Role of the non-neuronal cholinergic system in the eye: a review

Pharmacologically active preparations directed towards modulating muscarinic receptor activity in the eye have been used for over 2000 years when extracts from Atropa belladonna were first applied to enhance eye appearance through pupillary dilation. The first clinically active drugs targeting a specific eye disease were anticholinesterases (e.g. ecothiophate) applied as eye drops to treat glaucoma in the 1960's. However, cataract was soon detected as a relatively frequent side effect and such drugs are now only used to treat glaucoma as a last resort. As muscarinic agonists have been found to reduce intraocular pressure both by decreasing aqueous humour production (through Na,K-ATPase pump inhibition) and increasing outflow (by muscle contraction), it is likely that treatments will be developed that target specific muscarinic subtypes. Recently, it has been shown that the M1 receptor subtype predominates in the lens. It is therefore important that this subtype is not targeted in future ocular therapies so that the side-effect of cataract is avoided. Form-deprived myopia resulting from an increased axial length in the affected eye can be reduced by the application of atropine. This effect has been achieved both in a chick model system and in human clinical trials, and in the former system atropine has been shown to reduce the production of scleral extracellular proteins. Carbachol stimulates tear fluid production through the activation of muscarinic receptors. Interestingly, at least part of the stimulation occurs via epidermal growth factor (EGF) receptors and although the precise signalling mechanisms are not completely understood, it has been shown that calcium mobilisation plays a critical role in both muscarinic and EGF receptor activity. It should be noted that in the four examples described above, the cell types responsible for producing the physiological output are non-neuronal in origin. Therefore cholinergic receptor activation plays diverse roles in the eye and pharmacological intervention based on specific receptor sub-types has potential benefit in a number of ocular problems. However, potential side effects have also recently been identified.     

Endothelin B receptors contribute to retinal ganglion cell loss in a rat model of glaucoma

Glaucoma is an optic neuropathy, commonly associated with elevated intraocular pressure (IOP) characterized by optic nerve degeneration, cupping of the optic disc, and loss of retinal ganglion cells which could lead to loss of vision. Endothelin-1 (ET-1) is a 21-amino acid vasoactive peptide that plays a key role in the pathogenesis of glaucoma; however, the receptors mediating these effects have not been defined. In the current study, endothelin B (ET(B)) receptor expression was assessed in vivo, in the Morrison's ocular hypertension model of glaucoma in rats. Elevation of IOP in Brown Norway rats produced increased expression of ET(B) receptors in the retina, mainly in retinal ganglion cells (RGCs), nerve fiber layer (NFL), and also in the inner plexiform layer (IPL) and inner nuclear layer (INL). To determine the role of ET(B) receptors in neurodegeneration, Wistar-Kyoto wild type (WT) and ET(B) receptor-deficient (KO) rats were subjected to retrograde labeling with Fluoro-Gold (FG), following which IOP was elevated in one eye while the contralateral eye served as control. IOP elevation for 4 weeks in WT rats caused an appreciable loss of RGCs, which was significantly attenuated in KO rats. In addition, degenerative changes in the optic nerve were greatly reduced in KO rats compared to those in WT rats. Taken together, elevated intraocular pressure mediated increase in ET(B) receptor expression and its activation may contribute to a decrease in RGC survival as seen in glaucoma. These findings raise the possibility of using endothelin receptor antagonists as neuroprotective agents for the treatment of glaucoma.     

Control of lymph-drainage tracts of the eye by the use of dalargin

The objective was to study dalargin in experiment and its effect on drainage tracts in norm and with experimental hypertension, modulating secondary glaucoma. Investigations have been made on 82 rabbit eyes. The overall results show, that subconjunctival and electrophoretic dalargin injection of (0.1%-0.2-0.5 ml) causes the expansion of anterior and posterior lymph-drainage tracts of the eye and have hypotensive effect for ocular hypertension and secondary glaucoma.     

