Long-term bisphosphonate treatment coupled with ovariectomy in mice provokes deleterious effects on femoral neck fracture pattern and modifies tibial shape.

AIMS: The processes linking long-term bisphosphonate treatment to atypical fracture remain elusive. To establish a means of exploring this link, we have examined how long-term bisphosphonate treatment with prior ovariectomy modifies femur fracture behaviour and tibia mass and shape in murine bones. METHODS: Three groups (seven per group) of 12-week-old mice were: 1) ovariectomized and 20 weeks thereafter treated weekly for 24 weeks with 100 µm/kg subcutaneous ibandronate (OVX+IBN); 2) ovariectomized (OVX); or 3) sham-operated (SHAM). Quantitative fracture analysis generated biomechanical properties for the femoral neck. Tibiae were microCT scanned and trabecular (proximal metaphysis) and cortical parameters along almost its whole length measured. RESULTS: Fracture analyses revealed that OVX+IBN significantly reduced yield displacement (vs SHAM/OVX) and resilience, and increased stiffness (vs SHAM). OVX+IBN elevated tibial trabecular parameters and also increased cortical cross-sectional area and second moment of area around minor axis, and diminished ellipticity proximally. CONCLUSION: These data indicate that combined ovariectomy and bisphosphonate generates cortical changes linked with greater bone brittleness and modified fracture characteristics, which may provide a basis in mice for interrogating the mechanisms and genetics of atypical fracture aetiology.Cite this article: Bone Joint Open 2020;1-9:512-519.

Bone quality evaluation of experimental osteometabolic disease in Pantanal alligators (Caiman yacare) by High Resolution Computerized Microtomography (µCT) / Avaliação da qualidade óssea de doença osteometabólica experimental em jacarés do pantanal (Caiman yacare) por Microtomografia Computadorizada (µCT)

Computerized microtomography is the gold standard examination for the evaluation of the three-dimensional bone structure. This experiment was developed to evaluate the structure and bone quality of Caiman yacare with metabolic bone disease using high resolution computerized microtomography (μCT). The animals were distributed into four groups: G1 - hyperphosphatemic diet with sun exposure deprivation (n=4), G2 - hyperphosphatemic diet with sun exposure (n=4), G3 - balanced diet with sun exposure deprivation (n=4), and G4 - balanced diet with exposure to sunlight (n=4). The parameters for the trabecular bone (Trabecular Number, Trabecular Thickness, Trabecular Separation, Bone Pattern Factor, Fractal Dimension, Euler Number, Structural Model Index, Degree of Anisotropy, Eigenvalues 1, 2 and 3, and Centroides X, Y and Z), and cortical bone (Number of Closed Pores, Volume of Closed Pores, Surface of Closed Pores, Closed Porosity, Volume of Open Pores, Open Porosity and Total Porosity). The overall results showed that the structure and bone quality of group G3 and G4 were better than those of groups G1 and G2, and that the diet factor influenced more than the sun exposure factor. The computerized microtomography allowed to evaluate the quality of the cortical and trabecular bones of the Pantanal alligator tibia with osteometabolic disease. The diet and sun exposure factors influenced individually the results of the μCT parameters between the groups, demonstrating the functional and structural complexity. Thus, these parameters can contribute to the interpretation of the mechanical behavior of bones and correlate them with the risk of lesions and fractures associated with osteometabolic diseases.(AU)

