Background
Spinal cord injury (SCI) is followed by delayed secondary damage that occurs for days, weeks and even months following the initial insult [
1,
2]. Inflammation, including the activation and migration of microglia and macrophages, plays a significant role in this secondary injury [
3‐
9]. Microglia are the primary immune response cells in the CNS [
10] and can be activated by a number of pro-inflammatory cytokines and chemokines or other alterations in the CNS environment [
11,
12]. Microglia respond quickly, within minutes, to environmental changes such as increases in ATP concentration or injury [
13]. After SCI, microglia are the dominant monocyte occupying the injury site through 3 days post-injury, after which macrophages begin to invade the lesion site [
14]; immunocytochemically, the two cell types are indistinguishable.
We have shown that genes associated with inflammation, including those expressed primarily by microglia/macrophages, are strongly up-regulated immediately after injury and remain up-regulated for at least 7 days [
15]. Further, Popovich et al. [
16] has demonstrated that areas of blood-spinal cord barrier permeability 14 to 28 days post-injury are associated with OX42 (microglia/macrophage) labeling, suggesting extensive monocytic activity at delayed time points post-injury.
Our earlier work investigated the delayed up-regulation of expression of selected inflammation-related genes up to 7 days after SCI [
15]; these genes included C1qB, CD53, galectin-3 and p22
PHOX, among others. While these genes have not been studied extensively in SCI, they have all been shown to play important roles in post-injury inflammation. For example, p22
PHOX is a core component of the NADPH oxidase enzyme, which plays a key role in the production of reactive oxygen species (ROS). This enzyme is composed of 4 cytosolic subunits (p40
PHOX, p47
PHOX, p67
PHOX and GTP-binding protein p21-Rac1) and 2 membrane subunits (gp91
PHOX and p22
PHOX) [
17]. ROS and their derivatives can have severe cytotoxic effects [
18,
19], including induction of pro-inflammatory cytokine expression via MAPK and NFκB signaling [
20]. Reduction of NADPH oxidase activity can mitigate the microglial response and reduce neuronal cell death [
15,
21‐
25]. Diphenylene iodonium (DPI), a nonspecific, irreversible inhibitor of NADPH oxidase, operates by modifying the heme component of NADPH oxidase, disrupting the ability of the enzyme to generate ROS [
26,
27]. DPI blocks NFκB activation in microglia, which reduces iNOS and cytokine production [
24]. Inhibition of NADPH oxidase with DPI also impairs peroxynitrite production and suppresses microglial-induced oligodendrocyte precursor cell death [
28].
The goal of this work was to examine the chronic expression of microglial-related genes, examining up to 6 months after SCI, and to begin to assess the relationship and function of these proteins, particularly of NADPH oxidase. The characterization of inflammatory gene expression is important for understanding the role of inflammation, including microglial and macrophage activation, in secondary injury for the development of SCI therapeutics.
Methods
Spinal Cord Injury
Contusion SCI was performed in adult male Sprague Dawley rats as previously described [
29]. Briefly, rats (275 - 325 g) were anesthetized with sodium pentobarbital (67 mg/kg, I.P.) and mild, moderate or severe injury was induced using a weight drop method, in which a 10 g weight was dropped from 17.5, 30, or 50 mm, respectively, onto an impounder positioned on the exposed spinal cord at vertebral level T-9. Sham injured animals underwent the same experimental procedures, but received a laminectomy only, without weight drop. All experiments complied fully with the principles set forth in the "Guide for the Care and Use of Laboratory Animals" prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Resources, National Research Council (DHEW pub. No. (NIH) 85-23, 2985) and were approved by the Georgetown University IACUC.
Expression Profiling
Animals were deeply anesthetized with sodium pentobarbital (100 mg/kg, I.P.) and decapitated 4 hours, 24 hours, 7, 14 and 28 days and 3 or 6 months after injury. A 1 cm section of the spinal cord centered at the lesion epicenter, T-9, was dissected, and immediately frozen on dry ice. Two naïve controls (rats that did not undergo any surgical procedure) were also included in the analysis.
