http://informahealthcare.com/bij ISSN: 0269-9052 (print), 1362-301X (electronic) Brain Inj, Early Online: 1–7 ! 2015 Informa UK Ltd. DOI: 10.3109/02699052.2014.1002004
ORIGINAL ARTICLE
Neuroglobin up-regulation after ischaemic pre-conditioning in a rat model of middle cerebral artery occlusion Yichen Liu1,2, Baoquan Li2, Qiaojun Li1, & Liping Zou1 Department of Pediatrics, General Hospital of Chinese People’s Liberation Army, Beijing, PR China and 2Department of Pediatrics, 159th Hospital of Chinese People’s Liberation Army, Zhumadian, Henan Province, PR China
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Abstract
Keywords
Primary objective: Neuroglobin (NGB) is a known neuroprotector and is up-regulated after ischaemia-hypoxia brain damage. However, no studies have investigated NGB levels after ischaemic pre-conditioning and middle cerebral artery occlusion (MCAO). Methods and procedures: This study subjected rats to different ischaemic pre-conditioning and MCAO regimens and assayed NGB levels in the hippocampus, cortex and hypothalamus by immunohistochemistry, quantitative polymerase chain reaction (PCR) and western blot. Main outcomes and results: After 30 minutes of ischaemic pre-conditioning, the number of NGB-positive cells and NGB levels in the hippocampus, cortex and hypothalamus were increased with longer reperfusion times, peaked at 24-hours reperfusion and slightly decreased at 48-hours reperfusion. Similarly, the mRNA and protein expression levels of NGB were also up-regulated; they peaked at 24-hours reperfusion and slightly decreased at 48-hours reperfusion. Conclusions: NGB may regulate neuroprotection against ischaemia and hypoxia-mediated brain damage after ischaemic pre-conditioning. The results provide additional evidence supporting the utility of ischaemic pre-conditioning and help elucidate its potential regulatory mechanism.
Ischaemic pre-conditioning, middle cerebral artery occlusion, neuroglobin, neuroprotection, up-regulation
Introduction Ischaemic brain damage is a prevalent condition and a leading cause of morbidity and mortality worldwide; it is primarily the result of acute brain ischaemia-reperfusion injuries [1]. It occurs when excessive excitatory amino acid releases lead to intracellular calcium influx, electrophysiological and metabolic dysfunction, lipid peroxidation and other oxidative processes [2]. The identification of endogenous neuroprotective mechanisms in the brain, such as those that can be activated with ischaemic pre-conditioning [3] and postconditioning [4], have drawn increased attention due to the disappointing results of pharmacological neuroprotective strategies in a variety of clinical trials. Ischaemic pre-conditioning is an endogenous phenomenon induced by sub-lethal ischaemia that leads to adaptive responses that protect against future severe ischaemic injuries. It is comprised of acute and delayed phases [5]. The acute phase develops within a few minutes and wanes after 2–3 hours [6] and the delayed phase appears within a few hours and lasts for several days. There are several proteins involved in this process, including potassium channels, caspase-3, heat shock protein (HSP)70, N-methyl-D-aspartate (NMDA) Correspondence: Liping Zou, Department of Pediatrics, General Hospital of Chinese People’s Liberation Army, Beijing, PR China, 100853. Tel: +86-10-68025680. Fax: +86-10-68025680. E-mail: [email protected]
History Received 7 June 2014 Revised 6 November 2014 Accepted 20 December 2014 Published online 27 January 2015
receptors, protein kinase C, superoxide dismutase, tumour necrosis factor (TNF)- , nuclear factor (NF)-B, hypoxiainducible factor (HIF)-1 and erythropoietin. However, no study has described alterations in neuroglobin (NGB) levels following ischaemic pre-conditioning and ischaemic brain damage. The role of NGB as a neuroprotective factor against ischaemic-hypoxic brain damage was first described by Burmester et al. [7] in 2000. The monomeric protein is highly expressed in the central nervous system and exhibits high oxygen affinity. The oxygen-binding properties of NGB are comparable to those of a typical vertebrate myoglobin, suggesting a similar function of NGB in the brain [8]. Later studies revealed that NGB could reversibly bind oxygen and that it plays an important role in maintaining oxygen homeostasis in neural tissues [9]. One report found that NGB was up-regulated under hypoxic conditions and had neuroprotective effects in vitro and in vivo [10]. The existing evidence suggests that NGB might be involved in the pathophysiological course of ischaemic and hypoxic injury [11]. It was hypothesized that NGB would exert a significant neuroprotective effect against ischaemic brain injury and that ischaemic pre-conditioning can further up-regulate its expression. The results indicate that NGB expression is increased after ischaemic pre-conditioning and middle cerebral artery occlusion (MCAO) and they suggest that further studies should investigate the neuroprotective effects and underlying mechanisms of ischaemic pre-conditioning.
