浙江大学 文本 The augmentation of brain thioredoxin-1 expression after severe hypobaric hypoxia by the preconditioning in rats_生理学

Available online at www.sciencedirect.com SCIENCE DIRECT Neuroscience Letters ELSEVIER Neuroscience Letters 370(2004)224-229 www.elsevier.com/locate/neulet The augmentation of brain thioredoxin-1 expression after severe hypobaric hypoxia by the preconditioning in rats Serguei A.Stroeva.b,Ekaterina I.Tjulkovab,Tatjana S.Gluschenkob Elena A.Rybnikova,Michail O.Samoilov Markku Pelto-Huikkoa. Department of Developmental Biology.Tampere University Medical School and Department of Pathology. Tampere University Hospital,33014 Tampere,Finland Laboratory of Regulation of Brain Neuron Functions,Pavloy Institute of Physiology RAS.199034 St.Petersburg.Russia Laboratory of Neuroendocrinology.Pavloy Institute of Physiology RAS.199034 St.Petersburg.Russia Received 14 June 2004;received in revised form 12 August 2004;accepted 12 August 2004 Abstract Induction of endogenous antioxidants is one of the key molecular mechanisms of cell resistance to hypoxia/ischemia.The effect of severe hypoxia on the expression of cytosolic antioxidant thioredoxin-1(Trx)in hippocampus and neocortex was studied in preconditioned and non-preconditioned rats.The preconditioning consisted of three trials of mild hypobaric hypoxia(360 Torr,2 h)spaced at 24 h.Twenty-four hours after the last trial rats were subjected to severe hypobaric hypoxia(180 Torr,3h).Trx expression was studied by immunocytochemistry. In hippocampus severe hypobaric hypoxia rapidly induced Trx expression,which remained elevated still at 24 h.In neocortex the enhanced expression appeared only at 24 h.The preconditioning significantly augmented severe hypoxia-induced Trx-immunoreactivity at 3 h but not at 24 h.These findings point out that Trx contributes to mechanisms of brain tolerance to hypobaric hypoxia,especially in early periods after the exposure 2004 Elsevier Ireland Ltd.All rights reserved Keywords:Hypobaric hypoxia;Preconditioning;Antioxidants;Thioredoxin-1;Immunocytochemistry Severe hypoxia/ischemia can induce apoptotic and necrotic active disulfide/dithiol within the conserved active site se- neuronal cell death [3,27,61].The oxidative stress caused by quence-Cys-Gly-Pro-Cys-[22,23].The thioredoxin reduc- redox balance disruption and overproduction of reactive oxy- tase reduces the oxidized form using NADPH [38].Trx is in- gen species(ROS)is an important mechanism of cell dam- duced by hypoxia/ischemia [4,50]and protects cells against age produced by hypoxia/ischemia [7,8,46].Oxidative stress different kinds of oxidative stress [24,42,48]. and redox balance impairment are followed by a dysfunction Mild hypoxic/ischemic preconditioning increases the neu- of important redox-sensitive enzymes,membrane receptors ronal resistance to subsequent severe hypoxia/ischemia and ion channels [32,55],DNA damage [5,6,18,44],mem- [29,41].The 2-min ischemic preconditioning suppresses the brane lipid peroxidation [56.59]and cytochrome c release cytochrome c release from mitochondria induced by severe from mitochondria,which activate the caspases that result in 5-min ischemia in gerbil hippocampus [36].The expression cell death [28.541. of Trx and other antioxidants appears to provide one of the The thioredoxin and glutathione systems control the cel- neuroprotective mechanisms activated by the precondition- lular redox state.Thioredoxin-1 (Trx)is a small (about ing [2]. 12 kDa)multifunctional ubiquitous protein with a redox- We previously showed that hypobaric hypoxia increased the expression of mitochondrial Trx-2 in different rat brain *Corresponding author.Tel.:+35832156644;fax:+358 3 2156170. areas including hippocampus and sensory-motor neocortex. E-mail address:blmapel@uta.fi (M.Pelto-Huikko). The preconditioning significantly augmented this induction 0304-3940/S-see front matter 2004 Elsevier Ireland Ltd.All rights reserved. doi:10.1016M.neulet.2004.08.022

