Transplanted and repopulated retinal pigment epithelial cells on damaged Bruch's membrane in rabbits

AIMS: The authors studied how artificially damaged Bruch's membrane influenced growth and differentiation of transplanted embryonic retinal pigment epithelial (RPE) cells and of host RPE cells in rabbits. METHODS: Embryonic RPE cells obtained from pigmented rabbits were transplanted into the subretinal space of adult albino rabbits. The host RPE was removed with a silicone cannula, and Bruch's membrane was damaged by scratching with a microhooked 27 gauge needle under the detached retina in closed vitrectomy. The transplantation sites were examined 3, 7, and 14 days after surgery by light and electron microscopy. RESULTS: Varying degrees of damage in Bruch's membrane were observed. Pigmented and hypopigmented RPE cells showed a normal polarity and tight junctions were seen at the sites of mild to moderate damage 3-7 days after the surgery. In contrast, fibroblast-like cells with no such features of RPE cells formed multiple layers at the sites of severe damage involving the full thickness of Bruch's membrane and the choriocapillaris even 14 days after the surgery. Without transplantation, host RPE cells repopulated the damaged areas in the same way as transplanted RPE cells. CONCLUSIONS: Transplanted embryonic RPE cells as well as host RPE cells grew and differentiated on the moderately damaged Bruch's membrane, while the severely damaged Bruch's membrane did not allow differentiation of RPE cells although these cells could grow and cover the damaged areas.     

Host response to mycobacterial infection in the alcoholic rat: male and female dimorphism

BACKGROUND: The intravitreal injection of ornithine produces selective damage to the retinal pigment epithelium (RPE) and results in a loss of RPE, choriocapillaris and photoreceptor cells. To elucidate the mechanism of secondary retinal atrophy, we investigated the presence of apoptotic cells in a rat model of ornithine-induced retinopathy. METHODS: At 6 and 12 h and 1, 2, 4, 7, 14 and 28 days after an intravitreal injection of L-ornithine hydrochloride in rat eyes, we removed the eyes and subjected them to histopathological examination. We detected apoptotic cells by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate digoxigenin nick end labeling (TUNEL) assay, which stains the 3'-OH ends of fragmented DNA. We used electron microscopy to detect the apoptotic cells morphologically. RESULTS: RPE cells were selectively damaged immediately after ornithine administration. TUNEL-positive photoreceptor cells appeared exclusively in the photoreceptor cell layer 12 h after ornithine administration. The number of TUNEL-positive cells increased throughout the 2 days following the injection, then decreased markedly. TUNEL-positive cells remained until 28 days, when the photoreceptor cells had disappeared. The ganglion cell layer, inner nuclear layer and damaged RPE cells were negative for TUNEL staining during all stages. The electron microscopic study also revealed the pyknotic nuclei of apoptotic photoreceptor cells. CONCLUSION: An intravitreal injection of ornithine caused primary damage to the RPE, and subsequently some of the photoreceptor cells revealed apoptosis by TUNEL assay. These findings suggest the dysfunction of the RPE causes photoreceptor cell death according to the intrinsic program of an apoptotic mechanism.     

Culture of human retinal pigment epithelial cells from peripheral scleral flap biopsies

PURPOSE: We studied various methods for harvesting retinal pigment epithelium (RPE) biopsies from cadaver human eyes of donors over age 60 years. Our goal was to harvest cells for possible autologous RPE cell transplantation in patients with age-related macular degeneration and to test the viability of the RPE after isolation by evaluating explant growth in culture. METHODS: Choroid-RPE biopsies were excised from enucleated human eyes. The RPE was separated from the choroid by treatment with type IV collagenase. RPE patches were cultured. After 100-500 cells had grown out from the explant, the primary cultures were passaged. RESULTS: There was no clear effect of donor age on the ability to establish primary RPE cultures with good morphology from biopsies 2 x 2-10 x 10 mm2 in size. Biopsies 6 x 6 mm2 or larger produced satisfactory primary cultures more than 70% of the time. The number of viable RPE cells (defined as the number of cells adherent to the culture dish 24 h after plating) obtained after enzymatic separation of the RPE and choroid was an important determinant of our ability to establish primary cultures and passage the cells. Primary cultures with good cellular morphology were obtained 100% of the time when RPE explants > 4 mm2 in size were obtained from the biopsy specimen. Seventy-three percent of the biopsies yielding explants > 4 mm2 in size were successfully passaged. CONCLUSIONS: These results suggest that peripheral scleral flap biopsies in aging donors can be used to establish RPE explant primary cultures. These cultures may be suitable as a source for autologous RPE transplantation in patients.     

