Central Role of Microglia in Neonatal Excitotoxic Lesions of the Murine Periventricular White Matter

Abstract

Periventricular leukomalacia (PVL) is the main cause of neurologic handicap in pre-term infants. The understanding of cellular and molecular mechanisms leading to white matter damage is critical for development of innovative therapeutic strategies for PVL. The pathogenesis of PVL remains unclear but possibly involves glutamate excitotoxicity as an important molecular pathway. We previously described a neonatal mouse model of excitotoxic white matter lesion mimicking human PVL. In the present study, we used this experimental tool to investigate the cellular populations and the glutamate receptor subtypes involved in excitotoxic white matter lesions. Combined immunohistochemical, electron microscopic, and cell death detection data revealed that microglial activation and astrocytic death were the primary responses of white matter to excitotoxic insult. In vitro experiments suggested that microglia activated by ibotenate released soluble factors that kill astrocytes. The use of selective agonists and antagonists of glutamate receptors revealed that N-methyl-D-aspartate (NMDA) receptor activation was essential and sufficient to produce cystic white matter lesions. NMDA receptor immunohistochemistry labeled microglial cells in the neonatal periventricular white matter. The developing white matter displayed a window of sensitivity to excitotoxic damage that was paralleled by the transient presence of NMDA receptor-expressing white matter cells. Assuming that similar pathophysiologic mechanisms are present in human pre-term infants, microglia and NMDA receptors could represent key targets for treatment of PVL.ibotenate was administered at post-natal day (P) 0 to P2. In contrast, ibotenate injected at P5 or P10 killed neurons in all cortical layers. Furthermore, ibotenate at P2 and P5 induced periventricular white matter cystic lesions mimicking several aspects of human cystic PVL.Using our mouse paradigm of excitotoxic white matter lesions, the present study aimed to determine the cellular cascade leading to periventricular cystic lesions. The contribution of the different glutamate receptors was also addressed. Materials and MethodsAnimals and drug administration. Swiss pups (n = 456) and transgenic C57bl / 6xDBA 2 pups (n = 47) expressing the lacZ reporter gene under the control of plp regulatory sequences were used for the experiments. The plp gene encodes the proteolipid proteins and its alternatively spliced product DM-20, which are major proteins of central nervous system myelin. Previous work with this transgenic line indicates that, both in vivo and in vitro, the transgene is expressed by O4-positive pre-oligodendrocytes and by RIP-positive differentiated oligodendrocytes, but not by neuronal cells, astrocytes, or radial glial cells (46). Furthermore, the pattern of beta-galactosidase expression is similar and superimposable on the expression pattern of endogenous plp/dm-20 (46). These transgenic mice were used to study the outcome of the oligodendroglial lineage following ibotenate injections.

Vulnerability of white matter towards antenatal hypoxia is linked to a species-dependent regulation of glutamate receptor subunits

“…Several lines of evidence point to a role for neuronal glutamate receptors in many neurological disorders in neonates and adults (5,8,(24)(25)(26)(27). Here, we documented down-regulation just after birth of the AMPA-receptor subunits 1, 2, and 4; of the kainate-receptor subunit 7; and of the metabotropic receptor subunits 1, 2, 3, 5, and 7 in hypoxic mouse pups but not in hypoxic rat pups.…”

“…Because excitotoxicity and genetic regulation of glutamatereceptor expression are known to have a key role in brain damage (8,14,17,18), we investigated whether glutamate-receptor expression was altered by hypoxia, and whether these alterations differed between rats and mice, potentially explaining the phenotypic differences noted between these 2 species. We assessed the expression of glutamate-receptor subtypes in rat and mouse at early, intermediate, and late stages of brain development.…”

“…Excessive activation of glutamate receptors may cause cell vulnerability, in part as a result of intracellular calcium influx (5,6). Intracerebral injection of glutamate agonists into the neocortex and white matter of newborn rodents produces histological lesions that mimic the brain damage observed in preterm neonates (7)(8)(9)(10).…”

Glial activation in white matter following ischemia in the neonatal P7 rat brain

“…In our model, a significant increase of ameboid microglia occurred between 24 and 72 hours postischemia, during the window of vulnerability to selective HI white matter injury, as compared to controls. In the P7 rat, microglia have been implicated in the pathophysiology of neonatal injuries with a transient increase in microglial antigens between 24 hours and 3-4 days post HI in P7 rats (McRae et al, 1995), and after excitotoxic lesions of the developing murine periventricular white matter (Tahraoui et al, 2001). Here, we demonstrated that i) early NG2-positive OLs matured into O4-positive OLs in the cingulum and were also present at the vicinity of the cyst, and ii) increased macrophage/microglia phagocytosed O4-positive OLs, suggesting that the presence of activated microglia interferes with the maturation of immature OLs leading to impaired remyelination.…”

Microglia activation in the extremely preterm human brain

“…The PVWM regions are the most frequent site of preterm white matter injuries, such as periventricular leukomalacia, punctate lesions, and diffuse excessive high signal intensity, which are evident on magnetic resonance imaging (MRI). Animal studies have highlighted the critical role played by microglia in periventricular leukomalacia (16), lipopolysaccharide-induced inflammation (17,18), neonatal hypoxic-ischemic encephalopathy (17,19), and germinal matrix hemorrhage/intraventricular hemorrhage (GMH/IVH) (20). Despite a decrease in its incidence with routine use of both antenatal steroids and artificial surfactant at delivery, GMH/IVH remains one of the major complications of very preterm birth and brain injury.…”