接触式细胞共培养诱导人脐带华通胶间充质干细胞向类髓核细胞分化

目的:观察接触式细胞共培养条件下,人髓核细胞对脐带华通胶间充质干细胞(Wharton′s jelly-derived mesenchymal stem cells,WJMSCs)的诱导分化效应,为椎间盘退变性疾病的治疗寻找种子细胞来源。方法:取足月产健康新生儿脐带约30cm,分离、纯化、培养WJMSCs;取人椎间盘髓核组织,酶消化法分离培养髓核细胞。取第三代稳定增殖的WJMSCs,利用流式细胞方法检测细胞免疫表型CD73/CD90/CD105/CD34/CD45/HLA-ABC/HLA-DR。用羧基荧光素乙酰乙酸琥珀酰亚胺酯(CFSE)标记WJMSCs,与髓核细胞以1∶1比例进行混合,在6孔板中进行接触式细胞共培养;以单独培养的WJMSCs为对照组。培养7d后利用高速流式细胞仪分选荧光标记阳性的WJMSCs,提取细胞总RNA,进行反转录获得cDNA,利用Real-Time PCR方法检测其Ⅰ型胶原、Ⅱ型胶原、Ⅵ型胶原、蛋白多糖、SOX-9和多能聚糖基因的相对表达,管家基因GAPDH作为内参,单独培养的WJMSCs作为对照,利用2-ΔΔCt方法计算基因相对表达变化。结果:WJMSCs原代细胞接种24h可见部分细胞贴壁生长,形态为梭形和多角形,1周后形成集落,2周时细胞融合达到90%。流式细胞检测结果显示CD73/CD90/CD105/和HLA-ABC(+),CD34/CD45和HLA-DR(-)。与髓核细胞共培养7d后WJMSCs的Ⅱ型胶原、蛋白多糖和SOX-9基因相对表达较对照组显著性增加(P<0.01),Ⅰ型胶原、Ⅵ型胶原和多能聚糖基因相对表达未见显著性变化(P>0.05)。结论:通过接触式细胞共培养人脐带WJMSCs能够被髓核细胞诱导分化为类髓核细胞,可为组织工程技术和细胞治疗修复退变椎间盘提供种子细胞来源。     

Radiologic features compared to clinical findings in a prospective study of 153 patients with metastatic spinal cord compression treated by radiotherapy

Summary Plain radiography, myelography and post-myelographic CT-scan are described and related to clinical findings in a prospective study of 153 consecutive patients with myelographic signs of spinal cord compression. The majority of the metastatic tumours arise in the vertebral body or the pedicles. In 80% of the patients with total blockage to the contrast medium on myelography the post-myelographic-CT showed passage of the contrast medium. Ambulatory function at time of diagnosis was correlated to the degree and the localization of the epidural block. In 64 patients who underwent a second myelography, the post-treatment findings of sensory function were correlated to radiological regression.     

Effect of timing of surgery on neurological outcome and survival in metastatic spinal cord compression

Purpose

Metastatic spinal cord compression (MSCC) requires expeditious treatment. While there is no ambiguity in the literature about the urgency of care for patients with MSCC, the effect of timing of surgical intervention has not been investigated in detail. The objective of our study was to investigate whether or not the ‘timing of surgery’ is an important factor in survival and neurological outcome in patients with MSCC.

Methods

All patients with MSCC presenting to our unit from October 2005 to March 2010 were included in this study. Patients were divided into three groups—those who underwent surgery within 24 h (Group 1, n = 45), between 24 and 48 h (Group 2, n = 23) and after 48 h (Group 3, n = 53) from acute presentation of neurological symptoms. The outcome measures studied were neurological outcome (change in Frankel grade post-operatively), survival (survival rate and median survival in days), incidence of infection, length of stay and complications.

Results

Patients’ age, gender, revised Tokuhashi score, level of spinal metastasis and primary tumour type were not significantly different between the three groups. Greatest improvement in neurology was observed in Group 1, although not significantly when compared against Group 2 (24–48 h; (p = 0.09). When comparisons of neurological outcome were performed for all patients having surgery within 48 h (Groups 1 and 2) versus after 48 h (Group 3), the Frankel grade improvement was significant (p = 0.048) favouring surgery within 48 h of presentation. There was a negative correlation (−0.17) between the delay in surgery and the immediate neurological improvement, suggesting less improvement in those who had delayed surgery. There was no difference in length of hospital stay, incidence of infection, post-operative complications or survival between the groups.

