Research ArticleNEUROSCIENCE

Engineered glycomaterial implants orchestrate large-scale functional repair of brain tissue chronically after severe traumatic brain injury

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Science Advances  05 Mar 2021:
Vol. 7, no. 10, eabe0207
DOI: 10.1126/sciadv.abe0207
  • Fig. 1 eCS implants reduce lesion volume and improve gross motor function 20 weeks after sTBI.

    (A) Experimental schedule. All rats received an sTBI at week 0 (D0). eCS implants were intracortically administered 48 hours after sTBI (D2). Following 1 week of recovery, all rats underwent behavioral testing at weeks 1 and 2, and every other week thereafter (4, 6, 8, 10, 12, 14, 16, 18, and 20). (B) The rotarod test was used as a measure of balance and motor coordination. Two-way repeated-measures analysis of variance (ANOVA): PTreatment = 0.153; PTime < 0.001; PTreatmentxTime = 0.004. Post hoc multiple comparisons using Dunn-Sidak correction are shown above each time point for Sham versus TBI (red), Sham versus eCS (blue), and TBI versus eCS (black). (C) Representative T2-weighted (T2W) MRI images (top) for each treatment group Sham, TBI, and eCS (coronal section); scale bars, 500 μm; top view of the extracted brain (bottom); scale bars, 1 mm. D, dorsal; V, ventral; M, middle; L, lateral; A, anterior; P, posterior. (D) Average injury volume was quantified for each 1-mm slice around the injury and based on the T2W MRI images (mm3). Two-way repeated-measures ANOVA; treatment factor: P < 0.001; time factor: P = 0.53; treatment × time: P = 0.0279. (E) Representative Nissl-stained coronal sections of rat brain for the Sham, TBI, and eCS groups. Scale bar, 1 mm. (F) Lesion volume quantification using Nissl stain for Sham (six rats, four images per rat), TBI (six rats, four images per rat), and eCS (three rats, four images per rat) groups. One-way ANOVA; treatment: P < 0.001. Post hoc least significant difference (LSD), *P < 0.05, **P < 0.01, and ***P < 0.001. Graphs show means ± SEM.

  • Fig. 2 eCS implants promote NSC proliferation and neurotrophic factor expression 20 weeks after sTBI.

    (A) Representative tiled images of ipsilesional hemisphere (left coronal sections) of eCS rat brain tissue; scale bar, 1 mm. (A1 to A4) Representative magnification of dashed white square shown in (C) for DAPI (A1), Sox-1 (A2), Ki67(A3), and merged (A4); scale bar, 100 μm. (B) Representative tiled images of ipsilesional hemisphere (left coronal sections) of eCS rat brain tissue; scale bar, 1 mm. (B1 to B4) Representative magnification of dashed white square shown in (A) for DAPI (B1), CS56 (B2), FGF2 (B3), and merged (B4); scale bar, 100 μm. (C) Colocalization of Sox-1+ cells with Ki67+ cells as a percentage of Ki67+ cells for each treatment. Kruskal-Wallis, treatment: P < 0.001. (D) Colocalization of FGF2+ cells with DAPI+ cells as a percentage of DAPI+ cells for each treatment. Kruskal-Wallis, treatment: P < 0.05. (E) CS56+ percentage area for each treatment. Kruskal-Wallis, treatment: P < 0.001. Post hoc LSD Mann-Whitney U test, *P < 0.05, **P < 0.01, and ***P < 0.001. (F) At 20-week time point, brains were flash-frozen and tissue was laser capture microdissected. Total RNA was purified and used to synthesize cDNA. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed using prevalidated primers of interest for quantification of gene expression. (G) Relative expression of BDNF, FGF2, CXCL12, and CXCR4 transcripts in TBI and eCS treatment groups. Two-tailed t test, *P < 0.05, **P < 0.01, and ***P < 0.001. Graphs show means ± SEM.

  • Fig. 3 eCS implants promote neurogenesis and plasticity 20 weeks after sTBI.

    (A) Representative tiled images of ipsilesional hemisphere (left coronal sections) coronal sections with merged DAPI (blue), DCX (red), and BrdU (green) staining of eCS rat brain tissue; scale bar, 1 mm. (B) Colocalization of DCX+ and BrdU+ cells with DAPI+ cells as a percentage of DAPI+ cells for each treatment; Kruskal-Wallis, treatment: P < 0.001. (C) Representative tiled images of ipsilesional hemisphere (left coronal sections) with merged DAPI (blue), Syn1 (red), and NeuN (green) staining of eCS rat brain tissue; scale bar, 1 mm. Inset shows magnified images of DAPI+ and NeuN+ cells (merged; left) and Syn1+ cells (right) from the region represented by the dashed white square; scale bar, 100 μm. (D) Colocalization of Syn+ cells with NeuN+ cells as percentage of NeuN+ cells for each treatment. Kruskal-Wallis, treatment: P < 0.001. Post hoc LSD Mann-Whitney U test, *P < 0.05, **P < 0.01, and ***P < 0.001. Graphs show means ± SEM.

