Combined effect of extracellular matrices and growth factors on bovine corneal endothelial cells in culture.

PURPOSE: First, to confirm that corneal endothelial cells in the confluent state have the capability to form cellular covering. Second, to establish a method to study the combined effect of extracellular matrices (ECMs) and growth factors on the biological response in corneal endothelial cells in culture. METHODS: Bovine corneal endothelial cells were cultured inside a cylinder set on a plastic dish. They formed a confluent cell nest on the dish coated with type I or type IV collagen, laminin, or fibronectin. After the removal of the cylinder, hepatocyte growth factor (HGF), epidermal growth factor, transforming growth factor-alpha or transforming growth factor-beta(1) was added to the cultures. Each confluent cell nest enlarged outward, and its increased area size was measured. Cellular response in the nest, including cellular proliferation, was analyzed. RESULTS: The size of the increased area of the culture on type IV collagen plus HGF was the largest of all the combinations of ECMs and growth factors. The responses of component cells in the increased area consisted of cellular hypertrophy, proliferation, migration and giant cell formation. The treatment with type IV collagen plus HGF clearly promoted all the above responses. CONCLUSIONS: The biological response of corneal endothelial cells was regulated by ECMs and growth factors.

Retinal disease in patients with systemic lupus erythematosus.

OBJECTIVE: To investigate the incidence of retinopathy in systemic lupus erythematosus (SLE) and to clarify its significance in relation to other clinical manifestations. METHODS: A cross sectional study on lupus retinopathy was made in 69 patients with SLE. One expert ophthalmologist examined the ocular fundi of the lupus patients without any information of their disease state. Clinical and laboratory findings in the patients with retinopathy and those without were compared. RESULTS: Retinopathy was found in 7/69 (10%) patients. The findings included haemorrhages, vasculitis, cotton wool spots, and hard exudates, all of which were considered to reflect vascular damage. Retinopathy was found to be associated with the presence of anticardiolipin antibody (p<0.05) and with central nervous system lupus (p<0.01). The patients with retinopathy had higher levels of serum creatinine than the patients without retinopathy (p<0.01). The disease activity of lupus, as assessed by the maximum SLE disease activity index (SLEDAI) score of the patients, was also significantly higher in the patients with retinopathy (p<0.03). CONCLUSION: Incidence of retinopathy in SLE was similar to that in previous reports and it may reflect tissue microangiopathy, particularly associated with vasculitis or anticardiolipin antibodies, or both.

Traumatic Horner syndrome without anhidrosis.

In a patient with a traumatic avulsion of the arm, magnetic resonance imaging showed the exact site of the lesion that produced Horner syndrome with preservation of sweating on the face.

Multicenter clinical trial for evaluating methylprednisolone pulse treatment of idiopathic optic neuritis in Japan. Optic Neuritis Treatment Trial Multicenter Cooperative Research Group (ONMRG).

BACKGROUND: A randomized, controlled clinical trial was conducted in 1991 to compare an intravenous megadose of methylprednisolone with a control drug (mecobalamin) for treating acute idiopathic optic neuritis. CASES: Sixty-six cases from 22 clinical centers throughout Japan were examined to evaluate the treatment on visual function parameters, such as visual acuity, visual field, color vision, contrast sensitivity, and critical flicker frequency. OBSERVATIONS: The methylprednisolone pulse treatment group showed faster recovery of visual function, particularly the visual acuity at 1 week (P<.05), Humphrey field analyzer mean deviation at 3 weeks (P<.05), and color vision at 1 week (P<.05). Recovery of contrast sensitivity at several different spatial frequencies was significant in the pulse treatment group at 1 (P<.01), 2 (P<.05), and 4 weeks (P<.05) after the start of treatment. Visual function test results at 12 weeks and 1 year were essentially the same in the two treatment groups. Side effects appeared more frequently in the pulse treatment group than in the control (P<.05). CONCLUSIONS: Pulse treatment does not appear effective for idiopathic optic neuritis even though visual function in the pulse treatment group of this trial recovered more quickly during the initial phase compared to the controls. More effective and specific treatment should be established for optic neuritis.

