A search for food extracts inhibiting ferroptosis using immortalized mouse embryonic fibroblasts derived from xCT knockout mice.

Depleting glutathione by xCT inhibition induces iron-dependent ferroptotic cell death, which is suppressed by lipophilic antioxidants. We screened food extracts with xCTKO-MEFs, identifying garlic extracts as particularly potent in inhibiting ferroptosis among the food extracts examined in this study. xCTKO-MEFs can serve as a convenient tool for identifying find food extracts that are effective in inhibiting ferroptosis.

Proposal of Simplified Standardization of the Cell-Growth-Promoting Activity of Human Adipose Tissue Mesenchymal Stromal Cell Culture Supernatants.

The conditioned medium (CM) obtained from mesenchymal stromal cell (MSC) culture has excellent cell growth-promoting activity and is used for cosmetics and healthcare products. Unlike pharmaceuticals, strict efficacy verification is not legally required for these products. However, their efficacy must be substantiated as commercial products. We attempted to simplify CM production and to standardize the evaluation of the growth-promoting activity of CM. CM was obtained through the culturing of two lines of commercially available human adipose tissue-derived MSCs using MEMα with or without 10% fetal bovine serum (FBS) for 24 h. Non-CM control media were produced by the same protocol without MSCs. Growth-promoting activities of the CM were estimated by [3H]-thymidine pulse. CM were subjected to molecular weight fractionation with ultrafiltration using 10 k-, 30 k-, 50 k-, and 100 k-membranes. The FBS-free CMs showed 1.34- to 1.85-fold increases and FBS-containing CMs showed 1.45- to 1.67-fold increases in proliferation-promoting activity compared with non-CM controls, regardless of the source of the cell. The thymidine incorporation levels were approximately three times higher in FBS-containing CMs. Aged cells also showed 1.67- to 2.48-fold increases in the activity due to FBS-containing CM, but not to FBS-free CM. The CM activities were sustained even after 1 year at 4 °C. Molecular weight fractionation showed that the activity was recovered in the fraction above 100 k. Clear and stable cell-growth-promoting activity was confirmed with CMs of commercially available adipose tissue MSCs. The activity was detected in the fraction over 100 k. We propose here the importance of standardizing the production and evaluation of CMs to indicate their specific action.

Phosphoramide-Based Metal-Organic Frameworks for Effective Gas Adsorption.

Cost-effective and facile synthetic routes to organic ligands, along with porous materials that exhibit exceptional gas-storage properties, promise significant industrial applications. Here, a two-step synthesis of novel organophosphorus ligands without metal catalysts is reported. These ligands serve as versatile linkers for the construction of metal-organic frameworks (MOFs) incorporating various metal ions, including zinc and copper. One of the zinc-based MOFs demonstrates remarkable gas-storage properties, with a hydrogen (H2) capacity exceeding 2.5 wt% at 77 K and 100 kPa as well as a carbon dioxide (CO2) capacity exceeding 20 wt% at 298 K and 100 kPa. Furthermore, this zinc-based MOF can be synthesized through a solvothermal process on the gram scale that yields high-quality single crystals.

Genetic insights into ossification of the posterior longitudinal ligament of the spine.

Ossification of the posterior longitudinal ligament of the spine (OPLL) is an intractable disease leading to severe neurological deficits. Its etiology and pathogenesis are primarily unknown. The relationship between OPLL and comorbidities, especially type 2 diabetes (T2D) and high body mass index (BMI), has been the focus of attention; however, no trait has been proven to have a causal relationship. We conducted a meta-analysis of genome-wide association studies (GWASs) using 22,016 Japanese individuals and identified 14 significant loci, 8 of which were previously unreported. We then conducted a gene-based association analysis and a transcriptome-wide Mendelian randomization approach and identified three candidate genes for each. Partitioning heritability enrichment analyses observed significant enrichment of the polygenic signals in the active enhancers of the connective/bone cell group, especially H3K27ac in chondrogenic differentiation cells, as well as the immune/hematopoietic cell group. Single-cell RNA sequencing of Achilles tendon cells from a mouse Achilles tendon ossification model confirmed the expression of genes in GWAS and post-GWAS analyses in mesenchymal and immune cells. Genetic correlations with 96 complex traits showed positive correlations with T2D and BMI and a negative correlation with cerebral aneurysm. Mendelian randomization analysis demonstrated a significant causal effect of increased BMI and high bone mineral density on OPLL. We evaluated the clinical images in detail and classified OPLL into cervical, thoracic, and the other types. GWAS subanalyses identified subtype-specific signals. A polygenic risk score for BMI demonstrated that the effect of BMI was particularly strong in thoracic OPLL. Our study provides genetic insight into the etiology and pathogenesis of OPLL and is expected to serve as a basis for future treatment development.

