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Three-dimensional (3D) biomimetic systems hold great promise for the study of biological systems in vitro as well as for the development and testing of pharmaceuticals. Here, we test the hypothesis that an intact segment of lumbar rat spinal cord will form functional neuromuscular junctions (NMJs) with engineered, 3D muscle tissue, mimicking the partial development of the peripheral nervous system (PNS). Muscle tissues are grown on a 3D-printed polyethylene glycol (PEG) skeleton where deflection of the backbone due to muscle contraction causes the displacement of the pillar-like "feet." We show that spinal cord explants extend a robust and complex arbor of motor neurons and glia in vitro. We then engineered a "spinobot" by innervating the muscle tissue with an intact segment of lumbar spinal cord that houses the hindlimb locomotor central pattern generator (CPG). Within 7 days of the spinal cord being introduced to the muscle tissue, functional neuromuscular junctions (NMJs) are formed, resulting in the development of an early PNS in vitro. The newly innervated muscles exhibit spontaneous contractions as measured by the displacement of pillars on the PEG skeleton. Upon chemical excitation, the spinal cord-muscle system initiated muscular twitches with a consistent frequency pattern. These sequences of contraction/relaxation suggest the action of a spinal CPG. Chemical inhibition with a blocker of neuronal glutamate receptors effectively blocked contractions. Overall, these data demonstrate that a rat spinal cord is capable of forming functional neuromuscular junctions ex vivo with an engineered muscle tissue at an ontogenetically similar timescale.

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OBJECTIVES: The purpose of this study was to quantify and determine the degree to which dogs experience negative displacement of the paw during movement initiation on natural surfaces, the frequency of that displacement, and whether or not the negative displacement could yield injuries. METHODS: Seven retired racing Greyhound dogs were selected to participate in sprint starts on two natural (non-vegetated and vegetated) surfaces. Kinematic analysis was conducted to quantify the displacements. RESULTS: All dogs in all trials experienced a negative paw displacement in at least one or more limbs. Significant effects were found for negative displacement across surfaces, limb, and for the surface x limb interactions. Rear paw negative displacement was -6.68 ± 2.55% body length (BL) for the non-vegetated surface and -5.29 ± 1.92% BL for the vegetated surface. Front paw negative displacement was -21.42 ± 2.62% BL for the non-vegetated surface and -17.25 ± 3.82% BL for the vegetated surface. There was no significant difference between average torso velocity on the two track surfaces. This study verified that the paw does negatively displace (moves backwards) during movement initiation. The magnitude of the displacements suggests that multiple injury mechanisms could be present. CLINICAL RELEVANCE: These findings demonstrate the extreme kinematics placed on the canine body during movement initiation, which might further the understanding of the mechanism of injury and contribute to enhanced surgical and rehabilitation techniques.

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Glutamate, acting through its N-methyl-D-aspartate (NMDA) and non-NMDA receptors in the hypothalamus, regulates reproductive neuroendocrine functions via direct and indirect actions upon gonadotrophin-releasing hormone (GnRH) neurones. Previous studies indicate that the NMDA receptor subunit NR2b undergoes changes in protein and gene expression in the hypothalamus in general, and on GnRH neurones in particular, during reproductive ageing. In the present study, we examined whether the NR2b-expressing cell population, both alone and in association with the NR1 subunit (i.e. the latter subunit is necessary for a functional NMDA receptor), is altered as a function of age and / or steroid hormone treatment. Studies focused on the anteroventral periventricular (AVPV) nucleus of the hypothalamus, a region critically involved in the control of reproduction. Young (3-5 months), middle-aged (9-12 months), and aged (approximately 22 months) female rats were ovariectomised and, 1 month later, they were treated sequentially with oestradiol plus progesterone, oestradiol plus vehicle, or vehicle plus vehicle, then perfused. Quantitative stereologic analysis of NR2b-immunoreactive cell numbers in the AVPV showed an age-associated decrease in the density of NR2b-immunoreactive cells, but no effect of hormone treatment. In a second study, immunofluorescent double labelling of NR2b and NR1 was analysed by confocal microscopy of fraction volume, a semi-quantitative measure of fluorescence intensity. No effect of ageing was detected for immunofluorescent NR1 or NR2b alone, whereas the NR2b fraction volume increased in the oestradiol plus vehicle group. With ageing, the fraction volume of the NR2b/NR1-colocalised subunits increased. Together with the stereology results, this suggests that, although fewer cells express the NR2b subunit in the ageing AVPV, a greater percentage of these subunits are co-expressed with NR1. Our results suggest that the subunit composition of NMDA receptors in the AVPV undergo both age- and hormonal-regulation, which may be related to previous observations of changes in functional responses of reproductive neuroendocrine systems to NMDA receptor modulators with ageing.

