Post-traumatic osteoarthritis progression is diminished by early mechanical unloading and anti-inflammatory treatment in mice.

OBJECTIVE: Post-traumatic osteoarthritis (PTOA) is a degenerative joint disease initiated by injury. Early phase (0-7 days) treatments often include rest (unloading) and anti-inflammatory medications, but how those early interventions impact PTOA progression is unknown. We hypothesized that early unloading and anti-inflammatory treatment would diminish joint inflammation and slow PTOA progression. DESIGN: Mice were injured with non-invasive ACL rupture followed by hindlimb unloading (HLU) or normal cage activity (ground control: GC) for 7 days, after which all mice were allowed normal cage activity. HLU and GC mice were treated with daily celecoxib (CXB; 10 mg/kg IP) or vehicle. Protease activity was evaluated using in vivo fluorescence imaging, osteophyte formation and epiphyseal trabecular bone were quantified using micro-computed tomography, and synovitis and articular cartilage were evaluated using whole-joint histology at 7, 14, 21, and 28 days post-injury. RESULTS: HLU significantly reduced protease activity (-22-30% compared to GC) and synovitis (-24-50% relative to GC) at day 7 post-injury (during unloading), but these differences were not maintained at later timepoints. Similarly, trabecular bone volume was partially preserved in HLU mice at during unloading (-14-15% BV/TV for HLU mice, -21-22% for GC mice relative to uninjured), but these differences were not maintained during reloading. Osteophyte volume was reduced by both HLU and CXB, but there was not an additive effect of these treatments (HLU: -46%, CXB: -30%, HLU + CXB: -35% relative to vehicle GC at day 28). CONCLUSIONS: These data suggest that early unloading following joint injury can reduce inflammation and potentially slow PTOA progression.

The microbiome mediates epiphyseal bone loss and metabolomic changes after acute joint trauma in mice.

OBJECTIVE: To compare the early responses to joint injury in conventional and germ-free mice. DESIGN: Post-traumatic osteoarthritis (PTOA) was induced using a non-invasive anterior cruciate ligament rupture model in 20-week old germ-free (GF) and conventional C57BL/6 mice. Injury was induced in the left knees of n = 8 GF and n = 10 conventional mice. To examine the effects of injury, n = 5 GF and n = 9 conventional naïve control mice were used. Mice were euthanized 7 days post-injury, followed by synovial fluid recovery for global metabolomic profiling and analysis of epiphyseal trabecular bone by micro-computed tomography (µCT). Global metabolomic profiling assessed metabolic differences in the joint response to injury between GF and conventional mice. Magnitude of trabecular bone volume loss measured using µCT assessed early OA progression in GF and conventional mice. RESULTS: µCT found that GF mice had significantly less trabecular bone loss compared to conventional mice, indicating that the GF status was protective against early OA changes in bone structure. Global metabolomic profiling showed that conventional mice had greater variability in their metabolic response to injury, and a more distinct joint metabolome compared to their corresponding controls. Furthermore, differences in the response to injury in GF compared to conventional mice were linked to mouse metabolic pathways that regulate inflammation associated with the innate immune system. CONCLUSIONS: These results suggest that the gut microbiota promote the development of PTOA during the acute phase following joint trauma possibly through the regulation of the innate immune system.

Incident fracture is associated with a period of accelerated loss of hip BMD: the Study of Osteoporotic Fractures.

Bone loss following a fracture could increase the risk of future fractures. In this study, we found that elderly women who had an upper body fracture or multiple fractures lost more bone at the hip than those who did not fracture. This suggests a possible systemic bone loss response initiated by fracture. INTRODUCTION: A prior fracture is one of the strongest predictors of subsequent fracture risk, but the etiology of this phenomenon remains unclear. Systemic bone loss post-fracture could contribute to increased risk of subsequent fractures. Therefore, in this study, we investigated whether incident fractures, including those distant to the hip, are associated with accelerated loss of hip bone mineral density (BMD) in elderly women. METHODS: We analyzed data from 3956 Caucasian women aged ≥ 65 years who were enrolled in the Study of Osteoporotic Fractures and completed hip BMD measurements at study visit 4 (year 6) and visit 6 (year 10). Clinical fractures between visits 4 and 6 were ascertained from triannual questionnaires and centrally adjudicated by review of community radiographic reports. Subjects provided questionnaire information and clinical variables at examinations for known and potential covariates. Generalized linear models were used to calculate average annual percent change in total hip BMD between visits 4 and 6 for each incident fracture type and for upper body and lower body fractures combined. A subset of women (n = 3783) was analyzed for annual total hip BMD change between study visits 4 and 5 and between study visits 5 and 6 to evaluate change in total hip BMD during these 2-year intervals. RESULTS: Women with incident upper body fracture or incident lower body fracture exhibited reductions in total hip BMD of 0.89 and 0.77% per year, respectively, while women who did not fracture exhibited reductions in total hip BMD of 0.66% per year during the 4-year period. Accelerated loss of hip BMD was isolated to the 2-year time interval that included the fracture. Loss of total hip BMD was not affected by the number of days from fracture to follow up DXA. CONCLUSIONS: Systemic bone loss following fracture may increase the risk of future fractures at all skeletal sites. There is a need for improved understanding of mechanisms leading to apparent accelerated bone loss following a fracture in order to reduce subsequent fracture risk.

