It has been suggested recently that masticatory muscle size reduction in humans resulted in greater encephalization through decreased compressive forces on the cranial vault. Following this logic, if masticatory muscle size were increased, then a reduction in brain growth should also occur. The present study was designed to test this hypothesis using a myostatin (GDF-8) knockout mouse model. Myostatin is a negative regulator of skeletal muscle growth, and individuals lacking this gene show significant hypermuscularity. Sixty-two (32 wild-type and 30 GDF-8 −/− knockout), 1, 28, 56 and 180 day old CD-1 mice were used. Body and masseter muscle weights were collected following dissection and standardized lateral and dorsoventral cephalographs were obtained. Cephalometric landmarks were identified on the radiographs and cranial volume was calculated. Mean differences were assessed using a two-way ANOVA. KO mice had significantly greater body and masseter weights beginning at 28 days compared to WT controls. No significant differences in cranial volumes were noted between KO and WT. Muscle weight was not significantly correlated with cranial volume in 1, 28, or 180 day old mice. Muscle weights exhibited a positive correlation with cranial volume at 56 days. Results demonstrate that masticatory hypermuscularity is not associated with reduced cranial volume. In contrast, there is abundant data demonstrating the opposite, brain growth determines cranial vault growth and masticatory apparatus only affects ectocranial morphology. The results presented here do not support the hypothesis that a reduction in masticatory musculature relaxed compressive forces on the cranial vault allowing for greater encephalization.
Myostatin; hypermuscularity; brain evolution; knockout model
One important component of the cell-cell communication that occurs during regenerative patterning is bioelectrical signaling. In particular, the regeneration of the tail in Xenopus laevis tadpoles both requires, and can be initiated at non-regenerative stages by, specific regulation of bioelectrical signaling (alteration in resting membrane potential and a subsequent change in sodium content of blastemal cells). While standing gradients of transmembrane voltage and ion concentration can provide positional guidance and other morphogenetic cues, these biophysical parameters must be transduced into transcriptional responses within cells. A number of mechanisms have been described for linking slow voltage changes to gene expression, but recent data on the importance of epigenetic marks for regeneration suggest a novel hypothesis: that sodium/butyrate transporters link ion flows to influx of small molecules needed to modify chromatin state. Here, we briefly review the data on bioelectricity in tadpole tail regeneration, present a technique for convenient alteration of transmembrane potential in vivo that does not require transgenes, show augmentation of regeneration in vivo by manipulation of voltage, and present new data in the Xenopus tail consistent with the hypothesis that the monocarboxlyate transporter SLC5A8 may link regeneration-relevant epigenetic modification with upstream changes in ion content.
The paucity of mammalian adult cardiac myocytes (CM) proliferation following myocardial infarction (MI) and the remodeling of the necrotic tissue that ensues, result in non-regenerative repair. In contrast, zebrafish (ZF) can regenerate after an apical resection or cryoinjury of the heart. There is considerable interest in models where regeneration proceeds in the presence of necrotic tissue. We have developed and characterized a cautery injury model in the giant danio (GD), a species closely related to ZF, where necrotic tissue remains part of the ventricle, yet regeneration occurs. By light and transmission electron microscopy (TEM), we have documented four temporally overlapping processes: 1) a robust inflammatory response analogous to that observed in MI, 2) concomitant proliferation of epicardial cells leading to wound closure, 3) resorption of necrotic tissue and its replacement by granulation tissue, 4) regeneration of the myocardial tissue driven by 5-EDU and [3H]thymidine incorporating CMs. In conclusion, our data suggest that the GD possesses robust repair mechanisms in the ventricle, and can serve as an important model of cardiac inflammation, remodeling and regeneration.
