Intense peripheral electrical stimulation evokes brief and persistent inhibition of the nociceptive tail withdrawal reflex in the rat

Rrrun Research 761 ( 1997) 192-202

Research

report

Intense peripheral electrical stimulation evokes brief and persistent inhibition of the nociceptive tail withdrawal reflex in the rat

[n a study of mdull\tion

of nocicepuon by henbury inputs.

cffccts on the nociceptive tail withdrawul defiwd

sites in the hindlimb,

ms square pulses

given for

application of a noxious

reflex

meridian points j&~~~firtlr 20 min

heat stimulus

at 20 x

clcctrical stimulation

v.erc monitored

in the lightly

(ST-32).

the threshold

wit\ clpplicd to specific >ilr’\ in the hindlinlh

anucsthetizcd

Ji*,~g.dri (GB-3 1) and zmtrrrli (ST-36).

intensity

applied to the tip of the tail.

required Results

for

muscle

atimulution

only.

pohc-stimulutian T~CW

thtu

At 7 i\nd I4 days after

spinal

increase wus also observed. Tho

suggest thut high intensity.

prtrluces

both brief

und persistent

diffcrcm

mcchunihms.

The prsistcnt

ilkhciW

U makjor piWticipotion of supruspinill Elsevicr

Wucturc\

:I

plkc

of hindlimb

hut tllilt

chilngc III ccntrirl

spilld

known

rcflc~.

inhih~tory

hy

possible

us the cut off.

During

the inhibition

during the 4mulntion

only.

no p)st-~tilllul;ltiolI

effect

;I stn;~ll. hricf incrcaw

meridian

II~CC/liUli4111~Urc UISO

was provoked

stimulation.

the stimulrrtion:

htimululion

effects on the ncwiccptivc tuil withdr;l\vul

cftkct m;ry reprcscnt

@> lW7

&I~ulution

withdrawal

Following

in I~WIIO~ hut during

was grcatcr in lll~~gllitUd~ and u hrict

rcturn of (hc 01’this I;rttcr c‘ft’cct wus coincident

low l’rcqucncy clcctricitl

inhibitory

rchponsc during

It consisted of it 4 HZ train of I!

Hz evoked inhibition during

XKI

was applied 10 previously

us a percentage of the maximal

inhibition.

of meridian points rlicitcd

this

tr;rnscction,

Tail

latency othawise

which is achieved when the post-treatment

stimulation.

was produced. In ucutely transcctcd unimols ( I 48 h). .stimulution

twitch.

were expressed

latency is :! X the pre-treutment the latency of the withdrawni increased to = 70% of the tnaximal possible persisted for > 1 h. Stimulationat 2 or 6 Hz elicited similar effects but stimulation at 8 S(imulution applied to sites uwuy from defined mcridian points inhibited tail withdruwul inhibition

rut. Stimulntion

with puints

Thcsc

the rckn+n of hladdcr function. in the lightly cl’lcc~s

mcch;uli\ms.

Cil~Xlhll2

Ol’

q~pc’ar lhtu

~u4lainmg

anaeslhc~i/cd rut to

hc

rlicilcd

by

front \pin;d iulillluls Ilctth ljj)L’\ Ill CWCCl.

Science B.V.

1. Introduction Both high and low threshold sensory inputs are thought to modify central mechanisms of nociception. For eXiu~lplC. light vihrudon [30,X] and aversive stimulirtion [31,33] have both been used to idleviutc pain in humans. In unimid studies. vibration applied just outside the receptive field depresses c&vity of wide dynamic range neurones in rhe cat dorsni horn [44]. High intensity thermal stimulation applied to the same dermatome in the rat facilitates a nociccprive withdmwul reflex [14,15]. In contrast to this evoked fuciliturion, an inhibition has been observed when iI high intensity stimulus is upplicd ut iI different dcrmatomal level [36]. In human patients and subjects, high intensity electrical stimulation has been shown to attenuate chronic and expwimentul pain [2.3.17,32,47]. However, attempts to elicit a parallel effect of electrical stimulation in experimenral animals huve gcncrally led to un antinocicepdon which may be attributed to stress. In awake mice. low frequency electiiciil stimulation ut inknsilies subthreshold to produce vocalization evokes a naloxone-reversible, post-srimulation antinoclception which lasts up to 2 h [38,40]. This effect may have been related to stress because the intense stimulus was applied to

’ ~t~Sptiinp ;luthW. Dqxumlenl of Physiology. McGill University. 29X-7452; E-mail: mc’[email protected]

MO6-W93/97/Sl7.1W) 8 I997 Elscvicr Science B.V. All rights rewrvcd. PI/ s00u6-8’~93~97!00250-3

3655 Drummond Street. Montreal, Que.. C1.G IYh. Ctindu.

f-%x: + I (514)

awak.

rcstrainsd animals and strcskinduccd

t’nvir(9mnent.

which

allltinociccptiol~ ;ippcarb to 19~’ opioid mediated. g:~r cx;mlp~c. ral, II1 ci Zl(,, cl

is prcsumcd lo be mildly

>trcssful.

