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Journal of Cerebral Blood Flow & Metabolism logoLink to Journal of Cerebral Blood Flow & Metabolism
. 2010 Jun 30;31(1):305–314. doi: 10.1038/jcbfm.2010.94

Activation of the central histaminergic system is involved in hypoxia-induced stroke tolerance in adult mice

Yan-ying Fan 1,4, Wei-wei Hu 1,4, Hai-bin Dai 2,4, Jian-xiang Zhang 1, Lu-yi Zhang 1, Ping He 1, Yao Shen 1, Hiroshi Ohtsu 3, Er-qing Wei 1, Zhong Chen 1,*
PMCID: PMC3049494  PMID: 20588322

Abstract

We hypothesized that activation of the central histaminergic system is required for neuroprotection induced by hypoxic preconditioning. Wild-type (WT) and histidine decarboxylase knockout (HDC-KO) mice were preconditioned by 3 hours of hypoxia (8% O2) and, 48 hours later, subjected to 30 minutes of middle cerebral artery (MCA) occlusion, followed by 24 hours of reperfusion. Hypoxic preconditioning improved neurologic function and decreased infarct volume in WT or HDC-KO mice treated with histamine, but not in HDC-KO or WT mice treated with α-fluoromethylhistidine (α-FMH, an inhibitor of HDC). Laser-Doppler flowmetry analysis showed that hypoxic preconditioning ameliorated cerebral blood flow (CBF) in the periphery of the MCA territory during ischemia in WT mice but not in HDC-KO mice. Histamine decreased in the cortex of WT mice after 2, 3, and 4 hours of hypoxia, and HDC activity increased after 3 hours of hypoxia. Vascular endothelial growth factor (VEGF) mRNA and protein expressions showed a greater increase after hypoxia than those in HDC-KO or α-FMH-treated WT mice. In addition, the VEGF receptor-2 antagonist SU1498 prevented the protective effect of hypoxic preconditioning in infarct volume and reversed increased peripheral CBF in WT mice. Therefore, endogenous histamine is an essential mediator of hypoxic preconditioning. It may function by enhancing hypoxia-induced VEGF expression.

Keywords: histamine, hypoxic preconditioning, vascular endothelial growth factor

Introduction

Cerebral ischemic tolerance is an adaptive process in which a brief subtoxic stress, such as hypoxic or ischemic preconditioning, induces endogenous neuroprotection against a subsequent severe ischemia. Alterations in the release of neurotransmitters and expression or activation of numerous proteins are involved in this process (Dirnagl et al, 2003), although the mechanisms are poorly understood. Knowledge of tolerance mechanisms may lead to identification of therapeutic targets to protect the brain either before surgery or in patients at high risk of stroke.

Histamine is recognized as an important neurotransmitter or neuromodulator in the central nervous system, which is synthesized from histidine by the specific enzyme, histidine decarboxylase (HDC) (Haas and Panula, 2003). Histaminergic neurons are located in the tuberomammillary nucleus of the posterior hypothalamus, and its fibers are widely distributed in the entire brain. Both in vivo and in vitro studies have indicated that histaminergic neurotransmission mediates neuroprotective activity through histamine H1 or H2 receptors, which are two postsynaptic receptors of histamine in the brain. Inhibition of histamine signaling by α-fluoromethylhistidine (α-FMH, a selective inhibitor of HDC) aggravates cerebral ischemic injury (Adachi, 2005). Through H2 receptor activation, histamine prevents the severe damage to hippocampal CA1 pyramidal cells induced by transient forebrain ischemia (Adachi, 2005). In vitro, the H1 receptor antagonist terfenadine enhances the excitotoxic response to NMDA (N-methyl--aspartic acid) in cerebellar neurons, and this is inhibited by histamine (Diaz-Trelles et al, 2000). Recently, we also reported that histamine protects against NMDA-induced necrosis in cultured cortical neurons through the H2 receptor/cyclic AMP/PKA pathway (Dai et al, 2006). This evidence suggests that histamine has an important role in brain ischemia. In addition, an increase in histamine release occurs in synaptosomal preparations from rats with hypoxia (Waskiewicz et al, 1988), and in the rat cortex or striatum after focal cerebral ischemia (Adachi, 2005; Irisawa et al, 2008). However, the effect of histamine on hypoxia-induced stroke tolerance has not been examined.

