Beneficial Effects of Dietary Fiber in Young Barley Leaf on Gut Microbiota and Immunity in Mice.

The health benefits of young barley leaves, rich in dietary fiber, have been studied for several decades; however, their beneficial effects on the intestinal microenvironment remain to be elucidated. To investigate the effects of young barley leaf-derived dietary fiber (YB) on the gut microbiota and immunity, mice were fed an AIN-93G diet containing cellulose or YB and subjected to subsequent analysis. The population of MHC-II-positive conventional dendritic cells (cDCs) and CD86 expression in the cDCs of Peyer's patches were elevated in the YB-fed mice. MHC-II and CD86 expression was also elevated in the bone marrow-derived DCs treated with YB. 16S-based metagenomic analysis revealed that the gut microbiota composition was markedly altered by YB feeding. Among the gut microbiota, Lachnospiraceae, mainly comprising butyrate-producing NK4A136 spp., were overrepresented in the YB-fed mice. In fact, fecal butyrate concentration was also augmented in the YB-fed mice, which coincided with increased retinaldehyde dehydrogenase (RALDH) activity in the CD103+ cDCs of the mesenteric lymph nodes. Consistent with elevated RALDH activity, the population of colonic IgA+ plasma cells was higher in the YB-fed mice than in the parental control mice. In conclusion, YB has beneficial effects on the gut microbiota and intestinal immune system.

Surgical Predictors for Prevention of Postoperative Shoulder Imbalance in Lenke Type 2A Adolescent Idiopathic Scoliosis.

STUDY DESIGN: Multicenter, retrospective cohort study. OBJECTIVE: The aim of this study was to investigate the occurrence and surgical predictors of postoperative shoulder imbalance (PSI) in Lenke type 2A adolescent idiopathic scoliosis (AIS). SUMMARY OF BACKGROUND DATA: Although several studies have investigated the factors influencing PSI in Lenke type 2 curves, no studies have analyzed PSI-related factors considering upper instrumented vertebra (UIV) and lumbar modifier type simultaneously. METHODS: Patients with Lenke Type 2A AIS treated by spinal fusion were retrospectively identified and their data were extracted from six spine centers in Japan. Inclusion criteria were age between 10 and 20 years at surgery, UIV = T2, major curve 40° to 90°, and follow-up for 24 to 30 months after surgery. We analyzed patient characteristics, surgical characteristics, and preoperative and immediate-postoperative radiographic parameters. We defined patients with lower instrumented vertebra (LIV) equal or proximal to the last touching vertebra (LTV) as selective thoracic fusion (STF-LTV) and patients with LIV distal to the LTV as non-STF-LTV. t Tests, Mann-Whitney U test, χ2 tests, Fisher exact tests, and multivariate logistic regression were used for statistical analyses. RESULTS: Among the 99 consecutive patients with a mean follow-up of 25.6 months, PSI was seen in 27 (27.3%) patients immediately after and in 17 (17.2%) patients at 24 to 30 months. The univariate analysis revealed that the significant risk factors of PSI were preoperative radiographical shoulder height, non-STF-LTV, and high main thoracic curve (MTC) correction (immediate-postoperative MTC correction rate: ≥70%), with PSI incidence of 40.0%. The multivariate logistic regression analysis indicated that interaction term of non-STF-LTV and high MTC correction was an independent risk factor for PSI (non-STF-LTV and high MTC correction, odds ratio: 5.167, 95% confidence interval: 1.470-18.159, P = 0.010). CONCLUSION: To prevent PSI in Lenke Type 2A AIS patients, surgeons should avoid the combination of non-STF-LTV and high MTC correction in those surgeries with UIV as T2.Level of Evidence: 4.

Delayed notochordal cell disappearance through integrin α5ß1 mechanotransduction during ex-vivo dynamic loading-induced intervertebral disc degeneration.

