Journal of Bodywork & Movement Therapies 24 (2020) 215e221

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Journal of Bodywork & Movement Therapies

journal homepage: www.elsevier.com/jbmt

Fascia Science and Clinical Applications

Cranial osteopathic treatment and stress-related effects on autonomic

nervous system measured by salivary markers: A pilot study
A. Abenavoli *, F. Badi , M. Barbieri , M. Bianchi , G. Biglione , C. Dealessi , M. Grandini ,
C. Lavazza , L. Mapelli , V. Milano , L. Monti , S. Seppia , M. Tresoldi , A. Maggiani
AIMO Accademia Italiana di Medicina Osteopatica, Piazzale del Santuario 7, 21047, Saronno, (VA), Italy

a r t i c l e i n f o a b s t r a c t

Article history: Background: Variations in the concentration of salivary alpha amylase (sAA) may indicate a change in the
Received 12 February 2020 autonomic nervous system functionality. In osteopathic medicine it has long been stated that the
Received in revised form osteopathic manipulative treatment (OMT) can modulate the autonomic nervous system. Studies carried
3 July 2020
out on the compression of the fourth ventricle (CV4) have shown a positive effect in reducing the
Accepted 19 July 2020
sympathetic tone. The goal of this pilot study is measuring the physiological response of the sAA levels
after CV4 technique.
Methods: 90 subjects were randomly assigned to a sham, a control or a CV4 group. Randomization
accounted for sex and score in the STAI-2 (form Y) questionnaire. Each subject completed the STAI-1
(form Y) questionnaire to evaluate the anxiety of the moment. sAA activity and saliva flow rate were
measured. Saliva was collected before, immediately after and 30 min after treatment.
Results: Within group analysis revealed that sAA activity increased significantly immediately after the
technique application only in the CV4 group (p ¼ 0,05). Between groups analysis show a significant
difference of the sAA activity in the CV4 group respect the control group (p < 0,05), but no significant
difference between CV4 and sham group (p > 0,05). The effect in the CV4 group after the intervention is
highly variable and appeared to be related to the level of stress measured with the STAI-Y1 questionnaire
(p ¼ 0,002).
Conclusions: This study shows a positive effect of the CV4 procedure on sAA activity even if not signif-
icantly different from the sham procedure, probably due to the confounding effect of stress variability
between groups.
© 2020 Elsevier Ltd. All rights reserved.

1. Introduction Central Nervous System including also the Autonomous Nervous

Osteopathy cranial manipulative medicine (OCMM), which is
part of Osteopathic Manual Medicine (OMM), assumes that the
skull manifests intrinsic mobility (King, 2012). The biological model
usually called upon to explain the various diagnostic and thera-
peutic applications of OCMM has been given the name “craniosa-
cral mechanism” or “primary respiratory mechanism” (PRM) and
includes the inherent rhythmic motility of the brain and spinal
cord, the rhythmic fluctuation of cerebrospinal fluid (CFS), the
articular mobility of cranial bones, the mobility of intracranial and
intraspinal dural membranes and the mobility of the sacrum be-
tween the ilia. CFS is considered vital for the metabolism of the
* Corresponding author.
E-mail address: alessandra.abenavoli@aimoedu.it (A. Abenavoli).
System (ANS) (Sutherland, 1939; Upledger, 1983).
To date, no evidence has been made about the actual mobility of
the cranial bone, nor to the agreement between operators in
detecting this mobility (Guillaud et al., 2016). Nonetheless, cranial
osteopathy techniques are taught and practiced by most osteo-
paths. In the field of osteopathic research, in recent years, it has
been attempted to demonstrate that, irrespective of the demon-
stration of the validity of the assumptions, these techniques have a
measurable physiological or therapeutic effect (Ja €kel and von
Hauenschild, 2011; Sandhouse et al., 2010; Hanten et al., 1999). In
this regard, cranial manipulation is thought affecting physiological
parameters mediated by Autonomic Nervous System (ANS) through
parasympathetic and sympathetic activity, like blood-flow velocity
oscillation in its low-frequency Traube-Hering-Mayer components
(Nelson et al., 2001; Sergueff et al., 2002), cardiac autonomic

