Injury, Recovery and Death in Relation to Conductivity and Permeability

It may be taken for granted that vitality, whatever else it may signify, means ability to resist unfavorable influences. When organisms which are of the same kind, and similar in age, size and general characters, are placed under the same unfavorable conditions, the one which lives longest may be said to have the greatest vitality;15 the one which lives next longest may be rated second in this respect, and so on. Determination of the electrical resist- ance of these individuals enables us to predict at the outset which will live longest, which next longest, and so on through the entire group.

It is therefore obvious that determinations of electri- cal resistance afford a means of measuring vitality and in the course of an extensive series of experiments it has been found that this method may be relied upon to give accurate results. The fact that determinations of electrical resistance afford an accurate measure of vitality enables us to attach the same sort of quantitative significance to nor- mal vitality as we attach to normal size or to normal weight. For this purpose we may construct a variation curve and determine the mode in the usual way.

There is no reason to suppose that the vitality of an individual organism is constant any more than its weight is. There is probably some fluctuation which usually passes unperceived unless a quantitative method of detecting it exists. The writer finds that all substances (whether organic or inorganic) and all agents (such as excessive light, heat, electric shock, mechanical shock, partial drying, lack of oxygen, etc.) which alter conductivity of the protoplasm shorten the life of the organism. This is equally true whether the alteration consists in an increase

It might be expected that this individual would also excel in other respects. A discussion of these is unnecessary from our present stand- point: in so far as they can be quantitatively treated they form proper material for a supplementary investigation. of conductivity, or in a decrease of conductivity (followed by an increase), as is the case when certain reagents (such as CaCl2) are applied. This is a very striking fact and its significance in the present connection seems to be perfectly clear. It shows in a convincing manner, that electrical resistance is a delicate and accurate indicator of normal vitality.

Since it is evident that a fall of resistance indicates injury, it seems reasonable to assume that the amount of fall is a measure of the amount of injury. This may be expressed as per cent, of the total possible loss (this would correspond to the amount of loss of the substance, M, as previously discussed). If tissue which has been in- jured by exposure to a toxic solution be replaced in sea water, it may recover a part or all of the resistance which it had lost. If the resistance should fall to 70%, then recover in sea water to 90%, and remain stationary, we might call the temporary loss of resistance temporary injury and the permanent loss permanent injury. In this case the temporary injury16 would be 30-^90=33.33% and the permanent injury 10-^90=11.11%. If we calculate the protoplasmic resistance in this case we find that starting at 100% the resistance decreases to 62.50% and recovers to 86.4%. In the case of protoplasmic resistance the total possible loss17 is 92.65, the temporary injury is there- fore (100 — 62.5) -~ 92.65 = 40.5% and the permanent injury is (100 — 86.4) -^ 92.65 = 14.68%. In this manner we arrive at a quantitative basis for the study of injury and recovery.

We divide by 90 because if the resistance starts at 100 the total possible loss is 90. This is merely another way of saying that we It is evident from Fig. 37 that in the earlier stages of the death process, recovery may be complete (i. e., the normal resistance may be completely regained), but this is not the case in the later stages. In other words we see that as temporary injury increases, permanent injury also increases. Another interesting aspect of the subject18 is illus- trated by the results obtained in mixtures of NaCl and

FIG. 37. — Curves showing net electrical resistance of Laminaria agardhii in NaCl 0.52 M (unbroken line), and recovery in sea water (dotted lines). The figure attached to each recovery curve denotes the time of exposure (in minutes) to the solution of NaCl. CaCl2. Curve C in Fig. 38 shows the behavior of tissue placed in a solution containing 97.56 mols of NaCl to 2.44 of CaCl2 ; its electrical resistance falling in 37.5 hours to 72.87% of the original value in sea water. In a solution containing 85 mols of NaCl to 15 mols of CaClo (Curve A) the resistance fell in the same time to practically the same point (72.47%).

