Horst Helbig

Primary vitrectomy without scleral buckling for rhegmatogenous retinal detachment

Abstract• Background: Pars planta vitrectomy has evolved as an alternative method in the treatment of more complicated rhegmatogenous retinal detachments. We report a series of patients who underwent primary vitrectomy with gas tamponade without the use of additional scleral buckling. • Methods: A retrospective study of 53 patients with a follow-up of 6–45 months (mean 17.8 months) was carried out. Preoperative findings included unusual, multiple or large breaks, vitreous haemorrhage, proliferative vitreoretinopathy and bullous retinal detachment. Preoperative visual acuity was between light perception and 1.0, with 30% (16/53) of patients with 0.4 or better. • Results: Retinal reattachment was achieved in 64% of cases (34/53) with one and in 92% (49/53) with one or more operations. Final visual acuity was between light perception and 1.0, with 41% (22/53) of patients with 0.4 or better. Cataract formation occurred in 86% (37/43) of all patients with a clear lens preoperatively. Macular pucker was noted in 11 % (6/53) and postoperative proliferative vitreoretinopathy causing redetachment in 6% (3/53).• Conclusion: With primary vitrectomy, a high final anatomical success rate with few intraoperative complications can be achieved in more complicated forms of rhegmatogenous retinal detachment. The major drawback of the procedure is the high incidence of post-operative cataract formation.

Regulation of cytoplasmic pH of cultured bovine corneal endothelial cells in the absence and presence of bicarbonate

SummaryIntracellular pH (pHi) in confluent monolayers of cultured bovine corneal endothelial cells was determined using the pH-dependent absorbance of intracellularly trapped 5(and 6)carboxy-4′,5′-dimethylfluorescein. Steady-state pH was 7.05±0.1 in the nominal absence of bicarbonate, and 7.15±0.1 in the presence of 28mm HCO3−/5% CO2. Following an acid load imposed by a NH4Cl prepulse, pHi was regulated in the absence of HCO3− by a Na+-dependent process inhibitable to a large extent by 1mm amiloride and 0.1mm dimethylamiloride. In the presence of 28mm HCO3−/5% CO2, this regulation was still dependent on Na+, but the inhibitory potency of amiloride was less. DIDS (1mm) partially inhibited this regulation in the presence, but not in the absence of bicarbonate. With cells pretreated with DIDS, amiloride was as effective in inhibiting recovery from acid load as in the absence of HCO3−. The presence of intracellular Cl− did not appreciably affect this recovery, which was still sensitive to DIDS in the absence of Cl−. Removal of extracellular Na+ led to a fall of pHi, which was greatly attenuated in the absence of HCO3−. This acidification was largely reduced by 1mm DIDS, but not by amiloride. Cl removal led to an intracellular alkalinization in the presence of HCO3−. The presence of a Cl−/HCO3− exchanger was supported by demonstrating DIDS-sensitive36Cl− uptake into confluent cell monolayers. Thus, bovine corneal endothelial cells express three processes involved in intracellular pH regulation: an amiloride-sensitive Na+/H− antiport, a Na−−HCO3− symport and a Cl−/HCO3− exchange, the latter two being DIDS sensitive.

K+-conductance and electrogenic Na+/K+ transport of cultured bovine pigmented ciliary epithelium

SummaryUsing intracellular microelectrode technique, we investigated the changes in membrane voltage (V) of cultured bovine pigmented ciliary epithelial cells induced by different extracellular solutions. (1)V in 213 cells under steady-state conditions averaged −46.1±0.6 mV (sem). (2) Increasing extracellular K+ concentration ([K+]o) depolarizedV. Addition of Ba2+ could diminish this response. (3) Depolarization on doubling [K+]o was increased at higher [K+]o (or low voltage). (4) Removing extracellular Ca2+ decreasedV and reduced theV amplitude on increasing [K+]o. (5)V was pH sensitive. Extra-and intracellular acidification depolarizedV; alkalinization induced a hyperpolarization.V responses to high [K+]o were reduced at acidic extracellular pH. (6) Removing Ko+ depolarized, Ko+ readdition after K+ depletion transiently hyperpolarizedV. These responses were insensitive to Ba2+ but were abolished in the presence of ouabain or in Na+-free medium. (7) Na+ readdition after Na+ depletion transiently hyperpolarizedV. This reaction was markedly reduced in the presence of ouabain or in K+-free solution but unchanged by Ba2+. It is concluded that in cultured bovine pigmented ciliary epithelial cells K+ conductance depends on Ca2+, pH and [K+]o (or voltage). An electrogenic Na+/K+-transport is present, which is stimulated during recovery from K+ or Na+ depletion. This transport is inhibited by ouabain and in K+-or Na+-free medium.

