Study Reports of Research Stay
─ The Chihiro and Kiyoko Yokochi Fund ─2017 2020

The Ichiro Kanehara FoundationTokyo

Study Reports of Research Stay─The Chihiro and Kiyoko Yokochi Fund─ 2017-2020
Published by
The Ichiro Kanehara Foundation
IS Yumicho Bldg. 7F, 1-28-24 Hongo, Bunkyo-ku, Tokyo 113-0033, JAPANⒸ2022 The Ichiro Kanehara Foundation, Tokyo
Printed in Japan

Preface to the Study Reports (2017-2020)
The Chihiro and Kiyoko Yokochi Fund was established in January 1999 through adonation by Chihiro Yokochi, M. D., Emeritus Professor of Anatomy of the Kanagawa
Dental College, Yokosuka, Japan, in memory of his beloved wife, the late Kiyoko
Yokochi, who died in September 1995. It was her wish to organize a scholarship programfor German medical researchers. The roots of this intimate relationship trace back to the1970s, when an extraordinarily fruitful and successful co-project between Prof. Chihiro
Yokochi and Prof. Johannes W. Rohen at the University of Erlangen, Germany, wasinitiated. It finally resulted in the famous “Color Atlas of Anatomy: A Photographic
Study of the Human Body”, published by Igaku-Shoin (Tokyo) and F. K. Schattauer
Verlag (Stuttgart), which up to now provides unsurpassed insights into the delicatecomplexity of the human body to medical students all over the world. In the course of thisproject, Chihiro and Kiyoko Yokochi visited Erlangen several times, and a mutual
admiration of German and Japanese cultures developed.
The aim of the fund is to promote a special program to financially support young
German researchers who wish to pursue basic medical research in medical schools, dental
schools or institutes in Japan. This generous offer was well appreciated, and the turn ofthe millennium marked the beginning of what turned out to develop as an extraordinaryscientific and cultural scholarly program. Between 2000 and 2004, ten recipients of ascholarship visited basic research institutes and clinical departments of medical schools inJapan to conduct collaborative projects and deepen cultural interrelationship. The
scientific reports of these initial ten scholars have been published in a first volume in2007. In the upcoming next eleven years, grants have been awarded to another eleven
German researchers, selected on the basis of the scientific quality of their proposed
project to be conducted with their Japanese host. Their projects covered indeed the full
spectrum of medical research, spanning from basic questions in anatomy and physiology
to the immediate needs of patients in developing artificial organs, summarized in asecond volume of the study reports. To allow for competent review and judgement ofsuch a broad spectrum of proposals, the Scientific Evaluation Committee consisting ofboth Japanese and German scientist had been expanded in this period by including
another Chair, Prof. Stefan Bauer from the Institute of Immunology at the Philipps
University Marburg, Germany.
The present third study report volume documents the achievements of the last 4
German scholars visiting host institutions in Japan in the years 2017 to 2020, which
altogether resulted in 7 peer-reviewed joint scientific publications of the scholarship
holders together with their hosts. They addressed basic aspects of areas of utmost clinicalrelevance such as stem cell therapy after stroke, underlying mechanisms of dry eye
disease, and functional understanding of cartilaginous and connective tissue elements ofiii

Preface to the Study Reports

the pelvic ring that contribute so much to the hard-to-treat syndromes of pelvic girdle
pain.
Thus, over a period of 21 years, altogether 25 young German scientists enjoyed an
outstanding opportunity. A distinctive feature of this program from most others is that
fostering of new cultural bonds between the participating countries, Germany and Japan,
is as much intended and indeed achieved as the rationale aspect of research. This is
supported by the unusual approach to bring together people who share interests but nothave worked together extensively in the past, rather than just boosting pre-existing
collaborations. The reports published in this volume document not only the scientificsuccess but also the cultural exchange that developed, very much in the spirit of thesponsors. I wish to express my gratitude to the generous sponsors, the Ichiro Kanehara
Foundation for the very effective handling and support of the program, and of course all
those in both countries, scholars and hosts, who filled it with the spirit in which it wasestablished.

Wolfgang Kummer, M. D.

Professor of Anatomy and Cell Biology
Justus Liebig-University, Giessen
Chair of the Evaluation Committeeiv

History of The Chihiro and Kiyoko Yokochi Fund
The Chihiro and Kiyoko Yokochi Fund was established in January 1999 through adonation by Chihiro Yokochi, M.D., Emeritus Professor of Anatomy of the Kanagawa
Dental College, Yokosuka, Japan in memory of his beloved wife, the late Kiyoko
Yokochi, who died in September 1995. It was her wish to organize a scholarship programfor German medical students. The aim of the Fund is to promote a special program tofinancially support young German researchers who wish to study basic medical or dental
science in schools or research institutes in Japan.
Prof. Chihiro Yokochi was born in Morioka (Iwate Pref.) on October 25, 1918 andgraduated from Nippon Medical School (Tokyo) in 1942. After World War II he obtained
his M.D. degree from Keio University School of Medicine (Tokyo) and began his studiesof anatomy at the Gunma University School of Medicine in Maebashi (Gunma Pref.). He
then moved to Yokohama (Kanagawa Pref.) in 1953 to accept a position as Associate
Professor of Anatomy at the Yokohama City University School of Medicine. In 1965 he
was promoted to Full Professor and Chairman of Anatomy at Kanagawa Dental College
in Yokosuka (Kanagawa Pref.). He dedicated his efforts mostly to the study ofmacroscopic anatomy and student education in courses of anatomy in medical and dental
schools.
Prof. Yokochi wrote and edited four anatomy atlases for medical, dental and allied
health students. He published his first photographic atlas “Photographic Anatomy of the
Human Body” in Japanese (Igaku-Shoin,1962). The first English edition was publishedby Igaku-Shoin in 1969, and its subsequent editions were published in 1978 (second) and1989 (third). This “Small Atlas” was the milestone for a more extensive anatomy atlas for
medical students, which was to be co-authored later by Prof. Johannes Rohen (Marburg,
Erlangen).
In the mid 1970ʼs a co-publication project with Chihiro Yokochi was proposed byProfessor Rohen with the publishers Igaku-Shoin (Tokyo) and F. K. Schattauer Verlag(Stuttgart). During the preparation of this manuscript, Prof. Yokochi visited Erlangenseveral times for discussion of editorial matters and to dissect new specimens, with hiswife, Kiyoko, who accompanied him frequently. During these visits she developed anadmiration for German culture and a mutual admiration arose between herself and theGerman people. The first edition of the atlas appeared in 1983 under the title “Rohen/
Yokochi: Color Atlas of Anatomy; A Photographic Study of Human Body ” (IgakuShoin/F. K. Schattauer Verlag) and it has been revised periodically since then, the ninth
edition was published by Wolters Kluwer in 2021.
v

History of The Chihiro and Kiyoko Yokochi Fund

In 1997, during the preparation of its Fourth edition the Igaku-Shoinʼs share in thetitle was sold to Wavery Press [Lippincott, Williams and Wilkins (Baltimore, USA)] andthe new Fourth edition was published in 1998, inviting Prof. Elke Lütjen-Drecoll(Erlangen) as the third author.
Even after his retirement in 1986, Prof. Yokochi continued to make new dissections
for the revised editions of “Color Atlas of Anatomy”. In 1997 he published in Japanese asmall book called “Stereoscopic Atlas of Anatomy” (Igaku-Shoin) and its second revised
edition with 3D viewer in 2017.
A large portion of the donation by Prof. and Mrs. Yokochi includes royalties fromhis four books. Prof. Yokochi truly believes that this work “Color Atlas of Anatomy” isliterally a co-work between two academic institutes in two different countries, or amasterpiece produced by the magnificent collaboration of German and Japanese cultures(From the eighth edition published in 2016 the title has been changed to “Anatomy - A
Photographic Atlas”). This scholarship program was initially proposed by Mrs. Yokochi
and was verified in her last testament. In the 20 years since 2000, the Chihiro and Kiyoko
Yokochi Fund granted a total of 80,762,979 yen in subsidies to 25 researchers. We aresure that the Chihiro and Kiyoko Yokochi Scholarship has been a very useful program forthose who have studied in Japan from research institutes in Germany, and we areconvinced that the long-nurtured hope of Prof. and Mrs. Yokochi was fulfilled in thecreation of the Yokochi Fund and its successful operation over many years.
In accordance with the intention expressed by Prof. Yokochi, the founder, applications for the Chihiro and Kiyoko Yokochi Scholarship program was closed as of the 31st
day of March 2019. It has been 20 years after the establishment of the program. The
Ichiro Kanehara Foundation would like to express our deepest gratitude to everyone concerned for their efforts and cooperation over the years.

