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The iris is the most anterior portion of the uveal tract. The pupil is round opening near the center of the iris; it is displaced slightly downward and nasally with respect to the center of the cornea. The mammalian iris sphincter is considered to be innervated by cholinergic, and the dilator muscle by adrenergic excitatory nerve fibers, and both miosis and mydriasis are the result of contraction of the iris sphincter and the dilator muscles due to activation of these excitatory nerve fibers. Pharmacological and histological investigations also reveal that the sphincter muscle is innervated in part by inhibitory adrenergic nerve fibers, and that the dilator muscle is also innervated by inhibitory cholinergic nerve fibers. In addition to the release of acetylcholine and norepinephrine by these nerves, the peripheral nerves to the mammalian iris contain various neuropeptides, although the functional role of these pepetides is not clear. It has been known for more than 100 years that two types of photosensitive cells exist in man. However, some totally blind individuals maintain a normal circadian rhythm. Such a phenomenon cannot be explained by the rod and cone functions. Recently, a new photosensitive pigment, melanopsin, was found in the dermal melanophore cells of the frog. In 2002, melanopsin-containing retinal ganglion cells (mRGCs) were discovered and revealed that mRGCs would depolarize without input from the photoreceptors, meaning that these cells are photosensitive. In the human retina, mRGCs comprise only 0.2% of all ganglion cells. Electrophysiological studies show that light slowly depolarizes mRGCs but rapidly hyperpolarizes rods and cones. The mRGCs innervate the suprachiasmatic nucleus, which is the master circadian pacemaker in mammals, and the olivary pretectal nucleus of the midbrain. In addition to their role in circadian entrainment, the mRGCs mediate the pupillary light reflex. We investigated the mechanism of photoreception by retinal photoreceptor cells, and to evaluate the relative contribution of pupil light response using the control, instigated pharmacological blockade of neurotransmission (PB) model and a transgenic model of retinal degeneration (Tg) rabbit. Although rod and cone photoreceptors disappeared in the PB and Tg models, miosis was still induced during exposure to blue light (470 nm). The greater sustained constriction of pupils to blue light in eyes with outer retinal damage reflects mRGC activation. Our study also indicated that some histologically-identified RGCs were consistent with the characteristics and structures of mRGC. Clinically, in age-related macular degeneration patients, there was no reliable recordable pupil response to red light, even at the brightest intensity but a blue light evoked a sustained pupil constriction. However, in glaucoma patients, there was no reliable recordable pupil response to the brightest intensity of blue light. These preliminary recordings in human subjects demonstrate that changes in the pupil responses to chromatic stimuli are readily detectable and easily quantifiable with standard instruments of clinical testing. We hypothesize that changes in the transient pupil response to red light and low intensity blue light may be more sensitive to cone and rod disease, whereas changes in the sustained pupil response to bright blue light may be more sensitive to optic nerve disease. Ongoing studies of the pupil are aimed at optimizing stimulus conditions that elicit pupil responses that can better localize the site of damage to rods, cones, and RGCs, to quantify the extent of disease.

Purpose: We examined electron microscopically the tissues removed from the retinal surface in cases with idiopathic macular holes., Materials and Methods: We performed vitrectomy for idiopathic macular holes in 43 eyes from June 1994 to December 1998 at Fukuoka University Chikushi Hospital. The removed tissues were evaluated with transmission electron microscope in only 8 of the 43 eyes. All cases underwent 3 port vitrectomy. After occurrence of posterior vitreous detachment, epiretinal tissues were removed around the macular hole using a micro-hook needle., Results: All tissues were transparent under an operating microscope, and they curled naturally. Histopathological examination showed that the removed tissues were from 2 to 6.5 microns thick. All tissues were composed mainly of extracellular matrix. There were type 2 collagen fibers in 6 eyes and basement membrane-like structures in 2 eyes. There were few cell components. All cases had the macular hole closed by the first surgery. Four eyes had improved visual acuities of more than 2 lines. The remaining 4 eyes had the same visual acuity as before the operation., Conclusion: Residual vitreous on the retinal surface was the main component of the transparent tissues removed from the retinal surface during vitrectomy.

