Aus der Klinik und Poliklinik für Anästhesiologie – Anästhesie-, Intensiv-, Notfall- und Schmerzmedizin (Direktor Univ.- Prof. Dr. Klaus Hahnenkamp) der Universitätsmedizin der Universität Greifswald Thema: Anti-nozizeptive Effekte und Wirkungsweisen aurikularer transkutaner Vagusnervenstimulation Inaugural - Dissertation zur Erlangung des akademischen Grades Doktor der Wissenschaften in der Medizin (Dr. rer. med.) der Universitätsmedizin der Universität Greifswald 2018 vorgelegt von: Henriette Janner, geb. Hacker geb. am: 09.03.1991 in: Rostock
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Aus der Klinik und Poliklinik für Anästhesiologie – Anästhesie-, Intensiv-, Notfall- und
Schmerzmedizin
(Direktor Univ.- Prof. Dr. Klaus Hahnenkamp)�
der Universitätsmedizin der Universität Greifswald
Thema: Anti-nozizeptive Effekte und Wirkungsweisen aurikularer transkutaner
Vagusnervenstimulation
Inaugural - Dissertation
zur
Erlangung des akademischen
Grades
Doktor der Wissenschaften in der Medizin
(Dr. rer. med.)
der
Universitätsmedizin
der
Universität Greifswald�
2018
vorgelegt von:
Henriette Janner, geb. Hacker
geb. am: 09.03.1991
in: Rostock
2
Dekan: Prof. Dr. Max P. Baur
1. Gutachter: Prof. Dr. Taras I. Usichenko
2. Gutachter: Prof. Dr. Mathias Weymar
(3. Gutachter:)
Ort, Raum: Greifswald, Konferenzraum D 0.30 der Klinik für Neurologie/Neurochirurgie
Tag der Disputation: 17.06.2019
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Inhaltsverzeichnis
Zusammenfassung 4
1 Einleitung 6
2 Invasive Vagusnervstimulation (VNS) 6
3 Aurikulare transkutane Vagusnervstimulation (tVNS) 7 3.1 Die aurikulare Akupunktur als tVNS 8 3.2 Die Wirkung aurikularer elektrischer tVNS auf die Schmerzwahrnehmung 10 3.3 Die Wirkung aurikularer tVNS auf Stimmung, Affekt und Kognition 13
und anterioren cingularen Kortex assoziiert ist. Zum anderen wird sie mit verminderter
Aktivität in limbischen Regionen wie der Amygdala, dem Hypothalamus, dem Hippocampus,
dem Parahippocampus, dem mittleren und superioren Gyrus temporalis und dem posterioren
cingularen Kortex in Verbindung gebracht. Diese Hirnareale stehen in Verbindung mit der
Verarbeitung und Regulation von emotionalen und nozizeptiven Reizen (Dietrich et al., 2008;
Kraus et al., 2013; Frangos et al., 2015; Usichenko et al., 2017a; Yakunina et al., 2016).
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Untersucht wurde die tVNS bisher hauptsächlich zur Reduktion epileptischer Anfälle, zur
Verbesserung kognitiver Funktionen, zur Reduktion von situationaler Angst, zur
Stimmungsaufhellung und zur Schmerzlinderung (Ben-Menchanem et al., 2015; Frangos et al.
2017).
3.1 Die aurikulare Akupunktur als tVNS
Die aurikulare Akupunktur oder auch Ohrakupunktur ist eine komplementär-
medizinische Methode, die zur Behandlung von verschiedenen Krankheiten und Zuständen
angewandt wird (Usichenko & Anders, 2016). In ihrer differenziertesten Form geht sie als
therapeutische Methode auf den französischen Arzt Paul Nogier zurück. Dieser ersetzte um
1950 die bis dahin von „Heilern“ durchgeführte Kauterisierung bestimmter Punkte an den
Ohrmuscheln zur Behandlung von Krankheiten, wie z.B. Rückenschmerzen, durch die
mechanische Stimulation mittels Nadeln. In der Theorie von Nogier (1957) gehen
Dysfunktionen in Organen mit Veränderungen in bestimmten Arealen der Ohrmuschel einher,
wobei der gesamte menschliche Körper im externen Aurikel somatotop repräsentiert sei
(Nogier, 1957). Die Reizung dieser Areale, welche in Verbindung mit dem entsprechenden
Organ stünden, soll laut Nogier zur Verbesserung der Organfunktion führen bzw. Schmerz
lindern (Nogier, 1957; Usichenko & Anders, 2016). Tatsächlich konnte in zwei Studien erhöhte
Hautleitfähigkeit sowie reduzierte Gewebedichte an den von Nogier postulierten Punkten bei
Patienten mit Skelettmuskelschmerzen und koronaren Herzerkrankungen gefunden werden
(Oleson et al., 1980; Saku et al. 1993). Dennoch ist die genaue Art der Verbindung zwischen
den Arealen im Ohr und den Organen bisher nicht hinreichend in der Literatur beschrieben
worden (Usichenko & Anders, 2016; Usichenko et al., 2017b).
In randomisierten kontrollierten Studien konnte gezeigt werden, dass die
Ohrakupunktur im Vergleich mit Placebo- und Sham-Akupunktur insbesondere bei der
Behandlung von Schmerzen effektiv ist (Asher et al., 2010; Usichenko & Anders, 2016). Dabei
gibt es Befunde zur Senkung des post- und peri-operativen Opiod-Bedarfs sowie zur Linderung
akuter und chronische Schmerzen durch Ohrakupunktur (Asher et al., 2010). Als ein den
Effekten zugrundeliegender Wirkmechanismus kommt dabei die mechanische Stimulation
vagaler Nervenenden in der Ohrmuschel in Betracht. Zur Überprüfung dieser Hypothese haben
wir die Daten aus der bis zu diesem Zeitpunkt einzigen Meta-Analyse von ausreichender
wissenschaftlicher Qualität, veröffentlicht von Asher et al. (2010), hinsichtlich der
Innervationsgebiete der verwendeten Stimulationsorte analysiert (Usichenko et al., 2017b;
Manuskript 1). In die Meta-Analyse von Asher et al. (2010) wurden 17 randomisierte,
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kontrollierte Studien über die Effekte von Ohrakupunktur zur Behandlung von akuten und
chronischen Schmerzen einbezogen (Asher et al., 2010).
Wir extrahierten aus dem Artikel von Asher et al. (2010) sowie den originalen Arbeiten
die spezifischen Ohrakupunktur-Punkte, welche zur Behandlung von Schmerzen untersucht
wurden sowie die verwendeten Orte der Kontroll-Stimulationen. Abbildung 1 zeigt die
Lokalisation der verwendeten Akupunktur- und Kontroll-Punkte auf der schematischen
Zeichnung einer Ohrmuschel sowie die Innervationsgebiete der Ohrmuschel nach Peuker und
Filler (Peuker & Filler, 2002).
Unsere Analyse ergab, dass mit 15 von 20 Akupunktur-Punkten Areale stimuliert wurden,
die entweder exklusiv von Vagusnerv-Afferenzen oder sowohl von kranialen als auch von
zervikalen Nerven innerviert waren. Sham-Stimulationen hingegen erfolgten an der Helix oder
den Ohrläppchen, welche zervikal innerviert sind.
Demnach erscheint es möglich, dass Ohrakupunktur als mechanische Form der tVNS
betrachtet werden kann, wobei der Wirkmechanismus auf der Stimulation kranialer Nerven und
assoziierter Hirnareale beruht.
Abbildung 1: In den Studien verwendete Ohrakupunkturpunkte (schwarze Kreise), Sham-Stimulationspunkte (Dreiecke) und Innervationsgebiete der Ohrmuschel durch Vagusnerv (VN), Plexus Cervicale (PC) und Trigeminusnerv (TN).
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3.2 Die Wirkung aurikularer elektrischer tVNS auf die Schmerzwahrnehmung
Hinsichtlich der anti-nozizeptiven Effekte der tVNS wird angenommen, dass die
Stimulation der Vagusnerv-Afferenzen, durch deren Verbindungen zu den oben beschriebenen
Hirnarealen, zu einer Aktivierung des sogenannten endogenen absteigenden schmerz-
hemmenden Systems führt (Ossipov et al. 2010; Frangos et al., 2017; Usichenko et al., 2017a;
Usichenko et al., 2017b). Dieses beruht auf der Freisetzung von Monoaminen sowie einer „top-
down“ Inhibition der Weiterleitung nozizeptiver Reize auf Spinal-Ebene (Ossipov et al., 2010).
Elektrische tVNS erwies sich in klinischen Studien insbesondere bei der Behandlung
von Migräne und Cluster Kopfschmerz als wirksam (Silberstein et al, 2016a; Silberstein et al,
2016b; Frangos et al., 2017). Experimentelle Studien konnten zeigen, dass die elektrische
Stimulation der Conchae, Triangular fossa und des Tragus im Vergleich zur Stimulation der
Helix and Scapha des Ohrs Schmerzschwellen erhöhen bzw. wahrgenommene
Schmerzintensität während tonischer Schmerzstimulation senken konnte (Oliveri et al. 1985;
Simmons et al., 1991; Busch et al., 2013). Im Gegensatz dazu fanden Johnson et al. (1991)
keine Effekte von aurikularer elektrischer Stimulation auf die (elektrischen) Schmerzschwellen
(Johnson, 1991). Laqua et al. (2014) und Usichenko et al. (2017) fanden nur bei einem Teil der
untersuchten Probanden erhöhte Hitze-Schmerzschwellen unter tVNS (Laqua et al., 2014;
Usichenko et al., 2017a). Die inkonsistente Befundlage könnte auch dadurch begünstigt
werden, dass es herkömmlichen experimentellen Schmerz induzierenden Methoden oftmals an
Sensitivität und externer Validität mangelt, um moderate analgetische Effekte nachzuweisen
(Staahl et al., 2004).
