Depth-Dependent Changes in Collagen Organization in the Human Peripapillary Sclera

PurposeThe collagen structure of the human peripapillary sclera plays a significant role in determining optic nerve head (ONH) biomechanics, and is therefore of interest in the study of glaucoma. The aim of the current work was to map the anisotropic collagen structure of the normal human peripapillary sclera as a function of tissue depth.MethodsWide-angle x-ray scattering was used to quantify collagen fibril orientation at 0.5mm intervals across six 150μm-thick serial sections through the peripapillary sclera of eight normal European-derived human eyes. Two structural parameters were measured: 1) the relative number of fibrils preferentially aligned at a given angle within the tissue plane, 2) the degree of collagen alignment (anisotropy).ResultsThe inner-most one-third of the peripapillary scleral stroma (nearest to the choroid) was characterised by collagen fibrils either randomly arranged or preferentially aligned radially with respect to the ONH. In contrast, the outer two-thirds of the tissue was dominated by a circumferential arrangement of collagen encircling the ONH. In all tissue regions the degree of collagen anisotropy peaked in the mid-stroma and progressively decreased towards the tissue surfaces, with the largest depth variations occurring in the inferior-nasal quadrant, and the smallest occurring in the superior-nasal quadrant.ConclusionsSignificant, region-specific variations in collagen structure are present in the human peripapillary sclera as a function of depth. In normal eyes, the circumferential collagen fibril architecture is most prominent in the outer two-thirds of the stroma, possibly as a mechanical adaption to more effectively support the lamina cribrosa at the level of its insertion into the scleral canal wall.     

Structure-Function Correlation of the Human Central Retina

Background

The impact of retinal pathology detected by high-resolution imaging on vision remains largely unexplored. Therefore, the aim of the study was to achieve high-resolution structure-function correlation of the human macula in vivo.

Methodology/Principal Findings

To obtain high-resolution tomographic and topographic images of the macula spectral-domain optical coherence tomography (SD-OCT) and confocal scanning laser ophthalmoscopy (cSLO), respectively, were used. Functional mapping of the macula was obtained by using fundus-controlled microperimetry. Custom software allowed for co-registration of the fundus mapped microperimetry coordinates with both SD-OCT and cSLO datasets. The method was applied in a cross-sectional observational study of retinal diseases and in a clinical trial investigating the effectiveness of intravitreal ranibizumab in macular telangietasia type 2. There was a significant relationship between outer retinal thickness and retinal sensitivity (p<0.001) and neurodegeneration leaving less than about 50 µm of parafoveal outer retinal thickness completely abolished light sensitivity. In contrast, functional preservation was found if neurodegeneration spared the photoreceptors, but caused quite extensive disruption of the inner retina. Longitudinal data revealed that small lesions affecting the photoreceptor layer typically precede functional detection but later cause severe loss of light sensitivity. Ranibizumab was shown to be ineffective to prevent such functional loss in macular telangietasia type 2.

Conclusions/Significance

Since there is a general need for efficient monitoring of the effectiveness of therapy in neurodegenerative diseases of the retina and since SD-OCT imaging is becoming more widely available, surrogate endpoints derived from such structure-function correlation may become highly relevant in future clinical trials.     

视网膜单细胞成像技术研究

建立了一台基于37单元变形镜、Shack-hartman波前像差传感器和12位科研级CCD相机的自适应光学视网膜相机。采用一个中心波长为679nm的超辐射二极管(SLD)作为相机的光源,通过将超辐射二极管和多模光纤耦合,显著减小了SLD光源的空间相干性,从而消除了散斑噪声对成像的影响。多模光纤的输出提供了一种高亮度、均匀照明的光源,使人眼视网膜单细胞成像的速度达到4．8幅／秒。     

Label-Free Method Development for Hydroxyproline PTM Mapping in Human Plasma Proteome

The Protein Journal - Post-translational modifications (PTMs) impart structural heterogeneities that can alter plasma proteins' functions in various pathophysiological...     

