Brain Research, 590 (1992) 263-270 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00

263

BRES 18067

Blood-brain barrier integrity and brain water and electrolytes during hypoxia/hypercapnia and hypotension in newborn piglets B a r b a r a S. S t o n e s t r e e t

a,

G a r y H. B u r g e s s a a n d H e l e n F. Cserr

b

"Department of Pediatrics Women and Infants' Hospital of Rhode Island, Prot'idence, RI 02905 (USA) and I, Section of Physiology Brown University, Prot,idence, R! 02912 (USA) (Accepted 14 April 1992)

Key words: Blood-brain barrier; Brain; Brain water; Electrolyte; Hypoxia/hypercapnia; Newborn

This study examines the effects of hypoxia/hypercapnia and hypoxia/hypercapnia with hypotension (hypotensive-hypoxia/hypercapnia) on blood-to-brain transfer constants (K I) for sodium and mannitol and brain water and electrolyte contents in newborn piglets. Hypoxia/hypercapnia was induced for 60 rain with the piglets breathing a gas mixture of 15% carbon dioxide, 10-12% oxygen, and 73-75% nitrogen adjusted to achieve an arterial pH < 7.15, PO2 < 40, and pCO2 > 60 mmHg and hypotenstion for 20 min by rapid phlebotomy to achieve a mean arterial blood pressure < 40 mmHg. Piglets were studied during 1 h of, and 24 h after resuscitation from hypoxia/hypercapnia (arterial pH 6.9+0.18, pO 2 36+6 mmHg, pCO2 68+8 mmHg, mean + S.D.) and 10 min, and 24 h after resuscitation from hypotensive-hypoxia/hypercapnia (mean arterial blood pressure 28 + 10 mmHg, mean + S.D.). Values for K t for sodium and mannitol, measured using the integral technique were 15.9 and 5.2 ml. g- t. min- t × 104 respectively, in 2-4-day-old controls, suggesting that the barrier is fully developed in newborn piglets. Values were not different during or after hypoxia/hypercapnia or 24 h after hypotensive-hypoxia/hypercapnia. Ten to forty min after hypotensivehypoxia/hypercapnia, there was a proportional decrease in the K t for sodium and mannitol of about 40%. These results suggest that the newborn piglet is similar to the adult with respect to impermeability of the blood-brain barrier to ions and smail molecules and resistance of this barrier to systemic hypoxia/hypercapnia and hypotension. We suggest that acute decreases in K t for sodium and mannitol after hypotensive-hypoxia/hypercapnia reflect a reduction in capillary surface area,

INTRODUCTION Perinatal hypoxia-ischemia frequently results in neonatal brain damage. Although brain edema has been reported in human neonates dying after intrapartum asphyxia~, the role of changes in blood-brain barrier permeability and brain edema has not been firmly established. Because the brain capillary endothelium contains a large number of mitochondria, it has been assumed that the maintenance of low blood-brain barrier permeability to small molecules is energy dependent requiring a continuous supply of oxygen for metabolism27'2s. However, in adult animals, the blood-brain barrier is resistent to hypoxia2'29'32. Small increases in permeability to ions 2s are seen initially, followed by much later increases to larger molecules, which appear to be secondary to tissue necrosis t3'31'43. Hypoxia is thought to increase brain water through the production

of cytotoxic or vasogenic edema ~j. The former is characterized by an increase in intracellular water and sodium and the latter by increased permeability of the brain capillary endothelium tl. To evaluate the possible role of changes in bloodbrain barrier permeability and brain edema in the production and pathogenesis of perinatal hypoxicischemic brain damage, blood-brain barrier permeability and brain water and electrolytes should be evaluated simultaneously. Moreover, there is a need to evaluate permeability using tracers of different molecular size over a prolonged period after hypoxia-ischemia, as has been done in adults. Previous studies of the perinatal response to hypoxia-ischemia have yielded conflicting results. Some indicate either no change in brain water 36°37or barrier permeability 42, whereas others demonstrate the development of either brain edema 7,24,25,3°,33,3s or increased permeability 23'3'~. However, it appears that none of these studies have simul-

Correspondence: B.S. Stonestreet, Women and Infants' Hospital, 101 Dudley Street, Providence, RI 02905, USA. Fax: (i) (401) 453-1330.

