Acta pharmacol. et toxicol. 1962, 19, 101-106.

From the Department of Pharmacology, University of Copenhagen (Professor Knud 0. Merller, M.D.)

The Effects of Adrenalectomy and Administration of Desoxycorticosterone on Formation of Granulation Tissue

BY

Ole Jsrgensen (Received January 23, 1962)

It is generally accepted that the adrenocortical hormones of the gluco- corticoid type have an inhibitory effect on the process of healing. The roles of desoxycorticosterone (desoxycortone (WHO, NFN), deoxycortone (BP)) and the other mineralocorticoids in this process are less universally agreed upon.

In connection with the effect of the adrenal cortex on the process of healing it should be of interest to establish how healing proceeds after adrenalectomy, when the physiological secretion of adrenocortical hor- mones has been abolished.

The available results of investigations into this problem seem to be contradictory. The work recorded here has been carried out to throw light on the influence of adrenalectomy and administration of desoxycortone on the granulation tissue produced by the technique due to RUDAS (1960).

Methods Female rats weighing 80-100 g were used throughout.

The adrenalectomised animals were operated on 5 days before the experimental period. The operation was performed through an incision in the dorsal midline. The control animals were treated similarly, except that the glands were not removed. Post- operatively the controls and the adrenalectomised animals were given drinking-water containing 1 % NaCI, and all were kept at 27°C. Unless treated in this way, adrenalec- tomised animals die within a few days.

The desoxycortone treated animals were given daily injections of0.5 mg desoxycor- tone acetate dissolved in 0.6 ml of arachis oil. The injections were begun 5 days before the experimental period. The doses given were of pharmacological order.

The controls were given injections of arachis oil alone. After insertion of the plastic rings (vide infru), all the animals were kept in single

cages. Acta pharmacologica. vol. 19. fasc. 2. 7

102 OLE JQRGENSEN

Wet Weight s.e.m.

Formation of granulation tissue: The technique described by RUDAS (1960) was employed. With the rat under ether anaesthesia a circular piece of skin was excised medially and distally from its back. A plastic ring was inserted in the resulting gap. The diameter of the ring was larger than the gap, so that it remained fixed there. Further, the ring was provided with flanges to prevent its slipping. All the rings had exactly the same inner diameter (20 mm), so that quantitative analyses of the granula- tion tissue formed in the rings were possible. The animals were killed 8 days later, and the rings were removed. The granulation tissue formed was then easily removed by blunt dissection from the muscular fascia. The tissue was weighed on a torsion ba- lance immediately after excision and then dried.

Determination of water content: After weighing, the tissue was frozen at about -25" and dried in a desiccator over silica gel at a pressure of 1-3 mm Hg for 24 hours. This procedure was repeated until the tissue reached constant weight. The tissue was then defatted by shaking several times with a solvent in small perforated metal cases. First light petroleum was used twice for 45 minutes each time and then anhydrous ether for 45 minutes at a time, until constant weight was reached (usually four times).

Hexosamine determination: The hexosamine content was determined in dried de- fatted tissue by BLIX'S (1948) modification of ELSON & MORGAN'S method. The tissue was hydrolysed for 15 hours with 2 N-HCl in sealed glass ampoules at 100". The hexosamine content was expressed as milligram hexosamine per 100 g dried defatted tissue.

Collagen determination: In the granulation tissue the content of collagen was determined as hydroxyproline multiplied by the factor 7.46. The dried defatted tissue was hydrolysed with 6 N-HCl for 24 hours at 100" in sealed glass ampoules. The hydro- lysate was filtered, and the amount of hydroxyproline was measured by MARTIN & AXELROD'S (1955) modification of NEUMANN & LOGAN'S method.

Statistics: For statistical purposes we used the t-test.

Dry Weight mg s.e.m.

Results In table 1 are recorded the weights of wet and dry defatted granulation

tissue in the treated and the control groups. It is clear that there was no difference between the groups.

Table 2 shows the contents of hydroxyproline and collagen as well as the hexosamine concentration in the various animal groups. The hydroxy- proline content has been converted into collagen content by multiplying

Table 1. The weights of wet and dry defatted granulation tissue in the different groups.

S.e.m., standard error of the mean; n, number of animals.

ADRENOCORTICAL HORMONES A N D GRANULATION TISSUE 103

Table 2. The contents of hydroxyproline and collagen and the

hexosamine concentration i n granulation tissues. The values are expressed as mg, g, and mg per 100 g dry defatted tissue.

Hydroxy- ~ Collagen Hexosamine

Control animals . . . . . . ~ 14 1 1927 1 14.38 0.51 I 10 1 1069 29

proline nig

Desoxycortone treated . 1- 16-1-1766- ~!13.173.541-.9-11063pT3p Control animals . . . . . . 1 16 I 2179 I 16.26 0.43 1 8 I 1023 13

by 7.46. The collagen is expressed as grams collagen per 100 g dry defatted tissue. No changes in the parameters were found during the treatment given.

Discussion

All reported histological examinations appear to have shown that injec- tion of desoxycortone influences the process of wound healing. PIRANI, STREPTO & SUTHERLAND (1 95 1) administered the compound to guinea-pigs and found that in laparotomy wounds the hormone stimulated prolifera- tion of fibroblasts, and delayed maturation of the collagenous fibrils was seen at the same time. During the entire process of healing they observed an increased number of reticular fibrils and a reduced number of collage- nous fibrils.

TAUBENHAUS, TAYLOR & MORTON (1952) submitted to histological analysis granulation tissue produced by injecting turpentine and showed that desoxycortone counteracted the inhibitory influence of cortisone. Under the combined treatment with these two substances, abundant granulation tissue was formed but the tissue did not become normal, the fibroblasts lacking the regular orientation with cells lying parallel with the abscess cavity.

