Secukinumab and Dead Sea Climatotherapy Impact Resolved Psoriasis Skin Differently Potentially Affecting Disease Memory.

Secukinumab and Dead Sea treatment result in clear skin for many psoriasis patients, through distinct mechanisms. However, recurrence in the same areas after treatments suggests the existence of a molecular scar. We aimed to compare the molecular and genetic differences in psoriasis patients who achieved complete response from secukinumab and Dead Sea climatotherapy treatments. We performed quantitative immunohistochemical and transcriptomic analysis, in addition to digital spatial profiling of skin punch biopsies. Histologically, both treatments resulted in a normalization of the lesional skin to a level resembling nonlesional skin. Interestingly, the transcriptome was not normalized by either treatments. We revealed 479 differentially expressed genes between secukinumab and Dead Sea climatotherapy at the end of treatment, with a psoriasis panel identifying SERPINB4, SERPINB13, IL36G, IL36RN, and AKR1B10 as upregulated in Dead Sea climatotherapy compared with secukinumab. Using digital spatial profiling, pan-RAS was observed to be differentially expressed in the microenvironment surrounding CD103+ cells, and IDO1 was differentially expressed in the dermis when comparing the two treatments. The differences observed between secukinumab and Dead Sea climatotherapy suggest the presence of a molecular scar, which may stem from mechanistically different pathways and potentially contribute to disease recurrence. This may be important for determining treatment response duration and disease memory.

Pharmaceutical treatment of bone loss: From animal models and drug development to future treatment strategies.

Animal models are fundamental to advance our knowledge of the underlying pathophysiology of bone loss and to study pharmaceutical countermeasures against it. The animal model of post-menopausal osteoporosis from ovariectomy is the most widely used preclinical approach to study skeletal deterioration. However, several other animal models exist, each with unique characteristics such as bone loss from disuse, lactation, glucocorticoid excess, or exposure to hypobaric hypoxia. The present review aimed to provide a comprehensive overview of these animal models to emphasize the importance and significance of investigating bone loss and pharmaceutical countermeasures from perspectives other than post-menopausal osteoporosis only. Hence, the pathophysiology and underlying cellular mechanisms involved in the various types of bone loss are different, and this might influence which prevention and treatment strategies are the most effective. In addition, the review sought to map the current landscape of pharmaceutical countermeasures against osteoporosis with an emphasis on how drug development has changed from being driven by clinical observations and enhancement or repurposing of existing drugs to today's use of targeted anti-bodies that are the result of advanced insights into the underlying molecular mechanisms of bone formation and resorption. Moreover, new treatment combinations or repurposing opportunities of already approved drugs with a focus on dabigatran, parathyroid hormone and abaloparatide, growth hormone, inhibitors of the activin signaling pathway, acetazolamide, zoledronate, and romosozumab are discussed. Despite the considerable progress in drug development, there is still a clear need to improve treatment strategies and develop new pharmaceuticals against various types of osteoporosis. The review also highlights that new treatment indications should be explored using multiple animal models of bone loss in order to ensure a broad representation of different types of skeletal deterioration instead of mainly focusing on primary osteoporosis from post-menopausal estrogen deficiency.

Contemporary Advances in Computer-Assisted Bone Histomorphometry and Identification of Bone Cells in Culture.

Static and dynamic bone histomorphometry and identification of bone cells in culture are labor-intensive and highly repetitive tasks. Several computer-assisted methods have been proposed to ease these tasks and to take advantage of the increased computational power available today. The present review aimed to provide an overview of contemporary methods utilizing specialized computer software to perform bone histomorphometry or identification of bone cells in culture. In addition, a brief historical perspective on bone histomorphometry is included. We identified ten publications using five different computer-assisted approaches (1) ImageJ and BoneJ; (2) Histomorph: OsteoidHisto, CalceinHisto, and TrapHisto; (3) Fiji/ImageJ2 and Trainable Weka Segmentation (TWS); (4) Visiopharm and artificial intelligence (AI); and (5) Osteoclast identification using deep learning with Single Shot Detection (SSD) architecture, Darknet and You Only Look Once (YOLO), or watershed algorithm (OC_Finder). The review also highlighted a substantial need for more validation studies that evaluate the accuracy of the new computational methods to the manual and conventional analyses of histological bone specimens and cells in culture using microscopy. However, a substantial evolution has occurred during the last decade to identify and separate bone cells and structures of interest. Most early studies have used simple image segmentation to separate structures of interest, whereas the most recent studies have utilized AI and deep learning. AI has been proposed to substantially decrease the amount of time needed for analyses and enable unbiased assessments. Despite the clear advantages of highly sophisticated computational methods, the limited nature of existing validation studies, particularly those that assess the accuracy of the third-generation methods compared to the second-generation methods, appears to be an important reason that these techniques have failed to gain wide acceptance.

