Experimental Evaluation of Analgesia
Author: C. Radouco-Thomas, S. Radouco-Thomas, Gl. Nosal
Pages: 14 to 38
Creation Date: 1956/01/01
1. New algesimetric method 2.Comparative study of the analgesic action of morphine, pethidine, 1-methadone and levorphanolM.D. C. Radouco-Thomas Lecturer, University of Geneva, Faculty of Medicine
Ph.D. S. Radouco-Thomas
I. Experimental conditions 15
1. Painful stimulus 15
2. Pain receptors 15
3. Substances studied 16
II. Apparatus 16
1. Stimulating circuit 16
(A) Generator 16
(B) Electrodes 16
2. Accessories 17
III. Experimental procedure 17
1. Preliminary phase 17
2. Determination of the Pain-Response Threshold 17
3. Evaluation of analgesic potency by the increase of the painresponse threshold 18
IV. Biometric analysis of results 18
I. Biological response 18
1. Qualitative study 18
Somatic physiological reactions: R 0, R 1, R 2 19
2. Quantitative study of the Pain-Reaction Test (P.R.T. = R 1) 20
A. Threshold voltage and biological factors 20
(a) Inter-animal variations 20
(b) Intra-animal variations 21
B. Threshold voltage and biophysical factors (parameters of the current) 22
(a) Threshold voltage/impulse frequency ratio
(b) Voltage threshold/impulse duration ratio 22
II. Evaluation of analgesic action 22
1. Narcotic analgesics (morphine, pethidin, l-methadone, levorphanol) 22
A. Qualitative study 22
B. Quantitative study 22
(a) Dose/effect/time ratio 22
(b) Dose/effect ratio 25
Margin of safety 25
2. Other depressants of the central nervous system (ether, thiopental) 25
I. Reliability of the method 26
1. Physiological data 26
A. Are the reactions evidence of pain ? 27
B. Qualitative and quantitative stability of the P.R.T. 27
2. Pharmacological data 28
A. Reliability of the method 28
B. Selectivity of the method 29
Intensity of analgesia 29
Duration of analgesia 29
C. Electivity of the method 30
II. Validity of the method 30
1. Comparison with results of animal experimentation 30
A. Electro-dental methods 30
B. Thermo-cutaneous methods 32
2. Comparison with results obtained in man 33
A. Experimental algesimetry (superficial, deep and visceral pain) 33
B. Pathological pain 34
III. Efficiency of the method 34
Doch, einzig in der engen Hohle des Bakenzahnes verweilt die Seele.
The "pain complex" comprises a primary, sensory process-the sensation of pain-with, superadded, a series of "psycho-physiological" alarm and defence reactions.
Pain is not a normal condition and it is actually injurious when chronic. "Pain itself may initiate or perpetuate a biological destructive cycle", Wolff 1951 ( ). Man has always sought to suppress it.
Among the many monographs and works on the physiopathology of pain and analgesic medication, mention may be made of the following : Kruger, Eddy & Sumwalt, 1940 ( ); Lewis, 1942 ( ); Symposium on Pain, 1943 ( ); Wolff and Hardy, 1947 ( ); Symposium on New Synthetic Analgesics, 1948 ( ); Isbell, Fraser and al. ( ,  ,  ,  ); Wikler, 1950 ( ); Wolff & Wolf, 1951 ( ); Beckett, 1952 ( ); Dorpat, 1952 ( ); Schaumann, 1952 ( ); Bonica, 1953 ( ); Hardy, Wolff & Goodell, 1953 ( ); Schoen, 1953 ([137 ] ); Wikler, 1953 ( ,  ); Braenden, Eddy & Halbach, 1954 ( ); Jacob, 1954 ( ); Scherrer et al, 1954, 1955 ( ,  ); White & Sweet 1955 ([160 b] ); Radouco-Thomas C., Radouco-Thomas S. & Nosal, 1956 ( ,  , [124b] ). There are also recent works by Hazard ( ), Jacob ( ) and Scherrer ( ), published under the direction of Alajouanine, 1957 ( ), on pain and its pharmacodynamics.
A survey of antalgic medication as now practised shows that it is fundamentally symptomatic. Two groups have pride of place in the arsenal of pain therapeutics-the non-narcotic analgesics (antipyretics) and the narcotic analgesics (morphine and the morphinomimetics).
The first group has a moderate and partial antalgic action, often attended by toxic side-effects. The second comprises highly potent analgesics as effective as they possess addiction liability.
It is to pharmacology and experimental therapeutics that falls the difficult task of selecting by appropriate methods ever more potent and more easily tolerated drugs from the amorphous mass of new synthetics.
The much-debated problem ( ,  ,  , [160b] ) of the value of the algesimetric methods used in experiments with animals and on man will be discussed later on. Suffice it to state here that there are now more and more neuro-physiological and neuro-pharmacological arguments for the view that the real evidence for the sensation of pain in animals lies in a "chain" of physiological reactions (somatic-vegetative-humoral).
Many different methods are used for the experimental evaluation of analgesic action.
Chemical and physical stimuli (mechanical, thermal and electrical) have been applied at the level of the various pain receptors to reproduce the classic types of pathological pain (superficial, deep and visceral) in animals. A number of studies have been devoted to this problem by Seevers et al., 1936 ( ), 1937 ( ), Goetzl, Burril & Ivy, 1943 ( ); Pfeiffer et al., 1948 ( ); Miller, 1948 ( ); Wirth, 1952 ( ); Fleisch & Dolivo, 1953 ( ); and Jacob, 1954 ( ).
Among the multitude of algesimetric methods, our attention has been particularly drawn to the electro-dental stimulation introduced into experiments with animals (dogs) by Koll & Reffert in 1938 ( ), and later used and improved by Goetzl et al., 1943 ( ), Soehring et al., 1949 ( ) and Boreus & Sandberg, 1955 ( ). Though it has given remarkable results, it remains confined to specialized research: "Die Methode von Koll hat den besonderen Vorteil am hoher organisierten Tier zu arbeiten. Sie erfordert aber bezüglich Tierhaltung, operativer Vorbereitung, u.dgl. einen erheblichen Aufwand, so dass sie mehr für spezielle als für Reihenuntersuchungen in Frage kommt."-Wirth, 1952( ).
We venture to propose a new routine algesimetric method based on electro-dental stimulation of the pain-receptors of guinea-pigs.
This work * deals in turn with the reliability, validity and efficiency of the method in evaluating the analgesic power of morphine and of some synthetic morphinomimetics : pethidine, 1-methadone and levorphanol.
The problem of the neuro-physiological mechanism of pain ( ) and the manner in which narcotic analgesics act from the dual aspect of analgesia andaddiction liability will be studied elsewhere.
We shall describe in turn the experimental conditions, apparatus, experimental procedure ( ) and the methods of biometric analysis used.
Electrical stimulation was systematically used in our algesimetric study.
The most common types of current employed are faradic, sinusoidal and square-wave impulses and condenser discharges. Schroeder, 1907 ( ); Heinroth, 1926 ( ); Hesse & Reichelt, 1933 ( ); Freund, 1936 (44); and Jores & Frees, 1937 ( ) used faradic current. Goetzl et al., 1943 ( ); Harris & Blockus, 1952 ( ) and Fleisch & Dolivo, 1953 ( ) preferred alternating current. Stimulation by condenser discharges was used by Koll & Reffert, 1938 ( ) and by Kiesig-Orzechowski, 1941 ( ). Finally, square-wave impulses were selected by Björn, 1946 ( ), Soehring & Becher, 1949 ( ), Stellmach et al., 1952 ( ), Boreus & Sandberg, 1955 ( ) and Yim et al., 1955 ( ).
The parameters of the current (voltage duration and frequency of impulses), and the duration of stimulation and interval between stimulations vary with the type of current, the animal used, etc.
In most of the studies: Koll and Reffert, 1938 ( ); Goetzl et al., 1943 ( ); Stellmach, Schroeder & Soehring, 1952 ( ); Fleisch & Dolivo, 1953 ( ); Boreus & Sandberg, 1955 ( ); Yim et al., 1955 ( ), the voltage generally serves to define the threshold.
We used square-wave impulses with the following characteristics:
Frequency of impulses: 40 per second
Duration of impulse: 5 milliseconds
Length of stimulation: 0.5 sec.
We selected the afferent dental nervous system as the pain receptor field. Stimulation can take place: either, as indicated by Soehring ( ), through the section of the dentine between the physical electrode and the tooth pulp, Steinhausen’s "physiological electrode" ( ); or directly on the afferent nervous system in the dentine itself, the existence of such a system having been reported by Dependorf 1913 ( ), Mummery 1924 ( ), Münch 1927 ( ) and Berkelbach 1934 ( ). Recently Baud & Held, 1953 ( ,  ), by an improved technique revealed the existence of sensitive fibres in the dentinal tubules in man. Baud, Radouco-Thomas & Nosal are now making a similar study on the guinea-pig’s teeth. As to their nature, two types of sensitive and vegetative fibres have been described by Berkelbach 1934 ( ), Brashear 1936 ( ), Christensen 1940 ( ) and Zerosi 1946 ( ). Physiologically speaking, a sensation of pain is the afferent system’s sole response to any kind of stimulus.
