Inflammatory Pathways in Spinal Cord Injury

CHAPTER FIVE

Inflammatory Pathways in Spinal Cord Injury Samuel David1, Juan Guillermo Zarruk, Nader Ghasemlou2 Centre for Research in Neuroscience, The Research Institute of the McGill University Health Centre, Montreal, Quebec, Canada 1 Corresponding author: e-mail address: [email protected] 2 Current address: F.M. Kirby Neurobiology Center, Children’s Hospital Boston & Department of Neurobiology, Harvard Medical School, Boston, Massachusetts, USA

Contents 1. Introduction 2. The Inflammatory Response After SCI 2.1 Changes at the site of injury 2.2 Changes in areas undergoing Wallerian degeneration 3. Effects of Immune Cells on Secondary Damage 4. Identification of Novel Targets that Mediate Inflammatory Responses After SCI 4.1 MAPK-activated protein kinase 2 4.2 KCNN4/KCa3.1 4.3 Secretory leukocyte protease inhibitor 4.4 Phospholipase A2 5. Conclusions Acknowledgments References

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Abstract Injury to the spinal cord results in direct damage to axons, neuronal cell bodies, and glia that cause functional loss below the site of injury. In addition, the injury also triggers an inflammatory response that contributes to secondary tissue damage that leads to further functional loss. Reducing inflammation after spinal cord injury (SCI) is therefore a worthy therapeutic goal. Inflammation in the injured spinal cord is a complex response that involves resident cells of the central nervous system as well as infiltrating immune cells, and is mediated by a variety of molecular pathways and signaling molecules. Here, we discuss approaches we have used to identify novel therapeutic targets to modulate the inflammatory response after SCI to reduce tissue damage and promote recovery. Effective treatments for SCI will likely require a combination of approaches to reduce inflammation and secondary damage with those that promote axon regeneration.

International Review of Neurobiology, Volume 106 ISSN 0074-7742 http://dx.doi.org/10.1016/B978-0-12-407178-0.00006-5

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1. INTRODUCTION The adult mammalian central nervous system (CNS) has a very limited capacity to regenerate damaged axons through the CNS environment, and to replace damaged neurons. As a result, injuries to the spinal cord result in permanent loss of motor, sensory, and autonomic function. Damage to CNS tissue after spinal cord injury (SCI) occurs in two phases—the acute phase in which damage is caused as a direct result of the trauma itself, and a subacute phase in which secondary damage to CNS tissue occurs due to a variety of factors that include ischemia, hemorrhage, excitotoxic damage, and inflammation. The inflammatory response is thought to contribute importantly to secondary damage after SCI, and is mainly an innate immune response, involving infiltrating macrophages and neutrophils, and CNS-resident microglia. In experimental models of SCI, the size of the lesion increases over time, particularly over the first 2 weeks. Degenerative changes involving atrophy and loss of neurons and myelin also occur over a more prolonged course of several months that have been shown to be mediated in part by the adaptive immune response involving lymphocytes and antibody production (Ankeny & Popovich, 2009). Therapeutic strategies to promote axon regeneration (discussed in other chapters) to reconnect severed fiber pathways either by long distance axon growth (David & Lacroix, 2003) or short distance relays (Courtine et al., 2008; van den Brand et al., 2012) are required to restore motor and sensory function after SCI. However, the degree to which axon regeneration is required may be substantially reduced by minimizing the secondary damage to fiber tracts. Much attention has therefore been focused over the past decade in understanding the mechanisms underlying secondary damage. Here, we will focus our attention on one aspect that mediates secondary damage, namely, the role of inflammation. Cell and tissue damages that are caused by inflammation is generally a bystander effect of inflammation, the primary role of which is to control infections, clear cellular debris, and promote wound healing. Inflammation therefore plays an important and useful role. The aim in SCI would therefore be to curb certain harmful aspects of the inflammatory response while still allowing its useful beneficial effects in wound healing and tissue repair. In addition, inflammation in its natural course gets resolved and switched off. The resolution of inflammation is a combination of downregulation of proinflammatory molecules and the expression of proresolution factors that actively suppress inflammation (Serhan, Chiang, & Van Dyke, 2008).

