Sepsis otopathy: experimental sepsis leads to significant hearing … · sepsis leads to...

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INTRODUCTION The incidence of sepsis is still high, with up to three cases per 1000 population per year, more than half of them requiring intensive care management (Angus et al., 2001). Multi-organ failure includes involvement of the central nervous system, peripheral nerves and skeletal muscles (Latronico and Bolton, 2011), but so far systemic sepsis has not been linked to hearing impairment. However, hearing loss has been accepted as an unappreciated phenomenon in critical care. Postulated causes range from trauma, ototoxic medications, local infections, vascular and hematologic disorders, autoimmune disease and environmental noise (Halpern et al., 1999). Recently, our group demonstrated the involvement of the inner ear in murine cerebral malaria, a systemic inflammatory disease (Schmutzhard et al., 2010) showing apoptosis in the fibrocytes of the spiral ligament, which play an essential role in the electrolyte circulation of the cochlea, and a breakdown of the blood-labyrinth barrier (Schmutzhard et al., 2012). The fact that no malaria-typical alterations, like microhemorrhages or leukocyte sequestration, could be found in the temporal bones suggests that the pathological findings in the inner ear might be linked to the systemic inflammatory reaction. Comparable animal models exist for severe sepsis. A well- established form is the cecal ligation puncture (CLP) model. In this approach, sepsis is induced with a ligation of the cecum and an additional standardized puncture of the ligated part (Rittirsch et al., 2009). Activation of the apoptosis cascade is a frequently observed pathological reaction in the inner ear. In a guinea pig animal model for Menière’s disease, an activation of cleaved caspase-3 (an established apoptosis detection marker) has been described in the fibrocytes of the spiral ligament and the stria vascularis (Labbé et al., 2005). In perinatal asphyxia, a cleaved caspase-3 activation has been shown in the inner and outer hair cells. Additionally, downregulation of the anti-apoptotic pathways could be demonstrated with decreased Bcl-2 labeling (Schmutzhard et al., 2009). The upregulation of the Bcl-2 anti-apoptotic protein needs to be compared with the expression rate of the Bax protein, the pro- apoptotic antagonist of the Bcl-2 protein. A Bax/Bcl-2 ratio favoring Bax expression with additional downstream cleaved caspase-3 upregulation is a solid sign for induced apoptosis (Salakou et al., 2007). The aim of this experiment was to study whether in the standardized CLP mouse model sepsis leads to hearing impairment. Possible pathological alterations causing such a hearing loss were looked for by light microscopy, transmission electron microscopy (TEM) and immunohistochemistry for cleaved caspase-3, Bcl-2 and Bax, thus focusing on the activation of apoptotic and anti-apoptotic pathways. Disease Models & Mechanisms 745 Disease Models & Mechanisms 6, 745-754 (2013) doi:10.1242/dmm.011205 1 Department of Otorhinolaryngology, Innsbruck Medical University, Anichstrasse 35, 6020 Innsbruck, Austria 2 Department of Anatomy, Histology and Embryology, Division of Clinical and Functional Anatomy, Innsbruck Medical University, Müllerstrasse 59, 6020 Innsbruck, Austria 3 Department of Neurology, Innsbruck Medical University, Anichstrasse 35, 6020 Innsbruck, Austria 4 Department of Hygiene and Medical Microbiology, Innsbruck Medical University, Anichstrasse 35, 6020 Innsbruck, Austria *Author for correspondence ([email protected]) Received 22 October 2012; Accepted 11 February 2013 © 2013. Published by The Company of Biologists Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Share Alike License (http://creativecommons.org/licenses/by-nc-sa/3.0), which permits unrestricted non-commercial use, distribution and reproduction in any medium provided that the original work is properly cited and all further distributions of the work or adaptation are subject to the same Creative Commons License terms. SUMMARY Hearing loss is frequent in intensive care patients and can be due to several causes. However, sepsis has not been examined as a possible cause. The aim of this study is to assess the influence of experimental sepsis on hearing thresholds and to evaluate pathological changes in the cochlea. The cecal ligation puncture technique was used to induce sepsis in 18 mice. Results were compared with those from 13 sham-operated and 13 untreated control mice. The hearing thresholds of the animals were evaluated with auditory evoked brainstem responses prior to the induction of sepsis and again at the peak of the disease. Immediately after the second measurement, the mice were sacrificed and the inner ears harvested and prepared for further evaluation. The cochleae were examined with light microscopy, electron microscopy and immunohistochemistry for Bax, cleaved caspase-3 and Bcl-2. The mice with sepsis showed a significant hearing loss but not the control groups. Induction of apoptosis could be shown in the supporting cells of the organ of Corti. Furthermore, excitotoxicity could be shown at the basal pole of the inner hair cells. In this murine model, sepsis leads to significant hearing impairment. The physiological alteration could be linked to apoptosis in the supporting cells of the organ of Corti and to a disturbance of the synapses of the inner hair cells. Sepsis otopathy: experimental sepsis leads to significant hearing impairment due to apoptosis and glutamate excitotoxicity in murine cochlea Joachim Schmutzhard 1, *, Rudolf Glueckert 1 , Christian Pritz 1 , Michael J. F. Blumer 2 , Mario Bitsche 2 , Peter Lackner 3 , Manfred Fille 4 , Herbert Riechelmann 1 , Matthias Harkamp 1 , Thongrong Sitthisak 1 and Annelies Schrott-Fischer 1 RESEARCH ARTICLE Disease Models & Mechanisms DMM

Transcript of Sepsis otopathy: experimental sepsis leads to significant hearing … · sepsis leads to...

Page 1: Sepsis otopathy: experimental sepsis leads to significant hearing … · sepsis leads to significant hearing impairment. The physiological alteration could be linked to apoptosis

INTRODUCTIONThe incidence of sepsis is still high, with up to three cases per 1000population per year, more than half of them requiring intensivecare management (Angus et al., 2001). Multi-organ failure includesinvolvement of the central nervous system, peripheral nerves andskeletal muscles (Latronico and Bolton, 2011), but so far systemicsepsis has not been linked to hearing impairment. However, hearingloss has been accepted as an unappreciated phenomenon in criticalcare. Postulated causes range from trauma, ototoxic medications,local infections, vascular and hematologic disorders, autoimmunedisease and environmental noise (Halpern et al., 1999).

