Mouse Skin Tissue Research Articles

Effect of Smallanthus sonchifolius extracts on croton oil-induced oedema and neutrophil migration to the ear skin tissue of mice

Smallanthus sonchifolius (yacón) is an Andean species whose leaves have glandular trichomes rich in sesquiterpene lactones (STLs). The chemical profile of the leaf rinse extract (RLE) of yacón showed several STLs [1]. The major STL isolated were quantified [2] and the in vitro anti-inflammatory effect of the two major STLs was evaluated [1]. However, STLs are known for their oral toxicity and the study of topical applications can be an alternative to the use of these anti-inflammatory substances. Thus, the aim of this work was to evaluate the in vivo topic anti-inflammatory potential of yacón. The RLE was obtained from entire dried leaves rinsed with acetone. After liquid-liquid partition with n-hexane the resulting hydromethanolic fraction was evaluated using the croton oil ear oedema assay in mice [3]. Subsequently, fragments of ears were used for myeloperoxidase (MPO) determination in order to evaluate leukocyte migration to the subcutaneous tissue. IR spectrum and HPLC-UV-DAD analysis demonstrated that the RLE is a STL-rich extract, which showed anti-oedematogenic activity (Image 1A) followed by inhibition of neutrophil migration to the inflamed tissue in all tested doses (Image 1B). Plant extracts that decrease oedema and leukocytes migration can be an useful alternative to the treatment of topical inflammatory injuries. Thus, the STL-rich extract of yacón can be a promising phytotherapeutic agent for topic application in the inflammatory process.

Leishmania infantum proteophosphoglycans regurgitated by the bite of its natural sand fly vector, Lutzomyia longipalpis, promote parasite establishment in mouse skin and skin-distant tissues

We demonstrate that a proteophosphoglycan-rich gel secreted by Leishmania infantum inside the midgut of Lutzomyia longipalpis sand flies (promastigote secretory gel) is regurgitated along with an average dose of 500 L. infantum metacyclic promastigotes per infected bite. Using both low (10 3) and high (10 5) doses of parasites in the ears of BALB/c mice we show that the infections benefit from the presence of vector saliva and parasite gel in the skin. However, chronic infection of the spleen was only enhanced in high dose co-infections with gel. These results provide the framework for a more natural experimental model of visceral leishmaniasis.

Tumor Cell Response to Synchrotron Microbeam Radiation Therapy Differs Markedly From Cells in Normal Tissues

High-dose synchrotron microbeam radiation therapy (MRT) can be effective at destroying tumors in animal models while causing very little damage to normal tissues. The aim of this study was to investigate the cellular processes behind this observation of potential clinical importance. MRT was performed using a lattice of 25 mum-wide, planar, polychromatic, kilovoltage X-ray microbeams, with 200-microm peak separation. Inoculated EMT-6.5 tumor and normal mouse skin tissues were harvested at defined intervals post-MRT. Immunohistochemical detection of gamma-H2AX allowed precise localization of irradiated cells, which were also assessed for proliferation and apoptosis. MRT significantly reduced tumor cell proliferation by 24 h post-irradiation (p = 0.002). An unexpected finding was that within 24 h of MRT, peak and valley irradiated zones were indistinguishable in tumors because of extensive cell migration between the zones. This was not seen in MRT-treated normal skin, which appeared to undergo a coordinated repair response. MRT elicited an increase in median survival times of EMT-6.5 and 67NR tumor-inoculated mice similar to that achieved with conventional radiotherapy, while causing markedly less normal tissue damage. This study provides evidence of a differential response at a cellular level between normal and tumor tissues after synchrotron MRT.

