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Allodynia and hyperalgesia suppression by a novel analgesic in experimental neuropathic pain
BBRC Biochemical and Biophysical Research Communications 350 (2006) 358–363 www.elsevier.com/locate/ybbrc
Allodynia and hyperalgesia suppression by a novel analgesic in experimental neuropathic pain Jian-Guo Cui *, Xiong Zhang, Yu-Hai Zhao, Chu Chen, Nicolas Bazan
*
Neuroscience Center of Excellence, Louisiana State University, Health Sciences Center, 2020 Gravier Street, New Orleans, LA 70112-2272, USA Received 25 August 2006 Available online 22 September 2006
Abstract SCP-1, n-[a-(benzisothiazol-3(2ho-ona,1-dioxide-2yl)-acetyl]-p-aminophenol (100 nmol), when intrathecally injected, suppressed tactile allodynia and thermal hyperalgesia in a rat neuropathic pain model. The tactile allodynia suppression lasted for at least 4 h and SCPM1 (100 nmol), the main metabolite of SCP-1, displayed similar suppression as SCP-1, but shorter latency, indicating SCP-M1 may be the bioactive component of SCP-1. Acetaminophen was less potent than SCP-1 and SCP-M1. To study mechanisms underlying SCP-1 action, we recorded voltage-gated Ca2+ channel currents in acutely isolated dorsal root ganglion neurons using the whole-cell patchclamp technique. SCP-1 and SCP-M1 inhibited non-L-type calcium channel currents up to 23.0 ± 2.3% and 23.1 ± 3.5%, respectively, at a depolarized pulse to 10 mV from a holding potential of 80 mV. Acetaminophen only induced 6.8 ± 1.0% inhibition. The results suggest SCP-1 possesses anti-nociceptive activity in the rat model involving calcium channel blocking properties. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Neuropathic pain; Tactile allodynia; Thermal hyperalgesia; Behavioral test; Calcium channel; Patch-clamp; SCP-1; Rat
Neuropathic pain is often associated with peripheral nerve injury. The nerve injury can induce neuroma at the end of injured fibers [1], cross-talk between different nerve fibers [2], and both peripheral and sympathetic sprouting from intact adjacent nerve fibers and in the DRG [3,4], resulting in spinal hyperexcitability. The hyperexcitability subsequently leads to pathological changes in genes, neurotransmitters, and cellular structures [5,6]. Excitatory neurotransmitters and related genes, such as glutamate, aspartate, substance P, and calcitonin-gene related peptide, are up-regulated in dorsal root ganglia and spinal dorsal horn, while inhibitory transmitters, such as c-aminobutyric acid (GABA), adenosine, are down-regulated [7–9]. Furthermore, inflammatory factors (macrophages, interleukins, tumor necrosis factor, and cyclooxygenase) and growth factors (nerve growth factor, epidermal growth factor (EGF), and brain-derived neurotrophic factor *
Corresponding authors. Fax: +1 504 599 0891. E-mail addresses: [email protected] (J.-G. Cui), [email protected] (N. Bazan). 0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.09.055
(BDNF)) are also involved in neuropathic pain development [10–13]. In addition, ion channels, in particular calcium channels and N-methyl-D-aspartate (NMDA receptors, which mediate synaptic excitability and control various signal transductions), are important mechanisms for neuropathic hypersensibility [14]. However, the mediating mechanisms for neuropathic pain development are still not clear. Understanding of neuropathic pain development and modulation has advanced tremendously since animal models of neuropathic pain came into use for the study of its mechanisms. However, the treatments for neuropathic pain are far behind. Due to complicated developmental mechanisms of neuropathic pain [15], opioids have little effect on such pain and possess a high risk of addiction and constipation [16]. Anti-convulsants may attenuate neuropathic pain on a small number of patients [17]. but the effectiveness is often unsatisfactory [18]. Acetaminophen (APAP) is an over-the-counter analgesic, which has been shown to inhibit cyclooxygenases in pain modulations, but it displays liver and kidney toxicity under a variety of circum-
J.-G. Cui et al. / Biochemical and Biophysical Research Communications 350 (2006) 358–363
stances [19–23]. Therefore, effective analgesics with less adverse effects are needed for neuropathic pain. The present study investigates whether SCP-1 and its metabolite SCP-M1 have effect on neuropathic pain in a neuropathic pain rat model and explores possible mechanisms. SCP-1 is a novel analog of APAP with limited liver and kidney toxicity [24]. Materials and methods Animals and surgery. Male Sprague–Dawley rats (250–350 g) were used for the study approved by the Local Ethical Committee for animal research. The animals were housed under standard conditions of 12 h day/ night cycles and at a room temperature of 22 °C with free access to food and water. Two to three rats were kept in one cage. Surgical procedures were performed under general anesthesia of 1.25% isoflurane mixed with 50% oxygen and 50% nitric oxide via open mask system at 1.5 l/min. Body temperature was maintained at 38 ± 0.5 °C by an automatic heating device. The left sciatic nerve was exposed in the middle thigh, and four chromic gut (4/0) ligatures were loosely applied around the nerve so that the epineural circulation was preserved [25]. The tissues were closed in layers. After 5–10 days post-surgery, some of these rats displayed allodynia and hyperalgesia, accessed by the von Frey filament test and the hot plate test (see below). We installed a PE10 catheter into the dorsal spinal canal of these animals, introduced by a 21 G cannula between L5 and L6 lamina to the level of lumbar enlargement. The proximal part was tunneled under the skin out of the upper dorsal region. The catheter position was confirmed by the injection of Xylocanine (6 ll, 50 mg/ml), indicative of a transient, flaccid paralysis in the leg during anesthesia. To prevent the catheter from being bitten by other rats, the animals with catheters were housed in individual cages. Only the animals with a well-functioning catheter, an abnormally low withdrawal threshold, and without neurological sequelae were selected for
