Differential effects of brain-derived neurotrophic factor and neurotrophin-3 on hindlimb function in paraplegic rats methods
Aim. Evidence-backed execution summary for Differential effects of brain-derived neurotrophic factor and neurotrophin-3 on hindlimb function in paraplegic rats methods from Differential effects of brain-derived neurotrophic factor and neurotrophin-3 on hindlimb function in paraplegic rats.
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rat
Subject model for the experiment.
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- confirm full cohort details in the source paper
SCI and application of neurotrophins
reagent used in the protocol.
- Use
- All procedures were approved by the Institutional Animal Care and Use Committee at the State University of New York at Stony Brook. These experiments were carried out with female Sprague-Dawley rats (5-6 months old, 300-310 g). Under isoflurane anesthesia (1.5-2% in O 2 ) and with sterile pro...
SCI and application of neurotrophins
reagent used in the protocol.
- Use
- Rats then received 2.5 × 10 10 genomic copies of AAV5-BDNF ( n = 6), AAV5-NT-3 ( n = 6) or AAV5-GFP ( n = 5) injected into the stump of the distal cord at the lesion site and into the lesion epicenter (Gelfoam) via a 1-mL Hamilton syringe. After the spinal cord had been covered with Durafilm o...
Electrophysiology
reagent used in the protocol.
- Use
- Rats were anesthetized with an intraperitoneal injection of a ketamine hydrochloride (80 mg/kg) and xylazine (10 mg/kg) solution supplemented as required by intrajugular infusion of a 20% mixture of the ketamine/xylazine solution in dextran lactated Ringer's solution. End-tidal CO 2 (25-30 mmHg) was moni...
Histology
reagent used in the protocol.
- Use
- For immunohistochemical staining, tissue sections were rinsed in Tris-buffered saline (TBS), blocked in a 2% goat serum/TBS solution with 0.1% Triton-X, and incubated in the primary antibody overnight at 4 °C. For visualization of the antibody, sections were again rinsed in TBS, and incubated in a fluorescent s...
SCI and application of neurotrophins
All procedures were approved by the Institutional Animal Care and Use Committee at the State University of New York at Stony Brook. These experiments were carried out with female Sprague-Dawley rats (5-6 months old, 300-310 g). Under isoflurane anesthesia (1.5-2% in O 2 ) and with sterile pro...
- Use
- All procedures were approved by the Institutional Animal Care and Use Committee at the State University of New York at Stony Brook. These experiments were carried out with female Sprague-Dawley rats (5-6 months old, 300-310 g). Under isoflurane anesthesia (1.5-2% in O 2 ) and with sterile pro...
Behavioral assessments
Behavioral assessments were performed in all rats pre-injury to obtain baseline measures of locomotor ability and sensory responsiveness. Locomotor performance was evaluated with three stepping tasks: (i) walking over-ground across a 1.2-m stationary glass walkway enclosed by plexiglass panels, which were narrowed t...
- Use
- Behavioral assessments were performed in all rats pre-injury to obtain baseline measures of locomotor ability and sensory responsiveness. Locomotor performance was evaluated with three stepping tasks: (i) walking over-ground across a 1.2-m stationary glass walkway enclosed by plexiglass panels, which were narrowed t...
Hargreaves test
The Hargreaves apparatus (Ugo Basile) was used to determine the latency of hindpaw withdrawal in response to a noxious heat stimulus. Testing was performed pre-injury and at 6 weeks post-Tx. Rats were placed in a behavioral chamber and allowed to acclimate for 20 min. A radiant heat source was then placed under each...
- Use
- The Hargreaves apparatus (Ugo Basile) was used to determine the latency of hindpaw withdrawal in response to a noxious heat stimulus. Testing was performed pre-injury and at 6 weeks post-Tx. Rats were placed in a behavioral chamber and allowed to acclimate for 20 min. A radiant heat source was then placed under each...