Conclusions

Our results show that surgery should be performed sooner rather than later. Furthermore, earlier surgical treatment within 48 h in patients with MSCC resulted in significantly better neurological outcome. However, the timing of surgery did not influence length of hospital stay, complication rate or patient survival.     

Ambulation and survival following surgery in elderly patients with metastatic epidural spinal cord compression

Background and Context

Metastatic epidural spinal cord compression (MESCC) is a disabling consequence of disease progression. Surgery can restore or preserve physical function, improving access to treatments that increase duration of survival; however, advanced patient age may deter oncologists and surgeons from considering surgical management.

Spinal cord compression caused by plasmocytomas. A retrospective review of 14 cases

Plasmocytoma is a plasma cell tumor, which occurs in various structures of the body. When the spinal column is involved, it may cause cord compression. In this study, 14 cases of spinal plasmocytomas are presented. Seven of them were male and seven female. The major complaints were pain in twelve cases, motor weakness in eight cases, and bladder disturbance in six cases. On neurological examination, twelve of the patients had impairment of extremity movements (85.7%), and eight had sensory losses (57.1%). Compression was more frequent in the thoracic region. There was a total block in nine and partial block in five cases. All patients underwent surgery. In thirteen cases laminectomy was performed, in one thoracotomy.In this report, complaints, clinical and laboratory findings, neurological examination, and histopathology of our cases are reviewed and the results discussed.     

Evaluation of functional outcome and local control after radiotherapy for metastatic spinal cord compression in patients with prostate cancer

PURPOSE: Patients with prostate cancer with metastatic spinal cord compression have better survival prognosis than other patients with MSCC and may live long enough to develop a local recurrence of MSCC. This study investigates prognostic factors and radiation schedules for functional outcome and local control of MSCC after radiotherapy in patients with prostate cancer. MATERIALS AND METHODS: A total of 281 patients irradiated between January 1992 and December 2003 were included. Potential prognostic factors were investigated including age, performance status, number of involved vertebra, pretreatment ambulatory status, time of developing motor deficits before RT and radiation schedule--short course RT (1x8/5x4 Gy) vs long course RT (10x3/15x2.5/20x2 Gy). RESULTS: Overall response to RT was 86% (33% improvement of motor function, 53% no further progression). Of the nonambulatory patients 33% regained the ability to walk. On multivariate analysis functional outcome was significantly affected by the time of developing motor deficits before RT (more than 14 days better than 8 to 14 days and 1 to 7 days, p <0.001) and number of involved vertebrae (1 to 2 better than 3 or more, p = 0.013), but not by the radiation schedule (p = 0.859). The 2-year local control of MSCC was 84% depending on the radiation schedule (better after long course RT, p = 0.001). CONCLUSIONS: Functional outcome after RT was significantly influenced by the time of developing motor deficits before RT and number of involved vertebra. Local control was significantly better after application of long course RT. Patients with a poor expected survival could be treated with short course RT, because a short treatment time means less discomfort for the patient. For patients with good survival prognosis, long course RT should be applied to achieve better local control.     

转移瘤硬膜外脊髓压迫症运动功能障碍与影像学及临床特征相关性分析

目的：探讨转移瘤硬膜外脊髓压迫症（metastaticepiduralspinalcordcompression,MESCC）运动功能障碍与影像学及临床特征的相关性。方法：自2006年7月至2008年12月对连续收治的26例MESCC患者43个主要病变椎体进行MRI及CT扫描评估,并进行运动功能障碍评分。结果：26例MESCC患者中,12例发生内脏转移,其中10例发生运动功能障碍;14例无内脏转移,其中4例发生运动功能障碍（P=O．0079）。主要病变椎体连续组中,16椎发生运动功能障碍;主要病变椎体非连续组中,9椎发生运动功能障碍（P=0．1034）。主要病变椎体累及椎板组中,11椎发生运动功能障碍;主要病变椎体未累及椎板组中,14椎发生运动功能障碍（降0．0205）。主要病变椎体后壁向后突出组中,12椎发生运动功能障碍;主要病变椎体后壁无向后突出组中,13椎发生运动功能障碍（P=0．0334）。侵犯椎管内硬膜外组织组中,11椎发生运动功能障碍;未侵犯椎管内硬膜外组织组中,14椎发生运动功能障碍（P=O．0036）。转移瘤患者年龄、性别、术前接受正规化疗、转移灶部位腰背部疼痛程度、原发肿瘤已行根治手术、原发肿瘤治疗效果、脊柱外骨转移灶数目、主要受累脊椎数目、主要病变椎体节段、连续病变椎体节段、累及椎体、椎体前柱骨折、椎体后壁骨折、累及椎弓根等因素对MESCC运动功能障碍影响均无统计学意义（P〉0．05）。结论：发生内脏转移、主要病变椎体累及椎板、椎体后壁向后突出、转移瘤侵犯椎管内硬膜外组织的MESCC较易发生运动功能障碍,转移瘤连续病椎的发生率于颈椎和上胸椎组最高。     