Baseline features of idiopathic optic neuritis as determined by a multicenter treatment trial in Japan. Optic Neuritis Treatment Trial Multicenter Cooperative Research Group (ONMRG).

BACKGROUND: An optic neuritis treatment trial was conducted at 30 clinical centers in Japan using the same protocol. Patient participation was based on: age range of 14-55 years; acute symptoms indicative of unilateral optic neuritis of unknown or demyelinating origin; visual symptoms of 14-day duration or less; relative afferent pupillary defect in affected eye; and normal or swollen optic disc of affected eye. CASES: Initially, 102 patients qualified for participation; baseline data were obtained for analysis from 70 of these patients. Demographic characteristics of Japanese patients with optic neuritis were clarified and compared with those in a US study. OBSERVATIONS: The incidence of ocular or periocular pain and the presence of periventricular plaques were noted to be lower, and the incidence of disc swelling higher, in the Japanese patients, suggesting racial differences in the characteristics of the disease. Such differences may possibly be related to the lower incidence of multiple sclerosis in Japanese patients. The results of visual function tests were virtually the same in both studies. The nonaffected eyes of more than half the patients showed abnormal mean deviation in Humphrey field analysis, as also noted in the US study. CONCLUSIONS: The baseline clinical features of optic neuritis in the Japanese patients have been defined. Some racial differences in the characteristics of the disease may exist.

Effects of hepatocyte growth factor, transforming growth factor-beta1 and epidermal growth factor on bovine corneal epithelial cells under epithelial-keratocyte interaction in reconstruction culture.

In the cornea, corneal epithelial cells are in close contact with keratocytes: the epithelial cells organize thickened lamellar structure on a layer of keratocytes embedded in extracellular matrix (ECM). Thus, growth factors are expected to critically regulate corneal component cells under epithelial-keratocyte interaction. The purpose of this study is to clarify effects of hepatocyte growth factor (HGF), transforming growth factor-beta1 (TGF-beta1) or epidermal growth factor (EGF) on corneal epithelial cells under epithelial-keratocyte interaction. We examined proliferation and differentiation of the epithelial cells in a simple corneal reconstruction culture composed of an epithelial cell layer on the keratocyte-containing stromal layer, using three-dimensional collagen gel matrix culture. We observed the morphological change by phase contrast microscopy, and conducted histological and immunohistochemical examinations. The epithelial proliferation was examined by nuclear bromodeoxy-uridine (BrdU) uptake. In the reconstructed cornea under epithelial-keratocyte interaction, EGF-, TGF-beta1- and HGF-treated cells formed a thickened epithelial layer consisting of 5-6, 5-6 and 3-4 cells, respectively. In fact, both EGF and TGF-beta1 induced significantly higher intakes of nuclear BrdU of the epithelial cells than HGF. In lamellar differentiation of the epithelial cells, TGF-beta1- or HGF-treated cells formed a triple lamellar structure specific for the in vivo corneal epithelium: basal, middle and superficial layers are composed of cuboidal basal-like cells, spindle wing-like cells and flat superficial-like cells, respectively. TGF-beta1-treated cells formed a more markedly thickened epithelial layer than HGF-treated cells. In contrast, EGF formed a single lamellar structure consisting of cuboidal cells. These results suggest that those growth factors regulate proliferation and/or lamellar differentiation of corneal epithelial cells under epithelial-keratocyte interaction. The most interesting result was that TGF-beta1 promotes proliferation and lamellar differentiation of corneal epithelial cells through keratocyte-mediated stimulation.

Histopathological study of the effect of phacoemulsification-aspiration on iris muscles.

