Association of chronic sublethal hypoxia with ventriculomegaly in the developing rat brain

Developmental Brain Research 111 Ž1998. 197–203

Research report

Association of chronic sublethal hypoxia with ventriculomegaly in the developing rat brain Laura R. Ment a

a,b,)

, Michael Schwartz c , Robert W. Makuch d , William B. Stewart

e

Department of Pediatrics, Yale UniÕersity School of Medicine, 333 Cedar Street, New HaÕen, CT 06511, USA b Department of Neurology, Yale UniÕersity School of Medicine, New HaÕen, CT, USA c Department of Neurobiology, Yale UniÕersity School of Medicine, New HaÕen, CT, USA d Department of Epidemiology and Public Health, Yale UniÕersity School of Medicine, New HaÕen, CT, USA e Department of Surgery, Yale UniÕersity School of Medicine, New HaÕen, CT, USA Accepted 15 September 1998

Abstract Bronchopulmonary dysplasia remains a major cause of neurodevelopmental handicap in preterm infants. Because bronchopulmonary dysplasia may be associated with prolonged hypoxemia without obvious changes in systemic blood pressure, we developed an animal model of chronic sublethal hypoxia to test the hypothesis that this insult results in significant alterations in corticogenesis in the developing brain. Three groups of newborn rats were placed in a chamber with FIO 2 9.5% on postnatal day 3 ŽP3.. One group was sacrificed at P13; a second group was sacrificed at P33, and the third group was removed at P33 and reared in normoxia until sacrifice at P63. Control rats were those raised in room air for the corresponding periods of time. Rats were transcardially perfused and the brains were embedded in celloidin and prepared for morphometric analysis using standard stereology methods. Although experimental rat pups in the third group demonstrated ‘catch-up’ of body weight following return to normoxia, these studies demonstrated both failure of brain growth Ž p - 0.01. and progressive cerebral ventriculomegaly Ž p - 0.01.. Decreased subcortical white matter Ž p - 0.05. and corpus callosum size Ž p - 0.01. were noted at P63 in pups reared under conditions of chronic hypoxia. Decreases in cortical volume Ž p - 0.05. were noted at all three experimental time points for hypoxic-reared pups when compared to control animals. These data suggest that chronic sublethal hypoxia may lead to severe impairments in corticogenesis in an animal model of developing brain. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Ventriculomegaly; Chronic sublethal hypoxia; External capsule; Corpus callosum

1. Introduction Chronic hypoxia is a not uncommon problem of preterm infants w1,40x, and those infants who demonstrate oxygen dependence beyond 28 days of life and the pulmonary radiographic changes originally described by Northway et al. w33x are said to suffer bronchopulmonary dysplasia ŽBPD.. Bregman and Farrell w5x have estimated that as many as 40 to 70% of all infants - 1000 g birth weight who require respiratory support develop BPD, and their recent review of the same topic suggests that 25 to 40% of infants with BPD suffer major longterm neurodevelopmental handicap. Thus, the neurodevelopmental outcome of infants with PBD represents a major concern for those who

)

Corresponding author. Fax: q1-203-785-7194

care for these tiny and frequently critically ill preterm infants. Although Tay-Uyboco et al. w47x have suggested that the chronic, and sometimes undetected, episodes of severe hypoxia secondary to airway constriction common in infants with BPD are the causative factor of the developmental delay found in some of these children, other investigators have noted a strong association between duration of assisted ventilation and handicap w6,13,30,44x. Furthermore, although preterm infants with BPD are known to experience cerebral ventriculomegaly in the newborn period w37x, the mechanism by which bronchopulmonary dysplasia produces injury to the newborn brain remains unknown. The newborn rat brain has been demonstrated to provide a good model for that of the preterm infant at the end of the second trimester; neuronal generation is complete,

0165-3806r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 3 8 0 6 Ž 9 8 . 0 0 1 3 9 - 4

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axonal and dendritic branching is robust, and synaptogenesis is just beginning w8,10x. Since preterm infants with BPD suffer chronic sublethal hypoxemia and are at high risk for neurodevelopmental handicap, we raised newborn rat pups in FIO 2 9.5% and compared them to control pups reared under normoxic conditions to test the hypothesis that chronic sublethal hypoxia results in alterations in the normal corticogenetic schedule of the developing brain.

