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Ischemia-reperfusion injury (IRI) is a critical condition associated with serious clinical manifestations. Extensive research has focused on the strategies increasing organ tolerance to IRI. Preconditioning (PC) has been shown to provide protection to various organs toward IRI. However, the underlying mechanisms remain unknown. This study aimed to evaluate the role of PC on muscle regeneration after IRI and the potential underlying mechanisms. Three-month-old male UCP-1 reporter mice underwent unilateral hindlimb IRI with or without PC, the tissue viability and injury index were measured at 24 h after IRI. Hindlimb gait, muscle contractility, muscle histology were analyzed at 2 weeks after IRI. In another group of animals, β3 adrenergic receptor (β3AR) agonist amibegron and β3AR antagonist SR-59230A were administrated before PC/IRI, the hindlimb function and muscle regeneration were evaluated at 2 weeks after IRI. Our results showed that PC has little effect on improving the tissue viability at the acute phase of IRI, but it showed a long-term beneficial role of improving hindlimb function and muscle regeneration as evidenced by increased central nuclei regenerating myofibers. The effects of PC are related to inducing muscle fibro-adipogenic progenitor (FAP) brown/beige-like adipocyte (BAT) differentiation. Amibegron treatment displayed a similar role of PC while SR-59230A abolished the effect of PC. This study suggests PC has a beneficial role in promoting muscle regeneration after IRI through β3AR signaling pathway-stimulated FAP-BAT differentiation.

Rostro-caudal specificity of corticospinal tract (CST) projections from different areas of the cortex was assessed by retrograde labeling with fluorogold and retrograde transfection following retro-AAV/Cre injection into the spinal cord of tdT reporter mice. Injections at C5 led to retrograde labeling of neurons throughout forelimb area of the sensorimotor cortex and a region in the dorsolateral cortex near the barrel field (S2). Injections at L2 led to retrograde labeling of neurons in the posterior sensorimotor cortex (hindlimb area) but not the dorsolateral cortex. With injections of biotinylated dextran amine (BDA) into the main sensorimotor cortex (forelimb region), labeled axons terminated selectively at cervical levels. With BDA injections into caudal sensorimotor cortex (hindlimb region), labeled axons passed through cervical levels without sending collaterals into the gray matter and then elaborated terminal arbors at thoracic sacral levels. With BDA injections into the dorsolateral cortex near the barrel field, labeled axons terminated at high cervical levels. Axons from medial sensorimotor cortex terminated primarily in intermediate laminae and axons from lateral sensorimotor cortex terminated primarily in laminae III-V of the dorsal horn. One of the descending pathways seen in rats (the ventral CST) was not observed in most mice.

ObjectiveTransforming growth factor β (TGFβ) signaling plays a complex tissue-specific and nonlinear role in osteoarthritis (OA). This study was conducted to determine the osteocytic contributions of TGFβ signaling to OA.MethodsTo identify the role of osteocytic TGFβ signaling in joint homeostasis, we used 16-week-old male mice (n = 9-11 per group) and female mice (n = 7-11 per group) with an osteocyte-intrinsic ablation of TGFβ receptor type II (TβRIIocy-/- mice) and assessed defects in cartilage degeneration, subchondral bone plate (SBP) thickness, and SBP sclerostin expression. To further investigate these mechanisms in 16-week-old male mice, we perturbed joint homeostasis by subjecting 8-week-old mice to medial meniscal/ligamentous injury (MLI), which preferentially disrupts the mechanical environment of the medial joint to induce OA.ResultsIn all contexts, independent of sex, genotype, or medial or lateral joint compartment, increased SBP thickness and SBP sclerostin expression were spatially associated with cartilage degeneration. Male TβRIIocy-/- mice, but not female TβRIIocy-/- mice, had increased cartilage degeneration, increased SBP thickness, and higher levels of SBP sclerostin compared with control mice (all P < 0.05), demonstrating that the role of osteocytic TGFβ signaling on joint homeostasis is sexually dimorphic. With changes in joint mechanics following injury, control mice had increased SBP thickness, subchondral bone volume, and SBP sclerostin expression (all P < 0.05). TβRIIocy-/- mice, however, were insensitive to subchondral bone changes with injury, suggesting that mechanosensation at the SBP requires osteocytic TGFβ signaling.ConclusionOur results provide new evidence that osteocytic TGFβ signaling is required for a mechanosensitive response to injury, and that osteocytes control SBP homeostasis to maintain cartilage health, identifying osteocytic TGFβ signaling as a novel therapeutic target for OA.

