research article olfactory dysfunction in patients with neuromyelitis...

Hindawi Publishing CorporationMultiple Sclerosis InternationalVolume 2013 Article ID 654501 4 pageshttpdxdoiorg1011552013654501

Research ArticleOlfactory Dysfunction in Patients with Neuromyelitis Optica

Felix Schmidt12 Oumlnder Goumlktas3 Sven Jarius4 Brigitte Wildemann4

Klemens Ruprecht12 Friedemann Paul125 and Lutz Harms12

1 Department of Neurology Charite-Universitatsmedizin Berlin Chariteplatz 1 10117 Berlin Germany2 Clinical and Experimental Multiple Sclerosis Research Center Charite-Universitatsmedizin Berlin Berlin Germany3Department of Otolaryngology Charite-Universitatsmedizin Berlin Berlin Germany4Division of Molecular Neuroimmunology Department of Neurology University of Heidelberg Heidelberg Germany5NeuroCure Clinical Research Center and Experimental and Clinical Research Center Charite-Universitatsmedizin Berlin and MaxDelbrueck Center for Molecular Medicine Berlin Germany

Correspondence should be addressed to Felix Schmidt felixschmidtcharitede

Received 9 June 2013 Revised 22 August 2013 Accepted 23 August 2013

Academic Editor Wolfgang Bruck

Copyright copy 2013 Felix Schmidt et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Background Neuromyelitis optica (NMO) is a severely disabling autoimmune disorder of the CNS which mainly affects the opticnerves and spinal cord However recent studies have shown that extra-opticospinal are more common in NMO than previouslythought Objective To investigate olfactory function (OF) in patients with neuromyelitis optica (NMO) versus healthy controls(HC)Methods Psychophysical testing of the orthonasal OF was performed using theThreshold-Discrimination-Identification test(TDI) measuring different qualities of olfaction in 10 unselected NMO patients and 10HC Results Five of 10NMO patients (50)showed hyposmia while all 10HC were normosmic Moreover NMO patients had significantly lower mean TDI-scores comparedto HC based on a poorer performance in both the Discrimination and the Identification subtests Conclusions Our results suggestthat hyposmia might be part of the expanding clinical spectrum of NMO

1 Introduction

Neuromyelitis optica (NMODevicrsquos syndrome) is an autoim-mune central nervous system disorder that predominantlyaffects the optic nerves and the spinal cord [1 2] Impairedolfaction is increasingly recognized in neurodegenerativediseases such as Parkinsonrsquos and Alzheimerrsquos diseases andhas been reported in patients with multiple sclerosis (MS)[3 4] As the clinical presentation of NMO may extendbeyond relapses of optic neuritis and myelitis [2 5] we hereinvestigated whether olfactory function (OF) is altered inNMO

2 Patients and Methods

This pilot study was performed from July 2011 to October2012 Ten patients with NMO according to the 2006 diagnos-tic criteria [1] were prospectively recruited from the Chariteoutpatient clinics Aquaporin-4 antibodies were tested using

a commercially available cell-based assay employing recom-binant human target antigen (EUROIMMUN Luebeck Ger-many) [6] A healthy control group (HC) of 10 individualsclosely matched for gender and age (plusmn3 years) was recruitedamong the hospital staff Exclusion criteria for both groupswere olfactory disorders (postinfectious posttraumatic andsinunasal) infections of the upper respiratory tract tumourstreated with radiation or chemotherapy allergies majordepression and Parkinsonrsquos or Alzheimerrsquos disease All studyparticipants declared to be nonsmokers Patients takingdrugs that could cause olfactory dysfunction as for exam-ple amitriptyline and D-penicillamine were excluded fromthe study Furthermore patients receiving corticosteroidtreatment during the testing period were excluded becausecorticosteroids can have an effect on the OF Olfactionwas evaluated using the tripartite Threshold-Discrimination-Identification test (TDI) based on pen-like odour-dispensingdevices as recommended by the ldquoWorking Group Olfac-tology and Gustologyrdquo of the German ENT Society [7]

