Hai-Bo Chen

Effects of Isopropyl Unoprostone Ophthalmic Solution on Cultured Rabbit Corneal Epithelial Cells

Purpose: To investigate the effects of isopropyl unoprostone (referred to as unoprostone) ophthalmic solution on the barrier function of cultured rabbit corneal epithelium grown on permeable supports. Methods: Rabbit corneal epithelial cells cultured on collagen-coated filter inserts were administered one of the following for 30 min: unoprostone in vehicle solution (polysorbate 80), unoprostone in vehicle solution with a preservative (benzalkonium chloride), preservative only or vehicle only. For a control, no chemicals were added to the medium. After administration, the transepithelial electrical resistance (TER) measurement, a sensitive method by which to investigate the barrier function, and morphological observation using phase-contrast microscopy were performed before exposure and at 0.5, 1, 3, 6, 12, 24, 48 and 72 h after exposure. The transmission electron-microscopic observation was performed before and 72 h after exposure in all experimental conditions. Results: The cells exposed to unoprostone with the preservative showed a significant decrease in the TER, although no morphological changes were observed. The corneal epithelial cells exposed to unoprostone without preservative, the vehicle only or the preservative only did not show any differences from the control group at any measurements. Conclusion: The corneal barrier function is damaged by a combined solution of unoprostone and preservative, but not by a single solution of unoprostone, in vitro.

Ultrastructural Identification of Substance P Containing Nerves in the Guinea Pig Trabecular Meshwork

We aimed to provide a detailed analysis of substance P (SP)-containing nerves in the trabecular meshwork by ultrastructural immunohistochemistry and capsaicin treatment for chemical ablation of the sensory nerves. Numerous myelinated and unmyelinated nerves were observed inside the sheets and intertrabecular spaces of the trabecular meshwork, and the inner side of Schlemm’s canal. SP-like immunoreactive products were identified in some of these nerves and associated with numerous vesicles of different sizes, a few mitochondria and numerous neurotubili. After the capsaicin treatment, SP-like immunoreactive nerves persisted and no degenerated SP-like immunoreactive nerves were noted. On the basis of the ultrastructural features and the results of capsaicin treatment, it seems that these nerves are most probably autonomic in origin. Autonomic efferent SP-containing nerves are confirmed directly located in the trabecular meshwork; these have not been previously described at electron microscopic level. SP in autonomic efferent nerves might act as a neurotransmitter or neuromodulator for intraocular pressure regulation.

Histochemical study on rat tear film and ocular surface epithelial cells

PURPOSE To investigate the histochemistry of the tear film, conjunctival goblet cells and corneal superficial epithelial cells by an in vivo cryofixation with the freeze-substitution method. METHODS Following rapid freezing of the eyeballs by an isopentane-propane mixture cooled by liquid nitrogen directly, the specimens were freeze-substituted and then embedded in Lowicryl K4M resin at low temperature, or in epoxy resin for histochemical study, using light and transmission electron microscopy. RESULTS The tear film was weakly positive with periodic acid-Schiff staining, but negative with alcian blue at pH 2.5 or pH 1.0 and high iron diamine staining. Conjunctival goblet cells were positively stained by all these techniques. There was a gradually weakening staining intensity of mucus from the goblet cells to the conjunctival surface. With the periodic acid-thiocarbohydrazide-silver protein method the tear film was weakly stained, while glycocalices, vesicles, glycogen in epithelial cytoplasm and the mucus secretory granules of goblet cells were all intensely stained. CONCLUSIONS The tear film consists mainly of dilute mucus and contains either neutral or acid glycoproteins.

Ultrastructural Studies on the Corneal Superficial Epithelium of Rats by in vivo Cryofixation with Freeze Substitution

The method of in vivo cryofixation with freeze substitution (VC-FS) was used to examine the natural ultrastructure of the corneal superficial epithelium under the transmission electron microscope, and the results have been compared with the conventional method. With the VC-FS method, the ultrastructure down to 8 microns in depth was well preserved in the specimen. There was a layer of homogeneous material thought to be tear film on the corneal surface. Except for the intercellular spaces presenting under the first and sometimes also the second layers of the epithelial cells, other adjacent cells were in close contact without any spaces between them or between the desmosomes. In contrast, when using the conventional method, intercellular spaces were found between adjacent cells in all the layers of epithelium, and no tear film remained on the corneal surface. VC-FS is considered to be a better way to preserve the structure of tissue in its natural state than the conventional method and to be a useful method for the morphological study of the corneal epithelium and the tear film. Intercellular spaces, a kind of artifact, can be reduced by this method.

