Pituitary adenylate cyclase-activating peptide-immunoreactive nerve fibers in the cat eye

Purpose: To study the occurrence and distribution of the neuropeptide pituitary adenylate cyclase-activating peptide (PACAP) in ocular and orbital structures of the cat. Methods: Immunocytochemistry to localize PACAP and double immunostaining to detect co-localization of PACAP with other neuropeptides. Results: Numerous PACAP-immunoreactive nerve fibers were observed in the lacrimal gland, choroid and retroocular arteries. There was a sparse supply of PACAP-containing nerve fibers in the iris, ciliary body and conjunctiva. Subpopulations of PACAP-containing nerve fibers stored vasoactive intestinal peptide (VIP) or calcitonin gene-related peptide (CGRP). Around 10% of the ganglion cells in the sphenopalatine ganglion harbored PACAP immunoreactivity. In the trigeminal ganglion around 5% of the neuronal cell bodies and in the ciliary ganglion only occasional ganglion cells contained PACAP immunoreactivity. PACAP immunoreactivity co-localized with VIP in the sphenopalatine ganglion and with CGRP in the trigeminal ganglion. Conclusion: PACAP-containing nerves in the eye and associated structures demonstrate a distribution pattern resembling that of VIP. Subpopulations of nerve fibers containing PACAP immunoreactivity store VIP or CGRP immunoreactivity. Neuronal PACAP in the eye and orbit may take part in regulation of smooth muscle tone, glandular secretion and sensory processing.     

Immunocytochemical and ultrastructural evaluation of the distribution of nervous tissue and neuropeptides in the meibomian gland

Background: The ultrastructure of the meibomian gland, of its innervation and the localization of neuropeptides in the glandular tissue of the guinea pig and humans are incompletely known. Therefore they have been investigated in the present study. Methods: The ultrastructure of the tissue was examined using standard transmission electron microscopic techniques. Additional scanning electron microscopy was carried out on rabbit tissue. Antisera against the neuronal marker protein gene product were used to demonstrate the distribution pattern of the nerve fibers. The neuropeptides substance P (SP) and neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP) and calcitonin gene-related peptide (CGRP) and the neuronal enzyme tyrosine hydroxylase (TH) were identified by their specific antisera. Results: The glands were found to be composed of arrays of alveoli. The outer cells of the alveoli form a germinal layer. Toward the inside of the alveolus the cells are laden with a secretory substance. The cells disintegrate as they approach the excretory duct. Nerve fibers from a plexus around the alveoli. These nerve fibers form synapses à distance to the basal alveolar cells and enter the basal lamina of the capillaries. In guinea pigs many nerve fibers were positive for the neuropeptides SP and NPY and for VIP, and fewer for CGRP and TH; in humans only SP and CGRP were demonstrated. Conclusion: Both the density of nerve fibers and the presence of various neuropeptides suggest that the stimulation of the meibomian gland is subject to nervous control.Presented at the Joint European Research Meetings in Ophthalmology and Vision (JERMOV), October 1995, Montpellier, France     

Multiple ocular impairment in a patient affected by Waldenström's macroglobulinaemia

Background: We studied a bilateral tumefaction of the lacrimal gland in a female patient. Method: Ocular and general clinical examinations were carried out. Result: Computerized tomography (CT) of the cranial orbit showed a tumefaction of solid density in the lacrimal gland. Histological examination of material removed by needle aspiration revealed the presence of elements of a lymphoplasmacytoid nature. Fluorescein angiography showed dilatation of the veins, intraretinal flame haemorrhages and small ischaemic areas. Chest CT showed an increase in the size of the middle and upper mediastinal lymph nodes, and examination of a specimen of bone marrow from the chest revealed the presence of small lymphocytes with a plasmacytoid tendency. Conclusion: On the basis of the findings, we diagnosed Waldenström's disease with rare multiple ocular impairment (lacrimal gland and retina) in an early stage.     

The ultrastructure of parapapillary chorioretinal atrophy in eyes with secondary angle closure glaucoma

Background: The present study was performed to investigate the ultrastructure of deep retinal layers and choroid corresponding to the parapapillary chorioretinal atrophy in eyes with secondary angle-closure glaucoma. Methods: The glaucomatous eyes included two eyes enucleated due to iris ring melanoma with high intraocular pressure and one eye with neovascular glaucoma enucleated due to ocular pain. The control eyes included one eye enucleated due to choroidal malignant melanoma with normal intraocular pressure and one eye enucleated during surgery for supramandibular carcinoma. These eyes were studied with light and electron microscopy. Results: In the region of parapapillary chorioretinal atrophy of glaucomatous eyes, the retinal pigment epithelial cells showed degenerative changes, such as loss of basal in foldings and microvilli, degenerated mitochondria, vacuolar degeneration and irregular distribution of melanin granules. The photoreceptors were decreased in number in this area of glaucomatous eyes. The lumen of the choriocapillary vessels adjacent to the optic nerve was collapsed. Conclusion: These results elucidate the fine structures of deep retina and choroid in the region of parapapillary chorioretinal atrophy of glaucomatous eyes, and suggest that the reduced choroidal perfusion might be the pathogenetic mechanism of glaucomatous parapapillary chorioretinal atrophy.     