Recently, our group demonstrated the involvement of the innerear in murine cerebral malaria, a systemic inflammatory disease(Schmutzhard et al., 2010) showing apoptosis in the fibrocytes ofthe spiral ligament, which play an essential role in the electrolytecirculation of the cochlea, and a breakdown of the blood-labyrinth

barrier (Schmutzhard et al., 2012). The fact that no malaria-typicalalterations, like microhemorrhages or leukocyte sequestration,could be found in the temporal bones suggests that the pathologicalfindings in the inner ear might be linked to the systemicinflammatory reaction.

Comparable animal models exist for severe sepsis. A well-established form is the cecal ligation puncture (CLP) model. In thisapproach, sepsis is induced with a ligation of the cecum and anadditional standardized puncture of the ligated part (Rittirsch etal., 2009). Activation of the apoptosis cascade is a frequentlyobserved pathological reaction in the inner ear. In a guinea piganimal model for Menière’s disease, an activation of cleavedcaspase-3 (an established apoptosis detection marker) has beendescribed in the fibrocytes of the spiral ligament and the striavascularis (Labbé et al., 2005). In perinatal asphyxia, a cleavedcaspase-3 activation has been shown in the inner and outer haircells. Additionally, downregulation of the anti-apoptotic pathwayscould be demonstrated with decreased Bcl-2 labeling (Schmutzhardet al., 2009).

The upregulation of the Bcl-2 anti-apoptotic protein needs to becompared with the expression rate of the Bax protein, the pro-apoptotic antagonist of the Bcl-2 protein. A Bax/Bcl-2 ratio favoringBax expression with additional downstream cleaved caspase-3upregulation is a solid sign for induced apoptosis (Salakou et al.,2007).

The aim of this experiment was to study whether in thestandardized CLP mouse model sepsis leads to hearing impairment.Possible pathological alterations causing such a hearing loss werelooked for by light microscopy, transmission electron microscopy(TEM) and immunohistochemistry for cleaved caspase-3, Bcl-2 andBax, thus focusing on the activation of apoptotic and anti-apoptoticpathways.

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Disease Models & Mechanisms 6, 745-754 (2013) doi:10.1242/dmm.011205

1Department of Otorhinolaryngology, Innsbruck Medical University, Anichstrasse35, 6020 Innsbruck, Austria2Department of Anatomy, Histology and Embryology, Division of Clinical andFunctional Anatomy, Innsbruck Medical University, Müllerstrasse 59, 6020Innsbruck, Austria3Department of Neurology, Innsbruck Medical University, Anichstrasse 35, 6020Innsbruck, Austria4Department of Hygiene and Medical Microbiology, Innsbruck Medical University,Anichstrasse 35, 6020 Innsbruck, Austria*Author for correspondence ([email protected])

Received 22 October 2012; Accepted 11 February 2013

© 2013. Published by The Company of Biologists LtdThis is an Open Access article distributed under the terms of the Creative Commons AttributionNon-Commercial Share Alike License (http://creativecommons.org/licenses/by-nc-sa/3.0), whichpermits unrestricted non-commercial use, distribution and reproduction in any medium providedthat the original work is properly cited and all further distributions of the work or adaptation aresubject to the same Creative Commons License terms.

SUMMARY

Hearing loss is frequent in intensive care patients and can be due to several causes. However, sepsis has not been examined as a possible cause.The aim of this study is to assess the influence of experimental sepsis on hearing thresholds and to evaluate pathological changes in the cochlea.The cecal ligation puncture technique was used to induce sepsis in 18 mice. Results were compared with those from 13 sham-operated and 13untreated control mice. The hearing thresholds of the animals were evaluated with auditory evoked brainstem responses prior to the induction ofsepsis and again at the peak of the disease. Immediately after the second measurement, the mice were sacrificed and the inner ears harvested andprepared for further evaluation. The cochleae were examined with light microscopy, electron microscopy and immunohistochemistry for Bax, cleavedcaspase-3 and Bcl-2. The mice with sepsis showed a significant hearing loss but not the control groups. Induction of apoptosis could be shown inthe supporting cells of the organ of Corti. Furthermore, excitotoxicity could be shown at the basal pole of the inner hair cells. In this murine model,sepsis leads to significant hearing impairment. The physiological alteration could be linked to apoptosis in the supporting cells of the organ of Cortiand to a disturbance of the synapses of the inner hair cells.

Sepsis otopathy: experimental sepsis leads to significanthearing impairment due to apoptosis and glutamateexcitotoxicity in murine cochleaJoachim Schmutzhard1,*, Rudolf Glueckert1, Christian Pritz1, Michael J. F. Blumer2, Mario Bitsche2, Peter Lackner3, Manfred Fille4,Herbert Riechelmann1, Matthias Harkamp1, Thongrong Sitthisak1 and Annelies Schrott-Fischer1

RESEARCH ARTICLED

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RESULTSA total of 16 (out of 18) CLP mice were included in the evaluation.Two CLP mice died unobserved during the experiment and wereexcluded. One animal had an elevated pretreatment auditorybrainstem response (ABR) threshold on the left ear. In this animal,only the right ear was evaluated. The sham-operated group wascomposed of 13 mice. This left 16 CLP mice, 13 sham mice and13 untreated control mice for statistical evaluation.