cxbibit 3 p~tentiated inot?hjtl~-ir9~ila~~el ;ti~tllli:~,C~~‘:91ai,j, it)] ar:~

rcstruincd rtik subjcctcd to ;t non-avc‘rsi\ L‘i;919(-shoch Sht9w stimulus

[-I I]. Sonw 3nirn;i!

frequency ~~hle

yet

strrdic\: ha\~

intensity

ilP.Oilkd

tllC

a ncriouolle-s~nSili\

Cll’CCt\

e al~tin~9ci~~pti~9r~

clcctric

9roJucc4 illltii1~9~i~~~~(lOi? hut tlij4 ct‘ti.cl ix analgck~ produced in human\ hc high inten\&!. ICW, fr~~qucnc! cicctricltl \timulutioil. h9w

in ~~~tic~p~tio~~

iI1 IRC ;ln&!SthCil/cd

Of’ S?FC*S;

(9t a 19al,ltal

cat f-371 ;Ind :‘;1t [jyj hrjcf

arId

thcr&>

It >lgificant

Lo\\

&9~4

f9(jt

3,pcc1 C,l the

::n:algc\i:a rcportcd ir> human plflicnt\ ;1n9 subjecfh given high iljrcnsitv clcctric;ll ~imui;lrion i\ the prolonged II;IWI’C crf fh‘* to hours to cvcn days [17.32.47]. The closest model that reflects a pro]onged

analgesic effect. lasting several minutes. anlits&ceptivc

effect has been achieved in several animal studies by high intensity,

applied directly to peripheral nerves. This

produces ;m inhibition

which lasts IO-60

of the sural-gustrocnemius

min [I?],

and un inhibition

an inmbition

of spinothalamic

tract cells in anacsthaized,

animal models are all restricted to segmental mechanisms generalized stimulation

analgesia which [ 17.32.47).

‘non-meridian

n14ks.

stimulation-evoked

IIUI~~S

Analgesia is evoked by stimulation

mimill

reported [hat stimtkrtion

reflex in the decerebratea and spinalized rabbit [5~]

decerebrated and spinalized

monkeys [13]. However.

antinociccption.

thehe

as opposed to the more low frequency electrical

type of stimuli\tion in humans is oticn rcferrcd LO ;LS clectroacupuiicture.

points’ [I .4K]. Similar

humans. However.

low frequency electrical stimulalioll

reflex in decercbrated and spinalizcd c;tts

charac*tcrizcs the analgesia-induced in humans by high intensity.

This

One t’eitture which characterizes clcctroucupunctttr~ in of the stimulating

of

of the flexion

is the WC of tncridiiul points ah spccici~~sites for insertion at meridian points but not hy srimuMon

at other. .\o-called

meridian points hitvc been reported in experimental animals [4,5.37] based on p19iuts in

studies hnvc ~cIler:tlly

hilcd

of n1txidian iulcl non-mcridixn

to compare tncridiu

vs. non-mcridiatl

point stimulation

points achieves Gmilar It\ cls of’ :Intinocic~ption

Our objcctiv2 in the prccait Gudy \t’;l:%to in\tsii ‘1 ’ gt~tc the cffccts oi‘ high intensity,

or ha\x

[s].

IOW l~rc‘qt~~ncyckctrical \tll~~t~li~tit~~~ of

peripheral sites on nooiceptivc mechanisms in the rat. Aticr

cxplorin g a number of possible models. the one in which ;I persistent antinociception could be prodl;ced reliably and in which stress could be eliminated. wits the lightly tin:iesthetiLed

rut [18.23.46.X?].

We cscd

il

used clinici~lly for alleviation

high intensity, of pain in

low frequency train of clcctrical stimuli.

IIUI~~I~S

[Z,

17,X.47].

frequency tramcutaneous clectricul nerve sti.:?ulrrtion on activation

of Iargc diameter aff&en(s

Ms.

IWiiriin;iry

In

ill1 CiISCS.

versions of tllc

1llC guidclincs

Council on Anitnal McGill

University

c‘;irc

Animal

Male Sprague-Dawlcy

dillil

(TENS

or so-called ‘conventional TENS’)

(441. In addition.

;lcuplliicturL’-induCCd ontinociceprion [JS,SJ]

as lhesc lprtramctcrs rescnihlc those

Thcsc parnmctcrs arc very diffaat iiS

sc9mc sludies

liclvc2 inq~licated supmpinal

a series of experiments was done to include LL similar

IlilLC been prcscnted in 49strXt

I‘)Ilou~d.

hlorcovcr.

high

slructurc4

II1

study in spinal transclM

form [42,43].

dC4~iIlNXi in I’llc, C*ctt.(*trtkl ll.\c~ (!I’f::\/t~,t.itttc~tl/t// fIt/itttrt/.\.

were strictly

i’rz)m I(,w intcnsi,j.

which appc:\rs lo hc based

tlic culwitiicntal

\‘fjlr.

I (t/Id II, 01 tllC

(‘;lllidi~Ill

~~rotocol4 wcrc rcvicwcd ;urtl 49l91~ncd I9y lhc

C’are COIIIIIII~~CC.

rats (3%400

g) were lightly

iul;testhetizcd

for the duration

injection of a freshly prepared mixture of sodium pcntobarbital (20 q/kg, mg/kg, Fisher Scientific) in 50% propylene glycol and 30% physiological

of the cxpcrimcnr with

1111i.p.

