Shinshu Med J, 65⑴：37～44, 2017

Implantation of Autologous Bone Marrow-derived Cells Improves
Erectile Dysfunction in Spontaneously Hypertensive Rats
Masakuni Ishikawa1）＊, Tetsuya Imamura1）, Osamu Nishizawa2） and Osamu Ishizuka1）1）Department of Urology, Shinshu University School of Medicine2）Department of Urology, North Alps Medical Center, Azumi Hospital

Purpose : Erectile dysfunction (ED) decreases the quality of life. However, some patients are refractory to phosphodiesterase-5 (PDE-5) inhibitors. This preliminary study investigated the possibility that autologous bonemarrow-derived cells could improve the ED of spontaneously hypertensive rats (SHRs).
Materials and Methods : Erectile responses were induced by apomorphine. Bone marrow cells were harvested
from femurs of SHRs, and following culturing and labeling, they were implanted autologously into the corpus
cavernosum penis. Control SHRs received cell-free injections. At 7 days after the implantation, apomorphine-induced erectile responses were estimated. The presence of implanted bone marrow-derived cells in the corpora
cavernosa and the expression of neuronal nitric oxide synthase (nNOS) were determined by microscopy and reverse transcription polymerase chain reaction.
Results : The number of the negative reactions for apomorphine in the SHRs was significantly higher comparedto the Wister Kyoto (WKY) rats (P＝0.045). Ten of 15 cell-free control SHRs did not respond to apomorphine,
while 8 of 9 cell-implanted SHRs responded. Significantly more cell-implanted SHRs responded to apomorphine
than did cell-free SHRs (P＜0.013). The implanted cells formed clusters within the corpora cavernosa, and some
expressed nNOS mRNA and protein.
Conclusions : Bone marrow-derived cells autologously implanted into the corpus cavernosum penis significantly
increased the number of SHRs having erectile responses to apomorphine. Shinshu Med J 65 : 37―44, 2017
(Received for publication July 22, 2016 ; accepted in revised form September 14, 2016)
Key words : apomorphine, autologous bone marrow-derived cells, erectile dysfunction,neuronal nitric oxide synthase, spontaneously hypertensive rats

Ⅰ Introduction
Erectile dysfunction (ED) has a negative impact
1）
2）

on the quality of life

patients benefit from PDE-5 inhibition7）, many others do not1）6）. For those refractory patients, implantation of autologous bone marrow-derived cells,

over 50 ％ of men beyond 40

which are capable of differentiating into nerve, mus-years of age are affected by ED3）-6）. Administrationcle, and other tissues, may be an effective alternativeof phosphodiesterase-5 (PDE-5) inhibitors improves

treatment8）9）.

cyclic guanosine monophosphate (cGMP)-dependent

Spontaneously hypertensive rats (SHRs) are affect-smooth muscle and arteriole relaxation. This widely

ed with ED10）-13）. While the mechanism of the hyper-

accepted treatment for ED acts by amplification oftensive-related ED remains to be clarified14）-16）, these

the nitric oxide (NO) signaling level within the cor-

animals may serve as a suitable model to test thepus cavernosum penis. While approximately 70 ％ ofability of implanted autologous bone marrow-de-＊

Corresponding author : Masakuni Ishikawa
Department of Urology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan
E-mail : ishikawaof@shinshu-u.ac.jp

No. 1, 2017

rived cells to relieve ED. These cells, when implanted into freeze-injured urinary bladders or urethras,
differentiate into smooth and skeletal muscle cells
and restore physiological functions to these damaged
37

Ishikawa・Imamura・Nishizawa et al.

organs17）18）. Furthermore, considering future clinical

After confirming that each rat was alert, eachapplications, autologous cells are superior to allogen-catheter was attached to a pressure transducer

ic cells because the use of autologous cells is not(DX-100, Nihon Kohden, Tokyo, Japan). Intracavern-burdened with immunological rejection or ethicalous pressure signals were passed through a carrier

problems. Therefore in this preliminary study, we

amplifier (AP-601G, Nihon Kohden) and recorded byinvestigated the ability of implanted autologous bonea multichannel pen-recorder (Nihon Kohden). Aftermarrow-derived cells to remediate hypertensive-re-the pressures demonstrated stable traces, 0.1 mg/kg

lated erectile dysfunction in SHRs.

