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.

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.

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.

Crystalline lens changes in porcine eyes with implanted phakic IOL (ICL) with a central hole.

BACKGROUND: We calculated the smallest diameter of a hole in the center of the optic at which the optical character of a phakic IOL (ICL) may be maintained. The changes induced in the aqueous humor dynamics and the pathology of cataract development with such a hole were investigated. METHODS: A simulation was performed using ZEMAX software to calculate the hole diameter that makes possible the maintenance of a stable optical character of a phakic IOL. After a hole of calculated diameter was trepanned in the center of the optic of the ICL, the latter was implanted into one eye of a 5-month-old minipig, and an unperforated ICL into the other. The postoperative course was observed for 3 months. Then, Evans blue was injected into the vitreous body under general anesthesia to stain the anterior capsule of the crystalline lens. Within 30 min, the eye was enucleated and the tissues removed were fixed. RESULTS: The MTF of the perforated ICL (hole diameter, 1.0 mm) in the center of the optic resembled that of the unperforated ICL. In all cases with non-perforated ICLs, subcapsular turbidity developed, but no staining caused by EB was observed in the anterior capsule. On the other hand, the anterior capsules of the eyes fitted with ICLs with a 1.0-mm hole were stained, but exhibited no turbidity. CONCLUSION: An ICL with a central hole of diameter 1.0 mm in the optic is similar to an unperforated ICL. The size of the hole influenced the aqueous humor dynamics and increased the aqueous humor perfusion volume over the entire anterior surface of the crystalline lens. The possibility of preventing cataracts was therefore suggested.

Changes in the crystalline lens resulting from insertion of a phakic IOL (ICL) into the porcine eye.

BACKGROUND: Insertion of a phakic IOL offers these advantages: the corneal optical zone is not dissected or resected; preservation of the crystalline lens results in preservation of the accommodation force; and since the phakic IOL is removable, any error or change in refraction can be countered by exchanging it. However, the cause of secondary cataracts has never been clarified or discussed to date. METHODS: The following ICL lenses were inserted under general anaesthesia into 20 eyes of ten 3-month-old miniature pigs: a minus lens without holes into five eyes, a plus lens in two eyes, a minus lens with four holes around the lens haptics in three eyes, and a minus lens with a central hole in the optic in three eyes; and seven eyes were used as controls. The anterior segments were then photographed 1 week, 1 month and 3 months after surgery. At 3 months after surgery, Evans blue (EB) was infused into the vitreous under general anaesthesia, and after 30 min, the eyeball was enucleated, fixed and examined. RESULTS: In the case of the ICL without holes, the anterior subcapsular opacities were observed in all the eyes, and the anterior surfaces of the crystalline lenses were not stained with EB. Use of the ICL with four holes around the lens haptics resulted in light staining of the anterior surface of the crystalline lens, but the anterior subcapsular opacities observed were mild. As for the ICL with a hole in the centre of the optic, the anterior surface of the crystalline lens was stained and no anterior subcapsular opacities were observed. CONCLUSION: The results suggested that the insertion of an ICL brings about a change in the dynamics of the intraocular aqueous humour, reducing its circulation to the anterior surface of the crystalline lens. This is considered to cause a metabolic disturbance of the crystalline lens, resulting in anterior subcapsular opacification.

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.

[Experimental cataract models produced by repeated blunt mechanical stimulation and elucidation of the pathogenetic mechanism].