The loss of nucleus pulposus (NP) notochordal cells is one of the key initial hallmarks of age-related intervertebral disc degeneration. Although the transmembrane mechanoreceptor integrin α5ß1 is important in the process of disc degeneration, the relationship between integrin α5ß1 and notochordal cell disappearance remains unclear. The purpose of this study was to elucidate the role of integrin α5ß1 in the homeostasis of notochordal cells using an ex-vivo dynamic loading culture system that we developed. Rat tail functional spinal units (n = 80 from 40 rats) were cultured under unloading or 1.3-MPa, 1.0-Hz dynamic compressive loading for 48 or 144 h with or without an integrin α5ß1 inhibitor. Disc histomorphology, cell viability, apoptosis, senescence, and phenotypic expression were investigated. Consequently, histological degenerative disc changes with decreased cell viability and increased cell apoptosis and senescence were observed with an extended loading duration. Immunofluorescence revealed that the expression of notochordal cell markers, CD24 and brachyury, and chondrocyte markers, collagen type II and SRY-box 9, declined with loading. In particular, reduction in notochordal cell marker expression was more dramatic than that in chondrocyte marker expression. Apoptotic terminal deoxynucleotidyl transferase dUTP nick-end labeling positivity was also higher in brachyury-positive notochordal cells. Furthermore, all these changes were delayed by inhibiting integrin α5ß1. Findings of our dynamic loading regimen with a relatively high pressure suggest reproducibility of the cellularity and phenotypic disappearance of NP notochordal cells during adolescence, the susceptibility of notochordal cells to mechanical stimuli partially through the integrin α5ß1 pathway, and future potential treatment of integrin regulation for intervertebral disc disease.

Prospective Cohort Study of Performance Status and Activities of Daily Living After Surgery for Spinal Metastasis.

STUDY DESIGN: A prospective cohort study of performance status (PS) and activities of daily living (ADL) in patients with spinal metastasis. OBJECTIVE: To identify the effect of spinal surgery on PS and ADL in patients with spinal metastasis. SUMMARY OF BACKGROUND DATA: Spinal metastasis causes severe neurological deficits, resulting in drastic loss of patients' PS and ADL. However, the effect of spine surgery on PS and ADL is not well known. MATERIALS AND METHODS: Seventy patients with spinal metastasis were enrolled in this study. Forty-six patients desired and underwent spine surgery ("surgery" group) and 24 patients did not desire surgery ("nonsurgery" group). Both groups received optimal treatments, including radiation, chemotherapy, and palliative care services. Evaluation was performed at 1, 3, and 6 months after study enrollment using the Eastern Cooperative Oncology Group PS, the Barthel index (BI) for ADL, and Frankel classification for neurological status. RESULTS: There was no significant difference in baseline PS, the BI, or Frankel classification between the groups. The surgery group showed significant improvement in PS, maintaining grade 2 or less throughout the duration of the study, as well as in ADL, exceeding 70 points of the BI, compared with the nonsurgery group (P<0.05). Significantly improved neurological condition was also observed in the surgery group over the following 6 months. More than 95% of patients who underwent surgery improved their PS, the BI, and neurological status. Furthermore, >80% of these patients maintained improvement in PS, the BI, and neurological status for at least 6 months. In contrast, PS, the BI, and neurological status of patients in the "nonsurgery" group deteriorated throughout the study period. CONCLUSIONS: Spine surgery improves PS, ADL, and neurological status in patients with spinal metastasis for a minimum 6 months. This indicates that these patients can acquire an independent daily life.

C5 nerve palsy after posterior reconstruction surgery: predictive risk factors of the incidence and critical range of correction for kyphosis.

PURPOSE: It has been reported that the incidence of post-operative segmental nerve palsy, such as C5 palsy, is higher in posterior reconstruction surgery than in conventional laminoplasty. Correction of kyphosis may be related to such a complication. The aim of this study was to elucidate the risk factors of the incidence of post-operative C5 palsy, and the critical range of sagittal realignment in posterior instrumentation surgery. METHODS: Eighty-eight patients (mean age 64.0 years) were involved. The types of the disease were; 33 spondylosis with kyphosis, 27 rheumatoid arthritis, 17 athetoid cerebral palsy and 11 others. The patients were divided into two groups; Group P: patients with post-operative C5 palsy, and Group NP: patients without C5 palsy. The correction angle of kyphosis, and pre-operative diameter of C4/5 foramen on CT were evaluated between the two groups. Multivariate logistic regression analysis was used to determine the critical range of realignment and the risk factors affecting the incidence of post-operative C5 palsy. RESULTS: Seventeen (19.3 %) of the 88 patients developed C5 palsy. The correction angle of kyphosis in Group P (15.7°) was significantly larger than that in Group NP (4.5°). In Group P, pre-operative diameters of intervertebral foramen at C4/5 (3.2 mm) were significantly smaller than those in Group NP (4.1 mm). The multivariate analysis demonstrated that the risk factors were the correction angle and pre-operative diameter of the C4/5 intervertebral foramen. The logistic regression model showed a correction angle exceeding 20° was critical for developing the palsy when C4/5 foraminal diameter reaches 4.1 mm, and there is a higher risk when the C4/5 foraminal diameter is less than 2.7 mm regardless of any correction. CONCLUSIONS: This study has indicated the risk factors of post-operative C5 palsy and the critical range of realignment of the cervical spine after posterior instrumented surgery.

