Neuroscience Letters 563 (2014) 149–154
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Dose-dependent effect of sulfur dioxide on brain damage induced by recurrent febrile seizures in rats Ying Han 1 , Wenxia Yi 1 , Jiong Qin ∗ , Yang Zhao, Jing Zhang, Xingzhi Chang Department of Pediatrics, Peking University First Hospital, Beijing 100034, China
h i g h l i g h t s • The SO2 /AAT system is involved in febrile seizures (FS) and related toxicity injury. • In a rat FS model, preconditioning with a low concentration of SO2 alleviated neuronal damage and apoptosis. • An AAT inhibitor or high concentration of SO2 aggravated neuronal damage in FS rats.
a r t i c l e
i n f o
Article history: Received 23 October 2013 Received in revised form 15 December 2013 Accepted 18 December 2013 Keywords: Sulfur dioxide Aspartate aminotransferase Febrile seizures Brain damage
a b s t r a c t Sulfur dioxide (SO2 ) regulates many physiological processes. Little is known about its roles in neurological disorders. In this study, we investigated the role of endogenous SO2 in the development of febrile seizures (FS) and related brain damages. In the rat model of recurrent FS, we found that endogenous SO2 in the plasma and hippocampus was increased, accompanied by upregulation of aspartate amino-transferase 1 (AAT1) and AAT2, and neuronal apoptosis and mossy fiber sprouting (MFS) in the hippocampus. Preconditioning with low concentration of SO2 (1–10 mol/kg) alleviated the neuronal damage, and attenuated neuronal apoptosis and MFS, whereas preconditioning with high concentration of SO2 (100 mol/kg) or inhibition of AAT aggravated the neuronal damage, and promoted neuronal apoptosis and MFS in hippocampus of rats with recurrent FS. These data indicate that endogenous SO2 is involved in the development of FS and related brain damage. Preconditioning with low concentration of SO2 may protect neurons from toxicity caused by FS. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Febrile seizures (FS) are frequently encountered in pediatric populations, occurring in 2–4% of children between 6 months and 5 years of age [1]. Patient studies have suggested that recurrent FS may cause hippocampal damage, which is one of the most common neuropathological findings of temporal lobe epilepsy (TLE) [2]. It is important, therefore, to further investigate the pathogenesis of FS and related brain damage. Sulfur dioxide (SO2 ) is known to be an atmospheric pollutant, and studies concerning SO2 have mainly focused on its toxicity in animals [3,4]. However, SO2 has recently been shown to be produced endogenously by the normal mammalian metabolism of sulfur-containing amino acids [5], and l-cysteine in particular
Abbreviations: AAT, aspartate aminotransferase; FS, febrile seizures; HDX, laspartate--hydroxamate; MFS, mossy fiber sprouting. ∗ Corresponding author. Tel.: +86 13 701086050; fax: +86 10 66530532. E-mail addresses: [email protected], [email protected] (J. Qin). 1 These authors contributed equally to this work. 0304-3940/$ – see front matter © 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.12.042
[6]. l-Cysteine is catalyzed by cysteine dioxygenase (CDO) to lcysteinesulfinate, which converts to -sulfinylpyruvate through transamination by aspartate aminotransferase (AAT); it then spontaneously decomposes to pyruvate and SO2 [7]. Additionally, hydrogen sulphide (H2 S) can be catalyzed to SO2 by nicotinamide adenine dinucleotide phosphate oxidase [8] or by the reduction of thiosulphate [7]. SO2 is metabolized to sulfite and oxidized to sulfate by sulfite oxidase, and is finally excreted in urine [6]. The distribution of SO2 and its generating enzymes, AAT and CDO, in different tissues of the rat, and the biological effects of endogenous SO2 on cardiovascular diseases have recently been described [9–11]. By comparisons with other analogous endogenous gaseous molecules, such as nitric oxide (NO), carbon monoxide (CO) and H2 S, it has been suggested that SO2 plays a regulatory role for physiological functions [9–11]. Recent studies demonstrated that NO, CO and H2 S play important roles in some of the nervous system diseases, including FS and related brain damage [12–17]. However, it remains unknown whether endogenous SO2 is involved in the pathogenesis of FS and related brain injury. The present study was therefore intended to investigate the changes in SO2 plasma and hippocampal levels in a rat model of recurrent FS,