Two types of teeth have been used in electro-dental algesimetry- Limited growth teeth, such as the canine teeth of the cat ( ) and dog. The stimulation of the dental receptors of dogs has been, and still is, one of the more available methods for studying pain and analgesia. It is interesting to note that a similar technique has been used in man for some fifty years; Schroeder, 1907 ( ), Heinroth, 1926 ( ), Werz, 1932 ( ), Hesse & Reichelt, 1933 ( ) and Freund, 1936 ( ), having used it for physiological and pharmacological research. Koll & Reffert first applied it in 1938 ( ) to animal experiments to evaluate analgesic action, and it was subsequently used as such or with modifications by KiesigOrzechowski, 1941 ( ), Goetzl et al., 1943 ( ), Pfeiffer et al., 1948 (120), Soehring, 1949 ( ), Fleisch & Dolivo, 1953, ( ), Boreus & Sandberg, 1955 ( ), etc.
FIGURE 1 - Chemical structure of the synthetic analgesic narcotics studied
Continuous growth teeth such as the upper incisors of rodents This method, introduced by Gordonoff-Rückstuhl in 1939 ( ) was used in the rabbit for pharmacological research by Wilhemi, 1949 ( ), Domenjoz, 1952 ( ), 1953 ( ) Fleisch & Dolivo, 1953 ( ), Frommel et al., 1953 ( ) Sinniger, 1953 ( ) and Yim et al., 1955 ( ).
We have chosen as dental pain receptors the upper incisors of guinea-pigs. To our knowledge, there is no instance in medical literature of research on the dental organs of this species. The reasons for our choice are many. The guineapig gives to an electrical stimulus visible evidence of pain in the form of a clear-cut and constant biological response, similar to that of the dog. Furthermore, the response can be used for serial experiments, since the subject is a small laboratory animal, inexpensive and easy to handle.
We chose adult guinea-pigs of both sexes and average weight (500 g) living under standardized conditions (living space, food, temperature, etc.).
We studied the analgesic action of morphine and some synthetic morphinomimetics: pethidine, 1-methadone and levorphanol (fig. 1). The doses are given in Table II (p. 29) and were administered hypodermically.
At the same time we studied the analgesic effect of some depressants of the central nervous system: Thiopental (pentothal) 10-30 mg/kg, Officinal ether, inhalated: 7 cc for 5 and 15 minutes in a bell-glass.
The doses given are expressed in milligrammes per kilo of animal weight (mg/kg). The effect of each dose was traced on 4 to 10 animals.
The apparatus used for the algesimetric studies generally consists of a stimulating circuit and a few accessories.
This comprised a current generator and a system of electrodes.
The generator may vary in type according to the form of current selected. Some authors ( ,  ,  ) include a strong resistance in this circuit to counteract variations in the subject's resistance. For our experiments, we used a squarewave impulse generator-the Grass stimulator. An oscillograph was connected in parallel to check the stimuli.
The electrodes constitute one of the most important elements in the apparatus, for on them depend the speed and accuracy of the method and the constancy of the results. Several types of electrode have been devised, varying in form, composition and above all, in mode of insertion.
In man, Schroeder, 1907 ( ), Heinroth, 1926 ( ), Hesse & Reichelt, 1933 ( ), Freund, 1936 ( ), Goetzl et al., 1943 ( ), Björn, 1946 ( ), Pfeiffer et al., 1948 ( ) Sonnenschein and Ivy, 1949 ( ), Harris & Blockus, 1952 ( ) and Boreus et al., 1955 ( ), used an active dental electrode and an indifferent electrode of the E.C.G. type.
For animal experiments two different types of electrode are used:
(a) Fixed electrodes for animals with limited-growth teeth. The prototype of this type of electrode, employed by various authors on dogs and cats, was evolved by Koll & Reffert in 1938 ( ). They are of silver, inserted into a canine tooth, and connected by two insulated conductor wires the free ends of which are led through a facial fistula. They are connected in series to the stimulating circuit at the moment of stimulation. Soehring et al., 1949 ( ), Stellmach et al., 1952 ( ) and recently Boreus et al., 1955 ( ) made a number of improvements on this technique.
(b) Removable electrodes, used on animals with continuous growth teeth. For rabbits, the Fleisch & Dolivo's ( ) type of electrode was generally adopted. This consists of an insulated clip supporting two pointed metal electrodes (0.8 mm in diameter) which fit into holes drilled in the incisors. On guinea- pigs we used a system of buccodental removable electrodes (figure 2).
The indifferent electrode. At the beginning of our experiments, a cutaneous indifferent electrode was fixed to one guinea-pig's fore-paws. After comparative study, however, we found it preferable to fix the indifferent electrode in the mouth. This ensured constant biophysical parameters and also prevented the animal from biting the research worker or dislodging the dental electrode with its tongue.
FIGURE 2. Bucco-dental electrodes A. Active electrode (dental) B. Indifferent electrode (buccal)
FIGURE 3. Animal with the two electrodes inserted
The bucal electrode consisted of a nickel-silver plate of about 1 sq cm soldered to a semi-rigid insulated wire which followed the line of the animal's jaw. This type of electrode has already been used by Radouco-Thomas et al. ( ,  ) in a system of bucco-occipital electrodes for' electrically induced epilepsy in animal experiments.
The active electrode. The active or monodental electrode is constituted by a 4/10 mm nickel-silver wire held by a microclip fixed perpendicularly on the upper incisors of the animal just before stimulation (figure 3).
The indifferent electrode remains in place throughout the experiment. The two electrodes are connected with the generator and the cathode-ray oscillograph. To permit of a serial experiment (nine animals) the wires to the electrode system are run from a bracket connected by a switch to the stimulating circuit.
Such electrodes are easy to devise and rapidly inserted (it takes about five minutes to insert the buccal electrode for a group of 6-9 animals) and remain very steady.
These consist of a dental drill, boards for attaching the animals, etc. To perforate the teeth, we used an ordinary dentist's drill, but fitted with 0.5 mm drills. Larger drills (0.8 mm) of the type employed for drilling the teeth of rabbits were used at the beginning of our research, but these split the incisors, thus putting the animals out of action until the new teeth grew.
The algesimetric experiments were in three phases:
Our research showed that, to ensure a stable threshold voltage, it was preferable to drill the teeth the day before the experiment. The tooth selected must be sound, clean and long enough to bear a microclip (active electrode). The hole is drilled on the lateral upper side of one incisor about 0.5 mm below the gingival margin and to a depth of some 1/10 mm. The animal, unattached, is held still by the experimenter during the drill. Nine animals can be thus prepared in a quarter of an hour.
The animals are attached to boards, lying in "decubitus ventral ", and arranged in a row (6-9 animals) in a quiet room free from any sensitivo-sensorial stimuli. The insertion of the buccal electrodes is followed by some agitation. The guinea-pigs go through chewing motions and try to rid themselves of the electrode. After 10-15 minutes, however, they calm down and the threshold may be determined. The holes must be scrupulously cleaned before the pain measurements are made, as the tiniest particles of food affect the resistance of the tooth and cause an aspecific biological response or an abnormally high threshold value.
The threshold voltage is then determined by trial and error. All animals are first subjected to a voltage (3 volts) near to the average voltage (x = 3.16). Then, according to their response they are subjected to rising or decreasing voltages until the pain-reaction test is obtained. The threshold voltage thus determined is checked twice by applying a series of three stimuli and repeating them after twenty minutes. The threshold grading in a series of animals saves the time that would otherwise be lost by observing the necessary interval of three minutes between each two stimulations. The determination is accurate to 0.1 volts.
After grading, the animals receive the selected dose of the substance to be studied by whatever route has been chosen, and they are then subjected to increasing stimuli until the pain-reaction test is obtained.
The gradation of the stimuli for each animal is based on its control threshold voltage. The voltages selected were not in arithmetical progression (addition of a fixed number of volts or a percentage of the threshold voltage), but in geometrical progression: log V N + 1 = log V N + k. Each
Excitation Unit (Ex. U.) represents an increase of 1/10 on the previous voltage:
Ex. U. = 0 : V 0 = Threshold voltage
Ex. U. = 1 : V 1 = V 0 + V 0/10
Ex.U.=N+1 :V N+l=V N+V N/10
The interval between Excitation Units 0,1,2 ..... N is therefore smaller for low voltages than for high ones. This progression was chosen on the basis of Fechner's law of sensorial physiology: response = log (stimulus) + k. Whatever their control threshold voltage, the animals were sensitive to the interval chosen (1/10) and when stimulation was one Excitation Unit less than it should have been the result was always an incomplete response.
We therefore established a scale of supraliminal voltages for each animal derived from its threshold voltage by progression V N + 1 = V N + V N/10, which the experimentor was bound to follow. This procedure adopted in conjunction with serial animal experiments with different threshold voltages, helps to counteract the subjectivity of the experimenter while he is working only on volts. We always observed an interval of three minutes between each stimulation and twenty minutes between each determination (three stimulations). If this time-table is not adhered to, too frequent stimulations cause an artificial rise in the threshold voltage.
The determinations were made in turn on two groups of four and five animals. This arrangement enabled measurements to be taken for each group in ten minutes.
Tests were made every thirty minutes or every hour according to the metabolism of the drugs, until the pain-reaction test was again forthcoming at the initial threshold voltage.
Each animal was used only once a week.
