Human Neural Stem Cells Differentiate and Promote Locomotor Recovery in an Early Chronic Spinal coRd Injury NOD- scid Mouse Model methods

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    "name": "Human Neural Stem Cells Differentiate and Promote Locomotor Recovery in an Early Chronic Spinal coRd Injury NOD- scid Mouse Model methods",
    "description": "Evidence-backed execution summary for Human Neural Stem Cells Differentiate and Promote Locomotor Recovery in an Early Chronic Spinal coRd Injury NOD- scid Mouse Model methods from Human Neural Stem Cells Differentiate and Promote Locomotor Recovery in an Early Chronic Spinal coRd Injury NOD- scid Mouse Model.",
    "totalTime": "PT161760M",
    "step": [
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        "@type": "HowToStep",
        "position": 1,
        "name": "Methods and Findings",
        "text": "hCNS-SCns were transplanted into immunodeficient NOD- scid mice 30 days post spinal cord contusion injury. hCNS-SCns transplanted mice demonstrated significantly improved locomotor recovery compared to vehicle controls using open field locomotor testing and CatWalk gait analysis. Transplanted hCNS-SCns exhibited long-term engraftment, migration, limited proliferation, and differentiation predominantly to oligodendrocytes and neurons. Astrocytic differentiation was rare and mice did not exhibit mechanical allodynia. Furthermore, differentiated hCNS-SCns integrated with the host as demonstrated by co-localization of human cytoplasm with discrete staining for the paranodal marker contactin-associated protein."
      },
      {
        "@type": "HowToStep",
        "position": 2,
        "name": "Group design",
        "text": "Female mice were used in this study to avoid bladder complications and urolithiasis that frequently occur in male mice following SCI,. Animal surgeries were done in parallel over multiple days. Prior to surgery, NOD- scid mice (n = 48) were tested by the Basso Mouse Scale (BMS) to ensure normal locomotor function. One animal was excluded at this time because of abnormal gait. During the initial SCI surgery, one animal died and three additional animals were excluded because of surgical error. Pre-hoc criteria were established to exclude animals if any of the following conditions occurred: cord bruised or tearing of dura during laminectomy (n = 1); visual slippage of the mouse from the vertebral stabilization clamps (n = 0); abnormal time versus force curve indicating a bone hit or clamp slip (n = 1); or unilateral bruising of the cord (..."
      },
      {
        "@type": "HowToStep",
        "position": 3,
        "name": "Spinal Cord Injury",
        "text": "Female NOD- scid mice (transferred from StemCells Inc, the Jackson Laboratory, stock # 001303), 8-10 weeks of age were anesthetized with tribromoethanol (312.5 mg/kg i.p. bolus) and vertebral T9 exposed by laminectomy as previously described. Mice received a 50-kDyne-contusion injury using the IH device (Precision Systems and Instrumentation LLC). After surgery, mice recovered overnight in cages with Alpha-Dri bedding (Newco Distributors Inc) placed on water-jacketed heating pads at 37°C. Mice were maintained on twice daily manual bladder expression for 2 weeks, followed by once daily manual bladder expression for the remaining survival period. Post-operative care included buprenorphine twice a day for 2 days, lactated ringers once a day for 4 days, and antibiotic daily for the duration of the study. All animals were maintained on rotating schedule of antibiotics; Baytril,..."
      },
      {
        "@type": "HowToStep",
        "position": 4,
        "name": "Transplantation surgery",
        "text": "Isolation and culture of hCNS-SCns from fetal brain tissue (gestational 16-20 weeks) have been previously described,,. Briefly, FACS-sorted single cell suspensions were cultured in neurosphere initiation media consisting of Ex-Vivo 15 media with N2 supplement, FGF, EGF, LIF, neural survival factor-1, and NAC. Cells were propagated as neurospheres, fed weekly, and passaged every 2-3 weeks,,. Human fibroblasts derived from fetal liver were grown to confluence in Iscove's modified Dulbecco's medium/10% FBS, dissociated with trypsin, washed and concentrated to 50,000 cells per µl. Thirty days post-injury, mice were anesthetized with tribromoethanol (312.5 mg/kg i.p. bolus), the laminectomy site re-exposed and hCNS-SCns or hFbs were transplanted. Four injections of 250 nl each, bilateral from the midline, both rostral and caudal to the injury epicenter were performed..."
