Journal of Neuroimmunology 267 (2014) 28–34

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Journal of Neuroimmunology

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Cellular bioenergetics changes in magnocellular neurons may affectcopeptin expression in the late phase of sepsis

Gabriela R. Oliveira-Pelegrin ⁎, Paulo J. Basso, Maria José A. RochaDepartamento de Morfologia, Fisiologia e Patologia Básica, Faculdade de Odontologia de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil

⁎ Corresponding author at: Faculdade de Odontologiado Café s/n, CEP, 14040-904 Ribeirão Preto, SP, Brazil. Tel16 3633 0999.

E-mail address: gabi.pelegrin@gmail.com (G.R. Oliveir

0165-5728/$ – see front matter © 2013 Elsevier B.V. All rhttp://dx.doi.org/10.1016/j.jneuroim.2013.12.006

a b s t r a c t

a r t i c l e i n f o

Article history:Received 20 June 2013Received in revised form 8 October 2013Accepted 4 December 2013

Keywords:Supraoptic nucleusOxidative stressNitric oxide synthaseInterleukin-1βHypoxia-induced factor-1αApoptosis

We investigated whether inflammatory mediators during cecal ligation and puncture (CLP)-induced sepsis maydiminish copeptin expression inmagnocellular neurons, thus affecting arginine-vasopressin (AVP) synthesis. Thetranscript abundance of IL-1β, IL-1R1, iNOS andHIF-1αwas continuously elevated. IL-1β, iNOS and cytochrome cprotein levels progressively increased until 24 h. Immunostaining for these proteins was higher at 6 and 24 h, asalso seen in the annexin-V assay, while copeptin was continuously decreased. This suggests that increased IL-1βand NO levels may cause significant bioenergetics changes in magnocellular neurons, affecting copeptin expres-sion and compromising AVP synthesis and secretion in the late phase of sepsis.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Clinical studies report that high arginine-vasopressin (AVP) plasmalevels can be found in patients in the early phase of sepsis, in an attemptto restore blood pressure, which tends to decrease due to inflammatorymediators. Nonetheless, in the late phase, despite progressive hypoten-sion, the plasma AVP levels are low, contributing to septic shock anddeath (Landry et al., 1997; Sharshar et al., 2003a). Moreover, infusionof a low dose of exogenous AVP decreases norepinephrine requirementin septic patients, while maintaining or increasing blood pressure, sys-temic resistance and urine output in vasodilatory shock (Holmes et al.,2001). There are also clinical studies indicating impaired baroreflex sen-sitivity (Holmes et al., 2001), depletion of neurohypophyseal hormonecontent (Holmes et al., 2001; Sharshar et al., 2002), overproduction ofnitric oxide (NO) and oxidative stress in AVP neurons (Holmes et al.,2001) as reasons for theAVP secretion impairment. Correspondingfind-ings were also seen in experimental sepsis in previous work from ourgroup (Correa et al., 2007; Pancoto et al., 2008; Oliveira-Pelegrin et al.,2009, 2013).

During sepsis, the excessive production and release of inflammatorymediators may affect AVP synthesis. By using cecal ligation and punc-ture (CLP) to induce sepsis, we in fact saw a decrease in AVP expressionin the supraoptic (SON) and paraventricular (PVN) nuclei of the hypo-thalamus (Oliveira-Pelegrin et al., 2010a, 2010b). Septic patients and

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rats were also reported to show changes in AVP content, as well asneuronal and glial apoptosis in regions related to autonomic control, in-cluding SON and PVN hypothalamic nuclei (Sharshar et al., 2003b;Sonneville et al., 2010). We recently reported an increased expressionof cleaved caspase-3 in SON magnocellular neurons of CLP-inducedseptic rats (Oliveira-Pelegrin et al., 2013) suggesting that apoptosiswas occurring in these neurons. Increased cytokine levels, particularlyinterleukin-1β (IL-1β), are thought to trigger the inducible isoformof NO synthase (iNOS) gene expression in the hypothalamus (Wonget al., 1996b, 1997). Once induced, iNOS produces large NO levels,which may act dually on mitochondrial bioenergetics affecting oxy-gen consumption and enhancing the generation of superoxide anionsby decreasing the electron flow through cytochrome c oxidase.These changes may result in a “metabolic hypoxia” and hydrogen per-oxide formation (Mander and Brown, 2004; Mander et al., 2005;Erusalimsky and Moncada, 2007), which may further stimulate iNOSexpression and, consequently, an increase in NO levels (Guix et al.,2005). This metabolic hypoxia may also induce the expression and sta-bility of the α subunit of hypoxia-induced factor 1 (HIF-1α) (Chavezet al., 2000; Sharp and Bernaudin, 2004; Erusalimsky and Moncada,2007). Dimerization of HIF-1α with the constitutive HIF-1β subunitgenerates the functional transcription factor HIF-1, which regulatesthe expression of various genes involved in cellular energy metabolismand in the apoptosis pathway (Bruick, 2000; Sharp and Bernaudin,2004).

