Rodent Skin Research Articles

Histological Assessment of Cutaneous Acute Graft-Versus-Host Disease in a Preclinical Swine Model of Hematopoietic Cell Transplantation and Vascularized Skin Flap Tolerance

Abstract 1894Graft-versus-host disease (GVHD) remains a frequent complication of hematopoietic cell transplantation with skin being a principal target organ. Murine models have provided some insight into the mechanisms of this complex disease process. However, mouse skin differs from human skin, and results of studies in rodents may not translate well to the clinic. Miniature swine is a well-recognized animal model for preclinical studies of skin including dermal toxicology, transdermal drug delivery and wound healing. Unlike skin of rodents, dogs or non-human primates, porcine skin is similar to human skin in terms of structure of epidermal rete ridges (Figure 1A), hair follicle structure and density, and presence of sweat glands and subcutaneous fat.We have developed a miniature swine model of haploidentical hematopoietic cell transplantation that results in stable stem cell engraftment, hematopoietic chimerism and donor skin flap tolerance without GVHD. Swine leukocyte antigen (SLA)ad animals receive 100cGy total body irradiation and T cell depletion using porcine CD3 immunotoxin prior to cytokine mobilized peripheral blood hematopoietic cell transplantation from a haploidentical SLAac donor. Daily cyclosporine is administered at doses adjusted to maintain serum levels between 400–800 ng/ml starting one day prior to transplantation and continued for 30 days with taper until day 45.Mixed hematopoietic cell chimerism persists in these animals and donor vascularized skin flaps are accepted without the need for maintenance immunosuppression regardless of whether they are placed at the time of hematopoietic cell transplantation or greater than 100 days later. Despite the initial resistance to GVHD in this model, acute cutaneous GVHD could be induced through infusion of a high dose of donor leukocytes (DLI) including 15 million donor T cells/kg recipient body weight administered 463 days post hematopoietic cell transplantation and 314 days post vascularized donor skin flap transplantation. Conversion to full donor hematopoiesis was observed between 3–4 weeks post DLI. At this time a mild skin rash was evident in both donor and host skin which appeared to progress specifically within host skin. The presence of both donor and host skin on a tolerant animal whose peripheral blood consists of a mixture of both donor and host derived hematopoietic cells provides a novel model to evaluate specific targets of acute GVHD.In this study we assess histologically the manifestations of acute GVHD in both donor and host skin and compare it to typical histological manifestations of acute GVHD observed in patients. Specifically, recipient skin developed superficial lymphoid infiltrates that progressed to confluence along the dermal-epidermal junction by day 21. These infiltrates were associated with intraepidermal migration, prominent basal cell layer keratinocyte apoptosis, and satellitosis virtually indistinguishable from grade 3–4 human cutaneous acute GVHD (Figure 1B). Superficial portions of hair follicles were involved, as also seen in human disease. Of interest, mild disease was detected in the donor skin grafts by day 62 post-DLI, where selective lymphoid infiltration in the tips of the epidermal rete ridges and in association with target cell apoptosis/satellitosis was documented, a finding remarkably similar to early targeting events in humans.These findings emphasize the value of the porcine model for translational studies of cutaneous GVHD. Preliminary data suggest differences in cellular targeting between recipient and donor skin whereby the latter may be triggered by the presence of chimeric recipient-derived hematopoietic elements that have populated donor grafts. Further studies are now possible to explore the effector-target cell interactions in this clinically-relevant model of human cutaneous GVHD. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

The application of Gaussian processes in the predictions of permeability across mammalian and polydimethylsiloxane membranes

The problem of predicting the rate of percutaneous absorption of a drug is an important issue, particular with the increasing use of the skin as a means of moderating and controlling drug delivery. One key feature of this problem domain is that human skin permeability to penetrants (often characterised by Kp, the permeability coefficient) has been shown to be inherently non-linear when mathematically related to the key physicochemical parameters of penetrants. The aims of the current study were to apply and validate Gaussian process regression methods to datasets for membranes other than human skin, and to explore how the nature of the dataset may influence its analysis. Permeability data for absorption across rodent and pig skin, and polydimethylsiloxane Silastic® membranes was collected from the literature. Two QSPR methods were applied to compare to the Gaussian process models. The results demonstrated that Gaussian process models with different covariance functions outperform the QSPR model for human, pig and rodent datasets, but in general are not good for Silastic® membranes. These results suggest that the physicochemical parameters employed in this study might not be appropriate for developing models that represent this membrane. In addition, the results show the size of the datasets, in both absolute and comparative senses, appears to influence model quality.

