The structure and function of the human choroid.

In this manuscript, the structure of the human choroid is reviewed with emphasis of the macro- and microscopic anatomy including Bruch's membrane, choriocapillaris, Sattler's and Haller's layer, and the suprachoroid. We here discuss the development of the choroid, as well as the question of choroidal lymphatics, and further the neuronal control of this tissue, as well as the pathologic angiogenesis. Wherever possible, functional aspects of the various structures are included and reviewed.

Hypertrophic cardiomyopathy is characterized by alterations of the mitochondrial calcium uniporter complex proteins: insights from patients with aortic valve stenosis versus hypertrophic obstructive cardiomyopathy.

Introduction: Hypertrophies of the cardiac septum are caused either by aortic valve stenosis (AVS) or by congenital hypertrophic obstructive cardiomyopathy (HOCM). As they induce cardiac remodeling, these cardiac pathologies may promote an arrhythmogenic substrate with associated malignant ventricular arrhythmias and may lead to heart failure. While altered calcium (Ca2+) handling seems to be a key player in the pathogenesis, the role of mitochondrial calcium handling was not investigated in these patients to date. Methods: To investigate this issue, cardiac septal samples were collected from patients undergoing myectomy during cardiac surgery for excessive septal hypertrophy and/or aortic valve replacement, caused by AVS and HOCM. Septal specimens were matched with cardiac tissue obtained from post-mortem controls without cardiac diseases (Ctrl). Results and discussion: Patient characteristics and most of the echocardiographic parameters did not differ between AVS and HOCM. Most notably, the interventricular septum thickness, diastolic (IVSd), was the greatest in HOCM patients. Histological and molecular analyses showed a trend towards higher fibrotic burden in both pathologies, when compared to Ctrl. Most notably, the mitochondrial Ca2+ uniporter (MCU) complex associated proteins were altered in both pathologies of left ventricular hypertrophy (LVH). On the one hand, the expression pattern of the MCU complex subunits MCU and MICU1 were shown to be markedly increased, especially in AVS. On the other hand, PRMT-1, UCP-2, and UCP-3 declined with hypertrophy. These conditions were associated with an increase in the expression patterns of the Ca2+ uptaking ion channel SERCA2a in AVS (p = 0.0013), though not in HOCM, compared to healthy tissue. Our data obtained from human specimen from AVS or HOCM indicates major alterations in the expression of the mitochondrial calcium uniporter complex and associated proteins. Thus, in cardiac septal hypertrophies, besides modifications of cytosolic calcium handling, impaired mitochondrial uptake might be a key player in disease progression.

Distribution of the cysteinyl leukotriene system components in the human, rat and mouse eye.

The cysteinyl leukotrienes (CysLTs) have important functions in the regulation of inflammation and cellular stress. Blocking the CysLT receptors (CysLTRs) with specific antagonists is beneficial against progression of retinopathies (e.g. diabetic retinopathy, wet AMD). However, the exact cellular localization of the CysLTRs and their endogenous ligands in the eye have not been elucidated in detail yet. It is also not known whether the expression patterns differ between humans and animal models. Therefore, the present study aimed to describe and compare the distribution of two important enzymes in CysLT biosynthesis, 5-lipoxygenase (5-LOX) and 5-lipoxygenase-activating protein (FLAP), and of CysLTR1 and CysLTR2 in healthy human, rat and mouse eyes. Human donor eyes (n = 10) and eyes from adult Sprague Dawley rats (n = 5) and CD1 mice (n = 8) of both sexes were collected. The eyes were fixed in 4% paraformaldehyde and cross-sections were investigated by immunofluorescence with specific antibodies against 5-LOX, FLAP (human tissue only), CysLTR1 and CysLTR2. Flat-mounts of the human choroid were prepared and processed similarly. Expression patterns were assessed and semiquantitatively evaluated using a confocal fluorescence microscope (LSM710, Zeiss). We observed so far unreported expression sites for CysLT system components in various ocular tissues. Overall, we detected expression of 5-LOX, CysLTR1 and CysLTR2 in the human, rat and mouse cornea, conjunctiva, iris, lens, ciliary body, retina and choroid. Importantly, expression profiles of CysLTR1 and CysLTR2 were highly similar between human and rodent eyes. FLAP was expressed in all human ocular tissues except the lens. Largely weak immunoreactivity of FLAP and 5-LOX was observed in a few, yet unidentified, cells of diverse ocular tissues, indicating low levels of CysLT biosynthesis in healthy eyes. CysLTR1 was predominantly detected in ocular epithelial cells, supporting the involvement of CysLTR1 in stress and immune responses. CysLTR2 was predominantly expressed in neuronal structures, suggesting neuromodulatory roles of CysLTR2 in the eye and revealing disparate functions of CysLTRs in ocular tissues. Taken together, we provide a comprehensive protein expression atlas of CysLT system components in the human and rodent eye. While the current study is purely descriptive and therefore does not allow significant functional conclusions yet, it represents an important basis for future studies in diseased ocular tissues in which distribution patterns or expression levels of the CysLT system might be altered. Furthermore, this is the first comprehensive study to elucidate expression patterns of CysLT system components in human and animal models that will help to identify and understand functions of the system as well as mechanisms of action of potential CysLTR ligands in the eye.

