Cathleen V. Allen

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

Blood-brain barrier (BBB) 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 (K1) 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, 14C-dextran (70 kDa) was used to measure BBB transcapillary transport; K1 was increased 90-fold after probe insertion. In the 3rd group, 14C-AIB (alpha-aminoisobutyric acid) was used to evaluate BBB transport after insertion of a 27 gauge cannula, which was used to infuse 1 microliter of saline over 5 min. The K1 of AIB was increased 25 times control values. We conclude that BBB transcapillary transport function is disturbed in response to insertion of brain cannulas and/or 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.

Comparison of cytosine arabinoside delivery to rat brain by intravenous, intrathecal, intraventricular and intraparenchymal routes of administration

We evaluated the delivery of 14C-cytosine arabinoside (AraC) to rat brain by: 1) intravenous (IV) bolus, by 2) intrathecal (IT) and 3) intraventricular (IVT) infusion, and by 4) convection-enhanced delivery (CED) into the caudate nucleus. Plasma and brain AraC metabolites were measured with HPLC, and distribution and concentration of 14C-AraC in brain sections were measured by quantitative autoradiography. After IV administration, the alpha and beta plasma half-lives were 1.9 and 46.5 min, respectively. The blood-to-brain transfer constant of AraC was 2.5+/-1.4 microliter g(-1) min(-1), compatible with high water solubility. After IT and IVT administration, tissue levels were high at the brain and ventricular surfaces, but declined exponentially into brain. After CED, maximum brain levels were up to 10,000 times higher than the IV group, and the distribution pattern was one of high 14C-AraC concentration in the convective component, with exponentially declining concentrations outside this region. The rate loss constant from brain was 0.002+/-0.0004 min(-1), suggesting that AraC was accumulating in brain cells. AraC was metabolized into uracil arabinoside within the brain. 14C-AraC was infused into 1 dog and distributed widely in the ipsilateral hemisphere. These studies suggest that delivery of AraC to brain parenchyma by the IV, IT or IVT routes will be subtherapeutic. Delivery by CED can achieve, and maintain, therapeutic levels of AraC in the brain, and should be further evaluated as a potential method of drug delivery.

Efflux of Drugs and Solutes from Brain: The Interactive Roles of Diffusional Transcapillary Transport, Bulk Flow and Capillary Transporters:

We examined the roles of diffusion, convection and capillary transporters in solute removal from extracellular space (ECS) of the brain. Radiolabeled solutes (eight with passive distribution and four with capillary or cell transporters) were injected into the brains of rats (n = 497) and multiple-time point experiments measured the amount remaining in brain as a function of time. For passively distributed compounds, there was a relationship between lipid:water solubility and total brain efflux:diffusional efflux, which dominated when kp, the transcapillary efflux rate constant, was >100h−1; when 10−1< kp< 10−2h−1 both diffusion and convection contributed, and when kp< 10−3h−1, convective efflux dominated. Para-aminohippuric acid (PAH) experiments (n = 112) showed that PAH entered the brain passively, but had efflux transporters. The total efflux rate constant, keff, was the sum of a passive component (kp = 0.0018 h−1), a convective component (kcsf = 0.2 h−1), and a variable, concentration-dependent component (kx = 0 to 0.45 h−1). Compounds with cell membrane transporters had longer clearance half times as did an oligonucleotide, which interacted with cell surface receptors. Manipulation of physiologic state (n = 35) did not affect efflux, but sucrose efflux half time was longer with pentobarbital anesthesia (24 h) than with no anesthesia or ketamine-xylazine anesthesia (2 to 3 h). These results show that solute clearance from normal brain ECS may involve multiple physiologic pathways, may be affected by anesthesia, and suggests that convection-mediated efflux may be manipulated to increase or decrease drug clearance from brain.

Therapeutic implications of tumor interstitial fluid pressure in subcutaneous RG-2 tumors

Increased interstitial fluid pressure (IFP) in brain tumors results in rapid removal of drugs from tumor extracellular space. We studied the effects of dexamethasone and hypothermia on IFP in s.c. RG-2 rat gliomas, because they could potentially be useful as means of maintaining drug concentrations in human brain tumors. We used dexamethasone, external hypothermia, combined dexamethasone and hypothermia, and infusions of room temperature saline versus chilled saline. We measured tumor IFP and efflux half-time of 14C-sucrose from tumors. In untreated s.c. tumors, IFP was 9.1 +/- 2.1 mmHg, tumor temperature was 33.7 degrees C +/- 0.7 degrees C, and efflux half-time was 7.3 +/- 0.7 min. Externally induced hypothermia decreased tumor temperature to 8.9 degrees C +/- 2.9 degrees C, tumor IFP decreased to 3.2 +/- 1.1 mmHg, and efflux half-time increased to 13.5 min. Dexamethasone decreased IFP to 2.4 +/- 1.0 mmHg and increased efflux half-time to 15.4 min. Combined hypothermia and dexamethasone further increased the efflux half-time to 17.6 min. We tried to lower the tumor temperature by chilling the infusion solution, but at an infusion rate of 48 mul/min, the efflux rate was the same for room temperature saline and 15 degrees C saline. The efflux rate was increased in both infusion groups, which suggests that efflux due to tumor IFP and that of the infusate were additive. Since lowering tumor IFP decreases efflux from brain tumors, it provides a means to increase drug residence time, which in turn increases the time-concentration exposure product of therapeutic drug available to tumor.