Organotypic Modeling of Human Keratinocyte Response to Peroxisome Proliferators

Peroxisome proliferators (PPs) are a diverse chemical group including hypolipidemic drugs and some fatty acids. Their stimulation of PP-activated receptors (PPARs) and subsequent control of gene expression regulates metabolism and differentiation in many cells. PPs have multiple opportunities to target human epidermal keratinocytes because of delivery through dietary, clinical, and/or topical exposure routes. PPAR knockout mice and PP treatment of mouse skin or human keratinocytes in monolayer culture have established some effects for PPs in cutaneous differentiation. However, incomplete epidermal maturation characteristic of monolayer keratinocytes and rodent-specific effects may limit our full understanding of human keratinocyte responses to PPs. To address these issues, we investigated PP influence on primary human keratinocytes in organotypic cultures that recapitulate biochemical markers of epidermis. We found that the PPARα agonists clofibrate, docasohexaenoic acid, and WY-14,643 produced mild to moderate keratinocyte hyperplasia, increased stratification (particularly of granular and cornified layers), and enhanced levels of the differentiation markers filaggrin, ABCA12, and phosphorylated HSP27. Several PP effects generated in the organotypic system, however, were distinct from those previously reported for rodent skin and human keratinocyte monolayer cultures, suggesting that the species and growth context of target cells can impact exposure outcomes. Given the utility of organotypic cultures for modeling the epidermis, studies in this system may bridge the gap between the rodent assays and clinical studies of human epidermal responses to PPs.

Suppression of cytokine gene expression and improved therapeutic efficacy of microemulsion-based tacrolimus cream for atopic dermatitis.

Tacrolimus ointment being occlusive is known to give higher dermal penetration but offers limited patient acceptance in treatment of atopic dermatitis, especially in tropical countries. Hence, the aim of this study was to develop, characterize, and evaluate a microemulsion-based cream formulation of tacrolimus against ointment in hapten-induced murine model of dermatitis. Tacrolimus-loaded microemulsion having mean globule size below 25nm was mixed with cetomacrogol cream base. The microemulsion-based cream exhibited a significantly faster drug release through semipermeable cellulose acetate membrane in comparison to commercially available ointment. The drug retention in rodent and human cadaver skin with cream was almost twofold greater in comparison to the commercially available ointment. Further, in vivo evaluation using a fluorescent marker revealed a greater and deeper accumulation of marker in skin with cream. In vivo studies in mice revealed a prompt and significant reduction in ear swelling. The reduction in inflammatory cytokine gene expression as evaluated by semiquantitative reverse transcriptase polymerase chain reaction was also significantly higher with cream. The better efficacy of cream was reflected in histopathology as well as in morphological observations at the site of application. Thus, microemulsion-based cream presents a possibility of development of an efficacious cream vehicle and a scope for dose reduction which needs to be confirmed in clinical studies.

Exploratory Study on the Effectiveness of a Standardized Extract fromAgeratina pichinchensisin Patients with Chronic Venous Leg Ulcers

The aerial parts of the species Ageratina pichinchensis, popularly known as "axihuitl", have been empirically used in Mexico for wound-healing purposes. The evaluation of an extract from A. pichinchensis (in an in vivo model) demonstrated its capacity to reduce, in a significant manner, the time required for wound healing in rodent skin. This same extract showed (in vitro) a capacity of encouraging proliferation of normal human fetal lung fibroblasts (MRC-5). This study's objective was to evaluate, in a clinical trial, the effectiveness and tolerability of the topical administration of a product elaborated with the standardized extract from the aerial parts of A. pichinchensis in patients with chronic venous leg ulcers, and to compare the effect with a control group treated with 7 % propylene glycol alginate. In this study, a total of 34 patients were included (50 % in each group). Six patients of the control group withdrew from the treatment, three of these because of lack of recovery. In the experimental group, 2 participants withdrew from the study, but none of these due to treatment or clinical-evolution reasons. The A. pichinchensis extract showed therapeutic effectiveness in one hundred percent of the patients treated with it, while the control treatment achieved this condition in 81.8 % of the control group patients. Ulcer size reduction resulted significantly higher (p < 0.010) in the group of patients administered the experimental treatment, which allows us to assure that the A. pichinchensis standardized extract is effective in the treatment of chronic venous leg ulcers and, compared with the 7 % propylene glycol alginate formula, achieves a significant reduction of the time required for the ulcers to heal.