In vivo response of the rat's retinal pigment epithelium to azide: changes induced by light damage

Functional changes in retinal pigment epithelium (RPE) associated with light-induced retinal damage were studied by measuring transocular potential changes evoked by injections of azide and thiocyanate (SCN-). The retinal damage by light in the rat is classified into two types: Type 1, rod cell death associated with RPE deterioration; Type 2, the loss of rod cells without RPE deterioration. To study the type 1 damage, littermate pairs of long-term dark-adapted adult albino rats were tested at 1 h and 10 d after the exposure to green light of 1,200 lx for 1/2 to 24 h. Time course of the damage progress was also followed for 12 h. We found that 1) RPE was affected rapidly by the damaging light, 2) the exposure length determined the ultimate degree of RPE damage, 3) damaging effects on RPE proceeded slower and weaker after exposure than during continuous light, 4) progress of the damage in RPE was two-phasic; during the first phase, the SCN- response was enhanced and the azide response was reduced; both responses were decreased rapidly in the second phase. The first phase was assumed to indicate a depolarization of the basolateral membrane of RPE, and the second phase to manifest the structural deterioration of RPE. The type 2 damage was studied in young rats with exposure to weak light for 28 d. At 30 d after the exposure, a-wave of the ERG and number of rod cells were substantially reduced but azide and SCN- responses were affected slightly.     

241Am removal by DTPA vs. occurrence of skeletal malignancy

PURPOSE: The distribution of hyaluronan (HA) and the cellular response after photokeratitis induced by different ultraviolet (UV) wavelengths in the rabbit cornea was examined to help understand the mechanism of corneal injury and repair after UV damage. HA is a high molecular weight disaccharide polymer capable of binding considerable amounts of water. It is not normally found in the rabbit corneal stroma. The production of HA represents a generalized corneal response to injury. METHODS: Twenty-four albino rabbit corneas were exposed to 270, 290, and 310 nm of UV radiant energy in 8-nm full wavebands in doses producing biomicroscopically significant keratitis (three corneal thresholds for keratitis (Hc): 0.016 J/cm2 for 270 nm, 0.04 J/cm2 for 290 nm, and 0.14 J/cm2 for 310 nm) and in subkeratitis doses (0.7 Hc: 0.004 J/cm2 for 270 nm, 0.008 J/cm2 for 290 nm, and 0.03 J/cm2 for 310 nm). The rabbits exposed to 270 and 290 nm of UV radiation were sacrificed 3 days after exposure. The rabbits exposed to 310 nm of UV radiation were sacrificed 3, 7, and 14 days after exposure, respectively. The corneal tissue specimens were double stained and examined morphologically and histochemically for HA by light microscopy. RESULTS: Evaluation of corneas exposed to 270 and 290 nm of UV radiant energy in both subkeratitis and keratitis doses and those corneas exposed to 310 nm of radiant energy in subkeratitis dose showed neither stromal changes nor production of HA by corneal cells. Corneas exposed to 310 nm of UV radiant energy in keratitis dose at 3 days after exposure showed disappearance of keratocytes in entire thickness of central cornea. Cells bordering this damaged area were staining for HA. By 7 days after exposure almost the whole damaged area, except one fourth of anterior stroma, was repopulated by new keratocytes staining positive for HA. The corneal structures became normal and HA almost completely disappeared 14 days after exposure. CONCLUSIONS: A keratitis dose of 310 nm of UV light irradiation is needed to cause keratocyte damage. A keratitis dose of the shorter wavelengths does not cause keratocyte cell damage at the light microscopic level. The keratocyte production of HA appears to be a sign of cell readiness to repopulate the damaged stroma devoid of keratocytes.     