Laboratory data, body weight and temperatureLaboratory data, weight loss, low body temperature and bacteremiaindicated that sepsis had been successfully induced in CLP mice.Blood cultures for all 16 CLP mice were positive for Escherichia coli.Klebsiella oxytoca grew in the blood culture of one CLP animal. Allcontrol animals had negative blood cultures. Sufficient volumes ofblood for the analysis of pH levels could be drawn in 9/16 CLPanimals, 8/13 sham and in 4/13 controls. In CLP mice, the meanblood pH level was 7.15±0.08 (all values are given as mean ± s.d.),for sham animals it was 7.28±0.05 (CLP versus sham P=0.001) andfor controls it was 7.39±0.07 (CLP versus control P=0.0028)(supplementary material Fig. S1A). The pretreatment body weightof the CLP mice was 21.58±1.18 g, the sham animals 20.8±1.6 g (CLPversus sham P=0.147) and the controls 21.45±1.67  g (CLP versuscontrol P=0.895) (supplementary material Fig. S1C). The post-treatment body weight of the CLP mice was 19±1.64  g (pre-CLPversus post-CLP P<0.001), the sham animals 21.07±1.3 g (pre-shamversus post-sham P=0.521) and in controls 23.12±2.02 g (pre-controlversus post-control P=0.03) (supplementary material Fig. S1D).Lactate levels were evaluated for 9/16 CLP animals, 2/13 shamanimals and 4/13 controls. The lactate value for the CLP mice was21.68±12.7  mg/dl, the sham animals 13.95±7.42  mg/dl and thecontrols 10.65 ±3.23 mg/dl (supplementary material Fig. S1B). Bodytemperature was evaluated for 16/16 CLP animals, 6/13 sham

animals and 5/13 control animals. At inclusion, the values for theCLP animals ranged from 38.31±0.9°C, the sham group 38.8±1.3°Cand the controls 37.8 ±0.5°C (supplementary material Fig. S1E). Atsacrifice, the temperature had dropped significantly in the CLPanimals to 33.19±2.13°C (CLP inclusion versus CLP sacrificeP<0.0001). No change of the temperature could be noted in the shamgroup with 38.2±0.8°C (sham inclusion versus sham sacrifice P=0.47)and the control animals with 38.42±0.92°C (control inclusion versuscontrol sacrifice P=0.28) (supplementary material Fig. S1F).

ABR measurementsPretreatment ABR thresholds could be identified in all 42 animalsat sound pressure levels ranging from 15 to 80 dB (supplementarymaterial Fig. S2). The average pretreatment thresholds of left andright ears for all 42 animals were 54.8±7.3 dB at 4 KHz, 31.6±6.3 dBat 8 KHz, 25.9±5.6 dB at 16 KHz and 46.2±15.12 dB at 32 KHz. Thepost-treatment threshold averages of the left and right ears were57.7±7.67 dB at 4 KHz, 35.7±8.5 dB at 8 KHz, 27.8±7.3 dB at 16 KHzand 50.8±13.2  dB at 32  KHz. The mean pretreatment pure toneaudiometry for all 42 animals was 39.6±6.8 dB (sound pressure level;SPL), the mean post-treatment pure tone audiometry was 43±6.9 dB.

Sepsis resulted in a significant increase in ABR thresholds (i.e.hearing loss), although ABR thresholds remained stable in shamand control animals (Fig. 1). Adjusted for pretreatment ABRthresholds, the mean post-treatment pure tone audiometryestimated marginal mean for CLP animals was 47.4  dB [95%confidence interval (CI) 45.12 dB to 49.7 dB], for the sham group40.8 dB (95% CI 38.3 dB to 43.3 dB) and for control mice 39.7 dB

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TRANSLATIONAL IMPACT

Clinical issueHearing loss is known to be an unpleasant side effect of intensive caretreatment. Various ototoxic factors such as medication, trauma andautoimmune response have been identified as potential causes. However, thepossible effects of sepsis – a frequent cause of critical care hospitalization – oninner ear function have not yet been examined.

ResultsIn this prospective animal study, the effects of sepsis on the inner ear wereinvestigated in C57BL/6 laboratory mice. Regularly hearing animals wereinfected using the cecal ligation puncture technique, and the hearing of themice was assessed at the peak of the disease. Statistical evaluation revealedsignificant hearing loss, at all frequencies, in the mice with sepsis. Subsequenthistological and immunohistochemical analysis indicated an induction ofapoptosis in a specialized cell subtype of the organ of Corti, as well asglutamate excitotoxicity at the basal pole of the inner hair cell.

Implications and future directionsThis study indicates, for the first time, that sepsis can be causative of hearingloss in the absence of other ototoxic influences. In light of these data,specialists should be encouraged to perform post-therapeutic hearing tests inpatients suffering from sepsis syndrome. Nonetheless, this finding awaitsvalidation in humans. Therefore, further studies, including objective hearingtests such as auditory evoked brainstem responses and otoacoustic emissions,need to be performed in patients with septicemia.

Fig. 1. Spaghetti plot of the audiological results for the control, CLP andthe sham group. All audiological results were averaged at the time-point ofinclusion as pure tone average 1 (PTA_T1). The audiological results at the time-point of sacrifice were averaged as pure tone average 2 (PTA_T2). Each line inthe graph stands for one individual, connecting PTA_T1 and PTA_T2. Ahorizontal line, as seen in the control and the sham group, indicates nohearing loss; a steep line indicates a change in the hearing threshold betweenthe first and second measurements.

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(95% CI 37.4 dB to 42.1 dB). Post-treatment thresholds of the CLPgroup differed significantly from the control group (P<0.001) andfrom sham-operated animals (P=0.002).

Light microscopyFour temporal bones of mice with a hearing loss of up to 20 dB invarious frequencies were further examined with light microscopy.The structural analysis revealed a well-preserved spiral ganglionin all examined temporal bones, independently of any hearing loss.Furthermore, the stria vascularis and the spiral ligament did notshow any morphological alterations (supplementary material Fig.S3). The organ of Corti showed the following morphologicalalterations: (i) The inner hair cells showed extensive vacuolizationat the basal pole, which could be correlated to hearing loss.Temporal bones of mice without hearing impairment did not showthe basal vacuolization. Hair cells of mice with hearing loss of 15-20 dB showed a basal vacuolization ranging from the basal pole ofthe inner hair cell to the basal membrane. Hearing loss of 10 dBcould be correlated to a minor basal vacuolization not reaching the

basal membrane. No basal vacuolization was found in temporalbones of mice without hearing loss. (ii) The apical pole of the innerhair cells was well preserved. (iii) Except for the vacuolization atthe basal pole, the outer hair cells showed an identicalmorphological presentation. (iv) The Deiters’ cells (supporting cellsof the outer hair cells) showed an intensive staining of thecytoplasm, a rough configuration of the cell and a chromatincondensation with nuclear shrinkage. (v) Similar cell morphologycould be shown in the Claudius’ and Hensen’s cells.

