NMR IN BIOMEDICINE, VOL. 6, 339-344 (1993)

Diffusion into Rat Brain of Contrast and Shift Reagents for Magnetic Resonance Imaging and

~

Spectroscopy

Edward Preston* and David 0. Foster Biosystems Group, Institute for Biodiagnostics, National Research Council of Canada, Ottawa, Ontario, Canada K1A OR6

A sensitive radiotracer technique was used to measure transfer constants (K#) for blood to brain diffusion of the MR contrast reagent gadolinium diethylenetriaminepentaacetate (GdDTPA*-) and the MR shift reagent dysprosium triethylenetetraminehexaacetate (DyTTHA3-) across the normal and the ischemically injured blood-brain barrier (BBB) of rats. In rats with a normal BBB mean Kg (nL/g/s) for these reagents ranged from 0.3 to 1.4 across eight brain regions and were significantly lower in each region than Kg for sucrose (1.5-3.2), a substance known to be a poor permeant of the intact BBB. Kis measured 6 h after a 10min period of normothennic forebrain ischemia were increased to 4.06.2 (reagents) and 6.6-7.5 (sucrose) in two brain regions, striatum and hippocampus, known to be especially vulnerable to ischemic injury. Measurements of BBB permeability to DyTTHA3- after osmotic opening of the barrier with hypertonic arabinose gave Kis of 25-30 in forebrain regions. Estimates of reagent concentrations in brain interstitial fluid 30 min after dosing the animals indicated that both an extremely high dose of Dyl"l'HA5- and severe disruption of the BBB would be required to shift the resonance frequency of extracellular Na' appreciably. With the moderate degrees of BBB injury produced by short-term ischemia, a dose of GdDTPA'- about 25 times the usual clinical dose of 0.1 mmoUkg would be required to quantify the injury by dynamic MRI.

INTRODUCTION

MRI following i .v. administration of the paramagnetic contrast reagent gadolinium diethylenetriaminepenta- acetate (GdDTPA2-) is used extensively to reveal neurological disease as areas of enhanced signal inten- sity on TI-weighted images.' The enhancement occurs because breakdown of the blood-brain barrier (BBB) within lesions allows GdDTPA2- to leak into the inter- stitium where it shortens the TI of water protons. The degree of signal enhancement is a function of the interstitial concentration of the contrast reagent, a major determinant of which is the leakiness of the microvasculature. By employing dynamic MRI to obtain the relation between signal enhancement and time, the leakiness of the BBB within a lesion can be quantified in terms of a transfer constant (Ki) indicative of the rate of blood to brain diffusion of contrast reagent. GdDTPA2- Kis obtained by dynamic MRI have been reported for human brain tumors and sclero- tic lesi0ns,2*~ and for experimental tumors in rat brain.4 The magnitudes of the Kis for these lesions indicate that their microvasculature is very leaky, and it remains to be seen whether moderate degrees of BBB leakiness associated with other brain pathologies can be quanti- fied by the dynamic MRI technique.

To this end, experiments were carried out in rats to measure Kis for transfer of GdDTPA2- across the BBB

* Author to whom correspondence should be addressed. Abbreviations used: GdDTPA*-, gadolinium diethylenetriamine- pentaacetate; BBB, blood-brain barrier; Ki, transfer constant; 2V0, two-vessel occlusion; DyTTHA3-, dysprosium triethylene- tetraminehexaacetate.

before and after producing moderate barrier injury by temporary brain ischemia. A radiotracer technique' that enables sensitive quantitation of very low Kis and minor changes in BBB permeability was employed. For comparison, K,s were also obtained for radiolabeled sucrose, which is commonly used to assess alterations in BBB permeability. Ischemic brain injury was produced by 10 min of bilateral carotid artery occlusion combined with arterial hypotension.6 This popular model of ische- mic stroke, known as the two-vessel occlusion (2VO) model, is designed to mimic the global ischemia that occurs during temporary cardiac arrest. The procedure evokes regionally selective, delayed neuronal death in the striatum, hippocampus and neocortex,' and moder- ate degrees of damage to the BBB.8,9 The connection between BBB injury and neuronal death, although not yet understood, suggests possible application of non- invasive, dynamic MRI measurements of BBB permea- bility in both laboratory and clinical work on stroke.

