Changes in blood-brain barrier permeability associated with insertion of brain cannulas and microdialysis probes1

Brain Research 803 Ž1998. 218–230

Interactive report

Changes in blood-brain barrier permeability associated with insertion of brain cannulas and microdialysis probes 1 Dennis R. Groothuis a,b,c,) , Sherman Ward b,c , Kurt E. Schlageter a,b,c , Andrea C. Itskovich Susan C. Schwerin b,c , Cathleen V. Allen a , Cynthia Dills d , Robert M. Levy c,d a

b,c

,

Department of Neurology, Northwestern UniÕersity Medical School, EÕanston Northwestern Healthcare, EÕanston, IL 60201, USA b Department of Neurobiology and Physiology, Northwestern UniÕersity, EÕanston, IL 60208, USA c Northwestern UniÕersity Institute for Neuroscience, Northwestern UniÕersity, EÕanston, IL 60208, USA d Department of Surgery (Neurosurgery), Northwestern UniÕersity Medical School, Chicago, IL, USA Accepted 25 May 1998

Abstract Blood-brain barrier ŽBBB. transcapillary transport was studied after insertion of cannulas and microdialysis probes into the brains of three groups of rats. Quantitative autoradiography was used to measure changes in BBB permeability around the insertion site. In the first group, BBB function was measured with 14C-sucrose at times from immediately, and up to 28 days, after insertion of a microdialysis probe. BBB function was disrupted biphasically: a 19-fold increase in the influx constant Ž K 1 . of sucrose occurred immediately after insertion with a second 17-fold increase at 2 days, followed by a slow decline to 5 times normal values at 28 days. In the second group, 14 C-dextran Ž70 kDa. was used to measure BBB transcapillary transport; K 1 was increased 90-fold after probe insertion. In the 3rd group, 14 C-AIB Ž a-aminoisobutyric acid. was used to evaluate BBB transport after insertion of a 27 gauge cannula, which was used to infuse 1 m l of saline over 5 min. The K 1 of AIB was increased 25 times control values. We conclude that BBB transcapillary transport function is disturbed in response to insertion of brain cannulas andror microdialysis probes, that BBB dysfunction is maximal at the cannula or probe tip, varies with time after insertion, may persist for at least 28 days after insertion, and occurs over a wide molecular range of solutes. These results suggest caution when using microdialysis as a method to study normal BBB function, and suggest that microdialysis may overestimate the rate of transfer into and out of the brain. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Blood-brain barrier; Brain; Drug delivery; Microdialysis; Permeability

1. Introduction Because the blood-brain barrier ŽBBB. effectively restricts the rate that water-soluble compounds enter the brain w28x, accurate quantitative methods to study BBB permeability are critical for evaluating the time course and distribution of drugs that can potentially be used for therapy of brain diseases. Microdialysis, in which extracellular fluid can be temporally sampled, holds promise for studying the time course of brain influx and efflux of intravenously administered drugs as well as changes in brain concentrations of neurotransmitters in response to both experimental paradigms and disease states w33,45,53x. )

Corresponding author: Division of Neurology, Burch Hall, Evanston Hospital, 2650 Ridge Avenue, Evanston, IL 60201, USA. Tel: q1-847570-1989; Fax: q1-847-570-1934; E-mail: [email protected] 1 Published on the world wide web on 10 July 1998. 0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 5 7 2 - 1

Furthermore, microdialysis can be used in species as diverse as mice and men w14,31x. Alternatively, cannulas may be inserted into the brain w17x to study the efflux of drugs from the brain or to administer drugs by convection-enhanced delivery w13,36,37,40x. In reviewing published studies of transcapillary transport across the BBB in association with the use of brain cannulas andror microdialysis probes, we have been unable to determine with confidence whether the BBB is affected by insertion of cannulas or probes into the brain, the spatial distribution of such effects, or the time course of alterations in BBB permeability. If BBB permeability is increased by the act of probe or cannula insertion, the normal relationships between solute concentration in the vasculature and in the brain extracellular fluid ŽECF. will be altered. In this situation, when a drug or solute is administered intravenously in the presence of increased BBB permeability, the concentration in the ECF will be

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increased. Consequently, ECF, which is the source fluid from which microdialysis obtains it measurements, will contain an artifactually high concentration. Conversely, if the drug or solute is administered through a cannula or microdialysis probe into the ECF, the drug or solute will leave the brain at an artifactually high rate and lead to the conclusion of higher than expected efflux rates from brain. Many studies have reported the lack of significant perturbation in BBB transport function by cannularprobe insertion w5,7,17,24,49x, while others have reported potentially significant changes w39,52x. The situation is complicated by the variety of methods that have been used to insert cannulas and probes, the methods used to document BBB function, and the variable temporal relationships between insertion of the cannula andror probe and when BBB function was studied. Because of these concerns, we performed experiments that were designed to measure the unidirectional blood-to-brain transfer of nonmetabolized compounds. We evaluated regional changes in BBB transcapillary transport function after insertion of brain cannulas and microdialysis probes, using water-soluble solutes in single time point experiments, in which tissue radioactivity concentrations were measured with quantitative autoradiography ŽQAR.. Unlike methods that sample tissue directly, QAR can measure changes in tissue radioactivity concentrations in tissue volumes as small as 50 = 50 = 20 m m Ž5 = 10y5 mm3 . w8,24x, which allows BBB transport function to be determined in the brain immediately surrounding the cannula and probe insertion sites.

