Locomotor recovery following contusive spinal cord injury does not require oligodendrocyte remyelination methods
Aim. Evidence-backed execution summary for Locomotor recovery following contusive spinal cord injury does not require oligodendrocyte remyelination methods from Locomotor recovery following contusive spinal cord injury does not require oligodendrocyte remyelination.
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mouse
Subject model for the experiment.
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- confirm full cohort details in the source paper
Methods
reagent used in the protocol.
- Use
- Procedures involving live animals were approved by the University of British Columbia, in accordance with guidelines from the Canadian Council on Animal Care (A13-0328). Experiments were initiated in 8-10-week old mice that were group housed, fed a standard chow ad libitum and maintained on a 12 h revers...
Methods
reagent used in the protocol.
- Use
- To determine the extent of new myelin produced by OPCs, Myrf fl/fl PDGFRα-CreERT2 or Myrf wt/wt PDGFRα-CreERT2 mice were crossed with Rosa26-mGFP (mT/mG) mice (JAX # 007576), which induces GFP expression that is tethered to the membrane following tamoxifen induced Cre-mediated recombination (mGFP). Myrf I...
Spinal cord injury and animal care
reagent used in the protocol.
- Use
- Prior to surgery, mice were anaesthetized for 3 min with a 3% isofluorane (Fresenius Kabi, Toronto, Canada, CPO40602 ) to oxygen mixture. Anesthesia was maintained at 1.5-2% isofluorane as needed during surgery. Each animal received 1 ml of Ringer's solution (Braun, Montreal, Canada, L7500) a...
Perfusion and tissue processing
reagent used in the protocol.
- Use
- To collect spinal cords for immunohistochemical analysis, mice were transcardially perfused with 20 ml of PBS followed by 40 ml of freshly prepared 4% paraformaldehyde (PFA) (Fisher Scientific, Ward Hill, MA A11313) at two or six WPI. The injury site was identified, then one cm of the spinal cord f...
Perfusion and tissue processing
reagent used in the protocol.
- Use
- Spinal cords were collected for electron microscopy at six WPI. Mice were transcardially perfused with 20 mL of 0.01 M PBS followed by 40 ml of 4% PFA with 1% glutaraldehyde chilled to 4 °C (Electron Microscopy Sciences, Hatfield, PA, 16220). The injury site was identified, then segments...
Immunohistochemistry
reagent used in the protocol.
- Use
- To prepare for antibody staining, slides were thawed then rehydrated in PBS. In order to effectively stain myelin proteins, tissue was put through ascending, then descending ethanol dilutions (50, 70, 90, 95, 100, 95, 90, 70, 50%), followed by three washes of PBS. Tissue was then blocked with 10% normal donkey serum...
Toluidine blue staining and electron microscopy
reagent used in the protocol.
- Use
- Spinal cords embedded in resin were sectioned to 1 µm thickness on an ultramicrotome (Ultracut E, Reichert-Jung). Ultra- and semithin sections were collected every 20 µm and semithin sections were viewed under a light microscope to find the injury epicenter, defined by the lowest number of myel...
Schwann cell myelination is unaltered by Myrf ICKO after SCI
The majority of new (GFP+) myelin sheaths in Myrf ICKO mice were found in the dorsal column, a location of extensive Schwann cell myelination following dorsal SCI,. Given that MBP is not only expressed in oligodendrocyte myelin but also Schwann cell myelin, we determined whether the new myelin produced in Myrf IC...
- Use
- The majority of new (GFP+) myelin sheaths in Myrf ICKO mice were found in the dorsal column, a location of extensive Schwann cell myelination following dorsal SCI,. Given that MBP is not only expressed in oligodendrocyte myelin but also Schwann cell myelin, we determined whether the new myelin produced in Myrf IC...
Motor recovery when oligodendrocyte remyelination is blocked
Given the large amount of oligodendrogenesis and remyelination that occurred in control mice after moderate thoracic SCI, we wanted to understand if oligodendrocyte remyelination was causative in locomotor recovery. In contrast to mice with functional MYRF, Myrf ICKO mice were almost completely unable to produce new...
