Deep tissue injury in development of pressure ulcers: a decrease of inflammasome activation and changes in human skin morphology in response to aging and mechanical load.

Molecular mechanisms leading to pressure ulcer development are scarce in spite of high mortality of patients. Development of pressure ulcers that is initially observed as deep tissue injury is multifactorial. We postulate that biomechanical forces and inflammasome activation, together with ischemia and aging, may play a role in pressure ulcer development. To test this we used a newly-developed bio-mechanical model in which ischemic young and aged human skin was subjected to a constant physiological compressive stress (load) of 300 kPa (determined by pressure plate analyses of a person in a reclining position) for 0.5-4 hours. Collagen orientation was assessed using polarized light, whereas inflammasome proteins were quantified by immunoblotting. Loaded skin showed marked changes in morphology and NLRP3 inflammasome protein expression. Sub-epidermal separations and altered orientation of collagen fibers were observed in aged skin at earlier time points. Aged skin showed significant decreases in the levels of NLRP3 inflammasome proteins. Loading did not alter NLRP3 inflammasome proteins expression in aged skin, whereas it significantly increased their levels in young skin. We conclude that aging contributes to rapid morphological changes and decrease in inflammasome proteins in response to tissue damage, suggesting that a decline in the innate inflammatory response in elderly skin could contribute to pressure ulcer pathogenesis. Observed morphological changes suggest that tissue damage upon loading may not be entirely preventable. Furthermore, newly developed model described here may be very useful in understanding the mechanisms of deep tissue injury that may lead towards development of pressure ulcers

Loading induces sub-epidermal separation and alters orientation of collagen fibers in aged individuals.

<p>H&E staining of unloaded and loaded young (<b>A</b>) and aged (<b>B</b>) skin for 0.5, 1, 2 and 4 hrs (n = 15 specimens per n = 3 experimental replicates). Breaks in a near proximity to basement membrane, as indicated by black arrows, are observed 4 hrs after loading in young and 2 and 4 hrs after loading in aged skin. Inserts represent enlarged images of the areas indicated by black arrows. Magnification 20×. Scale bar 100 µm. Orientation of collagen fibers in a dermis of unloaded and loaded young (<b>C</b>) and aged (<b>D</b>) skin. Quantification of collagen orientation in a plane of section indicates significantly higher mean value in loaded aged skin after 2 and 4 hours of loading. Values are expressed as mean ± SD (n = 15 specimens per n = 3 experimental replicates). * Indicates p<0.05 by paired Student t-test.</p

Maximum contact pressure measurements.

<p>An EMED-X sensor array was used to measure contact pressure with sub-cm² resolution on a seated individual. A maximum pressure map is shown in the horizontal view (<b>A</b>) and rotated to illustrate the spatial pressure distribution in 3-D (<b>B</b>). The graph shows the pressure in N/cm² (1 N/cm² = 10 kPa) as a function of time for the region of peak pressure(<b>C</b>). METS system used for skin loading (<b>D</b>).</p

Aged skin has significantly lower levels of IL-1β as compared to young skin regardless of loading, whereas loading induces NLRP3 and caspase-1 protein levels in young human skin.

<p>IL-1β levels in loaded and unloaded young and aged skin was determined by Western blots (<b>A</b>). Quantification of these immunoblots by densitometry using β-actin as a control (n = 3) (<b>B</b>) shows that loading decreased IL-1β expression in young skin. Levels of IL-1β in aged skin are negligible as compared to young skin regardless of load. Levels of NLRP3 inflammasome proteins quantified by Western blot are significantly lower in aged skin as compared to young skin. Caspase-1(<b>C</b>), ASC (<b>D</b>) and NLRP3(<b>E</b>) protein levels, quantified by densitometry using β-actin as a control (n = 3), show that loading induced a significant increase in NLRP3 and caspase-1 expression in young skin 0.5 and 4 hrs upon loading, but not in aged skin. Values are expressed as mean ± SEM. * Indicates p<0.05 by Student t-test.</p

Workshop on Transboundary Issues in Marine Spatial Planning (WKTBIMP)

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