Footprint analysis
Footprint analysis was performed with the CatWalk device (Noldus) pre-Tx and at 2, 4 and 6 weeks post-Tx. These sessions were recorded in the dark, and the footfall patterns from each rat were captured by an infrared camera positioned under the glass walkway as the rats walked across it. Stepping behavior was record...
- Use
- Footprint analysis was performed with the CatWalk device (Noldus) pre-Tx and at 2, 4 and 6 weeks post-Tx. These sessions were recorded in the dark, and the footfall patterns from each rat were captured by an infrared camera positioned under the glass walkway as the rats walked across it. Stepping behavior was record...
Over-ground locomotion
Paint markers were placed on the skin over the ilium, femoral head, center of rotation of the knee, ankle, metatarsophalangeal (MTP) joint, and tip of the second digit of the hindlimb. Markers were also placed over the acromion process (shoulder), center of rotation of the elbow, metacarpophalangeal joint, and tip o...
- Use
- Paint markers were placed on the skin over the ilium, femoral head, center of rotation of the knee, ankle, metatarsophalangeal (MTP) joint, and tip of the second digit of the hindlimb. Markers were also placed over the acromion process (shoulder), center of rotation of the elbow, metacarpophalangeal joint, and tip o...
Treadmill evaluation
The recovery of treadmill locomotion was evaluated at 2, 4 and 6 weeks post-injury at treadmill speeds of 7.4 and 15 cm/s; locomotor performance for 10 steps at 15 cm/s is reported. The rats were tested with and without perineal stimulation (squeezing the base of the tail). Perineal stimulation was included in the t...
- Use
- The recovery of treadmill locomotion was evaluated at 2, 4 and 6 weeks post-injury at treadmill speeds of 7.4 and 15 cm/s; locomotor performance for 10 steps at 15 cm/s is reported. The rats were tested with and without perineal stimulation (squeezing the base of the tail). Perineal stimulation was included in the t...
Intracellular recording
Glass electrodes (20-25-mΩ resistance) filled with 3 m potassium acetate were used for intracellular motoneuron recording. Motoneuron identity was established by the response to antidromic stimulation of the ipsilateral MG or LGS nerves at twice threshold. Measures of motoneuron rheobase (Rh) and input re...
- Use
- Glass electrodes (20-25-mΩ resistance) filled with 3 m potassium acetate were used for intracellular motoneuron recording. Motoneuron identity was established by the response to antidromic stimulation of the ipsilateral MG or LGS nerves at twice threshold. Measures of motoneuron rheobase (Rh) and input re...
Histology
Sections were imaged with confocal microscopy (Olympus). Optical sections were captured at 1-µm increments and flattened in the z -axis to give a two-dimensional image. I mage- J software ( ) was then used to obtain the optical density and counts of c-Fos-positive nuclei in the dorsal (laminae I-V), inter...
- Use
- Sections were imaged with confocal microscopy (Olympus). Optical sections were captured at 1-µm increments and flattened in the z -axis to give a two-dimensional image. I mage- J software ( ) was then used to obtain the optical density and counts of c-Fos-positive nuclei in the dorsal (laminae I-V), inter...
Statistical analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- The post-Tx/pre-Tx hindlimb withdrawal latency ratio (Hargreaves) was compared between groups with a one-way repeated measures anova and the Student-Neuman-Keuls post hoc test. Plantar index was compared between the groups with a Kruskal-Wallis anova on ranks with Dunn's post hoc test. Planta...
Motoneuron physiology
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Other variables measured in these motoneurons were affected by NT-3 but not by BDNF. In AAV-GFP-control rats, AHP amplitude averaged 2.4 ± 1.0 mV (21 cells), similar to the mean value (2.6 ± 1.5 mV) in untreated control transected adult rats ( ). Kruskal-Wallis one-way anova on ranks was used t...
