A Hypothalamic-Controlled Neural Reflex Promotes Corneal Inflammation.

Purpose: To test whether an acute corneal injury activates a proinflammatory reflex, involving corneal sensory nerves expressing substance P (SP), the hypothalamus, and the sympathetic nervous system. Methods: C57BL6/N (wild-type [WT]) and SP-depleted B6.Cg-Tac1tm1Bbm/J (TAC1-KO) mice underwent bilateral corneal alkali burn. One group of WT mice received oxybuprocaine before alkali burn. One hour later, hypothalamic neuronal activity was assessed in vivo by magnetic resonance imaging and ex vivo by cFOS staining. Some animals were followed up for 14 days to evaluate corneal transparency and inflammation. Tyrosine hydroxylase (TH), neurokinin 1 receptor (NK1R), and neuronal nitric oxide synthase (nNOS) expression was assessed in brain sections. Sympathetic neuron activation was evaluated in the superior cervical ganglion (SCG). CD45+ leukocytes were quantified in whole-mounted corneas. Noradrenaline (NA) was evaluated in the cornea and bone marrow. Results: Alkali burn acutely induced neuronal activation in the trigeminal ganglion, paraventricular hypothalamus, and lateral hypothalamic area (PVH and LHA), which was significantly lower in TAC1-KO mice (P < 0.05). Oxybuprocaine application similarly reduced neuronal activation (P < 0.05). TAC1-KO mice showed a reduced number of cFOS+/NK1R+/TH+ presympathetic neurons (P < 0.05) paralleled by higher nNOS expression (P < 0.05) in both PVH and LHA. A decrease in activated sympathetic neurons in the SCG and NA levels in both cornea/bone marrow and reduced corneal leukocyte infiltration (P < 0.05) in TAC1-KO mice were found. Finally, 14 days after injury, TAC1-KO mice showed reduced corneal opacity and inflammation (P < 0.05). Conclusions: Our findings suggest that stimulation of corneal sensory nerves containing SP activates presympathetic neurons located in the PVH and LHA, leading to sympathetic activation, peripheral release of NA, and corneal inflammation.

PEDF Gene Deletion Disrupts Corneal Innervation and Ocular Surface Function.

Purpose: The cornea is richly innervated by the trigeminal ganglion (TG) and its function supported by secretions from the adjacent lacrimal (LG) and meibomian glands (MG). In this study we examined how pigment epithelium-derived factor (PEDF) gene deletion affects the cornea structure and function. Methods: We used PEDF hemizygous and homozygous knockout mice to study effects of PEDF deficiency on corneal innervation assessed by beta tubulin staining, mRNA expression of trophic factors, and PEDF receptors by adjacent supporting glands, corneal sensitivity measured using a Cochet-Bonnet esthesiometer, and tear production using phenol red cotton thread wetting. Results: Loss of PEDF was accompanied by reduced corneal innervation and sensitivity, increased corneal surface injury and tear production, thinning of the corneal stroma and loss of stromal cells. PEDF mRNA was expressed in the cornea and its supporting tissues, the TG, LG, and MG. Deletion of one or both PEDF alleles resulted in decreased expression of essential trophic support in the TG, LG, and MG including nerve growth factor, brain-derived neurotrophic growth factor, and GDNF with significantly increased levels of NT-3 in the LG and decreased EGF expression in the cornea. Decreased transcription of the putative PEDF receptors, adipose triglyceride lipase, lipoprotein receptor-related protein 6, laminin receptor, PLXDC1, and PLXDC2 was also evident in the TG, LG and MG with the first three showing increased levels in corneas of the Pedf+/- and Pedf-/- mice compared to wildtype controls. Constitutive inactivation of ERK1/2 and Akt was pronounced in the TG and cornea, although their protein levels were dramatically increased in Pedf-/- mice. Conclusions: This study highlights an essential role for PEDF in corneal structure and function and confirms the reported rescue of exogenous PEDF treatment in corneal pathologies. The pleiotropic effects of PEDF deletion on multiple trophic factors, receptors and signaling molecules are strong indications that PEDF is a key coordinator of molecular mechanisms that maintain corneal function and could be exploited in therapeutic options for several ocular surface diseases.

Long term observation of ocular surface alkali burn in rabbit models: Quantitative analysis of corneal haze, vascularity and self-recovery.

