Neonatal Injury Evokes Persistent Deficits in Dynorphin Inhibitory Circuits within the Adult Mouse Superficial Dorsal Horn.

Neonatal tissue damage induces long-term deficits in inhibitory synaptic transmission within the spinal superficial dorsal horn (SDH) that include a reduction in primary afferent-evoked, feedforward inhibition onto adult projection neurons. However, the subpopulations of mature GABAergic interneurons which are compromised by early-life injury have yet to be identified. The present research illuminates the persistent effects of neonatal surgical injury on the function of inhibitory SDH interneurons derived from the prodynorphin (DYN) lineage, a population that synapses directly onto lamina I spinoparabrachial neurons and is known to suppress mechanical pain and itch in adults. The results demonstrate that hindpaw incision at postnatal day 3 (P3) significantly decreased the strength of primary afferent-evoked glutamatergic drive onto DYN neurons within the adult mouse SDH while increasing the appearance of afferent-evoked inhibition onto the same population. Neonatal injury also dampened the intrinsic membrane excitability of mature DYN neurons, and reduced their action potential discharge in response to sensory input, compared with naive littermate controls. Furthermore, P3 incision decreased the efficacy of inhibitory DYN synapses onto adult spinoparabrachial neurons, which reflected a prolonged reduction in the probability of GABA release. Collectively, the data suggest that early-life tissue damage may persistently constrain the ability of spinal DYN interneurons to limit ascending nociceptive transmission to the adult brain. This is predicted to contribute to the loss of feedforward inhibition onto mature projection neurons, and the "priming" of nociceptive circuits in the developing spinal cord, following injuries during the neonatal period.SIGNIFICANCE STATEMENT Neonatal injury has lasting effects on pain processing in the adult CNS, including a reduction in feedforward inhibition onto ascending projection neurons in the spinal dorsal horn. While it is clear that spinal GABAergic interneurons are comprised of multiple subpopulations that play distinct roles in somatosensation, the identity of those interneurons which are compromised by tissue damage during early life remains unknown. Here we document persistent deficits in spinal inhibitory circuits involving dynorphin-lineage interneurons previously implicated in gating mechanical pain and itch. Notably, neonatal injury reduced the strength of dynorphin-lineage inhibitory synapses onto mature lamina I spinoparabrachial neurons, a major output of the spinal nociceptive network, which could contribute to the priming of pain pathways by early tissue damage.

Nano-Sized Extracellular Matrix Particles Lead to Therapeutic Improvement for Cutaneous Wound and Hindlimb Ischemia.

Cell-derived matrix (CDM) has proven its therapeutic potential and been utilized as a promising resource in tissue regeneration. In this study, we prepared a human fibroblast-derived matrix (FDM) by decellularization of in vitro cultured cells and transformed the FDM into a nano-sized suspended formulation (sFDM) using ultrasonication. The sFDM was then homogeneously mixed with Pluronic F127 and hyaluronic acid (HA), to effectively administer sFDM into target sites. Both sFDM and sFDM containing hydrogel (PH/sFDM) were characterized via immunofluorescence, sol-gel transition, rheological analysis, and biochemical factors array. We found that PH/sFDM hydrogel has biocompatible, mechanically stable, injectable properties and can be easily administered into the external and internal target regions. sFDM itself holds diverse bioactive molecules. Interestingly, sFDM-containing serum-free media helped maintain the metabolic activity of endothelial cells significantly better than those in serum-free condition. PH/sFDM also promoted vascular endothelial growth factor (VEGF) secretion from monocytes in vitro. Moreover, when we evaluated therapeutic effects of PH/sFDM via the murine full-thickness skin wound model, regenerative potential of PH/sFDM was supported by epidermal thickness, significantly more neovessel formation, and enhanced mature collagen deposition. The hindlimb ischemia model also found some therapeutic improvements, as assessed by accelerated blood reperfusion and substantially diminished necrosis and fibrosis in the gastrocnemius and tibialis muscles. Together, based on sFDM holding a strong therapeutic potential, our engineered hydrogel (PH/sFDM) should be a promising candidate in tissue engineering and regenerative medicine.

