Injury, Recovery and Death in Relation to Conductivity and Permeability

4. If the fall of resistance in CaCl2 is due to coagula- tion, or to some other structural change, it might be expected to be irreversible almost from the start; but this is not the case. Only when it has proceeded a good way toward the death point, does it become irreversible. On the other hand, the fall in NaCl (due to liquefaction) might be expected to be reversible at every stage. But it ceases to be wholly reversible after it has proceeded one sixth of the way (or less) to the death point.

5. Since the changes in viscosity occur in dead as well as in living tissue we should expect to find in both cases similar changes in resistance. It is found that in tissue which has been killed in such a manner as not to alter the properties of the cell wall, the decrease in vis- cosity in NaCl produces no appreciable effect on resis- tance. Even when the process goes so far that the tissue is reduced to a very soft jelly, there is little or no change in resistance.51 The hardening in CaCl2 produces some rise in resistance, but it is much too small to account for the great changes which occur in living tissue.

It might be supposed that the reason that no change in resistance occurs in dead tissue is because the hard- ening and softening do not proceed as far as in living plants, but this is not the case. Moreover, it is found that the decrease of viscosity in NaCl is accompanied by absorption of water, while the increase of viscosity in CaCl2 is accompanied by loss of water, and these pro- cesses take place in the same way in living and dead tissue. It would seem that these and other important objec- tions must be removed before we can accept the idea that changes in permeability are determined by changes in viscosity.52

81 In a liquid a change of viscosity alters the resistance, but this is not necessarily the case in a gel. Where a gelatin gel is converted to a sol, the change in resistance is very slight. M It would appear that the term viscosity is loosely applied to a variety of phenomena which may be produced in different ways. Spaeth (1916) to account for variations in permeability under the influence of salts. The great variations in the electrical resistance pro- duced in living protoplasm by the action of salts seem to the writer to depend on the fact that living protoplasm is in a state of dynamic equilibrium so that the material of which it is composed is constantly chang- ing. This constant change is due to a succession of chemical processes which may be easily influenced so as to produce great changes in electrical resistance which appear to become irreversible if carried beyond a certain point.53

In dead protoplasm, as in gelatin, such processes do not occur, or at least they go on much more slowly. As a result we cannot expect such great variations in electri- cal conductivity. If we wish to imitate these it would seem advisable to work with systems, which, like living protoplasm, are in dynamic equilibrium. Clowes (1918), states that he has prepared emulsions of oil in soap, which change their electrical resistance under the influence of NaCl and CaCl2, in a manner similar to that observed in Laminaria. It remains to be seen whether this parallel extends to the effect of other substances.

The writer obtained similar results some years ago,54 with the shells of the Horse Chestnut (Aesculus) which had been killed by boiling or by soaking for 24 hours in 5% formaldehyde. He was not able to convince himself, how- ever, that the factors involved here are the same as in living protoplasm. 63 This apparent reversibility finds a ready explanation on the theory of successive reactions. See page 121. M A brief account of these was given at the Boston meeting of the American Physiological Society, in 1915.

The writer has experimented with a great variety of materials in order to determine whether it is possible to imitate by means of non-living materials, the change in permeability found in living cells. In some cases membranes have been found which show an increase of conductivity when transferred from sea water to NaCl and a decrease when transferred from sea water to CaCL or LaCl3.55 But in no case was the alteration great enough, nor produced by a, sufficient variety of substances, to justify the author in concluding that the effects were really the same as those found in living material. The relatively small changes found in dead material, in so far as they are due to the cell walls (or intercel- lular substance), must in the living conditions be superimposed on the changes due to the activities of the protoplasm.