Vascular endothelial growth factor (VEGF) has a key role in hypoxia-induced tolerance (Bernaudin et al, 2002; Laudenbach et al, 2007; Wick et al, 2002), moderates cerebral ischemic damage (Hayashi et al, 1998), and protects cultured neurons from hypoxia and glucose deprivation (Jin et al, 2000). Histamine is one of the main mediators of VEGF expression in various tissues and cells. In the granulation tissue, histamine enhances the content of the VEGF protein through the H2 receptor (Ghosh et al, 2001), and HDC knockout (HDC-KO) mice show lower VEGF levels than do wild-type (WT) mice (Ghosh et al, 2002). Similarly, histamine increases VEGF production in cyclooxygenase-2-positive HT29 and Caco-2 cells, and this effect is prevented by H2 antagonists (Cianchi et al, 2005). Thus, we hypothesized that endogenous histamine is involved in stroke tolerance induced by hypoxic preconditioning, and that this effect is associated with VEGF, although the relationship between them in the central nervous system remains unknown.

Materials and methods

Animals

Both WT and HDC-KO male mice (both are C57BL/6 strain) weighing 22 to 25 g were used, which were kindly provided by Professor Ohtsu (Liu et al, 2007). All experiments were conducted in accordance with the ethical guidelines of the Zhejiang University Animal Experimentation Committee and were in complete compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Every effort was made to minimize any pain or discomfort, and the minimum number of animals was used.

Hypoxic Preconditioning and Drug Treatment In Vivo

Normobaric hypoxia (8% O2) was achieved by replacing oxygen by nitrogen in an airtight chamber (5,500 mL). Hypoxic preconditioning was performed 48 hours before ischemia. In the α-FMH treatment group, α-FMH (25 mg/kg) was injected intraperitoneally 3 hours before hypoxia in WT mice. In the histamine treatment group of HDC-KO mice, histamine was administered intracerebroventricularly at a dose of 5 μg/2 μL per 6 h per animal for 3 injections in all, and the first injection was administered 30 minutes before hypoxia (i.e., 51.5, 45.5, and 39.5 hours before ischemia). The corresponding vehicle group and hypoxic preconditioning group of HDC-KO mice were injected with artificial cerebrospinal fluid three times by 2 μL/6 h per animal. SU1498 which is a VEGF receptor (VEGFR)2/Flk1 antagonist was administered intracerebroventricularly at a dose of 250 ng/2 μL per 12 h per animal for 3 injections in all WT mice, and the first injection was administered 30 minutes before hypoxia (i.e., 51.5, 39.5, and 27.5 hours before ischemia). The corresponding vehicle group and hypoxic preconditioning group of WT mice were injected with artificial cerebrospinal fluid containing 1% dimethyl sulfoxide three times by 2 μL/12 h per animal.

Transient Focal Cerebral Ischemia

Both WT and HDC-KO mice were anesthetized with an intraperitoneal injection of sodium pentobarbital (45 mg/kg). Cerebral blood flow (CBF) was determined in the territory of the middle cerebral artery (MCA) by laser Doppler flowmetry (Periflux System 5010; Perimed, Jarfalla, Sweden) as described previously (Inaba et al, 2009; Iwanami et al, 2007). A flexible fiberoptic probe was affixed to the skull over the cortex supplied by the proximal part (core; 2 mm caudal to the bregma and 6 mm lateral to midline) or the peripheral part (periphery; 2 mm caudal to the bregma and 3 mm lateral to midline) of the right MCA. The CBF in the core of the MCA territory was monitored in all animals subjected to MCA occlusion (MCAO), and in a part of animals, CBF in the periphery of the MCA territory was also monitored. Cerebral blood flow was expressed as a percentage of the value before MCAO. Animals with <80% reduction in CBF in the core of the MCA territory were excluded from the study. The exclusion criteria used to eliminate mice from further consideration are shown in Supplementary Table 1.

Transient focal cerebral ischemia was induced by MCAO as described previously (Yu et al, 2005). Briefly, a 6-0 nylon monofilament suture, blunted at tip, and coated with 1% poly--lysine, was advanced 10 mm into the internal carotid to occlude the origin of MCA. Reperfusion was allowed after 30 minutes by monofilament removal. Body temperature was maintained at 37°C by a heat lamp (FHC, Bowdoinham, ME, USA) during surgery and for 2 hours after the start of reperfusion. Systolic blood pressure and heart rate were measured by a noninvasive tail cuff (ML 125 NIBP system, ADInstruments Pty Ltd., Castle Hill, NSW, Australia) connected to a PowerLab system (ADInstruments Pty Ltd., Castle Hill, NSW, Australia). Arterial pH, pO2, and pCO2 were monitored using an ABL700 series blood gas analyzer (Radiometer, Copenhagen, Denmark).