Heart; Regeneration; giant danio; Remodeling; Zebrafish; Inflammation; cardiomyocytes
In cardiomyocytes of rats, two distinct mitochondrial division processes are in operation. The predominant process involves extension of a single crista until it spans the full width of a mitochondrion. Ingrowth of the outer membrane ultimately results in scission. The second division process involves “pinching”, in which narrowing of the organelle at specific surface locations leads to its attenuation. When limiting membranes from opposite sides meet, mitochondrial fission ensues. When pinching is the operative mode, elements of sarcoplasmic reticulum always are associated with the membrane constrictions. The nuclear control mechanisms that determine which modality of mitochondrial division will prevail are unknown.
mitochondrial division; mitochondrial pinching; cardiac; heart
Aging decreases oxidative phosphorylation through cytochrome oxidase (COX) in cardiac interfibrillar mitochondria (IFM) in 24-month old (aged) rats compared to 6-month old adult Fischer 344 rats, whereas subsarcolemmal mitochondria (SSM) located beneath the plasma membrane remain unaffected. Immunoelectron microscopy (IEM) reveals in aged rats a 25% reduction in cardiac COX subunit VIIa in cardiac IFM, but not in SSM. In contrast, the content of subunit IV remains unchanged in both SSM and IFM, irrespective of age. These subunits are localized mainly on cristae membranes. In contrast, semi-quantitative immunoblotting, which detects denatured protein, indicates that the content of COX VIIa is similar in IFM and SSM from both aged and adult hearts. IEM provides a sensitive method for precise localizing and quantifying specific mitochondrial proteins. The lack of immunoreaction of COX VIIa subunit by IEM in aged IFM is not explained by a reduction in protein, but rather by a masking phenomenon or by an in situ change in protein structure affecting COX activity.
immunoelectron microscopy; cytochrome c; mitochondria; oxidative phosphorylation; electron transport chain; aged heart
The biliary tree is a complex network of conduits that begins with the canals of Hering and progressively merges into a system of interlobular, septal, and major ducts which then coalesce to form the extrahepatic bile ducts, which finally deliver bile to the gallbladder and to the intestine. The biliary epithelium shows a morphological heterogeneity that is strictly associated with a variety of functions performed at the different levels of the biliary tree. In addition to funneling bile into the intestine, cholangiocytes (the epithelial cells lining the bile ducts) are actively involved in bile production by performing both absorbitive and secretory functions. More recently, other important biological properties restricted to cholangiocytes lining the smaller bile ducts have been outlined, with regard to their plasticity (i.e., the ability to undergo limited phenotypic changes), reactivity (i.e., the ability to participate in the inflammatory reaction to liver damage), and ability to behave as liver progenitor cells. Functional interactions with other branching systems, such as nerve and vascular structures, are crucial in the modulation of the different cholangiocyte functions.
cholangiocytes; normal liver; bile secretion; peribiliary vascular plexus; ductular reaction
Because both androgens and estrogens have been implicated in penile morphogenesis, we evaluated penile morphology in transgenic mice with known imbalance of androgen and estrogen signaling using scanning electron microscopy (SEM), histology, and immunohistochemistry of androgen and estrogen receptors α/β. Penises of adult wild-type, estrogen receptor-α knockout (αERKO), estrogen receptor-β knockout (βERKO), aromatase knockout (Arom-KO), and aromatase overexpression (Arom+) mice were evaluated, as well as adult mice treated with diethylstilbestrol (DES) from birth to day 10. Adult penises were examined because the adult pattern is the endpoint of development. The urethral orifice is formed by fusion of the MUMP (male urogenital mating protuberance) with the MUMP ridge, which consists of several processes fused to each other and to the MUMP. Similarly, the internal prepuce is completed ventrally by fusion of a ventral cleft. In adult murine penises the stromal processes that form the MUMP ridge are separated from their neighbors by clefts. αERKO, βERKO, and Arom-KO mice have penises with a MUMP ridge clefting pattern similar to that of wild-type mice. In contrast, Arom+ mice and neonatally DES-treated mice exhibit profound malformations of the MUMP, MUMP ridge clefting pattern, and internal prepuce. Abnormalities observed in Arom+ and neonatally DES-treated mice correlate with the expression of estrogen receptor-beta (ERβ) in the affected structures. This study demonstrates that formation of the urethal orifice and internal prepuce is due to fusion of separate epithelial-surfaced mesenchymal elements, a process dependent upon both androgen and estrogen signaling, in which ERβ signaling is strongly implicated.
