Nalbuphine Attenuates Microglial Activation and Inhibits Morphine-Induced Scratching via Regulation of PKCδ and p38 Signaling in Mice

Morphine-induced scratching (MIS) is a common issue in patients receiving clinical postoperative morphine intrathecal injections. The kappa opioid receptor (KOR) agonist nalbuphine is known to prevent and reduce MIS, but the underlying mechanism has remained unclear. Here, we found that protein kinase C (PKCβ) in the spinal cord dorsal horn was co-expressed with ionized calcium binding adapter molecule 1 (Iba1) and increased after morphine intrathecal (i.t.) injections in mice. Although knockdown PKCβ inhibited microglial activation and attenuated MIS, but nalbuphine can blocked MIS by promoting PKCδ expression rather than PKCβ expression, suggesting that antipruritic effect of nalbuphine is due to activation of the KOR/PKCδ pathway and that MIS is both PKCβ-dependent and closely related to microglial activation. Moreover, we also found that PKCδ could retroegulation microglial activation, thereby reversing nalbuphine’s inhibition of MIS and resulting in increased itching behaviors. Notably, microglial activation is required for p-p38 signaling in MIS. These data together suggest that nalbuphine activates KOR, so as to inducing the activation of PKCδ, which may in turn retroegulation microglial function and decrease phosphorylate p38, ultimately inhibiting MIS. Our research therefore indicates that a previously unknown KOR-PKCδ-microglia-p38 pathway in the spinal cord may underlie nalbuphine’s antipruritic effect.


Introduction
Pruritus, or itching, is de ned as an unpleasant sensation that elicits the desire or re ex to scratch [1]. As an evolutionarily conservative behavior, itching reminds people to avoid harmful environmental stimuli. However, not every itch can be prevented, and itching sensation is often unpleasant. Itching induced by opioid intrathecal injections is an uncomfortable sensation that occurs during postoperative analgesia, with a reported incidence of 18.7% [2]. Although intrathecal injections of morphine can effectively block postoperative pain, opioid-induced pruritus also signi cantly in uences patients' analgesia experience and limits the successful application of opioids [3].
Nalbuphine is a kappa opioid receptor (KOR) agonist and mu opioid receptor antagonist; it has a demonstrated analgesic effect and is also known to inhibit pruritus [4]. Moreover, intravenous nalbuphine has been shown to not only attenuate the incidence of pruritus but also to decrease total morphine consumption [5]. However, the mechanism underlying nalbuphine's mediation of morphine-induced scratching (MIS) is still not clearly understood.
Microglia are macrophages that reside in the central nervous system (CNS); they are known to play immunoregulatory roles within the CNS. Notably, they are involved in the regulation of both pain [6,7] and itching. For example, recent research found that compound 48/80 and 5'-guanidinonaltrindole (GNTI) elicited pronounced scratching behaviors in mice in conjunction with microglial activation in the mice's spinal cords [8]. Other research has also indicated the involvement of spinal microglia in both pruritogeninduced and chronic itching [9]. However, the precise role of microglia in MIS is still unclear, and it is unknown whether MIS is related to microglial activity or whether nalbuphine's inhibition of MIS is dependent on a microglial mechanism.
In the present study, we investigated i.t. injections of nalbuphine inhibited scratching behaviors in mice and examined the changes in microglial activation in the mice's spinal cords. We found that microglial activation mediated MIS and that nalbuphine inhibited MIS are by upregulating PKCδ expression, while inhibited microglial activation and in turn affected the expression of phosphorylated-p38 (p-p38). Overall, our study de ned the functional relationship between the KOR agonist nalbuphine and microglial activation in the modulation of MIS.

Materials And Methods
Animals Male C57BL/6J mice between 7 and 12 weeks of age were used for experiment.
They were purchased from the Guangdong Medical Laboratory Animal Center (GDMLAC).
All experiments were performed in accordance with the National Institutes of Health

