An anatomical study of the subarachnoid space surrounding the trigeminal ganglion in horses-in preparation for a controlled glycerol rhizotomy in equids.

Introduction: Equine trigeminal-mediated headshaking is a painful neuropathic disorder comparable to trigeminal neuralgia in humans. The selective destruction of pain fibers within the trigeminal ganglion, called rhizotomy, is the surgical treatment of choice for idiopathic trigeminal neuralgia refractory to medical treatment in humans. The human trigeminal ganglion is enclosed by a dural recess called the Meckel's or trigeminal cave, in which the ganglion is surrounded by a cerebrospinal fluid (CSF)-filled subarachnoid space. During glycerol rhizotomy, glycerol is percutaneously injected in this CSF-filled space. Until now, information about the anatomy of the dural recess and the subarachnoid space surrounding the trigeminal ganglion is lacking in horses. The aim of this study was to explore if a CSF-filled subarachnoid space around the trigeminal ganglion exists in horses. Materials and methods: Six equine cadaver heads were investigated for CSF accumulation around the ganglion with a 3 Tesla MRI. After anatomical dissection to expose the trigeminal root, a polymer-based radiopaque contrast agent was injected through the porus trigeminus into the subarachnoid space (cisternography). The exact delineation and the volume of the contrast agent accumulation were determined on subsequent micro-computed tomographic scans and segmentation. Finally, the distribution of the contrast agent within the subarachnoid space was examined histologically in three specimens. Results: In all 12 specimens included in this study, the trigeminal ganglion was surrounded by a subarachnoid space forming a trigeminal cistern. The mean volume of the trigeminal cave in this study was 0.31 mL (±SD: 0.11 mL). Distribution of the contrast agent along the peripheral nerves (i.e., ophthalmic, maxillary and/or mandibular nerve) was observed in 7 out of 12 specimens. Discussion/conclusion: A subarachnoid space surrounding the trigeminal ganglion exists in the horse and could be targeted for glycerol rhizotomy in horses suffering from trigeminal-mediated headshaking. However, the clinical relevance of contrast agent distribution along the peripheral nerves remains to be assessed.

LEKTI domain 6 displays anti-inflammatory action in vitro and in a murine atopic dermatitis model.

BACKGROUND: Lympho-epithelial Kazal-type-related inhibitor (LEKTI) is a serine protease inhibitor consisting of multiple domains. A loss of function mutation is described in Netherton patients that show severe symptoms of atopic lesions and itch. OBJECTIVES: LEKTI domain 6 (LD6) has shown strong serine protease-inhibitory action in in vitro assays and thus it was tested in vitro and in vivo for potential anti-inflammatory action in models of atopic skin disease. METHODS: Human skin equivalents were treated with LD6 and an inflammatory reaction was challenged by kallikrein-related endopeptidase 5 (KLK5). Furthermore, LD6 was tested on dorsal root ganglia cells stimulated with KLK5, SLIGRL and histamine by calcium imaging. The effect of topically administered LD6 (0.4-0.8%) in lipoderm was compared to a topical formulation of betamethasone-diproprionate (0.1%) in a therapeutic setting on atopic dermatitis-like lesions in NC/Nga mice sensitized to house dust mite antigen. Endpoints were clinical scoring of the mice as well as determination of scratching behaviour. RESULTS: KLK5 induced an upregulation of CXCL-8, CCL20 and IL-6 in skin equivalents. This upregulation was reduced by pre-incubation with LD6. KLK5 as well as histamine induced calcium influx in a population of neurons. LD6 significantly reduced the calcium response to both stimuli. When administered onto lesional skin of NC/Nga mice, both LD6 and betamethasone-dipropionate significantly reduced the inflammatory reaction. The effect on itch behaviour was less pronounced. CONCLUSION: Topical administration of LD6 might be a new therapeutic option for treatment of lesional atopic skin.

Application of an artificial intelligence for quantitative analysis of endothelial capillary beds in vitro.