mouse penis; prepuce; androgen receptor; estrogen receptor
Locus coeruleus (LC) consists of a densely packed nuclear core and a surrounding plexus of dendritic zone which is further divided into several sub-regions. Whereas many limbic-related structures topographically target specific sub-regions of the LC, the precise projections from two limbic areas, i.e., medial prefrontal cortex (mPFC) and dorsal raphe (DR), have not been investigated. The goal of the present study is to identify and compare the distribution patterns of mPFC and DR afferent terminals to the LC nuclear core as opposed to specific pericoerulear dendritic regions (Peri-LC). To address these issues, anterograde tracer injections were combined with dopamine-β-hydroxylase (DBH) immunofluorescent staining in order to reveal the distribution patterns around the LC nuclear complex. Our data suggest that both mPFC-LC and DR-LC projections exhibit selective afferent terminal patterns. More specifically, mPFC-LC projecting fibers mainly target the rostromedial Peri-LC, whereas DR-LC projecting fibers demonstrate a preference to the caudal juxtaependymal Peri-LC. Thus, our present findings provide further evidences that afferents to the LC are topographically organized. Understanding the relationship among different inputs to the LC may help to elucidate the organizing principle which likely governs the interactions between the broad afferent sources of the LC and its global efferent targets.
Medial prefrontal cortex; Dorsal raphe; Locus coeruleus; Serotonin; Norepinephrine
Craniofacial sutures are bone growth fronts that respond and adapt to biomechanical environments. Little is known of the role sutures play in regulating the skull biomechanical environment during patency and fusion conditions, especially how delayed or premature suture fusion will impact skull biomechanics. Tgf-β3 has been shown to prevent or delay suture fusion over the short term in rat skulls, yet the long-term patency or its consequences in treated sutures is not known. It was therefore hypothesized that Tgf-β3 had a long-term impact to prevent suture fusion and thus alter the skull biomechanics. In this study, collagen gels containing 3 ng Tgf-β3 were surgically placed superficial to the posterior interfrontal suture and deep to the periosteum in postnatal day 9 (P9) rats. At P9, P24, and P70, biting forces and strains over left parietal bone, posterior interfrontal suture, and sagittal suture were measured with masticatory muscles bilaterally stimulated, after which the rats were sacrificed and suture patency analyzed histologically. Results demonstrated that Tgf-β3 treated sutures showed less fusion over time than control groups, and strain patterns in the skulls of the Tgf-β3 treated group were different from that of the control group. While bite force increased with age, no alterations in bite force were attributable to Tgf-β3 treatment. These findings suggest that the continued presence of patent sutures can affect strain patterns, perhaps when higher bite forces are present as in adult animals.
Cranial sutures; cranial bone; suture fusion; Tgf-β3; biomechanics
In the prenatal heart, right-to-left atrial shunting of blood through the foramen ovale is essential for proper circulation. After birth, as the pulmonary circulation is established, the foramen ovale functionally closes as a result of changes in the relative pressure of the two atrial chambers, ensuring the separation of oxygen depleted venous blood in the right atrium from the oxygenated blood entering the left atrium. Little is known regarding the process of anatomical closure of the foramen ovale in the postnatal heart. Genetically engineered mouse models are powerful tools to study heart development and to reveal mechanisms underlying cardiac anomalies, including defects in atrioventricular septation. Using three-dimensional reconstructions of serial sectioned hearts at early postnatal Days 2–7, we show a progressive reduction in the size of the interatrial communication throughout this period and complete closure by postnatal Day 7. Furthermore we demonstrate that fusion of the septum primum and septum secundum occurs between 4 weeks and 3 months of age. This study provides a standard timeline for morphological closure of the right– left atrial communication and fusion between the atrial septa in normal mouse hearts.
mouse; heart defects; congenital; ASD; PFO
Whereas upregulation of protein degradation pathways contributes to the development of muscle weakness in response to muscle injury and inflammation in the adult diaphragm, less is known about the preterm diaphragm. Muscle development during the antenatal and early postnatal periods normally results in net growth. However, the structural and functional immaturity of the preterm diaphragm may predispose it to injury and inflammation induced by adverse antenatal and postnatal exposures. Characterization of the ontogeny of diaphragm protein degradation pathways in early life is essential to recognise altered signaling pathways under pathologic conditions in preterm babies.
