I.T. Injection of BIRT-377 reverses enduring allodynia resulting from minor CCI in PAE rats
Previous studies show that PAE-induced sensitivity to allodynia is enduring for at least 28 days after CCI [42]. Thus, we want to determine whether blocking the activated LFA-1 altered enduring PAE induced sensitivity to allodynia. To accomplish this, i.t. BIRT-377 is administered at Day 28 post-surgery. Interestingly, all rats reveal similar hindpaw sensitivity at BL (ipsilateral, F1,43 = 1.568, p = 0.217), indicating that PAE alone does not induce allodynia, but rather, it is not until a second insult is applied that the effects of PAE are unmasked, which replicates prior findings [42, 49, 63] (Fig. 1). Following CCI, a main effect of alcohol exposure (ipsilateral, F1,43 = 152.120, p < 0.0001), surgery (ipsilateral, F1,43 = 404.296, p < 0.0001) and an interaction between alcohol exposure and surgery (ipsilateral, F1,43 = 170.740, p < 0.0001) is observed. Normal sensitivity is maintained throughout the 28-day time-course following sham or minor (1-suture) CCI surgery in healthy Sac control rats (Fig. 1a). In contrast, compared to PAE rats with sham surgery that maintain normal sensitivity through day 28 post-surgery, minor CCI results in robust and enduring unilateral allodynia (Fig. 1b) (ipsilateral, F1,43 = 165.47, p < 0.0001). At Day 28, all rats received BIRT-377 or vehicle injection where a significant interaction is seen in the ipsilateral hindpaw between alcohol exposure, surgery, and injection (ipsilateral, F1,43 = 26.280, p < 0.0001). Chronic allodynia is reversed in PAE rats with hindpaw responses similar to BL levels following BIRT-377, with maximal reversal seen by day 4 post-injection (Fig. 1b). No changes in hindpaw sensitivity are observed in sham groups or Sac-minor CCI. Additionally, no significant differences are seen in contralateral hindpaw responses (Fig. 1c-d) after surgery (F1,43 = 0.953, p = 0.334) and following injection (F1,343 = 0.181, p = 0.672). These data suggest that the blockade of activated LFA-1 may alter the pronociceptive glial and immune cytokine/chemokine spinal milieu such that a full reversal from chronic allodynia in PAE rats occurs.
Spinal BIRT-377 treatment blunts spinal glial activation
To determine the mechanism by which BIRT-377 leads to allodynic reversal (Day 32 post-CCI surgery; Day 4 post-injection) in PAE rats, spinal cords are collected and processed for IHC and subsequent image analysis from all rat groups (rats from data shown in Fig. 1) for evaluation of glial activation (Fig. 2), cytokine expression (Fig. 3) and LFA-1 expression (Fig. 4) immediately following behavioral characterization of the effects of i.t. BIRT-377 (BIRT) on allodynia. Astrocyte and microglial activation demonstrate a significant interaction between surgery and injection (GFAP, F1,32 = 9.237, p = .005; Iba1, F1,27 = 6.775, p = 0.015). Analysis of GFAP immunoreactivity reveal elevated spinal astrocyte responses in Sac and PAE rats with minor CCI given a vehicle (Veh) injection compared to their corresponding sham groups (Fig. 2a), with the greatest increase of astrocyte activation observed from PAE rats with neuropathy induced by minor CCI. However, spinal cord astrocyte responses are decreased in spinal BIRT-377 treated rats (Fig. 2a), with the greatest magnitude decrease observed in PAE that revealed full reversal from allodynia. Interestingly, spinal microglial responses were increased in both PAE and Sac rats with minor CCI given vehicle compared to their corresponding sham groups (Fig. 2b) despite ongoing allodynia only in PAE rats. Spinal BIRT-377 injection results in significant decreases in microglial Iba-1 immunoreactivity (Fig. 2b). Given the lack of allodynia observed in Sac rats with minor CCI and the robust allodynia in PAE rats with minor CCI, the elevation in Iba-1 immunoreactivity may not reflect how microglia are playing a key role in the enduring allodynia observed only in PAE rats with minor CCI. Representative images (Fig. 2c-d) for GFAP and Iba1 are shown. These data suggest that PAE-induced allodynia requires mechanisms in addition to simply microglial activation. From these data, it is plausible that astrocyte actions play a greater role in PAE susceptibility to allodynia from minor injury.
