Neuroanatomy and neurochemistry of rat cornea: Changes with age

PurposeTo characterize the entire rat corneal nerve architecture, the changes that occur with aging, and its sensory, sympathetic, and parasympathetic fiber distribution.MethodsSprague-Dawley rats (aged 1 day to 2 years old) of both sexes were euthanized, and the whole corneas were immunostained with protein gene product 9.5 (PGP9.5). The specimens were double-labeled with antibodies against calcitonin gene-related peptide (CGRP) and substance P (SP) as sensory nerve markers, vasoactive intestinal peptide (VIP) as a parasympathetic nerve marker, and neuropeptide Y (NPY) and tyrosine hydroxylase (TH) as markers of sympathetic fibers. Relative nerve density positive for each antibody was assessed by computer-assisted image analysis.ResultsThick nerve trunks enter the cornea in the middle of the stroma and run towards the anterior stroma, subsequently dividing into smaller branches that penetrate upwards into the epithelium to form the subbasal nerve bundles. There was no significant difference in corneal innervation between sexes. CGRP and SP were the major sensory neuropeptides with 47.6% ± 3.5% and 34.9% ± 5.1%, respectively, of the total nerves. VIP was 18.4% ± 5.7%, and NPY and TH positive fibers took up 6.92% ± 2.66% and 2.92% ± 1.52%, respectively. Epithelial nerve density increased with age, reached full development at 5 weeks, and decreased at 120 weeks.ConclusionThis study provides a complete nerve architecture and content of components of sensory, parasympathetic, and sympathetic nerves in the rat cornea. The normal innervation pattern described here will provide an essential baseline for investigators who use the rat model for assessing corneal pathologies that involve nerve alterations.     

Impaired GSH biosynthesis disrupts eye development,lens morphogenesis and PAX6 function

PurposeThe purpose of this study was to elucidate the role and molecular consequences of impaired glutathione (GSH) biosynthesis on eye development.MethodsGSH biosynthesis was impaired in surface ectoderm-derived ocular tissues by crossing Gclc^f/f mice with hemizygous Le-Cre transgenic mice to produce Gclc^f/f/Le-Cre^Tg/- (KO) mice. Control mice included Gclc^f/f and Gclc^wt/wt/Le-Cre^Tg/- mice (CRE). Eyes from all mice (at various stages of eye development) were subjected to histological, immunohistochemical, Western blot, RT-qPCR, RNA-seq, and subsequent Gene Ontology, Ingenuity Pathway Analysis and TRANSFAC analyses. PAX6 transactivation activity was studied using a luciferase reporter assay in HEK293T cells depleted of GSH using buthionine sulfoximine (BSO).ResultsDeletion of Gclc diminished GSH levels, increased reactive oxygen species (ROS), and caused an overt microphthalmia phenotype characterized by malformation of the cornea, iris, lens, and retina that is distinct from and much more profound than the one observed in CRE mice. In addition, only the lenses of KO mice displayed reduced crystallin (α, β), PITX3 and Foxe3 expression. RNA-seq analyses at postnatal day 1 revealed 1552 differentially expressed genes (DEGs) in the lenses of KO mice relative to those from Gclc^f/f mice, with Crystallin and lens fiber cell identity genes being downregulated while lens epithelial cell identity and immune response genes were upregulated. Bioinformatic analysis of the DEGs implicated PAX6 as a key upstream regulator. PAX6 transactivation activity was impaired in BSO-treated HEK293T cells.ConclusionsThese data suggest that impaired ocular GSH biosynthesis may disrupt eye development and PAX6 function.     