Neuroscience Letters 370 (2004) 224–229 The augmentation of brain thioredoxin-1 expression after severe hypobaric hypoxia by the preconditioning in rats Serguei A. Stroeva,b, Ekaterina I. Tjulkovab, Tatjana S. Gluschenkob, Elena A. Rybnikovac, Michail O. Samoilovb, Markku Pelto-Huikkoa,∗ a Department of Developmental Biology, Tampere University Medical School and Department of Pathology, Tampere University Hospital, 33014 Tampere, Finland b Laboratory of Regulation of Brain Neuron Functions, Pavlov Institute of Physiology RAS, 199034 St. Petersburg, Russia c Laboratory of Neuroendocrinology, Pavlov Institute of Physiology RAS, 199034 St. Petersburg, Russia Received 14 June 2004; received in revised form 12 August 2004; accepted 12 August 2004 Abstract Induction of endogenous antioxidants is one of the key molecular mechanisms of cell resistance to hypoxia/ischemia. The effect of severe hypoxia on the expression of cytosolic antioxidant thioredoxin-1 (Trx) in hippocampus and neocortex was studied in preconditioned and non-preconditioned rats. The preconditioning consisted of three trials of mild hypobaric hypoxia (360 Torr, 2 h) spaced at 24 h. Twenty-four hours after the last trial rats were subjected to severe hypobaric hypoxia (180 Torr, 3 h). Trx expression was studied by immunocytochemistry. In hippocampus severe hypobaric hypoxia rapidly induced Trx expression, which remained elevated still at 24 h. In neocortex the enhanced expression appeared only at 24 h. The preconditioning significantly augmented severe hypoxia-induced Trx-immunoreactivity at 3 h but not at 24 h. These findings point out that Trx contributes to mechanisms of brain tolerance to hypobaric hypoxia, especially in early periods after the exposure. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Hypobaric hypoxia; Preconditioning; Antioxidants; Thioredoxin-1; Immunocytochemistry Severe hypoxia/ischemia can induce apoptotic and necrotic neuronal cell death [3,27,61]. The oxidative stress caused by redox balance disruption and overproduction of reactive oxy￾gen species (ROS) is an important mechanism of cell dam￾age produced by hypoxia/ischemia [7,8,46]. Oxidative stress and redox balance impairment are followed by a dysfunction of important redox-sensitive enzymes, membrane receptors and ion channels [32,55], DNA damage [5,6,18,44], mem￾brane lipid peroxidation [56,59] and cytochrome c release from mitochondria, which activate the caspases that result in cell death [28,54]. The thioredoxin and glutathione systems control the cel￾lular redox state. Thioredoxin-1 (Trx) is a small (about 12 kDa) multifunctional ubiquitous protein with a redox- ∗ Corresponding author. Tel.: +358 3 2156644; fax: +358 3 2156170. E-mail address: blmapel@uta.fi (M. Pelto-Huikko). active disulfide/dithiol within the conserved active site se￾quence –Cys–Gly–Pro–Cys– [22,23]. The thioredoxin reduc￾tase reduces the oxidized form using NADPH [38]. Trx is in￾duced by hypoxia/ischemia [4,50] and protects cells against different kinds of oxidative stress [24,42,48]. Mild hypoxic/ischemic preconditioning increases the neu￾ronal resistance to subsequent severe hypoxia/ischemia [29,41]. The 2-min ischemic preconditioning suppresses the cytochrome c release from mitochondria induced by severe 5-min ischemia in gerbil hippocampus [36]. The expression of Trx and other antioxidants appears to provide one of the neuroprotective mechanisms activated by the precondition￾ing [2]. We previously showed that hypobaric hypoxia increased the expression of mitochondrial Trx-2 in different rat brain areas including hippocampus and sensory-motor neocortex. The preconditioning significantly augmented this induction 0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.08.022

