Molecular Vision 2007; 13:2073-2082 <http://www.molvis.org/molvis/v13/a235/> Received 3 April 2007 | Accepted 18 October 2007 | Published 3 November 2007

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Demonstration and use of nanoliter sampling of in vivo rat vitreous and vitreoretinal interface

Sumith Kottegoda,¹ Jose S. Pulido,² Kongthong Thongkhao-on,¹ Scott A. Shippy¹
 
 

¹University of Illinois at Chicago, ²Department of Ophthalmology and Visual Sciences, Chicago, IL

Correspondence to: Scott A. Shippy, Department of Chemistry, M/C 111, 845 W Taylor Street, Room 4500, Chicago, IL 60607-7056; Phone: (312) 355-2426; FAX: (312) 996-0431; email: sshippy@uic.edu
 
Dr. Pulido is now at the Department of Ophthalmology, Mayo Clinic, Rochester, MN, and Dr. Kottegoda is now at the University of North Carolina at Chapel Hill, Department of Chemistry, Chapel Hill, NC

Abstract

Purpose: An understanding of the chemical microenvironments at different locations on the retina can provide unique insights into retinal neurochemistry and pathology. The anatomical shape and the small volumes available from a spatially restricted volume greatly complicate these types of measurements. The aim of this study was to demonstrate an in vivo sampling system to probe different regions of the rat retina.

Methods: A low-flow push-pull perfusion probe was developed with concentric fused-silica capillaries. It was designed to fit through a 29-gauge needle for placement in the vitreous and at the vitreoretinal interface of the rat eye. Physiological saline was perfused and withdrawn through outer and inner capillaries, respectively, at flow rates between 10-50 nl/min. Samples of 500 nl were collected for amino acid analysis by capillary electrophoresis. Perfusion of a potent and selective inhibitor of the excitatory amino acid transporters was performed through the probe with the tip located 1-2 mm away from the optic nerve head.

Results: Ten amino acids were quantified from the perfusates of vitreous and the vitreoretinal interface. Sampling through time showed the use of this system to monitor retinal changes in these amino acids. The infusion of a transport protein antagonist shows a statistically significant increase in the glutamate concentration in collected samples when the probe tip is placed peripheral to but not over the optic nerve head.

Conclusions: We demonstrate a new method for following neurochemical changes at the retina with spatial resolution. This in vivo method is widely applicable to the site-specific study of states of normal and dysfunctional retinal neurochemistry.

Introduction

Understanding neurochemical activity at the retina is a major topic in vision research. Amino acid neurochemistry in normal and anomalous retinas is of particular interest because of the important role these compounds play in neurotransmission, metabolic and cellular function, osmoregulation, and protein synthesis. They also appear to contribute to retinal degeneration [1,2], glaucoma [3,4], diabetic retinopathy [5-7], retinitis pigmentosa [8], and other retinal disorders.

Glutamate, γ-aminobutyric acid (GABA) and glycine are the principle neurotransmitters in the vertebrate retina [9,10]. Over 90% of the synapses in the retina contain these three amino acids as neurotransmitters [10]. Glutamate is the primary neurotransmitter for photoreceptors, bipolar cells, and ganglion cells in the retina [11]. However, glutamate accumulation may alter glutamate metabolism and lead to neurotoxic effects at the retina in glaucoma [12] and diabetic retinopathy [5]. Glutamate is also the immediate precursor to GABA, a major inhibitory neurotransmitter. GABA is mainly localized in amacrine cells and horizontal cells [10,13], and basal levels are elevated in retinal degeneration [8]. Glycine is a classic inhibitory neurotransmitter and is localized within approximately 50% of the amacrine cells and 20% of the bipolar cells in mammalian retinas [14]. Taurine, the most abundant free amino acid in the retina [2,15], is involved in many physiological functions including regulating calcium binding and transport [16], retinal degeneration [1,2,17], maintaining structural integrity of cell membranes [18], and as an osmolyte [19]. Glutamine and aspartate are important amino acids for glutamate formation and metabolism [8,20]. In experimentally induced glaucoma, amino acids have been found to be elevated due to intraocular inflammation and the action of proteolytic enzymes [4]. Excitatory amino acids are also reported to be elevated in diabetic retinopathy [21-23]. Comprehensive studies of the chemical composition at the retina contribute to an understanding of the retinal physiology and provide a way to probe the relationships with various genetic factors and biologically important molecules.

