New animal models of inflammatory disease workshop.
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Animals↗
Biomedical subjects
D E Griswold
Publications and source records attributed to D E Griswold.
Time-related changes in myeloperoxidase activity and leukotriene B4 receptor binding reflect leukocyte influx in cerebral focal stroke.
In previous studies, we have used histological methods to characterize cellular changes, and validated the use of the myeloperoxidase (MPO) activity assay to quantitate increased neutrophil infiltration in ischemic stroke. We also identified increased leukotriene B4 (LTB4) binding sites as a potential marker for neutrophil infiltration into focal ischemic tissue. However, these studies were conducted at only one time-point, 24 h after ischemia. In the present study, we examined the full time-course of MPO activity and LTB4 receptor binding following middle cerebral artery occlusion (MCAO) made permanently (PMCAO) or transiently (160 min followed by reperfusion; TMCAO) in spontaneously hypertensive rats, and compared the results to previously characterized histologic changes in these models. Ischemic and contralateral (control) cortical tissue samples were assayed for MPO (U/g wet wt) and [3H]LTB4 receptor binding (fmol/mg protein). Following PMCAO, MPO activity significantly increased as early as 12 h and continued to increase over the next 5 d (p < 0.05). Following TMCAO, MPO activity was significantly elevated already after only 6 h of reperfusion and also continued to increase over the next 5 d of reperfusion (p < 0.05). LTB4 receptor binding and MPO activity were highly correlated during periods when both measures increased together (i.e., 0.5-5 d; p <0.01). However, by 15 d post-MCAO, LTB4 receptor binding remained elevated after MPO activity levels had returned to normal. This persistent LTB4 binding was associated with the significant gliosis that was characterized previously to persist in these models. The time-course of increased MPO activity and initially increased LTB4 binding post-MCAO correspond very well to our previous histological data that characterized the time-course for leukocyte infiltration under these conditions. Therefore, the increased MPO activity over time was associated with initial neutrophil and later mononuclear cell infiltration into ischemic tissue in these models. In addition, the present studies utilized histochemical analysis to demonstrate peroxidase activity in macrophages within the cerebral infarct following MCAO, thus validating that MPO activity originates from the later infiltrating mononuclear cells in addition to the early infiltrating neutrophils that had been previously characterized in the same manner. TMCAO produces a significantly larger and earlier increase in ischemic cortex MPO activity and a similar later increase in MPO activity compared to PMCAO treatment.(ABSTRACT TRUNCATED AT 400 WORDS)
Analysis of Variance↗
SB 209247, a high affinity LTB4 receptor antagonist demonstrating potent antiinflammatory activity.
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12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
Pharmacological characterization of SB 202235, a potent and selective 5-lipoxygenase inhibitor: effects in models of allergic asthma.
The peptidoleukotrienes and leukotriene B4, formed from arachidonic acid through the action of 5-lipoxygenase (5-LO), exert a spectrum of biological effects. It has been proposed that potent and selective 5-LO inhibitors will be effective therapy in diseases in which the peptidoleukotrienes and leukotriene B4 have been implicated, such as asthma and arthritis. The novel compound (S)-N-hydroxy-N-(2,3-dihydro-6-phenylmethoxy-3-benzyofuranyl )urea (SB 202235) was evaluated as a selective inhibitor of 5-LO in a cell-free system as well as in various cellular assays. In addition, the potential therapeutic value of SB 202235 was assessed in preclinical models of allergic asthma. The activity of the 5-LO enzyme isolated from rat basophilic leukemia-1 cells was inhibited by SB 202235 in a concentration-dependent manner with an IC50 value of 1.9 microM. Consistent with its ability to inhibit 5-LO, SB 202235 inhibited the production of leukotriene B4 by human monocytes and in human whole blood (IC50 values of 1.5 microM and 1.1 microM, respectively). The selectivity of SB 202235 was confirmed by its lack of effect against several other enzymes and receptors. SB 202235 potently and effectively inhibited the contraction produced by a single concentration of ovalbumin in guinea pig trachea (IC50 = 20 microM) and of anti-IgE in human bronchus (IC50 = 2 microM). SB 202235 (3-30 microM) also inhibited the contraction of guinea pig trachea in response to increasing concentration of ovalbumin. When administered orally (30 mg/kg) to conscious guinea pigs, SB 202235 attenuated antigen-induced broncho-constriction and the subsequent eosinophil influx.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
(E)-3-[[[[6-(2-carboxyethenyl)-5-[[8-(4- methoxyphenyl)octyl]oxy]-2-pyridinyl]-methyl]thio]methyl]benzoic acid and related compounds: high affinity leukotriene B4 receptor antagonists.