Immunohistochemical localization of epidermal growth factor receptor and epithelial antigen in tumors of the human conjunctiva,eyelid, lacrimal gland,and orbit

Background: Increased numbers of epidermal growth factor (EGF) receptors are observed in squamous cell carcinomas of human lung, head, neck, and cervix. We studied the presence of EGF receptors and epithelial antigen in some ophthalmic lesions. Methods: Immunohistochemical staining for EGF receptors was assessed in tumors of human conjunctiva, eyelid, lacrimal gland, and orbit with monoclonal antibodies (EGF-R1 and clone 29.1). Reactivity of Ber-EP4, which reocgnizes epithelial antigen, was also examined. Results: Strong staining of EGF-R1 and clone 29.1 and weak to moderate staining of Ber-EP4 were demonstrated in conjunctival squamous cell carcinomas. Cell membranes of conjunctival papilloma were moderately or strongly stained with these antibodies. Ductal components in sebaceous gland adenoma of the eyelid and pleomorphic adenoma of the lacrimal gland were positively stained. The antibodies did not bind to reactive lymphoid hyperplasia of the orbit and Wegener's granulomatosis. Relatively good correlation for immunostaining reaction was observed among EGF-R1, clone 29.1, and Ber-EP4 in each tumor. Conlcusion: Immunostaining using EGFR1, clone 29.1, and Ber-EP4 may be useful in differentiating epithelial tumors from non-epithelial lesions. Strong immunostaining for EGF receptor may be the hallmark of epidermoid malignancy.     

Management of fibrous histiocytoma of the corneoscleral limbus: Report of a case and review of the literature

Background: Fibrous histiocytomas of the corneoscleral limbus are rare tumors. We present an additional case and review the treatment approaches in the literature. So far, only light and electron microscopic studies have been performed. We used immunohistochemical stains to further characterize the cellular composition. Methods: The excised lesion was routinely fixed and processed for light microscopy and immunohistochemical studies. For comparison, three dermal fibrous histiocytomas were also examined and processed similarly. Results: The corneolimbal tumor was mainly composed of fibroblasts and histiocytes with a large amount of interstitial collagen, arranged in a storiform pattern. Immunohistochemical stains were positive for histiocytes, fibroblasts, and a marker for mesenchymal cells. The staining pattern of the dermal lesions was similar. Conclusion: Fibrous histiocytomas of the corneolimbal region are morphologically benign, slowly growing and infiltrative tumors. Complete resection, especially of the deep margin, is suggested. The immunohistochemical staining pattern is similar to that of dermal fibrous histiocytomas, which behave in a benign manner.     

HLA antigens in a Japanese family with Behçet's disease

Background: Familial Behçet's disease is rare. Methods: HLA antigens in a Japanese family with Behçet's disease were examined. Results: The affected patients had HLA B51, and unaffected family members also had the same antigen. Conclusion: It is likely that not only HLA B51 but also other factors may be involved in the pathogenesis of Behçet's disease in Japanese patients.     

Course of the optic nerve fibers through the lamina cibrosa in human eyes

Background: This study investigated whether regional variations in the course of the optic nerve fibers through the lamina cribrosa may be one of the reasons why the local susceptibility for glaucomatous optic nerve fiber loss differs among the various regions of the optic disc. Methods: The study included 34 human eyes enucleated because of a malignant melanoma of the peripheral choroid without involvement of the anterior chamber angle or the optic nerve. Anterior-posterior sections through the pupil and the optic disc were histomorphometrically evaluated. In the central region and the peripheral part of the optic disc, we measured the thickness of the lamina cribrosa and the length of the lamina cribrosa channels through which the nerve fibers pass. Results: In the peripheral parts of the disc, compared with its central region, the lamina cribrosa was significantly thicker (P<0.0001, Wilcoxon test), the lamina cribrosa channels with the nerve fibers passing through were significantly longer (P<0.0001), and the ratio of length of the fiber channels to the thickness of the lamina cibrosa was significantly higher (P=0.0001). Conclusion: The lamina cribrosa is thicker and the course of the optic nerve fibers through the lamina cribrosa is more curvilinear in the disc pheriphery than in the disc center. These variations in the anatomy of the lamina cribrosa may be one of several factors influencing the regional susceptibility for glaucomatous optic nerve fiber loss within the optic nerve head.     

