Targeting the β2-integrin LFA-1, reduces adverse neuroimmune actions in neuropathic susceptibility caused by prenatal alcohol exposure

Animals and study group strategy

All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of New Mexico Health Sciences Center and closely adhered to guidelines from the International Association for the Study of Pain for the use of animals in research. Long-Evans rat breeders purchased from Harlan Industries (Indianapolis, IN) were maintained in a breeding colony on a 12:12-h reverse light/dark schedule (lights on from 2100 to 0900 h), and fed Harlan Teklad rat chow and water, available ad libitum. For all experiments, 5–8-month old male prenatal alcohol or saccharin exposed (described below) rat offspring derived from 56 litters were used. Offspring were transferred to a standard light/dark cycle (lights on from 0600 h to 1800 h) and allowed to acclimate to these conditions for at least 30 days before commencing experiments. Additionally, rats were kept in these conditions for the duration of the study.

Chronic constriction injury (CCI)

In adult male Sac or PAE rats, sham or CCI aseptic surgical procedures are performed as previously described [4, 42, 49] and modified, as described here. Under isoflurane anesthesia (3% volume in oxygen), the sciatic nerve is carefully isolated with sterile glass prongs and snuggly ligated with 1 (minor CCI) segment of sterile 4–0 chromic gut suture (Ethicon, Somerville, NJ) without pinching into the nerve. Sterile isotonic saline (0.9%) is applied to the nerve during the procedure to prevent dehydration. Sham surgery involves isolation of the sciatic nerve similar to CCI but without nerve ligation. The nerve is then gently placed back into position and the overlying muscle is sutured closed with two 3–0 sterile silk sutures (Ethicon, Somerville, NJ). Rats fully recover from anesthesia within approximately 5 min and are monitored daily following surgery for any post-operative complications. Two rats (1.8%) were excluded from the study due to the presence of hindpaw autotomy.

Behavioral assessment of allodynia

Allodynia was assessed using the von Frey fiber test as previously described [10, 49]. Briefly, habituation to the testing environment required placing rats atop 2-mm thick parallel bars spaced 8-mm apart allowing full access to the plantar hindpaw. Habituation occurres for approximately 45 min/day for 4 sequential days within the first 3 h of the light cycle (inactive phase) in a sound- and temperature-controlled dimly lit section of the home colony room. Baseline (BL) responses are then assessed using the von Frey behavioral test. A total of 13 calibrated monofilaments are used in these studies. Additionally, these calibrated monofilaments (3.61–5.18 log stimulus intensity) are applied to the plantar surface of the left and right hindpaw for a maximum of 8 s per application. Random order between left and right hindpaw assessment is conducted. A metronome placed in the room provided guidance at 1 tick/sec. Lifting, licking, or shaking of the paw is considered a response. In a similar manner to BL evaluation, one group of rats is re-assessed following CCI or sham surgery through Day 10 prior to drug treatment. A second group of rats is assessed through Day 28 prior to drug treatment. The experimental tester was blind to the prenatal and surgical treatment groups. At either Day 10 or Day 28, rats are given an i.t. injection of drug or vehicle (described below) and behavioral re-assessment occurs daily for 4 sequential days post-injection to examine the effects of i.t. BIRT-377, or at 1, 2, 2.5, 3, and 24 h post-injection to examine the effects of i.t. IL-1RA.

Drug preparation

BIRT-377 is a small molecule characterized as a non-competitive inhibitor of leukocyte function-associated antigen-1 (LFA-1) that minimizes the active conformation of this transmembrane β₂-adhesion receptor expressed on leukocytes (e.g. T cell and myeloid cells). BIRT-377 (Tocris, Bristol, UK) was initially reconstituted in 100% ethanol as a stock solution at a concentration of 22.156 mg/ml, aliquoted into 0.5 ml sterile Eppendorf tubes and stored in a clean sealed container at 4 °C for later use. On the day of i.t. injection, sterile ddH₂0 is warmed in a water bath and BIRT-377 is added to reach a concentration of 500 ng/20 μl for i.t. injection and vortexed for 2 min. Vehicle consisted of sterile ddH₂O. Animals are injected within the hour following drug dilution.