BackgroundSpinal cord injury elicits widespread inflammation that can exacerbate long-term neurologic deficits. Neutrophils are the most abundant immune cell type to invade the spinal cord in the early acute phase after injury, however, their role in secondary pathogenesis and functional recovery remains unclear. We have previously shown that neutrophil functional responses during inflammation are augmented by spleen tyrosine kinase, Syk, a prominent intracellular signaling enzyme. In this study, we evaluated the contribution of Syk towards neutrophil function and long-term neurologic deficits after spinal cord injury.MethodsContusive spinal cord injury was performed at thoracic vertebra level 9 in mice with conditional deletion of Syk in neutrophils (Sykf/fMRP8-Cre). Hindlimb locomotor recovery was evaluated using an open-field test for 35 days following spinal cord injury. Long-term white matter sparing was assessed using eriochrome cyanide staining. Blood-spinal cord barrier disruption was evaluated by immunoblotting. Neutrophil infiltration, activation, effector functions, and cell death were determined by flow cytometry. Cytokine and chemokine expression in neutrophils was assessed using a gene array.ResultsSyk deficiency in neutrophils improved long-term functional recovery after spinal cord injury, but did not promote long-term white matter sparing. Neutrophil activation, cytokine expression, and cell death in the acutely injured spinal cord were attenuated by the genetic loss of Syk while neutrophil infiltration and effector functions were not affected. Acute blood-spinal cord barrier disruption was also unaffected by Syk deficiency in neutrophils.ConclusionsSyk facilitates specific neutrophil functional responses to spinal cord injury including activation, cytokine expression, and cell death. Long-term neurologic deficits are exacerbated by Syk signaling in neutrophils independent of acute blood-spinal cord barrier disruption and long-term white matter sparing. These findings implicate Syk in pathogenic neutrophil activities that worsen long-term functional recovery after spinal cord injury.

Which areas of the neocortex are involved in the control of movement, and how is motor cortex organized across species? Recent studies using long-train intracortical microstimulation demonstrate that in addition to M1, movements can be elicited from somatosensory regions in multiple species. In the rat, M1 hindlimb and forelimb movement representations have long been thought to overlap with somatosensory representations of the hindlimb and forelimb in S1, forming a partial sensorimotor amalgam. Here we use long-train intracortical microstimulation to characterize the movements elicited across frontal and parietal cortex. We found that movements of the hindlimb, forelimb, and face can be elicited from both M1 and histologically defined S1 and that representations of limb movement types are different in these two areas. Stimulation of S1 generates retraction of the contralateral forelimb, while stimulation of M1 evokes forelimb elevation movements that are often bilateral, including a rostral region of digit grasping. Hindlimb movement representations include distinct regions of hip flexion and hindlimb retraction evoked from S1 and hip extension evoked from M1. Our data indicate that both S1 and M1 are involved in the generation of movement types exhibited during natural behavior. We draw on these results to reconsider how sensorimotor cortex evolved.

Background. Epidural stimulation of the spinal cord can reorganize and change the excitability of the neural circuitry to facilitate stepping in rats with a complete spinal cord injury. Parkinson's disease results in abnormal supraspinal signals from the brain to the spinal cord that affect the functional capacity of the spinal networks. Objective. The objective was to determine whether epidural stimulation (electrical enabling motor control, eEmc) of the lumbosacral spinal cord can reorganize the spinal networks to facilitate hindlimb stepping of rats with parkinsonism. Methods. A unilateral 6-OHDA (6-hydroxydopamine) lesion of the nigrostriatal pathway was used to induce parkinsonism. Sham rats (N = 4) were injected in the same region with 0.1% of ascorbic acid. Stimulation electrodes were implanted epidurally at the L2 and S1 (N = 5) or L2 (N = 5) spinal levels. Results. The 6-OHDA rats showed severe parkinsonism in cylinder and adjusting step tests and were unable to initiate stepping when placed in a running wheel and dragged their toes on the affected side during treadmill stepping. During eEmc, the 6-OHDA rats initiated stepping in the running wheel and demonstrated improved stepping quality. Conclusion. Stepping was facilitated in rats with parkinsonism with spinal cord stimulation. The underlying assumption is that the normal functional capacity of spinal networks is affected by supraspinal pathology associated with Parkinson's disease, which either generates insufficient or abnormal descending input to spinal networks and that eEmc can appropriately modulate spinal and supraspinal networks to improve the motor deficits.

