Mouse Ear Research Articles

Anti-Inflammatory Effects of Ribes diacanthum Pall Mediated via Regulation of Nrf2/HO-1 and NF-κB Signaling Pathways in LPS-Stimulated RAW 264.7 Macrophages and a TPA-Induced Dermatitis Animal Model.

Ribes diacanthum Pall (RDP) is a Mongolian traditional medicine used to treat renal inflammation. In the present study, we initially investigated the anti-inflammatory effects and mechanisms of action of ethylacetate extract of RDP (EARDP) in RAW 264.7 macrophages stimulated by lipopolysaccharide (LPS) and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced dermatitis in mice. We demonstrated that EARDP protected against LPS-induced cell death by inhibiting intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) production, as well as the synthesis of pro-inflammatory mediators and cytokines, such as nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1β. EARDP inhibited the phosphorylation and degradation of inhibitory κB-α (IκB-α) and the activation of nuclear factor (NF)-κB, indicating that the anti-inflammatory effect of EARDP was mediated via the suppression of NF-κB nuclear translocation. In addition, EARDP induced the heme oxygenase-1 (HO-1) expression and nuclear translocation of nuclear factor-E2-related factor 2 (Nrf2), indicating that EARDP induced HO-1 via the Nrf2 pathway in RAW 264.7 cells. Furthermore, EARDP significantly suppressed the protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in LPS-stimulated RAW 264.7 macrophages. However, ZnPP, a specific inhibitor of HO-1, reversed the EARDP-mediated inhibition of NO and TNF-α production in LPS-stimulated RAW 264.7 macrophages. EARDP blocked the phosphorylation of mitogen-activated protein kinase (MAPK) and Akt in LPS-stimulated RAW 264.7 cells. In the in vivo animal model, EARDP significantly and dose-dependently reduced TPA-induced secretion of TNF-α and IL-6 in mouse ear. Based on these results, EARDP represents a promising natural compound, protective against oxidative stress and inflammatory diseases.

Methotrexate-Loaded Nanostructured Lipid Carrier Gel Alleviates Imiquimod-Induced Psoriasis by Moderating Inflammation: Formulation, Optimization, Characterization, In-Vitro and In-Vivo Studies.

IntroductionMethotrexate exhibits poor cutaneous bioavailability and systemic side effects on topical administration, so there is an unmet need for a novel carrier and its optimized therapy. Methotrexate-loaded nanostructured lipid carriers (MTXNLCs) were formulated and characterized to determine in vitro drug release and evaluate the role of MTXNLC gel in the topical treatment of psoriasis.MethodsA solvent diffusion technique was employed to prepare MTXNLCs, which was optimized using 32 full factorial designs. The mean diameter and surface morphology of MTXNLCs was evaluated. The crystallinity of lyophilized MTXNLCs was characterized by differential scanning calorimetry (DSC) and powder X-ray diffraction (XRD). MTXNLCs were integrated in 1% w/w Carbopol 934 P gel base, and in vitro skin deposition studies in human cadaver skin (HCS) were carried out.ResultsThe optimized MTXNLCs were rod-shaped, with an average particle size of 253 ± 8.65 nm, a zeta potential of −26.4±0.86 mV, and EE of 54.00±1.49%. DSC and XRD data confirmed the formation of NLCs. Significantly higher deposition of MTX was found in HCS from MTXNLC gel (71.52 ±1.13%) as compared to MTX plain gel (38.48±0.96%). In vivo studies demonstrated significant improvement in therapeutic response and reduction in local side effects with MTXNLCs-loaded gel in the topical treatment of psoriasis. Anti-psoriatic efficacy of MTXNLCs 100 ug/cm2 compared with plain MTX gel was evaluated using imiquimod (IMQ)-induced psoriasis in BALB/c mice. The topical application of MTXNLCs to the mouse ear resulted in a significant reduction of psoriatic area and severity index, oxidative stress, inflammatory cytokines like TNF-α, IL-1β, and IL-6 and IMQ-induced histopathological alterations in mouse ear samples.ConclusionDeveloped formulation of MTXNLC gel demonstrated better anti-psoriatic activity and also displayed prolonged and sustained release effect, which shows that it can be a promising alternative to existing MTX formulation for the treatment of psoriasis.