PURPOSE: The pathogenesis of cataract produced by mechanical stimulation of the eye was studied using experimental models. METHODS: The eyes of rabbits (in vivo) and the extracted lenses of rats (in vitro) were subjected to vibration from an electric massage machine and tapping. The histological changes, aqueous flare score, protein and prostaglandin (PG) E2 levels in aqueous humor, and the rate of vitreous liquefaction were measured, and the involvement of apoptosis was examined in the rabbit model. RESULTS: In both models, mechanical stimulation resulted in a high frequency of opacification of the lens in the anterior or posterior subcapsular region. In the in vivo model, histopathological examination revealed vacuolar changes in the lens epithelium and swelling of lens fibers. Increases in aqueous flare score, protein level, PGE2 level, and the rate of vitreous liquefaction were confirmed. The involvement of apoptosis was not proven. CONCLUSION: Repeated mechanical stimulation of the eye produced lens opacification. In patients with atopic dermatitis, mechanical stimulation such as a habit of eye tapping may be the cause of or a factor in promoting cataract development.

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.

Central corneal haze increased by radial keratotomy following photorefractive keratectomy.

PURPOSE: To report a case of central corneal haze induced by minimally invasive radial keratotomy (mini-RK) after photorefractive keratectomy (PRK) and subsequent deep lamellar keratoplasty. METHODS: We report a case (one eye of one patient) of central corneal haze that worsened after mini-RK was performed 2 years following PRK. Four years later, a second PRK was done, myopic regression was subsequently observed, and corneal haze persisted. Deep lamellar keratoplasty was performed and a corneal graft was taken, which was examined by light and electron microscopy. RESULTS: In the ablated area, irregularity of the basal membrane and hypertrophy of the corneal epithelium were observed. In the stromal layer, collagen fibers showed disorder in their disposition. Aggregated activated keratocytes were observed. An epithelial plug filling the gap of the RK incision persisted for 6 years after the mini-RK. The RK incision was easily divided when deep lamellar keratoplasty was performed and the patient obtained a stable visual outcome. CONCLUSIONS: It is possible that mini-RK enhancement after PRK induces central corneal haze and reduces corneal integrity. Deep lamellar keratoplasty for refractory corneal haze after refractive surgery was useful in this eye.

The effect of continuous low doses of X-ray irradiation on the rat lens.

PURPOSE: To clarify the morphological changes in the rat lens after irradiation with continuous low doses of x-ray at different intervals. METHODS: Male Wistar rats at the age of 8 weeks were irradiated with three doses of 2 Gy at intervals of either 1 week or 1 day. Over the period from the first week to the eighth week after irradiation, the eyeballs of the rats were enucleated progressively and changes in the lenses were examined morphologically. A comparison was made with specimens from control rats at each time of enucleation. RESULTS: (1) Three doses of weekly irradiation: 1 week later, the lens epithelium and fibers at the equator showed mild swelling. Bow configuration was slightly disturbed. Eight weeks later, swelling and uneven height of epithelial cells in the equatorial area, irregular bow configuration, swelling of cortical fibers and epithelial loss and deformed nuclei at the central epithelium were observed. (2) Three doses of daily irradiation: 1 week later, mild changes in the lens such as uneven height of epithelial cells, irregular bow structures, and swelling of cortical fibers were observed. Eight weeks later, irregular bow configuration, posterior dislocation of nuclei, severe epithelial loss and marked swelling of cortical fibers were observed at the equatorial area. Epithelial loss and deformed nuclei of the epithelium were observed in the central area. CONCLUSION: The lens was damaged by continuous irradiation even though the dose was low. The damage to the lens caused by daily irradiation was more severe than that by weekly irradiation. The main symptoms were degeneration and loss of epithelial cells and swelling of cortical fibers.

Repair and reconstruction of the mouse lens after perforating injury.

