Generation of porcine endometrial organoids and their use as a model for enhancing embryonic attachment and elongation.

In brief: Porcine endometrial organoids (EOs) were isolated and characterized, revealing distinctive features such as unique extracellular matrix formation, fusion into uterine bud-like structures, and facilitation of embryo elongation. The yield of EOs was significantly enhanced by cryopreservation medium supplemented with the rock inhibitor (Y-27632), resulting in reduced expression of apoptotic mRNAs and microRNAs. Abstract: Endometrial organoids (EOs) are acceptable models for understanding maternal-embryonic cross talk. This study was conducted to generate EOs and optimize their cryopreservation and provide coculture modeling with embryos. The endometrial tissues were used for culturing the organoids inside domes of Matrigel®. To improve the long-term storage of EOs, 10 µM ROCK inhibitor (RI) was added to the cryopreservation medium. Day 7 parthenogenetically activated embryos were cocultured with EOs or EO outgrowths, and embryonic cell numbers and embryo attachment were monitored. Spherical EOs 100-300 µm in size can be retrieved on day 7 of culture, and larger EOs, approximately 1.5 mm in diameter, can be maintained in the Matrigel® dome for 21 days. The nuclear expression of Ki67 indicates that more than 80% of EOs nuclei were proliferative. EOs exhibit unique novel characters such as formation of extracellular matrix and ability for fusion. RI increased the yield and quality of organoids after freezing or thawing. The cell number of cocultured embryos increased five-fold, and the proportion of trophoblast outgrowths increased seven-fold compared with those of control embryos. The embryos cultured with EO-conditioned medium showed a better attachment rate than the other models, and - for the first time - we report embryonic elongation. Immunofluorescence staining of the attached embryos showed CDX2 in the periphery of EOs outgrowths. The 3D assembly and cryopreservation of EOs was optimized, and EO coculture supported embryo attachment, trophoblast outgrowth, and elongation, which would provide a valuable tool for studying the intricate processes involved in porcine embryo implantation.

MicroRNA profiling of royal jelly extracellular vesicles and their potential role in cell viability and reversing cell apoptosis.

MiRNAs are small non-coding RNA molecules that play important regulatory roles in diverse biological processes. Royal jelly, a milky-white substance produced by nurse honeybees (Apis mellifera), is the primary food of queen bees and plays a crucial role in their development. However, little is known about the microRNA (miRNAs) content of royal jelly and their potential functions. In this study, we isolated extracellular vesicles from the royal jelly of 36 samples through sequential centrifugation and targeted nanofiltration and performed high-throughput sequencing to identify and quantify the miRNA content of honeybee royal jelly extracellular vesicles (RJEVs). We found a total of 29 known mature miRNAs and 17 novel miRNAs. Through bioinformatic analysis, we identified several potential target genes of the miRNAs present in royal jelly, including those involved in developmental processes and cell differentiation. To investigate the potential roles of RJEVs in cell viability, RJEVs were supplemented to apoptotic porcine kidney fibroblasts induced by ethanol 6% exposure for 30 min. TUNEL assay showed a significant reduction in the apoptosis percentage after RJEV supplementation when compared with the non-supplemented control group. Moreover, the wound healing assay performed on the apoptotic cells showed a rapid healing capacity of RJEV-supplemented cells compared to the control group. We observed a significant reduction in the expression of the miRNA target genes such as FAM131B, ZEB1, COL5A1, TRIB2, YBX3, MAP2, CTNNA1, and ADAMTS9 suggesting that RJEVs may regulate the target gene expression associated with cellular motility and cell viability. Moreover, RJEVs reduced the expression of apoptotic genes (CASP3, TP53, BAX, and BAK), while significantly increasing the expression of anti-apoptotic genes (BCL2 and BCL-XL). Our findings provide the first comprehensive analysis of the miRNA content of RJEVs and suggest a potential role for these vesicles in the regulation of gene expression and cell survival as well as augmenting cell resurrection or anastasis.