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Serotonin (5-HT) is an intrinsic modulator of neural network excitation states in gastropod molluscs. 5-HT and related indole metabolites were measured in single, well-characterized serotonergic neurons of the feeding motor network of the predatory sea-slug Pleurobranchaea californica. Indole amounts were compared between paired hungry and satiated animals. Levels of 5-HT and its metabolite 5-HT-SO4 in the metacerebral giant neurons were observed in amounts approximately four-fold and two-fold, respectively, below unfed partners 24 h after a satiating meal. Intracellular levels of 5-hydroxyindole acetic acid and of free tryptophan did not differ significantly with hunger state. These data demonstrate that neurotransmitter levels and their metabolites can vary in goal-directed neural networks in a manner that follows internal state.

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Elevated thromboxane levels are associated with a number of disease states, including dermal pressure ulcers. When dazmegrel was orally administered to greyhound dogs wearing leg casts, it resulted in a sparring effect on the skin areas of potential pressure ulcer development. The objective of this research was to determine if bioelastic matrices could provide controlled release of thromboxane A2 synthetase inhibitor (dazmegrel) at tissue concentrations sufficient for inhibition of thromboxane synthesis. The animal used for these studies was the greyhound, which has thin skin, angular conformation, limited body fat and is predisposed to pressure ulcers similar to those occurring in humans. In vivo skin penetration studies showed that epidermal exposure to bioelastic thromboxane synthetase inhibitor (TSI) matrix resulted in local tissue concentrations of TSI sufficient for thromboxane synthetase inhibition. There were no significant differences between dazmegrel in the skin layers (epidermis, dermis and subcutaneous layers) on 1, 7 and 14-day exposures.

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REASONS FOR PERFORMING STUDY: Analgesia of the palmar digital (PD) nerves has been demonstrated to cause analgesia of the distal interphalangeal (DIP) joint as well as the sole. Because the PD nerves lie in close proximity to the navicular bursa, we suspected that that analgesia of the navicular bursa would anaesthetise the PD nerves, which would result in analgesia of the DIP joint. OBJECTIVES: To determine the response of horses with pain in the DIP joint to instillation of local anaesthetic solution into the navicular bursa. METHODS: Lameness was induced in 6 horses by creating painful synovitis in the DIP joint of one forefoot by administering endotoxin into the joint. Horses were videorecorded while trotting, before and after induction of lameness, at three 10 min intervals after instilling 3.5 ml local anaesthetic solution into the navicular bursa and, finally, after instilling 6 ml solution into the DIP joint. Lameness scores were assigned by grading the videorecorded gaits subjectively. RESULTS: At the 10 and -20 min observations, median lameness scores were not significantly different from those before administration of local anaesthetic solution into the navicular bursa (P > or = 0.05), although lameness scores of 3 of 6 horses improved during this period, and the 20 min observation scores tended toward significance (P = 0.07). At the 30 min observation, and after analgesia of the DIP joint, median lameness scores were significantly improved (P < or = 0.05). CONCLUSIONS: These results indicate that pain arising from the DIP joint can probably be excluded as a cause of lameness, when lameness is attenuated within 10 mins by analgesia of the navicular bursa. POTENTIAL RELEVANCE: Pain arising from the DIP joint cannot be excluded as a cause of lameness when lameness is attenuated after 20 mins after analgesia of the navicular bursa.

