Characterization of Inflammation in Delayed Cortical Transplantation

Abstract

We previously reported that embryonic motor cortical neurons transplanted 1-week after lesion in the adult mouse motor cortex significantly enhances graft vascularization, survival, and proliferation of grafted cells, the density of projections developed by grafted neurons and improves functional repair and recovery. The purpose of the present study is to understand the extent to which post-traumatic inflammation following cortical lesion could influence the survival of grafted neurons and the development of their projections to target brain regions and conversely how transplanted cells can modulate host inflammation. For this, embryonic motor cortical tissue was grafted either immediately or with a 1-week delay into the lesioned motor cortex of adult mice. Immunohistochemistry (IHC) analysis was performed to determine the density and cell morphology of resident and peripheral infiltrating immune cells. Then, in situ hybridization (ISH) was performed to analyze the distribution and temporal mRNA expression pattern of pro-inflammatory or anti-inflammatory cytokines following cortical lesion. In parallel, we analyzed the protein expression of both M1- and M2-associated markers to study the M1/M2 balance switch. We have shown that 1-week after the lesion, the number of astrocytes, microglia, oligodendrocytes, and CD45+ cells were significantly increased along with characteristics of M2 microglia phenotype. Interestingly, the majority of microglia co-expressed transforming growth factor-β1 (TGF-β1), an anti-inflammatory cytokine, supporting the hypothesis that microglial activation is also neuroprotective. Our results suggest that the modulation of post-traumatic inflammation 1-week after cortical lesion might be implicated in the improvement of graft vascularization, survival, and density of projections developed by grafted neurons.

Keywords: cortical lesion; delay; embryonic transplantation; motor cortex; neuroinflammation.

Figure 1

Timeline of the study. Timeline of the study representing the different groups and the different time points of mice sacrifices. Dpl, days post-lesion; dpt, days post-transplantation.

Figure 2

Characterization of the environment surrounding cortical lesion. The effect of cortical lesion on the number of resident immune cells and peripheral infiltrating cells in the injured cortex was evaluated in three groups of mice: control without lesion, Day 0 of lesion or Day 7 after lesion. Black squares show area of interest in Control (A), Day 0 (B) or Day 7 (C) groups. Immunostaining using antibodies against Glial fibrillary acidic protein (GFAP; D–F), Iba1 (G–I), Olig2 (J–L) or CD45 (M–O) allowed the detection of astrocytes, microglia, oligodendrocytes or hematopoietic cells in control, day 0, and day 7 after cortical lesion; respectively. (P) Histogram showing the quantification of the number of Iba1+, GFAP+, Olig2+ or CD45+ cells in different groups. The p-value was determined using two-way analysis of variance (ANOVA) followed by Bonferroni test. Values for ***p < 0.0001 were considered significant. Results are expressed as mean ± SEM. Results are representatives of n = 6 animals per group. Scale bar: (A,B) 2 mm (D–O) 80 μm.

Figure 3

Differential expression of IL-1β and transforming growth factor-β1 (TGF-β1) following lesion of the motor cortex. The presence of IL-1β and TGF-β1 mRNAs was tested in the cortex without lesion (Ctrl; A,B) at the day of lesion (D0; C,D), 4 days (D4; E,F) and 7 days (D7; G,H) after the lesion (in blue, black arrowheads). Quantification analysis of the mean number of IL-1β and TGF-β1-expressing cells, in Ctrl, D0, D4 and D7 groups (I). Co-labeling of IL-1β (J) or TGF-β1 (K) transcripts (in blue) with Iba1 macrophage/microglia marker (in brown) 4 days after lesion (black arrowheads). Red squares in inserts show area of interest. Data are presented as group mean ± SEM and asterisk indicates statistically significant differences (Student’s T-test, ***p < 0.0001). Scale bar: 20 μm.

Figure 4

Temporal kinetics of microglia/macrophage polarization after cortical lesion. Western blot analysis for CD86, CD206, and Arg1 protein expression from the cell lysate of control and lesioned motor cortex sacrificed at different time points: day 0, day 7 and day 21 (A). Quantification analysis of CD86, CD206, and Arg1 expression by normalizing to α tubulin level in ctrl, day 0, day 7, and day 21 groups (B). The p-value was determined using Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Values for *p < 0.05 were considered significant. Results are expressed as mean ± SEM. Results are representatives of n = 4 animals per group.