Phacoemulsification-aspiration (PEA) followed by peripheral iridectomy was performed in 10 patients, and the dissected iris fragments were examined by electron microscopy. Organic changes such as cytoplasmic vacuolation and lamellar formation were observed in both the sphincter and dilator muscles of the pupil. No changes in the axons were detected. The same findings were observed when a simian eye was examined after PEA. These results indicate that PEA causes injury to iris muscles.

The effects of phaco-emulsification-aspiration and intra-ocular lens implantation on the pupil: pupillographic and pharmacologic study.

The authors report postoperative change in pupil diameter and pupil dynamics after phaco-emulsification-aspiration and intra-ocular lens implantation by comparing the operated eye and non-operated eye in 20 patients who received unilateral surgery. Miosis was noted in the operated eye throughout the postoperative period in the dark, and also in the light at an early stage after the operation. Both the maximal constriction velocity and maximal dilatation velocity of the light reflex decreased postoperatively, but the constriction velocity alone was accelerated 1 or 2 weeks after the operation. In the drug instillation test, pupillary responses to both neosynephrine and pilocarpine were decreased, indicating damage both to the dilator and sphincter muscles. Since no hypersensitivity of the pupil was noted, lesions in the axonal fibres and denervation were judged to be absent. Postoperative miosis is believed to be explained by irreversible damage to the dilator muscle, as well as by the transient effects of inflammatory substances.

Occurrence of Borrelia garinii oculo-neuroborreliosis in Japan.

We report a 64-year-old Japanese man with oculo-neuroborreliosis. His clinical features consisted of polyarthralgia, keratoconjunctivitis, chorioretinitis, optic neuritis, confusion, and polyradiculitis. Assay of antibodies to Borrelia species detected IgG-antibody to B. garinii in both serum and CSF. Progressive declining of serum IgG antibody titer against Borrelia garinii, in parallel with clinical improvement, was observed after administration of ceftriaxone.

Seronegative myasthenia gravis associated with atonic urinary bladder and accommodative insufficiency.

We report a 20-year-old female with generalized myasthenia gravis (MG) who developed atonic urinary bladder and accommodative insufficiency. Although her sera did not contain antibodies to either nicotinic acetylcholine receptor (AChR) or voltage-gated calcium channels, a positive intravenous edrophonium test and a waning phenomenon on electromyographic studies indicated a diagnosis of seronegative MG. Myasthenic symptoms as well as urinary incontinence and impaired near vision disappeared with slight sequelae after corticosteroid therapy and total removal of the hyperplastic thymus. These symptoms recurred during a tapering course of corticosteroids, and improved again with an increased dose. Therefore, in this patient, the neuromuscular transmission of muscarinic type of AChR in the smooth muscles was also affected. This patient provides insight into the mechanisms by which some smooth muscles are involved in MG.

Hyperosmolality rapidly reduces atrial-natriuretic-peptide-dependent cyclic guanosine monophosphate production in cultured rat inner medullary collecting duct cells.

The present study was undertaken to explore the acute effect of hyperosmolality on the response of cultured rat inner medullary collecting duct (IMCD) cells to atrial natriuretic peptide (ANP). In contrast to the stimulatory effect of chronic incubation (12 h) in hypertonic medium, it was found that short-term incubation (< 2 h) reversibly suppressed the ANP-dependent cyclic guanosine monophosphate (cGMP) production. Urea, NaCl and mannitol were equi-potent as the osmolyte in suppressing the ANP-dependent cGMP production. Receptor binding assay revealed that hyperosmolality induced a rapid and marked reduction of the maximum binding (Bmax) of ANP without a significant change of the dissociation constant (Kd). Pretreatment with protein kinase C inhibitors (calphostin-C, staurosporin) or with cytoskeleton modulators (cytochalasin-B, colchicine) did not affect the inhibitory effect of hyperosmolality. In conclusion, acute hypertonicity inhibited the ANP-induced cGMP production in contrast to chronic hypertonicity, and reduction of the number of ANP binding sites was considered to be a mechanism responsible for the inhibitory effect of hypertonicity.

