Closed-loop control of propofol anaesthesia

Closed-loop control of propofol anaesthesia
Closed-loop control of propofol anaesthesia

British Journal of Anaesthesia83(2):223–8(1999)

Closed-loop control of propofol anaesthesia

G.N.C.Kenny1*and H.Mantzaridis2

1University Department of Anaesthesia,Glasgow Royal In?rmary,8–16Alexandra Parade, Glasgow G312ER,UK.2Department of Anaesthetics,Victoria In?rmary NHS Trust,Langside,

Glasgow G429TY,UK

*Corresponding author

We describe the use of a closed-loop system to control depth of propofol anaesthesia

automatically.We used the auditory evoked potential index(AEP index)as the input signal of

this system to validate it as a true measure of depth of anaesthesia.Auditory evoked potentials

were acquired and processed in real time to provide the AEP index.The AEP index was used in a

proportional integral(PI)controller to determine the target blood concentration of propofol

required to induce and maintain general anaesthesia automatically.We studied100spon-

taneously breathing patients.The mean AEP index before induction of anaesthesia was73.5(SD

17.6),during surgical anaesthesia37.8(4.5)and at recovery of consciousness89.7(17.9).

Twenty-two patients required assisted ventilation before incision.After incision,ventilation

was assisted in four of these22patients for more than5min.There was no incidence of

intraoperative awareness and all patients were prepared to have the same anaesthetic in future.

Movement interfering with surgery was minimal.Cardiovascular stability and overall control

of anaesthesia were satisfactory.

Br J Anaesth1999;83:223–8

Keywords:anaesthesia,depth;anaesthesia,general;anaesthetics i.v.,propofol;memory;brain,

evoked potentials;monitoring,evoked potentials

Accepted for publication:March2,1999

Anaesthesia has been de?ned as‘that state which ensures the suppression of the somatic and visceral sensory components, and thus the perception of pain’.1Before the introduction of neuromuscular blocking drugs into anaesthetic practice, movement of the patient provided a clear indication of depth of anaesthesia.However,when neuromuscular blocking drugs are administered,the availability of patient movement as a valuable sign of the inadequacy of anaesthe-sia is lost and reliance has been placed on indirect signs. There have been many reports of patients being aware during surgery2–5and considerable efforts have been made to develop a reliable index of depth of anaesthesia. Auditory evoked potentials(AEP)have been reported to ful?l many of the requirements for measurement of the level of anaesthesia.6–10In particular,the AEP has been shown to provide good discrimination of the transition from asleep to awake and vice versa.1011We have developed a system to obtain a single index which represents the morphology of the AEP12–15and used this index as the input signal for closed-loop anaesthesia(CLAN)during surgery in patients who did not receive neuromuscular blocking drugs.

Patients and methods

After obtaining approval from the Hospital Ethics Commit-tee for evaluation of the CLAN system and written informed consent,we studied100ASA I or II patients,mean age 50(range19–83)yr and mean weight66(40–108)kg, undergoing body surface surgery.All were able to under-stand the purpose of the study.There were no other exclusion criteria.Patients received temazepam for premedication approximately1h before surgery.Young patients received temazepam30mg and older patients received temazepam 20mg.Day-case patients did not receive premedication. Auditory evoked potential acquisition

In the operating theatre,patients were connected to the CLAN system.AEP were obtained using a system described previously10–14from three electrodes placed at the right mastoid(?)and middle forehead(–),with Fp2as the reference.The ampli?er was custom-built with a5-kV medical grade isolation.It had a common mode rejection ratio(CMRR)of170dB with balanced source impedance, input voltage noise of0.3μV(10Hz–1kHz rms)and current input noise of4pA(0.05Hz–1kHz rms).A third-order Butterworth analogue band-pass?lter with a bandwidth of1–220Hz was used.The clicks were70dB above the normal hearing level and had a duration of1ms. They were presented at a rate of6.9s–1to both ears.The ampli?ed EEG was sampled at a frequency of1778Hz by a12-bit analogue-to-digital converter and was processed in real-time by a microcomputer.

