Jacques Damas

Biocompatibility of thermosensitive chitosan-based hydrogels: an in vivo experimental approach to injectable biomaterials.

Chitosan, an amino-polysaccharide obtained from the alkaline deacetylation of chitin, presents an interest as a drug vehicle. Indeed, chitosan solutions containing glycerol-2-phosphate (beta-GP) undergo sol-gel transition at a temperature close to 37 degrees C, which make them suitable for the parenteral administration of drugs. However, before using these chitosan derivatives for biomedical applications, it is important to evaluate their biocompatibility, and particularly to test their inflammatory effects. When injected in the hindpaw of the rat, we have shown that: (i) four chitosan/beta-GP solutions tested triggered a non-specific response, with solutions prepared with chitosans of higher deacetylation degrees yielding a lesser inflammatory reaction and (ii) systemic pretreatment of animals with icatibant, apafant and diphenhydramine did not significantly diminish this response; dexamethasone practically abolished it for all solutions and ketanserine only slightly decreased it in one preparation at two different times. In conclusion, it appears that a higher degree of deacetylation of the chitin chain is desirable for superior biocompatibility.

The inflammatory reaction induced by formalin in the rat paw

The involvement of bradykinin and some other inflammatory mediators in formalin-induced oedema and plasma extravasation was examined. Formalin was injected in rat paws at two doses, 1.75% or 5%. The lower dose induced the development of an immediate oedema associated with a progressive accumulation of 125I-labelled albumin in the paws. These changes were suppressed by pretreatment with capsaicin or xylocaine. They were abolished by RP67580, a NK1 receptor antagonist, and increased by phosphoramidon or diprotin A. They were not affected by HOE140, a bradykinin B2 antagonist, captopril, methysergide, mepyramine, indomethacin, ketoprofen or l-NG-nitroarginine. The higher dose of formalin induced a swelling of the paws which took place in two phases associated with two periods of increase in vascular permeability. This oedema was reduced by pretreatment with capsaicin but not with xylocaine. It was reduced by RP67580 injected before or 30 min after formalin. It was inhibited by mepyramine, methysergide, indomethacin and NS-398, a cyclooxy-genase-2 inhibitor. It was not modified by HOE140. Its development was similar in normal and kininogen-deficient rats. We concluded that formalin administered at a low dose induces an oedema which mainly results from a neurogenic inflammation mediated by neuropeptides such as substance P. At higher doses, formalin induces an oedema which mainly depends on the release of substance P, prostanoids, 5-hydroxytryptamine and histamine. Bradykinin plays no significant role in the vascular changes whereas this peptide has been reported to participate in the stimulation of nociceptive afferent neurons. This discrepancy could be explained by a difference in the threshold of stimulation of the nociceptive neurons and that of the cells of the vascular walls, or by a formation of kinins in close contact of the neurons.

Further studies of the mechanism of counter irritation by turpentine

SummaryThe influence of counter irritation by turpentine (0.2 ml) on zymosan-and carrageenan-oedemas was investigated in the rat. Zymosan-oedema was inhibited by mepyramine and methysergide and by leucopenia. It was not modified by captopril and developed normally in kininogendeficient Brown Norway rats. Leucocytes and mast cell amines but not kinins are thus involved in zymosan-oedema. The last phase of this reaction was inhibited by counter irritation alone, but the odema was largely depressed by counter irritation in rats pretreated with mepyramine and methysergide.Carrageenan-oedema was increased by kininase inhibitors and inhibited by leucopenia in normal rats. This inflammatory reaction had a small developement and was not increased by kininase inhibitors in kininogen-deficient BN rats. Leucocytes and kinins participate in the developement of this inflammatory reaction in normal rats while kinins are lacking in deficient rats. Counter irritation depressed carrageenan-oedema in deficient Brown Norway rats and suppressed the potentiating effect of kininase inhibitors in normal rats. Carrageenan oedema was nearly abolished in turpentine-treated leucopenic rats.These results suggest that the anti-inflammatory effect of counter irritation by turpentine could depend on a reduction of leucocyte accumulation into zymosan-oedema and on a reduction of both kinin formation and of leucocyte accumulation into carrageenan-oedema. The significance of T-kininogen as acute phase reactant is discussed.