https://doi.org/10.1016/j.jbmt.2020.07.017
1360-8592/© 2020 Elsevier Ltd. All rights reserved.
216 A. Abenavoli et al. / Journal of Bodywork & Movement Therapies 24 (2020) 215e221
function (Milnes and Moran, 2007; Fornari et al., 2017), sleep la-
tency (Cutler et al., 2005), cerebral tissue oxygenation (Shi et al.,
2011) and activity of the hypothalamic-pituitary-adrenocortical
axis (Fornari et al., 2017). The compression of the fourth ventricle
(CV4) is one of the more well-known procedures in the cranial
manipulation curriculum and practice. It is also one of the most
clinically tested technique and is has been postulated to be asso-
ciated with decreased sympathetic tone (Miana et al., 2013; Nelson
et al., 2006; Milnes and Moran, 2007; Cutler et al., 2005).
Salivary markers are parameters that are easy to measure,
noninvasive and useful in monitoring ANS activity (Nater and
Rohleder, 2009). Salivary alpha-amylase (sAA) is one of the major
salivary enzymes in humans and it is produced by salivary glands,
mostly in the parotid gland. It belongs to the family of glycosyl
hydrolases and its biological function is the enzymatic digestion of
macromolecules and the mucosal immunity in the oral cavity
(Proctor and Carpenter, 2007). The production of saliva occurs in
acinar cells under the control of neural stimuli and sAA is secreted
in response to autonomic nervous system activity (Van Stegeren
et al., 2006). All salivary glands are provided by cholinergic para-
sympathetic nerves: acetylcholine binds to muscarinic receptors,
evoking the secretion of saliva by acinar cells in the ends of the
ductal tree of the salivary gland. Most salivary glands also receive a
variable innervation from sympathetic nerves: noradrenaline tends
to evoke a greater release of stored proteins, mainly from acinar
cells but also ductal cells (Proctor and Carpenter, 2007). Currently
sAA has been used as a non-invasive bio marker for measuring
stress-induced activity of the sympathetic nervous system (Takai
et al. 2004, 2007; Bosch et al., 2003). In raising the levels of sAA,
in addition to stress, the role of exercise, caffeine intake and diurnal
rhythm was also acknowledged (Bishop et al. , 2006, Rohleder, N., &
Nater, 2009). Studies have shown the relationship between physi-
ological stressors and the increased response of sAA activity sug-
gesting that it can function as a useful biological marker in acute
and chronic stress studies (Schumacher et al., 2013; Nater and
Rohleder, 2009; Nater et al., 2006). In raising the levels of sAA, in
addition to stress, the role of exercise, caffeine intake and diurnal
rhythm was also acknowledged (Bishop et al. , 2006; Rohleder, N., &
Nater, 2009).
Despite these results, the relationship between alpha amylase
and activity of the two branches of the ANS is still debated. In rats,
b-adrenergic stimulation increases sAA and the block of b2 re-
ceptors decrease sAA activity (Bush et al., 2002), suggesting a direct
involvement of the sympathetic group in sAA secretion, while
parasympathetic activity has been shown to be associated with an
increase in flow rate of saliva secretion (Anderson et al., 1984). In
fact, the response of the salivary glands reflects the sum and the
interactions of sympathetic and parasympathetic activity (Asking
and Proctor, 1989; Asking, 1985) and a synergistic interaction be-
tween the two ANS groups, whereby parasympathetic activity
amplifies sympathetic effects (Bosch et al., 2011; Proctor and
Carpenter, 2007). Even if the direct correlation is not clear with
sAA and parasympathetic and sympathetic activity, these results
suggest that sAA might be regarded as an indirect indicator of ANS
activity. For this reason, it could be a good marker in treatment
studies, such as testing the effect of osteopathic treatments
affecting the ANS activity.
Therefore, the goal of this pilot study is to evaluate if the tech-
