Currently, there are noeffective treatments that actively promote tissue repair and regeneration postmoderate-to-severe TBIs. While there have been efforts to mitigate thesecondary damage post-TBI, these measures have been proven clinicallyunsuccessful. At present, there is no cure for neither the initial damage northe secondary loss of tissue post the biomechanical insult. In this study, wesought to devise a mechanism that would provide neuroprotective factors andfacilitate tissue repair post moderate-to-severe TBIs. We developed a sulfatedCS-GAG matrix, a biomaterial native to the brain’s ECM, capable of regulatingNSC behavior.
29 As part of this study, we acutely delivered thesesulfated CS-GAG matrices into the rat cortex after subjecting it to amoderate-to-severe TBI, to investigate the tissue repair capability of thesematrix constructs 20 weeks post-TBI. We demonstrate that these CS-GAG matricesprovide neuroprotection and enhance brain tissue repair subacutely 20 weeks post-TBI.Previous studies havedocumented the neuroprotective factors of transplanted NSCs post-TBI. NSCs areknown to secrete neuroprotective factors such as NGF, BDNF, GDNF, and/or totrigger host immune responses, thereby serving as a “bystander,” where theypromote tissue regeneration. Contrary to the current therapeutic approachesfocused on NSC differentiation, we proposed that the maintenance of NSCs intheir undifferentiated state promotes chronic repair and recovery post thepolytrauma. In this study, we evaluated the effect that endogenous NSCs have ontissue repair post-TBI. Sox1 staining indicated agreater presence of endogenous NSCs in the CS-GAG treated animals, while theBrdU staining demonstrated enhanced cellular proliferation of the NSCs in theanimals with the CS-GAG constructs. Double staining for Reca-1 and Collagen IVwas conducted to evaluate the extent of angiogenesis among the GAG treatedanimals.
The expression of Collagen IV against Reca-1 was investigated to studythe presence of newly formed vasculatures amongst all the vascular structurespresent. CS-GAG treated animals displayed significantly greater angiogenesiswhen compared to the TBI-only controls. There was a local upregulation of BDNF inthe GAG treated animals; BDNF is known to promote angiogenesis.30 Inaddition, SDF-1? also promotes angiogenesis by elucidating homing mechanisms thatfacilitate hematopoietic progenitor cell migration to the injury site post-TBI.31Previous studies indicate that blocking the CXCL12/CXCR4 axis results in abolishedhematopoietic progenitor cell homing and decreased angiogenesis.31 OurCS-GAG treated animals demonstrated an upregulation of both the CXCL12 ligand andits corresponding CXCR4 receptor.
The CS-GAG treated animals also exhibitedupregulation of locally produced FGF2 which is a growth factor that ispro-mitogenic and pro-angiogenic. Hence, the enhanced presence of NSCs in the sulfatedGAG matrices depict the “bystander effect” explained earlier by promotingtissue repair through cellular mitogenesis and angiogenesis. The amalgamationof NSC proliferation and growth factor retention in the sulfated GAG constructspromote repair post-TBI. We have previously demonstrated that CS-GAGs have highbinding affinity to FGF2,26 and additionally the local upregulationof FGF2 in the lesion site post-TBI of the treated animals suggests thepotential role of CS-GAGs in the maintenance of undifferentiated NSCs.
BDNF is a neurotrophicfactor that has demonstrated neural regeneration, reconnection, and improvedsynaptic 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 andsynaptic connections in the TBI-GAG-TF animals when compared to the animals whoonly received the CCI.
Therefore, the upregulation of localized BDNF, howeverinsignificant, may contribute to the increased synaptophysin expression in theGAG treated animals demonstrating the ability of the sulfated GAGs to promotetissue repair via mediating synaptic plasticity. Astroglial scarring is arampant consequence of the secondary phase of injury post-traumatic insults inthe CNS that prevents neuroregeneration and neuroprotection. GFAP, a biomarkerthat is an essential component of the astrocytic cytoskeleton, was used toevaluate the extent of cellular degeneration.32 GFAP marked for hypertrophicreactive astrocytes. After TBIs, the brain releases these astrocytes into theinterstitial fluid and blood, crossing the blood-brain barrier.
32 Theseastrocytes express nerve inhibitory CSPGs forming astroglial scars which serveas molecular barriers to repair post-injury. The TBI-only animals depictedgreater presence of astroglial scarring as compared to the TBI-GAG-TF treatedanimals. Hence, the GAG matrices demonstrate a role in downregulating the accumulationof reactive astrocytes in the lesion site post-injury, resulting in a lower expressionof the inhibitory CSPGs. Our resultsreinforced previous studies indicating that the CS-GAGsmediate astrocytic destruction caused by the insult. Theextent of the inflammatory response was evaluated by measuring the number ofCD68+cells in the lesion territory. The CS-GAG treated animalsexhibited significantly greater presence of the activated macrophages,indicating a stronger inflammatory response. However, currently, we do not knowthe effects of these macrophages.
