Masato Ohkuma

Kuhnt intermediary tissue as a barrier between the optic nerve and retina

Utilizing adult albino rats and lanthanum nitrate as a tracer, electron microscopic studies were done to provide additional information on the blood-optic nerve barrier. Following injection into the cervical artery, lanthanum was observed to fill the intercellular space of the optic nerve parenchyma. Diffusion from the prelaminar optic nerve into the juxta-optic nerve retina was, however, prevented by the Kuhnt tissue at the level of the rod and cone layer. In this region, tight junctions were found between the glial cells of the Kuhnt intermediary tissue and were continuous from the tight junctions of the retinal pigment epithelial cells to the junctions (zonulae adhaerentes) of the outer limiting membrane. From the level of the outer limiting membrane to the inner limiting membrane there was no diffusion of lanthanum into the adjacent retina despite the absence of tight junctions, and the lanthanum which had diffused from the choroid was never observed in the area of the inner limiting membrane of the optic nerve head. It is in this region that the functional barrier may exist.

Experimental Chloroquine Retinopathy

Chloroquine retinopathy was produced experimentally in the eye of the albino corydoras (one of the tropical fish) by daily administration of chloroquine (0.1 mg per os). The enucleated eyes were examined from the 14th day to 3 months after the beginning of drug administration under light and electron microscopy. The first change of retina was the appearance of membraneous cytoplasmic body (MCB) in the cytoplasm of ganglion, amacrine, bipolar and horizontal cells. MCB might be degenerated lysosome. They showed lamellar figures or crystalline lattice-like structures. Secondarily, these MCB appeared in the inner segments of photoreceptor cells. The outer segments of rod cells disappeared, and then those of cone cells. Although photoreceptor cells were diminished in number in advanced degeneration, the cells of inner nuclear layer and ganglion cells were maintained in number. The presence of MCB dose not mean death of cells. The retinal pigment epithelial cells contained MCB in its cytoplasm only in severe degenerative cases, and did not show other remarkable changes. MCB also appeared in the cytoplasm of pericytes of retinal vessels. Chloroquine is considered to damage directly photoreceptor cells most severely.

DISRUPTION OF BLOOD-RETINAL BARRIER AT THE RETINAL PIGMENT EPITHELIUM AFTER SYSTEMIC UREA INJECTION

Electron microscopic studies were done on the structural alterations of retinal pigment epithelial cells occurring under osmotic stress utilizing rhesus monkeys and lanthanum nitrate as a tracer. Fluorescein fundus angiography revealed leaking points of fluorescein from the choroid to the retina, and various degenerative alterations of the retinal pigment epithelial cells were observed in these leaking areas. Some cells included many vacuoles in their cytoplasm, but diffusion of lanthanum from the choroid to the outer segment of visual cell was obstructed by the tight junctions between the retinal pigment epithelial cells. Other cells were severely damaged with ruptured membranes. Abundant tracer material diffused through the cytoplasm into the subretinal space. Other cells were broken down in an isolated manner and here marked extravasation of lanthanum into the subretinal space was observed.

Freeze-fractured replica of Schlemm's canal and the trabecular meshwork in the primate.

Using a freeze-fracture technique, the fine structure of the Schlemms canal and trabecular meshwork in the monkey was studied with special reference to intercellular junctions. Gap junctions were observed between the endothelial cells of Schlemms canal and between the trabecular meshwork cells. Maculae occludentes were rarely observed between the endothelial cells of the inner wall of Schlemms canal. The trabecular meshwork cells appeared to act in an electrotonic and metabolic syncytium. Die vorliegende Arbeit beschreibt mit Hilfe der Gefrierätzmethode die Feinstruktur des Schlemmschen Kanals und des Trabekelwerkes unter besonderer Berücksichtigung der Zellverbindungen. “Gap junctions” fanden sich zwischen den Endothelzellen des Schlemmschen Kanals und zwischen den Zellen des Trabekelwerkes. Vereinzelt fanden sich Maculae occludentes zwischen den Endothelzellen der Innenfläche des Schlemmschen Kanals. Wahrscheinlich kommt den Zellen des Trabekelwerkes die Funktion eines elektrotonischen und metabolischen Synzytiums zu.

Synaptic vesicle exocytosis in goldfish photoreceptor cells.

