Rehberg who also repeated with an improved technique many of the earlier experiments.

Transcription

1 STUDIES ON THE PHYSIOLOGY OF CAPILLARIES. III. The innervation of the blood vessels in the hind legs of the frog. BY A. KROGH, G. A. HARROP AND P. BRANDT REHBERG. (From the Laboratory of Zoophysiology, Copenhagen University.) THE investigations recorded in the present paper have arisen out of the study of the local reactions of the cutaneous vessels by one of us (1) and were undertaken primarily with a view to elucidate the mechanism of these reactions. As the experiments progressed it became necessary to modify and enlarge the scope of the research, and to repeat from new points of view experiments already done. The experiments were begun in January, 1920, by Krogh, carried on by Harrop, whose observations were partly checked by Dr v. Liebermann, and concluded by Rehberg who also repeated with an improved technique many of the earlier experiments. We propose to deal: 1. With the effects on the vessels of stimulating the peripheral ends of the different fibres supplying the skin and the muscles of the hind legs. 2. With the effect of section and degeneration of these fibres upon the state of the vessels, the local reactions and the effects produced by stimulation in the sciatic of the remaining fibres. 3. With experiments on the effect of cocaine on the local reactions, and 4. With the existence or otherwise of true vascular reflexes causing changes in the calibre of vessels and in the circulation at and about the point stimulated. Finally we shall attempt to synthesise our results into a picture of the innervation. Methods. We have generally narcotised the frogs for the operations with urethane giving curari later when necessary. Operations, from which the animal is left to recover, require a number of precautions which were only gradually and, we fear, incompletely made out. Rana esculenta appears to stand the operations better than R. temp., but for

2 180 A. KROGH, G. A. HARROP AND P. B. REHBERG. observations on the web we had to use R. temp. Quite small frogs of 5-15 gm. weight were found to be the most suitable. The operations must be performed as far as possible under aseptic conditions and with the least possible damage to muscles and other tissues. Touching with adrenaline 1: 1000 proved very useful to prevent excessive bleeding. We have filled up the defects produced in the vertebral column with soft, sterile paraffin wax and believe this procedure to be beneficial. Muscle sutures are to be avoided as far as possible, but the skin must be very carefully sutured and it is important to avoid any contact of the external surface with the internal or with the wound. The operated frogs were kept isolated in moist chambers with a little water. The water was changed frequently so as to remove the urethane by the end of 24 hours at least. Those which recovered (about 40 p.c.) were transferred afte'f about a week to suitable terraria where they had access to water and were regularly fed. Several, especially among the smallest, gained considerably in weight during the following months. By the frequent examination of the web on these operated frogs the use of anaesthetics was avoided as far as possible. The frog, except the leg to be examined, was in these cases put into a small box and immobilised with wet cotton wool Ṫo stimulate the nerves we have used single induction shocks repeated at the rate of about one per second, tetanisations at a rate of 5-10 per sec. and in several cases current from a small dynamo giving variations of a type like ^, the frequency of which could be varied from 10 to 30 per sec. In some cases we have stimulated mechanically by pinching. In stimulating electrically sympathetic ganglia or the posterior roots of spinal nerves we have been able to work on animals which were narcotised only, since we could easily avoid the production of muscular movements and the absence of such served as a useful control, but in stimulating anterior roots we had of course to curarise. In such cases we have sometimes given both urethane and curari. The reactions obtained have been studied exclusively by microscopic examination, in most cases with a binocular microscope and a magnification of 40-60, but in the later stages of the investigation and where the reactions of capillaries were specially concerned usually with an ordinary microscope and magnifications of We have also made some trials with plethysmographic methods, but we find that the interpretation of the results is so difficult and generally so uncertain that the direct visual method is at the present stage to be preferred.