PAE blunts normal IL-10 responses following minor CCI
Both IL-1β and IL-10 immunoreactivity in the dorsal horn of the spinal cord are assessed (Fig. 3a-b) for changes following i.t. BIRT-377 injection (Day 32; rats from data shown in Fig. 1). Analysis of IL-1β and IL-10 immunoreactivity reveal an interaction between surgery and injection (IL-1β, F1,27 = 9.559, p < 0.001; IL-10, F1,32 = 7.066, p = 0.012). Compared to sham groups, PAE rats with minor CCI given vehicle reveal elevated levels of IL-1β (Fig. 3a). Interestingly, this elevation is also seen in Sac rats with minor CCI given vehicle injection despite the absence of allodynia (Figs. 1a and 3a). Furthermore, BIRT-377 is sufficient to significantly decrease spinal IL-1β immunoreactivity (Fig. 3a). In healthy Sac control rats, elevated levels of IL-10 immunoreactivity are measured following minor CCI given vehicle compared to Sac-sham rats given vehicle (Fig. 3b). Interestingly, PAE CCI rats with ongoing allodynia (e.g. given vehicle) do not show a significant increase in IL-10 immunoreactivity expression, an observation that is present in Sac CCI rats without allodynia. The elevated IL-10 immunoreactivity in Sac rats with CCI suggests the increase occurs in response to minor damage, possibly to maintain homeostasis and consequent non-neuropathy. In contrast, it is possible that in PAE rats, IL-10 fails to upregulate, thereby allowing for the development of allodynia following minor CCI (Fig. 3b). Intrathecal BIRT-377 results in slight non-significant increases in IL-10 immunoreactivity. Representative IHC images of IL-1β (Fig. 3c) and IL-10 (Fig. 3d) are shown. These results demonstrate that compared to Sac control rats following minor sciatic nerve injury, PAE alters healthy IL-10 responses important for controlling allodynia long after the minor insult has occurred (32 days after minor injury). Additionally, the data suggest that enduring allodynia in PAE rats following minor CCI (Fig. 1) may be a result of chronic increases in glial activation (Fig. 2) resulting in IL-1β expression (Fig. 3a) that goes unchecked by diminished IL-10 responses (Fig. 3b). Furthermore, the data demonstrate that BIRT-377 may reverse chronic allodynia by reducing glial activation (Fig. 2a-b) and decreasing IL-1β expression (Fig. 3a).
LFA-1 expression is dysregulated in PAE rats
Immunoreactivity for Cd11a (LFA-1) is analyzed in the dorsal horn of the spinal cord in rats behaviorally verified for allodynia (Day 32; rats from data shown in Fig. 1). Data show a significant increase in LFA-1 immunoreactivity in PAE-sham rats given vehicle compared to Sac-sham rats given vehicle (Fig. 4) (p = 0.0188). Importantly, PAE-sham rats have not undergone a second insult, suggesting that the effects of PAE alone alter the LFA-1basal levels. No significant differences are observed between PAE-sham rats given vehicle and PAE-minor CCI given vehicle (Fig. 4). Additionally, a main effect of BIRT-377 (F1,42 = 5.529, p = 0.027) is revealed. It is possible that PAE the elevated basal expression of LFA-1 is a consequence of chronic aberrant ICAM-1 expression on vascular endothelial cells, which may be an underlying neuroimmune factor in the “primed” spinal cord of PAE rats leading to susceptibility upon a second challenge. In support of this possibility, a mouse model PAE lead to significantly lowered brain microvascular glucose transporter (GLUT-1) expression, a marker of functional brain microvessels [58]. BIRT-377 significantly decreases LFA-1 in PAE rats to levels similar to those measured from non-PAE Sac controls (Fig. 4), suggesting that “resetting” LFA-1 to basal levels may be sufficient to ultimately control PAE-induced sensitivity to allodynia.
