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Regional cerebral blood flow response to acute hypoxia changes with postnatal age in the rat
Developmental Brain Research, 76 (1993) 197-205 © 1993 Elsevier Science Publishers B.V. All rights reserved 0165-3806/93/$06.00
197
BRESD 51716
R e s e a r c h Report
Regional cerebral blood flow response to acute hypoxia changes with postnatal age in the rat A n n e Bilger and Astrid Nehlig * INSERM U272, Universitd de Nancy I, 30 rue Lionnois, B.P. 3069, 54013 Nancy Cedex, France (Accepted 6 July 1993)
Key words: Cerebral blood flow; [14C]lodoantipyrine; Autoradiography; Hypoxia; Postnatal development
The quantitative autoradiographic [14C]iodoantipyrine technique was applied to measure the effects of an acute hypoxic exposure on rates of local cerebral blood flow (LCBF) in the 10 (P10)-, 14 (P14)- and 21 (P21)-day-old rat. The animals were exposed to hypoxic (7% 0 2/93% N 2) or control gas mixtures (21% 0 2/79% N 2) for 40 min before the initiation of the l-min LCBF measurement. At P10, hypoxia induced a 142-415% increase in LCBF over control levels, which affected the 45 structures studied. The highest increases in LCBF were noticed in posterior midbrain and brainstem regions. These increases are in good accordance with hypoxia-induced increases in LCBF recorded during acute hypoxia exposure in both newborn and adult animals. At P14 and P21, rates of LCBF decreased with hypoxia. These decreases were significant in 23 and 21 brain regions, respectively, belonging to all systems studied. These changes in LCBF are in quite good correlation with our previous data on the effects of acute hypoxia exposure on cerebral glucose utilization but the decrease in LCBF is of higher amplitude than the one in cerebral glucose utilization translating into a relative hypoperfusion at a constant metabolic level at P14 and P21. However. arterial blood pressure was reduced by 16 mmHg and arterial pCO 2 was significantly decreased at the two latter ages in hypoxic animals compared to controls. These two systemic factors, and mainly hypocapnia, are rather responsible for the cerebral hypoperfusion recorded at PI4 and P21 in hypoxic rats whereas the circulatory response seems to be predominantly hypoxic at P10.
INTRODUCTION
Acute perinatal hypoxic-ischemic cerebral injury in the term newborn is a major cause of long-term neurologic abnormalities in childhood 22'56. The extent of hypoxic-ischemic cerebral injury is determined principally by the maturity of the brain at the time of insult and the severity and duration of the insult 22. Indeed, in a previous study, we have shown that the cerebral metabolic response to acute, severe hypoxia varies as a function of the age of the animal 5. In 10-day-old rats, which cerebral maturity is close to the one of a human term newborn t,43, acute hypoxia induces a general increase in glucose utilization in all brain areas studied. At 14 and 21 days, the metabolic response to hypoxia varies according to the region and shows regional increases, decreases or no change in the rates of cerebral glucose utilization compared to control levels. These data are in good accordance with the better resistance of the immature brain to anoxia/asphyxia11't3'23,25 and show the relative vulnerability of some brain areas to
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that kind of insults. Indeed, thalamus, brainstem and auditory structures, which have high metabolic demands, appear to have increased susceptibility to injury 5'1°'42. However, the precise relationship between regional metabolic factors and hypoxic neuronal injury has not yet been defined. The immediate circulatory response to hypoxia or asphyxia in the newborn involves redistribution of systemic blood flow with increased perfusion of more vital organs, e.g. brain, heart, adrenal glands and corresponding decreased perfusion of lungs, kidneys and gastrointestinal tract. However, when the hypoxicischemic insult lasts, this homeostatic hemodynamic mechanism fails, leading to systemic hypotension and impaired cerebral autoregulation. Thus, systemic hypotension may result in decreased cerebral blood flow and consequently cause ischemic cerebral injury 22.56 Therefore, in the present study, we examined the effects of an acute hypoxic exposure on rates of local cerebral blood flow (LCBF) measured by the quantitative autoradiographic [14C]iodoantipyrine technique in
198 rats aged 10 to 21 days after birth. The hypoxic exposure was performed in the same conditions as in our previous study of the effects of acute hypoxia on cerebral glucose utilization in immature rats 5. MATERIALS
AND METHODS
Animals Adult S p r a g u e - D a w l e y rats (Iffa-Credo Breeding Laboratories, l'Arbresle, France) were housed together in mating groups of one male and two females and constantly maintained u n d e r standard laboratory conditions on a 12/12 h l i g h t / d a r k cycle (lights on at 06.00 h). After delivery, litter sizes were reduced to 10 pups for homogeneity. The experiments were performed on a total n u m b e r of 26 rats, 14 hypoxic and 12 control animals at 10 (P10), 14 (P14), and 21 days after birth (P21). Rats from 3 or 4 different litters were used in each age group. All animal experimentation was conducted in conformity with the 'Guiding Principles for Research Involving Animals and H u m a n Beings'. A femoral artery and vein were catheterized with polyethylene catheters (Clay-Adams PE-10, i.d. of 0.28 mm, o.d. of 0.61 m m ) under light halothane anesthesia. Both catheters were threaded under the skin, up to the neck through a small opening in the skin. A loop was made with the end of the catheters, which were put back under the skin, and the small opening in the skin was sewn. The animals were returned back to their m o t h e r in their normal environment. T h e experiments were performed on the following day. Only those animals that did not suffer from surgery and did not lose weight (suckling rats which were sometimes rejected by the mother) were used for the study.
Hypoxic conditions O n the day of the experiment, the threads on the skin in the neck were cut and the catheters were pulled out. The rats were transferred to a rectangular plastic box with 6-1 capacity. Hypoxic conditions were obtained by circulation of an O 2 / N 2 ( 7 % / 9 3 % ) gas mixture. Control rats were exposed to the mixture O 2 / N 2 ( 2 1 % / 79%). The circulation of the gas inside the box was calculated to avoid any overpressure. Two gas entries placed on the side of the box were connected to distribution ramps in order to assure a homogenous distribution of the gas mixture inside the box. A central lengthwise split on the cover of the box allowed the release of excess gas and easy access to the catheters of the freely moving rats. The temperature inside the box was adjusted in order to maintain body temperature of Suckling rats in the normal physiological range. The experiments were performed 40 min after the onset of exposure to the gas mixture and the animals were constantly maintained in the same gas environment throughout the whole experimental period.
Measurement of local cerebral blood flow LCBF rates were measured by m e a n s of the [I4C]iodoantipyrine (lAP) method described by Sakurada et al. 46 and adapted to the immature rat by Nehlig et al. 36. The 4-iodo-N-methyl-[14C]-iodoan tipyrine (spec. act. 1.85-2.2 G B q / m m o l ; A m e r s h a m , Little Chalfont, Buckinghamshire, UK) was injected into the animals through the femoral vein at a concentration of 925 B q / m l . T h e period of measurement of LCBF was approximately 1 rain in duration, during which variable a m o u n t s of the [InC]IAP solution were administred to the rats. The volumes injected ranged from 130 to 270/xl according to the age studied. The intravenous infusion was conducted at a progressively increasing rate to produce a rising arterial concentration of the tracer approximating a ramp input function. This ramp input function serves to delay or to prevent the equilibration of rapidly perfused tissues with the arterial blood during the period of measurement. Throughout the period of 114C]IAP administration, timed arterial blood samples, freely flowing from the arterial catheter, were collected in glass capillary tubes. The last sample was taken at the time of killing and as long as blood could be withdrawn from the arterial catheter. The volume of blood withdrawn at each age was
approximately equal to the w)lume ol lAP solution itljccted, m ordc~ to avoid a normotensive shock and any change in physiological variables. The rats were killed by decapitation at tJbout 1 rain after the beginning of [x4C]IAP infusion and brains were removed within i rain, frozen in methylbutane chilled to - 4 0 ° C , coated with chilled embedding matrix (carboxymethylcellulose 4~,: in water~. ~md stored at - 8 0 ° C in plastic bags until sectioned. The content of each capillary tube was transferred to a preweighed scintillation vial that was immediately covered and reweighed aftel blood collection. The blood samples were then treated with 0.5 ml of tissue solubilizer (Optisolv, FSA Laboratory Supplies) and 0.5 ml oi hydrogen peroxide (30%). Blood concentration of [14C]IAP was then determined by liquid scintillation counting in 1{) ml scintillation mixture (Optiphase Hisafe, FSA Laboratory Supplies) in a Beckman scintillation counter (model LS 18(ll. Beckman Instruments, Fullerton, USA). The concentration of tracer per unit w)lume of blood in each sample was calculated from the measured amount of t4C, the weight of the blood sample, and an assumed specific gravity of 1.06 g / m l for blood. The frozen brains were cut into 20-/xm coronal sections at - 2 2 ° C in a cryostat. Sections were picked up on glass coverslips and dried on a hot plate (60°C). Sections were autoradiographed on Kodak SB5 film along with calibrated [14C]methylmethacrylate standards (Amersham) using a special set of low t4C concentration standards for animals 10 and 14 days old. All standards were calibrated for their ~4C concentration in brain sections, as previously described 4°. Adjacent sections were fixed and stained with thionin for histological identification of specific nuclei. The autoradiographs were analyzed by quantitative densitometry with a computerized image-processing system (Biocom 200, France) or a manual microdensitometer (Macbeth, T D 901, Kollmorgen Co., Newburg, USA). Optical density m e a s u r e m e n t s for each structure anatomically defined according to the developing rat brain atlas of Sherwood and Timiras 4s were made bilaterally in a m i n i m u m of four brain sections. Tissue ~4C concentrations were determined from the optical densities of the autoradiographic representations of the tissues and a calibration curve obtained from the autoradiographs of the calibrated standards.