Blackleg is an infectious disease of cattle and rarely other ruminants, produced by Clostridium chauvoei and characterized by necrotizing myositis. In most cases of blackleg, the large muscles of the pectoral and pelvic girdles are affected, with other skeletal muscles and the heart involved less frequently. We studied 29 blackleg cases selected from the archives of the California Animal Health and Food Safety Laboratory, 1991-2015. Immunohistochemistry was also evaluated to detect C. chauvoei in formalin-fixed, paraffin-embedded (FFPE) tissues of cattle. Nineteen animals had gross and/or microscopic lesions in both skeletal muscle and heart, 9 had lesions in the skeletal musculature alone, and 1 in the heart alone. Gross lesions in the skeletal musculature involved the following muscle groups: hindquarters ( n = 8), forequarters ( n = 5), neck ( n = 5), lumbar area ( n = 3), brisket ( n = 2), diaphragm ( n = 2), abdominal wall ( n = 1), thoracic wall ( n = 1), and tongue ( n = 1). Of the 20 animals that had lesions in the heart, 11 had pericarditis and myocarditis; 7 had pericarditis, myocarditis, and endocarditis; and 1 each had pericarditis and myocarditis. Immunohistochemistry was 100% sensitive to detect C. chauvoei in FFPE skeletal muscle and/or heart of cattle with blackleg. Simultaneous lesions in skeletal musculature and heart were relatively common in blackleg cases in California; the most affected skeletal muscles were those of the hindlimbs.

Vasovagal syncope (VVS) is characterized by hypotension and bradycardia followed by lowering of cerebral blood flow. Remote limb ischemic preconditioning (RIPC) is well documented to provide cardio- and neuroprotection as well as to improve cerebral blood flow. We hypothesized that RIPC will provide protection against VVS-induced hypotension, bradycardia, and cerebral hypoperfusion. Second, because endothelial nitric oxide synthase has been reported as a mediator of cerebral blood flow control, we hypothesized that the mechanism by which RIPC primes the vasculature against VVS is via the α1-adrenoceptor-protein kinase Cε-endothelial nitric oxide synthase pathway. We utilized sinusoidal galvanic vestibular stimulation in rats as a model of VVS. RIPC attenuated the lowerings of mean arterial pressure, heart rate, and cerebral blood flow caused by sinusoidal galvanic vestibular stimulation, as well as improving behavior during, and recovery after, stimulation. RIPC induced elevated serum norepinephrine, increased expression of brain α1-adrenoceptors, and reduced brain expression of norepinephrine transporter 1. Antagonizing adrenoceptors and norepinephrine transporter 1 prevented RIPC protection of cerebral perfusion during sinusoidal galvanic vestibular stimulation. Taken together, this study indicates that RIPC may be a potential therapy that can prevent VVS pathophysiology, decrease syncopal episodes, and reduce the injuries associated with syncopal falls. Furthermore, the α1-adrenoceptor-protein kinase Cε-endothelial nitric oxide synthase pathway may be a therapeutic target for regulating changes in cerebral blood flow.

OBJECTIVE To identify potential risk factors for digit injuries in dogs training and competing in agility events. DESIGN Internet-based, retrospective, cross-sectional survey. ANIMALS 1,081 dogs training or competing in agility events. PROCEDURES Data were collected for eligible animals via retrospective surveys distributed electronically to handlers of dogs participating in agility-related activities. Variables evaluated included demographic (handlers) and signalment (dogs) information, physical characteristics of dogs, and injury characteristics. A separate survey of dogs competing in similar agility-related activities but without digit injuries was also administered. Multivariable logistic regression was used to develop a model for assessment of risk factors. RESULTS Data were collected from 207 agility dogs with digit injuries and 874 agility dogs without digit injuries. Factors associated with significantly increased odds of injury included Border Collie breed (OR, 2.3; 95% confidence interval [CI], 1.5 to 3.3), long nails (OR, 2.4; 95% CI, 1.3 to 4.5), absence of front dewclaws (OR, 1.9; 95% CI, 1.3 to 2.6), and greater weight-to-height ratio (OR, 1.5; 95% CI, 1.1 to 2.0). Odds of injury decreased with increasing age of the dog (OR, 0.8; 95% CI, 0.76 to 0.86). CONCLUSIONS AND CLINICAL RELEVANCE Results should be cautiously interpreted because of potential respondent and recall bias and lack of review of medical records. Nevertheless, results suggested that retaining healthy dewclaws, maintaining lean body mass, and trimming nails short for training and competition may decrease the likelihood of digit injuries. Research to investigate training practices, obstacle construction specifcations, and surface considerations for dogs competing in agility activities is indicated.