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Materials and methods
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Experimental animals and groups A total of 36 adult specific pathogen-free Sprague Dawley rats (SD, male, 250–330 g) were purchased and raised in the Laboratory Animal Center of the General Hospital of the People’s Liberation Army (House temperature: 25 ± 2 C, Humidity: 40–60%, 12-hour light/dark cycle) and randomly divided into six groups, including control MCAO (2-hour/24hour ischaemic and reperfusion), 30 minutes + 2-hours/2hours (30 minutes ischaemic pre-conditioning plus 2-hours ischaemia and 2-hours reperfusion), 30 minutes + 2-hours/6hours (30 minutes ischaemic pre-conditioning plus 2-hours ischaemia and 6-hours reperfusion), 30 minutes + 2-hours/12hours (30 minutes ischaemic pre-conditioning plus 2-hours ischaemia and 12-hours reperfusion), 30 minutes + 2-hours/ 24-hours (30 minutes ischaemic pre-conditioning plus 2-hours ischaemia and 24-hours reperfusion), 30 minutes + 2-hours/ 48-hours (30 minutes ischaemic pre-conditioning plus 2-hours ischaemia and 48-hours reperfusion). This experiment was approved by the Experiment and Chinese People’s Liberation Army General Hospital Ethics committees. Ischaemic pre-conditioning and MCAO Ischaemic pre-conditioning of SD rats was performed as previously described [12]. The SD rats were anaesthetized with diethyl ether and the four limbs fixed before an incision was made along the neck midline. The thyroid, vein and nerve tissues were stripped to expose the left carotid artery communis, which was ligated with 5/0 surgical line for 30 minutes of ischaemia and then the line was removed to allow reperfusion for 24 hours. Subsequently, the animals were subjected to ischemia for 2 hours followed by 2-, 6-, 12-, 24-, or 48-huor reperfusion according to the method described earlier. The control group was just subjected to 2 hours of ischemia and 24-hour reperfusion. Afterwards, the rats killed by dislocation to prepare brain tissues for slicing and further identification. TTC staining The rats’ brain tissues were collected and washed with normal saline for 5 minutes and frozen for 20 minutes at 20 C. Then, the brain were transferred to a brain sectioning mould and cut into slices (2 mm) and the sections were put in 1% 2,3,5-triphenyl four azole nitrogen chloride (TTC) at 37 C for 30 minutes (turn once per 5 minutes). Finally, the sections were washed with ddH2O three times and the images gathered for further analysis. Immunohistochemistry (IHC) The rats’ brain tissues were cut into slices (1 mm) and the sections were deparaffinized, rehydrated, post-fixed with 4% paraformaldehyde for 10 minutes and then washed three times with 0.01 M phosphate-buffered saline (PBS). Endogenous peroxidase was inactivated by incubating the sections in 3% H2O2 for 30 minutes. The sections were subjected to sequential incubations with 10% normal goat serum in 0.01 M PBS for
Brain Inj, Early Online: 1–7
30 minutes at room temperature. They were then incubated in rabbit anti-NGB monoclonal (SC-30144, 1: 200; Santa Cruz Biotechnology, Santa Cruz, CA) in PBS containing 0.3% Triton X-100 overnight at 4 C. The sections were washed three times for 5 minutes each with PBS and then incubated in peroxidase-conjugated goat anti-rabbit IgG (1:200; Zymed, South San Francisco, CA) for 1 hour at room temperature. Finally, the sections were developed with diaminobenzidine (Sigma, St. Louis, MO) in 0.1 M Tris-buffered saline (TBS) containing 0.001% H2O2 for 30 minutes. The hippocampus, cortex and hypothalamus were observed under a microscope (Olympus, Tokyo, Japan) and five specific areas in each region were captured. The numbers of NGB-positive cells and the integrated optical density (IOD) values of NGB labelling in the hippocampus, cortex and hypothalamus were measured by Image-Pro Plus 7.0 software (http://www.mediacy.com/ index.aspx?page¼IP_Premier; Media Cybernetics, Bethesda, MD) and histogram analysis using Origin 9.0 software (http:// www.originlab.com/; Northampton, MA). Each assessment was repeated at least three times. Quantitative PCR (Q-PCR) The hippocampus and cortex of the rats were dissected and total RNA was extracted using TRIzol reagent (Invitrogen/Life Technologies, Carlsbad, CA) and reverse transcribed using a Total RNA transcription kit (OMEGA, Japan) following the manufacturers’ instructions. A total of 100 ng mRNA was used to amplify the NGB gene by Q-PCR using the following primer pairs: NGB: forward 50 -AAGGGCGGTTCTCTGGGAGCTT30 , reverse 50 -AGAGGATGTGCAGGGCCAGCTT-30 ; -actin (internal loading control): forward 50 -GATGCAGCACGATC TCGGCGAA-30 , reverse 50 -TGGGAGCTCACGTTGTGGGG AA-30 . The relative quantification of NGB was performed with SDS 1.4 software according to the 2DDCT method. Western blot assay The hippocampus and cortex were separated and total protein was extracted and quantified with bicinchoninic (BCA) kits. Approximately 35 mg of each protein sample was fractionated by electrophoresis through 15% polyacrylamide gels and transferred to a polyvinylidene difluoride membrane following the manufacturer’s instructions. The membrane was probed with a rabbit-derived anti-NGB antibody (1:500 in TBS with Tween-20 [TBST], Santa Cruz Biotechnology) and a mouse-derived anti- -actin antibody (1:1,500 in TBST; Beijing Zhongshan Biotechnology, Beijing, China) for 1.5 hours at room temperature. Then, the membrane was incubated with horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit secondary antibody (1:5,000 in TBST; Beijing Zhongshan Biotechnology, at room temperature for 1 hour. Signals were developed with a chemiluminescence substrate luminol reagent (GE Healthcare, Little Chalfont, UK) and the blots were exposed to X-ray film to reveal the immunolabelled bands. The optical densities of the bands were scanned and quantified using ImageJ 1.46 software (http://rsb.info.nih.gov/ij/download.html; National Institutes of Health, Bethesda, MD). Each test was repeated at least three times.
DOI: 10.3109/02699052.2014.1002004
NGB up-regulation after ischaemic pre-conditioning
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Figure 1. TTC staining of brain slices. The lesion size was decreased with reperfusion time prolonging when compared with that of control.
Figure 2. Hippocampal NGB expression. (A): IHC assay of hippocampal NGB expression. (B): Histogram analysis of the number of NGB-positive hippocampal cells. (C): Histogram analysis of the IODs of NGB. The number of NGB-positive cells was significantly increased in animals subjected to 0.5-h ischemic preconditioning (*p50.05 and **p50.01 compared to 2-h ischemia/24-h reperfusion).
Statistical analysis All data are expressed as the mean ± standard deviation (SD). Statistical analysis was performed with one-way ANOVA using SPSS software (version 21.0, http://spss.en.softonic.
com/; Chicago, IL) and Student’s t-tests were performed in a group of two samples and p50.05 and p50.01 were considered to indicate significant differences and highly significant differences, respectively.
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Figure 3. Cortical NGB expression. (A): IHC assay of cortical NGB expression. (B): Histogram analysis of the number of NGB-positive cortical cells. (C): Histogram analysis of the IODs of NGB. The number of NGB-positive cellswas significantly increased in animals subjected to 0.5-h ischemic preconditioning (*p50.05 and **p50.01 compared to 2-h ischemia/24-h reperfusion).
