Revealing the Meissner Corpuscles in Human Glabrous Skin Using In Vivo Non-Invasive Imaging Techniques.

The presence of mechanoreceptors in glabrous skin allows humans to discriminate textures by touch. The amount and distribution of these receptors defines our tactile sensitivity and can be affected by diseases such as diabetes, HIV-related pathologies, and hereditary neuropathies. The quantification of mechanoreceptors as clinical markers by biopsy is an invasive method of diagnosis. We report the localization and quantification of Meissner corpuscles in glabrous skin using in vivo, non-invasive optical microscopy techniques. Our approach is supported by the discovery of epidermal protrusions which are co-localized with Meissner corpuscles. Index fingers, small fingers, and tenar palm regions of ten participants were imaged by optical coherence tomography (OCT) and laser scan microscopy (LSM) to determine the thickness of the stratum corneum and epidermis and to count the Meissner corpuscles. We discovered that regions containing Meissner corpuscles could be easily identified by LSM with an enhanced optical reflectance above the corpuscles, caused by a protrusion of the strongly reflecting epidermis into the stratum corneum with its weak reflectance. We suggest that this local morphology above Meissner corpuscles has a function in tactile perception.

Analysis of the morphometric parameters of pig ear hair follicles.

BACKGROUND: Porcine ear skin is used in studies of percutaneous penetration as a substitute for human skin. The objective of the present study was to determine the structure of the hair follicles on the dorsal area of porcine ear skin and make a morphometric comparison with the hair follicles of human skin. MATERIALS AND METHODS: Sections of frozen biopsies were cut vertically to the skin surface in longitudinal sections using a cryotome and were investigated using microscopy. For each hair follicle, various parameters were determined. RESULTS: The follicular density in porcine ear skin varies according to the area studied, and the length of most of the follicles was approximately 1458 ± 286 µm. The size of the follicular orifice was also determined in a total of 305 follicles. It showed a diameter of roughly 113 ± 43 µm. CONCLUSION: The results showed a very good similarity between human and pig hair follicles. Therefore, porcine ear skin can be considered as a very suitable model of human skin in dermal and especially follicular penetration studies.

Solvent-Containing Closure Material Can Be Used to Prevent Follicular Penetration of Caffeine and Fluorescein Sodium Salt on Porcine Ear Skin.

AIM: The skin represents a drug delivery portal. The establishment of a skin model capable of distinguishing between the follicular and intercellular penetration pathways remains a challenge. The study described herein was aimed at showing the influence of two nail varnishes as closure material and four application techniques to spread the active pharmaceutical ingredient (API) on a successful follicular closure without inducing penetration-enhancing effects. MATERIALS AND METHODS: For all experiments, ex vivo porcine ear skin was used. In study design A, a standard and a solvent-free nail varnish were compared. It was tested whether the different application techniques (spreading with pipette, careful finger massage, 5-Hz finger massage, 5-Hz automatic massage) potentially destroy an intact follicular closure. Laser scanning microscopy imaging was used to measure if the model drug (fluorescein sodium salt) penetrated into the hair follicles. Study design B investigated how the penetration is affected when applying standard nail varnish containing solvents to skin. It was tested if the varnish blocks the API (caffeine) on completely covered areas and if adjacent areas show increased penetration. Furthermore, lateral diffusion of the API was investigated. After 20 h, the skin layers were separated by tape stripping and heat separation. The tissue samples were homogenized. Caffeine was quantified by chromatography. RESULTS: In study design A, the standard nail varnish showed a secure follicular closure, while the solvent-free nail varnish was not able to prevent follicular penetration. Moreover, rapid application techniques were found to destroy an intact follicular closure. Only the two most gentle application techniques kept the follicular closing intact. In study design B, no caffeine was detected in both skin areas that were completely covered. Since no significant difference in caffeine penetration between the two uncovered groups was found, any influence of the applied closure material on adjacent areas was excluded. CONCLUSION: This study clearly demonstrates that a standard nail varnish in combination with a gentle application technique of the API provides a secure follicular closure. The presented study only investigated the closure for the substances caffeine and fluorescein sodium salt. The results might not be transferable to all kinds of APIs.

