Impact of Obesity and Age on Mouse Corneal Innervation at the Epithelial-Stromal Interface.

Purpose: The corneal epithelium is the most highly innervated structure in the body. Previously, we reported a novel event whereby stromal axons fuse with basal epithelial cells, limiting nerve penetration into the epithelium. Although corneal-epithelial nerves undergo changes in sensitivity and distribution throughout life and in response to an obesogenic diet, it is unknown if neuronal-epithelial cell fusion is altered. Here, we sought to determine if neuronal-epithelial cell fusion frequency correlates with obesogenic diet consumption and age. Methods: Corneas were collected from C57BL/6 mice and evaluated for neuronal-epithelial cell fusion frequency using serial block-face scanning electron microscopy. To assess the correlation between diet-induced obesity and fusion frequency, 6-week-old mice were fed either a normal diet or an obesogenic diet for 10 weeks. To assess changes in fusion frequency between young and adult mice under normal dietary conditions, 9- and 24-week-old mice were used. Results: Mice fed a 10-week obesogenic diet showed 87% of central-cornea stromal nerves engaged in fusion compared with only 54% in age-matched controls (16 weeks old). In 9-week-old normal-diet animals, 48% of central-cornea stromal nerves contained fusing axons and increased to 81% at 24 weeks of age. Corneal sensitivity loss correlated with increased body weight and adiposity regardless of age and diet. Conclusions: Neuronal-epithelial cell fusion positively correlates with age and obesogenic diet consumption, and corneal nerve sensitivity loss correlates with increased body weight and adiposity, regardless of age and diet. As such, neuronal-epithelial cell fusion may play a role in corneal nerve density and sensitivity regulation.

Examination of Subbasal Nerve Plexus and Central Corneal Stromal Microstructure in Subjects With Congenital Aniridia, Using in Vivo Confocal Laser Scanning Microscopy.

PURPOSE: During life up to 70% of aniridia subjects develop aniridia-associated keratopathy (AAK). AAK is characterized by limbal stem cell insufficiency, impaired corneal epithelial cell differentiation and abnormal cell adhesion, which leads to centripetal spreading vascularization, conjunctivalization, and thickening of the cornea. Our aim was to examine the subbasal nerve plexus and central corneal stromal microstructure in subjects with congenital aniridia, using in vivo confocal laser scanning microscopy CLSM. METHODS: 31 eyes of 18 patients (55.6% males, mean age: 25.22 ± 16.35 years) with congenital aniridia and 46 eyes of 29 healthy subjects (41.4% males, mean age 30 ± 14.82 years) were examined using the Rostock Cornea Module of Heidelberg Retina Tomograph-III. At the subbasal nerve plexus, corneal nerve fiber density (CNFD), corneal nerve fiber length (CNFL), corneal total branch density (CTBD), and corneal nerve fiber width (CNFW) were analyzed using ACCMetrics software. Keratocyte density in the anterior, middle and posterior stroma was assessed manually. RESULTS: The CNFD (2.02 ± 4.08 vs 13.99 ± 6.34/mm2), CNFL (5.78 ± 2.68 vs 10.56 ± 2.82 mm/mm2) and CTBD (15.08 ± 15.62 vs 27.44 ± 15.05/mm2) were significantly lower in congenital aniridia subjects than in controls (p < 0.001 for all). CNFW was significantly higher in aniridia subjects than in controls (0.03 ± 0.004 vs 0.02 ± 0.003 mm/mm2) (p = 0.003). Keratocyte density was significantly lower in all stromal layers of aniridia subjects than in controls (p < 0.001 for all). Stromal alterations included confluent keratocytes, keratocytes with long extensions and hyperreflective dots between keratocytes in aniridia. CONCLUSIONS: Decrease in CNFD, CNFL, and CTBD, as well as increase in CNFW well refer to the congenital aniridia-associated neuropathy. The decreased keratocyte density and the stromal alterations may be related to an increased cell death in congenital aniridia, nevertheless, stromal changes in different stages of AAK have to be further analyzed in detail.

Nerve influence on the metabolism of type I and type II diabetic corneal stroma: an in vitro study.

