膜性高分子材料在兔眼部的生物相容性

目的评价聚羟基脂肪酸(polyhydroxyalkanoates,PHA)、聚乳酸(polylacetic acid,PLA)和聚己内酯(polycaprolactone,PCL)三种膜性高分子材料在兔眼部的生物相容性。方法将24只新西兰兔随机分为4组,每组6只。PHA、PLA、PCL为实验组,材料植入兔右眼结膜下。假手术组结膜下钝性分离,但不植入任何高分子材料。使用裂隙灯显微镜观察并记录植入后不同时间手术眼的反应并评分。裂隙灯下观察材料的吸收时间。术后4周和16周取眼球,行HE染色、Masson染色和天狼猩红染色分别定性观察组织结构和炎症细胞、胶原纤维和胶原纤维的亚型与排列方向。结果术眼刺激性评分等级各组均不高于"轻度刺激性"。结膜下吸收时间PHA、PLA和PCL组分别是16周,12周和大于16周。组织学观察术后4周PHA、PLA和PCL组均形成材料包裹囊腔,囊壁以纤维组织为主,伴有毛细血管形成和炎性细胞浸润,以中性粒细胞为主。胶原纤维染色与假手术组无明显差异,以Ⅰ型和Ⅲ型为主,大致呈平行排列。术后16周PHA和PLA组材料已不可查及,包裹囊腔结构不规则,而PCL材料整体可查及,包裹囊腔规则。各组未见毛细血管,偶见淋巴细胞浸润。胶原纤维与假手术组无明显差异,以Ⅰ型和Ⅲ型为主,仍大致呈平行排列。结论 PHA、PLA和PCL三种膜性高分子材料在兔眼具有较好的生物相容性,结膜下吸收时间分别是16周,12周和大于16周。     

儿童过敏性结膜炎与变应性鼻炎的相关性及鼻眼联合防治效果分析

目的：探讨儿童过敏性结膜炎与变应性鼻炎的相关性研究及鼻眼联合防治的临床效果。方法：回顾性分析300 例儿童过敏性 结膜炎与310 例儿童变应性鼻炎患者的临床资料,对儿童过敏性结膜炎与变应性鼻炎的相关性进行分析后将所有患儿随机均分 为对照组与观察组,对照组采用常规点眼的方法进行治疗,观察组则采用鼻朗喷鼻联合人工泪液点眼进行治疗。比较两组临床疗 效及不良反应情况。结果：（1）300 例过敏性结膜炎患儿中,50 例（16.67%）并发变应性鼻炎;310 例变应性鼻炎患儿中,59 例 （19.03%）并发过敏性结膜炎（P＞0.05）;（2）109 例同时并发两种疾病患儿中,均进行眼结膜与鼻粘膜的刮片检查嗜酸性粒细胞, 其中60 例（55.05%）结膜刮片与67 例（61.47%）鼻粘膜刮片检测到嗜酸性粒细胞（P＞0.05）;（3）两组治疗前后BUT 及角膜荧光素 染色评分、症状评分、临床总有效率比较差异明显（P＜0.05）。结论：儿童过敏性结膜炎与变应性鼻炎具有一定的相关性;鼻朗喷鼻 联合人工泪液点眼治疗儿童合并变应性鼻炎的临床疗效显著。     

电针对佐剂性关节炎大鼠踝关节周围皮肤及皮下组织NK-1受体与IL-1β荧光双标免疫反应性的影响

目的探讨电针治疗佐剂性关节炎的外周神经免疫机制.方法用佐剂性关节炎模型,采用免疫荧光双标技术,观察了致炎后第3d佐剂性关节炎大鼠踝关节周围皮肤及皮下组织NK-1受体与IL-1β荧光双标免疫反应性,及电针胆经环跳穴、阳陵泉穴(刺激参数为0.5～1.5V,4～16Hz,30min)对其是否具有调控作用.结果 (1)各组大鼠炎症侧外踝关节周围皮肤及皮下组织均可见IL-1β/NK-1受体阳性双标细胞.(2)致炎后第3d,炎症组大鼠外踝关节周围皮肤及皮下组织IL-1β/NK-1受体阳性双标细胞数目较生理盐水组显著升高(P<0.01).电针组大鼠双标细胞数目较生理盐水组、炎症组显著降低(P<0.01).(3)电针组双标细胞占NK-1受体阳性细胞百分比显著低于生理盐水组、炎症组(P<0.05,P<0.01).(4)电针组双标细胞占IL-1β阳性细胞百分比较生理盐水组、炎症组显著降低(P<0.05).结论电针可抑制炎症侧外踝关节周围皮肤及皮下组织NK-1受体阳性的免疫细胞中IL-1β的合成,并下调IL-1β阳性的免疫细胞中NK-1受体表达,提示电针可能通过阻断炎症灶局部炎症因子、致痛物质相互促生的恶性循环,从而起到消炎镇痛的作用.     

