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Anesth Analg 1999;88:559
© 1999 International Anesthesia Research Society

NEUROSURGICAL ANESTHESIA

The Influence of Anesthetic Choice, PaCO2, and Other Factors on Osmotic Blood-Brain Barrier Disruption in Rats with Brain Tumor Xenografts

Laura G. Remsen, DVM, PhD*, Michael A. Pagel, BS*,§, Christopher I. McCormick, AHT, LATG*,§, Steven A. Fiamengo, MD{ddagger}, Gary Sexton, PhD{dagger},{dagger},{dagger}, and Edward A. Neuwelt, MD*,§

Departments of *Neurology, {dagger}Surgery, {dagger}Medicine, {dagger}Preventive Medicine and Public Health, and {ddagger}Anesthesiology, Oregon Health Sciences University and §Veterans Administration Medical Center, Portland, Oregon

Address correspondence and reprint requests to Edward A. Neuwelt, MD, Oregon Health Sciences University, Department of Neurology, L603, 3181 SW Sam Jackson Park Rd., Portland, OR 97201. Address e-mail to neuwelte{at}ohsu.edu


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Increasing the delivery of therapeutic drugs to the brain improves outcome for patients with brain tumors. Osmotic opening of the blood-brain barrier (BBB) can markedly increase drug delivery, but achieving consistent, good quality BBB disruption (BBBD) is essential. We evaluated four experiments compared with our standard isoflurane/O2 protocol to improve the quality and consistency of BBBD and drug delivery to brain tumor and normal brain in a rat model. Success of BBBD was assessed qualitatively with the large molecular weight marker Evans blue albumin and quantitatively by measuring delivery of the low molecular weight marker [3H]-methotrexate. With isoflurane/O2 anesthesia, the effects of two BBBD drugs of different osmolalities were evaluated at two different infusion rates and infusion durations. Arabinose was superior to saline (P = 0.006) in obtaining consistent Evans blue staining in 16 of 24 animals, and it significantly increased [3H]-methotrexate delivery compared with saline in the tumor (0.388 ± 0.03 vs 0.135 ± 0.04; P = 0.0001), brain around the tumor (0.269 ± 0.03 vs 0.035 ± 0.03; P = 0.0001), brain distant to the tumor (0.445 ± 0.05 vs 0.034 ± 0.07; P = 0.001), and opposite hemisphere (0.024 ± 0.00 vs 0.016 ± 0.00; P = 0.0452). Forty seconds was better than 30 s (P = 0.0372) for drug delivery to the tumor. Under isoflurane/O2 anesthesia (n = 30), maintaining hypocarbia was better than hypercarbia (P = 0.025) for attaining good BBBD. A propofol/N2O regimen was compared with the isoflurane/O2 regimen, altering blood pressure, heart rate, and PaCO2 as covariates (n = 48). Propofol/N2O was superior to isoflurane/O2 by both qualitative and quantitative measures (P < 0.0001). Neurotoxicity and neuropathology with the propofol/N2O regimen was evaluated, and none was found. These data support the use of propofol/N2O along with maintaining hypocarbia to optimize BBBD in animals with tumors.

Implications: Propofol/N2O anesthesia may be better than isoflurane/O2 for optimizing osmotic blood-brain barrier disruption for delivery of chemotherapeutic drugs to brain tumor and normal brain.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
In normal brain, the primary factor limiting access of water-soluble drugs to the central nervous system (CNS) is the blood-brain barrier (BBB). Osmotic opening of the BBB by rapid intraarterial (IA) infusion of hyperosmolar solutions (BBBD) has been used experimentally (1,2), and clinically (3–6) in children and adults to increase the delivery of chemotherapy to intracerebral (IC) tumors. BBBD is performed under general anesthesia at Oregon Health Sciences University because it affords cerebral protection, allows for mild hyperventilation during the administration of the osmotic drug, and because IA hypertonic infusion is painful. The most impressive results have been in adults with primary CNS lymphoma. In a recent report of 58 patients with non-autoimmune deficiency syndrome primary CNS lymphoma, the 39 nonirradiated patients had a median survival of 41 mo after initial treatment with BBBD chemotherapy. This study included patients who were disease-free for >10 y and is the first report of complete and durable responses with enhanced chemotherapy delivery without radiation and without cognitive loss in patients with malignant brain tumors (5). This can be compared with an expected median survival of 12–15 mo, with no 5-yr survivors, after radiation with or without chemotherapy (7).