Purpose: The morphological observation of the human retinal changes caused by severely elevated intraocular pressure (IOP) in congenital glaucoma., Method: Light and electron microscopy., Subjects: One eye of a 15-year-old boy diagnosed as having congenital glaucoma was obtained after he had suffered from severe visual disturbance and ocular pain because of extensive elevated IOP, corneal leukoma, and exophthalmos., Results: Ganglion cells and nerve fibers in the inner layer were lost and replaced by Müller cell processes. The cellular elements in the outer layer were regularly arranged, but these cytoplasmic organelles were not very well developed. Most of the cellular elements of the whole retina contained many dense bodies. The retinal capillaries in the nerve fiber layer were lost or severely damaged, but some of these in the inner plexiform layer were intact. In the retinal arteries and veins marked thickening of the adventitial tissues was observed. These tissues consisted of remarkably increased and irregularly arranged collagen fibers, and a few elastic fibers scattered in some parts., Conclusion: It appeared that the pathological changes in the inner layer of the retina were caused by ischemia. The retinal vascular abnormality was thought to be the result of protective reaction against the severely elevated IOP.

50 eyes of 30 Sprague-Dawley rats were subjected to 60 minutes of pressure-induced ischemia, then fixed for light and electron microscopy with no reperfusion, or reperfusion after 30 minutes, 1, 2 or 4 hours, and 1 or 3 days from the time ocular ischemia was relaxed. The TdT-mediated dUTP-biotin nick end labeling (TUNEL) method revealed apoptotic signs at the inner retina as early as 1 hour after reperfusion. However, the incidence of apoptotic signs with the TUNEL method did not accord with the results of electron microscopic examination. During the time after the reperfusion started, especially after more than 4 hours, apoptotic signs became obvious and extended from the inner to the outer retina. These apoptotic findings could be seen with both the TUNEL method and electron microscopy. By 3 days after the reperfusion, necrotic cells in the ganglion cell layer, and the inner and outer nuclear layer became more prominent than apoptotic cells. These results may provide a baseline for therapeutic strategy and the prognosis of ischemia-reperfusion injury in the retina.

We used light and electron microscopy to examine detailed ultrastructural changes in the course of the development and degeneration of the rds mouse (rds/rds) retina with particular reference to the relationship between the photoreceptor and retinal pigment epithelial cells. During the first 6 postpartum days, the mutant retina showed the same developmental pattern as in normal mice. After that, however, rds mouse retinas failed to develop the photoreceptor outer segments. After 14 days, the photoreceptor inner segments and the cilia were all arranged parallel in the same direction in which the pigment epithelial cells extend their long microvilli. The inner segments and the cilia were very long and surrounded by the microvilli of the retinal pigment epithelial cells in the mutant retinas 21 days postpartum or latter. Some inner segments and cilia showed cytoplasmic degeneration in the part surrounded by the microvilli, while the other part appeared normal. There were pigment epithelial cells that contained fragments of the inner segments. The photoreceptor nucleus and inner segment were reduced in number after 21 days. These findings indicate that the pigment epithelial cells play a role in photoreceptor degeneration in rds mice.

Experimental retinal detachment was produced by injecting various fluids into the subretinal space in pigmented rabbits. We selected autologous aqueous humor, autologous vitreous, and autoserum as substitutes for subretinal fluid. Retinal detachment disappeared within 24 hours. The eyes were enucleated 7 days after surgery for histopathological study. Destructive changes in photoreceptor layer occurred most severely when the eye was treated with serum. There was a decrease in the number of outer nuclear layer cells. Macrophages and fibroblastic cells had migrated into the subretinal space. Histopathological changes with vitreous were less pronounced than with serum. The cytoplasm of Mueller cells appeared to compensate for the loss of photoreceptor cells in the outer nuclear layer. Mild decrease in the number of photoreceptor cells was the sole pathological feature when the eye was treated with aqueous humor. The retina thus showed different reactions to the subretinal fluids of differing nature.