Residual Neuropathic Pain in Postoperative Patients With Cervical Ossification of Posterior Longitudinal Ligament.

STUDY DESIGN: A prospective multi-institutional observational study. OBJECTIVE: To investigate and identify risk factors for residual neuropathic pain after surgery in patients with cervical ossification of posterior longitudinal ligament (c-OPLL). SUMMARY OF BACKGROUND DATA: Patients with c-OPLL often require surgery for numbness and paralysis of the extremities; however, postoperative neuropathic pain can considerably deteriorate their quality of life. METHODS: Out of 479 patients identified from multicenter c-OPLL registries between 2014 and 2017, 292 patients who could be followed up for 2 years postoperatively were reviewed, after excluding patients with nervous system comorbidities. Demographic details; medical history; radiographic factors including the K-line, spinal canal occupancy rate of OPLL, cervical kyphosis angle, and presence of spinal cord myelomalacia; preoperative Japanese Orthopaedic Association (JOA) score; surgical procedure (fusion or decompression surgery); postoperative neurological deterioration; and the visual analogue scale for pain and numbness in the upper extremities (U/E) or trunk/lower extremities (L/E) at baseline and at 2 years postoperatively were assessed. Patients were grouped into residual and non-residual groups based on a postoperative visual analogue scale ≥40 mm. Risk factors for residual neuropathic pain were evaluated by multiple logistic regression analysis. RESULTS: The prevalence of U/E and L/E residual pain in postoperative c-OPLL patients was 51.7% and 40.4%, respectively. The U/E residual group had a poor preoperative JOA score and longer illness duration, and fusion surgery was more common in the residual group than in non-residual group. The L/E residual group was older with a poorer preoperative JOA score. On multivariate analysis, risk factors for U/E residual pain were long illness duration and poor preoperative JOA score, whereas those for L/E residual pain were age and poor preoperative JOA score. CONCLUSIONS: The risk factors for residual spinal neuropathic pain after c-OPLL surgery were age, long duration of illness, and poor preoperative JOA score. LEVEL OF EVIDENCE: IV.

Risk Factors for Cervical Deformity After Posterior Cervical Decompression Surgery: A Multicenter Study.

STUDY DESIGN: Retrospective multicenter study. OBJECTIVE: Posterior decompression surgery for cervical spondylotic myelopathy (CSM) and ossification of the posterior longitudinal ligament (OPLL) is a common surgery; however, it can cause postoperative cervical deformity (CD). The purpose of this study was to investigate the risk factors for CD. METHODS: The participants were 193 patients underwent laminoplasty or laminectomy for CSM or OPLL. CD was defined as a C2-7 sagittal vertical axis (SVA) ≥ 40 mm or a cervical lordosis angle (CL) ≤ -10°. The participants were divided into 2 groups: NCD (without CD before surgery), CD (with CD before surgery). NCD group was divided based on the presence of CD 1 year after surgery as follows: postoperative CD (PCD) and no PCD (NPCD). RESULTS: There were 153 patients (NCD), 40 (CD), 126 (NPCD), and 27 (PCD). There was significant difference in the number of decompressed lamina (NPCD: PCD = 4.1:4.5), the presence of C2 decompression (2: 11%), and C5 palsy (0: 11%). The risk factors for onset of CD, PCD, and CL ≤ -10° as assessed by multiple logistic regression analysis were preoperative C2-7 SVA ≥ 30 mm (odds ratio [OR]: 19.0), decompression of C2 or C7 lamina (OR 3.1), and preoperative CL ≤ 2° (OR 42.0), respectively. CONCLUSIONS: To prevent postoperative CD, it is important to avoid decompression of the C2 or C7 lamina. Moreover, in case with C2-7 SVA ≥ 30 mm or CL ≤ 2° before surgery, it is important to explain the risks and consider adding fusion surgery.

