Marina Hovakimyan

Imaging corneal crosslinking by autofluorescence 2-photon microscopy, second harmonic generation, and fluorescence lifetime measurements

PURPOSE: To evaluate the use of 2‐photon microscopy (TPM), which excites tissue autofluorescence, in detecting and calculating the grade of collagen corneal crosslinks, which are not visible through the slitlamp and in vivo confocal microscopy. SETTING: Departments of Ophthalmology, University of Lübeck, Lübeck, and University of Rostock, Rostock, Germany. DESIGN: Experimental study. METHODS: Corneas of rabbits were treated with different crosslinking (CXL) protocols. Two weeks after treatment, the corneas were evaluated in vivo by confocal microscopy. Eyes were enucleated and TPM was performed at 710 nm and 826 nm excitation wavelengths to detect tissue autofluorescence, second harmonic generation, and fluorescence lifetime measurements (FLIM). Eyes were then fixed and analyzed by histology. RESULTS: Crosslinking following the standard protocol generated a strong autofluorescence signal in the stroma that was detected by TPM. This signal was weakly present in the control specimens, and a sharp transition zone between the peripheral zone and the CXL zone was seen. On FLIM, an increase in corneal crosslinks was measured when the standard protocol was used. CONCLUSIONS: Two‐photon microscopy, a noninvasive method, was able to detect the effects of therapeutic CXL and measure the grade of CXL. In addition to postoperative treatment control, the technique has possibilities for use in online dosimetry during 2‐photon triggered CXL. Financial Disclosure: No author has a financial or proprietary interest in any material or method mentioned.

Results of Confocal Microscopy Examinations after Collagen Cross-Linking with Riboflavin and UVA Light in Patients with Progressive Keratoconus

Purpose: Keratoconus is a predominantly bilateral form of corneal degeneration that is associated with central thinning and cone-shaped bulging of the cornea usually accompanied by a progressive reduction in visual acuity. A recent therapeutic option is cross-linking, a procedure designed to prevent the progression of keratoconus by the photochemical cross-linkage of collagen fibers. Patients and Methods: Eight eyes in 8 patients with progressive keratoconus were treated by the photochemical cross-linking method using riboflavin and UVA light. In addition to the usual ophthalmological examinations, patients were examined pre- and postoperatively by confocal in vivo laser scanning microscopy. Follow-up examinations were performed at 2 weeks and at 2, 4, 6 and 12 months postoperatively. Results: Complete regeneration of corneal epithelium was detected by 2 weeks after therapy at the latest. The sub-basal nerve plexus could not be visualized by confocal microscopy after treatment. Immediately after treatment, the anterior corneal stroma had a honeycombed appearance but without the typical hyperreflective keratocyte nuclei. At about 6 months postoperatively, the corneal stroma had virtually regained its normal configuration. After therapy, confocal microscopy revealed that corneal endothelium was normal in terms of cell density and morphology at every time point. Conclusions: Confocal in vivo laser scanning microscopy is an investigative technique that permits reproducible visualization of structural changes in the cornea (epithelium, stroma and endothelium) following collagen cross-linking with riboflavin and UVA light. Once epithelial healing is complete, the epithelium and endothelium appear to be unaffected by the treatment. The most noteworthy structural changes, which are detected on confocal microscopy shortly after treatment, involve the anterior and middle corneal stroma. Over the course of time, up to 12 months postoperatively, these changes show a definite tendency to regress.

In vivo visualisation of murine corneal nerve fibre regeneration in response to ciliary neurotrophic factor.

The aim of this study was to examine the murine subbasal nerve fibre plexus (SNP) regeneration altered by surgical dissection. Investigations in the mouse model addressed the regeneration capabilities of the SNP, and the influence of local ciliary neurotrophic factor (CNTF) application on the regeneration process. In preliminary experiments, the healthy mouse cornea was monitored using in vivo confocal laser-scanning microscopy (CLSM) from the age of 8-52 weeks, to reveal and rule out the age-dependent changes in SNP. Nerve fibre density (NFD) was determined with the semi-automatic nerve tracing program NeuronJ. No quantitative or qualitative changes in NFD were detected in untreated animals over time; mean NFD in mice aged 8 weeks (28.30 ± 9.12 mm/mm2), 16 weeks (29.23 ± 7.28 mm/mm2), 30 weeks (26.31 ± 8.58 mm/mm2) and 52 weeks (26.34 ± 6.04 mm/mm2) showed no statistically significant differences between time points (p > 0.05). For regeneration studies a circular incision through corneal epithelium and anterior stroma of minimum 60 μm depth was generated with a custom-made guided trephine system to cut the subbasal corneal nerves in adult mice. The corneal nerve pattern was monitored and NFD was measured before and up to 8 weeks after surgery. Animals were divided in three groups each comprising 6 mice. The CNTF group received eye drops containing CNTF (25 ng/ml) 3 times daily for 3 weeks, whereas the control group received no further medication. In the sham group the same treatment schedule was applied as in CNTF group, using vehicle. The regenerating subbasal nerve fibres sprouted out of stromal nerves within the cut and additionally regrew over the scar rim from outside. They showed parallel orientation but were thinner than before incision. Whorl patterning was observed after 4 weeks. All three groups revealed a marked NFD reduction starting at one week after incision, followed by continuous recovery. After 8 weeks the NFD reached 23.5 ± 2.4 mm/mm2 (78% of baseline), 21.9 ± 1.6 mm/mm2 (73% of baseline) and 29.2 ± 3.4 mm/mm2 (93% of baseline) in the control, sham and CNTF group, respectively. By comparison with control and sham group, the CNTF group demonstrated significantly higher NFD at every observation time point. The mouse cornea provides a practicable animal model for in vivo CLSM monitoring of corneal nerve behaviour over time and following injury. Non-penetrating trephination generated a severe reduction in the NFD of the SNP, but murine corneas recovered to pre-injury NFD levels within 8 weeks. Local application of CNTF served merely to temporarily accelerate the recovery of NFD.