Supplemental cerclage wiring in angle stable plate fixation of distal tibial spiral fractures enables immediate post-operative full weight-bearing: a biomechanical analysis.

PURPOSE: Distal tibial fractures generally require post-operative weight-bearing restrictions. Especially geriatric patients are unable to follow these recommendations. To increase post-operative implant stability and enable early weight-bearing, augmentation of the primary osteosynthesis by cerclage is desirable. The purpose of this study was to identify the stabilizing effects of a supplemental cable cerclage following plate fixation of distal tibial spiral fractures compared to solitary plate osteosynthesis. METHODS: In eight synthetic tibiae, a reproducible spiral fracture (AO/OTA 42-A1.1c) was stabilized by angle stable plate fixation. Each specimen was statically loaded under combined axial and torsional loads to simulate partial (200 N, 2 Nm) and full (750 N, 7 Nm) weight-bearing. Tests were repeated with supplemental cable cerclage looped around the fracture zone. In a subsequent stepwise increased dynamic load scenario, construct stiffness and interfragmentary movements were analyzed. RESULTS: With supplemental cable cerclage, construct stiffness almost tripled compared to solitary plate osteosynthesis (2882 ± 739 N/mm vs. 983 ± 355 N/mm; p < 0.001). Under full weight-bearing static loads, a supplemental cerclage revealed reduced axial (- 55%; p = 0.001) and shear movement (- 83%; p < 0.001), and also lowered shear movement (- 42%; p = 0.001) compared to a solitary plate under partial weight-bearing. Under dynamic loads supplemental cerclage significantly reduced axial (p = 0.005) as well as shear movements (p < 0.001). CONCLUSION: Supplemental cable cerclage significantly increases fixation stiffness and reduces shear movement in distal tibial spiral fractures. This stabilizing effect enables from a biomechanical point of view immediate mobilization without any weight-bearing restrictions, which may improve the quality of care of orthopedic patients and may trigger a change towards early weight-bearing regimes, especially geriatric patients would benefit from.

Biomechanical comparison of different cerclage types in addition to an angle stable plate osteosynthesis of distal tibial fractures.

BACKGROUND: Different stand-alone cerclage configurations and their optimal twisting techniques have been investigated over the years. This study tests for the stabilizing effect of different supplemental cerclage materials in combination with locked plating of distal tibia fractures. METHODS: Locking plate fixation of a distal tibial spiral fracture was tested as stand-alone and with supplemental cerclage materials (one cable, two cables, wire, fiber tape). Construct stiffness and fracture gap movements were investigated under quasi-static and dynamic loads and compared to the stand-alone locking plate. RESULTS: With each of the tested cerclages, stiffness was significantly higher than for a solitary plate osteosynthesis. Most reduction in fracture gap movement was achieved by cable cerclages, followed by double-looped wire and double-looped fiber tape cerclages. Under dynamic loading an additional cable cerclage reduces excessive gap movement. CONCLUSION: Compared to solitary plate osteosynthesis all supplemental cerclage materials were generally superior with reduced fracture gap movements whereas cable cerclages showing the greatest stabilizing effect.

Alarin in different human intestinal epithelial cell types.

