NBQX attenuates excitotoxic injury in developing white matter

Abstract

The excitatory neurotransmitter glutamate is released from axons and glia under hypoxic/ischemic conditions. In vitro, oligodendrocytes (OLs) express non-NMDA glutamate receptors (GluRs) and are susceptible to GluR-mediated excitotoxicity. We evaluated the role of GluR-mediated OL excitotoxicity in hypoxic/ischemic white matter injury in the developing brain. Hypoxic/ischemic white matter injury is thought to mediate periventricular leukomalacia, an age-dependent white matter lesion seen in preterm infants and a common antecedent to cerebral palsy. Hypoxia/ischemia in rat pups at postnatal day 7 (P7) produced selective white matter lesions and OL death. Furthermore, OLs in pericallosal white matter express non-NMDA GluRs at P7. Unilateral carotid ligation in combination with hypoxia (6% O(2) for 1 hr) resulted in selective, subcortical white matter injury with a marked ipsilateral decrease in immature and myelin basic protein-expressing OLs that was also significantly attenuated by 6-nitro-7-sulfamoylbenzo(f)quinoxaline-2,3-dione (NBQX). Intracerebral AMPA demonstrated greater susceptibility to OL injury at P7 than in younger or older pups, and this was attenuated by systemic pretreatment with the AMPA antagonist NBQX. These results indicate a parallel, maturation-dependent susceptibility of immature OLs to AMPA and hypoxia/ischemia. The protective efficacy of NBQX suggests a role for glutamate receptor-mediated excitotoxic OL injury in immature white matter in vivo.

Fig. 1.

Developmental expression of MBP-positive cells in pericallosal white matter of immature rat brain. A, B, Lack of mature MBP-producing OLs at P4 (A) and the initial appearance of sparse MBP expression at P7 (B). C, D, Rapid progression to numerous MBP-producing OLs by P11 (C) and a less dense but mature-appearing pattern at P18 (D). Scale bar, 100 μm.

Fig. 2.

Selective white matter injury follows hypoxia/ischemia to immature rat brain at P7. A, Coronal section through the dorsal hemisphere of a rat killed 48 hr after carotid ligation and hypoxia at P7, demonstrating the absence of injury to the overlying cortex. The arrow points to tissue loss in pericallosal white matter. Top of figure represents cortical surface. B, High-power view of dying cells in an adjacent coronal section. ISEL-positive cells are common in pericallosal white matter 48 hr after hypoxic/ischemic insult. Scale bars: A, 100 μm; B, 10 μm.

Fig. 3.

Loss of immature OLs in subcortical white matter after hypoxia/ischemia. A, B, O1 staining of subcortical white matter tracts in coronal sections of a P11 rat. Sections are contralateral (A) and ipsilateral (B) to the unilateral carotid ligation that was followed by hypoxia at P7. Arrows point to an area of much reduced O1 staining after hypoxia/ischemia. Scale bar, 100 μm.

Fig. 4.

Effect of NBQX on MBP expression in cerebral white matter after hypoxia/ischemia. A–D, MBP expression in the subcortical white matter of a P11 rat after unilateral carotid ligation and hypoxia at P7, with and without NBQX treatment. MBP staining of white matter tracts contralateral (A) and ipsilateral (B) to the ligation in a vehicle-treated control and contralateral (C) and ipsilateral (D) to the ligation in a littermate post-treated with NBQX demonstrates significant attenuation of myelin loss with treatment. Scale bar, 100 μm.

Fig. 5.

Immature OLs expressing O1 antibody double-label for non-NMDA GluRs in vivo at P7. D, E, O1-expressing immature OLs in the periventricular white matter of a P7 rat (D) and GluR4 subunit expression in the same region (E). F, Superimposed images demonstrating the colocalization of receptor protein with OLs at P7.Arrows point to individual O1+ immature OLs also expressing GluR4. A, B, O4-expressing OL progenitors (A) and GluR4 subunit expression (B) in the periventricular white matter of a P4 rat. G, H, MBP-expressing OLs (G) and GluR4 subunit expression (H) in the periventricular white matter of a P11 rat. C, I, Superimposed images at P4 (C) and P11 (I) showing the absence of colocalization at the younger and older ages. Scale bar, 50 μm.

Fig. 6.

Evaluation of white matter injury with the OL-specific markers O1 and MBP. Comparison of the severity of white matter injury 96 hr after hypoxia/ischemia at P7 in treated (n = 7) and untreated (n = 9) pups shows significant attenuation of injury with NBQX post-treatment (20 mg/kg, i.p.; every 12 hr for 48 hr). NBQX treatment attenuates the loss of O1+ OLs (A; *p < 0.005) and MBP expression (B; *p < 0.001) ipsilateral to the ligation. CTL, Control.

Fig. 7.

White matter injury after intracerebral injections of AMPA. A, Injury after injection with 5 mol AMPA plus 5 nmol MK-801 (n = 8) was significantly more severe than with MK-801 alone (control, CTL; n = 7; **p < 0.001)B, The effect on rats of different ages. Significantly greater injury severity is shown in white matter at P7 (n = 8; one-way ANOVA; *p < 0.001) compared with that at younger (P4; n = 4) and older (P11; n = 6) ages and with MK-801 alone at P7 (n = 7).