3 BLOOD VESSELS OF FROG. Stimulation of peripheral nerve fibres. Stimulation of the sympathetic ganglia nos. 8 to 10 according to Gaupp's nomenclaturel gives constantly a contraction, generally very pronounced, of the arteries and capillaries in the web, both when the nerves behind them are left intact and when they are divided. Induction currents appear to be the most suitable stimulus, but the effect of the dynamo current is distinct also. After a latent period of secs. the stimulation produces constriction often to obliteration of the arteries2. It is worthy of note that all the arteries are affected including those branches which do not respond to adrenaline. The capillaries become constricted generally 5-10 secs. after the contraction of the arteries, but usually the contraction is not nearly complete; thus, a capillary of 15u diameter contracted to 13,u, another of 17 5,u to 11,u. The contracted vessels rapidly relax again when the stimulus ceases, and the reaction can be provoked several times in succession with intervals of about a minute. In some cases the effect is uniform, as far as can be judged, over the whole field, but in others some of the capillaries appear to escape stimulation. This is probably not due to any lack of sympathetic innervation but to the obvious difficulty of stimulating with sufficient intensity all the fibres or cells at the level of the ganglia. When the cut sciatic nerve is stimulated by induction shocks the effect is essentially the same as that of stimulating the sympathetic ganglia alone. When a capillary is watched with a magnification of 200 while the sympathetic is stimulated it can be observed that the contraction starts from certain definite points along its course and spreads from these in both directions. At the same time a curious reaction appears to take place in the surrounding tissue which appears to become less transparent. While this latter phenomenon must be reserved for further study the meaning of the first observation will be fully explained in a paper by Vi mtrup on the contractile elements in the capillary wall which is to be published shortly. Stimulation of the sympathetic produces contraction also of muscle arteries and capillaries. In some cases the arterial effect only has been observed, bringing the blood flow in the capillaries to a standstill without producing any measurable contraction; in other cases several open ' Gaupp, Anat. d. Fro8che8, H. p. 212, The latent periods given, are approximate and have only some relative.significance. The absolute time of latency depends upon temperature and is probably subject also to other influences. The' effect of sympathetic stimulation upon arteries is of course well known. See the paper by Langley(2) in which the literature is discussed.

4 182 A. KROGH, G. A. HARROP AND P. B. REHBERG. capillaries have disappeared from view by contraction while others have remained open but with a diminished diameter as shown by the following measurements: Diameter in (before stimulation *5 17 Dduring stimulation Since a fall of arterial blood pressure even to 0 causes only the very slightest diminution in diameter of the capillaries there can be no doubt that the influence upon them of the sympathetic stimulus is direct. Electrical stimulation of the posterior roots of the 9th and 10th spinal nerves produces distinct dilation of the web arteries as shown by the following examples: Before stimulation ,u During stimulation , These changes occur after a latency of secs. The vessels contract slowly when the stimulus ceases and the reaction can be repeated a few times only. In a few cases it has been noted that the peripheral arterial branches which do not respond to adrenaline are especially sensitive to the stimuli from the posterior roots and may during stimulation become wider than the stems from which they spring. Some capillaries also become dilated but a considerable number of these vessels do not respond to any stimulus from the posterior roots. In several cases we have like D oi(3) found it convenient to apply a small dose of acetyl-choline to the web to insure the filling of any capillaries the tonus of which might relax during the subsequent stimulation of the posterior roots. This procedure abolishes also the source of possible error which the dilatation of arteries and arterioles might otherwise constitute. The arteries dilated by acetyl-choline do not dilate further under the stimulation and the dilatation observed in capillaries cannot therefore be due to any increase in the blood pressure. Those capillaries which do dilate under the stimulus from the posterior roots often do so to a very considerable degree as shown by the following measurements: Before stimulation ,u During stimulation ,u As shown by Doi(3) and confirmed by us mechanical stimulation of posterior roots will also produce, dilatation of web capillaries, but we feel sure that in any case only a limited (though large) number of these vessels can be influenced through posterior root fibres. Mechanical stimulation by pinching of the sciatic nerve produces dilatation of the skin and web vessels as found by Bayliss in the case of mammals.

5 BLOOD VESSELS OF FROG. 183 In muscles the effects of stimulating the posterior root fibres are not very marked, though in several cases a slight jncrease in the diameter of capillaries and the opening of a few new capillaries has been noticed. The effect on arteries appears generally to be very slight or even absent, but in a single case marked dilatation occurred. In some experiments we have urethanised and curarised the frogs, extirpated the 8th-lOth sympathetic ganglia and stimulated the 9th and 10th anterior roots with induction or dynamo currents. No trace of muscular contraction can be observed by the microscope, but after a latency of secs. some widening of arterioles takes place and there seems to be a slight opening up and dilatation of capillaries. We have never been able to imitate by stimulation of nerves the great increase in the number of open capillaries and in the current of blood through them which accompany actual contraction of the muscles. In the web no effect has been observed from stimulation of the anterior roots after extirpation of the sympathetic ganglia. In Part 11(1) it was shown that weak mechanical stimulation of one point in a web artery can produce dilatation of the artery over a length of one or more mm. after a latency of about 15 secs. while a strong stimulus produces contraction over a similar distance. In the light of the results given above these reactions must be taken to be due to stimulation of posterior root fibres and sympathetic fibres respectively, but more precise information regarding the mechanisms of these local reactions is obtained by the following experiments. The effects of section and degeneration of nerve fibres. In one case the conduction of nervous impulses along the sciatic was temporarily blocked by freezing the nerve: the local arterial and capillary reactions remained perfectly undisturbed. In numerous other cases the sciatic nerve or certain of its roots were cut through, but apart from the resulting changes in arterial and capillary tone, presently to be described, the local reactions were unaffected. The whole of the reaction mechanisms must therefore be local and we must have to do either with axon reflexes or with short reflex arcs over local ganglion cells. The vascular tone in the web has in our experiments never been affected by section of posterior or anteriorl nerve roots or by removal of the 9th and 10th spinal ganglia, but removal of the sympathetic 1 We cannot confirm the observations of Lapinsky(4) giving dilatation of bloodvessels in the web after section of the anterior roots of the sciatic. Since, according to Langley(2) these roots do not carry preganglionic sympathetic fibres, the effects observed by Lapinsky are difficult to understand.

