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Protein phosphatase 2A regulates central sensitization in the spinal cord of rats following intradermal injection of capsaicin

Protein phosphatase 2A regulates central sensitization in the spinal cord of rats following... Background: Intradermal injection of capsaicin into the hind paw of rats induces spinal cord central sensititzation, a process in which the responsiveness of central nociceptive neurons is amplified. In central sensitization, many signal transduction pathways composed of several cascades of intracellular enzymes are involved. As the phosphorylation state of neuronal proteins is strictly controlled and balanced by the opposing activities of protein kinases and phosphatases, the involvement of phosphatases in these events needs to be investigated. This study is designed to determine the influence of serine/threonine protein phosphatase type 2A (PP2A) on the central nociceptive amplification process, which is induced by intradermal injection of capsaicin in rats. Results: In experiment 1, the expression of PP2A protein in rat spinal cord at different time points following capsaicin or vehicle injection was examined using the Western blot method. In experiment 2, an inhibitor of PP2A (okadaic acid, 20 nM or fostriecin, 30 nM) was injected into the subarachnoid space of the spinal cord, and the spontaneous exploratory activity of the rats before and after capsaicin injection was recorded with an automated photobeam activity system. The results showed that PP2A protein expression in the spinal cord was significantly upregulated following intradermal injection of capsaicin in rats. Capsaicin injection caused a significant decrease in exploratory activity of the rats. Thirty minutes after the injection, this decrease in activity had partly recovered. Infusion of a phosphatase inhibitor into the spinal cord intrathecal space enhanced the central sensitization induced by capsaicin by making the decrease in movement last longer. Conclusion: These findings indicate that PP2A plays an important role in the cellular mechanisms of spinal cord central sensitization induced by intradermal injection of capsaicin in rats, which may have implications in clinical pain therapy. Introduction kinases and phosphatases (PP), respectively. Such reac- The phosphorylation and dephosphorylation of proteins tions seem to modulate the function of several proteins are reversible processes, catalyzed by opposing protein involved in synaptic transmission, including voltage- Page 1 of 12 (page number not for citation purposes) Molecular Pain 2006, 2:9 http://www.molecularpain.com/content/2/1/9 Strong noxious stimulation of peripheral tissues, such as intradermal injection of capsaicin into the hind paw of PP2A 36 KD rats, induces spinal cord central sensititzation, a process in which an amplified responsiveness of central nociceptive β-actin 42 KD neurons is found. It has been verified that signal transduc- 5 15 30 60 90 5 15 30 60 90 tion pathways involved in central sensitization are com- Vehicle injection Capsaicin injection posed of several cascades of enzymes that activate intracellular protein kinases. As the phosphorylation state of neuronal proteins is strictly controlled and balanced by the opposing activities of protein kinases and phos- phatases, the involvement of phosphatases in these events needs to be investigated. In the present study, we evaluated the effects of noxious 5 m i n 15 m i n 3 0 m i n 60 m i n 90 m i n stimulation of peripheral tissues on the expression of Tim e s PP2A protein in the spinal cord of rats. We also tested the E and p Figure 1 xpression of PP2A pr eriphery 90 minutes following vehicle otein in the spinal cor or capsaicin injection in d at 5, 15, 30, 6 the 0, effects of two inhibitors of PP2A, okadaic acid and fos- Expression of PP2A protein in the spinal cord at 5, 15, 30, 60, triecin, on the induction and maintenance of central sen- and 90 minutes following vehicle or capsaicin injection in the sitization induced by capsaicin injection, using the periphery. (A) Immunoreactive bands of rat spinal cord tissue spontaneous exploratory activity test. The inhibitors were (L3–L6) against PP2A antibody at 5, 15, 30, 60, and 90 min- administered through an intrathecal catheter to detect the utes after capsaicin or vehicle injection; the lower row shows role of the spinal cord in changes in these behaviors. the bands produced by the antibody to β-actin as a loading control at the same times. (B) Bar graph summarizing the rel- Results ative density of the immunoblots of rat spinal cord tissue Increased expression of PP2A in spinal cord after capsaicin (L3–L6) with the PP2A antibody to that of β-actin at 5, 15, injection 30, 60, and 90 minutes after capsaicin or vehicle injection. Open bar: vehicle injection, filled bar: capsaicin injection. N = To determine if the expression of PP2A protein in the lum- 6 in each group; the relative densities are expressed as mean ber spinal cord of rats changes following noxious stimula- ± SEM. *p < 0.05. tion with intradermal injection of capsaicin, a Western blot analysis was performed. The Western blots in Fig. 1A show that the expression of PP2A in the lumbar segments (L -L ) of the spinal cord tissue was detected at around 36 3 6 kDa. The relative density of immunoblots of PP2A protein gated and ligand-gated channels, ionotropic and metabo- from rat spinal cord tissue after intradermal vehicle or tropic neurotransmitter receptors, proteins involved in capsaicin injection is summarized in Fig 1B. Statistical neurotransmitter release and transport, and cytoskeletal analysis of these data showed that intradermal injection proteins [1-8]. These proteins play an important role in of capsaicin causes a significant increase in expression of the control of many intracellular events. Serine/threonine PP2A protein in a time-dependent manner compared protein phosphatase, which dephosphorylates serine and with the vehicle injection group. The PP2A protein started threonine protein residues, can be divided into PP1, to increase in spinal cord tissue by 15 min, peaked at 30 PP2A, PP2B, PP2C, PP4, PP5, PP6 and PP7 [2]. Among min, and the increase continued for more than 60 min them, PP2A is the most abundant phosphatase in mam- following intradermal injection of capsaicin. Vehicle malian cells and is expressed at high levels in the central injection did not result in an obvious increase in the nervous system [1,6]. PP2A regulates many fundamental expression of PP2A protein in the lumbosacral spinal cellular processes, such as cell division, signal transduc- cord. In addition, the expression of β-actin did not show tion, gene expression, development, the cell cycle, exocy- any significant change in any of the groups. tosis, and apoptosis [2,6,9-12]. The function of this protein phosphatase can be inhibited by several drugs, The effect of intrathecal catheter implantation on such as okadaic acid (OA), which is a cell permeable mol- exploratory behavior ecule that inhibits PP2A in vitro at much lower concentra- The spontaneous activity of rats and all six behavioral tions than PP1 (1:100) [13,14]. Fostriecin is another cell parameters were examined both before and 5 days after permeable protein phosphatase inhibitor. Since this mol- intrathecal implantation of a catheter. Fig. 2 demonstrates 4 5 ecule is on the order of 10 –10 times more selective for that motor function was not impacted by the intrathecal PP2A than PP1, fostriecin is the most selective inhibitor injection procedure. Compared to the naïve rats, the gen- known for any member of this class of phosphatases [13]. eral exploratory behaviors of rats with intrathecal implan- Page 2 of 12 (page number not for citation purposes) Relative optical density Molecular Pain 2006, 2:9 http://www.molecularpain.com/content/2/1/9 A. Total Activity B. Distance Traveled Naive i.t. catheter 3000 1000 Capsaicin 0 0 C. Active Time D. Resting Time * 1500 E. Rearing Time F. Rearing Events n Figure 2 Eo xploratory a vel chamber ctivity during a consecutive period of 45 minutes (9 intervals, 5 min/interval) after introduction of the rat into the Exploratory activity during a consecutive period of 45 minutes (9 intervals, 5 min/interval) after introduction of the rat into the novel chamber. Compared to naïve rats (n = 14), intrathecal catheter implanted rats (n = 14) do not have significant alterations in exploratory activity. Rats with intradermal injection of capsaicin (1%, 50 µl) (n = 6) demonstrated a reduction of exploratory activity, including decreased number of photobeams broken (A), shortened travel distance (B), decreased active time (C), increased resting time (D), shortened rearing time (E), and decreased number of rearing events (F). The capsaicin-injected group was compared to the other two groups. *p < 0.05. Page 3 of 12 (page number not for citation purposes) Active Time (sec.) Number of Beams Broken Rearing Time (sec.) Resting Tiem (sec.) Distance Traveled (inches) Number of Rearing Molecular Pain 2006, 2:9 http://www.molecularpain.com/content/2/1/9 B. Distance Traveled A. Total Activity i.t. catheter i.t. catheter+OA C. Active Time Resting Time D. E. Rearing Time F. Rearing Events 800 500 0 0 E (9 inter Figure 3 ffect of th vals, 5 min/interval) e PP2A inhibitor, okadaic acid (OA, 20 nM) on exploratory activity of rats during a consecutive period of 45 minutes Effect of the PP2A inhibitor, okadaic acid (OA, 20 nM) on exploratory activity of rats during a consecutive period of 45 minutes (9 intervals, 5 min/interval). Compared to the intrathecal catheter implanted rats (n = 14), pretreatment with OA through an intrathecal catheter (n = 7) did not produce significant alterations in exploratory activity. tation, such as the total activity (1874 ± 146), distance in consecutive 45-minute intervals (n = 13) were not sig- traveled (668 ± 