The experiments with analgesics were mainly performed with three doses of three narcotic analgesics. To eliminate day-to-day variations (animals' diet, weather, ambient temperature), the nine animals in each pharmacological experiment were divided into groups of three, each of which received a different dose of the drugs. Each point on the graphs therefore represents the average of the results obtained under the same conditions ( ,  ). The guinea-pigs, after administration of the drugs, were lined up at random to increase the objectivity of the experimenter in assessing the increase of the threshold ( , ).
The analgesic effect was expressed in "Excitation Units" and not in volts, to eliminate the influence of the sensitivity of the normal animal to pain. Biometric analysis of the results was also made on the basis of these values. In the graphs reproduced in this document, we have added a scale showing the increase in the threshold value as a percentage of the initial threshold voltage, to permit of comparison with other data in medical literature.
Determination of the equiactive doses of the drug studied was based on the quantal response of the animal ( ,  ). Analgesia was considered to have been induced in the animals when they showed no Pain-Reaction Test to a stimulus of 5 units (over 50% increase in the threshold).
Lethality was expressed in terms of the percentage kill in the groups of 5-10 animals receiving increasingly toxic doses of each product. The ED 50 and LD 50 doses were determined by the Miller-Tainter method. The margin of safety was fixed by the Index 50 and the therapeutical index ( ,  ,  ).
Our results are presented in two sections:
I. Study of the biological response to stimulation of the dental pain receptors in guinea-pigs;
II. Evaluation of the analgesic action.
Under the experimental conditions just described-i.e., taking as constant parameters a frequency of 40 impulses per sec., an impulse duration of 5 msec. and a stimulation duration of 0.5 sec. and subjecting the animal to increasing voltages-we obtained several types of response:
Reaction R 0 (opening of the mouth)
At a low voltage (less than 1 volt), the animal responded by a chewing movement or a slight tremor of the jaw, sensitive to the touch but difficult to see.
With stronger stimuli (x = 2.4 volts ± 0.1), the animal responded by a slight, abrupt opening of the mouth (figure 4). This reaction, R 0, is constant, and its threshold regularly recorded.
Increases in voltage elicited an ever-wider opening of the mouth, subsequently accompanied by un-coordinated defensive movements on the part of the animal, as if it wished to rid itself of the tooth electrode.
At higher voltages (x = 3,16 volts ± 0.06) a characteristic response ( ) emerged which we have selected as the PainReaction Test (R 1 = P.R.T). The main feature of this response was a rapid upward thrust of the head. Response is immediate, brisk and performed in a single smooth movement (figures 5 and 6).
In some animals, the angle of tilt was more than 90°, in others, less. With some the movement was accompanied by a contralateral or ipsilateral twist (fig. 7). These individual variations are extremely slight with tied animals, but quite definite with those in freedom. In conditions of freedom (fig 7a), changes in attitude were mainly as regards the intensity of the head movement. The upward thrust of the head is so violent that the animal loses balance and fills over backwards (figure 7b). We never observed any tendency to flight.
Pain Reaction Test (P.R.T.=R 1) front view (tied animal)
Although the response of the free animal is richer in physiological information, we performed all our experiments on tied animals. This position standardizes individual responses and makes it easier to differentiate subliminal responses (jerky upward thrust, in one or two jerks or undulating) from the Pain Reaction Test, and above all makes it possible to perform experiments in series.
At even higher voltages, the animal's response was of the same type, but more violent and often accompanied by a cry.
At very high voltages (about 10 volts), the upward thrust of the head disappeared completely and was replaced by a "horrified" recoil of the head, accompanied by a cry (R 2). Mydriasis, a certain bristling of the hair and trembling of the hind-quarters were also noted. This stimulus was followed by a few groans and a state of excitation. In free animals, the same increase in voltage elicits first an agitated movement of recoil and then flight.
Pain Reaction Test, profile (tied animal)
Pain Reaction Test, contralateral (tied animal)
Pain Reaction Test, free animal, front view and profile
Such were the various aspects of the somatic responses obtained from guinea-pigs by electro-dental stimulation.
The extent to which they can be regarded as an objectivation of the sensation of pain in guinea-pigs following dental stimulation will be considered in the discussion chanter.
Of the various responses, we chose the single upward thrust of the head as the Pain Reaction Test (PRT) owing to its clear-cut nature, regularity and reproducibility. This choice, as we shall see later, was also justified by a number of neuro-physiological and pharmacological criteria.
Before passing to the quantitative study of the P.R.T., it may be advisable to give some information on the muchdebated question of training the animals.
No training period was required to obtain the P.R.T. with guinea-pigs. There were no variations between the first and later determinations. At the threshold voltage, the animals showed the same type of P.R.T. in the various series of experiments.
With animals already subjected to the test, the threshold voltage appeared, however, to be easier to determine than with new animals, the upward thrust of the head attaining more swiftly a stable threshold with a characteristically clean movement.
The only factor requiring training is the experimenter, who has to become used to recognizing the P.R.T. and distinguishing it from subliminal responses (jerky or undulating movement) and supraliminary responses (more violent movements).
It should, however, be pointed out that during the various sessions to determine the control values prior to each pharmacological experiment, we sometimes obtained for one or other of the animals a regular non-typical response or a response of type R 2, whatever the voltage used. These animals were excluded from the current experiment, but gave a typical P.R.T. again on following days or after a new drilling. Such unexpected and passing deficiency appears to have been due to some anomaly in the conditions of the experiment.
Subsequently we avoided this drawback by drilling holes the day before in two incisors. Then, if the stimulation of one tooth brought a non-typical response or one of type R 2, it was enough to stimulate the other incisor in order to obtain a typical P.R.T. The animal could then be used for the experiment.
FIGURE 7 b
Pain Reaction Test, free animal. The intensity of the reaction is such that the animal loses balance
Under this heading, we analysed the P.R.T. and, occasionally, responses R 0 and R 2. The lowest voltage eliciting a characteristic P.R.T. was taken as the threshold voltage or threshold for reaction.
We will now present the variations in threshold voltages in their relation first to the animal and then to the parameters of the stimulus.
The study of inter-animal variations mainly concerns the Pain Reaction Test. A series of supplementary measurements were made of the R 0 response. Figure 8 gives a block diagram of the P.R.T. threshold voltages for 221 determinations and shows that guinea-pigs have an average threshold voltage of 3.16 volts ± 0.06.
Block diagram of the threshold voltage of the Pain Reaction Test x axis : voltage (logarithmic scale) y axis : number of animals
In 167 cases we also registered the voltages for R 0 and R 1 during the same experiment. These are shown in the double block diagram in Figure 9. It will be seen that the average voltage for R 1 shows an increase of 50% over that for R 0. x R0 = 2,4 ± 0,1 x R1 = 3,7 ± 0,1
This ratio, regularly encountered when establishing the individual control values, was confirmed by statistical study, the correlation coefficient "r" having the highly significant value of 0.879.
Block diagram of the threshold voltages of R 0 and R 1 (P.R.T.) x axis : voltage (logarithmic scale) y axis : number of animals
We studied the variations of the threshold voltage in the same animal on the same day with one drill-hole and with several drill-holes.
On any single day, the response threshold of the animals proved remarkably constant.
During the pharmacological experiments, the control animals were subjected for the same time (3-7 hours) to stimulation at the same rate as the drugged animals. Their thresholds proved constant (figure 20), a subliminal and a superliminal stimulus (± 1 stimulation unit) eliciting respectively a subliminal and a superliminal response.
We also obtained a consistent return to the initial threshold voltage with drugged animals. This stability of the threshold of response in the course of a single experiment is the basis for the pharmacological study of analgesic action. As pointed out in the section on method, the increases in the threshold value recorded in all the pharmacological experiments were always related to the individual premedication threshold voltages.
Thus each point represents not the average increase in the voltage, but the average of the elevations of the individual threshold voltages. This explains why, despite inter-animal variations in the threshold voltage, all the curves (figures 13, 14, 15 and 20) have a common point of departure, V 0, expressing all the initial threshold voltages.
Variations of the threshold voltage of the Pain Reaction Test during one week for one drilling (4 animals) x axis : time i n days y axis : voltage
As a matter of interest we also recorded variations in the threshold voltage during the days following the drilling. For the first two or three days it remained constant. After this, however, there was a steady rise in the threshold voltage, inversely proportional to the downward movement of the hole (figure 10) Any given hole could be used only during the 8 to 10 days after drilling, owing to its gradually moving farther away from the tooth pulp and to the increasing difficulty of fitting the micro-clip.
The variations in the threshold voltage in the same animal from one drill to another are quite unrelated.
For example, the following variations were registered on one animal with 8 perforations : 2.5, 2.7, 3.4, 3.6 2.9, 2.5, 1.4, 2.4 volts. This variability of threshold voltages in the same animal brings out the fact that the threshold, voltage value depends more on the electrode-receptor complex than on the threshold of the animals' sensitivity to pain.
We studied the variations in the threshold voltage of the P.R.T. in relation to the frequency and duration of impulses.
With a constant impulse duration of 5 m.sec., we subjected five animals to frequencies varying from 7.5 to 100 imp/sec. The threshold voltages required to elicit the P.R.T. under such conditions are shown in figure 11.
It will be seen that the voltage/frequency ratio values can be plotted as a hyperbola.
Threshold voltage/Impulse frequency ratio x axis : impu lse frequency y axis : voltage
FIGURE 12. Threshold voltage/Impulse duration ratio x axis: impulse duration (msec.) y axis: voltage
In the 9-100 imp/sec. zone, the quality of the P.R.T. remained unchanged.