We assessed the relative role of the major proteolytic pathways and antioxidant capacity during muscle maturation in ovine fetuses and lambs from 75 d to 200 d post-conceptual age. Gene expression and protein content of calpain and caspase 3 exhibited a similar profile with advancing gestation, increasing from 75 d to 100 d/128 d and subsequently decreasing gradually toward the end of gestation. In contrast, ubiquitin conjugating and ligase genes do not change during gestation. All proteolytic genes examined (except Ubiquitin) are up-regulated rapidly after delivery, with a similar developmental trend observed in calpain II protein content as well as calpain protease activity. In contrast, antioxidant gene expression demonstrated a steady increase from 75 d gestation until 24 h after birth, followed by a significant reduction at 7 w of postnatal age (p ≤ 0.002). The proteolytic signaling and antioxidant capacity patterns reflect the adaptive process to metabolic change and muscle maturity with development.
ontogeny; diaphragm; calpain; caspase 3; ubiquitin-proteasome pathway; reactive oxygen species
The current working model of primate auditory cortex is constructed from a number of studies of both New and Old World monkeys. It includes three levels of processing. A primary level, the core region, is surrounded both medially and laterally by a secondary belt region. A third level of processing, the parabelt region, is located lateral to the belt. The marmoset monkey (Callithrix jacchus jacchus) has become an important model system to study auditory processing, but its anatomical organization has not been fully established. In previous studies, we focused on the architecture and connections of the core and medial belt areas (de la Mothe et al., 2006a,b). In the current study the corticocortical connections of the lateral belt and parabelt were examined in the marmoset. Tracers were injected into both rostral and caudal portions of the lateral belt and parabelt. Both regions revealed topographic connections along the rostrocaudal axis, where caudal areas of injection had stronger connections with caudal areas, and rostral areas of injection with rostral areas. The lateral belt had strong connections with the core, belt and parabelt, whereas the parabelt had strong connections with the belt but not the core. Label in the core from injections in the parabelt was significantly reduced or absent, consistent with the idea that the parabelt relies mainly on the belt for its cortical input. In addition, the present and previous studies indicate hierarchical principles of anatomical organization in the marmoset that are consistent with those observed in other primates.
primate; auditory cortex; interhemispheric; superior temporal sulcus
The mammary gland is one of the best-studied examples of an organ whose structure and function are influenced by reciprocal signalling and communication between cells and their microenvironment. The mammary epithelial cell microenvironment includes stromal cells and the extracellular matrix (ECM). Abundant evidence shows that the ECM and growth factors cooperate to regulate cell cycle progression, and that the ECM is altered in breast tumors. In particular, mammographically dense breast tissue is a significant risk factor for developing breast carcinomas. Dense breast tissue is associated with increased stromal collagen and epithelial cell content. In this article we overview recent studies addressing the effects of ECM composition on the breast cancer cell cycle. While the normal breast ECM keeps the mammary epithelial cell cycle in check, the ECM remodeling associated with breast cancer positively regulates the mammary epithelial cell cycle. ECM effects on the downstream biochemical and mechanosignalling pathways in both normal and tumorigenic mammary epithelial cells will be reviewed.
