Regional differences in susceptibility to hypoxic-ischemic injury in the preterm brain: exploring the spectrum from white matter loss to selective grey matter injury in a rat model

Abstract

Models of premature brain injury have largely focused on the white matter injury thought to underlie periventricular leukomalacia (PVL). However, with increased survival of very low birth weight infants, injury patterns involving grey matter are now recognized. We aimed to determine how grey matter lesions relate to hypoxic-ischemic- (HI) mediated white matter injury by modifying our rat model of PVL. Following HI, microglial infiltration, astrocytosis, and neuronal and axonal degeneration increased in a region-specific manner dependent on the severity of myelin loss in pericallosal white matter. The spectrum of injury ranged from mild, where diffuse white matter abnormalities were dominant and were associated with mild axonal injury and local microglial activation, to severe HI injury characterized by focal MBP loss, widespread neuronal degeneration, axonal damage, and gliosis throughout the neocortex, caudate putamen, and thalamus. In sum, selective regional white matter loss occurs in the preterm rat concomitantly with a clinically relevant spectrum of grey matter injury. These data demonstrate an interspecies similarity of brain injury patterns and further substantiates the reliable use of this model for the study of preterm brain injury.

Figure 1

Periventricular white matter loss following hypoxia-ischemia (HI) in postnatal day 6 (P6) rats. Seventy-two hours following HI at P6, myelin basic protein (MBP) is significantly depleted in the hemisphere ipsilateral to carotid ligation. Representative photomicrographs show MBP in hemispheres both ipsilateral (a₁–d₁) and contralateral (a₂–d₂) to carotid ligation in animals with Grade 0 MBP loss (no discernable white matter injury, a₁-a₂), Grade 1 MBP loss (mild white matter injury, b₁-b₂), Grade 2 MBP loss (moderate white matter injury, c₁-c₂), and Grade 3 MBP loss (severe white matter injury, d₁-d₂). Histogram shows average percent change in MBP for each group (e).

Figure 2

Astrogliosis and microgliosis in relation to severity of white matter injury in hypoxic-ischemic (HI) neonatal rats. Seventy-two hours following HI at postnatal day 6 (P6), numerous reactive astrocytes (a–d) and activated microglia (e–h) are present throughout the brains of neonatal rats with significant white matter injury. Representative photomicrographs depict GFAP-positive astrocytes and mild (a), moderate (b), and severe (c) astrogliosis following HI. Histogram in (d) displays the region-specific pattern and degree of reactive astrocytosis as a function of MBP loss. Lower panels depict CD68-positive microglia and mild (e), moderate (f), and severe (g) microgliosis following HI. Histogram in (h) shows the region-specific pattern and degree of microglial activation as a function of white matter injury severity. Magnification 40x.

Figure 3

Significant grey matter injury accompanies myelin basic protein (MBP) loss in postnatal day 6 (P6) neonatal rats. Grey matter injury, as determined by neuronal and axonal degeneration, increases in hypoxic-ischemic neonatal rats with the severity of white matter injury. Representative photomicrographs show Grade 1 MBP loss (a), with mild cortical neuronal degeneration (FJB staining, (b)) and axonal injury (fractin immunoreactivity, (c)). With Grade 2 MBP loss (d), the density and distribution of FJB-positive neuronal cell bodies (e) and fractin-positive axons (f) increase in the cortex overlying the periventricular white matter. With Grade 3 MBP loss (g), the density of FJB-positive neuronal cell bodies (h) and fractin-positive spheroids (i) are further increased and encompasses the majority of the cortical mantle, as well as the hippocampus, thalamus, caudate putamen, and internal capsule. Histograms display region-specific neurodegeneration (j) and axonal injury (k) in relation to the severity of white matter loss. Magnification 100x for MBP and 25x for FJB and fractin.