In vitro modeling of skeletal muscle ischemia-reperfusion injury based on sphere differentiation culture from human pluripotent stem cells.

Skeletal muscle ischemia-reperfusion (IR) injury poses significant challenges due to its local and systemic complications. Traditional studies relying on two-dimensional (2D) cell culture or animal models often fall short of faithfully replicating the human in vivo environment, thereby impeding the translational process from animal research to clinical applications. Three-dimensional (3D) constructs, such as skeletal muscle spheroids with enhanced cell-cell interactions from human pluripotent stem cells (hPSCs) offer a promising alternative by partially mimicking human physiological cellular environment in vivo processes. This study aims to establish an innovative in vitro model, human skeletal muscle spheroids based on sphere differentiation from hPSCs, to investigate human skeletal muscle developmental processes and IR mechanisms within a controlled laboratory setting. By eticulously recapitulating embryonic myogenesis through paraxial mesodermal differentiation of neuro-mesodermal progenitors, we successfully established 3D skeletal muscle spheroids that mirror the dynamic colonization observed during human skeletal muscle development. Co-culturing human skeletal muscle spheroids with spinal cord spheroids facilitated the formation of neuromuscular junctions, providing functional relevance to skeletal muscle spheroids. Furthermore, through oxygen-glucose deprivation/re-oxygenation treatment, 3D skeletal muscle spheroids provide insights into the molecular events and pathogenesis of IR injury. The findings presented in this study significantly contribute to our understanding of skeletal muscle development and offer a robust platform for in vitro studies on skeletal muscle IR injury, holding potential applications in drug testing, therapeutic development, and personalized medicine within the realm of skeletal muscle-related pathologies.

The gut microbiota and metabolite profiles are altered in patients with spinal cord injury.

BACKGROUND: Metabolites secreted by the gut microbiota may play an essential role in microbiota-gut-central nervous system crosstalk. In this study, we explored the changes occurring in the gut microbiota and their metabolites in patients with spinal cord injury (SCI) and analyzed the correlations among them. METHODS: The structure and composition of the gut microbiota derived from fecal samples collected from patients with SCI (n = 11) and matched control individuals (n = 10) were evaluated using 16S rRNA gene sequencing. Additionally, an untargeted metabolomics approach was used to compare the serum metabolite profiles of both groups. Meanwhile, the association among serum metabolites, the gut microbiota, and clinical parameters (including injury duration and neurological grade) was also analyzed. Finally, metabolites with the potential for use in the treatment of SCI were identified based on the differential metabolite abundance analysis. RESULTS: The composition of the gut microbiota was different between patients with SCI and healthy controls. At the genus level, compared with the control group, the abundance of UBA1819, Anaerostignum, Eggerthella, and Enterococcus was significantly increased in the SCI group, whereas that of Faecalibacterium, Blautia, Escherichia-Shigella, Agathobacter, Collinsella, Dorea, Ruminococcus, Fusicatenibacter, and Eubacterium was decreased. Forty-one named metabolites displayed significant differential abundance between SCI patients and healthy controls, including 18 that were upregulated and 23 that were downregulated. Correlation analysis further indicated that the variation in gut microbiota abundance was associated with changes in serum metabolite levels, suggesting that gut dysbiosis is an important cause of metabolic disorders in SCI. Finally, gut dysbiosis and serum metabolite dysregulation was found to be associated with injury duration and severity of motor dysfunction after SCI. CONCLUSIONS: We present a comprehensive landscape of the gut microbiota and metabolite profiles in patients with SCI and provide evidence that their interaction plays a role in the pathogenesis of SCI. Furthermore, our findings suggested that uridine, hypoxanthine, PC(18:2/0:0), and kojic acid may be important therapeutic targets for the treatment of this condition.

WKYMVm/FPR2 Alleviates Spinal Cord Injury by Attenuating the Inflammatory Response of Microglia.

