thyroidectomy delayed healing by 4-40 days. thyroxin or by homo- or hetero-transplantation of thyroid gland, and

Transcription

1 : : THE INFLUENCE OF METABOLIC STIMULANTS ON WOUND HEALING; THE INFLUENCE OF THYROID and 2-4-a DINITROPHENOL. By T. H. C. BARCLAY,' D. P. CUTHBERT- SON, and A. IsAACs, Institute of Physiology, University of Glasgow. (Received for publication 29th November 1943.) THE daily subcutaneous injection of an alkaline extract of the anterior lobe of ox pituitary gland into the rat has no appreciable effect on the rate of healing of experimental wounds, in spite of its definite anabolic effect [Cuthbertson, Shaw, and Young, 1941]. Davidson and Waymouth [1943] found that similar extracts, which had shown growth accelerating activity in young rats, did not show a growth-promoting effect on chick heart fibroplast cultures. Since this extract of the anterior pituitary gland had no significant action on the rate of wound healing, the influence of catabolic stimulants (e.g. thyroid gland extract and 2-4-a dinitrophenol) on the rate of wound healing was investigated. The results are recorded below. Lauber [1930] has drawn attention to the value of thyroid administration clinically in wound healing. Kosdoba [1934], as the result of a study of incised wounds made on a variety of species, has come to the conclusion that, as judged by macroscopic and microscopic appearances, the healing times are definitely reduced in all species by the injection of thyroxin or by homo- or hetero-transplantation of thyroid gland, and that this takes place in wounds healing both by first and second intention. Further, he found that partial (up to 3) resection of the thyroid or total thyroidectomy delayed healing by 4-40 days. EXPERIMENTAL. The technique of wounding and measurement of healing was that previously described by Cuthbertson et al. [1941]. This involves the removal of discs of skin from the outer aspects of the thighs. The criterion of healing was based on the complete epithelialisation of the wounded area (macroscopic observation) with subsequent confirmation by histological methods. By the latter confirmatory test it was possible to demonstrate that the healing process was shared by the whole thick- James Reid Stewart post-graduate Bursar. 309

2 310 Barclay, Cuthbertson, and Isaacs ness of the skin and that the apparent healing on naked-eye view was not merely a covering of epithelium over incompletely healed deeper layers. Young adult male rats were used in all the experiments. Each rat received two circular wounds each of 12.5 mm. diameter. Throughout the experiment the animals were given, ad libitum. the stock diet described by Thomson [1936]. They were weighed at regular intervals in order to determine that the activity of the catabolic stimulant was being maintained. Sepsis was observed in none of the wounds. Experimental Notes. Expt. 1.-The thyroid-fed group diet was made up as follows: Ext. thyroid sicc. B.P. was added in the proportion of 1 g. to 200 g. of stock food Ṫhere were 24 wounds (12 animals) in the control group and there would have been the same number in the thyroid group, had one animal not died and one wound been chewed by its owner. In the thyroid group (T), consumption of this. diet started 8 days prior to operation and was continued throughout the experimental period. Expt. 2.-In this experiment the proportion of ext. thyroid sicc. B.P. added to the stock diet was reduced to 0-75 g. per 200 g. With the above exception in quantity of thyroid extract, the thyroid group (1) was similar to that of Expt. 1 with regard to the pre-operative feeding. In thyroid group (2) the animals began their thyroid diet on the day of operation. There were 24 wounds in the control group (12 animals), 18 wounds in thyroid group (1), and 22 wounds in thyroid group (2). Since the conditions of the experiment, as far as the thyroid group (1) were concerned, were the same as in Expt. 1, with the exception of dosage, it was felt that to use the animals available to the best advantage it would be preferable to have controls and the unknown thyroid group (2) as nearly as possible the same, and allow the deficiency, due to the insufficient number of animals available, to exist in thyroid group (1). Unfortunately one of the thyroid group (2) animals died, hence there were only 22 wounds to investigate. Expt. 3.--The technique was identical with that of Expt. 2, T(1). There were 26 wounds (13 animals) in the control group. Originally the experiment had 32 wounds in each group (16 animals per group). Three rats died in the thyroid group, and the same number in the control group. One wound of the thyroid group was chewed and was in consequence discounted. In all three experiments (1, 2, and 3) the thyroid treated groups

