It has been established that a postsynaptic scaffolding protein, gephyrin, is essential for anchoring two main groups of inhibitory receptors, GABA_A receptors (GABA_ARs) and glycine receptors (GlyRs), to the postsynaptic sites of neurons. The present study was primarily attempted to examine if expression patterns of gephyrin might be different between jaw-closing (JC) and jaw-opening (JO) motoneurons. The JC- and JO-motoneurons in the rat trigeminal motor nucleus (Vm) were located in the dorsolateral (Vm.dl) and ventromedial (Vm.vm) divisions, respectively (Mizuno et al.,1975). Thus, immunoreactivity (IR) for gephyrin was investigated in the Vm: immunofluorescence histochemistry for gephyrin was combined with retrograde tract-tracing of fluorogold (FG), which was injected into nerves innervating JC-muscles or nerves innervating JO-muscles; neuronal cells were counterstained with propidium iodide (PI). The Vm.dl was discriminated from the Vm.vm by the presence of vesicular glutamate transporter 1 (VGLUT1)-immunopositive axon terminals, which were distributed in the Vm.dl but not in the Vm.vm (Pang et al., J Comp Neurol2009;512:595–612). Gephyrin-IR showed a punctate pattern of fluorescence, and motoneuronal profiles were coated with small clusters of gephyrin-immunopositive puncta throughout the Vm. The distribution density of such clusters was apparently higher in the Vm.dl than in the Vm.vm; this was confirmed quantitatively by a method similar to that described by Lorenzo et al. (Eur J Neurosci2006;23:3161–3170). On the basis of the present results, possible correlation between the distribution density of gephyrin clusters in the submembrane region of Vm motoneurons and that of axon terminals making inhibitory synapses on Vm motoneurons was discussed. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.

The clouded leopard (N. nebulosa) is an endangered felid native to Southeast Asia. It is considered to be the largest and most acrobatic of the arboreal cats. To date, published studies have focused on cranial morphology, particularly osteology and dentition. This study describes the hip and thigh morphology of N. nebulosa and compares it to the domestic cat (F. catus). A number of statistically significant differences were observed between N. nebulosa and F. catus that were primarily associated with muscle attachment surface areas rather than differences in overall morphology. F. catus had proportionally larger attachment areas for Mm. gluteus profundus, tensor fasciae latae, and biceps femoris, while N. nebulosa had larger attachment areas for Mm. gluteus medius, rectus femoris, and semitendinosus. By comparison, surface area analysis of nonfelid species (C. latrans and V. vulpes) showed more differences when compared to felids demonstrating that this analytical technique is useful for differentiating closely-related species from more distant ones. These results suggest that muscle map area analysis may be useful for comparing phylogenetic relationships between and within groups of different species and may also suggest variations in locomotor habits. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.

Mammals are unique in being the only group of amniotes that can hear sounds in the upper frequency range (>12 kHz), yet details about the evolutionary development of hearing patterns remain poorly understood. In this study, we used high resolution X-ray computed tomography to investigate several functionally relevant auditory structures of the inner ear in a sample of 21 fossil primate species (60 Ma to recent times) and 25 species of living euarchontans (primates, tree shrews, and flying lemurs). The structures examined include the length of the cochlea, development of bony spiral lamina and area of the oval window (or stapedial footplate when present). Using these measurements we predicted aspects of low-frequency and high-frequency sensitivity and show that hearing patterns in primates likely evolved in several stages through the first half of the Cenozoic. These results provide temporal boundaries for the development of hearing patterns in extant lineages and strongly suggest that the ancestral euarchontan hearing pattern was characterized by good high-frequency hearing but relatively poor low-frequency sensitivity. They also show that haplorhines are unique among primates (extant or extinct) in having relatively longer cochleae and increased low-frequency sensitivity. We combined these results with additional, older paleontological evidence to put these findings in a broader evolutionary context. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.

The current study used MR imaging to determine the volume of the cerebellum and its component parts in the brain of three adult male African elephants (Loxodonta africana) and compared this with published data from Asian elephants and other mammalian species including odontocete cetaceans, primates, chiropterans, insectivores, carnivores, and artiodactyls. The cerebellum of the adult elephant has a volume of ∼925 mL (average of both African and Asian species). Allometric analysis indicates that the elephant has the largest relative cerebellum size of all mammals studied to date. In addition, both odontocete cetaceans and microchiropterans appear to have large relative cerebellar sizes. The vermal and hemispheric components of the African elephant cerebellum are both large relative to other mammals of similar brain size, however, for odontocete cetaceans the vermal component is small and the hemispheric component is large. These volumetric observations are related to life-histories and anatomies of the species investigated. The current study provides context for one aspect of the elephant brain in the broader picture of mammalian brain evolution. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.