Influence of a long-acting bradykinin antagonist, Hoe 140, on some acute inflammatory reactions in the rat

We studied the influence of Hoe 140, a bradykinin antagonist, on inflammatory reactions induced in rats. Hoe 140 reduced paw oedema induced by bradykinin alone, bradykinin plus prostaglandin (PG) E1, carrageenan, urate crystals or urate crystals plus captopril. The inhibitory effect of Hoe 140 lasted for at least 4 h. Hoe 140 also reduced plasma exudation in sponges implanted in the back of the rat. However it did not modify paw oedema induced by zymosan or by heating the paw at 55 degrees C for 30 s. Carrageenan oedema developed to a small extent in kininogen-deficient rats while the swelling induced by heating the paw of kininogen-deficient rats was the same as that measured in normal animals. Hoe 140 had no effect on the slight swelling induced by carrageenan in kininogen-deficient rats. We conclude that the kinin system is involved in inflammatory reactions induced by carrageenan, urate crystals, sponge implantation but not by zymosan and scalding.

Inhibitory effects of proanthocyanidins from Ribes nigrum leaves on carrageenin acute inflammatory reactions induced in rats.

BackgroundThe anti-inflammatory effects of proanthocyanidins (PACs), isolated from blackcurrant (Ribes nigrum L.) leaves, were analysed using carrageenin-induced paw oedema and carrageenin-induced pleurisy in rats.ResultsPretreatment of the animals with PACs (10, 30, 60 and 100 mg/kg, i.p.) reduced paw oedema induced by carrageenin in a dose and time-dependent manner. PACs also inhibited dose-dependently carrageenin-induced pleurisy in rats. They reduced (A) lung injury, (B) pleural exudate formation, (C) polymorphonuclear cell infiltration, (D) pleural exudate levels of TNF-α, IL-1β and CINC-1 but did not affect IL-6 and IL-10 levels. They reduced (E) pleural exudate levels of nitrite/nitrate (NOx). In indomethacin treated rats, the volume of pleural exudate was low, its content in leukocytes and its contents in TNF-α, IL-1β, IL-6 and IL-10 but not in NOx were reduced. These data suggest that the anti-inflammatory properties of PACs are achieved through a different pattern from those of indomethacin.ConclusionThese results suggest that the main mechanism of the anti-inflammatory effect of PACs mainly lies in an interference with the migration of the leukocytes. Moreover, PACs inhibited in vivo nitric oxide release.

Kinins and peritoneal exudates induced by carrageenin and zymosan in rats.

1 Kinins were measured by a radioimmunoassay in the inflammatory exudates induced by carrageenin or zymosan in the peritoneal cavity of normal Wistar rats and of kininogen‐deficient Brown Norway rats. 2 After administration of carrageenin to normal rats, levels of immunoreactive kinins showed a single peak during the first two hours and then decreased. The presence of kinins preceded and accompanied the exudation of 125I‐labelled albumin. Kinins were identified as bradykinin by chromatography. 2 Captopril, an inhibitor of kininase 2, increased the level of kinins and the volume of the exudates after carrageenin treatment. In Brown Norway rats, the volume of the exudates was small and contained little or undetectable amounts of immunoreactive kinins. 4 During zymosan‐induced peritonitis, the exudates were devoid of immunoreactive kinins in both species. The volume of the exudates was larger in kininogen‐deficient rats than in normal rats. 5 We conclude that in rats, the kinin system is a major factor responsible for the development of the inflammatory reactions induced by carrageenin, but is not involved in the reactions induced by zymosan.

Insulin Sensitivity, Clearance and Release in Kininogen-Deficient Rats

Insulin sensitivity of kininogen‐deficient rats was compared with that of normal rats using euglycaemic hyperinsulinaemic glucose clamping. Anaesthetized animals were infused with 2‐50 mU kg−1 min−1 of insulin and the glucose infusion rates needed to maintain euglycaemia were determined. Maximum glucose uptake, insulin sensitivity index and insulin clearance were reduced in kininogen‐deficient rats. Captopril increased the amount of glucose needed to maintain euglycaemia during infusion of 2 and 10 mU kg−1 min−1 of insulin in normal rats, but had no effect in kininogen‐deficient rats. Anaesthetized rats of both strains were given an intraperitoneal injection of glucose and the evolution of blood glucose was followed for 120 min. The peak increase was higher in kininogen‐deficient rats. Similar larger increases in blood glucose were observed after glucose injection in normal rats previously treated with HOE 140, a bradykinin B2 receptor antagonist. After glucose injection, plasma insulin increased in both groups of rats but reached lower levels in kininogen‐deficient animals. These results suggest that bradykinin is involved not only in the clearance of glucose and insulin by the tissues during insulin infusion but also that bradykinin can affect the release of insulin after a glucose load.

