Mouse Leg Research Articles

Anti-inflammatory Effect of n-Hexane Fraction of Kentut leaves (Paederia scandens L.) on Male White Rats (Rattus novergicus)

The anti-inflammatory effect of fart plant leaf extract (Paederia scandens L.) on white rats (Rattus norvegicus) has been carried out. This research shows that the effective dose of the n-hexane fraction of P. scandens leaf extract provides the best anti-inflammatory effect on white rats' feet soles. The study used an artificial edema formation method and periodically measured the volume of white mice's legs using a plethysmometer. The doses used for the extract are 100mg/200g BW, 150mg/200g BW, and 200mg/200g BW. The research data was observed visually. The effective dose of P. scandens leaf extract, which has the best anti-inflammatory effect, is 150mg/200g BW.

Two-dimensional time-resolved scintillating sheet monitoring of proton pencil beam scanning FLASH mouse irradiations.

Dosimetry in pre-clinical FLASH studies is essential for understanding the beam delivery conditions that trigger the FLASH effect. Resolving the spatial and temporal characteristics of proton pencil beam scanning (PBS) irradiations with ultra-high dose rates (UHDR) requires a detector with high spatial and temporal resolution. To implement a novel camera-based system for time-resolved two-dimensional (2D) monitoring and apply it in vivo during pre-clinical proton PBS mouse irradiations. Time-resolved 2D beam monitoring was performed with a scintillation imaging system consisting of a 1mm thick transparent scintillating sheet, imaged by a CMOS camera. The sheet was placed in a water bath perpendicular to a horizontal PBS proton beam axis. The scintillation light was reflected through a system of mirrors and captured by the camera with 500 frames per second (fps) for UHDR and 4 fps for conventional dose rates. The raw images were background subtracted, geometrically transformed, flat field corrected, and spatially filtered. The system was used for 2D spot and field profile measurements and compared to radiochromic films. Furthermore, spot positions were measured for UHDR irradiations. The measured spot positions were compared to the planned positions and the relative instantaneous dose rate to equivalent fiber-coupled point scintillator measurements. For in vivo application, the scintillating sheet was placed 1cm upstream the right hind leg of non-anaesthetized mice submerged in the water bath. The mouse leg and sheet were both placed in a 5cm wide spread-out Bragg peak formed from the mono-energetic proton beam by a 2D range modulator. The mouse leg position within the field was identified for both conventional and FLASH irradiations. For the conventional irradiations, the mouse foot position was tracked throughout the beam delivery, which took place through repainting. For FLASH irradiations, the delivered spot positions and relative instantaneous dose rate were measured. The pixel size was 0.1mm for all measurements. The spot and field profiles measured with the scintillating sheet agreed with radiochromic films within 0.4mm. The standard deviation between measured and planned spot positions was 0.26mm and 0.35mm in the horizontal and vertical direction, respectively. The measured relative instantaneous dose rate showed a linear relation with the fiber-coupled scintillator measurements. For in vivo use, the leg position within the field varied between mice, and leg movement up to 3mm was detected during the prolonged conventional irradiations. The scintillation imaging system allowed for monitoring of UHDR proton PBS delivery in vivo with 0.1mm pixel size and 2ms temporal resolution. The feasibility of instantaneous dose rate measurements was demonstrated, and the system was used for validation of the mouse leg position within the field.

Stretchable and environment-resistant doubly crosslinked hydrogel for all-in-one supercapacitor and strain sensors

Despite their importance as components for flexible electronics, most stretchable hydrogels suffer from incomplete recovery after deformation, are prone to failure upon long-term repeated stretching, and cannot be exploited at subzero temperatures because of the freezing of their constituent water. Consequently, strategies for circumventing these drawbacks are highly sought after. This study describes the synthesis of a doubly (chemically and physically) crosslinked hydrogel from gelatin and methacrylic acid and demonstrates the suitability of this material for the fabrication of high-performance stretchable and environment-resistant supercapacitors and strain sensors. The performance of this supercapacitor (areal capacitance = 1,210.2 mF/cm2 at a current density of 1 mA/cm2, maximum energy density = 158.8 μW⋅h/cm2, maximum power density = 659.5 μW/cm2) was superior to that of most of integrated supercapacitors reported to date and was hardly affected by stretchable, low temperatures, bending, ice-cold water and strong acid/alkali solutions or long-term storage. Additionally, a strain sensor based on the above hydrogel was capable of accurately capturing human body motions when affixed to skin and recognising mouse movement (even in humid environments) after implantation into mouse legs. Our work may pave the way to high-performance stretchable and environment-resistant wearable electronics.