The Brazilian free-tailed bat (Tadarida brasiliensis) exhibits a highly vascularized, hairless thermal window (or “radiator”) on the proximal ventral surfaces of extended wings and body. We identified this character using thermal infrared imaging and investigated the vasculature using barium sulfate enhanced microcomputed tomography (micro-CT). Micro-CT images revealed unique arrangements of arteries and veins in the region of the radiator positioned perpendicular to the axis of the body. Coupling micro-CT imaging with analysis of surface temperature profiles, we concluded that radiators aid in thermoregulation during flight in variable environments. This study represents the first application of contrast enhanced micro-CT to visualize vasculature of bats and thus exhibits a promising technique for further investigations of cardiovascular function and anatomy in bats. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.

The Slit–Robo GTPase-activating proteins (srGAPs) play an important role in neurite outgrowth and axon guidance; however, little is known about its role in nerve regeneration after injury. Here, we studied the expression of srGAPs in mouse dorsal root ganglia (DRG) following sciatic nerve transection (SNT) using morphometric and immunohistochemical techniques. Reverse transcriptase polymerase chain reaction and Western blot analysis indicated that srGAP1 and srGAP3, but not srGAP2, were expressed in normal adult DRG. Following unilateral SNT, elevated mRNA and protein levels of srGAP1 and srGAP3 were detected in the ipsilateral relative to contralateral L_3–4 DRGs from day 3 to day 14. Immunohistochemical results showed that srGAP1 and srGAP3 were largely expressed in subpopulations of DRG neurons in naïve DRGs. However, after SNT, srGAP3 in neurons was significantly increased in the ipsilateral relative to contralateral DRGs, which peaked at day 7 to day 14. Interestingly, DRG neurons with strong srGAP3 labeling also coexpressed Robo2 after peripheral nerve injury. These results suggest that srGAPs are differentially expressed in murine DRG and srGAP3 are the predominant form. Moreover, srGAP3 may participate in Slit–Robo signaling in response to peripheral nerve injury or the course of nerve regeneration. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.

The mammary gland is one of the best-studied examples of an organ whose structure and function are influenced by reciprocal signaling and communication between cells and their microenvironment. The mammary epithelial cell (MEC) microenvironment includes stromal cells and extracellular matrix (ECM). Abundant evidence shows that the ECM and growth factors co-operate 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. Although the normal breast ECM keeps the MEC cycle in check, the ECM remodeling associated with breast cancer positively regulates the MEC cycle. ECM effects on the downstream biochemical and mechanosignaling pathways in both normal and tumorigenic MECs will be reviewed. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.

The fine structures, enzyme histochemical, and immunohistochemical characterization of liver in zebrafish were investigated using light microscopy and electron microscopy. The results showed that liver was separated into three lobes and each lobe had a central vessel comparable to the mammalian central vein. However typical hepatic lobules, portal areas, and hepatic arteries were not observed. A portal vein entered the liver and its tributaries were connected directly to the sinusoids, which then converged to the central vessel. Three central vessels in lobes finally carried the blood out of liver. The polygonal and bilayered hepatocytes were arranged as twisting, branching, and anastomosing cords. Ultrastructurally, they showed apparent morphological features of protein synthesis and secretion. Bile entered the biliary tree through the intracellular canaliculus, the ramifications of intercellular canaliculi that originated near the hepatic nucleus and then extended to the hepatocyte surface where two adjacent hepatocyte membranes formed intercellular canaliculi, and then ran sequentially through bile preductules, bile ductules, and bile ducts to be secreted out of the liver. Bile preductular epithelial cells (BPs) were cells located between bilayered hepatocytes in one hepatic cord. Occasionally, some tight junctions were detected forming the link between BPs and hepatocytes, which led us to assume that BPs might have a close relationship with hepatocytes during evolution. The present results indicate that zebrafish liver has its own specific fine structure. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.

