Particle Hydrogels Decrease Cerebral Atrophy and Attenuate Astrocyte and Microglia Macrophage Reactivity After Stroke

RESEARCH ARTICLE
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Particle Hydrogels Decrease Cerebral Atrophy and Attenuate

Astrocyte and Microglia/Macrophage Reactivity after Stroke
Elias Sideris, Sophia Kioulaphides, Katrina L Wilson, Aaron Yu, Jun Chen,
S. Thomas Carmichael, and Tatiana Segura*
the injury leaves them with a serious dis-

Increasing numbers of individuals live with stroke related disabilities. ability. There are currently no US food and
Following stroke, highly reactive astrocytes and pro-inflammatory microglia drug administration (FDA) approved thera-
can release cytokines and lead to a cytotoxic environment that causes further peutics to treat long-term disability, leaving
brain damage and prevents endogenous repair. Paradoxically, these same physical therapy as their only medical treat-
ment.
cells also activate pro-repair mechanisms that contribute to endogenous
Immediately following stroke onset, the
repair and brain plasticity. Here, it is shown that the direct injection of a lack of oxygen and nutrients causes sig-
hyaluronic acid-based microporous annealed particle (MAP) hydrogel into the nificant cell death, a large influx of mi-
stroke core in mice reduces the percent of highly reactive astrocytes, increases croglia/macrophages, and the activation of
the percent of alternatively activated microglia, decreases cerebral atrophy, highly reactive astrocytes, which release
pro-inflammatory cytokines,[4,5] and lead to
and preserves NF200 axonal bundles. Further, MAP hydrogel is shown to
further neuronal death and a clearance of
promote reparative astrocyte infiltration into the lesion, which directly cellular debris.[6] Over time, the brain’s de-
coincides with axonal penetration into the lesion. This work shows that the fense mechanism is to compartmentalize
injection of a porous scaffold into the stroke core can lead to clinically relevant the injured tissue from the surrounding tis-
decrease in cerebral atrophy and modulates astrocytes and microglia toward a sue via an astrocytic border and fibrotic scar,
pro-repair phenotype. with tissue fibrosis preventing repair in the
stroke core.[7] With time, the stroke core is
devoid of vessels and axons and cerebral at-
rophy occurs (brain shrinkage). Atrophy in
the motor cortex accounts for at least a portion of the motor deficit
1. Introduction in stroke patients[8] and occurs at a rate of 0.95% of initial vol-
Stroke is the leading cause of adult long-term disability and af- ume in the stroke hemisphere.[9] There are currently no thera-
fects 795 000 Americans every year.[1] Strokes occurs in two main pies to prevent or treat cerebral atrophy, which is correlated with
forms, hemorrhagic or ischemic. Ischemic strokes are caused by dementia,[10] depression,[11] and reduced motor function.[8]
an obstruction within a blood vessel, and account for 87% of all Astrocytes can both aid and obstruct stroke recovery,[12,13] with
strokes and have a 32% mortality.[2,3] For some stroke survivors complete ablation of astrocytes resulting in a worse outcome after
stroke.[14] Astrocytes are able to communicate with multiple neu-
rons via secreted and contact-mediated signals, can coordinate
the development of synapses[15–19] and neural circuits,[20] yet as-
E. Sideris, A. Yu
Department of Chemical and Biomolecular Engineering trocytes can limit long term repair and regeneration when a pro-
University of California Los Angeles inflammatory phenotype is adopted.[12,13] Recently, others[21–23]
Los Angeles, CA 90095, USA and we[24,25] have published a number of publications inject-
S. Kioulaphides, K. L Wilson, J. Chen, T. Segura ing hydrogels into the stroke cavity. These studies have demon-
Departments of Biomedical Engineering strated that the stroke core can accept a significant gel injec-
Neurology, and Dermatology
Duke University
tion volume without damage and that these gels can vary widely
Durham, NC 27708, USA in composition ranging from hydrogels formed from decellu-
E-mail: tatiana.segura@duke.edu larized native tissue,[26] to peptide derived hydrogels,[27] to syn-
S. T. Carmichael thetic hydrogels.[28] Although biomaterial strategies for brain re-
Department of Neurology pair have investigated astrocytes after stroke in the context of the
David Geffen School of Medicine glial scar, no biomaterial has been described that can modulate
University of California Los Angeles
Los Angeles, CA 90095, USA the astrocyte phenotype from inflammatory to pro-regenerative.
Herein, we explore the use of hyaluronic acid (HA)-based granu-
The ORCID identification number(s) for the author(s) of this article lar hydrogels to modulate astrocyte and microglia phenotype to-
can be found under https://doi.org/10.1002/adtp.202200048 ward a pro-repair phenotype and the evaluation of the resulting
DOI: 10.1002/adtp.202200048 reparative response.
Adv. Therap. 2022, 5, 2200048 2200048 (1 of 15) © 2022 Wiley-VCH GmbH

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Figure 1. A) Flow-focusing microfluidic device used to produce hydrogel microparticles (microgels) via a water-in-oil emulsion. Inset shows flow focusing
region. B) Image of microgels in aqueous solution. Scale bar = 100 𝜇m. C) Histogram of microgel diameter. D) Instron mechanical compression testing of
MAP scaffold after annealing of microgels. E) Image of high molecular weight fluorescent dextran between the MAP void spaces showing interconnected
porosity. F) Injection schematic showing direct injection into the stroke cavity. G–K) Images of serial sections of a single stroked brain treated with MAP
with each progressive section ≈300 𝜇m apart.
2. Characterization of MAP Hydrogel and Stroke tion (Figure 1F,G).[41] MAP fills the stroke core as can be seen on
Tissue the serial sections (Figure 1H–K).
Microporous annealed particle (MAP) scaffolds are granular hy-

drogels generated from interlinked hydrogel microparticles (mi- 2.1. Injection of MAP Hydrogel Preserves Long Term Structural
crogels). HA microgels were generated using a previously de- Integrity and Function
scribed microfluidic flow focusing device[29] (Figure 1A). HA (70–
90 kDa) was utilized as our based material because it is a brain To expand from our previous work using MAP scaffolds to treat
extracellular matrix (ECM) component. Because microgels con- stroke,[24] we wanted to understand how long the gel lasts in the
tained cross-linked HA, it behaves as high molecular weight, and brain after implantation and if there are any detrimental effects
thus is not expected to induce inflammation as has been reported of hydrogel injection long term. Our previous work focused on
for low molecular weight HA.[30–32] Microgels used in this study only local tissue assessment at 2 weeks post stroke.[24] As previ-
had 80 𝜇m diameter (Figure 1B,C) because we have previously ously done, we injected MAP hydrogel 5 days after stroke[41] and
found this diameter to be injectable and result in MAP scaffolds assessed for hydrogel degradation by monitoring hydrogel fluo-
that were biocompatible in brain.[24] The Young’s modulus of rescence at days 2, 10, 30, and 120 days post injection or 1, 2, 4,
our MAP hydrogel is 927 Pa (Figure 1D), which was chosen to and 16 weeks post stroke (Figure 2A,B). We find that hydrogel
match the Young’s modulus of brain cortex.[33] Last, a key feature fluorescence is visible at 4 weeks post stroke but not 16 weeks
of MAP is interconnected void structure, which can be observed post stroke, indicating that the hydrogel can provide mechanical
by filling the void space with fluorescently labeled dextran (Fig- support for at least 30 days post injection.
ure 1E). Others and we have utilized MAP scaffolds for cell cul- Atrophy is commonly observed in stroke patients and is known
ture in vitro[24,34–40] and support tissue ingrowth in vivo.[24,35–37] to cause effects at regions far away from the stroke core.[9] In
In this study, MAP scaffolds were injected into the stroke core at human patients’ cerebral atrophy in the motor cortex accounts
5th day post wounding because a significant fluid filled cavity has for at least a portion of the worsening motor deficit in stroke
been formed by this time that can accept a large volume of injec- patients over time.[8] Thus, we wanted to get a sense if there are