  • Fig. 4 eCS matrices promote vascularization and increased CBF 20 weeks after sTBI.

    (A) Representative tiled image of ipsilesional hemisphere of the eCS group; scale bar, 1 mm. (B) Colocalization of Reca1+ cells with Col IV+ cells as a percentage of Col IV+ cells for each treatment. Kruskal-Wallis, treatment: P < 0.001. Post hoc LSD Mann-Whitney U test, *P < 0.05, **P < 0.01, and ***P < 0.001. (C) Phase gradient was estimated from MRI. First, the wrapped phase map (top left) was used to generate phase gradient maps (PGMs; top right) for each slice by calculating the magnitude of the phase gradients determined in the readout and phase encoding directions (see Materials and Methods for details). Bottom: nPG differences between a vessel and the surrounding tissue were measured across all distinguishable vessels at the coronal cross section (inset, white line). Note: The rate of flow in a blood vessel is inversely proportional to the change in phase between the vessel and the surrounding tissue (see Materials and Methods). (D) Distribution of nPG for all identified blood vessels in Sham (n = 84), TBI (n = 121), and eCS (n = 432) groups. Dashed line represents the median nPG for Sham (black), TBI (red), and eCS (blue). One-way ANOVA, F(2) = 230.56, P = 8.57 × 10−51/measure effect size P < 0.001. (E) Representative segmentation of MRI coronal brain slice for regional and hemispheric comparison of blood flow. IL, ipsilesional; CL, contralesional. (F) nPG for the perilesional region of interest (ROI) and matching ROI in the contralateral hemisphere. Two-way ANOVA, treatment: P < 0.0001. Post hoc one-sample t test; post hoc LSD, *P < 0.05, **P < 0.01, and ***P < 0.001. Graph shows means ± SEM.

  • Fig. 5 eCS matrix–implanted animals demonstrate enhanced recovery of reach-to-grasp–specific motor function 8 to 10 weeks after sTBI.

    (A) Experimental schedule of SRT preference (PREF) and forced left (FL) limb training before TBI induction and eCS matrix implantation (top). Volumetric mapping of lesion from iDisco+ cleared and NeuN+ stained Sham-treated (left), TBI-treated (middle), and eCS-treated (right) brains (bottom). Contra- and ipsilesional hemispheres are colored blue and red, respectively. (B) Overall SRT performance of Sham (n = 6), TBI (n = 6), and eCS (n = 9) groups after sTBI and treatment. Repeated-measures ANOVA, time: P < 0.05, group: P < 0.01, time × group: P = 0.1306; post hoc LSD, *P < 0.05 and **P < 0.01. Color-coded group comparisons indicate Sham versus TBI (SvT; red), Sham versus eCS (SvG; blue), and TBI versus eCS (TvG; magenta). RFAa, RFA anterior; RFAp, RFA posterior. (C) ROI analysis of Arc+ NeuN+ colocalization and reach-to-grasp function mapping. The ipsi- and contralesional sides are marked in red and blue, respectively. (D) Ipsilesional (top) and contralesional (bottom) quantification of Arc+ NeuN+ colocalized cells after SRT assay. Sham (n = 3), TBI (n = 3), and eCS (n = 3). Line plots and bar graphs show means ± SEM. *P < 0.05.

  • Fig. 6 eCS matrix implants promote changes in intra- and perilesional vascular architecture 8 weeks after sTBI.

    (A) Image processing step for vasculature tracing. Using Imaris software on volumetric images of iDisco+ cleared brains, we first reconstructed volumetric images from Reca1+ staining (green, A1) and performed surface mapping (A2), surface-based masking and Gaussian filtering (A3), and finally filament tracing (A4). Representative vascularization in eCS implanted region; original Reca1+ images (left) and ROIs corresponding to vasculature tracing (right). (B) Representative images of Sham (top), TBI (middle), and eCS (bottom) rat brain tissue for the reconstructed original volume image (left) and vasculature tracing (right). (C) Quantification of blood vessel segment density per 500 μm3 for Sham (n = 15), TBI (n = 15), and eCS (n = 15) groups in all four ROIs. (D) Quantification of blood vessel length density per micrometer/500 μm3 for Sham (n = 15), TBI (n = 15), and eCS (n = 15) groups in all four ROIs. (E) Quantification of mean blood vessel diameter (volume: 500 μm3) for Sham (n = 15), TBI (n = 15), and eCS (n = 15) groups in all four ROIs. Post hoc LSD Mann-Whitney U test, *P < 0.05, **P < 0.01, and ***P < 0.001. Bar graphs show means ± SEM. RFAa, RFA anterior; RFAp, RFA posterior/perilesional. Graphs show means ± SEM.