Corneal innervation plays a major role in the pathobiology of diabetic corneal disease. However, innervation impact has mainly been investigated in the context of diabetic epitheliopathy and wound healing. Further studies are warranted in the corneal stroma-nerve interactions. This study unravels the nerve influence on corneal stroma metabolism. Corneal stromal cells were isolated from healthy (HCFs) and diabetes mellitus (Type1DM and Type2 DM) donors. Cells were cultured on polycarbonate membranes, stimulated by stable Vitamin C, and stroma-only and stroma-nerve co-cultures were investigated for metabolic alterations. Innervated compared to stroma-only constructs exhibited significant alterations in pyrimidine, glycerol phosphate shuttle, electron transport chain and glycolysis. The most highly altered metabolites between healthy and T1DMs innervated were phosphatidylethanolamine biosynthesis, and pyrimidine, methionine, aspartate metabolism. Healthy and T2DMs main pathways included aspartate, glycerol phosphate shuttle, electron transport chain, and gluconeogenesis. The metabolic impact on T1DMs and T2DMs was pyrimidine, purine, aspartate, and methionine. Interestingly, the glucose-6-phosphate and oxaloacetate was higher in T2DMs compared to T1DMs. Our in vitro co-culture model allows the examination of key metabolic pathways corresponding to corneal innervation in the diabetic stroma. These novel findings can pave the way for future studies to fully understand the metabolic distinctions in the diabetic cornea.

In Vivo Confocal Microscopy of Stromal Lenticule Addition Keratoplasty for Advanced Keratoconus.

PURPOSE: To investigate the in vivo corneal microscopic changes after femtosecond laser-assisted stromal lenticule addition keratoplasty in keratoconus by means of in vivo confocal microscopy. METHODS: Patients affected by advanced keratoconus were included in the study. Negative meniscus-shaped stromal lenticules, produced with a femtosecond laser (VisuMax; Carl Zeiss Meditec) from eye bank corneas were transplanted into a stromal pocket dissected in the recipient cornea at a depth of 120 µm. In vivo confocal microscopy was performed during the 12-month follow-up to investigate changes of the corneal and lenticule structure. RESULTS: Ten patients were enrolled in the study. No changes of the dendritic cell population were documented during the follow-up period. Mild edema and stromal keratocyte activation gradually decreased during the first month. Subbasal nerve density returned to preoperative values after 6 months. Donor-recipient interfaces appeared hyperreflective but gradually improved over time with significantly reduced reflectivity after 3 months. No evidence of stromal inflammatory cell migration or matrix opacification was observed. Endothelial and keratocyte density remained stable over time. A variable degree of stromal radially distributed folds, not visible on biomicroscopy, was observed in the lenticule and in the posterior recipient stroma. CONCLUSIONS: Stromal lenticule addition keratoplasty produces transitory nerve plexus density reduction and minor inflammatory reaction that rapidly decreases during the first month. Donor-recipient interface reflectivity is comparable to a femtosecond laser refractive procedure with no sign of stromal opacification or stromal rejection in 1 year of follow-up. [J Refract Surg. 2020;36(8):544-550.].

Corneal nerve healing after in situ laser nerve transection.

PURPOSE: We have previously reported that lamellar dissection of the cornea transects stromal nerves, and that regenerating neurites form a dense net along the surgical plane. In these experiments, we have disrupted the stromal nerve trunks in situ, without incising the cornea, to determine the regeneration events in the absence of a surgical plane. METHODS: Thy1-YFP mice were anesthetized and in vivo images of the corneal nerves were obtained with a wide-field stereofluorescent microscope. A far infrared XYRCOS Laser attached to 20X objective of an upright microscope was used to perform in situ transection of the stromal nerves. 3 types of laser transections were performed (n = 5/group): (i) point transection (a single cut); (ii) segmental transection (two cuts enclosing a segment of nerve trunk); and (iii) annular transection (cuts on all nerve trunks crossing the perimeter of a 0.8 mm diameter circular area centered on the corneal apex). Mice were imaged sequentially for 4 weeks thereafter to assess nerve degeneration (disappearance or weakening of original fluorescence intensity) or regeneration (appearance of new fluorescent fronds). Beta-3-tubulin immunostaining was performed on corneal whole-mounts to demonstrate nerve disruption. RESULTS: The pattern of stromal nerves in corneas of the same mouse and in corneas of littermates was dissimilar. Two distinct patterns were observed, often within the same cornea: (i) interconnected trunks that spanned limbus to limbus; or (ii) dichotomously branching trunks that terminate at the corneal apex. Point transections did not cause degeneration of proximal or distal segment in interconnected trunks, but resulted in degeneration of distal segment of branching trunks. In segmental transections, the nerve segment enclosed within the two laser cuts degenerated. Lack of beta-3 tubulin staining at transection site confirmed nerve transection. In interconnected trunks, at 4 weeks, a hyperfluorescent plaque filled the gap created by the transection. In annular transections, some nerve trunks degenerated, while others regained or retained fluorescence. CONCLUSIONS: Interconnected stromal nerves in murine corneas do not degenerate after in situ point transection and show evidence of healing at the site of disruption. Presence or absence of a surgical plane influences corneal nerve regeneration after transection.