6 184 A. KROGH, G. A. HARROP AND P. B. REHBERG. ganglia or section of the sciatic usually produces relaxation of arteries -and capillaries. Sometimes this relaxation is observed at once, but more often it develops slowly and becomes pronounced after a few hours. The arteries usually regain their normal tone after a few days and in some cases appear not to lose it at all, but the capillaries often remain dilated for a long period extending even to 100 days. Very often however the arteries in the web of the operated side are found in a dilated state while the arteries of the normal side are narrow. The tone which develops *after cutting off sympathetic influences appears to be very variable and the arteries have only a slight power of resistance against dilator influences. The local arterial reactions have been followed in a number of operated frogs. It will be convenient to describe a few examples in some detail before proceeding to summarise the results. In a small male frog, the right sciatic of which had been cut (with resection of about 1 cm.) on March 22nd, the examination on the 12th day showed contracted arteries and dilated capillaries (the left web was normal). The contraction reaction on strong mechanical stimulation of arteries was distinct, but in spite of the contracted state of the arteries the attempt to obtain dilatation failed. On the 19th day both arteries and capillaries were of normal appearance. The arteries were now hypersensitive, showing dilatation upon the slightest touch and contraction after stimuli which would normally produce dilatation. The capillaries reacted normally to mechanical stimulation, showing dilatation after weak, contraction after strong, stimuli. On the 28th day a number of abnormal reactions were observed after strong mechanical stimulation; the arteries sometimes showing contraction in the place stimulated and dilatation over a considerable distance in both directions, in other cases dilatation only, and in others again sharply localised contractions without any dilatation. The capillary reactions were still normal and if anything perhaps extended over a larger area than normally. When tested on the 42nd and 53rd day no dilatation of the arteries in the right web could be obtained by mechanical stimulation, though the arteries reacted promptly to acetyl-choline, but on the 60th day the dilatation reaction reappeared and remained distinct afterwards, while the contraction reaction became very indistinct at about this time though probably never completely absent until the observations were discontinued on the 123rd day. On some occasions a sharply local contraction appearing without any measurable period of latency could be elicited on most of the arteries, while a more or less normal reaction

7 BLOOD VESSELS OF FROG. 185 over a short distance was obtained on a few. On the 95th day the capillary reactions to mechanical stimulation were tested. Dilatation only could be observed. In four similar experiments the dilatation reaction was absent or very doubtful for a variable period, but reappeared in the two cases in which the animal lived long enough, after 100 to 150 days. On a female frog of 20 gm. weight operated on April 2nd the spinal ganglia 9 and 10 were removed on the right side. The local reactions remained nearly normal for 50 days, but later the dilatation reaction became distinctly weaker and reduced in extent and failed to appear in several experiments between the 50th and 190th days. After 230 days it was, however, quite distinct, and remained so. The contraction reaction was normal throughout. In another similar experiment begun in June and lasting 140 days the dilatation was very slight on the 60th day, could not be elicited on the 101st, and reappeared on the 140th. Cutting of the anterior and posterior spinal roots 9 and 10 (one case) and degeneration for 145 days had no effect on the local reactions-as was to be expected. Removal of the sympathetic ganglia 8 to 10 on one side (three cases) only diminished the contraction reactions to some extent and for a somewhat variable period between the 50th and 120th day. In one case which was only examined once after 77 days no contraction of arteries could be elicited. As mentioned above, the web arteries of these frogs were often strongly dilated and on such vessels it is very difficult and sometimes impossible to elicit the dilation reaction. It was observed however whenever the state of the vessels was suitable, and there is no reason to believe that it is in any way affected by removal of the sympathetic ganglia. The operations were controlled in all cases where that was possible by examination of the sensitivity and reflex movements of the corresponding leg and in most of the cases by post-mortem inspection of the nerves. In one of the cases of resection of the sciatic a certain amount of regeneration had taken place after about 180 days, but the nerve was evidently abnormal and did not appear to function. In the cases in which the ganglion cells themselves had been removed regeneration was obviously impossible. The results of these degeneration experiments are fairly consistent, but they differ greatly from those which we had to expect on the basis of current views on innervation. According to the nerve stimulation and nerve section experiments we confidently assumed that we had to

