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BEHAVIORAL PHARMACOLOGY

Effects of Spinal ₂-Adrenoceptor and I₁-Imidazoline Receptor Activation on Hindlimb Movement Induction in Spinal Cord-Injured Mice

Neuroscience Unit, Laval University Medical Centre, Quebec City, Quebec, Canada (N.P.L., R.-V.U., P.R., P.A.G.); and Department of Anatomy and Physiology, Laval University, Quebec City, Quebec, Canada (P.A.G.)

Received November 30, 2007; accepted March 24, 2008.

	Abstract

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A partial recovery of locomotor functions has been shown in spinal cord-transected (Tx) cats after regular treadmill training and repeated administration of clonidine, an ₂-adrenoreceptor agonist. However, clonidine has generally failed to show prolocomotor effects in other models (e.g., rat or mudpuppy in vitro-isolated spinal cord preparations). The reasons for this discrepancy remain unclear, but they may suggest condition- or species-specific effects induced by clonidine. This study is aimed at examining both the acute (at 6 or 41 days post-Tx) and chronic effects of repeated (once a week for one month) clonidine administration (0.25–5.0 mg/kg i.p.) on hindlimb movement generation in Tx mice (thoracic segment9/10). Locomotor-like (LM) and nonlocomotor movements (NLM) were assessed both in open-field and treadmill conditions. The results show that clonidine consistently failed, in both conditions, to induce LM and NLM at all time points even though control experiments revealed hindlimb movements steadily induced by 8-hydroxy-2-(di-N-propylamino)-tetralin (8-OH-DPAT), a serotonin receptor agonist. In turn, clonidine acutely suppressed (I₁-imidazoline receptor-mediated) the frequency of spontaneously occurring LM and NLM but apparently increased spinal excitability over time, because the frequency of spontaneous LM and NLM was significantly greater in clonidine-treated (before an injection) than vehicle-treated animals after repeated administration for a few weeks. The results clearly show that clonidine can not acutely induce hindlimb movements in untrained and otherwise nonstimulated (e.g., no tail or perineal pinching) Tx mice, although repeated administration may progressively facilitate the expression of spontaneous hindlimb movements.

In the field of spinal cord injury (SCI) research, clonidine has been reported to induce promising effects in early or late chronic spinal cord-transection (Tx) cats (Forssberg and Grillner, 1973; Barbeau et al., 1987). Indeed, combined with sensory stimulation (e.g., perineal and/or tail pinching), clonidine (i.v. or i.p. administered) was found to evoke locomotor movements or to increase functional recovery (larger movement amplitude) after regular treadmill training with body weight-support assistance in Tx cats (reviewed in Rossignol et al., 2001). Further supporting a centrally mediated action on central pattern generator (CPG) neurons, intrathecal (lumbar level) or intraspinal (L3–L4) administration of clonidine was shown to acutely (within a few minutes) evoke hindlimb stepping movements or comparable neurographic activity in Tx cats (Chau et al., 1998; Marcoux and Rossignol, 2000).

However, clonidine administration (intrathecally or orally administered) has always failed to trigger "reflex" stepping in complete SCI patients, although it has been found to facilitate ambulation by decreasing spasticity in incomplete SCI individuals (Stewart et al., 1991; Dietz et al., 1995; Rémy-Néris et al., 1999). Along this line of evidence, bath application of clonidine was reported not to induce fictive locomotion in in vitro-isolated spinal cord preparations from neonatal rats or mudpuppies (Sqalli-Houssaini and Cazalets, 2000; Fok and Stein, 2002). Therefore, in contrast with the results in cats, results from studies in men, rats, or mudpuppies tend to support the idea that clonidine can neither activate CPG neurons nor induce prolocomotor effects. This suggests that the prolocomotor effects of clonidine found in cats may depend upon specific paradigms, species, or treatments.

The present study aimed at examining the acute and chronic effects of clonidine on locomotor movement generation in complete Tx mice. In particular, we analyzed the effects of repeated (once a week for 4 weeks) clonidine administration (0.25–5.0 mg/kg i.p.) versus control (vehicle-treated) on hindlimb movement in Tx mice. We also compared the effects of clonidine in open-field versus treadmill (8–10 cm/s) conditions, to investigate the contribution of treadmill-associated peripheral afferent inputs to drug-induced effects. As additional control experiments, we examined the acute effects of clonidine in nonpreviously treated late chronic Tx mice (at 41 days post-Tx) and tested the effects of 8-OH-DPAT (a known CPG-activating drug; Landry et al., 2006) in the same animals (i.e., at 43 days). Hindlimb movements assessed after drug administration were also compared with those found (if any) just before drug administration to distinguish spontaneously occurring hindlimb movements from those acutely induced by clonidine or 8-OH-DPAT (Guertin, 2005; Landry et al., 2006; Lapointe et al., 2006a). Preliminary results have been reported in abstract form (Guertin et al., 2002; Lapointe et al., 2006b).

	Materials and Methods

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Surgical Procedures. This work was done in accordance with the Canadian Guide for the Care and Use of Laboratory Animals. All procedures were also approved by the Animal Care and Use Committee of the Laval University Medical Center. Experiments were performed on adult male mice (n = 74, CD1 strain; Charles River Laboratories, Montreal, QC) weighing 30 to 35 g at the time of surgery. Mice were housed four or five per cage in a controlled-temperature environment maintained under a 12-h light/dark cycle with access to food and water ad libitum. Preoperative care comprised administration of lactate Ringer's solution (1.0 ml s.c.), analgesic (buprenorphine, 0.1 mg/kg s.c.; Schering-Plough Ltd., Montreal, QC), and antibiotic (Baytril, 5 mg/kg s.c.; Bayer, Toronto, ON) given 30 min before the surgery. Then, a complete transection (intervertebrally) of spinal cord was performed by experimented surgeons under general anesthesia using isoflurane (2.5%) as described elsewhere (Landry et al., 2006; Lapointe et al., 2006a). In brief, after having exposed the area between the 6th and the 12th thoracic segments, an intervertebral transection was performed using microscissors inserted between the 9th and the 10th thoracic vertebrae. To ensure that complete transection was achieved, the inner vertebral walls were entirely scrapped several times with scissor tips to sever all remaining fibers that had not been previously transected. After suturing the opened skin area, mice were placed for a few hours on heating pads. Postoperative care including daily administration of lactate-Ringer's solution (2 x 1.0 ml s.c.), buprenorphine (2 x 0.1 mg/kg, s.c.), and Baytril (5 mg/kg s.c.) was provided for the following 3 days. Bladders were emptied manually twice a day for the first few days. Data were analyzed only from Tx mice that displayed flexion reflexes (withdrawal of the paw after toe pinching) and for which complete transection was confirmed by postmortem histological examination of the spinal cord (cresyl violet and fast blue staining).