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SCI and application of neurotrophins
All procedures were approved by the Institutional Animal Care and Use Committee at the State University of New York at Stony Brook. These experiments were carried out with female Sprague-Dawley rats (5-6 months old, 300-310 g). Under isoflurane anesthesia (1.5-2% in O 2 ) and with sterile procedures, the T10 spinal cord was exposed by dorsal laminectomy. The dura was opened with microscissors, and 0.1 mL of lidocaine (2% solution) was added dropwise to the surface of the cord to minimize the injury discharge that accompanies spinal Tx. The spinal cord was then completely transected at T10 with microscissors. As a result of Tx, the ends of the cord were separated by approximately 0.5-1 mm. With the surgical microscope and glass probes, this cavity was carefully inspected visually to confirm the absence of spared spinal tissue, and then filled with Gelfoam....
SCI and application of neurotrophins
Rats then received 2.5 × 10 10 genomic copies of AAV5-BDNF ( n = 6), AAV5-NT-3 ( n = 6) or AAV5-GFP ( n = 5) injected into the stump of the distal cord at the lesion site and into the lesion epicenter (Gelfoam) via a 1-mL Hamilton syringe. After the spinal cord had been covered with Durafilm or parafilm, muscle and skin layers were closed with 4.0 vicryl (Ethicon) and surgical staples, respectively. Isoflurane was discontinued, and the rats were transferred to their home cages, where Baytril (2.5 mg/kg), buprenorphine (0.1-0.5 mg/kg) and lactated Ringer's solution were given via subcutaneous injection. The rats were housed individually and closely monitored during the recovery period. During this time, bladders were manually voided three times daily. Reflexive bladder voiding generally began within 2 weeks post-surgery.
Hargreaves test
The Hargreaves apparatus (Ugo Basile) was used to determine the latency of hindpaw withdrawal in response to a noxious heat stimulus. Testing was performed pre-injury and at 6 weeks post-Tx. Rats were placed in a behavioral chamber and allowed to acclimate for 20 min. A radiant heat source was then placed under each hindpaw and triggered to deliver a calibrated stimulus of 90 W/cm 2. Hindpaw withdrawal stopped the automated timer, and the latency was recorded. This process was repeated for five trials, alternating between the left and right hindpaws of each rat. The highest and lowest values were discarded, and the remaining three latencies were averaged to give the mean withdrawal latency for each hindpaw.
Footprint analysis
Footprint analysis was performed with the CatWalk device (Noldus) pre-Tx and at 2, 4 and 6 weeks post-Tx. These sessions were recorded in the dark, and the footfall patterns from each rat were captured by an infrared camera positioned under the glass walkway as the rats walked across it. Stepping behavior was recorded for three trials per rat at each time point. In order to obtain kinematic measures of this stepping, the CatWalk was modified such that the rats were filmed as they moved across the walkway (see Over-ground locomotion).
Over-ground locomotion
Paint markers were placed on the skin over the ilium, femoral head, center of rotation of the knee, ankle, metatarsophalangeal (MTP) joint, and tip of the second digit of the hindlimb. Markers were also placed over the acromion process (shoulder), center of rotation of the elbow, metacarpophalangeal joint, and tip of the second digit of the forelimb. The rats were videotaped through a plexiglass panel as they walked across the CatWalk platform to their home-cage on the other end. Balance support was provided in one of two ways: by the experimenter holding the tail, or by narrowing the lateral walls of the CatWalk such that the rats remained upright. These video recordings were digitized with M ax T raq P ro and analyzed with M ax M ate (Innovision Systems). The evaluation of treadmill locomotion, over-ground locomotion and footprint analysis were compared at 6 weeks post-injury, just...
Treadmill evaluation
Paint markers were placed over forelimb and hindlimb joints as previously discussed. Assessments were performed in one of two ways: quadrupedal stepping to examine interlimb coordination, and hindlimb-only locomotion, where the forelimbs were placed on a stationary platform 3 mm above the treadmill belt and only hindlimb stepping was evaluated. The experimenter provided balance support by holding the tail.
