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Stroke. 1997;28:2281-2289

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(Stroke. 1997;28:2281-2289.)
© 1997 American Heart Association, Inc.

Articles

Vulnerability to Cerebral Hypoxic-Ischemic Insult in Neonatal but Not in Adult Rats Is in Parallel With Disruption of the Blood-Brain Barrier

Kanji Muramatsu, MD, PhD; Atsuo Fukuda, MD, PhD; Hajime Togari, MD, PhD; Yoshiro Wada, MD, PhD; Hitoo Nishino, MD, PhD

From the Departments of Pediatrics (K.M., H.T., Y.W.) and Physiology (K.M., A.F., H.N.), Nagoya City University Medical School, Mizuho-cho, Mizuho-ku, Nagoya, Japan.

Correspondence to Kanji Muramatsu MD, PhD, Department of Pediatrics, Nagoya City University Medical School, Mizuho-cho, Mizuho-ku, Nagoya 467, Japan. E-mail muramatu{at}med.nagoya-cu.ac.jp


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Background and Purpose Vulnerability to cerebral hypoxic-ischemic (H-I) insult and its relation to disruption of the blood-brain barrier were investigated in postnatal rats.

Methods Pups of postnatal day (P) 7, P14, and P21 underwent ligation of a unilateral carotid artery and were exposed to hypoxic conditions. For the detection of early-phase deterioration, brains were perfusion-fixed 24 hours after H-I insult and examined by argyrophil III method. For the detection of later infarction, animals were fixed at 72 hours after the H-I insult.

Results In either case, tissue damage was detected in the striatum, parietal cortex, and hippocampus. The vulnerability of P7 and P21 rats was remarkable, as compared with P14 rats. Although the developmental status of the vasculature was not significantly different at each age, the permeability of IgG after H-I injury was prominent in P7 rats and to a lesser extent in P14 rats. In P21 rats, however, there was little IgG leakage even 24 hours after the insult. Dexamethasone pretreatment blocked the extravasation of IgG and reduced the damaged tissue in P7 and P14 rats but not in P21 rats. Percentages of reduction in infarcted areas by the dexamethasone became smaller in proportion to ages.

Conclusions The results suggest that in younger rats vulnerability to H-I insult was in parallel with permeability of the blood-brain barrier, whereas in adults it might be more dependent on cellular vulnerability.


Key Words: blood-brain barrier • dexamethasone • hypoxia • rats


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
The extent of an H-I injury depends principally on the degree of maturation of the brain as well as on the severity and duration of the insult.1 It is generally accepted that immature neurons tolerate a much longer period of oxygen deprivation and/or ischemia than do those of adults. Using electrophysiological techniques, acutely dissociated hippocampal neurons2 and neocortical slices3 demonstrated the higher sensitivity of mature neurons to hypoxic conditions. The rate of ATP use in slices from adult rats was about fourfold that from P4 rats.4 The energy consumption rate correlated well with age and paralleled a susceptibility to cell death.5 Thus, the greater resistance of immature neurons to oxygen deprivation and/or ischemia would be largely dependent on their lower energy consumption.

In a previous study,6 using the argyrophil III method, we examined histopathological changes in the developing rat brain after H-I injury with unilateral carotid artery ligation at P7, P14, and P21. In the early phase after H-I insult, argyrophil (deteriorated) neurons appeared in the ipsilateral cortex, hippocampus, and striatum. The topographic distribution of deteriorated cells after the insult was not different by age, but the duration of insult needed to cause damage differed according to age (P14>P7>P21). These results suggest that the pathophysiological process triggered in H-I situations has a characteristic topographical distribution that does not change during development but that there are differences in susceptibility relative to age. However, since P14 rats were the most resistant to damage, this difference could not be correlated simply with maturation. Since younger neurons have more resistance to hypoxia in vitro than do adult neurons,2 3 we postulated that our results might be modified by some other factors acting specifically in vivo.