vi

Contents
Preface to the Study Reports (2017-2020) 
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History of The Chihiro and Kiyoko Yokochi Fund
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List of Contributors 
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1. Transient receptor potential channel expression in lacrimal gland andassociation with the aqueous deficient form of Dry Eye Disease
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1
Anna Kanewska
2. Electromagnetic field preconditioning of neural stem cells for improved stem
cell therapy after stroke 
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Jasmin Matuszak
3. The topographical anatomy of the symphysis pubis ─ insights in theligamentʼs morphometry, the musculotendinous connections and vascularsupply 
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Philipp Pieroh
4. Immunohistochemical observations of neurovascular structures associatedwith the human sacrospinous ligament
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20
Johann Zwirnervii

List of Contributors
Anna Kanewska, M.D.
Institute of Functional and Clinical Anatomy II,
Friedrich Alexander University Erlangen-Nürnberg,
Universitätsstraße 19, 91054 Erlangen, Germany
E-mail address: anna.kanewska@fau.deJasmin Matuszak, Dr. rer. nat.
Molecular Cardiology-Medicine 2,
University Hospital Erlangen,
Schwabachanlage 12, 91054 Erlangen, Germany (2018)
E-mail address: jasmin_matuszak@yahoo.de

Philipp Pieroh, M.D.
Department of Orthopaedics, Trauma and Plastic Surgery,
University of Leipzig,
Liebigstraße 20, 04103 Leipzig, Germany
Department of Anatomy and Cell Biology,
Martin Luther University Halle-Wittenberg,
Grosse Steinstraße 52, 06097 Halle (Saale), Germany
E-mail address: Philipp.Pieroh@medizin.uni-leipzig.deJohann Zwirner, M.D., Dr. med.
Department of Legal Medicine,
University Medical Center Hamburg-Eppendorf,
Butenfeld 34, 22529 Hamburg, Germany
E-mail address: medijo@gmx.deviii

1. Transient receptor potential channel

expression in lacrimal gland andassociation with the aqueous deficient
form of Dry Eye Disease
Anna Kanewska
Scientific Background

The Dry Eye Disease (DED) as a disease which has a higher prevalence in the elderly
population [1], has been gaining importance due to the demographic changes. There areseveral attempts on identifying the causes, which concentrate on different parts of thelacrimal functional unit. In my studies, I focused on the lacrimal gland.
Aside, there had been studies in the role of transient receptor potential channels(TRP-channels) in the ocular surface for the development of DED [2]. TRP-channels are
ion channels somewhat permeable for Ca2+ and mediate sensory sensation activated by e.
g. changes in the osmolarity [3].
Our group has already shown the expression of TRP channels in human corneal [4]and conjunctival epithelial cells [5], as well as human corneal endothelial cells [6].
However, the expression of TRP-channels in the lacrimal gland has not been exploredyet. Since the lacrimal gland plays an essential role in the tear production and, therefore,
might be affected in case of DED, the goal of the studies was to characterize the
TRP-channel expression and relevance in the lacrimal gland.

Results

Gene expression results
The aim was to provide an overview of TRP-channel expression in the murine lacrimal
gland (LG), submandibular gland (SMG) and Harderian gland (HG). For this, we chose
representatives of the TRPM-family based on preliminary microarray results of Prof. Dr.
Ito’s laboratory at the Department of Developmental Anatomy and Regenerative Biology,
National Defense Medical College, as well as TRPV1 based on own preliminary work.
【Keywords】lacrimal gland; developmental anatomy; ocular diseases; TRPM3; TRP channels
Institution in Japan: Department of Developmental Anatomy and Regenerative Biology, National Defense
Medical College, Tokorozawa, Saitama, Japan
Supervisor: Prof. Masataka Ito, M.D., Ph.D.
Period of Stay: From April 1, 2017 to January 3, 2018
Submission Date of Manuscript: June 22, 20181

Anna Kanewska

Figure 1. Relative TRPM3-gene expression in murine lacrimal gland at different
stages of development. (A) shows the expression of TRPM3 in the murine lacrimal
gland (LD) at different stages (E=embryonic, p=postnatal). For each stage, four mice (n=4) were analyzed. There is a significant (p＜0.001) 1.6-fold increase in TRPM3 gene
expression from E16 (0.80±0.13) to E18 (1.31±0.37) and a significant (p＜0.01)
reduction by about two thirds (69%) from 4d (0.68±0.10) to 7d (0.21±0.10). (B) showsa significant (N=3, P＜0.01) reduction by 77% in TRPM3 expression in the murine
submandibular gland (SMG) from day 0 ( 1. 02±0. 20 ) to day 10 ( 0. 27±0. 10 ). For
statistical analyses, Bonferroni multiple-t-test was used.

Figure 2. Relative TRPM3 gene expression in p0d, p4d and p7d old mice kept
under different light conditions. The statistical analyses with Bonferroni multiple-t-test(N=4) reveals a significant (p＜0.01) 87% decrease from p0 (1.19±0.27) to 7d (0.15±0.04) and a 78% decrease from 4d (0.71±0.27) to 7d in normal mice and a decrease by
two thirds (67%) from p0 (0.84±0.39) to 7d (0.28±0.18) in light deprived mice.

The TRPM2-, TRPM4-, TRPM6- and TRPV1-gene expression in LG, SMG and HG
did not reveal any conspicuous pattern. TRPM3-gene expression, however, showed a significant and continuous decrease after the stage E18. This developmental pattern was
similar for the submandibular gland (Figure 1).
The decrease might be unrelated to light conditions: Since the decrease of TRPM3-gene

expression occurred after birth and seemed to continue significantly until about the stage2

1. Developmental TRPM3-expression in the murine LG

Figure 3: TRPM3 expression pattern in murine LG in different age stages. Immunohistochemical analyses with paraffin embedded sections of murine lacrimal gland. Brown signal shows reactivity
with αTRPM3 antibody. In mice of embryonic age 18 (A), newborn (B) and 7d postnatal (C) there
seems to be a positive signal in the apical membranes of ducts and acini (black arrow in A). In older
stages, 8W (D) and 6M (E), the signal is either membranous in bigger ducts or diffusive cytoplasmic.
Nuclei are counterstained with Hematoxylin.

of eye opening (10d), light-presence was a hypothetical influence factor for TRPM3
expression. Therefore, an animal model of light deprived mice was established andanalyzed. The gene expression in LG of model mice, which were born and grew up incomplete darkness, was compared with the expression in LG of age-matched mice, whichwere held under normal light conditions. To make sure whether eye opening itself had an
impact, only age stages before eye opening, p0, 4d and 7d were used. Despite of thesignificant decrease from p0 to the other stages in both, model and normal mice, light
exposure was not identified as a relevant factor for TRPM3-gene expression (Figure 2).

Histology
Membranous expression of TRPM3 in younger stages: Due to its character as an ion

channel, TRPM3 cellular localization was expected to be in the cell membrane of thelacrimal gland cells. This could be observed for the younger stages (Figure 3). For theolder stages, where qPCR-results indicate a low expression of TRPM3, this was not thecase. These stages contained weak, diffuse cytoplasmic signals for TRPM3 channel in theLG.