(E)-3-[[[[6-(2-Carboxyethenyl)-5-[[8-(4- methoxyphenyl)octyl]oxy]-2-pyridinyl]methyl]thio]methyl]benzoic acid (11, SB 201993) is a novel, potent LTB4 receptor antagonist. Compound 11 arose from a structure-activity study of a series of trisubstituted pyridines that demonstrated LTB4 receptor antagonist activity. The placement of an additional methylene unit in the sulfur containing chain linking the pyridine and benzoic acid moieties of lead compound 8 (K(i) = 80 nM) resulted in a greater than 10-fold increase in receptor affinity. Additionally, in this new series of compounds, the oxidation state of the sulfur was found to be critical to the activity, i.e., the sulfoxide and sulfone showed substantially lower affinity for the LTB4 receptor. Compound 11 competitively inhibits the binding of [3H]LTB4 to LTB4 receptors on human polymorphonuclear leukocytes with a Ki of 7.1 nM and blocks both the LTB4-induced calcium mobilization and the LTB4-induced degranulation responses in these cells with IC50 values of 131 and 271 nM, respectively. Compound 11 demonstrated oral LTB4 antagonist activity as well as topical antiinflammatory activity in the mouse.
Benzoates↗
The soluble form of E-selectin is an asymmetric monomer. Expression, purification, and characterization of the recombinant protein.
The gene coding for a soluble form of human E-selectin (sE-selectin) has been expressed in Chinese hamster ovary (CHO) cells. Cells seeded into a hollow fiber reactor secreted protein at a level of 160 mg/liter. The protein was purified to > 95% pure and low endotoxin (< 2 ng/mg), using physiological pH and buffers. The amino acid composition and N-terminal sequence were as predicted from the cDNA sequence. HL-60 cells bound to sE-selectin-coated plates in a dose-dependent manner, and this binding could be blocked up to 100% by pretreatment of HL60 cells with sE-selectin. The concentration of sE-selectin required for 50% inhibition was 1 microM. This value puts an upper limit for the affinity of E-selectin for its natural receptor. sE-selectin also inhibited inflammatory migration of neutrophils in a selective fashion. Purified sE-selectin exhibited a broad band of M(r) approximately 75,000 on nonreducing SDS-PAGE. sE-selectin eluted with M(r) approximately 310,000 from size exclusion chromatography at physiological pH and buffers, suggesting an oligomeric state. Matrix-assisted laser-desorption MS gave a molecular weight of 80,000, while the minimum monomer molecular weight from the gene sequence should be 58,571, demonstrating that the monomeric molecule thus expressed had 27% carbohydrate. Equilibrium analytical ultracentrifugation gave an average solution molecular weight of 81,600 (+/- 4,500). Velocity ultracentrifugation gave a sedimentation coefficient of 4.3 S and, from this, an apparent axial ratio of 10.5:1, assuming a prolate ellipsoid of revolution. An analysis of the NMR NOESY spectra of sE-selectin, sialyl-Lewis X, and sE-selectin with sialyl-Lewis X demonstrates that the recombinant protein binds sialyl-Lewis X productively. Hence, in solution, sE-selectin is a functional elongated monomer.
Amino Acid Sequence↗
Pyridinyl imidazoles inhibit the inflammatory phase of delayed type hypersensitivity reactions without affecting T-dependent immune responses.