Calculation of local cerebral blood flow Throughout the period of m e a s u r e m e n t of LCBF, blood samples were collected at the distal end of an arterial catheter to determine the continuously changing [14C]IAP concentration in the arterial blood at the proximal end of the catheter. The short duration of the experiment requires extremely precise timing of the samples. Moreover, the length of the arterial catheter was quite high compared to the size of the animal. Therefore, two types of arterial sampling distorsion were taken into account. The first one was related to the time necessary for the blood from the proximal end to reach the distal end of the arterial catheter. The other one was due to a dilution of the true arterial concentration of the tracer at the proximal end of the catheter by the dead space inside the catheter, i.e. the so-called washout effect. The correction for both the time lag and the washout effect was taken into account for final calculations of LCBF, as previously described 36. Finally, LCBF values were calculated according to the Fick equation using a brain-blood partition coefficient of 0 . 8 4 6 .
Physiological variables Just prior to gas exposure and prior to infusion of [14C]IAP, m e a n blood pressure of the animals was measured with an air-damped mercury m a n o m e t e r and the hematocrit value was determined on 5 /zl samples. Arterial pH, p O 2, and p C O 2 were measured on 40 ,~1 blood samples by m e a n s of a blood gas analyzer (Coming, Model 158, Le Vesinet, France) just before gas exposure and before the onset of LCBF procedure.
Statistical analysis LCBF values were determined in 45 cerebral structures in 3 age groups of control, and 3 similar age groups of hypoxie rats. LCBF values underwent three statistical analyses. First, a two-way analysis
199
TABLE I Effects qf hypoxia on physiological variables in det,eloping rats Values are m e a n s + S . E . M , the number of animals in parentheses. " P < I).115, b p <0.01, statistically significant differences from control. * P < 0.05, * * P < 0.01, statistically significant differences from one developmental age to the preceding one. ° P < 11.115, °° P
Arterial blood presure (mmHg) Before gas exposure 40 rain after gas exposure Hematocrit ( ~ ) Arterial pH Before gas exposure 40 rain after gas exposure Arterial p O 2 (mmHg) Before gas exposure 40 min after gas exposure Arterial p C O 2 (mmHg) Before gas exposure 40 min after gas exposure
C Hyp C Hyp C Hyp C Hyp C Hyp
PIO (n = 4)
PI4 (n = 4)
P21 (n 5)
43.7 46.0 41.3 38.6 29.3 29.6
51.5 52.2 53.11 42.8 26.8 28.//
79.5 75.3 75.8 59.4 29.5 31.3
_+2.1 _+1.3 ±2.6 _+1,2 °° + 1.9 +1.0
7.41 +0.02 7.39 _+0.02 7.40 _+/I.01 7.45 ±/I.03
_+1.8" ±1.5" +2.11" _+2.8 b°° + 1.4 +2.7
7.41 +0.02 7.40 _+/I.I/1 7.39 _+11.111 7.41 ± 11.112
±1.7 ** +1.7 ** +3.3"* _+2.2 b**°° + 1.1 +0.6
7.40+0.01 7.42 _+I1.111 7.41 + 1/.111 7.46 + 11.112
C Hyp C Hyp
97.8 87.9 95.7 33.6
±2.5 + 3.3 ~' ±3.9 ±4.8 boo
88.0 92.3 93.9 34.8
+4.1 ± 1.5 +3.0 ±2.3 bo
73.5 81/.3 73.0 32.5
±3.3 * + 1.8 ** ±2.3"* + 2.3 boo
C Hyp C Hyp
29.6 29.9 30.4 28.1
+0.9 _+ 1.0 L0.8 _+1.5
29.2 32.2 34.2 25.2
_+3.0 ±2.5 ±2.5 +_2.0 ~'
35.6 36.6 36.2 22.1
_+ 1.6 + 1.3 +2.3 +1.8 b°°
TABLE II Effects of hypoxia on local cerebral blood flow in sensory systems of deceloping rats Values, expressed as ml/100 g/rain, are means_+ S.E.M. of the number of animal in parentheses. ~' P < I).05, b P < 0.01, statistically significant differences from control. * P < 0.05, ** P < 0.01, statistically significant differences from one developmental age to the preceding one. Brain structure Auditory system Auditory cortex Medial geniculate body Inferiorcolliculus Superior olive Cochlear nucleus Visual system Visualcortex Lateral geniculate body Superior colliculus Olfactory system Olfactory cortex Somatic system Frontoparietal cortex somatosensory area Vestibular system Vestibular nucleus
PlO (n = 4)
P14 (n = 4)
P21 (n = 5)
58.1± 9.2 ** 20.7+ 6.0 b 63.9± 9.7 ** 34.5± 6.2 ~' 53.0± 6.8 ** 29.6_+ 6.8 ~** 51.7± 8.3 * 35.4_+ 9.4 ** 60.6_+ 11.4 * 36.3+12.4"
80.7+ 7.1 45.9+ 10.1 ~' 911.7+ 5.7 * 54.0+ 10.6 " 911.2+ 4.4 ** 56.7_+ 9.2 ~' 93.6+ 1.6 ** 67.9± 9.7 1113.3+ 6.3 ** 86.6+11.6"
C Hyp C Hyp C Hyp C Hyp C Hyp
10.7± 37.6_+ 15.1 _+ 58.11± 16.8± 74.9_+ 22.9± 98.2_+ 24.3_+ 97.3+
1.3 3.7 2.2 9.3 1.6 7.6 2.5 7.8 3.2 7.5
C Hyp C Hyp C Hyp
11.7-+ 60.0+ 15.1 _+ 54.8+ 12.0± 57.7+
1.1 4.0 b 1.9 8.8 b 0.9 4.8 b
C Hyp
22.7-+ 2.5 73.1-+ 4.6 b
57.2+ 2 . 9 " * 33.4+ 3.4 b**
81.5+ 8 . 4 * * 49.4± 3 . 3 * *
C Hyp
16.0+ 2.0 53.6+ 8.0b
81.5±13.9"* 19.9± 6.8b
86.1+ 4.9 51.3+10.6a
C Hyp
28.1_+ 2.9 95.2+_12.0 b
83.5_+ 8.1 ** 49.7+ 4.6 b*
99.4_+_ 5.3 83.8-+ 9.9
b
b b b
43.3_+ 22.9+ 50.9_+ 32.0+ 42.7_+ 24.2+
5.9"* 3.2 b** 9.5 ** 7.4 8.7 ** 4.3 *
76.6± 43.3_+ 81.4_+ 50.7 + 56.8_+ 38.4_+
4.2 ** 8.11 ~'* 2.1 ** 9.7 " 2.9 9.2
20O TABLE 111 Effects of hypoxia on local cerebral blood flow in limbic and tunctionally non-specific areas of det,eloping rats Values, expressed as ml/100 g / m i n , are m e a n s ± S.E.M. of the number of animals in parentheses. ~ P < 0.05, ~' P < 0.01, statiscally significant differences from control. * P < 0.05. ** P < 0.01, statistically significant differences from one developmental age to the preceding one. Brain structure Nucleus accumbens Medial septum Medial amygdala Dorsal hippocampus, CA3 area Ventral hippocampus, C A 3 a r e a Mediodorsal thalamus Medial habenula Lateral habenula Medial raphe Locus coeruleus
C Hyp C Hyp C Hyp C Hyp C Hyp C Hyp C Hyp C Hyp C Hyp C Hyp
PIO (n = 4)
P14 (n = 4)
P21 (n ~ 5)
14.0± 1.9 50.1+ 6.2 b 14.5_+ 2.0 54.2± 6.1 b 13.4± 1.3 41.4_+ 4.0 I' 13.2_+ 1.9 39.4-+ 1.5 b 9.5_+ 0.9 33.0± 6.2 ~' 15.6 ± 2.5 53.6+ 7.2 b 19.5_+ 2.9 59.0_+ 5.5 b 19.5± 3.3 62.2+ 5.5 b 18.1_+ 2.0 78.3_+ 4.9 b 18.6+- 1.8 84.9+11.5h
33.3_+ 4.8 * 32.2_+ 2.7* 36.9± 1.3 ** 30.1± 6.3 * 54.7± 9.7 ** 25.8_+ 6.3 a 50.4_+ 8.8 ** 12.5± 3.9 b** 36.3_+ 7.7 ** 21.0± 3.9 89.3 _+ 15.9 * * 35.9± 8.3 ~ 66.3-+ 12.3 ** 14.8+_ 7.0 b** 70.8_+ 11.5 ** 13.3± 4.1 b** 50.9± 2 . 9 " * 24.0± 9.7 a** 51.9_+ 9.6 ** 28,5_+ 8.2 **
76.5_+ 52.4_+_ 54.7± 42.2_+ 47.4_+ 39.1± 58.6_+
of variance was performed. Thereafter, LCBF in each group of rats were compared with those in the immediate preceding age by means of Bonferroni's multiple comparison procedures 26. Moreover, at each age, values of LCBF in the hypoxic rats were compared with those in the control animals by means of a non-paired Student's t-test. To compare physiological values before gas exposure and before sacrifice, a paired Student's t-test was used.