Itch models
Acute itch of morphine-induced model The mice skin of back were shaved 3 days before 0.06 nmol morphine was i.t injected after nalbuphine(2 nmol) injection, the control group was i.t injected normal saline (NS), according to experiment design, after injection, each mice was return to the animal arena and videotape recording for 30 min immediately.
Itch behavioral Scratching behaviors were performed as previously described [10]. Behavioral tests were videotaped (HDR-CX190, Canon) at the animal plastic arena. The videos were played back on computer and the quanti cation of mice scratching behaviors were nished by observer who was blinded to the treatments. Hind paw scratching behaviors were recorded for 30 min with 5 min intervals. A battery of scratches were de ned as a lifting of the hind limb to the painting (nape) or injection site and then a replacing of the limb back to the oor or to the mouth, regardless of how many scratching strokes take place in between [11].
Mechanical sensitivity test The mice of the two groups were tested for paw withdrawal mechanical threshold (PWMT) before and after given intrathecal (i.t.) injection of morphine. PWMTs were measured on day 0, 1 and 3. The mechanical allodynia test method was described previously [12]. Mechanical sensitivity to mechanical stimulation was measured on a Dynamic Plantar Anesthesiometer apparatus (Ugo Basile, Comeria, Italy) according to the manufacturer's manual. Place the mice into the enclosure of Dynamic Plantar Anesthesiometer at least 30 min before the experiment to habituate them to the measurement conditions. The Dynamic Plantar Anesthesiometer pushed a thin rod (diameter 0.5 mm) with increasing force through a wire-grated oor against the plantar surface of a hind paw from beneath. The force increased from 0 to 5 g within 10 s (ramp 0.5 g/s) and was then held at 5 g for an additional 10 s. It stopped automatically and recorded the latency time when the animal withdraws the paw. Paw withdrawal mechanical threshold time was calculated as the mean of 6 consecutive stimulations of the hind paw at intervals of at least 10 s.
Thermal hyperalgesia evaluation For the heat pain test, the mice were placed in the plate apparatus. The rst ipsilateral hind paw exion re ex was considered the nociceptive endpoint, which was recorded as paw withdrawal thermal latency (PWTL). The response latency was recorded before the morphine treatment and at the 1st and 3rd day post-administration morphine. The maximum latency (cut-off) was set at 20 s to avoid tissue damage. To obtain data purely derived from the treatment, the inhibition values are presented as the difference between the basal values of vehicle or drug-treated animals and the respective controls.
PKC siRNA treatment Before observe PKC isoform role in MIS or in nalbuphine treatment. The experiment group divided into three groups: Control group, treatment with saline plus scramble siRNA; morphine group, treatment with PKC siRNA rst then i.t. morphine; Nalbuphine group, treatment with PKC siRNA rst then i.t. nalbuphine plus morphine. PKCα, PKCβ, PKCδ siRNA (Thermo Fisher) were delivered to the lumbar region of the spinal cord via i.t. injection as described [13]. In vivo jetPEI® as the transfection median was diluted by steriled DEPC·PBS·ddH 2 O. The three kinds of PKC siRNA were diluted with steriled DEPC·PBS·ddH 2 O in 0.5ug/ul concentration, then mixed up the PEI solution with siRNA, and put into room temperature for 20 min, total 2.5 µg/10 µl mixture siRNA was i.t. injected into mice, the scramble siRNA was injected into another control group mice. Mice were injected twice a day for 5 consecutive days.
Immunohistochemistry Mice were deeply anaesthetized with 10% chloralhydrate and perfused through right auricle with saline followed by 4% paraformaldehyde. After the perfusion, the spinal cords were removed and post-xed in 4% paraformaldehyde overnight. Then, dehydration according to 10%, 20%, 30% sucrose concentration gradient. Spinal cord tissues were cut into 20 µm thickness. Free oating sections were incubated in blocking solution containing 2% donkey serum and 0.1% Triton X-100 in PBS (PBS-T) for 1 h at room temperature. Then incubated with primary antibodies overnight at 4 °C, washed three times in PBS-T, incubated with the secondary antibodies for 2 h at room temperature and washed three times. Sections were mounted on slides with Fluoromount G (Southern Biotech) and coverslips. The following primary antibodies were used: Rabbit anti-Iba1(1:400; Sigma), rabbit anti-KOR (1:200; Sigma), mouse anti-phosphorylate-p38(1:200; Cell Signaling), mouse anti-Iba1 (1:400, Thermo Fisher). The secondary antibodies were purchased from Jackson Immuno Research Laboratories including Dylight 488 goat anti-rabbit antiserum (1:500, 1.25 µg/ml) and Dylight 594 goat anti-mouse(0.33 µg/ml). Images were taken using a Nikon Eclipse Ti-U microscope.
Western Blot Brie y, tissues were homogenized at 4 °C with an electric homogenizer (COYOTE, China) in RIPA with complete protease inhibitor cocktail (Selleck, USA). The homogenate was centrifuged at 12000 × g for 5 min at 4 °C and retained the supernatant. Protein concentration was determined using a Micro-BCA protein assay kit (Pierce, cat. 23235, Rockford, IL, USA) and BCA test method. Protein samples were diluted with the 2 × electrophoresis sample buffer, boiled for 5 min and rapid cooling on ice. For western blotting, proteins were electrophoretically transferred from resolving gels to nitrocellulose membranes Quanti cation and statistics Statistical comparisons were performed with GraphPad Prism 7. All data are presented as means ± SEM. Student t test was used for inter-group comparison, and one way-ANOVA was used for intra-group comparison. P values < 0.05 were considered to be signi cant.