      },
      {
        "@type": "HowToStep",
        "position": 5,
        "name": "BMS",
        "text": "Open-field locomotor recovery was assessed using the BMS locomotor rating scale prior to injury, 2 days after injury, weekly for 4 weeks until transplantation, then weekly following transplantation until sacrifice. Briefly, mice were observed in the open-field for 4 minutes each by two individuals blinded to the experimental groups,. Motor function of the hind limbs was rated, recorded, and converted to a score according to the published scale."
      },
      {
        "@type": "HowToStep",
        "position": 6,
        "name": "CatWalk Analysis",
        "text": "CatWalk video was recorded at 16 weeks post-transplantation. Individuals blinded to experimental groups analyzed video using CatWalk software version 6.13 for Windows."
      },
      {
        "@type": "HowToStep",
        "position": 7,
        "name": "Mechanical Allodynia",
        "text": "Von Frey hair testing of hind limbs was done at 15 weeks post-transplantation. Animals were tested for a withdrawal response to successively higher force filaments (Touch-Test Sensory Evaluators, North Coast Medical). The lowest filament from which an animal withdrew from was the threshold assigned for that animal. After a positive response was elicited, the previous filament was tested to confirm a lack of response and the next filament was tested to confirm a positive response."
      },
      {
        "@type": "HowToStep",
        "position": 8,
        "name": "Histological Assessment",
        "text": "Sixteen weeks post-transplantation, mice were anesthetized using pentobarbital (100 mg/kg) and transcardially perfused with 15 mls of PBS followed by 100 mls of 4% phosphate-buffered paraformaldehyde. A T6-T12 segment of the spinal cord was dissected based on counting the dorsal spinal roots for all mice to obtain an anatomically consistent region of the spinal cord for stereological analysis. Spinal roots were identified by visualization of the features of C6, C7 and C8 root brachial plexus and exposure of the distal thoracic roots. Dissected T6-T12 spinal cord segments were post-fixed in a 20% sucrose/4% paraformaldehyde solution for cryoprotection overnight at 4°C. Following cryoprotection, spinal cords were frozen in isopentane at -65°C, and serial sliding microtome sections were collected free floating for immunocytochemical staining. Parasagittal sections (n̴..."
      }
    ],
    "tool": [
      {
        "@type": "HowToTool",
        "name": "Methods and Findings"
      },
      {
        "@type": "HowToTool",
        "name": "Transplantation surgery"
      },
      {
        "@type": "HowToTool",
        "name": "CatWalk Analysis"
      },
      {
        "@type": "HowToTool",
        "name": "Stereological Quantification"
      },
      {
        "@type": "HowToTool",
        "name": "Quantification of human cell differentiation"
      },
      {
        "@type": "HowToTool",
        "name": "Statistical Analysis"
      },
      {
        "@type": "HowToTool",
        "name": "hCNS-SCns successfully engraft and migrate"
      }
    ],
    "supply": [
      {
        "@type": "HowToSupply",
        "name": "Transplantation surgery"
      },
      {
        "@type": "HowToSupply",
        "name": "Histological Assessment"
      },
      {
        "@type": "HowToSupply",
        "name": "hCNS-SCns survive, engraft, and migrate within the injured spinal cord"
      },
      {
        "@type": "HowToSupply",
        "name": "hCNS-SCns do not affect lesion volume, spared tissue volume, or glial scar area"
      },
      {
        "@type": "HowToSupply",
        "name": "hCNS-SCns successfully engraft and migrate"
      },
      {
        "@type": "HowToSupply",
        "name": "hCNS-SCns successfully engraft and migrate"
      }
    ],
    "isBasedOn": {
      "@type": "ScholarlyArticle",
      "headline": "Human Neural Stem Cells Differentiate and Promote Locomotor Recovery in an Early Chronic Spinal coRd Injury NOD- scid Mouse Model",
      "datePublished": "2010",
      "author": [
        {
          "@type": "Person",
          "name": "Desirée L. Salazar"
        },
        {
          "@type": "Person",
          "name": "Nobuko Uchida"
        },
        {
          "@type": "Person",
          "name": "Frank P. T. Hamers"
        },
        {
          "@type": "Person",
          "name": "Brian J. Cummings"
        },
        {
          "@type": "Person",
          "name": "Aileen J. Anderson"
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      "identifier": "10.1371/journal.pone.0012272"
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