Knockout of beta-2 microglobulin reduces stem cell-induced immune rejection and enhances ischaemic hindlimb repair via exosome/miR-24/Bim pathway.

Generating universal human umbilical mesenchymal stem cells (UMSCs) without immune rejection is desirable for clinical application. Here we developed an innovative strategy using CRISPR/Cas9 to generate B2M- UMSCs in which human leucocyte antigen (HLA) light chain ß2-microglobulin (B2M) was deleted. The therapeutic potential of B2M- UMSCs was examined in a mouse ischaemic hindlimb model. We show that B2M- UMSCs facilitated perfusion recovery and enhanced running capability, without inducing immune rejection. The beneficial effect was mediated by exosomes. Mechanistically, microRNA (miR) sequencing identified miR-24 as a major component of the exosomes originating from B2M- UMSCs. We identified Bim as a potential target of miR-24 through bioinformatics analysis, which was further confirmed by loss-of-function and gain-of-function approaches. Taken together, our data revealed that knockout of B2M is a convenient and efficient strategy to prevent UMSCs-induced immune rejection, and it provides a universal clinical-scale cell source for tissue repair and regeneration without the need for HLA matching in the future.

Methods for the reliable induction of heterotopic ossification in the conditional Alk2Q207D mouse.

OBJECTIVES: Conditional Alk2Q207D-floxed (caALK2fl) mice have previously been used as a model of heterotopic ossification (HO). However, HO formation in this model can be highly variable, and it is unclear which methods reliably induce HO. Hence, these studies report validated methods for reproducibly inducing HO in caALK2fl mice. METHODS: Varying doses of Adex-cre and cardiotoxin (CTX) were injected into the calf muscles of 9, 14, or 28-day-old caALK2fl/- or caALK2fl/fl mice. HO was measured by planar radiography or microCT at 14-28 days post-injury. RESULTS: In 9-day-old caALK2fl/- or caALK2fl/fl mice, single injections of 109 PFU Adex-cre and 0.3 µg of CTX were sufficient to induce extensive HO within 14 days post-injury. In 28-day-old mice, the doses were increased to 5 x 109 PFU Adex-cre and 3.0 µg of CTX to achieve similar consistency, but at a slower rate versus younger mice. Using a crush injury, instead of CTX, also provided consistent induction of HO. Finally, the Type 1 BMPR inhibitor, DMH1, significantly reduced HO formation in 28-day-old caALK2fl/fl mice. CONCLUSIONS: These data illustrate multiple methods for reliable induction of localized HO in the caALK2flmouse that can serve as a starting point for new laboratories utilizing this model.

Changes in BMP-2 expression and mechanical properties during treatment of rats with osteoporotic hindlimb fracture with strontium ranelate.

OBJECTIVES: This study aims to investigate the changes in bone morphogenetic protein-2 (BMP-2) expression and mechanical properties in the healing process of rats with osteoporotic hindlimb fracture. METHODS: 120 rat models of osteoporotic hindlimb fracture were established and randomly divided into experimental group and control group. Quantitative real-time polymerase chain reaction (PCR) used to detect the BMP-2 expression in the rat's callus tissue on the fractured side. The mechanical properties of rat's hindlimb skeleton were examined using a universal material mechanics testing machine. RESULTS: The BMP-2 expression in the experimental group was higher than that in the control group (p<0.05). The linear correlation analysis showed that the BMP-2 was positively correlated with healing time (r=0.87, p<0.05). The mechanical properties were markedly improved at T2, T3 and T4, which peaked at T4 (p<0.05). However, the mechanical properties in the rats in the experimental group were notably superior to those in the control group at T2, T3, and T4 (p<0.05). CONCLUSIONS: The treatment with strontium ranelate can effectively improve the BMP-2 and bone mechanical properties of the rats with osteoporotic hindlimb fracture in the healing stage and accelerate the healing progress, which could be proved to be an efficacious means in treating osteoporotic fracture.