Anti-inflammatory and immunological effects of Centaurea cyanus flower-heads

Centaurea cyanus flower-heads are used in European phytotherapy for the treatment of minor ocular inflammations. Different pharmacological experiments (inhibition of carrageenan, zymosan and croton oil-induced oedemas, inhibition of plasma haemolytic activity, induction of anaphylatoxin activity) showed that polysaccharides extracted from C. cyanus flower-heads had anti-inflammatory properties and interfered with complement. Moreover, these polysaccharides were found to be mainly composed of galacturonic acid, arabinose, glucose, rhamnose and galactose.

Proanthocyanidins, from Ribes nigrum leaves, reduce endothelial adhesion molecules ICAM-1 and VCAM-1

BackgroundThe effects of proanthocyanidins (PACs), isolated from blackcurrant (Ribes nigrum L.) leaves, on neutrophil accumulation during inflammatory processes were investigated in vivo and in vitro.MethodsIn vivo studies were performed using carrageenin-induced pleurisy in rats pre-treated with PACs. Exudate volume and PMNs accumulation were measured. Leukocyte cell adhesion molecules (LFA-1, Mac-1 and VLA-4) mobilization in circulating granulocytes were analysed by flow cytometry and endothelial cell adhesion molecules (ICAM-1 and VCAM-1) were detected by immunohistochemistry on lung sections.In vitro studies were conducted on endothelial LT2 cells, stimulated with TNF-α, to evaluate ICAM-1, IL-8 and VEGF mRNA expression upon PACs treatment.Data sets were examined by one-way analysis of variance (ANOVA) followed by a Scheffe post-hoc test.ResultsPretreatment of the animals with PACs (10, 30 and 60 mg/kg) inhibited dose-dependently carrageenin-induced pleurisy in rats by reducing pleural exudate formation and PMNs infliltration. Leukocyte cell adhesion molecules mobilization was not down-regulated on granulocytes by PACs. Immunohistochemistry on lung sections showed a decreased production of endothelial cell adhesion molecules.In vitro experiments demonstrated that PACs were able to significantly inhibit ICAM-1 but not IL-8 and VEGF165 mRNA expression. Moreover, VEGF121 mRNA expression was dose-dependently enhanced.ConclusionThis study provides evidence to support the anti-inflammatory activity of proanthocyanidins is related to an inhibition of leukocyte infiltration which can be explained at least in part by a down-regulation of endothelial adhesion molecules, ICAM-1 and VCAM-1 and that these compounds are capable of modulating TNF-α-induced VEGF transcription.

Platelet-activating factor and the vascular effects of zymosan in rats

Platelet-activating factor (PAF; 2.5 micrograms/kg) injected in the tail vein of anaesthetized rats increased the vascular permeability of the duodenum, paws, skin and muscles, as measured by the extravasation of 125I-labelled albumin. It did not affect the permeability of the lungs or the presence of labelled albumin in the liver and spleen. The effects of PAF were dose dependently inhibited by WEB 2086 (ID50: 1.39 to 2.09 mg/kg) and SM-12502 (ID50: 7.17 to 8.36 mg/kg). Zymosan, an activator of the alternative complement pathway (10 or 16 mg/kg), induced protein extravasation in the lungs, duodenum, paws and skin, and the accumulation of labelled albumin in the liver. The effects of zymosan on the duodenum and liver were dose dependently inhibited by WEB-2086 and SM-12502. Both PAF antagonists increased the effects of zymosan in the paws but they did not affect protein extravasation in the lungs. The hypotensive effect of PAF (0.5 micrograms/kg) was inhibited by WEB 2086 (ID50: 1.21 mg/kg) and SM-12502 (ID50: 13.4 mg/kg). Both PAF antagonists reduced the hypotensive effects of zymosan (4 or 16 mg/kg) with a similar relative inhibitory potency. PAF is the major mediator involved in the hypotensive effect of zymosan but plays only a minor role in the permeability-enhancing effect of zymosan, mostly in the splanchnic area.