Drug Treatment. Intraperitoneal administration of 0.25, 0.5, 1.0, 2.5, or 5.0 mg/kg clonidine (2,6-dichloro-N-2-midazolidinylidenebenzenamine; Tocris Cookson Inc., Ellisville, MO) was performed once a week for four consecutive weeks or, alternatively, at 41 days in nonpreviously treated animals. This range of doses was chosen based upon the prolocomotor effects (i.e., brain-mediated) reported elsewhere in DSP4-treated mice (Archer and Fredriksson, 2000). Eight groups were constituted (8 animals per group initially), 5 of which received varying doses (from 0.25 to 5.0 mg/kg) of clonidine. One group received saline (vehicle) as the control. As additional controls, two other groups were treated instead with 0.25 or 5.0 mg/kg clonidine (n = 8 and n = 7 mice per group, respectively) at 41 days post-Tx (nonpreviously treated) and, at 43 days post-Tx with 1.0 mg/kg 8-OH-DPAT (Tocris Cookson Inc.), a serotonin-type 1A and 7 receptor agonist (5-HT_1A/7 agonist) known to display CPG-activating effects (Landry et al., 2006). For the five groups receiving weekly repeated administration of clonidine, a varying dose regimen was chosen to avoid dose-dependent side-effects in some animals (e.g., that would have received only the highest doses). None of the animals received the same dose twice during 4 weeks (i.e., 0.25, 0.5, 1.0, 2.5, or 5.0 mg/kg). Yet, in the end, each group was constituted of animals that received overall the same average dose of clonidine. This arbitrary decision of using a varying dose regimen was based upon preliminary observations made in this laboratory, showing progressive health degradation and, in some cases, death after repeated administration of relatively high doses of clonidine (2.5 mg/kg). In fact, despite this measure used to protect the animals, a number of them repeatedly treated with clonidine died after the 2nd (n = 8), 3rd (n = 4), and 4th (n = 8) week, which has forced the regrouping of some of the groups for analysis and statistical reasons. An attempt was made to assess and compare with clonidine the effects induced by moxonidine (I₁-imidazoline receptor agonist with lower affinity to ₂-adrenoceptor, 1–5 mg/kg i.p.) in a group (n = 6) of Tx mice. Two other groups of mice (n = 16) were used to pharmacologically dissect the mechanisms of action of clonidine-induced effects found in this study using selective antagonists (Ernsberger et al., 1987). These animals were either pretreated with yohimbine (₂-adrenoceptor antagonist, 1.0 mg/kg i.p.) (Buerkle and Yaksh, 1998; Zarrindast et al., 2002; Galeotti et al., 2004) or efaroxan (I₁/I₃-imidazoline receptor antagonist and ₂-adrenoceptor antagonist, 1.0 mg/kg i.p.) (Shannon and Lutz, 2000; Sabetkasaie et al., 2007) 15 min before clonidine administration (1.0 mg/kg i.p.).

Locomotor Assessment. Hindlimb movement was assessed once a week for 4 weeks (i.e., at 6, 13, 20, and 27 days post-Tx). We chose not to collect data more frequently (e.g., three times a week) to prevent cumulative training-induced effects. On each testing day, assessment was performed by two well trained observers immediately before clonidine (or vehicle) or 8-OH-DPAT administration and 20 min after drug administration because additional effects have not been reported in this laboratory beyond this postinjection delay (Guertin et al., 2002; Lapointe et al., 2006b). Hindlimb movement assessment was performed for 4 min in open-field condition and for 4 min in treadmill condition (randomly ordered). For open-field condition, we used a circular arena (60 cm in diameter) made of transparent Plexiglas. For treadmill condition, each mouse was placed on a treadmill equipped with a motor-driven rubber belt (8–10 cm/s). Note that no significant body weight-support assistance was provided when performing trials in treadmill condition because the harness was used only to maintain some lateral stability.

Two different assessment methods designed for complete paraplegic rodents were used. The first method called Average Combined Score (ACOS) (Guertin, 2005; Lapointe et al., 2006a; Ung et al., 2007; Zhang et al., 2007) is a semiquantitative method developed in this laboratory that reports amplitude, incidence, and frequency (number per minute) of nonlocomotor movements (NLM) and locomotor-like movements (LM) distinctively. In brief, 1 LM is defined as a flexion followed by an extension or vice versa (involving one or several articulations) occurring in both hindlimbs in alternation (out-of-phase relationship). In turn, 1 NLM is defined as a unilateral (or nonbilaterally alternating) movement that may qualify either as fast paw shaking, jerk, twitch, or any other type of nonlocomotor movements. Movement amplitude was reported as small or large and given the arbitrary numerical value of 1 or 2, respectively. Movement incidence, defined as the number of animals in which LM or NLM were observed, was also reported. ACOS represents the arithmetic sum of the above values (ACOS = [NLM/min + (2 x LM/min)] x amplitude), which was used to ease comparisons between groups (Guertin, 2005).

We used a second assessment method called AOB developed by Antri, Orsal, and Barthe (Antri et al., 2002). This locomotor rating scale was also designed specifically to assess hindlimb movement in Tx rodents. For instance, it does not assess forelimb versus hindlimb coordination, which is irrelevant in complete paraplegic animals (i.e., without regeneration-promoting treatments). In brief, the AOB scale consists of 22 discriminative scores, mainly grouped into 4 categories. Relevant scores for our study are the following: 0, no movement; 1, weak limb jerks; 2, weak rhythmic movements with no bilateral alternation; 3, large rhythmic movements with no bilateral alternation; 4, weak rhythmic movements with occasional bilateral alternation; 5, large rhythmic movements with occasional bilateral alternation; and 6, weak rhythmic movements with frequent bilateral alternation (for higher levels, see Antri et al., 2002).