The effects of pyridinyl imidazoles, specifically SK&F 105809 and its metabolite, on normal T-cell and B-cell mediated immune responses were examined and compared to the fungal macrolide immunosuppressives, cyclosporin A, FK506 and rapamycin and to the corticosteroid, dexamethasone. The orally active prodrug SK&F 105809 [2-(4-methylsulfinylphenyl)-3-(4-pyridyl)-6,7-dihydro-[5H]-pyrrolo [1,2-a] imidazole[ and its metabolite, SK&F 105561 [2-(4-methylthiophenyl)-3-(4-pyridyl)-6,7-dihydro-[5H]-pyrrolo[1,2 -a] imidazole] are dual 5-lipoxygenase (5-LO) and cycloxygenase (CO) inhibitors with potent anti-inflammatory and cytokine (IL-1/TNF) suppressive activities. The anti-inflammatory activity of SK&F 105809 and its metabolite were evaluated in an antigen-specific murine model of delayed type hypersensitivity (DTH) response, where they were found to affect only the inflammatory and not the induction phase of this response. In contrast, these compounds and other pyridinyl imidazoles (SK&F 86002 and its analog, SK&F 104351) exhibited no immunosuppressive activity under conditions where the macrolide rapamycin and the corticosteroid dexamethasone abrogated both the cellular and humoral immune responses. Thus, the ability of pyridinyl imidazoles to attenuate independently the inflammatory components of the disease without causing generalized immunosuppression enhances their profile as candidates for therapy of chronic inflammatory diseases, specifically those mediated by cytokines (e.g. IL-1, TNF) and eicosanoids.
Animals↗
Interleukin-8 production is regulated by protein kinase C in human keratinocytes.
Interleukin-8 (IL-8) is a potent pro-inflammatory molecule present in high amounts in psoriatic skin. Here it may play an important role in the keratinocyte hyperproliferation and the neutrophil and T-lymphocyte infiltration associated with the disease. In this study the effect of protein kinase C inhibitors on IL-8 production by human keratinocytes in vitro was investigated. The anti-inflammatory and immunomodulatory compound auranofin ([1-thio-beta-D-glucopyranose-2,3,4,6-tetraacetato-S] [triethylphosphine] gold) is known to inhibit protein kinase C. In addition, auranofin has been shown to inhibit skin inflammation. As such, auranofin was also studied for its effect on IL-8 production. Auranofin and staurosporine, inhibitors of protein kinase C, inhibited phorbol-myristate-acetate-stimulated IL-8 production. Northern analysis of IL-8 mRNA revealed that the inhibition of IL-8 production was associated with an inhibition of IL-8 mRNA expression. In contrast, these compounds potentiated the minimal IL-8 protein and mRNA seen in response to interleukin-1 beta or tumor necrosis factor-alpha. These findings suggest that IL-8 synthesis may be either positively or negatively regulated by protein kinase C depending on the stimulus.
Adjuvants, Immunologic↗
Low-molecular-weight TNF biosynthesis inhibitors: strategies and prospectives.
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Adenosine↗
Conjunctival leukocyte infiltration evoked by leukotrienes: differing responses among rodent species.
Application of leukotrienes (LT) B4 or D4 to the guinea-pig eye evokes conjunctival eosinophil infiltration, providing an in vivo model of tissue eosinophilia. To determine if other rodent species would respond similarly, LTB4 or LTD4 were applied to the right eye of male mice and rats (three per group) at doses of 250, 1000 or 5000 ng/eye (mice) and 1000 or 5000 ng/eye (rats). Left eyes received the same volume (mice, 5 microliters; rats, 20 microliters) of vehicle (isotonic saline). Conjunctivae were evaluated histologically 6 h after application. In mice, 1000 or 5000 ng/eye LTB4 resulted in mild conjunctival neutrophilia, but no leukocyte response to LTD4 was observed. In rats, no leukocyte responses, either to LTB4 or to LTD4, were observed. The results illustrate interspecies differences in leukocyte responsiveness to LT and indicate specifically that, unlike the guinea pig, neither mouse nor rat responds to LT with conjunctival eosinophilia.
Animals↗
Effect of selective phosphodiesterase type IV inhibitor, rolipram, on fluid and cellular phases of inflammatory response.