Results The lesion size was decreased with reperfusion time prolong after 30 minutes of ischaemic pre-conditioning As TTC staining results display, the lesion size was decreased with reperfusion time after 30 minutes of ischaemic preconditioning and slightly increased at 48-hours reperfusion after 30 minutes of ischaemic pre-conditioning (Figure 1). Ischaemic pre-conditioning increased hippocampal NGB expression The number of positive cells in the hippocampus increased with reperfusion time and peaked at 12-hours reperfusion (Figure 2(A)), then slightly decreased at 48 hours reperfusion (Figure 2(B), p50.05, compared with 24-hours reperfusion after 30 minutes of ischaemic pre-conditioning). Likewise, in ischaemic pre-conditioning groups, hippocampal NGB expression was significantly increased with increasing reperfusion time and peaked at 24-hours reperfusion (p50.05 and p50.01 compared to 2-hours ischemia and 24-hours reperfusion), before it slightly decreased at 48-hours reperfusion (Figure 2(C), p50.05 and p50.01 compared with 24-hours reperfusion after 30 minutes ischaemic preconditioning).
Ischaemic pre-conditioning increased cortical NGB expression after reperfusion The number of NGB-positive cells in the cortex was increased with reperfusion time and peaked at 12-hours reperfusion (p50.05 and p50.01 compared to 2-hours of ischemia and 24hours reperfusion, Figure 3(A)), then slightly decreased at 48-hours reperfusion (Figure 3(B), p50.05 compared with 24-hours reperfusion after 30 minutes of ischaemic preconditioning). Likewise, NGB expression significantly increased in the pre-conditioning groups with increasing reperfusion time. It peaked at 24-hours reperfusion (p50.05 and p50.01 compared to 2-hours of ischaemia and 24-hours reperfusion) and then slightly decreased at 48-hours reperfusion (Figure 3(C), p50.05 and p50.01 compared with 24-hours reperfusion after 30 minutes of ischaemic pre-conditioning). Ischaemic pre-conditioning increased hypothalamic NGB expression In the hypothalamus, the number of NGB-positive cells was increased with reperfusion time and peaked at 24 hours reperfusion, p50.05 and p50.01, compared to 2-hours of ischaemia and 24-hours reperfusion (Figure 4(A)), then slightly decreased at 48-hours reperfusion (Figure 4(B), p50.05 compared with 24-hours reperfusion after 30 minutes
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DOI: 10.3109/02699052.2014.1002004
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Figure 4. Hypothalamic NGB expression. (A): IHC assay of hypothalamic NGB expression. (B): Histogram analysis of the number of NGB-positive cells. (C): Histogram analysis of the IOD of NGB. The number of NGB-positive cells was significantly increased in animals subjected to 0.5-h ischemic preconditioning (*p50.05 and **p50.01 compared to 2-h ischemia/24-h reperfusion).
of ischaemic pre-conditioning). Similarly, NGB expression in the ischaemic pre-conditioning groups was significantly increased with reperfusion time and peaked at 24 hours reperfusion (p50.05 and p50.01 compared to 2-hours of ischaemia and 24-hours reperfusion), then slightly decreased after 48-hours reperfusion (Figure 4(C), p50.05 and p50.01 compared with 24-hours reperfusion after 30 minutes of ischaemic pre-conditioning). Ischaemic pre-conditioning increased NGB mRNA and protein levels in the hippocampus and cortex In the hippocampus and cortex, NGB mRNA levels were significantly increased, with longer reperfusion time in animals subjected to 30 minutes of ischaemic pre-conditioning (p50.05 and p50.01 compared to 2-hours of ischemia and 24-hours reperfusion), before slightly decreasing at 48hours reperfusion (Figure 5, p50.05 compared with 24-hours reperfusion after 30 minutes of ischaemic pre-conditioning). NGB protein levels showed similar patterns in the hippocampus and cortex. In the hippocampus, NGB was upregulated after 30 minutes of ischaemic pre-conditioning when compared with control (2-hours ischemia and 24-hours reperfusion, p50.01) and levels increased with reperfusion time until 24-hours reperfusion, then slightly decreased at 48hours reperfusion (Figure 6(A), p50.01). In the cortex, NGB was upregulated after 30 minutes of ischaemic pre-conditioning when compared with control (2-hours ischemia and
24-hours reperfusion, p50.01), increased with reperfusion time up to 24-hours and then slightly decreased at 48-hours reperfusion (Figure 6(B), p50.01). This finding implies that ischaemic pre-conditioning up-regulates NGB, which may exert significant neuroprotection against ischaemic-hypoxic brain damage.
Discussion The results of the present study demonstrate that NGB expression was significantly up-regulated in the hippocampus, cortex and hypothalamus of MCAO rats that also underwent 30 minutes of ischaemic pre-conditioning until 24 hours reperfusion. mRNA and protein expression levels were also up-regulated in MCAO rats, peaked after 24-hours reperfusion and slightly decreased at 48-hours reperfusion. Thus, ischaemic pre-conditioning may increase NGB, which could have a neuroprotective effect against ischaemic brain damage. Ischaemic pre-conditioning was originally described by Murry et al. [13] in 1986; it is defined as the ability of short periods of ischaemia to make the myocardium more resistant to a subsequent ischaemic insult [14]. Murry et al. [13] reported that four 5-minute episodes of regional ischaemia in the canine myocardium, each followed by a 5-minute period of reperfusion, resulted in preservation of high-energy phosphates and significantly delayed or protected against myocardial necrosis during a subsequent prolonged period of ischaemia. Another study demonstrated that the human
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Figure 5. NGB mRNA levels in the hippocampus and cortex determined by Q-PCR. (A): NGB mRNA levels in the hippocampus. (B): NGB mRNA levels in the cortex. NGB mRNA transcription was significantly increased with increasing reperfusion time in animals subjected to 0.5-h ischemic preconditioning (*p50.05 and **p50.01 compared to 2-h ischemia/24-h reperfusion) and then slightly decreased after 48-h reperfusion (*p50.05 compared with 24-h reperfusion after 0.5-h ischemic preconditioning). (B)
(A)
β-actin (42 kD) NGB (17 kD)
(D)
(C)
β-actin (42 kD) NGB (17 kD)
Figure 6. NGB expression levels in the hippocampus and cortex by western blot and histogram analysis. (A): Western blot assay of hippocampal NGB expression. (B): Histogram analysis of hippocampal NGB expression in hippocampus. (C): Western blot assay of cortical NGB expression. (D): Histogram analysis of cortical NGB expression. It NGB expression was significantly increased with increasing reperfusion time in animals subjected to 0.5-h ischemic preconditioning (*p50.05 and **p50.01 compared to 2-h ischemia/24-h reperfusion) and then slightly decreased after 48-h reperfusion (*p50.05compared with 24-h reperfusion after 0.5-h ischemic preconditioning).