In the low-frequency zone (below 9 imp/sec.), we were unable to obtain a typical response, whatever the elevation of the threshold. The animals made as if to thrust their heads upward and back in a movement consisting of several jerks synchronized with the impulses received by the animal during stimulation.
We registered the voltage threshold values required to elicit a P.R.T. with various impulse durations at constant frequency (40 imp/sec). A typical response was obtained only for durations from 0.2 to 7 m.sec. The points calculated for five animals can be plotted as a hyperbola (figure 12).
The results obtained for morphine and three synthetic narcotic analgesics, pethidine, l-methadone and levorphanol, are given below. For purposes of comparison, a few results are appended for two central nervous system depressants, ether and thiopental.
We will consider the effect of the narcotic analgesics first on the form (qualitative study) and then on the threshold (quantitative study) of the Pain-Reaction Test ( ).
The animals' response remained unchanged in form under the influence of the narcotic analgesics, there being only an increase in the threshold voltage value.
With the doses administered, we observed no excito-depressive effects (narcotic effect, respiratory depression, vomiting, etc.). The animals remained brisk and wide-awake.
We first studied, substance by substance, the intensity and duration of the analgesic action with increasing doses (dose/ effect/time ratio).
Analgesic action of morphine. Curve: dose/effect/time ratio:
I = 0.8 mgr/kg II = 2.5 mgr/kg III = 7.5 mgr/kg x axis : time in hours y axis : rise in the threshold of response in Excitation Units (Ex.U.) and in %
We then made a comparative study in connexion with the dose/effect ratio (percentage of animals showing analgesia or percentage killed) of the analgesic effect and the margin of safety of the drugs.
Morphine. Three doses, in a ratio of 1/3 (0.83 mg/kg, 2.50 mg/kg and 7.50 mg/kg), were administered to the guinea-pigs by subcutaneous injection. A significant effect was already observed with the 0.83 mg/kg dose. Whatever the dose, the analgesic action developed gradually, reaching its peak towards the second hour after administration (figure 13).
The duration of effect increased with the dose, varying from three hours with 0.83 mg/kg to six with 7.5 mg/kg.
Pethidine. This was administered in 3, 10 and 20 mg/kg doses. Pethidine, as can be seen from figure 14, is characterised by its rapid but brief action. The 3 mg/kg dose caused a very significant rise in the threshold of response. With the 10 mg/kg dose, the action practically reached its peak about one hour after the subcutaneous injection, gradually declining from then on. The curve for the 20 mg/kg dose flattened out from one hour to one and a half hours. The slopes of the onset and waning of the analgesic effect were very steep. Whatever the dose, action was only very weak in the third hour.
Analgesic action of pethidine. Curve: dose/effect/time ratio:
I = 3 mgr/kg II = 10 mgr/kg III = 20 mgr/kg x axis : time in hours y axis : rise in the threshold of response in Excitation Units (Ex.U.) and in %
Analgesic action of 1-methadone. Curve: dose/effect/time ratio:
I = 0.4 mgr/kg II = 1.25 mgr/kg III = 3.75 mgr/kg
x axis : time in hours y axis : rise in the threshold of response in Excitation Units (Ex.U.) and in %
Analgesic and lethal effect of morphine, p ethidine and l-methadone. Curve: dose/effect x axis : doses in mgr/kg (logarithmic scale) y axis : % of animals in which analgesia was induced; or dead animals (probits)
Methadone. As for morphine and pethidine, three doses in increasing quantities, namely 0.41, 1.25 and 3.75 mg/kg were administered subcutaneously. The analgesic action increased steadily with the dose. Methadone is reaching its peak similarly to morphine towards the second hour after administration (figure 15). Duration of effect was four to six hours according to dose.
Levorphanol. With this drug, we made only a few experiments which showed it to have an action of the morphine type. A systematic study will be submitted later. The 3.75 mg/kg dose had an effect equivalent in intensity and duration to that of 7.5 mg/kg of morphine (figure 20).
In order to compare the analgesic potency of the three drugs studied, we tried to ascertain their equiactive doses. Calculation of these can be based either on the threshold value, or on the absence or presence of analgesia for a given intensity of stimulation. We selected the quantal method for preference, as it is also the basis for determining the lethal dose and thus permits of subsequent comparison of zones of activity and lethality.
Lots of 5-10 animals, each given progressively larger doses of the analgesics under study, were subjected during the hour of peak effect of each product to a standard stimulus representing five stimulation units (an increase of 50 per cent over the control threshold voltage). We noted the percentage of animals in each lot in which analgesia was induced-i.e., those not giving the Pain Reaction Test.
The straight-line curves delimiting the zone of activity of each product, are shown in figure 16.
The doses inducing analgesia in 50 per cent of the animals (analgesic dose 50 = ED 50), calculated by the Miller-Tainter method ( ) are given in table I.
We also determined the toxicity of the same products in guinea-pigs. Once the analgesic 50 and lethal 50 doses are known, one can work out the corresponding index 50. These indices (table I) illustrate the very broad margin of safety in the guinea-pig, between the analgesic and the lethal effect of the narcotic analgesics. The analgesic action does not interfere with the toxic side-effects either. This question will be dealt with again during the discussion.
The straight-line curves of effect and toxicity not being parallel, the therapeutical index is a more accurate means of estimating the margin of safety ( ). We have none the less given the more widely used Index 50 in table I to permit of comparison with data in medical literature. In view of the breadth of the margin of safety, the differences between the values of the two indices are of only very minor importance.
Analgesic ac tion of ether. Curve: effect/time x axis: time in minutes y axis: rise in the threshold of response in excitation units (Ex.U.)
We also studied the effect on the P.R.T. of two central nervous system depressants in the general anaesthetics group: ether and thiopental.
Etherwas administered by inhalation:
On an initial control lot (six animals), we recorded a second-degree general anaesthesia of an average duration of 35 minutes (figure 17).
The animals in the second lot were left 5 minutes in a bell-glass. We stopped the experiment in the pre-anaesthesia phase in the case of ether. We immediately checked the P.R.T. threshold and found no significant increase in the value (figure 17).
The animals in the third lot were left 15 minutes in the bell-glass and then subjected to electro-dental stimulation. The results are plotted in figure 17. This shows that as long as the anaesthetic sleep lasts, the P.R.T. is completely absent; even very high voltages (about 20 volts) provoke only a R 0 type reaction.
Analgesic action of thiopental. Curve: dose/effect/time ratio: I = 10 mgr II = 25 mgr x axis: time in hours y axis: rise in the threshold of response in excitation units (EX.U.)
On waking, the animals at first give a P.R.T. only at very high voltages (120%). The response returns to the initial threshold after about 50 minutes.
Parallel with the elevation of the threshold, there is a change in the quality of the P.R.T. It loses its vigour, becoming feeble and sluggish.
In the six animals studied, administration of thiopental in doses of 10 mg/kg induced sedation accompanied by a slight increase in the threshold value (about 15%), for an average duration of 60 minutes. The response, as in the case of ether, was feeble and sluggish. Return to normal was gradual and took a few hours.
With a hypnotic dose, 25 mg/kg (on four animals) it was, as in the case of ether, impossible to obtain a P.R.T. a long as the anaesthetic sleep lasts. The initial threshold value was returned to after about 7 hours (figure 18).
For an algesimetric method to be used in experimental medicine and pharmacology, it must meet a series of basic requirements with respect to reliability validity and efficiency.
For the method to be reliable:
( ) It must be certain to elicit in the animal a sensation of pain which can be identified by physiological reactions (somato-vegetativo-humoral).
( ) The responses must be easily and clearly definable, qualitatively speaking, and capable of quantitative evaluation.
( ) It must be able to demonstrate the analgesic action of substances of established clinical efficacy.
We will first discuss the physiological and then the pharmacological data which ensure the reliability of the method.
There are as yet no methods of neuro-physiological investigation of sufficient accuracy for the direct evaluation of pain phenomena. As Jacob, 1954 ( ), points out, the most objective biological response would be the electrical disturbances recorded at the centres and along the path of the pain. Little work has been done in this field: Nims et al. 1941 ( ), Andrews 1942 ( ), 1943 ( ), Cahen & Wikler, 1944 ( ), Leimdorfer 1948 ( ), Toman & Davis, 1949 ( ), Wikler 1950 ( ), Scherrer et al., 1954 ( ), 1955 ( ), and Monnier & Gangloff, 1955 ( ), 1956 ([114b] ). Such research is just beginning.
For this reason, algesimetric studies have still to be made through the medium of indirect biological responses. In man, information on the spontaneous (pathological) or induced (experimental) sensation of pain is obtained from the patient's own description.
In animal experiments, a series of somatic reactions of the quantal type may be said to provide evidence of the presence and intensity of the sensation of pain. In the guinea-pig, we induced by progressive electric stimulation of the dental receptors a series of reactions (R 0, R 1, R 2) described in the chapter on results.
The fundamental problem which arose before the analgesics could be studied was whether the reactions were really the visible expression of a pain phenomenon and, if so, whether the vigour of the pain reactions increased regularly in proportion to the intensity of the stimulus.
Response R 0.-Response R 0, opening of the mouth (figure 4), is characterized by its uniformity and monotony. Is it a response to a minimum sensation of pain or a simple reflex without pain ?