The mdx mouse is an experimental model widely used for the study of Duchenne muscular dystrophy, which is characterized by the lack of dystrophin and cycles of muscle degeneration/regeneration. Studies demonstrated elevated levels of growth factors and accelerated skin wound repair in these animals. We therefore raised the hypothesis that the bone repair process might also be altered in these animals. Thus, the objective of this study was to evaluate the spontaneous healing of calvarial defects in mdx mice by histomorphometric analysis. Animals (45 days old) were divided into mdx and control groups. A defect measuring 2 mm in diameter was produced surgically in the right parietal bone of each animal. The animals were sacrificed 15, 30, and 60 days after surgery, and the skulls were processed by routine histological procedures. No difference in the volume of new bone inside the defect was observed between the two groups at any of the three postoperative time points. There was also no difference between the different periods of healing when each group was analyzed separately. The lower quality of femoral and calvarial bone in mdx mice reported in previous studies and the similar bone regeneration rates seen in two groups suggest that the healing capacity of calvarial defects was more expressive in mdx mice than in control animals. An increase in the amount of osteogenic factors released by damaged myofibers may have favored osteogenesis during bone defect healing in mdx mice. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.

It is now well documented that androgen and estrogen signaling during early development cause a sexual dimorphism in second-to-fourth digit length ratio (2D:4D). It is also well documented that males of mammalian species have a smaller 2D:4D than females. Although there are discrepancies among 2D:4D studies in birds, the consensus is that birds exhibit the opposite pattern with males having a larger 2D:4D than females. The literature currently lacks substantial information regarding the phylogenetic pattern of this trait in amphibians and reptiles. In this study, we examined 2D:4D in two species of frogs (Oophaga pumilio and Craugastor bransfordii) and two species of lizards (Anolis humilis and Anolis limifrons) to determine the existence and the pattern of the sexual dimorphism. Male O. pumilio and C. bransfordii displayed larger 2D:4D than females in at least one of their two forelimbs. Male A. humilis had larger 2D:4D than females in both hindlimbs, but smaller 2D:4D than females in both forelimbs. Male A. limifrons may also have smaller 2D:4D than females in the right forelimb. Finally, digit ratios were sometimes positively related to body length, suggesting allometric growth. Overall, our results support the existence of the 2D:4D sexual dimorphism in amphibians and lizards and add to the knowledge of 2D:4D trait patterning among tetrapods. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.

Skeletal myoblast (SKM) transplantation is a promising approach to regenerate tissue and improve the function of the injured heart. However, the number of survival cells transplanted to host myocardium is quite poor due to high rate of apoptosis; diazoxide (DZ) is a highly selective mito-KATP channel opener that may reduce cell apoptosis by relieving reactive oxygen species (ROS) damage. The aim of this study is to explore the protective effects of DZ on L6 SKM damage induced by hydrogen peroxide (H₂O₂) in vitro. Different dose and time of H₂O₂ and DZ treatment were performed and only 24 hr of 1.00 mmol/L H₂O₂ treatment and 200 μmol/L DZ pretreatment for 30 min were used for further experiment. L6 SKMs were cultured and divided into control group (no treatment), H₂O₂ group (24 hr of 1.00 mmol/L H₂O₂ treatment) and DZ + H₂O₂ group (pretreated with 200 μmol/L DZ for 30 min before 24 hr of 1.00 mmol/L H₂O₂ treatment). Compared with control group, H₂O₂ treatment caused cell damage, increased lactate dehydrogenase release, cell apoptosis, and bax gene expression, while reduced cell proliferation and decreased bcl-2 expression. DZ pretreatment may protect cells from damage induced by H₂O₂ and reduce cell apoptosis by increasing bcl-2 and decreasing bax expression. DZ pretreatment may also promote cell proliferation measured by both PCNA expression and flow cytometry method. These results suggest that DZ may protect L6 SKMs from damage induced by H₂O₂ by maintaining integrity of cell membrane, reducing apoptosis and increasing proliferation in vitro. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.

The avian pineal is a directly photosensory organ taking part in the organization of the circadian and seasonal rhythms. It plays an important role in regulation of many behavior and physiological phenomena including migration. The aim of the study was to investigate morphology of the pineal organ in the common gull (Larus canus). The light and electron microscopic studies were performed on the pineals of juvenile birds living in natural conditions of the Baltic Sea coast, which have been untreatably injured during strong storms in autumn and qualified for euthanasia. The investigated pineals consisted of a wide, triangular, superficially localized distal part and a narrow, elongated proximal part, attached via the choroid plexus to the intercommissural region of the diencephalon. The accessory pineal tissue was localized caudally to the choroid plexus. Based on the histological criteria, the organ was classified as the solid-follicular type. Two types of cells of fotoreceptory line were distinguished: rudimentary–receptor pinealocytes and secretory pinealocytes. Both types of cells were characterized by unusual features, which have been not previously described in avian pinealocytes: the presence of paracrystalline structures in the basal processes and their endings, the storage of glycogen in the form of large accumulations and the arrangement of mitochondria in clusters. Further studies on other species of wild water birds dwelling in condition of cold seas are necessary to explain if the described features of pinealocytes are specific for genus Larus, family Laridae or a larger group of water birds living in similar environmental conditions. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.