2. Materials and methods The use of animals in this project was reviewed and approved by the Institutional Animal Care and Use Committee of Evanston Northwestern Healthcare. Three groups of male Sprague–Dawley rats, 350–400 g, were used to evaluate changes in BBB transcapillary transport associated with placement of cannulas and microdialysis probes into the brain under different experimental conditions. In the first group, changes in BBB transcapillary transport were examined as a function of time after insertion of commercially available intracerebral guide cannulas and microdialysis probes. BBB transcapillary transport was examined with 14 C-sucrose and quantitative autoradiography ŽQAR.. The second group also used commercially available intracerebral guide cannulas and microdialysis probes, but transcapillary transport function was evaluated with a 70 kDa 14 C-dextran and QAR with 1 week between insertion of the guide cannula and the microdialysis probe. In the third group, a 27 gauge needle was inserted into the brain and 1 m l of buffered saline was infused over 5 min; BBB transcapillary transport function was evaluated with 14 C-AIB Ž a-aminoisobutyric acid. and QAR. In the first group, BBB changes associated with insertion of a commercial cannula and probe were examined as

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a function of time. The rats were anesthetized with ketamine Ž50 mgrkg. and xylazine Ž7.5 mgrkg.. A rat was placed in a Kopf stereotaxic frame, a skin incision made lateral to the midline, and a burr hole made through the skull with a dental drill. An O-ring intracerebral guide cannula ŽMD-2250, Bioanalytical Systems, Lafayette, IN. was stereotaxically inserted into the caudate nucleus Ž2.5 mm lateral and 1 mm anterior to the bregma, and at a depth of 5 mm. according to the atlas of Paxinos and Watson w47x, and the cannula affixed to the skull with dental cement. A total of 11 groups of 3–4 rats per group were used to assess BBB transcapillary transport function associated with insertion of the cannula andror probe. The first group was used for experiments immediately following insertion of the cannula. The second group was used 1 h after cannula insertion, but before the microdialysis probe was inserted. In the remaining groups, a microdialysis probe ŽBR-2, Bioanalytical Systems, Lafayette, IN. was filled with saline ŽpH s 7.4., the inlet and outlet cannulas were sealed, and the probe was inserted 1 h after the guide cannula. Once the probe was in place, the skin surrounding the cannula was surgically closed. Rats were used for experiments at the following time points: immediately after probe insertion at 2, 4, 24 h, and at 2, 7, 14, 21, and 28 days. To evaluate BBB transcapillary transport function, the rats were prepared for single label quantitative autoradiographic experiments as previously described w8,30x. Unilateral femoral arterial and venous polyethylene catheters ŽPE-50. were inserted. Blood pressure and rectal temperature were monitored and heat lamps used to maintain body temperature at 378C. At the beginning of an experiment, 50 m Ci 14 C-sucrose ŽNew England Nuclear, Wilmington, DE; 632 mCirmmol. was injected intravenously over 30 s. Arterial blood samples were obtained at selected intervals, centrifuged, and plasma used to determine radioactivity concentration by liquid scintillation counting ŽLSC. with appropriately quenched 14 C standards. At the conclusion of each 15 min experiment the rat was decapitated and the brain rapidly removed and frozen in liquid freon Žy408C.. Each brain was serially sectioned at 20 m m thickness on a cryostat and used to prepare autoradiographic images along with 14 C-methylmethacrylate standards, as previously described w8,30x. After preparation of the autoradiographs, the brain sections were stained with hematoxylinreosin. In the second experimental group, guide cannulas were stereotaxically inserted into the caudate nucleus of 5 rats, as described above; the burr hole was made with a dental drill. One week later, the rats were again anesthetized with ketamine and xylazine and a microdialysis probe that had been filled with normal saline, pH s 7.41, was inserted through the guide cannula. An experiment to evaluate BBB transcapillary transport function was performed as described above, except that 100 m Ci 14 C-dextran ŽMW s 70 kDa, 1.7 mCirg, Sigma, St. Louis, MO. was infused intravenously over 30 s immediately after insertion of the

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Fig. 1. Diagram of quantitative autoradiographic sampling. The target site of the microdialysis probe or infusion cannula was the caudate nucleus Žindicated by the solid dot.. The image analysis system was used to collect a profile of tissue radioactivity concentrations in a 2 cm wide by 1 mm tall bar, with 50 m m subdivisions, as illustrated by the horizontal rectangle. A similar rectangle, oriented vertically from the insertion site in the cortex, through the cannularprobe tip, and to the ventral surface of the brain was used to collect a profile of tissue radioactivity concentrations.