A diode laser, emitting light in the near-infrared range (803 nm), was used for photocoagulation at mild, moderate, and strong intensities with or without intravenous administration of indocyanine green (ICG) in 7 eyes of 4 rhesus monkeys. The retinochoroidal lesions were studied histopathologically from 24 hours to 3 months after treatment, and choroidal vascular casts were studied by electron microscopy. Mild diode laser burns caused occlusion of the choriocapillaris and slight outer retinal damage. Moderate and severe burns caused more marked occlusion of the choriocapillaris, deep choroidal vessels, and choroidal nerves. There was little difference between ICG-enhanced and non-enhanced mild burns, but ICG-enhanced moderate and severe burns showed larger retinochoroidal lesions and more complete obstruction of the choriocapillaris than did burns without ICG enhancement. It seems that at moderate and strong levels, ICG-enhanced diode laser burns have more apparent effect on the retina and choroid, particularly on the choroidal vessels.

We reported previously selective damage of the outer segment of the photoreceptors and retinal pigment epithelium after intravitreal injection of netilmicin sulfate in the rabbit. The cellular damage was thought due to active damage of phospholipase C enzyme, preventing it from performing phospholipid inositol metabolism, and to the presence of calcium. Therefore we investigated the retinal damage following after intravitreal injection of calcium and netilmicin sulfate in the rabbit. Morphologically, the retina did not show any structural change after intravitreal injection of CaCl2 at any concentration. After an intravitreal injection of netilmicin sulfate mixed with CaCl2, the damage in the photoreceptor outer segment was reduced at a concentration of 200 micrograms/0.1 ml and in the retinal pigment epithelium at a concentration of 1,000 micrograms/0.1 ml. This showed that retinal damage due to netilmicin sulfate can be reduced by the addition of calcium.

In the first stage of this study, peri- or intravascular unmyelinated nerve axon which was clearly different from that of the optic nerve was confirmed in the rabbit retina transmission electron microscopically. In the second stage, the correlation of these unmyelinated nerves and autonomic nerve was studied in rabbits and monkeys. For the purpose, wheat germ agglutinin horseradish peroxidase (WGA-HRP) was injected into the superior cervical ganglion. Electron microscopic observation of the retina 48 hrs after the injection of WGA-HRP showed deposit of its reaction product in the axon of the unmyelinated nerve located around the vascular wall and in the adventitia and smooth muscle cell in both rabbits and monkeys. This result clearly reveals the existence of an autonomic vasomotor in the retinal vessels at least in rabbits and monkeys.

Using light and electron microscopy, we studied the histopathological findings of retinopathy of prematurity (zone II, stage 3). The infant was born at 32-week gestational period and the birth weight was 1,255 g. He suffered from intracranial hemorrhage and hydrocephalus, and died at 78 days old. The ophthalmoscopic findings of both eyes at 72 days after birth showed that the intermedia was clear. The optic disc and posterior pole showed normal findings. Ridge formation with non-vascularized retina was seen at the equator of all quadrants of the fundus except the nasal quadrant of retina. Light and electron microscopic studies showed the following; in the most periphery of the vascularized retina, endothelial cell proliferation with the capillary lumen of glomeruloid tufts (rearguard) were seen. There were the aggregation of immature mesenchymal cells (vanguard) near to the rearguard. The cytoplasmic organelles of spindle cells in the non-vascularized retina were different from those of surrounding glial cell. Therefore, they were thought to be mesenchymal cells in the non-vascularized retina. Between the vanguard and the rearguard, the proliferative tissues composed of immature endothelial cells and mesenchymal cells extended into the vitreous body through the inner limiting membranes, and they formed the fibrovascular membranes on the retina. It was concluded that fibrovascular proliferation on the retinal surface in the active case of retinopathy of prematurity was composed of immature endothelial cells with vascular lumen and mesenchymal cells.

