Electrophysiological, behavioral and histological characterization of paclitaxel, cisplatin, vincristine and bortezomib-induced neuropathy in C57Bl/6 mice methods
Aim. Evidence-backed execution summary for Electrophysiological, behavioral and histological characterization of paclitaxel, cisplatin, vincristine and bortezomib-induced neuropathy in C57Bl/6 mice methods from Electrophysiological, behavioral and histological characterization of paclitaxel, cisplatin, vincristine and bortezomib-induced neuropathy in C57Bl/6 mice.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Methods
reagent used in the protocol.
- Use
- A total of 80 nine-week-old male C57BL/6J mice from Charles River (Sulzfeld, Germany) were used for this study. Animals were assigned to cages with the help of randomly generated numbers to reduce possible litter effects. Mice were housed in groups of five and allowed food and water ad libitum. A 12:12 hour light/d...
Drug Injection protocol
reagent used in the protocol.
- Use
- Solutions of paclitaxel (Sigma-Aldrich, Taufkirchen, Germany), cisplatin (Sigma-Aldrich, Taufkirchen, Germany), vincristine sulfate (Tocris, Bristol, UK) and bortezomib (Selleck Chemicals, Houston, Texas, USA) were prepared before each treatment. All substances were injected intraperitoneally (i.p.); animals in the...
Drug Injection protocol
reagent used in the protocol.
- Use
- Paclitaxel was dissolved in Cremophor EL:Ethanol (1:1) with a concentration of 2 mg/ml and diluted in sterile 0.9% NaCl solution to a final concentration of 0.1 mg/ml, a dose previously used in rat and mouse studies. Cisplatin was dissolved in sterile 0.9% NaCl solution with a concentration of 0.23̳...
Behavioral alterations
The drugs used in this study are known to mainly affect the sensory nervous system, however in high doses they can sometimes also cause motor symptoms. We tested for an impaired motor performance with the rotarod test and observed that performance in animals treated with cytostatic drugs was similar to that in the...
- Use
- The drugs used in this study are known to mainly affect the sensory nervous system, however in high doses they can sometimes also cause motor symptoms. We tested for an impaired motor performance with the rotarod test and observed that performance in animals treated with cytostatic drugs was similar to that in the...
Analysis of sciatic nerve histology
After completion of behavioral and electrophysiological analysis, animals were killed and sciatic nerves were dissected, fixed, stained for myelin and cut as semi-thin sections ( ). Light microscopy was performed to capture an image of the entire nerve which was then subjected to semi-automatic software-based nerve...
- Use
- After completion of behavioral and electrophysiological analysis, animals were killed and sciatic nerves were dissected, fixed, stained for myelin and cut as semi-thin sections ( ). Light microscopy was performed to capture an image of the entire nerve which was then subjected to semi-automatic software-based nerve...
Behavioral alterations
Automated gait analysis with the catwalk technique has been used previously to detect gait alterations induced by dose-dense paclitaxel treatment. In particular, an increase in the swing phase was observed as well as a decrease in the stance phase and duty cycle. The parameter duty cycle expresses the stance phase...
- Use
- Automated gait analysis with the catwalk technique has been used previously to detect gait alterations induced by dose-dense paclitaxel treatment. In particular, an increase in the swing phase was observed as well as a decrease in the stance phase and duty cycle. The parameter duty cycle expresses the stance phase...
Methods
A total of 80 nine-week-old male C57BL/6J mice from Charles River (Sulzfeld, Germany) were used for this study. Animals were assigned to cages with the help of randomly generated numbers to reduce possible litter effects. Mice were housed in groups of five and allowed food and water ad libitum. A 12:12 hour light/d...
- Use
- A total of 80 nine-week-old male C57BL/6J mice from Charles River (Sulzfeld, Germany) were used for this study. Animals were assigned to cages with the help of randomly generated numbers to reduce possible litter effects. Mice were housed in groups of five and allowed food and water ad libitum. A 12:12 hour light/d...
Drug Injection protocol
Solutions of paclitaxel (Sigma-Aldrich, Taufkirchen, Germany), cisplatin (Sigma-Aldrich, Taufkirchen, Germany), vincristine sulfate (Tocris, Bristol, UK) and bortezomib (Selleck Chemicals, Houston, Texas, USA) were prepared before each treatment. All substances were injected intraperitoneally (i.p.); animals in the...
