(Received 12 April 1938)

Transcription

1 206 J. Physiol. (I938) 93, 206-2I4 6II.83:6II.314:6I2.392.OI3 A DEGENERATIVE CHANGES IN THE AXIS CYLINDERS OF THE DENTAL NERVES, DUE TO DIETS DEFICIENT IN VITAMIN A AND CAROTENE BY J. D. KING, W. LEWINSKY AND D. STEWART University of Sheffield, and the From the Field Laboratories, Department of Anatomy, University of Manchester (Received 12 April 1938) DURING recent years the investigations of E. Mellanby [ ] and his co-workers have firmly established the truth of the observation that marked degenerative changes occur in the nerve fibres of both the central and peripheral nervous systems of animals which have been kept on a diet deficient in vitamin A. The histological methods which have been used to demonstrate these changes have been concerned mainly with the alterations found in the myelin sheath of the nerve, and the Marchi technique has been used for the most part for this purpose. On the other hand, little is known of the influence of such a diet on the structure of the axis cylinders themselves. Our lack of knowledge on this subject is due principally to the capriciousness of the methods which have to be employed in staining these structures. The silver impregnation techniques of Bielschowsky and Cajal are very uncertain in their action, and it is difficult with them to obtain consistent results. A study of the literature reveals that very few of the workers in this field have employed an axis cylinder stain. Zimmerman & Burack [1934] used Bielschowsky to stain the axis cylinders of dogs which had been fed on diets deficient in vitamin B2. Apparently they were unable to detect any changes in these structures even in fibres which showed myelin degeneration. Grinker & Kandel [1933] utilized both Cajal and Bielschowsky, and found no changes in the axis cylinders. It is also to be noted that these investigators were unable to detect any changes in the central nervous system in animals fed on diets deficient in vitamin A or vitamin B complex. Culley [1927], in an investigation into the polyneuritis of fowls, says that axis cylinders undergo changes at a very late

2 DENTAL NERVES AND DIET 207 period, long after the myelin degeneration, hut he gives no illustration of such changes nor does any description of them appear in his paper. Woollard [1927] utilized Bielschowsky to demonstrate nerve changes in starvation and beri-beri, and found degenerative alterations of the axis cylinders in both these conditions. He noticed particularly that marked changes occurred in the muscular nerve endings. These structures lost much of their complicated form, and indeed some of the nerve endings seemed to have disappeared altogether. More commonly, however, only the finer branchings were lost. The myelin sheaths of the fibres connecting the endings were often swollen and fragmented and the axis cylinders themselves sometimes became fragmented and even disappeared but, as a rule, they persisted even when the myelin was greatly altered. This worker also used the intravital methylene-blue stain, and obtained similar results with this method. The changes in the nerve endings affected the muscle spindles as well as the motor nerve endings. Woollard also found that the degenerative processes were more marked peripherally and became less distinct when the nerve fibres were traced centrally towards the spinal cord. In the larger trunks it was difficult to detect any affected fibres. Mellanby [1935] experimented with several silver methods, but he states that the results which he obtained, especially in the early stages of axis cylinder degeneration, were not sufficiently definite to warrant publication. However, in rabbits fed on vitamin A and carotene-deficient diets, changes in the axis cylinders were definitely present before advanced myelin degeneration occurred. From this brief survey of the literature, it will be seen that little is known of the degenerative processes which occur in the axis cylinder of nerves of animals on a vitamin-deficient diet. Recently, Lewinsky & Stewart [1936a, b, 1937a, b] have made extensiveinvestigations into the innervation ofthe dentinal structures andthe neighbouring tissues and, in these investigations, they relied practically entirely upon the Cajal silver impregnation method to demonstrate the course and distributions of the nerve fibres. They found that the decalcification that is necessary before it is possible to prepare histological specimens from this material had profound effects upon the staining properties of this method. These effects were investigated by Gooding & Stewart [1934] who discovered that nitric acid, which is normally employed as a decalcifying agent, delayed the action of the silver nitrate, and it took longer to impregnate the nerve fibres. On the other hand, it had one very useful characteristic. It checked to a very marked degree the formation of deposit in the non-nervous tissue, which so commonly 14-2