Apoptosis can be triggered by various stimuli that activate theextrinsic and/or intrinsic pathway upstream of the caspase cascade.The extrinsic apoptosis pathway is induced by the activation of deathreceptors, which in turn belong to the tumor necrosis factor receptor

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superfamily (TNFRS). On the other hand, the intrinsic apoptosis path-way is mainly associated with mitochondrial and other intracellularstress signals (Sola et al., 2013). Oxidative stress promotes the move-ment of pro-apoptotic proteins to the mitochondrial surface, whichchanges the permeability of the mitochondrial membrane leading totransient pore formation and consequent release of proteins related tothe activation of the intrinsic apoptosis pathway, such as cytochromec (Mignotte and Vayssiere, 1998; Erusalimsky and Moncada, 2007). Anearly signal at this stage of the apoptosis process is the exposure ofphosphatidylserine (PS), which can be detected by its affinity forannexin-V (van Engeland et al., 1998).

On the background of all this information we hypothesized that thecellular bioenergetics changes seen during sepsis could trigger alter-ations in synthesis of the AVP precursor, including that of copeptin, aC-terminal glycopeptide in the AVP precursor preprovasopressin.Copeptin plays an important role in the correct structural formationand proteolytic maturation of AVP (Barat et al., 2004; Struck et al.,2005;Morgenthaler et al., 2008).With this inmind,we analyzed the ex-pression of oxidative stress and apoptosis markers in copeptin-AVPneurons of the SON and associated these with changes in AVP synthesisand basal plasma concentrations typically seen in the late phase ofsepsis.

2. Material and methods

2.1. Animals

MaleWistar rats (250 ± 30 g) provided by theAnimal Facility of theCampus of Ribeirão Preto, University of São Paulo, were housed in con-trolled temperature (25 ± 1 °C) and photoperiodic (12:12 h night: daycycle) conditions, with food (Nuvilab CR-1, NUVITAL) and tap wateravailable ad libitum. All experimental protocols were approved and per-formed according to the guidelines of the Ethics Committee of the Uni-versity of São Paulo—Campus Ribeirão Preto. Humane endpoints inshock research (Nemzek et al., 2004) were used as criterion to eutha-nize CLP-animals in high suffering, immediately before or soon afterthe studied time-points defined in this study.

2.2. Cecal ligation and puncture surgery

Animals were randomly assigned to one of two groups, CLP or con-trol (sham-operated or non-manipulated animals). All experimentswere performed at the same timeof day (08:00–10:00 AM). Severe sep-sis was induced by a cecal ligation and puncture (CLP) procedure. Brief-ly, rats were anesthetized with a short-acting anesthetic agent(tribromoethanol; 2.5%, 250 mg/kg i.p.; Acros Organics) to minimizedeleterious effects of anesthesia on cardiovascular functions. The ani-malswere subjected to amidline laparotomydoneunder sterile surgicalconditions. The cecum was carefully isolated to avoid damage to bloodvessels. Subsequently, the cecum was ligated below the ileocecalvalve, without causing bowel obstruction, and punctured ten timeswith a 16-gauge needle allowing fecal contents to spill into the perito-neum. The abdominal cavity was closed in two layers, and all animalsreceived a subcutaneous injection of saline (20 mL/kg body weight).Sham-operated animals were submitted to laparotomy, the cecumwas manipulated, but neither ligated nor punctured. The animals wereallowed to recover in their cages with free access to food and water.

2.3. Experimental protocol

Following sham-operation or CLP surgery, the animals were decapi-tated at 4 or 6 (early phase of sepsis) or 24 or 48 h (late phase of sepsis)for removal of the brain, whichwas snap-frozen on dry ice and stored at−80 °C until SON dissection. The hypothalamic nuclei were carefullymicrodissected and processed for reverse transcription (RT) and quan-titative polymerase chain reaction (qPCR), or Western blot detection.