Neurologic deficit scores were evaluated as described previously (Longa et al, 1989) at 24 hours of reperfusion as follows: 0, no deficit; 1, flexion of the contralateral forelimb on lifting of the whole animal by the tail; 2, circling to the contralateral side; 3, falling to contralateral side; and 4, no spontaneous motor activity.

Infarct volume was determined 24 hours after MCAO. The brains were quickly removed, sectioned coronally at 2-mm intervals, and stained by immersion in the vital dye 2,3,5-triphenyltetrazolium hydrochloride (TTC; 0.25%) at 37°C for 30 minutes. Extents of the normal and infarcted areas were analyzed using the ImageJ software (National Institutes of Health, Bethesda, MD, USA) and determined by the indirect method, which corrects for edema (contralateral hemisphere volume minus nonischemic ipsilateral hemisphere volume). The percentage of the corrected infarct volume was calculated by dividing the infarct volume by the total contralateral hemispheric volume, and this ratio was then multiplied by 100.

High-Performance Liquid Chromatography Determination of Histamine Concentration

Wild-type mice were killed immediately after 3 hours of hypoxia or normoxia, the brain was quickly removed, and the cerebral cortex was isolated and stored at −80°C until assay. The cerebral cortex was homogenized with 0.4 mol/L perchloric solution and centrifuged at 15,000 g for 20 minutes at 4°C, and the supernatant was collected. Analysis of histamine in each sample was performed by high-performance liquid chromatography (HPLC) as described previously (Jin et al, 2005). The HPLC was controlled, and data were acquired and analyzed using CoulArray software (ESA, Chelmsford, MA, USA). All equipments were obtained from ESA (Chelmsford, MA, USA).

Measurements of Histidine Decarboxylase Activity

Histidine decarboxylase activity was measured as described before (Shen et al, 2007). In short, the cerebral cortex was homogenized in 10-fold volume of ice-cold HDC buffer (0.1 mol/L potassium phosphate buffer, pH 6.8, 0.01 mmol/L pyridoxal-5′-phosphate, 0.2 mmol/L dithiothreitol, 1% polyethyleneglycol with average molecular weight of 300, 100 μg/mL phenylmethane sulfonylfluoride). The homogenates were centrifuged at 10,000 g for 15 minutes at 4°C, and 100 μL of the supernatant was transferred into a microcentrofilter tube (Ultracel YM-30, Millipore, Billerica, MA, USA). After centrifugation at 5,000 g for 40 minutes, 100 μL of the phosphate buffer was added to the pellet, which was gently suspended. This process was repeated twice to remove endogenous histamine. The enzymatic reaction was then started by adding 100 μL 1 mmol/L -histidine at 37°C and the histamine produced during a 3-hour reaction was measured using HPLC.

Quantitative Real-Time PCR

Total RNA was isolated using a total RNA extraction kit, according to the manufacturer's instructions (Sangon, Shanghai, China). We then prepared cDNA using a Prime Script RT Reagent kit (Takara, Dalian, China) and performed quantitative real-time PCR with an ABI 7500 instrument (Applied Biosystems, Foster City, CA, USA) and a SYBR Green Real-time PCR Master Mix (Toyobo, Osaka, Japan). The following mouse primers were used: VEGF, 5′-GTAACGATGAAGCCCTGGAGT-3′ and 5′-TCACATCTGCTGTGCTGTAGGA-3′ erythropoietin, 5′-CCCACCCTGCTGCTTTTACT-3′ and 5′-ACAACCCATCGTGACATTTTCT-3′ HDC, 5′-ACCCCATCTACCTCCGACAT-3′ and 5′-ACCGAATCACAAACCACAGC-3′. Glyceraldehyde-3-phosphate dehydrogenase, 5′-GTCGGTGTGAACGGA;TTTGG-3′ and 5′-GCTCCTGGAAGATGGTGATGG-3′.

Immunoblotting

The cerebral cortex was homogenized, and total proteins were purified using cell and tissue protein extraction reagents according to the manufacturer's instructions (KC-415; KangChen, Shanghai, China). In all, 42μg protein equivalent of each sample was electrophoresed on polyacrylamide gel, transferred onto nitrocellulose, and probed with monoclonal anti-VEGF antibodies (1:1,000; Abcam, Cambridge, UK), followed by IRDye 700-coupled anti-mice IgG (1:10,000; LI-COR Biosciences, Lincoln, NE, USA) secondary antibodies. Anti-glyceraldehyde-3-phosphate dehydrogenase antibody (1:5,000; KangChen, Shanghai, China) was used as the control. Blots were visualized using an Odyssey infrared imaging system (LI-COR Biosciences) and analyzed using Odyssey software. Relative optical densities were obtained by comparing measured values with the mean values from the WT control group.

