Comparative morphology and scaling of the femur in yangochiropteran bats.

Better known by their remarkable forelimb morphology, bats are also unique among mammals with respect to their hindlimbs. Their legs are rotated through 180°, generally reduced in size, and in some extant taxa particular bones (e.g. fibula) can even be absent. The femur is the main leg bone, but to date few bat studies have considered its morphology in detail, none in a wide-scale comparative study. Yangochiroptera is the largest bat taxon, spans nearly three orders of magnitude in body mass, and is highly diverse both in ecology and behavior, representing a good model for comparative analyses. Here, we describe the anatomy of the femur in a large sample of yangochiropteran bats (125 species, 70 genera, and 12 families), and explore major trends of morphological variation and scaling patterns in this bone. We used 13 categorical characters in the anatomical description and five linear dimensions in the quantitative analyses. Based on the categorical data, each family studied here was diagnosed, and those from the Neotropical region were included in an identification key. From the phylogenetic principal component analysis (pPCA) we showed that, in addition to size, major axes of variation in bat femur are related to robusticity and head morphology, features that are clearly distinct among some families. We also generated a phylomorphospace based on pPCA scores, highlighting convergences in femur shape. Molossidae, Mystacinidae, and Desmodontinae were grouped based on their greater robusticity, a pattern that was also recovered from categorical data. In these families, we found anatomical features (e.g. presence of tubercles and posterior ridges on the greater trochanter, long or medially/distally displaced lateral ridges on the shaft) that are well-known from their functional link with quadrupedal locomotion. Using phylogenetic regressions, we found out that compared with body mass, femur length scaled with negative allometry, as expected, but that femur width scaled isometrically, counter to expectations. As a result, robusticity index (the ratio of width to length), scaled with positive allometry - larger bats tended to have more robust hindlimbs. At species level, our most remarkable finding was related to Myotis simus, which presented the most robust femur (for its size) among yangochiropterans. Our results reinforce the informative potential of the chiropteran femur from both taxonomic and functional perspectives. Furthermore, the allometric trends seen in this bone may help understand the strategies adopted by flying vertebrates to deal with the high energetic cost of flight and, at the same time, evolve diversified foraging behaviors.

Endochondral ossification in hindlimbs during bufo gargarizans metamorphosis: A model of studying skeletal development in vertebrates.

Background Endochondral ossification, the process by which most of the skeleton is formed, is accurately regulated by many specific groups of molecules and extracellular matrix components. The molecular mechanisms of endochondral ossification have been extensively investigated in mammals. However, there are few studies about it in amphibians. Results Ossification of femur and tibiofibula was observed at Gs 40, and tarsals and metatarsals were ossified at Gs 42. Most of the skeletons in hindlimbs were completely ossified except for articular cartilages at Gs 46. Thirty-two genes related to endochondral ossification were found in the differentially expressed genes (DEGs) library of hindlimbs, and nine of these genes were validated by qRT-PCR during metamorphosis. Sox9 was expressed in the columnar, prehypertrophic, and upper hypertrophic zones, and the expression of Ihh was observed in prehypertrophic chondrocyte zone in hindlimbs of B. gargarizans. Conclusions The ossification of hindlimbs increased gradually, and the ossification sequence was from proximal to distal in B. gargarizans during metamorphosis. Thirty-two genes found in the DEGs library were related to the regulation of endochondral ossification of hindlimbs in amphibians. The present study will provide a valuable genomic resource for the future study of endochondral ossification in amphibian. Developmental Dynamics 247:1121-1134, 2018. © 2018 Wiley Periodicals, Inc.

Whole-body 3D kinematics of bird take-off: key role of the legs to propel the trunk.

