In vivo nonlinear imaging of corneal structures with special focus on BALB/c and streptozotocin-diabetic Thy1-YFP mice.

Two-photon microscopy (TPM) allows high contrast imaging at a subcellular resolution scale. In this work, the microscopy technique was applied to visualize corneal structures in two mouse models (BALB/c and B6.Cg-Tg(Thy1-YFP)16Jrs/J) in vivo. In particular, the transgenic Thy1-YFP mice expressing the yellow fluorescent protein (YFP) in all motor and sensory neurons had been used for investigating the nerve fiber density in healthy and streptozotocin-diabetic mice. This model is clinically relevant since patients suffering from diabetes mellitus have a high risk to develop small fiber neuropathy. Nonlinear laser scanning microscopy displayed a reduction of nerve fiber density in streptozotocin-diabetic versus healthy mice and confirmed data obtained by confocal laser scanning microscopy (CLSM). In recent years, corneal CLSM was proved to be an appropriate non-invasive tool for an early diagnosis of diabetic neuropathy. Nevertheless, validation of the CLSM method for the clinical routine is currently a matter of investigation and requires confirmation by further studies and complementary techniques. Thus, the present study provides further evidence of corneal confocal microscopy as a promising technique for non-invasive detection of diabetic neuropathy. Information derived from these experiments may become clinically relevant and help to develop new drugs for treatment of diabetic neuropathy.

Age-Related Changes in Murine Corneal Nerves.

PURPOSE: The aim of this study is to determine age-related morphological changes in the corneal subbasal nerve plexus (SNP) in two inbred mouse strains. MATERIALS AND METHODS: The corneal SNP was investigated by in vivo confocal laser scanning microscopy (CLSM) in 0.5-, 1-, 1.5-, and 2-year-old C57BL/6J mice and in 0.5- and 1-year-old BALB/c mice (n = 4 per age category and strain; 10 images per mouse). Fixed corneal samples from C57BL/6J mice were also analyzed after PGP9.5 staining. Nerve fiber density (NFD) was determined using the semi-automated NeuronJ program. In addition, a new custom-designed, fully automated computerized technique based on oriented multiscale matched filtering was tested to objectify and accelerate image analysis. RESULTS: C57BL/6J mice showed low NFD (11.7 ± 0.5 mm/mm2). Aging from 0.5 to 1, 1.5, and 2 years resulted in significant reductions in subbasal NFD by 34%, 49%, and 66%, respectively. The decline in nerve fibers revealed by in vivo CLSM together with NeuronJ quantification was confirmed by ex vivo immunohistochemical analyses. Subbasal NFD in BALB/c mice (30.0 ± 1.4 mm/mm2) was 3-fold higher than in C57BL/6J mice. Aging from 0.5 to 1 year resulted in a significant 17% reduction in NFD. With the automated approach, NFD of 22.6 ± 2.9 mm/mm2 and a 45% reduction during aging was determined from the same images. CONCLUSIONS: An age-related reduction in subbasal corneal nerve fibers was observed. The differing extent of reduction in the two mouse strains may be accounted for by genetic factors. Automated NFD quantification of corneal nerve fibers in mice appears to be a useful, reliable, objective, and time-saving tool.

Diabetes mellitus leads to accumulation of dendritic cells and nerve fiber damage of the subbasal nerve plexus in the cornea.

