不同角膜屈光手术对基底膜下神经的影响

目的：观察角膜屈光不同术式后基底膜下神经恢复情况。
　　方法：角膜激光共焦显微镜观察术后1 mo 的 LASEK 12眼、LASIK 12眼、FLEx 12眼、SMILE 12眼,并对部分其它术后时段进行了对比观察。
　　结果：术后1 mo SMILE组基底膜下神经均未见明显破坏,走行规律,完整,切口处被切断神经对合良好;LASEK, LASIK和FLEx基底膜下神经仍现不同程度损害,其它时段眼显示随着时间推移,逐渐形成交通支。
　　结论：SMILE手术对基底膜下神经影响小,相对于其他几种角膜屈光手术,在术后神经恢复方面可能有一定优势。     

In vivo corneal confocal microscopic analysis in patients with keratoconus

AIM:To quantify corneal ultrastructure using laser scanning in vivo confocal microscopy (IVCM) in patients with keratoconus and control subjects.METHODS: Unscarred corneas of 78 keratoconic subjects without a history of contact lens use and 36 age-matched control subjects were evaluated with slit-lamp examination (SLE), corneal topography and laser scanning IVCM. One eye was randomly chosen for analysis. Anterior and posterior stromal keratocyte, endothelial cell and basal epithelial cell densities and sub-basal nerve structure were evaluated.RESULTS: IVCM qualitatively demonstrated enlarged basal epithelial cells, structural changes in sub-basal and stromal nerve fibers, abnormal stromal keratocytes and keratocyte nuclei, and pleomorphism and enlargement of endothelial cells. Compared with control subjects, significant reductions in basal epithelial cell density (5817±306 cells/mm² vs 4802±508 cells/mm², P<0.001), anterior stromal keratocyte density (800±111 cells/mm² vs 555±115 cells/mm², P<0.001), posterior stromal keratocyte density (333±34 cells/mm² vs 270±47 cells/mm², P<0.001), endothelial cell density (2875±223 cells/mm² vs 2686±265 cells/mm², P<0.001), sub-basal nerve fiber density (31.2±8.4 nerves/mm² vs 18.1±9.2 nerves/mm², P<0.001), sub-basal nerve fiber length (21.4±3.4 mm/mm² vs 16.1±5.1 mm/mm², P<0.001), and sub-basal nerve branch density (median 50.0 (first quartile 31.2 - third quartile 68.7) nerve branches/mm² vs median 25.0 (first quartile 6.2 - third quartile 45.3) nerve branches/mm², P<0.001) were observed in patients with keratoconus.CONCLUSION: Significant microstructural abnormalities were identified in all corneal layers in the eyes of subjects with keratoconus using IVCM. This non-invasive in vivo technique provides an important means to define and follow progress of microstructural changes in patients with keratoconus.     

Up-regulation of connexin45 in heart failure

INTRODUCTION: Heart failure is associated with reduced expression of the major gap junction protein connexin43 (Cx43), which may contribute to arrhythmias and sudden cardiac death in this patient population. Other cardiac connexins may be altered as well. Because connexin45 (Cx45) has been shown to colocalize with Cx43, we determined whether the number, size, or distribution of Cx45 gap junctions is altered in the failing heart. METHODS AND RESULTS: Cx45 expression levels were measured by immunoblotting and quantitative immunostaining in failing and control human left ventricles. Total Cx45 protein was significantly (P = 0.021) up-regulated 1.8-fold in failing hearts. Cx45 immunohistochemical signal was increased by 80% (P = 0.005) due to a 3.5-fold increase in the number of gap junctions containing Cx45. Cx45 mRNA was not altered in failing hearts, suggesting reduced degradation of Cx45 protein in the failing heart. Cx43 signal, on the other hand, was reduced by 49% in failing hearts. Double-label experiments demonstrated colocalization of Cx45 and Cx43 in the same gap junctions. CONCLUSION: Cx45 is markedly enhanced in the failing heart. Up-regulation of Cx45 in conjunction with down-regulation of Cx43 could result in abnormal impulse propagation and generation of ventricular arrhythmias, thereby predisposing patients in heart failure to sudden cardiac death.     

