Ocular responses to superoxide generated by intraocular injection of xanthine oxidase.

Xanthine oxidase (XaO) was injected into the anterior chamber of rabbit eyes by a closed circuit perfusion system. Doses of 1.5 milliunits (mU) or greater produced a maximal leucocyte accumulation after 4 hr, with an initial elevation of ocular pressure in the first 15 min. Similar experiments on rats with intravitreal injections of 0.1-1.5 mU of XaO resulted in a significant accumulation of leucocytes after 5 hr which, at the highest dose of XaO, was partly due to traces of bacterial endotoxin in the XaO. However, in endotoxin-desensitized rats the response to 1.5 milliunits XaO was seven-fold greater than the response to endotoxin alone. Simultaneous administration of xanthine (Xa) substrate with XaO was not required to elicit cell infiltration into the anterior chamber. Dialyzed enzyme was also effective but boiling abolished the response. Addition of XaO to rabbit aqueous humor in vitro decreased the ascorbate content, consistent with the generation of superoxide from an endogenous substrate. The results suggest that enzymatically active XaO, which can cause intraocular generation of superoxide from an XaO substrate present in aqueous humor, initiates the chemotactic response. A chemotactic agent may be generated from superoxide reacting with endogenous precursors in aqueous humor or by selective activation of the lipoxygenase pathway of arachidonic acid metabolism in adjacent tissues.

Ascorbic acid inhibits the activity of polymorphonuclear leukocytes in inflamed ocular tissues.

Myeloperoxidase (MPO) is present at high levels in polymorphonuclear leukocytes (PMNs) and has been used as a marker to quantify the accumulation of PMNs in inflamed tissues. MPO activity in inflamed ocular tissues was inhibited by aspirates of aqueous humor. This inhibition could be duplicated by the addition of ascorbic acid at concentrations equivalent to those present in the aliquots of aqueous humor. Similarly, aqueous humor and ascorbic acid inhibited MPO from isolated rabbit leukocytes. Therefore, ascorbic acid appears to inhibit the functional activity of the peroxidase in PMNs, thus preventing potential tissue damage by this enzyme when released during leukocyte degranulation in inflammation. Ascorbic acid might fulfill a role as an endogenous anti-inflammatory agent in the eye.

Different humidification systems for high-frequency jet ventilation.

This study examined the effect of several different high-frequency jet ventilation (HFJV) humidification techniques on tracheobronchial mucosa. Six groups (2 in each group) of mongrel dogs, chronically instrumented, were cared for in an intensive care-like setting for data acquisition. Four groups received HFJV with different humidification systems during 72 h of continuous ventilation. Two groups served as controls (conventional ventilation with and without humidity). Although there was significant damage to tracheal mucosa during ventilation without humidification, there were no significant pathologic differences in bronchial mucosa between humidified and nonhumidified groups. Several techniques of HFJV humidification produced no pathologic evidence of mucosal damage and could be clinically useful.

A comparison of the ocular anti-inflammatory activity of steroidal and nonsteroidal compounds in the rat.

The anti-inflammatory activities of steroidal and nonsteroidal compounds have been evaluated in the rat model of ocular inflammation induced by subcutaneous injection of lipopolysaccharides. Dexamethasone sodium phosphate, BW755C, flurbiprofen, indomethacin, and benoxaprofen were administered orally or topically for 24 or 48 hrs. Oral administration of dexamethasone, BW755C, and flurbiprofen inhibited iris-vasodilatation and leukocyte accumulation in the anterior chamber in a dose-dependent manner. Indomethacin and benoxaprofen were active only at high doses. Topical administration of these compounds inhibited the inflammatory responses in a similar manner. The inhibitory effect on leukocyte accumulation by these compounds was greater than their effect on vasodilatation. BW755C, a phenyl pyrazoline derivative, which is an inhibitor of both the cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism was the most active nonsteroidal compound and had an anti-inflammatory profile similar to dexamethasone. The results of this study also indicate that the model of rat ocular inflammation induced by subcutaneous injection of endotoxin can be used satisfactorily for comparative evaluation of anti-inflammatory agents.

Biosynthesis of lipoxygenase products by ocular tissues.