3 208 J. D. KING, W. LEWINSKY AND D. STEWART occurs in specimens which do not require decalcification. This deposit is sometimes very marked, and may conceal the finer fibres and make it difficult or even impossible to trace their course and distribution. When, however, nitric acid was employed for decalcification, these other tissues were stained uniformly yellow or orange in colour, and gave a marked contrast to the black- or darkly brown-stained axis cylinder. Also, the coarse deposit was either absent or much reduced in amount. They also found that 48 hr. immersion of the tissues in 5 p.c. nitric acid seemed to be the optimum time to prevent deposit, without unduly checking the impregnation of the nerve fibres. These authors therefore recommended as a modification of the Cajal technique, that after the tissues had been fixed, they should be immersed in nitric acid for 48 hr. before being placed in the silver nitrate solution. The results obtained by this method were so satisfactory and consistent that it was felt that this was a suitable medium to employ as a routine in the investigation of the axis cylinder changes which occur in vitamin A deficiency. Rats were selected as the experimental animals and, from the histological viewpoint, they had this great advantage, that the tissues of rodents stain particularly well with this modification, and secondly, the lower jaws of these animals took about 48 hr. to decalcify. METHODS Experimental feeding of the rats was begun at weaning, when the animals were 3 weeks old and weighed about 30 g., and continued for varying periods up to 384 days. Basal diet. Oatmeal, 89 g., bakers' yeast, 5 g., heated alfalfa,' 2 g.,' calcium carbonate, 1-5 g., sodium pyrophosphate, 1-65 g., sodium chloride, 085 g. Each rat received g. of this mixture daily, to which was added 0 5 c.c. of heated olive oil,' 0 3 c.c. of orange juice and 125 international units of irradiated ergosterol (vitamin D). Instead of the above, a few animals were given the casein-starch basal diet previously used by M. Mellanby & King [1934]. Vitamin A in the form of mammalian liver oil was added to the rations of some of the rats as shown in Table I. After the animals had been killed with chloroform, half of the lower jaw was retained at Sheffield and fixed in 10 p.c. formol saline to determine the general tissue changes, and the other half was placed in a tube containing ammoniated alcohol and forwarded to Manchester. After 48 hr. in this solution, the half jaws were placed in nitric acid for a further 48 hr. and were then found to be completely decalcified. I Heated and oxygenated so that any traces of vitamin A were eliminated.

4 DENTAL NERVES AND DIET 209 The specimens were next passed through the various stages of the Cajal silver impregnation method and finally dehydrated and embedded in paraffin. Longitudinal serial sections at a thickness of 12,u were cut parallel to the vertical plane of the half jaw, with the teeth in situ. The cutting of these serial sections offers little difficulty, and they are particularly valuable because the course of the fibres and small bundles can be followed from one section to another for long distances. Having the half jaw, it was also possible to study differences in the condition of the axis cylinders in different regions and tissues of that part. RESULTS The general clinical findings just before death and the vitamin A content of the liver are shown together with the dietary variations and experimental period in Table I. Confirmation has been obtained of the previous observation of M. Mellanby & King [1934] with regard to the breaking down of the myelin sheaths of the afferent dental nerves of rats fed on diets deficient in vitamin A and carotene, when stained by the modified Marchi technique of R. J. Stewart [1936]. On histological examination of the nerves, it was found that the condition of the axis cylinders was not uniform throughout the tissues. In the pulps of the molar teeth, degenerative changes of the nerves were particularly marked, and in different specimens many stages could be observed from quite early ones up to those of an advanced degree. In those pulps in which the degenerative conditions of the axones were most pronounced, the connective tissue was also undergoing change. Signs of degeneration were also detected in the nerves of the periodontal membrane, but these changes were not so marked as those found in the pulps themselves. On the other hand, no changes of any kind could be detected in the nerve supply of the mucous membrane of the gum. In some of our specimens, small portions of the muscles still remained, and although not sufficient to give indications of alterations in the nerve endings themselves, there was sufficient tissue present to show that the fibres of the motor nerves themselves were little, if at all, affected. As the process of degeneration was most clearly seen in the pulps of the molar teeth, our description of these changes is confined to the nerves of this tissue. The earliest signs which were observed during the course of this investigation consisted in a definite thickening of the axis cylinders. This is clearly demonstrated in P1. I, Figs. 1 and 2, which are photomicrographs of nerve bundles running in the pulp tissue and are of the same magnification, namely 233, as are all the photomicrographs in