The degree and consistency of BBBD varies with species (1), tumor presence (8), anesthetic technique (9), and hemodynamic status (9). In experiments from this laboratory, >95% of normal Long-Evans rats had a good or excellent BBBD when infused with 1.4 molal (25%) mannitol under pentobarbital or isoflurane/O2 anesthesia. In contrast, drug delivery studies have shown that the BBBD procedure is less predictable in rats with brain tumor xenografts (8). In addition, patients with tumors receiving general anesthesia to facilitate BBBD and administration of chemotherapy demonstrated only a 60% incidence of good or excellent disruption as assessed by contrast-enhanced computed tomographic scans (10).

The disruption of the BBB is a threshold event that is dependent on the osmolality of the solute, which normally does not cross the BBB, as well as the duration and flow rate of the infused IA hyperosmolar solution (10–12). Rapoport et al. (13) observed that osmolalities <1.4–1.6 molal or IA infusion times <20 s resulted in essentially no modification of the BBB in normal brain.

Physiologic and pharmacologic factors that might influence the degree and consistency of BBBD include blood pressure (BP), heart rate (HR), PaCO2, and the choice of anesthetic. Because isoflurane has vasodilatory effects on normal cerebral vasculature (17) and variable effects on vascular tone in cerebral tumor (18), the use of an anesthetic such as propofol (19,20), without such variable vasoactivity, may improve the quality of BBBD in animals with tumors.

Hyperventilation is a standard means of decreasing intracranial pressure (ICP). The resultant decrease in PaCO2 causes cerebral vasoconstriction and, thus, decreased intracranial blood volume. An earlier study (14) showed that when a PaCO2 of 24–30 mm Hg was established in normal animals at the time of BBBD, methotrexate (MTX) delivery to ipsilateral barrier-modified brain was enhanced compared with normocarbia (PaCO2 of 34–44 mm Hg); however, marked hypocarbia alone (PaCO2 <20 mm Hg) caused pathologic opening of the BBB. Bradbury (15) and Rapoport (16) found that hypercarbia could result in modest reversible changes in BBB permeability.

We undertook the present study to elucidate pharmacologic or physiologic factors that may be important in optimizing osmotic BBBD in a well characterized nude rat brain tumor xenograft model. A series of four experiments was designed to 1) evaluate 2.0 molal arabinose and our standard 1.4 molal mannitol, at different infusion flow rates and times, under isoflurane/O2 anesthesia; 2) evaluate the effect of altering PaCO2 under isoflurane/O2 anesthesia on the success rate of BBBD; 3) compare propofol/N2O anesthesia with isoflurane/O2 anesthesia on quality and consistency of BBBD; and 4) to evaluate neurotoxicity and neuropathology with propofol/N2O anesthesia and BBBD. The high molecular weight marker (Evans blue albumin; Mr 68k) was used to document BBBD, and the low molecular weight marker 3H-methotrexate ([3H]-MTX; Mr 454) was used to document delivery to the tumor (T) and surrounding brain.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
The care and use of the animals was approved by our institutional animal care and use committee and was under the supervision of the Department of Animal Care. All studies were performed in female nude rats (rnu/rnu) (200–220 g) with intracerebral LX-1 (human small-cell lung carcinoma) xenografts. Tumors were implanted as previously reported (21), and animals were treated on Tumor Day 6. BBBD was performed as previously described (22). Except as described in the anesthesia study below, anesthesia was induced with 5% isoflurane in 100% oxygen. The trachea was intubated and the lungs were ventilated with 2% isoflurane in O2. Tidal volume was 2.5 mL, with a respiratory rate of 50 breaths/min. Using aseptic technique, a ventral neck incision was made, and the right external carotid artery was isolated at the level of the carotid bifurcation. A polyethylene catheter (PE-50) was inserted into the external carotid artery retrograde toward the bifurcation with the common carotid. To provide a qualitative measure of the degree of disruption, Evans blue dye (2%, 2 mL/kg) was administered IV to all animals 5 min before BBBD. Evans blue binds to serum albumin and therefore does not cross an intact BBB. It acts as a visual marker for opening of the BBB and was assessed after the animals were killed. Evans blue albumin staining was graded as either nil (0–1+) or good (2–3+) (1).