Previous studies showed that birds primarily use their hindlimbs to propel themselves into the air in order to take-off. Yet, it remains unclear how the different parts of their musculoskeletal system move to produce the necessary acceleration. To quantify the relative motions of the bones during the terrestrial phase of take-off, we used biplanar fluoroscopy in two species of birds, diamond dove (Geopelia cuneata) and zebra finch (Taeniopygia guttata). We obtained a detailed 3D kinematics analysis of the head, the trunk and the three long bones of the left leg. We found that the entire body assisted the production of the needed forces to take-off, during two distinct but complementary phases. The first one, a relatively slow preparatory phase, started with a movement of the head and an alignment of the different groups of bones with the future take-off direction. It was associated with a pitch down of the trunk and a flexion of the ankle, of the hip and, to a lesser extent, of the knee. This crouching movement could contribute to the loading of the leg muscles and store elastic energy that could be released in the propulsive phase of take-off, during the extension of the leg joints. Combined with the fact that the head, together with the trunk, produced a forward momentum, the entire body assisted the production of the needed forces to take-off. The second phase was faster with mostly horizontal forward and vertical upward translation motions, synchronous to an extension of the entire lower articulated musculoskeletal system. It led to the propulsion of the bird in the air with a fundamental role of the hip and ankle joints to move the trunk upward and forward. Take-off kinematics were similar in both studied species, with a more pronounced crouching movement in diamond dove, which can be related to a large body mass compared to zebra finch.

Alteration of pentylenetetrazole-induced seizure threshold by chronic administration of ginger (Zingiber officinale) extract in male mice.

CONTEXT: Zingiber officinale Roscoe (Zingiberaceae), or ginger, used in traditional Chinese medicine, has antioxidant activity and neuroprotective effects. The effects of this plant on clonic seizure have not yet been studied. OBJECTIVE: The present study evaluated the anticonvulsant effect of ginger in a model of clonic seizures induced with pentylenetetrazole (PTZ) in male mice. MATERIALS AND METHODS: The anticonvulsant effect of Z. officinale was investigated using i.v. PTZ-induced seizure models in mice. Different doses of the hydroethanolic extract of Z. officinale (25, 50, and 100 mg/kg) were administered intraperitonal (i.p.), daily for 1 week before induction of PTZ. Phenobarbital sodium (30 mg/kg), a reference standard, was also tested for comparison. The effect of ginger on to the appearance of three separate seizure endpoints, e.g., myoclonic, generalized clonic, and tonic extension phase, was recorded. RESULTS: Hydroethanolic extract of Z. officinale significantly increased the onset time of myoclonic seizure at doses of 25-100 mg/kg (55.33 ± 1.91 versus 24.47 ± 1.33 mg/kg, p < 0.001) and significantly prevented generalized clonic (74.64 ± 3.52 versus 47.72 ± 2.31 mg/kg, p < 0.001) and increased the threshold for the forelimb tonic extension (102.6 ± 5.39 versus 71.82 ± 7.82 mg/kg, p < 0.01) seizure induced by PTZ compared with the control group. DISCUSSION AND CONCLUSION: Based on the results, the hydroethanolic extract of ginger has anticonvulsant effects, possibly through an interaction with inhibitory and excitatory systems, antioxidant mechanisms, and oxidative stress inhibition.

Beyond head and wings: Unveiling influence of diet, body size, and phylogeny on the evolution of the femur in phyllostomid bats.

Phyllostomidae, the most diverse family of Neotropical bats, encompass 230 species with varied dietary habits and food acquisition methods. Their feeding niche diversification has shaped skull and wing morphologies through natural selection, reflecting food processing and flight strategies. Yet, evolution of bat hindlimbs, especially in phyllostomids, remains little understood. Previous studies highlighted the femur's morphology as a key to understanding the evolution of quadrupedalism in yangochiropteran bats, including the adept walking observed in vampire bats (Desmodontinae). Here, we aimed to describe the femoral morphological variation in Phyllostomidae, correlating this with body size and assessing the effects of phylogenetic history, dietary habits, and hindlimb usage. Analyzing 15 femoral traits from 45 species across 9 subfamilies through phylogenetically informed methods, we discovered a significant phylogenetic structure in femoral morphology. Allometric analysis indicated that body mass accounts for about 85% of the variance in phyllostomid femoral size and about 11% in femoral shape. Relatively smaller femurs showed to be typical in Stenodermatinae, Lonchophyllinae, and Glossophaginae, in contrast to the larger femurs of Phyllostominae, Desmodontinae, Micronycterinae, and Lonchorrhininae. Furthermore, extensive femur shape variation was detected, with the most distinct morphologies in vampire bats, followed by frugivorous species. Adaptive evolutionary models related to diet more effectively explained variations in femoral relative size and shape than stochastic models. Contrary to the conventional belief of limited functional demand on bat femurs, our findings suggest that femoral morphology is significantly influenced by functional demands associated with diet and food capture, in addition to being partially structured by body size and shared evolutionary history.

