Morphological Changes and Potential Mechanisms of Intraocular Pressure Reduction after Micropulse Transscleral Cyclophotocoagulation in Rabbits.

INTRODUCTION: Micropulse transscleral cyclophotocoagulation (MP-TSCPC) is a method for intraocular pressure (IOP) reduction in patients with glaucoma; however, the specific mechanisms underlying its ability to reduce IOP remain unclear. We therefore investigated the morphological changes and mechanisms of IOP reduction after MP-TSCPC. METHODS: The right eyes of 4 pigmented rabbits were treated with MP-TSCPC with power setting corresponding to those used in glaucoma patients (1 power: 2,000 mW; time: 160 s; duty cycle: 31.3%). Power settings of 1, 1/8, 1/16, and 1/32 power were applied to the right eyes. The left eyes were used as controls. A light microscope and electron microscope were used to observe morphological findings after 1 week of MP-TSCPC. IOP and IOP reduction rate were compared before and after MP-TSCPC application on days 1, 3, and 5, and at 1 week. RESULTS: In the pre-MP-TSCPC, IOP was 16.7 ± 0.6 mm Hg. The IOP of rabbit treated with the 1 power was 3 mm Hg, with an IOP reduction rate of 80%; however, the eyes developed phthisis bulbi. The IOP was 7.0 ± 0.0 mm Hg 1 week after MP-TSCPC (IOP reduction rate: 59%) in rabbit treated with the 1/8 power. Reduction in IOP was observed, but there was significant tissue invasion to the ciliary body. The IOP was 10.3 ± 0.6 mm Hg (IOP reduction rate: 40%) 1 week after MP-TSCPC in rabbit treated with the 1/16 power, which was more effective to reduce IOP than that with the 1/8 power. Tissue invasion to the ciliary body was negligible, nonpigmented epithelial cells of the pars plicata were damaged, basal infoldings were destroyed, and repair was accompanied by proliferating tissue. No IOP reduction or tissue change was observed in rabbit treated with the 1/32 power. CONCLUSION: A potential mechanism for IOP reduction in pigmented rabbits is aqueous humor transport dysfunction due to damage to the nonpigmented epithelial cells of the pars plicata and destruction of basal infoldings. The power of MP-TSCPC was consistent with both morphological changes and IOP reduction.

Effects of 0.4% ripasudil hydrochloride hydrate on morphological changes in rabbit eyes.

We evaluated the cellular structure changes after continuous use of ripasudil hydrochloride hydrate in rabbit eyes which might affect its own efficacy and adverse effects. Two pigmented Dutch rabbits and 1 Japanese white rabbit were instilled with 0.4% ripasudil hydrochloride hydrate to the left eye twice daily. The right eye was observed as the control. Both eyes of all 3 rabbits were then enucleated for histopathologic examination by light and electron microscope at 1mo in 1 of the pigmented Dutch rabbits, 3mo in the other pigmented Dutch rabbit, and in the Japanese white rabbit after instillation. Microscopic observations showed increase intercellular space in trabecular meshwork, ciliary body, and iris stoma, increase pigmented granule number and size in iris epithelial cells, and decrease actin filament in iris muscle fiber cells. Consequently, ripasudil hydrochloride hydrate decreases the intraocular pressure by improving the conventional outflow and may also facilitate the unconventional outflow via intercellular space widening without serious side effects.

Epidemiologic and Clinical Characteristics of Optic Neuritis in Japan.