S.A.Stroev et al.Neuroscience Letters 370 (2004)224-229 225 [45].The aim ofpresent research is to investigate the cytosolic Trx expression in rat neocortex and hippocampus in identical experimental model. Male Wistar rats weighing 200-250g were subjected to hypobaric hypoxia.Severe and repetitive mild precondition- ing hypoxias were produced in a hypobaric chamber by main- taining the pressure at 160-180 Torr (equivalent to 5%nor- mobaric oxygen)for 3h,and 360 Torr (equivalent to 10% normobaric oxygen)for 2h daily for 3 days,respectively. The severe hypoxia produced in such a paradigm caused ex- tensive neuronal damage in hippocampus and neocortex,but the preliminary preconditioning prevented severe hypoxia- induced neuronal damage [39].All the animals were divided into three groups (four to six rats per group):(i)rats subjected (B) to severe hypoxia;(ii)rats subjected to preconditioning hy- poxia 24 h prior to the severe hypoxia;(iii)control rats placed in the chamber for 3h with no hypoxia produced.The Trx immunoreactivity was studied 3 and 24 h following severe hypoxia. For immunocytochemistry the rats were anaesthetized and perfused transcardially first with 100 ml of saline followed by 4%paraformaldehyde in 0.1 M phosphate-buffered saline (PBS;pH 7.3)for 4-5 min.After perfusion the brains were excised and subsequently fixed by immersion in the same solution for 60 min.The samples were cryoprotected with 15%sucrose in PBS and stored at +4C until sectioning in the cryostat.Immunocytochemistry was performed us- ing ABC-method.Coronal sections (11 um)of the brain (about-2.80 mm from bregma [37])were mounted onto the poly-L-lysine(Sigma)covered slides and then incubated with affinity-purified rabbit antiserum against mouse cytosolic Trx [48](dil.1:500 in PBS containing 1%BSA and 0.3%Triton X-100)at +4C overnight.After several washes,the sec- tions were incubated with biotinylated goat antirabbit (Vector Labs)antibodies (dil.1:300)and ABC complex for 30min each.Diaminobenzidine was used as a chromogen to visual- (E) ize the sites expressing Trx immunoreactivity.The sections were dehydrated,mounted and assayed with image analysis Fig.1.Trx-immunoreactivity in the CAl area of hippocampus.Photomicro- system consisting of IBM PC,Nikon Microphot-FXA mi- graphsofcontrol hippocampal CAl field(arrowspoint non-labeled neurons) croscope,SensiCam digital camera(PCO Computer Optics (A),hippocampal CAl field at 3(arrows show lightly labeled neurons)(B), GmbH),Image-Pro Plus(Media Cybernetics)program. and 24 h(few neurons are moderately labeled)(D),after severe hypobaric hypoxia and after precondioned severe hypoxia at 3(most of the neurons Trx expression was examined in fronto-parietal cortex, are strongly stained)(C),and 24 h(some neurons are strongly labeled)(E). CAl,CA2,CA3 hippocampal fields and dentate gyrus.The Scale bar:50μm. Trx-immunoreactive cells were quantified in the area of 500 um in length (in hippocampus)or in square 300 um and the number of intensely-labeled cells as a percent of con- x 300 pm (in neocortex),using Videotest Morphology pro- trol (Ni).One-way ANOVA was used for statistical analysis gram.Six sections were analyzed from each brain;one field of data of each brain area studied was measured per each slice.The Immunocytochemistry revealed that Trx expression in hip- intensity of staining was expressed as conventional value of pocampus and neocortex was affected by severe hypoxia and optical density scale from 0(absolute white)to 100(abso- preconditioning.A notable increase in Trx immunoreactiv- lute black).Immunoreactive cells were divided in 2 relative ity in all hippocampal areas examined but not in neocortex classes:slightly-labeled(staining intensity was at 1-10 con- was detected 3 h after severe hypoxia(Figs.1-3).The ex- ventional units above the background)and intensely-labeled posure to severe hypoxia significantly increased N+in CAl (more than 10 units above the background).Trx immunoreac- (129%)and CA2(145%)(Fig.2).The number of intensely- tivity was assayed using following criteria:the total number labeled cells (Ni)was essentially elevated in CAl (238%), of immunoreactive cells shown as a percent of control (N) CA2(776%),CA3(469%),and DG(259%)(Fig3)