Prediction of neurologic outcome in patients with spinal cord injury by using hyperbaric oxygen therapy

The effectiveness of hyperbaric oxygen therapy (HBO) in predicting neurological recovery in patients with spinal cord injury was evaluated. HBO has been used to treat spinal cord injury, but HBO does not appear to greatly alter the neurological outcome. This is the first report of the use of HBO as a diagnostic tool to evaluate neurological recovery after spinal cord injury. The study group consisted of 22 patients, aged 21–73 years, with spinal cord injuries. The effect of HBO was evaluated on admission and categorized as one of four grades (excellent, good, fair, or poor). The neurological status was evaluated on admission and at the time of follow-up, according to Frankel grade and the American Spinal Injury Association (ASIA) motor score. Correlations between the HBO effect and Frankel grade recovery and correlations between the HBO effect and recovery rate of the ASIA motor score were evaluated. The recovery in Frankel grade from admission to the final follow-up became better as the effectiveness of HBO increased (r = 0.445; P = 0.0414). The Frankel grade (r = 0.036; P = 0.871) and ASIA motor score (r = 0.029; P = 0.893) on admission did not correlate with the recovery in Frankel grade. There was a significant correlation between the HBO effect and the recovery rate of the ASIA motor score (r = 0.586; P = 0.0072), but this correlation was weaker than that for the ASIA motor score on admission (r = 0.752; P = 0.0006). We conclude that HBO can be employed to assess the status of spinal cord function recovery after spinal cord injury. Received: January 27, 2001 / Accepted: May 11, 2001     

Surgery and survival outcomes of 22 patients with epidural spinal cord compression caused by thyroid tumor spinal metastases

Purpose

The aim of this study was to discuss the clinical presentation, imaging findings, treatments received, and outcome of therapies for patients with epidural spinal cord compression caused by thyroid spinal metastases, with the goal of emphasizing the importance of surgery in this setting and discussing therapeutic plan for treating these patients.

Methods

A total of 22 patients with spinal cord compression due to thyroid tumor spinal metastases who received surgery in our department were identified from 2004 to 2011. The series of 22 patients collected from our institution over the past 7 years was used to discuss treatment options for thyroid cancer spinal metastases on the basis of literature review and our own extensive experience.

Results

The mean age of the patients in this study was 57 years (range 37–78 years). The duration of the preoperative symptoms was 1–24 months, with an average of approximately 6 months. All patients attained improvement of at least one level of the Frankel classification after surgery. Two patients received more than one operation at our institution. Two patients died during follow-up, two patients had stable disease, and all other patients maintained a disease-free status during follow-up.

Conclusions

As thyroid tumor spinal metastases have a favorable prognosis, a radical therapeutic attitude should be considered in decision-making. Dorsal spinal decompression through curettage and stabilization can preserve or restore neurological function for most patients. For patients who have more than one metastatic lesion of the spine, surgeries can be sequentially performed based on the urgency of the case. In addition to treatment of primary disease, surgery and bisphosphonate treatment are the most important therapies for these patients.     

