Effect of vitamin D on bone morphometry and stability of orthodontic tooth movement in rats.

INTRODUCTION: Vitamin D (VitD) maintains bone health and may influence orthodontic tooth movement (OTM). The objective was to evaluate the VitD effect on bone morphometry and the rate and stability of OTM. METHODS: Thirty-two male Sprague Dawley rats were assigned into 2 experimental groups, treated with VitD by gavage (systemic) or injection (local), and 2 respective control groups treated with phosphate-buffered saline for 47 days. OTM was performed for 7 days with a nickel-titanium coil bonded between the maxillary first molar and incisors. Microcomputed tomography scanning was performed at 5 time points: before administration of VitD, the start of OTM, the end of OTM, 7 days post-OTM, and 30 days post-OTM. The rate and stability of OTM were assessed. Bone morphometry was analyzed by bone mineral density, bone volume/total volume, total porosity, trabecular pattern factor, structure model index, and connectivity density. RESULTS: The systemic VitD group showed a lower OTM rate and a lower relapse than the control (P <0.05). It also demonstrated increased bone mineral density, bone volume/total volume, and a decrease in total porosity (P <0.05). The bone structure appeared more fragmented and presented a lower connectivity density than the control (P <0.05). No statistical difference was found between VitD local administration and the other groups for the rate and stability of OTM or bone morphometry. CONCLUSIONS: The systemic administration of VitD caused a decrease in the OTM rate by generating more bone resistance but also contributed to a lower relapse with a higher bone mineral density.

Alteration of the brain methylation landscape following postnatal inflammatory injury in rat pups.

Preterm infants are vulnerable to inflammation-induced white matter injury (WMI), which is associated with neurocognitive impairment and increased risk of neuropsychiatric diseases in adulthood. Epigenetic mechanisms, particularly DNA methylation, play a role in normal development and modulate the response to pathological challenges. Our aims were to determine how WMI triggered DNA methylation alterations in brains of neonatal rats and if such changes persisted over time. We used a robust model of WMI by injecting lipopolysaccharide (LPS) or sterile saline in the corpus callosum of 3-day-old (P3) rat pups. Brains were collected 24 hours (P4) and 21 days post-injection (P24). We extracted genomic DNA from the brain to establish genome-wide quantitative DNA methylation profiles using reduced representation bisulfite sequencing. Neonatal LPS exposure induced a persistent increased methylation of genes related to nervous system development and a reduced methylation of genes associated with inflammatory pathways. These findings suggest that early-life neuroinflammatory exposure impacts the cerebral methylation landscape with determining widespread epigenetic modifications especially in genes related to neurodevelopment.

Assessing therapeutic response non-invasively in a neonatal rat model of acute inflammatory white matter injury using high-field MRI.

Perinatal infection and inflammatory episodes in preterm infants are associated with diffuse white matter injury (WMI) and adverse neurological outcomes. Inflammation-induced WMI was previously shown to be linked with later hippocampal atrophy as well as learning and memory impairments in preterm infants. Early evaluation of injury load and therapeutic response with non-invasive tools such as multimodal magnetic resonance imaging (MRI) would greatly improve the search of new therapeutic approaches in preterm infants. Our aim was to evaluate the potential of multimodal MRI to detect the response of interleukin-1 receptor antagonist (IL-1Ra) treatment, known for its neuroprotective properties, during the acute phase of injury on a model of neonatal WMI. Rat pups at postnatal day 3 (P3) received intracerebral injection of lipopolysaccharide with systemic IL-1Ra therapy. 24 h later (P4), rats were imaged with multimodal MRI to assess microstructure by diffusion tensor imaging (DTI) and neurochemical profile of the hippocampus with 1H-magnetic resonance spectroscopy. Astrocyte and microglial activation, apoptosis and the mRNA expression of pro-inflammatory and necroptotic markers were assessed. During the acute phase of injury, neonatal LPS exposure altered the concentration of hippocampus metabolites related to neuronal integrity, neurotransmission and membrane integrity and induced diffusivity restriction. Just 24 h after initiation of therapy, early indication of IL-1Ra neuroprotective effect could be detected in vivo by non-invasive spectroscopy and DTI, and confirmed with immunohistochemical evaluation and mRNA expression of inflammatory markers and cell death. In conclusion, multimodal MRI, particularly DTI, can detect not only injury but also the acute therapeutic effect of IL-1Ra suggesting that MRI could be a useful non-invasive tool to follow, at early time points, the therapeutic response in preterm infants.