In the present study, we have hypothesized that the degree of stability of the BBB as well as the maturation of vasculature could be responsible for the differences in neuronal susceptibility to injury during development. The BBB has three major functions: (1) protection of the brain from the blood milieu, (2) selective transport, and (3) metabolism or modification of blood- or brain-borne substances.7 The tight junction between brain endothelial cells and the wrapping of capillaries by astrocytic end-feet is thought to be fundamental to the optimal functioning. The development of the BBB is thought to be parallel with age.8 A disruption of the BBB can cause leakage of large molecules after an ischemic episode in adult rats9 that might be cytotoxic.10 Therefore, it was thought worthwhile to observe developmental differences in the susceptibility to BBB disruption with respect to how these differences affect neuronal vulnerability after H-I insult, since they may play a critical role in the brain damage that follows.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Hypoxia-Ischemia
Wistar rats (Chubu Kagaku Shizai, Nagoya, Japan) of either sex were used at P7, P14, and P21. Animal care and handling were done according to the guidelines of the Institute for Experimental Animal Science, Nagoya City University Medical School. Cerebral H-I injury was produced by a modification of the Levine method.11 12 These pups were anesthetized with diethyl ether, and the right common carotid artery was permanently ligated with surgical thread via neck incision. After the operation, they were returned to the dam for 1 to 2 hours of recovery. The rats were exposed for 1.5 hours to humidified 8% O2/92% N2 gas at a flow rate of 8 L/min in a 2000-cm3 polyethylene chamber at 37°C. Animals of each age were divided into two groups: a control group receiving saline (0.9% NaCl, 10 mL/kg) and another group given dexamethasone (0.1 mg/kg), and both these agents were administered intraperitoneally 24 hours before hypoxic exposure. After hypoxia was induced, animals were returned to their dams and later killed. Mortalities of the groups were compared using Fisher's exact probability test.

Neuronal Deterioration by the Argyrophil III Method
Saline- and dexamethasone-treated groups of P7, P14, and P21 animals (n=55) were evaluated 24 hours after hypoxia. Under deep anesthesia with pentobarbital (50 mg/kg), pups were perfused with saline and then with a fixative consisting of 4% paraformaldehyde and 2% glutaraldehyde in a cacodylate buffer (pH 7.5). The brains were removed and immersed in the same fixative at room temperature. After cryoprotection, 50-µm-thick coronal sections were made with a freezing microtome, and argyrophil III staining of deteriorated neurons was performed.13 14 Briefly, sections were treated with 1% acetic acid and with 50%, 75%, and 100% 1-propanol for 5 minutes each. They were then esterified with 1-propanol containing 2% distilled water and 1.2% sulfuric acid at 56°C for 16 hours. The sections were rehydrated with 50% and 25% 1-propanol for 5 minutes with each solution, washed with 8% acetic acid for exactly 10 minutes, and then submerged in silicotungstate developer containing 0.125% NH4NO3, 0.1% AgNO3, 1.0% tungstosilicic acid, 0.1% formaldehyde, and 5.0% Na2CO3. When the background turned brown, development was stopped by a 30-minute treatment with 1% acetic acid. For evaluation of argyrophil neurons, cells with clearly stained soma and dendrites/axons were considered positive. Contralateral hemispheres were used as controls for background staining (Fig 1Down). For each animal, two coronal sections, at the caudate-putamen and at the middle of dorsal hippocampus, were observed.



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  		Figure 1. Photomicrographs of brain sections stained with argyrophil III and H-E. Representative sections from P7 (A through D), P14 (E through H), and P21 (I through K) rats after H-I insult. Argyrophil neuron–positive areas were apparent in the ipsilateral (right) cerebral cortex (A and B), striatum (B), and hippocampus (A). At a higher magnification, the argyrophil pyramidal neurons were found to have the characteristic shape of shrunken somata and corkscrew-like apical dendrites (C). In P14 sections, argyrophil neurons were not detected at either low (E and F) or high magnification (G). In P21 sections, argyrophil neurons appeared in the ipsilateral cortex (I and J), striatum (J), and hippocampus (I). In the sections stained with H-E, large necrotic areas were apparent in the cortex of P7 (D) and P21 (K) rats, but only a small necrotic area was found in the cortex of P14 rats (H). Scale bars: A, B, D through F, and H through K, 900 µm; C, 40 µm; G, 50 µm.