2 Multiple Sclerosis International

TheThreshold test (119879-test) consists of 48 ldquosniffing sticksrdquo witha 16-stage dilution series of n-butanol for determining theolfactory perception threshold of a patient The Discrimi-nation test (119863-test) consists of 48 sniffing sticks to test thedistinction of different smells Everyday smells have to beidentified with the Identification test (119868-test) Low scores ineach subtest demonstrate low olfactory performance Thescores of all three subtests are summed up to the TDI-score A TDI value of less than 16 means anosmia up to 30points hyposmia and above 30 points normosmia Statisticalanalysis was performed with GraphPad Prism 401 (SanDiego USA)TheMann-Whitney119880 test was used to comparemedians of olfactory test results between NMO patientsand the control group Proportions of hyposmic patientsbetween both groups were compared by Fisherrsquos exact testThe significance level was defined at 119875 lt 005 Owing tothe exploratory nature of this pilot study no adjustments formultiple testing were made The study was approved by theethics committee of the Charite and all participants gavewritten informed consent

To assess the binding of NMO-IgG to AQP4 expressed inthe olfactory bulb 7 120583m adult rat and mouse olfactory bulbcryosections (10 120583m) were incubated with 10 phosphate-buffered formalin for 4 minutes After three washes inphosphate-buffered saline (PBS) detergent (1 CHAPS inPBS) was applied for 4 minutes After three additionalwashes in PBS PBS containing 10 normal goat serum wasapplied for 60 minutes The sections were then incubatedwith patient and control sera (1 60) for 60 minutes at roomtemperature A commercial Alexa Fluor 488-conjugated goatanti-huIgG antibody (Invitrogen Darmstadt Germany) wasused to detect bound patient IgG After 60 minutes the wellswere washed thoroughly in PBS and a glass coverslip wasapplied to each slide with ProLong Gold mounting medium(Invitrogen)

3 Results

Five out of ten patients (50) with NMO showed hyposmianone was anosmic The remaining patients had a normalolfactory function By contrast all control subjects werenormosmic (Table 1) Two of the hyposmic patients werenot aware of their olfactory deficit Moreover both meantotal TDI-scores andD- and I-subscores were lower in NMOpatients compared toHC (Table 1) indicating lower olfactorycapacityThe hyposmic NMOpatients had amean TDI-scoreof 26 with a standard deviation of 20 Four out of five (80)hyposmic patients were positive for AQP4 antibodies Theprevalence of olfactory dysfunctionwas higher inAQP4-IgG-positive patients (67) than in AQP4-IgG-negative patients(25) though the difference was not significant There wasno association between OF and disease duration

Immunohistochemistry In accordance with the NMO-IgGstaining pattern originally described on cerebellum tissuesections strong staining of the astrocytic endfeet adjacent tothe pia mater and the microvasculature was found (Figures 1and 2) Only mild if any staining of the olfactory glomeruli

was detected No significant binding was present in theperipheral olfactory nerves the connective tissue next tothe medial surface of the olfactory bulb or (apart fromperivascular staining) any of the inner olfactory bulb layersOverall twenty tissue sections (10x mouse 10x rat) werestained The staining pattern described above was observedwith 55 unselected AQP4-Ab-positive NMO samples butnot with three unselected AQP4-Ab-negative NMO samplesand not with two healthy control samples Identical patternswere observed whenmouse instead of rat tissue sections wereemployed though use of rat tissue resulted in a more intensestaining (not shown)

4 Discussion

Our study revealed orthonasal hyposmia detected by psy-chophysical testing in five out of ten NMO patients Fourout of five hyposmotic patients were positive for AQP4antibodies Our findings are in line with the broadeningspectrum of clinical manifestations of NMO that goes farbeyond the classical bouts of optic neuritis and myelitis [5]and now encompasses various brain stem symptoms painand the posterior reversible encephalopathy syndrome