  • Table 1 Primary antibody panel for immunostaining.

    Primary antibody panel for immunostaining..

    PurposeAntibodyManufacturerCatalog no.HostDilutionUsage
    Neuronal presence/
    marker
    NeuNEMD MilliporeMAB377Mouse1:500Slices
    Neuronal presence/
    marker
    NeuNEMD MilliporeABN91Chicken1:500iDisco+
    Neuronal
    proliferation
    Doublecortin (DCX)Abcamab18723Rabbit1:500Slices
    MyelinationOlig2Abcamab109186Rabbit1:100Slices
    Neural progenitorSox-1Abcamab87775Rabbit1:1000Slices
    Ki67Santa Cruzsc-7846Goat1:500Slices
    NeuroinflammationGFAPDAKOz0334Rabbit1:1000Slices
    CD68Bio-Radmca341RMouse1:500Slices
    Growth factor
    presence
    FGF2Abcamab8880Rabbit1:200Slices
    Chondroitin sulfate
    GAG presence
    CS56Sigma-AldrichC8035Mouse1:200Slices
    PlasticitySynapsin-1Abcamab14692Rabbit1:500Slices
    Neuronal activation
    marker
    ArcSynaptic Systems156 003Rabbit1:1000Slices/iDisco+
    VasculatureRECABio-RadMCA970RMouse1:500Slices/iDisco+
    Col IVAbcamAb19808Rabbit1:500Slices
  • Table 2 Secondary antibody panel.

    Secondary antibody panel..

    Alexa FluorHostReactivityIsotypeCatalog no.Company
    488GoatMouseIgG (H+L)A11006Life Technologies
    488GoatChickenIgY (H+L)A11039Life Technologies
    647GoatRabbitIgG (H+L)A21244Life Technologies
    488DonkeyGoatIgG (H+L)A11055Life Technologies
    594GoatMouseIgG (H+L)A11005Life Technologies
  • Table 3 iDisco+tissue clearing steps.

    iDisco+tissue clearing steps.. For primary and secondary antibody concentration, please refer to Tables 1 and 2. RT, room temperature; DCM, dichloromethane; DBE, dibenzyl ether; DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline; PTx2, 0.05% Triton X-100 in 1× PBS; PTwh, 0.0.5% Tween 20, 0.5 ml of heparin (10 mg/ml) in 1× PBS.

    ProcessConditionReagentSolvent
    Initial wash15 min 3× in eachPTx2 and PTwh1× PBS
    Serial dehydration1 hour each20/40/60/80/100% methanol1× PBS
    DefattingOvernightDCM (66%, v/v)Methanol
    BleachingOvernightH2O2 (5%, v/v)Methanol
    Serial rehydration1 hour each20/40/60/80/100% PBSMethanol
    Permeabilization4 daysTriton X-100, 20% DMSO1× PBS
    Blocking2 daysGoat serum (6%, v/v) and DMSO
    (10%, v/v)
    PTx2
    Primary antibody incubation7 days, 37°C, 100 rpmGoat serum (3%, v/v) and DMSO (5%,
    v/v)
    PTx2
    WashOvernightPTwh
    Secondary antibody incubation6 days, 37°C, 100 rpmGoat serum (3%, v/v) and DMSO (5%,
    v/v)
    PTx2
    Final dehydration1 hour each20/40/60/80/100% methanolDiH2O
    Defatting3 hour, RTDCM (66%, v/v)Methanol
    DefattingOvernightDCM 100%DCM
    Final defatting/clearing30 min—hold indefinitely100% DBEDBE
  • Time (min)%A%B
    0973
    108020
    307525
    550100
    650100

Supplementary Materials

  • Supplementary Materials

    Engineered glycomaterial implants orchestrate large-scale functional repair of brain tissue chronically after severe traumatic brain injury

    Charles-Francois V. Latchoumane, Martha I. Betancur, Gregory A. Simchick, Min Kyoung Sun, Rameen Forghani, Christopher E. Lenear, Aws Ahmed, Ramya Mohankumar, Nivedha Balaji, Hannah D. Mason, Stephanie A. Archer-Hartmann, Parastoo Azadi, Philip V. Holmes, Qun Zhao, Ravi V. Bellamkonda, Lohitash Karumbaiah

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