Evidence for Na/H exchange and Cl/HCO3 exchange in A10 vascular smooth muscle cells

In the present study we used the pH sensitive absorbance of 5(and6)-carboxy-4′,5′-dimethylfluorescein to investigate intracellular pH (pHi) regulation in A10 vascular smooth muscle cells: (1) The steady state pHi in A10 cells averaged 7.01±0.1 (mean±SEM,n=26) at an extracellular pH of 7.4 (28 mM HCO3/5% CO2). (2) Removal of extracellular sodium led to an intracellular acidification of 0.36±0.07 pH-units (mean±SEM,n=8). (3) pHi-Recovery after an acute intracellular acid load (by means of NH4Cl-prepulse) was reversibly blocked by 1 mM amiloride and was dependent on the presence of sodium. The velocity of pHi recovery increased with increasing sodium concentrations with an apparentKm for external sodium of about 30 mM and aVmax of about 0.35 pH units/min. These findings are compatible with a Na/H exchanger being responsible for pHi recovery after an acid load. (4) Removal of extracellular chioride induced an intracellular alkalinization of 0.23±0.03 pH-units (mean±SEM,n=10). The alkalinization was dependent on the presence of extracellular bicarbonate (5) Removal of chloride during pHi recovery from an alkaline load (imposed by acetate prepulse) stopped and reversed pHi backregulation. Chloride removal had no effect in the absence of bicarbonate or in the presence of 10−4 M DIDS, suggesting that the effects were mediated by a Cl/HCO3 exchanger. In conclusion we have demonstrated evidence for a Na/H exchanger and a Cl/HCO3 exchanger in A10 vascular smooth muscle cells.

Endothelin depolarizes membrane voltage and increases intracellular calcium concentration in human ciliary muscle cells

The ciliary muscle which is involved in accommodation and regulation of aqueous humour outflow resistance resembles smooth muscle in other parts of the body. In the present investigation we used an established primary cell line (H7CM) to study the effects of endothelin, a novel vasoconstrictor peptide, on membrane voltage (V) and intracellular calcium in cultured human ciliary muscle cells. Membrane voltage was measured in confluent monolayers of H7CM cells using conventional microelectrodes. Intracellular calcium concentration [( Ca]i) was measured in single H7CM cells using the fluorescent calcium indicator fura-2. Under resting conditions V averaged -66.9 +/- 0.7 mV (mean +/- SEM, n = 125). Endothelin (10(-10)-10(-6)M) induced a dose-dependent reversible membrane voltage depolarization and a dose-dependent rise in [Ca]i. The initial calcium peak was followed by a recovery phase during which oscillations of [Ca]i occurred. The initial calcium peak was not dependent on the presence of extracellular calcium and was not abolished in the presence of the calcium antagonist verapamil (10(-4)M). Thus it is probably mediated by a release of calcium from intracellular reservoirs. We conclude that cultured human ciliary muscle cells express a functional endothelin receptor.

Characterization of Cl−HCO3− exchange in cultured bovine pigmented ciliary epithelium

Many recent data indicate that transport of Cl- across the ciliary epithelium plays an important role in aqueous humor formation. We used 36Cl to investigate the pathways for Cl- transport in confluent monolayers of cultured bovine pigmented ciliary epithelial cells. Cl- uptake mainly occurred via a mechanism with typical characteristics of an anion exchanger, and could be stimulated by an outwardly directed HCO3- gradient. One mM SITS and 1 mM DIDS inhibited Cl- uptake by some 80-90%, the latter with an IC50 of about 20 microM. HCO3- stimulated Cl- uptake could be partly inhibited for furosemide and to a lesser extent by bumetanide, indicating an action of loop-diuretics on the anion exchanger. 36Cl- uptake was cis-inhibited by the halides Cl-, I- and Br-, by NO3-, formate and acetate. Inhibition of Cl- uptake by extracellular HCO3- was less effective in the absence of extracellular Na+, suggesting that not only HCO3- but also NaCO3- binds to the carrier. SO2/4-, cyclamate and gluconate did not significantly reduce Cl- uptake via the anion exchanger. DIDS-senstive Cl- uptake showed saturation kinetics with respect to the Cl- concentration with an apparent Km of 8 mM. Cl- efflux could be stimulated by external Cl- and HCO3- and was inhibited by DIDS. Thus, cultured bovine pigmented ciliary epithelial cells express a Cl-/HCO3- exchanger. A possible role of this carrier system for aqueous humor formation is discussed [corrected].

Rubeosis iridis after vitrectomy for diabetic retinopathy.