Until we succeed in finding a membrane (or other static system) which imitates qualitatively and quanti- tatively the permeability of the living protoplasm, the author is inclined to regard a dynamic equilibrium as essential. 55 The solutions of NaCl, CaCL and LaCl3 had the same conductivity as sea water. When transferring from sea water to another solution a temporary rise or fall may occur which is due to diffusion. See page 28. In order to ascertain the effect of anesthetics on con- ductivity, experiments were performed with ether,1 chloroform, chloral hydrate and alcohol.2 Subsequently alkaloids were employed.3

The method may be illustrated by the following experiment with ether. Tissue was transferred from sea water to a mixture consisting of 990 c.c. sea water + 10 c.c. ether + 5 c.c. sea water which had been concentrated by evaporation until its conductivity was about double that of ordinary sea water. This mixture contained approx- imately 1% by volume of ether (= .099 M) and had the conductivity of sea water. In 10 minutes the resistance had risen to 113.4%, 4 but, in 10 minutes more it had fallen to 109.4%. It continued to fall until it had reached 98.8%, after which it fell very slowly (at about the same rate as the control). The fact that it fell below the starting point is not necessarily to be attributed to any injury, but rather to the fact that the exaporation of the ether increases the conductivity of the sea water, which is contained in the apparatus, and in the cell walls between the protoplasmic masses. The results of the experi- ment are shown in Fig. 80.

1 Since ether, chloroform, and alcohol deteriorate on standing, espe- cially when in contact with metal or with cork stoppers, special care must be taken to obtain pure reagents. Those used were Kahlbaum's or Squibb's. Of. Baskerville (1913). * All readings were made at 18° C. or corrected to this temperature. from the solution influenced the result, another experi- ment was performed in which the solution was renewed every 5 minutes during the first 60 minutes, and there- after every 15 minutes. In this way the concentration of ether was kept more nearly constant. It was then found that the resistance rose as before, but did not fall during

Fia. 80. — Curve A shows the net electrical resistance of Laminaria agardhii in sea water; B in sea water containing 1% ether by volume (.099 M) from which the ether was allowed to evaporate in an open dish; C in the same mixture in which the concentration of ether was the first 80 minutes, and after this fell very slowly, so that after 300 minutes it was still 80 ohms above that of the control. At this point, the experiment was discon- tinued. The results are shown in Fig. 80.

In order to see whether the effect of the anesthetic could be quickly reversed, some tissue was kept in sea water containing 0.099 M ether for 50 minutes (the solution being renewed every 5 minutes). During this time the resistance rose to 113.7%. It was then placed in sea water. At the end of 10 minutes the resistance had fallen to 100%. It was then left in sea water contain- ing 0.099 M ether (the solution being renewed every 15 minutes). The resistance promptly rose to 113.7%, and

remained there for an hour ; 240 minutes later, when the experiment was discontinued, the resistance was 111.4%. The results are shown in Fig. 81. The effect of higher concentrations of ether was next investigated. Tissue was placed in a mixture of 970 c.c. sea water + 30 c.c. ether + 15 c.c. of concentrated sea Fia. 81. — Curve A shows the net electrical resistance of Laminaria agardhii in sea water, Curve B, unbroken line, in sea water containing 1% ether (.099 M), the solution being fre- quently renewed, broken line in sea water; Curve C, unbroken line in sea water containing

water, which was added to make the conductivity of the mixture equal to that of sea water. The concentration of the ether was therefore 2.96% by volume (= 0.293 If). In the course of 10 minutes the resistance rose to 112% ; dur- ing the next 10 minutes it fell to 105.3% ; it continued to fall rapidly during the next 40 minutes, reaching 89.5% at the end of this period. The tissue was then placed in sea water; in the next 10 minutes, the resistance fell to 87%. This fall in resistance was doubtless due to the continued action of the ether, which required time to diffuse out of the tissue. During the next 10 minutes, there was a rise

of 2.5%, which was probably due, either wholly or in part, to the fact that the resistance of the sea water was greater than that of the mixture from which the ether had partly evaporated. During the next 400 minutes no rise occurred. The results are shown in Fig. 81. This outcome is very significant, for it shows that the increase of permeability produced by ether is not reversible, while, as we have seen, the decrease of permeability is easily reversed. Since the essential characteristic of an anesthetic is the reversibility of its action, we must conclude that anesthesia is associated with the reversible decrease of permeability and not with the irreversible increase of permeability.

