Cerebral blood flow andmorphological changes after hypoxic-ischaemicinjury in preterm lambs

ENRIQUE HILARIO1, MARI CARMEN REY-SANTANO2, FELIPE GONI-DE-CERIO1,

FRANCISCO JOSE ALVAREZ2, ELENA GASTIASORO2, VICTORIA EUGENIA MIELGO2,

AMALE CABALLERO3, ADOLFO VALLS-I-SOLER4, SONIA GOMEZ-URQUIJO3

& ANTONIA ALVAREZ1

1Department of Cell Biology and Histology, 3Department of Neuroscience, and 4Department of Paediatrics, School of Medicine

and Dentistry, University of the Basque Country, Leioa, Bizkaia, Spain, and 2Research Unit on Experimental Respiratory

Physiology, Hospital de Cruces, 48903 Barakaldo, Vizcaya, Spain

AbstractAim: To evaluate the effect of cerebral hypoxia-ischaemia induced by partial occlusion of the umbilical cord on therelationship of the regional cerebral blood flow and the cerebral cell death in near-term fetal lambs.Methods: Fifteen near-termlambs were assigned to two hypoxic-ischaemic groups with or without life support (3 h), and a healthy one. Hypoxia-ischaemia was induced by partial occlusion of the umbilical cord (60 min). Routine light and electron microscopy, and theTUNEL method for apoptosis were performed. Regional cerebral blood flow was measured by coloured microspheres.Cardiovascular, gas exchange and pH parameters were also evaluated. Results: Both hypoxic-ischaemic groups produced atransient acidosis and a decrease of base excess in comparison to the healthy group. Cortical and cerebellar zones, where theregional cerebral blood flow values were similar to baseline, showed an increased number of oligodendrocyte-like apoptoticcells. In contrast, in the inner zones, where regional cerebral blood flow was increased, the number of apoptotic cells did notincrease. Necrotic neurons were observed in the basal nuclei, mesencephalon, pons and deep cerebellar nuclei.

Conclusion: Our results suggest that regional cerebral blood flow and the presence of apoptotic cells, 3 h after hypoxic-ischemic injury, are correlated.

Key Words: Apoptosis, brain damage, cell death, cellular lesion, perinatal asphyxia

Introduction

The reduction in oxygenation (hypoxia) and/or

blood flow (ischaemia) results in a perinatal asphyctic

event [1]. Cerebral hypoxic-ischaemic (HI) lesion

is one of the major causes of mortality and neuro-

logical morbidity both in the premature and in the

term newborn. Some 2 to 4 of 1000 term live births

suffer asphyxia in the prenatal or intrapartum

periods, representing up to 90% of neonatal HI

lesions [2].

At term, the most frequent intrapartum cause of

neonatal brain injury is asphyxia, which can be divided

into two types [3]. The first is the interruption of the

fetal oxygen supply by some acute obstetric events that

interfere either with the maternal or fetal blood flow

(acute abruption, umbilical cord accident, etc.). In all

cases, the maternal blood flow supply to the placenta is

compromised, and a sudden interruption of fetal

oxygenation is present. The second asphyctic mech-

anism is denominated chronic intrapartum asphyxia

[3]. During labour, each contraction of the uterus

compresses the maternal blood vessels of the placenta,

reducing the maternal blood flow and the oxygen

supply, and probably stopping it altogether. In normal

labour, the fetus is unaffected due to the short duration

of the hypoxia and the presence of a maternal blood

pool within the placenta. However, the intermittent

interruption of the maternal blood supply may be

enough to compromise the fetal acid-base balance in an

abnormal placenta. Thus, the intensity and duration of

HI injury determine the degree of cerebral damage

[4,5], which induce the appearance of a significant

proportion of cells susceptible of undergoing apoptosis

Correspondence: Enrique Hilario, Department of Cell Biology and Histology, School of Medicine and Dentistry, University of the Basque Country, 48940 Leioa,

Vizcaya, Spain. Fax: +34 944648966. E-mail: gcphiroe@lg.ehu.es

(Received 14 June 2004; revised 3 January 2005; accepted 17 January 2005)

Acta Pædiatrica, 2005; 94: 903–911

ISSN 0803-5253 print/ISSN 1651-2227 online # 2005 Taylor & Francis Group Ltd

DOI: 10.1080/08035250510031151

that significantly contributes to perinatal cerebral

damage. Moreover, the damage worsens during the

reperfusion phase [6], and continues for hours after

reoxygenation/reperfusion.