MYH13, a superfast myosin expressed in extraocular, laryngeal and syringeal muscles.

MYH13 is a unique type of sarcomeric myosin heavy chain (MYH) first detected in mammalian extraocular (EO) muscles and later also in vocal muscles, including laryngeal muscles of some mammals and syringeal muscles of songbirds. All these muscles are specialized in generating very fast contractions while producing relatively low force, a design appropriate for muscles acting against a much lower load than most skeletal muscles inserting into the skeleton. The definition of the physiological properties of muscle fibres containing MYH13 has been complicated by the mixed fibre type composition of EO muscles and the coexistence of different MYH types within the same fibre. A major advance in this area came from studies on isolated recombinant myosin motors and the demonstration that the affinity of actin-bound human MYH13 for ADP is much weaker than those of fast-type MYH1 (type 2X) and MYH2 (type 2A). This property is consistent with a very fast detachment of myosin from actin, a major determinant of shortening velocity. The MYH13 gene arose early during vertebrate evolution but was characterized only in mammals and birds and appears to have been lost in some teleost fish. The MYH13 gene is located at the 3' end of the mammalian fast/developmental gene cluster and in a similar position to the orthologous cluster in syntenic regions of the songbird genome. MYH13 gene regulation is controlled by a super-enhancer in the mammalian locus and deletion of the neighbouring fast MYH1 and MYH4 genes leads to abnormal MYH13 expression in mouse leg muscles.

High-throughput screening of ancient forest plant extracts shows cytotoxicity towards triple-negative breast cancer

According to the World Health Organization, women's breast cancer is among the most common cancers with 7.8 million diagnosed cases during 2016–2020 and encompasses 15 % of all female cancer-related mortalities. These mortality events from triple-negative breast cancer are a significant health issue worldwide calling for a continuous search of bioactive compounds for better cancer treatments. Historically, plants are important sources for identifying such new bioactive chemicals for treatments. Here we use high-throughput screening and mass spectrometry analyses of extracts from 100 plant species collected in Chinese ancient forests to detect novel bioactive breast cancer phytochemicals. First, to study the effects on viability of the plant extracts, we used a MTT and CCK-8 cytotoxicity assay employing triple-negative breast cancer (TNBC) MDA-MB-231 and normal epithelial MCF-10A cell lines and cell cycle arrest to estimate apoptosis using flow cytometry for the most potent three speices. Based on these analyses, the final most potent extracts were from the Amur honeysuckle (Lonicera maackii) wood/root bark and Nigaki (Picrasma quassioides) wood/root bark. Then, 5 × 106 MDA-MB-231 cells were injected subcutaneously into the right hind leg of nude mice and a tumour was allowed to grow before treatment for seven days. Subsequently, the four exposed groups received gavage extracts from Amur honeysuckle and Nigaki (Amur honeysuckle wood distilled water, Amur honeysuckle root bark ethanol, Nigaki wood ethanol or Nigaki root bark distilled water/ethanol (1:1) extracts) in phosphate-buffered saline (PBS), while the control group received only PBS. The tumour weight of treated nude mice was reduced significantly by 60.5 % within 2 weeks, while on average killing 70 % of the MDA-MB-231 breast cancer cells after 48 h treatment (MTT test). In addition, screening of target genes using the Swiss Target Prediction, STITCH, STRING and NCBI-gene database showed that the four plant extracts possess desirable activity towards several known breast cancer genes. This reflects that the extracts may kill MBD-MB-231 breast cancer cells. This is the first screening of plant extracts with high efficiency in 2 decades, showing promising results for future development of novel cancer treatments.

