Effects of FGF-2 on E11-mediated osteocytogenesis in skeletal health and disease

Fibroblast growth factor 2 (FGF-2) is known to be released from cartilage upon injury and is able to influence chondrocyte gene expression in vitro. In cartilage, FGF-2 regulates E11/podoplanin expression in murine joints following surgical destabilisation (DMM model of osteoarthritis (OA)), and in cartilage explant injury models. In bone, E11 is critical for the early stages of osteocytogenesis and is responsible for the acquisition of the osteocyte dendritic phenotype. This dendritic phenotype is dysregulated in OA and given the known role of the osteocyte in controlling bone remodelling, this may contribute to the subchondral bone thickening observed in OA. Hence, the aim of this study was to elucidate the nature of FGF-2- mediated E11 expression and osteocytogenesis in skeletal health and disease. This thesis has shown that FGF-2 dose-dependently increased E11 mRNA expression in MC3T3 cells, primary osteoblasts and in primary calvaria organ cultures, which was confirmed by E11 protein western blotting data. The FGF-2 induced changes in E11 expression were accompanied by significant increases in the mRNA expression of the osteocyte markers Phex and Dmp1, and significant decreases in the mRNA expression of the osteoblast markers Col1a1, Postn, Bglap and Alpl expression. This thus supports the hypothesis that FGF-2 drives osteocytogenesis. The acquisition of osteocyte phenotype involves the re-organisation of the cytoskeleton, such as F-actin. This step is important for the transition of cuboidal-shaped osteoblasts to the stellate-shaped osteocyte phenotype. FGF-2 stimulation of MC3T3 cells and primary osteoblasts revealed more numerous and longer dendrites, as visualised by phalloidin staining for F-actin and indicative of the acquisition of the osteocyte phenotype. In contrast, control cells had a typical rounded morphology with fewer and shorter dendrites. Furthermore, immunofluorescence labelling for E11 in control cells revealed uniform distribution throughout the cytoplasm, especially in the perinuclear region. In contrast, FGF-2 treated cells showed a modified distribution where E11 was negligible in the cytoplasm, but concentrated in the dendrites. The use of siRNA knockdown of E11 achieved a 70% reduction of basal E11 mRNA expression. This knockdown also effectively abrogated FGF-2-related changes in E11 expression and dendrite formation as disclosed by mRNA and protein expression, immunofluorescence and F-actin staining with phalloidin. Despite these FGF-2 driven increases in E11 and osteocyte dendrite formation in vitro, immunohistochemical labelling revealed no differences in E11 expression in subchondral, trabecular and cortical osteocytes from naïve Fgf-2 deficient mice in comparison to wild-type mice. Similar results were observed upon sclerostin immunolabelling. FGF-2 stimulation of MC3T3 cells elicited activation of ERK1/2, Akt and p38 MAPK. However, inhibition of the aforementioned pathways failed to reduce FGF-2- mediated E11 expression and as such, the specific signalling pathway responsible remains unclear. Upstream, the expression of Fgfr1 was increased (>10-fold) over 24 h time point, while a reduction was seen in Fgfr2/3 expression over same time point especially in the FGF-2 treated cultures. This suggests that increased E11 expression and the acquisition of the osteocyte phenotype may be speculatively though upregulation of Fgfr1. The expression of E11 and sclerostin in OA pathology in mice, human and dogs were investigated. Initially sequence homology using the Clustal Omega alignment program showed both proteins to be homologous in the domestic animals under study. A comparative study using canine subchondral bone osteocytes revealed increased E11 expression in the OA samples relative to the control. This feature may be related to newly embedded osteocytes during sclerosis. However, E11 and sclerostin were unchanged in both murine (DMM) and human OA subchondral bone osteocytes in comparison to controls. In mice, this may be due to limited OA development; whilst in humans the sample size, age, stage of the disease and sourcing from same diseased joint may be important in the interpretation of the results. The expression of E11 and sclerostin during OA pathology was also investigated in Fgf-2 deficient mice in which OA was induced using the DMM model. There was no difference in E11 expression between the OA and control (sham-operated) samples, suggesting that compensation of E11 expression may be mediated by growth factors from the FGF family. Surprisingly, increased E11 expression was observed in the control Fgf-2 deficient mice, in comparison to the wild-type control mice. This suggests a potential adjustment to loading by the contralateral knee, as this was not observed in naïve mice from both groups. Together, these data show that FGF-2 promotes the osteocyte phenotype, and that this is mediated by increased E11 expression. These results may help explain (1) the altered osteocyte phenotype and (2) increased subchondral bone thickening observed in OA. This knowledge will be of interest in the search for disease modifying therapeutics for skeletal health, including OA and osteoporosis