The metabolism of arachidonic acid via the lipoxygenase pathway has been investigated in conjunctival and iris tissue taken from eyes of various species. In addition we have also studied two inhibitors of arachidonate metabolism, BW 755 and indomethacin, on albino rabbit ocular tissues. The ocular tissues of most species (monkey, dog, cat, rabbit, guinea-pig and rat) formed lipoxygenase products from exogenous arachidonic acid. The exception was the albino rabbit iris, where no lipoxygenase product was detected. The major lipoxygenase product found was 12-HETE, although 5-HETE and 5,12-diHETE were formed to a lesser extent by the conjunctiva and iris of the Dutch rabbit. The rat ocular tissues and guinea pig conjunctiva also formed 5-HETE. In the conjunctiva of the albino rabbit, indomethacin was a relatively specific inhibitor of the cyclo-oxygenase pathway whereas BW 755 inhibited both the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism. In view of the possible roles of lipoxygenase products in inflammatory reactions and the ability of ocular tissues to synthesize these products, dual inhibitors of cyclo-oxygenase and lipoxygenase pathways may be useful agents to control ocular inflammatory responses.

Quantification of ocular inflammation: evaluation of polymorphonuclear leucocyte infiltration by measuring myeloperoxidase activity.

Myeloperoxidase (MPO) activity was measured in rabbit cornea and iris-ciliary body to quantitate the infiltration and accumulation of polymorphonuclear leucocytes (PMN's) following an inflammatory stimulus. Following injection of clove oil into the cornea, MPO activity could be detected in the cornea at 6 hr, reaching a maximum at 12 hr, and falling to non-detectable levels at 72 hr. MPO activity was only detected in the iris-ciliary body 24 hr after intracorneal clove oil injection. MPO activity in the iris-ciliary body increased in a dose-dependent manner following intravitreal injection of endotoxin. No MPO activity could be detected in cornea. Topical administration of dexamethasone inhibited MPO activity in cornea and iris-ciliary body 24 hr after intracorneal clove oil and intravitreal endotoxin injection, respectively. Measurement of MPO activity in ocular tissues could provide a useful tool to quantitatively evaluate the severity and time course of inflammation.

Chemotactic response to some arachidonic acid lipoxygenase products in the rabbit eye.

The effects of arachidonic acid, its cyclo-oxygenase and lipoxygenase products and the synthetic chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (FMLP) on leukocyte accumulation in the aqueous humour and intraocular pressure in the rabbit were studied in vivo. Substances were injected into the anterior chamber of the eyes of anaesthetised rabbits using a closed circuit perfusion system. Injection of arachidonic acid, prostaglandins E1 and E2, and the monohydroperoxy and hydroxy acids of the lipoxygenase pathway did not result in any significant accumulation of leukocytes in the anterior chamber. In contrast, FMLP and 5,12-diHETE (Leukotrine B4) resulted in significant dose dependent accumulation of leukocytes into the aqueous humour. Leukocytes appeared in the aqueous humour between 2 and 3 h after the injection of either FMLP or LTB4 and the response was maximal at 4 h. None of the lipoxygenase products tested had any effect on intraocular pressure in contrast to the profound effects observed with arachidonic acid and the E type prostaglandins. FMLP had a small but significant effect on intraocular pressure at the highest dose tested for leukocyte accumulation. These results indicate that the effects of the cyclo-oxygenase products of arachidonate metabolism are mainly vascular in the rabbit eye in contrast to the predominantly cellular effects of lipoxygenase products. Thus in the eye, the interaction of cyclo-oxygenase and lipoxygenase products of arachidonate metabolism may be important in the development of both acute and chronic ocular inflammation.

Anti-inflammatory effects of betamethasone phosphate, dexamethasone phosphate and indomethacin on rabbit ocular inflammation induced by bovine serum albumin.

Following the induction of immunogenic uveitis betamethasone and dexamethasone reduced conjunctiva and iris hyperemia and aqueous flare. Indomethacin reduced iris hyperemia, but potentiated conjunctival hyperemia. All three agents inhibited the increase in the protein content of the aqueous that follows induction of immunogenic uveitis. Betamethasone was slightly more potent than dexamethasone and indomethacin. White cell entry into the aqueous was inhibited by betamethasone and dexamethasone. Paradoxically, however, indomethacin significantly potentiated the number of polymorphonuclear leukocytes (PMNs) entering the aqueous. The anti-inflammatory effects of steroidal agents may be due to the inhibition of the release of arachidonic acid (AA) which is a precursor of both lipoxygenase and cyclooxygenase products. The potentiation of the PMN response by indomethacin in immunogenic uveitis may be due to an inhibition of cyclooxygenase product formation and a facilitation of lipoxygenase products (which are potent chemotactic agents) from AA.

Inhibition of arachidonic acid cyclo-oxygenase and lipoxygenase activities of leukocytes by indomethacin and compound BW755C.