probe, and plasma samples collected over a 60 min experimental period. The animal was decapitated and the brain used to prepare serial autoradiographs as described above. In the third experimental group, 8 rats were first pre-

pared for single label quantitative autoradiographic experiments as described above; a dental drill was used to thin, but not penetrate, the skull. A 27 gauge needle was stereotaxically inserted into the caudate nucleus over a period of 1 min, using the procedures outlined above. An intravenous injection of 14 C-AIB Ž50 m Ci, w1-14 Cxalpha aminoisobutyric acid, 40–60 mCirmol, New England Nuclear, Boston, MA. was given over 30 s. At the conclusion of the 14 C-AIB injection, 1 m l of phosphate buffered saline ŽpH s 7.41. was infused through the 27 gauge needle over 5 min. The needle was left in place for an additional 5 min and then slowly removed over 1 min. Plasma samples were collected at timed intervals, the animal decapitated 15 min after the start of the AIB infusion, the brain removed and serial autoradiographs prepared as described above. Autoradiographic sections from all experiments were analyzed with the same methods. The autoradiographic sections and hematoxylinreosin stained histologic sections were reviewed and those through the center of the insertion site were selected for analysis and digitized with a video digitizing system at a resolution of 50 m m. Some data analysis was performed on a Macintosh computer using the public domain NIH Image program Žversion 1.60, developed at the US National Institutes of Health.. Preliminary examination of the autoradiograms showed a distribution pattern in which the highest tissue radioactiv-

Fig. 2. Influx constant of 14 C-sucrose at different times relative to insertion of a microdialysis probe. The value of the influx constant of sucrose, m l gy1 miny1 , is shown on the vertical axis. The bars on the horizontal axis represent the mean" SD of the top 10% of the K 1 values in the area of brain with disrupted BBB function for each experimental group of animals. A guide cannula was first inserted, followed by insertion of a microdialysis probe 1 h later. The groups are identified relative to insertion of the microdialysis probe, e.g. P s y1H means that the guide cannula had been inserted and that the BBB studies were performed 1 h before the microdialysis probe was inserted. The solid line at the bottom represents the mean K 1 of sucrose in the contralateral, unoperated caudate nucleus, bracketed by a dotted line that represents "1 SD.

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ity concentrations were around the probercannula insertion pathway, and decreased outward from this location. The autoradiograms were analyzed with two different methods. First, the brain area in which tissue radioactivity values were increased above normal brain values was determined by thresholding the image and outlining the area with increased values; the area and mean tissue radioactivity content Ž"SD. was determined. To express changes reflecting maximal breakdown of the BBB, we recorded the mean " SD of the highest 10% of values within the region in which BBB function was altered. The with maximal BBB disruption was generally about 1 mm in diameter and was centered around the infusion site. This method avoided skewing data when isolated pixels with very high values were present, e.g. when inflammatory cells were present, and gave an estimate of maximal BBB changes in the immediate vicinity of the cannula or microdialysis probe. The mean " SD tissue radioactivity was also determined from a brain area of the same size in the contralateral caudate nucleus, and served as a measure of normal BBB function. Areas of disturbed BBB function along the cannularprobe path and in the cortex were not included in the area calculations. In the second method, a radioactivity distribution profile was created by using an image processor to construct a rectangle, 2 cm long and 1.0 mm tall with 50 m m subdivisions, extending through

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the infusion site and across the coronal brain section ŽFig. 1.. Data were returned as a histogram of tissue radioactivity ŽnCirg; mean " SD. in each 50 m m subdivision. Another rectangle was constructed with a vertical orientation, extending from the cortical location of the insertion site, through the insertion site and tip of the cannularprobe and extending to the ventral surface of the brain. In the vertically oriented rectangles, information about the magnitude of BBB disruption was collected in cortex immediately beneath the burr hole, which was used to relate BBB disturbance in the cortex to the method used to produce the burr hole. The location of the infusion site was used to align and combine data from multiple experiments within an experimental group. For final data analysis, the tissue radioactivity concentration was used to express a blood-to-tissue transfer constant for each subdivision, according to the following expression: K1 s

C b Ž T . y Vp Cp Ž T . T

Ž 1.

H0 C Ž t . d t p

where K 1 was the transfer constant with units of ml gy1 miny1 , C b ŽT .was the tissue radioactivity concentration ŽnCirg. at the end of the experiment ŽT ., Cp was the

Fig. 3. Volume of brain with increased BBB permeability. The y-axis represents the volume of brain Žmean" SD. in which BBB permeability was increased over background values, in units of mm3. The bars on the horizontal axis are the same as in Fig. 2 and are identified relative to the time of insertion of the microdialysis probe.

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plasma tissue radioactivity concentration ŽnCirml., and Vp was the fractional tissue plasma space reported for the caudate nucleus. Throughout this report for purposes of consistency, K 1 was reported in units of m l gy1 miny1 . The octanol water partition coefficient for sucrose and the 70 kDa dextran was determined as previously reported w10x. Briefly, 1 m Ci of 14 C-sucrose or 14 C- dextran was added to each of 3 vials containing 10 ml of n-octanol and 10 ml saline. Each vial was vortexed for 3 min, allowed to sit for 30 min, and then triplicate samples taken from the octanol and water phases and counted by liquid scintillation counting ŽLSC.. The octanolrwater partition coefficient was calculated as the mean of LSC counts in octanol divided by the mean of the counts in the saline.