Mechanobiological analysis of porcine spines instrumented with intra-vertebral staples.

OBJECTIVE: To characterize growth plate histology of porcine spines instrumented with a new intra-vertebral staple. METHODS: Spinal segments (T7-T9) previously instrumented with an intra-vertebral staple (experimental group, n=7) or non-instrumented (control group, n=4) underwent average growth rate (AGR), and histomorphometric measurements: heights of proliferative (PZH) and hypertrophic (HZH) growth plate zones, hypertrophic cells height (CH), and the number of proliferative chondrocytes per column (CC). These measurements were done over three regions: (1) left side; (2) middle; (3) right side (instrumented side). The two groups were analyzed by comparing the difference between results for regions 1 and 3 (Dif-R1R3). RESULTS: A significantly higher Dif-R1R3 was found for AGR and HZH for the experimental group as compared with controls. This Dif-R1R3 was also significantly higher for CC at T8 level, CH at T7 level and PZH at both levels. No significant changes for the Dif-R1R3 were observed in the adjacent vertebrae (T11-T12). CONCLUSIONS: This study confirmed the local growth modulation capacity of the intra-vertebral staple, translated at the histomorphometric level by a significant reduction in all parameters, but not in all spinal levels. Further analyses are needed to confirm the regional effect, especially for the intervertebral disc and other connective tissues.

Persistent reduction in sialylation of cerebral glycoproteins following postnatal inflammatory exposure.

BACKGROUND: The extension of sepsis encompassing the preterm newborn's brain is often overlooked due to technical challenges in this highly vulnerable population, yet it leads to substantial long-term neurodevelopmental disabilities. In this study, we demonstrate how neonatal neuroinflammation following postnatal E. coli lipopolysaccharide (LPS) exposure in rat pups results in persistent reduction in sialylation of cerebral glycoproteins. METHODS: Male Sprague-Dawley rat pups at postnatal day 3 (P3) were injected in the corpus callosum with saline or LPS. Twenty-four hours (P4) or 21 days (P24) following injection, brains were extracted and analyzed for neuraminidase activity and expression as well as for sialylation of cerebral glycoproteins and glycolipids. RESULTS: At both P4 and P24, we detected a significant increase of the acidic neuraminidase activity in LPS-exposed rats. It correlated with significantly increased neuraminidase 1 (Neu1) mRNA in LPS-treated brains at P4 and with neuraminidases 1 and 4 at P24 suggesting that these enzymes were responsible for the rise of neuraminidase activity. At both P4 and P24, sialylation of N-glycans on brain glycoproteins decreased according to both mass-spectrometry analysis and lectin blotting, but the ganglioside composition remained intact. Finally, at P24, analysis of brain tissues by immunohistochemistry showed that neurons in the upper layers (II-III) of somatosensory cortex had a reduced surface content of polysialic acid. CONCLUSIONS: Together, our data demonstrate that neonatal LPS exposure results in specific and sustained induction of Neu1 and Neu4, causing long-lasting negative changes in sialylation of glycoproteins on brain cells. Considering the important roles played by sialoglycoproteins in CNS function, we speculate that observed re-programming of the brain sialome constitutes an important part of pathophysiological consequences in perinatal infectious exposure.

Changes in growth plate extracellular matrix composition and biomechanics following in vitro static versus dynamic mechanical modulation.

