In Vivo Confocal Microscopy of Prominent Conjunctival and Corneal Nerves in Multiple Endocrine Neoplasia Type 2B.

OBJECTIVE: To report a case of a patient affected by multiple endocrine neoplasia type 2B (MEN 2B) with imaging of conjunctival neuromas by in vivo confocal microscopy (IVCM). METHODS: Case report. RESULTS: A 48-year-old patient affected by MEN2B complained of progressive visual loss in his right eye and severe red, dry and itchy eyes. Best-corrected visual acuity was 20/63 OD and 20/25 OS. Slit lamp exam showed thickened and turned out lid margins, significant blepharitis, conjunctival injection, multiple presumed subconjunctival neuromas at the bulbar conjunctiva and at the limbus, marked prominence of corneal nerves, exposure keratopathy due to incomplete blinking and corneal hypoesthesia, subepithelial corneal neovascularization and scarring in the mid inferior part of both corneas and bilateral iris nodules. We performed IVCM on conjunctival neuromas, revealing large, thick bundles of nerves with disorganization, prominent loops, bifurcations and dilations measuring as much as 1 mm. The IVCM of corneal nerves demonstrated hypertrophic sub basal plexus. CONCLUSIONS: To date, this is the first report which documents conjunctival neuromas by confocal microscopy in MEN2B.

In Vivo Small Molecule Delivery to the Optic Nerve in a Rodent Model.

Small molecule delivery to the optic nerve would allow for exploration of molecular and cellular pathways involved in normal physiology and optic neuropathies such as glaucoma, and provide a tool for screening therapeutics in animal models. We report a novel surgical method for small molecule drug delivery to the optic nerve head (ONH) in a rodent model. In proof-of-principle experiments, we delivered cytochalasin D (Cyt D; a filamentous actin inhibitor) to the junction of the superior optic nerve and globe in rats to target the actin-rich astrocytic cytoskeleton of the ONH. Cyt D delivery was quantified by liquid chromatography and mass spectrometry of isolated optic nerve tissue. One day after Cyt D delivery, anterior ONH filamentous actin bundle content was significantly reduced as assessed by fluorescent-tagged phalloidin labeling, relative to sham delivery. Anterior ONH nuclear counts and axon-specific beta-3 tubulin levels, as well as peripapillary retinal ganglion cell layer nuclear counts were not significantly altered after Cyt D delivery relative to sham delivery. Lastly, the surgical delivery technique caused minimal observable axon degeneration up to 10 days post-surgery. This small molecule delivery technique provides a new approach to studying optic neuropathies in in vivo rodent models.

Topography of the supraorbital nerve with reference to the lacrimal caruncle: danger zone for direct browplasty.

PURPOSE: To elucidate the course of the supraorbital nerve (SON) with reference to the lacrimal caruncle in order to facilitate safer direct browplasty by preventing nerve injury. METHODS: Thirty-four hemifaces from 18 embalmed Korean cadavers were dissected. A vertical line through the upmost point of the lacrimal caruncle and a horizontal line through the supraorbital margin were used as the horizontal and vertical reference positions, respectively. The course of the SON in the frontal view and the point at which it pierced the overlaying musculature were examined. RESULTS: The SON divides into a superficial branch and a deep branch just after exiting the orbit. In all cases, the deep SON remains in the subgaleal plane deep to the corrugator and frontalis muscles. The superficial SON travels under the corrugator muscle dividing into three branches (medial, intermediate and lateral) and pierced the frontalis muscle at 19-32 mm above the supraorbital margin. However, in 11 cases (32%) the medial branch of the superficial SON pierced the lower portion of the corrugator muscle at 3.6 mm above the supraorbital margin and ran in front of the muscle along with the vertical line through the upmost point of the lacrimal caruncle. CONCLUSIONS: One-third of the medial branch of the superficial SON without corrugator muscle protection is vulnerable to iatrogenic injury during direct browplasty. Therefore, the oculofacial surgeon must bring the dissection plane of the forehead tissue more superficially around the vertical line through the upmost point of the lacrimal caruncle in order to avoid nerve injury.