Effects of the Aminophenol Analogue p-Dodecylaminophenol on Mouse Skin

p-Dodecylaminophenol (p-DDAP) was designed on the basis of structure-activity relationship studies on N-(4-hydroxyphenyl)retinamide (fenretinide, 4-HPR), a synthetic derivative of retinoic acid (RA). p-DDAP exhibits antioxidative activities greater than those of RA and 4-HPR. RA shows biological effects in epidermal cells that include the inhibition of differentiation to the squamous phenotype. In the current study, we examined the effects of topical p-DDAP treatment on the skin of hairless mice as compared with those of RA treatment. p-DDAP caused an increase in epidermal thickness and decreased matrix metalloprotease and hyaluronidase activities in mouse skin tissues to the same extent that RA did. p-DDAP did not induce desquamation, erythema, or inflammatory cytokine expression as observed with RA treatment. Two-dimensional polyacrylamide gel electrophoresis patterns of proteins from skin treated with p-DDAP were distinct from those treated with RA. A protein induced by both p-DDAP and RA was identified as cytokeratin 16. p-DDAP did not elevate transcriptional activities of RA nuclear receptors. These results suggest that p-DDAP improves skin as potently as RA without causing the desquamation and erythema that the latter does. An increase in cytokeratin 16 expression might be essential for the effects of both p-DDAP and RA in skin healing and maintenance.

Weight Loss via Exercise with Controlled Dietary Intake May Affect Phospholipid Profile for Cancer Prevention in Murine Skin Tissues

Exercise has been linked to a reduced cancer risk in animal models. However, the underlying mechanisms are unclear. This study assessed the effect of exercise with dietary consideration on the phospholipid profile in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse skin tissues. CD-1 mice were randomly assigned to one of the three groups: ad libitum-fed sedentary control; ad libitum-fed treadmill exercise at 13.4 m/min for 60 min/d, 5 d/wk (Ex+AL); and treadmill-exercised but pair-fed with the same amount as the control (Ex+PF). After 14 weeks, Ex+PF but not Ex+AL mice showed approximately 25% decrease in both body weight and body fat when compared with the controls. Of the total 338 phospholipids determined by electrospray ionization-tandem mass spectrometry, 57 were significantly changed, and 25 species could distinguish effects of exercise and diet treatments in a stepwise discriminant analysis. A 36% to 75% decrease of phosphatidylinositol (PI) levels in Ex+PF mice occurred along with a significant reduction of PI 3-kinase in TPA-induced skin epidermis, as measured by both Western blotting and immunohistochemistry. In addition, approximately 2-fold increase of the long-chain polyunsaturated fatty acids, docosahexaenoic and docosapentaenoic acids, in phosphatidylcholines, phosphatidylethanolamines, and lysophosphatidylethanolamines was observed in the Ex+PF group. Microarray analysis indicated that the expression of fatty acid elongase-1 increased. Taken together, these data indicate that exercise with controlled dietary intake, but not exercise alone, significantly reduced body weight and body fat as well as modified the phospholipid profile, which may contribute to cancer prevention by reducing TPA-induced PI 3-kinase and by enhancing omega-3 fatty acid elongation.

Gadolinium toxicity: epidermis thickness measurement by magnetic resonance imaging at 500 MHz

Regional epidermal thickening and hair follicle width measurement by delayed gadolinium contrast magnetic resonance imaging (MRI) may assess the contrast agent gadolinium toxicity on mice skin. Delayed contrast in vivo MRI was performed in mice. Six mice skin samples were removed and exposed to a gadolinium contrast agent at different times after 2, 4, 6 and 8 h. The relaxation constants of each skin structure were measured. The thickness of the epidermis and hair follicle on follow-up ex vivo delayed-contrast MRI served as an index of gadolinium toxicity on the skin. In vivo MRI by fast low-angle shot imaging technique showed distinct skin layers. High-resolution gradient echo T1-weighted and multislice multiecho proton density-weighted MRI intensities in the epidermis and hair follicle showed a positive correlation with delayed gadolinium-enhanced MRI hyperintensities (Pearson's correlation coefficient r(2)=0.81, P<0.0001) in the excised mice skin tissues. Delayed contrast-enhanced mice skin MRI after 2-4 h showed epidermis swelling and hair follicle regions with a size measurement accuracy of 65%, a sensitivity of 95%, a specificity of 25%, a positive predictive value of 65% and a negative predictive value of 65%. Areas under the receiver operating characteristic curves by MRI were 0.92-0.94 for hair and epidermis as good discriminators. MRI visualized distinct relaxation constants of the epidermis, sebaceous gland, skin papillary and reticular dermis layers and hair follicle. Gadolinium contrast-enhanced MRI may visualize the thickening of the epidermis wall and hair follicle as an index of viable mice skin. Gadolinium enhanced the MRI visibility of skin structures. Gadolinium treatment showed skin toxicity as epidermis thickening the first time due to the undesirable use of high concentrations of gadolinium in microimaging.

