Injury, Recovery and Death in Relation to Conductivity and Permeability

By E. M. EAST and D. F. JONES, Bussey Institution, Harvard University THE rapidly increasing specialization makes it im- possible for one author to cover satisfactorily the whole field of modern Biology. This situation, which exists in all the sciences, has induced English authors to issue series of monographs in Biochemistry, Physiology, and Physics. A number of American biologists have decided to provide the same opportunity for the study of Experimental Biology.

Biology, which not long ago was purely descriptive and speculative, has begun to adopt the methods of the exact sciences, recognizing that for permanent progress not only experiments are required but that the experi- ments should be of a quantitative character. It will be the purpose of this series of monographs to emphasize and further as much as possible this development of Biology. Experimental Biology and General Physiology are one and the same science, by method as well as by contents, since both aim at explaining life from the physico-chemical constitution of living matter. The series of monographs on Experimental Biology will therefore include the field of traditional General Physiology.

THIS volume endeavors to treat certain aspects of biology according to the spirit and methods of the exact sciences. The treatment is confined to certain funda- mental problems which have been studied quantitatively. These studies lead to a theory of some aspects of injury, recovery, and death, as well as of antagonism and per- meability. The behavior of the organism in these respects may be predicted with a satisfactory degree of accuracy by means of the equations which express the theory in mathematical form.

The author is under great obligation to the Marine Biological Laboratory at Woods Hole for the facilities generously placed at his disposal. He desires to make grateful acknowledgement to Mr. F. S. Mathews and Mr. G. B. Ray for the preparation of drawings and to Mr. Lee Morrison for technical assistance in conducting the experiments. 1. Apparatus for Measuring the Electrical Conductivity of Muscle 2. Apparatus for Measuring the Electrical Conductivity of Sea

3. Apparatus for Determining the Electrical Conductivity of Living 9. Two Glass Cells each Provided with an Electrode with Strip of 10. A Disk of Hard Rubber, One of Tissue and One of Celluloid, 11. Disk of Rubber with a Mass of Tissue Wedged in the Central 13. Curves of Net Electrical Resistance of Laminaria Agardhii ....... 41 14. Curves of Net Electrical Resistance of Laminaria Agardhii ...... 42 16. Curve Showing Changes in the Hydrogen Ion Concentration of

17. Curves of Net Electrical Resistance of Laminaria Agardhii ....... 45 18. Curve Showing the Net Electrical Resistance of Laminaria Agardhii 46 19. Curve Showing the Net Electrical Resistance of Laminaria Agardhii 47 20. Curves Showing the Net Electrical Resistance of Laminaria Agardhii 48 22. Curves Showing Net Electrical Resistance of Laminaria Agardhii 51 23. Curves of Net Electrical Resistance of Laminaria Agardhii ....... 52 24. Curves Showing Net Electrical Resistance of Laminaria Agardhii. 53

25. Curves of Net Electrical Resistance of Laminaria Agardhii 54 26. Curve Showing Rise in Net Electrical Resistance of Laminaria 29. Curve Showing the Net Electrical Resistance of Laminaria Agardhii 64 31. Curves Showing Changes in the Net Electrical Resistance of Tissues 71 32. Curve Showing Value of M under Various Velocity Constants 76 33. Graph Showing the Fall of Net Electrical Resistance of Laminaria 34. Graph Showing Loss of Net Electrical Resistance of Laminaria

35. Rise of Net Electrical Resistance of Laminaria Agardhii 84 36. Extreme Alterations of Net Electrical Resistance of Laminaria 37. Curves Showing Net Electrical Resistance of Laminaria Agardhii .... 92 38. Curves Showing Net Electrical Resistance of Laminaria Agardhii .... 93 39. Curves Showing Rate of Respiration of Laminaria Agardhii 96 40. Curves Showing Rate of Respiration of Laminaria Agardhii 97 41. Curves Showing Fall of Net Electrical Resistance of Laminaria

