Introduction: Observation of sub-clinical neurological abnormalities has led to the proposal of a neuro-developmental etiologic model for adolescent idiopathic scoliosis (AIS). We have previously demonstrated prolonged latency in somatosensory evoked potentials (SSEP) and impaired balance control in subjects with AIS. Furthermore we have compared regional brain volumes in right thoracic AIS subjects and normal controls. Significant neuro-anatomic regional differences were observed in the corpus callosum, premotor cortex, proprioceptive and visual centers of the AIS subjects compared to control subjects. Most of these regional differences involved the brain unilaterally, indicating there may be abnormal asymmetrical development in the brain of subjects with right thoracic AIS. Methods: Following ethical committee approval a total of 29 subjects with AIS were recruited. Patients with congenital, neuromuscular or syndromic scoliosis were excluded from the study. Twenty-eight age- and sex-matched controls were recruited from local schools. All recruits underwent three-dimensional isotropic magnetization prepared rapid acquisition gradient echo (3D_MPRAGE) magnetic resonance (MR) imaging of the brain. Modern morphometric analyses of the MR images were carried out including classification of tissue into grey matter (GM), white matter (WM) and cerebrospinal fluid (CSF). Tissue densities were compared between AIS subjects and controls. Comparisons were made between those subjects with left thoracic AIS (n=9) and age and sex-matched controls (n=11) and those subjects with right thoracic AIS (n=20) and age and sex-matched controls (n=17). Results: For subjects with left thoracic curves the mean Cobb angle was 19 degrees. For subjects with right thoracic curves the mean Cobb angle was 33.8 degrees There was no significant differences observed between AIS subjects and normal controls when comparing both absolute and relative (i.e. adjusted for brain size) volumes of GM and WM. However voxel-based morphometric analysis identified significant differences in the density of WM in the genu of the corpus callosum, the left internal capsule and WM underlying the left orbitofrontal cortex when comparing those subjects with left thoracic scoliosis to controls. The above differences were not not observed when those subjects with right thoracic scoliosis were compared to controls. Discussion: This controlled study of regional brain tissue density has demonstrated important differences in the corpus callosum, the left internal capsule and the left orbitofrontal cortex when the brain of those subjects with left thoracic scoliosis is compared to age and sex matched controls. In this study significant regional brain differences have not been identified in those subjects with right thoracic scoliosis. Further studies are warranted to ascertain whether these morphologial differences in the brain are linked with the etiopathogenisis of left sided thoracic scoliosis. A larger sample and a longitudinal study are required to establish whether brain abnormalities are predictive of curve progression

Aim: To investigate whether there is any difference in regional brain volumes between AIS patients and age matched, sex matched control subjects. Method and Materials: 20 adolescent idiopathic scoliosis (AIS) female patients (age ranged from 11 yrs to 18 yrs, mean age of 14.5 yrs) and 20 sex matched, age matched controls have undergone MRI brain examination performed with a 1.5T scanner (Sonata, Siemens Medical Solutions, Erlanger, Germany). A Magnetization Preparation Rapid Acquisition Gradient Echo (MPR) sequence was used. Volumes of neuroanatomical regions were quantitated automatically by using whole brain segmentation technique of atlas-based hybrid warping. The whole brain was classified into 100 fine anatomical regions. The results were taken as significant when p value was less than 0.05. Results: Significant unilateral regional differences were found in the following regions:. Left thalamus and left postcentral gyrus of AIS patients were significantly larger than the control subjects. Anterior and posterior limb of right internal capsule, right caudate nucleus, right cuneus and left middle occipital gyurs of AIS patients were significantly smaller than the control subjects. Some regions were bilaterally involved: Perirhinal and hippocampus regions were larger in AIS while inferior occipital gyrus and precuneus were smaller than the corresponding regions in the control subjects. In the midline, the volumes of corpus callosum and brainstem in AIS patients were significantly larger than the control subjects. Conclusion: Our study found that significant differences in particular regional brain volumes exist between AIS patients and the controls. Most of these regions involved the brain unilaterally, indicating that there might be abnormal asymmetrical development of the brain in AIS. This is the first study of its kind to show the presence of L-R asymmetry of regional brain volume difference in AIS patients as compared to normal controls. The findings might also help to explain the reported poor performance in the combined visual and proprioceptive test, spatial orientation test, abnormal nystagmus response to calorie test, and impaired postural balance in AIS patients