Assessment of the morphological development of the caecal tonsil in turkey (Meleagris gallopavo)

The post hatch (PH) development of the caecal tonsil in turkey (Meleagris gallopavo) was studied from day (D) 1 post-hatch to D 140. The tonsils appeared as nodular structure on the surface of the proximal part of the caecal base. The caecal wall of all ages was composed of four histological layers. The mucosa consisted of simple columnar epithelium with goblet cells. At D 1 PH the tonsil was composed of minute aggregates of proliferating lymphoid cells. By D 7, Leiberkuhn glands were observed in the stroma of the tonsil, and at D 28, nodules packed with lymphocytes and some plasma cells were observed. Between D 28 and D 133, there were gradual increase in dimension of the tonsils and proliferation of lymphatic nodules. At D 140, there was depletion of lymphatic nodules especially at the apical regions bordering the caecal lumen, and increase in thickness of the intervening connective tissue was very obvious.Key words: caecal tonsil, turkey (Meleagris gallopavo ), morphology, post-hatch, developmen

Comparative cytoarchitecture of the midbrain colliculi of defined age groups in the African giant pouched rat (Cricetomys gambianus)

Background: The rostral and caudal colliculi of mammalian midbrain are pivotal to vision and audition, respectively. Cytoarchitecture of these midbrain  colliculi in neonate, juvenile and adult African giant pouched rats is dearth. Objective: This study compared the histology of the nuclei, neurons and  laminations of the colliculi in these age groups. Methods: Thirty-six captive African giant pouched rats consisting of twelve neonates, juveniles and adults were used. Thickness of the histologic layers of  the rostral colliculi and lengths of the central nucleus of the caudal colliculi were compared among the groups. Results: the rostral colliculus  composed of an outermost stratum zonale, middle stratum griseum superficiale and innermost stratum griseum  profundus; migratory immature neurons of radial glial cells were observed in the stratum zonale of neonates, which disappeared in juveniles and adults;  the stratum griseum superficiale of neonates was characterized of already formed neurites, similar to those of the juveniles and adults. However, this  layer in adults had more oligodendrocytes than in juveniles. There was significant increase in thickness of the stratum griseum superficiale and stratum  griseum profundus, with increasing age (P < 0.05). The caudal colliculus contained a central nucleus, dorsal and lateral cortices, with significant increase  in the length of the central nucleus with increasing age (P < 0.05); there was transformation of fibre shaft in the lateral cortex of neonates into a mixture  of chain like and marshy matrix in the juveniles which completely disappeared in adults, indicative of mature lateral cortex in the later. Conclusion: The adult African giant pouched rat may have the best auditory and visual senses, followed by juvenile. The neonate has the least acuity of  these senses

Comparative morphology of the visual and auditory reflex centres in the African grasscutter (Thryonomys swinderianus – Temminck, 1827)

The African grasscutter is a nocturnal rodent, currently bred as a minilivestock, and research model. The present study was aimed at describing the structure of the auditory and visual reflex centres, with a view to understanding the nocturnal behaviour of the rodent. The rostral and caudal colliculi of the mesencephalic tectum were harvested from neonate, juvenile and adult African grasscutters and subjected to basic neuro-anatomical techniques. On each postnatal period, the corpora quadrigemina was bound rostrally by the 3rd ventricle and caudally by the 4th ventricle, and the caudal colliculus was grossly bigger than the rostral colliculus. The heights of the rostral and caudal colliculi in the neonates were 6.38 ± 0.28 mm and 8.78 ± 0.08 mm, respectively, and the difference was significant (P < 0.05). The values in the juveniles were 5.94 ± 0.21 mm and 11.45 ± 0.17 mm, respectively, and the difference was significant (P < 0.05). The values in the adults were 8.08 ± 0.35 mm and 16.16 ± 0.30 mm, respectively, and the difference was also significant (P < 0.05). The relative weight of rostral colliculus in the adult was significantly (P < 0.05) higher than that of the neonates, but not the juveniles. The relative weight of the caudal colliculus in the adults was significantly (P < 0.05) higher than that of the juveniles. In conclusion, the relatively bigger caudal colliculi, than the rostral colliculi, observed at all postnatal periods studied, suggests a more acute auditory than visual ability in the African grasscutter

STRUCTURE AND FUNCTIONAL SIGNIFICANCE OF BRANCHED ANASTOMOSING MUCOSAL FOLDS IN THE PROXIMAL INTESTINE OF THE FARMED AFRICAN CATFISH (Clarias gariepinus BURCHELL 1822)