d-Cysteine supplementation partially protects against ferroptosis induced by xCT dysfunction via increasing the availability of glutathione.

Glutathione (GSH) is synthesized from three amino acids and the overall process is highly dependent on the availability of l-cysteine (l-Cys). GSH serves as an essential cofactor for glutathione peroxidase 4 (Gpx4), which reduces phospholipid hydroperoxides. The inactivation of Gpx4 or an insufficient supply of l-Cys results in the accumulation of lipid hydroperoxides, eventually leading to iron-dependent cell death, ferroptosis. In this study, we investigated the anti-ferroptotic properties of d-cysteine (d-Cys) under conditions of dysfunction in cystine transporter, xCT. l-Cys supplementation completely rescued ferroptosis that had been induced by the erastin-mediated inhibition of xCT in Hepa 1-6 cells. Upon d-Cys supplementation, the erastin-treated cells remained completely viable for periods of up to 24 h but eventually died after 48 h. d-Cys supplementation suppressed the production of lipid peroxides, thereby ferroptosis. The addition of d-Cys sustained intracellular Cys and GSH levels to a certain extent. When Hepa 1-6 cells were treated with a combination of buthionine sulfoximine and erastin, the anti-ferroptotic effect of d-Cys was diminished. These collective results indicate that, although d-Cys is not the direct source of GSH, d-Cys supplementation protects cells from ferroptosis in a manner that is dependent on GSH synthesis via stimulating the uptake of l-Cys.

The concerted elevation of conjugation reactions is associated with the aggravation of acetaminophen toxicity in Akr1a-knockout mice with an ascorbate insufficiency.

AIMS: Acetaminophen (APAP) is a relatively safe analgesic drug, but overdosing can cause acute liver failure. Ingested APAP is detoxified by metabolic conversion through conjugation reactions with glucuronate, sulfate, or glutathione (GSH). The consumption of GSH through conjugation as well as mitochondrial dysfunction is considered to be responsible for the increased susceptibility to APAP-induced hepatotoxicity. Compared to wild-type (WT) mice, Akr1a-knockout (KO) mice are vulnerable to developing hepatotoxicity due to the fact that ascorbate synthesis is attenuated. We used such KO mice to investigate how these conjugation reactions are involved in the hepatotoxicity caused by an overdose of APAP under ascorbate-deficient conditions. MAIN METHODS: APAP (400 mg/kg) was intraperitoneally administered to WT mice and KO mice. In addition to histological and blood biochemical analyses, metabolites in the liver, blood plasma, and urine were measured at several time points by liquid chromatography-mass spectrometry. KEY FINDINGS: Liver damage occurred earlier in the KO mice than in the WT mice. The levels of APAP-Cys, a final metabolite of GSH-conjugated APAP, as well as glucuronidated APAP and sulfated APAP were all higher in the KO mice compared to the WT mice. Treatment of the APAP-administered KO mice with N-acetylcysteine or supplementation of ascorbate suppressed the conjugation reactions at 6 h after APAP had been administrated, which mitigated the degree of liver damage. SIGNIFICANCE: An ascorbate deficiency coordinately stimulates conjugation reactions of APAP, which, combined with the mitochondrial damage caused by APAP metabolites, collectively results in the aggravation of the acute liver failure.

Methionine Deprivation Reveals the Pivotal Roles of Cell Cycle Progression in Ferroptosis That Is Induced by Cysteine Starvation.