BACKGROUND: The use of endothelial cell cultures has become fundamental to study angiogenesis. Recent advances in artificial intelligences (AI) offer opportunities to develop automated assessment methods in medical research, analyzing larger datasets. OBJECTIVE: The aim of this study was to compare the application of AI with a manual method to morphometrically quantify in vitro angiogenesis. METHODS: Co-cultures of human microvascular endothelial cells and fibroblasts were incubated mimicking endothelial capillary-beds. An AI-software was trained for segmentation of endothelial capillaries on anti-CD31-labeled light microscope crops. Number of capillaries and branches and average capillary diameter were measured by the AI and manually on 115 crops. RESULTS: The crops were analyzed faster by the AI than manually (3 minutes vs 1 hour per crop). Using the AI, systematically more capillaries (mean 48/mm2 vs 27/mm2) and branches (mean 23/mm2 vs 11/mm2) were counted than manually. Both methods had a strong linear relationship in counting capillaries and branches (r-capillaries = 0.88, r-branches = 0.89). No correlation was found for measurements of the diameter (r-diameter = 0.15). CONCLUSIONS: The present AI reduces the time required for quantitative analysis of angiogenesis on large datasets, and correlates well with manual analysis.

Microvascular changes following exposure to iodinated contrast media in vitro. A qualitative comparison to serum creatinine concentrations in post-cardiac catheterization patients.

INTRODUCTION: Contrast-associated acute kidney injury (CA-AKI) is characterized as a loss of renal function following radiological contrast media administration. While all contrast media induce variable changes in microvascular endothelial cells in vitro, only few studies report clinical significance of their findings. A comprehensive assessment of the effect of iodinated contrast media on the renal function in vitro and in vivo is essential. The aim of our study was to morphometrically quantify the effect of two different contrast media (Iobitridol and Iodixanol) on vascular endothelial capillaries in vitro and to analyze their effect on the renal function of patients who underwent cardiac catheterization including the intra-arterial administration of contrast media, by measuring serum creatinine concentration (SCr), a byproduct of muscle metabolism, primarily excreted by the kidneys. Our hypothesis suggests that conducting a qualitative comparison of both outcomes will enable identification of differences and similarities between in vitro and in vivo exposure. MATERIAL AND METHODS: In vitro, co-cultures of human dermal fibroblasts and human dermal microvascular endothelial cells forming capillary beds were exposed to a mixture of phosphate buffered saline and either Iobitridol, Iodixanol, or one of their supplements EDTA or Trometamol for 1.5 or 5 min. Negative control co-cultures were exposed exclusively to phosphate buffered saline. Co-cultures were either directly fixed or underwent a regeneration time of 1, 3 or 7 days. An artificial intelligence software was trained for detection of labeled endothelial capillaries (CD31) on light microscope images and measurements of morphometric parameters. In vivo, we retrospectively analyzed data from patients who underwent intra-arterial administration of contrast media and for whom SCr values were available pre- and post-contrast exposition (1, 3, and 7 days following procedure). Temporal development of SCr and incidence of CA-AKI were assessed. Both exposure types were qualitatively compared. RESULTS: In vitro, Iobitridol, Iodixanol and EDTA induced a strong decrease of two morphometric parameters after 3 days of regeneration. In vivo, a significant increase of SCr and incidence of CA-AKI was observed 3 days following procedure in the post-contrast media patients. No difference was observed between groups. DISCUSSION: Two of the morphometric parameters were inversely proportional to the SCr of the patients. If the endothelial damages observed in vitro occur in vivo, it may result in renal hypoxia, inducing a loss of kidney function clinically translated into an increase of SCr. Further development of our in vitro model could allow closer replication of the internal structure of a kidney and bridge the gap between in vitro studies and their clinical findings.

Efficient skin interactions of graphene derivatives: challenge, opportunity or both?

Interactions between graphene, with its wide deployment in consumer products, and skin, the body's largest organ and first barrier, are highly relevant with respect to toxicology and dermal delivery. In this work, interaction of polyglycerol-functionalized graphene sheets, with 200 nm average lateral size and different surface charges, and human skin was studied and their potential as topical delivery systems were investigated. While neutral graphene sheets showed no significant skin interaction, their positively and negatively charged counterparts interacted with the skin, remaining in the stratum corneum. This efficient skin interaction bears a warning but also suggests a new topical drug delivery strategy based on the sheets' high loading capacity and photothermal property. Therefore, the immunosuppressive drug tacrolimus was loaded onto positively and negatively charged graphene sheets, and its release measured with and without laser irradiation using liquid chromatography tandem-mass spectrometry. Laser irradiation accelerated the release of tacrolimus, due to the photothermal property of graphene sheets. In addition, graphene sheets with positive and negative surface charges were loaded with Nile red, and their ability to deliver this cargo through the skin was investigated. Graphene sheets with positive surface charge were more efficient than the negatively charged ones in enhancing Nile red penetration into the skin.

