Cell culture and treatments
CT26 (mouse colon carcinoma) cells, purchased from the ATCC (CRL-2638), were grown in Dulbecco’s Modified Eagle Medium (Gibco, Sigma-Aldrich) with 10% FBS (Gibco, Sigma-Aldrich), penicillin/streptomycin, and L-glutamine supplementation. N2a (mouse brain neuroblastoma) cells, purchased from the ATCC (CCL-131), were grown in Eagle Minimum Essential Medium supplemented with 10% FBS, penicillin/streptomycin, 2% sodium pyruvate, 1% ciprofloxacin, non-essential amino acids, and L-glutamine supplementation.
Animal models and treatments
Five-week-old male BALB/cJRj mice were purchased from Janvier Laboratory (Le Genest Saint Isle, France). All mice were housed in ventilated cages (n = 7 to 10 animals per cage) with sterile food and water ad libitum and were exposed to a standard light cycle of 12 h on and 12 h off. All experiments were performed in accordance with European and French institutional guidelines (Directive 2010/63/EU, Ethics committee CEEA 34 – APAFIS authorization #8394).
Model of oxaliplatin-induced peripheral neuropathies
Both models of oxaliplatin-induced peripheral neuropathies, namely, the acute form and the chronic form were induced by protocols with timeframes that were analogous to the clinical protocols which results in the induction of either of these forms in patients, and were previously used in mice.
The acute form of oxaliplatin peripheral neuropathy was induced in mice (n = 7 per group) by daily low doses intraperitoneal injections of oxaliplatin (3 mg/kg diluted in phosphate-buffered saline (PBS 1X)) for 5 days followed by 5 days of rest and another 5 days of oxaliplatin at the same dosage, as previously described [16, 18, 79]. Benztropine (10 mg/kg diluted in PBS) was administered as daily intraperitoneal injections 6 h after oxaliplatin injection, 5 days a week during the 2 cycles of oxaliplatin injection and the rest period.
The chronic form of oxaliplatin peripheral neuropathy mice (n = 25 per group for all the experiments) was induced by intraperitoneal injections of oxaliplatin (10 mg/kg diluted in PBS) once a week each Mondays for 6–8 weeks as previously described [16, 18, 89]. Benztropine (10 mg/kg diluted in PBS) was administered as daily intraperitoneal injections 6 h after oxaliplatin injection, 5 days a week for 6–8 weeks.
Model of streptozotocin-induced diabetic peripheral neuropathies
Poor survival over the course of preliminary tests was observed with a 3-h food withdrawal before and after a single dose of 200 mg/kg of Streptozotocin (STZ) . This dose was therefore lowered to 180 mg/kg (dissolved in 10 mmol/L sodium citrate buffer, pH 4.5) with 3-h food fasting before and after the injection (n = 8 per group). STZ was diluted to allow a final volume of 0.1 ml/10 g body weight for intraperitoneal injections. One week after STZ injection, diabetes was confirmed by blood glucose levels using a Cobas 8000 modular analyzer (Roche) in blood samples taken under isoflurane 2,5% (Isovet, #ISO005, Centravet, Plancoët, France) from mice retro-orbital sinus. Mice with blood glucose levels ≥12 mM were considered diabetic and included in the study .
Prior to baseline measurements for every behavioral test, mice underwent 2 weeks of acclimation sessions with the specific training protocols to familiarize them with the experimental environments and testing procedures, without probing with plastic fibers nor temperature setting. Before each behavioral test, mice were allowed to acclimate to the experimental room for 10 min in their home cages. Mice were only tested once on any given test day to avoid any possible stress, anesthetic or tissue damage effects that could result from repeated exposure to the cold surface.
In vivo cold hyperalgesia
Mice from the acute model of oxaliplatin-induced peripheral neuropathies were submitted to weekly tests for cold hyperalgesia. Mice were put on a cold/hot plate (Ugo Basile, Comerio, Italy) set at the temperature of 2 °C ± 0.2 °C [16, 18, 79]. The total number of brisk lifts from either hind paw or jumps, considered as a painful response to cold, was counted simultaneously by two observers to ensure accuracy and averaged for the two observers’ counts. A maximal cut-off time of 5 min was imposed to prevent tissue damage. Results are expressed as the mean ± SEM of the observers’ counts, with 7 different mice under each condition.
