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Acta Biochim Biophys
Sin 2008, 40: 803-810 |
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doi:10.1111/j.1745-7270.2008.00461.x |
Up-regulation of heme oxygenase-1 by isoflurane preconditioning during tolerance� against neuronal injury induced by oxygen glucose deprivation
Qifang Li, Yesen Zhu, Hong Jiang*, Hui Xu, and Heping Liu
Department of Anesthesiology, Shanghai Ninth
People�s Hospital Affiliated to Shanghai Jiao Tong University School of
Medicine, Shanghai 200011, China
Received: March 16,
2008�������
Accepted: June 19,
2008
This work was supported
by grants from the Nature Science Foundation of Shanghai Science Committee
(Nos. 07ZR14069 and 074119626), the Science Foundation of Shanghai Education
Committee (No. 08YZ35), the Youth Science Foundation of Shanghai Health Bureau
(No. 2007Y34) and Shanghai Rising-Star Program (No. 08QA14044)
*Corresponding
author: Tel, 86-21-63138341; Fax, 86-21-53591385; E-mail, [email protected]
Heme oxygenase (HO) is the rate-limiting
enzyme in the degradation� of heme to produce bile pigments and carbon monoxide.
The HO-1 isozyme is induced by a variety of factors� such as heat, heme,
ischemia, and hydrogen peroxide. In recent� years, mounting findings have
suggested that HO-1 has a neuroprotective activity against ischemic injury. The
neuroprotective role of isoflurane, a commonly used anesthetic, has been well
documented, but little is known about the underlying mechanisms involved.
Recently, isoflurane has been shown to up-regulate HO-1 in the liver. In this
study, we show that isoflurane preconditioning promotes the survival� of
cultured ischemic hippocampal neurons by increasing the number of surviving
neurons and their viability. Further study by reverse transcription-polymerase
chain reaction and Western� blot analysis showed that isoflurane preconditioning�
significantly increases HO-1 expression in oxygen glucose deprivation
(OGD)-induced neuronal injury. Furthermore, inhibition of HO activity by tin
protoporphyrin partially abolishes� isoflurane preconditioning�s protective
effect as measured by lactate dehydrogenase release in OGD neurons. These
findings indicated that the neuroprotective role of isoflurane preconditioning
against OGD-induced injury might be associated with its role in up-regulating
HO-1 in ischemic neurons.
Keywords������� isoflurane; heme oxygenase-1; ischemia; primary hippocampal neuron
It is well known that volatile anesthetics can protect the brain. This effect can be exploited clinically during neurosurgical� procedures and is a well-known confounding� factor in cerebral ischemia research, in which experimental� ischemia has been induced with volatile anesthetics [1]. The fluorinated volatile anesthetic isoflurane protects the brain when administered during or immediately before an ischemic insult to the brain [2]. Isoflurane can induce the expression of immediate-early genes in several organs including� the brain [3]. Since many immediate-early genes act as transcription factors, it is tempting to speculate that isoflurane anesthesia induces transcriptional and translational� events that affect the organism long after termination� of anesthesia and that washout or metabolize the compound. Accumulating evidence has suggested that the heme oxygenase (HO) enzyme system plays a pivotal role in the maintenance of cellular function in nearly all organ systems, including the brain, after a sublethal stress [4].
HO catalyzes the conversion of heme to carbon monoxide, free ferrous iron, and biliverdin; the latter is rapidly converted to bilirubin by biliverdin reductase [5]. Among HO�s three isoforms, HO-1 is an ubiquitous and redox-sensitive inducible stress protein that is strongly induced� by various stimuli, including heme, heavy metal, cytokines, hormones, endotoxins, heat shock, and hypoxic-ischemic� injury, while HO-2 is the constitutive form [6]. HO-3 has been identified, but its function is still unknown [7]. Both neuronal and non-neuronal brain cells exhibit the ability to rapidly up-regulate HO-1 at the transcriptional and posttranscriptional level in response to noxious stimuli [8]. Furthermore, HO-1 has been shown to protect� the brain from acute excitotoxicity [9]. Reduced HO-1 protein� expression is associated with more severe neuro�degeneration after cortical compression-induced transient� ischemia in diabetic Goto-Kakizaki rats [10]. It has also been reported that overexpression of HO-1 is neuro�protective in a model of permanent middle cerebral artery occlusion in transgenic mice [11].