There are several methods currently available for studying the chemical nature of vitreous and retina, including direct sampling [3,4,6], homogenization of tissues [1,5,15], efflux from an isolated retina in a chamber [24,25], and ocular microdialysis [26-29]. Direct sampling and tissue analysis provide only a broad understanding of the chemical composition, and this information is available for only a single time point. By measuring the efflux of an isolated retina and performing microdialysis sampling it is possible to determine changes in chemical composition over time [24]. Microdialysis sampling is especially powerful in application to in vivo sampling where it is commonly used in studies of pharmacokinetics [27,30,31], ischemia [26,32,33] and fundamental studies for monitoring amino acids levels [28], metabolites of nitric oxide [34,35], ascorbic acid [29], and dopamine [36]. However, the dialysis probe is made with a semipermeable membrane around the probe tip that prevents direct contact between the infusion saline and the tissue during sampling. The probe size creates a relatively large depletion region through which diffusion is limited such that recoveries are usually reported in the 7-25% range [31]. The relatively large probe size also limits the spatial resolution; neurochemicals are collected along the entire active length of the dialysis membrane, which has been reported to span 1-5 mm. Dialysis probes are commonly inserted into the eye by the relatively large 15-25 gauge needle guides which complicates their use with smaller animals. Considering the aforedescribed limitations with ocular sampling techniques, there is a need for new methods that can produce higher recovery of vitreal analytes with improved spatial resolution and less tissue disturbance for use with smaller animal models.

Push-pull perfusion is another in vivo sampling technique that has a long history for use in the brain [37-39] or spinal cord [37,40]. Recently, we introduced a new low-volume sampling technique that has addressed problems associated with previously reported push-pull perfusion [41]. Our previous studies with low-flow push-pull perfusion showed high collection efficiencies, minor tissue damage to the sampling area, high in vitro recovery, and nano-volume in vivo collection [41]. It has been used for the recovery of amino acids, biogenic amines, metabolites of nitric oxide, and peptide and proteins [42-44].

High spatial resolution is an important consideration for understanding the neurochemical microenvironment at different points over the retina. Rat models of induced glaucoma have revealed that there is regional specificity during retinal ganglion cell death [45] or optic nerve damage [46]. The anatomical shape of the retina has largely precluded determining in vivo chemical changes in different locations. This goal is further complicated by the small size of the rat eye and the relatively large lens that occupies most of the posterior chamber [47]. Pharmacological manipulation of the in vivo tissue being studied is one of the best methods to evaluate the accuracy of the probe placement. L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) is a potent and selective inhibitor of the excitatory amino acid transporters that remove glutamate from the synaptic cleft. Extracellular glutamate concentrations are increased as result of PDC infusion in the brain and at the retina [41]. In this article we report the development of a new sampling method based on low-flow push-pull perfusion for nanoliter sampling from the rat vitreoretinal interface. This method minimizes tissue damage due to a miniaturized construction of a push-pull perfusion probe using fused-silica capillary. Basal level amino acid concentrations were determined from the rat vitreous as well as the vitreoretinal interface and compared to concentrations obtained over time and to levels following infusion of PDC. We also show that there is an asymmetric release of glutamate into the vitreous depending upon where PDC is delivered and where the levels are measured.

Methods

Chemicals and reagents

Unless specified otherwise chemicals were obtained from Sigma-Aldrich (St. Louis, MO). Buffer salts, histamine, sodium tetraborate, sodium phosphate, sodium dodecyl sulfate (SDS), β-cyclodextrin (β-CD), sodium hydroxide, potassium cyanide (KCN), and 2-propanol were purchased from Fisher Scientific (Itasca, IL). Potassium chloride was purchased from Malinckrodt Baker, Inc. (Paris, KY). Proparacaine hydrochloride was purchased from Bausch & Lomb Pharmaceuticals, Inc. (Tampa, FL). Cyclopentolate hydrochloride (1%) was obtained from Alcon Laboratories Inc. (Fort Worth, TX). The primary amine derivatizing agent 3-(4-carboxybenzoyl) quinoline-2-carboxyladehyde (CBQCA) was purchased from Molecular Probes (Eugene, OR).