In the present work, we use a neonatal model of HI

injury at term in which the injury intensity could be

appreciated and quantified at an early stage. Also, the

injury model used to produce brain damage is similar

to the above related conditions in humans, in which

the decrease of blood flow and hypoxia are due to an

umbilical cord compression. Therefore, although our

study is developed under a less physiological condition

than fetal chronic preparation under spontaneous

behaviour as previously reported [7–12], our model

seems to be close to the clinical situation of asphyxiated

neonates in neonatal intensive care units, and it has

low cost. Also, this model could be used to evaluate

the crucial effect of neuroprotective treatments and

resuscitation techniques (air versus pure oxygen,

surfactant, etc.) to mitigate the HI injury during the

transition from fetal to neonatal life.

The aim of the present work was to study the effect of

cerebral hypoxia-ischaemia induced by partial occlu-

sion of the umbilical cord on the relationship of the

regional cerebral blood flow and the cerebral cell death

in near-term fetal lambs after resuscitation and 3 h of

neonatal life support.

Materials and methods

Animals

The study was carried out on 15 fetal lambs at 86–92%

of gestation (124–133 d of developmental age; term:

145 d). At 120 d (86%) of gestation, the develop-

mental stage of the brain of the fetal sheep is considered

to be similar to that of the full-term newborn infant

[13]. The experimental protocol met European regu-

lations for animal research (EU 86/609), and was

approved by the Experimental Research Committee.

Surgical preparation

Fifteen ewes were used for each experiment and,

independently of multiple gestation, only one fetal

lamb per ewe was used. Sheep were sedated with

xylazine (1 mg/kg) prior to surgery. Anaesthesia was

induced by an intravascular injection of ketamine

hydrochloride (5 mg/kg), and maintained with

continuous infusion of Propofol1 (10 mg/kg/h).

Hydro-electrolyte balance was adjusted by continuous

infusion of Ringer lactate (10 mg/kg/h).

At right lateral recumbent, animals were immedi-

ately intubated with an endotracheal tube (8 mm ID),

and controlled ventilation (CPU1, Omeda, Riverside,

CA, USA) was used to maintain normoxia (16.0–

20.0 kPa with inspired O2 fraction: 0.21–0.60) and

normocapnia (percentage of expired CO2: 4.5–6.0%).

An arterial catheter (Insyte 20GA Catheter, Becton

Dickinson, Drogheda, Ireland) was inserted in a

peripheral artery to monitor arterial pressure.

Fetal lambs were exposed by left laparotomy. The

head was exteriorized and an endotracheal tube (Hi-Lo

Jet Tracheal tube ID 4 mm, Mallinckrodt Medical,

St. Louis, MO, USA) was inserted. Catheters (XRO

umbilical catheter, Vygon, France) were placed in the

right jugular vein (to heart and brain directions), and

the arterial catheter was inserted in the left axillary

artery, advanced through the subclavian artery to place

the tip in the brachiocephalic trunk (Figure 1). The left

carotid artery was isolated, and a non-invasive flow

Doppler sensor was allocated to measure cerebral

blood flow in real time (T106, Transonics, Ithaca, FL,

USA).

The umbilical cord was isolated to determine both

venous and arterial flows using non-invasive flow

Doppler sensors (T106, Transonics, Ithaca, FL,

USA). Core temperature was monitored and kept

between 37 and 38�C with a temperature blanket

Figure 1. Regional cerebral blood flow was determined by coloured

microspheres infused in the left subclavian artery. In the ovine animal

model, the left and right subclavian arteries arise from the brachio-

cephalic trunk, which divides equally into left and right carotid

arteries. Arrows represent blood flow through arterial vessels.

LCA/RCA: left and right carotid arteries; DS: Doppler sensor; LSA/

RSA: left and right subclavian arteries; MI: microspheres injection;

C: catheter; BT: brachiocephalic trunk; A: aorta; DA: ductus

arteriosus; PA: pulmonary artery; PT: pulmonary trunk; RA: right

atrium.

904 E. Hilario et al.

(Digiterm S542, JP Selecta, Barcelona, Spain). At this

moment, the basal point (B) was established in all

animals and an arterial blood sample was taken and all

parameters were recorded.

