CHEMOTHERAPY80

ON THE

TakeruSEPT.1992

PHARMACOLOGICALEFFECTSOF CEFCLIDINCARDIOVASCULAR
AND AUTONOMICNERVOUSSYSTEMS

Kaneko, Masatoshi Fujimoto, Kanemasa Katsu and Hiroshi Yamauchi
Tsukuba Research, Laboratories, Eisai Co., Ltd.

Algate,Department

D.R., Baines,

M.W., Jobling,of Pharmacology,

Huntingdon

C.M. and Munt, P.L.,
Research

CentreLimited,

The effects ofcefclidin(CFCL), a novel antibacterial agent, on the cardiovascular andautonomic nervous system were examined using a variety of in vivo and in vitro models.
The effects of cefazolin (CEZ) were also examinated for comparative purposes.
Neither CFCL nor CEZ caused any notable effects on the cardiovascular or autonomic
nervous system when administered intravenously to anaesthetised dogs and cats or in vitro
preparations. Some evidence of a possible transient stimulation of the sympathetic nervous
system was seen following high doses of both CFCL and CEZ.
Key words : cefclidin, general pharmacology, cardiovascular, autonomic nerve,
respiration.

Introduction
Cefclidin (CFCL) is a new parenteral cephalosporin synthesized by Eisai Co. Ltd, Japan. It has abroad antibacterial spectrum and potent activity
againstgram-negative
bacteria
includingPseudomonas aeruginosa. CFCL also showed highresistance to hydrolysis by and low affinity forvarious types of fi-lactamases.
This paper describes the results of the pharmacological studies with CFCL in comparison withcefazolin (CEZ). CFCL and CEZ were each evaluated at two dose levels using a battery of standard
laboratory tests designed to investigate the effects
on the cardiovascular and autonomic nervous systems.
Materials and Methods
1. Materials
1) Animals
Male Wistar rats (203-226 g), male New
Zealand White rabbits (2-3 kg) , male and female
cats (3.45-4.7 kg) and male and female beagle
dogs (9.5- 11.0 kg) were housed appropriately in
air-conditioned
temperatureand humidity
controlled rooms. The lighting schedule was 12: 12

light/dark.
The animals were fed an appropriate laboratory
diet or commercial pet food. Tap water was available ad libitum except where indicated.
2) Test compounds
CFCL and CEZ were dissolved in physiologicalsaline solution at different concentrations.
The other test materials used were cefazolin(CEZ) (Fujisawa Pharmaceutical Co. Limited),
noradrenaline, adrenaline, acetylcholine, pilocarpine, adenosine diphosphate, 1-1- dimethy1-4phenyl piperazinium iodide (DMPP)(Sigma Chemical Company):anaesthetic
ether, sodium
thiopentone (May Baker Limited): heparin sodium(Evans Medical Limited) chloralose (BDH Chemical Limited):collagen (Hormon Chemie) andtrisodium citrate (FSA Laboratory Supplies). The
materials were dissolved in a medium compatible
with the test procedure.
2. Methods
1) Effects on respiration, blood pressure, heart rate,
femoral blood flow and electrocardiogram
Three male dogs were used in this experiment.
Dogs were anaesthetised with sodium thiopentoneCefclidinの 呼 吸 ・循 環 ・自律 神 経 系 へ の 影 響
VOL.40181

S-4(25mg/kg i.v.) and maintained under a-chloralose(60-80mg/kg i.v.). Respiratory rate and tidal flow
were recorded with the aid of a respiratory transducer (Grass PT5A) connected to the cannulated
trachea. The blood pressure was recorded by meansof a pressure transducer (Bell and Howell) connected to a cannulated femoral artery. Heart rate
was determined by integration of pulse waves witha tachometer (Grass Instruments). The blood flow
in a femoral artery was recorded with the aid of anelectromagnetic flow meter (Carolina). In addition,
the ECG (Lead II) was monitored by appropriately

3)

IsolatedThe

perfusedmethodLangendorff
rabbits.