Alarin (AL), a new member of the galanin family, has been localized in various CNS regions, mainly in rodents. Among other effects, it modulates food intake. Therefore, we analyzed the immunohistochemical distribution pattern of AL in human intestinal epithelia. Cryosections of 12 human bowel samples were immunohistochemically double-stained for AL and α-defensin 5 (αD; first set). Two further sets of sections were quadruple-stained either (second set) for AL, chromogranin (CG), synaptophysin (SY), and somatostatin (SO) or (third set) for AL, CG, Peptide Y (PY), and 5-hydroxytryptamine (5-HT). Slides were digitized and quantitative analysis of co-localization rates was undertaken. Small bowel: most of AL-positive cells (56%) were αD-positive Paneth cells located within the base of the crypts (first set). In the second set, about 27% of AL-labeled cells were co-reactive for SY and CG, likely representing entero-endocrine cells. In the third set, the largest subpopulation of AL-positive cells was not co-reactive for other markers applied (89%); most of them were likely Paneth cells. Large bowel: co-localization of AL with αD was not detected (first set). In the second set, AL was frequently co-localized with the other three markers applied (68%). In the third set, AL was frequently co-localized with 5-HT and CG (31%) as well as with PY and 5-HT (22%). Due to its presence in various enteroendocrine as well as Paneth cells, AL may be involved in different physiological and pathological processes.

Bone surrogates provide authentic onlay graft fixation torques.

Onlay graft bone augmentation is a standard practice to restore the loss of height of the alveolar ridge following loss of a tooth. Cranial grafts, lifted from the parietal bone, are sandwiched and used to bridge the bony defect in the jaw by means of small screws. During the elevation of the covering gum and subsequent screw placement, care has to be taken in order to preserve underlying nerves. Therefore, to avoid harm to the patient, a solid education of surgeons is essential, which requires training and experience. A simulator for cranial graft-lift training was already developed and shall be expanded to train the augmentation of the lifted implants. Therefore, in this study, synthetic bones for onlay block graft screw placement with realistic haptics for the screw application training were evaluated and compared with human specimens. Six different polyurethane based bone surrogate composites, enriched with varying amounts of calcium-based mineral fillers and blowing agents, were developed. The haptical properties of these synthetic bones were validated for screw placement and compared with human parietal bone specimens. For that, bones were pre-drilled, screws were automatically inserted using a customized testbench and the slope of the screw-insertion torques were analyzed. The slope of the screw insertion torques of the human reference bones was 56.5 ±â€¯14.0 * 10-3 Nm/deg, Surrogates with lower amounts of mineral fillers and blowing agents showed lower torques than the human bone. Synthetic bones, validated for drilling, milling and sawing in an earlier study, also achieved significantly lower torques, which were only the half of the human parietal bones. Two intermediate stages of the aforementioned material compositions, consisting of 75% mineral filler with 0.75% blowing agent and 100% mineral filler with 1.00% blowing agent revealed results comparable with human bone (57.4 ±â€¯10.2 *10-3 Nm/deg, p = 0.893 and 54.9 ±â€¯11.1 *10-3 Nm/deg, p = 0.795, respectively). In conclusion, our findings suggest that, two newly developed polyurethane-based materials mimicking the haptical properties of an onlay bone graft screw fixation, have been identified. Thus, these surrogates are capable of mimicking real bone tissue in our simulator for the education of novice surgeons.

Characterization of polyurethane-based synthetic vertebrae for spinal cement augmentation training.

Vertebral augmentation techniques are used to stabilize impacted vertebrae. To minimize intraoperative risks, a solid education of surgeons is desirable. Thus, to improve education of surgeons as well as patient safety, the development of a high-fidelity simulator for the surgical training of cement augmentation techniques was initiated. The integrated synthetic vertebrae should be able to provide realistic haptics during all procedural steps. Synthetic vertebrae were developed, tested and validated with reference to human vertebrae. As a further reference, commercially available vertebrae surrogates for orthopedic testing were investigated. To validate the new synthetic vertebrae, characteristic mechanical parameters for tool insertion, balloon dilation pressure and volume were analyzed. Fluoroscopy images were taken to evaluate the bone cement distribution. Based on the measurement results, one type of synthetic vertebrae was able to reflect the characteristic parameters in comparison to human vertebrae. The different tool insertion forces (19.7 ± 4.1, 13.1 ± 0.9 N, 1.5 ± 0.2 N) of the human reference were reflected by one bone surrogate (11.9 ± 9.8, 24.3 ± 3.9 N, 2.4 ± 1.0 N, respectively). The balloon dilation pressure (13.0 ± 2.4 bar), volume (2.3 ± 1.5 ml) of the synthetic vertebrae were in good accordance with the human reference (10.7 ± 3.4 bar, 3.1 ± 1.1 ml). Cement application forces were also in good accordance whereas the cement distribution couldn't be reproduced accurately. Synthetic vertebrae were developed that delivered authentic haptics during transpedicular instrument insertion, balloon tamp dilation and bone cement application. The validated vertebra model will be used within a hybrid simulator for minimally invasive spine surgery to educate and train surgeons.