右美托咪定后处理对胸部撞击-失血性休克和复苏致急性肺损伤的影响

为探讨右美托咪定后处理对胸部撞击失血性休克和复苏致急性肺损伤的影响。本研究选取45只SPF级雄性健康大鼠依据随机数字表法将其分为3组,生理盐水组、THSR组(胸部撞击-失血性休克/复苏组)和治疗组(右美托咪定后处理组),THSR组和治疗组制备胸部撞击-失血性休克/复苏致急性肺损伤模型,治疗组模型建立后静脉注射右美托咪定10μg/kg。模型制备6 h后采血,并处死大鼠。比较3组大鼠血气分析指标(PaO_2,PaCO_2和氧合指数(OI))、肺组织病理形态、炎症因子水平(血浆IL-6和IL-1β水平,肺泡灌洗液(BALF)中蛋白浓度和白细胞数目)以及肺组织中TLR4和p-p38MAPK表达水平。与生理盐水组比较,THSR组和治疗组的PaO_2升高(p0.05),OI降低(p0.05),THSR组PaCO_2升高(p0.05),THSR组和治疗组的PaO_2、PaCO_2和OI差异具有统计学意义(p0.05);THSR组疗组大鼠肺组织病理学损伤评分、血浆炎症因子(BLAF蛋白,白细胞计数,IL-6和IL-1β)水平最高,治疗组次之,生理盐水组最低(p0.05);THSR组和治疗组大鼠肺组织中TLR4和p-p38MAPK表达较生理盐水组上调(p0.05),治疗组大鼠肺组织中TLR4和p-p38MAPK表达水平较THSR组显著下调(p0.05)。右美托咪定后处理可明显减轻胸部撞击-失血性休克和复苏致急性肺损伤,其起效机制可能与抑制TLR4/p-p38MAPK信号通路激活,降低机体炎性反应有关。     

左旋紫草素抗炎作用的实验研究

目的:探讨左旋紫草素的抗炎药理作用.方法:采用二甲苯涂抹小鼠耳面,致使毛细血管渗出导致耳肿胀和醋酸所致小鼠腹腔毛细血管通透性增高的炎症模型,观察左旋紫草素对急性炎症的对抗作用;采用大鼠腋窝皮下置无菌棉球的炎症模型,观察左旋紫草素对亚急性炎症的对抗作用.结果:与生理盐水组比较,左旋紫草素低、中、高三个剂量组和地塞米松组均能显著抑制二甲苯所致的小鼠耳廓肿胀和醋酸所致的小鼠腹腔毛细血管通透性增高(P<0.05),对大鼠棉球肉芽肿组织增生的炎症模型也有明显的抑制作用(P<0.05),且左旋紫草素高剂量组作用接近地塞米松组.结论:左旋紫草素对急性和亚急性炎症动物模型均有明显的对抗作用,这一作用可能是其作为抗炎药物应用的药理学基础.     