These pathological alterations could be found exclusively intemporal bones of mice with hearing loss (supplementary materialFig. S3A-D). Two sham animals were further evaluated with lightmicroscopy and did not show the basal vacuolization at the innerhair cells. The supporting cells, the outer hair cells, the stria vascularis,the spiral ligament and the spiral ganglion cells did not reveal anypathological alterations (supplementary material Fig. S3E-H).

Three animals of the control group were further evaluated withlight microscopy. No hearing impairment had been detected inthese animals. The spiral ganglion cells and the stria vascularis were

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Fig. 2. TEM of the inner hair cells and supporting cells.(A)Overview of the organ of Corti of a CLP mouse withhearing loss, focusing on the supporting cells. The outerhair cells appear normal. A Deiters’ cell has pathologicalalterations. (B)Close-up view of a Deiters’ cell withreduction in the cellular volume and chromatincondensation (asterix). (C)High magnification of a CLPmouse inner hair cell. The basal membrane of the cell iswell preserved. The vacuolization (arrows) is due to aswelling of the afferent nerve fibers, with membranedisruption and loss of cytoplasmic content. (D)Highermagnification of a basal swelling, which is marked with anasterix in C. The vacuole contains a membrane remnant(arrow). Furthermore, on the corresponding inner hair cellside a synaptic ribbon can be seen (bold arrow). (E)Organof Corti from a sham animal. The basal pole of the innerhair cell does not show any vacuolization. The Deiters’ cellshows a regular configuration with euchromatin-containing nucleus (arrow). (F)Close-up view of the innerhair cell shown in E. At the basal pole, regular afferentnerve fibers with intact membranes and cytoplasmaticcontent can be seen (asterix). OHC, outer hair cell; IHC,inner hair cell; DC, Deiters’ cells.

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well preserved. The organ of Corti showed regular inner and outerhair cells. No vacuolization was seen at the basal pole of the innerhair cells. The Deiters’, Claudius’ and Hensen’s cells (supportingcells of the organ of Corti) showed a smooth configuration andeuchromatin-containing nuclei.

Transmission electron microscopyTEM was performed on four temporal bones of septic mice withhearing loss and two sham animals without hearing loss. Examinationof the supporting cells revealed the following pathological alterations:Firstly, the Deiters’ cells of the hearing-impaired animals showed acellular shrinking and chromatin condensation (Fig. 2A,B). Secondly,examination of the Claudius’ cells revealed a cellular shrinking andchromatin condensation. The membranes (cellular, nuclear) of theexamined temporal bones were well preserved.

The inner hair cells of septic mice with hearing impairmentshowed a well-preserved basal pole. The vacuolization could beconnected to the afferent nerve fibers. The dendrites showed amassive swelling, with membrane disruption and loss ofcytoplasmic content (Fig. 2C,D). The sham animals did not showany basal vacuolization, which was similar to the findings from lightmicroscopy (Fig. 2E,F).

Cleaved caspase-3 reactionSeven cochleae from septic mice with varying levels of hearingloss were randomly included into the immunohistochemical

analysis. Distinct staining for the cleaved caspase-3 was seen inthe supporting cells of the cochlea (Deiters’ cells, Claudius’ cellsand Hensen’s cells) (Table 1; Fig. 3B). Animals without hearingloss did not show any reaction or only a very mild staining in the supporting cells of the cochlea (Table 1). Theimmunohistochemical evaluation of three control animals withoutany hearing loss did not reveal any immunolabeling for cleavedcaspase-3 (Table 1).

Bcl-2 and Bax reactionSeven cochleae from septic mice with varying levels of hearing losswere randomly included in the immunohistochemical Bcl-2evaluation. An intensive reaction could be detected in the fibrocytesof the spiral ligament. Furthermore, positive immunolabeling forBcl-2 could be detected in the supporting cells of the organ of Corti.No immunohistochemical reaction could be noted in the inner andouter hair cells. The spiral ganglion cells labeled positive for Bcl-2 (Table 2; Fig. 4A-D). The cochleae of three control animals didnot show any positive reaction for Bcl-2.

Bax immunohistochemistry was performed for all CLP mice. Anintensive upregulation of Bax could be seen in the supporting cellsof the organ of Corti, comparable with the Bcl-2 expression (Table2; Fig. 5A,B). One temporal bone did show a mild Bax reaction inthe stria vascularis and the fibrocytes of the spiral ligament (Table2; Fig.  5C,D). The remaining six cochleae did not show a Baxreaction in these two structures (Table 2).

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Table 1. Immunohistochemical results for cleaved caspase-3

Animal

number

Time of

sacrifice*

(hours) Cochlea turn

Hearing loss

(dB SPL)

Cleaved caspase-3

IHC OHC HC CC DC Stria vasc. Fibro SL

CLP 1 14 Apical –10 – – + – – – –

Middle 10 – – + + + – –

Basal 15 – – ++ + + – –

CLP 2 39 Apical 0 – – ++ – ++ – –

Middle 20 – – ++ + ++ – –

Basal 15 – – ++ ++ ++ – –

CLP 3 20 Apical 0 – – + – – – –

Middle –10 – – – – + – –

Basal –5 – – – – + – –

CLP 4 37 Apical –5 – – + – – – –

Middle 5 – – – – – – –

Basal –5 – – – – – – –

CLP 5 19 Apical 20 – – – + + – –

Middle 15 – – – + + – –

Basal 10 – – + + + – –

CLP 6 33 Apical –10 – – – – – – –

Middle 0 – – – – – – –

Basal –10 – – + – + – –

CLP 7 48 Apical 20 – – + ++ ++ – –

Middle 20 – – ++ ++ ++ – –

Basal 50 – – ++ + ++ – –

*Time after CLP that animals showed induction of sepsis and were sacrificed. IHC, inner hair cell; OHC, outer hair cell; HC, Hensen’s cells; CC, Claudius’ cells; DC, Deiters’ cells; Stria

vasc, stria vascularis; Fibro SL, fibrocytes of the spiral ligament; ++, intensive reaction; +, mild reaction; –, no reaction.

