Dynamics of N6-methyladenosine modification during aging and their potential roles in the degeneration of intervertebral disc.

Background: The N6-methyladenosine (m6A) dynamics in the progression of intervertebral disc (IVD) aging remain largely unknown. This study aimed to explore the distribution and pattern of m6A modification in nucleus pulpous (NP) tissues of rats at different ages. Methods: Histological staining and MRI were performed to evaluate the degeneration of IVD. The expression of m6A modifiers was analyzed using qRT-PCR and western blot. Subsequently, methylated RNA immunoprecipitation next generation sequencing and RNA-seq were conducted to identify differences in m6A methylome and transcriptome of NP tissues. Results: Compared to 2-month-old rats, we found significant changes in the global m6A level and the expression of Mettl3 and FTO in NP tissues from 20-month-old rats. During the progression of NP aging, there were 1126 persistently differentially m6A peaks within 931 genes, and 51 persistently differentially expressed genes. GO and KEGG analyses showed that these m6A peaks and m6A modified genes were mainly engaged in the biological processes and pathways of intervertebral disc degermation (IDD), such as extracellular matrix metabolism, angiogenesis, inflammatory response, mTOR and AMPK signaling pathways. Meanwhile, conjoint analyses and Venn diagram revealed a total of 405 aging related genes contained significant methylation and expression levels in 20-month-old rats in contrast to 2-month-old and 10-month-old rats. Moreover, it was found that four aging related genes with hypermethylated modification including BUB1, CA12, Adamts1, and Adamts4 depicted differentially expressed at protein level, of which BUB1 and CA12 were decreased, while Adamts1 and Adamts4 were increased during the progression of NP aging. Conclusion: Collectively, this study elucidated the distribution and pattern of m6A modification during the aging of IVD. Furthermore, the m6A modified genes were involved in the IDD related biological processes and pathways. These findings may provide novel insights into the mechanisms and therapies of IDD from the perspective of aging.

Lactic acid promotes nucleus pulposus cell senescence and corresponding intervertebral disc degeneration via interacting with Akt.

The accumulation of metabolites in the intervertebral disc is considered an important cause of intervertebral disc degeneration (IVDD). Lactic acid, which is a metabolite that is produced by cellular anaerobic glycolysis, has been proven to be closely associated with IVDD. However, little is known about the role of lactic acid in nucleus pulposus cells (NPCs) senescence and oxidative stress. The aim of this study was to investigate the effect of lactic acid on NPCs senescence and oxidative stress as well as the underlying mechanism. A puncture-induced disc degeneration (PIDD) model was established in rats. Metabolomics analysis revealed that lactic acid levels were significantly increased in degenerated intervertebral discs. Elimination of excessive lactic acid using a lactate oxidase (LOx)-overexpressing lentivirus alleviated the progression of IVDD. In vitro experiments showed that high concentrations of lactic acid could induce senescence and oxidative stress in NPCs. High-throughput RNA sequencing results and bioinformatic analysis demonstrated that the induction of NPCs senescence and oxidative stress by lactic acid may be related to the PI3K/Akt signaling pathway. Further study verified that high concentrations of lactic acid could induce NPCs senescence and oxidative stress by interacting with Akt and regulating its downstream Akt/p21/p27/cyclin D1 and Akt/Nrf2/HO-1 pathways. Utilizing molecular docking, site-directed mutation and microscale thermophoresis assays, we found that lactic acid could regulate Akt kinase activity by binding to the Lys39 and Leu52 residues in the PH domain of Akt. These results highlight the involvement of lactic acid in NPCs senescence and oxidative stress, and lactic acid may become a novel potential therapeutic target for the treatment of IVDD.

The sonic hedgehog pathway suppresses oxidative stress and senescence in nucleus pulposus cells to alleviate intervertebral disc degeneration via GPX4.

Disruption of intervertebral disc (IVD) homeostasis caused by oxidative stress and nucleus pulposus cell (NPC) senescence is a main cause of intervertebral disc degeneration (IDD). The sonic hedgehog (Shh) pathway plays an important role in IVD development, but its roles in IDD are unknown. This study aimed to investigate the effects of the Shh pathway on the alleviation of IDD and the related mechanisms. In vivo, the effect of the Shh pathway on IVD homeostasis was studied by intraperitoneal injection of recombinant Shh (rShh) and GANT61 based on puncture-induced IDD. GANT61, lentivirus-coated sh-Gli1 and rShh were used to investigate the role and mechanism of the Shh pathway in NPCs based on senescence induced by Braco19 and oxidative stress induced by TBHP. Shh pathway expression decreased, and senescence and oxidative stress increased with age. Intraperitoneal injection of rShh activated the Shh pathway to suppress oxidative stress and NPC senescence and consequently alleviated needle puncture-induced IDD. In vitro, the Shh pathway upregulated glutathione peroxidase 4 (GPX4) expression to suppress oxidative stress and senescence in NPCs. Moreover, GPX4 suppression in NPCs by si-GPX4 significantly reduced the protective effect of the Shh pathway on oxidative stress and senescence in NPCs. Our results demonstrate for the first time that the Shh pathway plays a key role in the alleviation of IDD by suppressing oxidative stress and cell senescence in NP tissues. This study provides a new potential target for the prevention and reversal of IDD.