Ocular Cubosome Drug Delivery System for Timolol Maleate: Preparation,Characterization, Cytotoxicity, <Emphasis Type="Italic">Ex Vivo</Emphasis>, and <Emphasis Type="Italic">In Vivo</Emphasis> Evaluation

Glaucoma is an ocular disease featuring increased intraocular pressure (IOP) and its primary treatment strategy is to lower IOP by medication. Current ocular drug delivery in treating glaucoma is confronting a variety of challenges, such as low corneal permeability and bioavailability due to the unique anatomical structure of the human eye. To tackle these challenges, a cubosome drug delivery system for glaucoma treatment was constructed for timolol maleate (TM) in this study. The TM cubosomes (liquid crystalline nanoparticles) were prepared using glycerol monooleate and poloxamer 407 via high-pressure homogenization. These constructed nanoparticles appeared spherical using transmission electron microscopy and had an average particle size of 142 nm, zeta potential of ?6.27 mV, and over 85% encapsulation efficiency. Moreover, using polarized light microscopy and small-angle X-ray scattering (SAXS), it was shown that the TM cubosomes have cubic liquid crystalline D-type (Pn3m) structure, which provides good physicochemical stability and high encapsulation efficiency. Ex vivo corneal permeability experiments showed that the total amount of TM cubosomes penetrated was higher than the commercially available eye drops. In addition, in vivo studies revealed that TM cubosomes reduced the IOP in rabbits from 27.8～39.7 to 21.4～32.6 mmHg after 1-week administration and had a longer retention time and better lower-IOP effect than the commercial TM eye drops. Furthermore, neither cytotoxicity nor histological impairment in the rabbit corneas was observed. This study suggests that cubosomes are capable of increasing the corneal permeability and bioavailability of TM and have great potential for ocular disease treatment.     

Neurotrophic rationale in glaucoma: a TrkA agonist, but not NGF or a p75 antagonist, protects retinal ganglion cells in vivo

Glaucoma is a major cause of vision impairment, which arises from the sustained and progressive apoptosis of retinal ganglion cells (RGC), with ocular hypertension being a major risk or co-morbidity factor. Because RGC death often continues after normalization of ocular hypertension, growth factor-mediated protection of compromised neurons may be useful. However, the therapeutic use of nerve growth factor (NGF) has not proven effective at delaying RGC death in glaucoma. We postulated that one cause for the failure of NGF may be related to its binding to two receptors, TrkA and p75. These receptors have distinct cellular distribution in the retina and in neurons they induce complex and sometimes opposing activities. Here, we show in an in vivo therapeutic model of glaucoma that a selective agonist of the pro-survival TrkA receptor was effective at preventing RGC death. RGC loss was fully prevented by combining the selective agonist of TrkA with intraocular pressure-lowering drugs. In contrast, neither NGF nor an antagonist of the pro-apoptotic p75 receptor protected RGCs. These results further a neurotrophic rationale for glaucoma.     

Analysis of Pulsatile Retinal Movements by Spectral-Domain Low-Coherence Interferometry: Influence of Age and Glaucoma on the Pulse Wave

Recent studies have shown that ocular hemodynamics and eye tissue biomechanical properties play an important role in the pathophysiology of glaucoma. Nevertheless, better, non-invasive methods to assess these characteristics in vivo are essential for a thorough understanding of degenerative mechanisms. Here, we propose to measure ocular tissue movements induced by cardiac pulsations and study the ocular pulse waveform as an indicator of tissue compliance. Using a novel, low-cost and non-invasive device based on spectral-domain low coherence interferometry (SD-LCI), we demonstrate the potential of this technique to differentiate ocular hemodynamic and biomechanical properties. We measured the axial movement of the retina driven by the pulsatile ocular blood flow in 11 young healthy individuals, 12 older healthy individuals and 15 older treated glaucoma patients using our custom-made SD-OCT apparatus. The cardiac pulse was simultaneously measured through the use of an oximeter to allow comparison. Spectral components up to the second harmonic were obtained and analyzed. For the different cohorts, we computed a few parameters that characterize the three groups of individuals by analyzing the movement of the retinal tissue at two locations, using this simple, low-cost interferometric device. Our pilot study indicates that spectral analysis of the fundus pulsation has potential for the study of ocular biomechanical and vascular properties, as well as for the study of ocular disease.     

The role of cytokines in the pathogenesis of inflammatory eye disease.

A coherent view of the role of cytokines in inflammatory eye disease is emerging as a result of studies both in man and experimental animals. Cytokines have been demonstrated in ocular tissue obtained from patients with intraocular inflammation (uveitis) (gamma interferon, IL-2) and have been shown to induce inflammation in experimental animals after intraocular injection [(IL-1, IL-6, IL-8, tumour necrosis factor (TNF), granulocyte macrophage-colony stimulating factor (GM-CSF)]. Several unique features of the immunology of the eye such as the immunosuppression associated with anterior chamber associated immune deviation (ACAID) may be due to the effects of cytokines. Similarly, common complications of ocular inflammation such as glaucoma, keratic precipitates, retinal (macular) oedema and neovascularization may be mediated by cytokines. Understanding of the role of cytokines in inflammatory eye disease has the potential to lead to the development of therapies to abrogate the effects of these important mediators of the inflammatory response.     

