Efficient aseptic and automatable vitrification of human Embryonic Stem Cells using bio-safe & chemically defined media

Conference (2013, March 23)

Long term culture and characterization of chicken primordial germ cells

Poster (2012, November)

Avian primordial germ cells (PGCs), can keep their germ cells properties and are foreseen as promising tools for developing avian transgenesis and preservation of genetic resources of endangered species ... [more ▼]

Avian primordial germ cells (PGCs), can keep their germ cells properties and are foreseen as promising tools for developing avian transgenesis and preservation of genetic resources of endangered species. We have developed original methods that allow long term (20 month) expansion of primary cultures of undifferentiated PGCs and their efficient cryopreservation. Blood samples were collected from stage 13-18 embryos, pooled, deposited in cell culture inserts and co-cultivated in the presence of irradiated BRL cells. This physically separated co-culture system along with selective culture medium promoted emergence, selection and proliferation of undifferentiated PGCs lines. Overall, 35% of blood samples gave rise to PGCs cell lines originating from three commercial layer breeds and two Belgian endangered breeds. PGCs lines were first characterised for the expression of the stem cells and PGCs characteristic marker SSEA-1 by FACS (expression rate: 90-99%). RT-PCR confirmed expression of germ-line specific markers (CVH, CDH, DAZL), pluripotency markers (cPouV, cSox2, cNanog), telomerase and CXCR4 receptor. In addition, by means of a quantitative PCR amplification of a chromosome W specific sequence, we demonstrated a drift of all our lines towards the male sex (WL), while they were initially isolated from pooled blood samples with statistically equivalent numbers of male and female embryos (35 females: 29 males). PGCs were subsequently efficiently cryopreserved by slow freezing or by a newly developed vitrification method. Labelled PGCs from 10 lines were injected in recipient embryos. Colonization of the genital ridges confirmed that PGCs retain their gonadal migratory ability, both after long-term culture (min 3, max 20 month) and after cryopreservation. [less ▲]

The three-dimensional reconstruction of the innervation pattern in the lymphoid compartment of the ovine pharyngeal tonsil highlighted a possible way of neuro-invasion by the scrapie agent.

in Proceedings of the 2nd Scientific Meeting of the Faculty of Veterinary Medicine, ULg, Belgium, October 19, 2012 (2012, October 19)

Morphological and morphometric analyses of the suspensory ligament in Standardbreds

Poster (2012, October)

Ultrasound techniques allow examination of some parts of the suspensory ligament (SL) but "anomalies" are regularly observed. Their significance is not known. Few studies have described the relationship ... [more ▼]

Ultrasound techniques allow examination of some parts of the suspensory ligament (SL) but "anomalies" are regularly observed. Their significance is not known. Few studies have described the relationship between ultrasonographic appearance and the exact morphology in histological sections. The aim of this study is to develop good techniques for cutting, staining, and showing the variation in the tissue composition within the SL. The SLs from the right limbs of 11 horses were collected. Samples were taken from cross-sections at six levels of the SL and they were embedded in paraffin or in Tissue-Tek®. Most of the paraffin sections were shredded. By using the cryosection technique, some freezing artifacts (holes) appeared. Therefore, a technique of freezing with cryoprotection was carried out, which produced the best results. Hematoxylin-phloxine-saffron gives a good contrast of colors between the tissues observed allowing the use of an image analysis program. The percentage of each tissue within the SL for each section and for six levels of the ligament was calculated. Results were analyzed by SAS software. The muscle tissue (PMT) and adipose tissue (PAT) decreased significantly (p < 0.0001), whereas the connective tissue (PCT) increased significantly (p < 0.0001) with age and when descending from the proximal to the distal level of the SL. The PMT was significantly higher (p < 0.0001) in females than males, while the PCT was significantly higher (p < 0.0001) in males than females. The PAT was significantly higher (p = 0.0278) in hindlimbs than in forelimbs. [less ▲]

Neuroimmune connections in ovine pharyngeal tonsil: potential site for prion neuroinvasion

in Cell & Tissue Research (2012)

Recent studies have proved the possible implication of nasal associated lymphoid tissues, mainly the pharyngeal tonsil, in prion pathogenesis. However, the mechanisms of this neuroinvasion are still being ... [more ▼]