IL-1 receptor antagonist (IL-1RA) (R&D systems, Minneapolis, MN) is reconstituted in sterile ddH₂0 at a stock concentration of 50 mg/ml, aliquoted into 0.5 ml sterile Eppendorf tubes and stored at − 20 °C for later use. On day of i.t. drug injection, IL-1RA is diluted in 0.1% rat serum albumin (RSA) in sterile phosphate buffered saline (PBS) to a concentration of 1 μg/μl containing 0.01% RSA. Therefore, each rat received 20 μg of IL-1RA in 20 μl i.t. Vehicle consists of 0.1% RSA in sterile PBS.

Intrathecal (i.T.) injection

BIRT-377, IL-1RA or vehicle is administered using an acute i.t. catheter on Day 28 (BIRT-377) or Day 10 (BIRT-377 or IL-1RA) post-surgery as described previously [36]. Briefly, under isoflurane anesthesia (3% volume in oxygen), the beveled needle end of an 18-gauge sterile (Bectin Dickson, Franklin Lakes, NJ) needle with the plastic hub removed is inserted between lumbar vertebrae L5-L6. Because the beveled tip of the needle is wider than L5-L6 intervertebral foramen, only ~ 1 mm of the beveled needle tip is capable of puncturing the dura mater to access the underlying arachnoid space. Consequently, spinal tissue or rootlets from the cauda equina remain uninjured and intact. PE-10 tubing (Fisher Scientific, Hampton, NH) is fitted with a 30-gauge sterile needle (Bectin Dickson, Franklin Lakes, NJ) containing a Hamilton syringe at one end and labeled with a marking at 7.7 cm at the opposite end. Drug or equivolume of vehicle (20 μl) is withdrawn from the open side of the PE-10 tubing. The open end of the tubing is then threaded into the 18-gauge needle until the 7.7 cm marking is reached. This position corresponds to the lower lumbar L4-L5 region of the spinal cord. Correct placement of the PE-10 is determined by twitching of the hindleg and/or tail during catheter advancement, as well as cerebrospinal fluid efflux from the externalized portion of the needle. Drug is administered over a 25 s interval. Immediately following drug or vehicle administration, tubing is removed followed by removal of the guide 18-g needle and rats are allowed to recover before being placed in their home cage. Rats fully recover from anesthesia within 5 min and 100% motor recovery is observed following injection.

Immunohistochemical (IHC) tissue sample preparation

All tissues from rats behaviorally verified for allodynia on Day 4 (Either Day 14 or Day 32 post-surgery) after i.t. BIRT-377 injection are collected for IHC processing as described previously [42, 49]. Briefly, rats are overdosed with sodium phenobarbital (390 mg/ml Sleepaway, Fort Dodge Animal Health, Fort Dodge, IA) and transcardially perfused with 0.1 M phosphate buffered saline (PBS; pH = 7.4), initially at a rate of 28 ml/min, then increased to 32 ml/min (~ 10 min), followed by 4% paraformaldehyde (PFA; pH = 7.4; 28–34 ml/min, 8 min). Immediately following transcardial perfusion, the spinal vertebral column from C2-L6 with the spinal cord intact within the vertebral column are collected and cut at vertebra T7 into a rostral and caudal half of the spinal vertebral column. All spinal cord sections undergo 48-h post-fixation in 4% PFA at 4 °C.

Following post-fixation, the entire spinal cord (T7- L6) is carefully isolated, and separately, the corresponding L4-L6 dorsal root ganglia (DRG), making sure to maintain tissue integrity. The isolated spinal cords are then subsequently paraffin processed. At a later time, DRGs are individually dissected and paraffin processed. Paraffin-embedded blocks are then subsequently sliced on a microtome with adjacent 7 μm tissue sections mounted onto vectabond-treated slides.