The present study also examined the permeability of the normal and the damaged BBB to dysprosium tri- ethylenetetraminehexaacetate (Dy'TTHA3-), an NMR shift reagent" that is employed in =Na spectrosco y of tissues and organs, both in uiuo" and in uitro , to differentiate between intra- and extracellular Na+ . In this application it is essential that the reagent distri- butes readily and uniformly into the total extracellular space of the tissue (vascular and interstitial space) so that all extracellular Na+ is shifted sufficiently in reso- nance frequency to distinguish it from intracellular Na'. For brain, adequate permeation of the shift rea- gent through the BBB into the interstitial space would seem unlikely, and is one reason that the interpretati~n'~ of 23Na NMR spectra from the brains of rats infused with DyTTHA3- has been questioned.14

R

0952-3480/93/050339-06 $08.00 0 1993 by John Wiley & Sons, Ltd.

Received 20 January 1993 Accepted (revised) 3 June 1993

340 E. PRESTON AND D. 0. FOSTER

Osmotic opening of the BBB was tested as a means of obtaining an adequate interstitial concentration of DyTl"HA3-.

METHODS

Chemicals

Gadolinium (111) oxide (Gd,O,, 99.9%) and diethyle- netriaminepentaacetic acid (H,DPTA, 98%) were pur- chased from Sigma Chemical Co., St Louis, MO. Dysprosium (111) oxide (Dy,03, 99.9%), triethylene- tetraminehexaacetic acid (H,mHA, 98%), and N-methyl-D-ghcamine (meghmine, 990/0) were pro- ducts of Aldrich Chemical Co., Milwaukee, WI. '47Pr~methium chloride [18.5 GBq (0.5 Ci) per mg Pm] was from Amersham Corp., Arlington Heights, TL. [Fructose-l-'H]sucrose [99.8%, 462 GBq (12.5 Ci) per mmol] was obtained from DuPont NEN Products, Boston, MA. Soluene@ 350 and Hionic-Fluor@ were products of Packard Instrument Co., Downers Grove, IL. All other chemicals employed were of standard reagent grade, and all solutions were prepared with ultrapure water.

Preparation of radiolabeled contrast or shift reagents

Both the contrast reagent, GdDTPA2-, and the shift reagent, DyTTHA'-, were radiolabeled by incorporat- ing minute amounts of the p--emitting lanthanide, 14'Pm. It was assumed that the physicochemical proper- ties (e.g., stabilities, BBB transfer constants) of the DTPA or TTHA complexes with I4'Pm are practically the same as those of the complexes formed with Gd or Dy. Details on preparation of the reagents were obtained from the paper by Chu et d."' Contrast rea- gent [175 mM; ca 530 mOsm (ideal), ca 32 pCi/mL] was prepared as the N-methyl-D-glucamine salt [i.e., (megl~rnine),Gd('~~Pni)DTPA]. Gd20, (solid), HsDTPA (solid) and '47PmC13 were reacted ( 5 5 4 0 "C) in aqueous suspension in the molar ratios of 0.5:1.01:2.5 x lo-, until a clear solution was obtained (12-18 h). The solution was then titrated to pH 7.4 with 2 M N-methyl-D-glucamine. Shift reagent [150 mM, ca 620 mOsm (ideal), ca 21.5 pCi/mL] was prepared as the monosodium dimeglumine salt [i.e., Na(megl~mine),Dy('~~Prn)TTHA]. Dy203 (solid), H,TTHA (solid) and 147PmC13 were reacted (55-60 "C) in aqueous suspension in the molar ratios of 0.5:1.01:1.25 x 10-' until a clear solution was obtained (12-18 h). NaOH was then added to give a final "a+] of 150 meq/L, and the solution was titrated to pH 7.4 with N-methyl-D-glucamine. When pH reached 6.5-7.0 CaC12 was added to give a final [Ca'+] of 4 meq/L.