 Epidermolytic palmoplantar keratoderma (EPPK) is generally associated with dominant-negative mutations of the Keratin 9 gene (KRT9), and rarely with the Keratin 1 gene (KRT1). To date, a myriad of mutations has been reported with a high frequency of codon 163 mutations within the first exon of KRT9 in different populations. Notably, a distinct phenotypic heterogeneity, digital mutilation, was found recently in a 58-year-old female Japanese EPPK patient with p.R163W. Here, we report the most common mutation, c.C487T (p.R163W) of KRT9, in two large EPPK pedigrees from southeast China. The arginine residue in peptide position 163 remains almost constant in at least 47 intermediate filament proteins ranging from snail to human. A substitution in arginine alters both the charge and shape of the 1A rod domain and disrupts the function of the helix initiation motif of keratins, finally compromising the integrity of filaments and weakening their stability in the epidermis of palms and soles. We summarize the clinical symptoms of EPPK in Chinese and show that knuckle pads are associated with KRT9 mutations. We suggest that the frequency of p.R163W in Chinese EPPK patients (31.03%) is consistent with that in the general population (29.33%), and that codon 163 is truly a hotspot mutational site of KRT9. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.

The histology and ultrastructure of porcine tonsils were studied. The porcine tonsils were lymphoepithelial organs situated at the opening of both the digestive and respiratory tracts. The tonsil of the soft palate in the oropharyngeal tract and the paraepiglottic tonsil in the laryngopharynx were mainly consisted of secondary lymphoid follicles encapsulated by connective tissue. The stratified squamous epithelia covering the tonsils and their crypts were frequently heavily infiltrated by lymphoid cells. The pharyngeal and tubal tonsils (TT) were situated in the nasopharyngeal tract. The cells of the pseudostratified columnar epithelia of the pharyngeal and TT were loosely connected, with large intercellular space. They consisted of scattered lymphoid follicles, aggregations of lymphoid cells and diffuse lymphoid tissues. Many high endothelial venules, specialized for the diapedesis of lymphoid cells into the tonsillar tissue, were detected in the four porcine tonsils. Therefore, the overall structures of the tonsils (the tonsil of the soft palate, the paraepiglottic tonsil, the pharyngeal and the TT) reflect their immune functionality in the oral and intranasal immunity. Anat Rec, 2011. © 2011 Wiley Periodicals, Inc.

Stromal cells appear as paraductal networks in the human parotid gland. Stromal cells are immunostained with vimentin. See Figure 1 (left panel) in Nicolescu et al., on page 380, in this issue.

The discovery of a nonphagocytotic process of cell-in-cell phenomena can be traced to over a century ago. However, its biological significance remains poorly understood. Three types of cell-in-cell phenomena have been described so far, termed “cannibalism,” “emperipolesis,” and “entosis.” These three kinds of cell-in-cell phenomena, apart from a common feature of one cell internal to another, are distinct both cytologically and biologically. In this review, we discussed them in their morphology, cell recognition, penetration mechanisms, and physiological roles, respectively. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.

The parotid histological structure includes acinar, ductal, and myoepithelial cells, surrounded by a connective stromal component. The parotid stroma is mostly regarded as an inert shell, consisting of septa, which divide the parenchyma. Telocytes were recently identified as a new stromal cell type in various organs, including exocrine pancreas. We aimed to evaluate telocytes presence in parotid stroma and whether their topographical features might support an involvement in parotid function modulation. Serial ultrathin sections of human and rat parotid glands were studied and compared by transmission electron microscopy. Two-dimensional concatenation of sequenced micrographs allowed the ultrastructural identification of parotid telocytes, with their specific long, thin, and moniliform prolongations (telopodes). Telocyte location appeared frequently as a strategic one, in close contact or vicinity of both secretory (acini and ducts) and regulatory (nerves and blood vessels) apparatuses. They were also found in the interacinar and the subductal stroma. Two previously reported telocyte markers (c-kit/CD117 and vimentin) were assayed by immunohistochemistry. Actin expression was also evaluated. Telocytes are making a network, especially by branching of their long telopodes. Elements of this telocyte network are interacting with each other (homocellular connections) as well as with other cell types (heterocellular connections). These interactions are achieved either by direct contact (stromal synapse), or mediated via shed microvesicles/exosomes. Since telocyte connections include both neurovascular and exocrine elements (e.g., acini and ducts), it is attractive to think that telocytes might mediate and integrate neural and/or vascular input with parotid function. Anat Rec, 2012. © 2011 Wiley Periodicals, Inc.

