Dual-function injectable angiogenic biomaterial for the repair of brain tissue following stroke

Abstract

Stroke is the primary cause of disability due to the brain's limited ability to regenerate damaged tissue. After stroke, an increased inflammatory and immune response coupled with severely limited angiogenesis and neuronal growth results in a stroke cavity devoid of normal brain tissue. In the adult, therapeutic angiogenic materials have been used to repair ischaemic tissues through the formation of vascular networks. However, whether a therapeutic angiogenic material can regenerate brain tissue and promote neural repair is poorly understood. Here we show that the delivery of an engineered immune-modulating angiogenic biomaterial directly to the stroke cavity promotes tissue formation de novo, and results in axonal networks along thee generated blood vessels. This regenerated tissue produces functional recovery through the established axonal networks. Thus, this biomaterials approach generates a vascularized network of regenerated functional neuronal connections within previously dead tissue and lays the groundwork for the use of angiogenic materials to repair other neurologically diseased tissues.

Figure 1. Post-stroke angiogenic response and vascular remodeling

(A) Fluorescent images of vessels (Glut-1) with Dapi, (B) a marker of proliferation (BrdU), and (C) pericyte/smooth muscle cells (PDGFR-β) in and around the stroke site (*) at day 10 after gel transplantation. Quantification of the vascular density (% Glut-1 area) in the infarct (D1) and peri-infarct (D2), angiogenesis (Glut-1/BrdU cells) in the infarct (E1) and peri-infarct (E2) and pericyte vascular coverage (% PDGFR-β area) in the infarct (F1) and peri-infarct area (F2). No gel = stroke only condition, empty gel = HA hydrogel, gel + Vs = HA hydrogel loaded with 200ng of soluble VEGF, gel + nH = HA hydrogel with 1μg heparin nanoparticles (nH), gel + lcV = HA hydrogel with 1μg nH loaded with 200 ng VEGF, gel + hcV = HA hydrogel with 0.01 μg nH loaded with VEGF and 0.99 μg unloaded nH. Data is presented using a min to max box plot. Each dot in the plots represents one animal and p values were determined by One-way ANOVA with a Tukey’s post-hoc test, with *, ** and **** indicating p < 0.05, p < 0.01 and p < 0.0001, respectively. Scale bar: 100 μm.

Figure 2. Long-term post-stroke vascular growth

(A) Fluorescent images of vessels (Glut-1) in and around the stroke site (*) 16 weeks after gel transplantation. (B) Quantification of the vascular density (% Glut-1 area) in and around the stroke site (*), Quantification of the vessel morphology: vessel tortuosity = total vessel length/shortest distance (C1) and diameter (C2), Quantification of vessel growth with number of ramifications = the number of branches/vessel (D1) and maximum infiltration distance of the vessels into the stroke site (D2). In all plots, the dotted red line and red number indicates the value for the give quantification of the contralateral side. Empty gel = gel = HA hydrogel, Vs = 200 ng of soluble VEGF, lcV = 1μg nH loaded with 200 ng VEGF, hcV = 0.01 μg nH loaded with VEGF and 0.99 μg unloaded nH. Data is presented using a min to max box plot. Each dot in the plots represents one animal and p values were determined by One-way ANOVA with a Tukey’s post-hoc test, with *, ** and **** indicating p < 0.05, p < 0.01 and p < 0.0001, respectively. Scale bar: 100 μm.

Figure 3. Post-stroke neurogenesis and axonal sprouting

(A) Fluorescent images of neuroblasts (Dcx) and the proliferation marker BrdU, and (B) axonal neurofilaments (NF200) in and around the stroke site (*) at 2 weeks and (G) at 16 weeks after gel transplantation. (C) Quantification of neuroblasts (Dcx) and proliferating neuroblasts (Dcx/BrdU) in the ipsilateral ventricle, (D) the number of neuroblasts migrating and their migration distance and number, (E) the axonal area (NF200) in and around the stroke site, and (F) infiltration distance and penetration angle in the stroke site. (H) Quantification of axonal area (NF200) in and (I) around the stroke site, (J) infiltration distance and (K) axonal penetration angle 16 weeks after gel injection. In all plots, the dotted red line and red number indicates the value for the give quantification of the contralateral side. Empty gel = gel = HA hydrogel, Vs = 200 ng of soluble VEGF, lcV = 1μg nH loaded with 200 ng VEGF, hcV = 0.01 μg nH loaded with 200 ng VEGF and 0.99 μg unloaded nH, Endo = a daily i.p injection of endostatin day 5 to 15. Data is presented using a min to max box plot. Each dot in the plots represents one animal and p values were determined by One-way ANOVA with a Tukey’s post-hoc test, with *, ** and **** indicating p < 0.05, p < 0.01 and p < 0.0001, respectively. Scale bar: 100 μm.

