Sodium heparin

Twenty-four hours
before the experiment,
mice were anesthetized with isoflurane gas (Vetflurane, Virbac, France)
allowing the shaving of their hind limbs after a 3 min-long application
of a commercial depilatory cream. Then, mice were returned to their
cages to allow recovery and complete anesthesia washout. At the day
of experimentation, mice received intraperitoneal (i.p.) injection
of oxime at 100 μmol/kg by analogy with the previous studies
conducted in our department. At various times (0, 2, 5, 10, 15, 30,
60, and 180 min after oxime injection), the saphenous vein was drilled
with a needle, and approximately 20 μL of blood were collected
with a heparinized capillary tube and put in a collection tube containing
2 μL of sodium heparin (Choay, Sanofi, France). Plasma was next
isolated from erythrocytes by centrifugation at 4 °C, 3000g for 10 min. Plasma samples were then heated 30 min at
56 °C and treated as previously described to obtain the percentage
of reactivated enzyme (%E_react) with a one-compartment
model. T_max corresponds to the time when
the curve reaches the peak of %E_react. The areas under
the percentage of the reactivation curve (AUC) and the first moment
curve (AUMC) were calculated using the trapezoidal rule. Mean residence
time (MRT) was calculated as the ratio of AUMC to AUC.⁴⁷

Blood was collected from the tip of the tail (sectioned at its soft nonbone level with scissors) using 20-µL heparinized capillaries (Minicap Capillary Pipette, Hirschmann Instruments, ref 11772533). Blood was recovered 1, 2, 4, and 7 hours after p.o. administration of amitriptyline or THN101. Immediately after collection, the blood was mixed with 10 µL of 5000 IU/mL sodium heparin (Sanofi, France) and stored at −80°C. After the last blood collection, mice were anesthetized with ketamine/xylazine (100/10 mg/kg) and killed by cardiac exsanguination. The blood collected in vacuumed lithium-heparinized tubes (BD Vacutainer LH PST II REF 367374) was centrifuged at 425g for 10 minutes at 4°C, and plasma was frozen at −80°C. In parallel, the brain and spinal cord were recovered in Eppendorf tubes on dry ice and stored at −80°C.