2. Methods All studies were performed at Yale University School of Medicine and the following protocols and procedures were reviewed and approved by the Yale University Animal Care and Use Committee. Rats were reared under either hypoxic or normoxic conditions from postnatal day ŽP. 3 to P13 or to P33. On P3, litters of rat pups were culled to ten pups and placed with the dam in a Plexiglas chamber in which ambient O 2 levels were continuously monitored and controlled. Oxygen levels were monitored using a Cameron Instrument Dual Channel Oxygen Monitor attached to O 2 electrodes placed at each end of the chamber. O 2 levels were then fed to a laboratory computer to provide a record of levels throughout the experimental period. Rats raised in hypoxia were maintained at an ambient O 2 level of 9.5 " 1.0%. Normoxic animals were exposed to ambient levels of O 2 in the range of 22–23%. A small fan was used to continuously circulate air and appropriate filter canisters were used to remove excessive levels of CO 2 and humidity within the box. Twice weekly the box was opened for less than 5 min and the cages, water and food changed. All pups were weaned on P23 and the mothers were removed from the box. Subgroups of rat pups were removed from the chamber on either P13 or P33. All rats removed at P13 and a portion of those removed on P33 were immediately sacrificed. The P33 rats not sacrificed on P33 were returned to ambient oxygen concentrations and subsequently sacrificed at P63. 2.1. Fostering study Prior to beginning the analyses of corticogenesis, fostering studies were performed to assess the availability of

Fig. 1. Body and brain weights for control and hypoxic-reared rat pups at postnatal days 13, 33 and 63. Hypoxic pups were placed in FIO 2 9.5% from P3 through P33. A first group of animals was sacrificed at P13; a second group was sacrificed at P33. A third group of hypoxic-reared pups was removed from hypoxic conditions at P33 and returned to normoxic rearing conditions until sacrifice at P63.

maternal nutritional supply to rat pups reared under conditions of chronic sublethal hypoxia. For these studies, we performed daily weights on four groups of newborn rat pups. The first group was placed with their own dam in a chamber with FIO 2 9.5% on postnatal day 3 ŽP3.. Dams rotated into the hypoxia chamber for 8-h intervals were used to nurture groups two and three. Group two rats were those placed in the hypoxia chamber but fed by a rotating cycle of ‘foster dams’ who remained in the chamber for 8 h of each 24 h day; the third group of rat pups were those reared in normoxic conditions and nurtured by the ‘foster dams’ during the 16 h each day during which they were not in the hypoxia chamber. The fourth group of animals was raised with their own dam under normoxic conditions. These data demonstrated no difference in weights between rat pups reared with their own dam compared to those who were nourished by a rotating cycle of ‘foster mothers’ when all pups were exposed to chronic sublethal hypoxia. Similarly, no differences in weights were noted between the normoxic reared litters nourished by either their own dam or those nourished by the foster dams. The data suggest that adequate nutrition was available for the experimental pups but do not rule out nutritional deficits which may be a consequence of the effects of hypoxia on rat pup feeding behavior. 2.2. Subcortical white matter, Õentricular and cortical Õolume measurements

Table 1 Chronic sublethal hypoxia animal data

P13 P33 P63

Body hypoxia

Weight control Žg.

Ns5

Ns5

Ns5

Ns5

16.2"2.2) 66.1"9.8) 244.5"52

35.4"4.8 80.4"7.9 247.8"46

1.1"0.05 1.3"0.04) 1.5"0.06)

1.3"0.06 1.5"0.1 2.0"0.08

) p- 0.01.

Brain hypoxia

Weight control Žg.