In vivo cold hypoesthesia
Mice from the chronic model of oxaliplatin-induced peripheral neuropathies were submitted to weekly tests for cold hypoesthesia which was evaluated using temperature settings previously described [16, 18]. Briefly, mice were put on the cold/hot plate set at 4 °C ± 0.2 °C for 5 min. Similar, to the test assessing cold hyperalgesia, the total number of brisk lifts from either hind paw or jumps was counted simultaneously by two observers to ensure accuracy and averaged for the two observers’ counts. Results are expressed as the mean ± SEM of the observers’ counts, with 8 different mice under each condition.
In vivo tactile hypoesthesia
Mice from both the chronic oxaliplatin-induced and the diabetic models underwent weekly tests for mechanical allodynia with the von Frey method , a standardized protocol assessing tactile sensitivity . A set of 20 monofilaments based on the Semmes Weinstein monofilament set was used (Model: Bio-VF-M, Bioseb, USA/Canada). The Semmes Weinstein set of monofilaments provides an approximate logarithmic scale of actual force, and a linear scale of perceived intensity. Briefly, mice were put on a mesh grid, an enclosed by a clear plexiglass barrier with a top cover and left to calm down for 5 min. After the settling phase, mice are motionless allowing the experimenter to touch their hind paws with a flexible plastic fiber of a fixed diameter. The fiber is pressed through the mesh against the plantar surface at a right angle. The force of application increases as long as the investigator pushes the probe and until the fiber bends. The scale of force used ranged from 0.008 to 1.400 g. The threshold to perception of probing was asserted to the movement of pulling back of either hind paw of 8 different mice under each condition.
In vivo hot hyperesthesia
Mice from the diabetic model of peripheral neuropathies underwent a weekly assessment of thermal hyperalgesia according to a previously published study protocol . The same equipment as for cold sensitivity was used for this experiment. The plate was set at 55 °C ± 0.2 °C. Latency to hind paw licking or jumping was recorded simultaneously by two observers with a timer and time to response was averaged for the two counts, for 8 different mice under each condition. To prevent tissue damage a cutoff time of 30 s was implemented.
In vivo electrophysiological exploration of neuromuscular and sensory excitability
The sensory and/or neuromuscular excitability was assessed in vivo on mice under isoflurane (AErrane®) anesthesia by minimally invasive electrophysiological methods using the Qtrac© software (Prof. H. Bostock, Institute of Neurology, London, England), as previously described . Briefly, an anaesthetized mouse (n = 10) was placed on a heating pad to maintain body temperature (from 36.02 ± 0.06 to 36.13 ± 0.04, n = 85) throughout the experiments to avoid non-specific modifications of excitability variables.
For neuromuscular excitability exploration, electrical stimulation was delivered to the sciatic motor nerve by means of surface electrodes, and the compound muscle action potential (CMAP) was recorded using needle electrodes inserted into the plantar muscle. Each mouse was systematically submitted to one session of excitability measurements (TRONDE protocol), which consisted of five different excitability tests performed together: (C0) The stimulus-response relationship (i.e. the CMAP amplitude as a function of the intensity of a 1-ms stimulation) evaluated notably both the CMAP maximal amplitude and the stimulation intensity that had to be applied to evoke a CMAP of 50% maximal amplitude, giving information on the global neuromuscular excitability state. (C1) The current-threshold relationship evaluated the threshold changes at the end of 200-ms conditioning subthreshold depolarizing and hyperpolarizing currents ranging from 50 to 100% thresholds, giving information on axonal accommodation capacities to depolarizations and hyperpolarizations. (C2) The strength-duration relationship (i.e., the intensity in relation to the duration of a stimulus necessary to evoke a given amplitude of CMAP) evaluated the minimal intensity of infinitely long duration stimulation necessary to evoke a CMAP (rheobase) and the intensity duration of twice the rheobase stimulation necessary to evoke a CMAP (chronaxis), giving information on the axonal resting potential at the nodal membrane. (C3) The threshold electrotonus (i.e., the threshold changes during and after 100-ms conditioning subthreshold depolarizing and hyperpolarizing currents applied at ±40% thresholds) evaluated the electrotonic changes in membrane potential, giving also information on axonal accommodation capacities to depolarizations and hyperpolarizations. (C4) The recovery cycle (i.e., the excitability changes that occur following a CMAP) evaluated the refractory periods (during which membrane excitability is either nil or markedly decreased) followed by the supernormal and late subnormal periods (during which membrane excitability is increased and decreased, respectively). As a whole, more than 30 variables were determined from these excitability tests and analyzed. Most of them provide specific and complementary information on the density and functional state of ion channels, receptors and pumps, as well as on the passive membrane properties of the neuromuscular system [45, 46].