Enhanced recovery after microdiscectomy: reductions in opioid use, length of stay and cost.

BACKGROUND: Enhanced recovery after surgery (ERAS) protocols are widely used worldwide. Recently, studies of the ERAS program in spinal surgery subspecialties have been reported. The aim of this study was to evaluate the impacts of ERAS in minimally invasive microdiscectomy (MD) surgery. METHODS: This was a retrospective cohort study of patients undergoing MD at a single center. From March 2018 to March 2021, 286 patients were in the ERAS group. A total of 140 patients from March 2017 to February 2018 were in the conventional group. The outcomes included length of stay (LOS), the postoperative numeric rating scale (NRS), complications, 30-day readmission rate, 30-day reoperation rate and cost. Moreover, perioperative factors were also evaluated. RESULTS: Compared with the conventional group, the LOS and cost were reduced in the ERAS group. There were no significant differences in the NRS, complication rate, 30-day readmission or reoperation rates between the groups. Furthermore, postoperative drainage volume, and postoperative opioid use were lower in the ERAS group. CONCLUSIONS: The ERAS protocol for MD surgery reduces LOS, cost and opioid use and accelerates patient recovery.

Gli1 depletion induces oxidative stress and apoptosis of nucleus pulposus cells via Fos in intervertebral disc degeneration.

Background: Intervertebral disc degeneration (IDD) is the most common chronic disease. Oxidative stress and apoptosis of nucleus pulposus (NP) cells disrupt intervertebral disc (IVD) homeostasis, which is the main cause of IDD. Glioma-associated oncogene 1 (Gli1) is an important transcription factor in the Hedgehog (Hh) pathway. Depletion of Gli1 accelerates the occurrence and development of degenerative diseases. This study aimed to explore the role of aging related Gli1 depletion in the progression of IDD. Methods: The relationship between aging related Gli1 depletion and IDD was studied in the NP tissues of human and rats of different ages, and the levels of oxidative stress and NP cell apoptosis during IDD were explored. Gli1 depletion of NP cells were established by targeting inhibitor GANT61 or lentivirus-coated Gli1 sh-RNA (sh-Gli1) to explore the role of Gli1 in NP cells and underlying mechanism. Exogenous Gli1 depletion induced IDD of rats was established by intraperitoneal injection of GANT61. Also, the roles of Fos in the Gli1 depletion induced NP cell oxidative stress, apoptosis and IDD were investigated. Results: Gli1 was down-regulated in the tissues of degenerative NP, and the level of Gli1 was negatively correlated with the severity of aging related IDD in human and rats. Furthermore, we found enhanced oxidative stress and apoptosis in degenerative NP tissues. Gli1 depletion promoted oxidative stress and apoptosis of NP cells and resulted in the degradation of extracellular matrix (ECM) and decreased ECM synthesis. Transcriptome sequencing showed that Gli1 depletion caused Fos activation in NP cells. the effect of Gli1 depletion on the oxidative stress and apoptosis of NP cells were retarded by Fos inhibitor. In vivo, Fos inhibition alleviated the IDD induced by exogenous Gli1 depletion. Conclusions: This study revealed for the first time that Gli1 is gradually depleted in NP with IDD progression. Exogenous Gli1 depletion causes oxidative stress and apoptosis of NP cells both in vivo and in vitro. Fos suppression effectively retards the destructive effects of Gli1 depletion on IVD homoeostasis.The translational potential of this article: This study may provide new potential targets for preventing and reversing IDD. Maintaining Gli1 expression in NP and suppressing Fos activation may be an effective treatment strategy for IDD.

Application of metagenomic next-generation sequencing in the detection of pathogens in spinal infections.