Critical protein-protein interactions within the CARMA1-BCL10-MALT1 complex: Take-home points for the cell biologist.

The CBM complex, which is composed of the proteins CARMA1, BCL10, and MALT1, serves multiple pivotal roles as a mediator of T-cell receptor and B-cell receptor-dependent NF-κB induction and lymphocyte activation. CARMA1, BCL10, and MALT1 are each proto-oncoproteins and dysregulation of CBM signaling, as a result of somatic gain-of-function mutation or chromosomal translocation, is a hallmark of multiple lymphoid malignancies including Activated B-cell Diffuse Large B-cell Lymphoma. Moreover, loss-of-function as well as gain-of-function germline mutations in CBM complex proteins have been associated with a range of immune dysregulation syndromes. A wealth of detailed structural information has become available over the past decade through meticulous interrogation of the interactions between CBM components. Here, we review key findings regarding the biochemical nature of these protein-protein interactions which have ultimately led the field to a sophisticated understanding of how these proteins assemble into high-order filamentous CBM complexes. To date, approaches to therapeutic inhibition of the CBM complex for the treatment of lymphoid malignancy and/or auto-immunity have focused on blocking MALT1 protease function. We also review key studies relating to the structural impact of MALT1 protease inhibitors on key protein-protein interactions.

Melatonin and resveratrol alleviate molecular and metabolic toxicity induced by Bisphenol A in endometrial organoids.

Bisphenol A (BPA), a widespread environmental contaminant, poses concerns due to its disruptive effects on physiological functions of the uterine endometrium. In contrast, melatonin (MT) and Resveratrol (RSV) are under scrutiny for their potential protective roles against BPA-induced damage. For the efficacy and ethical concerns in the animal test, endometrial organoids, three-dimensional models mimicking endometrium, serve as crucial tools for unraveling the impact of environmental factors on reproductive health. This study aimed to comprehensively characterize the morphological, molecular and metabolic responses of porcine endometrial organoids to BPA and assess the potential protective effects of MT and RSV. Porcine uteri were prepared, digested with collagenase, mixed with Matrigel, and incubated at 38°C with 5 % CO2. Passaging involved dissociation through trypsin-EDTA treatment and subculturing. The culture medium was refreshed every 2-3 days. To investigate the environmental impact on reproductive health, endometrial organoids were treated with BPA (0.5 µM), MT (with/without BPA at 0.1 µM), and/or RSV (10 µM). Various molecular screening using gene expression, western blotting, immunofluorescence staining, and metabolites profiling were assessed the effects of BPA, MT, and RSV in terms of cell viability, morphology, reproductivity, and metabolism alteration in the endometrial organoids. As expected, BPA induced structural and molecular disruptions in organoids, affecting cytoskeletal proteins, Wnt/ß-catenin signaling, and epithelial/mesenchymal markers. It triggered oxidative stress and apoptotic pathways, altered miRNA expression, and disrupted the endocannabinoid system. The level of glucose, galactose, and essential amino acids were increased or decreased by approximately 1.5-3 times in BPA-treated groups compared to the control groups (p-value < 0.05), indicating metabolic changes. Moreover, MT and RSV treated groups exhibited protective effects, mitigating BPA-induced disruptions across multiple pathways. For the first time, our study models endometrial organoids, advancing understanding of environmental impacts on reproductive health.

The combination of rolipram and cilostamide improved the developmental competence of cloned porcine embryos.

