JOURNAL OF SURGICAL RESEARCH 19, 1 IS-119 (1975)

Anesthesia in Rabbits by lntrahepatic Sodium Pentobarbitall

ROGER JACOBS, PH.D. AND DAVID L. KROHN, M.D.

Institute for Medical Research and Studies, New York and the Department of Ophthalmology, New York University Medical Center, New York, New York 10016

Submitted for publication September 20, 1974

Sodium pentobarbital has been commonly used for production of general “anesthesia” in animals [1, 31 for about 40 yr [3]. Intravenous administration is most com- monly used in rabbits, using the marginal ear vein. However, this requires considerable restraint, is frequently painful enough to cause movement and loss of position of the needle, and often renders the ear vein useless for administration of other drugs. Most im- portantly, it has the disadvantage that the toxic-therapeutic range of the agent in rab- bits is so narrow that inadvertent anesthetic death is quite frequent. The speed of induc- tion is so rapid that the use of life-support maneuvers in case of respiratory failure are rarely possible.

The effect of sodium pentobarbital in rab- bits for useful dose range is not true anesthesia, but deep sedation with obtund- ation of all reflexes, allowing the use of block, infiltration, or topical anesthesia. Be- cause of tradition and convenience, however, the use of the word “anesthesia” in this report will be used to describe the pen- tobarbital effect.

The problem and inconvenience of intra- venous barbiturate for rabbit anesthesia, in addition to the short span of effective ob- tundation, encouraged a search for another method of administration which combines greater safety and longer duration of effect.

Intravenously administered Innovar-Vet (0.4 mg fentanyl and 20 mg droperidol per

Supported by U.S. Public Health Service Grant CA 12523 from the National Cancer Institute, National In- stitutes of Health, United States Public Health Service.

Reprint requests to D. L. Krohn, M.D. Dept. Ophth., NYU Medical Center, 550 1st Ave., New York, NY 10016.

ml, Pitman-Moore) has many of the same disadvantages as sodium pentobarbital in rabbits with the added disadvantage of short duration of action [4].

Volatile agents are probably more efficient as anesthetics, but their use in labo- ratory rabbits requires relatively elaborate equipment and some inconvenience.

Combinations of barbiturates with secon- dary agents such as preanesthetic phenothi- azine or with synthetic antimuscarinic drugs allows a smaller total dose of barbiturate, but introduces extra steps.

Intraperitoneal barbiturate has been used, but the response is slow and there is wide variation in effective dose with consequent frequent overdosage.

The use of an intrathoracic method has been described [5, lo]. The method has been criticized because of production of various lesions including hemorrhage [ 121, subse- quent scarring [ 131, and pleural infection [5, 12, 141. In addition, it has been pointed out that the intrathoracic method may be lethal if the entire calculated dose is administered at once [ 141.

This paper reports an intrahepatic method for rabbit anesthesia which features the ad- vantages of the intrathoracic method but ap- pears to avoid the disadvantages.

METHODS AND MATERIALS Albino female New Zealand rabbits

weighing 1.5-2.0 kg, fed rabbit chow and water ad lib., were used.

Sodium pentobarbital was obtained as Nembutal, 50 mg/ml (Abbott). All needles and syringes were standard disposable sup- plies (Becton-Dickinson).

The best dose formula was found to be

115 Copyright c: 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

116 JOURNAL OF SURGICAL RESEARCH, VOL. 19, NO. 2, AUGUST 1975

weight in kg multiplied by 0.66 ml and calcu- lated to the nearest 0.05 ml. Occasionally, additional anesthetic is required, the dose estimated by extrapolation based on the depth of sedation initially obtained. This never exceeded one half of the original dose and was administered by the same intrahe- patic route.

METHOD OF ADMINISTRATION Administration of the anesthetic solution

is made with a 3-ml syringe (or a l-ml tu- berculin syringe for the smaller rabbits) and a 25gauge 5/8-inch-long needle. The rabbit

is permitted to sit on its haunches. The lo- cation of the injection site is determined by grasping the rabbit gently with the left hand and palpating the end of the rib cage on the right flank of the animal with the left thumb. Once the caudad extremity of the rib cage has been located, a small forward sliding movement of the thumb identifies the two adjacent intercostal spaces (Fig. 1). The needle is inserted as fully as possible between the second and third rib, counting (for con- venience) from the caudad extremity of the rib cage, and at a point one third the vertical distance from the spine to the ventral ex-

FIG. 1. Diagrams of intrahepatic injection technique. The entry angle and position of the needle are important in obtaining the correct site for hepatic infiltration. The thumb rests on the most caudal points of the last rib. The needle is inserted between the second and third rib counting (for convenience) from the caudal extremity at a position 2/3 the distance from the ventral extremity of the rib cage to the spine (A). The needle is pointed 20” cephalad (B) and 10” ventrally (C). The large diagram in the figure shows entry of the needle into the dorsal part of the right inferior lobe of the liver, which is not covered (dark shading) by other liver tissue.