Results
Nalbuphine signi cantly inhibited MIS but had no effect on mechanical and thermal pain behaviors MIS has been previously used to study itching [14], and KOR agonists, including nalbuphine, have been previously used to suppress itch, including MIS [15]. In the present study, we created an acute MIS model to more precisely determine the relevant effects of nalbuphine and its underlying mechanisms. Consistent with previous research [16], we found that nalbuphine signi cantly reduce MIS behaviors in 10-15 min after intrathecal injections when recording 30 min (Fig. 1A). Meanwhile, we observed that nalbuphine's inhibition of MIS was dose-dependent, with maximum inhibition observed at a dose of 2 nmol (Fig. 1B). We thus con rmed that nalbuphine dose-dependent inhibitory effect on MIS. However, the withdrawal thresholds for both mechanical and thermal pain were not signi cantly different between the morphine i.t. and nalbuphine treatment groups (Fig. 1C, 1D).
Microglial activation was involved in MIS and Iba1 was co-expressed with KOR Although previous research has observed that microglia mediate itch sensations [8], there has been a lack of research regarding microglial involvement in MIS. In order to address this research gap, we gave i.t.
injections of morphine to mice and then used immunohistochemical and western blot analyses to observe the mice's microglial expressions during their immediate MIS responses. We found that ionized calcium-binding adapter molecule-1 (Iba1), which is a marker of microglial, was signi cantly increased in the spinal cord laminae I-V of mice in the treatment group; in the control group, Iba1 was only present in laminae I and II (Fig. 2C, 2D). Morphologically, the microglial cell bodies were hypertrophic, with thickened and retracted processes, and their cell numbers were higher than in control mice. Increased CD11b, another marker of microglial, was also observed in mice exhibiting MIS ( Fig. 2A, 2B). Furthermore, after our administration of 2 nmol nalbuphine, the expression of Iba1 decreased signi cantly and even similarly closed located in laminae I and II (Fig. 2C). Western blot analyses also showed decreased CD11b expression in the nalbuphine treatment group.
We therefore next sought to investigate whether KORs activation inhibited microglial activation directly or indirectly. Using immuno uorescence, we found that KORs and Iba1 were co-expressed in the mice's spinal cords after nalbuphine treatment, as expected (Fig. 2C) [17]. More speci cally, KOR expression increased 20 min post-injection of nalbuphine, following a reduction of activated microglia. Moreover, although the total number of microglia decreased, most of the remaining microglia were co-expressed with KORs. These results suggest that nalbuphine may activate KORs, which in turn inhibit microglial activation, thereby diminishing MIS behaviors.
The antipruritic effects of nalbuphine were PKCδ-dependent and MIS was PKCβ-dependent Previous research has shown that PKCs mediate both KOR activation and a non-canonical opioid signaling mechanism in which Gastrin releasing peptide receptor (GRPR) activity is attenuated by KORmediated cross-signaling in mouse spinal cords [13,18]. These results led us to investigate which PKC isoform mediated KOR activation and inhibited itching. We thus performed three PKC knockdown studies in which mice were treated with either PKCα, PKCβ, or PKCδ siRNA [19].
We found that the PKCδ isoform was involved in KOR-related itching inhibitions: when we performed i.t. injections of PKCδ siRNA twice a day for 3 days, the PKCδ siRNA knockdown blocked nalbuphine's inhibition of MIS, as compared with the other siRNA treatment mice (Fig. 3A). However, PKCδ knockdowns had no effect on mice that received only morphine (Fig. 3B). Similarly, we found that the expression of PKCδ decreased after an initial i.t. morphine treatment, as compared with C57/BL6 control mice, but increased after subsequent treatment with nalbuphine (Fig. 3E), suggesting that nalbuphine's observed antipruritic effect was PKCδ-dependent.
We next tested the effects of PKCβ siRNA on MIS and found that knockdown PKCβ mice had attenuated MIS; they scratched signi cantly less than scramble siRNA control mice (Fig. 3C). However, nalbuphine's effects were the same in the scramble siRNA control mice and PKCβ knockdown mice (Fig. 3D). Similarly, western blot analyses showed that PKCβ were signi cantly increased in the morphine-treated mice and, accordingly, decreased after injection with nalbuphine (Fig. 3F). These results suggest that MIS is PKCβdependent.