Non-invasive mouse models of post-traumatic osteoarthritis.

Animal models of osteoarthritis (OA) are essential tools for investigating the development of the disease on a more rapid timeline than human OA. Mice are particularly useful due to the plethora of genetically modified or inbred mouse strains available. The majority of available mouse models of OA use a joint injury or other acute insult to initiate joint degeneration, representing post-traumatic osteoarthritis (PTOA). However, no consensus exists on which injury methods are most translatable to human OA. Currently, surgical injury methods are most commonly used for studies of OA in mice; however, these methods may have confounding effects due to the surgical/invasive injury procedure itself, rather than the targeted joint injury. Non-invasive injury methods avoid this complication by mechanically inducing a joint injury externally, without breaking the skin or disrupting the joint. In this regard, non-invasive injury models may be crucial for investigating early adaptive processes initiated at the time of injury, and may be more representative of human OA in which injury is induced mechanically. A small number of non-invasive mouse models of PTOA have been described within the last few years, including intra-articular fracture of tibial subchondral bone, cyclic tibial compression loading of articular cartilage, and anterior cruciate ligament (ACL) rupture via tibial compression overload. This review describes the methods used to induce joint injury in each of these non-invasive models, and presents the findings of studies utilizing these models. Altogether, these non-invasive mouse models represent a unique and important spectrum of animal models for studying different aspects of PTOA.

In vivo fluorescence reflectance imaging of protease activity in a mouse model of post-traumatic osteoarthritis.

OBJECTIVE: Joint injuries initiate a surge of inflammatory cytokines and proteases that contribute to cartilage and subchondral bone degeneration. Detecting these early processes in animal models of post-traumatic osteoarthritis (PTOA) typically involves ex vivo analysis of blood serum or synovial fluid biomarkers, or histological analysis of the joint. In this study, we used in vivo fluorescence reflectance imaging (FRI) to quantify protease, matrix metalloproteinase (MMP), and Cathepsin K activity in mice following anterior cruciate ligament (ACL) rupture. We hypothesized that these processes would be elevated at early time points following joint injury, but would return to control levels at later time points. DESIGN: Mice were injured via tibial compression overload, and FRI was performed at time points from 1 to 56 days after injury using commercially available activatable fluorescent tracers to quantify protease, MMP, and cathepsin K activity in injured vs uninjured knees. PTOA was assessed at 56 days post-injury using micro-computed tomography and whole-joint histology. RESULTS: Protease activity, MMP activity, and cathepsin K activity were all significantly increased in injured knees relative to uninjured knees at all time points, peaking at 1-7 days post-injury, then decreasing at later time points while still remaining elevated relative to controls. CONCLUSIONS: This study establishes FRI as a reliable method for in vivo quantification of early biological processes in a translatable mouse model of PTOA, and provides crucial information about the time course of inflammation and biological activity following joint injury. These data may inform future studies aimed at targeting these early processes to inhibit PTOA development.

The effect of noggin interference in a rabbit posterolateral spinal fusion model.