Clinical applications of NGF in ocular diseases

Nerve Growth Factor (NGF) and its receptors TrkA and p75 are expressed in physiological states in the anterior and posterior segments of the human eye, where they exert several tissue-specific functions. The roles played by NGF in the homeostasis of the eye and in vision are, therefore, crucial and have been widely investigated both in vitro and in vivo, with growing evidence of an NGF-pathway alteration in several ocular diseases. In this review we describe the functions of NGF in health and diseases states of the eye, and discuss the potential therapeutic effectiveness of NGF in preliminary clinical reports performed in severe ocular diseases unresponsive to any standard treatment. In fact, pharmacodynamic studies showing that NGF administered topically on the ocular surface affects not only the ocular surface but is also able to reach the retina, optic nerve and brain, recently opened new perspectives for the treatment of challenging ocular surface diseases, optic nerve diseases, and degenerative diseases of the retina currently lacking an effective therapy.     

The potential impact of recent insights into proteomic changes associated with glaucoma

Introduction: Glaucoma, a major ocular neuropathy, is still far from being understood on a molecular scale. Proteomic workflows revealed glaucoma associated alterations in different eye components. By using state-of-the-art mass spectrometric (MS) based discovery approaches large proteome datasets providing important information about glaucoma related proteins and pathways could be generated. Corresponding proteomic information could be retrieved from various ocular sample species derived from glaucoma experimental models or from original human material (e.g. optic nerve head or aqueous humor). However, particular eye tissues with the potential for understanding the disease’s molecular pathomechanism remains underrepresented.

Areas covered: The present review provides an overview of the analysis depth achieved for the glaucomatous eye proteome. With respect to different eye regions and biofluids, proteomics related literature was found using PubMed, Scholar and UniProtKB. Thereby, the review explores the potential of clinical proteomics for glaucoma research.

Expert commentary: Proteomics will provide important contributions to understanding the molecular processes associated with glaucoma. Sensitive discovery and targeted MS approaches will assist understanding of the molecular interplay of different eye components and biofluids in glaucoma. Proteomic results will drive the comprehension of glaucoma, allowing a more stringent disease hypothesis within the coming years.     

Using zebrafish to study the complex genetics of glaucoma

The overall goal of this review is to highlight the power of zebrafish as a model system for studying complex diseases which involve multiple genetic loci. We are interested in identifying and characterizing genes implicated in the blinding condition of glaucoma. Glaucoma is a complex disease that often involves multiple genetic loci. Most disease causing and modifying genes for glaucoma remain unidentified. However, several genes that regulate various aspects of ocular development have been shown to associate with glaucoma. With zebrafish, forward and reverse genetic approaches can be combined in order to identify critical genetic interactions required for normal and pathological events in the development and maintenance of the eye.     

Toll-like receptors and corneal innate immunity

The ocular surface is constantly exposed to a wide array of microorganisms. The ability of the cornea to recognize pathogens as foreign and eliminate them is critical to retain its transparency, hence preservation of sight. In the eye, as in other parts of the body, the early response against invading pathogens is provided by innate immunity. Corneal innate immune system uses a series of pattern recognition receptors to detect the presence of pathogens thus allowing for rapid host defense responses to invading microbes. A key component of such receptors is the "Toll-like receptors" (TLRs), which have come to occupy the center stage in innate immunity against invading pathogens. An increasing number of studies have shown that TLRs are expressed by a variety of tissues and cells of the eye and play an important role in ocular defense against microbial infection. Here in this review we summarize the current knowledge about TLR expression in human eye with main emphasis on the cornea, and discuss the future directions of the field.     

Proteases in eye development and disease

The eye is one of the classical systems in developmental biology. Furthermore, diseases of the eye, many of which have a developmental basis, have devastating effects that often result in blindness. Proteases have diverse roles in ocular physiology and pathophysiology. Here, a broad overview is provided of the recent literature pertaining to the involvement of proteases in various aspects of eye development and disease: lens development (focusing on apoptosis and lens fiber cell denucleation and organelle loss) and cataract progression, cornea development and disease, retina development and degeneration, sclera development and myopia, and the trabecular meshwork and glaucoma. Proteases discussed include caspases, calpains, matrix metalloproteases (MMPs), a disintegrin and metalloproteinases (ADAMs) and ADAM with thrombospondin motifs (ADAMTS), the ubiquitin-proteasome pathway (UPP), tissue plasminogen activator (tPA), and secretases. It is clear that proteases have diverse and important roles in ocular development and disease, and represent, in many cases, useful therapeutic targets for treating ocular conditions, which would otherwise lead to visual impairment.     