Endoscopic detection of early upper GI cancers

The detection of early-stage neoplastic lesions in the upper GI tract is associated with improved survival and the potential for complete endoscopic resection that is minimally invasive and less morbid than surgery. Despite technological advances in standard white-light endoscopy, the ability of the endoscopist to reliably detect dysplastic and early cancerous changes in the upper GI tract remains limited. In conditions such as Barrett's oesophagus, practice guidelines recommend periodic endoscopic surveillance with multiple biopsies, a methodology that is hindered by random sampling error, inconsistent histopathological interpretation, and delay in diagnosis. Early detection may be enhanced by several promising diagnostic modalities such as chromoendoscopy, magnification endoscopy, and optical spectroscopic/imaging techniques, as these modalities offer the potential to identify in real-time lesions that are inconspicuous under conventional endoscopy. The combination of novel diagnostic techniques and local endoscopic therapies will provide the endoscopist with much needed tools that can considerably enhance the detection and management of early stage lesions in the upper GI tract.     

Optimizing tissue clearing and imaging methods for human brain tissue

ObjectivesTo identify optimum sample conditions for human brains, we compared the clearing efficiency, antibody staining efficiency, and artifacts between fresh and cadaver samples.MethodsFresh and cadaver samples were cleared using X-CLARITY™. Clearing efficiency and artifact levels were calculated using ImageJ, and antibody staining efficiency was evaluated after confocal microscopy imaging. Three staining methods were compared: 4-day staining (4DS), 11-day staining (11DS), and 4-day staining with a commercial kit (4DS-C). The optimum staining method was then selected by evaluating staining time, depth, method complexity, contamination, and cost.ResultsFresh samples outperformed cadaver samples in terms of the time and quality of clearing, artifacts, and 4′,6-diamidino-2-phenylindole (DAPI) staining efficiency, but had a glial fibrillary acidic protein (GFAP) staining efficiency that was similar to that of cadaver samples. The penetration depth and DAPI staining improved in fresh samples as the incubation period lengthened. 4DS-C was the best method, with the deepest penetration. Human brain images containing blood vessels, cell nuclei, and astrocytes were visualized three-dimensionally. The chemical dye staining depth reached 800 µm and immunostaining depth exceeded 200 µm in 4 days.ConclusionsWe present optimized sample preparation and staining protocols for the visualization of three-dimensional macrostructure in the human brain.     

Confocal Laser Scanning Microscopy of Liesegang Rings in Odontogenic Cysts: Analysis of Three-Dimensional Image Reconstruction

Liesegang rings are concentric noncellular lamellar structures, occasionally found in inflammatory tissues. They have been confused with various parasites, algas, calcification, and psammoma bodies. The authors examined Liesegang rings from oral inflammatory cysts by both optical and confocal laser scanning microscopy, and perfomed a three-dimensional reconstruction. These investigations indicate that Liesegang rings are composed of multiple birefringent concentric rings, resulting from a progressive deposition of organic substances, with an unclear pathogenesis.     

Retinal ganglion cell topography and spatial resolution in the Japanese smelt Hypomesus nipponensis (McAllister, 1963)

