Conditional deletion of E11/podoplanin in bone protects against load-induced osteoarthritis.

BACKGROUND: Subchondral bone (SCB) thickening is one of the earliest detectable changes in osteoarthritic joints and is considered a potential trigger for subsequent articular cartilage degeneration. In this manuscript, we examine whether disruption to the SCB osteocyte network contributes to the initiation and pathogenesis of osteoarthritis. METHODS: We examined expression patterns of the glycoprotein E11/podoplanin by immunohistochemical labelling in murine, human and canine osteoarthritis models. We also examined the effects of twice-weekly administration of Bortezomib, a proteasome inhibitor which stabilises osteocyte E11 levels, to C57/BL6 wild-type male mice (1 mg/kg/day) for 8 weeks after surgical destabilisation of the medial meniscus. By inducing osteoarthritis-like changes in the right knee joint of 12-week-old male E11 hypomorphic mice (and corresponding controls) using a post-traumatic joint loading model, we also investigated whether a bone-specific E11 deletion in mice increases joint vulnerability to osteoarthritis. Articular cartilage degradation and osteophyte formation were assessed by histology and in line with the OARSI grading system. RESULTS: Our studies reveal increased E11 expression in osteocytes of human and canine osteoarthritic SCB. We found that Bortezomib administration had no effect on surgically-induced osteoarthritis, potentially due to a lack of the expected stabilisation of E11 in the SCB. We also found, in concordance with our previous work, wild-type mice exhibited significant load-induced articular cartilage lesions on the lateral femoral condyle (p < 0.01) and osteophyte formation. In contrast, E11 hypomorphic mice did not develop osteophytes or any corresponding articular lesions. CONCLUSIONS: Overall, these data suggest that an intact osteocyte network in the SCB contributes to the development of mechanically-driven osteoarthritis. Further, the data presented here indicate that the molecular pathways that preserve the osteocyte network, such as those driven by E11, may be targeted to limit osteoarthritis pathogenesis.

FGF-2 promotes osteocyte differentiation through increased E11/podoplanin expression.

E11/podoplanin is critical in the early stages of osteoblast-to-osteocyte transitions (osteocytogenesis), however, the upstream events which regulate E11 expression are unknown. The aim of this study was to examine the effects of FGF-2 on E11-mediated osteocytogenesis and to reveal the nature of the underlying signaling pathways regulating this process. Exposure of MC3T3 osteoblast-like cells and murine primary osteoblasts to FGF-2 (10 ng/ml) increased E11 mRNA and protein expression (p < 0.05) after 4, 6, and 24 hr. FGF-2 induced changes in E11 expression were also accompanied by significant (p < 0.01) increases in Phex and Dmp1 (osteocyte markers) expression and decreases in Col1a1, Postn, Bglap, and Alpl (osteoblast markers) expression. Immunofluorescent microscopy revealed that FGF-2 stimulated E11 expression, facilitated the translocation of E11 toward the cell membrane, and subsequently promoted the formation of osteocyte-like dendrites in MC3T3 and primary osteoblasts. siRNA knock down of E11 expression achieved >70% reduction of basal E11 mRNA expression (p < 0.05) and effectively abrogated FGF-2-related changes in E11 expression and dendrite formation. FGF-2 strongly activated the ERK signaling pathway in osteoblast-like cells but inhibition of this pathway did not block the ability of FGF-2 to enhance E11 expression or to promote acquisition of the osteocyte phenotype. The results of this study highlight a novel mechanism by which FGF-2 can regulate osteoblast differentiation and osteocyte formation. Specifically, the data suggests that FGF-2 promotes osteocytogenesis through increased E11 expression and further studies will identify if this regulatory pathway is essential for bone development and maintenance in health and disease.

Hypomorphic conditional deletion of E11/Podoplanin reveals a role in osteocyte dendrite elongation.

