indicates that VEGF may be involved in post-trauma brain vasogenic edema. Previous studies showed that HIF-1α or hypoxia mediates VEGF expression43–45 however, a significant decrease in the level of upregulated VEGF expression in the injured hemisphere
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Fig. 6. Expression of AQP4 protein determined by immunofluorescence after FPI. AQP4 was hardly found in group A and peaked on post-injury day 1 in groups B and C.
was found in the ethanol-pretreated FPI rats relative to the saline-pretreated FPI rats. However, the mechanism needs to be investigated further . In conclusion, we found that acute ethanol intake exacerbates cytotoxic brain edema through AQP4, which is activated by HIF-1α. In our experiment, the dose of alcohol for rats approximately equals to one bottle of spirits (700 ml, 40% vol) for 70-kg adult. These results provide a better understanding of how ethanol augments post-trauma brain edema and identify a potential therapeutic target. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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Ethanol promotes edema after TBI 8. Kaur C, Sivakumar V, Zhang Y, Ling EA . Hypoxia-induced astrocytic reaction and increased vascular permeability in the rat cerebellum. Glia 2006;54:826–39. 9. Papadopoulos MC, Krishna S, Verkman AS. Aquaporin water channels and brain edema. Mt Sinai J Med 2002;69:242–8. 10. Nielsen S, Nagelhus EA , Amiry-Moghaddam M, et al. Specialized membrane domains for water transport in glial cells: highresolution immunogold cytochemistry of aquaporin-4 in rat brain. J Neurosci 1997;17:171–80. 11. Kimelberg HK . Current concepts of brain edema. Review of laboratory investigations. J Neurosurg 1995;83:1051–9. 12. LaManna JC, Chavez JC, Pichiule P. Structural and functional adaptation to hypoxia in the rat brain. J Exp Biol 2004;207: 3163–9. 13. Zhang ZG, Zhang L, Jiang Q, et al. VEGF enhances angiogenesis and promotes blood-brain barrier leakage in the ischemic brain. J Clin Invest 2000;106:829–38. 14. Chodobski A , Chung I, Kozniewska E, et al. Early neutrophilic expression of vascular endothelial growth factor after traumatic brain injury. Neuroscience 2003;122:853–67. 15. Chen X, Zhang M, Jiang C, et al. Estrogen attenuates VEGF-initiated blood-retina barrier breakdown in male rats. Horm Metab Res 2011;43:614–8. 16. Greiffenstein P, Mathis KW, Stouwe CV, Molina PE. Alcohol binge before trauma/hemorrhage impairs integrity of host defense mechanisms during recovery. Alcohol Clin Exp Res 2007;31: 704–15. 17. Crews F, Nixon K, Kim D, et al. BHT blocks NF-kappaB activation and ethanol-induced brain damage. Alcohol Clin Exp Res 2006; 30:1938–49. 18. Katada R, Nishitani Y, Honmou O, et al. Prior ethanol injection promotes brain edema after traumatic brain injury. J Neurotrauma 2009;26:2015–25. 19. Allansson L, Khatibi S, Olsson T, Hansson E. Acute ethanol exposure induces [Ca2]i transients, cell swelling and transformation of actin cytoskeleton in astroglial primary cultures. J Neurochem 2001;76:472–9. 20. Haorah J, Knipe B, Leibhart J, Ghorpade A , Persidsky Y. Alcohol-induced oxidative stress in brain endothelial cells causes blood-brain barrier dysfunction. J Leukoc Biol 2005;78: 1223–32. 21. Haorah J, Ramirez SH, Floreani N, et al. Mechanism of alcoholinduced oxidative stress and neuronal injury. Free Radic Biol Med 2008;45:1542–50. 22. Haorah J, Ramirez SH, Schall K , et al. Oxidative stress activates protein tyrosine kinase and matrix metalloproteinases leading to blood-brain barrier dysfunction. J Neurochem 2007;101: 566–76. 23. Gottesfeld Z, Moore AN, Dash PK . Acute ethanol intake attenuates inflammatory cytokines after brain injury in rats: a possible role for corticosterone. J Neurotrauma 2002;19:317–26. 24. Ding JY, Kreipke CW, Speirs SL, et al. Hypoxia-inducible factor1alpha signaling in aquaporin upregulation after traumatic brain injury. Neurosci Lett 2009;453:68–72. 25. Yu R, Gao L, Jiang S, Guan P, Mao B. Association of HIF-1alpha expression and cell apoptosis after traumatic brain injury in the rat. Chin J Traumatol 2001;4:218–21. 26. Chodobski A , Chung I, Kozniewska E, et al. Early neutrophilic expression of vascular endothelial growth factor after traumatic brain injury. Neuroscience 2003;122:853–67.