Localization of anionic constituents in mast cell granules of brachymorphic (bm/bm) mice by using avidin-conjugated colloidal gold

We used the egg avidin gold complex as a polycationic probe for the localization of negatively charged sites in the secretory granules of mouse mast cells. We compared the binding of this reagent to mast cell granules in wild-type mice and in congenic brachymorphic mice in which mast cell secretory granules contained undersulfated proteoglycans. We localized anionic sites by post-embedding labeling of thin sections of mouse skin and tongue tissues fixed in Karnovsky's fixative and OsO(4) and embedded in Araldite. Transmission electron microscopy revealed that the mast cell granules of bm/bm mice had a lower optical density than those of wild-type mice (P<0.001) and a lower avidin gold binding density (by approximately 50%, P<0.001). The latter result provided additional evidence that the contents of mast cell granules in bm/bm mice were less highly sulfated than in those of wild-type mice. In both wild-type and bm/bm mast cells, the distribution of granule equivalent volumes was multimodal, but the unit granule volume was approximately 19% lower in bm/bm cells than in wild-type cells (P<0.05). Thus, bm/bm mast cells develop secretory granules that differ from those of wild-type mice in exhibiting a lower optical density and slightly smaller unit granules, however the processes that contribute to granule maturation and granule-granule fusion in mast cells are operative in bm/bm cells.

Synergistic Effects of Combined Phytochemicals and Skin Cancer Prevention in SENCAR Mice

The purpose of our study was to determine the inhibitory effect of combined phytochemicals on chemically induced murine skin tumorigenesis. Our hypothesis was that concurrent topical and dietary treatment with selected compounds would lead to more efficient prevention of skin cancer. We tested ellagic acid and calcium D-glucarate as components of diets, while resveratrol was applied topically; grape seed extract was applied topically or in the diet. The 4-week inflammatory-hyperplasia assay based on the 7,12-dimethylbenz[a]anthracene (DMBA)-induced skin carcinogenesis model in SENCAR mice was used. We have found that all the selected combinations caused a marked decrease of epidermal thickness compared with the DMBA-treated group and also with groups treated with a single compound and DMBA. All combinations of resveratrol with other compounds showed a synergistic effect on hyperplasia and Ha-ras mutations. Skin tissue of mice receiving the combinations showed decreased cell proliferation and Bcl2 expression; decreased p21, a regulator of cell cycle; and decreased marker of inflammation cyclooxygenase-2. All the selected combinations diminished the DMBA-induced mRNA expression of the CYP1B1 level, and also caused a marked decrease of proto-oncogenes c-jun and c-fos, components of transcription factor activator protein. In conclusion, all combinations showed either additive or synergistic effects and their joint actions allowed for decreasing the doses of the compounds. Especially, resveratrol combinations with ellagic acid, grape seed extract, and other phytochemicals are very potent inhibitors of skin tumorgenesis, based on the suppression of epidermal hyperplasia as well as on the modulation of intermediate biomarkers of cell proliferation, cell survival, inflammation, oncogene mutation, and apoptosis.

Cytokeratins 16 and 10 bind to retinoic acid covalently in skin tissue of mice

Retinoic acid (RA) has various biological effects in mammalian cells and tissues. In epidermal cells, RA is an inhibitor of differentiation to the squamous phenotype. The molecular mechanisms underlying the effects of RA on epidermal cells and other cell types are mediated by RA nuclear receptors and retinoylation (acylation by RA) of proteins. To understand the components responsible for RA effects via RA nuclear receptors and retinoylation. We examined for the first time RA-binding proteins in mouse skin in vivo by immunoblotting using anti-RA monoclonal antibodies and identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. We identified eight RA-binding proteins in the skin of hairless mice that were increased by topical RA treatment. Three of these proteins were identified as cytokeratin 10, cytokeratin 16 and serum albumin. These results raise the possibility that RA binding to cytokeratins in vivo may be involved in the effect of RA on keratinocytes in mouse skin.