Alteration of normal SGC and cytokine responses is seen in DRGs of PAE rats following chronic minor CCI
It is possible that L4-L6 DRGs of damaged sciatic nerve axons may respond to damage signals in an exaggerated manner, contributing to elevated glial and IL-1β occurring in the spinal cord. GFAP immunoreactivity for satellite glial cells (SGCs), IL-1β, and IL-10 in the L4-L6 DRGs from behaviorally verified rats (Day 32; rats from data shown in Fig. 1) is analyzed. The goal of this experiment is to identify neuroimmune factors influenced by PAE that may underlie susceptibility. Therefore, rats treated with BIRT-377 were intentionally omitted from this analysis. Statistical analysis demonstrate a main effect of PAE (SGCs, F1,20 = 17.74, p = 0.0004; IL-1β, F1,16 = 17.16, p < 0.0008; IL-10, F1,16 = 12.58, p = 0.0027), surgery (SGCs, F1,20 = 77.53, p < 0.0001; IL-1β, F1,16 = 24.87, p < 0.0001; IL-10, F1,16 = 25.7, p = 0.0001), and an interaction of PAE and surgery is seen (SGCs, F1,20 = 27.81, p < 0.0001; IL-1β, F1,16 = 30.67, p < 0.001; IL-10, F1,16 = 26.81, p < 0.001). Large and significant increases in the expression of GFAP in PAE rats with minor CCI compared to Sac controls with minor CCI are observed (Fig. 5a), suggesting that SCGs from PAE are more reactive/activated only following an injury, thereby unmasking satellite glial cell sensitization. The data show significant increases in IL-1β only in PAE rats following minor CCI (Fig. 5b). Interestingly, compared to Sac-sham rats displaying basal levels of IL-10 immunoreactivity, Sac rats with minor CCI, PAE-sham rats and PAE rats with minor CCI all display dramatically reduced basal levels of IL-10 immunoreactivity (Fig. 5c). Curiously, following minor CCI in Sac control rats, a reduction in IL-10 expression is observed despite a lack of allodynia (Fig. 5c), which is in contrast to the upregulation observed in the spinal cord (Fig. 3b). Interestingly, diminished IL-10 expression in the DRG following a 28-day CCI has previously been demonstrated in the DRG [42, 61], albeit the current CCI is a dramatically diminished injury. It is possible that the lack of elevated IL-10 in the Sac rats with minor injury may be a result of the significant reduction of damage applied to the sciatic nerve in the current report, as the diminished injury may be insufficient to elicit a modest “normal” compensatory IL-10 response at the peripheral nerve. Representative images (Fig. 5d-f) of SGCs, IL-1β, and IL-10 are shown.
Spinal IL-1β is necessary for induction of a 10-day allodynia in PAE rats
Several prior reports indicate the action of spinal IL-1β is necessary for the early and chronic phase of allodynia induced by the standard CCI model [36, 38]. The current report reveals that following a minor 32-day CCI, both Sac and PAE rats display elevated spinal IL-1β despite the absence of allodynia in Sac-minor CCI rats (Fig. 3a). leaving the role of spinal IL-1β ambiguous in PAE allodynia. The goal of the current experiment was to assess whether spinal IL-1β is necessary for a 10-day allodynia in PAE rats induced by minor peripheral nerve injury. Pharmacological intervention of spinal IL-1β with intrathecal (i.t.) IL-1 receptor antagonist (IL-1RA) was examined to determine if changes in PAE allodynia could be observed. To accomplish this, a separate group of PAE rats were assessed prior to surgery to determine BL responses, which demonstrated no significant differences between groups (Fig. 6a-b) (Ipsilateral, F1,12 = 4.523, p = 0.055; Contralateral, F1,12 = 0.014, p = 0.907). Following minor CCI, all PAE rats developed allodynia relative to their BL values (Fig. 6), replicating our prior observations (Figs. 1 and 7) (Ipsilateral, F1,12 = 0.743, p = 0.406; Contralateral, F1,12 = 2.213, p = 0.163). On Day 10 after minor CCI immediately following hindpaw threshold values, i.t. IL-1RA reversed allodynia almost to pretreated BL levels. Compared to vehicle, allodynia is reversed with hindpaw sensitivity initially changing within 1 h after the injection. This reversal is sustained for 3 h post-injection (Fig. 6) (Ipsilateral, F1,12 = 139.459, p < 0.001; Contralateral, F1,12 = 1.089, p = 0.317). Full allodynia returns by 24 h post-injection, replicating prior reports of the duration of action of IL-1RA (Ipsilateral, F1,12 = 0.887, p = 0.365; Contralateral, F1,12 = 0.743, p = 0.406) [14, 27, 52]. No significant differences are seen in sensitivity within the contralateral hindpaw throughout the behavioral time-course (Fig. 6b). This study establishes that in PAE rats with minor CCI, the susceptibility of developing allodynia is mediated, at least in part, by the actions of IL-1β in the spinal cord [45]. However, it remains unclear whether IL-1β acts in concert with other proinflammatory cytokines such as TNF-α that ultimately generates the observed allodynia. The role of spinal TNF-α has previously been shown to participate in the development of allodynia in a rat model of local spinal glial activation [37], and thus, may be an important consideration in PAE generating susceptibility to neuropathy.