Statistical Analysis

All data were collected and analyzed in a blind manner. Data are presented as mean±s.e.m. One-way ANOVA (analysis of variance) with least significant difference (LSD) or Dunnett's T3 post hoc test (in which equal variances were not assumed) was applied for multiple comparisons, whereas Student's t-test was used for comparisons between two groups. Neurologic deficit scores were analyzed with the nonparametric Mann–Whitney U-test. P<0.05 was considered statistically significant.

Results

Hypoxic Preconditioning Induces Tolerance to Transient Focal Cerebral Ischemia in Wild-Type but Not in Histidine Decarboxylase Knockout Mice

During normobaric hypoxic preconditioning (8% O2), animals displayed increased respiratory rate and reduced spontaneous movement. After preconditioning, they recovered within minutes. Two or three hours of hypoxia, performed 48 hours before transient MCAO, reduced infarct volume to 68.8% (P<0.05) or 50.5% (P<0.05) of the control group, respectively (Figure 1A). However, after a longer period of hypoxia (4 hours), the protection became weaker. The reduction in infarct volume in preconditioned animals was paralleled by functional changes in neurologic deficits. Animals preconditioned for 2 or 3 hours exhibited lower neurologic deficit scores at 24 hours after transient MCAO (P<0.05) relative to control animals (Figure 1B).

Figure 1.

Figure 1

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Effect of hypoxic preconditioning (8% O2) on infarct volume and neurologic scores after transient MCAO in wild-type mice. Mice were exposed to hypoxia for 2, 3, or 4 hours. Forty-eight hours later, MCAO was induced for 30 minutes. (A) Infarct volume and (B) neurologic scores were determined 24 hours after the insult. Infarct volume was expressed as the percentage of the infarcted tissue in reference to the contralateral hemisphere. Values show mean±s.e.m. n=10 to 15 for each group; *P<0.05 and **P<0.01. MCAO, middle cerebral artery occlusion.

To determine whether histamine is involved in hypoxic preconditioning, we used HDC-KO mice, which lack the synthetic enzyme for histamine. Three hours of hypoxic preconditioning induced the strongest neuroprotection; therefore, we compared the effects of 3 hours of preconditioning on WT and HDC-KO mice. No significant differences in infarct volume and neurologic deficit scores were found between the control groups of WT and HDC-KO mice 24 hours after 30 minutes of MCAO (Figure 2). In HDC-KO mice, hypoxic preconditioning did not decrease the infarct volume or improve neurologic function compared with the nonpreconditioned group. Similarly, the histamine synthesis enzyme inhibitor α-FMH reversed the neuroprotection induced by hypoxic preconditioning in infarct volume and neurologic scores in WT mice (P<0.05; Figures 3A and 3B), whereas α-FMH alone had no effect on infarct volume without hypoxic preconditioning (infarct volume: control group, 23.63%±3.72%, n=4; α-FMH-treated group, 22.72%±5.15%, n=5). In contrast, hypoxic preconditioning combined with histamine (5 μg/2 μL per 6 h per animal for 3 injections, intracerebroventricularly) in HDC-KO mice significantly improved neurologic function and decreased infarct volume (P<0.05; Figures 3C and 3D), whereas histamine alone had no effect on infarct volumes without hypoxic preconditioning (infarct volume: vehicle group, 26.59%±4.36%, n=4; histamine-treated group, 23.44%±2.60%, n=6). In addition, mean arterial blood pressure, heart rate, and arterial blood gas (pH, PaO2, PaCO2) before and after ischemia did not differ among the six groups (Supplementary Table 2).

Figure 2.

Figure 2

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Effect of hypoxic preconditioning (HPC; 8% O2) on transient MCAO-induced injury in wild-type (WT) and histidine decarboxylase knockout (HDC-KO) mice. Forty-eight hours after 3 hours of hypoxic preconditioning or normoxia, MCAO was induced for 30 minutes. (A) Infarct volume and (B) neurologic scores were determined 24 hours after MCAO in WT and HDC-KO mice. Values show mean±s.e.m. n=8 to 12 for each group; *P<0.05. MCAO, middle cerebral artery occlusion.

Figure 3.

