Effects of Exogenous Lipopolysaccharide Exposure on Bone Outcomes in Rodent Models

Chronic low-grade inflammation has been identified as a potential contributor to the pathophysiology of osteoporosis. A key mediator may be lipopolysaccharide (LPS) released from gram-negative bacteria in the gut that can enter circulation stimulating an inflammatory response and upregulate bone resorption. Since rodent models mimic the loss of bone mineral density (BMD) and structure that occurs in humans, rodents offer an accelerated model for studying these inflammation-mediated changes. Therefore, the objective of this thesis was to characterize a rodent model of LPS-induced bone loss using repeated in vivo μCT scans to establish a time course effect of LPS longitudinally and for this purpose three studies were conducted. Study 1 & 2 were run simultaneously using the same control mice. Study 1 demonstrated that repeated irradiation had a negative impact on trabecular bone in both male and female CD-1 mice, while cortical bone was only negatively impacted in the females. In study 2, continuous delivery of exogenous LPS via osmotic pumps for 12 weeks elevated serum LPS in both male and female CD-1 mice but did not alter trabecular or cortical bone structure or BMD at any of the scanning timepoints. Results from Study 2 may in part have been influenced by the effects of repeated irradiation from the in vivo μCT scans at 4-week intervals for a total of 4 scans analyzed in Study 1. In study 3, a systematic review was conducted to better characterize a model of LPS induced bone loss and identify factors that may impact the effects of LPS on bone outcomes in rodent models. Regardless of study duration, exogenous LPS negatively impacted trabecular bone structure and BMD but not cortical bone structure, due to an upregulation in bone resorption. Together these data suggest that exogenous LPS can induce alterations in bone structure and BMD in rodent models, however a clearly defined model of exogenous LPS induced bone loss has yet to be fully characterized

The effects of a high fat diet on musculoskeletal health in aged male C57BL/6J mice

Aging and obesity are two major aspects that can negatively impact musculoskeletal structure and function. It is important to study these aspects because of current high rates of obesity and the increasing proportion of seniors in North America. This study investigated the effects of a long-term high fat and sucrose diet (HFS) superimposed with aging on bone and muscle structure and function. Male C57BL/6J mice were randomized 1 of 2 diets: control (AGE, AIN93M, 10.3% kcal fat, 100 g/kg sucrose) or HFS (HFS-AGE, 45.3% kcal fat 200 g/kg sucrose) for 13 weeks starting at 20 weeks of age to represent middle age. Trabecular bone structure and volumetric bone mineral density (vBMD), body composition, and grip strength were measured longitudinally at 20, 24, 28, and 32 weeks of age. In vitro contractile measures were performed on isolated soleus and extensor digitorum longus (EDL) muscles at baseline (BSL, 20 weeks of age, n=11) and at the end of the 13-week intervention when AGE (n=12) and HFS-AGE (n=12) mice were 33 weeks of age. Both the AGE and HFS-AGE groups had similar declines in trabecular bone (bone structure and vBMD). For muscle contractile function, HFS+AGE resulted in increased soleus cross-sectional area (CSA) compared AGE (p=0.0008), but this did not translate to greater twitch or tetanus peak force. The ratio of outcomes of bone to muscle declined in both the AGE and HFS-AGE groups as a result of a greater decline in key measures of bone structure (BV/TV) than muscle function (soleus and EDL peak tetanus and CSA) and was not altered by feeding HFS. In conclusion, beginning a HFS diet during middle age did not exacerbate age-related declines in bone or muscle, but these tissues do not decline in a coordinate manner with aging as bone structure declined at a greater rate than muscle function

Evaluation of neuropathological effects of a high-fat high-sucrose diet in middle-aged male C57BL6/J mice