Melanopsin Bistability: A Fly's Eye Technology in the Human Retina

In addition to rods and cones, the human retina contains light-sensitive ganglion cells that express melanopsin, a photopigment with signal transduction mechanisms similar to that of invertebrate rhabdomeric photopigments (IRP). Like fly rhodopsins, melanopsin acts as a dual-state photosensitive flip-flop in which light drives both phototransduction responses and chromophore photoregeneration that bestows independence from the retinoid cycle required by rods and cones to regenerate photoresponsiveness following bleaching by light. To explore the hypothesis that melanopsin in humans expresses the properties of a bistable photopigment in vivo we used the pupillary light reflex (PLR) as a tool but with methods designed to study invertebrate photoreceptors. We show that the pupil only attains a fully stabilized state of constriction after several minutes of light exposure, a feature that is consistent with typical IRP photoequilibrium spectra. We further demonstrate that previous exposure to long wavelength light increases, while short wavelength light decreases the amplitude of pupil constriction, a fundamental property of IRP difference spectra. Modelling these responses to invertebrate photopigment templates yields two putative spectra for the underlying R and M photopigment states with peaks at 481 nm and 587 nm respectively. Furthermore, this bistable mechanism may confer a novel form of “photic memory” since information of prior light conditions is retained and shapes subsequent responses to light. These results suggest that the human retina exploits fly-like photoreceptive mechanisms that are potentially important for the modulation of non-visual responses to light and highlights the ubiquitous nature of photoswitchable photosensors across living organisms.     

Expression of Toll-Like Receptors in the Early Prenatal Development of the Human Retina

Russian Journal of Developmental Biology - The expression of Toll-like receptors TLR2 and TLR4 in the human retina during early prenatal development is shown for the first time. It was found that...     

Changes in Bioelectric Activity in the Human Retina after Transcranial Magnetic Stimulation

The human electroretinogram (ERG) was recorded before and after transcranial magnetic stimulation (TMS) in the region of the cortical link of the visual analyzer. TMS at the frequency of 5 Hz in the projection of the cortical link of the visual analyzer significantly increased the amplitude of the ERG b wave; this was most likely associated with changes in the state of neurons of the central links of the visual analyzer, which enter the retina with efferent fibers of the optic nerve and are actively involved in lateral inhibition.     

Characterization of a Human Fovea cDNA Library and Regional Differential Gene Expression in the Human Retina

The primate fovea is the region of the retina responsible for acute vision. This region constitutes less than 5% of the total area of the retina and has not been intensely studied at the molecular level. As a first step in the molecular characterization of the fovea, we have constructed a primary human fovea cDNA library. Experiments confirm that our cDNA library reflects a nonbiased distribution of foveal expressed sequences. Single-pass sequencing was performed on 209 randomly isolated clones from this library. Analysis of the sequences generated reveals that the distributions of fovea clones with either human mitochondrial gene sequences or repetitive elements are different than those observed in cDNA libraries made from other tissues. A significant number of the fovea expressed sequence tags (ESTs) (88, 42.1%) represent novel human ESTs. This suggests that the library will be useful in identifying new human genes. Northern analysis of specific fovea ESTs defined in this study suggests that there are significant quantitative differences in gene expression that distinguish the fovea from the rest of the retina.     

Label-Free Quantitative Proteomics and N-terminal Analysis of Human Metastatic Lung Cancer Cells

Proteomic analysis is helpful in identifying cancer-associated proteins that are differentially expressed and fragmented that can be annotated as dysregulated networks and pathways during metastasis. To examine meta-static process in lung cancer, we performed a proteomics study by label-free quantitative analysis and N-terminal analysis in 2 human non-small-cell lung cancer cell lines with disparate metastatic potentials—NCI-H1703 (primary cell, stage I) and NCI-H1755 (metastatic cell, stage IV). We identified 2130 proteins, 1355 of which were common to both cell lines. In the label-free quantitative analysis, we used the NSAF normalization method, resulting in 242 differential expressed proteins. For the N-terminal proteome analysis, 325 N-terminal peptides, including 45 novel fragments, were identified in the 2 cell lines. Based on two proteomic analysis, 11 quantitatively expressed proteins and 8 N-terminal peptides were enriched for the focal adhesion pathway. Most proteins from the quantitative analysis were upregulated in metastatic cancer cells, whereas novel fragment of CRKL was detected only in primary cancer cells. This study increases our understanding of the NSCLC metastasis proteome.     

低亲和性神经营养素受体p75在人胚胎视网膜中的表达

p75神经营养素受体在视网膜的发育以及再生过程中发挥着重要的作用,而在人类视网膜中的分布状况尚未被研究. 利用免疫组织化学方法,在光镜水平下确定了p75在人胚胎发育5、6和7个月的视网膜中的分布情况. 在视网膜神经节细胞层出现最强的p75免疫阳性反应,在其他各层也有较弱的免疫阳性反应. 在胚胎6、7月的视网膜中,主要由Müller细胞的终足构成的内界膜上出现了比较强的p75表达. p75在人胚胎视网膜中的分布情况与大鼠视网膜中很类似,主要表达在Müller细胞, 在神经节细胞上也可能有表达.     