42. Curves Showing the Rise and Fall of Net Electrical Resistance in 43. Curves Showing the Value of 0+10 in Various Solutions 109 44. Curves Showing the Net Electrical Resistance of Laminaria Agardhii 111 45. Curves Showing the Net Electrical Resistance of Laminaria Agardhii 116 46. Curves Showing the Net Electrical Resistance of Laminari a Agardhii 117 47. Curves Showing the Net Electrical Resistance of Laminaria Agardhii 118 48. Curves Showing the Net Electrical Resistance of Laminaria Agardhii 120

49. Curves Showing the Growth of Roots in Toxic Solutions 125 51. Curves Showing Growth in Mixtures of Unequally Toxic Solutions . . 129 53. Diagram Representing the Composition of Various Mixtures 132 54. Solid Model Showing the Forms of the Antagonism Curves 134 56. Effect of Dilutions on the Forms of Antagonism Curves 136 58. Solid Model Showing Antagonism Affected by Altered Solutions.. 139 59. Curves Showing the Net Electrical Resistance of Laminaria Agardhii 140

60. Curves of Net Electrical Resistance of Laminaria Agardhii 143 61. Curve of Net Electrical Resistance of Laminaria Agardhii 144 62. Curve of Net Electrical Resistance of Laminaria Agardhii. 145 63. Curve of Net Electrical Resistance of Laminaria Agardhii 146 66. Curves Showing the Rise of Resistance After Exposure to Toxic 67. Curves Showing Calculated Value of s in Various Solutions 155 68. and 69. Curves Showing the Net Electrical Resistance of Laminaria

70. Curves Showing the Net Electrical Resistance of Laminaria Agardhii 157 72. Curves Showing Antagonism Between NaCl and Na-taurocholate . . 169 75. Curves Showing Antagonism Between NaCl and Cevadine Sulfate. . 172 78. Electrical Resistance of Laminaria Agardhii in Sodium Acetate 177 83. Curves Showing the Net Electrical Resistance of Laminaria Agardhii 190 89. Apparatus for Testing Rate of Diffusion of Salts through Living Tissue 206 90. Diffusion of Various Solutions through Laminaria Agardhii 207

91. Exosmosis into Distilled Water from Taraxacum Officinale 208 92. Recovery of Taraxacum Officinale from Effect of Various Hypertonic SOME of the fundamental ideas of biology are most difficult to define with precision. This is especially true of such conceptions as life, vitality, injury, recovery, and death. To put these conceptions on a more definite basis it is necessary to investigate them by quantita- tive methods. To illustrate this we may consider some researches on the electrical conductivity of organisms. These ex- periments show that the electrical resistance of a plant or animal is an excellent indicator of what may be called its normal condition of vitality. Injurious agents in- variably change its electrical resistance. For example, if the marine plant, Laminaria, is taken out of its normal environment of sea water and placed in a solution of pure Nad it is at once injured, and if the exposure be sufficiently prolonged it is killed. During the whole time of exposure to the solution of NaCl its electrical resistance falls steadily until the death- point is eventually reached; after this there is no further change. A study of the time curve of this process shows that it corresponds to a monomolecular reaction (slightly inhibited at the start). This may be expressed in the form of an equation which can be utilized to predict the curve of death under various con-

ditions. We find that in testing these predictions we must ascertain when the death process reaches a definite stage, (i. e., when it is one-fourth or one-half completed). This can be determined experimentally with a satisfac- tory degree of accuracy. We can therefore follow the process of death in the same manner that we follow the progress of a chemical reaction in vitro ; in both cases we obtain curves which may be subjected to mathematical analysis, from which we may draw conclusions regarding the nature of the process. This method has been fruitful in chemistry and it is possible that it may prove equally so in biology.

Studies undertaken from this point of view lead us to look upon the death process as one which is always going on, even in a normal, actively growing cell.1 In other words we regard the death process as a normal part of the life process, producing no disturbance unless unduly accelerated by an injurious agent which up- sets the normal balance and causes injury so that the life- process comes to a. standstill. The process of death which occurs in a solution of NaCl may be checked by adding a little CaCl2 to the solution. In this case we speak of antagonism between sodium and calcium. When the calcium is added in the proper proportion the fall of resistance is very slow and the tissue lives for a long time. Any deviation from this optimum proportion hastens death.

