Amelioration of motor/sensory dysfunction and spasticity in a rat model of acute lumbar spinal cord injury by human neural stem cell transplantation methods
Aim. Evidence-backed execution summary for Amelioration of motor/sensory dysfunction and spasticity in a rat model of acute lumbar spinal cord injury by human neural stem cell transplantation methods from Amelioration of motor/sensory dysfunction and spasticity in a rat model of acute lumbar spinal cord injury by human neural stem cell transplantation.
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rat
Subject model for the experiment.
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- confirm full cohort details in the source paper
Grafting procedure
reagent used in the protocol.
- Use
- For the intraparenchymal injections, the animals were placed in the stereotactic frame as described above. The L3 spinal cord (that is, the dura mater) was then re-exposed at the previous laminectomy site. Injections were performed using a 33 gauge beveled needle and 100 µL Nanofil syringe (World Precision Inst...
Axon counting in plastic semi-thin sections
reagent used in the protocol.
- Use
- After MRI imaging, spinal cords were dissected from the spine and a transverse (1.5-mm-thick) spinal cord block cut from the injury epicenter and prepared for plastic embedding as previously described [ ]. Briefly, dissected tissue blocks were treated with 0.1% osmium tetroxide in 0.1 M non-saline phosphate buffer (...
Axon counting in plastic semi-thin sections
reagent used in the protocol.
- Use
- Mosaic images were taken of two sections per animal at 20X using a Zeiss Imager. M2 fitted with a Zeiss MRm camera (Carl Zeiss Microscopy, Thornwood, NY, USA), a BioPrecision2 stage (Cat# 96S100, Ludl Electronic Products, Hawthorne, NY, USA), and Stereo Investigator software (MBF Biosciences, Williston, VT, USA). Co...
Methods
All animal studies were approved by the University of California, San Diego Institutional Animal Care and Use Committee (Protocol No.: S01193). The study design is outlined in Figure. Twelve-week-old Female SD rats were used. The rationale for choosing female rats was based on our previous experience which de...
- Use
- All animal studies were approved by the University of California, San Diego Institutional Animal Care and Use Committee (Protocol No.: S01193). The study design is outlined in Figure. Twelve-week-old Female SD rats were used. The rationale for choosing female rats was based on our previous experience which de...
Axon counting in plastic semi-thin sections
After MRI imaging, spinal cords were dissected from the spine and a transverse (1.5-mm-thick) spinal cord block cut from the injury epicenter and prepared for plastic embedding as previously described [ ]. Briefly, dissected tissue blocks were treated with 0.1% osmium tetroxide in 0.1 M non-saline phosphate buffer (...
- Use
- After MRI imaging, spinal cords were dissected from the spine and a transverse (1.5-mm-thick) spinal cord block cut from the injury epicenter and prepared for plastic embedding as previously described [ ]. Briefly, dissected tissue blocks were treated with 0.1% osmium tetroxide in 0.1 M non-saline phosphate buffer (...
Axon counting in plastic semi-thin sections
Mosaic images were taken of two sections per animal at 20X using a Zeiss Imager. M2 fitted with a Zeiss MRm camera (Carl Zeiss Microscopy, Thornwood, NY, USA), a BioPrecision2 stage (Cat# 96S100, Ludl Electronic Products, Hawthorne, NY, USA), and Stereo Investigator software (MBF Biosciences, Williston, VT, USA). Co...
- Use
- Mosaic images were taken of two sections per animal at 20X using a Zeiss Imager. M2 fitted with a Zeiss MRm camera (Carl Zeiss Microscopy, Thornwood, NY, USA), a BioPrecision2 stage (Cat# 96S100, Ludl Electronic Products, Hawthorne, NY, USA), and Stereo Investigator software (MBF Biosciences, Williston, VT, USA). Co...
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Methods
All animal studies were approved by the University of California, San Diego Institutional Animal Care and Use Committee (Protocol No.: S01193). The study design is outlined in Figure. Twelve-week-old Female SD rats were used. The rationale for choosing female rats was based on our previous experience which demonstrates better tolerability of female rats to spinal trauma-related side effects, such as urinary retention. Animals were anesthetized with isoflurane (5% induction, 1.5% to 2% maintenance; in room air) and placed into a Lab Standard Stereotaxic frame (Stoelting, Cat# 51600, Wood Dale, IL, USA). The animal was elevated 2 cm by placing it on a homeothermic heating blanket (set at 37°C with feedback from a rectal thermometer (Harvard Apparatus, Cat# 507214, Holliston, MA, USA) which sits on a plastic rectangular block. The animal was then placed in Spine Adaptors (Sto...