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the subsequent tests. After catheter installation, the rats were allowed at least one day for recovery before the experiments started. Drug and behavioral test. SCP-1 n-[a-(benzisothiazol-3(2ho-ona-1,1dioxide-2yl-acetyl]-p-aminophenol is a derivative of APAP [24]. Sodium saccharin is inserted into APAP at C4 (Fig. 1). SCP-1, SCP-M1, and APAP were dissolved in 45% 2-hydroxypropyla-cyclodetrin in saline at a final concentration of 20 mM. All of the experiments were performed in blind fashion for the experimenters. The drugs were coded as A, B, C, and D. Ten microliter of each solution was prewarmed to 39 °C and injected into the spinal canal via the catheter at 2 ll/min, followed by 10 ll warmed artificial cerebrospinal fluid (CSF) to rinse the catheter. Then the animals were subjected to behavioral tests. In a quiet room, von Frey filaments or hot plate tests were performed from 9:00 to 16:00. The rats were placed in a row of square plastic boxes with a wire mesh floor for 15 min before withdrawal threshold measurements were taken. A set of von Frey filaments with stiffness corresponding to 1.0, 2.0, 4.0, 6.0, 8.0,. . .. . ., 30.0 g (Stelgent USA) was applied to the mid-plantar surface of both hind paws. The test always started by assessing the withdrawal threshold in the right intact paw. The filament was pressed against the paw for 1–2 s until it bent. A brisk withdrawal of the hind limb was considered a positive response. Each filament was applied 5 times starting with the softest and continuing in ascending order of stiffness. If a filament induced two or more withdrawal responses, the filament strength value in grams was designated as the 50% withdrawal threshold [26]. A filament corresponding to 30 g was selected as the cut-off. The basal withdrawal thresholds were taken at least twice before intrathecal drug injection. After the injection, the thresholds were checked every 15-min for 4 h in 4 groups. Hyperalgesia testing was performed on a hot plate (Life Science model), which is composed of plastic boxes with a heated glass floor, a light beam guider, and a stronger light beam as a heating source. The glass floor was warmed to 28 °C. The rats were left in the box for 15-min intervals. A light beam guider was projected on the mid-plantar of a paw followed by the stronger light beam for thermal stimuli at a 30% rate of intensity. When heat was accumulated up to a certain degree on the paw
Fig. 1. SCP-1 structure, n-[a-(benzisothiazol-3(2ho-ona1,1-dioxide-2yl)-acetyl]-p-aminophenol [24]. Sodium saccharin combines acetaminophen (APAP) at C4 bond, forming SCP-1.
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surface, the rat would withdraw its paw and sometimes lick the paw. The time lapse between the stronger light onto the paw and paw withdrawal was recorded as paw withdrawal latency. The withdrawal latency was performed twice, starting from intact paws at 10-min intervals. If a latency of an injured paw was 30% shorter than that of an intact paw the rat was considered hyperalgesic. The average of the two latency measurements was designated as the latency value. After intrathecal injection of the drugs the latency was measured every 30 min until the activity of the drug declined. In order to avoid interactions between drugs each rat was used only once and injected with one drug only. DRG neuron dissociation and whole-cell patch-clamp recording. The rats were deeply anesthetized and decapitated. DRGs were dissected out and minced before being incubated for 35 min at 37 °C in 10 ml DMEM containing 0.3 mg/ml trypsin (type III), 1 mg/ml collagenase (type I), and 0.1 mg/ml deoxyribonuclease (type IV) in a heated water bath shaker. Soybean trypsin inhibitor (type I) 1 mg/ml was added to terminate trypsin activity. Then cells were dissociated by pipette triturating into cell suspension and were centrifuged down at 1500 rpm for 5 min. Finally, cells suspended in fresh DMEM were plated on poly-L-lysine-coated 3.5 mm Petri dishes and used for whole-cell patch-clamp recording within 2–10 h of plating. Whole-cell patch-clamp recordings were made on the small neurons using an AXOPATCH-200B amplifier at room temperature (22–24 °C). Cell membrane capacitance was obtained by reading the value for whole cell input capacitance neutralization directly from the amplifier. Data were filtered at 5 kHz by a lowpass Bessel filter. The external solution contained (in mM): 130 tetraethylammonium chloride, 8 NaCl, 10 CsCl, 2.5 BaCl2, 1 MgCl2 10 Hepes, and 10 D-glucose with pH adjusted to 7.35 with CsOH. TTX (0.5 lM) and nimodipine (10 lM) were included in external solutions to eliminate voltage-gated Na+ and L-type Ca2+ currents. The internal solution contained (in mM): 125 CsCl, 8 NaCl, 2 MgCl2, 2 MgATP, 0.5 Tris–GTP, 10 Hepes, and 10 EGTA with pH adjusted to 7.25 with CsOH. Calcium channel currents were recorded with Ba2+ as the charge carrier and were elicited by a 90 ms depolarizing pulse from a holding potential of 80 mV. Drugs, SCP-1, SCP-M1 or APAP (100 lM, respectively), were applied via bath perfusion for more than 5 min to obtain a stable effect. Current–voltage relationship was checked from a holding potential of 70 mV in the absence and presence of SCP-1, SCP-M1 or acetaminophen (100 lM, respectively). The current was evoked by a 90 ms voltage pulse starting from 70 mV and was recorded with the amplifier. The voltage pulse was reduced 10 mV for each stimulation until it reached 70 mV. Statistics. Changes of the withdrawal thresholds or latencies induced by a drug were first analyzed with a one-way ANOVA. Comparisons between the effects of different drugs were then subjected to t-tests for unpaired means. A value of P < 0.05 was considered significant.