For sensory excitability exploration, the compound nerve action potential (CNAP) was recorded using needle electrodes inserted into the base of the tail, in response to stimulation of the caudal nerve applied at the distal part of the tail by means of surface electrodes. Each mouse was systematically and only submitted to the first session of excitability measurements (TRONDE protocol) to establish the stimulus-response relationship (i.e., the CNAP amplitude as a function of the intensity of a 1-ms stimulation) and thus, evaluate notably the CNAP maximal amplitude, the stimulation intensity that had to be applied to evoke a CNAP of 50% maximal amplitude and the latency measured from stimulation onset to peak amplitude, giving information on the global sensory excitability state.
In vitro electrophysiological exploration of sensory excitability
In vitro electrophysiological exploration of sensory excitability was performed by recording the action potential from primary cultures of mouse dorsal root ganglia (DRG) sensory neurons, using the patch-clamp technique. After being removed from the spinal cord of euthanized adult female Swiss mice (10–12 weeks of age and 28–32 g body weight, purchased from Janvier Elevage and housed at the CEA animal facility), DRG were placed in iced-Ham’s F-12 medium (Sigma-Aldrich, Saint-Quentin Fallavier, France) and enzymatically dissociated with collagenase type IA (2 mg/mL; Sigma-Aldrich) and dispase (5 mg/mL; Gibco, Thermo Fisher Scientific, Villebon-sur-Yvette, France). Neurons were then plated on 12-mm glass coverslips placed in a 24-wells plate coated with 10 μg/mL of poly-D-lysine and 100 μg/mL of murin laminin (Sigma-Aldrich). The cells were maintained in culture at 37 °C (in 95% air and 5% CO2) in Neurobasal A medium (Gibco) containing horse serum (5%; Gibco), penicillin/streptomycin (47.64 U/mL; Gibco), nerve growth factor (83.33 ng/mL; Sigma-Aldrich), N2 supplement (3.18x; Gibco), Dulbecco’s PBS (1X) w/o CaCl2 and MgCl2 (1.68%; Gibco), bovine serum albumin (16.83 μg/mL; Sigma-Aldrich), corticosteron (214.85 nM; Sigma-Aldrich), T3 hormone (56.06 nM; Sigma-Aldrich) and L-glutamine (1.90 mM; Sigma-Aldrich). Cytosine β-D-arabinofuranoside (2 μM; Sigma-Aldrich) was added to the culture medium, 24 h later, to stop astrocyte proliferation. Experiments were carried out within 2 to 6 days after cell plating. The day of their use, the neurons plated on coverslips were transferred, for a minimum of 30 min at 37 °C prior to patch-clamp recordings, in 35-mm Petri dishes filled with a standard physiological medium of the following composition (in mM): NaCl 134, KCl 3, CaCl2 1, MgCl2 1, D-glucose 20, and HEPES 20 (pH 7.35, adjusted with NaOH), and then in the recording bath filled with the standard physiological medium.
Whole-cell patch-clamp experiments were performed under current-clamp condition, by using a MultiClamp 700B integrating patch-clamp amplifier and the pClamp10.6 software (Molecular Devices, Sunnyvale, CA, USA), as previously described . The signals, acquired at a 4-kHz sample rate, were filtered at 2 kHz with a low-pass Bessel filter and digitized with the aid of a computer equipped with an analog-to-digital converter (Digidata-1440A model; Molecular Devices). The patch-clamp pipettes were filled with a medium composed of (in mM): KCl 134, NaCl 10, MgCl2 2, EGTA 2, ATP-Na2 4, and HEPES 20 (pH 7.32, adjusted with KOH), and had 2.71 ± 0.25 MΩ resistance (n = 18) in the standard physiological medium. A fast superfusion system allowed changing the solution [standard physiological medium without or with oxaliplatin (25–50 μM) alone or oxaliplatin (25–50 μM) plus benztropine (10 μM)] around the recorded cell within a few seconds. The experiments were carried out at constant room temperature (22 °C). Action potentials were elicited, at a frequency of 0.5 Hz, by 100-ms current test-pulses of − 0.2 to 1 nA (in 0.1-nA increments) applied 200 ms after 200-ms current pre-pulses of − 0.1 nA (to check the membrane passive properties of neurons, mainly membrane capacitance).