Hypoxic-ischaemic injury

In the hypoxic-ischaemic experimental groups, the HI

injury was induced by partial occlusion of the umbilical

blood flow for 60 min. A rubber occluder was placed

around the umbilical cord, and the flow Doppler

sensor was maintained in the umbilical arteries in order

to continuously measure and display, in real time, the

blood flow. The occluder was properly compressed to

produce a reduction of the umbilical arterial blood flow

lower than 50% of basal value, as the flow Doppler

sensor monitor displayed. During HI injury, fetal

arterial pH, blood gases, mean systemic arterial pres-

sure and heart rate were determined at 15-min inter-

vals. The hypoxic-ischaemic endpoint was established

after 60 min of partial occlusion (HI point), and an

arterial blood sample was taken and all parameters

were recorded. Previous studies have demonstrated

that, at this point, a partial occlusion of the umbilical

cord produces an arterial pH less than 7.1, a 50%

decrease in arterial O2 content (in relation to basal

value) and a base excess less than 715 mmol/l [8].

In animals without HI injury, 60 min after basal

point, an arterial blood sample was also taken and all

parameters were recorded, in order to obtain reference

values for the hypoxic-ischaemic experimental groups

in the statistical study.

Neonatal care

All lambs received i.v. ketamine hydrochloride (8 mg)

and pancuronium bromide (0.4 mg), and then the

umbilical cord was cut. Animals were weighed (SECA

727, Snoqualmie, Germany) and placed in an open

incubator, and the endotracheal tube was connected to

a pressure-limited ventilator (Bourns BP 200, Beard

Med. Sys. Inc., Riverside, CA, USA) with the follow-

ing settings: rate 50 breath/min; peak inspiratory

pressure: 2.9 kPa; positive end-expiratory pressure:

0.5 kPa; inspiratory expiratory ratio 1 : 2; inspired O2

fraction: 1.0; and flow: 10 l/min. Anaesthesia and

paralysis were maintained by a continuous infusion of

ketamine hydrochloride (5 mg/kg/h) and pancuronium

bromide (1 mg/kg/h) in 5% dextrose.

Groups

Lambs were randomly assigned to three different

experimental groups by means of the closed envelope

method. Healthy group: after caesarean section with-

out hypoxia-ischaemia, lambs were managed on

conventional mechanical ventilation for 3 h (n=6) and

sacrificed. Hypoxic-ischaemic control group: after HI

endpoint (60 min of hypoxia-ischaemia), lambs (n=3)

were sacrificed without resuscitation or conventional

mechanical ventilation. Hypoxic-ischaemic injured

group: after 60 min of hypoxia-ischaemia, lambs were

resuscitated and managed on conventional mechanical

ventilation for 3 h (n=6) and sacrificed.

Measurement

Blood samples were obtained from the brachiocephalic

trunk for haematocrit, acid-base balance and gas

exchange. Core temperature, mean systemic arterial

pressure, heart rate and carotid blood flow were also

measured. All parameters were determined and regis-

tered at basal point (B), every 15 min during 60 min of

HI injury, during conventional mechanical ventilation

(1 h) and at 3 h (3 h). Animals were sacrificed by

means of an intravascular overdose of barbiturate.

Necropsy

After sacrifice, the fetal brains were prepared for

regional cerebral blood flow (RCBF) and histopatho-

logical studies. First, carotid arteries were isolated and

catheters were placed to perfuse Ringer lactate solution

at 4�C at a previously registered flow (100–150 ml/

min). After that, the brain was perfused with 500 ml of

4% paraformaline and removed from the skull. The

brain was divided into different cerebral regions in

order to perform RCBF or morphological studies (see

below).

Regional cerebral blood flow

Measurement of RCBF at different intervals was

determined using four coloured microspheres (Dye

Track1, Triton Technology Inc., San Diego, USA),

which were infused through the left subclavian artery

because, in the ovine animal model, the left and right

subclavian arteries arise from the brachiocephalic

trunk which divides equally into the left and right

carotid arteries (Figure 1). The infusion of coloured

microspheres was realized within 30–45 s at B, HI, 1-h

and 3-h intervals as previously described [14]. As

previously mentioned, the flow Doppler sensor and its

monitor display the value of flow at real time, so the

mean carotid blood flow displayed by the monitor

during the 30–45 s of microsphere infusion was used as

a “reference flow” value in the equation below. The

brain was divided into nine cerebral regions: cortical

zones (frontal, parietal, temporal and occipital cortex),

inner zones (thalamus, striatum and hippocampus),

cerebellum and medulla oblongata [9]. Each piece

was weighed and digested with alkali (KOH, 4 M)