Characterization of an artificial skull cap for cranio-maxillofacial surgery training.

Cranial grafts are favored to reconstruct skeletal defects because of their reduced resorption and their histocompatibility. Training possibilities for novice surgeons include the "learning by doing" on the patient, specimens or simulators. Although the acceptance of simulators is growing, the major drawback is the lack of validated bone models. The aim of this study was to create and validate a realistic skull cap model and to show superiority compared to a commercially available skull model. Characteristic forces during machinery procedures were recorded and thickness parameters from the bony layers were obtained. The thickness values of the bone layers of the developed parietal bone were comparable to the human ones. Differences between drilling and sawing forces of human and artificial bones were not detected using statistical analysis. In contrast the parameters of the commercially available skull model were significantly different. However, as a result, a model-based simulator for tabula externa graft lift training, consisting of a brain, skull bone cap and covering soft tissues was created. This simulator enables the training of all procedural steps of a "split thickness graft lift". In conclusion, an artificial skull cap suitable for parietal graft lift training was manufactured and validated against human parietal bones.

Procedure-Specific Validation of Artificial Vertebrae.

OBJECTIVE: The development of a novel hybrid patient simulator was initiated to provide a safe training possibility for novice surgeons. Integrated artificial vertebrae should be able to realistically mimic the haptics of transpedicular vertebroplasty instrument insertion and pedicle screw placement. Therefore, new open-celled material compositions were developed, tested, and validated with reference to elderly human vertebrae. METHODS: Vertebroplasty tool insertion force and pedicle screw torque measurements were performed. To validate the new bone surrogates for transpedicular tool insertion, a novel parametric model of the procedure was developed identifying three characteristic insertion parameters (weighting factors, cutting, and clamping forces). Furthermore, the slope of the insertion torque was used to validate the new materials against the human vertebrae for pedicle screw placement. RESULTS: A relative error less than 6% confirmed the suitability of the parametric model for validation. The weighting factors () and the clamping forces ( ) of the human reference were met by the bone surrogate with 1.25% of blowing agent ( and , respectively). However, no material was able to reflect the instrument cutting forces. The slope obtained during pedicle screw placement in human vertebrae was  Nm/m. The material composition with 1% blowing agent achieved similar results ( N m/m). CONCLUSION: Two suitable materials that deliver realistic haptics during both instrument insertions were validated. The parametric model suitably modeled the transpedicular instrument insertion. SIGNIFICANCE: These newly developed models provide a realistic haptic feedback during transpe-dicular instrument insertions with the potential of cement application during surgical skill training.

Retinal Pericytes: Characterization of Vascular Development-Dependent Induction Time Points in an Inducible NG2 Reporter Mouse Model.

Purpose/aim of the study: In the retina, defects in pericytes (PCs) function/loss are associated with various complications; however, the exact pathological mechanisms are still not fully elucidated. Following the behavior of retina-resident PCs during health and disease will reveal new insights for both the understanding of pathological mechanisms and the development of new regenerative therapies for the treatment of retinopathies. The main goal of this study is to determine whether the NG2-reporter mouse (NG2CreERTM-eGFP) is a suitable model to study the fate of retina-resident PCs. MATERIAL AND METHODS: Vascular development-dependent reporter induction in retinal PCs was evaluated at different time points [(a) > P21, (b) < P21, and (c) P1 to > P21)] and additionally four different modes of application were tested. Reporter expression was evaluated by enhanced green fluorescent protein (eGFP) immunofluorescence by confocal microscopy and induction efficiency was calculated by analyzing NG2-expressing PCs in comparison to eGFP-labeled PCs in the three capillary layers. RESULTS: eGFP-positive PCs were detected in the three retinal capillary layers at all time points and administration routes tested. Multiple tamoxifen (TAM) applications in adult (> P21) NG2CreERTM-eGFP mice resulted in 3.59% eGFP-positive PCs. 2.37% eGFP-labeled PCs were detected after single intraperitoneal TAM injections at early postnatal days (P2/P5); however, just 1.61% PCs revealed reporter expression upon activation via the lactating mother (P4-P7). The highest number of eGFP-labeled PCs (7.09%) was detected following triple TAM administrations (P10-P12). The number of reporter-positive PCs doubled using homozygous animals. CONCLUSION: Despite low recombination efficiency in the used PC-specific fate mapping mouse model, changes in NG2 promoter activity of PCs during vascular development are indicated by single and multiple TAM inductions at different developmental time points. Nevertheless, these findings need further confirmation in up-coming studies by using homozygous NG2CreERTM-eGFP mice and additionally by mating the NG2CreERTM with a different reporter mouse to increase the low recombination efficiency.