I.T. BIRT-377 reverses PAE induced allodynia at day 10 post-surgery
Prior reports and the initial studies detailed in the present report demonstrate robust unilateral allodynia through Day 28 [49] or Day 32 after minor CCI in PAE rats. To further explore the spinal mechanisms underlying early-established allodynia following minor injury, adult PAE and Sac rats are subjected to either sham or minor CCI surgery, and upon established allodynia observed on Day 10, the effect of i.t. BIRT-377 on allodynia was assessed. As before (Fig. 1), BL light touch sensory thresholds are similar between all groups (Fig. 7) (ipsilateral, F1,35 = 0.687, p = 0.413; contralateral, F1,35 = 0.041, p = 0.840). Furthermore, following CCI, a main effect of alcohol exposure (ipsilateral, F1,35 = 52.497, p < 0.0001; contralateral, F1,35 = 1.929, p = 0.174), surgery (ipsilateral, F1,35 = 148.009, p < 0.0001; contralateral, F1,35 = 3.118, p = 0.086) and an interaction between alcohol exposure and surgery (ipsilateral, F1,35 = 87.448, p < 0.0001; contralateral, F1,35 = 0.387, p = 0.538) is seen only in the ipsilateral hindpaw. Additionally, while normal sensitivity throughout the timecourse is maintained near BL thresholds in Sac groups with sham or minor CCI, a pronounced unilateral allodynia is observed ipsilateral to minor sciatic CCI in PAE rats while no change in contralateral hindpaw sensitivity is observed (Fig. 7a-b) (ipsilateral, F1,35 = 89.118, p < 0.0001; contralateral, F1,35 = 0.710, p = 0.405). Following i.t. BIRT-377 or vehicle injection in Sac rats, hindpaw thresholds remain stable throughout the 4-day timecourse, hovering close to pretreatment BL values. However, i.t. BIRT-377 induces complete reversal of ipsilateral allodynia for the remainder of the timecourse while contralateral hindpaw thresholds continue to remain stable and non-allodynic (Fig. 7a-b) (ipsilateral, F1,35 = 9.808, p = 0.003; contralateral, F1,35 = 0.032, p = 0.858). These data demonstrate that LFA-1 blockade during early-established (10-day) allodynia may alter factors similar to those driving enduring (Day 28 or longer) allodynia. Following the completion of behavioral verification of i.t. BIRT-377 efficacy on Day 10, sciatic nerve, DRG and lumbar spinal cord were dissected and candidate protein and mRNA for cytokines and chemokines were examined, as detailed below.