Sports Participation and Osteoarthritis in Females: A Systematic Review.

Sports participation and the risk of osteoarthritis (OA) have been a concern for decades. Few research efforts have been dedicated to clarify this issue for females, although they are considered at greater risk of developing OA than males. In contrast, several reviews have established an association between sports participation and OA for males. The aim of the systematic review was to assess the association between OA and participation in popular sports for females. PubMed, Embase, and Google Scholar were searched and yielded 578 articles. Nine eligible studies were included and covered ballet (age range: 19-54 years), running or tennis (age range: 40-65 years), Olympic sports (age range: not specified), volleyball (age range: 16.0 ± 0.8 to 46.8 ± 5.1 years), and cross-country skiing (age range: 15 to ≥60 years). For females, participating in sports at an elite level was associated with a higher risk of OA and an increased need for surgical treatment. At non-elite level, it was associated with a higher risk of OA, but it did not materialize to an increased risk for surgical treatment. Few studies compared females and males, and these studies suggested that sex did not affect the risk of developing OA from participating in sports. Nevertheless, to isolate the precise effect of sports participation on the development of OA remains difficult as injuries are common among athletes and are independently associated with an increased risk of OA.

Drill-Hole Bone Defects in Animal Models of Bone Healing: Protocol for a Systematic Review.

BACKGROUND: Bone fractures are common conditions of the musculoskeletal system. Several animal models of bone fractures have been established to help elucidate the complex process of bone healing. In the last decades, drill-hole bone defects have emerged as a method to study bone healing. Animal models of drill-hole defects are easy to standardize and do not require external fixation of the bone. However, current studies of drill-hole bone defects lack detailed descriptions of techniques and interstudy standardization. OBJECTIVE: This systematic review aims to present a detailed description of the different methods used to induce drill-hole bone defects in long bones of laboratory animals and to provide a comprehensive overview of their methodology and potential for investigation of bone healing. METHODS: A systematic search of PubMed and Embase will be performed of abstracts containing variations of the following four keywords: "long bone," "drill-hole," "regeneration," and "animal model." Abstract screening and full-text screening will be performed independently by 2 reviewers, and data will be extracted to a predesigned extraction protocol. The primary outcome of the included studies is the technique used to create the drill-hole bone defect, and secondary outcomes are any measurements or analyses of bone defect and regeneration. A narrative synthesis will be used to present the primary outcome, while information on secondary outcomes will be displayed graphically. The study protocol follows the PRISMA-P (Preferred Reporting Items for Systematic Review and Meta-analysis Protocols) guidelines. RESULTS: Abstract and full-text screening is ongoing and is expected to be completed by October 2022. Data extraction will commence immediately after, and the manuscript is expected to be completed by December 2023. The systematic review will follow the PRISMA statement. CONCLUSIONS: The strength of this systematic review is that it provides a comprehensive methodological overview of the different drill-hole methods and their advantages and disadvantages. This will assist researchers in choosing which model to use when studying different aspects of bone healing. TRIAL REGISTRATION: International Prospective Register of Systematic Reviews CRD42020213076; https://tinyurl.com/bp56wdwe. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): PRR1-10.2196/34887.

Quantification of Immunohistochemically Stained Cells in Skin Biopsies.

