Ex Vivo Micro-CT in Ophthalmology: Preparation and Contrasting for Non-invasive 3D-Visualisation. / Ex-vivo-Mikro-CT in der Augenheilkunde: Probenbehandlung und -kontrastierung für die 3D-Darstellung.

X-ray-based micro-computed tomography (micro-CT) is a largely non-destructive imaging method for the visualisation and analysis of internal structures in the ex vivo eye and affords high resolution. In contrast to other high-resolution imaging methods, micro-CT enables spatial recording of larger and more complex tissue structures, such as the anterior chamber of the eye. Special contrasting methods help to enhance the absorption properties of soft tissue, that is otherwise only weakly radiopaque. Critical point drying (CPD), as primarily used in scanning electron microscopy, offers an additional tool for improving differential contrast properties in soft tissue. In the visualisation of intraosseous soft tissue, such as the efferent lacrimal ducts, sample treatment by decalcification with ethylenediaminetetraacetic acid and subsequent CPD provides good results for micro-CT. Micro-CT can be used for a wide range of questions in 1. basic research, 2. application-related studies in ophthalmology (e.g. evaluation of the preclinical application of microstents for glaucoma treatment or analysis of the positioning of intraocular lenses) but also 3. as a supplement to ophthalmological histopathology.

Micro-CT in ophthalmology: ex vivo preparation and contrasting methods for detailed 3D-visualization of eye anatomy with special emphasis on critical point drying.

Background: Micro-computed tomography (micro-CT) provides detailed 3-dimensional (3D) visualization of anatomical structures and encourages morphological reinvestigation of organs with delicate features. The low radiodensity of soft tissues necessitates preceding sample preparation to conduct X-ray imaging with decent contrast between different tissues. In this study, we demonstrate the preparation with three radiopaque agents in combination with elimination of liquids by critical point drying (CPD) introduced for ocular samples. Methods: Enucleated porcine eyes were prepared with ethanolic iodine (EI), aqueous iodine-potassium iodide, or ethanolic phosphotungstic acid (EPTA). Micro-CT scans of the samples were conducted in a moist environment with an isotropic resolution of 9.2-12.5 µm voxel size. Subsequently, samples were chemically dehydrated and critical point (CP) dried to conduct a second scan in a dry environment with a resolution up to 4.7-5.4 µm in voxel size. The visualization effects were qualitatively and semi-quantitatively evaluated with regard to the generated contrast between different ocular tissues. Results: All three contrast agents accumulated well in most of the investigated ocular tissues and lead to an increased X-ray attenuation which allowed for differentiated visualization of ocular structures. Problematic agent penetration into the lens was obvious for iodine-potassium iodide and EPTA. Artificial damages of the lens and thickness reduction for the cornea and sclera due to CPD were noticed. The effects of the different contrasting treatments are described and compared with regard to the effects of CPD. Exclusively CP dried samples that were not treated with contrast agents could also be visualized excellently with a good distinction of different ocular structures from each other. Conclusions: All ocular structures can be visualized by micro-CT. To contrast moist samples, the best results were achieved with iodine potassium iodide (IPI). CPD improved the scan quality in all cases. Even without pretreatment with contrasting agents, the CP dried samples showed a contrast similar to the IPI treated samples.

Transmembrane protein 119 is neither a specific nor a reliable marker for microglia.

Microglia are the resident innate immune cells of the central nervous system (CNS) parenchyma. To determine the impact of microglia on disease development and progression in neurodegenerative and neuroinflammatory diseases, it is essential to distinguish microglia from peripheral macrophages/monocytes, which are eventually equally recruited. It has been suggested that transmembrane protein 119 (TMEM119) serves as a reliable microglia marker that discriminates resident microglia from blood-derived macrophages in the human and murine brain. Here, we investigated the validity of TMEM119 as a microglia marker in four in vivo models (cuprizone intoxication, experimental autoimmune encephalomyelitis (EAE), permanent filament middle cerebral artery occlusion (fMCAo), and intracerebral 6-hydroxydopamine (6-OHDA) injections) as well as post mortem multiple sclerosis (MS) brain tissues. In all applied animal models and post mortem MS tissues, we found increased densities of ionized calcium-binding adapter molecule 1+ (IBA1+ ) cells, paralleled by a significant decrease in TMEM119 expression. In addition, other cell types in peripheral tissues (i.e., follicular dendritic cells and brown adipose tissue) were also found to express TMEM119. In summary, this study demonstrates that TMEM119 is not exclusively expressed by microglia nor does it label all microglia, especially under cellular stress conditions. Since novel transgenic lines have been developed to label microglia using the TMEM119 promotor, downregulation of TMEM119 expression might interfere with the results and should, thus, be considered when working with these transgenic mouse models.