myocardium could be pre-conditioned and that ischaemic preconditioning may occur as a part of some naturally occurring ischaemic syndromes [15]. Furthermore, in addition to enhanced tolerance to lethal cell injury, pre-conditioning was
found to be protective against other end-points of ischaemiareperfusion injury, including post-ischaemic contractile dysfunction [16] and ischaemia- and reperfusion-induced ventricular arrhythmias [17–19]. Ischaemic pre-conditioning
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DOI: 10.3109/02699052.2014.1002004
also decreased apoptosis in animal models [20, 21], highlighting another mechanism that can contribute to cell death after myocardial ischaemia and reperfusion [22–24]. Since the original study, ischaemic pre-conditioning has been reproducibly demonstrated in many animal species, including rabbits [25], rats [26], mice [27] and pigs [28, 29]. In the present study, the ischaemic pre-conditioning was performed before rat MCAO. After 30 minutes of successive ischaemic preconditioning treatment, animals exhibited a normal behaviour and no or only mild hemiplegia; yet after 2 hours of subsequent ischaemia, the animals exhibited poor behaviour and contralateral hemiplegia. Notably, brain infarct size in the preconditioning group was lower than that of the 2-hour ischaemic and 24-hour reperfusion groups as assessed with triphenyl tetrazolium chloride (TTC) staining (data not shown). This suggests that ischaemic pre-conditioning had a significant neuroprotective effect against ischaemic brain damage. In conclusion, it was found that NGB was up-regulated after ischaemic pre-conditioning and MCAO. The results provide a basis for further studies into the neuroprotective mechanism(s) of ischaemic pre-conditioning.
Declaration of interest The authors report no conflicts of interest. This research project was sponsored by and supported by the International Science and Technology Cooperation Foundation of the Ministry of Science and Technology of China, (No.2008DFA 31850), the International Cooperation of Science and Technique Foundation of Beijing (2007G05), the Beijing Chinese medicine projects (Grant no. JJ2005-17) and the National Natural Science Foundation of China (Grant no.81100862).
References 1. Chen Y, Yi Q, Liu G, Shen X, Xuan L, Tian Y. Cerebral white matter injury and damage to myelin sheath following whole-brain ischemia. Brain Research 2013;1495:11–17. 2. Hagberg H, Lehmann A, Andine P, Hamberger A, Sandberg M, Nordborg C, Kjellmer I, Thiringer K. [Excitatory amino acids and brain damage in ischemia and asphyxia]. Lakartidningen 1988;85: 1427–1429. 3. Liu XQ, Sheng R, Qin ZH. The neuroprotective mechanism of brain ischemic preconditioning. Acta Pharmacologica Sinica 2009;30: 1071–1080. 4. Galle AA, Jones NM. The neuroprotective actions of hypoxic preconditioning and postconditioning in a neonatal rat model of hypoxic-ischemic brain injury. Brain Research 2013;1498:1–8. 5. Raval AP, Lin HW, Dave KR, Defazio RA, Della Morte D, Kim EJ, Perez-Pinzon MA. Resveratrol and ischemic preconditioning in the brain. Current Medicine and Chemistry 2008;15:1545–1551. 6. Kersten JR, Schmeling TJ, Pagel PS, Gross GJ, Warltier DC. Isoflurane mimics ischemic preconditioning via activation of K(ATP) channels: reduction of myocardial infarct size with an acute memory phase. Anesthesiology 1997;87:361–370. 7. Burmester T, Weich B, Reinhardt S, Hankeln T. A vertebrate globin expressed in the brain. Nature 2000;407:520–523. 8. Venis S. Neuroglobin might protect brain cells during stroke. Lancet 2001;358:2055. 9. Garry DJ, Mammen PP. Neuroprotection and the role of neuroglobin. Lancet 2003;362:342–343. 10. Brunori M, Vallone B. Neuroglobin, seven years after. Cell Molecular Life Science 2007;64:1259–1268. 11. Jin K, Mao Y, Mao X, Xie L, Greenberg DA. Neuroglobin expression in ischemic stroke. Stroke 2010;41:557–559.