Pfeiffer et al., 1948 ( ) report that it is possible to induce in man by dental stimulation a faint pain with a very low threshold (T 1) and a severe pain with a higher threshold (T 2). According to Harris and Blockus, 1952 ( ), the first sensation is not painful, and it is only when the stimulus becomes more intense that a clear sensation of pain emerges. Koll et al., 1938 ( ), record the emergence of uniform reactions at low voltages: muscular jerks in the floor of the mouth, licking movement of the lip on the side of the stimulated tooth, but do not regard these as pain-responses. Soehring and Becher, 1949 ( ), describe a response, syndrome Z, in dogs, similar to R 0 in the guinea-pig, which is constant and monotonous in form and development. They regard it as a mere reflex representing the earlier stage in the motor effect provoked by the dental stimulus.
Response R 1 ( P.R.T.).-The qualitative study shed some light on the concrete evidence of pain furnished by the testing reaction. Although this reaction takes the form of a primary movement-a clear-cut and easily identifiable single upward thrust of the head as far as it will go (figures 5 and 6)-each animal shows individual secondary variations. Moreover, there is often a cry at the threshold voltage or, more often, at superliminal voltages. The individual characteristics, more marked when the animal is free, are probably, as reported by Koll & Reffert in the case of dogs, the sign of a painresponse and not of a simple reflex without pain.
The quantitative study, based on variations in the frequency and duration of the impulses of the stimulus, show, as Soehring observed in the case of dogs ( ), that the test response of the guinea-pig may be regarded as a pain reaction. The voltage/frequency ratio can be graphically represented as a hyperbola (figure 11), which should be an indication of a summation phenomenon. This phenomenon, studied by Magun, 1948 ( ), is said to be characteristic of the pain sensation in man. Scherrer et al., 1954 ( ), 1956 ( ), have recently made an important contribution to study of the pain sensation phenomenon.
Similarly, the voltage duration of stimulus ratio in the guinea-pig can be expressed as a hyperbola (fig. 12), thereby indicating that the threshold of the test reaction, as in the case of dogs ( ), follows the law of Weiss-Hoorweg. Moreover, the relation between the emergence of the test reaction and the quantity of electricity required takes the form of a straight-line function. Similar results have been obtained on the sensation of pain in man as described by the patient. There is therefore parallelism between the test response in guinea-pigs and dogs and the sensation of pain in man. All these data together may thus be said to provide a sound basis for regarding the test reaction of the guinea-pig as somatic evidence of the sensation of pain. That is why we have called it the "Pain Reaction Test" (P.R.T).
Response R 2.-A pain syndrome also appears to be clearly present in this response. The "horrified" recoil, struggle and sudden flight, the phenomena connected with the sympathetic nervous system and the cry are all symptoms indicating the presence of a painful stimulus.
It is to be concluded from this discussion that response R 1(Pain Reaction Test) and response R 2 may be regarded as reactions to pain. Furthermore these reactions seem to present responses graded according to the intensity of the stimulus. The Pain Reaction Test would correspond to a bearable pain, whereas the flight response R 2 would be evidence of excessive, paroxystic pain. As for response R 0, we can neither disprove nor confirm its painful character. We can only point out the close relation R 0/R 1 (r = 0.86) in a constant ratio of 50% (figure 9).
Additional proof of the fact that these reactions are alike in character will be provided by the analgesia tests.
Thus, within the complex biological response system of the guinea-pig, reactions R 1 and R 2 may be regarded as somatic evidence of a painful sensation, a " sichtbare Ausdruck empfundenen Schmerzes" ( ). For analgesimetric studies we have kept the Pain Reaction Test (P.R.T. = R 1), which lends itself to both qualitative and quantitative study.
Along what paths does the Pain Reaction Test travel and what is their relation to the pain centres ?
According to Soehring & Becher ( ), Pain Reaction Test and the syndrome R in dogs are defensive movements controlled by the diencephalon and the brain stem. Great similarity has been found by these authors between the test response in dogs and the affective defence reaction (" Affektive Abwehrreaction ") of Hess & Magnus, 1943 ( ,  ) elicited from cats by excitation of the diencephalon. The experiments of Amsler, 1921 ( ) and Keller, 1932 ( ) on thalamic animals seem to support the same view. One objection may be raised-namely, that in the tooth stimulation of cats Boreus could obtain no reaction similar to the complex defensive movement. Be that as it may, the correlation reported by Soehring is really convincing and offers a very interesting working hypothesis for new research.
On the basis of these data and the results set out under the heading of biological response, the P.R.T. of the guinea-pig may be regarded as a long supraspinal circuit. Our results with analgesia and the work of Winder et al., 1946 ( ) and 1947 ( ), Houde & Wikler, 1951 ( ), Irwin & Houde, 1951 ( ), Winter & Flataker, 1953 ([169a] ) and Jacob, 1954 ( ) on defensive reactions to nociceptive thermal stimuli would confirm this view.
Research is in progress on these circuits and their exact relation to the pain centres. The results will be published later ( ).
As already pointed out, for an algesimetric method to be reliable the pain-reaction selected as the test response must be capable of qualitative definition and of measurement.
The qualitative study of the P.R.T. of the guinea-pig-single upward thrust of the head as far as it will go accompanied by some minor individual vatriations-shows that response to be clear, easily identifiable, steady and reproducible in that form.
It may be of value to compare the biological response obtained by stimulating the tooth pain-receptors of other animals. We have found a biological response similar to the Pain Reaction Test of the guinea-pig only in work on dogs. This reaction was considered as characteristic of a highly organized animal ( ). Its presence in the guinea-pig seems to suggest that it is not confined to any animal species and is quite likely to appear in other laboratory animals (cats or rabbits) under suitable experimental conditions.
Even in dogs it does not always appear. Koll et al. ( ,  ,  ) found it with its qualitative and quantitative characteristics in all the animals studied. Soehring and al. ( ,  ,  ) did not encounter it regularly. It may be replaced by other types of response (Z syndrome and R syndrome). Wirth ( ) working on dogs, managed to single out four, which gave constant threshold values for a year. One dog developed a conditioned reflex, giving the test response merely on hearing sound made by the apparatus. Boreus and Sandberg ( ) succeeded in eliciting the Koll reaction consistently. But they had to select the animals somewhat, as with some dogs the reaction was difficult to recognize, while with others there were day-to-day variations of more than 15 volts in the threshold value.
The responses of the R 0 and R 2 type only, which we sometimes encountered (c.f. chapter on method) in one or more of the animals tested and which gave way after a new drilling to a typical P.R.T. might perhaps provide a partial explanation for the exceptions found among dogs. Through the use of removable electrodes with guinea-pigs it was possible to show that the absence of the test response was purely apparent, but due to the experimental conditions.
There is another problem which can be raised. In guineapigs the Pain Reaction Test was from the beginning constant in appearance and could be reproduced without any training. Koll and Reffert ( ) report that, in dogs, the "Reaction" appeared from the beginning with its qualitative and quantitative characteristics. Soehring and Becher ( ), on the other hand, report that the characteristic biological response developed only in the course of the experiments. Similarly, Boreus and Sandberg ( ) claim that 4-5 weeks' training is necessary to obtain a steady response.
The biological response in rabbits after dental stimulation is characterized by a chewing movement ( ,  ). Being easy to reproduce and constant in form, it tends sometimes to become a conditioned reflex. As for the somatic reactions of the cat, qualitative defects make them of little use in algesimetry ( ).
From the quantitative standpoint, the reliability of the tooth-pulp algesimetric method with guinea-pigs is ensured by the stability of the potential stimulus and, above all, of the actual stimulus.
Use of electrical stimulation and the apparatus described permits of accurate measurement and reproduction of the parameters of the potential stimulus. Although with guineapigs continuous growth type teeth involved the use of removable physical electrodes (figure 2), we were able to obtain a physiological electrode (stimulus-receptor complex) of great stability. This stability of the actual stimulus is reflected in the remarkable steadiness of the Pain Response Threshold (threshold voltage) in any particular experiment (figure 20).
The progressive increase in the threshold value in the days following the hole drilling indicate that the stimulus-receptor contact is becoming steadily weaker. Whatever its level, however, the threshold voltage always stays constant during the day of the experiment. Physiological researches and pharmacological experiments with substances of rapid metabolism (thiopental) could thus be made on the same animal for a whole week after drilling the hole.
The slight dispersion of individual response thresholds and their distribution along a Gauss curve (figure 8) may be taken as an indication of a fairly uniform physiological electrode and of slight variability in the thresholds of sensitivity to pain (receptor-pain centres complex) in the various animals. For 221 gradings, we have had an average threshold voltage of 3.16 ± 0.06 volts. The slightness of the dispersion facilitates grading.
Koll & Reffert ( ), with fixed electrodes, obtained a constant threshold voltage throughout several months of testing. With certain dogs, the value was absolutely steady, but with others some variations in threshold were recorded. They are of the same order of magnitude (32-40 volts) as the interindividual variations recorded with four dogs. Boreus and Sandberg ( ) recorded day-to-day variations of more than 15 volts in the threshold value with certain dogs. With rabbits and removable electrodes, Fleish & Dolivo obtained an average threshold voltage of 18 volts with a standard error of 7.5 volts, in the course of 1,600 experiments.
