Neural stem cell mediated recovery is enhanced by Chondroitinase ABC pretreatment in chronic cervical spinal cord injury methods
Show snapshot details
On this page
This experiment, in seven questions
Jump straight to the part of the recipe you need. Data and provenance labels stay close to the action they support.
Shopping and prep list
What do I need before I start?
Biological model pending
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Before you run
What should be confirmed before execution?
First confirmation
Species or subject information is missing.
Confirm before execution
Protocol does not list structured equipment.
Confirm before execution
This page shows passages quoted from the paper. Equipment, materials, times, and temperatures are hidden until the page is rebuilt from the full paper and checked by a reviewer. Check the paper before using this method.
Confirm before execution
Open the source paper before finalizing run-specific details.
Procurement checkpoint
Use source-stated vendors where present. Treat mapped products as sourcing options unless the page marks an exact source match.
Open quote workflowStep-by-step procedure
What do I do, in order?
Spinal cord injury
Mice were anesthetized with inhaled isoflurane (2% induction; 1% maintenance) in a 1:1 mixture of nitrous oxide and oxygen. Using strict aseptic technique, a C6 and C7 laminectomy was completed using a dorsal midline approach. The dura was gently dissected circumferentially to allow the ventral blade of a modified aneurysm clip (FEJOTA mouse clip, University Health Network, Canada) to be passed at C6/7 and closed on the cord as described in our previous reports ( )[ ]. A closing force of 6.5g for 40 seconds was used. Sham injured mice received a laminectomy only without dural dissection or clip injury. 1mL Normal Saline and 0.05mg/kg buprenorphine were administered subcutaneously immediately after the operation. Animals were recovered in sterile cages under heating lamps and then housed in a temperature-controlled room (27°C) for the length of the study. Food and water were provi...
Experimental procedures
All experimental protocols were approved by the Animal Care Committee at Krembil Research Institute (Toronto, Canada). Guidelines from the Canadian Council on Animal Care were strictly followed. All animals received food and water ad libitum throughout the study. A total of 70 adult (8-10 weeks old), female, wild-type C57BL/6 mice (15 to 20g) from Jackson Laboratories (Bar Harbor, ME, USA) were the subjects of this study. All surgeries were performed under deep anesthesia with isofluorane (2% induction; 1% maintenance) in a 1:1 mixture of oxygen and nitrous oxide. Ten mice received a C6/7 laminectomy alone (sham control group). Sixty mice received a C6/7 clip-contusion injury as described below. Six weeks after injury, mice were randomized into four experimental groups (control, ChABC, iPS-NSCs, & ChABC + iPS-NSCs; ). Grip strength measurements at six weeks were not statisticall...
Experimental procedures
Treatments are in green; assessments are in blue. (A) A C6 clip-contusion spinal cord injury (SCI) was performed at 0 weeks in C57/BL6 mice. Seven weeks post-injury, a mini-osmotic pump was implanted rostral to the injury site. The pump delivered either Chondroitinase ABC (ChABC) in artificial cerebral spinal fluid (aCSF) or aCSF alone. One week later the pump was explanted and induced pluripotent stem cell-derived neural stem cells (iPS-NSC) were transplanted into the cord parenchyma at two rostral and two caudal sites. 50,000 cells in 1µL were delivered to each site. Cyclosporine A immunosuppression was delivered from 2 days prior to transplantation until the end of the study. Behavioral assessments were performed across all 16 weeks of the study. At 16 weeks electrophysiologic assessments were completed followed by whole-animal perfusion, fixation and immunohistochemical analy...
