Effect of physical therapy on joint range of motion and muscle collagen deposition in the golden retriever muscular dystrophy (GRMD) model / Efeito da fisioterapia na amplitude de movimento articular e deposição de colágeno muscular no modelo golden retriever muscular dystrophy (GRMD)

OBJECTIVE: To elucidate the effect of physical therapy on joint range of motion (ROM) and muscle fibrosis in GRMD animals. METHODS: This was a nonrandomized blinded study with a control group, with six months of intervention evaluated beforehand and afterwards. Six dystrophic male Golden Retrievers of mean age 10.16±3.46 months and weight 17.75±6.01 kg were divided into a treated group (n=3) and an untreated group. These groups of dogs were named: G1=treated group before treatment; G2=treated group after treatment; G3=untreated group before treatment; and G4=untreated group after treatment. G1 underwent a physical therapy program that consisted of a 300-meter circuit with obstacles. Stifle, tarsal, elbow and carpal ROM were assessed using a goniometer before and after treatment. The area of collagen in the vastus lateralis muscle was measured using histomorphometry. The locations of collagen types I, III and IV were studied using immunohistochemistry. RESULTS: The tarsal ROM values in G2 presented an increasing trend. The area of muscle collagen differed between the groups after treatment and an increasing trend in these values was observed in G4. Collagen types I and III were the ones most frequently observed, forming broad bands in the perimysium of both G2 and G4. Type I collagen was observed in the endomysium more than type III collagen. Type IV collagen was observed only in the basal layer. CONCLUSION: Physical Therapy seemed to improve tarsal ROM in the treated group without increasing muscular fibrosis.

OBJETIVO: Elucidar o efeito da fisioterapia na Amplitude de Movimento Articular (ADM) e na fibrose muscular em animais GRMD. MÉTODOS: Estudo não randomizado, com grupo controle, cego, seis meses de intervenção, avaliação antes e depois da intervenção. Seis animais da raça Golden Retriever, distróficos, machos, média de idade 10,16±3,46 meses e peso de 17,75±6,01 kg foram separados em grupo tratado (n=3) e não tratado. Esses grupos de animais foram nomeados: G1=grupo tratado antes do tratamento; G2=grupo tratado após tratamento; G3=grupo não tratado antes do tratamento; G4=grupo não tratado após tratamento. O G1 participou do programa de fisioterapia que consistiu em um circuito de 300 metros com obstáculos. As ADMs do joelho, tarso, cotovelo e carpo foram avaliadas com goniômetro antes e após o tratamento. A área de colágeno do músculo vastus lateralis foi mensurada por histomorfometria, e a localização dos tipos de colágeno I, III e IV foi estudada por Imuno-histoquímica (IHC). RESULTADOS: Os valores da ADM do tarso do G2 apresentaram uma tendência a aumentar. A área de colágeno muscular foi diferente entre os grupos após o tratamento, e uma tendência ao aumento desses valores no G4 foi observada. Os colágenos dos tipos I e III foram os mais observados, constituindo feixes largos no perimísio nos dois grupos (G2 e G4). O colágeno do tipo I foi mais observado no endomísio do que o colágeno do tipo III. O colágeno do tipo IV foi observado apenas na lâmina basal. CONCLUSÃO: A Fisioterapia parece aumentar a ADM do tarso dos animais do grupo tratado sem aumentar a fibrose muscular.

Ultrastructure of the maternal-fetal interface of the yolk sac placenta in sharks.

The blacknose shark, Carcharhinus acronotus, is a viviparous anamniote that develops an epitheliochorial yolk sac placenta. The fetal portion of the uteroplacental complex consists of a proximal portion that forms saccular evaginations. The cells are bilayered stratified squamous with surface microvilli and a high concentration of cytoplasmic filaments. The tertiary egg envelope intervenes between the distal portion of the placenta and uterus. It has delaminations on the uterine surface and is compacted on the placental surface. The uterine epithelium is cuboidal to columnar and is characterized by prominent RER, Golgi, and secretion vesicles. The capillary endothelium is continuous. Nutrient and respiratory exchange is effected between the uterus and distal portion of the yolk sac. The distal portion of the placenta is a bilayer. An elaborate array, of microvilli forms an interface with the egg envelope. Dense non-membrane bound granules occur in the interspace between the egg envelope and the distal placenta. This material, presumably of uterine origin, is endocytosed in smooth-walled vesicles of the placental cells. The endothelium of the capillaries is fenestrated.