6.9 ** 3.7 b* 3.8 ** 3.2 ~ 2.3 4.2 4.5 50,8± 4.5 ** 52.5± 2.8 36.6± 6.3 89.2 ± 3.8 55.0_+11.4 ~ 77.4+_ 5.9 58.5_+ 3.3 a** 86.4± 8.1 63.9± 3.2a** 69.4± 6.1" 66.8± 6.5 ** 67.4± 4.4 67.1+_ 8 . 4 "
RESULTS
Physiological values Arterial blood pressure significantly increased between P10 and P14 and between P14 and P21 in both
TABLE IV Effects of hypoxia on local cerebral blood flow in motor areas of developing rats" Values, expressed as ml/100 g / m i n , are means :t: S.E.M. of the number of animals in parentheses, a p < 0.05, b p < 0.01, statistically significant differences from control. * P < 0.05, ** P < 0.01, statistically significant differences from one developmental age to the preceding one. Brain structure Frontoparietal cortex motor area Dorsomedial caudate nucleus Globus pallidus Substantia nigra, pars reticulata Substantia nigra, pars compacta Ventrolateral thalamus Red nucleus Cerebellar cortex Cerebellar nuclei Dentate nucleus Fastigial nucleus Interpositus nucleus
PIO (n = 4)
P14 (n = 4)
C Hyp C Hyp C Hyp C Hyp C Hyp C Hyp C Hyp C Hyp
17.8 + 1.6 66.5± 9.0 b 13.4 _+ 2.l 32.5± 3.1 b l l . 6 + 1.3 33.0 ± 5.9 h 15.3± 1.7 44.9± 3.8 b 16.3 ± 1.5 53.8_+ 3.6 b 15.9 ± 1.3 72.3_+ 11.7 b 20.8_+ 1.7 58.5 ± 4.0 b 11.5± 1.1 54.3± 5.9 b
54.1 _+ 7.0 31.2± 6.4 34.5 ± 4.7 25.5+ 9.5 32.7± 7.9 20.3 ± 4.5 46.1 ± 7.7 13.9± 5.6 51.2 ± 8.8 29.3± 8.6 62.8 ± 9.1 20.7± 4.8 65.3 ± 10.6 40.1 + 10.0 26.6± 2.8 27.8± 7.5
C Hyp C Hyp C Hyp
19.4 ± 1.4 63.3_+ 10.0 b 21.5 ± 1.9 75.7+ 6.9 b 21.4 4- 2.1 59.8± 8.5 b
64.5 ± 14.9 30.8± 8.6 79.8 ± 10.8 41.0±12.8 77.6 ± 10.0 38.3_+ 9.2
P21 (n = 5) ** **
** b** ** * b** **
* ** * ** * **
80.8_+ 0.7 47.8± .5.8 61.7 ± 3.2 46.8± 5.0 47.4+ 2.5 34.2 ± 4.9 63.1 + 3.2 55.4± 3.8 65.1 ± 4.8 61.2± 4.9 89.8 ± 5.4 55.2+ 10.2 71.3 ± 3.9 69.3± 4.8 67.3± 10.3 44.7± 5.3 96.0 ± 67.6± 92.5 ± 82.9± 92.9 ± 68.2±
5.0 5.2 4.2 11.9 4.5 9.9
**
b **
* ** ** * a, * **
* b, * a
201 TABLE V
Effects of hypoxia on local cerebral blood flow in hypothalamus and white matter areas of developing rats Values, expressed as m l / 1 0 0 g / r a i n , are means +_S.E.M. of the number of animals in parentheses, a p < 0.05, b p < 0.01, statistically significant differences from control. * P < 0.05, ** P < 0.01, statistically significant differences from on developmental age to the preceding one.
Brain structure Hypothalamus Supraoptic nucleus Anterior hypothalamus Paraventricular nucleus V e n t r o m e d i a n hypothalamus Mammillary body
White matter areas Genu of the corpus callosum Anterior commissure Cerebellar white matter
PI O (n = 4)
PI 4 (n = 4)
P21 (n = 5)
C Hyp C Hyp C Hyp C Hyp C Hyp
19.3 i 2.3 57.8+-4.8 ~ 14.5 +_ 1.8 45.0_+4.0 b 15.8 + 2.0 46.3_+1.3b 14.8+_ 1.5 50.5+-3.6 b 19.9+_3.9 72.6+-6.1 b
64.1 _+8.7 * * 38.3+_9.3 51.7 + 7.5 * * 37.3+_2.0 45.8 +_5.8 * * 8.0+_2.8~** 42.7_+9.3 * 21.8+-7.1 ** 55.4±9.4 ** 18.4+-1.8 ~**
52.6 ± 3.(/ 45.8+_ 5.1 54.7 +_ 2.2 41.9+_ 4.6 ~ 53.4 +_ 7.4 46.1+_ 3 . 8 " * 59.1 + 3.5 39.1 + 4.2 ~' 87.6_+ 1.2 ** 54.8+- 10.4 ~*
C Hyp C Hyp C Hyp
9.0 +_2.6 28.2+_5.1 b 9.3 +- 1.2 24.4+-1.7 b 7.0+- 1.0 24.0+_2.5 b
28.5 +_ 1.6 20.7_+3.4 33.2 +- 3.3 15.8_+3.5 29.3 +-5.6 23.9+_5.2
48.4 +_ 24.2_+ 42.2 _+ 48.5_+ 49.0+41.4+_
groups of animals, except between P10 and PI4 in hypoxic animals (Table I). It decreased significantly at the end of hypoxic gas exposure as compared to preexposure in hypoxic rats at the 3 ages studied. It was also significantly lower at P14 and P21 in hypoxic animals as compared to controls 40 min after the onset of gas exposure. Hematocrit value and arterial pH were the same in both hypoxic and control animals at the three ages studied. Between P14 and P21, p O 2 significantly decreased in the control group at both times and also in the hypoxic group before any gas exposure. At all ages studied, p O 2 was decreased in hypoxic animals at the end of gas exposure compared with preexposure,
* ** b **
8.5 3.2 1.0 8.1 6.0 3.0
* b * ** * *
and before sacrifice compared with control groups. At preexposure, p O 2 was significantly lower at P21 in hypoxic compared to control animals, p C O 2 was significantly lower at P14 and P21 just prior sacrifice in hypoxic than in control animal. Moreover, at P21, p C O 2 significantly decreased between the beginning and the end of hypoxic gas exposure.
Local cerebral blood flow rates Two-way analysis of variance. The data of the two-way analysis of variance which are not in the Tables show that there was an age effect in 40 structures out of the 45 studied with 10 -5 < P < 0.02. The age effect was not
T A B L E V1
Effects of hypoxia on local cerebral blood flow m respiratory areas of developing rats Values, expressed as m l / 1 0 0 g / m i n , are means+_S.E.M, of the numbe r of animals in parentheses, a P < 0.05, b p < 0.01, statistically significant differences from control. * P < 0.05, * * P < 0.01, statistically significant differences from one developmental age to the preceding one.
Brain structure Gigantocellularis nucleus Parvocellularis nucleus Ambiguus nucleus Parabrachial nucleus Nucleus of the solitary tract Whole brain weighted average
C Hyp C Hyp C Hyp C Hyp C Hyp C Hyp
PIO (n = 4)
P14 (n = 4)
P21 (n = 5)
21.7 +_2.7 79.1 +_2.4 b 19.1 +_2.3 71.9+_7.8 b 20.7 +_0.9 71.6+_8.1 b 17.4 +_2.4 92.8_+5.3 b 16.8 +- 1.6 59.0+_6.9 b 16.5 :t: 2.7 58.8+-3.8 b
67.9 +- 8.9 * * 29.9+_ 3.9 b** 58.7 ± 7.7 * * 28.3+_ 5.1b* 53.7 +_ 11.0 * 30.1+_ 5.3 ** 56.3 _+ 7.5 ** 26.4+- 8.4 a** 54.3 +- 8.5 * * 25.5+_ 3.0 b** 53.8 4-_ 4.2 * * 27.1+_ 3.3 b**
60.5 +- 6.9 55.4+_ 5 . 8 " * 65.1 ± 8.5 57.4 ± 11.7 79.6+ 6.1 59.9 +_ 9.0 59.8 _+ 8.3 69.4+_ 12.3 * 57.3 ± 7.6 67.0+_ 9.9 * 71.1+_ 4.4 * 54.1+_ 4.0 b**
202 significant in superior colliculus (Table II), supraoptic nucleus (Table III), ventromedian hypothalamus (Table V), gigantocellularis and parvocellularis nuclei (Table VI). The effect of the gas exposure was significant in 20 brain regions with 10 5< p < 0.05. These were auditory and frontoparietal cortex, inferior and superior collicuti, cochlear nucleus (Table II), dorsal hippocampus, mediodorsal thalamus, medial and lateral habenula, medial raphe and locus coeruleus (Table III), substantia nigra pars reticulata and compacta, ventrolateral thalamus, red nucleus and the 3 cerebellar nuclei (Table IV), as well as gigantocellularis and parabrachial nuclei (Table VI). The interaction between age and treatment was significant in all brain areas with 10 -5 < P < 0.015.