iPS-NSC generation
Mouse embryonic fibroblasts (MEF) for use as an iPS feeder layer were prepared from CD1 embryos following a protocol described by Nagy et al.[ ] iPS cells were generated from CD1 mouse fibroblasts using the piggyBac (PB) transposon system reported by Woltjen et al.[ ] In brief, fibroblasts were plated on gelatinized 6-well plates at 7.5x10 4 cells/well. After 24 hours they were transfected using FugeneHD (Roche, Basel, Switzerland) with 2µg/well of a DNA mixture containing expression vectors for the PB transposase and PB transposons. These contained a CAG promoter driven rtTA, a CAG-GFP, and a set of tetracycline inducible promoter (Tet-On) driven reprogramming factors (polycistronic cMyc, Klf4, Oct4 and Sox2) linked by self-cleaving 2A sequences. 24hrs after transfection, the medium was supplemented with 1.5µg/ml doxycycline and 48hrs after transfection the cells were trans...
iPS-NSC generation
Definitive NSCs were generated using a well-established protocol previously described by our lab [, ]. Briefly, iPS colonies were picked and dissociated to single cells using TrypLE. Cells were seeded at 10 cells/µL in low-attachment culture flasks contain serum-free media (SFM) with LIF to yield primitive neurospheres after 7 days. Definitive neurospheres containing iPS-NSC were generated by dissociating and expanding primitive neurospheres in SFM containing 10µg/mL fibroblast growth factor (FGF; Sigma-Aldrich), 2µg/mL heparin (Sigma-Aldrich), B-27 supplement (Gibco, Grand Island, NY) and Delta-like 4 Notch ligand (Dll4; R&D Systems Inc., Minneapolis, MN).
Chondroitinase ABC injection
7 weeks after injury and 1 week prior to cell transplantation mice were randomly assigned to receive either artificial cerebrospinal fluid (aCSF) or aCSF with ChABC (3.64mU/mL; amsbio, Abingdon, UK) ( ). Under isofluorane anesthetic (as above) the injured spinal cord was surgically exposed and a fine (0.23 mm OD; 0.09 mm ID) intrathecal catheter (Mouse IT catheter 0007743; Alzet, Cupertino, CA; ) was implanted and connected to an osmotic mini-pump (Model 1007D, Alzet) as described by Karimi-Abdolrezaee et al.[ ] The treatment fluid was delivered at 0.5µl/hr for 7 days followed by surgical explantation at the time of cell transplant.
iPS-NSC transplantation
iPS-NSC transplantations were performed as described by Salewski et al.[ ] Briefly, under isofluorane anesthetic the injured spinal cord was exposed and the intrathecal catheter and osmotic pump were removed. Mice were randomly assigned to receive iPS-NSC or cell-free PBS (control; ). Four injection sites were mapped bilaterally adjacent to the midline dorsal vein at 1mm rostral and 1mm caudal to the injury epicenter. The dura at each site was punctured using a fine needle. A pulled glass micropipette attached to a 5µL Hamilton syringe on a computer-controlled syringe pump (World Precision Instruments) was used to stereotactically transplant 1µL (at 0.2µl/min) of PBS alone or 50,000 cells in PBS into the underlying spinal cord at each of the four sites. Immunosuppression with subcutaneous Cyclosporine A (20mg/kg daily) was started 2 days prior to transplantation and con...
Behavioral testing
The BMS is a global measure of hindlimb function in mice [ ] scored from 0 (no function) to 9 (normal movement, coordination, and stability). Mice were placed in an open field and evaluated once per week at the same day and time for 16 weeks by each observer.
Measurement outputs
What raw and processed outputs should exist?
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
Review raw outputs for quality, remove unusable captures, and organize the data into a comparison-ready table or image set.
inferred from protocolScoring or quantification
Quantify the primary endpoint described in the methods before moving into any statistical comparison.
inferred from protocolStatistical comparison
Statistical method not yet structured for this page.
inferred from protocolReporting output
Report representative outputs alongside the summary comparison used to support the conclusion.
inferred from protocolSource and audit
What supports the facts on this page?