后路椎板切除椎管减压钉棒系统内固定术治疗脊柱转移瘤硬膜外脊髓压迫症疗效分析

目的探讨后路椎板切除椎管减压钉棒系统内固定术治疗脊柱转移瘤硬膜外脊髓压迫症的应用价值。方法对64例脊柱转移瘤硬膜外脊髓压迫症患者采用后路椎板切除椎管减压钉棒系统内固定术治疗。比较治疗前后患者的疼痛评分、生活质量及身体一般状况评分。结果治疗后患者的疼痛评分、生活质量评分及身体一般状况评分均较治疗前显著改善,差异有统计学意义(P0.05)。结论采用后路椎板切除椎管减压钉棒系统内固定术治疗脊柱转移瘤硬膜外脊髓压迫症,疗效确切,可显著缓解患者疼痛感,改善其身体一般状况,提高生活质量。     

临床症状轻微型重度结核性脊髓压迫症

[目的]探讨临床症状轻微型重度结核性脊髓压迫症的致病机理、临床特点及外科治疗方法。[方法]对15例临床症状轻微的重度结核性脊髓压迫症患者采用彻底病灶清除、植骨及选择性前路或后路器械内固定手术,分析手术适应证、疗效及外科治疗特点。[结果]术后随访6个月-7年,平均3．8年,15例患者结核病灶清除彻底,结核治愈无复发。合并不全瘫5例中,术前Frankel分级C级1例恢复至E级,D级4例中恢复至E级2例,1例无变化,1例术后脊髓功能损伤加重。[结论]临床症状轻微型结核性脊髓压迫因发病过程缓慢,脊髓有一定自行调整与适应时间和空间,但脊髓仍处于危象状态,早期手术可有效降低脊髓进行性损伤。     

甲基强的松龙在脊柱转移瘤所致恶性脊髓压迫症围手术期的应用

目的：观察围手术期应用甲基强的松龙（MP）对脊柱转移瘤所致恶性脊髓压迫症患者脊髓神经功能恢复的作用。方法：2002年1月～2007年5月，在我院手术治疗的脊柱转移瘤所致恶性脊髓压迫症患者22例，13例采用一期后路椎管减压、肿瘤姑息切除手术治疗，减压前30min给予MP1000mg冲击（用药组），术后第1天起予MP80～120mg静脉点滴，每日2次，共应用3～5d；同期9例未使用MP而其余处理相同的同类型患者作为对照组。对术前、术后1d、术后1周和术后1个月时两组患者的脊髓功能进行ASIA评分。结果：两组患者无围手术期死亡病例，全部病例随访2～27个月，用药组与对照组术前的ASIA评分无显著性差异（P〉0．05），术后1d、1周和1个月时两组ASIA评分有显著性差异（P〈0．05）。结论：对于脊柱转移瘤所致恶性脊髓压迫症患者，围手术期应用MP有利于保护脊髓神经功能。     

Prognostic value of changes in spinal cord signal intensity on magnetic resonance imaging in patients with cervical compressive myelopathy

Background contextSignal intensity on preoperative cervical magnetic resonance imaging (MRI) of the spinal cord has been shown to be a potential predictor of outcome of surgery for cervical compressive myelopathy. However, the prognostic value of such signal remains controversial. One reason for the controversy is the lack of proper quantitative methods to assess MRI signal intensity.PurposeTo quantify signal intensity and to correlate intramedullary signal changes on MRI T1- and T2-weighted images (WIs) with clinical outcome and prognosis.Study designRetrospective case study.Patient samplePatients (n=148; cervical spondylotic myelopathy, n=102 and ossified posterior longitudinal ligament, n=46) who underwent surgery for cervical compressive myelopathy and had high signal intensity change on sagittal T2-WI MRI before surgery between 2006 and 2010.Outcome measureNeurologic assessment was conducted with the Japanese Orthopedic Association (JOA) scoring system for cervical myelopathy. The rate of neurologic improvement was calculated with the use of preoperative and postoperative JOA scores.MethodsQuantitative analysis of MRI signal on both T1- and T2-WIs via use of the signal intensity ratio (SIR; signal intensity of lesion relative to that at C7-T1 disc level) was performed. Correlations between SIR on T1- and T2-WIs and preoperative JOA score, JOA improvement rate, disease duration, and MRI morphologic classification (cystic or diffuse type) were analyzed. Multivariate regression analysis for JOA improvement rate was also analyzed. In a substudy, 25 patients underwent follow-up MRI starting from 6 months after surgery to analyze the relationship between changes in SIR on follow-up MRI and clinical outcome.ResultsSIR on T1-WIs, but not SIR on T2-WIs, correlated with postoperative neurologic improvement. The disease duration correlated negatively with SIR on T1-WIs and JOA improvement rate but not with SIR on T2-WIs. SIR on T2-WIs of “cystic type” was significantly greater than of “diffuse type,” but SIR on T1-WI and JOA improvement rate were not different in the two types. Stepwise multivariate regression analysis indicated that SIR on T1-WIs and long disease duration were significant predictors of postoperative neurologic outcome. SIR on follow-up T1-WI and changes in SIR on T1-WI after surgery correlated positively with postoperative improvement rate. SIR on follow-up T2-WI and changes on T2-WI correlated negatively with postoperative neurologic improvement.ConclusionsOur results suggest that low intensity signal on preoperative T1-WIs but not T2-WIs correlated with poor postoperative neurologic outcome. Furthermore, decreased signal intensity on postoperative T1-WIs and increased signal intensity on postoperative T2-WIs are predictors of poor neurologic outcome.     