Under acute hypoxic conditions, the muscle oxygen uptake (mV˙O2) during exercise is reduced by the restriction in oxygen-supplied volume to the mitochondria within the peripheral tissue. This suggests the existence of a factor restricting the mV˙O2 under hypoxic conditions at the peripheral tissue level. Therefore, this study set out to test the hypothesis that the restriction in mV˙O2 is regulated by the net decrease in intracellular oxygen tension equilibrated with myoglobin oxygen saturation (∆PmbO2) during muscle contraction under hypoxic conditions. The hindlimb of male Wistar rats (8 weeks old, n = 5) was perfused with hemoglobin-free Krebs-Henseleit buffer equilibrated with three different fractions of O2 gas: 95.0%O2, 71.3%O2, and 47.5%O2 The deoxygenated myoglobin (Mb) kinetics during muscle contraction were measured under each oxygen condition with a near-infrared spectroscopy. The ∆[deoxy-Mb] kinetics were converted to oxygen saturation of myoglobin (SmbO2), and the PmbO2 was then calculated based on the SmbO2 and the O2 dissociation curve of the Mb. The SmbO2 and PmbO2 at rest decreased with the decrease in O2 supply, and the muscle contraction caused a further decrease in SmbO2 and PmbO2 under all O2 conditions. The net increase in mV˙O2 from the muscle contraction (∆mV˙O2) gradually decreased as the ∆PmbO2 decreased during muscle contraction. The results of this study suggest that ΔPmbO2 is a key determinant of the ΔmV˙O2.

Diabetes is associated with a deficit of circulating endothelial progenitor cells (EPCs), which has been attributed to their defective mobilization from the bone marrow. The basis for this mobilization defect is not completely understood, and we sought to determine if hyperglycemic conditions enhanced EPC adhesion. We found that culturing EPCs in high glucose media increased adhesion to bone marrow stromal cells. This enhanced adhesion was associated with decreased expression of protein kinase A regulatory subunit 1β (PRKAR1β), activation of protein kinase A (PKA), and phosphorylation of α4-integrin on serine 988. This potentiated adhesion was reversed by treatment with a PKA inhibitor, overexpression of PRKAR1β, or expression of a phosphorylation-defective α4-integrin variant (α4[S988A]). Using a model of type 1 diabetes, we showed that α4(S988A)-expressing mice have more circulating EPCs than their wild-type counterparts. Moreover, diabetic α4(S988A) mice demonstrate enhanced revascularization after hind limb ischemia. Thus, we have identified a novel signaling mechanism activating PKA in diabetes (downregulation of an inhibitory regulatory subunit) that leads to deficits of circulating EPCs and impaired vascular repair, which could be reversed by α4-integrin mutation.

We propose that the extracellular matrix (ECM) signals CD44, a hyaluronan receptor, to increase the responsiveness to mechanical stimulation in the rat hind paw. We report that intradermal injection of hyaluronidase induces mechanical hyperalgesia, that is inhibited by co-administration of a CD44 receptor antagonist, A5G27. The intradermal injection of low (LMWH) but not high (HMWH) molecular weight hyaluronan also induces mechanical hyperalgesia, an effect that was attenuated by pretreatment with HMWH or A5G27. Pretreatment with HMWH also attenuated the hyperalgesia induced by hyaluronidase. Similarly, intradermal injection of A6, a CD44 receptor agonist, produced hyperalgesia that was inhibited by HMWH and A5G27. Inhibitors of protein kinase A (PKA) and Src, but not protein kinase C (PKC), significantly attenuated the hyperalgesia induced by both A6 and LMWH. Finally, to determine if CD44 receptor signaling is involved in a preclinical model of inflammatory pain, we evaluated the effect of A5G27 and HMWH on the mechanical hyperalgesia associated with the inflammation induced by carrageenan. Both A5G27 and HMWH attenuated carrageenan-induced mechanical hyperalgesia. Thus, while LMWH acts at its cognate receptor, CD44, to induce mechanical hyperalgesia, HMWH acts at the same receptor as an antagonist. That the local administration of HMWH or A5G27 inhibits carrageenan-induced hyperalgesia supports the suggestion that carrageenan produces changes in the ECM that contributes to inflammatory pain. These studies define a clinically relevant role for signaling by the hyaluronan receptor, CD44, in increased responsiveness to mechanical stimulation.

