Fibrosis comparable to IPF was induced into isolated lung segments, without compromising the respiratory functioning of the animal. This model may have potential for investigating novel therapies for IPF by allowing direct comparison of multiple treatments with internal controls, and sampling and drug delivery that are clinically relevant.

BackgroundIdiopathic pulmonary fibrosis (IPF) is a severe and progressive respiratory disease with poor prognosis. Despite the positive outcomes from recent clinical trials, there is still no cure for this disease. Pre-clinical animal models are currently largely limited to small animals which have a number of shortcomings. We have previously shown that fibrosis is induced in isolated sheep lung segments 14 days after bleomycin treatment. This study aimed to determine whether bleomycin-induced fibrosis and associated functional changes persisted over a seven-week period.MethodsTwo separate lung segments in nine sheep received two challenges two weeks apart of either, 3U bleomycin (BLM), or saline (control). Lung function in these segments was assessed by a wedged-bronchoscope procedure after bleomycin treatment. Lung tissue, and an ex vivo CT analysis were used to assess for the persistence of inflammation, fibrosis and collagen content in this model.ResultsFibrotic changes persisted up to seven weeks in bleomycin-treated isolated lung segments (Pathology scores: bleomycin12.27 ± 0.07 vs. saline 4.90 ± 1.18, n = 9, p = 0.0003). Localization of bleomycin-induced injury and increased tissue density was confirmed by CT analysis (mean densitometric CT value: bleomycin −698 ± 2.95 Hounsfield units vs. saline −898 ± 2.5 Hounsfield units, p = 0.02). Masson’s trichrome staining revealed increased connective tissue in bleomycin segments, compared to controls (% blue staining/total field area: 8.5 ± 0.8 vs. 2.1 ± 0.2 %, n = 9, p < 0.0001). bleomycin-treated segments were significantly less compliant from baseline at 7 weeks post treatment compared to control-treated segments (2.05 ± 0.88 vs. 4.97 ± 0.79 mL/cmH20, n = 9, p = 0.002). There was also a direct negative correlation between pathology scores and segmental compliance.ConclusionsWe show that there is a correlation between fibrosis and correspondingly poor lung function which persist for up to seven weeks after bleomycin treatment in this large animal model of pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive disease of increasing prevalence marked by poor prognosis and limited treatment options. Ca-activated K3.1 potassium channels have been shown to play a key role in the aberrant activation and responses to injury in both epithelial cells and fibroblasts, both considered key drivers in the fibrotic process of IPF. Pharmacological inhibition of IPF-derived fibroblasts is able to somewhat prevent TGF-β- and basic fibroblast growth factor-dependent profibrotic responses. In the current study, we investigated whether blockade of the K3.1 ion channel in vivo with a selective inhibitor, Senicapoc, was able to attenuate both histological and physiological outcomes of early fibrosis in our large animal (sheep) model for pulmonary fibrosis. We also determined whether treatment was targeting the profibrotic activity of sheep lung fibroblasts. Senicapoc was administered in established fibrosis, at 2 weeks after bleomycin instillation, and drug efficacy was assessed 4 weeks after treatment. Treatment with Senicapoc improved pre-established bleomycin-induced changes compared with vehicle control, leading to improved lung compliance, reduced extracellular matrix and collagen deposition, and a reduction in both α-smooth muscle actin expression and proliferating cells, both in vivo and in vitro. These studies show that inhibiting the K3.1 ion channel is able to attenuate the early fibrogenic phase of bleomycin-dependent fibrosis and inhibits profibrotic behavior of primary sheep lung fibroblasts. This supports the previous research conducted in human IPF-derived fibroblasts and suggests that inhibiting K3.1 signaling may provide a novel therapeutic approach for IPF.

BackgroundThe Ca2+-activated K+ channel KCa3.1 is expressed in several structural and inflammatory airway cell types and is proposed to play an important role in the pathophysiology of asthma. The aim of the current study was to determine whether inhibition of KCa3.1 modifies experimental asthma in sheep.Methodology and Principal FindingsAtopic sheep were administered either 30 mg/kg Senicapoc (ICA-17073), a selective inhibitor of the KCa3.1-channel, or vehicle alone (0.5% methylcellulose) twice daily (orally). Both groups received fortnightly aerosol challenges with house dust mite allergen for fourteen weeks. A separate sheep group received no allergen challenges or drug treatment. In the vehicle-control group, twelve weeks of allergen challenges resulted in a 60±19% increase in resting airway resistance, and this was completely attenuated by treatment with Senicapoc (0.25±12%; n = 10, P = 0.0147). The vehicle-control group had a peak-early phase increase in lung resistance of 82±21%, and this was reduced by 58% with Senicapoc treatment (24±14%; n = 10, P = 0.0288). Senicapoc-treated sheep also demonstrated reduced airway hyperresponsiveness, requiring a significantly higher dose of carbachol to increase resistance by 100% compared to allergen-challenged vehicle-control sheep (20±5 vs. 52±18 breath-units of carbachol; n = 10, P = 0.0340). Senicapoc also significantly reduced eosinophil numbers in bronchoalveolar lavage taken 48 hours post-allergen challenge, and reduced vascular remodelling.ConclusionsThese findings suggest that KCa3.1-activity contributes to allergen-induced airway responses, inflammation and vascular remodelling in a sheep model of asthma, and that inhibition of KCa3.1 may be an effective strategy for blocking allergen-induced airway inflammation and hyperresponsiveness in humans.