All solutions were prepared with ultrafiltered, deionized water from a U.S. Filter Purelab Plus water purification system (Lowell, MA) and filtered with a 0.2-mm acrodisc filter (Gelman Science, Ann Arbor, MI) immediately before use. The composition of the Krebbs-Ringer buffer (KRB) was 3-mM KCl, 145-mM NaCl, 1.2-mM CaCl₂, 1-mM MgCl₂, 1.61-mM Na₂HPO₄, 0.4-mM NaH₂PO₄. Proparacaine hydrochloride was diluted 1:10 with KRB.

Probe construction and instrument setup

The concept of the concentric style push-pull perfusion probe construction and the method of sample collection have been previously described [41]. Because of the spatial limitations of the rat vitreous, we designed a miniaturized concentric style probe, consisting of fused silica capillaries that had an average length of 2.5-cm and an outer diameter of 170 mm. The basic instrumental set up is shown schematically in Figure 1A. The concentric style eye probe was made of an inner withdrawing capillary (15-cm long, 20/90-mm inner diameter/outer diameter) and an outer capillary (2.5-cm long, 100/170-mm inner diameter/outer diameter). The outer capillary is connected to an infusion capillary (43-cm long, 50/150-mm inner diameter/outer diameter) via a Tygon tube connection for continuous infusion flow (see Figure 1D). The PHD 2000 programmable syringe pump (Harvard Apparatus, Holliston, MA) generates an infusion flow of 40-50-nl/min of physiological saline. At the tip of the probe saline passes through the outer capillary, mixes with vitreous fluid and is then collected via the inner withdrawing capillary. The withdrawal capillary was connected to a vacuum pump (Barnant Co. Barrington, IL) to provide a pressure that resulted in a flow of 40-50 nl/min.

Animals

All animal procedures were performed in accordance with ARVO statement for the use of animals in ophthalmic and visual research and were approved by the University of Illinois at Chicago Animal Care Committee. Male Sprague-Dawley rats weighing 350-450 g, were obtained from a colony maintained by the Department of Psychology, University of Illinois at Chicago. Long-Evans rats (Harlan Inc., Indianapolis, IN) in the same weight range were used for all vitreoretinal interface experiments. All subjects were kept under anesthesia using sodium pentobarbital (50 mg/kg) intraperitoneal. Additional doses were given as needed to maintain anesthesia. Sampling experiments were performed at ambient light levels.

Probe placement

Anesthetized rats were positioned in a stereotaxic apparatus to stabilize probe placement in the posterior cavity. Prior to probe insertion, a drop of 0.2% cyclopentolate hydrochloride was given for dilation. A single drop of local anesthetic 0.05% proparacaine hydrochloride was applied to the cornea every 20 min from a segment of tygon tubing connected to a syringe. An extra drop was given if there was any noticeable movement of the eyelid. This dose was based upon the results of a series of tests to eliminate the blink reflex in anesthetized animals due to the periodic application of a drop of refrigerated KRB over the course of an hour. A 29-gauge guide needle was vertically moved through the edge of the sclera and conjunctiva and placed in the middle of the vitreous cavity (see Figure 1B). The position of the needle tip was verified by indirect ophthalmoscopy. The push-pull perfusion probe was then inserted through the guide and the tip of the probe was placed 1 mm below the end of the needle.

Vitreoretinal interface probe placements required a slight modification. After the guide needle was passed through the sclera and conjunctiva, the needle was moved medially and pushed down toward the retina. This allowed the probe to be placed either close to the optic nerve head or in a more peripheral region of the vitreoretinal interface, which was 1-2 mm away from the optic nerve head. Before the push-pull perfusion probe was inserted through the needle, the placement of the guide needle at 1-2 mm from the retina was verified by indirect ophthalmoscopy of the needle in the posterior chamber. The relative size of the needle, the angle of the needle across the field of view, and the location of the tip relative to the optic nerve head were landmarks used to ensure successful push-pull probe placement. Once the push-pull probe was inserted, both the probe and needle were then gently moved until the probe tip was barely touching the retinal surface (Figure 1C).