It has been shown that a cognitive function (CF) loss can occur after hip or knee arthroplasty procedures, with an incidence of 40 to 70%. The pathogenesis remains unclear but studies suggest some form of brain emboli; although both trans-cranial doppler and trans-oesophageal doppler have both shown emboli per-operatively a correlation has never been shown with CF loss post-operatively. In contrast, in the cardiothoracic literature an embolic cause is widely accepted for detectable post-operative CF drop. The purpose of this study was to ascertain whether MRI could show evidence of embolic phenomena in patients undergoing hip or knee arthroplasty. Twenty-five patients presenting for hip or knee arthroplasty procedures were consented for this study. Brain MRI scans and MR angiograms were performed 1 week pre-operatively and within 1 week post-operatively using a Phillips 1.5Tesla MRI unit. All scans recorded were independently reviewed by 2 radiologists. A series of tests to examine several modes of cognitive function were carried out by a clinical psychologist pre-operatively, and at 1 week post-operatively. The CF tests showed a clinically significant drop following surgery in 64% of cases – this is in keeping with other recently published data. None of the post-operative scans or angiograms showed overt evidence of new lesions. Three Scans had equivocal tiny brainstem hypodensities on a single slice with no correlating abnormality on diffusion images to support the presence of new ischaemia. We conclude that either the aetiology of post-operative CF drop following arthroplasty is not embolic in nature, or that with current technology MRI brain scans even with angiograms are not sensitive enough to show the corresponding abnormality. With currently available equipment there appears to be no benefit from using MRI as a tool to evaluate post-operative CF loss in this group of patients

Acute pain is one of the most common symptoms shared among patients who have suffered from an orthopedic trauma such as an isolated upper limb fracture (IULF). Development of interventions with limited side effects aiming to prevent the installation of chronic pain is critical as persistent pain is associated with an increased risk of opioid dependence, medical complications, staggering financial burdens and diminished quality of life. Theta burst stimulation (TBS), a non-invasive magnetic brain stimulation technique with minimal side effects, has shown promising results in patients experiencing various types of chronic pain conditions as it precisely targets brain regions involved in pain processing. Surprisingly, its impact on acute pain has never been investigated. This study aims to assess longitudinal effects of a 10-day continuous TBS (cTBS) protocol applied in the acute phase of an IULF on key functional outcomes. Patients with an IULF aged between 18 to 60 years old were recruited within 7 days post-accident at a Level I Trauma Center. Exclusion criteria included a history of brain injury, neurological disorders, musculoskeletal complications, and open fractures. In order to assess longitudinal changes, questionnaires measuring intensity and characteristics of pain (Numerical Rating Scale, NRS; McGill Pain Questionnaire, MPQ) as well as functional disability (DASH) were completed by all patients at three time points, namely prior to the start of the TBS program as well as 72 hours and 3 months post-intervention. Patients were randomly attributed to the active TBS protocol (active group) or to the placebo protocol (sham group). The stimulation site for each participant corresponded to the contralateral motor cortex of the injured arm. Fifty patients were recruited (female: 24; age: 40.38 years old), of which 25 were in the active group and 25 were in the sham group. Both groups were equivalent based on age, sex, type of injury, and surgical procedures (p>0.05). The intervention protocol was introduced on average 6.18 days post-accident. In comparison to the sham group, the active group showed a significant decrease in pain intensity (NRS) at 72h (F=6.02; p=0.02) and 3-month (F=6.37; p=0.02) post-intervention. No group difference was found early-on (72h post) in regard to pain characteristics (MPQ; F=3.90; p=0.06) and functional disabilities (DASH; F=0.48; p= 0.49). At three-month post-intervention, the active group showed statistically significant improvement on the MPQ (F=5.02; p=0.04) and the DASH (F=5.88; p=0.02) compared to the placebo group. No complications related to the treatment were reported. Results from this study show that patients who underwent active cTBS reported less pain and better functional states shortly after the end of the TBS protocol compared to sham patients and treatment effects were maintained at three months post-intervention. Given that acute pain intensity is an excellent predictor of chronic pain development, this safe technique available in numerous centers in Canada may help prevent chronic pain development when administered during the acute post-injury phase. Future studies should continue to investigate mechanisms involved to optimize this technique among the orthopedic trauma population and to reduce opioid consumption