Can Intra-articular 1α, 25-Dihydroxyvitamin D3 Administration Be Therapeutical in Joint Cartilage Damage?

INTRODUCTION Vitamin D-deficiency is known to cause nerve conduction impairments, cancer and chronic diseases, as well as the pathogenesis of osteoarthritis. Our goal with this study is to evaluate the cartilage healing by applying intraarticular 1α, 25 (OH) 2D3 at different doses in rats with normal vitamin D levels and metabolism, which we made focal chondral damage model in the knee joint. MATERIAL AND METHODS 35 male Sprague-Dawley rats aged 20-24 weeks were used in our study. Both knees of rats were cartilage defected surgically on day 0. Joint injections performed at 06:00 am on 0th and 2nd days and after second injection others performed on days 9-16 and 23 following a weekly period. RESULTS In the fourth week, hematoxylin eosin staining measurements showed statistically significant difference according to the groups (p < 0.01) Metalloproteinase-13 (MMP-13) in histological staining for evaluating cartilage healing and healing levels showed statistically significant differences between the groups at first week and fourth week (p < 0.05). DISCUSSION Vitamin D, which affects many tissues through its receptors, is believed to be chondroprotective and neuroprotective by decreasing the expression of MMP in cartilage fibroblast, macrophage, lymphocyte through its intracellular receptors. To the best of our knowledge, this is the first study known to be intraarticular use of 1α, 25-dihydroxyvitamin D3. Our study has been found to be safe and successful in terms of weight, systemic PTH and 1α, 25-dihydroxyvitamin D3 levels in rats during treatment as well as better healing of cartilage damage. Key words: vitamin D3 receptor, articular cartilage, orthopedics, nerve conduction.

The Use of Bone Marrow-Derived Stem Cells in the Treatment of a Comminuted Tibiotarsal Fracture in a Juvenile Red-legged Seriema (Cariama cristata).

An adult red-legged seriema (Cariama cristata) presented with a comminuted fracture of the tibiotarsus and fibula. Surgery was performed, and a type II external fixator, with 2 distal and 2 proximal pins, was used to stabilize the fracture. After a 10-day stabilization period, the bird developed a second fracture on the same bone, proximal to the first fracture site. Another surgery was performed on the seriema similar to the first one. However, in this second surgical procedure a single pin, instead of 2 perpendicular pins, was placed proximally to the fracture site. After the second surgical procedure, bone marrow stem cells (BMSCs) from the seriema's left ulna were collected. Twenty-seven days after the second surgery, the BMSCs were transplanted, into the fracture sites. Twenty-four days after the stem cells were injected into the fractures (51 days after the second surgical procedure), radiographic images revealed healing bone calluses at the fracture sites. The fracture healing was relatively long for this case (a total of 75 days). The addition of bone marrow stem cell therapy to the use of external fixation may have contributed to the healing observed radiographically 24 days after administration; therefore, bone marrow stem cell therapy, in addition to traditional surgical fracture reduction and stabilization, may be a promising therapeutic approach for avian cases with similar injuries and bone anatomy. However, as this is a single case, this therapeutic modality deserves further application and study. Moreover, we suggest modifications in the bone marrow stem cell collection and therapy, which may be useful for future studies and application involving birds.

Effects of induced weight shift in the hind limbs on claw loads in dairy cows.