Isoflurane preconditioning has been shown to provide neuroprotection against hypoxic-ischemic injury in rats and neurons [2]. However, the molecular mechanisms underlying� this phenomenon are still poorly understood. Recently, studies have shown that isoflurane leads to an expression of HO-1 in vivo and in vitro [12-14]. So it is tempting to speculate that HO-1 could be involved in neuroprotection. To elucidate the role of HO-1 during isoflurane-induced neuroprotection against ischemic injury, we therefore characterized the effects of isoflurane preconditioning on the expression of HO-1 as well as cell viability and lactate dehydrogenase (LDH) release� in ischemic neurons.
Materials and Methods
Animals
The experimental protocol used in this study was approved by the Ethics Committee for Animal Experimentation of Shanghai Jiao Tong University School of Medicine (Shanghai, China) and was conducted according to the Guidelines for Animal Experimentation of Shanghai Jiao Tong University School of Medicine. Wistar rats were purchased within 24 h of their birth from the Laboratory Animal Center, Shanghai Jiao Tong University School of Medicine.
Hippocampal neuron culture
Brain cell cultures were obtained from the cerebral hippocampi� of newborn Wistar rats [15,16]. Newborn rats were euthanized, and the hippocampi were removed from the brain and dissected in ice-cold neurobasal medium. They were digested with 0.125% trypsin for 20 min at 37 �C. Then, each hippocampus was dissociated by repeated� pipetting and filtered through a Falcon cell strainer (BD Biosciences, San Jose, USA). Cells were plated at a density of 5�105/ml on poly-D-lysine-coated dishes and maintained in neurobasal medium, serum-free B-27 supplement� (Life Technologies/BRL, Rockville, USA), 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine. Glutamate (25 mM) was added during the first 3 d in vitro. The cultures consisted of more than 92% neurons as identified by double immunostaining with antibodies� against microtubule-associated protein-2 and glial fibrillary acidic protein (Zymed, San Francisco, USA). Cultures were kept at 36.5 �C in a 5% CO2 humidified incubator with culture the neurobasal medium, but without� glutamate, and half of the medium was replaced twice a week. The cultures were used at 10 d in vitro.
Isoflurane preconditioning
Isoflurane preconditioning was performed with the neuron�-enriched cultures after 10 d in vitro at 36.5 �C in a closed chamber in an atmosphere of 1.4% isoflurane (one minimum� alveolar concentration for rat), 5% CO2, 20% O2, and N2 (remainder) for a 3 h period [17]. In the present study, cells were divided into four groups: isoflurane only, isoflurane+oxygen-glucose deprivation (OGD), control only and control+OGD. Cells treated identically but without� the addition of isoflurane served as controls. OGD was then performed with the pretreated or control cells 24 h later.
OGD treatment
To mimic cerebral ischemia in vitro, OGD was performed with the pretreated or control cells 24 h after isoflurane preconditioning [17]. For OGD, medium was first removed from the cultures and stored, and then the cultures were rinsed twice with phosphate-buffered saline without Ca2+/Mg2+. In an anoxia chamber, cultures were subjected to OGD for 2 h by rinsing twice in glucose-free balanced salt solution as described below and covered with the same glucose-free balanced salt solution (121.7 mM NaCl, 0.8 mM MgSO4, 20.7 mM NaHCO3, 5.5 mM KHCO3, 1 mM NaH2PO4, 1.8 mM CaCl2, 0.01 mM glycine, and 10 mM HEPES at pH 7.4) preequilibrated with the atmosphere of the chamber (95% N2 and 5% CO2). Control cells were incubated in the same solution with glucose under normoxic conditions (in a CO2 incubator). After OGD, cultures were removed from the anoxia chamber, and 1:1 mixture of the preserved pre-OGD medium� and fresh neurobasal medium with B-27 supplement replaced the balanced salt solution. Cultures were maintained in a CO2 incubator for the next 24 h.