Structure and glycosylation of the term yolk sac placenta and uterine attachment site in the viviparous shark Mustelus canis.

The viviparous shark Mustelus canis nurtures its young within the uterus by means of a modified yolk sac which functions as a placenta. Two term specimens have been examined with a panel of 21 biotinylated lectins to assess whether glycans form a prominent interface between fetal and maternal tissues as in their therian counterparts. The yolk sac placenta was lined by a thin egg envelope which apposed though did not make contact with the uterine epithelium, and expressed fucosyl, N-acetyl glucosamine/lactosamine residues and some complex N-glycan, while the attached, thin ectoderm cells stained selectively with lectins from Anguilla anguilla and Arachis hypogaea indicating fucosyl and beta-galactosyl residues; other lectins bound to a variable degree. Large yolk sac endoderm cells were heavily glycosylated and expressed a wide range of glycans. The apposing uterine epithelium had two epithelial layers with distinctive patterns of glycosylation, the apical layer stained strongly with Anguilla anguilla lectin and the basal cells with lectins from Wisteria floribunda and Helix pomatia, the latter indicating plentiful N-acetyl galactosamine though both layers stained variably with other lectins also. A population of sparse, large, globular cells expressed alpha2,3-linked sialic acid and N-acetyl glucosamine oligomers. Fetal and maternal vessels were heavily glycosylated as in their therian counterparts. These data indicate a prominent role for glycans at the fetomaternal interface of these chondrichthyan fishes.

Sperm storage in the oviduct of the internal fertilizing frog Ascaphus truei.

This study provides the first descriptions of sperm storage at the tissue and cellular levels in a female frog or toad. Oviducal anatomy was studied by light and electron microscopy in Ascaphus truei from north coastal California. Ascaphus truei is one of the few species of anurans in which fertilization is internal. Unlike other anurans with internal fertilization, however, mating in A. truei consists of a unique combination of amplectic and copulatory mechanisms that we term "copulexus." Posterior to a short, aglandular infundibular region, the oviduct possesses: 1) a proximal, convoluted ampullary region where intrinsic tubular glands secrete gelatinous envelopes around eggs; 2) a middle ovisac region where fertilization occurs; and 3) a distal oviducal sinus formed by medial junction of the ovisacs. Sperm storage tubules (SSTs) occur in the anterior portions of the ovisacs and consist of simple tubular glands. SSTs and the rest of the oviducal lining stain positively with the periodic acid-Schiff's procedure for neutral carbohydrates and this reaction is especially intense in reproductively active females. Sperm were found in the SSTs of gravid females as well as some nonvitellogenic females. The sperm are in orderly bundles in the SSTs, and although occasionally sperm nuclei were embedded in the epithelium, no evidence for spermiophagy was found. Oviducal sperm storage in A. truei is homoplastic, with closest structural similarities to squamate reptiles. Oviduct/sperm design constraints appear to limit the options for expression of features associated with oviducal sperm storage.

Ultrastructural analysis of folliculogenesis in the ovary of the yellow spotted stingray, Urolophus jamaicensis.

The ovary of the yellow spotted ray, Urolophus jamaicensis, is embedded in the epigonal gland, a lymphomyeloid organ. The covering of the ovary is composed of a germinal epithelium that is cuboidal and dome-shaped with microvilli. Adjacent cells have elaborate intercellular folds that create dilated intercellular spaces. In previtellogenic follicles, the follicle cells are simple cuboidal and contain modest amounts of synthetic or transport organelles. As vitellogenesis proceeds, the epithelium becomes multilaminar. Follicle cells are columnar as yolk precursors are transported from the maternal circulation, through the follicle cell cytoplasm, to the oocyte. Large, round cells occur in the follicle wall that contain lipid-like substances. These cells decrease in size and number as folliculogenesis proceeds and eventually disappear prior to ovulation. Columnar follicular cells and the oocyte have cellular extensions that impinge upon the zona pellucida. Transosomes are follicle cell extensions that indent the oocyte membrane. Tips of transosmes become enclosed by a layer of oocyte plasmalemma. The tips of transosomes pinch off and become resident in the ooplasm. Dense staining material occurs on the inner surface of the transosome membrane derived from the follicle cell. In Other animals, this material has been described as ribosome-like. This study is the first to document the presence of transosomes in a group other than Aves or reptiles. Follicle cells are supported by an extremely thick basal lamina. Subjacent to the lamina is the vascularized theca with fibroblasts embedded in a collagenous network. There is no differentiation into definitive theca interna and externa. In vitellogenic eggs, extensive inward folding of the follicular epithelium occur thereby generating more surface area for the transport of yolk precursors to the oocyte. Atretic follicles are common.

