Increased Wound Healing Effect of 111 Hertz Sound Frequency in Male ICR Mice

Full text

Increased Wound Healing Effect of 111 Hertz Sound
Frequency in Male ICR Mice
Jake Wilson Binaday, Al Christine Gobres, Adrian Abbarientos, Katherine Diesta,
Kathleen Marquez, Rushaine Palomillo, Athena Heart Lobos, Christopher Lita
Department of Biology, College of Science, Bicol University, Legazpi City, Albay, Philippines
Abstract
The sound frequency 111 Hz is known for cell rejuvenation which is associated in cell
regeneration. The use of this frequency in wound healing has been explored. After the experiment
was conducted, we have found out that 111 Hz frequency made the wound healing process faster.
The 111 Hz frequency is known to induce the release of beta-endorphins which in turn induces
the expression of the cytokeratin 16 gene. This gene produces the protein Keratin 16 (KRT16)
which speeds up the wound healing process. Therefor the use of this sound frequency in healing
wounds could possibly be of use to increase the wound healing process. However, ANOVA test
showed that there was no significant difference between the percentage wound closure of
exposed and unexposed mice to 111 Hz.
Keywords: sound frequency, wound healing, beta-endorphins, cytokeratin 16
Introduction
Wound healing is a complex process
which involves the activity of various cell
types. This process is tightly orchestrated by
specific cytokines (Grochot-Przeczek et al.
2009). This process involves orderly but
temporarily overlaid stages: inflammation,
cell proliferation and tissue generation (Park
and Barbul 2004, Zhai et al. 2009). After
wounding, the inflammation phase occurs
immediately, characterized by hypoxia with
fibrin clot formation, as well as recruitment of
neutrophils and platelets. After 2 to 10 days,
formation of granulation tissue and new
blood vessels with the addition of the
accumulation of macrophage and fibroblasts
(Uchiyama et al. 2014).
During the process of wound healing,
many biomolecules are involved. Among
them is Beta-endorphin. It is a peptide
neurotransmitter that is mainly produced in
the central nerve system where it causes an
analgesic effect and a feeling of euphoria
after binding to opiate receptors. However,
recent studies have discovered that it also
has effects on the process of wound healing.
(Schmid and Zulle 2005)
Mice is a widely used animal for
different biological experiments. It has been
the subject for most of wound healing studies
(Wong et al. 2011). There are different
models for mouse wound. This includes
excisional, incisional, burn and fibrosis
models (Park et al. 2014). However, wound
healing in mice is different from humans
(Fang and Mustoe 2008). The reason is that
unlike humans, mice have pamiculus
carnosus and a loose skin layer which
provides them an increased contractile
capacity. These anatomical features
promote wound closure primarily by
contraction, while human wounds heal
mainly by re-epithelialization and granulation
tissue formation (Lindbald 2008).
Rejuvenation is the process of
reversing the aging process. It involves the
repair of damage that is associated with
aging or replacement of damage tissue with

new tissue, which is associated with cell
regeneration. There are a lot of studies on
the use of alternative ways of curing
diseases. One of which is the use of sound
in medicine. The sound frequency 111 Hertz
(Hz) shows cell rejuvenation property, which
we could associate with cell regeneration.
The main objective of this study is to
be able to know whether the sound
frequency 111 Hz will increase the healing
process of wounds inflicted on mice.
Methodology
Experimental Design
A total of 20 six-week old male ICR
mice were obtained from the Food and Drug
Administration. They were first acclimatized
for 4 days. After which, 10 mice were chosen
as the experimental animals. Five of those
were the control, where in these are mice
that were have wounds but were not
exposed to the sound frequency, this was
set-up one. The other five were the ones that
were wounded and exposed to the sound
frequency 111 Hz, this was set-up two.
For set-up two, speakers were placed
on top of the cage. The speakers were then
connected to an android phone where in the
Frequency Generator v.1.71 software was
used to produce the sound frequency 111
Hz. This was run for 24 hours each day.
Wound Infliction
After acclimation, the mice were
anesthetized with 0.1mL Lidocaine®. Then,
the antero-lateral portion near the thighs of
the mice were shaved and wiped with 75%
ethyl alcohol to prevent infection. A circular-
shaped wound with a diameter of 8mm
millimetres, using a heated head of a
sterilized nail, were induced in the shaved
part.
Everyday, the wounds were treated
with povidine-iodine (Betadine) for each set-
up. After 5 days, data from the wounds were
recorded. This was done by measuring the
diameter of the wounds with a ruler. This was
done by one person only to avoid inter-
observer bias.
Statistical Analysis
The approximate area of the wound
were computed using the formula for the
area of a circle:
   