The significance of thrombophilia in paediatric thromboembolism.

The role of thrombophilia in paediatric thromboembolic disease remains controversial. The objective of the present study was to investigate the prevalence of thrombophilia and other risk factors among children with a thromboembolic event. We systematically examined the medical records and laboratory results of 126 children (≤15 years) referred to Aarhus University Hospital, Denmark with a recent thromboembolic event during 2005-2016. Among the 126 children with thrombosis, 99 (79%) had arterial thrombosis, 66 (52%) being in the neonatal period. Remarkably, 48 (38%) children had occurrence of acute illness or underlying disease prior to the thromboembolic event. In total, 11 (11%) children with arterial thrombosis had a hereditary or acquired thrombophilia with a non-significant odds ratio compared with the general population, while 9 of the 27 (33%) children with venous thrombosis had a hereditary or acquired thrombophilia being significantly higher than in the general population. Acute illness or underlying diseases seem to induce increased risk of thromboembolic disease in children. The present study does not support that thrombophilia is associated with arterial thrombosis. However, the results support thrombophilia testing in children with venous thromboembolism.

Prevalence of incontinence, attention deficit/hyperactivity disorder and oppositional defiant disorder in preschool children.

Externalizing disorders as attention deficit/hyperactivity disorder (ADHD) and oppositional defiant disorder (ODD) are common in children with nocturnal enuresis (NE), daytime urinary incontinence (DUI) and faecal incontinence (FI). We examined the prevalence rates of ADHD, ODD and incontinence in a defined geographical area and analysed the association between externalizing disorders and subtypes of incontinence. 1,676 parents of children who were presented at the mandatory school-entry medical examination completed a questionnaire with all DSM-IV items of ADHD, ODD and six questions regarding incontinence. 50.2% were male and mean age was 5.7 years. 9.1% had at least one subtype of incontinence (8.5% had NE, 1.9% DUI and 0.8% FI). Boys were significantly more affected by incontinence overall, NE, FI and ADHD than girls. 6.4% had ADHD, 6.2% had ODD and 2.6% were affected by ADHD and ODD. 10.3% of the children with incontinence had ADHD and 10.3% ODD. Children with FI were significantly more affected by externalizing disorders (50%) than children with isolated NE (14.5%), children with DUI (9.5%) and continent children (9.5%). Children with incontinence, especially those with FI, are at much higher risk of externalizing disorders. An additional effect of children with both ADHD and ODD having higher rates of incontinence than children with only one disorder could not be found. However, these children represent a high-risk group with lower compliance to treatment and worse outcome. Therefore, screening not only for ADHD but also for ODD should be implemented for all children with incontinence.

Elimination disorders and anxious-depressed symptoms in preschool children: a population-based study.

The prevalence rates of elimination disorders and anxious/depressed symptoms of a representative sample of young children and their associations were assessed. 2,079 children in a defined geographical area were examined at school-entry. A parental questionnaire with 4 questions referring to symptoms of incontinence and 14 items of the anxious/depressed scale of the Child Behavior Checklist (CBCL) was administered. 9.5 % of 6-year-old children wet at night, 2.7 % wet during day and 1.2 % had faecal incontinence. Significantly more boys wet at night (11.7 %) than girls (7.2 %; OR 0.58, 95 % CI 0.43-0.78). 12.7 % had clinically relevant anxious/depressed symptoms. Children with at least one elimination disorder had significantly higher T values of the 'anxious/depressed' CBCL than continent controls. Children with faecal incontinence had highest T values of the 'anxious/depressed' CBCL syndrome scale, significantly higher than those of children with other elimination disorders and controls. Elimination disorders, as well as anxious/depressed symptoms are common at a young age. Boys are more affected by elimination disorders than girls, but not by anxious/depressed symptoms. Children with faecal incontinence have the highest rate of anxious/depressed symptoms.

