Injury, Recovery and Death in Relation to Conductivity and Permeability

Attention may be called to a further difficulty in deter- mining toxicity. If tissue of Laminaria be transferred from sea water to pure solutions of toxic salts their relative toxicity sometimes appears to be different from that which is observed when the same substances are added directly to the sea water. Similar considerations may be found to apply to animals and plants which live on land or in fresh water, in which cases Ringer's solu- tion or the water of soils and rivers may play the same role as the sea water in experiments with marine forms. These differences depend largely on the antagonistic action of salts, which will be discussed in Chapter IV.

It may be added that in some cases variations in the supply of oxygen may cause changes in relative toxicity; and in view of the fact that the temperature coefficient is not the same in all cases of toxic action it seems desirable to carry out determinations as far as possible at a standard temperature, preferably at 18° C. In conclusion, attention may be drawn to the effects of temperature48 upon consecutive reactions such as are here assumed to be responsible for the phenomena with which we are dealing. The temperature coefficient of death in NaCl 0.52 M, and CaCl2 0.278 M is not far from 2.

The temperature coefficients of life processes have within the last few years attracted a good deal of atten- tion. Interest has chiefly centred about the question whether life processes have the temperature coefficients of ordinary chemical reactions and whether investiga- tions of this sort enable us to distinguish between chemical and physical processes (on the ground that in general, the latter possess lower temperature coeffi- cients than the former).

In these discussions of life processes it is generally assumed that we are dealing with simple chemical reac- tions. A little consideration shows that this cannot always (or even commonly) be the case. Most substances formed in the organism are also broken down, and the amount present must depend on the relative rates of formation and of decomposition. Change of temperature may affect consecutive reactions in an entirely different manner from simple reactions (in which the substance formed is not at once broken down). This may be made clear by a concrete illustration.

Let us take for this purpose the death curve in NaCl (Curve I, Table III) and consider the effect of raising the temperature 10° C. If both reactions have the tempera- ture coefficient 2, K± becomes 0.036 and K2 becomes 1.080. 48 Cf. Osterhout (1917, E). For the temperature coefficients of living and dead tissues in sea water see p. 37. The values of M under these conditions are given in Table III (Curve II). Inspection of the table, and of the curves in Fig. 32, shows that at the higher temperature it re-

FIG. 32. — Curves showing the value of M when the velocity constants have the valuee Curves II, III and IV are derived from Curve I by assuming that the temperature is raised 10° C.; if the two reactions have the temperature coefficient 2 we obtain Curve II; if the coefficients are 1.2 and 2 respectively we obtain Curve III; if the coefficients are 2 aud 1.2 quires just half as long to produce the same amount of chemical action as at the lower. Hence the consecutive reaction appears to behave in this instance like a simple reaction.

The result will be quite different if the two reac- tions have different temperature coefficients. Let us suppose that the speed of the reaction A — > M is deter- mined by diffusion (as happens in some heterogeneous reactions) and has in consequence a low temperature coefficient which we will assume to be 1.2. Assuming that the reaction M — *~B has a temperature coefficient 2 we find than on raising the temperature 10° C., K1 becomes 0.0216 and K^ becomes 1.080. The values under these conditions are given in Table III (Curve III).

Let us now consider the effect when the temperature coefficient of the first reaction is 2 and that of the second is 1.2, On raising the temperature 10° C. Kl becomes 0.036 and K2 becomes 0.648. The values are given in Table III (Curve IV). The form of the curve is quite different from that of the others in that there is first a rise followed by a fall. In experimental work a short period of rise might be overlooked or regarded as due to experimental error or some disturbing ("inhibiting") factor, such as is commonly assumed to account for delay at the beginning of a reaction.

simple reaction of the type M — >B and proceeded to calculate the velocity constant, he wonld obtain the values given in Table IV. A consideration of these values is very instructive. It is evident that when the relation K 2 -f- K l has a certain value (as in Curves I and II where K 2 -r- K l = 30) the Apparent velocity constants obtained on the supposition that the process is a reaction appears to proceed as a monomolecular reaction which is somewhat "inhibited" at the start,49 while with other values it may appear to be greatly inhibited at the start (Curve IV, K 2 ~- K 1 = 18) or to go much faster in the beginning than is expected (Curve III, K2-^-K 1=50). These facts deserve consideration in interpreting the temperature coefficients of consecutive reactions, to which category many life processes undoubtedly belong.60

