CaWRKY01-10 and CaWRKY08-4 Confer Pepper's Resistance to Phytophthora capsici Infection by Directly Activating a Cluster of Defense-Related Genes.

Phytophthora blight of pepper, which is caused by the notorious oomycete pathogen Phytophthora capsici, is a serious disease in global pepper production regions. Our previous study had identified two WRKY transcription factors (TFs), CaWRKY01-10 and CaWRKY08-4, which are prominent modulators in the resistant pepper line CM334 against P. capsici infection. However, their functional mechanisms and underlying signaling networks remain unknown. Herein, we determined that CaWRKY01-10 and CaWRKY08-4 are localized in plant nuclei. Transient overexpression assays indicated that both CaWRKY01-10 and CaWRKY08-4 act as positive regulators in pepper resistance to P. capsici. Besides, the stable overexpression of CaWRKY01-10 and CaWRKY08-4 in transgenic Nicotiana benthamiana plants also significantly enhanced the resistance to P. capsici. Using comprehensive approaches including RNA-seq, CUT&RUN-qPCR, and dual-luciferase reporter assays, we revealed that overexpression of CaWRKY01-10 and CaWRKY08-4 can activate the expressions of the same four Capsicum annuum defense-related genes (one PR1, two PR4, and one pathogen-related gene) by directly binding to their promoters. However, we did not observe protein-protein interactions and transcriptional amplification/inhibition effects of their shared target genes when coexpressing these two WRKY TFs. In conclusion, these data suggest that both of the resistant line specific upregulated WRKY TFs (CaWRKY01-10 and CaWRKY08-4) can confer pepper's resistance to P. capsici infection by directly activating a cluster of defense-related genes and are potentially useful for genetic improvement against Phytophthora blight of pepper and other crops.

Auto-segmentation of important centers of growth in the pediatric skeleton to consider during radiation therapy based on deep learning.

BACKGROUND: Routinely delineating of important skeletal growth centers is imperative to mitigate radiation-induced growth abnormalities for pediatric cancer patients treated with radiotherapy. However, it is hindered by several practical problems, including difficult identification, time consumption, and inter-practitioner variability. PURPOSE: The goal of this study was to construct and evaluate a novel Triplet-Attention U-Net (TAU-Net)-based auto-segmentation model for important skeletal growth centers in childhood cancer radiotherapy, concentrating on the accuracy and time efficiency. METHODS: A total of 107 childhood cancer patients fulfilled the eligibility criteria were enrolled in the training cohort (N = 80) and test cohort (N = 27). The craniofacial growth plates, shoulder growth centers, and pelvic ossification centers, with a total of 19 structures in the three groups, were manually delineated by two experienced radiation oncologists on axial, coronal, and sagittal computed tomography images. Modified from U-Net, the proposed TAU-Net has one main branch and two bypass branches, receiving semantic information of three adjacent slices to predict the target structure. With supervised deep learning, the skeletal growth centers contouring of each group was generated by three different auto-segmentation models: U-Net, V-Net, and the proposed TAU-Net. Dice similarity coefficient (DSC) and Hausdorff distance 95% (HD95) were used to evaluate the accuracy of three auto-segmentation models. The time spent on performing manual tasks and manually correcting auto-contouring generated by TAU-Net was recorded. The paired t-test was used to compare the statistical differences in delineation quality and time efficiency. RESULTS: Among the three groups, including craniofacial growth plates, shoulder growth centers, and pelvic ossification centers groups, TAU-Net had demonstrated highly acceptable performance (the average DSC = 0.77, 0.87, and 0.83 for each group; the average HD95 = 2.28, 2.07, and 2.86 mm for each group). In the overall evaluation of 19 regions of interest (ROIs) in the test cohort, TAU-Net had an overwhelming advantage over U-Net (63.2% ROIs in DSC and 31.6% ROIs in HD95, p = 0.001-0.042) and V-Net (94.7% ROIs in DSC and 36.8% ROIs in HD95, p = 0.001-0.040). With an average time of 52.2 min for manual delineation, the average time saved to adjust TAU-Net-generated contours was 37.6 min (p < 0.001), a 72% reduction. CONCLUSIONS: Deep learning-based models have presented enormous potential for the auto-segmentation of important growth centers in pediatric skeleton, where the proposed TAU-Net outperformed the U-Net and V-Net in geometrical precision for the majority status.

miR-21 Targets Long Noncoding RNA PCAT29 to Promote Cell Proliferation in Neuroblastoma.