Sulfur mustard analog induces oxidative stress and activates signaling cascades in the skin of SKH-1 hairless mice

A monofunctional analog of the chemical warfare agent sulfur mustard (HD), 2-chloroethyl ethyl sulfide (CEES), induces tissue damage similar to HD. Herein we studied the molecular mechanisms associated with CEES-induced skin inflammation and toxicity in SKH-1 hairless mice. Topical CEES exposure caused an increase in oxidative stress as observed by enhanced 4-hydroxynonenal and 5,5-dimethyl-2-(8-octanoic acid)-1-pyrroline N-oxide protein adduct formation and an increase in protein oxidation. The CEES-induced increase in the formation of 8-oxo-2-deoxyguanosine indicated DNA oxidation. CEES exposure instigated an increase in the phosphorylation of mitogen-activated protein kinases (MAPKs; ERK1/2, JNK, and p38). After CEES exposure, a significant increase in the phosphorylation of Akt at Ser473 and Thr308 was observed as well as upregulation of its upstream effector, PDK1, in mouse skin tissue. Subsequently, CEES exposure caused activation of AP-1 family proteins and the NF-κB pathway, including phosphorylation and degradation of IκBα in addition to phosphorylation of the NF-κB essential modulator. Collectively, our results indicate that CEES induces oxidative stress and the activation of the transcription factors AP-1 and NF-κB via upstream signaling pathways including MAPKs and Akt in SKH-1 hairless mouse skin. These novel molecular targets could be supportive in the development of prophylactic and therapeutic interventions against HD-related skin injury.

Peak Multiphoton Excitation of mCherry Using an Optical Parametric Oscillator (OPO)

mCherry is a red fluorescent protein which is bright, photostable, and has a low molecular weight. It is an attractive choice for multiphoton fluorescence imaging; however, the multiphoton excitation spectrum of mCherry is not known. In this paper we report the two photon excitation spectrum of mCherry measured up to 1190 nm in the near infrared (NIR) region. Skin tissues of transgenic mice that express mCherry were used in the experiments. mCherry in the tissues was excited with a Titanium:Sapphire laser and an optical parametric oscillator pumped by the Titanium:Sapphire laser. We found that the peak excitation of mCherry occurs at 1160 nm.

Antigen non-specific tissue damage in T cell-mediated experimental autoimmune orchitis: preliminary characterization of a testis-specific T-cell line by using dermal tissue and cells.

A previous study demonstrated the establishment of a murine testicular antigen (mTA)-specific CD4+ T-cell line (designated BT.1) which was capable of transferring experimental autoimmune epididymo-orchitis to naive recipient mice. The disease transfer was antigen-specific, because no inflammatory lesion was observed in any other organs and tissues of the recipients. In this study, to investigate the local environment of BT.1 cells, the effect of the cells and their culture supernatant on a local tissue integrity was studied. When BT.1 cells were seeded on cultured fibroblastoid cell monolayers, the cells completely disrupted these monolayers in spite of the absence of the specific antigens. Moreover, the culture supernatant of BT.1 cells induced non-specific dermal inflammation when injected into skin tissue of normal syngeneic mice. Therefore, BT.1 cells were shown to devastate a tissue integrity and cause attraction and activation of inflammatory cells of the recipient origin in a local environment. These results suggest that the transferred BT.1 cells will specifically home to the testis and epididymis of recipients but the following devastation of seminiferous tubules and epididymal ducts might be non-specifically produced by the inflammatory cells of both donor and recipient origin in the lesion.