apomorphine was administered subcutaneously justⅡ
A

Materials and Methods

below the back of the neck. Our previous studies
confirmed that the dose was able to induce erectile
responses in healthy rats22）. We quantified the fol-

Animals
Male Wister Kyoto (WKY) rats and SHRs at post-

lowing parameters : blood pressure (cmH2O), time tonatal week 25 (Japan SLC Inc., Shizuoka, Japan)

first response (TFR ; minutes), number of peak pres-were used to assess the erectile responses. In sepa-sures per 30 minutes (PP30), tonic peak pressure

rate experiments, SHRs were used to assess the ef-(TPP ; cmH2O), and burst peak pressure (BPP ; cm-fects of implanted autologous bone marrow-derived

H2O).

cells. All animals were treated in accordance withD

Harvest and culture of autologous bone mar-

National Institutes of Health Animal Care Guidelines

row-derived cells

and the guidelines approved by the Animal Ethics

Twenty-four SHRs were anesthetized by inhala-

Committee of Shinshu University School of Medicine.

tion of sevoflurane (Abbot Japan Co., Ltd.). In eachBanimal, two pediatric bone marrow needles were in-

DrugThis study used apomorphine (Sigma-Aldrich,serted approximately 1 cm apart into a femur. The

Inc., St. Louis, MO, USA), which is the central dopa-

bone marrow cells were flushed out with saline andmine D1/D2-recepter agonist in the central nervous

collected through the other needle11）23）. After centrif-system to induce erectile responses19）-21）. The apo-

ugation, the cells were suspended with culture medi-

morphine was dissolved to the desired concentrationum composed of Dulbeccoʼs modified eagle medium

with 0.9 ％ saline.

(Invitrogen Life Technologies, Carlsbad, CA, USA)C

supplemented with 15 ％ fetal bovine serum (Biow-

Measurements of intracavernous pressure
The apomorphine-induced erectile responses of

est, Paris, France) and 0.1 ％ penicillin-streptomycin

untreated SHRs (n＝15) and WKY rats (n＝16) were

(Invitrogen). The harvested bone marrow cells wereassessed by intracavernous pressure measurements.

cultured on type I collagen-coated culture dishes

22）

As previously described , a 22-gauge needle at-(Asahi Glass Co., Ltd., Tokyo, Japan). During the cul-tached to a polyethylene catheter (PE-50) was in-ture, the medium was completely replaced every

serted into the corpus cavernosum penis of animals

day, and non-attached cells were discarded. At 5

anesthetized by inhalation of sevoflurane (Sevof-

days, the Qtracker 525 Cell Labeling Kit (Invitrogen)rane®, Abbot Japan Co., Ltd., Tokyo, Japan). The

was used for 24 hours to label the cytoplasm withcatheter was sealed and brought out subcutaneously

nanocrystals. The cells taking the marker during theto the back of the neck. For measurements of blood

culture period were easily detected within the recip-pressure, another single catheter was inserted into

ient tissues by a Leica DAS Microscope (Leica Mi-

the right carotid artery and then brought subcuta-

crosystems GmbH, Wetzlar, Germany) at 488 nm ex-

neously to the back of the neck. The skin incisioncitation. At two days after labeling, the adherent,

was closed. After the surgery, each animal was placed

proliferating, labeled bone marrow-derived cells

without any restraint in a cage to recover for over 2

were collected after brief incubation with a 0.25 ％hours.

trypsin solution17）18） and then suspended in culture

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Autologous bone marrow-derived cells improves erectile dysfunction

medium at 1.0×107 cells/ml (viability ＞ 80 ％).

synthase (nNOS, 1 : 300, goat polyclonal, Osenses Pty

ELtd, Flagstaff Hill, SA, Australia). The sections were

Implantation of autologous bone marrow-derived cells

rinsed with PBS at 4 ℃ , and then incubated with

Seven days after the culture, the SHRs from which

secondary antibody consisting of donkey anti-goat

the bone marrow cells were collected were randomly

IgG conjugated with Alexa Fluor 594 (1 : 250, Molec-

divided into cell implantation (n＝9) and cell-freeular Probes, Eugene, OR, USA) for 1 hr at 4 ℃. Neg-

injection control (n＝15) groups. After re-anesthetizing

ative controls were performed without the primary

the SHRs, the corpus cavernosum penis was exposed.