S.A. Stroev et al. / Neuroscience Letters 370 (2004) 224–229 225 [45]. The aim of present research is to investigate the cytosolic Trx expression in rat neocortex and hippocampus in identical experimental model. Male Wistar rats weighing 200–250 g were subjected to hypobaric hypoxia. Severe and repetitive mild precondition￾ing hypoxias were produced in a hypobaric chamber by main￾taining the pressure at 160–180 Torr (equivalent to 5% nor￾mobaric oxygen) for 3 h, and 360 Torr (equivalent to 10% normobaric oxygen) for 2 h daily for 3 days, respectively. The severe hypoxia produced in such a paradigm caused ex￾tensive neuronal damage in hippocampus and neocortex, but the preliminary preconditioning prevented severe hypoxia￾induced neuronal damage [39]. All the animals were divided into three groups (four to six rats per group): (i) rats subjected to severe hypoxia; (ii) rats subjected to preconditioning hy￾poxia 24 h prior to the severe hypoxia; (iii) control rats placed in the chamber for 3 h with no hypoxia produced. The Trx immunoreactivity was studied 3 and 24 h following severe hypoxia. For immunocytochemistry the rats were anaesthetized and perfused transcardially first with 100 ml of saline followed by 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS; pH 7.3) for 4–5 min. After perfusion the brains were excised and subsequently fixed by immersion in the same solution for 60 min. The samples were cryoprotected with 15% sucrose in PBS and stored at +4 ◦C until sectioning in the cryostat. Immunocytochemistry was performed us￾ing ABC-method. Coronal sections (11
m) of the brain (about −2.80 mm from bregma [37]) were mounted onto the poly-l-lysine (Sigma) covered slides and then incubated with affinity-purified rabbit antiserum against mouse cytosolic Trx [48] (dil. 1:500 in PBS containing 1% BSA and 0.3% Triton X-100) at +4 ◦C overnight. After several washes, the sec￾tions were incubated with biotinylated goat antirabbit (Vector Labs) antibodies (dil. 1:300) and ABC complex for 30 min each. Diaminobenzidine was used as a chromogen to visual￾ize the sites expressing Trx immunoreactivity. The sections were dehydrated, mounted and assayed with image analysis system consisting of IBM PC, Nikon Microphot-FXA mi￾croscope, SensiCam digital camera (PCO Computer Optics GmbH), Image-Pro Plus (Media Cybernetics) program. Trx expression was examined in fronto-parietal cortex, CA1, CA2, CA3 hippocampal fields and dentate gyrus. The Trx-immunoreactive cells were quantified in the area of 500
m in length (in hippocampus) or in square 300
m × 300
m (in neocortex), using Videotest Morphology pro￾gram. Six sections were analyzed from each brain; one field of each brain area studied was measured per each slice. The intensity of staining was expressed as conventional value of optical density scale from 0 (absolute white) to 100 (abso￾lute black). Immunoreactive cells were divided in 2 relative classes: slightly-labeled (staining intensity was at 1–10 con￾ventional units above the background) and intensely-labeled (more than 10 units above the background). Trx immunoreac￾tivity was assayed using following criteria: the total number of immunoreactive cells shown as a percent of control (N+) Fig. 1. Trx-immunoreactivity in the CA1 area of hippocampus. Photomicro￾graphs of control hippocampal CA1 field (arrows point non-labeled neurons) (A), hippocampal CA1 field at 3 (arrows show lightly labeled neurons) (B), and 24 h (few neurons are moderately labeled) (D), after severe hypobaric hypoxia and after precondioned severe hypoxia at 3 (most of the neurons are strongly stained) (C), and 24 h (some neurons are strongly labeled) (E). Scale bar: 50