29), and length of active time (1675 ± 42) nificantly different. In addition, no significant decreases Page 4 of 12 (page number not for citation purposes) Active Time (sec.) Number of Beams Broken Rearing Time (sec.) Resting Time (sec.) Distance Traveled (inches) Number of Rearing Molecular Pain 2006, 2:9 http://www.molecularpain.com/content/2/1/9 Total Activity B. Distance Traveled A. ACSF+CAP. OA+CAP. Fostriecin+CAP. D. Resting Time C. Active Time 900 * E. Rearing Time F. Rearing Events E dur Figure 4 ffect ing s of two PP2A a consecutive per inhibitor iod of 45 mi s, OA an nutes d fostriecin, on the reduced (9 intervals, 5 min/interval) exploratory activity of rats initiated by capsaicin injection Effects of two PP2A inhibitors, OA and fostriecin, on the reduced exploratory activity of rats initiated by capsaicin injection during a consecutive period of 45 minutes (9 intervals, 5 min/interval). Compared to the rats in the ACSF-pretreated group (n = 7), the rats with either OA (20 nM, n = 7) or fostriecin (30 nM, n = 5) pretreatment displayed significantly decreased explor- atory activity, including a decreased number of photobeams broken (A), shortened distance traveled (B), decreased active time (C), increased resting time (D), shortened rearing time (E), and decreased number of rearing events (F). *p < 0.05. Page 5 of 12 (page number not for citation purposes) Active Time (sec.) Number of Beams Broken Rearing Time (sec.) Resting Time (sec.) Distance Traveled (inches) Number of Rearing Molecular Pain 2006, 2:9 http://www.molecularpain.com/content/2/1/9 were found in other parameters. On the average, rearing Spontaneous exploratory activity in rats with OA pretreat- events and rearing times of 267 ± 16/45 min and 451 ± ment was greatly affected compared to the control group. 77/45 min were found in naïve rats; they were 221 ± 19/ Total activity significantly decreased from 1123 ± 160 to 45 min and 424 ± 61/45 min, respectively, in implanted 570 ± 89; consequently the distance traveled decreased rats. greatly from 426 ± 41 to 225 ± 37; active time decreased from 1200 ± 97 to 797 ± 123; and the resting time Intradermal injection of capsaicin changes exploratory increased from 1500 ± 97 to 1903 ± 123 in consecutive behavior of rats 45-minute intervals. Rearing time in the OA group was After noxious stimulation by intradermal injection of cap- also reduced greatly from 193 ± 26 to 39 ± 9, and the rear- saicin in the left hind paw of rats that were implanted ing events were reduced from 125 ± 33 to 36 ± 9. The intrathecally with a catheter, spontaneous exploratory measurements in the fostriecin-pretreated group were also activity was severely affected compared to the naïve and significantly reduced compared to the control. For com- intrathecally implanted rats (Fig. 2). Total activity greatly parison, the total activity, distance traveled, active time, decreased from 1874 ± 147 to 1022 ± 81, and distance resting time, rearing time, and rearing events in fostriecin traveled decreased significantly from 668 ± 29 to 495 ± 9 group were 674 ± 73, 286 ± 28, 900 ± 60, 1800 ± 60, 65 ± in consecutive 45-minute intervals (Fig. 2A and 2B). 21, 60 ± 18 respectively. Active time decreased from 1675 ± 42 to 1401 ± 25, whereas resting time increased from 1025 ± 42 to 1299 ± The line graphs in Fig. 5 demonstrate the changes in 25 (Fig. 2C and 2D). Rearing activity was also significantly exploratory behavior at each 5-min interval. When rats impacted by capsaicin injection. Rearing time decreased were placed in a novel environment, the spontaneous greatly from 424 ± 61 to 206 ± 6, and rearing events exploratory activity was high initially and slowly declined decreased from 221 ± 19 to 116 ± 13 (Fig. 2E and 2F). over the first 30 min. The activity in all groups of rats in the current study showed this same temporal pattern. The The effect of intrathecal administration of a general total entries, distance traveled, active time, rearing time, inhibitor of PP2A on exploratory behavior and rearing events were high in the first 20–25 min and OA, a nonselective inhibitor of PP2A and PP1, was given then declined. In the groups treated with PP2A inhibitors, intrathecally in a group of seven rats. Fig. 3 demonstrates the exploratory activity was decreased compared with the that administration of OA had no effect on the spontane- ACSF-treated group. This reduction lasted throughout the ous exploratory activity of the rats. None of the activity 45-min block. parameters in OA-administered rats, such as total activity (1628 ± 170), distance traveled (596 ± 40), active time Discussion (1603 ± 76), resting time (1097 ± 76), rearing time (373 The results of this study showed that PP2A in the spinal ± 58), and rearing events (198 ± 18) in consecutive 45- cord plays an important role in the signal cascades that are minute intervals was significantly different when com- activated following intradermal injection of capsaicin in pared to the intrathecally implanted group. the hind paw of rats. Based on the data presented, expres- sion of PP2A in the spinal cord of rat starts to increase by Decreased exploratory activity induced by capsaicin was 15 minutes after capsaicin injection and starts to return to potentiated by intrathecal administration of PP2A baseline by about 1 hour. inhibitors The bar graphs in Fig. 4 show the effects of PP2A inhibi- With capsaicin injection, the spontaneous exploratory tors on the spontaneous exploratory activity of rats fol- activity of rats was significantly reduced due to nocicep- lowing capsaicin injection. Reduced exploratory behavior tion. Following the injection, the number of rearing induced by capsaicin was aggravated by both the general events and the rearing time were decreased. Measures of inhibitor of PP1 and PP2A, OA, and the specific inhibitor active time, total entries, and distance traveled were also of PP2A, fostriecin. decreased despite the fact that hind limb locomotor func- tion was not impacted. In this model, secondary mechan- Three groups of rats were used in this experiment. Pre- ical allodynia and hyperalgesia and primary thermal treatment with ACSF administered through the intrathe- hyperalgesia would have occurred following capsaicin cal catheter (n = 7) was done in the control group; OA (n injection. So, during the process of central sensitization, a = 9) or fostriecin (n = 5) was administered in the two concurrent decrease in exploratory behavior would be experimental groups. Thirty minutes after the pretreat- expected, as seen in the current study. The reduction in ment with drugs infused through the intrathecal catheter, exploratory activity seen in our model is in agreement capsaicin was injected intradermally in the left hind paw with previous reports of exploratory behavior in other of the rats. Animals were put into an activity chamber 30 pain models. Palecek et al. [15] found a decrease in min after the injection to monitor the movements. exploratory behavior following pain induced by colon Page 6 of 12 (page number not for citation purposes) Molecular Pain 2006, 2:9 http://www.molecularpain.com/content/2/1/9 A Total Activity B Distance Traveled ACSF+CAP. OA+CAP. Fostriecin+CAP. ^ 90 * * * ^ 60 * ^ ^ ^ * * * * ^ ^ ^ * ^ 100 ^ C Active Time D Resting Time * ^ * * 250 250 ^ ^ * ^ * ^ 200 200 * * ^ ^ 150 ^ * * ^ ^ 100 100 50 50 0 0 E Rearing Time F Rearing Events 100 60 ^ 30 40 * * ^ ^ * * * ^ ^ * * 20 ^ 0 0 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 Time (min) Time (min) ity induced by Figure 5 Effect of intrath capsaic ecal infusion in (0 of the phosphatase inhibitors okadaic acid and fostriecin, or ACSF on decreased exploratory activ- Effect of intrathecal infusion of the phosphatase inhibitors okadaic acid and fostriecin, or ACSF on decreased exploratory activ- ity induced by capsaicin (0.1%, 50 µl) injection. Thirty min after capsaicin injection, okadaic acid (20 nM), fostriecin (30 nM), or ACSF was infused into the subarachnoid space of the spinal cord at the L5 segmental level of the rats. Compared to ACSF (n = 7), six parameters of spontaneous activity of the rats with okadaic acid (n = 7) and fostriecin (n = 5) infusion showed a signifi- cant decrease at every time interval. Each point represents the mean ± S.E.M. *p < 0.05. Page 7 of 12 (page number not for citation purposes) Rearing Time (sec.) Active Time (sec.) Number of Beams Broken Resting Time (sec.) Distance Traveled (inches) Number of Rearing Molecular Pain 2006, 2:9 http://www.molecularpain.com/content/2/1/9 inflammation and distention. Zhang et al. [16] showed Capsaicin injection reduces exploratory activity of rats reduced exploratory behavior in a model of pancreatitis. In the present study, an automated photobeam activity The decrease of movement with a normal ability to move system was used to test the rodent exploratory activity to results in less active time and more resting time, implying evaluate behavioral changes induced by intradermal the development of pain following capsaicin injection. injection of capsaicin. The activity system was a useful Intrathecal administration of PP2A inhibitors signifi- tool for measurement of the exploratory activity for com- cantly aggravates pain, leading to a decrease in the total parisons of control rats and rats with noxious stimulation activity, traveled distance, active time, rearing time, rear- of peripheral tissues, with or without pharmacological ing events, and an increase in the resting time of rats. treatments. Capsaicin injection reduced the exploratory activity of rats by decreasing the total activity, traveled dis- PP2A expression in the spinal cord after noxious stimuli tance, active time, rearing time, and rearing events, while Intradermal injection of capsaicin is a useful and reversi- increasing the resting time in a 45-min period. Intrader- ble experimental model for the study of an inflammatory mal injection of