The modern dairy industry is plagued by a high prevalence of claw horn lesions in cows, which cause lameness, affect well-being, limit milk production, and are responsible for premature removal of cows from the herd. The lateral hind claws are primarily affected, and this has been linked to a relatively higher load being exerted on the lateral claws when cows shift weight from one hind limb to the other. The vertical ground reaction forces and mean and maximum pressures under the claws were measured in 40 nonlame dairy cows before and during a shift in weight from one hind limb to the other, which was accomplished by applying pressure manually to one side of the pelvis. During square standing on firm ground, about two-thirds of the entire hind limb load was exerted on the 2 lateral claws, and the remaining one-third was exerted on the medial claws combined. At the moment of maximum weight shift, the lateral claw of the loaded limb bore almost two-thirds of the entire load of both hind limbs, with the heel zone bearing almost half of the load of both hind limbs. Subsequently, the load of the lateral claw of the contralateral hind limb decreased, as did the load of both medial claws. Thus, the weight redistribution had occurred predominantly between the lateral hind claws. The high load exerted on a lateral hind claw during weight shift and at maximum weight shift is assumed to play a role in the pathogenesis of claw horn lesions, particularly when accentuated by a softened claw horn and hard flooring.

Human primary CD34+ cells transplantation for critical limb ischemia.

BACKGROUND: The goal of this study was to characterize the properties of human CD34+ cells in culture and investigate the feasibility and efficacy of CD34+ transplantation in a mouse model of limb ischemia and in patients with no-option critical limb ischemia. METHODS: Human CD34+ cells isolated from peripheral blood and grown in culture for up to four passages stained positively for the surface markers CD34 and CD133 and showed high viability after cryopreservation and recovery. Seven days after surgery to induce limb ischemia, ischemic muscles of nude mice were injected with CD34+ cells. Two weeks later, mice were scored for extent of ischemic injury, and muscle tissue was collected for immunohistochemical analysis of vascular endothelial cells and RT-PCR analysis of cytokine expression. RESULTS: Injury scores of CD34+ -treated, but not control, mice were significantly different before and after transplantation. Vascular density and expression of VEGF and bFGF mRNAs were also significantly increased in the treated mice. Patients with severe lower extremity arterial ischemia were injected with their own CD34+ cells in the affected calf, foot, or toe. Significant improvements were observed in peak pain-free walking time, ankle-brachial index, and transcutaneous partial oxygen pressure. These findings demonstrate that growth of human CD34+ cells in vitro and cryopreservations are feasible. CONCLUSION: Such cells may provide a renewable source of stem cells for transplantation, which appears to be a feasible, safe, and effective treatment for patients with critical limb ischemia.

Can Hyaluronidase Be an Alternative Postoperative Anti-edema Agent to Dexamethasone? Preliminary Results of an Animal Study.

PURPOSE: Recombinant human hyaluronidase (rHuPH20) is widely used as a spreading factor, which enhances the absorption of subcutaneously injected medicines. The anti-inflammatory and anti-edema effects of the enzyme were demonstrated in previous studies. In the present study, the anti-edema effect of rHuPH20 was compared with that of dexamethasone in a traumatic rat paw edema model. MATERIALS AND METHODS: Twenty-four Sprague-Dawley rats (weight 200 to 450 g) were divided into 3 groups: control (group 1), rHuPH20 (group 2), and dexamethasone (group 3). Traumatic edema was induced in the right hind paws of the rats using Feeney's weight-drop model. After edema induction, 0.4 mL of rHuPH20 (100 U/kg = 0.88 µg/kg dose) and 0.4 mL of dexamethasone (0.5 mg/kg dose) were injected into the right hind paws of the rats in groups 2 and 3. The paw volumes were measured before edema induction and at 3, 6, 12, 24, 48, and 72 hours after induction using a plethysmometer. The Mann-Whitney U test was used for the statistical analyses. Probabilities < .05 were accepted as statistically significant. RESULTS: The between percentage change in the edema mean values of groups 1 and 3 showed no significant difference at all time points; however, group 2 showed significantly less change in the edema mean values at 3, 6, 12, 24, and 48 hours after edema induction (P < .05) compared with group 1. The change in the edema mean value for group 2 was significantly less than that for group 3 at 3, 6, 12, 24, and 48 hours after edema induction (P < .05). CONCLUSIONS: Local rHuPH20 injection more effectively reduced the edema that was induced traumatically in rat paws than did dexamethasone. However, further clinical studies are needed regarding the use of rHuPH20 as a postoperative anti-edema agent in place of dexamethasone.