PURPOSE: To evaluate whether nerve fibers of the subbasal nerve plexus (SNP) and dendritic cells (DCs) are in association with each other leading to neuropathy in the diabetic cornea. METHODS: BALB/c mice were injected with streptozotocin (STZ) for 5 days for induction of diabetes mellitus (DM) or with vehicle solution (control). B6.VLep(ob/ob) (ob/ob) mice served as an obese and glucose-intolerant DM type 2 (DM II) model and lean B6.VLep(ob/+) (ob/+) mice as respective controls. Using in vivo corneal confocal microscopy (CCM), nerve fibers and DCs were quantified over a period of 9 weeks and additionally analyzed by in vitro immunofluorescence whole-mount staining. RESULTS: In STZ-diabetic mice, CCM revealed an increase of DC density (DCD) in contrast to controls, whereas nerve fiber density (NFD) was decreased with duration of DM. In ob/ob mice, DCD was 3-fold higher than in both ob/+ mice and STZ-diabetic mice. Whole-mount staining displayed CD11c(+) and major histocompatibility complex (MHC) class II(+) mature DCs in colocalization with class III ß-tubulin(+) nerve fibers in the cornea. CONCLUSIONS: Hyperglycemia leads to corneal DC infiltration, and obesity aggravates this immune response. The direct contact between DCs and the SNP can be assumed to be a trigger of nerve fiber damage and thus a contributing factor to polyneuropathy in diabetic corneas.

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.

Non-invasive in vivo imaging by confocal laser scanning microscopy of gingival tissues following natural plaque deposition.

AIM: Imaging with Confocal Laser Scanning Microscopy (CLSM) generates high-resolution images and may be well suited for basic research in Periodontology and Implant Dentistry. The present study was aimed to explore the in vivo application of CLSM in experimentally induced gingivitis. MATERIALS AND METHODS: Ten subjects were recruited and were advised to stop any oral hygiene of the upper front teeth for 7 days. The gingival tissues were observed using a Heidelberg Retina Tomograph combined with a Rostock Cornea Module at baseline and day 7. The system used a laser of 670 nm and the contrast was given by backscattering from different tissues. Each examination created 800-1200 images that were descriptively analysed. RESULTS: After 7 days of abandoned oral hygiene, plaque scores and bleeding frequencies increased. By using CLSM images tooth hard substances, cells and plaque deposits were distinguishable. Increased epithelial cell irregularities, the apical migration of the sulcular epithelium, cellular infiltrates within the sulcus and plaque deposits were observed at day 7. CONCLUSIONS: The present study showed for the first time that CLSM is suitable for in vivo imaging of the gingival sulcus and adjacent tissues.

Distribution changes of epithelial dendritic cells in canine cornea and mucous membranes related to hematopoietic stem cell transplantation.

BACKGROUND/AIM: Dendritic cells (DCs) are important immune mediators following allogeneic hematopoietic stem cell transplantation (HSCT). We screened for DC frequency in the cornea and oral mucous membranes after HSCT by confocal laser scanning microscopy (CLSM) in a canine model. MATERIALS AND METHODS: In vivo CLSM images of the epithelia were taken the day before and on days 28, 56 and 112 following HSCT. Peripheral blood counts and chimerism were determined. RESULTS: An increase of DCs after HSCT was detected in each animal in both investigated tissue types. Highest DC numbers in the flew and the gingiva were detected on day 28 and in the corneal epithelium on day 56 after HSCT, respectively. CONCLUSION: Changes of DCs in ocular and non-ocular mucous membranes can be monitored by CLSM in vivo. The DC frequency in the cornea and mucosa changes following HSCT. DC recovery is rapid and their numbers correlate with the development of chimerism of peripheral blood mononuclear cells.

Corneal alterations during combined therapy with cyclodextrin/allopregnanolone and miglustat in a knock-out mouse model of NPC1 disease.