Methods
Schematic diagram of experimental design. A: To induce spinal cord injury, a 35 g circular rod was placed on the exposed L3 spinal segment and the spinal cord compressed in the dorso-ventral direction for 15 minutes. B: Three days after injury, the animals were randomly assigned to experimental groups and received a spinal graft of HSSC or media only. A total of 12 injections were performed targeting the injury epicenter and adjacent areas (see Spinal Injection Map). C: After spinal injections, the animals survived for two months while being continuously immunosuppressed and periodically tested for recovery of motor/sensory functions, changes in motor evoked potentials (MEPs) and gastrocnemius muscle spasticity response evoked by computer-controlled ankle rotation. D: At two months after treatment, animals were perfusion fixed with 4% PFA and spinal cord MRI-imaged in situ before hist...
Grafting procedure
For the intraparenchymal injections, the animals were placed in the stereotactic frame as described above. The L3 spinal cord (that is, the dura mater) was then re-exposed at the previous laminectomy site. Injections were performed using a 33 gauge beveled needle and 100 µL Nanofil syringe (World Precision Instruments, Cat# NF33BV and Nanofil-100, Sarasota, FL, USA) connected to a microinjection unit (Kopf Instruments, Cat# 5000 and 5001, Tujunga, CA, USA). The duration of each injection was ≥45 seconds followed by a ≥30 second pause before slow needle withdrawal. The center of the injection was targeted intermediate of the ventral and dorsal horn and close to the lateral funiculus (distance from the dorsal surface of the spinal cord at the L3 level: 0.80 mm). Twelve injections (20,000 cells/µL) were done; four injections (0.5 µL each, 0.8 to 1.0 mm apart, r...
Axon counting in plastic semi-thin sections
After MRI imaging, spinal cords were dissected from the spine and a transverse (1.5-mm-thick) spinal cord block cut from the injury epicenter and prepared for plastic embedding as previously described [ ]. Briefly, dissected tissue blocks were treated with 0.1% osmium tetroxide in 0.1 M non-saline phosphate buffer (pH 7.4) for 12 hours, followed by adequate rinsing in non-saline phosphate buffer. This was followed by progressive alcohol dehydration according to standard procedures up to 100% ethanol, with the addition of further dehydration in a 1:1 solution of ethanol/propylene oxide, and lastly in 100% propylene oxide. Dehydrated blocks were then prepared for resin infiltration by incubation in a 1:1 solution of resin/propylene oxide on a rotator in a fume hood overnight. The resin solution used consisted of: Eponate 12, Araldite 502, dodecenyl succinic anhydride, and 2,4,6-tri (dim...
Axon counting in plastic semi-thin sections
Mosaic images were taken of two sections per animal at 20X using a Zeiss Imager. M2 fitted with a Zeiss MRm camera (Carl Zeiss Microscopy, Thornwood, NY, USA), a BioPrecision2 stage (Cat# 96S100, Ludl Electronic Products, Hawthorne, NY, USA), and Stereo Investigator software (MBF Biosciences, Williston, VT, USA). Complete mosaic images were loaded into ImageJ 1.45s. Axonal quantification involved manual definition of pixel threshold (0 to 255, grayscale; using the Triangle method). Next, ImageJ's Analyze Particles option was used to find particles with a size of 0.20 to 250 µm 2 and a circularity of 0.5 to 1.0 (which corresponded to axons). All acquisition and analysis values were held consistent throughout the study. Final measurements acquired were the minimal diameter (Feret's) of each particle (and particle counts). Particles with a minimum diameter >10 µm we...
Effective suppression of muscle spasticity in HSSC-grafted SCI animals
Independent of SCI group (control or HSSC-injected), two quantitatively different EMG patterns and corresponding resistance response (EMG/RES) patterns were recorded in spinally-injured animals. First, if compared to control non-injured animals, little or no change in EMG/RES response was seen at 1.5 weeks after SCI. Second, SCI induced an increased spasticity response in a portion of the animals at 1.5 weeks after injury. A K-Means clustering method was used to group all 44 (SCI and non-injured) animals into two groups based on the magnitude of resistance to ankle rotation at 1.5 weeks post-injury (or equivalent time point in non-injured animals). Seven animals of each SCI group (that is, control or HSSC-injected) were found to be clustered in the high 'spasticity' group (HIGH), which had a 31.7 ± 3.9 g increase in measured muscle resistance during ankle rotation, co...
Measurement outputs
What raw and processed outputs should exist?
