Multiplex analysis of mass imaging data: Application to the pathology of experimental myocardial infarction.

AIM: Imaging mass cytometry (IMC) affords simultaneous immune-labelling/imaging of multiple antigens in the same tissue. Methods utilizing multiplex data beyond co-registration are lacking. This study developed and applied an innovative spatial analysis workflow for multiplex imaging data to IMC data determined from cardiac tissues and revealed the mechanism(s) of neutrophil-mediated post-myocardial-infarction damage. METHODS: IMC produced multiplex images with various redox/inflammatory markers. The cardiac peri-infarct zone (PIZ) was determined to be up to 240 µm from the infarct border based on the presence of neutrophils. The tissue region beyond the infarct was defined as the remote area (RA). ImageJ was used to quantify the immunoreactivity. Functional assessments included infarct size, cell necro/apoptosis, total thiol assay and echocardiogram. RESULTS: Expression of damage markers decreased in order from the infarct area to PIZ and then RA, reflecting the neutrophil density in the regions. Concentrically spaced "shoreline contour analysis" around the cardiac infarct extending into the PIZ showed that immunoreactivity for damage markers decreased linearly with increasing distance from the infarct, concomitant with a decreasing neutrophil-myeloperoxidase (MPO) gradient from the infarct to the PIZ. Stratifying by concentric bands around individual MPO+ -signal identified that the immunoreactivity of haem-oxygenase-1 (HO-1) and phosphorylated-p38 mitogen-activated protein kinase (pP38) peaked near neutrophils. Furthermore, spatial dependence between neutrophils and markers of cardiac cellular damage was confirmed by nearest-neighbour distance analysis. Post-infarction tissue exhibited declined functional parameters that were associated with neutrophil migration from the infarct to PIZ. CONCLUSION: This image-based quantitative protocol revealed the spatial association and provided potential molecular pathways responsible for neutrophil-mediated damage post-infarction.

Pro-Inflammatory Serum Amyloid a Stimulates Renal Dysfunction and Enhances Atherosclerosis in Apo E-Deficient Mice.

Acute serum amyloid A (SAA) is an apolipoprotein that mediates pro-inflammatory and pro-atherogenic pathways. SAA-mediated signalling is diverse and includes canonical and acute immunoregulatory pathways in a range of cell types and organs. This study aimed to further elucidate the roles for SAA in the pathogenesis of vascular and renal dysfunction. Two groups of male ApoE-deficient mice were administered SAA (100 µL, 120 µg/mL) or vehicle control (100 µL PBS) and monitored for 4 or 16 weeks after SAA treatment; tissue was harvested for biochemical and histological analyses at each time point. Under these conditions, SAA administration induced crosstalk between NF-κB and Nrf2 transcriptional factors, leading to downstream induction of pro-inflammatory mediators and antioxidant response elements 4 weeks after SAA administration, respectively. SAA treatment stimulated an upregulation of renal IFN-γ with a concomitant increase in renal levels of p38 MAPK and matrix metalloproteinase (MMP) activities, which is linked to tissue fibrosis. In the kidney of SAA-treated mice, the immunolocalisation of inducible nitric oxide synthase (iNOS) was markedly increased, and this was localised to the parietal epithelial cells lining Bowman's space within glomeruli, which led to progressive renal fibrosis. Assessment of aortic root lesion at the study endpoint revealed accelerated atherosclerosis formation; animals treated with SAA also showed evidence of a thinned fibrous cap as judged by diffuse collagen staining. Together, this suggests that SAA elicits early renal dysfunction through promoting the IFN-γ-iNOS-p38 MAPK axis that manifests as the fibrosis of renal tissue and enhanced cardiovascular disease.

The Synthetic Myeloperoxidase Inhibitor AZD3241 Ameliorates Dextran Sodium Sulfate Stimulated Experimental Colitis.