BACKGROUND CONTEXT: The precise diagnosis and treatment of spinal infections (SI) remains challenging for spine surgeons. Identifying the pathogens of SI through metagenomic next-generation sequencing (mNGS) may be a key approach to addressing this challenge. PURPOSE: To evaluate the accuracy and applicability of mNGS in determining the etiology of SI. STUDY DESIGN: Diagnostic test study. PATIENT SAMPLE: Twenty-five patients who had a clinical suspicion of SI and underwent mNGS testing. OUTCOME MEASURES: The specificity, sensitivity, and time cost of mNGS and bacterial culture were compared. Clinical outcomes were assessed using the numeric rating scale (NRS) score, Oswestry Disability Index (ODI), and the Japanese Orthopedic Association (JOA) score. Demographic data and laboratory results (blood cell count (WBC), erythrocyte sedimentation rate (ESR), neutrophil percentage (NEUT%), and C-reactive protein level (CRP) were also evaluated. METHODS: In this retrospective study, samples were obtained from 25 eligible patients via surgery or CT-guided puncture and subjected to histopathological examination, bacterial culture, and mNGS. The sensitivity and specificity of the bacterial cultures and mNGS were calculated with respect to the histopathological results as a reference. Postoperative antibiotics or antituberculosis drugs were administered on the basis of mNGS results, combined with clinical manifestations, imaging examination, and histopathology. The changes of clinical outcomes and laboratory results after treatment were observed. RESULTS: Of the 25 patients, 21 had a positive pathology, of which 10 showed a tuberculous pathology, and the remaining 11 showed a nontuberculous inflammatory pathology. The sensitivity of mNGS was higher than that of the bacterial culture. However, the difference in specificity between bacterial culture and mNGS was not significant. Moreover, the time needed to perform mNGS was significantly lower than that of bacterial culture and pathology. All patients were followed up for more than three months, and CRP and NEUT% significantly decreased by three months after treatment. There was no significant difference in WBC and ESR. The ODI, NRS and JOA scores were significantly improved after treatment. CONCLUSION: Metagenomic next-generation sequencing technology can play an important role in the detection of pathogens in SI and should be further investigated and applied in future studies.

An enhanced recovery after surgery pathway: LOS reduction, rapid discharge and minimal complications after anterior cervical spine surgery.

BACKGROUND: Enhance recovery after surgery (ERAS) is a new and promising paradigm for spine surgery. The purpose of this study is to investigate the effectiveness and safety of a multimodal and evidence-based ERAS pathway to the patients undergoing anterior cervical discectomy and fusion (ACDF). METHODS: The patients treated with the ACDF-ERAS pathway were compared with a historical cohort of patients who underwent ACDF before ERAS pathway implementation. Primary outcome was length of stay (LOS). Secondary outcomes included cost, MacNab grading, complication rates and 90-day readmission and reoperation. And perioperative factors and postoperative complications were reviewed. RESULTS: The ERAS protocol was composed of 21 components. More patients undergoing multi-level surgery (n ≥ 3) were included in the ERAS group. The ERAS group showed a shorter LOS and a lower cost than the conventional group. The postoperative satisfaction of patients in ERAS group was better than that in conventional group. In addition, the rate of overall complications was significantly higher in the conventional group than that in the ERAS group. There were no significant differences in operative time, postoperative drainage, or 90-day readmission and reoperation. CONCLUSIONS: The ACDF-tailored ERAS pathway can reduce LOS, cost and postoperative complications, and improve patient satisfaction without increasing 90-day readmission and reoperation.

Roles of multimodal intra-operative neurophysiological monitoring (IONM) in percutaneous endoscopic transforaminal lumbar interbody fusion: a case series of 113 patients.

BACKGROUND: Despite the wide use of intraoperative neurophysiological monitoring (IONM) in spinal surgeries, the efficacy of IONM during percutaneous endoscopic transforaminal lumbar interbody fusion (PE-TLIF) surgery in detecting postoperative neurological deficits has not been well characterized. METHODS: MIONM data from 113 consecutive patients who underwent PE-TLIF surgeries between June 2018 and April 2020 were retrospectively reviewed. Postoperative neurological deficits were documented and analyzed, and the efficacy and specificity of various IONM techniques were compared. RESULTS: Of the 113 consecutive patients, 12 (10.6%) with IONM alerts were identified. The MIONM sensitivity and specificity were 100 and 96.2%, respectively. The frequency of neurological complications, including minor deficits, was 6.2% (n = 7); all of the neurological complications were temporary. The ability of single IONM modalities to detect neurological complications varied between 25.0 and 66.6%, whereas that of all modalities was 100%. CONCLUSIONS: MIONM is more effective and accurate than unimodal monitoring in assessing nerve root function during PE-TLIF surgeries, reducing both neurological complications and false-negative findings. We recommend MIONM in PE-TLIF surgeries.

Cartilage intermediate layer protein affects the progression of intervertebral disc degeneration by regulating the extracellular microenvironment (Review).