明目羊肝丸联合普拉洛芬滴眼液治疗围绝经期干眼症的临床研究

摘要 目的：探讨明目羊肝丸联合普拉洛芬滴眼液治疗围绝经期干眼症的临床疗效。方法：选取2019年7月至2020年8月在我院就诊的围绝经期干眼症患者103例206眼,根据随机数字表法将患者分为对照组（普拉洛芬滴眼液治疗）和实验组（对照组的基础上联合明目羊肝丸治疗）,分别为51例102眼和52例104眼,两组均治疗2个月。对比两组疗效、症状评分、泪液炎症因子、生存质量、Schirmer I试验（SIT）、泪膜破裂时间（BUT）、角膜荧光素染色（FL）评分及不良反应。结果：实验组治疗2个月后的临床总有效率优于对照组（P<0.05）。治疗2个月后,实验组眼干涩、视疲劳、异物感、灼热感症状评分、FL评分低于对照组（P<0.05）。治疗2个月后,实验组BUT、SIT高于对照组（P<0.05）。治疗2个月后,实验组泪液白介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）水平低于对照组（P<0.05）。治疗2个月后,实验组心理健康、眼痛、社会活动、一般健康评分高于对照组（P<0.05）。两组治疗过程中均无明显不良反应。结论：明目羊肝丸联合普拉洛芬滴眼液治疗围绝经期干眼症,可改善患者症状和泪膜稳定性,降低泪液炎症因子水平,提高患者生存质量,安全有效。     

大气细颗粒物致炎动物模型系统评价

目的 探索大气污染对动物的致病机制,对BALB/c小鼠采用无创性气管滴注PM2.5颗粒悬浮液的方法,构建大气污染致炎动物模型。方法 将150只SPF级BALB/c小鼠随机分成空白对照组、生理盐水组、PM2.5低度组(2.5 mg/kg)、PM2.5中度组(5 mg/kg)和PM2.5高度组(10 mg/kg)共5组,各剂量组气管滴注第3天,第7天、第21天、第35天、第49天,气管滴注操作完成后24 h采取组织样本,采用ELISA、肺组织病理HE染色的方法,来验证无创性气管滴注方法的可行性和致炎模型构建成功与否。结果 本建模方法,成功率高达96%。采用气管滴注法,建模小鼠肺组织炎症评分与气道滴注时间的延长和剂量呈正相关。PM2.5暴露后,肺内有大量淋巴细胞聚集及吞噬颗粒的巨噬细胞浸润,肺泡间隔增宽。各暴露组分别与生理盐水对照组、空白组比较,肺泡灌洗液中炎症因子IL-6、肺组织匀浆中TNF-α水平增高,高剂量组差异最显著。结论 本实验用气管滴注法建立小鼠致炎模型成功,并证明此方法简单、可靠,可广泛用于小鼠呼吸系统重复滴注,有利于进一步研究大气污染及其他致炎机制。     

眼色素层炎的免疫机制及免疫治疗

眼色素层由脉络膜、睫伏体、虹膜组成。这些部位的炎症反应称眼色素层炎。眼内的炎症主要有两类：抗原特异性免疫应答介导;非特异性炎症反应。本文重点综述抗原特异性免疫应答介导的眼色素层炎的发病机制及免疫治疗。过去认为梅毒螺旋体、结核杆菌是大多数眼色素层炎的病因,近几年来大量实验和临床研究证实,免疫应答在感染性和非感染性眼色素层炎的发生中起重要作用,对其机制有更深入的了解,以Ｔ 细胞介导的细胞免疫为主要损伤机制,根据免疫机制的各个环节建立了新的免疫治疗方法,包括环孢素Ａ 免疫抑制剂的使用、口服耐受的诱导以及细胞粘附分子疗法。     

蛇伤胶囊对竹叶青蛇伤t-PA、PAI-1和AT-Ⅲ的影响

目的研究蛇伤胶囊治疗竹叶青蛇伤凝血障碍的机制。方法根据前期实验复制竹叶青蛇伤的动物及细胞模型,均设置空白组(KB),模型组(MX),低、中、高剂量蛇伤胶囊药液组(DY、ZY、GY),共5组,每组10个样本。在动物实验中,KB组经兔右后腿皮下注射0.75ml/kg生理盐水,6h后灌胃10ml/kg生理盐水。MX、DY、ZY和GY组经兔右后腿皮下注射0.75ml/kg竹叶青蛇毒液,6h后分别灌胃10ml/kg生理盐水和10ml/kg低、中、高剂量蛇伤胶囊药液。各组均每日灌胃1次,连续灌胃1周。于末次灌胃后24h经耳缘静脉采血,分离血清。在细胞实验中,KB组加入10%正常兔血清培养,MX组在KB组的基础上加入5μg/ml竹叶青蛇毒液培养,DY、ZY、GY组在MX组基础上培养6h后分别加入5%、10%、15%的含中药兔血清继续培养,各组分别培养72h后,收集细胞培养液。以酶联免疫吸附法检测兔血清及细胞培养液中组织型纤溶酶原激活剂(tissue plasminogen activator,t-PA)、纤溶酶原激活物抑制物-1(plasminogen activator inhibitor-1,PAI-1)和抗凝血酶-Ⅲ(Antithrombin-Ⅲ,AT-Ⅲ)水平。结果 MX组的AT-Ⅲ和t-PA水平相对KB组均显著升高(P0.01),PAI-1水平较KB组显著降低(细胞实验中,P0.05;动物实验中,P0.01);DY、ZY和GY组的AT-Ⅲ及t-PA水平相对MX组均明显降低(动物实验:ZY组t-PA和DY组AT-Ⅲ,均P0.05;细胞实验:ZY组AT-Ⅲ,P0.05;其余均P0.01),DY、ZY和GY组的PAI-1水平较MX组明显升高(P0.01)。结论蛇伤胶囊具有促凝和抗纤溶、改善机体凝血功能的作用,是蛇伤胶囊治疗竹叶青蛇伤凝血障碍的部分机制。     