AN investigation of the process of death leads us naturally to a study of the power of the organism to recover from exposure to unfavorable influences. An interesting aspect of this subject is the connection between injury and permeability. In the opinion of some writers permeability is a relatively fixed property of the cell which changes only as the result of injury, and is then altered1 irreversibly, while others assume that reversible changes in permeability may form a normal part of the activities of the cell.2 In view of the fact that such changes may control metabolism it seemed desirable to the writer to investigate them by determining conduc- tivity, since (as will be shown in Chapter VI) an increase in conductivity indicates an increase in permeability, and since it is also possible to calculate the increase in proto- plasmic conductivity (and hence of permeability) as dis- tinguished from the increase in the conductivity of the tissue as a whole.

The following will serve to illustrate the method of experimentation.3 Tissue which had in sea water a net resistance of 770 ohms was placed in a solution of NaCl 0.52 M. In the course of 5 minutes the resistance fell to 580 ohms, or 75.32% of the original resistance.4 When 4 Complete recovery after such a large increase of conductance is not always obtainable unless the material is in good condition and is freshly collected. Even in such material a lot will occasionally be found in which recovery is poor.

the tissue was replaced in sea water the resistance soon rose to normal and so continued during the remainder of the day.5 In order to see whether this increase is accompanied by permanent injury, an experiment was made in which the same piece of tissue was exposed to the action of NaCl several times during the same day. The net resist- ance of the tissue in sea water was 810 ohms; after 5 minutes in NaCl the resistance fell to 84% of the original resistance; the tissue was then placed in sea water and a reading .10 minutes later showed that the resistance had risen to 100%. In this case the fall of protoplasmic resistance was (100 — 78.94) -+- 100 = 21.06% and the increase in permeability (conductance) was [(1-f- 78.94)

1 1t might be objected that this increase is not necessarily the result of increase in permeability, but may be due to the fact that the protoplasm is more permeable to NaCl than to CaCl2 and since the number of Na-ions is increased the conductance also increases. But the increase in Na-ions is much too small to account for the effect since it amounts to about 1%. — (1 -r- 100) ] -r- (1 ~ 100) = 26.68%. During the next 95 minutes it showed no change. It was then placed in NaCl for 5 minutes and the resistance fell to 82.8%. It was then replaced in sea water ; a reading taken 10 minutes later showed that it had returned to normal, where it remained for 90 minutes. It was then placed in NaCl for 5 minutes. The resistance fell to 86.42% and returned to normal dur- ing the ensuing 10 minutes in sea water. After 105

FIG. 33. — Graph showing the fall of net electrical resistance of Larrinaria agardhii in NaC 0.52 M (unbroken line) and recovery in sea water (broken line). All readings were made at 20° C. or corrected to this temperature. The graph represents a single experiment. minutes in sea water (during which no change occurred) it was again exposed to NaCl for 5 minutes. The resist- ance fell to 82.8% and returned again to normal during the following 10 minutes in sea water. On the following day its resistance was only 30 ohms below the resistance of the control, which at the beginning of the experiment was 810 ohms. The results are presented in Fig. 33.

The successful outcome of this experiment led to an attempt to carry on such an experiment for several days in succession, giving the tissue one treatment daily with NaCl. The material was selected with especial care. The fronds were fairly thick, without reproductive organs. The experiment was made at Woods Hole, Mass., in July, at which time such fronds may be easily obtained. The disks cut from these fronds were slightly curved, so that when placed in the apparatus they separated spontan-

eously, thus allowing the running sea water in which they were kept to circulate freely between them. Care was taken to keep them only about two-thirds submerged, so that they had free access to air, but ran no risk of drying up. The tissue in sea water had a net resistance of 780 ohms at 20° C. As the temperature of the FIG. 34. — Graph showing loss of "net electrical resistance of Laminaria agardhii in NaCl 0.52 M (unbroken lines) and recovery in sea water (broken lines) on 15 successive days. All readings were made at 20° C. or corrected to this temperature. Each graph represents a

sea water varied but slightly from this during the experi- ment, all readings were taken at 20° C. On being placed in NaCl 0.52 M, the resistance fell in 5 minutes to 83.3% ; the tissue was then placed in sea water, and a reading taken 10 minutes later showed that it had risen again to the normal. The tissue was then placed in run- ning sea water, with the precautions mentioned above. At the end of 22 hours the resistance was 780 ohms. An exposure of 5 minutes to NaCl resulted in a drop to 87.2%, with complete recovery within 10 minutes. The same treatment was given once each day for 15 days. On the tenth day the resistance began to fall off, but as this

