Insulin-transferrin-selenium as an alternative to foetal serum for epidermal equivalents.

OBJECTIVE: Organotypic skin models are powerful tools for research in development, ageing and diseases. They have become more and more complex with the use of multiple cell types. This requires a culture medium adapted to optimize the development of such in vitro skin. Foetal bovine serum (FBS) is the most complete supplement in existence at the moment, providing at once growth factors, vitamins, hormones and other circulating compounds. However, this cocktail suffers from batch variability and its animal origin is ethically questionable. More importantly, its biological activities may interfere with the study of certain signalling pathways. Here, we present a strategy for constructing an epidermal equivalent using a defined culture medium without serum. METHODS: An epidermal equivalent was constructed with primary human keratinocytes cultured using an insulin-transferrin-selenium (ITS) medium. Determination of steady-state gene expression levels and the immunohistological characterization of keratinocyte markers were performed to compare the ITS medium condition with a reference model, where keratinocytes were co-cultured with fibroblasts in the presence of FBS. RESULTS: The data show that the ITS medium promoted the expression of keratinocyte proliferation and differentiation markers at the protein and transcript levels in a similar way to that of the reference model. CONCLUSION: We show that culture using the ITS medium appears as a viable replacement for FBS in the construction of epidermal equivalents, opening the way to signal transduction studies.

Cutaneous neurovascular interaction involved in tactile sensation.

The sense of touch is one of the most vital; still, it is incompletely understood. We review the afferent function that allows for the relay of sensory information from the periphery (the skin) to the central nervous system. Within this afferent function, we examine the different integrating levels including several candidates for cutaneous transducers, the conduction of the information via the afferent nervous fibres and the transmission of the sensory stimuli to higher brain structures, resulting in the perception of the different senses. We then examine the efferent system that stimulates the skin by secreting neurotransmitters. Finally, we discuss the tools available to study the cutaneous neurovascular interaction and conclude on a novel test that assesses this interaction triggered by the application of a local non noxious pressure (tactile stimulation).

Deterioration in carotid baroreflex during carotid endarterectomy.

OBJECTIVE: Blood pressure instability after carotid endarterectomy (CEA) has been associated with a disturbance of the baroreflex control mechanism caused by the surgery in the carotid sinus region. The purpose of this study was to determine if a deterioration in carotid baroreceptors occurs during the surgery. METHOD: Heart rate (HR) and blood pressure (BP) were recorded continuously in 60 patients undergoing CEA as well as preoperatively and postoperatively at 2 days and 6 weeks. The baroreflex sensitivity was determined by cross-spectral analysis of HR and systolic blood pressure (SBP). During the surgery, three tests were used to assess the baroreflex response. The first test simulated a sudden fall in systemic blood pressure by clamping the common carotid artery. The second test simulated a rise in systemic blood pressure by applying pressure by using a rubbing action on the luminal surface of the carotid sinus region. The rub test was performed twice, once with the atheromatous plaque in situ and once when the plaque had been removed. The third test is clamp removal and restoration of blood flow through the carotid sinus. RESULTS: Carotid cross-clamping increased mean +/- standard error of the mean SBP from 117 +/- 3 mm Hg before clamping to 125 +/- 3 mm Hg (P <.05) at 30 beats after clamping. The first rub test with the plaque in situ decreased SBP from 121 +/- 3 mm Hg to 117 +/- 3 mm Hg (P <.01) at 10 beats after the rub test, indicating a functioning baroreceptor reflex. The second rub test increased SBP from 126 +/- 3 mm Hg to 128 +/- 3 mm Hg (P <.05). SBP dropped (P <.01) when unclamping suggesting a selective alteration of the baroreflex sensitivity. The baroreflex sensitivity was significantly reduced 2 days postoperatively when compared to preoperative values (P <.05). CONCLUSIONS: These findings suggest that the act of plaque removal could be associated with a partial disruption of baroreceptor mechanism in the carotid artery. This could affect type I baroreceptors.

Break excitation alone does not explain the delay and amplitude of anodal current-induced vasodilatation in human skin.

In iontophoresis experiments, a 'non-specific' current-induced vasodilatation interferes with the effects of the diffused drugs. This current-induced vasodilatation is assumed to rely on an axon reflex due to excitation of cutaneous nociceptors and is weaker and delayed at the anode as compared to the cathode. We analysed whether these anodal specificities could result from a break excitation of nociceptors. Break excitation is the generation of action potentials at the end of a square anodal DC current application, which are generally weaker than those observed at the onset of a same application at the cathode. In eight healthy volunteers, we studied forearm cutaneous laser Doppler flow (LDF) responses to: (1) anodal and cathodal 100 microA current applications of 1, 2, 3, 4 or 5 min; (2) 100 microA anodal applications of 3 min with a progressive ending over 100 s (total charge 23 mC); these were compared to square-ended 100 microA anodal applications of the same total charge (23 mC) or duration (3 min); (3) a 4 min 100 microA anodal application with a 333 msec break at half time. Results (mean +/- S.D.) are expressed as percentage of heat-induced maximal vasodilatation (%MVD). Onset (T(vd)) and amplitude (LDF(peak)) of vasodilatation were determined. We observed that: T(vd) was linearly related to the duration of current application at the anode (slope = 1.01, r(2) = 0.99, P < 0.0001) but not at the cathode (slope = 0.03, r(2) = 0.02, n.s.). Progressive ending of anodal current did not decrease LDF(peak) (63.3 +/- 24.6 %MVD) as compared to square-ending of current application of the same duration (36.9 +/- 22.2 %MVD) or the same total charge (57.1 +/- 23.5 %MVD). A transient break of anodal current did not allow for the vasodilatation to develop until current was permanently stopped. We conclude that, during iontophoresis, anodal break excitation alone cannot account for the delay and amplitude of the vascular response.