5 210 J. D. KING, W. LEWINSKY AND D. STEWART Rat no. Basal diet 2582 Oatmeal, etc. Diet addition 2585 Ditto Vitamin A, 1500 international units daily 2581 Ditto 2583 Ditto Vitamin A, 1500 international units daily 2579 Ditto Ditto 2577 Ditto 2580 Ditto 2584 Ditto Vitamin A, 1500 international units daily 2303 Casein, etc Ditto Vitamin A, 1500 international units daily TABLE I Duration of ex- periment (days) Condition of animal immediately prior to death 74 Stance and movements appear normal; eyelids slightly inflamed; lower incisors normally pigmented 74 No abnormalities seen 147 Slightly shaky; no eye symptoms; lower incisors normally pigmented 147 No abnormalities seen 203 Hind legs rather stiff; head shaky; movements slightly uncertain; lower incisors rather pale 234 Very high on legs, stiff and shaky; body hunched; eyes partly closed with sore lids; very weak 238 Highonlegs,stiffand mthershaky; eyes partly closed, cornea of right eye dull; cornea oflefteye slightly ulcerated; lower incisors pale 289 Very stiff, shaky and weak; both eyes almost sealed up; eyelids very sore; both corneas ulcerated; coat poor; lowerincisors very pale 289 No abnormalities seen 384 Shaky and unsteady; slight head movements; cornea of each eye opaque and swollen; lower incisors pale 384 No abnormalities seen Vitamin A contents of liver (blue units per g. liver)* Not estimated Not estimated 23 * As estimated by the antimony trichloride method, using a Rosenheim-Schuster (no. 63) colorimeter. this paper. Pi. I, Fig. 1, is the dentinal pulp from one of the control rats (no. 2585). As in other parts of the body, the nerve fibres in the pulp fall into two groups, namely, thick and thin fibres and the bundle which is illustrated in this figure is formed of thick fibres of a normal structure. P1. I, Fig. 2, is from rat 2581, which had been fed for 147 days on a diet deficient in vitamin A. This bundle also consists of the thicker type of fibre, and a glance is sufficient to show that its individual fibres have increased markedly in breadth and their diameters are far greater than those of any seen in a normal pulp. An examination of the cornual region of the pulp demonstrates the changes which occur in the finer