falling off was also shown by the control, which remained in sea water through the experiment, it was not due to NaCl but to other causes. The results are shown in Fig. 34. Electrolytes may also cause a reversible decrease in permeability. The simplest way of demonstrating this is by means of the following very striking experiment. The net resistance of a cylinder of living tissue in sea water was found to be 500 ohms. It was tested an hour later and found to be the same. Sufficient La(N03)3 was then added in solid form to make its concentration8 in the sea water 0.02 M. After 5 minutes the resistance rose to 130%. In order to ascertain whether this change in per- meability is reversible, the tissue was replaced in sea water. In the course of an hour its resistance returned again to the original value.9 The experiment was repeated three times on the same lot of material with practically the same result ; it was then allowed to stand over night in sea water. On the following day there was no appearance of injury, and the resistance was the same as that of the control, which had remained in sea water throughout the experiment. The tissue was then placed in the sea water plus lanthanum and left until its resist- ance had increased 100 ohms; it was then put back into sea water and left until the resistance fell to nearly normal. This was repeated three times, and the tissue was then allowed to stand over night in sea water. On the

8 The concentration was reduced by the precipitation of a small amount of La, (SO^a; this had practically no influence on the subsequent result, since the outcome is the same if we use in place of sea water a mixture of 1000 c.c. NaCl 0.52 M + 20 c.c. CaCl2 0.278 M, in which case no precipitate is formed. It should be noted that the addition of lanthanum chloride has the same effect as the addition of lanthanum nitrate. 9 If the material is left in sea water plus La(N03)3 the increased re- sistance is maintained for a long time.

third, fourth and fifth days, the same experiment was repeated four times. On the fifth day the tissue appeared to be in as good condition as the control, and had a resistance which was slightly higher. There was no reason, therefore, to suspect that the changes in permea- FIG. 35. — Rise of net electrical resistance of Laminaria aoardhii in 1000 cc. eea water pluB sufficient La(NOa)s to make the concentration 0.002 M (unbroken line) and subsequent fall on replacing in sea water (broken line). Lower horizontal broken line represents the control in sea water. The same lot of tissue was exposed four times daily on five successive days to the action of La(NOs)a. All readings were made at 20° C. or corrected to this temperature.

bility had been attended by any permanent injury. The results are shown in detail in Fig. 35. Similar experiments were performed in which CaCl2 was used in place of La (N03)3. In this case 3.3 gm. CaCl2 were added to each 1000 cc. of sea water. Owing to the fact that the rise in resistance took place more slowly10 'If in place of solid CaCl2 a strong solution is added, the rise is more rapid and reaches a higher figure. than when lanthanum was used, the experiment was per- formed twice daily on each of the five successive days. On the sixth day the material was in as good condition as the control, and had the same resistance.

It is evident, therefore, that the conductivity may be greatly decreased and then restored to the normal several times on successive days, without any trace of injury. Experiments on dead tissue (killed by heat or by formalin or allowed to die a natural death) showed that the results described above are due entirely to the living cells. A very marked decrease of permeability may be pro- duced by a considerable variety of other salts. The addition of these salts in solid form simultaneously increases the conductivity of the solution and decreases the conductivity of the tissue. This affords the most convincing proof that the change in the conductivity of the tissue in these experiments cannot be due to any cause other than a change in permeability ; for the concen- tration of the ions of the sea water remains unchanged, and if they were able to penetrate as freely as they did before the addition of the salt, the resistance wrould not increase. It would, in fact, diminish on account of the increased conductivity of the solution held in the cell walls, as is clearly shown by experiments on dead tissue.

It may be remarked incidentally that these experi- ments effectually dispose of the possible objection that the current passes between the cells, but not through them. Were this objection well founded, the decrease in con- ductivity could be explained only as the result of a decrease in the size of the spaces between the cells. This decrease could not be brought about except by greatly reducing the thickness of the cell walls. Both macroscopic and microscopic measurements show most conclusively that this does not occur. The contrary effect would be produced by the addition of salts in solid form, for they would tend to produce plasmolysis and thereby increase the space between the cells.