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and to evaluate the effects of endogenous SO2 on FS-induced brain damage. 2. Materials and methods Animal care and experimental protocols complied with the Animal Management Rule of the Ministry of Health, China and the Animal Care Committee of Peking University First Hospital, Beijing, China. Sprague–Dawley male rats were housed with their mothers under standard laboratory conditions until they were weaned at 21 days. Our studies started with animals aged 21 days. The FS model has been described previously in detail [15]. In brief, the control rats were placed into 37 ◦ C water for 5 min, and the rats in the other seven groups were placed into 45.2 ◦ C water until a seizure occurred. As described in detail previously [18], hyperthermia treatment initiated seizures in rats, manifested as facial clonus, head nodding, forelimb clonus, rearing of the animal to a standing posture aided by its tail and the laterally spreading of hindlimbs with increased tone, before falling back. Water immersion was carried out once every 2 days, for 10 times in total. Rats were then randomly divided into eight groups that received the following treatments: (1) control group, preconditioning with normal saline 30 min before water immersion (n = 24); (2) FS group, hyperthermia treatment as described previously, preconditioning with normal saline 30 min before hyperthermia treatment (n = 24); (3) FS + S1 group, preconditioning with 1 mol/kg of SO2 (n = 24); (4) FS + S5 group, preconditioning with 5 mol/kg of SO2 (n = 24); (5) FS + S10 group, preconditioning with 10 mol/kg of SO2 (n = 24); (6) FS + S50 group, preconditioning with 50 mol/kg of SO2 (n = 24); (7) FS + S100 group, preconditioning with 100 mol/kg of SO2 (n = 24); (8) FS + HDX group, preconditioning with 3.7 mg/kg of l-aspartate--hydroxamate (HDX, an inhibitor of the enzyme responsible for endogenous generation of SO2 ) 30 min before hyperthermia treatment (n = 24). For groups 3–7, a SO2 donor (NaHSO3 and Na2 SO3 , 1:3 MM ratio) was intravenously injected 30 min before each hyperthermia treatment. An equal volume of normal saline was injected intraperitoneally in control and FS groups. All rats were anaesthetized within 6 h after the final water immersion (Fig. 1B). Rats were anesthetized with 4% chloral hydrate (600 mg/kg, intraperitoneally) and perfused through the heart with 0.9% saline followed by 3% paraformaldehyde (PFA) and 1% glutaraldehyde in phosphate-buffered saline (PBS). The brain was removed and the hippocampus was isolated and cut into approximately 1 mm3 cubes after immersion in 3% glutaraldehyde in PBS. The tissue was washed three times in sucrose and post-fixed in 1% osmium tetroxide for 2 h, and then dehydrated in graded ethanol solutions and embedded overnight in Epon 812 at 37 ◦ C. Semi-thin sections (2 m) were stained with toluidine blue. These sections were used for selection of the centermost CA1 and CA3 subfields of the hippocampus. Ultrathin sections (60–90 nm) were stained with uranylacetate and lead citrate, and closely examined under a transmission electron microscope (JEM-100CX, JEOL, Japan). SO2 concentrations were measured using high-performance liquid chromatography (HPLC, Agilent 1200 series, Agilent Technologies, Palo Alto, CA, USA) [5]. The plasma and hippocampus samples for SO2 determination were prepared in the same manner, as previously reported [5]. Total RNA in rat tissues was extracted using Trizol reagent and reverse-transcribed by oligo(dT)15 primer and M-MLV reverse transcriptase. The reaction for the real-time PCR (final volume of 25 L) was mixed with 2.5 L of 10× PCR buffer, 1 L of 7.5 mol/L forward and reverse primer, 1 L of 2.5 mmol/L dNTP mixture, 0.25 L of Taq DNA polymerase, and 2 L of rat tissue cDNA. PCR products were amplified again using the PCR primers (AAT1 forward: 5 -CCAGGGAGCTCGGATCGT-3 , reverse:
Fig. 1. Graphical abstract and flow chart of the experimental steps. (A) Graphical abstract. (B) Flow chart of the experimental steps.
5 -GCCATTGTCTTCACGTTTCCTT-3 , TaqMan probe: 5 -CCACCACCCTCTCCAACCCTGA-3 ; AAT2 forward: 5 -GAGGGTCGGAGCCAGCTT-3 , reverse: 5 -GTTTCCCCAGGATGGTTTGG-3 , TaqMan probe: 5 -TTTAAGTTCAGCCGAGATGTCTTTC-3 ; -actin forward: 5 -ACCCGCGAGTACAACCTTCTT-3 , reverse: 5 -TATCGTCATCCATGGCGAACT-3 , TaqMan probe: 5 -CCTCCGTCGCCGGTCCACAC-3 ). The PCR condition was set to predenaturation at 95 ◦ C for 5 min, followed by 40 cycles of 95 ◦ C for 15 s and 60 ◦ C for 1 min. An extraction of the passaged viruses of all genotypes available was at 10-fold (1 × 10−1 to 1 × 10−6 ) dilution and was analyzed by both real-time and reverse-transcribed PCR methods. -Actin in each sample was used to calibrate the sample amount used for the determination. Animals (six for each group) were perfused transcardially with saline followed by the 4% paraformaldehyde for 30 min. Brains were removed, maintained in the same fixative for 20 h, successively dehydrated overnight in 20 and 30% sucrose in PBS, frozen in liquid nitrogen, and then stored at −70 ◦ C for further analysis. Brains coronal sections of 10 m thickness were incubated with AAT1 or AAT2 antibodies (dilutions of 1:50 and 1:300, respectively) overnight at 4 ◦ C, and then with a secondary biotinylated anti-rabbit or antimouse antibody (Zhongshan Goldenbridge Biotechnology, China) at 37 ◦ C for 15 min (AAT1) or 60 min (AAT2). Signals were visualized by diaminobenzidine (DAB).