Fig. 2. Dose responsive curve for SCP-1. SCP-1 of 100 and 200 nmol suppressed tactile allodynia with 30–45 min latency, while SCP-1 of 50 nmol had a very mild effect on the allodynia. One and 10 nmol had no such effect. All doses were given intrathecally.
10 nmol had no effect on the thresholds (Fig. 2). During testing, the animals maintained calm, free movement. SCP-1 suppressed tactile allodynia After opening the codes, results showed that when SCP1 was administerted intrathecally in a dose of 100 nmol in allodynic rats, tactile withdrawal thresholds started rising 45 min post-injection, as assessed by von Frey filaments (n = 14) (Fig. 3). The thresholds were increased to 28 + 2.4 g from the basal thresholds of 4.6 + 1.1 g at 90 min after the intrathecal injection. At 4 h post-injection, the thresholds were still at similar levels. SCP-M1, given intrathecally in the same dose, produced a quicker increase in thresholds (n = 12). Within 15 min, the average withdrawal thresholds were elevated more than 10 g. At 60 min post-injection, SCP-M1 reached its maximum effect.
Results The incidence for tactile allodynic development was about 38% from 142 rats subjected to the surgery. The average withdrawal threshold with the neuropathic rats used in the experiments was 4.2 + 0.4 g. The incidence for thermal hyperalgesia was 62%. Dose response for SCP-1 In order to find an effective dose of SCP-1 on tactile allodynia suppression, SCP-1 of 1, 10, 50, 100, and 200 nmol was tested on six allodynic rats. The results showed that SCP-1 of 100 and 200 nmol normalized the withdrawal thresholds, while 50 nmol had moderate effect, and 1 and
Fig. 3. Paw withdrawal threshold changes in grams were assessed by von Frey hairs on the left nerve-injured paw plantar following intrathecal drugs in Bennett neuropathic rats. Before the injection all the rats had abnormally low withdrawal thresholds averaging 4.6 g, indicative of tactile allodynia. SCP-1 i.t. elevated the thresholds starting at 45 min postinjection and normalized the thresholds during the period of time from 75 to 240 min. SCP-M1, a metabolite of SCP-1, displayed the same effect on the thresholds as SCP-1, but the latency was much shorter. Acetaminophen i.t. had mild threshold elevations and its effectiveness disappeared at 150 min post-injection. The vehicle in the same volume i.t. did not change the paw withdrawal thresholds. In the comparison between vehicle control and SCP-1, SCP-M1 was highly significant (**P < 0.01, t-test). There also was a significant difference between SCP-1, SCP-M1, and acetaminophen (**P < 0.01, t-test).
J.-G. Cui et al. / Biochemical and Biophysical Research Communications 350 (2006) 358–363
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The effect of both SCP-1 and SCP-M1 lasted at least for 4 h after injection. APAP administerted in the same dose produced a mild threshold increase. Its maximum increase for the thresholds was around 20 g (n = 10). The effect of APAP on the thresholds started to drop at 90 min after the injection. The withdrawal thresholds restored pre-injection levels at 150 min. Vehicle (the saline) injected intrathecally with the same volume had little effect on the withdrawal thresholds (n = 8). There was a significant difference in the threshold increase between SCP-1, or SCPM1, and APAP (P < 0.01, t-test). SCP-1 depressed thermal hyperalgesia With the same doses of the drugs as von Frey filament testing, SCP-M1 (n = 8) displayed the strongest effect on the paw withdrawal latency, which increased by almost onefold. SCP-1 (n = 9) had a less potent increase in latency than SCP-M1. APAP (n = 8) had a similar increase in the latency to that of SCP-1. However, the effect dropped to the initial latency value at 120 min post-injection, while SCP-M1 and SCP-1 had a longer effect. The comparison between SCP-1, or SCP-M1 and APAP at the 120 min check point was significant (P < 0.01, t-test) (Fig. 4). SCP-1 inhibited nimodipine-resistant Ca2+ channel currents Effects of SCP-1 on non-L-type calcium channel currents were examined in acutely dissociated DRG neurons. Fig. 5 shows the time course of inhibition of calcium channel currents by SCP-1 (100 lM), SCP-M1 (100 lM), and APAP (100 lM), respectively. SCP-1 inhibited the currents of 23.0 + 2.3% (n = 7) after 5 min of application. Similar to SCP-1, SCP-M1 reduced the currents by 23.1 ± 3.5% (n = 5). There was no significant difference in the latency
Fig. 5. SCP-1 and SCP-M1 inhibit non-L-type calcium channel currents in acutely isolated DRG neurons. Plots of calcium channel currents are shown versus time before and after the application of the drugs. SCP-1 inhibited the calcium channel currents by 23.0 ± 2.3% at a depolarizing pulse to 10 mV from a holding potential of 80 mV in the presence of 10 lM nimodipine, an L-type calcium channel blocker. SCP-M1 decreased the currents by 23.1 ± 3.5%, while acetaminophen changed the current by 6.8 ± 1.0%. The inhibition of the currents by SCP-1 and SCP-M1 was partially reversible when the drugs were washed out (the current was recorded at every 20 s).