Animals

Experiments were performed on 38 male Sprague-Dawley rats (323433 g) from the NRCC spe- cific pathogen-free colony. Prior to experimental pro- cedures, rats were anesthetized with sodium pentobar-

bital, 65 mg/kg i.p. For arterial blood sampling or i.v. injections, tapered polyethylene (PE50) cannulas were inserted into the femoral artery or vein. In some rats a functional nephrectomy was performed prior to radio- tracer injection to prevent tracer excretion and opti- mize the circulating plasma level which, along with permeability, governs 1 he amount of tracer diffusion into brain. A procedure similar to that described by Waynforth and Flecknell" was used, except that the kidneys were not excised.

Opening of the BBB by ischemia or osmotic shock

Cerebral ischemia was produced in rats by the method of combined carotid artery occlusion and hyp~tension.~.~ Briefly, anesthetized rats were intu- bated and mechanical ventilation was initiated ( 1 6 18 cm H 2 0 , 32-36 breathdmin). Blood gases and pH were monitored to ensure adequate ventilation, and tympanic and colonic temperatures were maintained between 37.5 "C and 38.0 "C by heating pad. Through a cannula in the tail artery, blood was drawn into a heparinized syringe to rapidly lower and maintain mean arterial pressure between 42 mmHg and 47 mmHg (monitored by pressure transducer and polygraph). Both carotid arteries were then clamped and hypoten- sion was maintained. After 10min had elapsed, the clamps were released and blood returned to the rat to restore blood pressure. The wounds were closed, venti- lation was removed, and the animal permitted to recover until re-anesthetized for the radiotracer experi- ment 6 h later.

Osmotic opening of the BBB was effected by infu- sion of a hypertonic solution into the right internal carotid arteryI6 via a cannula inserted into the right common carotid. The right external carotid artery was ligated. A syringe pump was used to infuse 3.6 mL of 1.6 molal L( + ) arabinose over a 30 s period.

Measurement of Kis for blood to brain diffusion of radiolabeled test substances

In experiments with [3H]sucrose and Gd( "'Pm)DTPA2- either substance was administered as an i.v. bolus. The former (60 yCi, 12.5 Wmmol) was given in 1 mL of saline and the latter (32 yCi, 175 ymol) consisted of 1 mL of the contrast reagent solution described above. For experiments with Dy(147Pm)TTHA3- either 5.1 mL of the shift reagent solution described above (110 pCi, 765 pmol) was infused by syringe pump over 10min (stroked rats or controls) or 2 mL (43 pCi, 300 pmol) was infused over 1.5 min (rats subjected to osmotic opening of the BBB).

With the onset of radiotracer bolus injection or infusion a syringe pump was started (time 0) to with- draw femoral arterial blood (0.039 mllmin) for 30 min. At time 0 + 25 min a cannula was inserted distally into the right common carotid. At 0+30min the head vasculature was cleared of blood by perfusion of 25 mL salineI7 through the cannula and the rat decapitated. The brain was removed, dissected and portions placed in tared vials and weighed. These samples and 50pL volumes of plasma from the syringe pump blood sample

DIFFUSION OF GdDTPAZ- AND DyTTHA' ACROSS BLOOD-BRAIN BARRIER 341

Table 1. Transfer constants (nL/g/s) for blood-to-brain diffusion of sucrose, contrast reagent and shift reagent in rats subjected to global brain ischemia or osmotic opening of the blood-brain barrier, and in controls

Brain region

Frontal cortex Parietal cortex Occipital cortex Striatum Hippocampus Diencephalon-mesencephalon Cerebellum Pons-medulla

Sucrose

1 2 Control Ischemic (intact) (intact)

2.3 f O . l a 3.4 f 0.4b 1.92 0.l8 5.3 f 0.6" 2.1 f O . l a 3.8f0.4bc 1.5f O.la 6.6 f0.8" 2.0+0.18 7.5k0.9" 1.920.1' 4.7k0.5' 3.2 f. 0.28 3.6 f 0.3b 3.0 f 0.2a 3.9 k 0.3b

3 Control (intact)

0.5 f 0.1 0.4 f 0.04 0.5 f 0.1 0.3 f 0.01 0.4f0.1 0.5 f 0.05 0.8 f 0.1 0.9f0.1

GdDTPA' '

4 Control

(nephrec.)