[Incidence of acute idiopathic optic neuritis and its therapy in Japan. Optic Neuritis Treatment Trial Multicenter Cooperative Research Group (ONMRG)].

Data on the incidence of and treatment for acute idiopathic optic neuritis were obtained by questionnaire sent to departments of ophthalmology, university hospitals, and general hospitals throughout Japan. Inquiry was made as to the number of cases which developed idiopathic optic neuritis from April 1992 to March 1993 along with their clinical features. The response rate was 53.6%. There were a total of 550 cases, and the male to female ratio was 1:1.22. Patients 14 to 55 years old were 65.9%; bilateral involvement: 28.2%; recurrence: 18.6%; positive past history of the other eye; 7.5%. Assuming the answering rate to be 100% and two thirds of the patients to be included, annual incidence of this disease (the annual number of patients) was determined to be 1.62 for an adult population of 100,000 (1.03 cases/100,000 people). Tochigi, Tokyo, Kanagawa, Hyogo, Wakayama, Okayama, Yamaguchi, Tottori, Shimane, Ehime, and Fukuoka showed an annual incidence exceeding 2.0/100,000 adults. At more than 95% of all medical institutions questioned, patients with optic neuritis were usually treated with systemic corticosteroids. Oral corticosteroid therapy, which was shown in a recent study in USA to be contraindicated, was still being used at 15% of the institutions.

[Heterogeneity in gastric carcinoma with special reference to DNA content and mitotic activity: histopathologic differentiation].

DNA ploidy was microspectrophotometrically investigated in 46 patients with gastric carcinoma. Measurements of DNA content and mitotic index (MI) were examined in the mucosal, submucosal, muscularis propria, and serosal layers of tumors, respectively. The frequency of cells with values exceeding hexaploid chromosome (6c) and mitotic counting analysis revealed a significantly higher value in the serosa than in the mucosa. This tendency was not evident in differentiated type adenocarcinoma but was noted in those with the undifferentiated type. There were 37 tumors (80.0%) with the same DNA distribution pattern in every layer of the stomach (homogeneous DNA ploidy). Heterogeneity of DNA ploidy was observed in nine tumors (20.0%). Carcinoma with heterogeneous DNA ploidy manifested a significantly higher incidence of metastasis to lymph nodes than did those with the homogeneous type. Characteristically, there was venous permeation preponderance in the differentiated type and peritoneal dissemination preponderance in the undifferentiated type. This evidence of DNA heterogeneity in gastric carcinoma tissue suggests a possible correlation with metastatic behavior.

Influence of simultaneous pattern-reversal electroretinogram recording on visual evoked potentials.

Little is known about the influence of the presence of a gold-foil electrode on pattern-reversal visual evoked potential recording, although simultaneous pattern-reversal electroretinography has been applied in several clinical investigations. We compared the results of pattern-reversal visual evoked potentials simultaneously recorded with pattern-reversal electroretinograms with those obtained during separate pattern-reversal visual evoked potential recording in 10 normal subjects. Transient response (reversal rate, 1 Hz; check size, 30') and steady-state response (reversal rate, 5 Hz; check size, 120', 60', 30', 15' and 7.5') were analyzed under the stimulus contrast condition of 90%. Neither P100 latency in transient responses nor amplitudes in steady-state responses exhibited significant change at any check size when they were recorded separately or simultaneously with pattern-reversal electroretinograms. The results suggest that the gold-foil electrode exerts no significant influence on pattern-reversal visual evoked potentials. Simultaneous pattern-reversal electroretinogram and visual evoked potential recording is therefore confirmed to be a clinically useful method. This procedure makes the synchronous recording of both responses possible under the identical stimulus conditions.

Reconstruction of cornea in three-dimensional collagen gel matrix culture.