The application and limitations of mathematical modelling in the prediction of permeability across mammalian skin and polydimethylsiloxane membranes

Predicting the rate of percutaneous absorption of a drug is an important issue with the increasing use of the skin as a means of moderating and controlling drug delivery. One key feature of this problem domain is that human skin permeability (as K(p)) has been shown to be inherently non-linear when mathematically related to the physicochemical parameters of penetrants. As such, the aims of this study were to apply and evaluate Gaussian process (GP) regression methods to datasets for membranes other than human skin, and to explore how the nature of the dataset may influence its analysis. Permeability data for absorption across rodent and pig skin, and artificial membranes (polydimethylsiloxane, PDMS, i.e. Silastic) membranes was collected from the literature. Two quantitative structure-permeability relationship (QSPR) models were used to compare with the GP models. Further performance metrics were computed in terms of all predictions, and a range of covariance functions were examined: the squared exponential (SE), neural network (NNone) and rational quadratic (QR) covariance functions, along with two simple cases of Matern covariance function (Matern3 and Matern5) where the polynomial order is set to 1 and 2, respectively. As measures of performance, the correlation coefficient (CORR), negative log estimated predictive density (NLL, or negative log loss) and mean squared error (MSE) were employed. The results demonstrated that GP models with different covariance functions outperform QSPR models for human, pig and rodent datasets. For the artificial membranes, GPs perform better in one instance, and give similar results in other experiments (where different covariance parameters produce similar results). In some cases, the GP predictions for some of the artificial membrane dataset are poorly correlated, suggesting that the physicochemical parameters employed in this study might not be appropriate for developing models that represent this membrane. While the results of this study indicate that permeation across rodent (mouse and rat) and pig skin is, in a statistical sense, similar, and that the artificial membranes are poor replacements of human or animal skin, the overriding issue raised in this study is the nature of the dataset and how it can influence the results, and subsequent interpretation, of any model produced for particular membranes. The size of the datasets, in both absolute and comparative senses, appears to influence model quality. Ideally, to generate viable cross-comparisons the datasets for different mammalian membranes should, wherever possible, exhibit as much commonality as possible.

Efficacy of linezolid against Staphylococcus aureus in different rodent skin and soft tissue infections models

&lt;p&gt;Linezolid is approved for complicated and uncomplicated skin and soft tissue infections. We have evaluated the efficacy of this drug in murine as well as in rat skin and soft tissue infection models using &lt;em&gt;Staphylococcus aureus&lt;/em&gt; ATCC and clinical strains. In thigh infection model the dose of linezolid required for more than 1 log&lt;sub&gt;10&lt;/sub&gt; kill from baseline inoculum in neutropenic mice and rats was 100 mg/kg and 50 mg/Kg BW bid /day, respectively, which was 5 and 4 folds more than that in immunocompetent animals, respectively. Dose required to achieve 1 log&lt;sub&gt;10&lt;/sub&gt; killing was similar against different strains of &lt;em&gt;S. aureus &lt;/em&gt;in immunocompetent mouse thigh infection model. However, in murine groin abscess infection model, a dose of 100 mg/kg, b.i.d/day of linezolid produce static effect in 2 days, but revealed to be superior in 4 days treatment and showed approximately 1 log&lt;sub&gt;10&lt;/sub&gt; killing from base line inoculums. Based upon pharmacokinetic profile, a 24-h AUC/MIC required for linezolid efficacy in murine groin abscess model was 91.5 for the strain used in this study. As linezolid is taken as a gold standard drug in the evaluation of new chemical entity, this data could be useful for comparing the preclinical efficacy of new anti-MRSA agents.&lt;/p&gt;

L-3-Phosphoserine phosphatase (PSPH) regulates cutaneous squamous cell carcinoma proliferation independent of l-serine biosynthesis