- Use
- Given the large amount of oligodendrogenesis and remyelination that occurred in control mice after moderate thoracic SCI, we wanted to understand if oligodendrocyte remyelination was causative in locomotor recovery. In contrast to mice with functional MYRF, Myrf ICKO mice were almost completely unable to produce new...
Motor recovery when oligodendrocyte remyelination is blocked
Mice also underwent footprint analysis using the Catwalk apparatus, which quantifies numerous aspects of gait and is capable of detecting subtle differences in locomotion (Fig. -f). The base of support, stride length, relative paw position, duty cycle and the percentage of time individual paws were on th...
- Use
- Mice also underwent footprint analysis using the Catwalk apparatus, which quantifies numerous aspects of gait and is capable of detecting subtle differences in locomotion (Fig. -f). The base of support, stride length, relative paw position, duty cycle and the percentage of time individual paws were on th...
Immunohistochemistry
To prepare for antibody staining, slides were thawed then rehydrated in PBS. In order to effectively stain myelin proteins, tissue was put through ascending, then descending ethanol dilutions (50, 70, 90, 95, 100, 95, 90, 70, 50%), followed by three washes of PBS. Tissue was then blocked with 10% normal donkey serum...
- Use
- To prepare for antibody staining, slides were thawed then rehydrated in PBS. In order to effectively stain myelin proteins, tissue was put through ascending, then descending ethanol dilutions (50, 70, 90, 95, 100, 95, 90, 70, 50%), followed by three washes of PBS. Tissue was then blocked with 10% normal donkey serum...
Cell counting and tissue analysis
All analyses were performed blinded to animal genotypes. A Zeiss Axio-Observer M1 inverted confocal microscope with a Yokogawa spinning disk and Zen 2 software (Zeiss) was used for imaging. For analysis of the area of spared tissue, images of whole spinal cord cross sections stained with GFAP were taken at 100 ×...
- Use
- All analyses were performed blinded to animal genotypes. A Zeiss Axio-Observer M1 inverted confocal microscope with a Yokogawa spinning disk and Zen 2 software (Zeiss) was used for imaging. For analysis of the area of spared tissue, images of whole spinal cord cross sections stained with GFAP were taken at 100 ×...
Toluidine blue staining and electron microscopy
Spinal cords embedded in resin were sectioned to 1 µm thickness on an ultramicrotome (Ultracut E, Reichert-Jung). Ultra- and semithin sections were collected every 20 µm and semithin sections were viewed under a light microscope to find the injury epicenter, defined by the lowest number of myel...
- Use
- Spinal cords embedded in resin were sectioned to 1 µm thickness on an ultramicrotome (Ultracut E, Reichert-Jung). Ultra- and semithin sections were collected every 20 µm and semithin sections were viewed under a light microscope to find the injury epicenter, defined by the lowest number of myel...
Toluidine blue staining and electron microscopy
For transmission electron microscopy, ultrathin sections of 100 nm thickness at the lesion epicenter were stained with Reynold's lead citrate and uranyl acetate to enhance contrast, and imaged at 5000 × primary magnification on a Zeiss EM910 equipped with a digital camera. At least ten nonoverlapping...
- Use
- For transmission electron microscopy, ultrathin sections of 100 nm thickness at the lesion epicenter were stained with Reynold's lead citrate and uranyl acetate to enhance contrast, and imaged at 5000 × primary magnification on a Zeiss EM910 equipped with a digital camera. At least ten nonoverlapping...
Behavioral assessments
All behavioral assessments were performed during the dark cycle to increase activity. The raters were blinded to animal genotype while running behavioral tests and during subsequent analyses. Behavioral assessements were run in mice lacking the mT/mG reporter to avoid any confounding effects of the expression of flu...
- Use
- All behavioral assessments were performed during the dark cycle to increase activity. The raters were blinded to animal genotype while running behavioral tests and during subsequent analyses. Behavioral assessements were run in mice lacking the mT/mG reporter to avoid any confounding effects of the expression of flu...
Statistical analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Statistical analyses were conducted using the Statistical Package for the Social Sciences (SPSS) or Graphpad 6.0 (Prism). Individual data points were displayed when possible and represent a single mouse. However, bar graphs were plotted for lesion size, the contribution of PDGFRα-cell derived myelin to total my...
