Hyperosmolar stress induces neutrophil extracellular trap formation: implications for dry eye disease.

PURPOSE: To determine if hyperosmolar stress can stimulate human neutrophils to form neutrophil extracellular traps (NETs) and to investigate potential strategies to reduce formation of NETs (NETosis) in a hyperosmolar environment. METHODS: Neutrophils were isolated from peripheral venous blood of healthy subjects and incubated in iso-osmolar (280 mOsM) or hyperosmolar (420 mOsM) media for 4 hours. Neutrophil extracellular traps were quantified using a PicoGreen dye assay to measure extracellular DNA. Two known inhibitors of NETosis, staurosporine and anti-ß2 integrin blocking antibody, and two proresolution formyl peptide receptor 2 (FPR2) agonists, annexin/lipocortin-1 mimetic peptide and 15-epi-lipoxin A4, were evaluated as possible strategies to reduce hyperosmolarity-induced NETosis. RESULTS: The amount of NETs induced by hyperosmolar medium (420 mOsM) increased linearly over time to 3.2 ± 0.3 times that induced by iso-osmolar medium at 4 hours (P < 0.05). NETosis increased exponentially with increasing osmolarity and was independent of the stimulus used to increase osmolarity. Upon neutrophil exposure to hyperosmolar stress, restoration of iso-osmolar conditions decreased NET formation by 52.7% ± 5% (P < 0.05) but did not completely abrogate it. Among the strategies tested to reduce NETosis in a hyperosmolar environment, annexin-1 peptide was the most efficacious. CONCLUSIONS: Hyperosmolarity induces formation of NETs by neutrophils. This NETosis mechanism may explain the presence of excessive NETs on the ocular surface of patients with dry eye disease. Because they reduce hyperosmolarity-induced NETosis, FPR2 agonists may have therapeutic potential in these patients.

Mouse strain variation in SMA(+) myofibroblast development after corneal injury.

The purpose of present study was to investigate differences in myofibroblast development after haze-generating injury in different commonly used strains of mice. The inbred mouse strains used in this study were Balb/c, C57BL/6, C3H/HeJ and DBA/1J. All mice had uniform irregular phototherapeutic keratectomy with an excimer laser according to a previously published method to generate stromal haze. DBA/1J mice generated significantly greater density of alpha smooth muscle actin (SMA)-positive myofibroblasts in the anterior stroma compared to Balb/c (p < 0.05), C57BL/6 (p < 0.05) and C3H/HeJ (p < 0.01) mice. The C3H/HeJ strain had significantly lower density of SMA-positive myofibroblasts compared to other three mouse strains. These results indicate that mouse strain must be considered in designing experiments and interpreting the results of experiments in which corneal haze and myofibroblast generation is studied in mice. Further investigation of genetics underlying mouse strain variation could provide insight into the corneal wound healing and haze generation processes.

Genetic ablation of calcium-independent phospholipase A(2)beta causes hypercontractility and markedly attenuates endothelium-dependent relaxation to acetylcholine.

Activation of phospholipases leads to the release of arachidonic acid and lysophospholipids that play prominent roles in regulating vasomotor tone. To identify the role of calcium-independent phospholipase A(2)beta (iPLA(2)beta) in vasomotor function, we measured vascular responses to phenylephrine (PE) and ACh in mesenteric arterioles from wild-type (WT; iPLA(2)beta(+/+)) mice and those lacking the beta-isoform (iPLA(2)beta(-/-)) both ex vivo and in vivo. Vessels isolated from iPLA(2)beta(-/-) mice demonstrated increased constriction to PE, despite lower basal smooth muscle calcium levels, and decreased vasodilation to ACh compared with iPLA(2)beta(+/+) mice. PE constriction resulted in initial intracellular calcium release with subsequent steady-state constriction that depended on extracellular calcium influx. Endothelial denudation had no effect on vessel tone or PE-induced constriction although the dilation to ACh was significantly reduced in iPLA(2)beta(+/+) vessels. In contrast, vessels from iPLA(2)beta(-/-) constricted by 54% after denudation, indicating smooth muscle hypercontractility. In vivo, blood pressure, resting vessel diameter, and constriction of mesenteric vessels to PE were not different in iPLA(2)beta(-/-) vessels compared with WT mouse vessels. However, relaxation after ACh administration in situ was attenuated, indicating an endothelial inability to induce dilation in response to ACh. In cultured endothelial cells, inhibition of iPLA(2)beta with (S)-(E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (BEL) decreased endothelial nitric oxide synthase phosphorylation and reduced endothelial agonist-induced intracellular calcium release as well as extracellular calcium influx. We conclude that iPLA(2)beta is an important mediator of vascular relaxation and intracellular calcium homeostasis in both smooth muscle and endothelial cells and that ablation of iPLA(2)beta causes agonist-induced smooth muscle hypercontractility and reduced agonist-induced endothelial dilation.

Skull burns resulting in calvarial defects: cognitive and affective outcomes.

OBJECTIVE: To determine the extent of cognitive and affective difficulties experienced in children with full thickness burns of the calvaria caused by flame and electrical sources. METHODS: Medical records were examined retrospectively to identify patients with full thickness burns of the calvaria. Data were reviewed from the acute period and long term for these patients and their matched controls. RESULTS: Fourteen children were identified who sustained full thickness burns. The average age of injury was 7.1+/-7.3 years and average percent TBSA was 39.7+/-26.3. Of these 14 patients, 8 were under the age of 4 years at the time of injury and suffered flame injuries. Six children were over 11 years old at injury and suffered electrical burns. Imaging was performed on 10 patients acutely and revealed 5 with major brain injury. The youngest patients suffering flame injuries experienced significant difficulty with developmental functioning acutely and a trend towards long-term cognitive difficulty. Older children did not differ from their matched controls at any time. CONCLUSIONS: Children with burns to the calvarium can be at risk for affective and cognitive sequalae. Therefore, we recommend comprehensive neuropsychological evaluations at multiple time points to adequately assess functioning across time.