Joachim B. Grammer

Ionic mechanisms of electrical remodeling in human atrial fibrillation

OBJECTIVES Atrial fibrillation (AF) is associated with a decrease in atrial ERP and ERP adaptation to rate as well as changes in atrial conduction velocity. The cellular changes in repolarization and the underlying ionic mechanisms in human AF are only poorly understood. METHODS Action potentials (AP) and ionic currents were studied with the patch clamp technique in single atrial myocytes from patients in chronic AF and compared to those from patients in stable sinus rhythm (SR). RESULTS The presence of AF was associated with a marked shortening of the AP duration and a decreased rate response of atrial repolarization. L-type calcium current (ICa,L) and the transient outward current (Ito) were both reduced about 70% in AF, whereas an increased steady-state outward current was detectable at test potentials between -30 and 0 mV. The inward rectifier potassium current (IKI) and the acetylcholine-activated potassium current (IKACh) were increased in AF at hyperpolarizing potentials. Voltage-dependent inactivation of the fast sodium current (INa) was shifted to more positive voltages in AF. CONCLUSIONS AF in humans leads to important changes in atrial potassium and calcium currents that likely contribute to the decrease in APD and APD rate adaptation. These changes contribute to electrical remodeling in AF and are therefore important factors for the perpetuation of the arrhythmia.

Ocular absorption of cyclosporine A from liposomes incorporated into collagen shields.

We investigated the ability of liposomes to deliver the immunosuppressive agent cyclosporine A (CsA) to the cornea, anterior sclera, aqueous humor, and vitreous in rabbit eyes. One drop (10 microliters) of liposome-encapsulated CsA (CsA-LIP) or olive oil drops containing an equivalent concentration of CsA (CsA-DR) were administered at 15-minute intervals within the first hour and then one hourly over a 6-hour period. In addition, collagen shields soaked for 30 minutes in the liposome preparation (CsA-LIP-CS) were tested in vitro and in vivo as a new drug delivery approach. CsA levels were measured by fluorescence-immunoassay after 1, 3, or 6 hours of drug administration. CsA levels in this study were highest in cornea and anterior sclera. In animals receiving either CsA-DR or CsA-LIP, CsA levels generally increased from 1 to 6 hours. In animals receiving a single application of CsA-CS-LIP, CsA levels peaked at 3 hours and declined at 6 hours in cornea and sclera. CsA-LIP and CsA-CS-LIP delivered significantly higher levels of CsA to the cornea and sclera at 1 and 3 hours than CsA-DR. In aqueous and vitreous humor, CsA levels increased from 1 to 6 hours in animals receiving either CsA-DR or CsA-LIP. On the other hand, animals receiving a single application of CsA-CS-LIP had lower levels of CsA at 6 hours than at the earlier time points. Animals receiving CsA-LIP or CsA-CS-LIP had significantly higher levels of CsA in aqueous and vitreous humor at 1, 3, and 6 hours than animals receiving CsA-DR.(ABSTRACT TRUNCATED AT 250 WORDS)

Retinal toxicity of liposome-incorporated and free ofloxacin after intravitreal injection in rabbit eyes.

Background: Ofloxacin (OFLX) is a fluoroquinolone-antibiotic with a broad antimicrobial spectrum that may have a potential role in the treatment of bacterial endophthalmitis. However, its elimination half life after intravitreal injection is short. To prolong the intravitreal antibacterial level OFLX was incorporated into liposomes. This study was performed to investigate the retinal toxicity of liposome-incorporated and free OFLX. Materials and methods: OFLX was incorporated into multilamellar large vesicles. 0,1 ml of this suspension (= 180.2 μg OFLX) was injected into the midvitreous of rabbit eyes (n = 6). Free OFLX in doses of 100 μg, 500 μg and 1,000 μg was injected into the midvitreous of a second group of rabbit eyes (n = 18). The other eye served as a control and received empty liposomes or normal saline solution, respectively. Before injection and at the end of follow-up an ERG was obtained. After a follow-up of 1 day, 14 and 28 days the animals were perfused with glutaraldehyde and the eyes were examined by light- and transmission electron microscopy. Results: The ERG as well as the histologic studies did not reveal any pathological changes after injection of liposome-incorporated OFLX compared to the control eyes. Significant reduction of the ERG was observed after 500 μg free OFLX in 2 out of 6 eyes after 1 and 14 days, respectively, and in 2 eyes 1 day after 1,000 μg free OFLX. Three days after injection of 1,000 μg OFLX the retina showed focal destruction in 1 out of 6 eyes. In another eye with the same dose 14 days after injection the photoreceptor outer segments showed disorganisation. Conclusion: This study shows that liposome-incorporated OFLX did not have any retinal toxicity in this animal model. Free OFLX appears to have no retinal toxicity in rabbit eyes at a dose of 100 μg after intravitreal injection. Injection of higher doses resulted in ERG changes and marked retinal damage.