Um klinische (chronische) Schmerzerfahrungen experimentell valider imitieren zu
können, ist die Untersuchung der Wahrnehmung von wiederholten schmerzhaften Reizen
besonders geeignet (Herrero, et al., 2000; Staahl et al., 2004). Wiederholte schmerzhafte
Reizung kann zu einer zunehmenden Schmerzintensität bei gleicher Reizstärke führen; ein
Phänomen, welches als temporale Summation von Schmerz bekannt ist (Herrero et al., 2000;
Granot et al., 2002). Temporale Summation von Schmerz gilt als perzeptives Korrelat zentraler
Sensibilisierung (Herrero et al., 2000; Granot et al. 2002; Anderson et al., 2013). Letztere ist
eine Voraussetzung für die Entwicklung chronischer Schmerzen (Herrero et al., 2000; Granot
et al. 2002; Anderson et al., 2013). Die experimentelle Untersuchung von zentraler
Sensibilisierung und dem Einfluss von hypo-/analgetischen Interventionen auf diese Prozesse
kann neue Aufschlüsse über biologische Schmerzmechanismen sowie die Behandlung von
chronischen Schmerzen geben. Herkömmliche Methoden zur Erzeugung dieses Phänomens
fallen jedoch durch große Variabilität hinsichtlich des Auftretens der temporalen Summation
von Schmerz auf (Anderson et al., 2013). Im vergangenen Jahr haben wir deshalb ein neues
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Paradigma entwickelt und in einer methodologischen Studie getestet. Mit unserer Methode
gelingt es uns zuverlässig temporale Summation von Schmerz zu erzeugen, während wir eine
Erhöhung der externen Validität gegenüber anderen Schmerzmodellen erreichten (Möller et al.,
2017).
Um weiteres Verständnis über die Wirksamkeit und die Wirkungsweisen transkutaner
Vagusnervstimulation zu erlangen, untersuchten wir in einer randomisierten kontrollierten
Crossover-Studie den Einfluss von elektrischer tVNS auf die Wahrnehmung von wiederholten
schmerzhaften Hitze-Reizen bei gesunden Probanden und Probandinnen. Dabei überprüften
wir, ob die wahrgenommene Intensität von wiederholten schmerzhaften Hitze-Reizen unter
dem Einfluss von tVNS geringer ist verglichen mit dem Einfluss von Sham- und Placebo-
Stimulation sowie der Schmerzintensität ohne Intervention. Darüber hinaus untersuchten wir,
ob tVNS das Ausmaß von temporaler Summation von Schmerz durch wiederholte schmerzhafte
Hitze-Reizung im Vergleich mit Sham- oder Placebo-Stimulation sowie ohne Intervention
reduziert (Janner et al., 2018; Manuskript 2).
49 Probanden (25 Frauen) nahmen dafür an vier Untersuchungen teil, während derer jeweils
90 pulsierende Hitze-Reize mit Schmerztoleranztemperatur mittels einer Thermode (CHEPS,
Medoc Advanced Medical Systems, Ramat Yishai, Israel) am Unterarm appliziert wurden. Die
Probanden bewerteten die Schmerzintensität des ersten und jeden 10. Reizes auf einer
numerischen Skala (0=keine Empfindung bis 100=unerträglicher Schmerz). In der ersten
Untersuchung wurde die wahrgenommene Schmerzintensität ohne Intervention erfasst. In den
drei folgenden wurde in randomisierter Reihenfolge tVNS bzw. Placebo-Stimulation (inaktives
Gerät) in den Ohrmuscheln und Sham-Stimulation an den Ohrläppchen für 20 min vor sowie
während der Hitze-Reizungen durchgeführt (Janner et al., 2018; Abbildung 2A).
Die wahrgenommene Schmerzintensität wurde durch alle Interventionen im Vergleich mit den
Werten ohne Intervention signifikant reduziert. Die hypoalgetischen Effekte von tVNS waren
in der Gesamtgruppe jedoch vergleichbar mit denen der Sham- und Placebo-Stimulationen. In
der Untergruppe der Männer war der Effekt der tVNS vor dem Beginn der temporalen
Summation des Schmerzes denen der Sham- und Placebo-Stimulation überlegen. Damit weisen
die Ergebnisse auf mögliche Geschlechterunterschiede hinsichtlich der Effekte von
Ohrstimulation hin, welche zukünftig Gegenstand systematischer Untersuchungen sein sollten.
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Wie in Abbildung 2B dargestellt war der Verlauf der wahrgenommenen
Schmerzintensitäten über die Zeit in allen Bedingungen sehr ähnlich: nach einer initialen
Abnahme des Schmerzes, nahm die Schmerzintensität mit zunehmender Stimulus-Anzahl
wieder substantiell zu (temporale Summation). Dieses Muster könnte eine adaptive Reaktion
auf die wiederholte Stimulation mit Schmerzreizen reflektieren. Ein initiales „Warnsignal“,
gekennzeichnet durch hohe wahrgenommene Reizintensität, wird dabei von Schmerz
regulierenden Prozessen gefolgt, währenddessen die Person an die Stimulation habituiert, z.B.
durch die Adaptation der peripheren Nozizeptoren und/oder eine zentrale „top-down“
Suppression der Antwortraten im primären nozizeptiven Neuron im Rückenmark, (Andrew &
Greenspan, 1999; Herrero et al., 2000). Die anhaltende Reizung könnte dann zu einer zentralen
Sensibilisierung mit zunehmender Schmerzintensität führen (Herrero et al., 2000), welche die
Person dazu drängt, der schmerzhaften Reizung entgegenzuwirken, um Verletzungen des
Gewebes zu verhindern.
Die temporale Summation des Schmerzes wurde nur in der Untergruppe der Frauen,
signifikant durch die tVNS reduziert im Vergleich mit den Werten ohne Intervention (Janner et
al., 2018). Auch dieses Ergebnis deutet auf mögliche Geschlechterunterschiede bei der Wirkung
von tVNS hin.
Die deutlichen Effekte der Kontrollbedingungen auf die Schmerzwahrnehmung könnten
aufgrund von methodologischen Besonderheiten, welche im Manuskript 2 genauer beschrieben
Abbildung 1 A: Stimulationsorte der transkutanen Vagusnervenstimulation (TVNS), aktiver Sham- bzw. inaktiver Placebo-Stimulation am Ohr. B: Veränderungen der Schmerzintensität bei wiederholter Hitze-Reizung. Daten sind dargestellt als Mittelwerte ± Standardfehler. ***p < .001 für den Haupteffekt „Bedingung“ in der Varianzanalyse.
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werden, auch auf eine unabsichtliche Stimulation vagaler Nervenenden (mechanisch durch
Druck der Elektroden oder elektrisch weitergeleitete Aktivierung benachbarter Nervenenden)
beruhen. Selbst eine unspezifische mechanische Stimulation vagaler Afferenzen könnte zur
Aktivierung bestimmter Hirnareale führen und eine veränderte Verarbeitung nozizeptiver oder
emotionaler Reize im Allgemeinen bewirken (Janner et al., 2018). Darüber hinaus ist es
möglich, dass tVNS eher die affektive und weniger die sensorische Komponente der
Schmerzwahrnehmung beeinflusst, wobei erstere in unserer Studie nicht isoliert erfasst wurde.
Für eine Beeinflussung der affektiven Komponente der Schmerzwahrnehmung durch tVNS
sprechen z.B. Befunde zu stimmungsaufhellender sowie angstreduzierender Wirkung von
elektrischer tVNS und Ohrakupunktur (Usichenko & Anders, 2016; Frangos et al., 2017).
3.3 Die Wirkung aurikularer tVNS auf Stimmung, Affekt und Kognition
Die stimmungsaufhellenden und angstlösenden Effekte von tVNS, welche sowohl bei
gesunden Probanden als auch Patienten mit depressiven Erkrankungen gefunden wurden,
werden mit einer durch tVNS aktivierten Steigerung der serotonergen und noradrenegen
Transmitterauschüttung im Locus coeruleus und den Raphe Kernen, welche durch deren enge
Verbindungen mit Strukturen des limbischen Systems maßgeblich an der Regulation
emotionaler Prozesse beteiligt sind, in Zusammenhang gebracht. Darüber hinaus wurden
Ergebnisse zum Einfluss von tVNS auf verschiedene kognitive Prozesse wie Aufmerksamkeit,
Gedächtnisleistung und Extinktionslernen veröffentlicht (Frangos et al., 2017).
Auch mechanische tVNS in Form von Ohrakupunktur erwies sich in verschiedenen
Studien als effektives Mittel zur Reduktion situationaler Angst in klinischen Settings z.B. bei
präoperativer Angst und Dentophobie (Wang et al., 2001; Karst et al., 2007; Michalek-Sauberer
et al., 2012). Darauf aufbauend untersuchten wir in einer weiteren randomisierten kontrollierten
Studie die Effektivität von Ohrakupunktur zur Reduktion von Prüfungsangst in gesunden
Medizinstudierenden (Klausenitz et al., 2016; Manuskript 3). Dafür erhielten 44
Medizinstudierende randomisiert Ohrakupunktur, Placebo-Akupunktur und keine Intervention
bevor sie drei aufeinander folgende, vergleichbare, mündliche Prüfungen absolvierten. So
genannte Ohrakupunktur-Dauernadeln wurden beidseitig an den nach WHO-Nomenklatur
bezeichneten Punkten MA-IC1, MA-TF1, MA-SC, MAAT1 und MA-TG (World Health
Organization, 1987; Abbildung 3) am Tag vor der Prüfung appliziert. Placebo-Nadeln wurden
als Kontrolle verwendet. Die Stärke der Angst wurde mittels visuellen Analogskala und State-
Anxiety Inventar vor und nach jeder Intervention sowie direkt vor der Prüfung erfasst
(KLausenitz et al., 2016). Im Vergleich mit den Werten ohne Intervention war die situationale
Angst sowohl nach der Ohrakupunktur als auch nach der Placebo-Akupunktur reduziert. Direkt
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vor der mündlichen Prüfung war die Reduktion der Angst durch die Ohrakupunktur jedoch
signifikant größer als die der Placebo-Akupunktur. Diese Ergebnisse sprechen für eine
angstlösende Wirkung der Ohrakupunktur und rücken diese als mögliche alternative
Behandlung zu nebenwirkungsreichen angstlösenden Medikamenten in den Fokus.