JACOBS AND KROHN: ANESTHESIA IN RABBITS 117

tremity of rib cage, that is, somewhat above the apparent horizontal midline of the rab- bit. This is easily identified by palpation as well as by visual estimation, since at this point, the sharp delineation between the ribs and the spaces is lost. The needle and syringe are angled so as to point about 20 degrees cephalad and 10 degrees ventrally (Fig. 1). The correct angle of the injection is im- portant in that pointing too cephalad may result in puncture of the lung followed by hemorrhage, and pointing too caudad may result in an intraperitoneal injection, yielding inadequate anesthesia. The lo-degree ventral direction of the needle results in penetration of the inferior lobe of the liver in its longest axis. The correct angle of insertion affords a high probability that the needle tip will be within the central portion of the inferior lobe. Instillation is made over a IO-set pe- riod. The animal becomes drowsy in 3-5 min and fully obtunded in 5-10 min, at which point a drop of 1% atropine sulfate solution is administered orally to minimize respira- tory secretions.

In occasional administrations, respiratory embarassment occurs. Shallow and ir- regular respiration or the development of Cheyne-Stokes respiration is treated by moderate pressure applied to the right side of the rib cage over the cardiac area manually. This significantly improves the rate and depth of respiration in most ani- mals. Breathing is also facilitated by placement of the rabbit on its right flank when practical. If these simple methods are insufficient, and cardiac action remains, pentylenetetrazole (as Metrazole, 100 mg/ ml-Knoll) in a dose of 0.1-0.2 ml is injected into the marginal ear vein. If no cardiac action remains, intracardiac epinephrine 0.1-0.2 ml is administered, followed by Metrazole and forced respiration via an animal mask, with use of 95% oxygen with 5% carbon dioxide. These pharmacologic methods of life support are seldom necessary.

Injection is accomplished easily by one person and without restraints. The rabbit

rarely reacts or attempts to move. Gentle pressure over the torso with the hand used to localize the rib cage position appears to se- date or distract the rabbit during entry and injection.

ANATOMICAL LOCALIZATION The rabbit liver on the right side features

two major lobes. The superior dorsal lobe is by far the largest and consists generally of three major leaves which overlap each other in a caudad direction, partially covering the smaller single inferior lobe except in its dorsal half. The long axis of this exposed part of the inferior lobe approximates the in- jection angle described above. To ascertain that the needle tip is located centrally in this lobe, four rabbits were injected with the cal- culated dose of sodium pentobarbital to which had been added one drop of India ink so as to allow visualization of the locus of in- jection with simultaneous confirmation that the standard and expected anesthesia effect was obtained.

Liver Chemistry Determination; Pathological Examination

After successful intrahepatic pentobar- bital anesthesia, cardiac blood was drawn at intervals of 1, 2, 3, and 10 days. The following determinations were made for each serum sample: bilirubin, SGOT, SGPT, alkaline phosphatase, and LDH. These were compared to a control group in which the taps were made 6 days prior to anesthesia. Not less than 34 individual samples were used for each type of determination and not less than six determinations of each type were done on any given day.

Six livers were examined histopathologi- tally at two different intervals (2 and 6 days) after anesthesia procedures.

RESULTS In 421 general anesthetic procedures by

the intrahepatic method, the anesthetic mortality rate was 2.9% over-all. In the last 200 procedures, the rate was under I%, the improvement attributed to refinement of

118 JOURNAL OF SURGICAL RESEARCH, VOL. 19, NO. 2, AUGUST 1975

TABLE 1 Fatalities Attributed to lntrahepatic Administration

of Sodium Pentobarbita?

Number of deaths Probable cause Probable reason

4 Respiratory failure Inability to monitor respiration rate

3 Puncture of lung Needle inserted too anteriorly

2 Puncture of lung Unusual position of lung lobule

1 Overdose Error in dose calculation

1 Overdose Pentobarbital intolerance

‘Given as 0.66 ml/kg total rabbit weight of a 50 mg/ml solution.

techniques both of administration and life support as described. Table 1 lists probable causes of deaths attributed to anesthesia in this study.