Abbott Lnboratorics) saline (0.9% NKI).

and chlorrtl hydrate ( IX This llrovidcs iI level of is anuesthesia sufficient to prevent any overt sign of discomfort to the rat during experimentation. yet 11 StilblL rcslxxw obtained in the tai! withdmwal test for = I h. To maintain this light state of imitcsthcsin throughout rhc durution of IIZC , fl)C initiitl JON! Of Illlil~StlWtiC Wllb experiment, subsequent injections of the mixture were given. Thus. ;m injection of ’ given 35.5 tnin after the first stimulation.

injection; this injection wits timed to occur I .S min prior to the hcpinning of clcctrical Subhcqucnt injections of \, : UKI I/6 the initial dose were given iIt 20 min inlcrvals.

The lcvcl of nokxption

was detormincd us the latency of tail withdrawal

of the tip of the tail was blrtckcncd to facilitate the absorption of heat. This bulb to elicit the tail withdrawal

reflex. This

withdraw;LI

giving reaction time measured to 0.01 s. The intensity the deviation wils time.

from a noxious radiant heat stimulus.

A portion

portion \viIS positioned abr)ve ;I focused prc!jcctor

cxposcd the light bcum to a photodctcctor which sl~ppcd 3 tinier

of the bulb was set so that the bnscline reaction time was 4-6

< 10%. The timer was iurned off automaticrtlly if II tail withdrawal

did not occur within

s iind

I2 s. the cut-off

At each ,sumpletime, two readings were taken, separated by JO-50 s. at two different sites roslral and caudal within the 2 cm bl;zcke& .segmentof the tail. Thus, the tail withdrawal latency was never measured twice from the samt! site within a 3-5 min pried. The mean of the two readings was calculated and the vdue expressed as I percentage of the m~~ximum possible inhibition (MPI) according to the formula MPI

=

(POST-TREATMENTLATENCY (CUT-OFFTIME

- PRE-TREATMENTLATENCY)

x 100

- PRE-TREATMENTLATENCY)

The MPI occurs when the post-treatment latency equals that of the cut-off time. To me&sure the latency of the tail withdrawal during the period of the electrical stimulation. the stimulator was temparhfy turned off, just long enough for the reading to be taken; this was necessary because the stimulus was above the threshold to elicit a direct contraction of muscles (vide infra). 2.3. Electricul stinuhtion Previous studies have defined meridian points in the hindlimb of the rat [4,S] znd cat [37]. Stimulation of these silts elicits an antinociceptive or analgesic effect. The present study is based on stimulation of thcsc sites as illusrrated in Fig. I. Two pairs of stainless steel insect pins were inserted in the vicinity of meridian points .t>~N1~~:fittli (ST-321 2nd ,fiwg.shi (GB-3 I) as defined by other investigators [4,5.37] or in non-meridian points in the nearby medial and latcrol gastrocnrmius. femur biceps and semitendinosus muscles of the hindlimb. To stimulate jiwztcr,fittu, the cathode was placed along the medial side of the knee and the anode was inserted along the lateral side of the knee so that it lay across ~~.vtn~li(ST-M). To stimulate j?n&i. the electrodes were inserted from the lateral aspect under the femur midway between the hip and the knee. The needles were connected 10 coupled Grass stimulators (SD9 and SD51 which passed 3 train of monophasic square pulses 2 ms in duration. Stimulation was applied to the respective sites at 20 X the threshold for n~uscfccontraction (20-30 mA; minimum I5 volts). Threshold was taken as the lowest intensity of stimulation which just produced muscle contr;lc(ion. The duration of the train was 20 min. The frequency was usuulfy 4 Hz. These parameters of stimulation never provoked any behavioural signs of discomfort. In control animals. electrodes were inserted into the respective areas of the hindlimb hut the stimulators were not switched on.

The lightly ;muestheWd rat WIS placed in a plastic restrnincr on the apparatus ~etf 10 mC:tsurctail WitlldlXW;ll IiltCllCy. needles were inserted tind threshold tfctermined. Refi~c testing \vils s;tiu+ttA,tf~ ;mimitfs were i~ffowcd to stohili/.c ti)r 30 Famu.FMu

I

hmu

FitZ.I. %&m;rtiCilluWilitmof hindlimb nlrrridirn and non-ttxridi;m points slimulered in rk ml as well as notable meridian point\ which wcn’;~voidedin fk slimulrtion ilt non-meridi;ur puinrh.0. meridian poinr; m. non-meridian point. Upper I& meridian points stimulated in reliltion to &V ~wS. l.0~~ I&. p)inl%slimulal&3svisurlizedfrom Iatcral aspect.Right, non-mcridirur poke. stimulatcvl as wdl BS meridian points avoided in this ~timuldon; tk dotted lk ttprchc?nl~ a mitilc line along the dorsal ctpec~of the hindlimb. The points were derived loom the litemture [4.5,.?7).

pl=inp &WWkS

In souw csperimcnt5,

qGna1 corcIs v.a’i Llat>Sciicd

elicited after spinal section. Rats Were ~~iesthetized Tribrisscn

249

CO.02 ml/ IO0 ,c; Trimethnprim

The spinal cord WHS

CX~OS~

to

JCtLYi.illillC

and Sulfodiazine),

nip/kg

ubscnd

i.p.,

FiShcr

III iI:1;Ld rsls i~ruld hi: Scientific).