6 THE JOURNAL OF PHYSIOLOGY, VOL. 93, No. 3 PLATE I Fig. 1. Fig. 2. Fig. 3. Fig. 4. To face p. 210

7 PLATE II THE JOURNAL OF PHYSIOLOGY, VOL. 93, No. 3 Fig. 5. Fig. 6. Fig. 7. Fig. 8.

8 DENTAL NERVES AND DIET 211 fibres. P1. I, Fig. 3, is a photomicrograph which was taken from this region in the same control rat (no. 2585). In this portion of the pulp the number of thick fibres is markedly reduced and the finer fibres greatly increased in number, largely due to the thick fibres dividing into arborizations. P1., Fig. 4, is also from the cornual region, but this rat (no. 2578) had been on diet for 234 days. A definite alteration will be seen of a similar nature to that already described in the main bundles lower down in the pulp. The main fibres are distinctly thickened, and there is a definite increase in breadth of many of the finer ones. Besides this general condition, there are numerous irregular swellings in the course of the finer fibres which bear a close resemblance to the end bulbs which were seen in normal nerves, but they are much larger. Some of these on examination were found to contain a lighter area in the centre, giving indications of the beginning of vacuole formation. A good example of such a swelling with a clear centre will be seen in P1. II, Fig. 4 itself. The next stage, foreshadowed in the last, is the actual formation of vacuoles in the course of the thicker fibres. This can be seen in P1. II, Fig. 5. This photomicrograph was obtained from the cornu of the pulp from rat This specimen is a further illustration of the marked thickness of the fibres which is clearly brought out when it is compared with the normal in Fig. 3. These vacuoles continue to increase in size, and in some examples appear to have lost the argentophil substance. In the next stage, many of the vacuoles break down altogether, leaving the fibres rough and serrated and, when several fibres are grouped together in a bundle, they have the appearance of a network, rather than a fascicule. An example of this is shown in P1. II, Fig. 6, where the curious networklike appearance of a small bundle in the pulp can be seen. It should be compared with a normal bundle in P1. I, Fig. 1. This degenerated bundle, after a short course, divides and the isolated individual fibres will in some parts be seen to contain large fibres, but in others the vacuoles have ruptured and given the fibres the serrated appearance mentioned above, which is very different from the smooth outline of the normal nerve. This is as far as it has been possible to trace the effects of vitamin A deficiency on the nerves in the molar pulps, with the material at present at our disposal, but it is quite possible that the nerve fibres may ultimately disappear altogether and further experiments are now in progress which, it is hoped, will supply the evidence to determine this point. It has already been mentioned that these changes were not uniform throughout the jaw, and that in our specimens they have been most strongly marked in the pulps of the teeth and have also been present to

9 212 J. D. KING, W. LEWINSKY AND D. STEWART a somewhat lesser extent in the periodontal membrane. The nerves supplying the gum, as distinct from the subgingival epithelium have, however, been unaffected (P1. II, Fig. 7), even in those specimens where degenerative changes in the nerve fibres of the pulp are far advanced (P1. II, Fig. 8, which is a photomicrograph of the pulp of a tooth from the same jaw as P1. II, Fig. 7). It will be noticed (Table I) that rat 2300 showed no clinical signs of vitamin A deficiency but that at death the vitamin content of the liver was decidedly low. Moreover, examination of the pulp nerves of this animal showed early degenerative changes in the axis cylinders. Since the rat had received a liberal supply of vitamin A throughout the experimental period, it would seem that some unknown factor had rendered defective the absorption or utilization of the vitamin. DISCUSSION The degeneration of the nerve fibres which have been separated from the nerve cell, has been fully described by Cajal [1928]. It is an acute process in which the degeneration occurs very rapidly and bears little resemblance to the slow chronic degeneration which has been described in this paper, and therefore requires no further discussion at this point. Of much greater significance is the work of Speidel [1935]. This author has written a valuable contribution to our knowledge of the changes which take place in the nerve fibres during the earliest stages of irritation and degeneration. The study was made on the nerves of the tails of living frog tadpoles, and the changes were observed by direct microscopic examination and cinephotomicrographs. He found that the irritation of a nerve fibre by an adjacent wound, among other interesting features, had a tendency to cause the axis cylinders to lose their smooth edge and become slightly wavy. This apparently is the earliest stage which takes place in the axon cylinder change, and has not been observed by us very clearly in the present investigation, but certain of our specimens do suggest that such a stage does occur before the swelling of the fibres. In the irritative changes in a myelin nerve after alcohol, Speidel found specimens in which fine fibres ended in spherical knobs containing small vacuoles. He gives an illustration of this in his paper and it resembles very strikingly the condition which we have described in P1. I, Fig. 4. When Speidel's animals were embedded in ice for over an hour, marked tissue irregularities developed in the more delicate parts of the tail fin. There was strong nerve irritation, including swelling, vacuolization and tortuous course of the axis cylinder. We have not