during 3 h in a shaking bath at 72�C. The obtained

sample was filtered through a filter of 10-mm pore to

recover the coloured microspheres. Filters were

dried, and dimethylformamide (1.2 ml, Sigma-Aldrich

Cerebral blood flow and cell damage 905

Co. Ltd, Gillingham, UK) was used to extract dye

from the microspheres. To determine the amount of

microspheres per sample, the optical density was

measured by spectrophotometry (HP 8452A, Hewlett

Packard, Silicon Valley, CA, USA) at four different

wavelengths. RCBF was calculated as previously

described [15] as follows: tissue flow=tissue micro-

spheresrreference flow/reference microspheres.

Histology

Fixation quality was scored on a 10-point scale [16].

Thus, 0 corresponds to a bad perfusion, with collapsed

microvessels and the presence of residual erythrocytes,

whereas 10 represents good tissue preservation, in

which residual erythrocytes are not detected, the

microvessels are open and disseminated cell swelling is

absent.

Light and electron transmission microscopy

Brains were placed on a table, and cut into 0.5-cm-

thick consecutive sections. Samples from different areas

were embedded in paraffin wax for light microscopy.

The sections (5-mm thickness) were stained for routine

histological examination with haematoxylin-eosin

(HE), and immunostained for apoptotic studies.

Samples were also obtained for ultrastructural exam-

ination.

Samples processed for transmission electron

microscopy were embedded in Epon by conven-

tional methods. Briefly, tissue blocks of 1 mm3 were

immersed in 2.5% glutaraldehyde in 0.1 M sodium

cacodylate/HCl buffer (pH 7.4) for 2 h. The samples

were then post-fixed in 2% osmium tetroxide in the

same buffer for 2 h and subsequently embedded

in Epon 812 (Fluka, Switzerland). Semithin sections

(1-mm thickness) were stained with Toluidine blue,

and ultra-thin sections (60–80-nm thickness) were lead

stained and observed under the electron microscope.

In HE-stained sections, we evaluated the following

regions and parameters:

(1) Regions: cerebral cortex (frontal, parietal,

temporal, occipital), basal nuclei, hypothal-

amus, thalamus, hippocampus, amygdaloid

body, mesencephalon, pons, cerebellum (cortex

and deep cerebellar nuclei), white matter

(2) Parameters: cell injury (hydropic change,

pyknotic nuclei, etc.), focal necrosis, increase

in cellularity, oedema, microhaemorrhage,

vacuolization.

In situ apoptotic cell labelling

DNA fragmentation was examined on the paraffin

sections using the TUNEL method [17] applying the

ApopTag Apoptosis Kit (Intergene, NY, USA). The

procedure was as follows: brain sections (5-mm thick-

ness) were deparaffined, hydrated and pretreated with

20 mg/ml proteinase K for 30 min at 37�C. Endoge-

nous peroxidase activity was blocked by incubating

slides in 3% hydrogen peroxidase for 15 min. After

washing in PBS and in equilibrating buffer, the sections

were incubated in working strength terminal deoxy-

nucleotidyl transferase enzymes at 37�C for 1 h. The

reaction was halted by stop wash buffer for 10 min.

After several washes in PBS, the reaction was revealed

by peroxidase-diaminobenzidine and finally counter-

stained with methyl green. Involuting mammary gland

sections were used as positive controls for the TUNEL

method.

In each animal, we scanned the presence of apop-

totic figures, did the counting in all consecutive fields

and repeated the procedure in three paraffin sections

(20 mm apart) using a BX50 Olympus microscope.

Only unquestionable apoptotic figures were counted as

such. The amount of apoptosis was expressed as the

number of cells labelled per high power magnification

(high power magnification is meant to imply a r10 eye

piece and a r40 objective).

Statistical analyses

Values are given as mean+standard deviation (SD).

Results were contrasted with a Levene test to confirm

the homogeneity of variance between the different

treatments and a Kolmogorov-Smirnoff test for

normality. One-factor analysis of variance (ANOVA)

with Bonferroni-Dunn’s correction was performed to

assess differences in acid-base balance, base excess,

PCO2, cardiovascular parameters, RCBF and apoptotic

count as a function of group (Statview, Abacus Corp.,

USA).