m. and the number of intensely-labeled cells as a percent of con￾trol (Ni). One-way ANOVA was used for statistical analysis of data. Immunocytochemistry revealed that Trx expression in hip￾pocampus and neocortex was affected by severe hypoxia and preconditioning. A notable increase in Trx immunoreactiv￾ity in all hippocampal areas examined but not in neocortex was detected 3 h after severe hypoxia (Figs. 1–3). The ex￾posure to severe hypoxia significantly increased N+ in CA1 (129%) and CA2 (145%) (Fig. 2). The number of intensely￾labeled cells (Ni) was essentially elevated in CA1 (238%), CA2 (776%), CA3 (469%), and DG (259%) (Fig. 3)

226 S.A.Stroey et al.Neuroscience Letters 370 (2004)224-229 180 800 700 64000040 600 500 400 300 200 100 0 C P S P (A) 3h 24h (A) 3h 24h 250 2000 1750 0 1500 1250 10 1000 750 50 500 250 C P S P Q S P P (B) 3h 24h (B) 3h 24h 160 1600 140 1400 120 1200 1000 000 800 600 0 400 20 200 0 0 P S P Q S P (c) 3h 24h (C) 3h 24h 400 180 16 128 器 200 400000 150 100 5 0 P C (D) 24h (D) 24h 700 140 120 600 100 500 80 400 300 0 200 100 S P (E) 3h 24h 眉 Control 3h 24h Fig.2.Graphs showing changes in the total number of Trx-immunoreactive Fig.3.Graphs showing changes in the number of intensely-labeled cells+ cells+S.E.M.expressed as a percentage of control (N)in different rat S.E.M.expressed as a percentage of control (Ni)in different rat brain areas brain areas at 3 and 24 h after severe hypobaric hypoxia(S)(n=4)and at 3 and 24 h after severe hypobaric hypoxia(S)(n=4)and preconditioned preconditioned severe hypoxia(P)(n=5),as compared to control group (C) severe hypoxia (P)(n=5),as compared to control group (C)(n=6).CAl (n=6).CAl field of hippocampus(A),CA2 field of hippocampus(B),CA3 field of hippocampus(A),CA2 field of hippocampus(B),CA3 field of hip- field of hippocampus(C),dentate gyrus(D)and neocortex(E).Statistically pocampus(C),dentate gyrus(D)and neocortex(E).Statistically significant significant (P 0.05)differences:(*as compared to control,(#between (P 0.05)differences:(*as compared to control,(#between non-and non-and preconditioned animals,(S)between 3 and 24 h time-point. preconditioned animals,(s)between 3 and 24 h time-point

226 S.A. Stroev et al. / Neuroscience Letters 370 (2004) 224–229 Fig. 2. Graphs showing changes in the total number of Trx-immunoreactive cells ± S.E.M. expressed as a percentage of control (N+) in different rat brain areas at 3 and 24 h after severe hypobaric hypoxia (S) (n = 4) and preconditioned severe hypoxia (P) (n = 5), as compared to control group (C) (n = 6). CA1 field of hippocampus (A), CA2 field of hippocampus (B), CA3 field of hippocampus (C), dentate gyrus (D) and neocortex (E). Statistically significant (P < 0.05) differences: (*) as compared to control, (#) between non- and preconditioned animals, (§) between 3 and 24 h time-point. Fig. 3. Graphs showing changes in the number of intensely-labeled cells ± S.E.M. expressed as a percentage of control (Ni) in different rat brain areas at 3 and 24 h after severe hypobaric hypoxia (S) (n = 4) and preconditioned severe hypoxia (P) (n = 5), as compared to control group (C) (n = 6). CA1 field of hippocampus (A), CA2 field of hippocampus (B), CA3 field of hip￾pocampus (C), dentate gyrus (D) and neocortex (E). Statistically significant (P < 0.05) differences: (*) as compared to control, (#) between non- and preconditioned animals, (§) between 3 and 24 h time-point

S.A.Stroev et al.Neuroscience Letters 370 (2004)224-229 227 Preconditioning with mild repetitive hypoxia markedly ditioning greatly promotes this protective reaction in hip- augmented severe hypoxia-induced Trx expression in all pocampus and induce it in neocortex.The augmentation brain areas studied at 3 h time-point(Figs.1-3).The increase of Trx expression at early period of reoxygenation critical in the number ofimmunoreactive cells(N)was considerably for apoptosis initiation provides one possible mechanism of higher in preconditioned rats then in non-preconditioned ones hypoxic/ischemic tolerance produced by the precondition- in CAl(159%from control),CA2 (207%),DG(154%),and ing neocortex(107%)(Fig.2).The