Low oxygen post conditioning prevents thalamic secondary neuronal loss caused by excitotoxicity after cortical stroke

Abstract

In the current study, we were interested in investigating whether Low oxygen post-conditioning (LOPC) was capable of limiting the severity of stroke-induced secondary neurodegeneration (SND). To investigate the effect of LOPC we exposed adult male C57/BL6 mice to photothrombotic occlusion (PTO) of the motor and somatosensory cortex. This is known to induce progressive neurodegeneration in the thalamus within two weeks of infarction. Two days after PTO induction mice were randomly assigned to one of four groups: (i) LOPC-15 day exposure group; (ii) a LOPC 15 day exposure followed by a 15 day exposure to normal atmosphere; (iii) normal atmosphere for 15 days and (iv) normal atmosphere for 30 days (n = 20/group). We observed that LOPC reduced the extent of neuronal loss, as indicated by assessment of both area of loss and NeuN⁺ cell counts, within the thalamus. Additionally, we identified that LOPC reduced microglial activity and decreased activity within the excitotoxic signalling pathway of the NMDAR axis. Together, these findings suggest that LOPC limits neuronal death caused by excitotoxicity in sites of secondary damage and promotes neuronal survival. In conclusion, this work supports the potential of utilising LOPC to intervene in the sub-acute phase post-stroke to restrict the severity of SND.

Figure 1

Experimental plan. Layout of the experimental design of LOPC and follow up protocol. Diagram illustrating the site of phototrombotic stroke induction (red arrow) at bregma 0 mm adapted from and representative cresyl violet stained coronal sections of a PTO stroke-affected hemisphere showing somatosensory cortex ablation (Bregma −0.6 to +1.5).

Figure 2

Secondary neuronal death is prevented by LOPC at 30 days. (A) Brain map adapted from and exemplificative NeuN staining showing the infarct region (*) and the thalamic regions VPL and PO. (B) Identified area of PO SND (dash line, scalebar = 400 μm) and representative images of the PO (scalebar = 70 μm) and VPL (scalebar = 40 μm) areas used for NeuN⁺ cells count. On the right, quantification shows that LOPC decreases the area of SND in PO and prevents NeuN⁺ cells decrease in the VPL and PO. Results are shown as the mean ± SD. *p < 0.05, **p < 0.01, 2-way ANOVA with Tukey’s multiple comparison test.

Figure 3

Thalamic microglia activation is resolved at 30 days after LOPC. (A) Decreased microglia activation area in thalamus of LOPC mice at 30 days, as estimated by Iba1 and CD68 (B) staining (scalebar = 300 μm). **p < 0.01, 2-way ANOVA with Tukey’s multiple comparison test.

Figure 4

Thalamic microglia expression of inflammatory marker CD11b. Representative western blot showing changes in CD11b expression (n = 8–10). Quantification revealed decreased expression of CD11b in LOPC mice at 30 days. Results are shown as the mean ± SD. *p < 0.05, 2-way ANOVA with Tukey’s multiple comparison test.

Figure 5

NMDAR mediated nNOS activation is prevented by LOPC at 15 days. (A) Calcium accumulation in the thalamus is not affected by LOPC at all pixel intensities as shown by cumulative tresholding analysis (scalebar = 400 μm). (B) The thalamic expression NR1, N2B and nNOS do not change in time or with treatment as assessed by WB. At 15 days, WB analysis show that the expression of synaptic marker PSD-95 is decreased by 50% in LOPC while Synapsin 1 expression is unchanged. (C) Co-immunoprecipitation of N2B and PSD-95 shows decreased interaction in LOPC samples between NMDAR and PSD-95 at 15 days (-Ab: beads not coated incubated in lysate; -Lysate: beads coated incubated in PBS). Images cropped from Supp. Fig. 3. Results are shown as the mean ± SD. *p < 0.05, Mann Whitney U test.