RationaleGenetic variation at the chromosome 9p21 cardiovascular risk locus has been associated with peripheral artery disease, but its mechanism remains unknown.ObjectiveTo determine whether this association is secondary to an increase in atherosclerosis, or it is the result of a separate angiogenesis-related mechanism.Methods and resultsQuantitative evaluation of human vascular samples revealed that carriers of the 9p21 risk allele possess a significantly higher burden of immature intraplaque microvessels than carriers of the ancestral allele, irrespective of lesion size or patient comorbidity. To determine whether aberrant angiogenesis also occurs under nonatherosclerotic conditions, we performed femoral artery ligation surgery in mice lacking the 9p21 candidate gene, Cdkn2b. These animals developed advanced hindlimb ischemia and digital autoamputation, secondary to a defect in the capacity of the Cdkn2b-deficient smooth muscle cell to support the developing neovessel. Microarray studies identified impaired transforming growth factor β (TGFβ) signaling in cultured cyclin-dependent kinase inhibitor 2B (CDKN2B)-deficient cells, as well as TGFβ1 upregulation in the vasculature of 9p21 risk allele carriers. Molecular signaling studies indicated that loss of CDKN2B impairs the expression of the inhibitory factor, SMAD-7, which promotes downstream TGFβ activation. Ultimately, this manifests in the upregulation of a poorly studied effector molecule, TGFβ1-induced-1, which is a TGFβ-rheostat known to have antagonistic effects on the endothelial cell and smooth muscle cell. Dual knockdown studies confirmed the reversibility of the proposed mechanism, in vitro.ConclusionsThese results suggest that loss of CDKN2B may not only promote cardiovascular disease through the development of atherosclerosis but may also impair TGFβ signaling and hypoxic neovessel maturation.

Recent rapid advances in experimental biology have expanded the opportunity for interdisciplinary investigations of the evolution of form and function in non-traditional model species. However, historical divisions of philosophy and methodology between evolutionary/organismal biologists and developmental geneticists often preclude an effective merging of disciplines. In an effort to overcome these divisions, we take advantage of the extraordinary morphological diversity of the rodent superfamily Dipodoidea, including the bipedal jerboas, to experimentally study the developmental mechanisms and biomechanical performance of a remarkably divergent limb structure. Here, we place multiple limb character states in a locomotor and phylogenetic context. Whereas obligate bipedalism arose just once in the ancestor of extant jerboas, we find that digit loss, metatarsal fusion, between-limb proportions, and within-hindlimb proportions all evolved independently of one another. Digit loss occurred three times through at least two distinct developmental mechanisms, and elongation of the hindlimb relative to the forelimb is not simply due to growth mechanisms that change proportions within the hindlimb. Furthermore, we find strong evidence for punctuated evolution of allometric scaling of hindlimb elements during the radiation of Dipodoidea. Our work demonstrates the value of leveraging the evolutionary history of a clade to establish criteria for identifying the developmental genetic mechanisms of morphological diversification.

Clinical spinal cord injury (SCI) is accompanied by comorbid peripheral injury in 47% of patients. Human and animal modeling data have shown that painful peripheral injuries undermine long-term recovery of locomotion through unknown mechanisms. Peripheral nociceptive stimuli induce maladaptive synaptic plasticity in dorsal horn sensory systems through AMPA receptor (AMPAR) phosphorylation and trafficking to synapses. Here we test whether ventral horn motor neurons in rats demonstrate similar experience-dependent maladaptive plasticity below a complete SCI in vivo. Quantitative biochemistry demonstrated that intermittent nociceptive stimulation (INS) rapidly and selectively increases AMPAR subunit GluA1 serine 831 phosphorylation and localization to synapses in the injured spinal cord, while reducing synaptic GluA2. These changes predict motor dysfunction in the absence of cell death signaling, suggesting an opportunity for therapeutic reversal. Automated confocal time-course analysis of lumbar ventral horn motor neurons confirmed a time-dependent increase in synaptic GluA1 with concurrent decrease in synaptic GluA2. Optical fractionation of neuronal plasma membranes revealed GluA2 removal from extrasynaptic sites on motor neurons early after INS followed by removal from synapses 2 h later. As GluA2-lacking AMPARs are canonical calcium-permeable AMPARs (CP-AMPARs), their stimulus- and time-dependent insertion provides a therapeutic target for limiting calcium-dependent dynamic maladaptive plasticity after SCI. Confirming this, a selective CP-AMPAR antagonist protected against INS-induced maladaptive spinal plasticity, restoring adaptive motor responses on a sensorimotor spinal training task. These findings highlight the critical involvement of AMPARs in experience-dependent spinal cord plasticity after injury and provide a pharmacologically targetable synaptic mechanism by which early postinjury experience shapes motor plasticity.

