Experimental modelling of the consequences of brief late gestation asphyxia on newborn lamb behaviour and brain structure

Brief but severe asphyxia in late gestation or at the time of birth may lead to neonatal hypoxic ischemic encephalopathy and is associated with long-term neurodevelopmental impairment. We undertook this study to examine the consequences of transient in utero asphyxia in late gestation fetal sheep, on the newborn lamb after birth. Surgery was undertaken at 125 days gestation for implantation of fetal catheters and placement of a silastic cuff around the umbilical cord. At 132 days gestation (0.89 term), the cuff was inflated to induce umbilical cord occlusion (UCO), or sham (control). Fetal arterial blood samples were collected for assessment of fetal wellbeing and the pregnancy continued until birth. At birth, behavioral milestones for newborn lambs were recorded over 24 h, after which the lambs were euthanased for brain collection and histopathology assessments. After birth, UCO lambs displayed significant latencies to (i) use all four legs, (ii) attain a standing position, (iii) find the udder, and (iv) successfully suckle - compared to control lambs. Brains of UCO lambs showed widespread pathologies including cell death, white matter disruption, intra-parenchymal hemorrhage and inflammation, which were not observed in full term control brains. UCO resulted in some preterm births, but comparison with age-matched preterm non-UCO control lambs showed that prematurity per se was not responsible for the behavioral delays and brain structural abnormalities resulting from the in utero asphyxia. These results demonstrate that a single, brief fetal asphyxic episode in late gestation results in significant grey and white matter disruption in the developing brain, and causes significant behavioral delay in newborn lambs. These data are consistent with clinical observations that antenatal asphyxia is causal in the development of neonatal encephalopathy and provide an experimental model to advance our understanding of neuroprotective therapies

Gestational age, and body weights at birth for lambs born after a normal pregnancy (Full-term control, n = 8), after experiencing 10 mins of cord occlusion <i>in utero</i> at 132 days gestation (UCO, n = 6), or after induction of preterm birth by treatmen

<p>Age at delivery shown as median (range), all other data shown as mean ± SEM.</p>*<p>indicates <i>p</i>&lt;0.05, compared to full-term controls.</p>†<p>indicates P&lt;0.05 UCO compared to preterm group. Abbreviation: UCO, umbilical cord occlusion.</p

Hematoxylin &amp; Eosin (A–C).

<p>Few degenerating cells were seen in the cortical gray matter of full-term (A) and preterm (B) control lambs. UCO lambs showed extensive neuronal injury displaying feature of apoptosis seen by H&amp;E staining as scattered dark, shrunken cells with pyknotic or small, densely staining nuclei and eosinophilic cytoplasm (black arrows C). The insert in panel C is a high power micrograph showing a neuron exhibiting ischemic morphology (eosinophilia, and nuclear pyknosis). Panels D–F show activated Caspase-3 immunoreativity in the cortex of a full term (D), preterm (E) and UCO (F) lambs. Note the increased immunoreactivity of activated Caspase-3 in the cortex of UCO lambs compared with both control groups. Scale bars –50 µm. Panel G: Quantitative results show cleaved caspase-3 cell counts in the corpus callosum, periventricular white matter (PVWM), external capsule, cortex and subventricular zone (SVZ) for full-term control (n = 5), preterm control (n = 5) and UCO lambs (n = 6) (G). Data are expressed as mean ± SEM. <i>p</i>&lt;0.05. White, gray and black bars represent full term, pre term and UCO lambs respectively.</p

Hematoxylin &amp; Eosin staining of full term control (A, D &amp; G), preterm control (B, E &amp; H), and UCO lambs (C, F &amp;I).