Suppression of endoplasmic reticulum stress reverses hindlimb unloading-induced hepatic cellular processes in mice.

BACKGROUND: The Hindlimb unloaded mouse, an animal model of simulated microgravity demonstrates significant metabolic and hepatic derangements. However, cellular and molecular mechanisms driving liver dysfunction in Hindlimb unloaded mice are poorly characterized. METHODS: We investigated the possible contribution of dysregulated protein homeostasis by endoplasmic reticulum, endoplasmic reticulum stress, to liver dysfunction during HU. C57BL/6j male mice were grouped into ground-based controls or Hindlimb unloaded groups treated daily with vehicle or 4-phenylbutyrate (4-PBA), a potent inhibitor of endoplasmic reticulum stress. Following three weeks of HU, mice were sacrificed, and liver tissues were dissected for further analysis. RESULTS: Hindlimb unloaded was associated with hepatic atrophy and elevated endoplasmic reticulum stress, which was restored by 4-PBA treatment. The Gene Ontology analysis revealed the downregulation of genes primarily involved in liver metabolic and Wingless-related integration site (WNT) signaling pathways, while those related to cytochrome P450, and liver fibrosis were upregulated. The Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed downregulation of several genes involved in metabolic pathways following treatment with 4-PBA, induced by HU. CONCLUSIONS: We report several differential and uniquely expressed genes associated with microgravity-induced elevated ER stress and liver injury. Our data has translational potential in unraveling novel molecular targets for pharmaceutical therapies of liver diseases. GENERAL SIGNIFICANCE: Our novel findings show a pathogenic role for elevated ER stress in liver injury in microgravity conditions.

Ontogeny of the meniscus in the anuran Xenopus laevis.

The anuran knee joint is subjected to the jump, one of the tetrapods' most demanding mechanical stresses. Consistent with this continuous effort, the knee of the anurans has a complex structure comparable to that of an amniote. Here, we describe the ontogeny of the Xenopus knee tissues and study the morphogenesis of the knee joint shape by performing a geometric morphometric analysis of specially selected anatomical structures: the menisci and the long bone epiphyses. A meniscus is a crescent-shaped fibrocartilaginous structure, with a triangular cross-section inserted between joints surfaces. A meniscus transmits load across the tibiofemoral joint by increasing congruity of the long bone epiphysis and decreasing the resulting stress exerted on the articular cartilage. We ask two questions: (1) what is the tissue composition along the ontogeny of the menisci of a swimming frog? (2) How do the menisci acquire the shape that will allow their adjustment? We studied the structures and tissue ontogeny of the knee of several specimens of Xenopus laevis and evaluated the congruity of the knee structures across the species ontogeny. Histological sections showed that the cavitation process responsible for separating the menisci and the epiphyses seems to be pivotal in shaping the conformity of these structures and the long bone epiphyses of the hindlimbs. The geometric morphometric analysis allowed us to interpret three phases of differentiation associated with limb functionality. The characteristic shape of the meniscus appears early in the ontogeny of the knee, simultaneously with the epiphysis contours.

FGF2 and FGF10 expression patterns in the epidermis and mesenchyme during homeotic transformation of tail into hindlimbs in frog tadpoles.

Limbs are trunk quintessential in tetrapods. Their development relies on the Retinoic acid (RA) gradient in association with the Fibroblast Growth Factors (FGFs). The role of various FGFs have been probed extensively and confirmed during the induction of ectopic limbs in vertebrates. On such factual backdrops, we studied the expression patterns of FGF2 and FGF10 in the epidermis and mesenchyme by immunohistochemical localization in the regenerating tails of tadpoles of the Indian tree frog, Polypedates maculatus. These tadpoles are known to exhibit a kind of homeotic transformation of tail to limbs during regeneration, whose exact mechanism is still to be established by scientific investigations. Here in this study, we provide the first evidence of the putative involvement of FGF2 and FGF10 during such ectopic appendage development.