Validation of a simulator for cranial graft lift training: Face, content, and construct validity.

PURPOSE: Surgical skills can be improved through practical exercise. The use of specimens, human as well as animal, or live animals for surgical training is limited due to ethical concerns. Drawbacks of simulators are costs, fidelity and creditibility. Thus, simulators must be evaluated objectively to determine their validity before they can be used as teaching modalities. The aim of this study was to verify the face content and construct validity of a novel model-based simulator for lifting tabula externa transplants from the parietal skull. MATERIALS AND METHODS: Participants were invited to perform a tabula externa graft lift during a training session on the simulator. Task performance was analyzed with a standardized assessment tool evaluating realism and appropriateness. Specialist ratings were used to evaluate the performance of the participants. This was an exploratory study using a questionnaire, at Kepler University Hospital, Linz, Austria, a university hospital. According to their expertise in craniomaxillofacial surgery, 17 participants were subdivided into 3 groups: 8 novices, 7 experts and 2 raters. RESULTS: The face validity (realism) obtained an average score of 4.2 of a maximum of 5 points. Likewise, the content validity (appropriateness as a teaching modality) obtained an average score of 4.8 of maximum 5 points. No differences were found between experts and novices concerning the recorded surgery completion times (p = 0.418) or the sizes of the lifted grafts (p = 0.110). During the evaluation of task performance, the expert surgeons (46.9 ± 3.7) were graded significantly better than the novices (36.4 ± 8.5), which proved the construct validity of the simulator (p = 0.001). CONCLUSION: All investigated validities were confirmed and approved the simulator as a valid training tool for parietal graft lift.

VIP changes during daytime in chicken intrinsic choroidal neurons.

PURPOSE: Ocular autonomic control is mediated by sympathetic and parasympathetic nerve fibres. Their interactions are complemented by primary afferent nerve fibers of and intrinsic choroidal neurons (ICN). As the vasodilatative neuropeptide, vasoactive intestinal peptide (VIP), is expressed in extrinsic and intrinsic ocular neurons, it is of special interest in ophthalmic research. Since circadian changes of ocular blood flow are known in humans and birds, this study aimed at investigating VIP expression at different daytimes in chicken choroid, the preferred model species in ICN research. METHODS: 12 eyes of 12 chickens were retrieved, slaughtered at 8.00-9.30 a.m. (n = 6) and 8.00 p.m. (n = 6), respectively, and choroidal wholemounts were prepared for immunofluorescence of VIP. VIP-positive ICN of both groups were quantified and density of VIP-positive axons assessed semi-quantitatively. In 28 additional eyes retrieved in the morning (n = 14) and evening (n = 14), choroidal VIP content was determined by ELISA. Morning and evening data were analyzed statistically. NADPH-diaphorase (NADPH-d, ICN cell marker) was done at additional 12 whole mount choroids of 12 chicken, retrieved in the morning (n = 6) and evening (n = 6). RESULTS: (1) Numbers of VIP positive neurons differed significantly between morning: (239.17 ±â€¯113.9) and evening: (550.83 ±â€¯245.7; p = 0.018). (2) Numbers of VIP-positive perikarya were significantly more accumulated in the temporal part of the choroid in the evening than in the morning (p = 0.026). (3) VIP positive axon density was found to be similar throughout the choroid in the morning and evening. (4) Number of NADPH-d positive neurons was not significantly different between morning (848.8 ±â€¯399.5) and evening (945.8 ±â€¯622.1, p > 0.05). (5) ELISA demonstrated a significant difference of VIP content (p = 0.012) in tissues harvested in the morning (145.41 ±â€¯43.3 pg/ml) compared to evening (221.44 ±â€¯106.3 pg/ml). CONCLUSIONS: As VIP positive axon density was similar in the morning and the evening throughout the choroid, PPG and ICN seemed to contribute equally to the axon network. Yet, changes in the total choroidal VIP content, the numbers of VIP positive perikarya, reflecting the intracellular VIP content, and their topographical distribution at two different days-times argue for a different status of activation of both neuronal sources in contrast to the equal amount of NADPHD-d positive neurons. The higher VIP content in the evening, compared to the morning, correlates with a known circadian rhythm of a lower IOP and a higher choroidal thickness at night. Thus, these changes may argue for a potential role of ICN in the regulation of ocular homeostasis and integrity.