Ex vivo confocal microscopy morphological study of sciatic nerves
The experiments were carried out on single myelinated axons isolated from the sciatic nerves of euthanized mice, as previously detailed . Briefly, sciatic nerve sections (n = 4 mice in each group) of about 2 cm in length were removed from their sheaths, dissected, and fixed for 1 h in PBS 1X with 2% paraformaldehyde, then rinsed three times with PBS. Sciatic nerves were deposited on microscope slides, myelinated axons were gently teased apart from the main trunk, and preparations were kept at − 20 °C until use. Just before the experiments, sciatic nerves were rehydrated for about 1 h with a standard physiological solution containing (in mM): NaCl 154, KCl 5, CaCl2 2, MgCl2 1, glucose 11, and HEPES 5 (pH 7.4, adjusted with NaOH. Preparations were then exposed for 30 min to the fluorescent dye FM1–43 (Molecular Probes) dissolved in a standard physiological solution to stain the plasma membranes of the myelinated axons, and washed with dye-free solution before imaging. A Zeiss LSM 510 META (Carl Zeiss) multiphoton scanning confocal microscope, mounted on an upright microscope and controlled with the manufacturer’s software and workstation, was used for optical sectioning of myelinated axons and subsequent 3D high-resolution digital reconstruction of their structure. Images were collected using a 63x oil-immersion objective with a 1.40 numerical aperture (Zeiss Plan-Apochromat); following excitation of FM1–43 with the 488 nm wavelength line of an Argon ion laser, and then digitized at 12-bit resolution into a 512 × 512 pixel array. Images were then analyzed using the ImageJ software (National Institutes of Health - NIH). Quantification of morphometric parameters of myelinated axons were performed by measuring the internodal diameter, nodal diameter (D) and nodal length (L). Assuming the simplest geometry in which a node of Ranvier approaches a cylinder, the nodal volume (V) was then determined as V = μL(D/2)2.
Ex vivo electronic microscopy morphological study of sciatic nerves
Mice were anesthetized by intraperitoneal injections of 100 mg/kg ketamine and 10 mg/kg xylazine and then intracardially perfused with first, 0.1 M PBS, pH 7.4, for 8 min and then 4% paraformaldehyde, 2.5% glutaraldehyde, and 0.1 M PBS, pH 7.4. Mice were euthanized after complete rigidity of the lower limbs and liver (endpoint of the perfusion). Tissues were dissected 24 h later and immersed in the fixative solution at 4 °C for 72 h, washed in PBS, post-fixed in 2% osmium tetroxide, dehydrated in graded ethanol, and embedded in epoxy resin. Ultrathin sections (50–90 nm) were cut on an ultramicrotome (8800 Ultrotome III; LKB Bromma) and collected on 300-mesh nickel grids. Staining was performed on drops of 4% aqueous uranyl acetate, followed by Reynolds lead citrate. Ultrastructural analyses were performed in a JEOL JEM-1011 electron microscope and digitalized with DigitalMicrograph software. G-ratios and axon diameters mice (n = 2 to 3 mice in each group) were calculated with the ImageJ software (National Institutes of Health) and the plugin g-ratio version 3.2 (Plug-in and source code available online at http://gratio.efil.de) according to previously published material . Diameters were calculated from enclosed areas, considering first, the diameter of the axon without the myelin and dividing it by the diameter of the axon plus the myelin sheaths surrounding it. The plugin allows for semi-automated analysis of randomly selected fibers with both diameters being considered and automatically processed through the algorithm to calculate the g-ratio. A minimum of 500 randomly selected axons were analyzed per experimental group, with at least 3 mice per group.
Ex vivo study of cutaneous nerve fiber density
Sections of skin from the hind paws of 8 mice per group were preserved in buffered 4% formol (VWR Chemicals, Labonord SAS, France) and immersed in successive baths of increasing concentrations of ethanol (50, 70, 90, 100%). Samples were then immersed in histosol before being fixed in paraffin, kept at 4 °C and cut into 6 μm-thick slices. Samples were then unwaxed in three successive baths of histosol for 3 min and rehydrated in ethanol baths for 1 min each, starting with two baths of pure ethanol followed with baths of 90, 70 and 30% ethanol and two baths of H2O. Slides were then washed in PBS for 2 min before being immersed in citrate buffer at 95 °C for 10 min. Samples were washed in 3 baths of PBS of 5 min each. Samples were then permeabilized (PBS, 0.25% Triton X-100) for 10 min before being rinsed in PBS in 3 baths of 5 min each and immersed in a blocking solution (PBS, 10% goat serum, 1% BSA) for at least 180 min. Samples were further incubated at 4 °C overnight in the primary antibody (Abcam, ab10404, rabbit polyclonal to PGP9.5, 1:1000) before being rinsed in PBS in 3 baths of 5 min each before incubating in the secondary antibody (Sigma, F9887, anti-rabbit IgG FITC-conjugate, 1:1000) at room temperature in the dark. Prior to mounting (Thermo Scientific, Shandon Immuno-Mount), slides were washed with PBS in 3 baths of 5 min each. Images were collected using a Nikon Eclipse 80i microscope with a Nikon PlanFluor 100x/1.30 oil-immersion DIC H/N2 objective.