Cooperation behavior of fore- And hindlimbs during jumping in Rana dybowskii and Xenopus laevis.

Frogs are characterized by their outstanding jumping ability, depending on the rapid extension of hindlimbs to propel their bodies into air. A typical jumping cycle could be broken into four phases: preparation, takeoff, flight, and landing. Considerable research has been performed to discuss the function of hindlimbs of frogs during takeoff phase, whereas the literature of limbs' motion in jumping between different species was limited. To profile the evolution of locomotion in anurans, it is necessary to investigate on the motion of fore- and hindlimbs of frogs within different taxa. In this work, we put forward a detailed description of jumping behavior of two frog species, Rana dybowskii and Xenopus laevis. High-speed cameras were used to explore the movement of different joints in fore- and hindlimbs of these two animals, and kinematic analysis was operated to identify both homologous behaviors and significant differences between them. We found that the Rana dybowskii's fore- and hindlimbs had good cooperation during jumping, while the Xenopus laevis' uncooperative behavior in limbs may give a functional explanation for the deficiency in terrestrial jumping; besides, the R. dybowskii's landing followed the "hands-belly-feet slap" strategy, and Xenopus laevis had clumsy landing with "belly-flops" sequence. The result gained here clarifies the cooperation behavior of anuran limbs and may supply a new insight into our understanding of the anuran's evolution.

Patterns of correlations and locomotor specialization in anuran limbs: association with phylogeny and ecology.

As anuran saltatory locomotion has specific functional requirements achieved through certain intra- and inter-limb proportions, we analyzed pattern and degree of morphological integration in limbs of ten anuran species to reveal the relationship of shared developmental programs of serially homologous structures and locomotor specialization. Our main objectives were (1) to examine if morphological and functional differences in forelimb and hindlimb were associated with reduced covariation between limbs, (2) and to reveal patterns of correlation between species and the roles played by evolutionary history (phylogeny) and ecology (lifestyle and habitat use). Species with different locomotor behaviours (walking, jumping, hopping, running, climbing, swimming and burrowing) were used. Partial correlations showed that species shared similar patterns of functionally based morphological integration, with increased correlations in elements within limbs and reduced correlations between limbs. This was mainly based on strong correlations between proximal elements, humerus-radioulna and femur-tibiofibula. To test the influence of phylogenetic relationships and ecological demands we used different matrices (correlation similarity matrix, ecological similarity matrix, matrices of phylogenetic distance and morphological distance). The changes in correlation patterns are shown to be dissociated from phylogeny. On the other hand, they are to some extent shaped by habitat use and locomotion, as the species with similar locomotor behaviour also tend to have stronger similarity in integration patterns. The results from this study provide insight into the processes underlying the evolutionary change of anuran limbs, highlighting function as the main factor that shaped morphological integration of the examined species.

Dye-Sensitized Rare Earth-Doped Nanoparticles with Boosted NIR-IIb Emission for Dynamic Imaging of Vascular Network-Related Disorders.

Real-time dynamic vascular network imaging can provide accurate hemodynamic and anatomical information, facilitating the diagnosis of blood circulatory system-related diseases and achieving precise evaluation of therapeutic effects. In vivo luminescence imaging in the NIR-IIb biological window (1500-1700 nm) has developed into a next generation of optical imaging method with significantly improved temporal-spatial resolution and penetration depth. Unfortunately, an imaging contrast agent capable of emitting NIR-IIb luminescence with sufficient brightness in this region is lacking. Herein, we designed and proposed a type of dye-sensitized rare earth-doped nanoparticle (RENPs@Alk-pi) with obviously boosted NIR-IIb emission and high biocompatibility, which can be used to realize the real-time NIR-IIb luminescence imaging with high temporal-spatial resolution and contrast. The dye sensitization process provides a 40-fold enhanced brightness of the NIR-IIb emission at 1525 nm of Er3+. Consequently, the RENPs@Alk-pi was not only able to depict a vascular network but also applicable in noninvasively monitoring the dynamic vascular processes and changes in the vascular anatomy of two blood circulatory system-related disorders, including hindlimbs ischemia and atherosclerosis. Our research provides a powerful tool for evaluating vascular network-related dysfunction and physiological processes.

