J. Garcia Leme

Pharmacological analysis of the acute inflammatory process induced in the rat's paw by local injection of carrageenin and by heating

1 Local injection of carrageenin in the rats paw produced oedema and leakage of dye which had been administered previously by the intravenous route. A net dissociation between both parameters was observed: while oedema developed slowly, maximal intensity being attained after 4–5 h, dye‐leakage was maximum after 1 hour. 2 Anti‐inflammatory drugs such as acetylsalicylic acid and indomethacin were effective in reducing oedema and dye‐leakage when given before the injection of carrageenin, but much less effective when given 30 or 60 min after carrageenin. Hexadimethrine bromide was effective in reducing dye‐leakage also when given 1 h after the injection of carrageenin. 3 Combined administration of benadryl and methysergide, before the injection of carrageenin caused only a slight reduction in oedema and dye‐leakage. 4 When the paws were heated (55° C for 30 s) as a noxious stimulus the dissociation between maximal oedema and maximal dye‐leakage was not observed, both phenomena running parallel. Pre‐treatment of the animals with indomethacin did not afford any protection. 5 These results suggest that the inflammatory reaction to mild stimuli (carrageenin in our experiments) develops through different phases: initially the increased vascular permeability involves extravasation of plasma proteins and that phase is followed by an increased permeability mainly to water. Stronger stimuli (heating in our experiments) produce an overlapping of both phases, probably by inflicting severe damage to the vascular bed of the affected area. 6 The anti‐inflammatory drugs employed affected chiefly the initial phase of the response.

Vascular reactivity in diabetes mellitus: possible role of insulin on the endothelial cell

1 The response to vasoactive agents of microvessels of the rat was tested in vivo by direct microscopic observation of the exteriorized mesentery and assessment of cutaneous vascular permeability changes with Evans blue. 2 The constrictor response to a standard amount of noradrenaline in mesenteric microvessels was fully antagonized by acetylcholine in normal, diabetic, adrenalectomized and diabetic‐adrenalectomized rats. In contrast, the minimum doses of histamine or bradykinin, effective in normal or adrenalectomized animals, had to be increased about 20 fold to be active in diabetic or diabetic‐adrenalectomized animals. 3 Topical application of insulin to mesenteric microvessels of diabetic animals, in amounts not causing any increase in serum insulin levels, improved or restored the capacity of the animals to respond to histamine or bradykinin, acting as antagonists of the vasoconstrictor response to noradrenaline. Topical insulin, however, was ineffective in normal animals given 2‐deoxyglucose, the acute effects of which result from cellular glucopaenia unrelated to insulin deficiency. 4 Vascular permeability responses to intracutaneous histamine or bradykinin were decreased in animals pretreated with 2‐deoxyglucose as much as in diabetic animals. 5 Pretreatment of normal animals with indomethacin produced no effect on the responses of these animals to histamine or bradykinin, tested as antagonists of noradrenaline on mesenteric microvessels, or as vascular permeability‐increasing factors in the skin. 6 Pretreatment of normal animals with chloroquine, mepacrine or dexamethasone had no effect on the reactivity of mesenteric microvessels to histamine and bradykinin, acting as antagonists to noradrenaline. 7 It is suggested that vasoactive substances, endowed with permeability‐increasing properties, evoke relaxation of microvessels through an insulin‐dependent action on endothelial cells, unrelated to the release of arachidonic acid metabolites. This action would lead to increased vascular permeability, with opening of interendothelial junctions, and temporary changes in composition of extravascular fluid, which in turn, would provide the basis for vasodilatation. Diabetes mellitus apparently impairs such responses as a result of the accompanying cellular glucopaenia. Adrenal corticosteroids are not involved in the impaired responses.

Influence of diabetes on the reactivity of mesenteric microvessels to histamine, bradykinin and acetylcholine.