The transmembrane glycoprotein E11/Podoplanin (Pdpn) has been implicated in the initial stages of osteocyte differentiation. However, its precise function and regulatory mechanisms are still unknown. Due to the known embryonic lethality induced by global Pdpn deletion, we have herein explored the effect of bone-specific Pdpn knockdown on osteocyte form and function in the post-natal mouse. Extensive skeletal phenotyping of male and female 6-week-old Oc-cre;Pdpnflox/flox (cKO) mice and their Pdpnflox/flox controls (fl/fl) has revealed that Pdpn deletion significantly compromises tibial cortical bone microarchitecture in both sexes, albeit to different extents (p < 0.05). Consistent with this, we observed an increase in stiffness in female cKO mice in comparison to fl/fl mice (p < 0.01). Moreover, analysis of the osteocyte phenotype by phalloidin staining revealed a significant decrease in the dendrite volume (p < 0.001) and length (p < 0.001) in cKO mice in which deletion of Pdpn also modifies the bone anabolic loading response (p < 0.05) in comparison to age-matched fl/fl mice. Together, these data confirm a regulatory role for Pdpn in osteocyte dendrite formation and as such, in the control of osteocyte function. As the osteocyte dendritic network is known to play vital roles in regulating bone modeling/remodeling, this highlights an essential role for Pdpn in bone homeostasis.

Gross morphological and morphometric study of the upper respiratory system of the African giant rat (Cricetomys gambianus, Waterhouse 1840).

The nose is a structurally and functionally complex organ in the upper respiratory tract. It not only serves as the principal organ for the sense of smell, but also functions to efficiently filter, warm, and humidify inhaled air before the air enters the more delicate distal tracheobronchial airways and alveolar parenchyma of the lungs. Despite the volume of published studies on the biology of rodents, there is no information on the gross upper respiratory morphology of the African giant rat (AGR) in the available literature. Hence, this study aimed to examine the anatomy of the turbinates, their meatuses, and the morphometry of the nasal cavity. The following were found and reported in this study: (a) There were three nasal conchae in AGR: the nasoturbinate, which was the largest; the ethmoturbinate, which was composed of one well-developed ectoturbinate and three well-developed endoturbinates; and the maxilloturbinate, which was fusiform, short, and branched. (b) Three major meatuses were observed: the dorsal nasal meatus, which was the longest and widest; the middle nasal meatus, which was without limbs but had a deep oval caudal recess; and the ventral nasal meatus, which directly continued caudally into the nasopharyngeal meatus. (c) Four ethmoturbinates with four slit-like meatuses were observed, each with dorsal and ventral limbs; the first contacted the middle nasal meatus but not the nasopharyngeal meatus. (d) There were three paranasal sinuses: one sphenoid, two frontal, and two palatine sinuses. The data obtained are relevant to pathologists and eco-morphologists, considering the burrowing habitat and behaviors of AGR, and provide baseline data for more investigative studies.

Comparative morphology and morphometry of the mesencephalic tectum in the African giant rat (Cricetomys gambianus).

The caudal colliculus serves as an integrative station and switchboard, controlling nucleus of lower auditory pathway and motor-auditory reflex production. The rostral colliculus coordinates reflexive movement of the head, neck, eye and focus the lens for visual tracking of objects. There is no information comparing mesencephalic tectum among neonates, juveniles and adults of African giant rat (AGR). Hence, this study aimed to compare the gross features and morphometric parameters of mesencephalic tectum postnatally in AGR. The following were found and reported: (a) Paired dorsal tips of caudal colliculi were observed through transverse fissure of the intact brain and so, corpora quadrigemina were partly occluded by cerebral cortex in neonates and juveniles. (b) The lateral and medial geniculate bodies were visible, though the lateral was grossly bigger than the medial in adults and juveniles but,  only the lateral was distinguishable in neonates. (c) Live body weight, absolute brain weight, caudal colliculus width, nose-rump and tail lengths increased as AGRs developed with age; mean values of rostral colliculus weight, caudal colliculus height and weight of caudal colliculus in neonates and juveniles were statistically same; while midbrain weight and rostral colliculus height tends to decrease as rats aged. (d) The mean weight of caudal colliculi and width of rostral colliculi were not affected by age. (e) Caudal colliculi were grossly wider than rostral in juveniles and adults, but not neonates. Established regression formulae are necessary to avoid future sacrifice of this rodent.