Spinal cord injury (SCI) is a common traumatic disease of the nervous system. The pathophysiological process of SCI includes primary injury and secondary injuries. An excessive inflammatory response leads to secondary tissue damage, which in turn exacerbates cellular and organ dysfunction. Due to the irreversibility of primary injury, current research on SCI mainly focuses on secondary injury, and the inflammatory response is considered the primary target. Thus, modulating the inflammatory response has been suggested as a new strategy for the treatment of SCI. In this study, microglial cell lines, primary microglia, and a rat SCI model were used, and we found that WKYMVm/FPR2 plays an anti-inflammatory role and reduces tissue damage after SCI by suppressing the extracellular signal-regulated kinases 1 and 2 (ERK1/2) and nuclear factor-κB (NF-κB) signaling pathways. FPR2 was activated by WKYMVm, suppressing the secretion of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1ß (IL-1ß) by inhibiting M1 microglial polarization. Moreover, FPR2 activation by WKYMVm could reduce structural disorders and neuronal loss in SCI rats. Overall, this study illustrated that the activation of FPR2 by WKYMVm repressed M1 microglial polarization by suppressing the ERK1/2 and NF-κB signaling pathways to alleviate tissue damage and locomotor decline after SCI. These findings provide further insight into SCI and help identify novel treatment strategies.

Ketogenic diet ameliorates inflammation by inhibiting the NLRP3 inflammasome in osteoarthritis.

BACKGROUND: The nucleotide-binding domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NLRP3) inflammasome has been reported to be involved in the pathological process of osteoarthritis (OA) inflammation. Here, we investigated the ketogenic diet (KD), which has been previously demonstrated to inhibit NLRP3 inflammasome activation, to elucidate its protective mechanism against OA in rats. METHODS: Anterior cruciate ligament transaction (ACLT) together with partial medial meniscectomy was used to create a rat knee joint OA model. After treatment with KD or standard diet (SD) for 8 weeks, the knee specimens were obtained for testing. RESULTS: The KD significantly increased the content of ß-hydroxybutyrate (ßOHB) in rats. Compared to the SD group, the KD significantly reduced the damage caused by OA in the articular cartilage and subchondral bone. The NLRP3 inflammasome and inflammatory cytokines interleukin-1 ß (IL-1ß) and IL-18 were significantly increased in the SD group compared with the sham group, while their expression was significantly decreased in rats treated with the KD. In addition, MMP13 was significantly decreased in the KD group compared to that in the SD group, while COL2 was significantly increased. CONCLUSIONS: KD can protect the articular cartilage and subchondral bone in a rat OA model by inhibiting NLRP3 inflammasome activation and reducing the OA inflammatory response.

Anatomical and behavioral outcomes following a graded hemi-contusive cervical spinal cord injury model in mice.

BACKGROUND: A graded hemi-contusion spinal cord injury produces complex anatomical deformation of the spinal cord parenchyma. The relationship between lesion severity and behavioral consequences in a novel contusion mouse model remains unknown. PURPOSE: We aimed to establish a graded cervical hemi-contusion spinal cord injury model in mice and investigate the correlation between graded anatomical damage to the spinal cord and resulting behavioral impairments. METHODS: Thirty-two mice were divided into groups of 1.2 mm, 1.5 mm and sham. The tip of an impactor with a diameter of 1 mm was utilized to compress the left dorsal cord of C5 by 1.2 mm or 1.5 mm at a speed of 300 mm/s. Forelimb motor function was evaluated using rearing, grooming and grip-strength tests before and after the injuries. Histologically the area of white matter sparing, gray matter sparing and lesion area were quantified at 6-week-post-injury. RESULTS: Behavioral assessments showed a more severe forelimb functional deficit in 1.5 mm contusion displacements relative to 1.2 mm contusion displacements after injury. The 1.2 mm hemi-contusion mainly caused damage to the dorsal fasciculus, ventral and dorsal horn, while the 1.5 mm hemi-contusion lead to additional damage extending to ventral fasciculus. Sparing of the gray and white matter at the epicenter was 36.8 ± 2.4% and 12.4 ± 2.6% in the 1.2 mm group, and 27.6 ± 4.0% and 4.1 ± 2.2% in the 1.5 mm group, respectively. Furthermore, the lesion area was 20.8 ± 3.0% and 36.0 ± 2.1% in the 1.2 mm and 1.5 mm groups, respectively. There was a significant correlation between the performance in the grooming test and white matter sparing, and between grip-test strength and gray matter sparing. CONCLUSION: The present study demonstrates that a hemi-contusion cervical spinal cord injury in mice can be graded by contusion displacement and that there is a correlation between anatomical and behavioral outcomes. This study provides a means for determining the severity of lesions in a contusion mouse model.