Impaired olfaction can result from damage to differentsections of the olfactory pathway Peripheral damage ismostly caused by anatomical abnormalities of the nasal cavi-ties especially the olfactory cleft and damage to the olfactoryepithelium and the olfactory nerve Olfactory dysfunctioncan also be a consequence of damage to or atrophy of theolfactory bulb the olfactory cortex and higher processingregions of the olfactory system such as the limbic system

In clinical routine examination of olfactory functionsis not regularly performed presumably owing to the time-consuming procedures required to objectify impaired olfac-tion However olfactory dysfunction has been describedin numerous neurodegenerative diseases [3] including MSHere olfactory dysfunction results from CNS damage asindicated by an association between olfactory function andlesion load in the olfactory brain [4] and a relation of olfac-tory functions to longitudinal changes in plaque numbers incentral olfactory structures [8] We could report a relationbetween the lesion load in the olfactory cortex and the volumeof the olfactory bulb and olfactory brain [9]

In NMO which is nowadays considered a disease entitydistinct from MS [10] recent neuroimaging studies haveconsistently reported brain abnormalities in more than 50of patients [5 11] however without particularly investigatinglesions or volume reductions in olfactory CNS structuresWe appreciate that neuropathological orMRI studies demon-strating typical NMO lesions in olfactory structures would berequired to formally prove the supposed link between NMOand OD in our patients Importantly however our resultsprovide a strong rationale for the inclusion of such analysesin future multicentre studies on NMO

AQP4 themain target antigen inNMO is expressed bothin the nasal olfactory mucosa including the basal cells thesupporting cells and the Bowman glands that are part ofthe olfactory epithelium as a microenvironment necessary

Multiple Sclerosis International 3

Table 1 Demographic data and means of olfactory test results in NMO patients and healthy controls

NMO (119873 = 10) Controls (119873 = 10) P valueAge (years mean plusmn SD) 451 plusmn 133 469 plusmn 1449 04Sex ratio (m f) 1 4 1 4 1AQP4 antibody (posneg119873) 64 na naDisease duration (months mean SD) 282 plusmn 435 na naSubjects with hyposmia (119873 ) 5 (50) 0 (0) 003TDI-score (mean plusmn SD) 301 plusmn 48 341 plusmn 19 004T-score (mean plusmn SD) 76 plusmn 14 68 plusmn 25 02D-score (mean plusmn SD) 99 plusmn 28 128 plusmn 22 002I-score (mean plusmn SD) 126 plusmn 20 145 plusmn 07 001

Figure 1 Binding of serum NMO-IgG to the olfactory bulb asdemonstrated by indirect immunohistochemistry on formalin-fixedrat tissue cryosections Binding of the patientrsquos IgG is shown ingreen (AF488) See method section for details The figure shows atransverse section through the right olfactory bulb near the medialsurface Arrowheads = pia mater arrows = microvasculature GL =olfactory glomeruli ON = peripheral olfactory nerves (ON) NL =nerve fibre layer CT = connective tissue next to the medial surfaceof the olfactory bulb

for olfaction [12] and in the astrocytic endfeet sealing themicrovasculature and the pial surface of the olfactory bulbaccordingly we found a strong staining of the olfactorybulb by AQP4-IgG-positive NMO sera (see Figures 1 and2) AQP4-IgG has been previously shown to cause tissuedamage by complement activation and antibody-dependentcell-mediated cytotoxicity [13 14] Olfactory dysfunctioncould thus occur due to tissue damage in olfactory structureswith AQP4 expression Whether mechanisms other thanAQP4-IgG were involved in the pathogenesis of hyposmiain the single ldquoseronegativerdquo patient (who otherwise met alldiagnostic criteria for NMO) remains unknown Alterna-tively ldquoseronegativityrdquo in this patient could be caused bylimited sensitivity of the currently available immunoassays[15] In myasthenia gravis (MG) another autoimmune dis-order with proven humoral pathogenesis it took almost25 years until a more sensitive class of immunoassays was