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Fig. 2. SO2 concentration in the plasma and hippocampus, and AAT protein and mRNA in the hippocampus of rats. (A) The concentration of SO2 in the plasma of rats. (B) The concentration of SO2 in the hippocampus of rats. (C) Expression of AAT1 protein in CA1 in control group. (D) Expression of AAT1 protein in CA1 in FS group. (E) Expression of AAT2 protein in CA3 in control group. (F) Expression of AAT2 protein in CA3 in FS group. Compared with those in control group, SO2 in the plasma and hippocampus was increased, accompanied by upregulation of AAT1 and AAT2 in FS group. (G) AAT1 mRNA level in the hippocampus in FS group was higher than that in control group. (H) AAT2 mRNA level in the hippocampus in FS group was clearly upregulated compared with that of control group (mean ± SEM). N P < 0.05, vs. control group; + P < 0.01, vs. control group; *P < 0.05, vs. FS group; # P < 0.01, vs. FS group.
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Fig. 3. Ultrastructure of hippocampal CA1 neurons stained with uranylacetate and lead citrate under transmission electron microscopy. (A) Control group: normal ultrastructure of hippocampal CA1 neurons. (B) FS group: swollen mitochondria, with dissolved and ruptured ridges, the appearance of vacuoles, aciculate rough endoplasmic reticulum (RER) and dilated Golgi body. (C) FS + S5 group: mitochondria were slightly swollen with relatively normal structures, and the RER and Golgi body were only slightly dilated. (D) FS + HDX group: mitochondria were highly swollen and the RER was extremely dilated.
Fig. 4. TUNEL staining showing apoptotic neurons in samples of rat hippocampus: (A) control group. (B) FS group. (C) FS + S5 group. (D) FS + HDX group. The number of apoptotic neurons was significantly increased in FS group compared with that of control group. Apoptotic cells were markedly decreased in the FS + S5 group while they were clearly increased in FS + HDX group compared with those in FS group.
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Fig. 5. Timm staining in the hippocampus of different groups. (A) Control group: no mossy fiber sprouting was found in the dentate gyrus. (B) FS group: mossy fiber sprouting was evident in the dentate gyrus. (C) FS + S5 group: mossy fiber sprouting was inconspicuous in the dentate gyrus. (D) FS + HDX group: mossy fiber sprouting was very apparent compared with that in FS group.
For the determination of apoptosis in the brain sections, TUNEL assay was performed using the In Situ Cell Death Detection Kit, Fluorescein (Roche Applied Science, Germany), according to the protocol provided by the manufacturer. The sections were stained with diaminobenzidine (DAB) to develop color. Animals (six for each group) were perfused transcardially with 0.9% saline followed by 1% sodium sulfide and 4% PFA in PBS. The brains were removed, post-fixed for 20 h and then equilibrated sequentially with 30% sucrose at 4 ◦ C for 72 h, and finally iced by liquid nitrogen and stored at −70 ◦ C for further analysis. Brains were sectioned into 40 m thick coronal sections and processed for Timm staining. The processing solutions consisted of 30% gum arabic, 3.825% citric acid, 3.525% sodium citrate, 3.525% hydroquinone, and 25.5% silver nitrate. The slides were incubated at 26 ◦ C for 70 min, and then washed in distilled water twice for 5 min, and dehydrated through alcohol to xylene. The intensity of sprouting in the hippocampus was evaluated by a subjective gradation score [19], which varies from 0 (no staining in the supragranular layer) to 3 (intense staining in the supragranular layer). Scoring was conducted by two observers blinded to the experimental condition of each animal. The mean of the values given by the two observers represented the score of each animal. Results were expressed as mean ± SEM. The analyses were performed using SPSS 13.0 (Chicago, IL, USA). ANOVA, followed by post hoc analysis (Newman–Keuls test) to compare differences between groups. P-values less than 0.05 were considered statistically significant.