Figure 3

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Effect of hypoxic preconditioning (HPC; 8% O2) on transient MCAO-induced injury in α-FMH-treated wild-type (WT) mice and histamine-treated histidine decarboxylase knockout (HDC-KO) mice. Forty-eight hours after 3 hours of hypoxic preconditioning or normoxia, MCAO was induced for 30 minutes. α-FMH (25 mg/kg) was injected intraperitoneally 3 hours before hypoxic preconditioning in WT mice, and (A) infarct volume and (B) neurologic scores were determined 24 hours after MCAO. Histamine (5 μg/2 μL per 6 h per animal for 3 injections; the first injection was administered 30 minutes before hypoxia.) was administered intracerebroventricularly with hypoxic preconditioning in HDC-KO mice, and (C) infarct volume and (D) neurologic scores were determined 24 hours after MCAO. Values show mean±s.e.m. n=8 to 12 for each group; *P<0.05 and **P<0.01. α-FMH, α-fluoromethylhistidine; MCAO, middle cerebral artery occlusion.

Hypoxic Preconditioning Ameliorated the Reduction in the Peripheral Cerebral Blood Flow During Transient Focal Cerebral Ischemia in Wild-Type Mice but not in Histidine Decarboxylase Knockout Mice

Cerebral blood flow was measured in the core and peripheral regions of the MCA territory. As shown in Figure 4A, no difference in CBF changes of the core region was detected among the five groups during and after MCAO. However, 33.7% increased blood flow was observed in the peripheral region during ischemia in the hypoxic preconditioned group of WT mice. In contrast, in HDC-KO and α-FMH-treated WT mice, hypoxic preconditioning aggravated the reduction in peripheral CBF by 42.5 and 40.5% of the control groups, respectively (Figure 4B).

Figure 4.

Figure 4

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Effect of hypoxic preconditioning (HPC; 3 hours, 8% O2) on cerebral blood flow (CBF) during transient MCAO in wild-type (WT) mice treated with or without α-FMH and histidine decarboxylase knockout (HDC-KO) mice. α-FMH (25 mg/kg) was injected intraperitoneally 3 hours before hypoxic preconditioning. CBF was measured in (A) the core and (B) the periphery of the MCA territory before, during, and after MCAO. CBF was expressed as a percentage of the value before MCAO. Values show mean±s.e.m. (Panel A) n=8 to 12 for each group; (panel B) n=8 to 9 for each group; *P<0.05, **P<0.01. α-FMH, α-fluoromethylhistidine; MCAO, middle cerebral artery occlusion.

Effect of Hypoxia on Histamine Content and Histidine Decarboxylase Activity in the Cerebral Cortex of Wild-Type Mice

After hypoxia, the cortex of WT mice was isolated for HPLC determination of histamine concentration and HDC activity. The histamine levels after 2, 3, and 4 hours of hypoxia were 69.3, 65.5, and 66.6% (all P<0.05) of the control group, respectively (Figure 5A). Histidine decarboxylase activity in the control group was 156.00±21.51 fmol/min per mg protein. Three hours of hypoxia significantly increased HDC activity (236.1±14.68 fmol/min per mg protein, P<0.0.5, Figure 5B). In addition, HDC mRNA of the hypothalamus where histamine-producing neurons are located after 3 hours of hypoxia was not significantly changed compared with the control group (Figure 5C).

Figure 5.

Figure 5

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Effect of hypoxic preconditioning (HPC, 8% O2) on histamine levels and histidine decarboxylase (HDC) activity of the cortex and HDC mRNA expression of the hypothalamus in wild-type mice. (A) Histamine levels in the cortex were assayed at 2, 3, and 4 hours of hypoxia. (B) HDC activity in the cortex and (C) HDC mRNA expression of the hypothalamus were assayed at 3 hours of hypoxia. Values show mean±s.e.m. n=4 to 6 for each group; *P<0.05 versus control group.

Vascular Endothelial Growth Factor is Induced by Hypoxic Preconditioning in Wild-Type but Not in Histidine Decarboxylase Knockout Mice

Both WT and HDC-KO mice were exposed to 8% O2 for 3 hours. After various times of reoxygenation in normal air, the cerebral cortex was collected for analysis of VEGF mRNA and protein. Real-time PCR analysis showed upregulated expression of VEGF mRNA (including VEGF165, VEGF121, and VEGF140 isoforms) after 3 hours of hypoxia (P<0.05), which then rapidly decreased to baseline after 3 hours of reoxygenation in both WT and HDC-KO mice (Figure 6A). However, the VEGF mRNA level in WT mice (6.59±1.46-fold control WT, P<0.05) was higher than that in HDC-KO mice (3.08±0.77-fold control WT, P<0.05) immediately after hypoxia (P<0.05). Otherwise, the cortex of both WT and HDC-KO mice not exposed to hypoxia showed similar levels of VEGF mRNA, indicating that histamine is not required for the constitutive expression of VEGF in the cortex under normal physiologic conditions. Similarly, VEGF protein expression (VEGF165 isoform) in WT mice increased at as early as 3 hours of hypoxia (P<0.01), remained upregulated after 8 hours of reoxygenation (P<0.01), and then normalized after 24 hours of reoxygenation. Interestingly, in HDC-KO- and α-FMH-treated WT mice, no significant increase in the VEGF protein occurred at various times of reoxygenation (Figures 6C and 6D). In addition, erythropoietin, which has also been found to be a key mediator in hypoxic preconditioning, responded similarly in both HDC-KO and WT mice (Figure 6B).