Variations with postnatal development (Bonferroni's ttest). In control rats, LCBF significantly increased between P10 and P14 in all areas studied, except in cerebellar cortex (Table IV). Between P14 and P21, LCBF significantly increased in 20 areas. These were 7 sensory regions (Table II), 3 limbic areas, accumbens nucleus, medial septum, medial raphe (Table III), 6 motors regions (Table IV), one hypothalamic area, mammillary body and the 3 white matter areas studied (Table V). There was no change in LCBF in respiratory areas between P14 and P21 (Table VI). In hypoxia-exposed animals, LCBF significantly decreased between P10 and P14 in 29 areas. Those decreases affected all systems studied (Tables II-VI). Between P14 and P21, LCBF significantly increased in 23 brain areas. Among those, 20 already decreased between P10 and P14 (Tables II-VI). Effect of hypoxia (Student's t-test). At P10, hypoxia induced a significant increase in LCBF ranging from 142 to 415%. This increase affected all the 45 structures studied. Within the areas (21)with higest LCBF rises ( > 250%) were all structures from auditory and visual systems (Table II), accumbens nucleus, medial septum, medial raphe, locus coeruleus (Table III), motor cortex, ventrolateral thalamus, cerebellar cortex, fastigial nucleus (Table IV), mammillary body (Table V) and all respiratory areas, except ambiguus nucleus (Table VI). At P14, hypoxia induced a general decrease in LCBF rates, ranging from 23 to 82% compared to control levels. However, because of a great interindividual variability, this decrease was significant only in 23 areas of the 45 studied but affected all systems. Among the areas significantly decreased by hypoxia at P14 were 6 sensory regions (Table II), 6 limbic areas (Table III), 2 motor structures, substantia nigra pars reticulata and ventrolateral thalamus (Table IV), paraventricular nucleus, mammillary body and anterior commissure (Ta-
ble V) and all respiratory regions, except ambiguus nucleus (Table VI). At P21, hypoxia induced a general decrease in LCBF rates, but, as at P14, it was only significant in 21 areas. This decrease in LCBF concerned the sensory areas already affected at P14, except vestibular nucleus and plus lateral geniculate nucleus (Table II). Hypoxia decreased LCBF rates in 5 limbic areas (Table Ill), 3 motor regions, the ventrolateral thalamus and 2 cerebellar nuclei (Table IV) and in 3 hypothalamic and one white matter area (Table V). DISCUSSION The results of the present study show that acute hypoxia induces cerebral blood flow changes which differ according to the age of the animals. Indeed, in the very immature rat, at P10, LCBF is widely and markedly increased by hypoxia, whereas at P14 and P21, LCBF is mostly decreased by the insult. These data are in quite good accordance with our previous results on the effects of acute hypoxia on local cerebral glucose utilization in rats at the same age 5. Until now, data on the effects of hypoxia on cerebral blood flow in immature animals have been mainly focused on newborns of different species, such as lambs 12'27'39'44, piglets 15'51, and dogs 6'2°'54. Data in newborns have been compared to results either from fetal 39 or adult animals 27. However, there is no study available on the effects of hypoxia on LCBF in rats or any other species at different postnatal ages. In the 10-day-old rat, which cerebral maturity corresponds to the human newborn 1'43, 40 min of acute hypoxia (7% 0 2 / 9 3 % N 2) induce a widespread and general increase in the rates of LCBF, as previously shown in newborn animals of different species 6'12'14'27'34'39'51. These increases in the rates of LCBF are quite high (142-415%) compared to animals of the same age breathing room air. Moreover, highest increases in LCBF are noticed in midbrain and brainstem relay nuclei of auditory and visual systems as well as in many other posterior regions (Tables II-VI). These results are in good accordance with previous data showing that the most prominent increases in LCBF in newborn animals during hypoxia are located in brainstem areas 2"6d4'15'27"29'5°'51. Only a few authors showed homogeneous responses of brain regions blood flow tO hypoxia 9'34. The often noticed redistribution of cerebral blood flow in favor of posterior regions is considered as a protective mechanism to autonomic functions e. Moreover, the preferential perfusion of the brainstem control centers for respiration and vasomotor tone might be a contributing cause of the greater
203 tolerance to asphyxia in immature animals compared to older ones 6~55. Conversely, in 14- and 21-day-old rats, LCBF is decreased by hypoxia in many areas as compared to control levels. These decreases are significant mainly in sensory and limbic areas. At P14, LCBF is also depressed in respiratory regions, whereas this is no longer true at P21. These results are in apparent contradiction with most data showing an increased LCBF in response to hypoxia in both newborn 2'6'9'12'14'27'34'39'51 and adult animals 4'z4'32'37. However, they are in quite good accordance with our previous data of the effects of acute hypoxia on the levels of energy metabolism in the rat at 14 and 21 days after birth, in which glucose utilization is reduced, but to a lesser extent than LCBF, by the hypoxic exposure compared to control levels 5. In both immature and adult animals, most studies on the effects of hypoxia on cerebral blood flow have been performed within a few minutes after the onset of hypoxia. At those early times, circulatory adjustements during hypoxia act to redistribute blood flow and maintain arterial pressure. Locally, hypoxia produces dilatation in coronary and cerebral vessels and vasoconstriction in peripheral vessels ~9. However, when hypoxia lasts, this homeostatic hemodynamic mechanism fails, leading to systemic hypotension and impaired cerebral autoregulation in the newborn, which in turn leads to ischemic cerebral injury 19"22'56. In the present study, at 40 rain after the onset of hypoxia, i.e. at the time of LCBF measurement, arterial blood pressure is reduced compared to the preexposure value (Table I). However, hypoxia-induced hypotension is of the same order of magnitude at the 3 postnatal ages studied (16-21% reduction from control values). Autoregulation of CBF has been shown to exist in newborns of several species, dogs 2°, piglets 3°'31 and lambs 38, but no data are available in the immature rat. It is likely that autoregulation of CBF exists in rats, already at P10, but there is no information regarding the limits of the autoregulation plateau as a function of postnatal age. From studies on immature animals from other species, it is known that the autoregulation plateau is effective over a narrower range relative to adults 2°'3°'3k38. In the absence of precise data, it is difficult to clearly ascertain whether the slight hypotension recorded in the present study may or not interfere with the lower limit of the autoregulation plateau and induce a decrease in CBF. Moreover, it is not certain that a 7.4-mm change in arterial blood pressure at P10 is equivalent to a 16-mm drop in arterial blood pressure at P21 (Table I). Therefore, hypotension consecutive to hypoxia in the present study may contribute, at least partly, to the decrease in LCBF recorded at P14 and P21. A better knowledge of
the limits of the autoregulation plateau would, however, be necessary to draw a clear conclusion on this aspect of CBF regulation in the immature rat. The other main difference in the effects of hypoxia on physiological variables in 10-, 14- and 21-day-old rats is the hypocapnia recorded at P14 and P21. The immature cerebral circulation has been shown to be responsive to changes in pCO2, in both dogs 16'17'21'47, monkeys 41, sheeps 45, pigs ts and rats 3. Therefore, the decrease in p C O 2 recorded at P14 and P21 may blunt the response of LCBF to hypoxia and be predominant at the time of measurement of LCBF, inducing a decrease in LCBF, as previously shown in both newborns and adults of several species 3'4°'41'45'49. Therefore, it is possible that, in the present study, the primary response in the first minutes following the onset of hypoxia may be an increase in LCBF at all ages, as recorded in adult and newborns animals of several species 6'12'14'27'34'39'51,followed by a secondary decrease in LCBF consecutive to hypoxia-induced hypocapnia. Thus, in the present study, at the time of measurement, i.e. at 40 min after the onset of hypoxia, it is likely that the response of LCBF is purely hypoxic at P10, translating into a general increase, whereas it may be predominantly hypocapnic at P14 and P21, inducing a decrease in LCBF. A temporal evolution of the same kind has been recorded for the effects of hypoxia on cerebral glucose utilization in rats z8'33"52'53 and explained in detail in our previous paper on the effects of hypoxic exposure on cerebral glucose utilization in immature rats 5. Finally, hypoxia-induced LCBF changes may also be a consequence of metabolic changes, as hypothesized by others 6. In our previous study on the effects of acute hypoxia on local cerebral glucose utilization, we have shown that the metabolic response to the hypoxic insult changes with age, mainly as a result of the maturation of brain energy metabolism and of the extent of cerebral metabolic needs 5. The cerebral metabolic level of the 10-day-old rat is quite low and at that age, the animal is able to cover its metabolic needs in hypoxia by increasing the rate of anaerobic degradation of glucose without building up very high concentrations of cerebral lactate. Indeed, in the immature brain, the monocarboxylic acid carrier concentration is high in the blood-brain barrier v, allowing an efficient flux of lactate from brain to blood 8, which prevents from building up the tissue-toxic amounts of lactate recorded in adult animals. Conversely, in mature animals, anaerobic degradation of glucose during hypoxia induces a great accumulation of lactate in cerebral tissue which in turn decreases tissue pH and depresses cerebral glucose utilization 27'54. Thus, after a first phase of
204 metabolic stimulation during oxygen deficiency 28'52, cerebral glucose utilization of mature animals comes back to normal values when hypoxia exposure lasts longer 33'53, and may even decrease under control levels in some brain areas if oxygen deprivation is more prolonged 35. The latter situation has also been recorded in both 14- and 21-day-old rats exposed to hypoxia in the same conditions as the present study 5. Therefore, it is likely that the changes in LCBF recorded in the present study at the three ages studied are, at least partly, a consequence of metabolic changes and a result of the more or less active accumulation of lactate and release of excitotoxic amino acids. As for lactate, the concentration of aspartate and glutamate released after an ischemic insult is related to age. It can only be noticed in rats older than 10 days, and progressively increases thereafter to adult levels 7. However, whereas the changes in LCMRglcs and LCBF rates are very similar at P10, the decreases in LCBF at P14 and P21 are more prominent than changes in LCMRglcs, showing the involvement of factors such as hypotension and hypocapnia in the regulation of LCBF levels in P14 and P21 rats exposed to hypoxia, and resulting in a relative cerebral hypoperfusion at the latter ages. In conclusion, it appears that, as for the metabolic response to acute severe hypoxia, the circulatory response of the immature brain to that kind of insult varies with age, the hypoxic response being predominant at P10 whereas the hypocapnic response seems to prevail at P14 and P21. Further studies would also be necessary to establish the contribution of hypotension to the decrease of LCBF recorded at the two older ages.
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Acknowledgements. This work was supported by the Institut National de la Sant~ et de la Recherche M~dicale (U. 272). The authors wish to thanks S. Boyet and V. Koziel for excellent technical assistance.