The adult heart has been reported to have an extensive lymphatic system, yet the development of this important system during cardiogenesis is still largely unexplored. The nuclear-localized transcription factor Prox-1 identified a sheet of Prox-1-positive cells on the developing aorta and pulmonary trunk in avian and murine embryos just prior to septation of the four heart chambers. The cells coalesced into a branching lymphatic network that spread within the epicardium to cover the heart. These vessels eventually expressed the lymphatic markers LYVE-1, VEGFR-3, and podoplanin. Before the Prox-1-positive cells were detected in the mouse epicardium, LYVE-1, a homologue of the CD44 glycoprotein, was primarily expressed in individual epicardial cells. Similar staining patterns were observed for CD44 in avian embryos. The proximity of these LYVE-1/CD44-positive mesenchymal cells to Prox-1-positive vessels suggests that they may become incorporated into the lymphatics. Unexpectedly, we detected LYVE-1/PECAM/VEGFR-3-positive vessels within the embryonic and adult myocardium which remained Prox-1/podoplanin-negative. Lymphatic markers were surprisingly found in adult rat and embryonic mouse epicardial cell lines, with Prox-1 also exhibiting nuclear-localized expression in primary cultures of embryonic avian epicardial cells. Our data identified three types of cells in the embryonic heart expressing lymphatic markers: (1) Prox-1-positive cells from an extracardiac source that migrate within the serosa of the outflow tract into the epicardium of the developing heart, (2) individual LYVE-1-positive cells in the epicardium that may be incorporated into the Prox-1-positive lymphatic vasculature, and (3) LYVE-1-positive cells/vessels in the myocardium that do not become Prox-1-positive even in the adult heart.
lymphatics; heart; Prox-1; LYVE-1; VEGFR-3; epicardium
Much has been learned regarding factors that specify joint placement, but less is known regarding how these molecular instructions are translated into functional joint tissues. Previous studies have shown that the matrix chondroitin sulfate proteoglycan, versican, exhibits a similar pattern of expression in the embryonic joint rudiment of chick and mouse suggesting conserved function during joint development. In the present study versican’s importance in developing joints was investigated by specific inhibition of its expression in the early joint interzone, tissue that gives rise to articular cartilages and joint cavity. In ovo microinjection of adenoviral shRNA constructs into the HH25 chick wing was employed to silence endogenous versican protein in developing appendicular joints. Results showed statistically significant (12–14%) reduction of non-chondrogenic elbow joint interzone area in whole mount specimens at HH36 in response to versican knockdown. Attenuated expression of key versican-associated molecules including hyaluronan, tenascin, CD44, and link protein was also noted by histochemical and immunohistochemical analysis. Versican knockdown also lowered collagen II expression in presumptive articular chondrocytes indicating possible delay in chondrogenesis. Results suggest that versican functions interactively with other matrix/cell surface molecules to facilitate establishment or maintenance of early joint interzone structure.
versican; extracellular matrix; immunohistochemistry; chick embryo; joint interzone; adenoviral gene transfer
Acid aspiration is a major cause of acute lung injury. However, the mechanisms that underlie this spatial expansion of the injury remain undefined. In current animal models of acid injury, intratracheal acid instillation replicates the lung injury. However intratracheal instillation causes a global effect, precluding studies of how the injury spreads. Here, we report an airway catheter-based method for localized acid delivery in the isolated blood-perfused rat lung. We co-instilled hydrochloric acid with evans blue through the catheter into one lung and determined blood-free extravascular lung water in tissue samples from regions that either received, or did not receive the instilled acid. Tissue samples from the non-catheterized contralateral lung were used as controls. Lung water increased both in the regions that received acid, as well as in adjacent regions that did not. Pre-treating the lung with vascular infusions of the gap junctional blocker, glycerrhetinic acid, blunted the acid-induced lung water increase at the adjacent regions. These findings indicate that endothelial gap junction communication causes spread of lung injury from regions that were directly acid injured, to adjacent sites that did not directly receive acid. Our new method for establishing localized acid injury provides evidence for a novel role for vascular gap junctions in the spatial expansion of acid injury.