Limbal Stem Cell Deficiency (LSCD), caused due to corneal injury, primarily by chemical/alkali burns, leads to compromised vision. Recently, several animal models of corneal alkali burn injury have become available. The majority of the studies with these animal models start interventions soon after the injury. However, in the clinical setting, there is a considerable delay before the intervention is initiated. Detailed knowledge of the molecular, histopathological, and clinical parameters associated with the progression of the injury leading to LSCD is highly desirable. In this context, we set out to investigate clinical, histopathological parameters of ocular surface alkali burn over a long period of time, post-injury. Limbal stem cell-deficient animal models of rabbits were created by alkali burn using sodium hydroxide, which was then assessed for their progression towards LSCD by grading the alkali burn, corneal haze, and vascularization. Additionally, cells present on the corneal surface after the burn was investigated by histology and immunophenotyping. Grading of rabbit eyes post-alkali burn had shown complete conjunctivalization in 80% (n = 12/15) of the rabbits with the alkali burn grade score of 3.88 ± 0.29 in three months and remained stable at four months (4.12 ± 0.24). However, ocular surface showed self-healing in 20% (n = 3/15) of the rabbits with a score of 1.67 ± 0.34 in four months irrespective of similar alkali injury. These self-healing corneas exhibited decreased opacity score from 2.51 ± 0.39 to 0.66 ± 0.22 (p = 0.002) and regressed vascularity from 1.66 ± 0.41 to 0.66 ± 0.33 in one to nine months, respectively. Restoration of the corneal phenotype (CK3+) was observed in central and mid-peripheral regions of the self-healing corneas, and histology revealed the localization of inflammatory cells to the peripheral cornea when compared to conjunctivalized and scarred LSCD eyes. Our study shows the essentiality to consider the time required for surgical intervention after the corneal alkali injury in rabbit models as evident from their tendency to self-heal and restore corneal phenotype without therapy. Such information on the possibility of self-healing should be useful in further studies as well as determining interventional timings and strategy during clinical presentation of corneal alkali burns.

Y-27632 Promotes the Repair Effect of Umbilical Cord Blood-Derived Endothelial Progenitor Cells on Corneal Endothelial Wound Healing.

PURPOSE: To investigate the proliferation of umbilical cord blood-derived endothelial progenitor cells (UCB EPCs) and the differentiation efficiency toward corneal endothelial cell (CEC)-like cells induced by rho-associated protein kinase (ROCK) inhibitor Y-27632 and to determine the most effective strategy for repairing corneal endothelium injuries in rabbits. METHODS: UCB EPCs were cultured in Endothelial Cell Growth Medium-2 (EGM-2) media or conditioned media (CM) from human CECs, with and without the addition of Y-27632. Bromo-deoxyuridine (BrdU) immunocytochemistry and cell counting kit-8 assays were used to examine the proliferation of the cells. Real-time polymerase chain reaction, western blot, and immunocytochemistry were used to detect the CEC markers. Nd:YAG laser was used to establish an appropriate endothelium injury model based on rabbit corneas. The following intracameral injections were then performed to repair the model: 100 µL Opti-MEM I reduced serum medium (model group), 2 × 105 UCB EPCs diluted in 100 µL Opti-MEM I reduced serum medium (EPC group), 100 µM Y-27632 diluted in 100 µL Opti-MEM I reduced serum medium (Y-27632 group), and 2 × 105 UCB EPCs supplemented with 100 µM Y-27632 (final volume 100 µL, EPC/Y-27632 group). The follow-up tests focused on corneal transparency, central corneal thickness, intraocular pressure, and in vivo confocal microscopy, which were performed to evaluate the healing of the wounds. RESULTS: Culturing UCB EPCs in CM supplemented with 10 µM Y-27632 resulted in higher proliferation rates compared with EGM-2 media and CM. There were significantly improved protein levels of Zona Occludens 1, N-cadherin, Na+-K+-ATPase α1, Na+-K+-ATPase ß1, and Pax6 and improved mRNA levels of collagen type IV and VIII and AQP1. The combined intracameral injection of Y-27632 and UCB EPCs accelerated the recovery of corneal transparency, regression of corneal edema, and healing of the corneal endothelium compared with the injections of Y-27632 and UCB EPCs on their own. CONCLUSIONS: Y-27632 not only promotes the proliferation of UCB EPCs but also contributes to differentiation of UCB EPCs toward CECs in the presence of CM. The intracameral injection of Y-27632 itself promotes the healing of corneal endothelium wounds. On this basis, supplementing UCB EPCs with Y-27632 accelerates the healing of corneal endothelium wounds.

Evaluation of Densitometric Analysis for Early Detection of Corneal Blood Staining in Hyphema.