Immunohistochemical quantification of inflammatory cells in skin biopsies is a valuable tool for diagnosing skin diseases and assessing treatment response. The quantification of individual cells in biopsies is time-consuming, tedious, and difficult. In this study, we presented and compared two methods for the quantification of CD8+ T cells in skin biopsies from patients with psoriasis using both commercial software (Adobe Photoshop) and open-source software (Qupath). In addition, we provided a detailed, step-by-step description of both methods. The methods are scalable by replacing the CD8 antibody with other antibodies to target different cells. Moreover, we investigated the correlation between quantifying CD8+ cells normalized to area or epidermal length and cell classifications, compared cell classifications in QuPath with threshold classifications in Photoshop, and analyzed the impact of data normalization to epidermal length or area on inflammatory cell densities in skin biopsies from patients with psoriasis. We found a satisfactory correlation between normalizing data to epidermal length and area for psoriasis skin. However, when non-lesional and lesional skin samples were compared, a significant underestimation of inflammatory cell density was found when data were normalized to area instead of epidermal length. Finally, Bland-Altman plots comparing Qupath and Photoshop to quantify inflammatory cell density demonstrated a good agreement between the two methods.

Anti-sclerostin antibodies and abaloparatide have additive effects when used as a countermeasure against disuse osteopenia in female rats.

Prolonged disuse and substantial mechanical unloading are particularly damaging to skeletal integrity. Preclinical studies in rodents and clinical studies have highlighted the need for potent bone anabolic drugs to counteract disuse-induced osteopenia. The aim of present study was to compare the efficacy of romosozumab (Scl-Ab) and abaloparatide (ABL), alone or in combination, to prevent botulinum toxin (BTX) induced bone loss in a rat model. Eighty female Wistar rats were divided into the following six groups: 1. Baseline (n = 12); 2. Control (Ctrl) (n = 12); 3. BTX (n = 12); 4. BTX + Scl-Ab (n = 16); 5. BTX + ABL (n = 12); and 6. BTX + Scl-Ab + ABL (n = 16). Disuse was achieved by injecting 4 IU BTX into the hind limb musculature at study start. Scl-Ab (25 mg/kg) was injected s.c. twice weekly, while ABL (80 µg/kg) was injected s.c. five days a week for four weeks. Hind limb disuse dramatically decreased muscle mass and skeletal integrity and deteriorated the cortical morphology and trabecular microstructure. Treatment with Scl-Ab alone prevented most of the adverse cortical and trabecular effects of disuse, while ABL monotherapy mainly attenuated the disuse-induced loss of femoral areal bone mineral density (aBMD). Moreover, the combination of Scl-Ab and ABL not only counteracted most of the negative skeletal effects of unloading, but also increased aBMD (+10% and +20%), epiphyseal trabecular bone volume fraction (BV/TV) (+25% and +73%), and metaphyseal bone strength (+18% and +30%) significantly above that of Scl-Ab or ABL monotherapy, respectively. The potent and additive osteoanabolic effect of Scl-Ab and ABL, when given in combination, is highly intriguing and underlines that an osteoanabolic bone gain can be maximized by utilizing two pharmaceuticals targeting different cellular signaling pathways. From a clinical perspective, a combination treatment may be warranted in patients where the osteoanabolic effect of either monotherapy is not sufficient, or if a dose-reduction is required due to adverse effects.

Effect of Acetazolamide and Zoledronate on Simulated High Altitude-Induced Bone Loss.