3 The Influence of Metabolic Stimulants on Wound Healing 311 lost weight. In Expt. 2 the loss was less in the thyroid (2) group than in thyroid (1) group. Expt. 4.-In this experiment the 2-4-a dinitrophenol was administered orally, beginning on the day of operation. The diet for this group was made up as follows: to 0-12 g. 2-4-a dinitrophenol N/NaOH was added until the solid dissolved and the solution was just alkaline, using phenolphthalein as indicator. This volume was made up to 50 ml. with ethyl alcohol and the solution added, drop by drop with constant stirring, to 1 kg. stock diet. The alcohol was then evaporated off in an incubator room with an air current. There were 16 wounds (8 animals) in the control group and the same number in the dinitrophenol group. The changes in body-weight were indeterminate. Expt. 5.-Since Murphy [1938] found that much larger doses of 2-4-a dinitrophenol could apparently be administered with safety to rats, the dosage in this experiment was increased approximately eightfold, 0 9 g. 2-4-a dinitrophenol being added to 1 kg. of stock diet. Originally there were 24 wounds (12 animals in each group), but 4 dinitrophenol treated animals died and 1 of the wounds of the control group was interfered with by the animal concerned. There was a definite loss of body-weight in the dinitrophenol group. Expt. 6.-In this experiment the dose of 2-4-a dinitrophenol was reduced to that of Expt. 4. The experiment was done in combination with Expt. 4. Originally there were 32 wounds in each group, but 3 control animals died (i.e. 6 wounds deficient). All the dinitrophenol treated animals remained alive. There was only a slight loss of bodyweight in the dinitrophenol group. EXPERIMENTAL DATA. Since the basis of comparison between the controls and the treated animals in these experiments is the average number of days taken by the wounds to heal, the latter has been calculated for each group and is given in column 3 of the table. As a criterion of significance the difference between the means must exceed twice the standard error of the difference between the means (in which case the odds are more than 100 to 5 against it having arisen by chance). It will be seen from the table that the difference between the means of the controls and treated group is significant except in Expt. 5. DIscUSSION. Kosdoba [1934] in his experiments on the white rat noted that, so far as macroscopic appearances were concerned, the period of wound

4 312 Barclay, Cuthbertson, and Isaacs Mean period required Difference between for healing. controls and treated. Percentage Experi- No. of difference ment. wounds. of mean Days. S.E. Days. o S.E. of periods. mean. Dy. duff. 1 C *38 ± ± o T *86 i049 2 C O*62 T (1) O ± % T (2) O ± % 3 C i *25 ± % I T ±O C *13 ±O ±O o" D.N.P *44 ± O ± O D.N.P *25 ± 0654 O58 ± O82 3 I 6 C ± h D.N.P *97 ± ±O7 5 C=Control. T=Thyroid treated. D.N.P.=dinitrohphenol treated. healing of his incised linear wounds was reduced from 6-8 days (controls) to 4-6 days in the injected or implanted series, a 19 per cent. reduction in time. Microscopically, the wounds were healed in days in the injected or implanted series, a 22 per cent. reduction from the time taken to heal the controls, viz days. It is to be noted that Kosdoba only used 29 animals in his rat series, and of these, controls appear to have amounted to somewhere in the region of one-quarter of the total. In the present series of experiments, thyroid gland and 2-4-a dinitrophenol have been shown to diminish significantly the mean time required for healing. The former, if given for some days before wounding, caused a reduction of per cent. in the mean time required for healing, but when the thyroid was given from the day of wounding till healing took place then the reduction of the mean time for healing was found to be less, viz. 11 per cent. With dinitrophenol, the reductions noted in the two favourable experiments were 15 and 27 per cent. These values for the thyroid-fed animals agreed remarkably well with Kosdoba's figures for linear wounds. Thyroid gland and dinitrophenol have the common property of increasing the rate of oxidation of the tissues. The evidence in respect of the thyroid gland is overwhelming, and Jeney and Valyi-Nagi [1938],

5 The Influence of Metabolic Stimulants on Wound Healing 313 and Freeman [1940], have satisfactory evidence in respect of dinitrophenol. Associated with this increase in oxygen uptake is a tendency for a slight rise in general body temperature. There is also ample evidence that the thyroid gland exerts a positive influence in speeding up cardiac output, circulation time, and, in consequence, the blood supply to the part. Kosdoba [1934] has attributed the beneficial effect of this hormone to its influence on the general metabolism, but, in particular, on fibrous tissue and the blood vascular system. There is no doubt but that measures which improve the blood supply to a part, influence favourably the healing of a wound of that part [Arey, 1936]. Although Simkins [1937] has found that "dinitrophenol is remarkable for its lack of significant effects on the cardiovascular system, even when the basal metabolic rate is raised to high levels," it is difficult to conceive of an increase in the metabolic rate without some parallel change in the circulation rate. Indeed, Cushny et al. [1941] have definitely stated that dinitrophenol causes a marked increase in the pulse rate. After the infliction of a wound, there appears to be a rapid fall in the metabolism of the tissues, the degree of fall being presumably related to the trauma. Green's [1943] and Bielschowsky and Green's [1943] recent observations on the reduction in body temperature which follows injection of muscle shock factor(s) from muscle is of special interest. There then follows a rise in metabolism above normal, followed finally by a decline to normal level [Gaza and Gissel, 1932]. One of us [Cuthbertson, 1942] has already discussed at some length the significance of this swing of the metabolic pendulum. It would appear that the action of the thyroid and dinitrophenol in enhancing the rate of wound healing may well depend on (a) a direct effect on the cells, in particular the oxidative phase of their metabolism, and which in consequence probably increases the blood supply to the part; or on (b) a stimulant action on cell proliferation, due to the products of the excess catabolism. These factors, singly or in conjunction, may prove to be the determining agents. In these experiments, only two concentrations of dinitrophenol were tested, one per cent., the other 0-09 per cent. With the latter dosage, the animals rapidly lost weight, and no favourable influence on wound healing was noted; indeed, rather the reverse was found. This was obviously a toxic dose. With the lower dosage no continued loss of body-weight took place. This was rather unexpected. It may have been that in proportion to the rise in metabolic rate the dinitrophenol treated animals ate more food than did the thyroid treated group. This is purely surmise since the intakes were not measured. It is of interest that despite loss in body-weight (6-17 per cent.), the thyroid treated animals exhibited a decrease in the average period of wound healing.