Statistical Analyses. Previous postinjection data were compared using paired sample t tests. A one-way repeated measures analysis of variance was performed to compare clonidine-induced effects (between time points) followed by a Dunnett's post hoc test when significant (P < 0.05). To compare data from control (saline-treated) versus clonidine-treated animals, t tests were also performed at each time point. All statistical analyses were done using SPSS 11.0 (SPSS Inc., Chicago, IL). Results were expressed as mean ± S.E.M. A value of P < 0.05 was considered statistically significant.

	Results

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Acute Effects Assessed in Open-Field Condition. As reported elsewhere, some spontaneously occurring NLM are generally found in this animal model within a few days to a few weeks post-Tx (Guertin, 2005; Lapointe et al., 2006a; Ung et al., 2007). Comparable results were found in this study because, at 6 days post-Tx, 16 of 40 animals displayed a few so-called "spontaneous" NLM (less than one per minute on average) (Fig. 1A, white bars) but no LM before clonidine injection (Fig. 1B). Average data from all animals revealed that 0.25 to 5.0 mg/kg clonidine did not induce or significantly (P > 0.05) change the frequency of NLM compared with preinjection (Fig. 1A, preinjection as white bars and postinjection as black bars). In addition, none of the animals displayed drug-induced LM at doses ranging between 0.25 and 5.0 mg/kg (Fig. 1B). Accordingly, at 6 days post-Tx, ACOS (<1) did not significantly (P > 0.05) change postinjection (0.25–5.0 mg/kg clonidine) compared with preinjection (Fig. 1C, amplitude and incidence values not shown). Comparable data were found with the second assessment method because nonsignificantly (P > 0.05) different AOB scores were found postinjection compared with preinjection (Fig. 1D). Note that after administration of 5.0 mg/kg clonidine, NLM and ACOS values were apparently increased although nonsignificantly (P = 0.08).

View larger version (29K): [in this window] [in a new window]   Fig. 1. Acute effects of clonidine at 6 days post-Tx assessed in open-field and treadmill conditions. Clonidine administration at doses ranging between 0.25 and 5.0 mg/kg generally failed to induce or increase NLM, LM, ACOS, and AOB scores in open-field (A–D, respectively) and treadmill (E–H, respectively) conditions. *, P < 0.05 compared with preinjection.

View larger version (36K): [in this window] [in a new window]   Fig. 2. Acute effects of clonidine at 13 days post-Tx assessed in open-field and treadmill conditions. Clonidine administration at doses ranging between 0.25 and 5.0 mg/kg failed to induce or increase NLM, LM, ACOS, and AOB scores in open-field (A–D, respectively) and treadmill (E–H, respectively) conditions. *, P < 0.05 and **, P < 0.01 compared with preinjection.

View larger version (37K): [in this window] [in a new window]   Fig. 3. Effects of clonidine at 20 days post-Tx assessed in open-field and treadmill conditions. Clonidine administration at doses ranging between 0.25 and 5.0 mg/kg failed to induce or increase NLM, LM, ACOS, and AOB scores in open-field (A–D, respectively) and treadmill (E–H, respectively) conditions. *, P < 0.05 compared with preinjection.

View larger version (32K): [in this window] [in a new window]   Fig. 4. Effects of clonidine at 27 days post-Tx assessed in open-field and treadmill conditions. Clonidine administration at doses ranging between 0.25 and 5.0 mg/kg failed to induce or increase NLM, LM, ACOS, and AOB scores in open-field (A–D, respectively) and treadmill (E–H, respectively) conditions. *, P < 0.05 compared with preinjection.

Acute Effects Assessed in Treadmill Condition. As in open-field condition, spontaneous movements assessed in treadmill condition progressively began to occur within a few days to a few weeks post-Tx. At 6 days, 17 of 40 animals displayed a few spontaneous NLM (0.71 ± 0.16 per min) but no LM before clonidine injection (0.25–5.0 mg/kg). As in the vehicle-treated group, data from all clonidine-treated animals showed that 0.25 to 5.0 mg/kg did not significantly augment the frequency of NLM compared with preinjection (Fig. 1E, preinjection as pale gray bars and postinjection as dark gray bars). In fact, at 0.25 mg/kg, NLMs were entirely suppressed (P < 0.05) after clonidine injection compared with preinjection (Fig. 1E). Regarding LMs, none were found before or after injection of 0.25 to 5.0 mg/kg clonidine. Accordingly, at 6 days post-Tx, ACOS (generally lower than 1) did not significantly change after drug injection (0.25–5.0 mg/kg clonidine) compared with preinjection (Fig. 1G, amplitude and incidence values not shown). Comparable data were found with the other method because nonsignificantly (P > 0.05) different AOB scores were found postinjection compared with preinjection (Fig. 1H). Although AOB and ACOS values were significantly (P < 0.05) increased at 5.0 mg/kg, no LM was induced and only a slight increase of NLM/min was observed. In turn, movement amplitude elicited by injection of 5.0 mg/kg clonidine was significantly (P < 0.01) increased, which was contributed to the rise of AOB and ACOS values. In addition, NLM/min, ACOS, and AOB values and scores were significantly (P < 0.05) depressed after injection of 0.25 mg/kg clonidine.

At 13 days post-Tx, an increasing number of spontaneous NLM as well as a few LM were found. We reported 8.96 ± 1.45 NLM/min and 4.35 ± 1.48 LM/min before clonidine injection. As in open-field condition, no significant (P > 0.05) increase in NLM and LM (frequency) was found after injection of 0.25 - 5.0 mg/kg clonidine (Fig. 2, E and F, dark gray bars). Instead, a significant (P < 0.05) reduction in NLM was found at all doses, with significant levels (P < 0.05) reached at doses ranging between 0.5 and 2.5 mg/kg compared with preinjection (0.53 ± 0.27 per min versus 8.00 ± 2.04 per min, respectively). Comparable results were reported with ACOS (significantly decreased at doses ranging between 0.5 and 2.5 mg/kg), whereas with AOB scores, a significant decrease was found at doses ranging between 0.25 and 2.5 mg/kg (P < 0.05; Fig. 2, G and H).