8 186 A. KROGH, G. A. HARROP AND P. B. REHBERG. do with two distinct axon reflexes, one along sympathetic fibrils causing contraction and one along posterior root fibrils causing dilatation. We expected therefore that degeneration of sympathetic or posterior root fibres would abolish the corresponding local reactions. While it is quite clear that the local reactions are temporarily inhibited by degeneration of the nerve fibres through the fibrillar branches of which we believe them to be conducted. it is equally certain that they are not completely abolished and return practically to normal sensitivity and intensity though no regeneration of the fibres belonging to the extirpated sympathetic or spinal ganglia can possibly take place. It will require experimentation along new lines, combined with histological investigation, to clear up this paradox, but a tentative explanation will be put forward at the end of this paper. On several of the operated frogs the sciatic was exposed and stimulated electrically by induction and dynamo current before the animals were killed. In one case the right spinal roots 9 and 10 had been cut through and the right sympathetic ganglia removed 67 days earlier, so that the only active fibres which could be present must be posterior root fibres from the 9th and 10th spinal ganglia. The stimulation produced dilatation of web arteries and of arteries in a muscle. In the muscle a slight widening of capillaries was observed and a corresponding widening of web capillaries had undoubtedly taken place but is not recorded in the protocol. In another frog the spinal and sympathetic ganglia had been removed 77 days. In this case no effect on the web vessels could be detected when the sciatic was stimulated. The examination of the muscles, which would have been of special importance, was unfortunately omitted. In several cases in which the sympathetic ganglia only had been removed stimulation of the sciatic caused dilatation of vessels both in muscles and in the web. These results confirm the stimulation experiments described above. Cocaine experiments. A rather large number of experiments have been made to test the effect of cocaine and allied substances in the hope of finding a definite difference between their influence upon the contraction and dilatation reaction respectively. The application of 1-2 p.c. chloride of cocaine or novocaine in large drops or by means of reaction basins generally shows very little influence upon the reactions during the first hour or half-hour, but later the dilatation reaction usually becomes diminished or abolished while the contraction reaction is sometimes unchanged, sometimes more or less diminished. Experiments on animals which had not been narcotised show clearly that cocaine must

9 BLOOD VESSELS OF FROG. 187 be absorbed very slowly through the skin since reflex movements can be induced by pinching of toes after one hour's bathing in 2 p.c. cocaine. When 2 p.c. cocaine or 1 p.c. novocaine is injected into the web by a micropipette with a very fine point the arteries after a very transitory contraction become more or less dilated and after a few minutes do not react at all to weak mechanical stimulation while strong stimuli sometimes produce a local dilatation and in other cases remain without any visible effect. Nothing definite can be concluded therefore from our cocaine experiments. Expertments on vascular reflexes. All the vascular reactions so far described as resulting from local stimulation of the tongue or skin of the frog depend on local mechanisms-nervous and others-but not upon true reflexes, and the problem very naturally suggests itself: Are the local mechanisms the only ones existing in the frog for the production of vascular reactions about a stimulated skin area, and are the vascular reflexes which are so conspicuous in the skin of mammals to be considered as belonging to a higher stage in the development of the central nervous system? We have found that chemical stimulation may cause dilatation of arteries and capillaries over a large area of the web even beyond the space between two toes where the stimulus is applied. Iodine may show this effect, but the most powerful stimulant appears to be nitrate of silver, which has the advantage that the direct corroding effect spreads over a very limited and well defined area only. When a small crystal of silver nitrate is placed on the web all the arteries between the two toes and often a large number between neighbouring toes will dilate after a latency of about 10 secs. A few seconds later a large number of capillaries will also show some dilatation which is certainly in the main independent of the increase in pressure and blood flow resulting from the widening of arteries. The arteries will sometimes show a rapid succession of changes in diameter, while thetcapillaries remain dilated for a long time. Example. Crystal placed near one of the toes. Observation of artery from opposite side. Initial diameter 30,A. After 10 secs. 35 /Aand thereupon 26-44, This response (which in appearance resembles closely the reflex erythema produced by mechanical, chemical or thermal stimulation of the human skin) depends upon a local mechanism, since it persists after section of the sciatic. In three frogs, the sciatics were cut on Sept. 26th and the reactions were examined on Oct. 8th at a period wben the