In view of the importance of this result the experi- ment was repeated many times, the fall of resistance (before placing in sea water) varying from 6 to 25%, but always with practically the same result. On placing in sea water there were sometimes irregular fluctuations (amounting to 5% or less) but no recovery. This result is the more striking inasmuch as material of which the resistance has fallen as much as 5 to 10% in NaCl recovers completely when placed in sea water, and may even undergo this treatment daily for several days in succession without injury.5

as a measure of the toxicity. The toxicity increases with the concentration, and it should be noted that it is greatly decreased if the material is allowed to stand in an open dish, owing to the evaporation of the ether. If the material be placed in a closed jar, oxygen must be sup- plied. The other alternative, frequent renewal of the solution, is usually preferable. A series of investigations on chloroform gave similar results, the chief difference being that chloroform is much more toxic, and that the concentration necessary for long continued decrease of permeability is much lower, being about 0.05% by volume (or 0.064 M). This is evident from Fig. 82, which shows the results of an experiment with a mixture containing 999.5 cc. sea water + 0.5 c.c. chloroform + 0.25 c.c. concentrated sea water (this mixture had the same conductivity as sea water). In this experiment the solution was renewed every 5 min- utes during the first 80 minutes, and every 15 minutes thereafter. j : ^

If we increase the concentration of chloroform to 0.1% by volume (=0.0128 M), the result is quite similar to that obtained with 0.293 M ether. This is shown in Fig. 82, which gives the results of an experiment contain- ing 999 c.c. sea water + 1 c.c. chloroform + 0.5 c.c. concen- trated sea water (this mixture had the conductivity of sea water).6 The solution was renewed every 5 minutes during the first 80 minutes, after which it was kept in sea water. There is no indication of recovery after the tissue is replaced in sea water.

Experiments with chloral hydrate gave results very similar to those obtained with chloroform, the corres- ponding effects being produced in both cases by approximately the same percentage concentrations,7 that is, chloral hydrate 0.1% (=0.006 M) acts similarly to chloroform 0.1% by volume (=0.0128 M). 8 Stiles and Jorgensen (1914) report a decrease of resistance as the result of exposure to chloroform. See also Waller, A. D. (1919). 7 No effort was made to find the exact percentages which are required to produce given effects, as this was not the primary object of the investi- gation. The actual concentration of chloral hydrate may have been somewhat lower than those given, owing to the presence of water in the chloral hydrate.

The experiments with alcohol lead to somewhat differ- ent results. In the first place, alcohol is not so toxic as ether, chloroform, or chloral hydrate, and higher concentrations must be used to produce the same effects on permeability. In sea water containing alcohol 0.051 M, or 2.955% by volume, (the solution being renewed every Fio. 82. — Curve B shows the net electrical resistance of Laminaria agardhii in sea water; Curve A in sea water containing 0.05% chloroform, Curve C placed for 80 minutes

in sea water containing 0.1% of chloroform and then put back into sea water. 15 minutes) the results were much the same as in 0.099 M ether (the solution being renewed every 5 minutes), except that the rise in resistance took place more slowly, sometimes occupying 30 minutes or more. It was found that 0.2385 M, or 13.8757o by volume, is decidedly toxic. An interesting feature of the results with alcohol is that the increase of permeability is reversible. If the increase be carried too far it is not reversible (or at

least recovery is incomplete) ; in the first experiments this condition was unintentionally realized and led the writer to suppose that alcohol behaves like ether. The course of a typical experiment is shown in Fig. 83. The tissue was first placed in a mixture containing 970 c.c. sea water + 30 c.c. Squibb 's absolute alcohol + about 15 Fio. 83. — Curves showing the net electrical resistance of Laminaria agardhii placed for 40 minutes in sea water containing 0.269% ethyl alcohol, then in 13.875% for 20 minutes and then

c.c. of concentrated sea water. The mixture had the conductivity of sea water; the concentration of the alcohol was 0.051 M (2.96% by volume). The net resist- ance rose to 110% in the course of 40 minutes. The tissue was then placed in sea water containing 0.2385 M alcohol (13.875% by volume) ; and in the course of 20 minutes the resistance fell to 87.6%. The tissue was then placed in sea water and the resistance again rose to 100%. The facts that recovery occurs in alcohol, and that irregular fluctuations are often observed in experiments on recovery from ether, suggest that the difference