Association between maternal social vulnerability and cesarean delivery: A cohort study.

OBJECTIVE: Although social vulnerability has been correlated to adverse obstetrical outcomes, its definition as well as its correlation to mode of delivery vary between different studies. The aim of this study is to evaluate the association between maternal social vulnerabilities and cesarean section with the inclusion of a wide range of social vulnerability characteristics. STUDY DESIGN: The current study is a retrospective single center cohort study in a tertiary care maternity unit between January 2020 and December 2021. All women who delivered after 24 gestational weeks were included. Multiple component analysis (MCA) grouped vulnerability characteristics in three independent vulnerability axes, named after their clinical relevance as administrative, psychological, and dependency axis. Multiple logistic regression was performed, controlling for obstetrical, medical factors as well as the Robson classification. RESULTS: In total, 7707 patients were identified. After adjustment for the aforementioned factors, a statistically significant association was shown between administrative vulnerability index and cesarean section before labor or during labor respectively (aOR 1.48 [1.23 - 1.78] and aOR 1.46 [OR 1.23 - 1.73]). In contrast, no significant correlation was found for the psychological vulnerability index (aOR 1.09 [0.86 - 1.38] and aOR 0.99 [0.78 - 1.25]) or the dependency vulnerability index (aOR 0.98 [0.76 - 1.26] and aOR 0.85 [0.64 - 1.12]). CONCLUSIONS: The current study provides new insight into the correlation between social vulnerabilities and the risk of cesarean section. It demonstrates that administrative vulnerability is an independent risk factor of cesarean delivery. These patients should be identified and offered an adapted pregnancy monitoring in order to reduce cesarean section rates.

Passive second stage of labor: Does a fourth hour increase maternal morbidity in nulliparous patients at term with epidural?

OBJECTIVES: Prolonging the passive second stage of labor could increase vaginal birth rate, but the data concerning maternal and fetal morbidity are contradictory. The French guidelines did not specify a maximum duration of the passive second stage. Our objective was to assess if allowing a 4th hour after full dilatation before pushing increased maternal morbidity, compared to 3 h after full dilatation. STUDY DESIGN: This single-center, retrospective, observational cohort study took place from January 1-December 31, 2020, in a tertiary maternity unit. All consecutive term nulliparous women who delivered under epidural anesthesia and without pathological fetal heart rate and reaching a second-stage passive phase of labor lasting at least 3 h were included. We compared 2 groups according to the duration of the passive second stage: "3-hour group" and "4-hour group". In the "3-hour group," featuring a second-stage passive phase of up to 3 h, pushing is initiated for favorable conditions, while a cesarean section is performed if conditions are deemed unfavorable. In the "4-hour group", obstetric conditions not justifying immediate pushing after three hours, and the obstetric team believed that an additional hour of expectant management could lead to a successful vaginal delivery. The principal endpoint was a composite criterion of maternal morbidity including obstetric anal sphincter injuries, postpartum hemorrhage, transfusion and intrauterine infection. RESULTS: We included 111 patients in the "4-hour group" and 349 in the "3-hour group". Composite maternal morbidity did not increase in the "4-hour group" compared to the "3-hour group" (21 (18.9 %) versus 61 (17.5 %); p = 0.73). Neonatal morbidity was similar between the two groups. In the "4-hour group, 91 (82 %) patients had vaginal deliveries", 62 (55,9 %) by spontaneous vaginal delivery and 29 (26,1 %) with instrumental assistance. CONCLUSION: For selected patients, waiting for 4 h at full dilation can be beneficial due to the high rate of vaginal delivery and low incidence of maternal and fetal complications.

The penetration efficiency of a dissolved model drug into hair follicles depends on the concentration of added nanoparticles.