PURPOSE: To investigate the possibility of detecting presumed corneal blood staining after traumatic hyphema with corneal densitometry and to evaluate corneal transparency after hyphema resolution. METHODS: Twenty-eight patients with uniocular nonpenetrating ocular trauma with hyphema were included in the study. Corneal densitometry measurements were performed at the first week and the first month after full resolution of blood in the anterior chamber and discontinuation of medication. The uninjured eyes were accepted as the control group. RESULTS: The corneal densitometry values at all zones of the posterior layer in the study eyes were significantly higher at the first week compared with the first month (P < 0.05 for all). Comparison of the corneal densitometry values of the study eyes at the first week with the fellow eyes showed significantly higher values at all zones of the posterior corneal layer (P < 0.05 for all). Comparison of the study eyes at the first month with the fellow eyes was significantly higher at the posterior 0- to 2-, 2- to 6-, and 6- to 10-mm zones (P = 0.030, P = 0.044, and P = 0.035, respectively). Although corneal densitometry values at the posterior 10- to 12-mm and posterior total zones were higher at the first month compared with those of the fellow eyes, these differences were not statistically significant (P = 0.197 and P = 0.085, respectively). CONCLUSIONS: Corneal densitometry at all zones of the posterior corneal layer significantly changed after traumatic hyphema. Corneal densitometry analysis could be used in clinically normal cases for possible early corneal blood staining detection.

Corneal nerves anatomy, function, injury and regeneration.

The cornea is a highly innervated tissue, exhibiting a complex nerve architecture, distribution, and structural organization. Significant contributions over the years have allowed us to come to the current understanding about the corneal nerves. Mechanical or chemical trauma, infections, surgical wounds, ocular or systemic comorbidities, can induce corneal neuroplastic changes. Consequently, a cascade of events involving the corneal wound healing, trophic functions, neural circuits, and the lacrimal products may interfere in the corneal homeostasis. Nerve physiology drew the attention of investigators due to the popularization of modern laser refractive surgery and the perception of the destructive potential of the excimer laser to the corneal nerve population. Nerve fiber loss can lead to symptoms that may impact the patient's quality of life, and impair the best-corrected vision, leading to patient and physician dissatisfaction. Therefore, there is a need to better understand preoperative signs of corneal nerve dysfunction, the postoperative mechanisms of nerve degeneration and recovery, aiming to achieve the most efficient way of treating nerve disorders related to diseases and refractive surgery.

Epithelial Cell Migration and Proliferation Patterns During Initial Wound Closure in Normal Mice and an Experimental Model of Limbal Stem Cell Deficiency.

Purpose: Establishing the dynamics of corneal wound healing is of vital importance to better understand corneal inflammation, pathology, and corneal regeneration. Numerous studies have made great strides in investigating multiple aspects of corneal wound healing; however, some aspects remain to be elucidated. This study worked toward establishing (1) if epithelial limbal stem cells (LSCs) are necessary for healing all corneal wounds, (2) the mechanism by which epithelial cells migrate toward the wound, and (3) if centrifugal epithelial cell movement exists. Methods: To establish different aspects of corneal epithelial wound healing we subjected mice lacking hyaluronan synthase 2 (previously shown to lack LSCs) and wild-type mice to different corneal debridement injury models. Results: Our data show that both LSCs and corneal epithelial cells contribute toward closure of corneal wounds. In wild-type mice, removal of the limbal rim delayed closure of 1.5-mm wounds, and not of 0.75-mm wounds, indicating that smaller wounds do not rely on LSCs as do larger wounds. In mice shown to lack LSCs, removal of the limbal rim did not affect wound healing, irrespective of the wound size. Finally, transient amplifying cells and central epithelial cells move toward a central corneal wound in a centripetal manner, whereas central epithelial cells may move in a centrifugal manner to resurface peripheral corneal wounds. Conclusions: Our findings show the dimensions of the corneal wound dictate involvement of LSCs. Our data suggest that divergent findings by different groups on the dynamics of wound healing can be in part owing to differences in the wounding models used.

Wounding Induces Facultative Opn5-Dependent Circadian Photoreception in the Murine Cornea.