A microvascular image analysis method for optical-resolution photoacoustic microscopy

Optical-resolution photoacoustic microscopy (OR-PAM) has been shown to be an excellent tool for high-resolution imaging of microvasculature, and quantitative analysis of the microvasculature can provide valuable information for the early diagnosis and treatment of various vascular-related diseases. In order to address the characteristics of weak signals, discontinuity and small diameters in photoacoustic microvascular images, we propose a method adaptive to the microvascular segmentation in photoacoustic images, including Hessian matrix enhancement and the morphological connection operators. The accuracy of our vascular segmentation method is quantitatively evaluated by the multiple criteria. To obtain more precise and continuous microvascular skeletons, an improved skeleton extraction framework based on the multistencil fast marching (MSFM) method is developed. We carried out in vivo OR-PAM microvascular imaging in mouse ears and subcutaneous hepatoma tumor model to verify the correctness and superiority of our proposed method. Compared with the previous methods, our proposed method can extract the microvascular network more completely, continuously and accurately, and provide an effective solution for the quantitative analysis of photoacoustic microvascular images with many small branches.

Influence of Elevated pH on Germination, Growth Attributes and ABA Contents of Sticky Mouse-Ear Chickweed (Cerastium glomeratum Thuill.)

Sticky mouse-ear chickweed (Cerastium glomeratum Thuill.) is a dominant annual weed of Korea. Control management of such an exotic weed is a challenging task for agronomist and researcher over the years. We evaluated the effect of elevated pH levels on the germination, growth, chlorophyll content, nutrients and hormonal regulation of sticky mouse-ear chickweed under green-house conditions. Our results showed that pH have no significant effect on seed germination except pH3, which reduced germination rate by 10%. Maximum hypocotyl and radicle growth was observed at pH5 and pH7, while hypocotyl failed to emerge at pH 3. Plant growth attributes showed that plant height, fresh biomass, number of leaves and chlorophyll content were higher at pH7 and pH9; while lowest number of leaves (24.8 ± 2.8) and chlorophyll content (313.2 ± 11.2 SPAD) were recorded for pH3. The endogenous ABA levels were significantly higher at pH3 (71.22±5.4ng/g) but lowered at pH7 and pH9 (6.6±0.8 ng/g). The weed nutrients uptake result showed higher Fe (858.47 mg/kg) and Al (835.05 mg/kg) at pH 3 and pH 5; while higher Ca content (4887.2 mg/kg) was observed at pH 5. It was concluded that use of varying pH can play a vital role in the weed management, although its possible adverse impact on endemic flora and fauna needs to be assessed.

Efficient In Utero Gene Transfer to the Mammalian Inner Ears by the Synthetic Adeno-Associated Viral Vector Anc80L65

Sensorineural hearing loss is one of the most common sensory disorders worldwide. Recent advances in vector design have paved the way for investigations into the use of adeno-associated vectors (AAVs) for hearing disorder gene therapy. Numerous AAV serotypes have been discovered to be applicable to inner ears, constituting a key advance for gene therapy for sensorineural hearing loss, where transduction efficiency of AAV in inner ear cells is critical for success. One such viral vector, AAV2/Anc80L65, has been shown to yield high expression in the inner ears of mice treated as neonates or adults. Here, to evaluate the feasibility of prenatal gene therapy for deafness, we assessed the transduction efficiency of AAV2/Anc80L65-eGFP (enhanced green fluorescent protein) after microinjection into otocysts in utero. This embryonic delivery method achieved high transduction efficiency in both inner and outer hair cells of the cochlea. Additionally, the transduction efficiency was high in the hair cells of the vestibules and semicircular canals and in spiral ganglion neurons. Our results support the potential of Anc80L65 as a gene therapy vehicle for prenatal inner ear disorders.