Ipsilesional versus contralesional postural deficits induced by unilateral brain trauma: a side reversal by opioid mechanism.

Unilateral traumatic brain injury and stroke result in asymmetric postural and motor deficits including contralateral hemiplegia and hemiparesis. In animals, a localized unilateral brain injury recapitulates the human upper motor neuron syndrome in the formation of hindlimb postural asymmetry with contralesional limb flexion and the asymmetry of hindlimb nociceptive withdrawal reflexes. The current view is that these effects are developed due to aberrant activity of motor pathways that descend from the brain into the spinal cord. These pathways and their target spinal circuits may be regulated by local neurohormonal systems that may also mediate effects of brain injury. Here, we evaluate if a unilateral traumatic brain injury induces hindlimb postural asymmetry, a model of postural deficits, and if this asymmetry is spinally encoded and mediated by the endogenous opioid system in rats. A unilateral right-sided controlled cortical impact, a model of clinical focal traumatic brain injury was centred over the sensorimotor cortex and was observed to induce hindlimb postural asymmetry with contralateral limb flexion. The asymmetry persisted after complete spinal cord transection, implicating local neurocircuitry in the development of the deficits. Administration of the general opioid antagonist naloxone and µ-antagonist ß-funaltrexamine blocked the formation of postural asymmetry. Surprisingly, κ-antagonists nor-binaltorphimine and LY2444296 did not affect the asymmetry magnitude but reversed the flexion side; instead of contralesional (left) hindlimb flexion the ipsilesional (right) limb was flexed. The postural effects of the right-side cortical injury were mimicked in animals with intact brain via intrathecal administration of the opioid κ-agonist (2)-(trans)-3,4-Dichloro-N-methyl-N-[2-(1-pyrrolidiny)-cyclohexyl]benzeneacetamide that induced hindlimb postural asymmetry with left limb flexion. The δ-antagonist naltrindole produced no effect on the contralesional (left) flexion but inhibited the formation of the ipsilesional (right) limb flexion in brain-injured rats that were treated with κ-antagonist. The effects of the antagonists were evident before and after spinal cord transection. We concluded that the focal traumatic brain injury-induced postural asymmetry was encoded at the spinal level, and was blocked or its side was reversed by administration of opioid antagonists. The findings suggest that the balance in activity of the mirror symmetric spinal neural circuits regulating contraction of the left and right hindlimb muscles is controlled by different subtypes of opioid receptors; and that this equilibrium is impaired after unilateral brain trauma through side-specific opioid mechanism.

Morphometric ratio analyses: Locomotor mode in anurans.

Current studies of anuran morphology use sacral vertebrae, hindlimbs as main predictor for locomotor modes, while forelimbs and their segments were not analysed in this context. Due the fact that the complex interplay of numerous morphological traits determines the locomotion ability of anurans, we used ratios for both fore- and hindlimbs to obtain useful information of the structural and functional properties of locomotor system. We examined patterns of variation in total length of fore-, and hindlimbs as well as sacral vertebrae width and diapophyseal expansion in order to position two locomotor modes (jumpers and walkers) in a morphological shape space. Further, we investigated patterns of variability in limbs proximal, medial and distal elements to highlight differences in intra- and inter-limb relations in two locomotor modes. Our results showed that the ratios between total hindlimb length and sacral width/diapophyseal expansion, as well as ratio between humerus and tibiofibula relate the most to locomotor modes. This implies that functional specialization in different locomotor modes is attained through modifications of ratios, not only within hindlimbs, but also through modifications of the whole integrated system, which consists of forelimb, hindlimb, and girdle elements.

Anatomical mechanism of spontaneous recovery in regions caudal to thoracic spinal cord injury lesions in rats.