antibody, and positive controls were performed with

For the cell implantation group, the suspended

rat lungs. Following rinsing, the nuclei were counter-6

autologous bone marrow-derived cells (1.0×10

stained with 4ʼ, 6-diamidino-2-phenylindole dihydro-

cells/100μl) were autologously implanted with achloride (DAPI, 5μg/ml, Molecular Probes), and then

29-gauge syringe into the corpus cavernosum penis

coated with Fluorescent Mounting Medium (Dakoat the 3- and 9-oʼclock positions. For the cell-free in-

Cytomation, Carpinteria, CA, USA). The slides were

jection control group, 100 μl of cell-free culture me-observed and photographed with a Leica DAS Mi-

dium was similarly injected at the same positions.

croscope (Leica Microsystems GmbH). The other

The implantation cell number and volume were cho-

sections from each sample were stained with hema-

sen to avoid further damaging the host tissues or

toxylin and eosin (H&E).

the implanted cells with shear stress

17）
18）

. At eachG

Real-time RT-PCR

operation, the retention of small swellings that in-

For real-time RT-PCR, the corpus cavernosum ofcluded the implanted cells or control media was vi-

SHRs were homogenized, and total RNA was ex-

sually confirmed.

tracted with the RNeasy Mini Kit (Qiagen Inc., Va-

At 7 days after these operations, the apomor-

lencia, CA, USA). Complementary DNA (cDNA)

phine-induced erectile responses of the cell-implant-

was synthesized from 0.2μg of total RNA with theed and control injected rats were estimated by intra-

High-Capacity cDNA Archive Kit (Applied Biosyste-

cavernous pressure measurements (described above).

ms, Foster City, CA, USA). Real time RT-PCR of

Following the measurement, the rats were eutha-

the cDNA was performed at 50 ℃ for 2 minutes fol-nized, and the corpus cavernosum penis was re-lowed by 95 ℃ for 10 minutes. These were followed

moved for immunohistochemical investigations andby 40 cycles at 95 ℃ for 15 seconds and 60 ℃ for 1

real-time reverse transcription polymerase chain re-minute. The primer of neuronal nitric oxide synthaseaction (RT-PCR) as described below.

1 (Rn00583793_m1, Applied Biosystems) was usedF

to measure the nNOS mRNA expression level. Gene

Immunohistochemistry
Tissue samples were fixed in 4 ％ paraformalde-activity was expressed as the ratio to the internal

hyde and 4 ％ sucrose in 0.1 M phosphate buffer (PB),

standard gene beta-actin (Rn00667869_m1).

pH 7.4, for 12 hr at 4 ℃, and then embedded in par-H

Statistical analysis

affin. The sections (3μm) were cut on a microtome

Results were expressed as means ± standard de-and then deparaffinized, rehydrated, rinsed threeviation. We determined statistical differences withtimes with 0.01 M phosphate buffer saline (PBS), andFisherʼs exact probablility test. Non-repeated mea-

immersed in 10 mM sodium citrate, pH 6.0. For anti-

sures ANOVA followed by the Tukey procedure

gen retrieval, they were then microwaved at 100 ℃

was used among experimental groups having thefor 5 min. The specimens were treated with 1.5 ％apomorphine-induced erectile responses. Also,

normal donkey serum (Chemicon International Inc.,

unpaired t-tests were used to compare between

Temecula, CA, USA) and 1.5 ％ non-fat milk in PBS

the cell-implantation and control injection group. P-

for 1 hr at 4 ℃. The sections were incubated for 12

values less than 0.05 were considered significant.

hr at 4 ℃ with antibody for neuronal nitric oxide

These tests were performed with GraphPad InStat®

No. 1, 201739

Ishikawa・Imamura・Nishizawa et al.
Table 1

Apomorphine-induced erectile responses in normal WKY rats and SHRs
Apomorphine-induced Erectile Response
Positive Response

Negative Response

14
7

2
9

WKY rats (n＝16)
SHRs (n＝15)

Fisherʼs exact probability test. P＝0.045, erectile response for WKY rats vs SHRs.
Table 2Intracavernous pressure measurements of rats having apomorphine-induced erectile responses
TFR (minutes)

PP30

TPP (cmH2O)

BPP (cmH2O)

9.6±5.6
8.5±3.2

2.4±1.3
2.7±2.176.4±47.8
49.2±30.0

159.6±97.0
93.5±63.1

Untreated
WKY rats (n＝14)
SHRs (n＝7)

TFR ; time to first response, PP30 ; number of peak pressures per 30 minutes,
TPP ; tonic peak pressure, BPP ; burst peak pressure.

