A Bipolar Theory of Living Processes

So from two points of view there could be less energy available on account of the brain and liver changing in their structure incident to fatigue and shock. Experimentally it has been shown that the cell structure in these organs breaks down from fatigue and shock, which would mean a lowering of the potential existent in the cell. Therefore, since the voltage has been decreased, the available energy output would also be decreased. Electrically one must have low internal resistance in order that a battery be given a chance to change its chemical potential energy to electrical energy within the internal structure of the battery, and finally transformed into heat, light or mechanical energy in the external circuit. An analogy is here presented in an old so-called dry battery. With age the internal resistance of a dry battery increases, so that one says it is dead. Fundamentally, the energy is still available, that is, the chemical energy in terms of the zinc plate is still available, but it cannot be made an energy output on account of the high internal resistance, so we say it is no good, it is dead.

If, therefore, there is an outstanding decrease in electrical conductivity in the two organs concerned, and also a lowering of potential due to a breaking down of cellular structure, both of these factors would contribute to the diminution in available electrical energy output. It is therefore definitely possible that reduced vitality incident to fatigue and shock might result not only from a lowering of electrical potential, but also from an increase in internal resistance of the body battery. In terms of an equation, one may visualize the above points.

A diminution in either factor, provided the other factor is constant, or a diminution in both factors simultaneously means less energy output. І believe, Dr. Crile, that this picture of conductivity within the body battery and its decrease is an additional point in which you will be interested. It is an experimental fact that lack of sleep produces death. This could result, as has been previously explained, by the possible decreasing potential or decrease in electrical conductivity. On the contrary, sleep restores the body, and is necessary in normal living. The amount of oxidation taking place in sleep is but fifty per cent of that while awake. As previously discussed, the cell structure in the organs concerned has been shown experimentally gradually to disintegrate, with the possible lowering of electrical potential and change in electrical conductivity, which means a decrease in the possibility of energy output. In sleep, the energy supply for muscular work is practically zero, so that very possibly a major portion of the oxidation which does take place could be very well utilized to build up the potential, which in turn means build up the available energy output for the trigger action previously discussed. To repeat, sleep is necessary for life, and it is therefore very possible from the above argument that during sleep nerve impulse energy is made available by the building up of electrical potential.

The above analogies and visualizations of body action seem to me to be a very plausible argument when considered from a purely physical and energy basis. Ву Сковсв W. Свше, HELEN В. Hosmer AND Amy Е. ROWLAND From the American Jowrnal of Physiology, Vol. 60, No. 1, March, 1922 Tur researches of Lillie, Loeb,? Osterhout,? Mathews,* McClendon, Hill? Lucas,” and of many other biophysicists and chemists ê indicate that the functions of the cells of living organisms are related to electrical processes; that the living cell, whether it exists alone or as an element in a complex organism, possesses a certain store of potential energy which is manifested by variations in polarity and by action currents; that variations in the permeability of the living cell to the electrically charged elements of the fluid which surrounds it parallel variations in irritability in response to stimulation; that factors which suspend or abolish irritability also suspend or abolish alterations in permeability.

Every activity of living tissue is accompanied by electrical currents; and many activities are also initiated by electrical currents. In fact, the work of the investigators referred to above shows how strong is the tendency to consider that vital processes depend upon electric energy, by means of which also the protoplasm is renewed, and the whole mechanism is constructed. In view of this trend of physiological conceptions, the electrical properties of living protoplasm become of vital interest. As this interest extends, the need of definite quantitative data increases. The laws which govern the action of electrical forces in inorganic systems are known exactly. It is possible to calculate exactly how much heat, or what chemical change, or how much work will result from the passage of a current of known strength through a known resistance during a definite period of time.

The two independent factors, current and resistance respectively, depend upon the amount of available electrical energy and the constitution of the system in which its conversion into heat, or chemical change, or other type of work is to be accomplished. The action of electrical energy in protoplasm, although all the conditions are far more complicated than in inorganic substances, is governed by the same laws. In protoplasm, as in inorganic matter, electrical curreuts will always choose the path over the lowest available resistance; and in protoplasm, as in inorganic matter, the current pays toll to the friction offered by the system through which it passes.