L-3-Phosphoserine phosphatase (PSPH) is a highly conserved and widely expressed member of the haloacid dehalogenase superfamily and the rate-limiting enzyme in l-serine biosynthesis. We previously found Psph expression to be uniquely upregulated in a α6β4 integrin transgenic mouse model that is predisposed to epidermal hyperproliferation and squamous cell carcinoma (SCC) formation implicating a role for Psph in epidermal homeostasis. We examined the status of PSPH in normal skin epidermis and skin tumors along with its sub-cellular localization in epidermal keratinocytes and its requirement for squamous cell carcinoma (SCC) proliferation. First, an immunohistochemical study was performed for PSPH in normal skin and skin cancer specimens and in cultured keratinocytes. Next, biochemical analyses were performed to confirm localization of PSPH and to identify candidate binding proteins. Finally, proliferation and apoptosis studies were performed in human SCC and normal keratinocytes, respectively, transduced with vectors encoding small hairpin RNAs targeting PSPH or overexpressing a phosphatase-deficient PSPH mutant. PSPH is expressed throughout the proliferative layer of the epidermis and hair follicles in rodent and human skin and is highly induced in SCC. In keratinocytes, PSPH is a cytoplasmic protein that primarily localizes to endosomes and is present primarily as a homodimer. Knock down of PSPH dramatically diminished SCC cell proliferation and cyclin D1 levels in the presence of exogenous of l-serine production suggesting a non-canonical role for PSPH in epithelial carcinogenesis. Psph is highly induced in proliferative normal keratinocytes and in skin tumors. PSPH appears to be critical for the proliferation of SCC cells; however, this phenomenon may not involve the phosphoserine metabolic pathway.

Inhibitory CB1 and activating/desensitizing TRPV1-mediated cannabinoid actions on CGRP release in rodent skin

Cannabinoid-induced antinociception relies on activation of inhibitory cannabinoid receptors (CB1) in the peripheral and central nervous system. However, most cannabinoids at higher concentration also activate excitatory ionotropic transient receptor potential (TRP) channels coexpressed with CB1 in primary nociceptive neurons that contain and release calcitonin gene-related peptide (CGRP) upon activation. Over a wide concentration range (0.01-100μM) we investigated the molecular action principles of the endocannabinoid anandamide and of the plant-derived Δ(9)-THC that can be prescribed for analgesia. Isolated rat and mouse skin preparations were used to measure CGRP release induced by noxious heat (47°C) and capsaicin (0.5μM), stimuli known to activate the capsaicin receptor TRPV1. At low concentration (0.1μM) both cannabinoids inhibited stimulated CGRP release by 34-65%, which effects were absent under CB1 block by AM 251 and in global CB1 but not TRPV1 knockout mice. At high concentration (100μM) both cannabinoids evoked CGRP release by themselves and desensitized subsequent heat responses, which effects were absent under TRPV1 block by BCTC and in global TRPV1 but not CB1 knockouts. A lower (0.01μM) and the intermediate concentrations (1 and 10μM) of cannabinoids were ineffective. Excitatory and desensitizing effects were not more expressed (disinhibited) in CB1(-/-), inhibitory effects not stronger in TRPV1(-/-). CGRP release induced by unspecific depolarization (KCl) was not modulated by cannabinoids. An incidental finding was that global CB1(-/-) showed reduced heat sensitivity, almost as low as TRPV1(-/-) and in accord with their behavioral phenotype. In conclusion, the antinociceptive potency of peripherally acting CB1 agonists is not restrained by opposing irritant effects through TRPV1 but by their own limited efficacy and narrow concentration-response relationship.

In-vitro permeability to salicylic acid of human, rodent, and shed snake skin.

In-vitro permeability to salicylic acid of human, rodent, and shed snake skin has been examined for the purpose of selecting model membranes for human skin corresponding to different anatomic sites, since a marked regional variation is suggested among the different sites. The greatest permeability to salicylic acid was observed in the scrotum, that of the sole was negligible. The cheek, neck, and inguinal skin seemed more permeable than the breast, back, thigh, lower leg, or foot skin. Shed snake and skin of hairless rat were found to show similar permeability to human breast and thigh skin. Wistar rat and nude mouse skin showed similar permeability to human cheek, neck, and inguinal skin.