Immediately after disruption, [3H]-MTX (12 µCi) was administered IA through the same catheter. A plasma sample was collected 10 min later to provide an estimate of peak plasma levels. The animals were killed with an intracardiac injection of pentobarbital, the brain was removed and immediately frozen (-20°C) until later processing. BBBD was quantitatively assessed by measuring delivery of [3H]-MTX to the T, BAT (1- to 2-mm section immediately surrounding the tumor), brain distant to the T (BDT; ipsilateral normal brain), and contralateral left hemisphere (LH).

Experiment 1: Two Hyperosmolar Solutions—The Effect of Drug, Rate, and Duration of Infusion Under Isoflurane/O2 Anesthesia
In this experiment, we evaluated the effect of 1.4 molal mannitol (our current clinical agent) and 2.0 molal arabinose with respect to infusion rate and infusion duration during an isoflurane/O2 anesthetic (Table 1). BBBD was otherwise performed as described above. Tidal volume was 2.5 mL, with a respiratory rate of 50 breaths/min. Tumor-bearing rats were assigned to one of the following groups: Group 1 = saline control (n = 16), Group 2 = mannitol (25% 1.4 molal) (n = 24), or Group 3 = arabinose (2.0 molal) (n = 24). Within each group, animals were randomly assigned to an infusion rate and infusion duration of either 0.09 mL/s for 30 or 40 s or 0.12 mL/s for either 30 or 40 s (n = 6 per group).


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  		Table 1. Experimental Design 1
 
Experiment 2: Effect of altering PaCO2 on BBBD Performed Under Isoflurane/O2 Anesthesia
We evaluated the effect of manipulating PaCO2 on drug delivery in tumor-bearing rats during isoflurane/O2 anesthesia (Table 2). Rats were assigned to one of two groups: Group 1 = hypocarbia (PaCO2 of 25–30 mm Hg) (n = 15) or Group 2 = moderate hypercarbia (PaCO2 of 45–50 mm Hg) (n = 15). Within each group, rats were randomized to mannitol (n = 5) or arabinose (n = 5) using infusion conditions of 0.12 mL/s for 30 s. Additionally, within each group, control animals received saline (n = 5) instead of mannitol or arabinose as a control for pathologic BBBD, which has been reported to occur with moderate hypo- and hypercarbia (14). Animals were surgically prepared as previously described, Evans blue dye was given, and a baseline blood gas sample was drawn and analyzed. The ventilatory rate for the animals in Group 1 under isoflurane/O2 anesthesia was manipulated to maintain PaCO2 values within the range of 25–30 mm Hg. In Group 2, it was necessary to deliver isoflurane in 5% CO2, 20% O2, and 75% N2 to maintain a PaCO2 of 45–50 mm Hg. The isoflurane concentration was unchanged at 2%. When PaCO2 values of 45–50 mm Hg were obtained, BBBD was performed as described above.


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  		Table 2. Experimental Design 2
 
Experiment 3: Anesthetics and BBBD
In preliminary studies, we had determined that mannitol was most effective at a flow rate of 0.12 mL/s with isoflurane/O2 (9); however, this flow rate resulted in excessive brain edema with propofol/N2O anesthesia in pilot study animals. Therefore, a lower flow rate (0.09 mL/s) was used with propofol/N2O (Table 3). Tumor-bearing rats were assigned to one of the following groups: Group 1 (n = 24) = isoflurane in 100% O2 with osmotic drug administered at 0.12 mL/s for 30 s or Group 2 (n = 24) = propofol/N2O 650 µg · kg-1 · min-1 IV with an inspiratory gas mixture of 50% O2 and 50% N2O with osmotic drug administered at a flow rate of 0.09 mL/s for 30 s (n = 24). Animals were ventilated at 50 breaths/min with a tidal volume of 2.5 mL, and baseline PaCO2 values were taken. Prior work by this laboratory suggested that, in nontumor-bearing animals, avoidance of hypotension and extreme tachycardia was important for reproducible BBBD (9). To test this observation in tumor-bearing animals, animals were randomized within each of the two anesthetic groups to one of the four following regimens: A = no pressor drug (n = 6), B = phenylephrine (1.5–24 µg · kg-1 · min-1 IV to increase BP by 25% above baseline (n = 6), C = propranolol (0.1–1.6 mg IV bolus to decrease HR to 25% below baseline (n = 6), or D = both (n = 6).