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Myrf ICKO prevents oligodendrocyte remyelination
We next determined whether Myrf knockout from resident OPCs was sufficient to halt new myelination in response to SCI. Six weeks after a moderate thoracic contusion injury, there was sparing of some ventrolateral white matter at the lesion epicenter (Fig. ), which contains both undamaged and regenerated myelin. To unequivocally differentiate newly generated myelin from surviving myelin, we crossed Myrf ICKO mice with Rosa26mGFP (mT/mG) mice. When administered tamoxifen, OPCs with active Cre recombinase express a membrane-anchored GFP that can be visualized within new myelin produced by oligodendrocytes which have differentiated from OPCs (Fig. ),,,. By six WPI, in the ventrolateral white matter, control mice had new myelin sheaths, which were indicated by GFP + colabeling within MBP + sheaths around NF-200/SMI312 positive axons (Fig. ). Co...
Motor recovery when oligodendrocyte remyelination is blocked
Mice also underwent footprint analysis using the Catwalk apparatus, which quantifies numerous aspects of gait and is capable of detecting subtle differences in locomotion (Fig. -f). The base of support, stride length, relative paw position, duty cycle and the percentage of time individual paws were on the platform were analyzed. These are outcome measures sensitive to motor dysfunction following SCI. In control mice and Myrf ICKO, injury induced profound impairments in stride length (Fig. ), base of support (Fig. ), and an increase in relative bilateral paw position (Fig. ), but did not alter hindlimb duty cycle in mice (time standing/time standing + time in swing) (Fig. ). After injury, there was also a decrease in the duration of time a mouse had one or two paws placed on the walkway (Fig. ) and an increase in the time in which...
Methods
To determine the extent of new myelin produced by OPCs, Myrf fl/fl PDGFRα-CreERT2 or Myrf wt/wt PDGFRα-CreERT2 mice were crossed with Rosa26-mGFP (mT/mG) mice (JAX # 007576), which induces GFP expression that is tethered to the membrane following tamoxifen induced Cre-mediated recombination (mGFP). Myrf ICKO and control mice were heterozygous for the mT/mG and PDGFRα-CreERT2 transgenes in genetic fate mapping experiments. A total of n = 23 mice were used in the study and n = 1 animal died during surgery bringing the total animals to n = 12 perfused at six WPI and n = 10 perfused 2-week postinjury. Genotyping was performed on ear clippings and DNA was extracted using the REDExtract-N-AMP Tissue Kit (Sigma, St. Louis, MO, R4775) and amplified with primers specific for the transgenes,,. Genotypes were visualized befor...
Spinal cord injury and animal care
Prior to surgery, mice were anaesthetized for 3 min with a 3% isofluorane (Fresenius Kabi, Toronto, Canada, CPO40602 ) to oxygen mixture. Anesthesia was maintained at 1.5-2% isofluorane as needed during surgery. Each animal received 1 ml of Ringer's solution (Braun, Montreal, Canada, L7500) and buprenorphine (0.05 mg/kg) (Reckitt-Benckiser Slough, Toronto, Canada) analgesic prior to surgery. The back was shaved and then disinfected using successive betadine (Purdue Pharma, 41731) and 70% alcohol washes. An incision and separation of the erector trunci muscles from the spine followed by a dorsal laminectomy of T9-10 was performed. The vertebral column was stabilized by clamping the exposed T8 and T10 vertebrae with forceps prior to positioning the animal under the Infinite Horizons Impactor (Precision Systems). The IH impactor tip was lowered until i...
Tamoxifen and EdU administration
Tamoxifen was dissolved in corn oil (Sigma, St. Louis, MO, C8267) at 20 mg ml -1 before administration. All mice received 100 mg kg -1 day -1 intraperitoneal injections of tamoxifen (Sigma, St. Louis, MO, T5648) beginning 9 days prior to SCI and continuing for 5 consecutive days. For the first two days after SCI, 5-ethynyl-2'-deoxyuridine (EdU) (Invitrogen, Eugene, OR A10044) was dissolved in sterile PBS and administered by intraperitoneal injection (5 mg kg -1 ). After two days, EdU (Carbosynth, San Diego, CA, 61135-33-9) was dissolved in drinking water (0.2 mg ml -1 ) with 1% d -glucose to encourage consumption. EdU water was changed every 2 days and the mice were administered EdU in their water until four WPI.