Neuron-specific enolase and embryology of the trabecular meshwork of the rat eye: an immunohistochemical study.

Neuron-specific enolase (NSE) is a unique form of the glycolytic enzyme enolase found exclusively in neurons and neuroendocrine tissues. Immunohistochemical techniques in which antineuron-specific enolase antibodies are used have made it possible to map out derivatives of the neural crest in humans. By using affinity-purified antibodies against NSE, we investigated whether the contribution of the neural crest cells to the development of the anterior ocular structures in the rat is similar to that in man. We found that filtration structures in rats show morphologically striking similarities with the analogous region of the human eye. Hence, the rat eye, with certain reservations, is a suitable model for experimental studies on ocular diseases that are characterized by chamber angle anomalies or congenital glaucoma.     

Notch signaling in the pigmented epithelium of the anterior eye segment promotes ciliary body development at the expense of iris formation

The ciliary body and iris are pigmented epithelial structures in the anterior eye segment that function to maintain correct intra‐ocular pressure and regulate exposure of the internal eye structures to light, respectively. The cellular and molecular factors that mediate the development of the ciliary body and iris from the ocular pigmented epithelium remain to be fully elucidated. Here, we have investigated the role of Notch signaling during the development of the anterior pigmented epithelium by using genetic loss‐ and gain‐of‐function approaches. Loss of canonical Notch signaling results in normal iris development but absence of the ciliary body. This causes progressive hypotony and over time leads to phthisis bulbi, a condition characterized by shrinkage of the eye and loss of structure/function. Conversely, Notch gain‐of‐function results in aniridia and profound ciliary body hyperplasia, which causes ocular hypertension and glaucoma‐like disease. Collectively, these data indicate that Notch signaling promotes ciliary body development at the expense of iris formation and reveals novel animal models of human ocular pathologies.     

Changes in retinal expression of neurotrophins and neurotrophin receptors induced by ocular hypertension

Open angle glaucoma is defined as a progressive and time-dependent death of retinal ganglion cells concomitant with high intraocular pressure, leading to loss of visual field. Because neurotrophins are a family of growth factors that support neuronal survival, we hypothesized that quantitative and qualitative changes in neurotrophins or their receptors may take place early in ocular hypertension, preceding extensive cell death and clinical features of glaucoma. We present molecular, biochemical, and phenotypic evidence that significant neurotrophic changes occur in retina, which correlate temporally with retinal ganglion cell death. After 7 days of ocular hypertension there is a transient up-regulation of retinal NGF, while its receptor TrkA is up-regulated in a sustained fashion in retinal neurons. After 28 days of ocular hypertension there is sustained up-regulation of retinal BDNF, but its receptor TrkB remains unchanged. Throughout, NT-3 levels remain unchanged but there is an early and sustained increase of its receptor TrkC in Müller cells but not in retinal ganglion cells. These newly synthesized glial TrkC receptors are truncated, kinase-dead isoforms. Expression of retinal p75 also increases late at day 28. Asymmetric up-regulation of neurotrophins and neurotrophin receptors may preclude efficient neurotrophic rescue of RGCs from apoptosis. A possible rationale for therapeutic intervention with Trk receptor agonists and p75 receptor antagonists is proposed.     

Alpha2-macroglobulin is a mediator of retinal ganglion cell death in glaucoma

Glaucoma is defined as a chronic and progressive optic nerve neuropathy, characterized by apoptosis of retinal ganglion cells (RGC) that leads to irreversible blindness. Ocular hypertension is a major risk factor, but in glaucoma RGC death can persist after ocular hypertension is normalized. To understand the mechanism underlying chronic RGC death we identified and characterized a gene product, alpha2-macroglobulin (alpha2M), whose expression is up-regulated early in ocular hypertension and remains up-regulated long after ocular hypertension is normalized. In ocular hypertension retinal glia up-regulate alpha2M, which binds to low-density lipoprotein receptor-related protein-1 receptors in RGCs, and is neurotoxic in a paracrine fashion. Neutralization of alpha2M delayed RGC loss during ocular hypertension; whereas delivery of alpha2M to normal eyes caused progressive apoptosis of RGC mimicking glaucoma without ocular hypertension. This work adds to our understanding of the pathology and molecular mechanisms of glaucoma, and illustrates emerging paradigms for studying chronic neurodegeneration in glaucoma and perhaps other disorders.     