3. Results and discussion All rats developed seizures within 5 min every time after being immersed in 45.2 ◦ C water, and no rats developed seizure in
37 ◦ C water. The endogenous SO2 /AAT pathway was upregulated significantly in the rats of FS groups, which exhibited an increase in endogenous SO2 content during the development of FS (Fig. 2A and B). Moreover, we identified changes of AAT expression in the hippocampus of FS rats. AAT has two isoenzymes named for their different intracellular location: AAT1 is located in the cell cytoplasm, and AAT2 is located in the cell mitochondria [5]. The expressions of AAT1 and AAT2 in the hippocampus in FS group were increased markedly (Fig. 2C–H). The results disclosed the involvement of the SO2 /AAT system in the pathogenesis of FS. The pathogenetic mechanism for FS remains obscure. It might be associated with the disordered regulation of ionic homeostasis [20]. SO2 derivatives might relieve calcium overload in association with upregulating the expression of sarcoplasmic reticulum Ca2+ ATPase and phosphorylation of phospholamban [9,11]. Changes in SO2 might affect ionic homeostasis in the brain of FS rats. In addition, FS-related brain damage might result from oxidative damage; thus, oxidative damage may be one factor in the genesis of brain damage that is induced by FS [21]. SO2 could increase the antioxidative capacity in rats [22]. Our previous studies demonstrated that NO plays an important regulatory role in recurrent FS [15,17]. SO2 has been found to regulate the expression of NO/NOS pathway [10]. We speculate that SO2 might also play a role in the pathogenesis of FS by regulating NO/NOS pathway. It remains unclear whether changes in SO2 are cytoprotective or detrimental to the brain. Previous studies explored SO2 derivatives (Na2 SO3 /NaHSO3 ) relaxed isolated artery rings in a concentrationdependent manner, and SO2 showed a dose-dependent effect on myocardial infarct size in rats with I/R [23,24]. These results prompted us to test whether SO2 has a dose-dependent effect in the pathogenesis of recurrent FS. Thus, different doses of Na2 SO3 /NaHSO3 , a donor of exogenous SO2 , and HDX, an inhibitor of AAT [25], were administered to rats in the present study. No
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differences in seizure development in FS + SO2 or FS + HDX groups were recorded compared with those in FS group. However, different ultrastructural changes in hippocampal neurons were demonstrated. In FS group, the mitochondria swelled, the ridges dissolved and ruptured, vacuoles and aciculate rough endoplasmic reticulum (RER) appeared and the Golgi body was dilated. Preconditioning with a low concentration of SO2 (1–10 mol/kg) alleviated this neuronal damage in rats with recurrent FS. Preconditioning with a high concentration of SO2 (100 mol/kg) or HDX aggravated the neuronal damage in rats with recurrent FS (Fig. 3). The ultrastructural differences in the hippocampus between FS and FS + S50 groups were not statistically significant. These results indicated that the endogenous SO2 system showed a dose-dependent effect on brain damage induced by recurrent FS in rats. FS, as a stressor, can induce toxicity in the brain and eventually triggers apoptosis to remove damaged cells. The results of the TUNEL assay showed neuronal apoptosis in the hippocampus had increased in FS + HDX and FS + S100 groups, decreased in FS + S1, FS + S5 and FS + S10 groups, and scarcely changed in FS + S50 group compared with those in FS group (Fig. 4). These findings suggested that preconditioning with a low concentration of SO2 could reduce the degree of neuronal apoptosis during the development of FS. As the detection of neuronal apoptosis is suitable for the evaluation of neuronal injury [26], the increased level of neuronal apoptosis in the hippocampus in FS + HDX and FS + S100 groups indicated that the downregulation of SO2 or preconditioning with a high concentration of SO2 may result in hippocampal injury. Meanwhile, the decreased level of neuronal apoptosis in the hippocampus in the groups preconditioned with a low concentration of SO2 indicated that a cytoprotective effect had been exerted on the hippocampal neurons in FS rats. In addition, Timm-stained sections showed that MFS was evident in FS group, more notable in FS + HDX and FS + S100 groups, and inconspicuous in FS + S1, FS + S5 and FS + S10 groups (Fig. 5). These findings further suggested that slight increases in SO2 levels might play a protective role in the molecular pathogenesis of FS-related brain damage. The mechanism by which the neurons of rats with recurrent FS benefited from low-dose SO2 remains unclear. One recent study reported that a slight upregulation of SO2 increased the antioxidative capacity in rats [22]. In the present study, preconditioning with SO2 at low concentrations may have enhanced the antioxidative capacity of FS rats, thereby playing a protective role against neuronal damage. In summary, we have demonstrated SO2 /AAT system is involved in FS related brain damage. Preconditioning with low concentration of SO2 may protect neurons from toxicity injury due to FS. Our study may help further elucidate the molecular and cellular mechanisms underlying FS related brain damage and identify potential treatment for brain injury caused by FS. Acknowledgments We thank Professor Junbao Du of Peking University First Hospital for providing AAT1 and AAT2 antibodies, and Professor Hongfang Jin of Peking University First Hospital for help in the determination of SO2 concentration in plasma and hippocampus by HPLC. This work was supported by grants from the National Natural Science Foundation of China (81200998), Beijing Natural Science Foundation (7092105 and 7112131), Key Clinical Project from the Ministry of Public Health (2010–12), and National Key Technology R&D Program (2012BAI03B02).