In einer Pilot-Studie haben wir deshalb die Methodologie der Ohrakupunktur von
Klausenitz et al. (2016) zur Reduktion von prä-operativer Angst hinsichtlich ihrer
Anwendbarkeit im klinischen Setting in Vorbereitung auf eine randomisierte kontrollierte
klinische Studie untersucht (Klausenitz et al., 2016; Wunsch et al., 2018; Manuskript 4). Dazu
wurden 32 Patientinnen vor einer ambulanten gynäkologischen Operation mit der oben
beschriebenen Ohrakupunktur-Methodik behandelt und mit 30 Patienten ohne weitere
Behandlung bezüglich des Ausmaßes an prä-operativer Angst verglichen. Während bei den
Patienten mit Ohrakupunktur die Angst vor der Operation signifikant sank, konnte in der
Vergleichsgruppe ein signifikanter Anstieg der Angst beobachtet werden. Die in dieser
Pilotstudie angewendete Methodik erwies sich als umsetzbar und vielversprechend für die
Untersuchung der Wirksamkeit von Ohrakupunktur zur Reduktion von prä-operativer Angst in
einer großen randomisierten kontrollierten Studie (Wunsch et al., 2018).
Abbildung 3: Verwendete Ohrakupunktur-Punkte bezeichnet nach WHO-Nomenklatur (schwarze Kreise) und Placebo-Stimulationspunkte (Dreiecke).
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4 Zusammenfassung und Ausblick
Thema dieser Arbeit waren die anti-nozizeptiven Effekte und möglichen
Wirkungsweisen der aurikularen transkutanen Vagusnervenstimulation. Dabei wurde die
Vagusnervenstimulation als ein möglicher Wirkmechanismus der Ohrakupunktur postuliert. Im
Rahmen unserer experimentellen Studie erwiesen sich sowohl tVNS als auch die Placebo- und
Sham-Stimulation als effektiv bei der Reduktion der Schmerzintensität, wobei die Effektstärken
der Interventionen vergleichbar waren. Weiter fanden wir Hinweise für mögliche
Geschlechterunterschiede bei der Wirkung von tVNS auf die Wahrnehmung von wiederholten
schmerzhaften Hitze-Reizen sowie die temporale Summation von Schmerz. Eine
unbeabsichtigte Stimulation vagaler Nervenenden in den Kontrollbedingungen konnte in
unserer Studie nicht ausgeschlossen werden und könnte auch einen Teil der Inkonsistenz
hinsichtlich der Befunde zur Wirksamkeit von tVNS in anderen Studien erklären. Deshalb ist
die Beachtung der methodologischen Besonderheiten bei aktiver Sham- und inaktiver Placebo-
Stimulation in zukünftigen Studien von zentraler Relevanz, um eine Verzerrung der Ergebnisse
zu verhindern. Darüber hinaus erscheint es möglich, dass die Stimulation der Vagusnerv-
Afferenzen keinen Einfluss auf die sensorischen Komponenten der neuronalen Verarbeitung
nozizeptiver Reize hat, sondern eher die affektive Verarbeitung der Schmerzreize verändert.
Diese Annahme wird unter anderem durch die Ergebnisse unserer Studien zu den Effekten von
mechanischer tVNS in Form von Ohrakupunktur zur Reduktion von Prüfungsangst sowie prä-
operativer Angst gestützt.
In zukünftigen Studien sollten demnach die schmerzlindernden Effekte von tVNS im
Vergleich zu einer aktiven Sham-Stimulation am Ohrläppchen jedoch mit sehr geringerer
Stromstärke durchgeführt werden, um eine ungewollte Stimulation kranialer Nerven zu
vermeiden. Zudem sollte in weiteren Studien darauf geachtet werden, die
Stimulationsparameter, wie z.B. den genauen Stimulationsort zu standardisieren, um die
Vergleichbarkeit zwischen den Studien zu erhöhen und möglicherweise Inkonsistenzen in den
Ergebnissen zu minimieren. Um die Stimulation zervikaler Nerven oder des Trigeminusnervs
zu vermeiden, bietet sich als Stimulationsort vor allem die Cymba Concha der Ohrmuschel an,
da diese laut Peuker und Filler (2002) ausschließlich vagal innerviert wird (Peuker & Filler,
2002). Einflüsse der tVNS auf die sensorische und affektive Komponente der
Schmerzverarbeitung sollten getrennt systematisch untersucht werden. Dabei sollten sowohl
Stichproben von gesunden Probanden als auch von Patienten mit z.B. chronischen
Schmerzerkrankungen untersucht werden, da sich Wirksamkeit und Wirkungsweisen der tVNS
in beiden Gruppen aufgrund von krankheits-assoziierten (neuronalen) Veränderungen
unterscheiden könnte.
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Zusammenfassend lässt sich feststellen, dass die Ergebnisse einer Vielzahl von Studien
darauf hindeuten, dass die Stimulation vor allem vagal-innervierter Gebiete der Ohrmuschel als
nicht-invasive Methode der Neuromodulation wirksam ist. Dennoch sind die genauen
Wirkungsweisen von tVNS bisher nicht hinreichend belegt und bedürfen weiterer
systematischer Untersuchungen. Bildgebende Verfahren und pharmakologische Blockaden
z.B. muskarinerger Rezeptoren können ferner helfen, weiteres Verständnis über die komplexen
Wirkungsweisen von tVNS zu erlangen.
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placebo-controlled investigation. Auton Neurosci 2014;185:120-2.
• Lindquist KA, Wager TD, Kober H, Bliss-Moreau E, Barrett LF. The brain basis of
emotion: a meta-analytic review. Behav Brain Sci 2012;35:121-43.
• Michalek-Sauberer A, Gusenleitner E, Gleiss A, Tepper G, Deusch E. Auricular
acupuncture effectively reduces state anxiety before dental treatment: a randomised
Clinical data suggest that electrical and acupuncture stimulation of certain areas on the external auricle can diminish acute and chronic pain.1–4 Moreover, several
experimental studies have shown increased pain thresholds during electrical stimulation applied to auricular (acupunc-ture) points situated at the concha, triangular fossa, and anti-tragus in comparison with stimulation at the helix and scapha of the auricle.5–9 It has been assumed that these hypoalgesic effects are based on a transcutaneous stimulation of cranial nerves, especially the vagus nerve,5,10,11 whose auricular affer-ent branches supply the external acoustic meatus and auricu-lar conchae.12 These afferents terminate in cerebral structures as the nucleus tractus solitarii and the locus coeruleus in the brainstem that are known to be involved in the transmission and processing of nociceptive stimulation.13–15 Moreover, pro-jections of the solitary tract and locus coeruleus also reach the ventromedial medulla, periaqueductal gray, hypothalamus, and limbic forebrain, regions that are involved in the modu-lation of arousal and pain perception.11,16
Noninvasive electrical transcutaneous vagus nerve stimulation (TVNS) has been shown to be a promising
KEY POINTS• Question: Does transcutaneous vagus nerve stimulation (TVNS) reduce the perceived inten-
sity of repetitive painful heat stimuli and temporal summation of pain processes?• Findings: Pain intensity but not temporal summation of pain decreased during TVNS, as well
as control interventions, and TVNS exerted differential effects in men and women at certain time points.
• Meaning: TVNS was indistinguishable from sham and placebo in the reduction of experimental heat pain and suggested that gender effects should be verified in appropriate investigations.