Cerebral blood flow measurements of the hypoxic-

ischaemic control group were used as references for

the hypoxic-ischaemic injured group to assess the

relationship between RCBF and apoptosis count.

Comparison between parameters was performed by

two-factor ANOVA for repeated measurements as a

function of brain zones and/or groups. A p40.05 was

considered significant.

Results

Physiological data

All animals were alive at the end of the experimental

period. At baseline, all groups demonstrated a mean

systemic arterial pressure of 55+3 mmHg. This value

was maintained without significant changes until the

end of experiment, in all groups. At 3 h, the values

were 55+3 mmHg in the healthy group and

55+4 mmHg in the hypoxic-ischaemic injured group.

Sixty minutes after HI injury, we observed an increase

906 E. Hilario et al.

in the heart rate in all hypoxic-ischaemic animals

(214+11 beats/min; n=9) in comparison to the

healthy group (159+5 beats/min; n=6). However, at

3 h, no difference was observed between the

hypoxic-ischaemic injured group (249+43 beats/min;

n=6) and the healthy one (180+37 beats/min; n=6).

In uteri, all animals showed (Figure 2) an adequate

gas exchange and acid-base balance by means of the

placenta (at baseline, arterial PO2: 4.3+0.3 kPa).

During HI injury, both HI groups showed a significant

decrease of arterial pH (Figure 2a) and base excess

(Figure 2b) in comparison to the healthy group. Only

transient changes in PCO2 (Figure 2c) and hypoxia

(2.7+0.9 vs 4.1+0.1 kPa) were observed. After 3 h,

no differences were shown between the healthy and

injured groups (Figure 2).

Regional cerebral blood flow

Data of RCBF are summarized in Table I. At baseline,

in all animals, the cerebral blood flow in the cortical

regions and in the inner zones of the brain were similar

(198+68 vs 216+112 ml/100 g/min). However, the

blood flow of the cerebellum only represented 11.5%

in comparison to the other zones.

In the healthy group, RCBF did not show significant

fluctuations during the fetal or neonatal period (at 3 h,

cortical RCBF: 170+58 ml/100 g/min; inner RCBF:

106+48 ml/100 g/min; cerebellar RCBF: 24+12 ml/

100 g/min), although inner cerebral blood flow showed

a downwards trend in time.

At the point of HI injury, both hypoxic-ischaemic

groups showed a significant increase in the inner

zones (320+86 vs 160+64 ml/100 g/min), although

changes in cortical and cerebellar RCBF were not

present (Table I). At 3 h, the increase of inner RCBF

was maintained (236+102 vs 106+48 ml/100

g/min). Similarly for the healthy group, neither

the cortical nor cerebellar flow showed significant

differences.

Neuropathological findings

Samples showed a good histological preservation.

Thus, the level of brain fixation corresponds to an 8–9

in the van Reempts’ scale [16].

In both hypoxic-ischaemic groups, mild perivascular

and interstitial oedema was observed; the mesence-

phalon, pons, deep cerebellar nuclei and cerebral

cortex being the most affected zones. Necrotic scat-

tered neurons (Figure 3) were observed in the mesen-

cephalon, pons, deep cerebellar nuclei and basal

nuclei. The ultrastructural study confirms this type of

cell death. Some cells showed a minimum degree of

damage, consisting of mild hydropic changes and

the regrouping of Nissl granules. Areas of necrosis,

haemorrhage or increased cellularity were not

observed.

Quantification of cell death by TUNEL stain

Small and round apoptotic cells were located in both

white and grey matter and not circumscribed to the

Figure 2. (a) Mean basal arterial pH of all animals (open square)

corresponds to baseline point (B). Mean values for both healthy

(open triangles) and hypoxic-ischaemic (open circles) groups are

represented as a function of time (HI, 1-h and 3-h intervals).

Differences at HI point (p50.05) are demonstrated by an asterisk.

Horizontal bars close to x-axis represent chronological sequence of

events. (b) Mean basal base excess of all animals (open square)

corresponds to baseline point (B). Mean values for both healthy

(open triangles) and hypoxic-ischaemic (open circles) groups are

represented as a function of time (HI, 1-h and 3-h intervals).

Differences at HI interval (p50.05) are demonstrated by an asterisk.