Figure 6.

Figure 6

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Effect of hypoxic preconditioning (3 hours, 8% O2) on expression of VEGF mRNA and protein in wild-type (WT) and histidine decarboxylase knockout (HDC-KO) mice. (A) VEGF mRNA expression, (B) erythropoietin mRNA expression, and (C) VEGF protein expression in the cerebral cortex of WT and HDC-KO mice at 0, 3, 8, and 24 hours after reoxygenation. (D) VEGF protein expression in the cerebral cortex of WT mice treated with or without α-FMH (25 mg/kg, intraperitoneally) at 0 and 8 hours after reoxygenation. Values show mean±s.e.m. n=5 to 6 for each group; *P<0.05 and **P<0.01. α-FMH, α-fluoromethylhistidine; R, reoxygenation; VEGF, vascular endothelial growth factor.

Effect of VEGFR2 Antagonist SU1498 on Neuroprotection Induced by Hypoxic Preconditioning

The VEGFR2 antagonist SU1498 or vehicle was administered intracerebroventricularly 30 minutes before hypoxic preconditioning, followed by two infusions (with an interval of 12 hours). SU1498 prevented the protective effect of hypoxic preconditioning in infarct volume in WT mice having transient focal cerebral ischemia (P<0.05; Figure 7A). It reversed the improved peripheral CBF induced by hypoxic preconditioning (P<0.05; Figure 7B). In addition, SU1498 alone had no effect on infarct volume in the absence of hypoxic preconditioning (infarct volume: vehicle group, 26.02%±6.18%, n=4; SU1498-treated group, 26.55%±5.55%, n=5).

Figure 7.

Figure 7

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Effect of hypoxic preconditioning (HPC; 8% O2) on transient MCAO-induced injury in wild-type mice treated with VEGFR2/Flk1 antagonist SU1498. Forty-eight hours after 3 hours of hypoxic preconditioning or normoxia, MCAO was induced for 30 minutes. (A) SU1498 (250 ng/2 μL per 12 h per animal for 3 injections; the first injection was administered 30 minutes before hypoxia) was administered intracerebroventricularly with hypoxic preconditioning in wild-type mice, and the infarct volume was determined 24 hours after MCAO. (B) CBF was measured in the peripheral region of the MCA territory before, during and after MCAO. CBF was expressed as a percentage of the value before MCAO. Values show mean±s.e.m. n=5 to 8 for each group; *P<0.05, **P<0.01. CBF, cerebral blood flow; MCAO, middle cerebral artery occlusion; VEGFR, vascular endothelial growth factor receptor.

Discussion

In this study, we showed for the first time that histamine is involved in hypoxia-induced ischemia tolerance in the adult brain. Three hours of hypoxic preconditioning performed 48 hours before transient MCAO afforded a significantly better outcome in WT mice, as shown by remarkable reductions in infarct volume and neurologic deficit. This result is in agreement with Miller et al (2001), and in their experiment, animals were exposed to 2 hours of hypoxia (11% O2) 48 hours before transient MCAO. However, in HDC-KO mice which chronically lack histamine, the protection induced by hypoxic preconditioning completely disappeared, and hypoxic preconditioning combined with histamine produced neuroprotection in HDC-KO mice. The infused histamine exerted in the central nervous system but not in the peripheral system, because histamine cannot cross the blood–brain barrier and 3 hours of hypoxic preconditioning combined with histamine treatment did not obviously influence the permeability of the blood–brain barrier in HDC-KO mice (data not shown). Alpha-FMH is a specific and irreversible inhibitor of HDC, and a single administration decreases the histamine content in the neuronal pool only, without affecting nonneuronal sources in the brain (Garbarg et al, 1980). Indeed, α-FMH also clearly reversed the neuroprotection induced by hypoxic preconditioning in our experiments. These data show that endogenous histamine is an important mediator in hypoxia-induced tolerance to stroke in adult mice. Furthermore, we found that histamine content significantly decreased in the cortex of WT mice at 2, 3, and 4 hours of hypoxia, and that HDC activity increased at 3 hours of hypoxia, which indicates that hypoxia increased the release of histamine in the mouse cortex (It must be noted that as extracellular histamine is rapidly degraded, massive release results in the depletion of intracellular histamine.) Taken together, our results support the hypothesis that an increase in histamine release resulting from activation of histaminergic neurons is necessary for the induction of hypoxic preconditioning.