3. Results The most extensive of these experiments were those in which a microdialysis cannula was first inserted, followed by insertion of a microdialysis probe 1 h later. These animals were then used for experiments to measure regional BBB function following intravenous injection of

14

C-sucrose. BBB experiments were conducted immediately after insertion of the guide cannula, immediately before and after insertion of the microdialysis probe, and at time points up to 4 weeks after insertion of the microdialysis probe ŽFig. 2.. Fig. 2 presents the mean Ž"SD. of the highest 10% of values of the blood-to-tissue transfer constant, K 1 , of 14 C-sucrose from each time point, from the brain area around the cannularprobe tip with altered BBB function. The diameter of the area included for these measurements was 0.4 " 0.05 mm at t - 2 d, and 0.1 " 0.02 mm at 28 d. For reference, the mean " SD of the transfer constant of sucrose in the contralateral caudate nucleus is also shown in Fig. 2 Ž0.46 " 0.09 m l gy1 miny1 .. In both groups studied after insertion of the guide cannula, but before the insertion of the microdialysis probe, the K 1 of sucrose was significantly increased compared to K 1 in the contralateral caudate nucleus Ž P - 0.01, Student’s t-test., even though the sampling location was beneath the tip of the guide cannula. Immediately following insertion of the microdialysis probe, the maximum value of the influx constant increased 19-fold over normal brain values Ž P - 0.01, Student’s t-test.. The maximum value of the influx constant was significantly increased

Fig. 4. Profile of BBB function through the microdialysis probe or cannula. The vertical axis represents the value of the influx constant, K 1 , in units of m l gy1 miny1 , for each compound. The plot profile of K 1 through the tip of the microdialysis probes is shown for the AIB experiments, for two groups from the sucrose experiments Žimmediately after probe insertion, P s q0, and at 28 days, P s 28D. and for the dextran experiments in which the guide cannula was inserted 1 week before the microdialysis probe. BBB experiments were done immediately after insertion of the probe. Distance is measured in mm relative to the center of the probe or cannula tip Ž0 mm.. For each compound, the dotted line at the bottom of the graph represents the mean value of K 1 in the contralateral, unoperated caudate nucleus, for the compound being shown.

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over that of the contralateral hemisphere for the duration of the 28 days. However, as evident in Fig. 2, the influx constant decreased rapidly by 2 h after insertion to a value 9 times that of normal caudate, and then increased a second time 2 days following probe insertion, to 17-fold that in contralateral caudate. From 7 to 28 days, the influx constant slowly decreased to about 5 times the K 1 value in the contralateral hemisphere. Fig. 5B shows the quantitative autoradiographic appearance of the cannularprobe

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pathway through the brain of a 28 day animal, including the site of microdialysis probe tip, and a plot of tissue radioactivity values made in the horizontal direction across the brain section Žas diagrammed in Fig. 1.. The volume of brain in which BBB transcapillary transport was altered also changed over time ŽFigs. 3 and 4.. The maximum volume of brain in which the BBB was damaged was found immediately after insertion of the microdialysis probe Ž7.1 " 2.0 mm3 ., which was followed

Fig. 5. Autoradiographic images of BBB function. ŽA. is from the AIB experiments, in which an infusion was made into the caudate and BBB function measured at the same time. The gray scale on the right represents the values of the influx constant, K 1 , in units of m l gy1 miny1 . The plot profile on the bottom represents the profile of K 1 values in a horizontal rectangle across the section, through the center of the infusion cannula as diagrammed in Fig. 1. ŽB. is from the 28 day group of the sucrose experiments in which the microdialysis guide cannula and probe were inserted 28 days before the BBB experiment was conducted. Note that the disturbances in BBB function are focal, and that the return to normal BBB values as a function of distance from the probe site occurs over a shorter distance in the 28 day animal than in the acute experiment.

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by a rapid decline over the next 4 h. The volume of brain with increased permeability then increased a second time, to a volume of 6.3 " 1.4 mm3 at 24 h, after which it again declined. At 4 weeks after insertion of the microdialysis probe, the volume of brain in which BBB function was disturbed was 1.7 " 0.2 mm3, and was centered upon the probe tip. In group 2, in which implantation of the guide cannula preceded insertion of the microdialysis probe by 1 week, there was a similar alteration of BBB function ŽFig. 4.. In these animals BBB function was studied by intravenous injection of 14 C-dextran Ž70 kDa. and a 1 h experimental duration. At the site of maximum disruption of the BBB, the K 1 of dextran Ž0.27 " 0.1 m l gy1 miny1 . was increased 90-fold over that in the contralateral caudate Ž0.003 " 0.0015 m l gy1 miny1 .. The diameter of tissue sampled to determine the highest 10% of K 1 values was 0.5 " 0.07 mm. In group 3 BBB transcapillary transport function was assessed after intravenous injection of AIB. In these experiments a microdialysis probe was not used. A 27 gauge cannula was inserted and used to infuse solutions into the brain. The changes in BBB function were similar to the previous two groups. Fig. 4 shows the averaged profile of

the transfer constant; the highest 10% of the influx constant values of AIB were 63.1 " 11.2 m l gy1 miny1 , compared to a value of 2.6 " 0.7 m l gy1 miny1 in the contralateral caudate. The diameter of brain included in the highest 10% measurements was 0.4 " 0.06 mm. Fig. 5A shows the autoradiographic image from a single animal in this group illustrating the peak increase in K 1 at the tip of the cannula, as well as the volume of affected brain surrounding the infusion site. The octanolrwater partition coefficient for sucrose was 7.0 " 0.2 = 10y4 ; that for the 70 kDa dextran was 2.7 " 0.3 = 10y5 .