Expression profiling was performed as described previously [
15,
30]. Briefly, 7 μg of total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) and used for complementary DNA (cDNA) and biotinylated complementary RNA (cRNA) synthesis. RNA was then hybridized to the Affymetrix rat U34A, B, and C arrays according to the manufacturer's protocol (Affymetrix, Santa Clara, CA). Collectively these chips include approximately 28,000 genes and ESTs. Samples were not combined; each gene chip was dedicated to a single spinal cord sample.
Microarray Quality control
We employed stringent quality control methods as previously published [
31]. Each array fulfilled the following quality control measures: cRNA fold changes between 5 to 10, scaling factor from 0.3-1.5, percentage of "present" (P) calls from 40-55%, average signal intensity levels between 900-1100, housekeeping genes and internal probe set controls showed > 80% present calls, consistent values and 5'/3' ratios were < 3.
Experimental normalization, data filtering and statistical analysis on gene expression profiles were generated with the dChip probe-set algorithm and GeneSpring software using a Welch ANOVA t-test p value <0.05 between sham and injured groups.
Pathway Analysis
The cluster of temporally correlated genes obtained from the microarray was inputted into the GeneGo MetaCore™ pathway analysis software (St. Joseph, MI). Using the Direct Interactions, Shortest Path, and Transcription Regulation algorithms, connectivity of the gene list was obtained.
Western Blot
At 28 days and 6 months post-injury, 4 moderate-contusion injured and 2 sham injured rats per time point were anesthetized (100 mg/kg sodium pentobarbital, I.P.) and decapitated. A 1 cm section of the spinal cord (approximately 50 mg of tissue weight) centered at the lesion epicenter, T-9, was dissected, and immediately frozen on dry ice and western blot was performed as described previously [
15]. Briefly, tissue was homogenized in RIPA Buffer and centrifuged to isolate protein. Twenty-five μg of protein were run in SDS polyacrylamide gel electrophoresis and blotted onto a nitrocellulose filter. The blot was then probed with antibodies against galectin-3 (1:1000; Abcam, Cambridge, MA), progranulin (1:1000; R&D Systems, Minneapolis, MN), gp91
PHOX (1:1000; BD Transduction Laboratories, San Jose, CA) and p22
PHOX (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA). Immune complexes were detected with appropriate secondary antibodies and chemiluminescence reagents (Pierce, Rockford, IL). β-actin or GAPDH were used as controls for gel loading and protein transfer. Scion Image Analysis (
http://www.scioncorp.com/) was used to assess pixel density of resultant blots to compare between sham-injured and injured spinal cord tissue.
Immunohistochemistry
At 28 days post-injury, 4 moderate-contusion injured and 2 sham injured rats per time point were anesthetized (100 mg/kg sodium pentobarbital, I.P.) and intracardially perfused with 100 ml of 0.9% saline followed by 300 ml of 10% buffered formalin. A 1 cm section of spinal cord centered at the lesion epicenter, T-9, was dissected, post-fixed in 10% buffered formalin overnight and cryoprotected in 30% sucrose for 48 hours. Standard fluorescent immunocytochemistry on serial, 20 μm thick coronal sections was performed as described previously [
15]. Antibodies included NeuN (1:200; Millipore, Billerica, MA), GFAP (1:100; Promega, Madison, WI), Iba-1 (1 μg/ml; Wako, Richmond, VA), C1q (1:100; US Biologicals, Swampscott, MA), galectin-3 (6 μg/ml), p22
PHOX (1:200), progranulin (1:200), and gp91
PHOX (1:100). Appropriate secondary antibodies linked to FITC or Cy3 fluorophores (Jackson Immunoresearch, West Grove, PA) were incubated with tissue sections for 1 hour at room temperature. Slides were coverslipped using mounting media containing DAPI to counterstain for nuclei (Vector Labs, Burlingame, CA). To ensure accurate and specific staining, negative controls were used in which the primary antibody was not applied to sections from injured tissue, and only staining that labeled cells that were double-labeled with expected cell markers or had expected labeling patterns (i.e., classic microglia morphology) was confirmed as positive labeling. Immunofluorescence was detected using confocal microscopy or an AxioPlan Zeiss Microscopy system (Carl Zeiss, Inc., Thornwood, NY).