Another set of animals was deeply anesthetized and perfused with 4%paraformaldehyde (4% PFA) in 0.1 M phosphate-buffered saline(0.1 M PBS) at 6 and 24 h after surgery. Brains were removed, post-fixed for 4 h and immersed in 30% sucrose in PBS for cryoprotection.Cryostat sections of 30 μm thickness containing the hypothalamuswere processed for IL-1β, IL-1R1, HIF-1α, iNOS, cytochrome c, andcopeptin immunohystochemistry and the annexin-V affinity assay.

2.4. Microdissection of supraoptic nucleus (SON)

The frozen brains were placed in a brain matrix (Insight EquipmentLTDA, Ribeirão Preto, Brazil) and cut based on the rat brain atlas coordi-nates (Swanson, 1998) with the optic chiasm as anatomical landmarkfor reproducibility among the dissections. A single section of approxi-mately 1 mm thicknesswas taken and the SON regionwas carefully dis-sected by using a punch needle of 1.2 mm diameter (Palkovits, 1973).

2.5. Primer design, RNA extraction and reverse transcription

Gene-specific primers for rat IL-1β (NM_031512), IL-1R1(Peinnequin et al., 2004), iNOS (Peinnequin et al., 2004), HIF-1α (NM_024359), GAPDH (NM_017008) and 18S rRNA (M11188) were usedas in the literature or designed based on GenBank sequences. The se-quences were as follows: IL-1β: (+) gca atg gtc ggg aca tag tt, (−) agacct gac ttg gca gag ga; IL-1R1: (+) gtt ttt gga aca ccc ttc agc c, (−) acgaag cag atg aac gga tag c; iNOS: (+) cat tgg aag tga agc gtt tcg, (−)cag ctg ggc tgt aca aac ctt; HIF-1α: (+) tca agt cag caa cgt gga ag, (−)tat cga ggc tgt gtc gac tg; GAPDH: (+) tca cca cca tgg aga agg c, (−)gct aag cag ttg gtg gtg ca; and 18S rRNA: (+) acg gaa ggg cac cac cagga, (−) cac cac cac cca cgg aat cg. The reference gene (GAPDH and 18SrRNA) primer combinations had already been validated in previousstudies (Oliveira-Pelegrin et al., 2010b). The tissue punches were ho-mogenized in 1 mL of TRIzol Reagent (Invitrogen, Carlsbad, CA, USA)for total RNA extraction. All samples were treated with RNase-freeDNase I (Invitrogen, Carlsbad, CA, USA) to remove any contaminant ge-nomic DNA. RNA purity and quantity were assessed by spectrophotom-etry using a Synergy H1 Take 3 system (BioTek) and Gen5 software.First-strand cDNA synthesis was carried out using the following proto-col. Two μg of total RNA, 1 μL of oligo(dT)12–18 primer (0.5 μg/μL,Invitrogen, Carlsbad, CA, USA) and 1 μL of dNTP mix (10 mM)(Invitrogen, Carlsbad, CA, USA) were incubated at 69 °C for 5 min andchilled on ice. Subsequently, 4 μL of 5× First Strand Buffer, 2 μL of DTT(0.1 M) and 1 μL of RNaseOUT Ribonuclease Inhibitor (Invitrogen,Carlsbad, CA, USA) were added and the samples incubated for 2 minat 42 °C. Next, SuperScript™ II Reverse Transcriptase (200 U,Invitrogen, Carlsbad, CA, USA) was added and the reaction incubatedat 42 °C for 50 min followed by 15 min at 70 °C. cDNAswere stored un-diluted at−20 °C until further use. All cDNA samples were diluted 1:3with DEPC-treated water before being used as templates in quantitativePCR assays.