Pkc May Affect Microglial Activation And Induce Kor Activation
To further evaluate the role of PKCs in MIS and in the KOR activation-induced inhibition of microglia, we rst examined the PKC isoform that in uenced microglial activation. Notably, we found that the microglia were activated after PKCδ siRNA treatment in our MIS model and that CD11b protein levels (a marker of microglial activation) were increased in both the morphine group and, especially, in the morphine plus nalbuphine group, as compared to the scramble siRNA control group (Fig. 4A). This is consistent with our previous results that the activation of microglia attenuated after nalbuphine treatment (Figs. 2A, 2B and 2C) but that, in knockdown PKCδ mice, CD11b increased more signi cantly after nalbuphine treatment for MIS (Fig. 4A). Furthermore, CD11b expression decreased in the morphine group after PKCβ siRNA treatment, thus con rming again that the morphine-induced microglial activation was PKCβ-dependent.
However, since microglial levels did not change signi cantly after treatment with nalbuphine in PKCβ siRNA mice (Fig. 4B), they may also be dependent on an additional underlying mechanism. Therefore, we next investigated whether the microglial activation marker Iba1 was co-expressed with either PKCβ or PKCδ in the morphine and nalbuphine treatment mice. We found that Iba1 expression was correlated with the expressions of both PKCδ and PKCβ (Figs. 4C, 4D); however, in the nalbuphine-treated MIS group, PKCδ expression increased while Iba1 expression decreased (Figs. 4C). This abnormal expression relationship thus indirectly con rmed that KORs may activate PKCδ but cannot trigger su cient microglial activation, thereafter, resulting in the inhibition of MIS. Moreover, we also found that PKCβ expression increased in laminae I and II after MIS was triggered, but decreased signi cantly after subsequent nalbuphine treatment (Fig. 4D). These results are consistent with our previously described western blot results (Fig. 3F).

The p38 mitogen-activated protein kinase (MAPK) microglial pathway was inhibited after KOR activation
Microglial activation after MIS led us to hypothesize that the p38 MAPK pathway was inhibited after nalbuphine-induced KOR activation. To investigate the involvement of the p38 MAPK pathway, we immunostained spinal cord sections for p-p38 expression after nalbuphine treatment. We found that p-p38 expression increased signi cantly in the morphine-treated mice as compared to the control mice; however, the positive p-p38 immunostained cells decreased after treatment with nalbuphine. Iba1 was also co-expressed with p-p38, and their expressions were consistent with each other in the spinal cord (Fig. 5B). We also examined p-p38 expression using western blot analyses and found that p-p38 protein levels in the spinal cord were increased in the morphine-treated mice but decreased after subsequent treatments of nalbuphine (Fig. 5A). Taken together, these results suggest that p-p38 expression levels in the activated microglia were involved in both MIS and in the KOR-mediated inhibition of itching.