Anatomy, histology, and development of the cardiac valvular system in elasmobranchs.

We report here on the anatomy, histology, and development of the three sets of cardiac valves in embryonic and adult elasmobranch fishes. The sinus venosus is the first segment of the heart to receive blood, and a pair of sinoatrial (SA) valves prevent backward flow of blood into the sinus venosus. The SA valves derive from two dorsolateral infoldings of the cardiac wall and consist of a simple endocardium covering transverse sheets rich in collagen. The SA valves are simple flaps of tissue without papillary muscles or chordae tendineae. Blood from the atrium passes the atrioventricular (AV; semilunar) valves, which are attached to papillary muscles in the ventricle by way of the chordae tendineae. A series of rows of conal or pocket valves (CV) in the conus arteriosus, equipped with chordae tendineae but no papillary muscles, prevent blood from reentering the ventricle. Chordae tendineae form in a similar fashion in both chambers. Elevations from the chamber wall emerge as a sheet covered on both surfaces with endocardium and separated by a core of connective tissue. Endocardial cells extend basal projections toward the opposing epithelium through their basal laminae. Basal cell projections make contact to create perforations that enlarge to produce spaces between the nascent chordae. Fibroblasts in the core of the chordae enlarge and strengthen the chordae by producing linear arrays of collagen fibers.

Subcellular organization of the placenta in the Atlantic sharpnose shark, Rhizoprionodon terraenovae.

The Atlantic sharpnose shark, Rhizoprionodon terraenovae, is a viviparous anamniote that develops a yolk sac placenta composed of: a) uterine mucosa, b) egg envelope and c) fetal yolk sac mucosa. The transporting uterine mucosa is a squamous epithelial bilayer with prominent lateral and basal infoldings between contiguous cells. The surface cells have prominent secretion vesicles that empty their contents to the exterior. Immediately beneath the epithelium is a basal lamina and a profuse vascular supply with a continuous endothelium. The epithelium of paraplacental uterine sites is mucous. The tertiary egg envelope is retained throughout gestation and separates the distal part of the yolk sac from the maternal uterine mucosa. The egg envelope is compact on the yolk sac surface but displays delaminations on the uterine surface. The fetal yolk sac is composed of two portions, viz., a proximal, saccular region and a heavily vascularized, rugose, distal portion. The proximal portion has ultrastructural characteristics of a steroid hormone producing tissue, including massive smooth endoplasmic reticulum frequently forming whorled arrays. However, definitive evidence that the yolk sac is an endocrine organ is lacking. The distal portion of the fetal yolk sac is composed of a squamous epithelial bilayer that is separated from the underlying vascular network by a continuous basal lamina. The endothelium of the vessels is fenestrated. Cytoplasmic characteristics of these cells include an extensive Golgi complex, smooth walled caveolae, vesicles with electron-dense contents that are presumably endocytotic in nature and dense bodies that are suggested to be lysosomes that are involved in the digestion of material that may be yolk metabolites.

Fine structure of the term umbilical cord in the Atlantic sharpnose shark, Rhizoprionodon terraenovae.