Purpose: Autonomous molecular circadian clocks are present in the majority of mammalian tissues. These clocks are synchronized to phases appropriate for their physiologic role by internal systemic cues, external environmental cues, or both. The circadian clocks of the in vivo mouse cornea synchronize to the phase of the brain's master clock primarily through systemic cues, but ex vivo corneal clocks entrain to environmental light cycles. We evaluated the underlying mechanisms of this difference. Methods: Molecular circadian clocks of mouse corneas were evaluated in vivo and ex vivo for response to environmental light. The presence of opsins and effect of genetic deletion of opsins were evaluated for influence on circadian photoresponses. Opn5-expressing cells were identified using Opn5Cre;Ai14 mice and RT-PCR, and they were characterized using immunocytochemistry. Results: Molecular circadian clocks of the cornea remain in phase with behavioral circadian locomotor rhythms in vivo but are photoentrainable in tissue culture. After full-thickness incision or epithelial debridement, expression of the opsin photopigment Opn5 is induced in the cornea in a subset of preexisting epithelial cells adjacent to the wound site. This induction coincides with conferral of direct, short-wavelength light sensitivity to the circadian clocks throughout the cornea. Conclusions: Corneal circadian rhythms become photosensitive after wounding. Opn5 gene function (but not Opn3 or Opn4 function) is necessary for induced photosensitivity. These results demonstrate that opsin-dependent direct light sensitivity can be facultatively induced in the murine cornea.

An immune response to the avascular lens following wounding of the cornea involves ciliary zonule fibrils.

The lens and central cornea are avascular. It was assumed that the adult lens had no source of immune cells and that the basement membrane capsule surrounding the lens was a barrier to immune cell migration. Yet, microfibril-associated protein-1 (MAGP1)-rich ciliary zonules that originate from the vasculature-rich ciliary body and extend along the surface of the lens capsule, form a potential conduit for immune cells to the lens. In response to cornea debridement wounding, we find increased expression of MAGP1 throughout the central corneal stroma. The immune cells that populate this typically avascular region after wounding closely associate with this MAGP1-rich matrix. These results suggest that MAGP1-rich microfibrils support immune cell migration post-injury. Using this cornea wound model, we investigated whether there is an immune response to the lens following cornea injury involving the lens-associated MAGP1-rich ciliary zonules. Our results provide the first evidence that following corneal wounding immune cells are activated to travel along zonule fibers that extend anteriorly along the equatorial surface of the lens, from where they migrate across the anterior lens capsule. These results demonstrate that lens-associated ciliary zonules are directly involved in the lens immune response and suggest the ciliary body as a source of immune cells to the avascular lens.

Corneal confocal microscopy identifies greater corneal nerve damage in patients with a recurrent compared to first ischemic stroke.

OBJECTIVES: Corneal nerve damage may be a surrogate marker for the risk of ischemic stroke. This study was undertaken to determine if there is greater corneal nerve damage in patients with recurrent ischemic stroke. METHODS: Corneal confocal microscopy (CCM) was used to quantify corneal nerve fiber density (CNFD), corneal nerve branch density (CNBD), corneal nerve fiber length (CNFL) and corneal nerve fiber tortuosity (CNFT) in 31 patients with recurrent ischemic stroke, 165 patients with a first acute ischemic stroke and 23 healthy control subjects. RESULTS: Triglycerides (P = 0.004, P = 0.017), systolic BP (P = 0.000, P = 0.000), diastolic BP (P = 0.000, P = 0.000) and HbA1c (P = 0.000, P = 0.000) were significantly higher in patients with first and recurrent stroke compared to controls. There was no difference in age, BMI, HbA1c, total cholesterol, triglycerides, LDL, HDL, systolic and diastolic BP between patients with a first and recurrent ischemic stroke. However, CNFD was significantly lower (24.98±7.31 vs 29.07±7.58 vs 37.91±7.13, P<0.05) and CNFT was significantly higher (0.085±0.042 vs 0.064±0.037 vs 0.039±0.022, P<0.05) in patients with recurrent stroke compared to first stroke and healthy controls. CNBD (42.21±24.65 vs 50.46±27.68 vs 87.24±45.85, P<0.001) and CNFL (15.66±5.70, P<0.001 vs 17.38±5.06, P = 0.003) were equally reduced in patients with first and recurrent stroke compared to controls (22.72±5.14). CONCLUSIONS: Corneal confocal microscopy identified greater corneal nerve fibre loss in patients with recurrent stroke compared to patients with first stroke, despite comparable risk factors. Longitudinal studies are required to determine the prognostic utility of corneal nerve fiber loss in identifying patients at risk of recurrent ischemic stroke.

Therapeutic Effects of Lyophilized Conditioned-Medium Derived from Corneal Mesenchymal Stromal Cells on Corneal Epithelial Wound Healing.