Effect of VIP on intracellular [Ca2+], extracellular regulated kinase 1/2, and secretion in cultured rat conjunctival goblet cells.

PURPOSE: To determine the intracellular signaling pathways that vasoactive intestinal peptide (VIP) uses to stimulate high molecular weight glycoconjugate secretion from cultured rat conjunctival goblet cells. METHODS: Goblet cells from rat bulbar and forniceal conjunctiva were grown in organ culture. Presence and localization of VIP receptors (VPAC1 and 2) were determined by RT-PCR, immunofluorescence microscopy and Western blot analysis. Intracellular [Ca(2+)] ([Ca(2+)]i) was measured using fura-2. Extracellular signal-regulated kinase (ERK)-1/2 activity was determined by Western blot analysis. High molecular weight glycoconjugate secretion was measured with an enzyme-linked lectin assay on cultured goblet cells that were serum-starved for 2 hours before stimulation with VIP, VPAC1-, or VPAC2-specific agonists. Inhibitors were added 30 minutes prior to VIP. Activation of epidermal growth factor receptor (EGFR) was measured by immunoprecipitation using an antibody against pTyr followed by Western blot analysis with an antibody against EGFR. RESULTS: Both VIP receptors were present in rat conjunctiva and cultured goblet cells. VIP- and VPAC-specific agonists increased [Ca(2+)]i and secretion in a concentration-dependent manner. VIP also increased ERK1/2 activity, VIP-stimulated increase in [Ca(2+)]i. Secretion, but not ERK1/2 activity, was inhibited by the protein kinase A inhibitor, H89. VIP-stimulated secretion was inhibited by siRNA for ERK2 but not by siRNA for EGFR. VIP did not increase the phosphorylation of the EGFR. CONCLUSIONS: In conclusion, in cultured rat conjunctival goblet cells, VPAC1 and 2 receptors are functional. VIP stimulates a cAMP-dependent increase in [Ca(2+)]i and glycoconjugate secretion, but not ERK1/2 activation. VIP does not activate with EGFR.

[Changes of the conjunctival nervous fillets in diabetic patients]. / Modificari ale filetelor nervoase in conjunctiva bolnavilor cu diabet zaharat.

PURPOSE: Our study suggested to reveal nervous fillets changes from conjunctiva structure in patients with diabetes. MATERIAL AND METHOD: We removed bulbar conjunctiva during cataract surgery on diabetic and non-diabetic patients from the same age group. The conjunctiva fragments were fixed in Lillie's solution, then studied by using optical microscopy with usual and histochemical stainings. RESULTS: Nervous fillets just like perinervium are better represented in younger diabetic patients without diabetic retinopathy or with early diabetic retinopathy and non-diabetic patients, too. In older patients and old diabetes, nervous fillets are much less in number with poor represented perinervium. CONCLUSIONS: Significant changes of the conjunctiva nervous fillets were found in patients with long-term diabetes.

A novel class of neurons at the trigeminal subnucleus interpolaris/caudalis transition region monitors ocular surface fluid status and modulates tear production.

Reflex tears are produced by many conditions, one of which is drying of the ocular surface. Although peripheral neural control of the lacrimal gland is well established, the afferent pathways and properties of central premotor neurons necessary for this reflex are not known. Male rats under barbiturate anesthesia were used to determine whether neurons at the ventral trigeminal subnucleus interpolaris- caudalis (Vi/Vc) transition or the trigeminal subnucleus caudalis-cervical cord (Vc/C1) junction region in the lower brainstem were necessary for tears evoked by noxious chemical stimulation (CO2 pulses) or drying of the ocular surface. Both the Vi/Vc transition and Vc/C1 junction regions receive a dense direct projection from corneal nociceptors. Synaptic blockade of the Vi/Vc transition, but not the Vc/C1 junction, by the GABA(A) receptor agonist muscimol inhibited CO2-evoked tears. Glutamate excitation of the Vi/Vc transition, but not the Vc/C1 junction, increased tear volume. Single units recorded at the Vi/Vc transition, but not at the Vc/C1 junction, were inhibited by wetting and excited by drying the ocular surface. Nearly all moisture-sensitive Vi/Vc units displayed an initial inhibitory phase to noxious concentrations of CO2 followed by delayed excitation and displayed an inhibitory surround receptive field from periorbital facial skin. Drying of the ocular surface produced many Fos-positive neurons at the Vi/Vc transition, but not at the Vc/C1 junction. This is the first report of a unique class of moisture-sensitive neurons that exist only at the ventral Vi/Vc transition, and not at more caudal portions of Vc, that may underlie fluid homeostasis of the ocular surface.