驻景丸加减方对形觉剥夺性近视小鼠视网膜厚度及细胞凋亡的影响

目的：探讨驻景丸加减方对形觉剥夺性近视小鼠视网膜厚度及细胞凋亡的影响。

方法：将72只3周龄C57BL/6J小鼠随机分为对照组1、模型组1、干预组1、对照组2、模型组2及干预组2,每组12只小鼠,前三组实验进行3wk,后三组实验进行6wk,除对照组1和对照组2外,所有小鼠均用半透明眼罩遮盖右眼诱导形觉剥夺性近视动物模型,干预组1在造模同时而干预组2则在实验3wk后灌胃驻景丸加减方混悬液0.546g/(kg·d)(0.1mL/d),其余组小鼠灌胃等量生理盐水0.1mL/d,实验前后均测眼轴,实验结束时取小鼠视网膜行HE染色用于观察视网膜各层厚度改变,免疫组织化学(IHC)和western blotting(WB)用于检测Bcl-2和Caspase3蛋白的表达。

结果：在实验结束时模型组1的眼轴比对照组1显著增加(P<0.01),干预组1的眼轴显著低于模型组1(P<0.01),模型组2的眼轴比对照组2显著增加(P<0.01),干预组2的眼轴明显低于模型组2(P<0.01); 模型组1的NFL和ONL的厚度比对照组1显著变薄(P<0.01),模型组1的INL厚度比对照组1显著变薄(P<0.05),干预组1的NFL、INL和ONL厚度较模型组1显著增加(P<0.05); 模型组2的NFL、INL和ONL的厚度比对照组1显著变薄(P<0.01); IHC检测显示Bcl-2蛋白平均光密度模型组1显著低于对照组1(P<0.05),干预组1显著高于模型组1(P<0.01),模型组2显著低于对照组2(P<0.01),干预组2显著高于模型组2(P<0.01); Caspase3蛋白平均光密度模型组1显著高于对照组1(P<0.01),干预组1显著低于模型组1(P<0.01),模型组2显著高于对照组2(P<0.05),干预组2显著低于模型组2(P<0.01); WB检测证明,Bcl-2的蛋白相对表达水平模型组1显著低于对照组1(P<0.01),干预组1显著高于模型组1(P<0.01),模型组2显著低于对照组2(P<0.01),干预组2显著高于模型组2(P<0.01); Caspase3的蛋白相对表达水平模型组1显著高于对照组1(P<0.01),干预组1显著低于模型组1(P<0.01),干预组2显著低于模型组2(P<0.05)。

结论：驻景丸加减方可通过调控凋亡相关蛋白Bcl-2和Caspase3的表达,减轻近视形成过程中及已形成近视小鼠视网膜细胞凋亡,从而起到干预形觉剥夺近视小鼠视网膜厚度变薄的作用。     

康柏西普联合不同手术方式对新生血管性青光眼疗效观察

目的：探讨玻璃体腔注射康柏西普后对新生血管性青光眼(NVG)患者房水中VEGF、IL-6、IL-8的影响,并观察其联合不同手术方式对NVG患者的疗效。

方法：选择2019-01/2020-02在中国人民解放军新疆军区总医院全军眼科中心就诊的NVG患者102例,均玻璃体腔注射康柏西普,3～5d后行手术治疗,按照随机数字抽签法分为小梁切除术组(50例50眼)及EX-PRESS引流器植入术组(52例52眼),运用酶联免疫吸附法分析房水中VEGF、IL-6、IL-8的变化情况,观察虹膜新生血管消退情况,比较两组手术疗效及术后眼压变化、视力改善情况及并发症发生情况。

结果：玻璃体腔注射康柏西普3～5d后房水中VEGF、IL-6、IL-8表达水平较术前降低(均P<0.05); 术后随访各时间点两组眼压水平均显著低于术前(均P<0.05),两组患者眼压在术后3、6、12mo组间比较有差异(均P<0.05); 术后6、12mo时EX-PRESS组视力优于小梁切除术组(P<0.05); 术后12mo,两组使用抗青光眼药物种类及数量无明显差异(均P>0.05); 随访12mo,EX-PRESS组患者手术成功率(86.5%)优于小梁切除术组(70.0%),且EX-PRESS组术后并发症发生率显著低于小梁切除术组(P<0.05)。

结论：玻璃体腔注射康柏西普后可以降低NVG患者房水中VEGF、IL-6、IL-8等因子表达,同时小梁切除术与EX-PRESS引流器植入术均可降低NVG患者眼压,但后者手术并发症较少、改善视力方面更有优势。     