When these two lots of tissue were replaced in sea water they behaved differently. The resistance of the first lot rose to 78.2% (Pig. 38, upper dotted line), but the resistance of the second fell (much more rapidly than if it had not been removed to sea water) and eventu- FIG. 38. — Curves showing net electrical resistance of Laminaria aoardhii in a solution con- taining 97.56 mols of NaCl to 2.44 mols of CaCh (Curve C) and in a solution containing 85 mols of NaCl to 15 mols of CaCh (Curve A). The dotted lines show recovery in sea water. Curves B and D show the levels to which the resistance rises when the tissue recovers in eea water after exposure to these mixtures; their abscissae denote the times of exposure. Curve B pertains to the first mixture (belonging with Curve C), while Curve D pertains to the

ally became practically stationary at 38.1% (Fig. 38, lower dotted line). If we plot the curve of permanent injury (i. e. level to which the resistance rises after replacing the tissue in sea water) after various periods of exposure to the first mixture, we get Curve B (and for the second mix- ture, Curve D). If we use the term recovery for the rise of resistance which occurs when tissue is transferred to sea water from certain solutions (such as the first mixture) there seems to be no good reason why it should not be applied to the fall of resistance which occurs when tissue is trans- ferred from certain other solutions (such as the second mixture) to sea water.19 The amount of recovery after any given period of exposure is equal to the vertical distance between Curves B and C, in the case of the first mixture, and between Curves A and D in the case of the second mixture.

It may be asked whether Curves B and D are better criteria of toxicity than Curves A and C. The question involves the definition of toxicity. Since this term is used in a variety of ways, it is desirable that it should always have a precise quantitative significance. In the present case it is evident that we need not only A and C but also B and D for a complete description of the facts. It seems possible that this may be generally true in the study of toxicity, although at present we may be unable to con- struct similar curves in many cases because suitable methods of measurement are lacking.

The fact that recovery is never complete except at the beginning (as shown by Curves B and D) might also be explained as due to the death of certain cells; for if some of the cells are killed by exposure to a solution of NaCl the complete recovery of the surviving cells cannot restore the resistance to its normal value. This hypoth- 19 Substances which cause increase of resistance commonly produce per- manent injury; this is apparent when the tissues are replaced in sea water.

It would therefore seem that any alteration of resistance (increase or decrease) may produce permanent injury if sufficiently prolonged. In spite of this it seems preferable to restrict the term temporary injury to the fall of resistance observed in toxic solutions without coining a new term to express the injurious action accompanying rise of resistance. esis would in no way invalidate the conception developed above, that an individual cell may lose part of its resistance and subsequently regain it, either partially or completely. But there are serious objections to this hypothesis. The appearance of the cells under the micro- scope indicates that they all die at about the same time. Moreover, Inman (1921, B) has recently obtained striking experimental evidence that recovery may be far from complete when practically all the cells are alive. In his experiments he employed a unicellular alga, Chlorella, which does not stain readily with methylene blue as long as it is alive, but stains intensely as soon as it dies. Cells were treated with hypertonic salt solutions until the rate of respiration was greatly diminished. When they were replaced in the normal culture medium, the respiration did not return to normal, but the rate appeared to be permanently lowered. In order to determine whether this was due to the death of a part of the cells they were carefully stained with methylene blue. The percentage of dead cells20 was practically the same as in the normal culture before treatment with the salt solution. In other words, the incompleteness of the recovery seems to be due to the fact that the metabolism of each cell is permanently lowered. Similar results were obtained when the cells were treated with chloroform; in this case a great depression of respiration was not followed by recovery, but by a greatly lowered metabolism which was perman- ent and which was not due to the death of a part of the cells.

Some recent experiments of Inman (1921, A) indicate that we obtain similar results whether we use electrical resistance or respiration as the criterion of partial recov- ery. He found that in NaCl 0.52 M the rate of production of carbon dioxide by Laminaria steadily decreased. If the tissue was replaced in sea water after exposure to NaCl the recovery (as judged by the rate of production of CO2) was either partial or complete according to the degree of depression which the rate had undergone. The results are shown in Fig. 39. It will be seen that they

FIG. 39. — Curves showing rate of respiration of Laminaria agardhii (expressed as per cent, of the normal). The normal rate represents a change from pH 7.78 to 7.36 in from 1^4 to 2 minutes, depending upon the amount of material used. The solid lines show rate of res- piration during one hour of exposure to isotonic sodium chloride (0.52 M for Woods Hole sea water). The dotted lines show stages of recovery after the tissue was put back in normal sea water. Each curve represents a typical experiment.

offer a striking parallel to those obtained by measuring the electrical resistance. Similar results are observed where we employ hyper- tonic or hypotonic solutions in place of NaCl. When Laminaria is placed in dilute sea water, or in sea water concentrated by evaporation, injury may occur, and recov- ery may be partial or complete. This is true whether we use electrical resistance or rate of production of C02 as the criterion of injury and recovery. The results obtained by Inman (1921, A) with hypertonic solutions are shown in Fig. 40.