Figure 2. A) Schematic of experimental timeline where arrow signifies stroke day, + signifies injection day (MAP only), and x signifies sacrifice and analysis
time point. Sham is stroke only with no injection. B) Hydrogel imaging over time after stroke (1, 2, 4, and 16 weeks post stroke). Scale bar = 100 𝜇m.
C) Global measurements of brain and cortex volume for healthy, sham, and MAP-treated groups. D) Ratios of hemisphere and cortex area comparing
ipsilateral to contralateral regions for healthy (no injury), sham (stroke only), and MAP (stroke + MAP)-treated groups. E) Schematic of NF200 axonal
bundles in hemispheres ipsilateral and contralateral to the lesion, showing ROI presented in (F). F) NF200 axonal bundles over time for healthy, sham,
and MAP. G) Ipsilateral to contralateral ratios of NF200 axonal bundles number for healthy, sham, and MAP-treated groups. All measurements were done
on FIJI. Statistical analysis was done in GraphPad Prism using two-way ANOVA analysis of variance followed by a multiple comparisons test comparing
each condition across time or between condition at each timepoint. The dots in each plot indicate the number of animals (n = 5 biological replicates)
that were analyzed per-group and were considered independent experiments. *, **, ***, and **** means p < 0.05, 0.01, 0.001, and 0.0001. ☆ represents
the stroke cavity.
other visible effects of a long-lasting hydrogel in the brain post changes in brain volume and cortex volume that are reversed
stroke and reduced cerebral atrophy in mice. We first wanted to when MAP hydrogel is injected in the stroke core (Figure 2C).
understand if changes are to be expected due to ageing. Healthy We next wanted to compare the ratio of the ipsilateral (injured
mice that did not experience stroke or any other procedure were hemisphere) and contralateral (non-injured hemisphere), which
sacrificed at 1, 2, 4, and 16 weeks (matching the age of the stroke directly address differences between the two hemispheres in the
mice). We find that in general, healthy mice brain size does same animal. As with the global brain and cortex volume, healthy
not change with age and thus any changes in brain size in our animals had no statistical difference between their two hemi-
experiments will be due to stroke related injury (Figure 2C). We spheres, indicating their symmetry and that again any changes
find that stroke (sham condition) induces statistically significant would be due to the stroke related injury and not age. We find

that both stroke only (sham) and stroke + MAP injection (MAP) sham condition, while only 25.2% of all astrocytes are reactive
results in a statistical different ratio at 16 weeks. However, the in the 3.5% MAP gel peri-infarct condition. Comparing the peri-
change for the sham group is statistically significant at 4th week, infarct of the MAP gel (≈25.2%) to the infarct of the MAP gel
but it is not for the MAP-treated groups (Figure 2D). Overall, we (≈10.7) once again further shows that the infiltrating astrocytes
find that MAP gel injection delays significant changes in brain are less reactive and suggests a pro-regenerative phenotype (Fig-
and cortex volume, which may have implications in delaying ure 3G). All these results indicate that the MAP gel can attenu-
atrophy related disability. ate astrocyte reactivity just 2 days after injection by reducing the
We next wanted to assess how NF200 axonal bundles in the number of highly reactive neurotoxic astrocytes by greater than
striatum vary across stroke only, and MAP-treated brains vary. twofold, promoting a less pro-inflammatory environment in the
These NF200 axonal bundles contain cortico-spinal, corticotha- peri-infarct area and stimulating pro-recovery astrocyte infiltra-
lamic, cortico-bulbar, and corticostriatal axonal projections. All tion in the infarct. Since inflammatory responses occur early af-
of these are the major output of the cortex to subcortical sites ter injury and initial inflammatory responses have long lasting
and can be damaged after injury to the cortex and due to atro- effects in the tissue,[4] we expect that early modulation of the as-
phy. We quantified the number of NF200 bundles over time us- trogliotic response will have long lasting effects.
ing a watershed method to segment individual bundles. We re-
port the ratio of number bundles between the two brain hemi-
2.3. Microglia Upregulate Pro-Repair Marker Arg1 in MAP
spheres. Although healthy animals have no statistical difference
in NF200 axonal bundles in the striatum, they trend to a decrease
Microglial polarization directly affects astrocyte reactivity, with
with age. We find that at 4th week post stroke the stroke only con-
microglia that exhibit the more reactive M1 phenotype in-
dition (sham) has significantly reduced number of bundles com-
fluencing astrocytes to a more highly reactive state.[13] Simi-
pared to aged-match healthy controls and MAP-treated brains
lar to the astrocyte experiments, MAP hydrogel was injected
suggesting that treatment with a hydrogel in the cortex can pre-
5 days post stroke and tissue analyzed 2 days later (Fig-
vent damage to cortical axonal projections. Taken together the re-
ure 3H). Though we recognize that microglia/macrophage exist
duced cerebral atrophy and preservation of NF200 axonal bundles
in a spectrum of activation, we broadly defined pro-repair mi-
for MAP-treated brains could reduce the long-term degenerative
croglia/macrophages as expressing Arg1 and pro-inflammatory
damage associated with these conditions.
microglia/macrophages-expressing iNOS.[44] Interestingly, the
percentage of microglia/macrophages expressing the M1 pro-
inflammatory phenotype is similar across both conditions in the
2.2. Highly Reactive, Scar Forming Astrocytes Are Reduced after
peri-infarct and in the infarct (Figure 3I). Further, the percent-
MAP Injection
age of microglia/macrophages expressing the M2 pro-repair phe-
notype is similar across both conditions in the peri-infarct area.
We next moved to explore the use of MAP gels to modulate astro-
However, the percentage of pro-repair microglia/macrophages
cyte phenotype. MAP gel was injected 5 days post stroke and tis-
in the infarct, where the cells are encapsulated within HA-MAP
sue was collected 2 days post injection to assess the early immune
during injection, is almost threefold higher in the MAP gel
response to the material (Figure 3A,B). We assessed both the peri-
condition compared to sham condition (Figure 3J). This sug-
infarct (area surrounding the stroke core) for both stroke and
gests that the infarct in the MAP gel has a more pro-reparative
stroke + MAP groups and the infarct area only for stroke + MAP
environment than the infarct in the sham condition. Confine-
because astrocytes do not infiltrate the stroke core in the absence
ment of macrophages has been shown to prevent LPS-polarized
of MAP injection. In the peri-infarct area, we observe a signifi-
M1 macrophages to activate late-stage inflammatory genes.[45]
cantly higher percentage of reactive astrocytes in the sham con-
These findings combined with our findings of increased percent-
dition expressing pERK1/2 (≈56% compared to ≈25% in MAP)
age of Arg1 positive cells, suggests that microporous hydrogels
(Figure 3C), an astrocytic downstream pathway for high reactiv-
formed using ≈80 𝜇m microgels have pore sizes that can spatially
ity. Moreover, significantly fewer infiltrating astrocytes express
confine the microglia and lower M1 polarization. Chondroitin
pERK1/2 compared to those in the peri-infarct (≈12% in the in-
sulfate proteoglycans (CSPGs) have been linked to decreased re-
farct compared to ≈25% in the peri-infarct) (Figure 3D). This
generative potential in the CNS.[28,46,47] We find that the amount
data suggests that the astrocytes that are infiltrating the MAP gel
of CSPGs in the HA-MAP-treated mice significantly decreases in
are less reactive and potentially more pro-reparative. We also ob-
both the infarct (≈5.3%) and peri-infarct (≈9.8%) compared to
serve a significantly higher percentage of reactive astrocytes ex-
sham infarct (≈59.7%) and peri-infarct (≈38%) (Figure S1, Sup-
pressing S100𝛽 in the sham condition (≈69%) compared to the
porting Information). Several studies have delivered Chondroiti-
3.5% MAP gel (≈26%), with high expression of S100𝛽 consid-
nase ABC to digest the high release of CSPG after brain injury
ered pathological[42,43] (Figure 3E). Like pERK expression, the in-
and show behavioral functional benefits.[46,47] Thus, a material
filtrating astrocytes expressing S100𝛽 (≈15%) were significantly
such as HA-MAP that can decrease the CSPG level without addi-
less than in the peri-infarct area (≈26%). Additionally, we used
tional enzyme delivery would be advantageous.
in situ hybridization to dive deeper into the reactive astrocyte
phenotype (Figure 3F). Using RNA markers, we probed for C3,
a marker shown to be upregulated in neurotoxic reactive astro- 2.4. Astrocytes Continue to Infiltrate Lesion over Time
cytes, and SLC1A2, a marker for all astrocytes.[13] Using a ratio of
C3/SLC1A2 to analyze what percentage of astrocytes are highly One key difference between injection of MAP into the stroke cav-
reactive, we observed 54.2% of all astrocytes are reactive in the ity compared to any other hydrogel that we have tested[25,48–51]