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27. McIntosh TK, Vink R, Noble L, et al. Traumatic brain injury in the rat: characterization of a lateral fluid-percussion model. Neuroscience 1989;28:233–44. 28. Park E, Bell JD, Siddiq IP, Baker AJ. An analysis of regional microvascular loss and recovery following two grades of fluid percussion trauma: a role for hypoxia-inducible factors in traumatic brain injury. J Cereb Blood Flow Metab 2009;29:575–84. 29. Chavez JC, Agani F, Pichiule P, LaManna JC. Expression of hypoxiainducible factor-1alpha in the brain of rats during chronic hypoxia. J Appl Physiol 2000;89:1937–42. 30. Sharp FR, Bernaudin M. HIF1 and oxygen sensing in the brain. Nat Rev Neurosci 2004;5:437–48. 31. Han SR, Yee GT, Choi CY, Lee CH. Cortical laminar necrosis in an infant with severe traumatic brain injury. J Korean Neurosurg Soc 2011;50:472–4. 32. Ding JY, Kreipke CW, Speirs SL, et al. Hypoxia-inducible factor1alpha signaling in aquaporin upregulation after traumatic brain injury. Neurosci Lett 2009;453:68–72. 33. Rao KV, Reddy PV, Curtis KM, Norenberg MD. Aquaporin-4 expression in cultured astrocytes after fluid percussion injury. J Neurotrauma 2011;28:371–81. 34. Oliva AA Jr, Kang Y, Truettner JS, et al. Fluid-percussion brain injury induces changes in aquaporin channel expression. Neuroscience 2011;180:272–9. 35. Jeong HJ, Hong SH, Park RK , An NH, Kim HM. Ethanol induces the production of cytokines via the Ca2, MAP kinase, HIF-1alpha, and NF-kappaB pathway. Life Sci 2005;77:2179–92. 36. Crews FT, Collins MA , Dlugos C, et al. Alcohol-induced neurodegeneration: when, where and why. Alcohol Clin Exp Res 2004;28:350–64. 37. Crews FT, Nixon K. Mechanisms of neurodegeneration and regeneration in alcoholism. Alcohol Alcohol. 2009;44:115–27. 38. Stroop R, Thomale UW, Pauser S, et al. Magnetic resonance imaging studies with cluster algorithm for characterization of brain edema after controlled cortical impact injury (CCII). Acta Neurochir Suppl 1998;71:303–5. 39. Chodobski A , Chung I, Kozniewska E, et al. Early neutrophilic expression of vascular endothelial growth factor after traumatic brain injury. Neuroscience 2003;122:853–67. 40. Park JH, Park SW, Kang SH, et al. Detection of traumatic cerebral microbleeds by susceptibility-weighted image of MRI. J Korean Neurosurg Soc 2009;46:365–9. 41. Lenzlinger PM, Saatman KE, Hoover RC, et al. Inhibition of vascular endothelial growth factor receptor (VEGFR) signaling by BSF476921 attenuates regional cerebral edema following traumatic brain injury in rats. Restor Neurol Neurosci 2004;22:73–9. 42. Kimura R, Nakase H, Tamaki R, Sakaki T. Vascular endothelial growth factor antagonist reduces brain edema formation and venous infarction. Stroke 2005;36:1259–63. 43. Fischer S, Wobben M, Marti HH, Renz D, Schaper W. Hypoxiainduced hyperpermeability in brain microvessel endothelial cells involves VEGF-mediated changes in the expression of zonula occludens-1. Microvasc Res 2002;63:70–80. 44. Shin YJ, Choi JS, Choi JY, et al. Induction of vascular endothelial growth factor receptor-3 mRNA in glial cells following focal cerebral ischemia in rats. J Neuroimmunol 2010;229:81–90. 45. Fischer S, Wiesnet M, Marti HH, Renz D, Schaper W. Simultaneous activation of several second messengers in hypoxia-induced hyperpermeability of brain derived endothelial cells. J Cell Physiol. 2004. 198(3): 359–69.