Corneal Neurotization for Neurotrophic Keratopathy: Clinical Outcomes and In Vivo Confocal Microscopic and Histopathological Findings.

PURPOSE: To describe the long-term outcomes and in vivo confocal microscopic (IVCM) and histopathological findings after corneal neurotization surgery. METHODS: We included 2 patients who underwent corneal neurotization surgery for severe unilateral neurotrophic keratopathy secondary to cerebellopontine angle meningioma. Corneal sensation was measured using the Cochet-Bonnet esthesiometer (CBE) (0-60 mm). IVCM was performed using the Heidelberg HRT3 Rostock Corneal Module. Histopathological examination was performed on the excised corneoscleral disc of patient 2. RESULTS: In patient 1, corneal sensation improved from 0 mm preoperatively to 60 mm in all 4 quadrants by 2 years postoperatively and was maintained at 5 years postoperatively with identifiable subbasal and stromal corneal nerves on IVCM. In patient 2, corneal sensation improved from 0 mm preoperatively to 10 mm in 3 quadrants (9 months postoperatively) but returned to 0 mm in all quadrants by 2 years postoperatively. IVCM failed to identify any subbasal and stromal corneal nerves. At 5 years postoperatively, evisceration was performed to ameliorate uncontrolled and persistent ocular pain and poor cosmesis. Histopathological examination of the excised corneoscleral disc confirmed the presence of normal-sized, central corneal stromal nerve fascicles but without direct continuity with the transplanted perilimbal nerve bundles. CONCLUSIONS: Our study elucidates the mechanism of corneal neurotization surgery at a cellular level. Although only 1 patient achieved long-term improvement in corneal sensation postoperatively, the findings on IVCM and histopathological examination suggest that partial regeneration/maintenance of corneal nerves after corneal neurotization surgery is likely attributed to the paracrine neurotrophic support, instead of direct sprouting, from the perilimbal transplanted nerve fascicles.

Survival of corneal nerve/sheath structures in organ-cultured donor corneas.

PURPOSE: To study the morphology of human corneal nerves in eye bank organ-cultured corneas and in corneal grafts post-transplantation. METHODS: Thirty-seven organ-cultured corneas were divided into: Group-A, anterior 300-400 µm of 20 corneas used for Descemets stripping endothelial keratoplasty, and Group-B, 17 full-thickness corneas unsuitable for transplantation. Corneas whole mounts were stained for nerves using acetylcholinesterase technique and examined by NanoZoomer digital pathology microscope. Central and sub-Bowman's stromal nerves and the sub-basal nerve plexus including perforation sites and terminal bulbs were studied. Ten eyes were imaged following penetrating keratoplasty using in-vivo confocal microscopy (IVCM) for the presence of sub-basal and stromal nerves at 1, 4-5 and 7-8 weeks postoperatively (five eyes) and in all the other five eyes, the final follow-up was at 12 weeks. RESULTS: Fifteen of twenty (75%) corneas had stromal nerves in Group-A and 15 of 17 (88.2%) in Group-B. Average number of stromal nerves entering peripherally were 9.1 (range: 1-36). 7.5 in Group-A and 10.8 in Group-B. Central stromal nerves were seen in eight samples in Group-A and nine in Group-B. Many stromal nerves terminated abruptly without demonstrable continuity through Bowman's membrane. No terminal bulbs or sub-basal nerves were detected. In-vivo confocal microscopy (IVCM) showed 4 of 5 in 9 of 10 (90%) donor corneas had stromal nerves 1 week postoperatively, which remained present in 8 of 10 (80%) corneas at 4-5 weeks and in 9 of 10 (90%) at 7-8 weeks postoperatively. All 5 corneas analysed at 12 weeks showed the same stromal nerves from 1 to 12 weeks postoperatively. Sub-basal nerves were absent in all corneas over the 12-week study period. CONCLUSION: This study provides further insight into the behaviour of corneal nerves in transplanted corneas. Corneal stromal nerves/nerve-sheaths are preserved in organ-cultured eye bank eyes and persist post-transplantation up to 3 months. These could provide directional guidance to regenerating nerves from host stroma.