Sampling

During push-pull perfusion probe insertion, an outward 500 nl/min flow rate was maintained from both infusion and withdrawing capillaries for 0.5-1 min to prevent any probe tip blockage. The flow rate was then linearly decreased to a sampling flow rate (40-50 nl/min) over 45 s and maintained for 5 min before the start of the sample collection. Next, the withdrawing capillary was disconnected from infusion flow and connected to the vacuum system via a 4-cm long, 250 μm i.d. Tygon tubing (see Figure 1E). Approximately 500 nl volume samples were collected into the Tygon tubing at 45-50 nl/min flow rate. After the desired volume was collected, the vial was changed out, typically within 30 s, and sampling continued. This process was repeated for up to 3 h. The length of the collected sample in the tubing was measured and converted to a volume. The volume collected and time of collection was noted in order to plot the withdrawal flow rate. The tubing segments were then stored in a sealed 250 μl centrifuge vial for later analysis.

Drug infusion

Probe placement and sample collection at the vitreoretinal interface was confirmed by infusing 1-mM PDC to the sampling site during sample collection. After 75-80 min of regular collection, 1-mM PDC was infused through the infusion line as described previously [41]. Briefly, a Tygon connection was placed between the probe infusion capillary and the infusion syringe pump; the probe infusion capillary was moved from a syringe filled with buffered saline to one with saline and dissolved PDC. The drug infusion was performed for 24-26 min, and then the infusion connection was returned to the perfusion saline. The estimated time for the drug solution to reach the probe tip after switching was 20-22 min.

Sample preparation and capillary electrophoresis

In vivo samples were derivatized with 1 μl of 10-mM CBQCA in DMSO and 1 μl of 10-mM of KCN solution and incubated for 2 h in the dark at room temperature. Derivatized samples were analyzed by capillary electrophoresis with laser-induced fluorescence (CE-LIF). The CE-LIF system and separation condition have been described previously [43]. Briefly, a home-built CE system employed a separation capillary (57-cm long, 360/50-mm inner diameter/outer diameter) placed between two run buffer vials in a plexiglass box. High voltages are applied across the separation capillary via platinum electrodes in the run buffer vials. The separation capillary is routed through a Zetalif LIF detector (Picometrics, Paris, France) with illumination via the 488 nm line from an Ar ion laser. A sample aliquot is injected into the capillary by placing the inlet end of the capillary in the sample solution for 5 s while raising the inlet 8 cm. The run buffer consists of 20 mM sodium tetraborate, 20 mM sodium chloride, 45 mM SDS and 55 mM β-CD. The applied potential was 360 V/cm. Samples are run in triplicate. In the study of probe placement by drug infusion, the CE separation conditions were optimized for a fast separation. Figure 2 presents a representative electropherogram. During optimization more attention was given to the separation of glutamate, aspartate, and arginine. The field strength was increased to 490 V/cm and the capillary was shortened to 41-cm (37-cm effective). As a result the complete separation was performed within 3 min. All samples were run in triplicate. Peaks were identified by spiking samples with standard amino acids labeled with CBQCA.

Data analysis

Detector output was exported to Microsoft Excel for analysis. The peak height of each analyte was related to a perfusate concentration from a calibration curve. Basal levels of particular amino acids were calculated by averaging the mean value of the observed concentration of the samples collected during the second hour of sampling or for time course studies samples collected between 30-60 min. The basal levels were presented as the mean±standard error (SEM). In time course studies, the average concentrations measured from electrophoretic separations were normalized for each sample from each subject relative to the calculated basal level (100%) for that subject. The relative amino acid level (relative percent of basal level) was plotted versus sample collection time for a single subject. The mean normalized relative percentage of basal level±standard deviation was plotted for multiple subjects. Statistical significance was determined by the calculation of the Student t-test (equal sample sizes) at 95% confidence levels.

Results

In vivo sampling

We modified the low-flow push-pull perfusion tool, which we previously described [41], for sampling the vitreous or vitreoretinal fluid from the rat eye for the characterization of in vivo chemical composition. Sample collection was performed routinely for more than 2.5 h. All perfusates were assayed for amino acids by CE-LIF.