Figure 2 Binding of serumNMO-IgG to the inner portion of the ratolfactory bulb as demonstrated by indirect immunohistochemistrySee results section for details Binding of the patientrsquos IgG is shownin green (AF488) cell nuclei are shown in blue (410158406-diamidino-2-phenylindole) Arrowheads = microvasculature ExtPL-externalplexiform layer ML-mitral cell layer IntPL = internal plexiformlayer GrL = granular layer

developed subsequently two-thirds of patients previouslyclassified as ldquoseronegative MGrdquo were found to harbour low-affinity acetylcholine receptor (AChR) serum autoantibodies[16]

In conclusion our results suggest that hyposmia mightbe part of the expanding clinical spectrum of NMO andprovide the rationale for large-scale multicentre studies onthe frequency of olfactory dysfunction in NMO

Conflict of Interests

The authors declare that there is no conflict of interests


Authorsrsquo Contribution

Study concept and coordination was performed by FelixSchmidt data acquisition and interpretation were performedby Felix Schmidt Onder Goktas Klemens Ruprecht Friede-mann Paul and Lutz Harms initial draft was performed byFelix Schmidt AQP4 antibody testing and immunohisto-chemistry were performed by Sven Jarius study supervisionLutz Harms critical revision of the manuscript for importantintellectual content was performed by Felix Schmidt OnderGoktas Sven Jarius Brigitte Wildemann Klemens RuprechtFriedemann Paul and Lutz Harms

Acknowledgments

This work was supported by the German Research Founda-tion and by the German Ministry of Education and Research(Competence Network Multiple Sclerosis) (DFG Exc 257 toFP) The authors would like to thank Mrs Anna Eschlbeckand theNikon ImagingCenter at theUniversity ofHeidelbergfor the excellent technical assistance

References

[1] D MWingerchuk V A Lennon S J Pittock C F Lucchinettiand B G Weinshenker ldquoRevised diagnostic criteria for neu-romyelitis opticardquo Neurology vol 66 no 10 pp 1485ndash14892006

[2] S Jarius K Ruprecht BWildemann et al ldquoContrasting diseasepatterns in seropositive and seronegative neuromyelitis opticaa multicentre study of 175 patientsrdquo Journal of Neuroinflamma-tion vol 9 article 14 2012

[3] R I Mesholam P J Moberg R N Mahr and R L DotyldquoOlfaction in neurodegenerative disease a meta-analysis ofolfactory functioning inAhlzheimerrsquos and Parkinsonrsquos diseasesrdquoArchives of Neurology vol 55 no 1 pp 84ndash90 1998

[4] R L Doty C Li L J Mannon et al ldquoOlfactory dysfunction inmultiple sclerosisrdquo The New England Journal of Medicine vol337 no 7 p 507 1997

[5] D MWingerchuk V A Lennon C F Lucchinetti S J Pittockand B GWeinshenker ldquoThe spectrum of neuromyelitis opticardquoThe Lancet Neurology vol 6 no 9 pp 805ndash815 2007

[6] S Jarius C Probst K Borowski et al ldquoStandardizedmethod forthe detection of antibodies to aquaporin-4 based on a highlysensitive immunofluorescence assay employing recombinanttarget antigenrdquo Journal of the Neurological Sciences vol 291 no1-2 pp 52ndash56 2010

[7] T Hummel G Kobal H Gudziol and A Mackay-Sim ldquoNor-mative data for the ldquoSniffinrsquo Sticksrdquo including tests of odoridentification odor discrimination and olfactory thresholdsan upgrade based on a group of more than 3000 subjectsrdquoEuropeanArchives of Oto-Rhino-Laryngology vol 264 no 3 pp237ndash243 2007