10 188 A. KROGH, G. A. HARROP AND P. B. REHBERG. arteries had regained their tone1. In one of these cases the crystal was applied between toes II and III near the end of III, while arteries were measured (1) in the web between the same toes, issuing near the end of II, (2) between III and IV issuing near the middle of III, and (3) issuing near the middle of IV. The following dilatations were observed: (1) 35,u- 61,u, (2) 26,u-44,u, (3) 35p-46,t. We have tried of course to stimulate the web of one foot with nitrate of silver and observe the web of the other but have been unable to find any response. When the skin of the femur is corroded with nitrate of silver a dilatation of arteries in both webs is often observed. The spreading of the impulses due to strong chemical stimulation over large areas of the web through purely local channels, which must necessarily be nervous in nature, points definitely to the existence in the web of a network of nerves or rather of nerve fibrils in which conduction can take place in any direction2, and reviewing the main facts brought together in the present and the preceding paper we arrive at the following conception which we put forward tentatively, being well aware to what extent it differs from generally accepted theories of innervation :--The arteries and capillaries are innervated through sympathetic fibres. The fibrils of these reach all the contractile elements to which they carry continuous (?) impulses maintaining their tonus. The fibres run along the arteries and arterioles which they supply with numerous fibrils responsible for the axon reflexes which can be set up along these vessels by strong mechanical stimulation. The fibrillar system does not degenerate completely after operative removal of the sympathetic ganglia and may regenerate independently; it probably forms some sort of a network which either contains a few cells or remains in connection through nerve paths other than the sciatic3 with sympathetic ganglion cells outside the web. Posterior root fibres are (directly?) connected with all the web arteries and with many of the contractile elements in the capillaries. No example is known of their carrying impulses from the central nervous system to the vessels, but their terminal fibrils form networks through which impulses can be carried from any spot all over the web and produce 1 A study of these reactions during the process of degeneration after section of the sciatic or its roots will be undertaken next summer. 2 The existence of such nets has been maintained by Bethe(5) and his school, and Erik MuIller(6) has shown that true nets are formed by nerve fibrils in the vagus system in the stomach of sharks. 3 Like Langley(2) we have been unable to find any evidence of fibres running along the femoral artery.

11 BLOOD VESSELS OF FROG. dilatation. These nets must be especially close meshed along the arteries, and they probably contain cells which prevent their complete degeneration after section of the fibres from the spinal ganglia and enable them to regenerate even after removal of these ganglia. SUMMARY. The capillaries and arteries in skin and muscles of the hind legs in frogs are innervated through sympathetic fibres from the 9th and 10th sympathetic ganglia, which -maintain the vessels in a state of (variable) tonic contraction. Stimulation of these fibres causes contraction. The arteries and many capillaries in the skin and likewise a number of vessels in the muscles are innervated through posterior root fibres the stimulation of which causes dilatation. There is no evidence of stimuli passing from the spinal cord through posterior root fibres to the vessels. Some arteries and capillaries in muscles appear to be innervated through anterior root fibres unconnected with sympathetic ganglia. The stimulation of these causes slight dilatation. The local reactions of skin vessels (arteries and capillaries) to mechanical and certain chemical stimuli are due to axon reflexes along the terminal fibrils of sympathetic and posterior root fibres respectively. They are not influenced by section of the corresponding nerve stems or roots. They are inhibited to a certain extent by degeneration of the fibres, but regeneration takes place independently of these. Chemical stimulation of a single spot in the web produces dilatationf over the whole web independently of the central nervous system, and, the existence of extensive networks made up of the terminal fibrils, belonging to fibres of the same category and probably in connection with local nerve cells is suggested to account for the phenomena observed. 189 REFERENCES. (1) A. Krogh. This Journ. 55. p (2) Langley. Ibid. 41. p (3) Doi. Ibid. 54. p (4) Lapinsky. Arch. f. Physiol. Supp. p (5) Bethe. AUg. Anat. u. Physiol. des Nervensystems. Leipzig, (6) E. Muller. Arch. mik. Anat. 94. p PH. LVI. 13