264

taneously examined changes in barrier permeability and brain water and electrolytes quantitatively in the perinatal subject. In this study, we examine normal blood-brain barrier permeability and the response to systemic hypoxia/hypercapnia and hypotension, using small molecular weight tracers immediately and 24 h after the insult. Permeability is assessed quantitatively using sodium and mannitol to measure blood-to-brain transfer constants. The transfer constant (K~) may vary because of changes in either permeability or surface area. Because changes in blood flow may be associated with changes in surface area, we measured blood flow in a separate series of piglets under similar conditions. We studied piglets during, and after resuscitation from hypoxia/hypercapnia and hypotension, because these combined insults render brain blood flow pressure-passive tT'2°'3s'4t potentially resulting in hypoxic-ischemic brain injury similar to that observed in sick neonates with severe cardiorespiratory compromise. MATERIALS A N D M E T H O D S Filty-one 2-4-day- old, farm-bred piglets obtained from a local breeder, who provided accurate times of farrowing, were the subjects of this study. The weight of the newborn piglets was 1.4±0.3 kg (mean + S.D.). The study was approved by our institutional animal review board. At)imal prei)aration. Two hours belore study, catheters were placed under nitrous oxide and local lidocaine anesthesia its previously descr!bed"". For the blood-brain barrier permeability studies, catheters were placed in the upper and lower abdominal aorta and inferior vena cava. For the measurement of brain blood flow, catheters were placed in the left ventricle via the right brachial artery for injection of radionuclide-labeled microspheres, the left brachial artery for withdrawal of reference blood samples, the abdominal aorta via a femoral artery for blood pressure measurement, heart rate, arterial blood gases, oxygen content and hematocrit values, and the inferior vena cava via a femoral vein for blood replacement with age matched donor piglet blood. Catheter placement was verified by pressure tracings and by autopsy. After catheter placement, the newborn piglets were allowed to recover for two h. Studies then proceeded as follows in the awake, spontaneously breathing newborn piglets. Study groups. The newborn piglets randomly assigned to 7 different groups were studied as follows: during I h of(n ,~ 6) and 24 h after I h of hypoxia/hypercapnia (n ~ 7), 10 rain (n = 6) and 24 h after (n = 7) I h of hypoxia/hypercapnia and with 2(1 rain of hypotension (n = 6). Two groups of age-matched, sham-control piglets were also studied after ! h (n = 12) and 24 h of sham-control treatment (n = 7). In a separate group (n = 6), brain blood flow was measured under conditions of hypoxia/hypercapnia and hypotensive-hypoxia/hyper. capnia.

E.ff~'rimental protocol,~ One hour of hylu~xia/hypercapnia. The newborn piglets were placed with their heads in a sealed hood, breathed air for 30 min, while their deep rectal temperature was maintained between 38.5 and 39°C zz via a heating pad. After the air breathing, hypoxia/hyper. capnia was induced for 60 rain by changing the gas mixture in the hood to 15% carbon dioxide, 10-12% oxygen and 73-75% nitrogen adjusted to achieve an arterial pH < 7.15, pO z < 40 and pCO 2 > 60 mmHg. Serial determinations of arterial blood gases, hematocrit, heart rate and mean arterial blood pressure were made during air

breathing and hypoxia/hypercapnia. Blood-brain barrier permeability measurements were made during the last 30 min of hypoxia/hypercapnia. After the study had been completed, the piglet was administered sodium thiamylal (200 mg/kg) and the piglet's head immediately removed with a guillotine. The brain was then removed from the cranium within 1 min. One half the brain was obtained for the blood-brain barrier permeability measurements and the other for brain tissue water and electrolytes. Twenty-four hours after hypoxia/ hypercapnia. This study protocol was as described above, except that, after 1 h of hypoxia/hypercapnia, the piglets were removed from the head hood and entered a recovery phase for 24 h. During this phase, they were placed in a box in which they were able to stand and move around. The catheters were filled with heparin (250 U. ml- i), knotted and secured, and the piglets received a continuous infusion of 0.2 NaCI and 5% dextrose and water at a rate of 8 m l . k g - l . h - I via an inferior vena cava catheter. The piglets recovered relatively rapidly from hypoxia/hypercapnia and were able to sit or stand within 2 h. Because of the possibility of hypoxia/hypercapnia related gastrointestinal injury, the piglets were not fed. However, their body weight remained unchanged compared with the pre-operative weight, in this group, blood-brain barrier permeability was measured 24 h after hypoxia/hypercapnia. All other measurements were as described above, except that additional measurements of arterial blood gases, hematocrit, heart rate and arterial blood pressure were obtained at the onset and completion of the permeability studies, i.e., 24 and 24.5 h after hypoxia/hypercapnia. Hypotensit'e.hypoxia/hypercapnia. The newborn piglets in this group were treated in a similar fashion to the 1 h of hypoxia/hypercapnia protocol, with an exception that hypotension was induced by rapid phlebotomy during the last 20 rain of hypoxia/hypercapnia via a catheter placed in the inferior vena cava until the mean arterial blood pressure was < 40 mmHg. This value was selected, because of previous work suggesting that this represents the lower limit of brain blood flow autoregulation in normal newborn pigletsIs. To achieve this reduction in mean arterial blood pressure, 22 ± 8 ml. kg- i (mean ± S.D.) of blood was removed from the piglets. A reduction in the mean arterial blood pressure below 40 mmHg was achieved for 15 ± I min (mean ± S.D.). After I h of hypoxia/hypercapnia with 20 min of