Microtomografia computadorizada é o exame padrão-ouro para a avaliação da estrutura tridimensional do osso. Este estudo experimental foi desenvolvido para avaliar a estrutura e a qualidade óssea de jacarés-do-pantanal (Caiman yacare) com doença óssea metabólica utilizando a microtomografia computadorizada (μCT) de Alta Resolução. Os animais foram distribuídos em quatro grupos, G1 - dieta hiperfosfatêmica com privação de luz solar (n=4), G2 - dieta hiperfosfatêmica com exposição à luz solar (n=4), G3 - dieta balanceada com privação de luz solar (n=4) e G4 - dieta balanceada com exposição à luz solar (n=4). Avaliaram-se os parâmetros para o osso trabecular (Número de Trabéculas, Espessura Trabecular, Separação Trabecular, Fator do Padrão Ósseo, Dimensão Fractal, Número de Euler, Índice do Modelo Estrutural, Grau de Anisotropia, Autovalores 1, 2 e 3 e Centroides X, Y e Z) e osso cortical (Número de Poros Fechados, Volume dos Poros Fechados, Superfície de Poros Fechados, Porosidade Fechada, Volume de Poros Abertos, Porosidade Aberta e Porosidade Total). Os resultados gerais evidenciaram que a estrutura e a qualidade óssea dos grupos G3 e G4 foram superiores aos dos grupos G1 e G2, sendo que o fator dieta influenciou mais do que o fator exposição solar. A Microtomografia Computadorizada permitiu avaliar a qualidade dos ossos cortical e trabecular da tíbia de jacarés do pantanal com doença osteometabólica. Os fatores dieta e exposição solar influenciaram individualmente no resultado dos parâmetros do μCT entre os grupos, demonstrando a complexidade funcional e estrutural. Assim, esses parâmetros podem contribuir na interpretação do comportamento mecânico dos ossos e correlacioná-los com o risco de lesões e fraturas associadas às doenças osteometabólicas.(AU)

Granulomatous meningoencephalitis due to toxoplasma gondii in a black-headed night monkey (Aotus nigriceps).

This report describes toxoplasmosis infection in a black-headed night monkey (Aotus nigriceps) from the Zoological Park of the Federal University of Mato Grosso, Brazil. The animal was submitted alive to the Veterinary Hospital of the Federal University of Mato Grosso presenting neurologic clinical signs. There were no improvements on the animal's clinical condition after treatment, so it was euthanized. At necropsy, the most significant gross lesions observed were hemorrhages diffusely scattered throughout the cerebral hemispheres. Histologic findings were characterized by nonsuppurative encephalitis and mild multifocal necrotizing nonsuppurative myocarditis. Groups of tachyzoites and cysts were observed surrounding the inflamed areas of cerebral parenchyma. Sections of the brain were submitted for immunohistochemistry examination for Toxoplasma gondii and Neospora caninum and also for polymerase chain reaction (PCR) to determine the presence of Toxoplasma gondii. The samples were positive for Toxoplasma gondii by PCR; however, cross reactivity was observed between Toxoplasma gondii and Neospora caninum antibodies. These findings were consistent with a toxoplasmosis infection in this animal.

The first case report of Leishmania (leishmania) chagasi in Panthera leo in Brazil.

We reported here the first known case of natural infection of a lion (Panthera leo-Linnaeus, 1758) with Leishmania (Leishmania) chagasi (L. chagasi) in Brazil. The specimen was created by a circus handler in the state of Mato Grosso and was donated to the zoological park of the Federal University of Mato Grosso. Infection by L. chagasi was detected using a PCR-RFLP test. It was known that the domestic felids can act as reservoir of infection of L. chagasi in endemic areas, making it important that studies demonstrate their participation in the epidemiological chain. We demonstrate in this work that wild animals can have an important role in the epidemiological chain and must be considered in order to plan methods of control of this zoonosis.

Leishmania(Leishmania) chagasi in captive wild felids in Brazil.

This study used a PCR-RFLP test to determine the presence of Leishmania (Leishmania) chagasi in 16 captive wild felids [seven Puma concolor (Linnaeus, 1771); five Panthera onca (Linnaeus, 1758) and four Leopardus pardalis (Linnaeus, 1758)] at the zoological park of the Federal University of Mato Grosso, Brazil. Amplification of Leishmania spp. DNA was seen in samples from five pumas and one jaguar, and the species was characterized as L. chagasi using restriction enzymes. It is already known that domestic felids can act as a reservoir of L. chagasi in endemic areas, and further studies are necessary to investigate their participation in the epidemiological chain of leishmaniasis.

