Drug Administration into the Third Ventricle of the Brain of Anesthetized and Conscious Cats

M. D. DAY, R. H. POYSER, AND J. SEMPIK

A method is described for the administration of drugs into the third ventricle of the brain of conscious and anesthetized cats. Noradrenaline administered by this route produced cardiovascular stimulant effects as against depressor effects when administered into a lateral cerebral ventricle. The pressor responses evoked by third ventricular administration of noradrenaline were antagonized by spinal section in anesthetized cats and by hexamethonium in conscious animals.

Conscious cats were more sensitive to the pressor effects of third ventricular noradrenaline than chloralose-anesthetized animals.

Key Words: Third ventricle; Lateral cerebral ventricle; Conscious cats; Nora- drenaline

INTRODUCTION

Many workers have administered drugs directly into the brains of either anes-

thetized or conscious animals in order to examine, for instance, central mechanisms involved in blood pressure control. In conscious animals the method usually em-

ployed involves drug administration into a lateral cerebral ventricle via a previously implanted cannula (for example, Carmichael et al., 1964). This method suffers from

the disadvantage that the drug solution is rapidly distributed throughout the entire brain ventricular system and the precise site of action of the drug is difficult to

assess. In an attempt to invoke cardiovascular responses involving a more restricted

area of the brain we have administered drugs into the third ventricles of both conscious and anesthetized cats. Preliminary pharmacological results from these

experiments have already been published (Day et al., 1976; Day and Sempik, 1978)

and the purpose of the present communication is to describe in detail the methods used.

METHODS

Cats of either sex weighing 2-3 kg were used. Anesthesia was induced by the intramuscular injection of a mixture comprising 4.5 mg/kg alphadolone and 13.5

mg/kg alphaxolone (Saffan, Glaxo Ltd.) and maintained using l-3% halothane (Flu-

From the Department of Physiology and Pharmacology, Queens Medical Centre, University of Not- tingham, Nottingham, U.K. and Beecham Research Laboratories, Harlow, Essex, U.K.

Address reprint requests to: Dr. J. Sempik, Fisons Ltd., Pharmaceutical Division, R and D Laboratory, Loughborough, Leicestershire, U.K.

Received June 8, 1979; revised and accepted November 16, 1979.

Journal of Pharmacological Methods 4, 71-78 (1980)

@ 1980 Elswier North Holland, Inc., 52 Vanderbilt Avenue, New York, NY 10017

II

016%5402/80/050071OlU3$02.25

72 M. D. Day et al.

othane, ICI) in equal volumes of nitrous oxide and oxygen administered via an

endotracheal tube (4.0 mm cuffed).

Catheters for the measurement of blood pressure and intravenous administration

of drugs were implanted in the descending aorta and in the jugular vein respectively

by the methods described by Day and Whiting (1972). Coordinates for the position

of the third ventricle of the cat were obtained from a stereotaxic atlas of the cat

brain (Snider and Niemer, 1961). These were quoted as follows:

A 10.51 L,,’ H+6 (figures in mm)

(A - anterior; L - lateral; H - height above the zero line). Cannulae were made from

3-cm lengths of 21 g stainless steel hypodermic needle tubing fitted with stainless

steel stillettes. These were clamped in the needle holder of a stereotaxic apparatus

(Precision Cinematographique or Baltimore Instruments) and, by adjusting the controls, the tip of a cannula was positioned to coincide with the tip of an ear bar;

this gave the stereotaxic coordinate:

Ao’ H-1,

(the H,, co-ordinate is defined as the position 10 mm above the interaural line,

therefore the position of an ear bar is H--1,,). The readings displayed on all scales of

the stereotaxic apparatus were noted and the cannula moved away. The cat’s head was then clamped in the stereotaxic frame, a midline incision was made in the skin

of the head and the skull cleared of connective tissue. The cannula was then moved

10.5 mm anterior of the ear bar into the A 1o.5 position and gently lowered until this

position could be marked on the sagital suture which was taken as the midline, i.e., L,,. A hole was drilled at this point using a dental drill (2.5 mm diameter). A

further three smaller holes (1 mm diameter) were drilled approximately 1 cm from

the hole for the cannula, and stainless steel screws (l/8”, 12 BA) were screwed into

these. The dura was then pierced with a sterile hypodermic needle and the cannula

lowered into position 16 mm above the position of the interaural line signified by

the position of the ear bar, i.e. H +6 (since the readings on the vertical scale of the stereotaxic apparatus were taken when the tip of the cannula was at H_Io, the scale

reading required for the position H +6 was deduced by subtraction). The cannula was fixed in position using acrylic dental cement (Sevriton, Amalgamated Dental

Trade Distributors Ltd); this also covered the stainless steel screws, thus giving firm adhesion to the skull. The cannula was then detached from the needle holder

which was moved away. A silicone rubber mould was placed over the external part

of the cannula and more dental cement poured into this. When set the mould was removed and a stainless steel cap was cemented in place. The skin was then sutured around the cannula with sterile silk sutures. A sectional view of this cannula in place is shown in Figure 1.

