Dual effects of Rho-kinase inhibitors on a rat model of inflammatory pain.

BACKGROUND: Rho-kinases (ROCKs), a family of small GTP-dependent enzymes, are involved in a range of pain models, and their inhibition typically leads to antinociceptive effects.
OBJECTIVES: To study the effects of inhibiting ROCKs using two known inhibitors, Y27632 and HA1077 (fasudil), administered locally, on nociception and paw edema in rats.
METHODS: A range of doses of Y27632 or HA1077 (2.5 μg to 1000 μg) were injected locally into rat paws alone or in combination with carrageenan, a known proinflammatory stimulus. Nociceptive responses to mechanical stimuli and increased paw volume, reflecting edema formation, were measured at 2 h and 3 h, using a Randall-Selitto apparatus and a hydroplethysmometer, respectively.
RESULTS: Animals treated with either ROCK inhibitor showed biphasic nociceptive effects, with lower doses being associated with pronociceptive, and higher doses with antinociceptive responses. In contrast, a monophasic dose-dependent increase in edema was observed in the same animals. Local injection of 8-bromo-cyclic (c)GMP, an activator of the nitric oxide⁄cGMP⁄protein kinase G pathway, also produced biphasic effects on nociceptive responses in rat paws; however, low doses were antinociceptive and high doses were pronociceptive. Local administration of cytochalasin B, an inhibitor of actin polymerization and a downstream mediator of ROCK activity, reversed the antinociceptive effect of Y27632.
CONCLUSIONS: The results of the present study suggest that ROCKs participate in the local mechanisms associated with nociception⁄antinociception and inflammation, with a possible involvement of the nitric oxide⁄cGMP⁄protein kinase G pathway. Also, drug effects following local administration may differ markedly from the effects following systemic administration. Finally, separate treatment of pain and edema may be needed to maximize clinical benefit in inflammatory pain.

Rho-kinase (ROCK) is a serine/threonine kinase identified as a GTP-Rho-binding protein belonging to the family of GTPases (1). Although many studies have focused on ROCKs in smooth muscle cells (2,3), many other activities have been associated with ROCKs in other cellular systems (3–5). Particularly, the participation of ROCKs in platelet aggregation, formation of stress fibres, focal adhesion, cellular motility (6–9) and monocyte transendothelial migration (10) has been described, which, collectively, suggest the involvement of ROCKs in inflammatory processes (11–13). Closer to the present context, ROCK activity has also been implicated in pain sensitivity responses such as those stimulated by cold (14), heat (15,16), formalin (17) and other stimuli (18–20). In addition, ROCKs have been implicated in the inhibition of acetylcholine release in peripheral cholinergic nerves (21).

In terms of underlying mechanisms, ROCK activity modifies cyto-skeletal structure and function by metabolism of intermediary filaments (vimentin [8]); phosphorylation of myristoylated alanine-rich C-kinase substrate (22); or, especially, via binding proteins that regulate microfilament polymerization (5,23,24). The imbalance in cyto-skeleton assembly caused by ROCK activation may interfere in crucial functions of the central nervous system (19) including inflammatory and neuropathic pain (22). Accordingly, ROCK inhibition increased neurite outgrowth (17,24,25), an important aspect of the cytoskeleton associated with augmented pain sensitivity (26); conversely, its disturbance was reflected in a decrease in the pain sensitivity response (15). Another mechanistic link between ROCKs and nociception (17) is their with the phosphorylation cascade of cofilin, an actindestabilizing protein (27), which implies an important role in nociception mediated via the nitric oxide (NO)/cyclic (c)GMP/protein kinase G (PKG) pathway. Other NO-ROCK interactions lead to a stimulatory activity of ROCKs on NO synthesis (28) and the inverse, activation of ROCK production by NO (29). Thus, although ROCK-NO interactions have been demonstrated, the mechanisms underlying these interactions are not completely understood. The NO/cGMP/PKG pathway is an important component of the peripheral analgesic effects associated with a range of known analgesic compounds (30–39). Despite the well-documented analgesic or, more properly, antinociceptive effects related to this cGMP pathway, nociceptive as well as antinociceptive responses have been observed following injections of NO donors, cGMP analogues or L-arginine, depending on the dose and agent used (40–45). Because biphasic effects are brought about by activation of the cGMP pathway (41,43) and by ROCK inhibition (46), and because both pathways could be involved in nociceptive responses in the periphery, we have analyzed the role(s) played by ROCK inhibitors in a model of peripheral nociception.

