Induction of anaesthesia in swine by thiopentone (27.1-35.7mg/kg, mean 29.9mg/kg) was followed by bolus doses and continuous infusion of midazolam and ...
Evaluation of thiopentone-midazolamfentanyl anaesthesia in pigs Eirik Sf.6fteland1, Tore Framstad2, Terje Thorsen1 & Holm Holmsen1 'Department of Biochemistry and Molecular Biology, University of Bergen, and 2Department of Reproduction and Forensic Medicine, Norwegian College of Veterinary Medicine, Oslo. Norway
Summary Induction of anaesthesia in swine by thiopentone (27.1-35.7mg/kg, mean 29.9mg/kg) was followed by bolus doses and continuous infusion of midazolam and fentanyl (0.90 mg/kg followed by 0.90 mg/kg/h and 0.025 mg/kg followed by 0.025 mg/kg/h, respectively). This produced good anaesthesia and analgesia for up to 2 h in 6 Norwegian Landrace pigs (wt: 17-42 kg), based on responses to painful stimuli elicited by pinching the nasal septum, the mouth, the forefoot and the perineal skin area. The first responses occurred after 110 min of anaesthesia. No significant drop in rectal temperature due to the regimen was noted during monitoring periods (140-180 min). This combined intravenous anaesthetic regimen gave good anaesthesia and analgesia to pigs for up to 2 h as monitored by clinical signs. The regimen may not be sufficient for longer time periods. We cannot advocate the incorporation of neuromuscular blocking agents in this regimen. Keywords
Pigs; anaesthesia; thiopentone; midazolam; fentanyl
We wanted to adapt a porcine model (Thorne et 01. 1984, Thorne et al. 1986) to investigate platelet activation in vivo and required an anaesthetic pattern that was simple to administer. In this model in vivo distribution of radio-labelled (llllndiumoxine) platelets were registered under a gamma scintillation camera. To obtain a dynamic scintigraphic image it was important that the experimental animals were motionless during registration periods. Minor surgery in preparing the animals for the study had to be carried out. Other requirements of the anaesthetic regimen were an absence of effects on platelet function (Gibbs 1991), provision of stable haemodynamic conditions and maintenance of body temperature for up to 2h. Pigs are widely used in biomedical research and a variety of experimental Correspondence to: Eirik S0freland MD, Department of Anaesthesia and Intensive Care, Haukeland Universiry Hospital, N·S021 Bergen, Norway Accepted 26 October 1994
models have been described (Tumbleson 1986, Swindle et al. 1992J. Several reviews addressing the use of anaesthesia for research purposes have been made (Becker et 01. 1972, Kyle et al. 1979, Riebold & Thurmon 1986, Thurmon & Tranquilli 1986, Swindle et 01. 19861. We decided to use an intravenous bolus of the barbiturate thiopentone for induction and a bolus then infusion of midazolam and fentanyl for continuous anaesthesia and analgesia. This regimen was based on recent haemodynamic studies using this combination in conjunction with a neuromuscular blocking agent (pancuronium) (Trouwborst et al. 1990, Van-Woerkens et al. 1992). From the literature there seemed to be no known effects on platelet function from either of the 3 anaesthetics (Gibbs 1991). According to some ethical guidelines (Council of The American Physiological Society 1986) neuromuscular blocking agents may only be used in research animals in conjunction with drugs known Laboratory Animals (1995) 29, 269-275
270
to produce analgesia. Anaesthetics to be used in research animals together with neuromuscular blocking agents must therefore be evaluated separately before the agent is applied. We decided to make such an evaluation to determine whether anaesthesia and analgesia was adequate, and if a neuromuscular blocking agent was necessary. During anaesthesia simple haemodynamic variables and rectal temperature were monitored. Materials and methods In this study 6 female Norwegian Landrace pigs were used lwt: 17-42 kg). They had a conventional microbiological status, and there was no acclimatization period. The animals were housed together with their siblings until the start of the experiments and kept on a normal diet. For the duration of the experiments each animal was kept in a separate cage. A permanent indwelling catheter was placed into the left jugular vein on the day before the reported study. For the jugular vein catheter insertion animals were anaesthetized using azaperone (Stresnil® Janssen) (3mg/kg) given by intramuscular injection into the neck muscles followed 15min later by metomidate (4mg/kg) (Hypnodil® Janssen) injected into an ear vein. Anaesthesia was maintained for the length of the procedure with incremental i.v. doses of metomidate (20-40 mg) every 5 or 10 min as judged necessary by clinical signs. The jugular vein catheter was tunnelled subcutaneously to the animal's back and used for blood sampling and anaesthesia the next day. The animals had no food overnight but free access to water. Blood was collected from the jugular vein catheter in one 50 and one 60 ml syringe from conscious animals the next morning. Saline (100ml 0.9%) was injected through the catheter to replace the volume taken. At the start of general anaesthesia 0.02 mg/kg of atropine was given into the jugular vein catheter. Anaesthesia was induced with thiopentone Lv. (20mg/kgj followed by incremental doses to provide suitable conditions for endotracheal
Sl1lfteland et al.