between the behavior of alcohol and the other anesthetics investigated may be only one of degree. It is probable that there is some recovery in ether, chloroform, and chloral hydrate, but that it is so slight and so transitory as to be difficult to detect, It is evident that suitable concentrations of anesthetics produce a marked decrease of permeability.8 This con- dition may be maintained for a long time if the concentra- tion is not too high; with higher concentrations the period is shortened and may become so short as to be observed with difficulty. This decrease of permeability can be easily and quickly reversed by replacing the tissue in sea water. It does not seem to produce any injury if the concentration is not too high. The relative con- centrations necessary to produce this result, correspond closely with those required to produce anesthesia, being least for chloral hydrate and greatest for alcohol.

On the other hand, the increase of permeability, (except in the case of alcohol, within certain limits) pro- duces permanent injury and is not reversible. It cannot be regarded, therefore, as the characteristic effect of the anesthetic. The characteristic effect must be regarded as in some way connected with decrease of permeability.9 8 The amount depends somewhat on the condition of the material. Material in poor condition generally shows less rise in resistance than good material.

8 Winterstein (1916) says that these experiments are not convincing because the anesthetic may act on the interior of the cell rather than on the surface of the protoplasm. This objection can hardly apply, since the interior of the cell is filled with cell sap: this is surrounded by a thin layer of protoplasm (see page 197). If the anesthetic decreased the con- ductivity of the cell sap to any marked degree, this effect would be observed in the material immediately after death: this, however, is not the case; if any rise in resistance occurs in dead tissue it is much less than in living tissue. Loewe, (1913) states that anesthetics decrease the con- ductivity of artificial lipoid membranes. See also Moore and Roaf (1905).

It is easy to see how a decrease of permeability to ions must hinder the production and the transmission of stimuli in so far as these are dependent on the move- ment of ions in the tissues, and there is abundant evidence that stimulation is always accompanied by such movements of ions in the protoplasm. It seems clear, therefore, that a decrease of permeability may result in the decrease of irritability, which is the characteristic effect of an anesthetic.10

These investigations are of interest in view of the fact that a number of writers hold the view that anesthet- ics increase permeability, while others believe that anesthetics bring about a decrease of permeability.11 It appeared desirable to clear up this uncertainty as a necessary step toward a satisfactory theory of anesthesia. In order to see whether these facts are generally true, the scope of the investigation was widened to include a variety of material. Similar results were obtained in experiments on frog skin,12 but the effect was much more striking. The increase of resistance was greater and occurred with lower concentrations.13 With respect to recovery, the same difference was found between alcohol, on the one hand, and ether, chloroform, and chloral hydrate on the other.

10 It might be expected on this basis that substances which decrease permeability, such as Ca, La, etc. would act as anesthetics. To what extent this is the case must be decided by future investigation. Experiments were also made14 to determine the effects of ether on a variety of plants. An increase of resistance (followed by a decrease) was observed in Laminaria and Ulva. In Rhodymenia ether (2.5, 3, 5 and 5.5% by volume), and alcohol (1, 3.5, 7, 8% by volume) added to sea water produced little or no rise. This is not surprising in view of the fact that these substances always produce less rise in Laminaria than does Ca and that even Ca produces very little rise in Rhodymenia. In respect to recovery from the injury caused by these sub- stances, Rhodymenia agrees with Laminaria in that recovery is practically complete in alcohol (if the fall in resistance has not gone too far), but is almost entirely absent in ether and chloroform.

While the writer has found no records of similar experiments made by other investigators, it may be desirable to refer briefly to the work of Joel on the con- ductivity of red blood corpuscles. When red blood cor- puscles are repeatedly washed in an isotonic solution of cane sugar and allowed to stand in this solution the conductivity of the suspension gradually increases. This is due in part to the exosmosis of electrolytes (which increases the conductivity of the solution) and probably in part to the fact that the permeability of the corpuscles to ions increases. The experiments of Joel,15 show that this increase in conductivity can be hindered by the addition of "indifferent' narcotics (at certain concentrations).