The Effect of Dose-Fractionation Mode on the Immune Response and Therapeutic Effect of Lewis Lung Cancer Mice Model Based on Tumor Microenvironment

The therapeutic effect of different groups of Lewis lung cancer mice and the expression of immune cells in peripheral blood and tumor tissues of mice were observed after different dose-segmentation modes of treatment, so as to select the most suitable for stimulating immune effect with good prognosis and small side effects. Female homologous inbred C57BL/6 male mice at 6-8 weeks were selected and inoculated subcutaneously on the right thigh according to LLC: L929: lymphocyte = tumor cell suspension with PBMC(inactivated lymphocyte) = 5:1:1 ratio; after tumor growth to the size of 100-200mm3, different dose-segmentation modes were used for irradiation, which was divided into 5 groups, group A: no radiotherapy; Group B: 2Gy/qd, continuous radiotherapy for 30 days; Group C: 2Gy/bid, continuous radiotherapy for 13 days; Group D: 3Gy/bid, continuous radiotherapy for 10 days; Group E: 6Gy/qd, continuous radiotherapy for 8 days. On the 3rd day after radiotherapy, some mice were sacrificed, peripheral blood lymphocytes of mice were isolated with lymphocyte separation solution, and the transplanted tumor of mice was prepared into single-cell suspension. Then, the number of CD3+T cells, CD3+B cells, NK1.1 cells, CD3+CD4+T cells, CD3+CD8+T cells, CD4+Foxp3+Treg cells in peripheral blood and single-cell suspension of mice was detected by flow cytometry. HE staining was used to observe the tumor tissue structure of mice leg transplantation. The expressions of CD4, CD8, Foxp3 and CD31 in tumor tissues of each radiotherapy group were observed by immunohistochemical staining (IHC) in paraffin sections. The survival time of mice in group C (2Gy/ bidx13d) was significantly improved compared with that in group B (2Gy/qdx30d), and the twin-daily segmentation mode could improve the elevation of Treg cells after once-daily segmentation mode radiotherapy in both peripheral blood and tumor tissues. There was no significant difference in the survival time of mice in group E (6Gy/qdx8d) and group D (3Gy/bidx10d), the number of Treg cells in peripheral blood of mice in group E was significantly lower than that in group D, but there were more Treg cells in tumor tissue of mice in group E than group D. Compared with the control mice, the overall immunity of the experimental group was inhibited. The twice-daily segmentation mode in the conventional dose radiotherapy group significantly improved Treg in peripheral blood and tumor immune microenvironment and promoted neovascularization. The twice-daily segmentation mode in the high-dose radiotherapy group improved the immunosuppression of lymphocytes in peripheral blood but increased immunosuppression in tumor microenvironment.

An experimental setup for proton irradiation of a murine leg model for radiobiological studies

Background The purpose of this study was to introduce an experimental radiobiological setup used for in vivo irradiation of a mouse leg target in multiple positions along a proton beam path to investigate normal tissue- and tumor models with varying linear energy transfer (LET). We describe the dosimetric characterizations and an acute- and late-effect assay for normal tissue damage. Methods The experimental setup consists of a water phantom that allows the right hind leg of three to five mice to be irradiated at the same time. Absolute dosimetry using a thimble (Semiflex) and a plane parallel (Advanced Markus) ionization chamber and Monte Carlo simulations using Geant4 and SHIELD-HIT12A were applied for dosimetric validation of positioning along the spread-out Bragg peak (SOBP) and at the distal edge and dose fall-off. The mice were irradiated in the center of the SOBP delivered by a pencil beam scanning system. The SOBP was 2.8 cm wide, centered at 6.9 cm depth, with planned physical single doses from 22 to 46 Gy. The biological endpoint was acute skin damage and radiation-induced late damage (RILD) assessed in the mouse leg. Results The dose–response curves illustrate the percentage of mice exhibiting acute skin damage, and at a later point, RILD as a function of physical doses (Gy). Each dose–response curve represents a specific severity score of each assay, demonstrating a higher ED50 (50% responders) as the score increases. Moreover, the results reveal the reversible nature of acute skin damage as a function of time and the irreversible nature of RILD as time progresses. Conclusions We want to encourage researchers to report all experimental details of their radiobiological setups, including experimental protocols and model descriptions, to facilitate transparency and reproducibility. Based on this study, more experiments are being performed to explore all possibilities this radiobiological experimental setup permits.