Serum-free media for culturing and serial-passaging of adult human retinal pigment epithelium

The ability of a chemically-defined serum-free culture medium to support the attachment, growth and serial passaging of primary adult human retinal pigment epithelial (RPE) cells was studied. Primary cultures of adult human RPE were established in a chemically-defined serum-free culture medium on both bare or bovine corneal endothelial extracellular matrix-coated tissue-culture plastic. Confluent cells were serially passaged in chemically-defined serum-free culture medium three times by trypsinization, and trypsin activity was quenched with aprotinin. First passage RPE cells were plated onto tissue-culture plastic precoated with bovine corneal endothelial extracellular matrix or uncoated tissue-culture plastic in 24 well plates at a density of 50 viable cells mm-2. Cells were maintained either in chemically-defined serum-free culture medium, DMEM without serum, or DMEM with 15% fetal bovine serum. For each medium plating, efficiencies were determined 24 hours after plating, and growth rates were determined on the first, third and seventh days after plating. Morphometric image analysis was performed on cells cultured for up to 6 weeks and three serial passages. Seeding efficiency on bovine corneal endothelial extracellular matrix-coated tissue-culture plastic and treated tissue-culture plastic were higher for chemically-defined serum-free culture medium (88.9+/-2.7% and 47.1+/-4.1%, respectively) and DMEM with serum (87.2+/-5.6% and 52.9+/-10.5%, respectively) than DMEM without serum (59.2+/-5.6% and 33.1+/-6.9%, respectively; P<0.01). The RPE proliferation rate in chemically-defined serum-free culture medium was comparable to DMEM with serum on both substrates within the first 3 days, although cells in DMEM with serum had a higher proliferation rate on day 7. Cells cultured in DMEM without serum, eventually decreased in number. RPE maintained in chemically-defined serum-free culture medium maintained a consistent proliferation rate, reached confluence, and retained an epitheloid morphology on either extracellular matrix or tissue-culture plastic for up to 6 weeks and three serial passages. Primary RPE reached confluence at 12+/-3 days on bovine corneal endothelial extracellular matrix-coated tissue-culture plastic and 21+/-5 days on treated tissue-culture plastic. Confluent cultures were composed of small hexagonal cells with epitheloid morphology on both substrates. We concluded that primary adult human RPE can be cultured in this chemically-defined serum-free culture medium. RPE will proliferate, reach confluence, retain their epitheloid morphology and can be serially passaged in the absence of serum.     

Retinal pigment epithelium cell culture on thin biodegradable poly(DL-lactic-co-glycolic acid) films

Thin films of 50:50 and 75:25 poly(DL-lactic-co-glycolic acid) (PLGA) were manufactured with a controlled thickness of less than 10 microm. The effect of PLGA copolymer ratio on in vitro cell attachment, proliferation, morphology, and tight junction formation was evaluated using a human D407 retinal pigment epithelium (RPE) cell line. Almost complete cell attachment was achieved on both PLGA films after 8 h of cell seeding, which was comparable to that on tissue culture polystyrene (TCPS) controls. The initial cell seeding density affected attachment, and the optimal value for 50:50 PLGA was 25000 cells cm(-2). After 7 days of in vitro culture, cell density on 50:50 and 75:25 PLGA films increased 45 and 40 folds, respectively, and a 34-fold increase was observed on TCPS. The RPE cells cultured on PLGA films at confluence had a characteristic cobblestone morphology. Confluent RPE cells also developed normal tight junctions in vitro which were concentrated mainly at the apical surfaces of cell-cell junctions. These results demonstrated that thin biodegradable PLGA films can provide suitable substrates for human RPE cell culture, and may serve as temporary carriers for subretinal implantation of organized sheets of RPE.     