[Study of the nervous-vascular reactivity of the bulbar conjunctiva in patients with primary open-angle glaucoma with normalized intraocular pressure]. / Izuchenie nervno-sosudistoi reaktivnosti bul'barnoi kon"iunktivy glaza u bol'nykh pervichnoi otkrytougol'noi glaukomoi s normalizovannym vnutriglaznym davleniem.

Clinical evaluation of adrenoreception was carried out in 96 patients with various clinical patterns of primary open-angle glaucoma with normalized intraocular pressure by changes in vascular reactivity in the bulbar conjunctiva capillaries in response to epinephrine and norepinephrine. Neuro-vascular reactions of the bulbar conjunctiva in response to norepinephrine and epinephrine were different in patients with different patterns of primary open-angle glaucoma with normalized intraocular pressure. A significant correlation between the rate of progress of optic disk glaucomatous atrophy and the parameters of ocular adrenoreceptor structures of the eye was detected.

Presence of nerves and their receptors in mouse and human conjunctival goblet cells.

PURPOSE: To determine whether neural pathways for controlling goblet cell secretion are present in mouse and human conjunctiva. METHODS: Mouse conjunctiva was homogenized and subjected to electrophoresis and Western blotting to detect PGP 9.5 (indicates nerves), muscarinic receptor subtypes (indicates parasympathetic pathway), and adrenergic receptors (indicates sympathetic pathway). Mouse eyes and human conjunctival tissue were analyzed by immunofluorescence microscopy. Antibodies to vasoactive intestinal peptide (VIP), tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), and muscarinic and alpha(1)- and beta-adrenergic receptor subtypes were used. RESULTS: Western blot demonstrated PGP 9.5, M(1), M(2), and M(3) muscarinic receptors and alpha(1A)-, beta(1)-, beta(2)-, and beta(3)-adrenergic receptors in mouse conjunctiva. Immunoreactivity for VIP, TH, and DBH was found adjacent to mouse and human goblet cells. M(1) and M(2) muscarinic receptors were identified throughout mouse conjunctiva, but M(3) receptor was predominantly on goblet cells. All three muscarinic receptor subtypes were detected on goblet cells in human conjunctiva. alpha(1A)-Adrenergic receptors were found on epithelial cells and on goblet cells in mouse and human conjunctiva. beta(1)- and beta(2)-Adrenergic receptors were found on both epithelial and goblet cells in mouse conjunctiva, but not on human conjunctival cells. beta(3)-Adrenergic receptors were found on both epithelial and goblet cells in human conjunctiva but not on mouse conjunctival cells. CONCLUSIONS: The following conclusions were drawn: parasympathetic nerves and M(1), M(2), and M(3) muscarinic receptors, as well as sympathetic nerves are present on mouse and human goblet cells. The adrenergic receptors beta(1) and beta(2,) but not alpha(1A) and beta(3) are present on mouse conjunctival goblet cells, whereas alpha(1A) and beta(3,) but not beta(1) and beta(2) are present on human conjunctival goblet cells, suggesting that these nerves and receptors could activate goblet cell secretion in mouse and humans.

Activation of neurons in rat trigeminal subnucleus caudalis by different irritant chemicals applied to oral or ocular mucosa.