Treatment of digital dermatitis using salicylic acid in European bison (Bison bonasus) reveals promising results.

Digital dermatitis (DD) associated with the presence of multiple Treponema spp. was recently described for the first time in European bison (Bison bonasus). DD is characterized by skin inflammation in the distal foot area in various ungulates. The objective of this proof of concept study was to test a treatment protocol adopted from cattle for its applicability in this wildlife species using five animals. Keratolytic salicylic acid paste was administered topically under bandages for seven days to enable removal of the affected skin. All interventions were performed under general anesthesia. To evaluate the treatment efficacy, photographs and biopsies were taken pre- and post-treatment. The biopsies were examined histologically, by PCR for the presence of different bacterial species, by Treponema-specific fluorescent in situ hybridization (FISH), and by transmission electron microscopy. Based on photographs, complete clinical healing of the 15 feet with macroscopical DD lesions was achieved. Histological examination showed mild to moderate dermatitis in 17/20 feet before, and in 12/20 feet after treatment. 17/20 feet were Treponema spp. PCR positive before, and none was positive after treatment. Dichelobacter nodosus, Fusobacterium necrophorum, and Porphyromonas levii could not be detected in any of the samples. By FISH and electron microscopy, Treponema spp. could be visualized in the stratum corneum before, but not after treatment. These results suggest that this treatment method can be applied as standard practice prior to transporting DD affected European bison to prevent the spread of this contagious disease.

Post-mortem Computed Tomographic Angiography in Equine Distal Forelimbs: A Feasibility Study.

In-depth understanding of pathophysiological processes occurring in the vasculature of the equine distal limb is of great importance to improve both diagnostic and therapeutic approaches to diseases. To gain further insights, a model allowing high-resolution 3D-visualization of the vasculature is necessary. This pilot study evaluated the feasibility of restoring vascular perfusion in frozen-thawed distal equine cadaver limbs without prior preparation using computer tomographic imaging (CT). Five frozen-thawed, radiographically normal forelimbs were perfused with a lipophilic contrast agent through the median artery and radial vein in three phases (arterial, venous, and arterial-venous combined (AVC) dynamic). For comparison, one additional limb was perfused with a hydrosoluble contrast agent. The CT-studies (16-slice MDCT, 140 kV, 200 mA, 2 mm slice thickness, 1 mm increment, pitch 0.688) were evaluated at 11 specified regions for visualization of the vasculature and presence of artifacts or anatomic variations. The protocol used in this study proved to be feasible and provided good visualization (93.1%) of vasculature with low rates of artifacts. During the different phases, vascular visualization was similar, but while filling defects decreased in the later phases, extravasation worsened in the 2 limbs where it was observed. Subjectively, the best quality of angiographic images was achieved during the AVC dynamic phase. Perfusion with hydrosoluble contrast resulted in significantly lower vascular visualization (74.0%) and higher artifact rates. This study shows that reperfusion of frozen-thawed equine distal limbs with a lipophilic contrast agent allows for high-quality 3D-visualization of the vasculature and may serve as a model for in situ vascular evaluation in the future.

Independent COL5A1 Variants in Cats with Ehlers-Danlos Syndrome.

We investigated four cats with similar clinical skin-related signs strongly suggestive of Ehlers-Danlos syndrome (EDS). Cases no. 1 and 4 were unrelated and the remaining two cases, no. 2 and 3, were reportedly siblings. Histopathological changes were characterized by severely altered dermal collagen fibers. Transmission electron microscopy in one case demonstrated abnormalities in the collagen fibril organization and structure. The genomes of the two unrelated affected cats and one of the affected siblings were sequenced and individually compared to 54 feline control genomes. We searched for private protein changing variants in known human EDS candidate genes and identified three independent heterozygous COL5A1 variants. COL5A1 is a well-characterized candidate gene for classical EDS. It encodes the proα1 chain of type V collagen, which is needed for correct collagen fibril formation and the integrity of the skin. The identified variants in COL5A1 are c.112_118+15del or r.spl?, c.3514A>T or p.(Lys1172*), and c.3066del or p.(Gly1023Valfs*50) for cases no. 1, 2&3, and 4, respectively. They presumably all lead to nonsense-mediated mRNA decay, which results in haploinsufficiency of COL5A1 and causes the alterations of the connective tissue. The whole genome sequencing approach used in this study enables a refinement of the diagnosis for the affected cats as classical EDS. It further illustrates the potential of such experiments as a precision medicine approach in animals with inherited diseases.