Discussion
Itching is a pathological process in the somatosensory system at the levels of the primary sensory neurons, spinal cord, and brain. Although there has been distinct progress in the itching research eld, the mechanisms underlying the sensation and transmission of itching, and in particular morphine-induced itch, are still unclear. Research over the past decade has increasingly shown that pain and itch are two clearly distinct sensations [20], and recent studies have identi ed separate neuronal pathways involved in each sensation [21].
One proposed mechanism for chronic itch involves an abnormal excitability of pruritic-speci c neurons, such as the GRPR and NPPA neurons in the spinal dorsal horn [13,22,23]. However, although GRPR has been shown to be involved in MIS [19], an increasing body of evidence suggests that synaptic hyperexcitability in the spinal dorsal horn may not be a consequence simply of neuronal changes but may also be involved in multiple glial cell alterations [6].
The important role of microglia in itching sensations has also been veri ed in a series of studies [8,[24][25][26]. As CNS-dwelling macrophages, it is known that microglia play an important immunoregulatory role within the CNS [27]. Moreover, it has been shown that microglia become activated in the spinal dorsal horn after peripheral nerve damage [8]. Previous studies have also revealed the involvement of spinal microglia in pruritogen-induced itching, and recent research has shown that compound 48/80 and GNTI can elicit pronounced scratching behaviors in mice in conjunction with microglial activation in mouse spinal cords [21]. However, it remains rather unclear exactly how microglia mediate MIS and how they affect the KOR activation process.
Previous evidence has suggested that PKCδ could play a speci c role as an intracellular modulator of itch in sensory neurons [28]. It is also known that KOR activation attenuates itch via PKCδ, although the broader role of PKCs in the development of acute itch has not yet been elucidated. We therefore set out to investigate PKC mechanisms, and we found that PKCδ affected the KOR-mediated inhibition of MIS. However, although PKCδ could still be activated by the administration of nalbuphine in mice treated with PKCδ siRNA, it had less effect on MIS. These observations are consistent with the hypothesis that PKCδ is activated in the KOR-mediated inhibition of MIS [13].
In addition, we also found that CD11b levels demonstrated an opposite trend to that of PKCδ. Since microglia were activated after treatment with PKCδ siRNA in the nalbuphine plus morphine group, it is clear that PKCδ had a negative feedback regulation of morphine, and this is also consistent with previous research. In contrast, mice treated with PKCβ siRNA demonstrated that PKCβ positively regulated microglia. The MIS model was therefore associated with increased PKCβ expression, which was in turn inhibited by nalbuphine. The expressions of both PKCδ and PKCβ were also both associated with microglial activation. However, whether PKCδ activates microglia directly via phosphorylation or indirectly via other kinases requires additional research [29]. We were also unable to identify any role for PKCα in acute itching.
Our research used knockdown PKC isoforms to observe the role of microglia in MIS and to explore the relationship between microglial activation and the KOR/PKC pathways in nalbuphine's antipruritic effect. Using a series of pharmacological, behavioral, immunochemical, and molecular biology experiments, we rst found that the KOR agonist nalbuphine inhibited MIS in a dose-dependent manner and that this inhibitory effect was PKCδ-dependent. Second, we demonstrated that MIS could induce microglial activation and that this activation was PKCβ dependent. Third and nally, we found that nalbuphine inhibited MIS via the retroegulation of microglial activation by the KOR-PKCδ pathway. Our research has thus identi ed a new mechanism underlying nalbuphine's antipruritic effect and provides novel insights about MIS that may lead to drug targets. Moreover, previous research has shown that KOR agonists are effective at suppressing scratching in mice treated with different pruritogens [30]. Nalbuphine is a nonscheduled KOR agonist and mu opioid receptor antagonist that has been approved by the FDA since the 1980s for clinical treatment of moderate to severe pain, including in postoperative pain management; more recently, it has also been studied in clinical trials as a treatment for chronic itch, or prurigo-nodularis. Although the use of nalbuphine as a treatment for the pruritus associated with neuraxial opioid use has not been approved by the FDA, it has nevertheless been used in this capacity. In fact, one study found that nalbuphine was a better treatment for MIS than either a placebo, a control, diphenhydramine, naloxone, or propofol in patients receiving neuraxial opioids for acute pain related to surgery or childbirth [31]. However, the mechanism underlying nalbuphine's antipruritic effects is still unclear.
There has been relatively little research on this topic to demonstrated that spinal KOR activation reduced itch transmission by inhibiting the function of GRPR. However, this is the only study to date that has investigated the role of a KOR agonist in MIS, and it investigated the role of neurons rather than microglia. The present research demonstrated that microglia's immunoregulatory roles may be involved in MIS and that nalbuphine may affect microglial functioning in some manner. As expected, we observed signi cantly increased microglial activation after morphine i.t. injections; moreover, this activation was related to PKCβ function. Since this is the rst time that this phenomenon has been observed in MIS, we used siRNA knockdowns to con rm that it was PKC isoform-speci c; we observed that, in knockdown PKCβ mice, microglial activation was dramatically inhibited following decreases in MIS, thus demonstrating PKCβ's importance in MIS.
Two recent photon in vivo imaging studies found that microglial processes are highly dynamic in the brain and spinal cord [32]: stimulated microglia rapidly move toward the site of an injury and directly appose synaptic regions, in response to neuronal activity, thereafter facilitating contact with highly active neurons [25]. Microglial activation had not been previously reported in MIS [33]. Moreover, KOR activation was thought to attenuate the expression of microglia via some other mechanism than the G-protein signaling pathway [34]. However, we found that microglia were activated in the spinal dorsal horns.
Microglia are known to dramatically alter gene expressions at a molecular level, including via Iba1, CD11, and p-p38, and our results demonstrate that microglia play an important role in physical condition monitoring, such as itching, regardless of the severity of an injury [35].
Research has also shown that various microglia signaling molecules, including cell-surface receptors, are increased in the spinal dorsal horn once microglia are activated following a nerve injury [36]. Following a nerve injury, the phosphorylation of p38 MAPK increases, although this is highly restricted to spinal microglia [37]. In vitro studies have furthermore shown that activated p38 MAPK is present in spinal microglia and participates in the signaling pathways that mediate nociception at the spinal level [38]. Microglia and p-p38 have also been shown to have a role in chronic itch [9].
Since we knew the potential roles of PKCs and microglia, we therefore hypothesized that p38 MAPK was also involved [39]. An examination of p-p38 expression showed increased levels of p-p38 after treatment with morphine that were furthermore inhibited by nalbuphine. Iba1 cells were also co-expressed with p-p38 in the spinal cord, con rming our western blot results. These results suggest that microglial activation may occur by phosphorylating p38 to maintain an acute MIS signal. Our previous research has also suggested that the microglial inhibitor minocycline inhibited the p-p38 MAPK pathway in our MIS model [40]. Overall, our present results suggest that, as the regular downstream microglial pathway, p38 MAPK precisely regulated microglia during the KOR-mediated inhibition of MIS. Although signi cant prior evidence existed that microglia play a role in chronic itch and pain, the eld has lacked a deep understanding of microglia's role in pruritus-induced itching and in neuraxial opioid-induced itching. Although our research observed an increased expression of microglia and the involvement of the p-p38 pathway, the exact microglial mechanisms underlying MIS still require further research.
Taken together, our acute itch mouse experiments have suggested a new regulation mechanism involving KORs and microglia. Moreover, the signaling pathway that we identi ed here as a mediator of acute MIS is similar to the one involved in pain, which also hinges on microglia and p-p38 activation. Ultimately, our ndings about PKC isoform involvement and the downstream effects of p38 MAPK and KOR activation suggest new ways to treat MIS.