Conclusions and Future Directions
TRPV1 and Dry Eye Disease

TRPV1 is a thermo-TRP-channel involved in inflammational processes, either as a
nociceptor in peripheral nerve endings or a contributor to the IL-6 release [7]. It has beenshown that IL-6 specifically is involved with ocular autoimmune diseases, as in Sjögren
3

Anna Kanewska

syndrome [8].
As the expression of TRPV1 in the murine lacrimal gland showed to be almost
below the threshold for qPCR analyses (results not shown) despite of positive signals inprior RT-PCR, we would like to focus more on the neuroimmunological role of TRPV1
in the lacrimal gland, which may be not necessarily associated within the LGEC itself.

Role of TRPM3 in the ocular surface
Our studies indicate a peak of TRPM3 expression in the murine lacrimal gland at E18. At
this point, there also is a crucial term for the development of the LG [9]. Since there areindications for the co-relation of TRPM3 and Pax-6, an essential transcription factor for
development of ocular structures [9], it is possible that TRPM3 plays an important role inthe development of the lacrimal gland.

References[1] Craig JP, Nichols KK, Akpek EK, Caffery B, Dua HS, Joo C-K, et al. TFOS DEWSII Definition and classification report. The Ocular Surface. 2017;15(3):276-283.
doi:10.1016/j.jtos.2017.05.008.
[2] Almaraz L, Manenschijn J-A, de La Pena E, Viana F. TRPM8. Handbook of Experimental Pharmacology. 2014;222:547-579. doi:10.1007/978-3-642-54215-2_22.
[3] Ramsey IS, Delling M, Clapham DE. An introduction to TRP channels. Annu Rev
Physiol. 2006;68:619-647. doi:10.1146/annurev.physiol.68.040204.100431.
[4] Mergler S, Garreis F, Sahlmüller M, Reinach PS, Paulsen F, Pleyer U. Thermosensitive transient receptor potential channels in human corneal epithelial cells. J
Cell Physiol. 2011;226(7):1828-1842. doi:10.1002/jcp.22514.
[5] Mergler S, Garreis F, Sahlmüller M, Lyras E-M, Reinach PS, Dwarakanath A, et al.
Calcium regulation by thermo- and osmosensing transient receptor potential
vanilloid channels (TRPVs) in human conjunctival epithelial cells. Histochem Cell
Biol. 2012;137(6):743-761. doi:10.1007/s00418-012-0924-5.
[6] Mergler S, Mertens C, Valtink M, Reinach PS, Székely VC, Slavi N, et al. Functional significance of thermosensitive transient receptor potential melastatin channel
8 (TRPM8) expression in immortalized human corneal endothelial cells. Exp Eye
Res. 2013;116:337-349. doi:10.1016/j.exer.2013.10.003.
[7] Seki N, Shirasaki Hideaki, Kikuchi M, Himi T. Capsaicin induces the production of
IL-6 in human upper respiratory epithelial cells. Life Sci. 2007;80(17):1592-1597.
doi:10.1016/j.lfs.2007.01.037.
[8] Zahir-Jouzdani F, Atyabi F, Mojtabavi N. Interleukin-6 participation in pathology
of ocular diseases. Pathophysiology: the official journal of the International Society
for Pathophysiology. 2017;24(3):123-131. doi:10.1016/j.pathophys.2017.05.005.
[9] Makarenkova HP, Ito M, Govindarajan V, Faber SC, Sun L, McMahon G, et al.
FGF10 is an inducer and Pax6 a competence factor for lacrimal gland development.
Development (Cambridge, England). 2000;127(12): 2563-2572.
4

1. Developmental TRPM3-expression in the murine LG

Publication of Project Results
 Kanewska A, Ito M, Karasawa Y, Inada M, Garreis F, Paulsen F, Takeuchi M.
Developmental change in the gene-expression of transient receptor potential melastatin channel 3 (TRPM3) in murine lacrimal gland. Ann Anat. 2020;231:151551.

Project-related Presentations
 Kanewska, Anna (2017): Transient receptor potential channel expression in lacrimal
gland and association with the aqueous deficient form of Dry Eye Disease, 27th
Tokyo ocular immunology meeting on 1st September 2017.
 Kanewska, Anna (2017): Transient receptor potential channel expression in lacrimal
gland, Meeting on the lacrimal gland at the Keio University, chair: Prof. Dr.
Tsubota, 25th September 2017.
 Kanewska, Anna (2018): Expression and function of TRP-channels in the murine
lacrimal gland, 46th Anatomical Colloquium at the Friedrich-Alexander University
Erlangen Nürnberg on 8th March 2018.

5

2. Electromagnetic field preconditioning ofneural stem cells for improved stem celltherapy after stroke
Jasmin Matuszak
Research BackgroundThe second leading cause of death is stroke, resulting in around 5 million deaths per year
all over the world [1]. At the moment only thrombolysis with tissue-plasminogenactivator is a successful treatment for ischemic stroke, however it needs to be admitted
within the short time of 4.5 h after occurrence of first symptoms, which is difficult for the
majority of patients. Therefore, a lot of research is dealing with possible therapies forstroke. One promising concept is the regeneration of the damaged brain area by stem celltherapy. There are several in vivo studies using neural [2], embryonic [3] or mesenchymal
stem cells [4] showing the feasibility of stem cell therapy. Although promising, only asmall percentage of transplanted cells survives, proliferates and differentiates in the
typical hostile environment of a stroke area [5]. It is known that electromagnetic fields(EMF) can have stimulatory as well as inhibitory influence on stem cells [6] and their
therapeutic use was evaluated in clinical trials as well [7]. The main idea of this project is
to us EMF for preconditioning of neural stem cells (NSC) before transplantation to
improve stem cell survival and differentiation, for better regeneration of stroke damaged
brain areas.

Project

The main goal of this project was to evaluate if the tested EMF settings have any beneficial effect on neural stem cells and therefore may improve stem cell transplantation.
During the project, murine neural stem cells from three different areas of the brain
were used. For each experiment the brain of 4 male mice was dissected. The olfactorybulb, the subventricular zone and the hippocampus were isolated, as these are the only
regions of the adult brain with stem cell niches. After dissociation, the single cellsuspensions were cultured in neurobasal medium for around 1-2 weeks, until neuro【Keywords】electromagnetic field (EMF); EMF preconditioning; neural stem cells; stroke;
stem cell differentiation
Institution in Japan: Cognitive and Molecular Research Institute of Brain Diseases, Kurume University Schoolof Medicine, Kurume- shi, Fukuoka, Japan
Supervisor: Prof. Hideki Harada, M.D., Ph.D.
Period of Stay: From January 3, 2018 to December 30, 2018
Submission Date of Manuscript: March 29, 20196