Amorphous Calcium Carbonate Shows Anti-Cancer Properties That are Attributed to Its Buffering Capacity.

Amorphous calcium carbonate (ACC) is a non-crystalline form of calcium carbonate, and it is composed of aggregated nano-size primary particles. Here, we evaluated its anti-cancer effect postulated relative to its buffering capabilities in lung cancer. Tumors were evaluated in vivo using the Lewis lung carcinoma (LLC) mouse cell line and A549 human lung cancer carcinoma cell line. LLC and A549 cells were injected subcutaneously into the right hind leg of mice. Treatments (ACC, cisplatin, vehicle, and ACC with cisplatin, all given via daily IP injections) started once tumors reached a measurable size. Treatments were carried out for 14 days in the LLC model and for 22 and 24 days in the xenograft model (two experiments). LLC tumors were resected from ACC at the end of the study, and vehicle groups were evaluated for cathepsin B activity. Differential gene expression was carried out on A549 cells following 8 weeks of in vitro culture in the presence or absence of ACC in a culture medium. The ACC treatment decelerated tumor growth rates in both models. When tumor volumes were compared on the last day of each study, the ACC-treated animal tumor volume was reduced by 44.83% compared to vehicle-treated animals in the LLC model. In the xenograft model, the tumor volume was reduced by 51.6% in ACC-treated animals compared to vehicle-treated animals. A more substantial reduction of 74.75% occurred in the combined treatment of ACC and cisplatin compared to the vehicle (carried out only in the LLC model). Cathepsin B activity was significantly reduced in ACC-treated LLC tumors compared to control tumors. Differential gene expression results showed a shift towards anti-tumorigenic pathways in the ACC-treated A549 cells. This study supports the ACC anti-malignant buffering hypothesis by demonstrating decelerated tumor growth, reduced cathepsin B activity, and altered gene expressions to produce anti-cancerous effects.

PO-2205 Histopathological examination of proton radiation-induced late effects in mice legs

PO-2205 Histopathological examination of proton radiation-induced late effects in mice legs

Development of an Animal Model for Traumatic Brain Injury Augmentation of Heterotopic Ossification in Response to Local Injury.

Heterotopic ossification (HO) is the abnormal growth of bone in soft connective tissues that occurs as a frequent complication in individuals with traumatic brain injury (TBI) and in rare genetic disorders. Therefore, understanding the mechanisms behind ectopic bone formation in response to TBI is likely to have a significant impact on identification of novel therapeutic targets for HO treatment. In this study, we induced repetitive mild TBI (mTBI) using a weight drop model in mice and then stimulated HO formation via a local injury to the Achilles tendon or fibula. The amount of ectopic bone, as evaluated by micro-CT analyses, was increased by four-fold in the injured leg of mTBI mice compared to control mice. However, there was no evidence of HO formation in the uninjured leg of mTBI mice. Since tissue injury leads to the activation of hypoxia signaling, which is known to promote endochondral ossification, we evaluated the effect of IOX2, a chemical inhibitor of PHD2 and a known inducer of hypoxia signaling on HO development in response to fibular injury. IOX2 treatment increased HO volume by five-fold compared to vehicle. Since pericytes located in the endothelium of microvascular capillaries are known to function as multipotent tissue-resident progenitors, we determined if activation of hypoxia signaling promotes pericyte recruitment at the injury site. We found that markers of pericytes, NG2 and PDGFRβ, were abundantly expressed at the site of injury in IOX2 treated mice. Treatment of pericytes with IOX2 for 72 h stimulated expression of targets of hypoxia signaling (Vegf and Epo), as well as markers of chondrocyte differentiation (Col2α1 and Col10α1). Furthermore, serum collected from TBI mice was more effective in promoting the proliferation and differentiation of pericytes than control mouse serum. In conclusion, our data show that the hypoxic state at the injury site in soft tissues of TBI mice provides an environment leading to increased accumulation and activation of pericytes to form endochondral bone.