A New, Rapid, Standardized Method for Harvesting Split Skin Grafts in Rodents

Harvesting split skin grafts in rodents has proven to be challenging, as the presence of panniculus carnosus tissue in rodent skin causes high flexibility and mobility of the upper skin layer. Thus, fixation is indispensable for granting precise manipulation during the grafting process. All techniques established in the literature achieve stability by dissecting a subcutaneous pocket beneath the panniculus carnosus. In this article, the authors present a method that excludes inflicting additional wounds other than that to the donor site but takes advantage of the elasticity of rat skin. A customized sturdy support plate is held laterally beside the skin area where the split skin is to be cut. The skin is mobilized, pulled over the support plate, and fixed with the nondominant hand on the bottom of the device. Using a dermatome, the split skin graft is cut on the metal plate, which provides a flat surface to permit a precise harvesting process. A total of 208 split skin grafts were harvested, of which nine had to be rejected. The average time to perform a split skin graft on one animal, not considering preparation time, was 2 minutes. All of the harvested grafts were 2 × 2.5 cm (0.79 × 0.98 inch), with a mean thickness of 0.3 mm. Reharvesting could be performed on postoperative day 7. Using this new standardized method, grafts of equal size and thickness can be obtained rapidly. It is cheap and highly reproducible and can be carried out by a single surgeon. It provides a rapid healing process with fewer complications and immensely reduced stress for the animals.

Expression of C4.4A, a structural uPAR homolog, reflects squamous epithelial differentiation in the adult mouse and during embryogenesis.

The glycosylphosphatidylinositol (GPI)-anchored C4.4A was originally identified as a metastasis-associated protein by differential screening of rat pancreatic carcinoma cell lines. C4.4A is accordingly expressed in various human carcinoma lesions. Although C4.4A is a structural homolog of the urokinase receptor (uPAR), which is implicated in cancer invasion and metastasis, no function has so far been assigned to C4.4A. To assist future studies on its function in both physiological and pathophysiological conditions, the present study provide a global survey on C4.4A expression in the normal mouse by a comprehensive immunohistochemical mapping. This task was accomplished by staining paraffin-embedded tissues with a specific rabbit polyclonal anti-C4.4A antibody. In the adult mouse, C4.4A was predominantly expressed in the suprabasal layers of the squamous epithelia of the oral cavity, esophagus, non-glandular portion of the rodent stomach, anus, vagina, cornea, and skin. This epithelial confinement was particularly evident from the abrupt termination of C4.4A expression at the squamo-columnar transition zones found at the ano-rectal and utero-vaginal junctions, for example. During mouse embryogenesis, C4.4A expression first appears in the developing squamous epithelium at embryonic day 13.5. This anatomical location of C4.4A is thus concordant with a possible functional role in early differentiation of stratified squamous epithelia.

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

Leishmania species: Detection and identification by nested PCR assay from skin samples of rodent reservoirs

Many rodent species act as reservoir hosts of zoonotic cutaneous leishmaniasis in endemic areas. In the present study a simple and reliable assay based on nested PCR was developed for the detection and identification of Leishmania parasites from rodent skin samples. We designed Leishmania-specific primers that successfully amplified ITS regions of Leishmania major, Leishmania gerbilli and Leishmania turanica using nested PCR. Out of 95 field collected Rhombomys opimus, 21 were positive by microscopic examination and 48 by nested PCR. The percentage of gerbils infected with L. major, L. gerbilli and L. turanica was 3.2%, 1.1% and 27.4%, respectively. In 15.8% of the rodents, we found mixed natural infections by L. major and L. turanica, 1.1% by L. major and L. gerbilli, and 2.1% by the three species. We concluded that this method is simple and reliable for detecting and identifying Leishmania species circulating in rodent populations.