hypc)tension, the piglets were removed from the head hood, breathed room air, and the previously removed heparinized blood returned to the piglet by a slow infusion into the inferior vena cava. After this resuscitation, the piglets were allowed a I(I rain rest, The rate constants for blood-to-brain transfer were measured between l0 and 40 rain alter resuscitation. Determinations of arterial blood gases, hematocrit, heart rate, and mean arterial blood pressure were obtained before hypoxia/hypercapnia, serially during hypoxia/hypercapnia, and hypotensive-hypoxia/hypercapnia and 10 and 40 min following the piglet's resuscitation. All other aspects of this protocol were as described for the ! h of hypoxia/hypercapnia protocol. in a separate group of similarly treated piglets (n = 6), brain blood flow was measured using radionuclide-labeled microspheres, during 5 different time periods. Blood flow was measured as follows: (!) during baseline, 10 rain before, and (2) after 40 rain of hypoxia/hypercapnia, and (3) 60 rain of hypoxia/hypercapnia with 20 min of hypotension, and (4) 10, and (5) 40 rain after resuscitation from these insults, in order to quantify brain blood flow during the acute studies. Twemy.fo,r hours after hypotensit'e.hypoxia / hypercapnia. Treatment of the newborn piglets during the acute phase of this protocol was as described for the hypotensive-hypoxia/hypercapniaprotocol. Following resuscitation, the piglets recovered for 24 h and thereafter the blood-brain barrier permeability studies were performed over 30 rain. During the 24 h recovery phase, the newborn piglets received the intravenous fluids described above. At the time of the study, the piglets body weight remained unchanged compared with that prior to surge,3'. Age-matched sham-control studies. The sham-control newborn piglets were treated in the same fashion as in the experimental protocols, except that the piglets breathed air and were not rendered hypotensire. Time matched blood-brain barrier permeability studies were performed after 60 min of air breathing and 24 h of sham-control

265 treatment with the same intravenous therapy as in the experimental piglets; these were the age-matched, sham-control subjects.

Methodology Measurement of the blood-to-brain transfer constant (K z) for sodium and mannitol. Radiolabeled tracers were injected intravenously, and the arterial plasma concentration monitored for 30 min, Based upon our previous analysis of rate constants and exposure times for sodium and mannitol tracers in adult rats 5, this time interval was determined to be adequate to detect changes in barrier permeability in the newborn piglets. Brain tracer concentration was determined at the end of the experiment. Knowledge of the plasma concentration profile, together with the concentration of tracer in the tissue parenchyma, permits calculation of blood-to-brain transport constants (see below), as described by Ohno et al. 26. In these experiments, unidirectional transfer constants for influx across the bloodbrain barrier (K t) were estimated for [t4C]mannitol and either 22Na or 24Na (Du Pont-New England Nuclear, Boston, MA). Brain vascular volume was determined individually for each animal using L~Slhuman serum albumin (RISA, Mallinckrodt) to determine residual brain blood volume. Details of the animal preparation, tracer injection, sample collection, assay and calculations are similar to those described for adult rats 5 with minor modification. Briefly, 40-60/~Ci 22Na or 24Na and 7.5 /~Ci [14C]mannitol in 0.5 ml of physiologic saline was injected into the inferior vena cava as a single bolus. Blood samples (0.15 ml) were withdrawn from an arteriovenous bypass inserted between the abdominal aorta and inferior vena cava. Samples were taken at 15 s intervals for the first 2 rain after isotope administration and subsequently at I rain intervals. Five minutes prior to sacrifice and the final sample, 3/~Ci of RISA was administered intravenously. At the end of the 30 rain experimental period, the piglet was decapitated, and the brain removed and homogenized. Aliquots of the homogenized whole brain were analyzed for radioactivity s and for water, sodium, potassium and chloride*; plasma samples for radioactivity only. The blood-to-brain transfer constant K~ (ml.g wet wt. -t. min - t ) is given by: K t - Ah,/f~icr,(T ) dT

(!)