Objectives: The conditioned-medium derived from corneal mesenchymal stromal cells (cMSCs) has been shown to have wound healing and immunomodulatory effects in corneal injury models. Here, the therapeutic effects of lyophilized cMSC conditioned-medium were compared with fresh conditioned-medium. Methods: The epithelial wound healing effects of fresh and lyophilized cMSC conditioned-medium were compared with conditioned-medium from non-MSC cells (corneal epithelial cells) using scratch assay. To evaluate the anti-inflammatory effects of fresh and lyophilized cMSC conditioned-media, macrophages were stimulated by a Toll-Like Receptor (TLR) ligand followed by treatment with the conditioned-media and measuring the expression of inflammatory genes. In vivo wound healing effects of fresh and lyophilized cMSC conditioned-media were assessed in a murine model of cornea epithelial injury. Results: Both fresh and lyophilized cMSCs-derived conditioned-medium induced significantly faster closure of in vitro epithelial wounds compared to conditioned-medium from non-MSC cells (P < .0001). Treating stimulated macrophages with fresh or lyophilized cMSCs-derived conditioned-media significantly decreased the expression of inflammatory genes compared to control (P < .0001). Murine corneal epithelial wounds were healed by 87.6 ± 2.7% and 86.2 ± 4.6% following treatment with fresh and lyophilized cMSC conditioned-media, respectively, while the control was healed by 64.7 ± 16.8% (P < .05). Conclusion: Lyophilized cMSC-derived conditioned-medium is as effective as fresh conditioned-medium in promoting wound healing and modulating inflammation. The results of this study support the application of lyophilized cMSCs-derived conditioned-medium, which allows for more extended storage, as a promising non-invasive option in the treatment of corneal wounds.

The IGF/Insulin-IGFBP Axis in Corneal Development, Wound Healing, and Disease.

The insulin-like growth factor (IGF) family plays key roles in growth and development. In the cornea, IGF family members have been implicated in proliferation, differentiation, and migration, critical events that maintain a smooth refracting surface that is essential for vision. The IGF family is composed of multiple ligands, receptors, and ligand binding proteins. Expression of IGF type 1 receptor (IGF-1R), IGF type 2 receptor (IGF-2R), and insulin receptor (INSR) in the cornea has been well characterized, including the presence of the IGF-1R and INSR hybrid (Hybrid-R) in the corneal epithelium. Recent data also indicates that each of these receptors display unique intracellular localization. Thus, in addition to canonical ligand binding at the plasma membrane and the initiation of downstream signaling cascades, IGF-1R, INSR, and Hybrid-R also function to regulate mitochondrial stability and nuclear gene expression. IGF-1 and IGF-2, two of three principal ligands, are polypeptide growth factors that function in all cellular layers of the cornea. Unlike IGF-1 and IGF-2, the hormone insulin plays a unique role in the cornea, different from many other tissues in the body. In the corneal epithelium, insulin is not required for glucose uptake, due to constitutive activation of the glucose transporter, GLUT1. However, insulin is needed for the regulation of metabolism, circadian rhythm, autophagy, proliferation, and migration after wounding. There is conflicting evidence regarding expression of the six IGF-binding proteins (IGFBPs), which function primarily to sequester IGF ligands. Within the cornea, IGFBP-2 and IGFBP-3 have identified roles in tissue homeostasis. While IGFBP-3 regulates growth control and intracellular receptor localization in the corneal epithelium, both IGFBP-2 and IGFBP-3 function in corneal fibroblast differentiation and myofibroblast proliferation, key events in stromal wound healing. IGFBP-2 has also been linked to cellular overgrowth in pterygium. There is a clear role for IGF family members in regulating tissue homeostasis in the cornea. This review summarizes what is known regarding the function of IGF and related proteins in corneal development, during wound healing, and in the pathophysiology of disease. Finally, we highlight key areas of research that are in need of future study.

Advanced Glycation End Products and Receptor (RAGE) Promote Wound Healing of Human Corneal Epithelial Cells.

Purpose: We used a human corneal epithelial cell (HCE) line to determine the involvement of the advanced glycation end products (AGEs) / receptor for AGEs (RAGE) couple in corneal epithelium wound healing. Methods: After wounding, HCE cells were exposed to two major RAGE ligands (HMGB1 and AGEs), and wound healing was evaluated using the in vitro scratch assay. Following wound healing, the HCE cells were used to study the influence of the RAGE ligands on HCE proliferation, invasion, and migration. Activation of the nuclear factor (NF)-κB signaling pathway by the AGEs/RAGE couple was tested using a luciferase reporter assay. Functional transcriptional regulation by this pathway was confirmed by quantification of expression of the connexin 43 target gene. For each experiment, specific RAGE involvement was confirmed by small interfering RNA treatments. Results: AGEs treatment at a dose of 100 µg/mL significantly improved the wound healing process in a RAGE-dependent manner by promoting cell migration, whereas HMGB1 had no effect. No significant influence of the AGEs/RAGE couple was observed on cell proliferation and invasion. However, this treatment induced an early activation of the NF-κB pathway and positively regulated the expression of the target gene, connexin 43, at both the mRNA and protein levels. Conclusions: Our results demonstrate that the RAGE pathway is activated by AGEs treatment and is involved in the promotion of corneal epithelial wound healing. This positive action is observed only during the early stages of wound healing, as illustrated by the quick activation of the NF-κB pathway and induction of connexin 43 expression.