Kenny and Mantzaridis

Table1Induction algorithm.Step2was repeated up to three times.The algorithm was interrupted and the system switched to the proportional integral control algorithm when the desired AEP index was obtained

Premedicated Unpremedicated

(n?67)(n?33)

Step1

Initial target propofol

concentration 2.0μg ml–1 4.0μg ml–1

Wait for50s40s

Step2

Increase target by 1.0μg ml–1 1.5μg ml–1

Wait for50s40s

Repeat up to3times3times

Step3

Thereafter increase target by 1.0μg ml–1 1.5μg ml–1

Every60s60s

AEP were produced by averaging256sweeps of144ms duration.The time required to have a full update of the signal was36.9s,but a moving time averaging technique allowed a faster response time to any change in the signal. AEP were obtained at3-s intervals and the AEP index (AEP index),a mathematical derivative re?ecting the morpho-logy of the AEP,was calculated automatically.The AEP index is de?ned as the sum of the square root of the absolute difference between every two successive segments of the AEP waveform.11–14

The3-s running average of the AEP index was entered into a proportional integral(PI)control algorithm.The algorithm calculated the required alteration in the target blood concen-tration of propofol from the difference between the measured AEP index and the control value of the AEP index(AEP index

control) selected by the anaesthetist.The new value for the target propofol concentration was transmitted to the target-con-trolled infusion(TCI)system which used a pharmacokinetic model16to achieve and maintain the required target concen-tration set by the PI control algorithm.

The value for the AEP index was recorded with the patient awake and the anticipated AEP index

control for satisfactory anaesthesia was entered into the CLAN system.Previous experience using the AEP index as a monitor of the adequacy of anaesthesia1115suggested a range of30–40for the AEP index

control.Anaesthesia was induced automatically by the CLAN system using a predetermined series of increasing target blood propofol concentrations(Table1)until the AEP index was equal to the selected AEP index

control?10.There-after,control of anaesthesia was achieved by transmitting the target blood propofol concentration calculated by the PI algorithm to the infusion system and maintaining the measured AEP index close to the selected AEP index

control.

A target plasma concentration of alfentanil15ng ml–1was achieved before induction and was maintained throughout surgery.17During maintenance of anaesthesia,patients breathed a mixture of66%nitrous oxide in oxygen.A laryngeal mask airway was inserted in all patients to maintain a clear airway and to allow monitoring of ventil-atory frequency and end-tidal carbon dioxide partial pres-sure.Arterial oxygen saturation was monitored continuously Table2Anaesthetic and surgical times.Durations are mean(SD)[range]min Duration of induction 3.7(1.4)[1.3–8.9]

Start of induction to incision12.4(3.8)[5.6–27.9]

Incision to end of surgery37.9(26.4)[4.4–123.8]

End of surgery to recovery 6.6(4.6)[0.0–17.9]

Duration of anaesthesia56.9(28.4)[19.7–156.3]

Table3Values of AEP index recorded in100patients(mean(SD)[range]) Before induction of anaesthesia73.5(17.6)[41–134]

During body surface surgery37.8(4.5)[31–57]

At recovery of consciousness89.7(17.9)[60–147]

and arterial pressure,heart rate,degree of sweating and tear formation were recorded at intervals of5min and used to calculate PRST scores.18Measurements of heart rate, arterial pressure,sweating and tear formation were com-pared with baseline values and scored to provide an estimate of the amount of sympathetic stimulation.18

Data analysis was performed on an IBM-compatible PC using Minitab for Windows95(version11).Statistical tests used were analysis of variance(ANOV A),t test and chi-square test,as appropriate.

Results

Duration of induction(de?ned as the time required for loss of verbal response and abolition of the eyelash re?ex)and other times are shown in Table2.

The mean AEP index before induction of anaesthesia was 73.5(SD17.6)and anaesthesia was induced smoothly in all patients.The mean value required to produce surgical anaesthesia was37.8(4.5)(Table3).The AEP index and the AEP index

control were not affected by age or sex.

Propofol concentrations selected by the system are shown in Table4.The number of changes in the AEP index

control is shown in Table5.Control of the AEP index was maintained within AEP index

control?5for an average of65.2%of the duration of anaesthesia(Table6).