Inter-animal variations, in any case, are of only relative importance. Even experimenters working with fixed electrodes in practice carry out grading before each pharmacological experiment. It therefore follows that the reliability of algesimetric results is ensured by extremely accurate control determination coupled with strict constancy of the threshold voltage during the experiment.
We have described the physiological condition ensuring the reliability of the algesimetric method using the tooth pulp of guinea-pigs-namely, the presence of a qualitatively and quantitatively identifiable response as evidence of pain. Let us now consider the degree of reliability of the method from the pharmacological standpoint in the light of the results obtained with narcotic analgesics.
Since one of the main objects of any algesimetric method is to evaluate the analgesic action of new products, it is essential first to check its reliability by studying drugs whose analgesic action has been confirmed by clinical experience. We will therefore consider in turn the results relating to the analgesic action of morphine, pethidine, methadone and levorphanol (figures 13, 14, 15 and 20).
* The effect of antipyretic analgesics has been studied at greater length elsewhere ( ). Let us merely mention that the action of 100 mg/kg of Aminopyrine and of 100 mg/kg of Butazolidine is similar in intensity to that of 2.5 mg/kg of morphine but is of longer duration (about 5 hours).
Dose in mg/kg →
Elevation of threshold
St.E of x
% of threshold voltage
The rise in the threshold of the Pain Reaction Test brought about by increasing the dose of the drugs provides a means of checking the sensitivity of the method and its selectivity in two main respects, intensity and duration.
The small doses required to obtain a minimum of analgesia bring out the sensitivity of the method. For the three drugs studied, there was a significant rise in the pain threshold with such tiny doses as 0.4 mg/kg for methadone and 0.8 mg/kg for morphine. With 3 mg/kg of pethidine a highly significant effect was obtained.
To study the selectivity of the method, we have grouped in table II below values showing the intensity of the analgesic effect obtained by increasing doses of the three drugs at peak effect period-i.e., the second hour in the case of morphine and l-methadone, and the first hour in the case of pethidine.
The intensity of the analgesic effect is expressed in terms of Excitation Units (x and o) and of the percentage rise in the threshold valul (x).
It will be seen that the elevation of the threshold is progressive and proportioned to the dose.
Figure 19, giving the above data in graphic form, shows a linear relation between the increase in dose and the analgesic effect, expressed in excitation units (log. of the stimulus). The progression did not reach a ceiling in the zone studied.
Application of the "t" test enabled us to check whether the ratios of 1: 3 and 1: 2 chosen for the increase in dose were sufficient to produce significant elevations of the threshold value (analgesic action). As table III shows, significant figures are obtained for methadone and highly significant ones for pethidine. In the case of morphine, it seems that it would have been preferable to have had a broader spread of doses. However, the difference between the doses is significant if comparison is made not at the point of peak effect, but over the whole of each curve (effect/time ratio).
The determination of the equiactive doses (figure 16) based on the percentage of animals showing analgesia (i.e., showing over 50 per cent increase in the threshold value) enabled us to evaluate the relative potency of the drugs studied (figure 16). If morphine is taken as the reference drug (ED 50 = 2.5 mg = 1), the relative potency of methadone is 1.92 (ED 50 = 1.30) and that of pethidine 0.32 (ED 50 = 7.7).
Figures 13, 14 and 15 show that it was also possible to evaluate, and establish a quantitative difference between the dura- tion of effect of the increasing doses of each drug by this method.
In the case of morphine, the slope of the rate-of-action curve increased proportionately to the dose, the peak effect being reached, whatever the dose, towards the second hour after administration. Return to normal was also gradual and varied from the third hour for a weak dose (0.8 mg/kg), the fourth hour for a medium dose (2.5 mg/kg), to the fifth hour for a strong dose (7.5 mg/kg).
Effect/dose ratio for morphine, pethidine and l-methadone x axis: doses in mgr/kg y axis: rise in the threshold of response in excitation units (Ex.U.)
Dose ratio → (mg/kg)
2.5 to 0.8
7.5 to 2.5
7.5 to 0.8
10 to 3
20 to 10
20 to 3
1.25 to 0.4
3.75 to 1.25
3.75 to 0.4
In the case of methadone there was also a progressive slope becoming steeper and steeper with the increase in dose, peak effect being reached, as with morphine, about two hours after administration, and remaining more or less at that level for the two larger doses. The effect lasted somewhat longer than with morphine, especially with the strong dose, for which the duration was more than five hours (figure 15).
In the case of pethidine, action was much more rapid, the slope being particularly sharp for the medium and strong doses. Peak effect was generally reached after one hour. Return to normal was fairly swift, so that by about the third hour a slight effect was still discernible only with the strong dose.
We have grouped in figure 20 the effect time curves of the maximum doses studied. This brings out the rate and brevity of the action of pethidine. The curve for the onset of analgesia has the same shape for morphine, l-methadone and levorphanol.
A last reliability factor which must be considered is the electivity of the method-i.e., the possibility of detecting the true analgesic potency of the substances tested. Analgesia is a general pharmacodynamic phenomenon which can be produced by the administration of quite a number of neurotropic drugs: general anaesthetics, hypnotic sedatives, central muscular relaxants, etc. But in the case of all these "pseudo-analgesic" substances, analgesia is only a side-effect. The characteristic of a true analgesic is the fact that the suppression of pain constitutes an isolated effect with an adequate margin of safety in relation to its other pharmacodynamic actions ( ).
Thus the post-discharge of the polysynaptic circuits following the painful stimuli is blocked by morphine and morphinomimetics. The same effect is obtained, as pointed out by Wikler et al. ( ,  ) with barbiturates and central muscular relaxants (myanesine), but whereas with the narcotic analgesics this effect is selective, in the case of the other depressants it is merely a side-effect to the inhibiting action on consciousness and postural integration.
This fundamental difference is also evident in the results obtained in the parallel study of narcotic analgesics and the central depressants. On the one hand we observed no hypnotic sedative effect with the analgesic doses of narcotic analgesics in guinea-pigs. The animals were brisk and wide awake. The quality of the P.R.T. was unchanged, and the movement was as clear-cut and swift as on grading. The margin of safety for toxic side-effects was I 50 = 6.8. There is thus every reason to believe that the increase in the threshold value is really due to analgesic action.
With the central depressants which were studied, on the other hand, analgesic action could be detected only with anaesthetizing doses. Only then could a large elevation of the threshold of response be observed. This side-effect analgesia was of short duration with ether (figure 17), but several hours long with thiopental (figure 18). This crosseffect is also evidenced by a change in the form of the P.R.T. The movement is sluggish, and even completely absent in certain animals. With non-hypnotic doses of thiopental and ether we obtained little or no analgesic effect (figures 17 and 18).
When discussing this question one must bear in mind that the responses are somatic defensive reactions and that a certain muscular relaxation induced by strong doses of anaesthetics may distort the analgesic action.
It follows that, on the whole, use of the electro-dental method on guinea-pigs revealed an undoubted electivity in analgesic action in the case of morphine and morphinomimetics.
With dogs, it seems that analgesic doses already tended to have a slight dormitive action. The doses of morphine studied by Koll & Reffert ( ) were 1.25 and 3.75 mg/kg, while those of Fleisch & Dolivo ( ) ran from 0.5 to 3.75 mg/kg. For dogs, however, the depressive dose of morphine is 2 mg/kg. Hatcher & Eggleston ( ), in a series of systematic studies, even put it at less than 1 mg/kg.
With the electro-dental method used on rabbits, the analgesic doses lie between 2.5 and 5 mg/kg ( ). Hypnotic effect is already present with 2-3 mg/kg doses ( ,  ). Respiratory depression is already evident at 0.15 mg/kg ( ), and the diminution in the minute volume reaches 50 per cent with a 3 mg/kg dose ( ). Such effects alter the experimental conditions, and the analgesic effect can be put down at least in part to hypnosis.
After studying the reliability of the method from the physiological and pharmacological standpoints, we must now test the validity of the results by comparing them with other data obtained in experiments with animals and in man. Any parallel with the whole gamut of mechanical, chemical and electro-cutaneous methods used in animal experiments being outside the scope of this work, our comparison will be confined to the electro-dental and thermo-cutaneous methods.
Analgesic action of morphine, pethidine, l-methadone and levorphanol. Curve: effect/time of maximum doses studied x axis: time in hours y axis : rise in the threshold of response in Excitation Units (Ex.U.) and in %
The analgesic effect of morphine and the morphinomimetics, as recorded by the electro-dental method, varies according to the author.
On dogs, Koll & Reffert ( ) studied the action of morphine and some semi-synthetic derivatives. With four dogs, the standard doses of morphine, 1.25 and 3.75 mg/kg, led to heavy increases in the threshold value, of 85 per cent and 171 per cent respectively. Two dogs showed a tendency to sleepiness which the author attributes not to the narcotic effect of the morphine, but, after checking with codeine, to a sluggishness of the motor system brought about by narcotic analgesics.
Soehring notes a very marked elevation of the threshold of response with 1 mg/kg of morphine and a limited rise in the Z syndrome value. Is it possible to strike a parallel with the results obtained in guinea-pigs where narcotic analgesics also show a distinctly stronger action on the Pain Reaction Test than on the R 0 response ? Whether this is a pharmacological proof that R 0, like Soehring's ( ) Z syndrome is not a reaction to pain, but the earliest stage in the motor effect induced by excitation, is a difficult question to answer. The data in medical literature regarding the action of morphine on simple reflexes are still contradictory. Mazoue, 1927 ( ), found that morphine reduced the reflex excitability of the marrow, while Luckard & Johnson, 1928 ( ), obtained inhibition of the patellar reflex in cats. Houde & Wickler ( ) and Wickler ( ), on the other hand, have recently reported that with spinal dogs morphine does not depress the monosynaptic (patellar) reflex or the bi-synaptic reflex (ipsi-lateral extensor reflex).