intubation (loss of laryngeal reflexes). After intubation the animal was ventilated temporarily with a ventilation bag (Laerdal®)while at the same time bolus doses of midazolam (Dormicum Roche®) (0.90 mg/kg) and fentanyl [Leptanal Janssen®) (0.025 mg/kg) were given i.v. The endotracheal tube was then connected to a ventilator (Oxylog Driiger®)and an oxygen/air mixture (50% oxygen in total) delivered. Flow was adjusted to a minute volume lMVl of 100-120ml/kg and a respiratory rate (RR)of 12-15 Imino A continuous infusion of midazolam, fentanyl and 5% dextrose was begun using a syringepump (ModelAS20A,BaxterHealthcare Corp®). Midazolam was given at a rate of 0.90 mg/kg/h and fentanyl at 0.025 mg/kg/h. Surgery began after the continuous infusion had been started and consisted of femoral vein catheterization for injections and bloodsampling and femoral arterial catheterization for invasive blood pressure (IBP)monitoring. Invasive blood pressure monitoring with heart rate (HR)determination was established as soon as the femoral arterial catheterization was complete. The level of anaesthesia was assessed at 10 min intervals following start of anaesthesia, during surgical procedures and monitoring periods. Responses to painful stimuli by pinching the nasal septum, an angle of the mouth, the interdigital spaces on the forefoot and the perineal skin on both sides of the tail with a surgical pincher (toothed forceps) were evaluated. A positive response was defined as a sudden, purposeful movement either in the region where the pinching was applied or by the head. The responses were graded 0-2 (0 = no response, 1 = slight response, 2 = forceful response) with a possible maximum score of 8. Heat loss was minimized using an isolating mattress (H. Hansen®), and by covering up the animals with a thick woollen blanket throughout the experiments. Rectal temperature (Tp) was monitored continuously during anaesthesia. The IBP, HR and Tp parameters were all measured using a patient monitor (Propaq 104 Protocol Systems®).
Intravenous
Table
1
Animal Score Score Score Score Score Score
anaesthesia
Sum score
no. 1 2 3 4 5 6
Score System:
271
in pigs
of response
Weight (kg)
Duration 10 30
40 42 17 17 21 22
0 0 0 0 0 0
of anaesthesia (min) 90 100 110 60
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 1 0 0 2 0
Sum Score of Response (Responses to pinching of the nasal septum, 0= No response 1 = Slight response 2 = Forceful response
0 1 0 0 2 0
120 0 3 0 2 1 0
130
140
0 2 0 0
1 4 0 2 1 0
angle of mouth, forefoot.
150
160
170
180
0 0
0 0
0
0 2 0
perineal skin)
Possible maximum Sum Score = 8
40,5
Animal
nr.
40 39,5
IT
39·
e
38,5
~
38
.a Ql
--1 ---u--
2
-3
a. E 37,5
~-4
Ql
';.
37
"~ 36,5
..
o
36 35,5 35
o Fig 1
20
Body temperature
40
during
60
80
Duration
of Anaesthesia
a combined
100
120
Results
Surgery for implanting the jugular venous catheters on the first day lasted for 65-95 min (mean: 78 min) requiring total supplemental doses of metomidate of 3.8-11.7mg/kg (mean: 9.0mg/kg). A time period of 22 h 15 min-27 h 2S min elapsed between ending this procedure and starting the thiopen tone-midazolam -fentan yI anaesthesia.
160
180
(min)
thiopentone-midazolam-fentanyl
Arterial blood samples for blood-gas analysis were taken at several points throughout the experiments.
140
anaesthesia
in 6 pigs
Blood samples taken from the jugular vein catheter the next morning were 3.04-8.01 % (mean: 6.13% J of the total blood volume (TBV) (Bossone & Hannon, 1986). There was an interval of 2 h 50 min4 h 40 min between blood sampling and induction of general anaesthesia. Total doses of thiopentone given were 27.1-35.7mg/kg (mean 29.9mg/kg, bolus + incremental doses). Total doses of midazolam and fentanyl given were 3.0-3.6 mg/kg and 0.08-0.10 mg/kg, respectively. The pigs were anaesthetized for periods of 140-180 min. The surgical procedures were commenced within 5 min
272
St/lfteland
120
et a/.
Animal nr.
110 _100··
c:
-+-1
'E
S
90·
--0-
2
III
CII
e .s
-+-3
80-
III
~ ~
--5
70 -
III
CII
J:
--- 6
60 50 40
o
20
40
60
80
100
120
140
160
180
Duration of Anaesthesia (min) Fig 2
Heart rate during
Table 2
a combined
thiopentone-midazolam-fentanyl
Blood gas analysis
Animal no.