Evaluation of Infarction
The areas of infarction were evaluated in animals 72 hours after the H-I insult. After cryoprotection, 10-µm-thick coronal sections were made on a cryostat and stained with H-E (n=56). Areas of H-I damage were evaluated at six coronal levels: nucleus accumbens, caudate-putamen, globus pallidus, anterior tip of the dorsal hippocampus, middle of dorsal hippocampus, and ventral hippocampus. Infarct areas were measured at these six coronal levels using charge-coupled device camera (Sony, model XC-57), and were analyzed with a computer using image analysis software (NIH Image) with a resolution of 5600 pixels/mm2. For each animal, measurements were done at all six levels in the cerebral cortex and at two levels of the striatum. The percentage of H-I necrosis in each animal was determined for the cortex and striatum by dividing the sum of each damaged area by the sum of the contralateral counterpart areas of each structure. These values may give a slight underestimation of the necrotic area, since tissue loss might not be taken into account. The extent of the areas of H-I brain damage in the cortex and striatum was tested by one-way ANOVA followed by a post hoc test (Fisher's protected least significant difference).

Observation of Vasculature
Animals of each age group (n=15) with no treatment were anesthetized and perfused with heparinized saline followed by PBS. A mixed solution of 60% barium sulfate and 1.5% gelatin was injected through the left ventricle. Brains were removed and immersed in PBS with 4% paraformaldehyde for 12 hours. Coronal sections (500 µm) made on the freezing microtome were dehydrated with ethanol and penetrated with xylene. The vasculature of the cortex and striatum was inspected with light microscope.15

Evaluation of Immunoreactivity of IgG
The saline- (n=90) or dexamethasone-treated (n=90) group of each age was subjected to H-I insult as described above. The animals were perfusion fixed at different times (3, 6, 9, 12, 18, and 24 hours) after hypoxia and postfixed with 4% paraformaldehyde in PBS (pH 7.4). For immunohistochemistry, purified biotinylated rabbit anti-rat IgG antibody (Vector Labs, x200) was used. After the reaction with biotinylated IgG and formation of avidin-biotin complex, visualization was carried out with diaminobenzidine enhanced with ammonium nickel sulphate.16 For each animal, two coronal sections at the caudate-putamen and at the middle of the dorsal hippocampus were evaluated. Total immunoreactive areas were measured at these coronal levels using charge-coupled device camera (FUJI, model FV-10D) and were analyzed with a computer using image analysis software (NIH Image). The percentage of immunoreactive area in each animal was determined by dividing the sum of each reactive area by the sum of the contralateral counterpart areas. The extent of immunoreactive areas was analyzed by the Mann-Whitney U test. Data are expressed as mean±SD.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
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Neuronal Deterioration and Infarction
The incidence of mortality during the experiments did not differ by age or by treatment (saline group P7 1/20, P14 1/20, and P21 4/20; dexamethasone group P7 1/20, P14 0/20, and P21 2/20). Animals were killed at 24 hours after hypoxia for argyrophil III staining (Fig 1AUp through 1C, 1E through 1G, 1I, and 1J) and at 72 hours for H-E staining (Fig 1DUp, 1HUp, and 1KUp). With argyrophil III staining, silver impregnated neurons were detected 24 hours after insult in the ipsilateral parietal cortex, striatum, and hippocampus in the sections of P7 and P21 animals (Fig 1AUp, 1BUp, 1IUp, and 1JUp). Pyramidal neurons of the cortex were clearly stained. Shrunken somata and corkscrew-like dendrites were the specific morphological features of the damaged neurons (Fig 1CUp, arrows). Topographical distribution of argyrophil neurons in sections from P7 and P21 rats was quite similar. Argyrophil neurons were not detected in sections from P14 animals, although the background became brown (Fig 1EUp through G). In control groups, severe damage (infarction) in the cortex and hippocampus was revealed in H-E–stained sections of P7 and P21 rats 72 hours after insult (Fig 1DUp and 1KUp). In sections from P14 animals, a small necrotic area was detected only in the cortex (Fig 1HUp).

Development of Vasculature
Representative sections after barium sulfate–gelatin injection showed well-developed vasculature in the cortex and the striatum at each age of rat (Fig 2Down). Vertical orientation of the vasculature to the pial surface could be seen in the cortex of each age. Even in P7, dense, radially penetrating vessels and homogeneously distributed capillary beds were observed in the cerebral cortex (Fig 2ADown). In P14 and P21 rats, the density of long, radially penetrating vessels did not change, although the capillary density increased with age (Fig 2CDown and 2EDown). The lateral striatal artery, running through the lateral striatum with vertical branches, displayed a similar morphology at any age (Fig 2BDown, 2DDown, and 2FDown), but the density of branch capillaries was higher in P14 and P21 rats than in P7 rats (Fig 2DDown and 2FDown). Avascular areas were not detected at any age of animal, and the capillary beds were well developed at all ages, even in the periventricular areas.