To investigate spinal augmented glial cell activation and cytokine profile, the expression of the astrocyte marker, glial fibrillary acidic protein (GFAP), the microglial activation marker ionized calcium-binding adapter molecule 1 (Iba1), the proinflammatory cytokine marker IL-1β, and the anti-inflammatory marker IL-10 in L4–6 spinal segments, as reported previously [61]. Slides containing tissue from lumbar (L4-L6) spinal cord and (L4-L6) DRG are chosen for staining to capture overall reactivity [61]. Paraffin-processed tissues undergo deparaffinization followed by rehydration and antigen retrieval procedures in a standard rice cooker at 94–96 °C allowing for gentle and equal distribution of temperature warming throughout tissue sections. For antigen retrieval, polyclonal GFAP required a Tris-based buffer at pH 9.5 (BioCare Medical, Concord, CA), Monoclonal GFAP, Iba1, and IL-1β required a Tris-based buffer at pH 9.0 (Vector, Burlingame, CA), and IL-10 required a citrate-based buffer at pH 6.0 (BioCare Medical, Concord, CA). All tissue sections are incubated with 5% normal donkey serum (NDS), PBS pH 7.4 for 2 h, followed by overnight primary antibody incubation using rabbit anti-rat polyclonal GFAP (Millipore, 1:1000), rabbit anti-rat monoclonal GFAP (Abcam. 1:400), rabbit anti-rat polyclonal Iba-1 (Wako, 1:300), or rabbit anti-rat polyclonal IL-1β (Santa Cruz, 1:100), or goat anti-rat polyclonal IL-10 (Vector Labs, 1:250) in a humidity chamber at 4 °C. Tissues are washed 3x with 0.1 M PBS pH 7.4. All tissues, except those containing IL-10 antibody, are incubated with donkey-anti rabbit FITC- or TRITC- conjugated secondary antibody incubation for 2 h in a humidity chamber at room temperature and rinsed in 0.1 M PBS. IL-10 spinal cord and DRG sections are incubated with a biotinylated donkey anti-goat secondary antibody (Vector Labs, 1:1300) for 1 h followed by a 1 h incubation with Vectastain ABC Elite kit (Vector Labs, Burlingame, CA). Sections are incubated for 10 min with TSA plus Fluorescein System (PerkinElmer Life Sciences, Waltham, MA,). All tissues are stained with the nuclear stain 4,6-diamidino-2-phenylindole (DAPI) (Vector Labs, Burlingame, CA) separately before cover slipping. These slides are left at room temperature overnight and then placed at 4 °C until proceeding with image acquisition and microscopy analysis [49, 61].

Microscope spectral imaging for immunofluorescent quantification

Image acquisition for spectral analyses is performed using the Nuance spectral imaging system (Perkin Elmer, Waltham, MA) as described previously [42, 49]. Briefly, images of dorsal horn spinal cord are obtained using either a 20X or 40X (IL-10 spinal cord sections) objective with a Zeiss Axioplan2 inverted fluorescence microscope. A flat-field correction is applied to correct for uneven illumination during image acquisition. Image cubes are obtained from multi-labeled tissue containing DAPI, conjugated secondary antibody, as well as autofluorescence. A spectral library is then created using single-labeled slides for each fluorophore (e.g. FITC or TRITC) and a label-free (autofluorescence) slide. A computed spectrum (420–720 nm) is obtained by separating the known spectrum (autofluorescence) from mixed spectrum (single labeled) to produce a pure spectrum of each fluorophore. This allowed the separation of spectrum from multi-labeled slides (e.g. DAPI + TRITC) to obtain composite images containing only labels of interest. These composite images are then used for further analysis using Slidebook 6 software (below). For dorsal horn spinal cord sections ipsilateral to sciatic manipulation, sixteen to thirty-two images per experimental group (4 sections per animal, 4–8 animals per condition) were acquired and analyzed. For L4-L6 DRG sections ipsilateral to the sciatic manipulation, thirty images per experimental group (6 sections/rat, 5 rats per condition) were acquired and analyzed. It should be noted that the 6 sections per animal were made up of the L4, L5, and L6 DRG and contained two images per DRG level. Representative acquired images from the Nuance spectral imaging system from the dorsal horn spinal cord of GFAP immunoreactive and Iba1 immunoreactive regions are shown in Fig. 2c-d, as well as IL-1β and IL-10 shown in Fig. 3c-d; and in DRG of satellite glial cells (SGCs), IL-1β, and IL-10 in Fig. 5. 20x and 40x objectives are used to produce representative images.