- Use
- Solutions of paclitaxel (Sigma-Aldrich, Taufkirchen, Germany), cisplatin (Sigma-Aldrich, Taufkirchen, Germany), vincristine sulfate (Tocris, Bristol, UK) and bortezomib (Selleck Chemicals, Houston, Texas, USA) were prepared before each treatment. All substances were injected intraperitoneally (i.p.); animals in the...
Behavior analysis
Behavior analysis was conducted on consecutive days: On day one mice were tested with the catwalk apparatus, allowed to rest and then tested on the rotarod. On day two, the mechanical withdrawal threshold was measured with the von Frey method, followed by electrophysiological tests in anesthesia. Animals were also t...
- Use
- Behavior analysis was conducted on consecutive days: On day one mice were tested with the catwalk apparatus, allowed to rest and then tested on the rotarod. On day two, the mechanical withdrawal threshold was measured with the von Frey method, followed by electrophysiological tests in anesthesia. Animals were also t...
Open Field
To determine general wellbeing of mice after injection of cytostatic agents, locomotion was evaluated in the open field test. Mice were placed in a box (120 cm × 120 cm × 40 cm) and monitored for 10 min at the early and late time points listed in. An automated video tracking system...
- Use
- To determine general wellbeing of mice after injection of cytostatic agents, locomotion was evaluated in the open field test. Mice were placed in a box (120 cm × 120 cm × 40 cm) and monitored for 10 min at the early and late time points listed in. An automated video tracking system...
Rotarod
Motor coordination was assessed using the rotarod performance test as described before. A baseline value was recorded on the last day of training by measuring the initial latency to fall off the rod. Subsequently testing was done at three time points to determine if motor coordination was affected (early, middle, a...
- Use
- Motor coordination was assessed using the rotarod performance test as described before. A baseline value was recorded on the last day of training by measuring the initial latency to fall off the rod. Subsequently testing was done at three time points to determine if motor coordination was affected (early, middle, a...
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Behavioral alterations
Automated gait analysis with the catwalk technique has been used previously to detect gait alterations induced by dose-dense paclitaxel treatment. In particular, an increase in the swing phase was observed as well as a decrease in the stance phase and duty cycle. The parameter duty cycle expresses the stance phase as a percentage of the step cycle (stance + swing phase). Given the limited experience with this technique in CIPN animal models, we were interested as to whether the drugs and treatment schedules used in this study would also lead to detectable gait alterations in mice. In light of previous publications including human studies, a symmetrical, distal and length-dependent neuropathy was to be expected. Fore- and hindpaws were therefore analyzed separately, while parameters for the right and left side were evaluated together.
Electrophysiological measurements
Previous studies noted a decreased NCV, indicative of demyelination, in animals treated with high doses of cisplatin, paclitaxel and bortezomib. We observed a significant decrease of the NCV for paclitaxel at the middle time point (-8% ± 2% compared to baseline, [10], p < 0.05; ), which had normalized 7 days later at the late time point. Cisplatin caused a more sustained reduction of the NCV which was observed at the middle (-8% ± 1%, [9], p < 0.05) and late time point (-9% ± 3%, [9], p < 0.05; ). No significant alterations were observed for vincristine and bortezomib. In summary, all of the tested drugs led to a sustained reduction of SNAP amplitudes, suggesting a predominantly axonal pathology. Only paclitaxel and cisplatin conferred a comparatively small but significant reduction of the NCV, and in the case of paclitaxel the NCV recovered quickly a...
Methods
A total of 80 nine-week-old male C57BL/6J mice from Charles River (Sulzfeld, Germany) were used for this study. Animals were assigned to cages with the help of randomly generated numbers to reduce possible litter effects. Mice were housed in groups of five and allowed food and water ad libitum. A 12:12 hour light/dark cycle (7 am-7 pm) was maintained and behavioral testing was conducted between 10 am and 6 pm. Injections administered on the same day as behavior tests, were given only after all testing had been completed. The general well-being of the mice was assessed each day and weight was recorded before and 24h after drug administration as well as before the Catwalk test. For each drug a corresponding control group treated with the appropriate vehicle was included. Baseline weights were comparable: 22.7 g ± 0.4 g (paclitaxel group, [10]), 22.4 g ±...