onary

circulation

%

The

heart

was

by

aorta
was

at

CO2

The

essentiallyThe

maintained

37•Ž

means

heartsaascannulated

perfusedwithaerateda

Watsonpressure

rate

of

mmHg

20-60

ducer

(Bell

and

was

measured

positioned limb leads. All recordings were made on
a Grass Instruments chart recorder. Drugs were
injected into the cephalic vein through an indwelling
catheter. The effects of the test compounds on the
cardiovascular and respiratory parameters wererecorded over a 30 minute observation period.
Vehicle and two doses of both CFCL and CEZ were
examined in each dog.
2) Effect on hind limb perfusion
One male and two female dogs were used for thisexperiment.
The dogs were anaesthetised with sodium
thiopentone (25mg/kg i.v.) and maintained underachloralose (60-80mg/kg).
Blood pressure wasrecorded by means of a pressure transducer (Belland Howell) connected to a cannulated femoral
artery. The trachea was cannulated to allow unrestricted ventilation.
The contralateral femoral artery was bicannulated. Blood from the central end of the artery wasstraingauge

attached

Heart

rate

was

derivedelectronicallywererecorded

using

a

the

nictitating

S88).

The

contraction

pumped via a Watson Marlow flow inducer into the
distal end. Perfusion pressure was measured bymeans of pressure transducer (Bell and Howell)connected to the perfused femoral artery by meansof a T connector. The pump was adjusted such thatthe maximum perfusion pressure was equivalent to
the resting systolic blood pressure. The dogs
received 25,000 units of heparin prior to the start ofthe experiment.
Vehicle and two doses of both CFCL and CEZ
were examined in each dog. The effects of the test
drugs on the hind limb perfusion pressure wererecorded over a 30 minute observation period . A
minimum dose interval of 30 minutes was used.

wasrecorded

with

by

of

The

means

a

pressure

force

of
to

a

and

of

to

trans-

contraction

Dynamometer

the

apex

5

pump.

sufficient

recorded

Howell).

02
flow

was

parameters

means

cor-

solution

95%

which

chart

by

the

Krebs

a

four

and

with

at

by

from

Marlow

perfused

perfusion

described

removed

was

and
of

were

provide

heart

was

1)

male

rabbit

used

UF1

of

the

heart.
andall

Lectromed

recorder.

4)

Effect

on

brane

and

the

drenaline

contraction

and

The

of

cardiovascularbilateralcarotid

methodology

mem-

response

was

to

nora-

occlusion

based

on

that

described

were

used

by

Bevan2).
One

maleexperiment.
andmaintainedv.).

Bipolarandtwofemale

The

cats

were

under

trunk

and

a

second

the

right

tion

of

nerve

were

of

the

beneathsuperior
thewassuperior

nerve.

nictitating

membranewithvoltage

of

Hz,
the

aid

prenerve

trunk
elicit1ofa contrac-

theappropriate

ms,

10

rectangular

(50

the

the

cervicalTostimulated

supramaximali.

beneathpost-ganglioniccervical

this
ether

(60-80mg/kg
sited

left
the

for

with

a-chloraloseelectrodes

ganglioniccatsanaesthetised

pulses

of

s

Grass

nictitating

membraneisotonictransducersof(Devices).
Responsesof

intravenous
enaline

and

were

also

ducer

(Bell

femoralatopressureandandthrough

noradrenaline

occlusion

(BCO)

of

a

connectedpressuretotrans-

a cannulated

wererecorded

chart

recorder.

The

test

injected

into

indwelling
on
nerve
were

a

catheter.
theThe

responses

stimulation
recordedtoandduringtonoradr-parameterscompounds

gangilionic

rate

All

were
an

means

heart

1pg/kgcarotidbyHowell)artery.

Lectromedofbilateral

recorded

noradrenaline

test

blood

administration

using

drugs

and

cephalicveineffects

of

pre-

and

to

BCO

each

30

the
postandminuteCHEMOTHERAPY

182

observation

period.

Vehicleand two doses of both

CFCL and CEZ were examinedin each cat.