nique CV4, can influence the sAA activity or saliva flow rate, related
to the stress level of the subjects.
2. Methods
This pilot randomised single blind clinical trial with three par-
allel groups (treatment, sham and control) was performed in AIMO,
Accademia Italiana di Medicina Osteopatica, of Saronno (Italy) in
July 2016. The ethical approval was obtained from the ethics
committee of the British College of Osteopathic Medicine (BCOM).
The study was registered on May 27, 2016, with the United States
National Institutes of Health's ClinicalTrials.gov registry and
assigned ID number NCT02785796.
2.1. Participants
Students (90) from AIMO, were recruited for the trial: subjects
were first- and second-year students, aged between 20 and 25, and
they were not yet formed in the craniosacral field to avoid influ-
encing the placebo effect during the application of the sham pro-
cedure. The health certifications of all the recruited subjects were
evaluated before including them in the study to avoid presenting
psychophysical pathologies that could affect the secretion of sAA.
All subjects were made to read and sign an informed consent by
which they were instructed to refrain from eating, smoking,
drinking and exercising 2 h before the experiment (Rohleder and
Nater, 2009; Harmon et al., 2008). Oral contraceptives, assump-
tion of drugs, and pathology of the oral cavity represented exclu-
sion criteria because they could affect enzymatic secretion (Nater
and Rohleder, 2009). All volunteers completed the Y form of STAI
questionnaire (Elwood et al., 2012) to assess state anxiety (STAI-Y1)
and trait anxiety (STAI-Y2). Subjects completed the STAI-Y1 form
the treatment day to evaluate the anxiety of the moment and the
STAI-Y2 form before the treatment day. Subjects were randomly
assigned to a group with a stratified procedure (two levels, sex and
STAI-Y2 score, with an online software) and are blinded to treat-
ment (CV4 or sham). Subjects were required to sign consent forms
for inclusion in the study.
2.2. Interventions
Operators were osteopaths trained in the cranial field, with two
years of experience (low experience) or more than 6 years of
experience (high experience). The application of the CV4 and
control protocols occurred in an examination room setting.
CV4 procedure. Participant were lying supine on a treatment
table. While sitting at the head of the table, the practitioner con-
tacted the participant's occiput (lateral to the external occipital
protuberances, but medial to the occipital-mastoid suture) with his
or her thenar eminences. The technique is then performed pro-
gressively inducing the phase of cranial extension perceived in the
occipital region with a movement directed towards the operator
until a phase of immobility (Still Point) is reached as described in
King (2012). Each operator was allowed to independently decide
the duration of the technique based on his perception of the Still
Point. Sham CV-4 Procedure. The subject was supine with the
operator at the head of the treatment table with forearms resting
on table. The operator overlapped the hands so that the thumbs
formed a “V”. The operator's thenar eminences contacted the pa-
rietal bones, cranially respect to the true position used in the CV4
procedure. Once placement was achieved the operator's hands
remained motionless for 10 min without applying any pressure.
Control group. Subjects in the control group just remained sitting
quietly in the examination room for 15 min.
2.3. Saliva collection
Sampling session were limited to 10.00 a.m. to 1.00 p.m. to
minimize the effect of diurnal variation.
Methods for the collection of saliva have been described
€schl, 2008; Rohleder and Nater, 2009). We used the passive
(Gro
drool method to collect whole saliva samples, which is more valid if
 A. Abenavoli et al. / Journal of Bodywork & Movement Therapies 24 (2020) 215e221 217