We have tested the effects of two ROCK inhibitors, Y27632 and HA1077 (fasudil), alone or in combination with carrageenan, a standard proinflammatory stimulus (47), in a well-established model of inflammatory pain (48), assessing both nociception and edema. Because most of the work using ROCK inhibitors in vivo cited above has used systemic or central administration of these compounds, and because we were particularly interested in their peripheral effects, we administered the ROCK inhibitors locally, by intraplantar (IPL) injection. We also examined the inflammatory effects of 8-bromo-cGMP, a cGMP analogue, to assess the contribution of the NO/cGMP/PKG pain pathway to this model and investigated the participation of the cytoskeleton (actin filaments) in the nociceptive responses to the ROCK inhibitor Y27632.

METHODS

Animals and preparation

All animal care and experimental procedures conformed to the regulations of the International Association for the Study of Pain on Ethical Issues and were approved by the Federal University of Minas Gerais Ethics Committee for Animal Use (Belo Horizonte, Brazil). Male Holtzman rats (150 g to 180 g) were supplied by the Bioterism Center of the Federal University of Minas Gerais. The animals were housed in groups of five animals with a 12 h light/12 h dark cycle and temperature of 24±2°C. They were allowed access to commercial food and water ad libitum. In the first experiment, groups of rats (n=3 to n=5) were injected with Y27632 (2.5 μg to 1000 μg), HA1077 (25 μg to 500 μg), 8-bromo-cGMP (25 μg to 1000 μg) or vehicle (2% dimethyl sulfoxide in saline) IPL at time zero. In a second experiment, groups of animals (n=5) were injected with Y27632 (25 μg or 500 μg) or vehicle 5 min before the proinflammatory stimulus (250 μg carrageenan, injected IPL), considered to represent time 0. In a third set of experiments, cytochalasin B (1 μg) or vehicle was injected IPL 15 min before Y27632 (25 μg or 500 μg; n=3 to n=5).The total volume of all IPL injections was never >100 μL and they were administered with sterile 1 mL syringes and 0.38×13 mm needles. The doses and dilutions of drugs and the times of administration used in the experiments were based on published data for the ROCK inhibitors (15,18) and for 8-bromo-cGMP (41,43).

Measurement of the nociceptive threshold

All assays were performed without knowledge of the treatments administered to the animals. The nociceptive response was evaluated using a pressure algesimeter (Ugo Basile, Italy), in which a progressive compression is applied to a rat’s hindpaw, essentially as proposed by Randall and Sellitto (48). This method consists of the measurement (in g) of the force applied to the pads necessary to trigger a defensive behaviour, such as hindpaw withdrawal, which is a sign of nociception. To avoid tissue damage, the maximum weight applied was fixed at 300 g. The intensity of the nociceptive response (Δ nociceptive threshold) was expressed as the mean (± SEM) difference between nociceptive thresholds (in g) obtained in the right (injected with the test substance) and left (injected or not injected with vehicle) paws at each timepoint. The difference (Δ) of nociceptive thresholds among saline, vehicle and naive-contralateral paws did not change significantly over the experimental period from the values at time 0 (Table 1).

TABLE 1

Effects of the Rho-kinase inhibitor Y27632 (Y) on nociceptive thresholds in rat paws

Treatment/dose, μg	Nociceptive threshold g applied/time, min
	0	30	60	120	180
Vehicle (n=5)
RP	134±9	126±15	126±5	128±8	136±9
LP	138±8	130±12	134±9	132±11	132±8
2.5 μg Y (n=4)
RP	127±9	117±20	125±13	125±6	130±12
LP	128±10	127±10	130±12	128±10	128±10
25 μg Y (n=7)
RP	133±8	83±17	81±13	84±14	101±12
LP	131±7	129±9	130±8	124±8	130±8
50 μg Y (n=6)
RP	130±9	88±8	86±20	100±31	125±14
LP	130±9	133±12	135±12	128±15	130±13
150 μg Y (n=5)
RP	130±10	124±11	130±7	124±11	128±11
LP	132±8	126±11	126±9	128±8	130±10
500 μg Y (n=7)
RP	126±5	197±17	191±31	151±19	131±12
LP	127±8	128±11	130±10	126±8	133±8
1000 μg Y (n=6)
RP	127±5	220±32	215±26	178±21	138±22
LP	125±5	117±5	118±4	123±10	120±6