Aim: To determine the range of cerebral blood flow velocities (CBFVs) and Doppler indices of cerebrovascular resistance in normal-term neonates as a baseline for a study of hypoxic-ischaemic encephalopathy. Methods: The CBFVs, resistive index (RI) and pulsatility index (PI) were measured in the anterior and middle cerebral arteries (ACA and MCA) of 38 normal neonates. Results: The mean peak systolic, end diastolic and time-averaged velocities (PSV, EDV and TAV) were 36.3 AE 6.6, 12.4 AE 3.9 and 22.0 AE 4.0 cm/s (ACA) and 41.4 AE 13.2, 13.0 AE 5.5 and 25.8 AE 7.9 cm/s (MCA), respectively. All CBFVs in the ACA correlated with gestation; only EDV was correlated to post-natal age. The RI in the ACA (0.67 AE 0.06) and MCA (0.68 AE 0.07) were correlated (r = 0.72, P < 0.001); RI correlated to postnatal age. Two infants with RI < 0.55 were both fed within 25 mins of the study; RI correlated with post-prandial time (dichotomous, pivot 25 min). The mean PI was 1.11 AE 0.18 (ACA) and 1.17 AE 0.23 (MCA). Correlations were observed with post-natal age and post-prandial time (dichotomous). The average angle of insonation was greater in the ACA than in the MCA (median of 5 vs. 18 ).Conclusions: Results corresponded with previous published studies. No correlation was observed between Doppler indices and gestation as component velocities all increase with advancing gestation. Less variation and smaller standard deviation of CBFV's was associated with a smaller angle of insonation. Low RIs (<0.55), without a pathological cause, warrants further study.Key words: cerebral arteries; cerebrovascular circulation; infant, newborn; ultrasonography, Doppler, transcranial; vascular resistance. What is already known on this topic1 The resistive index (RI) is an accepted indicator of cerebrovascular resistance. 2 In previous studies, the normal RI in term neonates has been established to be greater than 0.55. 3 An RI lower than 0.55 may be observed after periods of hypoxia. What this paper adds1 It is preferable to measure cerebral blood flows in the anterior cerebral artery as they exhibit less variance and are therefore more reproducible than middle cerebral artery measurements. 2 Low RIs that may be considered clinically significant (RI < 0.55) were observed in two normal infants without a known pathological cause. 3 The RI and PI can remain unchanged if component velocities decrease or increase together.The Doppler indices, the resistive index (RI) and the pulsatility index (PI) are considered a measure of downstream cerebrovascular resistance (CVR) and provide a non-invasive means of investigating the neonatal circulation. 1 These indices are calculated from the cerebral arterial velocities measured using Doppler in the neonate through the 'acoustic windows,' the fontanelles and sutures of the skull. Pourcelot's resistive (resistance) index (RI) is the ratio of the difference in pulsatile flow velocities, peak systolic velocity (PSV) -end diastolic velocity (EDV), divided by the PSV 5,6 :The PI of Gosling (PI) is a ratio of the dif...

Tumstatin, a protein fragment of the alpha-3 chain of Collagen IV, is known to be significantly reduced in the airways of asthmatics. Further, there is evidence that suggests a link between the relatively low level of tumstatin and the induction of angiogenesis and inflammation in allergic airway disease. Here, we show that the intra-segmental administration of tumstatin can impede the development of vascular remodelling and allergic inflammatory responses that are induced in a segmental challenge model of experimental asthma in sheep. In particular, the administration of tumstatin to lung segments chronically exposed to house dust mite (HDM) resulted in a significant reduction of airway small blood vessels in the diameter range 10+–20 μm compared to controls. In tumstatin treated lung segments after HDM challenge, the number of eosinophils was significantly reduced in parenchymal and airway wall tissues, as well as in the bronchoalveolar lavage fluid. The expression of VEGF in airway smooth muscle was also significantly reduced in tumstatin-treated segments compared to control saline-treated segments. Allergic lung function responses were not attenuated by tumstatin administration in this model. The data are consistent with the concept that tumstatin can act to suppress vascular remodelling and inflammation in allergic airway disease.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease with limited therapeutic options and poor prognosis. IPF has been associated with aberrant vascular remodelling, however the role of vascular remodelling in pulmonary fibrosis is poorly understood. Here, we used a novel segmental challenge model of bleomycin-induced pulmonary fibrosis in sheep to evaluate the remodelling of the pulmonary vasculature, and to investigate the changes to this remodelling after the administration of the KCa3.1 channel inhibitor, senicapoc, compared to the FDA-approved drug pirfenidone. We demonstrate that in vehicle-treated sheep, bleomycin-infused lung segments had significantly higher blood vessel density when compared to saline-infused control segments in the same sheep. These microvascular density changes were significantly attenuated by senicapoc treatment. The increases in vascular endothelial growth factor (VEGF) expression and endothelial cell proliferation in bleomycin-infused lung segments were significantly reduced in sheep treated with the senicapoc, when compared to vehicle-treated controls. These parameters were not significantly suppressed with pirfenidone treatment. Senicapoc treatment attenuated vascular remodelling through inhibition of capillary endothelial cell proliferation and VEGF expression. These findings suggest a potential new mode of action for the novel drug senicapoc which may contribute to its efficacy in combatting pulmonary fibrosis.

Our results show for the first time that the airways of sheep chronically exposed to HDM allergen undergo vascular remodeling. These findings show the potential of this large animal model for investigating airway angiogenesis in asthma.