Ex vivo study of myelin protein content in sciatic nerves
Nerves were extracted in lysis buffer containing a cocktail of proteinase and phosphatase inhibitors using a glass homogenizer. Protein lysate samples (40 g) were resolved on 10% SDS-PAGE gels and transferred by electrophoresis to nitrocellulose membranes. The membranes were blocked for non-specific binding sites at room temperature in TBS buffer containing 0.1% Tween 20 and 5% nonfat dried milk for 1 h. The membranes were then incubated overnight at 4 °C with a primary antibody against MBP (1:500; Merck). Subsequently, the membranes were incubated with an anti-rabbit IgG, horseradish peroxidase linked whole antibody from donkey (1:1000; GE Healthcare Life Sciences NA934, Little Chalfont, UK) and subjected to ECL reagent treatment. After film exposure, all membranes were washed and then incubated with a 1:50,000 dilution of mouse monoclonal anti-actin-peroxidase antibody (Sigma Aldrich; Saint Louis, Missouri, USA) as previously described .The images were captured using a CCD camera (LAS3000 from Fujifilm), and the bands were quantified using MultiGauge software from Fujifilm.
Ex vivo study of systemic inflammatory markers
Blood samples were taken under isoflurane from the retro-orbital sinus immediately before sacrifice (n = 8 per group), centrifuged at 10.000 rpm and 2 °C. Sera were diluted (1:4) in ELISA/ELISPOT diluent 1X before being distributed on ELISA 96-well plates specific of IL-6 and Tumor necrosis factor alpha (TNF-α; Mouse IL-6 ELISA Ready-SET-Go!® and Mouse TNF ELISA Ready-SET-Go!® - eBioscience, San Diego, CA, USA). Concentrations were calculated from a standard curve according to the manufacturer’s protocol.
In vivo study of antitumor activity of benztropine upon association with chemotherapy
A total of 1.106 viable CT26 cells, as determined by trypan blue staining, and resuspended in DMEM were injected subcutaneously into the back of the mice. When tumors reached a mean size of 200 to 500 mm3, animals were randomized and received a single weekly injection of either oxaliplatin (10 mg/kg) or vehicle. Concomitantly, mice received vehicle or benztropine (10 mg/kg) every day. Following randomization, tumor size was measured with a numeric caliper twice a week for 3 weeks. In order to comply with ethical guidelines, tumor growth experiments were stopped 3 weeks after the first oxaliplatin injection. Tumor volume was calculated as follows: TV (mm3) = (L x W2)/2, where L is the longest and W the shortest radius of the tumor in millimeters. Results are expressed as mean ± SD of tumor volumes (n = 7 in each group).
Viability and ROS assays
All cells (2 × 104 per well) were seeded in 96-well plates (Sigma-Aldrich, Saint-Quentin Fallavier France) and incubated for 24 h with 7.5 to 30 μM of benztropine (Sigma-Aldrich, Saint-Quentin Fallavier France) and treated with 0 to 100 μM of oxaliplatin (Accord Healthcare Limited, Lille, France). Cell viability was assessed by a crystal violet assay, and results are expressed as the mean percentage of viable cells ± SEM versus cells not exposed to oxaliplatin (100% viability). Cellular production of ROS and reduced glutathione (GSH) were assessed by spectrofluorimetry with 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA, #6883 Sigma-Aldrich, Saint-Quentin Fallavier France) and monochlorobimane (#69899 Sigma-Aldrich), respectively .
Statistical analysis was performed using GraphPad Prism 5. Artwork, was also created using GraphPad Prism 5, except for electrophysiological study artwork which was created using the Qtrac© software. Differences between values were tested using the unpaired Student’s t-test, two-way ANOVA, or the nonparametric Mann-Whitney U test, depending on the equality of variances estimated using Lilliefors test. They were considered significant when p < 0.05, p values being denoted as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; NS: non-significant.