Rapid intracerebroventricular injection assisted by an automatic syringe

Rapid lntracerebroventricular Automatic Syringe

Injection Assisted by an

JONATHAN J. LIPMAN* AND P. S. J. SPENCER

Rapid intracerebroventricular licv) injections in the conscious mouse are described, using a small volume gas-tight syringe with repeating dispenser device (Hamilton Bonadoz, Switzerland). The device gives reproducibility within 2% of chosen volume, while facilitating rapid icv injections to groups of animals. The technique is used to demonstrate in the mouse the antinociceptive activity of clonidine, using the tail-immersion test. Automatic activity;

Key Words: Antinociceptive

syringe; lntracerebroventricular Tail-immersion test

injections;

Clonidine;

INTRODUCTION The quires

study of drug effects on the behaviour of conscious laboratory animals that drug administration is carried out efficiently and with a minimum

stress

or restraint

upon the animals.

individual

animals

especially

when

macological

using

effects

may be followed macokinetic cordingly,

makes

smaller

by behavioural

change, while

the extent

variation

large numbers

and central

sites,

a large number

reasons-do

not

between

of animals,

such as rats or mice. Many drugs

at peripheral

physicochemical

both to limit

biological

to use relatively

species

simultaneously

and/or

Simultaneously,

it necessary

exert phar-

either

of which

of agents-for

reach higher

of drug action to the central

reof

phar-

centres.

Ac-

system

and

nervous

ensure that the best possible experimental conditions are achieved, the administration of drugs directly into the brain and other CNS regions is becoming increasingly popular. Methods for most

for the intracerebroventricular

laboratory

of the conscious

species.

When

dog (Haley and Dasgupta,

1953; 1954; 1955), it is clearly necessary indwelling

(icv) injection

injections

of drugs have been devised

are made into the cerebral

1958) or the cat (Feldberg

to prepare the recipient

animal with a chronic

icv cannula or guide, some weeks prior to injection.

(1971) have described

such a chronic

indwelling

ventricles

and Sherwood,

Sparkes

guide assembly

suitable

and Spencer for the rat.

Noble et al. (1967) have demonstrated a technique for acute icv administration of agents into the lightly anaesthetised rat that does not require prior surgery or implantation; From

The

Address of Pharmacy,

yet the necessity Diwsion

requests UWIST,

for light anaesthesia

of Pharmacology, for reprints Cardiff,

The Welsh

School

to Dr. P. S. 1. Spencer, CFI 3NU.

* Present

address:

Anesthesiology

Received

February

11, 1980;

revised

mitigates

of Pharmacy,

The Division

against the technique’s UWIST,

Cardiff,

of Pharmacology,

CF~ 3NU,

The Welsh

U.K.

School

U.K.

Dept.,

UTCHS,

and accepted

Memphis, May

Tennessee

38163.

2. 1980.

327 lournalof

Pharmacological

2 1980 Elsevter North

Methods

Holland,

4, 327-333 f19801

Inc., 52 Vanderbilt

Avenue. New York, NY 10017

Olh0-5402’8010803?7007’$02 25

328

j. j. Lipman and P. S. J. Spencer usefulness scious

in acute behavioural

mouse

activities,

commends

and Haley

nique for this

volume.

that requires

landmarks

of location

Brittain

and Handley

(1957)

as possessing

albino

strain,

readily

by the icv needle.

(1967) and Sewell

possesses

present

modification

by employing

reproducible

and accurate volumes

an icv injection

technique

needle

utilises

and thus

control

and Spencer

tech-

external

suffers

from

over the injected (1975) modified

adult mice from

thin

method

an automatic

of drug

loose

young

a particularly

The

the con-

This

only

by using

screening,

for acute evaluation

have described

no preparation.

technique

which

behavioural

ideal subject

to locate the icv syringe

as well

Haley and McCormick

derived

For routine

as the

and McCormick

species

morphological inaccuracy

studies.

itself

the

the ICI-derived

bregma that can be penetrated

simplifies

injector

the Sewell

apparatus

to the ventricular

system

for

and Spencer

rapid delivery

of the conscious

of ICI-

mouse.