Ketone Metabolite ß-Hydroxybutyrate Ameliorates Inflammation After Spinal Cord Injury by Inhibiting the NLRP3 Inflammasome.

Ketogenic diet (KD) has been shown to be beneficial in a range of neurological disorders, with ketone metabolite ß-hydroxybutyrate (ßOHB) reported to block the nucleotide oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome in bone marrow-derived macrophages. In this study, we show that pretreatment with KD or in situ ßOHB suppressed macrophages/microglia activation and the overproduction of inflammatory cytokines, while KD downregulated the expression of NLRP3 inflammasome. Moreover, KD promoted macrophages/microglia transformation from the M1 phenotype to the M2a phenotype following spinal cord injury (SCI) in the in vivo study. Rats in the KD group demonstrated improved behavioral and electrophysiological recovery after SCI when compared to those rats in the standard diet group. The in vitro study performed on BV2 cells indicated that ßOHB inhibited an LPS+ATP-induced inflammatory response and decreased NLRP3 protein levels. Our data demonstrated that pretreatment with KD attenuated neuroinflammation following SCI, probably by inhibiting NLRP3 inflammasome and shifting the activation state of macrophages/microglia from the M1 to the M2a phenotype. Therefore, the ketone metabolite ßOHB might provide a potential future therapeutic strategy for SCI.

A Cervical Spinal Cord Hemi-Contusion Injury Model Based on Displacement Control in Non-Human Primates (Macaca fascicularis).

Non-human primate (NHP) spinal cord injury (SCI) models can be informative in the evaluation of treatments that show promise in rodent models prior to translation to humans. In the present study, we aimed to establish a cervical spinal hemi-contusion model with controlled displacement and evaluate the abnormalities in behavior, electrophysiology, histology, and magnetic resonance imaging. Twelve adult NHPs were divided into an SCI group (n = 8, 24 and 48 weeks) and a control group (n = 4). An impactor (Φ = 4 mm) was driven to compress the left C5 cord at 800 mm/sec. The contusion displacement and peak force was 4.08 ± 0.17 mm and 19.8 ± 4.6 N. The behavioral assessment showed a consistent dysfunction below the wrist and spontaneous recovery of limb function after injury. Lesion length and lesion area at the epicenter based on T2 hyperintensity were 5.68 ± 0.47 mm and 5.99 ± 0.24 mm2 at 24 weeks post-injury (wpi), and 5.29 ± 0.17 mm and 5.95 ± 0.24 mm2 at 48 wpi. The spared spinal cord area immuno-positive for glial fibrillary acidic protein was significantly reduced, while the staining intensity increased at 24 wpi and 48 wpi, compared with the sham group. Ipsilateral somatosensory and motor evoked potentials were dynamic, increasing in latency and decreasing in amplitude compared with pre-operative values or the contralateral values, and correlated to varying degrees with behavioral outcomes. A shift in size-frequency distribution of sensory neurons of the dorsal root ganglia (DRG) was consistent with a loss of large-diameter cells. The present study demonstrated that the NHP SCI model resulted in consistent unilateral limb dysfunction and potential plasticity in the face of loss of spinal cord and DRG tissue.

Comparison of two modified methods of intrathecal catheterization in rats.

The study designed to compare two different methods of intrathecal catheterization in rats and to develop a simple and safe drug administration in cervical spinal canal of rats. The subarachnoid catheterization was performed via either atlanto-occipital membrane or laminectomy at L3-4 in rats. Body weight, Basso, Beattie, and Bresnahan (BBB) locomotion rating scores and forelimb locomotor rating scale (FLS) were measured on pre-operative day 1 and postoperative day 1, 7, 14, respectively. FLS score of 37.5% rats and BBB score of 50% rats in the atlanto-occipital approach (AOA) group decreased, but no rats showed locomotor impairment in the lumber approach (LA) group. The mean body weight of rats in AOA group reduced significantly compared with LA group. In LA group, 62.5% of catheter tips were located at T1, and in AOA group, the tips of catheter located at C2 in 62.5% cases. The PE10 catheter can be successfully inserted into the spinal intrathecal space for chronic delivery of drugs either via L3-L4 interlaminar space or via atlanto-occipital membrane. And the subarachnoid catheterization via L3-L4 interlaminar space could be easily placed at T1 with little complication.