0.6f 0.04 0.5 + 0.04 0.8 2 0.05 0.3 f 0.04 0.5 2 0.1 0.6 f 0.03 1 .o f 0.1 1.420.1

5 Ischemic

(nephrec.)

1.8 k 0.3bc 4.1 f 0.2' 2.2 k 0.04bC 6.2 f. 0.4" 6.1 f 0.7" 3.4 f. 0.lk 2.5 f 0.1 2.6 f. 0.2&

6 Control (intact)

0.6 f. 0.04 0.5 k 0.01 0.7 k 0.05 0.4 f 0.1 0.5 0.05 0.5 f 0.03 1.0+ 0.1 1.2 f 0.2

7 Control

Inephrec.)

0.7 f 0.1 0.6 f 0.05 0.7 f 0.1 0.4 f 0.03 0.5 * 0.05 0.6k 0.05 1.2 * 0.1 1.3 2 0.1

8 Ischemic

(nephrec.)

2.8 rt 0.4bC 1.8 k 0.2bC 4.9 rt 0.4" 4.0 f 0.6' 2.3 k 0.4bc 2.0 & O.Zb 1.7 f 0.2b

1.2 f 0.2b

9 Osmotic opening

26.4 f. 6.4d 30.2 f. 8.7d 28.0 f 5.7d - -

24.9 k 3.8d - -

Values are rneansf SEM for four rats per treatment group (five for sucrose groups). Brain ischemia was produced for 10 min at 6 h prior to i.v. injection of the test substance. If not left intact, rats were nephrectomized (nephrec.) ca 30 min before injection of reagent. Osmotic opening was induced 2 min after injection of shift reagent. Significant differences (p<0.05) between mean values are marked as follows: a within brain region, greater than for controls (intact or nephrec.) given contrast or shift reagent (columns 3, 4, 6 and 7);

within treatment group, less than for striatum or hippocampus; within region and test substance, greater than for controls; within region, greater than for controls or ischemic.

were digested in Soluene 350, and Hionic-Fluor was then added for liquid scintillation counting. The con- centration of tracer was determined in brain paren- chyma (C,,,,,, dpm/g) and plasma (Cplasma, d p d m l ) . The latter value was multiplied by the circulation time in seconds (1 800 s) to provide the time-integrated plasma concentration (J:8'1 C,,,,,, dt, d p d d m l ) . Regional Kis were calculated from the mathematical relation~hip:~

Ki = Cparcnj Cplasma dt 1: There were four protocols: (i) rat anesthetized, cannu- lated, and radiotracer experiment conducted (controls); (ii) same as protocol (i) but with nephrec- tomy prior to cannulation (nephrectomized controls) ; (iii) 10 min global ischemia followed 6 h later by proto- col (i) or (ii); (iv) 30s osmotic opening procedure beginning 2min after start of radiotracer injection in protocol (i). Sham stroked controls were not used, since previous studies'.' found no effect of a sham stroke protocol on BBB permeability.

Differences in mean Ki values between treatment groups or between brain regions within a group were analyzed by ANOVA followed by Tukey's post-hoc comparison. Statements regarding significant differ- ences are based on p<0.05.

RESULTS

Table 1 reports mean values of the K,s for permeation of blood-borne ['HH]sucrose, Gd('47Pm)DTPA2- and D Y ( ' ~ ~ P ~ ) T T H A ~ - into eight regions of the brain, under the various experimental conditions employed (columns 1-9). The magnitude of K, is directly pro- portional to the degree of BBB permeability to a given substance. An increase in K, signifies that, for a given plasma concentration of substance, there has been a proportionate increase in the total amount of it enter- ing brain parenchyma per unit time.

K,s for ['H]sucrose in each brain region of control rats (intact BBB) were significantly greater (column 1)

than corresponding regional values for either NMR reagent (columns 3, 4, 6 and 7). These results showed that although [-'H]sucrose permeated the intact RBB to a very limited degree (K,s>1000 nL/g/s would be found were permeation largely unimpeded) the NMR rea- gents permeated even more slowly, at only 2&430/0 the rate of sucrose. Regional K,s for permeation of GdDTPA2- in control groups (columns 3 and 4) did not differ significantly from corresponding values for DyTTHA"- (columns 6 and 7), and there were no significant differences in the regional K,s for the MR reagents between intact and nephrectomized controls (columns 3 vs 4, and 6 vs 7).