where At,., is the amount of tracer (dpm.g ' t ) that crossed the blood-brain barrier from the blood to brain during the tracer study, and cp is the concentration of the tracer in plasnm (dpm.ml ~m) at the time t (rain). Ahr is obtained by correcting the total amount of isotope measured in the tissue Am (dpm'g" t) for that residual part remaining in the brain vasculature space, which is measured by the t2Sl.serum albumin. Thus, At,r = A m - Vpcp, where Vp = A m/cp, where A m and % have the same definitions as above except that they apply to tZ'~l-serum albumins. Separate samples of the weighed homogenized whole brain tissue and volumetrically measured plasma were counted in a gamma counter (Trachor Analytic Sample Changer 1185, Elk Grove Village, IL) interfaced with a Multichannel Analyzer (8192 Channel Series 40 Processor, Canberra Industries, Inc., Meriden, CT) to determine 1:'51 and 2:'Na or 24Na activities. The gamma counter had been previously calibrated with pure standard for each isotope. All sample counts were corrected for background, sample decay, and for spillover as appropriate. Radioactivity of the beta admitter, t4C was assayed by liquid scintillation spectroscopy. Samples were counted in gelled Aquasol (Du Pont-New England Nuclear, Boston, MA) with double settings for t4C and t251. This permitted residual t2Sl in ~he extract of the final plasma and brain samples described below. Sample counts were corrected for counting efficiency, using the external-standard method, and for background. All experiments with ~4C were triple label experiments, including lac, Z4Na and t251. The plasma samples collected before the administration of the t2Sl-serum albumin bolus injection contained only t4C and 24Na. Aiiquots of these samples (50 ~1) were added directly to the aquasol for counting. The terminal plasma samples and the brain sample contained 14C, 12Sl and :'4Na. Before assay of these samples, the t25I-serum albumin was removed by precipitation with tricarboxylic acid (TCA) and the [t4C]mannitoi extracted as previ-

ously described 21. All samples were cou~ted for [t4C]mannitol after they had been stored for 1 w,~ek ( > 10 half-lives of 24Na) before radioassay to Dermi! the decay of 24Na. Experiments with :2Na did not contain [14C]mannitol. Measurement of brain blood flow. Randomly administered microspheres (15+5 p,m, Du Pont New England Nuclear, Boston, MA) labeled with 4'Sc, ~SNb, 57C0, tt3Sn and I°aRu, were used to measure brain blood flow according to previously established techniqueslzaT"ts'aa. The fresh pre-weighed brain tissue was placed in counting vials and counted along with the blood specimens in the gamma counter. Blood flow data were generated with a Digital PDP-11/34 computer system (Digital Equipment, Maynard, MA) which corrected for isotope spiilover and background counts. Blood flow wele calculated using the followingS2: blood flow (ml/min) counts per minute in individual tissues counts per minute in reference blood × rate of withdrawal of reference blood

(2)

All specimens had sufficient microspheres to ensure accuracy to +5% 12. Paired samples of right and left brain (data not shown) documented the absence of streaming. Arterial blood gases were measured using a Corning 175 Blood Gas Analyzer (Coming Scientific, Medford, MA), oxygen contents in duplicate on a Lex-O2-Con Instrument (Lexington Instruments, Waltham, MA), and blood pressure and heart rate with a pressure transducer (Hewlett-Packard, Model 1280 C, Lexington, MA), and recorded on a Hewlett-Packard 7754 Series Polygraph. Statistical analysis. All results are expressed as mean + S.D. Serial measurements over time were separately compared to the respective baseline values using two-way analysis of variance for repeated measures. When a significant difference was found by analysis of variance, the Dunnett's test was used to compare the means to the baseline values before hypoxia/hypercapnia~. Each experimental group was compared to the respective age matched control group for brain water and electrolyte contents, and the unidirectional influx coefficient for sodium and mannitol by the unpaired Student's t-test. P < ¢).05 was considered statistically significant.

RESULTS

The physiologic data including arterial blood gases, heart rate, mean arterial blood pressure and hematocrit values in the control, hypoxia/hypercapnia and hypotensive-hypoxia/hypercapnia groups of piglets ~re summarized in Table I. The baseline pH, blood gas, mean arterial blood pressure and hematocrit values were within the normal range for 2-4-day-old piglets 17.1~,22.Hypoxia/hypercapnia was associated with the expected decreases in arterial pH, pO 2 and base excess and increases in pCO2. Heart rate decreased significantly after 1 h of hypoxia/hypercapnia and mean arterial blood pressure was not changed. Similar changes in blood gas values were observed in the hypotensive-hypoxia/hypercapnia group. In this group, a reduction in heart rate was observed throughout the 1 h of hypoxia/hypercapnia. With phlebotomy, mean arterial blood pressure was reduced. Changes were not observed in the control piglets. In the separate group of piglets, in which brain blood flow was measured

266 TABLE I

pH,,, paCO.,, p,,O,, bast" excess, heart ntte, mean arterial blood pressun', and hematocrit cahies in the newborn piglets BP, blood pressure; H P / H C , hypoxia/hypercarbia; n.d., value not determined. * P < 0.05 vs. baseline. Hypot.-HP/HC, hypotensive-hypoxia/hypercarbia; Control group, n = 12; H P / H C group, subjected to 60 min of H P / H C , n = 6; H y p o t . - H P / H C group, subjected to 60 rain of H P / H C with 20 rain of hypotension, n = 6; mean -+ s.d.