Clival screw and plate fixation by the transoral approach for the craniovertebral junction: a CT-based feasibility study.

PURPOSE: A clivus screw and plate was invented and proved to strengthen the stability of the craniovertebral junction (CVJ). However, it is unclear whether the clivus screw and plate could be placed onto the CVJ by transoral approach. Therefore, the present study aims to evaluate the feasibility of clivus screw and plate placement by transoral approach and investigate its relative anatomic parameters. METHODS: A total of 80 normal adults (40 males/40 females) with an average age of 60.4 ± 11.6 years old were enrolled in this study. All parameters were measured in a supposed maximums mouth-opening status on computed tomography images, where the vertex of lower incisor was defined as Point A. The vertical intersection from Point A to extracranial clivus was defined as Point B, and its distance to the bottom of clivus was measured as B length. Point B was considered as ideal screw entry point. All the cases were divided into three types based on the location of Point B: above the top portion (Type 1), between the top and bottom portion (Type 2), and below the bottom portion (Type 3) of extracranial clivus. The B Length was defined as a minus value if the case belonged to Type 3. The anterior skull base angle, the angles between tangent of extracranial clivus and the lines from Point A to different parts of clivus, and distances between Point A and clivus and C1-3 vertebra were also measured. RESULTS: One in eighty cases (1.2%) belonged to Type 1 with a B Length of 32.12 mm. Most cases (61.3%) were Type 2 with a B Length of 8.7 mm, while Type 3's was - 9.7 mm occupying for 37.5%. Significant statistic differences were found in anterior skull base angle between these three types (128.9°, 122.7° and 118.5° for Type 1, 2 and 3, respectively). The distances from Point A to the top and bottom portion of the clivus and the pharyngeal tubercle were 97.5, 96.0 and 96.8 mm, respectively. The angles between the tangent of the clivus and the lines from Point A to the above three structures were 75.7°, 92.3° and 84.0°, respectively. The distances from Point A to the middle point of anterior margin of C1 anterior tubercle, C2 vertebra and C3 vertebra were 79.1, 73.4 and 61.5 mm, respectively. CONCLUSION: The clivus screw and plate placement could be accomplished with optimal screw angle by transoral approach in most of patients. Mandibular splitting would be needed in patients with greater anterior skull angle. These slides can be retrieved under Electronic Supplementary Material.

Anterior Atlanto-Occipital Transarticular Screw Fixation: A Radiological Evaluation.

BACKGROUND: In situations in which posterior atlanto-occipital fixation might not be possible or might require supplemental fixation, anterior fixation might add stability in obtaining arthrodesis. The present study aimed to provide a radiographic evaluation of the safety and feasibility in the anterior approach. METHODS: The bilateral craniocervical computed tomography slices of 60 patients were examined. The anterior screw entry point was the lowest point at the middle anterior aspect of C1 (atlas) lateral mass. To avoid hypoglossal canal and craniocerebral injury, the height of the hypoglossal canal and occipital condyle and occipital condyle width were obtained. The mandible occlusion angle (MOA), anterior screw trajectory above, under, and distal to the hypoglossal canal (AHA, UHA, and DHA) relative to the tangent line of C1 front border were measured, together with the maximum screw length under each angle (AHL, UHL and DHL). An independent samples t test was used for statistical analysis. RESULTS: The height of the hypoglossal canal and occipital condyle and occipital condyle width were all larger in the men than in the women. The MOA, AHA, UHA, and DHA were 55.0°, 18.7°, 41.0°, and 55.0°, respectively, and were similar between genders. The AHL, UHL, and DHL were 34.5, 30.9, and 31.3 mm, with the measurements for the men generally longer than those for the women by 3-4 mm. A total of 10 of 120 bilateral measurements showed the possibility of mandible occlusion, and the potential success rate of the anterior approach could reach 91.7%. CONCLUSIONS: The ideal entry angle for anterior atlanto-occipital fixation ranges from 41.0° to 55.0°, with a safe screw length from 30.9 to 31.3 mm. The potential success rate could reach >90%.