Data presented as mean ± SD. Animals received vehicle or Y in a volume of 100 μL injected intraplantarly into animal's right hind paw(RP). The left hind paw (LP) was injected only with vehicle and served as a control. Measurements of nociceptive thresholds were performed without knowledge of the treatments

The nociceptive responses were measured immediately before (time 0), and 15 min, 30 min, 60 min and 120 min after administration of ROCK inhibitors, and also 180 min after carrageenan administration. To facilitate data comparison, the results were transformed into area under the curve over 120 min or 180 min of observation for each group, using the trapezoidal rule from GraphPad Prism version 6.01 (GraphPad Software, USA).

Assessment of paw edema formation

Paw volumes of the same animals (see above) were measured using a hydroplethysmometer (Ugo Basile, Italy), and data were expressed as the difference (Δ) between right and left hindpaws in millilitres (mean ± SEM) for a group of animals, at the same time points as used in the nociceptive threshold experiments. To facilitate data comparison, these results were also transformed into the area under the curve using the trapezoidal rule, as described above.

Chemicals

The compounds Y27632 ([R]-[+]-trans-4-[1-aminoethyl]-N-[4-pyridyl] cyclohexanecarboxamide dihydrochloride) and HA1077 (fasudil) were purchased from Cayman Chemicals (USA) and LClabs (USA), respectively. Cytochalasin B, an inhibitor of actin polymerization (49), and 8-bromo-cGMP were purchased from Sigma (USA) and λ-carrageenan from Science Lab (USA). All compounds, except carrageenan, were dissolved in 1% to 20% dimethyl sulfoxide in sterile isotonic saline. Carrageenan was dissolved in sterile isotonic saline.

Statistical analysis

Data for each group of four to seven animals (mean ± SEM) were analyzed using one-way ANOVA. Differences between means were further examined using Bonferroni’s test. Differences between means were considered to be statistically significant at P<0.05.

RESULTS

Effects of single IPL injections of ROCK inhibitors or a cGMP analogue on nociceptive threshold and paw edema

To study the role of the ROCK inhibitors on nociception or paw edema, single injections of Y27632 or HA1077 were administered IPL to rat paws. As shown in Figure 1A and 1B, the lower doses of both ROCK inhibitors (25 μg/paw to 50 μg/paw) induced a decrease in the nociceptive threshold, equivalent to the development of hyper-algesia, ie, a pronociceptive response. Intermediate doses were associated with no significant changes in nociceptive thresholds, whereas higher doses (500 μg/paw to 1000 μg/paw) were associated with an elevation of nociceptive threshold above basal levels, equivalent to hypoalgesia or an analgesic response. However, IPL administration of the same doses of Y27632 and HA1077 evoked a continuous dose-response curve of paw edema formation, as shown in Figure 2A and 2B, respectively. It is worth emphasizing that the edema measurements were conducted in the same rats and at the same time as the nociceptive assays. The effects of a cGMP analogue given IPL on nociceptive threshold and paw edema were also studied. As shown in Figure 3, 8-bromo-cGMP also elicited a biphasic effect on nociceptive responses over the dose range used (25 μg to 1000 μg). However, the low dose of 8-bromo-cGMP (25 μg/paw) was associated with an elevation of nociceptive threshold above basal levels (hypoalgesia) whereas the highest dose (1000 μg/paw) was associated with a lowering of nociceptive thresholds (Figure 3). Moreover, the animals injected IPL with 8-bromo-cGMP showed no increases in paw volume, ie, no signs of paw edema (data not shown).

Figure 1)

Dual effects of two Rho-kinase (ROCK) inhibitors, Y27632 (A) and HA1077 (B), on nociceptive threshold in rats. Nociceptive thresholds were measured using a pressure algesimeter at 15 min, 30 min, 60 min and 120 min following single injections in rat hind paws. Data were transformed into area under the curve (AUC) over 120 min. Inhibitors were prepared in a solution of dimethyl sulfoxide diluted in iso-tonic saline: vehicle (Veh); Y27632 (Y), 2.5 μg to 1000 μg; HA1077 (HA), 25 μg to 500 μg.