2.6. Quantitative real-time PCR (qPCR)

Quantitative real-time PCR studies were performed using FastEvaGreen Master Mix (Biotium, Hayward, USA) in a Mastercycler eprealplex (Eppendorf, Hamburg, Germany). qPCR reactions, performedin 96-well 0.2 mL thin-wall PCR microplates (Axygen) sealed withfilm, consisted of 10 μL of Fast EvaGreen Master Mix, 0.8 μL of each for-ward and reverse primer (10 μM) and 2 μL of 1:3-diluted templatecDNA in a total volume of 20 μL completed with DEPC-treated water.Cyclingwas performedusing the optimized conditions in a four-step ex-perimental run protocol: (i) denaturation program(2 min at 96 °C); (ii)amplification and quantification program repeated 45 times (5 s at96 °C, 5 s at 58 °C, 25 s at 72 °C); melting curve program (60–95 °Cwith a heating rate of 0.5 °C/s and continuous fluorescence measure-ment); (iv) cooling down to 4 °C. Melting curves obtained after

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thermocycling assured that the primers produced a gene-specific prod-uct. The qPCR assays for each gene included a negative control (withoutcDNA template). All samples were run in triplicate. Standard curves foreach pair of primers were prepared using serial 1:10 dilutions of a pos-itive control cDNA sample. Primer amplification efficiency (E) was cal-culated according to the equation: E = 10(−1 / slope) − 1 (Bustin et al.,2009). Non-manipulated animals were used as control, and the normal-ization factor obtained from a combination of two different referencegenes (GAPDH and 18S) was used to normalize gene expression levels.The relative gene expression ratio (R) of mRNA was calculated on thebasis of PCR efficiency (E) and quantification cycle deviation (ΔCq) ac-cording to the equation: R = (Etarget)ΔCq target(Mean control − Mean samples) /(Eref)ΔCq ref(Mean control − Mean samples) (Pfaffl et al., 2002). Amplificationefficiencies, slopes, y-intercepts and correlation coefficients were obtain-ed directly from theMastercycler ep realplex Software showing similar Eand correlation coefficients for all analyzed genes.

2.7. Western blot analysis

Hypothalamus tissue punches were placed in RIPA buffer (R0278,Sigma-Aldrich) with 10% of diluted 1:10 of protease inhibitor cocktail(P2714, Sigma-Aldrich) and 0.5% of phenylmethylsulfonyl fluoride(PMSF, 78830, Sigma-Aldrich) 200 mM in methanol for homogeniza-tion by sonication. After shaking for 2 h on ice, the samples were centri-fuged at 3500 ×g for 20 min at 4 °C to collect the supernatant, whichcontained the cytoplasmic fraction. Total protein contentwasmeasuredat 592 nm by means of a bicinchoninic acid (BCA) assay following themanufacturer's instructions (Pierce BCA protein assay kit, #23225).Equal amounts of total protein (40 μg) were diluted in 2× Laemmlibuffer (S3401, Sigma-Aldrich) and separated by SDS-PAGE in a 10%polyacrylamide gel (125 V for 1.5 h). Molecular mass markers of 10–250 kDa (RPN800E GE Healthcare) were applied on the gel to visualizeseparation and transfer quality.

Following electrophoresis, the proteinswere blotted onto a nitrocel-lulose membrane (0.45 μm; Millipore) in a tank blotting system. West-ern blotting was performed under a current of 100 V for 2 h in transferbuffer containing 20% methanol. After remaining in blocking solution(5% bovine serum albumin [BSA] in 0.1 M PBS with 0.2% Tween20(P5927, Sigma-Aldrich) [PBS-T]) for 1 h, the membranes were rinsedin PBS-T and then incubated overnight under agitation at room temper-ature with specific antibodies diluted in PBS-T with BSA 1%. For IL-1βdetection, a rabbit polyclonal IgG antibody (ab9787, Abcam) was usedat a 1:2500 dilution, and for IL-1R1 detection, a rabbit monoclonal IgG(ab40774, Abcam) was used diluted 1:800. iNOS detection was donewith a rabbit polyclonal IgG antibody (ab15323, Abcam) at a 1:133 dilu-tion, and HIF-1α detection with a 1:500 diluted rabbit polyclonal IgGantibody (ab65979, Abcam). For cytochrome c detection, a mousemonoclonal IgG antibody (sc13156, Santa Cruz Biotechnology) wasused at a 1:10,000 dilution, and β-actin detection was done with amouse monoclonal IgG antibody (sc-47778, Santa Cruz Biotechnology)at a 1:5000 dilution. Secondary HRP-conjugated antibodies (anti-rabbit[Abcam] and anti-mouse [Santa Cruz Biotechnology]) were diluted1:10,000 in PBS-T with BSA 1% and the membranes incubated underagitation for 2 h at room temperature. A chemiluminescence reactionkit (enhanced chemiluminescence [ECL], GE Healthcare) and X-ray filmwere used for detection of proteins. X-ray films were photographedusing a light box and image acquisition system, and the ECL-detected pro-tein bands were quantified by Genetools software (Syngene, Cambridge,England). The resultswere transformed into arbitrary units of optical den-sity and expressed as their ratio to β-actin (internal control).