Evidence quotes (8)
Mice were anesthetized with inhaled isoflurane (2% induction; 1% maintenance) in a 1:1 mixture of nitrous oxide and oxygen. Using strict aseptic technique, a C6 and C7 laminectomy was completed using a dorsal midline approach. The dura was gently dissected circumferentially to allow the ventral blade of a modified aneurysm clip (FEJOTA mouse clip, University Health Network, Canada) to be passed at C6/7 and closed on the cord as described in our previous reports ( )[ ]. A closing force of 6.5g for 40 seconds was used. Sham injured mice received a laminectomy only without dural dissection or clip injury. 1mL Normal Saline and 0.05mg/kg buprenorphine were administered subcutaneously immediately after the operation. Animals were recovered in sterile cages under heating lamps and then housed in a temperature-controlled room (27°C) for the length of the study. Food and water were provided ad libitum. Water was supplemented with Clavamox antibiotic (60mg/L water; Pfizer Animal Health, New York, NY) from 2 days pre-operatively to 3 days post-operatively. Buprenorphine was administered twice daily for 2 days after surgery. The animals' bladders were manually expressed twice da...
All experimental protocols were approved by the Animal Care Committee at Krembil Research Institute (Toronto, Canada). Guidelines from the Canadian Council on Animal Care were strictly followed. All animals received food and water ad libitum throughout the study. A total of 70 adult (8-10 weeks old), female, wild-type C57BL/6 mice (15 to 20g) from Jackson Laboratories (Bar Harbor, ME, USA) were the subjects of this study. All surgeries were performed under deep anesthesia with isofluorane (2% induction; 1% maintenance) in a 1:1 mixture of oxygen and nitrous oxide. Ten mice received a C6/7 laminectomy alone (sham control group). Sixty mice received a C6/7 clip-contusion injury as described below. Six weeks after injury, mice were randomized into four experimental groups (control, ChABC, iPS-NSCs, & ChABC + iPS-NSCs; ). Grip strength measurements at six weeks were not statistically different between groups (ANOVA; p = 0.871).
Treatments are in green; assessments are in blue. (A) A C6 clip-contusion spinal cord injury (SCI) was performed at 0 weeks in C57/BL6 mice. Seven weeks post-injury, a mini-osmotic pump was implanted rostral to the injury site. The pump delivered either Chondroitinase ABC (ChABC) in artificial cerebral spinal fluid (aCSF) or aCSF alone. One week later the pump was explanted and induced pluripotent stem cell-derived neural stem cells (iPS-NSC) were transplanted into the cord parenchyma at two rostral and two caudal sites. 50,000 cells in 1µL were delivered to each site. Cyclosporine A immunosuppression was delivered from 2 days prior to transplantation until the end of the study. Behavioral assessments were performed across all 16 weeks of the study. At 16 weeks electrophysiologic assessments were completed followed by whole-animal perfusion, fixation and immunohistochemical analyses. (B) Outline of the control and treatment groups. For additional details please see.
Mouse embryonic fibroblasts (MEF) for use as an iPS feeder layer were prepared from CD1 embryos following a protocol described by Nagy et al.[ ] iPS cells were generated from CD1 mouse fibroblasts using the piggyBac (PB) transposon system reported by Woltjen et al.[ ] In brief, fibroblasts were plated on gelatinized 6-well plates at 7.5x10 4 cells/well. After 24 hours they were transfected using FugeneHD (Roche, Basel, Switzerland) with 2µg/well of a DNA mixture containing expression vectors for the PB transposase and PB transposons. These contained a CAG promoter driven rtTA, a CAG-GFP, and a set of tetracycline inducible promoter (Tet-On) driven reprogramming factors (polycistronic cMyc, Klf4, Oct4 and Sox2) linked by self-cleaving 2A sequences. 24hrs after transfection, the medium was supplemented with 1.5µg/ml doxycycline and 48hrs after transfection the cells were transitioned to mouse ESC medium containing 1,000U/mL leukemia inhibitory factor (LIF; Sigma-Aldrich, St Louis, MO) and 1.5µg/ml doxycycline. On day 10-14, colonies morphologically resembling ESCs were picked and expanded on mitomycin-c arrested MEF feeders. Lines typically became doxycycline-...