Chronic inflammatory bowel disease (IBD) is a condition with multifactorial pathophysiology. To date, there is no permanent cure and the disease is primarily managed by immunosuppressive drugs; long-term use promotes serious side effects including increased risk malignancies. The current study aimed to target neutrophil-myeloperoxidase, a key contributor to the pathogenesis of IBD, through the use of AZD3241that inhibits extracellular myeloperoxidase. Experimental colitis was induced in C57BL/6 male mice by 2% dextran sodium sulfate in drinking water ad libitum over 9 days. Mice received either normal drinking water and peanut butter (control), 2% DSS in drinking water and peanut butter or 2% DSS in drinking water and AZD3241 (30 mg/kg) dispersed in peanut butter daily for 9 days. Administered AZD3241 attenuated body weight loss (10% p<0.05) and improved clinical score (9 fold p<0.05; a score comprising the time-dependent assessment of stool consistency and extent of rectal bleeding), loss of colonic crypts (p<0.001), preserved surface epithelium (p<0.001) and enhanced expression of the transcription factor Nrf-2 (regulator of antioxidants) and enhanced expression of the downstream antioxidant response element haeoxygenase-1 (HO-1) in the colon tissue. Also, the concentration of fecal hemoglobin and the myeloperoxidase specific oxidative damage biomarker 3-chlorotyrosine in the colon were significantly decreased in the presence of AZD3241. This latter result was consistent with AZD3241 inhibiting MPO activity in vitro. Overall, AZD3241 ameliorated the course and severity of experimental colitis through ameliorating MPO derived tissue damage and could be considered a potential therapeutic option, subject to further validation in chronic IBD models.

The role of sodium thiocyanate supplementation during dextran sodium sulphate-stimulated experimental colitis.

Ulcerative colitis is a condition characterised by the infiltration of leukocytes into the gastrointestinal wall. Leukocyte-MPO catalyses hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN) formation from chloride (Cl-) and thiocyanous (SCN-) anions, respectively. While HOCl indiscriminately oxidises biomolecules, HOSCN primarily targets low-molecular weight protein thiols. Oxidative damage mediated by HOSCN may be reversible, potentially decreasing MPO-associated host tissue destruction. This study investigated the effect of SCN- supplementation in a model of acute colitis. Female mice were supplemented dextran sodium sulphate (DSS, 3% w/v) in the presence of 10 mM Cl- or SCN- in drinking water ad libitum, or with salts (NaCl and NaSCN only) or water only (controls). Behavioural studies showed mice tolerated NaSCN and NaCl-treated water with water-seeking frequency. Ion-exchange chromatography showed increased fecal and plasma SCN- levels in thiocyanate supplemented mice; plasma SCN- reached similar fold-increase for smokers. Overall there was no difference in weight loss and clinical score, mucin levels, crypt integrity and extent of cellular infiltration between DSS/SCN- and DSS/Cl- groups. Neutrophil recruitment remained unchanged in DSS-treated mice, as assessed by fecal calprotectin levels. Total thiol and tyrosine phosphatase activity remained unchanged between DSS/Cl- and DSS/SCN- groups, however, colonic tissue showed a trend in decreased 3-chlorotyrosine (1.5-fold reduction, p < 0.051) and marked increase in colonic GCLC, the rate-limiting enzyme in glutathione synthesis. These data suggest that SCN- administration can modulate MPO activity towards a HOSCN-specific pathway, however, this does not alter the development of colitis within a DSS murine model.

Virtual Reality interventions for acute and chronic pain management.

Virtual Reality (VR) is now consumer ready and nearing ubiquity. In terms of clinical applications, several studies suggest that VR can be effective as a complementary adjunct or alternative non-pharmacologic analgesic in a range of pain-inducing procedures and in management of chronic pain. The increasing affordability and quality of portable VR headsets and the ongoing utility of pain therapy signals an exciting future for the use of VR for analgesia. However, further research is needed to establish its long-term benefits if VR is to be adopted into mainstream protocols for analgesia management. This research requires a range of study designs with collection of patient self-report and clinical data together to develop bespoke interventions for different cohorts.