Sampling of orbital sinus blood closely reflects brain ethanol content in rats

Physiology & Behavior, Vol. 33, pp. 895-898. Copyright©Pergamon Press Ltd., 1984. Printed in the U.S.A.

0031-9384/84$3.00 + .00

Sampling of Orbital Sinus Blood Closely Reflects Brain Ethanol Content in Rats ' LINDA MATTUCCI-SCHIAVONE

A N D A N D R E W P. F E R K O 2

D e p a r t m e n t o f Pharmacology, H a h n e m a n n University, B r o a d and Vine Streets, Philadelphia, P A 19102 R e c e i v e d 27 A p r i l 1984 MATTUCCI-SCHIAVONE, L. AND A. P. FERKO. Sampling of orbital sinus blood closely reflects brain ethanol content in rats. PHYSIOL BEHAV 33(6) 895-898, 1984.--It was demonstrated that the aerial righting reflex can be used as an index of acute ethanol-induced impairment of motor coordination in rats, and was found to directly correlate with blood ethanol content from the infraorbital plexus. A study of ethanol within the blood and its distribution in brain regions showed that the ethanol content of orbital sinus blood closely reflected that in the cerebral cortex, midbraln, and cerebellum. Ethanol administration by intraperitoneal (IP) injection (2, 3 or 4 g/kg) produced the same distribution as 24 hr ethanol vapor inhalation (28 mg/1). Blood ethanol concentrations were slightly higher than brain ethanol concentrations when measured at 10, 30, and 60 min after IP injection and immediately following ethanol vapor administration. Also, in rats 48 hr following ethanol vapor inhalation when tolerance to ethanol is exhibited, the distribution and concentrations of ethanol in blood and brain from acute ethanol (2 g/kg, IP) were unaltered when compared with controls. These data suggest that ethanol distribution within the brain does not play a role in the phenomenon of tolerance to ethanol. Blood ethanol content

Brain ethanol content

Motor coordination

T H E effect of ethanol on impairment of motor coordination has been the subject of investigations examining the depressant properties of ethanol. Various methods for evaluating motor impairment include the tilting plane test [8], inhibition of bar holding [ 11], treadmill performance [9], and the aerial righting reflex [3]. The former tests may be measures of ataxia and learning behavior, in addition to motor coordination [7]. An attempt has been made in this investigation to confirm that the aerial righting reflex is an accurate measure of the ethanol-induced impairment of motor coordination. Frequently, the observed behavioral effect from ethanol administration is stated in dose administered and not in the actual concentration of ethanol measured in the blood or brain of the animal. In this work, ethanol-induced motor impairment in rats is correlated with blood ethanol content from the infraorbital plexus. The validity of using orbital sinus blood to estimate the ethanol content of the brain following acute ethanol intraperitoneal (IP) injection or subacute ethanol vapor inhalation is studied. Finally, a one day procedure of ethanol vapor inhalation which induces tolerance to ethanol in rats [1,12] is examined to note if cellular adaptation to ethanol in the central nervous system is responsible for the observed tolerance or if there is an altered regional distribution of ethanol in the brain. METHOD

Animals and Chemicals Male Sprague-Dawley rats (180-220 g) were obtained from Charles River Laboratories, Inc. (Wilmington, MA) and were housed 6 to a cage for 1 week prior to experi-

mentation at 22_ + I°C with a light cycle from 6:00 a.m. to 6:00 p.m. The animals had free access to Purina Laboratory Chow (Ralston Purina Co., St. Louis, MO) and water; however, they were fasted 18 hr prior to drug or saline administration but water was available ad lib. The bedding used in the cages was ground corn cob, 1/s in (Anderson Cob Division, Maumee, OH). Ethanol solutions (10 or 20% w/v) for injection were prepared from 95% ethanol.

Measurement of Motor Coordination The procedure described previously [4], was used to measure the alteration in the aerial righting reflex of rats as an index of motor coordination. Briefly, the rats were held in an inverted position above a foam rubber mat and released. The height was measured by an adjacent meter stick. The minimum height that the animal could successfully right himself on two consecutive releases was used as an index of motor impairment (usually 5 cm for control animals). A suecessful righting required that all four paws touched the mat simultaneously upon landing. Time required to test an animal was 10--20 sec. To note the effect of ethanol on motor coordination, animals were administered ethanol (I, 2, or 3 g/kg, IP) or saline (0.9% NaCI solution) one hr prior to testing. From each animal immediately after testing, blood samples (20/zl) were obtained from the orbital sinus to determine blood ethanol concentrations according to an enzymatic method [I0].

Blood and Brain Ethanol Content After IP Injection To study the distribution of ethanol after IP injection,

~This material is based on work supported under a National Science Foundation Graduate Fellowship. 2Requests for reprints should be addressed to Andrew P. Ferko, Ph.D.