In vitro maturation of porcine oocytes is characterized by asynchronous cytoplasmic and nuclear maturation, leading to less competent oocytes supporting embryo development. The purpose of this study was to evaluate the combined effect of rolipram and cilostamide as cyclic Adenine monophosphate (cAMP) modulators to find the maximum cAMP levels that temporarily arrest meiosis. We determined the optimal time to maintain functional gap junction communication during pre-in vitro maturation to be four hours. Oocyte competence was evaluated by the level of glutathione, reactive oxygen species, meiotic progression, and gene expression. We evaluated embryonic developmental competence after parthenogenetic activation and somatic cell nuclear transfer. The combined treatment group showed significantly higher glutathione and lower reactive oxygen species levels and a higher maturation rate than the control and single treatment groups. Cleavage and blastocyst formation rates in parthenogenetic activation and somatic cell nuclear transfer embryos were higher in two-phase in vitro maturation than in the other groups. The relative levels of BMP15and GDF9 expression were increased in two-phase in vitro maturation. Somatic cell nuclear transfer blastocysts from two-phase in vitro maturation oocytes showed a lower level of expression of apoptotic genes than the control, indicating better pre-implantation developmental competence. The combination of rolipram and cilostamide resulted in optimal synchrony of cytoplasmic and nuclear maturation in porcine in vitro matured oocytes and there by enhanced the developmental competence of pre-implantation embryos.

Isolation, characterization, proteome, miRNAome, and the embryotrophic effects of chicken egg yolk nanovesicles (vitellovesicles).

Egg yolk constitutes about a third of the structure of the chicken egg however, the molecular structure and physiological effects of egg yolk-derived lipid membranous vesicles are not clearly understood. In this study, for the first record, the egg yolk nanovesicles (vitellovesicles, VVs) were isolated, characterized, and used as a supplement for porcine embryo culture. Yolks of ten freshly oviposited eggs were filtered and ultracentrifuged at 100,000 × g for 3 h to obtain a pellet. Cryogenic transmission electron microscopy and nanoparticle tracking analysis of the pellet revealed bilipid membranous vesicles. Protein contents of the pellet were analyzed using tandem mass spectrometry and the miRNA content was also profiled through BGISEQ-500 sequencer. VVs were supplemented with the in vitro culture medium of day-7 hatched parthenogenetic blastocysts. After 2 days of blastocyst culture, the embryonic cell count was increased in VVs supplemented embryos in comparison to the non-supplemented embryos. TUNEL assay showed that apoptotic cells were increased in control groups when compared with the VVs supplemented group. Reduced glutathione was increased by 2.5 folds in the VVs supplemented group while reactive oxygen species were increased by 5.3 folds in control groups. Quantitative PCR analysis showed that VVs significantly increased the expression of lipid metabolism-associated genes (monoglyceride lipase and lipase E), anti-apoptotic gene (BCL2), and superoxide dismutase, while significantly reducing apoptotic gene (BAX). Culturing embryos on Matrigel basement membrane matrix indicated that VVs significantly enhanced embryo attachment and embryonic stem cell outgrowths compared to the non-supplemented group. This considers the first report to characterize the molecular bioactive cargo contents of egg yolk nanovesicles to show their embryotrophic effect on mammalian embryos. This effect might be attributed to the protein and miRNA cargo contents of VVs. VVs can be used for the formulation of in vitro culture medium for mammalian embryos including humans.

Effects of extracellular vesicles derived from steroids-primed oviductal epithelial cells on porcine in vitro embryonic development.

Extracellular vesicles (EVs) play an active role in regulating different physiological events, however, endocrine control of EVs cargo contents remain poorly understood. In this study, we aimed to isolate EVs from the porcine oviductal epithelial cells (POECs) that were primed with steroid hormones including estradiol (E2) and progesterone (P4), mimicking the in vivo conditions of the reproductive cycle and studied their effects on in vitro produced embryonic development. For this purpose, POECs were treated either with 0 concentration (control) or two different combinations of E2 and P4 including 50 pg/mL E2 + 0.5 ng/mL P4 (group H1), and 10 pg/mL E2 + 35 ng/mL P4 (group H2). Embryos were prepared after in vitro maturation either by parthenogenetic activation or somatic cell nuclear transfer (SCNT) technique. Treating parthenogenetic embryo with EVs, led a significantly higher rate of the blastocyst formation in the group supplemented with each EVs, compared to the control group. In addition, TUNEL assay and gene expression level analysis revealed that apoptosis was significantly reduced in the H2 EVs group. Furthermore, EVs from hormone-primed POECs improved the formation rate of porcine SCNT embryos compared to the control group. While in each EVs supplemented group (control EVs, H1 EVs, H2 EVs), the expression of cell reprogramming-related genes in cloned embryos showed a tendency of increase, the effect was stronger in H1 EVs and H2 EVs. In conclusion, EVs derived from POECs cultured in hormonal conditions simulating the in vivo environment had a positive effect on porcine blastocysts formation, which will likely facilitate in the production of cloned embryos.