PRST scores recorded during surgery did not exceed2 in any patient but15patients made some movement during surgery(Table7).These movements were mainly slight and,apart from two patients,did not interfere with surgery. The mean AEP index

control was38.8for patients who moved and37.7for non-movers(ns)–not statistically signi?cant. However,mean the AEP index was signi?cantly different between the two groups(mean39.9and37.4,respectively; P?0.005).

AEP index and target blood propofol concentrations recorded from one patient are shown in Figure1.

At the end of anaesthesia,the CLAN system was set to achieve recovery,and delivery of propofol was stopped automatically.Both nitrous oxide and alfentanil were discon-tinued.Anaesthesia lasted for a mean duration of56.9(SD 28.4)min and the mean time from the end of surgery to recovery,de?ned as obeying commands,was6.6(4.6)min (Table2).The mean AEP index when patients recovered

Closed-loop anaesthesia

Table4Target propofol concentrations selected by the CLAN system(μg ml–1)

At the end of Maximum during Minimum during Average during

induction maintenance maintenance maintenance At recovery Mean(SD) 5.7(2.1) 6.7(2.2) 2.6(1.5) 4.3(1.8) 1.3(0.6) Range 1.0–11.5 2.1–13.00.9–6.8 1.3–10.60.4–4.2

consciousness was89.7(17.9)(Table3).Each patient regained consciousness at an AEP index which was greater than the value at which consciousness was lost.

Cardiovascular stability

Changes in systolic arterial pressure and heart rate are shown in Figure2.The mean maximum decrease in systolic arterial pressure was22%of baseline.The mean maximum decrease in heart rate was5%of baseline.

Respiratory stability

Before incision,end-tidal carbon dioxide partial pressure increased to more than8kPa in22patients and ventilation was assisted until spontaneous respiration resulted in end-tidal carbon dioxide values of less than8kPa.Fourteen patients required assistance for less than5min while eight patients required assistance for5min or more.Only four of these patients required assistance for more than5min after skin incision.There was no signi?cant difference in the AEP index or the AEP index

control between patients who required respiratory assistance and those who did not.Six of the patients who required assistance made some movement during surgery and nine did not.Ventilatory frequency and end-tidal carbon dioxide measurements are given in Figure3.

Patient assessment

Seventy-seven patients completed a questionnaire on recall and acceptability of the anaesthetic technique(Table8). There were no occurrences of awareness during anaesthesia in any patient and all were prepared to have the same anaesthetic again.Six patients described the clicks presented to them before induction of anaesthesia as slightly annoying while the remainder were not concerned.Five patients had no recollection of hearing the clicks during the study. Although all patients were awake in the operating theatre, only nine patients remembered waking up in theatre;49 patients thought they woke in recovery and19in the ward.

Discussion

At present,monitoring of clinical signs is the only routine method for determining depth of anaesthesia.When no neuromuscular blocking drugs are used,skeletal muscle response to surgical stimulation and the frequency and depth of respiration re?ect accurately the depth of anaesthesia.If ‘adequate anaesthesia’is schematically illustrated as a Table5Number of changes in AEP index

control indexcontrol during CLAN

During1st During2nd Total number of

15-min period15-min period changes

Median20 3.5

Range0–90–60–17

Table6Control of AEP index as a percentage of total CLAN time(mean (SD)[range])

Within target value?5%65.2(14.1)[10.3–89.0]

Within target value?10%89.6(9.5)[46.7–100.0]

Within target value?15%98.7(4.6)[74.9–100.0]

Table7Adequacy of anaesthesia

Highest PRST scores0(0–2) Movement during surgery(n)15

Disturbance of surgery(n)2

(1)one patient received no drug for7min

(2)surgery stopped for45s in one patient

Awareness or recall(n)0

Table8Patient questionnaire.

Where did you awake after your operation?

Theatre9 Recovery49 Ward19

Do you remember any dreams or noises during your operation?

Yes0

No77 Would you be happy to have the same anaesthetic?

Yes77

No0

Do you remember the clicks before induction?

Yes72

No5

Did the clicks bother you?