Ketogenic diet compromises vertebral microstructure and biomechanical characteristics in mice.

Ketogenic diet (KD) compromised the microstructure of cancellous bone and the mechanical property in the appendicular bone of mice, while the effects of KD on the axial bone have not been reported. This study aimed to compare the changes in the microstructure and mechanical properties of the forth lumbar (L4) vertebra in KD and ovariectomized (OVX) mice. Forty eight-week-old female C57BL/6J mice were assigned into four groups: SD (standard diet) + Sham, SD + OVX, KD + Sham, and KD + OVX groups. L4 vertebra was scanned by micro-CT to examine the microstructure of cancellous bone, after which simulative compression tests were performed using finite element (FE) analysis. Vertebral compressive test and histological staining of the L4 and L5 vertebrae were performed to observe the biomechanical and histomorphologic changes. The KD + Sham and SD + OVX exhibited a remarkable declination in the parameters of cancellous bone compared with the SD + Sham group, while KD + OVX demonstrated the most serious bone loss in the four groups. The stiffness was significantly higher in the SD + Sham group than the other three groups, but no difference was found between the remaining groups. The trabecular parameters were significantly correlated with the stiffness. Meanwhile, the OVX + Sham and KD + OVX groups showed a significant decrease in the failure load of compressive test, while there was no difference between the KD + Sham and SD + Sham groups. These findings suggest that KD may compromise the vertebral microstructure and compressive stiffness to a similar level as OVX did, indicating adverse effects of KD on the axial bone of the mice.

Bone microstructure and metabolism changes under the combined intervention of ketogenic diet with intermittent fasting: an in vivo study of rats.

Ketogenic diet (KD) has been used in epilepsy for decades, but previous studies found it may cause severe bone loss. Every-other-day ketogenic diet (EODKD), the combination of KD with intermittent fasting, showed better potential for seizure control recently, while its effects on bone remain unknown. This study aims to establish different ketogenic rat models and compare the influence of EODKD with KD on bone microstructure and metabolism. Thirty male Sprague-Dawley rats were divided into Control, KD and EODKD groups, fed with standard diet, continuous and intermittent ketogenic diet respectively. After 12 weeks, bone mineral density (BMD) and body fat percentage were obtained by dual energy X-ray absorptiometry. Micro-CT and three-point bending test were used to evaluate the bone microstructure and mechanical properties. Activities of serum alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) were measured, together with the osteogenic capabilities of bone marrow stromal cells (BMSCs) tested by ALP activities and alizarin red stain in different osteogenic stage. Both EODKD and KD induced higher ketone and more fat percentage, but led to lower body weight compared with Control group. They both compromised bone mass and mechanical properties. Compared with KD, EODKD demonstrated higher ketone levels, but it also inhibited osteoclastic process as well as early osteogenic differentiation. In general, EODKD accelerated ketosis, but may not deteriorate bone microstructure and strength than KD.

The Anatomic Study of Intracranial Structures Related to Clival Screw Placement.

BACKGROUND: A clival plate and screw fitted to the craniovertebral junction (CVJ) were developed in our previous studies. However, the anatomy of intracranial structures related to clival screw placement has not been studied. OBJECTIVE: To measure the intracranial anatomic parameters delineating the basilar plexus and vertebrobasilar artery (VBA) course and to determine the relationship between brainstem and clivus for safer screw placement. METHODS: Morphometric analysis of the VBA was conducted on 95 patients (48 men, 47 women) by CT angiography (CTA) and of the basilar plexus and brainstem on 80 patients (44 men, 36 women) by magnetic resonance imaging (MRI) The narrowest distance between the VBA and the intracranial clivus and the distance between the VBA and the median line of the skull were measured on the axial plane at different parts. The relative positions of point I (intersection of vertebral artery and basilar artery) and the pharyngeal tubercle were analyzed. The distance between the clivus and the brainstem was measured at different levels. RESULTS: As the VBA ascended the distances to the intracranial clivus and the midline decreased. In 83.3% cases, point I was above the pharyngeal tubercle, and in only 9.5% cases was it located on the vertical line drawn from pharyngeal tubercle. The smallest safe space between the intracranial clivus and the brainstem was 7.46 mm at the lower part. CONCLUSION: Appropriate screw size at the lower clivus and the pharyngeal tubercle may help avoiding inadvertent injury to the brainstem and the VBA. Preoperative CTA and MRI of the CVJ are suggested to guide the surgeon in this regard.