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DISCUSSIONAn involvement of the inner ear in severe sepsis syndrome has onlybeen reported in a single case of sepsis with Capnocytophagacanimorsus following a dog bite (Widlund and Duberg, 2010). Inour study, a significant hearing loss could be demonstrated in micesuffering from sepsis experimentally induced with the cecal ligationpuncture technique. Furthermore, the morphological andimmunohistochemical evaluation revealed an induction ofapoptosis in the supporting cells of the organ of Corti and glutamateexcitotoxicity at the basal pole of the inner hair cells as keypathophysiological mechanisms potentially explaining thefunctional impairment. No hearing loss and no pathologicalalterations could be demonstrated in control animals.

The blood cultures of the CLP group showed a bacteremia withE. coli and K. oxytoca. No bacteria could be grown in the controlor sham animals. proving the ‘successful induction of sepsis’ in theCLP group.

Blood examinations showed pH levels ranging from 7.0 to 7.26and lactate levels ranging from 5.6 to 41.1 mg/dl in the CLP group.In the control and sham groups pH was 7.21-7.47 and lactate 7.2-19.2 mg/dl. Lactate acidosis is a typical pathological finding in sepsissyndrome (Eissa et al., 2010), thus confirming the disease in theCLP mouse group.

The first ABR measurement showed regular hearing thresholdsfor C57 BL/6J mice younger than 3 months (Zheng et al., 1999).

The mean hearing threshold in the septic mice deteriorated by anaverage of 8.1 dB SPL, in contrast to no change in the control groupand an average loss of 0.76 dB SPL in the sham mice. Adjusting fordifferences in the first measurement, the hearing loss between thefirst and the second measurements in the sepsis group wassignificantly different (CLP versus control P<0.001, CLP versussham P=0.002). No difference was detected between the first andthe second measurements for the control and the sham group. Thisfinding clearly supports the hypothesis that sepsis leads to asignificant hearing impairment. So far, no experimental data existsevaluating the inner ear in sepsis syndrome. Another severesystemic inflammatory disease, severe malaria, has been linked tohearing loss (Schmutzhard et al., 2010). No malaria-typicalalterations have been detected in the temporal bones of hearing-impaired malaria mice, suggesting that the hearing impairmentresults from the general inflammatory reaction (Schmutzhard etal., 2011). Thus, the detection of a significant hearing loss in sepsisis well in line with this experimental background.

The question of whether the observed significant hearingimpairment in mortally sick mice is transient or permanent damagecannot be answered with this study. In order to show a permanenthearing loss in mice with sepsis further studies using sepsis modelswith a lower mortality rate need to be applied.

Light microscopy showed a well-preserved cochlea with regularspiral ganglion cells, stria vascularis and sensory epithelium. The

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Fig. 3. Immunohistochemistry for cleaved caspase-3 in ahearing-impaired animal. Brown staining marks cleavedcaspase-3 activity. (A)Overview of the cochlea duct at themiddle turn. The box marks the organ of Corti. (B)Highmagnification of the organ of Corti. Positive labeling forcleaved caspase-3 can be detected in Deiters’ cells, Hensen’scells and Claudius’ cells. No staining can be detected in theinner and outer hair cells. (C)High magnification of the striavascularis and the spiral ligament. No reaction can bedetected. (D)Close-up of the spiral ganglion cells. Nolabeling can be visualized. OHC, outer hair cell; IHC, inner haircell; CC, Claudius’ cells; DC, Deiters’ cells; HC, Hensen’s cells;SL, spiral ligament; SV, stria vascularis.

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supporting cells in the organ of Corti showed morphologicalalterations, such as dark cytoplasm indicating high protein synthesisaccompanied by apoptotic activity, cell shrinkage and a chromatincondensation with shrunken nuclei. These alterations could bedetected in Deiters’ cells, Hensen’s cells and Claudius’ cells – thesesupporting cells of the organ of Corti are involved in potassiumrecirculation. A high potassium concentration in the endolymphis responsible for the endolymphatic potential, which is needed forsensory transduction in the inner and outer hair cells. Therefore,a lack of potassium in the endolymph results in a malfunction ofthe cochlea (Kikuchi et al., 2000). Electron microscopy of cellsinvolved with potassium recycling revealed cell shrinkage, withfolding of the plasma membrane and chromatin condensation.These morphological alterations are known to be typicalmorphological hallmarks of apoptosis (Häcker, 2000). These typicalmorphological findings support the notion that apoptosis may bethe primary cause of hearing impairment. Damage of themembranes (cell, nuclear) as typically seen in necrosis could notbe observed. Therefore, necrosis is unlikely to play an importantrole in the pathophysiology of this hearing impairment.

A further interesting finding is the vacuolization on the basalpole of the inner hair cells. The vacuolization could be linked tohearing loss in the CLP mice. Furthermore, the intensity of thevacuolization was associated with the severity of the hearing loss.No such pathological alteration could be found in the control andsham animals. Electron microscopy found the vacuolization to be

linked to a massive swelling of the radial dendrites. These alterationshave been reported to be induced by excessive AMPA, kainite andglutamate exposure. Furthermore, the identical pathologicalalteration has been shown in ischemia- and noise-induced hearingloss, which could be linked to an excitotoxic pathway (Ruel et al.,2007). With these pathological results in the CLP animals,excitotoxicity of glutamate at the radial dendrites is a furtherpossible pathomechanism of the observed hearing loss.

We applied immunohistochemical labeling for activated caspase-3 as a crucial executioner of apoptosis and a reliable method ofdetecting apoptosis, which could be visualized in Deiters’ cells,Claudius’ cells and Hensen’s cells (i.e. the supporting cells of theorgan of Corti). The positive labeling for cleaved caspase-3 wasexclusively detected in the hearing-impaired animals, whereasalmost no labeling could be shown in the CLP mice without hearingimpairment. The positively labeled temporal bones showeddifferences in the intensity of the labeling: mice with hearingimpairment and suffering from sepsis for 2 days showed a moreintensive reaction with cleaved caspase-3, whereas only mildstaining could be detected in mice being sacrificed within less than24  hours after sepsis induction. Similar time dependency of theupregulation of cleaved caspase-3 in the CLP model has beenpreviously described in intestinal cells (Coopersmith et al., 2002a).