Wing bone histology in three species of birds was characterized in order to test hypotheses related to the relationship between skeletal microstructure and inferred wing loading during flight. Data on the degree of laminarity (the proportion of circular vascular canals) and the occurrence of secondary osteons were obtained from three species that utilize different primary flight modes: the Double-crested cormorant, a continuous flapper; the Brown pelican, a static soarer; and the Laysan albatross, a dynamic soarer. Laminarity indices were calculated for four quadrants for each of the three main wing elements. Ulnae and carpometacarpi were predicted to exhibit quadrant specific patterns of laminarity due to hypothesized differences in locally applied loads related to the attachment of flight feathers. However, few differences among the quadrants were identified. No significant differences were identified among the three elements, which is notable as different bones are likely experiencing different loading conditions. These results do not support the concept of bone functional adaptation in the primary structure of the wing elements. Significant differences in laminarity were found among the three primary flight modes. The dynamic soaring birds exhibited significantly lower laminarity than the flapping and static soaring birds. These results support the proposed hypothesis that laminarity is an adaptation for resisting torsional loading. This may be explained by overall wing shape: whereas dynamic soaring birds have long slender wings, flappers and static soaring birds have broader wings with a larger wing chord that would necessarily impart a higher torsional moment on the feather-bearing bones. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.

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 this 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 nonchondrogenic 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. Anat Rec, 2012. © 2011 Wiley Periodicals, Inc.

ER60 protease, a 58-kDa molecular chaperone in the endoplasmic reticulum, is involved in glycoprotein synthesis. ER60 protease has been reported to be differentially expressed in various cancers including breast carcinoma. This study explored the relationship of ER60 protease with cell proliferation in breast cancer in vitro. ER60 protease expression was first determined in a panel of breast cell lines by real-time RT-PCR and Western blot analysis and found to be most abundantly expressed in T47D breast cancer cells. The ER60 protease gene was then successfully knocked down in T47D breast cancer cells using two different sequences of small-interfering RNA. The silencing efficiencies of siER-1 and siER-2 at 48-hr post-transfection were found to be >80% at the mRNA level with concomitant downregulation of the ER60 protease protein by >60% when compared with control T47D breast cancer cells. Downregulation of ER60 protease was also associated with inhibition of cell proliferation when assessed by the AlamarBlue assay. Cell cycle analysis performed on the siER-1- and siER-2-transfected cells, revealed an increase in G1 phase population and a decrease in the S and G2/M phase populations compared with control cells, implicating G1/S cell cycle arrest. It would appear that ER60 protease is involved in breast tumorigenesis and could therefore be a prospective target for cancer therapeutics. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.

To investigate the mechanism of oridonin (ORI)-induced autophagy in prostate cancer PC-3 cells, PC-3 cells cultured in vitro were treated with ORI, and the inhibitory ratio of ORI on PC-3 cells was assayed by 3-4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. The ultrastructural changes of the cells were observed under light microscope, scanning electron microscope (SEM), and transmission electron microscope (TEM). Acridine orange (AO) staining was used to observe the acidic vesicular organelles (AVOs). The level of autophagy-related proteins, MAP1-LC3, was detected by Western Blot, and RT-PCR was used to detect the level of mRNA of beclin 1. After ORI treatment, the proliferation of PC-3 cells was inhibited significantly in a concentration and time-dependent manner. SEM examination revealed cellular shrinkage and disappearance of surface microvilli in ORI-treated cells. Under TEM examination, the nuclei exhibited chromatin condensation and the appearance of a large number of autophagosomes with double-membrane structure in cytoplasm. AO staining showed the existence of AVOs. The expression of LC3 and the mRNA level of beclin 1 was increased by ORI. Furthermore, autophagy inhibitor 3-methyladenine reversed the increase of beclin 1 mRNA. The growth of PC-3 cells was inhibited, and autophagy was induced by ORI, indicating ORI may have a potential antitumor effect. Anat Rec, 2012. © 2011 Wiley Periodicals, Inc.