Treadmill evaluation
The recovery of treadmill locomotion was evaluated at 2, 4 and 6 weeks post-injury at treadmill speeds of 7.4 and 15 cm/s; locomotor performance for 10 steps at 15 cm/s is reported. The rats were tested with and without perineal stimulation (squeezing the base of the tail). Perineal stimulation was included in the testing paradigm, and applied in a manner that is consistent with other groups (;;; ), in order to fully assess the reorganization of the locomotor circuitry post-Tx and with the neurotrophic factor treatments. To avoid any training effect that might occur because of testing, rats were evaluated for only 60 s at each speed. Video footage was then digitized as above to generate kinematic measures of stepping behavior, such as step length and angular joint excursion. The number of plantar hindlimb steps executed during each evaluation session was also quantified (plantar in...
Electrophysiology
Rats were anesthetized with an intraperitoneal injection of a ketamine hydrochloride (80 mg/kg) and xylazine (10 mg/kg) solution supplemented as required by intrajugular infusion of a 20% mixture of the ketamine/xylazine solution in dextran lactated Ringer's solution. End-tidal CO 2 (25-30 mmHg) was monitored through a tracheal canula, which also provided access for emergency ventilation if necessary. Body temperature was maintained at 35-37 °C with a circulating water blanket.
Measurement outputs
What raw and processed outputs should exist?
Behavioral assessments were performed in all rats pre-injury to obtain baseline measures of locomotor ability and sensory responsiveness. Locomotor performance was evaluated wit...
- 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
The Hargreaves apparatus (Ugo Basile) was used to determine the latency of hindpaw withdrawal in response to a noxious heat stimulus. Testing was performed pre-injury and at 6 w...
- 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
Footprint analysis was performed with the CatWalk device (Noldus) pre-Tx and at 2, 4 and 6 weeks post-Tx. These sessions were recorded in the dark, and the footfall patterns fro...
- 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
Paint markers were placed on the skin over the ilium, femoral head, center of rotation of the knee, ankle, metatarsophalangeal (MTP) joint, and tip of the second digit of the hi...
- 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
The post-Tx/pre-Tx hindlimb withdrawal latency ratio (Hargreaves) was compared between groups with a one-way repeated measures anova and the Student-Neuman-Keuls post hoc test.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Behavioral assessments were performed in all rats pre-injury to obtain baseline measures of locomotor ability and sensory responsiveness. Locomotor performance was evaluated wit...; The Hargreaves apparatus (Ugo Basile) was used to determine the latency of hindpaw withdrawal in response to a noxious heat stimulus. Testing was performed pre-injury and at 6 w...; Footprint analysis was performed with the CatWalk device (Noldus) pre-Tx and at 2, 4 and 6 weeks post-Tx. These sessions were recorded in the dark, and the footfall patterns fro...; Paint markers were placed on the skin over the ilium, femoral head, center of rotation of the knee, ankle, metatarsophalangeal (MTP) joint, and tip of the second digit of the hi....
from paperStatistical comparison
The post-Tx/pre-Tx hindlimb withdrawal latency ratio (Hargreaves) was compared between groups with a one-way repeated measures anova and the Student-Neuman-Keuls pos...; Comparison of c-Fos immunoreactivty in L2 and L4/L5. Rats treated with AAV-BDNF (A) showed greater numbers of c-Fos-positive nuclei than AAV-NT-3-treated (B) or AAV&...; Release of BDNF into the lumbar cord would be expected to sensitize the nociceptive pathway in the dorsal horn (; ). This was examined by using the Hargreaves test, where rats...; Ratio of post-Tx and pre-Tx thermal nociceptive threshold measured in the same rats with the Hargreaves test. Note the significant reduction in hindlimb withdrawal latency with...
