Investigation of molecular mechanisms of experimental compounds in murine models of chronic allergic airways disease using synchrotron Fourier-transform infrared microspectroscopy

Abstract

the ovalbumin-induced (oVA) chronic allergic airways murine model is a well-established model for investigating pre-clinical therapies for chronic allergic airways diseases, such as asthma. Here, we examined the effects of several experimental compounds with potential anti-asthmatic effects including resveratrol (RV), relaxin (RLN), l-sulforaphane (LSF), valproic acid (VPA), and trichostatin A (TSA) using both a prevention and reversal model of chronic allergic airways disease. We undertook a novel analytical approach using focal plane array (fpA) and synchrotron fourier-transform infrared (S-ftiR) microspectroscopic techniques to provide new insights into the mechanisms of action of these experimental compounds. Apart from the typical biological effects, S-FTIR microspectroscopy was able to detect changes in nucleic acids and protein acetylation. Further, we validated the reduction in collagen deposition induced by each experimental compound evaluated. Although this has previously been observed with conventional histological methods, the S-FTIR technique has the advantage of allowing identification of the type of collagen present. More generally, our findings highlight the potential utility of S-ftiR and fpA-ftiR imaging techniques in enabling a better mechanistic understanding of novel asthma therapeutics. Animal models designed specifically for chronic lung diseases such as asthma are important for investigating the underlying mechanisms of the disease and the effectiveness of potential novel therapies 1. One of the most widely used models for asthma is the mouse chronic allergic airways disease model that utilises ovalbumin (OVA) 2,3. This model recapitulates many of the hallmark characteristics of chronic asthma including eosinophilic inflammation, airway hyperresponsiveness, epithelial wall thickening, goblet cell metaplasia, and fibrosis 4,5 .

Sulforaphane prevents and reverses allergic airways disease in mice via anti-inflammatory, antioxidant and epigenetic mechanisms

“…Mice were then challenged by whole body inhalation exposure (nebulization) to aerosolized 2.5% (w/v) OVA in saline for 30 minutes, three days per week for six weeks using an ultrasonic nebulizer (NE-U07, Omron Corporation, Tokyo, Japan), between days 21 and 63 to establish AAD. Following exposure to nebulized OVA, mice were treated with 5mg/kg LSF (OVA-LSF) or 100mg/kg SAHA (OVA-SAHA, n=6) or vehicle control (OVA-VEH, n=15) by i.p injection, three days per week for six weeks, which represent doses known to be effective and non-toxic 30,44 . A fourth group of mice were sensitized with 1mg alum in 0.5ml saline on days 0 and 14, and were challenged with saline aerosols three days per week for six weeks (n=15).…”

Sulforaphane prevents and reverses allergic airways disease in mice via anti-inflammatory, antioxidant, and epigenetic mechanisms

“…We examined the effects of LSF in the OVA-induced murine model of chronic AAD, and LSF was found to prevent and reverse the pathological features associated with the model (Figs 1 and 2). The OVA-induced murine model is well characterized and recapitulates several hallmarks of chronic asthma 24,26,30 . Our findings highlighted that the key features associated with the model, including epithelial thickening, goblet cell metaplasia, apoptosis in the bronchial epithelium, and inflammatory cell infiltration were prominent in OVA-VEH sensitized mice.…”

“…Mice were then challenged by whole body inhalation exposure (nebulization) to aerosolized 2.5% (w/v) OVA in saline for 30 minutes, three days per week for six weeks using an ultrasonic nebulizer (NE-U07, Omron Corporation, Tokyo, Japan), between days 21 and 63 to establish AAD. Following exposure to nebulized OVA, mice were treated with 5mg/kg LSF (OVA-LSF) or 100mg/kg SAHA (OVA-SAHA, n=6) or vehicle control (OVA-VEH, n=15) by i.p injection, three days per week for six weeks, which represent doses known to be effective and non-toxic 30,44 . A fourth group of mice were sensitized with 1mg alum in 0.5ml saline on days 0 and 14, and were challenged with saline aerosols three days per week for six weeks (n=15).…”

“…The sections were subsequently deparaffinized by two consecutive 5 min washes with xylene (Sigma-Aldrich) and stored in desiccator prior to FPA-FTIR microspectroscopy. The acquisition of FPA-FTIR chemical images was performed using an offline FPA-FTIR microspectroscopic system (Bruker Optik GmbH, Ettlingen, Germany) at the Australian Synchrotron Infrared Microspectroscopy (IRM) beamline (Clayton, VIC, Australia) as described previously 30 . Briefly, the FPA-FTIR images were acquired in transmission mode using a Bruker Hyperion 3000 FTIR microscope, with a liquid nitrogen cooled 64 x 64 element FPA detector and a matching 15x objective and condenser (NA = 0.40), coupled to a Bruker Vertex 70 FTIR spectrometer with an internal thermal (Globar TM ) IR source.…”