Recently Boreus, 1955 ( ), also made a comparative study of the analgesic effect on dogs of morphine and three synthetic morphinomimetics : l-methadone, pethidine and ketobemidone. He found that, for equal doses, the intensity of action of l-methadone was double that of morphine, while that of pethidine was only one-sixth.
The data derived from stimulating the tooth pulp in dogs show the method to be highly sensitive. Significant analgesic effects can be recorded with a dose of 1 mg/kg of morphine.
Study of the analgesic action of narcotic analgesics in rabbits by the electro-dental method gave the following results. Fleisch & Dolivo ( ), on administration of 2.5 and 5 mg/kg of morphine, obtained increases of 70-200 per cent in the threshold value. The relative potency of pethidine by comparison with morphine was 1/4 (2.5 mg/kg: 10 mg/kg). The analgesic action did not seem to be pure with such high doses, there being, according to the authors, some interference from the hypnotic effect.
Yim et al. ( ) made a parallel study of the analgesic and the bradypneic action of morphine and levorphanol. With the doses studied, the analgesic effect and the diminution of the minute volume was greater than 50 per cent, and their effect lasted more than 3 to 4 hours. The results obtained by this author with rabbits are similar to ours with respect to the levorphanol morphine analgesic action ratio.
In short, the results we obtained with narcotic analgesics in guinea-pigs by the electro-dental method may be said to tally from the standpoint of intensity and duration of effect with those recorded with the same method in dogs and rabbits. The order of relative potency was the same, though there was some discrepancy in the case of pethidine, the guinea-pig appearing to be twice as sensitive to this drug as the dog or the rabbit. A narcotic effect (dogs and rabbits) and other side-effects (rabbits) may, on the other hand, interfere with analgesia at the doses used.
As far as thermo-cutaneous methods are concerned, we dwell mainly on the work done with guinea-pigs. Although a different painful stimulus was used, this work had one factor in common with ours-the choice of animal. The thresholds of sensitivity to drugs and their metabolism were thus constants.
Hildebrandt, 1934 ( ), using the nociceptive direct-heat method, recorded a minimum analgesic action with a dose of 5 mg/kg of morphine, the duration of effect being 60 minutes. It was not until the dose was increased to 20 mg/kg that a 50 per cent increase in the threshold value (40 to 60) was obtained with a duration of effect of about six hours. Thus, with the same animal, the doses were 6-8 times as great as with dental stimulation.
We deal more fully with the radiant heat methods introduced by Hardy-Wolff-Goodell ( ), and d'Amour Smith ( ), as they now constitute the routine laboratory procedure in algesimetry. Apparently this popularity is primarily due to the fact that the heat ray, as pointed out by Jacob ( ), is the most selective of the painful stimuli in the mixed sensorial field. Furthermore, with this method parallel quantitative measurements can be made in man and animals.
Since Winder ( ,  ,  ), in a thorough statistical study, laid the algesimetric basis for using this method in guinea-pigs, observing the analgesic action and toxicity of two narcotic analgesics, morphine and pethidine, it might be of interest to compare their results with ours with respect to absolute analgesic effect and the margin of safety for toxic side-effects.
With morphine, the minimum dose is 3.2 mg/kg, intraperitoneally with the thermal method, as against 0.8 mg/kg subcutaneously (figure 9) with the electro-dental method. The analgesic doses (ED 50) are respectively 20.3 mg/kg and 2.5 mg/kg. The margin of safety for the commencement of toxic side-effects which may distort the results is I 50 =32/20 mg/kg = 1.6 with the thermal method and 32/2.5 mg/kg = 12.8 with the electrical method. The margin of safety for lethality is less important for algesimetry, especially as lethality in guinea-pigs (LD = 390 mg/kg) is much lower than in man.
With pethidine, 20 per cent analgesia is obtained with a 26 mg/kg dose using the thermal method and with 3 mg/kg using the electrical method. The analgesic doses (ED 50) are 80 mg/kg and 7.7 mg/kg respectively. The corresponding I 50 are 53/80 mg/kg = 0.6 and 53/7.7 = 6.8. The Index 50 values for morphine and pethidine bring out the difference in the margins of safety for the two methods in guinea-pigs.
It can thus be seen that despite Winder's remarkably strict experimental conditions, the radiant-heat method is less practical than the electrical one. The very heavy doses required in Winder's work, as Bianchi & Franceschini ( ) point out, may also be due to administration by the less effective intraperitoneal route. Narcotic analgesics thus injected are absorbed by the portal system and destroyed in the liver ( ,  ).
De Jong & Knoppers, 1952 ( ), obtained similar results with Winder's method. The lowest dose for which they recorded a significant elevation of the threshold (p = 0.002) was 5 mg/kg.
The results obtained in rats ( ,  ,  ,  ,  ,  ,  , [128b] ,  ,  ,  , [169a] , [169b] ,  ) by radiant and hot-plate heat methods show the average analgesic dose (ED 50) to be about 4 mg/kg (range: 2-10 mg/kg). The depressive dose in rats is estimated, according to the author, at 4.5 mg/kg ( ), 5 mg/kg ( ) and 2.1 mg/kg ( ).
When the same methods are used with mice ( ,  ,  ,  ,  ,  ,  ,  ,  ,  ), the average value of the analgesic doses is very near to that for rats: 3.8 mg/kg (range: 1.6-8.6 mg/kg). But with doses of less than 1 mg/kg, mice already show a psychoneurotic state which might interfere with the analgesic action, Radouco-Thomas C., Radouco-Thomas S. & Nosal Gl. ( ) have recently studied the relation between the analgesia and the psychomotor state induced by morphine and the synthetic morphinomimetics in animal experiments (cats and mice).
It emerges from all these comparisons that with radiant or hot-plate heat methods, whatever the animal (guinea-pig, rat or mouse), the side-effects, depression or hyper-excitability of the central nervous system, interfere with the analgesic action. Other drawbacks to these methods are:
Stimulation of two types of receptor;
Difficulty of obtaining thermal stability at the level of the pain receptors, which may alter the value of the actual stimulus and conceal the true degree of analgesia ( ,  ,  ,  );
Biological reactions which, though involving long supraspinal circuits ( ,  ), have not the complexity of the reactions obtained by the tooth pulp stimulation. Narcotic analgesics, however, act selectively on the complex reactions ( ) mainly of a preponderant type.
On the other hand, the way in which the potential stimulus is measured (the caloric energy applied), the steadiness of the threshold value (in terms of heat or reaction time), the reproducibility and the constancy of the biological response generally elicited from small laboratory animals make this method suitable for mass experiments.
Hence thermal methods, especially those using radiant heat, have become the most generally used in algesimetry.
Comparison of the algesimetric results obtained in experiments with animals by the two main groups of methods-the thermocutaneous and the electro-dental-seems, however, to suggest that the latter are to be somewhat preferred.
We do not thereby wish to imply that one group of methods should be chosen rather than the other for the study of analgesics. We merely wish to bring out the fact that when guinea-pigs are used for the stimulation of dental receptors, the method ceases to be a luxury suited only for pure research, and lends itself to routine laboratory work on a par with the other techniques.
Pathological pain is a subjective phenomenon which takes on several forms (superficial, deep and visceral pain) and is very complex in origin. The value of the experimental methods, though real, is limited, for they provide information only on the physiological reactions (somato-vegetativo-humoral) to a painful stimulus. This deficiency has to be remedied by using stimuli of the most varied kinds and sources. A whole battery of methods is therefore essential for analgesimetric studies.
We will now compare our results with the data obtained in man for induced (experimental) pain and natural (pathological) pain.
The main varieties of experimental (superficial, deep and visceral) pain will be considered. Schaumacher et al., 1940 ( ), Holland & Gross, 1946 ( ), Miller, 1948 ( ) and Christensen & Gross, 1948 ( ), obtained very favourable results by the radiant-heat method and administration of morphine and morphinomimetics. Christensen & Gross, 1948 ( ), using the Hardy-Wolff-Goodell method ( ), found methadone administered subcutaneously three times as potent as morphine and several times as potent as pethidine. The peak action effect comes about 90 minutes after administration, and duration of effect for the three analgesics ranges from 3 to 6 hours. The radiant-heat method, regarded as the best for studying superficial pain, has not, however, always given concordant results. The work of Dodds, 1945 ( ), Thorp, 1946 ( ), Denton & Beecher, 1949 ( ) and Kuhn & Bromiley, 1951 ( ), points to the uncertainty of the data in man.
In the study of deep pain, results have been obtained by the ischemic contraction of the muscle and by the electro-dental method. The method of ischemic contractions of muscle was introduced by Lewis et. al. ( ,  ), to study pain and adopted by Harrison & Bigelow, 1943 ( ), to evaluate analgesia. Hewer et al. 1948 ( ), 1949 ( ), made a statistical study of the effect of psychogenic factors and analgesic medication on this type of deep pain. They observed that, even with the very great variations caused by the former, morphine, pethidine and methadone had significant action (P = 0.02). Measurements show that 30 mg/kg of l-methadone have a similar analgesic action to 32 mg/kg of morphine and greater than that induced by 150 mg/kg of pethidine.