Rats were weighed and then anesthetized by an overdose of pentobarbital, a thoracotomy was performed and they were transcardially perfused with 4% paraformaldehyde. The brains were cut midsagittally and one hemisphere was embedded in celloidin and cut sagittally at 50 mm. All sections were mounted and stained with cresyl violet. All sections were projected onto a counting grid using a microscopic projector and the volumes and areas

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199

Fig. 2. Sagittal cresyl-violet stained sections from control Žtop. and experimental Žbottom. animals at postnatal day 63. Note considerable ventriculomegaly ŽV. in hypoxic-reared rat pups.

of selected structures were measured using standard stereology methods w14x. The volume of the lateral ventricles was determined to assess ventriculomegaly w39x. In addition, the total volume of the corpus callosum and external capsule from the frontal region to the occipital pole w39x was calculated as a measure of subcortical white matter volume. The area of the corpus callosum calculated from three consecutive sections from the midline was determined to assess interhemispheric white matter. Cortical volume measurements were performed as previously described w46x. Values presented are means for individual hemispheric data. 2.3. Statistical methods Statistical analyses were performed using StatView software. Two-tailed t-tests were performed for between group comparisons of continuous data. All p values in this report are of the two-sided type.

These data demonstrate significant decreases in body weight in hypoxic-reared rat pups at both P13 and P33 Ž p - 0.01 for both.. When returned to normoxic conditions, hypoxic-reared pups demonstrated ‘catch-up’ body weight growth and we found no differences between the mean body weights of the two groups at P63 ŽFig. 1.. Similarly, brain weights of hypoxic-reared pups were significantly lower than normoxic-reared animals at P33. In contrast to the body weight data, the brain weights of the hypoxic-reared animals remained significantly lower than those of control pups at P63 despite normoxic rearing from P33 through P63 Ž p - 0.01.. Gross inspection of the brains revealed ventricular enlargement and cortical thinning at P63, and sections of brains from hypoxic and control pups at P63 are shown in

Table 2 Ventricular volumes of control and hypoxic rats Žmm3 . Hypoxic

3. Results Body and brain weights for hypoxic animals compared to control pups at P13, P33 and P63 are shown in Table 1.

P13 P33 P63

Control

Ns5

Ns5

0.74"0.46 2.56"0.82 3.70"0.80

0.83"0.46 1.91"0.52 1.90"0.82

p value

0.60 0.32 - 0.01

200

L.R. Ment et al.r DeÕelopmental Brain Research 111 (1998) 197–203

Fig. 3. Ventricular volume Župper left panel., subcortical white matter volume Župper right panel., corpus callosum area Žlower left panel. and cortical volume at P3, P33 and P63 for control rat pups and those reared in hypoxia from P3 through P33 and, in the P63 group, returned to normoxia from P33 through P63.

Fig. 2. The ventricular volume of the control pups, shown in Table 2 and Fig. 3 Župper left panel., appeared to stabilize by P33 and there was no difference between ventricular volumes for control animals between P33 and P63. In contrast, the ventricular volume of the hypoxic-reared pups, although not statistically different, tended to be larger than that of the control pups at P33. At P63 the ventricular volume of hypoxic pups continued to increase and was almost twice the volume of the control animals at the same postnatal age Ž p - 0.01.. Volume measurements of the subcortical white matter and cross-sectional area measurements of the corpus callosum for normoxic- and hypoxic-reared animals at P13, P33 and P63 are shown in Table 3 and Fig. 3 Župper right and lower left panels, respectively.. These data demonstrated that the volume of the subcortical white matter included

within the external capsule and corpus callosum of control animals reaches adult values at P33, and, similar to the ventricular volume data, there is little change in the subcortical white matter of control animals between P33 and Table 3 Subcortical white matter and corpus callosum

P13 P33 P63

White matter Žmm3 .

Corpus callosum Žmm2 .

Hypoxia

Control

Hypoxia

Ns5

Ns5

Ns5

Ns5

2.00"0.47) 2.69"0.45)) 3.74"0.49)

2.74"0.47 5.35"0.57 5.51"0.95

0.93"0.19) 1.82"0.61) 1.68"0.57))

1.51"0.59 2.34"0.70 2.23"0.60

) p- 0.05. )) p- 0.01.