The impact of TP53 status of tumor cells including the type and the concentration of administered 10B delivery agents on compound biological effectiveness in boron neutron capture therapy.

Human head and neck squamous cell carcinoma cells transfected with mutant TP53 (SAS/mp53) or neo vector (SAS/neo) were inoculated subcutaneously into left hind legs of nude mice. After the subcutaneous administration of a 10B-carrier, boronophenylalanine-10B (BPA) or sodium mercaptododecaborate-10B (BSH), at two separate concentrations, the 10B concentrations in tumors were measured using γ-ray spectrometry. The tumor-bearing mice received 5-bromo-2'-deoxyuridine (BrdU) continuously to label all intratumor proliferating (P) tumor cells, then were administered with BPA or BSH. Subsequently, the tumors were irradiated with reactor neutron beams during the time of which 10B concentrations were kept at levels similar to each other. Following irradiation, cells from some tumors were isolated and incubated with a cytokinesis blocker. The responses of BrdU-unlabeled quiescent (Q) and total (= P + Q) tumor cells were assessed based on the frequencies of micronucleation using immunofluorescence staining for BrdU. In both SAS/neo and SAS/mp53 tumors, the compound biological effectiveness (CBE) values were higher in Q cells and in the use of BPA than total cells and BSH, respectively. The higher the administered concentrations were, the smaller the CBE values became, with a clearer tendency in SAS/neo tumors and the use of BPA than in SAS/mp53 tumors and BSH, respectively. The values for BPA that delivers into solid tumors more dependently on uptake capacity of tumor cells than BSH became more alterable. Tumor micro-environmental heterogeneity might partially influence on the CBE value. The CBE value can be regarded as one of the indices showing the level of intratumor heterogeneity.

Quantitative Ultrasound for Evaluation of Tumour Response to Ultrasound-Microbubbles and Hyperthermia.

Objectives: Prior study has demonstrated the implementation of quantitative ultrasound (QUS) for determining the therapy response in breast tumour patients. Several QUS parameters quantified from the tumour region showed a significant correlation with the patient's clinical and pathological response. In this study, we aim to identify if there exists such a link between QUS parameters and changes in tumour morphology due to combined ultrasound-stimulated microbubbles (USMB) and hyperthermia (HT) using the breast xenograft model (MDA-MB-231). Method: Tumours grown in the hind leg of severe combined immuno-deficient mice were treated with permutations of USMB and HT. Ultrasound radiofrequency data were collected using a 25 MHz array transducer, from breast tumour-bearing mice prior and post-24-hour treatment. Result: Our result demonstrated an increase in the QUS parameters the mid-band fit and spectral 0-MHz intercept with an increase in HT duration combined with USMB which was found to be reflective of tissue structural changes and cell death detected using haematoxylin and eosin and terminal deoxynucleotidyl transferase dUTP nick end labelling stain. A significant decrease in QUS spectral parameters was observed at an HT duration of 60 minutes, which is possibly due to loss of nuclei by the majority of cells as confirmed using histology analysis. Morphological alterations within the tumour might have contributed to the decrease in backscatter parameters. Conclusion: The work here uses the QUS technique to assess the efficacy of cancer therapy and demonstrates that the changes in ultrasound backscatters mirrored changes in tissue morphology.

Time-resolved dose rate measurements in pencil beam scanning proton FLASH therapy with a fiber-coupled scintillator detector system.