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  		Table 3. Experimental Design 3
 
For Group 1 animals, anesthesia was induced in a chamber with 5% isoflurane in O2, and the trachea was intubated. Anesthesia was maintained with 2% isoflurane in O2. A catheter was inserted into the right femoral artery, and initial measurements of BP, HR, and blood gases were taken before insertion of a catheter into the right external carotid artery for BBBD. Measurements also were taken at the following time points: after a catheter was placed into the carotid artery, 5 min after Evans blue dye was administered (baseline), after the pressor drugs were administered, and immediately after BBBD. BP and HR measurements were also taken 5 and 10 min after BBBD.

In pilot studies, the dose of propofol/N2O 650 µg · kg-1 · min-1 was determined to give a plane of anesthesia comparable to that of 2% isoflurane, as evaluated by standard criteria such as absence of withdrawal to pain and absence of hemodynamic response to surgical stimulus. Because of technical difficulties with the induction of propofol in an awake rat, isoflurane 5% in O2 was used for anesthetic induction. Group 2 animals, propofol/N2O anesthesia was induced with 5% isoflurane, the trachea was intubated, and anesthesia was initially maintained with 2% isoflurane. An infusion of propofol (650 µg · kg-1 · min-1) was started into the left femoral vein. Animals were given both propofol and isoflurane for 10 min, then isoflurane was discontinued and N2O (50%) was delivered by inhalation. It had been determined in pilot studies that 10 min of the propofol/N2O infusion was adequate for the animals to reach a surgical plane of general anesthesia. By airway gas analysis, it also was determined that end-tidal isoflurane was nearly zero after another 10 min. Animals were surgically prepared, and measurements identical to those described for Group 1 were taken. Immediately after infusion of osmotic drug, animals in each group received 12 µCi of [3H]-MTX as an IA bolus. A plasma sample was collected 10 min later, and the animals were killed. Their brain, liver, kidney, and spleen were removed and immediately frozen at -80°C until later processing.

Experiment 4: Neurotoxicity and Neuropathology of Propofol/N2O
Animals (n = 10) were anesthetized with propofol/N2O as described in Experiment 3 and surgically prepared for BBBD. Immediately after mannitol infusion, animals received an IA infusion of 2 mL of NaCl given at 0.2 mL/min. After the infusion, the catheter was removed, the wound was closed with sutures, and the animals were allowed to recover. They were weighed daily and observed for signs of neurotoxicity for 30 days, at which time they were killed and their brains examined for neuropathology with hematoxylin and eosin stains.

Tissues were partially thawed, and samples of T, BAT, BDT, and LH were dissected, weighed, and placed in scintillation vials containing 1 mL of Soluene-350 (Packard Instr. Co., Inc., Downers Grove, IL). Twenty-four hours later, after tissues were completely solubilized, 18 mL of Hionic-Fluor Scintillation Cocktail (Packard Instr. Co.) was added, and radioactivity was assessed by liquid scintillation analysis. Tissue results were expressed as percent injected dose normalized to 1 g of tissue.