Delayed environmental enrichment reverses sevoflurane-induced memory impairment in rats.

BACKGROUND: Anesthesia given to immature rodents causes cognitive decline, raising the possibility that the same might be true for millions of children undergoing surgical procedures under general anesthesia each year. We tested the hypothesis that anesthesia-induced cognitive decline in rats is treatable. We also tested if anesthesia-induced cognitive decline is aggravated by tissue injury. METHODS: Seven-day old rats underwent sevoflurane anesthesia (1 minimum alveolar concentration, 4 h) with or without tail clamping. At 4 weeks, rats were randomized to environmental enrichment or normal housing. At 8 weeks rats underwent neurocognitive testing, which consisted of fear conditioning, spatial reference memory, and water maze-based memory consolidation tests, and interrogated working memory, short-term memory, and early long-term memory. RESULTS: Sevoflurane-treated rats had a greater escape latency when the delay between memory acquisition and memory retrieval was increased from 1 min to 1 h, indicating that short-term memory was impaired. Delayed environmental enrichment reversed the effects of sevoflurane on short-term memory and generally improved many tested aspects of cognitive function, both in sevoflurane-treated and control animals. The performance of tail-clamped rats did not differ from those rats receiving anesthesia alone. CONCLUSION: Sevoflurane-induced cognitive decline in rats is treatable. Delayed environmental enrichment rescued the sevoflurane-induced impairment in short-term memory. Tissue injury did not worsen the anesthesia-induced memory impairment. These findings may have relevance to neonatal and pediatric anesthesia.

Long-term condylar remodelling after bimaxillary orthognathic surgery in skeletal Class III patients.

In this study, we aimed to evaluate long-term condylar remodelling in skeletal Class III patients who underwent bimaxillary orthognathic surgery using cone-beam computed tomography (CBCT). Twenty-three patients were studied retrospectively, and a total of 4 CBCT scans were obtained for each subject: 1 month before surgery (T0), immediately after the surgery (T1), 6 months after the surgery (T2), and 6.1 (±2.1 years after the surgery) (T3). Condylar remodelling was measured using the condylar volume, height, width, and depth. To examine a significant change, a one-way repeated measures analysis of variance was performed. The correlation between postoperative skeletal movement and condylar volume was assessed using Spearman's correlation analysis. Condylar volume and height showed a significant decrease from T1 to T2 but a significant increase from T2 to T3. Furthermore, a significant decrease was observed between T1 and T3. The condylar width and depth showed no significant changes. Postoperative skeletal movement showed no correlation with the change in condylar volume. The change in condylar volume mostly occurred as the condylar height changed. However, it did not contribute much to the postoperative skeletal movement.

GRK2 suppresses lymphomagenesis by inhibiting the MALT1 proto-oncoprotein.

Antigen receptor-dependent (AgR-dependent) stimulation of the NF-κB transcription factor in lymphocytes is a required event during adaptive immune response, but dysregulated activation of this signaling pathway can lead to lymphoma. AgR stimulation promotes assembly of the CARMA1-BCL10-MALT1 complex, wherein MALT1 acts as (a) a scaffold to recruit components of the canonical NF-κB machinery and (b) a protease to cleave and inactivate specific substrates, including negative regulators of NF-κB. In multiple lymphoma subtypes, malignant B cells hijack AgR signaling pathways to promote their own growth and survival, and inhibiting MALT1 reduces the viability and growth of these tumors. As such, MALT1 has emerged as a potential pharmaceutical target. Here, we identified G protein-coupled receptor kinase 2 (GRK2) as a new MALT1-interacting protein. We demonstrated that GRK2 binds the death domain of MALT1 and inhibits MALT1 scaffolding and proteolytic activities. We found that lower GRK2 levels in activated B cell-type diffuse large B cell lymphoma (ABC-DLBCL) are associated with reduced survival, and that GRK2 knockdown enhances ABC-DLBCL tumor growth in vitro and in vivo. Together, our findings suggest that GRK2 can function as a tumor suppressor by inhibiting MALT1 and provide a roadmap for developing new strategies to inhibit MALT1-dependent lymphomagenesis.