Development of Targeted Muscle Reinnervation Model in Hind Limb Amputated Rats.

BACKGROUND: Targeted muscle reinnervation (TMR) is a novel approach to postamputation neuroma pain; however, this has not been explicitly studied. The purpose of this study was to develop a TMR model in hind limb amputated rats. METHODS: Ten hind limbs from 5 Sprague Dawley cadaver rats were used. Sciatic nerve, main branches of the sciatic nerve (common peroneal, tibial, sural), motor branches from the sciatic nerve to the biceps femoris and cauda femoris, gluteal nerve and its motor branches to the semimembranosus, and biceps femoris and femoral nerve were dissected to look for consistent nerve anatomy that can be used for TMR in the rat hind limb amputation model. Transfemoral amputation was performed and two types of coaptations were made: common peroneal nerve to motor branch to biceps femoris and tibial nerve to motor branch to semimembranosus. RESULTS: The total surgical time for the dissection, amputation, and coaptation of nerves was ∼90 minutes. A total of 100 nerves were dissected in 10 rat hind limbs. Anatomical dissections were straightforward to perform. Anatomy of the dissected nerves was consistent. Hind limb amputations were performed without damaging the target muscles and nerves. Nerve lengths were sufficient for coaptation without any tension. CONCLUSIONS: To the best of our knowledge, this is the first report on TMR model in hind limb amputated rats. This model will allow for mechanical, electromyography (EMG), and histological analysis for future assessment of neuroma prevention.

Design and Use of a 3D Prosthetic Leg in a Red-lored Amazon Parrot ( Amazona autumnalis).

A three-dimensional (3D) prosthesis was designed and built for a red-lored Amazon parrot ( Amazona autumnalis) with a pre-existing amputation of the distal left leg at the tibiotarsal-tarsometatarsal joint and injuries to the right leg caused by cage companion aggression. The prosthesis consisted of a straight main imprint, with a round element at both ends to provide stability, and a bridge connecting this to a socket without a bottom where the stump could be accommodated and held securely with self-adhesive bandaging. Over a 4-month period, 3 different 3D prosthetic models were made and evaluated. The first model was fitted, but the parrot would only use the tip of the main imprint to stand and walk. The second model was designed with a semicircular imprint with only 1 round element at the cranial end, a different bridge to accommodate the change to the main imprint, and the same socket. With these changes, the parrot was able to place the imprint of the prosthesis on the floor to stand and move freely around its enclosure. To accommodate morphologic changes on the stump, a third model was created consisting of the same imprint and bridge, but the socket was cut vertically all the way on one side to allow distention on its diameter and provide a long-lasting fit to the stump over time.

Retrospective Evaluation of Tibiotarsal Fractures Treated With Tape Splints in Birds: 86 Cases (2006-2015).

Tibiotarsal fractures are a common presentation in small bird species and anecdotally have been reported to carry a good prognosis with proper treatment, such as external coaptation. For this retrospective study, the medical records of 5 institutions were reviewed for tibiotarsal fractures diagnosed in companion birds weighing less than 200 g. A total of 86 cases met the inclusion criteria. Cockatiels ( Nymphicus hollandicus) (24/86) and budgerigars ( Melopsittacus undulatus) (19/86) were the most frequently represented species. Median body weight of the birds included was 72 g (range, 16-182 g). Mid-diaphyseal (46/86) and closed (73/86) fractures with intact, deep pain sensation in the affected limb (69/76) were most frequent. A tape splint alone (79/86) or a tape splint in addition to an intramedullary pin (7/86) were applied in all cases. Median time to fracture stabilization based on palpation was 19 days (range, 7-49 days). In most cases (61/86), the initial splint applied was maintained until fracture healing was complete. A successful outcome was documented in 92% (79/86) of birds. Fractures caused by a dog or cat attack, birds presenting without deep pain sensation in the affected limb, and cases where the splint was removed before 14 days after fixation were associated with a significantly increased risk of complications, resulting in an unsuccessful outcome. The findings of this study indicated that a tape splint is an appropriate means for treatment of tibiotarsal fractures in birds weighing less than 200 g.