PAE may induce allodynia by altering proinflammatory factors at the sciatic nerve
To assess whether BIRT-377 changes the spinal proinflammatory environment resulting in the reversal of allodynia in PAE rats, ipsilateral sciatic nerve (SCN) as well as ipsilateral and contralateral (as a potential within animal biochemical control) lumbar spinal cord were collected at Day 4 post-injection (Day 14; rats from data shown in Fig. 7) at maximal i.t. BIRT-377 efficacy to examine protein using the V-plex immunoassay. Expression levels of IL-10, IL-1β, and TNFα were measured in the SCN and the ipsilateral L4-L6 dorsal lumbar spinal cord as well as CXCL1 in the SCN. Results for the SCN demonstrate that following vehicle injection, minor CCI induces a modest but significant increase in IL-10 expression in PAE rats compared to Sac rats (F7,35 = 1.672, p = 0.0271). These results are in stark contrast to our prior report demonstrating that protein IL-10 levels are dramatically blunted in PAE rats with CCI [42]. In the prior report, the standard CCI model was applied and the sciatic nerves from Sac-CCI rats revealed a 5-fold increase in IL-10 protein over PAE rats with standard CCI, with one distinction being that tissues were collected at Day 28 after CCI [42]. It is also possible that in the current study, minor CCI in Sac rats is insufficient to stimulate peri-sciatic immune cells to produce a significant IL-10 protein response. As such, IL-10 responses to minor CCI in Sac animals cannot unmask the IL-10 response-deficit to injury in PAE rats.
Despite the ambiguous results of IL-10 protein from sciatic nerve tissues, significant and reliable increases in protein IL-1β, CXCL1, and TNFα were observed following minor injury, with the magnitude of protein increases observed to be far greater in PAE rats with minor CCI (Fig. 8a) (IL-1β: F7,35 = 5.224, Sac minor CCI: p = 0.0060; PAE Sham Veh: p = 0.0008; CXCL1: F7,35 = 5.224, Sac minor CCI: p = 0.0158; PAE Sham Veh: p = 0.0004; TNFα: F7,35 = 5.224, Sac minor CCI: p = 0.0239; PAE Sham Veh: p = 0.0005). No significant differences were seen between PAE minor CCI vehicle groups and PAE minor CCI BIRT groups (F7,35 = 5.224, IL-10: p = 0.4716; IL-1β: p = 0.9904, TNFα: p = 0.5436, CXCL1: p = 0.2309). Ipsilateral lumbar spinal cord IL-10 data reveal no significant differences between groups.
As observed with protein collected from sciatic nerves, IL-10 expression in the spinal cord did not show blunted IL-10 responses to injury in PAE rats compared to Sac rats. These observations were surprising, as we predicted that the lack of allodynia observed in Sac rats with minor CCI was due to elevated spinal IL-10 at this early 10-day timepoint of chronic allodynia. Interestingly, elevated spinal IL-10 is observed in Sac rats with a 32-Day allodynia (Figs. 3b and 5c), suggesting that during the early spinal response to peripheral injury, significant changes in IL-10 protein levels is not requisite for controlling the development of neuropathy from a minor injury. Analysis of IL-1β expression demonstrated significant increases in PAE minor CCI vehicle injected rats compared to PAE sham vehicle rats (F7,32 = 5.224, Sac minor CCI: p = 0.0097). Also at this early timepoint, similar IL-1β protein responses are observed in both PAE and Sac rats with minor CCI and vehicle injection despite ongoing allodynia only observed in PAE rats (F7,32 = 5.224, Sac minor CCI: p = 0.9070). Taken together, these data suggest other neuroimmune factors may play a role in generating allodynic susceptibility either by synergizing with IL-1β or by acting downstream of IL-1β. Moreover, while a modest reduction of IL-1β in allodynic PAE rats treated with BIRT-377 is measured, the reduction is not significant.
Spinal TNFα is significantly increased in PAE allodynic rats compared to Sac non-allodynic rats with minor CCI (F7,32 = 5.224, Sac minor CCI: p = 0.0432). Following BIRT-377 treatment, a strong trend towards reduced levels of TNFα is observed in pain-reversed PAE rats (Fig. 8b) (p = 0.057). Lumbar spinal cord contralateral to the sciatic CCI did not show significant differences between rat groups (data not shown), which coincides with unaltered contralateral hindpaw threshold levels. As eluded to above, these data demonstrate that pronounced peri-sciatic immune reactivity may drive other neuroimmune factors in the spinal cord that synergize with IL-1β resulting in allodynia in PAE rats following a minor CCI. Indeed, IL-1β and TNFα have been demonstrated to act synergistically [34] and speculated to underlie pathological pain from heightened spinal glial activation [37]. It is clear, however, that BIRT-377 reverses early-phase PAE allodynia by altering the action of multiple cytokines.