Exposure to hypobaric hypoxia at high altitude puts mountaineers at risk of acute mountain sickness. The carbonic anhydrase inhibitor acetazolamide is used to accelerate acclimatization, when it is not feasible to make a controlled and slow ascend. Studies in rodents have suggested that exposure to hypobaric hypoxia deteriorates bone integrity and reduces bone strength. The study investigated the effect of treatment with acetazolamide and the bisphosphonate, zoledronate, on the skeletal effects of exposure to hypobaric hypoxia. Eighty 16-week-old female RjOrl : SWISS mice were divided into five groups: 1. Baseline; 2. Normobaric; 3. Hypobaric hypoxia; 4. Hypobaric hypoxia + acetazolamide, and 5. Hypobaric hypoxia + zoledronate. Acetazolamide was administered in the drinking water (62 mg/kg/day) for four weeks, and zoledronate (100 µg/kg) was administered as a single subcutaneous injection at study start. Exposure to hypobaric hypoxia significantly increased lung wet weight and decreased femoral cortical thickness. Trabecular bone was spared from the detrimental effects of hypobaric hypoxia, although a trend towards reduced bone volume fraction was found at the L4 vertebral body. Treatment with acetazolamide did not have any negative skeletal effects, but could not mitigate the altitude-induced bone loss. Zoledronate was able to prevent the altitude-induced reduction in cortical thickness. In conclusion, simulated high altitude affected primarily cortical bone, whereas trabecular bone was spared. Only treatment with zoledronate prevented the altitude-induced cortical bone loss. The study provides preclinical support for future studies of zoledronate as a potential pharmacological countermeasure for altitude-related bone loss.

Hypobaric hypoxia deteriorates bone mass and strength in mice.

Mountaineers at high altitude are at increased risk of acute mountain sickness as well as high altitude pulmonary and cerebral edema. A densitometric study in mountaineers has suggested that expeditions at high altitude decrease bone mineral density. Surprisingly, the in vivo skeletal effects of hypobaric hypoxia are largely unknown, and have not been studied using advanced contemporary methods to assess bone microstructure. Eighty-four 22-week-old female mice were divided into seven groups with 12 mice in each group: 1. Baseline; 2. Normobaric, 4 weeks; 3. Hypobaric hypoxia, 4 weeks; 4. Normobaric, 8 weeks; 5. Hypobaric hypoxia, 8 weeks; 6. Normobaric, 12 weeks; and 7. Hypobaric hypoxia, 12 weeks. Hypobaric hypoxia mice were housed in hypobaric chambers at an ambient pressure of 500 mbar (5500 m altitude), while normobaric mice were housed at sea level atmospheric pressure for 4, 8, or 12 weeks, respectively. Hypobaric hypoxia had a profound impact on femoral cortical bone and L4 trabecular bone, while the effect on femoral trabecular bone was less pronounced. Hypobaric hypoxia reduced the bone strength of the femoral mid-diaphysis and L4 at all time-points. At femoral cortical bone, hypobaric hypoxia reduced bone formation through fewer mineralizing surfaces and lower bone formation rate after 2 weeks. In addition, bone strength decreased, and C-terminal telopeptide of type I collagen (CTX-I) increased independently of the duration of exposure to simulated high altitude. At L4, hypobaric hypoxia resulted in a substantial reduction in bone volume fraction, trabecular thickness, and trabecular number after 4 weeks of exposure. Hypobaric hypoxia reduced bone strength and femoral bone mass, while femoral trabecular bone was much less affected, indicating the skeletal response to hypobaric hypoxia differ between cortical and trabecular bone. These findings provide initial preclinical support for future clinical studies in mountaineers to assess bone status and bone strength after exposure to prolonged high altitude exposure.

A review of the skeletal effects of exposure to high altitude and potential mechanisms for hypobaric hypoxia-induced bone loss.

Mountaineering and exposure to high altitude result in physiological adaptations to the reduced inspiratory oxygen availability. Acute mountain sickness (AMS), high altitude pulmonary edema (HAPE), and high altitude cerebral edema (HACE) are well-described harmful effects of exposure to high altitude. Common to AMS, HAPE, and HACE are distinct clinical signs and symptoms of impaired function. However, several studies have suggested that high altitude might result in a substantial bone loss, which usually does not produce any apparent symptoms. This review aims to provide a comprehensive overview of, and map current knowledge of the skeletal effects of hypobaric hypoxia and high altitude. PubMed and Embase were searched from inception to September 6, 2021, to identify studies investigating the skeletal effects of exposure to hypobaric hypoxia and high altitude. Three hundred sixty titles and abstracts were screened, and 20 full-text articles were included (16 in vivo studies and four real-world human studies). In rodents, simulated high altitude up to 2900 m did not result in any adverse skeletal effects. In contrast, studies exposing animals to very high altitude (3500-5500 m) reported substantial reductions in BMD, cortical morphology, and bone strength, as well as deteriorated trabecular microstructure. Detrimental microstructural effects were also reported in rats exposed to simulated extreme altitude (6000 m). Finally, real-world human studies in mountaineers suggested high altitude exposure reduced bone mineral density (BMD) and that the harmful skeletal effects of hypobaric hypoxia were not entirely recovered after 12 months. In conclusion, in vivo and real-world studies demonstrated high altitude exposure results in adverse skeletal effects. The underlying mechanism for hypobaric hypoxia-induced bone loss is not elucidated.