Influence of Age and Breed on Bovine Ovarian Capillary Blood Supply, Ovarian Mitochondria and Telomere Length.

Worldwide, dairy cows of the type of high-producing cattle (HPC) suffer from health and fertility problems at a young age and therefore lose productivity after an average of only three lactations. It is still contentious whether these problems are primarily due to genetics, management, feeding or other factors. Vascularization plays a fundamental role in the cyclic processes of reproductive organs, as well as in the regeneration of tissues. In a previous study, HPC were shown to have a greater ovarian corpus luteum vascularization compared to dual-purpose breeds. We hypothesize that this activated angiogenesis could likely lead to an early exhaustion of HPC's regenerative capacity and thus to premature reproductive senescence. The objective of this study was to investigate if a HPC breed (Holstein-Friesian, HF) exhibits higher ovarian angiogenesis than a dual-purpose breed (Polish Red cow, PR) and if this is related to early ovarian aging and finally reproductive failure. For this purpose, we assessed the degree of vascularization by means of ovarian blood vessel characterization using light microscopy. As indicators for aging, we measured ovarian mitochondrial size and telomere length in peripheral leukocytes. We report in this study that in both breeds the distance between capillaries became smaller with increasing age and that the mean telomere length decreased with increasing age. The only difference between the two breeds was that PR developed larger capillaries than HF. Neither a relationship between telomere length, nor the morphology of the mitochondrial apparatus and nor angiogenesis in HF was proven. Although the data trends indicated that the proportion of shortened telomeres in HF was higher than in the PR, no significant difference between the two breeds was detected.

Endothelial cells and angiogenesis in the horse in health and disease-A review.

The cardiovascular system is the first functional organ in the embryo, and its blood vessels form a widespread conductive network within the organism. Blood vessels develop de novo, by the differentiation of endothelial progenitor cells (vasculogenesis) or by angiogenesis, which is the formation of new blood vessels from existing ones. This review presents an overview of the current knowledge on physiological and pathological angiogenesis in the horse including studies on equine endothelial cells. Principal study fields in equine angiogenesis research were identified: equine endothelial progenitor cells; equine endothelial cells and angiogenesis (heterogeneity, markers and assessment); endothelial regulatory molecules in equine angiogenesis; angiogenesis research in equine reproduction (ovary, uterus, placenta and conceptus, testis); angiogenesis research in pathological conditions (tumours, ocular pathologies, equine wound healing, musculoskeletal system and laminitis). The review also includes a table that summarizes in vitro studies on equine endothelial cells, either describing the isolation procedure or using previously isolated endothelial cells. A particular challenge of the review was that results published are fragmentary and sometimes even contradictory, raising more questions than they answer. In conclusion, angiogenesis is a major factor in several diseases frequently occurring in horses, but relatively few studies focus on angiogenesis in the horse. The challenge for the future is therefore to continue exploring new therapeutic angiogenesis strategies for horses to fill in the missing pieces of the puzzle.

The TRPV3 channel of the bovine rumen: localization and functional characterization of a protein relevant for ruminal ammonia transport.