Label-Free Detection of Insulin and Glucagon within Human Islets of Langerhans Using Raman Spectroscopy

Intrahepatic transplantation of donor islets of Langerhans is a promising therapy for patients with type 1 diabetes. It is of critical importance to accurately monitor islet quality before transplantation, which is currently done by standard histological methods that are performed off-line and require extensive sample preparation. As an alternative, we propose Raman spectroscopy which is a non-destructive and label-free technique that allows continuous real-time monitoring of the tissue to study biological changes as they occur. By performing Raman spectroscopic measurements on purified insulin and glucagon, we showed that the 520 cm^-1 band assigned to disulfide bridges in insulin, and the 1552 cm^-1 band assigned to tryptophan in glucagon are mutually exclusive and could therefore be used as indirect markers for the label-free distinction between both hormones. High-resolution hyperspectral Raman imaging for these bands showed the distribution of disulfide bridges and tryptophan at sub-micrometer scale, which correlated with the location of insulin and glucagon as revealed by conventional immunohistochemistry. As a measure for this correlation, quantitative analysis was performed comparing the Raman images with the fluorescence images, resulting in Dice coefficients (ranging between 0 and 1) of 0.36 for insulin and 0.19 for glucagon. Although the use of separate microscope systems with different spatial resolution and the use of indirect Raman markers cause some image mismatch, our findings indicate that Raman bands for disulfide bridges and tryptophan can be used as distinctive markers for the label-free detection of insulin and glucagon in human islets of Langerhans.     

Quantification of Histone Deacetylase Isoforms in Human Frontal Cortex,Human Retina,and Mouse Brain

Histone deacetylase (HDAC) inhibition has promise as a therapy for Alzheimer’s disease (AD) and other neurodegenerative diseases. Currently, therapeutic HDAC inhibitors target many HDAC isoforms, a particularly detrimental approach when HDAC isoforms are known to have different and specialized functions. We have developed a multiple reaction monitoring (MRM) mass spectrometry assay using stable isotope-labeled QconCATs as internal standards to quantify HDAC isoforms. We further determined a quantitative pattern of specific HDACs expressed in various human and mouse neural tissues. In human AD frontal cortex, HDAC1,2 decreased 32%, HDAC5 increased 47%, and HDAC6 increased 31% in comparison to age-matched controls. Human neural retina concentrations of HDAC1, 2, HDAC5, HDAC6, and HDAC7 decreased in age-related macular degeneration (AMD)-affected donors and exhibited a greater decrease in AD-affected donors in comparison to age-matched control neural retinas. Additionally, HDAC concentrations were measured in whole hemisphere of brain of 5XFAD mice, a model of β-amyloid deposition, to assess similarity to AD in human frontal cortex. HDAC profiles of human frontal cortex and mouse hemisphere had noticeable differences and relatively high concentrations of HDAC3 and HDAC4 in mice, which were undetectable in humans. Our method for quantification of HDAC isoforms is a practical and efficient technique to quantify isoforms in various tissues and diseases. Changes in HDAC concentrations reported herein contribute to the understanding of the pathology of neurodegeneration.     

Alpha-Fetoprotein in Retina and Lens of the Human Eye at Early Stages of Prenatal Development

The presence of alpha-fetoprotein (AFP) was shown in the retina and lens of the human fetal eye at different stages of prenatal development. PCR analysis revealed AFP mRNA neither in the retina nor in the lens, whereas in the fetal liver (control) AFP mRNA was found to be expressed. The data obtained indicate that AFP is not synthesized in retinal and lens cells of the human fetal eye but is imported from elsewhere to be taken up by these cells. The presence of AFP in the retina and lens implies its involvement in early morphogenesis and differentiation of these ocular tissues during prenatal human development.     