A Rare Case of Facial Artery Branching-A Review of the Literature and a Case Report with Clinical Implications.

Background and Objectives: Vascular variations appear as morphologically distinct patterns of blood diverging from the most commonly observed vessel patterns. The facial artery is considered to be the main vessel for supplying blood to the anterior part of the face. An anatomical understanding of the facial artery, its course, its topography, and its branches is important in medical and dental practice (especially in neck and face surgery), and is also essential for radiologists to be able to interpret vascular imaging in the face following angiography of the region. A profound knowledge of the arteries in the region will aid in minimizing the risks to the patient. Materials and Methods: In our publication a narrative literature review and a case report are presented. Results: A rare case of a facial artery pattern has been described anatomically for the first time with respect to its course and branching. This variation was found on the left side of a 60-year-old male corpse during anatomical dissection. The anterior branch of the facial artery arched in the direction of the labial angle, and there divided into the inferior and superior labial arteries. At the same time, the posterior branch coursed vertically and superficially to the masseter muscle. It here gave off the premasseteric branch, and continued towards the nose, where it ran below the levator labii superioris and the levator labii superioris alaeque nasi muscles and terminated at the dorsum nasi. Conclusions: Our review of the literature and the case report add to knowledge on the facial artery with respect to its topographical anatomy and its branching and termination patterns, as well as the areas of supply. An exact knowledge of individual facial artery anatomy may play an important role in the planning of flaps or tumor excisions due to the differing vascularization and can also help to prevent artery injuries during aesthetic procedures such as filler and botulinum toxin injections.

Ultrahigh field MRI determination of water diffusion rates in ex vivo human lenses of different age.

BACKGROUND: The development of presbyopia is correlated with increased lens stiffness. To reveal structural changes with age, ultrahigh field magnetic resonance imaging (UHF-MRI) was used to analyze water diffusion in differently aged human lenses ex vivo. METHODS: After enucleation lens extractions were performed. Lenses were photographed, weighed, and embedded in 0.5% agarose dissolved in culture medium. UHF-MRI was conducted to analyze anatomical characteristics of the lens using T2-weighted Turbo-RARE imaging and to obtain apparent diffusion coefficients (ADC) measurements. A Gaussian fit routine was used to examine the ADC histograms. RESULTS: An age-dependent increase in lens wet weight, lens thickness, and lens diameter was found (P<0.001). T2-weighted images revealed a hyperintense lens cortex and a gradually negative gradient in signal intensity towards the nucleus. ADC histograms of the lens showed bimodal distributions (lower ADC values mainly located in the nucleus and higher ADC values mainly located in the cortex), which did not change significantly with age [ßPeak1=1.96E-7 (-20E-7, 10E-7), P=0.804 or ßPeak2=15.4E-7 (-10E-7, 40E-7), P=0.276; respectively]. CONCLUSIONS: Clinically relevant age dependent lens hardening is probably not correlated with ADC changes within the nucleus, which could be confirmed by further measurements.

The initial engraftment of tumor cells is critical for the future growth pattern: a mathematical study based on simulations and animal experiments.