Variable

Study period (rain)

Group

BP J, Baseline

HP / HC or Air breathing

- 20

7.48 + 0.04 7.53 + 0.04 7.45 _+ 0.06

Control HP/HC Hypot-HP/HC

pH (mmHg) PaO.~ (mmHg) paCO, (mmHg)

n.d. 7.05_+ 0.14 * 6.82 _+ 0.13 *

n.d.

n.d. 42 34

_+ 8" _+ 7 *

69 36 31

_+ 9 _+ 6" _+ 8*

n.d. n.d. 120

n.d. 65 61

_+ 7 " _+ 8 *

35 68 70

_+ 8 _+ 8 " _+ 5 *

74 6q 75

+ 10 -+ 13 + 9

n.d. 38 33

Control HP/tlC Hypot.-HP/HC

32

_+ 6 _+ ? + 3

n.d.

Base excess (mEq/l)

Control HP/HC ltypot.-HP/ttC

Heart rate (#/rain)

Control HP/ltC Hypot.-HP/HC

32

1.9 + 5.1 3.9 + 1.5 0.8 + 6.1

_+ 5*

n.d. - 12.6 _+ 8.0" - 2 7 . 3 _+ 7.1 *

81 87 79

76 87 75

± I11 ± 15 ± 8

! lematocrit (r;)

Control IIP/IIC Itypot,-llP/llC

23 27 25

± 2 ± 3 ± 5

ACUT

65

234 216 148

IIP/HC llypot.-HP/tlC

(mL,0=,,mm. i x 104 )

-+ 8 *

± 65 + 32 ± 26

Control

_+ 5 * + 10 *

66

235 262 223

Mean arterial blood pressure (mmHg)

20

40

Control HP/HC Hypot.-HP/HC

3O

+ 54 _+28 _+35 * + 12 -+ 14 + 9

n,d. n,d, n,d,

E

24Hrs AFTER INSULT t?l (?}{6}



O

,

SODIUM

,

MANhITOL

,,

Recocery

20

---.==--=:f,~~ ~ SODIUM/ MANNITOL

Fig. I. Blood-to-brain transfer constants, K I for sodium, mannitol and sodium/mannitol ratio for control, hypoxia/hypercarbia and hypotensive-hypoxia/hypercarbia groups of newborn piglets. Open bars designate control piglets. Hatched bars designate piglets exposed to hypoxia/hypercarbia. Closed bars designate piglets exposed to hypotensive-hypoxia/hypercarbia. * p < 0.05 vs. control; number is given between parentheses; mean +_S.D.

60 7.00_+ 0.15 * 6.62_+ 0.25 *

n.d. - 15.4 _+ 8.7 * - 32.5 _+ 8.2 * 234 222 154 79 86 72 n.d. n.d. n,d.

:t: 6(1 -+38 -/:27 * -+ 9 -+ 10 -t-15

7.51+ 0.04 6.93_+ 0.18" 6.50_+ 0.14 *

7O

100

n.d. n.d.

n.d. n.d. 6.6_+ 0.19 *

6.68_+ 0.24 *

_+ 7 *

n.d. n.d. 117

_+!0"

n.d. n.d. 19 _+ 6 *

n.d. n.d. 16

_+ 4 *

n.d. n.d. - 38.5 _+ 5.5 *

n.d. n.d. - 36.0 _+ 7.4 *

+ 70 +33 * +49"

n.d. n.d. 145

_+27"

n.d. n.d. 194

+43

76 81 29

-+11 -+ 12 +i0'

n.d. n.d. 70

-+11

n.d. n.d. 61

_+20

24 27 21

+ 2 -+ 5 -+ 5 '

n.d. n,d, 27

-+ 6

n.d, n,d. 27

_+ 7

1.3 _+ 4.2 - 19.0 _+ 8.6 * - 39.2 _+ 5.3 253 195 123

during hypoxia/hypercapnia and hypotensivehypoxia/hypercapnia, the blood gas and hemodynamic values (data not shown) were equivalent to that summarized in Table I for the similar hypotensive-hypoxia/hypercapnia group, in which blood-brain permeability was measured. Table !i summarizes the blood gas and hemodynamic values for the newborn piglets studied 24 h after hypoxia/hypercapnia and hypotensive-hypoxia/hypercapnia along with the age-matched control group. These resuscitated newborn piglets showed :,tmilar physiologic values during the acute phase of the studies, to acutely studied piglets shown in Table I. Twenty-four hours after resuscitation from the acute insults, most variables were similar to baseline. As shown in Fig. 1, sodium and mannitol blood-tobrain transfer constants were not altered by hypoxia/ hypercapnia. Acute hypotensive-hypoxia/hypercapnia was associated with a significant proportional reduction in sodium and mannitol blood-to-brain transfer constants. Sodium blood-to-brain transfer constants were not altered 24 h after hypoxia/hypercapnia or

267 TABLE II

p H a, Pat02, Pa02, base excess, heart rate, mean arterial blood pressure and hematocrit ralues it, the resuscitated newborn piglets surcicing 24 hours after HP / HC and Hypot.-HP / HC BP, blood pressure; H P / H C , hypoxia/hypercarbia; n.d., value not determined. * P < 0.05 vs. baseline. Hypot.-HP/HC, hypotensive-hypoxia/hypercarbia; Control group, n = 12; H P / H C group, subjected to 60 rain of H P / H C , n = 6; Hypot.-HP/HC group, subjected to 60 min of H P / H C with 20 rain of hypotension, n -- 6; mean ± S.D.