(GraphPad Software, Inc. San Diego, CA, USA).
Ⅲ

C

Effects of autologous bone marrow-derived
cells

Results

Blood pressure of the cell-implanted SHRs, 225.3±

The blood pressure in normal SHRs, 236.4 ± 26.4

16.9 cmH2O, was not significantly different from the

cmH2O, was significantly higher than that of the nor-cell-free injected control SHRs, 234.0±47.4 cmH2O (Pmal WKY rats, 154.4±21.7 cmH2O (P＜0.01).

＝0.623). Seven days after implantation, 8 of 9 cell-

A

Erectile functions of normal WKY rats and SHRs

implanted SHRs developed apomorphine-induced

Apomorphine induced an erectile response in 14

erectile responses. In the cell-free control SHRs, onlyof 16 WKY rats (Table 1). In contrast, 7 of 15 of the

5 of 15 rats (P＝0.013) responded (Table 3). Neither

untreated SHRs responded to the apomorphine, while

TFR nor PP30 for the cell-implanted SHRs differed

8 did not. Thus, the negative responses by the nor-significantly from the cell-free controls. While TPP

mal SHRs were significantly greater than that by

and BPP in the cell-implanted SHRs tended to be

the normal WKY rats (P＜0.05). For the 14 responsive

greater than in cell-free controls, the difference was

WKY rats and the 7 responsive SHRs, there were no

not significant (Table 4).

differences in TFR, PP30, TPP, or BPP (Table 2).

D

B

Cultured autologous bone marrow-derived cells

Histological and gene expression evaluation of
the corpus cavernosum penis

Immediately after seeding, the harvested bone

Seven days after implantation in the penile corpo-

marrow cells consisted of heterogeneous, spin-ra cavernosa of SHRs, the bone marrow-derived

dle-shaped, round, and polygonal cells, along withcells formed clusters (Fig. 1A) that contained numer-

red blood cells. After 7 days of culture, the adhered

ous Qtracker 525 nanocrystal-labeled cells (Fig. 1B).

proliferating cells reached approximately 90 ％ con-

In contrast, the cell-free control injected SHRs didfluence and were relatively homogenous in spin-

not have the cell clusters (Fig. 1C) or nanocrystal-la-dle-shaped appearance. The cultured autologous

beled cells (Fig. 1D). Some of the labeled implantedbone marrow-derived cells did not stain with anti-

cells were positive for nNOS antibody (Fig. 2). The

bodies for nNOS just prior to implantation. Thus,nNOS mRNA expression level of cell-implanted

this finding suggested that the cells might not differ-

SHRs, 1.18±0.43, tended to be higher than the con-entiate into nNOS-produced cells during the culture

trols, 0.93±0.25, but the difference was not signifi-

period.

cant (P＝0.296).

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Autologous bone marrow-derived cells improves erectile dysfunction
Table 3 Apomorphine-induced erectile responses in cell-implanted and cell-freecontrol SHRs
Erectile Response
Positive Response

Negative Response

5
8

10
1

Cell-free control SHRs (n＝15)
Cell-implanted SHRs (n＝9)

Fisherʼs exact probability test. P＝0.013, erectile response for cell-free control SHRs
vs cell-implanted SHRs.

Table 4 Intracavernous pressure measurements of rats having apomorphine-induced
erectile responses
TFR (minutes)

PP30

TPP (cmH2O)

BPP (cmH2O)

8.7±7.5

2.2±1.385.3±46.3

206.8±125.4

8.3±4.92.5±1.4

130.3±83.4

317.2±185.6

Cell-treatment
Cell-free control
group (n＝5)
Cell-implantationgroup (n＝8)

TFR ; time to first response, PP30 : number of peak pressures per 30 numites,
TPP ; tonic peak pressure, BPP ; burst peak pressure.

Fig. 1 Histological evaluation of the penile corpus cavernosum. (A) The cell-implanted corpus cavernosum contained clusters of cells (asterisks). (B) These cells contained the green fluorescent-labeled nanocrystals(green) taken up while the cells were in culture before implantation. (C) The cell-free control injected corpora cavernosa did not have any clusters or (D) nanocrystal-labeled cells. Blue, DAPI-labeled nuclei.
No. 1, 201741

Ishikawa・Imamura・Nishizawa et al.