Depression Prediction by Using Ecological Momentary Assessment, Actiwatch Data, and Machine Learning: Observational Study on Older Adults Living Alone.

BACKGROUND: Although geriatric depression is prevalent, diagnosis using self-reporting instruments has limitations when measuring the depressed mood of older adults in a community setting. Ecological momentary assessment (EMA) by using wearable devices could be used to collect data to classify older adults into depression groups. OBJECTIVE: The objective of this study was to develop a machine learning algorithm to predict the classification of depression groups among older adults living alone. We focused on utilizing diverse data collected through a survey, an Actiwatch, and an EMA report related to depression. METHODS: The prediction model using machine learning was developed in 4 steps: (1) data collection, (2) data processing and representation, (3) data modeling (feature engineering and selection), and (4) training and validation to test the prediction model. Older adults (N=47), living alone in community settings, completed an EMA to report depressed moods 4 times a day for 2 weeks between May 2017 and January 2018. Participants wore an Actiwatch that measured their activity and ambient light exposure every 30 seconds for 2 weeks. At baseline and the end of the 2-week observation, depressive symptoms were assessed using the Korean versions of the Short Geriatric Depression Scale (SGDS-K) and the Hamilton Depression Rating Scale (K-HDRS). Conventional classification based on binary logistic regression was built and compared with 4 machine learning models (the logit, decision tree, boosted trees, and random forest models). RESULTS: On the basis of the SGDS-K and K-HDRS, 38% (18/47) of the participants were classified into the probable depression group. They reported significantly lower scores of normal mood and physical activity and higher levels of white and red, green, and blue (RGB) light exposures at different degrees of various 4-hour time frames (all P<.05). Sleep efficiency was chosen for modeling through feature selection. Comparing diverse combinations of the selected variables, daily mean EMA score, daily mean activity level, white and RGB light at 4:00 pm to 8:00 pm exposure, and daily sleep efficiency were selected for modeling. Conventional classification based on binary logistic regression had a good model fit (accuracy: 0.705; precision: 0.770; specificity: 0.859; and area under receiver operating characteristic curve or AUC: 0.754). Among the 4 machine learning models, the logit model had the best fit compared with the others (accuracy: 0.910; precision: 0.929; specificity: 0.940; and AUC: 0.960). CONCLUSIONS: This study provides preliminary evidence for developing a machine learning program to predict the classification of depression groups in older adults living alone. Clinicians should consider using this method to identify underdiagnosed subgroups and monitor daily progression regarding treatment or therapeutic intervention in the community setting. Furthermore, more efforts are needed for researchers and clinicians to diversify data collection methods by using a survey, EMA, and a sensor.

MALT1 is a critical mediator of PAR1-driven NF-κB activation and metastasis in multiple tumor types.