一种新的可用于干细胞移植的新西兰白兔青光眼视神经损伤模型

青光眼是一种致盲且不可逆神经退行性疾病,目前还没有能够彻底解决其视野缺失的治疗方法。神经干细胞研究的兴起为该病的治疗带来了可能,且迫切需要一种适用于干细胞移植研究的青光眼造模方法。该实验对新西兰白兔球结膜下注射地塞米松给药,2.5mg/次,一周3次,持续8周。眼底照相显示造模眼眼球屈光间质保持清晰,视乳头凹陷明显扩大、血管呈屈膝状;病理切片显示造模眼视神经明显损伤;海德堡视网膜断层扫描仪(Heidelberg retina tomography,HRT)定量分析显示造模眼盘沿面积减小(1.10±0.88)mm2、杯/盘比增大0.17±0.13,视网膜神经纤维层平均厚度降低(0.44±0.31)mm等青光眼性质病理改变,且均达到极显著水平(P<0.001)。建模成功后,于角膜缘后4mm向造模眼玻璃体注入猕猴神经干细胞,植入5个月后摘取眼球切片,通过荧光显微镜观察到存活的移植细胞。该实验建立了一种简单可靠、重复性强且适用于神经干细胞移植研究的慢性青光眼造模方法。     

The natural histidine-containing dipeptide Nalpha-acetylcarnosine as an antioxidant for ophthalmic use

The naturally occurring compound Nalpha-acetylcarnosine is proposed as a prodrug of L-carnosine that is resistant to enzymatic hydrolysis by carnosinase. Eyes of rabbits were treated with 1% Nalpha-acetylcarnosine, L-carnosine, or placebo and extracts of the aqueous humor from the anterior eye chamber were analyzed for imidazole content by reverse-phase analytical high performance liquid chromatography (HPLC) and thin-layer (TLC) and ion-exchange chromatographic techniques. Topical administration of pure L-carnosine to the rabbit eye did not lead to accumulation of this compound in the aqueous humor over 30 min in concentration exceeding that in the placebo-treated matched eye. Nalpha-Acetylcarnosine showed dose-dependent hydrolysis in its passage from the cornea to the aqueous humor, releasing L-carnosine after l5-30 min of ocular administration of the prodrug in a series of therapeutic modalities: instillation < or = subconjunctival injection < or = ultrasound-induced phoresis. Different treatment techniques showed excellent toleration of 1%Nalpha-acetylcarnosine by the eye. Once in the aqueous humor, L-carnosine might act as an antioxidant and enter the lens tissue when present at effective concentrations (5-l5 mM). The advantage of the ophthalmic prodrug Nalpha-acetylcarnosine and its bioactivated principle L-carnosine as universal antioxidants relates to their ability to give efficient protection against oxidative stress both in the lipid phase of biological membranes and in aqueous environments. Nalpha-Acetylcarnosine is proposed for treatment of ocular disorders that have a component of oxidative stress in their genesis (cataracts, glaucoma, retinal degeneration, corneal disorders, ocular inflammation, complications of diabetes mellitus, and systemic diseases).     

Synthesis and evaluation of potent and selective MGL inhibitors as a glaucoma treatment

Monoacylglycerol lipase (MGL) inhibition provides a potential treatment approach to glaucoma through the regulation of ocular 2-arachidonoylglycerol (2-AG) levels and the activation of CB1 receptors. Herein, we report the discovery of new series of carbamates as highly potent and selective MGL inhibitors. The new inhibitors showed potent nanomolar inhibitory activity against recombinant human and purified rat MGL, were selective (>1000-fold) against serine hydrolases FAAH and ABHD6 and lacked any affinity for the cannabinoid receptors CB1 and CB2. Protein-based ¹H NMR experiments indicated that inhibitor 2 rapidly formed a covalent adduct with MGL with a residence time of about 6?h. This interconversion process “intrinsic reversibility” was exploited by modifications of the ligand’s size (length and bulkiness) to generate analogs with “tunable’ adduct residence time (τ). Inhibitor 2 was evaluated in a normotensive murine model for assessing intraocular pressure (IOP), which could lead to glaucoma, a major cause of blindness. Inhibitor 2 was found to decrease ocular pressure by ～4.5?mmHg in a sustained manner for at least 12?h after a single ocular application, underscoring the potential for topically-administered MGL inhibitors as a novel therapeutic target for the treatment of glaucoma.