Large quantities of ammonia (NH3 or NH4+) are absorbed from the gut, associated with encephalitis in hepatic disease, poor protein efficiency in livestock, and emissions of nitrogenous climate gasses. Identifying the transport mechanisms appears urgent. Recent functional and mRNA data suggest that absorption of ammonia from the forestomach of cattle may involve TRPV3 channels. The purpose of the present study was to sequence the bovine homologue of TRPV3 (bTRPV3), localize the protein in ruminal tissue, and confirm transport of NH4+. After sequencing, bTRPV3 was overexpressed in HEK-293 cells and Xenopus oocytes. An antibody was selected via epitope screening and used to detect the protein in immunoblots of overexpressing cells and bovine rumen, revealing a signal of the predicted ~ 90 kDa. In rumen only, an additional ~ 60 kDa band appeared, which may represent a previously described bTRPV3 splice variant of equal length. Immunohistochemistry revealed staining from the ruminal stratum basale to stratum granulosum. Measurements with pH-sensitive microelectrodes showed that NH4+ acidifies Xenopus oocytes, with overexpression of bTRPV3 enhancing permeability to NH4+. Single-channel measurements revealed that Xenopus oocytes endogenously expressed small cation channels in addition to fourfold-larger channels only observed after expression of bTRPV3. Both endogenous and bTRPV3 channels conducted NH4+, Na+, and K+. We conclude that bTRPV3 is expressed by the ruminal epithelium on the protein level. In conjunction with data from previous studies, a role in the transport of Na+, Ca2+, and NH4+ emerges. Consequences for calcium homeostasis, ruminal pH, and nitrogen efficiency in cattle are discussed.

Impact of intercellular crosstalk between epidermal keratinocytes and dermal fibroblasts on skin homeostasis.

Dermal fibroblasts seem critical for epidermal maturation and differentiation and recent work demonstrated that diseased fibroblasts may drive pathophysiological processes. Nevertheless, still very little is known about the actual crosstalk between epidermal keratinocytes and dermal fibroblasts and the impact of dermal fibroblasts on epidermal maturation and differentiation. Aiming for a more fundamental understanding of the impact of the cellular crosstalk between keratinocytes and fibroblasts on the skin homeostasis, we generated full-thickness skin equivalents with and without fibroblasts and subsequently analysed them for the expression of skin differentiation markers, their barrier function, skin lipid content and epidermal cell signalling. Skin equivalents without fibroblasts consistently showed an impaired differentiation and dysregulated expression of skin barrier and tight junction proteins, increased skin permeability, and a decreased skin lipid/protein ratio. Most interestingly, impaired Ras/Raf/ERK/MEK signalling was evident in skin equivalents without fibroblasts. Our data clearly indicate that the epidermal-dermal crosstalk between keratinocytes and fibroblasts is critical for adequate skin differentiation and that fibroblasts orchestrate epidermal differentiation processes.

Fatal Elephant Endotheliotropic Herpesvirus Infection of Two Young Asian Elephants.

Elephant endotheliotropic herpesvirus (EEHV) can cause a devastating haemorrhagic disease in young Asian elephants worldwide. Here, we report the death of two young Asian elephants after suffering from acute haemorrhagic disease due to EEHV-1A infection. We detected widespread distribution of EEHV-1A in various organs and tissues of the infected elephants. Enveloped viral particles accumulated within and around cytoplasmic electron-dense bodies in hepatic endothelial cells were detected. Attempts to isolate the virus on different cell cultures showed limited virus replication; however, late viral protein expression was detected in infected cells. We further showed that glycoprotein B (gB) of EEHV-1A possesses a conserved cleavage site Arg-X-Lys/Arg-Arg that is targeted by the cellular protease furin, similar to other members of the Herpesviridae. We have determined the complete 180 kb genome sequence of EEHV-1A isolated from the liver by next-generation sequencing and de novo assembly. As virus isolation in vitro has been unsuccessful and limited information is available regarding the function of viral proteins, we have attempted to take the initial steps in the development of suitable cell culture system and virus characterization. In addition, the complete genome sequence of an EEHV-1A in Europe will facilitate future studies on the epidemiology and diagnosis of EEHV infection in elephants.

Histological and functional comparisons of four anatomical regions of porcine skin with human abdominal skin.

Because of the shortage of human skin for research purposes, porcine skin has been used as a model of human skin. The aim of this study was to identify the region of German Landrace pig skin that could be used as the best possible substitute for human abdominal skin. Porcine samples were collected from the ear, flank, back and caudal abdomen; human abdominal skin samples were excised during plastic surgery. Histological and ultrastructural assessments were carried out on the epidermis and dermis, with emphasis on the dermo-epidermal interface length, dermo-epidermal thickness ratio as well as densities of; hair follicles, arrector pili muscles, blood vessels and sweat glands. In the pig, the barrier function of the four anatomical regions was assessed. Results showed that both histologically and ultrastructurally, all four regions of porcine skin were similar to human skin. These include the shapes of keratinocytes, structure of cell contacts and presence of Weibel Palade bodies in endothelial cells. Other parameters such as the thickness of epidermis, the thickness of stratum basale, spinosum and granulosum and the number of cell layers in the stratum corneum were similar in human abdominal and in all four regions of porcine skin. However, there were also significant differences especially in the thickness of the stratum corneum, the dermo-epidermal interface length and the blood vessel density.