Impact of herpetic stromal immune keratitis in corneal biomechanics and innervation.

PURPOSE: To study corneal innervation in eyes with history of herpetic keratitis and its correlation with corneal sensitivity and biomechanical properties. METHODS: A total of 56 eyes were included, of which 16 had a history of unilateral immune stromal herpetic keratitis, 16 were their contralateral eyes, and 20 were healthy controls. Structural analysis of corneal nerve plexus was performed by confocal microscopy. Biomechanical properties were measured with the Ocular Response Analyzer. Corneal sensitivity was assessed by contact (Cochet-Bonnet) and non-contact (Belmonte) esthesiometry. RESULTS: The eyes with a history of herpetic keratitis had reduced sensitivity for mechanical stimuli when compared to healthy eyes (1441.88 ± 83 ml/min vs. 67.9 ± 7.86 ml/min). Nerve fiber density in the corneas with a history of herpetic disease was lower (4.13 ± 2.19 U/image) than in the contralateral eyes (7.44 ± 2.9 U/image, p value = 0.01) and than in healthy controls (10.35 ± 2.01, p value < 0.0001). The best structural and functional correlation was established between the total length of nerves per section and mechanic threshold assessed by Belmonte esthesiometer (Coef. -0.58 p value < 0.0001) and between total length of nerves and corneal resistance factor (CRF) (Coef. -0.64, p value < 0.0001). CONCLUSIONS: The corneal sensitivity impairment in eyes with immune stromal herpetic keratitis can be explained by the loss of nerve fibers. Biomechanical corneal properties are affected as well. Corneal hysteresis (CH) and CRF are lower for the eyes with a history of herpetic keratitis, and also for the contralateral eye when compared to healthy controls.

Unravelling the stromal-nerve interactions in the human diabetic cornea.

Corneal defects due to diabetes mellitus (DM) may cause severe vision impairments. Current studies focus on the corneal epithelium and nerve defects neglecting the corneal stroma. The aim of this study was to develop a 3D in vitro model to examine the interactions between corneal stroma and nerves in the context of DM. Primary human corneal stromal fibroblasts isolated from healthy (HCFs), Type 1 (T1DM) and Type 2 (T2DM) patients were stimulated with stable ascorbic acid to secrete and assemble an extracellular matrix (ECM). Human neuronal cells were then seeded on top and differentiated to create the 3D co-cultures. Our data revealed successful co-culture of stromal fibroblasts and neuronal cells with large elongated neuron extensions. T2DM showed significant upregulation of Collagen III and IGF1 when compared to T1DM. Interestingly, upon nerve addition, those markers returned to HCF levels. Neuronal markers were also differentially modulated with T2DM co-cultures expressing high levels of ßIII tubulin where T1DM co-cultures expressed Substance P. . Overall, our unique 3D co-culture model provides us with a tool that can be utilized for both molecular and therapeutic studies for diabetic keratopathy.

A Central Role for Sympathetic Nerves in Herpes Stromal Keratitis in Mice.