All operative procedures were performed under aseptic conditions, and the cats were treated against possible infection by an intramuscular injection of 250 mg ampicillin suspension (Penbritin, Beecham Pharmaceuticals) given immediately after surgery and repeated daily for a further three days.

Third Ventricular Drug Administration in Cats 73

FIGURE 1. (Upper panel) Cannula for third ventricular administration of drugs in the con- scious cat. The individual components are: steel cap (A), moulded acrylic dental cement (B), initial application of dental cement to fix cannula position during implantation (C), skull (D), cannula (E). Normally kept patent by stilette not shown in diagram. (lower panel) X-ray photograph of the cannula in position on a cat skull.

74 M. D. Day et al.

Cannulation of a lateral Cerebral Ventricle

For infusion of drugs into a lateral cerebral ventricle, a similar stereotaxic and operative procedure was used except that the tip of the cannula was positioned at

the co-ordinates

A,,’ H+e.,’ L

i.e., in the left lateral ventricle. When a cannula was positioned in a lateral ventricle

as opposed to the third ventricle, small volumes of cerebrospinal fluid could be

withdrawn. The relative positions of the lateral and the third ventricular cannulae

in respect to the ventricular system are shown diagramatically in Figure 2.

Blood Pressure Recording and Drug Administration in Conscious Cats

For the purpose of recording blood pressure in conscious cats, the animals were

placed in a sound-proofed cabinet fitted with lighting and ventilation, and at am-

bient room temperature. Once in the cabinet the cats were not disturbed by the experimenter or by any movements or noises in the laboratory. Blood pressure was

recorded using a Bell and Howell physiological pressure transducer connected to

a Devices M-4 or M-19 recorder or a Grass polygraph. Heart rate was obtained by

using either a Devices instantaneous ratemeter (type 2751) or a Grass tachyograph

(type 7P 4F) triggered from the blood pressure pulse. The recording apparatus and

the pressure transducer were situated outside the cabinet and the arterial valve on

the cat was connected to the transducer by a length of approximately 3 meters of polyvinylchloride tubing (0.75 mm, Portex Ltd). The frequency response of the

system was measured and no damping of a standard pulsation was found up to 4

A B

FIGURE 2. Diagram of the brain ventricular system of the cat showing cannulae positioned in a lateral vent&al (A) and into the third ventricle (B). T represents the intermediate mass of the thalamus, HT the hypothalamus and HB the hind brain region (after Carmichael, Feldberg and Fleischhauer, 1964).

Third Ventricular Drug Administration in Cats 75

Hz. The venous and intraventricular catheters were connected to syringes outside

the cabinet by polyethylene tubing (PP30, Portex Ltd) so that intravenous injections

and intraventricular infusions could be made from the outside of the cabinet with

a minimum of disturbance to the animal. Drugs dissolved in normal (0.9% w/v) saline were infused into the third or lateral

ventricles in 25 PI volumes over a period of 90 seconds using an infusion pump

(Scientific and Research Instruments Ltd.). Intravenously administered drugs were dissolved in 0.5 ml of saline and were flushed into the vein with a further 0.5 ml

saline.

At the end of a series of experiments in a particular animal the position of the cannula was verified by infusing a solution of Pontamine Sky Blue dye into the

cannula, after which the animal was killed by an overdose of pentobarbitone sodium, the brain removed, fixed in formalin, sectioned and examined visually.

Third Ventricular Administration of Drugs in Chloralose-Anesthetized Cats

Cannulae were inserted into the third ventricles of chloralose (80mg/kg, i.v) anesthetized cats by a method similar to that used for conscious animals. In anes- thetized cats screws were not inserted into the skull, nor were protective caps for

the cannulae used. Blood pressure was recorded from a catheter inserted in the femoral artery, and heart rate was derived from the blood pressure pulse. Drugs were administered intravenously via a catherized femoral vein.

RESULTS

Administration of isotonic saline, in volumes used for drug administration, into

either third or lateral ventricular cannulae did not evoke cardiovascular responses. Administration of noradrenaline (15 and 30 pg) into the third ventricles of 12

anesthetized cats evoked rises in blood pressure lasting IO-20 min, and accom-

panied by variable effects on heart rate.

In conscious animals third ventricular administration of noradrenaline produced similar rises in blood pressure with doses of only 1.5 to 10 pg (Fig. 3). In anesthe-

tized animals the cardiovascular responses to third ventricular noradrenaline were abolished by spinal section (C2) and, in conscious animals, were markedly reduced

by prior administration of the autonomic ganglion-blocking substance hexame- thonium (I-10 mg/kg, IV).