In our preconditioning paradigm, hypoxia (8% O2) produced a bell-shaped curve for protection against a transient MCAO, having a peak at 3 hours. A longer period of hypoxia (4 hours) did not result in significant protection, although this did induce a release of histamine comparable with hypoxia for 2 or 3 hours. Prass et al (2003) reported similar results which showed a peak at 5 hours, whereas a longer period (6 hours) induced severe damage in the CA1 region. It is still unknown why the protection disappears with time. This is not likely to be related to the glutamate excitotoxicity induced by hypoxia, because the glutamate content after 4 hours of hypoxia was not significantly changed from that of the control group (data not shown), and glutamate in synaptic terminals is not reduced by hypoxia in rat hippocampal slices (Madl and Royer, 1999). Other factors, such as hypotension (Prass et al, 2003) and hypocapnia induced by hypoxia may be of relevance, because hypocapnia with hypotension causes hippocampal neuronal death (Ohyu et al, 2000), and hypocapnia activates caspase-3 to induce apoptosis (Xie et al, 2004). The narrow effective range for the duration of hypoxic preconditioning greatly restricts its direct clinical application. Therefore, aiming at effective targets in the protection process will be more productive. In our system, the release of histamine maintains a stable level during hypoxia, and histamine is a known neuroprotective factor. Thus, regulating histaminergic neuronal activity to maintain a specific level of histamine may be more controllable and safer against stroke, without the side effects of overlong hypoxia.

In the search for mechanisms involved in the effect of histamine on hypoxic preconditioning, we found that hypoxia-induced VEGF mRNA expression was higher in the cerebral cortex of WT mice than HDC-KO mice, and that VEGF165 protein expression was induced by hypoxia in WT mice but not in HDC-KO or α-FMH-treated WT mice, which strongly indicates that the lack of histamine can inhibit VEGF expression induced by hypoxia. Vascular endothelial growth factor protects the brain against ischemia and is involved in the establishment of hypoxic preconditioning (Bernaudin et al, 2002; Laudenbach et al, 2007; Wick et al, 2002). Bernaudin et al (2002) reported that hypoxia induces tolerance to cerebral ischemia in association with increased expression of VEGF in the adult mouse brain. Neuroprotection induced by hypoxic exposure is diminished by administration of the anti-VEGFR2/Flk1 blocking antibody or by the use of mutant mice lacking the hormone response element of the VEGF gene promoter in newborn mice (Laudenbach et al, 2007). Histamine enhances VEGF production in the granulation tissue and in cyclooxygenase-2-positive HT29 and Caco-2 cells through the H2 receptor (Cianchi et al, 2005; Ghosh et al, 2001, 2002). We also found that endogenous histamine induced the expression of VEGF mRNA and protein in the cerebral cortex on exposure to hypoxia. Furthermore, SU1498, a VEGFR2/Flk1 antagonist, inhibited the neuroprotection induced by hypoxic preconditioning in WT mice. As histamine is proved to be involved in hypoxic preconditioning-induced neuroprotection in this study, it is likely that the robust neuroprotection is established by VEGF production induced by histamine release.

In WT mice, the expression of hypoxia-inducible factor-1α, which is a main transcription factor involved in the induction of VEGF (Bernaudin et al, 2002), was higher than that in HDC-KO mice immediately after hypoxia, and α-FMH also inhibited hypoxia-inducible factor-1α production induced by hypoxia (Supplementary Figure 1). Hypoxia (O2 tension <0.2%) can increase HDC mRNA expression by induction of hypoxia-inducible factor-1α (Jeong et al, 2009). However, in our present system, HDC mRNA level of the hypothalamus, where histamine-producing neurons are located, was not significantly changed after hypoxia. This conflict may be attributed to different hypoxic conditions, because O2 tension <0.2% is much severer than 8% O2. These results at least imply that histamine mediated VEGF expression in hypoxia likely through regulating hypoxia-inducible factor-1α expression. In addition, erythropoietin has also been found to be a key mediator in hypoxic preconditioning (Prass et al, 2003). In this study, in HDC-KO mice, erythropoietin response was similar to that measured in WT mice. It suggests that erythropoietin may be not involved in histamine induced-protection in hypoxic preconditioning. As the exogenous treatment of recombinant human erythropoietin increases brain levels of VEGF after stroke (Wang et al, 2004), erythropoietin may also be involved in hypoxic preconditioning-induced VEGF production beside histamine.