capsaicin is a noxious stimulus that pain state that includes hyperalgesia and allodynia [17- evokes not only ongoing pain (which lasts for about 15– 20]. In the capsaicin model, central sensitization can be 20 minutes), but also a pain state in which there is initiated by neural activity originating in the area of injec- increased sensitivity to mechanical stimuli. Intradermal tion and carried by chemosensitive afferent fibers. The injection of capsaicin can cause changes in behavioral whole process relies on central pain mechanisms. It occurs responses of rats and increase responses of nociceptive after prolonged C-nociceptor input and depends on acti- projection neurons in dorsal horn of the spinal cord at a vation of wide dynamic range neurons in the dorsal horn cellular level [15,17-20,22]. In our experiment, the of the spinal cord [21,22]. During central sensitization, a exploratory activity of animals was tested 30 min after variety of signal transduction pathways in central neurons capsaicin injection. The changes of activity reflect the pain are activated, through which the excitability of central induced by mechanical stimulation of the paw during neurons is enhanced. It has been demonstrated that pro- movement in the activity chamber instead of the pain tein kinase C, NO/protein kinase G, protein kinase A, and evoked directly by the intradermal injection. In previous calcium/calmodulin dependent kinase II (CaMKII) are all studies in our laboratory, intradermal injection of capsai- involved in this process. In addition, several types of iono- cin did not cause any systemic effect, and the handling of tropic glutamate receptors become phosphorylated dur- rats during the injection had no effect [15]. On the other ing central sensitization [20,23-31]. hand, the exploratory activity was not affected by the sham injection of vehicle (data not shown), which is con- In this study, 15 minutes after capsaicin injection into the sistent with the results of Palecek et al. [15]. The behavio- hind paw of rats, PP2A expression in the spinal cord is ral changes in exploratory activity are presumably due to upregulated compared with the control group with a vehi- nociceptive signals induced by capsaicin injection, and cle injection. In such a the short time period, the increased the changes reflect primary and/or secondary mechanical expression may not only reflect an increase of PP2A pro- allodynia and hyperalgesia. tein synthesis but also the posttranslational modification of PP2A subunits. The catalytic subunits of PP2A (PP2Ac) PP2A inhibitors potentiate the reduction of exploratory can be regulated mainly by three posttranslational modi- activity that is induced by capsaicin injection In the present study, PP2A inhibitors, either OA or fos- fications: methylation, phosphorylation of tyrosine and phosphorylation of threonine. By these modifications, triecin, did not themselves reduce the exploratory activity PP2A activity and function can be regulated [32]. The anti- of the animals. On the other hand, pretreatment with body that we used in the Western Blot study recognizes PP2A inhibitors through an intrathecal catheter extended the PP2A at 36 KDa, and it preferentially recognizes the duration of the decreases of the behavior induced by demethylated PP2A. This suggests that the increased PP2A capsaicin. This suggests that without the peripheral nox- expression may partly reflect an increased demethylation ious stimuli, the reactions that PP2A modulated would of PP2A. not initiate central sensitization directly, but they may be involved in the process of central sensitization by limiting The increased PP2A protein expression peaked at 30 min the duration of this process. and it continued at least through 1 h following intrader- mal injection of capsaicin. This suggests that PP2A may It is well known that protein phosphatases function as key also be involved in central sensitization after intradermal molecules in essential regulatory mechanisms in many injection of capsaicin through modulation of the phos- cellular processes. Serine/threonine protein phosphatases phorylation state of some critical proteins in the spinal remove phosphate groups from either serine or threonine cord. sites following phosphorylation of target proteins and thus reverse protein phosphorylation [1,5,6,8,33,34]. Page 8 of 12 (page number not for citation purposes) Molecular Pain 2006, 2:9 http://www.molecularpain.com/content/2/1/9 Since protein dephosphorylation is an essential post- catheter was inserted into the subarachnoid space of the translational regulatory mechanism in signal transduction spinal cord for the administration of drugs at the L5 seg- in the central nervous system that reverses the process of mental level according to the protocol of Yaksh et al. [42] protein phosphorylation, protein phosphatases which as modified by LoPachin and Rudy [43]. To implant a mediate this process have gained more attention because catheter, the anesthetized rat was mounted in a stereotaxic their role in regulating protein phosphorylation is as instrument to hold the rat's head firmly. A midline inci- important as that of protein kinases. PP2A is one of the sion was made beginning at a line joining the ears and most important protein phosphatases in the central nerv- extending about 2 cm in a caudal direction. The fascia was ous system, and it can regulate the activities of many retracted from the skull. A superficial layer of neck mus- kinases both in vivo and in vitro, such as ERK/MAPKs, PKA, cles was exposed by the initial incision of the skin, and the PKC, IKB kinase, and Ca/calmodulin-dependent kinase muscles were separated by a midline incision beginning at [35-37]. Currently, glutamate receptors have become a the occipital crest and extending caudally about 2 cm. The main focus in pain research. The increases in responsive- separation of the superficial musculature exposed an ness of dorsal horn neurons to mechanical stimulation of underlying layer of muscles which could be easily sepa- the skin after intradermal injection of capsaicin are due in rated along the midline by blunt dissection. Then, the part to the upregulation of phosphorylated glutamate muscles were freed from their point of origin on the occip- receptors by a variety of protein kinases [38-41]. Our cur- ital bone for about 0.5 cm on either side of the midline by rent findings of the effect of PP2A inhibitors on capsaicin- using a scraping tool. While observing the back of the induced central sensitization suggest that PP2A has an skull, the neck musculature was gently retracted and the effect on the duration of central sensitization. Although atlanto-occipital membrane was exposed. In the midline, the mechanism is unclear, it may partially relate to the a small incision was made through the atlanto-occipital modulation of the phosphorylation of glutamate recep- membrane, using the tip of a fresh 18- or 20-ga disposable tors, or regulation of the function of protein kinases by needle as a cutting edge. Once the membrane was pierced, PP2A. there was ample outflow of clear cerebrospinal fluid. The correct incision had to be carried out with considerable Methods care to prevent cutting the dorsal surface of the medulla, Experimental animals which lies immediately beneath the membrane. The cath- Male Sprague-Dawley rats weighing 250–300 g were used eter was carefully advanced 7.3–7.5 cm into the spinal in this study. The protocols for these experiments were subarachnoid space from the incision. The tip of the cath- approved by the Institutional Animal Care and Use Com- eter was placed at the T12-L1 vertebral level, which was mittee. These experimental protocols were also consistent estimated to be the L4/5 level of the spinal cord. When the with the ethical guidelines of the National Institutes of catheter had been advanced 1–2 cm, a slight tension was Health and the International Association for the Study of applied to the tail of the animal to keep the animal's spine Pain. All animals used in the experiments were housed straight for the rest of the insertion. Once the catheter was and maintained in accordance with the guidelines of the inserted, the incision was sutured and the animal returned University of Texas Medical Branch (UTMB) Animal Care to the Animal Care Facility for 5 days to allow it to recover and Use Committee (ACUC). from the surgical procedure. The awake animals implanted with a catheter were checked for motor or sen- Intrathecal catheter implantation sory deficits after recovery. Animals with any sign of defi- The intrathecal catheters were made by joining three pol- cits were discarded. yethylene tubes of different diameters and had a total length of 16 cm. The smallest diameter PE32 tubing (7.3– Administration of drugs 7.5 cm in length) (MICOR Inc., PA, USA) was inserted The drugs used in these experiments were serine/threo- into the subarachnoid space of the spinal cord. The other nine protein phosphatase inhibitors: okadaic acid, a gen- end was connected with PE10 (3 cm) and then PE20 (8 eral inhibitor of PP2A and PP1 (20 nM, Calbiochem, cm) tubing (Becton Dickson, MD, USA) for a stepdown dissolved in ACSF) and fostriecin, a potent PP2A inhibitor connection to a Hamilton syringe. Each joint of the cath- (30 nM, Calbiochem, dissolved in ACSF). Concentrations eter was sealed with epoxy glue. The catheter was dried, of these drugs were based on published dose response sterilized by immersion in 70% ethanol before insertion, curves [44-46]. and fully flushed with sterile saline prior to use. A length of stainless steel wire, whose diameter just fit into the All drugs were administered through an intrathecal cathe- PE32 tubing, was used during insertion. ter implanted into the subarachnoid space of the spinal cord 30 minutes before injection of capsaicin. Drugs for Rats used in behavioral tests were anesthetized with