<p>Normal cytoarchitecture of the cerebral cortex was seen in both full-term (A) and preterm brains (B). In UCO brains (C) subtle alterations in the cellular composition and spatial arrangement of neurons was seen throughout the cortical gray matter. Extensive neuronal injury (arrows) in the cortical gray matter of UCO lambs as well as areas devoid of neurons (asterisks). D &amp; E show normal pathology in the cortex of full term and preterm control lambs respectively. In UCO lambs (F) some other cellular degenerative changes were observed, such as vacuolation of brain parenchyma (arrow). The insert in panel F shows a high power view of the vacuolar degeneration, histologic features consistent with hypoxic/ischemic changes. Inflammatory cell infiltration was seen in the periventricular white matter of UCO lambs (I), as marked by the box, which showed the morphological appearance of eosinophils (i), lymphocytes (ii) and neutrophils (iii) (inserts in I). These were not seen in full term (G) or preterm control lambs (H). Panels J, K &amp; L are representative images of glial fibrillary acidic protein (GFAP) staining in the periventricular white matter of full term control, preterm control and UCO lambs respectively. UCO lambs displayed reactive astrocytosis. Note the dense staining of the enlarged cell bodies and the highlighted cell processes shown in the high power insert in panel L. Scale bars: A–F = 100 µm, G–L = 50 µm.</p

Photomicrograph showing changes to the cerebrovasculature following UCO.

<p>Panels A–C show albumin staining in the cortical gray (CxGM) and subcortical white matter (CxWM) of a full term (A), a preterm (B) and a UCO lambs (C). Albumin extravasation (brown staining) consistent with blood brain barrier permeability disruption was observed throughout the brain in UCO lambs (C). Note the positive albumin staining around a blood vessels (circle), as well as in cells (arrows). The insert in C is a high power photomicrograph showing the albumin staining surrounding a blood vessel (BV), as well as albumin positive cells, We observed moderate levels of albumin extravasation in some pre tem control lambs (B), while no albumin staining was noted in brains of full term control lambs. Panels D, E and F show Mallory trichrome staining in the periventricular white matter of a full term (D), preterm (E) and UCO lamb (F). Microbleeds were seen in UCO brains shown by degradation products of hemorrhage staining a muddy brown. No microbleeds were detected in full term or preterm control brains. G–I show GFAP immunohistochemistry and show normal perivascular astrocytes in the periventricular white matter of a full term (G) and preterm lamb (H); while blood vessels in UCO lamb were often seen to be devoid of astrocytic contact (I). BV = blood vessel. Scale bars: A–C = 100 µm; D–I = 20 µm.</p

Luxol fast blue staining on brain sections of full term control (A), preterm control (B) and UCO lamb (C) in the corpus callosum.

<p>Myelin irregularities (disruption) seen as patchiness (asterisks in C) were detected only in UCO lambs. Panels D–F show CNPase immunohistochemistry in the corpus callosum of a full term (D), preterm (E) and UCO lamb, confirmed myelin disruption seen as patchy staining (asterisks). Myelin disruption was also seen in the periventicular white matter of UCO lambs both with luxol fast blue (I) and CNPase (L) (asterisks). Myelination in full term (G &amp; J) and preterm (H &amp; K) appeared to be intact with both stains. Scale bars = 50 µm. Quantitative results show densitometry analysis of CNPase stained myelination (M) and CNPase positive cell bodies (N) in the corpus callosum, subcortical (CxWM) and periventricular white matter (PVWM), external and internal capsule for full-term control (n = 8), preterm control (n = 4) and UCO lambs (n = 5). Data are expressed as mean ± SEM. <i>p</i>&lt;0.05. White, gray and black bars represent full term, pre term and UCO lambs respectively.</p

Postnatal lamb behavior was observed from the time that lambs fully cleared the birth canal (time 0).

<p>Time taken to use hind limbs (A), four legs (B), stand stable for &gt;5 sec (C), find the udder (D) and successfully suckle (E) for full-term control (n = 6), preterm control (n = 5) and UCO lambs (n = 5) were recorded. The percentage of time spent active from 4 to 23 h was also monitored (G). Data are expressed as mean ± SEM. <i>p</i>&lt;0.05.</p

Fetal arterial blood gases, pH, hematocrit, blood lactate, and arterial pressure and heart rate before (−1 h), at the end of (+10 mins), and 1 to 48 h after occlusion of the umbilical cord at 132 days gestation.

<p>Data shown as mean ± SEM, n = 5 throughout, except for +24 h and +48 h (n = 4).</p>*<p><i>p</i>&lt;0.05, compared to values at −1 h. (n/a = data not available).</p