BACKGROUND: Xenograft mouse tumor models are used to study mechanisms of tumor growth and metastasis formation and to investigate the efficacy of different therapeutic interventions. After injection the engrafted cells form a local tumor nodule. Following an initial lag period of several days, the size of the tumor is measured periodically throughout the experiment using calipers. This method of determining tumor size is error prone because the measurement is two-dimensional (calipers do not measure tumor depth). Primary tumor growth can be described mathematically by suitable growth functions, the choice of which is not always obvious. Growth parameters provide information on tumor growth and are determined by applying nonlinear curve fitting. METHODS: We used self-generated synthetic data including random measurement errors to research the accuracy of parameter estimation based on caliper measured tumor data. Fit metrics were investigated to identify the most appropriate growth function for a given synthetic dataset. We studied the effects of measuring tumor size at different frequencies on the accuracy and precision of the estimated parameters. For curve fitting with fixed initial tumor volume, we varied this fixed initial volume during the fitting process to investigate the effect on the resulting estimated parameters. We determined the number of surviving engrafted tumor cells after injection using ex vivo bioluminescence imaging, to demonstrate the effect on experiments of incorrect assumptions about the initial tumor volume. RESULTS: To select a suitable growth function, measurement data from at least 15 animals should be considered. Tumor volume should be measured at least every three days to estimate accurate growth parameters. Daily measurement of the tumor volume is the most accurate way to improve long-term predictability of tumor growth. The initial tumor volume needs to have a fixed value in order to achieve meaningful results. An incorrect value for the initial tumor volume leads to large deviations in the resulting growth parameters. CONCLUSIONS: The actual number of cancer cells engrafting directly after subcutaneous injection is critical for future tumor growth and distinctly influences the parameters for tumor growth determined by curve fitting.

Defining body-weight reduction as a humane endpoint: a critical appraisal.

In many animal experiments scientists and local authorities define a body-weight reduction of 20% or more as severe suffering and thereby as a potential parameter for humane endpoint decisions. In this study, we evaluated distinct animal experiments in multiple research facilities, and assessed whether 20% body-weight reduction is a valid humane endpoint criterion in rodents. In most experiments (restraint stress, distinct models for epilepsy, pancreatic resection, liver resection, caloric restrictive feeding and a mouse model for Dravet syndrome) the animals lost less than 20% of their original body weight. In a glioma model, a fast deterioration in body weight of less than 20% was observed as a reliable predictor for clinical deterioration. In contrast, after induction of chronic diabetes or acute colitis some animals lost more than 20% of their body weight without exhibiting major signs of distress. In these two animal models an exclusive application of the 20% weight loss criterion for euthanasia might therefore result in an unnecessary loss of animals. However, we also confirmed that this criterion can be a valid parameter for defining the humane endpoint in other animal models, especially when it is combined with additional criteria for evaluating distress. In conclusion, our findings strongly suggest that experiment and model specific considerations are necessary for the rational integration of the parameter 'weight loss' in severity assessment schemes and humane endpoint criteria. A flexible implementation tailored to the experiment or intervention by scientists and authorities is therefore highly recommended.

Continuous Blood Sampling in Small Animal Positron Emission Tomography/Computed Tomography Enables the Measurement of the Arterial Input Function.

For quantitative analysis and bio-kinetic modeling of positron emission tomography/computed tomography (PET/CT) data, the determination of the temporal blood time-activity concentration also known as arterial input function (AIF) is a key point, especially for the characterization of animal disease models and the introduction of newly developed radiotracers. The knowledge of radiotracer availability in the blood helps to interpret PET/CT-derived data of tissue activity. For this purpose, online blood sampling during the PET/CT imaging is advisable to measure the AIF. In contrast to manual blood sampling and image-derived approaches, continuous online blood sampling has several advantages. Besides the minimized blood loss, there is an improved resolution and a superior accuracy for the blood activity measurement. However, the major drawback of online blood sampling is the costly and time-consuming preparation to catheterize the femoral vessels of the animal. Here, we describe an easy and complete workflow for catheterization and continuous blood sampling during small animal PET/CT imaging and compared it to manual blood sampling and an image-derived approach. Using this highly-standardized workflow, the determination of the fluorodeoxyglucose ([18F]FDG) AIF is demonstrated. Further, this procedure can be applied to any radiotracer in combination with different animal models to create fundamental knowledge of tracer kinetic and model characteristics. This allows a more precise evaluation of the behavior of pharmaceuticals, both for diagnostic and therapeutic approaches in the preclinical research of oncological, neurodegenerative and myocardial diseases.