5) Effect on the cardiovascularresponse
stimulation, acetylcholine
and DMPP.
Three

to vagal

A femoral

arteryand cephalicbetween

7)

vein

Three

femoral artery cannula was connected to a heparin/
saline (250 IU/ml) -filled Bell and Howell pressure

redtransducer

coupled to a Grass Model 7E polygraph.

theThe heartrate was derived

prepared

EDTA

vagi

nm.

and 10,ug/kg DMPP at approximately

a

each

rabbitandplaced

into

tubes

washed
in

a 3%

to

the

at

37•Ž

for

in

on
of

Five
the

test

puncture

10

male

the

The

rats

methodthat a change

which was not within

respectively.

activated
of

iment

of

pupil

5

male

which

was

assessedsubjectively

ment. Ten minutes

adminis-1,

2

tration of the test drug, each rat received a rapid
intravenous
injection of 80mg/kg of pilocarpine

of

test

10)
The

and

five

munutesfollowing

andFollowingthesupernatantatwasdetermined

used

for

540

for

and

4

were

by

tail

time

bled

was

dete-

method

of

Dale

the
then

anaesthetised

removedfromofexperi-clotting

wereorbital

time

and

by

the

time
and

and
with

the

prothrombin

ProctorRapaport')

diameter
rabbits

pupil

were
in

used

after

for

a roomdiameter
one

hours

this

administration

rats

conducted

The

before

drug

using

and

on

intensity.

immediately

test

thromboplastin

Quick')

EffectGroupseye was measuredthesuspension

hours..

were

samplepartialJanssen and Niemegeers3).
Groups of ten male rats were used for this experi-of each

of

of

the

immediately

doses if it was judged

The pupil diameterwas' spectophotometerwhole-blood

cycles were included betweenhydrochloride.

cells

intravenous

compounds
andLaidlaw4).

9)

rats
after

determination

intravenous

redcoagulation

minutesforfollowing

the

cell
2

'EEL

blood

sinus

by

red

haemolysis

the same point in each subsequent cycle a dose of
one of the test articles was administered.
Extradescribed

repeated
Followingofabsorbanceana blood

used is based on that

the

by

centrifugation.

concentration

added

and

The method

times

suspenision

theGroupstest

earcontaining

saline.

ether

had occurredandexperiment.
marginal

centrifugation

threesalineisotonic

Effectrminedthe normal range.
6) Pilocarpine antagonism

used

Following

were

percentage

8)ly, over two minutes,

after

were

sample.

of

5 minutes

this

wash

The
each

10 minute intervals were repeated twice. Vehicle (5ml/kg 0.9% saline) was administered intravenouscommencingformeasuredment.

the second dose of DMPP and 5 minutes before avagal stimulation which initiated a third cycle. At

3 scalepilocarpine

fromcentrifugation,

Following equilibration, thirty minute cycles consisting of vagal stimulation,
injections of lug/kg
acetylcholine(or0following

taken

then

at 30 Hz, pulse width 5 ms,

6
7 volts for 30 seconds to elicit reproduciblechanges in blood pressure and heart rate.

rabbits

appropriate

was

was

nerves were stimulated

minutes

a

was

in

The

electrodes.

10

lacrimation
on

blood

final

stimulatedof bipolarandscoredeffectresuspension

incubatedpairs

salivation

anticoagulant.

were separated from connective tissure and severedat their cardiac
ends. The central
ends werebystereomicros-

of

cells

The

Both cervical

and

male

of

signal.
was catheterised.

of

Haemolytic

veinfrom theaeye-piece.

was5

the administrationof anaesthetic,
test compounds
respectively.
The

blood pressure
The trachea

using

1992

injection.

mlelectronicallya scaled

intensity

Five

and
and

measurementTheof bloodpressureagonists

to facilitateadministration

withchromodacryorrhoea)

male cats were used for this experiment.

were cannulated

pilocarpine
cope

Cats were anaesthetisedwith ether and maintained
witha-chloralose(60-80mg/kg)
administeredintravenously.

SEPT.

hour

of

with
both

beforeandintravenous

this
low

experlight

eyes

was

1/4,

1/2,

administration

drugs.
Effectmethodonplateletusedaggregation
was

based

on

that

described

bysample

(PRP)

was

Bore.
A

pooledplatelet-richplasmaCefclidinの 呼 吸 ・循 環 ・自律 神 経 系 へ の 影 響
VOL.40prepared

using

blood

teers

of eithered bycentrifuging

rpm

for

sex.

10

from

healthy

Platelet-richplasmacitrated

minutes

human

blood

and

volun-

wassamplespooling

preparat

the

1,000

resultantplasma.
The

concentration

300,000

perobtainedfrompooledsame

to aliquots

15 minutes

at

was

adjustedplatelet-poor

toplasmadonors.
of theof the

PRP

test

and

compounds

incubatedforaddition

of

37•Ž.