Acid Aspiration; Gap Junctions; Endothelial; Acute Lung Injury; Lung Water
An important hypothesis is that the degree of infilling of secondary osteons (Haversian systems) is controlled by the inhibitory effect of osteocytes on osteoblasts, which might be mediated by sclerostin (a glycoprotein produced by osteocytes). Consequently, this inhibition could be proportional to cell number: relatively greater repression is exerted by progressively greater osteocyte density (increased osteocytes correlate with thinner osteon walls). This hypothesis has been examined, but only weakly supported, in sheep ulnae. We looked for this inverse relationship between osteon wall thickness (On.W.Th) and osteocyte lacuna density (Ot.Lc.N/B.Ar) in small and large osteons in human ribs, calcanei of sheep, deer, elk, and horses, and radii and third metacarpals of horses. Analyses involved: (1) all osteons, (2) smaller osteons, either ≤150μm diameter or ≤ the mean diameter, and (3) larger osteons (>mean diameter). Significant, but weak, correlations between Ot.Lc.N/B.Ar and On.W.Th/On.Dm (On.Dm = osteon diameter) were found when considering all osteons in limb bones (r values −0.16 to −0.40, p<0.01; resembling previous results in sheep ulnae: r= −0.39, p<0.0001). In larger osteons, these relationships were either not significant (five/seven bone types) or very weak (two/seven bone types). In ribs, a negative relationship was only found in smaller osteons (r= −0.228, p<0.01); this inverse relationship in smaller osteons did not occur in elk calcanei. These results do not provide clear or consistent support for the hypothesized inverse relationship. However, correlation analyses may fail to detect osteocyte-based repression of infilling if the signal is spatially non-uniform (e.g., increased near the central canal).
The global biomechanical impact of cranial sutures on the face and cranium during dynamic conditions is not well understood. It is hypothesized that sutures act as energy absorbers protecting skulls subjected to dynamic loads. This hypothesis predicts that sutures have a significant impact on global patterns of strain and cranial structural stiffness when analyzed using dynamic simulations; and that this global impact is influenced by suture material properties. In a finite element model developed from a juvenile Rhesus macaque cranium, five different sets of suture material properties for the zygomaticotemporal sutures were tested. The static and dynamic analyses produced similar results in terms of strain patterns and reaction forces, indicating that the zygomaticotemporal sutures have limited impact on global skull mechanics regardless of loading design. Contrary to the functional hypothesis tested here, the zygomaticotemporal sutures did not absorb significant amounts of energy during dynamic simulations regardless of loading speed. It is alternatively hypothesized that sutures are mechanically significant only insofar as they are weak points on the cranium that must be shielded from unduly high stresses so as not to disrupt vitally important growth processes. Thus, sutural and overall cranial form in some vertebrates may be optimized to minimize or otherwise modulate sutural stress and strain.
vertebrate skulls; elastic properties; loading speed
Several Eph receptors, prominently EphA4 and EphA7, and their corresponding ligands are known to influence neocortical development, including topographic sorting of thalamocortical axons within primary somatosensory cortex (SI). The present study investigated postnatal expression of a ligand that can bind to these receptors, ephrin-A2. Quantitative methods revealed that expression of ephrin-A2 mRNA in SI reached maximum levels on postnatal day (P) 4 and dropped thereafter to background by P18. Ephrin-A2 mRNA expression assessed by in situ hybridization qualitatively revealed a similar time course and localized the expression pattern primarily in two broad laminae in SI, comprising the supragranular and infragranular layers, and with additional expression in the subplate. This expression pattern was investigated in greater detail using immunohistochemistry for ephrin-A2 protein. Immunoreactivity generally showed the same laminar distribution as seen with in situ hybridization, except that it persisted longer, lasting to approximately P14. Expression in the cortical plate was low or absent within presumptive layer IV, and it remained so as cortical lamination progressed. Double-labeling immunohistochemistry with confocal microscopy revealed that cortical neurons were the principal elements expressing ephrin-A2 protein. These findings are consistent with possible involvement of ephrin-A2, in concert with one or more Eph receptors, in influencing arbor development of thalamocortical axons at cortical layer IV boundaries.