10 DENTAL NERVES AND DIET 213 observed any increased tortuosity in the course of the nerve fibres in our specimens, but the swelling and the vacuolization of the fibres closely correspond to our findings. It would appear, therefore, that the degenerative changes in the axis cylinder in vitamin A deficiency correspond to those which occur when the nerve fibres are acted upon by irritants either of a chemical or of a physical nature. E. Mellanby [1935] found that when the amount of degeneration in the myelin was only "annular", the nerve fibres so affected could be brought back to normal fairly rapidly when adequate vitamin A was supplied in the diet. If, however, the degeneration was more advanced, and the myelin sheath had disintegrated, structural recovery required a much longer time. These changes described by Mellanby resemble the irritative changes in the myelin sheath seen by Speidel, and we have already shown that the changes in the axis cylinder after vitamin A deficiency also resemble those seen by Speidel. It is probable, therefore, that when there is only annular degeneration of the myelin, there will be but little alteration in the axis cylinder, probably confined to thickening. These conditions could be remedied by a diet adequate in vitamin A. It is probable that the later changes in the axis cylinder correspond with those cases in which the myelin is markedly degenerate, and do not respond so effectively to treatment by administration of vitamin A. Further investigations are in progress into the possible relationship between structural changes occurring in the epithelium and nerves of the subgingival tissue due to vitamin A deficiency. Abnormalities in the calcified tissues of the incisor teeth of A-deficient rats were demonstrated by one of us (J.D.K.) at the Annual Meeting of the British Dental Association [1937]. They will be described in a subsequent report together with changes found in the subgingival epithelium adjacent to the molar teeth and in the alveolar bone. A brief account has already been given of the effects of vitamin A deficiency on the development of the teeth and jaws in dogs [King, 1936]. SUMMARY 1. The effects of deficiencies of vitamin A and carotene upon the axis cylinders of the dental nerves of rats have been investigated. 2. The nerves of the pulp were markedly affected and the nerves of the periodontal membrane showed somewhat less severe changes. 3. An account is given of the degenerative changes which were found in the nerves of the pulp.

11 214 J. D. KING, W. LEWINSKY AND D. STEWART The expenses of a portion of this investigation have been defrayed by the Medical Research Council, and one of us has also received a whole-time grant from the Council, for which our thanks are due. REFERENCES Cajal, S. Ramon y (1928). Degeneration and Regeneration of the Nervou8 Sy8tem. Oxford and London. Culley, P. G. (1927). Quart. J. exp. Physiol. 17, 65. Gooding, H. & Stewart, D. (1932). Laboratory J. Gooding, H. & Stewart, D. (1934). Ibid. Grinker, R. R. & Kandel, E. (1933). Arch. Neurol. P8ychiat., Chicago, 30, King, J. D. (1936). J. Phy8iol. 88, 62. Lewinsky, W. & Stewart, D. (1936a). J. Anat., Lond., 70, 349. Lewinsky, W. & Stewart, D. (1936b). Ibid. 71, 98. Lewinsky, W. & Stewart, D. (1937a). Ibid. 71, 232. Lewinsky, W. & Stewart, D. (1937b). Proc. Roy. Soc. Med. 30, 75. Mellanby, E. (1926). J. Phy8iol. 61, 24 P. Mellanby, E. (1931). Brain, 25, 412. Mellanby, E. (1934). J. Path. Bact., Lond., 38, 391. Mellanby, E. (1935). Brain, 58, 141. Mellanby, M. & King, J. D. (1934). Brit. dent. J. 56, 538. Speidel, C. C. (1935). J. comp. Neurol. 61, 1. Stewart, R. J. (1936). J. Path. Bact., Lond., 43, 339. Woollard, H. H. (1927). J. Anat., Lond., 61, 283. Zimmerman, H. M. & Burack, E. (1934). J. exp. Med. 59, 21. EXPLANATION OF PLATES I AND 11 PLATE I Fig. 1. Rat 2585 x 233. Normal bundle of nerve fibres. Fig. 2. Rat 2581 x 233. Nerve bundle showing early thickening. Fig. 3. Rat 2585 x 233. Distribution of normal nerve fibres in the cornu. Fig. 4. Rat 2578 x 233. Thickening of nerve fibres, swelling and early vacuole formation in the cornu of the pulp. PLATE II Fig. 5. Rat 2579 x 233. Further thickening and more marked vacuole formation in the cornu. Fig. 6. Rat 2577 x 233. Late degenerative changes in nerve bundle in the pulp. Fig. 7. Rat 2302 x 233. The gum and normal nerve fibres. Fig. 8. Rat 2302 x 233. Late degenerative changes in nerve bundle in the pulp.