MATERIALS

AND

METHODS

ICV Injection GB, used

variants

of the ICI-derived

throughout

(Hamilton

(Triangle

Bonadoz,

dispenser

Switzerland)

device (Type

albino

strain Devon,

Mouseries,

PB-600-1

with

luer

Hamilton

of mouse, weighing 20-228, were U.K.). A 500 t.d gas tight syringe

fitting

was assembled

Bonadoz).

in a repeating

To this was fitted

a 27 gauge

(icv) needle (luer fitting). The syringe needle had previously been cut to a length of 3.25 t 0.05mm and polished to a fine bevelled tip, devoid of burrs. The device was then filled of clonidine to be injected and first gentle

either

with

but firm

pressure,

such that the dorsal assembly

found

skin

0.9%

w/v saline

or isotonic

wedge that formed

at the back of the neck with

an inclined

the icv needle

of the interorbital

the thumb

device being held in the other.

point.

Applying

by pressing

plane at 30” to the horizontal,

surface was in the horizontal

held vertically,

solutions

w/v Evans Blue dye. In use, the mouse

the head of the animal was then immobilised

skull

by estimation

the needle from midsagittal incomplete

1.0%

of one hand, the injector

against a polycarbonate

suture

apyrogenic

or with

was held by the loose

fingers

syringe

sterile

HCI (Boehringer)

plane (see Fig. I).

With

the

was located over the midsagittal

The

suture

was felt by gently

tracing

left to right and, when located, was then traced caudally along the

groove. Since the intersection of the midsagittal and coronal sutures is in the young adult ICI-derived mouse, this was readily located. It was

that the weight

cause the needle

of the hand-held

to penetrate

the skull

injector

assembly

to its fullest

(12Og) was adequate to

extent

when

this

point

was

reached (Fig. 2). At the fullest extent of the needle, (its boss preventing further penetration), the needle tip was located in the third ventricle. The dispenser was then operated, delivering one aliquot of solution over a period of about 0.5 seconds, into the region of the needle tip. The needle was left in place for 10 seconds

before

removal. Method

of Measuring

The tail-immersion A vessel

of water

Antinociceptive

Effect

test (48°C) was used as described (lOOmI) was water-jacketed

by Sewell and Spencer

and maintained

at 48

t

(1975).

0.5”C

by

Rapid lntracerebroventricular

Hamilton microlitre syringe

Injections

r

h-J--

nicrolitre syringe

f

driver

handle

Inclined plane I ICV syringe needle

FIGURE 1. Diagram of gas-tight syringe and dispenser assembly at point of icv injection of the conscious mouse. The inclined plane is used to achieve a horizontal plane for the upper frontal surface of the mouse skull.

means of a thermostatically controlled hot water circulator (type SC-lo, struments, Cambridge). Groups of IO animals were restrained individually

Grant Inin a semi-

recumbent

position

their

protruded. familiarised

These mice were restrained apparently without discomfort and were with the restraint condition for 30-60 minutes before nociceptive testing

using

ventilated

polypropylene

tubes

from

which

tails

commenced. Starting 40 minutes before drug or vehicle administration by either the icv or SC route, the tails of the restrained animals were immersed in the heated water and the latency of the tail-withdrawal reaction was measured by means of a

329

330

J. J. Lipman and P. S. I. Spencer

Injection

site

FIGURE 2. line diagram of the mouse skull, showing external sutures, outline surface (-----) and the cerebral ventricles (. . . . . .).

of brain

stopwatch. A 15 second cut-off time was imposed to prevent occurrence of tissue damage. The stimulus was presented thereafter to each mouse at successive 20 minute intervals until 100 minutes (5 presentations) after drug or vehicle administration. Calculation

of Antinociceptive

Effect

The data obtained over the O-100 minute period (6 determinations) was integrated with respect to duration of experiment (minutes) and reaction time (seconds). The

Rapid Intracerebroventricular areas under each minute-second the formula:

% antinociceptive

Injections

curve (the integrals) were then introduced (T-C) effect ( = %AE) = 7

into

x 100

where T is the test group’s integral and C is the integral of the parallel vehicle control group. Confirmation

of Ventricular

Perfusion

Periodic checks were made of the icv injection technique by administering Evans Blue dye solution (1.0% w/v) in place of saline or clonidine solution. Animals so injected were killed immediately and their brains removed, blocked optic lobes uppermost on the freezing stage of a sledge microtome (MSE Pelcool), and frozen prior to coronal sectioning (50pm) in a rostrocaudal direction.