BACKGROUND: Transcutaneous vagus nerve stimulation (TVNS) is a promising treatment for acute and chronic pain. However, experimental studies yielded controversial results. We exam-ined if TVNS reduces the perceived intensity of repetitive painful heat stimulation and temporal summation of pain (TSP) in healthy volunteers in comparison with placebo and sham stimula-tion, as well as no intervention.METHODS: In 4 sessions, 90 heat pulse stimuli at individual pain tolerance temperature were applied to the ventral forearm of 49 healthy volunteers (25 women) using a Contact Heat Evoked Potential Stimulator thermode (Medoc, Ramat Yishai, Israel). Pain intensity was assessed with verbal ratings on a numeric pain scale (0–100) at every tenth heat pulse. After the first session in which pain intensities without intervention were evaluated, participants completed 3 sessions in a single-blinded randomized crossover manner: (1) sham stimulation applied at the earlobes, (2) placebo stimulation (inactive device), or (3) TVNS applied at the cymbas conchae. Primary data were analyzed using analysis of variance for repeated measures and t test for paired samples.RESULTS: Pain intensity decreased during all interventions as compared to no intervention (ηp
2 = 0.22, P < .001; mean difference TVNS versus no intervention 9.5; 95% confidence interval [CI], 3.6–15.4; P < .001). Hypoalgesic effect of TVNS was better than that of placebo and sham in men before the onset of TSP (mean differences for TVNS versus placebo 6.2; 95% CI, 0.2–12.1; TVNS versus sham 6.2; 95% CI, 0.2–12.1; P < .05). In women, TSP response under TVNS was decreased if compared to no intervention (median difference, 7.5; 95% CI, 3.5–15.0; P = .003).CONCLUSIONS: TVNS, placebo, and sham stimulation exerted comparable effects under experi-mental heat pain stimulation. Only in male participants, TVNS was superior to sham and placebo conditions in the reduction of heat pain before the onset of TSP. (Anesth Analg 2018;126:2085–92)
Effects of Electrical Transcutaneous Vagus Nerve Stimulation on the Perceived Intensity of Repetitive Painful Heat Stimuli: A Blinded Placebo- and Sham-Controlled Randomized Crossover InvestigationHenriette Janner, MSc,* Catharina Klausenitz, MD,† Nancy Gürtler, BSc,* Klaus Hahnenkamp, MD, PhD,* and Taras I. Usichenko, MD, PhD*‡
Transcutaneous Vagus Nerve Stimulation and Heat Pain
auricular stimulation treatment for acute and chronic pain conditions3,10,17; however, some experimental and clinical studies examining the pain-reducing effects of electrical auricular stimulation yielded controversial results.5,6,11,18,19 Furthermore, changes of experimental pain thresholds associated with auricular stimulation do not provide an explanatory model for the impact of auricular stimula-tion techniques on chronic pain conditions. Therefore, the examination of changes in pain perceptions over time using repetitive painful stimulation seems a more appropriate and convenient experimental method for this purpose.20 Repetitive noxious stimulation can lead to an increase of the perceived intensity of painful stimuli in humans, a phenomenon known as temporal summation of pain (TSP).21–25 TSP is suggested as a sensitive experi-mental human pain model for the measurement of mod-erate analgesic effects of nonopioid analgesic drugs or hypoalgesic neuromodulation.20 Moreover, it is claimed to be a perceptual correlate of (experimentally induced) central sensitization processes21,22,25–27 that are thought to play a crucial role in the development of chronic pain disorders.22,24,25
To find further evidence and understanding for hypo-algesic effects of electrical auricular stimulation in an experimental setting, we examined the impact of TVNS on perceived pain intensity and TSP in comparison with a pla-cebo and sham stimulation, as well as no intervention using a modified experimental heat stimulation protocol based on previously established methods.28 We expected TVNS to decrease the perceived intensity of painful stimulation and reduce TSP processes in comparison with no intervention and sham and placebo stimulation.
METHODSDesign of the Investigation and Participants’ Selection CriteriaThe local Institutional Review Board of the University Medicine of Greifswald approved the study protocol (No: BB 083/15), and written informed consent was obtained from each participant. The article adheres to the Consolidated Standards of Reporting Trials (CONSORT) guidelines.
The prospective, single-blinded, crossover, placebo- and sham-controlled experimental investigation was performed in a laboratory room of the Department of Anesthesiology of the University Hospital Greifswald from July to December 2015. Fifty-one participants were recruited via an Internet announcement according to the following eligibility criteria: healthy volunteers (no physical or psychological disorders), ages 18–45 years, American Society of Anesthesiologists physical status score of I or II, no abnormal skin conditions (infection, scars, psoriasis, eczema) at the sites of stimula-tions, abstinence from analgesics (including over-the-coun-ter medications) or psychoactive substances for at least 3 days, and abstinence from caffeine for at least 3 hours before each visit. The subjects were paid for their participation.
The study consisted of 4 sessions, with an intersession interval of at least 48 hours to prevent carry-over effects. Each session lasted about 50 minutes and was performed at the same time of day.
In the first session, volunteers were acquainted to the environment, equipment, and study procedure to minimize the influence of fear or surprise on study outcomes. During the first visit, participants were informed about the study procedure. Subjects were told that the aim of the study was to test the influence of 3 different auricular stimula-tion methods on the perception of painful heat stimuli to blind them regarding the existence of an active treatment or placebo and sham condition. The investigator performing the stimulation procedures was aware of the allocation of conditions.
All participants signed the informed consent and completed the State-Trait-Anxiety-Inventory (STAI).29 Afterward, the heat stimulation procedure, described below, was performed without any auricular stimulation.
Thereafter, each subject participated in 3 randomly ordered sessions in a crossover manner, including TVNS, placebo, or sham stimulation (Figure 1). For randomiza-tion, the stimulation sides (left and right) and interven-tional conditions (TVNS, placebo, and sham) were coded with numbers. Random sequences of these numbers were created for each participant using an online random num-ber generator.30 Thus, 6 different sequences of 3 study
Heat-stimulation
(no intervention)
Heat-stimulation +
Placebo
Heat-stimulation +
TVNS
Heat-stimulation +
Sham
Assessed for eligibility (N = 51)
Final Analysis(N = 49 )
For each participant in randomized order
Discontinuedparticipation (n=1)
Session 1 Session 3Session 2 Session 4
Discontinuedparticipation (n=1)
Excluded from analysis due to
incomplete data sets
Figure 1. Flow of the study. Each par-ticipant underwent painful heat stimula-tion procedure without intervention (no intervention, session 1) and in random-ized order with transcutaneous vagus nerve stimulation (TVNS) at the cymbas conchae, placebo (inactive stimulation device), and sham stimulation (electri-cal stimulation at the earlobes) in a crossover manner (sessions 2–4).
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interventions, which followed the first session with no intervention, were created.
Heat Stimulation ProcedureHeat stimulation was applied using a “Contact Heat Evoked Potential Stimulator” (Medoc Advanced Medical Systems, Ramat Yishai, Israel), described elsewhere in detail.11 The thermode was placed in a randomized order on the left or right ventral forearm, 45 mm below the elbow crease, and was fixed with a blood pressure cuff of 10 cm width, manually inflated to a pressure of 40 mm Hg, to enable a standardized pressure at the stimulated site throughout the investigation.
For determination of the individual stimulation tempera-ture, the contact surface of the thermode was automatically heated with a rate of 1°C/s from a baseline temperature (32°C) until participants determined their pain tolerance temperature (the temperature at which they felt intolerable pain) by pressing the button of a response unit that imme-diately cooled down the contact surface. Pain tolerances were measured with 6 trials with an interval of 3 seconds in between. The mean temperature of the last 5 trials was used as a maximum stimulation temperature for the further investigation. The first pain tolerance value was excluded due to influences of novelty and fear.31
Afterward, the stimulation side was changed to the alter-nate ventral forearm, and the thermode was fixed again as described above. Then repetitive heat pulses with a fre-quency of 0.4 Hz were applied. Within 8 pulses at the begin-ning, the pulse temperature was gently raised until the peak temperature was reached to mimic the rising skin tempera-ture seen during repetitive contacts with a preheated probe in previous studies.24 Subjects were stimulated with 90 brief heat stimuli of their individual pain tolerance temperature. For each pulse, the temperature was increased with a rate of 20°C/s from a baseline temperature (38°C), held for 800 mil-liseconds at the peak temperature, and then returned to the baseline temperature with a cooling rate of 40°C/s.
The stimulation and acquisition parameters were chosen according to methodology of previous investigations21,23,24 and our methodological investigation,28 whereas repetitive heat stimulation is the most commonly used and validated model to elicit the TSP phenomenon in humans.25
Intervention ProcedureAuricular electrical stimulation was applied bilaterally using self-manufactured electrodes, described elsewhere in detail,32 that were connected to a transcutaneous electri-cal nerve stimulation device PuntoBravo (Medizintechnik Rostock GmbH, Rostock, Germany). The electrodes’ con-tact surfaces, wrapped in wool, were moistened with 0.9% sodium chloride solution before each stimulation proce-dure, and the stimulation site was cleaned with alcohol to achieve optimal conductivity.
TVNS was applied at the cymbas conchae (Figure 2A) using electrical square impulses delivered in blocks of 9 impulses with a frequency of 100 Hz and a pulse width of 200 μseconds emitted twice per second, resulting in mixed frequency pattern of 100 Hz/2 Hz that was shown to elicit maximal analgesic effects and minimal habituation to elec-trical stimulation in animal and clinical studies.6,9,33 Current intensity was individually adjusted for each ear separately until participants described it as “clearly perceptible tin-gling sensation that was not uncomfortable or painful.”11 The electrodes were fixed using commercially available antinoise earplugs made of polyurethane foam, mull pads, adhesive tape, and an elastic headband to ensure a consis-tent contact of the electrodes with the skin.
For sham stimulation, the electrodes were attached with adhesive tape to the earlobes (Figure 2A) that receive affer-ent supply from cervical nerves,12 and the stimulation proce-dure was performed with the same parameters as described for the TVNS condition.
For the placebo condition, electrodes were attached at the cymbas conchae (Figure 2A), as described for the TVNS condition. The participants were told to choose a
Figure 2. A, Sites of bilateral auricular stimulations. Transcutaneous vagus nerve stimulation (TVNS): mixed frequency pattern of 100 Hz/2 Hz, duration: 25 min; placebo: inactive stimulation device declared as current intensity under perception threshold (duration: 25 min); sham: electrical stimulation at the earlobes (mixed frequency pattern of 100 Hz/2 Hz, duration: 25 min). B, Changes in pain intensity ratings in dif-ferent conditions. x-axis indicates number of heat stimuli (above) over time in seconds. No intervention: without any intervention ***P < .001, interventional conditions versus no intervention; main effect analysis of variance.
Transcutaneous Vagus Nerve Stimulation and Heat Pain
current intensity that was “directly under their perception threshold.” Once the current intensity was adjusted, the device was switched off without participants’ knowledge. During the adjustment of current intensity, impulses with a pulse width of 175 μseconds were applied with a fre-quency of 10 Hz.
Auricular stimulation was started 20 minutes before the repetitive heat stimulation procedure (Figure 3). Meanwhile, the participants sat comfortably in an armchair and were asked to solve conventional cognitive tasks that included questions of reasoning, numerical series, and common knowledge in the style of the German Intelligence-Structure-Test 2000 R34 to standardize participants’ behavioral states during the interventions as much as possible. The partici-pants were assured that these tasks were a method for stan-dardization of activity, and results were not the object of further investigation.