Amino acid assay

Using the previously reported method for amino acid analysis [43], the amino acid content was quantified from push-pull perfusates. Table 1 summarizes the basal concentrations of the amino acids found in rat vitreous and vitreoretinal interface perfusates. These values have not been corrected for dilution in the sampling process. We observed in a previous report a 70-80% relative recovery in vitro [41]. We also found biogenic amines, phosphoethaloamine, GABA, taurine, glutamine, o-phosphoserine, and histamine in vitreal samples but were either not clearly resolved during the separation or the concentration was below the limit of detection. There were two fewer amino acids reported in the Table 1 here as compared to our previous study [43]. Upon further study, we observed the peaks previously identified as Lys and D-Ser were not consistently found in all samples from individual subjects and thus were not included in the table.

Basal level stability of glutamate at vitreoretinal interface

The stability of the glutamate basal level at the vitreoretinal interface during the sampling process is shown in Figure 3. During the first 45 min the basal levels were higher and more variable. These higher basal glutamate concentrations in the initial samples were likely due to tissue damage during the probe insertion as well as excess perfusion saline around the retina. After 30-40 min, lower and more consistent glutamate values were observed with less than a 30% relative standard deviation. The glutamate basal values fluctuations seen in Figure 3 after 1.75 h of sampling may have been a result of the long-term anesthesia. Therefore, the average amino acid concentrations were taken from samples collected from the relatively stable 0:30 to 1:45 h time period. The inset of Figure 3 shows the approximate sampling sites at vitreoretinal interface determined visually. These sample collections were performed close to the optic nerve head.

Confirmation of the probe placement

Glutamate concentrations in perfusates were increased at least 200% over baseline for all PDC infusions at the vitreoretinal interface. A representative result from a single animal is shown in Figure 4. The baseline levels of glutamate, aspartate and arginine are shown. For comparison, the inset of Figure 4A and Figure 4B represent the probe placement locations and the sample collection flow rate, respectively. With this particular example, the sample collection flow rate continuously decreased from 60 to 20 nl/min until the fifth sample and then stabilized at that level. The amino acid levels increased with the flow rate drop as has been reported previously [41]. Importantly, the response to the drug infusion was a considerably larger increase. Notably, only glutamate and aspartate levels increased but not arginine.

Two different regions of the retina were selected for drug infusion. The probe was placed either close to the optic nerve head or peripherally. The total effect from the infusion of the PDC containing saline on glutamate levels was different in each region, and the results are shown in Figure 5. The insets of Figure 5A and Figure 5B clearly indicate the probe placements (determined visually) and the placement dissimilarity. Close to the optic nerve head, the average increase due to drug stimulation was slightly higher 211±54%, but this difference was not significant. For regions 1-2 mm peripheral to the optic nerve head region, the average increase was 466±102%. This result was significantly different from both controls and the increase due to drug infusion seen at the optic nerve head. There was no change in amino acid levels in experiments performed with the probe peripheral to the optic nerve head, where the infusion line was switched between two drug-free KRB solutions.

Discussion

We demonstrated a novel approach for sampling from rat vitreous and vitreoretinal interface that we used to show spatial resolution of sampling. While in vivo sampling with microdialysis has been performed previously [26-28,30-36], the method presented here affords a number of advantages. Because the low-flow push-pull perfusion was smaller than microdialysis probes it was possible to minimize tissue disruption by the use of a 29-gauge guide needle for probe placement. For experiments here the probe was inserted at the extreme periphery of the retina which does not to lead to visible tissue disturbance. It is possible that the probe will make contact with the lens and a cataract can develop. Even with careful placement, this occurred in this study less than 5% of the time (n = 42). The small size of the probe was well suited to the dimensions of the rat eye. The advantages for the rat as a common model include the small size, low cost, easy handling, and ready availability. While the rat has been used for microdialysis sampling [32], the method here affords spatial resolution over the retina (see Figure 5). The perfusion fluid mixes and is collected from the tip of the capillary probe which greatly limits the tissue volume from which analytes are able to diffuse. Also, the ability to directly perfuse the tissue of interest allows both higher recovery [41] including higher molecular weight peptides [44].