1 Noradrenaline (NA) evoked a vasoconstrictor response in rat mesenteric microvessels in situ, the latency and nature of which was analogous in normal and alloxan‐diabetic animals. 2 Histamine and bradykinin (Bk) were capable of antagonizing the response to NA in normal but not in diabetic animals. In contrast, acetylcholine (ACh) was equally effective as an antagonist to NA in both groups of animals. 3 The altered responses to histamine and Bk were not associated with hyperglycaemia since fasting rendered the diabetic animals normoglycaemic and yet did not restore the reactivity of microvessels. Previous administration of insulin to diabetic animals corrected the impaired responses to histamine and Bk. 4 A similar condition of impaired responses to histamine and Bk was produced in normal animals by the intravenous injection of 2‐deoxyglucose although ACh remained fully active. 5 Apparently, the functional changes observed in the response to histamine or Bk, as antagonists of the vasoconstrictor reaction to NA, were not associated with a defective response of all smooth muscle. First, because ACh remained active in diabetic animals, and, second, because extravascular smooth muscles obtained from either normal or diabetic rats were equally relaxed by histamine or Bk in vitro. 6 It is suggested that histamine and Bk antagonized the vasoconstrictor response of microvessels to NA through an action on lining endothelial cells resulting in increased vascular permeability and hyperosmolarity of extracellular fluids. 7 The process depended on the availability of insulin, and, therefore, might be affected by intracellular glucopaenia as occurring in diabetes. 8 Intracellular glucopaenia markedly affected other structures. Reduced atria rates were observed in diabetes, despite the fact that the isolated preparation responded normally to NA, ACh or tyramine. Partial substitution of glucose in the bathing fluid by 2‐deoxyglucose or addition of NaF to the organ bath evoked similar changes in atria from normal animals. 9 ACh which has little effect on vascular permeability must exert its vasodilator effects through mechanisms which are different from those influenced by the biochemical changes occurring in diabetes.

Formation of a factor increasing vascular permeability during electrical stimulation of the saphenous nerve in rats

1 Increased vascular permeability following electric antidromic stimulation of the rat saphenous nerve was observed in the skin area supplied by the nerve, confirming previous results by other authors. 2 The phenomenon was not affected by pretreatment of the rats with diphenhydramine, burimamide or their combination; atropine, methysergide, methysergide plus diphenhydramine, carboxypeptidase B, acetylsalicylic acid, indomethacin or methiazinic acid. It was partially reduced by previous injection of cellulose‐sulphate, a kininogen‐depleting agent. 3 Perfusates from the subcutaneous tissue of the paw area supplied by the saphenous nerve contained permeability increasing activity as shown by intradermal tests in other rats. This activity was present in perfusates collected during nerve stimulation but not in those collected before stimulation. It was not destroyed by heating to 100°C, or by α‐chymotrypsin or trypsin. 4 Bradykinin‐like activity may appear later in the perfusates, depending on the intensity of the stimuli. 5 It is concluded that following electrical antidromic stimulation of the saphenous nerve a permeability increasing factor is released, possibly from nerves. It is dialysable and can be distinguished from acetylcholine, histamine, 5‐hydroxytryptamine, plasma kinins, substance P, prostaglandins and high molecular weight proteins. The increased vascular permeability induced by this factor leads to plasma exudation and activation of the kinin system.

STIMULATION OF THE HYPOTHALAMO‐PITUITARY‐ADRENAL AXIS BY COMPOUNDS FORMED IN INFLAMED TISSUE

1 Rat paws were injected with carrageenin, and their subcutaneous tissue perfused 135 min later. These perfusates were injected intravenously into receptor rats in which they caused an attenuation of inflammatory responses. 2 The effect was not observed in adrenalectomized receptor rats nor in receptors with electrolytic lesions in the median eminence of the hypothalamus but persisted in adrenal‐demedullated animals. 3 The active perfusates also induced eosinopenia in normal or adrenal‐demedullated animals, but not in adrenalectomized rats, and produced an increase in blood corticosterone with a concomitant decrease in the amounts of adrenal ascorbic acid. 4 The active perfusates did not affect the responses of isolated preparations to histamine, bradykinin, prostaglandins and 5‐hydroxytryptamine neither did they elicit changes of the arterial blood pressure in receptor animals. 5 The anti‐inflammatory activity present in perfusates from inflamed paws seems to be formed slowly at the site of the developing inflammatory reaction, since perfusates collected 30‐65 min after the injection of carrageenin were ineffective, as was plasma taken from donor rats at various time intervals after carrageenin injections. 6 It is suggested that the anti‐inflammatory factor present in the active perfusates exerts its action by stimulation of the hypothalamo‐pituitary‐adrenal axis.

Endogenous corticosteroids and insulin in acute inflammation

Carrageenin-induced inflammatory responses in the hindpaws of rats were quantitated by measuring: (1) alterations in volumes of the paws; and (2) alterations in concentration of dye, previously injected intravenously, which was recovered in perfusates from the paws. The inflammatory response in one paw was attenuated by previously inducing an inflammatory response in the contralateral paw. The effect was abolished by pretreatment with insulin. Indexes of adrenal activity were increased after the induction of the inflammatory response and they were not attenuated by pretreatment with insulin. Adrenal hyperactivity was characterized by increased serum corticosterone concentration, decreased adrenal ascorbic acid content, and reduced number of circulating eosinophils. It is concluded that inflammatory stimuli which lead to alterations in microvessels depend on a facilitatory effect of insulin. This effect is antagonized by glucocorticoids released in enhanced concentrations after the application of noxious stimuli. Therefore, endogenous insulin and glucocorticoids act as modulators of inflammatory responses.