Relationship between age and brainstem allometry in the African grasscutter (Thryonomys swinderianus Temminck, 1827).

Allometric values of brainstem structures were evaluated in African grasscutters Thryonomys swinderianus (n = 27). Brain samples were extracted from 9 animals each of 3 days (neonates), 72 days (juveniles) and 450 days of age (adults). The midbrain, pons and medulla oblongata were separated from each brain sample and dimensions and weights obtained. The weights of the midbrain in the neonate, juvenile and adult African grasscutters were 0.33 g ± 0.01 g, 0.47 g ± 0.01 g and 0.93 g ± 0.02 g, respectively. The increase from neonate to juvenile (p = 0.002) and adult (p = 0.003) was significant. The pons lengths in the neonate, juvenile and adult were 2.05 mm ± 0.05 mm, 3.86 mm ± 0.05 mm and 4.16 mm ± 0.22 mm, respectively. There was a significant increase in the length of the pons from the neonate to the juvenile (p = 0.002), but the increase from the juvenile to the adult period was not significant (p = 0.263). There was also a significant (p < 0.05) increase in the weights and lengths of the medulla oblongata from neonate to juvenile and adult periods. In adults, the nose-rump length and the length of the medulla were significantly negatively correlated (r² = 0.47; p = 0.043). The present study concluded that the postnatal development of some brainstem structures in the African grasscutter varies with age.

The Stomach of the Adult African Catfish (Clarias gariepinus, Siluriforme s: Clariidae) in Farm Conditions: A Morphological and Mucin Histochem istry Analysis / El Estómago del Bagre Africano Adulto (Clarias gariepinus, Siluriformes: Clariidae) en Condiciones de Granja: Análisis Morforlógico e Histoquímico por Mucina

The stomach of the farmed African catfish (Clarias gariepinus, Siluriformes: clariidae) was investigated in this study to establish its basic anatomy, as there is scanty information available on the literature. It produced to baseline data for further investigative research, help fish clinicians in pathology and aid nutritionists in feed management. After cropping from a commercial aquaculture, apparently healthy fish were immobilized and euthanized by using chloroform. The samples were dissected and slices of cardiac, fundic and pyloric stomach processed through routine histological procedures. Grossly, the stomach was J-shape. Cranial to the stomach was the oesophagus but no sphincter was seen separating them. Caudally, a pyloric sphincter separated the stomach from the proximal intestine. The stomach contained three regions cardiac, fundic and pyloric. The entire stomach surface was lined by a simple columnar epithelium containing mucin at the apical cytoplasm. Only the cardiac and fundic regions contained gastric glands in the lamina propria, but the glands were more developed in the fundic region. The tunica muscularis contained smooth muscle cells in an inner circular and outer longitudinal orientation. Mucin histochemistry revealed the presence of only neutral mucin in the stomach. The results obtained in this study suggest that the absence of gastric glands in the pylorus may be an adaptation of this species to reduce the quantity of acid entering the proximal intestine; hence it might help the alkaline medium to maximize pancreatic enzyme actions in the proximal intestine. The presence of only neutral mucin in the stomach will act as a buffer to neutralize the high acidic stomach content. Neutral mucins are also associated with transport of macromolecules.