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Absence or delay of this resolution phase results in chronic inflammation. We will first present a brief overview of the cellular changes in the immune response after SCI. This will be followed by a discussion of our recent studies on the identification and characterization of several molecules that regulate inflammation and mediate secondary damage after SCI.

2. THE INFLAMMATORY RESPONSE AFTER SCI 2.1. Changes at the site of injury The cells that respond most rapidly to SCI are those within the CNS itself. In situ hybridization and immunohistochemical analysis of cytokine expression in tissue sections indicate that in addition to microglia (the resident tissue macrophage in the CNS), other cells including astrocytes, neurons, and oligodendrocytes rapidly express proinflammatory cytokines that can influence the recruitment and activation of peripheral immune cells into the injured spinal cord. For example, IL-1b mRNA expression peaks at 12 h after SCI in mice and is expressed mainly by microglia and astrocytes (Pineau & Lacroix, 2007). A similar rapid expression of IL-1b is also detected in injured rat and human spinal cord at the protein level using immunostaining of tissue sections (Yang et al., 2004, 2005). Interestingly, in humans, IL-1b is detected in neurons at 30 min and in neurons and microglia at 5 h after injury (Yang et al., 2004). TNF-a mRNA expression after SCI in mice peaks at 1 h and decreases by 24 h, followed by a second prominent peak between 14 and 28 days (Pineau & Lacroix, 2007). At the early time-points (1–3 h), TNF-a mRNA is expressed by all CNS cell types—microglia, astrocytes, neurons, and oligodendrocytes (Pineau & Lacroix, 2007), and at later times (2 weeks), it is expressed mainly by activated microglia/macrophages (Pineau & Lacroix, 2007). In the injured human spinal cord, TNF-a is detected at 1–3 h at the protein level in neurons and in cells with a microglial morphology (Yang et al., 2005). A number of chemokines (macrophage inflammatory protein-1a and b [MIP-1a and MIP-1b], MIP-2, monocyte chemotactic protein-1 [MCP-1] and interferon-inducible protein 10 [IP10/CXCL10]) are also upregulated within 30 min after SCI and peak at 6 h (Rice, Larsen, Rivest, & Yong, 2007). Similar changes are also seen in other CNS conditions such as brain ischemia with some differences. For example, IL-1b mRNA and protein are upregulated between 15 and 30 min postischemia and have a biphasic behavior with a second peak between 6 and 24 h after ischemia/reperfusion. TNF-a mRNA expression

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also increases between 1 and 3 h after ischemia but its biphasic behavior is faster (24–36 h) than that seen after SCI (Denes, Thornton, Rothwell, & Allan, 2010; Wang, Tang, & Yenari, 2007). The first responders after SCI to express cytokines that initiate the immune response to injury are therefore the resident CNS cells including microglia, astrocytes, neurons, oligodendrocytes, and endothelial cells. It is also instructive to know the time course of the entry of different peripheral immune cell populations, and the activation of endogenous microglia to understand when and how to modulate these responses to reduce secondary damage and promote recovery. As mentioned above, resident microglia are the first immune cell population to respond to injury within minutes. Neutrophils are the first peripheral immune cell type to enter the injured spinal cord. They enter the cord within hours, reach a peak at 24 h and are markedly reduced by 3 days in rodents (Donnelly & Popovich, 2008; Yang et al., 2005) and humans (Fleming et al., 2006; Norenberg, Smith, & Marcillo, 2004). This differs from brain ischemia/reperfusion in which the first cells to enter the injured brain are macrophages starting at 12 h and peaking at 72 h, while neutrophils enter the brain only 72 h after the reperfusion and stay longer for 7 days (Gelderblom et al., 2009). By fluorescence-activated cell sorting (FACS) analysis, neutrophils comprise over 90% of the cellular infiltrates at 12 h after SCI (Stirling & Yong, 2008). Monocytes and macrophages from the peripheral circulation enter the spinal cord at around 3–4 days (Pineau, Sun, Bastien, & Lacroix, 2010). Peripheral macrophages become rapidly activated and along with activated microglia begin to phagocytose tissue debris. When they become activated and have phagocytosed cellular material, macrophages derived from the periphery cannot be distinguished from microglia based on their morphology or antigenic profile. They are therefore referred to as microglia/macrophages. Quantification by FACS analysis shows that microglia/macrophages constitute over 70% of the CD45þ population at 4 days after SCI in mice (Stirling & Yong, 2008). These cells reach peak numbers by 7–10 days after contusion injury in rodents and remain in the tissue for several weeks to months (reviewed by Donnelly & Popovich, 2008). The presence of and changes in the numbers of T and B lymphocytes after SCI vary in mice, rats, and humans (Fleming et al., 2006; Kigerl, McGaughy, & Popovich, 2006; Sroga, Jones, Kigerl, McGaughy, & Popovich, 2003). In C57BL/6 mice, they are found as early as 1 week after SCI, reaching their peak at 42 days, and contribute at the later stages to CNS pathology (Ankeny, Lucin, Sanders, McGaughy, & Popovich, 2006; Ankeny & Popovich, 2009; Sroga et al., 2003). In contrast, in the injured human spinal