2. EMF preconditioning of neural stem cells

spheres developed. The neurospheres were seeded in coated 6-well plates. Cells adhered
overnight and were treated with EMF (1.4 mT, 50 Hz) for 7 days, whereas the control
cells were kept in a normal incubator (37° C) without EMF. The total RNA was isolated
and transcribed with reverse transcriptase into cDNA. This cDNA was used to perform acomparative quantitative real-time polymerase chain reaction (PCR) with SYBR-green.
We pursued two different experimental set-ups:a) EMF preconditioning in undifferentiated NSC: NSC were cultured in medium withgrowth factors (P1). In this condition, cells are able to proliferate and maintain their stem
cell like character, but cannot differentiate. After 7 days of EMF preconditioning the
RNA was isolated immediately.
b) EMF preconditioning in differentiating neural stem cells: For the first 7 days of theexperiment, the cells were cultured with P1 medium and one part of the cells was exposed
to EMF. Afterwards the media was changed to media without growth factors (D1)allowing the cells to differentiate. The cells were kept for another 7 days at control
conditions for differentiation. After 14 days in total, the RNA was isolated.
The total RNA was isolated and transcribed with reverse transcriptase into cDNA. This
cDNA was used to perform a comparative quantitative real-time PCR with SYBR-green.
As an endogenous control the housekeeping gene GAPDH (glyceraldehyde-3-phosphate
dehydrogenase) was used. Measured values were normalized to endogenous control with
the Ct method and afterwards normalized to control cells (37° C, no EMF).
Comparison of the CT value of a target gene with that of the endogenous control gene
allows the gene expression level of the target gene to be normalized to the amount of
input RNA or cDNA.
During the project, a massive problem with the EMF system was detected. Because of ainsufficient cooling system, the whole system heated up, with hot spots of over 40° C andin-well temperatures of 38° C.
Therefore, the first two sets of experiments were performed under heating conditions. They will be referred to as “old” or “heating” setting/condition. To analyze if theeffect of the increased temperature was significantly influencing the experiment outcome,additional experiments were performed comparing the gene expression levels of therelated genes at 37 and 38° C.
After improving the cooling system, temperatures of around 36.8° C under EMF
were maintained throughout the whole experiment. This setting is described in this reportas “new setting”.
7Jasmin Matuszak

Results and Discussion

Gene expression in NSC after 7 days of EMF preconditioning under “heating”
conditionsa) Undifferentiated NSC: The results in Figure 1 display some significant changes in thegene expression of the tested genes after 7 days of EMF exposure for all areas. In the
NSC derived from the olfactory bulb Olig2 and klf4 were significantly reduced. Both of
those genes are expressed in very immature stem cells and progenitor cells. The decreaseof their expression could be a sign of a possible acceleration of differentiation due to
EMF. A similar result was observed for the NSC cultured from the subventricular zone,
where SSEA-1, a marker for immature NSC, was decreased. Interestingly, a distinct
increase of both marker typical for mature neurons, beta-III-tubulin and Map2, wasmeasured for NSC from the hippocampus. This suggests the acceleration of differentiation and even maturation.
b) Differentiated NSC: Although gene expression levels in hippocampus derived cells

were equally reduced, almost no changes could be observed for olfactory bulb or subventricular zone. In general, there were no significant changes in the gene expression
levels in comparison to the EMF untreated control for any of the tested areas.
Although the gene expression levels shown in Figure 2 give a first idea that EMF
probably had a distinct effect on the differentiation of NSC, it is unclear if this effect was
mainly due to EMF or also due to the temperature increase of 1° C, which unfortunately
were only detected after the performance of the first set of experiments. To analyze thepossible effect of increased temperature on NSC, some additional experiments were
performed (see Figure 4).

Figure 1: Gene expression of differentiation related genes in undifferentiated
NSC. NSC were exposed to EMF (1.4 mT, 50 Hz) for 7 days under non-differentiating
conditions. A RT-qPCR was used to determine the relative quantitation (RQ) values forthe respective genes. Values are normalized to untreated control cells. n=7. Shown are
mean±SEM. * p＜0.05 vs. control with student’s t-test.
8

2. EMF preconditioning of neural stem cells

Figure 2: Gene expression of differentiation related genes in differentiated NSC.
NSC were exposed to EMF (1.4 mT, 50 Hz) for 7 days under non-differentiating conditions. Afterwards EMF treatment was stopped and cells were cultured under differentiating conditions for additional 7 days. A RT-qPCR was used to determine the relative
quantitation (RQ) values for the respective genes. Values are normalized to untreatedcontrol cells. n=7. Shown are mean±SEM. *p＜0.05 vs. control with student’s t-test.

The problem of insufficient temperature control within the EMF systema) Improvement of the air cooling system: It is known that temperature is a crucial factor

in nature for regulating biological processes. The essential key temperature in humans isaround 37° C and scientific experiments in vitro are usually performed at defined conditions, such as 37° C, 5% CO2 and 95% humidity.
During the first cascade of experiments performed within this project, a strongly enhanced evaporation in the well plate under EMF in comparison to the plate under control
condition was observed. With a thermograph, several “hotspots” (areas with strongly
increased temperatures) up to 42° C were identified. Additional in-well measurementswith temperature sensors revealed temperatures over 38° C within the wells, corresponding to a temperature increase of more than 1° C in comparison to the baseline temperatureof 37° C (Figure 3, “old setting”). After several adjustments of the cooling system andrepeated measurements (data not shown) the temperature increase was avoided. The tubes
connecting the cooling system with the magnetic coil were replaced by bigger tubes,
allowing more cool air to reach the coil in shorter time and therefore more efficiently cool
down the whole system. After application of an electromagnetic field of 1.4 mT, temperature changes were now stable in a range of ± 0.3° C within the wells.
b) Change in temperatures effects gene expression in neural stem cells: To address thepossible effects of changes in temperature and inconsistent experiment set-up some
additional experiments were performed, comparing the gene expression levels of differentiation related genes after exposure to 37° C and 38° C. The graph in Figure 4 showsthat a few effects indeed were measured. Although for the majority of the genes nosignificant change in gene expression occurred, a distinct decrease was observed for βIII-tubulin in NSC from the hippocampus and subventricular zone. These findings ques9

Jasmin Matuszak

Figure 3: Change of temperature when using the EMF system before and after
adjustment of the cooling system. Temperature was measured with highly sensitive
sensors positioned at the electromagnetic coil, inside the well and in the middle of the
incubator (air) with an electromagnetic field of 1.4 mT. Graph shows change in temperature (in comparison to baseline temperature of 37° C) 2 h after turning on the EMF withrunning cooling system. “Old setting” refers to the use of the not yet improved coolingsystem and “New setting” describes the system after adjusting the cooling system. n=1.

Figure 4: Gene expression of differentiation related genes in relation to temperature. NSC were exposed to 37° C or 38° C for 7 days under non-differentiating conditions. A RT-qPCR was used to determine the relative quantitation (RQ) values for therespective genes. Values are normalized to untreated control cells. n=7. Shown are mean
± SEM. * p＜0.05 vs. control with student’s t-test.

tion the previous data obtained under “ heating ” conditions, because gene expression
patterns for β-III-tubulin are exactly opposite comparing EMF+ “heating” and only
“heating” (mimicked by 38° C). Whereas an increase of 1° C resulted in decreased βIII-tubulin expression, the combination of both EMF and temperature increase lead to anincrease of the gene expression.

Gene expression in NSC after 7 days of EMF with “new setting”a) Undifferentiated NSC: After repeating the experiments with proper temperature condi-

tions, no significant changes in the gene expression for NSC from olfactory bulb wereobserved (see Figure 5). For the NSC from subventricular zone, a significant increase was10

2. EMF preconditioning of neural stem cells

Figure 5: Gene expression of differentiation related genes in undifferentiated NSC(“New setting”). NSC were exposed to EMF (1.4 mT, 50 Hz) for 7 days under nondifferentiating conditions. A RT-qPCR was used to determine the relative quantitation(RQ) values for the respective genes. Values are normalized to untreated control cells. n=
7. Shown are mean ± SEM. * p＜0.05 vs. control with student’s t-test.

observed for SOX2 and nestin. Nestin is typically expressed in NSC and precursor cells.
SOX2 is important for maintaining pluripotency of stem cells. The increased expressionof both factors, also expressed at the same time in radial glial precursor cells, could beinterpreted as a non-differentiating effect of EMF by preserving the stem cell or
precursor state of the cell. In line with this goes the observed decreased expression of
MAP2 in the NSC from hippocampus.
b) Differentiated NSC: The gene expression levels measured after 7 days of EMF and 7

additional days of differentiation with the “new setting” (see Figure 6) look similar to theprevious observed results with the “old setting”. For olfactory bulb a significant decrease
in SOX2 was measured, although SOX2 is usually already lowly expressed in differentiated cells. Except that, no change in the gene expression pattern in NSC from other areas
in comparison to control was observed. An explanation could be a time dependency,
which means that possible effects of EMF are only exerting influence when EMF isactually applied or short time after.
Although it was observed that EMF influences many different cells in various ways,
the presented results are less clear. The hypothesis of potential beneficial effects likeacceleration of differentiation after EMF exposure on NSC for future stem cell therapy
was not affirmed. Actually, an increase of nestin and SOX2 in subventricular zone NSC
and a decrease of MAP2 in hippocampus derived NSC was measured, indicating a
non-differentiating effect of the EMF. Of course, there are several potential reasons forthese findings, like the EMF setting of 50 Hz and 1.4 mT. There are infinite possible
variations of the magnetic field and frequency that can have very different effects on
NSC. Maybe it is just a question of testing and a bit of luck to find the “one” combinationthat triggers the right gene expression patterns beneficial for stem cell therapy. Further,11