The

sntibiotic,

was injected S.C. 3 h prior 10 and 3 h after the surgery.

the 6h and 7th thoracic segments. After

at

tl?C CtfCdt

l\tl2tjldl'

with chioral hydrate (300

making a slit in the dorsal part of the dur:l mater.

= 2 mm crtudal and rostra1 to Ihe level of transection. Gelfoam and/or bone wax was placed into the empty vcrtcbrul column to reduce bieedin g and lo Seal the empty vertebral cavity. 3 h after surgery, the ruts had complctcly recovered from the annesthetic and produced a tail withdrawal to the noxious the cord was transected and aspirated by suction

heat stimulus

applied to the tip of the tuil at a cnnsisient

For testing, ench spinal anima: protruded. consistent

The

uuanae:;!hctized

piitced. unimaesthetizcd.

was seen in all spinaiized

h after

Wils covered with ;I black cloth to minimize

transection

experinlcnt~tion.

The

v,,cre introdu:od

protocol

and h~~bitui~ted

thcrc was no sign (:f distress

the

IO

(imps

At1 \‘ilillCS ~lli‘ct

i\ftCr spin;bi

rcstrlriner

ill’c

cxpx4~4

;L Lllc

IIlC;lIl

wcrc

:i S.E.M.

subtrxtion

group, to eliminutc Wits necessary. These

which rcccived was stimulation

IIO

stimulation

V;IIUC

W;IS

similar

fashion

III

2 dayr

prcvioiisly.

prior

tc:

Some ;Ininl&

C‘cmv!

rat4,

Stidiccl

nt

tk

points or stimulation

taken us the Within-subject

and from control ;mimuis The betwccu-subject factor

With one XI of pur~m~ctcrs vs. stimulittion

filctor. Tukey’s

wholly

periods, the mean MPI

significnnt

With imother

difference test ~3s used to make

was c~iculitted for the period of stimulation

periods. For the 20 min period of stimuiittion.

the meim MPI

wits citicuiOted

OVCI’ the

ittld fol first

!i

was calculated over the five readings taken in each

f-tests were used to compare the means between treittment qx~ups.

TO determine whether curdiov;rscuinr nnimuis

or

Ihe illtilCt control rats.

vitlues from treilted rats which received electricid stimuirttion

readings. For the three 25 min post-stimulation period. Non-paired

UsL’d

I

prcpitration.

c;lCll tiIlIc point in CilCtl group of ril1S. IU the ;lllileSltl~~li/Cd r;Il\. lllc Ucl

~os~-I~oc comparisons betweeu means. 11~some amdyses, the mean MPl subsequetlt post-stitnuintion

;LS

mit!

were unuiyscd using it two-Wily rmaiysis of viuiimce (ANOVA).

at meridian vs. non-meridian

set of parameters. Time

l’or

il

60-90

iulacsthetized

VillUC ilt 1llC rcSpt‘~IIvc OlllC 119 ull~ti~l\uii~t~d glc)up front tl1C 1111’;111 llw ~,uanacstllcti/.c:J. .spiu‘ii lath. no such in the etfects of udalilri~trHtiI,lt 01 imr\~~d~etic.

wilS l9btiliUed by ~tIbtr:lctmg the IIIC;UI

1lle S1ilnuk!d

trCi1tCl.l ill

to the animals yet

ii~tcncie~ at the doses used iii

Rats which were used expcrimcnti4ly

for

Wits li9li~~weJ as described abc~c for the lightly

tri~llsCcli~~l1.

withdr:tW~l

visual stimuli.

were used in more thau out_! group but ill no group wcrc more than hilit tlic tlllillliliS tlif(crcnt

animnls.

in ~1l~lilstic restrainer SO I~LII c~1ly the tail XI~ one hindtimh

rat wxs used ~L’c~~ISL’, in pilot studies.

baseline readings wcrc observed. The anaesthetic produced inconsistent

intact Tilts. The restrainer > 24

wits

latency. This

were used to determine

nnaesthesia (3(M) nip/kg

changes might hitvc contributed

;he effects of the stimulation

i.p.. Fisher Scientific),

h prior to the experiment.

Hepurin

the left fernor

sodium salt (150

U/ml.

to the effects seen (911withdmwiil

on arterial

latency. some

pressure and heart rate. Under chic~riti hydrate

artcry was cimnuiatcd with intramcdic PE-SO tubing 24-4X

Sigma) was used to tlush the catheter twice daily to maintain

patency. During d

the experiment,

the s;Lme protocol was followed

sodium pentobarbital and chlorul hydrate. The

P23-ID)

3s i\bove. Thus,

and arterini pressure was monitored via a Grass model 5 D.C.

calculated from these recordings by countins the rate in beats/min

anucsthe!i>~d with 11~~IIliXtUI’C

preamplifier

and driver

the number of beats in a 10 s period and nwilipiying

(bpm). Needles Were inserted into hindlimb

min period of stabilization.

ri\t< were lightly

:~rteriul catheter Wi\s connected to II Stathim

prcssurc tmnsducer (Gould mpiifier.