Figure 3. A) Schematic of experimental timeline where arrow signifies stroke day, + signifies injection day (MAP only), and x signifies sacrifice and
analysis time point. Sham is stroke only with no injection. B) Schematic of analysis setup showing where sections were imaged and analyzed. C) IHC
images showing astrocyte reactivity through pERK, S100𝛽, and GFAP. D) Quantification of pERK/GFAP percent of astrocytes which are highly reactive.
E) Quantification of S100𝛽/GFAP percent of astrocytes which are highly reactive. F) In situ hybridization images of astrocyte reactivity through C3 and
SLC1A2 probing. G) Quantification of C3/SLC1A2 percent of astrocytes which are highly reactive. H) IHC images of microglia reactivity through CD11b,
Arg1, and iNOS staining. I) Quantification of iNOS/DAPI for determination of microglial pro-inflammatory phenotype. J) Quantification of Arg1/DAPI
for determination of microglial pro-repair phenotype. All scale bar = 100 𝜇m. All measurements were done on FIJI. Statistical analysis was done in
GraphPad Prism using one-way ANOVA analysis of variance followed by a multiple comparisons test comparing each condition (Tukey). The dots in
each plot indicate the number of animals (n = 5 biological replicates) that were analyzed per-group and were considered independent experiments. *,
**, ***, and **** means p < 0.05, 0.01, 0.001, and 0.0001. Individual p-values in G indicate t-test between the condition indicated. ☆ represents the
stroke cavity.
is that it elicits astrocyte infiltration into the stroke cavity. Thus, morphology affects the scar thickness and if astrocyte infiltration
rather than astrocytes remaining in the area surrounding the is continuous over time, we injected HA-MAP 5 days after stroke
stroke core, as occurs in sham conditions, astrocytes change their and assessed scar thickness and astrocyte infiltration over time
morphology and begin to infiltrate. We observed this previously (7, 15, and 30 days post stroke) by quantifying the thickness of
using a middle cerebral artery occlusion model[24] and in data pre- the astrocyte (GFAP+) dense layer surrounding the stroke and
sented here with a PT stroke model, indicating the finding is not infiltration distance of astrocytes starting from the stroke border
model- or cortex location-specific. To investigate if this change in (Figure 4A,B). As expected, sham animals have scar thicknesses

Figure 4. A) IHC images showing reactive astrocytes and microglia of healthy tissue and 5 days post stroke. B) Schematic of experimental timeline where
arrow signifies stroke day, + signifies injection day (MAP only), and x signifies sacrifice and analysis time point. Sham is stroke only with no injection. C)
IHC images of sham stroke tissue showing reactive astrocytes and reactive microglia at 1, 2, and 4 weeks post stroke through GFAP and Iba1 staining.
D) IHC images of MAP gel-treated stroke tissue showing reactive astrocytes and reactive microglia at 1, 2, and 4 weeks post stroke through GFAP and
Iba-1 staining. E) Quantification of scar thickness over time through GFAP staining comparing sham and HA-MAP-treated brains. F) Quantification of
percent area of reactive astrocytes in the peri-infarct through GFAP staining comparing sham and HA-MAP-treated brains. G) Quantification of astrocyte
infiltration into infarct through GFAP staining comparing sham and HA-MAP-treated brains. H) Quantification of percent area of reactive microglia in
peri-infarct area through Iba-1 staining comparing sham and HA-MAP-treated brains. I) Quantification of percent area of reactive microglia in infarct
through Iba-1 staining comparing sham and HA-MAP-treated brains. All scale bars = 100 𝜇m. All measurements were done on FIJI. Statistical analysis
was done in GraphPad Prism using two-way ANOVA analysis of variance followed by a multiple comparisons test (Sidak) comparing between condition
at each timepoint. The dots in each plot indicate the number of animals (n = 5 biological replicates) that were analyzed per-group and were considered
independent experiments. *, **, ***, and **** means p < 0.05, 0.01, 0.001, and 0.0001. ☆ represents the stroke cavity.
and peri-infarct % glial fibrillary acidic protein (GFAP) area that stroke, reaching close to 500 𝜇m into the lesion by day 30 (Fig-
increases over time (7–30 days after stroke) (Figure 4C), indica- ure 4G). The infiltration pathway closely mimics what we expect
tive of a developing scar.[52] In contrast, injection of MAP hydro- is the void structure of the hydrogel (Figure 1E), indicating that
gel into the stroke cavity significantly reduces the scar thickness the cells infiltrate through the void space rather than through the
and peri-infarct % GFAP area within 2 days of implantation and microgels. This infiltration pattern further suggests that signifi-
remains low throughout (Figure 4D–F). cant gel degradation is not required for astrocyte infiltration and
We next examined astrocyte (GFAP+) cell infiltration over that any degraded ECM or new ECM deposition within the void
time. We find that astrocyte infiltration begins within 2 days of space of HA-MAP does not prevent infiltration of astrocytes. We
hydrogel injection and continues to increase from 7–30 days post believe that promoting infiltration of pro-regenerative astrocytes