Esc-1GN shows therapeutic potentials for acne vulgaris and inflammatory pain.

The inflammatory response plays important roles in acne vulgaris and pain pathogenesis. In previous study, Esc-1GN with anti-inflammatory, antimicrobial, and lipopolysacchride (LPS) binding activity was identified from the skin of the frog Hylarana guentheri. Here, we report its therapeutic potentials for acne vulgaris and inflammatory pain. Esc-1GN destroyed the cell membrane of Propionibacteria acnes in the membrane permeability assays. In addition, bacterial agglutination test suggested that Esc-1GN triggered the agglutination of P. acnes, which was affected by LPS and Ca2+ . Meanwhile, in vivo anti-P. acnes and anti-inflammatory effects of Esc-1GN were confirmed by reducing the counts of P. acnes in mice ear, relieving P. acnes-induced mice ear swelling, decreasing mRNA expression and the production of pro-inflammatory cytokines, and attenuating the infiltration of inflammatory cells. Moreover, Esc-1GN also displayed antinociceptive effect in mice induced by acetic acid and formalin. Therefore, Esc-1GN is a promising candidate drug for treatment of acne vulgaris and inflammatory pain.

Characterization of promoters for adeno-associated virus mediated efficient Cas9 activation in adult Cas9 knock-in murine cochleae

CRISPR/Cas9 gene editing enables the treatment of hearing loss in congenitally deaf neonatal mice via both viral and non-viral delivery. While adeno-associated virus (AAV)-mediated gene delivery systems have been shown to be effective tools for gene replacement in the inner ear, application of the AAV-mediated CRISPR/Cas9 gene-editing approach for this purpose is yet to be documented. Based on our previous findings, we focused on the effects of several AAVs delivered via canalostomy injection in adult mice. Among the AAVs examined, AAV8 showed the greatest efficiency and specificity in transducing inner hair cells (IHC). The ability of Cre-expressing AAV8 to activate Cas9 in floxed-Cas9 knock-in (Cas9 KI) mice was further evaluated. We compared the effects of six different promoters (CMV, CAG, hSyn, CaMKIIa, GFAP, and ALB) of AAV8 delivered to the inner ear of adult Cas9 KI mice. Our findings showed that three AAV groups (CMV, CAG and hSyn promoters) infected the inner ear efficiently with different tropisms. Notably, AAVs with CMV, CAG, and hSyn promoters infected diverse cell types in mature murine cochleae, including IHCs. In particular, AAV8-hSyn showed high affinity to IHCs and spiral ganglion neurons (SGN). Neither the AAV8 virus itself (except AAV8-CAG) nor the surgical procedures used caused damage to HCs or impaired normal hearing. Our findings indicated that injection of AAV-Cre into mature inner ear efficiently induces Cas9 activation to achieve safe and efficient gene editing and different constituent promoters confer diverse infection patterns in cochlea, expanding the repertoire of gene-editing tools for regulating gene expression in target cells of the inner ear as part of the collective effort to rescue genetic hearing loss and develop effective gene therapy techniques.