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  		Figure 2. Coronal sections (500 µm–thick) of P7 (A and B), P14 (C and D), and P21 (E and F) brains with barium sulfate–gelatin–filled vessels in the cerebral cortex (A, C, and E) and the striatum (B, D, and F). In P7 rats, there was already a high density of radially penetrating vessels toward the pial surface and homogeneously distributed capillary beds (A). There were no apparent differences in the capillary density of P14 rats (C). Note that the architecture of the arteries did not undergo any basic change, although capillary density in P21 rats increased, particularly in layers III through V (E). Even in P7 sections, the capillary beds were formed homogeneously by the branches of the lateral striatal artery (B). Scale bars: A, C, and E, 250 µm; B and D, 200 µm; F, 180 µm.

Extravasation of IgG
To investigate the permeability of the BBB during recovery from H-I insult, IgG immunoreactivity was investigated from 3 to 24 hours after the insult. Extravasation of IgG from the vascular compartment into the brain parenchyma was observed in P7 and P14 rats but not in P21 rats. In P7 rats, immunoreactivity to IgG was positive in the ipsilateral cortex and striatum as early as 6 hours after H-I insult (Fig 3ADown), whereas no immunoreactivity was detected at this stage in P14 rats. IgG reactivity became more intense with time and it became detectable in P14 rats at 12 hours after H-I insult (not shown). A very intense immunoreactivity was detected at 24 hours in P7 (Fig 3BDown) and P14 (Fig 3DDown) brains. Both parenchyma and neurons of the cortex were immunopositive (Fig 3CDown). On the other hand, there was little or no extravasation of IgG in P21 brain tissues even at 24 hours after insult (Fig 3EDown). In the dexamethasone-pretreated group, the extravasation of IgG was blocked in P7 and P14 brains, even at 24 hours after hypoxia (Fig 3FDown and 3GDown). As in control rats (Fig 3EDown), no apparent immunoreactivity was detected in dexamethasone-pretreated P21 rats (Fig 3HDown).



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  		Figure 3. Photomicrographs of IgG immunoreactivity at different times (A, 6 hours; B through H, 24 hours) after H-I insult in control (A through C, P7; D, P14; and E, P21) and dexamethasone-pretreated (F, P7; G, P14; and H, P21) rats. Note the moderate staining at 6 hours (A, arrowheads) and prominent reactivity at 24 hours (B) after hypoxia in the ipsilateral parietal cortex and lateral striatum in a control P7 rat. Higher magnification shows intense IgG immunoreactivity of not only the parenchyma but also the cell body of pyramidal neurons (C). A section from P14 control rats also had apparent immunoreactivity (D). P21 control sections revealed no detectable immunoreactivity (E). Dexamethasone-pretreated P7 (F) and P14 (G) rats did not show any immunoreactivity. No immunoreactivity was detected in dexamethasone-pretreated P21 rats (H). Scale bars: A, B, and D through H, 900 µm; C, 30 µm.

Evaluation of Immunoreactivity
The percentage area of IgG immunoreaction at the caudate putamen and the middle of dorsal hippocampus with saline or dexamethasone pretreatment was measured at different times after H-I insult (Fig 4Down). In the control group, immunoreactivity in P7 sections was clearly detected as early as 6 hours after H-I insult. In P14 rats, however, the similar percentage area of reaction appeared somewhat later, at around 18 hours. In P21 rats, only a faint immunoreactivity was found even at 24 hours. Dexamethasone pretreatment significantly decreased the area of immunoreaction in P7 and P14 brains as compared with control groups (Fig 4Down).



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  		Figure 4. The percentage area of IgG immunoreaction at different times after H-I insult. Two coronal sections were used and total immunoreactive areas were evaluated. In the saline-pretreated group, immunoreactivity in P7 sections was detected as early as 6 hours after H-I insult. In P14 rats, the immunoreaction was not apparent until 9 to 12 hours. In P21 rats, no apparent immunoreactivity was detected up to 24 hours. The number of animals in each column is five. Values are given as mean; error bars, SD. {dagger}P<.01, *P<.05, different from saline control, Mann-Whitney U test.