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BRESD 51716
R e s e a r c h Report
Regional cerebral blood flow response to acute hypoxia changes with postnatal age in the rat A n n e Bilger and Astrid Nehlig * INSERM U272, Universitd de Nancy I, 30 rue Lionnois, B.P. 3069, 54013 Nancy Cedex, France (Accepted 6 July 1993)
Key words: Cerebral blood flow; [14C]lodoantipyrine; Autoradiography; Hypoxia; Postnatal development
The quantitative autoradiographic [14C]iodoantipyrine technique was applied to measure the effects of an acute hypoxic exposure on rates of local cerebral blood flow (LCBF) in the 10 (P10)-, 14 (P14)- and 21 (P21)-day-old rat. The animals were exposed to hypoxic (7% 0 2/93% N 2) or control gas mixtures (21% 0 2/79% N 2) for 40 min before the initiation of the l-min LCBF measurement. At P10, hypoxia induced a 142-415% increase in LCBF over control levels, which affected the 45 structures studied. The highest increases in LCBF were noticed in posterior midbrain and brainstem regions. These increases are in good accordance with hypoxia-induced increases in LCBF recorded during acute hypoxia exposure in both newborn and adult animals. At P14 and P21, rates of LCBF decreased with hypoxia. These decreases were significant in 23 and 21 brain regions, respectively, belonging to all systems studied. These changes in LCBF are in quite good correlation with our previous data on the effects of acute hypoxia exposure on cerebral glucose utilization but the decrease in LCBF is of higher amplitude than the one in cerebral glucose utilization translating into a relative hypoperfusion at a constant metabolic level at P14 and P21. However. arterial blood pressure was reduced by 16 mmHg and arterial pCO 2 was significantly decreased at the two latter ages in hypoxic animals compared to controls. These two systemic factors, and mainly hypocapnia, are rather responsible for the cerebral hypoperfusion recorded at PI4 and P21 in hypoxic rats whereas the circulatory response seems to be predominantly hypoxic at P10.
INTRODUCTION
Acute perinatal hypoxic-ischemic cerebral injury in the term newborn is a major cause of long-term neurologic abnormalities in childhood 22'56. The extent of hypoxic-ischemic cerebral injury is determined principally by the maturity of the brain at the time of insult and the severity and duration of the insult 22. Indeed, in a previous study, we have shown that the cerebral metabolic response to acute, severe hypoxia varies as a function of the age of the animal 5. In 10-day-old rats, which cerebral maturity is close to the one of a human term newborn t,43, acute hypoxia induces a general increase in glucose utilization in all brain areas studied. At 14 and 21 days, the metabolic response to hypoxia varies according to the region and shows regional increases, decreases or no change in the rates of cerebral glucose utilization compared to control levels. These data are in good accordance with the better resistance of the immature brain to anoxia/asphyxia11't3'23,25 and show the relative vulnerability of some brain areas to
* Corresponding author. Fax: (33) 83.32.43.40.
that kind of insults. Indeed, thalamus, brainstem and auditory structures, which have high metabolic demands, appear to have increased susceptibility to injury 5'1°'42. However, the precise relationship between regional metabolic factors and hypoxic neuronal injury has not yet been defined. The immediate circulatory response to hypoxia or asphyxia in the newborn involves redistribution of systemic blood flow with increased perfusion of more vital organs, e.g. brain, heart, adrenal glands and corresponding decreased perfusion of lungs, kidneys and gastrointestinal tract. However, when the hypoxicischemic insult lasts, this homeostatic hemodynamic mechanism fails, leading to systemic hypotension and impaired cerebral autoregulation. Thus, systemic hypotension may result in decreased cerebral blood flow and consequently cause ischemic cerebral injury 22.56 Therefore, in the present study, we examined the effects of an acute hypoxic exposure on rates of local cerebral blood flow (LCBF) measured by the quantitative autoradiographic [14C]iodoantipyrine technique in
198 rats aged 10 to 21 days after birth. The hypoxic exposure was performed in the same conditions as in our previous study of the effects of acute hypoxia on cerebral glucose utilization in immature rats 5. MATERIALS
AND METHODS
Animals Adult S p r a g u e - D a w l e y rats (Iffa-Credo Breeding Laboratories, l'Arbresle, France) were housed together in mating groups of one male and two females and constantly maintained u n d e r standard laboratory conditions on a 12/12 h l i g h t / d a r k cycle (lights on at 06.00 h). After delivery, litter sizes were reduced to 10 pups for homogeneity. The experiments were performed on a total n u m b e r of 26 rats, 14 hypoxic and 12 control animals at 10 (P10), 14 (P14), and 21 days after birth (P21). Rats from 3 or 4 different litters were used in each age group. All animal experimentation was conducted in conformity with the 'Guiding Principles for Research Involving Animals and H u m a n Beings'. A femoral artery and vein were catheterized with polyethylene catheters (Clay-Adams PE-10, i.d. of 0.28 mm, o.d. of 0.61 m m ) under light halothane anesthesia. Both catheters were threaded under the skin, up to the neck through a small opening in the skin. A loop was made with the end of the catheters, which were put back under the skin, and the small opening in the skin was sewn. The animals were returned back to their m o t h e r in their normal environment. T h e experiments were performed on the following day. Only those animals that did not suffer from surgery and did not lose weight (suckling rats which were sometimes rejected by the mother) were used for the study.
Hypoxic conditions O n the day of the experiment, the threads on the skin in the neck were cut and the catheters were pulled out. The rats were transferred to a rectangular plastic box with 6-1 capacity. Hypoxic conditions were obtained by circulation of an O 2 / N 2 ( 7 % / 9 3 % ) gas mixture. Control rats were exposed to the mixture O 2 / N 2 ( 2 1 % / 79%). The circulation of the gas inside the box was calculated to avoid any overpressure. Two gas entries placed on the side of the box were connected to distribution ramps in order to assure a homogenous distribution of the gas mixture inside the box. A central lengthwise split on the cover of the box allowed the release of excess gas and easy access to the catheters of the freely moving rats. The temperature inside the box was adjusted in order to maintain body temperature of Suckling rats in the normal physiological range. The experiments were performed 40 min after the onset of exposure to the gas mixture and the animals were constantly maintained in the same gas environment throughout the whole experimental period.
Measurement of local cerebral blood flow LCBF rates were measured by m e a n s of the [I4C]iodoantipyrine (lAP) method described by Sakurada et al. 46 and adapted to the immature rat by Nehlig et al. 36. The 4-iodo-N-methyl-[14C]-iodoan tipyrine (spec. act. 1.85-2.2 G B q / m m o l ; A m e r s h a m , Little Chalfont, Buckinghamshire, UK) was injected into the animals through the femoral vein at a concentration of 925 B q / m l . T h e period of measurement of LCBF was approximately 1 rain in duration, during which variable a m o u n t s of the [InC]IAP solution were administred to the rats. The volumes injected ranged from 130 to 270/xl according to the age studied. The intravenous infusion was conducted at a progressively increasing rate to produce a rising arterial concentration of the tracer approximating a ramp input function. This ramp input function serves to delay or to prevent the equilibration of rapidly perfused tissues with the arterial blood during the period of measurement. Throughout the period of 114C]IAP administration, timed arterial blood samples, freely flowing from the arterial catheter, were collected in glass capillary tubes. The last sample was taken at the time of killing and as long as blood could be withdrawn from the arterial catheter. The volume of blood withdrawn at each age was
approximately equal to the w)lume ol lAP solution itljccted, m ordc~ to avoid a normotensive shock and any change in physiological variables. The rats were killed by decapitation at tJbout 1 rain after the beginning of [x4C]IAP infusion and brains were removed within i rain, frozen in methylbutane chilled to - 4 0 ° C , coated with chilled embedding matrix (carboxymethylcellulose 4~,: in water~. ~md stored at - 8 0 ° C in plastic bags until sectioned. The content of each capillary tube was transferred to a preweighed scintillation vial that was immediately covered and reweighed aftel blood collection. The blood samples were then treated with 0.5 ml of tissue solubilizer (Optisolv, FSA Laboratory Supplies) and 0.5 ml oi hydrogen peroxide (30%). Blood concentration of [14C]IAP was then determined by liquid scintillation counting in 1{) ml scintillation mixture (Optiphase Hisafe, FSA Laboratory Supplies) in a Beckman scintillation counter (model LS 18(ll. Beckman Instruments, Fullerton, USA). The concentration of tracer per unit w)lume of blood in each sample was calculated from the measured amount of t4C, the weight of the blood sample, and an assumed specific gravity of 1.06 g / m l for blood. The frozen brains were cut into 20-/xm coronal sections at - 2 2 ° C in a cryostat. Sections were picked up on glass coverslips and dried on a hot plate (60°C). Sections were autoradiographed on Kodak SB5 film along with calibrated [14C]methylmethacrylate standards (Amersham) using a special set of low t4C concentration standards for animals 10 and 14 days old. All standards were calibrated for their ~4C concentration in brain sections, as previously described 4°. Adjacent sections were fixed and stained with thionin for histological identification of specific nuclei. The autoradiographs were analyzed by quantitative densitometry with a computerized image-processing system (Biocom 200, France) or a manual microdensitometer (Macbeth, T D 901, Kollmorgen Co., Newburg, USA). Optical density m e a s u r e m e n t s for each structure anatomically defined according to the developing rat brain atlas of Sherwood and Timiras 4s were made bilaterally in a m i n i m u m of four brain sections. Tissue ~4C concentrations were determined from the optical densities of the autoradiographic representations of the tissues and a calibration curve obtained from the autoradiographs of the calibrated standards.