Histopathology of episcleral fibrosis after trabeculectomy with and without mitomycin C

Background: The aim of the study was to investigate the histopathologic features of scar tissue which have proliferated at the site of trabeculectomy of surgical failures after procedures with and without the use of the antimetabolite mitomycin C (MMC). Methods: We obtained seven surgical specimens after trabeculectomy without MMC and five specimens after trabeculectomy with MMC, which were compared with 23 controls. Sections were stained with hematoxylin and eosin, Verhoeff-van Gieson, Grocott methenamine silver, and alcian blue. An immunohistochemical stain was performed for -smooth muscle actin. Results: Specimens from eyes operated without MMC showed dense scar tissue with many fibroblasts, much ground substance, parallel-oriented collagen fibers, and contractile intracellular proteins within the fibroblasts. Specimens from eyes operated with MMC consisted of tissue with only few fibroblasts which did not exhibit contractile proteins. Collagen fibers were arranged randomly with less ground substance. Conclusion: Even after 1–10 months, the scar tissue was distinctly different in the two groups. These results suggest that the use of MMC has long-term effects in vivo. Surgical failures related to scar formation are possible and not reduced to zero.Presented in part at the Association for Research in Vision and Ophthalmology (ARVO) Meeting, Fort Lauderdale, USA, 14–19 May 1995     

Morphometry of the optic nerve and retinal vessels in children by computer-assisted image analysis of fundus photographs

Background: The retinal fundus in childhood has a different morphology than in adulthood. Existing methods are not suitable for evaluation of fundus photographs from children. Therefore, a new method for quantitative analysis of fundus morphology utilizing a personal computer-assisted digital mapping system was developed. Methods: A CCD flatbed scanner is used to digitize fundus photographs, producing computer images which are analyzed on an IBM/AT computer. Area measurements of the optic disc, excavation and peripapillary crescent are made, as well as determinations of the length, branching, tortuosity and distribution of the retinal vessels on the fundus surface. Results: Determination of the inter-and intra-observer variability of the computer-assisted image analysis technique demonstrated good reproducibility. The method is demonstrated using fundus photographs of six normal children and six children with the fetal alcohol syndrome. Typical variations in appearance of optic disc and retinal vessels are seen. Conclusion: The system is unique in measuring both the optic nerve head and the retinal vessels and is therefore especially useful for detailed studies of normal and abnormal development of these structures in children.     

Immunohistochemical study of leucine-enkephalin and its secretory effects in the isolated pig lacrimal gland

Background: In our previous studies immunohistochemical studies have demonstrated the presence of leucine-enkephalin (Leu-Enk) in the intrinsic nerves of the pig lacrimal gland, which are discernible in the interlobular and interacinar areas from where branches that innervate the acinar tissues are sent. Since the intrinsic nerves have been shown to contain Leu-Enk, this study aimed to investigate the secretory effects of this neuropeptide in isolated segments of the pig lacrimal gland and to reconfirm its presence in the neuronal tissue of the lacrimal gland. Methods: Leu-Enk was identified using immunohistochemical techniques, while total protein output was measured in the effluent samples by an automated on-line colorimetric method. The Ca²⁺ and Mg²+ concentrations in the effluent samples were determined using an atomic absorbance spectrophotometer. Results: Leu-Enk (10^–12 – 10^–7 M) evoked marked increases in total protein output from superfused lacrimal gland segments. The secretory effect of Leu-Enk was not blocked by pretreatment of the tissue with atropine but was substantially reduced by a combination of phentolamine and propranolol. The competitive antagonist, naloxone, has no effect on basal protein output, but when combined with Leu-Enk it caused a significant reduction in total protein output. Combining theophylline (10^–3 M) with Leu-Enk resulted in a marked potentiation of total protein output. In superfused lacrimal gland segments Leu-Enk (10^–8 M) evoked a net efflux of magnesium (Mg²⁺ release) and a net influx of calcium (Ca²⁺ uptake). Conclusion: The results indicated a physiological role for Leu-Enk in the regulation of protein secretion in the pig lacrimal gland.     