Profiling of glycan alterations in regrowing limb tissues of Cynops orientalis.

Glycans are known to play important roles in molecular recognition, cell-cell adhesion, molecular trafficking, receptor activation, and signal transduction during development and regeneration. Despite numerous investigations of regenerating salamander limbs, global analysis of the precise variation of glycans during the limb regeneration process has received little attention. Here, we have used lectin microarrays and lectin histochemistry to analyze the alterations and distribution of glycans during the early stages leading to blastema formation during Cynops orientalis limb regeneration in response to limb amputation. Compared with the control group, analysis at several time points (3, 7, and 14 days postamputation) using microarrays containing 37 lectins showed that limb tissues expressed significantly different complements of glycans recognized by 9 different lectins. Postamputation limb tissues showed higher expression of two glycan structures recognized by the lectins STL and LTL and lower expression of seven glycan structures recognized by PHA-E, MAL-I, SNA, UEA-I, PHA-E + L, VVA, and GNA. We also observed significant changes in glycans specifically at 7 days postamputation, including higher binding capacity by WFA, GSL-I, and NPA and lower binding capacity by PNA, HHL, ConA, LCA, GSL-II, and PWM. Next, we validated our lectin microarray data using lectin histochemistry in limb tissues. Glycans recognized by STL and GNA showed similar changes in signal intensity to those found in the lectin microarrays, with STL staining in the cytoplasm and GNA in the cytoplasm and nucleus. Our findings are the first report of significant postamputation changes in glycans in limb tissues and suggest that those glycans perform potentially important functions during the early stages of C. orientalis limb regeneration.

Level-specific amputations and resulting regenerative outcomes in the mouse distal phalanx.

Mouse digit tip regeneration involves an intricate coordinated regrowth of the terminal phalanx, nail, dermis and epidermis. During this time, regenerating digits undergo wound healing, blastema formation, and differentiation. However, the regenerative response of the digit is dependent on the level of the amputation. Amputation of <30% of the distal phalanx (P3), with part of the base nail remaining, results in extensive digit regeneration. In contrast, >60% P3 removal results in no regeneration. This level-dependent regenerative ability of the mouse digit provides a comparative model between regeneration and non-regeneration that may enable identification of specific factors critical to regeneration. Although the ability to create regenerating and non-regenerating conditions has been well established, the regenerative response between these regions ("intermediate" zone) has received less scrutiny, and may add insight to the regenerative processes, including the degree of histolysis, and the level of blastema formation. The objective of this study is then to compare the regeneration capacity between amputation levels within the regenerating (<30%), intermediate (40-59%), and non-regenerating (>60%) regions. Results indicated that regenerative and intermediate amputations led to significant histolysis and blastema formation of the distal phalanx 14 days post-amputation. Unlike the regenerating digits, intermediate amputations led to incomplete regeneration whereby regrowth of the digits were not to the levels of the intact or regenerating digits. Non-regenerating amputations did not exhibit significant histolysis or blastema formation. Remarkably, the histolytic process resulted in day 14 P3 lengths that were similar regardless of the initial amputation over 19%. The differences in histolysis, blastema formation and injury outcomes were also marked by changes in the number of proliferating cells and osteoclasts. Altogether, these results indicate that although intermediate amputations result in histolysis and blastema formation similar to regenerating digits, the resulting cellular composition of the blastema differs, contributing to incomplete regeneration.

The Development of Nociceptive Network Activity in the Somatosensory Cortex of Freely Moving Rat Pups.