The auricular stimulation was continued during the repetitive painful heat stimulation and was terminated directly afterward (Figure 3). The total time of auricular stimulation was approximately 25 minutes.
Outcome MeasuresThe intensity of pain, reported by the participants during the repetitive heat pain stimulation, was the primary out-come measure. The subjects were asked to verbally rate the perceived intensity of pain at the first and every tenth heat stimulus, with pain tolerance temperature on a numeric pain scale ranging from 0 = no sensation to 100 = intoler-able pain, which was used and described in previous stud-ies.21,24,25,28 Every rating was immediately transcribed to a computer by the investigator, resulting in total number of 10 ratings per subject and session. TSP was calculated as the difference between the last and lowest pain intensity rating for each participant and condition.
As a secondary outcome measure, the STAI in German29 was assessed before the first session. At the beginning of each subsequent session, participants completed the state form of the STAI to quantify situational anxiety as a pos-sible confounding factor influencing the evaluation of pain-ful stimuli. Furthermore, heart rate and blood pressure were measured noninvasively using a SC 7000 patient monitor (Siemens Healthcare GmbH, Erlangen, Germany) at the beginning of each session (time point I) after completing the STAI, as well as before (time point II) and after (time point III) the repetitive painful heat stimulation (Figure 3).
After each interventional session, subjects were asked if the auricular stimulation had influenced their pain percep-tion and in what way. To test the quality of blinding, after
the last session, participants were asked which stimulation technique was most effective in influencing their pain per-ception and whether they realized that the auricular stimu-lation device was switched off during the placebo session.
Statistical AnalysisTo test our hypothesis that TVNS decreases perceived pain intensity in comparison with no intervention, placebo stim-ulation, and sham stimulation, at first 2 separate repeated measures analysis of variance (ANOVA) were performed to detect possible systematic differences among the conditions regarding pain tolerance temperature and STAI scores that could have influenced the primary outcome because the session with no intervention was not included in the ran-domization process. Afterward, primary outcome measures were analyzed using repeated measures ANOVA, with the within factors condition with 4 levels (no intervention, pla-cebo, TVNS, sham) and time with 10 levels (pain ratings), as well as the between-subject factors gender with 2 lev-els (men, women) and order with 6 levels (orders of inter-ventional applications). The gender and order levels were included to account for possible differences in perception/processing of pain between genders35 and to test for a differ-ential carry-over effect due to the nonrandomized session with no intervention. In the case of a significant interaction with a between-subject factor, subgroup analyses were per-formed. Mean ratings at certain time points of the condi-tions were compared using paired sample t test, and mean differences with 95% confidence intervals (CIs) were given as treatment effect estimates.
To test the hypothesis that TVNS reduces TSP response in comparison with no intervention, placebo stimulation, and sham stimulation, TSP responses were quantified as the difference between the last pain rating and the low-est pain rating for each individual and condition. Within a subgroup, skewed TSP responses of each condition were then compared with the Wilcoxon signed rank test, and the median of differences with 95% CIs were given as treatment effect estimates.
Data are presented as mean ± standard deviation unless otherwise stated. Two-sided P values are reported. P values of multiple pairwise comparisons in the subgroup analysis were corrected using the Holm-Bonferroni method. Data analysis was performed using the Statistical Package for the Social Sciences, Version 22.0 (SPSS, IBM Corporation, New York, NY).
Sample size calculation was performed using the pro-gram G*power 3.136 for ANOVA for repeated measures with the within factors condition (4 levels) and time (10 levels),
Heat-stimulatio n
TVNS / Placebo / Sham
10 min 2 0 min 4 min(I) (II) (III)
Start End
Figure 3. Flow of 1 study session with auricular stimulation in minutes. Assessment of State-Trait-Anxiety-Inventory at time point (I) and heart rate and blood pressure at time points (I)–(III). Rating of perceived pain intensities at the first and every tenth heat pulse during heat stimula-tion. Placebo indicates inactive device declared as under perception threshold stimulation at cymba conchae; sham: electrical stimulation at earlobes; TVNS, transcutaneous vagus nerve stimulation at cymba conchae.
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with a 2-sided level of significance of 5%, a power of 90%, and an estimated population effect size of f = 0.1, resulting in a suggested sample size of 44 subjects. Expecting a drop-out rate of 15%, the sample size was set to 51 participants.
RESULTSFifty-one participants were enrolled in the study. One woman refused to participate during the third session (pla-cebo condition) because of painful pressure in the ears after fixation of the electrodes. One male volunteer could not complete the third and fourth sessions due to a sport injury of his right arm. The data of 49 subjects (25 women), ages 24.8 ± 3.3 years, all Caucasians, were included for final anal-ysis (Table 1). Individual pain tolerance temperatures and STAI scores were comparable among the study conditions.
All interventional conditions reduced pain intensity in comparison with no intervention (main effect for condition
with F3,111 = 10.7; P < .001; ηp2 = 0.22), whereas Bonferroni-
adjusted pairwise comparisons showed comparable hypo-algesic effects, if compared to no intervention, for TVNS (mean difference 9.5; 95% CI, 3.6–15.4; P < .001), placebo (mean difference 9.1; 95% CI, 3.3–14.9; P = .001), and sham condition (mean difference 10.1; 95% CI, 4.3–16.0; P < .001). After an initial decrease of pain intensity (from the beginning of stimulation to approximately 93 seconds) (Figure 2B), the participants reacted with an increase of pain intensity (temporal summation [TSP]; main effect for factor time with F2.7100 = 20.8; P < .001; ηp
2= 0.36) during all 4 condi-tions. There was no effect of order on the primary outcome. Possible differences in responses between male and female subjects were indicated by small interaction effects of time and gender (F2.7100 = 5.3; P = .003; ηp
2 = 0.13) and condition and gender (F3,111 = 2.9; P = .04; ηp
2 = 0.07). Therefore, fur-ther subgroup analyses for male and female subjects were performed.
In male participants, pain intensity during TVNS at pulse 20 (after 70 seconds) was lower than during no intervention (mean difference 8.9; 95% CI, 0.5–17.2; t23 = 2.2; P = .039) and sham intervention (mean difference 5.2; 95% CI, 1.4–9.0; t23 = 2.8; P = .02). At pulse 30 (after 95 seconds), pain inten-sity under TVNS was lower in comparison with no inter-vention (mean difference 10.7; 95% CI, 3.1–18.3; t23 = 2.9; P = .024) and sham (mean difference 6.2; 95% CI, 1.8–10.5; t23 = 2.9; P = .027), as well as placebo condition (mean differ-ence 6.2; 95% CI, 0.2–12.1; t23 = 2.2; P = .04) (Figure 4). Other pain ratings under TVNS, sham stimulation, and placebo stimulation were comparable. In women, pain intensities under TVNS, placebo stimulation, and no intervention were also comparable.
Abbreviation: STAI, State-Trait-Anxiety-Inventory.aData are presented as mean ± SD.bSTAI ranges from 20 to 80 points and quantifies anxiety as a state in a specific situation and as a general personal trait.
35
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A B
Figure 4. Changes in pain intensity ratings in different conditions for the male (A) and female (B) subgroups. x-axis indicates number of heat stimuli (above) over time in seconds. No intervention: without any intervention; transcutaneous vagus nerve stimulation (TVNS): with TVNS; sham: with electrical stimulation at the earlobes; placebo: with inactive stimulation device. *P < .05, Student t test revealed significantly reduced pain ratings at pulse 30 under TVNS compared with no intervention, placebo, and sham condition in the male but not in the female subgroup.
Transcutaneous Vagus Nerve Stimulation and Heat Pain
TSP responses were comparable among study condi-tions in male participants. In contrast, in women, TSP was decreased under TVNS (median difference = 7.5; 95% CI, 3.5–15.0; Z = 3.18; P = .003) and placebo condition (median difference = 8.0; 95% CI, 2.5–16.5; Z = 2.7; P = .012) (Table 2) in comparison with no intervention. However, no signifi-cant differences in TSP between TVNS, placebo, and sham condition were found.
Hemodynamic parameters were comparable among all conditions. No correlations between state or trait anxiety and TSP were found. None of the participants was aware that the device had been inactive in the placebo condi-tion, and there were no differences among the participants regarding their beliefs about the most effective intervention (χ2 = 3.6; P = .17), which indicates a successful blinding.
DISCUSSIONIn this randomized controlled single-blinded prospective crossover investigation, the effects of TVNS on the percep-tion of repetitive painful heat stimuli in comparison with no intervention, placebo stimulation, and sham stimulation were examined. TVNS and sham and placebo stimulation reduced the perceived intensity of the repetitive pain-ful stimulation about 15% if compared to no intervention. Busch et al5 also found reductions in pain ratings during the application of tonic heat for 5 minutes under TVNS and under sham stimulation if compared to baseline responses; in contrast, only the reduction under TVNS was signifi-cant in comparison with baseline (about 25%) and sham stimulation.
The pattern of pain intensity changes was similar in all conditions: after an initial decrease of pain, which is in line with findings of habituation of (first pain) experiences after repetitive stimulation,37 pain reincreased with a rising num-ber of applied heat stimuli. This increase indicates temporal summation processes that could be interpreted as an experi-mentally induced form of central sensitization.22–25,38
Furthermore, our results raise questions about possible differences in the impact of auricular stimulation in male and female subjects. In the male subgroup, TVNS was found to be more effective than placebo and sham stimula-tion, with a reduction of about 20% in perceived pain inten-sity compared to no intervention before the beginning of the
TSP. In women, no such difference was revealed, and the greatest pain-reducing effects were found under sham con-dition at all time points (Figure 4). However, TVNS reduced the TSP response substantially if compared to no interven-tion in female subjects.