FIG. 36. — Extreme alterations of net electrical resistance produced by placing Laminaria agardhii alternately in CaCh 0.278 M (unbroken line) and in NaCl 0.52 M (broken line) and then in sea water (broken line with dots). The experiment was repeated with the same lot of tissue on the second day. All readings were taken at 18° C. or corrected to thie tem- perature. The control in sea water remained constant during the two days. to produce no bad effects, it occurred to the writer to see whether the protoplasm could endure still more violent alterations without permanent injury. In order to test this the following experiment was performed. A lot of tissue was found to have in sea water a net resistance of 750 ohms. It was placed in CaCl2 0.278 M, which had the same conductivity as the sea water. At the end of 10 minutes a reading was taken which showed that the resistance had risen to 168%. The material was

then placed in NaCl 0.52 M, which had the same conduc- tivity as the sea water ; at the end of 10 minutes the resist- ance was 85.4%. The experiment was continued by placing the material for 10 minutes alternately in CaCl2 and NaCl, with the results shown in Fig. 36. After 80 minutes the material was placed in sea water, where it soon regained its normal resistance: 24 hours later the resistance was found to be unaltered, and the experiment was repeated. After 80 minutes of alternate exposure to CaCl2 and NaCl, the material was placed in sea water, where it soon regained its normal resistance, which it maintained for 3 days, when the experiment was discontinued.

Similar results11 were obtained with Ulva (sea let- tuce), Rhodymenia (dulse) and Zostera (eel grass). Recovery was also observed with frog skin.12 The fact that protoplasm is able to endure such vio- lent alterations of conductivity throws a new light on the normal life processes of the cell. In the course of met- abolism a great variety of substances are produced which affect the permeability of the protoplasm. Since it is clear that the permeability may be greatly increased or decreased without rendering a return to normal permea- bility impossible, it is evident that considerable fluctua- tions in permeability may form a normal part of the life processes of the protoplasm. In this way the whole course of metabolism may be controlled, since this depends on the exchange of substances between the cell and its environment.

It is a striking fact that normal specimens of Laminaria are quite uniform in respect to electrical resist- ance,13 but if plants have been subjected to unfavorable conditions14 their resistance is below the normal. This is of considerable practical value, enabling the experimenter to reject abnormal material, and is also theoretically important, for it provides us with a measure of what we may call the normal condition, or normal vitality, of the organism.

Although the idea of normal condition (or normal vitality) is one of the fundamental conceptions of biology, it has never been precisely formulated : nor does it seem possible to attempt this without the employment of quantitative methods. The writer's studies in this field have led to a quantitative treatment of injury and recov- ery, which may now be discussed. In practice, we determine the condition of material by measuring the resistance of 'pieces of tissue or of intact organisms. These investigations show that it is often difficult to judge of the condition of an organism by its appearance. Tissues were found to be capable of losing much of their vitality without betraying it by their appearance. (This was particularly the case with the eel grass, Zostera, which retained its normal green color and appearance for some days after electrical measurements showed it to be dead). On the other hand, material of doubtful appearance often turned out to be much better than that which looked to be in sound condition.

Material collected in the same locality and examined as soon as taken from the ocean gave a very uniform resistance. To make the comparison as accurate as possi- ble disks of the same average thickness were used in the experiments. Under these circumstances the net resist- ance at 18 °C. did not vary much from 1070 ohms. For example, in a series of determinations of 10 different lots of tissue, the highest reading was 1090 ohms, and the lowest 1055 ohms. These lots of tissue were allowed to remain in the laboratory under different conditions. Some were in running salt water, some in quiet salt water in pans of various sizes, a part being placed in direct sunlight (where the temperature rose to an injurious point) while others were kept in a cool place, in partial shade. At the end of 24 hours, there was no difference in the appearance of these lots, but their net electrical resistance varied from 200 ohms to 1090 ohms. All were then placed side by side in the same dish. Those with the lowest resistance were the first to die. The others died in the order indicated by their electrical resistance.

Determinations of the resistance made it evident that in no case did visible signs of death make their appear- ance until twenty-four hours after death occurred, and subsequent experiments showed that in some cases (espe- cially at low temperatures and in the presence of certain reagents) they may not appear until several days after death. It was found that material from one locality showed a low resistance, and subsequent examination showed that it was contaminated by fresh-water sewage. The appearance of the plants was not such as to lead to their rejection for experimental purposes. They did not sur- vive as long in the laboratory as plants of normal resist- ance taken from the other localities.