Data were summarized by computing percentages for nominal variables and mean values for continuous variables together with their SEs. Significant P values were taken to be <0.05. For Experiment 1, a 3 x 2 x 2 analysis of variance (ANOVA) with drug at three levels (mannitol, arabinose, and NaCl), rate at two levels (0.09 and 0.12 mL/s), and duration at two levels (30 and 40 s) was used. This ANOVA was repeated for each region evaluated (T, BAT, BDT, and LH) without adjusting the significance level for the number of regions. The outcome in the analysis of each region was percent injected dose. Inasmuch as there were, in general, no interactions among drug, rate, and duration of infusion on percent injected dose and the only significant main effect in each region of the brain was due to drug, animals were pooled across rate and duration for evaluation of BBBD by Evans blue staining. This resulted in 16, 24, and 24 animals in the NaCl, mannitol, and arabinose groups, respectively. A {chi}2 statistic was used for this evaluation. For Experiment 2, the effect of hypo- and hypercarbia and drug on drug delivery was evaluated using a 2 x 2 ANOVA with drug at two levels (mannitol and arabinose) and PaCO2 at two levels (25–30 and 45–50 mm Hg, respectively). The NaCl group was included for evaluation of pathologic opening of the BBB due to the effect of hypo- or hypercarbia and was not included in the [3H]-MTX delivery analysis. For Experiment 3, the effect of isoflurane/O2 versus propofol/N2O alone or in combination with phenylephrine, propranolol, phenylephrine/propranolol, and osmotic agent on [3H]-MTX delivery was analyzed using a 2 x 2 x 2 x 2 ANOVA with anesthetic at two levels (isoflurane/O2 vs propofol/N2O), pressor drug at two levels (phenylephrine yes or no), pressor drug at two levels (propranolol yes or no), and osmotic drug at two levels (mannitol versus arabinose). In the absence of significant interactions between osmotic and pressor drugs and because the only significant main effect was due to isoflurane/O2 versus propofol/N2O, data were pooled across pressor and osmotic drugs to evaluate [3H]-MTX delivery and physiologic variables (e.g., BP, HR, and PaCO2). In addition, the effects of isoflurane/O2 versus propofol/N2O on [3H]-MTX delivery were summarized by recalculating the ANOVA with the data pooled as above. Physiologic variables were compared at various time points by using Student’s t-test with Bonferroni corrections applied for multiple time points.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Experiment 1
Arabinose was superior to saline control (P = 0.006) in obtaining consistent Evans blue staining (Figure 1, Table 4). Arabinose significantly increased [3H]-MTX delivery compared with saline control in T, BAT, BDT (P = 0.0001, P = 0.001), and LH (P = 0.0452) (Figure 1). When comparing mannitol and NaCl or arabinose and mannitol, there was no statistical difference in drug delivery; however, there were too few animals with mannitol which received Evans blue staining of 2–3+ (6 of 24; 25%) to allow for meaningful statistical analysis. For mannitol and arabinose inclusive, the only effect of flow rate on [3H]-MTX delivery was to T, for which a 40-s duration was better than 30 s (0.513 ± 0.04 vs 0.223 ± 0.04 percent injected dose/g tissue; P = 0.0372). There was no difference with respect to duration of infusion for all other brain areas.



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  		Figure 1. A, Percent good blood-brain barrier disruptions (BBBD) by Evans blue staining (2–3+). B, Effect of osmotic drug on [3H]-methotrexate delivery to the tumor, brain around the tumor (BAT), brain distant to the tumor (BDT), and left hemisphere (LH). *Arabinose significantly increased [3H]-methotrexate delivery compared with saline control in the tumor, BAT, BDT (P = 0.0001, P = 0.001), and LH (P = 0.0452). Values are mean ± SE.

 

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  		Table 4. Effect of Osmotic Drug on Quality of Blood-Brain Barrier Disruption (BBBD) as Measured by Evans Blue-Albumin
 
Experiment 2
There was a significant effect of varying PaCO2 on quality of BBBD as determined by Evans blue staining (P = 0.025) (Figure 2). Staining (graded 2–3+) was not seen in any animal with either arabinose or mannitol with a PaCO2 >45 mm Hg. The proportion of "good" disruptions (2–3+) was 70% (7 of 10) in the hypocarbic animals when the mannitol and arabinose groups were combined. Arabinose was significantly better than mannitol for [3H]-MTX delivery to T (0.272 ± 0.04 vs 0.160 ± 0.04 percent injected dose; P < 0.05), BAT (0.163 ± 0.03 vs 0.085 ± 0.03 percent injected dose; P < 0.05), and BDT (0.174 ± 0.03 vs 0.085 ± 0.03 percent injected dose; P < 0.05), but not to the LH. Despite this, there was no significant effect of PaCO2 on [3H]-MTX delivery to any area of the brain (Figure 2). Control animals were used to evaluate pathologic BBBD due to the effects of hypo- (n = 5) or hypercarbia (n = 5). In addition to the lack of Evans blue staining in these two groups, there was minimal [3H]-MTX delivery, thus indicating lack of opening of the BBB.