Aggregation

was(2/2g/m1),

theninducedcollagen(5•~10-6M)

andgregometerandrvogorusing

theplatelets

concentrations

added

ADP

of

mm3

Appropriate
weremeasuredtheby(10pg/m1),

or

using

traceswereaadrenaline

Bryston

ag-

on

a Se-

heart

rate,

recordedrecorder.
Results

1)

Effects

femoralrespiration,

blood

blood

on

flow

and

electrocardiogram

pressure,

and

CEZ

were

examined

at

doses

tendedtotemporarily(Figures

1-3)
CFCL
and

1,000mg/kg.

CFCL

Fig. 1-a. Effect of intravenous administration
respiration rate in the anaesthetised

Fig.

183

S‑4

1-h. Effectof intravenous

respiration

ratein theof300of cefazolin
dogonadministrationofonanaesthetiseddog

cefclidin

increase blood pressure at a dose of 300mg/kg but
had variable effects after 1,000mg/kg. CEZ alsotended to cause temporary dose-related increases inblood pressure. Heart rate tended to be reduced by
CFCL at both doses. The effects were small andtransient. CEZ tended to cause temporary increases
in heart rate.
An effect on respiration was seen after bothcompounds characterised by a dose-related reduction in tidal volume and increase in respiration rate.
The magnitude of responses after each compound
were comparable however after 1,000mg/kg CEZ
the effect were sustained.
An increase in femoral arterial blood flow wasobserved after both CFCL and CEZ. In conjunction
with these increases, femoral resistance was
reduced. Recovery from these changes occurred
within 30 minutes.
The electrocardiogram was not affected by either
dose level of CFCL or CEZ2) Effect on hind limb perfusion (Figure 4)
CFCL and CEZ were administered intravenously

Fig.

Fig.

2-a.

2-b.

Effect of intravenous
administration
tidal volume
in the anaesthetised

Effecttidalof intravenous
volumeadministrationin the anaesthetisedof cefazolinonof cefclidin

on

dog

dog

CHEMOTHERAPY

184

at

300

and

1,000mg/kg.

CFCL

at

300mg/kg

either CFCL or CEZ failed to modify the cardiovasreducedpressure

andrecoverywasminutes.

CFCL

also

causedpressure

at

blood

pressureCEZ

a doseofanddoses.

sure

were

seen

dogs

after

1,000mg/kg.

dogs

exhibitedAll

completeinin two

hind

limb

after

tremorly unaffected.

increased

rate.

perfusion

diminishedfifteen

CFCL

pressureatperfusionpres-

300mg/kg

after

1,000mg/kg
thecourseIsolated

perfused

CFCL

and

of

2 •~ 10-5

tions

had

and
no

contraction
Effect

and

the

and

bilateral

heart

This

range

3-a.

3-h.

concentraforce

response

carotidofmembranetolowestof intravenous
blood

dose

(300mg/kg)

administrationof cefazolin

flow in the anaesthetised

Effectof intravenous

femoralflow in theadministration
anaesthetised

at 300 and 1 , 000mg/kg.
The responses of blood pressureand heart rate tovagal

or DMPPstimulation,ofone animal.

on

Fig.

dog

of ccfclidindog

to vagal

acetylcholine

were

unaffected by any of the treatments.
CEZ (1,000mg/kg)
induced mild muscle tremor innoradrenaline

occlusionof theEffectofpressure,

response

stimulation, acetylcholine
and DMPP.
CFCL and CEZ were administeredintravenously

concentrationsof the nictitating

to pre- andganglionic blockade following CEZ. Both CFCL and
CEZ evoked a slight inhibition of the response tobilateral carotid occlusion.

the

rate.

cardiovascularmembrane

5) Effect on the cardiovascularatperfusion

on contraction

femoralFig.

onof the nictitating

CEZ
of

heart

perfused

2 •~ 10-4M.

Administration

Fig.

were

effector

4)

rabbitCEZ

responses

had no effect on
In general the

post-ganglionicstimulation
were similarly unaffected although one cat showed signs of a weakexperiment.
3)

CFCL and CEZ (1,000mg/kg)
the responses to noradrenaline.

all

and

over

or bilateral carotid

occlusion. Contractions of the nictitating membrane
to pre- and post-ganglionicstimulation were similar-

in perfusion

heart

dogs

cular responses to noradrenaline

perfusion

withina reduction

limb

Reductions

gradually

limb

1,000mg/kg.

hind

both

which

hind

decreased

increased

SEPT.1992

4-a.