The fact that in the case of Laminaria and Chlorella recovery may be either partial or complete, according to circumstances, raises the question whether this is also true of other forms. It is certainly true of all the plants investigated by the writer, such as the green alga, Ulva (sea lettuce), the red alga, Rhodymenia (dulse), and the flowering plant, Zoster a, (eel grass). It seems to be also true of frog skin as far as the experiments of the writer have gone.21 In physiological literature it seems to be generally assumed that when recovery occurs at all it is practically complete, as though it obeyed an "all or none" law.22 It is evident that partial recovery might

FIQ. 40. — Curves showing rate of respiration of Laminaria agardhii (expressed as per cent. of the normal). The normal rate represents a change from pH 7.78 to 7.36 in from IK minutes to 2 minutes, depending upon the amount of material used. The solid lines show rate of respiration while tissue was exposed to hypertonic sea water (sp. gr. 1.130, A° = — 9.37° approximately). The dotted lines show stages of recovery after the tissue was put back in normal sea water. Each curve represents a typical experiment. The figure attached to each recovery curve denotes the time (in minutes) of exposure to the solution of hypertonic sea water; thus the uppermost curve represents recovery after an exposure of 5 minutes.

81 The recovery experiments on frog skin were few in number and dealt chiefly with the effects of anaesthetics. 22 There are indications in the literature that partial recovery occurs. Thus Leo Loeb and his collaborators Loeb, L. (1903) 1905; Corson-White, E. P. and Loeb, L. (1910) ; Fleischer, M. S., Corson-White, E. P., and Loeb, L. (1912) ; Ishii, O., and Loeb, L. (1914) observed that destruction of the corpora, lutea produces a permanently depressing effect on the ovary and

that the virulence of tumor tissue is permanently diminished by exposure to heat or certain reagents. In both cases a condition is produced which is intermediate between death and normal vigor. The diminution of the virulence of bacteria by various means cannot be cited as an illustration unless it is certain that it is not due to the selection of less viru- lent individuals. easily be overlooked except in cases where recovery can be measured with considerable accuracy, and it seems possible that further investigation may show that incom- plete recovery is a general phenomenon.

Let us now consider the cause of permanent injury. If we assume that the death process proceeds according to the scheme it is evident that in sea water A must be continually renewed. Let us assume that this occurs by means of the reactions 0 — >S — > A and that 0 (the origin of all the substances produced) is present in such large amount that its concentration does not appreciably change dur- ing the time of the experiment. If we start with 0 alone, it will produce all the other substances according to the scheme

and their amounts will increase until equilibrium is reached, i. e., until they are decomposed as rapidly as they are formed. Their values will then remain constant. We assume that when the tissue is placed in NaCl 0.52 Mj the reactions 0 — ** S — >A cease, while the reac- tions A — >M^ _ B continue. In consequence the values of A, Mj and B steadily fall. If the tissue is now replaced in sea water the reactions 0 — >$ — >A recommence and in consequence the values of A, M, and B will rise to their original level (the level which is normal for sea water). But if 0 is diminished by exposure to the solution of NaCl it can no longer restore these values to their original level. If, for example, it diminishes to one-half it can restore them only to one-half the normal values. In this case the permanent injury would amount to 50%. We therefore

see that the permanent injury is an index of the condition of 0. We may now calculate the curve of recovery23 after exposure to a solution of NaCl 0.52 M. We assume that when the tissue is transferred from sea water to the solution of NaCl the reactions 0 — >8 — >A cease and that the velocity constant KA of the reaction A — >M increases from 0.0036 to 0.0180 while the velocity constant We may then calculate the resistance in the solution of NaCl after any length of exposure by means of the formula

in which TE is the time of exposure in minutes, and e is the basis of natural logarithms. 10 is added in the for- mula because the base line is taken as 10 (not as 0) for the reason that the resistance sinks to 10 (as shown in Fig. 28) when the tissue dies. We assume that when the tissue is replaced in sea water the reactions 0 — >S — > A recommence and that the values of KA and JT^ become 0.0036 and 0.1080 respec- tively, while the other velocity constants likewise acquire the values which they normally have in sea water. Under these conditions M will be formed faster than it is decom- posed and the resistance will rise.