Horizontal bars close to x-axis represent chronological sequence

of events. (c) Mean basal PCO2 of all animals (open square) corre-

sponds to baseline point (B). Mean values for both healthy control

(open triangles) and hypoxic-ischaemic (open circles) groups

are represented as a function of time (HI, 1-h and 3-h intervals).

Horizontal bars close to x-axis represent chronological sequence of

events.

Cerebral blood flow and cell damage 907

regions where necrotic neurons were present, which

were negative to apoptag Kit (Figure 4). In some cases,

apoptotic cells were located beside medium- and

large-sized neurons.

Table II summarizes the apoptotic cell count in

different brain areas. In the hypoxic-ischaemic injury

group, TUNEL-positive cells were higher in compar-

ison to both healthy and hypoxic-ischaemic control

ones. This increase was observed in the cerebral cortex,

cerebellum as well as in the pons. In the hypoxic-

ischaemic injury group, a non-significant increase in

the apoptotic cell count was noted in the cerebral basal

nuclei.

Relationship between RCBF and apoptotic cell count

Both parameters demonstrated a relationship as a

function of experimental group (RCBF, p=0.02;

apoptotic count, p=0.0001), but not in regard to brain

zones (RCBF, p=0.11; apoptotic count, p=0.16).

However, a relationship between RCBF and apoptotic

Table I. Regional cerebral blood flow (ml/100 g/min).

Interval

Group

B HI 1 h 3 h

All animals

(n=15)

Healthy

(n=6)

Hypoxic-ischaemic

injury (n=9)

Healthy

(n=6)

Hypoxic-ischaemic

injury (n=6)

Healthy

(n=6)

Hypoxic-ischaemic

injury (n=6)

Frontal cortex 40+12 33+6 66+15 28+4 31+6 37+13 36+5

Parietal cortex 47+8 45+8 57+6 37+5 29+9 42+11 34+10

Temporal cortex 73+66 93+85 69+24 46+13 37+5 63+43 48+28

Occipital cortex 37+9 31+2 64+19 27+4 36+3 32+10 37+14

Mean flow of cortical

zones

198+68 202+88 256+64 138+28 138+14 170+58 152+40

Striatum 54+31 42+30 91+26 36+15 52+12 29+19 49+18

Hippocampus 92+55 67+22 85+29 44+3 66+23 43+13 105+70

Thalamus 90+61 50+16 119+51 42+3 57+18 34+18 81+35

Mean flow in inner

zones

216+112 160+64 320+86a 122+26 176+32a 106+48 236+102a

Medulla Oblongata 72+62 38+13 155+59 24+6 67+18 27+10 112+81

Cerebellum 24+16 18+4 46+27 20+5 27+15 24+12 26+13

Values are given as mean+SD. One-factor analysis of variance (ANOVA) was performed between groups at each interval (B, HI, 1 h and 3 h).a p50.05 vs healthy group.

Figure 3. Presence of neurons showing coagulative changes in the deep cerebellar nuclei of the hypoxic-ischaemic injury group. Cells show

shrinkage and eosinophilia of the cytoplasm (arrows point to cells with more severe damage). Hematoxilin-eosin stain; original

magnificationr100.

908 E. Hilario et al.

count was demonstrated ( p=0.04) as a function of

brain zones and groups.

Discussion

Our results suggest a relationship between regional

cerebral blood flow and histopathological changes in

the fetal lambs. These changes consist of mild peri-

vascular and interstitial oedema with necrotic scattered

neurons and apoptotic oligodendrocyte-like cells.

The morphological changes were more incipient

than grade 1 in the classification of Ikeda et al. [8] in

near-term fetal lambs after 72 h of the HI injury.

However, after 3 h of the HI injury, we observed the

presence of neurons showing coagulative cell changes,

characteristic of cell death [16,18], which corresponds

to a grade 3 of damage in the Ikeda classification. In the

fetal lamb brain, these changes have been previously

reported in the periventricular white matter and in the

parasagital cerebral cortex after 24 h of repetitive cord

occlusion [12], and in the Purkinje cells after 72 h of

severe asphyctic injury [9].

