Injectable Gelatin Hydrogel Suppresses Inflammation and Enhances Functional Recovery in a Mouse Model of Intracerebral Hemorrhage

Abstract

Intracerebral hemorrhage (ICH) is a devastating subtype of stroke with high morbidity and mortality. However, there is no effective therapy method to improve its clinical outcomes to date. Here we report an injectable gelatin hydrogel that is capable of suppressing inflammation and enhancing functional recovery in a mouse model of ICH. Thiolated gelatin was synthesized by EDC chemistry and then the hydrogel was formed through Michael addition reaction between the thiolated gelatin and polyethylene glycol diacrylate. The hydrogel was characterized by scanning electron microscopy, porosity, rheology, and cytotoxicity before evaluating in a mouse model of ICH. The in vivo study showed that the hydrogel injection into the ICH lesion reduced the neuron loss, attenuated the neurological deficit post-operation, and decreased the activation of the microglia/macrophages and astrocytes. More importantly, the pro-inflammatory M1 microglia/macrophages polarization was suppressed while the anti-inflammatory M2 phenotype was promoted after the hydrogel injection. Besides, the hydrogel injection reduced the release of inflammatory cytokines (IL-1β and TNF-α). Moreover, integrin β1 was confirmed up-regulated around the lesion that is positively correlated with the M2 microglia/macrophages. The related mechanism was proposed and discussed. Taken together, the injectable gelatin hydrogel suppressed the inflammation which might contribute to enhance the functional recovery of the ICH mouse, making it a promising application in the clinic.

Keywords: functional recovery; gelatin; hydrogel; inflammation; intracerebral hemorrhage.

FIGURE 1

(A) Synthesis pathway of thiolated gelatin. (B) Schematic presentation of gelatin hydrogel injection into the lesion site in a mouse model of ICH. (C) Experimental protocol and timeline.

FIGURE 2

(A) Injection operation of the gelation hydrogel (staining with phenol red). (B) SEM image of the hydrogel, scale bar = 100 μm. (C) The rheological curve of the hydrogel. (D) Live/dead staining of the cells within the hydrogel on day 1, and 5, scale bar = 100 μm.

FIGURE 3

(A) Body-weight change. (B) The statistical graph of the corner turn test. (C) The statistical graph of the neurological deficit testing. (D) Representative images for Immunofluorescence staining of neuron marker Neun, scale bar = 20 μm. (E) The statistical graph of the Neun positive cells. Data are present as mean ± SD (n = 4), *P < 0.05, ***P < 0.001.

FIGURE 4

H&E staining. (A–F) The overall morphology of the injured brain in different time-point after gelatin hydrogel injection, scale bar = 500 μm. (a–f) Magnified images show the host tissue-lesion or gelatin hydrogel interface, scale bar = 50 μm. (g) Local magnification image of hemosiderin deposition. *Represents the hydrogel.

FIGURE 5

Gelatin hydrogel affects the activation of microglia/macrophages and astrocytes. (A) Double immunofluorescence staining was performed with astrocyte marker GFAP (green) and microglia/macrophages marker Iba-1 (red) in brain sections, scale bar = 20 μm. (B) Representative images for Immunofluorescence staining of Iba-1, scale bar = 50 μm. (C) The percentage of Iba-1 positive area analysis. (D) Representative images for Immunofluorescence staining of GFAP, scale bar = 50 μm. (E) The percentage of GFAP positive area analysis. *Represents the hydrogel. Data are mean ± SD, n = 4 mice per group, *P < 0.05, ***P < 0.001.

FIGURE 6

Effects of gelatin hydrogel on microglia/macrophages polarization. (A) M1 pro-inflammatory microglia/macrophages marker iNOS⁺Iba-1⁺. (B) The percentage of iNOS⁺Iba-1⁺cells/Iba-1⁺cells in two groups. (C) M2 anti-inflammatory microglia/macrophages marker Arg-1⁺Iba-1⁺. (D) The percentage of Arg-1⁺Iba-1⁺ cells/Iba-1⁺cells in two groups. Scale bar = 20 μm. Data are mean ± SD, n = 4 mice per group, *P < 0.05, ***P < 0.001.

FIGURE 7

Effects of gelatin hydrogel on pro-inflammatory cytokine. (A) Immunohistochemical staining of IL-1β around the lesion. (B) The area percentage of IL-1β. (C) Immunohistochemical staining of TNF-α around the lesion. (D) The area percentage of TNF-α. Scale bar = 50 μm. *Represents the hydrogel. Data are mean ± SD, n = 4 mice per group, ***P < 0.001.

FIGURE 8

Gelatin hydrogel affects the expression of integrin β1. (A) Double immunofluorescence staining was performed with integrin β1 (red) and microglia marker/macrophages Iba-1 (green) in brain sections on day 7, scale bar = 20 μm. (B) Immunofluorescence staining of integrin β1 around the lesion, scale bar = 20 μm. (C) Fluorescence intensity (FI) analysis of the images. (D) The correlation analysis between the Arg⁺IBA⁺ double-positive cells positive percentage and fluorescence intensity of β1, the correlation coefficient is 0.73 (P < 0.01). *Represents the hydrogel. Data are mean ± SD, n = 4 mice per group, *P < 0.05, ***P < 0.001.

FIGURE 9

Schematic presentation of the effect of the injectable gelatin hydrogel on the host cells and proposed mechanism.