The antiinflammatory activity of rolipram, a selective inhibitor of the cyclic AMP-specific phosphodiesterase (PDE IV), was studied. Rolipram did not inhibit 5-lipoxygenase activity but did inhibit human monocyte production of leukotriene B4 (LTB4, IC50 3.5 microM). Likewise, murine mast cell release of leukotriene C4 and histamine was inhibited. In vivo, rolipram inhibited arachidonic acid-induced inflammation in the mouse, while the low Km-cyclic-GMP PDE inhibitor, zaprinast, did not inhibit. Rolipram had a modest effect on LTB4 production in the mouse, but markedly reduced LTB4-induced PMN infiltration. Beta-adrenergic receptor activation of adenylate cyclase was important for rolipram antiinflammatory activity since beta blockade abrogated arachidonic acid-induced inflammation. Thus, the antiinflammatory profile of rolipram is novel and may result from inhibition of PMN function and perhaps vasoactive amine release and leukotriene biosynthesis. These actions may be dependent upon endogenous beta-adrenergic activity and are likely mediated through inhibition of PDE IV.
3',5'-Cyclic-AMP Phosphodiesterases↗
Differentiation in vivo of classical non-steroidal antiinflammatory drugs from cytokine suppressive antiinflammatory drugs and other pharmacological classes using mouse tumour necrosis factor alpha production.
The stimulation of tumour necrosis factor alpha (TNF alpha) production by lipopolysaccharide (LPS) has been widely used, both in vitro and in vivo, to examine the biochemistry and pharmacology of inflammatory cytokine production. It appears that classical nonsteroidal antiinflammatory drugs (NSAIDs) (prostaglandin H synthase 1 (PGHS-1) inhibitors) do not inhibit but instead stimulate cytokine production. In the current study, the authors utilized LPS-induced TNF alpha production in the Balb/c mouse to evaluate the activity of a classical NSAID, a mixed inhibitor, and SmithKline Beecham cytokine suppressive antiinflammatory drugs (CSAID). The results corroborated the stimulation of TNF alpha production by NSAIDs (indomethacin, naproxen, ibuprofen) and indicated that the stimulation rank-ordered with the potency of inhibition of PGHS-1. Neither acetaminophen nor nabumetone was found to stimulate TNF alpha production significantly. Tenidap, a compound reported to inhibit 5-lipoxygenase, cyclooxygenase and cytokine production, also stimulated TNF alpha production while the 5-lipoxygenase inhibitor, phenidone, was inactive. The CSAID (exemplified by SK&F 86002, SK&F 105809 and SK&F 104351), strongly inhibited TNF alpha production in this model system (ED50s of 32, 48, and 34 mg/kg p.o., respectively). These results clearly differentiate CSAID from the other compounds tested and suggest that CSAID are relatively weak inhibitors of PGHS 1 while being potent inhibitors of inflammatory cytokine production.
Animals↗
Role of complement in endotoxin/platelet-activating factor-induced lung injury.
C receptor-1 is a protein involved in the regulation of C3 and C5-convertases. Recombinant human soluble C receptor-1 has recently been produced and shown to reduce infarct size in a rat model of myocardial ischemia/reperfusion injury. The present study aimed to investigate whether recombinant human soluble C receptor-1 exerts any protective effect on pulmonary injury produced in a rodent model of adult respiratory distress syndrome. In this model, Escherichia coli endotoxin (LPS, 0.1 microgram/kg) combined with platelet-activating factor (1 pmol/kg/min over 60 min, n = 10) caused microvascular lung injury characterized by elevation of myeloperoxidase activity, deposition of C3 and C5b-9 on the endothelium of pulmonary vessels, and pulmonary edema. Furthermore, bronchoalveolar lavage revealed increased neutrophil count and elevated protein concentration. These pulmonary responses were associated with elevated serum TNF-alpha. Pretreatment (10 min, i.v.) with recombinant human soluble C receptor-1 at 10 mg/kg (n = 13), but not at 1 mg/kg, prevented the LPS/platelet-activating factor-induced pulmonary edema (p less than 0.01) and changes in the bronchoalveolar lavage fluid cell count (p less than 0.01) and protein concentration (p less than 0.05), and attenuated the deposition of C3 and C5b-9 to lung vessels. There was no effect on lung myeloperoxidase activity and serum TNF-alpha. Also, C depletion by cobra venom factor (500 U/kg, i.v.) eliminated the pulmonary edema and elevated leukocyte count in bronchoalveolar lavage fluid, but had no effect on lung myeloperoxidase activity and serum TNF-alpha. These data suggest that C factors may play an important role in the pathophysiology of adult respiratory distress syndrome.