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ORIGINAL ARTICLE
A pre-injury high ethanol intake in rats promotes brain edema following traumatic brain injury Weichuan Wu1, Runfa Tian1, Shuyu Hao1, Feifan Xu1, Xiang Mao2 & Baiyun Liu1 1Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Beijing, P. R. China,
and 2Department of Clinical Medicine, Anhui Medical University, Hefei, P. R. China endothelial growth factor (VEGF) are downstream regulatory factors of HIF-1α,5–8 and both of these proteins are highly associated with brain edema after TBI. Aquaporins (AQPs) are a family of glial membrane water channel proteins that transport water in or out of glial cells.
Abstract Drinking is a risk factor for traumatic brain injury (TBI), and ethanol can aggravate the outcome by promoting brain edema. The mechanism involved is not fully understood. It has been confirmed that aquaporin-4 (AQP4) and vascular endothelial growth factor (VEGF) play pivotal roles in cytotoxic/vasogenic brain edema individually, and both of these proteins are downstream regulatory factors of hypoxiainducible factor-1a (HIF-1a). In this study, we used a fluid percussion injury (FPI) model in rats to determine the effects of acute ethanol intake on the expression levels of HIF-1a, AQP4, and VEGF prior to FPI. The animals were sacrificed 1, 2, 3, and 4 days post-injury. We found that the expression levels of HIF-1a and AQP4 were significantly upregulated in the ethanol-pretreated groups, whereas the VEGF expression level was not. In addition, there was a positive correlation between HIF-1a and AQP4. The results of this study indicate that cytotoxic brain edema may play an important role in the early stage of FPI in ethanol-pre-treated animals and that HIF-1a and AQP4 might be involved. Keywords: brain edema; ethanol intake; fluid percussion injury; traumatic brain injury
Introduction Traumatic brain injury (TBI) is a critical disease due to its high morbidity and mortality. As a major complication of TBI, brain edema is responsible for an increase in intracranial pressure, which if not treated promptly and effectively may eventually lead to brain herniation. Post-trauma brain edema consists of two different types: cytotoxic and vasogenic brain edema, both of which are involved in the pathophysiological development of TBI.1 Hypoxia-inducible factor-1α (HIF-1α), a transcription factor induced under hypoxic conditions, controls the downstream expression of some types of proteins. This protein is expressed at very low levels in cells but is prominently upregulated in TBI rats.2–4 Aquaporin-4 (AQP4) and vascular
Fig. 1. Expression of HIF-1α protein determined by Western blot analysis after FPI. ( ) Representative Western blot illustrating the different expression levels of HIF-1α at different time points. ( ) Densitometric analysis of HIF-1α expression. Values are the mean standard error of eight animals for each treatment and post-injury time point studied.
Correspondence: Baiyun Liu, Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Beijing 100050, P. R. China. Tel: 86010-67059175. E-mail: [email protected] Received for publication 4 April 2013; accepted 6 April 2014
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Fig. 2. Expression of HIF-1α protein determined by immunofluorescence after FPI. HIF-1α was hardly found in group A and peaked on post-injury day 2 in groups B and C.
The discovery of AQPs subverted the traditional opinion that simple diffusion is the prominent mode by which water enters cells.9 AQP4 is a key factor responsible for cytotoxic edema. This protein is primarily found in astrocyte foot processes that are attached to capillary walls, accounting for 85–90% of the surface area of these capillaries.10 Because of the proportion of astrocytes in the brain, astrocyte swelling plays an important role in cytotoxic edema.11 VEGF is a key factor in the regulation of angiogenesis, and expression of this protein is regulated by HIF-1α.5,12 HIF1α expression is up-regulated under hypoxic conditions, and this protein controls the expression of VEGF, which is responsible for the increase in vascular permeability.13,14 Recently, it has been reported that VEGF impaired the integrity of the blood-retina barrier, and estrogen, an inhibitor of VEGF, attenuates VEGF-initiated blood-retina barrier breakdown in rats.15 As a result of the increasing permeability of the blood-brain barrier, macromolecules and water move out of the vasculature.