Variable

Group

Study period (min) BP J, Baseline

HP / HC or Air brea thing

Recovery

20

40

n.d. 7.05 ± 0.26 * 7.13± 0.05 *

n.d. 6.92 ± 0.30 * 7.11± 0.07*

± 4 + 18 ± 9

n.d. 39 31

± 10 ± 6*

n.d. 40 28

+ 9* ± 2"

76 35 34

± 8" ± 9* ±12"

87 93 87

+ 7 ± 6 ±18"

84 86 91

+ 7 + 8 +20 *

± 2 ± 6 ± 4

n.d. 64 59

± 12 * + 6*

n.d. 72 63

+ 8* ± 5"

32 80 72

± 3 ± 8* ±14'

28 29 29

± 3" ± 6* ± 9

31 31 32

+ 3 ± 6 ± 13

- 20

pH

Control HP/HC Hypot.-HP/HC

PaO2 (mmHg)

Control HP/HC Hypot.-HP/HC

84 86 70

paCO~ (mmHg)

Control HP/HC Hypot.-HP/HC

32 30 29

Base excess (mEq/l)

Control HP/HC Hypot.-HP/HC

- 0 . 1 ± 4.6 - 1 . 3 ± 6.2 - 2 . 4 ± 4.0

Heart rate (#/rain)

Control HP/HC Hypot.-HP/HC

217 219 209

±36 ±54 ±72

199 199 223

Mean arterial blood pressure (mmHg)

Control HP/HC Hypot.-HP/HC

74 75 73

± 3 ± 14 + 7

68 86 80

Hematocrit (%)

Control HP/HC HyOOt.-HP/HC

24 24 25

± 2 + 3 ± 2

7.45 ± 0.07 7.47 ± 0.07 7.46± 0.06

n.d. - 1 0 . 6 +12.4 - 9 . 3 + 3.3" ± 15 +54 +37

60

n.d. -16.1 ± 1 3 . 0 " -9.1 ± 4.0*

2.4 ± 3.8 -17.9 + 1 4 . 9 " -21.6 ± 8.5*

24.5 (h)

(h)

7.40 + 0.06 7.43 ± 0.04 7.48± 0.06

7.43 + 0.03 7.45 + 0.0a 7.50+ 0.06

-5.1 ± 4.2 - 3 . 0 ± 4.8 - 2 . 0 ± 2.9

- 2 . 6 ± 3.0 - 0 . 7 ± 4.8 - 0 . 7 ± 2.6

217 194 216

±45 +47 +37

223 189 157

+50 ±37 ±55 *

217 195 175

±48 ±58 ±53

227 225 195

± 44 ± 61 ± 78

72 84 79

+ 7 ±13 ± 6

73 78 28

± 10 ±12 ±10*

80 69 65

± 8 ± 7 +10

82 69 63

± 10 ± 6 ± 13

± 2

25 24 18

± 2 ± 3 ± I*

25 23 22

± 3 + 3* ± 3

n.d. 22 20

+ 3 ± 2#

± 3* + 16 ± 7

n.d. n.d. 25

n.d. n.d. n.d.

24 7.46 ± 0.07 6.86 ± 0.34 * 6.88± 0.13 *

hypotensive-hypoxia/hypercapnia. Mannitol blood-tobrain transfer constants were also not altered 24 h after hypoxia/hypercapnia. Mannitoi blood-to-brain transfer constants were not measured after hypotensive-hypoxia/hypercapnia. The 1251-albumin brain vascular space (2.5 + 0.5%), taken as an estimate of brain blood volume for the solution of Equation 1, was similar among all groups of piglets.

Table I11 summarizes the brain water and electrolyte contents in the newborn piglets. Differences in brain water and electrolyte contents were not observed among the groups of newborn piglets. Fig. 2 illustrates the changes in arterial oxygen content, mean arterial blood pressure, brain blood flow and oxygen delivery to the brain during hypoxia/hypercapnia and hypotension. Brain perfusion was in-

TABLE Ill

Brain water and electrolyte contents in the newborn piglets Hypoxia/hypercarbia group subjected to 60 rain of hypoxia and hypercarbia; hypotensive-hypoxia/hypercarbia group subjected to 60 min of hypoxia/hypercarbia with 20 rain of hypotension; mean + S.D.