Ferroptosis, a type of iron-dependent necrotic cell death, is triggered by the accumulation of excessive lipid peroxides in cells. Glutathione (GSH), a tripeptide redox molecule that contains a cysteine (Cys) unit in the center, plays a pivotal role in protection against ferroptosis. When the transsulfuration pathway is activated, the sulfur atom of methionine (Met) is utilized to generate Cys, which can then suppress Cys-starvation-induced ferroptosis. In the current study, we cultured HeLa cells in Met- and/or cystine (an oxidized Cys dimer)- deprived medium and investigated the roles of Met in ferroptosis execution. The results indicate that, in the absence of cystine or Met, ferroptosis or cell cycle arrest, respectively, occurred. Contrary to our expectations, however, the simultaneous deprivation of both Met and cystine failed to induce ferroptosis, although the intracellular levels of Cys and GSH were maintained at low levels. Supplementation with S-adenosylmethionine (SAM), a methyl group donor that is produced during the metabolism of Met, caused the cell cycle progression to resume and lipid peroxidation and the subsequent induction of ferroptosis was also restored under conditions of Met/cystine double deprivation. DNA methylation appeared to be involved in the resumption in the SAM-mediated cell cycle because its downstream metabolite S-adenosylhomocysteine failed to cause either cell cycle progression or ferroptosis to be induced. Taken together, our results suggest that elevated lipid peroxidation products that are produced during cell cycle progression are involved in the execution of ferroptosis under conditions of Cys starvation.

Lifespan extension with preservation of hippocampal function in aged system xc--deficient male mice.

The cystine/glutamate antiporter system xc- has been identified as the major source of extracellular glutamate in several brain regions as well as a modulator of neuroinflammation, and genetic deletion of its specific subunit xCT (xCT-/-) is protective in mouse models for age-related neurological disorders. However, the previously observed oxidative shift in the plasma cystine/cysteine ratio of adult xCT-/- mice led to the hypothesis that system xc- deletion would negatively affect life- and healthspan. Still, till now the role of system xc- in physiological aging remains unexplored. We therefore studied the effect of xCT deletion on the aging process of mice, with a particular focus on the immune system, hippocampal function, and cognitive aging. We observed that male xCT-/- mice have an extended lifespan, despite an even more increased plasma cystine/cysteine ratio in aged compared to adult mice. This oxidative shift does not negatively impact the general health status of the mice. On the contrary, the age-related priming of the innate immune system, that manifested as increased LPS-induced cytokine levels and hypothermia in xCT+/+ mice, was attenuated in xCT-/- mice. While this was associated with only a very moderate shift towards a more anti-inflammatory state of the aged hippocampus, we observed changes in the hippocampal metabolome that were associated with a preserved hippocampal function and the retention of hippocampus-dependent memory in male aged xCT-/- mice. Targeting system xc- is thus not only a promising strategy to prevent cognitive decline, but also to promote healthy aging.

Is anterior decompression and fusion more beneficial than laminoplasty for K-line (+) cervical ossification of the posterior longitudinal ligament? An analysis using propensity score matching.