4. Discussion The most notable finding of these experiments was that, regardless of the size of cannularprobe inserted into the brain, regardless of the methods used to insert the cannularprobe, and regardless of the time point after insertion, BBB transcapillary transport function was abnormal compared to the unoperated, contralateral caudate. The insertion of a microdialysis guide cannula and probe produced a time-dependent increase in the influx constant of

Fig. 6. Logarithmic plot of influx constant vs. octanol-water coefficient % 6MW. The values for AIB, sucrose and 70 kDa dextran are shown in normal brain Žsolid line. and in the cannularprobe experiments Ždotted line.. There is a consistent upward shift in K 1 for all compounds in the cannularprobe experiments representing increased transport across the BBB. The value for sucrose is the median value from all experiments, and the error bar for sucrose represents the range of values from all experiments shown in Fig. 2. The values of the K 1 of sucrose from the experiments of Morgan et al. Ž1996. are also shown for normal brain Ž`. and after insertion of a microdialysis probe Žv ..

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C-sucrose that persisted over the 28 day study period, with a biphasic response, peaking immediately after insertion of the microdialysis probe and 2 days later ŽFig. 2.. The volume of brain in which BBB function was abnormal also showed a biphasic response, which peaked immediately after insertion of the probe and then 1–2 days later ŽFig. 3.. BBB disruption after insertion of cannulas and probes occurred over a wide range of water-soluble compounds, including a 70 kDa dextran, suggesting that the normal permeability of the BBB to large and water-soluble compounds was significantly altered ŽFigs. 4 and 6.. In all experiments, including insertion of cannulas andror probes, the autoradiographic images showed a similar cross-sectional profile ŽFigs. 4 and 5.; the maximum change in BBB function occurred along the cannularprobe path and at the tip of the cannularprobe. The disturbance in BBB function was spatially restricted and BBB function returned to normal values within 1–2 mm from the cannularprobe tip ŽFig. 4.. The insertion of a cannula or microdialysis probe into the brain produces a traumatic brain injury. Morgan et al. discussed the relationship between intercapillary distance in the brain, the size of the cannula andror probe, and the rate of insertion on physical injury to brain capillaries and concluded that traumatic capillary injury was almost inevitable w39x. There seems to be little controversy about the effects of the initial injury. The injury initially causes a decrease in local brain blood flow and glucose utilization w6x, and increases in the brain extracellular space w24x, ventricular pressure w25x, and capillary permeability. Our data ŽFigs. 2 and 3. suggests a biphasic response in the increased BBB permeability, with a prompt increase immediately after probe insertion, followed by a second increase 1–2 days after probe insertion. This biphasic response may be characteristic of traumatic brain injury. In a study in which the brain was subjected to a percussion injury and the time course of BBB injury evaluated by Evan’s blue extravasation, Baskaya et al. found that BBB injury peaked at 4 h and again at 3 days w2x. Westergren et al. found higher brainrblood tissue radioactivity ratios 24 h after probe insertion than at 3 h w52x. Graham and Gennarelli have discussed the neuropathology and cellular biology of traumatic brain injury w29x, which also support the concept of a biphasic response in BBB function. In addition to mechanical injury, Graham and Gennarelli attribute the initial events to large ion fluxes associated with injury of cell membranes, which may last for minutes or at most a few hours w29x. The initial events then trigger a complex cascade that develops over subsequent hours or days, which includes the influx of leukocytes and lymphocytes and the release of vasoactive cytokines. Our studies indicate that the maximum effect of this cascade on local BBB function is seen at 1–2 days. However, even at 28 days the influx constant of sucrose at the tip of the microdialysis probe was 5 times that of the contralateral caudate.

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We considered methodological issues that might have resulted in experimental errors in our studies. Aside from microdialysis many different methods have been developed to measure transcapillary transport across the BBB w18,26,27x. We selected conditions for our experiments to optimize our ability to detect regional, or geographic, changes in the unidirectional brain-to-tissue transcapillary transport with single time point experiments. Blasberg stated that conditions for measuring unidirectional brainto-tissue transcapillary transport in single time point experiments could be met if: Ž1. tissue-to-blood backflux was insignificant during the experimental period, Ž2. the distribution of the test substance in the blood vessels and extravascular tissue compartments that rapidly exchange with blood was negligible or accurately known, and Ž3. the experimental time was sufficiently long to permit an accurate determination of the amount of test solute that has actually crossed the BBB w11x. Our experiments were designed to meet these conditions. The radiolabeled compounds were administered by intravenous injection; consequently, the sole route of entry into brain was by passage from blood to the brain extracellular space. The final time point of the experiments was selected so that sufficient solute had crossed the BBB in order to be accurately measured and so that the solute concentration in the final plasma sample w Cp ŽT .x exceeded the expected concentration in the brain extracellular space w CeŽT .x, thus maintaining a concentration gradient from blood to brain to minimize the effects of efflux from brain. These conditions were met in all experiments. For AIB the radioactivity concentration values for the final plasma sample, normal caudate, and tissue around the microdialysis probe were 359.5 " 105.1, 8.4 " 2.5, and 48.7 nCirg Žmean " SD.. For sucrose, the values were 365.3 " 102.5, 4.1 " 2.7, and 59.5 " 33.8 nCirg, and for dextran the values were 902.9 " 308.7, 3.7 " 0.6, and 22.3 " 8.5 nCirg. If a value of Ve s 0.17 ml gy1 is assumed for the size of the brain extracellular space w43,46x, then the concentration in the brain extracellular space Ž Ce . was less than the plasma concentration Ž Cp . in each group. If, as suggested by Dykstra et al. w24x, Ve increased in size during microdialysis, then Ce would always be smaller than Cp in our experiments. Since none of these compounds is known to be actively transported by the BBB, the conditions necessary to maintain unidirectional diffusional movement were met for our experiments. We did not include an independent vascular space marker in these experiments. Since our tissue radioactivity measurements were corrected for vascular space ŽEq. Ž1.., it was possible that an error in the assumption about the tissue plasma space could have affected the calculated value of K 1 w15x, particularly if the true tissue plasma space was increased in size. If Vp was increased in size, then the vascular space correction in Eq. Ž1. would reduce the value of the numerator in Eq. Ž1. and yield a smaller calculated value for K 1. We used a plasma space correc-

