Long-term retention of ECM hydrogel after implantation into a sub-acute stroke cavity reduces lesion volume

Abstract

Salvaging or functional replacement of damaged tissue caused by stroke in the brain remains a major therapeutic challenge. In situ gelation and retention of a hydrogel bioscaffold composed of 8mg/mL extracellular matrix (ECM) can induce a robust invasion of cells within 24h and potentially promote a structural remodeling to replace lost tissue. Herein, we demonstrate a long-term retention of ECM hydrogel within the lesion cavity. A decrease of approximately 32% of ECM volume is observed over 12weeks. Lesion volume, as measured by magnetic resonance imaging and histology, was reduced by 28%, but a battery of behavioral tests (bilateral asymmetry test; footfault; rotameter) did not reveal a therapeutic or detrimental effect of the hydrogel. Glial scarring and peri-infarct astrocytosis were equivalent between untreated and treated animals, potentially indicating that permeation into host tissue is required to exert therapeutic effects. These results reveal a marked difference of biodegradation of ECM hydrogel in the stroke-damaged brain compared to peripheral soft tissue repair. Further exploration of these structure-function relationships is required to achieve a structural remodeling of the implanted hydrogel, as seen in peripheral tissues, to replace lost tissue and promote behavioral recovery.

Statement of significance: In situ gelation of ECM is essential for its retention within a tissue cavity. The brain is a unique environment with restricted access that necessitates image-guided delivery through a thin needle to access tissue cavities caused by stroke, as well as other conditions, such as traumatic brain injury or glioma resection. Knowledge about a brain tissue response to implanted hydrogels remains limited, especially in terms of long-term effects and potential impact on behavioral function. We here address the long-term retention of hydrogel within the brain environment, its impact on behavioral function, as well as its ability to reduce further tissue deformation caused by stroke. This study highlights considerable differences in the brain's long-term response to an ECM hydrogel compared to peripheral soft tissue. It underlines the importance of understanding the effect of the structural presence of a hydrogel within a cavity upon host brain tissue and behavioral function. As demonstrated herein, ECM hydrogel can fill a cavity long-term to reduce further progression of the cavity, while potentially serving as a reservoir for local drug or cell delivery.

Keywords: Behavior; Biodegradation; Biomaterial; Cell invasion; Extracellular matrix; Hydrogel; Implantation; Magnetic resonance imaging; Stroke; Tissue repair.

Figure 1. Experimental Design

A Diagrammatic representation of animals enrolled in the study charts the inclusion and exclusion of animals, as well as their random group allocation points. B. Overview of experimental time line with procedures performed on animals in relation to implantation of ECM hydrogel (week 0). C. A priori calculations to define a sufficient sample size as a function of statistical power (1-β) and effect size (f for ANOVA analyses). A power of 80% is considered sufficient to account for Type II errors, whereas a statistical significance of p<0.05 is considered a threshold for Type I errors. Considering these parameters, the 36 animals enrolled here will achieve sufficient power if a medium effect size of 0.25 is achieved.

Figure 2. Evolution of lesion volume and tissue deformation

A T₂-weighted MR images of experimental groups over time reveal the hyperintense lesion cavity in animals with stroke damage. B. Measurement of lesion volume (red region-of-interest) based on the hyperintense T₂-weighted signal on MR images defined by mean signal of the contralateral hemisphere + 1 standard deviation (scale bar 2 mm). Ipsilateral (green) and contralateral (light green) parenchymal tissue, lateral ventricles (yellow) were also segmented on MR images. Midline shift (blue line) was calculated by a ratio between distance of the ipsilateral and contralateral hemisphere midpoints. C. Lesion volume was calculated for a baseline pre-implantation time point, as well as for 4 and 12 weeks post-implantation. D. To account for variations in lesion volume at baseline, % change between baseline and 12 weeks were calculated. E. A ratio of parenchymal volume revealed a significant loss of parenchyma in stroke animals, which further declined a little over 12 weeks. F. A gradual shift of the midline was evident in both stroke groups, but was not impacted by ECM hydrogel implantation. G. An equivalent dramatic increase in ipsilateral lateral ventricle was evident in both stroke groups. H. The ratio between the ipsilateral and contralateral ventricle further reflect these gradual long-term changes in tissue structure after stroke. ECM hydrogel did not affect these tissue deformations.