In rats that had undergone 10min of global brain ischemia 6 h earlier (columns 2, 5 and 8), the resultant microvascular injury with increased BBB leakiness was apparent as elevated K,s for each of the three test substances. K,s that are significantly (p<0.05) greater than the relevant control value(s) are denoted by superscript c. For each substance, the largest increases in K, occurred in striatum and hippocampus. Most of the other regions exhibited post-ischemia K,s signifi- cantly less (denoted by superscript b) than those for striatum and hippocampus.

Table 1 (column 9) also shows regional K,s for DyTTHA'- measured in right cerebral tissues after infusion of hypertonic arabinose into the right internal carotid artery. The resultant osmotic opening of the BBB increased DyTTHA3- K,s as much as 60-fold above control values, and to levels that were signifi- cantly higher (denoted by superscript d) than those resulting from ischemic injury.

The K, measurements were based on determinations of the amounts of radioactivity in brain parenchyma (dpm/g) and in plasma (dpdmL) . Radioactivity meas- urements of the stock solutions of shift and contrast reagents gave specific activities of 406 dpdnmol GdDTPA2- and 319 dpdnmol DyTTHA'-. These values were then used to calculate the concentrations that the reagents achieved in plasma and brain intersti- tial fluid. In calculating the latter we assumed that the tracer distributed into a tissue interstitial fluid volume of 0.2 mWg. The results of these calculations, which provide a more practically oriented perspective on BBB permeability to the reagents than the K,s, are

342 E. PRESTON AND D. 0. FOSTER

presented in Table 2. The plasma concentrations shown represent the time-averaged values for the whole 30 min period of reagent circulation. Nephrectomy prior to reagent injection approximately doubled the average plasma level for a given dosage of reagent, and tissue uptake was increased accordingly. Additional features of Table 2 regarding dose and tissue concentra- tions of reagents are discussed below.

DISCUSSION

The radiotracer methodology used in this study provided a measurement, Ki, indicative of the rate at which GdDTPA2-, DyTTHA3- or sucrose moved from the bloodstream through the capillary endothelial cell layer into brain. The experimental conditions govern- ing the validity of a two-compartment (plasmdbrain) simple diffusion model, and factors influencing inter- pretation of Ki changes have been discussed in earlier paper^.^.'^

Kg for intact BBB

The regional Kis obtained under control conditions for both the contrast reagent and the shift reagent indi- cated that movement of these substances across the intact BBB was highly restricted. Specifically, the mean regional Kis for the reagents (Table 1, columns 3, 4, 6 and 7) were only 20-43% as large as corresponding Kis for sucrose (column l), which is known to permeate p ~ o r l y . ~ The Kis for GdDTPA2- and Dy3THA3- were similar to values for CoDTPAZ- (0.23-0.44 nL/g/s across brain regions) reported by Blasberg el ~ 1 . ‘ ~

That the MR reagents permeated the intact BBB more slowly than sucrose is probably attributable mainly to their negative charge, which presumably makes them even less lipid soluble than sucrose. Lipid solubility is considered important because endothelial cells of the brain vasculature are joined by tight junc- tions, which prevent paracellular diffusion, and

because endothelial pores or pinocytotic vesicles are rare or absent.m321 Therefore, to enter brain paren- chyma from the blood most solutes, with the notable exception of carrier-transported metabolites, must dis- solve in and diffuse across both the luminal and ablumi- nal membranes of the endothelial cells. This concept of BBB permeation by transmembrane diffusion is sup- ported by analyses showing that differences in Kis among solutes relate more to differences in lipid solubi- lity than to molecular size and diffusivity.z(’*22 Nevertheless, the mechanism for slow BBB permeation by non-nutrient polar solutes is still debated. Lucchesi and GosselinU recently proposed that such molecules cross the intact BBB by a form of vesicular transport (adsorptive-phase endocytosis) initiated by solute bind- ing to the glycocalyx on the luminal surface of endothe- lial cells. If this is correct, the low K,s for the MR reagents may reflect an inhibition of their binding due to the negatively charged sialic acid and other groups of the glyc~calyx.~~