Pygmy mice whistle for the audience

It might just be squeaking to you and me, but to the ears of northern pygmy mice (Baiomys taylori), the shrill cries of their own can be a romantic serenade. Describing how the feisty little rodents rear up on their hindlegs to summon others, Bret Pasch from Northern Arizona University, USA, quotes from a research paper dating back to 1941, saying, ‘The staccato-like song of pygmy mice is described as a “high-pitched, barely audible squeal” produced with the head “thrust forward and upward, stretching the throat”’. But there is an additional ultrasonic component of the rodent's ardent aria, stretching well beyond the limits of our hearing. Explaining that many small rodents use either their vocal chords to produce lower pitched calls or whistles to hit ear-splitting highs, Tobias Riede from Midwestern University, USA, and Pasch, teamed up to find out how the most diminutive rodent in North America produces its extraordinary vocal range.‘Pygmy mice are relatively rare’, says Pasch, describing how the tiny animals live in the desert grasslands of Arizona and New Mexico, and recalling how he often encountered rattlesnakes when retrieving the mini rodents from traps. Once the mice were safe in the Northern Arizona University lab, it was simply a case of recording the obliging creatures’ serenades. ‘Mice routinely sing spontaneously or if they encounter the voices or odour of a potential mate’, says Pasch. However, when the duo analysed the pygmy mouse's vocal range, they were surprised that the voices of the 10 g rodents were much deeper than they had expected, ranging from 16 to 40 kHz. ‘Traditionally, our understanding is that vocal pitch is driven by the size of the vocal organ’, says Riede, explaining that the voices of smaller animals are always squeakier than the voices of larger creatures. Yet, the highest tones in the pygmy mouse's range were only as high as those of lab rats, which are 40 times larger.Then, the team played a trick on the squeaky pygmy mice, replacing the air in their cages with heliox – a mixture of oxygen and helium inhaled by deep-sea divers – which shifts the pitch of whistles without altering the pitch of calls produced by vibrating vocal chords, to distinguish the origins of different tones in the rodent's repertoire. And when the mice switched to breathing heliox, every tone in their vocal range became squeakier. The duo realised that instead of the voluble rodents using their vocal chords to make calls over the lower range of their repertoire, every syllable they articulated was a whistle. In addition, each syllable of the serenade became shorter, probably because the flow of the lighter gas through the animal's tiny voice box is different from that of air, making the puffs of gas they exhale to whistle less effective.But how were the bijou animals able to produce such deep whistles when their frames are so tiny? Riede examined the structure of the tiny creature's voice box. Dissecting the minute vocal organs, which are the smallest that Riede has ever investigated, he discovered that pygmy mice have an unexpectedly large pouch that expands the size of the voice box, allowing the tiny creatures to produce the deeper tones (∼16 kHz) by whistling, instead of using their short vocal chords like other rodents.So, pygmy mice produce ultrasonic squeals by whistling, to evade the hearing of predators. Having discovered the secret of the maverick mammal's relatively deep voices, Pasch and Riede are eager to discover how the mouse's voice compares with those of other rodents to learn about the evolution of their communication.

Intravital confocal fluorescence lifetime imaging microscopy in the second near-infrared window.

We present confocal fluorescence lifetime imaging microscopy in the second near-infrared (NIR-II) window to assess the morphological and biochemical information of live samples. A home-built superconducting single-photon detector (SSPD) was used to facilitate the NIR-II fluorescence lifetime measurement. The SSPD has many advantages, including high sensitivity to NIR-II signals (detection efficiency >50%), fast temporal response (∼109ps), low timing jitter (∼50ps), and low dark count rate (<100cps). We demonstrate the feasibility of the developed microscopy system by comparing fluorescence lifetimes of a range of fluorophores with emission in the NIR-II window and by performing multicolor three-dimensional fluorescence lifetime imaging of a mouse ear in vivo. The biochemical properties of the cells and tissues probed by the fluorescence lifetimes of the fluorophores provide complementary information for biomedical studies, significantly benefiting diverse applications in life science.

Acacia Polyphenol Ameliorates Atopic Dermatitis in Trimellitic Anhydride-Induced Model Mice via Changes in the Gut Microbiota.