This study was designed to assess the impact of several molecular markers and clinicopathological characteristics on postoperative survival of patients with hepatocellular carcinoma (HCC). Postoperative clinical data of 64 patients with HCC were retrospectively analyzed. K-ras, PIK3CA, and BRAF gene mutations in surgically resected specimens of the 64 patients with HCC were detected by pyrosequencing. H-ras and XB130 protein expression was examined by immunohistochemistry. A Cox proportional hazards regression model was used for univariate and multivariate survival analyses of the clinical and pathological parameters. The mutation rates of K-ras, PIK3CA, and BRAF genes in HCC were found to be 4.69%, 1.56%, and 0%, respectively. Positive expression rate of XB130 and H-ras in HCC was 75.0% and 93.8%, respectively. Univariate analysis revealed that clinicopathological factors impacting postoperative prognosis of patients with HCC include clinical stage, tumor diameter, and postoperative transcatheter arterial embolization therapy for HCC. Meanwhile, multivariate analysis showed that clinical stage (relative risk [RR]: 6.420, P = 0.013) and tumor diameter (RR: 1.498, P = 0.014) were independent factors impacting postoperative survival of patients with HCC. These findings indicate that the clinical stage and tumor diameter are independent risk factors impacting postoperative survival of patients with HCC. Gene mutations of K-ras and PIK3CA and protein expression of XB130 and H-ras are not associated with the postoperative prognosis of patients with HCC. Anat Rec, 2012. © 2011 Wiley Periodicals, Inc.

Our previous study found that the artery interposed to vein did not develop atherosclerosis but rather underwent atrophic remodeling in hyperlipidemic rabbits, suggesting that local hemodynamic load was another important determinant for the development of atherosclerosis. This study focused on the cellular and molecular changes in autologous artery grafts derived from rabbits fed with high lipid diet for 1, 2, 4, 8, and 12 weeks. Thickness, area of vessel wall, and lumen area were measured and analyzed on the grafted common carotid artery (GCCA) interposed to vein and on the right common carotid artery. Apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling. Both elastin and collagen of GCCA were identified by the method of double stains of elastin and collagen. Reverse transcription polymerase chain reaction was used to observe matrix metalloproteinases (MMPs) mRNA expression changes in the examined arteries. The lumen area increased gradually in control common carotid artery and remained unchanged in GCCA 3 months later, since the surgery and the start of high lipid diet, while significantly increased apoptosis was evidenced from inner to outer part of GCCA. Collagen content decreased gradually and elastic fibers remained unchanged in GCCA. At 1 week after operation, the mRNA expression of MMP₂ and MMP₉ increased significantly and returned to baseline thereafter. The artery interposed to a vein underwent atrophy, characterized by increased apoptosis in the vessel wall from intima to adventitia, possibly due to low shear stress circumference and reduced vessel collagen resulting from postsurgical upregulated MMP₂ and MMP₉ expression. Anat Rec,, 2012. © 2011 Wiley Periodicals, Inc.

To speculate how the extrinsic cardiac nerve plexus (ECNP) evolves phyletically and ontogenetically within the primate lineage, we conducted a comparative anatomical study of the ECNP, including an imaging examination in the great apes using 20 sides from 11 bodies from three species and a range of postnatal stages from newborns to mature adults. Although the position of the middle cervical ganglion (MG) in the great apes tended to be relatively lower than that in humans, the morphology of the ECNP in adult great apes was almost consistent with that in adult humans but essentially different from that in the lesser apes or gibbons. Therefore, the well-argued anatomical question of when did the MG acquire communicating branches with the spinal cervical nerves and appear constantly in all sympathetic cardiac nerves during primate evolution is clearly considered to be after the great apes and gibbons split. Moreover, a horizontal four-chambered heart and a lifted cardiac apex with a relatively large volume in newborn great apes rapidly changed its position downward, as seen in humans during postnatal growth and was associated with a reduction in the hepatic volume by imaging diagnosis and gross anatomy. In addition, our observation using a range of postnatal stages exhibits that two sympathetic ganglia, the middle cervical and cervicothoracic ganglia, differed between the early and later postnatal stages. Anat Rec, 2012. © 2011 Wiley Periodicals, Inc.

One of the long-standing arguments about the evolution of bipedality centers on the locomotor pattern used by the last common ancestor (LCA) of apes and humans. In particular, knuckle-walking has been suggested as this locomotor pattern on the basis of shared morphology in the upper limb between African apes and humans and phylogenetic parsimony. Using three-dimensional geometric morphometrics, this study tests whether the distal radius of extant hominoids is sufficient for determining locomotor pattern and the affiliations of Plio-Pleistocene hominins to the extant taxa. Results indicate that while the entire radius differentiates the extant taxa very well by locomotor pattern, the distal radius fails to clearly differentiate the extant taxa. The sigmoid notch of the distal radius is the anatomical feature that differs most among the extant taxa, and its variability broadly correlates with necessary mobility at the wrist joint. Principal components and discriminant function analyses indicate that early hominins are affiliated with a variety of extant taxa with different locomotor patterns. Overall, the bony anatomy of the distal radius of early hominins points towards something adapted for a wide variety of locomotor postures. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.