At 20 days post-Tx, acute suppressing effects were also found after administration of clonidine (Fig. 3). It significantly (P < 0.05) decreased NLM specifically at doses ranging between 0.5 and 1.0 mg/kg (preinjection 7.25 ± 2.04 per min and postinjection 1.08 ± 0.82 per min), although clear reductions were found at all doses (Fig. 3E). LMs were also reduced at all doses (preinjection 4.35 ± 1.19 per min versus postinjection 0.77 ± 0.45 per min), although, statistically, values were either close to or above significant level (P = 0.13 and P = 0.05; Fig. 3F). A corresponding decrease in hindlimb motor scores was also found with ACOS and AOB methods (Fig. 3, G and H).

At 27 days, all values and scores were also acutely suppressed after clonidine injection (Fig. 4, E–H). On average, NLM decreased from 15.64 ± 3.45 per min before injection down to 9.07 ± 1.89 per min after injection. LM values decreased from 4.63 ± 1.58 per min down to 3.20 ± 1.82 per min after clonidine injection. A comparable decrease in ACOS and AOB scores was found with statistically significant (P < 0.05) levels reached with doses ranging between 0.25 and 1.0 mg/kg. On the other hand, nonsignificant increases of LM (frequency), ACOS, and AOB scores were detected after administration at 5.0 mg/kg. Therefore, as in open-field condition, clonidine was found in treadmill condition not to increase but to generally decrease NLM/minute, LM/minute, ACOS, and AOB values and scores at 13, 20, and 27 days post-Tx.

Again, as in open-field, amplitude and incidence values remained generally unchanged postinjection compared with preinjection, especially at 20 days post-Tx and subsequently.

Chronic Effects of Repeated Administration in Clonidine- versus Vehicle-Treated Animals. We showed, in the previous subsections, data revealing the existence of acute suppressing effects by clonidine (i.e., just after injection) on hindlimb movement expression in animals treated weekly with clonidine. In the following subsections, we will report data showing the existence of chronic changes (i.e., just before injection) by comparing results obtained from animals repeatedly treated with clonidine versus saline. In open-field condition, the results showed no significant (P > 0.05) change of NLM frequency (per minute) at all timepoints between clonidine-treated and vehicle-treated groups (Fig. 5A, comparing triangles versus circles). No significant change was found either in LM/minute, ACOS, or AOB scores when comparing data from the clonidine-treated and the vehicle-treated groups (Fig. 5, B–D). Significant differences (P < 0.001) within the clonidine-treated-groups were observed when comparing values at each time point with values obtained at 6 days post-Tx (Fig. 5, A–D, triangles). Significant (P < 0.05) increases were also found within the vehicle-treated groups, especially with NLM/minute and AOB scores (Fig. 5, A and D, circles). As mentioned already, although nonstatistically significant differences were found between both groups in open-field condition, this analysis revealed that spontaneously occurring hindlimb movements were greater (higher frequency) in the clonidine-treated groups than the control group (i.e., especially at 20 days post-Tx and subsequently), suggesting the existence of long-term effects (e.g., increased spinal cord excitability) induced by repeated clonidine administration (i.e., before injection but not immediately after injection where an acute decrease is found instead).

View larger version (31K): [in this window] [in a new window]   Fig. 5. Chronic effects by comparing values and scores preinjection in clonidine- versus vehicle-treated animals. Before clonidine administration of doses ranging between 0.25 and 5.0 mg/kg, time-dependent changes of NLM, LM, ACOS, and AOB scores in open-field (A–D, respectively) and treadmill (E–H, respectively) conditions were examined. *, P < 0.05, **, P < 0.01, and ***, P < 0.001.

In turn, statistically significant differences between the clonidine-treated and the vehicle-treated groups were found in treadmill condition (Fig. 5, E–H). Indeed, NLM/minute significantly increased (P < 0.001) with time in the clonidine-treated groups but not (P > 0.05) in the vehicle-treated group (Fig. 5E). LM/minute were also significantly greater at 13, 20, and 27 days post-Tx (*, P < 0.05 or **, P < 0.01) than at 6 days (Fig. 5F). Moreover, LM/minute were significantly greater (, P < 0.05) in the clonidine-treated groups than in the vehicle-treated group (Fig. 5F). In a similar manner, ACOS and AOB scores were also significantly (P < 0.001) increased at 13, 20, and 27 days post-Tx compared with scores at 6 days post-Tx in the clonidine-treated groups only. Despite a clear time-dependent increase of values and scores in the vehicle-treated group, only nonstatistically significant augmentations were found in this study (P > 0.05).

Late Chronic Mice Nonpreviously Treated That Were Tested at 41 and 43 Days Post-Tx Either with Clonidine or 5-HT_1A/7 Receptor Agonist. As additional control experiments, we formed two groups of Tx mice that were left untreated in their cage for nearly 6 weeks (41 days post-Tx) before testing. This was done to investigate the acute effects of clonidine in late chronic Tx animals in absence of possible repeatedly administered drug-induced effects and/or training-induced effects. Two doses were tested: a relatively small dose (0.25 mg/kg) in the first group and a high dose (5.0 mg/kg) in the second group (Fig. 6). In general, comparable effects were found (Fig. 6) compared with those reported in weekly treated animals (Figs. 1, 2, 3, 4, 5). No significant (P > 0.05) change or induction of NLM or LM was found in open-field (Fig. 6, A and B) or treadmill conditions at 0.25 or 5.0 mg/kg (Fig. 6, E and F). ACOS and AOB scores also remain unchanged (P > 0.05) after administration of 0.25 or 5.0 mg/kg clonidine, with the exception of AOB scores at 0.25 mg/kg, where a significant (P < 0.05) decrease was found after clonidine injection in open-field condition only (Fig. 6D).

View larger version (27K): [in this window] [in a new window]   Fig. 6. Effects of clonidine or 8-OH-DPAT in late chronic paraplegic mice nonpreviously treated. A single administration of low or high dose of clonidine (0.25 or 5.0 mg/kg i.p., respectively) did not induce or increase NLM or LM in open-field (A and B) and treadmill conditions (E–F). Comparable results were found with ACOS and AOB scores (C–D, open-field and G–H, treadmill). In contrast, 8-OH-DPAT was found to evoke or largely increase LM, NLM, ACOS, and AOB scores in both conditions. *, P < 0.05, **, P < 0.01, and ***, P < 0.001.