The facts already established regarding bio-electric currents are sufficient to indicate the importance of further investigation, especially along certain lines. For example: What is the range of the electric conductance of living tissue? How does that range compare with that of other electrical conductors? Is the range of conductance the same for all types of tissue, and in each tissue does it remain constant under all conditions? Is the electric conductance of each tissue a factor in the production of the activities of the organism, to which a fairly constant value can be assigned 2

These are questions which occur at once to the most casual student of bio-electric problems, and the fact that the literature offers no clear answer is sufficient reason for a detached study of this subject. During recent years various investigators have applied measurements of electrical conductivity to the determination of variations in the permeability of protoplasm' under varying conditions. In particular the work of Osterhout, Lillie and Loeb along these lines is too well known to be more than mentioned here.

Other investigators have used conductivity measurements as a means for estimating the volume of the corpuscles in blood, for determining the H-ion concentration in body fluids, for measuring the variations in the conductivity of muscle which result from contraction. But none of these investigators has attempted to determine the specific conductance of any tissue. Early in this century Galeotti® carried out a limited number of experiments for the purpose of studying the changes which occur at death. He utilized the tissues of dogs, rabbits, guinea pigs, frogs and turtles, making measurements at successive intervals after the removal of the tissue from the animal. In general, he observed a rapid decrease in conductivity during the first few minutes, followed by a more gradual decrease, which in some instances lasted for several hours. After reaching the minimum,

which he considered marked the death point of the tissue, the conductivity began to rise, gradually at first, and then very rapidly until a very high value was reached. His values for the normal conductivity of certain rabbit tissues may be of interest as compared with those observed in the work to be described later (Tables 1 and 2). Specific conductivity of certain rabbit tissues as determined by Galeotti. (Expressed in reciprocal ohms) Comparison of the specific conductivity of rabbit blood before and after coagulation as determined by Galeotti. (Hapressed in reciprocal ohms)

For the most part Galeotti worked with the firmer tissues, sections of which he introduced directly between platinum ејес- trodes which were clamped in place after the application of a greater or less degree of pressure. Не used only a few animals of each species and does not state that he measured more than one sample of each tissue from any one animal. The work to be described below was the direct outcome of preliminary measurements made by G. B. Obear of the Case School of Applied Science in the fall of 1917. In spite of inadequate apparatus and a limited number of observations, Doctor Obear’s results indicated such consistent relative values of the specific conductivities of certain tissues, especially the cerebrum and cere-

bellum, as to encourage a further research under better conditions. In the fall of 1918, therefore, the research was continued. Apparatus. The apparatus employed in this research includes that devised by Dr. E. W. Washburn and developed for the market by Leeds and Northrup. It consists of the typical Wheatstone bridge made up of a Kohlrausch slide wire and a resistance box of Curtis coils, with a telephone connected across the ends of the slide wire. Suitable capacities for tuning the circuit and for balancing the capacity of the conductivity cell were inserted in the current. A high frequency (1000 cycles) alternating current was obtained from a constant speed high frequency generator located in another room. All measurements were made with the cell partially immersed in a constant temperature bath. A Freas bath of 300 liters capacity was used, supplemented by a specially constructed cover to minimize fluctuations of temperature, to maintain a sufficiently humid atmosphere, and to insure maintenance of the leads and upper part of the conductivity cell at a uniform temperature.

On account of faulty construction of the glass parts, the automatie temperature regulator for this bath has never worked satisfactorily, but by hand regulation of the lights it has not been difficult to keep the temperature constant within 0.1? C. Washburn's recommendations in regard to magnetic shielding, grounding, etc., have been carefully observed. Electrodes. Аз the early part of this work was done at а time when it was a patriotie duty to conserve platinum, in the preliminary experiments all types of tissue were measured in the same set of electrodes, though it is obvious that this is far from an ideal mode of procedure.