Animals↗
Cardioprotective effects of the vasodilator/beta-adrenoceptor blocker, carvedilol, in two models of myocardial infarction in the rat.
The purpose of this study was to evaluate the cardioprotective effects of carvedilol, a beta-adrenergic blocker and vasodilator, in two models of ischemic myocardial damage in the rat. Following coronary artery occlusion for 0.5 h and reperfusion for 24 h (MI/R group), left ventricular (LV) injury was determined by planimetric analysis of triphenyltetrazolium chloride-stained tissue, and polymorphonuclear leukocyte infiltration was assessed by measuring myeloperoxidase (MPO) activity. In the vehicle-treated MI/R group, infarct size was 14.2 +/- 1.3% of the LV (n = 16), and MPO activity was increased to 2.8 +/- 0.7 from 0.14 +/- 0.03 U/g tissue in the vehicle-treated sham-occluded group (p less than 0.01). Carvedilol (1 mg/kg i.v., 15 min prior to coronary artery occlusion and at 3.5 h following reperfusion) reduced myocardial infarct size to 7.5 +/- 1.2% of the LV (n = 14; p less than 0.01) and attenuated the increase in MPO activity to 1.4 +/- 0.4 U/g tissue (p less than 0.05). A lower dose of carvedilol (i.e. 0.3 mg/kg i.v.) did not limit myocardial infarct size or the increase in MPO activity. In a model of permanent coronary artery occlusion, 24-hour survival was reduced from 85% in sham-occluded animals (n = 38) to 44% in the vehicle-treated MI group (n = 84; p less than 0.01). In comparison to the vehicle-treated MI group, carvedilol (0.3 mg/kg i.v., 15 min prior to coronary artery occlusion and 1 mg/kg 4 h after occlusion) improved survival by 55% (n = 64; p less than 0.05, compared to the vehicle-treated MI group), whereas the same dose of propranolol (n = 42) had no significant effect on survival. These results indicate that carvedilol reduces myocardial ischemia/reperfusion injury, and significantly improves survival in a permanent coronary artery occlusion model of myocardial infarction.
Adrenergic beta-Antagonists↗
Reperfusion increases neutrophils and leukotriene B4 receptor binding in rat focal ischemia.
BACKGROUND AND PURPOSE: Neutrophils are critically involved with ischemia and reperfusion injury in many tissues but have not been studied under conditions of reperfusion after focal cerebral ischemia. The present studies were conducted to confirm our previous observations quantifying neutrophils in rat permanent focal stroke using a myeloperoxidase activity assay and to extend them to transient ischemia with reperfusion. In addition, leukotriene B4 receptor binding in ischemic tissue was evaluated as a potential marker for inflammatory cell infiltration. METHODS: Histological, enzymatic, and receptor binding techniques were used to evaluate neutrophil infiltration and receptor binding in infarcted cortical tissue 24 hours after permanent middle cerebral artery occlusion (n = 25) or temporary occlusion for 80 (n = 12) or 160 (n = 22) minutes followed by reperfusion for 24 hours in spontaneously hypertensive rats. RESULTS: Sham surgery (n = 26) produced no changes in any parameter measured. After permanent middle cerebral artery occlusion, neutrophil accumulation was observed histologically, but the infiltration was moderate and typically within and adjacent to blood vessels bordering the infarcted cortex. After temporary middle cerebral artery occlusion with reperfusion, marked neutrophil infiltration was observed throughout the infarcted cortex. Myeloperoxidase activity was increased (p less than 0.05) after permanent occlusion and to a greater extent after temporary occlusion with reperfusion. Myeloperoxidase activity (units per gram wet weight) in ischemic cortex was increased over that in nonischemic (control) cortex 32.2-fold, 54.6-fold, and 92.1-fold for permanent occlusion and 80 and 160 minutes of temporary occlusion with reperfusion, respectively (p less than 0.05). Sham surgery produced no changes in myeloperoxidase activity. Leukotriene B4 receptor binding also was increased (p less than 0.05) after focal ischemia and paralleled the increases in myeloperoxidase activity. Ischemic cortex-specific receptor binding (femtomoles per milligram protein) was 3.87 +/- 0.63 in sham-operated rats and 4.57 +/- 0.98, 8.98 +/- 1.11, and 11.12 +/- 1.63 for rats subjected to permanent occlusion and 80 and 160 minutes of temporary occlusion with reperfusion, respectively (all p less than 0.05 different from sham-operated). Cortical myeloperoxidase activity was significantly correlated with the degree of cortical leukotriene B4 receptor binding (r = 0.66 and r = 0.79 in two different studies, p less than 0.01). CONCLUSION: These data indicate that neutrophils are involved in focal ischemia and that there is a dramatic accumulation of neutrophils in infarcted tissue during reperfusion that can be quantified using the myeloperoxidase activity assay. Leukotriene B4 receptor binding increases in infarcted tissue in a parallel manner, which suggests that the increased leukotriene B4 binding is to receptors located on the accumulating neutrophils.