Novel synthetic vertebrae provide realistic haptics for pedicle screw placement.

During vertebral surgery, misplaced pedicle screws can harm vital neural and vascular structures. Haptic distinction between cortical and cancellous bone structures is therefore essential for correct screw placement. This tactile experience during pedicle screw placement can be obtained by training on human or animal specimens even if expensive or ethically questionable. In this study, novel synthetic vertebrae were evaluated within a hybrid simulator to provide realistic haptics for the training of spine surgeries. Synthetic vertebrae were custommade of calcium powder-based composites imitating both, cancellous and cortical bone. The mechanical properties of synthetic surrogates were validated for pedicle screw placement and cement augmentation and were compared with those obtained from human vertebrae and insertion torques were analyzed. In human vertebrae pedicle screw torque measurements resulted in mean torque slopes of 82±33Nm/m. Calcium carbonate-based materials achieved lower torques than the human bone whereas calcium phosphate-based bone surrogates showed comparable results. A further differentiation of the calcium phosphate-based vertebrae revealed, that synthetic vertebrae with lower amounts of blowing agent, achieved suitable torques (83 ± 28Nm/m) in comparison to the human reference (p = 0.39). Cement application and subsequent fluoroscopy images confirmed, that the cancellous core of the synthetic vertebrae enabled cement augmentation. In conclusion, our findings suggest, that the artificial bone samples mimic the properties of human bone during pedicle screw placement and cement augmentation and are therefore suitable as synthetic vertebrae in a hybrid surgical simulator.

Novel bone surrogates for cranial surgery training.

Parietal graft lifts are trained on human or animal specimens or are directly performed on patients without extensive training. In order to prevent harm to the patient resulting from fast rotating machinery tools, the surgeon needs to apply appropriate forces. Realistic haptics are essential to identify the varying parietal bone layers and to avoid a penetration of the brain. This however, requires experience and training. Therefore, in this study, bone surrogate materials were evaluated with the aim to provide an anatomically correct artificial skull cap with realistic haptic feedback for graft lift training procedures. Polyurethane composites made of calcium carbonate and calcium phosphate were developed and were used to create customized bone surrogates, imitating both cancellous and cortical bone. Mechanical properties of these surrogates were validated for drilling, milling and sawing by comparison with human parietal bones. For that, surgical tool tips were automatically inserted into artificial and human bones in a customized test bench and the maximum axial insertion forces were analyzed. Axial tool insertion measurements in human parietal bones resulted in mean maximum forces of 1.8±0.5N for drilling, 1.7±0.3N for milling and 0.9±0.1N for sawing. Calcium carbonate-based materials achieved higher forces than the human bone for drilling and milling, and lower forces for sawing. The calcium phosphate-based bone surrogates showed comparable axial insertions forces for all investigated tools and were identified as a suitable surrogate for drilling (p=0.87 and 0.41), milling (p=0.92 and 0.63) and sawing (p=0.11 and 0.76) of the cortical layer and the cancellous bone, respectively. In conclusion, our findings suggest, that a suitable material composition for artificial parietal bones has been identified, mimicking the properties of human bone during surgical machinery procedures. Thus, these materials are suitable for surgical training and education in simulator training.

Distribution of the neuro-regulatory peptide galanin in the human eye.