Figure 2)

Dose-response curve of Y27632 (Y) (A) and HA1077 (HA) (B) on paw edema formation. The paw volumes (in mL) were measured using a plethysmometer (Ugo Basile, Italy) at 15 min, 30 min, 60 min and 120 min following single injections in rat hindpaws. Inhibitors were prepared in a solution of dimethyl sulfoxide diluted in isotonic saline: vehicle (Veh); Y27632 (Y), 2.5 μg to 1000 μg; HA1077 (HA), 25 μg to 500 μg.

Figure 3)

Dual effect of a cyclic (c)GMP analogue, 8-bromo-cGMP (8-B-cGMP) (25 μg to 1000 μg), on nociceptive thresholds in rat paws. Low doses of the cGMP analogue (25 μg) induced analgesia and higher doses induced hyperalgesia. *Significantly different from vehicle (P<0.01); n=4 animals per group. AUC Area under the curve; Veh Vehicle

Treatments administered to the right ipsilateral hindpaw that altered its nociceptive threshold or volume had no effects on the responses in the left contralateral paw that received only vehicle (Table 1).

Effects of Y27632 on responses to carrageenan in rat hindpaws

Because Y27632 administered alone altered the nociceptive threshold of rat paws, the effects of combining Y27632 with carrageenan, a standard proinflammatory stimulus (47), were subsequently examined. In these assays, Y27632 was injected IPL 5 min before carrageenan and nociceptive thresholds and paw volume measured over the subsequent 180 min. At the lower dose, Y27632 (25 μg per paw) which, when given alone, induced significant hyperalgesia (Figure 1A), did not add to the hyperalgesia induced by carrageenan (Figure 4A) or modify its time course (data not shown). At the higher dose (500 μg), however, Y27632 did modify carrageenan-induced hyperalgesia, decreasing the fall of nociceptive threshold (Figure 4A), ie, the ROCK inhibitor behaved as an antinociceptive agent. Hindpaw volumes from these animals injected with carrageenan and Y27632 were also measured (Figure 4B). Again, the low dose of Y27632 did not affect carrageenan-induced paw edema, whereas the higher dose of Y27632 clearly increased the carrageenan-induced edema. The cGMP analogue 8-bromo-cGMP combined with carrageenan showed effects similar to those of Y27632 on carrageenan-induced hyperalgesia, with the lower dose of 8-bromo-cGMP (25 μg) producing an antinociceptive effect (data not shown).

Figure 4)

Effects of the combination of Y27632 (Y) (25 μg or 500 μg) with carrageenan (CG) (250 μg) on rat hyperalgesia (A) and paw edema (B). The effects were assessed using a pressure algesimeter and a plethysmometer, respectively. Data were transformed to area under the curve (AUC) over 180 min.

Effect of cytochalasin B on the pro- and antinociceptive effects of Y27632

Cytochalasin B is known as an inhibitor of actin filament polymerization (49) and has recently been shown to be involved in analgesia induced by morphine in the pain model used in the present study (50). Local pretreatment of rat paws (15 min before) with cytochalasin B (1 μg IPL) did not affect hyperalgesia following the low dose of Y27632 (25 μg) but completely prevented the hypoalgesia induced by the high dose (500 μg) (Figure 5). Pretreatment with cytochalasin B did not affect the paw edema induced by the ROCK inhibitor in these animals (data not shown).