Heart riltc was

this Value by fi to obtain

meridian points and threshold WiIS determined. After 3 30 three baseline readings were taken at ?I min intervals: i nlin after the hst baseline reading.

hindlimb meridian points wert?stimulated. Arterial pressure and heart rate were then recorded ut 3 of stimulation and at S min intcrvuls thereafter. up to 60 min.

ilId

5

min after the onset

3. Results In control rats, i.e. those m which stimulating electrodes were inserted into meridian points but the stim&~or wil\ not turned on, reaction time in the tail withdrawal test remained relatively constant throughout the experiment, with the exception of slight increases in latency occurring as a result of the administration of the successive doses of anaesthetic. This provided the control for the use of anaesthetic. The data from these control animals for each paradigm are illustrated in the respective figures.

Electrical stimulation of hindlimb meridian points applied at 20 X threshold and at 4 Hz for a duration of 20 min (n = 8). increased withdrawal latency. Readings were significantly different from those in the control group (11 = 12) throughout the period of stimulation and for the next 75 min as illustrated in Fig. 2A. The response was characterized by what appeared to be two peaks occurring during the period of stimulation us well ~1s;I third peak occurring during the post-stimulation period; two peaks were similarly ohservcd during the period of stimulrnion in illI subsequent groups (vide infra). In this series. these two peaks were observed at 10 and 20 min nftcr the onset ot stimulation and were 70.6 + 7.45 and 68.0 f 6.96% of the MPI. respcctivcly. The third peak was 48.8 + 9.62% of the MPI and occurred at the reading taken 10 min after the end of the stimulation.

Fig. 3 illustrates the effects produced at different frequencies of stimulation which included 2.4. 6 and 8 Hz; in all cases. the parameters were otherwise the same as those in the previous series of experiments. It is also important to point otu that the group stimulated at 4 Hz was different from that in the previous series - the group of rats tested at 4 Hz was run within the same time period as the other groups in the present series to achieve consistency. Stimulation at 4 ()I= 7) and 6 (11 = 8) Hz produced the same type of response reported from the previous scrims of experiments. Stimulation at 2 Hz 01 = 7) produced a significant inhibition compared to the control group hut the post-stimulation effect was smaller in amplitude and shorter in durcttion when compared to the respcctivc cffcct in the groups stimulated at 4 und 6 Hz. Stimulation at 8 HI. (tl = 6) produced an effect only during the period of stimulation. It is important to note thut the wccgnitudcof the inhibition produced durin g Anulation \vilS the SiLIllt! ilt idI frcquencic~. It WilS only the persistent effect. observed following the pcricd of stitndation. which showed signit‘icunt dit’f’crcncch h~tw~n ths four groups,

nME mw

ftuE WN

A k!.

2. Effect un tail withdnwltl

~ka&i(~

imkrld

by &k%d

(UFB). Th~shold

puints f@mur-fitfu (ST-33 fh&dJi

B

latency of high intensity (III 20 x (GB-3 I) ml

WI

threshold), low frqucncy

(4 Hz) &ctric;~l

that which iuht elicited muscle twitch hy dirca simulation.

Jcsunli (ST-36).

dmulation

for 20 min (durdon

ot

A: stimulation WBL 8f hiudlimh mcridinn

Dc~cd lines indicate udministrcltion of aaac\rhciic.Tail withdrawal is cxprchred as a

plc-W?c of the Mpf. A. xk~ul;rml group (n = 8): 0. needlesonly 01 = 12): A, diffcrcncc between the two groups. 6: btimuI;ctic)nof non-mrridian F,inrx in * hincflimh El~dcx were inserted in the gilsttucnemius musdc~ and biceps of the hindlimb. A. \timulrttcd group (II = I)): 0. nccdlchonly (n = 12): A. differencehetwccn the two pmups. ’P < O.OS;* ’P < 0.01.

PERIOD1

80 :

PERIOD 2

T

60

40 MEAN PM’l 20

Stiniulalion was

11o

pulses min.

31 20

These

control

of nonmeridian

post-stimulation X

group

indicated

tht

threshold

peaks

wcrc

points ! II = 9: so2

ctl~ct. The ha

in ;I 20 min train. There 45.9

-t_

7.X:

(tt = 12: 1”< 0.01 1. I~C

inhibition

Fig.

XL‘ pnxmted

I) hi0

~~c)kcd hy

tail withdrawal

lillL?llC~.

ilMl0Ugh

nppesrcd IO be two peaks during the stimulation.

cud 47.3 4 7.X64 ‘Two-way

iilcRXd

in Fig. X3. The standard paran~eters of’ stimulation

ANOVA

of the MPI.

This

inhibition

(O.OI > P < 0.05)

1~111~-:11cri~~i~lt~iAnt

stimulation

was

signilicantly

r-test kss

~III

MPI 10

0

10 Jn) TIME

SD

40

-*o -10

0

10 $1 TIME

50

from

the

co~npariso~~s

that evoked hy meridian

0

-20 -10

diffcrcnt

( I’ < 0.001 1

point stiniu~ation.

