ENaC-mediated effects assessed by MRI in a rat model of hypertonic saline-induced lung hydration.

BACKGROUND AND PURPOSE: The epithelial sodium channel (ENaC) regulates airway mucosal hydration and mucus clearance. The lack of such regulation in cystic fibrosis patients leads to desiccation of the airway lumen, resulting in mucostasis that establishes the environment for infections. Osmotic agents and negative ENaC regulators can be used to restore mucosal hydration. We aimed to assess whether: (i) osmotically driven fluid flux into the rat lung could be quantified in vivo by magnetic resonance imaging (MRI); and (ii) the MRI signals could be modulated through the regulation of ENaC function. EXPERIMENTAL APPROACH: Lung images from spontaneously breathing rats were acquired following intra-tracheal (i.t.) administration of physiological or hypertonic saline (HS). Compounds known to modulate the ENaC function were given i.t. prior to saline. Volumes of fluid signals were quantified on the images. KEY RESULTS: A tonicity-dependent increase in lung fluid was demonstrated following HS administration. Pretreatment with the ENaC blockers, amiloride or 552-02, resulted in an enhancement of HS-induced lung fluid signals, which were detectable for up to 4 h, consistent with a role for ENaC in fluid clearance. Aprotinin, a serine protease inhibitor that attenuates ENaC function, likewise enhanced the HS-induced increase in lung fluid signal, while alpha(1)-anti-trypsin was without significant effect. CONCLUSIONS AND IMPLICATIONS: Proton MRI provides a non-invasive technique for studying modulators of lung fluid hydration in rat lung in vivo. The pharmacological sensitivity of MRI-detected fluid signals is consistent with ENaC-mediated fluid reabsorption after HS. This target-related readout may be used to characterize new ENaC modulators.

Activation of the lung S1P(1) receptor reduces allergen-induced plasma leakage in mice.

BACKGROUND AND PURPOSE: It has been suggested that intratracheal administration of the immunomodulator, FTY720, could have anti-inflammatory effects without causing a decrease in blood lymphocyte counts. However, the receptor responsible for this effect has not been defined. EXPERIMENTAL APPROACH: We have described, in a mouse model of allergen-induced inflammation, the use of proton magnetic resonance imaging to non-invasively assess lung fluid accumulation and inflammation. Here, we used this model to investigate the sphingosine-1-phosphate (S1P) receptor responsible for the anti-inflammatory effect of FTY720. KEY RESULTS: When given intranasally, FTY720 (3 and 10 microg.kg(-1)) inhibited by approximately 50% the allergen-induced accumulation of fluid in the lung detected by magnetic resonance imaging, but had no effect on the cellular inflammation in the airway space or on circulating blood lymphocytes. Inhibition of the infiltration of inflammatory cells into the airways was only observed at a dose of FTY720 that induced lymphopenia (100 microg.kg(-1)). Similar results were observed in S1P(3)-deficient mice. The effect of FTY720 was mimicked by intranasal treatment of wild-type mice with a S1P(1)-specific agonist, AUY954. CONCLUSIONS AND IMPLICATIONS: Thus, in contrast to previously published work, our results suggest that systemic exposure of FTY720 is necessary to obtain an airway anti-inflammatory effect. On the contrary, inhibition of the allergen-induced accumulation of fluid in the lung, via activation of the S1P(1) receptor, is obtainable without systemic effects.

Comparison of [18F]-tracers in various experimental tumor models by PET imaging and identification of an early response biomarker for the novel microtubule stabilizer patupilone.

PURPOSE: The suitability of [18F]FDG, [18F]FLT, [18F]FET, and [18F]FCH as non-invasive positron emission tomography (PET) biomarkers for monitoring response to chemotherapy was analyzed in various experimental tumor models. PROCEDURES: Tracer uptake into three syngeneic rodent tumor models and ten human xenograft models was evaluated using semiquantitative analysis of small-animal PET data. Murine RIF-1 fibrosarcomas and [18F]FLT were selected to monitor the effects of the novel cytotoxic patupilone. RESULTS: Except [18F]FCH, all tracers provided good tumor visualization. Highest [18F]FDG uptake was identified in syngeneic tumors. Xenograft models, however, showed low [18F]FDG SUVs and were better visualized by [18F]FLT. Monitoring the effects of patupilone on [18F]FLT uptake in RIF-1 tumors revealed a significant decrease of tracer uptake after 24 h, which strongly negatively correlated with apoptosis. CONCLUSION: [18F]FLT PET of experimental tumors is a viable complement to [18F]FDG for preclinical drug development. [18F]FLT may be an excellent biomarker for patupilone-induced apoptosis.

In vivo pharmacological evaluation of compound 48/80-induced airways oedema by MRI.