intrathecal injection were dissolved in a 10 µl volume of sodium pentobarbital (50 mg/kg i.p.). The intrathecal ACSF containing the desired concentration of the agent. Page 9 of 12 (page number not for citation purposes) Molecular Pain 2006, 2:9 http://www.molecularpain.com/content/2/1/9 All drugs were injected over a 1–2 minute time period. A Behavioral tests 10 µl injected volume has been shown to spread 2 cm ros- The exploratory activity of the animals was recorded 5 tral and caudally [42]. After intrathecal drug injection, the days after intrathecal catheter implantation. For monitor- catheter was flushed with a subsequent 10 µl injection of ing movements, two activity chambers (40 × 40 × 40 cm) ACSF. A Hamilton microinjection syringe (10 µl, Hamil- were used and the data for motor activity were collected in ton Co., Reno, NV) was used for all injections. the clear plastic chambers and analyzed using the software and hardware provided by the Photobeam Activity System Injection of capsaicin (PAS) with Flexfield (San Diego Instruments, Inc.) cou- Capsaicin (1%, 50 µl) was injected into the plantar surface pled to a 486 Compaq computer. The PAS allows moni- of the glabrous skin of the left paw of halothane anesthe- toring of the exploratory activity within the chamber by tized rats [15]. Capsaicin was dissolved in Tween 80 (7%), detecting the number of times photobeams are inter- alcohol (20%), and saline. rupted in an x, y, and z axis oriented grid system. There are 16 photobeams on each horizontal axis, which are Western blotting arranged 4 cm above the chamber floor. Obstruction of Animals used in the Western immunoblotting study were these beams can be recorded as movements in the x and y deeply anesthetized with sodium pentobarbital (50 mg/ planes. Movements along the z axis, which are called rear- kg, i.p.). The spinal cord was removed quickly at different ing events, can be detected by another set of beams timepoints after intradermal injection of capsaicin or located 12 cm above the chamber floor. When the animal vehicle. Laminectomy was done and the ipsilateral dorsal remains still for 1 second or longer, this is defined as rest- spinal cord tissue was placed in liquid nitrogen immedi- ing time. There are six different behavioral measures of ately and homogenized in 50 mM Tris buffer, pH 7.4 (0.1 exploratory activity to be recorded: the number of beams mol/l EGTA, 0.14 µl/ml β-mercaptoethanol, 100 mol/l broken (counts), traveled distance, active time, resting PMSF, and 0.2 mg/ml trypsin inhibitor). The homogenate time, rearing events, and rearing time. Since changes in was centrifuged at 13,000 g for 15 minutes, and the super- one parameter are not reflected in every other parameter, natant was decanted from the pellet and used for Western the changes are evaluated for each parameter individually. blot analysis. The concentration of protein in the The activity of each animal is recorded over nine consecu- homogenate was measured using a BCA kit (Pierce, Rock- tive 5-minute intervals for a total of 45 minutes. All the ford, IL). Equal amounts of protein (60 µg) were size frac- animals are tested at the same time during the day in a tionated by 7.5% (w/v) sodium dodecyl sulfate- separate room where no people or other animals are polyacrylamide gel electrophoresis (SDS-PAGE) and present. The room is controlled at a stable temperature transferred onto a PVDF membrane (Bio-Rad, Hercules, (70–72°F) and low level of noise. The animals are CA). After blocking in the blocking buffer for 1 hour at brought into the room just before the test, and the room room temperature, the blots are incubated with primary is dark during the test. Between tests, the enclosure is antibody for 1 hour at room temperature. The primary cleaned with Cavicide and alcohol to eliminate urine and antibody to PP2A was monoclonal from Upstate (Char- other olfactory cues from previous animals [15,16,47]. lottesville, VA) and primary antibody to β-actin was from Sigma (St. Louis, MO). Then the blots were washed three Statistical analysis times for 30 minutes each in washing buffer and incu- The relative densities of Western blots from the tissue of the lumbar segments of rats at different time points (5 bated with horseradish peroxidase conjugated IgG diluted in 2.5% (w/v) non-fat milk in washing buffer. The mem- min, 15 min, 30 min, 60 min, and 90 min) were com- branes were washed with buffer three times for 30 min- pared between vehicle and capsaicin groups. One-way utes and enhanced with a chemiluminescence reagent ANOVA was performed followed by post hoc compari- (ECL kit, Amersham, Arlington Heights, IL). Then the sons using Student-Newman-Keuls multiple comparisons blots were exposed to autoradiographic film (Kodak, test. All values are presented as mean ± S.E.M. A p value of Rochester, NY), and the intensity of specific immunoreac- less than 0.05 was considered significant. tive bands was quantified using densitometric scanning analysis. Densitometric units of specific bands were All six activity measures were compared during the 45- expressed relative to the values of background units. The min interval. Rats of the naïve group and intrathecal injec- relative density of the immunoblots from the spinal cord tion group were compared with Student's t-test. Student's tissue of rats with injection of vehicle or capsaicin was cal- t-test was also used to compare the responses of rats in the culated by dividing the density of the PP2A blots by the capsaicin group and the vehicle group. One-way ANOVA density of β-actin blots for the same conditions. was performed to assess behavioral changes followed by post hoc comparisons using Student-Newman-Keuls mul- tiple comparisons test. All treatment groups with capsai- cin injection were compared to vehicle-treated rats. All Page 10 of 12 (page number not for citation purposes) Molecular Pain 2006, 2:9 http://www.molecularpain.com/content/2/1/9 22. Willis WD: Role of neurotransmitters in sensitization of pain data were expressed as mean ± S.E.M. A p value of less than responses. Ann N Y Acad Sci 2001, 933:142-156. 0.05 was considered significant. 23. Fang L, Wu J, Lin Q, Willis WD: Calcium-calmodulin-dependent protein kinase II contributes to spinal cord central sensitiza- tion. J Neurosci 2002, 22:4196-4204. Acknowledgements 24. Hunt SP, Pini A, Evan G: Induction of c-fos like protein in spinal The authors would like to thank Kelli Gondesen and Griselda Gonzalez for cord neurons following sensory stimulation. Nature 1987, technical assistance. This effort is supported by NIH grants NS09743, 328:632-634. NS11255, and DE15814. 25. Lin Q, Peng YB, Willis WD: Possible role of protein kinase C in the sensitization of primate spinothalamic tract neurons. J Neurosci 1996, 16:3026-3034. References 26. Lin Q, Peng YB, Wu J, Willis WD: Involvement of cGMP in noci- 1. Chan SF, Sucher NJ: An NMDA receptor signaling complex ceptive processing by and sensitization of spinothalamic neu- with protein phosphatase 2A. J Neurosci 2001, 21:7985-7992. rons in primates. J Neurosci 1997, 17:3293-3302. 2. Herzig S, Neumann J: Effects of serine/threonine protein phos- 27. Lin Q, Wu J, Willis WD: Effects of protein kinase A activation phatases on ion channels in excitable membranes. 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Schonthal AH: Role of PP2A in intracellular signal transduc- 30. Tao YX, Hassan A, Haddad E, Johns RA: Expression and action of tion pathways. Front Biosci 1998, 15:1262-1273. cyclic GMP-dependent protein kinase Ialpha in inflammatory 7. Sistiaga A, Sanchez-Prieto J: Protein phosphatase 1 and 2A inhib- hyperalgesia in rat spinal cord. Neuroscience 2000, 95:525-533. itors prolong the switch in the control of glutamate release 31. Wu J, McAdoo DJ, Willis WD: Nitric oxide contributes to cen- by group I metabotropic glutamate receptors: characteriza- tral sensitization following intradermal injection of capsai- tion of the inhibitory pathway. J Neurochem 2000, 75:1566-1574. cin. Neuroreport 1998, 9:589-592. 8. Thiels E, Norman ED, Barrionuevo G, Klann E: Transient and per- 32. Gentry MS, Li Y, Wei H, Syed FF, Patel SH, Hallberg Rl, Pallas DC: A sistent increases in protein phsphatase activity during long- novel assay for protein phosphatase 2A (PP2A) complexes in term depression in the adult hippocampus in vivo. Neuro- vivo reveals differential effects of covalent modifications on science 1998, 86:1023-1029. different Saccharomyces cerevisiae PP2A heterotrimers. 9. Garcia A, Cayla X, Guergnon J, Dessauge F, Hospital V, Rebollo MP, Eukaryot Cell 2005, 4:1029-40. Fleischer A, Rebollo A: Serine/threonine protein phosphatases 33. Goldberg Y: Protein phosphatase 2A: who shall regulate the PP1 and PP2A are key players in apoptosis. Biochimie 2003, regulator? Biochem Pharmacol 1999, 57:321-328. 85:721-6. 34. Tapia R, Pena F, Arias C: Neurotoxic and synaptic effects of oka- 10. Hoof CV, Goris J: Phosphatases in apoptosis: to be or not to daic acid, an inhibitor of protein phosphatases. Neurochem Res be, PP2A is in the heart of the question. Biochimica et Biophysica 1999, 24:1423-1430. Acta 2003, 1640:97-104. 35. Janssens V, Goris J: Protein phosphatase 2A: a highly regulated 11. Lechward K, Awotunde OS, Swiatek W, Muszynska G: Protein family of serine/threonine phosphatases implicated in cell phosphatase 2A: variety of forms and diversity of functions. growth and signaling. Biochem J 2001, 353:417-439. Acta Biochim Pol 2001, 48:921-33. 36. Millword TA, Zolnierowicz S, Hemmings BA: Regulation of protein 12. Sim AT, Ludowyke RI: The complex nature of protein phos- kinase cascades by protein phosphatase 2A. Trends Biochem Sci phatases. IUBMB Life 2002, 53:283-6. 1999, 24:186-191. 