The objective of this study was to investigate the effects of mechanical modulation parameters on structural proteins biocomposition and mechanical properties of the growth plate. Establishing these parameters is a crucial step in the development of fusionless treatment of scoliosis. In this study, ulna explants from 4-weeks-old (pubertal) swines were used. The biocomposition was characterized using biochemical content evaluation and immunohistochemistry. Mechanical properties were characterized by fitting the data of the stress relaxation curves using a fibril reinforced biphasic model. For the mechanical loading, one static modulation condition and three different dynamic modulation conditions, with similar average stress but different amplitude and frequency values, were performed using a bioreactor. Results showed that static loading triggers a decrease in proteoglycan content and type X collagen in specific zones of the growth plate. These changes can be associated with the observed decrement of permeability in the static group. None of the three conditions evaluated for dynamic modulation affected the growth plate biocomposition and biomechanical responses. Results of this study provides an improved understanding of growth plate responses to mechanical environment, which will be useful in finding the optimal and non-damaging parameters for fusionless treatments based on the mechanical modulation of bone growth.

User-independent diffusion tensor imaging analysis pipelines in a rat model presenting ventriculomegalia: A comparison study.

Automated analysis of diffusion tensor imaging (DTI) data is an appealing way to process large datasets in an unbiased manner. However, automation can sometimes be linked to a lack of interpretability. Two whole-brain, automated and voxelwise methods exist: voxel-based analysis (VBA) and tract-based spatial statistics (TBSS). In VBA, the amount of smoothing has been shown to influence the results. TBSS is free of this step, but a projection procedure is introduced to correct for residual misalignments. This projection assigns the local highest fractional anisotropy (FA) value to the mean FA skeleton, which represents white matter tract centers. For both methods, the normalization procedure has a major impact. These issues are well documented in humans but, to our knowledge, not in rodents. In this study, we assessed the quality of three different registration algorithms (ANTs SyN, DTI-TK and FNIRT) using study-specific templates and their impact on automated analysis methods (VBA and TBSS) in a rat pup model of diffuse white matter injury presenting large unilateral deformations. VBA and TBSS results were stable and anatomically coherent across the three pipelines. For VBA, in regions around the large deformations, interpretability was limited because of the increased partial volume effect. With TBSS, two of the three pipelines found a significant decrease in axial diffusivity (AD) at the known injury site. These results demonstrate that automated voxelwise analyses can be used in an animal model with large deformations.

Neonatal microglia: The cornerstone of brain fate.

Microglia, mainly known for their role in innate immunity and modulation of neuroinflammation, play an active role in central nervous system development and homeostasis. Depending on the context and environmental stimuli, microglia adopt a broad spectrum of activation status from pro-inflammatory, associated with neurotoxicity, to anti-inflammatory linked to neuroprotection. Pro-inflammatory microglial activation is a key hallmark of white matter injury in preterm infants and is involved in developmental origin of adult neurological diseases. Characterization of neonatal microglia function in brain development and inflammation has allowed the investigation of promising therapeutic targets with potential long-lasting neuroprotective effects. True prevention of neuro-degenerative diseases might eventually occur as early as the perinatal period.

New means to assess neonatal inflammatory brain injury.

Preterm infants are especially vulnerable to infection-induced white matter injury, associated with cerebral palsy, cognitive and psychomotor impairment, and other adverse neurological outcomes. The etiology of such lesions is complex and multifactorial. Furthermore, timing and length of exposure to infection also influence neurodevelopmental outcomes. Different mechanisms have been posited to mediate the observed brain injury including microglial activation followed by subsequent release of pro-inflammatory species, glutamate-induced excitotoxicity, and vulnerability of developing oligodendrocytes to cerebral insults. The prevalence of such neurological impairments requires an urgent need for early detection and effective neuroprotective strategies. Accordingly, noninvasive methods of monitoring disease progression and therapy effectiveness are essential. While diagnostic tools using biomarkers from bodily fluids may provide useful information regarding potential risks of developing neurological diseases, the use of magnetic resonance imaging/spectroscopy has emerged as a promising candidate for such purpose. Various pharmacological agents have demonstrated protective effects in the immature brain in animal models; however, few studies have progressed to clinical trials with promising results.