To investigate the role of trigeminal subnucleus caudalis in neural mechanisms of irritation, we recorded single-unit responses to application of a variety of irritant chemicals to the tongue or ocular mucosa in thiopental-anesthetized rats. Recordings were made from wide dynamic range (WDR) and nociceptive-specific units in superficial layers of the dorsomedial caudalis (0-3 mm caudal to obex) responsive to mechanical stimulation and noxious heating of the ipsilateral tongue ("tongue" units) and from WDR units in ventrolateral caudalis (0-2 caudal to obex) responsive to mechanical and noxious thermal stimulation of cornea-conjunctiva and frequently also surrounding skin ("cornea-conjunctival" units). The following chemicals were delivered topically (0.1 ml) onto the dorsal anterior tongue or instilled into the ipsilateral eye: capsaicin (0.001-1% = 3.3 x 10(-2) to 3.3 x 10(-5) M), ethanol (15-80%), histamine (0.01-10% = 9 x 10(-1) to 9 x 10(-4) M), mustard oil (allyl-isothiocyanate, 4-100% = 4 x 10(-1) to 10 M), NaCl (0.5-5 M), nicotine (0.01-10% = 6 x 10(-1) to 6 x 10(-4) M), acidified phosphate buffer (pH 1-6), piperine (0.01-1% = 3.5 x 10(-2) to 3.5 x 10(-4) M), serotonin (5-HT; 0.3-3% = 1.4 x 10(-1) to 1.4 x 10(-2) M), and carbonated water. The dose-response relationship and possible tachyphylaxis were tested for each chemical. Of 32 tongue units, 31 responded to one or more, and frequently all, chemicals tested. The population responded to 75.3% of the various chemicals tested (

Degenerative changes in unmyelinated nerve fibers in late-infantile neuronal ceroidlipofuscinosis. A morphometric study of conjunctival biopsy specimens.

Late-infantile neuronal ceroidlipofuscinosis (LINCL) is an autosomal recessive disease involving rapidly progressive myoclonic epilepsy, mental and motor regression and progressive visual failure. Neurodegeneration and deposition of fluorescent lipid bodies are the neuropathological hallmarks of this disease. In this study we examined the conjunctival biopsy (CB) specimens of three siblings and two unrelated patients with LINCL. At the time of examination each of three siblings presented a different stage of the disease. The unrelated patients were examined at an advanced stage of LINCL. The findings in these patients are compared to the normal data derived from analysis of seven age-matched 9- to 41-month-old healthy subjects. In young children with LINCL the distribution of unmyelinated fiber (UF) diameter is unimodal. In advanced disease there is a bimodal distribution and a significant reduction of UF density and of relative UF area. As the disease progresses, degenerative changes can be demonstrated: at first a diffuse UF swelling, followed by a decrease of UF density and finally the increase of regenerates (microaxons). These changes, however, seem to reflect an unspecific reaction to nerve injury. They can be demonstrated in a variety of conditions of different pathophysiology such as diabetes mellitus, crush injury and normal aging. This is the first morphometric study of CB specimens. Normal data of UF distribution (unimodal, mode at 0.4-0.6 micron) and UF density (1447,760 +/- 19,347/mm2) in CB specimens correspond well to age-specific data published on the sural nerve.

Localization of nerves adjacent to goblet cells in rat conjunctiva.