PURPOSE: Herpes simplex virus type 1 (HSV-1) is a neurotrophic virus that can cause herpes stromal keratitis (HSK), a severe corneal inflammation that can lead to corneal scarring and blindness. This study identified neurologic changes that occur in HSV-1-infected corneas and related them to HSV-1-induced immunopathology. METHODS: Corneas of BALB/c and C57BL/6 mice were infected with HSV-1 strains that induce HSK. Changes in sensory nerves were identified by immunofluorescence staining of sensory and sympathetic nerves for substance P (SP) and tyrosine hydroxylase (TH), respectively, and confocal microscopic examination. Some mice received superior cervical ganglionectomy (SCGx) to eliminate sympathetic nerves from the cornea. RESULTS: Normal corneas exclusively expressed sensory nerves that entered the stroma as large nerve stalks, branched to form a plexus at the epithelial/stromal interface, and extended termini into the epithelium. These nerves completely retracted from the infected cornea and were replaced by sympathetic nerves that sprouted extensively to hyperinnervate the corneal stroma but failed to form a plexus or extend termini into the epithelium. The hyperinnervating nerves expressed the sympathetic nerve marker TH and their invasion was blocked by performing SCGx. Moreover, the corneal opacity and neovascularization that normally characterizes HSK in this mouse model were largely abrogated by SCGx. Sensory nerves reinnervated infected corneas following SCGx, reformed a nerve plexus, and extended termini into the epithelium resulting in recovery of corneal sensitivity. CONCLUSIONS: Sympathetic nerves have a central role in HSK in mice, preventing reinnervation by sensory nerves and promoting severe and persistent corneal inflammation.

Assessment of corneal alterations by confocal microscopy in vernal keratoconjunctivitis.

BACKGROUND: Vernal keratoconjunctivitis (VKC) is a bilateral chronic, seasonally exacerbated inflammation of the ocular surface that especially affects male children and young boys. AIM: To evaluate the corneal microscopic features of patients affected by VKC and to assess whether some corneal changes were associated with specific ocular symptoms and/or signs. METHODS: 20 children aged between 4 and 14 years were enrolled. All patients underwent corneal confocal microscopy by Confoscan CS3 (Nidek). 350 images of the central cornea of each eye were obtained with a ×40 noncontact lens 3,5 micron gap in automode. RESULTS: Some alterations of the sub-basal and stromal corneal nerves were detected. These alterations were more evident in patients with higher severity of photophobia. On the other hand, there were scarce other signs of the anterior segment of the eye. CONCLUSIONS: Our preliminary findings show that there is another group of patients affected by VKC, characterized by an intense photophobia caused by corneal damage and without other significant ocular alterations. Therefore confocal microscopy may be useful for an early identification of corneal alterations before the onset of severe ocular symptoms and to set an appropriate therapeutic management.

On the Cornea of Healthy Merino Sheep: A Detailed Ex Vivo Confocal, Histological and Ultrastructural Study.

Our study performed qualitative and quantitative studies on the corneal ultrastructure of healthy female Merino sheep of ages 4 months and 6 years old from the Argentinean Pampa. The corneas were evaluated using ex vivo laser-scanning confocal microscopy, light microscopy and transmission electron microscopy. Those studies allowed us to obtain detailed images of the corneal layers as well as quantitative data of the cellular and sub-basal nerve densities in the cornea from sheep of different ages. The density of the corneal cells was significantly different in the anterior versus the posterior epithelium and stroma. Moreover, the density of the epithelial, stromal cells and endothelial cells, as well as the sub-basal nerve density were significantly lower in adult than in young animals. Our work provided a wide-ranging description of the corneal ultrastructure of healthy female Merino sheep, which adds to the current knowledge about the ophthalmological aspects of this species and undoubtedly benefits veterinarians.

Análisis de nervios estromales en pacientes con queratocono / Comparison of stromal corneal nerves between normal and keratoconus patients using confocal microscopy

