Hypoxia induces giant osteoclast formation and extensive bone resorption in the cat.

Dental disease due to osteoclast (OC) overactivity reaches epidemic proportions in older domestic cats and has also been reported in wild cats. Feline odontoclastic resorptive lesions (FORL) involve extensive resorption of the tooth, leaving it liable to root fracture and subsequent loss. The etiopathogenesis of FORL remains unclear. Here, we explore the hypothesis that FORL is associated with hypoxia in the oral microenvironment, leading to increased OC activity. To investigate this, we developed a method of generating OCs from cat blood. Reducing O2 from 20% to 2% increased the mean area of OC eightfold from 0.01 to 0.08 mm2. In hypoxic cultures, very large OCs containing several hundred nuclei were evident (reaching a maximum size of approximately 14 mm2). Cultures exposed to 2% O2 exhibited an increase of approximately 13-fold in the area of bone slices covered by resorption lacunae. In line with this finding, there was a significant increase in cells differentiating under hypoxic conditions, reflected in increased expression of cathepsin K and proton pump enzymes. In conclusion, these results demonstrate that oxygen tension is a major regulator of OC formation in the cat. However, in this species, hypoxia induces the formation of "giant" OCs, which can be so large as to be visible with the naked eye and yet also actively resorb. This suggests that local hypoxia is likely to play a key role in the pathogenesis of FORL and other inflammatory conditions that are associated with bone resorption in cats.

Evidence that the canonical Wnt signalling pathway regulates deer antler regeneration.

Wnt signalling regulates many developmental processes, including the fate specification, polarity, migration, and proliferation of cranial neural crest. The canonical Wnt pathway has also been shown to play an important role in bone physiology and there is evidence for its recapitulation during organ regeneration in lower vertebrates. This study explores the role of the Wnt signalling pathway in deer antlers, frontal bone appendages that are the only mammalian organs capable of regeneration. Immunocytochemistry was used to map the distribution of the activated form of beta-catenin ((a)betaCAT). A low level of (a)betaCAT staining was detected in chondrocytes and in osteoblasts at sites of endochondral bone formation. However, (a)betaCAT was localised in cellular periosteum and in osteoblasts in intramembranous bone, where it co-localised with osteocalcin. The most intense (a)betaCAT staining was in dividing undifferentiated cells in the mesenchymal growth zone. Antler progenitor cells (APCs) were cultured from this region and when the canonical Wnt pathway was inhibited at the level of Lef/TCF by epigallocatechin gallate (EGCG), the cell number decreased. TUNEL staining revealed that this was as a result of increased apoptosis. Activation of the pathway by lithium chloride (LiCl) had no effect on cell number but inhibited alkaline phosphate activity (ALP), a marker of APC differentiation, whereas EGCG increased ALP activity. This study demonstrates that beta-catenin plays an important role in the regulation of antler progenitor cell survival and cell fate. It also provides evidence that beta-catenin's function in regulating bone formation by osteoblasts may be site-specific.

A feline assay using osteoclasts generated in vitro from peripheral blood for screening anti-resorptive agents.

Musculo-skeletal diseases are a major cause of pain and suffering in cats and several conditions involve increased bone resorption by osteoclasts. However, little is known about the biology of these cells in the cat. In this study we established a method to generate feline osteoclasts from blood mononuclear cells stimulated by macrophage colony stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL). Cultured osteoclasts are multinucleated, express tartrate resistant acid phosphatase (TRAP), form F-actin rings and resorb bone. They express alpha(v)beta3 vitronectin receptor and osteoclast enzymes, cathepsin K and MMP9; the myeloid antigen, CD18, and the megakaryocyte/platelet integrin, CD41, are absent. This phenotype is typical of osteoclasts from other species. Three resorption inhibitors were examined for activity against feline osteoclasts. Calcitonin, bisphosphonate and RGD integrin inhibitory peptide all reduced bone resorption at doses similar to those efficacious in rabbit or human. We conclude that blood-derived osteoclast cultures are a suitable in vitro system for assessing the ability of drugs to inhibit bone resorption in domestic cats.

Identification of a novel phosphonocarboxylate inhibitor of Rab geranylgeranyl transferase that specifically prevents Rab prenylation in osteoclasts and macrophages.