Conidiobolomicose em ovinos no Estado de Mato Grosso / Conidiobolomycosis in sheep in the state of Mato Grosso, Brazil

Relatam-se os achados epidemiológicos, clínicos, patológicos e micológicos de conidiobolomicose em ovinos no Estado de Mato Grosso. A doença ocorreu em uma propriedade no município de Nobres, em um rebanho com 40 ovelhas adultas, entre os meses de janeiro a junho de 2007. Aproximadamente 30 por cento dos ovinos da propriedade adoeceram e todos os doentes morreram em curso clínico de 2-5 semanas. O quadro clínico foi caracterizado por apatia, emagrecimento, dificuldade respiratória com dispnéia, respiração ruidosa e oral, secreção nasal mucosa ou sero-sanguinolenta, exoftalmia unilateral, por vezes com cegueira, e morte. Havia desaparecimento de etmoturbinados com substituição por tecido bran-cacento, finamente granular, multilobulado e friável infil-trando-se na lâmina cribiforme, no septo nasal e nas coa-nas em todos os ovinos necropsiados. Lesões similares foram encontradas em linfonodos regionais (2 casos), pulmões (3), encéfalo (2) e em linfonodos do abomaso (1). Microscopicamente havia inflamação granulomatosa da região rinocerebral, caracterizada por necrose, proliferação de tecido conjuntivo, infiltrado de neutrófilos, eosinófilos, células epitelióides e células gigantes multinucleadas freqüentemente circundando material Splendori-Hoeppli, onde havia imagens negativas de hifas. Na impregnação pela Prata-Metenamina, as hifas tinham septos e ramificações escassas e irregulares, com dilatação balonosa terminal e com forte demarcação de contornos. Iso-lou-se de tecido nasal de quatro ovinos Conidiobolus sp.

The epidemiological, clinical, pathological and mycological findings of an outbreak of conidiobolomycosis in a flock of 40 Santa Ines sheep, in the state of Mato Grosso, Brazil, are reported. The illness occurred in the municipality of Nobres during January-June, 2007, resulting in death of about 30 percent of the affected sheep within 2-5 weeks. The clinical signs were characterized by apathy, weight loss, labored and noisy breathing with dyspnea, and mucous or serohemorrhagic nasal discharge. In three sheep there was unilateral exophthalmia, keratitis and corneal ulceration. A firm whitish, multi-lobulated, friable growth was evident in the ethmoturbinate region at the coronal sections of the head from six affected sheep. In all sheep the choana had similar nodular infiltration which resulted in local obstruction. In three of those there was exophthalmia. There was infiltration in the cribiform plate and brain (2 cases), regional lymph nodes (2), lungs (3), and abomasums (1). Microscopic findings were granulomatous inflammation of the rhinoetimoidal region, with necrosis, lymphocytic infiltration, epithelioid multinucleated giant cells and fibrovascular tissue, surrounding Splendore-Hoeppli material wich contained unstained ghost images of hyphae. The methenamine-silver stain uncover fungi hyphae, rarely ramified with bulbous dilatation in their extremities. Conidiobolus sp. was isolated from nasal tissue lesions of four sheep.

Blockade of beta-adrenergic signaling does not influence the bone mechano-adaptive response in mice.