into the infarct can be beneficial toward recovery.[53] This data GFAP staining. Examining the MAP hydrogel condition at 4th
also shows that the MAP gel sustains astrocyte infiltration over month reveals that the close association of axons and NF200 is
time without the need for biologic delivery such as growth factors maintained and that it is also accompanied by progenitor cells
or small molecules. (SOX2+) (Figure 5I). Although these images cannot determine
the ratio of GFAP+, NF200+, and GFAP+/SOX2+ cells, we likely
have all three cell types present within MAP hydrogel at 4 months
2.5. Microglia Percent Decreases over Time for Hydrogel post injection. Both recruitment of endogenous neural progen-
Injection itor cells (NPCs) and transplantation of exogenous NPCs have
been used as strategies to promote stroke repair and showed
Next, we examined microglia/macrophage reactivity over time increase behavioral response.[54] This astrocyte/axon correlation
using Iba1 staining (Figure 4D). At 2 days post injection we suggests that the astrocytes are crucial for the axon penetra-
find a three- or six-fold decrease in microglia stain for the peri- tion and maintenance, supporting our rationale that promotion
infarct (Figure 4H) and infarct (Figure 4I) area, respectively, of pro-repair astrocyte infiltration will be beneficial for down-
when comparing sham to HA-MAP-treated strokes. As previ- stream tissue repair. As prior studies have shown, these pene-
ously mentioned, reactive microglia have been shown to con- trating astrocytes, in very near proximity to axons could be crucial
tribute to highly reactive astrocytes.[13] Further examination into in forming synapses[15,16,19] and neuronal circuits directly in the
microglial progression shows a very elevated total number of re- lesion site,[18,20] where such recovery is not normally observed
active microglia even 30 days post stroke (Figure 4H,I). Remark- after stroke.[4] Although other molecules and cell types maybe
ably, the total number of reactive microglia at 30th day post stroke needed to generate the desired circuitry, having astrocytes, axons
in the infarct of the sham condition is still threefold higher than and NPCs in this same space can be now further manipulated
the infarct of the HA-MAP condition at 7th day post stroke, the to achieve this. Our results demonstrate that injection of MAP
peak of inflammation. hydrogel into the stroke cavity is generating a permissible envi-
ronment in the peri-infarct and infarct spaces that leads to axonal
infiltration into the stroke core.
2.6. Astrocytes and Axons Co-Infiltrate the Stroke Core after MAP
Injection
2.7. Vessel Infiltration Does Not Coincide with Astrocyte/Axonal
Given the significant impact that MAP has on the phenotype of Infiltration
astrocytes and the number of microglia, we wanted to assess if
changes in the stroke environment were accompanied with in- Similar to axonal density, we observed an initial increase in ves-
creases in axonogenesis. As before, HA-MAP hydrogel was in- sel density (GLUT1 positive) in the peri-infarct space, which
jected into the stroke cavity 5 days after stroke and axonal area was statistically significant at the 15-day time-point compared
(NF200+) in the peri-infarct as well as infiltration distance in the to sham mice (Figure S2A–C, Supporting Information). This re-
infarct quantified (Figure 5A,B). The peri-infarct percent NF200+ sult is consistent with other reports of stroke induced angiogen-
area decreases from 53.2% to 20.4% comparing un-injured brain esis in the peri-infarct space.[55] However, by 30 days post stroke,
and stroke brain at 5th day, indicating the rapid loss of neu- there is no statistical significance in vessel density between MAP
rofilaments in the peri-infarct space after stroke (Figure 5C). hydrogel injection and sham. Thus, like our axonal sprouting
The stroke cavity is devoid of neurofilaments at this timepoint. data, the increase in angiogenesis suggests that the lower mi-
We find that axons (NF200+) increase in the peri-infarct space crophage/microglia number and reduced reactive astrocytes in
following injection of HA-MAP hydrogel, with a significant in- the peri-infarct space generates an environment that further pro-
crease at 7th day post stroke compared with sham (Figure 5D– motes angiogenesis early after MAP gel injection, but that cannot
F). However, this increase becomes non-significant at the 15- be maintained long term.
and 30-day timepoints. This data suggests that the lower num- After observing significant astrocyte/axonal infiltration into
ber of reactive astrocytes combined with the decreased number the stroke core, we tested if vessels also infiltrate the stroke
in macrophage/microglia in the MAP condition, promotes a pro- core and if they follow a similar infiltration pattern (Figure
reparative environment early after MAP injection that promotes S2D, Supporting Information). We find that vessels infiltrate
axonal sprouting, but this environment cannot be maintained. the stroke core, reaching a statistically significant difference of
In the infarct cavity we observe axonal infiltration for the MAP infiltrating vessels between brains treated with MAP and sham
hydrogel-treated groups, but not in the sham groups. The path groups at 30th day post injection. The infiltration path is similar
of infiltration is similar to what the astrocytes follow, navigat- to that of astrocytes and axons, following the void space between
ing between the microgels rather than through the microgels microgels, but there appears to be no correlation between
(Figure 5G). The infiltration distance of axons steadily increases astrocyte infiltration and vessel infiltration, unlike what was
over time reaching ≈450 𝜇m by day 30 (Figure 5G). Staining observed for axonal infiltration. The vessel stain Glut-1 does not
for both astrocytes (GFAP) and axons (NF200) revealed that not coincide with the astrocyte stain GFAP, indicating that vessels
only do the axons follow the same path, but the axons appear invade the stroke core independently. Further, we find that the
to localize closely with the infiltrating astrocytes (Figure 5H,I). vessel infiltration distance is significantly lower compared to
High magnification image analysis revealed that although GFAP what was found for astrocytes and axons (Figure S3, Supporting
stain exists without axonal stain, the reverse is not true; when- Information). The average infiltration distance at 30th day for
ever an axonal stain was present it was closely associated with vessels is ≈207 𝜇m while it is ≈467 𝜇m for axons and astrocytes.

Figure 5. A) IHC images showing reactive astrocytes and axons of healthy tissue and 5 days post stroke through GFAP and NF200 staining. B) Schematic
of experimental timeline where arrow signifies stroke day, + signifies injection day (MAP only), and x signifies sacrifice and analysis time point. Sham
is stroke only with no injection. C) IHC images of sham stroke tissue showing reactive astrocytes and axons at 1, 2, and 4 weeks post stroke through
GFAP and NF200 staining. D) IHC images of MAP gel-treated stroke tissue showing reactive astrocytes and axons at 1, 2, and 4 weeks post stroke
through GFAP and NF200 staining. E) Quantification of percent area of axons in the peri-infarct through NF200 staining comparing sham and HA MAP-
treated conditions. F) Quantification of percent area of axons in the infarct through NF200 staining comparing sham and HA MAP-treated conditions.
G) Quantification of axon infiltration distance into infarct through NF200 staining comparing sham and HA MAP-treated conditions. H) IHC images
of co-residing astrocytes and axons at 4th week post stroke through GFAP and NF200. I) IHC images of co-residing astrocytes and axons at 120 days
post stroke through GFAP and NF200. All scale bars = 100 𝜇m. All measurements were done on FIJI. Statistical analysis was done in GraphPad Prism
using two-way ANOVA analysis of variance followed by a multiple comparisons test (Sidak) comparing between condition at each timepoint. The dots
in each plot indicate the number of animals (n = 5 biological replicates) that were analyzed per-group and were considered independent experiments.
*, **, ***, and **** means p < 0.05, 0.01, 0.001, and 0.0001. ☆ represents the stroke cavity.
This difference in infiltration distance is perplexing as HA-MAP the brain,[35] skin,[35] and bone.[56] Thus, mechanical properties
hydrogel contains no releasable bioactive signals that would are not likely to be the reason. The porosity of the scaffold is an-
promote the infiltration of astrocytes and axons but not vessels. other possibility; HA-MAP gels with microgels of 80–100 𝜇m in
A few possibilities for the reduced infiltration distance for vessels diameter have a median pore area of ≈200𝜇m[2] which would cor-
are the physical properties of the gel, mechanical properties, and respond to 15–20 𝜇m when measured per z-stack.[35,57] Although
porosity. It is difficult to know what the mechanical properties of we have previously shown that polyethylene glycol (PEG)-MAP
the gel are over time; however, the initial HA-MAP gel Young’s gels with 80 𝜇m diameter microgels promote vascularization
modulus is ≈1000 Pa. It is possible that the mechanical modulus and rapid wound closure in skin wounds,[35] others have shown
of the gel is not optimal for vessel infiltration, though others, that in cardiac engineering there is limited vessel penetration in
including our lab, have used soft hydrogels for vascularization in scaffolds with 20 𝜇m pores.[58] Thus, the effect in pore size in