Perfusion and tissue processing
To collect spinal cords for immunohistochemical analysis, mice were transcardially perfused with 20 ml of PBS followed by 40 ml of freshly prepared 4% paraformaldehyde (PFA) (Fisher Scientific, Ward Hill, MA A11313) at two or six WPI. The injury site was identified, then one cm of the spinal cord flanking the injury was dissected. Spinal cords were fixed in PFA for 8 h, then incubated in ascending sucrose solutions (12, 18, and 24%) at 4 °C. Tissue was submerged in OCT compound (Tissue-Tek, Torrance, CA 4583) frozen on dry ice and stored at -80 °C. All spinal cords were sectioned using a cryostat (Thermo Scientific, Walldorf, Germany, HM-525) into 20 µm thick cross-sections, which were mounted in series on ten slides making each individual section on a slide 200 µm apart.
Immunohistochemistry
To prepare for antibody staining, slides were thawed then rehydrated in PBS. In order to effectively stain myelin proteins, tissue was put through ascending, then descending ethanol dilutions (50, 70, 90, 95, 100, 95, 90, 70, 50%), followed by three washes of PBS. Tissue was then blocked with 10% normal donkey serum dissolved in PBS with 0.1% Triton X-100 for 30 min. Primary antibodies were diluted in PBS with 0.1% Triton X-100 and applied to the slides overnight at room temperature in a humid chamber. The following morning, slides were washed and incubated with donkey Dylight or Alexa Fluor secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.) for 2 h, then washed again before being coversliped using Fluoromount-G (Southern Biotech, 0100-01). Antibodies used were raised against the following antigens: CC1 (1:300, Millipore, OP80), OLIG2 (1:500, Millipore, AB961...
Measurement outputs
What raw and processed outputs should exist?
We next determined if Myrf ICKO was effective at inhibiting the accumulation of new oligodendrocytes in response to SCI. Oligodendrocyte lineage cells (OLIG2+) were typically no...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Given the large amount of oligodendrogenesis and remyelination that occurred in control mice after moderate thoracic SCI, we wanted to understand if oligodendrocyte remyelinatio...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Mice also underwent footprint analysis using the Catwalk apparatus, which quantifies numerous aspects of gait and is capable of detecting subtle differences in locomotion (Fig.&...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
To determine the extent of new myelin produced by OPCs, Myrf fl/fl PDGFRα-CreERT2 or Myrf wt/wt PDGFRα-CreERT2 mice were crossed with Rosa26-mGFP (mT/mG) mice (JAX # 0...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
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
We next determined if Myrf ICKO was effective at inhibiting the accumulation of new oligodendrocytes in response to SCI.
from paperScoring or quantification
Quantify the primary readouts for this experiment: We next determined if Myrf ICKO was effective at inhibiting the accumulation of new oligodendrocytes in response to SCI. Oligodendrocyte lineage cells (OLIG2+) were typically no...; Given the large amount of oligodendrogenesis and remyelination that occurred in control mice after moderate thoracic SCI, we wanted to understand if oligodendrocyte remyelinatio...; Mice also underwent footprint analysis using the Catwalk apparatus, which quantifies numerous aspects of gait and is capable of detecting subtle differences in locomotion (Fig.&...; To determine the extent of new myelin produced by OPCs, Myrf fl/fl PDGFRα-CreERT2 or Myrf wt/wt PDGFRα-CreERT2 mice were crossed with Rosa26-mGFP (mT/mG) mice (JAX # 0....
from paperStatistical comparison
We next determined if Myrf ICKO was effective at inhibiting the accumulation of new oligodendrocytes in response to SCI. Oligodendrocyte lineage cells (OLIG2+) were typically no...; We next determined whether Myrf knockout from resident OPCs was sufficient to halt new myelination in response to SCI. Six weeks after a moderate thoracic contusion injury, ther...; The majority of new (GFP+) myelin sheaths in Myrf ICKO mice were found in the dorsal column, a location of extensive Schwann cell myelination following dorsal SCI,. Given that...; Given the large amount of oligodendrogenesis and remyelination that occurred in control mice after moderate thoracic SCI, we wanted to understand if oligodendrocyte remyelinatio...