Antibiotic susceptibility among aerobic gram-negative bacilli in intensive care units in 5 European countries. French and Portuguese ICU Study Groups

BACKGROUND: Many successful pigment epithelium transplantation studies involving pink-eyed Royal College of Surgeons (RCS) dystrophic rats showed highly pigmented transplanted cells forming a double layer with slightly pigmented cells, attached to Bruch's membrane. Since it is not clear whether transplanted pigmented cells can displace retinal pigment epithelial (RPE) host cells from Bruch's membrane, we suggested that RPE cells of RCS dystrophic rats can phagocytize melanin granules, possibly derived from perished transplanted cells. METHODS: In a series of three experiments, RPE cells of nine pink-eyed, 2 1/2-month-old RCS dystrophic rats were isolated by trypsinization and mechanical dissection and cultivated in Dulbecco's modified Eagles' medium. These cells were then fed with melanin granules, isolated from bovine RPE cells, double-trypsinized after phagocytosis and viewed by light and electron microscopy. We also transplanted iris pigment epithelial (IPE) cells of 20-day-old Long-Evans rats into the subretinal space of pink-eyed RCS dystrophic rats of the same age, shown in light-microscopic photography after 42 days. RESULTS: Living RPE cells were heavily pigmented after feeding with isolated melanin granules in all three experiments as viewed by light microscopy. In addition, we identified melanin granules phagocytized by dystrophic RPE cells in electron microscopy. After transplantation of pigmented IPE cells into the subretinal space of pink-eyed RCS dystrophic rats' eyes, a layer of slightly pigmented cells was seen on Bruch's membrane below the transplanted IPE cells, shown in light microscopy. CONCLUSION: We have shown by phagocytosis assay that dystrophic RPE cells can take up melanin granules in vitro. Our results assume that pigmented cells in transplantation studies, found as a monolayer, attached to Bruch's membrane, cannot automatically be identified as transplanted cells. Instead, the possibility of perished transplanted cells serving as melanin donors for RPE host cells must be taken into consideration.     

Minimum number of adult human retinal pigment epithelial cells required to establish a confluent monolayer in vitro

PURPOSE: To determine the minimum number of cells required to establish a confluent monolayer of retinal pigment epithelium (RPE) with an epitheloid morphology in vitro. METHODS: Primary or passaged human RPE were harvested by trypsinization from 6 donors and plated onto bovine corneal endothelium extracellular matrix-coated tissue culture plastic in 96-well plates. Plating densities ranged from 1 to 66,000 viable cells/well (0.03-2062 viable cells/mm2) for primary cells or 1 to 100,000 viable cells/well (0.03-3112 viable cells/mm2) for passaged cells. The time required to reach confluence was determined by monitoring the cultures daily until they reached confluence. Mean cell area and circularity index at confluence was calculated to determine the effect of different plating densities on final RPE morphology. RESULTS: Primary RPE plated at densities above 10 viable cells/mm2 (320 cells/well) and passaged RPE plated above 2 viable cells/mm2 (64 cells/well) reached confluence on every occasion. There was a negative correlation between the plating density and time required to reach confluence. Plating densities above 3 viable cells/mm2 (96 cells/well) and 50 viable cells/mm2 (1600 cells/well) yielded smaller, rounder cells at confluence for primary and passaged RPE, respectively. CONCLUSIONS: As few as 96 primary RPE cells and 1600 passaged RPE are required to obtain a confluent, 6mm (4-disc diameter) patch of RPE in vitro. This suggests that autologous RPE grafts can be prepared with high efficiency for subsequent transplantation into the subretinal space in vivo.     