vascularization maybe tissue specific. Regardless of the reason, MAP (Figure 6B). There is also no infiltration of astrocytes into
we have previously demonstrated that a robust vascular network the stroke cavity, suggesting that the microstructure of MAP is
within the stroke, promotes the formation of a neurovascu- critical to this process. Microglia/macrophage area was signifi-
lar niche that leads to effective axonogenesis and behavioral cantly decreased in the infarct but not the peri-infarct compared
improvement.[25] Thus, efforts should be made to introduce to sham; however, the decrease was not as great as that observed
pro-angiogenic cues into MAP to promote vascularization. with MAP.
An additional rationale for further promoting vascularization We next tested the effect of the integrin binding ligand RGD.
is the fact that astrocytes and axons stopped infiltrating 200 𝜇m Injection of HA-MAP(-RGD) revealed that the scar thickness was
away from vessels, which is the oxygen diffusion limit.[59] Thus, again lower than sham, but not as low as HA-MAP. Similar to HA
lack of vascular infiltration, likely limited the range of astrocyte non-porous there was no infiltration of astrocytes into the stroke
and axonal infiltration. cavity, pointing to the importance of RGD in this process. The
number of reactive microglia for HA-MAP(-RGD) is similar to
HA-MAP for the peri-infarct space, but significantly higher for
2.8. Moderate Hydrogel Stiffness Changes Do Not Affect infarct (Figure 6C). Overall, we see that RGD has a major effect
Astrocyte Behavior in the ability of HA-MAP to modulate astrocyte and microglia
phenotype.
Biomaterial and substrate stiffness has been previously shown Last, we tested the role of the hydrogel backbone and compared
to regulate astrocyte reactivity with softer substrates promoting HA to polyethylene glycol (PEG). PEG-vinyl sulfone (20 000 Da)
astrocyte quiescence, suggesting that softer substrates should was used to generate microgels using the same concentration of
be used to better modulate astrocytes following stroke.[60] Stiff- K, Q, and MMP peptides. PEG microgels were generated with
ness has also been implicated in axonal sprouting, vessel sprout- RGD or no RGD. PEG microgels have the same diameter as HA
ing, and microglia polarization. We tested a moderately stiffer microgels (Figure S4A, Supporting Information) and the storage
HA-MAP hydrogel (3.5% = 1000 Pa, 4.5% = 1500 Pa) to deter- modulus is the same as HA-MAP (Figure S4B, Supporting In-
mine if this stiffness change can affect astrocyte behavior (Figure formation). We find that having a PEG backbone in the micro-
S4A,B, Supporting Information). All other parameters, RGD con- gels significantly reduces the effect observed with HA-MAP. Scar
centration, microgel diameter, and void fraction were kept con- thickness is significantly thinner than sham, but larger than HA-
stant (Figure S4A,B, Supporting Information). Overall, the 4.5% MAP and there is no significant difference between PEG-MAP
MAP hydrogel produced very similar results compared to the and PEG-MAP(-RGD) (Figure 6D). Astrocyte infiltration in the
3.5% hydrogel for astrocyte infiltration and scar thickness (Fig- PEG-MAP or PEG-MAP(-RGD) conditions is also close to zero.
ure S4C,D, Supporting Information). However, we did observe Finally, the microphage/microglia area is significantly decreased
an increased number of reactive microglia in the infarct and in in the infarct for both PEG-MAP or PEG-MAP(-RGD), but not
the peri-infarct at 2nd week post stroke, but no significant differ- statistically different from sham in the peri-infarct space. Overall,
ences were observed at 4th week post stroke (Figure S4E,F, Sup- we find that HA is an essential component to promote infiltration
porting Information). Interestingly, these increased microglia re- of astrocytes into the infarct core.
sponse may have caused some downstream effects as we observe Taken together this data shows that HA-MAP composition and
a slight decrease in axon penetration compared to the 3.5% con- microstructure is essential for the observed astrocyte infiltration,
dition (Figure S4G, Supporting Information). It is possible that reduced scar thickness, and reduced microphage/microglia in
the increased number of microglia in the 4.5% condition created the peri-infarct and infarct spaces. No other condition tested was
a more cytotoxic environment that affected the axon penetration. equally effective as HA-MAP. Importantly, all the conditions that
used HA as the backbone significantly decreased the scar thick-
ness compared to conditions that used PEG as the backbone,
2.9. Porosity, Hyaluronic Acid, and RGD Are Crucial for Astrocyte demonstrating that the biological activity of HA plays an impor-
Infiltration tant role in this process. After stroke, CD44 expression is elevated
in reactive astrocytes.[61] Thus, receptor-mediated astrocyte/HA
We next wanted to examine if the observed responses are specific binding maybe responsible for the infiltration in HA-MAP but
to the MAP formulation tested. MAP are produced from ≈80 𝜇m not PEG-MAP. Previous studies implanting porous HA-RGD hy-
microgels, which contain HA (70 000 Da, 3.5%), RGD (500 𝜇m), K drogels into cortex, demonstrated that RGD increased astrocyte
and Q peptides (250 𝜇m), and 7.8 mm of matrix metalloproteinase infiltration into the cortex wound.[62] These results agree with
(MMP) cross-linker. The MAP gel is cross-linked using FXIIIa our finding that HA-MAP but not HA-MAP(-RGD) promoted as-
(5 U mL−1 ) and 1 U mL−1 of thrombin. To test the role of mi- trocyte infiltration. It is surprising that PEG-MAP and HA-MAP
crostructure on the observed findings, we compared the results resulted in different tissue responses. These materials have the
from MAP to those of a hydrogel with identical composition but same microstructures and same concentration of RGD. Given
cross-linked as a bulk gel (non-porous) (Figure 6A,B). As a com- the large number of studies conducted using PEG hydrogels for
parison we tested the ability of these gels to promote astrocyte tissue repair applications, it is possible that the results observed
infiltration, reduce scar thickness, and reduce the number of mi- here are particular to brain and not generalizable to all tissues. In
crophage/microglia in the peri-infarct and infarct spaces. We find our own laboratory, we have used PEG-MAP to treat skin wounds
that the scar thickness in the non-porous group is significantly and shown substantial cellular infiltration and wound closure
reduced compared to sham but significantly larger compared to just 5 days after implantation.[35]

Figure 6. A) Schematic of experimental timeline where arrow indicates day of stroke, + indicates day of injections, and x indicates day of sacrifice and
analysis. IHC images for sham and HA MAP-treated brains at 1st week post stroke staining for GFAP and Iba1. B) IHC images of non-porous-treated
brains staining for GFAP and Iba1. Quantification of reactive astrocytes and reactive microglia testing for scar thickness, astrocyte infiltration distance,
Iba1 peri-infarct percent area, and Iba1 infarct percent area to analyze the role of hydrogel microstructure through GFAP and Iba1 staining. C) IHC images
of HA MAP (-RGD)-treated brains staining for GFAP and Iba1. Quantification of reactive astrocytes and reactive microglia testing for scar thickness,
astrocyte infiltration distance, Iba1 peri-infarct percent area, and Iba1 infarct percent area to analyze the role of RGD integrin binding through GFAP and
Iba1 staining. D) IHC images of PEG-MAP and PEG-MAP (-RGD)-treated brains staining for GFAP and Iba1. Quantification of reactive astrocytes and
reactive microglia testing for scar thickness, astrocyte infiltration distance, Iba1 peri-infarct percent area, and Iba1 infarct percent area to analyze the
role of hydrogel backbone through GFAP and Iba1 staining. All scale bars = 100 𝜇m. All measurements were done on FIJI. Statistical analysis was done
in GraphPad Prism using one-way ANOVA analysis of variance followed by a multiple comparisons test comparing each condition (Tukey). The dots in
each plot indicate the number of animals (n = 5 biological replicates) that were analyzed per-group and were considered independent experiments. *,
**, ***, and **** means p < 0.05, 0.01, 0.001, and 0.0001. ☆ represents the stroke cavity.
3. Conclusions response. Vascularization is critical to providing oxygen and nu-