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Stipanuk, Metabolism of sulfur-containing amino acids, Annu. Rev. Nutr. 6 (1986) 179–209. [7] R. Shapiro, Genetic effects of bisulfite (sulfur dioxide), Mutat. Res. 39 (1977) 149–175. [8] H. Mitsuhashi, S. Yamashita, H. Ikeuchi, T. Kuroiwa, Y. Kaneko, K. Hiromura, K. Ueki, Y. Nojima, Oxidative stress-dependent conversion of hydrogen sulfide to sulfite by activated neutrophils, Shock 24 (2005) 529–534. [9] S. Chen, J.B. Du, Y.F. Liang, R. Zhang, C.S. Tang, H.F. Jin, Sulfur dioxide restores calcium homeostasis disturbance in rat with isoproterenol-induced myocardial injury, Histol. Histopathol. 27 (2012) 1219–1226. [10] W. Lu, Y. Sun, C.S. Tang, T. Ochs, J.G. Qi, J.B. Du, H.F. Jin, Sulfur dioxide derivatives improve the vasorelaxation in the spontaneously hypertensive rat by enhancing the vasorelaxant response to nitric oxide, Exp. Biol. Med. (Maywood) 237 (2012) 867–872. [11] R.Y. Zhang, J.B. Du, Y. Sun, S. Chen, H.J. Tsai, L. Yuan, L. Li, C.S. Tang, H.F. Jin, Sulfur dioxide derivatives depress L-type calcium channel in rat cardiomyocytes, Clin. Exp. Pharmacol. Physiol. 38 (2011) 416–422. [12] E. Marutani, S. Kosugi, K. Tokuda, A. Khatri, R. Nguyen, D.N. Atochin, K. Kida, K. Van Leyen, K. Arai, F. Ichinose, A novel hydrogen sulfide-releasing N-methyl-daspartate receptor antagonist prevents ischemic neuronal death, J. Biol. Chem. 287 (2012) 32124–32135. [13] K. Kida, M. Yamada, K. Tokuda, E. Marutani, M. Kakinohana, M. Kaneki, F. Ichinose, Inhaled hydrogen sulfide prevents neurodegeneration and movement disorder in a mouse model of Parkinson’s disease, Antioxid. Redox Signal. 15 (2011) 343–352. [14] N. Tyagi, K.S. Moshal, U. Sen, T.P. Vacek, M. Kumar, W.M. Hughes Jr., S. Kundu, S.C. Tyagi, H2 S protects against methionine-induced oxidative stress in brain endothelial cells, Antioxid. Redox Signal. 11 (2009) 25–33. [15] Z.X. Yang, J. 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Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Dose-dependent effect of sulfur dioxide on brain damage induced by recurrent febrile seizures in rats Ying Han 1 , Wenxia Yi 1 , Jiong Qin ∗ , Yang Zhao, Jing Zhang, Xingzhi Chang Department of Pediatrics, Peking University First Hospital, Beijing 100034, China
h i g h l i g h t s • The SO2 /AAT system is involved in febrile seizures (FS) and related toxicity injury. • In a rat FS model, preconditioning with a low concentration of SO2 alleviated neuronal damage and apoptosis. • An AAT inhibitor or high concentration of SO2 aggravated neuronal damage in FS rats.
a r t i c l e
i n f o
Article history: Received 23 October 2013 Received in revised form 15 December 2013 Accepted 18 December 2013 Keywords: Sulfur dioxide Aspartate aminotransferase Febrile seizures Brain damage
a b s t r a c t Sulfur dioxide (SO2 ) regulates many physiological processes. Little is known about its roles in neurological disorders. In this study, we investigated the role of endogenous SO2 in the development of febrile seizures (FS) and related brain damages. In the rat model of recurrent FS, we found that endogenous SO2 in the plasma and hippocampus was increased, accompanied by upregulation of aspartate amino-transferase 1 (AAT1) and AAT2, and neuronal apoptosis and mossy fiber sprouting (MFS) in the hippocampus. Preconditioning with low concentration of SO2 (1–10 mol/kg) alleviated the neuronal damage, and attenuated neuronal apoptosis and MFS, whereas preconditioning with high concentration of SO2 (100 mol/kg) or inhibition of AAT aggravated the neuronal damage, and promoted neuronal apoptosis and MFS in hippocampus of rats with recurrent FS. These data indicate that endogenous SO2 is involved in the development of FS and related brain damage. Preconditioning with low concentration of SO2 may protect neurons from toxicity caused by FS. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Febrile seizures (FS) are frequently encountered in pediatric populations, occurring in 2–4% of children between 6 months and 5 years of age [1]. Patient studies have suggested that recurrent FS may cause hippocampal damage, which is one of the most common neuropathological findings of temporal lobe epilepsy (TLE) [2]. It is important, therefore, to further investigate the pathogenesis of FS and related brain damage. Sulfur dioxide (SO2 ) is known to be an atmospheric pollutant, and studies concerning SO2 have mainly focused on its toxicity in animals [3,4]. However, SO2 has recently been shown to be produced endogenously by the normal mammalian metabolism of sulfur-containing amino acids [5], and l-cysteine in particular
Abbreviations: AAT, aspartate aminotransferase; FS, febrile seizures; HDX, laspartate--hydroxamate; MFS, mossy fiber sprouting. ∗ Corresponding author. Tel.: +86 13 701086050; fax: +86 10 66530532. E-mail addresses: [email protected], [email protected] (J. Qin). 1 These authors contributed equally to this work. 0304-3940/$ – see front matter © 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.12.042