Assessment of hippocampal neuron viability and injury
Neuronal survival rate was analyzed by trypan blue exclusion� as described [18]. Neuron death was determined by measuring reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). Briefly, after OGD in each group, 0.5 mg/ml MTT (final concentration) was added to the assays for 4 h at 37 �C. The amount of MTT formazan, quantified by calorimetrically determining its absorbance at 570 nm using a microplate reader (Tecan Group Ltd, M�nnedorf, Swiss), was dissolved by dimethyl� sulfoxide. Experiments were performed in triplicate and repeated at least three batches of cultures. The effects of OGD time course on the viability of the primary hippo�campal neuron cultures were also assessed. We quantitatively assessed neuronal injury by measuring LDH activity in the medium 24 h after the injury [19]. Enzyme standard was obtained from Sigma (St. Louis, USA). In each experiment, the results of the LDH measurements from the controls (without isoflurane or OGD treatment) were set at 100%. Isoflurane preconditioning alone did not change the LDH release compared with the control. The results from the sister cultures subjected to both isoflurane preconditioning and/or OGD were then calculated as a percentage of the control. In addition, 50 mM tin protoporphyrin (SnPP) (Frontier Scientific, Lancashire, UK), an inhibitor of HO activity, was added into the medium 30 min prior to isoflurane treatment to investigate the role of HO-1 in the isoflurane preconditioning-induced neuroprotection as measured by LDH release in OGD neurons� [20]. Morphological changes in the cells were also observed using an inverted research microscope (Nikon, Tokyo, Japan). After treatment, those cells were rinsed in phosphate-buffered saline and were traced at 100�total magnification (approximately the same number of cells in a given field). The traced neurons were chosen prior to the commencement of the analysis by an independent investigator blind to the experiment conditions in order� to minimize any bias. Once each neuron was traced, the analysis system calculated the total neuron volume and total length of dendrites.
Reverse transcriptase-polymerase chain reaction (RT-PCR)
TRIzol reagent (Gibco, Grand Island, USA) was used to extract the total cellular RNA from the neurons grown in 25 mm tissue culture plates according to the manufacturer�s instructions. The relative purity of the isolated� RNA was assessed spectrophotometrically. The total RNA was then quantified and the integrity was tested by gel electrophoresis. The total RNA (2 mg) from each sample was retro-transcribed to cDNA by using the reverse� transcription kit (Gibco). PCR amplification for the cDNA of HO-1 was 5�-ACTGCTGACAG�AGGAACA�CA�A�A-3� (forward) and 5�-CAACAGGAAACTGAGTG�T�GAGG-3� (reverse) (GenBank accession No. NW001084742), and of GAPDH, the loading control, it was 5�-TGAAGGCGGTG�TCA�AC�G�G�ATTTGGC-3� (forward) and 5�-CATGTAGGCCATG�A�G�GTCCACCAC-3� (reverse) (GenBank accession No. M17701). The amplification� cycle was at 95 �C for 1 min, 55 �C for 1 min, and 72 �C for 1 min, repeated for 30 cycles. RT-PCR products (8 ml) were separated by electrophoresis on 1.8% agarose gel containing ethidium bromide (181 bp for HO-1 and 352 bp for GAPDH). Expression of HO-1 mRNA was quantified by normalization to GAPDH mRNA (optical density).
Western blot analysis
Whole cell extracts were lysed with 200 ml lysis buffer [50 mM Tris-HCl (pH 8.0), 20 mM EDTA, 1% SDS, and 100 mM NaCl] [12]. A total of 100 mg protein was loaded on to a 10% sodium dodecylsulfate-polyacrylamide gel, and after electrophoresis, it was blotted onto nitro�cellulose membranes. The primary rabbit anti-rat HO-1 (AB1284; Chemicon, Temecula, USA) and hypoxia-inducible factor (HIF)-1a (AB1285; Abcam, Cambridge, UK) polyclonal antibodies were used at 1:500 or 1:1000 dilution, respectively, and anti-b-actin monoclonal antibody (A1978; Sigma) was used at 1:4000 dilution. The horseradish peroxidase�-conjugated anti-rabbit immunoglobulin G secondary� antibody (KPL, Gaithersburg, USA) was used at 1:2000 dilution, and the signal was detected by enhanced chemiluminescence kit (Chemicon). The abundance of HO-1 and HIF-1a proteins was normalized to b-actin using� software Quantity One-4.2.3 (Bio-Rad, Hercules, USA).
Statistical analysis
Statistical analyses were performed by using SPSS 11.0 (SPSS Inc, Chicago, USA). Data were presented as mean�SD. The difference between the means was determined� by one-way ANOVA followed by a Student-Neuman-Keuls� test for multiple comparisons. P<0.05 was statistically significant.