Effect of glycyrrhizin (GL) on β-defensin production in tissues surrounding the burn area of thermally injured mice (134.44)

Abstract The decreased production of antimicrobial peptides in tissues surrounding the burn area has been demonstrated in patients and animals with severe burn injury. Previously, sepsis stemming from burn wound infections with Pseudomonas aeruginosa was effectively controlled in burn mice treated with GL. Therefore, in this study, effects of GL on β-defensin production in tissues surrounding the burn area were examined. BALB/c mice with 25% TBSA of 3rd degree flame burn were treated i.p. with GL (Minophagen Pharmaceutical Co., Ltd., Tokyo, Japan) at various schedules after burn injury. Eight punch biopsies (8-mm diameter) per mouse were obtained from skin tissues surrounding the burn area of these mice 24 hrs after burn injury. The homogenates of 8 biopsies were assayed for MBD-1 and MBD-3 by ELISA. In the results, normal mouse skin homogenates consistently contained 0.8 ± 0.4 μg/ml of MBD-1 and 6.9 ± 1.8 μg/ml of MBD-3. However, MBD-1 and MBD-3 were not detected in homogenates of tissues surrounding the burn area. When 10 mg/kg of GL was administrated i.p. to mice 2 and 12 hrs after burn injury, the amounts of MBD-1 and MBD-3 in the homogenates of tissues surrounding the burn area recovered to the level shown in normal mouse tissue specimens. MBD production in skin tissues of normal mice was not influenced by GL treatment. These results indicate that, after treatment with GL, the decreased production of β-defensins in tissues surrounding the burn area is improved in thermally injured mice. This study was supported by SHC NA #8610.

Nonlinear Spectral Imaging Microscopy

The deep tissue penetration of nonlinear microscopy and the high detection efficiency of a spectrograph are utilized to record spectral images of the intrinsic emission of living mouse skin tissues. Visualization of the spectral images by wavelength-to-RGB color conversion allowed identification and discrimination of tissue structures Nonlinear spectral imaging microscopy (NSIM) can provide a wealth of information not easily obtainable with present conventional nonlinear imaging systems.

Detailed Analysis of Sequence Changes Occurring during vlsE Antigenic Variation in the Mouse Model of Borrelia burgdorferi Infection

Lyme disease Borrelia can infect humans and animals for months to years, despite the presence of an active host immune response. The vls antigenic variation system, which expresses the surface-exposed lipoprotein VlsE, plays a major role in B. burgdorferi immune evasion. Gene conversion between vls silent cassettes and the vlsE expression site occurs at high frequency during mammalian infection, resulting in sequence variation in the VlsE product. In this study, we examined vlsE sequence variation in B. burgdorferi B31 during mouse infection by analyzing 1,399 clones isolated from bladder, heart, joint, ear, and skin tissues of mice infected for 4 to 365 days. The median number of codon changes increased progressively in C3H/HeN mice from 4 to 28 days post infection, and no clones retained the parental vlsE sequence at 28 days. In contrast, the decrease in the number of clones with the parental vlsE sequence and the increase in the number of sequence changes occurred more gradually in severe combined immunodeficiency (SCID) mice. Clones containing a stop codon were isolated, indicating that continuous expression of full-length VlsE is not required for survival in vivo; also, these clones continued to undergo vlsE recombination. Analysis of clones with apparent single recombination events indicated that recombinations into vlsE are nonselective with regard to the silent cassette utilized, as well as the length and location of the recombination event. Sequence changes as small as one base pair were common. Fifteen percent of recovered vlsE variants contained “template-independent” sequence changes, which clustered in the variable regions of vlsE. We hypothesize that the increased frequency and complexity of vlsE sequence changes observed in clones recovered from immunocompetent mice (as compared with SCID mice) is due to rapid clearance of relatively invariant clones by variable region-specific anti-VlsE antibody responses.

In vivo nonlinear spectral imagingmicroscopy of visible and ultraviolet irradiated hairless mouse skin tissues

We demonstrate the capability of nonlinear spectral imaging microscopy (NSIM) in investigating ultraviolet and visible light induced effects on albino Skh:HR-1 hairless mouse skin non-invasively.