Ketamine and nifedipine protect cultured cortical neurons against injurious effect of glutamate]

The effects of glutamate on cultured cortical neurons and the protective effect of ketamine and nifedipine were studied. On day 10 after plating of the cortical cells from 16-18 day-old fetal rats, the cultures were exposed to 50 mumol.L-1 glutamate and low glucose (1 g.L-1) for 10 min-24 h. The results showed that a release of lactate dehydrogenase (LDH) into the culture supernatant was observed as a function of time. The values of LDH efflux in culture medium was significantly lower than those of controls when the cells were pretreated with ketamine or nifedipine 10 min prior to addition of glutamate. More significant decrease of LDH activity in culture medium was observed when the two drugs were used in combination. These results demonstrate that the dissociated cultured cortical neurons from fetal rat are seriously damaged by glutamate. Such damage could be attenuated by ketamine and nifedipine, suggesting that ketamine and nifedipine may protect neurons from the glutamate toxicity, and the effect of combining ketamine and nifedipine was greater than either ketamine or nifedipine alone.     

Iron-induced fluorescence in the retina: dependence on vitamin A

PURPOSE: Intravitreal iron injection induces fluorophore formation in the photoreceptor outer segments, followed by an accumulation of inclusions with lipofuscin-like fluorescence in the retinal pigment epithelium (RPE). The accumulation of RPE lipofuscin during aging is dependent on vitamin A availability. Experiments were conducted to determine whether iron-induced fluorophore formation in the outer segments and in RPE is also dependent on vitamin A, and thus whether oxidation promotes the participation of vitamin A in lipofuscin formation. METHODS: For 23 weeks, beginning at weaning, albino Fischer rats were fed diets containing vitamin A either in the form of retinyl palmitate (+A), which can be metabolically converted into the retinoids involved in vision, or retinoic acid (-A), which does not support visual function. After 23 weeks, when rhodopsin levels had decreased more than 90% in the -A rats, some animals in this group were given an intramuscular injection of all-trans retinol and were allowed to recover from retinoid deficiency for 7 days (-A+A). Animals in all three treatment groups were then given an intravitreal injection of ferrous sulfate. Both 1 day and 7 days after the iron injections, the retinas and RPEs were examined for fluorophores with excitation and emission properties similar to those of RPE lipofuscin fluorophores. RESULTS: In retina sections examined with fluorescence microscopy 24 hours after the ferrous sulfate treatment, the photoreceptor outer segments of rats in all of the treatment groups displayed a fluorescence with a blue emission maximum. This outer-segment fluorescence was not present in untreated eyes. The in situ outer-segment fluorescence was correlated with the appearance of blue-emitting fluorophores in organic solvent extracts of the retinas. One week after the iron injections, the RPE cells of the +A animals became filled with inclusions that displayed a golden-yellow fluorescence emission when excited by blue light. Very little of this lipofuscin-like fluorescence was observed in the RPE of the -A rats 1 week after iron treatment. However, in the -A rats that had been repleted with vitamin A, the ability of iron to induce the RPE fluorescence was restored. Several orange-emitting fluorophores were present in organic solvent extracts of the RPE-choroids of the +A rats. The amounts of these fluorophores were not appreciably affected by the iron treatment. These orange-emitting compounds were not observed in extracts of any eyes in the -A or -A+A groups. CONCLUSIONS: The results of this study suggest that oxidation of the photoreceptor outer-segment lipids generates blue-emitting fluorophores that are not directly involved in RPE lipofuscin fluorophore formation. The findings also indicate that retinoids are direct precursors of RPE lipofuscin fluorophores, and that oxidative stress to the retina promotes participation of vitamin A in the formation of some of the compounds responsible for RPE lipofuscin fluorescence.     