Groups

Time

Acute studies Control Hypoxia/hypercarbia Hypotensive-hypoxia/hypercarbia 24 h after resuscitation from each insult Control Hypoxia/hypercarbia Hypotensive-hypoxia/hypercarbia

(rain) 60 60 100 (h) 24 24 24

Number

Brain water (mi / g dry wt. - / )

Sodium Potassium Chloride (mEq / kg dry wt - I)

Sodium/potassium

12 6 6

4.77+0.22 4.57+0.21 4.69 + 0.24

304+19 292+ 15 306 + 21

552+18 531 ± 25 556 + 14

225+20 217+ 13 226 + 17

0.55+0.02 0.55 +0.01 0.55 ± 0.03

7 7 7

4.69+0.32 4.93 + 0.37 4.72 + 0.24

285+29 305 + 40 288 + 33

531 +40 537 + 39 520 + 17

199+21 22 i + 24 215 + 19

0.54+0.02 0.57 + 0.08 0.56 + 0.08

268 toregulation for newborn piglets~TJS; and indicate a decrease in blood-to-brain transfer constants for sodium and mannitol immediately after hypotensivehypoxia/hypercapnia.

CaO~ (mL/dL)

Blood-to-brahl transfer constants in control newborn piglets. Few quantitative measurements of blood brain-

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MABP (turn Hg)

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TIME (Minutes)

Fig,, 2, Arterial oxyg,cn cuntcn I ((',,O..), mean arterial bhn~d pressure (MABP), brain blood ll()w (O) and oxygen tlclivcry ( D O . ) tu the brain in pig.let.~ durint~ hypt~xia/hypcrcarbia, hypt)t~:nsivchypt)xia/hypercarbia and recovt:ry from the,~e in.~ult.~, Recovery incasurcmcnt.~ at 7(I and I(ll) reprt:scnt n.:a,~urement,~ at Ihu bt:ginnint~ and end of thu blot)tl=brain barri~:r pcrm¢¢Lbilily ,~tudi~:,~ indic~m:d by IIBBP, tbr a grtmp of ,~imilarly ,~lu,,ied pi~lel,~, Mean ± S,I),: ° P < (1,1).~v,~, ba,~dine,

creased during hypoxia/hypercapnia, similar to baseline during hypotensive-hypoxia/hypercapnia and increased with a reactive hyperemia 10 rain after resuscitation. The pattern of oxygen delivery to the brain was similar. DISCUSSION This study investigated the effects of hypoxia/hypercapnia and hypotensive-hypoxia/hypercapnia on blood-brain barrier permeabilit~ to sodium and mannitol and brain water and electrolyte contents, with the aim of evaluating the role of changes in these variables in the pathogenesis of perinatal hypoxic-ischemic brain damage. Results discussed separately below: relate to the maturity of the blood-brain barrier in newborn piglets; indicate maintenance of blood-brain barrier integrity and resistance to the development of brain edema in newborn piglets exposed to systemic hypoxia/hypercapnia, even when accompanied by reductions in blood pressure below the lower limit of au-

barrier permeability have been made in newborn and young mammals 3"4J4'23"34. Mean values of K~ (mean + S.D., m i . g - I. min-~ × 10 4) for sodium and mannitol in control newborn piglets (15.9 + 2.8 and 5.2 + 1.6, resp., Fig. 1) are in the same range as those for adult rats (26.3 and 3.4, resp.) 5 suggesting that the bloodbrain barrier to these compounds is fully developed in the newborn piglet. This is in agreement with earlier work based upon steady-state plasma-to-brain ratios of similar test compounds ~'~4 suggesting that the barrier is mature at birth. Our quantitative blood-to-brain transfer constants in non-anesthetized, awake, newborn pigs are similar to recent work by others 23 in anesthetized, ventilated pigs, when corrected for units of time, i.e., minutes vs. seconds 23. in this study, brain vascular volume was evaluated with albumin to determine residual blood radioactivity (for Equation 1). Since albumin underestimates the blood volume for sodium and mannitol, our control permeability measurements are probably somewhat higher than if vascular volume had been determined with a smaller molecule "~'~.Nevertheless, because brain vascular volume was measured individually in each animal and identically in all study groups, this would not effect the outcome of the studies.

b~tegrity o1' the blood-bra#z barrier t,~ hypoxia / hypercapnk~ and hypotension. As outlined above (Introduction), maintenance of low blood-brain barrier permeability is energy dependent '7''~. Although the adult blood-brain barrier is very resistent to hypoxia ''''LL' permeability to ions appears to increase slightly in response to severe hypoxia z", whereas changes in permeability to larger compounds occur much later (18-24 h), and are thought to be secondary to tissue necrosis 13'31'43 and not a direct effect of hypoxia on the vascular endothelium. Moreover, injury to the barrier is thought to correlate with the severity of the insult La. Consideration of our results, plus those of a recent study by Mirro et al. 2a, suggests that the neonatal blood-brain barrier in the newbon~ piglet is also simila:" to the adult with respect to its resistance to hypoxia. Both studies used multiple, small-molecular weight tracers to assess barrier permeability. The advantage of this approach is that it permits evaluation of the mechanism of permeability changes TM. Increases in permeability due to the opening of water-filled channels, or 'pores', at the blood-brain barrier will be associated