Ultrastructural and Molecular Analysis of Ribose-Induced Glycated Reconstructed Human Skin.

Aging depicts one of the major challenges in pharmacology owing to its complexity and heterogeneity. Thereby, advanced glycated end-products modify extracellular matrix proteins, but the consequences on the skin barrier function remain heavily understudied. Herein, we utilized transmission electron microscopy for the ultrastructural analysis of ribose-induced glycated reconstructed human skin (RHS). Molecular and functional insights substantiated the ultrastructural characterization and proved the relevance of glycated RHS beyond skin aging. In particular, electron microscopy mapped the accumulation and altered spatial orientation of fibrils and filaments in the dermal compartment of glycated RHS. Moreover, the epidermal basement membrane appeared thicker in glycated than in non-glycated RHS, but electron microscopy identified longitudinal clusters of the finest collagen fibrils instead of real thickening. The stratum granulosum contained more cell layers, the morphology of keratohyalin granules decidedly differed, and the stratum corneum lipid order increased in ribose-induced glycated RHS, while the skin barrier function was almost not affected. In conclusion, dermal advanced glycated end-products markedly changed the epidermal morphology, underlining the importance of matrix⁻cell interactions. The phenotype of ribose-induced glycated RHS emulated aged skin in the dermis, while the two to three times increased thickness of the stratum granulosum resembled poorer cornification.

Human and equine endothelial cells in a live cell imaging scratch assay in vitro.

BACKGROUND: Human and equine patients are known to frequently develop vascular complications, particularly thrombosis both in veins and arteries as well as in the microvasculature. OBJECTIVE: The aim of the present study was to investigate and compare the angiogenic response of human and equine endothelial cells to lesions in an in vitro scratch assay. METHODS: Endothelial cells from human umbilical vein (HUVEC), abdominal aorta (HAAEC) and dermal microvasculature (HDMEC) as well as equine carotid artery (EACEC) and jugular vein (EVJEC) were cultured and an elongated defect was created (scratch or "wound"). Cultures were monitored over a period of 90 hours in a live cell imaging microscope. RESULTS: In the human endothelial cell cultures, there was a uniform and continuous migration of the cells from the scratch fringe into the denuded area, which was closed after 17 (HUVEC), 15 (HAAEC) and 26 (HDMEC) hours. In the equine endothelial cell cultures, a complete closure of the induced defect occurred after 17 (EVJEC) and 35 (EACEC) hours. CONCLUSIONS: In the equine arterial cells, the delay in closure of the denuded area seems to be the results of a disoriented and uncoordinated migration of endothelial tip cells resulting in slow re-endothelialization.

A comparative study of ammonia transport across ruminal epithelia from Bos indicus crossbreds versus Bos taurus.

Absorption of ammonia from the rumen of cattle decreases nitrogen availability for fermentational protein synthesis, leading to increased competition of cattle with humans for protein and enhancing the release of toxic nitrogenous compounds into the environment. Given that differences in feeding and breeding might induce differences in ruminal ammonia transport, we compared electrophysiological, histological, and molecular biological characteristics of ruminal epithelia of Bos indicus crossbreds (Sahiwal-Mix, SWM) with those of Bos taurus (Holstein-Friesian, HF). As in HF, the stratified cornified epithelium of SWM expressed claudin 1 and 4. Measurements of ammonia flux (HF) and serosal pH (both breeds) suggested that at a mucosal pH of 6.4, net transport primarily occurred as NH4 + . As shown previously for HF, NH4 + induced a concentration-dependent rise in short circuit current (Isc ) in SWM that could be further stimulated by the TRP channel agonist menthol. Relative mRNA expression levels for TRPV3, TRPV4, TRPM6, and TRPM7 were significantly lower in SWM than in HF, with TRPA1 expression near the limit of detection. We conclude that uptake of ammonia from the rumen of both breeds occurs electrogenically as NH4 + with functional and molecular biological evidence pointing towards involvement of TRPV3 and TRPV4.