trients to neighboring cells as it has been shown that the oxygen
Astrocytes have previously been thought to exacerbate the in- diffusion limit is ≈200 𝜇m. We observe the astrocytes and axons
flammation after stroke and be detrimental to recovery,[5] how- to be infiltrating ≈200 𝜇m further than the infiltrating vessels.
ever, recent studies show that astrocytes with pro-regenerative This suggests that the limiting factor to further astrocyte/axonal
phenotypes can be beneficial to the repair process.[13] When in- infiltration is the vessel response. Likely introducing delivery of
jected directly into the stroke cavity, MAP drastically affects the angiogenic factors is required to improve the vasculature, infil-
phenotype and reactivity state of the astrocytes just 2 days after tration, and thereby, improving astrocyte/axonal infiltration.
injection. Using markers of highly reactive astrocytes, we show
that the MAP hydrogel significantly reduces highly reactive as-
trocytic phenotype compared to sham, while also promoting the 4. Experimental Section
infiltration of less reactive astrocytes into the lesion. Examina-
Microgel Production and Purification: Microfluidic devices[35] and 3.5%
tion into microglial polarity suggested a more pro-repair envi- microgels[65] were produced as previously described. Briefly, HA func-
ronment compared to sham. Taken together, the MAP is able tionalized with an acrylate was dissolved at 7% w/v in 0.3 m triethy-
to modulate the brain’s inflammatory response to the stroke by loamine pH 8.8 and pre-reacted with K-peptide (Ac-FKGGERCG-NH2 )
reducing highly reactive astrocytes and promoting infiltration of and Q-peptide (Ac-NQEQVSPLGGERCG-NH2 ) at a final hydrogel con-
cells with a pro-reparative phenotype. The MAP hydrogel is able centration of 250 𝜇m, and RGD (Ac-RGDSPGERCG-NH2 ) at a final hy-
to sustain decreased inflammation over time by maintaining a drogel concentration of 500 𝜇m. Concurrently, the cross-linker solution
was prepared by dissolving the di-thiol MMP sensitive peptide (Ac-
reduced astrocytic scar, preventing a large influx of reactive mi- GCRDGPQGIWGQDRCG-NH2 , Genscript) in distilled water at 7.8 mm
croglia/macrophages, while extending astrocyte infiltration into and reacted with 10 𝜇m Alexa-Fluor 647-maleimide (Life-Technologies)
the lesion. The ability of the MAP gel to modulate inflamma- for 5 min. These solutions were mixed in a flow focusing microfluidic de-
tion could transform treatments of severe inflammation in the vice and then immediately pinched by 1% span-80 in heavy mineral oil
brain. We hypothesize that spatial confinement and scaffold ge- to form microspheres. These microspheres were collected and allowed to
ometry are responsible for the observed changes. These effects of gel overnight at room temperature to form microgels. The microgels were
then purified by repeated washes with HEPES buffer (pH 7.4 containing
porous scaffolds have been observed in other tissues and in vitro
10 mm CaCl2 ) and centrifugation. 4.5% microgels were produced in the
assays.[45,63] Further experimentation will be needed to elucidate same manner, however, the HA-acrylate precursor solution was dissolved
the mechanism of MAP immune cell phenotype modulation. at 9% w/v. The HA without RGD microgels were produced with the same
By decreasing inflammation and promoting astrocytic infiltra- precursor solution as the 3.5% HA, however, no RGD was added. The PEG
tion, the MAP hydrogel is able to influence axonal penetration microgels were produced as previously described.[35] Briefly, 4-arm PEG-
at the injured site. Every axon penetrating in the lesion is ob- vinyl sulfone (Jenkem) was dissolved at 10% w/v and the peptide concen-
trations were the same as the HA solutions.
served to be co-residing next to an infiltrating astrocyte. Mean-
Generation of Scaffold from Microgels and Mechanical Testing: The mi-
while in the sham we observe minimal astrocyte and axon in- crogels were pelleted by centrifuging at 18 000 × g and the supernatant
filtration. This suggests that the less reactive astrocytes infiltrat- was discarded to form a concentrated solution of microgels. 5 U mL−1 of
ing into the lesion are partaking in the recovery process by sup- FXIII and 1 U mL−1 of Thrombin were combined in the presence of 10 mm
porting axonal infiltration. Thus, the anti-inflammatory effects of Ca2+ with the pelleted 𝜇gels and allowed to incubate at 37 °C for 90 min
the HA-MAP hydrogel have downstream benefits by promoting between two glass slides (1 mm thickness) surface coated with sigma-
increasing axonal penetration over time. Looking further down- cote (Sigma-Aldrich). The mechanical testing on the hydrogel scaffolds
was done using a 5500 series Instron. After annealing, the scaffolds were
stream, we observe that the injection of the MAP hydrogel can allowed to swell in HEPES buffer saline for 4 h at room temperature. A
provide mechanical support to the surrounding brain tissue by 2.5 N load cell with a 3.12 mm tip in diameter was used at a compression
preventing the fibrotic pulling of the ventricle known as cerebral strain rate of 1 mm min−1 and the hydrogel scaffold was indented 0.8 mm
atrophy. With the combination of early on decreased inflamma- or 80% of its total thickness.
tion and increased mechanical support provided by the MAP gel, Nanoporous Hydrogel Production: Nanoporous hydrogel precursor so-
the striatal pathway bundles, associated with motor function,[64] lutions were exactly the same as the microgel precursor solutions. Addi-
tionally, the same enzyme sensitive di-thiol cross-linker solution was pre-
are much better preserved compared to sham. This suggests that
pared. These two solutions were thoroughly mixed in an Eppendorf tube
the ability of the MAP gel to modulate inflammation and support by vortexing and pipetting. 5 U mL−1 of FXIII and 1 U mL−1 of Thrombin
the tissue long term has significant downstream benefits and can were added to the solution and the nanoporous hydrogel was allowed to
lead to improved motor function. gel in situ via the same Michael type addition in which the microgels were
Systematically testing effect of microstructure, RGD, and poly- individually formed.
mer backbone, we show that all three play crucial roles in mod- In Vivo Photothrombotic Stroke Model: Animal procedures were per-
formed in accordance with the US National Institutes of Health Animal
ulating astrocyte infiltration. All injected hydrogels decreased in-
Protection Guidelines, the University of California Los Angeles Chancel-
flammation to some extent; however, the MAP hydrogel signifi- lor’s Animal Research Committee, and Duke University Animal Research
cantly decreased the reactive microglial and astrocytic population Committee (A016-21-01). A cortical photothrombotic stroke was induced
the most. Moreover, only the MAP hydrogel was able to promote on 8–12 week male C57BL/6 mice obtained from Jackson laboratories
astrocyte infiltration into the lesion. This suggests that porosity (Bar Harbor, ME). The mice were anesthetized with 2.5% isoflurane and
and RGD are required for astrocyte infiltration, while receptor- placed onto a stereotactic setup. The mice were kept at 2.5% isoflurane in
mediated astrocyte/HA binding could also be critical to infiltra- N2 O:O2 for the duration of the surgery. A midline incision was made and
Rose Bengal (10 mg mL−1 , Sigma-Aldrich) was injected intraperitoneally
tion. at 10 𝜇L g−1 of mouse body weight. After 5 min of Rose Bengal injection,
The MAP hydrogel does have vascular limitations and further a 2-mm diameter cold fiberoptic light source was centered at 0 mm ante-
engineering is required of the material to improve the vascular rior/1.5 mm lateral left of the bregma for 18 min and a burr hole was drilled
through the skull in the same location. All mice were given sulfamethoxa- dles that might have merged, and then counted. Only bundles that were
zole and trimethoprim oral suspension (TMS, 303 mL TMS/250 mL H20, at least 400 𝜇m2 were counted.
Amityville, NY) every 5 days for the entire length of the experiment. 5 days Astrocyte Reactivity, IHC (Figure 3): Cryofrozen sections were allowed
following stroke surgery, microgels with FXIII were loaded into a Hamilton to thaw at room temperature. Sections were washed with PBS for 5 min
syringe (Hamilton Reno, NV) connected to a pump and 6 𝜇L of microgels with three repetitive washes. Sections were incubated with a 10% donkey
were injected into the stroke cavity using a 30-gauge needle at stereotaxic serum and PBS with 0.3% triton at room temperature for 1 h. The liquid