Recombinant human SIRT1 protects against nutrient deprivation-induced mitochondrial apoptosis through autophagy induction in human intervertebral disc nucleus pulposus cells.

INTRODUCTION: Nutrient deprivation is a likely contributor to intervertebral disc (IVD) degeneration. Silent mating type information regulator 2 homolog 1 (SIRT1) protects cells against limited nutrition by modulation of apoptosis and autophagy. However, little evidence exists regarding the extent to which SIRT1 affects IVD cells. Therefore, we conducted an in vitro study using human IVD nucleus pulposus (NP) cells. METHODS: Thirty-two IVD specimens were obtained from patients who underwent surgical intervention and were categorized based on Pfirrmann IVD degeneration grades. Cells were isolated from the NP and cultured in the presence of recombinant human SIRT1 (rhSIRT1) under different serum conditions, including 10 % (v/v) fetal bovine serum (FBS) as normal nutrition (N) and 1 % (v/v) FBS as low nutrition (LN). 3-Methyladenine (3-MA) was used to inhibit autophagy. Autophagic activity was assessed by measuring the absorbance of monodansylcadaverine and immunostaining and Western blotting for light chain 3 and p62/SQSTM1. Apoptosis and pathway analyses were performed by flow cytometry and Western blotting. RESULTS: Cells cultured under LN conditions decreased in number and exhibited enhanced autophagy compared with the N condition. Medium supplementation with rhSIRT1 inhibited this decrease in cell number and induced an additional increase in autophagic activity (P < 0.05), whereas the combined use of rhSIRT1 and 3-MA resulted in drastic decreases in cell number and autophagy (P < 0.05). The incidence of apoptotic cell death increased under the LN condition, which was decreased by rhSIRT1 (P < 0.05) but increased further by a combination of rhSIRT1 and 3-MA (P < 0.05). Under LN conditions, NP cells showed a decrease in antiapoptotic Bcl-2 and an increase in proapoptotic Bax, cleaved caspase 3, and cleaved caspase 9, indicating apoptosis induction via the mitochondrial pathway. These changes were suppressed by rhSIRT1 but elevated further by rhSIRT1 with 3-MA, suggesting an effect of rhSIRT1-induced autophagy on apoptosis inhibition. Furthermore, the observed autophagy and apoptosis were more remarkable in cells from IVDs of Pfirrmann grade IV than in those from IVDs of Pfirrmann grade II. CONCLUSIONS: SIRT1 protects against nutrient deprivation-induced mitochondrial apoptosis through autophagy induction in human IVD NP cells, suggesting that rhSIRT1 may be a potent treatment agent for human degenerative IVD disease.

Functional impact of integrin α5ß1 on the homeostasis of intervertebral discs: a study of mechanotransduction pathways using a novel dynamic loading organ culture system.