Figure 5

Histogram showing quantifications of GFAP+, Iba1+, Olig2+ or CD45+ cells at 4 days (A) and 7 days (B) after cortical transplantation. Results are expressed as mean ± SEM. Results are representatives of n = 6 animals per group. The p-value was determined using Student’s T-test. Values for ***p < 0.0001 and **p < 0.001 were considered significant.

Figure 6

Expression of astrocytes and microglia at 7 days after cortical transplantation. Low magnification photomicrographs of coronal sections illustrating the immunolabeled immune cells (red) in the GFP+ transplants (green) at day 7 after transplantation (A,E,I,M). Astrocytes (A,E) and microglia (I,M) after transplantation with no delay (A,I) or with delay of 1-week (E,M) after the cortical lesion. High magnification images from regions of interest showing immunolabeled astrocytes (B–D,F–H), microglia (J–L,N–P), in the GFP+ transplants (in green) after transplantation with no delay (B–D,J–L) or with delay (F–H,N–P) of 1-week after the cortical lesion. Scale bars: (A,E,I,M) 480 μm, (B–D,F–H,J–L,N–P) 80 μm.

Figure 7

Expression of oligodendrocytes and hematopoietic cells after cortical transplantation. Low magnification photomicrographs of coronal sections illustrating the immunolabeled immune cells (red) in the GFP+ transplants (green) at day 7 after transplantation (A,E,I,M). Oligodendrocytes (A,E) and hematopoietic cells (I,M) after transplantation with no delay (A,I) or with delay of 1-week (E,M) after the cortical lesion. High magnification images from regions of interest showing immunolabeled oligodendrocytes (B–D,F–H) and hematopoietic cells (J–L,N–P) in the GFP+ transplants (in green) after transplantation with no delay (B–D,J–L) or with delay of 1-week (F–H,N–P) after the cortical lesion. Scale bars: (A,E,I,M) 480 μm, (B–D,F–H,J–L,N–P) 80 μm.

Figure 8

Astrocytes polarization after cortical transplantation. Low magnification photomicrographs of coronal sections illustrating the astrocytes (red) and their phenotype (blue) in the GFP+ transplants (green) at day 7 after transplantation (A,D,G,J). A1 astrocytes (GFAP+/C3+; A,D) and A2 astrocytes (GFAP+/CD109+; G,J) after transplantation with no delay (A,G) or with 1-week delay (D,J) after the cortical lesion. High magnification images from regions of interest showing GFAP+/C3 cells (B,C,E,F), GFAP+/CD109 cells (H,I,K,L), in the GFP+ transplants (in green) after transplantation with no delay (B,C,H,I) or with delay (E,F,K,L) of 1-week after the cortical lesion. Scale bars: (A,D,G,J) 480 μm. Histogram showing quantifications of the percentage of C3+ or CD109+ cells of total GFAP+ cells. (M) Results are expressed as mean ± SEM. The p-value was determined using Kruskal-Wallis test and Dunn’s multiple comparison post hoc test, *p < 0.05.

Figure 9

Microglia/macrophage polarization after cortical transplantation. Low magnification photomicrographs of coronal sections illustrating the microglia cells (red) and their phenotype (blue) in the GFP+ transplants (green) at day 7 after transplantation (A,D,G,J). M2 microglia phenotype (Iba1+/Arg1+; A,D) and M1 microglia phenotype, (Iba1+/CD86+; G,J) after transplantation with no delay (A,G) or with delay of 1-week (D,J) after the cortical lesion. High magnification images from regions of interest showing Iba1+/Arg1+ cells (B,C,E,F), Iba1+/CD86+ cells (H,I,K,L), in the GFP+ transplants (in green) after transplantation with no delay (B,C,H,I) or with delay (E,F,K,L) of 1-week after the cortical lesion. Scale bars: (A,D,G,J) 480 μm. Histogram showing quantifications of the percentage of Arg1+ or CD86+ cells of total Iba1+ cells. (M) Results are expressed as mean ± SEM. The p-value was determined using Kruskal-Wallis test and Dunn’s multiple comparison post hoc test, *p < 0.05.