OBJETIVO: Evaluar las diferencias de los nervios del estroma de la córnea entre sujetos normales y pacientes con queratocono. MÉTODOS: Un total de 140 ojos de 70 sujetos normales (grupo A) y 122 ojos de 87 pacientes con queratocono (grupo B), fueron evaluados con el microscopio confocal, realizando un rastreo central del espesor total de la córnea. La morfología y el espesor de los nervios fueron evaluados utilizando el programa Navis V. 3.5.0. El espesor de los nervios se obtuvo del promedio de la porción más delgada y la más gruesa de cada nervio. RESULTADOS: Los nervios del estroma se observaron como estructuras lineales de alta reflexión e irregulares, con porciones gruesas y angostas en todos los casos. El promedio del espesor de los nervios en el grupo A fue de 5,7 ± 1,7 (rango de 3,3 a 10,4 μ), en el grupo B fue de 7,2 ± 1,9 (rango de 3,5 a 12,0 μ). La diferencia en el espesor de los nervios entre el grupo A y el grupo B fue estadísticamente significativa (p < 0,05). CONCLUSIONES: La morfología de los nervios del estroma de la córnea fue similar en ambos grupos; el espesor de estos fue mayor en los pacientes con queratocono

OBJECTIVE: To evaluate the differences in stromal corneal nerves between normal patients and keratoconus patients. MATERIAL AND METHODS: A total of 140 eyes of 70 normal patients (group A) and 122 eyes of 87 keratoconus patients (group B) were examined with the confocal microscope, with a central scan of the total corneal thickness being taken. The morphology and thickness of the corneal stromal nerves were evaluated by using the Navis V. 3.5.0. software. Nerve thickness was obtained from the mean between the widest and the narrowest portions of each stromal nerve. RESULTS: Corneal stromal nerves were observed as irregular linear hyper-reflective structures with wide and narrow portions in all cases. Mean corneal stromal nerves thickness in group A was 5.7 ± 1.7 (range from 3.3 to 10.4 μ), mean corneal stromal nerves thickness in group B was 7.2 ± 1.9 (range from 3.5 to 12.0 μ). There was a statistical significant difference (P<0.05) in stromal corneal nerves thickness between group A and group B. CONCLUSION: Stromal corneal nerves morphology was similar in both groups, but stromal nerves were thicker in keratoconus patients

Comparison of stromal corneal nerves between normal and keratoconus patients using confocal microscopy.

OBJECTIVE: To evaluate the differences in stromal corneal nerves between normal patients and keratoconus patients. MATERIAL AND METHODS: A total of 140 eyes of 70 normal patients (group A) and 122 eyes of 87 keratoconus patients (group B) were examined with the confocal microscope, with a central scan of the total corneal thickness being taken. The morphology and thickness of the corneal stromal nerves were evaluated by using the Navis v. 3.5.0. software. Nerve thickness was obtained from the mean between the widest and the narrowest portions of each stromal nerve. RESULTS: Corneal stromal nerves were observed as irregular linear hyper-reflective structures with wide and narrow portions in all cases. Mean corneal stromal nerves thickness in group A was 5.7±1.7 (range from 3.3 to 10.4 µ), mean corneal stromal nerves thickness in group B was 7.2±1.9 (range from 3.5 to 12.0 µ). There was a statistical significant difference (P<.05) in stromal corneal nerves thickness between group A and group B. CONCLUSION: Stromal corneal nerves morphology was similar in both groups, but stromal nerves were thicker in keratoconus patients.

A novel association between resident tissue macrophages and nerves in the peripheral stroma of the murine cornea.

PURPOSE: To characterize the interactions between resident macrophage populations and nerves in naïve and injured corneas of the mouse eye. METHODS: Corneas from wild-type (WT) C57BL/6J, BALB/cJ, and transgenic Cx3cr1-eGFP mice were subjected to a 1-mm central epithelial debridement injury. The eyes were fixed and immunostained as flat mounts with a range of antibodies to identify macrophages, neurons, and Schwann cells. Interactions between nerves and immune cells were analyzed and quantitated using three-dimensional reconstructions of confocal microscopy images. Naïve eyes acted as controls. RESULTS: A distinctive association between resident immune cells and corneal nerves was noted in the peripheral or perilimbal stromal nerve trunks. These epineurial cells were mostly Cx3cr1(+) Iba-1(+) major histocompatibility complex (MHC) class II(+) F4/80(+) CD11b(+) macrophages. The number of nerve-associated macrophages was greater in WT BALB/c mice than in C57BL/6J mice. There were no qualitative or quantitative differences in the circumferential distribution of nerve-associated macrophages in the cornea. Sterile corneal epithelial debridement led to a dissociation of macrophages from peripheral nerve trunks as early as 2 hours postinjury, with numbers returning to baseline after 72 hours. This dissociation was Cx3cr1 dependent. CONCLUSIONS: This study is the first to highlight a direct physical association between nerves and resident immune cells in the murine cornea. Furthermore, we reveal that this association in normal eyes is responsive to central corneal epithelial injury and is partly mediated by Cx3cr1 signaling. This association may serve as an indicator of malfunctioning neuroimmune communication in disease states such as neurotrophic keratitis and peripheral neuropathy.