Ganglion Cell and Displaced Amacrine Cell Density Distribution in the Retina of the Howler Monkey (Alouatta caraya)

Unlike all other New World (platyrrine) monkeys, both male and female howler monkeys (Alouatta sp.) are obligatory trichromats. In all other platyrrines, only females can be trichromats, while males are always dichromats, as determined by multiple behavioral, electrophysiological, and genetic studies. In addition to obligatory trichromacy, Alouatta has an unusual fovea, with substantially higher peak cone density in the foveal pit than every other diurnal anthropoid monkey (both platyrrhines and catarrhines) and great ape yet examined, including humans. In addition to documenting the general organization of the retinal ganglion cell layer in Alouatta, the distribution of cones is compared to retinal ganglion cells, to explore possible relationships between their atypical trichromacy and foveal specialization. The number and distribution of retinal ganglion cells and displaced amacrine cells were determined in six flat-mounted retinas from five Alouatta caraya. Ganglion cell density peaked at 0.5 mm between the fovea and optic nerve head, reaching 40,700–45,200 cells/mm². Displaced amacrine cell density distribution peaked between 0.5–1.75 mm from the fovea, reaching mean values between 2,050–3,100 cells/mm². The mean number of ganglion cells was 1,133,000±79,000 cells and the mean number of displaced amacrine cells was 537,000±61,800 cells, in retinas of mean area 641±62 mm². Ganglion cell and displaced amacrine cell density distribution in the Alouatta retina was consistent with that observed among several species of diurnal Anthropoidea, both platyrrhines and catarrhines. The principal alteration in the Alouatta retina appears not to be in the number of any retinal cell class, but rather a marked gradient in cone density within the fovea, which could potentially support high chromatic acuity in a restricted central region.     

A Comparison of Some Organizational Characteristics of the Mouse Central Retina and the Human Macula

Mouse models have greatly assisted our understanding of retinal degenerations. However, the mouse retina does not have a macula, leading to the question of whether the mouse is a relevant model for macular degeneration. In the present study, a quantitative comparison between the organization of the central mouse retina and the human macula was made, focusing on some structural characteristics that have been suggested to be important in predisposing the macula to stresses leading to degeneration: photoreceptor density, phagocytic load on the RPE, and the relative thinness of Bruch’s membrane. Light and electron microscopy measurements from retinas of two strains of mice, together with published data on human retinas, were used for calculations and subsequent comparisons. As in the human retina, the central region of the mouse retina possesses a higher photoreceptor cell density and a thinner Bruch’s membrane than in the periphery; however, the magnitudes of these periphery to center gradients are larger in the human. Of potentially greater relevance is the actual photoreceptor cell density, which is much greater in the mouse central retina than in the human macula, underlying a higher phagocytic load for the mouse RPE. Moreover, at eccentricities that correspond to the peripheral half of the human macula, the rod to cone ratio is similar between mouse and human. Hence, with respect to photoreceptor density and phagocytic load of the RPE, the central mouse retina models at least the more peripheral part of the macula, where macular degeneration is often first evident.     

Electrophysiological Measurements and Analysis of Nociception in Human Infants

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are based on behavioural and physiological body reactions, such as crying, body movements or changes in facial expression. While these measures demonstrate that infants mount a response following noxious stimulation, they are limited: they are based on activation of subcortical somatic and autonomic motor pathways that may not be reliably linked to central sensory processing in the brain. Knowledge of how the central nervous system responds to noxious events could provide an insight to how nociceptive information and pain is processed in newborns.The heel lancing procedure used to extract blood from hospitalised infants offers a unique opportunity to study pain in infancy. In this video we describe how electroencephalography (EEG) and electromyography (EMG) time-locked to this procedure can be used to investigate nociceptive activity in the brain and spinal cord.This integrative approach to the measurement of infant pain has the potential to pave the way for an effective and sensitive clinical measurement tool.     

Ink Perfusion for Displaying Capillaries in the Chicken

A chicken was anesthetized with Na-phenobarbital, 130 mg/kg, and the external ischiatic artery was exposed. Heparin, 90 units/kg, was injected into the brachial vein, after which a cannula was inserted into the exposed ischiatic artery. Heat was applied to the head to produce vasodilation. Perfusion through the cannula was made at about 150 mm Hg pressure and consisted of 30 ml of a biological type of Pelikan ink to which was added NaNO₂ to make a 0.02% solution. After the bird was killed by an overdose of anesthetic, the skin around the perimeter of the femoral feather tract was cauterized, the tract removed, attached to a stainless steel wire net, fixed for 1 hr in 10% formic acid, then dehydrated in 4 changes of acetone, 1 hr each, followed by a 5th change for 12 hr. The dehydrated specimens were placed in methyl benzoate, the 1st change for 1 hr and the 2nd until clear (3-15 hr). This procedure exhibits small vessels and capillary networks within the integument, but if only the vessels larger than capillaries are to be studied, they can be shown by slowly injecting 15-30 ml of the perfusion mixture into a peripheral vein. The still beating heart will distribute the ink to other regions of the body, thus allowing the vessels to be traced in excised tissue.