Results
Effects of OGD on the viability of primary hippocampal neuron cultures
To mimic cerebral ischemia in vitro, OGD was performed with hippocampal neurons. We first investigated the effects� of OGD time course on the viability of the primary hippocampal� neurons. The primary hippocampal neurons were treated with glucose-free balanced salt solution in an anoxia chamber for 0, 1, 2, 4 and 6 h, respectively. Neuron viability was then assessed. It was found that the viability of neurons decreased with the increase of OGD time. The neuron viability at 2, 4 and 6 h was significantly lower than that of the control group (Fig. 1). Based on these findings, 2 h of OGD was used in the following experiments.
Isoflurane preconditioning significantly increased the viability of
OGD neurons
The effects of isoflurane preconditioning on the viability of the OGD neurons were investigated. The primary hippocampal� neurons were pretreated with 1.4% isoflurane for 3 h, returned to control conditions for 24 h, and then exposed to the OGD condition for 2 h. The data showed that the 2 h OGD treatment of the hippocampal neurons led to a significant decrease in cell viability (P<0.05) (Fig. 2). However, cell viability was significantly higher in the OGD neurons pretreated with isoflurane than those without� isoflurane preconditioning (P<0.05). The decrease in the viability of the neurons induced by the OGD treatment was significantly recovered by the pretreatment of isoflurane, implying a protective role of isoflurane against OGD-induced injury in the hippocampal neurons.
The protective effects of isoflurane preconditioning were also observed by phase contrast microscopy. Purified� hippocampal neurons showed a well-rounded and phase-bright appearance with intact neuritis to form a network [Fig. 3(A)]. After exposure of hippocampal cultures to OGD for 2 h at 37 �C, microscopy revealed that the neurons� were destroyed. OGD induced neuronal loss and degeneration, which was characterized with karyopyknosis� or karyolysis and shortage of dendritic trees, while some neuronal cell bodies were swollen and others were replaced by debris [Fig. 3(B)]. Isoflurane preconditioning (1.4%) produced less neuronal degeneration and loss. The neuronal� network and neuronal cell bodies were partly preserved [Fig. 3(C)].
Isoflurane preconditioning significantly decreased the release of LDH from OGD neurons
We also investigated the effects of isoflurane preconditioning on the release of LDH from the OGD hippocampal neurons. The primary hippocampal neurons were pretreated� with 1.4% isoflurane for 3 h, returned to control� conditions for 24 h, and then exposed to OGD conditions for 2 h. LDH was then measured. OGD treatment of the neurons for 2 h induced a significant increase in LDH (P<0.05). The LDH in the OGD neurons pretreated with isoflurane was significantly lower than that in the OGD cells without isoflurane pretreatment (P<0.05). However, it was also found that the LDH in the neurons of the isoflurane+OGD group was still significantly higher than that in the control cells (P<0.05) (Fig. 4). This implied that isoflurane preconditioning has a role in significantly, though not completely, inhibiting the increased release of LDH from the primary cultured hippocampal neurons induced� by OGD. Isoflurane alone did not produce any significant changes in viability and LDH release in the neurons� (data not shown).
Isoflurane preconditioning significantly increased the expression of HO-1 mRNA in OGD neurons
To determine whether isoflurane preconditioning�s up-regulation of the HO-1 gene caused it to protect against OGD damage in neuronal cells, we investigated the effect of isoflurane preconditioning on HO-1 mRNA expression in the OGD neurons. The primary hippocampal neurons were treated with 1.4% isoflurane for 3 h, returned to control conditions for 24 h and then exposed to the OGD for 2 h. The expression of HO-1 mRNA was determined using RT-PCR analysis and quantified by normalization to GAPDH mRNA. The findings showed that the expression of HO-1 mRNA was significantly higher in the OGD neurons� than in the control cells (Fig. 5). The treatment of the OGD cells with isoflurane preconditioning led to a further enhancement of the level of HO-1 mRNA expression. There was a significant difference in the levels� of HO-1 mRNA expression between the OGD neurons treated with and without isoflurane pre�conditioning.