Figure 4. Association between the vascular and axonal network in the lesion site

(A) Fluorescent images of vessels (Glut-1, red) and axonal neurofilaments (NF200, green) in and around the stroke site (*) 16 weeks after gel transplantation. (B) Quantitative assessment of the proximity between the 2 networks with the quantification of NF200 positive signal on vessels and (C) positive area a distance of 50 μm from vessels. (D) Fluorescent images of the peri-infarct astrocytic scar (GFAP, green) and BDA-traced neurons (red) in the ipsilateral hemisphere of gel + hcV injected mice 16 weeks after gel transplantation. (E) Fluorescent images of astrocytes (GFAP) co-stained with vessels (Glut-1) and pericytes/smooth muscle cells (PDGFR-β), or (F) with end-feet astrocytes (Aquaporin-4) in the stroke site of hcV-treated mice, 16 weeks after gel transplantation. Empty gel = gel = HA hydrogel, Vs = 200 ng of soluble VEGF, lcV = 1μg nH loaded with 200 ng VEGF, hcV = 0.01 μg nH loaded with 200 ng VEGF and 0.99 μg unloaded nH, Endo = a daily i.p injection of endostatin days 5 to 15. Data is presented using a min to max box plot. Each dot in the plots represents one animal and p values were determined by One-way ANOVA with a Tukey’s post-hoc test, with ** and **** indicating p < 0.01 and p < 0.0001, respectively. Data represent the average. $$$ indicates p < 0.001 vs Gel+hcV. Scale bar: 100 μm.

Figure 5. Post-stroke neurological recovery

(A) Experiment timeline for the behavioral tests. Mice were injected 5 days after stroke with one of the following treatments: empty gel, gel + Vs, gel + lcV, and gel + hcV. Mice were subjected to different behavioral tests (illustrated by vertical grey boxes on the timeline) On week 0, 1, 4, 8, 12 and 16 after stroke. (B1) The Cylinder test was used to measure the dexterity of their contralateral forelimb, (B2) the Grid test for the contralateral hindlimb, and (B3, B4) the Pasta test for the contralateral forepaw, normally sensitive to post-stroke lateralized impairments. (C1–C4) In order to determine the role of gel+hcV -induced vascularization on behavioral recovery, a supplemental set of gel+hcV animals was administered with endostatin, a VEGF-independent angiogenic inhibitor for 10 days after the gel injection, and submitted to the same behavioral tests: Cylinder (C1), Grid (C2), and Pasta (C3–C4). (D1–D4) In order to determine the role of gel+hcV –induced axonal growth on recovery, a supplemental set of gel+hcV animals received a brain injection of an AAV5 viral construct expressing hM4 DREADD receptors (designer receptors exclusively activated by a designer drug) directly in the stroke area on week 13. Transfected neurons are silenced after i.p administration of the DREADD ligand, clozapine-N-oxide (CNO) on week 16. Mice are then submitted to the same behavioral tests: Cylinder (D1), Grid (D2), and Pasta (D3–D4). Empty gel = HA hydrogel, Vs = 200 ng of soluble VEGF, lcV = 2μg nH loaded with 200 ng VEGF, hcV = 0.01 μg nH loaded with 200 ng VEGF and 0.99 μg unloaded nH. Data represent the average ± SEM (n = 12 mice) and p values were determined by One-way ANOVA, Tukey’s post-hoc test, * indicating P < 0.05.

Figure 6. Role of naked heparin particles in the hcV treatment at 2 weeks post-stroke

Since the injected high and low cluster treatments (respectively hcV and lcV) were designed to contain equal amounts of heparin and VEGF, the VEGF clusterization in the hcV treatment was obtained on a low amount of heparin, leaving a high amount of naked particles. In order to understand the contribution of these naked particles in the pro-repair effect of the hcV, two supplemental group were studied: the hcV – nH where the naked particles from the hcV were removed, and the LcV + nH where additional naked particles were added to the low VEGF cluster condition. (A) Fluorescent images of vessels (Glut-1), astrocytic scar (GFAP), microglia (Iba-1) and axonal neurofilaments (NF200) in and around the stroke site (*) of gel + (hcV – nH) and LcV + nH) conditions, 2 weeks post-stroke. Quantitative assessment of the vascular area in the infarct (B1) and the peri-infarct area (B2), microglial area in the infarct (C1) and the peri-infarct area (C2), axonal area in the infarct (D1) and the peri-infarct area (D2), axonal infiltration distance (E1) and astrocytic scar (E2) at 2 weeks post-stroke. hcV – nH = 0.01 μg nH loaded with 200 ng VEGF, lcV = 1μg nH loaded with 200 ng VEGF and 0.99 μg unloaded nH. Data is presented using a min to max box plot. Each dot in the plots represents one animal and p values were determined by One-way ANOVA with a Tukey’s post-hoc test, with **, *** and **** indicating p < 0.01, p < 0.001 and p < 0.0001, respectively. Data represent the average. Scale bar: 100 μm.