PURPOSE: To investigate the process of repair in the epithelial cells and the reconstruction in lens fibers of a mouse lens that has developed opacity because of a large-scale perforating injury. METHODS: Lenses of 4-week-old mice were perforated with a needle through the cornea to induce the development of traumatic cataracts. Over the period from the first day to the fifth month after injury, eyeballs were extracted progressively from the mice and changes in the epithelial cells of these lenses were observed morphologically as well as histochemically. RESULTS: Following the injury, the epithelial cells of the lenses extended toward the center of the injury, while undergoing repeated proliferation and stratification. After a month, the epithelial cells completely covered the entire injury. Later, a basal lamina and collagen fibers developed among the epithelial cells that had proliferated, the intracellular space enlarged, but the number of cells decreased. Histochemically, a strong actin-positive finding was observed in the epithelial cells in the growth phase. On the other hand, an investigation by means of the TUNEL method revealed epithelial cell death and a decrease in cell number. Maximal cell death was observed in the second month. During this period, lens fibers regenerated, and the clear areas of the cortex increased. CONCLUSION: Although the lenses exhibited opacity over a large area, the epithelial cells eventually fully covered the injured area. Once the repair was completed, the number of epithelial cells decreased. At the same time, the lenses were found to have developed increased clarity, leading to reconstruction. The epithelial recovery and the residual posterior suture may be the key to the reconstruction of the lenses.

Response of the mouse lens to varying sizes of injured area.

PURPOSE: To examine the response of the lens to varying sizes of perforating injury. METHODS: Four-week-old mice were used. Injuries consisted of pricking in the central region of the lens by transcorneal insertion of needles of two different sizes. After injury, the eyeballs were removed sequentially at various intervals up to 30 days and examined morphologically. RESULTS: The mouse lens showed three patterns of reaction; retained transparency, posterior opacity, and anterior opacity. (1) When the lens remained clear, the injury was small in area. The damaged portion of the lens was repaired by epithelial proliferation. (2) When opacity occurred abruptly at the posterior cortical area, epithelial damage was mild and lens fiber damage was relatively severe. Evans blue dye moved toward the posterior polar region along the cortical fiber arrangement. (3) When opacity developed abruptly at the anterior cortex, the size of epithelial damage and the damage to lens fibers were extensive. The posterior cortex remained transparent. Evans blue dye remained in the anterior polar region just beneath the injured part. CONCLUSION: It was found that the size of the injured area is a determinant of repair or opacity, and the site of opacity is dependent on the severity of epithelial damage and the location of the liquefied area.

Morphological analysis of iodoacetic acid-induced cataract in the rat.

PURPOSE: To clarify the mechanism underlying the development of cataract in the rat lens after intraperitoneal administration of iodoacetic acid (IAA). METHODS: (1) The 2% IAA dissolved in saline solution was injected at a dose of 40 mg/kg body weight into the rat peritoneal cavity. The retina and lens were intermittently extirpated and were examined by light and electron microscopy. (2) Two kinds of tracer, Evans blue (EB) and horseradish peroxidase (HRP), were injected into the tail veins and anterior chamber, and were observed with dissecting and electron microscopes. RESULTS: (1) Four weeks after administration, a part of the lens epithelium at the lateral side of the lens was degenerated, and the lens nucleus developed faint turbidity after 8 weeks. After 16 weeks, the nuclear turbidity could not be observed because mild cortical opacity was developing. The epithelial degeneration recovered from around 12 weeks, and instead of spherical nuclei, elliptical nuclei appeared. (2) The EB dye injected into the tail vein significantly stained the ciliary body, where the anterior and posterior ciliary arteries anastomosed. EB injected from the lateral side of the lens was seen to move towards the lens nucleus. Electron microscopically, the epithelial degeneration of the ciliary body was observed. The incorporated HRP substance was found in the cytoplasm of the nonpigmented cells of the ciliary epithelium at an early stage after IAA administration. CONCLUSION: IAA injected intravenously first developed epithelial degeneration at the lateral side of the lens. This change induced swelling of the lens fibers in the lens nucleus. Recovered epithelial cells had a transformed nucleus, and in turn the cortical cataract was induced by a differentiation disorder of the lens fibers. These results indicate that the breakdown of the blood-aqueous barrier in the nonpigmented epithelium of the ciliary body is a trigger to cause the cataract. The IAA-induced cataract may be useful as an animal model of human age-related cataract.