Figure 5)

Reversal of the analgesic effect induced by Y27632 (Y) (25 μg or 500 μg) by cytochalasin B (CB) (1 μg) in rat paws. CB was injected 5 min before administration of a low (25 μg) or a high (500 μg) dose of Y locally. CB and Y were diluted in a solution of dimethyl sulfoxide and isotonic saline (vehicle [Veh]). #Significant difference between Veh-Y500 and CB-Y500 (P<0.05). n=3 to n=5 animals per group

DISCUSSION

Our experiments showed that Y27632, a ROCK inhibitor, induced a dose-dependent biphasic effect on nociceptive response, ie, hyper- and hypoalgesia, when administered locally to rat hindpaws. Because both the biphasic nature of the response and the clear pronociceptive effect were unexpected, we used another ROCK inhibitor, HA1077, in our system. Over a similar dose range (25 μg to 500 μg per paw), HA1077 also induced a decrease and an increase in nociceptive threshold, respectively, ie, hyperalgesia at the lowest dose and hypoalgesia at the highest dose used. Interestingly, we had previously shown that analgesic drugs, such as celecoxib and morphine, also induced hypoalgesic or antinociceptive responses under similar experimental conditions (50–52). Because the molecular weight of the two ROCK inhibitors are very close (approximately 300), the doses we used were comparable in molar terms, and the similarity in responses to Y27632 or HA1077 suggests that these biphasic nociceptive responses were more likely to be related to their common activity as ROCK inhibitors.

Our data are comparable with those reported by Chan et al (46), who used a model of cervical column transection, in that beneficial and detrimental effects, respectively, were also observed when a higher or a lower dose of Y27632 was used. However, the predominant effect associated with ROCK inhibitors was antinociception (14–17,53). In particular, HA1077 was associated with antinociceptive properties in a wide variety of pain models (18). In addition, another ROCK inhibitor (AS1892802) tested in a rat model of arthritis was similarly antinociceptive after oral (10 mg/kg) or intra-articular injection (3 μg) in inflamed knees (19,53). Yoshimi et al (20) observed no effect of this ROCK inhibitor, ie, neither nociception nor antinociception, after intra-articular injection in the noninflamed knee. One possible resolution to this paradox is to suggest that other kinases were inhibited by the doses of ROCK inhibitors that we were using (see below), that these other kinases were mediating these opposing effects and overcoming the pronociceptive effects of ROCK inhibition. Another possible solution, also suggested by Zulauf et al (17), is that ROCK, via microfilament modulation, has a role in the cGMP pathway, which has been described as inducing a biphasic effect on nociception (reviewed in Cury et al [45]).

Indeed, the cGMP analogue (8-bromo-cGMP) used in the present study under the same experimental conditions as that of the ROCK inhibitors showed a biphasic nociceptive threshold response, clearly demonstrating a role for this pathway under our conditions. However, this cGMP analogue, which is an agonist of the NO/cGMP/PKG pain pathway, showed antinociceptive effects at low doses, compatible with earlier reports of the involvement of the cGMP pathway in analgesia (33,34,44). The pronociceptive effects of high doses of 8-bromocGMP could be related to the pronociceptive effects reported for NO (43,44) or to effects of other unrelated systems. These data suggest that the NO/cGMP/PKG pathway may be involved in the antinociceptive effect of the ROCK inhibitors we studied.

Our results involving cytochalasin B demonstrated the dependence of the antinociceptive effect of ROCK inhibitors on cytoskeleton integrity, indicating the involvement of microfilaments in nociceptive pathways in the periphery. The present data are compatible with earlier findings using cytochalasin B and the cyclooxygenase-2 inhibitor celecoxib and morphine (50). Because ROCK is a possible target for PKG (54) and ROCK activation can lead to modulation of actin filaments and neurite outgrowth (17,55), it appears that ROCK and microfilaments in the cytoskeleton are important components of the modulation of peripheral pain.

One clear limitation to the present study is the specificity of the ROCK inhibitors. Signalling systems other than the ROCKs may be particularly relevant to the effects of high doses of ROCK inhibitors because both the inhibitors we have used, similar to most kinase inhibitors, are also inhibitors of kinases other than ROCK, usually with lower potencies. Thus, relative to ROCK, Y27632 has a half-maximal inhibitory concentration (IC50) 10-fold greater and HA1077 is approximately threefold less potent toward mitogen and stress activated kinase 1 (56). Potencies against protein kinase A or protein kinase C are also approximately 10- to 20-fold less than against ROCK, for both compounds (56). However, the IC50 values for ROCK inhibition range between 800 nM and 20 μM, concentrations well below those we have applied (78 nmol to 1560 nmol [25 μg to 500 μg] in 100 μL), but there are factors that would further reduce this margin. First, the reported IC50 values have been determined in vitro with cell-free systems, and we applied the compounds to the foot pad, so there will be both diffusion and binding in the extracellular space and passage through the cell membrane to dilute and delay the amounts actually reaching an intracellular kinase. Also, the IC50 measured using a whole-cell assay may be very different from that with cell-free enzymes. For instance, the IC50 for fasudil as an inhibitor of 95D lung carcinoma cells in culture was 0.79 mg/mL (equivalent to 2.1 mM [57]). Chan et al (46) used intrathecal infusions of 2 mM or 20 mM repeated over 14 days to show the effects of Y27632 on rat spinal cord injury. Nevertheless, and in spite of the similarity of the hyperalgesic responses to the two different ROCK inhibitors, we cannot exclude the possibility that the effects we observed were not causally related to inhibition of ROCK.