BACKGROUND: Niemann Pick disease type C1 is a neurodegenerative disease caused by mutations in the NPC1 gene, which result in accumulation of unesterified cholesterol and glycosphingolipids in the endosomal-lysosomal system as well as limiting membranes. We have previously shown the corneal involvement in NPC1 pathology in form of intracellular inclusions in epithelial cells and keratocytes. The purpose of the present study was to clarify if these inclusions regress during combined substrate reduction- and by-product therapy (SRT and BPT). METHODOLOGY/PRINCIPAL FINDINGS: Starting at postnatal day 7 (P7) and thereafter, NPC1 knock-out mice (NPC1(-/-)) and wild type controls (NPC1(+/+)) were injected with cyclodextrin/allopregnanolone weekly. Additionally, a daily miglustat injection started at P10 until P23. Starting at P23 the mice were fed powdered chow with daily addition of miglustat. The sham group was injected with 0.9% NaCl at P7, thereafter daily starting at P10 until P23, and fed powdered chow starting at P23. For corneal examination, in vivo confocal laser-scanning microscopy (CLSM) was performed one day before experiment was terminated. Excised corneas were harvested for lipid analysis (HPLC/MS) and electron microscopy. In vivo CLSM demonstrated a regression of hyperreflective inclusions in all treated NPC1(-/-)mice. The findings varied between individual mice, demonstrating a regression, ranging from complete absence to pronounced depositions. The reflectivity of inclusions, however, was significantly lower when compared to untreated and sham-injected NPC1(-/-) mice. These confocal findings were confirmed by lipid analysis and electron microscopy. Another important CLSM finding revealed a distinct increase of mature dendritic cell number in corneas of all treated mice (NPC1(-/-) and NPC1(+/+)), including sham-treated ones. CONCLUSIONS/SIGNIFICANCE: The combined substrate reduction- and by-product therapy revealed beneficial effects on the cornea. In vivo CLSM is a non-invasive tool to monitor disease progression and treatment effects in NPC1 disorder.

In vivo confocal laser-scanning microscopy to characterize wound repair in rabbit corneas after collagen cross-linking.

BACKGROUND: Collagen cross-linking using the photosensitizer riboflavin combined with ultraviolet A light was developed to stiffen the cornea by increasing its mechanical and biochemical stability. Investigation of post-treatment events, such as wound healing, is important to evaluate possible risks and to optimize treatment protocols. This in vivo confocal laser-scanning microscopy study in rabbits was conducted to provide a quantitative and qualitative analysis of corneal wound repair over 16 weeks following collagen cross-linking. METHODS: Six New Zealand White rabbits underwent riboflavin/ultraviolet A cross-linking. In vivo confocal laser-scanning microscopy using a Heidelberg Retina Tomograph equipped with a Rostock Cornea Module was performed preoperatively and at 2, 4, 8, 12 and 16 weeks postoperatively. RESULTS: From 2 weeks onwards the epithelium demonstrated no abnormalities. Evidence of inflammation was visualized in the intermediate, basal cells and Bowman's membrane. Nerve fibre regeneration was first noted at 12 weeks. Keratocyte activation and hyperreflective extracellular matrix were observed consistently, but by 16 weeks keratocyte activation was diminished, and extracellular matrix resumed normal reflectivity. Cell density in the posterior stroma and endothelium regained preoperative values by 4 weeks, although anterior stroma keratocyte cell density was still reduced by about 10% at 16 weeks. CONCLUSIONS: Complete qualitative and quantitative characterization of corneal wound repair was achieved by in vivo confocal laser-scanning microscopy over 16 weeks following collagen cross-linking in rabbits. In terms of assessing the ever-increasing range of cross-linking protocols, in vivo confocal laser-scanning microscopy may contribute to minimizing the number of experimental animals, because multiple examinations of the same cases are possible over time.

Confocal laser scanning microscopy for detection of Schistosoma mansoni eggs in the gut of mice.

BACKGROUND: The gold standard for diagnosing Schistosoma mansoni infections is the detection of eggs from stool or biopsy specimens. The viability of collected eggs can be tested by the miracidium hatching procedure. Direct detection methods are often limited in patients with light or early infections, whereas serological tests and PCR methods fail to differentiate between an inactive and persistent infection and between schistosomal species. Recently, confocal laser scanning microscopy (CLSM) has been introduced as a diagnostic tool in several fields of medicine. In this study we evaluated CLSM for the detection of viable eggs of S. mansoni directly within the gut of infected mice. METHODOLOGY/PRINCIPAL FINDINGS: The confocal laser scanning microscope used in this study is based on the Heidelberg Retina Tomograph II scanning laser system in combination with the Rostock Cornea Module (image modality 1) or a rigid endoscope (image modality 2). Colon sections of five infected mice were examined with image modalities 1 and 2 for schistosomal eggs. Afterwards a biopsy specimen was taken from each colon section and examined by bright-field microscopy. Visualised eggs were counted and classified in terms of viability status. CONCLUSIONS/SIGNIFICANCE: We were able to show that CLSM visualises eggs directly within the gut and permits discrimination of schistosomal species and determination of egg viability. Thus, CLSM may be a suitable non-invasive tool for the diagnosis of schistosomiasis in humans.