Drinking is a risk factor for TBI. It has been observed that high acute ethanol intake exacerbates the prognosis of individuals with TBI by promoting post-trauma brain edema.16–18 Cultured rat primary astrocytes and astroglia swell when exposed to ethanol in an acute manner.19 Recent studies have been conducted from the perspectives of oxidative stress,20–22 inflammatory responses,23 and cell survival.18 In this study, we examined the expression of HIF-1α, VEGF, and AQP4, which are highly correlated to either cytotoxic or vasogenic brain edema, to determine the mechanism by which the acute intake of a high dose of ethanol exacerbates post-trauma brain edema.
Materials and methods Materials HIF-1α and VEGF antibodies were purchased from Santa Cruz Biotechnology. Rabbit anti-beta-actin was purchased from Cell Signaling Technology. Mouse anti-beta-tubulin
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Fluid percussion injury Rats were anesthetized (10% chloral hydrate, 0.3 ml/100 g, i.p.) and were placed in a stereotaxic frame in the prone position. A median incision was made, and the right temporal muscle was reflected. A 4-mm hole was made in the parietal bone 1 mm from the sagittal line and between the bregma and lambda. A Luer-Lok cap was cemented over the hole to allow the attachment of the FP device via a plastic tube that was filled with saline in advance. The rats were subjected to injury of moderate severity (2.6–2.8 atm) by releasing the pendulum hammer to produce an impact that was conducted by the saline in the plastic tube to the brain surface, as previously described.27 The Luer-Lok cap was not removed and skin was not sutured until the rats had recovered spontaneous respiration for a few seconds. A heating pad was used to maintain the body temperature throughout all procedures. Sham-operated rats underwent all procedures except fluid percussion injury (FPI).
Histopathology After brain removal, they were cut from the contusion core into two parts. The anterior parts were used for histopathological analysis, and the posterior parts were used for biochemical analysis. The former were sectioned into 10-μm slices on a cryostat and were stored at 80°C for immunofluorescence staining.
Immunofluorescence staining Fig. 3. Expression of VEGF protein determined by Western blot analysis after FPI. ( ) Representative Western blot illustrating the different expression levels of VEGF at different time points. ( ) Densitometric analysis of VEGF expression. Values are the mean standard error of eight animals for each treatment and post-injury time point studied.
and anti-AQP4 antibodies were purchased from Abcam. All other chemicals were purchased from Zhongshan Goldenbridge Biotechnology Co., Ltd.
Animals Male Sprague–Dawley rats (270–300 g) were purchased from Vital River Laboratory Animal Technology Co. Ltd. and caged in a room with a 12-hour dark/light cycle. The rats had free access to food and water until 24 h before the experiment. All procedures that involved animals were approved by the local ethics committee for the use of experimental animals and were conducted in accordance with institutional guidelines. Rats were divided into three groups: A, sham operated; B, 0.9% saline at 10 ml/kg; and C, 3 g/kg ethanol at 10 ml/ kg. Saline and ethanol were administered intraperitoneally to rats in groups B and C 1 h prior to injury, as previously described.18 Animals in group B maintained a normal serum sodium and normal pathophysiological status (B, P, R, SPO2) before injury. Groups B and C both consisted of 32 animals. Group A consisted of 20 animals. Rats were decapitated, and their brains were harvested and rinsed in ice-cold artificial cerebrospinal fluid (CSF) at 1, 2, 3, or 4 days after injury. These time points were chosen based on previous studies that showed peak expression of HIF-1α and VEGF at 2 and 4 days post-injury, respectively.24–26
Coronal sections from the anterior parts of the contusion core were used for immunofluorescence staining. These sections were fixed with 4% paraformaldehyde for 20 min and washed with PBS three times. Sections that were used to detected HIF1α expression were treated with 0.3% Triton X-100 and then washed with PBS. Hydrogen peroxide (1%) was used to eliminate endogenous peroxidase activity. Then, the sections were blocked with goat serum at room temperature for 10 min. After that, the sections were incubated with primary antibodies against HIF-1α (sc-53546, 1:100; Santa Cruz Biotechnology), VEGF (sc-152, 1:200; Santa Cruz Biotechnology), or AQP4 (ab46182,1:100; Abcam) overnight at 4°C. After these sections were washed with PBS three times, they were incubated with the secondary antibodies that were diluted in 1 ml of reagentquality water (Cat. no. 03-18-06 and Cat. no. 02-15-06; KPL; 1:10) for 2 h at 37°C. Unconjugated secondary antibodies were washed away with PBS. Cells were exposed to DAPI (0.5 mg/ ml, Sigma) at room temperature for 5 min to stain the nuclei. Then, the sections were observed under a fluorescence microscope (DM14000B, Leica, Germany).