K,s post ischemia

The BBB injury and increase in sucrose and reagent Kis that developed most prominently in striatum and hip- pocampus after ischemia are consistent with previous findings. It was observed8q9 that after 10 min 2VO ische- mia, striatum and hippocampus exhibited a delayed increase in BBB permeability to sucrose, which was prominent at 6 h post ischemia and then largely sub- sided by 24 h. The relation between this microvascular damage and the phenomenon of delayed neuronal death in these brain regions’ remains to be determined. Further, the physical nature of the vascular injury is unknown. Since sucrose permeated the intact BBB of striatum and hippocampus at a rate four to five times that of the MR reagents (Table 1, column 1 vs 3, 4, 6 and 7), it is of interest that this difference largely disappeared after ischemic injury (column 2 vs 5 and 8). This suggests that, after injury, solute lipid solubility or solute binding to the endothelial cell surface may no longer be the major determinant of solute K,. This

Table 2. Putative concentrations (pM) of contrast or shift reagent in brain interstitial space following the treatments of Table 1 Gd DTPA2- DyTn-IA3- -

1 2 3 4 5 6 7 Control Control Ischemic Control Control Ischemic Osmotic (intact) fnephrec.) fnephrec.) fintact) (nephrec.) Inephrec.) opening

780 k 30 Dose (pmollkg) 507+19 475+24 469 f 6 1950+100 1850f30 1790f20 Plasma concn (1M) 910+30 1910f60 1690+240 3870f240 64605410 6570+270 2120+160

Brain region Frontal cortex Parietal cortex Occipital cortex Striatum Hippocampus Diencephalon-mesencephalon Cerebellum Pons-medulla

5.6 * 0.8 4.7 + 0.4 5.6 k 0.9 2.7 t- 0.1 4.6 + 0.5 5.1 + 0.4 8.5 f 0.9 9.7f1.0

14+ 1 12fl 17+2 7.6 + 0.7 1221 14f 1 24+ 1 32f2

35+5 80f10 41 +4 121 + 15 116f10 69+11 51 +9 54f 10

27+3 23f2 31 f 4 17+2 2321 22+ 1 47+4 5226

57f9 45+4 57f7 31 +3 41 +6 43+5 94+ 14 98+ 12

94+ 12 218f29 141 f 21 386 + 47 312+47 179f36 157f20 137f 19

680 f 160 760f210 710f 140

- 640+_100 -

Values are means + SEM for the same four rats per treatment group as used to obtain the data of ‘Table 1. The amount of intravenously administered reagent that accumulated in brain parenchyma during 30 min circulation was calculated from the specific activity of the reagent (dpmlpmol) and parenchymal radioactivity (dpmlg). An interstitial volume of 0.2 mL per g brain, and uniform distribution of reagent in this space were assumed in calculating interstitial concentrations. The stated concentrations of the reagents in plasma represent mean concentrations for the 30 min period of circulation, during which the concentrations declined non-linearly.

DIFFUSION OF GdDTPA2- AND D y m 3 - ACROSS BLOOD-BRAIN BARRIER 343

could be envisioned, for example, if the injury were to involve formation of endothelial pores or pinocytotic vesicles within which the solutes could move into brain without leaving aqueous solution.

Inapplicability of DyTTHA3- for resolving brain intra- and extracellular Na'

Our measurements of the permeability of the normal BBB to DylTHA'- do not support the proposal of Naritomi et al.13 that this shift reagent can be applied in conjunction with in uiuo 23Na NMR spectroscopy of the brain to distinguish between intra- and extracellular Na'. To obtain a minimally adequate shift of 3 4 ppm in the resonance frequency of extracellular Na' would require an interstitial concentration of DyTI'HA3- of 7-9 mM." Using nephrectomized rats to eliminate renal excretion of the shift reagent we obtained a mean plasma concentration of reagent (over 30 min) of ca 6.5 mM following a dose of ca 1.85 mmoUkg (Table 2, column 5). However, the calculated interstitial concen- tration of reagent in cortex after 30min was only ca 0.06 mM i.e., less than M O O that required.