Administration of low doses of noradrenaline (I-IO pug) into the lateral ventricles

of both conscious and anesthetized cats did not evoke cardiovascular responses, whereas doses of 30 pg produced prolonged (approximately 60 min) depressor

responses in both cases. Figure 4 illustrates experiments made in conscious animals in which the cardiovascular responses produced by third and lateral ventricular administration of noradrenaline have been compared.

DISCUSSION

In general, third ventricular administration of drugs in cats presented few prac- tical problems. Since the cannulae were inserted through the midline of the brain

76 M. D. Day et al.

(6) 75

ABP (systolii)

mml-tg

q conscious

cl anaesthetised

FIGURE 3. Effect of noradrenaline administered into the third ventricle of anesthetized and conscious normotensive cats. The upper cross-hatched columns represent the mean systolic responses to 15 and 30 pg doses of noradrenaline infused into the third ventricle of cholor- alose anaesthetized (8tl mg/kg) cats; the upper open columns represent the mean systolic responses to third ventricular noradrenaline 2.5 and 5 pg in conscious cats. The lower columns represent the diastolic responses. The values for n are given in parentheses, and the vertical bars above the columns denote the standard errors of the mean.

they occasionally punctured the sagital sinus, resulting in hemorrhage at the site of entry of the cannula. This bleeding was usually easily contained by swabbing,

except on a few occasions when it was severe and was only arrested by raising the head in the stereotaxic frame in order to lower the pressure in the sinus. The roof

of the third ventricle is composed of the corpus calosum and, in the cat, is com-

paratively thin (1-2 mm). In order to minimize erosion of this tissue by frequent

drug administration, drug solution volumes were kept to 25 ~1, and the number of administrations were normally limited to a maximum of 4 per experimental session

and sessions in the conscious animal to a maximum of two per week.

So far a total of 20 cats have been cannulated for administration of drugs into the third ventricle of the conscious animal. All of these survived the operative proce-

dures and subsequently recovered with no adverse effects. When a preparation was at the end of its useful experimental life (2-3 months) it became obvious that drugs administered into the third ventricular cannula were leaking into the systemic circulation. In these cases the pressor responses to noradrenaline administered into the third ventricular cannula became much larger and were further increased, rather than reduced, by administration of hexamethonium.

The results obtained from the third and lateral ventricular administration of noradrenaline indicate that qualitatively different responses of the cardiovascular

system may be elicited by administration of noradrenaline into different parts of

Third Ventricular Drug Administration in Cats 77

blood pressure mmHg

60- 160-

heart rate beats /min

q!w&wrw

min

C

160-

180- heart rate

beats jmin I, r*r*n*;yJLcr* 1”‘ “*SC ( 4m&&@unmk%.-J<-- m!wb 60-

1 . min

I NA 30 pg i.c.v. 90 min

FIGURE 4. Blood pressure recordings from two conscious unrestrained cats, one annu- lated for administration of drugs into a lateral ventricle (traces A and C) and the other for third ventricular administration (trace 8). Administration of 2.5 ccg noradrenaline into a lateral ventricle (icv) produced no effect on blood pressure or heart rate (trace A); this same dose administered into the third ventricle (Ill V) of another cat caused a substantial rise in blood pressure and a slight tachycardia (trace B). A larger dose of noradrenaline (36 @g) adminis- tered into a lateral ventricle produced a prolonged hypotension and bradycardia; recovery was seen after 90 min. (trace C).

the brain ventricular system. Thus, third ventricular administration of drugs, together with conventional lateral ventricle drug administration, may eventually prove useful in further understanding the central control of blood pressure.

The differences in sensitivity between conscious and anesthetized cats to the pressor effects of the third ventricularly administered noradrenaline suggest that cardiovascular regulating centers in the brain are depressed by chloralose. It would therefore appear preferable to use conscious animals for studying the effects of central administration of drugs on the cardiovascular system.

78 M. D. Day et al.

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Methods for perfusing different parts of the cat’s

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Day MD, Poyser RH, Sempik J (1976) Pressor re-

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conscious cats. Br) Pharmacol57:450.

Day MD, Sempik J (1978) Effect of centrally admin-

istered noradrenaline and isoprenaline on

splanchnic nerve activity in anaesthetized and

conscious cats. Br ) Pharmacol 64:393-394.

Day MD, Whiting RL (1972) An improved valve de-

vice for the continuous measurement of arterial

blood pressure in the conscious unrestrained

cat. ] Pharm Pharmacol24 : 263 -264.

Snider RS, Niemer WT (1961) A Stereotaxic Atlas of

the Cat Brain. Chicago: University of Chicago

Press.