Neuroprotection by dimethyloxalylglycine following permanent and transient focal cerebral ischemia in rats

Abstract

Dimethyloxalylglycine (DMOG) is an inhibitor of prolyl-4-hydroxylase domain (PHD) enzymes that regulate the stability of hypoxia-inducible factor (HIF). We investigated the effect of DMOG on the outcome after permanent and transient middle cerebral artery occlusion (p/tMCAO) in the rat. Before and after pMCAO, rats were treated with 40 mg/kg, 200 mg/kg DMOG, or vehicle, and with 40 mg/kg or vehicle after tMCAO. Serial magnetic resonance imaging (MRI) was performed to assess infarct evolution and regional cerebral blood flow (rCBF). Both doses significantly reduced infarct volumes, but only 40 mg/kg improved the behavior after 24 hours of pMCAO. Animals receiving 40 mg/kg were more likely to maintain rCBF values above 30% from the contralateral hemisphere within 24 hours of pMCAO. DMOG after tMCAO significantly reduced the infarct volumes and improved behavior at 24 hours and 8 days and also improved the rCBF after 24 hours. A consistent and significant upregulation of both mRNA and protein levels of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) was associated with the observed neuroprotection, although this was not consistently related to HIF-1α levels at 24 hours and 8 days. Thus, DMOG afforded neuroprotection both at 24 hours after pMCAO and at 24 hours and 8 days after tMCAO. This effect was associated with an increase of VEGF and eNOS and was mediated by improved rCBF after DMOG treatment.

Figure 1

Results of the magnetic resonance imaging experiments from the pMCAO study. (A) Examples of diffusion-weighted imaging data obtained from one representative animal per group; on diffusion-weighted images, infarcts can be detected as areas of hyperintensity and on the derived ADC maps as areas of hypointensity. Calculated ADC infarct volumes are shown in (B), with ^* indicating P<0.05 versus control. Mean ADC reductions after pMCAO are presented in (C) for the striatum and the cortex. The inlay indicates the regions of interest, which have been measured. ^*P<0.05 for both treatment groups versus control. (D) Representative CBF maps for each group obtained using ASL-PWI. The rCBF values were not significantly different between groups, but at 1 and 24 hours there was a trend towards higher rCBF values in the 40 mg/kg DMOG group compared with the other groups (E). ^#P<0.1. (F) The correlation between mean rCBF over time and final infarct volumes is significant for all animals. Four animals of the 40 mg/kg DMOG group had values above the critical threshold of 30% from normal (P<0.05). ADC, apparent diffusion coefficient; DMOG, dimethyloxalylglycine; pMCAO, permanent middle cerebral artery occlusion; rCBF, regional cerebral blood flow.

Figure 2

Results of the magnetic resonance imaging experiments from the tMCAO study. (A) Representative examples of diffusion-weighted imaging data from the 24-hour reperfusion experiment. (B) Graph showing the calculated ADC infarct volumes with $ indicating P<0.01 versus control. (C) Representative examples of T2W and pc-T1W imaging data obtained from the 8-day reperfusion experiment. (D) Graph showing the calculated T2W infarct volumes and the pcTW volumes of BBB breakdown, with ^* indicating P<0.05 versus control. (E) Representative examples of the ASL-PWI CBF maps after tMCAO; hyperintensities represent hyperperfusion. (F) Graph showing the calculated rCBF values of the ipsilateral hemisphere, with $ indicating P<0.01 in a paired test. The inter-group comparison showed no significant differences. (G) Graph of rCBF/infarct volume ratios, with $ indicating P<0.01 versus control in an unpaired test. ADC, apparent diffusion coefficient; ASL-PWI; arterial spin labeling perfusion-weighted imaging; tMCAO, transient middle cerebral artery occlusion; rCBF, regional cerebral blood flow.

Figure 3

(A) Cortical mRNA levels of selected genes of interest after 24 hours of pMCAO as measured by RT-PCR. Values are normalized for sham levels and ^* indicates P<0.05 versus control. (B) Representative Western blots after 24 hours of pMCAO. (C) The calculated immunoreactivities after correction for PonceauS staining and normalization for sham values. ^*P<0.05 versus control. pMCAO, permanent middle cerebral artery occlusion.

Figure 4

(A) Representative Western blots after 24 hours and 8 days of reperfusion in a 60-minute tMCAO experiment. (B) Calculated immunoreactivities after correction for PonceauS staining and normalization for sham values. ^*P<0.05 versus control. tMCAO, transient middle cerebral artery occlusion.