We have previously shown that acacia polyphenol (AP), which was extracted from the bark of Acacia mearnsii De Wild, exerts antiobesity, antidiabetic, and antihypertensive effects. In this study, we examined the effect of AP on atopic dermatitis. Trimellitic anhydride (TMA) was applied to the ears of mice to create model mice with atopic dermatitis. The frequency of scratching behavior in the TMA-treated group was significantly higher than that in the control group, and the expression levels of inflammatory markers (tumor necrosis factor-α, interleukin-6, inducible nitric oxide synthase, and cyclooxygenase-2) in the skin also increased. In contrast, both the frequency of scratching behavior and the expression levels of skin inflammatory markers in the AP-treated group were significantly lower than those in the TMA-treated group. The abundances of beneficial bacteria, such as Bifidobacterium spp. and Lactobacillus spp., increased in the AP-treated group compared with the TMA-treated group. Furthermore, the abundances of Bacteroides fragilis and Clostridium coccoides in the gut, which are known for anti-inflammatory properties, increased significantly with AP administration. The present results revealed that AP inhibits TMA-induced atopic dermatitis-like symptoms. In addition, the results also suggested that this effect may be associated with the mechanism of gut microbiota improvement.

5-Bromo-3,4-dihydroxybenzaldehyde from Polysiphonia morrowii attenuate IgE/BSA-stimulated mast cell activation and passive cutaneous anaphylaxis in mice

The present study investigates the anti-allergic activity of the marine algal bromophenol, 3-bromo-4,5-dihydroxybenzaldehyde (BDB), isolated from Polysiphonia morrowii Harvey in immunoglobulin (Ig)E/bovine serum albumin (BSA)-stimulated mouse bone marrow-derived cultured mast cells (BMCMCs) and a passive cutaneous anaphylaxis (PCA) mice ear model. BDB effectively inhibited β-hexosaminidase release (IC50 = 80.12 µM), in IgE/BSA-stimulated BMCMCs without a cytotoxic response. Also, BDB down-regulated the expression or secretion of cytokines, interleukin (IL)-1β, IL-4, IL-5, IL-6, IL-10, IL-13, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α and the chemokine (thymus and activation-regulated chemokine (TARC). The above effects could be attributed to the dose-dependent decrease of FcεRI expression on the surface of BMCMCs and its stable IgE binding. Moreover, BDB suppressed the nuclear factor (NF)-κB and spleen tyrosine kinase (SYK)-linker for T-cell activation (LAT)-GRB2 associated binding protein 2 (Gab2) signaling axis activated by IgE/BSA stimulation. Furthermore, oral administration of BDB to IgE-sensitized mice effectively attenuated IgE-triggered PCA reaction. Collectively, the anti-allergic effects of BDB suggest its potential applicability as a candidate for in-depth test trials.

Wide-field polygon-scanning photoacoustic microscopy of oxygen saturation at 1-MHz A-line rate

We report wide-field polygon-scanning functional OR-PAM that for the first time achieves 1-MHz A-line rate of oxygen saturation in vivo. We address two technical challenges. The first is a 1-MHz dual-wavelength pulsed laser that has sufficient pulse energy and ultrafast wavelength switching. The second is a polygon-scanning imaging probe that has a fast scanning speed, a large field of view, and great sensitivity. The OR-PAM system offers a B-scan rate of 477.5 Hz in a 12-mm range and a volumetric imaging rate of ∼1 Hz over a 12 × 5 mm2 scanning area. We image microvasculature and blood oxygen saturation in a 12 × 12 mm2 scanning area in 5 s. Dynamic imaging of oxygen saturation in the mouse ear is demonstrated to monitor fast response to epinephrine injection. The new wide-field fast functional imaging ability broadens the biomedical application of OR-PAM.

A Glycosaminoglycan-Rich Fraction from Sea Cucumber Isostichopus badionotus Has Potent Anti-Inflammatory Properties In Vitro and In Vivo.

Sea cucumber body wall contains several naturally occurring bioactive components that possess health-promoting properties. Isostichopus badionotus from Yucatan, Mexico is heavily fished, but little is known about its bioactive constituents. We previously established that I. badionotus meal had potent anti-inflammatory properties in vivo. We have now screened some of its constituents for anti-inflammatory activity in vitro. Glycosaminoglycan and soluble protein preparations reduced 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammatory responses in HaCaT cells while an ethanol extract had a limited effect. The primary glycosaminoglycan (fucosylated chondroitin sulfate; FCS) was purified and tested for anti-inflammatory activity in vivo. FCS modulated the expression of critical genes, including NF-ĸB, TNFα, iNOS, and COX-2, and attenuated inflammation and tissue damage caused by TPA in a mouse ear inflammation model. It also mitigated colonic colitis caused in mice by dextran sodium sulfate. FCS from I. badionotus of the Yucatan Peninsula thus had strong anti-inflammatory properties in vivo.

