All surgical and experimental procedures were approved by the institutional animal care and use committee. Female Sprague-Dawley rats (210–290 g) were used for the studies.
Animal Preparation for MRI
All rats were initially anesthetized with 3% isoflurane and an intraperitoneal (i.p.) dose of methohexital sodium (50 mg/kg) and were also pretreated with glycopyrolate (0.04 mg/kg i.p.). After oral endotracheal intubation, mechanical ventilation was initiated (Harvard Apparatus, Inspira ASV). Anesthesia was maintained with 1.5%–2% isoflurane delivered in a 1:1 air/O2 gas mixture. The femoral vein and a tail vein were catheterized for fluid and drug administration and the femoral artery for arterial blood pressure monitoring and collection of blood gas measurements during the study.
MRI and Forepaw Stimulation Protocol
All imaging was performed on a superconducting 9.4T/210 horizontal bore magnet (Magnex) controlled by an ADVANCE console (Bruker) using a 30-mm diameter surface radiofrequency coil secured above the head of the rat. All vital signs were continuously monitored during the experiment (SAM PC monitor, SA Instruments, Inc). Needle electrodes were inserted under the skin of each forepaw, between digits 2 and 3 and between 4 and 5. After positioning and insertion of needle electrodes, the anesthesia was switched from isoflurane to α-chloralose (Sigma); α-chloralose was administered first as an IV bolus of 40 mg/kg over a 10–15 min time period followed by a continuous infusion of 25 mg · kg−1 · h−1. Two to three trials of forepaw stimulations were administered to assure that the anesthetic plane was adequate. If the forepaw stimulation did not induce changes in mean arterial blood pressure (MABP), heart rate or the respiratory waveforms, the anesthetic depth was considered adequate. Muscle paralysis with vecuronium (0.01 mg/kg) was then achieved.
For fMRI a single-shot echo-planar sequence was used with the following parameters: TR = 1500; TE = 30 ms; effective bandwidth = 227272 Hz, field of view = 2.56 × 2.56 cm2; 64 × 64 matrix with a resulting in-plane resolution of 400 μm; 8 axial 1.4 mm-thick slices spaced 0.15 mm apart. Each scan was acquired in 3 s. To more accurately identify the anatomical location of the functional activation maps, higher resolution T2-weighted spin-echo images were acquired with identical spatial geometry using a RARE pulse-sequence.
The pulse train forepaw stimulations were generated using a commercial current stimulator (Isolated Pulse Stimulator 2100, A-M Systems). Negative short rectangular current pulses of 0.3 ms duration were applied at a frequency of 3 Hz to either right or left forepaw. Preliminary data from the literature indicate that a 2 mA forepaw electrical stimulus seems to be innocuous (i.e., brain fMRI activation is limited to the somatosensory cortex and does not include other pain-related brain regions), whereas stronger stimulation currents are not. We therefore divided the animals into three groups based on the stimulation current used: Group 1 (n = 4) 2 mA; Group 2 (n = 2) 4 mA; and Group 3 (n = 3) 8 mA. The 2 mA paradigm consisted of 23 scans acquired during rest, 10 scans acquired during stimulation (total stimulation time 30 s), and followed by a poststimulation rest period of 30 scans (total acquisition time 63 × 3 s = 3.15 min). To minimize painful exposures to the higher stimulation currents, we shortened the stimulation time for the 4 and 8 mA trials. The 4 mA paradigm consisted of 13 scans acquired during rest, 3 scans during stimulation (total stimulation time 9 s), and followed by a poststimulation rest period of 20 scans (total acquisition time = 36 × 3 s = 1.8 min). The 8 mA paradigm consisted of 13 prestimulation scans, 1 scan during stimulation (total stimulation time 3 s) and 22 scans acquired after stimulation (total acquisition time: 36 × 3 s = 1.8 min). For each of the stimulation current paradigms, the BOLD signal amplitude as well as the activation map distribution acquired during control conditions were established before LIDO administration. The baseline was obtained by repeating the stimulation paradigm (i.e., 2 mA, 4 mA or 8 mA) 4 times at an interval of 6–10 min (). After the baseline acquisitions, the animals were allowed to rest for approximately 20 min before administration of the first of the 3 escalating doses of LIDO (see below). The entire fMRI experiment required approximately 3.5 h.
Figure 1 Outline of experimental stimulation paradigm. The rat was allowed to rest 6–10 min between each forepaw simulation trial. For the 2 mA experiment the stimulation time was 30 s. For the 4 mA and 8 mA currents the stimulation time was 9 s and 3 (more ...)
IV LIDO Administration
In clinical studies using LIDO for treatment of neuropathic pain or acute nonneuropathic pain, LIDO was administered IV to achieve plasma levels between 1 to 5 μ
Thus, previous animal studies with systemic LIDO have used IV computerized infusions to achieve similar LIDO plasma concentration ranges to mimic the clinical studies and to keep the LIDO in a nontoxic range.8,9
We did not use a continuous infusion of LIDO. However, we performed experiments to determine the plasma LIDO concentration over time in rats anesthetized and prepared as described for the imaging experiments. The rats were similarly exposed to the escalating doses of LIDO as shown in . Blood samples were collected at set time intervals via the femoral artery and placed in heparinized microcentrifuge tubes. The blood volume removed from the animals was replaced with an equal volume of blood containing heparin to avoid hemodynamic instability secondary to blood loss. The collected blood was centrifuged at 3000 rpm for 5–10 min, and the plasma was extracted and stored at −80°C until analysis. The blood sampling times were 5 and 35 min after the 1 mg/kg LIDO dose and 5, 15, 25, and 35 min after the 10 mg/kg LIDO dose (no samples were collected for the 4 mg/kg/dose).