Association of Postfungal Keratitis Corneal Scar Features With Visual Acuity.

Importance: Corneal opacity is a leading cause of visual impairment worldwide; however, the specific features of corneal scars, which decrease visual acuity, have not been well characterized. Objective: To investigate which features of a postfungal keratitis corneal scar contribute to decreased visual acuity after an episode of infectious keratitis and evaluate whether any corneal features may be used as outcomes for clinical trials. Design, Setting, and Participants: In this ancillary, prospective cross-sectional study, a subset of study participants treated for fungal keratitis (n = 71) as part of the Mycotic Ulcer Treatment Trial I (MUTT I) underwent best spectacle-corrected visual acuity (BSCVA) and best contact lens-corrected visual acuity examination, Scheimpflug imaging, and anterior segment optical coherence tomography at a referral hospital in India approximately 2 years after enrollment. Data were collected from December 3, 2012, to December 19, 2012, and analyses were performed from December 2, 2013, to October 2, 2019. Main Outcomes and Measures: Linear regression models were used to evaluate the importance of various corneal features for BSCVA and to assess whether these features could be used to differentiate the 2 treatment arms of the MUTT I trial. Results: Seventy-one patients (42 men [59.1%]; median age, 48 [range, 39-60] years) were examined at a median (IQR) time of 1.8 (1.4-2.2) years after enrollment. The mean (SD) logMAR BSCVA was 0.17 (0.19) (Snellen equivalent, 20/32). In multivariable linear regression models, BSCVA was most associated with irregular astigmatism (1.0 line of worse BSCVA per 1-line difference between BSCVA and contact lens visual acuity; 95% CI, 0.6-1.4) and corneal scar density (1.5 lines of worse vision per 10-unit increase in the mean central corneal density; 95% CI, 0.8-2.3). The thinnest point of the cornea was the metric that best discriminated between the natamycin- and voriconazole-treated ulcers in MUTT I, with 29.3 µm (95% CI, 7.1-51.6 µm) less thinning in natamycin-treated eyes. Conclusions and Relevance: Both irregular astigmatism and corneal scar density may be important risk factors for BSCVA in a population with relatively mild, healed fungal corneal ulcers. The thinnest point of the corneal scar may be a cornea-specific outcome that could be used to evaluate treatments for corneal ulcers.

VEGF188 promotes corneal reinnervation after injury.

Vascular endothelial growth factor A (VEGF) induces angiogenesis and vascular hyperpermeability in ocular tissues and is therefore a key therapeutic target for eye conditions in which these processes are dysregulated. In contrast, the therapeutic potential of VEGF's neurotrophic roles in the eye has remained unexploited. In particular, it is not known whether modulating levels of any of the 3 major alternatively spliced VEGF isoforms might provide a therapeutic approach to promote neural health in the eye without inducing vascular pathology. Here, we have used a variety of mouse models to demonstrate differences in overall VEGF levels and VEGF isoform ratios across tissues in the healthy eye. We further show that VEGF isoform expression was differentially regulated in retinal versus corneal disease models. Among the 3 major isoforms - termed VEGF120, VEGF164, and VEGF188 - VEGF188 was upregulated to the greatest extent in injured cornea, where it was both necessary and sufficient for corneal nerve regeneration. Moreover, topical VEGF188 application further promoted corneal nerve regeneration without inducing pathological neovascularization. VEGF isoform modulation should therefore be explored further for its potential in promoting neural health in the eye.

Role of TRPM8 Channels in Altered Cold Sensitivity of Corneal Primary Sensory Neurons Induced by Axonal Damage.