Essential protective role attributed to the surface lipoproteins of Borrelia burgdorferi against innate defences

To initiate infection, a microbial pathogen must be able to evade innate immunity. Here we show that the Lyme disease spirochete Borrelia burgdorferi depends on its surface lipoproteins for protection against innate defences. The deficiency for OspC, an abundantly expressed surface lipoprotein during early infection, led to quick clearance of B. burgdorferi after inoculation into the skin of SCID mice. Increasing expression of any of the four randomly chosen surface lipoproteins, OspA, OspE, VlsE or DbpA, fully protected the ospC mutant from elimination from the skin tissue of SCID mice; moreover, increased OspA, OspE or VlsE expression allowed the mutant to cause disseminated infection and restored the ability to effectively colonize both joint and skin tissues, albeit the dissemination process was much slower than that of the mutant restored with OspC expression. When the ospC mutant was modified to express OspA under control of the ospC regulatory elements, it registered only a slight increase in the 50% infectious dose than the control in SCID mice but a dramatic increase in immunocompetent mice. Taken together, the study demonstrated that the surface lipoproteins provide B. burgdorferi with an essential protective function against host innate elimination.

Bio-heat transfer analysis during short pulse laser irradiation of tissues

The objective of this paper is to analyze the temperature distributions and heat affected zone in skin tissue medium when irradiated with either a collimated or a focused laser beam from a short pulse laser source. Experiments are performed on multi-layer tissue phantoms simulating skin tissue with embedded inhomogeneities simulating subsurface tumors and as well as on freshly excised mouse skin tissue samples. Two types of lasers have been used in this study – namely a Q-switched pulsed 1064 nm Nd:YAG short pulse laser having a pulse width of 200 ns and a 1552 nm diode short pulsed laser having a pulse width of 1.3 ps. Experimental measurements of axial and radial temperature distribution in the tissue medium are compared with the numerical modeling results. For numerical modeling, the transient radiative transport equation is first solved using a discrete ordinates method for obtaining the intensity distribution and radiative heat flux inside the tissue medium. Then the temperature distribution is obtained by coupling the bio-heat transfer equation with either hyperbolic non-Fourier or parabolic Fourier heat conduction model. The hyperbolic heat conduction equation is solved using MacCormack’s scheme with error terms correction. It is observed that experimentally measured temperature distribution is in good agreement with that predicted by hyperbolic heat conduction model. The experimental measurements demonstrate that converging laser beam focused directly at the subsurface location can produce desired high temperature at that location compared to that produced by collimated laser beam for the same laser parameters. Finally the ablated tissue removal is characterized using histological studies as a function of laser parameters.

Comparison of Protein and DNA Profiles of Mice Skin Tissue and Colon Cancer, Cells Extracted with the Precellys®24 Vs. Conventional Methods

Comparison of Protein and DNA Profiles of Mice Skin Tissue and Colon Cancer, Cells Extracted with the Precellys®24 Vs. Conventional Methods

Gr-1+CD11b+ cells as an accelerator of sepsis stemming fromPseudomonas aeruginosawound infection in thermally injured mice

Using a mouse model of thermal injury, we studied why antimicrobial peptides are not produced at the burn-site tissues and how this defect contributes to the increased susceptibility to Pseudomonas aeruginosa burn-wound infection. Logarithmic growth of P. aeruginosa was demonstrated locally (at the burn site) and systemically (in circulation) in thermally injured mice exposed to 10(2) CFU/mouse of the pathogen beneath the burn wound. However, neither systemic nor local growth of the pathogen was observed in sham burn mice when they were infected intradermally with 10(6) CFU/mouse P. aeruginosa. Murine beta-defensins (MBDs) were detected in the skin homogenates of sham burn mice. However, the amounts of MBDs were reduced greatly in the same tissue homogenates from thermally injured mice. Gr-1(+)CD11b(+) cells, with an ability to suppress antimicrobial peptide production by skin keratinocytes, were isolated from tissues surrounding the burn areas, and these cells were not obtained from skin tissues of sham burn mice. After intradermal inoculation of Gr-1(+)CD11b(+) cells, which were isolated from burn-site tissues, the production of antimicrobial peptides around the cell-inoculation site of sham burn mice decreased. Also, like thermally injured mice, these mice were shown to be susceptible to P. aeruginosa intradermal infection. These results indicate that sepsis stemming from P. aeruginosa burn-wound infection is accelerated by burn-induced Gr-1(+)CD11b(+) cells with abilities to suppress antimicrobial peptide production by epidermal keratinocytes.