PURPOSE: To elucidate the clinical and epidemiologic characteristics of optic neuritis in Japan. DESIGN: Multicenter cross-sectional, observational cohort study. PARTICIPANTS: A total of 531 cases of unilateral or bilateral noninfectious optic neuritis identified in 33 institutions nationwide in Japan. METHODS: Serum samples from patients with optic neuritis were tested for anti-aquaporin-4 antibodies (AQP4-Abs) and anti-myelin oligodendrocyte glycoprotein antibodies (MOG-Abs) using a cell-based assay and were correlated with the clinical findings. MAIN OUTCOME MEASURES: Antibody positivity, clinical and radiologic characteristics, and visual outcome. RESULTS: Among 531 cases of optic neuritis, 12% were AQP4-Ab positive, 10% were MOG-Ab positive, 77% were negative for both antibodies (double-negative), and 1 case was positive for both antibodies. Pretreatment visual acuity (VA) worsened to more than a median 1.0 logarithm of the minimum angle of resolution (logMAR) in all groups. After steroid pulse therapy (combined with plasmapheresis in 32% of patients in AQP4-Ab-positive group), median VA improved to 0.4 logMAR in the AQP4-Ab-positive group, 0 logMAR in the MOG-Ab-positive group, and 0.1 logMAR in the double-negative group. The AQP4-Ab-positive group showed a high proportion of females, exhibited diverse visual field abnormalities, and demonstrated concurrent spinal cord lesions on magnetic resonance imaging (MRI) in 22% of the patients. In the MOG-Ab-positive group, although posttreatment visual outcome was good, the rates of optic disc swelling and pain with eye movement were significantly higher than those in the AQP4-Ab-positive and double-negative groups. However, most cases showed isolated optic neuritis lesions on MRI. In the double-negative group, 4% of the patients had multiple sclerosis. Multivariate logistic regression analysis of all participants identified age and presence of antibodies (MOG-Ab and AQP4-Ab) as significant factors affecting visual outcome. CONCLUSIONS: The present large-scale cohort study revealed the clinicoepidemiologic features of noninfectious optic neuritis in Japan. Anti-aquaporin-4 antibody-positive optic neuritis has poor visual outcome. In contrast, MOG-Ab positive cases manifested severe clinical findings of optic neuritis before treatment, but few showed concurrent lesions in sites other than the optic nerve and generally showed good treatment response with favorable visual outcome. These findings indicate that autoantibody measurement is useful for prompt diagnosis and proper management of optic neuritis that tends to become refractory.

Histopathological Changes of Inner Retina, Optic Disc, and Optic Nerve in Rabbit with Advanced Retinitis Pigmentosa.

We observed the histopathological changes of retinal ganglion cells (RGCs), optic disc, and optic nerve in rabbit with advanced retinitis pigmentosa (RP). Wild-type (WT) and rhodopsin transgenic (Tg) of RP rabbits were used at age 24 months. Light and electron microscopy were used to observe the retina, optic disc, and optic nerve. RGCs were also confirmed by immunofluorescent staining with a TUJ-1 monoclonal antibody. In addition to the rod and cone degeneration, we observed the astrocyte infiltration of the optic disc due to the damage of small RGCs and nerve fibres and atrophy of small optic nerve fibres. They subsequently lead to the optic disc excavation and atrophy of the optic nerve. Consequently, our histopathological study clarified that not only the outer retina but also the inner retina, the optic disc, and the optic nerve were also affected in the late stages of RP rabbit.

Pre- and postsynaptic effects of brimonidine on isolated rabbit iris dilator muscles.

PURPOSE: Brimonidine is an imidazoline compound used for the treatment of glaucoma, but having very little effect on pupil diameter. Like para-aminoclonidine, most imidazoline compounds interact with postsynaptic α-adrenoceptors and cause pupil dilatation. Therefore, as part of an investigation of the mechanism of action of brimonidine on pupil diameter, the present study was initiated to measure, in vitro, the relative potency of brimonidine on the pre- and postsynaptic α-adrenoceptors of rabbit iris dilator muscle. METHODS: The contractile activity of brimonidine and its effect on twitch contraction evoked by electrical field stimulation were studied in isolated rabbit iris dilator muscles by isometric tension recording. RESULTS: Brimonidine significantly inhibited the twitch contraction of the dilator muscle caused by field stimulation, without affecting the response to exogenously applied phenylephrine. Compared to phenylephrine, brimonidine caused only a small contractile response with % maximum contraction values of <10%. CONCLUSION: These results suggest that brimonidine may act on nerve endings to inhibit adrenergic neurotransmission with very little effect on postsynaptic α-adrenoceptors. This may indicate that brimonidine reduced the pupil diameter just a little, thus improving night vision.

Functional and morphological study of retinal photoreceptor cell degeneration in transgenic rabbits with a Pro347Leu rhodopsin mutation.