1 The general conception that the death process goes on continually is in harmony with the ideas expressed by many physiologists from Claude Bernard (1879; I, 28), down to the present day. Cf. Lipschiitz, both sodium and calcium combine with a constituent of the protoplasm, forming a compound which inhibits the death process. This enables us to formulate an equa- tion by means of which the death curve in any mixture of sodium and calcium can be predicted with consider- able accuracy.

The changes in electrical conductivity which occur under the influence of reagents run parallel to changes in the permeability of the protoplasm. This is to be expected, since it is evident that when a current passes from a salt solution into living protoplasm, ions must enter the protoplasm, and if there is an increase in the permeability of the protoplasm to these ions its electri- cal conductivity must increase, and vice versa. The electrical conductivity of the protoplasm may therefore be regarded as a measure of its permeability to ions.

The resistance of the tissue does not depend upon the protoplasm alone, but also upon the cell wall and the cell sap. But we find, as a matter of fact, that the re- sistance of the protoplasm rises and falls with that of the tissue as a whole. Hence when we observe that the conductivity of the tissue increases in a solution of NaCl and decreases in a solution of CaCl2, we may con- clude that the permeability is increased by NaCl and decreased by CaCl2. This is in harmony with experiments in which permeability is measured by other methods (such as plasmolysis, specific gravity, exosmosis, tissue tension, and the diffusion of salts through living tissue). It is likewise confirmed by direct determinations, in which the penetration of various substances is ascertained by testing for their presence in the cell sap.

It has been observed in the course of these investi- gations that plants which have developed in a normal environment are fairly uniform in their electrical resis- tance, so that we may speak of a normal degree of resistance as indicating a normal condition. If the plant is injured and the resistance falls, we may consider that the loss of resistance gives a measure of the amount of injury. This enables us to place the study of injury upon a quantitative basis. As the result of this we are able to formulate a definite conception of the mechanism of recovery. We find that if injury in a solution of NaCl amounts to 5% the tissue recovers its normal resistance when replaced in sea water. But if the injury amounts to 25% recovery is incomplete: instead of returning to the normal the resistance rises to only 90% of the normal. The greater the injury the less complete the recovery. When injury amounts to 90% there is no recovery.

This is of especial interest, since in physiological literature it seems to be generally assumed that when recovery occurs it is always complete, or practically so, as if it obeyed an "all or none" law. But it is evident that partial recovery may be easily overlooked unless accurate measurements can be made. This fact may serve to illustrate the importance of quantitative methods in the study of fundamental problems. The significance of such methods is further shown by the fact that they have led to the development of equations which enable us to predict with a satisfactory degree of accuracy the recovery curves which are ob- served under a great variety of conditions.

As the result of these investigations we are led to look upon recovery in a somewhat different fashion from that which is customary. While recovery is usually regarded as due to the reversal of the reaction which produces injury, the conception here developed is funda- mentally different. It assumes that the reactions in- volved are irreversible (or practically so) and that injury and recovery differ only in the relative speed at which certain processes take place. The reasons for this are fully explained in the following pages.

The experiments of the writer lead to the view that life depends upon a series of reactions which normally proceed at rates bearing a definite relation to each other. If this is true it is clear that a disturbance of these rate- relations may have a profound effect upon the organism, and may produce such diverse phenomena as stimulation, development, injury, and death. Such a disturbance might be produced by changes of temperature (if the temperature coefficients of the reactions differ) or by chemical agents. The same result might be brought about by physical means, especially where structural changes occur which alter the permeability of the plasma membrane or of internal structures (such as the nucleus and plastids) in such a way as to bring together sub- stances which do not normally react.