Definitive NSCs were generated using a well-established protocol previously described by our lab [, ]. Briefly, iPS colonies were picked and dissociated to single cells using TrypLE. Cells were seeded at 10 cells/µL in low-attachment culture flasks contain serum-free media (SFM) with LIF to yield primitive neurospheres after 7 days. Definitive neurospheres containing iPS-NSC were generated by dissociating and expanding primitive neurospheres in SFM containing 10µg/mL fibroblast growth factor (FGF; Sigma-Aldrich), 2µg/mL heparin (Sigma-Aldrich), B-27 supplement (Gibco, Grand Island, NY) and Delta-like 4 Notch ligand (Dll4; R&D Systems Inc., Minneapolis, MN).
7 weeks after injury and 1 week prior to cell transplantation mice were randomly assigned to receive either artificial cerebrospinal fluid (aCSF) or aCSF with ChABC (3.64mU/mL; amsbio, Abingdon, UK) ( ). Under isofluorane anesthetic (as above) the injured spinal cord was surgically exposed and a fine (0.23 mm OD; 0.09 mm ID) intrathecal catheter (Mouse IT catheter 0007743; Alzet, Cupertino, CA; ) was implanted and connected to an osmotic mini-pump (Model 1007D, Alzet) as described by Karimi-Abdolrezaee et al.[ ] The treatment fluid was delivered at 0.5µl/hr for 7 days followed by surgical explantation at the time of cell transplant.
iPS-NSC transplantations were performed as described by Salewski et al.[ ] Briefly, under isofluorane anesthetic the injured spinal cord was exposed and the intrathecal catheter and osmotic pump were removed. Mice were randomly assigned to receive iPS-NSC or cell-free PBS (control; ). Four injection sites were mapped bilaterally adjacent to the midline dorsal vein at 1mm rostral and 1mm caudal to the injury epicenter. The dura at each site was punctured using a fine needle. A pulled glass micropipette attached to a 5µL Hamilton syringe on a computer-controlled syringe pump (World Precision Instruments) was used to stereotactically transplant 1µL (at 0.2µl/min) of PBS alone or 50,000 cells in PBS into the underlying spinal cord at each of the four sites. Immunosuppression with subcutaneous Cyclosporine A (20mg/kg daily) was started 2 days prior to transplantation and continued until animal sacrifice.
The BMS is a global measure of hindlimb function in mice [ ] scored from 0 (no function) to 9 (normal movement, coordination, and stability). Mice were placed in an open field and evaluated once per week at the same day and time for 16 weeks by each observer.
Machine-readable layer
[
{
"@context": "https://schema.org",
"@type": "HowTo",
"name": "Neural stem cell mediated recovery is enhanced by Chondroitinase ABC pretreatment in chronic cervical spinal cord injury methods",
"description": "Protocol for Neural stem cell mediated recovery is enhanced by Chondroitinase ABC pretreatment in chronic cervical spinal cord injury methods",
"step": [
{
"@type": "HowToStep",
"position": 1,
"name": "Spinal cord injury",
"text": "Mice were anesthetized with inhaled isoflurane (2% induction; 1% maintenance) in a 1:1 mixture of nitrous oxide and oxygen. Using strict aseptic technique, a C6 and C7 laminectomy was completed using a dorsal midline approach. The dura was gently dissected circumferentially to allow the ventral blade of a modified aneurysm clip (FEJOTA mouse clip, University Health Network, Canada) to be passed at C6/7 and closed on the cord as described in our previous reports ( )[ ]. A closing force of 6.5g for 40 seconds was used. Sham injured mice received a laminectomy only without dural dissection or clip injury. 1mL Normal Saline and 0.05mg/kg buprenorphine were administered subcutaneously immediately after the operation. Animals were recovered in sterile cages under heating lamps and then housed in a temperature-controlled room (27°C) for the length of the study. Food and water were provi..."