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12. Allcock JM. Occlusion of the middle cerebral artery: serial angiography as a guide to conservative therapy. Journal of Neurosurgery 1967;27:353–363. 13. Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 1986;74:1124–1136. 14. Murry CE, Richard VJ, Reimer KA, Jennings RB. Ischemic preconditioning slows energy metabolism and delays ultrastructural damage during a sustained ischemic episode. Circulation Research 1990;66:913–931. 15. Lim R, Laskey WK. Ischemic preconditioning in unstable coronary syndromes: evidence for time dependence. Journal of the American College of Cardiology 1997;30:1461–1465. 16. Cave AC, Apstein CS. Preconditioning-induced protection against post-ischemic contractile dysfunction: inhibitory effect of tissue washout. Basic Research in Cardiology 1996;91:16–19. 17. Dow J, Bhandari A, Simkhovich BZ, Hale SL, Kloner RA. The effect of acute versus delayed remote ischemic preconditioning on reperfusion induced ventricular arrhythmias. Journal of Cardiovascular Electrophysiology 2012;23:1374–1383. 18. Riha H, Neckar J, Papousek F, Netuka I, Pirk J, Kolar F, Ostadal B. Suppression of ischemic and reperfusion ventricular arrhythmias by inhalational anesthetic-induced preconditioning in the rat heart. Physiology Research 2011;60:709–714. 19. Kaneko H, Anzai T, Naito K, Kohno T, Maekawa Y, Takahashi T, Kawamura A, Yoshikawa T, Ogawa S. Role of ischemic preconditioning and inflammatory response in the development of malignant ventricular arrhythmias after reperfused ST-elevation myocardial infarction. Journal of Cardiac Failure 2009;15: 775–781. 20. Racay P, Chomova M, Tatarkova Z, Kaplan P, Hatok J, Dobrota D. Ischemia-induced mitochondrial apoptosis is significantly attenuated by ischemic preconditioning. Cell Molecualr Neurobiology 2009;29:901–908. 21. Iliodromitis EK, Lazou A, Kremastinos DT. Ischemic preconditioning: protection against myocardial necrosis and apoptosis. Vascular Health Risk Management 2007;3:629–637. 22. Ordonez AN, Jessick VJ, Clayton CE, Ashley MD, Thompson SJ, Simon RP, Meller R. Rapid ischemic tolerance induced by adenosine preconditioning results in Bcl-2 interacting mediator of cell death (Bim) degradation by the proteasome. International Journal of Physiology, Pathophysiology and Pharmacology 2010;2: 36–44. 23. Drognitz O, Liu X, Obermaier R, Neeff H, von Dobschuetz E, Hopt UT, Benz S. Ischemic preconditioning fails to improve microcirculation but increases apoptotic cell death in experimental pancreas transplantation. Transplant International 2004;17:317–324. 24. Zhang C, Rosenbaum DM, Shaikh AR, Li Q, Rosenbaum PS, Pelham DJ, Roth S. Ischemic preconditioning attenuates apoptotic cell death in the rat retina. Investigative Ophthalmology and Visual Science 2002;43:3059–3066. 25. Sang H, Cao L, Qiu P, Xiong L, Wang R, Yan G. Isoflurane produces delayed preconditioning against spinal cord ischemic injury via release of free radicals in rabbits. Anesthesiology 2006; 105:953–960. 26. Li H, Jin M, Lv T, Guan J. Mechanism of focal cerebral ischemic tolerance in rats with ischemic preconditioning involves MyD88and TRIF-dependent pathways. Experimental Therapy and Medicine 2013;6:1375–1379. 27. Stringa P, Romanin D, Lausada N, Machuca M, Raimondi JC, Cabanne A, Rumbo M, Gondolesi G. Ischemic preconditioning and tacrolimus pretreatment as strategies to attenuate intestinal ischemia-reperfusion injury in mice. Transplant Proceedings 2013;45: 2480–2485. 28. Kyrou IE, Papakostas JC, Ioachim E, Skoufi U, Koulouras V, Arnaoutoglou E, Angelidis C, Matsagkas MI. Spinal cord early ischemic preconditioning activates the stabilized fraction of betacatenin after thoracoabdominal aortic occlusion in pigs. Annals of Vascular Surgery 2013;27:480–486. 29. Pan MX, Kong FD, Wu ZF, Zhang Y, Wang HL, Li AH, Xie JM, Gao Y. [Protective effect of donator liver ischemic preconditioning on posttransplant liver graft function in Chinese miniature pigs]. Nan Fang Yi Ke Da Xue Xue Bao 2007;27:250–253.
ORIGINAL ARTICLE
Neuroglobin up-regulation after ischaemic pre-conditioning in a rat model of middle cerebral artery occlusion Yichen Liu1,2, Baoquan Li2, Qiaojun Li1, & Liping Zou1 Department of Pediatrics, General Hospital of Chinese People’s Liberation Army, Beijing, PR China and 2Department of Pediatrics, 159th Hospital of Chinese People’s Liberation Army, Zhumadian, Henan Province, PR China
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Abstract
Keywords
Primary objective: Neuroglobin (NGB) is a known neuroprotector and is up-regulated after ischaemia-hypoxia brain damage. However, no studies have investigated NGB levels after ischaemic pre-conditioning and middle cerebral artery occlusion (MCAO). Methods and procedures: This study subjected rats to different ischaemic pre-conditioning and MCAO regimens and assayed NGB levels in the hippocampus, cortex and hypothalamus by immunohistochemistry, quantitative polymerase chain reaction (PCR) and western blot. Main outcomes and results: After 30 minutes of ischaemic pre-conditioning, the number of NGB-positive cells and NGB levels in the hippocampus, cortex and hypothalamus were increased with longer reperfusion times, peaked at 24-hours reperfusion and slightly decreased at 48-hours reperfusion. Similarly, the mRNA and protein expression levels of NGB were also up-regulated; they peaked at 24-hours reperfusion and slightly decreased at 48-hours reperfusion. Conclusions: NGB may regulate neuroprotection against ischaemia and hypoxia-mediated brain damage after ischaemic pre-conditioning. The results provide additional evidence supporting the utility of ischaemic pre-conditioning and help elucidate its potential regulatory mechanism.