the subject can be properly instructed and supervised during Table 1

collection, as done in this study (Rohleder and Nater, 2009). Par- Baseline characteristics of the three groups: means (SD) are shown. No significant
differences are present between groups.
ticipants were in the seated position and rinsed their mouth with
clean water. They collected saliva and emptied their mouths by CV4 (n ¼ 23) Sham (n ¼ 21) Control (n ¼ 28) p
spitting in a 15 ml polypropylene tube, continuously for 5 min. Gender (F) 13 10 12 0,51a
Saliva was collected before (t0), immediately after (t1) and 30 min STAI-Y1 34,04 (10,77) 34,67 (7,60) 34,67 (7,44) 0,541b
(t2) after the application of the CV4 or the sham/control procedures. STAI-Y2 42,91 (10,41) 41,67 (8,93) 44 (10,05) 0,721b

For time requirements, samples were collected in the control group a

another day (one week later) compared to the other two groups and
subjects were aware of the lack of intervention when collecting
samples. Salivary flow rate was calculated by determining the saliva
volume expressed for 5 min (ml/min). At the end of the session, all
tubes were placed at 20  C.
Immediately before performing the assays, saliva aliquots were
thawed and centrifuged for 15 min at 3000 revolutions per minute
and 4  C to pellet the mucins. The supernatant was transferred to a
fresh tube and amylase assays were performed immediately. These
assays were performed with assay kits purchased from IBL inter-
national (Hamburg, Germany) according to the manufacturer's in-
structions. The volume of saliva produced was evaluated before the
enzymatic assay immediately after the thawing procedure, with
5 ml capillary tubes.
2.4. Outcomes and data analysis
The two primary outcomes of this clinical trial are the flow rate
of saliva production and the activity of the salivary marker alpha-
amylase. Data were tested for normality with a Shapiro test
(p > 0,05). Consequently, parametric (one-way ANOVA or repeated
one-way ANOVA) or non-parametric (Kruskal-Wallis ANOVA or
Freidman repeated measure) tests were performed for between
and within subject analysis; post hoc (Duncan test) is used if
necessary. A multifactorial ANOVA was used for revealing variables
contributing to high variability observed in the response of subjects
Chi squared.
b
Kruskal-Wallis ANOVA.
to treatment. Correlation between variable was tested with a
Pearson's product moment correlation coefficient.
3. Results
90 AIMO students were recruited for this RCT and divided into
three groups (treatment, sham and control) according to a stratified
randomization. Two variables were considered for the randomi-
zation procedure: gender and STAI-Y2 score. Some subjects (n ¼ 7)
were for not following the required procedures on the day of saliva
collection (see methods). Samples not analysed (n ¼ 10) were
discarded for different reasons (blood in the sample, missed sam-
ples or saturation during the enzymatic assay; Fig. 1).
In Table 1, subjects baseline characteristics are shown: no sig-
nificant differences are present between groups. A further analysis
of initial variables shows that there is a strong correlation between
the two STAI scores (STAI-Y1 and STAI-Y2, p < 0,0001), but there is
no correlation between sAA activity and STAI scores (STAI-Y1
p ¼ 0,66; STAI-Y2 p ¼ 0,77), nor between the flow rate and STAI
scores (STAI-Y1 p ¼ 0,58; STAI-Y2 p ¼ 0,49).
Data for sAA activity and flow rate over time are shown for the
three groups in Fig. 2 (Fig. 2A for sAA and Fig. 2B for flow rate) and
in Table 2. Between subject analysis revealed an effect of the spe-
cific treatment in sAA activity (p < 0,001) before (t0), immediately

Fig. 1. CONSORT diagram for transparency in reporting trials shows the randomization of subject. Seven of the ninety subjects recruited were discarded on the day of the
experiment because they had not complied with the indications. Samples not analysed were discarded for the presence of blood in the sample, missed samples or saturation during
the enzymatic assay.
218 A. Abenavoli et al. / Journal of Bodywork & Movement Therapies 24 (2020) 215e221

Fig. 2. Trend over time (t0 - before the treatment, t1 - immediately after the treatment, t2 - 30 min after the treatment) of the two primary outcomes, sAA (panel A) and flow rate
(panel B). Single measurements are shown as circles; line represents the mean value.