The use of tissue plasminogen activator in post-traumatic total hyphaema

Background: We wanted to evaluate whether intracameral injection of tissue plasminogen activator (tPA) is useful in managing traumatic hyphaema. Methods: Two eyes with total hyphaema after a severe penetrating injury were treated with a single intracameral injection of 25 g of tPA 5 and 14 days after the injury, respectively. Results: Most of the blood coagulum dissolved within 24 h, and in one of the two eyes the intraocular pressure decreased from 45 to 8 mmHg. The other eye was hypotonic. No re-bleeding or complications related to the use of tPA were noticed. Conclusion: The results in these two cases suggest that tPA is a useful adjunct in managing total hyphaema.     

Effect of histamine and substance P on the rabbit and human iris sphincter muscle

Background and methods: In an attempt to clarify the functional action of histamine and substance P on atropine-resistant miosis, we isolated rabbit and human iris sphincter muscles and investigated their mechanical properties using the isometric tension recording method. Results: Substance P dose-dependently contracted the rabbit iris sphincter, but had no effect on the human iris sphincter. In the rabbit iris sphincter, histamine reduced the amplitude of twitch contraction evoked by field stimulation but had no effect on carbachol-induced contraction. Thioperamide, but not mepyramine or cimetidine, partially antagonized the histamine-induced reduction in the amplitude of twitch contractions. In the human iris sphincter, on the other hand, histamine dose-dependently provoked contraction and the amplitude of histamine-induced contraction was affected neither by atropine nor by indomethacin. Conclusions: These results provide evidence that histamine has strong contractile effect on the human iris sphincter muscle; the rabbit iris sphincter muscle, however, apparently lacks functional histamine receptors. In rabbits, exogenously applied histamine only activates H₃ receptors located on the cholinergic nerve terminal, hence the excitatory neuro-effector transmission is suppressed. Thus, histamine may have an important roles in atropine-resistant miosis in humans, but not in rabbits.     

Tissue expression of lipocalins in human lacrimal and von Ebner's glands: colocalization with lysozyme

Background: Tear-specific prealbumin is a group of proteins recently renamed as the tear lipocalins. These proteins were initally described as unique to lacrimal fluid. The tissue distribution and localization have never been thoroughly studied. Methods: The distribution of purified tear lipocalins was studied in many human secretions and tissues by western blots, immunohistochemistry and immunoelectron microscopy. Results: Tear lipocalin species of the same molecular weights were observed in western blot lanes loaded with tears, saliva, and protein extracts from the lacrimal and lingual von Ebner's glands. Lacrimal and von Ebner's glands contained tear lipocalins; other human tissues and secretions, including other salivary glands and taste buds, did not. Tear lipocalins colocalized with lysozyme in serous acinar cells of lacrimal and von Ebner's glands. Ultrastructurally, tear lipocalins were present on polyribosomes, endoplasmic reticulum, and Golgi areas. Lipocalins were concentrated in lacrimal secretory granules in amounts commensurate with a regulated pathway. Conclusion: Tear lipocalins are expressed and truncated similarly in lingual von Ebner's and lacrimal glands, but not at all in other human tissues. Lipocalins are expressed and secreted with lysozyme. Lipocalins are concentrated in secretory granules in an amount consistent with a regulated secretory pathway.     

Adaptation to monocular torsion after macular translocation

Purpose: To document the functional outcome of two patients following successful macular translocation for the treatment of severe subretinal hemorrhage in age-related maculopathy. Methods: The retina was surgically rotated around the optic nerve with translocation of the fovea either upward or downward to an area of healthy retinal pigment epithelium. In the postoperative period, visual function was carefully studied with emphasis on adaptation to torsion. Results: Visual acuity in one patient improved from 2/200 to 20/80 and the other patient remained at 20/200. Both patients developed horizontal and vertical strabismus with torsion of up to 55°. After a prolonged period of occlusion of the unoperated eye, both patients were subjectively able to adapt to monocular torsion. However, adaptation under binocular conditions did not occur. Conclusion: Macular translocation was successful in improving visual acuity in one patient, with no improvement in the second. Both patients had significant ocular torsion and strabismus, but under monocular conditions they were successful in perceptually adapting to the change in the visual environment. Fear of cyclotorsion should not be a deterrent to considering macular translocation as a possible treatment option for severe subretinal macular hemorrhage if the patient is willing to accept monocular vision.     

Butterfly-shaped pigment dystrophy of the fovea associated with subretinal neovascularization

Background: The association of butterfly-shaped pigment dystrophy of the fovea, an uncommon inherited macular disease, with subretinal neovascularization has rarely been reported in the literature. Case report: We describe the clinical history of a patient affected with butterfly-shaped pigment dystrophy of the fovea, myopia, and optic nerve head dysplasia. She was followed up for 23 years. During the course of the disease, bilateral subretinal neovascularization in the macular area occurred. Fluorescein angiography confirmed the diagnosis. Recently, indocyanine green (CG) videoangiography was also performed. Because of the bilateral subfoveal localization no laser treatment was advised. Discussion: Usually, good visual acuity is maintained in this uncommon inherited macular disease. However, acute visual loss can be caused by the ingrowth of subretinal new vessels. Therefore, if visual acuity decreases or metamorphopsia develops in these patients, careful biomicroscopic examination and fluorescein/ICG angiography is advisable.     