SlideBook6 software image analysis

Composite images are analyzed using SlideBook6 software (Intelligent Imaging Innovations, Denver, CO, USA) as described previously [49]. Briefly, to discriminate signals originating from artifacts, the experimenter determined an acceptable threshold of very low-level emission fluorescence intensity per each experimental condition (polyclonal GFAP and Iba1 analysis), or per individual animal (monoclonal GFAP, IL-1β and IL-10 analysis). This was done by closely replicating the on-screen composite computer image with that observed through the microscope eyepiece, as described previously [42, 49]. In addition, for spinal cord sections, the dorsal horn (region of interest (ROI)) is outlined for analysis eliminating the surrounding white matter and peri-spinal blank space (intrathecal space). For DRG sections, ROI included all cell bodies found within the composite image. The image is refined to include the predetermined threshold within the outlined area. The ‘Sum Intensity’ (the total signal within the outlined area in the dorsal horn) is determined and divided by the ‘Area’ (total area in micrometers squared (μm²)) to obtain the ‘Fluorescence Intensity’. The average of the adjacent sections from a single slide (representing a single animal) is calculated to determine the value for each animal. Finally, averages for each slide within the same condition are calculated along with the standard deviation and standard error of the mean. Data are analyzed as “Sum Intensity of ROI/μm²”.

Tissue collection and total RNA isolation

Following behavioral assessment on Day 4 post-injury (Day 14 post-surgery), rats are deeply anesthetized with isoflurane (induction 3% vol. followed by 5% in oxygen) for 10–12 min. Rats are then transcardially perfused with ice cold 0.1 M PBS pH 7.4 for 3 min at a rate of 24 ml per minute. The perfused body is placed on a frozen gel refrigerant pack (Glacier Ice, Pelton Shepherd Industries, Columbus, OH). Laminectomy and tissue dissection is performed to collect both ipsilateral and contralateral lumber L4-L6 spinal cord, followed by L4-L6 dorsal root ganglia, and finally ipsilateral sciatic nerve. All samples are placed in DNase/RNAse-free 1.5 ml centrifuge tubes (VWR International, Radnor, PA), flash frozen on dry ice, and stored at − 80 °C for future analysis.

Flash frozen tissues are transferred to ice and 100 μl sterile 1 x PBS was added. It should be noted that, in contrast to spinal cord and DRG tissue, sciatic nerve is quickly chopped with scissors for ~ 2 min on ice. All tissues are then homogenized using a motorized VWR disposable Pellet Mixer and motor pestle system (VWR International, Radnor, PA) for about 30 s. 60% of the tissue homogenate is then processed for protein analysis (described below). The remaining 40% of the homogenate is added to chilled Qiazol Lysis Reagent (Qiagen, Hilden, Germany) for total RNA isolation (described below). It should be noted that, due to the small quantity of DRG tissue collected, 100% of tissue homogenate is used for RNA isolation and not protein analysis.

Tissue lysate preparation and Meso scale discovery (MSD) multi-spot assay system

Flash frozen ipsilateral sciatic nerve and ipsilateral and contralateral lower lumbar L4-L6 spinal cord sections collected from animals are prepared for MSD Multi-Spot Assay system as previously described [42]. Briefly, tissues are allowed to thaw on ice and homogenized using a Fisherbrand Disposable Pestle system (Fisher Scientific, Hampton, NH). Samples are then sonicated in buffer containing protease inhibitors per instructions of manufacturer. All samples are then centrifuged at 4200 g, at 4 °C, for 10 min. Lysate is collected from samples and Bradford protein assay (Biorad, Hercules, Ca) is used to determine protein concentrations. Using a V-PLEX immunoassay (MesoScale Discovery, Gauthersburg, MD), 100 μg protein of all samples is used to determine cytokine expression levels for TNFα, IL-1β, IL-10, and CXCL1 as previously validated [33, 42, 47].