Protease-activated receptor 1 (PAR1), a thrombin-responsive G protein-coupled receptor (GPCR), is implicated in promoting metastasis in multiple tumor types, including both sarcomas and carcinomas, but the molecular mechanisms responsible remain largely unknown. We previously discovered that PAR1 stimulation in endothelial cells leads to activation of NF-κB, mediated by a protein complex comprised of CARMA3, Bcl10, and the MALT1 effector protein (CBM complex). Given the strong association between NF-κB and metastasis, we hypothesized that this CBM complex could play a critical role in the PAR1-driven metastatic progression of specific solid tumors. In support of our hypothesis, we demonstrate that PAR1 stimulation results in NF-κB activation in both osteosarcoma and breast cancer, which is suppressed by siRNA-mediated MALT1 knockdown, suggesting that an intact CBM complex is required for the response in both tumor cell types. We identify several metastasis-associated genes that are upregulated in a MALT1-dependent manner after PAR1 stimulation in cancer cells, including those encoding the matrix remodeling protein, MMP9, and the cytokines, IL-1ß and IL-8. Further, exogenous expression of PAR1 in MCF7 breast cancer cells confers highly invasive and metastatic behavior which can be blocked by CRISPR/Cas9-mediated MALT1 knockout. Importantly, we find that PAR1 stimulation induces MALT1 protease activity in both osteosarcoma and breast cancer cells, an activity that is mechanistically linked to NF-κB activation and potentially other responses associated with aggressive phenotype. Several small molecule MALT1 protease inhibitors have recently been described that could therefore represent promising new therapeutics for the prevention and/or treatment of PAR1-driven tumor metastasis.

Novel intracellular N-terminal truncated matrix metalloproteinase-2 isoform in skeletal muscle ischemia-reperfusion injury.

Ischemia-reperfusion injury (IRI) occurs when blood returns to tissues following a period of ischemia. Reintroduction of blood flow results in the production of free radicals and reactive oxygen species that damage cells. Skeletal muscle IRI is commonly seen in orthopedic trauma patients. Experimental studies in other organ systems have elucidated the importance of extracellular and intracellular matrix metalloproteinase-2 (MMP-2) isoforms in regulating tissue damage in the setting of oxidant stress resulting from IRI. Although the extracellular full-length isoform of MMP-2 (FL-MMP-2) has been previously studied in the setting of skeletal muscle IRI, studies investigating the role of the N-terminal truncated isoform (NTT-MMP-2) in this setting are lacking. In this study, we first demonstrated significant increases in FL- and NTT-MMP-2 gene expression in C2C12 myoblast cells responding to re-oxygenation following hypoxia in vitro. We then evaluated the expression of FL- and NTT-MMP-2 in modulating skeletal muscle IRI using a previously validated murine model. NTT-MMP-2, but not FL-MMP-2 expression was significantly increased in skeletal muscle following IRI. Moreover, the expression of toll-like receptors (TLRs) -2 and -4, IL-6, OAS-1A, and CXCL1 was also significantly up-regulated following IRI. Treatment with the potent anti-oxidant pyrrolidine dithiocarbamate (PDTC) significantly suppressed NTT-MMP-2, but not FL-MMP-2 expression and improved muscle viability following IRI. This data suggests that NTT-MMP-2, but not FL-MMP-2, is the major isoform of MMP-2 involved in skeletal muscle IRI.

Direct Measurement of Tissue Oxygenation as a Method of Diagnosis of Acute Compartment Syndrome.

OBJECTIVE: The aim of this study was to investigate the utility of direct measurement of tissue oxygenation during compartment syndrome (CS) and tourniquet-induced ischemia in a large animal model. We hypothesize that as compartment pressure (CP) rises, circulation within the compartment will decrease resulting in a decreased level of oxygen in the muscle. METHODS: This study used a dog model of both CS- and tourniquet-based ischemia. In 15 animals, CS was induced in 1 hind limb with varying degrees of severity using an infusion model. Tourniquet ischemia was induced in the contralateral hind limb for varying durations. The partial pressure of oxygen (PmO2) was continuously monitored using a polarographic oxygen probe in the muscle of both hind limbs. CP was monitored in the CS limb. PmO2 and CP were analyzed after fasciotomy, performed after approximately 7 hours of warm ischemia, or release of tourniquet. RESULTS: With the application of tourniquet ischemia, PmO2 fell from 38.40 to 1.30 mm Hg (P < 0.001) and subsequently rose after release of the tourniquet to 39.81 mm Hg (P < 0.001). Elevated CP induced by infusion was relieved by fasciotomy (52.04-11.37 mm Hg postfasciotomy, P < 0.001). PmO2 readings in the infusion model were significantly higher in pre-CS than during CS (31.77 mm Hg vs. 3.88 mm Hg, P < 0.001) and rebounded after fasciotomy (50.24 mm Hg, P < 0.001), consistent with hyperemic response. CONCLUSIONS: Increased CP caused an observable decrease in PmO2 that was reversed by fasciotomy. PmO2 can be directly measured in real time with a polarographic tissue pO2 probe. This study is the first step of evaluating an alternative method for diagnosing acute CS.