},
{
"@type": "HowToStep",
"position": 2,
"name": "Experimental procedures",
"text": "All experimental protocols were approved by the Animal Care Committee at Krembil Research Institute (Toronto, Canada). Guidelines from the Canadian Council on Animal Care were strictly followed. All animals received food and water ad libitum throughout the study. A total of 70 adult (8-10 weeks old), female, wild-type C57BL/6 mice (15 to 20g) from Jackson Laboratories (Bar Harbor, ME, USA) were the subjects of this study. All surgeries were performed under deep anesthesia with isofluorane (2% induction; 1% maintenance) in a 1:1 mixture of oxygen and nitrous oxide. Ten mice received a C6/7 laminectomy alone (sham control group). Sixty mice received a C6/7 clip-contusion injury as described below. Six weeks after injury, mice were randomized into four experimental groups (control, ChABC, iPS-NSCs, & ChABC + iPS-NSCs; ). Grip strength measurements at six weeks were not statisticall..."
},
{
"@type": "HowToStep",
"position": 3,
"name": "Experimental procedures",
"text": "Treatments are in green; assessments are in blue. (A) A C6 clip-contusion spinal cord injury (SCI) was performed at 0 weeks in C57/BL6 mice. Seven weeks post-injury, a mini-osmotic pump was implanted rostral to the injury site. The pump delivered either Chondroitinase ABC (ChABC) in artificial cerebral spinal fluid (aCSF) or aCSF alone. One week later the pump was explanted and induced pluripotent stem cell-derived neural stem cells (iPS-NSC) were transplanted into the cord parenchyma at two rostral and two caudal sites. 50,000 cells in 1µL were delivered to each site. Cyclosporine A immunosuppression was delivered from 2 days prior to transplantation until the end of the study. Behavioral assessments were performed across all 16 weeks of the study. At 16 weeks electrophysiologic assessments were completed followed by whole-animal perfusion, fixation and immunohistochemical analy..."
},
{
"@type": "HowToStep",
"position": 4,
"name": "iPS-NSC generation",
"text": "Mouse embryonic fibroblasts (MEF) for use as an iPS feeder layer were prepared from CD1 embryos following a protocol described by Nagy et al.[ ] iPS cells were generated from CD1 mouse fibroblasts using the piggyBac (PB) transposon system reported by Woltjen et al.[ ] In brief, fibroblasts were plated on gelatinized 6-well plates at 7.5x10 4 cells/well. After 24 hours they were transfected using FugeneHD (Roche, Basel, Switzerland) with 2µg/well of a DNA mixture containing expression vectors for the PB transposase and PB transposons. These contained a CAG promoter driven rtTA, a CAG-GFP, and a set of tetracycline inducible promoter (Tet-On) driven reprogramming factors (polycistronic cMyc, Klf4, Oct4 and Sox2) linked by self-cleaving 2A sequences. 24hrs after transfection, the medium was supplemented with 1.5µg/ml doxycycline and 48hrs after transfection the cells were trans..."
},
{
"@type": "HowToStep",
"position": 5,
"name": "iPS-NSC generation",
"text": "Definitive NSCs were generated using a well-established protocol previously described by our lab [, ]. Briefly, iPS colonies were picked and dissociated to single cells using TrypLE. Cells were seeded at 10 cells/µL in low-attachment culture flasks contain serum-free media (SFM) with LIF to yield primitive neurospheres after 7 days. Definitive neurospheres containing iPS-NSC were generated by dissociating and expanding primitive neurospheres in SFM containing 10µg/mL fibroblast growth factor (FGF; Sigma-Aldrich), 2µg/mL heparin (Sigma-Aldrich), B-27 supplement (Gibco, Grand Island, NY) and Delta-like 4 Notch ligand (Dll4; R&D Systems Inc., Minneapolis, MN)."