PURPOSE: To report on an in vitro reconstruction of cornea composed of three main layers. METHODS: These layers were reconstructed from isolated bovine corneal cells in three-dimensional collagen gel culture conditions. RESULTS: The topmost layer, corneal epithelial cells on collagen gel, was treated by what was designated as the "air-liquid interface method," a method developed specifically for this study. The treatment induced proliferation and differentiation of this layer. Those epithelial cells were positively immunostained with anti-epithelial keratin (55-kD/64-kD) antibody, AE5, which was specific for in vivo corneal epithelial cells. The middle layer consisted of keratocytes in the three-dimensional collagen gel matrix. This layer was comparable to the stroma in vivo. The underlayer, the counterpart of in vivo endothelial layer, was composed of corneal endothelial cells. CONCLUSIONS: This culture system probably provides a physiologic environment for detailed investigations of corneal cells.

Pattern-reversal visual evoked potentials in Down's syndrome.

We examined pattern-reversal visual evoked potentials (PRVEPs) in 36 adult patients with Down's syndrome, and analyzed the effects of the ophthalmological abnormalities on results of PRVEPs. The P100 latency was significantly delayed in 24 eyes of 16 patients. The P100 latency was significantly longer and its amplitude was significantly smaller (P < 0.001) in Down's syndrome patients than in age-matched normal controls. In 9 patients without any ophthalmological abnormalities, P100 latency showed no significant difference from that in normal controls, but three of them showed a significant delay of P100. Their P100 amplitudes were significantly smaller than those in normal controls. Therefore, we considered that the ophthalmological abnormalities are one of the main factors causing PRVEPs abnormalities in Down's syndrome patients, but some unknown factors should be also responsible.

Distribution of the innervating neurons of pupillary dilator and tarsal muscles in the superior cervical ganglion.

In an experimental study using adult cats, we analyzed the distribution of the sympathetic postganglionic neurons in the superior cervical ganglion (SCG) which innervate the pupillary dilator and tarsal muscles to determine whether there are direct fiber projections from the spinal cord to the iridic and tarsal muscles. This study consisted of three experiments. In Experiments 1 and 2, respectively, horseradish peroxidase (HRP) was injected unilaterally into the iridic stroma or superior tarsal muscles. The SCG and spinal cord were then removed for microscopic analysis. In Experiment 3, the ganglion was examined microscopically after the injection of bisbezimide into the iris and of HRP into the ipsilateral superior tarsal muscle. Experiment 1 revealed more numerous HRP-labeled neurons in the rostral part than in any other region of the ipsilateral superior cervical ganglion. In Experiment 2, HRP-labeled neurons were evenly distributed over the whole ipsilateral SCG. No HRP-labeled neurons were detected in the spinal cord in either of these experiments. In Experiment 3, no doubly-labeled neurons were discernible in the ganglion. These results clearly indicate the localization of internal carotid nerve cells in a specific region of the SCG, and provide evidence for the absence of postganglionic neurons that innervate both the dilator muscle of the pupil and the tarsal muscles.

The pharmacokinetics of fluconazole during haemodialysis in uraemic patients.

We have studied the pharmacokinetics of fluconazole in five patients on long-term haemodialysis. The single-pass extraction rate of the dialyzer was 59 (3.5)% (n = 4), and the serum concentration was reduced by haemodialysis for 3 or 4 h by 26 (3.2)% (n = 5) and 39 (2.2)% (n = 9) respectively. The estimated amount extracted by a dialysis of 4 h was 33 (3.2)% (n = 4) of the dose. During repeated administration the serum fluconazole concentration increased, reaching a plateau at about 4 times the peak concentration after the first dose. After discontinuing administration the serum fluconazole concentration fell by 25% in every 3 h dialysis session. We conclude that fluconazole should be given in the usual dose of 100 or 200 mg at the end of every haemodialysis session.