ABSTRACT Histology has provided morphologist a powerful tool that enhances the nature, form and detailed structure of organs. The proximal intestine though a more muscular tube than the rest of the intestine presented a complex anastomosing mucosal fold resembling a honey comb-like structure. This specialization was not seen in the other intestinal segments. This specialization increases the surface area for nutrient absorption. This probably compensates for shortness of intestine and absence of pyloric caeca in this species. The glycoconjugates in the goblet cell of proximal intestine mucosal fold were subjected to mucin histochemical study. The study revealed the presence of neutral and acid mucin but acid mucin was predominant. The neutral mucin can be associated to buffering the effect of gastric acid, transport of small disaccharide molecules. The predominating acid mucin suggest air-breathing function since the acid will always make the epithelium moist for gaseous exchange between dissolved air in swallowed water and abundant blood vessels present in the tunica mucosa

Makroskopska građa malog mozga afričkoga divovskog trstičnog štakora (Thryonomys swinderianus - Temminck, 1827) tijekom fetalnog i postnatalnog razvoja

In Nigeria, the African grasscutter (Thryonomys swinderianus) is bred as a mini-livestock, and research model. This study provides some information on the ontogeny of its cerebellum through gross observations of external cerebellar features from foetuses on foetal days 60 and 90, neonates on postnatal day 3, juveniles on postnatal day 72 and adults on postnatal day 450. On foetal day 60, the cerebellum was a smooth, semi-circular mass, devoid of folia or sulci; cerebellar lobes and vermal lobules were not present. On foetal day 90, the vermis was distinct, but vermal lobules were indistinct. On postnatal day 3, the cerebellar cortex was convoluted with the folia and fissures of grossly distinct lobes. The cerebellar vermis was divided into all the lobules typical of mammalian vermis; while the lingula, central lobule, culmen and declive were on the cerebellar dorsum, the folium, tuber, pyramis, uvula and nodulus were caudally located. The culmen was undivided and the crus rostralis was unpaired. On postnatal day 72, the pre-central fissure was deeper than that observed on postnatal day 3; the culmen was divided into the pars rostralis and pars caudalis; the crus rostralis was bifoliated by a fissural line. On postnatal day 450, the cerebellar fissures were deeper than before; the vermal and hemispheric lobules were better delineated. The bifoliation of the crus rostralis was more evident. The intra-crural fissure was very distinct and referred to as the great horizontal fissure. The results of the present study will add to the data base of rodent neuroanatomy, necessary for teaching and research.U Nigeriji se afrički divovski trstični štakor (Thryonomys swinderianus) uzgaja kao mini stoka i model za istraživanje. U radu su prikazani podaci o razvoju njegova malog mozga, prikupljeni makroskopskim promatranjem vanjskih obilježja mozga kod fetusa u dobi 60 i 90 dana, novorođenčadi u dobi 3 dana, mladih životinja u dobi od 72 dana i odraslih životinja u dobi od 450 dana. Mali mozak fetusa u dobi od 60 dana bio je glatka, polukružna masa bez vijuga i brazda, na kojoj nisu bili prisutni moždani režnjevi ni vermisni režnjići. U dobi fetusa i u dobi od 90 dana bio je izražen vermis, ali vermisni su režnjići bili nejasni. Treći dan nakon porođaja kora maloga mozga je na vrlo izraženim režnjevima bila isprepletena s vijugama i usjeklinama. Vermis maloga mozga bio je podijeljen na sve režnjeve tipične za vermis sisavaca. Lingula, središnji režanj, kulmen i declive bili su na dorzumu maloga mozga, dok su folium, tuber, pyramis, uvula i nodulus smješteni kaudalno. Kulmen je bio nepodijeljen, a crus rostralis nesparen. 72 dana nakon porođaja predsredišnja usjeklina bila je dublja u odnosu na opažanja 3. dana nakon porođaja. Kulmen je bio podijeljen na pars rostralis i pars caudalis. Crus rostralis je linijom usjekline bio dvovijugav. Mali mozak životinja u dobi od 450 dana imao je dublje usjekline, a vermisni i hemisferični režnjevi bili su bolje razgraničeni. Dvovijugavost crus rostralis bila je još naglašenija. Intrakruralna usjeklina bila je vrlo izražena i prikazana kao velika vodoravna usjeklina. Rezultati ove studije nadopunit će bazu podataka o neuroanatomiji glodavaca koja je potrebna za poučavanja i istraživanja

THE KIDNEY AND ADRENAL GLAND OF THE AFRICAN PALM SQUIRREL Epixerus ebii: A Microanatomical Observation

Abstract   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.Resumen  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.&nbsp