Role of Bone Morphogenetic Protein 7 (BMP7) in the Modulation of Corneal Stromal and Epithelial Cell Functions.

In the cornea, healing of the wounded avascular surface is an intricate process comprising the involvement of epithelial, stromal and neuronal cell interactions. These interactions result to the release of various growth factors that play prominent roles during corneal wound healing response. Bone morphogenetic proteins (BMPs) are unique multi-functional potent growth factors of the transforming growth factor-beta (TGF-β) superfamily. Treatment of corneal epithelial cells with substance P and nerve growth factor resulted to an increase in the expression of BMP7 mRNA. Since BMP7 is known to modulate the process of corneal wound healing, in this present study, we investigated the influence of exogenous rhBMP7 on human corneal epithelial cell and stromal cell (SFs) function. To obtain a high-fidelity expression profiling of activated biomarkers and pathways, transcriptome-wide gene-level expression profiling of epithelial cells in the presence of BMP7 was performed. Gene ontology analysis shows BMP7 stimulation activated TGF-β signaling and cell cycle pathways, whereas biological processes related to cell cycle, microtubule and intermediate filament cytoskeleton organization were significantly impacted in corneal epithelial cells. Scratch wound healing assay showed increased motility and migration of BMP7 treated epithelial cells. BMP7 stimulation studies show activation of MAPK cascade proteins in epithelial cells and SFs. Similarly, a difference in the expression of claudin, Zink finger E-box-binding homeobox 1 was observed along with phosphorylation levels of cofilin in epithelial cells. Stimulation of SFs with BMP7 activated them with increased expression of α-smooth muscle actin. In addition, an elevated phosphorylation of epidermal growth factor receptor following BMP7 stimulation was also observed both in corneal epithelial cells and SFs. Based on our transcriptome analysis data on epithelial cells and the results obtained in SFs, we conclude that BMP7 contributes to epithelial-to-mesenchymal transition-like responses and plays a role equivalent to TGF-β in the course of corneal wound healing.

Molecular composition of the human primary visual cortex profiled by multimodal mass spectrometry imaging.

The primary visual cortex (area V1) is an extensively studied part of the cerebral cortex with well-characterized connectivity, cellular and molecular architecture and functions (for recent reviews see Amunts and Zilles, Neuron 88:1086-1107, 2015; Casagrande and Xu, Parallel visual pathways: a comparative perspective. The visual neurosciences, MIT Press, Cambridge, pp 494-506, 2004). In humans, V1 is defined by heavily myelinated fibers arriving from the radiatio optica that form the Gennari stripe in cortical layer IV, which is further subdivided into laminae IVa, IVb, IVcα and IVcß. Due to this unique laminar pattern, V1 represents an excellent region to test whether multimodal mass spectrometric imaging could reveal novel biomolecular markers for a functionally relevant parcellation of the human cerebral cortex. Here we analyzed histological sections of three post-mortem brains with matrix-assisted laser desorption/ionization mass spectrometry imaging and laser ablation inductively coupled plasma mass spectrometry imaging to investigate the distribution of lipids, proteins and metals in human V1. We identified 71 peptides of 13 different proteins by in situ tandem mass spectrometry, of which 5 proteins show a differential laminar distribution pattern revealing the border between V1 and V2. High-accuracy mass measurements identified 123 lipid species, including glycerolipids, glycerophospholipids and sphingolipids, of which at least 20 showed differential distribution within V1 and V2. Specific lipids labeled not only myelinated layer IVb, but also IVa and especially IVc in a layer-specific manner, but also and clearly separated V1 from V2. Elemental imaging further showed a specific accumulation of copper in layer IV. In conclusion, multimodal mass spectrometry imaging identified novel biomolecular and elemental markers with specific laminar and inter-areal differences. We conclude that mass spectrometry imaging provides a promising new approach toward multimodal, molecule-based cortical parcellation.