The present study used one model of inflammatory pain and one mode of stimulation; therefore, it is possible that in other peripheral pain models, such as IPL formalin (43) or rat arthritic knees (19,53), and using other stimuli, the effects of ROCK inhibitors could be different. However, we used a thermal stimulus (Hargreaves method) in our model with carrageenan inflammation (58) and obtained results in good agreement with those obtained with the mechanical stimulus we used here. It is also possible that the outcome of our experiments would be different in female rats because we used only male rats in the present study.

It is important to emphasize that even at the highest doses of the ROCK inhibitors, the contralateral paw that was not injected with Y27632 or HA1077 showed no changes in its nociceptive or edema status. This demonstrated very clearly that the effects we observed in the treated ipsilateral paw were, indeed, local effects and not due to leakage of the locally injected inhibitor into the systemic circulation. These data from the contralateral paw also serve to exclude the possibility of a central or spinal (28,46) action of the ROCK inhibitors, leading to sedation and consequent motor dysfunction. In another model, alterations in the activation of the RhoA-ROCK system were associated with changes in conduction velocity only in sensory, not motor, neurons (59).

An unexpected outcome of our experiments was that the ROCK inhibitors were inflammatory at the lower doses, when the compounds were most likely to be acting as selective inhibitors of ROCK. This action was expressed in our model as hyperalgesia (a pronociceptive effect), and the antinociceptive effects were only observed at the higher doses when selectivity would be less, even when the paw was inflamed by carrageenan. The proinflammatory effect of ROCK inhibition by Y27632 or HA1077 was also expressed as increased microvascular permeability leading to the formation of edema. This component of inflammation was directly dose-dependent, with no indication of a biphasic profile. Thus, at low doses, the ROCK inhibitors were primarily proinflammatory and only partially anti-inflammatory (in terms of pain) at the higher doses.

Our results have also contributed to the evidence for a mechanistic divergence between two classical components of inflammation – pain and edema. Both Y27632 and HA1077 clearly, and without any sign of a biphasic profile, induced paw edema dose-dependently, contrasting sharply with the biphasic effects on nociception. This division between edema and nociception in our model was emphasized by the effect of 8-bromo-cGMP, which elicited no signs of edema over a dose range that induced a biphasic effect on nociception. This discrepancy between the two inflammatory responses did, however, show very clearly that, in our model, changes in nociception and edema were coincident in time but were not mediated by the same pathways. Although these distinct pathways have not yet been fully elucidated, a similar separation has been observed earlier in our model (51) and under other experimental conditions (60).

CONCLUSION

We have demonstrated that the peripheral injection of two ROCK inhibitors exerted a dual effect on nociceptive responses. Biphasic nociceptive responses were also induced by 8-bromo-cGMP, an activator of the NO/cGMP/PKG pathway. Our results are in agreement with most previous reports of antinociceptive effects of ROCK inhibitors but in contrast to their more frequently described anti-inflammatory activity. This difference is most likely to reflect the local and peripheral, as distinct from systemic, application of the ROCK inhibitors. Our data also suggested the possible involvement of the actin filaments of the cytoskeleton in nociceptive responses, which was modulated by ROCK inhibition. Further studies, at the molecular level are warranted to confirm the involvement of ROCKs and the NO/cGMP/PKG pathway in the processes of peripheral nociception, in both inflamed and normal tissues. Such data, however, open new approaches to our understanding of the mechanisms involved in the genesis of pain at the structural level of the cells.