&lt;p&gt;&lt;strong&gt;Italian alien species in Caryophyllaceae: nomenclatural remarks&lt;/strong&gt;&lt;/p&gt;

The typification of the names Cerastium biebersteinii, Dianthus glumaceus, D. plumarius, Gypsophila collina, and G. elegans is discussed. Specimens from the herbaria G, LE, LINN, and P are designated as lectotypes, or neotype (G. elegans) of the concerned names.

Toxic and active material basis of Aconitum sinomontanum Nakai based on biological activity guidance and UPLC-Q/TOF-MS technology

BackgroundAs a folk medicine, Aconitum sinomontanum Nakai (Ranunculaceae, Gaowutou, in Chinese) is used by traditional healers to treat many disorders, including pain and inflammatory diseases, but it exhibits the toxic side effects. This study aimed to obtain toxic extract parts from A. sinomontanum roots and to further evaluate the antinociceptive and anti-inflammatory effects of toxic extract parts on mice. This work also aimed to identify various chemical compositions of the toxic and active extract parts and evaluate the safety profile of this plant. MethodsExperimental drugs (petroleum ether, chloroform, ethyl acetate, n-butanol, alcohol and water extracts) were obtained through systematic solvent extraction from 95 % ethanol extract from A. sinomontanum roots. An acute toxicity test was conducted to compare the toxicity of different extracts administered at the maximum dose to screen a highly toxic extract. In pharmacodynamic activity analysis, the antinociceptive activity of the A. sinomontanum toxic extract was assessed using an acetic acid-induced abdominal writhing model and a hot plate test. Anti-inflammatory activity was assessed in terms of xylene-induced inflammation. Ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) was performed to establish a chromatographic fingerprint and to identify various chemical components of the toxic and active extract. ResultsChloroform, water and n-butanol extracts elicited significant toxic effects and had LD50 of 89.65, 1805.40 and 24409.41 mg/kg, respectively. Antinociceptive and anti-inflammatory activities indicated that the chloroform extract significantly alleviated (p < 0.01) the pain induced by acetic acid with an inhibition rate of 44.7 % (5.9 mg/kg) and 50.4 % (17.7 mg/kg). The chloroform extract also significantly (p < 0.01) increased the latency time during the hot plate test. The latency time at 5.9 and 17.7 mg/kg increased from 15.6 ± 4.1 s to 47.3 ± 6.4 s and from 16.3 ± 3.8 s to 49.8 ± 7.6 s (p < 0.01), respectively, 2 h after treatment. In the inflammatory test, the chloroform extract significantly reduced (p < 0.01) the xylene-induced mouse ear oedema with an inhibition rate of 45.48 % (5.9 mg/kg) and 51.46 % (17.7 mg/kg), respectively. This result indicated that A. sinomontanum chloroform extract was also the active extract part of A. sinomontanum. Phytochemical analysis revealed the presence of alkaloids in the chloroform extract. A total of 30 compounds were detected, and 23 compounds, including lappaconine, ranaconidine, 8-O-acetylexcelsine, sinomontanine H, finaconitine, lappacontine, N-dacetyllappaconitine, ranaconitine and isolappaconitine, were identified. ConclusionsA. sinomontanum chloroform extract possesses antinociceptive and anti-inflammatory activities and exhibits significant toxic effects. Phytochemical analysis indicated that some alkaloids may be the main bioactive ingredient responsible for the toxicity and efficacy of A. sinomontanum. This work contributes to the determination of the safety of the medicinal use of A. sinomontanum roots.