LIDO and five major unconjugated metabolites were quantified using a specifically developed and validated liquid chromatography procedure. Reference standards for LIDO and xylidide (XYL) were purchased from Sigma Chemical Co. (St. Louis, MO) and Aldrich Chemical Co. (Milwaukee, WI), respectively. Metabolites monoethylglycinexylidide (MEGX), glycinexylidide, 4-hydroxyxylidide (4-OH-XYL), 3-hydroxymonoethylglycinexylidide, 3-hydroxylidocaine (3-OH-LIDO), and internal standard pipecoloxylidide (PPX) were generously supplied by AstraZeneca Pharmaceutical Co., (Sodertalje, Sweden). The extraction procedure involved a 0.2 mL sample of plasma (in 0.01 M HCl) and the addition of 25 μL (250 ng) PPX, 0.5 mL of 0.5 M NaOH, and 5.0 mL of ethyl acetate: methyl-tert-butyl ether (1:1). The mixture was shaken for 10 min at a slow speed on a tilt-platform rocker and then centrifuged at 2000 rpm for 15 min. The top organic layer was transferred to a conical centrifuge tube containing 150 μL of 0.1 M HCl. The contents were mixed for 10 min and then centrifuged for 10 min. The organic phase was aspirated to waste and the remaining acid layer evaporated to dryness using a vacuum centrifuge with moderate heat (approximately 45°C). The dried residue was reconstituted with 125 μL of mobile phase and transferred to the autosampler inserts for chromatography.
Separation of the extracted LIDO, its metabolites and internal standard was performed using an octadecylsilyl reversed phase column (Phenomenex Luna C-18, 3 μ, 150 × 4.6 mm, Torrance, CA) with a mobile phase consisting of 0.05 M monobasic potassium phosphate: acetonitrile (84:16) with the addition of 1 mL/L phosphoric acid (85%), 1.2 mL/L triethylamine, and 7 mL/L heptane sulfonate (20%). The eluted compounds were detected by ultraviolet absorbance at 210 nm. Using a flow rate of 1.5 mL/min with a column thermostated at 32°C, the retention times of the eluted compounds were: 4-OH-XYL, 3.2 min; 3-hydroxymonoethylglycinexylidide, 4.7 min; 3-OH-LIDO, 6.5 min; glycinexylidide, 13.7 min; XYL, 15.6 min; MEGX, 19.0 min; PPX, 26.0 min; LIDO, 28.5 min. Every set of samples was preceded with a 7-point calibration curve, which included the expected concentration range of samples, a blank, and three sets of quality controls. The limit of quantification for LIDO and the metabolites (except for 4-OH-XYL) was about 12.5 ng/mL.
shows the time course of the plasma LIDO concentration after 1 mg/kg and 10 mg/kg (other metabolites not shown). As can be seen, the plasma LIDO concentration is at the “therapeutic” target range of >1 μg/mL at the 5-, 15-, and 25-min time points after the 10 m/kg LIDO dose. For the 1 mg/kg dose, the plasma concentration is <1 μg/mL at both the 5-min and 35 min time point.
Figure 2 The time course of the average plasma lidocaine concentration after 1 mg/kg and 10 mg/kg in 3 rats. As can be seen, the plasma lidocaine concentration is at the “therapeutic” target range of >1 μg/mL at the 5-, 15-, and (more ...)
In all imaging experiments, the rats were exposed to 1 mg/kg, 4 mg/kg, and 10 mg/kg over the course of the study as illustrated in . Each dose of LIDO was administered IV over 3–5 min, which translated into approximately 0.5–0.1 mg/min. After each of the IV LIDO dose challenges, forepaw stimulation trials were conducted at 5-min, 15-min, 25-min, and 35-min (). One hour was allowed between each of the escalating doses of LIDO to assure that the LIDO plasma concentration was negligible before the next dose was administered.
MRI Data Analysis
The fMRI images were analyzed using Stimulate V6.01 (CMRR, University of MN). After a background masking, the intensity time course of each pixel during the scan was cross-correlated with a boxcar template according to the known stimulation profile. We used a correlation coefficient (r) ≥ 0.3 which corresponds to a P value of 0.01 to calculate the activation maps and detect the areas with a statistically significant BOLD signal. A 4-neighbor 2D clustering was performed to remove scattered false activation. For each rat, a sector-shape region of interest was delineated covering the entire forepaw area of the somatosensory cortex (which spanned 2–3 of the acquired slices and included both primary and secondary somatosensory cortex). Both the number of pixels (nominally, each pixel accounts for a volume of 0.4 × 0.4 × 1.5 × 0.24 mm3/pixel) and the average time course of the statistically significantly active pixels (pixels with r ≥ 0.3) within the region of interest were extracted for each trial, and used to represent the spatial and temporal profile of the BOLD response, respectively. Each time course was then normalized to its mean value during the prestimulation baseline and the peak value was extracted to assess the magnitude for each stimulation trial. All calculated and color-coded functional activity maps were overlaid on the echo-planar images and the anatomical location of activated pixels identified by comparing with the corresponding high resolution T2-weighted MRI. Intra-group differences in the baseline BOLD signal amplitude and magnitude of BOLD activation (number of pixels with statistically significant BOLD signal increases) were analyzed using an F-test. Statistical differences between the average baseline BOLD signal amplitude (average of the four individual stimulations before injection of LIDO) and amplitudes obtained after the LIDO administrations were analyzed using a paired t-test. Statistical differences between the cortical volume activated during baseline conditions (average of the 4 trials) was compared with cortical volumes activated 5-min (where the plasma LIDO concentration was highest) after each of the escalating doses of LIDO using a paired t-test.