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tion of 5 m l gy1 w41x; Cremer and Seville reported a tissue blood volume of 6.29 " 0.13 m l gy1 in the caudate nucleus of the adult rat w16x. Although insertion of microdialysis probes has been reported to transiently affect blood flow and glucose metabolism w6x, we are unaware of any measurements of blood or plasma volume in this setting. Two observations suggest that an error in the vascular space correction are unlikely to significantly change our conclusions. First, brain blood volume has been reported to decrease after traumatic brain injury w34,48x, although though the mechanism of trauma in these reports was different than insertion of microdialysis probes. Second, the effect of a variable Vp on the outcome of K 1 calculations with Eq. Ž1. can be modeled, using the experimentally determined measurements for C b and Cp . In the AIB experiments shown in Fig. 4, for each incremental increase of 0.5 m l gy1 in the value of Vp , there will be a corresponding decrease of 0.09 m l gy1 miny1 in the calculated value of K 1. A change of Vp from 5 m l gy1 to 20 m l gy1 would decrease K 1 from 64 to 60 m l gy1 miny1 . Thus, errors about the assumption of the size of Vp are not likely to significantly affect the conclusions of these experiments. The validity of the experimental methods used in our studies can be compared to other reports for values of the blood-to-tissue transfer constants for these compounds in normal brain. For normal brain, Ohno et al. reported an influx constant for sucrose of 4.3 = 10y4 miny1 in the caudate nucleus w44x, and Morgan reported a value of 2.6 = 10y4 miny1 w39x, both of which compare favorably with our measured value of 0.46 m l gy1 miny1 Ž' 4.6 =

10y4 ml gy1 miny1 .. Blasberg et al. reported a K 1 of 0.89 m l gy1 miny1 in caudate in their original studies with AIB w12x, although in subsequent studies we have reported a range of 0.5 to 4.0 m l gy1 miny1 in tumor-free caudate nucleus w9,30,38x; these compare favorably to the value of 2.6 m l gy1 miny1 in the present study. Nakano et al. reported a value of K 1 F 1 m l gy1 miny1 for a 70 kDa dextran in normal brain w42x. The behavior of the three water-soluble compounds that we used in this study is similar to results reported by others. Several studies have been designed and conducted to measure transcapillary transport across the BBB with microdialysis ŽTable 1.. The methods have differed widely and have included a variety of cannula andror probe insertion methods, variable intervals between insertion of the final probercannula and different methods for documenting BBB function. Some investigators have concluded that BBB permeability is altered acutely, if at all, while others have concluded that there were significant changes in permeability ŽTable 1.. There are two explanations that may account for an observation of apparently ‘normal’ BBB function when a focal BBB disturbance may have, in fact, been present. The first possibility concerns the effects of volume averaging, i.e. the part of the tissue with disturbed BBB function may be averaged with another part of the sample having normal BBB function. This is of particular concern in microdialysis experiments because the cannularprobe insertion causes a geographically restricted zone of BBB dysfunction around the cannularprobe ŽFigs. 4 and 5., which quickly returns to

Table 1 Comparison of studies of BBB function associated with insertion of microdialysis probes or cannulas Study

Probe type

Interval BBB examined

Tracer

Studies reporting no change or only acute changes in BBB permeability: 22 Cserr et al. Ž1981. V, C 7d Na 14 Ž . Benveniste et al. 1984 H, TC 30 min C-AIB Tossman et al. Ž1986. V, P 0h Na99m TcO4 , 3H 2 O Ž . Dykstra et al. 1992 V, P 1.5 h Evan’s Blue 14 Terasaki et al. Ž1992. H, TC 1, 48 h C-Sucrose de Lange et al. Ž1995. H, TC 24 h Atenolol Aasmundstad et al. Ž1995. V, C and P 3h Na99m TcO4 3 Ž . Sandouk et al. 1997 H, TC 24 h H-AIB Studies reporting altered BBB permeability: 51 Major et al. Ž1990. V, P 0–6 h Cr-EDTA 3 Westergren et al. Ž1995. V, PC 3 and 24 h H-Inulin Evan’s Blue 3 Morgan et al. Ž1996. V, C and P 2h H-Sucrose, 14 C-Urea