[6]. l-Cysteine is catalyzed by cysteine dioxygenase (CDO) to lcysteinesulfinate, which converts to -sulfinylpyruvate through transamination by aspartate aminotransferase (AAT); it then spontaneously decomposes to pyruvate and SO2 [7]. Additionally, hydrogen sulphide (H2 S) can be catalyzed to SO2 by nicotinamide adenine dinucleotide phosphate oxidase [8] or by the reduction of thiosulphate [7]. SO2 is metabolized to sulfite and oxidized to sulfate by sulfite oxidase, and is finally excreted in urine [6]. The distribution of SO2 and its generating enzymes, AAT and CDO, in different tissues of the rat, and the biological effects of endogenous SO2 on cardiovascular diseases have recently been described [9–11]. By comparisons with other analogous endogenous gaseous molecules, such as nitric oxide (NO), carbon monoxide (CO) and H2 S, it has been suggested that SO2 plays a regulatory role for physiological functions [9–11]. Recent studies demonstrated that NO, CO and H2 S play important roles in some of the nervous system diseases, including FS and related brain damage [12–17]. However, it remains unknown whether endogenous SO2 is involved in the pathogenesis of FS and related brain injury. The present study was therefore intended to investigate the changes in SO2 plasma and hippocampal levels in a rat model of recurrent FS,
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and to evaluate the effects of endogenous SO2 on FS-induced brain damage. 2. Materials and methods Animal care and experimental protocols complied with the Animal Management Rule of the Ministry of Health, China and the Animal Care Committee of Peking University First Hospital, Beijing, China. Sprague–Dawley male rats were housed with their mothers under standard laboratory conditions until they were weaned at 21 days. Our studies started with animals aged 21 days. The FS model has been described previously in detail [15]. In brief, the control rats were placed into 37 ◦ C water for 5 min, and the rats in the other seven groups were placed into 45.2 ◦ C water until a seizure occurred. As described in detail previously [18], hyperthermia treatment initiated seizures in rats, manifested as facial clonus, head nodding, forelimb clonus, rearing of the animal to a standing posture aided by its tail and the laterally spreading of hindlimbs with increased tone, before falling back. Water immersion was carried out once every 2 days, for 10 times in total. Rats were then randomly divided into eight groups that received the following treatments: (1) control group, preconditioning with normal saline 30 min before water immersion (n = 24); (2) FS group, hyperthermia treatment as described previously, preconditioning with normal saline 30 min before hyperthermia treatment (n = 24); (3) FS + S1 group, preconditioning with 1 mol/kg of SO2 (n = 24); (4) FS + S5 group, preconditioning with 5 mol/kg of SO2 (n = 24); (5) FS + S10 group, preconditioning with 10 mol/kg of SO2 (n = 24); (6) FS + S50 group, preconditioning with 50 mol/kg of SO2 (n = 24); (7) FS + S100 group, preconditioning with 100 mol/kg of SO2 (n = 24); (8) FS + HDX group, preconditioning with 3.7 mg/kg of l-aspartate--hydroxamate (HDX, an inhibitor of the enzyme responsible for endogenous generation of SO2 ) 30 min before hyperthermia treatment (n = 24). For groups 3–7, a SO2 donor (NaHSO3 and Na2 SO3 , 1:3 MM ratio) was intravenously injected 30 min before each hyperthermia treatment. An equal volume of normal saline was injected intraperitoneally in control and FS groups. All rats were anaesthetized within 6 h after the final water immersion (Fig. 1B). Rats were anesthetized with 4% chloral hydrate (600 mg/kg, intraperitoneally) and perfused through the heart with 0.9% saline followed by 3% paraformaldehyde (PFA) and 1% glutaraldehyde in phosphate-buffered saline (PBS). The brain was removed and the hippocampus was isolated and cut into approximately 1 mm3 cubes after immersion in 3% glutaraldehyde in PBS. The tissue was washed three times in sucrose and post-fixed in 1% osmium tetroxide for 2 h, and then dehydrated in graded ethanol solutions and embedded overnight in Epon 812 at 37 ◦ C. Semi-thin sections (2 m) were stained with toluidine blue. These sections were used for selection of the centermost CA1 and CA3 subfields of the hippocampus. Ultrathin sections (60–90 nm) were stained with uranylacetate and lead citrate, and closely examined under a transmission electron microscope (JEM-100CX, JEOL, Japan). SO2 concentrations were measured using high-performance liquid chromatography (HPLC, Agilent 1200 series, Agilent Technologies, Palo Alto, CA, USA) [5]. The plasma and hippocampus samples for SO2 determination were prepared in the same manner, as previously reported [5]. Total RNA in rat tissues was extracted using Trizol reagent and reverse-transcribed by oligo(dT)15 primer and M-MLV reverse transcriptase. The reaction for the real-time PCR (final volume of 25 L) was mixed with 2.5 L of 10× PCR buffer, 1 L of 7.5 mol/L forward and reverse primer, 1 L of 2.5 mmol/L dNTP mixture, 0.25 L of Taq DNA polymerase, and 2 L of rat tissue cDNA. PCR products were amplified again using the PCR primers (AAT1 forward: 5 -CCAGGGAGCTCGGATCGT-3 , reverse:
Fig. 1. Graphical abstract and flow chart of the experimental steps. (A) Graphical abstract. (B) Flow chart of the experimental steps.