Nahman-Averbuch et al39 demonstrated that higher para-sympathic activity was associated with higher efficiency of pain modulation in male but not in female subjects. The authors concluded that pain treatments that affect para-sympathetic activity (eg, via stimulation of the vagus nerve) may be more effective in male than in female subjects.39 Our findings could support this hypothesis. However, to the best of our knowledge, sex differences regarding the impact of TVNS on pain processing/modulation were not system-atically examined or discussed in previous studies.5,6,11,18
The interpretation of the data is limited due to several reasons: (1) our study was not a priori designed to detect gender differences in responses, and statistical analyses regarding these differences are underpowered; (2) the ses-sion without intervention was not randomized like the sub-sequent conditions; (3) despite the absence of the order effect on primary outcome, a potential differential carry-over effect cannot be completely excluded because the statistical analysis for this purpose was underpowered. Furthermore, even if the results of the STAI as a measurement instrument of situational anxiety did not indicate differences between anxiety levels among the conditions, influences of novelty or other confounding factors on the ratings of pain inten-sity cannot be entirely excluded. However, in contrast to the findings of Granot et al,21 neither state nor trait anxiety was associated with TSP responses in our study; (4) the fixation of electrodes for placebo condition exerted a mechanical pressure, which could have stimulated vagal nerve endings. Therefore, the substantial effects found in this condition may not only be based on a placebo effect. It would explain why perceived pain intensity was reduced during placebo condition without the clearly perceptible electrical stimula-tion, as in the TVNS and sham condition, which could have served as a distraction from the painful stimulation. Thus, our placebo condition might not have been a valid method for comparison that allows clear conclusions. (5) Because some participants reported the impression of their whole ear being electrically stimulated even in the sham condition, the intensity of current applied at the earlobes could have led to widespread neural activation, including cervical and cranial nerves. Regarding these uncertainties, at this point, we can-not differentiate whether or in which amount the reported hypoalgesic effects are based on distraction, expectations/beliefs of effectiveness, or genuine activation of vagal affer-ent fibers. Because the afferent fibers of the auricular branch of the vagal nerve (ABVN) terminate in the nuclei of the soli-tary tract, stimulation applied to ABVN could have led to an activation of the descending pain modulatory circuits.16 Indeed, the ascribed brainstem structures (eg, the nuclei of the solitary tract, which project to locus coeruleus [norad-renergic] and raphe [serotonergic] nuclei) are suggested to be part of the descending pain inhibitory system, exerting their antinociceptive effects through the (1) central release of monoamines, and (2) “top-down” inhibition of nociceptive transmission at the level of the spinal cord.16 The latter mech-anism may explain the expected antinociceptive effect of
Table 2. Temporal Summation of Pain Responses in Each Conditiona
Shamf 12 (5–25) 15 (9.25–25) 10 (5–25)aTemporal summation of pain, defined as difference between the last and the lowest pain rating on a numeric rating scale (0–100) during repetitive pain stimulation. Data are presented as median (interquartile range).bTVNS: transcutaneous vagus nerve stimulation at cymbas conchae.cP = .003 for comparison of TVNS versus no intervention; Holm-Bonferroni–adjusted Wilcoxon test.dPlacebo: inactive stimulation device.eP = .012 for comparison of placebo versus no intervention; Holm-Bonferroni–adjusted Wilcoxon test.fSham: electrical stimulation at earlobes.
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TVNS on TSP, which represents a perceptual correlate of the “wind-up” phenomenon (facilitation of neuronal responses in the dorsal horn of the spinal cord under direct repetitive stimulation of afferent unmyelinated C-fibers in experimen-tal animals).22,23,26 Moreover, in patients with chronic pain conditions, where descending pain inhibition may be weak-ened and descending pain facilitation enhanced,16 TVNS might reactivate the pain inhibitory pathways. This sug-gestion is supported by positive results from clinical stud-ies on auricular stimulation in patients with chronic pain and by findings from functional neuroimaging of TVNS effects, where decreased activation in limbic structures and hypothalamus (involved in regulation of emotional and autonomic components in cerebral processing of pain) was demonstrated.11,14,16 Because patients with chronic pain dem-onstrate enhanced TSP in comparison with healthy volun-teers,40 we expect TVNS to exert more pronounced effects in these patients, thus explaining the clinically relevant find-ings of auricular stimulation described previously in clini-cal trials.1–4,17 However, an involuntary stimulation of ABVN endings in sham and placebo conditions might be 1 explana-tion for controversial results in studies examining the pain-reducing effects of electrical auricular.5,6,11,18,19
However, this study also has some clear strengths because it was performed with a crossover design with 2 control conditions, whereas all interventional conditions and stimulation sides were randomized to prevent sys-tematic influences of the application order on primary out-comes. Possible confounding factors such as time of day and caffeine intake were controlled by standardization. Moreover, we used a different, complementing experi-mental heat stimulation paradigm developed to examine changes in perceived pain over time by applying a large number of pulsating heat stimuli, which results in a greater external validity. Because the course of changes in pain rat-ings was stable in all sessions, the method was shown to reliably induce TSP.
In future studies, the pain-reducing effects of TVNS should be examined in comparison with an active sham stimulation applied at the earlobes with lower current inten-sities, which is not likely to produce widespread neural acti-vation. Moreover, because verbal ratings can be biased due to an interaction with the investigator, subjects should rate their perceived pain on a continuous measurement device such as a computerized visual analog scale with a sliding controller. Intensity and unpleasantness of pain experience should be assessed. Female participants should be tested in standardized periods of their menstrual cycles to control influences of hormonal fluctuations.35 Equally, heart rate variability and skin conductance as indicators of activities in the autonomous system should be assessed in all partici-pants. An investigator who is not aware of the intervention allocation should analyze the data. Future studies should reveal the optimal parameters of TVNS that will exert the most specific effects on the parasympathetic nervous sys-tem and central pain inhibitory pathways. Finally, using improved methodology, the underlying effects of TVNS with regard to possible influences of gender and TVNS effects on TSP in patients with chronic pain conditions should be investigated.
CONCLUSIONSTVNS and sham and placebo stimulation exerted compa-rable effects in reducing the perceived pain intensity of repetitive painful heat stimulation. Evidence for the supe-rior effectiveness of TVNS in comparison with sham and placebo stimulation could only be found in male subjects before the beginning of TSP processes. These findings could be biased by involuntary stimulation of vagal nerve endings in the sham and placebo conditions. E
ACKNOWLEDGMENTSThe authors thank all volunteers for their participation; Dr James Paul, Department of Anesthesia, McMaster University, Canada, for carefully rechecking the article for spelling and grammar; and Mrs Nadin Möller, Department of Anesthesiology, University Medicine of Greifswald, Germany, for her support during the preparation of the study.
DISCLOSURESName: Henriette Janner, MSc.Contribution: This author helped with study design and concep-tion, data acquisition, data analysis, interpretation of data, drafting the article, and final approval of the article.Name: Catharina Klausenitz, MD.Contribution: This author helped with study design, interpretation of data, revising the article, and final approval of the article.Name: Nancy Gürtler, BSc.Contribution: This author helped with data acquisition, analysis of data, drafting the article, and final approval of the article.Name: Klaus Hahnenkamp, MD, PhD.Contribution: This author helped with study design and concep-tion, revising the article, and final approval of the article.Name: Taras I. Usichenko, MD, PhD.Contribution: This author helped with study design and concep-tion, data analysis, interpretation of the data, drafting the article, and final approval of the article.This manuscript was handled by: Honorio T. Benzon, MD.
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Auricular Acupuncture for Exam Anxiety in Medical Students-A Randomized
Crossover Investigation
Catharina Klausenitz, Henriette Janner (geb. Hacker), Thomas Hesse, Thomas
Kohlmann, Karlhans Endlich, Klaus Hahnenkamp & Taras Usichenko
Veröffentlicht in Plos One 2016
CK, KH und TU konzipierten das Studiendesign. CK, TH, KHE und TU erhoben die Daten.
HJ, TK und TU analysierten die Daten. CK, KH und TU erstellten den ersten Entwurf des
Manuskripts. Alle Autoren waren an der Interpretation der Daten und Revision des Manuskripts
beteiligt.
RESEARCH ARTICLE
Auricular Acupuncture for Exam Anxiety inMedical Students—A Randomized CrossoverInvestigationCatharina Klausenitz1,2, Henriette Hacker1, Thomas Hesse1, Thomas Kohlmann3,Karlhans Endlich4, Klaus Hahnenkamp1, Taras Usichenko1,5
1 Department of Anesthesiology, University Medicine of Greifswald, Greifswald, Germany, 2 Institute ofDiagnostic Radiology and Neuroradiology, University Medicine of Greifswald, Greifswald, Germany,3 Institute of Community Medicine, University Medicine of Greifswald, Greifswald, Germany, 4 Institute ofAnatomy, University Medicine of Greifswald, Greifswald, Germany, 5 Department of Anesthesia, McMasterUniversity, Hamilton, Canada
Auricular acupuncture (AA) is effective in the treatment of preoperative anxiety. The aim
was to investigate whether AA can reduce exam anxiety as compared to placebo and no
intervention. Forty-four medical students were randomized to receive AA, placebo, or no
intervention in a crossover manner and subsequently completed three comparable oral
anatomy exams with an interval of 1 month between the exams/interventions. AA was
applied using indwelling fixed needles bilaterally at points MA-IC1, MA-TF1, MA-SC, MA-
AT1 and MA-TG one day prior to each exam. Placebo needles were used as control. Levels
of anxiety were measured using a visual analogue scale before and after each intervention
as well as before each exam. Additional measures included the State-Trait-Anxiety Inven-
tory, duration of sleep at night, blood pressure, heart rate and the extent of participant blind-
ing. All included participants finished the study. Anxiety levels were reduced after AA and
placebo intervention compared to baseline and the no intervention condition (p � 0.003). AA
was better at reducing anxiety than placebo in the evening before the exam (p = 0.018). Par-
ticipants were able to distinguish between AA and placebo intervention. Both AA and pla-
cebo interventions reduced exam anxiety in medical students. The superiority of AA over
placebo may be due to insufficient blinding of participants.