Control

L.R. Ment et al.r DeÕelopmental Brain Research 111 (1998) 197–203 Table 4 Cortical volumes of control and hypoxic rats Žmm3 .

P13 P33 P63

Hypoxia

Control

Ns5

Ns5

31.6"2.8)) 52.4"7.2) 66.8"7.6)

45.6"4.1 84.8"9.3 85.0"8.2

) p- 0.05. )) p- 0.01.

P63. In contrast, the volume of the subcortical white matter of the hypoxic-reared animals is one-half that of the control pups at P33 Ž p - 0.01.. Although the volume of subcortical white matter of the hypoxic-reared rats increases between P33 and P63, it remains significantly less than control animals at P63 Ž p - 0.05.. Like the subcortical white matter, the corpus callosum of the control animals reaches adult size by P33 and little change was noted in this measurement at P63. Hypoxic-reared pups were found to have significantly smaller values for corpus callosal area beginning at P13 through P63 Ž p - 0.05 for P13 and P33, p - 0.01 for P63. with little evidence for ‘catch-up’ growth. Cortical volume measurements are found in Table 4 and Fig. 3 Žlower right panel.. Similar to the subcortical white matter and corpus callosum, the cortical volume of the control rat reaches adult size by P33. At P13, however, the cortical volume of the hypoxic-reared pups was significantly less than that of the control pups Ž p - 0.01.. This difference persisted throughout the experimental period Ž p - 0.05 for both P33 and P63., although some evidence for relative improvement in the cortical volume of the hypoxic-reared animals was noted at P63.

4. Discussion Review of the literature suggests that oxygen deprivation is correlated with neurodevelopmental impairments in preterm infants w50,51x, and we have designed our studies of chronic sublethal hypoxia as previously described to mimic the effects of bronchopulmonary dysplasia on the developing brain w16x. Cerebral development in the human fetus is characterized by sequential periods of cellular proliferation, migration of glia and neurons into appropriate cortical positions and the elaboration of synaptic connections with other cortical and subcortical regions of the brain, and recent data have demonstrated that the entire cerebral cortex matures as an integrated network w4,9,54x. By 25 weeks of gestation, a time of birth for which the survival rate was 60% at Yale University School of Medicine last year, almost all of the developing cortical neurons have been generated w23x, the elaboration of axonal and dendritic

201

arbors is at an active stage and many synaptic contacts are being formed in the developing cortex w19,22x. Although the establishment of these contacts may well occur independent of neuronal activity, pharmacological insults such as prenatal halothane, ethanol and vitamin B6 deprivation have all been shown to decrease synaptic density and impair learning in rats w21,25,29x. The influence of circulatory impairments on the developing preterm brain are dependent not only on the severity and duration of hypoxia to which the animal is exposed but also on the cerebral maturity of that animal w34,41,49x. Experimental animal data have included studies of both hypoxicrischemic insult and those of relatively brief periods of total anoxia. In contrast to models for the former insult, there have been few reports of gross neuropathologic changes following neonatal anoxia experiments w18,32,49x. In newborn rats, the first 20 postnatal days represent the period of rapid differentiation of axons and dendrites w8,36,42x. Laroia et al. w24x found a significant decrease in cortical volume in neonatal rats at P14 following a 3-h hypoxic insult on P7. Similarly, Dell’Anna et al. w7x noted inhibition of the normal development of the basal and apical dendrites of hippocampal pyramidal cells in neonatal rats exposed to a brief period of anoxia, and Nyakas et al. w34x reported that the number of dendritic spines of these cells remained significantly decreased 90 days following insult. Finally, rats exposed to brief periods of neonatal hypoxia have been shown to have relatively poor skills for food- and water-motivated mazes and hole-board learning paradigms when compared to control animals at various postnatal ages w34x. These data suggest that the damaging mechanisms of perinatal hypoxia may relate to subtle and yet long-lasting changes in axonal and dendritic elaboration and the formation of synaptic contacts. Since preterm infants with BPD are known to gain weight far less readily their gestational-aged matched peers with normal pulmonary function despite over-abundance of caloric input w20x, the level of chronic sublethal hypoxia which we employed, FIO 2 9.5%, was designed to result in a decrease in metabolic rate w17x. Oxygen consumption has been reported to decrease in young animals when exposed to moderate hypoxia such as we have chosen w15x, and may reflect the inability of the newborn animal to increase cardiac output to major organs during times of chronic hypoxic insult w48x. Although we have not performed studies of arterial blood gases during our study, others have evaluated these parameters w17x and noted that this level of hypoxia ŽFIO 2 9.5%. would compare to that of preterm infants with severe BPD w3,20x. When we hypothesized that the injury which preterm infants with BPD experience may be modeled by chronic sublethal hypoxia to the developing brain, the insult which we employed in newborn rats resulted in the relative failure of brain growth and progressive cerebral ventriculomegaly. These findings occurred despite ‘catch-up’