Effect of cold storage and freezing on the biomechanical properties of swine growth plate explants.

Ex vivo biomechanical testing of growth plate samples provides essential information about its structural and physiological characteristics. Experimental limitations include the preservation of the samples since working with fresh tissues involves significant time and transportation costs. Little information is available on the storage of growth plate explants. The aim of this study was to determine storage conditions that could preserve growth plate biomechanical properties. Porcine ulnar growth plate explants (n = 5 per condition) were stored at either 4 °C for periods of 1, 2, 3, and 6 days or frozen at -20 °C with slow or rapid sample thawing. Samples were tested using stress relaxation tests under unconfined compression to assess five biomechanical parameters. The maximum compressive stress (σmax) and the equilibrium stress (σeq) were directly extracted from the experimental curves, while the fibril-network reinforced biphasic model was used to obtain the matrix modulus (Em), the fibril modulus (Ef), and the permeability (k). No significant changes were observed in σeq and Em in any of the tested storage conditions. Significant decreases and increases, respectively, were observed in σmax and k in the growth plate samples refrigerated for more than 48 h and in the frozen samples, when compared with the fresh samples. The fibril modulus Ef of all stored samples was significantly reduced compared to the fresh samples. These results indicate that the storage of growth plates in a humid chamber at 4 °C for a maximum of 48 h is the condition that minimizes the effects on the measured biomechanical parameters, with only Ef significantly reduced. Refrigerating growth plate explants for less than 48 h maintains their maximal stress, equilibrium stress, matrix modulus, and permeability. However, cold storage at 4 °C for more than 48 h and freezing storage at -20 °C significantly alter the biomechanical response of growth plate samples. Appropriate growth plate sample storage will be beneficial to save time and reduce transportation costs to pick up fresh samples.

Zoledronate reduces loading-induced microdamage in cortical ulna of ovariectomized rats.

As a daily physiological mechanism in bone, microdamage accumulation dissipates energy and helps to prevent fractures. However, excessive damage accumulation might bring adverse effects to bone mechanical properties, which is especially problematic among the osteoporotic and osteopenic patients treated by bisphosphonates. Some pre-clinical studies in the literature applied forelimb loading models to produce well-controlled microdamage in cortical bone. Ovariectomized animals were also extensively studied to assimilate human conditions of estrogen-related bone loss. In the present study, we combined both experimental models to investigate microdamage accumulation in the context of osteopenia and zoledronate treatment. Three-month-old normal and ovariectomized rats treated by saline or zoledronate underwent controlled compressive loading on their right forelimb to create in vivo microdamage, which was then quantified by barium sulfate contrast-enhanced micro-CT imaging. Weekly in vivo micro-CT scans were taken to evaluate bone (re)modeling and to capture microstructural changes over time. After sacrifice, three-point-bending tests were performed to assess bone mechanical properties. Results show that the zoledronate treatment can reduce cortical microdamage accumulation in ovariectomized rats, which might be explained by the enhancement of several bone structural properties such as ultimate force, yield force, cortical bone area and volume. The rats showed increased bone formation volume and surface after the generation of microdamage, especially for the normal and the ovariectomized groups. Woven bone formation was also observed in loaded ulnae, which was most significant in ovariectomized rats. Although all the rats showed strong correlations between periosteal bone formation and microdamage accumulation, the correlation levels were lower for the zoledronate-treated groups, potentially because of their lower levels of microdamage. The present study provides insights to further investigations of pharmaceutical treatments for osteoporosis and osteopenia. The same experimental concept can be applied in future studies on microdamage and drug testing.

Segmentation of trabecular bone microdamage in Xray microCT images using a two-step deep learning method.