A type IV collagenase inhibitor, N-hydroxy-3-phenyl-2-(4-phenylbenzenesulfonamido) propanamide (BiPS), suppresses skin injury induced by sulfur mustard

Sulfur mustard (SM) is a highly toxic blistering agent thought to mediate its action, in part, by activating matrix metalloproteinases (MMPs) in the skin and disrupting components of the basement membrane zone (BMZ). Type IV collagenases (MMP-9) degrade type IV collagen in the skin, a major component of the BMZ at the dermal-epidermal junction. In the present studies, a type IV collagenase inhibitor, N-hydroxy-3-phenyl-2-(4-phenylbenzenesulfonamido) propanamide (BiPS), was tested for its ability to protect the skin against injury induced by SM in the mouse ear vesicant model. SM induced inflammation, epidermal hyperplasia and microblistering at the dermal/epidermal junction of mouse ears 24–168 h post-exposure. This was associated with upregulation of MMP-9 mRNA and protein in the skin. Dual immunofluorescence labeling showed increases in MMP-9 in the epidermis and in the adjacent dermal matrix of the SM injured skin, as well as breakdown of type IV collagen in the basement membrane. Pretreatment of the skin with BiPS reduced signs of SM-induced cutaneous toxicity; expression of MMP-9 mRNA and protein was also downregulated in the skin by BiPS. Following BiPS pretreatment, type IV collagen appeared intact and was similar to control skin. These results demonstrate that inhibiting type IV collagenases in the skin improves basement membrane integrity after exposure to SM. BiPS may hold promise as a potential protective agent to mitigate SM induced skin injury.

Expression of Laminin γ2 Proteolytic Fragments in Murine Skin Following Exposure to Sulfur Mustard.

Laminin-332 is a basement membrane protein composed of three genetically distinct polypeptide chains that actively promote both skin epidermal cell adhesion and migration. Proteolytic fragments of the laminin γ2 chain stimulate migration and scattering of keratinocytes and cancer cells. Sulfur mustard (SM) is a bifunctional alkylating agent that induces separation of basal keratinocytes from the dermal-epidermal junction and invokes a strong inflammatory response leading to delayed wound repair. In the present studies, the role of laminin γ2 in SM-induced skin injury and wound repair was investigated using the mouse ear vesicant model. We found that laminin γ2 chain mRNA was preferentially upregulated in mouse ear skin exposed to SM. In situ hybridization confirmed overexpression of laminin γ2 transcript. Western blot analysis showed increased protein expression of the full-length proform of laminin γ2 and smaller processed fragments of laminin γ2 in skin exposed to SM. Dual immunofluorescence labeling indicated that laminin γ2 fragments are prevalent in suprabasal keratinocytes behind the leading edge in areas of hyperplasia in injured skin. In addition, co-expression of laminin γ2 and the senescent marker, p16-INK4a was found to overlap with the hyperplastic migratory epithelial sheet. This observation is similar to hypermotile keratinocytes reported in invasive carcinoma cells. Overall, our studies indicate that laminin γ2 is preferentially expressed in skin post SM exposure and that protein expression appears to become progressively more fragmented. The laminin γ2 fragments may play a role in regulating SM-induced skin wound repair. Anat Rec, 2020. © 2020 American Association for Anatomy.

High-speed wide-field multi-parametric photoacoustic microscopy.

Capable of imaging blood perfusion, oxygenation, and flow simultaneously at the microscopic level, multi-parametric photoacoustic microscopy (PAM) has quickly emerged as a powerful tool for studying hemodynamic and metabolic changes due to physiological stimulations or pathological processes. However, the low scanning speed poised by the correlation-based blood flow measurement impedes its application in studying rapid microvascular responses. To address this challenge, we have developed a new, to the best of our knowledge, multi-parametric PAM system. By extending the optical scanning range with a cylindrically focused ultrasonic transducer (focal zone, 76µm×4.5mm) for simultaneous acquisition of 500 B-scans, the new system is 112 times faster than our previous multi-parametric system that uses a spherically focused transducer (focal diameter, 40 µm) and enables high-resolution imaging of blood perfusion, oxygenation, and flow over an area of 4.5×1mm2 at a frame rate of 1 Hz. We have demonstrated the feasibility of this system in the living mouse ear. Further development of this system into reflection mode will enable real-time cortex-wide imaging of hemodynamics and metabolism in the mouse brain.