The same animals were tested again 2days later (at 43 days post-Tx) with 1 mg/kg 8-OH-DPAT, an agonist belonging to another family of ligands, the 5-HT_1A/7 receptor agonists, known at this concentration for potent CPG-activating effects (Landry et al., 2006). This was done as another form of control to make sure that these animals were indeed capable of expressing drug-induced, CPG-mediated movements. Administration of 8-OH-DPAT clearly induced hindlimb movements. NLM significantly (P < 0.05) increased from 1.93 ± 0.81 per min (preinjection) to 14.14 ± 3.55 per min (postinjection), whereas LM significantly (P < 0.01) augmented from 0.07 ± 0.07 per min (preinjection) to 4.79 ± 1.05 per min (postinjection) in open-field condition (Fig. 6, A and B, respectively). In a similar manner, ACOS and AOB scores significantly (P < 0.01 and 0.001, respectively) increased also after 8-OH-DPAT injection compared with preinjection (Fig. 6, C and D).

Comparable large changes were found after 8-OH-DPAT administration assessed in treadmill condition. NLM/minute, LM/minute, ACOS, and AOB scores all significantly (P < 0.01) and largely increased (>5-fold), clearly indicating the existence of a functional CPG network in these animals (see Fig. 6, E–H).

Clonidine-Induced Effects in I₁-Imidazoline Receptor or ₂-Adrenoceptor Antagonist-Pretreated Tx Mice. An attempt was also made to pharmacologically dissect the mechanisms of action underlying the acute suppressive effect found in this study to be induced by clonidine. First of all, 1 to 5 mg/kg moxonidine, a preferred agonist of I₁-imidazoline receptors with low affinity for ₂-adrenoceptor, was administered in Tx mice previously treated at 6 days post-Tx with clonidine. Moxonidine was found to decrease, although nonsignificantly, LM values and ACOS scores in the open-field condition (Fig. 7, B and C). NLM values did not change, but AOB scores were significantly reduced (P < 0.05) after moxonidine administration (Fig. 7, A and D). Comparable results were found in the treadmill condition (Fig. 7, E–H). In addition, two groups of Tx mice received either 1.0 mg/kg yohimbine (₂-adrenoceptor antagonist with low affinity to I₁-imidazoline receptor) or 1.0 mg/kg efaroxan (I₁-imidazoline receptor antagonist with low affinity to ₂-adrenoceptor; Haxhiu et al., 1994) followed 15 min later by 1.0 mg/kg clonidine. As illustrated in Fig. 8, NLM (Fig. 7, A and E, white bars) and LM (Fig. 8, B and F, white bars), which are typically found to spontaneously occur in these animals (>2 weeks post-Tx), were not significantly changed in the yohimbine-pretreated group (Yoh + clonidine) in both open-field and treadmill conditions (Fig. 8, A, B, E, and F). A comparable lack of effects (compared with preinjection) was found with ACOS and AOB scores (Fig. 8, C, D, G, and H). We were surprised to find that yohimbine (Fig. 8, pale gray bars), before clonidine administration, significantly increased LM values (P < 0.05) as well as ACOS and AOB scores (P < 0.01) compared with preinjection (Fig. 8, white bars). In fact, a 2- to 3-fold increase in scores and values was reported after yohimbine administration. In clear contrast, the other group of animals that received clonidine but that were pretreated instead with efaroxan displayed a large increase in motor scores and values. Figure 9 shows that, in efaroxan (1.0 mg/kg i.p.)-pretreated mice, clonidine (1.0 mg/kg i.p.) administration significantly increased (a 2–3-fold increase), at least in treadmill conditions, both NLM and LM values (Fig. 9, black bars) compared with predrug injection (Fig. 9, white bars). In addition, the corresponding ACOS and AOB scores were significantly (P > 0.05) augmented after clonidine injection in the efaroxan-pretreated group both in open-field and treadmill conditions (Fig. 9, C, D, G, and H). Efaroxan (pale gray bars), just before clonidine administration, did not significantly change scores and values compared with preinjection.

View larger version (23K): [in this window] [in a new window]   Fig. 7. Effects of moxonidine in chronic paraplegic mice. At 13 days post-Tx, moxonidine (preferred I1-imidazoline agonist, 1.0–5.0 mg/kg) administered in early chronic Tx mice previously (at 6 days post-Tx) treated with 1.0 mg/kg clonidine failed to increase NLM, LM, and ACOS scores and values in open-field (A–C) and treadmill (E–G) conditions. AOB scores were significantly decreased both in open-field and treadmill conditions (D and H) after moxonidine injection. *, P < 0.05.

View larger version (26K): [in this window] [in a new window]   Fig. 8. Effects of clonidine in chronic paraplegic mice pretreated with yohimbine. At 13 days post-Tx, blockade of 2-adrenoceptors (yohimbine, 1.0 mg/kg) in early chronic Tx mice previously (at 6 days post-Tx) treated with 1.0 mg/kg clonidine increased LM, ACOS, and AOB scores and values in open-field (A–D) and treadmill (E–H) conditions. Subsequent injection of 1.0 mg/kg clonidine in those animals demonstrated a near-complete blockade of yohimbine-induced movement. However, no significant changes after clonidine administration were found compared with preinjection. *, P < 0.05, **, P < 0.01, and ***, P < 0.001.

View larger version (25K): [in this window] [in a new window]   Fig. 9. Effects of clonidine in chronic paraplegic mice pretreated with efaroxan. At 13 days post-Tx, blockade of I1-imidazoline receptors (efaroxan, 1.0 mg/kg) in early chronic Tx mice previously (at 6 days post-Tx) treated with 1.0 mg/kg clonidine did not increase movement induction in open-field (A–D) or treadmill (E–H) conditions. In contrast, subsequent injection of 1.0 mg/kg clonidine in those animals revealed a significant increase of NLM, LM, ACOS, and AOB values and scores, especially in treadmill condition. *, P < 0.05 and **, P < 0.01.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The results clearly show that systemic administration of clonidine, an ₂-adrenoceptor agonist, does not acutely induce LM or NLM, respectively, in the hindlimbs of adult mice completely spinal cord-transected at the low-thoracic level (thoracic segment9/10). Such a lack of hindlimb movement-inducing effects was reported at low or high doses (0.25–5.0 mg/kg i.p.) both in open-field and treadmill conditions. In fact, against all expectations, occasionally occurring hindlimb movements normally found within a few days to a few weeks post-Tx (e.g., preinjection; Figs. 1A and 2A) (see also Guertin, 2005; Lapointe et al., 2006a; Ung et al., 2007) were acutely suppressed after an injection of clonidine but generally enhanced (increased frequency just before an injection) after repeated administration for a few weeks (20 days post-Tx). Moreover, using animals pretreated with selective ₂-adrenoceptor or I₁-imidazoline receptor antagonist, we found evidence suggesting that the acute suppressive effects reported in this study were largely mediated by the I₁-imidazoline receptors. Along this line of evidence, administration of moxonidine (preferred I₁-imidazoline receptor agonist) was also found to acutely suppress spontaneous movements in both open-field and treadmill conditions.

