Hydrogel-delivered brain-derived neurotrophic factor promotes tissue repair and recovery after stroke

Abstract

Stroke is the leading cause of adult disability. Systemic delivery of candidate neural repair therapies is limited by the blood-brain barrier and off-target effects. We tested a bioengineering approach for local depot release of BDNF from the infarct cavity for neural repair in chronic periods after stroke. The brain release levels of a hyaluronic acid hydrogel + BDNF were tested in several stroke models in mouse (strains C57Bl/6, DBA) and non-human primate ( Macaca fascicularis) and tracked with MRI. The behavioral recovery effects of hydrogel + BDNF and the effects on tissue repair outcomes were determined. Hydrogel-delivered BDNF diffuses from the stroke cavity into peri-infarct tissue over 3 weeks in two mouse stroke models, compared with 1 week for direct BDNF injection. Hydrogel delivery of BDNF promotes recovery of motor function. Mapping of motor system connections indicates that hydrogel-BDNF induces axonal sprouting within existing cortical and cortico-striatal systems. Pharmacogenetic studies show that hydrogel-BDNF induces the initial migration of immature neurons into the peri-infarct cortex and their long-term survival. In chronic stroke in the non-human primate, hydrogel-released BDNF can be detected up to 2 cm from the infarct, a distance relevant to human functional recovery in stroke. The hydrogel can be tracked by MRI in mouse and primate.

Keywords: Axonal sprouting; bioengineering; hyaluronan; neural repair; neurogenesis.

Figure 1.

Hydrogel release of BDNF and visualization in MRI. (a) Top panel is cortical stroke in forelimb motor cortex; bottom panel shows location of subcortical stroke in striatum. (b) Timeline for studies. (c) ELISA for BDNF in mouse brain striatum, after striatal stroke and after striatal stroke with hydrogel, hydrogel-BDNF at two doses, and high dose BDNF alone (soluble). ***p < 0.001 versus stroke+hydrogel at 1 week, ###p < 0.001 versus striatal stroke at 1 week; ^^^p < 0.001 versus stroke+highBDNF at 1 week; + ++p < 0.001 versus control striatum; $$p < 0.001 versus all conditions at 3 weeks. (d) ELISA for BDNF in mouse cortex, after cortical stroke and after cortical stroke with hydrogel/medBDNF and hydrogel+BDNF (high dose, soluble). ^^^p < 0.005 versus cortical stroke cortex; ^^p < 0.05 versus control cortex, ###p < 0.005 versus cortical stroke+BDNF, ***p < 0.005 versus control cortex, **p < 0.01 versus control cortex. (e and f) T2-weighted MRI images of mouse with motor cortex stroke 15 d after hydrogel-BDNF injection in control stroke (e) and in stroke+hydrogel (f). Arrow points to bright signal of hydrogel. (g) Size of stroke, hydrogel, and total size of stroke+hydrogel as measured in MRI images. *p < 0.01, stroke+hydrogel versus stroke. Stroke only and stroke-hydrogel are separate cohorts of mice (n = 4). The hydrogel values are taken from the stroke+hydrogel mice.

Figure 2.

Behavioral effect of hydrogel-delivered BDNF. (a) Effect of hydrogel-BDNF on forelimb use in exploratory rearing after stroke (cylinder task). All stroke conditions are statistically different from control, non-stroke mice (*p < 0.05) except for stroke+hydrogel+medBDNF at weeks 5 and 9 after stroke. (b) Effect of hydrogel-BDNF on forelimb use in gait. Stroke causes an approximate doubling of the number of footfaults in walking on a grid at one week (before hydrogel injection), with no difference across stroke treatment groups. Beginning on weeks 5 and 9 only stroke+hydrogel+medBDNF is not different from control performance (*p < 0.05). (c) Effect of BDNF alone (no hydrogel) on exploratory rearing. There is no significant difference between stroke and stroke+BDNF or stroke+hydrogel groups. (d) Effect of BDNF alone on gridwalking. There is no significant difference between stroke and stroke+BDNF or stroke+hydrogel.

Figure 3.

Motor cortex axonal connections in stroke and stroke+BDNF. (a–d) Quantification of axonal label from BDA injection into the forelimb motor cortex ipsilateral to the stroke at 9 weeks after stroke. All values are normalized to the axonal label that is present in the control, naïve mouse brain. In (a–d), *p < 0.05 and **p < 0.01 versus naïve. (e and f) Two representative coronal sections through the frontal cortex in each condition. These sections are anterior to the stroke site. Arrowheads denote location of BDA injection. Arrows show highlight contralateral cortex (e) or contralateral striatal (f) projections in which the pattern of axonal labeling is similar in stroke+hydrogel+medBDNF to that of control (top row) or in which there is an increase in axonal label in contralateral striatum in stroke+hydrogel+medBDNF compared to stroke and to control (bottom row).