Hair follicles have recently emerged as promising drug delivery targets and gates for skin penetration. The so-called ratchet effect, which is based on an interaction between the hair shaft surface, the intrafollicular stratum corneum and nanoparticles, has proven to be very effective for the transport of active ingredients. Especially the nanoparticle-assisted decolonization of hair follicles constitutes an interesting new area of application. In a recently published work it was shown that small molecules as well as macromolecules solved in an outer phase of a formulation can be transported into the deeper parts of the hair follicles by adding nanoparticles to the formulation. In this case the nanoparticles constitute an entity independent of the drug and the transport is hypothesized to be based on an adhesion effect. In the present work, we focused on the impact of the particle concentration in the formulation on the transport efficiency of the model drug fluorescein sodium into hair follicles utilizing an ex vivo porcine skin model. It was observed that a particle concentration of 4% significantly enhances the transport efficiency of fluorescein as compared to 2% particle concentration. Doubling the concentration to 8% did not significantly increase the penetration depth. The effect evolved more efficiently when using 4 Hz circular motion massage as compared to 100 Hz oscillating massage. These results deliver interesting information on the optimal formulation as well as application parameters for a future application in clinical studies for e.g. skin antisepsis purposes.

Dissolvable microneedles in the skin: Determination the impact of barrier disruption and dry skin on dissolution.

Dissolvable microneedles (DMNs), fabricated from biocompatible materials that dissolve in both water and skin have gained popularity in dermatology. However, limited research exists on their application in compromised skin conditions. This study compares the hyaluronic acid-based DMNs penetration, formation of microchannels, dissolution, and diffusion kinetics in intact, barrier-disrupted (tape stripped), and dry (acetone-treated) porcine ear skin ex vivo. After DMNs application, comprehensive investigations including dermoscopy, stereomicroscope, skin hydration, transepidermal water loss (TEWL), optical coherence tomography (OCT), reflectance confocal laser scanning microscopy (RCLSM), confocal Raman micro-spectroscopy (CRM), two-photon tomography combined with fluorescence lifetime imaging (TPT-FLIM), histology, and scanning electron microscopy (SEM) were conducted. The 400 µm long DMNs successfully penetrated the skin to depths of ≈200 µm for dry skin and ≈200-290 µm for barrier-disrupted skin. Although DMNs fully inserted into all skin conditions, their dissolution rates were high in barrier-disrupted and low in dry skin, as observed through stereomicroscopy and TPT-FLIM. The dissolved polymer exhibited a more significant expansion in barrier-disrupted skin compared to intact skin, with the smallest increase observed in dry skin. Elevated TEWL and reduced skin hydration levels were evident in barrier-disrupted and dry skins compared to intact skin. OCT and RCLSM revealed noticeable skin indentation and pronounced microchannel areas, particularly in barrier-disrupted and dry skin. Additional confirmation of DMN effects on the skin and substance dissolution was obtained through histology, SEM, and CRM techniques. This study highlights the impact of skin condition on DMN effectiveness, emphasizing the importance of considering dissolvability and dissolution rates of needle materials, primarily composed of hyaluronic acid, for optimizing DMN-based drug delivery.

Significance of melanin distribution in the epidermis for the protective effect against UV light.

Melanin, the most abundant skin chromophore, is produced by melanocytes and is one of the key components responsible for mediating the skin's response to ultraviolet radiation (UVR). Because of its antioxidant, radical scavenging, and broadband UV absorbing properties, melanin reduces the penetration of UVR into the nuclei of keratinocytes. Despite its long-established photoprotective role, there is evidence that melanin may also induce oxidative DNA damage in keratinocytes after UV exposure and therefore be involved in the development of melanoma. The present work aimed at evaluating the dependence of UV-induced DNA damage on melanin content and distribution, using reconstructed human epidermis (RHE) models. Tanned and light RHE were irradiated with a 233 nm UV-C LED source at 60 mJ/cm2 and a UV lamp at 3 mJ/cm2. Higher UV-mediated free radicals and DNA damage were detected in tanned RHE with significantly higher melanin content than in light RHE. The melanin distribution in the individual models can explain the lack of photoprotection. Fluorescence lifetime-based analysis and Fontana-Masson staining revealed a non-homogeneous distribution and absence of perinuclear melanin in the tanned RHE compared to the in vivo situation in humans. Extracellularly dispersed epidermal melanin interferes with photoprotection of the keratinocytes.