Galanin (GAL) is a neuro-regulatory peptide involved in many physiological and pathophysiological processes. While data of GAL origin/distribution in the human eye are rather fragmentary and since recently the presence of GAL-receptors in the normal human eye has been reported, we here systematically search for sources of ocular GAL in the human eye. Human eyes (n=14) were prepared for single- and double-immunohistochemistry of GAL and neurofilaments (NF). Cross- and flat-mount sections were achieved; confocal laser-scanning microscopy was used for documentation. In the anterior eye, GAL-immunoreactivity (GAL-IR) was detected in basal layers of corneal epithelium, endothelium, and in nerve fibers and keratinocytes of the corneal stroma. In the conjunctiva, GAL-IR was seen throughout all epithelial cell layers. In the iris, sphincter and dilator muscle and endothelium of iris vessels displayed GAL-IR. It was also detected in stromal cells containing melanin granules, while these were absent in others. In the ciliary body, ciliary muscle and pigmented as well as non-pigmented ciliary epithelium displayed GAL-IR. In the retina, GAL-IR was detected in cells associated with the ganglion cell layer, and in endothelial cells of retinal blood vessels. In the choroid, nerve fibers of the choroidal stroma as well as fibers forming boutons and surrounding choroidal blood vessels displayed GAL-IR. Further, the majority of intrinsic choroidal neurons were GAL-positive, as revealed by co-localization-experiments with NF, while a minority displayed NF- or GAL-IR only. GAL-IR was also detected in choroidal melanocytes, as identified by the presence of intracellular melanin-granules, as well as in cells lacking melanin-granules, most likely representing macrophages. GAL-IR was detected in numerous cells and tissues throughout the anterior and posterior eye and might therefore be an important regulatory peptide for many aspects of ocular control. Upcoming studies in diseased tissue will help to clarify the role of GAL in ocular homeostasis.

Characterization of Antigen-Presenting Macrophages and Dendritic Cells in the Healthy Human Sclera.

PURPOSE: The sclera is mainly made of collagen and fibroblasts. The aim of this study was to analyze whether immune cells are present in the healthy human sclera. METHODS: Ten human anterior episcleral or stromal tissue samples from globe donors were immunohistochemically examined using confocal microscopy. The expression of the macrophage markers CD68, CD163 and CD11b, CD45 (a general leukocyte marker), MHCII (expressed by antigen-presenting cells [APCs]), CD11c (dendritic cell marker), lymphatic endothelium hyaluronan receptor-1 (LYVE1; expressed on lymphatic endothelium and macrophage subsets), chemokine receptor 7 (CCR7, a homing receptor for leukocytes), CXCL12 (expressed by activated leukocytes), CCR2 (a marker for inflammatory monocytes), and glial fibrillary acidic protein (GFAP; expressed by astrocytes) was analyzed and quantified. RESULTS: In the episclera, a high number of cells (≥40 cells/mm2) were immunoreactive for CD68, CD45, MHCII, CCR7, LYVE1, and CD11b. Lower numbers (<20 cells/mm2) were positive for CXCL12, CCR2, and GFAP. The episclera showed a significantly higher number of cells compared to the stroma (P = 0.008). MHCII+ cells could be double positive for CCR7, CD45, CD11c, or CD11b and seldom CXCL12. Macrophages were most likely from the M1 type (CD68+, CD163-). CONCLUSIONS: The healthy human sclera contains several macrophage populations, which can function as APCs, with the highest density being present in the episclera. Most cells express macrophage markers and may function as APCs. The presence of these cells might indicate that scleral immune cells are important for maintaining physiological functions in the eye and may potentially contribute to blood vessel homeostasis.

Retinal Vessel Diameter Responses to Central Electrical Stimulation in the Rat: Effect of Nitric Oxide Synthase Inhibition.

PURPOSE: Recent histological data suggest autonomic innervation of the central retinal artery. In the present study, we investigated the effect of electrical brain stem stimulation at the superior salivatory nucleus (SSN) on the retinal vessel diameter in rats and whether nitric oxide mediates a possible effect. METHODS: Sprague-Dawley rats (n = 12) were anesthetized using pentobarbital sodium (50 mg/kg intraperitoneally). The animals were artificially ventilated and the femoral artery and vein were cannulated for blood pressure measurement and drug administration. After a craniotomy was performed, a unipolar stainless steel electrode was inserted into the brainstem at the coordinates of the SSN. Stimulations were performed at 20 Hz, 9 µA, 1 ms pulse duration and 200 pulses. Retinal vessel diameters were measured continuously with the Imedos DVA-R, a noncontact fundus camera for rats with image analysis software. After control measurements, L-NAME, a nonspecific inhibitor of NO synthase, was applied intravenously (10 mg/kg), and the SSN stimulations were repeated. RESULTS: Stimulation at the SSN coordinates increased the retinal arterial diameter by 6.41% ± 1.65% and the venous diameter by 3.48% ± 1.93% (both P < 0.05). Application of L-NAME reduced the arterial response significantly to 2.93% ± 0.91%, but did not change the venous response. Mean arterial pressure, carotid blood flow, and heart rate remained unaltered (by the stimulation). CONCLUSIONS: The present study demonstrates that the retinal circulation reacts to electric stimulation at the SSN coordinates in rats. Nitric oxide is involved in the response, but it is not the sole neurotransmitter.