MPI 10

Cast’ there

were I Hz. 2 m

occurring al 10 and 20

was significantly

and paired

in this

40

(As there wa.. no significant diffcrencc in mean baseline values for withdrawal latencics in the control group for acute and chmnie spinal transectedanimals the data were pooled from both groups to yield an ‘n’ of IS.). In the group tested 24 h after transection (at = 6). the duration of the antinociceptive effect evoked by electrical stimulation was more prolonged than in the group tested at 4 h (Fig. JAI. Thus. at IO and IS min after the onset of the stimulation, the tail withdrawal latency increased to 9.36 f 2.77 and 14.2 f 4.58% of the MPI. respectively (P < 0.05 and P < 0.01, respectively), There was no post-stimulation effect in this group. 2 days after spinal transection(n = 71, stimulation resulted in a more pronounced effect with respect to amplitude and duration (Fig. 4A). The greatesteffect in this group was 24.8 f 8.57% of the MPI and occurred at I5 min after the onset of the stimulation. The withdrawal latencies were significantly different (P < 0.01) from those in the control group throughout the period of stimulation. A significant post-stimulation effect was seen but only at the reading taken 5 min after the stimulation was turned off (P < 0.05). In comparison with the effects seen in rats tested 4 h after transection, the mean increase in withdrawal latency in ritts tested 2 days after transection was greater at IS and 20 min after the onset of the stimulus (P C 0.01) and this effect remained significantly greater at 5 and IO min after the end of the stimulation (P < 0.09. in comparison with the effects seen in rats tested 24 h after transection, the latency in the rats tested 2 days after transection was greater only at the 20 min reading after the end of stimulation (P < 0.05).

The data from chronically spinal transected rats are illustrated in Fig. 4B. All testing was done after bladder function had returned. In the group of rats tested 7 days after spinal transection ()I= IO). stimulation produced an incrcasr in withdruwal latency throughout the period of stimulation (P < 0.01). Two peaks, occurring at 3 _ and I5 min after the onset of the stimulation, were 15.7 f 3.59 and 21.6 f 6.76% of the MPI, respectively. In contrast to the rats tested acutely after spinal transection (see Fig. 48) when the stimulation was turned off, the withdrawal latency remained elevated at = IO% of the MPI for the next I5 min (P < 0.09. In the group tested I4 days after spinal transection (n = 8; Fig. 4B), stimulation produced an increase in withdrawal latency similar to that seen in the rats tested 7 days after spinal transection. The first two peaks occurred during the stimulation at 2 and 15 min after the onset and were 14.5 f 3.95 and 17.3 3_ 4.06% of the MPI, respectively (P < 0.01). The post-stimulation effect persisted for IS min and was maximum at 17.5 f 5.87% of the MPI (P < 0.01).

In the group of animals tested for the effects of stimulation on artcriul prczsure UIIJ heart riltc (II = 5). the mcm busclinc heart rate. systolic prcssurc and diastolic pressure prior to stimulation were 402 f 5.7 hpm, 120 t_ I .X mm Hg and 85 f 4. I mm Hg, respectively. Stimulation of hindlimb meridian points applied for 20 min at 4 Hz und 2 ms fuiled to produce uny changein arterial prmsu~ or heart wtc during or after the stimulution.

4. DIscussIon Our experiments indicate that high intensity, low frequency electrical stimulation of previously detined sites in the rat hindlimb inhibits the nociccptive tail withdrawal rct‘lex provoked by noxious cutaneous heat. We interpret this to be an antinociceptive effect. The effect consists of a brief response during stimulation and a persistent rcsponsc which lasts > I h after the stimulus. Neither response appears to be secondary to a change in arterial pressure. It appears that the spinal cord can sustain both types of inhibition. However, the data also indicate an important role for supraspinal structures in eliciting both antinociceptive responses in the intact rat.

It is suggested that the brief and the persistent effects constitute two different responses because the brief effect couid be elicited independently of the prolonged effect: the brief effect was elicited alone at 8 Hz, with stimulation at non-meridian points and with stimulation in acutely spinal transected rats, Suggested mechanisms underlying each response will be elaborated separately in later sections but it is important to point out that other reports have not made this distinction between brief and persistent effects. Experimental studies have reported only a brief, non-persistent effect. Some lasting effects have bwn observed but, as discussed in detail below, in most cases these may be attributed to the decay of a brief response becaUSe recovery occurs relatively rapidly compared to the effect in the present study.

ral [5-t] ;mJ rabbit [I?]. 9(at r’!rlW.\i!\.. BiWi f’rCyuC;lcv \lirnill;!!ion produce.4 anlir.oclc ep[i”n u,hj& ]3g, x due to \1re4\;t\ ;i result of. using min. ahf~ou~h in this case the possibility remains that the ctfi’ct\ could have 1.6. <,._cn stimulation in rhe zrtzarzaesz/z~r;& mouse [&I showed an awake mimais. (3) .%udics using hi+@ zzztcmit~, Iuw ~wyucncy

tif~fo~crrs~lreri,-f,f~

IO-N

effect faring

up to 2 h but this effect was almost certainly due to stress

awake animals.