Declarations
Ethics approval and consent to participate The study was approved by the Animal Studies Committee at Guangzhou Medical University, China.

Consent for publication
Not applicable.

Availability of data and materials
The datasets generated and/or analyzed in this study are available from the author on reasonable request.

Funding
The research was supported by the National Natural Science Foundation of China (No. 81771182) to L Wan, by Natural Science Foundation of Guangdong Province (No.2016A030313599) to L Wan.

Competing interests
The authors report no con icts of interest in this work.
Authors' contributions LW designed the experiments, YT, NL and SH performed behavioral, immunohistochemistry and molecular biology experiments. YT performed molecular analysis, ZP contributed to the project, YT and LW wrote the manuscript and LW revised the writing. All authors read and approved the nal manuscript. Figure 1 Intrathecal administration of nalbuphine can attenuate morphine induced scratch, but have no effect on    PKCδ and PKCβ are in uenced in the express of CD11b. (A) Western blot and quanti ed data showed the protein level of CD11b was slightly increased after treat the PKCδ siRNA mice with morphine while it was signi cantly increased after nalbuphine treatment in contrast. **P<0.01, ***P<0.001; bars represent mean ± SEM; n=6 for each group. (B) Western blot and quanti ed data showed the protein level of CD11b decreased after PKCβ siRNA treatment in morphine model mice while had more effect on nalbuphinetreated mice in contrast. *P<0.05; bars represent mean ± SEM; n=6 for each group. (C) Representative images of spinal cord sections immunostained with anti-Iba1 and anti-PKCδ after morphine treatment; additional animals received concomitant treatment with nalbuphine following morphine treatment. Bar graph showing the number of PKCδ immunoreactive (IR) cells per spinal cord section from animals with treatments indicated before and the PKCδ-IR+ and Iba1-IR+ cells were expressed more in nalbuphine treatment mice than morphine alone treated mice. **P<0.01; bars represent mean ± SEM; n=6 for each group; Six sections per animal were selected for counting. (D) Representative images of spinal cord sections immunostained with anti-Iba1 and anti-PKCβ after morphine treatment; additional animals received concomitant treatment with nalbuphine following morphine treatment. Bar graph showing the number of PKCβ immunoreactive (IR) cells per spinal cord section from animals with treatments indicated before and the PKCβ-IR+ cells were expressed more in morphine treatment mice than combine nalbuphine treated mice, but the Iba1-IR+ expression was more in nalbuphine treated mice than morphine mice. **P<0.01, ***P<0.001; bars represent mean ± SEM; n=6 for each group; Six sections per animal were selected for counting.