Abaloparatide: A review of preclinical and clinical studies.

Osteoporosis is a debilitating disease characterized by reduced bone mineral density and an increased risk of fractures. This review aims to provide a comprehensive overview of, and map current knowledge, obtained from preclinical and clinical studies of the osteoanabolic agent abaloparatide. PubMed and Embase were meticulously searched from inception to May 4, 2021.178 titles and abstracts were screened, and 57 full-text articles were assessed for inclusion. A total of 55 articles were included; 5 (9%) in vitro studies, 21 (38%) in vivo studies, and 29 (53%) clinical studies. Preclinical in vitro studies have demonstrated receptor conformation preferability, structural insights into the receptor-agonist complex, and proliferative effects of abaloparatide on osteoblasts. Preclinical studies have shown abaloparatide to be similarly effective to teriparatide using comparable doses in both ambulating mice and rats challenged by disuse. Other animal studies have reported that abaloparatide effectively mitigates or prevents bone loss from ovariectomy, orchiectomy, and glucocorticoids and improves fracture healing. The pivotal clinical study ACTIVE demonstrated 18 months of treatment with abaloparatide substantially increase bone mineral density and reduce fracture risk in post-menopausal women compared with placebo. The extension study ACTIVExtend highlighted that subsequent treatment with alendronate sustained the bone gained by abaloparatide treatment and the reduced fracture risk for up to two years. Post-hoc sub-group analyses have also supported the efficacy and safety of abaloparatide treatment independent of various baseline risk factors. In conclusion, mounting evidence from preclinical and clinical studies has uniformly reported that abaloparatide increases bone mineral density and reduces fracture risk.

Artificial intelligence-assisted identification and quantification of osteoclasts.

Quantification of osteoclasts to assess bone resorption is a time-consuming and tedious process. Since the inception of bone histomorphometry and manual counting of osteoclasts using bright-field microscopy, several approaches have been proposed to accelerate the counting process using both free and commercially available software. However, most of the present alternatives depend on manual or semi-automatic color segmentation and do not take advantage of artificial intelligence (AI). The present study directly compare estimates of osteoclast-covered surfaces (Oc.S/BS) obtained by the conventional manual method using a bright-field microscope to that obtained by a new AI-assisted method. We present a detailed step-by-step guide for the AI-based method. Tibiae from Wistar rats were either enzymatically stained for TRAP or immunostained for cathepsin K to identify osteoclasts. We found that estimation of Oc.S/BS by the new AI-assisted method was considerably less time-consuming, while still providing similar results to the conventional manual method. In addition, the retrainable AI-module used in the present study allows for fully automated overnight batch processing of multiple annotated sections.•Bone histomorphometry•AI-assisted osteoclast identification•TRAP and cathepsin K.

Short-term glucocorticoid excess blunts abaloparatide-induced increase in femoral bone mass and strength in mice.