STUDY DESIGN: Noggin protein levels and spinal fusion rates were compared in a rabbit model after application of siRNA against BMP antagonist noggin in paraspinal muscle. OBJECTIVE: To test whether endogenous BMPs are sufficient to form bone in the absence of their antagonists, using noggin siRNA to interrupt the negative feedback loop on endogenous BMP within the paraspinal muscles in rabbits. Unused Posterolateral lumbar fusion is a standard surgical treatment for many spinal disorders, yet even under ideal conditions the rate of non-fusion approaches 25 %. BMPs are effective in promoting bone formation, and are inhibited by antagonists such as noggin. We have previously shown that in this model, endogenous BMPs are present and endogenous BMP antagonist noggin is strongly increased during spinal fusion. Previous studies have found that noggin siRNA enhanced spinal fusion in combination with supra-physiological amounts of exogenous BMP; however, the effect of the siRNA alone remains unknown. METHODS: A posterolateral intertransverse rabbit lumbar fusion was utilized, as established by Boden et al. SiRNA against noggin was electroporated into paraspinal muscle to determine its effect on fusion. Outcome measures included noggin protein expression, and assessment of spinal fusion at 6 weeks. RESULTS: SiRNAs were effective in reducing overexpressed noggin in vitro. Noggin protein was successfully knocked down in vivo for the initial 7 days in our rabbit model and returned to detectable levels by 4 weeks and to normal levels by 6 weeks. The overall fusion rate was not significantly enhanced compared to established controls from our earlier work (Tang et al.). CONCLUSIONS: Early noggin suppression does not appear to enhance the BMP activity sufficiently to significantly affect final fusion rates in our model.

Altered bone development in a mouse model of peripheral sensory nerve inactivation.

OBJECTIVES: The present study sought to determine the effects of decreased peripheral sensory nerve function on skeletal development and bone metabolism in mice. METHODS: C57BL/6 neonatal mice were treated with capsaicin to induce peripheral sensory nerve degeneration, and compared to vehicle-treated controls at 4, 8 and 12 weeks of age. Changes in bone structure were assessed using micro-computed tomography, mechanical properties and fracture resistance were assessed using three-point bending of radii, and bone turnover was assessed using dynamic histomorphometry and serum biomarkers. RESULTS: Capsaicin treatment resulted in small but significant decreases in bone structure, particularly affecting trabecular bone. Capsaicin-treated mice exhibited lower trabecular thickness at the femoral metaphysis and L5 vertebral body compared with vehicle-treated mice. However, capsaicin- and vehicle-treated mice had similar mechanical properties and bone turnover rates. CONCLUSION: Neonatal capsaicin treatment affected trabecular bone during development; however these small changes may not be meaningful with respect to bone strength under normal loading conditions. It is possible that capsaicin-sensitive neurons may be more important for bone under stress conditions such as increased mechanical loading or injury. Future studies will investigate this potential role of peripheral sensory nerves in bone adaptation.

Musculoskeletal changes following non-invasive knee injury using a novel mouse model of post-traumatic osteoarthritis.

OBJECTIVE: Post-traumatic osteoarthritis (PTOA) is a common consequence of traumatic joint injury, with 50% of anterior cruciate ligament (ACL) rupture patients developing PTOA within 10-20 years. Currently accepted mouse models of PTOA initiate symptoms using various methods, none of which faithfully mimic clinically-relevant injury conditions. In this study we characterize a novel non-invasive mouse model of PTOA that injures the ACL with a single load of tibial compression overload. We utilize this model to determine the time course of articular cartilage and subchondral bone changes following knee injury. DESIGN: Mice were euthanized 1, 3, 7, 14, 28, or 56 days after non-invasive knee injury. Knees were scanned using micro-computed tomography (µCT) in order to quantify subchondral trabecular bone, subchondral bone plate, and non-native bone formation (heterotopic ossification). Development of osteoarthritis (OA) was graded using the osteoarthritis research society international (OARSI) scale on histological sections of injured and uninjured knees. RESULTS: Following injury we observed a rapid loss of trabecular bone in injured knees compared to uninjured knees by 7 days post-injury, followed by a partial recovery of trabecular bone to a new steady state by 28 days post-injury. We also observed considerable non-native bone formation by 56 days post-injury. Grading of histological sections revealed deterioration of articular cartilage by 56 days post-injury, consistent with development of mild OA. CONCLUSIONS: This study establishes a novel mouse model of PTOA, and describes the time course of musculoskeletal changes following knee injury, helping to establish the window of opportunity for preventative treatment.

Reliability of vertebral fracture assessment using multidetector CT lateral scout views: the Framingham Osteoporosis Study.