In the ischemically injured rats, a mean plasma level of 6 . 6 m ~ shift reagent (Table 2, column 6) led to an estimated interstitial concentration of ca 0.4 mM in the most injured tissue, striatum. Thus, even after the appreciable degree of BBB injury seen in the 2VO model, unrealistic doses and plasma levels of reagent would be required to achieve adequate shift of striatal extracellular Na' . Furthermore, even the stratagem of osmotically opening the BBB, which augmented the shift reagent Ki 40 to 60-fold in cortex (Table 1, column 9), appears inadequate. On the basis of the data of Table 2 (column 7) obtaining 7 - 9 m ~ shift reagent in the interstitium of frontal cortex by osmotic opening of the BBB would require a plasma concentration of 21- 27 mM. In our experience with rats, plasma concentra- tions of Dy?THA3- in excess of ca 12mM have been associated with marked physiological instability (unpublished observations).

Practicality of assessing ischemic injury to BBB with GdDTPA2-

Comparison of the Kis for GdDTPA2- in ischemically injured hippocampus and striatum (ca 6 nUs/g; Table 1, column 5) with those obtained for other brain patho- logies emphasizes the moderate nature of the BBB injury produced in our study. For example, using dyna- mic MRI Tofts and Kermode3 obtained GdDTPAZ- Kis of 200-1200 nL/s/g for multiple sclerosis plaques in human brain, and Larsson et ~ 1 . ~ measured a GdDTPA2- Ki of 1880 nL/s/g in a human brain tumor. In measuring Kis of these magnitudes the normal clini- cal dose of GdDTPA2-, 0.1 mmoUkg, was sufficient. A

much larger dose would be required in using MRI to quantify moderate ischemic injury to the BBB.

The dose required to obtain measurements within ca 30min can be estimated using data of Kenney et ~ 1 . ~ showing that the T,-weighted signal intensity of gliomas is doubled at a GdDTPA2- level of ca 65ymoYkg tissue. For an assumed interstitial fluid volume of 0.2mUg7 this tissue level would correspond to a GdDTPA2- concentration of ca 0.33 mM in the intersti- tial fluid. This is about three times the interstitial concentration of GdDTPA2- that was attained in 30 min in the ischemically injured striatum and hippo- campus of nephrectomized rats dosed at 0.47 mmoVkg (Table 2, column 3). We estimate that a GdDTPAZ- dose of ca 1.3 mmoYkg would be required in nephrecto- mized rats to attain after a 10min 2VO stroke an interstitial GdDTPA2- concentration of 0.33 mM (and double the signal intensity) in the striatum and hippo- campus. Comparison of dosage and the resultant plasma concentrations achieved with and without neph- rectomy indicates that in non-nephrectomized rats a dose of ca 2.6mmoYkg (>25-fold the clinical dose) would be required. Since the LDSo of GdDTPA2- in rats is ca 10 mmoUkg,25 a dose of 2.6 mmoUkg should be tolerable and acceptable. Thus, in experimental ani- mals, it would seem feasible to use GdDTPA2- in conjunction with dynamic MRI to quantitate mild injury to the BBB. For similar measurements in humans it would seem essential to employ a contrast reagent much more efficacious on a molar basis than GdDTPA2-.

SUMMARY

The results of this study have shown that following short-term global brain ischemia in rats the permeabi- lity of the BBB to the contrast reagent, GdDTPA2-, increases sufficiently in certain regions (striatum and hippocampus) to make feasible the quantitative assess- ment of barrier injury by dynamic MRI. However, since the dose of the reagent required would be ca 25 times the usual clinical dose, applicability to patients would be unlikely. Permeability of the BBB to intra- venously administered DylTHA3- was far too low, even after major barrier injury, to obtain the high interstitial concentrations of the reagent that would be required to distinguish intra- and extracellular Na' , via shift in the resonance frequency of the latter.

Acknowledgement

The authors appreciate the excellent surgical and other assistance provided by Ariane Finsten during this study.

344 E. PRESTON AND D. 0. FOSTER

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