BACKGROUND AND PURPOSE: Allergen-induced airways oedema in actively sensitized rats has been studied earlier by magnetic resonance imaging (MRI). We used MRI to follow the consequences of non-immunological mast cell activation induced by compound 48/80 in the rat lungs in vivo. EXPERIMENTAL APPROACH: Male naïve rats were scanned by MRI prior to and at several time points following intratracheal administration of the mast cell secretagogue, compound 48/80. The effects of a range of drugs on the response induced by compound 48/80 were studied. KEY RESULTS: Strong fluid signals were detected by MRI in the lungs at 24 h after compound 48/80, correlating with increased protein concentration and inflammatory cell infiltration in bronchoalveolar lavage, and with perivascular oedema observed histologically. Pharmacological intervention demonstrated that the increase in MRI signal volume induced by compound 48/80 24 h after challenge was blocked by disodium cromoglycate and the glucocorticoid, budesonide. Pretreatment with wortmannin, capsazepine, DNK333 (a dual neurokinin (NK) 1 and NK2 antagonist) or the anti-allergy drug CGS8515, but not indomethacin, resulted in partial inhibition. CONCLUSIONS AND IMPLICATIONS: Compound 48/80 induced a complex inflammatory reaction which did not solely involve mast cell degranulation but also activation of sensory nerves and was qualitatively similar to allergen challenge. Changes observed by MRI correlated with decreases in protein concentration in BAL fluid. However, the magnitude of the changes detected was greater using MRI. Our results demonstrate that MRI is a sensitive and efficient tool to assess the effects of drugs on lung inflammation.

Capsaicin-induced mucus secretion in rat airways assessed in vivo and non-invasively by magnetic resonance imaging.

BACKGROUND AND PURPOSE: An up-regulation of the sensory neural pathways in the lung has been implicated in asthma and chronic obstructive pulmonary disease (COPD) and is thought to contribute to mucus hypersecretion, an essential feature of both diseases. The aim of this study was to assess non-invasively the acute effects (up to 60 min) of sensory nerve stimulation by capsaicin in the lung, using magnetic resonance imaging (MRI). EXPERIMENTAL APPROACH: Male Brown Norway rats were imaged prior to and 10, 30 and 60 min after intra-tracheal challenge with capsaicin (30 microg kg(-1)) or vehicle (0.5% ethanol solution). In subsequent studies, pre-treatment with the transient receptor potential vanilloid (TRPV)-1 antagonist, capsazepine; the dual neurokinin (NK) 1 and NK2 receptor antagonist, DNK333 and the mast cell stabilizer, di-sodium cromoglycate (DSCG) was used to modulate the effects of capsaicin. KEY RESULTS: Diffuse fluid signals were detected by MRI in the lung as early as 10 min after capsaicin, remaining constant 30 and 60 min after treatment. Broncho-alveolar lavage (BAL) fluid analysis performed 60 min after capsaicin revealed increased mucin concentration. Capsazepine (3.5 mg kg(-1)), DNK333 (10 mg kg(-1)) but not DSCG (10 mg kg(-1)) administered prophylactically were able to block the effect of capsaicin in the airways. CONCLUSIONS AND IMPLICATIONS: These observations suggest that the fluid signals detected by MRI after capsaicin administration reflected predominantly the release of mucus following activation of sensory nerves. They point to the opportunity of non-invasively assessing with MRI the influence of neuronal mechanisms in animal models of asthma and COPD.

Airway hyperresponsiveness to bradykinin induced by allergen challenge in actively sensitised Brown Norway rats.

The mechanism(s) of bradykinin-induced bronchoconstriction was investigated in the Brown Norway (BN) rat model of allergic asthma. Bronchoconstrictor responses to i.v. bradykinin in BN rats were maximally augmented 24 h following challenge with allergen and declined at later time points. Histological evaluation of the inflammatory status of the lungs after ovalbumin (OA) challenge showed a marked inflammatory response, which was maximal at 24 h and declined thereafter. However, pretreatment with budesonide did not inhibit the augmented bronchoconstrictor response to bradykinin 24 h after allergen challenge. The selective B1 receptor agonist, Lys-[desArg9]-BK had no bronchoconstrictor effects, whereas the selective B2 receptor antagonist, HOE 140, abolished the response to bradykinin in OA-challenged animals. The augmented response to bradykinin was not affected by methysergide, indomethacin, disodium cromoglycate, iralukast, the 5-lipoxygenase inhibitor, CGS8515, or the NK2 receptor antagonist, SR48968. It was, however, partially inhibited by atropine both in saline- and OA-challenged animals. Pretreatment with captopril and thiorphan markedly potentiated responses to bradykinin both in saline- and OA-challenged animals. Thus, augmentation of the bronchoconstrictor response to bradykinin occurs in actively sensitised BN rats 24 h after challenge with OA and is associated with marked pulmonary inflammation. The response is entirely B2 receptor mediated and approximately 50% of the response is cholinergic. However, mast cell activation, the products of the cyclooxygenase or 5-lipoxygenase pathways and tachykinins are not involved. Peptidase inhibition mimics the effect of allergen challenge on the bronchoconstrictor response to bradykinin and it remains possible that the mechanism of the augmented response to bradykinin following allergen challenge involves downregulation of peptidase activity as a consequence of the inflammatory response.

MRI-based monitoring of inflammation and tissue damage in acute and chronic relapsing EAE.