Introduction. Different subclinical neurological dysfunction has been reported in adolescent idiopathic scoliosis (AIS), including poor postural control and asymmetric otolith vestibulo-ocular responses when compared with normal controls. The objective of this pilot study is to establish whether abnormal MRI morphoanatomical changes arise in the CNS (brain and vestibular system), among left-thoracic versus right-thoracic AIS when compared with normal adolescent controls, with use of advanced computerised statistical morphometry techniques. Methods. We compared nine girls with left-thoracic AIS (mean age 14 years; mean Cobb angle 19°) with 11 matched controls, and 20 girls with right-thoracic AIS (mean age 15 years, mean Cobb angle 33·8°) with 17 matched controls. The statistical brain analysis was done with validated automatic segmentation and voxel-based morphometry (VBM). The T2W-MRI data for shape analysis of the vestibular system were obtained from 20 patients with right-thoracic AIS and 20 matched controls. A best-fit plane and a best-fit circle were calculated to approximate each semicircular canal. The shape of vestibular system was measured by: (1) the angle between each pair of best-fit planes; (2) the length; and (3) angle formed between the corresponding lines connecting the centres of each pair of circles. Statistical analysis was done with one-way ANOVA. Results. Patients with left-thoracic AIS had significantly lower white matter density in corpus callosum, left internal capsule, and white matter underlying orbitofrontal cortex of left hemisphere, which were not observed in patients with right-thoracic AIS. In the right-thoracic AIS group, the distance between centres of lateral and superior canals (p=0·0264) and the angle with vertex at the centre of posterior canal (p=0·02) of left-side vestibular system were significantly smaller than in the control group (figure). For vestibular analysis, there were no data for left-thoracic AIS. Conclusions. Findings from this pilot study have shown significant MRI morphoanatomical difference in CNS between patients with AIS and controls. In the brain analysis, corpus callosum (the principle commissural fibre bundle connecting left and right cerebral hemispheres, which might affect the coordination of left and right sides of the body), differed between left-thoracic and right-thoracic AIS. In the vestibular analysis, geometric morphological difference was detected on the left-side semicircular canals between right-thoracic AIS and healthy controls. These morphological changes are likely to be related to the subclinical postural, vestibular, and proprioceptive dysfunctions. Further association studies and longitudinal studies could help to further define the link between morphological and functional dysfunction, which might have important predictive and prognostic effect on curve development and progression

Study Design: Retrospective case review. Summary of Background Data: The Chiari malformation is a condition characterised by herniation of the posterior fossa contents below the level of the foramen magnum. Objectives: To present the long term outcome and complication rate following hindbrain decompression for this condition. Methods: We retrospectively analysed the results of patients who underwent hind brain decompression between 1994 and 2003. There were 70 cases with a mean age of 32 years. Follow up was carried out with clinical examination and repeat MRI scans. The mean follow up was 4.7 years. Thirty-six patients had associated syringomyelia. Patients underwent hind brain decompression through a small posterior fossa craniectomy, opening of the foramen magnum with or without removal of arch of C1. Results: One patient died and one had a stroke which resolved except for mild facial weakness. Long term follow up revealed that 50% of the patients were asymptomatic following surgery and another 26% had marked improvement in their symptoms. One patient deteriorated post-operatively and the remainder (23%) had unchanged condition. Of the patients presenting with scoliosis 67% had no further progression in their curve. Conclusion: This is the largest series presented from a single centre with pre- and postoperative MRI fol1ow up. Our results compare favourably with previously published literature