The fine structure of the umbilical cord and appendiculae in the Atlantic sharpnose shark, Rhizoprionodon terraenovae, is examined by light, scanning and transmission electron microscopy. During ontogeny of placental sharks, the yolk sac and stalk become progressively modified as a functional hematrophic placenta and umbilical cord respectively. In most placental sharks the umbilical cord is smooth. In the Atlantic sharpnose shark, the epithelial ectoderm of the somatopleure forms richly vascularized extensions termed appendiculae. Scanning electron microscopy reveals that the base and shaft of appendiculae are flattened while the distal portion may be expanded to form one to three lobes. The surface of appendiculae is composed of two distinct cell types, the most plentiful are microvillar cells. The second cell type contains prominent granules. These cells are much larger than the former and are partially submerged below the surface, except for the cell apex. These cells undergo secretory cycles ending in expulsion of their contents. The possible function of the granulated cells is discussed. The umbilical cord contains an umbilical vein, umbilical artery, ductus vitellointestinalis and extraembryonic coelom. The endodermal ductus initially conveys yolk from the yolk sac to the fetal gut by activity of ciliated cells lining it. The ductus persists in the adult. Microvillar cells, also present in the ductus, may play a role in the absorption of yolk metabolites early in development, prior to yolk depletion. Enteroendocrine cells are wedged between the ciliated and microvillar cells. These cells may exert paracrine regulation of local areas of the ductus.

Subcellular organization of the yolk syncytial-endoderm complex in the preimplantation yolk sac of the shark, Rhizoprionodon terraenovae.

The structure of the yolk syncytial-endoderm complex of the preimplantation yolk sac of the shark is examined by light- and transmission electron microscopy. The yolk syncytium is bounded by a membrane that is anchored to the plasmalemma of adjacent endoderm cells by desmosomes. Enlarged nuclei, rough endoplasmic reticulum, Golgi complexes, mitochondria, and other cellular organelles populate the syncytium. Microtubules and filamentous elements are also observed free in the syncytium. Yolk is present as pleomorphic droplets, the profiles of which are generally spherical but may be vesicular, especially at the periphery of large yolk droplets. Occasionally, large yolk droplets have a paracrystalline configuration. Small yolk droplets are modulated through the Golgi complex of the yolk syncytium, and it is suggested that acid hydrolases are added there. Small yolk droplets released from the maturing face of the Golgi complex are sequestered in membrane-limited packets. The membrane of the packets fuses with the membrane enveloping the yolk syncytium and the yolk droplets are released into the yolk syncytial-endoderm interspace. Subsequently, the yolk droplets are endocytosed by the endoderm. Yolk droplets disperse and fuse to form the large irregular yolk inclusions of the endoderm. Yolk metabolites are transported out of the endoderm through the yolk sac endothelium. The yolk sac endoderm thus mediates the transfer of metabolites from the yolk mass to the extraembryonic circulation.

Stingray placental analogues: structure of trophonemata in Rhinoptera bonasus.

The cownose ray, Rhinoptera bonasus, displays a non-placental form of viviparity since direct maternal-embryonic connections are lacking. Early stage embryos depend on yolk reserves for growth to 215 mm disc width; growth to term, 405 mm disc width, is effected by ingestion of uterine histotrophe. During late gestation, the maternal uterine epithelium possesses 2-3 cm long spatulate, villiform appendages, termed trophonemata. These secrete histotrophe, which is a viscous, nutrient fluid. Scanning electron microscopy of the trophonematal surface reveals branching ridges, each of which is supplied by a capillary. The secretory unit is composed of 8-10 cells joined by extensive junctional complexes. Characteristically, secretory cells have a rough endoplasmic reticulum whose irregular cisternae are grossly distended and filled with low density flocculent material. Uncoated vesicles are given off by an extensive juxtanuclear Golgi complex. Coated vesicles are also present, but are not directly associated with the Golgi complex. Electron dense granules, larger lysosome-like vesicles, and multivesicular bodies are in the vicinity of the Golgi.

Ultrastructure of the full-term shark yolk sac placenta. I. Morphology and cellular transport at the fetal attachment site.