UNLABELLED: Two radiologists evaluated images of the spine from computed tomography (CT) scans on two occasions to diagnose vertebral fracture in 100 individuals. Agreement was fair to good for mild fractures, and agreement was good to excellent for more severe fractures. CT scout views are useful to assess vertebral fracture. INTRODUCTION: We investigated inter-reader agreement between two radiologists and intra-reader agreement between duplicate readings for each radiologist, in assessment of vertebral fracture using a semi-quantitative method from lateral scout views obtained by CT. METHODS: Participants included 50 women and 50 men (age 50-87 years, mean 70 years) in the Framingham Study. T4-L4 vertebrae were assessed independently by two radiologists on two occasions using a semi-quantitative scale as normal, mild, moderate, or severe fracture. RESULTS: Vertebra-specific prevalence of grade ≥ 1 (mild) fracture ranged from 3% to 5%. We found fair (κ = 56-59%) inter-reader agreement for grade ≥ 1 vertebral fractures and good (κ = 68-72%) inter-reader agreement for grade ≥ 2 fractures. Intra-reader agreement for grade ≥ 1 vertebral fracture was fair (κ = 55%) for one reader and excellent for another reader (κ = 77%), whereas intra-reader agreement for grade ≥ 2 vertebral fracture was excellent for both readers (κ = 76% and 98%). Thoracic vertebrae were more difficult to evaluate than the lumbar region, and agreement was lowest (inter-reader κ = 43%) for fracture at the upper (T4-T9) thoracic levels and highest (inter-reader κ = 76-78%) for the lumbar spine (L1-L4). CONCLUSIONS: Based on a semi-quantitative method to classify vertebral fractures using CT scout views, agreement within and between readers was fair to good, with the greatest source of variation occurring for fractures of mild severity and for the upper thoracic region. Agreement was good to excellent for fractures of at least moderate severity. Lateral CT scout views can be useful in clinical research settings to assess vertebral fracture.

Expectancy for social facilitation from drinking: the divergent paths of high-expectancy and low-expectancy adolescents.

Using a 3-wave longitudinal design, adolescents were studied over a 2-year period during which many first began to drink. Covariance structure modeling showed that teens' expectancy for social facilitation from alcohol and their drinking experience influenced each other in a reciprocal, positive feedback fashion: the greater the expectancy endorsement, the higher subsequent drinking levels, and the higher the drinking levels, the greater the subsequent expectancy endorsement. This model fit the data quite well; comparison models, in which expectancy (or drinking) had no independent influence on future drinking (or expectancy), showed significantly poorer fit than the present model. Initial nondrinkers' social expectancy predicted individual differences in the rate of drinking increase over the 2 years. Results bolster the hypothesis that expectancy actively influences drinking and point to the importance of expectancy-based intervention efforts.

Latent inhibition of the rat eyeblink response: effect of hippocampal aspiration lesions.

The effect of hippocampal aspiration lesions on latent inhibition of eyeblink conditioning in the restrained rat preparation was examined. Rats received either sham, cortical control, or hippocampal aspiration lesions. Control animals, but not animals with hippocampal lesions, showed slower conditioning after being preexposed to the conditioned stimulus (latent inhibition). Together with previous results regarding the effect of hippocampal lesions in acquisition and extinction of delay conditioning, the present study suggests that the restrained rat preparation may serve as a reliable way of investigating hippocampal participation in different classical conditioning paradigms.

Hippocampectomy disrupts the topography of the rat eyeblink response during acquisition and extinction of classical conditioning.

The effect of hippocampal lesions on acquisition and extinction of eyeblink conditioning in rats was examined with the procedure described by Schmajuk and Christiansen (Physiol. Behav., 48 (1990) 755-758). Male Long-Evans rats received either sham, cortical control or hippocampal lesions. Animals were trained in a delay conditioning paradigm with a 500-ms, 98-dB, white noise conditioned stimulus; a 150-ms, 5-psi air puff unconditioned stimulus; a 350-ms interstimulus interval; and a 60-s average intertrial interval. Each animal received 50 trials daily until it reached a criterion of 80% conditioned responses (CR) on a given day. After acquisition criterion was reached, extinction began the next day and continued until each animal achieved a criterion of one CR in a block of 10 consecutive trials. Although hippocampal lesions affected neither acquisition nor extinction rates, hippocampal lesioned animals showed significantly shorter CR onset latency during acquisition and extinction, and larger CR peak amplitude during acquisition. The results are discussed in terms of hippocampal modulation of cerebellar learning.

Alcoholism and memory: broadening the scope of alcohol-expectancy research.

