Brain repair after stroke with injectable treatment

New injectable treatment helps the immune system to promote Brain repair 

An injectable biomaterial helped stroke-damaged brains recruit immune cells, regrow blood vessels, and restore movement in mice. Duke researchers developed an injectable scaffold which helped stroke-damaged brains grow new blood vessels, support nerve regrowth and recover movement in mice. The treatment appears to work partly by recruiting the body’s own immune cells, including neutrophils which may switch from damaging to helpful under the right conditions. Millions of people experience ischemic strokes each year. These strokes occur when a blood clot blocks blood flow to part of the brain. Biomedical engineers at Duke University have created an injectable biomaterial which may help the brain recover from damage left behind by an ischemic stroke. In experiments with mice, the material transformed the cavity created by lost brain tissue into a more favorable environment for healing. The treatment recruited the body's own immune cells, encouraged the formation of new blood vessels, supported changes in neural tissue, and improved motor function in the animals. The findings were published in Cell Biomaterials. Emergency treatments such as clot-dissolving drugs and procedures which physically remove the clot can restore circulation and help save brain tissue that is still viable. However, once brain tissue has died, restoring blood flow cannot replace what has already been lost. Severe strokes can destroy substantial amounts of tissue, leaving a cavity where healthy brain tissue once existed. After doctors remove the clot, recovery depends largely on rehabilitation. Rehabilitation can help surviving brain circuits adapt, but it does not directly rebuild the damaged region.

Delivered more than 24 hours after a stroke in mice, the biomaterial promoted vascular repair, neural remodeling and improved motor performance two-photon imaging at day 27 using Ly6G-green fluorescent protein (GFP) reporter mice to visualize neutrophils. IL-4/C1q-EV + MAPS implants exhibited dense vascularization and focal accumulation of GFP+ cells within scaffold pores. Injectable biomaterial that helps transform the cavity left behind after a stroke caused by a blood clot into an environment more supportive of repair. "Once brain tissue has been lost, restoring blood flow is no longer enough," said Tatiana Segura, the Robert Plonsey Distinguished Professor of Biomedical Engineering at Duke. "Our goal is to engineer the injured space so that immune, vascular and neural repair processes can begin to work together." Every year, millions of people experience strokes caused by blood clots, called ischemic strokes. Immediate treatments, including clot-dissolving drugs and mechanical removal of the clot, can restore blood flow and preserve threatened brain tissue. But these treatments cannot replace tissue that has already been lost. Large strokes can result in substantial tissue loss and leave behind a cavity at the site of the injury. After the clot is removed, recovery relies largely on rehabilitation, which helps surviving brain circuits adapt but does not directly repair the damaged area.

Segura and her team sought to create an environment inside the stroke cavity which could support several types of repair at once. They used MAPS, or microporous annealed particle scaffolds. These scaffolds consist of individual hydrogel microparticles that assemble into a porous structure. The open spaces provide cells with a framework they can enter and use while rebuilding neural tissue. Building on earlier successes with the biomaterial, the researchers wanted to determine whether they could also use the body's immune system to guide and strengthen the repair process. To pull in helpful immune cells, the team turned to astrocytes, star-shaped cells that support normal brain function and respond rapidly to injury. Astrocytes communicate with other cells in part by releasing extracellular vesicles, or EVs, which are nanoscale packages that carry proteins, lipids and genetic material. Once brain tissue has been lost, restoring blood flow is no longer enough. Main goal is to engineer the injured space so that immune, vascular and neural repair processes can begin to work together. The researchers collected EVs from lab-grown astrocytes and experimented with adding various signaling molecules to attract immune cells and promote vascular repair and functional improvement. Rather than simply injecting the EVs, the team used a chemical reaction to anchor them to the surfaces of the hydrogel microparticles. This kept the signals localized within the scaffold, where incoming cells could encounter them.

“We are not simply placing a material into the brain,” Segura said. “We are engineering a local environment that can coordinate several parts of the repair response.” One signaling combination stood out. IL-4 and C1q were particularly effective at drawing potentially helpful immune cells into the injured region. Those cells included macrophages as well as a surprisingly persistent population of neutrophils. Neutrophils are often linked to inflammation and tissue damage during the early stages of a stroke. The new results suggest their role may be more complicated. At a later point after injury, and when surrounded by the right signals and material environment, neutrophils may instead help support tissue repair. Researchers tested their importance by reducing the neutrophil-rich immune-cell population. When they did so, blood vessel formation declined substantially and the scaffold underwent less remodeling. The result indicated that these immune cells were playing an important role in the healing response. "This result changes how we think about neutrophils after stroke," said Shangjing Xin, lead scientist of the study and a postdoctoral fellow in the Segura Laboratory. "Their role appears to depend on when they arrive, where they are located, and the signals they receive from their surroundings. Our study demonstrates a potential engineering strategy to recruit and retain these cells at the right time."

One combination of signaling molecules, IL-4 and C1q, proved best at attracting helpful immune cells into the damaged region, including macrophages and an unexpectedly persistent population of neutrophils. Neutrophils are commonly associated with inflammation and tissue damage during the early stages of stroke. But the study suggests that, at a later stage and within the right material environment, these cells can also contribute to repair. When the researchers depleted the neutrophil-rich immune-cell population, blood vessel growth and scaffold remodeling were markedly reduced. This finding showed that the cells were important contributors to the repair response. “This result changes how we think about neutrophils after stroke,” said Shangjing Xin, lead scientists of the study and a postdoctoral fellow in the Segura Laboratory. “Their role appears to depend on when they arrive, where they are located and the signals they receive from their surroundings. Our study demonstrates a potential engineering strategy to recruit and retain these cells at the right time.” The immune response was accompanied by the formation of blood vessels throughout the treated cavity. The researchers also observed increased axonal fibers, which are key structures in brain cells, within and around the damaged region. Mice receiving the optimized scaffold also performed better on a grid-walking test which measures errors in forelimb placement. By eight weeks, their performance was statistically indistinguishable from that of healthy control mice, and the improvement was sustained throughout the study.

Importantly, EVs delivered without the MAP scaffold did not produce comparable vascular repair. This result showed that the biomaterial was not simply carrying a therapeutic cargo. Its porous structure and ability to localize the EV signals were essential to the response. While these findings are a step in the right direction, the findings remain preclinical. The evolving treatment was tested in mouse models by directly injecting it into the damaged site. Additional studies will be required to evaluate its safety, determine how the different immune-cell populations contribute to recovery, and test the approach in larger and more clinically representative stroke models. The current study used EVs collected from primary rat astrocytes. As a next step, the Segura laboratory is exploring EVs produced by human induced pluripotent stem cell-derived astrocytes. This approach could provide a more scalable and clinically relevant source of EVs while allowing the researchers to better control the signals they carry. The researchers also tested whether the extracellular vesicles could produce similar effects without the biomaterial scaffold. They could not.

Despite the promising results, the approach remains preclinical. So far, researchers have tested the treatment in mouse models by injecting the material directly into the damaged area of the brain. Further research will be necessary to assess safety, understand precisely how different immune-cell populations influence recovery, and determine whether the treatment works in larger models that more closely resemble human stroke. The researchers also currently obtain the EVs from primary rat astrocytes. “You do not restore an ecosystem simply by containing the initial damage,” Segura said. “You have to create the conditions that allow life to return. That is how we think about the stroke cavity. The material is not intended to reproduce the brain itself, but to create an environment where the body’s own cells can enter, communicate and participate in rebuilding vascularized tissue.”