The morphological traits and the localization (both

in grey and in white matter) of the positive TUNEL

cells suggest that these cells could correspond to

oligodendrocyte lineage. Oligodendrocytes are sus-

ceptible to a variety of injurious stimuli induced by

HI injury [1,19], and the intrinsic vulnerability of

oligodendrocyte precursors is considered central to the

pathogenesis of periventricular leukomalacia [20]. The

different mode of cell death observed in neurons and

oligodendrocyte-like cells suggests the activation of a

Figure 4. Presence of two apoptotic figures (arrows) in the basal ganglia of the hypoxic-ischaemic injury group. On the left (arrowhead), a

damaged neuron with a negative stain for TUNEL can also be observed. TUNEL counterstained with methyl green; original

magnificationr400.

Table II. Apoptosis count as a function of different brain regions and experimental groups represented as healthy, hypoxic-ischaemic control

and hypoxic-ischaemic injured groups.

Group

Healthy

(n=6)

Hypoxic-ischaemic control

(n=3)

Hypoxic-ischaemic injury

(n=6)

Parietal cortex 10+4 9+5 79+15a

Temporal cortex 12+2 16+14 82+16a

Basal nuclei 10+2 12+8 41+15

Hypothalamus 20+7 34+11 14+9

Hippocampus 30+3 31+9 23+3

Thalamus 18+3 24+16 24+9

Mesencephalon 39+6 10+1 38+10

Pons 17+10 20+10 55+5a

Cerebellum 27+6 29+10 104+15a

Three sections per zone and animal were evaluated and the mean value+SD expressed as the number of apoptotic cellsr1072 by high-power

microscopic field.a p50.05 vs healthy and hypoxic-ischaemic control groups.

Cerebral blood flow and cell damage 909

different death programme in these cells due to the HI

injury. In this respect, intracellular levels of ATP and

the intensity of the initial insult have been involved in

the fate of cell death by necrosis or apoptosis [5,21].

However, the existence of neuronal subpopulations

and glial cells with a differential sensitivity to asphyxia

or with different abilities to generate ATP via glycolysis

[22] could be also considered.

The fetal lamb model is ideal for the study of cerebral

metabolism and blood flow [7,23,24]. It has been used

in the study of the influence of several physiopa-

thological factors (hypoxia, PCO2 level, acidosis, etc.),

autoregulation and O2 delivery on cerebral blood flow

and on metabolic energy status. Cerebral blood flow is

regulated by the cerebral perfusion pressure and the

cerebrovascular resistance, which is controlled by

arteriolar tone under the influence of blood compo-

nents, blood gases (O2, CO2), pH, etc. [25,26]. In the

fetus, during the initial phase of hypoxia, the increase

of arterial PCO2 and the decrease of arterial PO2 induce

a cerebral vasodilatation. Later, the arterial blood

pressure and the cardiac output decrease with a loss of

cerebral autoregulation and, finally, a change in RCBF

occurs [27]. Also, the alteration of CO2 levels produces

a sudden fall in acid-base balance [28], which also

contributes. In our work, the observed alteration of

CO2, pH, heart rate, etc. could be responsible for the

changes of RCBF during HI injury. Also, we must

consider that the resuscitation and the postnatal

ventilatory management did not affect the auto-

regulation of RCBF, because these parameters were

close to basal (normal) values.

In our study, the cortical and cerebellar zones, where

the RCBF values were similar to baseline, showed an

increased number of apoptotic cells. In contrast, in the

zones where RCBF was increased (inner zones), the

number of apoptotic cells did not increase. Partial

umbilical cord occlusion produces a continuous

decrease of O2 content [8,9] and, consequently, those

brain areas, even with a non-altered flow, but receiving

less O2 content, are susceptible to producing brain cell

injury. In near-term fetal lambs, the failure in perfusion

and/or hypoxia by partial occlusion of the umbilical

flow produces a brain lesion that resembles periven-

tricular leukomalacia findings [8,19]. The differential

distribution between cerebral blood flow and the

apoptotic or necrotic areas observed remains to be

elucidated.

Acknowledgements

We thank Prof. David Hallett for his careful review of themanuscript. This work has been partially supported by grantsfrom Fondo de Investigacion Sanitaria, Ministerio deSanidad (FIS01/0110-1 and FIS01/0110-2), and from theUniversidad del Paıs Vasco (1/UPV075.327-E-14885/2002and 9/UPV00077.327-15330/2003 Hilario). The authorscarried out the work on behalf of the RESPIRA group of

Cooperative Research Networks of Fondo de InvestigacionSanitaria (C03/11).

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