Long noncoding RNA (lncRNA) PCAT29 has been characterized as a tumor suppressor in several types of cancer, although its involvement in neuroblastoma (NB) is unknown. In this study, we analyzed the role of PCAT29 in NB. In paired NB and nontumor tissues from 56 patients with NB, microRNA (miR)-21 and PCAT29 expression was determined with reverse transcription quantitative PCR. Correlation between miR-21 and PCAT29 was evaluated with linear regression. The interaction between miR-21 and PCAT29 was predicted by the IntaRNA 2.0 program. In NB cells, miR-21 and PCAT29 were overexpressed to explore their relationship. In NB cell proliferation, the roles of miR-21 and PCAT29 were analyzed with propidium iodide staining and Ki67 staining assays. The results showed that PCAT29 was downregulated and miR-21 was upregulated in NB. MiR-21 was inversely correlated with PCAT29. RNA-RNA interaction prediction revealed that miR-21 might target PCAT29. MiR-21 overexpression reduced PCAT29 expression and increased NB cell proliferation, whereas PCAT29 overexpression inhibited NB cell proliferation. PCAT29 overexpression promoted NB cell apoptosis, while miR-21 overexpression inhibited NB cell apoptosis and attenuated PCAT29 overexpression-mediated NB cell apoptosis. In conclusion, MiR-21 may target PCAT29 to promote cell apoptosis in NB.

A Novel Multimodal Radiomics Model for Predicting Prognosis of Resected Hepatocellular Carcinoma.

Objective: To explore a new model to predict the prognosis of liver cancer based on MRI and CT imaging data. Methods: A retrospective study of 103 patients with histologically proven hepatocellular carcinoma (HCC) was conducted. Patients were randomly divided into training (n = 73) and validation (n = 30) groups. A total of 1,217 radiomics features were extracted from regions of interest on CT and MR images of each patient. Univariate Cox regression, Spearman's correlation analysis, Pearson's correlation analysis, and least absolute shrinkage and selection operator Cox analysis were used for feature selection in the training set, multivariate Cox proportional risk models were established to predict disease-free survival (DFS) and overall survival (OS), and the models were validated using validation cohort data. Multimodal radiomics scores, integrating CT and MRI data, were applied, together with clinical risk factors, to construct nomograms for individualized survival assessment, and calibration curves were used to evaluate model consistency. Harrell's concordance index (C-index) values were calculated to evaluate the prediction performance of the models. Results: The radiomics score established using CT and MR data was an independent predictor of prognosis (DFS and OS) in patients with HCC (p < 0.05). Prediction models illustrated by nomograms for predicting prognosis in liver cancer were established. Integrated CT and MRI and clinical multimodal data had the best predictive performance in the training and validation cohorts for both DFS [(C-index (95% CI): 0.858 (0.811-0.905) and 0.704 (0.563-0.845), respectively)] and OS [C-index (95% CI): 0.893 (0.846-0.940) and 0.738 (0.575-0.901), respectively]. The calibration curve showed that the multimodal radiomics model provides greater clinical benefits. Conclusion: Multimodal (MRI/CT) radiomics models can serve as effective visual tools for predicting prognosis in patients with liver cancer. This approach has great potential to improve treatment decisions when applied for preoperative prediction in patients with HCC.

Bone Marrow Mesenchymal Stem Cells-Derived Extracellular Vesicles Promote Proliferation, Invasion and Migration of Osteosarcoma Cells via the lncRNA MALAT1/miR-143/NRSN2/Wnt/ß-Catenin Axis.