Novel mouse model of spinal cord injury-induced heterotopic ossification.

Heterotopic ossification (HO) develops in about 20% to 30% of patients with spinal cord injury (SCI) and significantly impairs their rehabilitation. There is no effective prevention or treatment for this condition at this time. Our current understanding of its etiology and pathophysiology is limited partially due to the lack of clinically relevant animal models. In this study, we report a novel mouse model of SCI-induced HO by administering a subthreshold dose of bone morphogenetic protein (BMP)-2 to muscles in mice after SCI. Micro-computed tomography scanning showed that an intramuscular injection of 0.25 micrograms of BMP-2 causes significant HO in mice with SCI but not in control (sham surgery) mice. Our analysis of gene expression showed significantly increased BMP signaling in quadriceps following SCI, suggesting that BMP signaling may play a role in SCI-induced HO. Administering 0.25 micrograms of BMP-2 to the front arms of the mice with SCI also results in the development of significant HO but not in control mice. This suggests that SCI causes a systematic osteogenic effect, which is not limited to paralyzed limbs. This novel mouse model will serve as a powerful tool in exploring the molecular mechanisms of SCI-induced HO, which may lead to novel treatment for this disease.

A novel mouse model of trauma induced heterotopic ossification.

Severe soft tissue trauma is associated with heterotopic ossification (HO), the abnormal deposition of bone at extra-skeletal sites. The pathophysiology of the development of trauma-induced HO remains largely unknown due in part to the lack of appropriate animal models. In this study, we sought to develop a new trauma-induced HO mouse model using muscle impact injury combined with low dose BMP-2. BMP-2 at doses ranging from 0 to 2 µg was injected into quadriceps muscles of adult male C57/BL6 mice. Animals then received a one-time quadriceps impaction injury to mimic the trauma associated with severe injuries. HO was monitored using in vivo microCT scanning at 1, 2, 4, and 8 weeks after treatment. After trauma, the expression of BMP-2, -4, BMP receptor 1, SOX9 and RUNX2 were increased in muscle. Although little or no HO was observed in mice receiving 1 µg BMP-2, combining this dose with muscle trauma produced an abundance of HO. At higher doses of BMP-2, trauma did not augment mineral deposition. These results suggest that BMP-2 signaling can sensitize muscle to trauma-induced HO. They also provide the basis for a new model to study the pathogenesis of trauma-induced HO.

Psychometric curve and behavioral strategies for whisker-based texture discrimination in rats.

The rodent whisker system is a major model for understanding neural mechanisms for tactile sensation of surface texture (roughness). Rats discriminate surface texture using the whiskers, and several theories exist for how texture information is physically sensed by the long, moveable macrovibrissae and encoded in spiking of neurons in somatosensory cortex. However, evaluating these theories requires a psychometric curve for texture discrimination, which is lacking. Here we trained rats to discriminate rough vs. fine sandpapers and grooved vs. smooth surfaces. Rats intermixed trials at macrovibrissa contact distance (nose >2 mm from surface) with trials at shorter distance (nose <2 mm from surface). Macrovibrissae were required for distant contact trials, while microvibrissae and non-whisker tactile cues were used for short distance trials. A psychometric curve was measured for macrovibrissa-based sandpaper texture discrimination. Rats discriminated rough P150 from smoother P180, P280, and P400 sandpaper (100, 82, 52, and 35 µm mean grit size, respectively). Use of olfactory, visual, and auditory cues was ruled out. This is the highest reported resolution for rodent texture discrimination, and constrains models of neural coding of texture information.