Combined therapy with cyclodextrin/allopregnanolone and miglustat improves motor but not cognitive functions in Niemann–Pick Type C1 mice

Niemann-Pick Type C1 (NPC1) is an autosomal recessive disorder characterized by the accumulation of cholesterol and glycosphingolipids. Combination-treatment utilizing cyclodextrin, allopregnanolone and miglustat (CYCLO/ALLO/miglustat) can ameliorate NPC1 disease in a mutant mouse model. The present study was designed to add behavioral analysis in NPC1 mutant mice upon CYCLO/ALLO/miglustat therapy. NPC1 mutant (BALB/cJ NPC1NIH) and control mice were used. For the combination treatment mice were injected with CYCLO/ALLO weekly, starting at P7. The miglustat injection was performed daily from P10 till P23. Starting at P23, miglustat was added to the powdered chow. For the sham treatment of control and mutant mice the same schedule was used with 0.9% NaCl injection. Locomotor activity was assessed in open field, elevated plus maze and accelerod tests. For assessment of spatial learning and memory the Morris water maze test was conducted. Electron microscopy has been performed to support the behavioral data. The sham-treated mutant mice exhibited motor impairments in all performed tests. In the water maze the sham-treated mutants exhibited impairment in remembering the location of the hidden platform. CYCLO/ALLO/miglustat treatment positively influenced motor dysfunction: total distance and number of visits significantly increased, and accelerod performance improved. The spatial learning, however, did not benefit from therapy. At the morphological level, an excessive accumulation of electron-dense material was seen in the cerebellar Purkinje cells of mutant mice. A regression of these autophagosomal inclusions was seen upon therapy. CYCLO/ALLO/miglustat therapy ameliorates motor but not cognitive deficits in NPC1 mutant mice, suggesting unequal vulnerability of different brain areas to the treatment.

Olfactory Deficits in Niemann-Pick Type C1 (NPC1) Disease

Background Niemann-Pick type C disease (NPC) is a rare autosomal recessive lipid storage disease characterized by progressive neurodegeneration. As only a few studies have been conducted on the impact of NPC on sensory systems, we used a mutant mouse model (NPC1−/−) to examine the effects of this disorder to morphologically distinct regions of the olfactory system, namely the olfactory epithelium (OE) and olfactory bulb (OB). Methodology/Principal findings For structural and functional analysis immunohistochemistry, electron microscopy, western blotting, and electrophysiology have been applied. For histochemistry and western blotting, we used antibodies against a series of neuronal and glia marker proteins, as well as macrophage markers. NPC1−/− animals present myelin-like lysosomal deposits in virtually all types of cells of the peripheral and central olfactory system. Especially supporting cells of the OE and central glia cells are affected, resulting in pronounced astrocytosis and microgliosis in the OB and other olfactory cortices. Up-regulation of Galectin-3, Cathepsin D and GFAP in the cortical layers of the OB underlines the critical role and location of the OB as a possible entrance gate for noxious substances. Unmyelinated olfactory afferents of the lamina propria seem less affected than ensheathing cells. Supporting the structural findings, electro-olfactometry of the olfactory mucosa suggests that NPC1−/− animals exhibit olfactory and trigeminal deficits. Conclusions/Significance Our data demonstrate a pronounced neurodegeneration and glia activation in the olfactory system of NPC1−/−, which is accompanied by sensory deficits.

Suppression of TGF-β 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-β (TGF-β) 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-β1, TGF-β2 and TGF-β3 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-β1 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-β1 induced expression of fibronectin and α-smooth muscle actin in fibroblast cultures, without impairing cell viability. To summarize, manipulation of the TGF-β 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.

Surface Modification of Biodegradable Polymers towards Better Biocompatibility and Lower Thrombogenicity.