The spatial and temporal dose rate distribution of pencil beam scanning (PBS) proton therapy is important in ultra-high dose rate (UHDR) or FLASH irradiations. Validation of the temporal structure of the dose rate is crucial for quality assurance and may be performed using detectors with high temporal resolution and large dynamic range. To provide time-resolved in vivo dose rate measurements using a scintillator-based detector during proton PBS pre-clinical mouse experiments with dose rates ranging from conventional to UHDR. All irradiations were performed at the entrance plateau of a 250MeV PBS proton beam. A detector system with four fiber-coupled ZnSe:O inorganic scintillators and 20μs temporal resolution was used for dose rate measurements. The system was first characterized in terms of precision and stem signal. The detector precision was determined through repeated irradiations with the same field. The stem signal contribution was quantified by irradiating two of the detector probes alongside a bare fiber (fiber without a coupled scintillator). Next, the detector system was calibrated against an ionization chamber (IC) with all four detector probes and the IC placed in a water bath at 2cmdepth. A scan pattern covering 9.6×9.6cmwas used. Multiple irradiations with different requested nozzle currents provided instantaneous dose rates at the detector positions in the range of 7-1270Gy/s.The correspondence of the detector signal (in Volts) to the instantaneous dose rate (in Gy/s) was found. The instantaneous dose rate was calculated from the beam current and the spot-to-detector distance assuming a Gaussian beam profile at distances up to 8mmfrom the spot. Afterwards, the calibrated system was used in vivo, in mouse experiments, where mouse legs were irradiated with a constant dose and varying field dose rates of 0.7-87.5Gy/s.Theinstantaneous dose rate was measured for each delivered spot and the delivered dose was determined as the integrated instantaneous dose rate. The spot dose profile and PBS dose rate map were calculated. The dose contamination to neighbouring mice were measured together with the upper limit of the dose to the mouse body. The detectors showed high precision with ≤0.4% fluctuations in the measured dose. The stem signal exceeded 10% for spots <5mmfromthe optical fiber and >18mmfromthe scintillator. It contributed up to 0.2% to the total dose, which was considered negligible. All four detectors showed a non-linear relation between signal and instantaneous dose rate, which was modelled with a polynomial response function. In the mouse experiments, the measured scintillator dose showed 1.8% fluctuations, independent of the field dose rate. The in vivo measured spot dose profile had tails that deviated from a Gaussian profile with measurable dose contributions from spots up to 85mm from the detector. Neighbour mouse irradiation contributed ∼1% of the total mouse dose. The upper limit of the mouse body dose was 6% of the mouse leg dose. A fiber-coupled inorganic scintillator-based detector system can provide high precision in vivo measurements of the instantaneous dose rate if correction for the non-linear dose rate dependency is applied.

Loss of 4E-BPs prevents the hindlimb immobilization-induced decrease in protein synthesis in skeletal muscle.

The present study was designed to test the hypothesis that upregulating protein synthesis attenuates the loss of muscle mass in a model of disuse atrophy. The studies compared the effect of unilateral hindlimb immobilization in wild-type (WT) mice and double-knockout (DKO) mice lacking the translational regulators 4E-BP1 and 4E-BP2. Immobilization-induced downregulation of protein synthesis occurred in both groups of mice, but protein synthesis was higher in gastrocnemius muscle from the immobilized hindlimb of fasted DKO compared with WT mice. Surprisingly, although protein synthesis was partially elevated in DKO compared with WT mice, atrophy occurred to the same extent in both groups of animals. This may be partially due to impaired leucine-induced stimulation of protein synthesis in DKO compared with WT mice due to downregulated eukaryotic initiation factor eIF4E expression in muscle of DKO compared with WT mice. Expression of the E3 ubiquitin ligases MAFbx and MuRF-1 mRNAs and total protein ubiquitylation was upregulated in the immobilized compared with the nonimmobilized hindlimb of both WT and DKO mice, with little difference in the magnitude of the upregulation between genotypes. Analysis of newly synthesized proteins revealed downregulation of several glycolytic enzymes in the gastrocnemius of DKO mice compared with WT mice, as well as in the immobilized compared with the nonimmobilized hindlimb. Overall, the results suggest that the elevated rate of protein synthesis during hindlimb immobilization in fasted DKO mice is insufficient to prevent disuse-induced muscle atrophy, probably due to induction of compensatory mechanisms including downregulation of eIF4E expression.NEW & NOTEWORTHY Basal rates of protein synthesis are elevated in skeletal muscle in the immobilized leg of mice lacking the translational repressors, 4E-BP1 and 4E-BP2 (knockout mice), compared with wild-type mice. However, disuse-induced muscle atrophy occurs to the same extent in both wild-type and knockout mice suggesting that compensatory mechanisms are induced that overcome the upregulation of muscle protein synthesis. Proteomic analysis revealed that mRNAs encoding several glycolytic enzymes are differentially translated in wild-type and knockout mice.

