White matter vulnerability to ischemic injury increases with age because of enhanced excitotoxicity

Abstract

Stroke incidence increases with age and this has been attributed to vascular factors. We show here that CNS white matter (WM) is intrinsically more vulnerable to ischemic injury in older animals and that the mechanisms of WM injury change as a function of age. The mouse optic nerve was used to study WM function. WM function in older animals (12 months) was not protected from ischemic injury by removal of extracellular Ca2+ or by blockade of reverse Na+/Ca2+ exchange, as is the case with young adults. Ischemic WM injury in older mice is predominately mediated by glutamate release and activation of AMPA/kainate-type glutamate receptors. Glutamate release, attributable to reverse glutamate transport, occurs earlier and is more robust in older mice that show greater expression of the glutamate transporter. The observation that WM vulnerability to ischemic injury is age dependent has possible implications for the pathogenesis of other age-related CNS conditions.

Figure 1.

Recovery of WM function depends on age and the duration of OGD. The CAP area recovery after 30, 45, or 60 min of OGD was either similar (a) reduced (b), or completely abolished (c) in 12-month-old MONs compared with 1-month-old MONs. Each point represents the CAP area averaged from the number of separate experiments indicated in parentheses. CAPs were elicited every 30 s by supramaximal stimulation. For simplifying the illustrations, the CAP area and SEMs are plotted every 3 min. d, Aging increased the vulnerability of axons to OGD, but the evoked CAPs preserved their characteristic forms. Representative traces show CAPs and the extent of recovery after 30, 45, or 60 min OGD in 12-month-old animals. Traces represent CAPs before OGD (1), at the end of OGD (2), and after recovery (3) as indicated in the plot (a). *p = 0.0185, **p = 0.0011, Student's t test. Calibration: 2 mV, 1 ms.

Figure 2.

Similar durations of OGD cause a greater extent of irreversible functional injury in older WM. The extent of CAP area recovery was drastically reduced after 30 (a), 45 (b), or 60 min (c) of OGD starting at ages 18, 12, and 12 months, respectively. *p < 0.05, **p < 0.01, ***p < 0.0016, one-way ANOVA. Error bars indicate SEM.

Figure 3.

OGD-induced injury in older WM is temperature dependent and evident in mice of different strains. a, The 45 min OGD reduced CAP area recovery in 20-month-old compared with 3-month-old MONs obtained from C57BL/6 mice. **p = 0.003, Student's t test. b, Lowering the temperature from 37 to 31°C dramatically restored the CAP area recovery in 24-month-old MONs after 45 min of OGD. The inset shows quantification of CAP area recovery after 45 min of OGD at 37°C (0.0 ± 5.6%; n = 6) or at 31°C (39.6 ± 5.6%; n = 7; ***p = 0.0004, Student's t test). Error bars indicate SEM.

Figure 4.

OGD-induced injury in older WM is Ca²⁺ independent. MONs were exposed to OGD in normal ACSF (containing 2 mm Ca²⁺) or in ACSF with no Ca²⁺ (plus 200 μm EGTA and 4 mm Mg²⁺). Superfusion conditions were maintained starting 30 min before, during, and 30 min after OGD. a, Pretreatment with Ca²⁺-free ACSF offered nearly complete recovery from 60 min OGD injury in 1-month-old MONs (95.6 ± 4.3%; n = 6). The CAP area recovery in 12-month-old MONs failed to show comparable recovery after 30 min (b) or 45 min of OGD. Reproduced from Tekkök et al. (2007). c, Under identical conditions. The stability of the CAP area recovery after OGD in 12-month-old MONs dropped below recovery levels in Ca²⁺ containing ACSF (with Ca²⁺, 60.5 ± 9%, without Ca²⁺, 8.9 ± 6.5% after 30 min of OGD; with Ca²⁺, 21.7 ± 1.8%, without Ca²⁺, 5.9 ± 6.8% after 45 min of OGD). *p < 0.05, **p < 0.002, ***p < 0.0001, Student's t test. Error bars indicate SEM.

Figure 5.

