Figure 1.
Experimental protocol and diagram of MRI session.
(A) Experimental protocol. Surgery consisted in either transient middle cerebral artery occlusion (tMCAO) or sham operation. Treatment consisted in intravenous injection of either 2 million M2 macrophages diluted in 1 ml phosphate-buffered saline (PBS) or the same volume of pure PBS. The number of animals included is given in Table 1. (B) Diagram of one MRI session. T1/T2-WI: T1/T2-weighted imaging; MSME: Multi-Slice Multi-Echo; USPIO: ultrasmall superparamagnetic particles of iron oxide. BBB: blood-brain barrier. The sequence parameters are given in Table 3.
Table 1.
Number of animals included.
Table 2.
Primers used in PCR reactions.
Table 3.
MR imaging parameters.
Figure 2.
Co-cultured macrophages prevent hypoxia-induced neuronal cell death in vitro.
A–F: NeuN staining was performed on control organotypic hippocampal cultures (control OHCs), OHCs subjected to oxygen-glucose deprivation (OGD) (hypoxia) or OHCs subjected to OGD and co-cultured with macrophages (hypoxia+macrophages). Low magnification views of hippocampal slices are shown in A, C and E. High magnification views of the dentate gyrus are shown in B, D and F. As compared to control hippocampal slices (A, B), neuron cell loss was demonstrated in oxygen/glucose-deprived OHCs (C, D) and was found to affect the dentate gyrus systematically. In contrast, oxygen/glucose-deprived OHCs that were co-cultured with macrophages appeared partially protected from OGD-induced neuron cell death. G: A quantitative analysis of neuron density was performed in the dentate gyrus by measuring the area covered by NeuN staining relative to the area analyzed. An arbitrary value of 100% was attributed to the measures obtained in control OHCs. Data showed a mean 35% ±8% neuron density relative to controls in oxygen/glucose-deprived OHCs (hypoxia) and that co-culture with macrophages (hypoxia+macrophages) allowed neuron density to reach 70% ±10% relative to controls (Mann-Whitney, n = 3 experiments in each group, ***: p<0.001).
Figure 3.
Hypoxic hippocampal slices trigger an alternatively-activated program in macrophages.
Bone marrow-derived macrophages were cultured in the presence of control (Control) or oxygen/glucose-deprived OHCs (Hypoxia) in 6-well culture plates. Macrophages from each experimental condition were pooled and assessed by Q-RTPCR for the expression of the following genes: inductible nitric oxide synthase (iNOS), IL-6, TNF-α and Arginase-1. As controls, analyses were performed in parallel on mRNAs extracted from M1 macrophages (stimulated with IFN-γ), M2 macrophages (stimulated with IL-4) or M0 macrophages (non-stimulated). Data showed that, as expected, iNOS and the pro-inflammatory cytokines IL-6 and TNF-α were highly expressed in M1 macrophages as compared to M0 or M2 macrophages. With regard to these M1-type pro-inflammatory genes, bone marrow-derived macrophages co-cultured with oxygen/glucose-deprived OHCs (Hypoxia) exhibited low levels of mRNA expression and a similar profile to bone marrow-derived macrophages co-cultured with control OHCs (Control). In contrast, the M2-type gene Arginase-1 was highly expressed in hypoxic macrophages (co-cultured with oxygen/glucose-deprived OHCs) as compared to control macrophages (co-cultured with control OHCs). As expected, Arginase-1 was also highly expressed in M2 macrophages as compared to M0 or M1 macrophages. Data shown are representative of two independent experiments.
Figure 4.
M2 macrophages do not improve functional outcome after ischemic stroke.
A- mNSS test. B- Adhesive removal test. There was no statistical difference in rat performance before treatment (D2) for either behavioral test. After treatment (D11 and D16), there was an improvement in neuroscores in both treatment groups, but no statistically significant difference between them (analysis of variance for repeated measures, N = 8 in each group, p = 0.505). Similarly, time to remove the adhesive was slightly improved in both groups, without any significant difference (analysis of variance for repeated measures, N = 8 in each group, p = 0.699). PBS: phosphate-buffered saline (used as vehicle for cell delivery).
Figure 5.
M2 macrophages do not reduce lesion size after ischemic stroke.
A- Typical T2-weighted images obtained at D3, D7 and D14 post-ischemia showing the lesion as a hyperintense signal. B- The ratio of hemisphere volumes [ipsilateral:contralateral] showed moderate brain swelling at D3, normalization at D7 and moderate atrophy at D14. No statistically significant difference was seen between treatment groups (analysis of variance for repeated measures, N = 8 in each group, p = 0.941). C- Before treatment (D3), lesion volume (corrected for brain swelling/atrophy) was not significantly different between groups. Likewise, after treatment (D7 and D14), there was no significant difference between groups (analysis of variance for repeated measures, N = 8 in each group, p = 0.346).
Figure 6.
M2 macrophages do not reduce cerebral USPIO uptake after ischemic stroke.
A- Typical T2-weighted images obtained before and 24 h after intravenous USPIO injection. The arrow shows the slight hyposignal detected in the perilesional area. B- Abundant ED-1+ cells were detected at the same location (arrow). C- Quantification of T2 difference between ipsilateral and contralateral hemisphere (%T2) showed that the T2 drop in the perilesional area was specific to tMCAO rats injected with USPIO (analysis of variance for repeated measures, *p<0.01). However, no effect of M2 treatment on T2 changes was found (analysis of variance for repeated measures, N = 5 in each group, p = 0.523).