Jasmin Matuszak

Figure 6: Gene expression of differentiation related genes in differentiated NSC(“New setting”). NSC were exposed to EMF (1.4 mT, 50 Hz) for 7 days under nondifferentiating conditions. Afterwards EMF treatment was stopped and cells were cultured under differentiating conditions for additional 7 days. A RT-qPCR was used todetermine the relative quantitation (RQ) values for the respective genes. Values are normalized to untreated control cells. n=7. Shown are mean ± SEM. * p＜0.05 vs. control
with student’s t-test.

the duration of EMF application can have a tremendous influence on the result outcome
as well. From our experiments with differentiated cells, it seemed that one week after
EMF almost no effect could be measured. An important task would be to examine indetail how long after preconditioning the EMF showed an effect. Important for further
studies is the fact that with all tested EMF settings no significant decrease in the viabilityof NSC was observed (data not shown).
Although unintentionally, the occurred problems and mistakes during the project
revealed how important it is to maintain proper experimental conditions during EMF andof course in general. Interestingly, the gene expression patterns observed with the wrong“heating” conditions were more beneficial for a potential use during stem cell therapy
then during the corrected “new setting”. Further, experiments only addressing temperature as a factor influencing the gene expression, resulted in partly contradictory results. It
seems that not the increased temperature itself led to the differences observed between“old” and “new” setting, but the combination of EMF and temperature increase. Thismight be an interesting point to future investigations of potential factors for preconditioning of stem cells.

References[1] Mozaffarian D, Benjamin EJ, Go AS, Arnett DK, Blaha MJ, Cushman M, Das SR,
de Ferranti S, Després JP, Fullerton HJ, Howard VJ, Huffman MD, Isasi CR,
Jiménez MC, Judd SE, Kissela BM, Lichtman JH, Lisabeth LD, Liu S, Mackey RH,
Magid DJ, McGuire DK, Mohler ER 3rd, Moy CS, Muntner P, Mussolino ME, Nasir
K, Neumar RW, Nichol G, Palaniappan L, Pandey DK, Reeves MJ, Rodriguez CJ,
Rosamond W, Sorlie PD, Stein J, Towfighi A, Turan TN, Virani SS, Woo D, Yeh
12

2. EMF preconditioning of neural stem cells

[2]

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RW, Turner MB. American Heart Association Statistics Committee; Stroke Statistics Subcommittee. “Heart Disease and Stroke Statistics-2016 Update: A Report
From the American Heart Association”. Circulation. 2016 Jan 26;133(4):e38-360.
Roitberg BZ, Mangubat E, Chen EY, Sugaya K, Thulborn KR, Kordower JH, Pawar
A, Konecny T, Emborg ME. Survival and early differentiation of human neural stem
cells transplanted in a nonhuman primate model of stroke. J Neurosurg. 2006 Jul;
105(1):96-102.
Nagai N, Kawao N, Okada K, Okumoto K, Teramura T, Ueshima S, Umemura K,
Matsuo O. Systemic transplantation of embryonic stem cells accelerates brain lesion
decrease and angiogenesis. Neuroreport. 2010 Jun 2;21(8):575-579.
Lee JS, Hong JM, Moon GJ, Lee PH, Ahn YH, Bang OY. A long-term follow-up
study of intravenous autologous mesenchymal stem cell transplantation in patientswith ischemic stroke. Stem Cells. 2010 Jun;28(6):1099-1106.
Thored P, Arvidsson A, Cacci E, Ahlenius H, Kallur T, Darsalia V, Ekdahl CT,
Kokaia Z, Lindvall O. Persistent production of neurons from adult brain stem cells
during recovery after stroke. Stem Cells. 2006 Mar;24(3):739-747.
Maziarz A, Kocan B, Bester M, Budzik S, Cholewa M, Ochiya T, Banas A. How
electromagnetic fields can influence adult stem cells: positive and negative impacts.
Stem Cell Res Ther. 2016 Apr 18;7(1):54.
Rohde CH, Taylor EM, Alonso A, Ascherman JA, Hardy KL, Pilla AA. Pulsed
electromagnetic fields reduce postoperative interleukin-1β, pain, and inflammation:
A double-blind, placebo-controlled study in TRAM flap breast reconstruction
patients. Plast Reconstr Surg. 2015 May;135(5):808e-817e.

13

3. The topographical anatomy of thesymphysis pubis―insights in the ligament’s
morphometry, the musculotendinous
connections and vascular supply
Philipp Pieroh
Background“Open book” injuries are rare occurring injuries of the pelvic ring and are defined by thesymphyseal disruption and a widening or disruption of the sacroiliac joint [1]. In general,these injuries are treated with a plate osteosynthesis and depending on the posterior injury
with sacroiliac screws or a triangular osteosynthesis. However, in 75% an implant failure
occurs [2] but only 8% of patients require revision surgery [3]. Probably, an improved understanding of the anatomy of the symphyseal region may decrease the rate of implantfailure and the related revision surgeries, e.g. by a more flexible osteosynthesis [4]. Evenin inflammatory diseases of this region, namely symphysitis or osteitis pubis, theimproved understanding of the anatomy might help to monitor the progress/regress of thedisease. Currently, a grading using magnetic resonance imaging is not possible [5].
Besides traumatic ruptures of the symphysis or inflammatory processes, the symphyseal
region is coming to the fore as a cause of pain, especially in sports medicine. Conflicting
reports exist on the structure and location of the layers within this region [6, 7], complicating the interpretation of magnetic resonance tomography and possibly lead to differences to intraoperative findings. In addition, a “new” anatomical concept was recently
presented [8]. These divergent data on the anatomy of the musculotendinous connectionsof the symphysis pubis underline the need for additional gross anatomical investigations
to improve the understanding of the region and the treatment of patients. In addition, thevascular supply of the symphysis pubis was investigated.

Materials and Methods

Sixty formaldehyde-embalmed hemipelvises were obtained from 30 cadavers. During【Keywords】pelvis; symphysis pubis; pubic ligaments; vascular anatomy; groin pain
Institution in Japan: Department of Anatomy, Tokyo Medical University, Shinjuku, Tokyo, Japan
Supervisor: Prof. Masahiro Itoh, M.D., Ph.D.
Period of Stay: From July 10, 2017 to September 30, 2017
Submission Date of Manuscript: December 31, 201814

3. The topographical anatomy of the symphysis pubis

Figure 1: Measurement of the pubic ligaments. (A) Sagittal measurement of the symphysis in thehemipelvis. (B) Measurement of the photographs using ImageJ after previous normalization to theadjacent reference.

medical dissection course the medical students removed the surrounding soft tissue and aclassic hemisection in the median plane was performed to receive two symmetrichemipelvises. Subsequently, a layered dissection was performed investigating the musculotendinous connections of the symphysis pubis. The following muscles were investigated:rectus abdominis, gracilis, adductor longus and brevis, obturatorius externus and internus.
The extension of the symphysis, the area and thickness of the pubic ligaments (anterior,posterior, inferior, superior) were determined applying a ruler parallel to the sagittal planeof the pubic bone and taking a perpendicular photo (Figure 1). Measurements were
performed using ImageJ (ImageJ 1.43, imagej.nih.gov/ij/).
Obtained data were confirmed during the preparation of 10 additional formaldehyde-embalmed, non-dissected cadavers. These cadavers were used to investigate thevascular anatomy of the symphysis pubis. Dissection was started with an anterior
intrapelvic approach followed by a layered dissection identifying the vessels giving avascular supply to the symphysis pubis. After all vessels were identified, the surrounding
muscles were exposed beginning from the middle of the pectineal muscle, the periosteum
was completely removed following a separation of the posterior and superior pubic
ligament. Afterwards the pubic ligaments including their attached muscles were extracted
and the muscles were step-step released from the “ specimen ” to get insights in thelayered organization of the symphysial region.