For demonstrating ultrastructurally the distribution of anionic sites (AS) in the inner limiting membrane (ILM) of the rabbit's retina, the immersion method with cationic tracer, polyethyleneimine (PEI) was applied. This method was found to be simple and useful for the observation of AS in the ILM. In the posterior pole of the eyeball (visual streak and medullary ray), PEI particles were arranged evenly in the lamina rara (LR) of the ILM. The mean diameter of PEI particles was 20 nm, and intervals between them (40-50 nm) were the same as those of fine strands in the LR. However, the peripheral retina, the distribution of PEI particles were sparse and random, while in the lamina densa (LD) of each ILM, PEI particles were absent. AS stained with PEI were present in the residual vitreous fibers. These results may provide the morphological basis for the theory that AS of the ILM act as a charge barrier between the retina and the vitreous cavity.

A transpupillary retinal photocoagulator utilizing GaAlAs diode laser which emits an 810 nm beam has been constructed. It has a continuous-wave mode with a maximum output of 400 mW at the cornea. Animal experiments with monkeys were carried out successfully at various intensities to produce controlled coagulations. Observation by ophthalmoscopy, light and electron microscopes at different time intervals proved diode laser photocoagulation produced more intensive effects in choroid than krypton red laser (647 nm). However, due to the greater absorption of the choroid, there is a tendency to over-coagulation unless conditions are carefully set. No serious side effects in the retina and choroid were observed during the experiments.

We clarified a role of the retinal pigment epithelium (RPE) in the regression of experimentally induced subretinal neovascularization (SRN) in monkey. Eight eyes of 5 rhesus monkeys were used in this study. Two weeks after intense krypton laser photocoagulation to the posterior pole of the fundus, 0.5M l-ornithine hydrochloride solution 0.03 ml was injected intravitreously for the purpose of selective RPE damage. After ornithine injection, SRN continued without any evidence of spontaneous regression over 8 weeks following photocoagulation. Histopathologically, SRN developed with wide lumen in the subretinal space accompanied with serous detachment of the sensory retina, and new vessels were not enveloped completely by the proliferating RPE cells. We already showed that experimentally induced subretinal neovascularizations naturally regress spontaneously by envelopment of RPE cells 5 to 8 weeks after photocoagulation. Our results suggested that SRN persist actively without regression due to incomplete enclosure by RPE by selective damage of RPE at the active stage of SRN. We have confirmed that the RPE cells played an important role at the involution stage of SRN.

It has been pointed out that the silicone oil tamponade can result in complications such as corneal damage, elevation of intraocular pressure and retinal toxicity against. The effect of silicone oil on the ocular tissues was investigated histopathologically by injecting silicone oil into the anterior chamber of the eyes of rabbits. In addition, in order to study its effect on the retina, silicone oil was injected into the vitreous cavity after vitrectomy. The eyes were extracted 3, 6, 9, 12 and 18 months after injection and various tissues were observed by light and electron microscopy. Silicone oil particles were first observed in the retro-corneal membrane 18 months after injection. In the trabecular meshwork silicone oil particles were seen for the first time 12 months after injection. Migrating cells engulfing silicone oil particles were attached to the internal limiting membrane of the retina three months after injection. Twelve and 18 months after injection, silicone oil particles passed through the internal limiting membrane and were engulfed by Mueller cells.

In order to clarify the ultrastructural details, electron microscopic observations of the inner limiting membrane (ILM), critical point drying and surface replication methods with collagenase digestion were performed on rabbit's ILM. The results were as follows,: 1) This method was simple, the electron microscopic image is highly reproducible and a bird's-eye view of the ILM is possible. 2) The ILM was composed of a fine three-dimensional meshwork structure with numerous pores (sieves). 3) The diameter of the pores varied from 10 to 25nm. Mean diameter of the pores is 13.43nm in the visual streak, 13.59nm in the medullary ray, 13.40nm in the peripheral retina, and there were no statistical differences among these three regions. 4) In the peripheral retina, it was difficult to observe the ILM because vitreous fibrils lay densely on it. The pore's shape in the peripheral retina was different from that in the posterior pole retina. These results may provide the morphological basis for the theory that the pores of the ILM act as the main filtration barrier between the retina and the vitreous cavity. It is supposed that the bulk of substances are transported from the retina mainly through the ILM of the posterior pole, because dense vitreous fibrils of the peripheral retina disturb that transport.