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  		Figure 2. A, Percent good blood-brain barrier disruptions (BBBD) by Evans blue staining (2–3+). B, Effect of manipulation of PaCO2 on [3H]-methotrexate delivery to the tumor, brain around the tumor (BAT), brain distant to the tumor (BDT), and left hemisphere (LH). Data only include animals which received mannitol or arabinose to open the blood-brain barrier. Manipulation of PaCO2 had no statistically significant effect on drug delivery to any part of the tumor or normal brain, regardless of whether arabinose or mannitol was used as the osmotic drug. Values are mean ± SE.

 
Experiment 3
There was no effect of either phenylephrine, propranolol, or the combination on the number of good disruptions as determined by Evans blue staining of 2–3+ (7 of 12 [58%], 7 of 14 [50%], and 6 of 11 [54%], respectively). There was no difference in Evans blue staining when evaluating mannitol versus arabinose (Fisher’s exact test P = 0.933). However, by Evans blue staining, propofol/N2O was superior to isoflurane/O2 in obtaining a good BBBD (19 of 24 [79%] vs 6 of 24 [24%]; P = 0.0007) (Figure 3). With identical constitution and because there were no differences with pressor drug or osmotic drug administration, all groups were combined for analysis of [3H]-MTX delivery with propofol/N2O versus isoflurane/O2 (n = 24 per group). Propofol/N2O was superior to isoflurane/O2 for [3H]-MTX delivery (P = 0.0001). In all parts of the brain evaluated, the propofol/N2O combination yielded approximately 2 times more [3H]-MTX delivered (Figure 3).



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  		Figure 3. A, Percent good blood-brain barrier disruptions (BBBD) by Evans blue staining (2–3+). B, Effect of anesthetics on [3H]-methotrexate delivery to the tumor, brain around the tumor (BAT), brain distant to the tumor (BDT), and left hemisphere (LH). Because there was no difference among those animals which received mannitol and arabinose, all animals that underwent osmotic BBBD are included. *Propofol/N2O anesthesia was superior to isoflurane/O2 anesthesia for drug delivery to all areas measured (P = 0.0001). Values are mean ± SE.

 
BP values were consistently and significantly higher at all time points when BBBD could be documented by Evans blue staining (data not shown). When comparing isoflurane/O2 with propofol/N2O, significant differences in BP also were noted at all time points (P = 0.0001) (Table 5). BP values were lower immediately after the osmotic drug infusion; however, these values approached baseline after 10 min. At 5 and 10 min after BBBD, BP in propofol/N2O-anesthetized animals was still significantly higher than that in isoflurane/O2-anesthetized animals (P = 0.0001) (Table 5).


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  		Table 5. Values at Various Time Points Around BBBD
 
HR (range 290–410 bpm) was not different between the two groups, except that immediately after BBBD, a more significant slowing of the HR was noted in isoflurane/O2 animals (17% ± 1% vs 10% ± 1%; P = 0.001). PaCO2 was significantly higher immediately before the osmotic drug was given in animals receiving propofol/N2O versus those receiving isoflurane/O2 (31 ± 1 vs 24 ± 1; P = 0.0005), and it was still significantly higher 5 min after BBBD (44 ± 1 vs 38 ± 1; P = 0.0147). Immediately after osmotic drug delivery, animals were hypercarbic (PaCO2 >40 mm Hg), but the percent change was not different between isoflurane/O2 and (49% ± 7%) and propofol/N2O (47% ± 7%). Animals had significant decreases in arterial pH (P = 0.0001) immediately postinfusion (from 7.40 to 7.16 for isoflurane/O2 and from 7.37 to 7.12 for propofol/N2O).