Vision plays a crucial role in the life of the vast majority of vertebrate species. The spatial arrangement of retinal ganglion cells has been reported to be related to a species’ visual behavior. There are many studies focusing on the ganglion cell topography in bony fish species. However, there are still large gaps in our knowledge on the subject. We studied the topography of retinal ganglion cells (GCs) in the Japanese smelt Hypomesus nipponensis, a highly visual teleostean fish with a complex life cycle. DAPI labeling was used to visualize cell nuclei in the ganglion cell and inner plexiform layers. The ganglion cell layer was relatively thin (about 6-8 μm), even in areas of increased cell density (area retinae temporalis), and was normally composed of a single layer of cells. In all retinal regions, rare cells occurred in the inner plexiform layer. Nissl-stained retinae were used to estimate the proportion of displaced amacrine cells and glia in different retinal regions. In all retinal regions, about 84.5% of cells in the GC layer were found to be ganglion cells. The density of GCs varied across the retina in a regular way. It was minimum (3990 and 2380 cells/mm² in the smaller and larger fish, respectively) in the dorsal and ventral periphery. It gradually increased centripetally and reached a maximum of 14,275 and 10,960 cells/mm² (in the smaller and larger fish, respectively) in the temporal retina, where a pronounced area retinae temporalis was detected. The total number of GCs varied from 177 × 10³ (smaller fish) to 212 × 10³ cells (larger fish). The theoretical anatomical spatial resolution (the anatomical estimate of the upper limit of visual acuity calculated from the density of GCs and eye geometry and expressed in cycles per degree) was minimum in the ventral periphery (smaller fish, 1.46 cpd; larger fish, 1.26 cpd) and maximum in area retinae temporalis (smaller fish, 2.83 cpd; larger fish, 2.75 cpd). The relatively high density of GCs and the presence of area retinae temporalis in the Japanese smelt are consistent with its highly visual behavior. The present findings contribute to our understanding of the factors affecting the topography of retinal ganglion cells and visual acuity in fish.     

组织蛋白酶D和嗜铬颗粒素A在人原发性肝细胞癌的表达意义

目的探讨组织蛋白酶D和嗜铬颗粒素A在人原发性肝细胞癌(HCC)的表达及其意义.方法免疫荧光双标记染色技术结合激光扫描共聚焦显微镜观察HCC 85例.结果组织蛋白酶D在正常肝细胞有弱表达,但在HCC组织,组织蛋白酶D的表达显著增强,而且癌细胞荧光染色强度存在区域性差别,通常临床恶性程度越高、肿瘤分化越差,荧光染色较强的癌细胞越多. HCC的组织学分级越高,组织蛋白酶D的表达越多. 85例HCC中有71例癌细胞呈组织蛋白酶D强阳性,阳性率83.5%. 在组织学分级为3～4级的28例中,组织蛋白酶D阳性的占26例(阳性率92.9%);组织学分级为2级的53例中,有46例阳性(阳性率86.8%);而组织学分级为1级的4例中,仅有1例阳性(阳性率25.0%). 组织蛋白酶D阳性细胞的分布,在组织学分级不同的HCC中有明显差别(P＜0.01). 我们还发现组织蛋白酶D的表达与患者年龄无明显关系. 有组织蛋白酶D表达的患者平均年龄为51.2岁±2.8岁(32岁～68岁),无组织蛋白酶D表达的患者平均年龄为51.2岁±4.5岁(28岁～71岁)(P＞0.05). 组织学分级为1级的HCC CgA阳性率为75.0%(3/4),2级的阳性率71.7%(38/53),3～4级的阳性率为71.4%(20/28). 经统计学分析表明,CgA阳性细胞的分布,在组织学分级不同的HCC没有明显差别(P＞0.05). 免疫荧光双标记显示,在大部分癌组织中,二者同时表达. 结果提示HCC的癌细胞对组织蛋白酶D的加工出现障碍,因此组织蛋白酶D在细胞中积聚. 由于组织蛋白酶D的蛋白水解酶活性和自分泌性丝裂原活性,因此组织蛋白酶D的表达增加可能在肿瘤的浸润和转移中有一定作用. 这些发现表明组织蛋白酶D对HCC有预后诊断价值.结论组织蛋白酶D和CgA在绝大部分HCC都可同时表达. 也提示肝癌细胞中组织蛋白酶D可能与CgA的加工有关. 因为其潜在的临床应用价值,组织蛋白酶D在HCC表达显然是值得进一步研究的课题.