Current biopsychosocial research on the etiology of alcoholism has begun to focus on memory processes as a possible common pathway for drinking decisions. The alcohol-expectancy construct is rooted both in cognitive psychology and alcohol research and can serve as a vehicle for this study. Reexamination of one recent review of issues in alcohol-expectancy research provides an opportunity to broaden the scope of this research with theoretical and methodological alternatives to those suggested in that review. Most importantly, this article shows that expectancy findings, discussed by Leigh (1989a) as reflecting "psychometric" limitations, are instead quite consistent with recent network models of memory structure. Such models can provide an informative guide to future research activities. It is also recommended that alcohol-expectancy research remain open to inputs from expectancy theories already developed in several psychological domains, as well as to theories of social cognition and attitude structure in addition to those advanced by Leigh.

Eyeblink conditioning in rats.

Acquisition of eyeblink conditioned responses to a tone was studied in restrained male albino rats. Animals were placed in a modified restraining cage with their heads immobilized by surgically implanting a small bolt at the top of the skull and fastening it to the cage. Eyeblink responses were measured by a phototransistor which detected changes in the level of light reflected by eyelid closure. The conditioned stimulus was a 2,000-Hz, 90-dB, 500-ms tone; the unconditioned stimulus was a 4-psi, 150-ms air puff directed at the cornea; and the interstimulus interval was 350 ms, with a 60-s intertrial interval. Animals received 50 trials daily. All conditioned animals reached a criterion of 80% conditioned responses by Day 10. Pseudoconditioned control animals showed few conditioned responses by Day 10. The topography of the conditioned responses was comparable to those responses found with the nicitating membrane response in the rabbit. The data suggest that eyeblink conditioning in the rat can be used as a preparation to examine the attributes of classical conditioning as well as the neuroanatomical basis of associative learning.

Using alcohol expectancies to predict adolescent drinking behavior after one year.

An accumulating literature has shown the influence of childhood experiences associated with alcohol use on later drinking practices. Recent studies have suggested that alcohol-related expectancy may serve as an intervening variable to connect these early experiences with the later, proximal decision to drink when opportunities for actual alcohol consumption arise. Those studies, however, have collected expectancy and drinking data concurrently, whereas the present study for the first time reports on the power of expectancies measured in early adolescents (seventh and eighth grades) to predict self-reported drinking onset and drinking behavior measured a full year later. Results show that five of seven expectancy scores readily discriminated between nonproblem drinkers and those subsequently beginning problem drinking and accounted for a large portion of the variance in a continuous quantity/frequency index and a problem drinking index. The strength of these timelagged relations strengthens the case for inferring that expectancies have causal power on drinking behavior and suggests prevention strategies.

Cross-cultural comparisons of Irish and American adolescent drinking practices and beliefs.

The drinking behavior and alcohol expectancies of 168 Irish adolescents aged 15-18 were compared with those of a group of American adolescents matched on age and sex. The Irish adolescents reported less frequent social drinking and less problematic drinking. However, unlike American adolescents, those Irish youth who did drink in a social, frequent manner also reported drinking-related problems Irish adolescents expect less social benefit, less improvement of cognitive and motor functioning and less sexual enhancement, but greater increase in aggression as a consequence of drinking. These findings are discussed as possible etiological clues to established differences between Irish adult drinking and drinking by adults in other countries.

The Alcohol Expectancy Questionnaire: an instrument for the assessment of adolescent and adult alcohol expectancies.

Alcohol-related expectancies can influence the behavioral effects of alcohol and decisions regarding alcohol use. A standard measure of expectancies is needed for research and clinical work in the alcohol arena; the development and current status of the Alcohol Expectancy Questionnaire (AEQ) is summarized. The adolescent and adult forms of this instrument are designed to measure the degree to which individuals expect alcohol to produce a variety of general and specific effects. Research with the AEQ indicates a consistent relationship between alcohol expectancies and alcohol consumption, alcohol abuse and behavior while drinking. Data bearing on the psychometric properties and clinical and research utility of the AEQ are discussed.

The differential development of adolescent alcohol expectancies may predict adult alcoholism.

To investigate changes in adolescent's alcohol expectancies as a function of increasing age and drinking experience, we compared the degree to which 12-14, 15-16, and 17-19 year old adolescents from normal seventh to twelfth grade classrooms (N = 1580) affirmed items comprising seven alcohol-expectancy scales. Results showed that adolescents increasingly believe alcohol improves social behavior, increases arousal, and decreases tension as they age. In contrast, the belief that alcohol improves cognitive and motor functioning increased and then decreased in a general adolescent sample, but remained high in problem drinking adolescents. The subsequent discovery of this same factor in 305 hospitalized alcoholics suggests that strong affirmation of this expectancy in late adolescence may have prognostic, and perhaps etiologic significance for the development of alcoholism.