Immunofluorescence was detected and quantified in twelve 20 μm sections, selected with a random start and consistent interslice distance, using confocal microscopy as described previously [
32]. In brief, the proportional area of tissue occupied by immunohistochemically stained cellular profiles within a defined target area (the lesion site and surrounding tissue) was measured using the Scion Image Analysis system using a method modified from Popovich and colleagues [
33].
NADPH Oxidase Activity Assay
At 3 and 6 months post-injury, 4 moderate-contusion injured and 2 sham injured rats per time point were anesthetized (100 mg/kg sodium pentobarbital, I.P.) and decapitated. A 1 cm section of the spinal cord (approximately 50 mg of tissue weight) centered at the lesion epicenter, T-9, was dissected, and immediately frozen on dry ice for NADPH oxidase activity assessment as previously described [
34]. Briefly, tissue was homogenized in lysis buffer (50 mM Tris, 0.1 mM EDTA, 0.1 mM EGTA, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride) and centrifuged at 16,000G for 15 minutes. The supernatant was then centrifuged at 100,000G for 1 hr for cell fractionation. Forty μg of protein from the membrane fraction was added to lysis buffer and NADPH oxidase buffer, along with DHE and NADPH and the plate was assessed at an emission of 485 nm and absorption of 590 nm.
NADPH Oxidase Inhibition
For NADPH oxidase inhibition experiments, a polyethylene catheter (P-100, 1.52 outside diameter) attached to an Alzet mini-osmotic pump (Alzet, Cupertino, CA; Model 2001) was inserted into the intrathecal space at 30 minutes post-injury and advanced toward the lesion site, resting 1 - 2 mm below the lesion site, as described previously [
35]. The mini-osmotic pump was loaded with either DPI (Sigma, St. Louis, MO; 100 μM in 1% DMSO in saline; n = 3) or vehicle (1% DMSO in saline; n = 3) and administered 1 μl of drug (representing 0.1 pmoles/day) or vehicle per hour for 7 days (7 day infusing pump).
MRI Analysis
At 28 days post-injury, rats underwent magnetic resonance imaging (MRI) using a 7 Tesla 20 cm bore MRI (Bruker Biospin Billerica, MA). Rats underwent a 2D T2 weighted imaging protocol, in which the field of view was 90 × 90 mm. The TR = 3640 msec, TE = 121 msec, MTX = 256 × 256. Hyperintense areas on the 9 slices of each 90 × 90 mm MRI image was assessed using Image J analysis software, as previously described [
36].
Histological Analysis
The day after MRI imaging, spinal cord tissue was excised and processed for cresyl violet staining and lesion volume analysis as previously described {Byrnes, 2010 #3024}. Briefly, a 1 cm section of the spinal cord centered at the lesion epicenter, T9, was dissected and 20 μm thick coronal sections were collected and stained with cresyl violet. The Cavalieri method of stereology was used to estimate lesion volume, using a random start section followed by lesion volume measurement, including the cavity and surrounding damaged tissue, every 400 μm.
Statistical Analysis
Quantitative data are presented as mean +/- standard error of the mean. Lesion volume, western blot, and immunohistochemical data were obtained by an investigator blinded to treatment group. All data were analyzed using Student's t test or one-way ANOVA, where appropriate. All statistical tests were performed using the GraphPad Prism Program, Version 3.02 for Windows (GraphPad Software, San Diego, CA). A p value < 0.05 was considered statistically significant.
Discussion
This work is a continuation and expansion of our previous findings demonstrating the existence of a delayed expression cluster of inflammation-related genes [
15]. Here we demonstrate that SCI results in a marked chronic up-regulation of the expression of a cluster of inflammation-related genes. Secondary injury, including chronic demyelination, also lasts for weeks to months after SCI [
39]. A recent study by Naphade et al. [
40] demonstrated a secondary peak of inflammation as late as 2 months post-injury. It is possible that this chronic inflammatory response may contribute to the continuation of damage in the injured cord.
Further, certain of these genes have been found to be up-regulated in other CNS injury and neurodegenerative models. For example, C1qB expression is increased in areas of demyelination in amyotrophic lateral scleroris (ALS) patients [
41]. Both C1qB and galectin-3 are up-regulated after traumatic brain injury [
42]. Galectin-3 has also been shown to be increased after hypoxia/ischemia from 72 hours [
43] to at least 2 months post-injury [
44]. Progranulin, which has recently been shown to have delayed expression after SCI [
40], has also been shown to be increased in microglia in Alzheimer's [
45] and ALS disease cases [
46].