},
{
"@type": "HowToStep",
"position": 6,
"name": "Chondroitinase ABC injection",
"text": "7 weeks after injury and 1 week prior to cell transplantation mice were randomly assigned to receive either artificial cerebrospinal fluid (aCSF) or aCSF with ChABC (3.64mU/mL; amsbio, Abingdon, UK) ( ). Under isofluorane anesthetic (as above) the injured spinal cord was surgically exposed and a fine (0.23 mm OD; 0.09 mm ID) intrathecal catheter (Mouse IT catheter 0007743; Alzet, Cupertino, CA; ) was implanted and connected to an osmotic mini-pump (Model 1007D, Alzet) as described by Karimi-Abdolrezaee et al.[ ] The treatment fluid was delivered at 0.5µl/hr for 7 days followed by surgical explantation at the time of cell transplant."
},
{
"@type": "HowToStep",
"position": 7,
"name": "iPS-NSC transplantation",
"text": "iPS-NSC transplantations were performed as described by Salewski et al.[ ] Briefly, under isofluorane anesthetic the injured spinal cord was exposed and the intrathecal catheter and osmotic pump were removed. Mice were randomly assigned to receive iPS-NSC or cell-free PBS (control; ). Four injection sites were mapped bilaterally adjacent to the midline dorsal vein at 1mm rostral and 1mm caudal to the injury epicenter. The dura at each site was punctured using a fine needle. A pulled glass micropipette attached to a 5µL Hamilton syringe on a computer-controlled syringe pump (World Precision Instruments) was used to stereotactically transplant 1µL (at 0.2µl/min) of PBS alone or 50,000 cells in PBS into the underlying spinal cord at each of the four sites. Immunosuppression with subcutaneous Cyclosporine A (20mg/kg daily) was started 2 days prior to transplantation and con..."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Behavioral testing",
"text": "The BMS is a global measure of hindlimb function in mice [ ] scored from 0 (no function) to 9 (normal movement, coordination, and stability). Mice were placed in an open field and evaluated once per week at the same day and time for 16 weeks by each observer."
}
],
"tool": [],
"supply": [],
"isBasedOn": {
"@type": "ScholarlyArticle",
"headline": "Neural stem cell mediated recovery is enhanced by Chondroitinase ABC pretreatment in chronic cervical spinal cord injury",
"datePublished": "2017",
"author": [
{
"@type": "Person",
"name": "Hidenori Suzuki"
},
{
"@type": "Person",
"name": "Christopher S. Ahuja"
},
{
"@type": "Person",
"name": "Ryan P. Salewski"
},
{
"@type": "Person",
"name": "Lijun Li"
},
{
"@type": "Person",
"name": "Kajana Satkunendrarajah"
},
{
"@type": "Person",
"name": "Narihito Nagoshi"
},
{
"@type": "Person",
"name": "Shinsuke Shibata"
},
{
"@type": "Person",
"name": "Michael G. Fehlings"
}
],
"identifier": "10.1371/journal.pone.0182339"
}
},
{
"@context": "https://schema.org",
"@type": "BreadcrumbList",
"itemListElement": [
{
"@type": "ListItem",
"position": 1,
"name": "Experiments",
"item": "https://replicatescience.com/experiments"
},
{
"@type": "ListItem",
"position": 2,
"name": "Neural stem cell mediated recovery is enhanced by Chondroitinase ABC pretreatment in chronic cervical spinal cord injury methods",
"item": "https://replicatescience.com/experiments/neural-stem-cell-mediated-recovery-is-enhanced-by-chondroitinase-abc-pretreatment-in-chronic-cervical-spinal-cord-injury-methods-hidenori-suzuki-pmc5542671/neural-stem-cell-mediated-recovery-is-enhanced-by-chondroitinase-abc-pretreatment-in-chronic-cervica-mlph8xwj"
}
]
}
]