Beneficial Effects of Hyaluronan-Based Hydrogel Implantation after Cortical Traumatic Injury

Abstract

Traumatic brain injury (TBI) causes cell death mainly in the cerebral cortex. We have previously reported that transplantation of embryonic cortical neurons immediately after cortical injury allows the anatomical reconstruction of injured pathways and that a delay between cortical injury and cell transplantation can partially improve transplantation efficiency. Biomaterials supporting repair processes in combination with cell transplantation are in development. Hyaluronic acid (HA) hydrogel has attracted increasing interest in the field of tissue engineering due to its attractive biological properties. However, before combining the cell with the HA hydrogel for transplantation, it is important to know the effects of the implanted hydrogel alone. Here, we investigated the therapeutic effect of HA on host tissue after a cortical trauma. For this, we implanted HA hydrogel into the lesioned motor cortex of adult mice immediately or one week after a lesion. Our results show the vascularization of the implanted hydrogel. At one month after HA implantation, we observed a reduction in the glial scar around the lesion and the presence of the newly generated oligodendrocytes, immature and mature neurons within the hydrogel. Implanted hydrogel provides favorable environments for the survival and maturation of the newly generated neurons. Collectively, these results suggest a beneficial effect of biomaterial after a cortical traumatic injury.

Keywords: biomaterial; hyaluronan; neuroinflammation; traumatic brain injury.

Figure 1

Timeline of the study representing the different groups and the different time points of mice hydrogel implantation and sacrifice. dpl: days post-lesion; mpl: month post-lesion, dpi: days post-implantation, mpi: month post-implantation.

Figure 2

Cortical tissue loss one week and one month after the lesion. (A–F) Representative images of cells labeled with DAPI (blue) in lesioned (A,B) or implanted mice without delay (C,D) or with delay (E,F). Dashed lines indicate hydrogel localization. Scale bar: 200 μm. (G) Quantitative analysis of the cortical tissue loss over time in lesioned (red) or implanted mice without delay (green) or with delay (blue). Two-way ANOVA ** p < 0.01 **** p < 0.0001.

Figure 3

Hydrogel vascularization. (A–D) Representative immunofluorescence staining of blood vessels labeled with CD31 (red) in implanted mice in the no-delay group (A,B) or in delay group (C,D). Dashed lines indicate hydrogel localization. Scale bar: 200 μm. (E) Quantitative analysis of the blood vessels density over time in implanted mice in no-delay (green) or delay groups (blue). Two-way ANOVA * p < 0.05.

Figure 4

Neuroblasts migration to the cortex. (A–C) Representative immunofluorescence staining of neuroblasts labeled with DCX (green) in lesioned (A) or no-delay implanted (B) or delay implanted groups (C). Dashed lines indicate hydrogel localization. The squares indicate the area of magnification. Scale bar: 200 μm. (A′–C′) High magnification images of the neuroblast in the cortex. Scale bar: 20 μm. (D) Quantitative analysis of the number of the neuroblast over time in lesioned (red) or implanted mice without delay (green) or with delay (blue). Two-way ANOVA.

Figure 5

Neuroblasts migration into hydrogel. (A–C) Representative immunofluorescence staining of neuroblasts labeled with DCX (green) in delay (A,B) or no-delay implanted groups (C,D). Dashed lines indicate hydrogel localization. The squares indicate the area of magnification. Scale bar: 20 μm. (E,F) Quantitative analysis of the neuroblast number, one week (E) or one month (F) after implantation, in implanted no-delay (green) or delay implanted groups (blue). Mann–Whitney test.

Figure 6

Mode of neuroblast migration towards cortical lesion site. (A–F) Representative immunofluorescence staining of neuroblasts labeled with DCX (green) and blood vessels with CD31 ((A–C), red) or astrocytes with GFAP ((D–F), red), in lesioned (A,D) or implanted mice without delay (B,E) or with delay (C,F). Dashed lines indicate hydrogel localization. The squares indicate the area of magnification. Scale bar: 20 μm.