Effectof intravenous

hind-limb

Fig.

4-b. Effectperfusionof intravenous

hind-limb

perfusion

administrationpressure

in theadministrationpressure

in theofcefazolinonanaesthetised

dogof cefclidinonanaesthetiseddogCefclidinの 呼 吸 ・循 環 ・自律 神 経 系 へ の 影 響
VOL.40

6)

185

S‑4

Pilocarpine-antagonism
Thepossible

CEZ

were

1,000

and

test

examined

CFCL

followinghighestinhibition
A weakdecrease

ved.

Generalsigns

at this

CEZ

7)

the

10-4M)

ofofcausedwaspotentiationon

10-5M)

on

also

human

plateletofpilocarpine-andhaemolytic

CEZ

were

2 •~

coagulation

CFCL

and

CEZ

at

of

devoid

any

300

and

1,000mg/kg

any

overt

effects

pupil

diameter

ADP,

No(Table(1 x 10-4M

andadrenaline-induced

aggregationwere

effectsof1.

Effectschangesof intravenouswasnotablevalue
of

Statisticalsignificanceof differencefrom*: p<0.05**: p<0.01***
p<0.001

Table

2.

controlof cefclidin

and salivation

group

analysis

Effectsof cefclidin

inducedby ADP, collagenusing

and cefazolin

on in vitro humanand adrenaline

was

upon

where

recorded

at

of

was

a

a
con-

effectCEZ

uponplateletaggregation

where

of

19%

was

recordedateffectsof notewereobservedforon pilocarpine-inducedin the ratof variance

CFCL

a

a
con-

1 •~ 10-4M.

and cefazolin

lacrimation

1 •~1 •~ 10-4M.

compound.

administrationin pupil diameter,andaggregation

30%

parameters.

Table

2)

examined.

ofplatelet

most

other

rabbit.

CEZ

notableatsignifi-andcollagencollagen-inducedonin theCEZaggregationof CFCLpotentiation

cause

no

value

centration

doses
to

and

of

The

effects.

blood

CFCL

produced

1,000mg/kg

adrenaline-induced(2 x 10-5and
of

of

and

plateletmost

centration
examineddiameteradministrationonpotentiationon

coagulation

effectThe

Effectfailedcantobser-

were

pupil

300

effects

effects.

CFCL

intravenously

of

The

effect

both

doses

10) Effectalevel.

a weakonlacrimaalso

however,

EffectIntravenous

and

doses

(2,000mg/kg)

toxicity,

concentrations

noteworthy

blood

CFCL

pilocarpine-inducedin salivation

cholinergic

Haemolytic
At

8)

dose

causedinducedofintravenous

dose
of

tion.

noted

9)

1)

effect2,000mg/kg.
at themarked(Tableanticholinergic(Student's

platelet't' distribution):aggregation

either

CHEMOTHERAPY

186

Discussion
CFCL and CEZ were examinedin a variety

of invivo and in vitro models selected to investigate
effects on the cardiovascularand autonomic nervous systems.
In the anaesthetised

dog preparation

CFCL andhowever,
evoked a transient
increasein meanfemoral arterial blood flow. At the low dose thethat seen with vehicle. The effect, however
more marked at the higher dose.
The apparent

vasodilation

causedtobecameIn aseparate anaesthetised
dog experiment,
where perfusion of the hind limb was maintained constant,intravenous
decreaseadministrationin perfusionofpressure

CFCL

suggestive

evokedasupportsympathetic
respiration

for a transientin theanaesthetised

revealstimulation

doses of 1, 000 mg/kg ofcat

any effects on the efferent

to induceparasympathetic

any remarkablecoagulation
parametersof erythrocytes.
The

induced any notableeffects

on the cardiovascularand autonomic
nervous system altoughtransientstimulationof the sympathetic

a slight
nervoussystem may be associatedadminis-trationwith intravenousof very high doses.
References1.

Langendorff,E.,(1895),
Physiol., 190, 280.

2.

Bevan, J.A., Selected

Pfugers

PharmacologicalIn the rabbit, CFCL at the highest dose (2,000 mg/
kg)
inhibitedpilocarpine-induced
lacrimati on.