Eph receptors; cortical lamination; thalamocortical axons; axon guidance molecules; barrel cortex
Amphetamine-like psychostimulant drugs have been used for decades to treat a variety of clinical conditions. Methylphenidate (MPH) - RitalinR, a compound that blocks reuptake of synaptically released norepinephrine (NE) and dopamine (DA) in the brain, has been used for more than 30 years in low dose, long-term regimens to treat attention deficit-hyperactive disorder (ADHD) in juveniles, adolescents, and adults. These agents are now also becoming increasingly popular among healthy individuals from all walks of life (e.g. military, students) and age groups (teenagers thru senior citizens) to promote wakefulness and improve attention. While there is agreement regarding the primary biochemical action of MPH, the physiological basis for its efficacy in normal individuals and ADHD patients is lacking. Study of the behavioral and physiological actions of clinically and behaviorally relevant doses of MPH in normal animals provides an opportunity to explore the role of catecholamine transmitters in prefrontal cortical function and attentional processes as they relate to normal operation of brain circuits and ADHD pathology. The goal of ongoing studies has been to: 1) assess the effects of low dose MPH on rodent performance in a well characterized sensory-guided sustained attention task, 2) examine the effects of the same low-dose chronic MPH administration on task-related discharge of prefrontal cortical (PFC) neurons and 3) investigate the effects of NE and DA on membrane response properties and synaptic transmission in identified subsets of PFC neurons. Combinations of these approaches can be used in adolescent, adult and aged animals to identify the parameters of cell and neural circuit function that are regulated by MPH and to establish an overarching explanation of how MPH impacts PFC operations from cellular through behavioral functional domains.
Structural data about the human lung fine structure are mainly based on stereological methods applied to serial sections. As these methods utilize 2D images, which are often not contiguous, they suffer from inaccuracies which are overcome by analysis of 3D micro-CT images of the never-sectioned specimen. The purpose of our study was to generate a complete data set of the intact 3-dimensional architecture of the human acinus using high-resolution synchrotron-based micro-CT (synMCT). A human lung was inflation-fixed by formaldehyde ventilation and then scanned in a 64-slice CT over its apex to base extent. Lung samples (8-mm diameter, 10-mm height, n = 12) were punched out, stained with osmium tetroxide, and scanned using synMCT at (4μm)3 voxel size. The lung functional unit (acinus, n = 8) was segmented from the 3D tomographic image using an automated tree-analysis software program. Morphometric data of the lung were analyzed by ANOVA. Intraacinar airways branching occurred over 11 generations. The mean acinar volume was 131.3 ± 29.2 mm3 (range 92.5 – 171.3 mm3) and the mean acinar surface was calculated with 1012 ± 26 cm2. The airway internal diameter (starting from the bronchiolus terminalis) decreases distally from 0.66 ± 0.04 mm to 0.34 ± 0.06 mm (p < 0.001) and remains constant after the 7th generation (p < 0.5). The length of each generation ranges between 0.52 – 0.93 mm and did not show significant differences between the second and 11th generation. The branching angle between daughter branches varies between 113–134° without significant differences between the generations (p < 0.3). This study demonstrates the feasibility of quantitating the 3D structure of the human acinus at the spatial resolution readily achievable using synMCT.
Imaging; Human Lung; Acinus; Micro-CT
The Xenopus tadpole has the capacity fully to regenerate its tail after amputation. Previously, we have established that this regeneration process requires the operation of several signaling pathways including the bone morphogenic protein, Wnt, and Fgf pathways. Here, we have addressed the signaling requirements for spinal cord and muscle regeneration in a tissue-specific manner. Two methods were used namely grafts of transgenic spinal cord to a wild type host, and the use of the Tet-on conditional transgenic system to express inhibitors in the individual tissues. For the grafting experiments, the tail was amputated through the graft, which contained a temperature inducible inhibitor of the Wnt-β-catenin pathway. For the Tet-on experiments, treatment with doxycycline was used to induce cell autonomous inhibitors of the Wnt-β-catenin or the Fgf pathway in either spinal cord or muscle. The results show that both spinal cord and muscle regeneration depend on both the Wnt-β-catenin and the Fgf pathways. This experimental design also enables us to observe the effect of inhibition of regeneration of one tissue on the regeneration of the others. Regardless of the method of inhibition, we find that reduction of spinal cord regeneration reduces regeneration of other parts of the tail, including the myotomal muscles. In contrast, reduction of muscle regeneration has no effect on the regeneration of the spinal cord. In common with other regeneration systems, this indicates that soluble factors from the spinal cord are needed to promote the regeneration of the other tissues in the tail.