INTRODUCTION: Osteosarcoma is a malignant primary bone tumor. Bone marrow-derived mesenchymal stem cells-derived extracellular vesicles (BMSC-EVs) bear repair function for bone and cartilage. This study investigated the mechanism of BMSC-EVs in osteosarcoma cell proliferation, migration and invasion. METHODS: BMSC-EVs were isolated and identified. The effects of different concentrations of EVs on osteosarcoma cell proliferation, migration and invasion were evaluated. LncRNA MALAT1 expression in osteosarcoma cells was detected. BMSCs were transfected with si-MALAT1 or si-NC. The binding relationships between MALAT1 and miR-143, and miR-143 and NRSN2 were verified. Levels of NRSN2 and Wnt/ß-catenin pathway key proteins were detected. miR-143 mimic was transfected into EVs-treated osteosarcoma cells. Nude mice were injected with MG63 cells to verify the effect of EVs on osteosarcoma growth in vivo. RESULTS: BMSC-EVs facilitated proliferation, invasion and migration of osteosarcoma cells. BMSC-EVs carried MALAT1 into osteosarcoma cells. BMSC-EVs-treated osteosarcoma cells showed increased MALAT1 and NRSN2 expressions, decreased miR-143 expression, and activated Wnt/ß-catenin pathway. miR-143 mimic or si-MALAT1 reversed the effects of BMSC-EVs on osteosarcoma cells. In vivo experiment confirmed that BMSC-EVs promoted tumor growth in nude mice. DISCUSSION: BMSC-EVs promoted proliferation, invasion and migration of osteosarcoma cells via the MALAT1/miR-143/NRSN2/Wnt/ß-catenin axis. This study might offer new insights into osteosarcoma management.

Clinical and Pathological Study of Tumor Border Invasion-Is Narrow Resection Margin Acceptable in Hepatoblastoma Surgery?

Aim: We aim to study clinically and pathologically whether narrow resection margin (<1 cm) is acceptable in hepatoblastoma surgery. Methods: A total of 42 patients who underwent surgery for hepatoblastoma were selected, and these patients were divided into two groups according to whether or not they underwent preoperative chemotherapy (CHT). The general characteristics of the patients were summarized, the resection margin distance was recorded, and the event-free survival rates were followed up. Pathologically, H&E staining and immunochemical staining were used to study the invasion distance outside the tumor capsule in the tumor border. Results: Clinically, the event-free survival rates were not significantly different between the patients with wide resection margin (>1 cm) and narrow resection margin (<1 cm) of the two groups. Pathologically, the tumor of all 42 patients had capsules surrounding the tumor. Of the patients in Group 1 (without preoperative CHT), 9% (2/22) had micrometastatic cancer nests outside the capsule, and the farthest distance from the cancer nests to the capsule was 4.6 mm. Of the patients in Group 2 (with preoperative CHT), 75% (15/20) showed residual cancer nests in the paratumor liver tissue, and the farthest distance was 9.6 mm; three and two cases, respectively, showed extracapsular intravascular microtumorous thrombi. Conclusion: Clinically and pathologically, narrow resection margin is acceptable in hepatoblastoma surgery.

LncRNA cancer susceptibility candidate (CASC7) upregulates phosphatase and tensin homolog by downregulating miR-10a to inhibit neuroblastoma cell proliferation.

Long non-coding (lncRNA) cancer susceptibility candidate (CASC7) plays a tumor-suppressive role in several malignancies. In this study, the role of CASC7 in neuroblastoma was investigated for the first time. We observed the downregulation of CASC7 in neuroblastoma tissues compared to non-cancer tissues of neuroblastoma patients. Across neuroblastoma tissues, CASC7 was inversely correlated with microRNA-10a (miR-10a) but positively correlated with phosphatase and tensin homolog mRNA. In neuroblastoma cells, CASC7 overexpression led to downregulated miR-10a but upregulated phosphatase and tensin homolog. Furthermore, miR-10a overexpression led to downregulated phosphatase and tensin homolog and reduced effects of CASC7 overexpression. CASC7 overexpression resulted in inhibition, while miR-10a overexpression resulted in increased proliferation rate of neuroblastoma cells. We therefore concluded that lncRNA CASC7 may upregulate phosphatase and tensin homolog by downregulating miR-10a to inhibit neuroblastoma cell proliferation.

MicroRNA-429 inhibits neuroblastoma cell proliferation, migration and invasion via the NF-κB pathway.