13. Lewy DS, Gauss CM, Soenen DR, Boger DL: Fostriecin: chemistry 37. Ramachandran C, Goris J, Waelkens E, Merlevede W, Walsh DA: and biology. Curr Med Chem 2002, 22:2005-32. The interrelationship between cAMP-dependent α and β 14. Moncada A, Cendan CM, Baeyens JM, Del Pozo E: Effects of serine/ subunit phosphorylation in the regulation of phosphorylase threonine protein phosphatase inhibitors on morphine- kinase activity: studies using subunit specific phosphatases. J induced antinociception in the tail flick test in mice. Eur J Phar- Biol Chem 1987, 262:3210-3218. macol 2003, 465:53-60. 38. Fang L, Wu J, Zhang X, Lin Q, Willis WD: Increased phosphoryla- 15. Palecek J, Paleckova V, Willis WD: The roles of pathways in the tion of the GluR1 subunit of spinal cord alpha-amino-3- spinal cord lateral and dorsal funiculi in signaling nociceptive hydroxy-5-methyl-4-isoxazole propionate receptor in rats somatic and visceral stimuli in rats. Pain 2002, 96:297-307. following intradermal injection of capsaicin. Neuroscience 2003, 16. Zhang LP, Zhang X, Westlund KN: Restoration of spontaneous 122:237-45. exploratory behaviors with an intrathecal NMDA receptor 39. 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Simone DA, Baumann TK, Collins JG, LaMotte RH: Sensitization of receptor 1 subunits in spinal cord dorsal horn and spinotha- cat dorsal horn neurons to innocuous mechanical stimula- lamic tract neurons after intradermal injection of capsaicin tion after intradermal injection of capsaicin. Brain Res 1989, in rats. J Neurosci 2000, 20:6989-97. 486:185-189. 42. Yaksh TL, Rudy TA: Chronic catheterization of the spinal sub- 20. Sluka KA, Rees H, Chen PS, Tsuruoka M, Willis WD: Capsaicin- arachnoid space. Physiol Behav 1976, 17:1031-1036. induced sensitization of primate spinothalamic tract cells is 43. LoPachin RM, Rudy TA: An improved method for chronic cath- prevented by a protein kinase C inhibitor. Brain Res 1997, eterization of the rat spinal subarachnoid space. Physiol Behav 772:82-86. 1981, 27:559-561. 21. Dubner R, Ruda MA: Activity-dependent neuronal plasticity following tissue injury and inflammation. Trends Neurosci 1992, 15:96-103. Page 11 of 12 (page number not for citation purposes) Molecular Pain 2006, 2:9 http://www.molecularpain.com/content/2/1/9 44. McCluskey A, Sakoff JA: Small molecule inhibitors of serine/ threonine protein phosphatases. Mini Rev Med Chem 2001, 1:43-55. 45. McCluskey A, Sim AT, Sakoff JA: Serine-threonine protein phos- phatase inhibitors: development of potential therapeutic strategies. J Med Chem 2002, 45:1151-75. 46. Walsh AH, Cheng A, Honkanen RE: Fostriecin, an antitumor antibiotic with inhibitory activity against serine/threonine protein phosphatases types 1 (PP1) and 2A (PP2A), is highly selective for PP2A. FEBS Lett 1997, 416:230-234. 47. Mills CD, Grady JJ, Hulsebosch CE: Changes in exploratory behavior as a measure of chronic central pain following spi- nal cord injury. J Neurotrauma 2001, 18:1091-1105. Publish with Bio Med Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical researc h in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright BioMedcentral Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 12 of 12 (page number not for citation purposes) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Molecular Pain Springer Journals

Protein phosphatase 2A regulates central sensitization in the spinal cord of rats following intradermal injection of capsaicin

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Springer Journals
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Copyright © 2006 by Zhang et al; licensee BioMed Central Ltd.
Subject
Medicine & Public Health; Pain Medicine; Molecular Medicine; Neurobiology
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1744-8069
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1744-8069
DOI
10.1186/1744-8069-2-9
pmid
16549018
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Abstract

Background: Intradermal injection of capsaicin into the hind paw of rats induces spinal cord central sensititzation, a process in which the responsiveness of central nociceptive neurons is amplified. In central sensitization, many signal transduction pathways composed of several cascades of intracellular enzymes are involved. As the phosphorylation state of neuronal proteins is strictly controlled and balanced by the opposing activities of protein kinases and phosphatases, the involvement of phosphatases in these events needs to be investigated. This study is designed to determine the influence of serine/threonine protein phosphatase type 2A (PP2A) on the central nociceptive amplification process, which is induced by intradermal injection of capsaicin in rats. Results: In experiment 1, the expression of PP2A protein in rat spinal cord at different time points following capsaicin or vehicle injection was examined using the Western blot method. In experiment 2, an inhibitor of PP2A (okadaic acid, 20 nM or fostriecin, 30 nM) was injected into the subarachnoid space of the spinal cord, and the spontaneous exploratory activity of the rats before and after capsaicin injection was recorded with an automated photobeam activity system. The results showed that PP2A protein expression in the spinal cord was significantly upregulated following intradermal injection of capsaicin in rats. Capsaicin injection caused a significant decrease in exploratory activity of the rats. Thirty minutes after the injection, this decrease in activity had partly recovered. Infusion of a phosphatase inhibitor into the spinal cord intrathecal space enhanced the central sensitization induced by capsaicin by making the decrease in movement last longer. Conclusion: These findings indicate that PP2A plays an important role in the cellular mechanisms of spinal cord central sensitization induced by intradermal injection of capsaicin in rats, which may have implications in clinical pain therapy. Introduction kinases and phosphatases (PP), respectively. Such reac- The phosphorylation and dephosphorylation of proteins tions seem to modulate the function of several proteins are reversible processes, catalyzed by opposing protein involved in synaptic transmission, including voltage- Page 1 of 12 (page number not for citation purposes) Molecular Pain 2006, 2:9 http://www.molecularpain.com/content/2/1/9 Strong noxious stimulation of peripheral tissues, such as intradermal injection of capsaicin into the hind paw of PP2A 36 KD rats, induces spinal cord central sensititzation, a process in which an amplified responsiveness of central nociceptive β-actin 42 KD neurons is found. It has been verified that signal transduc- 5 15 30 60 90 5 15 30 60 90 tion pathways involved in central sensitization are com- Vehicle injection Capsaicin injection posed of several cascades of enzymes that activate intracellular protein kinases. As the phosphorylation state of neuronal proteins is strictly controlled and balanced by the opposing activities of protein kinases and phos- phatases, the involvement of phosphatases in these events needs to be investigated. In the present study, we evaluated the effects of noxious 5 m i n 15 m i n 3 0 m i n 60 m i n 90 m i n stimulation of peripheral tissues on the expression of Tim e s PP2A protein in the spinal cord of rats. We also tested the E and p Figure 1 xpression of PP2A pr eriphery 90 minutes following vehicle otein in the spinal cor or capsaicin injection in d at 5, 15, 30, 6 the 0, effects of two inhibitors of PP2A, okadaic acid and fos- Expression of PP2A protein in the spinal cord at 5, 15, 30, 60, triecin, on the induction and maintenance of central sen- and 90 minutes following vehicle or capsaicin injection in the sitization induced by capsaicin injection, using the periphery. (A) Immunoreactive bands of rat spinal cord tissue spontaneous exploratory activity test. The inhibitors were (L3–L6) against PP2A antibody at 5, 15, 30, 60, and 90 min- administered through an intrathecal catheter to detect the utes after capsaicin or vehicle injection; the lower row shows role of the spinal cord in changes in these behaviors. the bands produced by the antibody to β-actin as a loading control at the same times. (B) Bar graph summarizing the rel- Results ative density of the immunoblots of rat spinal cord tissue Increased expression of PP2A in spinal cord after capsaicin (L3–L6) with the PP2A antibody to that of β-actin at 5, 15, injection 30, 60, and 90 minutes after capsaicin or vehicle injection. Open bar: vehicle injection, filled bar: capsaicin injection. N = To determine if the expression of PP2A protein in the lum- 6 in each group; the relative densities are expressed as mean ber spinal cord of rats changes following noxious stimula- ± SEM. *p < 0.05. tion with intradermal injection of capsaicin, a Western blot analysis was performed. The Western blots in Fig. 1A show that the expression of PP2A in the lumbar segments (L -L ) of the spinal cord tissue was detected at around 36 3 6 kDa. The relative density of immunoblots of PP2A protein gated and ligand-gated channels, ionotropic and metabo- from rat spinal cord tissue after intradermal vehicle or tropic neurotransmitter receptors, proteins involved in capsaicin injection is summarized in Fig 1B. Statistical neurotransmitter release and transport, and cytoskeletal analysis of these data showed that intradermal injection proteins [1-8]. These proteins play an important role in of capsaicin causes a significant increase in expression of the control of many intracellular events. Serine/threonine PP2A protein in a time-dependent manner compared protein phosphatase, which dephosphorylates serine and with the vehicle injection group. The PP2A protein started threonine protein residues, can be divided into PP1, to increase in spinal cord tissue by 15 min, peaked at 30 PP2A, PP2B, PP2C, PP4, PP5, PP6 and PP7 [2]. Among min, and the increase continued for more than 60 min them, PP2A is the most abundant phosphatase in mam- following intradermal injection of capsaicin. Vehicle malian cells and is expressed at high levels in the central injection did not