Tissue effects of subclinical diode laser treatment of the retina

OBJECTIVE: To determine whether consistent tissue effects are obtained when laser lesions are produced with a commercially available diode laser that are near the limit of clinical detection at the time of treatment. METHODS: Continuous-wave or micropulse diode laser was used to produce clinically undetectable (subthreshold) or barely detectable (threshold) retinal lesions in pigmented rabbits. Tissue effects at intervals after treatment were determined in retinal pigment epithelial (RPE) whole mounts by fluorescence microscopy, and in sections of retina and RPE by light and electron microscopy. RESULTS: Continuous-wave and micropulse laser lesions that were originally clinically undetectable were detectable as zones of pigment mottling after 5 days. By microscopy, compaction and/or swelling was seen in the outer retina, and cells in the RPE monolayer became heterogeneous in size, shape, and pigmentation, but the tissue responses in the outer retina and RPE were variable even within and among lesions in the same eye. CONCLUSIONS: Subthreshold energies used to create both continuous-wave and micropulse laser lesions produced variable effects on the RPE and the overlying neurosensory retina. It appears that, near the minimum effective dose of laser irradiation, individual RPE cell heterogeneity becomes detectable as variability in sensitivity to laser injury. CLINICAL RELEVANCE: As laser energy is reduced to limit collateral tissue damage in clinical applications, it may be difficult to generate reproducible lesions because of heterogeneity among individual cells.     

Sudden death in implantable cardioverter-defibrillator recipients: clinical context, arrhythmic events and device responses

Reports regarding the effect of all-trans-retinoic acid (RA) on the cell growth of retinal pigment epithelial cells (RPE) have been contradictory. The aims of this study are to clarify the in vitro effect of RA on RPE cells and to examine polyamine metabolism after RA stimulation. A 4-day incubation of fetal-calf-serum (FCS)-stimulated RPE cells with 10 or 25 microM RA significantly increased both cell number and [3H]thymidine incorporation. RPE cells grown over an extended period for 8 days also increased in number and reached full confluency. However, if the incubation was further extended to 12 days, no further increase in cell number was detected. RA treatment of FCS-stimulated RPE cells shifted the peak of ornithine decarboxylase (ODC) activity from 16 to 4 h. S-adenosylmethionine decarboxylase (SAMDC) activity and spermidine/spermine N1-acetyltransferase (SAT) activity of RA-treated RPE cells were significantly greater until 8 and 16 h after incubation, respectively. The putrescine content was significantly increased in RA-treated RPE cells up until 24 h, while spermidine, spermine and N1-acetylspermidine contents were significantly increased until 16 h. Our findings suggest that RA treatment increases the intracellular polyamine concentration of RPE cells via activation of ODC, SAMDC and SAT and that this results in the promotion of RPE cell growth until the cells reach full confluency.     

Descemet''s membrane as membranous support in RPE/IPE transplantation

PURPOSE: The correct orientation of retinal pigment epithelium (RPE) cells is necessary for the integrity and proper function of the retina. For transplantation of RPE/iris pigment epithelium (IPE) grafts to the subretinal space in age-related macular degeneration, this cellular orientation is most effectively provided by a membranous support. The goal of this study was to establish an autologous or homologous membrane as a substratum for the growth of RPE/IPE. METHODS: Porcine and bovine RPE and IPE were placed in primary culture on a dissected sheet (5 x 5 mm) of autologous porcine and bovine Descemet's membrane in slide chambers and grown to confluence. RESULTS: RPE and IPE cells cultured on Descemet's membrane form an intact monolayer. Light and electron microscopy showed the formation of both an intact monolayer and microvilli in both cell types. CONCLUSION: Since the slow host-graft rejection appears to play an important role in the failure of RPE transplantation in the subretinal space, it is critical to be able to transplant autologous materials. The techniques presented here establish a novel means to culture RPE or IPE cells on autologous Descemet's membrane where they form a "cell monolayer patch," consisting of a fragment of Descemet's membrane with cultured RPE or IPE, which can be easily manipulated and transplanted, using an established glass pipette method.     