5 -GCCATTGTCTTCACGTTTCCTT-3 , TaqMan probe: 5 -CCACCACCCTCTCCAACCCTGA-3 ; AAT2 forward: 5 -GAGGGTCGGAGCCAGCTT-3 , reverse: 5 -GTTTCCCCAGGATGGTTTGG-3 , TaqMan probe: 5 -TTTAAGTTCAGCCGAGATGTCTTTC-3 ; -actin forward: 5 -ACCCGCGAGTACAACCTTCTT-3 , reverse: 5 -TATCGTCATCCATGGCGAACT-3 , TaqMan probe: 5 -CCTCCGTCGCCGGTCCACAC-3 ). The PCR condition was set to predenaturation at 95 ◦ C for 5 min, followed by 40 cycles of 95 ◦ C for 15 s and 60 ◦ C for 1 min. An extraction of the passaged viruses of all genotypes available was at 10-fold (1 × 10−1 to 1 × 10−6 ) dilution and was analyzed by both real-time and reverse-transcribed PCR methods. -Actin in each sample was used to calibrate the sample amount used for the determination. Animals (six for each group) were perfused transcardially with saline followed by the 4% paraformaldehyde for 30 min. Brains were removed, maintained in the same fixative for 20 h, successively dehydrated overnight in 20 and 30% sucrose in PBS, frozen in liquid nitrogen, and then stored at −70 ◦ C for further analysis. Brains coronal sections of 10 m thickness were incubated with AAT1 or AAT2 antibodies (dilutions of 1:50 and 1:300, respectively) overnight at 4 ◦ C, and then with a secondary biotinylated anti-rabbit or antimouse antibody (Zhongshan Goldenbridge Biotechnology, China) at 37 ◦ C for 15 min (AAT1) or 60 min (AAT2). Signals were visualized by diaminobenzidine (DAB).
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Fig. 2. SO2 concentration in the plasma and hippocampus, and AAT protein and mRNA in the hippocampus of rats. (A) The concentration of SO2 in the plasma of rats. (B) The concentration of SO2 in the hippocampus of rats. (C) Expression of AAT1 protein in CA1 in control group. (D) Expression of AAT1 protein in CA1 in FS group. (E) Expression of AAT2 protein in CA3 in control group. (F) Expression of AAT2 protein in CA3 in FS group. Compared with those in control group, SO2 in the plasma and hippocampus was increased, accompanied by upregulation of AAT1 and AAT2 in FS group. (G) AAT1 mRNA level in the hippocampus in FS group was higher than that in control group. (H) AAT2 mRNA level in the hippocampus in FS group was clearly upregulated compared with that of control group (mean ± SEM). N P < 0.05, vs. control group; + P < 0.01, vs. control group; *P < 0.05, vs. FS group; # P < 0.01, vs. FS group.
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Fig. 3. Ultrastructure of hippocampal CA1 neurons stained with uranylacetate and lead citrate under transmission electron microscopy. (A) Control group: normal ultrastructure of hippocampal CA1 neurons. (B) FS group: swollen mitochondria, with dissolved and ruptured ridges, the appearance of vacuoles, aciculate rough endoplasmic reticulum (RER) and dilated Golgi body. (C) FS + S5 group: mitochondria were slightly swollen with relatively normal structures, and the RER and Golgi body were only slightly dilated. (D) FS + HDX group: mitochondria were highly swollen and the RER was extremely dilated.
Fig. 4. TUNEL staining showing apoptotic neurons in samples of rat hippocampus: (A) control group. (B) FS group. (C) FS + S5 group. (D) FS + HDX group. The number of apoptotic neurons was significantly increased in FS group compared with that of control group. Apoptotic cells were markedly decreased in the FS + S5 group while they were clearly increased in FS + HDX group compared with those in FS group.
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Fig. 5. Timm staining in the hippocampus of different groups. (A) Control group: no mossy fiber sprouting was found in the dentate gyrus. (B) FS group: mossy fiber sprouting was evident in the dentate gyrus. (C) FS + S5 group: mossy fiber sprouting was inconspicuous in the dentate gyrus. (D) FS + HDX group: mossy fiber sprouting was very apparent compared with that in FS group.
For the determination of apoptosis in the brain sections, TUNEL assay was performed using the In Situ Cell Death Detection Kit, Fluorescein (Roche Applied Science, Germany), according to the protocol provided by the manufacturer. The sections were stained with diaminobenzidine (DAB) to develop color. Animals (six for each group) were perfused transcardially with 0.9% saline followed by 1% sodium sulfide and 4% PFA in PBS. The brains were removed, post-fixed for 20 h and then equilibrated sequentially with 30% sucrose at 4 ◦ C for 72 h, and finally iced by liquid nitrogen and stored at −70 ◦ C for further analysis. Brains were sectioned into 40 m thick coronal sections and processed for Timm staining. The processing solutions consisted of 30% gum arabic, 3.825% citric acid, 3.525% sodium citrate, 3.525% hydroquinone, and 25.5% silver nitrate. The slides were incubated at 26 ◦ C for 70 min, and then washed in distilled water twice for 5 min, and dehydrated through alcohol to xylene. The intensity of sprouting in the hippocampus was evaluated by a subjective gradation score [19], which varies from 0 (no staining in the supragranular layer) to 3 (intense staining in the supragranular layer). Scoring was conducted by two observers blinded to the experimental condition of each animal. The mean of the values given by the two observers represented the score of each animal. Results were expressed as mean ± SEM. The analyses were performed using SPSS 13.0 (Chicago, IL, USA). ANOVA, followed by post hoc analysis (Newman–Keuls test) to compare differences between groups. P-values less than 0.05 were considered statistically significant.