During ontogeny, the yolk sac of some viviparous sharks differentiates into a yolk sac placenta that persists to term. The placenta is non-invasive and non-deciduate. Hematrophic transport is the major route of nutrient transfer from mother to fetus. The placental unit consists of: (1) an umbilical stalk; (2) the smooth, proximal portion of the placenta; (3) the distal, rugose portion; (4) the egg envelope; and (5) the maternal uterine tissues. Exchange of metabolites is effected through the intervening egg envelope. The distal rugose portion of the placenta is the fetal attachment site. It consists of: (1) surface epithelial cells; (2) a collagenous stroma with vitelline capillaries; and (3) an innermost boundary cell layer. The columnar surface epithelial cells are closely apposed to the inner surface of the egg envelope. Wide spaces occur between the lateral margins of adjacent cells. Surface epithelial cells contain an extensive apical canalicular-tubular system and many whorl-like inclusions in their basal cytoplasm. Capillaries of the vitelline circulation are closely situated to these cells. A well-developed collagenous stroma separates the surface epithelium from an innermost boundary cell layer. In vitro exposure of full-term placentae to solutions of trypan blue and horseradish peroxidase (HRP) reveals little uptake by the smooth portion of the placenta but rapid absorption by the surface epithelial cells of the distal, rugose portion. HRP enters these cells by an extensive apical system of smooth-walled membranous anastomosing canaliculi and tubules. Prominent whorl-like inclusions that occupy the basal cytoplasm of the surface cells, adjacent to the pinocytotically active endothelium of the vitelline capillaries, are hypothesized to be yolk proteins that are transferred from the mother to embryo throughout gestation.

Ultrastructure of the full-term shark yolk sac placenta. II. The smooth, proximal segment.

The smooth, proximal portion of the yolk sac placenta of the sandbar shark, Carcharhinus plumbeus is comprised of: (1) An outermost epithelial ectoderm; (2) an intervening collagenous stroma; and (3) an inner mesothelium. The surface epithelium may be one to three cell layers thick. The surface epithelium comprises two cell types. A cuboidal cell that has a dome-like apical surface covered with microvilli and an ovoid nucleus predominate. These cells contain lipid inclusions, many cytoplasmic filaments, and are joined by desmosomes. The second cell type has a convoluted nucleus and a flattened cell apex with microvilli, cilia, and paddle cilia. Golgi complexes and elements of the endoplasmic reticulum are relatively uncommon in the cytoplasm of both cell types. Microplicae also occur on the surface of some cells. The smooth, proximal portion of the placenta is sparsely vascularized. The innermost cellular elements of the surface epithelium rest on a prominent basal lamina. A collagenous zone separates the epithelial basal lamina from the basal lamina of the mesothelium. The mesothelial cells are squamous with a fusiform nucleus, many pinocytotic pits and vesicles, and a large number of cytoplasmic filaments. The endoplasmic reticulum, except for occasional patches of the rough type, and the Golgi complex are poorly developed. Ultrastructural tracer studies show that this portion of the placenta does not absorb horseradish peroxidase (HRP) and trypan blue.

Ultrastructure of the full-term shark yolk sac placenta. III. The maternal attachment site.

During mid- and late gestation, the uterus of sandbar sharks possesses specialized sites for exchange of metabolites between the mother and fetus. Attachment sites are highly vascular, rugose elevations of the maternal uterine lining that interdigitate with the fetal placenta. The maternal epithelium remains intact and there is no erosion. The attachment site consists of a simple, low columnar juxtaluminal epithelium underlain by an extensive vascular network. Juxtaluminal epithelial cells possess branched microvilli, saccular invaginations of the apical surface, and coated pits. They contain numerous coated vesicles, lipid-like inclusions, a prominent rough endoplasmic reticulum, and many free ribosomes. Tight junctions join the luminal aspect of adjacent cells. Lateral cell boundaries are highly folded and interdigitated. Capillaries are closely apposed to the basal cell surfaces. The endothelium is pinocytotically active. Comparison with the uterine epithelium of non-placental sharks, mammalian epitheliochorial placentae, and selected transporting epithelia reveals that the structure of the maternal shark placenta is consistent with its putative multiple functions, viz: (1) nutrient transfer; (2) transport of macromolecules, e.g., immunoglobulins; (3) respiration; and (4) osmotic and ionic regulation.

Permeability of external gill filaments in the embryonic shark. Electron microscopic observations using horseradish peroxidase as a macromolecular tracer.