Isoflurane preconditioning significantly increased the expression of HO-1 and HIF-1a proteins in OGD neurons
Previously, we found that prolonged hypoxia activates HO-1 and HIF-1a, a major transcription factor of HO-1 during the development of hypoxia-induced pulmonary hypertension [21-23]. Therefore, we checked whether the expression of these two proteins would also be affected� by isoflurane preconditioning. Western blot analysis showed that the expression of both HIF-1a and HO-1 proteins was significantly increased in the OGD conditions� compared to that in the controls. The treatment of OGD neurons with isoflurane preconditioning also led to a further� increase in the protein levels of HIF-1a and HO-1 (Fig. 6), suggesting that isoflurane may up-regulate not only the expression of HO-1 mRNA but also HO-1 protein and its upstream transcription factor HIF-1a protein. Furthermore, the administration of SnPP, an inhibitor of HO activity, prior to isoflurane incubation did not alter the expression pattern of HO-1 protein, but partially abolished the protective effect of isoflurane as measured by LDH release (Fig. 7). Interestingly, isoflurane did not appear to increase HO-1 in OGD neurons in Fig. 7. We believe that the elevated exposure band of HO-1 that occurred in the presence of SnPP, OGD and isoflurane prevented us from detecting the effect of the latter on HO-1 levels. SnPP alone did not alter the LDH release in OGD neurons.
Discussion
Recently, multiple studies have shown that pretreatment of rats with isoflurane induces acute and delayed phases of ischemic tolerance in the brain [24,25]. Surprisingly little attention has been paid so far to the effects of isoflurane on gene expression in neurons. In the present study, we found that isoflurane preconditioning could significantly� increase the expression of HO-1 mRNA and HO-1 protein with an increase in cell viability and a decrease� in the release of LDH in OGD neurons. The findings� support our hypothesis that the neuroprotective role of isoflurane preconditioning is associated with its role in inducing HO-1.
It has been generally accepted that the development of delayed phase of neuroprotection requires new protein synthesis [26]. In the normal mammalian central nervous system, HO-2 is constitutively, abundantly and fairly ubiquitously� expressed, whereas HO-1 mRNA and HO-1 protein are confined to small populations of scattered neurons� and neuroglia. Unlike HO-2, the HO-1 gene in neural tissues is extremely sensitive to a host of pro-oxidant� and other noxious stimuli. HO-1 was shown to protect vessels against heme and hemoglobin-mediated injury [27], to protect kidneys against ischemia/perfusion-induced injury and to reduce hyperoxia-induced lung injury� in rats [28,29]. Furthermore, HO-1 was also shown to confer neuroprotection against ischemic injury in different� experimental models [11,10]. HO-1 and carbon monoxide suppress the pathogenesis of experimental central malaria [30]. Positive findings in the literature linking HO-1 to neuroprotection against ischemia prompted us to select it as a potential candidate for a neuroprotective role induced by isoflurane preconditioning. When the neurons were preconditioned with isoflurane 24 h before OGD treatment, the exposure to OGD produced less neuronal degeneration� and loss. The neuronal network was partly preserved by isoflurane preconditioning. These results indicated that isoflurane exerts some protective effects on the morphological changes of OGD observed in hippocampal cultures.
In this study, we showed that isoflurane preconditioning� induced HO-1 mRNA and HO-1 protein expression in OGD-treated neurons. HO-1 represents an important endogenous� antioxidative defense mechanism against post-ischemic tissue damage [31]. It has been shown to be important in weakening overall reactive oxygen species production through its ability to degrade heme, to produce carbon monoxide and biliverdin/bilirubin, and to release free iron. Biliverdin and bilirubin produced by HO-1 may act as physiological antioxidants and potent scavengers of oxygen radicals [32]. Moreover, recent findings have led to a redefinition of the HO pathway; it is not only an antioxidative mechanism, but it is also a more complex and better coordinated cytoprotective system, with effects on several signal transduction pathways [33]. Increased HO-1 gene expression following neuron ischemia and isoflurane preconditioning may reflect an elevation of antioxidant� defense mechanisms as a response to ischemia-induced oxidative stress. This is also implied by the inhibition� of HO activity partially abolishing the neuroprotective effect of isoflurane. In the current study, we also found that isoflurane preconditioning induced the expression of HIF-1a protein, a major transcription factor� of HO-1 gene under ischemic/hypoxic conditions. However, additional investigations are needed to elucidate the complete pathway.
In summary, the results indicated that administration of one minimum alveolar concentration of isoflurane before OGD protects hippocampal neurons from OGD-induced injury. The results further demonstrated that isoflurane preconditioning significantly increases HO-1 gene expression in the OGD-induced neuronal injury. The findings suggested that the neuroprotective role of isoflurane preconditioning� against ischemia-induced injury might be associated with its role in the up-regulation of HO-1 in ischemic neurons. Additional research is required to ascertain� the role of HO-1 in isoflurane preconditioning and to clarify the mechanisms responsible for this interesting� phenomenon.
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