兔眼增殖性玻璃体视网膜病变模型的建立

目的探讨兔眼增殖性玻璃体视网膜病变模型的建立方法。方法①体外培养兔眼视网膜色素上皮细胞;②兔眼3组,每组6只,分别在玻璃体内注射0.1 mL的生理盐水、1×106细胞及2×106细胞,在不同时间段进行裂隙灯显微镜、间接检眼镜、眼底照像和B超检查,观察成模情况。结果注射后28 d,生理盐水组成模0眼;1×106细胞组成模5眼,其中Ⅰ级眼1只,Ⅱ级眼3只,Ⅲ级眼1只;2×106细胞组成模6眼,其中Ⅱ级眼2只,Ⅲ级眼4只。结论兔眼玻璃体内注射2×106同种视网膜色素上皮细胞建立增殖性玻璃体视网膜病变模型,符合病变发展规律,而且稳定可靠,成模较快,简单易行。     

Reduced bone mineral density and radial bone growth in young rabbits treated with dexamethasone eye drops

OBJECTIVE: To investigate the effect of dexamethasone eye drops on bone metabolism in newborn rabbits. METHODS: Thirty-four 3-week-old rabbits had unilateral clear lens extraction and were randomized into three groups. Postoperatively, group 1 received high-dose and group 2 low-dose dexamethasone eye drops (average doses 0.27 and 0.10 mg/kg body weight/day, respectively). These rabbits also received a postoperative subconjunctival injection of betamethasone. Group 3 (control) received vehicle eye drops only. After 8 weeks of treatment, all animals were killed and the left femurs were isolated and subjected to peripheral quantitative computerized tomography (pQCT) and dual X-ray absorptiometry (DXA) analyses. RESULTS: DXA showed that rabbits treated with either a high or low dose of dexamethasone eye drops had significantly reduced areal bone mineral density (BMD), area and total bone mineral content (BMC) of the femur. Measurements with pQCT demonstrated a dose-dependent reduction in cortical BMC, cortical volumetric BMD and cortical area. These effects were associated with an inhibition of radial femur growth, cortical thickness and periosteal and endosteal circumferences. CONCLUSION: Dexamethasone eye drops have systemic effects affecting several bone parameters in young rabbits. Any long-term systemic effects of ocular glucocorticoids need to be further studied.     

Influence of anesthesia on ocular effects and temperature in rabbit eyes exposed to microwaves

To investigate the effect of systemic anesthesia on ocular effects and temperature in rabbit eyes exposed to microwaves, one eye each of 43 male pigmented rabbits (Dutch, 1.8-2.2 kg) was exposed at 2.45 GHz for 60-20 min (300 mW/cm2; 108 W/kg), either under anesthesia (ketamine hydrochloride (5 mg/kg) + xylazine (0.23 mg/kg)) or without anesthesia. Changes in the anterior segment were evaluated by image analysis utilizing a Scheimpflug camera, specular microscopy, and a laser flare cell meter. Temperatures within the eye were measured during microwave exposure by a Fluoroptic thermometer. The exposed eyes showed miosis, conjunctival congestion, corneal edema, and an increase in the light scattering of the anterior shallow cortex in the pupillary area of the lens. The group under systemic anesthesia showed much stronger symptoms than those treated without anesthesia. All of the anterior ocular changes disappeared within a week. The highest temperature during exposure was in the vitreous, followed by the anterior chamber, and the retrobulbar cavity of the orbit. The ocular temperatures of the rabbits under systemic anesthesia were 2-9 degrees C higher than those without anesthesia. Body temperature showed an increase of 1 degrees C during the exposure. Acute high intensity microwave exposure temporarily induced anterior segments inflammation and lens changes. The more pronounced ocular effects in the anesthetized rabbits were associated with the significantly higher ocular temperatures in the anesthetized animals. The influence of systemic anesthesia on ocular changes should be considered.     