(3) To

crnccc~.stltc~~i,edanimal will

our

only

[23): the anlinociccption

be elaborated further

efkct

knowledge.

the high intensity stimulus was applied to low frequency stimulation in the the period of stimulation by 15 min. This persistent effect because

one report has used

cficilcd

outfnsted

/zig/t intmsit~,

bcfow but after II survey oi the litcrnture it is clear that most previous studies focused on a brief

only.

The

observation

that stimulation

at 8 Hz elicits

mechrtnisms mediating this rcsponsc arc relatively tflosc mcditrting the persistent inhibition

inhibiiion.

only the brief

inhibition

in this cuntcxt, post-stimufntion

stimulation

just

[3] and the pcrceptuai rcsponsc IO activation of C lihres

toferabfc lo human subjects has hcca reported in an experimental

Therefore.

WC suggest that the aotiilociccption

activation of spinnf inputs and excitation of‘ firs

While

it is difficult

relatively

III orhcr words, the pcripheraf or ccntraf mechanisms

may be fimitcd by the frcqucncy of the stimufittion.

stimulation [27.53].

suggests that the peripheral and/or

independent ol’ frequency and follow

lo draw spccif’ic paaffcfs.

central

higher frcquoncics than mediating the pcnistent

analgesia evoked by intense

pain study with

2 but not

11) Hz

in human pcriphcraf ncrvcs begins to fail ut S HL

ob>rrved durin, 17,aimufation

may he due to continuous

synaptic

order ncuroncs.

lhc f”“st-stinlufriti(,n

rtntinociceptioil

reported here is unique among animaf

studies in thut the duration of’ the effects resemble ~~OSCof cfcctroh~upuncturc-e\loked iu~afgesiit in fsuminls which can Ias scvcraf hours to a few days clinically [ 17,32.47,5 I]. Animal studies on cfectroacupuncturc in which stress was not a t\clor have gcncralfy Mcd notable.

to show

In one study.

rmursthetkctf

r;it [%I

long-fasting

a pcrsistcnt

but this type of stimulation

in the other study. 11 long-lasting ncurones has iIfs Clinicill

effects

Hun

comf>arabfc to those produced in our paradigm. Two

ct’fcct was obtained using inhibitory

rcscmbfcs TENS

frequency

stimulation

at 10 X

cxccptions ;trc

thrcshofd

in the

and. thus. is different from that used in the present study.

effecr of high inttmsity,

low

tixqurncy

s~i~nufi~~i~~non spinal nociccpti\c

reported [ I31 hut in (his USC sCnwf;ltic~n with uppfkd directly to ;I peripflcral ncrvc.

reports tend IO indicntc that higfl intensity

unitfgesiit in humitns.

higfl

efcctricaf stimulation

but this unirfgcsiit IZ On the other h;mtf. stimulation at S-X X tflc flcrccption threshold incrcilscs toot11 f>;tin rfuahofd I.?. IO] itnd ~timufi~Ii()n ;11 3- S X fwccption thw.hofd. dcscribcd us just toleritblc. products itnttlgesiu in chronic flitin patients who otherwise luck suf’fkicnt pain rcliot’with low intensity. high f’rcqucncy stimultltion [47]. Thus, the clinicid data arc in general agreement with our view from the prcscnr study that low fiqucncy, high infcnsity sfimul;\tion products ;I generalized antinociccption or anulgcsiu, including u Antifgcsiil

IOW intensity.

itI ,ju)rt t~~f~ri~hk kvcfs c~ckcb it l(jng-i;t>ting

hits been rcportctf with

merginrrffy cff’cclivc in (hilt only IO-N’%

of’paticnls cxpcricncc attlicicn1

high L’rccfucncy stimuliItion puin rriicf

[x!c)].

prolonged aticrcfIizct.

Our data indicate that the frequency of stimulation the persistent inhibition. frequency of 2-6

Hz

Few reports have systematically

studied the optimal frequency for electroucupuncture. Usuitfly,

is used and most studies have used only one frequency within

compared effects of I5 Hz vs. f(H) Hz [2l] 2 HZ stimulation

lasted only

central inhibitory

mechanisms

persistent efk~t.

is tin important factor in triggering the spinal mechanisms mcdiuting u

this low range [I 1,371. Some have

or even 2 kHz vs. 5 kHz [26]. In our experiments. the ;tntinociccpGon elicited by

I5 min. Thus,

it is possible tfliit iit such a low frequency. optimul

is not achieved. Frequencies of 4-O Hz appar

temporal summation

of

to bc necessary to produce the maximum

AS discussed above. the luck of il response a~ X Hz may bc due to the filifure

of the synapses of’ small

inhibition UI this higher frequency of stimulalion. Therefore, we propose that activation of small diameter afferent fibres for 20 min at 2-6 Hz triggers mechanisms which evoke a plastic change giving rise to the persistent depression of the tail withdrawal

diameter afferenls to follow

this frequency and/or

inability

of central mechanisms to cvokc the post-slimulation

reflex.

The

selection

antinociception

of

points

for

stimulation

was

bused upon sites

in our study was dependent on stimulation

reported

in the fitcraturc

I4.5.371.

