Eiichi Shirasawa

Effects of the cytokines on the proliferation of and collagen synthesis by human cataract lens epithelial cells.

AIMS: To assess the effects of the cytokines, interleukin-1 (IL-1), IL-1 receptor antagonist (IL-1ra), transforming growth factor-beta 2 (TGF-beta 2) and basic fibroblast growth factor (b-FGF), on the mitosis of and collagen synthesis by lens epithelial cells (LECs) of human cataracts. METHODS: The anterior lens capsule with attached LECs was obtained by capsulotomy during cataract surgery and cultured. The cultures at 2 to 3 weeks before confluency were used for the experiments. To quantify the mitosis and collagen synthesis, the incorporation of 3H-thymidine and 3H-proline, respectively, into the LECs was measured by a scintillation counter at 48 hours and 24 hours, respectively, after addition of the cytokine at various concentrations into the incubation medium. RESULTS: IL-1 and b-FGF increased the mitosis and collagen synthesis significantly, but IL-1ra significantly decreased the mitosis while leaving the collagen synthesis intact. TGF-beta 2 decreased the mitosis significantly, but increased the collagen synthesis significantly. CONCLUSION: These cytokines may play an important role in an autocrine or paracrine pathway in the proliferation of residual LECs after cataract surgery. Elucidation of the role of these cytokines may lead to the development of new therapies for the prevention of secondary cataract.

Development of Electroretinographic Alterations in Streptozotocin-lnduced Diabetes in Rats

Development of electroretinographic alterations in 9-week-old experimental diabetic rats was studied for up to 6 weeks after a single intraperitoneal injection of streptozotocin (STZ, 60 mg/kg). The amplitudes and the peak latencies of the a and b waves in the diabetic rats did not differ significantly from those in the control rats. In contrast, the diabetic rats showed a significantly smaller amplitude of the second oscillatory potential (OP) 6 weeks after STZ treatment, and furthermore significantly delayed OP peaks as early as 2-3 weeks after STZ treatment. Vitreous fluorophotometric abnormality developed 6 weeks after STZ treatment. None of the diabetic rats had fundus angiographic changes. These results suggest that hyperglycemia or its related changes rapidly affects the light-induced electrical activities of the retina.

Effect of intraocular sustained release of indomethacin on postoperative inflammation and posterior capsule opacification

Purpose: To assess whether the sustained release of indomethacin significantly reduces postoperative inflammation and posterior capsule opacification (PCO). Setting: Nishi Eye Hospital, Jinshikai Medical Foundation, Osaka, Japan. Methods: A 7 mm diameter, 1 mm thick polylactic‐polyglycolic acid disk containing 7 mg of indomethacin was implanted in five rabbit eyes after continuous curvilinear capsulorhexis and phacoemulsification. The disk and an IOL placed above it were implanted in the capsular bag. The contralateral eyes, which served as controls, received a disk without indomethacin and the same type IOL in the same manner. Results: The indomethacin was fully released within 3 weeks in vitro, a release rate of about 14 &mgr;g/h. Postoperatively, aqueous flare intensity was significantly lower at days 2, 3, and 4 and at weeks 1, 2, and 3. Prostaglandin E2 was not detectable in the aqueous humor of the indomethacin‐treated eyes on day 3 and at week 4. In the control eyes, mean concentration was 491 pg/ml ± 54 (SD) and 990 ± 243 pg/ml, respectively. Histopathological examination showed no significant decrease in PCO. Conclusion: Although sustained release of indomethacin significantly decreased inflammation, it did not reduce PCO.

Effects of diclofenac sodium and indomethacin on proliferation and collagen synthesis of lens epithelial cells in vitro

Abstract We investigated the effects of diclofenac sodium and indomethacin on the proliferation of and collagen synthesis by lens epithelial cells (LECs) of human cataracts in culture. The anterior capsule with attached LECs, obtained by anterior capsulotomy during cataract surgery, was cultured directly without cell dispersion. When the culture became almost confluent, diclofenac sodium or indomethacin at various concentrations was added to the incubation medium. The incorporation of 3H‐thymidine and 3H‐proline into the LECs was measured after the cells were labeled with these radioactive materials. Both drugs greatly suppressed the incorporation of 3Hthymidine and of 3H‐proline, indicating that they inhibit cell division and collagen synthesis by LECs. Both drugs may help prevent posterior capsule opacification.