Total RNA isolation and mRNA analysis by quantitative real-time PCR

Total RNA is extracted from ipsilateral sciatic nerve, ipsilateral and contralateral spinal cord, and ipsilateral DRG. Tissue is placed in chilled Qiazol Lysis Reagent (Qiagen, Hilden, Germany) and homogenized for 15 s. Total RNA is then extracted using a miRNeasy Micro Kit (Qiagen, Hilden, Germany) per manufacturer’s instructions. It should be noted that an additional buffer RPE and 80% EtOH wash steps is added to remove any additional salt contaminants added from the PBS step. Tissue is centrifuged at 15,000 rpm for 1.5 min at 4 °C. PBS is aspirated and 35 μl of protease inhibitor solution (MesoScale Discovery System) is added and samples were stored on dry ice and later stored at − 80 °C for future analysis (described below).

RNA concentration and quality is assessed using Nanodrop (ThermoFisher Scientific, Walham, MA). RNA samples are then diluted to standardized RNA concentrations of 195 ng/μl for ipsilateral DRG, 137 ng/μl for ipsilateral and contralateral lumbar spinal cord, and 100 ng/μl for ipsilateral sciatic nerve. Standardized samples are then transcribed to cDNA at 2145 ng for ipsilateral DRG, 950 ng for ipsilateral SCN, and 1438.5 ng for ipsilateral and contralateral lumbar spinal cord. Reverse transcription is performed for all tissue types using a Superscript IV VILO cDNA Synthesis Kit (Invitrogen, Carlsbad, CA).

Levels of mRNA expression is measured and analyzed as described previously [35]. For assessment of transcripts, a dilution factor of 1:2.5 is applied to cDNA samples for ipsilateral DRG, 1:2 for ipsilateral SCN, and 1:2.2 for ipsilateral and contralateral lumbar spinal cord. cDNA dilution for assessment of rat 18S rRNA normalizer is 1:200 for all tissue types. Levels of mRNA and “Normalizer” rat 18 s rRNA (Rn18s, Taqman Assay ID#: Mm03928990_g1), is assayed in triplicate using quantitative real-time PCR (qRT-PCR) with Taqman Gene Expression Assays (ThermoFisher Scientific, Waltham, MA). Gene expression assays are deemed to be “best coverage” assay by the manufacturer and is analyzed with the formula C = 2^CT^Normalizer/2^CT^Target in order to exclude detection of genomic DNA [31, 51]. Chemokines chosen for qRT-PCR include CCL2 (Taqman Assay ID#: Rn00580555_m1), CXCL10 (Taqman Assay ID#: Rn01413889_g1), CXCL1 (Taqman Assay ID#: Rn00578225_m1), and CX3CL1 (Taqman Assay ID#: Rn00593186_m1). These chemokines have been previously shown to be upregulated during neuropathic pain conditions [2, 21, 29, 64], and additionally, CCL2 and CX3CL1 have been shown to be altered following PAE [42, 46].

Statistical analysis

SPSS (IBM, Chicago, IL, USA) is used for all behavioral analysis. At baseline (BL), a 3-way (2 × 2 × 2) analysis of variance (ANOVA) is applied to assess differences between prenatal exposure (Sac versus PAE), surgical treatment (Sham versus minor CCI) and drug injection (vehicle versus BIRT-377). Additionally, a 3-way (2 × 3) repeated measures ANOVA is used for analysis of the between-subject factors of prenatal exposure, surgery, and injection for days post-surgery as well as days post-injection. Furthermore, 3-way (2 × 2 × 2) ANOVA is used for analysis of all IHC immunoreactivity data. Data acquired from V-Plex immunoassay and qRT-PCR are analyzed using GraphPad Prism version 7 software (GraphPad Software Inc., San Diego, CA, USA). Post-hoc examination of IHC immunoreactivity is analyzed using Fisher’s LSD test. The threshold for statistical significance was set a priori at α = 0.05 for all sets of multiple comparisons. In order to minimize unnecessary duplication, we use the minimum number of animals possible to make statistically significant conclusions, which is based on our previous publications [10, 42, 49, 60, 61]. Outliers were removed following Grubbs’ Z-test [16]. In all cases, the data are presented as the mean ± SEM. All figures are created in GraphPad Prism version 7 software (GraphPad Software Inc., San Diego, CA, USA).