Arachidonic acid is metabolized via two pathways in leukocytes: cyclo-oxygenase, leading to the stable prostaglandins, and lipoxygenase, leading to hydroxyacids. Indomethacin inhibits the cyclo-oxygenase selectively, whereas compound BW755C (3-amino-1-(m-(trifluoromethyl)phenyl)-2-pyrazoline) inhibits both pathways equally. This offers a possible explanation for the differing activities of these two compounds in inflammatory models in vivo. The patterns of product inhibition by the two compounds support the suggestion that 11-HETE (hydroxy-eicosate-traenoic acid) and 15-HETE can arise by incomplete operation of the cyclo-oxygenase pathway.

Chemokinetic activity of arachidonic and lipoxygenase products on leuocyctes of different species.

A number of hydroperoxy (HPETE) and hydroxy (HETE) products of the lipoxygenase pathway of arachidonic acid metabolism are chemotactic and chemokinetic for human neutrophils. We have investigated the relative chemokinetic potency of some of these products on human, rat and rabbit neutrophils. The most potent lipoxygenase product studied was 5,12-dihydroxy-6,8,10-14-eicosatetraenoic acid (5,12-diHETE), which was maximally chemokinetic and chemotactic between 0.1 and 1.0ng/ml for the three species. The 5, 11 and 12-HPETEs and HETEs were chemokinetic, but less active by at least two orders of magnitude, for human and rabbit neutrophils at concentrations between 0.1 and 10micrograms/ml. 15-HPETE and 15-HETE were inactive on human leucoctes, and none of the monosubstituted products studied were chemokinetic for rat neutrophils. These results indicate that 5,12-diHETE may be an important mediator in the local accumulation of leucocytes in the inflammatory response.

Effects of prostaglandins and thromboxane A2 on the coronary circulation of adult dogs and puppies.

Intracoronary injections of prostaglandin endoperoxide (PGH2), prostacyclin (PGI2) and thromboxane A2 (TXA2) were given to adult dogs and puppies three months or younger. Both PGH2 and PGI2 caused dose-related coronary vasodilation in the adult as well as in the puppy dogs; and PGH2 was about twice as potent as PGI2. The threshold dose of PGH2 for coronary vasodilation, 0.01-0.02 microgram, was the same for the adult and puppy dogs although control coronary blood flow of the adult dog was 3-10 times higher. In the pupply, maximal coronary vasodilation was effected with high doses (1.0 microgram or more) of PGI2, but not with PGH2. TXA2 prepared from PGH2 up to 1.0 microgram, had no effects on the coronary circulation of the adult dog. In contrast, both the vasocon-stricting and platelet aggregating actions of TXA2 were demonstrated in the puppy. Mechanisms for the observed age-dependent differences of the canine coronary circulation to PGH2 quantitatively, and to TXA2 qualitatively, remain to be determined.

The effects of non-steroid anti-inflammatory drugs on leukocyte migration in carrageenin-induced inflammation.

Some non-steroid anti-inflammatory drugs which inhibit arachidonate cyclo-oxygenease have been examined for their effects on leukocyte migration, prostaglandin production and oedema formation in carrageenin-induced inflammation in the rat. At doses which inhibited oedema, all the drugs tested caused a dose-dependent reduction in numbers of leukocytes and prostaglandin concentrations in 24-h inflammatory exudates. At lower doses, indomethacin, aspirin, sodium salicylate, flurbiprofen and phenylbutazone significantly potentiated leukocyte migration by 20-70%. Ibuprofen, naproxen and BW755C reversed the indomethacin-induced increase in leukocyte accumulation. BW755C inhibits the generation of chemotactic lipoxygenase products and it is possible that the effects of all these drugs on leukocyte migration are mediated through the lipoxygenase pathway of arachidonic acid metabolism.

Inhibition of thromboxane A2 biosynthesis in human platelets by burimamide.

1 Burimamide selectively inhibited the formation of thromboxane A2 from prostaglandin endoperoxides by human platelet microsomes in a dose-dependent manner (IC50 = 2.5 x 10(-5) M). Burimamide was found to be equipotent to imidazole as a thromboxane synthetase inhibitor. 2 Metiamide, cimetidine and a series of compounds either bearing a structural or pharmacological relationship to histamine caused little or no inhibition of thromboxane A2 biosynthesis by human platelet microsomes. 3 Burimamide (5 x 10(-4) to 2.3 x 10(-3) M) did not inhibit either the cyclo-oxygenase or the prostacyclin synthetase of sheep seminal vesicles or the prostacyclin synthetase of dog aortic microsomes. 4 Burimamide (2.5 x 10(-5) to 1.2 x 10(-4) M) inhibited sodium arachidonate-induced human platelet aggregation; the degree of inhibition was dependent upon the concentration of arachidonic acid used to aggregate the platelets.