increase in Ni was obviously In neocortex hypoxia without preconditioning induced Trx higher in preconditioned rats then in non-preconditioned ones expression only at 24 h time-point.In hippocampus at this in CAl (536%),CA2(1158%),CA3(898%),and neocortex period the Trx expression remained enhanced.Hence the re- (244%(Fig.3). sponse in hippocampus is faster then in neocortex.In the At 24 h after severe hypoxia,Trx immunoreactiv- hippocampus of preconditioned rats the Trx expression in- ity remained enhanced in all hippocampal areas studied duction ceased by 24 h whereas in neocortex it continued to (Figs.1-3).N+was markedly increased in CA2 (159%), increase. CA3 (131%),and DG(129%)as compared to control.Ni Thiol redox status is one of the key factors of the apop- was increased in all brain areas studied:CAl (421%),CA2 tosis regulation [43].The protective functions of Trx dur- (1027%).CA3(1185%).DG(223%)and neocortex(337%) ing oxidative stress are diverse.One of the key Trx de- When compared to 3 h time-point,Trx immunoreactivity at fense function is the buffering of ROS [35,51]and inhi- 24 h was significantly elevated only in CA3(Ni but not N+) bition of cytochrome c release from mitochondria [2].By and in neocortex (N as well as Ni)(Figs.2 and 3). this way Trx can inhibit the apoptosis triggering.On the At 24 h after severe hypoxia there was no remarkable other hand Trx can switch necrosis to apoptosis by the difference in the Trx immunoreactivity (N+as well as Ni) regulation of redox-sensitive caspase activity [51,52].In between pre-and non-preconditioned animals (Figs.1-3) addition,Trx appears to function as a potent activator of except in CA3 where N+was found to be higher in non- other antioxidant systems,e.g.Mn-superoxide dismutase preconditioned ones.When compared to 3h time-point,a [10]. decrease of the immunoreactivity in hippocampal fields of Trx is translocated from cytoplasm to nucleus upon stress preconditioned rats was apparent at 24h time-point:the [33]and activates the transcriptional factors by enhancing changes of Ni were not significant but N+noticeably de- their binding activity to the target DNA:NF-KB,AP-1, creased in CAl (136%from control),CA2 (157%),and CREB,PEBP2/CBF,Myb,and HIF-1 [1,9,16,19-21,57,58], DG(132%).On the contrary,in neocortex Trx immunore- estrogen [17]and glucocorticoid [30,31]receptors.Trx also activity (Ni but not N.)of preconditioned rats was sub- augmented the DNA binding activity of p53 [53].Oxida- stantially elevated at 24h as compared to 3h time-point tive stress induced p53 [51]can in its turn activate a Gl (Fig.3). cyclin-dependent kinase inhibitor p21Cipl/WAFI that cause Trx provides an important defense of brain neurons dur- the cell-cycle arrest,presumable to allow an opportunity ing various hypoxic/ischemic events.Trx protein and mRNA for DNA repair [12,51].But p53 also can induce apopto- expression was down-regulated in the ischemic core regions sis 3]by an activation of proapoptotic protein Bax,result- but up-regulated in the perifocal ischemic regions since 4h ing in cytochrome c release [34].Trx augments the p53- after focal brain ischemia [15.4749]:the induced Trx was dependent p21 transcriptional activity and protein expression translocated into the nucleus after ischemia and ischemia- and thereby switch apoptosis triggering to DNA reparation reperfusion.It is important that changes in Trx expression way [53]. were observed in the earliest period after the insult because Trx is a negative regulator of apoptosis signal-regulating the first 2-4 h after the exposure to severe hypoxia are sup- kinase 1 (ASK1)[40].ASKI was identified as one of posed to be crucial for cytochrome c release [11].Transient the mitogen-activated protein (MAP)kinase kinase ki- global ischemia induced Trx in glial cells of the gerbil hip- nases,which activates the c-Jun N-terminal kinase (JNK) pocampus [50].Overexpression of the Trx in transgenic mice and p38 MAP