Selective prostaglandin D2 receptor stimulation elicits ocular hypotensive effects in rabbits and cats

The effects of the selective prostaglandin D2 (DP) receptor agonists, 572C85 ((+/-)-5-(3-carboxypropylthio)-1-(2-cyclohexyl-2-hydroxyethyl- amino)hexahydrocyclopenta(d)imidazol-2(1H)-one) and 192C86 ((+/-)-5-(3-carboxypropylthio)-1-(2-cyclohexyl-2-hydroxyethylidene - amino)-3-ethylhexahydrocyclopenta(d)imidazol-2(1H)-one), were determined on intraocular pressure regulation in rabbits and cats. 572C85 (50 micrograms) in rabbits maximally lowered intraocular pressure by 4.3 mm Hg, and significantly for 4 h compared to control. In cats 572C85 had a similar effect. 192C86 (50 micrograms) reduced intraocular pressure by 2.8 mm Hg for 2 h in rabbits. Following exposure to the specific DP receptor antagonist, BW A868C ((+/-)-3-benzyl-5-(6-carboxyhexyl)-1-(2-cyclohexyl-2-hydroxyethylamin o)- hydantoin; 50 micrograms), which had no effect on intraocular pressure by itself, 572C85 (50 micrograms) did not reduce intraocular pressure in rabbits and cats. The intraocular pressure lowering effect of the mixed DP and EP receptor agonist, BW245C (5-(6-carboxyhexyl)-1-(3-cyclohexyl-3-hydroxypropyl)-hydantoin; 50 micrograms), in cats was suppressed by only 64% by BW A868C (50 micrograms). These results clearly show that the DP receptors in rabbit and cat eyes are involved in intraocular pressure regulation. However, under baseline conditions DP receptor activity does not contribute to this regulation.

Types of collagen synthesised by the lens epithelial cells of human cataracts.

AIMS/BACKGROUND--Residual lens epithelial cells (LECs) undergo fibrous proliferation after cataract surgery, resulting in capsular fibrosis. The purpose of this study was to determine the types of collagen produced in cultured LECs derived from human cataract LECs. METHODS--A circular section of the anterior capsule, about 5 mm in diameter, with LECs attached was obtained by anterior capsulotomy during cataract surgery and cultured directly without dispersion of the cells in a well, on the bottom of which a disc-shaped, thin plate of poly(methyl methacrylate) had been placed. At 5 to 6 weeks of culture, the proliferated cells of the culture were stained immunohistochemically with antibodies against human collagens I-VI by the avidin-biotin-peroxidase complex method. RESULTS--Collagens I, IV, V, and VI were positive in the cultured cells. Types IV and V were strongly present in almost all the cells whereas types I and VI were only observed in a few cells. Collagens II and III were negative. CONCLUSIONS--Since the lens capsule is known to be comprised of collagen IV, collagens I, V, and VI seem to be produced newly in culture. The capsular fibrosis seen after cataract surgery in vivo as a wound healing process of the lens capsule, may contain these types of collagens. The present culture model is useful for studying secondary cataract formation.

Effect of anti-inflammatory agents on corneal wound-healing process after surface excimer laser keratectomy

PURPOSE To investigate the effect of anti-inflammatory agents on conjunctival inflammation and corneal haze formation after excimer laser keratectomy. SETTING Department of Ophthalmology, University of Tokyo Faculty of Medicine, Tokyo, Japan. METHODS After excimer laser keratectomy was performed in 21 rabbits (42 eyes), saline, betamethasone 0.1%, or diclofenac 0.1% was topically applied 6 times a day for 4 weeks and then 3 times a day for 8 weeks. The degree of conjunctival inflammation was determined 1, 2, 3, and 7 days after the keratectomy. The degree of corneal haze was quantitatively measured using a digital analyzer before and once a week after the keratectomy. The expression of type IV collagen in the corneas at baseline and 4 and 12 weeks after the keratectomy was examined immunohistochemically. RESULTS Compared with saline, betamethasone and diclofenac significantly decreased early-phase conjunctival inflammation. Betamethasone significantly inhibited corneal haze formation compared with saline at 3 to 5 and 8 to 12 weeks. Diclofenac did not inhibit corneal haze formation significantly. Although betamethasone tended to be more effective in inhibiting corneal haze formation and deposition of type IV collagen than diclofenac, there was no statistical difference between the 2 anti-inflammatory agents. CONCLUSION Topically applied betamethasone effectively suppressed corneal haze formation after excimer laser keratectomy. Diclofenac was not statistically effective in inhibiting corneal haze formation.