Can Recognition of Spinal Ischemia Be Improved? Application of Motor-Evoked Potentials, Serum Markers, and Breath Gas Analysis in an Acutely Instrumented Pig Model.

BACKGROUND: Paraplegia due to spinal cord ischemia (SCI) is a serious complication after repair of thoracoabdominal aortic aneurysms. For prevention and early treatment of spinal ischemia, intraoperative monitoring of spinal cord integrity is essential. This study was intended to improve recognition of SCI through a combination of transcranial motor-evoked potentials (tc-MEPs), serum markers, and innovative breath analysis. METHODS: In 9 female German Landrace pigs, tc-MEPs were captured, markers of neuronal damage were determined in blood, and volatile organic compounds (VOCs) were analyzed in exhaled air. After thoraco-phrenico-laparotomy, SCI was initiated through sequential clamping (n = 4) or permanently ligating (n = 5) SAs of the abdominal and thoracic aorta in caudocranial orientation until a drop in the tc-MEPs to at least 25% of the baseline was recorded. VOCs in breath were determined by means of solid-phase microextraction coupled with gas chromatography-mass spectrometry. After waking up, clinical and neurological status was evaluated (Tarlov score). Spinal cord histology was obtained in postmortem. RESULTS: Permanent vessel ligature induced a worse neurological outcome and a higher number of necrotic motor neurons compared to clamping. Changes of serum markers remained unspecific. After laparotomy, exhaled acetone and isopropanol showed highest concentrations, and pentane and hexane increased during ischemia-reperfusion injury. CONCLUSIONS: To mimic spinal ischemia occurring in humans during aortic aneurysm repair, animal models have to be meticulously evaluated concerning vascular anatomy and function. Volatiles from breath indicated metabolic stress during surgery and oxidative damage through ischemia reperfusion. Breath VOCs may provide complimentary information to conventional monitoring methods.

[Ultra-high-field MRI in the Chicken Embryo in Ovo - a Model for Experimental Ophthalmology]. / Ultrahochfeld-MRT am Hühnerembryo in ovo ­ ein Modell für die experimentelle Ophthalmologie.

Ultra-high-field MRI (UHF-MRI) is an outstanding technique for non-invasive and non-destructive imaging of soft tissues and can provide versatile contrasts and high resolution in the µm range. In vivo imaging of the embryonal chick eye with its filigree anatomical structures imposes these requirements. However, due to the short embryonal development cycle, chicken are a favourite animal model for embryonal research studies. Ultra-high-field MRI allows repeated and longitudinal in ovo investigations on the same embryo. In the present study, the limitations and opportunities of in ovo MR-imaging at 7 T were evaluated and the process of eye growth was described in detail.

Suppression of TGF-ß pathway by pirfenidone decreases extracellular matrix deposition in ocular fibroblasts in vitro.

In glaucoma surgery, fibrotic processes occur, leading to impairment of liquid outflow. Activated fibroblasts are responsible for postoperative scarring. The transforming growth factor-ß (TGF-ß) pathway plays a key role in fibroblast function, differentiation and proliferation. The aim of this study was the characterization of the fibrotic potential of two subtypes of primary human ocular fibroblasts and the attempt to inhibit fibrotic processes specifically, without impairing cell viability. For fibrosis inhibition we focused on the small molecule pirfenidone, which has been shown to prevent pulmonary fibrosis by the decrease of the expression of TGF-ß1, TGF-ß2 and TGF-ß3 cytokines. For in vitro examinations, isolated human primary fibroblasts from Tenon capsule and human intraconal orbital fat tissues were used. These fibroblast subpopulations were analyzed in terms of the expression of matrix components responsible for postoperative scarring. We concentrated on the expression of collagen I, III, VI and fibronectin. Additionally, we analyzed the expression of α-smooth muscle actin, which serves as a marker for fibrosis and indicates transformation of fibroblasts into myofibroblasts. Gene expression was analyzed by rtPCR and synthesized proteins were examined by immunofluorescence and Western blot methods. Proliferation of fibroblasts under different culture conditions was assessed using BrdU assay. TGF-ß1 induced a significant increase of cell proliferation in both cell types. Also the expression of some fibrotic markers was elevated. In contrast, pirfenidone decreased cell proliferation and matrix synthesis in both fibroblast subpopulations. Pirfenidone slightly attenuated TGF-ß1 induced expression of fibronectin and α-smooth muscle actin in fibroblast cultures, without impairing cell viability. To summarize, manipulation of the TGF-ß signaling pathway by pirfenidone represents a specific antifibrotic approach with no toxic side effects in two human orbital fibroblast subtypes. We presume that pirfenidone is a promising candidate for the treatment of fibrosis following glaucoma surgery.

