Samuel C. Harvey

Effect of Absolute and Partial Vitamin C Deficiency on Healing of Wounds.

It is now well established that the intercellular substances in general, and the collagen of all fibrous tissue structures in particular require ascorbic acid for their production and maintenance. Aschoff and Koch 1 made thorough postmortem studies of World War soldiers who had died of scurvy, and demonstrated constant pathological changes in the supporting tissues of the body. Höjer 2 found a general atrophy of the connective tissue in scorbutic guinea pigs, and pointed out for the first time a general deficiency in the formation of collagen. In a series of more recent communications Wolbach 3 4 5 and his collaborators confirmed the observations of Höjer, and conclusively showed that ascorbic acid is intimately concerned with the synthesis and maintenance of intercellular supporting materials. Jeney and Törö, 6 when they added ascorbic acid to the nutrient medium of a culture of fibroblasts in vitro, found a marked increase in the number of collagen fibrils that were produced. Since the repair and tensile strength of soft tissue wounds are direct functions of fibroblastic proliferation and collagen formation, it is at once apparent that Vitamin C may play a prominent rôle in the phenomenon of wound healing. Saitta 7 reported a delayed healing of exposed surface wounds in guinea pigs that had been kept on a Vitamin C deficient diet for more than 15 days. On the other hand, when a Vitamin C containing extract was applied directly to the wound, the healing time was appreciably diminished, regardless of whether the animals were maintained on a normal or a scorbutic dietary regime. He determined the rate of healing by actual daily measurements of the length and breadth of the wound.

An experimental study of the origin of the meninges.

As a result of experimental work concerning the healing of the meninges following injury, it was found by one of us (S. C. H), 1 that the dura in the presence of an intact lepto meninx heals without adhesion to the underlying membrane. This is brought about, not by the ingrowth of mesothelium from the surrounding normal dura, but by the direct transformation of mesenchymal cells into mesothelium which comes to lie against the uninjured and impervious lepto-meninx. On the other hand, 2 when the lepto-meninx is injured with the overlying dura remaining undamaged, an analogous healing without adhesions does not take place. The lining mesothelium of the dura disappears and this membrane enters intimately into the reparative process in the subjacent lepto-meninx, the final result being dense adhesions. This seems to indicate some fundamental histogenetic difference between these two structures. Harrison 3 has shown by experimental methods that the nerve sheath cells are derived from the neural crest. Inasmuch as these cells are analogous to the lepto-meninx in their relationship to the nerve structures which they surround, it seemed possible that this membrane was likewise derived from the neural crest while the dura probably developed from the mesenchyme. An examination of pig and chick embryos at and previous to the time of the outgrowth of the nerve root fibers from the neural tube, confirmed this belief. It seemed possible to verify this supposition by experimentation. This work was carried out by the transplantation of sections of the neural tube of the amblystoma with and without neural crest cells. If the lepto-meninx develops from neural crest cells, then transplanting a portion of the neural tube free from such cells should lead to the growth of neural tissues surrounded only by dura, while such transplantation carrying with it nerve crest cells should lead to the formation of both the dura and the lepto-meninx about the neural transplant. Such experiments were carried out in the Spring of 1921, with the following results.

Effect of Sulfanilamide on Wound Healing.

Bricker and Graham 1 reported that sulfanilamide had an inhibitory effect upon the healing of stomach wounds in dogs. Their determinations were made on the 3rd, 5th and 7th postoperative days. In view of the widespread and growing use of sulfanilamide and its related compounds, it seemed important to repeat these studies and to carry the determinations through all the normal phases of healing. The experiments were performed on adult white rats weighing about 250 g. The tensile strength of the wound was used as an index of healing. In addition comparative histological studies were made of the wounds. I. Control Group—51 animals. This group was maintained throughout the duration of the experiment on a diet of Purina dog chow. On the 3rd day of the diet a longitudinal wound measuring about one cm in length was made under ether anesthesia through the anterior wall of the stomach at its cardiac end. The wound edges were immediately re-approximated in one layer with a running continuous Connell suture of No. 000 plain catgut which, as has been previously shown, loses its tensile strength well within the 4th day. The abdominal wall was closed with 2 layers of fine No. A silk. On each of the 4th, 6th, 8th, 10th, 12th and 14th postoperative days 5 to 8 animals were sacrificed, and the strength of the wounds immediately determined by distending the stomach with air and noting its bursting point. The details of this method were described by Harvey and Howes. 2 Inasmuch as it has been shown that for approximately 4 days after the injury the wound has only the strength contributed by the holding power of the sutures, no studies were made during this first phase of healing.