INTRODUCTION: One of the current approaches to improve our understanding of osteoporosis is to study the development of bone microdamage under mechanical loading. The current practice for evaluating bone microdamage is to quantify damage volume from images of bone samples stained with a contrast agent, often composed of toxic heavy metals and requiring long tissue preparation. This work aims to evaluate the potential of linear microcracks detection and segmentation in trabecular bone samples using well-known deep learning models, namely YOLOv4 and Unet, applied on microCT images. METHODS: Six trabecular bovine bone cylinders underwent compression until ultimate stress and were subsequently imaged with a microCT at a resolution of 1.95 µm. Two of these samples (samples 1 and 2) were then stained using barium sulfate (BaSO4) and imaged again. The unstained samples (samples 3-6) were used to train two neural networks YOLOv4 to detect regions with microdamage further combined with Unet to segment the microdamage at the pixel level in the detected regions. Four different model versions of YOLOv4 were compared using the average Intersection over Union (IoU) and the mean average precision (mAP). The performance of Unet was also measured using two segmentation metrics, the Dice Score and the Intersection over Union (IoU). A qualitative comparison was finally done between the deep learning and the contrast agent approaches. RESULTS: Among the four versions of YOLOv4, the YOLOv4p5 model resulted in the best performance with an average IoU of 45,32% and 51,12% and a mAP of 28.79% and 46.22%, respectively for samples 1 and 2. The segmentation performance of Unet provided better IoU and DICE score on sample 2 compared to sample 1. The poorer performance of the test on sample 1 could be explained by its poorer contrast to noise ratio (CNR). Indeed, sample 1 resulted in a CNR of 7,96, which was worse than the average CNR in the training samples, while sample 2 resulted in a CNR of 10,08. The qualitative comparison between the contrast agent and the deep learning segmentation showed that two different regions were segmented by the two techniques. Deep learning is segmenting the region inside the cracks while the contrast agent segments the region around it or even regions with no visible damage. CONCLUSION: The combination of YOLOv4 for microdamage detection with Unet for damage segmentation showed a potential for the detection and segmentation of microdamage in trabecular bone. The accuracy of both neural networks achieved in this work is acceptable considering it is their first application in this specific field and the amount of data was limited. Even if the errors from both neural networks are accumulated, the two-steps approach is faster than the semantic segmentation of the whole volume.

Impact loading intensifies cortical bone (re)modeling and alters longitudinal bone growth of pubertal rats.

Physical exercise is important for musculoskeletal development during puberty, which builds bone mass foundation for later in life. However, strenuous levels of training might bring adverse effects to bone health, reducing longitudinal bone growth. Animal models with various levels of physical exercise were largely used to provide knowledge to clinical settings. Experiments from our previous studies applied different levels of mechanical loading on rat tibia during puberty accompanied by weekly in vivo micro-CT scans. In the present article, we apply 3D image registration-based methods to retrospectively analyze part of the previously acquired micro-CT data. Longitudinal bone growth, growth plate thickness, and cortical bone (re)modeling were evaluated from rats' age of 28-77 days. Our results show that impact loading inhibited proximal bone growth throughout puberty. We hypothesize that impact loading might bring different growth alterations to the distal and proximal growth plates. High impact loading might lead to pathological consequence of osteochondrosis and catch-up growth due to growth inhibition. Impact loading also increased cortical bone (re)modeling before and after the peak proximal bone growth period of young rats, of which the latter case might be caused by the shift from modeling to remodeling as the dominant activity toward the end of rat puberty. We confirm that the tibial endosteum is more mechano-sensitive than the periosteum in response to mechanical loading. To our knowledge, this is the first study to follow up bone growth and bone (re)modeling of young rats throughout the entire puberty with a weekly time interval.

Severe central nervous system demyelination in Sanfilippo disease.