3. Results and discussion All rats developed seizures within 5 min every time after being immersed in 45.2 ◦ C water, and no rats developed seizure in
37 ◦ C water. The endogenous SO2 /AAT pathway was upregulated significantly in the rats of FS groups, which exhibited an increase in endogenous SO2 content during the development of FS (Fig. 2A and B). Moreover, we identified changes of AAT expression in the hippocampus of FS rats. AAT has two isoenzymes named for their different intracellular location: AAT1 is located in the cell cytoplasm, and AAT2 is located in the cell mitochondria [5]. The expressions of AAT1 and AAT2 in the hippocampus in FS group were increased markedly (Fig. 2C–H). The results disclosed the involvement of the SO2 /AAT system in the pathogenesis of FS. The pathogenetic mechanism for FS remains obscure. It might be associated with the disordered regulation of ionic homeostasis [20]. SO2 derivatives might relieve calcium overload in association with upregulating the expression of sarcoplasmic reticulum Ca2+ ATPase and phosphorylation of phospholamban [9,11]. Changes in SO2 might affect ionic homeostasis in the brain of FS rats. In addition, FS-related brain damage might result from oxidative damage; thus, oxidative damage may be one factor in the genesis of brain damage that is induced by FS [21]. SO2 could increase the antioxidative capacity in rats [22]. Our previous studies demonstrated that NO plays an important regulatory role in recurrent FS [15,17]. SO2 has been found to regulate the expression of NO/NOS pathway [10]. We speculate that SO2 might also play a role in the pathogenesis of FS by regulating NO/NOS pathway. It remains unclear whether changes in SO2 are cytoprotective or detrimental to the brain. Previous studies explored SO2 derivatives (Na2 SO3 /NaHSO3 ) relaxed isolated artery rings in a concentrationdependent manner, and SO2 showed a dose-dependent effect on myocardial infarct size in rats with I/R [23,24]. These results prompted us to test whether SO2 has a dose-dependent effect in the pathogenesis of recurrent FS. Thus, different doses of Na2 SO3 /NaHSO3 , a donor of exogenous SO2 , and HDX, an inhibitor of AAT [25], were administered to rats in the present study. No
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differences in seizure development in FS + SO2 or FS + HDX groups were recorded compared with those in FS group. However, different ultrastructural changes in hippocampal neurons were demonstrated. In FS group, the mitochondria swelled, the ridges dissolved and ruptured, vacuoles and aciculate rough endoplasmic reticulum (RER) appeared and the Golgi body was dilated. Preconditioning with a low concentration of SO2 (1–10 mol/kg) alleviated this neuronal damage in rats with recurrent FS. Preconditioning with a high concentration of SO2 (100 mol/kg) or HDX aggravated the neuronal damage in rats with recurrent FS (Fig. 3). The ultrastructural differences in the hippocampus between FS and FS + S50 groups were not statistically significant. These results indicated that the endogenous SO2 system showed a dose-dependent effect on brain damage induced by recurrent FS in rats. FS, as a stressor, can induce toxicity in the brain and eventually triggers apoptosis to remove damaged cells. The results of the TUNEL assay showed neuronal apoptosis in the hippocampus had increased in FS + HDX and FS + S100 groups, decreased in FS + S1, FS + S5 and FS + S10 groups, and scarcely changed in FS + S50 group compared with those in FS group (Fig. 4). These findings suggested that preconditioning with a low concentration of SO2 could reduce the degree of neuronal apoptosis during the development of FS. As the detection of neuronal apoptosis is suitable for the evaluation of neuronal injury [26], the increased level of neuronal apoptosis in the hippocampus in FS + HDX and FS + S100 groups indicated that the downregulation of SO2 or preconditioning with a high concentration of SO2 may result in hippocampal injury. Meanwhile, the decreased level of neuronal apoptosis in the hippocampus in the groups preconditioned with a low concentration of SO2 indicated that a cytoprotective effect had been exerted on the hippocampal neurons in FS rats. In addition, Timm-stained sections showed that MFS was evident in FS group, more notable in FS + HDX and FS + S100 groups, and inconspicuous in FS + S1, FS + S5 and FS + S10 groups (Fig. 5). These findings further suggested that slight increases in SO2 levels might play a protective role in the molecular pathogenesis of FS-related brain damage. The mechanism by which the neurons of rats with recurrent FS benefited from low-dose SO2 remains unclear. One recent study reported that a slight upregulation of SO2 increased the antioxidative capacity in rats [22]. In the present study, preconditioning with SO2 at low concentrations may have enhanced the antioxidative capacity of FS rats, thereby playing a protective role against neuronal damage. In summary, we have demonstrated SO2 /AAT system is involved in FS related brain damage. Preconditioning with low concentration of SO2 may protect neurons from toxicity injury due to FS. Our study may help further elucidate the molecular and cellular mechanisms underlying FS related brain damage and identify potential treatment for brain injury caused by FS. Acknowledgments We thank Professor Junbao Du of Peking University First Hospital for providing AAT1 and AAT2 antibodies, and Professor Hongfang Jin of Peking University First Hospital for help in the determination of SO2 concentration in plasma and hippocampus by HPLC. This work was supported by grants from the National Natural Science Foundation of China (81200998), Beijing Natural Science Foundation (7092105 and 7112131), Key Clinical Project from the Ministry of Public Health (2010–12), and National Key Technology R&D Program (2012BAI03B02).
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