2.8. Immunohistochemistry

Free floating brain sections were washed 3 times in PBS (0.01 M–

pH 7.4) and immediately thereafter an antigen retrieval protocolwas per-formed in two steps: a 5 min incubation in a Tris/EDTA (10 mM/1 mM,

pH 9.0) solution, followed by heating for 30 min in a water bath at70 °C in 10 mM sodium citrate buffer (pH 6.0). After reaching room tem-perature, the sectionswerewashed in PBS for 15 min. Endogenousperox-idaseswere blocked by treatmentwith hydrogen peroxide (1% in PBS) for10 min. After several rinses in PBS for 15 min, nonspecific binding siteswere blocked for 60 min in PBS containing 5% normal goat serum(NGS) and 0.3% Triton X-100 (nonionic detergent, T8532, Sigma-Aldrich). Subsequently, the sections were incubated for 24 h at 4 °Cwith specific antibodies (IL-1β, ab9787 [Abcam], rabbit polyclonal IgG,1:500; IL-1R1, ab40774 [Abcam], rabbit monoclonal IgG, 1:500; iNOS,sc-727 [Santa Cruz Biotechnology], mouse monoclonal, IgG, 1:200; HIF-1α, sc-53546 [Santa Cruz Biotechnology], mouse monoclonal IgG2b,1:200; cytochrome c, A-8 SS-13156, [Santa Cruz Biotechnology], mousemonoclonal IgG2b, 1:1000; copeptin, #IPVS-1050 [RheaBiotech, Campi-nas, Brazil], rabbit polyclonal, IgG, 1:400) diluted in PBS containing 1%BSA, 1% NGS and 0.3% Triton X-100. After rinsing, the sections were incu-bated for 90 min at room temperature with the appropriate biotinylatedsecondary antibody (goat anti-rabbit, ABC Elite kit, Vectastain; goat anti-mouse, Pierce) diluted 1:2000 in PBS containing 1% BSA, 1% NGS and0.3% Triton X-100. After washing in PBS, the sections were placed inavidin–biotin peroxidase complex for 30 min (ABC, Vectastain) andonce again rinsed in PBS before incubation with 0.05% 3-3′-diaminoben-zidine (DAB) and 0.015% hydrogen peroxide for 10 min. Finally, thesections were mounted on gelatin-coated slides, dehydrated andcoverslipped with Entellan (Merck). The sections were analyzed bylight microscopy using a Zeiss KS300 microscope and images of labeledneurons captured using an AxioCam MRc system (Zeiss) coupled to themicroscope. The anatomical description of brain regions follows that ofSwanson (Swanson, 1998).

2.9. Annexin-V affinity assay

Coronal brain sections of 20 μm thickness containing the hypothala-mus were mounted on silanized slides and kept in a dark and humidchamber during the entire procedure. To each slide was added 500 μLof a fluorochrome-labeled Annexin-V solution (kindly provided byProf. Dr.Marcelo Dias Baruffi, University of Sao Paulo) diluted in bindingbuffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2—pH 7.4; 1:500).After 5 min of reaction, the slides were rinsed 10 times in PBS (0.01 M)and coveredwith coverslips usingmountingmedium suitable for fluores-cence detection (Prolong Gold, Invitrogen). The sections were analyzedby epifluorescence microscopy and images captured using an AxioCamMRc system (Zeiss) coupled to the microscope.

2.10. Statistical analysis

The data are presented as mean ± S.E.M. Statistical analysis wasperformed by two-way analysis of variance (ANOVA) and a post hocStudent–Newman–Keuls (SNK) test. mRNA relative expression ratioswere calculated using the Relative Expression Software Tool—MultipleCondition Solver (REST-MCS©-version 2) (Pfaffl et al., 2002). Thresholdquantification cycle (Cq) values were considered significant at 10 timesthe standard deviation above baseline noise. A positive ratio means up-regulation and a negative one down-regulation. P values of≤0.05 wereconsidered as significant.