肠风散的镇痛作用及皮肤安全性研究

为研究肠风散的镇痛作用及皮肤急性毒性、皮肤刺激性和皮肤过敏反应,采用醋酸扭体法和热板法观察肠风散的镇痛作用;用ELISA法测定肛肠病术后疼痛模型大鼠血清白介素-6（IL-6）和白介素-10（IL-10）含量,研究镇痛作用机理;观察小鼠皮肤急性毒性反应、豚鼠皮肤刺激性和过敏反应,研究肠风散局部使用的皮肤安全性。结果显示,肠风散大、中剂量（分别为100％和50％）局部外涂能明显提高小鼠足底痛阈（P〈0．05或P〈0．01）,亦能明显抑制冰醋酸所致小鼠扭体反应（P〈0．05）;肠风散大、中剂量（分别为100％和50％）局部外涂能使肛肠病术后疼痛模型大鼠血清IL-6含量显著降低、IL-10含量明显升高（P〈O．05或P〈O．01）;肠风散经皮给药,未见明显急性毒性、皮肤刺激性及过敏反应。结果表明,肠风散局部给药具有一定的镇痛作用,局部皮肤给药安全性较高。     

The Proposal and Early Results of Capitate Forage as a New Treatment Method for Kienböck's Disease

Kienböck's disease is a type of avascular necrosis which disrupts the biomechanics of the wrist as a result of the changes it creates in the lunate bone. Its treatment generally consists of osteotomies intended to relieve the pressure on the bone, pedicle bone grafting applications aiming to increase bone blood supply, and salvage procedures. Capitate forage is a safe and simple-to-apply surgical treatment method which is intended to enhance neovascularization of the lunate much like a radius osteotomy or core decompression     

雌激素对骨髓细胞白细胞介素-6及骨髓源性破骨细胞形成的影响

目的 观察雌激素对骨髓细胞白细胞介素-6(IL-6)及骨髓源性破骨细胞的影响.方法 将90只雌性SD大鼠均分为3组:对照组(N组)、去卵巢组(OVX组)及雌激素组(OVX+ E2组).OVX组与OVX+ E2组大鼠行双侧卵巢切除术.术后OVX+ E2组按照0.02 ml/kg的剂量每周肌肉注射苯甲酸雌二醇1次.术后第1、3、5周取骨组织培养骨髓细胞,检测骨组织培养液及血清中IL-6含量,对破骨细胞进行计数.结果 (1)术后第1、3、5周OVX组骨组织培养液IL-6含量分别为(62.01±12.38)、(95.63±16.79)、(139.00±20.11) ng/L,OVX+E2组分别为(27.99±9.59)、(30.74±10.17)、(36.29±11.28) ng/L,两组含量均高于N组(P＜0.05),且两组含量随时间逐渐增加(P＜0.05);(2)各组各时间点血清中IL-6含量差异无统计学意义(P＞0.05);(3)术后第1、3、5周OVX组破骨细胞数分别为(16.55±1.78)、(28.02±3.99)、(29.57±2.86)个/高倍视野,OVX+ E2组分别为(10.99±1.52)、(20.02±3.52)、(18.01±2.17)个/高倍视野,OVX+ E2组各时间点破骨细胞数均小于OVX组(P＜0.05),但均大于N组(P＜0.05).结论 雌激素对骨髓细胞IL-6具有一定抑制作用,但对血清中IL-6无明显影响;雌激素对骨髓源性破骨细胞形成具有一定抑制作用.     