896

M A T T U C C I - S C H I A V O N F AND F E R K O TABLE 1 EFFECT OF ACUTE ETHANOL ADMINISTRAFION(IP) ON BLOOD ETHANOl. CONTENT AND IMPAIRMENTOF AERIAL RIGHTING 60 MINUTES AFTER INJECTION Ethanol Dose (g/kg)

N

Blood Ethanols (mg/ml}

Height of Aerial Righting (cm)

0.(1" 1.0 2.0 3.0

8 6 13 15

0.00 /I.71 + 0.116§ t.72 ± 0.07§ 2.54 +_ 0.08§

5.3 :+ 0.3+ 8.0 +_ 1.2 17.7 + 1.7§ 33.0 _+ 2.2§

*Saline administered to controls. CMeans _+ SE. $Orbital sinus blood. §Significantly different from all other doses q~<0.01: ANOVA followed by Scheffe's test).

other groups of animals were administered 2, 3, or 4 g/kg ethanol IP. At various time intervals after injections (10, 30, or 60 min), blood samples were obtained from the infraorbital plexus and collected in a 20/~1 pipette. The rats were then decapitated and the whole brains were immediately rinsed with ice cold saline. The brains were dissected, on ice, into the regions of cerebral cortex, midbrain, and cerebellum [6]. Ethanol concentrations in blood and brain tissue were determined enzymatically [10]. The ethanol concentration in brain was expressed as mglml of tissue water and the ethanol concentration in blood was expressed as mg/ml of blood water, as the water content of brain and blood was determined to be 77 and 80%, respectively.

were administered 2 g/kg ethanol IP. At 10, 30, or 60 min following injection, blood (20/xl) was sampled from the infraorbital plexus, and brain sections excised as described. Ethanol concentrations in blood and brain sections were determined.

Distribution of Ethanol After Subacute Administration by Ethanol Vapor Inhalation

Effect of Ethanol on Aerial Righting Reflex

Statistical Analysis Significant differences were determined by analysis ot variance (ANOVA). All multiple comparisons with a control were done by A N O V A followed by Scheffe's test. All data were analyzed using an Apple IIe computer. RESULTS

The distribution of ethanol in brain regions caused by ! day exposure to ethanol vapor was compared with that produced by IP injection. Animals (group of 4) were exposed to ethanol vapor in a chamber (nominal concentration: 28 mg/liter) by inhalation for a period of 24 hr as previously described [1, 2, 12]. Briefly, ethanol (95%) was delivered into a warmed flask by an infusion pump (Harvard Apparatus Peristaltic Pump) at a calibrated rate of 150 mg/min. An air flow of 5.35 liters/rain through the flask maintained an ethanol vapor concentration of 28 mg/liter in the chamber. A two stage regulator valve and a Gilmont flow meter controlled the air flow. Temperature inside the chamber was 25+1°C. Ethanol vapor concentrations were determined twice daily in duplicate at 4 hr into the procedure and at 24 hr. F o o d and water were available during the period of ethanol vapor exposure. Control animals were exposed to air only in the chamber and food was restricted to match that consumed by the ethanol treated animals in order to reflect the weights attained by the treated group [1, 12, 13]. At the conclusion of the 24 hr inhalation period, animals were removed from the chamber. Orbital sinus blood samples (20 /xl) and brain tissue were obtained from some ethanol vapor treated animals immediately after removal from the chamber to determine the distribution of ethanol produced by this procedure. In order to assess any changes in the distribution of IP ethanol produced by this procedure, at 48 hr after removal from the air or ethanol vapor infused chamber, the animals

The effect of ethanol (1, 2, or 3 g/kg, IP) on motor coordination as measured by the aerial righting reflex is shown in Table 1. Both the orbital sinus blood ethanol content and aerial righting index resulting from 2 and 3 g/kg ethanol are significantly different from all other doses, as well as controls; F(3,35)=247, F(3,35)=44 for blood content and aerial righting, respectively.

Distribution of Ethanol Following IP Injection The concentrations of ethanol in orbital sinus blood, cerebral cortex, midbrain, and cerebellum were measured at various time periods following the IP injection of ethanol (2, 3, or 4 g/kg) (Fig. 1). The ethanol concentrations in orbital sinus blood and brain sections following each dose were not different at the time periods tested following IP administration, Also, the ethanol concentrations in the cortex, midbrain, and cerebellum were similar to each other following IP administration of each dose. Both blood and brain ethanol concentrations peaked at 10 rain after IP injection. Blood concentrations remained higher (although not statistically different) than brain concentrations at all time periods studied.