Ischaemic pre-conditioning, middle cerebral artery occlusion, neuroglobin, neuroprotection, up-regulation
Introduction Ischaemic brain damage is a prevalent condition and a leading cause of morbidity and mortality worldwide; it is primarily the result of acute brain ischaemia-reperfusion injuries [1]. It occurs when excessive excitatory amino acid releases lead to intracellular calcium influx, electrophysiological and metabolic dysfunction, lipid peroxidation and other oxidative processes [2]. The identification of endogenous neuroprotective mechanisms in the brain, such as those that can be activated with ischaemic pre-conditioning [3] and postconditioning [4], have drawn increased attention due to the disappointing results of pharmacological neuroprotective strategies in a variety of clinical trials. Ischaemic pre-conditioning is an endogenous phenomenon induced by sub-lethal ischaemia that leads to adaptive responses that protect against future severe ischaemic injuries. It is comprised of acute and delayed phases [5]. The acute phase develops within a few minutes and wanes after 2–3 hours [6] and the delayed phase appears within a few hours and lasts for several days. There are several proteins involved in this process, including potassium channels, caspase-3, heat shock protein (HSP)70, N-methyl-D-aspartate (NMDA) Correspondence: Liping Zou, Department of Pediatrics, General Hospital of Chinese People’s Liberation Army, Beijing, PR China, 100853. Tel: +86-10-68025680. Fax: +86-10-68025680. E-mail: [email protected]
History Received 7 June 2014 Revised 6 November 2014 Accepted 20 December 2014 Published online 27 January 2015
receptors, protein kinase C, superoxide dismutase, tumour necrosis factor (TNF)- , nuclear factor (NF)-B, hypoxiainducible factor (HIF)-1 and erythropoietin. However, no study has described alterations in neuroglobin (NGB) levels following ischaemic pre-conditioning and ischaemic brain damage. The role of NGB as a neuroprotective factor against ischaemic-hypoxic brain damage was first described by Burmester et al. [7] in 2000. The monomeric protein is highly expressed in the central nervous system and exhibits high oxygen affinity. The oxygen-binding properties of NGB are comparable to those of a typical vertebrate myoglobin, suggesting a similar function of NGB in the brain [8]. Later studies revealed that NGB could reversibly bind oxygen and that it plays an important role in maintaining oxygen homeostasis in neural tissues [9]. One report found that NGB was up-regulated under hypoxic conditions and had neuroprotective effects in vitro and in vivo [10]. The existing evidence suggests that NGB might be involved in the pathophysiological course of ischaemic and hypoxic injury [11]. It was hypothesized that NGB would exert a significant neuroprotective effect against ischaemic brain injury and that ischaemic pre-conditioning can further up-regulate its expression. The results indicate that NGB expression is increased after ischaemic pre-conditioning and middle cerebral artery occlusion (MCAO) and they suggest that further studies should investigate the neuroprotective effects and underlying mechanisms of ischaemic pre-conditioning.
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Materials and methods
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Experimental animals and groups A total of 36 adult specific pathogen-free Sprague Dawley rats (SD, male, 250–330 g) were purchased and raised in the Laboratory Animal Center of the General Hospital of the People’s Liberation Army (House temperature: 25 ± 2 C, Humidity: 40–60%, 12-hour light/dark cycle) and randomly divided into six groups, including control MCAO (2-hour/24hour ischaemic and reperfusion), 30 minutes + 2-hours/2hours (30 minutes ischaemic pre-conditioning plus 2-hours ischaemia and 2-hours reperfusion), 30 minutes + 2-hours/6hours (30 minutes ischaemic pre-conditioning plus 2-hours ischaemia and 6-hours reperfusion), 30 minutes + 2-hours/12hours (30 minutes ischaemic pre-conditioning plus 2-hours ischaemia and 12-hours reperfusion), 30 minutes + 2-hours/ 24-hours (30 minutes ischaemic pre-conditioning plus 2-hours ischaemia and 24-hours reperfusion), 30 minutes + 2-hours/ 48-hours (30 minutes ischaemic pre-conditioning plus 2-hours ischaemia and 48-hours reperfusion). This experiment was approved by the Experiment and Chinese People’s Liberation Army General Hospital Ethics committees. Ischaemic pre-conditioning and MCAO Ischaemic pre-conditioning of SD rats was performed as previously described [12]. The SD rats were anaesthetized with diethyl ether and the four limbs fixed before an incision was made along the neck midline. The thyroid, vein and nerve tissues were stripped to expose the left carotid artery communis, which was ligated with 5/0 surgical line for 30 minutes of ischaemia and then the line was removed to allow reperfusion for 24 hours. Subsequently, the animals were subjected to ischemia for 2 hours followed by 2-, 6-, 12-, 24-, or 48-huor reperfusion according to the method described earlier. The control group was just subjected to 2 hours of ischemia and 24-hour reperfusion. Afterwards, the rats killed by dislocation to prepare brain tissues for slicing and further identification. TTC staining The rats’ brain tissues were collected and washed with normal saline for 5 minutes and frozen for 20 minutes at 20 C. Then, the brain were transferred to a brain sectioning mould and cut into slices (2 mm) and the sections were put in 1% 2,3,5-triphenyl four azole nitrogen chloride (TTC) at 37 C for 30 minutes (turn once per 5 minutes). Finally, the sections were washed with ddH2O three times and the images gathered for further analysis. Immunohistochemistry (IHC) The rats’ brain tissues were cut into slices (1 mm) and the sections were deparaffinized, rehydrated, post-fixed with 4% paraformaldehyde for 10 minutes and then washed three times with 0.01 M phosphate-buffered saline (PBS). Endogenous peroxidase was inactivated by incubating the sections in 3% H2O2 for 30 minutes. The sections were subjected to sequential incubations with 10% normal goat serum in 0.01 M PBS for