Introduction
Exam (or test) anxiety is a type of situational anxiety and is reported to be a common problemamong university students [1,2]. Exam anxiety often leads to undesirable physiological andmental symptoms and may negatively influence academic performance [2,3]. Various mind-fulness-based behavioral interventions have been shown to be effective in reducing exam anxi-ety and stress among university students [4,5]. Some of these methods (e.g. expressive writing)have even been claimed to improve academic performance [6]. However, all of these methods
PLOS ONE | DOI:10.1371/journal.pone.0168338 December 29, 2016 1 / 9
Citation: Klausenitz C, Hacker H, Hesse T,Kohlmann T, Endlich K, Hahnenkamp K, et al.(2016) Auricular Acupuncture for Exam Anxiety inMedical Students—A Randomized CrossoverInvestigation. PLoS ONE 11(12): e0168338.doi:10.1371/journal.pone.0168338
Editor: Hong-Liang Zhang, National NaturalScience Foundation of China, CHINA
Received: September 30, 2016
Accepted: November 24, 2016
Published: December 29, 2016
Copyright: � 2016 Klausenitz et al. This is an openaccess article distributed under the terms of theCreative Commons Attribution License, whichpermits unrestricted use, distribution, andreproduction in any medium, provided the originalauthor and source are credited.
Data Availability Statement: All relevant data arewithin the paper and its Supporting Informationfiles. The raw data is given in SPSS table as S3 File.
Funding: The authors received no specific fundingfor this work.
Competing Interests: The authors have declaredthat no competing interests exist.
are time consuming, which makes the routine use of cognitive and behavioural interventionsin the treatment of anxiety immediately before an upcoming exam difficult [4,5].
Auricular acupuncture (AA) is a complementary medicine technique, which is physiologi-cally based on the mechanical stimulation of cranial nerves [7]. AA has already successfullybeen used to treat situational anxiety in clinical settings, such as dental and preoperative anxi-ety [8–10]. For exam anxiety, AA was studied in only one prospective observational study inmedical students without a control group [11]. Since this study did not provide sufficientinformation for further research, we tested the methodology of the AA intervention as well asthe outcome assessment in treatment of pre-exam anxiety using a preliminary pilot investiga-tion [12]. This pilot investigation informed the final study design and provided data to calcu-late the sample size for a subsequent randomized controlled study.
The aim of the present study was to investigate whether AA can reduce exam anxiety inmedical students in comparison with placebo and no intervention conditions in a randomizedcrossover investigation.
Methods
Study design and randomization
This prospective randomized, placebo controlled, single blinded crossover trial was performedbetween April and July 2012 at the University of Greifswald, Germany. The participants wererecruited via announcement in March 2012 before the first anatomy exam in April accordingto the following eligibility criteria: undergraduate medical students in their first year of studywith no fundamental knowledge about and experiences with acupuncture, undergoing threeoral anatomy exams within one month, without any history of alcohol abuse or use of opioidor psychotropic medication and with an American Society of Anesthesiologists physical statusscore of I-II. None of the students were taking any medications or recreational drugs at thetime of the study and all of them were paid for their participation. The follow-up was finishedon the day of the last anatomy exam in July 2012.
The research project was approved by the Institutional Ethics Committee of the UniversityMedicine of Greifswald (reference no. BB 49/12). The trial was registered at clinicaltrials.gov(registration number NCT02920164) after the enrollment of the participants was started sinceinitially the authors regarded the project as an experimental investigation. The authors con-firm that all ongoing and related trials for this drug/intervention are registered. The writteninformed consent was obtained from each participant after the nature of the study procedureswas explained. As all students took three comparable anatomy exams with an interval of onemonth, each of them was randomly assigned to the AA, placebo or no intervention conditionat the evening prior to a scheduled exam by drawing slips of paper with the numbers 1, 2 or 3out of a hat. Each number corresponded to an intervention, as determined a priori: 1 = AA,2 = placebo, 3 = no intervention before the first exam (Fig 1).
Before the second exam, participants were randomly assigned to one of the two remainingconditions by flipping a coin. The investigator, who performed the randomization, ensuredthat the participants could not have been randomized again to the condition they had before.Before the last exam, no further randomization was necessary. The investigator informed theacupuncturist about the assignment of the next participant immediately after the randomiza-tion procedure and prior to any intervention.
Study interventions
A licensed acupuncturist with more than five years of experience with this technique appliedAA at the acupuncture points MA-IC1 (Lung), MA-TF1 (ear Shenmen), MA-SC (Kidney),
Auricular Acupuncture for Exam Anxiety
PLOS ONE | DOI:10.1371/journal.pone.0168338 December 29, 2016 2 / 9
MA-AT1 (Subcortex) and MA-TG (Adrenal gland) bilaterally according to the methodology,which was previously described in detail elsewhere [12]. Indwelling fixed ’New Pyonex’ nee-dles (length 1.5mm, diameter 0.22mm; Seirin Corp, Shizuoka City, Japan) embedded in askin-colored adhesive tape were used for AA. The participants were instructed by the acupunc-turist to stimulate the auricular needles for 3–5 minutes, if they felt anxious.
For the placebo procedure, ’New Pyonex’ placebo needles were attached to five sites on thehelix of the auricle bilaterally. ’New Pyonex’ placebo needles have the same appearance as AAneedles but consist of self-adhesive tape only [13]. In order to avoid potential physiologiceffects of acupressure, the participants were not instructed to stimulate the attached ’New Pyo-nex’ placebo needles. AA and placebo needles were left in situ until the next day and wereremoved out of sight of the participants after the exam by the investigator, who was notinvolved in acupuncture procedure (Fig 2).
If the participants were assigned to the no intervention condition, they remained seated inthe examination room for 10 to 15 min, which is the same amount of time an application ofthe needles would have taken. During that time, the investigator conducted a conversation
Fig 1. Flow of the study. First randomization (R1) was performed by drawing wrapped pieces of paper with hidden numbers ranging from 1to 3 before the first exam. Second randomization (R2) was performed before the second anatomy exam by flipping a coin; no furtherrandomization was necessary before the last exam in July. R: randomization; AA: auricular acupuncture; NI: no intervention.
doi:10.1371/journal.pone.0168338.g001
Fig 2. Timeline of the investigation with endpoint measurements. Time I: baseline; time II: evening of the day before exam; time 3:immediately before the anatomy exam; time IV: after exam. Auricular acupuncture (AA) was performed in the evening before the day ofexam (time I) using indwelling fixed needles, which remained in situ and were removed after the exam (time IV). Exam anxiety wasmeasured using the German version of Spielberger’s State-Trait-Anxiety Inventory (STAI) and 100 mm visual analogue scale (VAS-100) attimes I, II and III, as was heart rate and blood pressure. Duration and quality of sleep (over the course of the preceding 1 night, 1 week and 6months) were enquired about at time III. Immediately after the exam, at time point IV, exam performance (passed or failed) and the quality ofparticipants’ blinding were recorded.
doi:10.1371/journal.pone.0168338.g002
Auricular Acupuncture for Exam Anxiety
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with the participants about leisure activities, place of birth, and opinions on the study facility,thereby, avoiding the topic of the upcoming exam.
For blinding purposes, participants were told that the study’s aim was to investigate two dif-ferent combinations of AA points as treatment methods for pre-exam anxiety in comparisonwith no intervention. Participants had no knowledge which condition they had been random-ized to, except for the no intervention condition.
Outcome measures
Pre-exam anxiety was measured in the evening prior to the exam; before the intervention(Time I); after the intervention (Time II); and immediately before the exam (Time III, Fig 2)using a 100 mm visual analogue scale (VAS-100; from 0 = no anxiety to 100 = maximum imag-inable anxiety) as primary outcome. Additionally state and trait anxiety levels assessed withthe German version of Spielberger’s State-Trait-Anxiety Inventory (STAI; ranging from20 = no anxiety to 80 = maximum imaginable anxiety; [14]) were registered at all three timepoints. In the morning of the exam the participants were asked to fill out a questionnaireabout the quality (6-point-scale ranging from 1 = excellent sleep to 6 = no sleep at all) andduration of sleep the night before as well as the duration of sleep in the preceding i) week andii) in the previous six months. Blood pressure and heart rate were measured before and aftereach intervention as well as before and after each exam (Time I-IV, Fig 2). Immediately afterthe exam, at Time IV, exam performance (passed or failed) and the quality of participantblinding were recorded.
Statistical analysis
The sample size was calculated based on a prospective pilot study [12] by determining the two-sided level of significance at 0.015 (three-period crossover investigation) and power at 85% fora paired sample t-test. Expecting to find a 25% difference in anxiety level between the differentstudy conditions and using the mean and standard deviation values measured in the pilotinvestigation using STAI State-anxiety, the number of participants needed was calculated to be43. Taking into account potential drop-out/withdrawal cases, the sample size was inflated to atotal of 46 volunteers.
Baseline characteristics as well as the differences between the study conditions at differenttime points were analyzed using paired sample t-tests, Holm-Bonferroni adjusted for multiplecomparisons. Fisher’s exact test was used to analyze the success of volunteer blinding. Dataanalysis was performed using IBM SPSS Statistics Software for Mac (Version 19.0.0, IBMCorp., New York, USA). All data are presented as mean (standard deviation) unless otherwisestated, two-sided Holm-Bonferroni-adjusted P-values < 0.05 were regarded as significant.