It is currently unclear if the protein products of the delayed expression cluster play beneficial or detrimental roles after SCI, as recent studies have sparked interest in the pro-inflammatory M1 and anti-inflammatory M2 phenotypes of microglia and macrophages [
47]. While the M1/M2 status of cells expressing genes of interest was not explored in this study, it is important to note that many of the genes in the delayed expression cluster can have both advantageous and deleterious effects after injury. For example, progranulin is reportedly associated with both pro- and anti-inflammatory responses, depending upon the availability of the serine protease elastase [
45,
48]. Elastase is produced by cells of myeloid lineage, such as microglia; in areas with large amounts of elastase, progranulin is cleaved into granulins. Granulins, in turn, can be chemoattractants for macrophages and other inflammatory cells and can induce cytokine production [
49,
50]. Galectin-3 is also related to both deleterious and regenerative responses after injury. For example, it can directly induce the expression of cytokines, such as IL6 and TNFα [
51]. After nerve transection, galectin-3 knockout resulted in significant increases in both the number of regrowing axons and the rate at which function was recovered [
52]. Further, knockout of the gene in mice exposed to hypoxia-ischemia reduced white matter loss and markers of apoptosis [
43]. However, galectin-3 knockout can reduce phagocytosis, which may impede debris clearance and regeneration [
53].
The amount of interaction amongst the genes of the delayed expression cluster is also currently unknown. Interactions have been noted in the literature, including evidence of increased ROS production following C1q or galectin-3 administration to cells [
54‐
56]. Computational pathways analysis revealed that, while there are few direct interactions between genes of the delayed-expression group, these genes may be intimately connected with similar up-stream and down-stream signaling pathways. Common transcription factors may partially explain the similar patterns of up- and down-regulation over time seen within a cluster. In this regard, a small group of transcription factors, notably ETS1 and SP1, were associated with many of the genes within the delayed-expression cluster. It is interesting to note that ETS1 and SP1 activity have not been identified previously in SCI models, despite their integral roles in the expression of several components of the inflammatory pathway [
57,
58], such as NAPDH oxidase components [
59,
60]. It is also possible that this chronic up-regulation of gene expression is a result of stimulation due to a positive feedback loop. For example, it has been shown that transcription factors, such as AP1 and SP1, are sensitive to ROS activation [
61]. In fact, knockout of components of the NAPDH oxidase enzyme inhibit AP1 transcriptional activity [
62].
Supporting this theory was the finding that DPI administration reduced the expression of progranulin, galectin-3 and p22
PHOX. Moreover, T2-weighted MRI and histology revealed significantly reduced lesion volume in DPI-treated rats, suggesting that these inflammatory responses may be related to secondary injury and expansion of the lesion site after SCI. Our previous work has demonstrated that there is a significant correlation between MRI-based lesion volume and histological findings [
36,
38]. These data are in line with previous findings, where administration of apocynin, an alternative NADPH oxidase inhibitor, after transient middle cerebral artery occlusion reduced infarct volume [
63]. Apocynin was also found to limit microglial and astrocytic activity in the hippocampus after ischemia, suggesting reductions in overall inflammatory responses [
64]. It is important to note that DPI is a nonspecific inhibitor of NADPH oxidase, and has been shown to have actions on other flavin-containing enzymes. Therefore, future work will explore the effects of more specific NADPH oxidase inhibitors on SCI recovery, including motor functional recovery and axonal preservation.
In summary, these findings show that SCI in the rodent is followed by a delayed up-regulation of pro-inflammatory genes that may play a role in secondary injury. These genes are related by similar up-stream regulators, including both transcription factors and inducers. NADPH oxidase, in particular, may play a significant role in propagating chronic expression of these genes, and may serve as a target for therapeutic intervention after SCI.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
KB carried out or directed the confirmation studies and the DPI study and drafted the manuscript. PW carried out the immunohistochemistry for both the confirmation study and the DPI study. SK carried out the microarray analysis and provided all of the microarray data. EH participated in the design of the study and completion of the microarray analysis. AIF conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.