Tissue sampling method

Expression of BBB function

Method for burr hole

DTS QAR MD Inspection MD MD MD DTS

Brain efflux constant Brain influx constant PerfusaterBlood ratio PerfusaterBlood ratio AUCbrrAUCpl PerfusaterBlood ratio Brain influx constant

Drill Drill NA NA NA Drill NA Drill

MD DTS Tissue Sections DTS, MD

Brain influx constant Brain Blood ratio Immunhistochemistry Brain influx constant

Drill Drill Drill NA

Listed are the direction of insertion for each study ŽV s vertical insertion, H s horizontal insertion., and cannularprobe type ŽC s cannula, TC s transcranial, single-lumen probe, P s single-lumen probe, PC s double-lumen probe without guide cannula, C and P s double lumen probe with separate guide cannula., the interval between insertion of the cannula or probe and performance of the BBB studies, the tracer used in the studies, the tissue sampling method ŽDTS s direct tissue sampling, MD s microdialysis, QAR s quantitative autoradiography, sectionss serial histological sections., the method used to express BBB function ŽAUCs area under the curve., and finally the method used to produce the burr hole. Most notable is that a wide variety of methods have been used in almost every category, indicating one possible basis for the variability in the reported effects of cannularprobe insertion on BBB function.

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normal away from the cannularprobe. However, it is precisely this restricted volume in which the microdialysis probe is used to obtain samples. The effect of volume averaging is such that if a volume of brain with abnormal BBB function is averaged with an equal volume of brain with normal BBB function, the result is slightly more than half the focally abnormal value, and decreases to one tenth the focally abnormal value if 10 volumes of normal brain are averaged. The volume of brain with an abnormal BBB was 7.1 mm3 immediately after probe insertion and fell to 2 mm3 4 h after probe insertion ŽFig. 3.. These volumes would correspond to brain sample sizes of 7.1 and 2 mg, respectively, and emphasize the geographically restricted nature of changes in BBB permeability around a microdialysis probe. The investigators in Table 1 who used direct tissue sampling either used whole brain or did not define their tissue sampling methods. Cserr et al. stated that they used whole brain sampling because of the difficulty in working with 3–20 mg brain samples from around the cannula path w17x. Benveniste et al. used QAR to measure BBB dysfunction with 14 C-AIB w7x; however, since resolution of their digitizing system was not stated and since they did not define the size of the tissue areas from which they obtained K 1 values for AIB, it is possible that volume averaging may have occurred and have obscured a focal increase. They reported a K 1 value in normal cortex of 1.7 " 0.5 m l gy1 miny1 for AIB and in hippocampus, through which their probe passed, of 3.7 " 2.4 m l gy1 miny1 . They stated that these values were within the normal range reported by Blasberg et al. w12x. However, Blasberg et al. did not report a value for hippocampus, and reported a range of values from 0.89 Žcaudate. to 2.1 m l gy1 miny1 Žcortex.. It is possible that the hippocampal value of 3.7 m l gy1 miny1 reported by Benveniste et al., which is 1.7 to 4 times the normal values reported by Blasberg et al., was the result of volume averaging. Elsewhere, Benveniste says that additional studies were done w4x, but we have been unable to find the final report of this data. A second possible explanation that may pertain to reports of normal BBB function around cannulas and probes when a focal disturbance was actually present, is the use of relative or semi-quantitative, rather than quantitative physiological expressions, for reporting BBB function. For example, Tossman and Ungerstedt reported a microdialysis perfusate-to-blood ratio of 0.0016 after Na99m TcO4 was administered intravenously, compared to a ratio of 0.51 for 3 H 2 O w50x. Aasmundstad et al. reported a microdialysis perfusate-to-blood ratio of 0.0011 for Na99m TcO4 , compared to morphine and morphine 6-glucuronide, which were 350 and 90-fold higher w1x. Both groups used the difference between the perfusaterblood ratio of Na99m TcO4 , as compared to those of the much more permeable water and morphine, as evidence of an intact BBB. However, to compare permeability-limited solutes such as Na99m TcO4 , to blood flow-limited solutes such as