Calculation of local cerebral blood flow Throughout the period of m e a s u r e m e n t of LCBF, blood samples were collected at the distal end of an arterial catheter to determine the continuously changing [14C]IAP concentration in the arterial blood at the proximal end of the catheter. The short duration of the experiment requires extremely precise timing of the samples. Moreover, the length of the arterial catheter was quite high compared to the size of the animal. Therefore, two types of arterial sampling distorsion were taken into account. The first one was related to the time necessary for the blood from the proximal end to reach the distal end of the arterial catheter. The other one was due to a dilution of the true arterial concentration of the tracer at the proximal end of the catheter by the dead space inside the catheter, i.e. the so-called washout effect. The correction for both the time lag and the washout effect was taken into account for final calculations of LCBF, as previously described 36. Finally, LCBF values were calculated according to the Fick equation using a brain-blood partition coefficient of 0 . 8 4 6 .
Physiological variables Just prior to gas exposure and prior to infusion of [14C]IAP, m e a n blood pressure of the animals was measured with an air-damped mercury m a n o m e t e r and the hematocrit value was determined on 5 /zl samples. Arterial pH, p O 2, and p C O 2 were measured on 40 ,~1 blood samples by m e a n s of a blood gas analyzer (Coming, Model 158, Le Vesinet, France) just before gas exposure and before the onset of LCBF procedure.
Statistical analysis LCBF values were determined in 45 cerebral structures in 3 age groups of control, and 3 similar age groups of hypoxie rats. LCBF values underwent three statistical analyses. First, a two-way analysis
199
TABLE I Effects qf hypoxia on physiological variables in det,eloping rats Values are m e a n s + S . E . M , the number of animals in parentheses. " P < I).115, b p <0.01, statistically significant differences from control. * P < 0.05, * * P < 0.01, statistically significant differences from one developmental age to the preceding one. ° P < 11.115, °° P
Arterial blood presure (mmHg) Before gas exposure 40 rain after gas exposure Hematocrit ( ~ ) Arterial pH Before gas exposure 40 rain after gas exposure Arterial p O 2 (mmHg) Before gas exposure 40 min after gas exposure Arterial p C O 2 (mmHg) Before gas exposure 40 min after gas exposure
C Hyp C Hyp C Hyp C Hyp C Hyp
PIO (n = 4)
PI4 (n = 4)
P21 (n 5)
43.7 46.0 41.3 38.6 29.3 29.6
51.5 52.2 53.11 42.8 26.8 28.//
79.5 75.3 75.8 59.4 29.5 31.3
_+2.1 _+1.3 ±2.6 _+1,2 °° + 1.9 +1.0
7.41 +0.02 7.39 _+0.02 7.40 _+/I.01 7.45 ±/I.03
_+1.8" ±1.5" +2.11" _+2.8 b°° + 1.4 +2.7
7.41 +0.02 7.40 _+/I.I/1 7.39 _+11.111 7.41 ± 11.112
±1.7 ** +1.7 ** +3.3"* _+2.2 b**°° + 1.1 +0.6
7.40+0.01 7.42 _+I1.111 7.41 + 1/.111 7.46 + 11.112
C Hyp C Hyp
97.8 87.9 95.7 33.6
±2.5 + 3.3 ~' ±3.9 ±4.8 boo
88.0 92.3 93.9 34.8
+4.1 ± 1.5 +3.0 ±2.3 bo
73.5 81/.3 73.0 32.5
±3.3 * + 1.8 ** ±2.3"* + 2.3 boo
C Hyp C Hyp
29.6 29.9 30.4 28.1
+0.9 _+ 1.0 L0.8 _+1.5
29.2 32.2 34.2 25.2
_+3.0 ±2.5 ±2.5 +_2.0 ~'
35.6 36.6 36.2 22.1
_+ 1.6 + 1.3 +2.3 +1.8 b°°
TABLE II Effects of hypoxia on local cerebral blood flow in sensory systems of deceloping rats Values, expressed as ml/100 g/rain, are means_+ S.E.M. of the number of animal in parentheses. ~' P < I).05, b P < 0.01, statistically significant differences from control. * P < 0.05, ** P < 0.01, statistically significant differences from one developmental age to the preceding one. Brain structure Auditory system Auditory cortex Medial geniculate body Inferiorcolliculus Superior olive Cochlear nucleus Visual system Visualcortex Lateral geniculate body Superior colliculus Olfactory system Olfactory cortex Somatic system Frontoparietal cortex somatosensory area Vestibular system Vestibular nucleus
PlO (n = 4)
P14 (n = 4)
P21 (n = 5)
58.1± 9.2 ** 20.7+ 6.0 b 63.9± 9.7 ** 34.5± 6.2 ~' 53.0± 6.8 ** 29.6_+ 6.8 ~** 51.7± 8.3 * 35.4_+ 9.4 ** 60.6_+ 11.4 * 36.3+12.4"
80.7+ 7.1 45.9+ 10.1 ~' 911.7+ 5.7 * 54.0+ 10.6 " 911.2+ 4.4 ** 56.7_+ 9.2 ~' 93.6+ 1.6 ** 67.9± 9.7 1113.3+ 6.3 ** 86.6+11.6"
C Hyp C Hyp C Hyp C Hyp C Hyp
10.7± 37.6_+ 15.1 _+ 58.11± 16.8± 74.9_+ 22.9± 98.2_+ 24.3_+ 97.3+
1.3 3.7 2.2 9.3 1.6 7.6 2.5 7.8 3.2 7.5
C Hyp C Hyp C Hyp
11.7-+ 60.0+ 15.1 _+ 54.8+ 12.0± 57.7+
1.1 4.0 b 1.9 8.8 b 0.9 4.8 b
C Hyp
22.7-+ 2.5 73.1-+ 4.6 b
57.2+ 2 . 9 " * 33.4+ 3.4 b**
81.5+ 8 . 4 * * 49.4± 3 . 3 * *
C Hyp
16.0+ 2.0 53.6+ 8.0b
81.5±13.9"* 19.9± 6.8b
86.1+ 4.9 51.3+10.6a
C Hyp
28.1_+ 2.9 95.2+_12.0 b
83.5_+ 8.1 ** 49.7+ 4.6 b*
99.4_+_ 5.3 83.8-+ 9.9
b
b b b
43.3_+ 22.9+ 50.9_+ 32.0+ 42.7_+ 24.2+
5.9"* 3.2 b** 9.5 ** 7.4 8.7 ** 4.3 *
76.6± 43.3_+ 81.4_+ 50.7 + 56.8_+ 38.4_+
4.2 ** 8.11 ~'* 2.1 ** 9.7 " 2.9 9.2
20O TABLE 111 Effects of hypoxia on local cerebral blood flow in limbic and tunctionally non-specific areas of det,eloping rats Values, expressed as ml/100 g / m i n , are m e a n s ± S.E.M. of the number of animals in parentheses. ~ P < 0.05, ~' P < 0.01, statiscally significant differences from control. * P < 0.05. ** P < 0.01, statistically significant differences from one developmental age to the preceding one. Brain structure Nucleus accumbens Medial septum Medial amygdala Dorsal hippocampus, CA3 area Ventral hippocampus, C A 3 a r e a Mediodorsal thalamus Medial habenula Lateral habenula Medial raphe Locus coeruleus
C Hyp C Hyp C Hyp C Hyp C Hyp C Hyp C Hyp C Hyp C Hyp C Hyp
PIO (n = 4)
P14 (n = 4)
P21 (n ~ 5)
14.0± 1.9 50.1+ 6.2 b 14.5_+ 2.0 54.2± 6.1 b 13.4± 1.3 41.4_+ 4.0 I' 13.2_+ 1.9 39.4-+ 1.5 b 9.5_+ 0.9 33.0± 6.2 ~' 15.6 ± 2.5 53.6+ 7.2 b 19.5_+ 2.9 59.0_+ 5.5 b 19.5± 3.3 62.2+ 5.5 b 18.1_+ 2.0 78.3_+ 4.9 b 18.6+- 1.8 84.9+11.5h
33.3_+ 4.8 * 32.2_+ 2.7* 36.9± 1.3 ** 30.1± 6.3 * 54.7± 9.7 ** 25.8_+ 6.3 a 50.4_+ 8.8 ** 12.5± 3.9 b** 36.3_+ 7.7 ** 21.0± 3.9 89.3 _+ 15.9 * * 35.9± 8.3 ~ 66.3-+ 12.3 ** 14.8+_ 7.0 b** 70.8_+ 11.5 ** 13.3± 4.1 b** 50.9± 2 . 9 " * 24.0± 9.7 a** 51.9_+ 9.6 ** 28,5_+ 8.2 **
76.5_+ 52.4_+_ 54.7± 42.2_+ 47.4_+ 39.1± 58.6_+
of variance was performed. Thereafter, LCBF in each group of rats were compared with those in the immediate preceding age by means of Bonferroni's multiple comparison procedures 26. Moreover, at each age, values of LCBF in the hypoxic rats were compared with those in the control animals by means of a non-paired Student's t-test. To compare physiological values before gas exposure and before sacrifice, a paired Student's t-test was used.
