Long Term Cyclic Pamidronate Reduces Bone Growth by Inhibiting Osteoclast Mediated Cartilage-to-Bone Turnover in the Mouse.

Bisphosphonates, used to treat diseases exhibiting increased osteoclast activity, reduce longitudinal bone growth through an as yet undefined mechanism. Pamidronate, an aminobisphosphonate, was given weekly to mice at 0, 1.25, or 2.50 mg/kg/wk beginning at 4 weeks of age. At 12 weeks of age, humeral length, growth plate area, regional chondrocyte cell numbers, chondrocyte apoptosis, TRAP stained osteoclast number, and osteoclast function assessed by cathepsin K immunohistochemistry were quantified. Humeral length was decreased in pamidronate treated mice compared to vehicle control mice, and correlated with greater growth plate areas reflecting greater proliferative and hypertrophic chondrocyte cell numbers with fewer hypertrophic cells undergoing apoptosis. Pamidronate treatment increased TRAP stained osteoclast numbers yet decreased cathepsin K indicating that pamidronate repressed osteoclast maturation and function. The data suggest that long term cyclic pamidronate treatment impairs bone growth by inhibition of osteoclast maturation thereby reducing cartilage-to-bone turnover within the growth plate.

Spatial distribution of growth hormone receptor, insulin-like growth factor-I receptor and apoptotic chondrocytes during growth plate development.

Linear bone growth depends upon proliferation, maturation, and apoptosis of growth plate chondrocytes, processes regulated by growth hormone (GH) and insulin-like growth factor-I (IGF-I). To investigate the contribution of GH, IGF-I and apoptosis to growth plate function, the expression of GH receptor (GHR) and IGF-I receptor (IGF-IR) mRNA were evaluated by in situ hybridization in fractionated costochondral growth plates of growing rats (at 2, 4, and 7 weeks). Apoptosis was determined by TUNEL assay and morphology in histological sections. GHR mRNA was greatest in resting cells with hypertropic cells increasing GHR expression with increasing age. Hypertropic and resting cell IGF-IR mRNA declined over the ages studied. Receptor mRNA expression was altered by exposing cells to GH or IGF-I. GH and IGF significantly decreased GHR mRNA in proliferative cells. GH and IGF also decreased IGF-IR mRNA in resting cells and the 2- and 4-week-old proliferative and hypertropic cells. Treating cells in culture with GH increased the number of apoptotic cells across all ages and zones. Histologically, apoptotic cells were observed at the chondro-osseous junction and within actively proliferating chondrocytes but not in resting cells. Apoptosis was highest at 4 weeks of age with lateral regions displaying the greatest number of cells undergoing apoptosis. These data indicate that apoptosis plays a role in growth plate function, particularly spatial configuration as indicated by the preferential lateral cell apoptosis. The susceptibility of proliferative cells to GHR and IGF-IR down regulation during the period of greatest apoptosis supports a role for the GH-IGF axis in both proliferation and apoptosis during growth plate development.

The genetics of epilepsy in the Belgian tervuren and sheepdog.

Idiopathic epilepsy is characterized by recurrent seizure activity without an identifiable underlying anatomic defect. Dogs experiencing repeated bouts of severe seizures are given therapeutic medication to control their frequency and severity. Idiopathic epilepsy has been reported in many dog breeds and was identified as the predominant health issue facing dog breeds in a recent survey by the American Kennel Club. A growing body of evidence supports a hereditary basis for idiopathic epilepsy, with a variety of genetic inheritance models proposed. In the Belgian tervuren and sheepdog, epilepsy is highly heritable with a polygenic mode of inheritance, though apparently influenced by a single autosomal recessive locus of large effect. In an effort to establish molecular linkage between the epileptic phenotype and the locus of large effect, we have screened genomic DNA from families of affected tervuren and sheepdogs with 100 widely dispersed, polymorphic canine microsatellite markers (0.595 average PIC value). Although not significant (LOD scores <3.0), three genomic regions have shown nominal linkage between markers and the epileptic phenotype. Additional related dogs are being screened with these and additional markers to increase the power to detect the presence of a linked locus.

Alternatives to blood as a source of DNA for large-scale scanning studies of canine genome linkages.

Participation and compliance are critical to the success of any large-scale study of canine disease using DNA markers. Most canine genetic studies rely upon DNA extracted from peripheral blood samples. We assessed the utility of buccal swab epithelial cells and toe nails as a source of DNA for use in genomic screening studies. Using eight multiplexed canine microsatellite markers, amplified DNA obtained from peripheral blood, and from freshly extracted buccal epithelial cells, and buccal swab DNA extracted and stored at 20 degrees C for 27 months or extracted from toe nails were compared for three dogs. The accuracy of the genotyping at each locus was identical for each preparation. Buccal swab DNA samples were readily and uniformly amplified and could be stored for years without loss of integrity. Each buccal swab provided sufficient DNA for more than 200 individual PCR reactions. Toe nails provided ample DNA for thousands of PCR reactions and had the added advantage of ease of storage of the original tissues. These studies demonstrate the potential utility of DNA derived from buccal swabs or nails in large-scale genomic scanning and marker linkage studies.