Experiment 4
BBBD under propofol/N2O anesthesia was performed in nontumor-bearing animals to evaluate neurotoxicity and neuropathology. Rats were treated with BBBD with mannitol at 0.09 mL/s for 30 s, followed by IA NaCl, allowed to recover, observed daily for neurological signs (seizures, circling, abnormal behavior, etc.) and killed at 30 days. No evidence of neurotoxicity or neuropathology was found.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The success rate of BBBD with isoflurane/O2 in rats with brain tumor xenografts is less than that in normal rats. The reasons for this are not clear. We speculate that there may be multiple factors responsible, including increased intratumoral pressure (23), increased ICP (24), or abnormal vasculature in tumor (25). We designed this series of experiments to determine the impact of these various factors on osmotic BBBD in the brain of rats with tumors.

Early studies determined that osmotic BBBD was a threshold event and that differing osmolalities were not important as long as solutions of a minimal osmolality of 1.4 were used (13). We tried to assess the impact of osmolality in tumor-bearing animals by using arabinose as the osmotic drug, because it can be prepared with osmolality as high as 2.0 molal. We found that arabinose was significantly better than saline in allowing [3H]-MTX delivery to all areas of the brain but was not statistically different from mannitol. With mannitol, very few good quality BBBD were obtained, as determined by brain staining with Evans blue albumin (6 of 24; 25%), whereas with arabinose, 16 of 24 (67%) demonstrated good/excellent Evans blue staining. Evans blue is not a sensitive indicator for delivery of smaller molecules, such as chemotherapeutic drugs, to the brain after BBBD.

We tested the quality of BBBD and the toxicity of BBBD when using other hyperosmolar drugs for BBBD in this rat model (data not shown). Although sucrose and glycerol could both be administered at high osmolalities, they were ineffective at opening the BBB and were also unsuitable due to high mortality (75% and 100%, respectively). Galactose was effective at opening the BBB but resulted in 100% mortality. Arabinose 1.8 molal seemed to be very similar to mannitol in terms of mortality and Evans blue staining.

Arabinose may show some promise for increased BBBD in tumor-bearing rats; however, arabinose is not approved by the Food and Drug Administration for use in patients, which limits its clinical utility. Because the enhancement of drug delivery with arabinose, compared with mannitol, was not substantial, further studies with arabinose or other osmotic drugs are not currently planned. Similarly, although increasing duration of osmotic drug infusion increased BBBD, this is not likely to be clinically useful because of patient health concerns (e.g., cerebral herniation, congestive heart failure); therefore, further experiments are not planned.

With moderate hypocarbia, Evans blue albumin crossed the BBB after arabinose or mannitol administration, but it did not cross the BBB with hypercarbia. There was increased [3H]-MTX delivery when the drug delivery data from animals which received arabinose and mannitol were combined and compared with data from animals which received NaCl. There was, however, no difference in [3H]-MTX delivery under either hypo- or hypercarbic conditions. This is in disagreement with a previous BBBD study under pentobarbital anesthesia in normal rats (14), in which drug delivery was significantly increased in animals with a PaCO2 of 25–30 mm Hg compared with that in animals with a PaCO2 of 35–40 mm Hg (P = 0.025). Brain vasculature is very sensitive to changes in PaCO2 tension. A decrease in PaCO2 produces vasoconstriction with a resultant decrease in intracranial blood volume and increased cerebral perfusion pressure. Although drug delivery was not affected by PaCO2 in tumor-bearing rats, the Evans blue staining data, as well as the previous results in normal rat brain, point out the importance of maintaining a low to normal PaCO2 and preventing hypercarbia in patients, especially when attempting to deliver high molecular weight markers. We did not see Evans blue staining with hypercarbia because, under these conditions, the BBB was not permeable to such a large molecular weight molecule (Evans blue Mr 68,000); it was only permeable to the low molecular weight [3H]-MTX.