The photoreceptor cell endings of goldfish retinas were examined using ultrathin and freeze-fracture techniques. The synaptic vesicles could be seen lying closely packed in rows on each side of the synaptic ribbon, and the vesicles nearest to the end of the ribbon could be seen to be in direct contact with the presynaptic membrane. The openings of the synaptic vesicles to the synaptic cleft could also be observed at the presynaptic membrane nearest to the end of the ribbon, that is, 100 nm distant from the center of the apex. In the freeze-fractured replicas of the presynaptic membrane, the band-shaped membrane particle aggregation was seen at the apex and a row of the crater-like or circular protuberances were observed at each side of the apex. These structures were considered to be changes of the presynaptic membrane organizations according to the synaptic vesicle exocytosis. It may be possible that the synaptic vesicle exocytosis in the photoreceptor cells occurs at the sites of the presynaptic membrane nearest to the apical end of the synaptic ribbon, and that the exocytotic sites form a line on each side of the apex. Die Fotorezeptorzellendigungen in der Goldfischretina wurden mit Ultradünnschnitten und unter Anwendung der Gefrierätztechnik untersucht. Die synaptischen Vesikel finden sich dicht gepackt in Reihen auf jeder Seite der synaptischen Bänder; die Vesikel am Ende der synaptischen Bänder besitzen dabei einen direkten Kontakt mit der präsynaptischen Membran. Öffnungen zwischen synaptischen Vesikeln und der präsynaptischen Membran konnten auch nahe der Enden der synaptischen Bänder beobachtet werden: dies entspricht etwa einer Distanz von ca. 100 nm vom sog. “Apex’. In Gefrierbrüchen der präsynaptischen Membran kann eine bandartige Aggregation von membrangebundenen Partikeln im Bereich des ‘Apex’ nachgewiesen werden; zusätzlich findet sich eine Reihe von kraterartigen bzw. zirkulären Protuberanzen auf jeder Seite des ‘Apex’. Diese Strukturen werden als Veränderungen in der Organisation der präsynaptischen Membran im Sinne der Exocytose von synaptischen Vesikeln angesehen. Es ist möglich, daß die Exocytose von synaptischen Vesikeln in Fotorezeptorzellen in Bereichen der synaptischen Membran stattfindet, die nahe dem aphikalen Ende der synaptischen Bänder liegen und daß die Bereiche, in denen die Exocytose stattfindet, Linien auf jeder Seite des ‘Apex’ bilden.

Electron microscopic studies on celestial goldfish retina-a possible new type of retinal degeneration in experimental animals.

The celestial goldfish (Carassius auratus) is one of the varieties of telescopic-eye goldfish, and a product of artificial selection. Their eyes are soft and telescopic anterior to dorsal. Abnormally thin retinas of celestial goldfish have only a small number of photoreceptor cells, and show various stages of retinal degeneration. The outer segments are short and markedly disorganized or often missing. Macrophages lies close to the outer or inner segments in single or clumped states, and sometimes replace the photoreceptor layer and/or the pigment epithelial layer. In a highly degenerated retina, the macrophages migrate to the inner layers or to around retinal vessels on the vitreous surface of the retina. The morphology of the celestial goldfish retina is regionally highly variable and also varies greatly between individuals. It is possible that this is a new type of primary retinal degeneration in experimental animals, although accompanying ocular enlargement.

FREEZE‐FRACTURE REPLICA OF THE RAT CORNEA

The line structures of the rat cornea, with special reference to their intercellular junctions, were studied using the freeze‐fracture technique. At the corneal epithelium, gap junctions could be observed between the adjacent cells. At the stroma, crater‐shaped depressions (between 300 and 500 Ä in diameter) with pipe‐like appearing structures connecting the lamellae were found. Intercellular junctions existing between the endothelial cells at the area near the anterior chamber are postulated to be the fascia (macula) occludens.

The developmental changes of ERGs on spontaneous retinal degeneration of Celestial goldfish

The Celestial goldfish, which belongs to the family of telescope-eye goldfish, displays unique spontaneous retinal degeneration associated with developmental anterodorsal protrusion of the eyeball. We observed concurrent changes in the electroretinogram. At the age of 75 days, the retinal layer was developed fully and the electroretinogram exhibited b-wave dominance similar to that of adult common goldfish. At the age of 105 days, when the eyeball began to protrude laterally, the b-wave amplitude decreased to 52% of its earlier developmental stage, with a prolonged peak time. Histologic change was observed in the retinal pigment epithelium and photoreceptor layers. At the age of 135 days, when the eyeball protruded further anterodorsally, the histologic changes extended to all retinal layers and the ERG b-wave was extinguished. Electrophysiologic and histologic changes in the Celestial goldfish eye were found to be proportional to the grade of eye protrusion. Since similar goldfish do not show these changes, however, the Celestial goldfish may be a new model of hereditary retinal degeneration.