Ultrastructural Study of Axonal Cytoskeletons in the Optic Nerve Damaged by Acutely Elevated Intraocular Pressure Using the Quick-Freezing and Deep-Etching Technique

The aim of this study was to examine the ultrastructure of axonal cytoskeletons in guinea pig optic nerve damage induced by acutely elevated intraocular pressure (IOP) employing the quick-freezing and deep-etching method. An IOP of 60 mm Hg was maintained for 4 h. The unmyelinated axoplasm of the optic nerve at the normal IOP was filled with longitudinally oriented neurofilaments and bundled microtubules, which were cross-linked by numerous cross-bridges. Membranous organelles, such as mitochondria and vesicles, were scattered in the axoplasm. These organelles were similarly interconnected with microtubules or neurofilaments by the cross-bridges. The unmyelinated axoplasm of the optic nerve following acutely elevated IOP was shown to include intraaxonal collections of various membranous organelles and reduction of microtubules and neurofilaments. The cross-bridges linking microtubules, neurofilaments and membranous organelles in such axoplasm appeared to be dramatically reduced in number. Thus, it is suggested that the ultrastructural changes of axonal cytoskeletons in some optic nerves following elevated IOP might include the reduction of microtubules, neurofilaments and their associated cross-bridges.

Ultrastructural Study of Basal Lamina of Retinal Pigment Epithelium

The ultrastructure of basal lamina (BL) of retinal pigment epithelium (RPE) in guinea pigs was studied by the quick-freezing, deep-etching (QF-DE) method. The retinal tissue was quickly frozen, fractured and deeply etched. A replica was prepared by shadowing with platinum and carbon. Electron micrographs of the BL of RPE provide a three-dimensional (3-D) ultrastructure. The lamina lucida was filled with traversing filamentous structures, which connected the basal infolding of RPE with the lamina densa. The lamina densa consisted of a 3-D meshwork structure. It is suggested that the traversing filamentous structures in the lamina lucida might play a role in anchoring the basal infolding of RPE and in maintaining the normal architecture.

Three-Dimensional Arrangement of Collagen Fibrils in Human Ciliary Body

The purpose of this study is to visualize the three-dimensional arrangement of collagen fibrils in aged human ciliary body and discuss their significance. The ciliary bodies obtained from two human eyes were treated with a NaOH cell-maceration method for 7 days, then prepared conventionally for light and scanning electron microscopy. The general morphology of the collagen tissue in the ciliary body appeared almost the same as that normally observed. Cellular elements were completely removed, but collagen fibrils were well preserved. In the stroma of the ciliary body, collagen fibrils were arranged irregularly. In the areas of the radial and circular ciliary muscles, considerable numbers of collagen fiber bundles were observed running in a circular direction. A honeycomb structure was seen in the pars plana, the walls and base of which were formed by interweaving collagen fibrils. The results suggested that collagen fibrils in the aged human ciliary body may be largely involved in the presbyopia.

Autonomic nerves containing substance P in the aqueous outflow channels and scleral spur of the guinea pig.

PURPOSE To study the innervation of the aqueous outflow channels and scleral spur by autonomic nerves containing substance P. METHODS The experiments were conducted on guinea pigs. Immunohistochemical techniques and capsaicin-ablation of the sensory nerves were used to investigate nerves containing substance P at the light and electron microscopic level. RESULTS Nerves containing substance P were observed in the aqueous outflow channels and scleral spur regions. The fine structures of these nerves had a similar pattern in those regions, and the labeled elements had abundant small vesicles, a few large vesicles, and numerous neurotubuli. Following capsaicin treatment, these nerves remained intact and no degenerated substance P-like immunoreactive nerves were found. CONCLUSIONS Nerves containing substance P are most likely of autonomic origin in view of their ultrastructural features. These nerves innervate the aqueous outflow channels and scleral spur, and are probably important for neurogenic influences on the intraocular pressure by the autonomic nervous system.

Acid Phosphatase Localization in Accumulated Membranous Organelles of Optic Nerve Axons Following Acute Elevation of Intraocular Pressure

Acid phosphatase localization in accumulated membranous organelles of optic nerve axons of guinea pigs following acute elevation of intraocular pressure (IOP) was determined, employing light and electron microscopic enzymic cytochemistry with beta-glycerophosphate as a substrate. Positive reaction products appeared to accumulate in the region of the lamina cribrosa, as revealed with light microscopic enzyme cytochemistry. Electron microscopic enzyme cytochemistry also demonstrated that such reaction products mainly localized on multivesicular or multilamellar bodies and myelin-like structures in the unmyelinated optic nerve axons. Following an acutely elevated IOP, retrograde-moving membranous organelles in the optic nerve axons were found to contain AcPase, suggesting that these organelles could be degraded in the axons through the lysosomal pathway.

Ultrastructural Study of the Cytoskeleton of Optic Nerve Axons in Guinea Pigs as Revealed by a Quick-Freezing, Deep-Etching Method

The ultrastructure of the cytoskeleton of optic nerve axons in guinea pigs was examined by the quick-freezing and deep-etching (QF-DE) method. The optic nerve tissues were treated with 0.5% saponin before QF. In the replicas, the axoplasm, as observed with conventional ultrathin sections, was seen to be composed of longitudinally oriented microtubules (MT) and neurofilaments (NF). Thin and elaborate cross-linking structures were observed in the interstices between MTs, NFs, and membranous organelles. They consisted of two different types: NF-associated cross-linking structures (about 20-50 nm in length) and MT-associated structures (about 10-20 nm in length), and may play a role in the slow transport of NF and MT and fast transport of organelles, respectively.