RESULTS In Vitro

Reproducibility

The in vitro accuracy of the injector apparatus was estimated gravimetrically by delivering 10 aliquots of distilled water to a dry ceramic boat that was then weighed. This procedure was repeated ten times and calculation revealed the average dispensed volume to be 9.95 5 O.lSj.J, representing reproducibility greater than 2.0% per delivery, nominally 1Oj.d.

150t

2 100

‘:/

ICV

SC

1

50

/

0

10

CLO&NE

DOSE ( ug,;z

FIGURE 3. Antinociceptive effect (expressed as % AE) of clonidine in the mouse, using the tail-immersion test. The lines illustrate dose-response relationships following intracerebroventricular (icv) and subcutaneous (SC) injections.

331

332

J. J. Lipman and P. S. J. Spencer

Location Gross

examination

of the entire

of coronally-sectioned

ventricular

system

of the lateral ventricles

brains revealed that complete

was obtained

and the fourth

in all cases, including

caudal ventricle.

Subarachnoid

apparent around

the site of the needle track, caused apparently

from

tip.

the needle

time of injection

In some

had caused

cases,

lateral

the needle

movement

perfusion

frontal

aspects

leakage was

by retrograde

of the mouse’s

flow

head at the

to enter the brain at an angle of about

15”,

but at this anterioposterior level, the needle tip nevertheless entered the curvatures of the left or right lateral ventricles (see Figure 2), so that complete ventricular perfusion

was obtained

Antinociceptive

in such cases.

Activity of Clonidine

Figure

3 illustrates

clonidine

produced

these

findings.

After ICV or SC Administration

In these determinations,

an antinociceptive

effect of 100%

icv administration

the dose required to produce (AE 100“4,dose); after SC administration, about 600 kg/kg (a ten-fold increase in potency in the icv route). This tends to confirm that the site of action of the antinociceptive idine

is

evoked

located

centrally,

profound

effects

because when

subanalgesic

centrally

of

at a dose of about 60 kg/kg

peripheral

the effect was effect of clon-

doses

(400

kg/kg)

administered.

CONCLUSIONS The major criteria for seeking to modify to increase the speed at which injections reproducibility.

The use of the present

apparatus fulfils

reducing

the accuracy of the injection

designed

to deliver

a nominal

this was reproducibly

the technique of Sewell and Spencer was could be made reliably and to improve volume.

10 ~1 volume

standard,

drug dosage must be constantly

these requirements

Although

the present

was in fact found to deliver

and hence adequate for behavioural reproducible

rather than accurately

without apparatus,

less (+0.15),

analysis known.

where In view

of the relatively large size (9.95 t 0.15 ~1) of the injection volume compared to the total volume of the ventricles, it might be considered that leakage of drug through the needle

track,

followed

by absorption

from

the subarachnoid

space into

the

systemic circulation could take place. As has similarly been pointed out by Haley and McCormick (1957), exudation of the drug from the foramina is likely to activate neurons proximal injection

of the external

surfaces

of the brain,

in addition

to the delivery site. These criticisms volume to the minimum compatible

to periventricular

systems

might be answered by reducing the with isotonicity and reproducibility.

In the system described here, such reduced volume may be achieved by the use of a smaller compatible syringe, although volumes of 5 ~1 and below present significant solution and tonicity problems. The possible advantages of using smaller injection

volumes

(where

possible)

are being studied.

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W,

Sherwood

SL (1953)

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333