Comparison of continuous spinal and continuous epidural anaesthesia for lower limb surgery in elderly patients A retrospective study

This retrospective study compared continuous spinal anaesthesia with continuous epidural anaesthesia for lower limb orthopaedic surgery in the elderly. The anaesthetic records of 457 patients who received continuous spinal anaesthesia and 274 who received continuous epidural anaesthesia over a 5-year period were analysed. The patients who had continuous spinal anaesthesia were at a higher anaesthetic risk (ASA 3-4, 76% as compared with 37%, p less than 0.001), but the incidence of failures was significantly lower (1.7%, as compared with 9%, p less than 0.001) and fewer patients showed a decrease in mean arterial pressure of more than 30% (44%, as compared with 65%, p less than 0.001) and (or) received vasopressors (65%, as compared with 77%, p less than 0.01). Our data show continuous spinal anaesthesia to be more reliable and to provide better cardiovascular stability.     

无脊髓压迫症状颈椎后纵韧带骨化患者的影像学特点及临床意义

目的:探讨无脊髓压迫症状颈椎后纵韧带骨化(OPLL)患者的影像学特点及临床意义。方法:分析42例无脊髓压迫症状颈椎OPLL患者初次就诊的影像学资料,男25例,女17例,年龄40～78岁,平均57岁。根据影像学表现对OPLL进行分型,观察椎管最大受压处骨化物占位率(OPLL占位率)与椎管最大受压节段活动范围(ROM)的相关性,同时观察MRI T2像上脊髓内信号的变化并随访患者症状进展情况。结果:根据Tsuyama分型标准,本组连续型24例,混合型10例,节段型8例。OPLL占位率20%～64%,平均38.4%;最大受压节段ROM平均4.5°。线性回归显示OPLL椎管占位率与ROM呈负相关(P<0.01)。所有患者未出现MRI T2相脊髓内信号改变。随访2年～5年6个月,平均3年8个月,所有患者末次随访查体均未发现脊髓压迫症临床表现。结论:无脊髓压迫症状的颈椎OPLL,以连续型骨化多见,椎管最大受压节段活动范围较小可能是其无脊髓压迫症状的原因之一。     

急性重度颈脊髓损伤患者临床特征分析及远期死亡危险因素初探

目的:总结重度创伤性颈脊髓损伤患者临床特征,分析影响其远期死亡的相关因素。方法:回顾性分析我院2010年1月1日～2018年5月31日收治的207例重度颈脊髓损伤患者临床特点,统计其人口学特征(性别、年龄等)、致伤因素、损伤特点(责任节段、AISA分级、合并伤等)、住院治疗情况(是否手术、手术方式、手术时机等)和并发症以及远期死亡情况,建立二元Logistic回归模型分析远期死亡的危险因素。结果:男性183例,女性24例,男女比例为7.63∶1,年龄15～74岁,平均44.7±13.7岁,以高能量损伤(包括高处坠落、车祸、重物砸伤以及其他复合伤)多见(159例,76.8%),50岁以下患者134例(64.73%),损伤节段C1～C2(3例,1.4%)、C3(28例,13.5%)、C4～C5(124例,58%)、C6(25例,12.1%)、C7(26例,12.6%)、C8(5例,2.4%),所有患者治疗受伤至住院时间3.6±3.5d,住院时间为16.2±16.9d;住院期间以呼吸系统(63例,30.4%)和水电解质紊乱(25例,12.1%)为主要并发症。8年间总死亡率为30.91%(64例),多元Logistic回归分析显示,年龄≥50岁、C1-C4损伤、保守治疗、需要呼吸机辅助呼吸是远期死亡的危险因素(P0.05)。结论:重度创伤性颈脊髓损伤常见于中青年男性患者,损伤部位好发于C4、C5节段,以高能量暴力创伤为主;高龄、上颈椎损伤、未行手术减压及合并呼吸功能障碍易导致患者远期死亡。     