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OBJECTIVE: To determine effects of intraincisional bioactive glass on healing of sutured skin wounds in dogs. ANIMALS: 9 purpose-bred mature female Beagles. PROCEDURE: 3 small matched bilateral (treated vs control) full-thickness truncal skin incisions were made and sutured. Treated wounds received intraincisional particulate bioactive glass prior to closure. Laser Doppler perfusion imaging was used to assess percentage change in tissue perfusion 3 and 5 days after incision on 1 set of 2 matched wounds, and skin and subcutaneous tissue-cutaneous trunci breaking strength were assessed at 5 days. The other 2 sets of wounds were used for histologic evaluation at 5 and 21 days, respectively. RESULTS: Subjective signs of gross inflammatory reaction were not detected in treated or control wounds. At 5 days, median subcutaneous tissue-cutaneous trunci breaking strength was significantly higher in treated wounds than in control wounds-(188.75 vs 75.00 g). At 5 days, median scores were significantly higher for neutrophils (1 vs 0), macrophages (2 vs 1), and necrosis (1 vs 0) for treated wounds than for control wounds. At 21 days, median macrophage scores were significantly higher for treated wounds than for control wounds (2 vs 1). CONCLUSIONS AND CLINICAL RELEVANCE: Bioactive glass in soft tissues does not cause a gross inflammatory reaction but causes an increase in histologic signs of inflammation, which decreases with time. Bioactive glass has potential for increasing tissue strength. Increased subcutaneous breaking strength could be beneficial in treating wounds in which early healing strength is needed.

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Quantitative analysis of signaling molecules from single cells and cellular materials requires careful validation of the analytical methods. Strategies have been investigated that enable single neurons and neuronal tissues to be stored before being assayed for many low-weight, biologically active molecules, such as serotonin, dopamine, and citrulline. Both metacerebral cell and pedal ganglia homogenates isolated from Pleiuohbrain-Chae californica have been studied by capillary electrophoresis with two complimentary laser-induced fluorescence detection methods. For homogenized ganglia samples, several cellular analytes (such as arginine and citrulline) are unaffected by standing at room temperature for days. Many other analytes in the biological matrix, including the catecholamines and indolamines, degrade by 20% within 10 h at room temperature. Rapidly freezing samples or preserving them with ascorbic acid preserves more than 80% of the dopamine and about 70% of the serotonin even after five days. In addition, serotonin and dopamine remain completely stable for at least five days by combining the ascorbic acid preservation and freezing at -20 degrees C. The timing of preservation is critical in maintaining the original composition of the biological samples. Using our optimum storage protocol of freezing the sample within 2 h after isolation, we can store frozen homogenate ganglia samples for more than four weeks before assay while still obtaining losses less than 10% of the original serotonin and dopamine. The nanoliter-volume single cell samples, however, must be analyzed within 4 h to obtain losses of less than 10% for serotonin related metabolites.

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OBJECTIVE: To determine the effects of a hydrolyzed bovine collagen dressing (HBCD) on healing of open wounds in healthy dogs. ANIMALS: 9 female Beagles. PROCEDURES: 2 full-thickness skin wounds were made bilaterally on the trunk of each dog. Wounds on 1 side were treated with powdered HBCD covered with a semiocclusive nonadherent bandage. Wounds on the other side (control wounds) were covered with a semiocclusive nonadherent bandage only. Wound healing was subjectively assessed, and percentage increase in tissue perfusion was assessed by use of laser Doppler perfusion imaging (LDPI). Planimetry was performed to determine the percentages of contraction, epithelialization, and total wound healing. Biopsy specimens were examined microscopically to evaluate histologic changes. RESULTS: The HBCD did not induce a strong inflammatory reaction, as reflected by results of LDPI and histologic examination. Moreover, HBCD appeared hydrophilic and provided an environment to keep wounds clean and enhance early epithelialization. After treatment for 7 days, treated wounds had a significantly greater percentage of epithelialization than control wounds (12.13 vs. 7.03%). CONCLUSION AND CLINICAL RELEVANCE: The hydrophilic property of HBCD may cleanse contaminated wounds with the body's homeostatic fluids and enhance early wound epithelialization.

