With the approval of the Institutional Animal Experimental Ethic Committee of Sichuan University (Chengdu, Sichuan, China), adult (200-300 g) and natal (7-12 days) Sprague-Dawley rats were used in experiments in vivo and in vitro, respectively. All the animals were housed in standard conditions.
EI, containing pure liquid isoflurane and 30% Intralipid, was prepared by our laboratory (18). Briefly, 8% (v/v) EI 100 ml contained pure liquid isoflurane 8 ml. Isoflurane was supplied by the Abbott Pharmaceutical Co. Ltd. (Shanghai, China). 30% (w/v) Intralipid was from the Sino-Swed Pharmaceutical Corp. Ltd. (Wuxi, Jiangsu, China). F6 was purchased from Johnson Matthey Co. (Tianjin, China). Lidocaine was purchased from the Fortune Zhaohui Pharmaceutical Co. Ltd. (Shanghai, China).
Subarachnoid Anesthetic Effects of EI and Emulsified F6 in Rats
The subarachnoid catheter (PE-10, Scientific Commodities INC. Lake Havasu City, Arizona, US) was inserted between L5-L6 segments and about 2 cm advanced in the cephalic direction to place the distal end of the catheter at about lumbar intumescentia. The injection volume was 150 μl/kg. Two days before formal experiment, 1% (w/v) lidocaine was injected to confirm the place of the catheter.
Spinal anesthetic median effective concentration (EC50) of EI was measured. The rats with both successful motor and sensory blockades were defined as spinal anesthesia. Motor function was evaluated according to the score scale as 0-normal posture; 1-intact dorsiflexion of foot with impaired ability to splay toes when elevated by the tail; 2-toes and foot plantar flexed with no splaying ability; 3-loss of dorsiflexion, flexion of toes, and impairment of gait but could try to withdraw when noxious stimulus applied; 4-complete paralysis of hind limbs (19). Motor function blockade at score 4 was regarded as successful motor blockade, and no aversive response to tail-clamping stimulus (alligator clip, 10 A, type 85, length 2-1/8 inches, Newark Electronics, Dublin, CA) was considered as successful sensory blockade. Rats were randomly divided into 7 groups (N=8 for each concentration group) receiving EI at concentrations of 6.4% (0.53 mol/L), 5.1% (0.42 mol/L), 4.1% (0.34 mol/L), 3.3% (0.27 mol/L), 2.6% (0.24 mol/L), 2.1% (0.17 mol/L), and 1.7% (0.14 mol/L), respectively.
Our previous studies demonstrated that 8% (v/v) EI was similar to 1% (w/v) lidocaine in regional anesthesia (7, 8). In the present study, 8% (v/v) EI, 2% (v/v) emulsified F6, 1% (w/v) lidocaine and 30% (w/v) Intralipid were intrathecally administrated in rats (N=10 for each group). Onset time and duration of motor and sensory blockades were observed. Duration of motor blockade was the time from complete blockade (score 4) to recovery (under score 1). Sensory function was evaluated by tail-flick test (Tail-Flick Unit 7360; Ugo Basile, Comerio, Italy) and duration of sensory blockade was the time from complete blockade to tail-flick latency recover to baseline.
Preparation of Acute Isolated Spinal Neurons
The isolation of spinal neurons was described as our previous study (12) and rats (7-12 days) were used. Spinal cord at L3-L5 was quickly removed and placed in iced artificial cerebrospinal fluid (ASCF) containing (in mmol/L) 117 NaCl, 26 NaHCO3, 5 KCl, 1 NaH2PO4, 2.5 CaCl2, 3.4 MgCl2, 10 glucose, pH at 7.30, saturated with O2/CO2 (95%:5%). Then the spinal cord was cut into pieces about 1 mm3 and placed into a digestion solution containing (in mmol/L) 117 NaCl, 26 NaHCO3, 5 KCl, 1 NaH2PO4, 10 glucose, pH at 7.30, containing 1 mg/ml collagenase I, at 37 centidegree for about 70 minutes.
Spinal neurons with diameter at 15-30 μm were selected for recording. After spinal neurons settled to the bottom of 35 mm Petri dish, whole-cell patch clamp technique was applied to examine the inhibition by EI and emulsified F6 to peak sodium currents and their effects on activation and inactivation of channel gating. Voltage-gated sodium channel currents were measured with holding potential at -90 mV and depolarized from -60 to 40 mV (12). Inactivation of sodium channel gating was recorded by pre-pulse potential from -100 to 0 mV at holding potential of -90 mV and tested at -10 mV. The neurons were bathed with extracellular solution containing (in mmol/L) 95 NaCl, 26 NaHCO3, 5.6 KCl, 1 NaH2PO4, 0.1 CaCl2, 5 MgCl2, 20 TEA, 11 glucose, pH at 7.40. Electrode resistance was 3-5 MΩ. The pipette electrode solution contained (in mmol/L) 140 CsCl, 5 EGTA, 1 MgCl2, 10 HEPES, 3 MgATP, pH at 7.30. Currents were sampled at 10 kHz and filtered at 1-3 kHz by Axon 200B amplifier, digitized using a Digidata 1440A interface, and analyzed by pClamp 10.0 (Axon/Molecular Devices, Sunnyvale, CA). Isoflurane and F6 were diluted from 500 mmol/L emulsion with extracellular solution and applied locally by gravity (at the speed of 100-150 μl/minute through a perfusion pipette with diameter of 0.2 mm, positioned 30-50 μm away from the patched neurons). Data analysis and curve fitting were utilized by the Clampfit 10.0 (Axon/Molecular Devices, Sunnyvale, CA), Origin 8.0 (OriginLab Corp., Northampton, MA) and Excel 2007 (Microsoft).
The data were analyzed by SPSS 16.0 (SPSS Inc., Chicago, IL). Probit analysis was applied to determine EC50 of spinal anesthesia of EI. One-way analysis of variance (ANOVA) with S-N-K post hoc test was applied to compare the onset time and the duration of motor and sensory blockade among 1% lidocaine, 8% EI, 2% emulsified F6 and 30% Intralipid. Data were expressed as mean ± SD.
Concentration-dependent effect of EI and emulsified F6 on voltage-gated sodium channel currents was calculated by least squares fitting of data to the Hill equation as: Y=1/ (1+10((logIC50 - X) × h)), in which X is concentration of isoflurane or F6, Y is the inhibition effect, IC50 is median inhibitory concentration, and h is Hill slope. Activation curves of sodium channel were fitted to the Boltzmann equation as the form: G/Gmax=1/ (1+e(V1/2–V)/k), in which Gmax is maximum conductance, G/Gmax is normalized channel conductance, V1/2 is voltage activation of half-maximum, and k is slope factor. Channel conductance was calculated as: GNa=INa/ (Vt – Vr), where INa is peak current, Vt is test potential, and Vr is sodium channel reversal potential. Inactivation curves of sodium channels were also fitted to the Boltzmann equation. Data were expressed as mean ± SD. Statistical significance was assessed by paired or unpaired t test (20). In all cases, P<0.05 was considered as statistically significant.