Nitrogen-containing bisphosphonate drugs inhibit bone resorption by inhibiting FPP synthase and thereby preventing the synthesis of isoprenoid lipids required for protein prenylation in bone-resorbing osteoclasts. NE10790 is a phosphonocarboxylate analogue of the potent bisphosphonate risedronate and is a weak anti-resorptive agent. Although NE10790 was a poor inhibitor of FPP synthase, it did inhibit prenylation in J774 macrophages and osteoclasts, but only of proteins of molecular mass approximately 22-26 kDa, the prenylation of which was not affected by peptidomimetic inhibitors of either farnesyl transferase (FTI-277) or geranylgeranyl transferase I (GGTI-298). These 22-26-kDa proteins were shown to be geranylgeranylated by labelling J774 cells with [(3)H]geranylgeraniol. Furthermore, NE10790 inhibited incorporation of [(14)C]mevalonic acid into Rab6, but not into H-Ras or Rap1, proteins that are modified by FTase and GGTase I, respectively. These data demonstrate that NE10790 selectively prevents Rab prenylation in intact cells. In accord, NE10790 inhibited the activity of recombinant Rab GGTase in vitro, but did not affect the activity of recombinant FTase or GGTase I. NE10790 therefore appears to be the first specific inhibitor of Rab GGTase to be identified. In contrast to risedronate, NE10790 inhibited bone resorption in vitro without markedly affecting osteoclast number or the F-actin "ring" structure in polarized osteoclasts. However, NE10790 did alter osteoclast morphology, causing the formation of large intracellular vacuoles and protrusion of the basolateral membrane into large, "domed" structures that lacked microvilli. The anti-resorptive activity of NE10790 is thus likely due to disruption of Rab-dependent intracellular membrane trafficking in osteoclasts.

Fibrillar amyloid-beta production, accumulation, and recycling in transgenic mice pancreatic acinar cells and macrophages.

Amyloid-beta (A beta) production, accumulation, and recycling were examined by light and electron microscopy in the pancreas of transgenic mice (from 45 days to 22 months of age) that express the gene for the carboxy-terminal fragment of the human amyloid-beta protein precursor. Ultrastructural immunocytochemistry revealed four types of cells accumulating fibrillar A beta 1-40 in cytoplasmic vacuoles: acinar pancreatic cells, macrophages infiltrating stroma, epithelial cells of pancreatic ducts, and blood monocytes/macrophages in the lumen of pancreatic vessels. The ultrastructure of amyloid deposits suggests that each of these four types of cells produces fibrillar A beta. Three basic types of amyloid deposits were distinguished: primary vacuoles in different stages of amyloid aggregation and fibrillization, secondary vacuoles that are the product of fusion of primary vacuoles, and phagosome-like vacuoles with morphologically intact fibrillar amyloid and residues of ingested cells. Amyloid production in acinar pancreatic cells starts in mice younger than 45 days, progresses in 2- to 7-month-old mice, and plateaus in the second year of life. In macrophages, amyloid appears in 60-day-old mice, and the increase in the number of macrophages and the amount of amyloid in their cytoplasm correlates with age.

Ultrastructure of myelinated fibers of sciatic nerve in acute vincristine intoxication.

Numerous clinical reports documented that in some patients vincristine (VCR) may cause a risk for life (stridor) and painful symptoms of acute intoxication. The present study was undertaken to examine ultrastructural changes in the sciatic nerve of rabbits following acute vincristine intoxication. Our results show, that in a few hours, some axons of this nerve may undergo degeneration and atrophy. These changes were initially followed by the cleavage of adaxonal myelin sheets, that spontaneously formed concentric scrolls and tangled threads. Finally, myelin sheath collapsed around the atrophied axon. The results provide evidence that acute VCR intoxication primarily affects the axons. Myelin changes are caused by the loss of supportive role of atrophied axon for the myelin sheath. The effect of intoxicated Schwann cells on the myelin in acute VCR intoxication is not clear and requires further study.

DNA fragmentation and ultrastructural changes in the brain of rabbits treated with vincristine.

Numerous anticancer drugs, which are in current use, have been shown to induce apoptosis in susceptible cells of some tissues. The aim of the study was to examine whether vincristine injected parenterally to rabbits may affect DNA and ultrastructure of cells in the brain. Vincristine 0.25 mg/kg of body weight was injected to immature (seven day-old) and adult (90 day-old) rabbits. Two, four and six hours after treatment, the significant increased number of cells with internucleosomal fragmentation of DNA was found. At the same time, in some glial cells characteristic for apoptosis condensation of chromatin was observed. Numerous apoptotic bodies were scattered throughout the tissue or digested by neighboring cells (astroglia, oligodendroglia, microglia and pericytes). The result provided evidence that vincristine treatment may induce apoptosis and trigger phagocytic clearance of apoptotic bodies in the brain. Presumably, both of these processes participate in neurotoxic effect of the drug on the CNS during chemotherapy.

Apoptosis, programmed cell death in the central nervous system.