Bone loss and biomechanical reduction of appendicular and axial bones under ketogenic diet in rats.

A ketogenic diet (KD) is composed of low-carbohydrate, high-fat and adequate levels of protein. It has been used for decades as a method to treat pediatric refractory epilepsy. However, recently, its side effects on the bones have received increasing attention. In order to comprehensively evaluate the effect of KD on the microstructures and mechanical properties of the skeleton, 14 male Sprague-Dawley rats were equally divided into two groups and fed with a KD (ratio of fat to carbohydrate and protein, 3:1) or a standard diet for 12 weeks. Body weight, as well as blood ketone and glucose levels, were monitored during the experiment. Bone morphometric analyses via micro-computerized tomography were performed on cortical and trabecular bone at the middle L4 vertebral body, the proximal humerus and tibia. The compressive stiffness and strength of scanned skeletal areas were calculated using micro-finite element analysis. The KD led to higher ketone levels and lower glucose levels, with reduced body weight and total bone mineral density (TBMD). After 12 weeks, the diet reduced the bone volume fraction, the trabecular number of cancellous bone, cortical thickness, total cross-sectional area inside the periosteal envelope and the bone area of cortical bone in the tibia and humerus, while increasing trabecular separation. However, KD may not affect the L4 vertebral body. The serum calcium or phosphate concentrations in the blood remained unchanged. In addition, bone stiffness and strength were clearly decreased by the KD, and significantly correlated with the BMD and bone area at all scanned sites. In conclusion, KD led to significant bone loss and reduced biomechanical function in appendicular bones, with a lesser impact on axial bones.

Metformin Alleviates the Bone Loss Induced by Ketogenic Diet: An In Vivo Study in Mice.

Metformin (Met), an anti-diabetes drug, has also shown therapeutic effects for ovariectomy-induced (OVX) osteoporosis. However, similar effects against bone loss induced by a ketogenic diet (KD) have not been tested. This study was aimed to evaluate the microarchitectures and biomechanics of KD-induced osteoporosis with and without administration of Met, and compare the effect of Met on bone loss induced by KD with OVX. Forty female C57BL/6J mice were randomly divided into Sham, OVX, OVX + Met (100 mg/kg/day), KD (3:1 ratio of fat to carbohydrate and protein), and KD + Met (100 mg/kg/day) groups. After 12 weeks, the bone mass and biomechanics were measured in distal cancellous bone and in mid-shaft cortical bone of the femur. The activities of serum alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP), together with immunohistochemistry staining of osteocalcin (OCN) and TRAP, were evaluated. Both OVX and KD induced significant bone loss and compromised biomechanical properties in the cancellous bone, but no effect was found in cortical bone. The administration of Met increased the cancellous bone volume fraction (BV/TV) from 3.78 to 5.23% following the OVX and from 4.04 to 6.33% following the KD, it also enhanced the compressive stiffness from 47 to 160 N/mm following the OVX and from 35 to 340 N/mm with the KD. Met effectively increased serum ALP in the KD group while decreased serum TRAP in the OVX group, but up-regulated expression of OCN and down-regulated expression of TRAP in both OVX and KD groups. The present study demonstrated that Met effectively attenuated the cancellous bone loss induced by KD and maintained the biomechanical properties of long bones, providing evidence for Met as a treatment of by KD-induced osteoporosis in teenage skeleton.

Estrogen Deficiency Exacerbates Intervertebral Disc Degeneration Induced by Spinal Instability in Rats.