External gill filaments of sharks are purely transient embryonic structures. They contain a single vascular sinusoidal loop that is continuous with the afferent and efferent branchial arteries. Each filament is comprised of a squamous epithelial bilayer that rests upon a prominent basal lamina. A collagenous stroma separates the epithelium from the underlying endothelium. The epithelium, from an embryo 4.5 cm in total length, is characterized by microvilli with smooth walled vesicles at their bases, a luminal glycocalyx, prominent tubular and vesicular elements, rough endoplasmic reticulum, a Golgi complex, a flattened nucleus, coated vesicles, lipid-like inclusion bodies and sparse cytoplasmic fibrils. Adjacent epithelial cells are joined by a zonula occludens, a zonula adherens and up to five maculae adherentes. The endothelium possesses mitochondria, rough endoplasmic reticulum, a Golgi complex, coated vesicles and many micropinocytotic vesicles on both the adluminal and abluminal surfaces. The endothelium has no basal lamina and is not associated with smooth muscle. After exposure to horseradish peroxidase (HRP) for 10 min, reaction product nearly occludes the cytoplasm of some surface epithelial cells. The deeper epithelial cells have reaction product in smooth walled vesicular and tubular elements. Reaction product is also present in smooth walled endothelial vesicles. Gill filaments from a 10 cm embryo show marked changes from earlier stages. In the epithelial cells, there is an increase in the number of cytoplasmic filaments and the formation of a dense terminal web. Fewer vesicles, tubules, rough endoplasmic reticulum and mitochondria, and a less elaborate Golgi complex characterize the epithelium. The endothelium remains unchanged. The amount of collagen increases and fibroblasts are observed in the stroma. These modifications contribute to the strength of the filaments and allow the gills to withstand increased abrasion by the developing skin denticles. These experiments establish the capability of external gill filaments to take up a macromolecular tracer in the form of horseradish peroxidase. Later in development, the yolk sac of R. terraenovae becomes modified as a yolk sac placenta which functions both in respiration and hematrophic nutrition. In viviparous sharks, the uterus elaborates nutrient-rich secretions. External gill filaments may thus serve as a nutrient absorptive membrane before the establishment of the yolk sac placenta as well as perform its respiratory function.

Morphogenesis of chordae tendineae. I: Scanning electron microscopy.

The formation of the chordae tendineae of the left atrioventricular valve in the chick embryo is described using scanning electron microscopy. These supportive structures for the valve cusps develop between days 6 and 13 of incubation. Elevations which represent the primitive papillary muscles form on the ventricular wall. These elevations bifurcate into thin, web-like folds which are attached to the primitive valve cusps. The folds are the primordia of the chordae tendineae. Linear ridges develop on the web between the cusp and papillary muscle. These ridges alternate with depressions. The depressions become perforate to create the individual chorda from the linear ridges. Multiple perforations form initially but they typically consolidate to create one large aperture between two chordae. Some interchordal connections of tissue do persist throughout the period studied. During the period of perforation, prominent rounded cells are typical of the endocardium between the chordae. These cells are similar at the scanning electron microscope level to those present in the formation of the foramina secunda of the atrial septum. Primary, secondary, and tertiary chordae tendineae appear to develop in the same manner. First order chordae (those attached at the free margin of a cusp) are not found in the chick embryo. The majority of the chordae are second order, which insert into the ventricular surface of the cusp a short distance from the free edge. These chordae typically have a horizontal banding or grooving along their length. Third order chordae which extend from the papillary muscle to the ventricular wall are also present. It is suggested that chordal development is a programmed cellular and hemodynamic event.

Ultrastructure of the pre-implantation shark yolk sac placenta.

During ontogeny, the yolk sac of viviparous sharks differentiates into a yolk sac placenta which functions in gas exchange and hematrophic nutrient transport. The pre-implantation yolk sac functions in respiration and yolk absorption. In a 10.0 cm embryo, the yolk sac consists of six layers, viz. (1) somatic ectoderm; (2) somatic mesoderm; (3) extraembryonic coelom; (4) capillaries; (5) endoderm; and (6) yolk syncytium. The epithelial ectoderm is a simple cuboidal epithelium possessing the normal complement of cytoplasmic organelles. The endoplasmic cisternae are dilated and vesicular. The epithelium rests upon a basal lamina below which is a collagenous stroma that contains dense bodies of varying diameter. They have a dense marginal zone, a less dense core, and a dense center. The squamous mesoderm has many pinocytotic caveolae. The capillary endothelium is adjacent to the mesoderm and is delimited by a basal lamina. The endoderm contains yolk degradation vesicles whose contents range from pale to dense. The yolk syncytium contains many morphologically diverse yolk granules in all phases of degradation. Concentric membrane lamellae form around yolk bodies as the main yolk granules begin to be degraded. During degradation, yolk platelets exhibit a vesicular configuration.