Purpose Drug-eluting stents (DES) based on permanent polymeric coating matrices have been introduced to overcome the in stent restenosis associated with bare metal stents (BMS). A further step was the development of DES with biodegradable polymeric coatings to address the risk of thrombosis associated with first-generation DES. In this study we evaluate the biocompatibility of biodegradable polymer materials for their potential use as coating matrices for DES or as materials for fully bioabsorbable vascular stents. Materials and Methods Five different polymers, poly(L-lactide) PLLA, poly(D,L-lactide) PDLLA, poly(L-lactide-co-glycolide) P(LLA-co-GA), poly(D,L-lactide-co-glycolide) P(DLLA-co-GA) and poly(L-lactide-co-ε-caprolactone), P(LLA-co-CL) were examined in vitro without and with surface modification. The surface modification of polymers was performed by means of wet-chemical (NaOH and ethylenediamine (EDA)) and plasma-chemical (O2 and NH3) processes. The biocompatibility studies were performed on three different cell types: immortalized mouse fibroblasts (cell line L929), human coronary artery endothelial cells (HCAEC) and human umbilical vein endothelial cells (HUVEC). The biocompatibility was examined quantitatively using in vitro cytotoxicity assay. Cells were investigated immunocytochemically for expression of specific markers, and morphology was visualized using confocal laser scanning (CLSM) and scanning electron (SEM) microscopy. Additionally, polymer surfaces were examined for their thrombogenicity using an established hemocompatibility test. Results Both endothelial cell types exhibited poor viability and adhesion on all five unmodified polymer surfaces. The biocompatibility of the polymers could be influenced positively by surface modifications. In particular, a reproducible effect was observed for NH3-plasma treatment, which enhanced the cell viability, adhesion and morphology on all five polymeric surfaces. Conclusion Surface modification of polymers can provide a useful approach to enhance their biocompatibility. For clinical application, attempts should be made to stabilize the plasma modification and use it for coupling of biomolecules to accelerate the re-endothelialization of stent surfaces in vivo.

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 Tenons 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.

Reduced Cerebellar Neurodegeneration After Combined Therapy With Cyclodextrin/Allopregnanolone and Miglustat in NPC1: A Mouse Model of Niemann-Pick Type C1 Disease

Niemann‐Pick type C1 (NPC1) disease is a lysosomal storage disease characterized by a deficiency of NPC1 gene function. The malfunction of protein results in a progressive accumulation of lipids in many organs. A combined approach with substrate‐reduction therapy (SRT) and byproduct therapy (BPT) has been shown to ameliorate the disease course in a mutant mouse model (NPC1–/–). The present study examines the morphological parameters underlying these changes. For the combined SRT/BPT treatment, NPC1–/– mutant mice (NPC1–/–SRT/BPT) were injected with allopregnanolone/cyclodextrin weekly, starting at postnatal day (P) 7. Starting at P10, a miglustat injection was administered daily until P23. Thereafter, miglustat was added to the powdered chow. For the sham treatment, both mutant NPC1–/– (NPC1–/–sham) and wild‐type (NPC1+/+sham) mice received an NaCl injection and were fed powdered chow without miglustat. Analysis was performed on cerebellar slices by histology and immunohistochemistry. The volumes and cell counts of cerebellar structures were quantified. Additionally, ultrastructural analysis was performed with transmission electron microscopy. In agreement with previous studies, the current study demonstrates Purkinje cell degeneration in the mutant mice, which was partially abrogated by SRT/BPT. The volumes of cerebellar white matter and molecular layer were reduced as well. Also, the number of neurons was reduced in granular and molecular layers. However, only the molecular layer benefited from the therapy, as shown by an increase in the volume and the amount of neurons. The volume and number of neurons of the deep cerebellar nuclei were significantly decreased in mutant mice; an appreciable therapeutic benefit could be demonstrated for the nucleus interpositus.

Chemical activation and changes in surface morphology of poly(ε-caprolactone) modulate VEGF responsiveness of human endothelial cells

The high degree of clinical routine in percutaneous transluminal coronary angioplasty (PTCA) with and without stenting has not changed the fact that a large number of coronary heart disease patients are still affected by post-operative complications such as restenosis and thrombosis. Because re-endothelialization is the crucial aspect of wound healing after cardiovascular implant surgery, there is a need for modern biomaterials to aid endothelial cells in their adhesion and functional recovery post-stenting. This study systematically examines the potential of numerous chemical polymer modifications with regard to endothelialization. Poly(ε-caprolactone) (PCL) and its chemically activated forms are investigated in detail, as well as the impact of polymer surface morphology and precoating with matrix protein. Human umbilical vein endothelial cells (HUVECs) are used to characterize endothelial cell responses in terms of in vitro viability and adhesion. As a potential component in drug eluting implants, VEGF is applied as stimulus to boost endothelial cell proliferation on the polymer. In conclusion, plasma chemical activation of PCL combined with VEGF stimulation best enhances in vitro endothelialization. Examining the impact of morphological, chemical and biological modifications of PCL, this study makes an important new contribution towards the existing body of work on polymer endothelialization.