Sections of each tissue were packed into small glass tubes of various sizes, each of which was accurately ground to insure uniform dimensions throughout. The tubes were packed with a sufficient excess of material to procure a slight projection from each end, and were placed between thin platinum electrodes, reinforced by brass backings. Sufficient pressure was applied to bring the electrodes flush with the ends of the tubes, when the electrodes were firmly clamped into place. Great pains were taken to avoid air spaces within the tubes and to insure uniform contact of the tissues with the electrodes. This was not difficult with the softer tissues, such as brain and liver, but with tougher tissues such as muscle and thyroid it was impossible to exclude considerable error from imperfect contact and other variations. The effect of these faults is plainly evident in the greater variation in the conductance values obtained for the latter tissues.

The tubes used for the measurement of the conductivity of the brain, the liver and voluntary and involuntary muscle, were approximately 5 mm. in diameter and 5 mm. in length, while those used in the measurement of the adrenals and the thyroid were of the same length with a diameter of approximately 2.5 mm. Special hard rubber containers were devised for the spinal cord. The conductance capacities or cell constants of these tubes were determined by repeated measurements of their conductance when filled with 0.01 NKCI, at the same temperature as that used for the tissue measurements.

In the later work larger tubes 1 cm. long X 1 cm. in diameter were used. These were only partially filled with tissue, and an upper electrode 1 cm. in diameter and pierced by slits was used. The tube was partially filled with closely packed material, and carefully placed in position on the lower electrode, after which the upper electrode was inserted within the tube and carried down until contact was made with the upper surface of the tissue, care being taken, however, to avoid sufficient pressure to cause the extrusion of material through the slits. This upper electrode was then clamped in place and the distance between the two electrodes was accurately measured and recorded.

Every precaution was taken to avoid any undue pressure with the consequent reduction of the fluid content of the tissue and resultant lowering of the conductance, although this danger was lessened by the use of the pierced electrode. Various measurements of different types of tissue were made to determine the effect of varying the pressure, the results of which are illustrated by the groups of measurements shown in Table 3. In each case the successive measurements were made upon the same sample of tissue, the distance between the electrodes, and consequently the pressure, being changed between each two measurements. Тһе results indicate that a greater error is to be feared from the application of too much pressure than from too light a contact.

The cell constants for the tubes used in the later experiments, as for those used in the earlier series, were determined by measurements with 0.01 ХКСІ with the electrodes at different distances apart, these results being plotted for convenient use in estimating the value of the tissue measurements. The electrodes were carefully cleansed after each measurement and were replatinized at intervals with a very light coating of platinum black. The electrodes and holders were always placed in the bath long enough before use to insure the thorough warming up of the metal and glass parts, and during the insertion of tissue

were exposed as little as possible to lower temperatures, and even | then they were given an opportunity to reach the bath temperature Effect of variations in pressure wpon the electrical conductivity of animal tissues before measurements were made. It was possible to exercise these precautions with but little loss of time by using two sets of electrodes and holders. АП measurements were either made at 39° С. or were corrected to that value from temperatures not over a degree removed from it.

Special points in technic. The object of this investigation being to determine the electric conductivity of animal tissues under conditions as nearly as possible identical with those existing in the living animal, every effort was made to keep the sections of each tissue always at a normal body temperature; to avoid any loss of moisture; and to measure each at the earliest possible moment after Conductivity measurements made at varying periods after death to deter- тате onset of post-mortem changes

the death of the animal and the removal of the tissue from the body. A series of measurements was made to determine the onset of post-mortem changes in the different tissues as evidenced by changes in the electric conductivity. We found that the conductivity of all tissues remained practically unchanged during the first hour after removal from the body; i.e., provided the section had been kept at a constant temperature in a humid atmosphere. The earliest post-mortem changes in conductivity were found in the liver and the brain; the latest in voluntary muscle. No significant change was noted in the brain in less than one hour after removal. In two instances liver changes began in approximately one-half hour after the death of the animal (Table 4). The observation of these changes led to the

adoption of an unvarying routine, in which the liver was always the first tissue to be removed, sectioned and measured. As a corollary to the measurements made specifically for the purpose of determining the onset of post-mortem changes it may be pertinent to note here that observations of the electric conductivity of the liver after it had been tied off for four hours, but left in situ, to produce an effectual hepatectomy, showed the conductivity was increased far above that observed in any experiment in which immediate measurements of the conductivity of the liver were made.