Glucocorticoids (GCs), such as prednisolone, are widely used to treat inflammatory diseases. Continuously long-term or high dose treatment with GCs is one of the most common causes of secondary osteoporosis and is associated with sarcopenia and increased risk of debilitating osteoporotic fragility fractures. Abaloparatide (ABL) is a potent parathyroid hormone-related peptide analog, which can increase bone mineral density (aBMD), improve trabecular microarchitecture, and increase bone strength. The present study aimed to investigate whether GC excess blunts the osteoanabolic effect of ABL. Sixty 12-13-week-old female RjOrl:SWISS mice were allocated to the following groups: Baseline, Control, ABL, GC, and GC + ABL. ABL was administered as subcutaneous injections (100 µg/kg), while GC was delivered by subcutaneous implantation of a 60-days slow-release prednisolone-pellet (10 mg). The study lasted four weeks. GC induced a substantial reduction in muscle mass, trabecular mineral apposition rate (MAR) and bone formation rate (BFR/BS), and endocortical MAR compared with Control, but did not alter the trabecular microarchitecture or bone strength. In mice not receiving GC, ABL increased aBMD, bone mineral content (BMC), cortical and trabecular microarchitecture, mineralizing surface (MS/BS), MAR, BFR/BS, and bone strength compared with Control. However, when administered concomitantly with GC, the osteoanabolic effect of ABL on BMC, cortical morphology, and cortical bone strength was blunted. In conclusion, at cortical bone sites, the osteoanabolic effect of ABL is generally blunted by short-term GC excess.

Teriparatide and Abaloparatide Have a Similar Effect on Bone in Mice.

Three bone anabolic pharmaceuticals are currently approved for treatment of osteoporosis, teriparatide (PTH (1-34)), the parathyroid hormone-related protein analog abaloparatide (ABL), and romosozumab. The present study compared the effect of intermittent PTH (1-34) and ABL on bone tissue directly mole-to-mole in female mice. Forty-seven C57BL/6 mice were randomly allocated to the following groups: Baseline (n = 11), Control (Ctrl) (n = 12), PTH (n = 12), and ABL (n = 12). The mice were injected s.c. with PTH (100 µg/kg), ABL (96 µg/kg), or saline (Ctrl) five days a week for three weeks. To assess the effect of PTH and ABL, the hindlimb bones were analyzed with DXA, µCT, mechanical testing, dynamic bone histomorphometry, and histological quantification of bone cells. In addition, serum calcium concentration was determined. PTH and ABL significantly increased femoral areal bone mineral density (aBMD) (borderline significant p = 0.06 for PTH), femoral mid-diaphyseal bone strength, femoral metaphyseal and epiphyseal and vertebral bone volume fraction (BV/TV), connectivity density, volumetric bone mineral density (vBMD), and bone formation rate (BFR/BS) compared to Ctrl. In addition, ABL also significantly increased mid-diaphyseal cortical thickness and bone area compared to Ctrl. Neither PTH nor ABL significantly increased bone strength at the femoral neck. In conclusion, abaloparatide and PTH have similar bone anabolic properties when compared directly mole-to-mole in mice.

The Effect of Normobaric Intermittent Hypoxia Therapy on Bone in Normal and Disuse Osteopenic Mice.

Bromer, Frederik Duch, Mikkel Bo Brent, Michael Pedersen, Jesper Skovhus Thomsen, Annemarie Brüel, and Casper Bindzus Foldager. The effect of normobaric intermittent hypoxia therapy on bone in normal and disuse osteopenic mice. High Alt Med Biol. 22: 225-234, 2021. Background: Systemic intermittent hypoxia therapy (IHT) has been shown to elicit beneficial effects on multiple physiological systems. However, only few studies have investigated the effect of long-term normobaric IHT on bone mass and mechanical and microstructural properties. The aim of the present study was to examine the effect of IHT on bone in both healthy and osteopenic mice. Materials and Methods: Thirty mice were stratified into four groups: Ctrl, Ctrl+IHT, Botox, and Botox+IHT. Osteopenia was induced by injecting Botox into the right hindlimb of the mice causing paralysis and disuse. IHT animals were placed in a normobaric hypoxia-chamber (10% oxygen) for 1 hour twice daily 5 days/week. Animals were sacrificed after 21 days, and DEXA, micro-computed tomography, and mechanical testing were performed on the femora. Results: As expected, Botox resulted in a significant reduction of bone mineral content (-23.4%), area bone mineral density (-19.1%), femoral neck strength (Fmax: -54.7%), bone volume fraction (bone volume/tissue volume: -41.8%), and trabecular thickness (-32.4%). IHT had no measurable effect on the bone properties in either healthy or osteopenic mice. Conclusion: The study confirmed that Botox led to loss of bone mass, deterioration of trabecular microstructure, and loss of bone strength. These changes were not influenced by IHT. Notably, IHT had no detrimental effect on bone in either healthy or osteopenic mice. This indicates that IHT of ailments outside of the skeletal system may be administered without causing harm to the bone.