Painful OA is linked to CNS changes in pain processing. Temporal summation of pain (TSP) is a measure of one such CNS change, central sensitization. TSP is defined using a series (≥0.33Hz) of painful stimuli and is a predictor of postoperative pain, experienced by 20% of patients after total knee replacement (TKR) surgery. This study has developed a protocol to use functional MRI to assess CNS changes in OA pain processing. This pilot includes 3 participants with chronic knee OA pain awaiting TKR (62 ± 4.4) and 5 healthy volunteers (50 ± 13.6). 3-Tesla BOLD fMRI brain scans were recorded during short series of one second painful stimuli, applied using an automated inflatable cuff to the calf muscle of the leg with the affected knee or left side in healthy volunteers. The pain intensity at onset and during the 10 painful stimuli were recorded using a numerical rating scale. The pattern of brain activation was averaged across noxious stimuli, and the differential activation compared the 1st vs. 10th (last) stimulus. Bone marrow lesions (BMLs), synovitis and effusion size were scored from 3-Tesla knee MRI's using MOAKS scoring. TSP was raised in OA patients compared to control group (p=0.023). TSP brain activity in the chronic OA patients displayed higher signal within the subgenual anterior cingulate (sgACC) compared to healthy volunteers. Knee MRI identified OA patient's exhibited higher BML scores (p=0.038) and more knee effusion (p=0.018), but the lack of synovitis did not differ from control group (p=0.107). Enhanced TSP in chronic knee OA pain may be linked with augmented responses in emotional circuitry. BMLs and effusion size appear to contribute more with pain than synovitis. These results may help understand sensitization to improve outcomes for patients with knee OA undergoing TKR surgery

Aims. Due to the recent rapid expansion of scooter sharing companies, there has been a dramatic increase in the number of electric scooter (e-scooter) injuries. Our purpose was to conduct a systematic review to characterize the demographic characteristics, most common injuries, and management of patients injured from electric scooters. Methods. We searched PubMed, EMBASE, Scopus, and Web of Science databases using variations of the term “electric scooter”. We excluded studies conducted prior to 2015, studies with a population of less than 50, case reports, and studies not focused on electric scooters. Data were analyzed using t-tests and p-values < 0.05 were considered significant. Results. We studied 5,705 patients from 34 studies. The mean age was 33.3 years (SD 3.5), and 58.3% (n = 3,325) were male. The leading mechanism of injury was falling (n = 3,595, 74.4%). Injured patients were more likely to not wear a helmet (n = 2,114; 68.1%; p < 0.001). The most common type of injury incurred was bony injuries (n = 2,761, 39.2%), of which upper limb fractures dominated (n = 1,236, 44.8%). Head and neck injuries composed 22.2% (n = 1,565) of the reported injuries, including traumatic brain injuries (n = 455; 2.5%), lacerations/abrasions/contusions (n = 500; 7.1%), intracerebral brain haemorrhages (n = 131; 1.9%), and concussions (n = 255; 3.2%). Standard radiographs comprised most images (n = 2,153; 57.7%). Most patients were treated and released without admission (n = 2,895; 54.5%), and 17.2% (n = 911) of injured patients required surgery. Qualitative analyses of the cost of injury revealed that any intoxication was associated with higher billing costs. Conclusion. The leading injuries from e-scooters are upper limb fractures. Falling was the leading mechanism of injury, and most patients did not wear a helmet. Future research should focus on injury characterization, treatment, and cost. Cite this article: Bone Jt Open 2022;3(9):674–683

Aims

Deciphering the genetic relationships between major depressive disorder (MDD) and osteoarthritis (OA) may facilitate an understanding of their biological mechanisms, as well as inform more effective treatment regimens. We aim to investigate the mechanisms underlying relationships between MDD and OA in the context of common genetic variations.

Aim of investigation: Fear of movement (avoidance) has been implicated as an obstacle to recovery in back pain. We have argued that the concept of fear-avoidance needs clarifying, to identify sub-groups of avoiders. This study explored the patterns of activation during exposure to previously reported feared movement in patients with chronic back pain. The aim was to explore activation in areas associated with catasrophizing.

Method: 13 chronic back pain patients, who scored above a cut-point on the Tampa Scale of Kinesiophobia selected photographic images representing 5 movements they feared most and five movements they hadno concerns with carrying out. Stimuli were therefore individually selected. Two other sets of stimuli included generally threatening images, and neutral images. These four stimulus types were presented in blocks in a fMRI scanner in random order. Ratings of pain were taken after the presentation of each block.

Results: Analysis of contrasts-of-interest showed that the highly feared movements caused selective activation in areas related to preparation for action, and attentional modulation. The canonical ‘pain matrix’, and areas associated with catasrophizing was not activated.

Conclusion: The activation seen may indicate the involvement of heightened attentional processing and/or response processes (bracing and protecting) when viewing pictures of feared movements. The absence of activity in affective pain areas in the contrast analysis will be discussed in reference to theoretical developments and methodological limitations.