Low glutathione and high iron govern the susceptibility of oligodendroglial precursors to oxidative stress

We have previously shown, using qualitative approaches, that oligodendroglial precursors are more readily damaged by free radicals than are astrocytes. In the present investigation we quantified the oxidative stress experienced by the cells using oxidation of dichlorofluorescin diacetate to dichlorofluorescein as a measure of oxidative stress; furthermore, we have delineated the physiological bases of the difference in susceptibility to oxidative stress found between oligodendroglial precursors and astrocytes. We demonstrate that (a) oligodendroglial precursors under normal culture conditions are under six times as much oxidative stress as astrocytes, (b) oxidative stress experienced by oligodendroglial precursors increases sixfold when exposed to 140 mW/m2 of blue light, whereas astrocytic oxidative stress only doubles, (c) astrocytes have a three times higher concentration of GSH than oligodendroglial precursors, (d) oligodendroglial precursors have > 20 times higher iron content than do astrocytes, and (e) oxidative stress in oligodendroglial precursors can be prevented either by chelating intracellular free iron or by raising intracellular GSH levels to astrocytic values. We conclude that GSH plays a central role in preventing free radical-mediated damage in glia.     

Preservation of red cell properties after virucidal phototreatment with dimethylmethylene blue

BACKGROUND: All published reports have described methods for virus photoinactivation which significantly alter red cell (RBC) properties during storage. In order to improve virucidal activity and reduce damage to RBCs, a series of phenothiazine derivatives were either synthesized or purified and screened for bacteriophage inactivation and red cell potassium efflux. One compound, 1,9-dimethylmethylene blue (dimethyl-methylene blue), had superior screening results and was chosen for further characterization. STUDY DESIGN AND METHODS: White cell reduced RBC suspensions (30% hematocrit) were deliberately inoculated with extracellular virus or virus-infected VERO cells, incubated with 4 microM dimethyl-methylene blue and illuminated with cool-white fluorescent light. Control and treated samples were titered for virus inactivation. In parallel studies, RBC suspensions were exposed to dimethylmethylene blue and light under identical conditions and assayed for in vitro RBC storage properties. RESULTS: Phototreatment of RBC suspensions inactivated > 4.4 log10 of extracellular vesicular stomatitis virus (VSV), > 3.0 log10 of intracellular VSV, > 5.0 log10 of extracellular pseudorabies virus (PRV), > 4.8 log10 of intracellular PRV, > 4.7 log10 of extra-cellular bovine virus diarrhea virus, 5.8 log10 of bacterio-phage phi 6 and > 7 log10 of bacteriophage R17. Encephalo-myocarditis virus, a nonenveloped picornavirus, was resistant to photoinactivation. Virucidal conditions resulted in no detectable IgG binding in 11 of 13 samples, unchanged RBC morphology, normal banding patterns of RBC membrane proteins on SDS PAGE, and unaltered characteristics of 12 of 13 RBC antigens during storage as measured by antibody titrations. In addition, minimal changes were observed in RBC osmotic fragility, lysis, potassium efflux, ATP and 2,3-DPG levels, and the strength of one RBC antigen during storage of phototreated samples compared with controls. CONCLUSION: Dimethylmethylene blue photo-treatment can inactivate several intracellular and extracellular model viruses under conditions which minimally alter RBC properties during 42 days storage at 1-6 degrees C.     