of onset of the inhibition between SCP-M1 and SCP-1. In contrast, APAP had little effect on the current (6.8 ± 1.0%, n = 4). To confirm that the isolated currents were voltage-gated Ca2+ channels currents, CdCl2 was applied. At a concentration of 0.5 mM, CdCl2 almost completely suppressed the currents (data not shown), indicating that ionic currents were mainly carried by calcium channels. The preferential blockade of calcium channel currents by SCP-1, SCP-M1, and APAP was further confirmed by the current–voltage (I–V) curve as shown in Fig. 6. Both SCP-1- and SCP-M1-induced inhibition occurred mostly at the peak of the curves ( 10 mV), and became less pronounced at the more negative voltage of 20 mV. In addition, both SCP-1 and SCP-M1 had no apparent effects on the steady state activation curves derived from the tail currents (data not shown), indicating the inhibition may not be voltage-dependent. Discussion
Fig. 4. Paw withdrawal latency in seconds was evaluated by hot plate on the left nerve-injured paw following intrathecal drugs in Bennett neuropathic rats. The latency for intact paws was as baseline. The nerve-injured paw latencies were at least 30% shorter than the intact ones, indicative of thermal hyperalgesia. SCP-1 i.t. significantly prolonged the latency, compared to vehicle controls (**P < 0.01, t-test). SCP-M1 displayed a stronger effect on latency prolongation than SCP-1. Acetaminophen had a similar effect on the latency of SCP-1, but the effect lasted shorter. There was no significant difference between SCP-1 and acetaminophen on hot plate test, except for the last time check point.
SCP-1, a derivative of APAP, displayed nice suppressive effects on allodynia and hyperalgesia when given intrathecally and the effect lasts at least 4 h. During the experiments, no adverse effects were observed. Interestingly, SCP-M1, which is a main metabolite of SCP-1, suppressed tactile allodynia with the same pattern as SCP-1, but the latency was just about 15 min shorter. The results indicate that SCP-M1 may be the effective component of SCP-1.
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Fig. 6. Changes in current–voltage (I–V) relationship induced by SCP-1, SCP-M1, and acetaminophen. (A–C) Representative current traces evoked with 90 ms voltage pulse to 30, 20, and 10 mV from a holding potential of 80 mV in the absence or presence of SCP-1, SCP-M1, and acetaminophen (each 100 lM), respectively. (D–F) Current–voltage relationship curves indicated that the greatest inhibition of calcium channel currents induced by SCP1 and SCP-M1 occurred near the peak of the I–V curves, and that acetaminophen had little effect on calcium channel currents.
APAP had a mild effect on the tactile allodynia, and the difference between APAP and SCP-1, or SCP-M1 was highly significant. The vehicle had little effect on the allodynia. However, for hyperalgesia induced by the hot plate, SCP-M1 displayed the strongest suppression, while SCP-1 had a similar effect on the hyperalgesia to that of APAP, but the effect of APAP lasted the shortest. It seems that SCP-1 suppresses A-fiber conducted allodynia [27] better than C-fiber, Ad fiber conducted hyperalgesia [19,28]. Why did SCP-M1 suppress hyperalgesia well, but SCP-1 did not? One possible explanation is that suppressing hyperalgesia may require more drug strength than suppressing allodynia. SCP-M1 is metabolite of SCP-1. One hundred nmol of SCP-1 may not produce 100 nmol SCPM1 in the animal bodies. Thus, SCP-M1 suppressed hyperalgesia better than SCP-1. Our whole-cell patch-clamp data demonstrated that SCP-1 100 lM decreased in calcium channel current by 23.0 + 2.3% at 10 mV when the holding potential was 70 mV in the presence of an L-type calcium channel blocker, nimodipine. SCP-M1 displayed a similar inhibition of the calcium channel current, while APAP had little effect on this channel current. At present, voltage dependent calcium channels of L-, N-, P-, Q-, R-, and T-type have been classified according to their electrophysiological and pharmacological features. It was shown that N-, P-, Q-, T-, but not L-type, voltage dependent calcium channel (VDCC) antagonists could block experi-
mental tactile allodynia when given intrathecally [29,30]. In the DRG, N-type VDCCs are distributed in all cells [31], L-type VDCCs are linked to small cells, T-type VDCCs are in the medium cells, while P- and Q-type VDCCs are in the medium and large cells [29,32]. After allodynia and hyperalgesia are developed, the allodynic pain signals activated by low threshold receptors are conducted via highly myelinated Ab fibers to large- and medium-size neurons in the DRG, while hyperalgesic signals are transducted via non-myelinated C-fiber and thinly myelinated Ad fiber to small- and medium-size neurons [19]. The results are that SCP-1 mainly suppressed tactile allodynia, which is associated with low threshold receptor activation, and Ab fiber conduction to large- and medium-size neurons in the DRG, which are consistent with the inhibition of low voltage-dependent calcium channel currents. Although present data cannot tell which kind of calcium channels SCP-1 and SCP-M1 blocked, SCP-1 did block allodynic and hyperalgesic behaviors as well as calcium current in the experiments without adverse effects. So they are promising for pain management, especially for neuropathic pain. Acknowledgments The work was supported by DARPA HR0011-04-C0068 and by the Neurobiotechnology Program of Louisiana.