from paperReporting output
Report representative outputs alongside summary comparisons for We next determined if Myrf ICKO was effective at inhibiting the accumulation of new oligodendrocytes in response to SCI. Oligodendrocyte lineage cells (OLIG2+) were typically no..., Given the large amount of oligodendrogenesis and remyelination that occurred in control mice after moderate thoracic SCI, we wanted to understand if oligodendrocyte remyelinatio..., Mice also underwent footprint analysis using the Catwalk apparatus, which quantifies numerous aspects of gait and is capable of detecting subtle differences in locomotion (Fig.&..., To determine the extent of new myelin produced by OPCs, Myrf fl/fl PDGFRα-CreERT2 or Myrf wt/wt PDGFRα-CreERT2 mice were crossed with Rosa26-mGFP (mT/mG) mice (JAX # 0....
inferred from protocolStructured statistical methods
We next determined if Myrf ICKO was effective at inhibiting the accumulation of new oligodendrocytes in response to SCI. Oligodendrocyte lineage cells (OLIG2+) were typically no...; We next determined whether Myrf knockout from resident OPCs was sufficient to halt new myelination in response to SCI. Six weeks after a moderate thoracic contusion injury, ther...; The majority of new (GFP+) myelin sheaths in Myrf ICKO mice were found in the dorsal column, a location of extensive Schwann cell myelination following dorsal SCI,. Given that...; Given the large amount of oligodendrogenesis and remyelination that occurred in control mice after moderate thoracic SCI, we wanted to understand if oligodendrocyte remyelinatio...
source structuredSource and audit
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Evidence quotes (7)
We next determined whether Myrf knockout from resident OPCs was sufficient to halt new myelination in response to SCI. Six weeks after a moderate thoracic contusion injury, there was sparing of some ventrolateral white matter at the lesion epicenter (Fig. ), which contains both undamaged and regenerated myelin. To unequivocally differentiate newly generated myelin from surviving myelin, we crossed Myrf ICKO mice with Rosa26mGFP (mT/mG) mice. When administered tamoxifen, OPCs with active Cre recombinase express a membrane-anchored GFP that can be visualized within new myelin produced by oligodendrocytes which have differentiated from OPCs (Fig. ),,,. By six WPI, in the ventrolateral white matter, control mice had new myelin sheaths, which were indicated by GFP + colabeling within MBP + sheaths around NF-200/SMI312 positive axons (Fig. ). Conversely, in Myrf ICKO mT/mG mice, GFP processes wrapped axons, but were almost always negative for MBP (Fig. ). After 6 weeks, the Myrf ICKO mice had generated only 248 ± 56 new myelin sheaths mm -2 in contrast to control mice which had 4664 ± 674 sheat...
Mice also underwent footprint analysis using the Catwalk apparatus, which quantifies numerous aspects of gait and is capable of detecting subtle differences in locomotion (Fig. -f). The base of support, stride length, relative paw position, duty cycle and the percentage of time individual paws were on the platform were analyzed. These are outcome measures sensitive to motor dysfunction following SCI. In control mice and Myrf ICKO, injury induced profound impairments in stride length (Fig. ), base of support (Fig. ), and an increase in relative bilateral paw position (Fig. ), but did not alter hindlimb duty cycle in mice (time standing/time standing + time in swing) (Fig. ). After injury, there was also a decrease in the duration of time a mouse had one or two paws placed on the walkway (Fig. ) and an increase in the time in which three or four paws were simultaneously in contact with the walkway (Fig. ). However, we did not find at any time point a difference between between Myrf ICKO and controls on any of these or other parameters. These same analyses were run on an additional cohort of Myrf ICKO and control mice wit...