Immunohistochemical Detection of CTGF in the Human Eye.

Purpose/Aim of the study: Connective tissue growth factor (CTGF) is a key player in the control of extracellular matrix remodeling, fibrosis, and angiogenesis. It is also involved in the modification of the trabecular meshwork, thus potentially modulating outflow facility and intraocular pressure (IOP). As a consequence, CTGF might be relevant for the development of elevated IOP, a major risk factor in glaucoma-pathogenesis. While comprehensive information on the origins of CTGF in the human eye is not available, the goal of this study is to identify ocular sources of CTGF using morphological methods. MATERIALS AND METHODS: Human donor eyes were prepared for immunohistochemical analysis of CTGF, α-smooth muscle-actin (ASMA), and CD31. Confocal laser scanning microscopy was used for documentation. RESULTS: In the cornea, CTGF-immunoreactivity (CTGF-IR) was detected in the epithelium, mainly in basal layers, stromal keratinocytes, and endothelial cells. Adjacent conjunctiva showed also CTGF-IR in epithelial cells. In the iris, both, the sphincter and dilator muscles displayed CGTF-IR, as did iris and ciliary body vessels, deriving at this location from the vascular endothelium, as detected with CD31, but not from vascular smooth muscle cells, as detected with ASMA. In the ciliary body, CTGF-IR was detected in smooth-muscle cells of the ciliary muscle and further in the non-pigmented epithelium. In the retina, CTGF-IR was detected in the NFL and weakly in the IPL/OPL. In the choroid, the choriocapillaris and blood vessels displayed CTGF-IR. Further, few cells in the optic nerve head and the lamina cribrosa were CTGF-positive. CONCLUSION: CTGF was detected in various structures of the human eye. Since CTGF has been also described in aqueous humor, the identified structures might be the sources of CTGF in the aqueous humor. By means of aqueous flow, CTGF is transported into the trabecular meshwork, where it could change outflow facility and therefore affecting IOP homeostasis.

Disturbed mitochondrial and peroxisomal dynamics due to loss of MFF causes Leigh-like encephalopathy, optic atrophy and peripheral neuropathy.

BACKGROUND: Mitochondria are dynamic organelles which undergo continuous fission and fusion to maintain their diverse cellular functions. Components of the fission machinery are partly shared between mitochondria and peroxisomes, and inherited defects in two such components (dynamin-related protein (DRP1) and ganglioside-induced differentiation-associated protein 1 (GDAP1)) have been associated with human disease. Deficiency of a third component (mitochondrial fission factor, MFF) was recently reported in one index patient, rendering MFF another candidate disease gene within the expanding field of mitochondrial and peroxisomal dynamics. Here we investigated three new patients from two families with pathogenic mutations in MFF. METHODS: The patients underwent clinical examination, brain MRI, and biochemical, cytological and molecular analyses, including exome sequencing. RESULTS: The patients became symptomatic within the first year of life, exhibiting seizures, developmental delay and acquired microcephaly. Dysphagia, spasticity and optic and peripheral neuropathy developed subsequently. Brain MRI showed Leigh-like patterns with bilateral changes of the basal ganglia and subthalamic nucleus, suggestive of impaired mitochondrial energy metabolism. However, activities of mitochondrial respiratory chain complexes were found to be normal in skeletal muscle. Exome sequencing revealed three different biallelic loss-of-function variants in MFF in both index cases. Western blot studies of patient-derived fibroblasts indicated normal content of mitochondria and peroxisomes, whereas immunofluorescence staining revealed elongated mitochondria and peroxisomes. Furthermore, increased mitochondrial branching and an abnormal distribution of fission-mediating DRP1 were observed. CONCLUSIONS: Our findings establish MFF loss of function as a cause of disturbed mitochondrial and peroxisomal dynamics associated with early-onset Leigh-like basal ganglia disease. We suggest that, even if laboratory findings are not indicative of mitochondrial or peroxisomal dysfunction, the co-occurrence of optic and/or peripheral neuropathy with seizures warrants genetic testing for MFF mutations.