result in an obvious increase in the nervous system [1,6]. PP2A regulates many fundamental expression of PP2A protein in the lumbosacral spinal cellular processes, such as cell division, signal transduc- cord. In addition, the expression of β-actin did not show tion, gene expression, development, the cell cycle, exocy- any significant change in any of the groups. tosis, and apoptosis [2,6,9-12]. The function of this protein phosphatase can be inhibited by several drugs, The effect of intrathecal catheter implantation on such as okadaic acid (OA), which is a cell permeable mol- exploratory behavior ecule that inhibits PP2A in vitro at much lower concentra- The spontaneous activity of rats and all six behavioral tions than PP1 (1:100) [13,14]. Fostriecin is another cell parameters were examined both before and 5 days after permeable protein phosphatase inhibitor. Since this mol- intrathecal implantation of a catheter. Fig. 2 demonstrates 4 5 ecule is on the order of 10 –10 times more selective for that motor function was not impacted by the intrathecal PP2A than PP1, fostriecin is the most selective inhibitor injection procedure. Compared to the naïve rats, the gen- known for any member of this class of phosphatases [13]. eral exploratory behaviors of rats with intrathecal implan- Page 2 of 12 (page number not for citation purposes) Relative optical density Molecular Pain 2006, 2:9 http://www.molecularpain.com/content/2/1/9 A. Total Activity B. Distance Traveled Naive i.t. catheter 3000 1000 Capsaicin 0 0 C. Active Time D. Resting Time * 1500 E. Rearing Time F. Rearing Events n Figure 2 Eo xploratory a vel chamber ctivity during a consecutive period of 45 minutes (9 intervals, 5 min/interval) after introduction of the rat into the Exploratory activity during a consecutive period of 45 minutes (9 intervals, 5 min/interval) after introduction of the rat into the novel chamber. Compared to naïve rats (n = 14), intrathecal catheter implanted rats (n = 14) do not have significant alterations in exploratory activity. Rats with intradermal injection of capsaicin (1%, 50 µl) (n = 6) demonstrated a reduction of exploratory activity, including decreased number of photobeams broken (A), shortened travel distance (B), decreased active time (C), increased resting time (D), shortened rearing time (E), and decreased number of rearing events (F). The capsaicin-injected group was compared to the other two groups. *p < 0.05. Page 3 of 12 (page number not for citation purposes) Active Time (sec.) Number of Beams Broken Rearing Time (sec.) Resting Tiem (sec.) Distance Traveled (inches) Number of Rearing Molecular Pain 2006, 2:9 http://www.molecularpain.com/content/2/1/9 B. Distance Traveled A. Total Activity i.t. catheter i.t. catheter+OA C. Active Time Resting Time D. E. Rearing Time F. Rearing Events 800 500 0 0 E (9 inter Figure 3 ffect of th vals, 5 min/interval) e PP2A inhibitor, okadaic acid (OA, 20 nM) on exploratory activity of rats during a consecutive period of 45 minutes Effect of the PP2A inhibitor, okadaic acid (OA, 20 nM) on exploratory activity of rats during a consecutive period of 45 minutes (9 intervals, 5 min/interval). Compared to the intrathecal catheter implanted rats (n = 14), pretreatment with OA through an intrathecal catheter (n = 7) did not produce significant alterations in exploratory activity. tation, such as the total activity (1874 ± 146), distance in consecutive 45-minute intervals (n = 13) were not sig- traveled (668 ± 29), and length of active time (1675 ± 42) nificantly different. In addition, no significant decreases Page 4 of 12 (page number not for citation purposes) Active Time (sec.) Number of Beams Broken Rearing Time (sec.) Resting Time (sec.) Distance Traveled (inches) Number of Rearing Molecular Pain 2006, 2:9 http://www.molecularpain.com/content/2/1/9 Total Activity B. Distance Traveled A. ACSF+CAP. OA+CAP. Fostriecin+CAP. D. Resting Time C. Active Time 900 * E. Rearing Time F. Rearing Events E dur Figure 4 ffect ing s of two PP2A a consecutive per inhibitor iod of 45 mi s, OA an nutes d fostriecin, on the reduced (9 intervals, 5 min/interval) exploratory activity of rats initiated by capsaicin injection Effects of two PP2A inhibitors, OA and fostriecin, on the reduced exploratory activity of rats initiated by capsaicin injection during a consecutive period of 45 minutes (9 intervals, 5 min/interval). Compared to the rats in the ACSF-pretreated group (n = 7), the rats with either OA (20 nM, n = 7) or fostriecin (30 nM, n = 5) pretreatment displayed significantly decreased explor- atory activity, including a decreased number of photobeams broken (A), shortened distance traveled (B), decreased active time (C), increased resting time (D), shortened rearing time (E), and decreased number of rearing events (F). *p < 0.05. Page 5 of 12 (page number not for citation purposes) Active Time (sec.) Number of Beams Broken Rearing Time (sec.) Resting Time (sec.) Distance Traveled (inches) Number of Rearing Molecular Pain 2006, 2:9 http://www.molecularpain.com/content/2/1/9 were found in other parameters. On the average, rearing Spontaneous exploratory activity in rats with OA pretreat- events and rearing times of 267 ± 16/45 min and 451 ± ment was greatly affected compared to the control group. 77/45 min were found in naïve rats; they were 221 ± 19/ Total activity significantly decreased from 1123 ± 160 to 45 min and 424 ± 61/45 min, respectively, in implanted 570 ± 89; consequently the distance traveled decreased rats. greatly from 426 ± 41 to 225 ± 37; active time decreased from 1200 ± 97 to 797 ± 123; and the resting time Intradermal injection of capsaicin changes exploratory increased from 1500 ± 97 to 1903 ± 123 in consecutive behavior of rats 45-minute intervals. Rearing time in the OA group was After noxious stimulation by intradermal injection of cap- also reduced greatly from 193 ± 26 to 39 ± 9, and the rear- saicin in the left hind paw of rats that were implanted ing events were reduced from 125 ± 33 to 36 ± 9. The intrathecally with a catheter, spontaneous exploratory measurements in the fostriecin-pretreated group were also activity was severely affected compared to the naïve and significantly reduced compared to the control. For com- intrathecally implanted rats (Fig. 2). Total activity greatly parison, the total activity, distance traveled, active time, decreased from 1874 ± 147 to 1022 ± 81, and distance resting time, rearing time, and rearing events in fostriecin traveled decreased significantly from 668 ± 29 to 495 ± 9 group were 674 ± 73, 286 ± 28, 900 ± 60, 1800 ± 60, 65 ± in consecutive 45-minute intervals (Fig. 2A and 2B). 21, 60 ± 18 respectively. Active time decreased from 1675 ± 42 to 1401 ± 25, whereas resting time increased from 1025 ± 42 to 1299 ± The line graphs in Fig. 5 demonstrate the changes in 25 (Fig. 2C and 2D). Rearing activity was also significantly exploratory behavior at each 5-min interval. When rats impacted by capsaicin injection. Rearing time decreased were placed in a novel environment, the spontaneous greatly from 424 ± 61 to 206 ± 6, and rearing events exploratory activity was high initially and slowly declined decreased from 221 ± 19 to 116 ± 13 (Fig. 2E and 2F). over the first 30 min. The activity in all groups of rats in the current study showed this same temporal pattern. The The effect of intrathecal administration of a general total entries, distance traveled, active time, rearing time, inhibitor of PP2A on exploratory behavior and rearing events were high in the first 20–25 min and OA, a nonselective inhibitor of PP2A and PP1, was given then declined. In the groups treated with PP2A inhibitors, intrathecally in a group of seven rats. Fig. 3 demonstrates the exploratory activity was decreased compared with the that administration of OA had no effect on the spontane- ACSF-treated group. This reduction lasted throughout the ous exploratory activity of the rats. None of the activity 45-min block. parameters in OA-administered rats, such as total activity (1628 ± 170), distance traveled (596 ± 40), active time Discussion (1603 ± 76), resting time (1097 ± 76), rearing time (373 The results of this study showed that PP2A in the spinal ± 58), and rearing events (198 ± 18) in consecutive 45- cord plays an important role in the signal cascades that are minute intervals was significantly different when com- activated following intradermal injection of capsaicin in pared to the intrathecally implanted group. the hind paw of rats. Based on the data presented, expres- sion of PP2A in the spinal cord of rat starts to increase by Decreased exploratory activity induced by capsaicin was 15 minutes after capsaicin injection and starts to return to potentiated by intrathecal administration of PP2A baseline by about 1 hour. inhibitors The bar graphs in Fig. 4 show the effects of PP2A inhibi- With capsaicin injection, the spontaneous exploratory tors on the spontaneous exploratory activity of rats fol- activity of rats was significantly reduced due to nocicep- lowing capsaicin injection. Reduced exploratory behavior tion. Following the injection, the number of rearing induced by capsaicin was aggravated by both the general events and the rearing time were decreased. Measures of inhibitor of PP1 and PP2A, OA, and the specific inhibitor active time, total entries, and distance traveled were also of PP2A, fostriecin. decreased despite the fact that hind limb locomotor func- tion was not impacted. In this model, secondary mechan- Three groups of rats were used in this experiment. Pre- ical allodynia and hyperalgesia and primary thermal treatment with ACSF administered through the intrathe- hyperalgesia would have occurred following capsaicin cal catheter (n = 7) was done in the control group; OA (n injection. So, during the process of central sensitization, a = 9) or fostriecin (n = 5) was administered in the two concurrent decrease in