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Published on: November 16, 2016
Antibiotics Reduce Retinal Cell Survival In Vitro
Amy E Lindsey1,2, Ellen Townes-Anderson3,4
1Department of Pharmacology, Physiology, and Neuroscience, New Jersey Medical School, Rutgers Biomedical and Health Sciences, Newark, NJ, 07103, USA. alindse@utica.edu.
Abstract:
Antibiotics such as gentamicin (an aminoglycoside) and penicillin (a beta-lactam antibiotic) are routinely used in retinal cell and explant cultures. In many cases, these in vitro systems are testing parameters regarding photoreceptor transplantation or preparing cells for transplantation. In vivo, milligram doses of gentamicin are neurotoxic to the retina. However, little is known about the effects of antibiotics to retina in vitro and whether smaller doses of gentamicin are toxic to retinal cells. To test toxicity, retinal cells were dissociated from tiger salamander, placed in culture, and treated with either 20 μg/ml gentamicin, 100 μg/ml streptomycin, 100 U/ml antibiotic/antimycotic, 0.25 μg/ml amphotericin B, or 100 U/ml penicillin G. All dosages were within manufacturer's recommended levels. Control cultures had defined medium only. Cells were fixed at 2 h or 7 days. Three criteria were used to assess toxicity: (1) survival of retinal neurons, (2) neuritic growth of photoreceptors assessed by the development of presynaptic varicosities, and (3) survival and morphology of Mueller cells. Rod cells were immunolabeled for rod opsin, Mueller cells for glial fibrillary acidic protein, and varicosities for synaptophysin. Neuronal cell density was reduced with all pharmacological treatments. The number of presynaptic varicosities was also significantly lower in both rod and cone photoreceptors in treated compared to control cultures; further, rods were more sensitive to gentamicin than cones. Penicillin G (100 U/ml) was overall the least inhibitory and amphotericin B the most toxic of all the agents to photoreceptors. Mueller cell survival was reduced with all treatments; reduced survival was accompanied by the appearance of proportionally fewer stellate and more rounded glial morphologies. These findings suggest that even microgram doses of antibiotic and antimycotic drugs can be neurotoxic to retinal cells and reduce neuritic regeneration in cell culture systems.
Insights
Antibiotics commonly used in retinal cell cultures, including gentamicin and penicillin, can be toxic to retinal cells even at low doses. These drugs reduce neuronal survival and neuritic regeneration, impacting research on photoreceptor transplantation.
Area of Science:
- Ophthalmology
- Neuroscience
- Cell Biology
Background:
- Antibiotics like gentamicin and penicillin are standard in retinal cell cultures for transplantation research.
- In vivo, high-dose gentamicin is known to be neurotoxic to the retina.
- The in vitro toxicity of commonly used antibiotic concentrations on retinal cells remains largely uncharacterized.
Purpose of the Study:
- To investigate the in vitro toxicity of various antibiotics on retinal cells.
- To determine if commonly used doses of gentamicin and other antibiotics affect retinal cell survival, neuritic growth, and Mueller cell health.
- To assess the impact of these antibiotics on neuronal regeneration in cell culture.
Main Methods:
- Retinal cells from tiger salamanders were cultured and treated with gentamicin, streptomycin, antibiotic/antimycotic (containing amphotericin B), and penicillin G at manufacturer-recommended levels.
- Toxicity was assessed at 2 hours and 7 days by evaluating retinal neuron survival, photoreceptor neuritic growth (presynaptic varicosities), and Mueller cell survival and morphology.
- Immunolabeling for rod opsin, glial fibrillary acidic protein, and synaptophysin was used to quantify cell types and structures.
Main Results:
- All tested antibiotics significantly reduced neuronal cell density and the number of presynaptic varicosities in both rod and cone photoreceptors compared to controls.
- Rod photoreceptors showed greater sensitivity to gentamicin than cone photoreceptors.
- Penicillin G exhibited the least inhibition, while amphotericin B was the most toxic. Mueller cell survival and healthy morphology were compromised by all treatments.
Conclusions:
- Even microgram doses of commonly used antibiotics and antimycotics can exert neurotoxic effects on retinal cells in vitro.
- These drugs can inhibit vital cellular processes like neuritic regeneration, potentially confounding results in retinal cell culture studies.
- Researchers should carefully consider the potential toxicity of antibiotics when designing in vitro experiments involving retinal cells, particularly for transplantation research.
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