To determine the extent of new myelin produced by OPCs, Myrf fl/fl PDGFRα-CreERT2 or Myrf wt/wt PDGFRα-CreERT2 mice were crossed with Rosa26-mGFP (mT/mG) mice (JAX # 007576), which induces GFP expression that is tethered to the membrane following tamoxifen induced Cre-mediated recombination (mGFP). Myrf ICKO and control mice were heterozygous for the mT/mG and PDGFRα-CreERT2 transgenes in genetic fate mapping experiments. A total of n = 23 mice were used in the study and n = 1 animal died during surgery bringing the total animals to n = 12 perfused at six WPI and n = 10 perfused 2-week postinjury. Genotyping was performed on ear clippings and DNA was extracted using the REDExtract-N-AMP Tissue Kit (Sigma, St. Louis, MO, R4775) and amplified with primers specific for the transgenes,,. Genotypes were visualized before and after the experiment by running PCR solutions on a 1.5% agarose (Invitrogen, Carlsbad, CA, 16500) gel. Primer sequences were:
Prior to surgery, mice were anaesthetized for 3 min with a 3% isofluorane (Fresenius Kabi, Toronto, Canada, CPO40602 ) to oxygen mixture. Anesthesia was maintained at 1.5-2% isofluorane as needed during surgery. Each animal received 1 ml of Ringer's solution (Braun, Montreal, Canada, L7500) and buprenorphine (0.05 mg/kg) (Reckitt-Benckiser Slough, Toronto, Canada) analgesic prior to surgery. The back was shaved and then disinfected using successive betadine (Purdue Pharma, 41731) and 70% alcohol washes. An incision and separation of the erector trunci muscles from the spine followed by a dorsal laminectomy of T9-10 was performed. The vertebral column was stabilized by clamping the exposed T8 and T10 vertebrae with forceps prior to positioning the animal under the Infinite Horizons Impactor (Precision Systems). The IH impactor tip was lowered until it just contacted the exposed spinal cord, raised 1 cm, and set to deliver 70 kilodynes of force. Following surgery, the skin and overlying musculature were sutured with 6-0 nylon sutures (Ethicon, San Lorenzo, Peurto Rico, 667G) and the mice were placed into a temperature and humidity controll...
Tamoxifen was dissolved in corn oil (Sigma, St. Louis, MO, C8267) at 20 mg ml -1 before administration. All mice received 100 mg kg -1 day -1 intraperitoneal injections of tamoxifen (Sigma, St. Louis, MO, T5648) beginning 9 days prior to SCI and continuing for 5 consecutive days. For the first two days after SCI, 5-ethynyl-2'-deoxyuridine (EdU) (Invitrogen, Eugene, OR A10044) was dissolved in sterile PBS and administered by intraperitoneal injection (5 mg kg -1 ). After two days, EdU (Carbosynth, San Diego, CA, 61135-33-9) was dissolved in drinking water (0.2 mg ml -1 ) with 1% d -glucose to encourage consumption. EdU water was changed every 2 days and the mice were administered EdU in their water until four WPI.
To collect spinal cords for immunohistochemical analysis, mice were transcardially perfused with 20 ml of PBS followed by 40 ml of freshly prepared 4% paraformaldehyde (PFA) (Fisher Scientific, Ward Hill, MA A11313) at two or six WPI. The injury site was identified, then one cm of the spinal cord flanking the injury was dissected. Spinal cords were fixed in PFA for 8 h, then incubated in ascending sucrose solutions (12, 18, and 24%) at 4 °C. Tissue was submerged in OCT compound (Tissue-Tek, Torrance, CA 4583) frozen on dry ice and stored at -80 °C. All spinal cords were sectioned using a cryostat (Thermo Scientific, Walldorf, Germany, HM-525) into 20 µm thick cross-sections, which were mounted in series on ten slides making each individual section on a slide 200 µm apart.
To prepare for antibody staining, slides were thawed then rehydrated in PBS. In order to effectively stain myelin proteins, tissue was put through ascending, then descending ethanol dilutions (50, 70, 90, 95, 100, 95, 90, 70, 50%), followed by three washes of PBS. Tissue was then blocked with 10% normal donkey serum dissolved in PBS with 0.1% Triton X-100 for 30 min. Primary antibodies were diluted in PBS with 0.1% Triton X-100 and applied to the slides overnight at room temperature in a humid chamber. The following morning, slides were washed and incubated with donkey Dylight or Alexa Fluor secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.) for 2 h, then washed again before being coversliped using Fluoromount-G (Southern Biotech, 0100-01). Antibodies used were raised against the following antigens: CC1 (1:300, Millipore, OP80), OLIG2 (1:500, Millipore, AB9610), MYRF (1:300, N-terminus, generously provided by Dr. Michael Wegner), GFP (1:4000, Abcam, ab13970), GFAP (1:1000, Sigma, G3893), PDGFRα (1:200, R and D Systems, AF-307-NA), NF200 (1:1000, Sigma, N0142), SMI312 (1:1000, Covance, SMI-312R-100) and P0 (1:100, Aveslabs, PZO).