Intervertebral disc degeneration (IDD), which is caused by multiple factors, affects the health of individuals and contributes to low back pain. The pathology of IDD is complicated, and changes in the extracellular microenvironment play an important role in promoting the process of degeneration. Cartilage intermediate layer protein (CILP) is a matrix protein that resides in the middle of human articular cartilage and is involved in numerous diseases that affect cartilage. However, there is no detailed review of the relationship between CILP and degenerative disc disease. Growing evidence has revealed the presence of CILP in the extracellular microenvironment of intervertebral discs (IVDs) and has suggested that there is a gradual increase in CILP in degenerative discs. Specifically, CILP plays an important role in regulating the metabolism of the extracellular matrix (ECM), an important component of the extracellular microenvironment. CILP can combine with transforming growth factor­ß or insulin­like growth factor­1 to regulate the ECM synthesis of IVDs and influence the balance of ECM metabolism, which leads to changes in the extracellular microenvironment to promote the process of IDD. It may be possible to show the correlation of CILP with IDD and to target CILP to interfere with IDD. For this purpose, in the present study, the current knowledge on CILP was summarized and a detailed description of CILP in discs was provided.

A positive feedback loop between EZH2 and NOX4 regulates nucleus pulposus cell senescence in age-related intervertebral disc degeneration.

BACKGROUND: The senescence of nucleus pulposus (NP) cells plays a vital role in the pathogenesis of intervertebral disc (IVD) degeneration (IDD). NADPH oxidase 4 (NOX4)-associated oxidative stress has been shown to induce premature NP cell senescence. Enhancer of zeste homolog 2 (EZH2) is a crucial gene regulating cell senescence. The aim of this study was to investigate the roles of EZH2 in NOX4-induced NP cell senescence and a feedback loop between EZH2 and NOX4. RESULTS: The down-regulation of EZH2 and the up-regulation of NOX4 and p16 were observed in the degenerative discs of aging rats. EZH2 regulated NP cell senescence via the H3K27me3-p16 pathway. Also, EZH2 regulated the expression of NOX4 in NP cells through the histone H3 lysine 27 trimethylation (H3K27me3) in the promoter of NOX4 gene. Furthermore, NOX4 down-regulated EZH2 expression in NP cells via the canonical Wnt/ß-catenin pathway. CONCLUSIONS: A positive feedback loop between EZH2 and NOX4 is involved in regulating NP cell senescence, which provides a novel insight into the mechanism of IDD and a potential therapeutic target for IDD.

Molecular basis of degenerative spinal disorders from a proteomic perspective (Review).

Intervertebral disc degeneration (IDD) and ligamentum flavum hypertrophy (LFH) are major causes of degenerative spinal disorders. Comparative and proteomic analysis was used to identify differentially expressed proteins (DEPs) in IDD and LFH discs compared with normal discs. Subsequent gene ontology term enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of the DEPs in human IDD discs or LFH samples were performed to identify the biological processes and signaling pathways involved in IDD and LFH. The PI3K­AKT signaling pathway, advanced glycation endproducts­receptor for advanced glycation endproducts signaling pathway, p53 signaling pathway, and transforming growth factor­b signaling pathway were activated in disc degeneration. This review summarizes the recently identified DEPs, including prolargin, fibronectin 1, cartilage intermediate layer protein, cartilage oligomeric matrix protein, and collagen types I, II and IV, and their pathophysiological roles in degenerative spinal disorders, and may provide a deeper understanding of the pathological processes of human generative spinal disorders. The present review aimed to summarize significantly changed proteins in degenerative spinal disorders and provide a deeper understanding to prevent these diseases.

Establishment and Implementation of an Enhanced Recovery After Surgery (ERAS) Pathway Tailored for Minimally Invasive Transforaminal Lumbar Interbody Fusion Surgery.

PURPOSE: The concept of enhanced recovery after surgery (ERAS) spread to different surgical specialties to minimize surgical stress response and to reduce length of hospital stay (LOS) and cost. Recently, several studies have reported experience with the ERAS program for spine surgery. The aim of this study is to introduce the establishment and implementation of the ERAS pathway for minimally invasive surgery (MIS) transforaminal lumbar interbody fusion (TLIF). METHODS: A multidisciplinary ERAS team was created to develop and implement a multimodal and evidence-based ERAS protocol for patients undergoing MIS-TLIF at a single spine center from January 2018. Fourty four cases in the ERAS group were compared with a historical cohort of 30 cases (from January 2017 to December 2017) who underwent MIS-TLIF before the pathway implementation (pre-ERAS group). We reviewed the compliance with ERAS components. The primary outcome was LOS. The secondary outcomes included 30-day readmission rate, 30-day reoperation rate, and financial cost. Perioperative factors and perioperative complications were also assessed. RESULTS: The protocol was composed of 11 ERAS components. The ERAS group showed high compliance with the ERAS program. The ERAS group manifested shorter LOS and lower cost compared with the the pre-ERAS group. There were no significant differences in complication rate, 30-day readmission and reoperation rates. Furthermore, the blood loss, operative time, intraoperative fluid infusion and postoperative drainage of the ERAS group decreased. CONCLUSIONS: Our ERAS program tailored for MIS-TLIF is able to reduce LOS and cost with minimal complications. The ERAS pathway expedites recovery in patients undergoing lumbar spine fusion.