kinase and induces stress-mediated apop- attenuates focal ischemic brain damage [48],on the contrary, tosis signaling [25].ASKI stimulates cytochrome c re- its inhibition increases oxidative stress [60].In addition,Trx lease and executes apoptosis mainly by mitochondria- reduces hypoxia-reoxygenation injury in cell culture in vitro dependent caspase activation [14].The negative regulation [261. of ASKI appears to be one of the Trx cytoprotective ef- In present study we for the first time showed that cy- fects.Trx also negatively regulates TNF-induced activa- tosolic Trx involved in neuronal responses to hypobaric hy- tion of p38 MAP kinase [13]activated by oxidative stress poxia.The expression of Trx in the brain of preconditioned 251. and non-preconditioned animals at 3 and 24 h following In conclusion,brain expression of thioredoxin-1 is en- severe hypoxia was studied by immunocytochemistry.Se- hanced after severe hypobaric hypoxia;the hypoxic precondi- vere hypoxia up-regulated the Trx expression in hippocam- tioning considerably up-regulates this enhancement.Present pus at 3h time-point;this induction appears to represent findings suggest a possible role for cytosolic antioxidant an adaptive neuronal response to oxidative stress.Precon- thioredoxin-1 in the induction of brain hypoxic tolerance

S.A. Stroev et al. / Neuroscience Letters 370 (2004) 224–229 227 Preconditioning with mild repetitive hypoxia markedly augmented severe hypoxia-induced Trx expression in all brain areas studied at 3 h time-point (Figs. 1–3). The increase in the number of immunoreactive cells (N+) was considerably higher in preconditioned rats then in non-preconditioned ones in CA1 (159% from control), CA2 (207%), DG (154%), and neocortex (107%) (Fig. 2). The increase in Ni was obviously higher in preconditioned rats then in non-preconditioned ones in CA1 (536%), CA2 (1158%), CA3 (898%), and neocortex (244%) (Fig. 3). At 24 h after severe hypoxia, Trx immunoreactiv￾ity remained enhanced in all hippocampal areas studied (Figs. 1–3). N+ was markedly increased in CA2 (159%), CA3 (131%), and DG (129%) as compared to control. Ni was increased in all brain areas studied: CA1 (421%), CA2 (1027%), CA3 (1185%), DG (223%) and neocortex (337%). When compared to 3 h time-point, Trx immunoreactivity at 24 h was significantly elevated only in CA3 (Ni but not N+) and in neocortex (N+ as well as Ni) (Figs. 2 and 3). At 24 h after severe hypoxia there was no remarkable difference in the Trx immunoreactivity (N+ as well as Ni) between pre- and non-preconditioned animals (Figs. 1–3) except in CA3 where N+ was found to be higher in non￾preconditioned ones. When compared to 3 h time-point, a decrease of the immunoreactivity in hippocampal fields of preconditioned rats was apparent at 24 h time-point: the changes of Ni were not significant but N+ noticeably de￾creased in CA1 (136% from control), CA2 (157%), and DG (132%). On the contrary, in neocortex Trx immunore￾activity (Ni but not N+) of preconditioned rats was sub￾stantially elevated at 24 h as compared to 3 h time-point (Fig. 3). Trx provides an important defense of brain neurons dur￾ing various hypoxic/ischemic events. Trx protein and mRNA expression was down-regulated in the ischemic core regions but up-regulated in the perifocal ischemic regions since 4 h after focal brain ischemia [15,47,49]; the induced Trx was translocated into the nucleus after ischemia and ischemia￾reperfusion. It is important that changes in Trx expression were observed in the earliest period after the insult because the first 2–4 h after the exposure to severe hypoxia are sup￾posed to be crucial for cytochrome c release [11]. Transient global ischemia induced Trx in glial cells of the gerbil hip￾pocampus[50]. Overexpression of the Trx in transgenic mice attenuates focal ischemic brain damage [48], on the contrary, its inhibition increases oxidative stress [60]. In addition, Trx reduces hypoxia-reoxygenation