Machine-readable layer
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"name": "Locomotor recovery following contusive spinal cord injury does not require oligodendrocyte remyelination methods",
"description": "Evidence-backed execution summary for Locomotor recovery following contusive spinal cord injury does not require oligodendrocyte remyelination methods from Locomotor recovery following contusive spinal cord injury does not require oligodendrocyte remyelination.",
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"name": "Myrf ICKO prevents oligodendrocyte remyelination",
"text": "We next determined whether Myrf knockout from resident OPCs was sufficient to halt new myelination in response to SCI. Six weeks after a moderate thoracic contusion injury, there was sparing of some ventrolateral white matter at the lesion epicenter (Fig. ), which contains both undamaged and regenerated myelin. To unequivocally differentiate newly generated myelin from surviving myelin, we crossed Myrf ICKO mice with Rosa26mGFP (mT/mG) mice. When administered tamoxifen, OPCs with active Cre recombinase express a membrane-anchored GFP that can be visualized within new myelin produced by oligodendrocytes which have differentiated from OPCs (Fig. ),,,. By six WPI, in the ventrolateral white matter, control mice had new myelin sheaths, which were indicated by GFP + colabeling within MBP + sheaths around NF-200/SMI312 positive axons (Fig. ). Co..."
},
{
"@type": "HowToStep",
"position": 2,
"name": "Motor recovery when oligodendrocyte remyelination is blocked",
"text": "Mice also underwent footprint analysis using the Catwalk apparatus, which quantifies numerous aspects of gait and is capable of detecting subtle differences in locomotion (Fig. -f). The base of support, stride length, relative paw position, duty cycle and the percentage of time individual paws were on the platform were analyzed. These are outcome measures sensitive to motor dysfunction following SCI. In control mice and Myrf ICKO, injury induced profound impairments in stride length (Fig. ), base of support (Fig. ), and an increase in relative bilateral paw position (Fig. ), but did not alter hindlimb duty cycle in mice (time standing/time standing + time in swing) (Fig. ). After injury, there was also a decrease in the duration of time a mouse had one or two paws placed on the walkway (Fig. ) and an increase in the time in which..."
},
{
"@type": "HowToStep",
"position": 3,
"name": "Methods",
"text": "To determine the extent of new myelin produced by OPCs, Myrf fl/fl PDGFRα-CreERT2 or Myrf wt/wt PDGFRα-CreERT2 mice were crossed with Rosa26-mGFP (mT/mG) mice (JAX # 007576), which induces GFP expression that is tethered to the membrane following tamoxifen induced Cre-mediated recombination (mGFP). Myrf ICKO and control mice were heterozygous for the mT/mG and PDGFRα-CreERT2 transgenes in genetic fate mapping experiments. A total of n = 23 mice were used in the study and n = 1 animal died during surgery bringing the total animals to n = 12 perfused at six WPI and n = 10 perfused 2-week postinjury. Genotyping was performed on ear clippings and DNA was extracted using the REDExtract-N-AMP Tissue Kit (Sigma, St. Louis, MO, R4775) and amplified with primers specific for the transgenes,,. Genotypes were visualized befor..."