The cornea is extensively innervated by trigeminal ganglion cold thermoreceptor neurons expressing TRPM8 (transient receptor potential cation channel subfamily M member 8). These neurons respond to cooling, hyperosmolarity and wetness of the corneal surface. Surgical injury of corneal nerve fibers alters tear production and often causes dry eye sensation. The contribution of TRPM8-expressing corneal cold-sensitive neurons (CCSNs) to these symptoms is unclear. Using extracellular recording of CCSNs nerve terminals combined with in vivo confocal tracking of reinnervation, Ca2+ imaging and patch-clamp recordings of fluorescent retrogradely labeled corneal neurons in culture, we analyzed the functional modifications of CCSNs induced by peripheral axonal damage in male mice. After injury, the percentage of CCSNs, the cold- and menthol-evoked intracellular [Ca2+] rises and the TRPM8 current density in CCSNs were larger than in sham animals, with no differences in the brake K+ current IKD Active and passive membrane properties of CCSNs from both groups were alike and corresponded mainly to those of canonical low- and high-threshold cold thermoreceptor neurons. Ongoing firing activity and menthol sensitivity were higher in CCSN terminals of injured mice, an observation accounted for by mathematical modeling. These functional changes developed in parallel with a partial reinnervation of the cornea by TRPM8(+) fibers and with an increase in basal tearing in injured animals compared with sham mice. Our results unveil key TRPM8-dependent functional changes in CCSNs in response to injury, suggesting that increased tearing rate and ocular dryness sensation derived from deep surgical ablation of corneal nerves are due to enhanced functional expression of TRPM8 channels in these injured trigeminal primary sensory neurons.SIGNIFICANCE STATEMENT We unveil a key role of TRPM8 channels in the sensory and autonomic disturbances associated with surgical damage of eye surface nerves. We studied the damage-induced functional alterations of corneal cold-sensitive neurons using confocal tracking of reinnervation, extracellular corneal nerve terminal recordings, tearing measurements in vivo, Ca2+ imaging and patch-clamp recordings of cultured corneal neurons, and mathematical modeling. Corneal nerve ablation upregulates TRPM8 mainly in canonical cold thermoreceptors, enhancing their cold and menthol sensitivity, inducing a rise in the ongoing firing activity of TRPM8(+) nerve endings and an increase in basal tearing. Our results suggest that unpleasant dryness sensations, together with augmented tearing rate after corneal nerve injury, are largely due to upregulation of TRPM8 in cold thermoreceptor neurons.

Coordinated Modulation of Corneal Scarring by the Epithelial Basement Membrane and Descemet's Basement Membrane.

PURPOSE: To provide an overview of the importance of the coordinated role of the epithelial basement membrane (EBM) and Descemet's basement membrane (DBM) in modulating scarring (fibrosis) in the cornea after injuries, infections, surgeries, and diseases of the cornea. METHODS: Literature review. RESULTS: Despite their molecular and ultrastructural differences, the EBM and DBM act in a coordinated fashion to modulate the entry of transforming growth factor beta (TGF-ß) and other growth factors from the epithelium/tear film and aqueous humor, respectively, into the corneal stroma where persistent levels of these modulators trigger the development and persistence of myofibroblasts that produced disordered, opaque extracellular matrix not usually present in the corneal stroma. The development of these myofibroblasts and the extracellular matrix they produce is often detrimental to visual function of the cornea after penetrating keratoplasty, LASIK buttonhole flaps, persistent epithelial defects, microbial keratitis, Descemet stripping automated endothelial keratoplasty, or Descemet membrane endothelial keratoplasty, while being beneficial in other situations such as the scarred edge of LASIK flaps and donor-recipient interface in penetrating keratoplasty. Efforts to modulate the repair or replacement of the EBM and DBM, and thereby the development or disappearance of myofibroblasts, should be a major emphasis of treatments provided by refractive and corneal surgeries, infections, trauma, or diseases of the cornea. CONCLUSIONS: The EBM and DBM are critical modulators of the localization of profibrotic growth factors, such as TGF-ß, that modulate the development and persistence of myofibroblasts that produce corneal scars (stromal fibrosis). Therapeutic efforts to regenerate or repair EBM and/or DBM, and interfere with the development of myofibroblasts or facilitate their disappearance are often the key to clinical outcomes. [J Refract Surg. 2019;35(8):506-516.].

Induction of Fibroblast Senescence During Mouse Corneal Wound Healing.