from paperReporting output
Report representative outputs alongside summary comparisons for Behavioral assessments were performed in all rats pre-injury to obtain baseline measures of locomotor ability and sensory responsiveness. Locomotor performance was evaluated wit..., The Hargreaves apparatus (Ugo Basile) was used to determine the latency of hindpaw withdrawal in response to a noxious heat stimulus. Testing was performed pre-injury and at 6 w..., Footprint analysis was performed with the CatWalk device (Noldus) pre-Tx and at 2, 4 and 6 weeks post-Tx. These sessions were recorded in the dark, and the footfall patterns fro..., Paint markers were placed on the skin over the ilium, femoral head, center of rotation of the knee, ankle, metatarsophalangeal (MTP) joint, and tip of the second digit of the hi....
inferred from protocolStructured statistical methods
The post-Tx/pre-Tx hindlimb withdrawal latency ratio (Hargreaves) was compared between groups with a one-way repeated measures anova and the Student-Neuman-Keuls pos...; Comparison of c-Fos immunoreactivty in L2 and L4/L5. Rats treated with AAV-BDNF (A) showed greater numbers of c-Fos-positive nuclei than AAV-NT-3-treated (B) or AAV&...; Release of BDNF into the lumbar cord would be expected to sensitize the nociceptive pathway in the dorsal horn (; ). This was examined by using the Hargreaves test, where rats...; Ratio of post-Tx and pre-Tx thermal nociceptive threshold measured in the same rats with the Hargreaves test. Note the significant reduction in hindlimb withdrawal latency with...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
All procedures were approved by the Institutional Animal Care and Use Committee at the State University of New York at Stony Brook. These experiments were carried out with female Sprague-Dawley rats (5-6 months old, 300-310 g). Under isoflurane anesthesia (1.5-2% in O 2 ) and with sterile procedures, the T10 spinal cord was exposed by dorsal laminectomy. The dura was opened with microscissors, and 0.1 mL of lidocaine (2% solution) was added dropwise to the surface of the cord to minimize the injury discharge that accompanies spinal Tx. The spinal cord was then completely transected at T10 with microscissors. As a result of Tx, the ends of the cord were separated by approximately 0.5-1 mm. With the surgical microscope and glass probes, this cavity was carefully inspected visually to confirm the absence of spared spinal tissue, and then filled with Gelfoam. Completeness of the lesion was also verified electrophysiologically and histologically (see Results).
Rats then received 2.5 × 10 10 genomic copies of AAV5-BDNF ( n = 6), AAV5-NT-3 ( n = 6) or AAV5-GFP ( n = 5) injected into the stump of the distal cord at the lesion site and into the lesion epicenter (Gelfoam) via a 1-mL Hamilton syringe. After the spinal cord had been covered with Durafilm or parafilm, muscle and skin layers were closed with 4.0 vicryl (Ethicon) and surgical staples, respectively. Isoflurane was discontinued, and the rats were transferred to their home cages, where Baytril (2.5 mg/kg), buprenorphine (0.1-0.5 mg/kg) and lactated Ringer's solution were given via subcutaneous injection. The rats were housed individually and closely monitored during the recovery period. During this time, bladders were manually voided three times daily. Reflexive bladder voiding generally began within 2 weeks post-surgery.
The Hargreaves apparatus (Ugo Basile) was used to determine the latency of hindpaw withdrawal in response to a noxious heat stimulus. Testing was performed pre-injury and at 6 weeks post-Tx. Rats were placed in a behavioral chamber and allowed to acclimate for 20 min. A radiant heat source was then placed under each hindpaw and triggered to deliver a calibrated stimulus of 90 W/cm 2. Hindpaw withdrawal stopped the automated timer, and the latency was recorded. This process was repeated for five trials, alternating between the left and right hindpaws of each rat. The highest and lowest values were discarded, and the remaining three latencies were averaged to give the mean withdrawal latency for each hindpaw.