Effect of Local Application of Sulfanilamide upon Wound Healing.

The efficacy of the sulfonamides in the systemic treatment of certain types of infection has been definitely established. During the past few years several communications 1 , 2 , 3 , 4 , 5 , 6 have described and recommended the application of these drugs directly into raw wounds as a prophylactic or therapeutic measure against infection. Most of these reports based their contentions upon qualitative impressions gathered from the observation of a small series of patients rather than upon controlled studies. Before indiscriminately depositing these drugs into a raw wound it is essential to determine first of all whether they have any noxious effect upon the tissues involved in the healing of the wound. Bricker and Graham 7 reported that the systemic administration of sulfanilamide had an inhibitory effect upon the healing of stomach wounds in dogs during the first 7 postoperative days. Taffel and Harvey 8 found that sulfanilamide did not affect the healing of stomach wounds in rats during all of the normal phases of healing. The drug was given orally in doses sufficient to maintain an adequate and sustained “therapeutic” blood level. This series of experiments was carried out to determine the effect of the local application of sulfanilamide upon the healing of a soft tissue wound. Adult white rats weighing about 250 g were used. The tensile strength of the wound served as an index of healing. In addition, histological studies were made of the wounds. I. Experimental Group. 57 animals. This group was maintained throughout the duration of the experiment on a diet of Purina Dog Chow. On the 3rd day of the diet the stomach was delivered through an incision in the anterior abdominal wall and 0.2 cc of a 3% suspension of sulfanilamide in normal saline were injected through a fine needle into the wall of the cardia.

Development of the Meninges in the Chick.

Harvey and Burr 1 , 2 have presented evidence that in the salamander, the neural crest was a necessary requisite for the formation of the leptomeninx. We have continued the study by means of chorioallantoic grafts in the chick. Two series of operations were performed by Dr. Van Campenhout. In the first, the prosencephalon of 50-hour chicks was transplanted adjacent to an allantoic vessel of a 9-day host and allowed to develop from 7 to 9 days. In the second, an entire segment of the thorax or abdomen from a 50- to 98-hour chick was transplanted on the chorio-allantoic membrane of 9-day hosts. These also were permitted to develop for a period of approximately 9 days. Our knowledge of the distribution of the neural crest cells indicates that at the time of operation no such cells have as yet reached the region of the forebrain. It could then be transplanted devoid of neural crest investiture. On the other hand, during the period immediately following the 50-hour incubation period, the neural crest has begun its migration about the spinal cord in the thoracic and abdominal segments. Thus we have the nervous system transplanted without neural crest in the first series and with neural crest in the second. Since all the other conditions were as nearly constant as possible any difference in the structure of the meninges could be attributed to the presence or absence of the neural crest. The material from the above experimental procedure was prepared for microscopic study using chiefly the Masson stain. A critical examination shows that there is a marked difference in the membranous investment of the nervous system in the 2 series. In series one, without neural crest, the nervous system is surrounded by a series of fibrous laminae of obvious mesenchymal origin.

A physiological study of the development of the collateral circulation in the leg of the dog.