Let us now consider the algesimetric results obtained by the electro-dental method in man, the forerunner of that which we used with animals.
Heinroth, 1926 ( ), obtained a marked elevation of the threshold (90 per cent) with 10 mg of morphine administered subcutaneously. The effect reached its acme after about one hour and lasted about five hours.
Goetzl, 1943 ( ), found a marked elevation of the threshold with narcotic analgesics, which was not confirmed under similar technical and pharmacological conditions by Harris & Blockus, 1952 ( ).
Pfeiffer et al., 1948 ( ), using this method, made a distinction between the threshold of perception (minimum pain) and the threshold of response (severe pain). They observe that the action of narcotic analgesics is much stronger at the threshold of response.
Boreus, 1955 ( ), evaluating analgesic action on the central incisors by the electro-dental method, obtained for doses equivalent to 10 mg, the strongest effect with morphine (P = 0.01-0.001) followed by l-methadone (P = 0.02-0.01). The peak action of morphine was towards the second hour. Pethidine, in a 65 mg dose, produced no significant effect. The action was recorded on the first distinct painful sensation. It is to be noted that a pronounced decrease in the threshold values of the controls was recorded during the experiment.
Visceral algesimetry is still in its infancy. Little work has been done on it ( ,  ,  ,  ). Gaensler et al., 1948 ( ), 1950 ( ), 1951 ( ), determined the analgesic action of morphine (10 mg) and the morphinomimetics pethidine (100 mg), l-methadone (7.5 mg) and levorphanol (3 mg), on the threshold of visceral pain induced by distension of the biliary ducts. With the doses administered, morphine proved most active (56 per cent), followed by dromoran (42 per cent), l-methadone (33 per cent) and pethidine (23 per cent). The acme was reached after about 30 minutes with morphine and meperidine (pethidine) and about one hour with the other substances. Duration of effect, in rising order of magnitude, was three hours for pethidine, five for morphine and six for l-methadone.
Apart from the difference with regard to the potency of morphine and its precocious peak action, a fairly good concordance is to be observed between the algesimetric data in guinea-pigs and the action of analgesics on visceral pain in man. In the same work, the author brings out the strong influence of psychological and physiological components on analgesic activity by studying simultaneously the threshold of response when asleep and that of perception when awake.
This brief review of the algesimetric methods used to induce experimental pain in man shows that there are many irregularities and even contradictions in the results obtained. This defect is probably due to too great variability in the biophysical parameters (insufficient regularity in the actual stimulating force) and the psychological parameters.
With regard to the former, Björn ( ) and Harris ( ) have succeeded, with electro-dental methods, in obtaining relatively high stability of the actual stimulating force. With thermal methods the problem seems more difficult to solve owing to the thermal instability of the stimulated place itself.
Whatever the method used, however, there still remain the psychological variables ( ,  ,  ,  ) which are a serious handicap in experiments with man. When pain is induced in man, the threshold of psychic, anticipative and associative reactivity, unlike that of perception, shows great variability ( ,  ,  ,  ,  ,  ,  , , ) due more to the affective tonus of the subject than the strength of the stimulus. The use of selected and trained subjects, the administration of placebos and the device of "dry runs" are only partial remedies. The objective study of analgesia seems to call for subjects with a constant threshold of response, in other words subjects in which a state of analgesia has already been induced.
In experiments with animals, the somatic reactions "visible and measurable evidence of pain" are less varied than the somatic reactions of man. Yet narcotic analgesics seem to act less on the perception factor than on the psychic and somatic reactions. The great variation in the thresholds of psychic response in man and the steadiness of the somatic response thresholds in animals explain the inconsistent results in human experiments and the great consistency of those obtained in experiments with animals - which have, furthermore, the advantage of great numbers. The results of animal experiments, paradoxical as that may seem, may therefore be regarded as coming closer to clinical analgesic action than those of human experiments, always assuming that the somatic reactions of the animal are accepted as genuine evidence of pain.
Let us now consider the data of human pathology which give final sanction to any experimental result. In Table IV, the analgesic action of the three substances studied is expressed in terms of effective doses and their relative potency by comparison with morphine. The experimental data are the ED 50 while the clinical data are the traditionally accepted average values, regardless of individual factors such as the intensity, duration (acute or chronic) or seat of pain (superficial, deep or visceral).
ED (mg. total)
ED 50 (mg./kg.)
It will thus be seen that the analgesic intensity met with in clinical practice is similar to that obtained in guinea-pigs. This similarity applies both to the sequence and the order of magnitude in relation to morphine. Some qualification must be made in the case of pethidine, to which the guinea-pig, unlike the dog ( ), is more sensitive than man.
As for the duration of effect, the progressive series, pethidine, morphine, l-methadone of clinical practice was also encountered in our experiments. The maximum doses studied have a duration of effect of the same order of magnitude as that of average clinical doses.
After the reliability and the validity of the electro-dental method in guinea-pigs, there remains a last criterion to consider - that of efficiency.
With dogs, the electro-dental method, although probably the best algesimetric method, has been replaced as a routine laboratory procedure by the thermo-cutaneous methods. "The disadvantages of the tooth pulp algesimetry as used in animals is that it is too time-consuming, cumbersome and expensive. Consequently, it cannot be recommended as a routine laboratory procedure" ( ).
The method we advocate avoids these disadvantages. The choice of a small laboratory animal, inexpensive and easy to handle, together with the use of a system of electrodes easily inserted and of simple design makes it possible for a series of experiments to be performed by a single experimenter.
achieve greater accuracy of results (systematic error x =
Electro-dental stimulation in guinea-pigs, therefore, seems to meet all the requirements, and may be regarded as a routine algesimetric procedure. There is, however, another bridge to be crossed before final judgment can be passed. We must wait until the results of the method will be reproduced and confirmed by other research workers.
The systematic reproduction of pain and evaluation of the analgesic potency of morphine and some synthetic morphinomimetics; pethidine, l-methadone and levorphanol, have been effected in animal experiments (with guinea-pigs) by a new electro-dental algesimetric method.
The reliability of the method has been checked from the physiological and pharmacological standpoints.
A series of "somatic physiological responses" R 0, R 1, R 2, has been induced by electrical stimulation of the tooth pain receptors of guinea-pigs. Response R 1 (upward thrust of the head) was selected as the Pain Reaction Test (P.R.T. = R 1). Neuro-physiological data make it possible to regard this as true evidence of a sensation of pain.
The P.R.T. is capable of qualitative and quantitative definition. It is easy to identify, constant and of reproducible form. Quantitatively, the P.R.T. is defined by the threshold voltage. The stability of the potential stimulus and, what is more, of the actual stimulus (stimulus-receptor complex) has been confirmed by the almost complete constancy of the threshold of response during the same experiment.
The analgesic potency of morphine and the three synthetic morphinomimetics, pethidine, l-methadone and levorphanol, has been evaluated from the increase in the threshold of the P.R.T. and expressed in the dose/effect/time ratio.
The sensitivity of the method has been proved by the weakness of the doses required to produce minimal analgesia.
The selectivity of the method has been revealed in two respects: intensity and duration of effect.
In descending order of potency we have : l-methadone, levorphanol, morphine and pethidine.
The relative potency, by comparison with morphine (ED 50 = 2.5) taken as one, is 1.9 for methadone (ED 50 = 1.3) and 0.32 for pethidine (ED 50 = 7.7).
The peak effect is reached after one hour with pethidine and after two hours with the three other drugs.
Duration of effect varies with the dose from about 2-3 hours for pethidine to 4-6 hours for morphine, levorphanol and l-methadone.
The electivity of the method when used with narcotic analgesics has been demonstrated by a double check: the inducing of analgesia as an isolated effect in the case of morphine and the morphinomimetics (wide margin of safety for toxic side effects and lethality); the inducing of analgesia as a side-effect of the depressants studied-i.e., ether and thiopental (cross-effect of general anaesthesia and analgesic action).
The validity of the method has been demonstrated by a comparison with the electro-dental and thermo-cutaneous methods used in animal experiments. Its validity has been finally established by comparison with the results obtained in man with induced pain (superficial, deep and visceral) and with spontaneous (pathological) pain.
The efficiency of the method is disputed. The use of a laboratory animal lending itself to statistical study, of simple apparatus and simple laboratory technique makes it possible to regard the electro-dental stimulation of guinea-pigs as a routine algesimetric procedure for studying new analgesics.
The authors wish to thank Dr. A. Linder, Professor of Statistics in Geneva for his valuable advice with the biometric analysis of the results we obtained.
We wish also to express our deep appreciation to Dr. E. Le Breton, Professor of Physiology at the University of Paris, who made it possible for us to do the present work, and to Dr. Ed. Frommel, Professor of Pharmacodynamics in Geneva, who permitted us to conduct part of our experiments in his laboratory. We wish, too, to convey our thanks to Mr. Ph. Gold, chemical Engineer, and to Mr. L. Strassberger, Technical Assistant at the Institute of Therapeutics of Geneva, for their valuable collaboration.
We wish to convey our thanks also to Dr. J. Scherrer, Professeur agrégé at the Faculty of Medicine at the University of Paris, for his kindness in placing at our disposal before their publication the manuscripts of the Symposium on Pain.
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