Recent studies have proved the possible implication of nasal associated lymphoid tissues, mainly the pharyngeal tonsil, in prion pathogenesis. However, the mechanisms of this neuroinvasion are still being debated. To determine the potential sites for prion neuroinvasion inside the ovine pharyngeal tonsil, the topography of neurofilaments heavy (200 kDa) (NFH), neurofilaments light (70 kDa) (NFL) and glial fibrillar acidic protein (GFAP) was semi-quantitatively analysed inside the different compartments of the tonsil. The results showed that the most innervated areas were the interfollicular area and the connective tissue located beneath the respiratory epithelium. Even if the germinal centre of the lymphoid follicles was poorly innervated, the existence of rare follicular dendritic cell-nerve synapses inside the germinal centre indicates that this mechanism of neuroinvasion is possible but unlikely to be unique. The host PRNP genotype did not influence the pattern of innervation in these different tonsil compartments, unlike age: an increase of nerve endings in a zone of high trafficking cells beneath the respiratory epithelium occurred with ageing. A minimal age-related increase of innervation inside the lymphoid follicles was also observed. An increase in nerve fibre density around the lymphoid follicles, in an area rich in mobile cells able to transport PrPd, could ensure a more efficient infectivity, not in the early phase but in the advanced phase of lymphoinvasion after amplification of PrPd, or could act as direct site of entry during neuroinvasion. [less ▲]

Prevalence of naturally occuring cartilage defects in the ovine stifle

Poster (2012)

Morphological and histological studies of sheep’s brain

Poster (2011, December)

Introduction: The study of normal structures of the sheep’s brain is very important to understand pathological changes caused by the bluetongue virus in the fetus’s brain at various stages of the ... [more ▼]

Introduction: The study of normal structures of the sheep’s brain is very important to understand pathological changes caused by the bluetongue virus in the fetus’s brain at various stages of the gestation. Bluetongue is an arthropod-borne viral disease of domestic and wild ruminants. The serotype 8 is responsible for outbreaks in Northern Europe in 2006. This virus causes lesions in the brain of fetuses as hydrancephaly and porencephaly. The aim of this work is to improve knowledge of anatomy and histology of the central nervous system of the sheep. Methods: Seven heads of adult sheep and one from a fetus aged 4,5 months were used. All heads were first opened in the frontal area using bone’s saw and immerged in a formalin solution for 10 days. After a good fixation, the brains were extracted and sectioned. Transversal, frontal and sagittal sections were realized. The sections of two brains were stained with Berlin-blue and treated to be embedded in methylmetacrylate for gross morphology. The different parts of the 6 resting brains were then embedded in paraffin, cut and the histological sections were stained with haematoxylin/eosin, cresyl violet or by use of silver impregnation. Results: Gross morphological examination of the brains embedded in methylmetacrylate showed the detailed anatomy of the different parts. The staining with haematoxylin/eosin permitted to differentiate the grey matter, the different nucleus and the layers of cerebral and cerebellum cortex. The cresyl violet technique permitted to visualize the Nissl bodies and the silver impregnation revealed nerve fibers. In the fetus brain, blood vessels were very numerous in the brainstem, the cerebellum and the cerebrum. The grey matter was less organized and looser. Conclusion: This work establishes an anatomical and histological approach allowing future studies in ovine fetuses with and without brain lesions potentially caused by the bluetongue virus. [less ▲]

Morphology of the suspensory ligament (interosseous muscle III) of the horse

Poster (2011)

Introduction: The injuries of the suspensory ligament (SL) are important causes of lameness and financial losses in the equine industry. Ultrasound examination permitted to visualize some parts of the SL ... [more ▼]

Introduction: The injuries of the suspensory ligament (SL) are important causes of lameness and financial losses in the equine industry. Ultrasound examination permitted to visualize some parts of the SL. The significance of “abnormal” findings is however not sufficiently known. Until now, few studies described the relationship between the ultrasonographic appearance and the exact morphology in histological sections. The aim of this study is to develop good techniques for cutting and staining the SL and to improve knowledge about the normal morphology of the SL. Methods: In this study, the SL of eight <sound> horses were collected. The body of the SL was divided in 3 thirds and sampling was realised within each third and between the thirds. The samples were embedded in paraffin or in Tissue-Tek for cryosections. The sections were stained with hematoxylin/eosin or Masson's trichrome. For 3 SL, ultrasounds were performed before sampling. The digital tip was maintained in physiological position owing to a press. Results: Most of the paraffin sections were shredded because of the hardness of the tissue. Cryosection revealed a better preservation of tissues. Only some freezing artifacts (holes) appeared on a few sections. Muscles fibers surrounded by adipose tissue containing blood vessels were present mainly in the proximal and medium third of the SL whereas they were not found in the distal third. The remaining structure look like a tendon and was composed of collagen fibers, stained in green with the Masson's trichrome coloration. Conclusions: This study permitted to develop cutting and staining techniques for the SL and helped to map the adipose, muscular and tendinous parts within the SL. It lays down the bases of subsequent studies that will concern ultrasonographically examined digital tips of sound and pathological horses of different breeds and ages. [less ▲]

Développement d'un nouveau modèle ex vivo pour l'étude de l'invasion de l'épiderme félin par Microsporum canis

Conference (2011)

Effects of formoterol and ipratropium bromide on repeated cadmium inhalation -induced pulmonary inflammation and emphysema in rats

in European Journal of Pharmacology (2010), 25(647), 178-187