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30 minutes at room temperature. They were then incubated in rabbit anti-NGB monoclonal (SC-30144, 1: 200; Santa Cruz Biotechnology, Santa Cruz, CA) in PBS containing 0.3% Triton X-100 overnight at 4 C. The sections were washed three times for 5 minutes each with PBS and then incubated in peroxidase-conjugated goat anti-rabbit IgG (1:200; Zymed, South San Francisco, CA) for 1 hour at room temperature. Finally, the sections were developed with diaminobenzidine (Sigma, St. Louis, MO) in 0.1 M Tris-buffered saline (TBS) containing 0.001% H2O2 for 30 minutes. The hippocampus, cortex and hypothalamus were observed under a microscope (Olympus, Tokyo, Japan) and five specific areas in each region were captured. The numbers of NGB-positive cells and the integrated optical density (IOD) values of NGB labelling in the hippocampus, cortex and hypothalamus were measured by Image-Pro Plus 7.0 software (http://www.mediacy.com/ index.aspx?page¼IP_Premier; Media Cybernetics, Bethesda, MD) and histogram analysis using Origin 9.0 software (http:// www.originlab.com/; Northampton, MA). Each assessment was repeated at least three times. Quantitative PCR (Q-PCR) The hippocampus and cortex of the rats were dissected and total RNA was extracted using TRIzol reagent (Invitrogen/Life Technologies, Carlsbad, CA) and reverse transcribed using a Total RNA transcription kit (OMEGA, Japan) following the manufacturers’ instructions. A total of 100 ng mRNA was used to amplify the NGB gene by Q-PCR using the following primer pairs: NGB: forward 50 -AAGGGCGGTTCTCTGGGAGCTT30 , reverse 50 -AGAGGATGTGCAGGGCCAGCTT-30 ; -actin (internal loading control): forward 50 -GATGCAGCACGATC TCGGCGAA-30 , reverse 50 -TGGGAGCTCACGTTGTGGGG AA-30 . The relative quantification of NGB was performed with SDS 1.4 software according to the 2DDCT method. Western blot assay The hippocampus and cortex were separated and total protein was extracted and quantified with bicinchoninic (BCA) kits. Approximately 35 mg of each protein sample was fractionated by electrophoresis through 15% polyacrylamide gels and transferred to a polyvinylidene difluoride membrane following the manufacturer’s instructions. The membrane was probed with a rabbit-derived anti-NGB antibody (1:500 in TBS with Tween-20 [TBST], Santa Cruz Biotechnology) and a mouse-derived anti- -actin antibody (1:1,500 in TBST; Beijing Zhongshan Biotechnology, Beijing, China) for 1.5 hours at room temperature. Then, the membrane was incubated with horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit secondary antibody (1:5,000 in TBST; Beijing Zhongshan Biotechnology, at room temperature for 1 hour. Signals were developed with a chemiluminescence substrate luminol reagent (GE Healthcare, Little Chalfont, UK) and the blots were exposed to X-ray film to reveal the immunolabelled bands. The optical densities of the bands were scanned and quantified using ImageJ 1.46 software (http://rsb.info.nih.gov/ij/download.html; National Institutes of Health, Bethesda, MD). Each test was repeated at least three times.
DOI: 10.3109/02699052.2014.1002004
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Figure 1. TTC staining of brain slices. The lesion size was decreased with reperfusion time prolonging when compared with that of control.
Figure 2. Hippocampal NGB expression. (A): IHC assay of hippocampal NGB expression. (B): Histogram analysis of the number of NGB-positive hippocampal cells. (C): Histogram analysis of the IODs of NGB. The number of NGB-positive cells was significantly increased in animals subjected to 0.5-h ischemic preconditioning (*p50.05 and **p50.01 compared to 2-h ischemia/24-h reperfusion).
Statistical analysis All data are expressed as the mean ± standard deviation (SD). Statistical analysis was performed with one-way ANOVA using SPSS software (version 21.0, http://spss.en.softonic.
com/; Chicago, IL) and Student’s t-tests were performed in a group of two samples and p50.05 and p50.01 were considered to indicate significant differences and highly significant differences, respectively.
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Figure 3. Cortical NGB expression. (A): IHC assay of cortical NGB expression. (B): Histogram analysis of the number of NGB-positive cortical cells. (C): Histogram analysis of the IODs of NGB. The number of NGB-positive cellswas significantly increased in animals subjected to 0.5-h ischemic preconditioning (*p50.05 and **p50.01 compared to 2-h ischemia/24-h reperfusion).
Results The lesion size was decreased with reperfusion time prolong after 30 minutes of ischaemic pre-conditioning As TTC staining results display, the lesion size was decreased with reperfusion time after 30 minutes of ischaemic preconditioning and slightly increased at 48-hours reperfusion after 30 minutes of ischaemic pre-conditioning (Figure 1). Ischaemic pre-conditioning increased hippocampal NGB expression The number of positive cells in the hippocampus increased with reperfusion time and peaked at 12-hours reperfusion (Figure 2(A)), then slightly decreased at 48 hours reperfusion (Figure 2(B), p50.05, compared with 24-hours reperfusion after 30 minutes of ischaemic pre-conditioning). Likewise, in ischaemic pre-conditioning groups, hippocampal NGB expression was significantly increased with increasing reperfusion time and peaked at 24-hours reperfusion (p50.05 and p50.01 compared to 2-hours ischemia and 24-hours reperfusion), before it slightly decreased at 48-hours reperfusion (Figure 2(C), p50.05 and p50.01 compared with 24-hours reperfusion after 30 minutes ischaemic preconditioning).
Ischaemic pre-conditioning increased cortical NGB expression after reperfusion The number of NGB-positive cells in the cortex was increased with reperfusion time and peaked at 12-hours reperfusion (p50.05 and p50.01 compared to 2-hours of ischemia and 24hours reperfusion, Figure 3(A)), then slightly decreased at 48-hours reperfusion (Figure 3(B), p50.05 compared with 24-hours reperfusion after 30 minutes of ischaemic preconditioning). Likewise, NGB expression significantly increased in the pre-conditioning groups with increasing reperfusion time. It peaked at 24-hours reperfusion (p50.05 and p50.01 compared to 2-hours of ischaemia and 24-hours reperfusion) and then slightly decreased at 48-hours reperfusion (Figure 3(C), p50.05 and p50.01 compared with 24-hours reperfusion after 30 minutes of ischaemic pre-conditioning). Ischaemic pre-conditioning increased hypothalamic NGB expression In the hypothalamus, the number of NGB-positive cells was increased with reperfusion time and peaked at 24 hours reperfusion, p50.05 and p50.01, compared to 2-hours of ischaemia and 24-hours reperfusion (Figure 4(A)), then slightly decreased at 48-hours reperfusion (Figure 4(B), p50.05 compared with 24-hours reperfusion after 30 minutes
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DOI: 10.3109/02699052.2014.1002004
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Figure 4. Hypothalamic NGB expression. (A): IHC assay of hypothalamic NGB expression. (B): Histogram analysis of the number of NGB-positive cells. (C): Histogram analysis of the IOD of NGB. The number of NGB-positive cells was significantly increased in animals subjected to 0.5-h ischemic preconditioning (*p50.05 and **p50.01 compared to 2-h ischemia/24-h reperfusion).