Vasoactive intestinal peptide induces vascular endothelial growth factor production in human HaCaT keratinocytes via MAPK pathway

Psoriasis is a chronic skin disease characterized by abnormal keratinocyte proliferation and differentiation, inflammation, and angiogenesis. Although dysfunction of the immune system is known to be an important factor in the pathogenesis of psoriasis, there is also strong evidence that psychological stresses are involved. Neuropeptides are thought to be main mediators of neurogenic inflammation, presumably involved in the pathogenesis of psoriasis. Vasoactive intestinal peptide (VIP) is one of the major neuropeptides in human and rodent skin. In the present study, we examined the effect and mechanism of VIP on vascular endothelial growth factor (VEGF) production by HaCaT cells which is a spontaneous, immortalized, human keratinocyte cell line. Our data indicate the mRNA and protein levels of VEGF by VIP were increased in a concentration-dependent manner. However, this increase was abrogated by pretreatment with an extracellular signal-regulated kinase (ERK) inhibitor PD98059 or p38MAPK inhibitor SB203580; pretreatment with c-Jun N-terminal kinase (JNK) inhibitor SP600125 did not attenuate the effects of VIP on the expression of VEGF. In addition, VIP treatment induced rapid phosphorylation of ERK1/2 and p38MAPK, and PD98059 and SB203580 were able to inhibit VIP-induced phosphorylation of ERK1/2 and p38MAPK, respectively. These results suggest that VIP increases the expression of VEGF through the ERK1/2 and p38MAPK signaling pathway in human HaCaT cells.

Intravital microscopy imaging of macrophage localization to immunogenic particles and co-localized tissue oxygen saturation

Well-designed biomaterial polymer particle-based vaccines will optimally promote immune cell antigen-presenting behavior while minimizing adverse inflammatory responses to the particles and encapsulated drugs or adjuvants. It is important in the design of particle-based vaccines to consider possible harmful effects of immune response on tissue at the vaccination site. Intravital microscopy with rodent dorsal skin window chambers enables in vivo serial observations in the same animal, and such models which have been used to study angiogenesis and macrophage response to implanted biomaterials may also be useful for the development of particle-based vaccines. To our knowledge there have been no reports where intravital microscopy has documented real-time immune cell localization and potentially harmful co-localized tissue effects. In this proof-of-principle study we used fluorescence and spectral imaging intravital microscopy of mouse window chambers to measure macrophage localization and co-localized tissue microvessel hemoglobin saturation changes in response to an immunogenic stimulus from polymer particles loaded with lipopolysaccharide (LPS) serving as a model vaccine/adjuvant system. We observed greater and faster macrophage localization to stronger inflammatory stimuli from LPS-loaded particle doses, a trend of decreased microvessel oxygenation with increased macrophage accumulation and, in an extreme case, complete microvessel collapse accompanied by tissue necrosis. Our technique may be useful for optimizing design of particle-based vaccines and may give insight into the use of hemoglobin saturation as a biomarker of tissue inflammation for clinical investigations of particle-based vaccines.

Impact of Renal Impairment on Long-Term Retention of Gadolinium in the Rodent Skin Following the Administration of Gadolinium-Based Contrast Agents

Several publications have suggested a possible association between Gd-based contrast agents (GBCAs) and the development of nephrogenic systemic fibrosis, a rare but serious disease. To date, nephrogenic systemic fibrosis has been observed only in patients with severe renal insufficiency.The aim of this study was to determine the impact of a prolonged circulation time of GBCAs caused by reduced renal clearance on the long-term retention of Gd in the skin of rats after administration of different GBCAs. Renally impaired Han Wistar Rats (5/6-nephrectomized rats) were injected with Omniscan, OptiMARK, Magnevist, or Gadovist. The contrast agents were administered once daily for 5 consecutive days into the tail vein at a dose of 2.5 mmol Gd/kg b.w. Skin biopsies were taken at various time points, and the gadolinium (Gd) concentration was determined by inductive coupled plasma mass spectrometry (ICP-MS) over an observation period of 168 days post injection (p.i.). Differences in the skin Gd concentrations were observed between the 4 investigated GBCAs. For the nonionic linear compounds, Omniscan and OptiMARK, high Gd concentrations were maintained in the skin over the observation period of up to 168 days p.i. For the ionic linear compound, Magnevist, comparatively lower Gd retention in the skin was observed over time. For the macrocyclic compound, Gadovist, the Gd values in the skin were even lower, and significantly lower than Gd values in the skin in Omniscan and OptiMARK treated animals. The results of this preclinical study support the use of 5/6-nephrectomized rats as a model for prolonged circulation time of GBCAs as seen in patients with severe renal impairment. Surgically induced severe renal impairment resulted in delayed clearance of the administered GBCAs in the study animals. The highest amount of Gd was observed in the skin after treatment with the nonionic linear GBCAs, whereas the lowest Gd values were observed after treatment with the macrocyclic agent. This suggests that the difference in the Gd values observed in rat skin tissue after treatment with the different GBCAs is caused of a different propensity of the different GBCAs to release Gd in vivo. However, the analytical method used does not distinguish between chelated and unchelated Gd.