Though dentin matrix protein 1 (Dmp1) is known to play critical role in mediating bone mineralization, it has also been validated to be expressed in brain and helps maintain blood brain barrier (BBB). Our study aims to clarify the expression pattern of Dmp1 in mouse brain and explore whether intercellular mitochondrial transfer occurs between Dmp1 positive astrocytes (DPAs) and endothelial cells, and thus acting as a mechanism in maintaining BBB during aging. Single cell RNA sequencing (scRNAseq) of 1 month, 6 month, and 20 month old mice brain (n=1, respectively) was employed to identify Dmp1 positive cell types. Dmp1. Cre. -mGmT and Dmp1. Cre. -COX8a fluorescent mice were generated to visualize DPAs and investigate their mitochondrial activities. A 3D noncontact coculture system and mitochondrial transplantation were applied to study the role of mitochondrial transfer between astrocytes and bEnd.3 endothelial cells. Dmp1. Cre. -Mfn2. f/f. mice were generated by depleting the ER-mitochondria tethering protein Mfn2 in DPAs. Dmp1 was mainly expressed in astrocytes at different ages. GO analysis revealed that cell projection and adhesion of DPAs were upregulated. Confocal imaging on Dmp1. Cre. -mGmT mice indicated that DPAs are a cluster of astrocytes that closely adhere to blood vessels (n=3). Bioinformatics analysis revealed that mitochondrial activity of DPAs were compromised during aging. Enriched scRNAseq of fluorescent cells from Dmp1. Cre. -COX8a mice (n=2) and immunofluorescent imaging (n=3) validated the acquisition of extrinsic mitochondria in endothelial cells. 3D coculture of astrocytes and bEnd.3 and direct mitochondrial transplantation revealed the rescue effect of mitochondrial transfer on damaged bEnd.3. BBB was impaired after depleting Mfn2 in DPAs, expressing a similar phenotype with aging brain. Astrocytes that express Dmp1 play a significant role in maintaining BBB via transferring mitochondria to vascular endothelial cells. Compromised mitochondrial transfer between DPAs and endothelial cells might be the potential mechanism of impaired BBB during aging

Both total joint arthroplasty (TJA) and Alzheimer's Disease (AD) are prevalent in elderly populations. It is the goal of this study to determine if the presence of implant metals originating from TJA correlates with the onset with higher implant metal content in the brain and AD pathology. Tissue samples from four brain regions of 701 (229 with TJA) participants from an ongoing longitudinal cohort study (Rush Memory and Aging Project) was analyzed including the inferior-temporal-cortex (ITC), which is associated with early onset of AD. Implant metal (Co, Cr, Mo, Ti, Al) content was determined by ICP-MS. Comparisons were conducted between the no-TJA-group and a TJA group. Due to the higher likelihood of Co release the TJA group was further differentiated in a THA (N=146) and a TKA/TSA (N=83) group. Diffuse and neuritic amyloid plaques and phosphorylated tau were assessed and summarized as standard measures of AD pathology. We used separate linear regression models adjusted for age, sex, education, and APOɛ4-status for the associations of all metals (log-transformed) with global AD pathology, amyloid plaques, and phosphorylated tau. The THA group had higher cobalt content across all brain regions (p=0.003) and within the ITC (p=0.051) compared to the no-TJA group, whereas the TKA/TSA group did not. Across all tissue samples, Co was associated with higher amyloid load (β=0.35, p=0.027), phosphorylated tau (β=0.47, p=0.011), and global AD pathology (β=0.19, 0.0004) in the ITC. The presence of TJA itself was not associated with AD pathology. We showed that only Co content was higher within the ITC in persons with THA. We found among all tested metals that Co was consistently associated with AD pathology. Although we found an association of cobalt with AD pathology, the cross-sectional nature of this study does not allow the determination of cause and effect