Pretreatment With Dexamethasone Attenuates Neuronal Deterioration
After pretreatment with dexamethasone, argyrophil neurons were detected only in P21 rats in the cortex, striatum, and hippocampus (Fig 5GDown and 5HDown). On the other hand, dexamethasone pretreatment of P7 rats dramatically suppressed the appearance of argyrophil neurons in each area examined (Fig 5ADown and 5BDown). Thus, dexamethasone was effective in P7 but not in P21 rats at preventing neuronal deterioration after H-I insult. The infarct areas in P7 rat brain, as revealed by H-E staining, were strikingly reduced by dexamethasone pretreatment (Fig 5CDown). In P14 rat brain, infarction became undetectable with dexamethasone pretreatment (Fig 5FDown). In contrast, dexamethasone had no effect on infarction in P21 rats (Fig 5IDown). P21 sections of both the control and dexamethasone groups revealed severe damage, and large necrotic areas were apparent in the cortex and striatum (Figs 1KUp and 5IDown). Topographical pattern of argyrophil neuron–positive areas was almost the same as that of saline-injected control rats (Fig 1IUp and 1JUp).



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  		Figure 5. Representative argyrophil III–and H-E–stained sections at the levels of the middle of dorsal hippocampus and striatum, demonstrating the effect of dexamethasone. Sections were from P7 (A through C), P14 (D through F), and P21 (G through I) rats. The ipsilateral hemisphere of P7 and P14 tissues stained by argyrophil III had no apparent argyrophil neuron–positive areas (A, B, D, and E). In P21 rats, argyrophil neurons were detected in the cortex (G and H), striatum (G and H), and hippocampus (G). H-E staining of P7 (C) and P14 (F) brains revealed almost intact tissue without any sign of necrosis. Sections from P21 rats had necrotic areas in the cortex and striatum (I). Scale bars: 900 µm.

The percentage area of infarction in the cortex and striatum of P14 rats was significantly less than in P7 and P21 rats (Fig 6Down). With the same H-I insult, P14 rats developed smaller areas of necrosis than did P7 or P21 rats. Percentages of reduction in infarcted areas of the cortex by the dexamethasone pretreatment were 81%, 50%, and 17% in P7, P14, and P21 rats, respectively. In the striatum, the percentage reductions of infarct areas were 53%, 26%, and -19%, in P7, P14, and P21 rats, respectively. The protective effect of dexamethasone was statistically significant in P7 animals (P<.01; cortex and striatum) and in P14 animals (P<.05; cortex).



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  		Figure 6. Percentage of the area of infarction in cerebral cortex and striatum of saline- and dexamethasone-pretreated rats. Measurement of infarct areas was performed on H-E–stained sections. Data represent mean values; error bars, SD. Numbers in parentheses are numbers of animals. {dagger}P<.01,*P<.05; analyzed by one-way ANOVA followed by Fisher's protected least significant difference test.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
In the present study, susceptibility to H-I insult was investigated in P7, P14, and P21 rats, and differences in vulnerability were discussed from the standpoint of BBB function. There have been many studies on H-I brain injury in neonatal (P7) rats,12 17 18 but only a few deal with other developing periods.19 20 21 Observations of degenerating neurons at different ages (P2 through P80) with H-I injury by Ikonomidou et al indicated that the peak severity of morphological change occurred at P6.21 In the present study, the degree of injury decreased between P7 and P14, in agreement with the study of Ikonomidou et al,21 but increased between P14 and P21, in disagreement with the results of Ikonomidou et al. This discrepancy in the results might be due to differences in the experimental method in which they used hypobaric conditions to induce hypoxia/ischemia. Indeed, H-I injury for 1 hour with our experimental method on P35 rats resulted in 92% mortality (data not shown) unlike the experience reported by Ikonomidou et al. Another difference of these two models was the recovery period. In their experiment, histology was performed at only 2 hours of recovery, whereas histology was performed at up to 72 hours in the present study.

Argyrophil III silver impregnation technique is a very sensitive method for detecting "early phase" morphological changes in neuronal damage.6 22 "Argyrophil positive" implies a deteriorated neuron state with damage to the involved cytoskeleton, especially in microtubules, although details of the pathophysiological processes remain unknown.23 At 24 hours after H-I insult with unilateral carotid artery occlusion, deteriorated neurons (argyrophil neurons) appeared in the parietal cortex, striatum, and hippocampus in P7 and P21 rats, but only a few in P14 rats (see Fig 1Up). Areas of infarction detected at 72 hours after H-I insult were also not proportional to age (P14<P7=P21, see Figs 1Up and 6Up), with the resistance of P14 brain tissues to H-I insult much greater than that of P7 and P21. However, sites of damage determined at 24 hours by argyrophil III staining and at 72 hours by H-E staining were similar; specifically the cortex, striatum, and hippocampus.