Percentage wound closure (Park et
al. 2014) will be computed using the
following formula:
   

Where in:
WA0: Wound Area in Day 0
WA5: Wound Area in Day 5
Analysis of Variance (ANOVA) will be
used to test the significance of the result at
5% significance level.
Results
The use of alternative ways in curing
diseases has become more useful in the field
of medicine. Among these, is the use of
sound. In this experiment, we have proven
that sound does have an effect in the healing
process of wound.
Table 1. Diameter of wounds during the 5th
day from wound infliction
MICE
Wound Diameter (mm)
Control
111 Hertz
1
3
4.5
2
5
4.5
3
4
3.5
4
6
5.5
5
6.5
4
Mean
(SD)
4.9
(1.4)
4.4
(0.74)

Figure 1. Diameter of wounds on the 5th day
Mice that were exposed to sound
have their wounds healed faster compared to
those that were not. As seen in Table 1 and
Figure 1, most of the diameter of the wounds
of mice are smaller in those that were
exposed to 111 Hz compared to those not
exposed (control), except for Mice 1. It could
also be observed that the mean wound
diameter of all the mice that are exposed to
111 Hz is smaller than those in the control
group.
Table 2. Percentage wound closure of mice
exposed and unexposed to 111 Hz
MICE
Wound Closure %
Control
111 Hertz
1
62.5
43.75
2
37.5
43.75
3
50
56.25
4
25
31.25
5
18.75
50
Mean
(SD)
38.75
(17.90)
45
(9.27)
The amount of wound closure was
computed and showed positive results. As
seen in Table 2 and Figure 2, mice that were
exposed to 11 Hz have greater wound
closure compared to those that were not
exposed except for Mice 1. This is more
reflected in the mean where in those mice
that were exposed to 111 Hz had their
wounds closed by almost half. However,
ANOVA test showed that there is no
significant difference between exposed and
unexposed mice.
Figure 2. Percentage wound closure
Discussion
Wound healing is complex process
where the body repairs itself after injury
(Nguyen et al. 2009) involving multiple
factors (Rieger et al. 2014). These factors
include a set of complex biochemical events
that takes place in a closely orchestrated
cascade in repairing the damage
(Stadelmann et al. 1998).
The sound frequency 111 Hz has
been known to induce the release of Beta-
endorphins (Dorian 2014). This endogenous
opioid neuropeptide found in the neurons of
both the central and peripheral nervous
system. This neuropeptide was proven to
have analgesic effects, which reduces the
pain experienced during the effect. However,
studies have shown that beta-endorphins
does not only plays a role in reducing pain.
In the study of Cheng et al. (2007),
they have observed that beta-endorphins
where weakly expressed in nerve terminal at
the border of dermis and epidermis,
keratinocyte in some epidermis, and in the
fibroblast in dermis of unwounded tissues
from rats. However, the concentration of this
neuropeptide increased on tissues that are
0
1
2
3
4
5
6
7
1 2 3 4 5
WOUND DIAMETER (MM)
MICE
Control
111 Hz
0
10
20
30
40
50
60
70
1 2 3 4 5
PERCENT WOUND CLOSURE
MICE
Control
111 Hz