With this method, probe stability is an important factor for continuous sampling. Any movement of the probe leads to a considerable decrease of flow rate or complete obstruction of the probe. After an initial infusion at a higher flow rate to prevent tissue entering the probe, the flow rate was ramped down to the desired sampling flow rate. Infusion at the target flow rate was continued for 5-7 min before the start of sample collection to create a region of less viscous media around the probe tip which helps to initiate smooth collection. If the collection flow rate went down 30-40% during sampling, the infusion was increased by 10-25% immediately. The vacuum pressure was increased gradually if greater infusion flow failed to improve decreased collection flow. Notably, complete obstruction of the tip was observed with an initial acceleration of vacuum without increasing the infusion flow. These results support the classical idea of a perfusion "drop" of infused saline at the tip of the push pull probe [37,38]. It is possible, although admittedly speculative, an estimate of 200-400 nl volume for this drop would be reasonable accounting for infusion during ramp times during probe placement and the 5-minute pause before beginning withdrawal flow.

In this study, in vivo samples from vitreous and vitreoretinal interface were analyzed for amino acids by CE-LIF. There were few reported values for basal amino acid levels in rat vitreous but none for the levels at the vitreoretinal interface. There were no significant differences between the observed amino acid levels from the vitreous and vitreoretinal interface in Table 1. The peak for valine appeared to approach a difference as well as a general trend for all peaks excluding glutamate and glycine to be higher near to the vitreoretinal interface. These results suggest that with more careful probe placement in the mid-vitreous, these differences might become statistically significant. Compared to our previous report [43], the average level of many of the amino acids measured from the perfusates were comparable but the variances reported here were higher. The reason for this is not entirely clear but may be related to the lower (1:10 diluted) dose of local anesthetic that was applied to the eye in this work.

Comparison of these data to previously published work is difficult because of the dissimilarity of assay methods as well as differing model systems. Homogenized tissues are not applicable as this represents both intracellular and extracellular contributions. There are a number of reports of glutamate being measured from vitreous expressed from enucleated eyes of rats [4,48,49]. These reports are uniformly higher than the levels observed here and are likely due to lower tissue trauma with low-flow push-pull sampling and the 70-90% recovery of the vitreous concentration for perfusate levels. There is good agreement with our glutamate level in the one report where vitreous amino acids were measured by microdialysis [32]. The vitreoretinal interface level observed here was somewhat lower, but this difference was within the margin of error for the reported values. There are a number of published reports for vitreous amino acids levels of clinical samples [21,50,51] including data from our laboratory [23]. With the exceptions of gluatamate, aspartate, and glycine, the amino acid concentrations found from clinical samples are about 10-times higher. These higher values may similarly be related to a greater extent of tissue trauma required for collection, the sample stability of whole vitreous, the differences between the rat and human vitreous, and the dilution of perfusates due to recovery. Notably, the levels of glutamate, aspartate, and glycine compare well with the rat perfusate values reported here.

Sampling from the vitreoretinal interface is a key achievement presented in this paper. This low-flow push-pull perfusion method allowed the recovery of amino acids from vitreoretinal interface, and the placement was confirmed by drug infusion. Further, the data demonstrated a clear difference when sampling from different regions of the retina. Figure 4 and Figure 5 both provide evidence that the in vivo collection was performed at vitreoretinal interface and that there was spatial resolution for sample collection. Spatial resolution could be important for fundamental studies of neurochemical signaling at the retina as well as for probing disease models. For example in a rat model of glaucoma, the retinal ganglion cells undergo apoptosis from the periphery to the central retina [45].

The elevated glutamate levels, a result of the PDC infusion, confirmed that sample was collected near retinal tissue. The expected increase was seen because PDC is a potent and selective inhibitor of excitatory amino acid transporters, which remove glutamate from the synaptic cleft. Further, there is a consistent pattern of increase with respect to the timing of the drug infusion. There is likely some drug in the perfusion saline in the samples directly preceding and following the sample that the glutamate level increase is seen. It appears that there is not a high enough local concentration for a sufficient time period for the glutamate levels to be elevated except when the drug is delivered to the tissue for the entire 20 min required for the collection of one sample. Experiments where the switching was performed between two KRB infusion lines demonstrated no change to amino acid levels. Notably, the process of switching the infusion line can introduce slight movement of the probe that may complicate sample collection. Because the total contact area at the tip of these probes is less than 0.023-mm², even a slight shift in this highly viscous media can easily lead to complete or partial obstruction. The use of a nanoliter fluid switch would greatly improve the process of flow switching.