The involvement of the sympathetic nervous system (SNS) in the modulation of bone adaptation to its load-bearing demand remains controversial. This study tested the involvement of SNS in the adaptive response of trabecular and cortical bone to either external loading or disuse. External loading consisted of cyclic strain (40 cycles, peak 1500 microstrain) applied for 7 min, 3 days/week, while disuse was induced by unilateral sciatic neurectomy (SN). C57Bl/J6 mice, female, 9 weeks old, were subjected to loading or disuse for 2 weeks. Half of the loaded and SN mice were injected with the beta-adrenergic antagonist, propranolol (PRO, 20 mug/g) 1 week before the start of loading or disuse and during all the duration of the experiment. MicroCT analysis of the tibiae showed that the applied load induced significant changes on both trabecular architecture and cortical geometry compared to the contralateral controls, indicating increased bone mass. In contrast, disuse markedly reduced trabecular and cortical indexes. However, these adaptive responses were not altered by PRO treatment. We further tested whether the lack of protective effect of PRO against disuse-induced bone loss was due to the very short duration of treatment by blocking SNS signaling for 8 weeks with either PRO (0.5 mg/ml in drinking water) or guanethidine sulfate (GS, 40 mug/g, injected). At the end of fourth week of treatment, mice underwent SN surgery so that disuse was induced for the remaining 4 weeks. Again, neither PRO nor GS treatments altered the disuse-induced bone loss in the neurectomized tibia. In addition, blockade of SNS signaling for either 3 or 8 weeks did not affect the basal trabecular bone architecture in control tibiae and in L4 vertebrae. This study shows that the mechano-adaptive response occurring in trabecular and cortical bone upon loading or disuse is not altered by inactivation of beta-adrenergic signaling. Furthermore, sympathectomy had no effect on trabecular bone at different skeletal sites. This suggests that the osteo-regulatory action of beta-adrenergic signaling is not involved in the bone mechano-adaptive response and must therefore affect other bone regulatory pathways.

Osteocytes use estrogen receptor alpha to respond to strain but their ERalpha content is regulated by estrogen.

UNLABELLED: The role of mechanical strain and estrogen status in regulating ERalpha levels in bone cells was studied in female rats. OVX is associated with decreased ERalpha protein expression/osteocyte, whereas habitual strain and artificial loading has only a small but positive effect, except on the ulna's medial surface, where artificial loading stimulates reversal of resorption to formation. INTRODUCTION: Osteoporosis is the most widespread failure of bones' ability to match their architectural strength to their habitual load bearing. In men and women, the severity of bone loss is associated with bioavailability of estrogen. This association could result from the estrogen receptor (ER) involvement in bone cells' adaptive response to loading. MATERIALS AND METHODS: In vivo semiquantitative analysis of the amount of ERalpha protein per osteocyte was performed in immuno-cytochemically stained sections from control and loaded rat ulna, as well as tibias of ovariectomy (OVX) and sham-operated female rats. In vitro, the effect of exogenous estrogen (10(-8) M) and mechanical strain (3400 microepsilon, 1 Hz, 600 cycles) on the expression of ERalpha mRNA levels was assessed in ROS 17/2.8 cells in monolayers using real-time PCR and ER promoter activity. ERalpha translocation in response to exogenous estrogen and mechanical strain was assessed in both ROS 17/2.8 and MLO-Y4 cells. RESULTS: More than 90 percent of tibial osteocytes express ERalpha, the level/osteocyte being higher in cortical than cancellous bone. OVX is associated with decreased ERalpha protein expression/osteocyte, whereas in the ulna habitual strain and that caused by artificial loading had only a small but positive effect, except on the medial surface, where loading stimulates reversal of resorption to formation. In unstimulated osteocytes and osteoblasts in situ, and osteocyte-like and osteoblast-like cells in vitro, ERalpha is predominantly cytoplasmic. In vitro, both strain and estrogen stimulate transient ERalpha translocation to the nucleus and transient changes in ERalpha mRNA. Strain but not estrogen also induces discrete membrane localization of ERalpha. CONCLUSIONS: Bone cells' responses to both strain and estrogen involve ERalpha, but only estrogen regulates its cellular concentration. This is consistent with the hypothesis that bone loss associated with estrogen deficiency is a consequence of reduction in ERalpha number/activity associated with lower estrogen concentration reducing the effectiveness of bone cells' anabolic response to strain.