This inflammatory response may serve a rolein wound sterilization and necrotic tissue clearing essential for wound repair;however, it may also detrimentally extend to neuronal cell death that impairstissue recovery post-TBI.33 Further testing must be conducted toevaluate the types of macrophages and the effects they have on tissue repairand recovery post moderate-to-severe TBIs. Inaddition to the mitogenic and angiogenic roles of FGF2, mentioned above, FGF2is also known inhibit cell excitotoxicity34 and dampen theinflammatory response after the mechanical insult to improve recovery.35The hydrogel matrices demonstrated their role in providing a microenvironment thatbinds neurotrophic factors. The local upregulation of FGF2 and the high bindingaffinity of growth factors in the CS-GAG treated animals as compared to the TBIcontrol animals depicts the neuroprotective role that the GAG matrices have in promotingtissue repair while minimizing the detrimental effects of astroglial scarring.
The chemokine stromalcell-derived factor-1 CXCL-12 (SDF-1) and its corresponding chemokine receptor CXCR4are essential regulatory components of molecular inflammation and immunity.36The SDF-1/CXCR4 ligand/receptor pair is known to recruit stem cells, promotecellular proliferation, differentiation, and angiogenesis.36,37,38We demonstrated an upregulation of both ligand and receptor in the CS-GAGtreated animals, however, they did not show a correlated increase.Interestingly, there was a greater upregulation of the CXCR4 chemokine receptorthan the corresponding ligand. Both CXCL12 and CXCR4 are constitutivelyexpressed in the mature CNS; CXCR4 mainly expressed in the stem cell nichesenabling progenitor cells to survive and proliferate.39 While theexpression of CXCL12 and its receptor are complementary, in the mature CNSCXCL12 is expressed in relatively lower levels.39 This supports ourresults from the qRT-PCR trials, where we noticed a greater than two-foldincrease in both CXCL12 and CXCR4, however, there was a greater upregulation ofthe chemokine receptor in comparison to the SDF-1 ligand. Hence, this indicatesthat the CS-GAG constructs influence cell receptor profile.
The CS-GAG matriceselicit a greater upregulation of the CXCR4 receptor than the CXCL12 ligand as expectedin the mature CNS. The local expression ofCXCL12/CXCR4 suggests the role of the ligand/receptor pair in homing stem cellsto the lesion site post-TBI. Guidance signals released by the ligand tofacilitate stem cell migration to the penumbra of the lesion site can result intissue recovery and repair post-injury.37 CS-GAGs serve as anintegral component with essential stem cell homing signaling molecules andtrophic factors that promote stem cell proliferation leading to a recovery postthe mechanical insult. CXCL12 and CXCR4 upregulation in the CS-GAG hydrogelsmay also play a role in the upregulation of Collagen IV expression in thehydrogel treated animals mentioned above.
Therefore, the chemokine pair notonly promotes stem cell migration but also enhance vascularization for enhancedtissue recovery. We proposed that CS-GAGswill ameliorate tissue repair and recovery post TBI by maintaining NSCs intheir undifferentiated state; however, interestingly we noticed a significantlyhigher presence of the olig2 transcription factor in the CS-GAG treatedanimals. The animals subjected to the CS-GAG hydrogels displayed greater olig2expression when compared to the TBI animals’ expression indicative of NSCsdifferentiating into the oligodendroglial lineage.
To evaluate CS-GAG’s abilityto keep the NSCs in their undifferentiated state, we must conduct further studieswith other markers such as NeuN, for neuronal differentiation, and compare thecolocalization of Sox1 displaying endogenous NSCs with these differentiationmarkers. In addition, for futurestudies, we can conduct TUNEL assays and Propidium Iodide staining assays toevaluate the extent of apoptosis and necrosis, respectively, in the injury areapost-TBI. These tests will allow for us to evaluate the extent of cellulardeath post-TBI and investigate the effect of the CS-GAG hydrogels in mitigatingcell death.
Previous studies in thelab investigated the functional recovery of animals subjected tomoderate-to-severe TBIs. Beam-walk, rotarod, and open field tests wereconducted 20 weeks post-TBI to evaluate the motor recovery in hydrogel treatedanimals. The results indicate enhanced motor recovery and ambulatory activitylongitudinally in the CS-GAG treated animals when compared to the TBI-onlycontrols. This data in addition to our current findings strengthen the notionthat CS-GAG hydrogels can serve as neuroprotective mechanism post-TBI fortissue recovery and repair both functionally and biochemically.