An in vitro study of hippocampal slices using electrophysiological techniques revealed a significant age-dependent increase in sensitivity to hypoxia.24 Synaptic responses in immature hippocampal slices up to 2 to 3 weeks of postnatal life were much more resistant to anoxia-aglycemia than those of adults.25 Kawai et al4 reported that the decay time of the postsynaptic field potential by hypoxia in P10 animals was similar to that of adult animals, indicating that maturation occurs at around this age. Compared with results of their in vitro experiments in which the potential differences in maturation of the circulatory system are excluded, our in vivo results indicated that neuronal susceptibility to H-I insult may be affected by vascular factors.

The vascular system extends itself by the sprouting (branching) and elongation of endothelial cells.26 Intracerebral vascularization in the early stage is characterized by the formation of new sprouts originating from these cells. The density of capillaries in rat brain increases several-fold during development,27 and no regional variation in capillary density has been reported.28 The present results are compatible with these observations, since irregular development of vasculature was not detected in the cortex or striatum from P7 to P21 animals.

The brain capillary is a structural unit consisting of endothelial cells, pericytes, and associated basement membrane surrounded by foot processes of adjacent astrocytes.26 In a developmental study, brain capillaries from animals younger than P15 were shown to lack a mature barrier in terms of permeability.29 Ultrastructural observations have revealed that a functional BBB is established as early as P13.30 In a study in which electrical resistance was evaluated, ion permeability was demonstrated to be completed by P28 to P33.31 The ages of animals used in this study seem to be in the process of BBB completion. We thought that the brain of P7 animals did not have mature BBBs, although BBBs of P21 rats have developed comparatively. Transient global cerebral ischemia increased vascular permeability,32 and caused remarkable changes in endothelial cells, as revealed by electron microscopic observation.33 Although the H-I conditions of the present study might result in a milder form of injury than ischemia, hypoperfusion would be sufficient to induce vasogenic edema in the brain34 via disruption of the BBB.

The present study showed that IgG immunoreactivity was apparent as early as 6 hours after H-I insult in P7 rats (Fig 3AUp) and 12 hours in P14 rats, indicating a leakage of IgG from vessels to brain parenchyma. Thus, it was found that 1.5 hours of hypoxia with unilateral carotid artery occlusion was sufficient to induce dysfunction of the BBB in P7 and P14 rat pups. Since IgG immunoreactivity might be correlated with the transfer of compounds of different sizes and charges, the early changes of the BBB, such as permeability changes for molecules smaller than IgG, may not be detected. Thus, the BBB dysfunction might begin in P21 rats too. Furthermore, leakage of such smaller molecules including excitatory amino acids, might occur in P7 and P14 rats even before 6 hours and 12 hours, respectively. In any case, BBB function in P21 rats proved to be more resistant than younger ones in terms of IgG permeability, although the actual H-I insult may not be precisely identical because of potential differences in ventilatory drive or arterial pressure. We hypothesize that the earlier onset of the increased BBB permeability, hence longer period of exposure to blood-borne molecules, could be the cause of the greater infarct volume in P7 rats than P14 rats with the comparable area of IgG immunoreaction.

The administration of glucocorticoids was ineffective in controlling neuronal damage, and even facilitated it to some extent, after transient ischemia in adults.35 36 However, recent studies have indicated that pretreatment with dexamethasone ameliorated brain damage after H-I insult of neonatal rats.37 38 The present study confirmed the previous reports showing that the effects of dexamethasone are age dependent: the dexamethasone pretreatment was only effective in protecting younger rats. Our results are also compatible with recent findings indicating that the size of infarction was reduced by dexamethasone pretreatment in P14 rats but not in 1-month-old rats.20