Comparative pharmacokinetics of 14C-sucrose in RG-2 rat gliomas after intravenous and convection-enhanced delivery

We compared tissue and plasma pharmacokinetics of 14C-sucrose in subcutaneous RG-2 rat gliomas after administration by 3 routes, intravenous bolus (i.v.-B; 50 microCi over 30 s), continuous i.v. infusion (i.v.-C, 50 microCi at a constant rate), and convection-enhanced delivery (CED, 5 microCi infused at a rate of 0.5 microl/min), and for 3 experimental durations, 0.5, 2, and 4 h. Plasma, tumor, and other tissue samples were obtained to measure tissue radioactivity. Plasma radioactivity in the CED group increased exponentially and lagged only slightly behind the IV-C group. After 90 min, plasma values were similar in all. Mean tumor radioactivity was 100 to 500 times higher in the CED group at each time point than in the i.v.-B and i.v.-C groups. Tumor radioactivity was homogeneous in the i.v. groups at 0.5 h and inhomogeneous at 1 and 2 h. In CED, radioactivity distribution was inhomogeneous at all 3 time points; highest concentrations were in tissue around tumor and in necrosis, while viable tumor contained the lowest and sometimes negligible amounts of isotope. Systemic tissue radioactivity values were similar in all groups. Efflux of 14C-sucrose from tumors was evaluated in intracerebral tumors (at 0.5, 1, 2, and 4 h) and subcutaneous tumors (at 0 to 0.5 h). Less than 5% of 14C activity remained in intracerebral tumors at each time point. The efflux half-time from the subcutaneous tumors was 7.3 +/- 0.7 min. These results indicate rapid efflux of drug from brain tumor and marked heterogeneity of drug distribution within tumor after CED administration, both of which may be potentially limiting factors in drug delivery by this method.

Noninvasive measurement of arterial blood plasma concentration of iodinated contrast agents from CT scans of human brain

Objective Our goal was to assess the accuracy of estimating the time course of the arterial plasma concentration of meglumine iothalamate from cranial CT images of different vascular structures in the brain. Materials and Methods Dynamic CT studies of transcapillary transport in various brain lesions were analyzed. Vascular structures in the brain were identified and classified in three categories: arteries, veins, and venous sinuses. Systemic venous blood samples were taken prior to the infusion of meglumine iothalamate and 10 min after completion of the infusion and used as a calibration for the volume averaging fraction of the image of each vascular structure. A time course of plasma meglumine iothalamate concentration for each of the vascular categories in the CT images was obtained and compared with a variety of methods. Results Significant differences were found for measurement of plasma meglumine iothalamate concentration from different vascular categories. There was also a disparity between the volume averaging fraction that we calculated and what would be expected due to the measured systemic hematocrit for all vascular structures. Conclusion The use of images of veins and venous sinuses consistently underestimated the arterial concentration around the peak values. Correcting the imaged venous sinus values with the measured systemic hematocrit was even less reliable. The most accurate method of determining arterial plasma concentration of meglumine iothalamate from CT images of brain was to correct the identified arterial vessels for volume averaging.

A new head holder for reducing axial movement and repositioning errors during physiological CT imaging

Objective We designed a new head holder for immobilization and repositioning in dynamic CT studies of the brain. Materials and Methods A customized thermoplastic face mask and foam head rest were made to restrict movement of the head in all directions, but particularly out of the axial plane (z-movement). Results This design provided a rigid, detailed mold of the face and back of the head that minimized motion during lengthy CT studies and enabled accurate repositioning of the head for follow-up studies. Markers applied directly to the skin were used to quantify z-movement. Conclusion When tested on 12 subjects, immobilization was limited to <2.0 mm under worst-case conditions when the subject was asked to attempt forced movements. Repositioning was accurate to <1.5 mm when the subject was removed from the head holder and then placed back into it.

Requirements for accurate anatomical imaging of the rat for electromagnetic modeling

Modeling electromagnetic energy distribution in laboratory test animals, such as the rat, requires accurate imaging of the animals anatomy. Accuracy, in this sense, refers to the proper positioning and shape of the various organs of the animal as they are situated in the laboratory setting. A number of factors related to the accuracy of bioelectromagnetic studies dictate the required animal positioning. The authors performed whole body imaging of Sprague-Dawley rats in an X-ray computed tomography (CT) scanner. By necessity the rats were anesthetized during imaging and a whole body thermoplastic cast was made to support the organs in the same position as they were in an awake rat. Comparisons were made to unsupported anesthetized rats and dead rats. It is concluded that it is necessary for rats to be supported in a whole-body cast to best simulate the anatomy of the awake rat. In addition, the authors note changes in abdominal structures due to death that necessitate the analysis of the animal while it is still alive.