Results

The pyramidal muscle and the inguinal ligament are connected and reach the superficial
fibers of the adductor longus and rectus abdominis muscle as well as the anterior pubic
ligament (Figure 2).
The adductor brevis muscle has a connection to both the superficial and the deepparts of the anterior pubic ligament. The adductor longus muscle (Figure 3) and rectus
15

Philipp Pieroh

Figure 2: M. pyramidalis. (A) In the hemipelvis laterally mobilized (red arrow) without release ofstronger fibers. (B) in the specimen mobilized with superficial attachment site (red arrow). (C)Plastinate sagittal view with red arrow marking the M. pyramidalis and M. rectus abdominis deep
below (light green arrow).

Figure 3: Adductor longus muscle. (A) In the hemipelvis, adductor longus muscle opened withconnection to the anterior pubic ligament (red arrow). (B) Illustration of the connection between the
adductor longus muscle and the anterior pubic ligament (red arrow) in the specimen.

Figure 4: M. rectus abdominis. (A) In the hemipelvis attachment (red arrow) of the M.
rectus abdominis. (B) In the specimen, the direct connection of the M. rectus abdoministo the anterior pubic ligament (red arrow) is visible.
163. The topographical anatomy of the symphysis pubis

Figure 5: Measurements of area (left) and thickness (right) of the pubic ligaments
in hemipelvises. * p＜0.05.

abdominis muscle (Figure 4) are directly connected to each other and to the anterior pubic
ligament.
The obturatorius externus and internus had no direct connection to the pubic
ligaments. The gracilis muscle is connected to the anterior and inferior pubic ligament.
The posterior pubic ligament is connected to the medial pubovesical/puboprostaticum
ligament. The posterior pubic ligament has the smallest thickness (1.51 ± 0.51 mm) andsmallest area (45.60 ± 17.76 mm2) of the pubic ligaments. The inferior pubic ligament(7.59 ± 3.03 mm) is thicker than the anterior pubic ligament (5.00 ± 1.58 mm, Figure5). The area of the inferior pubic ligament (75.38 ± 27.75 mm2) is smaller than that of
the anterior pubic ligament (104.60 ± 38.91 mm2) only in women. The anterior pubic
ligament (5.23 ± 1.56 mm) is thinner than the superior pubic ligament (6.92 ± 2.19 mm)
but only in men. The superior and inferior pubic ligaments are thicker in men than inwomen.

Arterial Supply
The superior part of the symphysis pubis got its arterial supply from a small artery
running on the back of the inguinal ligament and originating from the inferior epigastricartery. Posteriorly, a vessel from the obturator artery supplies the symphysis pubis and theperiosteum of the pubic bones. At the inferior portion, small branches from the dorsal
penile or dorsal artery of the clitoris enter the inferior pubic ligament laterally. The
anterior portion is the most complex regarding the course of the small arteries of the deep
pudendal artery. Moreover, during the dissections multiple variations were detected.

Summary

Besides the function as anchor for several muscles also the thickness and area data of theanterior pubic ligament suggest a pivotal role for the stability of the symphysis pubis.
Hence, apart from the superior and inferior ligament a (functional) reconstruction of theanterior pubic ligament should be considered. Our data reveal a direct connection of the17Philipp Pieroh

rectus abdominis, adductor longus and brevis and gracilis muscles to the pubic ligaments(anterior pubic ligament) as well as a superficial connection of the inguinal ligament andpyramidalis muscle. Furthermore, the vascular supply by multiple arteries supports the
hypothesis that symphysitis pubis may occur hematogenous.

Acknowledgement
I would like to express my sincere gratitude to the Kanehara Foundation and the Yokochi
Found offering me the opportunity to perform this project and granting me with thiswonderful scholarship. I would like to thank me teacher in Japan, Masahiro Itoh who
spend his time with me to discuss the project and introduces me in Japan. Furthermore, I
would like to thank Zhong-Lian Li, Shin Kawata and Yuki Ogawa for the interminable
help with the project and administrative affairs, the whole laboratory members as well as
Michiyo Miyanaga and Toshiyuki Saito for their loving hospitality and Hanno Steinke,
Christoph Josten and Faramarz Dehghani for their support.

References[1] Pennal GF, Tile M, Waddell JP, Garside H. Pelvic disruption: assessment andclassification. Clin Orthop Relat Res. 1980 Sep;151:12-21.
[2] Collinge C, Archdeacon MT, Dulaney-Cripe E, Moed BR. Radiographic changes of
implant failure after plating for pubic symphysis diastasis: an underappreciatedreality? Clin Orthop Relat Res. 2012 Aug;470(8):2148-2153.
[3] Morris SA, Loveridge J, Smart DK, Ward AJ, Chesser TJ. Is fixation failure after
plate fixation of the symphysis pubis clinically important? Clin Orthop Relat Res.
2012 Aug;470(8):2154-2160.
[4] Kiskaddon EM, Wright A, Meeks BD, Froehle AW, Gould GC, Lubitz MG, Prayson
MJ, Horne BR. A biomechanical cadaver comparison of suture button fixation toplate fixation for pubic symphysis diastasis. Injury. 2018 Nov;49(11):1993-1998.
[5] Larbi A, Pesquer L, Reboul G, Omoumi P, Perozziello A, Abadie P, Loriaut P,
Copin P, Ducouret E, Dallaudière B. MRI in patients with chronic pubalgia: Is
precise useful information provided to the surgeon? A case-control study. Orthop
Traumatol Surg Res. 2016 Oct;102(6):747-754.
[6] Gamble JG, Simmons SC, Freedman M. The symphysis pubis. Anatomic andpathologic considerations. Clin Orthop Relat Res. 1986 Feb;203:261-272.
[7] Norton-Old KJ, Schache AG, Barker PJ, Clark RA, Harrison SM, Briggs CA.
Anatomical and mechanical relationship between the proximal attachment ofadductor longus and the distal rectus sheath. Clin Anat. 2013 May;26(4):522-530.
[8] Schilders E, Bharam S, Golan E, Dimitrakopoulou A, Mitchell A, Spaepen M,
Beggs C, Cooke C, Holmich P. The pyramidalis-anterior pubic ligament-adductor
longus complex (PLAC) and its role with adductor injuries: a new anatomical
concept. Knee Surg Sports Traumatol Arthrosc. 2017 Dec;25(12):3969-3977.
18

3. The topographical anatomy of the symphysis pubis

Journal Articles Originated from the Study Period in Japan Kawata S, Marutani E, Hirai S, Hatayama N, Omotehara T, Nagahori K, Li ZL,
Miyaso H, Pieroh P, Naito M, Itoh M (accepted) Spraying urea solution reduces
formaldehyde levels during gross anatomy course. Anat Sci Int. In press.
 Yamamura S, Hayashi S, Li ZL, Kawata S, Pieroh P, Nagahori K, Omotehara T,
Miyaso H, Itoh M. Investigations of cortical and cancellous clavicle bone patterns
reveal an explanation for the load transmission and the higher incidence of lateralclavicle fractures in the elderly: a CT-based cadaveric study. Anat Sci Int. 2018 Sep;
93(4):479-486. PMID: 29654552.
 Shiboi R, Hayashi S, Kawata S, Li ZL, Pieroh P, Koga H, Takano Y, Inanami H,
Itoh M. Anatomical relation between the accessory process and pedicle in the lumbar
vertebrae. Anat Sci Int. 2018 Sep;93(4):430-436. PMID: 29427147.
 Pieroh P, Li ZL, Kawata S, Ogawa Y, Josten C, Steinke H, Dehghani F, Itoh M. The
topography and morphometrics of the pubic ligaments. Ann Anat. 2021 Jul;236:
151698. doi:10.1016/j.aanat.2021.151698. Epub 2021 Feb 11. PMID: 33582299.
 Pieroh P, Li ZL, Kawata S, Ogawa Y, Josten C, Steinke H, Dehghani F, Itoh M. The
arterial blood supply of the symphysis pubis - Spatial orientated and highly variable.
Ann Anat. 2021 Mar;234:151649. doi:10.1016/j.aanat.2020.151649. Epub 2020 Nov
20. PMID: 33227373.