The effect of high-dose methylprednisolone on laser-induced retinal injury in primates: an electron microscopic study

Background: Previously we reported an ameliorative effect of high-dose methylprednisolone in laser injury to monkey retinas. The ultrastructural modification by methylprednisolone has not been examined. Methods: Cynomolgus monkeys were given severe kgrade III) retinal laser burns and treated with an intravenous megadose of methylprednisolone. Pathologic features of the retinal lesions with or without methylprednisolone treatment were evaluated by light and electron microscopy. Results: Ultrastructurally, the treated lesions showed rapid recanalization of choriocapillaris; proliferation of retinal pigment epithelium to replace the necrotic and damaged cells, resulting in rapid re-establishment of blood retinal barrier; mild macrophagic activity; and rapid reformation of the outer limiting membrane by Mueller cells. Conclusion: A high dose of methylprednisolone affected the responses of the choriocapillaris, retinal pigment epithelium, photoreceptor cells and Mueller cells to laser injury, showing an overall beneficial effect. These modifications might be ascribed to methylprednisclone's anti-inflammatory action, protection of the microcirculation and anti-lipid peroxidation effect. Proprietary interest: none     

An immunocytochemical study of isolated human retinal Müller tells in culture

Background: We report a modified method for the isolation and propagation of adult human Müller cells in culture. Methods: The retina of postmortem human eyes was mechanically dissociated and cultured. Using immunocytochemical techniques, these cells were stained with monoclonal antibodies specific for Müller cells, glial fibrillary acidic protein (GFAP), vimentin, glutamine synthetase (GS) and keratin. Transmission electron microscopy (TEM) was also performed. Results: The dissociated and cultured cells expressed vimentin and GS, but not GFAP. At least 85% of these cells stained with a Müller tell-specific monoclonal antibody. Using TEM, flat cells containing 13-nm intermediate filaments and glycogen were identified. Conclusion: Human retinal Müller cells tan be isolated and propagated in culture. Purified cell cultures are required for controlled studies of the normal physiology and pathologie responses of Müller cells.     

Optic nerve fiber count and diameter of the retrobulbar optic nerve in normal and glaucomatous eyes

Background: The caliber of the retrobulbar optic nerve and the count of optic nerve fibers vary considerably in normals. The diameter of the retrobulbar optic nerve also decreases with optic nerve atrophy. This study aimed to determine the relationship between the caliber of the optic nerve and the optic nerve fiber count. Methods: We counted the optic nerve fibers and measured the diameter of histological cross sections of the optic nerve for 56 normal subjects and 23 patients with absolute glaucoma. Results: The optic nerve fiber count increased significantly (P<0.0001) by 777 000 fibers for every millimeter increase in retrobulbar optic nerve diameter, starting at a baseline diameter of 1.89 mm. Conclusion: Using this linear regression equation, the optic nerve fiber count can be estimated in routine histology by measuring the optic nerve diameter. Taking into account a fixation-induced tissue shrinkage, this method may also give some indication of the optic nerve fiber count intravitally, when, for eyes with opaque optic media, the diameter of the retrobulbar optic nerve has been measured by imaging techniques. Greater retrobulbar optic nerve caliber may indicate greater structural reserve capacity.     

Employment of bioluminescence for the quantification of adenosine phosphates in the human cornea

Background: Quantification of adenosine phosphates in human corneal extracts has been performed using spectrophotometry. We employed the bioluminescence technique to obtain a more sensitive assay for adenosine phosphates and to reduce the volume of the test sample. Methods: The bioluminescence assay for ATP, already known from sterility control, was modified and expanded. Standard curves were established using a standard solution with equimolar concentrations of ATP, ADP and AMP. To monitor the method, adenosine phosphates were measured in 35 human corneal extracts using both spectrophometry and bioluminescence. Results: Linear standard curves ranging from 1 to 45 pmol were established. The two methods yielded comparable results despite the use of a basic dilution of 1: 100 for the new technique. Conclusion: Bioluminescence provides a highly sensitive quantification of adenosine phosphates in the human cornea and facilitates an extremely detailed evaluation of the metabolic status of the cornea.Dedicated to Prof. Martin Reim on the occasion of his 65th birthday