Very much0 Slightly6

No66

range of acceptable responses(Fig.4),its upper limit(i.e. insuf?cient anaesthesia)is determined by movement to surgical stimuli and the lower limit(i.e.excessive anaesthe-sia)by respiratory depression.

If the patient’s lungs are ventilated mechanically but the patient is not paralysed,the upper limit remains the same but the lower limit is now determined by circulatory depression.When neuromuscular blocking drugs are used, an indication of the depth of anaesthesia is given by the combination of cardiovascular changes,sweating and lac-rimation,and pupil signs.The lower limit is still circulatory depression,but there is no clear upper limit as the patient

Kenny and

Mantzaridis

Fig 1AEP index and predicted target blood concentrations of propofol recorded in one

patient.

Fig 2Systolic arterial pressure (SAP)and heart rate (HR)during CLAN (mean,SD

).

Fig 3Ventilatory frequency and end-tidal carbon dioxide partial pressure during CLAN (mean,SD ).

Fig 4Signs relating to depth of anaesthesia in a spontaneously breathing patient.

cannot move.The only available signs are those which arise from stimulation of the sympathetic nervous system.However,these are not speci?c and are affected by many drugs used routinely in clinical practice.119–23Clearly,if for any reason anaesthesia becomes lighter,the patient may regain consciousness without necessarily providing the anaesthetist with any indication,especially if high opioid concentrations are administered.

There is no accepted standard by which depth of anaesthe-sia should be assessed but any signal used to measure depth of anaesthesia must provide suf?cient information to enable satisfactory general anaesthesia to be produced during surgery.Any monitor of anaesthetic depth should enable the anaesthetist to provide satisfactory conditions in a patient breathing spontaneously while undergoing a surgical procedure.This form of anaesthesia requires that good control must be maintained throughout the procedure.Satis-factory anaesthesia requires:

d adequat

e cardiovascular and respiratory stability d no or minimal patient movement

d no awareness or recall of events during th

e procedure.These conditions form the basis o

f a standard against which any monitor of depth of anaesthesia may be judged.In addition,the monitor should:

d provid

e similar signals when different anaesthetic agents are used

d alter its signal appropriately during surgical stimulation d produc

e a similar signal when the patient has recovered from anaesthesia to that recorded before induction o

f anaesthesia

d b

e unaffected by cardioactive drugs

Closed-loop anaesthesia

d produc

e a marked signal difference during the transition from awake to asleep and vice versa.

Attempts to provide an accurate assessment of depth of anaesthesia have included scoring systems for arterial pressure,heart rate,sweating and tear formation(PRST)18; movement of a non-paralysed hand24;measurement of lower oesophageal contractility25;and various assessments of the electroencephalogram(EEG).26–32

Closed-loop control of propofol anaesthesia in volunteers has been described previously using the median frequency of the EEG as the input signal.33However,anaesthesia was not always adequate even to abolish the corneal re?ex and this level would not be expected to produce satisfactory conditions for surgery.A similar system was used in11 patients undergoing surgery to control administration of alfentanil during nitrous oxide–oxygen anaesthesia with neuromuscular blocking drugs.34Systolic arterial pressure has also been used to control the level of anaesthesia by altering administration of iso?urane during surgery and to advise on the need for supplementary morphine.35Patients were anaesthetized to a satisfactory level before the control system was used and again,the system was evaluated only in patients who had received neuromuscular blocking drugs. In that study,9%of patients did not have satisfactory control of anaesthesia.

Ours is the?rst report describing closed-loop control of anaesthesia from induction through surgery to recovery in patients who did not receive neuromuscular blocking drugs and were breathing spontaneously.The PI algorithm controlled the AEP index within acceptable limits during the maintenance phase.The AEP index

control was initially set at35. Thereafter it was changed by the anaesthetist depending on patient response.The aim was to achieve adequate anaesthesia in terms of respiratory and cardiovascular stability and to avoid patient movement to surgical stimula-tion.All patients had satisfactory PRST scores,while movement interfering with surgery was minimal.Most of the patients had satisfactory values for end-tidal carbon dioxide concentration,although eight required assisted ventilation for more than5min.It is possible that excessively low values of the AEP index

control were selected in an attempt to avoid patient movement after the initial surgical incision. Electrical interference during diathermy overloaded the EEG ampli?er and prevented acquisition of a satisfactory signal.Therefore,during the use of diathermy,the system was set automatically to maintain the target blood propofol concentration at the previous value until a new valid signal was obtained.This did not appear to adversely affect control of depth of anaesthesia.