OBJECTIVE: It is unclear whether anterior cervical decompression and fusion (ADF) or laminoplasty (LMP) results in better outcomes for patients with K-line-positive (+) cervical ossification of the posterior longitudinal ligament (OPLL). The purpose of the study is to compare surgical outcomes and complications of ADF versus LMP in patients with K-line (+) OPLL. METHODS: The study included 478 patients enrolled in the Japanese Multicenter Research Organization for Ossification of the Spinal Ligament and who underwent surgical treatment for cervical OPLL. The patients who underwent anterior-posterior combined surgery or posterior decompression with instrumented fusion were excluded. The patients with a follow-up period of fewer than 2 years were also excluded, leaving 198 patients with K-line (+) OPLL. Propensity score matching was performed on 198 patients with K-line (+) OPLL who underwent ADF (44 patients) or LMP (154 patients), resulting in 39 pairs of patients based on the following predictors for surgical outcomes: age, preoperative Japanese Orthopaedic Association (JOA) score, C2-7 angle, and the occupying ratio of OPLL. Clinical outcomes were assessed 1 and 2 years after surgery using the recovery rate of the JOA score. Complications and reoperation rates were also investigated. RESULTS: The mean recovery rate of the JOA score 1 year after surgery was 55.3% for patients who underwent ADF and 42.3% (p = 0.06) for patients who underwent LMP. Two years after surgery, the recovery rate was 53.4% for those who underwent ADF and 38.7% for LMP (p = 0.07). Although both surgical procedures yielded good results, the mean recovery rate of JOA scores tended to be higher in the ADF group. The incidence of surgical complications, however, was higher following ADF (33%) than LMP (15%; p = 0.06). The reoperation rate was also higher in the ADF group (15%) than in the LMP group (0%; p = 0.01). CONCLUSIONS: Clinical outcomes were good for both ADF and LMP, indicating that ADF and LMP are appropriate procedures for patients with K-line (+) OPLL. Clinical outcomes of ADF 1 and 2 years after surgery tended to be better than LMP, but the analysis did not detect any significant difference in clinical outcomes between the groups. Conversely, patients who underwent ADF had a higher incidence of surgery-related complications. When considering indications for ADF or LMP, benefits and risks of the surgical procedures should be carefully weighed.

Comparison of laminoplasty and posterior fusion surgery for cervical ossification of posterior longitudinal ligament.

This prospective multicenter study, established by the Japanese Ministry of Health, Labour and Welfare and involving 27 institutions, aimed to compare postoperative outcomes between laminoplasty (LM) and posterior fusion (PF) for cervical ossification of the posterior longitudinal ligament (OPLL), in order to address the controversy surrounding the role of instrumented fusion in cases of posterior surgical decompression for OPLL. 478 patients were considered for participation in the study; from among them, 189 (137 and 52 patients with LM and PF, respectively) were included and evaluated using the Japanese Orthopaedic Association (JOA) scores, the JOA Cervical Myelopathy Evaluation Questionnaire (JOACMEQ), and radiographical measurements. Basic demographic and radiographical data were reviewed, and the propensity to choose a surgical procedure was calculated. Preoperatively, there were no significant differences among the participants in terms of patient backgrounds, radiographical measurements (K-line or cervical alignment on X-ray, OPLL occupation ratio on computed tomography, increased signal intensity change on magnetic resonance imaging), or clinical status (JOA score and JOACMEQ) after adjustments. The overall risk of perioperative complications was found to be lower with LM (odds ratio [OR] 0.40, p = 0.006), and the rate of C5 palsy occurrence was significantly lower with LM (OR 0.11, p = 0.0002) than with PF. The range of motion (20.91° ± 1.05° and 9.38° ± 1.24°, p < 0.0001) in patients who had PF was significantly smaller than in those who had LM. However, multivariable logistic regression analysis showed no significant difference among the participants in JOA score, JOA recovery rate, or JOACMEQ improvement at two years. In contrast, OPLL progression was greater in the LM group than in the PF group (OR 2.73, p = 0.0002). Both LM and PF for cervical myelopathy due to OPLL had resulted in comparable postoperative outcomes at 2 years after surgery.

Severity of Myelopathy is Closely Associated With Advanced Age and Signal Intensity Change in Cervical Ossification of the Posterior Longitudinal Ligament: A Prospective Nationwide Investigation.