A Systematic Review of Animal Models of Disuse-Induced Bone Loss.

OBJECTIVE: Several different animal models are used to study disuse-induced bone loss. This systematic review aims to give a comprehensive overview of the animal models of disuse-induced bone loss and provide a detailed narrative synthesis of each unique animal model. METHODS: PubMed and Embase were systematically searched for animal models of disuse from inception to November 30, 2019. In addition, Google Scholar and personal file archives were searched for relevant publications not indexed in PubMed or Embase. Two reviewers independently reviewed titles and abstracts for full-text inclusion. Data were extracted using a predefined extraction scheme to ensure standardization. RESULTS: 1964 titles and abstracts were screened of which 653 full-text articles were included. The most common animal species used to model disuse were rats (59%) and mice (30%). Males (53%) where used in the majority of the studies and genetically modified animals accounted for 7%. Twelve different methods to induce disuse were identified. The most frequently used methods were hindlimb unloading (44%), neurectomy (15%), bandages and orthoses (15%), and botulinum toxin (9%). The median time of disuse was 21 days (quartiles: 14 days, 36 days) and the median number of animals per group subjected to disuse was 10 (quartiles: 7, 14). Random group allocation was reported in 43% of the studies. Fewer than 5% of the studies justified the number of animals per group by a sample size calculation to ensure adequate statistical power. CONCLUSION: Multiple animal models of disuse-induced bone loss exist, and several species of animals have successfully been studied. The complexity of disuse-induced bone loss warrants rigid research study designs. This systematic review emphasized the need for standardization of animal disuse research and reporting.

Activin type IIA decoy receptor and intermittent parathyroid hormone in combination overturns the bone loss in disuse-osteopenic mice.

Damage of the lower motor neuron cell bodies or their axons results in reduced or abolished voluntary movement accompanied by a substantial loss of bone and muscle mass. Intermittent parathyroid hormone 1-34 (PTH) (teriparatide) is one of the most potent bone-anabolic treatment regimens. ActRIIA-mFc is an activin type IIA decoy receptor that increases bone mass mediated by inhibition of the activin receptor signaling pathway. We investigated whether PTH or ActRIIA-mFc alone or in combination could prevent loss of bone and muscle mass induced by injecting botulinum toxin A (BTX) into the right hind limb in mice. Seventy-two 16-week-old female C57BL/6 mice were allocated to the following groups: Baseline, Control, BTX, BTX + ActRIIA-mFc (10 mg/kg), BTX + PTH (100 µg/kg), and BTX + ActRIIA-mFc + PTH. The mice were sacrificed after three weeks of disuse and treatment. In contrast to monotherapy with PTH, ActRIIA-mFc alone or in combination with PTH was able partly or completely to prevent disuse-induced loss of whole femoral bone mass, trabecular thickness, and bone strength. Moreover, an additive effect of ActRIIA-mFc and PTH on areal bone mineral density and trabecular bone volume was found. In summary, ActRIIA-mFc and PTH in combination were more effective in preventing disuse-induced bone loss and deterioration of trabecular micro-architecture than either treatment alone.

The Efficacy of PTH and Abaloparatide to Counteract Immobilization-Induced Osteopenia Is in General Similar.