The?rst requirement for any closed-loop system is a valid input signal which can be used to alter the output of the controlling agent.AEP has been reported previously to re?ect accurately the level of anaesthesia,especially during the transition from awake to asleep and vice versa.1011By using the AEP as the input signal for the closed-loop control system in patients undergoing surgery while breathing spontaneously,we believe that we have validated this measurement technique as an assessment of the level of anaesthesia during maintenance with propofol.An important ?nding was that no patient recovered consciousness at an AEP index which was less than the value at which anaesthesia had been induced.This supports previous reports of the ability of AEP to distinguish clearly between awake and unconscious states.101113–15It would appear that if the AEP index is maintained below the value at which con-sciousness is lost during propofol anaesthesia,awareness should not occur.Clearly,other anaesthetic agents and patients must be assessed to determine if this is a universal?nding.

References

1Prys-Roberts C.Anaesthesia:a practical or impractical construct?

Br J Anaesth1987;59:1341–5

2Winterbottom EH.Insuf?cient anaesthesia.BMJ1950;1:247–8 3Hutchinson R.Awareness during surgery:a study of its incidence.

Br J Anaesth1960;33:463–9

4Bogetz MS,Katz JA.Recall of surgery for major trauma.

Anesthesiology1984;61:6–9

5Liu WH,Thorp TA,Graham SG,Aitkenhead AR.Incidence of awareness with recall during general anaesthesia.Anaesthesia1991;

46:435–7

6Thornton C,Heneghan CP,James MF,Jones JG.Effects of halothane or en?urane with controlled ventilation on auditory evoked potentials.Br J Anaesth1984;56:315–23

7Heneghan CP,Thornton C,Navaratnarajah M,Jones JG.Effect of iso?urane on the auditory evoked response in man.Br J Anaesth 1987;59:277–82

8Thornton C,Konieczko K,Jones JG,Jordan C,Dore CJ,Heneghan CP.Effect of surgical stimulation on the auditory evoked response.

Br J Anaesth1988;60:372–8

9Thornton C,Konieczko KM,Knight AB,et al.Effect of propofol on the auditory evoked response and oesophageal contractility.

Br J Anaesth1989;63:411–17

10Davies FW,Mantzaridis H,Kenny GN,Fisher AC.Middle latency auditory evoked potentials during repeated transitions from consciousness to unconsciousness.Anaesthesia1996;51:107–13 11Mantzaridis H,Kenny GN.Auditory evoked potential index:a quantitative measure of changes in auditory evoked potentials during general anaesthesia.Anaesthesia1997;52:1030–6

12Mantzaridis H.Closed-Loop Control Of Anaesthesia,PhD Thesis.

Glasgow:University of Strathclyde,1996

13Doi M,Gajraj RJ,Mantzaridis H,Kenny GN.Effects of cardiopulmonary bypass and hypothermia on electroencephalo-graphic variables.Anaesthesia1997;52:1048–55

14Doi M,Gajraj RJ,Mantzaridis H,Kenny GN.Relationship between calculated blood concentration of propofol and electrophysio-logical variables during emergence from anaesthesia:comparison of bispectral index,spectral edge frequency,median frequency and auditory evoked potential index.Br J Anaesth1997;78:180–4 15Gajraj RJ,Doi M,Mantzaridis H,Kenny GN.Analysis of the EEG bispectrum,auditory evoked potentials and the EEG power spectrum during repeated transitions from consciousness to unconsciousness.Br J Anaesth1998;80:46–52

16Marsh B,White M,Morton N,Kenny GN.Pharmacokinetic model driven infusion of propofol in children.Br J Anaesth1991;67:41–8