Results
46 students agreed to participate; two of them did not fulfill the inclusion criteria (Fig 1). 44students (all Caucasian, 35 females) aged 23 (3) were enrolled in the study. Four female partici-pants missed the third session because of illness (Fig 1), their data were treated as missed data.
The baseline anxiety levels (Time I) were comparable among all three trial conditions. Anx-iety levels measured with VAS 5 hours (2) after an intervention in the evening prior to theanatomy exam (Time II) decreased after AA in comparison with baseline values at Time I(mean difference (MD) = 10.5; 95% CI 5.3, 15.8; t40 = 4.0, P< 0.001, d = 0.6, 95% CI 0.3, 1.0;Table 1, Fig 3).
VAS-100 anxiety level at Time II was lower than after no intervention (MD = 13.4; 95% CI5.6, 21.1; t32 = 3.5; P = 0.003, d = 0.6) as well as lower after AA than after placebo (MD = 10.2;
Auricular Acupuncture for Exam Anxiety
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Table 1. Outcome measures of the investigation presented as mean (SD).
Passed exam, N (%) After exam 34 (81) 32 (79) 33 (84)
Statistically significant differences between 3 study conditions (in bold letters) revealed with paired sample t-tests with Holm-Bonferroni-adjustment for
multiple comparisons. VAS-100: Visual Analogue Scale 100 mm.
* P 0.05 for comparisons of auricular acupuncture (AA) vs. placebo
** P � 0.01 for comparisons of AA vs. no intervention.
doi:10.1371/journal.pone.0168338.t001
Fig 3. Exam anxiety measured using Visual Analogue Scale 100mm. Time I: baseline; time II: evening of the day before exam;time 3: immediately before the anatomy exam. Holm-Bonferroni adjusted P = 0.018 for AA vs. placebo; ** Holm-Bonferroni adjustedP = 0.003 for AA vs. no intervention at time II and *** P � 0.003 for AA vs. no intervention at time III. Data given as mean (standarderror of mean).
doi:10.1371/journal.pone.0168338.g003
Auricular Acupuncture for Exam Anxiety
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95% CI 2.7, 17.7; t40 = 2.7; P = 0.018, d = 0.4). On the morning of the exam (Time III), VAS-100 anxiety level after AA was also lower than after no intervention (MD = 12.3; 95% CI5.8,18.8; t36 = 3.9; P< 0.003, d = 0.6) and in comparison with placebo (MD = 9.65; 95% CI1.3,18.0; t39 = 2.3; P = 0.05; d = 0.4).
In line with the findings of the primary outcome, state anxiety assessed with STAI at TimeII was also reduced after AA in comparison with placebo (MD = 4.4; 95% CI 0.7, 8.1; t40 = 2.4;P = 0.021, d = 0.4) and as compared to no intervention (MD = 9.9; 95% CI 6.0,13.9; t33 = 5.1;P = 0.003; d = 0.9). State Anxiety after placebo at Time II was also lowered if compared to nointervention (MD = 5.2; 95% CI 1.2, 9.2; t36 = 2.6; P = 0.024, d = 0.4). At Time III, state anxietyafter AA (MD = 6.7; 95% CI 3.5,9.9; t37 = 4.2; P = 0.003, d = 0.7) and after placebo (MD = 5.1;95% CI 1.9,8.2; t40 = 3.3; P = 0.004, d = 0.5) was reduced as compared to no intervention. Thetrait anxiety, assessed with STAI was 44 (11), which significantly exceeds the mean found inthe norm sample for the female population aged between 15 and 29 years (36 (10); t35 = 4.1;p< 0.001, d = 0.7; 14, Table 1).
The duration and quality of sleep, blood pressure, heart rate and exam performance werecomparable among the three study conditions (Table 1 and S1 Table). Being asked after theexam about their opinion on allocation to the study condition, the participants could distin-guish between AA and placebo intervention: for the AA condition, 34 participants thoughtthat they had received acupuncture vs. 7 for the placebo condition (P< 0.001; Table 2).
Conclusion
This randomized crossover trial demonstrated that both auricular acupuncture (AA) and pla-cebo reduced exam anxiety in comparison with no intervention in medical students, whereasAA yielded stronger effects than placebo procedure.
Without any intervention, the level of anxiety, measured with both STAI and VAS-100,increased constantly before the upcoming exam (Fig 3). These results are in line with the find-ings of Brockmeyer et al. [15]. Exam anxiety decreased by up to 20% from baseline after AA incomparison to placebo procedure and no intervention. The largest effect size of AA over pla-cebo procedure and no intervention was measured using VAS-100 in the evening after theintervention on the day before the exam. The effect size observed in the present study is com-parable to the findings of our pilot investigation [12] and other previous studies of AA and sit-uational anxiety [8–11]. For example, Karst et al. [9] reported that state anxiety scoresdecreased by about 18% from baseline to after AA treatment of dental anxiety in 19 patients.Likewise, Michalek-Sauberer et al [10] demonstrated a reduction in STAI state anxiety levelsby about 15% from baseline to after AA in 61 patients for dental procedures. However, bothinvestigations failed to find the difference between verum AA and placebo procedures.
In our investigation, placebo procedure was found to reduce exam anxiety in comparisonto no intervention in medical students as well, although this effect was not as strong as thereduction of pre-exam anxiety through AA. Even if the majority of participants could
Table 2. Participants’ opinion about the allocation to study condition/intervention.
Intervention
Auricular Acupuncture Placebo
It was verum 34 (77) 7 (15)
It was placebo 6 (14) 21 (48)
Do not know 1 (2) 8 (18)
Data is presented as number of participants (%).
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distinguish between verum and placebo procedure, we are hesitant to say that the differencebetween AA and placebo occurred due to a bias of potential “unblinding” because of twoaspects: i) due to “unblinding”, the placebo effect should have disappeared, however there wassustained effect of placebo over the no intervention condition, as measured by both VAS-100and STAI (Table 1, Fig 3); ii) questioning about the opinion on the allocation to the study con-dition took place at the end of the study after the exam, meaning that the exact time of poten-tial “unblinding” is unclear. We could not determine the time of “unblinding” in this studyand this fact remains the main limitation of our investigation. Moreover, since the participantswere instructed to stimulate the needles in case they start to feel anxious only before the AAcondition, this action could not be controlled in the placebo condition and may have dimin-ished the difference between AA and placebo due to the weak physiological effect of acupres-sure applied to ’New Pyonex’ placebo needles [16]. Furthermore, on the day before the exam, 3participants did not document their anxiety levels. They reported to have forgotten about itbecause of their high stress levels immediately before the exam.
As expected, STAI trait anxiety scores did not change during the course of the investigation.This is unsurprising as they are thought to reflect a stable personal characteristic that remainsconstant over time and between events [14]. The mean value of trait anxiety in the study groupsignificantly exceeded the mean found in the general female population aged between 15 and29 years [14]. This is in agreement with previous findings about increased anxiety levels andlower self-confidence among female medical students [17] and explains the “natural” selectionof predominantly anxious females that volunteered to participate in our study.
Despite the expectations based on previous results [6, 8–12], we could not observe the bene-ficial effect of AA or placebo on quality and duration of sleep as well as exam performance inparticipants of our study. The stability of hemodynamic parameters and the absence of sideeffects and complications confirm the respective findings of previous investigations [6, 8–13].
This trial followed the CONSORT guidelines for specific requirements of acupuncture stud-ies [18,19]. The randomized crossover design and the use of a formulaic auricular acupuncture(constant pattern of cranial nerves stimulation) rather than individualized acupuncture haveminimized potential biases. The dropout rate of 9% was low.
Regarding the above-mentioned limitations of the present investigation we suggest thatfuture studies should examine larger samples to compensate for dropout rates and incompletedata. The “parallel arms” approach might be considered in order to prevent any exchange ofparticipant experiences and intra-individual “carry-over” experience, which may have contrib-uted to the potential “unblinding” in our investigation. Also the number of participants, whohave stimulated the needles by pressing, if they felt anxious, should be verified in future inves-tigations, since the stimulation (or not stimulation of the needles) might have caused the effectbias. Furthermore, the measurement of non-invasive stress biomarkers as salivary cortisol, sali-vary ċ-amylase or catecholamines in urine might strengthen the findings of any future investi-gations, giving further insights into the biological mechanisms of AA.
In order to evaluate the clinical significance of the AA effect, this technique should be com-pared with methods that are commonly used for treatment of exam anxiety, such as relaxationtechniques, biofeedback and systematic desensitization [4–6, 20–22]. Moreover, after appro-priate investigations, AA might be used to treat pre-operative anxiety in surgical patients, con-stituting serious alternative for benzodiazepines, commonly used for this purpose in clinicalpractice [9,10,23].
In conclusion, both auricular acupuncture and placebo procedure were shown to be effec-tive in reducing levels of exam anxiety in medical students. The superiority of verum AA overplacebo AA and no intervention is considered to be due to stimulation of cranial nerves, butmay have been increased in effect by insufficient participant blinding.
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Supporting Information
S1 Table. Blood pressure and heart rate during the study conditions given as mean (SD).(DOCX)
S1 File. Ethics commission application.(PDF)
S2 File. Pilot investigation.(PDF)
S3 File. Study Data.(SAV)
S4 File. Consort Checklist.(PDF)
Acknowledgments
The authors would like to thank Bianca Leutzow for her assistance in organization of thestudy, Franziska Miller for re-checking the manuscript for mistakes and the students who par-ticipated in this study.
Author Contributions
Conceptualization: CK TU.
Formal analysis: HH TK TU.
Investigation: CK TH KHE TU.
Methodology: CK HH TK TU.
Project administration: CK KH.
Resources: TK KHE KH.
Supervision: KHE KH.
Validation: HH TK.
Writing – original draft: CK KH TU.
Writing – review & editing: TH HH KHE.
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