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Escape swimming in the predatory sea slug Pleurobranchaea is a dominant behavior that overrides feeding, a behavioral switch caused by swim-induced inhibition of feeding command neurons. We have now found distinct roles for the different swim interneurons in acute suppression of feeding during the swim and in a longer-term stimulation of excitability in the feeding network. The identified pattern-generating swim neurons A1, A3, A10, and their follower interneuron A-ci1, suppress feeding motor output partly by excitation of the I1 feeding interneurons, which monosynaptically inhibit both the feeding command neurons, PC(P), PSE, and other major interneurons, the I2s. This mechanism exerts broad inhibition of the feeding network suitable to an escape response; broader than feeding suppression in learned and satiation-induced food avoidance and acting through a different presynaptic pathway. Four intrinsic neuromodulatory neurons of the swim network, the serotonergic As1-4, add little to direct suppression of feeding. Rather, they monosynaptically excite the serotonergic metacerebral giant (MCG) neurons of the feeding network, themselves intrinsic neuromodulators of feeding, as well as a cluster of adjacent serotonergic feeding neurons, with both fast and slow EPSPs. They also provide mild neuromodulatory excitation of the PC(P)/PSE feeding command neurons, and I1 and I2 feeding interneurons, which is masked by inhibition during the swim. As1-4 also excite the serotonergic pedal ganglion G neurons for creeping locomotion. These observations further delineate the nature of the putative serotonergic arousal system of gastropods and suggest a central coordinating role to As1-4.

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Hunger/satiation state interacts with appetitive and noxious stimuli to determine feeding and avoidance responses. In the predatory marine snail Pleurobranchaea californica, food chemostimuli induced proboscis extension and biting at concentration thresholds that varied directly with satiation state. However, food stimuli also tended to elicit avoidance behavior (withdrawal and avoidance turns) at concentration thresholds that were relatively low and fixed. When the feeding threshold for active feeding (proboscis extension with biting) was exceeded, ongoing avoidance and locomotion were interrupted and suppressed. Noxious chemostimuli usually stimulated avoidance, but, in animals with lower feeding thresholds for food stimuli, they often elicited feeding behavior. Thus, sensory pathways mediating appetitive and noxious stimuli may have dual access to neural networks of feeding and avoidance behavior, but their final effects are regulated by satiation state. These observations suggest that a simple cost-benefit computation regulates behavioral switching in the animal's foraging behavior, where food stimuli above or below the incentive level for feeding tend to induce feeding or avoidance, respectively. This decision mechanism can weigh the animal's need for nutrients against the potential risk from other predators and the cost of relative energy outlay in an attack on prey. Stimulation of orienting and attack by low-level noxious stimuli in the hungriest animals may reflect risk-taking that can enhance prey capture success. A simple, hedonically structured neural network model captures this computation.

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Sixteen sound Labrador retriever and Labrador retriever cross-breed adult dogs were evaluated for symmetry while in a trot gait using a two-dimensional motion analysis system. Reflective markers were placed at selected joint centers. Each dog had the right side and then the left side videotaped while in the trot gait. The markers on the videotape were then digitized for analysis. There was no significant difference (p less than 0.05) between the movements of the two sides. It was concluded that the trot gait is symmetrical and that a two-dimensional system can be used to analyze gait in the dog.

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NOS immunoreactivity was assayed in CNS and peripheral tissues of the sea slugs Pleurobranchaea californica, Tritonia diomedea and Aplysia californica using different antisera against mammalian nitric oxide synthase in Western blots. Polyclonal anti-nNOS labeled at 250, 185, 170, 155, 100, 75, and 65 kD in extracts of Pleurobranchaea CNS, salivary gland and esophagus but not of gills or muscle. The labeling pattern for Tritonia in bands at 250, 200, 120/110, 100, 69, 65, and 60 kD differed somewhat. Anti-nNOS labeling in Aplysia was markedly different, with bands labeled only at 69 and 60 kD in CNS extracts, and at 200, 190, 69 and 60 kD in salivary and esophagus extracts. The wide variation in NOS immunoreactivity is consistent with species differences in tissue localization and biochemical properties of molluscan NOS isoforms.

