Rationale and Objectives Evaluating and Comparing the Predictive Ability of computed tomography (CT)-based Hounsfield units (HU), Magnetic resonance imaging (MRI)-based vertebral bone quality score (VBQ) and modified VBQ scores for pedicle screw loosening (PSL) in patients with lumbar degenerative diseases. Materials and Methods Retrospective study of patients undergoing transforaminal lumbar interbody fusion (TLIF) for degenerative diseases (Dec 2019 – Jan 2021). Screw loosening was assessed using 12-month postoperative lumbar radiographs and defined by a ≥1 mm radiolucent halo surrounding the screw. Various VBQ scores and HU value were measured using preoperative MRI and CT, respectively. Predictive performance was evaluated using receiver operating characteristic (ROC) curve analysis. Results Among all patients (n = 185), several modified VBQ scores showed higher areas under the curve (AUCs) than the VBQ score (0.776, 95% CI: 0.701–0.851) and CT HU value (0.770, 95% CI: 0.684–0.857), without statistical significance (PAUC>0.05). No significant differences were observed in lumbar disc herniation (LDH) or spondylolisthesis (LS) subgroups. In lumbar spinal stenosis (LSS) patients, the AUC of T1-VBQFAT (0.876, 95% CI: 0.788–0.965) was significantly higher than that of CT HU value (0.718, 95% CI: 0.583–0.852) (PAUC=0.040). Conclusion In the overall cohort and other subgroups, HU values and several modified VBQ scores showed numerically higher AUCs without statistical significance (PAUC>0.05). In the LSS subgroup, T1-VBQFAT demonstrated superior predictive performance compared with HU values (PAUC=0.040); notably, this finding did not remain statistically significant after correction for multiple comparisons (PAUC>0.0026). These exploratory results suggest that T1-VBQFAT may serve as a potential adjunct for predicting PSL in patients with LSS.

Following spinal cord injury (SCI), the transcriptional regulator yes-associated protein 1 (YAP1) is upregulated and accumulates in the nuclei of astrocytes, where it promotes reactive astrogliosis-a process that critically influences wound healing and neurological function recovery. However, the mechanisms regulating YAP1 in reactive astrocytes after SCI remain largely unclear. This study, we identify the E3 ubiquitin ligase NEDD4 as a critical regulator of astrocyte reactive proliferation. NEDD4 enhances astrogliosis by suppressing YAP1 degradation. Conditional deletion of Nedd4 in astrocytes markedly attenuates reactive astrogliosis in vivo, and results in heightened inflammation, exacerbated neuronal injury, and impaired functional recovery following SCI. Importantly, YAP1 overexpression is sufficient to reverse the pathological and functional consequences of Nedd4 deficiency. Mechanistically, NEDD4 interacts with YAP1 and mediates K63-linked ubiquitination at lysine 254, thereby preventing its degradation via the chaperone-mediated autophagy (CMA) pathway involving HSC70. Furthermore, we demonstrate that the ROS-FOXM1 signaling cascade drives NEDD4 expression, thereby stabilizing YAP1 and promoting astrocyte proliferation. In summary, our findings underscore the pivotal role of the ROS-FOXM1-NEDD4-YAP1 signaling cascade in controlling astrocytic activation and tissue regeneration post-SCI, positioning NEDD4 as a viable target to regulate astrogliosis and facilitate neurological restoration after SCI.

BACKGROUND:Sleep disturbance delays surgical pain recovery. This impact is associated with gene dysregulation in the dorsal root ganglion (DRG) and spinal dorsal horn (SDH). However, the mechanisms underlying this dysregulation remain unclear. METHODS:Expression of ten-eleven translocation methylcytosine dioxygenase 1 (TET1) was examined in the DRG and SDH following plantar incision in rats subjected to short-term rapid eye movement sleep disturbance. A herpes simplex virus expressing Tet1 mRNA (HSV-TET1) was microinjected into the ipsilateral L4 and L5 DRGs or SDH, and its effects on μ-opioid receptor (MOR) expression, TET1 binding to the Oprm1 promoter, and promoter-associated 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) levels in microinjected regions were assessed. Pain behaviours were evaluated, and the effect of Tet1 siRNA microinjection, with or without a lentivirus expressing Oprm1 mRNA (LV-MOR), on MOR expression in microinjected DRGs and SDH and nociceptive threshold in naïve rats was assessed. RESULTS:Short-term rapid eye movement sleep disturbance downregulated TET1 in the ipsilateral L4 and L5 DRGs and SDH and prolonged incisional pain. HSV-TET1 microinjection restored MOR expression, TET1 binding activity to the Oprm1 promoter, and 5hmC levels at the promoter, while reducing 5mC accumulation, in microinjected L4 and L5 DRGs or SDH and prevented short-term rapid eye movement sleep disturbance-induced prolongation of incisional pain. Conversely, Tet1 siRNA microinjection reduced MOR expression in microinjected L4 and L5 DRGs or SDH and induced nociceptive hypersensitivity, effects abolished by LV-MOR co-microinjection. CONCLUSIONS:TET1 downregulation is required for short-term sleep disturbance to delay surgical pain recovery, likely by reducing μ-opioid receptor expression in the dorsal root ganglion and spinal dorsal horn.