Confocal microscopy in combination with commercial software is frequently used to generate three-dimensional images of tissue architecture. Here we report a novel, whole-mount imaging protocol technique that allows detailed three-dimensional imaging of adult pancreatic structures. This technique provides an improved appreciation of the anatomical detail of pancreatic structures and of the relationship between the pancreatic ducts and islets. In addition, imaging of the pancreatic ducts revealed a previously unappreciated high degree of hypervascularity. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.

Fukomys anselli, also known as Ansell's mole rat, is a subterranean, highly social (so-called eusocial) rodent that lives in Africa. These mole rats typically form multigenerational families consisting of a single monogamous breeding pair and their nonreproductive offspring. Research on other mammals suggests that oxytocin (OT) and vasopressin (VP) as well as the distribution of OT- and VP-receptors may influence social behavior and pair bonding. Recent studies on eusocial naked mole rats have shown a possible relation between sociality and OT-immunoreactive (OT-ir) processes. In this study, we examined expression patterns of OT and VP in the brains of F. anselli and the common Sprague-Dawley (SD) laboratory rat. As in other species, the majority of OT-ir and VP-ir neurons was found in the paraventricular (Pa) and supraoptic (SO) nuclei, and scattered labeling throughout the preoptic and anterior hypothalamic areas. We found no difference in either quality or quantity of OT- and VP-ir neurons between individuals of different social and reproductive ranks. Equally unexpected was the finding of specific OT-immunoreactivity in neurons of the mammillary complex of F. anselli that was not found in SD rats. Further studies are needed to determine whether these mammillary OT-ir neurons are causally related to monogamy in F. anselli and whether these correlates of monogamy are found in other species. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.

Many vertebrates have two anatomically distinct olfactory organs—the olfactory epithelium and the vomeronasal organ—to detect chemicals such as general odorants and pheromones in their environment. The vomeronasal organ is not present in fish but is present in vertebrates of a higher order than amphibians. Among all extant fishes, the lungfish is considered to be genetically and phylogenetically closest to tetrapods. In this study, we examined the olfactory organs of African lungfish, Protopterus annectens, by lectin histochemistry, immunohistochemistry, and transmission electron microscopy. Two types of sensory epithelia were identified in the olfactory organ—the olfactory epithelium covering the surface of lamellae and the sensory epithelium lining the recesses both at the base of lamellae and in the wall of the nasal sac—and designated here as the lamellar olfactory epithelium and the recess epithelium, respectively. Based on analysis of G-protein expression and ultrastructure, the lamellar olfactory epithelium resembled the olfactory epithelium of ordinary teleosts and the recess epithelium resembled the vomeronasal organ of tetrapods. Furthermore, lectin histochemistry demonstrated that the axons from the recess epithelium converge and project to the ventrolateral part of the olfactory bulb, suggesting that lungfish possess a region homologous to the accessory olfactory bulb of tetrapods. Based on these results, it seems appropriate to refer to the recess epithelium as “a primordium of the vomeronasal organ.” This study may provide important clues to elucidate how the vomeronasal organ emerged during the evolution of vertebrates. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.

Hspb8 is a member of the Hspb family of chaperone-like proteins. It is involved in several neural disorders such as Alzheimer's disease, amyotrophic lateral sclerosis, hereditary distal motor neuropathy, and Charcot-Marie-Tooth's disease. In this work, we aimed to characterize its expression pattern in the mouse brain, by using the information available at online databases of high-throughput in situ hybridization. Therefore, we downloaded and analyzed the image series from these databases showing Hspb8 mRNA expression from embryonic to adult and aging stages. In early gestational embryos, Hspb8 was expressed in the hippocampal anlagen and in the ventricular layer of rhombomere 4. At perinatal stages, there appeared transitory expression in the dentate gyrus and the cerebellar cortex. From perinatal to aging stages, the neurons of the mesencephalic trigeminal nucleus and cranial motor nuclei displayed stable and strong Hspb8 expression. Additionally, along these stages there was moderate and relatively homogenous expression in the anterodorsal thalamic, lateral mammillary, arcuate hypothalamic and medial habenular nuclei, and in the locus coeruleus. In its turn, the basal ganglia, cerebellar inner granular layer and diverse sensory and reticular formation nuclei of the hindbrain contained scattered cells with strong expression. In conclusion, Hspb8 mRNA is constitutively expressed in specific brain structures across ontogeny, so that eventually they could be affected by the malfunction or deregulation of this molecule. Anat Rec, 2012. © 2011 Wiley Periodicals, Inc.