Requirement for secondary dose occurred in 15.2% of procedures. Minor external postinjection bleeding at the site of injection was encountered in 1.6%.

Injections made with India ink mixed with the anesthetic agent were found on post- mortem examination 30 min after the in- jection to have localized mainly in the right inferior lobe. The India ink covered a rela- tively dense sphere of about 1.3-cm diameter at the injection locus and decreased in density outward therefrom. Most of the right inferior lobe was to some extent darkened. In one case the India ink was traced to the juncture of the inferior and su- perior dorsal and extended slightly into the latter. The injecting needle had encountered only the exposed portion of the inferior lobe in each case. No grossly observable India ink was found outside liver tissue.

The liver chemistry study showed no significant variation in average values ob- tained from the postpentobarbital injection samples, as compared with the prepento- barbital control specimens.

Histological examination of one of the 2- day postinjection group and one of the 6-day group showed small focal areas of necrosis in relation to the needle tract. In each case,

the histological change was consistent with chemical necrosis, but was limited to a cylinder about 4 mm in diameter, extending 9-10 mm into the lobe. No hemorrhage and no infectious process were seen. In relation to the total volume of the liver, the volume of the necrotic tissue was extremely small. There was no evidence of significant mechanical trauma such as would be produced by excursions of the needle as re- sult of respiratory movements.

No symptoms characteristic of liver dys- function were observed in the postoperative period. Periods of observation were minimally 3 wk but extended to 3 mo in some animals.

Although the length of effective anesthesia varied with the supplemental local or block anesthesia and with the procedure, the method afforded between 45 and 75 min for reaction-free surgery.

The depth of anesthesia produced by intrahepatic pentobarbital is comparable to that obtained by intravenous administration. Obtundation but not obliteration is obtained for such reflexes as reaction to ear pinching, whisker twitch and palpebral fissure reaction to strong light. Complete loss of these reflexes from pentobarbital is, however, pre- terminal in rabbits. Surgical procedures routinely required supplementary local or block anesthesia. After these supplements, which were well tolerated, ordinarily ex- tremely painful procedures such as extensive ocular surgery produced no reaction.

DISCUSSION

General anesthesia by intrahepatic in- jection has the advantages both of con- venience, since there is no requirement for restraint, and reliability of dosage. The range between dosage effective for surgery and the lethal dose is appreciably widened, relative to intravenous administration. In- duction is rapid enough for convenience but slow enough for life-supporting measures to be taken in case of apparent overdose. In common with the intravenous route, the method has the disadvantage that true

JACOBS AND KROHN: ANESTHESIA IN RABBITS 119

anesthesia is achieved only at the risk of respiratory collapse, so that the drug is best supplemented by local agents. However, the method appears to have considerably greater safety and reliability than the intravenous injection, in combination with relatively prolonged effective narcosis.

In contrast to the intrathoracic method of anesthetic administration, the risk of pulmonary puncture is minimal since the point of insertion is generally well caudal to the pleural cavity. In addition, the complica- tions encountered in the intrathoracic method, including interpleural hemorrhage and scarring sufficient to cause physical signs, are absent in the intrahepatic method. In most cases (85%), the single initial dose, administered with convenient speed, is sufficient. The blood chemistry results do not demonstrate impairment in liver function or liver damage as a result of intra- hepatic anesthesia. Although some trauma occurs, it is insufficient to cause chemical or gross clinical evidence of liver damage or functional impairment.

SUMMARY A reliable method for anesthesia of labo-

ratory rabbits by intrahepatic injection of sodium pentobarbital was developed. Ana- tomical dissection was the basis for designation of external landmarks for in- jection. The advantages of the method have been simplicity, accuracy of dosage based on body weight, and an induction speed great enough for convenience but consistent with life support measures in case of respiratory collapse. No restraint was required and the injections were easily made without assistance. Supplemental block or local infiltration anesthesia was usually required for surgical procedures. In 421 anesthetic procedures, the fatality rate was 2.9% from anesthesia-related events but the rate was much less in the last 200 procedures due to refinement of technique. Liver chemistry de-

terminations and histopathology demon- strated insignificant hepatic damage and there was no gross morbidity.

ACKNOWLEDGEMENTS We are grateful to Dr. Frederick Becker, Director,

Department of Laboratory Services, and to Dr. Roger Schinella, Pathologist, New York University Medical Center, for the blood chemistry determinations and for the pathological studies, respectively.

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