Sensitization of spinal nociceptive circuits plays a crucial role in neuropathic pain. This sensitization depends on new gene expression that is primarily regulated via transcriptional and translational control mechanisms. The relative roles of these mechanisms in regulating gene expression in the clinically relevant chronic phase of neuropathic pain are not well understood. Here, we show that, in mice, changes in gene expression in the spinal cord during the chronic phase of neuropathic pain are substantially regulated at the translational level. Downregulating spinal translation at the chronic phase alleviated pain hypersensitivity. Cell type-specific profiling revealed that spinal inhibitory and excitatory neurons exhibited substantial changes in translation after peripheral nerve injury. Notably, increasing translation selectively in all inhibitory neurons or parvalbumin-positive (PV+) interneurons, but not excitatory neurons, promoted mechanical pain hypersensitivity. Furthermore, increasing translation in PV+ neurons decreased their intrinsic excitability and spiking activity. Conversely, reducing translation in spinal PV+ neurons prevented the nerve injury-induced decrease in excitability but did not alleviate mechanical hypersensitivity. Together, these findings advance our understanding of translational control mechanisms in the spinal cord during neuropathic pain and highlight their cell type- and phase-specific contributions to gene expression and pain hypersensitivity.

Spinal cord injury (SCI) is a debilitating disorder characterized by intricate pathological processes that result in severe motor and sensory deficits. Existing therapeutic approaches remain insufficient to achieve comprehensive functional restoration, indicating the necessity of alternative treatment strategies. In this study, an advanced nanoparticle-based drug delivery system was established using extracellular vesicles (EVs) modified with a matrix metalloproteinase (MMP)-responsive peptide, ACPP, to achieve the targeted delivery of paclitaxel (PTX). The ACPP-EVs@PTX formulation integrates the drug loading capacity of EVs, the lesion-targeting capability conferred by ACPP, and the neuroprotective properties of PTX. Enhanced accumulation of PTX at the SCI lesion site was achieved, accompanied by a reduction in the off-target distribution. Both in vitro and in vivo experiments demonstrated marked therapeutic efficacy of ACPP-EVs@PTX through modulation of the SCI microenvironment, including stimulation of angiogenesis, attenuation of inflammatory responses, alleviation of oxidative stress, and promotion of axonal regeneration. In addition, the activation of PINK1-Parkin-mediated mitophagy was observed, leading to improved mitochondrial function and enhanced neuronal repair. Behavioral evaluations further confirmed significant recovery of neurological function, supporting the translational potential of this multitarget, synergistic therapeutic strategy. Collectively, this work establishes an integrated therapeutic strategy for spinal cord repair and supports its translational potential.

Nerve injury-induced gene dysregulation in the dorsal root ganglion (DRG) is considered a key molecular basis for neuropathic pain genesis. Circular RNA is emerging as a critical regulator of gene expression. Here, we reported a novel circular RNA circNrip1 formed by back-splicing from exon 3 to exon 2 of the Nrip1 pre-RNA. Peripheral nerve injury upregulates circNrip1, but not Nrip1 mRNA, in injured DRG neurons, at least in part due to increased binding of the RNA-binding protein FUS to Nrip1 pre-RNA, thereby promoting circNrip1 formation. Blocking this upregulation attenuates nerve injury-induced increases in toll-like receptor 2 (Tlr2) mRNA and TLR2 protein levels in injured DRG, as well as nerve injury-induced nociceptive hypersensitivity. Conversely, mimicking this upregulation elevates DRG Tlr2 mRNA and TLR2 protein expression and produces neuropathic pain-like symptoms in naïve mice. Mechanistically, upregulated circNrip1 enhances its binding to the 3'- untranslated region (UTR) of Tlr2 mRNA and to the RNA-binding protein SYNCRIP, thereby recruiting more SYNCRIP to the Tlr2 mRNA 3'-UTR and stabilizing Tlr2 mRNA in injured DRG neurons. Thus, circNrip1 contributes to neuropathic pain by promoting SYNCRIP-triggered stabilization of TLR2 in DRG neurons and represents a promising therapeutic target for intervention.