Neural encoding of the passage of time to produce temporally precise movements remains an open question. Neurons in several brain regions across different experimental contexts encode estimates of temporal intervals by scaling their activity in proportion to the interval duration. In motor cortex the degree to which this scaled activity relies upon afferent feedback and is guided by motor output remains unclear. Using a neural reward paradigm to dissociate neural activity from motor output before and after complete spinal transection, we show that temporally scaled activity occurs in the rat hindlimb motor cortex in the absence of motor output and after transection. Context-dependent changes in the encoding are plastic, reversible, and re-established following injury. Therefore, in the absence of motor output and despite a loss of afferent feedback, thought necessary for timed movements, the rat motor cortex displays scaled activity during a broad range of temporally demanding tasks similar to that identified in other brain regions.

The mechanisms of delayed damage and recovery after intracerebral hemorrhage (ICH) remain poorly defined. Two rodent models of ICH are commonly used: injection of the enzyme collagenase (cICH) and injection of autologous blood (bICH). In mice, we compared the effects of these two models on initial and delayed tissue damage, motor system connections, and behavioral recovery. There is no difference in lesion size between models. Injection of autologous blood causes greater mass effect and early mortality. However, cICH produces greater edema, inflammation, and cell death. Injection of the enzyme collagenase causes greater loss of cortical connections and secondary shrinkage of the striatum. Intracerebral hemorrhage occurs within the motor system connections of the striatum. Mapping of the projections of the forelimb motor area shows a significant sprouting in motor cortex projections only in cICH. Both models of ICH produce deficits in forelimb motor control. Behavioral recovery occurs by 5 weeks in cICH and 9 weeks in bICH. In summary, cICH and bICH differ in almost every facet of initial and delayed stroke pathophysiology, with cICH producing greater initial and secondary tissue damage and greater motor system axonal sprouting than bICH. Motor recovery occurs in both models, suggesting that motor system axonal sprouting in cICH is not causally associated with recovery.Journal of Cerebral Blood Flow & Metabolism advance online publication, 11 June 2014; doi:10.1038/jcbfm.2014.107.

Motor sequences are formed through the serial execution of different movements, but how nervous systems implement this process remains largely unknown. We determined the organizational principles governing how dirty fruit flies groom their bodies with sequential movements. Using genetically targeted activation of neural subsets, we drove distinct motor programs that clean individual body parts. This enabled competition experiments revealing that the motor programs are organized into a suppression hierarchy; motor programs that occur first suppress those that occur later. Cleaning one body part reduces the sensory drive to its motor program, which relieves suppression of the next movement, allowing the grooming sequence to progress down the hierarchy. A model featuring independently evoked cleaning movements activated in parallel, but selected serially through hierarchical suppression, was successful in reproducing the grooming sequence. This provides the first example of an innate motor sequence implemented by the prevailing model for generating human action sequences.

To understand the principles of taste coding, it is necessary to understand the functional organization of the taste organs. Although the labellum of the Drosophila melanogaster head has been described in detail, the tarsal segments of the legs, which collectively contain more taste sensilla than the labellum, have received much less attention. We performed a systematic anatomical, physiological, and molecular analysis of the tarsal sensilla of Drosophila. We construct an anatomical map of all five tarsal segments of each female leg. The taste sensilla of the female foreleg are systematically tested with a panel of 40 diverse compounds, yielding a response matrix of ∼500 sensillum-tastant combinations. Six types of sensilla are characterized. One type was tuned remarkably broadly: it responded to 19 of 27 bitter compounds tested, as well as sugars; another type responded to neither. The midleg is similar but distinct from the foreleg. The response specificities of the tarsal sensilla differ from those of the labellum, as do n-dimensional taste spaces constructed for each organ, enhancing the capacity of the fly to encode and respond to gustatory information. We examined the expression patterns of all 68 gustatory receptors (Grs). A total of 28 Gr-GAL4 drivers are expressed in the legs. We constructed a receptor-to-sensillum map of the legs and a receptor-to-neuron map. Fourteen Gr-GAL4 drivers are expressed uniquely in the bitter-sensing neuron of the sensillum that is tuned exceptionally broadly. Integration of the molecular and physiological maps provides insight into the underlying basis of taste coding.