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cord, CD8þ and a small number of CD4þ T cells are only found months after injury; B cells however are not detected (Fleming et al., 2006).

2.2. Changes in areas undergoing Wallerian degeneration The inflammatory response at the epicenter of injury results in a rapid and robust inflammatory response that leads to secondary damage and loss of gray and white matter. In contrast, the microglial/macrophage response in areas undergoing Wallerian degeneration in lesioned white matter tracts distal to the injury is very slow. Wallerian degeneration can take several weeks to months depending on the region of the CNS affected (David & Ousman, 2002; George & Griffin, 1994; Stoll, Trapp, & Griffin, 1989); while in peripheral nerves, Wallerian degeneration is rapid and occurs within the first week after injury (David & Ousman, 2002; Rotshenker, 2011; Stoll, Griffin, Li, & Trapp, 1989). The slow time course of Wallerian degeneration in the CNS is in part due to reduced levels of chemokines and cytokines as compared to injured peripheral nerves in which this response is robust (David & Ousman, 2002; Perrin, Lacroix, AvilesTrigueros, & David, 2005; Rotshenker, 2011). We have shown that microinjection of MCP-1/MIP-1a or IL-1b into the dorsal column white matter 10 mm distal to a dorsal hemisection 5 days after lesion results in increased recruitment and activation of microglia/macrophages and the rapid clearance of myelin assessed 9 days later (Perrin et al., 2005). These chemokines and cytokine may therefore play a role in microglia/ macrophage recruitment and activation required for Wallerian degeneration in white matter tracts. We have also shown that microinjection of lysophosphatidylcholine (LPC), the hydrolytic product of phospholipase A2 (PLA2), into the spinal cord white matter induces rapid expression within minutes to hours of MCP-1, MIP-1a, GM-CSF, and TNF-a that mediate microglial/macrophage recruitment and activation, and myelin clearance (Ousman & David, 2000, 2001). We will focus our attention in the rest of this chapter on the inflammatory response at the site of injury and its effects on secondary damage after SCI.

3. EFFECTS OF IMMUNE CELLS ON SECONDARY DAMAGE Neutrophils and microglia/macrophages are the main immune cell types that respond acutely after SCI. Why neutrophils enter the cord in sterile SCI lesions in experimental animals is not entirely clear as one of the