Neural stimulation of the cornea induces conjunctival goblet cell mucous secretion. Immunofluorescence microscopy was used to determine if nerves are present near conjunctival goblet cells and what types of nerves are present. In euthanized rats, the local anesthetic lidocaine (1%) was placed topically on the ocular surface for 10 min to prevent goblet cell mucous secretion. The ocular surface tissues were removed and either fixed in formaldehyde and then frozen, or frozen first and then post-fixed in formaldehyde. Tissue was sectioned and nerves localized by indirect immunofluorescence microscopy, using antibodies to synaptophysin (indicates nerve, independent of type), vasoactive intestinal peptide (VIP, indicates parasympathetic nerves), tyrosine hydroxylase (TH, indicates sympathetic nerves), dopamine beta-hydroxylase (DBH, indicates sympathetic nerves), phenylethanolamine-N-methyltransferase (PNMT, indicates sympathetic nerves), and calcitonin gene-related peptide (CGRP, indicates sensory nerves). Goblet cells were identified by phase-contrast microscopy. Synpatophysin-containing nerves were present in the basolateral region of conjunctival goblet cells clusters. Nerve fibers immunoreactive to VIP were found in the conjunctiva along the epithelial-stromal junction and around the basolateral aspect of goblet cell clusters. Nerve fibers immunoreactive to TH and DBH were detected surrounding goblet cells and in the conjunctival stroma. Nerve fibers immunoreactive to CGRP were detected in the epithelium and at the epithelial stromal junction, but were not localized near goblet cell clusters. CGRP-containing nerve fibers were also detected in the conjunctival stroma under the epithelium. We conclude that efferent parasympathetic and sympathetic, but not afferent sensory, nerves appear to be located adjacent to conjunctival goblet cell clusters. Activation of efferent parasympathetic and sympathetic nerves could directly stimulate conjunctival goblet cell mucous secretion. Antidromic activation of afferent sensory nerves releasing neurotransmitters could stimulate goblet cell secretion by a paracrine mechanism.

Neuronal pathways to the rat conjunctiva revealed by retrograde tracing and immunocytochemistry.

The origin and neuropeptide content of nerve fibres in the rat conjunctiva were studied by retrograde tracing and denervations in combination with immunocytochemistry. Immunocytochemistry revealed nerve fibres containing neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI), helospectin, substance P (SP), and calcitonin gene-related peptide (CGRP) around blood vessels, smooth muscle bundles and glands. Removal of the sphenopalatine ganglion eliminated the majority of the VIP-, PHI- and helospectin-containing fibres. Sympathectomy eliminated the majority of the NPY-containing fibres in the conjunctiva. Removal of both the sphenopalatine ganglion and the superior cervical ganglion led to further reduction of the NPY fibres. Injection of the retrograde tracer True Blue into the conjunctiva resulted in the appearance of labelled cell bodies in the trigeminal ganglion, the sphenopalatine ganglion, the superior cervical ganglion, and the ciliary ganglion. Judging from the number of labelled nerve cell bodies, the superior cervical ganglion contributes most to the innervation. These results indicate that the majority of NPY-containing nerve fibres in the conjunctiva derives from the superior cervical ganglion (sympathetic nerve supply). Most of the VIP- and a minor population of the NPY-containing fibres in the conjunctiva originate in the sphenopalatine ganglion (parasympathetic nerve supply). A sparse amount of VIP- and NPY-containing fibres derives from the ciliary ganglion. Nerve fibres containing SP and/or CGRP emanate from the trigeminal ganglia (sensory nerve supply). Together the findings indicate that several ganglia project to the conjunctiva and that many neuropeptides may be involved in the control of conjunctival activity.

Krause's end-bulb microtumor of the conjunctiva: optic and ultrastructural description of a case.

Ultrastructural examination of a conjunctival biopsy of a 90-year-old woman with a history of chronic lymphatic leukemia showed numerous densely packed structures located below the epithelial conjunctival layer. They were composed of concentrical flattened lamellae arranged around one or several clear cores containing a large number of mitochondria. The plasma membranes of the lamellae displayed large numbers of pinocytotic vesicles and resembled perineurial cell processes. The central areas were thought to be axons. Because of their conjunctional location and morphological features. The structures were categorized as nerve endings of the Krause's end-bulb type. The aberrant and profuse growth of these structures led to the diagnosis of Krause's end-bulb microtumor of the conjunctiva. We compare our findings with mucosal neuromas, paraneoplastic lesions and age-related alterations are discussed, although they differ morphologically from Krause's end-bulb microtumor.

Conjunctival nerve pathology in multiple endocrine neoplasia. A case report.