Table 2 A slight increase in sAA over time is observed in all groups, and
Mean and standard deviation for sAA and flow rate in the three groups over time. the within group analysis revealed that in control (p ¼ 0,008) and in
Results of the between and within group analysis are shown.
the CV4 group (p ¼ 0,021) this increase is statistically significant
CV4 (n ¼ 23) Sham (n ¼ 21) Control (n ¼ 28) p (Table 2). Post hoc test show a significant effect at t1 respect t0 for
aamylase (U/ml) the CV4 group (p ¼ 0,05) but no significant effect at t2 (p ¼ 0,25 at t2
t0 94,02 (39,55) 79,50 (47,72) 55,73 (29,98) <0,001b respect t0; p ¼ 0,89 at t2 respect t1) and a significant effect at t2 for
t1 197,95 (229,56) 90,04 (77,79) 58,96 (30,12) <0,001b the control group, either respect t0 (p ¼ 0,01) either respect t1
t2 174,68 (146,46) 131,29 (138,63) 82,99 (68,89) 0,003b
(p ¼ 0,03). Both the placebo group and the control group did not
P 0,021a 0,348a 0,008a
Flow rate (ml/min) show a significant increase in sAA immediately after treatment
t0 0,65 (0,28) 0,66 (0,32) 0,57 (0,34) 0,984d (p ¼ 0.491; p ¼ 0.599). No effect of time is relevant for the flow rate
t1 0,74 (0,33) 0,75 (0,37) 0,73 (0,45) 0,874d (Table 2).
t2 0,78 (0,37) 0,72 (0,30) 0,77 (0,42) 0,876d
Table 3 and Fig. 3 explicitly show sAA activity differences
P 0,911c 0,400c 0,151c
(mean ± CI95%) between t1 and t0 (D1), between t2 and t1 (D2) and
a
b
Friedman test. between t2 and t0 (D3) It can be noted that D1 in CV4 group is
Kruskal Wallis.
c significantly different from zero (p ¼ 0,045) and from the variation
RM one way ANOVA.
d
One way ANOVA. in the control group (p ¼ 0,02), but not significantly different from
the variation recorded in the sham group (p ¼ 0,09), consistently
with the ANOVA analysis. Thirty minutes after the treatment ses-
after (t1) and after 30 min; the only significant difference is be- sion, the effect of CV4 application seems to vanish (D2 < 0), even if
tween the CV4 and the control group (p < 0.05) at each detection not significantly (p ¼ 0,59). In the other groups (control and sham),
time. No significant differences are observed between CV4 and a slight increase over time is clear, which is significant only in the
sham group (p > 0,05). Flow rate doesn't show any significant dif- control group in the last measurement (D2 > 0; p ¼ 0,03). The lack
ference between the three groups at any time (see Table 2). of significance between CV4 and sham group, is probably due to the

Table 3
Differences between sAA activity at different time (mean and CI95%). Only in CV4 group D1 is significantly different from zero (p ¼ 0,045). Between group analysis
demonstrated a significant group effect only for D1. Post hoc analysis shows a significant difference only between D1 in the control and in the CV4 group (p ¼ 0,02). P-values
from one-way ANOVA test.