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, F_1,43 = 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, F_1,43 = 152.120, p < 0.0001), surgery (ipsilateral, F_1,43 = 404.296, p < 0.0001) and an interaction between alcohol exposure and surgery (ipsilateral, F_1,43 = 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, F_1,43 = 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, F_1,43 = 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 (F_1,43 = 0.953, p = 0.334) and following injection (F_1,343 = 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, F_1,32 = 9.237, p = .005; Iba1, F_1,27 = 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, F_1,27 = 9.559, p < 0.001; IL-10, F_1,32 = 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 (F_1,42 = 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, F_1,20 = 17.74, p = 0.0004; IL-1β, F_1,16 = 17.16, p < 0.0008; IL-10, F_1,16 = 12.58, p = 0.0027), surgery (SGCs, F_1,20 = 77.53, p < 0.0001; IL-1β, F_1,16 = 24.87, p < 0.0001; IL-10, F_1,16 = 25.7, p = 0.0001), and an interaction of PAE and surgery is seen (SGCs, F_1,20 = 27.81, p < 0.0001; IL-1β, F_1,16 = 30.67, p < 0.001; IL-10, F_1,16 = 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, F_1,12 = 4.523, p = 0.055; Contralateral, F_1,12 = 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, F_1,12 = 0.743, p = 0.406; Contralateral, F_1,12 = 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, F_1,12 = 139.459, p < 0.001; Contralateral, F_1,12 = 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, F_1,12 = 0.887, p = 0.365; Contralateral, F_1,12 = 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, F_1,35 = 0.687, p = 0.413; contralateral, F_1,35 = 0.041, p = 0.840). Furthermore, following CCI, a main effect of alcohol exposure (ipsilateral, F_1,35 = 52.497, p < 0.0001; contralateral, F_1,35 = 1.929, p = 0.174), surgery (ipsilateral, F_1,35 = 148.009, p < 0.0001; contralateral, F_1,35 = 3.118, p = 0.086) and an interaction between alcohol exposure and surgery (ipsilateral, F_1,35 = 87.448, p < 0.0001; contralateral, F_1,35 = 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, F_1,35 = 89.118, p < 0.0001; contralateral, F_1,35 = 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, F_1,35 = 9.808, p = 0.003; contralateral, F_1,35 = 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 (F_7,35 = 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: F_7,35 = 5.224, Sac minor CCI: p = 0.0060; PAE Sham Veh: p = 0.0008; CXCL1: F_7,35 = 5.224, Sac minor CCI: p = 0.0158; PAE Sham Veh: p = 0.0004; TNFα: F_7,35 = 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 (F_7,35 = 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 (F_7,32 = 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 (F_7,32 = 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 (F_7,32 = 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 F_7,34 = 5.32, p = 0.0242, CXCL1 F_7,34 = 5.32, p = 0.0033, CXCL10 F_7,34 = 5.32, p = 0.0117; PAE: CCL2 F_7,34 = 5.32, p = 0.0027, CXCL1 F_7,34 = 5.32, p = 0.0038, CXCL10 F_7,34 = 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) (F_7,34 = 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: F_7,35 = 5.26, p = 0.0014; PAE: F_7,35 = 5.26, p = 0.0168). Only PAE rats with BIRT-377 display significant increases in CXCL1 and CX3CL1 (Fig. 9b) (CXCL1: F_7,34 = 2.377, p = 0.0057; CX3CL1: F_7,35 = 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: F_7,35 = 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: F_7,35 = 3.879, p = 0.0321; CXCL10: F_7,35 = 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) (F_7,35 = 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.