Enhancing Techniques for Determining Inflammatory Edema Formation and Neutrophil Accumulation in Murine Skin.

Inflammatory edema formation and polymorphonuclear leukocyte (neutrophil) accumulation are common components of cutaneous vascular inflammation, and their assessment is a powerful investigative and drug development tool but typically requires independent cohorts of animals to assess each. We have established the use of a mathematical formula to estimate the ellipsoidal-shaped volume of the edematous wheal or bleb after intradermal injections of substances in mice pretreated intravenously with Evans blue dye (which binds to plasma albumin) to act as an edema marker. Whereas previous extraction of Evans blue dye with formamide is suitable for all strains of mice, we report this quicker and more reliable assessment of edema volume in situ. This therefore allows neutrophil accumulation to be assessed from the same mouse using the myeloperoxidase assay. Importantly, we examined the influence of Evans blue dye on the spectrometry readout at the wavelength at which myeloperoxidase activity is measured. The results indicate that it is feasible to quantify edema formation and neutrophil accumulation in the same mouse skin site. Thus, we show techniques that can assess edema formation and neutrophil accumulation at the same site in the same mouse, allowing paired measurements and reducing the total use of mice by 50%.

Elucidating the Ability of CGRP to Modulate Microvascular Events in Mouse Skin.

Oedema formation and polymorphonuclear leukocyte (neutrophil) accumulation are involved in both acute and chronic inflammation. Calcitonin gene-related peptide (CGRP) is a sensory neuropeptide that is released from stimulated sensory nerves. CGRP is a potent vasodilator neuropeptide, especially when administered to the cutaneous microvasculature, with a long duration of action. Here, we have investigated the ability of vasodilator amounts of CGRP to modulate oedema formation and neutrophil accumulation induced in the cutaneous microvasculature of the mouse. To learn more about the mechanism of action of endogenous CGRP, we have investigated the response to the inflammatory stimulants tumour necrosis factor alpha (TNFα) and carrageenan in three different murine models: a model where sensory nerves were depleted by resiniferatoxin (RTX); a pharmacological method to investigate the effect of a selective CGRP receptor antagonist; and a genetic approach using wildtype (WT) and αCGRP knockout (KO) mice. Our results show that exogenous CGRP potentiates oedema formation induced by substance P (SP) and TNFα. This is further supported by our findings from sensory nerve-depleted mice (in the absence of all neuropeptides), which indicated that sensory nerves are involved in mediating the oedema formation and neutrophil accumulation induced by TNFα, and also carrageenan in cutaneous microvasculature. Furthermore, endogenous CGRP was shown to contribute to this inflammatory response as carrageenan-induced oedema formation is attenuated in WT mice treated with the CGRP receptor antagonist, and in αCGRPKO mice. It is therefore concluded that CGRP can contribute to inflammation by promoting oedema formation in skin, but this response is dependent on the pro-inflammatory stimulus and circumstance.

(-)-Englerin-A Has Analgesic and Anti-Inflammatory Effects Independent of TRPC4 and 5.

Recently, we found that the deletion of TRPC5 leads to increased inflammation and pain-related behaviour in two animal models of arthritis. (-)-Englerin A (EA), an extract from the East African plant Phyllanthus engleri has been identified as a TRPC4/5 agonist. Here, we studied whether or not EA has any anti-inflammatory and analgesic properties via TRPC4/5 in the carrageenan model of inflammation. We found that EA treatment in CD1 mice inhibited thermal hyperalgesia and mechanical allodynia in a dose-dependent manner. Furthermore, EA significantly reduced the volume of carrageenan-induced paw oedema and the mass of the treated paws. Additionally, in dorsal root ganglion (DRG) neurons cultured from WT 129S1/SvIm mice, EA induced a dose-dependent cobalt uptake that was surprisingly preserved in cultured DRG neurons from 129S1/SvIm TRPC5 KO mice. Likewise, EA-induced anti-inflammatory and analgesic effects were preserved in the carrageenan model in animals lacking TRPC5 expression or in mice treated with TRPC4/5 antagonist ML204.This study demonstrates that while EA activates a sub-population of DRG neurons, it induces a novel TRPC4/5-independent analgesic and anti-inflammatory effect in vivo. Future studies are needed to elucidate the molecular and cellular mechanisms underlying EA's anti-inflammatory and analgesic effects.

Vascular Endothelial Primary Cilia: Mechanosensation and Hypertension.

Primary cilia are sensory organelles that extend from the cell surface and sense extracellular signals. Endothelial primary cilia protruding from the inner surface of blood vessel walls sense changes in blood flow and convert this mechanosensation into an intracellular biochemical/molecular signal, which triggers a cellular response. Primary endothelial cilia dysfunction may contribute to the impairment of this response and thus be directly implicated in the development of vascular abnormalities such as hypertension and aneurysms. Using both in vitro techniques as well as in vivo animal models, we and others have investigated fluid flow mechanosensory functions of endothelial cilia in cultured cells, animal models and autosomal dominant polycystic kidney disease (ADPKD) patients. More in-depth studies directed at identification of the mechanisms of fluid flow sensing will further enhance our knowledge of cilia-dependent vascular pathology. Although the current treatments aimed at treating the cardiovascular symptoms in ADPKD patients successfully slowed the progression of cyst growth, there is growing evidence which suggests that drugs which interfere with primary cilia function or structure could reduce cardiovascular complications in ADPKD. This review is to summarize the most recent studies on primary endothelial cilia function in the vascular system and to present primary cilia as a novel therapeutic target for vascular hypertension.