The most important observation in this study is that propofol/N2O was superior to isoflurane/O2, both for occurrence of successful BBBD, as evidenced by Evans blue staining (79% vs 24%), and for [3H]-MTX delivery to T, BAT, BDT, and LH. BP was significantly increased with propofol/N2O compared with isoflurane/O2 at all time points measured. Nevertheless, the increase in BP does not seem to be the etiological mechanism for the increased incidence of good BBBD. Logistic regression failed to confirm an independent BP effect in the quality of BBBD. That is not to say that BP variations cannot enhance or diminish the incidence of an adequate (>=2+) disruption. However, the difference in BP between isoflurane/O2 and propofol/N2O (Table 5) is not of the magnitude usually associated with BP-induced BBB dysfunction. In a previous study in normal rats (9), maintenance of HR and BP within a specific range was important in obtaining 2–3+ Evans blue albumin staining. We did not find this to be true in tumor-bearing rats. Gumerlock et al. (9) merely observed which HR and BP ranges were associated with better BBBD, whereas we attempted to assess the effects of HR and BP on BBBD. Manipulating HR with propranolol and BP with phenylephrine failed to alter the incidence of quality BBBD. Although high concentrations of propranolol may produce membrane-stabilizing activity, it is doubtful that this was significant at the doses used in our experiments (26). Specifically, it was the anesthetic used (propofol/H2O) that was important in obtaining consistent quality disruptions.

Immediately after osmotic drug infusion, animals were severely hypotensive, although the hypotension started to resolve immediately and approached baseline values after 10 min. This is in agreement with the results in normal animals found by in Gumerlock et al. (9). However, in our study, BP recovery was slower in tumor-bearing animals. This hypotension was probably due to a direct adverse affect of mannitol on the rat myocardium. In this rodent model, osmotic drug is directed cephalad up the internal carotid by the direction of blood flow in the common carotid. Toward the end of the osmotic drug infusion, the pressure of mannitol overcomes the blood flow, and mannitol is then perfused retrograde toward the heart. When instilled directly into coronary arteries, the mannitol—like contrast dye during coronary angiography—can be expected to cause transient myocardial dysfunction.

Isoflurane produces dose-dependent increases in both cerebral blood flow (CBF) and ICP (27,28), although it is generally thought to have less cerebral vasodilatory properties than other halogenated anesthetics at equipotent concentrations. It is possible that isoflurane-induced "luxury perfusion" to the contralateral hemisphere caused decreased CBF to the side of the brain being disrupted. The direct effect of isoflurane on the cerebral circulation smooth muscle is vasodilation, with resultant immediate ICP increases in both animals and humans (29,30). This potential reduction in flow and possible increase in ICP may have resulted in a lower incidence of adequate disruptions and decreased delivery of MTX.

Analogous to the barbiturates, propofol can reduce ICP, decrease cerebral metabolic requirement for O2, decrease cerebral perfusion pressure, and provide cerebral protection. It also causes a decrease in CBF secondary to propofol’s effect on the cerebral metabolic rate (CMR). Decreasing CMR affords greater cerebral protection if either focal or global cerebral ischemia should occur during the administration of propofol (20,31,32). Clinically, propofol is occasionally substituted for an isoflurane-based anesthetic for neurosurgical procedures in patients with reduced intracranial compliance due to intracranial mass lesions because of propofol’s reduction of CMR and resultant cerebral vasoconstriction (31–33). The use of N2O with propofol may also affect BBBD. Although N2O alone can induce substantial increases in CBF and ICP (34,35), when N2O is combined with IV drugs, these effects are often markedly diminished. Eng et al. (36) observed no change in CBF velocity when N2O was introduced in patients anesthetized with propofol.

With propofol/N2O anesthesia, excellent drug delivery to T and BAT can be obtained in a high percentage of animals (75%) without toxicity. Osmolality, infusion rate, and infusion duration, as well as manipulation of PaCO2, seem to be secondary and less important factors affecting BBBD and delivery of chemotherapeutic drugs when compared with anesthetics. We theorize that regional blood flow differences between the two anesthetic protocols will result in differences in the success of BBBD and quality of drug delivered to T and BAT.


   Acknowledgments
 
This work was supported by a Veterans Administration Merit Review Grant and National Institutes of Health Grants CA31770, NS34608, and NS33618.

The authors express their appreciation to Gail Engles for her clerical assistance.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Accepted for publication December 11, 1998.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins and Stanford University Libraries' HighWire Press®. Copyright 1999 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press