Haemophilus influenzae Protein D antibody suppression in a multi-component vaccine formulation.

Nontypeable Haemophilus influenzae (NTHi) has emerged as a dominant mucosal pathogen causing acute otitis media (AOM) in children, acute sinusitis in children and adults, and acute exacerbations of chronic bronchitis in adults. Consequently, there is an urgent need to develop a vaccine to protect against NTHi infection. A multi-component vaccine will be desirable to avoid emergence of strains expressing modified proteins allowing vaccine escape. Protein D (PD), outer membrane protein (OMP) 26, and Protein 6 (P6) are leading protein vaccine candidates against NTHi. In pre-clinical research using mouse models, we found that recombinantly expressed PD, OMP26, and P6 induce robust antibody responses after vaccination as individual vaccines, but when PD and OMP26 were combined into a single vaccine formulation, PD antibody levels were significantly lower. We postulated that PD and OMP26 physiochemically interacted to mask PD antigenic epitopes resulting in the observed effect on antibody response. However, column chromatography and mass spectrometry analysis did not support our hypothesis. We postulated that the effect might be in vivo through the mechanism of protein vaccine immunologic antigenic competition. We found when PD and OMP26 were injected into the same leg or separate legs of mice, so that antigens were immunologically processed at the same or different regional lymph nodes, respectively, antibody levels to PD were significantly lower with same leg vaccination. Different leg vaccination produced PD antibody levels quantitatively similar to vaccination with PD alone. We conclude that mixing PD and OMP26 into a single vaccine formulation requires further formulation studies.

An unusual complication of ear punching in a laboratory mouse

AbstractEar punching and notching are common methods of identification of laboratory rodents. Concerns have been raised regarding pain and discomfort, infection, thermoregulation and tearing. However, the evidence regarding these potential adverse events is currently limited. In this case report, we describe an unusual complication of ear punching in a laboratory mouse. The mouse had its left ear punched with a single hole at 3 weeks of age. At 11 weeks of age, the mouse caught its left foreleg through its left ear punch up to the level of the shoulder. The mouse's leg was released and the pinna cut to form a notch rather than a hole. To avoid this situation, we suggest that an ear notch be considered rather than an ear punch. This case also highlights the value of daily checks to ensure that the risk of any adverse effects from such unpredictable events is minimised.

In-situ sampling of lipids in tissues using a porous membrane microprobe for direct mass spectrometry analysis.

Direct sampling of lipids from tissues for direct mass spectrometry (MS) analysis allows a quick profiling of lipidome, which is important for biomedical applications. In this work, we developed a polyporous polymeric membrane (PPM) microprobe for highly efficient sampling of lipids directly from tissue samples. The PPM was prepared by polypropylene with pores as large of 10 ​μm, facilitating the permeation of lipids from tissue surfaces. The PPM was coated onto a stainless steel wire with a thickness of ∼100 ​μm. The entire analysis procedure includes sampling of the lipids in tissue, washing the probe, and extraction spray ionization for MS analysis. The effectiveness was validated by analyzing mouse brain tissue samples. It showed high recoveries for a series of lipid classes in comparison with total lipid extraction method. Further demonstration was carried out with analysis of tissue samples from mouse liver, stomach, kidney and legs. With high physical strength and good chemical stability, the microprobe was also demonstrated for sampling lipids inside mouse kidney tissue samples. By incorporating a photochemical derivatization, a workflow was also developed for fast detection of lipid C Created by potrace 1.16, written by Peter Selinger 2001-2019 ]]> C isomers in tissue samples. Finally, a microprobe array was also developed for simultaneous sampling of lipids from multiple sites on tissue surfaces.