PURPOSE: To investigate the process of retinal degeneration by analyzing the functional and morphological findings in transgenic rabbits with a Pro347Leu rhodopsin mutation. METHODS: Wild-type (WT) and transgenic (Tg) rabbits at ages 4, 8 and 12 months were used. We conducted functional evaluation by recording the changes in the pupil response to red and blue light stimulation and the amplitude of the electroretinography (ERG). Morphologically, rod and cone distribution was examined using light and electron microscopy. Immunostaining for the identification of retinal ganglion cells (RGCs) was also confirmed by injecting a TUJ-1 monoclonal antibody. RESULTS: Pupil constriction for infrared pupillography and the a- and b-waves for ERG in Tg rabbits decreased with increasing age; the differences were compared to the age-matched WT rabbits. The subnormal ERG in the Tg rabbits, especially the a-wave decrease and pupil constriction with a long latency time, was induced only during exposure to blue light stimulation at 12 months. Light and electron microscopic findings showed a progressive loss of photoreceptor cells over time manifesting by 8 months in the peripheral retina. Moreover, pyknotic nuclei of the outer nuclear layer in the center of the visual streak were observed. At 12 months, there was disappearance of the rods and ballooning degeneration of the cones. Some remaining RGCs had large cell bodies with long branching dendrites. CONCLUSIONS: The changes in the pupil light response and amplitude of the ERG could be used to predict the state of retinal degeneration in the Tg rabbit.

Effects of tamsulosin and silodosin on isolated albino and pigmented rabbit iris dilators: possible mechanism of intraoperative floppy-iris syndrome.

PURPOSE: To determine the mechanism of intraoperative floppy-iris syndrome (IFIS) by examining the binding affinity of tamsulosin and silodosin to α-receptors and melanin pigment using control and α(2)-blocker chronically administered in rabbit models. SETTING: Department of Ophthalmology, Kitasato University School of Medicine, Kanagawa, Japan. DESIGN: Experimental study. METHODS: The study was performed in isolated albino and pigmented rabbit iris dilators using pharmacologic and morphologic examinations. RESULTS: For pharmacologic examinations, the mean pK(B) values (pK(B) = -log K(B), where -log K(B) is the equilibrium dissociation constant of the antagonist-receptor complex) of tamsulosin in albino and pigmented rabbits were 9.10 and 8.08 and those of silodosin, 10.3 and 8.11, respectively. The pK(B) values of tamsulosin and silodosin in albino rabbits were significantly higher than in pigmented rabbits. In the isolated rabbit iris dilator, the maximum contraction evoked by 10(-3) mol/L phenylephrine gradually decreased by repetitive application in the chronic α-blocker-administered models. For morphologic examinations, the sizes of the pigment granules of pigment epitheliums for the α-blocker-administered models were irregular. The shape of shared nucleus of dilator muscles and pigment epitheliums changed to lobular, and the dilator muscle layer was thinner than in the control. CONCLUSIONS: The high affinity of α-blockers for α(1)-adrenoreceptors is important in the analysis of the mechanism of IFIS. However, IFIS should not be attributed to long-term binding with receptors alone; the drug-melanin interaction causing dilator muscle atrophy is probably the other important factor in the mechanism of IFIS.

Pharmacological vascular reactivity in isolated diabetic rabbit ciliary artery.

Impairment of the ocular circulation induced by diabetes mellitus has not been fully defined, but is thought to be related to hemodynamic changes in the ocular circulation. The purpose of the present study is to investigate the functional and morphological changes occurring in the ciliary artery wall of rabbits with alloxan-induced diabetes mellitus. A single intravenous bolus injection of alloxan (100 mg/kg) was given to each of 26 10-week-old rabbits and 16 sham-injected control rabbits. Twenty weeks later, control rabbits and diabetic rabbits were sacrificed, and their ciliary arteries were mounted in a myograph system. The responses of these arteries to high K+ solution (K-Krebs solution), phenylephrine and carbachol were investigated using isometric tension recording. L-NAME (NG-nitro-l-arginine methyl ester; 100 microM) and indomethacin (1 microM) were also used to test the mechanism causing the carbachol induced relaxation. The arteries were also examined morphologically. The maximum tensions induced by K-Krebs solution in this tissue were not significantly different: 17.2+/-0.8 mN (n=16) in the control rabbits and 17.6+/-0.8 mN (n=23) in the diabetic rabbits (P=0.36). Phenylephrine caused dose-dependent contraction with EC50 values of 1.3+/-0.4 microM (n=6) in the control and 5.1+/-2.3 microM (n=6) in the diabetic rabbits, but there was no significant difference between the two (P=0.36). Carbachol induced dose-dependent relaxations in segments precontracted with K-Krebs solution. These relaxations were significantly reduced in the diabetic rabbits. The maximum relaxation induced by carbachol was 77.0+/-2.4% (10 microM) and 66.4+/-2.5% (100 microM) in the control and diabetic rabbits, respectively. These values were significantly different (P=0.0076). The IC(50) value for carbachol was 396.3+/-58.4 nM (n=16) in the control, and 443.6+/-141.1 nM (n=23) in the diabetic rabbit (P=0.87). Application of a 100 microM nitric oxide synthase inhibitor, L-NAME, significantly inhibited the amplitude of relaxations evoked by carbachol (P=0.0066). However, these relaxations were not inhibited by pretreatment with 1 microM indomethacin (P=0.60). Histologically, the frequency of invaginations was less in the diabetic arterioles with a flattening of the lamina in the diabetic rabbits than in the controls. The cytoplasm of endothelial cells contained large vacuoles, indicating weak adhesion to the lamina. Some endothelial cells even showed vacuolar degeneration due to breakdown of the cell membranes. However, the smooth muscle cells were well preserved in the diabetic rabbit. These results suggest that the mechanism of impairment of ocular circulation induced by diabetes mellitus is mainly the reduction of NO synthase due to endothelial cell dysfunction. Furthermore, the characteristics of rabbits with alloxan-induced diabetes mellitus probably make them a useful model for investigating ocular complications induced by diabetic mellitus.