Drug Injection protocol
Paclitaxel was dissolved in Cremophor EL:Ethanol (1:1) with a concentration of 2 mg/ml and diluted in sterile 0.9% NaCl solution to a final concentration of 0.1 mg/ml, a dose previously used in rat and mouse studies. Cisplatin was dissolved in sterile 0.9% NaCl solution with a concentration of 0.23 mg/ml and administered according to the protocol published by. Vincristine sulfate was dissolved in sterile 0.9% NaCl solution with a concentration of 20 µg/ml; the treatment protocol and dose were adapted from. Bortezomib was dissolved in dimethylsulfoxide (DMSO) with a concentration of 1 mg/ml and diluted in sterile 0.9% NaCl solution to a final concentration of 40 µg/ml; the protocol was modified from.
Electrophysiology
NCV and SNAP amplitudes of the caudal nerve were recorded with a Dantec Keypoint electromyography system (Natus Medical Inc., Planegg, Germany) in animals anesthetized with isoflurane (1.3% to 1.5% in O 2 ). The protocol was adapted from. At the base of the tail stimulation electrodes were placed with the recording electrodes five cm distal and a ground electrode in-between the stimulation and recording electrodes. 50 stimuli (0.1 ms) with supramaximal stimulation intensity and a frequency of 1 Hz were averaged to measure SNAP and NCV at three timepoints (baseline, middle, and late).
Measurement outputs
What raw and processed outputs should exist?
After completion of behavioral and electrophysiological analysis, animals were killed and sciatic nerves were dissected, fixed, stained for myelin and cut as semi-thin sections...
- 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
Automated gait analysis with the catwalk technique has been used previously to detect gait alterations induced by dose-dense paclitaxel treatment. In particular, an increase in...
- 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 electrophysiological evidence for axonal damage, especially at the late time point, we were interested in whether changes in axon density or the distribution of axon d...
- 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 conclusion, these findings suggest substance specific patterns of axonal damage. While paclitaxel and vincristine affect large more than small myelinated fibers, the opposite...
- 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
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
One hallmark of murine CIPN is mechanical allodynia (reviewed by ).
from paperScoring or quantification
Quantify the primary readouts for this experiment: After completion of behavioral and electrophysiological analysis, animals were killed and sciatic nerves were dissected, fixed, stained for myelin and cut as semi-thin sections...; Automated gait analysis with the catwalk technique has been used previously to detect gait alterations induced by dose-dense paclitaxel treatment. In particular, an increase in...; Given the electrophysiological evidence for axonal damage, especially at the late time point, we were interested in whether changes in axon density or the distribution of axon d...; In conclusion, these findings suggest substance specific patterns of axonal damage. While paclitaxel and vincristine affect large more than small myelinated fibers, the opposite....
from paperStatistical comparison
One hallmark of murine CIPN is mechanical allodynia (reviewed by ). In line with previous reports, all treatments led to a marked reduction of the mechanical withdrawal threshol...; Compared to baseline, the stance phase of the hindpaws at the late timepoint in paclitaxel-, cisplatin- and vincristine-treated mice was significantly (p < 0.05) reduced by W...; To further characterize sensory neuropathy, we measured nerve conduction velocity (NCV) and sensory nerve action potential (SNAP) in the caudal nerve. Antidromic stimulation of...; Previous studies noted a decreased NCV, indicative of demyelination, in animals treated with high doses of cisplatin, paclitaxel and bortezomib. We observed a significant decre...
from paperReporting output
Report representative outputs alongside summary comparisons for After completion of behavioral and electrophysiological analysis, animals were killed and sciatic nerves were dissected, fixed, stained for myelin and cut as semi-thin sections..., Automated gait analysis with the catwalk technique has been used previously to detect gait alterations induced by dose-dense paclitaxel treatment. In particular, an increase in..., Given the electrophysiological evidence for axonal damage, especially at the late time point, we were interested in whether changes in axon density or the distribution of axon d..., In conclusion, these findings suggest substance specific patterns of axonal damage. While paclitaxel and vincristine affect large more than small myelinated fibers, the opposite....