BACKGROUND: MicroRNAs (miRNAs or miRs) can participate in the development and progression of neuroblastoma. Many studies have indicated that miR-429 can participate in tumor development. However, the mechanism underlying miR-429-mediated progression of neuroblastoma remains largely unclear. METHODS: Colony formation and apoptosis assays were used to determine the effect of miR-429 on cell proliferation. Its impact on cell migration was determined using the wound-healing and Transwell assays. The target gene of miR-429 was confirmed via western blotting and luciferase reporter assays. A nude mouse xenograft model with miR-429 overexpression was used to assess the effect on tumor growth. RESULTS: Our findings indicate that miR-429 is downregulated in neuroblastoma cell lines. We also found that it can induce apoptosis and inhibit proliferation in cells of those lines. MiR-429 can bind to the 3'-UTR of IKKß mRNA and overexpression of IKKß can reverse cell proliferation, blocking the effect of miR-429. Furthermore, miR-429 overexpression inhibited neuroblastoma growth in our nude mouse xenograft model. CONCLUSION: We provide important insight into miR-429 as a tumor suppressor through interaction with IKKß, which is a catalytic subunit of the IKK complex that activates NF-κB nuclear transport. Our results demonstrate that miR-429 may be a new target for the treatment of neuroblastoma.

Spontaneous regression of stage III neuroblastoma: A case report.

BACKGROUND: Neuroblastoma (NB) is the most common type of extracranial solid tumour in children. The overall prognosis of NB is poor, but at the same time, NB shows significant clinical diversity. NB can demonstrate spontaneous regression or can differentiate into benign ganglioneuroma. CASE SUMMARY: This study retrospectively analyzed the clinical data of a patient with spontaneous regression of stage III NB who was admitted in May 2015. Studies of the spontaneous regression of NB published from October 1946 to September 2019 were retrieved through PubMed. The clinical manifestations, diagnosis, treatment, and follow-up results were analysed. CONCLUSION: Spontaneous regression of stage III NB is rare in the clinic. The report of this case is an important supplement to the study of the spontaneous regression of NB.

Imaging and Pathology Study of the Chemotherapy Regression Area of Hepatoblastoma - A Prospective Single-Center Study.

Objective: The objective of this study is to determine the frequency of no residual cancer tissue in the chemotherapy regression area (CRA) of hepatoblastoma after preoperative chemotherapy and to measure the distance between the tumor capsule and the residual cancer nests. Methods: All the tissues in the CRAs of the resected specimens were excised. HE staining and immunohistochemical staining were performed to determine the frequency of residual cancer tissue in the CRA, and the distances between the residual cancer nests and the tumor capsule were measured. Results: A total of 30 patients were included in the study. The tumor volume decreased after chemotherapy by an average of 619 ml. Of the 30 patients, the CRAs of 18 still had residual cancer nests. The longest distance between the residual cancer nest and tumor capsule was 11.2 mm. Conclusions: After chemotherapy, 60% of patients still had residual cancer nests in CRAs, the furthest distance was 11.2 mm.

3D simulation assisted resection of giant hepatic mesenchymal hamartoma in children.

OBJECTIVE: To evaluate the usefulness of Hisense Computer Assisted Surgery System (Hisense CAS) in pre-operative surgical planning and intra-operative navigation for resection of pediatric giant hepatic mesenchymal hamartoma (HMH). METHODS: Five children with HMH underwent hepatectomy in our hospital. Pre-operative abdominal enhanced CT was performed for diagnosis and treatment planning. Using CT DICOM files, three-dimensional reconstruction was performed in three cases for operation planning and intra-operative navigation, with SID carrying out precise liver resection during the operation with Hisense CAS. RESULT: Two patients underwent right and left lobe hepatectomy, respectively, based only on enhanced CT. In 3 patients, by using the Hisense CAS system, three-dimensional reconstruction of the liver and tumors was successfully completed, and virtual hepatectomy performed successfully according to surgical plans. Hisense CAS could clearly and directly indicate the HMH location and shape, as well as its relationship with the intra-hepatic Glisson system, assisting safe hepatectomy. All five patients recovered well from surgery without any complications, and pathological examinations confirmed that all cases were HMH. No recurrence was observed during the follow-up period of 3 months to 5 years. CONCLUSION: Hisense CAS system is useful for preoperative planning and intra-operative navigation, assisting safer hepatectomy.

Usefulness of three-dimensional(3D) simulation software in hepatectomy for pediatric hepatoblastoma.