3
4
5
6
Min. after stan of anaesthesia
65
116
45
35
7.41 4.65 -1.8 21.5 38.07 99.8
7.56 3.85 4.9 25 29.33 99.8
7.53 4.48 6.1 27.4 38.46 99.9
7.47 4.99 4.1 26.9 35.49 99.8
pH pco2 kPa BE mmol/l HC03 mmol/l p02 02 sat%
after anaesthesia had been established and lasted for 30 min in all animals. Responses to pinching during surgical procedures and through the registration periods are noted as Sum Scores (Table 1). The first positive response occurred after 110 min of anaesthesia when pinching the forefoot in animal 2 produced slight withdrawal (SumScore= 1) and animalS reacted to pinching of the forefoot (SumScore = 2). At 120min the responses in animal 2 had increased and in addition there were positive responses in animal 4 (when pinching the nasal septum and the perineal skin). A positive response in animal 1(to pinching the forefoot) occurred at 130 min. When positive responses
anaesthesia
in 6 pigs
occurred there was a simultaneous transient increase in HR and IBP. Changes in rectal temperature and heart rate (HR) are shown in Figs 1 and 2, respectively. In animal 4 there was a significant drop in rectal temperature (36.7 to 3S.4°C) and in animal 1 and 5 a significant rise (37.7 to 39.6 and 38.7 to 40.2°C, respectively). All animals tended towards a lower HR during the first hour of monitoring. Results of blood gas analysis are shown in Table 2.
Discussion The reported induction doses of thiopentone with bolus and infusion of midazolam and fentanyl produced anaesthesia for 2 h using clinical signs considered acceptable (Hoyt et al. 1986). Minor surgery could be performed during the first half hour. When more than 2 h elapsed, positive responses to painful stimuli increased and were present in 4 animals at 130min. Barbiturates (including thiopentone) have been used as sole agents for anaesthesia in swine (Thurmon & Tranquilli 1986).Unstable haemodynamic conditions were produced by the large doses necessary to obtain sufficient analgesia (Worek et ai. 1988).
Intravenous
anaesthesia
in pigs
Midazolam provided stable haemodynamic and respiratory conditions in spontaneously breathing pigs when i.v. bolus doses of up to 1.0 mg/kg were used (Smith et 01. 1991).. In Smith et o1.'s study the pigs had already received repeated hourly doses accumulating to total doses of 3.2 mg/kg over 6 h without a measurable effect on cardiac output (CO). In this study total doses of midazolam were 3.0-3.6 mg/kg over a shorter time period (140-180min) and this might have had an effect on CO, but the simple haemodynamic parameters (IBP, HR) were stable. Fentanyl has been evaluated for its effect in combination with other drugs. Doses of fentanyl largely vary in the range from 0.0125 to 0.05 mg/kg for induction and from 0.0125 to 0.05 mg/kg/h for continuous infusion (Thurmon & Tranquilli 1986). The technique described satisfied most criteria. The animals were motionless, minor surgery could be performed and a stable haemodynamic situation was present. The agents had no previously reported effects on platelet function (Gibbs 1991). The doses and continuous infusion regimen were based on recent studies when this combination had been used in conjunction with pancuronium (Trouwborst et a1. 1990, Van-Woerkens et ai. 1992). Sedation with midazolam 0.3 mg/kg and ketamine 10mg/kg i.m. had preceded induction with thiopentone 5 mg/kg Lv. for intubation. Bolus doses of 0.45 mg/kg of midazolam and 0.0125 mg/kg of fentanyl were then given followed by a continuous infusion of 0.45 mg/kg/h of midazolam and 0.0125 mg/kg/h of fentanyl. At the same time a bolus dose and continuous infusion of pancuronium were given. In pilot studies we used these reported bolus and continuous infusion doses of midazolam and fentanyl without a neuromuscular blocking agent and they did not produce sufficient anaesthesia, repeated additional doses of thiopentone were necessary from the onset to avoid positive responses to pinching and we decided to double the doses of midazolam and fentanyl.