STUDY DESIGN: Prospective, nationwide case series. OBJECTIVE: To identify preoperative factors associated with myelopathy and neurological impairment in patients with cervical ossification of the posterior longitudinal ligament (OPLL). SUMMARY OF BACKGROUND DATA: Various studies have reported clinical outcomes following the surgical treatment of OPLL. However, there has been no large-scale study of preoperative clinical features in patients with cervical OPLL. MATERIALS AND METHODS: Data were prospectively collected from 28 institutions nationwide in Japan. In total, 512 patients with neurological impairment caused by cervical OPLL requiring surgery were enrolled. Basic demographic and clinical data, including age, sex, diabetes status, body mass index, smoking history, and disease duration were collected. C2-7 lordotic angle, canal narrowing ratio, range of motion in flexion-extension at C2-7, and type of OPLL were evaluated on lateral radiographs to identify factors influencing the clinical features of patients with OPLL in whom surgery was planned. RESULTS: Complete documentation was available for 490 patients (362 male, 128 female). In total, 34 patients had the localized type, 181 had the segmental type, 64 had the continuous type, and 211 had the mixed type. Although there were no significant differences in age, body mass index, disease duration, Japanese Orthopedic Association (JOA) score, and lordotic angle at C2-7 according to the type of OPLL, significant differences were observed in a range of motion at C2-7 and the canal narrowing ratio among the 4 types. Multiple regression analysis revealed that the JOA score was significantly associated with age and signal intensity change on magnetic resonance imaging. CONCLUSIONS: This is the first large-scale, prospective, multicenter case series study to investigate factors influencing preoperative neurological status in patients with OPLL. Age and signal intensity change on magnetic resonance images were significantly associated with JOA score in patients requiring surgery. LEVEL OF EVIDENCE: Level II.

The impact of diabetes mellitus on spinal fracture with diffuse idiopathic skeletal hyperostosis: A multicenter retrospective study.

BACKGROUND: Patients with diffuse idiopathic skeletal hyperostosis (DISH) are susceptible to spinal column injuries with neurological deterioration. Previous studies indicated that the prevalence of diabetes mellitus (DM) in patients with DISH was higher than that in patients without DISH. This study investigates the impact of DM on surgical outcomes for spinal fractures in patients with DISH. METHODS: We retrospectively evaluated 177 spinal fractures in patients with DISH (132 men and 45 women; mean age, 75 ± 10 years) who underwent surgery from a multicenter database. The subjects were classified into two groups according to the presence of DM. Perioperative complications, neurological status by Frankel grade, mortality rate, and status of surgical site infection (SSI) were compared between the two groups. RESULTS: DM was present in 28.2% (50/177) of the patients. The proportion of men was significantly higher in the DM group (DM group: 86.0% vs. non-DM group: 70.1%) (p = 0.03). The overall complication rate was 22.0% in the DM group and 19.7% in the non-DM group (p = 0.60). Poisson regression model revealed that SSI was significantly associated with DM (DM group: 10.0% vs. non-DM group: 2.4%, Relative risk: 4.5) (p = 0.048). Change in neurological status, mortality rate, instrumentation failure, and nonunion were similar between both groups. HbA1c and fasting blood glucose level (SSI group: 7.2% ± 1.2%, 201 ± 67 mg/dL vs. non-SSI group: 6.6% ± 1.1%, 167 ± 47 mg/dL) tended to be higher in patients with SSI; however, there was no significant difference. CONCLUSIONS: In spinal fracture in patients with DISH, although DM was an associated factor for SSI with a relative risk of 4.5, DM did not negatively impact neurological recovery. Perioperative glycemic control may be useful for preventing SSI because fasting blood glucose level was high in patients with SSI.

Factors Significantly Associated with Postoperative Neck Pain Deterioration after Surgery for Cervical Ossification of the Posterior Longitudinal Ligament: Study of a Cohort Using a Prospective Registry.

Postoperative neck pain has been reported as an unsolved postoperative complication of surgery for cervical ossification of the posterior longitudinal ligament (OPLL). The aim of the present study was to elucidate factors having a significant association with postoperative deterioration of neck pain in cervical OPLL patients. We studied a cohort of patients in a prospective registry of 478 patients who had undergone cervical spine surgery for cervical OPLL. We excluded those without evaluation of preoperative neck pain. Therefore, 438 patients were included in the present study. Neck pain was evaluated with the visual analogue scale (VAS, 0-100 mm). Postoperative neck pain deterioration was defined as a ≥20 mm increase of VAS neck pain. Patient factors, neurological status, imaging factors and surgical factors were assessed. Univariate analyses followed by multivariate analysis using stepwise logistic regression was performed. Six months after surgery, 50 (11.6%) patients showed postoperative neck pain deterioration and 76 (17.4%) patients showed postoperative neck pain deterioration 2 years after surgery. Six months after surgery, the rate of neck pain deterioration was significantly higher in patients who had undergone posterior surgery. Two years after surgery, the number of levels fused was significantly correlated with neck pain deterioration.