A Comparative Morphometric Analysis of Three Cranial Nerves in Two Phocids: The Hooded Seal (Cystophora cristata) and the Harbor Seal (Phoca vitulina).

While our knowledge about the senses of pinnipeds has increased over the last decades almost nothing is known about the organization of the neuroanatomical pathways. In a first approach to this field of research, we assessed the total number of myelinated axons of three cranial nerves (CNs) in the harbor (Phoca vitulina, Pv) and hooded seal (Cystophora cristata, Cc). Axons were counted in semithin sections of the nerves embedded in Epon and stained with toluidine blue. In both species, the highest axon number was found within the optic nerve (Pv 187,000 ± 8,000 axons, Cc 481,600 ± 1,300 axons). Generally, considering absolute axon numbers, far more axons were counted within the optic and trigmenial nerve (Pv 136,700 ± 2,500 axons, Cc 179,300 ± 6,900 axons) in hooded in comparison to harbor seals. The axon counts of the vestibulocochlear nerve are nearly identical for both species (Pv 87,100 ± 8,100 axons, Cc 86,600 ± 2,700 axons). However, when comparing cell density, the cell density is almost equal for all nerves for both species except for the optic nerve in which cell density was particularly higher than in the other nerves and higher in hooded in comparison to harbor seals. We here present the first comparative analysis of three CNs in two phocid seals. While the CNs of these closely related species share some general characteristics, pronounced differences in axon numbers/densities are apparent. These differences seem to reflect differences in e.g. size, habitat, and/or functional significance of the innervated sensory systems.

Novel subventricular zone early progenitor cell-specific adenovirus for in vivo therapy of central nervous system disorders reinforces brain stem cell heterogeneity.

Neural stem/progenitor cells (NSPCs) have the potential to self-renew and to generate all neural lineages as well as to repopulate damaged areas in the brain. Our previous targeting strategies have indicated precursor cell heterogeneity between different brain regions that warrants the development of NSPC-specific delivery vehicles. Here, we demonstrate a target-specific adenoviral vector system for the in vivo manipulation of progenitor cells in the subventricular zone of the adult mouse brain. For this purpose, we identified a series of peptide ligands via phage display. The peptide with the highest affinity, SNQLPQQ, was expressed in conjunction with a bispecific adaptor molecule. To verify the targeting potential of the specific peptide, green fluorescent protein-expressing Ad vectors were coupled with the adaptor molecule and injected into the subventricular region of adult mice by stereotaxic surgery. An efficient and selective transduction of NSPCs in the SVZ was achieved, whereas hippocampal NSPCs were negative. Our results offer an expeditious and simple tool to produce retargeted viral vectors for a specific and direct in vivo manipulation of these progenitor cells. This powerful technique provides an opportunity to develop innovative strategies and express therapeutic genes in specific types of neural progenitor cells to allow success in treatment of brain disorders.

Development of a novel injectable drug delivery system for subconjunctival glaucoma treatment.