273
Sedation with ketamine and midazolam reduced the thiopentone required for induction. Without previous sedation, thiopentone 29.9 mg/kg was required to perform endotracheal intubation. The effect of a single dose of ketamine would probably have subsided within the first 30-60 min (Riebold & Thurmon 1986). Pigs in the present study received a higher total dose of midazolam (1.80 vs. 1.20mg/kg in the first hour), but animals were responsive to painful stimuli within 2 h even when the fentanyl doses had also been doubled. It is possible that immobilization of the animals with pancuronium obscured an insufficient level of anaesthesia and analgesia when the lower doses of midazolam and fentanyl were used. In this experiment significant responses occurred after 120min, indicating inadequate anaesthesia and that the infusion rates were not optimal for longer periods. These responses can also be explained by a gradually subsiding effect of the large induction dose of thiopentone during the same time period. Any neuromuscular blocking agent would have obscured these responses, and we cannot advocate the use of such agents in conjunction with the anaesthetic technique described. It is difficult to draw any conclusion with certainty as to haemodynamic stability, but the values seem to be within the normal physiological range in pigs of this size, HR = 90-107 (Hannon et ai. 1990), HR = 110± 7 (Smith et 01. 1991). The HR tended to fall in each of the animals during the first hour, however. Atropine was used initially to avoid salivation. A waning of effect of atropine may have explained this HR fall as well as a direct effect of the anaesthetics. These observations should ideally have been compared to baseline values from conscious animals. The blood loss of up to 8% of the estimated blood volume as a result of samples taken on the second day seemed to have had no serious adverse effects on the haemodynamic situation. The saline volume given after the samples had been
Scpfteland et a/.
274
taken would only partly compensate the loss and an auto transfusion of blood from the spleen blood-pool due to induced blood loss cannot be excluded (Hannon et al. 1985). Only one animal (number 4) showed a significant drop in body temperature. This dramatic drop most probably resulted from cold ventilation gas due to a mishap with an oxygen bottle being left in free air over night at near freezing point. The lowered body temperature might have had an anaesthetic effect on the animal. In 2 of the animals (numbers 1 and 5) there was a considerable temperature rise. The explanation could be that body heat was too well conserved under the woollen blanket. There were no clinical signs of malignant hyperthermia in these 2 animals (muscle rigidity, hypermetabolism, respiratory insufficiency) (Riebold & Thurmon 1986, MacLennan & Phillips 1992). During our pilot studies we encountered severe respiratory depression when this drug combination was used and decided controlled ventilation was necessary. We had no access to end-tidal pC02 monitoring and the ventilator settings were determined by using clinical variables for the MV and kept at a constant value throughout. Blood gas analysis 14 animals I indicated arterial pH, pC02, and p02 values within acceptable limits, with a tendency to alkalosis, at least partially explained by a low pC02, due to some hyperventilation. Anaesthesia for animals in biomedical research is often developed from experience gained in veterinary medical practice, and the latter takes into account the subsequent use of the animals. Attention must be paid to avoiding drug residues in tissues if animals are used for human consumption (Heinritzi & Konig 1988, Haagsma et al. 1988, Ganter et al. 1990). Recent developments in anaesthesia for use in biomedical research are mainly based on anaesthetics for human use, but little attention appears to be paid to pharmacological variations between species. Doses required and responses may differ considerably. Therefore an anaesthetic which produces sufficient
anaesthesia and analgesia in man has to be evaluated carefully before being used in an animal model. Several other injectable forms of anaesthesia regimens for pigs without the use of inhalational agents and neuromuscular blocking agents have been described. A combination of pentobarbitone, fentanyl, and morphine chloralose gave a stable haemodynamic situation (Lang et al. 1992). A 2-stage intramuscular regimen consisting of mepheridine/ azaperone followed by hourly ketamine/morphine injections rendered stable anaesthesia with spontaneous respiration for 2h (Hoyt et al. 1986). A combined anaesthesia of guaifensin, ketamine and xylazine has also been used (Thurmon et al. 1986). A ketamine/ metomidine combination given into the left atrium gave a marked but transient suppression of haemodynamics (Vainio et al. 1992).
Conclusions The thiopentone-midazolam-fentany 1 regimen described in this report gave satisfactory anaesthesia and analgesia for up to 2 h. Minor surgery could safely be performed during the first half hour of the regimen and we found the regimen quite useful in this model. Response to painful stimuli returned in several of the animals after 2 h of continuous anaesthesia, however. The regimen was not sufficient for longer time periods. This suggests that the doses were not optimal for longer use. Any neuromuscular blocking agent would obscure these responses and we cannot advocate the use of such agents with this dose regimen. There were no significant reductions in rectal temperature when attention was paid to preventing heat loss. Acknowledgments We wish to thank The Norwegian Army, Drager Norway, Nycomed Jean Mette (Propaq 104), Abbot Norway (Transducers and cables to Propaq 104), Baxter A/S Norway, Helly Hansen A/S Norway, and the Norwegian Air Ambulance for supporting us with equipment. We have had invaluable help from the Stend Agricultural School, the Stend Veterinary Clinic,
Intravenous anaesthesia in pigs the Anaesthesia and Intensive Care Department and the Technical Medical Department of Haukeland University Hospital. We thank Trygve Gjerde for supporting us with transport. This research was financially supported by the Norwegian Air Ambulance and The Lxrdal Foundation for Acute Medicine.
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