Blockade of the reversal of NCX does not reduce OGD-induced injury in WM from older animals. a, KB-R (10 μm), a specific blocker of the reversal of NCX, reduced CAP loss during OGD and substantially improved CAP area recovery after 60 min of OGD (43.3 ± 6.9%) in 1-month-old MONs. b, KB-R prevented complete loss of the CAPs during OGD but did not improve recovery after 45 min of OGD in 12-month-old MONs (23.1 ± 14.8%). **p < 0.008, Student's t test. Error bars indicate SEM.

Figure 6.

Blockade of AMPA/kainate receptors reduces OGD-induced injury in WM from all ages. Application of NBQX (30 μm) reduced CAP loss during OGD and substantially improved CAP area recovery in 12-month-old MONs after 45 min (71.2 ± 6.8%) (a), 60 min (63.5 ± 6.9%) (b), and 45 min of OGD in 24-month-old MONs (70.9 ± 5.5%) (c). d, Quantification of CAP area recovery after 45 or 60 min of OGD in MONs pretreated with NBQX indicated comparable protection of WM function independent of age or duration of OGD. ***p < 0.0001, two-way ANOVA. Error bars indicate SEM.

Figure 7.

Blockade of NMDA receptors does not reduce OGD-induced injury in WM from older animals. The NMDA receptor glycine binding site blocker, 7-CKA (50 μm), did not improve CAP area recovery after 30 min (a) or 45 min (b) of OGD in 12-month-old MONs. NMDA receptor blockade caused delayed loss of CAP area after OGD only in 12-month-old MONs. c, Quantification of CAP area recovery after 30, 45, or 60 min of OGD in the 1-month-old MONs pretreated with 7-CKA neither improved nor hindered recovery. In 12-month-old MONs, the CAP area recovered less in 7-CKA compared with ACSF only. Error bars indicate SEM. d, NMDA receptors (red) were expressed in all age groups and mostly on glial cell bodies (blue). ***p < 0.001, two-way ANOVA. Scale bar, 10 μm.

Figure 8.

Blockade of the Na⁺-dependent glutamate transporters reduces OGD-induced injury in WM from all ages. The potent and competitive blocker of Na⁺-dependent glutamate transport, dl-TBOA (200 μm), effectively improved the CAP area recovery after 45 min (a) or 60 min (b) OGD in 12-month-old MONs and 45 min OGD in 24-month-old MONs (c). d, Quantification of CAP area recovery after pretreatment with dl-TBOA revealed comparable protection of WM function, independent of age or duration of OGD. ***p < 0.001, two-way ANOVA. The selective blocker of Na⁺-dependent glutamate transporter GLT1, DHKA (200 μm), improved the CAP area recovery after 60 min OGD in 1-month-old (20.5 ± 5.1, n = 12; vs 39.4 ± 6.6, n = 6) (e) and 12-month-old MONs (0 ± 1, n = 6; vs 57.2 ± 7.2, n = 6) (f). *p = 0.04, ***p < 0.0001, Student's t test. Error bars indicate SEM.

Figure 9.

Increases in GLT1 expression in older WM is correlated with increased release of glutamate during OGD. a, Baseline glutamate release in 1- or 12-month-old MONs was low and remained stable under control conditions. Glutamate release steadily increased starting at 30 min after the onset of OGD and reached a level fivefold greater than baseline in 1-month-old MONs (n = 7). Under identical conditions, glutamate release started 20 min after the onset of OGD and was significantly higher at every time point during OGD in 12-month-old compared with 1-month-old MONs (n = 7). Note the relatively sustained glutamate levels after the conclusion of OGD in 12-month-old MONs. b, GLT1 (red) and GFAP (green) labeling indicated that GLT1 was mainly expressed on astrocytes (merged) in 1-month-old MONs. c, There was a twofold increase in GLT1 labeling pixel intensity (188.6 ± 18.5%) and expression (211.8 ± 45.1%) with age in MONs but not in the hippocampus. The pattern of GFAP expression in MONs changed with age but without increase either in MONs (102.0 ± 9.9; n = 3; p = 0.8906) or in hippocampus (127.9 ± 14.3; n = 3; p = 0.2068). *p < 0.05, **p < 0.005, ***p < 0.001, Student's t test. The arrows indicate GLT1 and GFAP overlap; the arrowheads indicate GLT1 labeling on structures other than GFAP (+). Scale bar, 10 μm. Error bars indicate SEM.