[8] R L Doty C Li L J Mannon and D M Yousem ldquoOlfac-tory dysfunction in multiple sclerosis relation to longitudinalchanges in plaque numbers in central olfactory structuresrdquoNeurology vol 53 no 4 pp 880ndash882 1999

[9] F A Schmidt O Goktas L Harms et al ldquoStructural correlatesof taste and smell loss in encephalitis disseminatardquo PLoS ONEvol 6 no 5 Article ID e19702 2011

[10] S Jarius and B Wildemann ldquoThe history of neuromyelitisopticardquo Journal of Neuroinflammation vol 10 article 8 2013

[11] S J Pittock V A Lennon K Krecke D M Wingerchuk CF Lucchinetti and B G Weinshenker ldquoBrain abnormalities inneuromyelitis opticardquo Archives of Neurology vol 63 no 3 pp390ndash396 2006

[12] A Ablimit T Matsuzaki Y Tajika T Aoki H Hagiwara andK Takata ldquoImmunolocalization of water channel aquaporins inthe nasal olfactory mucosardquo Archives of Histology and Cytologyvol 69 no 1 pp 1ndash12 2006

[13] S Jarius F Paul D Franciotta et al ldquoMechanisms of diseaseaquaporin-4 antibodies in neuromyelitis opticardquo Nature Clini-cal Practice Neurology vol 4 no 4 pp 202ndash214 2008

[14] S Jarius and BWildemann ldquoAQP4 antibodies in neuromyelitisoptica diagnostic and pathogenetic relevancerdquo Nature ReviewsNeurology vol 6 no 7 pp 383ndash392 2010

[15] S Jarius and B Wildemann ldquoAquaporin-4 antibodies (NMO-IgG) as a serological marker of neuromyelitis optica a criticalreview of the literaturerdquo Brain Pathology In press

[16] M I Leite S Jacob S Viegas et al ldquoIgG1 antibodies toacetylcholine receptors in ldquoseronegativerdquo myasthenia gravisrdquoBrain vol 131 no 7 pp 1940ndash1952 2008

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Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


TheThreshold test (119879-test) consists of 48 ldquosniffing sticksrdquo witha 16-stage dilution series of n-butanol for determining theolfactory perception threshold of a patient The Discrimi-nation test (119863-test) consists of 48 sniffing sticks to test thedistinction of different smells Everyday smells have to beidentified with the Identification test (119868-test) Low scores ineach subtest demonstrate low olfactory performance Thescores of all three subtests are summed up to the TDI-score A TDI value of less than 16 means anosmia up to 30points hyposmia and above 30 points normosmia Statisticalanalysis was performed with GraphPad Prism 401 (SanDiego USA)TheMann-Whitney119880 test was used to comparemedians of olfactory test results between NMO patientsand the control group Proportions of hyposmic patientsbetween both groups were compared by Fisherrsquos exact testThe significance level was defined at 119875 lt 005 Owing tothe exploratory nature of this pilot study no adjustments formultiple testing were made The study was approved by theethics committee of the Charite and all participants gavewritten informed consent

To assess the binding of NMO-IgG to AQP4 expressed inthe olfactory bulb 7 120583m adult rat and mouse olfactory bulbcryosections (10 120583m) were incubated with 10 phosphate-buffered formalin for 4 minutes After three washes inphosphate-buffered saline (PBS) detergent (1 CHAPS inPBS) was applied for 4 minutes After three additionalwashes in PBS PBS containing 10 normal goat serum wasapplied for 60 minutes The sections were then incubatedwith patient and control sera (1 60) for 60 minutes at roomtemperature A commercial Alexa Fluor 488-conjugated goatanti-huIgG antibody (Invitrogen Darmstadt Germany) wasused to detect bound patient IgG After 60 minutes the wellswere washed thoroughly in PBS and a glass coverslip wasapplied to each slide with ProLong Gold mounting medium(Invitrogen)