19

4. Immunohistochemical observations ofneurovascular structures associated withthe human sacrospinous ligament
Johann ZwirnerPART 1:

MAIN STUDY

Introduction

Sacrospinous ligament (SSL) suspension is a surgical procedure to treat pelvic organ
prolapse and can be performed either through a vaginal [1] or laparoscopic approach [2,3]. Despite the high subjective (more than 84%) and objective (more than 67%) successrates of SSL suspension, especially postoperative perineal, gluteal or lower extremity
pain [3, 4] forms an unpleasant side effect, which sometimes even necessitates the sutures
to be released [5]. The entrapment of nerves adjacent to the SSL such as the pudendal
nerve, inferior rectal nerve, inferior gluteal nerve, sacral nerve branches, posterior
femoral cutaneous or the sciatic nerve [6, 7] seems to be the most obvious reason for a
postoperative pain occurrence. Moreover, to avoid postoperative gluteal pain it wassuggested to keep the depth of needle penetration during SSL suspension to a minimum[6], essentially assuming a nociceptive function of the SSL. Studies on the proprioception
and nociception of ligaments are currently emerging [8, 9, 10, 11, 12, 13, 14] as thesefunctions are suspected to be involved in the pathogenesis of pain syndromes [15]. Little
is known about these qualities in the human SSL [16, 17] with missing qualitative andquantitative analyses according to the widely accepted Freeman and Wyke classification[18]. With the SSL being essential for the biomechanical integrity of the pelvic girdle[19] its proprioceptive function might be involved in various pelvic pain syndromes suchas pelvic girdle pain, sacroiliac joint dysfunction or pubic symphysis dysfunction. A
thorough analysis of the nociceptor distribution within the SSL might allow to define‘safe zones’, which might decrease the likelihood of trapping these structures during SSL
suspension and equally the number of women experiencing post-operative pain. The
given study aims at a qualitative and quantitative immunohistochemical assessment of the【Keywords】free nerve endings; immunohistochemistry; pain; prolapse surgery; sacrospinous ligament
Institution in Japan: Department of Clinical Anatomy, Tokyo Medical and Dental University, Tokyo, Japan
Supervisor: Prof. Keiichi Akita, M.D., Ph.D.
Period of Stay: From October 1, 2019 to January 29, 2020
Submission Date of Manuscript: March 2, 202020

4. Human sacrospinous ligament immunohistochemistry

neurovascular structures of the sacrospinous ligament. As previous studies of otherligament-associated mechanoreceptors indicated a polar distribution close to the bony
attachments [13, 20, 21] with a potential positive influence on their sensitivity [22], westate the following hypothesis:
The density of mechanoreceptors of the central human SSL is significantly less comparedto the medial and lateral polar areas, implicating that sutures in SSL suspensions shouldbe placed centrally from a proprioceptive perspective.

Materials and Methods

Retrieval and handling of human sacrospinous ligaments
A total of 19 chemically unfixed right human SSLs were retrieved during forensic
autopsy at the Institute of Legal Medicine, University of Leipzig, Germany. The cadavers
were stored at 4° C after arrival at the mortuary. Study approval was obtained through the
ethics committee of the University of Leipzig, Germany (protocol number 486/16-ek).
The SSL retrieval was done as follows: Firstly, the surrounding connective tissue except
for the coccygeus muscle was removed by blunt dissection. Secondly, the SSL wasdetached from the ischium laterally by sharply cutting off the tip of the ischial spine using
a hammer and a gouge. Lastly, the SSL was carefully detached from the sacrum frommedial to lateral using a scalpel. After retrieval, the SSLs were fixed in premixed 4%paraformaldehyde for 24 hours before being transferred to a self-mixed phosphate buffered saline solution (PBS; pH=7.4) for storage. When further processed, the SSLs were
cut into 3 equispaced subsamples (medial, central and lateral) perpendicular to a line
between the tip of the ischial spine and the proximodistal midpoint along the medialdetachment of the SSL.

Histology, immunohistochemistry and scanning electron microscopy
The SSL subsamples were embedded in paraffin and cut into sections of 20 mm thickness
using a rotary microtome (Leica RM 2235, Wetzlar, Germany). Cutting was performed ina transverse plane from superficial (coccygeus muscle facing side) to deep at 3 levels foreach individual subsample. The mounted sections were deparaffinized using xylene andrehydrated with a decreasing ethanol series. For haematoxylin and eosin (H&E) staining,
the sections were incubated as follows: Gill’s haematoxylin for 4 min, tap water for 2
min, Scott’s tap water for 2 min, tap water for 2 min, eosin for 30 s, tap water for 1 min
and then dehydrated using an ethanol-xylene series. For the immunolabelings with antiS100, anti-neurofilament (NF) and anti-von Willebrand Factor (vWF), the following
protocol was used: sections were rinsed in PBS-Tween (PBS-T; pH=7. 4) 3x7 min,
2%-hydrogen peroxide diluted in 60% methanol for 60 min, PBS-T for 10 min, blocker
consisting of 10% bovine serum albumin (Serva, Heidelberg, Germany), 0.5% donkey
serum (Jackson Immuno Research, West Grove, PA) and 0.3% casein (Carl Roth GmbH,
Karlsruhe, Germany) in PBS-T for 60 min, primary rabbit antibody overnight at 4° C,
21

Johann Zwirner

PBS-T for 3x7 min, secondary anti-rabbit-Ig-horseradish peroxidase-conjugated antibody, PBS-T 3x7 min, trishydroxymethylamin(Tris)-buffered hydrogen chloride for 5min, diaminobenzidine substrate kit (Cell marque, Rocklin, CA), PBS-T for 2x7 min andfinally left in distilled water for 2 min. The following primary antibodies were used:anti-S100-protein (diluted 1:500; BioGenex, Fremont, CA), anti-NF 200 (diluted 1:200;
Sigma, Saint Louis, MO) and anti-vWF (diluted 1: 400; DakoCytomation, Glostrup,
Denmark). All stained sections were coverslipped with entellan (Merck, Darmstadt,
Germany). Subsequent to the immunolabeling, the sections were counterstained using thefollowing protocol: Gill’s haematoxylin for 30 s, tap water for 2 min, Scott’s tap water for
2 min, tap water for 1 min and finally dehydrated using an ethanol-xylene series.
Negative controls without adding the primary antibodies were performed for the
immunolabeled sections. All steps were performed at room temperature unless specified
differently. Scanning electron microscopy was conducted on one lateral SSL subsample
using a JEOL 6700F field emission scanning electron microscope (JEOL, Peabody, MA).
Sample coating was performed in a K575X sputter coater with a 5-nm layer of gold
palladium (Emitech Technologies, Kent, England).