STUDY DESIGN: An experimental animal study of osteoporosis (OP) and intervertebral disc degeneration (IDD). OBJECTIVE: The aim of this study was to clarify the effects of estrogen deficiency and supplement on cervical IDD induced by bilateral facetectomy in rats. SUMMARY OF BACKGROUND DATA: The relationship between IDD and OP is still controversy with the wide prevalence in aged people. METHODS: Seventy-two Sprague-Dawley female rats were randomly divided into ovariectomy (OVX) group, facet joints resection of C4-6 (FR), FR-OVX group, estrogen replacement therapy (ERT, based on the FR-OVX group) group, and sham group. Specimens of C4-6 segment were harvested at 12 and 24 weeks. The microstructures of C5 vertebrae, vertebral endplate lesions and calcification, and IDD of C5/6 disc were evaluated by micro-computed tomography (micro-CT) and histology. The protein and gene levels of aggrecan, Col2α1, matrix metalloprotease (MMP)-3, and MMP-13 in the C5/6 and C4/5 discs were measured. RESULTS: Microstructures of C5 vertebral body were weakened significantly after ovariectomy, while restored effectively with estradiol supplementation. The facetectomy led to significant IDD, and the IDD was aggravated when combined with OVX. The IDD of the ERT group was alleviated effectively and similar to that of the FR group in intervertebral disc height, vertebral endplate lesions and calcification, and disc degeneration scores. In addition, the estrogen supplement maintained the extracellular matrix by decreasing MMP-3 and MMP-13, and increasing aggrecan and Col2α1 expression. CONCLUSION: The present study demonstrated that estrogen deficiency exacerbated IDD induced by spinal instability, while estrogen supplementation alleviated the progression of disc degeneration related to osteoporosis. LEVEL OF EVIDENCE: N/A.

Ketogenic diet delays spinal fusion and decreases bone mass in posterolateral lumbar spinal fusion: an in vivo rat model.

BACKGROUND: Ketogenic diet (KD), a low-carbohydrate-and-high-fat diet, causes a metabolic state of ketogenesis and has been used to treat drug-resistance epilepsy. Our recent studies showed KD neuroprotective after spinal cord injury and causing bone loss. Effects of KD on spinal fusion were still unknown. This study was aimed to evaluate effects of KD on spinal fusion in rats. METHODS: Thirty-two Sprague-Dawley rats were randomly divided into KD and standard diet (SD) groups. The KD group was fed with food of 1:4 carbohydrates to fat. All rats were subjected to L4/5 posterolateral lumbar spinal fusion. The blood ketone, and serum calcium, phosphorus, and insulin-like growth factor-1 (IGF-1) were measured, as well as the fusion rates, bone mass (BV), and bone mineral contents (BMC) of fusion sites were estimated at 4 and 8 weeks. RESULTS: There was no significant difference in serum calcium or phosphorus levels between groups at 4 or 8 weeks. However, there was a significant increase of blood ketone (1.02 mmol/L vs 0.38 mmol/L at 4 weeks; 0.83 mmol/L vs 0.32 mmol/L, at 8 weeks) and decrease of serum IGF-1 (339.4 ng/mL vs 630.6 ng/mL at 4 weeks; 418.8 ng/mL vs 628.6 ng/mL, at 8 weeks) in the KD group compared with the SD group. The spinal fusion occurred less in the KD group (1/16 vs 6/16 at 4 weeks; 7/16 vs 10/16, at 8 weeks), particularly at 4 weeks after surgery. The BV and BMC were lower in the KD group than that in the SD group at 4 weeks, but not different between groups at 8 weeks. CONCLUSIONS: This study demonstrated that KD delayed spinal fusion and decreased bone mass in posterolateral lumbar spinal fusion in rats.

A Computed Tomography Study of the C7 Vertebra Screws Fixation in Children.

STUDY DESIGN: Retrospective analysis of computed tomographic images of 92 normal pediatric cervical vertebrae. OBJECTIVE: The aim of this study was to ascertain the feasibility of the pedicle, intralaminar, and lateral mass screws fixation in children. SUMMARY OF BACKGROUND DATA: Fixation techniques of C7 vertebrae are technically challenging in the pediatric population. Although the techniques offer alternatives to screws placement at C7 spine in adult, there is no consensus to apply these screws in children. METHODS: A total of 92 patients (59 males and 33 females) from 3- to 16-year old divided into three groups (group 1, 1-6 years; group2, 7-10 years; group 3, 11-16 years) were included. Measurements were taken through computed tomography (CT) imaging without evidence of spinal trauma. Length, width, and angle of lamina and pedicle were measured, and lateral mass length was also assessed. RESULTS: There was no statistical difference between the left and right sides, as well as the sexes. In 1 to 6 years' age group, 83.3% of the laminas and 87.5% of the pedicles measured have thicknesses of >3.5 mm. In 7 to 10 years' age group, 84.0% of the laminas and all the pedicles observed have thicknesses of >3.5 mm. In 11 to 16 years' age group, 88.4% of the laminas and 97.7% of the pedicles measured have thicknesses of >3.5 mm. Most of the spinolaminar angles were between 42.5° and 56°. There are three cases (3.3%) having lateral mass length >12 mm. CONCLUSION: It is feasible to insert a screw (Ø 3.5 mm) in C7 lamina in children, and the intralaminar screw fixation is a safe and reliable technique. Around 49°, the spinolaminar angle with axial position, can be used as reference for placing screws in the C7 lamina. Preoperative CT scan will help surgeons to insert the laminar screw safely and accurately. LEVEL OF EVIDENCE: N/A.