These results confirm preliminary data that were previously obtained in Dr. Rossignol's (Guertin et al., 2002) laboratory reporting comparable effects in a paraplegic mouse model. They had indeed found a lack of clonidine-induced hindlimb movements in early or late Tx mice. Yet, at 6.0 mg/kg, clonidine had been shown not to induce LM at 6 days post-Tx but to generate small amplitude NLM in a few animals tested in treadmill condition. This nicely fits with the effects detected here with 5.0 mg/kg clonidine at 6 days post-Tx, where NLMs were apparently induced (although nonstatistically significant levels were reached) in both open-field and treadmill conditions (see Fig. 1, A and D). However, this may reflect non-₂-mediated effects caused by increasingly unspecific binding at high doses. Otherwise, at all other time points, clonidine (even at 5.0 mg/kg) was not found to induce or to acutely increase spontaneously occurring NLM and LM.

A similar lack of acute CPG-activating effects has been reported in several other animal models. For instance, in an in vitro-isolated spinal cord preparation from neonatal rats, Sqalli-Houssaini and Cazalets (2000) have clearly shown that clonidine can not trigger motoneuronal rhythms and activity when administered alone or can completely suppress locomotor rhythms when coapplied with N-methyl-D-aspartate. In mudpuppies, bath application of 1 to 250 µM clonidine has also consistently failed to modulate fictive locomotor rhythms induced by N-methyl-D-aspartate in an in vitro-isolated spinal cord preparation (Fok and Stein, 2002). In a murine spinal cord preparation, clonidine was found to disrupt fictive locomotion evoked by electrical stimulation of primary afferents (Gordon and Whelan, 2006). In acutely Tx monkeys, clonidine combined with naloxone was found to trigger some nonlocomotor activity, i.e., bilaterally alternating rhythmicity in flexors but not in extensor nerves (Fedirchuk et al., 1998). Further supporting a lack of acute CPG-activating effects, clonidine has also failed to trigger reflex stepping in complete SCI individuals (Stewart et al., 1991; Dietz et al., 1995; Rémy-Néris et al., 1999).

The other striking effect reported here is the clonidine-induced suppression of spontaneously occurring movements. Although, the detailed mechanisms underlying this effect remain unknown, it may involve direct and indirect inhibition of motoneurons and sensorimotor integration. Indeed, clonidine was found to decrease, and even to antagonize, the excitatory effects of 5-hydroxytryptophan and thyrotropin-releasing hormone in adult Tx rats (Tremblay and Bedard, 1995). It was shown to also suppress synaptic transmission (mono- and poly-synaptic) to motoneurons when bath-applied in a slice preparation from adult rats (Ono and Fukuda, 1995). However, it is unclear how these mechanisms interact with those associated instead with plateau potentials and oscillations found in spinal cat motoneurons after clonidine administration (Conway et al., 1988).

In contrast, a number of studies essentially in cats have reported acute CPG-activating effects after administration of clonidine. Rossignol and collaborators (Barbeau et al., 1993; Chau et al., 1998) have shown that Tx cats trained and repeatedly treated with clonidine display enhanced locomotor recovery (larger movement amplitude) compared with animals trained only. These authors have suggested that repeated administration of clonidine may facilitate or improve the effects of training by acting upon plasticity-induced changes in sublesional spinal cord networks. The detailed mechanisms underlying these effects may be associated with increased c-fos expression in the dorsal and ventral horn areas found after chronic clonidine administration (Luo et al., 1995).

Because clonidine was administered systemically in this study, we can not also exclude the possibility of peripherally mediated actions (e.g., antihypertension; Seedat et al., 1969). In addition, we can not exclude the possibility of differential effects in other conditions as suggested by the time- and dose-dependent results reported elsewhere in DSP4-treated mice (Archer and Fredriksson, 2000). However, taken together, results from Tx cats showing prolocomotor effects at 0.15 mg/kg i.p. (e.g., Chau et al., 1998) and data from Tx mice (lack of prolocomotor effects up to 50 min postinjection; Guertin et al., 2002) suggest no significant dose-dependent (i.e., prolocomotor effects at high doses) or time-dependent effects (i.e., prolocomotor effects at 30 or 45 min postinjection) induced by clonidine after spinal cord transection.

A possible explanation and a reconciliating point of view may be that clonidine displays different effects, including reflex modulation (e.g., decreasing spasticity, as clearly shown in incomplete SCI patients (Stewart et al., 1991; Dietz et al., 1995; Rémy-Néris et al., 1999) and partial CPG activation, when combined with training-induced plasticity, and/or unspecific but intense sensory stimulation (i.e., with modalities other than those used in this study such as phasic instead of tonic). Although some of the stimuli traditionally used with clonidine in cats (e.g., tail or perineal pinching and/or abdominal stimulation) (see Forssberg and Grillner, 1973; Chau et al., 1998; Marcoux and Rossignol, 2000) are difficult to perform in smaller animal species such as in mice, an attempt was made in this study to examine at least the contribution of sensory inputs associated with the motor-driven treadmill in clonidine-treated animals. However, the results revealed comparable suppressing effects on hindlimb movement expression acutely induced by clonidine in open-field versus treadmill conditions, suggesting that natural sensory activation associated with the treadmill belt does not significantly contribute to clonidine-induced effects in Tx animals. On the other hand, the effects obtained with selective antagonists provided clear evidence suggesting that the suppressive effects of clonidine were mediated by the I₁-imidazoline receptors. Indeed, a blockade of these acute suppressive effects normally induced by clonidine was found in the I₁-imidazoline-pretreated group (Fig. 8) and not in the yohimbine-pretreated group (Fig. 7). This said, it remains unclear why more NLM were found in the yohimbine-treated group just before clonidine administration. Interspecies differences in the level of expression of I₁-imidazoline receptors have been reported (higher levels in mouse versus rat brains; Tolentino-Silva et al., 2000), which may perhaps also explain that clonidine-induced suppressive effects have never been found in Tx cats (i.e., if similar differences were to exist between other species versus cats). This said, different experimental conditions used generally in the cat model (tail or perineal pinching, body weight support, etc.) compared with other models may also explain this lack of suppressive effects in cats. Other explanations may include a slightly different role played by these receptors in cats versus mice. For instance, the well known hypotensive effect induced by clonidine was reported to be mediated by ₂-adrenoceptors in cats (Ally, 1997) and by imidazoline receptors in mice (Ernsberger et al., 1990).