Effect of anti‐proteases and hexadimethrine bromide on the release of a bradykinin‐like substance during heating (46° C) of rat paws

1 The conditions in which the release of an active, bradykinin‐like agent occurred when rat paws were heated to 46° C were studied by means of the double coaxial perfusion of the subcutaneous spaces. 2 The active material thus released stimulated the isolated rat uterus, produced a relaxing effect on the isolated rat duodenum, was destroyed by incubation with chymotrypsin and was potentiated by bradykinin‐potentiating factor. LSD‐25, in doses sufficient completely to block 5‐hydroxytryptamine, did not affect the responses of the isolated uterus to the active material. 3 The effects on this release of anti‐proteases and hexadimethrine bromide, atropine and diphenhydramine were studied. 4 Soy‐bean trypsin inhibitor and hexadimethrine bromide added to the perfusion fluid produced a potent and reversible inhibition of the release of the active material; aprotinin and Kunitz inhibitor caused a temporary block. 5 When administered intravenously, much larger doses of the substances were necessary to produce a similar block. 6 Pretreatment of the animals with atropine plus diphenhydramine did not affect the release of the active kinin(s). 7 Ligature of one iliac artery was followed by disappearance of the active material in the perfusate from the corresponding paw. 8 These facts suggest that heating elicits a process leading to plasma extravasation and that the subcutaneous tissue is the chief site of release of the active material.

CORTICAL POTENTIALS EVOKED BY NOCICEPTIVE STIMULI: THEIR DEPRESSION BY A FACTOR RELEASED DURING SAPHENOUS NERVE STIMULATION IN THE RAT

1 The amplitudes of the first positive and the first negative waves of cortical potentials evoked by electrical stimulation of the dental pulp in the rat were decreased following electrical stimulation of the peripheral cut end of the saphenous nerve. 2 This effect was greatly diminished when the stimulation of the saphenous nerve was performed with the saphenous or femoral veins ligated. 3 During stimulation of the saphenous nerve of a donor rat, the subcutaneous tissue in the area supplied by the nerve was perfused; when the perfusate (in which a permeability‐in creasing factor was detected) was injected intravenously into a recipient animal, a decrease in the amplitude of the evoked cortical potential of the recipient rat was also observed. 4 Intravenous injections of 5‐hydroxytryptamine, histamine, bradykinin, prostaglandins or adenosine‐triphosphate produced no effect on the evoked cortical potential, whereas large doses of acetylsalicylic acid caused a decrease. 5 It is suggested that a humoral factor, released during sensory nerve stimulation, may help to modulate the processing of afferent inputs from pain receptors.

ANTI‐INFLAMMATORY ACTION OF GLUCAGON IN RATS

1 Subcutaneous administration of glucagon (1 and 0.5 mg/kg) 30 min before the injection of carrageenin or dextran into the rats paw reduced oedema and the local exudation of Evans blue previously given intravenously. 2 The effect persisted after removal of the adrenal medulla but not after adrenalectomy. 3 When glucagon (1 mg/kg, s.c.) was given daily after a local reaction to Freunds adjuvant injected into the paw had developed, a decrease in the reaction was observed up to 12 days. Blood sugar levels remained within the normal range. 4 Glucagon may exert an anti‐inflammatory effect through the release of adrenal corticosteroids and thus help modulate inflammatory reactions.

Experimental diabetes and inflammatory reactions in the rat.

The colloidal carbon technique used in rats to study vascular permeability changes shows two main pictures: (1) Predominant capillary accumulation of carbon, observed in pulmonary edemas produced by many different methods. (2) Predominant venular and arteriolar labeling, as seen in alpha-naphtylthioureainduced pulmonary edema. The ultrastructural alterations of the lung blood vessels in acute immunological pulmonary edemas of rats, mice and guinea-pigs are characterized by vacuolization, swelling and sloughing of the endothelium and epithelium, by occlusion of small vessels by platelet thrombi, by opening of the endothelial and epithelial junctions and by interstitial edema. Separation of the endothelium from the epithelium, forming channels, may be observed in the alveolar vessels, suggesting drainage of fluids through these channels from the alveolar level to larger interstitial spaces.