BACKGROUND: Hepatoblastoma (HB) is the most common malignant liver tumor in childhood. Complete HB surgical resection which is technically demanding is the cornerstone of effective therapy with a good prognosis. The aim of our study is to evaluate the usefulness of 3D simulation software in assisting hepatectomy in pediatric patients with HB. METHODS: 21 children with HB who underwent hepatectomy were enrolled in this study. All patients underwent computer tomography (CT) imaging preoperatively. CT images from 11 cases (from September 2013 to August 2015) were reconstructed with Hisense CAS, and performed hetpatectomy. While 10 cases (from September 2011 to August 2013) without 3D simulation were token as the control group. The clinical outcome were analyzed and compared between the 2 groups. RESULTS: All the HB were successfully removed for all patients and there was no positive margins in the surgical specimens, no complications, and no recurrences. For the reconstructing group, 3D simulation software successfully reconstructed the 3D images of liver and were used as a navigator in the operation room during hepatectomy. Anatomic hepatectomy were successfully completed for all patients after operation planning using the software. There was no obvious discrepancy between the virtual and the actual hepatectomy. The mean operation time was shorter (142.18 ± 21.87 min VS. the control group, 173.5 ± 54.88 min, p = 0.047) and intraoperative bleeding was less (28.73 ± 14.17 ml VS. 42.8 ± 41.12 ml, p = 0.011) in the reconstructing group. Moreover, postoperative hospital stay tended to be shorter in the reconstructing group (11.18 ± 2.78d VS. the control group 13 ± 3.46d, P = 0.257). CONCLUSIONS: 3D simulation software facilitates the investigation of the complex liver structure, contributes to the optimal operation planning, and enables an individualized anatomic hepatectomy for each pediatric patient with HB.

Clinical application of a three-dimensional imaging technique in infants and young children with complex liver tumors.

INTRODUCTION: Three-dimensional (3D) imaging instead of two-dimensional (2D) computed tomography (CT) for diagnosis and preoperative planning in infants and young children with complex liver tumors is a promising technique for precision hepatectomy. METHODS: This study was a retrospective analysis of 26 infants and young children with giant liver tumors involving the hepatic hilum who underwent precise hepatectomy at the Affiliated Hospital of Qingdao University between February 2012 and January 2015. All patients received upper abdominal contrast-enhanced CT scanning before surgery. 16 patients used Hisense CAS system for 3D reconstruction as the reconstruction group. While ten patients underwent 3D CT reconstruction by the CT Workstation as the control group. The clinical outcomes were analyzed and compared between the two groups. The 3D reconstruction of abdominal organs and blood vessels was generated using the Hisense CAS system. Diagnosis and preoperative planning assisted by the system was used for preoperative and intraoperative decision-making for precise hepatectomy. RESULTS: All patients underwent successful surgery. The 3D models clearly demonstrated the association of liver tumors with the intrahepatic vascular system and provided a preoperative assessment of resectability, assisting surgeons in preoperative procedural planning. Anatomic hepatectomy was successfully completed in the reconstruction group. The mean operation time was shorter in the reconstruction group (137.81 ± 17.51 min) than in the control group (192 ± 34.66 min) (P < 0.01). The mean intraoperative blood loss was lesser in the reconstruction group (21.81 ± 14.05 ml) than in the control group (53.50 ± 21.35 ml) (P < 0.01). The difference was statistically significant. DISCUSSION: 2D CT scan images cannot accurately display the spatial relationship between the tumor and surrounding vasculature. The 3D reconstruction model used in this study gave detailed and accurate anatomical information and allowed for the assessment of tumor resectability and provided a detailed road map for preoperative decision-making and predicted the postoperative liver function. CONCLUSIONS: 3D visualization technology provides preoperative assessment and allows individualized surgical planning. Surgical controllability, accuracy, and safety can be improved in infants and young children undergoing precise hepatectomy for complex liver tumors.

Application value of computer assisted surgery system in precision surgeries for pediatric complex liver tumors.