It is not clearly known whether the effect of dexamethasone in preventing H-I damage has a direct or an indirect interaction with neurons. However, evidence that glucocorticoids exacerbate hypoxic and hypoglycemic neuronal damage in vitro39 suggests that dexamethasone works indirectly to prevent neuronal injury in vivo. Disruption of the BBB, or even transient opening by hypertension40 or hyperosmolarity,41 may cause neuronal damage. Thus, the extensive extravasation of IgG might indicate vessel leakage of numerous materials such as excitatory amino acids, some of which could be cytotoxic. With dexamethasone pretreatment, the leakage of IgG in P7 and P14 brain tissues was mostly blocked (Figs 3Up and 4Up), whereas neuronal deterioration (argyrophil neurons) in P7 animals and necrosis in P7 and P14 rats could be prevented (Figs 5Up and 6Up). These protective effects of dexamethasone suggest that the disruption of the BBB may exacerbate H-I brain damage in younger rats. In P21 rats, however, dexamethasone pretreatment had no effect. Since there was no leakage of IgG after H-I insult in P21 rats, neuronal damage in adult rats might not be significantly related to the magnitude of BBB disruption. Although the exact mechanism by which dexamethasone strengthens the BBB is not known, a recent report has indicated that neurotrophic factors were induced by dexamethasone in glial cells,42 suggesting that dexamethasone might act by protecting astrocytes rather than by directly affecting BBB function. The vulnerability of the BBB to H-I insult might be due to the immaturity of the BBB itself, since its permeability after H-I insult decreased with maturation in the present study.

Glucocorticoids have been used to induce various effects such as hyperglycemia,38 reduction of free radical formation,43 and inhibition of the expression of inducible nitric oxide synthase,44 all of which may influence H-I damage. Elevation of the blood glucose level has been shown to reduce ischemic neuronal damage in neonatal models38 45 as well as under in vitro conditions.39 Since the blood glucose was not measured in the present study, we could not exclude the possible influence of the induced hyperglycemia. However, Tuor et al showed the prevention of H-I damage with dexamethasone was not influenced by fasting,20 indicating that a mechanism other than elevated blood glucose level is responsible for the protective effect.

Cytotoxic oxygen free radicals are generated during H-I insult.46 Dexamethasone was found to induce the production of lipocortin, which suppresses the activity of phospholipase A2 and the production of arachidonic acid.47 This may lead to the suppression of immunological cytotoxic events after H-I insult.48 Nitric oxide also plays a cytotoxic role in H-I brain damage, and an inhibitor of nitric oxide synthase was shown to reduce this damage.49 Dexamethasone can inhibit the expression of inducible nitric oxide synthase,45 and can thereby block the production of nitric oxide. However, even if such events occur, they cannot be used to account for the relationship between age and the protective effect of dexamethasone seen in the present study. Dexamethasone exacerbates hypoxic-hypoglycemic injury in cultured neurons in which vascular effects are negligible.39 Thus, our present results suggest that the effect of dexamethasone on in vivo models is related to its vascular site of action, specifically the protection of BBB function.

In summary, the neuronal susceptibility to H-I insult correlated with the extent of insult-induced BBB permeability that was closely related to maturation of the animal. When dexamethasone was used to suppress the development of BBB dysfunction, the younger brains showed a greater resistance to insult. These results indicate that the BBB dysfunction that results from H-I insult initiates neuronal deterioration followed by necrosis. Since dexamethasone could prevent, or at least reduce, neonatal H-I brain damage, our results provide evidence of the therapeutic potential of dexamethasone for the prevention of perinatal hypoxic brain damage.


*    Selected Abbreviations and Acronyms
 
BBB = blood-brain barrier
H-E = hematoxylin-eosin
H-I = hypoxic-ischemic
P = postnatal day
PBS = phosphate-buffered saline


*    Acknowledgments
 
This work was supported by Ministry of Education, Science, Sports and Culture (Japan) grants-in-aid 07680897, 09260226, and 09680817 to Dr Fukuda. We would like to thank Dr William Campbell (Choju Medical Institute, Fukushimura Hospital, Toyohashi, Japan) for reading this manuscript.

Received January 29, 1997; revision received July 29, 1997; accepted July 30, 1997.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Hill A. Current concepts of hypoxic-ischemic cerebral injury in the term newborn. Pediatr Neurol. 1991;7:317-325.[Medline] [Order article via Infotrieve]
  2. Cummins TR, Donnelly DF, Haddad GG. Effect of metabolic inhibition on the excitability of isolated hippocampal CA1 neurons: developmental aspects. J Neurophysiol. 1991;66:1471-1482.[Abstract/Free Full Text]
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