Footprint analysis was performed with the CatWalk device (Noldus) pre-Tx and at 2, 4 and 6 weeks post-Tx. These sessions were recorded in the dark, and the footfall patterns from each rat were captured by an infrared camera positioned under the glass walkway as the rats walked across it. Stepping behavior was recorded for three trials per rat at each time point. In order to obtain kinematic measures of this stepping, the CatWalk was modified such that the rats were filmed as they moved across the walkway (see Over-ground locomotion).
Paint markers were placed on the skin over the ilium, femoral head, center of rotation of the knee, ankle, metatarsophalangeal (MTP) joint, and tip of the second digit of the hindlimb. Markers were also placed over the acromion process (shoulder), center of rotation of the elbow, metacarpophalangeal joint, and tip of the second digit of the forelimb. The rats were videotaped through a plexiglass panel as they walked across the CatWalk platform to their home-cage on the other end. Balance support was provided in one of two ways: by the experimenter holding the tail, or by narrowing the lateral walls of the CatWalk such that the rats remained upright. These video recordings were digitized with M ax T raq P ro and analyzed with M ax M ate (Innovision Systems). The evaluation of treadmill locomotion, over-ground locomotion and footprint analysis were compared at 6 weeks post-injury, just before the terminal electrophysiological procedure.
Paint markers were placed over forelimb and hindlimb joints as previously discussed. Assessments were performed in one of two ways: quadrupedal stepping to examine interlimb coordination, and hindlimb-only locomotion, where the forelimbs were placed on a stationary platform 3 mm above the treadmill belt and only hindlimb stepping was evaluated. The experimenter provided balance support by holding the tail.
The recovery of treadmill locomotion was evaluated at 2, 4 and 6 weeks post-injury at treadmill speeds of 7.4 and 15 cm/s; locomotor performance for 10 steps at 15 cm/s is reported. The rats were tested with and without perineal stimulation (squeezing the base of the tail). Perineal stimulation was included in the testing paradigm, and applied in a manner that is consistent with other groups (;;; ), in order to fully assess the reorganization of the locomotor circuitry post-Tx and with the neurotrophic factor treatments. To avoid any training effect that might occur because of testing, rats were evaluated for only 60 s at each speed. Video footage was then digitized as above to generate kinematic measures of stepping behavior, such as step length and angular joint excursion. The number of plantar hindlimb steps executed during each evaluation session was also quantified (plantar index = number of plantar steps executed per second during the evaluation session). The plantar index was then used to normalize step length [plantar step length = mean (stride length × plantar index)].
Rats were anesthetized with an intraperitoneal injection of a ketamine hydrochloride (80 mg/kg) and xylazine (10 mg/kg) solution supplemented as required by intrajugular infusion of a 20% mixture of the ketamine/xylazine solution in dextran lactated Ringer's solution. End-tidal CO 2 (25-30 mmHg) was monitored through a tracheal canula, which also provided access for emergency ventilation if necessary. Body temperature was maintained at 35-37 °C with a circulating water blanket.
Machine-readable layer
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"text": "All procedures were approved by the Institutional Animal Care and Use Committee at the State University of New York at Stony Brook. These experiments were carried out with female Sprague-Dawley rats (5-6 months old, 300-310 g). Under isoflurane anesthesia (1.5-2% in O 2 ) and with sterile procedures, the T10 spinal cord was exposed by dorsal laminectomy. The dura was opened with microscissors, and 0.1 mL of lidocaine (2% solution) was added dropwise to the surface of the cord to minimize the injury discharge that accompanies spinal Tx. The spinal cord was then completely transected at T10 with microscissors. As a result of Tx, the ends of the cord were separated by approximately 0.5-1 mm. With the surgical microscope and glass probes, this cavity was carefully inspected visually to confirm the absence of spared spinal tissue, and then filled with Gelfoam...."