},
{
"@type": "HowToStep",
"position": 4,
"name": "Spinal cord injury and animal care",
"text": "Prior to surgery, mice were anaesthetized for 3 min with a 3% isofluorane (Fresenius Kabi, Toronto, Canada, CPO40602 ) to oxygen mixture. Anesthesia was maintained at 1.5-2% isofluorane as needed during surgery. Each animal received 1 ml of Ringer's solution (Braun, Montreal, Canada, L7500) and buprenorphine (0.05 mg/kg) (Reckitt-Benckiser Slough, Toronto, Canada) analgesic prior to surgery. The back was shaved and then disinfected using successive betadine (Purdue Pharma, 41731) and 70% alcohol washes. An incision and separation of the erector trunci muscles from the spine followed by a dorsal laminectomy of T9-10 was performed. The vertebral column was stabilized by clamping the exposed T8 and T10 vertebrae with forceps prior to positioning the animal under the Infinite Horizons Impactor (Precision Systems). The IH impactor tip was lowered until i..."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Tamoxifen and EdU administration",
"text": "Tamoxifen was dissolved in corn oil (Sigma, St. Louis, MO, C8267) at 20 mg ml -1 before administration. All mice received 100 mg kg -1 day -1 intraperitoneal injections of tamoxifen (Sigma, St. Louis, MO, T5648) beginning 9 days prior to SCI and continuing for 5 consecutive days. For the first two days after SCI, 5-ethynyl-2'-deoxyuridine (EdU) (Invitrogen, Eugene, OR A10044) was dissolved in sterile PBS and administered by intraperitoneal injection (5 mg kg -1 ). After two days, EdU (Carbosynth, San Diego, CA, 61135-33-9) was dissolved in drinking water (0.2 mg ml -1 ) with 1% d -glucose to encourage consumption. EdU water was changed every 2 days and the mice were administered EdU in their water until four WPI."
},
{
"@type": "HowToStep",
"position": 6,
"name": "Perfusion and tissue processing",
"text": "To collect spinal cords for immunohistochemical analysis, mice were transcardially perfused with 20 ml of PBS followed by 40 ml of freshly prepared 4% paraformaldehyde (PFA) (Fisher Scientific, Ward Hill, MA A11313) at two or six WPI. The injury site was identified, then one cm of the spinal cord flanking the injury was dissected. Spinal cords were fixed in PFA for 8 h, then incubated in ascending sucrose solutions (12, 18, and 24%) at 4 °C. Tissue was submerged in OCT compound (Tissue-Tek, Torrance, CA 4583) frozen on dry ice and stored at -80 °C. All spinal cords were sectioned using a cryostat (Thermo Scientific, Walldorf, Germany, HM-525) into 20 µm thick cross-sections, which were mounted in series on ten slides making each individual section on a slide 200 µm apart."
},
{
"@type": "HowToStep",
"position": 7,
"name": "Immunohistochemistry",
"text": "To prepare for antibody staining, slides were thawed then rehydrated in PBS. In order to effectively stain myelin proteins, tissue was put through ascending, then descending ethanol dilutions (50, 70, 90, 95, 100, 95, 90, 70, 50%), followed by three washes of PBS. Tissue was then blocked with 10% normal donkey serum dissolved in PBS with 0.1% Triton X-100 for 30 min. Primary antibodies were diluted in PBS with 0.1% Triton X-100 and applied to the slides overnight at room temperature in a humid chamber. The following morning, slides were washed and incubated with donkey Dylight or Alexa Fluor secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.) for 2 h, then washed again before being coversliped using Fluoromount-G (Southern Biotech, 0100-01). Antibodies used were raised against the following antigens: CC1 (1:300, Millipore, OP80), OLIG2 (1:500, Millipore, AB961..."
}
],
"tool": [
{
"@type": "HowToTool",
"name": "Schwann cell myelination is unaltered by Myrf ICKO after SCI"
},
{
"@type": "HowToTool",
"name": "Motor recovery when oligodendrocyte remyelination is blocked"
},
{
"@type": "HowToTool",
"name": "Motor recovery when oligodendrocyte remyelination is blocked"
},
{
"@type": "HowToTool",
"name": "Immunohistochemistry"
},
{
"@type": "HowToTool",
"name": "Cell counting and tissue analysis"
},
{
"@type": "HowToTool",
"name": "Toluidine blue staining and electron microscopy"
},
{
"@type": "HowToTool",
"name": "Toluidine blue staining and electron microscopy"
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{
"@type": "HowToTool",
"name": "Behavioral assessments"
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"name": "Spinal cord injury and animal care"
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"@type": "HowToSupply",
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"@type": "HowToSupply",
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"headline": "Locomotor recovery following contusive spinal cord injury does not require oligodendrocyte remyelination",
"datePublished": "2018",
"author": [
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"name": "Greg J. Duncan"
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"name": "Jie Liu"
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"name": "Aaron Moulson"
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"@type": "Person",
"name": "Jason R. Plemel"
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{
"@type": "Person",
"name": "Wolfram Tetzlaff"
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"identifier": "10.1038/s41467-018-05473-1"
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