BACKGROUND: The nerve fibre circuits around a lesion play a major role in the spontaneous recovery process after spinal cord hemisection in rats. The aim of the present study was to answer the following question: in the re-control process, do all spinal cord nerves below the lesion site participate, or do the spinal cord nerves of only one vertebral segment have a role in repair? METHODS: First we made a T7 spinal cord hemisection in 50 rats. Eight weeks later, they were divided into three groups based on distinct second operations at T7: ipsilateral hemisection operation, contralateral hemisection, or transection. We then tested recovery of hindlimbs for another eight weeks. The first step was to confirm the lesion had role or not in the spontaneous recovery process. Secondly, we performed T7 spinal cord hemisections in 125 rats. Eight weeks later, we performed a second single hemisection on the ipsilateral side at T8-T12 and then tested hindlimb recovery for another six weeks. RESULTS: In the first part, the Basso, Beattie, Bresnahan (BBB) scores and the electrophysiology tests of both hindlimbs weren't significantly different after the second hemisection of the ipsilateral side. In the second part, the closer the second hemisection was to T12, the more substantial the resulting impairment in BBB score tests and prolonged latency periods. CONCLUSIONS: The nerve regeneration from the lesion area after hemisection has no effect on spontaneous recovery of the spinal cord. Repair is carried out by all vertebrae caudal and ipsilateral to the lesion, with T12 being most important.

Therapy induces widespread reorganization of motor cortex after complete spinal transection that supports motor recovery.

Reorganization of the somatosensory system and its relationship to functional recovery after spinal cord injury (SCI) has been well studied. However, little is known about the impact of SCI on organization of the motor system. Recent studies suggest that step-training paradigms in combination with spinal stimulation, either electrically or through pharmacology, are more effective than step training alone at inducing recovery and that reorganization of descending corticospinal circuits is necessary. However, simpler, passive exercise combined with pharmacotherapy has also shown functional improvement after SCI and reorganization of, at least, the sensory cortex. In this study we assessed the effect of passive exercise and serotonergic (5-HT) pharmacological therapies on behavioral recovery and organization of the motor cortex. We compared the effects of passive hindlimb bike exercise to bike exercise combined with daily injections of 5-HT agonists in a rat model of complete mid-thoracic transection. 5-HT pharmacotherapy combined with bike exercise allowed the animals to achieve unassisted weight support in the open field. This combination of therapies also produced extensive expansion of the axial trunk motor cortex into the deafferented hindlimb motor cortex and, surprisingly, reorganization within the caudal and even the rostral forelimb motor cortex areas. The extent of the axial trunk expansion was correlated to improvement in behavioral recovery of hindlimbs during open field locomotion, including weight support. From a translational perspective, these data suggest a rationale for developing and optimizing cost-effective, non-invasive, pharmacological and passive exercise regimes to promote plasticity that supports restoration of movement after spinal cord injury.

Gene expression of Hsps in normal and abnormal embryonic development of mouse hindlimbs.

Heat shock proteins (Hsps), which have important biological functions, are a class of highly conserved genetic molecules with the capacity of protecting and promoting cells to repair themselves from damage caused by various stimuli. Our previous studies found that Hsp25, HspB2, HspB3, HspB7, Hsp20, HspB9, HspB10, and Hsp40 may be related to all-trans retinoic acid (atRA)-induced phocomelic and other abnormalities, while HspA12B, HspA14, Trap1, and Hsp105 may be forelimb development-related genes; Grp78 may play an important role in forelimb development. In this study, the embryonic phocomelic, oligodactylic model of both forelimbs and hindlimbs was developed by atRA administered per os to the pregnant mice on gestational day 11, and the expression of 36 members of Hsps family in normal and abnormal development of embryonic hindlimbs was measured by real-time fluorescent quantitative polymerase chain reaction (qRT-PCR). It is found that HspA1L, Hsp22, Hsp10, Hsp60, Hsp47, HspB2, HspB10, HspA12A, Apg1, HspB4, Grp78, and HspB9 probably performs a major function in limb development, and HspA13, Grp94 and Hsp110 may be hindlimb development-related genes.