Metabolic dysfunction related to diet-induced obesity has recently been linked to the pathogenesis of sporadic Alzheimer’s disease (AD). However, the underlying mechanisms linking obesity and AD remain unclear. The purpose of this study was to examine early alterations in brain insulin signaling, inflammatory/stress markers, and energetic stress in a model of diet-induced obesity during middle age. Male C57BL/6J mice were randomized to either a control diet (AGE n = 12) or high-fat and sucrose diet (AGE-HFS n = 12) for 13-weeks from 20-weeks of age. Prefrontal cortex and hippocampal samples were collected at 20-weeks of age (BSL n = 11) and at 33-weeks of age (AGE and AGE-HFS). The HFS diet resulted in increased body weight (30%; P = 0.0001), increased %fat mass (28%; P = 0.0001), and decreased %lean mass (33%; P = 0.0001) compared to aged controls. In the prefrontal cortex, AGE-HFS resulted in increased 50 adenosine monophosphate – activated protein kinase (AMPK) phosphorylation (P = 0.045). In the hippocampus, AGEHFS resulted in increased extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) phosphorylation and protein kinase B (Akt) serine473 and glycogen synthase kinase (GSK) phosphorylation (P < 0.05). Results from this study demonstrate that aging combined with a HFS diet results in increased inflammation (pERK and pJNK) and energetic stress (pAMPK) in the hippocampus and prefrontal cortex, respectively. Together these novel results provide important information for future targets in early AD pathogenesis.Brock University Library Open Access Publishing Fun

Higher PLIN5 but not PLIN3 content in isolated skeletal muscle mitochondria following acute in vivo contraction in rat hindlimb

Contraction-mediated lipolysis increases the association of lipid droplets and mitochondria, indicating an important role in the passage of fatty acids from lipid droplets to mitochondria in skeletal muscle. PLIN3 and PLIN5 are of particular interest to the lipid droplet–mitochondria interaction because PLIN3 is able to move about within cells and PLIN5 associates with skeletal muscle mitochondria. This study primarily investigated: 1) if PLIN3 is detected in skeletal muscle mitochondrial fraction; and 2) if mitochondrial protein content of PLIN3 and/or PLIN5 changes following stimulated contraction. A secondary aim was to determine if PLIN3 and PLIN5 associate and whether this changes following contraction. Male Long Evans rats (n = 21;age, 52 days; weight = 317 6 g) underwent 30 min of hindlimb stimulation (10 msec impulses, 100 Hz/3 sec at 10–20 V; train duration 100 msec). Contraction induced a ~50% reduction in intramuscular lipid content measured by oil red-O staining of red gastrocnemius muscle. Mitochondria were isolated from red gastrocnemius muscle by differential centrifugation and proteins were detected by western blotting. Mitochondrial PLIN5 content was ~1.6-fold higher following 30 min of contraction and PLIN3 content was detected in the mitochondrial fraction, and unchanged following contraction. An association between PLIN3 and PLIN5 was observed and remained unaltered following contraction. PLIN5 may play a role in mitochondria during lipolysis, which is consistent with a role in facilitating/regulating mitochondrial fatty acid oxidation. PLIN3 and PLIN5 may be working together on the lipid droplet and mitochondria during contraction-induced lipolysis

Effects of company and season on blood fluke (Cardicola spp.) infection in ranched Southern Bluefin Tuna: preliminary evidence infection has a negative effect on fish growth

Aporocotylid blood flukes Cardicola forsteri and C. orientalis are an ongoing health concern for Southern Bluefin Tuna (SBT), Thunnus maccoyii, ranched in Australia. Therapeutic application of praziquantel (PZQ) has reduced SBT mortalities, however PZQ is not a residual treatment therefore reinfection can occur after the single treatment application. This study documents the epidemiology of Cardicola spp. infection in ranched SBT post treatment over three ranching seasons (2018, 2019 and 2021). Infection prevalence (percentage of SBT affected) and intensity (parasite load) was determined by adult fluke counts from heart, egg counts from gill filaments and the use of specific quantitative polymerase chain reaction (qPCR) for detection of C. forsteri and C. orientalis ITS-2 DNA in SBT hearts and gills. SBT Condition Index decreased as intensity of Cardicola spp. DNA in SBT gills increased, suggesting blood fluke infection had a negative effect on SBT growth (Spearman’s r = −0.2426, d.f. = 138, p = 0.0041). Prevalence and intensity of infection indicated PZQ remained highly effective at controlling Cardicola spp. infection in ranched SBT, 10 years after PZQ administration began in this industry. Company A had the highest prevalence and intensity of Cardicola spp. infection in 2018, and Company G had the highest in 2019. No consistent pattern was seen in 2021. Overall, intensity of infection did not increase as ranching duration increased post treatment. Results from this study improve our knowledge of the biology of blood flukes and helps the SBT industry to modify or design new blood fluke management strategies to reduce health risks and improve performance of SBT

Trabecular and cortical bone are unaltered in response to chronic lipopolysaccharide exposure via osmotic pumps in male and female CD-1 mice.