Analysis of interactions between the corneal epithelium and liposomes: Qualitative and quantitative fluorescence studies of a corneal epithelial cell line

Transcorneal drug transport is normally limited by the intrinsic permeation characteristics of the corneal epithelium. However, liposomes, i.e., phospholipid vesicles composed of phospholipid membranes, have recently attracted attention as carriers of topically applied agents. The present study therefore describes a qualitative and quantitative laboratory investigation of interactions between corneal epithelial cells and liposomes. The lipid bilayers and interior spaces of liposomes were labelled with different fluorophores. Fluorescence microscopy revealed a rapid uptake of rhodamine B-labelled liposome bilayer components by the epithelial cell membrane and the cytoplasm. Simultaneously, intracellular uptake of aqueous liposome content was indicated by uniform fluorescence of the cytoplasm due to carboxyfluorescein (CF). The fluorimetric experiments showed that the uptake of liposomes by SIRC cells depended on liposome concentrations and the time of exposure of the cells to the liposomes, and that saturation effect characteristics were present. Cell fluorescence dropped by approximately 45% when the incubation temperature of the cells was reduced from 37 degrees C to 4 degrees C. Both this phenomenon and a significant reduction in liposome uptake (p < 0.05 and p < 0.01) after incubation with the metabolic inhibitors 2-deoxyglucose and sodium azide indicated active, energy-dependent processes. Phagocytosis in cell-liposome interactions was directly shown by a significant reduction in cell fluorescence (p < 0.05) after application of the actin inhibitor cytochalasin B. The results presented here give concrete data on interactions between liposomes and superficial cells of the eye in vitro.

Experimental Intravitreal Application of Ciprofloxacin in Rabbits

Background: Ciprofloxacin (CFLX) is a fluoroquinolone antibiotic with a broad antimicrobial spectrum. This study was performed to examine the retinal toxicity of free and liposome-incorporated CFLX in rabbit eyes after intravitreal injection. Materials and Methods: Free CFLX in doses of 100, 250, 500, 1,000 and 2,000 µg was injected into the midvitreous of rabbit eyes (n = 28). To prolong the intravitreal antibacterial level, CFLX was incorporated into multilamellar liposomes: 0.1 ml of this suspension (≙ 273.6 µg CFLX) was injected into the midvitreous of a second group of rabbit eyes (n = 6). The other eye served as a control and received normal saline solution or empty liposomes, respectively. Before injection and at the end of follow-up an electroretinogram (ERG) was obtained. After a follow-up of 1, 14 and 28 days the animals were perfused with glutaraldehyde and the eyes were examined by light and transmission electron microscopy. Results: Significant reduction of the ERG was observed after 2,000 µg free CFLX in 4 out of 6 eyes after 14 days. Fourteen days after injection of 2,000 µg CFLX the central retina showed pigmentary changes in 4 out of 6 eyes. In the second group the ERG as well as the histologic studies did not reveal any pathologic changes after injection of liposome-incorporated CFLX compared to the control eyes. Conclusion: In therapeutic doses of 100–500 µg, free CFLX does not have retinal toxicity in rabbit eyes. No retinal toxicity was observed after intravitreal injection of liposome-incorporated CFLX.

Molecular and electrophysiological evidence for “remodeling” of the L-type Ca2+ channel in persistent atrial fibrillation in humans

