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Monocarboxylate transporter 2 and stroke severity in a rodent model of sleep apnea - PubMed

  • ️Sat Jan 01 2011

Monocarboxylate transporter 2 and stroke severity in a rodent model of sleep apnea

Yang Wang et al. J Neurosci. 2011.

Abstract

Stroke is not only more prevalent but is also associated with more severe adverse functional outcomes among patients with sleep apnea. Monocarboxylate transporters (MCT) are important regulators of cellular bioenergetics, have been implicated in brain susceptibility to acute severe hypoxia (ASH), and could underlie the unfavorable prognosis of cerebrovascular accidents in sleep apnea patients. Rodents were exposed to either intermittent hypoxia (IH) during sleep, a characteristic feature of sleep apnea, or to sustained hypoxia (SH), and expression of MCT1 and MCT2 was assessed. In addition, the functional recovery to middle cerebral artery occlusion (MCAO) in rats and hMCT2 transgenic mice and of hippocampal slices subjected to ASH was assessed, as well as the effects of MCT blocker and MCT2 antisense oligonucleotides and siRNAs. IH, but not SH, induced significant reductions in MCT2 expression over time at both the mRNA and protein levels and in the functional recovery of hippocampal slices subjected to ASH. Similarly, MCAO-induced infarcts were significantly greater in IH-exposed rats and mice, and overexpression of hMCT2 in mice markedly attenuated the adverse effects of IH. Exogenous pyruvate treatment reduced infarct volumes in normoxic rats but not in IH-exposed rats. Administration of the MCT2 blocker 4CN, but not the MCT1 antagonist p-chloromercuribenzene sulfonate, increased infarct size. Thus, prolonged exposures to IH mimicking sleep apnea are associated with increased CNS vulnerability to ischemia that is mediated, at least in part, by concomitant decreases in the expression and function of MCT2. Efforts to develop agonists of MCT2 should provide opportunities to ameliorate the overall outcome of stroke.

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Figures

Figure 1.
Figure 1.

Hippocampal slices were exposed to ASH for 10 min after which reemergence of fEPSPs was assessed. No differences in ASH susceptibility emerged in SH-exposed rats after 3, 7, or 14 d exposures (red columns; n = 24 slices from 6 different rats per group) when compared with RA-exposed rats (black column at time 0; n = 48 slices from 12 different rats per group). In contrast, although no changes occurred after 3 d of IH, the percentage of slices harvested from rats exposed to either 7 or 14 d IH showing functional recovery was reduced (*p < 0.0001 vs time 0; n = 24 slices from 6 different rats per group).

Figure 2.
Figure 2.

Infarct size after MCAO in adult rats after either 3 or 14 d (n = 12/group). Infarct volume was significantly larger at both time points after IH exposures when compared with normoxic controls (RA; *p < 0.001).

Figure 3.
Figure 3.

Behavioral test performances in rats subjected to MCAO (filled symbols) or sham (open symbols) after IH (square symbols) or RA (normoxic controls; triangle symbols). A, Eighteen-point neurological scores were significantly lower in IH rats subjected to MCAO (*p < 0.01, IH vs RA; n = 12). B, Whisker stimulation and paw placement test showed decreased responses in IH-exposed rats after MCAO compared with RA controls (#p < 0.04, ANOVA). C, Foot fault test performance tests showed increased number of misplacements in IH-exposed rats after MCAO (#p < 0.04, ANOVA). D, Rotarod test performances showed that rats subjected to IH exposures and MCAO had significantly shorter latencies than normoxic controls (*p < 0.01 vs RA).

Figure 4.
Figure 4.

Quantitative RT-PCR for MCT2 in rat cortical lysates harvested from animals exposed to SH (black columns) or IH (red columns) for 1–30 d. Significant downregulation of MCT2 mRNA expression emerged at 7 d and continued to progress until 30 d of IH; however, no significant changes in MCT2 expression occurred in SH-exposed animals (*p < 0.01; n = 6/time point). All values are reported as ratios between condition (SH or IH) and time-matched room air controls.

Figure 5.
Figure 5.

A, Representative immunoblots for MCT1 and MCT2 of cortical lysates after either IH or SH. B, MCT2 protein expression changes over time in rat cortex after IH or SH exposures expressed as ratios against corresponding β-actin densitometric values (n = 6/group; *p < 0.01, SH vs RA; #p < 0.01, IH vs RA).

Figure 6.
Figure 6.

A, Immunohistochemical staining of MCT2 (red) and NeuN (green) and overlay in the hippocampus showing the high degree of colocalization of MCT2 in neurons. B, Immunohistochemical staining of MCT2 (red) and GFAP (green) and overlay in the hippocampus showing the relative absence of colocalization of MCT2 in glia. C, Immunohistochemical staining of MCT2 (red) and NeuN (green) in two different rats exposed to IH for14 d showing marked reductions in the expression of MCT2.

Figure 7.
Figure 7.

Infarct volumes in normoxic rats subjected to MCAO after intracerebroventricular treatment with MCT blockers 4-CN, pCMBS, or vehicle (Veh) (**p < 0.01, 4-CN vs vehicle; #p < 0.01, pCMBS vs 4-CN; n = 6/group).

Figure 8.
Figure 8.

Top, Western blots of cortical lysates harvested from rats 24, 48, and 72 h after receiving MCT2 antisense ON (AS) or scrambled ON (S), showing effective reductions in the expression of MCT2. P, Vehicle control; B, lysate from primary neuronal cell culture; K, control peptide. Bottom, Infarct volumes after MCAO in rats treated with either MCT2 antisense ON (AS) or scrambled ON (S) (*p < 0.02; n = 5/group).

Figure 9.
Figure 9.

A, Representative distribution of siRNAs after treatment in rat cortex. B, Western blot analysis of MCT2 protein expression in cortical tissues harvested from rats treated with MCT2 siRNAs or negative scrambled RNA and delivered by intraventricle injection and infusion. Negative control siRNA had no effect on MCT2 expression, whereas siRNA achieved marked reductions in MCT2 expression. Each lane represents an individual animal cortex harvested from the same side of intracerebroventricular injection. C1 represents an untreated animal cortex. C, Cerebral infarct volume 48 h after MCAO in adult rats pretreated with either a negative control siRNA (negRNA) or MCT2 siRNAs showed significant increases after MCT2 siRNAs (*p < 0.01 vs negRNA; n = 4/group).

Figure 10.
Figure 10.

Infarct volumes in rats exposed to either IH for 14 d (CIH) or RA, and receiving intraperitoneal 500 mg/kg pyruvate (Pyr) or vehicle (Veh) within 1 h from reperfusion after MCAO (**p < 0.01, RA–Pyr vs RA–Veh, n = 6/group; #p < 0.02, CIH–Pyr or CIH–Veh vs RA–Veh; p value not significant, CIH–Pyr vs CIH–Veh, n = 6/group).

Figure 11.
Figure 11.

Infarct volumes in transgenic mice that overexpress human monocarboxylate transporter 2 and their wild-type littermates subjected to MCAO after 14 d of IH exposures or normoxia. Significant reductions in infarct size emerged in normoxic hMCT2 Tg mice compared with wild-type (WT) littermates (*p < 0.01, n = 8/group). However, IH exposures induced increases in infarct volume in wild-type mice (**p < 0.001 vs RA–WT) that were markedly attenuated in hMCT2 Tg mice (ρp < 0.01, #p < 0.01, n = 8/group; p value not significant, IH–TG vs RA–WT).

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