The induction of apoptosis in the CLP model is a well-describedeffect in various organs, such as gastrointestinal cells, hepatocytesand lung epithelial cells (Coopersmith et al., 2002b; Kim et al., 2000;

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Table 2. Immunohistochemical results for Bax and Bcl-2

Animal

number

Time of

sacrifice*

(hours) Cochlea turn

Hearing loss

(dB SPL)

Bax or Bcl-2

IHC OHC HC CC DC Stria vasc Fibro SL

CLP 1 14 Apical –10 –/– –/– –/+ +/+ +/+ –/– –/+

Middle 10 –/– –/– +/++ +/+ ++/++ –/– –/++

Basal 15 –/– –/– +/++ +/+ ++/++ –/– –/++

CLP 2 39 Apical 0 –/– –/– +/++ +/++ +/++ –/– –/+

Middle 20 –/– –/– +/++ +/++ ++/++ –/– –/++

Basal 15 –/– –/– +/++ +/+ ++/++ –/– –/++

CLP 3 20 Apical 0 –/– –/– +/+ +/+ +/++ –/– –/+

Middle –10 –/– –/– +/+ +/– ++/++ –/– –/–

Basal –5 –/– –/– +/+ +/– ++/++ –/– –/+

CLP 4 37 Apical –5 –/– –/– +/+ +/+ +/++ –/– –/–

Middle 5 –/– –/– +/+ +/+ ++/++ –/– –/+

Basal –5 –/– –/– +/+ –/+ +/++ –/– –/+

CLP 5 19 Apical 20 –/– –/– –/+ +/+ +/+ –/– –/+

Middle 15 –/– –/– –/– +/+ ++/++ –/– –/+

Basal 10 –/– –/– –/– +/+ +/++ –/– –/++

CLP 6 33 Apical –10 –/– –/– +/+ +/+ +/+ –/– –/–

Middle 0 –/– –/– +/++ +/++ +/++ –/– –/+

Basal –10 –/– –/– +/++ +/++ +/++ –/– –/++

CLP 8 14 Apical 10 –/– –/– +/++ ++/+ ++/++ –/– +/+

Middle 5 –/– –/– +/++ ++/+ ++/++ –/– +/++

Basal 15 –/– –/– +/++ ++/+ ++/++ –/– +/++

*Time after CLP that animals showed induction of sepsis and were sacrificed. IHC, inner hair cell; OHC, outer hair cell; HC, Hensen’s cells; CC, Claudius’ cells; DC, Deiters’ cells; Stria

vasc, stria vascularis; Fibro SL, fibrocytes of the spiral ligament; ++, intensive reaction; +, mild reaction; –, no reaction.

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Mutunga et al., 2001). Kafa and colleagues suspected that the sepsis-associated encephalopathy was caused by an activation of theapoptosis cascade (Kafa et al., 2010). The inner ear belongs to thesame intracranial blood circuit as the brain, and the blood-labyrinthbarrier of the inner ear has a micro-anatomical structurecomparable to the blood-brain barrier (Choi and Kim, 2008).Therefore, a similar reaction of the inner ear as that described inthe brain can be expected. Our findings of apoptosis in thefibrocytes of the spiral ligament and a breakdown of the blood-labyrinth barrier as pathological mechanisms of the hearing lossin the malaria mouse model support this hypothesis. In malaria,the systemic inflammatory reaction (comparable with the sepsisinflammatory response) has been suspected to cause thesepathological alterations (Schmutzhard et al., 2012). Surprisingly,apoptosis in the CLP inner ears cannot be found in the fibrocytesof the spiral ligament as described in the malaria inner ears(Schmutzhard et al., 2012). The mice with sepsis, however, showedan induction of apoptosis in the supporting cells (Deiters’, Hensen’sand Claudius’ cells). In an animal model of Menière’s disease, theinduction of apoptosis in these supporting cells could be associatedwith oxidative stress, a well-known and obviously more relevantreaction in sepsis (Labbé et al., 2005; Wang et al., 2012).

The third focus of this study was the anti-apoptotic activity. Bcl-2 is an accepted anti-apoptotic protein (Pfannenstiel and Praetorius,2008). Immunohistochemical labeling for Bcl-2 in the temporal

bones of the CLP mice revealed an anti-apoptotic activity in thesupporting cells and the fibrocytes of the spiral ligament. Theintensity of this upregulation could not be linked to the level ofhearing loss. Comparing the cleaved caspase-3 labeling and the Bcl-2 labeling in the supporting cells, a coexpression of the anti-apoptotic activity and the pro-apoptotic activity could be found.The upregulation of Bcl-2 is often observed simultaneously withan upregulation of the Bax protein, the pro-apototic antagonist ofthe Bcl-2 family proteins. Simultaneous labeling for both Bax andBcl-2 in the supporting cells strongly supports a pro-apoptotic cellactivity. This is further supported by the observed cleaved caspase-3 activation, which is mediated by the Bax protein (Salakou et al.,2007).

In contrast to this finding, the anti-apoptotic activity wasupregulated in the fibrocytes of the spiral ligament. Bcl-2overexpression is known to lead to a downregulation of cleavedcaspase-3 (Coopersmith et al., 2002a). This explains the lack ofupregulation in the fibrocytes of the spiral ligament, as describedin the malaria mouse model (Coopersmith et al., 2002b). Thesefindings are further supported by a lack of Bax activity in thefibrocytes of the spiral ligament.

The final cause of the above described pathological alterationsis not fully understood. On the one hand, a disturbance of themicrocirculation as seen in sepsis encephalopathy could affect theinner ear perfusion, resulting in an ischemic stimulus that could

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Fig. 4. Immunohistochemical results for Bcl-2. Brownstaining marks Bcl-2 activity. (A)Overview of the cochleaduct at the middle turn. The box marks the organ of Corti,with intensive Bcl-2 staining. Further reactivity can be seenin the spiral ligament. (B)Close-up of the organ of Corti.With the differential interference contrast mode, the innerand outer hair cells do not show any Bcl-2 activity. HighBcl-2 activation is seen for the Deiters’ cells, Hensen’s cells,Claudius’ cells and other supporting cells. (C)Highmagnification of the stria vascularis and the spiralligament. Upregulation of Bcl-2 in the fibrocytes of thespiral ligament can be noted. No reaction is found in thestria vascularis. (D)Bcl-2 activation can be seen in thespiral ganglion cells. OHC, outer hair cell; IHC, inner haircell; CC, Claudius’ cells; DC, Deiters’ cells; HC, Hensen’s cells;SL, spiral ligament; SV, stria vascularis.