injury in cell culture in vitro [26]. In present study we for the first time showed that cy￾tosolic Trx involved in neuronal responses to hypobaric hy￾poxia. The expression of Trx in the brain of preconditioned and non-preconditioned animals at 3 and 24 h following severe hypoxia was studied by immunocytochemistry. Se￾vere hypoxia up-regulated the Trx expression in hippocam￾pus at 3 h time-point; this induction appears to represent an adaptive neuronal response to oxidative stress. Precon￾ditioning greatly promotes this protective reaction in hip￾pocampus and induce it in neocortex. The augmentation of Trx expression at early period of reoxygenation critical for apoptosis initiation provides one possible mechanism of hypoxic/ischemic tolerance produced by the precondition￾ing. In neocortex hypoxia without preconditioning induced Trx expression only at 24 h time-point. In hippocampus at this period the Trx expression remained enhanced. Hence the re￾sponse in hippocampus is faster then in neocortex. In the hippocampus of preconditioned rats the Trx expression in￾duction ceased by 24 h whereas in neocortex it continued to increase. Thiol redox status is one of the key factors of the apop￾tosis regulation [43]. The protective functions of Trx dur￾ing oxidative stress are diverse. One of the key Trx de￾fense function is the buffering of ROS [35,51] and inhi￾bition of cytochrome c release from mitochondria [2]. By this way Trx can inhibit the apoptosis triggering. On the other hand Trx can switch necrosis to apoptosis by the regulation of redox-sensitive caspase activity [51,52]. In addition, Trx appears to function as a potent activator of other antioxidant systems, e.g. Mn-superoxide dismutase [10]. Trx is translocated from cytoplasm to nucleus upon stress [33] and activates the transcriptional factors by enhancing their binding activity to the target DNA: NF-B, AP-1, CREB, PEBP2/CBF, Myb, and HIF-1 [1,9,16,19–21,57,58], estrogen [17] and glucocorticoid [30,31] receptors. Trx also augmented the DNA binding activity of p53 [53]. Oxida￾tive stress induced p53 [51] can in its turn activate a G1 cyclin-dependent kinase inhibitor p21Cip1/WAF1 that cause the cell-cycle arrest, presumable to allow an opportunity for DNA repair [12,51]. But p53 also can induce apopto￾sis [3] by an activation of proapoptotic protein Bax, result￾ing in cytochrome c release [34]. Trx augments the p53- dependent p21 transcriptional activity and protein expression and thereby switch apoptosis triggering to DNA reparation way [53]. Trx is a negative regulator of apoptosis signal-regulating kinase 1 (ASK1) [40]. ASK1 was identified as one of the mitogen-activated protein (MAP) kinase kinase ki￾nases, which activates the c-Jun N-terminal kinase (JNK) and p38 MAP kinase and induces stress-mediated apop￾tosis signaling [25]. ASK1 stimulates cytochrome c re￾lease and executes apoptosis mainly by mitochondria￾dependent caspase activation [14]. The negative regulation of ASK1 appears to be one of the Trx cytoprotective ef￾fects. Trx also negatively regulates TNF-induced activa￾tion of p38 MAP kinase [13] activated by oxidative stress [25]. In conclusion, brain expression of thioredoxin-1 is en￾hanced after severe hypobaric hypoxia; the hypoxic precondi￾tioning considerably up-regulates this enhancement. Present findings suggest a possible role for cytosolic antioxidant thioredoxin-1 in the induction of brain hypoxic tolerance.

228 S.A.Stroev et al.Neuroscience Letters 370 (2004)224-229 Acknowledgments the mitochondria-dependent caspase activation,J.Biol.Chem.275 (2000)26576-26581. Authors are deeply grateful to Dr.Yumiko Nishinaka and [15]I.Hattori,Y.Takagi,K.Nozaki,N.Kondo,J.Bai,H.Nakamura,N. Hashimoto,J.Yodoi,Hypoxia-ischemia induces thioredoxin expres- Prof.Junji Yodoi for kindly granting us the antibodies against sion and nitrotyrosine formation in new-born rat brain,Redox Rep. 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