inferred from protocolStructured statistical methods
One hallmark of murine CIPN is mechanical allodynia (reviewed by ). In line with previous reports, all treatments led to a marked reduction of the mechanical withdrawal threshol...; Compared to baseline, the stance phase of the hindpaws at the late timepoint in paclitaxel-, cisplatin- and vincristine-treated mice was significantly (p < 0.05) reduced by W...; To further characterize sensory neuropathy, we measured nerve conduction velocity (NCV) and sensory nerve action potential (SNAP) in the caudal nerve. Antidromic stimulation of...; Previous studies noted a decreased NCV, indicative of demyelination, in animals treated with high doses of cisplatin, paclitaxel and bortezomib. We observed a significant decre...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (5)
Automated gait analysis with the catwalk technique has been used previously to detect gait alterations induced by dose-dense paclitaxel treatment. In particular, an increase in the swing phase was observed as well as a decrease in the stance phase and duty cycle. The parameter duty cycle expresses the stance phase as a percentage of the step cycle (stance + swing phase). Given the limited experience with this technique in CIPN animal models, we were interested as to whether the drugs and treatment schedules used in this study would also lead to detectable gait alterations in mice. In light of previous publications including human studies, a symmetrical, distal and length-dependent neuropathy was to be expected. Fore- and hindpaws were therefore analyzed separately, while parameters for the right and left side were evaluated together.
Previous studies noted a decreased NCV, indicative of demyelination, in animals treated with high doses of cisplatin, paclitaxel and bortezomib. We observed a significant decrease of the NCV for paclitaxel at the middle time point (-8% ± 2% compared to baseline, [10], p < 0.05; ), which had normalized 7 days later at the late time point. Cisplatin caused a more sustained reduction of the NCV which was observed at the middle (-8% ± 1%, [9], p < 0.05) and late time point (-9% ± 3%, [9], p < 0.05; ). No significant alterations were observed for vincristine and bortezomib. In summary, all of the tested drugs led to a sustained reduction of SNAP amplitudes, suggesting a predominantly axonal pathology. Only paclitaxel and cisplatin conferred a comparatively small but significant reduction of the NCV, and in the case of paclitaxel the NCV recovered quickly after the last injection.
A total of 80 nine-week-old male C57BL/6J mice from Charles River (Sulzfeld, Germany) were used for this study. Animals were assigned to cages with the help of randomly generated numbers to reduce possible litter effects. Mice were housed in groups of five and allowed food and water ad libitum. A 12:12 hour light/dark cycle (7 am-7 pm) was maintained and behavioral testing was conducted between 10 am and 6 pm. Injections administered on the same day as behavior tests, were given only after all testing had been completed. The general well-being of the mice was assessed each day and weight was recorded before and 24h after drug administration as well as before the Catwalk test. For each drug a corresponding control group treated with the appropriate vehicle was included. Baseline weights were comparable: 22.7 g ± 0.4 g (paclitaxel group, [10]), 22.4 g ± 0.4 g (paclitaxel control group, [10]), 22 g ± 0.2 g (cisplatin group, [10]), 22.7 g ± 0.2 g (cisplatin control group, [10]), 22.5 g ± 0.2 g (vincristine group, [10]), 21.8 g ± 0.2 g (vincristine control group, [10]), 21.6&#...
Paclitaxel was dissolved in Cremophor EL:Ethanol (1:1) with a concentration of 2 mg/ml and diluted in sterile 0.9% NaCl solution to a final concentration of 0.1 mg/ml, a dose previously used in rat and mouse studies. Cisplatin was dissolved in sterile 0.9% NaCl solution with a concentration of 0.23 mg/ml and administered according to the protocol published by. Vincristine sulfate was dissolved in sterile 0.9% NaCl solution with a concentration of 20 µg/ml; the treatment protocol and dose were adapted from. Bortezomib was dissolved in dimethylsulfoxide (DMSO) with a concentration of 1 mg/ml and diluted in sterile 0.9% NaCl solution to a final concentration of 40 µg/ml; the protocol was modified from.
NCV and SNAP amplitudes of the caudal nerve were recorded with a Dantec Keypoint electromyography system (Natus Medical Inc., Planegg, Germany) in animals anesthetized with isoflurane (1.3% to 1.5% in O 2 ). The protocol was adapted from. At the base of the tail stimulation electrodes were placed with the recording electrodes five cm distal and a ground electrode in-between the stimulation and recording electrodes. 50 stimuli (0.1 ms) with supramaximal stimulation intensity and a frequency of 1 Hz were averaged to measure SNAP and NCV at three timepoints (baseline, middle, and late).
Machine-readable layer
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