Mosaic images were taken of two sections per animal at 20X using a Zeiss Imager. M2 fitted with a Zeiss MRm camera (Carl Zeiss Microscopy, Thornwood, NY, USA), a BioPrecision2 s...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
In order to define the effect of spinal injection itself in modulating the functional recovery profile (that is, potential worsening in neurological outcome) in L3-injured anima...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
In our study, we assessed the sensory function below the level of injury (hind paws) by measuring the mechanical and thermal thresholds for supraspinally mediated escape behavio...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
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Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
In order to define the effect of spinal injection itself in modulating the functional recovery profile (that is, potential worsening in neurological outcome) in L3-injured animals, we first compared the effect of spinal media injection only with spinal injury animals that rece...
from paperScoring or quantification
Quantify the primary readouts for this experiment: Mosaic images were taken of two sections per animal at 20X using a Zeiss Imager. M2 fitted with a Zeiss MRm camera (Carl Zeiss Microscopy, Thornwood, NY, USA), a BioPrecision2 s...; In order to define the effect of spinal injection itself in modulating the functional recovery profile (that is, potential worsening in neurological outcome) in L3-injured anima...; In our study, we assessed the sensory function below the level of injury (hind paws) by measuring the mechanical and thermal thresholds for supraspinally mediated escape behavio....
from paperStatistical comparison
In order to define the effect of spinal injection itself in modulating the functional recovery profile (that is, potential worsening in neurological outcome) in L3-injured anima...; Independent of SCI group (control or HSSC-injected), two quantitatively different EMG patterns and corresponding resistance response (EMG/RES) patterns were recorded in spinally...; In our study, we assessed the sensory function below the level of injury (hind paws) by measuring the mechanical and thermal thresholds for supraspinally mediated escape behavio...
from paperReporting output
Report representative outputs alongside summary comparisons for Mosaic images were taken of two sections per animal at 20X using a Zeiss Imager. M2 fitted with a Zeiss MRm camera (Carl Zeiss Microscopy, Thornwood, NY, USA), a BioPrecision2 s..., In order to define the effect of spinal injection itself in modulating the functional recovery profile (that is, potential worsening in neurological outcome) in L3-injured anima..., In our study, we assessed the sensory function below the level of injury (hind paws) by measuring the mechanical and thermal thresholds for supraspinally mediated escape behavio....
inferred from protocolStructured statistical methods
In order to define the effect of spinal injection itself in modulating the functional recovery profile (that is, potential worsening in neurological outcome) in L3-injured anima...; Independent of SCI group (control or HSSC-injected), two quantitatively different EMG patterns and corresponding resistance response (EMG/RES) patterns were recorded in spinally...; In our study, we assessed the sensory function below the level of injury (hind paws) by measuring the mechanical and thermal thresholds for supraspinally mediated escape behavio...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (6)
All animal studies were approved by the University of California, San Diego Institutional Animal Care and Use Committee (Protocol No.: S01193). The study design is outlined in Figure. Twelve-week-old Female SD rats were used. The rationale for choosing female rats was based on our previous experience which demonstrates better tolerability of female rats to spinal trauma-related side effects, such as urinary retention. Animals were anesthetized with isoflurane (5% induction, 1.5% to 2% maintenance; in room air) and placed into a Lab Standard Stereotaxic frame (Stoelting, Cat# 51600, Wood Dale, IL, USA). The animal was elevated 2 cm by placing it on a homeothermic heating blanket (set at 37°C with feedback from a rectal thermometer (Harvard Apparatus, Cat# 507214, Holliston, MA, USA) which sits on a plastic rectangular block. The animal was then placed in Spine Adaptors (Stoelting, Cat# 51695, Wood Dale, IL, USA) and a wide Th13 laminectomy was performed using an air-powered dental drill and binocular microscope (exposing the dorsal surface of spinal segment L3). An acrylic rod (Ø 2.9 mm, length 15 cm; 35 g) was then slowly lowered onto the exposed L3 segment unti...
Schematic diagram of experimental design. A: To induce spinal cord injury, a 35 g circular rod was placed on the exposed L3 spinal segment and the spinal cord compressed in the dorso-ventral direction for 15 minutes. B: Three days after injury, the animals were randomly assigned to experimental groups and received a spinal graft of HSSC or media only. A total of 12 injections were performed targeting the injury epicenter and adjacent areas (see Spinal Injection Map). C: After spinal injections, the animals survived for two months while being continuously immunosuppressed and periodically tested for recovery of motor/sensory functions, changes in motor evoked potentials (MEPs) and gastrocnemius muscle spasticity response evoked by computer-controlled ankle rotation. D: At two months after treatment, animals were perfusion fixed with 4% PFA and spinal cord MRI-imaged in situ before histological processing. E: After MRI imaging, spinal cords were dissected from the spinal column and spinal blocks prepared for plastic embedding (injury epicenter region) or cryostat sectioning and used for immunofluorescence staining (the regions just above and below the injury epicenter). HSSC, huma...