Cerebral Palsy (CP) is a group of disorders that affect movement and posture caused by injury to the developing brain. While prematurity and low birth weight are common causes in developed countries, birth asphyxia, kernicterus, and infections have been identified as predominant aetiologies in Africa. There is, however, very little information on the aetiology of CP in South Africa. The purpose of this study was to determine the aetiology, severity, and topographical distribution of CP in children undergoing orthopaedic surgery at our tertiary paediatric unit. A retrospective folder review was performed for patients with CP that underwent orthopaedic surgery from July 2018 to June 2022. Data was collected on perinatal circumstances, aetiology or risk factors for developing CP, severity of disability as classified by the Gross Motor Function Classification Scale (GMFCS) and topographical distribution. Descriptive analysis was performed. Two-hundred-and-thirty-four patients were included in the analysis. No specific aetiology could be identified in 51 (21.9%) patients. Hypoxic ischaemic encephalopathy (HIE) accounted for 23.6% of patients and was the most common aetiology across the different categories except for patients graded as GMFCS 2, in whom prematurity was the most common aetiology. Congenital brain malformations (10.5%) and cerebral infections, including HIV encephalopathy (11.4%) were the next most frequent aetiologies, followed by prematurity (7.6%), ischaemic stroke (6.8%) and intraventricular haemorrhage (6.3%). Fifty-two percent of patients were classified as GMFCS 4 or 5. There was a predominance of quadriplegic patients (37%) compared to hemiplegics (29%), diplegics (30%) and monoplegics (4%). Most patients undergoing orthopaedic surgery for musculoskeletal sequelae of CP were severely disabled quadriplegic patients in whom HIE was the predominant cause of CP. This emphasises the need for intervention at a primary care level to decrease the incidence of this frequently preventable condition

Based on Ilizarov's law of tension-stress principle, distraction histogenesis technique has been widely applied in orthopaedic surgery for decades. Derived from this technique, cranial bone transport technique was mainly used for treating cranial deformities and calvarial defects. Recent studies reported that there are dense short vascular connections between skull marrow and meninges for immune cells trafficking, highlighting complex and tight association between skull and brain. Alzheimer's disease (AD) is a progressive neurodegenerative disease and the most common cause of dementia without effective therapy. Meningeal lymphatics have been recognized as an important mediator in neurological diseases. The augmentation of meningeal lymphatic drainage might be a promising therapeutic target for AD. Our proof-of-concept study has indicated that cranial bone transport can promote ischemic stroke recovery via modulating meningeal lymphatic drainage function, providing a rationale for treating AD using cranial bone maneuver (CBM). This study aims to investigate the effects of CBM on AD and to further explore the potential mechanisms. Transgenic 5xFAD mice model was used in this study. After osteotomy, a bone flap was used to perform CBM without damaging the dura. Open filed test, novel object recognition test and Barn's maze test were used to evaluate neurological functions of 5xFAD mice after CBM treatment. Congo red and immunofluorescence staining were used to evaluate amyloid depositions and Aβ plaques in different brain regions. Lymphangiogenesis and the level of VEGF-C were examined after CBM treatment. OVA-A647 was intra-cisterna-magna injected to evaluate meningeal lymphatic drainage function after CBM treatment. CBM significantly improved memory functions and reduced amyloid depositions and Aβ plaques in the hippocampus of 5xFAD mice. A significant increase of meningeal lymphatic vessels in superior sagittal sinus and transverse sinus, and the upregulation of VEGF-C in meninges were observed in 5xFAD mice treated with CBM. Moreover, CBM remarkably enhanced meningeal lymphatic drainage function in 5xFAD mice (n=5-16 mice/group for all studies). CBM may promote meningeal lymphangiogenesis and lymphatic drainage function through VEGF-C-VEGFR3 pathway, and further reduce amyloid depositions and Aβ plaques and alleviate memory deficits in AD