Specialized intramembrane organizations of the cone presynaptic membrane in the pigeon retina

The presynaptic membranes of the cone cell endings of the pigeon retina were investigated using the freeze-fracture technique.En face views of the cytoplasmic leaflet (P-face) of the split presynaptic membrane revealed several specialized membrane organizations, 1. membrane particle aggregates composed of 10–20 particles which were larger than the usual ones seen in the cell membrane, 2. fenestration-like circular structures of 30–50 nm in diameter which were not surrounded by membrane particles. 3. similiar circular structures as described above but which were accompanied by a few membrane particles on the circular margin and were considered to be an intermediate form of the first and second membrane structures. These three structures appeared simultaneously in one fracture plane of the presynaptic membrane; were situated at the same intervals from one another and were approximately equal in size to synaptic vesicles (30–50 nm). These findings strongly suggested that these three structures were serial events in presynaptic membrane organization. When fortuitous cross fractures exposed both the P-face of the presynaptic membrane and the adjacent cytoplasm of the cone ending, fusion of the synaptic vesicles to the presynaptic membrane was observed, and was considered to be the opening of the synaptic vesicle to the synaptic cleft. These openings were also situated at the same distance as the structures described above. These findings demonstrate the process of exocytosis of the synaptic vesicles by which the chemical transmitter is probably released to the synaptic cleft. Mit Hilfe der Gefrierätzungsmethode wurde die präsynaptische Membran von Zapfen-Endkolben der Taubenretina untersucht. Spaltbilder der dem Zapfen-Zytoplasma aufliegenden präsynaptischen Membran lassen mehrere spezialisierte Membranstrukturen erkennen: 1. Ansammlungen von ca. 10–20 Partikeln, die größer sind als die Partikel die man üblicherweise im Bereich von Membranen antrifft 2. Fenestrationsartige kreisförmige Strukturen mit einem Durchmesser von ca. 30–50 nm. Im Bereich dieser Strukturen finden sich keine Partikel. 3. Kreisförmige Strukturen wie unter 2) beschrieben, an deren Rand sich jedoch wenige Partikel finden. Ansammlungen von ca. 10–20 Partikeln, die größer sind als die Partikel die man üblicherweise im Bereich von Membranen antrifft Fenestrationsartige kreisförmige Strukturen mit einem Durchmesser von ca. 30–50 nm. Im Bereich dieser Strukturen finden sich keine Partikel. Kreisförmige Strukturen wie unter 2) beschrieben, an deren Rand sich jedoch wenige Partikel finden. Die dritte Struktur wird als Zwischenform zwischen der ersten Struktur und der zweiten Struktur aufgefaßt. Alle drei Strukturen erscheinen simultan auf einem Gefrierbruch der präsynaptischen Zapfenmembran, sind in regelmäßigen Abständen zueinander angeordnet und von annähernd gleicher Größe wie die synaptischen Vesikel (30–50 nm). Diese Befunde lassen vermuten, daß es sich bei den genannten Strukturen um ineinander übergehende Organisationsformen der präsynaptischen Zapfen-Membran handelt. In Gefrierbrüchen die sowohl das oben beschriebene Spaltbild der präsynaptischen Zapfen-Membran als auch das daran angrenzende Zytoplasma der Zapfen darstellen, konnte die Fusion der synaptischen Vesikel mit der präsynaptischen Membran beobachtet werden. Sie werden als Öffnung der synaptischen Vesikel in den synaptischen Spalt angesehen. Diese Öffnungen waren in gleichen Abständen wie die oben beschriebenen Strukturen der Zapfen-Membran angeordnet. Die Ergebnisse der vorliegenden Untersuchungen zeigen den Prozeß der Exozytose von synaptischen Vesikeln, in deren Verlauf wahrscheinlich der chemische Transmitter in den synaptischen Spalt freigesetzt wird.