C6、C7椎弓根置钉的应用解剖学研究

目的:通过对C6、C7椎弓根的解剖学测量,设计一种以“峡部”为参考的C6、C7椎弓根置钉的方法.方法:15具经福尔马林浸泡的成人颈椎标本,不分性别、年龄,排除畸形及破坏.取C6、C7共30个椎体.先测量椎弓根宽度(PW)、椎弓根高度(PH).将C6、C7侧块中的一部分定义为“峡部”,即过上关节突下缘最低点、下关节突上缘最高点的水平线与过上关节突内外侧缘的垂线所围成区域.过上关节突内外侧缘之间划两条垂线,将“峡部”分为3等份,中份为“峡部”的后侧面,外份为“峡部”的后外侧面.取过横突根部中点的水平线与过“峡部”中外1/3垂线的交点为螺钉的进钉点.选择3.5mm直径及合适长度的螺钉,直视下沿椎弓根中轴线方向置入,使螺钉中轴线与椎弓根中轴线重合,螺钉前端穿出椎体或上终板.沿椎弓根中轴线的水平面锯开标本,可看到螺钉处在椎弓根中轴线上.螺钉在水平面上与“峡部”的后外侧面形成的角为横向角(E角);螺钉在矢状面上与“峡部”后侧面形成的角为纵向角(F角).测量椎弓根钉道全长(FSC).对数据进行t检验及类聚分析.结果:各指标同一节段左右侧数据比较无统计学差异(P＞0.05),故将同节段左右侧数据合并后进行统计分析.C6的PW为6.12±0.78mm,PH为7.48±0.81mm;C7的PW为6.85±0.73mm,PH为8.03 ±0.38mm;PW与PH均为C6＜C7(P＜0.05),同一节段PW＜PH(P＜0.05).利用聚类分析中的Hierarchical cluster过程对数据进行聚类分析,结果显示E角趋向于两类,即E1和E2.C6的E1角、E2角、F角及FSC与C7比较均无统计学差异(P＞0.05),将同一指标C6与C7数据合并统计结果为FSC 30.83±0.91mm,E1角89.61°±1.24°,E2角59.71°±1.10°,F角75.86°±112°.结论:在C6、C7以过横突根部中点的水平线与过“峡部”中外1/3垂线的交点为螺钉的进钉点,沿椎弓根中轴线方向,在水平面上按照E角、在矢状面上按照F角进钉,行椎弓根置钉具有可行性.     

Influence of Different Stages of Experimental Chronic Kidney Disease on Rats Locomotor and Postural Skeletal Muscles Microcirculation

Background. Chronic kidney disease (CKD) is associated with muscle excess fatigue and diminished maximal whole body oxygen consumption, which in part could be depended on poor muscle microcirculatory network. The aim of this study was to assume the influence of different stages of CKD on microcirculation vessels in functionally different skeletal muscles—locomotor, the gastrocnemius muscle, and postural, the longissimus thoracis muscle. Methods. Male Wistar rats underwent sham operation (CON), uninephrectomy (CKD 1/2) and subtotal nephrectomy (CKD 5/6). Muscle samples were stained for an alkaline phosphatase to differentiate capillaries. The number of capillaries was estimated by a single observer in 10 μm transverse sections by point counting at a magnification of ×125 using an Image Analysis System Q 500 MC of Leica. Blood pressure and serum creatinine, haptoglobin, MCP-1, VEGF, and PDGF were measured. Results. There were significant differences (p < 0.05) in CD (number of capillaries per 1 mm² of muscle tissue), C:F (capillary to fiber ratio), and CC/F (capillary contact per fiber). The CKD 1/2 group in gastrocnemius and longissimus muscle had 53% and 33% lower C:F; 56% and 33% lower CD; and 44% and 20% less CC/F than CON, respectively. The CKD 5/6 group in gastrocnemius and longissimus muscle had 46% and 20% lower C:F; 47% and 11% lower CD; and 48% and 25% less CC/F versus control, respectively. Blood pressure was higher in CKD 5/6 vs. CKD 1/2 and CON (145/95 vs. 107/87 and 119/77 mmHg, p < 0.05, respectively). CKD 5/6 had higher creatinine than CKD 1/2 and CON (1.22 vs. 0.83 and 0.74 mg/dL, p < 0.05, respectively). Haptoglobin was higher in CKD 1/2 and CKD 5/6 versus CON (1.68 and 1.63 vs. 0.70 mg/mL, p < 0.05, respectively). MCP-1 was higher in CKD 5/6 and CKD 1/2 versus CON (609 and 489 vs. 292 pg/mL, p < 0.05, respectively). There were no significant differences in serum growth factors concentration between groups. Conclusion. Capillary rarefaction is present in early stages of CKD. These changes are independent of blood pressure and progression of CKD. We suspected that muscle function has a big impact on microvasculature as capillaries rarefaction has been reduced more in locomotor than postural skeletal muscle.