Experimental autoimmune encephalomyelitis (EAE) is a commonly used animal model that in several respects mimics human multiple sclerosis (MS), and can be used to design or validate new strategies for treatment of this disease. In the present study, different MRI techniques (macrophage tracking based on labeling cells in vivo by ultrasmall particles of iron oxide (USPIO), blood-brain barrier (BBB) breakdown, and magnetization transfer imaging (MTI)), as well as immunohistological staining were used to study the burden of disease in Lewis rats immunized by guinea pig myelin. The resulting imaging data was compared with behavioral readouts. Animals were studied during the acute phase and the first relapse. Activated monocytes were detected during both episodes in the brain stem or cortex. These areas coincided in part with areas of BBB breakdown. Significant changes of the magnetization transfer ratios (MTRs) of up to 35% were observed in areas of USPIO accumulation. This suggests that infiltrating monocytes are the major source of demyelination in EAE, but monocyte infiltration and breakdown of the BBB are temporally or spatially independent inflammatory processes.

Macrophage labeling by SPIO as an early marker of allograft chronic rejection in a rat model of kidney transplantation.

Anatomical and functional information (renography, perfusion) was obtained by MRI in a life-supporting transplantation model, in which Lewis rats received kidneys from Fisher 344 donors. Renography and perfusion analyses were carried out with Gd-DOTA and small particles of iron oxide (SPIO), respectively. Starting 12 weeks posttransplantation, images from grafts of untreated recipients exhibited distinctive signal attenuation in the cortex. Animals treated with cyclosporin (Sandimmune Neoral; Novartis Pharma, Basel, Switzerland) to prevent acute rejection showed a signal attenuation in the cortex at 33 weeks posttransplantation, while kidneys from rats treated additionally with everolimus (Certican; Novartis), a rapamycin derivative, had no changes in anatomical appearance. A significant negative correlation was found between the MRI cortical signal intensity and the histologically determined iron content in macrophages located in the cortex. Renography revealed a significantly reduced functionality of the kidneys of untreated controls 33 weeks after transplantation, while no significant changes in perfusion were observed in any group of rats. These results suggest the feasibility, by labeling macrophages with SPIO, of detecting signs of graft rejection significantly earlier than when changes in function occur. Monitoring early changes associated with chronic rejection can have an impact in preclinical studies by shortening the duration of the experimental period and by facilitating the investigation of novel immunomodulatory therapies for transplantation.

Transplantation-induced endothelial dysfunction as studied in rat aorta allografts.

BACKGROUND: Clinical evidence indicates that vascular endothelial cell (EC) dysfunction occurs early after transplantation (Tx) and initiates chronic graft vasculopathy. This study explored this phenomenon in rat aorta Tx using the stringent Dark Agouti (DA)-to-Lewis (LEW) and the weak Fischer 344 (F344)-to-LEW strain combinations. METHODS: Donor abdominal aortae were orthotopically grafted into LEW rats. At post-Tx days 7, 14, 28, and 56, grafts were collected to assess changes in EC morphology (en face silver staining) and EC function, i.e., vasodilatory response to acetylcholine (ACH) after phenylephrine (PHE) precontraction; changes in vascular smooth muscle cell (VSMC) alpha-actin (western blotting), degree of apoptosis (caspase-3 activity), and morphology. RESULTS: In DA allografts, VSMC and EC dysfunctions developed concomitantly and were completed at 14 days post-Tx, most likely due to the EC and alpha-actin-positive VSMC loss. Meanwhile, allografts revealed markedly increased caspase-3 activity. Neointima formation, restricted to the edges of allografts at day 28, covered the entire allografts by day 56 post-Tx. In F344-allografts, VSMC function was maintained up to day 14 post-Tx, whereas ACH-induced relaxation was reduced by 50% at day 7 and abolished at day 14. EC denudation was not seen up to 56 days post-Tx, despite prominent leukocyte adhesion. Neointima formation was not detected at day 28 post-Tx but appeared along the entire allografts at day 56 post-Tx. CONCLUSIONS: These results confirm that Tx-induced EC dysfunction precedes the development of vasculopathy in rat aorta allografts and suggest that this early phenomenon can be best studied in the F344-to-LEW strain combination.

[Comparative study of the antiglaucomatous activity of Glauplex 2 and pilocarpine nitrate on alpha-chymotrypsin-induced experimental glaucoma]. / Etude comparative de l'activité antiglaucomateuse de Glauplex 2 et du nitrate de pilocarpine sur le glaucome expérimental à l'alpha-chymotrypsine.

The antiglaucomatous effects of Glauplex 2 and pilocarpine nitrate on alpha-chymotrypsine-induced experimental glaucoma were studied in 8 rabbits. Changes in intraocular pressure were measured over a period of 12 hours after a single instillation of Glauplex 2 or two instillations of 2.6 p. cent pilocarpine nitrate at t = 0 and t = 6 hours. The antihypertensive effect of a single instillation of Glauplex 2 was shown to be approximately equivalent to that of two instillations of 2.6 p. cent pilocarpine nitrate.