The hypothalamus is critical for regulating behaviors essential for survival and locomotion, but how it integrates internal needs and transmits locomotion commands to the spinal cord (SC) remains unclear. We found that glutamatergic neurons in lateral hypothalamic area (LHA) are essential for regulating motivated locomotor activity. Using single-neuron projectome analysis, trans-synaptic tracing, and optogenetic manipulation, we showed that LHA facilitates motivated locomotion during food seeking via pontine reticular nucleus, oral part (PnO) projection neurons, rather than direct SC projections or indirect stress signaling via medial septum and diagonal band. Activating PnO-SC projection neurons also initiated locomotion. Importantly, LHA-PnO projection neurons were crucial for regulating locomotor recovery following mouse spinal cord injury (SCI). Motor cortex signals gated LHA deep brain stimulation treatment markedly promoted long-term restoration of hindlimb motor functions after severe SCI. Thus, we have identified a hypothalamic-pontine-spinal pathway and the stimulation paradigm for potential therapeutic intervention after SCI.

Mice lacking two neuropeptides thought to be essential for processing pain show no change in how they respond to a wide range of harmful stimuli.

Retrospective cohort. The aim of this study is to evaluate the potential of using MRI-based Vertebral Bone Quality (VBQ) scores and CT-derived Hounsfield Units (HU) as predictive tools for cervical screw loosening (CSL) in patients with anterior cervical discectomy and fusion (ACDF). Additionally, it analyzes the relationship between VBQ scores, HU values, and DXA T scores. A total of 112 patients who underwent anterior cervical discectomy and fusion (ACDF) between December 2016 and January 2023 were followed for over a year and divided into CSL and non-CSL. The radiological and clinical parameters investigated included age, sex, body mass index, diabetes, VBQ scores, HU values, and DXA T scores. Among the 112 patients included in the study, 18 patients developed cervical screw loosening postoperatively (16.07

Spinal microglia play a pivotal role in the development of neuropathic pain. Peripheral nerve injury induces changes in the transcriptional profile of microglia, including increased expression of components of the translational machinery. Whether microglial protein synthesis is stimulated following nerve injury and has a functional role in mediating pain hypersensitivity is unknown. Here, we show that nascent protein synthesis is upregulated in spinal microglia following peripheral nerve injury in both male and female mice. Stimulating mRNA translation in microglia by selectively ablating the translational repressor eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) promoted the transition of microglia to a reactive state and induced mechanical hypersensitivity in both sexes, whereas spontaneous pain was increased only in males. Conversely, inhibiting microglial translation by expressing a mutant form of 4E-BP1 in microglia attenuated their activation following peripheral nerve injury and alleviated neuropathic pain in both sexes. Thus, stimulating 4E-BP1-dependent translation promotes microglial reactivity and mechanical hypersensitivity, whereas inhibiting it alleviates neuropathic pain.

Ferroptosis is one of the cell death programs occurring after spinal cord injury (SCI) and is driven by iron-dependent phospholipid peroxidation. However, little is known about its underlying regulation mechanism. The present study demonstrated that lipid peroxidation was promoted in patients with SCI. Neurons affected by ferroptosis following SCI had a high expression of ferroptotic protein ACSL4. The E3 SUMOylase TRIM28 promoted neuronal ferroptosis by enhancing ACSL4 expression. Genetic deletion of Trim28 significantly attenuated neuronal ferroptosis and improved mouse hindlimb motor function following SCI. In contrast, mice with Trim28 overexpression demonstrated poor neurological function after SCI, which was attenuated by ferroptosis inhibitor Liproxstatin-1. Mechanistically, TRIM28 bound to ACSL4, promoted SUMO3 modification at lysine (K) 532, and inhibited K63-linked ACSL4 ubiquitination, thereby suppressing OPTN-dependent autophagic degradation. Additionally, SENP3 was identified as the deSUMOylation enzyme that can reverse this process and compete with TRIM28, which was transcriptionally upregulated due to excessive oxidative stress. These data unveiled a mechanism by which TRIM28-mediated SUMOylation regulated neuronal ACSL4 levels and ferroptosis, identified interactions and correlations involved in ACSL4 SUMOylation, ubiquitination, and autophagic degradation, and discovered a positive feedback loop where oxidative stress transcriptionally upregulated Trim28, and conversely TRIM28 promoted ferroptosis and oxidative stress. Notably, screening of the FDA-approved drug library revealed that pharmacological TRIM28/ACSL4 axis interventions with Rutin hydrate inhibited neuronal ferroptosis and improved hindlimb motor function in mice after SCI, thus providing a promising therapeutic strategy for its treatment.