Chronic low-grade inflammation has been identified as an underlying cause of many diseases including osteoporosis. Lipopolysaccharide (LPS) is a potent inducer of the inflammatory response that can negatively affect bone outcomes by upregulating bone resorption and inhibiting bone formation. The objective of this study was to assess the longitudinal response of trabecular and cortical bone structure and bone mineral density to LPS continuously administered for 12 weeks in male and female CD-1 mice. Mice were assigned to one of four LPS groups at 8-weeks of age: placebo (0.0 μg/d), low (0.9 μg/d), mid (3.6 μg/d) and high (14.4 μg/d) dose. Trabecular and cortical bone outcomes were measured at 8, 12, 16, and 20 weeks of age using in vivo micro-computed tomography. The anticipated serum LPS dose-dependent response was not observed. Therefore, the low, mid, and high LPS groups were combined for analysis. Compared to the placebo group, endpoint serum LPS was elevated in both males (p < 0.05) and females (p < 0.05) when all LPS treatment groups were combined. However, there was no significant change in trabecular or cortical bone outcomes in the combined LPS groups compared to the placebo following the 12-week LPS intervention for either sex. This suggests that although serum LPS was elevated following the 12-week LPS intervention, the dosages administered using the osmotic pumps was not sufficient to negatively impact trabecular or cortical bone outcomes in either male or female CD-1 mice

Skeletal site-specific effects of endurance running on structure and strength of tibia, lumbar vertebra, and mandible in male Sprague-Dawley rats

Bone microarchitecture, bone mineral density (BMD), and bone strength are positively affected by impact activities, such as running, however there are discrepancies in the magnitude of these changes. These inconsistencies are mainly a result of varying training protocols, analysis techniques, and whether skeletal sites measured are weight bearing or not. This studies purpose was to determine the effects of endurance running on sites that experience different weight bearing and load. Eight week old male Sprague-Dawley rats (n = 20) were randomized to either a progressive treadmill running protocol (25 m/min for 1 hour, incline of 10%) or a non-trained control group for 8-weeks. Trabecular structure of the tibia, lumbar vertebra (L3), and mandible, and cortical structure at the tibia midpoint were measured using μCT to quantify bone volume fraction, trabecular number, trabecular thickness, trabecular separation, and cortical thickness. BMD at the proximal tibia, lumbar vertebra (L1-3), and mandible was measured using DXA. Tibia midpoint strength was measured by three-point bending using a materials testing system. Endurance running resulted in superior bone structure at the proximal tibia (12% greater BV/TV, p = 0.03, 14% greater Tb.N, p = 0.01, and 19% lower Tb.Sp, p = 0.05) but not other sites. Contrary to our hypothesis, mandible bone structure was altered following endurance training (8% lower BV/TV, pThe accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

The Southern Bluefin Tuna Mucosal Microbiome Is Influenced by Husbandry Method, Net Pen Location, and Anti-parasite Treatment.

Aquaculture is the fastest growing primary industry worldwide. Marine finfish culture in open ocean net pens, or pontoons, is one of the largest growth areas and is currently the only way to rear high value fish such as bluefin tuna. Ranching involves catching wild juveniles, stocking in floating net pens and fattening for 4 to 8 months. Tuna experience several parasite-induced disease challenges in culture that can be mitigated by application of praziquantel (PZQ) as a therapeutic. In this study, we characterized the microbiome of ranched southern Bluefin Tuna, Thunnus maccoyii, across four anatomic sites (gill, skin, digesta, and anterior kidney) and evaluated environmental and pathological factors that influence microbiome composition, including the impact of PZQ treatment on microbiome stability. Southern bluefin tuna gill, skin, and digesta microbiome communities are unique and potentially influenced by husbandry practices, location of pontoon growout pens, and treatment with the antiparasitic PZQ. There was no significant relationship between the fish mucosal microbiome and incidence or abundance of adult blood fluke in the heart or fluke egg density in the gill. An enhanced understanding of microbiome diversity and function in high-value farmed fish species such as bluefin tuna is needed to optimize fish health and improve aquaculture yield. Comparison of the bluefin tuna microbiome to other fish species, including Seriola lalandi (yellowtail kingfish), a common farmed species from Australia, and Scomber japonicus (Pacific mackerel), a wild caught Scombrid relative of tuna, showed the two Scombrids had more similar microbial communities compared to other families. The finding that mucosal microbial communities are more similar in phylogenetically related fish species exposes an opportunity to develop mackerel as a model for tuna microbiome and parasite research