LAT1 inhibitor JPH203 sensitizes cancer cells to radiation by enhancing radiation-induced cellular senescence.

L-type amino acid transporter 1 (LAT1) is important for transporting neutral amino acids into cells. LAT1 expression is correlated with cancer malignancy, suggesting that LAT1 is a promising target for cancer therapy. JPH203, a potential novel drug targeting LAT1, has been shown to suppress tumor growth in various cancer cell lines. However, a combination study of JPH203 and radiation therapy has not been reported. Here, we examined the effects of JPH203 on radiosensitivity after irradiation in A549 and MIA Paca-2 cells. We showed that X-irradiation increased cellular neutral amino acid uptake via LAT1 in both cell lines. JPH203 inhibited the radiation-induced increase in neutral amino acid uptake. We demonstrated that JPH203, at minimally toxic concentrations, significantly sensitized cancer cells to radiation. JPH203 significantly downregulated mTOR activity and enhanced cellular senescence post-irradiation without reducing ATP and GSH levels. These results indicate that LAT1 inhibition by JPH203 sensitizes cancer cells to radiation by enhancing cellular senescence via mTOR downregulation. Thus, JPH203 may be a potent anti-cancer drug in combination with radiation therapy.

Perioperative Complications in Posterior Surgeries for Cervical Ossification of the Posterior Longitudinal Ligament: A Prospective Nationwide Investigation.

STUDY DESIGN: This was a prospective multicenter study. OBJECTIVE: The aim of this study was to investigate the perioperative complications of posterior surgeries for the treatment of cervical ossification of the posterior longitudinal ligament (OPLL). SUMMARY OF BACKGROUND DATA: Surgical treatment for cervical OPLL has a high risk of various complications. Laminoplasty (LAMP) and posterior decompression and instrumented fusion (PDF) are effective for multilevel cervical OPLL; however, few studies have focused on the surgical complications of these 2 procedures. MATERIALS AND METHODS: We prospectively included 380 patients undergoing posterior surgeries for cervical OPLL (LAMP: 270 patients, PDF: 110 patients), and investigated the systemic and local complications, including neurological complications. We further evaluated risk factors related to the neurological complications. RESULTS: Motor palsy was found in 40 patients (10.5%), and motor palsy in the upper extremity was most frequent (8.9%), especially in patients who received PDF (14.5%). Motor palsies involving the lower extremities was found in 6 patients (1.6%). Regarding local complications, dural tears (3.9%) and surgical site infections (2.6%) were common. In the univariate analysis, body mass index, preoperative cervical alignment, fusion surgery, and the number of operated segments were the factors related to motor palsy. Multivariate analysis revealed that fusion surgery and a small preoperative C2-C7 angle were the independent factors related to motor palsy. Motor palsy involving the lower extremities tended to be found at early time points after the surgery, and all the patients fully recovered. Motor palsy in the upper extremities occurred in a delayed manner, and 68.8% of patients with PDF showed good recovery, whereas 81.3% of patients with LAMP showed good recovery. CONCLUSIONS: In posterior surgeries for cervical OPLL, segmental motor palsy in the upper extremity was most frequently observed, especially in patients who received PDF. Fusion and a small preoperative C2-C7 angle were the independent risk factors for motor palsy. LEVEL OF EVIDENCE: Level III.

Impact of Diabetes Mellitus on Cervical Spine Surgery for Ossification of the Posterior Longitudinal Ligament.

