Tumour necrosis factor blockade after asphyxia in foetal sheep ameliorates cystic white matter injury

Abstract

Cystic white matter injury is highly associated with severe neurodevelopmental disability and cerebral palsy in preterm infants, yet its pathogenesis remains poorly understood and there is no established treatment. In the present study, we tested the hypothesis that slowly evolving cystic white matter injury after hypoxia-ischaemia is mediated by programmed necrosis initiated by tumour necrosis factor. Tumour necrosis factor blockade was begun 3 days after hypoxia-ischaemia to target the tertiary phase of injury, when most secondary cell death is thought to be complete. Chronically instrumented preterm foetal sheep (0.7 gestation) received 25 min of hypoxia-ischaemia induced by complete umbilical cord occlusion or sham-umbilical cord occlusion (controls, n = 10), followed by intracerebroventricular infusion of the soluble TNF inhibitor, Etanercept, at 3, 8 and 13 days after umbilical cord occlusion (n = 9) or vehicle (n = 9). Foetal brains were processed for histology at 21 days after umbilical cord occlusion. Umbilical cord occlusion with vehicle was associated with a spectrum of macroscopic white matter degeneration, including white matter atrophy, ventriculomegaly and overt temporal lobe cystic white matter injury. Oligodendrocyte maturational arrest and impaired labelling of myelin proteins, characteristic of diffuse white matter injury, was observed in the parietal lobe and surrounding the cystic lesions in the temporal lobe. Etanercept markedly attenuated cystic white matter injury on the side of the intracerebroventricular infusion, with partial contralateral protection. Further, Etanercept improved oligodendrocyte maturation and labelling of myelin proteins in the temporal and parietal lobes. The present study shows that cystic white matter injury reflects late-onset tertiary cell death mediated by delayed neuroinflammation through the tumour necrosis factor pathway. Delayed tumour necrosis factor blockade markedly attenuated cystic white matter injury and restored oligodendrocyte maturation and deficits in myelin protein expression. These data suggest that delayed tumour necrosis factor blockade may represent a viable therapeutic strategy to reduce the risk of cystic and diffuse white matter injury and potentially cerebral palsy after preterm birth, with a surprisingly wide therapeutic window.

Keywords: Etanercept; hypoxia-ischaemia; periventricular leukomalacia; preterm; white matter injury.

Figure 1

Macroscopic WMI. Examples of coronal sections of the right hemisphere and enlargement of the temporal lobe after 21 days recovery from UCO labelled for MBP-positive myelin proteins (left column) and Iba-1-positive microglia (right column). Note the cystic white matter lesions observed in the temporal lobe in the UCO-vehicle group (arrows), and absence of cystic lesions in the control and UCO-Etanercept groups. Images were taken at ×2.5 magnification. The top left coronal section shows the five white matter regions assessed for microscopic analysis. Scale bar = 10 mm for the right hemisphere and 1 mm for enlarged sections. Bottom graph shows that no relationship was found between the severity of hypotension during UCO (assessed as the area of arterial pressure under 20 mmHg) and the intact white matter area.

Figure 2

Temporal lobe WMI. Top panels show the numbers of total (Olig-2) and mature (CC1) oligodendrocytes and the area fractions of two myelin proteins (MBP and CNPase) in the TWM regions (TWM1 and TWM2). Asterisk denotes significant difference of P < 0.05 between the two groups indicated. Data are means ± SEM. Bottom panels show the representative photomicrographs of MBP-positive and CNPase-positive labelling and total (Olig2) and mature (CC1) oligodendrocytes in the TWM2 region. Note the sparse labelling of myelin proteins with abnormal morphology in the UCO-vehicle group. Improved myelin protein morphology is observed in the UCO-Etanercept group, more closely resembling that observed in the control group. All images were taken at ×20 magnification, insets are ×3 enlargements. Scale bar = 100 µm.

Figure 3

Temporal lobe white matter neuroinflammation. Top panels show the numbers of microglia (Iba-1) and astrocytes (GFAP) in the TWM regions (TWM1 and TWM2). Asterisk denotes significant difference of P < 0.05 between the two groups indicated. Data are means ± SEM. Bottom panels show the representative photomicrographs of microglia (Iba-1) and astrocytes (GFAP) in the TWM2 region. Note the profound decrease in microglia density, as well as a more subtle decrease in astrocyte density in the UCO-Etanercept group. All images were taken at ×20 magnification, insets are ×3 enlargements. (A) Example of ramified microglia in the control group. (B) Example of amoeboid microglia in the UCO-vehicle group. (C) Examples of ramified microglia and (D) amoeboid microglia in the UCO-Etanercept group. Scale bar = 100 µm.

Figure 4

Parietal lobe WMI. Top panels show the numbers of total (Olig-2) and mature (CC1) oligodendrocytes and the area fractions of two myelin proteins (MBP and CNPase) in the parietal white matter regions (IGWM1/2 and PVWM). Asterisk denotes significant difference of P < 0.05 between the two groups indicated. Data are means ± SEM. Bottom panels show the representative photomicrographs of MBP-positive and CNPase-positive labelling in the IGWM2 region. Note the near-normal intensity and morphology of myelin protein labelling in both the UCO-vehicle and UCO-Etanercept groups, closely resembling that observed in the control group. All images were taken at ×20 magnification, insets are ×3 enlargements. Scale bar = 100 µm.

Figure 5

Parietal lobe white matter neuroinflammation. Top panels show the numbers of microglia (Iba-1) and astrocytes (GFAP) in the parietal white matter regions (IGWM1/2 and PVWM). Asterisk denotes significant difference of P < 0.05 between the two groups indicated. Data are means ± SEM. Bottom panels show the representative photomicrographs of Iba-1-positive microglia and GFAP-positive astrocytes in the IGWM2 region. Note the profound reduction in both microglia and astrocyte density in the UCO-Etanercept group back to control levels. Micrographs also display a similar proportion of ramified to amoeboid microglia morphology between UCO-Etanercept and the control group. All images were taken at ×20 magnification, insets are ×3 enlargements. Scale bar = 100 µm.