Minor CCI alters CCL2 and CXCL1 in the ipsilateral SCN and DRG
Previous reports demonstrate that following sciatic nerve injury, the chemokines CCL2, CXCL1, CX3CL1, and CXCL10 become upregulated [2, 21, 29, 64]. Furthermore, separate studies show that astrocytes may mediate allodynia through the release of CXCL1 [9], CX3CL1, and CXCL10 [30]. Interestingly, minor CCI results in the activation of spinal astrocytes but not microglia in aged rats [49]. In addition, PAE alters the release of CCL2 [42]. Together, these reports suggest that susceptibility to allodynia in PAE rats may result from alterations in spinal cord chemokine production and release due to primed spinal glial activation. Thus, in order to characterize the chemokine profile following minor CCI, qRT-PCR is used to assess the gene activation of CCL2, CXCL1, CX3CL1, and CXCL10 in the ipsilateral SCN (Fig. 9a), DRG (Fig. 9b), and ipsilateral (Fig. 10a) and contralateral lumbar spinal cord (Fig. 10b) (Day 14; tissue collected from behaviorally verified rats from Fig. 7). SCN data show that only following CCI are significant increases seen in expression of CCL2, CXCL1, and CXCL10 in both Sac and PAE rats (Fig. 9a) (Sac: CCL2 F7,34 = 5.32, p = 0.0242, CXCL1 F7,34 = 5.32, p = 0.0033, CXCL10 F7,34 = 5.32, p = 0.0117; PAE: CCL2 F7,34 = 5.32, p = 0.0027, CXCL1 F7,34 = 5.32, p = 0.0038, CXCL10 F7,34 = 5.32, p = 0.0133). Interestingly, significant differences in CXCL1 from SCN are seen between PAE and Sac rats with CCI that received BIRT-377 (Fig. 9a) (F7,34 = 5.32, p = 0.0007). Assessment of the DRG demonstrated that CCL2 is upregulated following CCI in both Sac and PAE rats (Fig. 9b) (Sac: F7,35 = 5.26, p = 0.0014; PAE: F7,35 = 5.26, p = 0.0168). Only PAE rats with BIRT-377 display significant increases in CXCL1 and CX3CL1 (Fig. 9b) (CXCL1: F7,34 = 2.377, p = 0.0057; CX3CL1: F7,35 = 1.593, p = 0.0079). Overall, data suggest that minor CCI upregulates pain relevant chemokines from peri-sciatic immune cells, as one would predict, but that the magnitude of the immune cell response is greater in PAE rats.. Gene activation, as measured by mRNA analysis, reveals that immune signals known to drive leukocyte trafficking at the SCN and DRG during chronic neuropathy are similar between rats exposed to Sac or PAE.
I.T. BIRT-377 alters chemokine expression only in PAE rats with minor CCI within the spinal cord
Assessment of mRNA within the ipsilateral and contralateral spinal cord reveals that minor CCI increases CCL2 gene activation in Sac rats compared to sham conditions (Fig. 10a) irrespective of BIRT-377 (Sac: F7,35 = 3.879, p = 0.0139). Interestingly, CCL2 gene activation is blunted in PAE rats with CCI in that no significant differences seen when compared to Sham rats (Fig. 10b). Significant differences in CCL2 and CXCL10 gene expression are only observed in PAE rats with minor CCI that received BIRT-377 (Fig. 10a) (CCL2: F7,35 = 3.879, p = 0.0321; CXCL10: F7,35 = 3.879, p = 0.0373). It should be noted that these increases are not significantly different when compared to Sac rats with minor CCI and BIRT-377 injection (Fig. 10a). Contralateral data demonstrated no significant differences in CCL2, CXCL1, and CXCL10 gene activation are seen between groups, which correspond with normal and stable sensitivity observed in the contralateral hindpaw (Figs. 1c-d and 7b) (F7,35 = 1.289, p = 0.0182). Interestingly, PAE alone was sufficient to induce increases in CXCL1 gene expression (Fig. 10b). The lack of changes in chemokines assessed in this report at the lumbar spinal cord may suggest that BIRT-377 may not work primarily by transcriptional regulation of these chemokines.