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L.R. Ment et al.r DeÕelopmental Brain Research 111 (1998) 197–203

growth in body weight for the pups reared under conditions of chronic sublethal hypoxia. Examination of the cortex at both P13 and P33 demonstrated decreased subcortical white matter volume and cross-sectional area of the corpus callosum. At P63, the volume of subcortical white matter of hypoxic animals had improved, but remained significantly less than control values. Values for corpus callosum area did not increase in hypoxic-reared pups at P63 and remained significantly low; ventricular volumes were almost two times that of control animals. Finally, cortical volumes were relatively less at all ages studied in hypoxic-reared animals. Reduction in the volume of the subcortical white matter and corpus callosum suggests that hypoxia has a profound effect on the pattern and level of cortico–cortical and cortico–fugal connectivity. Recent studies have begun to emphasize the critical role of the extrinsic circuitry interconnecting areas of the cerebral cortex in the highest levels of information processing w12x. These data extend our previous findings which demonstrated decreased neuronal size and markedly diminished neuronal apical dendritic spine counts in animals with chronic sublethal hypoxia w35,43,46x. An unexpected finding, the presence of cerebral ventriculomegaly in our model system surprisingly mimicked that found in preterm infants. Available clinical data suggest that ventriculomegaly represents a symptom of both cortical and white matter insult w2,31x, rather than a diagnosis. Preterm infants with not only bronchopulmonary dysplasia w37x, but also parenchymal involvement of intraventricular hemorrhage w52,53x, migrational abnormalities w27x and arrested hydrocephalus w26x, among other conditions, have all been shown to suffer ventriculomegaly. Finally, Gilles et al. w11x have demonstrated endotoxin-induced leukoencephalopathy in newborn kittens which has shown similarity to the ventriculomegalyrleukoencephalopathy complex found in preterm neonates following bacterial sepsis and meningitis w28x. Taken as a group, preterm infants with ventriculomegaly of all etiologies experience a high frequency of cognitive and motor handicaps w38,45,53x. The MRI study of Stewart and Kirkbride w45x of 14 year old children with a history of preterm birth demonstrated a strong correlation between both ventriculomegaly and ‘atrophy’ of the corpus callosum and adverse school performance. Stewart and Kirkbride hypothesized that the decreased callosal sizes found in their patients represented underlying alterations in hemispheric connectivity and thus provided a basis for the cognitive impairments in the study population. The present data suggest that ventriculomegaly is a consequence of the vulnerability of the preterm brain. Although the development of ventriculomegaly may reflect prior insults of many different types, our experimental studies demonstrate that chronic sublethal hypoxia is associated with alterations in white matter development, callosal connectivity and cortical volume, and secondarily with ventriculomegaly in developing brain.

Acknowledgements This study benefited greatly from the assistance of the following individuals: Karol Katz, M.S., Jonathan Leonard, B.A., Carl Seashore, B.A., and Patrice Yang. This study is supported by NS 27116 and NS 32578 of the National Institute of Neurologic Disorders and Stroke and RR 06022 of the National Center of Research Resources

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