a-amylase (U/ml)
CV4 IC (95%) Sham IC (95%) Control IC (95%) p

D1 ¼ (t1-t0) 103,93 2,52e205,35 10,54 20,84-41,92 0,87 15,49-13,76 0,03

D2 ¼ (t2-t1) 30,48 147,59e86,62 46,03 2,62e94,68 24,03 2,97-45,10 0,28
D3 ¼ (t2-t0) 82,67 22,65e142,69 52,13 7,97e112,23 27,25 5,70-48,81 0,18
 A. Abenavoli et al. / Journal of Bodywork & Movement Therapies 24 (2020) 215e221
Fig. 3. Differences in sAA measurements over time. The difference between sAA ac-
tivity measured after the treatment (t1) and before the treatment (t0) is indicated with
D1. Similarly, D2 represent the difference between the values of sAA activity 30 min
after the treatment (t2) and immediately after the treatment (t1), and D3 the difference
between t2 and t0. Mean ± CI95% are shown.
presence of a high variability in the response of subject to CV4 and
sham treatment. We test which variables could give this variability,
including experience of the operator (low experience or high
experience), group of treatment, gender, STAI-Y1 and STAI-Y2
scores. Significant factors result to be STAI-Y1 score (p ¼ 0,001),
the class of experience (p ¼ 0,01) and the treatment (p ¼ 0,05).
In Fig. 4, STAI-Y1 score is plotted related to the variation in sAA
activity before and after the treatment (D1); for each subject, class
of experience of the operator is also shown by a code colour: low-
experienced operators are represented in red, high-experienced
operators in black. The factor probably determining the response
variability seems to be the STAI-Y1 score: subjects with low values
in the score (corresponding to lower stress levels) react strongly to
treatment. Dependence on the class of experience is accidental, as
younger operators have not dealt with low-level in STAI score
subjects.
4. Discussion
This study aims to measure saliva flow rate and salivary
markers, sAA, in the context of a cranial osteopathic treatment (CV4
procedure) that is postulated to stimulate the activity of the para-
sympathetic nervous system (Miana et al., 2013; Nelson et al., 2006;
Fig. 4. Relationship between STAI Y1 and differences in sAA (D1 ¼ t1-t0) in the three groups. Red dots represent low-experienced operators, black dots high-experienced operators.
Dotted line represents an arbitrary value above which subjects seem not to respond to the treatment. . (For interpretation of the references to colour in this figure legend, the reader
is referred to the Web version of this article.)
219
Milnes and Moran, 2007; Cutler et al., 2005).
Salivary alpha-amylase activity increased over time between t0
and t2 in all groups, but only in subjects in the CV4 group the in-
crease immediately after application of the technique was signifi-
cant. The high variability of the enzymatic activity present in CV4
and sham group subjects, perhaps due to the confounding stress
factor, did not however allow to identify significant differences
between the two groups at each detection time.
A problem found in all studies using salivary markers is the high
variability of the parameter. There are several factors that may
affect the secretion of this parameter: among others, exercise, drink
and food intake, smoking, age, hours of day, medication use (Nater
and Rohleder, 2009). In this study, attempts were made to mini-
mize the contribution of factors that could generate variability,
requiring subjects to refrain from eating and drinking, up to 2 h
before taking part in the study. In addition, harvesting of saliva
samples was restricted in the morning to avoid excessive variations
due to the circadian rhythm (Rohleder and Nater, 2009; Harmon
et al., 2008). However, initial sAA measurements of our subjects
appeared very scattered.
Given the relationship between physiological stressors and the
increased response of sAA activity (Takai et al. 2004, 2007; Bosch
et al., 2003), it was also attempted to achieve group baseline ho-
mogeneity for this variable by randomizing subjects with the score
of the STAI-Y2 questionnaire, a measure of the trait anxiety. The
groups were homogeneous for this variable, but not for baseline
sAA activity. In fact, the values of the control group have an average
value of sAA activity lower than the other two groups, and statis-
tically not comparable with the intervention group. This may be
since, for schedule requirements, the control group samples were
harvested in a different day compared to other samples, and the
subject were aware about the assigned group. This could be
considered a methodological bias, because these subjects lack the
expectation present in the other two groups, and this could influ-
ence their psychological state. Despite this initial discrepancy, the
variation of the parameter immediately after treatment was only
significant in the CV4 group, and there was no significant correla-
tion between this variation and the initial value of sAA (p ¼ 0,34).
The observed variability suggests, for future studies, the use of
stratified randomization for the level of stress at the moment of
samples collection.
In our study the flow rate of the whole collected saliva does not
increase after treatment in any of the three groups (Fig. 2B). The
sAA activity, on the other hand, shows a slight increase in the three
groups (Fig. 2A). This increase is probably due to the influence of
220 A. Abenavoli et al. / Journal of Bodywork & Movement Therapies 24 (2020) 215e221
the circadian rhythm on sAA secretion. To avoid this instability, it
would have been preferable to take the samples in the afternoon
when the release curve tends to flatten (Harmon et al., 2008). The
sAA increase in subjects undergoing CV4 is however evident
immediately after treatment (Figs. 2A and 3). The difference be-
tween marker values after and before treatment (D1) is in fact
significantly different from zero only in CV4 group (Fig. 3). How-
ever, the high variability of the outcome in the CV4 group does not