Skin optical properties from 200 to 300 nm support far UV-C skin-safety in vivo.

The growing threat of multi-drug resistant pathogens and airborne microbial diseases has highlighted the need to improve or develop novel disinfection methods for clinical environments. Conventional ultraviolet C (UV-C) lamps effectively inactivate microorganisms but are harmful to human skin and eyes upon exposure. The use of new 233 nm far UV-C LEDs as an antiseptic can overcome those limitations. In this research, the light penetration into the skin was elucidated for the UV-C region (<300 nm) by measuring the scattering and absorption of skin layers and inverse Monte Carlo simulation, and further confirmed by the first clinical pilot trial in which healthy volunteers were irradiated with a dose of 60 mJ/cm2 at 233 nm. The radiation is strongly absorbed in the stratum corneum, resulting in minimal skin damage without inducing inflammatory responses. The results suggest that 233 nm far UV-C light emitting diodes (LEDs) could effectively inactivate microorganisms, while being safe and soft for the skin.

Evaluation of DNA lesions and radicals generated by a 233 nm far-UVC LED in superficial ex vivo skin wounds.

The application of a far-ultraviolet C (UVC) light emitting diode (LED) of 233 nm showed significant bactericidal efficacy at an applied dose between 20 and 80 mJ cm-2 as reported recently. In addition, only minor epidermal DNA lesions were observed in ex vivo human skin and in vitro epidermal models <10% of the minimal erythema dose of UVB radiation. To broaden the potential range of applications of such systems, e.g. to include postoperative application on wounds for the purpose of decontamination, we assessed how a disruption of normal anatomic skin structure and function influences the skin damage induced by light from 233 nm far-UVC LEDs. Thus, we induced superficial skin wounds by mechanical detachment of the stratum corneum in ex vivo human skin. Barrier-disruption of the skin could be successfully determined by measuring an increase in the transepidermal water loss (TEWL) and the stratum corneum loss could be determined morphologically by 2-photon microscopy (2-PM). After far-UVC irradiation of the skin, we screened the tissue for the development of cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs). The abundance of DNA lesions was elevated in wound skin in comparison to intact skin after irradiation with far-UVC. However, no increase in DNA lesions was detected when artificial wound exudate consisting of cell culture medium and serum was applied to the disrupted skin surface prior to irradiation. This effect agrees with the results of ray tracing simulations of the absorption of far-UVC light incident on a superficial skin wound. Interestingly, no significant deviations in radical formation between intact skin and superficially wounded skin were detected after far-UVC irradiation as analyzed by electron paramagnetic resonance (EPR) spectroscopy. In conclusion, 233 nm LED light at a dose of 60 mJ/cm2 could be applied safely on superficial wounds for the purpose of skin antisepsis as long as the wounds are covered with wound fluid.

Primary Human Renal Proximal Tubular Epithelial Cells (pHRPTEpiCs): Shiga Toxin (Stx) Glycosphingolipid Receptors, Stx Susceptibility, and Interaction with Membrane Microdomains.