Introduction: Chronic progressive neuroinflammation is a hallmark of neurological lysosomal storage diseases, including mucopolysaccharidosis III (MPS III or Sanfilippo disease). Since neuroinflammation is linked to white matter tract pathology, we analyzed axonal myelination and white matter density in the mouse model of MPS IIIC HgsnatP304L and post-mortem brain samples of MPS III patients. Methods: Brain and spinal cord tissues of human MPS III patients, 6-month-old HgsnatP304L mice and age- and sex-matching wild type mice were analyzed by immunofluorescence to assess levels of myelin-associated proteins, primary and secondary storage materials, and levels of microgliosis. Corpus callosum (CC) region was studied by transmission electron microscopy to analyze axon myelination and morphology of oligodendrocytes and microglia. Mouse brains were analyzed ex vivo by high-filed MRI using Diffusion Basis Spectrum Imaging in Python-Diffusion tensor imaging algorithms. Results: Analyses of CC and spinal cord tissues by immunohistochemistry revealed substantially reduced levels of myelin-associated proteins including Myelin Basic Protein, Myelin Associated Glycoprotein, and Myelin Oligodendrocyte Glycoprotein. Furthermore, ultrastructural analyses revealed disruption of myelin sheath organization and reduced myelin thickness in the brains of MPS IIIC mice and human MPS IIIC patients compared to healthy controls. Oligodendrocytes (OLs) in the CC of MPS IIIC mice were scarce, while examination of the remaining cells revealed numerous enlarged lysosomes containing heparan sulfate, GM3 ganglioside or "zebra bodies" consistent with accumulation of lipids and myelin fragments. In addition, OLs contained swollen mitochondria with largely dissolved cristae, resembling those previously identified in the dysfunctional neurons of MPS IIIC mice. Ex vivo Diffusion Basis Spectrum Imaging revealed compelling signs of demyelination (26% increase in radial diffusivity) and tissue loss (76% increase in hindered diffusivity) in CC of MPS IIIC mice. Discussion: Our findings demonstrate an important role for white matter injury in the pathophysiology of MPS III. This study also defines specific parameters and brain regions for MRI analysis and suggests that it may become a crucial non-invasive method to evaluate disease progression and therapeutic response.

Severe kidney dysfunction in sialidosis mice reveals an essential role for neuraminidase 1 in reabsorption.

Sialidosis is an ultra-rare multisystemic lysosomal disease caused by mutations in the neuraminidase 1 (NEU1) gene. The severe type II form of the disease manifests with a prenatal/infantile or juvenile onset, bone abnormalities, severe neuropathology, and visceromegaly. A subset of these patients present with nephrosialidosis, characterized by abrupt onset of fulminant glomerular nephropathy. We studied the pathophysiological mechanism of the disease in 2 NEU1-deficient mouse models, a constitutive Neu1-knockout, Neu1ΔEx3, and a conditional phagocyte-specific knockout, Neu1Cx3cr1ΔEx3. Mice of both strains exhibited terminal urinary retention and severe kidney damage with elevated urinary albumin levels, loss of nephrons, renal fibrosis, presence of storage vacuoles, and dysmorphic mitochondria in the intraglomerular and tubular cells. Glycoprotein sialylation in glomeruli, proximal distal tubules, and distal tubules was drastically increased, including that of an endocytic reabsorption receptor megalin. The pool of megalin bearing O-linked glycans with terminal galactose residues, essential for protein targeting and activity, was reduced to below detection levels. Megalin levels were severely reduced, and the protein was directed to lysosomes instead of the apical membrane. Together, our results demonstrated that desialylation by NEU1 plays a crucial role in processing and cellular trafficking of megalin and that NEU1 deficiency in sialidosis impairs megalin-mediated protein reabsorption.

Validation of an in vivo micro-CT-based method to quantify longitudinal bone growth of pubertal rats.