Distribution of Ethanol Following Subacute Administration Table 2 lists the concentration of ethanol in blood and brain tissue achieved by the procedure of 24 hr ethanol vapor inhalation (N=10). Although higher ethanol levels were produced by this procedure, the distribution within brain

BLOOD AND BRAIN ETHANOL CONTENT

897 4.0

TABLE 2 ETHANOL CONCENTRATION (mg/ml) IN BLOOD AND BRAIN IMMEDIATELY FOLLOWING REMOVAL FROM 24 HR ETHANOL VAPOR INHALATION (28 mW1) Blood 4.07 _+ 0.28*

Cortex

Midbrain

Cerebellum

3.45 _+ 0.26

3.46 _+ 0.28

3.42 _+ 0.24

3.0

*Means _+ S.E. 2.8

i2.O

regions and the ratio o f brain ethanol c o n c e n t r a t i o n to blood ethanol c o n c e n t r a t i o n are similar to that p r o d u c e d by IP inj e c t i o n o f ethanol. Table 3 s u m m a r i z e s the effect o f 24 hr ethanol v a p o r or air inhalation on the distribution o f ethanol (2 g/kg, IP) given 48 hr after r e m o v a l from the chamber. The ethanol c o n t e n t in the orbital sinus blood and brain regions was similar in the controls (air-treated) and ethanol vapor-treated animals at e a c h time period.

1.5

1.0

DISCUSSION 0.6

E x p e r i m e n t s designed to m e a s u r e an effect of ethanol using a behavioral endpoint should be interpreted with the k n o w l e d g e o f the distribution o f ethanol as a function of time following ethanol administration. The results o f Table 1 indicate the aerial righting reflex test can be used as an index of the d e p r e s s a n t effect o f acute ethanol on m o t o r coordination, due to the direct relationship b e t w e e n test score and orbital sinus blood ethanol content. Based on the data from Fig. 1, sampling blood f r o m the infraorbital plexus illustrates a direct correlation with the ethanol content in the cortex, midbrain, and c e r e b e l l u m at the time periods and doses tested. Thus, orbital sinus bleeding can be a simple and reliable m e t h o d for estimating the concentration o f ethanol in the brain which results in the o b s e r v e d behavioral effect of ethanol. The distribution of ethanol in blood and brain appears to be consistent for the two routes o f administration studied: IP injection and 24 hr ethanol v a p o r inhalation (Fig. I and Table

• ~*J_--~.-

BLOOD

.............

¢on'rlx

.......

III~IIII~N CEIILqlULLUM

10

I0 TIME (IdlN)

FIG. 1. Ethanol concentration (mg/ml) in blood and brain at various time periods after acute ethanol (2, 3, or 4 g/kg, IP) administration. The range of standard errors was between 0.03 and 0.42 for all values. Each value represents N=6 to 9.

2). Blood ethanol c o n c e n t r a t i o n remains higher than brain content of ethanol. Samples o f orbital sinus blood collected as early as 10 min after IP ethanol injection reflect the ethanol conditions a c h i e v e d in the brain. In contrast, o t h e r reports indicate a higher c o n c e n t r a t i o n o f ethanol in brain than in v e n o u s blood from the tail vein for the first 60 min

TABLE 3 ETHANOL CONCENTRATION (mg/ml) IN BLOOD AND BRAIN FOLLOWING ACUTE ETHANOL (2 g/kg) 48 HR AFTER REMOVAL FROM CHAMBERS Time

N

Blood

Cor

Mid

Cer

1.54 _+ 0.13 1.55 + 0.08 1.63 _+ 0.05

1.45 ± 0.14 1.52 _+ 0.10 1.52 _+ 0.02

1.35 _+ 0.26 1.62 __+0.13 1.58 _+ 0.05

1.33 ± 0.27 1.49 ± 0.14 1.54 ± 0.05

Controls (Air-Treated) (24 hr) 10 rain 30 rain 60 rain

6 6 6

1.72 _+ 0.19" 1.74 _+ 0.10 1.76 ± 0.05

1.57 _+ 0.13 1.62 _+ 0.11 1.66 _+ 0.05

Ethanol Vapor Treated (24 hr) 10 min 30 rain 60 min

5 7 9

*Means _+ S.E.

1.49 _+ 0.30 1.78 _+ 0.15 1.78 ± 0.05

gO

1.38 + 0.26 1.65 _.+ 0.13 1.67 _+ 0.04

898

M A T T U C C I - S C H I A V O N E AND F E R K O

after IP administration. After 90 rain, equilibration occurs and the tail vein blood ethanol concentration exceeds that in brain [7]. Since blood ethanol concentration from the infraorbital plexus has been shown to peak more rapidly (within 5 to 10 min) than tail blood (15 to 30 min) after an IP ethanol injection [5], sampling of the orbital sinus blood may be a more precise index of brain ethanol when testing is performed at early time periods after ethanol administration. In earlier investigations [1,12], functional tolerance to the acute effect of ethanol was produced, in rats, 48 hr following 1 day of ethanol vapor inhalation. Although data on the disappearance of ethanol from blood showed no alteration in the kinetics of ethanol at the time tolerance was assessed,

this present study confirmed that ethanol vapor inhalation had no effect on the distribution of ethanol within brain regions following acute ethanol (IP) administration. Thus, tolerance appears to be functional in nature, and not the result of a modified or decreased ethanol distribution within the brain.

ACKNOWLEDGEMENTS The authors wish to express their appreciation to Dr. W. S. Chernick for his assistance and to Ms. Joanne Addario and Mrs. Linda Bush for help in the preparation of this manuscript.

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