The

persistent

of meridian points. Clinical studies indicate that meridian point

stimulation is important [ 1,481 although few such studies actually compart~dua from meridian vs. non-meridian points, In animal studies, high intensity stimulation of meridian points but not of non-meridian points in the rat products a persistent inhibition of the jaw-opening reflex W. The importance of meridian points has not always been supported by data from animal experiments [s]. possibly due to the fact that most studies have focused on the antinociception which occurs during the stimulation. Some studies huvc reported a difference between stimulation of meridian vs. non-meridian points in the rat [49] or mouse [M] but stimulation was only of large diameter afferents. the effects produced were only brief in duration and the experiments were done on awake animals. Thus, the antinociception studied was not similar to the persistent elect we are reporting here. The literature on human pain may be considered to support the importance of meridian points. In a study on pain rating upon immersion of one hand in ice-water, electroacupunctureproduced analgesia only from meridian points [I]. In another study, only meridian point stimulation increased tolerance to thermal pain [48]. Unfortunately, in neither of these studies were details of the stimulation provided and the duration of the pain relief was not mentioned. There are clinical reports on chronic pain in humans in which non-meridian points produce the same relief as meridian points [ 19.20.251. rlowever. in none of these studies were the parameters of stimulation reported, so it is not clear that high threshold afferents were activated,details of what constituted meridian or non-meridian points were not mentioned and the duration of the effect was not reported (in most cases. assessment was done immediately following stimulation). On the basis of our results. showing ;I brief antinociceptive effect which is independentof stimulation of meridian points, it would be predictable Illiit there would be no difference between meridian point and non-meridian point stimulation in clinical studies which d:, not report persistent analgesia. Therefore. support for a difference between meridian point vs. non-meridian point stimulation ma) come from only those clinical and basic studies in which a persistent rather than just ;I brief mtinociceptive cffc~.-t i> elicited by high intensity stimulation.

We interpret our data from spinal transected animals to indicate that there is a supraspinal component to both the brie! and persistent responses. However, as both effects can also be produced in the spinal rat. it appears that the spinal cord is capableof sustaining at least a component of each response. Data from our acutely transected animlh arc in aprcenxnt with the finding that noxious stimulation depresses activity of dorsal horn ncurones for several minutes in intact rats. yet shortly itftcr spinal transection this stimulation produces a weak inhibition [8]. In addition, clcctrical stimulation of the tail evokes it brief antinociception in the acutely spinal rat [56], Several studies have shown that high intensity stimulation applied cithcr cutaneously or to peripheral ncrvcs products scgmentid inhibition of spinal nociceptive rcflcxcs. lasting IO min to I h [ I%SO]. High intensity stimuli also inhibit convcrgcnt dorsal horn ncuroncs [K I .J] in acutclg \pinal transected 3ninla1s and noxious stimulation produces an cxtrascgmcntul antinociccptiorr in spinal ~Ulilllid!, [34-36). ‘I’hus. i~lttlUU&$l SllptWpilli~l structures appear to huvc a dominant role in eliciting antinociccption induced by activ;ltion of A is and C lihres [tl], thcrc is sufficient evidence to suggest that intruspinal mechanisms may also provide an important contribution to inhibition of nociceptivr?mechanisms, ‘l”he persistent antinociception occurred in our chronically but not acutely spinal animals. The temporal appearanceof this persistent rqmse was coincident with the return of bladder function. Our data, thus, indicate that intraspinal mechanisms contributing to the antinociceptive effects evoked by high intensity electrical stimulation of hindlimb meridian points my be derived in part from u spinal source and may be acting with or independently from supraspinul structures thus far implicated in stimulation-evoked antinociception [6,7,16.24], After spinal transection, these mechanisms may require a Period of RXOVCty to be cxpmssed. indicating adaptive changes in spinal analgesic mechanisms.

The data presented suggest that high intensity. low frequency clcctrical stimulation elicits two antinociceptivc responses. one which occurs during the stimulation and is dcpcndenton continuous synaptic activation and the other which can persist for > I h after the end of afferent stimulation. It is also suggested that the persistent antinociception is evoked by activation of high threshold, smail diameter sensory afferents which trigger long-term changes in spinal and supraspinal mechanisms. This persistent response appears to be dependent upon central temporal summation because 2 Hz stimulation evokes a limited post-stimulation effect while 4 or 6 Hz stimulation evokes a persistent antinociception lasting > 1 h after the termination of the stimulation. In our model. the post-stimulation antinociception is evoked only from meridian points. In addition to supraspinal mechanisms. spinal mechanisms appear to be able to produce antinociception in response to prolonged activation of high threshold sensory inputs.

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G.M. Pitchr,

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III rat\. Hr;un KC+..

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IV4

(Ed\.). Texthnjk ot Pain. Churchill Livingstonc. Edinhurgh. UK. 10X9. pp. XX4-XL)h. C.J. Woolf. G.D. BurrCtt. I). Mitchrll. ~1077~3Il-3IJ.

R.A. Mycrk. N;rlo~,,iic-rcvsr\ihl~ pcriphcrid clcctroanalgza~ IU inti(Lq;111tl~NII;II rat\,. f!ur J. f’h,irnl;lctll 4.~