Immobilization results in a substantial bone loss and increased fracture risk. Powerful bone anabolic therapies are necessary to counteract the bone loss and reduce fracture risk during periods with immobilization. Intermittent parathyroid hormone 1-34 (PTH) (teriparatide) and PTH related peptide analog abaloparatide (ABL) are potent bone anabolic therapies acting through the same receptor, but induce different durations of signaling response. We investigated the efficacy of PTH or ABL in preventing immobilization-induced bone loss in rats in a direct mole-to-mole comparison. Immobilization was achieved by injecting botulinum toxin type A (BTX) into the right hindlimb musculature. Sixty 14-week-old female Wistar rats were allocated to the following groups: Baseline, Control, BTX, BTX + PTH (80 µg/kg/day), and BTX + ABL (77 µg/kg/day). Immobilization resulted in a substantial and significant reduction in bone mineral density (aBMD), metaphyseal and epiphyseal trabecular bone volume fraction (BV/TV) and trabecular thickness (Tb.Th), metaphyseal trabecular number (Tb.N), and femoral neck bone strength. Both PTH and ABL prevented the immobilization-induced decrease in aBMD, metaphyseal and epiphyseal Tb.Th, and metaphyseal Tb.N. In addition, PTH rescued the reduction in metaphyseal BV/TV and femoral neck strength, while ABL did not. However, the effect of PTH and ABL did not differ significantly for serum calcium, aBMD, metaphyseal, and epiphyseal BV/TV, Tb.Th, or Tb.N. In conclusion, in a mole-to-mole comparison the efficacy of PTH and ABL is similar in counteracting immobilization-induced reduction in bone mineral density, deterioration in trabecular microarchitecture, and decrease in bone strength.

Rodent model of disuse-induced bone loss by hind limb injection with botulinum toxin A.

Bone loss materializes rapidly after immobilization or mechanical unloading. Hind limb injection with botulinum toxin A (BTX) is a highly reproducible animal model for disuse-induced bone loss. Here we describe an easy-to-use and enhanced version of the method employing multiple hind limb injections with BTX to induce a pervasive muscle paralysis and thereby disuse of the hind limb. Thirty-six 12-14-week-old female Wistar rats were stratified into three groups: Baseline (Base), Control (Ctrl), and BTX. Disuse was achieved by injecting BTX directly into the right quadriceps femoris muscle, the hamstring muscles, and the posterior calf muscles. The rats were sacrificed after six weeks, and the right rectus femoris muscle and femur were isolated and analyzed. Hind limb disuse resulted in a significant and substantial loss of both muscle mass and bone mass. The loss of bone mass was accompanied by a reduction of trabecular bone mass and a deterioration of the trabecular micro-architecture with a reduction of trabecular thickness and trabecular number compared to Ctrl. In addition, the trabeculae changed from a more plate-like towards a more rod-like shape as indicated by an increase in the structure model index.•Multiple injections with BTX targeting muscles on both the anterior and posterior thigh and the calf ensure a uniform and pervasive muscle paralysis and hind limb disuse.•Hind limb injections with BTX results in a substantial loss of muscle and bone mass and deterioration of the trabecular micro-architecture.•The induction of hind limb disuse with BTX is highly reproducible.

Animal models of disuse-induced bone loss: study protocol for a systematic review.

BACKGROUND: Disuse is a cardinal sign of various neurological diseases like stroke, cerebral palsy, and amyotrophic lateral sclerosis. Disuse leads to reduced mechanical loading of the skeleton, and a substantial and significant loss of bone mass quickly materializes. Several animal models have been proposed to investigate the pathogenesis of disuse-induced bone loss and to test new pharmaceutical targets to counteract it. As animal models may overcome several of the limitations in observational studies conducted in patients and allow for measurements not possible in humans, the primary objective of the present study is to provide a comprehensive overview of the available animal models of disuse-induced bone loss. METHODS/DESIGN: This is a protocol for a systematic review of animal models of disuse-induced bone loss. An exhaustive search will be performed on PubMed and Embase in order to identify relevant studies. The primary outcome will be the method of disuse induction. The secondary outcomes will be related to bone samples and anatomical sites investigated, methods used to analyze and quantify bone loss, and bibliographic information. The protocol adheres to the current guiding principles of the Preferred Reporting Items for Systematic Review and Meta-analysis Protocols (PRISMA-P) 2015 statement. Extracted data will be analyzed with descriptive statistics, and all the methods used to induce disuse will be described in detail with a narrative synthesis. DISCUSSION: This systematic review will provide an overview of available animal models of disuse-induced bone loss and discuss the different methods used to quantify and analyze the bone loss. Since bone loss caused by disuse is a hallmark of various diseases from different medical specialties, this overview will be of great benefit for all researchers planning to conduct disuse animal studies in the future. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42020157452 .