A 31-year-old male with typical clinical features of multiple endocrine neoplasia type 2b (MEN 2b) was subjected to conjunctival biopsy. The patient had previously been operated for pheochromocytoma and medullary carcinoma of the thyroid. He came for ophthalmologic consultation because of redness and irritation of the eye due to dry eye syndrome. The conjunctival biopsy taken from his left eye revealed not only an increase in the number of conjunctival nerves but also an increase of their axons. The ultrastructure of the nerves was mostly normal, but the perineurium was often incomplete, and the interaxonal space appeared to be enlarged. Immunohistochemically, SP-, neurofilament- and, unexpectedly, leucine-enkephalin-positive nerve fibres were demonstrated in the conjunctival stroma. The rat is the only species in which enkephalin-like immunoreactivity has been described in the anterior segment of the eye. Thus, the presence of enkephalin-positive nerves in the conjunctiva of our MEN 2b patient may reflect a profound neural alteration.

[Intraocular pressure and the trigeminal ganglion]. / Pression intraoculaire et ganglion trigéminal.

The way in which aqueous humor secretion and intraocular pressure are regulated is not well known. In order to shed light on the role played by the sensory nervous system in this regulation, hourly intraocular pressure measurements were made on patients who had undergone surgery of the first branch of the trigeminal nerve.

Vasoactive intestinal polypeptide immunoreactive nerve fibres in the human eye.

Immunohistochemistry applied to whole-mount preparations was used to investigate the presence and distribution of vasoactive intestinal polypeptide (VIP) immunoreactive nerves in the non-retinal part of the human eye. The choroid has a dense perivascular supply of VIP immunoreactive nerve fibers, and some free nerve endings within the stroma. These nerves enter the choroid in ciliary nerves and also as perivascular networks around the ciliary arteries. Occasional choroidal VIP immunoreactive nerve cell bodies are seen. The ciliary body stroma, close to the iris root has a dense circumferential plexus of VIP immunoreactive nerve fibers that occur both singly and in bundles. The iris root has a circumferential arrangement of bundles from which VIP immunoreactive nerve fibres travel radially in the stroma. They supply the pupillary region with numerous free nerve endings; the sphincter pupillae is not supplied by these nerves. The cornea is devoid of VIP immunoreactive nerves. These findings, together with existing knowledge of the physiological actions of VIP, indicate that VIP immunoreactive nerves are likely to be involved in the functioning of several ocular tissues.

Skin and conjunctival nerve biopsies in adrenoleukodystrophy and its variants.

This study demonstrates that skin or conjunctival biopsies are capable of diagnosing adrenoleukodystrophy and its variants. Electron microscopy of cutaneous nerve twigs in eleven patients showed characteristic curved clefts and leaflets in Schwann cells surrounding myelinated axons. In addition, three conjunctival biopsies were done, two of which were positive. The two peripheral nerve trunk biopsies performed also showed characteristic Schwann cell changes.

Electron microscopic examination of skin and conjunctival biopsy specimens in neuronal storage diseases.

Skin and conjunctival biopsy specimens from fourteen patients with neuronal storage diseases were investigated using an electron microscope. The diseases were Tay-Sachs disease, ceroid-lipofuscinosis (Jansky-Bielschowsky type), Niemann-Pick disease (type B), highly suspected adrenoleukodystrophy, I-cell disease, mucolipidosis of the beta-galactosidase deficient type, Hurler disease, Hunter disease and Morquio disease. This examination provided valuable diagnostic information on some neuronal storage diseases but not on Morquio disease or highly suspected adrenoleukodystrophy. False negative results may sometimes occur using this examination method. However, this examination suggests the usefulness of skin and conjunctival biopsy specimens as a diagnostic tool in some neuronal storage diseases.

Diagnosis of infantile neuroaxonal dystrophy by conjunctival biopsy.

Conjunctival biopsy and ultrastructural examination of conjunctival nerves, showing the presence of spheroids within axons, led to the confirmation of the diagnosis of infantile neuroaxonal dystrophy in two children with progressive mental deterioration. Conjunctival biopsy, which is simple to perform, even in young children, and does not require general anaesthesia or admission to hospital, is presented as a reliable and very convenient technique for the diagnosis of infantile neuroaxonal dystrophy.