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account for the swelling of the radial dendrites (Taccone et al., 2010).On the other hand, the induction of apoptosis in various organshas been observed as a further pathophysiological mechanism inmulti-organ dysfunction (Iwata et al., 2011), rendering an inductionof apoptosis in the inner ear likely. Further studies on mice thatsurvive CLP sepsis are necessary to evaluate the long-term effectof these pathological alterations.

These results show that sepsis induced by CLP leads to asignificant hearing loss in mice. Furthermore, the functional deficitcould be linked on one hand to an activation of apoptosis in thesupporting cells of the organ of Corti, disrupting the potassiumcirculation in the inner ear, and on the other hand to glutamateexcitotoxicity at the radial dendrites on the base of the inner haircells (Kikuchi et al., 2000; Ruel et al., 2007). To verify the effect ofsystemic sepsis on the human inner ear, prospective evaluation ofthe hearing capacity of patients with sepsis is urgently necessary.Nevertheless, this animal experiment should alert professionals tothe possibility that sepsis might directly lead to a significanthearing impairment.

MATERIALS AND METHODSThe animal studies conformed to the Austrian guidelines for thecare and use of laboratory animals and were approved by theAustrian Ministry Science with the reference number BMWF-66.011/0082-II/3b/2011.

Study designForty-four 2-month-old C57BL/6 mice (Charles River, Sulzfeld,Germany) were used in this study. An initial objective hearing testwas performed with auditory evoked brainstem responses (ABR).Animals with thresholds below 40 dB at 16 kHz were eligible. Sepsiswas induced in 18 mice with cecal ligation puncture; 13 miceunderwent sham surgery (laparotomy, no ligation, no puncture)and 13 mice served as control (identical housing and treatment,but no intervention). The course of sepsis was monitored for signsof prostration, loss of temperature (Brooks et al., 2007) and weightloss (Xiao et al., 2006) at 4-hour intervals. Body temperature below34°C, severe prostration and a significant weight loss indicated‘successful’ induction of sepsis and the animals were sacrificed at

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Fig. 5. Immunohistochemical results for Bax. Brownstaining marks Bax activity. (A)Overview of the cochleaduct at the middle turn. The box marks the organ of Corti,with intensive Bax staining. Further reactivity can be seenin the spiral ligament and the spiral ganglion cells.(B)Close-up of the organ of Corti. High Bax-2 activation isseen for Deiters’ cells, Hensen’s cells, Claudius’ cells andother supporting cells. (C)High magnification of the striavascularis and the spiral ligament. Upregulation of Bax inthe fibrocytes of the spiral ligament can be noted. Noreaction is found in the stria vascularis. (D)Bax activationcan be seen in the spiral ganglion cells. OHC, outer hair cell;IHC, inner hair cell; CC, Claudius’ cells; DC, Deiters’ cells; HC,Hensen’s cells; SGC, spiral ganglion cells; SL, spiral ligament;SV, stria vascularis.

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this point of time, immediately after the second ABR measurement.The control animals were sacrificed after 96 hours.

Immediately thereafter, blood samples for lactate and pHdetermination and for culture were obtained by cardiac puncture.If cardiac puncture was not successful within 60  seconds, theprocedure was stopped in order to preserve the vulnerable innerear structures. Animals were then sacrificed and both inner earsharvested within 6 minutes.

Prior to the audiological evaluation of the ABR data, eightrandomly selected sepsis inner ears and three controls wereanalyzed morphologically and immunohistochemically. The leftside was examined by semi-thin sectioning and TEM. The rightside was examined with immunohistochemistry for cleavedcaspase-3, Bax and Bcl-2.

Cecal ligation puncture mouse modelThe mice were deeply anesthetized with intraperitoneal ketaminehydrochloride (Graeub®, Senden-Bösensell, Germany) (67.5 mg/kgbody weight), xylazine hydrochloride (Bayer®, Leverkusen, Germany)(5.4  mg/kg body weight) and atropine sulfate (Nycomed®, Linz,Austria) (0.085 mg/kg). After fixation of the animal, the fur of thebelly was trimmed and disinfected with 90% alcohol. A medianincision was done and the linea alba was incised. The cecum wasmobilized and ligated with a 4.0 Vicyl suture (Ethicon, Norderstedt,Germany) including approximately three-quarters of the cecum inorder to induce a high-grade sepsis with an anticipated mortality of100% after 4 days (Rittirsch et al., 2009). The perforation, saving theblood vessels, was done with a 21 gauge needle, exposing smalldroplets of feces on both perforation sides. The cecum wasrepositioned and the incision closed in layers with 4.0 Vicryl sutures.Afterwards, the animals were rescued with a subcutaneous injectionof 37°C warm sodium chloride (1 ml/20 g body weight). After theprocedure, the animals were kept on a 37°C warm plate with freeaccess to unlimited food and water. Two hours after the intervention,the 4-hourly periodical assessment of body weight, body temperatureand grade of prostration started (Rittirsch et al., 2009).

Auditory evoked brain stem responsesAnesthesia was performed with an intraperitoneal injection ofketamine hydrochloride (67.5  mg/kg body weight), xylazinehydrochloride (5.4  mg/kg body weight) and atropine sulfate(0.085 mg/kg). The ABR measurement (PNS 2, Tübingen, Germany)was done in an electrically shielded sound attenuated chamber forthe frequencies 4, 8, 16 and 32 kHz. The potentials were recordedvia three subcutaneous needle electrodes, which were placed at thevertex (positive electrode), the bulla (negative electrode) and theipsilateral leg (ground electrode). The stimuli started at 90 dB andgradually decreased from 90 dB to 5 dB. The ABR thresholds aredetermined as the minimum stimulation level that produces a clearlyrecognizable potential. Electrical signals were averaged over 64repetitions of stimulus pairs. The ABR results were evaluated by twoindependent ear, nose and throat specialists in a blinded manner.