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Escape swimming in the notaspid opisthobranch Pleurobranchaea is an episode of alternating dorsal and ventral body flexions that overrides all other behaviors. We have explored the structure of the central pattern generator (CPG) in the cerebropleural ganglion as part of a study of neural network interactions underlying decision making in normal behavior. The CPG comprises at least eight bilaterally paired interneurons, each of which contributes and is phase-locked to the swim rhythm. Dorsal flexion is mediated by hemiganglion ensembles of four serotonin-immunoreactive neurons, the As1, As2, As3, and As4, and an electrically coupled pair, the A1 and A10 cells. When stimulated, A10 commands fictive swimming in the isolated CNS and actual swimming behavior in whole animals. As1-4 provide prolonged, neuromodulatory excitation enhancing dorsal flexion bursts and swim cycle number. Ventral flexion is mediated by the A3 cell and a ventral swim interneuron, IVS, the soma of which is yet unlocated. Initiation of a swim episode begins with persistent firing in A10, followed by recruitment of As1-4 and A1 into dorsal flexion. Recurrent excitation within the As1-4 ensemble and with A1/A10 may reinforce coactivity. Synchrony among swim interneuron partners and bilateral coordination is promoted by electrical coupling among the A1/A10 and As4 pairs, and among unilateral As2-4, and reciprocal chemical excitation between contralateral As1-4 groups. The switch from dorsal to ventral flexion coincides with delayed recruitment of A3, which is coupled electrically to A1, and with recurrent inhibition from A3/IVS to A1/A10. The alternating phase relation may be reinforced by reciprocal inhibition between As1-4 and IVS. Pleurobranchaea's swim resembles that of the nudibranch Tritonia; we find that the CPGs are similar in many details, suggesting that the behavior and network are primitive characters derived from a common pleurobranchid ancestor.

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Understanding the role of the gaseous messenger nitric oxide (NO) in the nervous system is complicated by the heterogeneity of its nerve cells; analyses carried out at the single cell level are therefore important, if not critical. Some invertebrate preparations, most especially those from the gastropod molluscs, provide large, hardy and identified neurons that are useful both for the development of analytical methodologies and for cellular analyses of NO metabolism and its actions. Recent modifications of capillary electrophoresis (CE) allow the use of a small fraction of an individual neuron to perform direct, quantitative and simultaneous assays of the major metabolites of the NO-citrulline cycle and associated biochemical pathways. These chemical species include the products of NO oxidation (NO2-/NO3-), l-arginine, l-citrulline, l-ornithine, l-argininosuccinate, as well as selected NO synthase inhibitors and cofactors such as NADPH, biopterin, FMN and FAD. Diverse cotransmitters can also be identified in the same nitrergic neuron. The sensitivity of CE methods is in the femtomole to attomole range, depending on the species analysed and on the specific detector used. CE analysis can be combined with prior in vivo electrophysiological and pharmacological manipulations and measurements to yield multiple physiological and biochemical values from single cells. The methodologies and instrumentation developed and tested using the convenient molluscan cell model can be adapted to the smaller and more delicate neurons of other invertebrates and chordates.

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Intracellular concentrations of L-citrulline (Cit) and its metabolites are related to nitric oxide synthase (NOS) activity, an enzyme producing the intercellular messenger NO in animal tissues including the nervous system. A capillary electrophoresis system using laser-induced fluorescence detection is described, and methods are developed to monitor the levels of L-arginine (Arg), Cit, and related molecules in identified neurons of the marine slugs, Pleurobranchaea californica and Aplysia californica. The limits of detection for Arg, Cit, L-arginino-succinate, L-ornithine, and L-arginine phosphate range from 50 amol to 17 fmol (5 nM to 17 microM in the neurons under study); these detection limits are significantly lower than actual intracellular levels of the metabolites, allowing the direct assay of single cells. The levels of NOS metabolites in individual neurons varied form 6 (Arg) and 4 mM (Cit) in putative NOS-containing neurons down to < 1 microM (undetectable) levels in many putative NOS-negative cells. The Arg/Cit ratio is independent of cell volume, correlates with NADPH-diaphorase staining, and appears to be a characteristic parameter for the presence of NOS activity in identified neurons.

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