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Allodynia and hyperalgesia suppression by a novel analgesic in experimental neuropathic pain Jian-Guo Cui *, Xiong Zhang, Yu-Hai Zhao, Chu Chen, Nicolas Bazan
*
Neuroscience Center of Excellence, Louisiana State University, Health Sciences Center, 2020 Gravier Street, New Orleans, LA 70112-2272, USA Received 25 August 2006 Available online 22 September 2006
Abstract SCP-1, n-[a-(benzisothiazol-3(2ho-ona,1-dioxide-2yl)-acetyl]-p-aminophenol (100 nmol), when intrathecally injected, suppressed tactile allodynia and thermal hyperalgesia in a rat neuropathic pain model. The tactile allodynia suppression lasted for at least 4 h and SCPM1 (100 nmol), the main metabolite of SCP-1, displayed similar suppression as SCP-1, but shorter latency, indicating SCP-M1 may be the bioactive component of SCP-1. Acetaminophen was less potent than SCP-1 and SCP-M1. To study mechanisms underlying SCP-1 action, we recorded voltage-gated Ca2+ channel currents in acutely isolated dorsal root ganglion neurons using the whole-cell patchclamp technique. SCP-1 and SCP-M1 inhibited non-L-type calcium channel currents up to 23.0 ± 2.3% and 23.1 ± 3.5%, respectively, at a depolarized pulse to 10 mV from a holding potential of 80 mV. Acetaminophen only induced 6.8 ± 1.0% inhibition. The results suggest SCP-1 possesses anti-nociceptive activity in the rat model involving calcium channel blocking properties. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Neuropathic pain; Tactile allodynia; Thermal hyperalgesia; Behavioral test; Calcium channel; Patch-clamp; SCP-1; Rat
Neuropathic pain is often associated with peripheral nerve injury. The nerve injury can induce neuroma at the end of injured fibers [1], cross-talk between different nerve fibers [2], and both peripheral and sympathetic sprouting from intact adjacent nerve fibers and in the DRG [3,4], resulting in spinal hyperexcitability. The hyperexcitability subsequently leads to pathological changes in genes, neurotransmitters, and cellular structures [5,6]. Excitatory neurotransmitters and related genes, such as glutamate, aspartate, substance P, and calcitonin-gene related peptide, are up-regulated in dorsal root ganglia and spinal dorsal horn, while inhibitory transmitters, such as c-aminobutyric acid (GABA), adenosine, are down-regulated [7–9]. Furthermore, inflammatory factors (macrophages, interleukins, tumor necrosis factor, and cyclooxygenase) and growth factors (nerve growth factor, epidermal growth factor (EGF), and brain-derived neurotrophic factor *
Corresponding authors. Fax: +1 504 599 0891. E-mail addresses: [email protected] (J.-G. Cui), [email protected] (N. Bazan). 0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.09.055
(BDNF)) are also involved in neuropathic pain development [10–13]. In addition, ion channels, in particular calcium channels and N-methyl-D-aspartate (NMDA receptors, which mediate synaptic excitability and control various signal transductions), are important mechanisms for neuropathic hypersensibility [14]. However, the mediating mechanisms for neuropathic pain development are still not clear. Understanding of neuropathic pain development and modulation has advanced tremendously since animal models of neuropathic pain came into use for the study of its mechanisms. However, the treatments for neuropathic pain are far behind. Due to complicated developmental mechanisms of neuropathic pain [15], opioids have little effect on such pain and possess a high risk of addiction and constipation [16]. Anti-convulsants may attenuate neuropathic pain on a small number of patients [17]. but the effectiveness is often unsatisfactory [18]. Acetaminophen (APAP) is an over-the-counter analgesic, which has been shown to inhibit cyclooxygenases in pain modulations, but it displays liver and kidney toxicity under a variety of circum-
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stances [19–23]. Therefore, effective analgesics with less adverse effects are needed for neuropathic pain. The present study investigates whether SCP-1 and its metabolite SCP-M1 have effect on neuropathic pain in a neuropathic pain rat model and explores possible mechanisms. SCP-1 is a novel analog of APAP with limited liver and kidney toxicity [24]. Materials and methods Animals and surgery. Male Sprague–Dawley rats (250–350 g) were used for the study approved by the Local Ethical Committee for animal research. The animals were housed under standard conditions of 12 h day/ night cycles and at a room temperature of 22 °C with free access to food and water. Two to three rats were kept in one cage. Surgical procedures were performed under general anesthesia of 1.25% isoflurane mixed with 50% oxygen and 50% nitric oxide via open mask system at 1.5 l/min. Body temperature was maintained at 38 ± 0.5 °C by an automatic heating device. The left sciatic nerve was exposed in the middle thigh, and four chromic gut (4/0) ligatures were loosely applied around the nerve so that the epineural circulation was preserved [25]. The tissues were closed in layers. After 5–10 days post-surgery, some of these rats displayed allodynia and hyperalgesia, accessed by the von Frey filament test and the hot plate test (see below). We installed a PE10 catheter into the dorsal spinal canal of these animals, introduced by a 21 G cannula between L5 and L6 lamina to the level of lumbar enlargement. The proximal part was tunneled under the skin out of the upper dorsal region. The catheter position was confirmed by the injection of Xylocanine (6 ll, 50 mg/ml), indicative of a transient, flaccid paralysis in the leg during anesthesia. To prevent the catheter from being bitten by other rats, the animals with catheters were housed in individual cages. Only the animals with a well-functioning catheter, an abnormally low withdrawal threshold, and without neurological sequelae were selected for
