Currently, matrices into the rat cortex after subjecting it

Currently, there are no
effective treatments that actively promote tissue repair and regeneration post
moderate-to-severe TBIs. While there have been efforts to mitigate the
secondary damage post-TBI, these measures have been proven clinically
unsuccessful. At present, there is no cure for neither the initial damage nor
the secondary loss of tissue post the biomechanical insult. In this study, we
sought to devise a mechanism that would provide neuroprotective factors and
facilitate tissue repair post moderate-to-severe TBIs. We developed a sulfated
CS-GAG matrix, a biomaterial native to the brain’s ECM, capable of regulating
NSC behavior.29 As part of this study, we acutely delivered these
sulfated CS-GAG matrices into the rat cortex after subjecting it to a
moderate-to-severe TBI, to investigate the tissue repair capability of these
matrix constructs 20 weeks post-TBI. We demonstrate that these CS-GAG matrices
provide neuroprotection and enhance brain tissue repair subacutely 20 weeks post-TBI.

Previous studies have
documented the neuroprotective factors of transplanted NSCs post-TBI. NSCs are
known to secrete neuroprotective factors such as NGF, BDNF, GDNF, and/or to
trigger host immune responses, thereby serving as a “bystander,” where they
promote tissue regeneration. Contrary to the current therapeutic approaches
focused on NSC differentiation, we proposed that the maintenance of NSCs in
their undifferentiated state promotes chronic repair and recovery post the
polytrauma. In this study, we evaluated the effect that endogenous NSCs have on
tissue repair post-TBI.

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Sox1 staining indicated a
greater presence of endogenous NSCs in the CS-GAG treated animals, while the
BrdU staining demonstrated enhanced cellular proliferation of the NSCs in the
animals with the CS-GAG constructs. Double staining for Reca-1 and Collagen IV
was conducted to evaluate the extent of angiogenesis among the GAG treated
animals. The expression of Collagen IV against Reca-1 was investigated to study
the presence of newly formed vasculatures amongst all the vascular structures
present. CS-GAG treated animals displayed significantly greater angiogenesis
when compared to the TBI-only controls. There was a local upregulation of BDNF in
the GAG treated animals; BDNF is known to promote angiogenesis.30 In
addition, SDF-1? also promotes angiogenesis by elucidating homing mechanisms that
facilitate hematopoietic progenitor cell migration to the injury site post-TBI.31
Previous studies indicate that blocking the CXCL12/CXCR4 axis results in abolished
hematopoietic progenitor cell homing and decreased angiogenesis.31 Our
CS-GAG treated animals demonstrated an upregulation of both the CXCL12 ligand and
its corresponding CXCR4 receptor. The CS-GAG treated animals also exhibited
upregulation of locally produced FGF2 which is a growth factor that is
pro-mitogenic and pro-angiogenic. Hence, the enhanced presence of NSCs in the sulfated
GAG matrices depict the “bystander effect” explained earlier by promoting
tissue repair through cellular mitogenesis and angiogenesis. The amalgamation
of NSC proliferation and growth factor retention in the sulfated GAG constructs
promote repair post-TBI. We have previously demonstrated that CS-GAGs have high
binding affinity to FGF2,26 and additionally the local upregulation
of FGF2 in the lesion site post-TBI of the treated animals suggests the
potential role of CS-GAGs in the maintenance of undifferentiated NSCs.

BDNF is a neurotrophic
factor that has demonstrated neural regeneration, reconnection, and improved
synaptic efficacy.23 In our qRT-PCR results, we indicated a slight upregulation
(less than two-fold increase) of BDNF in the CS-GAG treated subjects.
Synaptophysin immunostaining indicated enhanced synaptic plasticity and
synaptic connections in the TBI-GAG-TF animals when compared to the animals who
only received the CCI. Therefore, the upregulation of localized BDNF, however
insignificant, may contribute to the increased synaptophysin expression in the
GAG treated animals demonstrating the ability of the sulfated GAGs to promote
tissue repair via mediating synaptic plasticity.

Astroglial scarring is a
rampant consequence of the secondary phase of injury post-traumatic insults in
the CNS that prevents neuroregeneration and neuroprotection. GFAP, a biomarker
that is an essential component of the astrocytic cytoskeleton, was used to
evaluate the extent of cellular degeneration.32 GFAP marked for hypertrophic
reactive astrocytes. After TBIs, the brain releases these astrocytes into the
interstitial fluid and blood, crossing the blood-brain barrier.32 These
astrocytes express nerve inhibitory CSPGs forming astroglial scars which serve
as molecular barriers to repair post-injury. The TBI-only animals depicted
greater presence of astroglial scarring as compared to the TBI-GAG-TF treated
animals. Hence, the GAG matrices demonstrate a role in downregulating the accumulation
of reactive astrocytes in the lesion site post-injury, resulting in a lower expression
of the inhibitory CSPGs.  Our results
reinforced previous studies indicating that the CS-GAGs
mediate astrocytic destruction caused by the insult.