Western blot analysis Protein extracts were obtained from the posterior part as described above. Homogenates were centrifuged at 13 000 rpm for 15 min at 4°C. Supernatants were diluted in loading buffer and heated for 5 min at 95°C. Then, 50 μg/10 μl of protein was loaded onto a 10% (HIF-1α) or 12% (VEGF, AQP4) SDS-PAGE gel. After separation, the proteins were transferred to a nitrocellulose membrane at room temperature for 90 min or 50 min at 7 V, respectively. After being blocked with 5% skim milk, the membranes
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Fig. 4. Expression of VEGF protein determined by immunofluorescence after FPI. VEGF was hardly found in group A and peaked on post-injury day 4 in groups B and C.
were incubated overnight with a primary antibody against HIF-1α (1:2000, sc-53546, lot# 10910), VEGF (1:4000, sc-152, lot# E2411, Santa Cruz Biotechnology), AQP4, or rabbit beta-actin (1:2000, lot:6, #4967S, Cell Signaling) at 4°C. After an 1-hour incubation with the secondary antibody at room temperature, the bands were visualized by the addition of HRP Substrate Peroxide Solution using a FluorChem Imaging System (FC2, Cell Biosciences). The relative band intensities were determined using Odyssey software, version 2.0 (LI-COR).
Statistical analysis Values are expressed as the mean standard error and were analyzed by a one-way analysis of variance, followed by the Student Newman–Keuls test. A value of p 0.05 was considered significant.
Results Consistent with previous studies, this study showed that the expression of HIF-1α is significantly up-regulated in
both FPI rats and ethanol-pre-treated injured rats (Fig. 1). HIF-1α expression was significantly up-regulated after FPI on post-injury day 1 in both groups (p 0.01) and peaked on post-injury day 2 (p 0.001). HIF-1α expression was then down-regulated on post-injury day 3 and day 4. HIF-1α expression was significantly higher in ethanolpretreated injured rats than in the FPI rats on post-injury days 1 (p 0.01), 2 (p 0.01), and 3 (p 0.01). However, no significant difference was observed between the ethanolpretreated injured rats and the FPI rats on post-injury day 4 (p 0.069). Immunofluorescence staining for HIF-1α indicated that this protein was located in the cytoplasm in most HIF-1α-positive cells (Fig. 2). VEGF, responsible for vasogenic brain edema after FPI, is a major target regulated by HIF-1α. After FPI, VEGF expression was significantly up-regulated on post-injury days 2 and 3 and peaked on post-injury day 4 in both groups (p 0.05) (data not shown). No difference was observed in the expression of VEGF between FPI rats and ethanol-pre-treated injured rats on post-injury day 1 (p 0.68) or 2 (p 0.523). However, VEGF expression was significantly reduced in the
Ethanol promotes edema after TBI
Fig. 5. Expression of AQP4 protein determined by Western blot analysis after FPI. ( ) Representative Western blot illustrating the different expression levels of AQP4 at different time points. ( ) Densitometric analysis of AQP4 expression. Values are the mean standard error of eight animals for each treatment and post-injury time point studied.
ethanol-pretreated injured rats compared with the FPI rats on post-injury days 3 and 4 (p 0.05) (Fig. 3). Immunofluorescence staining for VEGF indicated that this protein was located in the cytoplasm in most VEGF-positive vascular endothelial cells and glial cells (Fig. 4). AQP4, responsible for cytotoxic brain edema after FPI, is also a major target regulated by HIF-1α. After FPI, AQP4 expression was significantly up-regulated on postinjury day 1 (p 0.001) in both groups and then gradually returned to the basal level on post-injury day 4 (group A vs. group C, p 0.055) (Fig. 5). On post-injury days 1 and 2, AQP4 expression was significantly higher in the ethanol-pretreated injured group than in the FPI group (p 0.03, p 0.001). No significant differences were observed on post-injury day 3 between the ethanolpretreated injured group and the FPI group (p 0.097). Immunofluorescence staining for AQP4 indicated that this protein was located in the cytoplasm in most AQP4positive cells (Fig. 6).