Abstract · Background: Iris rubeosis and neovascular glaucoma (NVG) are serious complications of vitrectomy for proliferative diabetic retinopathy. The present study analyzes incidence and risk factors of these complications. · Methods: Preoperative and postoperative iris rubeosis were compared in 389 diabetic eyes after vitrectomy. Minimum follow-up was 6 months (median 26 months). Risk factors were studied using multivariate logistic regression analysis. · Results: Following vitrectomy, in 8.5% of the eyes stromal iris rubeosis developed de novo; NVG occurred in 5%. Significant risk factors for postoperative rubeosis were preexisting iris neovascularizations and postoperative retinal detachment. Six months after surgery, regression of preexisting iris rubeosis was observed in 57% of the eyes. In eyes without preoperative iris rubeosis, progression was found in 13% of cases 6 months postoperatively. · Conclusion: With current surgical techniques iris rubeosis is more commonly regressive than progressive after vitreous surgery in diabetic eyes.

Kinetic properties of Na+/H+ exchange in cultured bovine pigmented ciliary epithelial cells

Uptake studies with22Na were performed in cultured bovine pigmented ciliary epithelial cells, in order to characterize mechanisms of Na+ transport. A large part of Na+ uptake was sensitive to amiloride, quinidine and harmaline. Na+ uptake was stimulated by intracellular acidification (using the NH4+ prepulse technique), and was inhibited with increasing extracellular proton concentration. Decreasing extracellular pH from 7.5 to 7.0 increased the apparentKM for Na+ from 38 to 86 mM without considerable changes inVmax. In the presence of 5 mM Na+ half maximal inhibition of amiloride sensitive Na+ uptake by extracellular protons was observed at a hydrogen concentration of 50 nM. In the presence of 50 mM Na+ the proton concentration necessary for 50% inhibition was 139 nM. Thus, the mode of inhibition of extracellular H+ seemed to be competitive with aKi of 20–40 nM. 10 μM amiloride increased the apparentKM for Na+ from 33 mM to 107 mM, whileVmax remained nearly unchanged. IC50 for amiloride was 6 μM at 5 mM Na+ and 36 μM in the presence of 150 mM Na+. Thus, amiloride behaves as a competitive inhibitor with aKi of about 5 μM. The affinities of Na+ to the transport site (KM≈16 mM), to the inhibitory site for protons (KM≈21 mM), and to the inhibitory site for amiloride (KM≈26 mM) were in the same order of magnitude.In summary, we have presented evidence for the presence of a Na+/H+ exchanger in cultured bovine pigmented ciliary epithelial cells. The kinetic data suggest the presence of only one common extracellular binding site for Na+, H+ and amiloride.

Sodium bicarbonate cotransport in cultured pigmented ciliary epithelial cells

Uptake of 22Na+ was studied in cultured bovine pigmented ciliary epithelial cells (PE) in HCO3-containing media. Two components of Na+-uptake were stimulated by intracellular acidification (NH4+-prepulse): One was amiloride-sensitive, the other DIDS-sensitive. The amiloride-sensitive component of Na+-uptake probably represents Na+/H+-exchange, which has previously been characterized in PE. The second, DIDS-sensitive component stimulated by intracellular acidification, was Cl- and HCO3--dependent. We conclude that a stilbene-sensitive, Cl--dependent Na+-HCO3--cotransport is present in PE. This transport could play an important role in aqueous humor formation.

Coupling of 22Na and 36Cl uptake in cultured pigmented ciliary epithelial cells: a proposed role for the isoenzymes of carbonic anhydrase.

Uptake studies with 22Na and 36Cl were performed in cultured bovine pigmented ciliary epithelial cells (PE) to investigate interdependence of Na+ and Cl- transport. (1) 22Na uptake into NaCl depleted cells was stimulated by Cl-. This stimulation was abolished by the simultaneous application of amiloride (1 mM) and bumetanide (0.1 mM), indicating two independent mechanism for Cl- stimulated Na+ uptake: loop diuretic sensitive Na+/Cl- symport and an indirect stimulation of Na+/H+ exchange by Cl-. The latter component of Cl- stimulated Na+ uptake was HCO3- dependent. (2) 36Cl uptake was increased by extracellular Na+. Na+-stimulated Cl- uptake also consisted of two components. One was bumetanide sensitive and the other was blockable by amiloride and partly inhibited by the carbonic anhydrase (CA) inhibitor methazolamide (0.1 mM). (3) Homogenized PE cells were tested for biochemical CA activity using an electrometric method. The cytoplasmic as well as the membrane fraction contained specific CA activity. (4) A model is presented for Na+ and Cl- transport into PE: in addition to Na+/Cl- symport, Na+/H+ and Cl-/HCO3- double exchange may operate in the ciliary epithelium. The latter mechanism provides NaCl uptake into the cell in exchange for H+ and HCO3-, which recycle as CO2 across the membrane. This recycling of CO2 and HCO3-/H+ (and hence indirectly NaCl uptake) is facilitated by the cooperation between membrane bound and cytoplasmic CA.