A low-power stretchable neuromorphic nerve with proprioceptive feedback.

By relaying neural signals from the motor cortex to muscles, devices for neurorehabilitation can enhance the movement of limbs in which nerves have been damaged as a consequence of injuries affecting the spinal cord or the lower motor neurons. However, conventional neuroprosthetic devices are rigid and power-hungry. Here we report a stretchable neuromorphic implant that restores coordinated and smooth motions in the legs of mice with neurological motor disorders, enabling the animals to kick a ball, walk or run. The neuromorphic implant acts as an artificial efferent nerve by generating electrophysiological signals from excitatory post-synaptic signals and by providing proprioceptive feedback. The device operates at low power (~1/150 that of a typical microprocessor system), and consists of hydrogel electrodes connected to a stretchable transistor incorporating an organic semiconducting nanowire (acting as an artificial synapse), connected via an ion gel to an artificial proprioceptor incorporating a carbon nanotube strain sensor (acting as an artificial muscle spindle). Stretchable electronics with proprioceptive feedback may inspire the further development of advanced neuromorphic devices for neurorehabilitation.

Deep-tissue two-photon microscopy with a frequency-doubled all-fiber mode-locked laser at 937 nm

In two-photon microscopy, low illumination powers on samples and a high signal-to-noise ratio (SNR) of the excitation laser are highly desired for alleviating the problems of photobleaching and phototoxicity, as well as providing clean backgrounds for images. However, the high-repetition-rate Ti:sapphire laser and the low-SNR Raman-shift lasers fall short of meeting these demands, especially when used for deep penetrations. Here, we demonstrate a 937-nm laser frequency-doubled from an all-fiber mode-locked laser at 1.8 μm with a low repetition rate of ∼9 MHz and a high SNR of 74 dB. We showcase two-photon excitations with low illumination powers on multiple types of biological tissues, including fluorescence imaging of mouse brain neurons labeled with green and yellow fluorescence proteins (GFP and YFP), DiI-stained and GFP-labeled blood vessels, Alexa Fluor 488/568-stained mouse kidney, and second-harmonic-generation imaging of the mouse skull, leg, and tail. We achieve a penetration depth in mouse brain tissues up to 620 μm with an illumination power as low as ∼10 mW, and, even for the DiI dye with an extremely low excitation efficiency of 3.3%, the penetration depth is still up to 530 μm, indicating that the low-repetition-rate source works efficiently for a wide range of dyes with a fixed excitation wavelength. The low-repetition-rate and high-SNR excitation source holds great potential for biological investigations, such as in vivo deep-tissue imaging.

The Efficacy of Radiation is Enhanced by Metformin and Hyperthermia Alone or Combined Against FSaII Fibrosarcoma in C3H Mice.

The effects of mild-temperature hyperthermia (MTH) and metformin, alone or in combination, on the efficacy of high-dose hypofractionated radiation against experimental tumors were investigated. FSaII fibrosarcoma grown subcutaneously in the hind legs of C3H mice was irradiated with a single 15 Gy dose using a 60Co irradiator. The radio frequency capacitive method was used to heat the tumors at 41.0°C for 30 min. Metformin was intraperitoneally (i.p.) administered daily to tumor-bearing mice at a dose of 150 mg/kg. The expression levels of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), and programmed cell death-ligand 1 (PD-L1) were determined by immunohistochemical staining of the excised tumor tissues. The apoptosis of tumor cells in vivo was quantified by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and cleaved caspase-3 staining of the excised tumor tissues. Irradiation of tumors markedly increased the expression of HIF-1α, VEGF, and PD-L1, and MTH and metformin used either alone or in combination significantly abrogated the radiation-induced upregulation of these proteins. MTH and metformin alone or combined increased the radiation-induced apoptosis in tumor cells and enhanced the radiation-induced suppression of tumor growth. The findings indicated that the increased tumor response to 15 Gy irradiation by MTH and metformin alone or in combination was due, in part, to the abrogation of the radiation-induced upregulation of HIF-1α and its downstream targets VEGF and PD-L1.