Another important finding in this study is that clonidine-treated animals displayed greater spontaneous recovery levels than vehicle-treated ones. This was found by comparing recovery levels (NLM, LM, ACOS, and AOB values and scores) at 6, 13, 20, or 27 days just before drug versus vehicle injection. To our knowledge, comparable experiments and analyses have never been done in untrained and nonstimulated Tx animals. These results strongly suggest that this greater level of spontaneous recovery (increased movement frequencies and motor scores) can be attributed mainly to repeated administration of clonidine and not to training-induced plasticity (i.e., since untrained animals) or sensory stimulation-induced effects (i.e., nonstimulated animals). In turn, this may also suggest that some of the outstanding effects in repeatedly clonidine-treated cats (i.e., a few weeks post-Tx) are due to clonidine-induced plasticity phenomena that may, as shown here, facilitate the spontaneous re-expression of hindlimb movements.

In conclusion, this study clearly shows that clonidine (0.25–5.0 mg/kg i.p.) administered in early or late chronic spinal cord-transected mice acutely suppresses spontaneously occurring hindlimb movements. However, before injection, animals repeatedly treated with clonidine could display greater spontaneous recovery levels than controls, strongly suggesting that, in the absence of training-induced or sensory stimulation-induced effects, clonidine may nonetheless promote sublesional plasticity changes that can facilitate the re-expression of spontaneous CPG and/or motoneuron activity.

	Footnotes

 
This study was supported by the Canadian Institutes of Health Research and the Fond de Recherche en Sante du Quebec (FRSQ). N.P.L. and R.-V.U. received studentships from the FRSQ.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.134874.

ABBREVIATIONS: SCI, spinal cord injury; Tx, spinal cord-transection; CPG, central pattern generator; 8-OH-DPAT, 8-hydroxy-2-(di-N-propylamino)-tetralin; ACOS, Average Combined Score; NLM, nonlocomotor movement; LM, locomotor-like movement; AOB, Antri, Orsal, Barthe locomotor scale; 5-HT_1A/7, serotonin-type 1A and 7 receptor.

Address correspondence to: Dr. Pierre A. Guertin, Neuroscience Unit, RC-9800, Laval University Medical Center, 2705 Laurier Blvd., Quebec City, Quebec, Canada G1V 4G2. E-mail: Pierre.Guertin{at}crchul.ulaval.ca

	References

Top Abstract Materials and Methods Results Discussion References

 

Ally A (1997) Cardiovascular effects of central administration of clonidine in conscious cats. Brain Res 761: 283-289.[CrossRef][Medline]

Antri M, Orsal D, and Barthe JY (2002) Locomotor recovery in the chronic spinal rat: effects of long-term treatment with a 5-HT2 agonist. Eur J Neurosci 16: 467-476.[CrossRef][Medline]

Archer T and Fredriksson A (2000) Effects of clonidine and -adrenoceptor antagonists on motor activity in DSP4-treated mice I: dose-, time-, and parameter-dependency. Neurotox Res 1: 235-247.[Medline]

Barbeau H, Julien C, and Rossignol S (1987) The effects of clonidine and yohimbine on locomotion and cutaneous reflexes in the adult chronic spinal cat. Brain Res 437: 83-96.[CrossRef][Medline]

Barbeau H, Chau C, and Rossignol S (1993) Noradrenergic agonists and locomotor training affect locomotor recovery after cord transection in adult cats. Brain Res Bull 30: 387-393.[CrossRef][Medline]

Buerkle H and Yaksh TL (1998) Pharmacological evidence for different alpha 2-adrenergic receptor sites mediating analgesia and sedation in the rat. Br J Anaesth 81: 208-215.[Abstract/Free Full Text]

Chau C, Barbeau H, and Rossignol S (1998) Effects of intrathecal alpha-2 and alpha-2-noradrenergic agonists and norepinephrine on locomotion in chronic spinal cats. J Neurophysiol 79: 2941-2963.[Abstract/Free Full Text]

Conway BA, Hultborn H, Kiehn O, and Mintz I (1988) Plateau potentials in alpha-motoneurones induced by intravenous injection of L-dopa and clonidine in the spinal cat. J Physiol 405: 369-384.[Abstract/Free Full Text]

Dietz V, Colombo G, Jensen L, and Baumgartner L (1995) Locomotor capacity of spinal cord in paraplegic patients. Ann Neurol 37: 574-582.[CrossRef][Medline]

Ernsberger P, Meeley MP, Mann JJ, and Reis DJ (1987) Clonidine binds to imidazole binding sites as well as alpha 2-adrenoceptors in the ventrolateral medulla. Eur J Pharmacol 134: 1-13.[CrossRef][Medline]

Ernsberger P, Giuliano R, Willette RN, and Reis DJ (1990) Role of imidazole receptors in the vasodepressor response to clonidine analogs in the rostral ventrolateral medulla. J Pharmacol Exp Ther 253: 408-418.[Abstract/Free Full Text]

Fedirchuk B, Nielsen J, Petersen N, and Hultborn H (1998) Pharmacologically evoked fictive motor patterns in the acutely spinalized marmoset monkey (Callithrix jacchus). Exp Brain Res 122: 351-361.[CrossRef][Medline]

Fok M and Stein RB (2002) Effects of cholinergic and noradrenergic agents on locomotion in the mudpuppy (Necturus maculatus). Exp Brain Res 145: 498-504.[CrossRef][Medline]

Forssberg H and Grillner S (1973) The locomotion of the acute spinal cat injected with clonidine i.v. Brain Res 50: 184-186.[CrossRef][Medline]