Figure 3. Behavioral assessment

A Bilateral asymmetry test (BAT) analyses revealed a significant sensorimotor bias in the left forepaw, but not in the right forepaw in untreated and treated animals. B. Treatment did not affect sensorimotor bias compared to untreated animals. The footfault test measured motor integration, which was significantly impaired in the left affected forepaw. Treated animals performed comparably to untreated animals on this task. C. Amphetamine-induced rotation bias on the rotameter reveals striatal integrity and dopamine responsiveness. Treated and untreated rats exhibited a comparable asymmetry in rotation.

Figure 4. ECM hydrogel retention and glial scarring

A A histological comparison between controls, untreated and treated stroke animals reveals a major tissue loss in the ipsilateral hemisphere, as well as the presence of ECM hydrogel within the lesion cavity. B. A quantification of parenchymal volume in these 3 groups indicated a 33% tissue loss in animals with stroke, as well as a dramatic increase in ipsilateral ventricular volume. A treatment effect was evident on stroke lesion volume with a 19.8% decrease in cavity volume. C. ECM hydrogel was retained inside the lesion cavity in all treated animals as indicated by collagen I staining surrounded by host tissue. D. Along the anterior-posterior axis, ECM hydrogel was identifiable as a morphologically distinct entity aided by collagen I staining. E. A comparison between injection volume of ECM gel precursor and volume of hydrogel at 3 months revealed a decrease of 32%, but due to individual variability this did not reach statistical significance. F. To evaluate the impact of ECM hydrogel on glial scarring at the tissue interface, whole brain slice images covering the lesion cavity along the anterior-posterior axis were acquired for all 3 groups to measure the level of astrocytic (GFAP) reactivity inside striatal and cortical tissue. G. A marked increase in GFAP staining was evident at the border of the lesion cavity that gradually decreased further inside the tissue. GFAP reactivity in the stroke-damaged striatum was higher than in the cortex. However, there was no significant difference between untreated and treated animals. H. Peri-infarct astrocytosis (i.e. the area occupied by reactive astrocytes) in the ipsilateral and contralateral parenchyma was measured by an 8-bit conversion of whole slice images to apply a threshold that created a binary map. I. This binary image afforded quantification of area occupied and provided a comparison of the 3 experimental groups to indicate that there was no significant difference between untreated and treated animals, although both had significantly more astrocytosis than normal controls. Scale bar 2 mm.

Figure 5. Presence of host cells in ECM hydrogel

A A sharp boundary of DAPI cells defined the interface between biomaterial and host brain with some cells being present within the ECM hydrogel. B. Using collagen I staining, a region-of-interest for the ECM hydrogel was defined and applied to the DAPI image to provide a quantification of the number of cells present within the hydrogel at 12 weeks post-implantation. A mean invasion of 65,050 cells with a range of 44,820-78,740 cells was measured. C. A non-significant (n.s.) correlation between injection volume of ECM or histological volume of ECM at 12 weeks indicates that the number of invading cells was not directly related to the volume of ECM injected into the brain. The regression line in the scatter plot reflects the steepness of the correlation between the two data sets. D. There was also no significant association between the degree of stroke damage (i.e. lesion volume) and the number of invading cells, further highlighting that other factors play an important role in host cell invasion. E. Host cells that invaded and remained within the ECM hydrogel appear to be localized along topological grooves revealed by collagen I staining. Scale bars 100 μm.

Figure 6. Phenotypic characterization of cells in ECM hydrogel

A Immunohistochemical characterization of individual cell phenotypes within the ECM hydrogel at different magnifications. B. A phenotypic analysis of cells present within the hydrogel were predominantly microglia (ionized calcium binding adapter molecule 1, Iba-1), as well as fewer (p<0.01) cells from the oligodendrocyte lineage (2′,3′-cyclic-nucleotide 3′-phosphodiesterase, CNPase). Significantly (p<0.001) fewer neurons (Fox3), neural progenitors (doublecortin, DCX), astrocytes (glial fibrillary acid protein, GFAP) or endothelial cells (rat endothelial cell antigen 1) were found. C. A comparison of monocyte polarization indicated that these mostly co-expressed M1 (CD86) and M2 (CD206) markers, with significantly fewer cell expressing only CD86 (p<0.01) or CD206 (p<0.001). Scale bars 100 μm.