It has been known for many years that on ligating the main vessel in the leg of an animal, the temperature in that limb will fall, and that at some later time it may return to normal with the development of a collateral circulation. In reviewing the literature, however, we have been unable to discover any work which would show just how soon such a response takes place, and in just what manner. Consequently, we undertook the following experiments. As a means of determining the flow of the blood in the limb, and the efficiency of the circulation, continuous record was kept of the temperature of the foot after tying off the superficial femoral artery on the one side, using the opposite limb as a control. This was done by means of two thermo-couple junctions soldered within needles and pushed into the foot pads of the two hind legs, according to the method of Barney Brooks. 1 For these junctions, German silver and copper wires were used. The copper wire from each junction was connected with one pole of a two-way switch. The German silver wire from each needle was joined to another piece of copper wire to form the “cold” junction, and the two were passed through a glass tube in the cork of a thermos bottle. From this cold junction in each case connection was made by the copper wire to the corresponding pole of the switch. By throwing the switch to the right or left, the therrno-electric current from one or the other needle could be sent into a galvanometer whose deflections were first calibrated according to known variations of temperature in order to transform the results into temperature readings.

Effect of Sulphur-Amino Acid Deficiency on Wound Healing.∗

From the results of his numerous plant and animal experiments, Hammett 1 2 3 arrived at the conclusion that the sulphydryl radicle, naturally present in all cells, was the essential and universal stimulus to growth by increase in the number of cells. This hypothesis did not long remain unchallenged. Others, 4 5 after carrying out similar investigations, have either not been able to reproduce Hammetts results, or have preferred to lend to them another interpretation. Reimann 6 7 8 translated Hammetts work into the clinic and reported instances of chronic indolent ulcers which healed with dramatic swiftness following the direct application of sulphydryl compounds. He described a rapid proliferation of both the fibroblastic and epithelial elements of the wound. In recent years Rose 9 10 has demonstrated methionine and not cystine to be the indispensable sulphur-containing amino-acid. Cystine stimulated growth only in the presence of methionine. In the absence of the latter from the ration, however, the animals rapidly lost weight and died, even though an abundant amount of cystine had been supplied. The purpose of these experiments was to determine whether a deprivation of cystine and methionine in a degree sufficient to abruptly arrest growth, had any effect on the healing of soft tissue wounds. The tensile strength of the wound was used as an index of healing. Young growing rats weighing between 90 and 100 g were divided into 2 groups: (1) normal control, (2) sulphur-ammo-acid deficient. The experimental diet used was that described by Dyer and du Vigneaud, 11 and consisted of: This is a full diet, adequate in all the essential constituents except the sulphur-bearing amino-acids. It contains cystine and methionine sufficient only for the maintenance of the animal but not for normal growth and increase in weight.

STUDIES ON THE COURSE OF VASOMOTOR FIBERS AS MEASURED BY THERMIC CHANGES IN THE FEET AFTER ARTERIAL LIGATION AND SECTION OF THE SPINAL CORD AT VARIOUS LEVELS

In a previous paper (7) it was shown that in dogs under amytal anesthesia, ligation of both femoral arteries proximal to the profunda branch caused a decrease in the temperature of the feet which was interpreted as indicating a diminution of blood flow. The temperature of the feet remained at this lower level for several hours following which there was a spontaneous rapid rise indicating the development of collateral circulation. The rapidity of this response was taken as evidence suggesting that the reestablishment of former conditions by collateral circulation is a vasomotor phenomenon. It was found furthermore that decrease in the temperature of the feet following ligation of the femoral artery as above, could be prevented by removal of the tributary sympathetic ganglia, or if the temperature had already decreased to the level of that of the room, removal of the sympathetic ganglia immediately induced a rise in the feet to the previous level. This suggested that the rapid spontaneous return of the circulation following ligation of the femoral artery was a vasomotor phenomenon controlled by means of the sympathetic nervous system. In another paper (3) experiments were reported in which the same method was used. Following transection of the spinal cord at the level of the first lumbar vertebra, no increase in the temperature of the limb was observed. Gaskell (2), Bayliss and Bradford (1) and Langley (4, 5, 6) have stated that in an animal with seven lumbar vertebrae the connector fibers to the sympathetic ganglia supplying the posterior extremity are contained in the 11th, 12th and 13th thoracic and the 1st, 2nd and 3rd lumbar spinal roots. Since the results of the above (3) experiments did not coincide with their findings it seemed desirable to conduct further experiments to determine the effect of transection of the spinal cord at various levels.