Galeotti N, Bartolini A, and Ghelardini C (2004) Alpha-2 agonist-induced memory impairment is mediated by the alpha-2A-adrenoceptor subtype. Behav Brain Res 153: 409-417.[CrossRef][Medline]

Gordon IT and Whelan PJ (2006) Monoaminergic control of cauda-equina-evoked locomotion in the neonatal mouse spinal cord. J Neurophysiol 96: 3122-3129.[Abstract/Free Full Text]

Guertin PA (2005) Semiquantitative assessment of hindlimb movement recovery without intervention in adult paraplegic mice. Spinal Cord 43: 162-166.[CrossRef][Medline]

Guertin PA, Leblond H, L'Esperance M, Provencher J, Lebel F, Orsal D, and Rossignol S (2002) Effects induced by serotonergic and -2 noradrenergic drugs on locomotor recovery in paraplegic mice; 2nd Invitational Annual Meeting; 2002 Jun 9–12; Ottawa, ON, Canada. Human Frontier Science Program, Strasbourg, France.

Haxhiu MA, Dreshaj I, Schafer SG, and Ernsberger P (1994) Selective antihypertensive action of moxonidine is mediated mainly by I1-imidazoline receptors in the rostral ventrolateral medulla. J Cardiovasc Pharmacol 24: S1-S8.

Landry ES, Lapointe NP, Rouillard C, Levesque D, Hedlund PB, and Guertin PA (2006) Contribution of spinal 5-HT1A and 5-HT7 receptors to locomotor-like movement induced by 8-OH-DPAT in spinal cord-transected mice. Eur J Neurosci 24: 535-546.[CrossRef][Medline]

Lapointe NP, Ung RV, Bergeron M, Cote M, and Guertin PA (2006a) Strain-dependent recovery of spontaneous hindlimb movement in spinal cord transected mice (CD1, C57BL/6, BALB/c). Behav Neurosci 120: 826-834.[CrossRef][Medline]

Lapointe NP, Ung RV, and Guertin P (2006b) The -2 adrenergic agonist, clonidine, does not acutely or chronically restore locomotor function in complete paraplegic mice. 36th Annual Meeting of Society for Neuroscience; 2006 Oct 14–18; Atlanta, GA. Abstract no. 646.13, Society for Neuroscience, Washington, DC.

Luo L, Ji RR, Zhang Q, Iadarola MJ, Hokfelt T, and Wiesenfield-Hallin Z (1995) Effect of administration of high dose intrathecal clonidine or morphine prior to sciatic nerve section on c-Fos expression in rat lumbar spinal cord. Neuroscience 68: 1219-1227.[CrossRef][Medline]

MacDougall AI, Addis GJ, MacKay N, Dymock IW, Turpie AG, Ballingall DL, MacLennan WJ, Whiting B, and MacArthur JG (1970) Treatment of hypertension with clonidine. Br Med J 3: 440-442.[Abstract/Free Full Text]

Marcoux J and Rossignol S (2000) Initiating or blocking locomotion in spinal cats by applying noradrenergic drugs to restricted lumbar spinal segments. J Neurosci 20: 8577-8585.[Abstract/Free Full Text]

Martin TJ and Eisenach JC (2001) Pharmacology of opioid and nonopioid analgesics in chronic pain states. J Pharmacol Exp Ther 299: 811-817.[Abstract/Free Full Text]

Ono H and Fukuda H (1995) Pharmacology of descending noradrenergic systems in relation to motor function. Pharmacol Ther 68: 105-112.[CrossRef][Medline]

Rémy-NérisO, Barbeau H, Daniel O, Boiteau F, and Bussel B (1999) Effects of intrathecal clonidine injection on spinal reflexes and human locomotion in incomplete paraplegic subjects. Exp Brain Res 129: 433-440.[CrossRef][Medline]

Rossignol S, Giroux N, Chau C, Marcoux J, Brustein E, and Reader TA (2001) Pharmacological aids to locomotor training after spinal injury in the cat. J Physiol 533: 65-74.[Abstract/Free Full Text]

Sabetkasaie M, Khansefid N, and Ladgevardi MA (2007) Possible role of NMDA receptors in antinociception induced by rilmenidine in mice in the formalin test. Eur J Pain 11: 535-541.[CrossRef][Medline]

Seedat YK, Vawda EI, Mitha S, and Ramasar R (1969) Clonidine. Lancet 2: 591.[Medline]

Shannon HE and Lutz EA (2000) Effects of the I(1) imidazoline/alpha(2)-adrenergic receptor agonist moxonidine in comparison with clonidine in the formalin test in rats. Pain 85: 161-167.[CrossRef][Medline]

Sqalli-Houssaini Y and Cazalets JR (2000) Noradrenergic control of locomotor networks in the in vitro spinal cord of the neonatal rat. Brain Res 852: 100-109.[CrossRef][Medline]

Stewart JE, Barbeau H, and Gauthier S (1991) Modulation of locomotor patterns and spasticity with clonidine in spinal cord injured patients. Can J Neurol Sci 18: 321-332.[Medline]

Tolentino-Silva FP, Haxhiu MA, Waldbaum S, Dreshaj IA, and Ernsberger P (2000) ₂-Adrenergic receptors are not required for central anti-hypertensive action of moxonidine in mice. Brain Res 862: 26-35.[CrossRef][Medline]

Tremblay LE and Bedard PJ (1995) Action of 5-hydroxytryptamine, substance P, thyrotropin releasing hormone and clonidine on spinal neuron excitability. J Spinal Cord Med 18: 42-46.[Medline]

Ung RV, Lapointe NP, Tremblay C, Larouche A, and Guertin PA (2007) Spontaneous recovery of hindlimb movement in completely spinal cord transected mice: a comparison of assessment methods and conditions. Spinal Cord 45: 367-379.[Medline]

Zarrindast MR, Ramezani-Tehrani B, and Ghadimi M (2002) Effects of adrenoceptor agonists and antagonists on morphine-induced Straub tail in mice. Pharmacol Biochem Behav 72: 203-207.[CrossRef][Medline]

Zhang Y, Ji SR, Wu CY, Fan XH, Zhou HJ, and Liu GL (2007) Observation of locomotor functional recovery in adult complete spinal rats with BWSTT using semiquantitative and qualitative methods. Spinal Cord 45: 496-501.[CrossRef][Medline]

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