Methamphetamine-induced toxicity in cultured adult rat cardiomyocytes

In an attempt to differentiate the direct effects of methamphetamine from the indirect sympathomimetic effects on the myocardium, primary culture of adult rat myocytes were established under serum-free conditions, and they were exposed to methamphetamine (1 x 10(-5) and 1 x 10(-3) M) for 1 to 24 h in the presence and absence of 1 x 10(-6) M propranolol. Cardiotoxicity was evaluated by light and ultramicroscopy, release of cytoplasmic enzymes (Lactate dehydrogenase: LDH and Creatine phosphokinase: CPK) and change in membrane permeability (Trypan blue stain). After 24 h methamphetamine treatment, light microscopy exhibited cellular granulation and swelling, myocyte hypercontraction, broken cellular membrane and cellular destruction. After the same time, electron microscopy revealed swelling and irregular mitochondria with disrupted cristaes, clump of sarcomeres with nearly complete loss of organized contractile elements, injury of intracellular membrane system and dissolution of myofibrils. These injurious features were more severe with the 1 x 10(-3) M methamphetamine. Propranolol (1 x 10(-6) M), a beta-adrenergic antagonist, failed to protect the myocytes against methamphetamine-induced cell injury. Release of LDH from methamphetamine (1 x 10(-5) and 1 x 10(-3) M)-treated myocytes increased significantly only after 24 h, while significant CPK release was observed in 1 x 10(-3) M methamphetamine-treated myocytes at 4 h. These findings suggest that methamphetamine exerts direct toxic effects on adult rat myocytes rather than indirect ones via receptors, although further experiments on more concentrations of propranolol are required.     

Radiation response of porcine RPE cells in vitro

PURPOSE: To investigate the antiproliferative effect of ionizing radiation on retinal pigment epithelial (RPE) cells that are supposed to play a major role in the pathogenesis of proliferative vitreoretinopathy (PVR). METHODS: RPE cells from pig eyes were irradiated with doses ranging from 4 to 16 Gy (1 Gray = 1 Joule/kilogram). Cells were counted at 1, 2, 3, and 4 weeks (Experiment 1) or 1, 2, 4, and 6 weeks (Experiment 2) after treatment. In Experiment 3, cells were trypsinized 24 h after radiation and seeded again. Colonies were counted 10 days later, and the surviving fraction was determined. RESULTS: The numbers of cells and colonies were inversely correlated to the doses applied. In Experiment 2, cell numbers of radiated cultures remained stable during the time of follow-up, whereas, in Experiment 1, significant proliferation occurred in treated cultures as well as in controls. This may be due to the higher growing rate that was found in the cultures of Experiment 2, compared to those of Experiment 1, at the time of radiation. In Experiment 3, a D0 value of 0.72 Gy was found. CONCLUSIONS: Proliferation of RPE cells can be suppressed by irradiation in a dose-dependent manner. Therefore, radiotherapy may be useful in the treatment of PVR. Its effect probably depends on the stage or activity of PVR at the time of radiation.     

Effects of basic fibroblast growth factor on hippocampal neurons after axonal injury

OBJECTIVE: Axons of adult central nervous system neurons fail to regenerate after diffuse axonal injury in head trauma. Basic fibroblast growth factor (bFGF) has been reported to enhance neuritic extensions after neuronal injury in immature nerve cells. To investigate the effects of bFGF on adult neurons and axonal reoutgrowth, differentiated nerve cells were axonally transected and bFGF was applied. DESIGN: Cell culture study with primary rat hippocampal neurons. MATERIALS AND METHODS: After axotomy, hippocampal cultures were maintained untreated or in the presence of 0.5, 1, 10, or 20 ng/mL bFGF and evaluated over a 7-day period after injury. MEASUREMENTS AND MAIN RESULTS: Seven days after injury, axotomy decreased cell survival to 65%, increased [3H]arachidonic acid release 1.8-fold from prelabeled cells, and showed negligible effects on neuronal dendrites. bFGF reduced this neurodegeneration at all doses applied. bFGF at 10 ng/mL most efficiently increased live cells to 85% and decreased [3H]arachidonic acid release from prelabeled cells to control values (p < 0.01, vs. damaged cells). Furthermore, 10 ng/mL bFGF induced axonal branching and the longest axonal re-extensions from 60 +/- 8 to 377 +/- 10 microns 7 days after injury (p < 0.01, vs. damaged cells). CONCLUSIONS: bFGF increased cell survival and supported axonal re-elongations in adult hippocampal neurons in vitro when applied after axotomy. bFGF may play a role in new therapeutic concepts for the management of axonal injury after head trauma.