3 Results

Five out of ten patients (50) with NMO showed hyposmianone was anosmic The remaining patients had a normalolfactory function By contrast all control subjects werenormosmic (Table 1) Two of the hyposmic patients werenot aware of their olfactory deficit Moreover both meantotal TDI-scores andD- and I-subscores were lower in NMOpatients compared toHC (Table 1) indicating lower olfactorycapacityThe hyposmic NMOpatients had amean TDI-scoreof 26 with a standard deviation of 20 Four out of five (80)hyposmic patients were positive for AQP4 antibodies Theprevalence of olfactory dysfunctionwas higher inAQP4-IgG-positive patients (67) than in AQP4-IgG-negative patients(25) though the difference was not significant There wasno association between OF and disease duration

Immunohistochemistry In accordance with the NMO-IgGstaining pattern originally described on cerebellum tissuesections strong staining of the astrocytic endfeet adjacent tothe pia mater and the microvasculature was found (Figures 1and 2) Only mild if any staining of the olfactory glomeruli

was detected No significant binding was present in theperipheral olfactory nerves the connective tissue next tothe medial surface of the olfactory bulb or (apart fromperivascular staining) any of the inner olfactory bulb layersOverall twenty tissue sections (10x mouse 10x rat) werestained The staining pattern described above was observedwith 55 unselected AQP4-Ab-positive NMO samples butnot with three unselected AQP4-Ab-negative NMO samplesand not with two healthy control samples Identical patternswere observed whenmouse instead of rat tissue sections wereemployed though use of rat tissue resulted in a more intensestaining (not shown)

4 Discussion

Our study revealed orthonasal hyposmia detected by psy-chophysical testing in five out of ten NMO patients Fourout of five hyposmotic patients were positive for AQP4antibodies Our findings are in line with the broadeningspectrum of clinical manifestations of NMO that goes farbeyond the classical bouts of optic neuritis and myelitis [5]and now encompasses various brain stem symptoms painand the posterior reversible encephalopathy syndrome

Impaired olfaction can result from damage to differentsections of the olfactory pathway Peripheral damage ismostly caused by anatomical abnormalities of the nasal cavi-ties especially the olfactory cleft and damage to the olfactoryepithelium and the olfactory nerve Olfactory dysfunctioncan also be a consequence of damage to or atrophy of theolfactory bulb the olfactory cortex and higher processingregions of the olfactory system such as the limbic system

In clinical routine examination of olfactory functionsis not regularly performed presumably owing to the time-consuming procedures required to objectify impaired olfac-tion However olfactory dysfunction has been describedin numerous neurodegenerative diseases [3] including MSHere olfactory dysfunction results from CNS damage asindicated by an association between olfactory function andlesion load in the olfactory brain [4] and a relation of olfac-tory functions to longitudinal changes in plaque numbers incentral olfactory structures [8] We could report a relationbetween the lesion load in the olfactory cortex and the volumeof the olfactory bulb and olfactory brain [9]

In NMO which is nowadays considered a disease entitydistinct from MS [10] recent neuroimaging studies haveconsistently reported brain abnormalities in more than 50of patients [5 11] however without particularly investigatinglesions or volume reductions in olfactory CNS structuresWe appreciate that neuropathological orMRI studies demon-strating typical NMO lesions in olfactory structures would berequired to formally prove the supposed link between NMOand OD in our patients Importantly however our resultsprovide a strong rationale for the inclusion of such analysesin future multicentre studies on NMO

AQP4 themain target antigen inNMO is expressed bothin the nasal olfactory mucosa including the basal cells thesupporting cells and the Bowman glands that are part ofthe olfactory epithelium as a microenvironment necessary














Acknowledgments


References






















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























Acknowledgments


References






















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article olfactory dysfunction in patients with neuromyelitis...

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