Animals↗
Pharmacology of the pyrroloimidazole, SK&F 105809--I. Inhibition of inflammatory cytokine production and of 5-lipoxygenase- and cyclooxygenase-mediated metabolism of arachidonic acid.
SK&F 105809 [2-(4- methylsulfinylphenyl)-3-(4-pyridyl)-6,7-dihydro-[5H]-pyrrolo[1,2- a] imidazole] was determined to be a prodrug for the sulfide metabolite SK&F 105561 [2-(4- methylthiophenyl)-3-(4-pyridyl)-6,7-dihydro-[5H]-pyrrolo[1,2-a] imidazole] which inhibited interleukin-1 (IL-1) production in vitro and both 5-lipoxygenase (5-LO) and prostaglandin H (PGH) synthase activities in vitro and ex vivo. SK&F 105561 inhibited partially purified 5-LO with a half-maximal concentration (IC50) of 3 microM. This inhibition was reversible, independent of preincubation time, and dependent on the concentration of the substrate arachidonic acid. SK&F 105561 also inhibited purified PGH synthase with the potency dependent on the level of peroxidase activity. The IC50 was 100 microM in the absence of peroxidase activity, whereas an IC50 of 3 microM was observed in the presence of peroxidase activity. Using human monocytes, SK&F 105561 inhibited A23187-stimulated prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) production with IC50 values of 0.1 and 2 microM, respectively. In addition, IL-1 production by lipopolysaccharide-stimulated human monocytes was also inhibited (IC50 2 microM). Oral administration of SK&F 105809 to rats resulted in a dose-related generation of SK&F 105561 and in the inhibition of thromboxane B2 and LTB4 production ex vivo with a half-maximal dose (ED50) of 15 and 60 mg/kg, respectively. SK&F 105561 showed weak inhibitory activity on 12-lipoxygenase with an IC50 of greater than 200 microM. Neither SK&F 105561 nor SK&F 105809 inhibited the stimulated-turnover of arachidonic acid-containing phospholipids in human monocytes or the activity of cell-free phospholipases A2 and C. Moreover, neither SK&F 105561 nor SK&F 105809 antagonized the binding of LTB4 or leukotriene D4 to membrane receptors. From these results, SK&F 105561, the active principle of SK&F 105809, acts as an inhibitor of both inflammatory cytokine and eicosanoid production.
Animals↗
Pharmacology of the pyrroloimidazole, SK&F 105809--II. Antiinflammatory activity and inhibition of mediator production in vivo.