In this study we present the development of an injectable polymeric drug delivery system for subconjunctival treatment of primary open angle glaucoma. The system consists of hyaluronic acid sodium salt (HA), which is commonly used in ophthalmology in anterior segment surgery, and an isocyanate-functionalized 1,2-ethylene glycol bis(dilactic acid) (ELA-NCO). The polymer mixtures with different ratios of HA to ELA-NCO (1/1, 1/4, and 1/10 (v/v)) were investigated for biocompatibility, degradation behavior and applicability as a sustained release system. For the latter, the lipophilic latanoprost ester pro-drug (LA) was incorporated into the HA/ELA-NCO system. In vitro, a sustained LA release over a period of about 60days was achieved. In cell culture experiments, the HA/ELA-NCO (1/1, (v/v)) system was proven to be biocompatible for human and rabbit Tenon's fibroblasts. Examination of in vitro degradation behavior revealed a total mass loss of more than 60% during the observation period of 26weeks. In vivo, LA was continuously released for 152days into rabbit aqueous humor and serum. Histological investigations revealed a marked leuko-lymphocytic infiltration soon after subconjunctival injection. Thereafter, the initial tissue reaction declined concomitantly with a continuous degradation of the polymer, which was completed after 10months. Our study demonstrates the suitability of the polymer resulting from the reaction of HA with ELA-NCO as an injectable local drug delivery system for glaucoma therapy, combining biocompatibility and biodegradability with prolonged drug release.

Corneal epithelial and neuronal interactions: role in wound healing.

Impaired corneal innervation and sensitivity are the main causes of corneal neurotrophic keratopathy which simultaneously also leads to poor epithelial wound healing. Restoration of the diminished communication between the corneal epithelium and trigeminal nerve is indispensable for the proper functioning of the epithelium. The present study aims to investigate corneal epithelial and trigeminal neuron interactions to shed light on corneal wound healing during neurotrophic keratopathy. Mouse trigeminal neurons and corneal epithelial cells were cultured according to standard methods. To study the effect of corneal epithelial cells on trigeminal neurons as well as the effect of trigeminal neurons on corneal epithelial cells during wound healing, conditioned media from the cultures of pure trigeminal neurons (CNM) and corneal epithelial cells (CEM) were collected freshly and applied on the other cell type. Neurite outgrowth assay and RT-PCR analysis using primers specific for substance P (SP), Map1a, Map1b were performed on trigeminal neurons in the presence of CEM. We observed an increase in the neurite outgrowth in the presence of CEM and also in co-culture with corneal epithelial cells. Increase in the expression of SP mRNA and a decrease in the expression of Map1b mRNA was observed in the presence of CEM. We also observed the presence of epithelial-to-mesenchymal transition (EMT)-like phenomenon during wound healing using a scratch assay in primary corneal epithelial cultures. This system was further employed to study the effect of CNM on corneal epithelial cells in the context of wound healing to find the effect of trigeminal neurons on epithelial cells. RT-PCR analysis of Pax6 expression in corneal epithelial cell cultures with scratch served as a positive control. Further, we also show the expression of bone morphogenetic protein 7 (BMP7) mRNA in corneal epithelial cells which is decreased gradually along with Pax6 mRNA when cultured together in the presence of CNM. The expression and down regulation of BMP7 in the presence of CNM was further confirmed at the protein level by western blotting. From this study it seems that the epithelial and neuronal interactions in the cornea may contribute to the corneal innervation as well as recovery of corneal epithelial cells during injury. Appraising the differences in the expression of various signalling molecules during EMT of epithelial cells in the presence of SP and BMP7 gives an insight into the detailed dissection of the involved signalling pathways to develop future therapeutics.

Morphological analysis of quiescent and activated keratocytes: a review of ex vivo and in vivo findings.

Keratocytes are specialized, neural crest-derived mesenchymal cells occupying approximately 3% of the corneal stromal volume. They reside between the collagen lamellae and are responsible for the secretion of extracellular matrix macromolecules, thus contributing to the corneal transparency and integrity. During the regeneration process after infection, traumata and refractive surgery, the keratocytes undergo transition into divergent phenotypes, which are referred to as "activated keratocytes". Quite shortly after injury, the keratocytes lose their quiescence, enter into the cell cycle and migrate toward the site of injury. In certain types of injury, which affect the integrity of basement membrane, activated keratocytes also participate in wound closure by production of α-smooth muscle actin (α-SMA). Since the activated keratocytes are the major cell type contributing to tissue repair during corneal wound healing, their morphological and biochemical properties have been studied in details in experimental studies using light and electron microscopy. More recently, emerging of in vivo microscopy techniques has opened new possibilities to investigate cornea in vivo. The non-invasive nature of this imaging modality enables repeated examination of the same tissue over time and is an ideal tool to rapidly and accurately investigate corneal wound healing. However, the in vivo data on activated keratocytes are not as uniform as data from experimental ex vivo studies. There is still inconsistency in the literature findings on activated phenotypes, and often the described morphologies cannot be appreciated in in vivo images. In this article, a literature review was performed in order to interpret the morphology of different activated phenotypes, based on biological processes underlying the morphological alterations.