Purpose: To investigate the presence and role of fibroblast senescence in the dynamic process of corneal wound healing involving stromal cell apoptosis, proliferation, and differentiation. Methods: An in vivo corneal wound healing model was performed using epithelial debridement in C57BL/6 mice. The corneas were stained using TUNEL, Ki67, and α-smooth muscle actin (α-SMA) as markers of apoptosis, proliferation, and myofibroblastic differentiation, respectively. Cellular senescence was confirmed by senescence-associated ß-galactosidase (SA-ß-gal) staining and P16Ink4a expression. Mitogenic response and gene expression were compared among normal fibroblasts, H2O2-induced senescent fibroblasts, and TGF-ß-induced myofibroblasts in vitro. The senescence was further detected in mouse models of corneal scarring, alkali burn, and penetrating keratoplasty (PKP). Results: The apoptosis and proliferation of corneal stromal cells were found to peak at 4 and 24 hours after epithelial debridement. Positive staining of SA-ß-gal was observed clearly in the anterior stromal cells at 3 to 5 days. The senescent cells displayed P16Ink4a+ vimentin+ α-SMA+, representing the major origin of activated corneal resident fibroblasts. Compared with normal fibroblasts and TGF-ß-induced myofibroblasts, H2O2-induced senescent fibroblasts showed a nonfibrogenic phenotype, including a reduced response to growth factor basic fibroblast growth factor (bFGF) or platelet-derived growth factor-BB (PDGF-BB), increased matrix metalloproteinase (MMP)1/3/13 expression, and decreased fibronectin and collagen I expression. Moreover, cellular senescence was commonly found in the mouse corneal scarring, alkali burn, and PKP models. Conclusions: Corneal epithelial debridement induced the senescence of corneal fibroblasts after apoptosis and proliferation. The senescent cells displayed a nonfibrogenic phenotype and may be involved in the self-limitation of corneal fibrosis.

Corneal injury: Clinical and molecular aspects.

Currently, over 10 million people worldwide are affected by corneal blindness. Corneal trauma and disease can cause irreversible distortions to the normal structure and physiology of the cornea often leading to corneal transplantation. However, donors are in short supply and risk of rejection is an ever-present concern. Although significant progress has been made in recent years, the wound healing cascade remains complex and not fully understood. Tissue engineering and regenerative medicine are currently at the apex of investigation in the pursuit of novel corneal therapeutics. This review uniquely integrates the clinical and cellular aspects of both corneal trauma and disease and provides a comprehensive view of the most recent findings and potential therapeutics aimed at restoring corneal homeostasis.

Is sex a biological variable in corneal wound healing?

Wound healing differs significantly between men and women in a tissue-dependent manner. Dermal wounds heal faster in women whereas mucosal wounds heal faster in men. However, the effect of sex as a variable in corneal wound healing is largely unknown. The primary objective of this study was to test whether sex is a biological variable in corneal wound healing activated by the trauma or injury using an established in vivo rabbit model with male and female New Zealand White rabbits. Corneal wounds in rabbits were produced by a single topical alkali (0.5N Sodium hydroxide) application. Serial slit-lamp, stereo biomicroscopy, and applanation tonometry evaluated corneal opacity, anterior segment ocular health, and intraocular pressure (IOP), respectively, at various times during the study. Fourteen days after alkali-wound, corneal tissues were collected after humane euthanasia to examine cellular and molecular wound healing parameters. Quantitative PCR (qPCR) and immunofluorescence were used to quantify changes in the extracellular modeling protein levels of alpha-smooth muscle actin (α-SMA), Fibronectin (FN), Collagen-I (Col-I), and Transforming growth factor beta 1 (TGFß1) involved in corneal healing. Hematoxylin and Eosin (H&E) staining was used to study histopathological changes in morphology and TUNEL assay to evaluate levels of apoptotic cell death. Male and female rabbits showed no significant differences in corneal opacity (Fantes score) or intraocular pressure (IOP) values (9.5 ±â€¯0.5 mm Hg) in live animals. Likewise, no statistically significant sex-based differences in the mRNA levels of α-SMA (male = 5.95 ±â€¯0.21 fold vs. female = 5.32 ±â€¯0.043), FN (male = 3.02 ±â€¯0.24 fold vs. female = 3.23 ±â€¯0.27), Col-I (male = 3.12 ±â€¯0.37 fold vs. female = 3.31 ±â€¯0.24), TGFß1 (male = 1.65 ±â€¯0.06 fold vs. female = 1.59 ±â€¯0.053); and protein levels of α-SMA (male = 74.16 ±â€¯4.6 vs. female = 71.58 ±â€¯7.1), FN (male = 60.11 ±â€¯4.6 vs. female = 57.41 ±â€¯8.3), Col-I (male = 84.11 ±â€¯2.8 vs. female = 84.55 ±â€¯3.6), TGFß1 (male = 11.61 ±â€¯2.8 vs. female = 9.5 ±â€¯3.04) were observed. Furthermore, H&E and TUNEL analyses found no statistically significant differences in cellular structures and apoptosis, respectively, in male vs. female corneas. Consistent with earlier reports, wounded corneas showed significantly increased levels of these parameters compared to the unwounded corneas. Our data suggest that sex is not a major biological variable during active early stages of corneal wound healing in rabbits in vivo.