Figure 7

Oligodendrocytes into hydrogel. (A–D) Representative immunofluorescence staining of oligodendrocytes labeled with Olig2 (red) in no-delay (A,B) or delay implanted groups (C,D). Scale bar: 20 μm. (E) Quantitative analysis of the oligodendrocyte number into the corpus callosum over time in lesioned (red) or implanted hydrogel in no-delay (green) or delay groups (blue). Two-way ANOVA ** p < 0.01.

Figure 8

Newly generated oligodendrocytes and astrocytes into hydrogel. (A–N) Representative immunofluorescence staining of newly generated BrdU cells (red) colocalized with Olig2 (blue, (A–G)) or with GFAP (green, (H–N)) in delay (A–C,H–J) or no delay hydrogel implanted groups (E–G,L–N). (D,K) Quantitative analysis of the BrdU+/Olig2+ cells (D) or BrdU+/GFAP+ cells (K) in no-delay (green) or delay (blue) hydrogel implanted groups. Mann–Whitney test.

Figure 9

Newly generated neuroblasts and mature neurons into hydrogel. (A–N) Representative immunofluorescence images of newly generated BrdU+ cells (red) colocalized with DCX (green, (A–G)) or with NeuN (green, (H–N)) in hydrogel in no delay (A–C,H–J) or in delay (E–G,L–N) hydrogel implanted groups. (D,K) Quantitative analysis of the BrdU+/DCX+ neuroblasts (D) or BrdU+/NeuN+ neurons (K) within hydrogel delay (green) or no-delay (blue) groups. Mann–Whitney test.

Figure 10

Neurons into hydrogel (A–D). Representative immunofluorescence staining of neurons labeled with βIII-tubulin (green, arrows, (A,C)) or with NeuN (red, arrows, (B,D)) in no-delay (A,B) or delay hydrogel implanted groups (C,D). Scale bar: 20 μm. (E,F) Quantitative analysis of the number of βIII-tubulin neurons (E) or NeuN+ neurons (F) in no-delay (green) or delay (blue) hydrogel implanted groups. One-way ANOVA.

Figure 11

Glial scar thickness one month after the lesion/implantation. (A–C) Representative immunofluorescence images of GFAP+ cells (red) in lesioned (A) or delay (B) or no-delay (C) hydrogel implanted groups. Dashed lines indicate the limit of the glial scar (A) or hydrogel localization (B,C). The squares indicate the area of magnification. Scale bar: 200 μm. (A′–C′) High magnification images of the glial scar. Scale bar: 20 μm. (D) Quantitative analysis of the glial scar thickness in lesioned (red) in delay (green) or in no-delay (blue) hydrogel implanted groups. Groups were compared using one-way ANOVA, p = 0.07.

Figure 12

Anti and pro-inflammatory astrocytes in the cortex one month after the lesion/implantation. (A–F) Representative immunofluorescence staining of GFAP (green) colocalized with S100A10 ((A–C), red) or C3 ((E,F), red) in lesioned (A,D) in no-delay (B,E) or in delay (C,F) hydrogel implanted groups. Arrows indicate colocalization. Scale bar: 20 μm. (G) Representation of pro (red) and anti (green) -inflammatory astrocytes subtype proportion per condition. One-way ANOVA per subtype.

Figure 13

Anti and pro-inflammatory microglial cells in the cortex lesion one month after the lesion/implantation. (A–F) Representative immunofluorescence staining of Iba1+ cells (green) colocalized with Arg1 ((A–C), red) or CD86 ((D–F), red) in lesioned (A,D) or in no-delay (B,E) or delay (C,F) hydrogel implanted groups. Arrows indicate colocalization. Scale bar: 20 μm. (G) Representation of pro (red) and anti (green)-inflammatory microglia subtype proportion per condition. One-way ANOVA per subtype.