Cortical perception of noxious stimulation is an essential component of pain experience but it is not known how cortical nociceptive activity emerges during brain development. Here we use continuous telemetric electrocorticogram (ECoG) recording from the primary somatosensory cortex (S1) of awake active rat pups to map functional nociceptive processing in the developing brain over the first 4 weeks of life. Cross-sectional and longitudinal recordings show that baseline S1 ECoG energy increases steadily with age, with a distinctive beta component replaced by a distinctive theta component in week 3. Event-related potentials were evoked by brief noxious hindpaw skin stimulation at all ages tested, confirming the presence of functional nociceptive spinothalamic inputs in S1. However, hindpaw incision, which increases pain sensitivity at all ages, did not increase S1 ECoG energy until week 3. A significant increase in gamma (20-50 Hz) energy occurred in the presence of skin incision at week 3 accompanied by a longer-lasting increase in theta (4-8 Hz) energy at week 4. Continuous ECoG recording demonstrates specific postnatal functional stages in the maturation of S1 cortical nociception. Somatosensory cortical coding of an ongoing pain "state" in awake rat pups becomes apparent between 2 and 4 weeks of age.

Clinical findings, treatment, and outcome in 11 dairy heifers with breakdown injury due to interosseous medius muscle rupture.

OBJECTIVE: To describe the diagnosis, treatment, and prognosis of fetlock breakdown due to interosseus medius muscle rupture in cattle. STUDY DESIGN: Retrospective clinical study. ANIMALS: Dairy heifers with unilateral or bilateral interosseus medius muscle rupture (n = 11). METHODS: Breakdown injury due to rupture of the interosseus medius muscle was documented clinically, radiographically, and ultrasonographically. Breakdown was bilateral in 4 heifers (3 in forelimbs, 1 in hind limbs) and unilateral in 7 (all hind limbs). One heifer with severe bilateral hind limb breakdown was slaughtered and the remaining 10 were treated by transfixation pin cast (1 heifer), box rest (n = 2), and/or a cast and splint (7). RESULTS: Hyperextension of the fetlock and hyperflexion of the proximal interphalangeal joints during weight bearing were characteristic for interosseus muscle breakdown. Ultrasonographically, the origin and body of the interosseus muscle and the branches to the sesamoid bones were primarily affected by the rupture. Conservative treatment was successful (used for their intended purpose) in 8 of 9 heifers with a median lifespan of 32 months after discharge from the clinic (range 6-83). CONCLUSION: Rupture of the interosseus medius muscle in young cattle may be more common than previously suggested in the literature. Imaging with ultrasound allowed more detailed localization of lesions of the musculo-tendinous structure. Interosseus medius muscle rupture had a favorable prognosis when treated conservatively in these heifers.

Characterization of incisional and inflammatory pain in rats using functional tools of MRI.

Underlying mechanisms of hyperalgesia differ with regard to the pain entities, which are well-modeled in animals for systematic studies. However, neuroimaging in different animal pain models often lacks clinical relevance and consistency with behavioral studies, which hinders the translation of results. Whereas mechanical stimulation is commonly used to explore hyperalgesia in animals and humans, functional magnetic resonance imaging (fMRI) studies frequently use electrical or heat stimuli to evaluate brain responses relevant to pain and hyperalgesia. To characterize the magnetic resonance (MR) representations of mechanical hyperalgesia after incision and inflammation, we aimed to investigate whole brain functional activities during innocuous and noxious mechanical or electrical stimulation (IMS/NMS; IES/NES), as well as metabolite levels in the thalamus of rats at rest and during electrical stimulation. In behavioral experiments, animal models of pain showed significant mechanical hyperalgesia, with a peak 24h after both injuries, but lasting longer after inflammation. In imaging experiments, mechanical and electrical stimulation revealed a biphasic BOLD response upon noxious stimulation in pain models. Analyses of the BOLD signal changes revealed significantly higher activation in pain models compared to sham animals. Furthermore, significant differences were present upon NMS (but not NES) between incision and inflammation models in all the studied regions except for contralateral somatosensory cortex (S1) and cerebellum (Cb) (F's>4.14, p's<0.05). Additionally, MS (but not ES) induced unexpected bilateral activation of S1 in all three animal groups. Finally, MR spectroscopy (MRS) in the thalamus showed higher concentrations of gamma-aminobutyric acid in both pain models at rest and during stimulation. We conclude that employment of MS in fMRI studies could provide an informative correlate of mechanical hyperalgesia in inflammatory and incisional pain models and might be used to further assess mechanisms and treatments relevant for these clinical pain states.