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water and morphine has significant limitations. Since radiolabeled compounds were used, it should have been possible to obtain normal brain tissue and determine an influx constant Ž K 1 . for Na99m TcO4 in these experiments, which may have been a much better control. In other experiments, Dykstra et al. used visual inspection to determine the presence of extravasated Evan’s blue-albumin complex, the sensitivity of which may be questioned w24x. De Lange et al. evaluated the ability of microdialysis to detect a difference between a microdialysis probe in normal brain and one subjected to hyperosmotic BBB disruption w19x. Although the microdialysis techniques could distinguish between the two physiological states, an independent method was not used to validate the BBB measurements. A minor issue that was separately evaluated in our studies was the impact of the method used to produce the burr hole on BBB function in the underlying cortex. We observed a large increase in BBB permeability in the underlying cortex ŽFig. 5., as did Benveniste w7x. These disturbances in BBB function lie some distance from the tip of the microdialysis probe andror cannula, but nonetheless could perturb brain function and experimental results. Almost all studies that have inserted cannulas and microdialysis probes have drilled the burr hole, although the precise details were not included in many of the studies ŽTable 1.. We evaluated several approaches, including: Ž1. a drill to penetrate the skull; Ž2. a drill to thin, but not penetrate, the skull; Ž3. a trephine to remove a bone plug from the skull; and, Ž4. a scalpel to slowly thin and penetrate the skull at the insertion site by placing the tip of the scalpel blade over the target site on the skull and then rotating the scalpel. Drilling consistently produced a zone of increased BBB permeability in the underlying cortex, regardless of whether the skull was penetrated or not. We assume, but have not documented, that this is the result of local tissue heating caused by the effect of the drill on the skull. The underlying cortical injury may be due to local hyperthermia. The trephine method was also associated with focal cortical BBB disturbances. Using a scalpel to slowly thin the skull was associated with the least disturbance of BBB function in underlying cortex, and is now the method that we routinely use for penetrating the skull. It may be important to note several issues that were not addressed in this study. In addition to changes in BBB permeability, there are several other factors that will determine equilibrium concentrations of drug in the extracellular space of the brain. In modeling the tissue concentration profiles associated with microdialysis, Dykstra et al. found that increasing the tissue extracellular volume from 0.15 to 0.4 ml gy1 best explained the tissue radioactivity concentration profiles around a microdialysis probe w24x. This is apparently a consequence of bulk-flow of proteins from blood into brain and associated changes in local osmotic pressure. Basser also predicted a significant increase in extracellular tissue volume near the injection site of brain infusions w3x. The changes that occur in association with

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insertion of cannulas andror probes into the brain, which include at least increases in blood-to-tissue influx, tissueto-blood efflux, and tissue extracellular space, need to be further evaluated in terms of their effects on BBB studies with microdialysis. We have not addressed any of the issues associated with obtaining data with microdialysis probes, including issues of standardization, which are also important for proper interpretation of this data w21,45x. Finally, it is important to state that our concern in this study has been the quantitative measurement of BBB function. The use of microdialysis to measure relative changes in neurotransmitter concentration in the brain w23x, the use of microdialysis or convection-enhanced delivery systems to deliver drugs locally in the brain for therapeutic purposes w35,36,51x, and the use of microdialysis in other tissues, or in brain tumors, in which capillary permeability is different from that of normal brain w22x, all raise issues different from those we addressed. In these settings, the use of cannulas and microdialysis probes may be a perfectly valid technique. However, in studies where the effect of inserting a cannula or probe into the brain may alter the pharmacokinetics of drug delivery and distribution, we would strongly suggest that independent measures of BBB function be included to document the magnitude and distribution of this effect, or to document that BBB permeability is normal. When the primary intent of experiments using microdialysis probes andror cannulas is to study the BBB, we believe that extra caution is warranted. The effect of disturbed BBB transcapillary transport around probes and cannulas is illustrated in Figs. 4–6. Fig. 4 shows the profile of the influx constant for AIB, sucrose, and a 70 kDa dextran at the cannularprobe tip. When the studies were done in close temporal proximity to cannularprobe insertion, the maximum disruption was always at the cannularprobe tip and BBB; BBB function returned to normal values within 1–2 mm. At 28 days, BBB function returned to normal within ; 0.5 mm. However, since the most active exchange zone of a microdialysis probe is at its surface, it is precisely from the volume brain with altered transcapillary transport function that measurements are being made experimentally. Fig. 5 graphically illustrates the appearance of the autoradiographic images, both in the acute insertion phase ŽFig. 5A. and 28 days later ŽFig. 5B., showing the highly focal nature of the BBB disturbance. This altered relationship can also be illustrated in a different way: Fig. 6 shows the relationship between the influx constant and a measure of lipid-water solubility wŽoctanolrwater solubility.r6MWx for the three watersoluble compounds used in our study, both in normal brain and in the area of disturbed BBB function around the cannularprobe. The acute insertion phase was associated with a marked shift in the curve that defines this relationship in normal brain w28x. For comparison, the values of sucrose in normal brain and after microdialysis probe insertion as reported by Morgan et al. are also shown in

Fig. 6w39x. The shift in this curve may explain the conclusions reached by Hammarlund-Udenaes, in which they questioned the classic relationship between the rate of blood-to-brain entry and lipid-water solubility w32x. Unless normalcy of BBB function has been documented, microdialysis studies may be measuring BBB function that has been perturbed by traumatic injury and run a risk of overestimating BBB permeability in this setting. We agree with de Lange et al. who said: ‘‘If intracerebral microdialysis could indeed provide information on local differences in the concentration of a compound in the interstitial fluid, it would be a very powerful tool to investigate BBB transport at different sites within the brain’’ w20x. However, we would caution that erroneous conclusions about BBB transport may result if insertion of a microdialysis probe alters normal BBB permeability, and consequently perturbs measurements of drug concentration in the interstitial fluid.

Acknowledgements This work was supported by NIH grants R01-NS12745 and S10-RR03321, by the Mark Moritz and Richard M. Lilienfeld Memorial Funds, and by a grant from Medtronic Corporation.

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