6 314 Barclay, Cuthbertson, and Isaacs It is to be noted, however, that there is some evidence to suggest that fat is the main material catabolised under the influence of dinitrophenol [Cushny et al., 1941]. Thus, there would be less change in body-weight per calorie of energy liberated than would probably occur under the influence of thyroid. Further, the end products of metabolism from the action of these two catabolites would be quantitatively and probably qualitatively different. Although not clear-cut, the evidence does suggest that the dominant factor in speeding up the wound healing is the direct stimulant action on the cells rather than the effect of the end products of that action. Young, Fisher, and Young [1941] found that a second wound inflicted some days after the primary, healed at a significantly more rapid rate than the initial wound. They deduced that some accelerating factor must operate in the closure or healing of secondary wounds which is lacking in relation to primary wounds. This substance might be a " growth-promoting substance " liberated by primary wounds or a by-product of immunity, or some other principle. The simplest explanation is that in the Young et al. experiments the second wound was produced during the final phase of the enhanced metabolism which follows injury, and which has been shown to be accompanied by marked evidence of an increased catabolism and traumatic fever [Cuthbertson, 1932, 1942]. This enhanced metabolism which follows the initial phase of depressed activity would appear to be due in the first instance to substances set free from the traumatised cells. The need to label these as "active wound-healing substances " is not necessary, and tends to deflect attention from the reactive mechanism which is the essential thing. In conclusion, the writers do not consider it wise to attempt to reduce the normal rate of wound healing in patients by such means as have been tried in these experiments. On the other hand, with indolent wounds the question of the administration of thyroid might be considered SUMMARY. 1. Dried thyroid gland, when fed to young adult male albino rats throughout the period of healing of circular skin wounds, caused a significant reduction in the mean time required for healing (11 per cent. reduction). When the rats were, in addition, given a pre-period of thyroid feeding before operation, the mean time required for healing was reduced by 12 and 22 per cent. in the two experiments performed a dinitrophenol fed as a component of the diet (0a012 per cent.) also caused a significant reduction in the mean time required for healing (15 to 27 per cent. reduction). Larger doses (0 09 per cent.) lacked this stimulant effect and the weight loss was marked. 3. The significance of these findings is discussed. It is not con-

7 The Influence of Metabolic Stimulants on Wound Healing 315 sidered wise to attempt to influence the rate of normally healing wounds of patients by such stimulants. In conclusion, we wish to thank Dr. E. Lewis-Faning and Dr. P. L. McKinlay for their assistance in the statistical treatment of this investigation. We are also greatly indebted to the Medical Research Council, and to the Rankin Medical Research Fund of the University of Glasgow, for grants in aid of this work. REFERENCES. AREY, L. B. (1936). Physiol. Rev. 16, 327. BIELSCHOWSKY, M., and GREEN, H. N. (1943). Lancet, ii, 153. CUSHNY, A. R., EDMUNDS, C. W., and CUNN, J. A. (1941). Therapeutics, Churchill, London. CUTHBERTSON, D. P. (1932). Quart. J. Med. 1 (new series), 387. CUTHBERTSON, D. P. (1942). Lancet, i, 433. Pharmacology and CUTHBERTSON, D. P., SHAW, G. B., and YOUNG, F. G. (1941). J. Endocrinology, 2, 475. DAVIDSON, J. N., and WAYMOUTH, G. (1943). Biochem. J. 37, 271. FREEMAN, H. (1940). Arch. Neurol. Psychiat. 43, 456. GAZA and GISSEL (1932). Cited by Arey (1936). GREEN, H. N. (1943). Lancet, ii, 147. JENEY, A. V., and VALYI NAGI, T. (1938). Arch. exp. Path. Pharmacol. 191, 423. KOSDOBA, A. S. (1934). Arch. klin. Chir. 179, 551. LAUBER, H. J. (1930). MIliunch. med. Wschr MURPHY, R. (1938). Amer. J. Physiol. 121, 107. SIMKINS (1937). J. Amer. med. Ass. 108, THOMSON, W. (1936). J. Hyg. Camb. 36, 24. YOUNG, J. S., FISHER, J. A., and YOUNG, M. (1941). J. Path. Bact. 52, 225.