Sympathetic nervous system does not mediate the load-induced cortical new bone formation.

UNLABELLED: The contribution of the SNS to bone's response to mechanical loading is unclear. Using a noninvasive model of axial loading of the murine tibia, we found that sciatic neurectomy enhances load-induced new cortical bone formation and that pharmacological blockade of the SNS does not affect such responses, indicating that the SNS does not mediate the osteogenic effects of loading in cortical bone. INTRODUCTION: There is increasing evidence that the sympathetic nervous system (SNS) contributes to the regulation of bone mass and may influence remodeling by modulating bones' response to mechanical load-bearing. The aim of this study was to examine the effect of sciatic neurectomy (SN) on the changes in cortical bone formation induced in response to mechanical loading and to investigate whether the SNS is directly involved in such load-induced responses. MATERIALS AND METHODS: Accordingly, load-induced responses were compared in tibias of growing and adult control C57Bl/J6 mice and in mice submitted to unilateral SN; noninvasive axial loading that induced 2,000 microstrain on the tibia lateral midshaft cortex was applied cyclically, 5 or 100 days after surgery, for 7 minutes, 3 days/week for 2 weeks, and mice received calcein on the third and last days of loading. Tibias were processed for histomorphometry, and transverse confocal images from diaphyseal sites were analyzed to quantify new cortical bone formation. Chemical SNS inactivation was achieved by prolonged daily treatment with guanethidine sulfate (GS) or by the introduction of propranolol in drinking water. RESULTS: Our results show that new cortical bone formation is enhanced by loading in all tibial sites examined and that load-induced periosteal and endosteal new bone formation was greater in the SN groups compared with sham-operated controls. This SN-related enhancement in load-induced cortical bone formation in tibias was more pronounced 100 days after neurectomy than after 5 days, suggesting that longer periods of immobilization promote a greater sensitivity to loading. In contrast, the increases in new bone formation induced in response to mechanical loading were similar in mice treated with either GS or propranolol compared with controls, indicating that inactivation of the SNS has no effect on load-induced cortical new bone formation. CONCLUSIONS: This study shows that SN, or the absence of loading function it entails, enhances loading-related new cortical bone formation in the tibia independently of the SNS.

Non-invasive axial loading of mouse tibiae increases cortical bone formation and modifies trabecular organization: a new model to study cortical and cancellous compartments in a single loaded element.

Systematic study of bones' responses to loading requires simple non-invasive models in appropriate experimental animals where the applied load is controllable and the changes in bone quantifiable. Herein, we validate a model for applying axial loads, non-invasively to murine tibiae. This allows the effects of mechanical loading in both cancellous and cortical bone to be determined within a single bone in which genetic, neuronal and functional influences can also be readily manipulated. Using female C57Bl/J6 mice, peak strains at the tibial mid-shaft were measured during walking (<300 micro epsilon tension) and jumping (<600 micro epsilon compression) with single longitudinally oriented strain gauges attached to the bone's lateral and medial surfaces. Identically positioned gauges were also used to determine, for calibration, the strains engendered by external applied compressive tibial loading between the flexed knee and ankle ex vivo. Applied loads between 5 and 13 N produced strains of 1150-2000 micro epsilon on the lateral surface, and in vivo repetitions of these loads on alternate days for 2 weeks produced significant load magnitude-related increases in cortical bone formation that were similar in mice at 8, 12 and 20 weeks of age. Micro-CT scans showed that loading significantly increases trabecular bone volume in 8 week old mice, but modifies trabecular organization with decreases in trabecular bone volume in 12 and 20 week old mice. This model for loading the tibia has several advantages over other approaches, including scope to study the effects of loading in cancellous as well as cortical bone, against a background of either disuse or of treatment with osteotropic agents within a single bone in normal, mutant and transgenic mice.