Electrophysiological, behavioral and histological characterization of paclitaxel, cisplatin, vincristine and bortezomib-induced neuropathy in C57Bl/6 mice methods
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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).
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Gait recovery and locomotor function
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- Per-run gait capture with paw placement, timing, and stride features for each animal
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- Group comparisons of gait indices, stride metrics, or recovery curves
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Quantify the primary readouts for this experiment: Gait recovery and locomotor function.
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Report representative outputs alongside summary comparisons for Gait recovery and locomotor function.
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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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