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the subsequent tests. After catheter installation, the rats were allowed at least one day for recovery before the experiments started. Drug and behavioral test. SCP-1 n-[a-(benzisothiazol-3(2ho-ona-1,1dioxide-2yl-acetyl]-p-aminophenol is a derivative of APAP [24]. Sodium saccharin is inserted into APAP at C4 (Fig. 1). SCP-1, SCP-M1, and APAP were dissolved in 45% 2-hydroxypropyla-cyclodetrin in saline at a final concentration of 20 mM. All of the experiments were performed in blind fashion for the experimenters. The drugs were coded as A, B, C, and D. Ten microliter of each solution was prewarmed to 39 °C and injected into the spinal canal via the catheter at 2 ll/min, followed by 10 ll warmed artificial cerebrospinal fluid (CSF) to rinse the catheter. Then the animals were subjected to behavioral tests. In a quiet room, von Frey filaments or hot plate tests were performed from 9:00 to 16:00. The rats were placed in a row of square plastic boxes with a wire mesh floor for 15 min before withdrawal threshold measurements were taken. A set of von Frey filaments with stiffness corresponding to 1.0, 2.0, 4.0, 6.0, 8.0,. . .. . ., 30.0 g (Stelgent USA) was applied to the mid-plantar surface of both hind paws. The test always started by assessing the withdrawal threshold in the right intact paw. The filament was pressed against the paw for 1–2 s until it bent. A brisk withdrawal of the hind limb was considered a positive response. Each filament was applied 5 times starting with the softest and continuing in ascending order of stiffness. If a filament induced two or more withdrawal responses, the filament strength value in grams was designated as the 50% withdrawal threshold [26]. A filament corresponding to 30 g was selected as the cut-off. The basal withdrawal thresholds were taken at least twice before intrathecal drug injection. After the injection, the thresholds were checked every 15-min for 4 h in 4 groups. Hyperalgesia testing was performed on a hot plate (Life Science model), which is composed of plastic boxes with a heated glass floor, a light beam guider, and a stronger light beam as a heating source. The glass floor was warmed to 28 °C. The rats were left in the box for 15-min intervals. A light beam guider was projected on the mid-plantar of a paw followed by the stronger light beam for thermal stimuli at a 30% rate of intensity. When heat was accumulated up to a certain degree on the paw
Fig. 1. SCP-1 structure, n-[a-(benzisothiazol-3(2ho-ona1,1-dioxide-2yl)-acetyl]-p-aminophenol [24]. Sodium saccharin combines acetaminophen (APAP) at C4 bond, forming SCP-1.
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surface, the rat would withdraw its paw and sometimes lick the paw. The time lapse between the stronger light onto the paw and paw withdrawal was recorded as paw withdrawal latency. The withdrawal latency was performed twice, starting from intact paws at 10-min intervals. If a latency of an injured paw was 30% shorter than that of an intact paw the rat was considered hyperalgesic. The average of the two latency measurements was designated as the latency value. After intrathecal injection of the drugs the latency was measured every 30 min until the activity of the drug declined. In order to avoid interactions between drugs each rat was used only once and injected with one drug only. DRG neuron dissociation and whole-cell patch-clamp recording. The rats were deeply anesthetized and decapitated. DRGs were dissected out and minced before being incubated for 35 min at 37 °C in 10 ml DMEM containing 0.3 mg/ml trypsin (type III), 1 mg/ml collagenase (type I), and 0.1 mg/ml deoxyribonuclease (type IV) in a heated water bath shaker. Soybean trypsin inhibitor (type I) 1 mg/ml was added to terminate trypsin activity. Then cells were dissociated by pipette triturating into cell suspension and were centrifuged down at 1500 rpm for 5 min. Finally, cells suspended in fresh DMEM were plated on poly-L-lysine-coated 3.5 mm Petri dishes and used for whole-cell patch-clamp recording within 2–10 h of plating. Whole-cell patch-clamp recordings were made on the small neurons using an AXOPATCH-200B amplifier at room temperature (22–24 °C). Cell membrane capacitance was obtained by reading the value for whole cell input capacitance neutralization directly from the amplifier. Data were filtered at 5 kHz by a lowpass Bessel filter. The external solution contained (in mM): 130 tetraethylammonium chloride, 8 NaCl, 10 CsCl, 2.5 BaCl2, 1 MgCl2 10 Hepes, and 10 D-glucose with pH adjusted to 7.35 with CsOH. TTX (0.5 lM) and nimodipine (10 lM) were included in external solutions to eliminate voltage-gated Na+ and L-type Ca2+ currents. The internal solution contained (in mM): 125 CsCl, 8 NaCl, 2 MgCl2, 2 MgATP, 0.5 Tris–GTP, 10 Hepes, and 10 EGTA with pH adjusted to 7.25 with CsOH. Calcium channel currents were recorded with Ba2+ as the charge carrier and were elicited by a 90 ms depolarizing pulse from a holding potential of 80 mV. Drugs, SCP-1, SCP-M1 or APAP (100 lM, respectively), were applied via bath perfusion for more than 5 min to obtain a stable effect. Current–voltage relationship was checked from a holding potential of 70 mV in the absence and presence of SCP-1, SCP-M1 or acetaminophen (100 lM, respectively). The current was evoked by a 90 ms voltage pulse starting from 70 mV and was recorded with the amplifier. The voltage pulse was reduced 10 mV for each stimulation until it reached 70 mV. Statistics. Changes of the withdrawal thresholds or latencies induced by a drug were first analyzed with a one-way ANOVA. Comparisons between the effects of different drugs were then subjected to t-tests for unpaired means. A value of P < 0.05 was considered significant.