make this difference statistically relevant respect to the sham
group. The variables responsible for this dispersion in the response
to CV4 treatment, as demonstrated by the ANOVA analysis, were
the level of stress measured by the STAI-Y1 questionnaire and the
operator's class of experience. Fig. 4 suggests (but the sample is not
big enough to perform an ancillary analysis) that the dependence
on the operator's experience is accidental and that it’s caused by
randomization procedure (all participants in the CV4 group treated
by the operators with low experience had a higher STAI-Y1 score).
For future studies that intend to detect the possible contribution of
the operator's experience, stratified randomization taking into ac-
count the stress level and the operator's experience is
recommended.
The sAA marker baseline level does not correlate with the stress
level measured with the score of STAI questionnaires, both Y1 and
Y2. This result was already present in literature (Takai et al., 2004).
Instead, the correlation between treatment response and stress
level of the subject is significant. This inverse proportion relation-
ship is shown in Fig. 4: subjects with low stress levels, approxi-
mately with a score lower than 30, react more to CV4 technique,
with an increase in sAA secretion. Future studies to confirm these
results should therefore take this trend into account. The data also
shows that, unlike what we would expect, the subjects that react
significantly to treatment are those with a low level of stress. The
protocol envisaged the application of a single technique: this type
of approach may not be enough, in subjects with higher stress
levels, to stimulate a consistent reaction.
Traditionally, the CV4 technique has been associated with an
increased relaxation, and several studies have attempted to
demonstrate an increase in parasympathetic nervous system ac-
tivity in response to the application of this specific technique
(Miana et al., 2013; Nelson et al., 2006; Milnes and Moran, 2007;
Cutler et al., 2005). Flow rate measurements, hypothetically
directly related to the activity of the parasympathetic nervous
system, did not detect any time-related or treatment-related ef-
fects. Although other studies have confirmed the same result (Takai
et al., 2004), this could be due to the low sensitivity of the mea-
surement. There are no studies that assess the sensitivity and val-
idity of salivary volume measurements, nor clear procedures for
measuring this parameter that can allow methodological repro-
ducibility. The mucopolysaccharides contained in the saliva make
accurate assessment of the volume difficult even with low-capacity
capillaries. It has been suggested that more cycles of freezing and
thawing may reduce the content of mucopolysaccharides. In this
study the samples have undergone only one freezing and thawing
cycle: it is advisable to increase the number of these cycles to
facilitate pipetting and to evaluate the volume. Nonetheless, for
these reasons the authors are not very confident about the results
regarding the flow rate. In literature, sAA activity is usually used to
verify the effect of stressor test, which, stimulating the sympathetic
nervous system increases sAA secretion from salivary glands
innervated by noradrenergic fibres. Few studies have specifically
attempted to measure sAA activity in humans as a marker for the
activity of the parasympathetic nervous system (Thoma et al.,
2013). The only result that seems to show a decrease in the
marker in response to a relaxing stimulus is the Takai's studio
(Takai et al., 2004) in which sAA activity diminishes in response to
the vision of a relaxing video. However, in vitro studies on animal
models seem to indicate that both the sympathetic and para-
sympathetic nervous system lead to increased sAA secretion, with
synergistic effect (Nater and Rohledere, 2009). In fact, the sAA ac-
tivity measure could not be used specifically to distinguish the
activity of the two branches of the autonomic nervous system, but
undoubtedly an increased activity, as measured in our study, may
be associated with a change in the general activity of the system. To
demonstrate an effect of osteopathic techniques on the ANS activity
through salivary markers, an approach that measures recovery
following a stressful event could be more effective (Fornari et al.,
2017).
5. Conclusion
This study shows a variation on sAA secretion as a possible effect
of CV4 cranial technique particularly in subjects with low levels of
stress suggesting a change of the autonomic nervous system ac-
tivity. However the increase in the CV4 group was not significantly
different from that in the sham group, probably due to the con-
founding effect of stress variability between groups. Further studies
are recommended to confirm these preliminary results.
CRediT authorship contribution statement
A. Abenavoli: Conceptualization, Methodology, Formal analysis,
Investigation, Writing - original draft, Writing - review & editing,
Visualization, Supervision. F. Badi: Investigation. M. Barbieri:
Investigation. M. Bianchi: Investigation. G. Biglione: Investigation.
C. Dealessi: Investigation. M. Grandini: Investigation. C. Lavazza:
Investigation. L. Mapelli: Investigation. V. Milano: Investigation. L.
Monti: Investigation. S. Seppia: Investigation. M. Tresoldi: Inves-
tigation. A. Maggiani: Conceptualization, Investigation, Writing -
original draft, Writing - review & editing, Project administration.
Declaration of competing interest
Authors have no conflict of interest or financial disclosure
relevant to the topic of the submitted manuscript.
Support: This work was supported by AIMO.
Acknowledgments
We thank Dr. Marco Ferrario for the enzymatic analysis of sali-
vary samples and Dr. Dario Truffelli for statistical assistance.
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