Tubular epithelial cells of the human kidney are considered as targets of Shiga toxins (Stxs) in the Stx-mediated pathogenesis of hemolytic-uremic syndrome (HUS) caused by Stx-releasing enterohemorrhagic Escherichia coli (EHEC). Analysis of Stx-binding glycosphingolipids (GSLs) of primary human renal proximal tubular epithelial cells (pHRPTEpiCs) yielded globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer) with Cer (d18:1, C16:0), Cer (d18:1, C22:0), and Cer (d18:1, C24:1/C24:0) as the dominant lipoforms. Investigation of detergent-resistant membranes (DRMs) and nonDRMs, serving as equivalents for the liquid-ordered and liquid-disordered membrane phase, respectively, revealed the prevalence of Gb3Cer and Gb4Cer together with cholesterol and sphingomyelin in DRMs, suggesting lipid raft association. Stx1a and Stx2a exerted strong cellular damage with half-maximal cytotoxic doses (CD50) of 1.31 × 102 pg/mL and 1.66 × 103 pg/mL, respectively, indicating one order of magnitude higher cellular cytotoxicity of Stx1a. Surface acoustic wave (SAW) real-time interaction analysis using biosensor surfaces coated with DRM or nonDRM fractions gave stronger binding capability of Stx1a versus Stx2a that correlated with the lower cytotoxicity of Stx2a. Our study underlines the substantial role of proximal tubular epithelial cells of the human kidney being associated with the development of Stx-mediated HUS at least for Stx1a, while the impact of Stx2a remains somewhat ambiguous.

Microdialysis on Ex Vivo Porcine Ear Skin Can Validly Study Dermal Penetration including the Fraction of Transfollicular Penetration-Demonstrated on Caffeine Nanocrystals.

Common ex vivo methods for penetration investigations often fail to monitor transfollicular penetration appropriately. In the present investigation, the validity of dermal microdialysis on the ex vivo porcine ear skin to investigate penetration kinetics, including transfollicular penetration, was studied. In setup A, a caffeine nanocrystal formulation was compared to a non-particular caffeine gel formulation. In setup B, two caffeine nanocrystal formulations of different sizes (200 nm, 700 nm) were compared to each other. Microdialysis samples were collected for 46 h. After sampling, the skin layers were separated, homogenized, and caffeine was quantified in all samples. In setup A the area under the curve (AUC) after crystal gel formulation application was 12 times higher than after non-particular formulation application. Setup B showed an increased AUC of 42% in the microdialysis data when the 700 nm caffeine crystals were applied compared to the 200 nm crystals. The microdialysis data was supported by the separation, homogenization and extraction data. Microdialysis performed on ex vivo porcine ear skin is a novel experimental setup. It is of high interest for further investigations since it is able to also capture the impact of follicular and transfollicular penetration kinetics as no other ex vivo setup can.

Investigation of transfollicular caffeine penetration using microdialysis on ex vivo porcine ear skin.

The aim of this study was to develop an ex vivo method that allows to quantify the transfollicular penetration of topically applied substances by combining microdialysis and selective follicular closure with varnish. An experimental setup with three skin areas on ex vivo intact porcine ear skin was designed (varnish on hair follicle, varnish next to hair follicle, no varnish). On each area, 10 µl/cm2 caffeine-hydroxyethyl-cellulose-gel was applied. Samples were collected for 22 h by microdialysis. After sampling, the skin layers were separated, homogenized and caffeine was quantified by high pressure liquid chromatography (HPLC) in all samples. Potential impact of the varnish placed next to the follicle by tension on the follicle during the drying process was monitored by a microscopic setup and could be excluded. The microdialysis and homogenization study showed a significantly reduced penetration of caffeine when the hair follicles were closed. In areas with open hair follicles caffeine was detected already in the first ten minutes after application. The reported novel combination of two methods is suitable to investigate ex vivo transfollicular penetration. Possible impact of the closure material in the control area can be ruled out by adjusting the design of the control area in future studies.

Three consecutive episodes of acute retinal necrosis due to herpes simplex-1 over twelve years following herpetic encephalitis.

PURPOSE: To report a case of multiple delayed recurrence of acute retinal necrosis syndrome occurring in the same eye following initial herpetic encephalitis. METHODS: Case report and review of literature. RESULTS: An otherwise healthy 67-year-old woman experienced three episodes of acute retinal necrosis in the left eye 3 months, 3 years, and 12 years, respectively, following initial herpes simplex-1 encephalitis. This is the first case report of more than one such recurrence, moreover in the same eye, and with such long interval. CONCLUSION: This case illustrates that extremely long intervals between initial herpetic encephalitis and acute necrosis syndrome recurrence can occur, thus warranting a long follow-up of these patients.