Fig. 2. Dose responsive curve for SCP-1. SCP-1 of 100 and 200 nmol suppressed tactile allodynia with 30–45 min latency, while SCP-1 of 50 nmol had a very mild effect on the allodynia. One and 10 nmol had no such effect. All doses were given intrathecally.
10 nmol had no effect on the thresholds (Fig. 2). During testing, the animals maintained calm, free movement. SCP-1 suppressed tactile allodynia After opening the codes, results showed that when SCP1 was administerted intrathecally in a dose of 100 nmol in allodynic rats, tactile withdrawal thresholds started rising 45 min post-injection, as assessed by von Frey filaments (n = 14) (Fig. 3). The thresholds were increased to 28 + 2.4 g from the basal thresholds of 4.6 + 1.1 g at 90 min after the intrathecal injection. At 4 h post-injection, the thresholds were still at similar levels. SCP-M1, given intrathecally in the same dose, produced a quicker increase in thresholds (n = 12). Within 15 min, the average withdrawal thresholds were elevated more than 10 g. At 60 min post-injection, SCP-M1 reached its maximum effect.
Results The incidence for tactile allodynic development was about 38% from 142 rats subjected to the surgery. The average withdrawal threshold with the neuropathic rats used in the experiments was 4.2 + 0.4 g. The incidence for thermal hyperalgesia was 62%. Dose response for SCP-1 In order to find an effective dose of SCP-1 on tactile allodynia suppression, SCP-1 of 1, 10, 50, 100, and 200 nmol was tested on six allodynic rats. The results showed that SCP-1 of 100 and 200 nmol normalized the withdrawal thresholds, while 50 nmol had moderate effect, and 1 and
Fig. 3. Paw withdrawal threshold changes in grams were assessed by von Frey hairs on the left nerve-injured paw plantar following intrathecal drugs in Bennett neuropathic rats. Before the injection all the rats had abnormally low withdrawal thresholds averaging 4.6 g, indicative of tactile allodynia. SCP-1 i.t. elevated the thresholds starting at 45 min postinjection and normalized the thresholds during the period of time from 75 to 240 min. SCP-M1, a metabolite of SCP-1, displayed the same effect on the thresholds as SCP-1, but the latency was much shorter. Acetaminophen i.t. had mild threshold elevations and its effectiveness disappeared at 150 min post-injection. The vehicle in the same volume i.t. did not change the paw withdrawal thresholds. In the comparison between vehicle control and SCP-1, SCP-M1 was highly significant (**P < 0.01, t-test). There also was a significant difference between SCP-1, SCP-M1, and acetaminophen (**P < 0.01, t-test).
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The effect of both SCP-1 and SCP-M1 lasted at least for 4 h after injection. APAP administerted in the same dose produced a mild threshold increase. Its maximum increase for the thresholds was around 20 g (n = 10). The effect of APAP on the thresholds started to drop at 90 min after the injection. The withdrawal thresholds restored pre-injection levels at 150 min. Vehicle (the saline) injected intrathecally with the same volume had little effect on the withdrawal thresholds (n = 8). There was a significant difference in the threshold increase between SCP-1, or SCPM1, and APAP (P < 0.01, t-test). SCP-1 depressed thermal hyperalgesia With the same doses of the drugs as von Frey filament testing, SCP-M1 (n = 8) displayed the strongest effect on the paw withdrawal latency, which increased by almost onefold. SCP-1 (n = 9) had a less potent increase in latency than SCP-M1. APAP (n = 8) had a similar increase in the latency to that of SCP-1. However, the effect dropped to the initial latency value at 120 min post-injection, while SCP-M1 and SCP-1 had a longer effect. The comparison between SCP-1, or SCP-M1 and APAP at the 120 min check point was significant (P < 0.01, t-test) (Fig. 4). SCP-1 inhibited nimodipine-resistant Ca2+ channel currents Effects of SCP-1 on non-L-type calcium channel currents were examined in acutely dissociated DRG neurons. Fig. 5 shows the time course of inhibition of calcium channel currents by SCP-1 (100 lM), SCP-M1 (100 lM), and APAP (100 lM), respectively. SCP-1 inhibited the currents of 23.0 + 2.3% (n = 7) after 5 min of application. Similar to SCP-1, SCP-M1 reduced the currents by 23.1 ± 3.5% (n = 5). There was no significant difference in the latency
Fig. 5. SCP-1 and SCP-M1 inhibit non-L-type calcium channel currents in acutely isolated DRG neurons. Plots of calcium channel currents are shown versus time before and after the application of the drugs. SCP-1 inhibited the calcium channel currents by 23.0 ± 2.3% at a depolarizing pulse to 10 mV from a holding potential of 80 mV in the presence of 10 lM nimodipine, an L-type calcium channel blocker. SCP-M1 decreased the currents by 23.1 ± 3.5%, while acetaminophen changed the current by 6.8 ± 1.0%. The inhibition of the currents by SCP-1 and SCP-M1 was partially reversible when the drugs were washed out (the current was recorded at every 20 s).