269 with size-dependent changes in tracer influx into brain, whereas other mechanisms (e.g. pinocytosis) should be relatively independent of molecular size. While both studies used similar approaches to study barrier permeability, the severity of the hypoxic-ischemic insult was clearly greater in the study of Mirro et al. ~3, namely 20 min of global ischemia 23. The brain remained relatively well perfused in our study, despite the fact that hypoxia/hypercapnia renders brain blood flow pressure-passive ~7"2°'4°'4t and mean arterial pressure was reduced (Fig. 2) below the lower limit of autoregulation for normal newborn piglets~8; whereas, complete cessation of blood flow =9'23 and tissue anoxia can be assumed in the case of global ischemia =~'23. We saw no increase in permeability to either sodium or mannitol during or up to 24 h following hypoxia/hypercapnia and hypotension (Fig. 1), whereas there was a differential, size-dependent increase in influx of the 3 tracers used following global ischemia 23. Blood-to-brain transfer constants for the two smaller molecular weight tracers (sodium and urea) increased 30 min after ischemia, followed by a subsequent increase for the largest tracer (sucrose) at 2 h 23. These results, in neonatal piglets, are consistent with the effects of hypoxia-ischemia in adults; namely, formation of a paracellular leak across the blood-brain barrier with a pore diameter which increases with time and the severity of the initial insult j'a. There appear to be only 3 additional, earlier studies in perinatal subjects which evaluated barrier permeability in response to hypoxia-ischemia, two studies which showed an increase in permeability 2c~''~'~and one which did not 4'. Results are difficult to interpret, because of the experimental design. Asphyxia was associated with hypertension in all 3 studies 2°''a'h42, which in itself might cause barrier openingt'S; and furthermore, only one study 3x used a small molecular weight tracer to evaluate barrier function. Blood-to-brain transfer constants following resuscitation from hypotensive-hypoxia / hypercapnia. A proportional reduction of 30 and 45% in K~ for mannitol and sodium, respectively (Fig. 1), was observed immediately after resuscitation from hypotensive-hypoxia/hypercapnia. The rate constant for tracer influx across the blood-brain barrier, K t, is dependent both on the permeability-surface area product, PS, and on blood flow. When PS is much lower than blood flow, a condition that is satisfied for sodium and mannitol, transfer of tracer into the tissue is independent of tissue blood flow and K t is approximately equal to PSt°. Thus, the observed decreases in K~ for sodium and mannitol could be mediated by a decrease in capillary permeability or surface area. Given the nor-

mal impermeability of the blood-brain barrier, it seems highly unlikely that they could arise from a further decrease in permeability. Thus, we speculate that the proportional decreases in K x observed between 10 and 40 min following resuscitation from hypotensive-hypoxia/hypercapnia is due to a reduction in brain capillary surface area. Resistance to the det,elopment of brain edema. In the adult subject, hypoxia is thought to increase brain water by the production of cytotoxic or vasogenic edema TM. The former is characterized by an increase in intracellular water and sodium ~j, and the latter by increased permeability of the brain capillary and endothelial cells as a result of tissue necrosis ~=.=t,.Therefore, evaluation of brain edema in the newborn requires measurement of both barrier permeability and brain water. Some studies have measured permeability, as summarized above (Integrity of the blood-brain barrier to hypoxia/hypercapnia and hypotension) and others have measured brain water and electrolytes 7'24''~°'3"~'3.'a7, but there do not appear to be any studies prior to this one, which have quantitatively measured both in the same study. The lack of brain edema in our piglets exposed to hypoxia/hypercapnia and hypotension is consistent with our findit,g of resistance of the blood-brain barrier to these insults. In summary, blood-brain barrier integrit; is maintained and brain edema does not develop in the brain of the newborn piglet exposed to severe systemic hypoxia/hypercapnia and hypotension.

Acknowledgements. We acknowledge the skillful technical assistance of M. DePasquale, and Donna L.A. Piva. Supported by the American Heart Association, Rhode island Affiliate, Inc. REFERENCES 1 Anderson, J,M. and Belton, N.R., Water and electrolyte abnormalities in the human brain after severe intrapartum asphyxi;i, J.

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hypercapnia and hypotension in newborn piglets.

This study examines the effects of hypoxia/hypercapnia and hypoxia/hypercapnia with hypotension (hypotensive-hypoxia/hypercapnia) on blood-to-brain tr...
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