BackgroundPlatelet-derived growth factor C (PDGF-C) has been reported to promote angiogenesis independently of vascular endothelial growth factor (VEGF), although its significance in postnatal angiogenesis in vivo remains poorly understood. VEGF has been employed as a major molecular tool to induce therapeutic angiogenesis. However, VEGF therapy is not very effective in models of cardiovascular diseases associated with diabetes, and the mechanisms of this phenomenon still remain to be elucidated.MethodsWe used a murine model of hind limb ischemia and of streptozotocin-induced diabetes.ResultsExpression of PDGF-C and its receptor PDGFR-α were markedly upregulated in ischemic limbs. Treatment with a neutralizing antibody against PDGF-C significantly impaired blood flow recovery and neovascularization after ischemia almost to the same extent as a VEGF-neutralizing antibody. Mice deficient in PDGF-C exhibited reduced blood flow recovery after ischemia compared with wild-type mice, confirming a strong proangiogenic activity of PDGF-C. Next, we injected an expression vector encoding PDGF-C into ischemic limbs. Blood flow recovery and neovascularization after ischemia were significantly improved in the groups treated with PDGF-C compared with controls. Attenuation of angiogenic responses to ischemia has been reported in patients with diabetes even after VEGF treatment, although a precise mechanism remains unknown. We hypothesized that PDGF-C might relate to the impaired angiogenesis of diabetes. We tested this hypothesis by inducing diabetes by intraperitoneal injection of streptozotocin. Expression levels of PDGF-C at baseline and after ischemia were significantly lower in limb tissues of diabetic mice than in those of control mice, whereas expression levels of other members of the PDGF family and VEGF were not changed or were even higher in diabetic mice. Introduction of VEGF complementary DNA expression plasmid vector into ischemic limbs did not improve blood flow recovery. However, these changes were effectively reversed by additional introduction of the PDGF-C complementary DNA plasmid vector.ConclusionsThese results indicate that downregulation of PDGF-C expression in limb tissues of diabetic mice contributes to impaired angiogenesis and suggest that introduction of PDGF-C might be a novel strategy for therapeutic angiogenesis, especially in the diabetic state.Clinical relevanceAngiogenesis and arteriogenesis after ischemia are attenuated in most diabetic patients, although the precise mechanisms remain unclear. Platelet-derived growth factors (PDGFs) have a variety of functions on many cell types, and PDGF-C stimulates angiogenesis and revascularizes ischemic tissues. This study indicates the role for PDGF-C as a critical regulator of impaired angiogenesis of diabetes and suggests that PDGF-C might be a novel target for the treatment of ischemic cardiovascular diseases in diabetes.

Endothelin-1 (ET-1) is unique among a broad range of hyperalgesic agents in that it induces hyperalgesia in rats that is markedly enhanced by repeated mechanical stimulation at the site of administration. Antagonists to the ET-1 receptors, ET(A) and ET(B), attenuated both initial as well as stimulation-induced enhancement of hyperalgesia (SIEH) by endothelin. However, administering antisense oligodeoxynucleotide to attenuate ET(A) receptor expression on nociceptors attenuated ET-1 hyperalgesia but had no effect on SIEH, suggesting that this is mediated via a non-neuronal cell. Because vascular endothelial cells are both stretch sensitive and express ET(A) and ET(B) receptors, we tested the hypothesis that SIEH is dependent on endothelial cells by impairing vascular endothelial function with octoxynol-9 administration; this procedure eliminated SIEH without attenuating ET-1 hyperalgesia. A role for protein kinase Cε (PKCε), a second messenger implicated in the induction and maintenance of chronic pain, was explored. Intrathecal antisense for PKCε did not inhibit either ET-1 hyperalgesia or SIEH, suggesting no role for neuronal PKCε; however, administration of a PKCε inhibitor at the site of testing selectively attenuated SIEH. Compatible with endothelial cells releasing ATP in response to mechanical stimulation, P2X(2/3) receptor antagonists eliminated SIEH. The endothelium also appears to contribute to hyperalgesia in two ergonomic pain models (eccentric exercise and hindlimb vibration) and in a model of endometriosis. We propose that SIEH is produced by an effect of ET-1 on vascular endothelial cells, sensitizing its release of ATP in response to mechanical stimulation; ATP in turn acts at the nociceptor P2X(2/3) receptor.

Stereotypical locomotor movements can be made without input from the brain after a complete spinal transection. However, the restoration of functional gait requires descending modulation of spinal circuits to independently control the movement of each limb. To evaluate whether a brain-machine interface (BMI) could be used to regain conscious control over the hindlimb, rats were trained to press a pedal and the encoding of hindlimb movement was assessed using a BMI paradigm. Off-line, information encoded by neurons in the hindlimb sensorimotor cortex was assessed. Next neural population functions, or weighted representations of the neuronal activity, were used to replace the hindlimb movement as a trigger for reward in real-time (on-line decoding) in three conditions: while the animal could still press the pedal, after the pedal was removed and after a complete spinal transection. A novel representation of the motor program was learned when the animals used neural control to achieve water reward (e.g. more information was conveyed faster). After complete spinal transection, the ability of these neurons to convey information was reduced by more than 40%. However, this BMI representation was relearned over time despite a persistent reduction in the neuronal firing rate during the task. Therefore, neural control is a general feature of the motor cortex, not restricted to forelimb movements, and can be regained after spinal injury.