Figure 4.

Cortical motor maps in stroke and stroke+hydrogel+medBDNF. (a) Schematic view of mouse brain with location of BDA tracer injection (black) and stroke in motor cortex (dotted circle). (b) Flattened map of tangential sections of the entire ipsilateral cortex to the stroke. Red is the location of projections from motor cortex in stroke+hydrogel+medBDNF animals (n = 4). Light blue represents motor cortex projections in stroke-only (n = 4) and dark blue is the region of dense overlap of the two projections. “Inj” is the region of the tracer injection and dense neuronal labeling. The dotted circle is the schematic location of the stroke. There is no significant difference between the spatial mapping of motor cortex projection maps in stroke-only and stroke+hydrogel+med/BDNF. (Hotelling's t2 test; p > 0.05). (c and d) Photomicrographs of axonal label in motor cortex in stroke-only (c) and stroke+hydrogel/BDNF (d). Bar=20 µm. (e) Quantification of the total axonal label in the ipsilateral hemisphere across conditions. **p < 0.01 (n = 4). (f) Infarct size in studies with quantitative mapping of connections in (a)–(d).

Figure 5.

Post-stroke neurogenesis in stroke and stroke+hydrogel+medBDNF. The initial response of migration of immature neurons from the SVZ to peri-infarct tissue was measured (a–f) as well as the long term survival of these cells (j–l). (a–c) Images from C57Bl/6 mice stained for doublecortin (white) 2 weeks after stroke/one week after hydrogel injection. (d)–(f) doublecortin positive cells in DCX-RFP mouse under same time conditions. Arrows in (b and e) show area of DCX + cells in peri-infarct cortex. Arrowhead in (f) shows DCX + cells in striatum in DCX-RFP+hydrogel/BDNF. In (a), v, ventricle; cc, corpus callosum; ctx, cortex. (g) Guantification of doublecortin positive cells in both C57Bl6 and DCX-RFP lines. ***p < 0.001 versus C57 stroke, DCX-RFP. (h and i) Photomicrographs with higher magnification of area of DCX + cells in peri-infarct cortex in stroke alone (h) and stroke+hydrogel/BDNF (i). These images are taken from region adjacent to arrows in (b and e). (j) Representative confocal image stack showing a double positive NeuN/BrdU cell (yellow, arrow) in maximum projection of a Z stack in peri-infarct cortex, panels on right and bottom show y/z and x/z stacked images. In (j) and (k), green is NeuN and red is BrdU. (k) Three-dimensional reconstruction of double positive cell for NeuN and BrdU from confocal image stack. Note that BrdU and NeuN are tightly co-localized. (l) The number of NeuN/BrdU double positive cells per mouse in peri-infarct cortex at 9 weeks after stroke. Stroke * p < 0.05 versus control, **p < 0.01 versus control. Bar in F = 100 µm. Bar in I = 20 µm.

Figure 6.

Effect of hydrogel/BDNF on reactive tissue changes after stroke. Brains were analyzed 5 weeks after hydrogel injection/6 weeks after stroke. First column is from control (no stroke), second column is from stroke only, third column is from stroke+hydrogel, and fourth column is from stroke+hydrogel+medBDNF. (a)–(d) Immunohistochemical stain for GFAP to demarcate reactive astrocytes. *Edge of infarct. Scale bar in A = 40 µm and applies to all photomicrographs. (e) Quantification of GFAP immunoreactivity across conditions. *p < 0.05 versus naïve, **p < 0.01 versus naïve, ***p < 0.005 versus naïve, ****p < 0.001 versus naive. (f)–(i) Immunohistochemical stain for the microglial/macrophage marker IBA-1 in peri-infarct cortex. (j) Quantification of IBA-1 immunoreactivity across conditions. There are no statistically significant differences across conditions. (k)–(n) Immunohistochemical stain for PECAM to indicate endothelial cells in peri-infarct cortex. (o) Quantification of PECAM immunoreactivity across conditions. There are no statistically significant differences across conditions.

Figure 7.

Release of BDNF from hydrogel in non-human primate stroke. Hydrogel+medBDNF or hydrogel alone was injected into the infarct core of a stroke in the non-human primate 3 months after the infarct and BDNF levels measured 2 weeks later in regions close to the infarct (a–c) or more distant to the infarct in the ipsilesional hemisphere (d and e) *p = 0.05. (f) Soluble BDNF was injected into the infarct core in the non-human primate 3 months after stroke and BDNF levels measured 2 weeks later. (g) MRI of non-human primate in vivo with stroke alone (left panel) or stroke+hydrogel+medBDNF (right panel). The hydrogel appears as dark mass in infarct.