Generation of full-thickness skin equivalents using hair follicle-derived primary human keratinocytes and fibroblasts.

Skin equivalents are increasingly used as human-based test systems for basic and preclinical research. Most of the established skin equivalents are composed of primary keratinocytes and fibroblasts, isolated either from excised human skin or juvenile foreskin following circumcisions. Although the potential of hair follicle-derived cells for the generation of skin equivalents has been shown, this approach normally requires microdissections from the scalp for which there is limited subject compliance or ethical approval. In the present study, we report a novel method to isolate and cultivate keratinocytes and fibroblasts from plucked hair follicles that were then used to generate skin equivalents. The procedure is non-invasive, inflicts little-pain, and may allow easy access to patient-derived cells without taking punch biopsies. Overall, minor differences in morphology, ultrastructure, expression of important structural proteins, or barrier function were observed between skin equivalents generated from hair follicle-derived or interfollicular keratinocytes and fibroblasts. Interestingly, improved basal lamina formation was seen in the hair follicle-derived skin equivalents. The presented method here allows easy and non-invasive access to keratinocytes and fibroblasts from plucked hair follicles that may be useful particularly for the generation of skin disease equivalents.

Subcellular Interactions during Vascular Morphogenesis in 3D Cocultures between Endothelial Cells and Fibroblasts.

BACKGROUND: Increasing the complexity of in vitro systems to mimic three-dimensional tissues and the cellular interactions within them will increase the reliability of data that were previously collected with in vitro systems. In vivo vascularization is based on complex and clearly defined cell-matrix and cell-cell interactions, where the extracellular matrix (ECM) seems to play a very important role. The aim of this study was to monitor and visualize the subcellular and molecular interactions between endothelial cells (ECs), fibroblasts, and their surrounding microenvironment during vascular morphogenesis in a three-dimensional coculture model. METHODS: Quantitative and qualitative analyses during the generation of a coculture tissue construct consisting of endothelial cells and fibroblasts were done using transmission electron microscopy and immunohistochemistry. RESULTS: Dynamic interactions were found in cocultures between ECs, between fibroblasts (FBs), between ECs and FBs, and between the cells and the ECM. Microvesicles were involved in intercellular information transfer. FBs took an active and physical part in the angiogenesis process. The ECM deposited by the cells triggered endothelial angiogenic activity. Capillary-like tubular structures developed and matured. Moreover, some ECM assembled into a basement membrane (BM) having three different layers equivalent to those seen in vivo. Finally, the three-dimensional in vitro construct mirrored the topography of histological tissue sections. CONCLUSION: Our results visualize the importance of the physical contact between all cellular and acellular components of the cocultures.

Isolation of equine endothelial cells and life cell angiogenesis assay.

Arterial or venous thromboses are frequent clinical complications with the risk of fatal progression. Recent studies suggest the disruption of angiogenesis in the course of thrombus resolution as the underlying pathomechanism. Very similar to the situation in human patients, equine vessels have been described to be particularly susceptible to thrombosis. In contrast to humans, equine donors are readily available to obtain organs and tissues for isolation of endothelial cells. Objective of this study was to isolate equine endothelial cells and develop an angiogenesis assay from primary cultures. Macrovascular endothelial cells were obtained from jugular veins and carotid arteries of nine horses, one of which suffered from inflammatory processes. After enzymatic isolation, the cells were incubated in different selective primary media. Phenotypic identification of endothelial cells was accomplished by morphology and positive staining to von Willebrand factor. The reliable, inexpensive, and standardized combination of methods presented here resulted in pure endothelial cultures for angiogenesis assays that can be used in any cell culture laboratory. Inverted phase microscopy and life cell imaging was used to characterize the stages of the angiogenic cascade of the endothelial cells. Life cell imaging gave new insights into the in vitro formation of capillary like structures including exocytosis of microparticles from endothelial cells before integration into the three-dimensional structure. We hypothesize that a specific population of endothelial cells showing a highly active migration pattern in life cell imaging might play a role in the resolution of thrombosis.