coordinates 0.26 mm anterior/posterior, 3 mm medial/lateral, and 1 mm was wicked away and primary antibodies of GFAP (Abcam, Cambridge,
dorsal/ventral with an infusion speed of 1 𝜇L min−1 . The needle was with- MA, USA) for astrocytes and pERK (Cell Signaling) for highly reactive as-
drawn from the mouse brain 5 min after the injection to allow for microgel trocytes at 1:100 dilution in PBS with 0.3% triton and 10% donkey serum
annealing. For each condition a minimum of five mice was used. were added and incubated overnight at 4 °C. The next day the primaries
1, 2, 4, and 16 weeks following stroke, mice were sacrificed via tran- were washed with three repeated PBS washes of 5 min each. Secondary an-
scranial perfusion of 0.1 m PBS followed by 40 mL of 4% w/v PFA. tibodies with donkey hosts along with Dapi at 1:1000 dilution in PBS with
The brains were isolated and post-fixed in 4% PFA overnight and sub- 0.3% triton and 10% donkey serum were added and incubated at room
merged in 30% w/v sucrose solution for 24 h. Tangential cortical sec- temperature for 2 h. After 2 h, the secondary antibodies were washed away
tions of 30 𝜇m-thickness were sliced using a cryostat and directly with three repeated PBS washes of 5 min each. The sections were allowed
mounted on gelatin-subbed glass slides for immunobiological staining to dry at room temperature and mounted using DPX mounting medium.
of GFAP (Abcam, Cambridge, MA, USA) for astrocytes, IBA-1 (ionized Analyses were performed on microscope images of three coronal brain
calcium binding adaptor molecule, Abcam, Cambridge, MA, USA) for levels at +0.80, −0.80, and −1.20 mm according to bregma, which consis-
microglial cells, Glut-1 (Glucose Transporter-1, Abcam, Cambridge, MA, tently contained the cortical infarct area. Each image represented a max-
USA) for endothelial cells, NF200 (Neurofilament 200, Abcam, Cam- imum intensity projection of 10 to 12 Z-stacks, 1 𝜇m apart, captured at
bridge, MA, USA) for axonal processes, pERK (Cell Signaling) for highly a 20× magnification with a Nikon C2 confocal microscope using the NIS
reactive astrocytes, S100𝛽 (Thermo Fisher) for highly reactive astrocytes, Element software. For the sham sections, using ImageJ and converting to
CD11b (Abcam) for immune cells, Arginase 1 (Santa Cruz) for Pro-repair 8-bit, a ratio of positive pERK area divided by positive GFAP area within the
microglia/macrophages, NOS2 (Santa Cruz) for pro-inflammatory mi- same area was taken to get percent of reactive astrocytes that were highly
croglia/macrophages, Sox2 (Santa Cruz) for NPCs, and DAPI (1:500 In- reactive in the peri-infarct area 0–300 𝜇m from the infarct border. For the
vitrogen) for nuclei. Primary antibodies (1:100) were incubated overnight HA MAP sections, the peri-infarct was analyzed similarly to sham. The in-
at 4 °C and secondary antibodies (1:1000) were incubated at room temper- farct was analyzed by taking 0–100 𝜇m infiltration into the lesion. S100𝛽
ature for 2 h. A Nikon C2 confocal microscope was used to take fluorescent (Thermo Fisher) was stained, imaged, and analyzed similarly to pERK.
images. Astrocyte Reactivity, HCR (Figure 3): In situ hybridization probes for
Image Analysis: The IBA-1, GFAP, pERK, S100𝛽, CD11b, Arg1, iNOS, C3 and SLC1A2 were purchased from Molecular Instruments and the pro-
CSPG, NF200, and Glut-1 astrocytic (GFAP) and positive area in the infarct tocol published on the molecular instrument website was used to probe
and peri-infarct areas were quantified in four to eight randomly chosen the sections. Analyses were performed on microscope images of three
regions of interest (ROI of 0.3 mm2 ) at a maximum distance of 300 𝜇m coronal brain levels at +0.80, −0.80, and −1.20 mm according to bregma,
from the infarct for the peri-infarct and infarct analysis. In each ROI, the which consistently contained the cortical infarct area. Large scale 40× im-
positive area was measured. ages were taken using a Nikon C2 confocal along the stroke border. The
Ventricular Hypertrophy and Nigrostriatal Bundles Analysis (Figure 2): images were than analyzed similar to pERK and S100𝛽.
Cryofrozen sections were allowed to thaw at room temperature. Sections Microglial Reactivity (Figure 3): Cryofrozen sections were allowed to
were washed with PBS for 5 min with three repetitive washes. Sections thaw at room temperature. Sections were washed with PBS for 5 min with
were incubated with a 10% donkey serum and PBS with 0.3% triton at three repetitive washes. Sections were incubated with a 10% donkey serum
room temperature for 1 h. The liquid was wicked away and primary anti- and PBS with 0.3% triton at room temperature for 1 h. The liquid was
bodies of GFAP (Abcam, Cambridge, MA, USA) for astrocytes and NF200 wicked away and primary antibodies of CD11b (Abcam) for immune cells,
(Neurofilament 200, Abcam, Cambridge, MA, USA) for axonal processes Arginase 1 (Santa Cruz) for Pro-repair microglia/macrophages, NOS2
at 1:100 dilution in PBS with 0.3% triton and 10% donkey serum were (Santa Cruz) for pro-inflammatory microglia/macrophages at 1:100 dilu-
added and incubated overnight at 4 °C. The next day the primaries were tion in PBS with 0.3% triton, and 10% donkey serum were added and in-
washed with three repeated PBS washes of 5 min each. Secondary antibod- cubated overnight at 4 °C. The next day the primaries were washed with
ies with donkey hosts along with Dapi at 1:1000 dilution in PBS with 0.3% three repeated PBS washes of 5 min each. Secondary antibodies with don-
triton and 10% donkey serum were added and incubated at room temper- key hosts along with Dapi at 1:1000 dilution in PBS with 0.3% triton and
ature for 2 h. After 2 h, the secondary antibodies were washed away with 10% donkey serum were added and incubated at room temperature for
three repeated PBS washes of 5 min each. The sections were allowed to dry 2 h. After 2 h, the secondary antibodies were washed away with three re-
at room temperature and mounted using DPX mounting medium. Anal- peated PBS washes of 5 min each. The sections were allowed to dry at
yses were performed on microscope images of three coronal brain levels room temperature and mounted using DPX mounting medium. Analy-
at +0.80, −0.80, and −1.20 mm according to bregma, which consistently ses were performed on microscope images of three coronal brain levels
contained the cortical infarct area. Large scale 10× images of each sec- at +0.80, −0.80, and −1.20 mm according to bregma, which consistently
tion was taken and analyzed for ventricular hypertrophy. Cross-sectional contained the cortical infarct area. Each image represented a maximum
areas in mm2 were measured on FIJI and multiplied by 0.3 mm to cal- intensity projection of 10 to 12 Z-stacks, 1 𝜇m apart, captured at a 20×
culate individual section volumes, and the three volumes of the ROI sec- magnification with a Nikon C2 confocal microscope using the NIS Ele-
tions were added together to find the ROI volumes; each brain section was ment software. In the peri-infarct for both sham and HA MAP, the ratio
0.03 mm thick, and there were ten slides of brain sections per brain, thus of the positive area of iNOS or Arg1 was divided by the positive area for
one section was representative of ten sections. Large scale 20× images CD11b from 0–300 𝜇m from the infarct border. In the infarct, the ratio
were taken by the side of ventricle to analyze for nigrostriatal bundle area. of the positive area of iNOS or Arg1 was divided by the positive area for
Pixel threshold was used on 8-bit converted images using ImageJ (Image CD11b from 0–300 𝜇m from the infarct border.
J v1.43, Bethesda, Maryland, USA) and expressed as the area fraction of Astrocyte and Microglial Progression Analysis (Figure 4): Cryofrozen sec-
positive signal per area (%). Values were then averaged across all areas tions were allowed to thaw at room temperature. Sections were washed
and sections, and expressed as the average positive area per animal. The with PBS for 5 min with three repetitive washes. Sections were incubated
number of NF200-stained bundles was analyzed in a ROI of 1 mm × 1 mm with a 10% donkey serum and PBS with 0.3% triton at room tempera-
(Figure S5, Supporting Information). These ROI were then converted into ture for 1 h. The liquid was wicked away and primary antibodies of GFAP
8-bit images, then to binary images, were then watershed to split up bun- (Abcam, Cambridge, MA, USA) for astrocytes, IBA-1 (ionized calcium