Kenny and Mantzaridis

17Davies FW,White M,Kenny GN.Postoperative analgesia using

a computerised infusion of alfentanil following aortic bifurcation

graft surgery.Int J Clin Monit Comput1992;9:207–12

18Evans JM.Pain and awareness during general https://www.360docs.net/doc/ee10332177.html,ncet 1987;2:1033

19Artusio JF jr.Ether analgesia during major surgery.JAMA1955;

157:33–6

20Parkhouse J.Awareness during surgery.Postgrad Med J1960;36: 674–7

21Robson JG.Measurement of depth of anaesthesia.Br J Anaesth 1969;41:785–8

22Saunders D.Anaesthesia,awareness and automation.Br J Anaesth 1981;53:1–3

23Breckenridge JL,Aitkenhead AR.Awareness during anaesthesia:

a review.Ann R Coll Surg Engl1983;65:93–6

24Tunstall ME.The reduction of amnesic wakefulness during caesarean section.Anaesthesia1979;34:316–19

25Evans JM,Davies WL,Wise CC.Lower oesophageal contractility:

a new monitor of https://www.360docs.net/doc/ee10332177.html,ncet1984;1:1151–4

26Gibbs FA,Gibbs EL,Lennox WG.Effect on the electroencephalogram of certain drugs which in?uence nervous activity.Arch Intern Med1937;60:154–66

27Martin JT,Faulconer A,Bickford RG.Electroencephalography in anesthesiology.Anesthesiology1959;20:359–76

28Edmonds HL jr,Paloheimo https://www.360docs.net/doc/ee10332177.html,puterized monitoring of the EMG and EEG during anesthesia.An evaluation of the anesthesia

and brain activity monitor(ABM).Int J Clin Monit Comput1985;

1:201–10

29Arden JR,Holley FO,Stanski DR.Increased sensitivity to etomidate in the elderly:initial distribution versus altered brain response.Anesthesiology1986;65:19–27

30Schwilden H,Stoeckel H.Quantitative EEG analysis during anaesthesia with iso?urane in nitrous oxide at1.3and1.5MAC.

Br J Anaesth1987;59:738–45

31Kearse LA jr,Manberg P,DeBros F,Chamoun N,Sinai V.

Bispectral analysis of the electroencephalogram during induction of anesthesia may predict hemodynamic responses to laryngoscopy and intubation.Electroencephalogr Clin Neurophysiol1994;90: 194–200

32Kearse LA jr,Manberg P,Chamoun N,DeBros F,Zaslavsky A.

Bispectral analysis of the electroencephalogram correlates with patient movement to skin incision during propofol/nitrous oxide anesthesia.Anesthesiology1994;81:1365–70

33Schwilden H,Stoeckel H,Schuttler J.Closed-loop feedback control of propofol anaesthesia by quantitative EEG analysis in humans.

Br J Anaesth1989;62:290–6

34Schwilden H,Stoeckel H.Closed-loop feedback controlled administration of alfentanil during alfentanil–nitrous oxide anaesthesia.Br J Anaesth1993;70:389–93

35Robb HM,Asbury AJ,Gray WM,Linkens DA.Towards a standardized anaesthetic state using iso?urane and morphine.Br J Anaesth1993;71:366–9

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氢化物,同时失去荧光,因而样品的荧光背景可以被测定。 OHHO OHH3CHNOOHOHHOOHH3CH3C-2HH3CH二氢化物在空气中易重新氧化,恢复其荧光,其反应如下: 核黄素的激发光波长范围约为440—500nm(一般为440nm),发射光波长范围约为510—550nm(一般为520nm)。利用核黄素在稀溶液中荧光的强度与核黄素的浓度成正比,由还原前后的荧光差数可进行定量测定。根据核黄素的荧光特性亦可进行定性鉴别。 注意:在所有的操作过程中,要避免核黄素受阳光直接照射。 三. 仪器与试剂 1. 实验试剂:核黄素标准品;冰醋酸;核黄素药片;连二亚硫酸钠(保险粉)或亚硫酸钠 2. 实验仪器:荧光光度计(F-2500型)天平(感量0.0001g) 3. 实验器材 普通试管: 容量瓶:

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