Introduction and Objective. Klinefelter Syndrome (KS, karyotype 47,XXY) is the most frequent chromosomal aneuploidy in males, as well as the most common cause of infertility in men. Patients suffer from a lack of testosterone, i.e. hypergonadotropic hypogonadism provoking infertility, but KS men also show an increased predisposition to osteoporosis and a higher risk of bone fracture. In a mouse model for human KS, bone analysis of adult mice revealed a decrease in bone mass that could not be rescued by testosterone replacement, suggesting a gene dosage effect originating from the supernumerary X-chromosome on bone metabolism. Usually, X chromosome inactivation (XCI) compensates for the dosage imbalance of X-chromosomal genes between sexes. Some studies suggested that expression of genes that escape silencing of the supernumerary X-chromosome (e.g. androgen receptor) has an impact on sex differences, but may also cause pathological changes in males. As a promising new such candidate for a musculoskeletal escape gene, we identified the integral membrane protein (ITM) 2a, which is encoded on the X-chromosome and related to enchondral ossification. The aim of the project was to characterize systemic bone loss in the course of aging in our KS mouse model, and whether the supernumerary X-chromosome causes differences in expression of genes related to bone development. Materials and Methods. Bone structure of 24 month (=aged) old male wild type (WT) and 41, XXY mice (B6Ei.Lt-Y) were analysed by μCT. Afterwards bones were paraffin embedded and cut. In addition, tissue of brain, liver, kidney, lung and heart were also isolated and embedded for IHC staining. Using an anti-ITM2a antibody, expression and cellular localization of ITM2a was evaluated. IHC was also performed on musculoskeletal tissue of WT embryos (E18.5) and neonatal mice to determine possible age-related differences. Results. In 24 month old mice, the analysis of the lumbar vertebrae revealed a significantly lower BV/TV, trabecular bone volume and trabecular number in the XXY- group compared to WT. Trabecular thickness appeared lower but did not reach significance, with the cortical thickness being significantly higher in the XXY- group. High expression of ITM2a was detected in bone slices of both karyotypes in the chondrocytes inside the growth plate, as well as in megakaryocytes and leucocytes as well as endothelial cells of blood vessels inside the bone marrow. Osteocytes, along with erythrocytes and erythropoetic stem cells were negative for ITM2a. Other organs that showed ITM2a positive staining were kidney (blood vessels), heart (muscle) and brain (different structures). Liver and lung tissue were negative for ITM2a. No obvious difference in the intensity of the ITM2a-expression was observed between the WT and the XXY-karyotype. Analyses of embryotic bone tissue (WT) showed high expression of ITM2a in proliferating, hypertrophic and resting chondrocytes in the growth plates of tibia and femur. In comparison, the neonatal animals (WT) did not show any protein-expression in chondrocytes. Furthermore, within the metaphysis of both, embryotic and neonatal bones, endothelial cells and osteoblasts were ITM2a-positive. Further analyses of bones and tissues from young mice (4–6 month) are ongoing. Conclusions. Bone analyses revealed a significant reduction in trabecular bone mass along with fewer and thinner trabeculae in XXY mice compared to the WT, especially in the spine. ITM2a expression was visible in different cell types inside the bone, and in addition, different expression patterns at different stages of development (embryonic/neonatal) were observed. However, we have not found a significant difference in the quantity of ITM2a between tissues of XXY-karyotypes and WT. Further analyses of X-chromosomal encoded and therefore dysregulated modulators in XXY-karyotype mice and patients may reveal new sex chromosomal effector proteins in bone metabolism

Serial section electron microscopy (SSEM) was initially developed to map the neural connections in the brain. SSEM eventually led to the term ‘Connectomics’ to be coined to describe process of following a cell or structure through a volume of tissue. This permits the true three-dimensionality to be appreciated and relationships between cells and structures. The purpose of this study was to utilize this methodology to interrogate S. aureus infected bone. Bone samples were harvested from mice tibia infected with S. aureus and were fixed, decalcified, and osmicated. The samples were paraffin embedded and 5-micron sections were cut to identify regions of bacterial invasion into the osteocyte-lacuna-canalicular-network (OLCN). This area was cut from the paraffin block, deparaffinized, post-fixed and reprocessed into epoxy resin. Serial sections were cut at 60nm and collected onto Kapton tape utilizing the Automated Tape-collecting Ultramicrotome (ATUMtome) system. Samples were mounted onto 4” silicon wafers and post-stained with 2% uranyl acetate followed by 0.3% lead citrate and carbon coated. A ZEISS GeminiSEM 450 scanning electron microscope fitted with an electron backscatter diffusion detector was used to image the sections. The image stack was aligned and segmented using the open-source software, VASTlite. 264 serial sections were imaged, representing approximately 40 × 45 × 15-micron (x, y, z) volume of tissue. 70% of the canaliculi demonstrated infiltration by S. aureus. This study demonstrates that SSEM can be applied to the skeletal system and provide a new solution to investigate the OLCN system. It is feasible that this methodology could be implemented to investigate why some canaliculi are resistant to colonization and potentially opens up a new direction for the prevention of chronic osteomyelitis. In order to make this a realistic target, automated segmentation methodologies utilizing machine learning must be developed and applied to the bone tissue datasets