The
extent of the inflammatory response was evaluated by measuring the number of
CD68+cells in the lesion territory. The CS-GAG treated animals
exhibited significantly greater presence of the activated macrophages,
indicating a stronger inflammatory response. However, currently, we do not know
the effects of these macrophages. This inflammatory response may serve a role
in wound sterilization and necrotic tissue clearing essential for wound repair;
however, it may also detrimentally extend to neuronal cell death that impairs
tissue recovery post-TBI.33 Further testing must be conducted to
evaluate the types of macrophages and the effects they have on tissue repair
and recovery post moderate-to-severe TBIs. 

In
addition to the mitogenic and angiogenic roles of FGF2, mentioned above, FGF2
is also known inhibit cell excitotoxicity34 and dampen the
inflammatory response after the mechanical insult to improve recovery.35
The hydrogel matrices demonstrated their role in providing a microenvironment that
binds neurotrophic factors. The local upregulation of FGF2 and the high binding
affinity of growth factors in the CS-GAG treated animals as compared to the TBI
control animals depicts the neuroprotective role that the GAG matrices have in promoting
tissue repair while minimizing the detrimental effects of astroglial scarring.

The chemokine stromal
cell-derived factor-1 CXCL-12 (SDF-1) and its corresponding chemokine receptor CXCR4
are essential regulatory components of molecular inflammation and immunity.36
The SDF-1/CXCR4 ligand/receptor pair is known to recruit stem cells, promote
cellular proliferation, differentiation, and angiogenesis.36,37,38
We demonstrated an upregulation of both ligand and receptor in the CS-GAG
treated animals, however, they did not show a correlated increase.
Interestingly, there was a greater upregulation of the CXCR4 chemokine receptor
than the corresponding ligand. Both CXCL12 and CXCR4 are constitutively
expressed in the mature CNS; CXCR4 mainly expressed in the stem cell niches
enabling progenitor cells to survive and proliferate.39 While the
expression of CXCL12 and its receptor are complementary, in the mature CNS
CXCL12 is expressed in relatively lower levels.39 This supports our
results from the qRT-PCR trials, where we noticed a greater than two-fold
increase in both CXCL12 and CXCR4, however, there was a greater upregulation of
the chemokine receptor in comparison to the SDF-1 ligand. Hence, this indicates
that the CS-GAG constructs influence cell receptor profile. The CS-GAG matrices
elicit a greater upregulation of the CXCR4 receptor than the CXCL12 ligand as expected
in the mature CNS.   

The local expression of
CXCL12/CXCR4 suggests the role of the ligand/receptor pair in homing stem cells
to the lesion site post-TBI. Guidance signals released by the ligand to
facilitate stem cell migration to the penumbra of the lesion site can result in
tissue recovery and repair post-injury.37 CS-GAGs serve as an
integral component with essential stem cell homing signaling molecules and
trophic factors that promote stem cell proliferation leading to a recovery post
the mechanical insult. CXCL12 and CXCR4 upregulation in the CS-GAG hydrogels
may also play a role in the upregulation of Collagen IV expression in the
hydrogel treated animals mentioned above. Therefore, the chemokine pair not
only promotes stem cell migration but also enhance vascularization for enhanced
tissue recovery.

We proposed that CS-GAGs
will ameliorate tissue repair and recovery post TBI by maintaining NSCs in
their undifferentiated state; however, interestingly we noticed a significantly
higher presence of the olig2 transcription factor in the CS-GAG treated
animals. The animals subjected to the CS-GAG hydrogels displayed greater olig2
expression when compared to the TBI animals’ expression indicative of NSCs
differentiating into the oligodendroglial lineage. To evaluate CS-GAG’s ability
to keep the NSCs in their undifferentiated state, we must conduct further studies
with other markers such as NeuN, for neuronal differentiation, and compare the
colocalization of Sox1 displaying endogenous NSCs with these differentiation
markers.

In addition, for future
studies, we can conduct TUNEL assays and Propidium Iodide staining assays to
evaluate the extent of apoptosis and necrosis, respectively, in the injury area
post-TBI. These tests will allow for us to evaluate the extent of cellular
death post-TBI and investigate the effect of the CS-GAG hydrogels in mitigating
cell death.

Previous studies in the
lab investigated the functional recovery of animals subjected to
moderate-to-severe TBIs. Beam-walk, rotarod, and open field tests were
conducted 20 weeks post-TBI to evaluate the motor recovery in hydrogel treated
animals. The results indicate enhanced motor recovery and ambulatory activity
longitudinally in the CS-GAG treated animals when compared to the TBI-only
controls. This data in addition to our current findings strengthen the notion
that CS-GAG hydrogels can serve as neuroprotective mechanism post-TBI for
tissue recovery and repair both functionally and biochemically.