Bone growth is an essential part of skeletal development during childhood and puberty. Accurately characterizing longitudinal bone growth helps to better understand the determining factors of peak bone mass, which has impacts on bone quality later in life. Animal models were largely used to study longitudinal bone growth. However, the commonly used histology-based method is destructive and unable to follow up the growth curve of live animals in longitudinal experiments. In this study, we validated an in vivo micro-CT-based method against the histology-based method to quantify longitudinal bone growth rates of young rats non-destructively. CD (Sprague Dawley) IGS rats aged 35, 49 and 63 days received the same treatments: two series of repeated in vivo micro-CT scans on their proximal hind limb at a five-day interval, and two calcein injections separated by three days. The longitudinal bone growth rate was quantified by registering time-lapse micro-CT images in 3D, calculating the growth distance on registered images, and dividing the distance by the five-day gap. The growth rate was also evaluated by measuring the 2D distance between consecutive calcein fluorescent bands on microscopic images, divided by the three-day gap. The two methods were both validated independently with reproducible repeated measurements, where the micro-CT-based method showed higher precision. They were also validated against each other with low relative errors and a strong Pearson sample correlation coefficient (0.998), showing a significant (p < 0.0001) linear correlation between paired results. We conclude that the micro-CT-based method can serve as an alternative to the histology-based method for the quantification of longitudinal growth. Thanks to its non-invasive nature and true 3D capability, the micro-CT-based method helps to accommodate in vivo longitudinal animal studies with highly reproducible measurements.

Modulatory effect of IL-1 inhibition following lipopolysaccharide-induced neuroinflammation in neonatal microglia and astrocytes.

INTRODUCTION: Inflammation-induced white matter injury (WMI) in preterm infants is characterized by microglia activation, astrogliosis, oxidative stress and neurodevelopmental impairments. Microglia and astrocytes activation can be described under a broad spectrum of activation profile with extremes described as pro-inflammatory/neurotoxic (M1 microglia or A1 astrocyte) or anti-inflammatory/neuroprotective (M2 microglia or A2 astrocyte) in response to stimuli including lipopolysaccharide (LPS) and interleukin 1 (IL-1). As IL-1 signalling pathway has been posited as a major driver of inflammation-induced perinatal WMI, our aim was to evaluate the contribution of IL-1 modulation in LPS-induced microglia and astrocyte activation. METHODS: Primary neonatal cell co-cultures of astrocytes and microglia were treated with LPS (100 ng/ml) for 8 h or 24 h. Two distinct IL-1 receptor antagonists, Rytvela or Kineret (1 µg/ml), were added simultaneously with LPS to respectively modulate or block IL-1 receptor. Medium was collected to measure levels of IL-1ß. Expression of markers related to pro- and anti-inflammatory microglia and astrocyte activation profiles and antioxidant genes were assessed. RESULTS: At 8 h, LPS exposure induced pro- (M1/A1) and anti-inflammatory (M2/A2) marker expression and inhibited antioxidant gene expression in microglia and astrocytes. By 24 h, continuous LPS exposure increased pro-inflammatory and neurotoxic microglial and astrocytic markers (M1/A1), as well as antioxidant genes. Administration of IL-1 antagonists Rytvela and Kineret with continuous LPS exposure had no significant effect on modulation of specific microglia and astrocyte activation pathways. DISCUSSION/CONCLUSION: We show that LPS effects on in vitro neonatal microglia and astrocytes co-cultures are time dependent eliciting a number of pro- and anti-inflammatory responses during the acute phase of inflammation (8 h), which shift towards pro-inflammatory and neurotoxic factors by 24 h. Although LPS-induced inflammation led to abundant IL-1 expression, IL-1 inhibition had no significant impact on in vitro modulation of microglia and astrocyte activation pathways towards M2 and A2 activation profile.

Non-invasive in vivo MRI detects long-term microstructural brain alterations related to learning and memory impairments in a model of inflammation-induced white matter injury.