SK&F 105809 [2-(4-methylsulfinylphenyl)-3-(4-pyridyl)- 6,7-dihydro-[5H]-pyrrolo[1,2,a] imidazole] demonstrated unique antiinflammatory activities in murine models that are resistant to selective cyclooxygenase (CO) inhibitors. Both edema and inflammatory cell infiltration induced by the topical application of arachidonic acid to the mouse ear were decreased by SK&F 105809 (ED50 values of 44 mg/kg, p.o.). Polymorphonuclear leukocyte (PMN) infiltration following the intraperitoneal injection of either monosodium urate crystal or carrageenan was inhibited with ED50 values of 64 and 72 mg/kg, p.o., respectively. These inflammatory responses were unaffected by the selective cyclooxygenase inhibitor naproxen. SK&F 105809 also inhibited leukotriene B4 (LTB4) and prostaglandin E2 production in vivo in arachidonic acid-induced inflammatory exudates (ED50 values of 41 and 15 mg/kg, p.o., respectively). The inhibition of LTB4 production preceded the inhibition of PMN infiltration. The impact of inhibition of both 5-lipoxygenase (5-LO) and CO was seen with platelet-activating factor-induced vascular permeability which was inhibited markedly by SK&F 105809. However, the 5-LO inhibitor, phenidone, only strongly inhibited when coadministered with the selective CO inhibitor, indomethacin. In spite of a short half-life (14-18 min) for both SK&F 105809 and the active metabolite SK&F 105561 [2-(4- methylthiophenyl)-3-(4-pyridyl)-6,7-dihydro-[5H]-pyrrolo[1,2-a] imidazole], the pharmacological activity lasted at least 1.5 hr. The biochemical evidence of inhibition of interleukin-1 (IL-1) production and 5-LO and CO activity, in vitro, by the metabolite (SK&F 105561) seen in the companion paper (Marshall PJ, Griswold DE, Breton J. Webb EF, Hillegass LM, Sarau HM, Newton J Jr, Lee JC, Bender PE and Hanna N, Pharmacology of the pyrroloimidazole, SK&F 105809--I. Inhibition of inflammatory cytokine production and of 5-lipoxygenase- and cyclooxygenase-mediated metabolism of arachidonic acid. Biochem Pharmacol 42: 813-824, 1991) and inhibition of the fluid and cellular phases of the inflammatory response, in vivo, by SK&F 105809 suggest that this compound possesses a unique profile of activity.
Animals↗
Polymorphonuclear leukocyte infiltration into cerebral focal ischemic tissue: myeloperoxidase activity assay and histologic verification.
Two different techniques were utilized to identify the infiltration of polymorphonuclear leukocytes (PMN) into cerebral tissue following focal ischemia: histologic analysis and a modified myeloperoxidase (MPO) activity assay. Twenty-four hours after producing permanent cortical ischemia by occluding and severing the middle cerebral artery of male spontaneously hypertensive rats, contralateral hemiparalysis and sensory-motor deficits were observed due to cerebral infarction of the frontal and parietal cortex. In hematoxylin-and-eosin-stained histologic sections, PMN, predominantly neutrophils, were identified at various stages of diapedesis from deep cerebral and meningeal vessels at the periphery of the infarct, into brain parenchyma. When MPO activity in normal brain tissue was studied initially, it could not be demonstrated in normal tissues extracted from non-washed homogenates. However, if tissue was homogenized in phosphate buffer (i.e., washed), MPO activity was expressed upon extraction. Utilizing this modified assay, MPO activity was significantly increased only in the infarcted cortex compared to other normal areas of the brain. This was observed in non-perfused animals and after perfusion with isotonic saline to remove blood constituents from the vasculature prior to brain removal. The increased PMN infiltration and MPO activity were not observed in forebrain tissue of sham-operated control rats. Also, MPO activity was not increased in the ischemic cortex of MCAO rats perfused immediately after middle cerebral artery occlusion, indicating that blood was not trapped in the ischemic area. By using a leukocyte histochemical staining assay, activity of peroxidases was identified within vascular-adhering/infiltrating PMN in the infarcted cortex 24 hr after focal ischemia. An evaluation of several blood components indicated that increased MPO activity was selective for PMN. The observed increase of approximately 0.3 U MPO/g wet weight ischemic tissue vs. nonischemic cerebral tissues probably reflects the increased vascular adherance/infiltration of approximately 600,000 PMN/g wet weight infarcted cortex 24 hr after focal ischemia. This combined biochemical and histological study strongly suggests that PMN adhere within blood vessels and infiltrate into brain tissue injured by focal ischemia and that the associated inflammatory response might contribute to delayed progressive tissue damage in focal stroke. This modified MPO assay is a useful, quantitative index of PMN that can be utilized to elucidate the potential deleterious consequences of neutrophils infiltrating into the central nervous system after cerebral ischemia, trauma, or other pro-inflammatory stimuli.
Animals↗