exploratory behavior would be experimental groups. Thirty minutes after the pretreat- expected, as seen in the current study. The reduction in ment with drugs infused through the intrathecal catheter, exploratory activity seen in our model is in agreement capsaicin was injected intradermally in the left hind paw with previous reports of exploratory behavior in other of the rats. Animals were put into an activity chamber 30 pain models. Palecek et al. [15] found a decrease in min after the injection to monitor the movements. exploratory behavior following pain induced by colon Page 6 of 12 (page number not for citation purposes) Molecular Pain 2006, 2:9 http://www.molecularpain.com/content/2/1/9 A Total Activity B Distance Traveled ACSF+CAP. OA+CAP. Fostriecin+CAP. ^ 90 * * * ^ 60 * ^ ^ ^ * * * * ^ ^ ^ * ^ 100 ^ C Active Time D Resting Time * ^ * * 250 250 ^ ^ * ^ * ^ 200 200 * * ^ ^ 150 ^ * * ^ ^ 100 100 50 50 0 0 E Rearing Time F Rearing Events 100 60 ^ 30 40 * * ^ ^ * * * ^ ^ * * 20 ^ 0 0 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 Time (min) Time (min) ity induced by Figure 5 Effect of intrath capsaic ecal infusion in (0 of the phosphatase inhibitors okadaic acid and fostriecin, or ACSF on decreased exploratory activ- Effect of intrathecal infusion of the phosphatase inhibitors okadaic acid and fostriecin, or ACSF on decreased exploratory activ- ity induced by capsaicin (0.1%, 50 µl) injection. Thirty min after capsaicin injection, okadaic acid (20 nM), fostriecin (30 nM), or ACSF was infused into the subarachnoid space of the spinal cord at the L5 segmental level of the rats. Compared to ACSF (n = 7), six parameters of spontaneous activity of the rats with okadaic acid (n = 7) and fostriecin (n = 5) infusion showed a signifi- cant decrease at every time interval. Each point represents the mean ± S.E.M. *p < 0.05. Page 7 of 12 (page number not for citation purposes) Rearing Time (sec.) Active Time (sec.) Number of Beams Broken Resting Time (sec.) Distance Traveled (inches) Number of Rearing Molecular Pain 2006, 2:9 http://www.molecularpain.com/content/2/1/9 inflammation and distention. Zhang et al. [16] showed Capsaicin injection reduces exploratory activity of rats reduced exploratory behavior in a model of pancreatitis. In the present study, an automated photobeam activity The decrease of movement with a normal ability to move system was used to test the rodent exploratory activity to results in less active time and more resting time, implying evaluate behavioral changes induced by intradermal the development of pain following capsaicin injection. injection of capsaicin. The activity system was a useful Intrathecal administration of PP2A inhibitors signifi- tool for measurement of the exploratory activity for com- cantly aggravates pain, leading to a decrease in the total parisons of control rats and rats with noxious stimulation activity, traveled distance, active time, rearing time, rear- of peripheral tissues, with or without pharmacological ing events, and an increase in the resting time of rats. treatments. Capsaicin injection reduced the exploratory activity of rats by decreasing the total activity, traveled dis- PP2A expression in the spinal cord after noxious stimuli tance, active time, rearing time, and rearing events, while Intradermal injection of capsaicin is a useful and reversi- increasing the resting time in a 45-min period. Intrader- ble experimental model for the study of an inflammatory mal injection of capsaicin is a noxious stimulus that pain state that includes hyperalgesia and allodynia [17- evokes not only ongoing pain (which lasts for about 15– 20]. In the capsaicin model, central sensitization can be 20 minutes), but also a pain state in which there is initiated by neural activity originating in the area of injec- increased sensitivity to mechanical stimuli. Intradermal tion and carried by chemosensitive afferent fibers. The injection of capsaicin can cause changes in behavioral whole process relies on central pain mechanisms. It occurs responses of rats and increase responses of nociceptive after prolonged C-nociceptor input and depends on acti- projection neurons in dorsal horn of the spinal cord at a vation of wide dynamic range neurons in the dorsal horn cellular level [15,17-20,22]. In our experiment, the of the spinal cord [21,22]. During central sensitization, a exploratory activity of animals was tested 30 min after variety of signal transduction pathways in central neurons capsaicin injection. The changes of activity reflect the pain are activated, through which the excitability of central induced by mechanical stimulation of the paw during neurons is enhanced. It has been demonstrated that pro- movement in the activity chamber instead of the pain tein kinase C, NO/protein kinase G, protein kinase A, and evoked directly by the intradermal injection. In previous calcium/calmodulin dependent kinase II (CaMKII) are all studies in our laboratory, intradermal injection of capsai- involved in this process. In addition, several types of iono- cin did not cause any systemic effect, and the handling of tropic glutamate receptors become phosphorylated dur- rats during the injection had no effect [15]. On the other ing central sensitization [20,23-31]. hand, the exploratory activity was not affected by the sham injection of vehicle (data not shown), which is con- In this study, 15 minutes after capsaicin injection into the sistent with the results of Palecek et al. [15]. The behavio- hind paw of rats, PP2A expression in the spinal cord is ral changes in exploratory activity are presumably due to upregulated compared with the control group with a vehi- nociceptive signals induced by capsaicin injection, and cle injection. In such a the short time period, the increased the changes reflect primary and/or secondary mechanical expression may not only reflect an increase of PP2A pro- allodynia and hyperalgesia. tein synthesis but also the posttranslational modification of PP2A subunits. The catalytic subunits of PP2A (PP2Ac) PP2A inhibitors potentiate the reduction of exploratory can be regulated mainly by three posttranslational modi- activity that is induced by capsaicin injection In the present study, PP2A inhibitors, either OA or fos- fications: methylation, phosphorylation of tyrosine and phosphorylation of threonine. By these modifications, triecin, did not themselves reduce the exploratory activity PP2A activity and function can be regulated [32]. The anti- of the animals. On the other hand, pretreatment with body that we used in the Western Blot study recognizes PP2A inhibitors through an intrathecal catheter extended the PP2A at 36 KDa, and it preferentially recognizes the duration of the decreases of the behavior induced by demethylated PP2A. This suggests that the increased PP2A capsaicin. This suggests that without the peripheral nox- expression may partly reflect an increased demethylation ious stimuli, the reactions that PP2A modulated would of PP2A. not initiate central sensitization directly, but they may be involved in the process of central sensitization by limiting The increased PP2A protein expression peaked at 30 min the duration of this process. and it continued at least through 1 h following intrader- mal injection of capsaicin. This suggests that PP2A may It is well known that protein phosphatases function as key also be involved in central sensitization after intradermal molecules in essential regulatory mechanisms in many injection of capsaicin through modulation of the phos- cellular processes. Serine/threonine protein phosphatases phorylation state of some critical proteins in the spinal remove phosphate groups from either serine or threonine cord. sites following phosphorylation of target proteins and thus reverse protein phosphorylation [1,5,6,8,33,34]. Page 8 of 12 (page number not for citation purposes) Molecular Pain 2006, 2:9 http://www.molecularpain.com/content/2/1/9 Since protein dephosphorylation is an essential post- catheter was inserted into the subarachnoid space of the translational regulatory mechanism in signal transduction spinal cord for the administration of drugs at the L5 seg- in the central nervous system that reverses the process of mental level according to the protocol of Yaksh et al. [42] protein phosphorylation, protein phosphatases which as modified by LoPachin and Rudy [43]. To implant a mediate this process have gained more attention because catheter, the anesthetized rat was mounted in a stereotaxic their role in regulating protein phosphorylation is as instrument to hold the rat's head firmly. A midline inci- important as that of protein kinases. PP2A is one of the sion was made beginning at a line joining the ears and most important protein phosphatases in the central nerv- extending about 2 cm in a caudal direction. The fascia was ous system, and it can regulate the activities of many retracted from the skull. A superficial layer of neck mus- kinases both in vivo and in vitro, such as ERK/MAPKs, PKA, cles was exposed by the initial incision of the skin, and the PKC, IKB kinase, and Ca/calmodulin-dependent kinase muscles were separated by a midline incision beginning at [35-37]. Currently, glutamate receptors have become a the occipital crest and extending caudally about 2 cm. The main focus in pain research. The increases in responsive- separation of the superficial musculature exposed an ness of dorsal horn neurons to mechanical stimulation of underlying layer of muscles which could be easily sepa- the skin after intradermal injection of capsaicin are due in rated along the midline by blunt dissection. Then, the part to the upregulation of phosphorylated glutamate muscles were freed from their point of origin on the occip- receptors by a variety of protein kinases [38-41]. Our cur- ital bone for about 0.5 cm on either side of the midline by rent findings of the effect of PP2A inhibitors