Specimen preparationImmediately after sacrifice, both cochleae were harvested anddissected within 6 minutes. The round and the oval windows wereopened and fixed with submersion fixation and subsequentlyrinsed with fixans. The left cochleae were fixed with Karnovsky’s

fixative (2.5% glutaraldehyde and 4% paraformaldehyde in 0.1 Mcacodylate buffer, pH 7.4) for semi-thin sectioning and TEM. Theright cochleae were treated with 4% paraformaldehyde in phosphatebuffered saline (PBS), pH 7.4, for further immunohistochemistry.Decalcification was performed with an 8-hour exposure of tissuein 20% EDTA (Titriplex III; Merck, Darmstadt, Germany) in PBS,pH 7.4, at 37°C.

After being thoroughly washed in PBS, Karnovsky-fixedcochleae were embedded in Embed 812 Kit© (Electron MicroscopyScience, Hatfield, PA) according to the recommended procedure.Semi-thin sections (1 μm) were cut on a Leica Ultracut microtome(Wetzlar, Germany) and then stained with Toluidine Blue at 60°Cfor light microscopic evaluation. Paraformaldehyde-fixedspecimens were cryoembedded following a published protocol(Coleman et al., 2009). For immunohistochemical examination,cryoembedded serial sections (10  μm) were cut on a cryostat(LEICA CM 3050), mounted on glass slides (SuperFrost®Plus,Menzel-Gläser, Braunschweig, Germany), air-dried for 1 hour andstored at −20°C.

Laboratory examinationImmediately before sacrifice ~0.7  ml of blood was drawn bycardiac puncture. Approximately 300 μl was used to measure lactateand pH levels (Blood Gas Analyzer Rapidlab 865 Siemens®, Munich,Germany).

The remaining blood was cultured. The BacT/ALERT* CultureMedia – Pediatric FAN bioMérieux France was used as culturevessel. The analysis of the bacteriaemia was done withBacT/ALERT® 3D Microbial Detection System bioMérieux(Craponne, France).

Light microscopyThe semithin sections (1 μm) and the immunohistochemical slideswere examined using an Image-pro® 6.0 analysis system (MediaCybernetics®, Silver Spring, MD) linked to a 3 CCD color videocamera (Sony DXC-950P, Tokyo, Japan) on an Olympus BX 50 lightmicroscope (Olympus, Tokyo, Japan).

Transmission electron microscopyTEM evaluation was done with the block surface method. Ultrathinsections (90  nm) were cut with a Leica UC6 microtome andtransferred to pioloform F (polyvinylacetate)-coated (1.5% pioloformin chloroform) slot grids. Staining was performed by an automatedsystem (Leica EM Stain) with uranyl acetate (5 g/l; 30 minutes) andlead citrate (5 g/l, 50 minutes) at 25°C. The ultrathin sections wereexamined with a Philips CM 120 transmission electron microscope(FEI, Eindhoven, The Netherlands) equipped with a MORADAdigital camera (Olympus SIS, Münster, Germany). The softwareOlympus TEM Imaging Platform was used.

ImmunohistochemistryImmunohistochemistry was performed on frozen sections in aVentana Roche Discovery XT Immunostainer (Mannheim,Germany) according to the DAB-MAP discovery research standardprocedure. After incubating the sections with primary antibodies[cleaved caspase-3 1:400 (Asp175), Cell Signaling Technology,Danvers, MA; Bcl-2 (N-19), sc-492 1:200 Santa Cruz Biotechnology,Santa Cruz, CA; and Bax (P-19) rabbit polyclonal IgG492 1:50 Santa

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Cruz Biotechnology] for 1 hour at 37°C, a biotinylated donkey anti-rabbit IgG secondary antibody (1:400, Jackson ImmunoResearch711-065-152) was applied for 30 minutes at room temperature. Thedetection was achieved using the DAB-MAP Detection Kit(Ventana 760-124) according to the diaminobenzidine developmentmethod followed by lightly counterstaining with hematoxylin(Ventana 760-2021) for 4  minutes. Sections were then manuallydehydrated, cleared in xylene and cover-slipped. Theimmunohistochemical staining reaction was referred to positivecontrols (tonsil) that were added to each experiment. In addition,for control purposes, representative sections were processed in thesame way as previously described by substituting them for isotypematching immunoglobulins (1:200, rabbit polyclonal IgG, Abcam,ab27478).

The sections were evaluated independently by two blindedauthors in a semi-quantitative manner: ++ intensive reaction, + mildreaction, – no reaction.

Statistical evaluationABR measurements were performed in each animal beforetreatment at the start (T1) and after the treatment at the end (T2)of the observation period. Pure tone average (PTA) was calculatedas the mean of the left and right ear ABR-thresholds for thefrequencies 4, 8, 16 and 32 kHz for each animal. To test treatmenteffects (CLP versus sham or control), one-way analysis of covariancewas used with post-treatment PTA (PTA_T2) as the outcomevariable adjusted for pretreatment PTA (PTA_T1) as a covariate.Homogeneity of slopes was assessed graphically and by testing theinteraction term treatment*PTA_T1 in a preliminary model.Covariate adjusted marginal means of PTA at T2 with 95%confidence intervals are provided.COMPETING INTERESTSThe authors declare that they do not have any competing or financial interests.

AUTHOR CONTRIBUTIONSJ.S. did the conceptual work, established the CLP model, evaluated the ABR andthe histological slices and wrote the manuscript. R.G., C.P. and T.S. prepared theimmunohistochemical slices. M.J.F.B. and M.B. prepared the electron microscopicslices and did the electron microscopy. M.F. made the blood cultures. P.L. and H.R.calculated the statistics. M.H. measured the ABRs. A.S.-F. was instrumental inhelping with the conceptual work, evaluated the ABR and the histological slicesand helped with the manuscript

FUNDINGThe work has been supported by the intramural funding program of the MedicalUniversity Innsbruck (MUI) for young scientists, MUI-Start [project number2012032018].

SUPPLEMENTARY MATERIALSupplementary material for this article is available athttp://dmm.biologists.org/lookup/suppl/doi:10.1242/dmm.011205/-/DC1

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