Natural products that elicit discomfort or pain represent invaluable tools for probing molecular mechanisms underlying pain sensation. Plant-derived irritants have predominated in this regard, but animal venoms have also evolved to avert predators by targeting neurons and receptors whose activation produces noxious sensations. As such, venoms provide a rich and varied source of small molecule and protein pharmacophores that can be exploited to characterize and manipulate key components of the pain-signalling pathway. With this in mind, here we perform an unbiased in vitro screen to identify snake venoms capable of activating somatosensory neurons. Venom from the Texas coral snake (Micrurus tener tener), whose bite produces intense and unremitting pain, excites a large cohort of sensory neurons. The purified active species (MitTx) consists of a heteromeric complex between Kunitz- and phospholipase-A2-like proteins that together function as a potent, persistent and selective agonist for acid-sensing ion channels (ASICs), showing equal or greater efficacy compared with acidic pH. MitTx is highly selective for the ASIC1 subtype at neutral pH; under more acidic conditions (pH < 6.5), MitTx massively potentiates (>100-fold) proton-evoked activation of ASIC2a channels. These observations raise the possibility that ASIC channels function as coincidence detectors for extracellular protons and other, as yet unidentified, endogenous factors. Purified MitTx elicits robust pain-related behaviour in mice by activation of ASIC1 channels on capsaicin-sensitive nerve fibres. These findings reveal a mechanism whereby snake venoms produce pain, and highlight an unexpected contribution of ASIC1 channels to nociception.

PurposeThe aim was to investigate that a bio-degradable alginate and poly lactide-co-glycolide (PLG) system capable of delivering growth factors sequentially would be superior to single growth factor delivery in promoting neovascularization and improving perfusion.MethodsThree groups of apoE null mice underwent unilateral hindlimb ischemia surgery and received ischemic limb intramuscular injections of alginate (Blank), alginate containing VEGF(165) (VEGF), or alginate containing VEGF(165) combined with PLG microspheres containing PDGF-BB (VEGF/PDGF). Vascularity in the ischemic hindlimb was assessed by morphologic and immunohistochemical end-points, while changes in blood flow were assessed by Laser Doppler Perfusion Index. Muscle VEGF and PDGF content was assessed at multiple time points.ResultsIn the VEGF/PDGF group, local tissue VEGF and PDGF levels peaked at week 2 and 4, respectively, with detectable PDGF levels at week 6. At week 6, mean vessel mean diameter was significantly greater in the VEGF/PDGF group compared to the VEGF or Blank groups with evidence of well-formed smooth muscle-lined arterioles.ConclusionsSequential delivery of VEGF and PDGF using an injectable, biodegradable platform resulted in stable and sustained improvements in perfusion. This sustained, control-released, injectable alginate polymer system is a promising approach for multiple growth factor delivery in clinical application.

In this study, we hypothesized that non-invasive continuous wave near-infrared spectroscopy (CWNIRS) can determine the severity or reversibility of muscle damage due to ischemia/reperfusion (I/R), and the results will be highly correlated with those from physical examination and histological analysis. To test this hypothesis, we performed CWNIRS measurements on two groups of male Sprague-Dawley rats ( approximately 400 g) that underwent 2 h (n = 6) or 3 h (n = 7) of pneumatic tourniquet application (TKA). Tissue oxyhemoglobin [HbO(2)] and deoxyhemoglobin [Hb] concentration changes were monitored during the 2 h or 3 h of 250 mmHg TKA and for an additional 2 h post-TKA. Rats were euthanized 24 h post-TKA and examined for injury, edema and viability of muscles. Contralateral muscles served as controls for each animal. In both groups, [HbO(2)] dropped immediately, then gradually decreased further after TKA and then recovered once the tourniquet was released. However, releasing after 2 h of TKA caused [HbO(2)] to overshoot above the baseline during reperfusion while the 3 h group continued to have lower [HbO(2)] than baseline. We found a significant correlation between the elapsed time from tourniquet release to the first recovery peak of [HbO(2)] and the muscle weight ratio between tourniquet and contralateral limb muscles (R = 0.86). Hemodynamic patterns from non-invasive CWNIRS demonstrated significant differences between 2 h and 3 h I/R. The results demonstrate that CWNIRS may be useful as a non-invasive prognostic tool for conditions involving vascular compromise such as extremity compartment syndrome.