Morphological alterations of the cornea in the mouse model of niemann-pick disease type c1.

PURPOSE: Niemann-Pick disease type C1 (NPC1) is a genetic neurovisceral disorder characterized by abnormalities in intracellular sterol trafficking. A knockout mouse model (NPC1) is an important tool for the study of pathogenesis and treatment strategies. In the present study, NPC1 mice were examined for pathological changes in the cornea. METHODS: Fifteen inbred homozygous NPC1 knockout mice (NPC1, 5-10 weeks old), 5 age-matched heterozygous mice (NPC1), and 14 wild-type control mice (NPC1) were examined. In vivo confocal laser scanning microscopy (CLSM) was performed on both eyes of each animal; afterward, the eyes were processed for histology, electron microscopy, and lipid analysis. RESULTS: In vivo CLSM disclosed hyperreflective intracellular deposits in the intermediate and basal cell layers of corneal epithelium in all NPC1 mice. At the electron microscopy level, however, vacuolated cytoplasmic structures, 200-500 nm in diameter, with electron-dense material appeared in all structures investigated, including all epithelial layers and stromal keratocytes. These deposits were negative for filipin, a marker for unesterified cholesterol. Lipid analysis showed a marked increase in disialotetrahexosylganglioside 2 (GM2) level in NPC1 mice corneas, whereas no changes were detected in free cholesterol and disialotetrahexosylganglioside 3 (GM3) levels when compared with controls. CONCLUSIONS: Morphological changes characteristic for the NPC1 mouse cornea were visualized in all epithelial layers and keratocytes. In vivo CLSM findings were confirmed by other techniques. In vivo detection of ocular manifestations and analysis of ocular tissue have the potential to aid the diagnosis of NPC1 disease and to monitor the efficacy of treatment.

Comparative in vivo confocal microscopical study of the cornea anatomy of different laboratory animals.

PURPOSE: The aim of the present study was to analyze and compare in vivo morphology of healthy cornea of six different laboratory animals. MATERIALS AND METHODS: One Pomeranian Coarsewool sheep, 5 Beagle dogs, 1 Norwegian and 2 Domestic Short-haired cats, 20 New Zealand White rabbits, 6 Wistar rats, and 10 Balb/c mice were included. The examination was performed bilaterally, using Heidelberg Retina Tomograph equipped with Rostock Cornea Module. The morphology of living corneal layers was visualized and compared between species. The central corneal thickness, density of keratocytes, and endothelial cells were quantified. RESULTS: The epithelial multilayer showed a similarity in morphology between animal types, displaying three clearly distinguishable layers: superficial, intermediate, and basal. Subbasal nerve fibers were displayed as hyperreflective structures underneath basal cells. The subbasal fibers were confirmed in all species, however, the density varied between species. A pronounced Bowman's membrane was visualized in sheep. In all other species, however, a thin acellular layer with overlying nerve fibers could be seen between basal epithelial cells and anterior stroma. The keratocytes nuclei could be demonstrated in all species except for mice, where no nuclei but only reflective structures resembling keratocytes cell bodies were detected. Overall, the density of keratocytes nuclei was significantly higher in the anterior than in the posterior stroma. Besides endothelial cells density, the endothelial cells morphology was very similar among all species, except for sheep. The endothelial cells were displayed as polygonal structures with bright cytoplasm and dark borders. In sheep, the appearance of the endothelium was very poor because of a thick hyperreflective Descemet's membrane. CONCLUSIONS: The present study will help researchers consider appropriate models for animal experiments, depending on focus of investigation. In vivo CLSM can be used for the characterization of the living cornea over time, thus, reducing the number of animal experiments.