The distribution of neuronal nitric oxide synthase (nNOS) in the process of normal mouse brain growth from embryonic (E) Day 11 to postnatal (P) Day 1 was investigated by means of immunohistochemical and immunofluorescent methods. Our results demonstrated that nNOS positive neurons appeared early in superficial cortex at E11. At E13, nNOS positive neurons were located in lateral hypothalamus and amygdala, and temporarily in medullar and ventral hypothalamic neuroepithelia. From E15 to P0, nNOS positive neurons were distributed in superior and inferior colliculi, positive staining could also be seen in superior and inferior tectal neuroepithelium at E15. From E17 to birth, the medial geniculate nucleus had a high density of nNOS labeling. The distribution of nNOS gradually increased and extended laterally in embryo brain, which in turn implies that NO might be involved in the development of mouse brain. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.

The ultrastructure of the optic nerve, brain, and some associated structures of larval zebrafish, grown under three different light regimens were studied. Fish grown under cyclic light (control), constant dark (CD), and constant light (CL) were studied for 4 and 8 days postfertilization (dpf). We also studied the control and CD fish at 15 dpf. The brains of the control and CL fish were larger at 4 dpf than at 8 dpf. In all 4 dpf fish, the brain occupied the entire expanse between the two retinas and the optic nerve extended the shortest distance between the retina and the brain. The 15 dpf zebrafish had the smallest brain size. Groups of skeletal muscle cells associated with the optic nerves became visible in all older larvae. In the 15 dpf larvae, bulges and dilations in the optic nerve occurred as it reached the brain and optic chiasms occurred proximal to the brain. Electron microscopy yielded information about myelinated and unmyelinated axons in the optic nerve, the dimensions of neurotubules, neurofilaments, and myofilaments, including a unique variation in actin myofilaments, and a confirmation of reported myosin myofilament changes (but with dimensions). We also describe the ultrastructure of a sheath-like structure that is confluent over the optic nerve and the brain, which has not been described before in zebrafish. Also presented are images of associated fibroblasts, epithelial cells lining the mouth, cartilage plates, blood vessels, nerve bundles, and skeletal muscle cells, most of which have not been previously described in the literature. Anat Rec,, 2012. © 2012 Wiley Periodicals, Inc.

This study aimed to explore the effect of high glucose concentration on the expression of glial cell line-derived neurotrophic factor (GDNF) and its family ligand receptors (GFRs) GFRα1 and GFRα2 in Müller cells and the protective role of GDNF in cultured Müller cells under high glucose circumstance. Cultured Müller cells (untreated or treated with 200 ng/mL of GDNF) were exposed to high glucose conditions (20 mmol/L glucose). We found that the expression levels of GDNF and GFRα1 mRNA and protein increased gradually over time under high glucose and exogenous GDNF-treated conditions, whereas the upregulation in GFRα2 expression was observed only in the early stage of high glucose conditions. Exogenous GDNF not only decreased apoptosis in cultured Müller cells under high glucose circumstance, but also accelerated the levels and speed of synthesis of GDNF and GFRα1 proteins in Müller cells. These results suggest that Müller cells can synthesize GDNF and GFRs under high glucose conditions, and GDNF may play important role in protecting Müller cells during the early stage of diabetic retinopathy. The difference in GFRs expression indicated that GDNF and neurturin may exert different effects on Müller cells under high glucose circumstance. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.

The pecten oculi is a highly vascular and pigmented organ placed in the vitreous body of the avian eye. As no data are currently available on the morphological organization of the pecten in the Psittaciformes, the pecten oculi of the budgerigar (Melopsittacus undulatus) was studied. The eyes from adult male budgerigars were examined by light, transmission, and scanning electron microscopy and a morphometric study on both light and transmission electron microscopy specimens was also performed in the different parts of the organ. In the budgerigar, the type of the pecten oculi was pleated. Its basal part had a cranio-caudal and postero-anterior course; its body consisted of 10–12-folds joined apically by a densely pigmented bridge. The pecten showed many capillaries, whose wall was thick and formed by pericytes and endothelial cells. These latter had a large number of microfolds, rectilinear on their luminal surface and tortuous on their abluminal surface. Interstitial pigment cells were placed among the capillaries, filled with melanin granules and showed many cytoplasmic processes. The morphometric analysis demonstrated significant differences among the three parts of the organ relative to the length of the endothelial processes and to the number and size of the pigment granules. The morphological and morphometric analysis showed that the bridge of the budgerigar, different from the other birds, had a large number of capillaries, so that this part of the organ could also play a trophic role for the retina in addition to the choriocapillaris. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.