of onset of the inhibition between SCP-M1 and SCP-1. In contrast, APAP had little effect on the current (6.8 ± 1.0%, n = 4). To confirm that the isolated currents were voltage-gated Ca2+ channels currents, CdCl2 was applied. At a concentration of 0.5 mM, CdCl2 almost completely suppressed the currents (data not shown), indicating that ionic currents were mainly carried by calcium channels. The preferential blockade of calcium channel currents by SCP-1, SCP-M1, and APAP was further confirmed by the current–voltage (I–V) curve as shown in Fig. 6. Both SCP-1- and SCP-M1-induced inhibition occurred mostly at the peak of the curves ( 10 mV), and became less pronounced at the more negative voltage of 20 mV. In addition, both SCP-1 and SCP-M1 had no apparent effects on the steady state activation curves derived from the tail currents (data not shown), indicating the inhibition may not be voltage-dependent. Discussion
Fig. 4. Paw withdrawal latency in seconds was evaluated by hot plate on the left nerve-injured paw following intrathecal drugs in Bennett neuropathic rats. The latency for intact paws was as baseline. The nerve-injured paw latencies were at least 30% shorter than the intact ones, indicative of thermal hyperalgesia. SCP-1 i.t. significantly prolonged the latency, compared to vehicle controls (**P < 0.01, t-test). SCP-M1 displayed a stronger effect on latency prolongation than SCP-1. Acetaminophen had a similar effect on the latency of SCP-1, but the effect lasted shorter. There was no significant difference between SCP-1 and acetaminophen on hot plate test, except for the last time check point.
SCP-1, a derivative of APAP, displayed nice suppressive effects on allodynia and hyperalgesia when given intrathecally and the effect lasts at least 4 h. During the experiments, no adverse effects were observed. Interestingly, SCP-M1, which is a main metabolite of SCP-1, suppressed tactile allodynia with the same pattern as SCP-1, but the latency was just about 15 min shorter. The results indicate that SCP-M1 may be the effective component of SCP-1.
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Fig. 6. Changes in current–voltage (I–V) relationship induced by SCP-1, SCP-M1, and acetaminophen. (A–C) Representative current traces evoked with 90 ms voltage pulse to 30, 20, and 10 mV from a holding potential of 80 mV in the absence or presence of SCP-1, SCP-M1, and acetaminophen (each 100 lM), respectively. (D–F) Current–voltage relationship curves indicated that the greatest inhibition of calcium channel currents induced by SCP1 and SCP-M1 occurred near the peak of the I–V curves, and that acetaminophen had little effect on calcium channel currents.
APAP had a mild effect on the tactile allodynia, and the difference between APAP and SCP-1, or SCP-M1 was highly significant. The vehicle had little effect on the allodynia. However, for hyperalgesia induced by the hot plate, SCP-M1 displayed the strongest suppression, while SCP-1 had a similar effect on the hyperalgesia to that of APAP, but the effect of APAP lasted the shortest. It seems that SCP-1 suppresses A-fiber conducted allodynia [27] better than C-fiber, Ad fiber conducted hyperalgesia [19,28]. Why did SCP-M1 suppress hyperalgesia well, but SCP-1 did not? One possible explanation is that suppressing hyperalgesia may require more drug strength than suppressing allodynia. SCP-M1 is metabolite of SCP-1. One hundred nmol of SCP-1 may not produce 100 nmol SCPM1 in the animal bodies. Thus, SCP-M1 suppressed hyperalgesia better than SCP-1. Our whole-cell patch-clamp data demonstrated that SCP-1 100 lM decreased in calcium channel current by 23.0 + 2.3% at 10 mV when the holding potential was 70 mV in the presence of an L-type calcium channel blocker, nimodipine. SCP-M1 displayed a similar inhibition of the calcium channel current, while APAP had little effect on this channel current. At present, voltage dependent calcium channels of L-, N-, P-, Q-, R-, and T-type have been classified according to their electrophysiological and pharmacological features. It was shown that N-, P-, Q-, T-, but not L-type, voltage dependent calcium channel (VDCC) antagonists could block experi-
mental tactile allodynia when given intrathecally [29,30]. In the DRG, N-type VDCCs are distributed in all cells [31], L-type VDCCs are linked to small cells, T-type VDCCs are in the medium cells, while P- and Q-type VDCCs are in the medium and large cells [29,32]. After allodynia and hyperalgesia are developed, the allodynic pain signals activated by low threshold receptors are conducted via highly myelinated Ab fibers to large- and medium-size neurons in the DRG, while hyperalgesic signals are transducted via non-myelinated C-fiber and thinly myelinated Ad fiber to small- and medium-size neurons [19]. The results are that SCP-1 mainly suppressed tactile allodynia, which is associated with low threshold receptor activation, and Ab fiber conduction to large- and medium-size neurons in the DRG, which are consistent with the inhibition of low voltage-dependent calcium channel currents. Although present data cannot tell which kind of calcium channels SCP-1 and SCP-M1 blocked, SCP-1 did block allodynic and hyperalgesic behaviors as well as calcium current in the experiments without adverse effects. So they are promising for pain management, especially for neuropathic pain. Acknowledgments The work was supported by DARPA HR0011-04-C0068 and by the Neurobiotechnology Program of Louisiana.
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