Mesenchymoangioblast-derived mesenchymal stromal cells inhibit cell damage, tissue damage and improve peripheral blood flow following hindlimb ischemic injury in mice.

BACKGROUND AIMS: Existing treatments have limited success in modifying the course of peripheral artery disease, which may eventually lead to limb-threatening ulcers and amputation. Cellular therapies have the potential to provide a new treatment option for this condition, but isolation of cells by conventional means has limitations with respect to reproducibility and scalability. METHODS: Induced pluripotent stem cells (iPSCs) were differentiated into precursor cells known as mesenchymoangioblasts (MCAs) and subsequently into mesenchymal stromal cells (MSCs). Hindlimb ischemia in mice was created by ligating both the iliac and femoral arteries of one hindlimb. Immediately after surgery, each animal received intramuscular injections of 5 × 10(6) cells or media in the ischemic limb. Toe necrosis was assessed visually, and hindlimb blood flow was measured by laser Doppler using a set region of interest (ROI) and by tracing the entire foot. Myofiber heterogeneity, nuclear centralization, fatty degeneration, fibrosis and capillary angiogenesis in the gastrocnemius muscle were assessed histologically. RESULTS: Blood flow in the MCA-derived MSC-treated animals was higher at each day (P <0.006), and these mice recovered faster than control animals (3.6 vs. 2.5 for set ROI; 7.5 vs. 4.1 foot tracing; slope; P <0.001). There was significantly less myofiber heterogeneity, nuclear centralization, fatty degeneration and fibrosis in MCA-derived MSC-treated animals, indicating less tissue damage. DISCUSSION: MCA-derived MSCs improved limb blood flow, reduced necrosis and maintained muscle mass and gross muscle appearance. We conclude that MCA-derived MSCs have a significant and protective effect against ischemic insults.

Functional MMP-10 is required for efficient tissue repair after experimental hind limb ischemia.

We studied the role of matrix metalloproteinase-10 (MMP-10) during skeletal muscle repair after ischemia using a model of femoral artery excision in wild-type (WT) and MMP-10 deficient (Mmp10(-/-)) mice. Functional changes were analyzed by small animal positron emission tomography and tissue morphology by immunohistochemistry. Gene expression and protein analysis were used to study the molecular mechanisms governed by MMP-10 in hypoxia. Early after ischemia, MMP-10 deficiency resulted in delayed tissue reperfusion (10%, P < 0.01) and in increased necrosis (2-fold, P < 0.01), neutrophil (4-fold, P < 0.01), and macrophage (1.5-fold, P < 0.01) infiltration. These differences at early time points resulted in delayed myotube regeneration in Mmp10(-/-) soleus at later stages (regenerating myofibers: 30 ± 9% WT vs. 68 ± 10% Mmp10(-/-), P < 0.01). The injection of MMP-10 into Mmp10(-/-) mice rescued the observed phenotype. A molecular analysis revealed higher levels of Cxcl1 mRNA (10-fold, P < 0.05) and protein (30%) in the ischemic Mmp10(-/-) muscle resulting from a lack of transcriptional inhibition by MMP-10. This was further confirmed using siRNA against MMP-10 in vivo. Our results demonstrate an important role of MMP-10 for proper muscle repair after ischemia, and suggest that chemokine regulation such as Cxcl1 by MMP-10 is involved in muscle regeneration.