Our previous in vitro studies of low-flow push-pull perfusion indicate that recovery is inversely proportional to flow rate [41]. A similar effect can be observed in Figure 4, the relative percentage of each amino acid was increased with a decline of the flow rate. This is somewhat surprising given that the infusion flow rate was increased by 25% during the course of this sample collection flow rate drop. With the decreased withdrawal and increased infusion, the concentrations of amino acids found in the perfusates would be expected to decrease due to increased dilution. Yet observed levels consistently increased with decreased withdrawal flow. Another observation was that increased infusion sometimes did not prevent probe occlusion during sampling which is suggestive of a regional disconnect of the infusion flow and the perfusion drop. If infusion was not increased at the perfusion drop at the end of the probe then there was an overall lower perfusion flow rate which would result in higher recoveries and increased amino acid levels similar to that seen with low-flow microdialysis [52]. Also, the extent of the slow increase was variable and was seen only slightly in averaged data in Figure 5A and not at all in Figure 5B. While the slow basal increase may complicate the interpretation of the infusion data in Figure 4, the infusion of the glutamate reuptake antagonist gave a visibly larger increase in the level of glutamate which also decreased to normal levels after returning to the non-drug containing perfusion saline.

The drug infusion study was extended to determine if there were tissue differences at two sites on the retina. Average glutamate elevations were 211±54% for central regions of the vitreoretinal interface and 466±102% for peripheral regions. It is notable that the change due to the experimental treatment (signal) was rather large in comparison to random fluctuations in the glutamate level (noise) such that statistical significance was achieved with relatively low replications per position (n=3). The observed regional differences may reflect that there is a lower synaptic density close to the optic nerve head than outer area of the retina; therefore, fewer transport proteins are blocked by PDC. The PDC infusion then results in less glutamate accumulation near to the optic nerve head. These regional differences may also be due to higher blood flow at optic nerve head compared to sites further away [53]. The higher blood flow near the optic nerve head may lead to a greater clearing of the drug or in the same way, glutamate may be washed out before recovery by eye probe. Further studies are necessary to explore these hypotheses. More importantly, the method provides the ability to explore the chemical microenvironment at distinct locations over the vitreoretinal interface.

In conclusion, this miniaturized probe and low-flow push-pull perfusion technique demonstrates new abilities for in vivo analysis of the vitreoretinal interface. In vivo collection was performed for more than 2 h and amino acid basal levels were quantitated for 10 amino acids from both the rat vitreous and the vitreoretinal interface. While there were trends for higher levels in the midvitreous, there were no statistically significant differences found between these regions. Glutamate levels did compare well with one report of microdialysis of the rat vitreous [32]. Sample collection at the vitreoretinal interface was confirmed by a tissue response due to a glutamate reuptake antagonist infusion. The small probe tip size relative to the retina allowed probe placement in the precise regions of the retina that generated statistically different responses to a drug infusion. Glutamate levels were significantly increased by PDC infusion above baseline levels at peripheral sites but the increase in glutamate levels over the optic nerve head was not significant. While the development of a novel in vivo sampling tool and the demonstration of qualitative and quantitative studies of retinal free amino acids is shown here, this technique may be much more widely applicable. The technique has already been demonstrated for the sampling of vitreous peptides [44]. The expected high recoveries and non-mass limited collection is applicable to nearly any molecule found in the vitreous. This powerful tool to describe the neurochemistry of the retina is also site-specific which may open doors to many fundamental studies of the in vivo retina that were not previously possible. Further applications include studying the chemical changes at the pathological retina in order to better understand the nature of tissue dysfunction and identify potential treatments.

Acknowledgements

This work was supported by the NIH grant EY014908 and the Research for the Prevention of Blindness Foundation. The authors thank David Wirtshafter for the use of laboratory facilities for some experiments. These results appeared in part at the 2005 annual meeting of the Association for Research in Vision and Ophthalmology in Fort Lauderdale, FL.

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