Fig. 2 Immunohistochemistry for nNOS antibodies.
The Qtracker-labeled implanted cells (green)differentiated into nNOS-expressing cells (red,arrows) within the penile corpora cavernosa.

phine-induced erectile responses of SHRs might be

Ⅳ Discussion

mediated by not only the nNOS-related NO-depen-

Along with aging, hypertension, diabetes mellitus,

dent mechanisms, but also by other neurogenic and/and hyperlipemia are considered to be risk factors

or muscular systems to induce smooth muscle and1）
5）

for ED. The SHRs are used as an animal model ofarteriole relaxations.

hypertension as well as models for lower urinary

Bone marrow-derived cells are capable of differ-tract symptoms such as C-fiber related detrusor

entiating both in vitro and in vivo along multiple

24）
-26）

27）

pathways that include nerve, muscle, bone, cartilage,. In our study, theadipose tissue, tendon, and connective tissue 8）9）.

blood pressure of the sham-operated SHRs was sig-

These cells, when situated in areas of tissue damage,nificantly higher than that of the normal sham-oper-

have paracrine effects that produce cytokines andated WKY rats. The frequency of erectile response

growth factors based on available signals from theto apomorphine in SHRs was significantly less thansurrounding tissues29）30）. Thus, the paracrine effects

that in WKY rats. These results are consistent withof the implanted bone marrow-derived cells might

damage to the nervous system, microcirculation,

contribute to the reconstruction of healthy peripher-and/or smooth muscle of the corpus cavernosum inal nervous system elements, microcirculation, and/orSHRs.

smooth muscle systems within the penile corpora

overactivity, benign prostatic hypertrophy ,and hypertension-related ED

12）
-15）

The percent of SHRs having apomorphine-induced

cavernosa.

erections was significantly greater in the cell-im-

This study had some limitations. While our results

planted rats compared to the cell-free injected ones.

showed that the implantation of bone marrow-de-

The erectile response is initiated by NO produced

rived cells improved the apomorphine-induced erec-

by nNOS and liberated from nonadrenergic and non-tions, we have not thoroughly demonstrated thecholinergic neurons in the corpora cavernosa28）. We

mechanisms of the recovery. In this study, we per-showed that the implanted bone marrow-derived

formed cell direct-injection methods to implant thecells formed clusters within the corpora cavernosa.

cells. While we detected the implanted cells within

Prior to implantation, these cells did not express

the penile corpora cavernosa, the cell numbers were

nNOS, but one week afterwards, some of them did.

very small. Thus, we need to improve the survival

The implanted cells differentiated into nerve-likerates of the implanted cells in the penile corpora

cells that produced nNOS, while this study did notcavernosa by using the novel techniques, such as

identify these cell types. However, the overall level

cell sheet engineering31）. In our next study, we willof nNOS mRNA expression was similar between thetest these effects and the safety over a long period.

cell-free injected tissues and the cell-implanted

In this study, we conducted autologous cell im-

ones. Thus, our results suggest that the apomor-

plantation by harvesting bone marrow cells from a

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Autologous bone marrow-derived cells improves erectile dysfunction

femur of each anesthetized animal. The cultured au-

Ⅴ Conclusions

tologous cells were similar to the cells harvested

The proportion of normal SHRs with an erectile

from femurs of the euthanized SHRs according to10）

our previous methods . The majority of patients

response to apomorphine was significantly lower

with ED are elderly or have other diseases that may

than that of normal WKY rats. SHRs implanted withaffect the developmental potential of bone marrowautologous cultured bone marrow-derived cells had

cells. However, our results suggest that bone mar-a higher frequency of response than did cell-free in-row-derived cells harvested from the SHRs behave

jected control SHRs. Therefore, the implantation ofwith the same potential as those from healthy ani-

autologous bone marrow-derived cells has the po-mals. Therefore, the bone marrow-derived cells har-tential for development into a treatment for refracto-

vested from aging and/or diseased patients also

ry ED.

might retain their developmental potential and beⅥavailable in treatments for ED.

Conflict of Interest

There are no conflicts of interest to declare.
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