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"text": "Rats then received 2.5 × 10 10 genomic copies of AAV5-BDNF ( n = 6), AAV5-NT-3 ( n = 6) or AAV5-GFP ( n = 5) injected into the stump of the distal cord at the lesion site and into the lesion epicenter (Gelfoam) via a 1-mL Hamilton syringe. After the spinal cord had been covered with Durafilm or parafilm, muscle and skin layers were closed with 4.0 vicryl (Ethicon) and surgical staples, respectively. Isoflurane was discontinued, and the rats were transferred to their home cages, where Baytril (2.5 mg/kg), buprenorphine (0.1-0.5 mg/kg) and lactated Ringer's solution were given via subcutaneous injection. The rats were housed individually and closely monitored during the recovery period. During this time, bladders were manually voided three times daily. Reflexive bladder voiding generally began within 2 weeks post-surgery."
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"text": "Footprint analysis was performed with the CatWalk device (Noldus) pre-Tx and at 2, 4 and 6 weeks post-Tx. These sessions were recorded in the dark, and the footfall patterns from each rat were captured by an infrared camera positioned under the glass walkway as the rats walked across it. Stepping behavior was recorded for three trials per rat at each time point. In order to obtain kinematic measures of this stepping, the CatWalk was modified such that the rats were filmed as they moved across the walkway (see Over-ground locomotion)."
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"text": "Paint markers were placed on the skin over the ilium, femoral head, center of rotation of the knee, ankle, metatarsophalangeal (MTP) joint, and tip of the second digit of the hindlimb. Markers were also placed over the acromion process (shoulder), center of rotation of the elbow, metacarpophalangeal joint, and tip of the second digit of the forelimb. The rats were videotaped through a plexiglass panel as they walked across the CatWalk platform to their home-cage on the other end. Balance support was provided in one of two ways: by the experimenter holding the tail, or by narrowing the lateral walls of the CatWalk such that the rats remained upright. These video recordings were digitized with M ax T raq P ro and analyzed with M ax M ate (Innovision Systems). The evaluation of treadmill locomotion, over-ground locomotion and footprint analysis were compared at 6 weeks post-injury, just..."
},
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"name": "Treadmill evaluation",
"text": "Paint markers were placed over forelimb and hindlimb joints as previously discussed. Assessments were performed in one of two ways: quadrupedal stepping to examine interlimb coordination, and hindlimb-only locomotion, where the forelimbs were placed on a stationary platform 3 mm above the treadmill belt and only hindlimb stepping was evaluated. The experimenter provided balance support by holding the tail."
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"name": "Treadmill evaluation",
"text": "The recovery of treadmill locomotion was evaluated at 2, 4 and 6 weeks post-injury at treadmill speeds of 7.4 and 15 cm/s; locomotor performance for 10 steps at 15 cm/s is reported. The rats were tested with and without perineal stimulation (squeezing the base of the tail). Perineal stimulation was included in the testing paradigm, and applied in a manner that is consistent with other groups (;;; ), in order to fully assess the reorganization of the locomotor circuitry post-Tx and with the neurotrophic factor treatments. To avoid any training effect that might occur because of testing, rats were evaluated for only 60 s at each speed. Video footage was then digitized as above to generate kinematic measures of stepping behavior, such as step length and angular joint excursion. The number of plantar hindlimb steps executed during each evaluation session was also quantified (plantar in..."
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"name": "Electrophysiology",
"text": "Rats were anesthetized with an intraperitoneal injection of a ketamine hydrochloride (80 mg/kg) and xylazine (10 mg/kg) solution supplemented as required by intrajugular infusion of a 20% mixture of the ketamine/xylazine solution in dextran lactated Ringer's solution. End-tidal CO 2 (25-30 mmHg) was monitored through a tracheal canula, which also provided access for emergency ventilation if necessary. Body temperature was maintained at 35-37 °C with a circulating water blanket."
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