6.9 ** 3.7 b* 3.8 ** 3.2 ~ 2.3 4.2 4.5 50,8± 4.5 ** 52.5± 2.8 36.6± 6.3 89.2 ± 3.8 55.0_+11.4 ~ 77.4+_ 5.9 58.5_+ 3.3 a** 86.4± 8.1 63.9± 3.2a** 69.4± 6.1" 66.8± 6.5 ** 67.4± 4.4 67.1+_ 8 . 4 "
RESULTS
Physiological values Arterial blood pressure significantly increased between P10 and P14 and between P14 and P21 in both
TABLE IV Effects of hypoxia on local cerebral blood flow in motor areas of developing rats" Values, expressed as ml/100 g / m i n , are means :t: S.E.M. of the number of animals in parentheses, a p < 0.05, b p < 0.01, statistically significant differences from control. * P < 0.05, ** P < 0.01, statistically significant differences from one developmental age to the preceding one. Brain structure Frontoparietal cortex motor area Dorsomedial caudate nucleus Globus pallidus Substantia nigra, pars reticulata Substantia nigra, pars compacta Ventrolateral thalamus Red nucleus Cerebellar cortex Cerebellar nuclei Dentate nucleus Fastigial nucleus Interpositus nucleus
PIO (n = 4)
P14 (n = 4)
C Hyp C Hyp C Hyp C Hyp C Hyp C Hyp C Hyp C Hyp
17.8 + 1.6 66.5± 9.0 b 13.4 _+ 2.l 32.5± 3.1 b l l . 6 + 1.3 33.0 ± 5.9 h 15.3± 1.7 44.9± 3.8 b 16.3 ± 1.5 53.8_+ 3.6 b 15.9 ± 1.3 72.3_+ 11.7 b 20.8_+ 1.7 58.5 ± 4.0 b 11.5± 1.1 54.3± 5.9 b
54.1 _+ 7.0 31.2± 6.4 34.5 ± 4.7 25.5+ 9.5 32.7± 7.9 20.3 ± 4.5 46.1 ± 7.7 13.9± 5.6 51.2 ± 8.8 29.3± 8.6 62.8 ± 9.1 20.7± 4.8 65.3 ± 10.6 40.1 + 10.0 26.6± 2.8 27.8± 7.5
C Hyp C Hyp C Hyp
19.4 ± 1.4 63.3_+ 10.0 b 21.5 ± 1.9 75.7+ 6.9 b 21.4 4- 2.1 59.8± 8.5 b
64.5 ± 14.9 30.8± 8.6 79.8 ± 10.8 41.0±12.8 77.6 ± 10.0 38.3_+ 9.2
P21 (n = 5) ** **
** b** ** * b** **
* ** * ** * **
80.8_+ 0.7 47.8± .5.8 61.7 ± 3.2 46.8± 5.0 47.4+ 2.5 34.2 ± 4.9 63.1 + 3.2 55.4± 3.8 65.1 ± 4.8 61.2± 4.9 89.8 ± 5.4 55.2+ 10.2 71.3 ± 3.9 69.3± 4.8 67.3± 10.3 44.7± 5.3 96.0 ± 67.6± 92.5 ± 82.9± 92.9 ± 68.2±
5.0 5.2 4.2 11.9 4.5 9.9
**
b **
* ** ** * a, * **
* b, * a
201 TABLE V
Effects of hypoxia on local cerebral blood flow in hypothalamus and white matter areas of developing rats Values, expressed as m l / 1 0 0 g / r a i n , are means +_S.E.M. of the number of animals in parentheses, a p < 0.05, b p < 0.01, statistically significant differences from control. * P < 0.05, ** P < 0.01, statistically significant differences from on developmental age to the preceding one.
Brain structure Hypothalamus Supraoptic nucleus Anterior hypothalamus Paraventricular nucleus V e n t r o m e d i a n hypothalamus Mammillary body
White matter areas Genu of the corpus callosum Anterior commissure Cerebellar white matter
PI O (n = 4)
PI 4 (n = 4)
P21 (n = 5)
C Hyp C Hyp C Hyp C Hyp C Hyp
19.3 i 2.3 57.8+-4.8 ~ 14.5 +_ 1.8 45.0_+4.0 b 15.8 + 2.0 46.3_+1.3b 14.8+_ 1.5 50.5+-3.6 b 19.9+_3.9 72.6+-6.1 b
64.1 _+8.7 * * 38.3+_9.3 51.7 + 7.5 * * 37.3+_2.0 45.8 +_5.8 * * 8.0+_2.8~** 42.7_+9.3 * 21.8+-7.1 ** 55.4±9.4 ** 18.4+-1.8 ~**
52.6 ± 3.(/ 45.8+_ 5.1 54.7 +_ 2.2 41.9+_ 4.6 ~ 53.4 +_ 7.4 46.1+_ 3 . 8 " * 59.1 + 3.5 39.1 + 4.2 ~' 87.6_+ 1.2 ** 54.8+- 10.4 ~*
C Hyp C Hyp C Hyp
9.0 +_2.6 28.2+_5.1 b 9.3 +- 1.2 24.4+-1.7 b 7.0+- 1.0 24.0+_2.5 b
28.5 +_ 1.6 20.7_+3.4 33.2 +- 3.3 15.8_+3.5 29.3 +-5.6 23.9+_5.2
48.4 +_ 24.2_+ 42.2 _+ 48.5_+ 49.0+41.4+_
groups of animals, except between P10 and PI4 in hypoxic animals (Table I). It decreased significantly at the end of hypoxic gas exposure as compared to preexposure in hypoxic rats at the 3 ages studied. It was also significantly lower at P14 and P21 in hypoxic animals as compared to controls 40 min after the onset of gas exposure. Hematocrit value and arterial pH were the same in both hypoxic and control animals at the three ages studied. Between P14 and P21, p O 2 significantly decreased in the control group at both times and also in the hypoxic group before any gas exposure. At all ages studied, p O 2 was decreased in hypoxic animals at the end of gas exposure compared with preexposure,
* ** b **
8.5 3.2 1.0 8.1 6.0 3.0
* b * ** * *
and before sacrifice compared with control groups. At preexposure, p O 2 was significantly lower at P21 in hypoxic compared to control animals, p C O 2 was significantly lower at P14 and P21 just prior sacrifice in hypoxic than in control animal. Moreover, at P21, p C O 2 significantly decreased between the beginning and the end of hypoxic gas exposure.
Local cerebral blood flow rates Two-way analysis of variance. The data of the two-way analysis of variance which are not in the Tables show that there was an age effect in 40 structures out of the 45 studied with 10 -5 < P < 0.02. The age effect was not
T A B L E V1
Effects of hypoxia on local cerebral blood flow m respiratory areas of developing rats Values, expressed as m l / 1 0 0 g / m i n , are means+_S.E.M, of the numbe r of animals in parentheses, a P < 0.05, b p < 0.01, statistically significant differences from control. * P < 0.05, * * P < 0.01, statistically significant differences from one developmental age to the preceding one.
Brain structure Gigantocellularis nucleus Parvocellularis nucleus Ambiguus nucleus Parabrachial nucleus Nucleus of the solitary tract Whole brain weighted average
C Hyp C Hyp C Hyp C Hyp C Hyp C Hyp
PIO (n = 4)
P14 (n = 4)
P21 (n = 5)
21.7 +_2.7 79.1 +_2.4 b 19.1 +_2.3 71.9+_7.8 b 20.7 +_0.9 71.6+_8.1 b 17.4 +_2.4 92.8_+5.3 b 16.8 +- 1.6 59.0+_6.9 b 16.5 :t: 2.7 58.8+-3.8 b
67.9 +- 8.9 * * 29.9+_ 3.9 b** 58.7 ± 7.7 * * 28.3+_ 5.1b* 53.7 +_ 11.0 * 30.1+_ 5.3 ** 56.3 _+ 7.5 ** 26.4+- 8.4 a** 54.3 +- 8.5 * * 25.5+_ 3.0 b** 53.8 4-_ 4.2 * * 27.1+_ 3.3 b**
60.5 +- 6.9 55.4+_ 5 . 8 " * 65.1 ± 8.5 57.4 ± 11.7 79.6+ 6.1 59.9 +_ 9.0 59.8 _+ 8.3 69.4+_ 12.3 * 57.3 ± 7.6 67.0+_ 9.9 * 71.1+_ 4.4 * 54.1+_ 4.0 b**
202 significant in superior colliculus (Table II), supraoptic nucleus (Table III), ventromedian hypothalamus (Table V), gigantocellularis and parvocellularis nuclei (Table VI). The effect of the gas exposure was significant in 20 brain regions with 10 5< p < 0.05. These were auditory and frontoparietal cortex, inferior and superior collicuti, cochlear nucleus (Table II), dorsal hippocampus, mediodorsal thalamus, medial and lateral habenula, medial raphe and locus coeruleus (Table III), substantia nigra pars reticulata and compacta, ventrolateral thalamus, red nucleus and the 3 cerebellar nuclei (Table IV), as well as gigantocellularis and parabrachial nuclei (Table VI). The interaction between age and treatment was significant in all brain areas with 10 -5 < P < 0.015.