injured and as the wound heals, it
continuously increases. This was further
supported by the study of Bigliardi et al.
(2002; 2003; 2004), where they found
evidence that beta-endorphins stimulate the
expression of cytokeratin 16 in a dose-
dependent manner. That is, the higher the
amount of beta-endorphins the greater
cytokeratin 16 will be expressed. The
cytokeratin 16 gene, also known as KRT16
gene produces the protein Keratin 16 or K16
which is involve in the process of wound
healing (KRT16 2012).They have also
showed that beta-endorphins are involved
not only in tissue regeneration but also in the
final reepethilialization of wounded tissues
(Schmid and Zulli 2005).
Since the frequency 111 Hz induces
the production of beta-endorphins, there will
also be an increase in the amount of
cytokeratin 16 protein which will increase the
wound healing process.
Conclusion
The sound frequency 111 Hz has
been tested if it has an increased wound
healing effect. After the experiment, positive
results were obtained. Therefor the sound
frequency 111 Hz is an effective way of
decreasing the days it take for a wound to
heal.
References
Bigliardi P.L., Büchner S., Rufli T. & Bigliardi-
Qi M. (2002). Specific Stimulation
of Migration of Human
Keratinocytes by μ-Opiate
Receptor Agonists. Journal of
Receptors and Signal Transduction
22: 191-199
Bigliardi P. L., Sumanovski L. T., Büchner S.,
Rufli T. & Bigliardi-Qi M. (2003).
Different Expression of μ-Opiate
Receptor in Chronic and Acute
Wounds and the Effect of β-
Endorphin on Transforming
Growth Factor β Type II Receptor
and Cytokeratin 16 Expression. J
Invest Dermatol 120: 145-152.
Bigliardi-Qi M., Sumanovski L.T., Büchner
S., Rufli T. & Bigliardi P.L. (2004).
Mu-Opiate Re- ceptor and Beta-
Endorphin Expression in Nerve
Endings and Keratinocytes in
Human Skin. Dermatology 209: 183-
189.
Cheng, B. , H.W. Liu, X.B. Fu, Z.Y. Sheng,
J.Y. Qiu, R. Cao (2007). Expression
of betaendorphin and microopioid
receptor during wound healing
process in rat with deep
partialthickness scald. Zhonghua
Shao Shang Za Zhi: 23(1), p.36-39.
Dorian (2014). Nerve Regeneration – ANTI
AGING – Through Isotonic Tones
and Binaural Beats. Reverse
Aging:Life Extension Science &
Research Videos. Retrieved from:
http://reverseaging.info/nerveregene
rationantiagingthroughisotonictonesa
ndbinauralbeats/. (Retieval date:
March 28, 2015).
Fang, R.C. and T.A. Mustoe (2008). Animal
models of wound healing: utility in
transgenic mice. J Biomatter Sci
Polym Ed. Vol. 19: 989-1005.
Grochot-Przeczek, Anna et al. (2009). Heme
oxygenase-1 accelerates
cutaneous wound healing in mice.
PloS ONE. Vol.4 (6): e5803.
KRT 16. (2012). Genetics Home
Reference: Genes. Retrieved from:
http://ghr.nlm.nih.gov/gene/KRT16
(Retrieval date: March 29, 2015).
Linbald, W. J. (2008). Considerations for
selecting the correct animal model
for dermal wound-healing studies.
J. Biomatter Sci Polym Ed. Vol.
19:1087-96.

Nguyen, D.T., Orgill D.P., Murphy G.F.
(2009). Chapter 4: The
Pathophysiologic Basis for Wound
Healing and Cutaneous
Regeneration. Biomaterials For
Treating Skin Loss. Retrieved from:
http://www.docin.com/p109651173.h
tml. Woodhead Publishing
(UK/Europe) & CRC Press (US),
Cambridge/Boca Raton, p.
2557.(ISBN 9781420099898/ISBN
9781845693633).
Park, J.E. and Barbul, A. (2004).
Understanding the role of immune
regulation in wound healing.
American Journal of Surgery.
Vol.187:11S-16S.
Park, Shin Ae et al. (2014). Full-thickness
splinted skin wound healing
models in db/db and heterozygous
mice: Implications for wound
heling impairment. Wound Repair
Regeneration. Vol.22:368-380.
Rieger, S.; Zhao, H.; Martin, P.; Abe, K.;
Lisse, T.S. (2014). "The role of
nuclear hormone receptors in
cutaneous wound repair". Cell
Biochemistry and Function. Vol.33
(1): 1–13. doi:10.1002/cbf.3086.
Schmid, D. and F. Zulli. (2005). Role of
Beta-endorphin in the skin. SOFW-
Journal: 131(4).
Stadelmann, WK; Digenis, AG; Tobin, GR
(1998). "Physiology and healing
dynamics of chronic cutaneous
wounds". American Journal of
Surgery. Vol.176 (2A Suppl): 26S–
38S.doi:10.1016/S00029610(98)001
834.
Uchiyama, Akahikp et al. (2014). MFG-E8
regulates angiogenesis in
cutaneous wound healing. The
American Journal of Pathology.
Vol.184 (7):1981-1991.
Wong, V. W. et al. (2011). Surgical
approaches to create murine
models of human wound healing.
Journal of Biomedicine and
Biotechnology. 2011: 969618.
Zhai, Zili et al. (2009). Alcohol extract of
Echninacea pallida reverses stress-
delayed wound healing in mice.
Phytomedicine. Vol.16 (6-7):669-
678.

Appendix
Appendix 1. ANOVA Table for the Percentage Wound Closure
Source of
variation
Sum of squares
Degrees of
freedom
MSS
F value
Between-
column
97.656
1
97.656
0.230627
Within-column
1625
8
203.125
Total
1,722.656
9
Plates
Plate 1. Injection of anesthesia Plate 2. Shaving of area to be wounded
Plate 3. Set-up 2 (speakers producing 111 Hz) Plate 4. Infliction of wound using nail.

Plate 5. Frequency generator software Plate 6. Male ICR mice
Plate 7. Freshly wounded mouse Plate 8. Measuring of wound diameter
Plate 9. Identity marks on mouse Plate 10. Wound of mouse after 5 days