3. Results

Within fewhours after surgery, all CLP animals developed the typicalclinical signs of sepsis, such as, lethargy, piloerection anddiarrhea. Shamanimals remained active in their cages, as expected. The fold changes oftranscripts for IL-1β, IL-1R1, iNOS and HIF-1α in magnocellular neuronsof the SON following CLP-induced sepsis were higher than in shamanimals, mainly so in the initial phase of sepsis (4 and 6 h) (Table 1).Protein levels assayed by Western blot analysis showed an increase forIL-1β (F(1,41) = 21.74; P b 0.001), iNOS (F(1,55) = 19.55; P b 0.001) and

Table 1Fold change in interleukin-1β (IL-1 β), interleukin-1 receptor type-1 (IL-1R1), induciblenitric oxide synthase (iNOS) and hypoxia-inducible factor-1α (HIF-1 α) transcript levelscomparing to control group (non-manipulated animals). Number of animals was 5 eachgroup.

Group Time IL-1β IL-1R1 iNOS HIF-1α

Sham 4 h 1.66 1.38 1.18 1.626 h 1.36 −1.08 1.18 1.69

24 h 1.16 −1.30 −3.82 2.0348 h 2.02 −1.10 1.13 1.68

CLP-induced sepsis 4 h 3.64 2.06 9.14 3.056 h 4.08 3.01 2.61 4.30

24 h 2.12 1.34 1.02 1.6548 h 2.81 1.34 −1.15 2.02

Fig. 1. Protein levels of inflammatory and nitric oxide-related factors and of cytochrome c in theblot analysis of target proteins and β-actin (internal control) in sham and septic rats (CLP); B–Inducible nitric oxide synthase (iNOS), Hypoxia-inducible factor-1α (HIF-1α) and cytochromwas performed using ANOVA followed by a SNK test. *P b 0.05 compared to sham group. +P

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cytochrome c (F(1,36) = 20.29; P b 0.001) at 4 h, and these remained sountil 24 h after CLP. For IL-1R1 (F(1,39) = 6.45; P = 0.015) and HIF-1α(F(1,41) = 8.99; P = 0.005), the respective protein levels in the septicgroup were higher only at 6 h (Fig. 1). The immunohistological analysesdone in serial independent experiments confirmed the increased expres-sion of IL-1β, HIF-1α and iNOS at 6 and 24 h after CLP, while IL-1R1 onlyshowed a veryweak immunostaining signal (Fig. 2). For cytochrome c,weobserved a different staining pattern between the experimental groups.Whereas in the control group the staining pattern was granular or punc-tate, in the CLP group certain neurons exhibited a diffuse staining pattern,indicating translocation or leakage of cytochrome c frommitochondria tothe cytoplasm (Fig. 2). Furthermore, the annexin-V affinity assay showeda progressive signal increase, while copeptin immunostaining was much

supraoptic nucleus (SON) of CLP-induced septic rats and sham-operated ones. A:WesternF: Quantitative analysis of Interleukin-1β (IL-1β), Interleukin-1 receptor type-1 (IL-1R1),e c. The data show means ± S.E.M. of the ratio target protein/β-actin. Statistical analysisb 0.05 within the group. Number of animals is 4–7 in each group.

Fig. 2. Photomicrographs illustrating interleukin-1β (IL-1β), interleukin-1 receptor type-1 (IL-1R1), inducible nitric oxide synthase (iNOS), hypoxia-inducible factor-1α (HIF-1α) andcytochrome c immunoreactivity in coronal sections through the supraoptic nucleus (SON) of control and septic animals at 6 and 24 h. Images were obtained by light microscopy. Thearrows show immunoreactive neurons. Original magnification: 20× and 100×. Scale bar = 50 μm.

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less intense in the CLP group at 24 h,when compared to the control group(Fig. 3).

4. Discussion

In the initial stage of AVP synthesis, a precursor protein of 164 aminoacid residues is produced. This precursor protein is structurally com-posed of the signal peptide, the AVP peptide, neurophysin II (a carrierresponsible for axonal transport of the hormone to the nucleus for neu-rohypophysis), and copeptin. The latter is a glycopeptide present in theC-terminal part of the AVP precursor. Copeptin plays an important role

in the correct structural formation and proteolytic maturation of AVP,and is also liberated into the plasma in equimolar proportions to AVP.Despite occasional clinical reports on a discrepancy between the ratiocopeptin/AVP (Lee et al., 2013), there is reasonable support thatcopeptin could be a surrogate marker for measuring AVP levels, sinceit is more stable in the circulation and apparently easier to determinethan AVP (Morgenthaler et al., 2008; Seligman et al., 2008).