Throughout these investigations the aim has been to apply to the study of living matter the methods which have proved useful in physics and chemistry. In this attempt no serious difficulty was encountered after ac- curate methods of measurement had been devised: nor does there seem to be any real obstacle to an extensive use of methods which lead biology in the direction of the exact sciences. It is evident from what has been said that we may investigate such fundamental conceptions as vitality, in-

jury, recovery, and death by quantitative methods and obtain a set of equations by which they can be predicted. It may be added that the predictive value of these equa- tions is quite independent of the assumptions upon which they were originally based. The importance of such equations is fully as great in biology as in physics or chemistry. The measurements described in this volume, and the accompanying mathematical analysis, lead to a quantita- tive theory of the mechanism which underlies certain important phenomena. The theory can be tested by exact methods and, as far as experiments have gone, appears to be sound. This investigation of certain fun- damental life processes seems to show that they obey the laws of chemical dynamics: it likewise illustrates a method which promises to throw light upon the under- lying mechanism of these processes and to assist in the analysis and control of life-phenomena.

SINCE the conclusions set forth in this work depend largely upon researches on the electrical conductivity of organisms it seems desirable to give an account of the methods of conducting such investigations. In the experiments of some investigators1 platinum electrodes have been applied directly to the tissue. It is difficult to obtain good contact by this method and there is danger that some of the platinum black may be rubbed off. Kodis (1901) states that it is impossible to obtain trustworthy results in this manner. He there- fore placed the tissue in a U-tube in each arm of which was a funnel plugged at the bottom with plaster of Paris. Each funnel was filled with a solution of NaCl into which an electrode dipped (Fig. 1).

In measuring the conductivity of red blood corpuscles or of unicellular organisms the electrodes are placed directly in a suspension of the cells, either with or with- out previous centrifugation.2(Fig. 2.) In experiments of the writer on unicellular plants (such as Euglena and Chlorella) the organisms were placed in a centrifuge tube near the bottom of which platinum electrodes were inserted (through the walls of the tube) a short distance apart. The material was then centrifugated and the resistance was measured. The supernatant liquid was then poured off and replaced by a different solution. The material was agitated in order

FIG. 1. — Apparatus for measuring the electrical conductivity of muscle (Kodis) : E, electrode, contained in a funnel (tilled with a solution of NaCl) plugged at the bottom with plaster of Paris CP); M, muscle: the whole is placed in a water bath, I. to disperse it through the solution and the process of centrifugation and washing was repeated until the first solution was removed. This must be done frequently since otherwise the organism may change the conduc- tivity of the external solution (by absorbing or giving out electrolytes) and this may be confused with a change in the conductivity of the cells.

A method of measuring the electrical conductivity of bacteria has recently been proposed by Thornton (1912), who states that it depends upon the principle that in an electric field bacteria orient themselves in a solution having a lower conductivity than the bacteria, but not in a solution of the same conductivity. The method therefore consists in placing the bacteria in an electric field and increasing the strength of the salt solution until they cease to orient. The results indicate that the conductivity of living bac- teria is usually greater than that of the medium in which they grow. This is opposed to the results of Shearer (1919, A) in which the conductivity was measured in the usual manner. On theoretical grounds there are ser- ious objections to Thornton's tech- nique as well as to his conclusions.

The method3 used by the writer gives under the most favorable condi- tions, measurements which are accurate to within 1%. This degree of accuracy may be regarded as satis- factory for biological purposes. In the original method4 aquatic plants with leaf-like fronds were employed, particularly one of the common kelps of the Atlantic coast (Laminaria agardhii) : disks were cut from this by means of a cork borer and packed together, like a roll oi corns, in an apparatus which is shown in Fig. 3. It consists of two platinum electrodes (covered with platinum black), A, sealed into glass tubes, B, which are filled

•This was developed without reference .to the methods previously used and differs somewhat from them. with mercury and into which dip copper wires, C, which go to the Wheatstone bridge. These tubes are contained in electrode holders of hard rubber, D, through which pass a rod, E, and a long screw, F, by means of which the electrode holders may be drawn toward each other and held firmly in any desired posi- FIG. 3. — Apparatus for determining the electrical conductivity of living tissue. The disks