Variations with postnatal development (Bonferroni's ttest). In control rats, LCBF significantly increased between P10 and P14 in all areas studied, except in cerebellar cortex (Table IV). Between P14 and P21, LCBF significantly increased in 20 areas. These were 7 sensory regions (Table II), 3 limbic areas, accumbens nucleus, medial septum, medial raphe (Table III), 6 motors regions (Table IV), one hypothalamic area, mammillary body and the 3 white matter areas studied (Table V). There was no change in LCBF in respiratory areas between P14 and P21 (Table VI). In hypoxia-exposed animals, LCBF significantly decreased between P10 and P14 in 29 areas. Those decreases affected all systems studied (Tables II-VI). Between P14 and P21, LCBF significantly increased in 23 brain areas. Among those, 20 already decreased between P10 and P14 (Tables II-VI). Effect of hypoxia (Student's t-test). At P10, hypoxia induced a significant increase in LCBF ranging from 142 to 415%. This increase affected all the 45 structures studied. Within the areas (21)with higest LCBF rises ( > 250%) were all structures from auditory and visual systems (Table II), accumbens nucleus, medial septum, medial raphe, locus coeruleus (Table III), motor cortex, ventrolateral thalamus, cerebellar cortex, fastigial nucleus (Table IV), mammillary body (Table V) and all respiratory areas, except ambiguus nucleus (Table VI). At P14, hypoxia induced a general decrease in LCBF rates, ranging from 23 to 82% compared to control levels. However, because of a great interindividual variability, this decrease was significant only in 23 areas of the 45 studied but affected all systems. Among the areas significantly decreased by hypoxia at P14 were 6 sensory regions (Table II), 6 limbic areas (Table III), 2 motor structures, substantia nigra pars reticulata and ventrolateral thalamus (Table IV), paraventricular nucleus, mammillary body and anterior commissure (Ta-
ble V) and all respiratory regions, except ambiguus nucleus (Table VI). At P21, hypoxia induced a general decrease in LCBF rates, but, as at P14, it was only significant in 21 areas. This decrease in LCBF concerned the sensory areas already affected at P14, except vestibular nucleus and plus lateral geniculate nucleus (Table II). Hypoxia decreased LCBF rates in 5 limbic areas (Table Ill), 3 motor regions, the ventrolateral thalamus and 2 cerebellar nuclei (Table IV) and in 3 hypothalamic and one white matter area (Table V). DISCUSSION The results of the present study show that acute hypoxia induces cerebral blood flow changes which differ according to the age of the animals. Indeed, in the very immature rat, at P10, LCBF is widely and markedly increased by hypoxia, whereas at P14 and P21, LCBF is mostly decreased by the insult. These data are in quite good accordance with our previous results on the effects of acute hypoxia on local cerebral glucose utilization in rats at the same age 5. Until now, data on the effects of hypoxia on cerebral blood flow in immature animals have been mainly focused on newborns of different species, such as lambs 12'27'39'44, piglets 15'51, and dogs 6'2°'54. Data in newborns have been compared to results either from fetal 39 or adult animals 27. However, there is no study available on the effects of hypoxia on LCBF in rats or any other species at different postnatal ages. In the 10-day-old rat, which cerebral maturity corresponds to the human newborn 1'43, 40 min of acute hypoxia (7% 0 2 / 9 3 % N 2) induce a widespread and general increase in the rates of LCBF, as previously shown in newborn animals of different species 6'12'14'27'34'39'51. These increases in the rates of LCBF are quite high (142-415%) compared to animals of the same age breathing room air. Moreover, highest increases in LCBF are noticed in midbrain and brainstem relay nuclei of auditory and visual systems as well as in many other posterior regions (Tables II-VI). These results are in good accordance with previous data showing that the most prominent increases in LCBF in newborn animals during hypoxia are located in brainstem areas 2"6d4'15'27"29'5°'51. Only a few authors showed homogeneous responses of brain regions blood flow tO hypoxia 9'34. The often noticed redistribution of cerebral blood flow in favor of posterior regions is considered as a protective mechanism to autonomic functions e. Moreover, the preferential perfusion of the brainstem control centers for respiration and vasomotor tone might be a contributing cause of the greater
203 tolerance to asphyxia in immature animals compared to older ones 6~55. Conversely, in 14- and 21-day-old rats, LCBF is decreased by hypoxia in many areas as compared to control levels. These decreases are significant mainly in sensory and limbic areas. At P14, LCBF is also depressed in respiratory regions, whereas this is no longer true at P21. These results are in apparent contradiction with most data showing an increased LCBF in response to hypoxia in both newborn 2'6'9'12'14'27'34'39'51 and adult animals 4'z4'32'37. However, they are in quite good accordance with our previous data of the effects of acute hypoxia on the levels of energy metabolism in the rat at 14 and 21 days after birth, in which glucose utilization is reduced, but to a lesser extent than LCBF, by the hypoxic exposure compared to control levels 5. In both immature and adult animals, most studies on the effects of hypoxia on cerebral blood flow have been performed within a few minutes after the onset of hypoxia. At those early times, circulatory adjustements during hypoxia act to redistribute blood flow and maintain arterial pressure. Locally, hypoxia produces dilatation in coronary and cerebral vessels and vasoconstriction in peripheral vessels ~9. However, when hypoxia lasts, this homeostatic hemodynamic mechanism fails, leading to systemic hypotension and impaired cerebral autoregulation in the newborn, which in turn leads to ischemic cerebral injury 19"22'56. In the present study, at 40 rain after the onset of hypoxia, i.e. at the time of LCBF measurement, arterial blood pressure is reduced compared to the preexposure value (Table I). However, hypoxia-induced hypotension is of the same order of magnitude at the 3 postnatal ages studied (16-21% reduction from control values). Autoregulation of CBF has been shown to exist in newborns of several species, dogs 2°, piglets 3°'31 and lambs 38, but no data are available in the immature rat. It is likely that autoregulation of CBF exists in rats, already at P10, but there is no information regarding the limits of the autoregulation plateau as a function of postnatal age. From studies on immature animals from other species, it is known that the autoregulation plateau is effective over a narrower range relative to adults 2°'3°'3k38. In the absence of precise data, it is difficult to clearly ascertain whether the slight hypotension recorded in the present study may or not interfere with the lower limit of the autoregulation plateau and induce a decrease in CBF. Moreover, it is not certain that a 7.4-mm change in arterial blood pressure at P10 is equivalent to a 16-mm drop in arterial blood pressure at P21 (Table I). Therefore, hypotension consecutive to hypoxia in the present study may contribute, at least partly, to the decrease in LCBF recorded at P14 and P21. A better knowledge of
the limits of the autoregulation plateau would, however, be necessary to draw a clear conclusion on this aspect of CBF regulation in the immature rat. The other main difference in the effects of hypoxia on physiological variables in 10-, 14- and 21-day-old rats is the hypocapnia recorded at P14 and P21. The immature cerebral circulation has been shown to be responsive to changes in pCO2, in both dogs 16'17'21'47, monkeys 41, sheeps 45, pigs ts and rats 3. Therefore, the decrease in p C O 2 recorded at P14 and P21 may blunt the response of LCBF to hypoxia and be predominant at the time of measurement of LCBF, inducing a decrease in LCBF, as previously shown in both newborns and adults of several species 3'4°'41'45'49. Therefore, it is possible that, in the present study, the primary response in the first minutes following the onset of hypoxia may be an increase in LCBF at all ages, as recorded in adult and newborns animals of several species 6'12'14'27'34'39'51,followed by a secondary decrease in LCBF consecutive to hypoxia-induced hypocapnia. Thus, in the present study, at the time of measurement, i.e. at 40 min after the onset of hypoxia, it is likely that the response of LCBF is purely hypoxic at P10, translating into a general increase, whereas it may be predominantly hypocapnic at P14 and P21, inducing a decrease in LCBF. A temporal evolution of the same kind has been recorded for the effects of hypoxia on cerebral glucose utilization in rats z8'33"52'53 and explained in detail in our previous paper on the effects of hypoxic exposure on cerebral glucose utilization in immature rats 5. Finally, hypoxia-induced LCBF changes may also be a consequence of metabolic changes, as hypothesized by others 6. In our previous study on the effects of acute hypoxia on local cerebral glucose utilization, we have shown that the metabolic response to the hypoxic insult changes with age, mainly as a result of the maturation of brain energy metabolism and of the extent of cerebral metabolic needs 5. The cerebral metabolic level of the 10-day-old rat is quite low and at that age, the animal is able to cover its metabolic needs in hypoxia by increasing the rate of anaerobic degradation of glucose without building up very high concentrations of cerebral lactate. Indeed, in the immature brain, the monocarboxylic acid carrier concentration is high in the blood-brain barrier v, allowing an efficient flux of lactate from brain to blood 8, which prevents from building up the tissue-toxic amounts of lactate recorded in adult animals. Conversely, in mature animals, anaerobic degradation of glucose during hypoxia induces a great accumulation of lactate in cerebral tissue which in turn decreases tissue pH and depresses cerebral glucose utilization 27'54. Thus, after a first phase of
204 metabolic stimulation during oxygen deficiency 28'52, cerebral glucose utilization of mature animals comes back to normal values when hypoxia exposure lasts longer 33'53, and may even decrease under control levels in some brain areas if oxygen deprivation is more prolonged 35. The latter situation has also been recorded in both 14- and 21-day-old rats exposed to hypoxia in the same conditions as the present study 5. Therefore, it is likely that the changes in LCBF recorded in the present study at the three ages studied are, at least partly, a consequence of metabolic changes and a result of the more or less active accumulation of lactate and release of excitotoxic amino acids. As for lactate, the concentration of aspartate and glutamate released after an ischemic insult is related to age. It can only be noticed in rats older than 10 days, and progressively increases thereafter to adult levels 7. However, whereas the changes in LCMRglcs and LCBF rates are very similar at P10, the decreases in LCBF at P14 and P21 are more prominent than changes in LCMRglcs, showing the involvement of factors such as hypotension and hypocapnia in the regulation of LCBF levels in P14 and P21 rats exposed to hypoxia, and resulting in a relative cerebral hypoperfusion at the latter ages. In conclusion, it appears that, as for the metabolic response to acute severe hypoxia, the circulatory response of the immature brain to that kind of insult varies with age, the hypoxic response being predominant at P10 whereas the hypocapnic response seems to prevail at P14 and P21. Further studies would also be necessary to establish the contribution of hypotension to the decrease of LCBF recorded at the two older ages.
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Acknowledgements. This work was supported by the Institut National de la Sant~ et de la Recherche M~dicale (U. 272). The authors wish to thanks S. Boyet and V. Koziel for excellent technical assistance.
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