Ossification of the posterior longitudinal ligament (OPLL) is commonly associated with diabetes mellitus (DM); however, the impact of DM on cervical spine surgery for OPLL remains unclear. This study was performed to evaluate the influence of diabetes DM on the outcomes following cervical spine surgery for OPLL. In total, 478 patients with cervical OPLL who underwent surgical treatment were prospectively recruited from April 2015 to July 2017. Functional measurements were conducted at baseline and at 6 months, 1 year, and 2 years after surgery using JOA and JOACMEQ scores. The incidence of postoperative complications was categorized into early (≤30 days) and late (>30 days), depending on the time from surgery. From the initial group of 478 patients, 402 completed the 2-year follow-up and were included in the analysis. Of the 402 patients, 127 (32%) had DM as a comorbid disease. The overall incidence of postoperative complications was significantly higher in patients with DM than in patients without DM in both the early and late postoperative periods. The patients with DM had a significantly lower JOA score and JOACMEQ scores in the domains of lower extremity function and quality of life than those without DM at the 2-year follow-up.

Nitric oxide protects against ferroptosis by aborting the lipid peroxidation chain reaction.

Ferroptosis is a type of iron-dependent necrotic cell death, which is typically triggered by the depletion of intracellular glutathione (GSH), which is associated with increased lipid peroxidation. Nitric oxide (NO) is a highly reactive gaseous radical mediator with anti-oxidation properties that terminates lipid peroxidation reactions. In the current study, we report the anti-ferroptotic action of NOC18, an NO donor that spontaneously releases NO, in cells under various ferroptotic conditions in vitro. Our results indicate that, when mouse hepatoma Hepa 1-6 cells are incubated with NOC18, cell death induced by various ferroptotic stimuli such as cysteine (Cys) starvation, the inhibition of glutathione peroxidase 4 (GPX4) and treatment with tertiary-butyl hydroperoxide (TBHP) is significantly reduced. Treatment with NOC18 failed to improve the decrease in the levels of Cys or GSH and the accumulation of ferrous iron upon ferroptotic stimuli. The fluorescent intensity of C11-BODIPY581/591, a probe that is used to detect lipid peroxidation products, was increased somewhat by treatment with NOC18 under conditions of Cys starvation, and the accumulation of lipid peroxidation end-products, as evidenced by the levels of 4-hydroxynonenal, were effectively suppressed. The pre-incubation of TBHP with NOC7, a short-lived NO donor completely eliminated its ability to trigger ferroptosis. These collective results indicate that NO exerts a cytoprotective action against various ferroptotic stimuli by aborting the lipid peroxidation chain reaction.

Carnosine dipeptidase II (CNDP2) protects cells under cysteine insufficiency by hydrolyzing glutathione-related peptides.

The knockout (KO) of the cystine transporter xCT causes ferroptosis, a type of iron-dependent necrotic cell death, in mouse embryonic fibroblasts, but this does not occur in macrophages. In this study, we explored the gene that supports cell survival under a xCT deficiency using a proteomics approach. Analysis of macrophage-derived peptides that were tagged with iTRAQ by liquid chromatography-mass spectrometry revealed a robust elevation in the levels of carnosine dipeptidase II (CNDP2) in xCT KO macrophages. The elevation in the CNDP2 protein levels was confirmed by immunoblot analyses and this elevation was accompanied by an increase in hydrolytic activity towards cysteinylglycine, the intermediate degradation product of glutathione after the removal of the γ-glutamyl group, in xCT KO macrophages. Supplementation of the cystine-free media of Hepa1-6 cells with glutathione or cysteinylglycine extended their survival, whereas the inclusion of bestatin, an inhibitor of CNDP2, counteracted the effects of these compounds. We established CNDP2 KO mice by means of the CRISPR/Cas9 system and found a decrease in dipeptidase activity in the liver, kidney, and brain. An acetaminophen overdose (350 mg/kg) showed not only aggravated hepatic damage but also renal injury in the CNDP2 KO mice, which was not evident in the wild-type mice that were receiving the same dose. The aggravated renal damage in the CNDP2 KO mice was consistent with the presence of abundant levels of CNDP2 in the kidney, the organ prone to developing ferroptosis. These collective data imply that cytosolic CNDP2, in conjugation with the removal of the γ-glutamyl group, recruits Cys from extracellular GSH and supports redox homeostasis of cells, particularly in epithelial cells of proximal tubules that are continuously exposed to oxidative insult from metabolic wastes that are produced in the body.