The fact that the rise does not reach as high a level after a long exposure as after a short one indicates that during the exposure 0 gradually diminishes ; we assume that this takes place by the reactions tion of NaCl the amount of S changes by means of the reactions and that on transferring to sea water S is rapidly con- verted into A. In order to calculate the rate of recovery we find by trial the most satisfactory values of the ve- locity constants. The values thus found are given in Table V.

As an example of the method of calculation we may take the case of tissue exposed for 15 minutes to a solu- tion of 0.52 M NaCl at 17 °C. The net resistance in sea water at the start was 960 ohms ; in the course of 15 min- utes in the solution of NaCl it fell to 775 ohms, which is 80.69% of the original resistance. The fall of resistance is a little more rapid than in the ' ' standard curve' ' previous- ly obtained. If we assume that this is due to the difference in temperature (these measurements were made at 17 °C. while those on which the standard curve is based were obtained at 15 °C.) we may introduce a correction by mul- tiplying the abscissa by the factor24 1.06, which makes it

24 This agrees closely with the temperature coefficient as determined else- where. See page 37. 15.9 minutes, and causes it to agree with the standard curve. All the abscissae are multiplied by the same factor.25 The effect of this is to make the process appear to proceed at 15 instead of at 17 °C. If the difference between the two curves is due wholly to difference in temperature this introduces no error, and if the difference is due in part to other factors, the error, if any, is less than the usual experimental error.

The advantages of this procedure are that we can employ for our calculations the constants already obtained for the standard curve and also compare the theoretical curves which start from the same points. This procedure has therefore been followed throughout and the corrected results (i. e., the figures multiplied by a suitable factor) are employed in the following description. When the tissue was replaced in sea water the resist- ance began to rise. At the end of 10 minutes it had risen from 80.69 to 89.10%.28 Since, however, the abscissa? of the death curve have been multiplied by 1.06 the same thing must be done for the recovery curve and in place of 10 minutes we must put 10.6 minutes. Proceeding in this manner we obtain the recovery curve which is labeled 15.9 in Fig. 41.

In order to calculate the course of the recovery we must consider the reactions which determine the amount of electrical resistance. When the tissue is placed in the 83 This procedure may displace the points on the curve so that where several curves are averaged it may be necessary to employ interpolation in order to average points on the same ordinate. In many cases curves were obtained by averaging the ordinates of death curves and recovery curves before multiplying by the factor.

M In earlier experiments it was found that complete recovery was pos- sible after the resistance had fallen to about 80%. This was not the case in the present series; the difference may be due to differences in material or in technique. Cf. Osterhout (1915, B). Let us first consider the reactions A — > M — > B. The value of A in sea water is taken as 2,700 and that of M as 90. As previously explained the value of A will diminish FIG. 41. — Curves showing the fall of net electrical resistance of Laminaria agardhii in 0.52 M NaCl (descending curve) and recovery in sea water (ascending curves). The figure attached to each recovery curve denotes the time of exposure (in minutes) to the solution of NaCl. In the recovery curves the experimental results are shown by dotted lines, the calculated results by the unbroken lines (the curves are extended beyond the last observed point shown because of later observations which cannot be shown in the figure). The observed points represent the average of eight or more experiments; probable error of the mean less than 10%

in which T£is the time of exposure to the solution. Since K^ = 0.018 (see Table V) the value of A after 15.9 minutes'in NaCl 0.52 M is The value of M at the end of 15.9 minutes is the observed resistance 80.69 less 10 (since the base line of the curve is not 0 but 10). On replacing the tissue in sea water, therefore, we start with M = 70.69 and A =2,027.96, but this value of A is at once augmented by the conversion of 8 into A. In order to find the amount of this augmentation we must know the value of S.