Counting of neurovascular structures and data analysis
All sections were digitalized using a slide scanner. The mechanoreceptors were analysed
according to the five specified types of the Freeman and Wyke classification [18]. Ruffini
endings (type I), Pacini corpuscles (type II), Golgi-like endings (type III), free nerve
endings (FNEs, type IV) and unclassifiable corpuscles (type V) were analysed in theanti-S100 and anti-NF immunolabeling. Vessels were identified using the anti-vWF
immunolabeling. For counting of mechanoreceptors the fatty and ligamentous parts weremanually outlined using ZEN lite 3.1 software (Carl Zeiss, Jena, Germany) and counted
according to the following rules: Brown DAB-highlighted spots of less than 5 micrometers were counted as free FNEs in the S-100 stain. It was assessed whether thesestructures were associated with blood vessels by scanning the area of interest in the antivWF immunolabeling. Also, S-100-stained structures of more than 5 micrometers werecounted to assess the number of larger nerve branches. Type I-III and V endings wereassessed using the mentioned stains and confirmed by the anti-NF stain. Data evaluation
was done separated for fat and ligaments to investigate the presence of mechanoreceptors
per tissue type. The obtained data was statistically evaluated using Excel Version 16.15(Microsoft Corporation, Redmond, WA) and GraphPad Prism version 7 (GraphPad So
ware, La Jolla, CA). P-values equal to or smaller than 0. 05 were considered to bestatistically significant.

Preliminary Results

Mechanoreceptors of all types were predominantly found in fat islands associated with
the SSL (Figure 1). The weight-bearing highly aligned collagens of the SSL only contained various FNEs, but did not surround any other receptor type according to the22

4. Human sacrospinous ligament immunohistochemistry

A

B

D

C

E

Figure 1: Impressions of the sacrospinous ligament and associated neuromechanics. Several
Pacinian corpuscules are shown in H&E (A), anti-S-100 (B) and anti-vWF (von Willebrand Factor)
stain (C). (D) shows a scanning electron microscopy image of the sacrospinous ligament and the ischialspine with related fat islands. (E) shows the sacrospinous ligament separated into the 3 different subregions (M, medial; C, central; L, lateral), which where compared in this study. White asterisk, ischialspine.

Freeman and Wyke classification. FNEs were almost exclusively associated with bloodvessels. Detailed analyses of the mechanoreceptor density in various sub-regions of the
SSL are currently being performed based on the obtained data.

Preliminary Conclusions

Mechanoreceptors of all types should be referred to as ‘associated with’ the SSL rather
than calling them part of the ligament itself. The present study highlights the importanceof fat as a ‘space holder’ or cushion for neurovascular structures.

PART 2:TIME IN JAPAN

Being the last student to receive the subsidy after 20 years the Yokochi Find was granted
to emerging researchers, I felt deep gratitude when the committee informed me that myproject had been approved. During my time at the Department of Clinical Anatomy ofTokyo Medical and Dental University (TMDU) I was surrounded by the outstanding teamof Prof Keiichi Akita making life very enjoyable for me and supporting me wherever they
could. In the future it will probably be very difficult to get a view out of my office thatcomes close to the one of the 18th floor of TMDU (Figure 2). Apart from studying I wasable to dive deep into the Japanese culture taking a few days of holiday over New Year23

Johann Zwirner

Figure 2: Impressions of my study period at Tokyo Medical and Dental University (TMDU). The
picture (A) shows the incredible view from Prof Akita’s lab at the 18th floor of TMDU. (B) was taken
at my welfare right before I left together with a couple of team members of Prof Akita’s lab. (C) shows
the venue, where the ceremony for body donors is held in Tokyo. (D) shows the TMDU building beinga landmark close to Ochanomizu station.

2020 and begin a new decade full of exciting research challenges in Japan. I enjoyed thestunning mountains in Kamikochi, Osaka’s delicious Okonomiyaki, the Golden Temple
in Kyoto and the calming sunset in Nagasaki overlooking the harbor. After four monthsof studying in Tokyo there is barely a street I haven’t walked or a food I haven’t eaten andI definitely felt this special attraction that fascinates Europeans about the Land of the
Rising Sun. Arigato!

Ceremony to honor body donors ─ a special experienceAs an integral part of the body donation program, researchers and students around theglobe honor the body donors with a special ceremony to acknowledge the irreplaceable
gift that science received through their donation. It was a special experience for me to bepart of this ceremony 9000 km from my hometown in Leipzig, Germany, and to observe
the way respect is shown to the body donors in Japan.

Research in Prof Akita’s lab ─ a German-Japanese success story
Soon after my arrival in Prof Akita’s lab I realized that German culture influenced
Japanese anatomists for a very long time. The library of Prof Akita’s department includes
a lot of anatomical treasures such as Walther Thiel’s ‘ Photographischen Atlas der
Praktischen Anatomie ’ (‘ Photographic Atlas of Practical Anatomy ’) or ‘ Studien der
Anatomie des Nervensystems und der Bindegewebes’ (‘Anatomical Studies of the Ner24

4. Human sacrospinous ligament immunohistochemistry

A

C

B

D

E

Figure 3: Several examples of the library of the Department of Clinical Anatomy of Tokyo
Medical and Dental University. (A) Several old German anatomical textbooks are shown. (B) The
title page of ‘Anatomical Studies of the Nervous System and Connective Tissue’ of Gustav Retzius isshown. The table of contents of book is shown (C) as well as impressions of Pacini corpuscules (D) andhandwritings (E).

vous System and Connective Tissue’). The latter was published in the last quarter of the
19th century by Gustav Retzius, the son of Anders Retzius, who gave his name to theretropubic space (‘space of Retzius’). This book contained numerous impressive drawings and detailed descriptions of mechanoreceptors of various species including humans(Figure 3). It is remarkable that 150 years after these studies were performed onmechanoreceptors by Axel Key and Gustav Retzius still so much about their function andmorphological environment remains unclear to date. Thanks to the Yokochi Fund andProf Akita’s team we were able to write another chapter of this story.

A little study on the side ─ biomechanics of the Achilles tendon-calcaneusplantar fascia complex
Apart from the main study on the neuromechanics of the sacrospinous ligament, Prof
Akita and I worked together on the topic of the structural continuity of the Achilles
tendon (AT) via the outer calcaneus to the plantar fascia (PF), which is a basic morphological and functional consideration of the structures related to the clinically-relevant
stretching treatment for plantar fasciitis patients. The study was based on plastination,
histology and biomechanical materials testing (Figure 4). We found out that there is
structural evidence for a morpho-logical connection between the AT and PF, which is inline with the concept of an integration and continuous remodeling of a tendinous AT-PF
connection during life. Moreover, in this study a robust testing protocol for the AT25

Johann Zwirner

(B)

(A)

(C)

Figure 4: Macroscopic observations during separation of the Achilles tendon- calcaneus- plantar fascia complex (AT- CAL- PF complex) of the small morphological/biomechanical sidestudy during my time in Prof Akita’s lab. (A) AT-CAL-PF complex viewed from proximal to distal,(B) The calcaneal tuberosity (white arrow) is shown. (C) AT-calcaneal periosteum-PF complex after
the calcaneus had been removed. AT, Achilles tendon; black arrows, transverse insertion line; CAL,
calcaneus; CP, calcaneal periosteum; PF, plantar fascia; RCB, retrocalcaneal bursa; white asterisk,
most proximal point of the calcaneal tuberosity; d, distal; m, medial; l, lateral; p, proximal.

calcaneus-PF complex was established that allows for a uniform tensile testing setup to
be used for both soft and hard tissues. This led to a preliminary biomechanical map of the
AT-calcaneus-PF complex from a materials testing perspective. This manuscript has
recently been submitted to the journal of Clinical Anatomy (Zwirner et al., 2020 [refer to
Publication Derived from This Study]).

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Publications Derived from This Study Zwirner J, Zhang M, Ondruschka B, Akita K, Hammer N. An ossifying bridge - on
the structural continuity between the Achilles tendon and the plantar fascia. Sci Rep.
2020;10:14523.

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