of ischaemic pre-conditioning). Similarly, NGB expression in the ischaemic pre-conditioning groups was significantly increased with reperfusion time and peaked at 24 hours reperfusion (p50.05 and p50.01 compared to 2-hours of ischaemia and 24-hours reperfusion), then slightly decreased after 48-hours reperfusion (Figure 4(C), p50.05 and p50.01 compared with 24-hours reperfusion after 30 minutes of ischaemic pre-conditioning). Ischaemic pre-conditioning increased NGB mRNA and protein levels in the hippocampus and cortex In the hippocampus and cortex, NGB mRNA levels were significantly increased, with longer reperfusion time in animals subjected to 30 minutes of ischaemic pre-conditioning (p50.05 and p50.01 compared to 2-hours of ischemia and 24-hours reperfusion), before slightly decreasing at 48hours reperfusion (Figure 5, p50.05 compared with 24-hours reperfusion after 30 minutes of ischaemic pre-conditioning). NGB protein levels showed similar patterns in the hippocampus and cortex. In the hippocampus, NGB was upregulated after 30 minutes of ischaemic pre-conditioning when compared with control (2-hours ischemia and 24-hours reperfusion, p50.01) and levels increased with reperfusion time until 24-hours reperfusion, then slightly decreased at 48hours reperfusion (Figure 6(A), p50.01). In the cortex, NGB was upregulated after 30 minutes of ischaemic pre-conditioning when compared with control (2-hours ischemia and
24-hours reperfusion, p50.01), increased with reperfusion time up to 24-hours and then slightly decreased at 48-hours reperfusion (Figure 6(B), p50.01). This finding implies that ischaemic pre-conditioning up-regulates NGB, which may exert significant neuroprotection against ischaemic-hypoxic brain damage.
Discussion The results of the present study demonstrate that NGB expression was significantly up-regulated in the hippocampus, cortex and hypothalamus of MCAO rats that also underwent 30 minutes of ischaemic pre-conditioning until 24 hours reperfusion. mRNA and protein expression levels were also up-regulated in MCAO rats, peaked after 24-hours reperfusion and slightly decreased at 48-hours reperfusion. Thus, ischaemic pre-conditioning may increase NGB, which could have a neuroprotective effect against ischaemic brain damage. Ischaemic pre-conditioning was originally described by Murry et al. [13] in 1986; it is defined as the ability of short periods of ischaemia to make the myocardium more resistant to a subsequent ischaemic insult [14]. Murry et al. [13] reported that four 5-minute episodes of regional ischaemia in the canine myocardium, each followed by a 5-minute period of reperfusion, resulted in preservation of high-energy phosphates and significantly delayed or protected against myocardial necrosis during a subsequent prolonged period of ischaemia. Another study demonstrated that the human
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Figure 5. NGB mRNA levels in the hippocampus and cortex determined by Q-PCR. (A): NGB mRNA levels in the hippocampus. (B): NGB mRNA levels in the cortex. NGB mRNA transcription was significantly increased with increasing reperfusion time in animals subjected to 0.5-h ischemic preconditioning (*p50.05 and **p50.01 compared to 2-h ischemia/24-h reperfusion) and then slightly decreased after 48-h reperfusion (*p50.05 compared with 24-h reperfusion after 0.5-h ischemic preconditioning). (B)
(A)
β-actin (42 kD) NGB (17 kD)
(D)
(C)
β-actin (42 kD) NGB (17 kD)
Figure 6. NGB expression levels in the hippocampus and cortex by western blot and histogram analysis. (A): Western blot assay of hippocampal NGB expression. (B): Histogram analysis of hippocampal NGB expression in hippocampus. (C): Western blot assay of cortical NGB expression. (D): Histogram analysis of cortical NGB expression. It NGB expression was significantly increased with increasing reperfusion time in animals subjected to 0.5-h ischemic preconditioning (*p50.05 and **p50.01 compared to 2-h ischemia/24-h reperfusion) and then slightly decreased after 48-h reperfusion (*p50.05compared with 24-h reperfusion after 0.5-h ischemic preconditioning).
myocardium could be pre-conditioned and that ischaemic preconditioning may occur as a part of some naturally occurring ischaemic syndromes [15]. Furthermore, in addition to enhanced tolerance to lethal cell injury, pre-conditioning was
found to be protective against other end-points of ischaemiareperfusion injury, including post-ischaemic contractile dysfunction [16] and ischaemia- and reperfusion-induced ventricular arrhythmias [17–19]. Ischaemic pre-conditioning
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DOI: 10.3109/02699052.2014.1002004
also decreased apoptosis in animal models [20, 21], highlighting another mechanism that can contribute to cell death after myocardial ischaemia and reperfusion [22–24]. Since the original study, ischaemic pre-conditioning has been reproducibly demonstrated in many animal species, including rabbits [25], rats [26], mice [27] and pigs [28, 29]. In the present study, the ischaemic pre-conditioning was performed before rat MCAO. After 30 minutes of successive ischaemic preconditioning treatment, animals exhibited a normal behaviour and no or only mild hemiplegia; yet after 2 hours of subsequent ischaemia, the animals exhibited poor behaviour and contralateral hemiplegia. Notably, brain infarct size in the preconditioning group was lower than that of the 2-hour ischaemic and 24-hour reperfusion groups as assessed with triphenyl tetrazolium chloride (TTC) staining (data not shown). This suggests that ischaemic pre-conditioning had a significant neuroprotective effect against ischaemic brain damage. In conclusion, it was found that NGB was up-regulated after ischaemic pre-conditioning and MCAO. The results provide a basis for further studies into the neuroprotective mechanism(s) of ischaemic pre-conditioning.
Declaration of interest The authors report no conflicts of interest. This research project was sponsored by and supported by the International Science and Technology Cooperation Foundation of the Ministry of Science and Technology of China, (No.2008DFA 31850), the International Cooperation of Science and Technique Foundation of Beijing (2007G05), the Beijing Chinese medicine projects (Grant no. JJ2005-17) and the National Natural Science Foundation of China (Grant no.81100862).
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