Vascular endothelial growth factor itself has a direct neuroprotective effect on neurons. Vascular endothelial growth factor protects cultured hippocampal neurons and several neuronal cell lines against cell death induced by ischemia or excitotoxic stimuli through the VEGFR2/phosphoinositide 3-kinase/Akt and VEGFR2/MEK/ERK signaling pathways (Jin et al, 2000; Matsuzaki et al, 2001; Qiu et al, 2003). In cultured cerebellar granule neurons, hypoxia-induced tolerance also depends on VEGF/VEGFR2 activation and Akt phosphorylation (Wick et al, 2002). However, once the dose of VEGF exceeds the limit, adverse effects of VEGF might surpass the neuroprotective effects and deteriorate neuronal survival and function in vivo and in vitro (Laudenbach et al, 2007; Manoonkitiwongsa et al, 2004; Yasuhara et al, 2005). Interestingly, we also found that 3 hours of hypoxia (8% O2) induced a moderate upregulation (34%) of the VEGF protein in WT mice. These results indicate the importance of maintaining VEGF levels in a suitable range for neuroprotection against ischemia, and that low levels of VEGF expression in response to mild hypoxia may be necessary for neuroprotection. A moderate regulator of VEGF such as histamine may be safer for the treatment of stroke.

In addition, an increase in CBF is considered to contribute to protection against brain ischemia. Our previous study has shown that histamine elicits an increase in CBF in the rat hippocampus through both the postsynaptic H1, H2 receptors and the presynaptic H3 receptor (Chen, 2001; Suzuki et al, 1999). Therefore, we measured CBF in the core and peripheral regions of the MCA territory. Although CBF in the core region showed no significant difference among the groups, hypoxic preconditioning elevated CBF in the peripheral region of WT mice. In HDC-KO mice or WT mice treated with α-FMH, hypoxic preconditioning aggravated the reduced CBF in the peripheral region during ischemia compared with nonpreconditioned animals, whereas histamine administered in HDC-KO mice reversed the decreased peripheral CBF after hypoxic preconditioning (data not shown). These data suggest that histamine mediates the enhanced CBF produced by hypoxia preconditioning. Besides the direct vasodilation effect of histamine, VEGF may be another mechanism conferring increased CBF. Vascular endothelial growth factor is a regulator of CBF by inducing nitric oxide production or enhancing angiogenesis (Vogel et al, 2003; Zhang et al, 2000). It was interesting to find that SU1498 reversed the increased peripheral CBF induced by hypoxic preconditioning in WT mice, and histamine elevated VEGF expression in this study or in other reports (Ghosh et al, 2001, 2002), suggesting that histamine release induced by hypoxic preconditioning might also improve peripheral CBF in ischemia partly through the VEGF/VEGFR2/Flk1 pathway.

In conclusion, this study indicates that endogenous histamine has an essential role in hypoxia-induced ischemic tolerance in brain. The beneficial effects of histamine in hypoxic preconditioning may occur through upregulating VEGF expression. Further studies will need to be conducted to understand the mechanisms of action of histamine in hypoxic preconditioning that lead to protection of the brain against ischemia and neurodegenerative disorders.

Acknowledgments

We are very grateful to Dr Iain C. Bruce for reading the manuscript.

The authors declare no conflict of interest.

Footnotes

Supplementary Information accompanies the paper on the Journal of Cerebral Blood Flow & Metabolism website (http://www.nature.com/jcbfm)

This project was supported by the National Natural Science Foundation of China (30725047, 30572176, 30600757, 30801392), the National Basic Research of China 973 Program, (2009CB521906), and partly by the New Century Excellent Talents Program, Ministry of Education, China (NCET-06-0511), the Zhejiang Provincial Natural Science Foundation of China (Z207289), Youth foundation of the Innovative Scientific Research of Zhejiang University (2009QNA7007).

Supplementary Material

Supplementary Figure 1
Click here for additional data file. (3.8MB, tif)
Supplementary Figure 1 Legend
Click here for additional data file. (28KB, doc)
Supplementary Table 1
Click here for additional data file. (78KB, doc)

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Associated Data

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Supplementary Materials

Supplementary Figure 1
Click here for additional data file. (3.8MB, tif)
Supplementary Figure 1 Legend
Click here for additional data file. (28KB, doc)
Supplementary Table 1
Click here for additional data file. (78KB, doc)

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