We discussed the diagnostic and treatment value and clinical significance of computer assisted surgery system (Higemi) in precision surgeries for pediatric complex liver tumors. A total of 21 pediatric cases receiving hepatectomy for tumors in the portal vein and giant liver tumors from June 2012 to January 2015 were analyzed. Higemi was used for 3-dimensional (3D) reconstruction of thin-slice CT images and surgical planning. Tumors were precisely located and blood vessel neighborhood was determined so as to evaluate surgical feasibility. In addition, pathological classification, surgical time, intraoperative blood loss, transfusion rate and complications were predicted. After 3D reconstruction using Higemi, the neighboring relationship of tumors with blood vessels and the running direction of the blood vessels were clearly visualized. Of 21 cases, 10 cases had tumors located in the left lobe, 5 cases in the right lobe, 3 cases showing involvement of right trilobes, and 3 cases in the middle lobe. Lobes exceeding one third of the total liver volume were resected in 18 cases. Postoperative pathological examination indicated 10 cases of hepatoblastoma, 3 cases of hepatocellular carcinoma, 3 cases of hamartoma, 3 cases of infantile hemangioendothelioma, 1 case of teratoma and 1 case of undifferentiated malignant mesenchymoma. The surgical time was 90-240 min with an average of 130 min; the medium intraoperative blood loss was 60 ml and the minimum blood loss was 3 ml; the transfusion rate was 42.9% (9/21). Surgeries were successful in 20 cases, who were discharged after recovery. However, one case had giant liver tumor combined with severe obstructive jaundice and hepatic insufficiency and died of postoperative liver failure and DIC. 3D reconstruction of CT data using Higemi can clearly visualize the running direction of blood vessels and the neighboring relationship with tumors. Higemi can improve the precision and safety of complex hepatectomy.

NF-κB signaling pathway confers neuroblastoma cells migration and invasion ability via the regulation of CXCR4.

BACKGROUND: Accumulating evidence implicates the transcription factor NF-κB as a positive mediator of tumor metastasis, but the molecular mechanism(s) involved in this process remains largely unknown. In this study, we investigated the role of NF-κB signaling pathway in the regulation of CXC chemokine receptor-4 (CXCR4) in neuroblastoma metastasis. MATERIAL AND METHODS: NF-κB, CXCR4 mRNA and protein expression were measured by RT-PCR, and Western blot. Tumor necrosis factor-α (TNF-α) was used to induce the upregulation of NF-κB and CXCR4. The knockdown of NF-κB and CXCR4 was achieved by PDTC. Transwell assay was used to investigate the role of NF-κB (P65) in neuroblastoma cell migration and invasion. An in vitro co-culture system was established to investigate the role of tumor microenvironment in regulation of the NF-κB signaling pathway. RESULTS: Over-expression of NF-κB (p65) promoted tumor migration and invasion through the upregulation of CXCR4; however, knockdown of NF-κB(P65) inhibited tumor migration and invasion through blocking the expression of CXCR4. Consistently, in the co-culture system, the expression of CXCR4 was partly dependent on the expression of NF-κB (p65). CONCLUSIONS: Our studies reveal critical roles for the NF-κB signaling pathway in neuroblastoma migration and invasion. The mechanism may be through up-regulation of CXCR4, mediated by the NF-κB signaling pathways. Targeting NF-κB signalling pathways and ultimately CXCR4 could be a strategy in neuroblastoma therapy.

Down-regulation of spinal D-amino acid oxidase expression blocks formalin-induced tonic pain.

A series of inhibitors of d-amino acid oxidase (DAAO) are specific in blocking chronic pain, including formalin-induced tonic pain, neuropathic pain and bone cancer pain. This study used RNA interference technology to further validate the notion that spinal DAAO mediates formalin-induced pain. To target DAAO, a siRNA/DAAO formulated in polyetherimide (PEI) complexation and a shRNA/DAAO (shDAAO, with the same sequence as siRNA/DAAO after intracellular processing) expressed in recombinant adenoviral vectors were designed. The siRNA/DAAO was effective in blocking DAAO expression in NRK-52E rat kidney tubule epithelial cells, compared to the nonspecific oligonucleotides. Furthermore, multiple-daily intrathecal injections of both siRNA/DAAO and Ad-shDAAO for 7 days significantly inhibited spinal DAAO expression by 50-80% as measured by real-time quantitative PCR and Western blot, and blocked spinal DAAO enzymatic activity by approximately 60%. Meanwhile, both siRNA/DAAO and Ad-shDAAO prevented formalin-induced tonic phase pain by approximately 60%. Multiple-daily intrathecal injections of siRNA/DAAO and Ad-shDAAO also blocked more than 30% spinal expression of GFAP, a biomarker for the activation of astrocytes. These results further suggest that down-regulation of spinal DAAO expression and enzymatic activity leads to analgesia with its mechanism potentially related to activation of astrocytes in the spinal cord.