Changes in the micromorphology of the corneal subbasal nerve plexus in patients after plaque brachytherapy.

BACKGROUND: To quantify the development of radiation neuropathy in corneal subbasal nerve plexus (SNP) after plaque brachytherapy, and the subsequent regeneration of SNP micromorphology and corneal sensation. METHODS: Nine eyes of 9 melanoma patients (ciliary body: 3, iris: 2, conjunctiva: 4) underwent brachytherapy (ruthenium-106 plaque, dose to tumour base: 523 ± 231 Gy). SNP micromorphology was assessed by in-vivo confocal microscopy. Using software developed in-house, pre-irradiation findings were compared with those obtained after 3 days, 1, 4 and 7 months, and related to radiation dose and corneal sensation. RESULTS: After 3 days nerve fibres were absent from the applicator zone and central cornea, and corneal sensation was abolished. The earliest regenerating fibres were seen at the one-month follow-up. By 4 months SNP structures had increased to one-third of pre-treatment status (based on nerve fibre density and nerve fibre count), and corneal sensation had returned to approximately two-thirds of pre-irradiation values. Regeneration of SNP and corneal sensation was nearly complete 7 months after plaque brachytherapy. CONCLUSIONS: The evaluation of SNP micromorphology and corneal sensation is a reliable and clinically useful method for assessing neuropathy after plaque brachytherapy. Radiation-induced neuropathy of corneal nerves develops quickly and is partly reversible within 7 months. The clinical impact of radiation-induced SNP damage is moderate.

Variation in the hypothenar muscles and its impact on ulnar tunnel syndrome.

Compression of the ulnar nerve at Guyon's canal can be caused not only by tumor-like structures, a fibrotic arch, a ganglion, lipoma, aneurysm or thrombosis but also by anomalous hypothenar muscles which are reviewed here. For the search of relevant papers, PubMed and crucial anatomical textbooks were consulted. The abductor digiti minimi is the most variable hypothenar muscle. It can possess one to three muscle bellies. Additional heads can arise from the flexor retinaculum, the palmaris longus tendon, the pronator quadratus tendon or the deep fascia of the palmar side of the forearm. Our own case of an aberrant abductor digiti minimi appearing like connective tissue and originating in the antebrachial fascia is included here. Hematoxylin and eosin staining revealed that macroscopically non-muscle-like tissue contained skeletal muscle tissue. The muscle itself resembled other described cases. In addition, at the flexor digiti minimi accessory heads with origin from the flexor retinaculum, the antebrachial fascia or the long flexor muscles of the forearm can be detected. By contrast, the opponens digiti minimi mostly lacks variations and is sometimes missing. In our opinion, this is due to its hidden location. However, in few cases an additional head can arise from the lower arm aponeurosis. Furthermore, additional (fourth) hypothenar muscles might be expressed. These muscles are characterized by origins in the forearm and insertions on the head of the 5th metacarpal bone or on the 5th proximal phalanx. It must be noted that accessory hypothenar muscles might look like connective tissue at first glance. Often their origin extends to the antebrachial fascia. This can be explained by the phylogenetic fact that all intrinsic muscles of the hand are derived from muscle masses that originated in the forearm. In the opinion of several authors, ulnar nerve compression mostly is evoked by hyper trophied variant hypothenar muscles due to overuse as for example in carpenters. In some rare cases, an aberrant hypothenar muscle can also evoke median nerve compression.