Cell death by apoptosis occurs in a number of normal tissue systems including the nervous system. It is a highly specific process, which requires new gene expressions and the production of new proteins. The genetic information determines biochemical and morphological sequence of events in the cell undergoing apoptosis. This review focuses on the recent informations concerning the participation of the genes in the survival and death of neurons in the central nervous system and the effect of the anti-cancer drugs on the induction of apoptosis in the cell.

The features of peripheral nerve lesions in young and adult rabbits after vincristine administration.

The comparison of neuropathological picture of vincristine induced neuropathy immediately after the end of its administration and after three months of survival was performed. The adult rabbits and six litters of newborns received vincristine once a week in increasing doses 0.05-0.25 mg/kg of body weight. The early changes consisted in degeneration of axons and myelin sheaths and lesions of the Schwann cells. The features of changes allow to suspect that vincristine may induce, in addition to the well known primary axonal damage, a toxic effect on the Schwann cells with secondary damage of myelin sheaths. The changes were more severe in adults than in young animals. The observations after three months of survival allow to think that the administered doses of vincristine did not impede the improvement when treatment was interrupted.

Ultrastructure of brain vessel in vincristine treated rabbits.

Ultrastructure of the brain blood vessels has been examined in rabbits treated with a single intraperitoneal injection of vincristine (VCR). The results provided evidence that some microvessels in these animals undergo vasoconstriction concomitant with projection of numerous endothelial microvilli into the vessel lumen. Vasoconstriction was followed by microvessel loss of regular shape and degeneration of some endothelial cells resulting in the digestion of apoptotic bodies by perivascular pericytes. Most of those changes (eg. vasoconstriction) appeared transient and were not observed seven days after treatment.

Phagocytic activity of polymorphonuclear leukocytes lavaged from the lungs of horses with clinically diagnosed chronic pulmonary disease.

The aim of this study was to compare phagocytic activity of polymorphonuclear cells (PMNs) from the bronchoalveolar lavage of clinically healthy horses and those with severe chronic bronchiolitis. Research was carried out on 28 horses. Chronic inflammation of the lower airways was diagnosed in nine horses. Cells from the respiratory tract were lavaged according to accepted methods. For comparison, PMNs were isolated from peripheral blood of all investigated horses. The phagocytic activity of PMNs was determined in relation to two standard strains of Staphylococcus aureus, Staph, aureus Smith which was phagocytized after previous opsonization, and Staph, aureus 305, phagocytized without opsonization. From the investigations, it is shown that the PMNs present in the terminal airways of horses with severe chronic bronchiolitis are characterized by decreased phagocytic activity in relation to opsonized Staphylococcus aureus Smith and increased activity in relation to non-opsonized Staphylococcus aureus 305, as compared to the PMNs lavaged from the terminal airways of clinically healthy horses. No changes in the phagocytic activity of the peripheral blood PMNs were observed between clinically diseased horses and healthy horses.

Ultrastructure of the Ranvier's node in vincristine neuropathy.

Paranode-node-paranode regions of the nerve fibre interact in a fundamental manner with the transport of axoplasmic material. The aim of the study was to examine the ultrastructure of Ranvier's node in sciatic nerve of rabbits treated with vincristine. The results showed accumulation of organelles in Ranvier's nodes of affected animals. Organelles arrested in this region disturbed each other and degenerated. Some of them were sequestered from the axon by Schwann cell fingers and terminal loops, but often this process was not sufficient to prevent nerve fibre against degeneration. In conclusion our results provided evidence that Ranvier's node become locus minoris resistentiae in vincristine affected nerve fibers.

Neurotoxic effect of vincristine on ultrastructure of hypothalamus in rabbits.

The neurotoxic side-effects are a striking accompaniment of the therapy with vincristine (VCR). Data concerning the influence of VCR on the central nervous system are controversial. In the present study a schedule of VCR treatment was employed to develop the neurotoxic side effects in New Zealand rabbits. In all treated animals neurons were primarily and most severely affected cells. Most of them underwent degeneration. The consecutive stages of the increasing condensation of chromatin and cytoplasm of these cells were observed. Degeneration of the synaptic connections and secondary changes in myelin sheaths of degenerating axons were found. Debris of some death cells were localized intracellularly or as typical apoptotic bodies ingested by some glial cells. Astroglia underwent swelling and their processes separated dying neurons from the surroundings. All the results lead to conclusion that the VCR treatment concomitant with neurotoxic symptoms may affects regions of CNS lacking of blood-brain barrier. Neurons are most vulnerable cells of the brain parenchyma. The changes induced in neurons by the drug are characteristic in many aspects to those described in many cells undergoing apoptosis.