Se efectuó un estudio de tipo histológico e histoquímico con mucina, en el estómago del bagre africano (Clarias gariepinus, Siluriformes: clariidae) cultivado en granja acuícola, para establecer su anatomía básica, debido a la escasa información disponible en la literatura. La investigación generó datos para ulteriores investigaciones, que contribuirán con los ictiopatólogos y ayudarán a los nutricionistas en el manejo alimentario. Después de su cultivo en una granja acuícola comercial, los peces aparentemente sanos, fueron inmovilizados y sacrificados, usando cloroformo. Se tomaron muestras de las regiones cardial, fúndica y pilórica, y se procesaron histológicamente. En su conjunto, el estómago presentaba una apariencia de “J”. El esófago estaba situado cranealmente al estómago, pero no se observó un esfínter que los separara. Caudalmente, un esfínter pilórico separaba al estómago del intestino proximal. El estómago contenía tres regiones: cardiaca, fúndica y pilórica. La totalidad de la superficie estomacal se encontraba recubierta por un epitelio cilíndrico, que contenía mucina en el citoplasma apical. Solamente las regiones cardiaca y fúndica tenían glándulas gástricas en la lámina propia, que eran más desarrolladas en la región fúndica. La túnica muscular poseía células musculares lisas con una orientación interna circular y externa longitudinal. La histoquímica mediante la mucina reveló la presencia solo de mucina neutra en el estómago. Los resultados obtenidos en este estudio sugieren que la ausencia de glándulas gástricas en el píloro, pudiera ser el resultado de una adaptación de esta especie para reducir la cantidad de ácido que penetra en el intestino proximal; por tanto, pudiera ayudar al medio alcalino a maximizar las acciones de las enzimas pancreáticas en el intestino proximal. La sola presencia de mucina neutra en el estómago actuaría como amortiguador contra el elevado contenido ácido del estómago. Las mucinas neutras también están asociadas con el transporte de micromoléculas.

The Kidney and Adrenal Gland of the African Palm Squirrel Epixerus ebii: A Microanatomical Observation / El Riñón y la Glándula Adrenal de la Ardilla de la Palma Africana Epixerus ebii: Una Observación Microanatómica

The kidneys and adrenal glands of the African palm Squirrel (Epixerus ebii), were subjected to histological and mucin histochemical studies. The kidney was covered by a capsule of periodic acid of shiff (PAS) positive connective tissue fibres. The kidney parenchyma was composed of large peripheral cortex and smaller centrally placed medulla. The cortex contained renal corpuscle, proximal convoluted tubules, distal convoluted tubules and juxtaglomerullar complex. At the hilus, the renal artery and vein, ureters, and nerve fibres were surrounded by adipose tissue. The renal medullar was composed of Henles’ loop and collecting ducts. The adrenal gland cortex contained zona glomerulosa, zona fasciculate and zona reticulata. The zona fasciculata was the largest of the zones containing polyhedrally shaped cells with less basophilic nuclei and very foamy cytoplasm. Some cells of zona fasciculate were binucleated. The adrenal medulla contained mostly glandular cells, few ganglion cells and capillaries. This study establishes that the organs investigated are typical of mammals. It will fill the knowledge gap, help wildlife clinicians in disease diagnosis of these organs in this species.

Los riñones y las glándulas adrenales de la ardilla de la Palma Africana (Epixerus ebii) fueron objeto de estudios histológicos y de estudios histoquímicos con mucina. El riñón estaba cubierto por una cápsula de fibras de tejido conectivo que resultaron positivas a la coloración con ácido peryódico de Schiff (PAS). El parénquima renal estaba constituido por una gran corteza periférica y una médula más pequeña, ubicada centralmente. La corteza contenía el corpúsculo renal, los túbulos contorneados proximales, los túbulos contorneados distales y un complejo yuxtaglomerular. A nivel del hilio del órgano, la arteria y vena renales, los uréteres y las fibras nerviosas estaban rodeados de tejido adiposo. La médula renal estaba compuesta por el asa de Henle y los túbulos colectores. La corteza de la glándula adrenal contenía las zonas glomerular, fasciculada y reticular. La zona fasciculada era la mayor de las tres y contenía células con forma poliédrica con núcleos menos basofilícos y citoplasma muy esponjoso. Algunas células de la zona fasciculada eran binucleadas. La médula adrenal contenía principalmente células glandulares y pocas células ganglionares y capilares. Los resultados de este estudio establecieron que los órganos investigados son típicos de los mamíferos. Esta investigación llenará la brecha de conocimiento existente y ayudará a los clínicos de fauna silvestre en el diagnostico de enfermedades de estos órganos en esta especies.