Xenopus tadpole; tail; regeneration; Wnt; Fgf; spinal cord; muscle; tissue interaction
For studies of vertebrate limb regeneration it is often desirable to visualize the regenerated skeleton, which is mostly cartilage, and also section the specimen for histological or immunohistochemical visualization of other tissues. However, the normal skeletal staining techniques are incompatible with immunohistochemistry. Here, we describe a contrast-based micro-computed tomography (microCT) method for direct and nondestructive observation of regenerated cartilage spikes in Xenopus frog limbs. In addition, we show that contrast based microCT imaging is compatible with immunohistochemistry protocols. This approach provides versatile and high contrast images of the cartilage allowing us to measure the regenerated skeletal structure in detail as well as carrying out the other types of analysis. It opens a wide range of potential microCT applications in research on vertebrate limb regeneration.
limb regeneration; regenerated skeleton; Xenopus frog; microCT; Hexabrix
Reduced sociability is a core feature of autism spectrum disorders (ASD) and is highly disabling, poorly understood, and treatment refractory. To elucidate the biological basis of reduced sociability, multiple laboratories are developing ASD-relevant mouse models, in which sociability is commonly assessed using the Social Choice Test. However, various measurements included in that test sometimes support different conclusions. Specifically, measurements of time the “test” mouse spends near a confined “stimulus” mouse (chamber scores) sometimes support different conclusions from measurements of time the test mouse sniffs the cylinder containing the stimulus mouse (cylinder scores). This raises the question of which type of measurements are best for assessing sociability. We assessed the test-retest reliability and ecological validity of chamber and cylinder scores. Compared with chamber scores, cylinder scores showed higher correlations between test and retest measurements, and cylinder scores showed higher correlations with time spent in social interaction in a more naturalistic phase of the test. This suggests that cylinder scores are more reliable and valid measures of sociability in mouse models. Cylinder scores are reported less commonly than chamber scores, perhaps because little work has been done to establish automated software systems for measuring the former. In this study, we found that a particular automated software system performed at least as well as human raters at measuring cylinder scores. Our data indicate that cylinder scores are more reliable and valid than chamber scores, and that the former can be measured very accurately using an automated video analysis system in ASD-relevant models.
sociability; behavior; mouse; model; autism; endophenotype
We have demonstrated that neonatal exposure to selective serotonin reuptake inhibitors has lasting effects on behavior and serotonergic neurons in Long Evans rats. Hyperserotoninemia and altered sensory processing are reported in autistic spectrum disorders (ASD). We hypothesized that early life exposure to SSRIs alters sensory processing, disrupts responses to novelty and impairs social interactions in a manner similar to that observed in ASD. Male and female Long-Evans rat pups were administered citalopram, buproprion, fluoxetine, or saline from postnatal day (P) 8 to 21. Rats were tested for response to a novel tone before weaning (P25). Later, rats were tested 2× for response to a novel object (P39), and to a novel conspecific (P78, P101). In addition, rats were assessed for juvenile play behaviors (P32–P34) and later, we assessed sexual response to an estrus female in male rats (P153–184). Antidepressant exposure increased freezing after tone, diminished novel object exploration and reduced conspecific interaction up to 3× compared to saline exposed rats. Juvenile play was profoundly reduced in antidepressant-exposed males when compared to saline exposed groups. Exposure to the SSRIs, but not bupropion disrupted male sexual behaviors. Moreover, specific male responses to female proceptive behaviors were disrupted in SSRI, but not bupropion exposed rats. We conclude that neonatal exposure to antidepressants in rats results in sensory and social abnormalities that parallel many of those reported in ASD.