Hopane-type triterpenes from Cnidoscolus spinosus and their bioactivities.

Chemical investigation of the aerial parts of Cnidoscolus spinosus resulted in the isolation of relatively infrequent hopane-type triterpenes, 3β-acetoxy-hop-22(29)-ene (1), first reported here as natural product, together with 3-oxo-hop-22(29)-ene (2), and 3β-hydroxy-hop-22(29)-ene (3). β-Amyrin palmitate and three phytosterols were also characterized. The structures of the compounds were established using spectroscopic methods, and those of 1 and 2 were confirmed by crystallographic analysis. Selected biological activities for the isolated hopane-type triterpenes were tested through a series of assays for determining the cytotoxic, anti-inflammatory, α-glucosidase inhibition and antiparasitic activities. Compounds 1-3 did not show cytotoxic activity, compound 1 displayed an important inhibitory effect in the mouse ear induced inflammation assay, and significantly inhibited the yeast α-glucosidase activity in vitro and in silico. Additionally, compounds 2 and 3 showed marginal activities against Trypanosoma cruzi and Leishmania mexicana. Therefore, the bioactivities of hopane-type triterpenes deserve further investigation, particularly their anti-inflammatory properties.

Systemic Dermatitis Model Mice Exhibit Atrophy of Visceral Adipose Tissue and Increase Stromal Cells via Skin-Derived Inflammatory Cytokines.

Adipose tissue (AT) is the largest endocrine organ, producing bioactive products called adipocytokines, which regulate several metabolic pathways, especially in inflammatory conditions. On the other hand, there is evidence that chronic inflammatory skin disease is closely associated with vascular sclerotic changes, cardiomegaly, and severe systemic amyloidosis in multiple organs. In psoriasis, a common chronic intractable inflammatory skin disease, several studies have shown that adipokine levels are associated with disease severity. Chronic skin disease is also associated with metabolic syndrome, including abnormal tissue remodeling; however, the mechanism is still unclear. We addressed this problem using keratin 14-specific caspase-1 overexpressing transgenic (KCASP1Tg) mice with severe erosive dermatitis from 8 weeks of age, followed by re-epithelization. The whole body and gonadal white AT (GWAT) weights were decreased. Each adipocyte was large in number, small in size and irregularly shaped; abundant inflammatory cells, including activated CD4+ or CD8+ T cells and toll-like receptor 4/CD11b-positive activated monocytes, infiltrated into the GWAT. We assumed that inflammatory cytokine production in skin lesions was the key factor for this lymphocyte/monocyte activation and AT dysregulation. We tested our hypothesis that the AT in a mouse dermatitis model shows an impaired thermogenesis ability due to systemic inflammation. After exposure to 4 °C, the mRNA expression of the thermogenic gene uncoupling protein 1 in adipocytes was elevated; however, the body temperature of the KCASP1Tg mice decreased rapidly, revealing an impaired thermogenesis ability of the AT due to atrophy. Tumor necrosis factor (TNF)-α, IL-1β and interferon (INF)-γ levels were significantly increased in KCASP1Tg mouse ear skin lesions. To investigate the direct effects of these cytokines, BL/6 wild mice were administered intraperitoneal TNF-α, IL-1β and INF-γ injections, which resulted in small adipocytes with abundant stromal cell infiltration, suggesting those cytokines have a synergistic effect on adipocytes. The systemic dermatitis model mice showed atrophy of AT and increased stromal cells. These findings were reproducible by the intraperitoneal administration of inflammatory cytokines whose production was increased in inflamed skin lesions.