4.

amacologia,1961, 11, 231-254.
Dale, H. H. and Laidlaw, P. P.

Whilst general

5.

volume seen in the dog after drug administration.

were also observed

P.

and

Niemegeers

In 'The haemorrhagicof Haemostasis',

Proctor,sympathetic receptors. The effect would seem to be
associatedonly with very high dose levels as no7.

Path ., 1961, 36, 212.
Born, G.V.R., (1962), Natureof an effect on pupil diameter

was obser-

J.Path.

physiology
Illinois.
6.

evidence

Testing
Inc.,

Psychophar-

Quick, A.J.

para-

post-ganglionic

ges.

Bact.,

1911, 16, 351.

this effect could also be
stimulation rather than anof the cholinergic

Arch.

Methods, ed. Burger, A., Mrcel Dekker
New York, (1968) ,p245.
Janssen,inhibition

drugs

or to disrupt the structureapparent
potentiationof3.

at this very high dose,
attributed to sympathetictwoeffects on blood

platelet aggregationinduced by adrenaline or collagen by the highest concentrationis unlikely to beof thenervous system was the increase inrate with concurrent
decrease in tidal

signs of toxicity

failed topathway by either CFCL or CEZ.
Intravenous
administrationof theof a sym-pathetically mediated action. CEZ similarly induced
a reduction in perfusion pressure, which was however subsequently followed by a vasoconstriction.
Further

receiving

CFCL or CEZ.
Experimentsof any biological significance.
It may be concluded that neither CFCL nor CEZ

by CFCL and

CEZ may not have been due to a local action.

ved in rabbits

failedCEZ showed few noteworthy
effects on systemic
blood pressure, heart rate or ECG. Both compounds,

effect of both CEZ and CFCL was comparable

SEPT.1992

R.R. and Rapaport,

disease and

1942,
S. I.

Thomas,Am. J. Clin.

No. 4832, p927.

Cefclidinの 呼 吸 ・循 環.自
VOL.

40

律 神 経系へ の影 響

187

S-4

Cefclidin一

般 薬 理 作 用:呼吸 ・循 環.自律 神 経系 へ の影 響

金 子 武 稔,藤 本 昌俊,勝鎌 政,山エーザ イ株 式会社 筑波研究所

Algate, D.R., Baines, M.W., Jobling,
Departmentof Pharmacology,

新 規 の 抗 生 剤,cefclidin(CFCL)の(CEZ)と内博

C.M. and Munt, P.L.,

Huntingdon

Research

Centre Limited,

呼吸 ・
心 脈 管 ・自律 神 経 系 に 対 す る影 響 に つ い て,cefazolin

比 較 検 討 した 。

麻 酔 ビ‑グル 犬 に お い て,CFCLは300mg/kgおよ び1,000mg/kgで,用量依 存的 に呼吸数 と分 時 換 気 量 が 増 加 した 。 但 し1回 換 気 量 は 変 化 しな か っ た。 血 圧 は 一 過 性 に 軽 度 上 昇 し,そ れに伴 う心 拍 数 の 減 少 が み られ た 。 心 電 図 に は 影 響 し な か っ た 。 また 後 肢 還 流圧 の 一 過 性 低 下 が
認 め られ た 。 さ ら にnoradrenaline,acetylcholine,DMPP,両脈 管 反 応,あ側 頚 動 脈 閉 塞,迷 走 神 経 刺 激 時 の 心

る い は神 経 節 刺 激 時 の 瞬 膜 収 縮 に 影 響 しな か っ た。 一 方,CEZに変 化 が 認 め ら れ,CFCLよつ い て も 同様 のり比 較 的 持 続 的 で あ っ た 。そ の 他,特 に 麻 酔 動 物 に お い て も振 せ ん が

観 察 され た 。
CFCLは2,000mg/kgでpilocarpineに
x10‑4Mでadrenaline惹

よ る 流 涙 反 応 を 抑 制 し た。 そ の 他,血液 系 に 対 し,1

起 血 小 板 凝 集 を軽 度 増 強 した 以 外,血 液 凝 固,溶 血 作 用 な どへ の 影 響 はな か っ た。
以 上,CFCLは*〒300‑26茨心 脈 管 あ る い は 自律 神 経 系 に 対 して,問城 県 つ くば 市 東 光 台5‑1‑3

題 と な る影 響 は な い と思 わ れ る 。