Spinal cord injury (SCI) is a condition with severe consequences, and its treatment poses significant challenges. The local inflammatory microenvironment and neuronal death following SCI are primary factors contributing to recovery difficulties. In this study, we combined exosomes that inhibit the inflammatory response with deferoxamine, a compound that chelates excessive iron ions and mitigates ferroptosis, using methacrylated chitosan as a scaffold. These two factors were integrated through two distinct methods: ionic interaction and covalent binding, allowing for the sustained release of both components. Our in vivo and in vitro results indicate that this hydrogel scaffold can enhance the inflammatory microenvironment post injury and diminish neuronal death by inhibiting ferroptosis. Consequently, it can facilitate tissue and functional recovery after SCI, presenting a novel and viable strategy for SCI repair.

Spinal cord injury (SCI) causes high morbidity, disability, and mortality, while current surgical and pharmacological treatments provide limited benefit. Ferroptosis, a newly recognized form of regulated cell death, contributes critically to SCI pathology, and targeting this process may enhance neuronal survival. Extracellular vesicles, key mediators of intercellular communication, are emerging as promising therapeutic agents for central nervous system injury. Here, we examined the role of athlete-derived plasma extracellular vesicles (AEVs) in neuronal ferroptosis and motor function recovery after SCI. In a murine model, AEVs markedly inhibited ferroptosis and improved motor outcomes. Mechanistically, AEVs delivered RNF216, which promoted ubiquitination and degradation of NOX1, thereby reducing ferroptotic damage and facilitating recovery. Moreover, RNF216-enriched vesicles enhanced synaptic plasticity, supporting neuronal regeneration and network reestablishment. These findings reveal a previously unrecognized RNF216-NOX1 axis in SCI and highlight AEVs as a previously unidentified therapeutic strategy.

Fibromyalgia is a prevalent syndrome characterized by widespread pain in the absence of evident tissue injury or pathology, making it one of the most mysterious chronic pain conditions. The composition of the gut microbiota in individuals with fibromyalgia differs from that of healthy controls, but its functional role in the syndrome is unknown. Here, we show that fecal microbiota transplantation from fibromyalgia patients, but not from healthy controls, into germ-free mice induces pain and numerous molecular phenotypes that parallel known changes in fibromyalgia patients, including immune activation and metabolomic profile alterations. Replacing the fibromyalgia microbiota with a healthy microbiota substantially alleviated pain in mice. An open-label trial in women with fibromyalgia (Registry MOH_2021-11-04_010374) showed that transplantation of a healthy microbiota is associated with reduced pain and improved quality of life. We conclude that altered gut microbiota has a role in fibromyalgia pain, highlighting it as a promising target for therapeutic interventions.

Astrocytic metabolic reprogramming is an adaptation of metabolic patterns to meet increased energy demands, although the role after spinal cord injury (SCI) remains unclear. Analysis of single-cell RNA sequencing (scRNA-seq) data identified an increase in astrocytic glycolysis, while PFKFB3, a key regulator of glycolytic flux, was significantly upregulated following SCI. Loss of PFKFB3 in astrocytes prohibited neuronal energy supply and enhanced neuronal ferroptosis in vitro and expanded infiltration of CD68+ macrophages/microglia, exacerbated neuronal loss, and hindered functional recovery in vivo after SCI. Mechanistically, deubiquitinase UCHL1 plays a crucial role in stabilizing and enhancing PFKFB3 expression by cleaving K48-linked ubiquitin chains. Genetic deletion of Uchl1 inhibited locomotor recovery after SCI by suppression of PFKFB3-induced glycolytic reprogramming in astrocytes. Furthermore, the UCHL1/PFKFB3 axis increased lactate production, leading to enhanced histone lactylation and subsequent transcription of Uchl1 and several genes related to glycolysis, suggesting a glycolysis/H4K8la/UCHL1 positive feedback loop. These findings help to clarify the role of the UCHL1/PFKFB3/H4K8la loop in modulation of astrocytic metabolic reprogramming and reveal a potential target for treatment of SCI.