In our experiments, employing a CLP-sepsis model in rats, we ob-served that in the late phase the copeptin immunostaining was de-creased in SON, while annexin-V affinity was increased. As in someclinical studies copeptin plasma concentrations are reported to increase

Fig. 3. Photomicrographs illustrating copeptin immunoreactivity and annexin-V affinity in coronal sections through the supraoptic nucleus (SON) of control and septic animals at 6 and24 h. Images were obtained by light microscope and epifluorescent microscopies, respectively. The arrows show the affinity for annexin-V in neurons. Original magnification: 20× and100×. Scale bar = 50 μm.

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during sepsis (Morgenthaler et al., 2007, 2008; Seligman et al., 2008)one explanation could be that this reflects the emptying of copeptinstorage. In previous work we could, however, show that in the latephase of sepsis there is a decrease in c-fos expression, indicating an in-hibition of neuronal activation in the SON, as well as increased expres-sion of cleaved caspase-3 (Correa et al., 2007; Oliveira-Pelegrin et al.,2013). This, together with the present annexin-V results, strongly sug-gests apoptosis of AVP-producing magnocellular neurons as one of thefactors responsible for the observed impairment of AVP synthesis andrelease observed in our previous work (Correa et al., 2007; Pancotoet al., 2008; Athayde et al., 2009; Oliveira-Pelegrin et al., 2010a,2010b;Martins et al., 2011). We furthermore had observed that CLP-inducedpolymicrobial sepsis led to increased expression of IL-1R1 and IL-1β inthe hypothalamus, mainly in the initial phase of sepsis (4 and 6 h).

The circumventricular organs (CVOs), which are highly vascularizedstructures lacking a blood–brain barrier, promote mutual communica-tion between the brain and the immune system (Wong et al., 1996a,

1997; Rivest et al., 2000). The vascular walls of the CVOs andperivascular glia constitutively express IL-1R1, which, when activated,induces IL-1β expression (Wong et al., 1996a, 1997). Additionally inthis context, IL-1β may activate iNOS expression (Wong et al., 1996b,1997) thus contributing to oxidative stress. In line with the increasedexpression of IL-1β and IL-1R1 we observed herein that CLP-inducedpolymicrobial sepsis was accompanied by a progressive increased ex-pression of the iNOS encoding gene in the SON until 24 h. We thus in-ferred that a consequent increase in NO production may cause adisturbance in mitochondrial bioenergetics.

The oxidative-nitrosative stress condition, togetherwith the increasedIL-1β level, may induce the expression of HIF-1α in neurons (Chavezet al., 2000; Sharp and Bernaudin, 2004; Erusalimsky and Moncada,2007). Surprisingly,wedid notfind an increase inHIF-1α transcript levelsfollowing CLP-induced sepsis, but, nonetheless, we observed an increasedexpression of HIF-1α protein in magnocellular neurons at 4 h of sepsis,suggesting cellular changes related to pro-apoptosis in certain neurons.

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These alterations promote transient pore formation, which facilitates therelease of cytochrome c and of other pro-apoptoticmolecules from the in-termembranemitochondrial space (Mignotte andVayssiere, 1998;Green,2000; Kiang and Tsen, 2006; Tam et al., 2010; Berg et al., 2011). Following6 and 24 h of CLP surgery we saw an increase in cytochrome c. Addition-ally we saw a change in the immunostaining pattern, from a granular orpunctuate (indicating the cytochrome restriction to mitochondria) to adiffuse staining in some of themagnocellular neurons, this being a strongindicator of its release frommitochondria and translocation into the cyto-plasm. In other words, cellular bioenergetics changes occurring in certainmagnocellular neurons may result in their apoptosis. Together withthe decrease in copeptin expression in the SONof septic animals, thefind-ings are strong evidence that the apoptosis of certain AVP-producingmagnocellular SON neurons may affect AVP synthesis and impairmentof hormone secretion seen during the late phase of sepsis.

Disclosure statement

The authors have nothing to disclose.

Acknowledgments

The authors thank, Aline de Souza Soares, Antonio Zanardo, MarianaRossin Martinez and Nadir Martins Fernandes for the technical assis-tance. Sérgio Akira Uyemura provided the infrastructure for quantita-tive PCR. Financial support from Fundação de Amparo à Pesquisa doEstado de São Paulo (FAPESP) is gratefully acknowledged.

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