Effect of the cytokines on the prostaglandin E2 synthesis by lens epithelial cells of human cataracts.

BACKGROUND--Lens epithelial cells (LECs) derived from human cataracts have been reported to produce various cytokines and prostaglandin E2 (PGE2) in culture. The effects of IL-1, TGF-beta, and b-FGF on the PGE2 synthesis by LECs have been studied. METHODS--A circular piece of the anterior capsule with attached LECs was obtained by capsulotomy during cataract surgery and cultured. The primary, almost confluent, cultures were used for the study. The PGE2 concentration of the culture media for 24 h was measured after the addition of recombinant human IL-1 alpha, TGF-beta 2, or b-FGF at various concentrations. The PGE2 concentration was also measured in the media to which each cytokine and rabbit polyclonal anti-human antibodies against the corresponding cytokine had been added. RESULTS--The PGE2 concentration of the culture media after addition of IL-1 alpha at the concentration of 100 or 500 pg/ml (1765 (768) and 3071 (1121) pg/10(4) cells) or TGF-beta 2 at the concentration of 10 or 100 ng/ml (689 (264) and 750 (189) pg/10(4) cells) was significantly increased compared with that in the controls (67 (20) pg/10(4) cells). These effects were suppressed by the corresponding anticytokine antibodies. Basic FGF and anti-human b-FGF showed no significant effect on the PGE2 concentration. IL-1 and TGF-beta increased but b-FGF did not affect the PGE2 synthesis by LECs in culture. CONCLUSION--IL-1 and TGF-beta may participate in postoperative inflammation after cataract surgery by increasing PGE2 synthesis by residual LECs.

Characterization of Esterases Involved in the Hydrolysis of Dipivefrin Hydrochloride

We characterized the interaction of the prodrug dipivefrin hydrochloride (DPE) with esterase activity in the rabbit cornea. The esterases which were identified included: (1) cholinesterase, (2) acetylcholinesterase, (3) a mixture containing carboxylesterase, acetylesterase and arylesterase, and (4) a non-specific esterase. DPE suppressed all of their activities as well as that of the mixture containing carboxylesterase, acetylesterase and arylesterase, and a nonspecific esterase. However, its effect on cholinesterase was larger than on any of the other activities, suggesting that DPE is a better substrate for cholinesterase than for any of the other esterases. These measurements along with those of substrate-dependent inhibition of 14C-DPE hydrolysis indicated that the DPE-esterase interaction was competitive based on changes in the apparent Km values which were extracted from Lineweaver-Burk plots of esterase activity. The substrate for cholinesterase competed with DPE most strongly among substrates. These results seem to suggest that DPE is hydrolyzed by various corneal esterases, mainly cholinesterase.

Involvement of both L- and N-type voltage-dependent Ca2+ channels in KCl- and veratridine-evoked transmitter release from non-adrenergic, non-cholinergic nerves in the rabbit iris sphincter muscle

Abstract To determine which types of voltage-dependent Ca2+ channels mediate tachykinin release in the isolated rabbit iris sphincter muscle, we examined the effects of several Ca2+ channel modulators on contractions induced by either an elevation of the extracellular KCl concentration or application of the Na+ channel activator veratridine. Contractions caused by either 45.9mMKCl or veratridine (10μM) were inhibited by spantide (10μM), a tachykinin receptor antagonist, and capsaicin (10μM), a tachykinin-depleting agent, but were not changed by atropine. Nicardipine, an L-type Ca2+ channel blocker, inhibited contractions induced by KCl and veratridine in a concentration-dependent manner. ω-Conotoxin GVIA (1μM), an N-type Ca2+ channel blocker, inhibited only contractions induced by lower concentrations of KCl, both when applied alone and when combined with nicardipine. Bay K8644, an L-type Ca2+ channel activator, caused a spantide- and nicardipine-sensitive contraction in muscles partially depolarized with 15.9mMKCl, and enhanced contractions induced by 15.9mMKCl and veratridine (2μM). ω-Agatoxin IVA (0.3μM), a P-type voltage-dependent Ca2+ channel blocker, did not affect contractions induced by either KCl or veratridine. Contractions induced by exogenous substance P were not modified by any of the Ca2+ channel blockers or by Bay K8644. These results suggest that, in the rabbit iris sphincter muscle, L- and N-type voltage-dependent Ca2+ channels are involved in neurotransmitter release from tachykininergic nerves elicited by high KCl and by veratridine.