Evaluation of corneal microstructure in keratoconus: a confocal microscopy study.

PURPOSE: To compare the corneal microstructure in patients with manifest keratoconus (KCN), subclinical KCN, and topographically normal relatives of patients with KCN and in healthy controls. DESIGN: Prospective and cross-sectional study. METHODS: We enrolled 145 subjects in the study. The participants were divided into 4 groups, based on clinical and topographical evaluation: the manifest KCN group (n = 30), the subclinical KCN group (n = 32), the KCN relatives group (n = 53), and the control group (n = 30). Corneal microstructure was assessed by corneal in vivo confocal microscopy in all of the individuals. Mean outcome measures were basal epithelial cell density, endothelial cell density, anterior keratocyte density, posterior keratocyte density, sub-basal nerve density, sub-basal nerve diameter, and stromal nerve diameter. RESULTS: The mean basal epithelial cell density, endothelial cell density, and sub-basal nerve diameter were not significantly different among the 4 groups (P = 0.057, P = 0.592, and P = 0.393, respectively). The mean anterior and posterior stromal keratocyte densities were significantly lower in the manifest group, in the subclinical group, and in the relatives group when compared with the control group (for both parameters; P < 0.001, P < 0.001, and P< 0.001, respectively). The mean stromal nerve diameter in the manifest group, subclinical group, and relatives group was significantly higher than in the control group (P = 0.001, P = 0.049, and P = 0.004, respectively). CONCLUSION: The anterior and posterior stromal keratocyte densities were statistically lower and stromal nerve diameter was statistically higher in patients with manifest KCN, subclinical KCN, and topographically normal KCN relatives compared with controls. Confocal microscopy may be useful for the determination of early corneal microstructural changes before manifestation of typical or subtle topographic signs.

Subbasal nerve morphology, corneal sensation, and tear film evaluation after refractive femtosecond laser lenticule extraction.

BACKGROUND: The purpose of this study was to compare corneal subbasal nerve morphology, corneal sensation, and tear film parameters after femtosecond lenticule extraction (FLEX) and small-incision lenticule extraction (SMILE). METHODS: A prospective, randomized, single-masked, paired-eye design clinical trial of 35 patients treated for moderate to high myopia with FLEX in one eye and SMILE in the other. In both techniques, an intrastromal lenticule was cut by a femtosecond laser and manually extracted. In FLEX, a LASIK-like flap allowed removal of the lenticule, whereas in SMILE, it was removed through a small incision. In-vivo confocal microscopy was used to acquire images of the central corneal subbasal nerve plexus, from which nerve density, total nerve number, and nerve tortuosity were analyzed. Corneal sensation was measured using Cochet-Bonnet esthesiometry. A visual analog scale, tear osmolarity, non-invasive tear film break-up time (keratograph) tear meniscus height (anterior segment OCT), Schirmer's test, and fluorescein tear film break-up time were used to evaluate tear film and ocular surface symptoms. Patients were examined before and 6 months after surgery. RESULTS: There were no statistically significant differences in baseline parameters between FLEX and SMILE (p > 0.050). With regard to changes from before to 6 months after surgery, mean reduction in subbasal nerve density was 14.22 ± 6.24 mm/mm(2) in FLEX eyes, and 9.21 ± 7.80 mm/mm(2) in SMILE eyes (p < 0.05). The total number of nerves decreased more in FLEX eyes than in SMILE eyes (p < 0.05). No change was found when comparing tortuosity (p > 0.05). Corneal sensation was reduced with 0.38 ± 0.49 cm in FLEX eyes, and 0.10 ± 0.34 cm in SMILE eyes (p < 0.01). No differences were found between FLEX and SMILE in tear film evaluation tests (p > 0.05). Significantly more patients felt postoperative foreign body sensation in the FLEX eye within the first days after surgery, as compared to the SMILE eye. CONCLUSIONS: Six months after surgery, the less invasive SMILE technique seemed better at sparing the central corneal nerves as compared to FLEX. Corneal sensation was only significantly reduced in FLEX eyes. There were no differences between FLEX and SMILE when comparing tear film evaluation tests 6 months after surgery.