Post-mortem change was not the only factor to be considered, however. It is obvious that animal tissues present no such ideally uniform material as is ordinarily subjected to conductivity measurements. Even if it were possible to secure sections of exact uniformity throughout, completely filling the space between the electrodes, an ideal that thus far has not been satisfactorily attained, it would still be necessary to consider variations in the structure of different parts of the same organ, as well as variations between individual animals.

It was obvious at once that at best we could expect only to establish the limits of variations for each tissue, and that if the results of these measurements were to be of any general physiological value, they must represent the findings in a large number of individuals. For all these reasons it seemed inadvisable to employ any technie which would delay the prompt measurement of sections after their removal from the animal; от would prevent the examination of a large number of animals, even ‘though such a technic might markedly increase the precision of any single measurement. The precision of a single measurement is of little significance if it is obtained after such a lapse of time as to permit a change from the conditions in the living animal. Also if the normal variation in the conductivity of the same tissue in different animals is several per cent, as one would expect to be the case, it becomes important to multiply the number of normal measurements as well as to try for a high degree of precision in single measurements.

On the other hand the absolute maintenance of constancy of method is imperative and no effort has been spared to insure this requisite. Uniformity of technic in the choice of animals, the method of killing, the nature of treatment, the lapse of time and conditions under which the tissue was kept between the killing of the animal and the actual measurement, in the method of measurement and in all other factors has been maintained just as far as possible.

Animals which showed any abnormal condition either before or during treatment or at autopsy were always rejected. The weight and temperature of each animal were recorded. The animals received their last feeding the evening before they were used, as the measurements were always made during the morning; calculations, testing and adjusting of apparatus and so forth being done during the afternoon. The animals were killed by stunning with a blow on the head and the immediate severance of the veins and arteries.

The liver was at once removed, sectioned, packed in warm tubes and placed in the saturated atmosphere of the constant temperature bath for immediate measurement. In the earlier experiments the block of liver tissue was removed and kept as nearly intact as possible. As it proved difficult to secure sufficient uniformity of filling of the tubes by this method, in the later work the liver substance was separated from the connective tissue and the resultant soft mass was carefully transferred to the tubes. Histologic examination shows that this treatment separates the lobules from the connective tissue, but as the size of the individual liver cell is 5 to 8 microns there is no reason to believe that more than a very small percentage of the liver cells themselves has been destroyed.

The other tissues were removed and sectioned in turn—always in the same order—and measured immediately. The time between the removal of a tissue until its measurement seldom exceeded 20 minutes and under no circumstances was the section allowed to cool perceptibly. By careful training and close coöperation between principals and assistants, the technie was developed to such a point that it was possible to prepare and measure two sections each from the liver, cerebrum, cerebellum, spinal cord, voluntary muscle and involuntary muscle (heart), and usually one section only from the adrenals and from the thyroid, within 45 minutes after the death of the animal In the later work, when in the majority of animals measurements were made of only the brain and the liver, it was possible to make measurements of a number of animals each day, and thus secure nearly uniform conditions in the different animals.

At the beginning of the research as many sections as possible were made of each type of tissue until a minimum percentage of variation for each was established. Thereafter, whenever the measurements of different sections of the same organ failed to agree, the higher value was in general the one accepted, for the reason that all sources of error under the conditions employed in this work were such as would diminish the conductivity. Thus if the tubes were incompletely filled, that is, if they contained air spaces; if there was imperfect contact with the electrodes; if the material protruded beyond the tubes used in the earlier work, so that the length of the section was greater than that of the containing tube; if too great pressure was exerted; if the temperature of the section was reduced below the normal—any of these conditions would produce a measurement below the true conductivity value.

Two readings of each section were made, an interval of one minute being allowed between the successive readings. The magnitude of the current used was varied according to the resistance of the individual section. The relation of the area of the cross section to the length of the section of each type of tissue was kept within the range that experience showed would give the most favorable minima on the telephone. Range of electrical conductivity of normal rabbit tissues. The accompanying table (Table 5) gives the average measurements of the electrical conductivity of tissues from 91 normal rabbits. In addition to these measurements preliminary studies made during the development of the technic, bring the total number of normal rabbits studied to over one hundred.