Discussion This study showed that the expression levels of HIF-1α and AQP4 are significantly up-regulated in FPI rats after the acute intake of a high dose of ethanol, whereas the expression of
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VEGF was down-regulated. We also found there is a positive correlation between HIF-1α and AQP4. A previous study showed that both the density and diameter of the microvasculature are significantly decreased in fluid-percussion-injured cortical hemispheres of rats.28 As a result of microvascular impairment, cerebral blood flow is decreased, and tissue oxygen delivery is reduced. Under hypoxic conditions, a series of molecular biological changes occur in astrocytes. HIF1α, the expression of which is one of the most prominent cellular responses to hypoxia,29 regulates the expression of a broad range of genes that participate in angiogenesis, vasomotor control, iron metabolism, and cell death.30 It has been observed that hypoxia could induce cortical laminar necrosis in infants with severe TBI.31 We found that HIF-1α expression is significantly upregulated in FPI rats compared with sham-operated rats and is significantly higher in ethanol-pretreated injured rats than in saline-pretreated injured rats. Consistent with our study, a previous study demonstrated that the expression of AQP4 is significantly upregulated in FPI rats.32 In addition, that study found that the pharmacological inhibition of HIF-1α with 2-methoxyestradiol, which can down-regulate HIF-1α expression, significantly reduced the up-regulated levels of AQP4. These results suggest that AQP4 expression is regulated by HIF-1α. It also has been observed that AQP4 expression was significantly activated in cultured astrocytes in the early stage after FPI.33,34 In our study, we found that AQP4 expression was detected in both groups B and C and that this expression is significantly higher in ethanol-pretreated rats than in saline-pretreated rats after FPI. This result suggests that AQP4 is responsible for ethanol’s aggravating effect on brain edema after FPI. In addition, AQP4 expression was positively correlated with HIF-1α expression. The expression of both proteins peaked at 24 h after FPI. It has been observed that ethanol activates the expression of HIF-1α in mast cells35 and induces oxidative stress in the brain cells of FPI rats18,36,37 Oxidative stress is an oxygenconsuming process that will contribute to hypoxia. Therefore, we hypothesize that ethanol augments the expression of HIF-1α in FPI rats by inducing oxidative stress. The detailed mechanism needs to be investigated further. The up-regulated expression of VEGF has been considered to be the predominant cause of post-trauma brain edema for many years due to the blood-brain barrier destruction in and around the contusion.38,39 Previous research detected cerebral microbleeds in mild TBI.40 Lenzlinger et al. found that inhibiting VEGF significantly reduces regional cerebral edema in TBI rats.41 Kimura et al. also found that a VEGF antagonist reduces the development of brain edema and venous infarction in a 2-vein occlusion model in rats.42 We also found that VEGF expression is significantly upregulated in FPI rats, which indicates that VEGF may be involved in post-trauma brain vasogenic edema. Previous studies showed that HIF-1α or hypoxia mediates VEGF expression43–45 however, a significant decrease in the level of upregulated VEGF expression in the injured hemisphere
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Fig. 6. Expression of AQP4 protein determined by immunofluorescence after FPI. AQP4 was hardly found in group A and peaked on post-injury day 1 in groups B and C.
was found in the ethanol-pretreated FPI rats relative to the saline-pretreated FPI rats. However, the mechanism needs to be investigated further . In conclusion, we found that acute ethanol intake exacerbates cytotoxic brain edema through AQP4, which is activated by HIF-1α. In our experiment, the dose of alcohol for rats approximately equals to one bottle of spirits (700 ml, 40% vol) for 70-kg adult. These results provide a better understanding of how ethanol augments post-trauma brain edema and identify a potential therapeutic target. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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