复明片联合曲伏前列素滴眼液对原发性开角型青光眼患者眼部血流动力学和房水EPO、sCD44的影响

摘要 目的：探讨复明片联合曲伏前列素滴眼液对原发性开角型青光眼（POAG）患者眼部血流动力学和房水促红细胞生成素（EPO）、可溶性CD44（sCD44）的影响。方法：选择2017年4月~2020年11月在本院接受治疗的112例193眼的POAG患者,根据随机数字表法分为对照组（n=56,96眼）和研究组（n=56,97眼）,对照组患者接受曲伏前列素滴眼液治疗,研究组接受复明片联合曲伏前列素滴眼液治疗,对比两组疗效、眼部血流动力学和房水EPO、sCD44,视力、视野、眼压,观察治疗期间不良反应发生状况。结果：研究组的临床总有效率高于对照组（P<0.05）。治疗12周后,研究组平均光敏感度（MS）、视力高于对照组,24 h眼压平均值小于对照组（P<0.05）。治疗12周后,研究组舒张末期流速（EDV）、收缩期峰值流速（PSV）高于对照组,血流阻力系数（RI）小于对照组（P<0.05）。治疗12周后,研究组房水EPO、sCD44水平低于对照组（P<0.05）。两组不良反应发生率组间对比无统计学差异（P>0.05）。结论：复明片联合曲伏前列素滴眼液治疗POAG患者,可促进患者视力、视野、眼压改善,可能与调节眼部血流动力学和房水EPO、sCD44水平有关。     

杞菊地黄汤联合玻璃酸钠滴眼液对肝肾阴虚型干眼症患者眼表功能和泪液MMP-2、MMP-9水平的影响

摘要 目的：观察杞菊地黄汤联合玻璃酸钠滴眼液治疗肝肾阴虚型干眼症的疗效及对眼表功能和泪液基质金属蛋白酶-2（MMP-2）、基质金属蛋白酶-9（MMP-9）的影响。方法：病例来源于2018年6月~2021年3月期间就诊于湖南省直中医医院眼科门诊的干眼症患者,共98例。采用随机数字表法分为对照组和实验组,两组均49例。对照组给予玻璃酸钠滴眼液治疗,实验组在对照组基础上接受杞菊地黄汤治疗,两组均治疗4周。对比两组疗效、中医证候积分、眼表功能和泪液MMP-2、MMP-9水平变化,观察两组治疗期间不良反应发生率。结果：实验组的临床总有效率高于对照组（P<0.05）。实验组治疗4周后眼部干涩不爽、畏光、双目频眨、口干少津、白眼泛红、舌红、苔薄、脉细证候积分均低于对照组（P<0.05）。实验组治疗4周后角膜荧光素染色（FL）评分低于对照组,泪膜破裂时间（BUT）、泪液分泌试验（SIT）水平高于对照组（P<0.05）。实验组治疗4周后泪液MMP-2、MMP-9水平低于对照组（P<0.05）。两组间不良反应发生率无统计学差异（P＞0.05）。结论：杞菊地黄汤联合玻璃酸钠滴眼液治疗肝肾阴虚型干眼症,可促进患者症状缓解,改善眼表功能,降低泪液MMP-2、MMP-9水平,安全可靠。     

Anti-inflammatory, antioxidant and antimicrobial activity of Ophthacare brand, an herbal eye drops.

In the present study, the herbal preparation of Ophthacare brand eye drops was investigated for its anti-inflammatory, antioxidant and antimicrobial activity, using in vivo and in vitro experimental models. Ophthacare brand eye drops exhibited significant anti-inflammatory activity in turpentine liniment-induced ocular inflammation in rabbits. The preparation dose-dependently inhibited ferric chloride-induced lipid peroxidation in vitro and also showed significant antibacterial activity against Escherichia coli and Staphylococcus aureus and antifungal activity against Candida albicans. All these findings suggest that Ophthacare brand eye drops can be used in the treatment of various ophthalmic disorders.     