on capsaicin- using a scraping tool. While observing the back of the induced central sensitization suggest that PP2A has an skull, the neck musculature was gently retracted and the effect on the duration of central sensitization. Although atlanto-occipital membrane was exposed. In the midline, the mechanism is unclear, it may partially relate to the a small incision was made through the atlanto-occipital modulation of the phosphorylation of glutamate recep- membrane, using the tip of a fresh 18- or 20-ga disposable tors, or regulation of the function of protein kinases by needle as a cutting edge. Once the membrane was pierced, PP2A. there was ample outflow of clear cerebrospinal fluid. The correct incision had to be carried out with considerable Methods care to prevent cutting the dorsal surface of the medulla, Experimental animals which lies immediately beneath the membrane. The cath- Male Sprague-Dawley rats weighing 250–300 g were used eter was carefully advanced 7.3–7.5 cm into the spinal in this study. The protocols for these experiments were subarachnoid space from the incision. The tip of the cath- approved by the Institutional Animal Care and Use Com- eter was placed at the T12-L1 vertebral level, which was mittee. These experimental protocols were also consistent estimated to be the L4/5 level of the spinal cord. When the with the ethical guidelines of the National Institutes of catheter had been advanced 1–2 cm, a slight tension was Health and the International Association for the Study of applied to the tail of the animal to keep the animal's spine Pain. All animals used in the experiments were housed straight for the rest of the insertion. Once the catheter was and maintained in accordance with the guidelines of the inserted, the incision was sutured and the animal returned University of Texas Medical Branch (UTMB) Animal Care to the Animal Care Facility for 5 days to allow it to recover and Use Committee (ACUC). from the surgical procedure. The awake animals implanted with a catheter were checked for motor or sen- Intrathecal catheter implantation sory deficits after recovery. Animals with any sign of defi- The intrathecal catheters were made by joining three pol- cits were discarded. yethylene tubes of different diameters and had a total length of 16 cm. The smallest diameter PE32 tubing (7.3– Administration of drugs 7.5 cm in length) (MICOR Inc., PA, USA) was inserted The drugs used in these experiments were serine/threo- into the subarachnoid space of the spinal cord. The other nine protein phosphatase inhibitors: okadaic acid, a gen- end was connected with PE10 (3 cm) and then PE20 (8 eral inhibitor of PP2A and PP1 (20 nM, Calbiochem, cm) tubing (Becton Dickson, MD, USA) for a stepdown dissolved in ACSF) and fostriecin, a potent PP2A inhibitor connection to a Hamilton syringe. Each joint of the cath- (30 nM, Calbiochem, dissolved in ACSF). Concentrations eter was sealed with epoxy glue. The catheter was dried, of these drugs were based on published dose response sterilized by immersion in 70% ethanol before insertion, curves [44-46]. and fully flushed with sterile saline prior to use. A length of stainless steel wire, whose diameter just fit into the All drugs were administered through an intrathecal cathe- PE32 tubing, was used during insertion. ter implanted into the subarachnoid space of the spinal cord 30 minutes before injection of capsaicin. Drugs for Rats used in behavioral tests were anesthetized with intrathecal injection were dissolved in a 10 µl volume of sodium pentobarbital (50 mg/kg i.p.). The intrathecal ACSF containing the desired concentration of the agent. Page 9 of 12 (page number not for citation purposes) Molecular Pain 2006, 2:9 http://www.molecularpain.com/content/2/1/9 All drugs were injected over a 1–2 minute time period. A Behavioral tests 10 µl injected volume has been shown to spread 2 cm ros- The exploratory activity of the animals was recorded 5 tral and caudally [42]. After intrathecal drug injection, the days after intrathecal catheter implantation. For monitor- catheter was flushed with a subsequent 10 µl injection of ing movements, two activity chambers (40 × 40 × 40 cm) ACSF. A Hamilton microinjection syringe (10 µl, Hamil- were used and the data for motor activity were collected in ton Co., Reno, NV) was used for all injections. the clear plastic chambers and analyzed using the software and hardware provided by the Photobeam Activity System Injection of capsaicin (PAS) with Flexfield (San Diego Instruments, Inc.) cou- Capsaicin (1%, 50 µl) was injected into the plantar surface pled to a 486 Compaq computer. The PAS allows moni- of the glabrous skin of the left paw of halothane anesthe- toring of the exploratory activity within the chamber by tized rats [15]. Capsaicin was dissolved in Tween 80 (7%), detecting the number of times photobeams are inter- alcohol (20%), and saline. rupted in an x, y, and z axis oriented grid system. There are 16 photobeams on each horizontal axis, which are Western blotting arranged 4 cm above the chamber floor. Obstruction of Animals used in the Western immunoblotting study were these beams can be recorded as movements in the x and y deeply anesthetized with sodium pentobarbital (50 mg/ planes. Movements along the z axis, which are called rear- kg, i.p.). The spinal cord was removed quickly at different ing events, can be detected by another set of beams timepoints after intradermal injection of capsaicin or located 12 cm above the chamber floor. When the animal vehicle. Laminectomy was done and the ipsilateral dorsal remains still for 1 second or longer, this is defined as rest- spinal cord tissue was placed in liquid nitrogen immedi- ing time. There are six different behavioral measures of ately and homogenized in 50 mM Tris buffer, pH 7.4 (0.1 exploratory activity to be recorded: the number of beams mol/l EGTA, 0.14 µl/ml β-mercaptoethanol, 100 mol/l broken (counts), traveled distance, active time, resting PMSF, and 0.2 mg/ml trypsin inhibitor). The homogenate time, rearing events, and rearing time. Since changes in was centrifuged at 13,000 g for 15 minutes, and the super- one parameter are not reflected in every other parameter, natant was decanted from the pellet and used for Western the changes are evaluated for each parameter individually. blot analysis. The concentration of protein in the The activity of each animal is recorded over nine consecu- homogenate was measured using a BCA kit (Pierce, Rock- tive 5-minute intervals for a total of 45 minutes. All the ford, IL). Equal amounts of protein (60 µg) were size frac- animals are tested at the same time during the day in a tionated by 7.5% (w/v) sodium dodecyl sulfate- separate room where no people or other animals are polyacrylamide gel electrophoresis (SDS-PAGE) and present. The room is controlled at a stable temperature transferred onto a PVDF membrane (Bio-Rad, Hercules, (70–72°F) and low level of noise. The animals are CA). After blocking in the blocking buffer for 1 hour at brought into the room just before the test, and the room room temperature, the blots are incubated with primary is dark during the test. Between tests, the enclosure is antibody for 1 hour at room temperature. The primary cleaned with Cavicide and alcohol to eliminate urine and antibody to PP2A was monoclonal from Upstate (Char- other olfactory cues from previous animals [15,16,47]. lottesville, VA) and primary antibody to β-actin was from Sigma (St. Louis, MO). Then the blots were washed three Statistical analysis times for 30 minutes each in washing buffer and incu- The relative densities of Western blots from the tissue of the lumbar segments of rats at different time points (5 bated with horseradish peroxidase conjugated IgG diluted in 2.5% (w/v) non-fat milk in washing buffer. The mem- min, 15 min, 30 min, 60 min, and 90 min) were com- branes were washed with buffer three times for 30 min- pared between vehicle and capsaicin groups. One-way utes and enhanced with a chemiluminescence reagent ANOVA was performed followed by post hoc compari- (ECL kit, Amersham, Arlington Heights, IL). Then the sons using Student-Newman-Keuls multiple comparisons blots were exposed to autoradiographic film (Kodak, test. All values are presented as mean ± S.E.M. A p value of Rochester, NY), and the intensity of specific immunoreac- less than 0.05 was considered significant. tive bands was quantified using densitometric scanning analysis. Densitometric units of specific bands were All six activity measures were compared during the 45- expressed relative to the values of background units. The min interval. Rats of the naïve group and intrathecal injec- relative density of the immunoblots from the spinal cord tion group were compared with Student's t-test. Student's tissue of rats with injection of vehicle or capsaicin was cal- t-test was also used to compare the responses of rats in the culated by dividing the density of the PP2A blots by the capsaicin group and the vehicle group. One-way ANOVA density of β-actin blots for the same conditions. was performed to assess behavioral changes followed by post hoc comparisons using Student-Newman-Keuls mul- tiple comparisons test. All treatment groups with capsai- cin injection were compared to vehicle-treated rats. All Page 10 of 12 (page number not for citation purposes) Molecular Pain 2006, 2:9 http://www.molecularpain.com/content/2/1/9 22. Willis WD: Role of neurotransmitters in sensitization of pain data were expressed as mean ± S.E.M. A p value of less than responses. Ann N Y Acad Sci 2001, 933:142-156. 0.05 was considered significant. 23. Fang L, Wu J, Lin Q, Willis WD: Calcium-calmodulin-dependent protein kinase II contributes to spinal cord central sensitiza- tion. 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Molecular PainSpringer Journals

Published: Mar 20, 2006

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