Chronisches Vorhofflimmern (AF) geht einher mit einer verkürzten Aktionspotentialdauer (APD) und einer verkürzten atrialen Refraktärzeit. Das “Remodeling” von Ionenströmen, die für den Verlauf des Aktionspotentials verantwortlich sind, wird als zugrundliegender Mechanismus bei chronischem Vorhofflimmern angesehen. In der vorliegenden Arbeit untersuchten wir die Aktivität des kardialen L-Typ-Ca2+-Kanals und die mRNA-Transkription der kardialen L-Typ-Ca2+-Kanal-Untereinheitenl bei Patienten, bei denen Sinusrhythmus (SR) vorlag. Rechte Herzohren von 10 Patienten in SR und von 5 Patienten mit AF wurden zur Isolation von Myocyten herangezogen, um mittels Patch-Clamp-Technik den L-Typ-Ca2+-Strom aufzuzeichnen. Rechte Herzohren von 16 Patienten in Sinusrhythmus und von 5 Patienten mit AF dienten zur Bestimmung der mRNA-Expresion der L-Typ-Ca2+-Kanal-Untereinheiten α1c, α2/δ, βa und βb/βc durch semiquantitative RT-PCR. Die Ionenstromdichte von ICa,L war bei Patienten mit AF gegenüber der Sinusrhythmusgruppe um 70% (p < 0,001) reduziert. Die Zellgrößen, ausgedrückt als Zellmembrankapazität, waren in beiden Gruppen identisch. Die mRNA-Expression der α1c-und der βb/βc-Untereinheiten war bei den AF-Patienten um 18,9% (p < 0,05) bzw. um 77,7% (p < 0,005) reduziert, während sich die mRNA Expression der α2/δ- und der βa-Untereinheit bei den SR- und AF-Patienten nicht signifikant unterschieden. Ein Rückgang funktioneller L-Typ-Ca2+-Kanäle bei AF-Patienten, verursacht durch reduzierte mRNA-Expression der α1c-Untereinheit und einem starken Rückgang der βb/βc mRNA-Transkription, scheint demnach zur Verkürzung der APD und der atrialen Refraktärzeit bei AF beizutragen und dadurch eine erhöhte Erregungsrate und die Aufrechterhaltung von AF zu begünstigen. Persistent atrial fibrillation (AF) is associated with shortened action potential duration (APD) and reduced atrial refractoriness. Remodeling of ion currents responsible for AP morphology has been proposed as a major mechanism in persistent AF. In the present study we investigated the activity of the cardiac L-type Ca2+ channel and the mRNA transcription of the cardiac L-type Ca2+ channel subunits in patients with persistent AF compared to patients in sinus rhythm (SR). Right atrial appendages of 10 patients in SR and of 5 patients with AF were used for myocyte isolations to record L-type Ca2+ currents (ICa,L) by the patch-clamp technique. Right atrial appendages of 16 patients in SR and of 5 patients with AF served as sources for determining the mRNA expression of the L-type Ca2+ channel α1c-, α2/δ-, βa-, and βb/βc-subunits by semiquantitative RT-PCR. ICa,L density was reduced by 70% (p < 0.001) in AF patients compared to the sinus rhythm group. Cell sizes, expressed as cell capacitance, were identical in both groups. mRNA expressions of the α1c-subunit and the βb/βc-subunits were reduced in AF patients by 18.9% (p < 0.05) and 77.7% (p < 0.005), respectively, while mRNA transcriptions of the α2/δ- and the βa-subunits were not significantly different between SR and AF patients. A decrease in the availability of functional L-type Ca2+ channels in AF patients, due to reduced α1c-subunit and substantial lack of βb/βc-subunit transcription seems to contribute to the shortening pf APD and refractory periods in AF, thereby favoring increased atrial excitation rate and perpetuation of AF.

Impregnation of collagen corneal shields with liposomes: Uptake and release of hydrophilic and lipophilic marker substances

PURPOSE Liposomes and collagen corneal shields (CCS) have been used as ophthalmic drug delivery devices. With regard to a possibly combined application, we studied the effects of surface charge and bilayer fluidity of liposomes on their uptake and release by CCS. METHODS 12-hours-CCS were soaked in large unilamellar liposomes, which had been labelled with 4,5-carboxyfluorescein (CF) and N-(lissamine rhodamine B sulfonyl)-diacyl-phosphatidylethanolamine (PE-RhB) in the aqueous space and in the liposome bilayer, respectively. Released fluorophores were determined fluorometrically in the elution buffer at intervals from 1 to 240 min after immersion. RESULTS The CF concentration in the CCS soaked in a CF solution was two to seven times higher than immersion in the liposome suspensions. Among those, the negatively charged, cholesterol-containing preparation led to the highest CF concentration in the CCS. The PE-RhB concentration was highest after soaking the CCS in neutral, cholesterol-free liposomes. All types of liposomes were found inside the CCS by freeze fracture electron microscopy. The release kinetics data indicate a first order release. More than 90% of CF was released by the CCS within the first 30 min. This was equal after soaking the CCS in the CF solution or in liposomes. With DOPC-liposomes, the maximal release was already attained after 10 min. In general, the differences in the release kinetics of both hydrophilic and lipophilic markers, obtained by the various liposome types were small. CONCLUSIONS Our results indicate that surface charge and bilayer fluidity are of minor importance for the interaction with collagen corneal shields. However, since the release kinetics of a liposome-encapsulated hydrophilic or lipophilic substance are similar to the release of a non-encapsulated drug, the combination of liposomes with collagen shields may be useful mainly with respect to the encapsulation of drugs which do not penetrate the ocular surface as well as to prolong corneal contact time of the liposomes.