Transepithelial corneal collagen crosslinking for keratoconus: qualitative investigation by in vivo HRT II confocal analysis.

PURPOSE: This was a qualitative investigation of corneal microstructural modifications in keratoconic patients undergoing experimental transepithelial crosslinking (TE CXL). METHODS: Ten patients with keratoconus intolerant to gas-permeable rigid contact lenses were enrolled. Corneal thickness was in the range 350-390 µm at the thinnest point measured by Visante AC optical coherence tomography system (Zeiss, Jena, Germany). All patients underwent TE CXL with 0.1% riboflavin-15% dextran solution supplemented with TRIS plus sodium EDTA (Ricrolin TE, Sooft Italia) according to Siena protocol. In vivo Heidelberg retinal tomograph II laser scanning confocal analysis (Rostock Cornea Module, Heidelberg, Germany) was performed with the following follow-up: preoperative and postoperative assessments at 1, 3, and 6 months. The following morphologic parameters were evaluated: epithelium, subepithelial, and anterior stroma nerve plexi, keratocytes apoptosis, stromal changes, and the endothelium. RESULTS: After TE CXL, epithelial cells showed apoptosis, with mosaic alterations gradually disappearing. Keratocytes apoptosis was variable, superficial, and uneven, with a maximum depth of penetration at about 140 µm, measured from the surface of epithelium. Treatment respected subepithelial and stromal nerves that did not disappear. No variation in cell count or endothelial mosaic was observed. CONCLUSIONS: In vivo confocal analysis of corneal modifications induced by TE CXL showed a limited apoptotic affect of this treatment, about one-third of classic epi-off crosslinking procedure. The TE CXL respected sub-basal and anterior stroma nerve fibers, resulting safe for corneal endothelium. According to limited penetration, its mid- to long-term efficacy needs to be determined in different clinical settings related to patient age and keratoconus progression.

Mapping the nerve architecture of diabetic human corneas.

OBJECTIVE: To investigate the entire human corneal nerve architecture of donors with different durations of insulin-dependent diabetes mellitus (IDDM). DESIGN: Experimental study. PARTICIPANTS AND CONTROLS: Sixteen fresh human eyes from 8 diabetic donors (aged 43-66 years, with IDDM for 2-17 years) and 12 eyes from 6 normal donors (aged from 44-67 years) were obtained from the National Disease Research Interchange (NDRI). METHODS: After fixation, corneas were stained with mouse monoclonal anti-ß-Tubulin III antibody, and images were acquired to build a whole view of the corneal nerve architecture. The same corneas were used for both whole-mount and cross-section examination. MAIN OUTCOME MEASURES: Corneal epithelial nerve density was calculated on the basis of the whole-mount view of the central area. The number of stromal nerves was calculated by counting the nerve trunks at the corneoscleral limbus of the entire cornea. Differences between diabetic and normal corneas in epithelial nerve densities and main stromal nerve numbers were compared by paired-samples t test. RESULTS: The diabetic eyes presented numerous neuropathies in areas where the epithelial nerve bundles emerged. A striking pathologic change was the presence of abundant nerve fiber loops in the stroma. These loops seemed to form by resistance presented by the basement membrane, which may prevent penetration of stromal nerve branches into epithelia. There was no difference in the numbers of main stromal nerve trunks between corneas from diabetic and normal donors, but there was a significant decrease in central epithelial nerve density in the diabetic corneas. We did not find an age effect on this decrease. Instead, it was significantly affected by 5 or more years of IDDM. CONCLUSIONS: This is the first study to show an entire view of the nerve architecture in human diabetic corneas. The decreased epithelial nerve density may result from the abnormalities of stromal nerve architecture and is affected by 5 or more years of IDDM. Although compensation for some nerve regeneration takes place, the alterations in the stromal nerves can explain the poor healing and persistent epithelial defects seen in diabetic patients.