binding adaptor molecule, Abcam, Cambridge, MA, USA) for reactive mi- infarct, the percent positive for Glut1 was measured 0–300 𝜇m from the
croglia/macrophages at 1:100 dilution in PBS with 0.3% triton and 10% infarct border.
donkey serum were added and incubated overnight at 4 °C. The next day Statistical Analysis: Minimum animal group sample size was deter-
the primaries were washed with three repeated PBS washes of 5 min each. mined to be 5 by using a statistical power analysis that compares the mean
Secondary antibodies with donkey hosts along with Dapi at 1:1000 dilu- value and standard deviation of a selected experiment (microglia surface
tion in PBS with 0.3% triton and 10% donkey serum were added and in- % in the peri-infarct area 2 weeks after stroke) in a selected animal con-
cubated at room temperature for 2 h. After 2 h, the secondary antibodies dition (HA-MAP) performed in a pilot study compared with a previously
were washed away with three repeated PBS washes of 5 min each. The sec- published finding on the same experiment, condition, and time point.[48]
tions were allowed to dry at room temperature and mounted using DPX The alpha value (Type 1 error) was fixed at the level of 5% (P = 0.05) and
mounting medium. Analyses were performed on microscope images of the power at 80%. There was no preprocessing of the data to remove
three coronal brain levels at +0.80, −0.80, and −1.20 mm according to outliers. Some analyses involve comparing the injured (ipsilateral) and
bregma, which consistently contained the cortical infarct area. Each im- non-injured (contralateral) brain regions. In these cases, quantification of
age represented a maximum intensity projection of 10–12 Z-stacks, 1 𝜇m the ipsilateral region was divided by the contralateral region. A value of
apart, captured at a 20× magnification with a Nikon C2 confocal micro- 1 would indicate no difference between the injured and non-injured re-
scope using the NIS Element software. Scar thickness was analyzed as gions. In plots comparing groups for a particular day, standard box plots
distance from stroke border using astrocyte morphology changes to sig- were used, which displayed the minimum and maximum value, the me-
nify end of scar. Astrocyte infiltration was measured as longest infiltrating dian quartile 1, quartile 3, and each individual values. Plots that showed
astrocyte from stroke border. In the peri-infarct for both sham and HA trends over time were plotted as the mean with standard deviation. Sig-
MAP, the percent positive for IBA-1 was measured 0–300 𝜇m from the in- nificance was analyzed using alpha = 0.05 and p < 0.05 was considered
farct border. In the infarct, the percent positive for IBA-1 was measured significant. One-way ANOVA, two-way ANOVA, and t-test were used as de-
0–300 𝜇m from the infarct border. tailed in the figure legends. GraphPad Prism 9 for MacOS was used for all
Axon Progression (Figure 5): Cryofrozen sections were allowed to thaw statistical analyses.
at room temperature. Sections were washed with PBS for 5 min with three
repetitive washes. Sections were incubated with a 10% donkey serum and
PBS with 0.3% triton at room temperature for 1 h. The liquid was wicked Supporting Information
away and primary antibodies of GFAP (Abcam, Cambridge, MA, USA) for
astrocytes and NF200 (Neurofilament 200, Abcam, Cambridge, MA, USA) Supporting Information is available from the Wiley Online Library or from
for axonal processes at 1:100 dilution in PBS with 0.3% triton and 10% the author.
donkey serum were added and incubated overnight at 4 °C. The next day
the primaries were washed with three repeated PBS washes of 5 min each.
Secondary antibodies with donkey hosts along with Dapi at 1:1000 dilu-
Acknowledgements
tion in PBS with 0.3% triton and 10% donkey serum were added and in- The authors would like to acknowledge Dr. Amy Gleichman for her exper-
cubated at room temperature for 2 h. After 2 h, the secondary antibodies tise on astrocytes and helpful discussion. The authors would like thank
were washed away with three repeated PBS washes of 5 min each. The sec- Dr. Irene Llorente for her help on quantification methods. The authors
tions were allowed to dry at room temperature and mounted using DPX would like to acknowledge Dr. Lina Nih for her assistance with tissue col-
mounting medium. Analyses were performed on microscope images of lection and helpful discussions. The authors would like thank Katrina Wil-
three coronal brain levels at +0.80, −0.80, and −1.20 mm according to son for her help producing microgels. The authors would like to thank
bregma, which consistently contained the cortical infarct area. Each im- the entire Carmichael lab at UCLA for their stroke expertise and help. The
age represented a maximum intensity projection of 10–12 Z-stacks, 1 𝜇m authors would like to acknowledge the National Institutes of Health and
apart, captured at a 20× magnification with a Nikon C2 confocal micro- the National Institute for Neurological Diseases and Stroke for funding
scope using the NIS Element software. Axon infiltration was measured (R01NS094599, 1R01NS112940, and 1R01NS079691).
as longest infiltrating neurofilament from stroke border. In the peri-infarct
for both sham and HA MAP, the percent positive for NF200 was measured
0–300 𝜇m from the infarct border. In the infarct, the percent positive for Conflict of Interest
NF200 was measured 0–300 𝜇m from the infarct border.
Vessel Progression (Figure 2, Supporting Information): Cryofrozen sec- The authors declare no conflict of interest.
tions were allowed to thaw at room temperature. Sections were washed
with PBS for 5 min with three repetitive washes. Sections were incu-
bated with a 10% donkey serum and PBS with 0.3% triton at room tem- Author Contributions
perature for 1 h. The liquid was wicked away and primary antibodies
of GFAP (Abcam, Cambridge, MA, USA) for astrocytes and Glut-1 (Glu- E.S., S.T.C., and T.S. designed the experiments. E.S., A.Y., S.K., K.W., and
cose Transporter-1, Abcam, Cambridge, MA, USA) for endothelial cells at J.C. performed experiments and analyzed data. E.S., S.T.C., and T.S. wrote
1:100 dilution in PBS with 0.3% triton and 10% donkey serum were added the manuscript with input from all authors.
and incubated overnight at 4 °C. The next day the primaries were washed
with three repeated PBS washes of 5 min each. Secondary antibodies with
donkey hosts along with Dapi at 1:1000 dilution in PBS with 0.3% triton
Data Availability Statement
and 10% donkey serum were added and incubated at room temperature The data that support the findings of this study are available from the cor-
for 2 h. After 2 h, the secondary antibodies were washed away with three responding author upon reasonable request.
repeated PBS washes of 5 min each. The sections were allowed to dry at
room temperature and mounted using DPX mounting medium. Analy-
ses were performed on microscope images of three coronal brain levels Keywords
at +0.80, −0.80, and −1.20 mm according to bregma, which consistently
contained the cortical infarct area. Each image represented a maximum in- astrocytes, brain repair, granular hydrogels, microporous annealed parti-
tensity projection of 10–12 Z-stacks, 1 𝜇m apart, captured at a 20× magni- cle hydrogels, microglia, particle hydrogels, strokes
fication with a Nikon C2 confocal microscope using the NIS Element soft-
ware. Vessel infiltration was measured as longest infiltrating vessel from Received: March 8, 2022
stroke border. In the peri-infarct for both sham and HA MAP, the percent Revised: May 25, 2022
positive for Glut1 was measured 0–300 𝜇m from the infarct border. In the Published online: June 28, 2022
[27] A. Hong, M.-I. Aguilar, M. P. Del Borgo, C. G. Sobey, B. R. S.

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Particle Hydrogels Decrease Cerebral Atrophy and Attenuate Astrocyte and Microglia Macrophage Reactivity After Stroke

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Particle Hydrogels Decrease Cerebral Atrophy and Attenuate Astrocyte and Microglia Macrophage Reactivity After Stroke

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RESEARCH ARTICLE

Particle Hydrogels Decrease Cerebral Atrophy and Attenuate

the injury leaves them with a serious dis-

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Microporous annealed particle (MAP) scaﬀolds are granular hy-

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3. Conclusions response. Vascularization is critical to providing oxygen and nu-

[27] A. Hong, M.-I. Aguilar, M. P. Del Borgo, C. G. Sobey, B. R. S.

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