Pharmacological vascular reactivity in isolated hypercholesterolemic rabbit ciliary artery.

We have investigated functional and morphological changes occurring in the wall of the hypercholesterolemic rabbit ciliary artery. A mutant rabbit with hypercholesterolemia and atherosclerosis was created by serial breeding. Ciliary arteries from hypercholesterolemic, age-matched control and young control rabbits' eyes were mounted in a myograph system. The effects of phenylephrine (PE), carbachol and electrical field stimulation on this artery were investigated using isometric tension recording methods. The arteries were also examined morphologically. PE caused dose-dependent contraction in young control, age-matched control and hypercholesterolemic rabbits. The EC(50) values were 1.0 microm (0.2-2.1, n = 6), 1.4 microm (0.4-2.4, n = 6) and 4.7 microm (1.8-7.7, n = 8) in the young, age-matched controls and in the Kurosawa and Kusanagi-hypercholesterolemic (KHC) rabbits, respectively. The EC(50) values of the KHC rabbit were significantly different from those of control. Electrical field stimulation evoked contraction only in the control rabbits. On the other hand, electrical field stimulation evoked relaxation when the ciliary artery was pre-contracted by 10 microm histamine in each type of rabbit equally. Carbachol also induced approximately equal dose-dependent relaxation after pre-contraction. The morphological findings of KHC rabbit ciliary artery revealed irregular contours on the internal elastic lamina and deformation of the shape of the medial smooth muscle cells with irregularity in size and widening of the intercellular spaces. However, the endothelial cells were well preserved. Compared with the ciliary artery, typical atherosclerotic changes existed in the intima, not the media, in the KHC rabbit aorta. In the hypercholesterolemic rabbit ciliary artery, the vasoconstricting function was reduced but vasodilatation was well preserved. Morphological findings supported this. The pharmacological vascular reactivity in the hypercholesterolemic rabbit ciliary artery is quite different from that of the large arteries.

Pharmacological effects of latanoprost, prostaglandin E2, and F2alpha on isolated rabbit ciliary artery.

BACKGROUND: Latanoprost is a prostaglandin (PG)F2alpha analogue widely recognized in the treatment of glaucoma. To investigate the action of this drug on the ocular circulation, we have studied its effects on isolated rabbit ciliary artery. The data obtained on this drug are compared with the data from PGE2 and PGF2alpha. METHODS: Under the microscope, ciliary artery specimens were prepared from rabbit eyes and mounted in a myograph system. The effects of latanoprost, PGE2, and PGF2alpha on the isolated rabbit ciliary artery were investigated in vitro using isometric tension recording methods. RESULTS: Exogenously applied PGF2alpha, but not latanoprost, evoked contraction in the rabbit ciliary artery. After precontraction with excess-[K]0 solution, latanoprost evoked relaxation dose-dependently. Latanoprost at a concentration of 100 microM induced maximum relaxation, which was not blocked by 10 microM L-nitro-L-arginine methylester (L-NAME), 1 microM 8-37 calcitonin gene-related peptide (CGRP) or 10 microM indomethacin. Moreover, latanoprost induced relaxation even in preparations without endothelium. The maximum relaxation obtained with PGE2 was somewhat less than 50% of that with latanoprost. CONCLUSIONS: These results indicate that latanoprost and PGE2 relaxed rabbit ciliary artery to different degrees. The relaxation provoked by latanoprost was not dependent on endothelium and was not caused by intrinsic PG, CGRP or nitric oxide. The mechanism of this relaxation is not yet clear.