Radiological Evaluation of Craniocervical Region in Patients with Basilar Invagination.

STUDY DESIGN: A retrospective analysis of collected data. OBJECTIVE: Our study aims to present the morphology of cranial-cervical spinal canal in basilar invagination (BI) patients. SUMMARY OF BACKGROUND DATA: BI is characterized by protrusion of the odontoid process into the foramen magnum (C0), leading to compression of the cervicomedullary junction. However, no study has ever clarified the anatomical diameters of spinal canal in patients with BI. METHODS: The study retrospectively examined computed tomography (CT)-based anatomical characteristics in a cohort of 84 patients with and without BI. We measured the anteroposterior diameter (APD) and transversal diameter (TVD) of spinal canal from C0 to C4, together with the area of vertebral canal (Area). Independent samples t test was used for statistical analysis. RESULTS: The APD in the BI group was shorter than the control group from C0 to C2 (C0: 27.98 vs. 35.11 mm, P < 0.001; C1: 11.87 vs. 16.91 mm, P < 0.001; C2: 12.91 vs. 14.84 mm, P < 0.001), but it became longer from C3 to C4. The TVD of the BI group was significantly wider from C0 to C3 (C0: 30.59 vs. 28.54 mm, P < 0.001; C1: 31.31 vs. 25.98 mm, P < 0.001; C2: 21.56 vs. 20.40 mm, P = 0.01; C3: 22.45 vs. 21.23 mm, P = 0.013), and it had no significance at C4. The Area showed no difference between the two groups from C1 to C2, but it turned larger at C3 and C4 in BI patients. CONCLUSION: BI patients may have shorter APD from C0 to C2, which could be the leading cause of neurological compression, necessitating decompression on sagittal plane. Below the pathological levels, BI patients have larger spinal canal than general population. LEVEL OF EVIDENCE: 3.

Ketogenic Metabolism Inhibits Histone Deacetylase (HDAC) and Reduces Oxidative Stress After Spinal Cord Injury in Rats.

The aim of this study is to investigate the effect of ketogenic metabolism, induced by different diet interventions, on histone acetylation and its potential antioxidant capacity to injured spinal cord tissue in rats. 72 male Sprague-Dawley rats were randomly divided into 4 groups, fed with ketogenic diet (KD), every other day fasting (EODF), every other day ketogenic diet (EODKD) and standard diet (SD) respectively for 2 weeks. ß-Hydroxybutyrate (ßOHB) concentration was measured both in serum and cerebrospinal fluid (CSF). C5 spinal cord tissue was harvested before, at 3 h and 24 h after injury for analysis of HDAC activity, histone acetylation and oxidative makers. All three dietary interventions resulted in a significant increase of ßOHB level in both serum and CSF, and inhibited HDAC activity by 31-43% in spinal cord. Moreover, the expressions of acetylated histone AcH3K9 and AcH3K14 were significantly increased. Anti-oxidative stress genes Foxo3a and Mt2 and related proteins, such as mitochondrial superoxide dismutase (SOD), FOXO3a, catalase were increased in dietary intervention groups. After SCI, high ketogenic metabolism demonstrated significant reduction of the expression of lipid peroxidation factors malondialdehyde (MDA), and this might contribute to the reported neuroprotection of the spinal cord from oxidative damage possibly mediated by increasing SOD. The result of this study suggested that by inhibiting HDAC activity and modifying related gene transcription, ketogenic metabolism, induced by KD, EODF or EODKD, might reduce oxidative damage in the spinal cord tissue after acute injury.