BACKGROUND CONTEXT: Intervertebral disc (IVD) degeneration, a major cause of low back pain, is considered to be induced by daily mechanical loading. Mechanical stress is widely known to affect cell survival and extracellular matrix metabolism in many cell types. Although the involvement of integrin α5ß1 transmembrane mechanoreceptor in IVD degeneration has been reported, the precise function of integrin α5ß1 remains obscure. PURPOSE: To reflect IVD tissue response to mechanical stress using a dynamic loading organ culture system and elucidate the functional impact of integrin α5ß1 on the pathomechanism of IVD degeneration. STUDY DESIGN: An ex vivo study using a dynamic loading organ culture system. METHODS: Ninety-six rat IVD explants were examined. Intervertebral discs were subjected to 1.3 MPa, 1.0 Hz dynamic compressive load in the presence or absence of an Arg-Gly-Asp (RGD) peptide with affinity to the fibronectin binding-site of integrin α5ß1. Cell viability and histomorphology were assessed. The localization of integrin α5ß1 in the IVD was assessed by immunohistochemistry. Gene expression levels of IVD cells were evaluated using real-time reverse transcription-polymerase chain reaction. RESULTS: In the nucleus pulposus (NP), cell density and viability were reduced by dynamic compressive load. Histologic degenerative alterations, mainly seen in the NP, were the morphologic changes of NP cells. In both NP and annulus fibrosus (AF), immunohistochemistry revealed localization of integrin α5ß1 and that the messenger-RNA expression of integrin α5ß1 was increased by dynamic load. Dynamic load induced a catabolic effect, the stimulation of matrix metalloproteinase-3 and -13 gene expressions by NP and AF cells. The RGD peptide partially blocked the histologic alterations and the catabolic effect. CONCLUSIONS: The dynamic loading organ culture system simulated cellular responses to mechanical loading of the IVD. Our results suggest that IVD cells recognize the mechanical stress through RGD integrins, particularly the α5ß1 subtype that is highly expressed in NP and AF cells. Further experiments using this system will provide information about pathomechanisms of IVD degeneration through the mechanotransduction pathways.

A rat tail temporary static compression model reproduces different stages of intervertebral disc degeneration with decreased notochordal cell phenotype.

The intervertebral disc nucleus pulposus (NP) has two phenotypically distinct cell types-notochordal cells (NCs) and non-notochordal chondrocyte-like cells. In human discs, NCs are lost during adolescence, which is also when discs begin to show degenerative signs. However, little evidence exists regarding the link between NC disappearance and the pathogenesis of disc degeneration. To clarify this, a rat tail disc degeneration model induced by static compression at 1.3 MPa for 0, 1, or 7 days was designed and assessed for up to 56 postoperative days. Radiography, MRI, and histomorphology showed degenerative disc findings in response to the compression period. Immunofluorescence displayed that the number of DAPI-positive NP cells decreased with compression; particularly, the decrease was notable in larger, vacuolated, cytokeratin-8- and galectin-3-co-positive cells, identified as NCs. The proportion of TUNEL-positive cells, which predominantly comprised non-NCs, increased with compression. Quantitative PCR demonstrated isolated mRNA up-regulation of ADAMTS-5 in the 1-day loaded group and MMP-3 in the 7-day loaded group. Aggrecan-1 and collagen type 2α-1 mRNA levels were down-regulated in both groups. This rat tail temporary static compression model, which exhibits decreased NC phenotype, increased apoptotic cell death, and imbalanced catabolic and anabolic gene expression, reproduces different stages of intervertebral disc degeneration.

Fas ligand plays an important role for the production of pro-inflammatory cytokines in intervertebral disc nucleus pulposus cells.

It is suggested that pro-inflammatory cytokines, which are produced by interaction of the intervertebral nucleus pulposus cells and macrophages, may be linked to the cause of pain of the intervertebral disc herniation. This study carries out the in vitro experiments to examine the mechanism, with the use of the co-culture of an immortalized cell line of nucleus pulposus of the human intervertebral disc and the macrophage cell line. As a result, it is found that the production of pro-inflammatory cytokines is significantly larger at the co-culture group than at the independent culture group. Also, at the co-culture group of macrophages and intervertebral nucleus pulposus cells with over-expression of fas ligand (FasL), the production of pro-inflammatory cytokines is found to be far larger. Furthermore, it is found that these pro-inflammatory cytokines are produced mainly by the intervertebral nucleus pulposus cells with over-expression of FasL, and that the expression of a disintegrin and metalloproteinase (ADAM) 10, which controls the expression of FasL and activates reverse signaling inside cells, also increases. From these findings, it is suggested that FasL and ADAM10 play an important role in the production of pro-inflammatory cytokines coming from interaction of the intervertebral nucleus pulposus cells and macrophages.