In previous studies TAUBENHAUS & AMROMIN (1949, 1950), using the same analytical technique, found that treatment with desoxycortone given during the development of the turpentine abscesses had little effect on granulation tissue, whereas pretreatment with the substances resulted in formation of many multipolar fibroblasts, but with few collagenous fibrils.

ROBERTSON & SANBORN (1958), on the other hand, by quantitative measurements of collagen in carrageenin-provoked granulomas in guinea- pigs, found that the amount of collagen was increased after treatment

'la

104 OLE J0RGENSEN

with desoxycortone. They state that the amount of granulation tissue seemed to be more abundant after treatment with desoxycortone, though they did not weigh the tissue formed. However, VAN CAUWENBERGE & LECOMTE (1957), after having inserted a cotton plug, observed no change in its weight during treatment of the animals with desoxycortone, com- pared with the conditions in controls.

In agreement with the latter result, our experiments gave no change in weight, either of the wet of the dry defatted tissue (table l), and the collagen content was also found to remain unaltered after desoxycortone treatment .

Histological studies by TAUBENHAUS & AMROMIN (1950) on the effect of adrenalectomy on granulation tissue in the walls of turpentine provoked abscesses revealed an inhibited fibroblast reaction, with small and scarce fibroblasts and slight collagen production. The collagen lay arranged partly in fibres and partly in masses.

In contrast with these results, ROBERTSON & SANBORN (1958) found a significant increase in collagen production by quantitative examination of carrageenin provoked granulomas in adrenalectomised guinea-pigs. PERNOKAS, EDWARDS & DUNPHY (1957) inserted ivalon sponges subcu- taneously into rats and measured the amount of tissue formed and also determined the hydroxyproline content. The values did not alter after adrenalectomy. The results of these investigations accord completely with those of our work.

Investigations into the hexosamine concentrations in granulation tissue during treatment with desoxycortone and after adrenalectomy have not been previously undertaken. PIRANI, STREPTO & SUTHERLAND (1 95 1) claim to have found an increased amount of intercellular substance in abdominal wounds under desoxycortone treatment, and they state that it showed increased staining. In our work neither adrenalectomy nor desoxycortone treatment caused alterations in hexosamine concentration (table 2). However, this by no means precludes the occurrence of changes in the ground substance, since hexosamine concentration is no reliable indicator of changes in the connective tissue ground substance during wound healing (vide SCHMIDT 1960 and JACKSON, FLICKINGER & DUNPHY 1960).

The force required to disrupt laparotomy wounds in rats seems not to have changed significantly after adrenalectomy (CHASSIN et al. 1954).

Considering the large doses of adrenocortical hormone required to inhibit the healing of wounds, cessation of the normal secretion by the adrenal glands seems unlikely to result in appreciably improved healing. A comparison of our results and those of PERNOKAS, EDWARDS & DUNPHY with those of ROBERTSON & SANBORN makes it surprising that they found

ADRENOCORTICAL HORMONES A N D GRANULATION TISSUE 105

increased collagen production after adrenalectomy. It may, perhaps, be explained by the fact that the latter workers had difficulty in keeping the animals alive without desoxycortone. Administration of this hormone stimulated the collagen production in their experiments when given to non-adrenalectomised animals.

With stresses of different kinds, such as artificially provoked fractures, excision of large pieces of skin and infliction of burns, the tensile strength of 5 day old laparoatomy wounds was impaired, while at the same time the weight of the adrenal glands was significantly increased. This fact argues in favour of the view that the adrenocortical secretion may sometimes be a factor of importance in the healing process (CHASSIN et al. 1953). It is not certain, however, that the increased weight of the adrenal glands is the direct cause of the retarded healing. The possibility must be considered that the injury causing the weight increase noted directly influenced the general state of the animals to such an extent that this alone interfered with healing. The hypothesis of an inhibitory effect of a former stress on the process of healing is also in disagreement with the results of previous investigations by TAFFEL, DONOVAN & LAPINSKY (1951). These workers showed that a previous soft tissue lesion did not interfere with the healing of subsequent wounds in the rat stomach.

The histological findings reported after adrenalectomy and administra- tion of desoxycortone seem to be inconsistent with the biochemical demonstrations of an increased or unchanged content of hydroxyproline in granulation tissue during these treatments. We cannot deny, however, that fibroblasts displaying an abnormal morphological picture can never- theless take a normal part in collagen production. Histological changes in the collagen, manifesting themselves by reduced fibril formation, need not be inconsistent with the results of biochemical determination of the collagen content of granulation tissue, measured as hydroxyproline. A possible inhibition of the extracellular maturation of collagen may result in a reduced number of collagenous fibrils without the hydroxyproline content being diminished, as the immature forms of the collagen contain hydroxyproline in the same amounts as the final protein. As, however, wound strength after adrenalectomy is not impaired, inhibited fibril for- mation is not expected in this condition.

To conclude, we would say that, under the experimental conditions described, neither adrenalectomy nor injection of desoxycortone seems to have influenced granulation tissue formation in general or, more particularly, collagen production.

106 OLE J0RGENSEN

Summary. The effects of adrenalectomy and administration of desoxycortone on

the formation of granulation tissue have been studied. No signs have been found to indicate that these procedures influence granulation tissue forma- tion, when assessed neither on the basis of the total amount of tissue formed or by the contents of collagen and hexosamine.

Acknowledgement . This investigation was carried out in >)The Laboratory for Investiga-

tion of the Pharmacology of the Connective Tissue(( with a grant from the Danish National Association against Rheumatic Diseases.

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