Magnetic resonance imaging (MRI) is currently under investigation as a non-invasive tool to monitor neurodevelopmental trajectories and predict risk of cognitive deficits following white matter injury (WMI) in very preterm infants. In the present study, we evaluated the capacity of multimodal MRI (high-resolution T2-weighted imaging and diffusion tensor imaging)to assess changes following WMI and their relationship to learning and memory performance in Wistar rats as it has been demonstrated for preterm infants. Multimodal MRI performed at P31-P32 shown that animals exposed to neonatal LPS could be classified into two groups: minimal and overt injury. Animals with overt injury had significantly enlarged ventricles, hippocampal atrophy, diffusivity changes in hippocampal white and gray matter, in the striatum and the cortex. Following neonatal LPS exposure, animals presented learning and memory impairments as shown at the fear conditioning test at P36-P38. The severity of learning and memory deficits was related to increased mean diffusivity in the hippocampal region. In conclusion, non-invasive multimodal MRI (volumetric and DTI) assessed and classified the extent of injury at long-term following neonatal LPS exposure. Microstructural changes in the hippocampus at DTI were associated to learning and memory impairments. This further highlights the utility of multimodal MRI as a non-invasive quantitative biomarker following perinatal inflammation.

Isolated Cyclic Loading During Adolescence Improves Tibial Bone Microstructure and Strength at Adulthood.

Bone is a unique living tissue, which responds to the mechanical stimuli regularly imposed on it. Adolescence facilitates a favorable condition for the skeleton that enables the exercise to positively influence bone architecture and overall strength. However, it is still dubious for how long the skeletal benefits gained in adolescence is preserved at adulthood. The current study aims to use a rat model to investigate the effects of in vivo low- (LI), medium- (MI), and high- (HI) intensity cyclic loadings applied during puberty on longitudinal bone development, morphometry, and biomechanics during adolescence as well as at adulthood. Forty-two young (4-week-old) male rats were randomized into control, sham, LI, MI, and HI groups. After a 5 day/week for 8 weeks cyclic loading regime applied on the right tibia, loaded rats underwent a subsequent 41-week, normal cage activity period. Right tibias were removed at 52 weeks of age, and a comprehensive assessment was performed using µCT, mechanical testing, and finite element analysis. HI and MI groups exhibited reduced body weight and food intake at the end of the loading period compared with shams, but these effects disappeared afterward. HI cyclic loading increased BMD, bone volume fraction, trabecular thickness, trabecular number, and decreased trabecular spacing after loading. All loading-induced benefits, except BMD, persisted until the end of the normal cage activity period. Moreover, HI loading induced enhanced bone area, periosteal perimeter, and moment of inertia, which remained up to the 52nd week. After the normal cage activity at adulthood, the HI group showed increased ultimate force and stress, stiffness, postyield displacement and energy, and toughness compared with the sham group. Overall, our findings suggest that even though both trabecular and cortical bone drifted through age-related changes during aging, HI cyclic loading performed during adolescence can render lifelong benefits in bone microstructure and biomechanics. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Association of AMP-activated protein kinase subunits with glycogen particles as revealed in situ by immunoelectron microscopy.

Immunogold cytochemistry was applied to reveal the intracellular location of AMP-activated protein kinase (AMPK) subunits in liver tissue of normal rats fed ad libitum. AMPK alpha and beta subunits were located both in the cytosol and in close association with rosettes of glycogen particles (alpha particles). To reveal their true in situ association with glycogen, particular tissue processing conditions that retain glycogen in the cells were required. These included fixation with a combination of glutaraldehyde and paraformaldehyde, followed by postfixation with osmium tetroxide and lead citrate and embedding in Epon. Processing by less-stringent fixation conditions and embedding in Lowicryl led to the extraction of the glycogen deposits, which in turn resulted in the absence of any labeling. This indicates that the loss of glycogen deposits leads to the loss of closely associated proteins. Labeling for the alpha(1) and alpha(2) subunits of AMPK was found to be about 2-fold greater over glycogen than over cytosol, whereas labeling for beta(1) was 8-fold higher over the glycogen particles than over the cytosol. Immunogold combined with morphometric analysis demonstrated that the beta(1) subunits are located at the periphery of the glycogen rosettes, consistent with a recent hypothesis developed via biochemical approaches.