Long-term retention of gadolinium in the skin of rodents following the administration of gadolinium-based contrast agents

Several publications suggest a potential association between the administration of Gadolinium-based contrast agents (GBCAs) and the onset of a rare but serious disease, Nephrogenic Systemic Fibrosis (NSF). The aim of this study was to determine the elimination time-course of Gadolinium (Gd) from skin tissue after application of GBCAs in rats. Seven different marketed GBCAs were injected on five consecutive days at a dose of 2.5 mmol/kg bodyweight into the tail vein of Han-Wistar rats and the Gd concentrations were determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) in skin biopsies taken at various time-points up to a year after the last injection. Most of the administered Gd was eliminated from the skin within a time-period of about 2 months. However, the repeated administration of linear GBCAs resulted in long-term retention of a small portion of the administered Gd in the skin tissue of rats, with substantially higher values observed in animals treated with non-ionic linear agents than in those that received ionic linear GBCAs. Following treatment with macrocyclic GBCAs, Gd values in the skin were in the same range as observed in the controls from day 24 post-injection onwards. In summary, we observed a correlation between the complex stability of GBCAs and the amount of residual Gd in the skin up to a year after application of GBCAs.

Further development of the EpiDerm™ 3D reconstructed human skin micronucleus (RSMN) assay

The upcoming ban on testing of cosmetics in animals by the European Union's 7th Amendment to the Cosmetics Directive will require genotoxicity safety assessments of cosmetics ingredients and final formulations to be based primarily on in vitro genotoxicity tests. The current in vitro test battery produces an unacceptably high rate of false positives, and used by itself would effectively prevent the use and development of many ingredients that are actually safe for human use. To address the need for an in vitro test that is more predictive of genotoxicity in vivo, we have developed an in vitro micronucleus assay using a three-dimensional human reconstructed skin model (EpiDerm™) that more closely mimics the normal dermal exposure route of chemicals. We have refined this model and assessed its ability to predict genotoxicity of a battery of chemicals that have been previously classified as genotoxins or non-genotoxins based on in vivo rodent skin tests. Our reconstructed skin micronucleus assay correctly identified 7 genotoxins and 5 non-genotoxins, demonstrating its potential to have a higher predictive value than currently available in vitro genotoxicity tests, and its utility as part of a comprehensive in vitro genotoxicity testing strategy.

Neural Precursors from Canine Skin: A New Direction for Testing Autologous Cell Replacement in the Brain

Recent work indicates that neural progenitors can be isolated from the skin of rodents and humans. The persistence of these cells in accessible adult tissue raises the possibility of their exploitation for research and therapeutic purposes. This study reports on the derivation, culture, and characterization of homogenous canine skin-derived neuroprecursor cells (SKiNPs) from mature animals. Canine tissue was used because naturalistic brain diseases in community-dwelling dogs are emerging as ecologically sound models for a range of neurological conditions. Adult SKiNPs were initially isolated as neurospheres and then cultured for 10-15 passages in an adherent monolayer assay. Serumfree expansion conditions contained B-27, 20 ng/mL EGF, and 40 ng/mL bFGF. Gene expressions by PCR indicated expression of nestin, CD133, NCAM, and FGF2R, but not GFAP. Highly uniform expression of nestin (76 +/- 8.3%), NCAM (84 +/- 3.3%), betaIII-tubulin (96 +/- 4.3%), and CD133 (68 +/- 13.5%) was also observed. Directed differentiation of SKiNPs in the presence of serum induced betaIIItubulin, NSE, NCAM, and MAP2 in >90% of differentiated cells by immunophenotype analysis. Our culture system rapidly induces canine skin cells into neural precursors, maintains nestin expression in more than 75% of proliferating cells, and generates an almost universal neuronal-like phenotype after 7 days of in vitro differentiation. Their biological characteristics are suggestive of transiently amplifying fate-restricted neuroprecursors rather than true neural stem cells. This system may be an effective alternative for autologous neurorestorative cell replacement in canine models for further translational research.