Fragile X syndrome is caused by the loss of the Fmr1 gene expression. Deletion of Fmr1 in various neuronal and non-neuronal subpopulations in the brain of mice leads to cell-type-specific effects. Microglia, immune cells critical for the refinement of neuronal circuits during brain development, have been implicated in various neurodevelopmental disorders, including fragile X syndrome. However, it is unknown whether reduced Fmr1 expression in microglia leads to molecular and behavioral phenotypes. We downregulated Fmr1 in microglia during early and late postnatal development and studied the effect on microglial morphology and distinct behaviours. Female, but not male, adult mice with downregulation of Fmr1 in microglia during early development exhibited reactive microglia and behavioral phenotypes, including enhanced self-grooming and alterations in social interaction. Downregulation of Fmr1 in microglia during late development induced a milder phenotype, characterized by impaired preference for social novelty without affecting microglia morphology. The downregulation of Fmr1 and its encoded protein FMRP in microglia contributes to behavioural phenotypes in a sex-specific manner.

Crosstalk between the central nervous system (CNS) and the immune system has recently gained increased attention; however, the interaction between innate and adaptive immunity after CNS injury remains unclear. Here, using single-cell RNA sequencing, we identified accumulation of CD8+ T lymphocytes in the cerebrospinal fluid of patients with spinal cord injury (SCI) and in spinal cords of injured mice, thus indicating poor neurological function. Furthermore, through genetic or pharmacologic interruption strategies, we found that CXCL16 chemokines derived from injury-activated microglia and macrophages (IAMs) recruited CXCR6+CD8+ T cells and further contributed to neuronal loss after SCI. Mechanistically, glycolytic reprogramming in IAMs enhanced histone-lactylation-mediated Cxcl16 transcription, whereas suppressing glycolysis through Pkm2 deletion partially reversed this effect. Notably, a pharmacologic intervention targeting the CXCL16-CXCR6 axis with Rutin promoted locomotor restoration after SCI. Our study highlights the crucial role of glycolytically reprogrammed IAM-derived CXCL16 chemokines in modulating a maladaptive innate/adaptive immune axis and reveals several potential therapeutic strategies.

The fluorescent non-canonical amino acid tagging (FUNCAT) technique has been used to visualize newly synthesized proteins in cell lines and tissues. Here, we present a protocol for measuring protein synthesis in specific cell types in the mouse brain using in vivo FUNCAT. We describe steps for metabolically labeling newly synthesized proteins with azidohomoalanine, which introduces an azide group into the polypeptide. We then detail procedures for binding a fluorophore-conjugated alkyne to the azide group to allow its visualization. For complete details on the use and execution of this protocol, please refer to tom Dieck et al. (2012)1 and Hooshmandi et al. (2023).2

A novel H-shaped miniplate (HSM) was specifically designed for restorative laminoplasties to restore patients’ posterior elements after laminectomies. A validated finite element (FE) model of L2/4 was utilized to create a laminectomy model, as well as three restorative laminoplasty models based on the fixation of different miniplates after a laminectomy (the RL-HSM model, the RL-LSM model, and the RL-THM model). The biomechanical effects of motion and displacement on a laminectomy and restorative laminoplasty with three different shapes for the fixation of miniplates were compared under the same mechanical conditions. This study aimed to validate the biomechanical stability, efficacy, and feasibility of a restorative laminoplasty with the fixation of miniplates post laminectomy. The laminectomy model demonstrated the greatest increase in motion and displacement, especially in axial rotation, followed by extension, flexion, and lateral bending. The restorative laminoplasty was exceptional in preserving the motion and displacement of surgical segments when compared to the intact state. This preservation was particularly evident in lateral bending and flexion/extension, with a slight maintenance efficacy observed in axial rotation. Compared to the laminectomy model, the restorative laminoplasties with the investigated miniplates demonstrated a motion-limiting effect for all directions and resulted in excellent stability levels under axial rotation and flexion/extension. The greatest reduction in motion and displacement was observed in the RL-HSM model, followed by the RL-LSM model and then the RL-THM model. When comparing the fixation of different miniplates in restorative laminoplasties, the HSMs were found to be superior to the LSMs and THMs in maintaining postoperative stability, particularly in axial rotation. The evidence suggests that a restorative laminoplasty with the fixation of miniplates is more effective than a conventional laminectomy due to the biomechanical effects of restoring posterior elements, which helps patients regain motion and limit load displacement responses in the spine after surgery, especially in axial rotation and flexion/extension. Additionally, our evaluation in this research study could benefit from further research and provide a methodological and modeling basis for the design and optimization of restorative laminoplasties.