For the intraparenchymal injections, the animals were placed in the stereotactic frame as described above. The L3 spinal cord (that is, the dura mater) was then re-exposed at the previous laminectomy site. Injections were performed using a 33 gauge beveled needle and 100 µL Nanofil syringe (World Precision Instruments, Cat# NF33BV and Nanofil-100, Sarasota, FL, USA) connected to a microinjection unit (Kopf Instruments, Cat# 5000 and 5001, Tujunga, CA, USA). The duration of each injection was ≥45 seconds followed by a ≥30 second pause before slow needle withdrawal. The center of the injection was targeted intermediate of the ventral and dorsal horn and close to the lateral funiculus (distance from the dorsal surface of the spinal cord at the L3 level: 0.80 mm). Twelve injections (20,000 cells/µL) were done; four injections (0.5 µL each, 0.8 to 1.0 mm apart, rostrocaudally) at each lateral boundary of the injury (eight in total), plus two (bilateral) injections (0.5 µL each) 1.5 mm caudal from the previous, most caudal injections, and two injections at the core of the epicenter (1 µL at each side of the dorsal vein, bilaterally; see diagram in...
After MRI imaging, spinal cords were dissected from the spine and a transverse (1.5-mm-thick) spinal cord block cut from the injury epicenter and prepared for plastic embedding as previously described [ ]. Briefly, dissected tissue blocks were treated with 0.1% osmium tetroxide in 0.1 M non-saline phosphate buffer (pH 7.4) for 12 hours, followed by adequate rinsing in non-saline phosphate buffer. This was followed by progressive alcohol dehydration according to standard procedures up to 100% ethanol, with the addition of further dehydration in a 1:1 solution of ethanol/propylene oxide, and lastly in 100% propylene oxide. Dehydrated blocks were then prepared for resin infiltration by incubation in a 1:1 solution of resin/propylene oxide on a rotator in a fume hood overnight. The resin solution used consisted of: Eponate 12, Araldite 502, dodecenyl succinic anhydride, and 2,4,6-tri (dimethylamino-methyl) phenol (DMP-30; Ted Pella, Inc., Redding, CA, USA), mixed in ratios of 10:10:25:1, respectively. The blocks were then transferred to 100% resin for subsequent overnight infiltration on a rotator. Finally, the tissue blocks were embedded using fresh resin in multi-chamber silicone...
Mosaic images were taken of two sections per animal at 20X using a Zeiss Imager. M2 fitted with a Zeiss MRm camera (Carl Zeiss Microscopy, Thornwood, NY, USA), a BioPrecision2 stage (Cat# 96S100, Ludl Electronic Products, Hawthorne, NY, USA), and Stereo Investigator software (MBF Biosciences, Williston, VT, USA). Complete mosaic images were loaded into ImageJ 1.45s. Axonal quantification involved manual definition of pixel threshold (0 to 255, grayscale; using the Triangle method). Next, ImageJ's Analyze Particles option was used to find particles with a size of 0.20 to 250 µm 2 and a circularity of 0.5 to 1.0 (which corresponded to axons). All acquisition and analysis values were held consistent throughout the study. Final measurements acquired were the minimal diameter (Feret's) of each particle (and particle counts). Particles with a minimum diameter >10 µm were excluded. Employment of this parameter allowed for further axonal analysis, in which axons were divided into empirically-derived caliber sizes of small, medium, and large axons (0.3 to 1.0 µm, 1.0 to 2.5 µm, and 2.5 to 10 µm, respectively). Data were acquired per spinal region (tha...
Independent of SCI group (control or HSSC-injected), two quantitatively different EMG patterns and corresponding resistance response (EMG/RES) patterns were recorded in spinally-injured animals. First, if compared to control non-injured animals, little or no change in EMG/RES response was seen at 1.5 weeks after SCI. Second, SCI induced an increased spasticity response in a portion of the animals at 1.5 weeks after injury. A K-Means clustering method was used to group all 44 (SCI and non-injured) animals into two groups based on the magnitude of resistance to ankle rotation at 1.5 weeks post-injury (or equivalent time point in non-injured animals). Seven animals of each SCI group (that is, control or HSSC-injected) were found to be clustered in the high 'spasticity' group (HIGH), which had a 31.7 ± 3.9 g increase in measured muscle resistance during ankle rotation, compared to the low 'spasticity' group (LOW) showing 8.9 ± 1.5 g resistance (Student's t -test: P <0.0001). No difference in the incidence of this high 'spasticity' response was noted between SCI control versus cell-treated groups (incidence: X 2: P = 0.53; extend:...
Machine-readable layer
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