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J. S. Pritchett, E. E. Patterson, II, S. A. Shippy; Low-flow Push-pull Perfusion System for in vivo Monitoring of the Extracellular Chemical Content of the Rat Vitreoretinal Interface. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4323.
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Low-flow push-pull perfusion sampling (LFPS) allows the characterization of the chemical content from the intact, in vivo retina of an anesthetized rat. Multiple sample collection through a sampling period provides a means to monitor chemical changes on the order of 5 to 10 min. More frequent sample collection and analysis would better match expected extracellular content changes. A novel system is presented that couples LFPS to capillary electrophoresis (CE) to provide high temporal resolution monitoring of the in vivo rat retina. The system provides results for monitoring the main nitric oxide (NO) metabolite, nitrate, or ascorbate at the rat retina is performed on a time scale 25-times faster than previously reported off-line detection methods.
Once the animal is anesthetized, the push-pull probe is positioned visually at the vitreoretinal interface through a guide needle with an indirect ophthalmoscope and condensing lens. A flow-gated interface is utilized to connect the withdraw end of the LFPS probe directly to a separation capillary. All of the components are held at a low pressure to drive the sample solution from the probe to the interface. Sample volumes of 7.5 nL are injected onto the separation capillary and mobilized by electrophoresis to the detector for absorbance measurements of ascorbate at 265 nm with an online UV detector. Peak heights were converted to concentration after calibration. Additionally, the on-line system was employed to record in vivo nitrate, the major NO metabolite, absorbance levels at 214 nm. The in vivo presence of the enzymatic family, nitric oxide synthases (NOS), was also explored by inclusion of NOS inhibitors in the perfusion saline for delivery to the sampling site.
The temporal resolution was limited by the injection time and is less than 16 sec. This is a 3-fold improvement compared to other established online in vivo sampling systems. Basal values of ascorbate were found to be 86 ± 18 µM (n=3) that are comparable to values observed by other methods. Upon high K+ stimulation (145 mM), there was a 25-100% increase in observed ascorbate levels that demonstrates that the system can be used to monitor stimulated chemical changes at the retina. Additionally, upon the delivery of the NOS inhibitors, a significant decrease in baseline values of nitrate was observed.
The flow-gate assisted online detection system, which directly couples LFPS with CE, is capable of closely monitoring extracellular changes occurring at the in vivo retina. The improved temporal resolution will likely aid in studying retinal physiology and characterizing retinal diseases.
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