Extension of the rank sum test for clustered data: two-group comparisons with group membership defined at the subunit level

The Wilcoxon rank sum test is widely used for two-group comparisons for nonnormal data. An assumption of this test is independence of sampling units both between and within groups. In ophthalmology, data are often collected on two eyes of an individual, which are highly correlated. In ophthalmological clinical trials, randomization is usually performed at the subject level, but the unit of analysis is the eye. If the eye is used as the unit of analysis, then a modification to the usual Wilcoxon rank sum variance formula must be made to account for the within-cluster dependence. For some clustered data designs, where the unit of analysis is the subunit, group membership may be defined at the subunit level. For example, in some randomized ophthalmologic clinical trials, different treatments may be applied to fellow eyes of some patients, while the same treatment may be applied to fellow eyes of other patients. In general, binary eye-specific covariates may be present (scored as exposed or unexposed) and one wishes to compare nonnormally distributed outcomes between exposed and unexposed eyes using the Wilcoxon rank sum test while accounting for the clustering. In this article, we present a corrected variance formula for the Wilcoxon rank sum statistic in the setting of eye (subunit)-specific covariates. We apply it to compare ocular itching scores in ocular allergy patients between eyes treated with active versus placebo eye drops, where some patients receive the same eye drop in both eyes, while other patients receive different eye drops in fellow eyes. We also present comparisons between the clustered Wilcoxon test and each of the signed rank tests and mixed model approaches and show dramatic differences in power in favor of the clustered Wilcoxon test for some designs.     

Evaluation of a Prednisolone Acetate-Loaded Subconjunctival Implant for the Treatment of Recurrent Uveitis in a Rabbit Model

Aim

To assess the efficacy of a biodegradable, prednisolone acetate implant in a rabbit uveitis model.

Methods

Randomized, controlled study of biodegradable microfilms preloaded with prednisolone acetate (PA) in a rabbit uveitis model. Experimental uveitis was induced by unilateral intravitreal injection of Mycobacterium tuberculosis H37Ra antigen (50 ug; 1 ug/uL) in preimmunized rabbits. PA-loaded poly[d,l-lactide-co-ε-caprolactone] (PLC) microfilms (n = 10) and blank microfilms (n = 6) were implanted subconjunctivally. An estimate of PA release in vivo was calculated from measured residual PA amounts in microfilms after the rabbits were sacrificed. The eyes were clinically monitored for ocular inflammation for 28 days. Histopathological examination of the enucleated eyes was performed at the end of the study period.

Results

In vitro studies revealed that sandwich PA-loaded microfilm formulations exhibited higher release kinetic compared to homogenous PA-loaded microfilms. The 60–40–60% microfilm released an average of 0.034 mg/day of PA over the period of 60 days in vitro; and we found that approximately 0.12 mg/day PA was released in vivo. Animals implanted with the PA-loaded microfilms exhibited significantly lowered median inflammatory scores when compared against the control group in this model for recurrent uveitis (P<0.001). The implants were clinically well tolerated by all the animals. Histology results showed no significant scarring or inflammation around the PA-loaded microfilms.

Conclusion

Our pilot study demonstrated that a subconjunctival PA-loaded implant is effective in suppressing inflammation in the rabbit model of uveitis, by providing therapeutic levels of PA that attenuated the inflammatory response even after a rechallenge. Longer term studies are now needed to establish the therapeutic potential of such a delivery system for treatment of ocular inflammation.     

Aqueous, oil, and ointment formulations of ketorolac: efficacy against prostaglandin E2-induced ocular inflammation and safety: A technical note

The efficacy of aqueous, oil, and ointment formulations of ketorolac against PGE₂-induced ocular inflammation in rabbits was evaluated, with monitoring of blinking rate and PMN and protein migration in tear fluid, following topical PGE₂ instillation. Ketorolac ophthalmic formulations protected the eye against inflammatory insult. Chronic topical administration of formulations for 10 days into rats' eyes did not lead to any appreciable gastrointestinal ulceration, which indicates that the formulations are safe for long-term use.