Demographic and clinical characteristics of patients with transverse myelitis and traumatic spinal cord injury: A comparative retrospective study

ObjectivesTo identify the demographic and clinical characteristics of patients with transverse myelitis (TM) and to compare functional status between those patients and a matched group with traumatic spinal cord injury (T-SCI).Study DesignRetrospective study.SettingA tertiary rehabilitation hospitalParticipantsThe demographic and clinical characteristics of 484 T-SCI patients and 25 TM patients were compared. Functional status was further analyzed by matching the two groups.Outcome MeasurementsThe matched patients were compared in terms of motor and sensory functions, bladder and bowel symptoms, ambulation level, the Rivermead Mobility Index, and SCI-related medical complications.ResultsThe mean age of the TM patients was 35.6 years and was similar to that of the T-SCI patients. There were significantly more females in the TM group (P = 0.017). Individuals with TM had fewer cervical injuries (P = 0.032) and a higher rate of paraplegia (P = 0.047) and were more often incomplete (P = 0.009) than those with T-SCI. Sensory function was significantly better in the TM group compared to the matched T-SCI group (P = 0.05). Independent ambulation frequency was higher in the TM patients. The SCI-related complications seen in the TM group were as common as those in the T-SCI group.ConclusionThe TM and T-SCI groups differed in terms of the demographic and clinical characteristics recorded. Additionally, when matched for these differences, functional status was slightly better in the TM group. However, like T-SCI, TM was a significant cause of disability and SCI-related complications were common.     

Venous thromboembolic complications with and without intermittent intraoperative and postoperative pneumatic compression in patients with glioblastoma multiforme using intraoperative magnetic resonance imaging. A retrospective study

Objective

To evaluate the effectiveness of intraoperative and postoperative intermittent pneumatic compression (IPC) as a method used to decrease the incidence of deep venous thrombosis (DVT), in comparison to the standard use of graduated compression stockings, low-molecular weight heparin (LMWH) and physiotherapy during the hospital stay. All patients in this study underwent intracranial surgery for glioblastoma multiforme (GBM) using intraoperative magnetic resonance imaging (MRI) guidance.

A retrospective review of safety using a nursing driven protocol for autonomic dysreflexia in patients with spinal cord injuries

Objective/Background: Autonomic dysreflexia is a potentially life-threatening condition which afflicts a significant proportion of individuals with spinal cord injuries (SCI). To date, the safety and efficacy of several commonly used interventions for this condition have not been studied.

Design: A retrospective chart review of the safety of a previously implemented nursing driven inpatient autonomic dysreflexia protocol.

Methods: Seventy-eight male patients with SCI who experienced autonomic dysreflexia while inpatient at our Veterans Affairs SCI unit over a 3–1/2-year period were included. The safety of a nursing driven protocol utilizing conservative measures, nitroglycerin paste, and oral hydralazine was evaluated.

Outcome Measures: Occurrence of adverse events and relative hypotensive events during all episodes treated with the protocol, and efficacy of attaining target blood pressure for all episodes with protocol adherence and for initial episode experienced by each patient.

Results: Four hundred forty-five episodes of autonomic dysreflexia were recorded in the study period, with 92% adherence to the protocol. When the protocol was followed, target blood pressure was achieved for 97.6% of all episodes. Twenty-three total adverse events occurred (5.2% of all episodes). All adverse events were due to hypotension and only 0.9% required interventions beyond clinical monitoring. Of each patient's initial autonomic dysreflexia episode, 97.3% resolved using the protocol without need for further escalation of care.

Conclusion: This inpatient nursing driven-protocol for treating autonomic dysreflexia utilizing conservative measures, nitroglycerin paste and oral hydralazine achieved target blood pressure with a high success rate and a low incidence of adverse events.