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major functions of neutrophils is to combat pathogens. They may play a role in remodeling the damaged area to promote wound healing or serve to recruit monocytes to the injured site. Several interventions that led to reduction in the number of neutrophils in the injured spinal cord have been shown to reduce tissue damage and improve functional recovery (Bao, Chen, Dekaban, & Weaver, 2004; Bao, Chen, Schneider, & Weaver, 2008; Gris et al., 2004; Naruo et al., 2003; Taoka et al., 1997). However, these effects cannot be attributed only to neutrophils as macrophages are also affected by these treatments. Nevertheless, as neutrophils release a variety of proteases and other factors contained in cytoplasmic granules that are cytotoxic as well as produce free radicals, they are likely to have some detrimental effect and contribute to secondary damage. However, there is recent evidence questioning this view (Stirling, Liu, Kubes, & Yong, 2009). The depletion of peripheral macrophages using a systemically administered toxin (clodronate) contained in liposomes, which is taken up by macrophages and kills them, was shown to reduce tissue damage at the site of contusion injury in rats and lead to some improvement in locomotor control (Popovich et al., 1999). In addition, microglia have also been shown to have cytotoxic properties capable of killing neurons in vitro and in vivo (Fordyce, Jagasia, Zhu, & Schlichter, 2005; Kaushal, Koeberle, Wang, & Schlichter, 2007; Kaushal & Schlichter, 2008). This cytotoxic effect has been shown to be mediated by peroxynitrite formation via the generation of superoxide and nitric oxide. Therefore, activated microglia may also be detrimental in the injured spinal cord. On the other hand, some studies show that macrophages have a protective and beneficial effect under certain conditions after CNS injury. Macrophages recruited to the vitreal space by lens injury release factors that promote survival of retinal ganglion cells and axon regeneration after optic nerve injury in rats (Leon, Yin, Nguyen, Irwin, & Benowitz, 2000; Yin et al., 2003). These effects have been reported to be mediated by a Ca2þ binding growth factor called oncomodulin (Yin et al., 2003, 2006), as well as by other factors such as ciliary neurotrophic factor (Muller, Hauk, & Fischer, 2007) and b and g crystallins (Fischer, Hauk, Muller, & Thanos, 2008). In addition, other work has shown that macrophages activated in vitro with segments of peripheral nerve and then transplanted into different regions of the injured CNS such as the spinal cord or optic nerve, are protective and promote functional recovery (LazarovSpiegler, Solomon, & Schwartz, 1998; Lazarov-Spiegler et al., 1996; Rapalino et al., 1998; Schwartz, Moalem, Leibowitz-Amit, & Cohen, 1999; Schwartz & Yoles, 2006). Although there has been much debate in the past

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about whether microglia/macrophages are good or bad after SCI, recent evidence from the broader literature suggests that these differences are likely to be due to differences in polarization states (David & Kroner, 2011). Macrophages polarized to an M1 state are proinflammatory and cytotoxic while M2-polarized macrophages are anti-inflammatory and protective. See review by David and Kroner (2011) for further discussion on macrophage activation and polarizing signals. Interestingly, recent work has shown that the environment of the injured spinal cord favors polarization to a proinflammatory M1 state (Kigerl et al., 2009).

4. IDENTIFICATION OF NOVEL TARGETS THAT MEDIATE INFLAMMATORY RESPONSES AFTER SCI Several studies done over the past decade have led to the identification of molecular targets that modulate inflammation after SCI. These include proinflammatory cytokines such as TNF-a and IL-1b (Genovese et al., 2008; Zong, Zeng, Wei, Xiong, & Zhao, 2012), integrins such as aDb2 and a4b1 involved in the migration of immune cells into the injured cord (Fleming, Bao, Cepinskas, & Weaver, 2010; Gris et al., 2004), free radical generation (Bao et al., 2004, 2008), and others. We have recently reported on the identification of several novel inflammatory mediators that play a role in SCI. As inflammation in SCI is a complex process involving a number of cellular and molecular pathways, we used different strategies to identify novel molecular targets. (i) One approach was to carry out an Affymetrix gene array screen of spinal cord tissue taken at the peak period of the macrophage response to identify genes of interest, which led to the identification of MAPK-activated protein kinase 2 (MK2). (ii) Another approach was to look at other models of CNS injury to identify potential mediators of secondary damage. This led to studies on KCNN4/KCa3.1, an intermediate conductance Ca2þ-activated potassium channel which we found is expressed by astrocytes and may mediate secondary damage after SCI. (iii) As part of the inflammatory response in SCI deals with wound healing, we looked at wound healing in the skin. This led to studies on secretory leukocyte protease inhibitor (SLPI), a serine protease inhibitor produced by neutrophils and macrophages that has both anti-inflammatory and wound-healing properties. (iv) We also identified several lipolytic enzymes belonging to the PLA2 superfamily that generate metabolites that give rise to a wide

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array of inflammatory mediators such as prostaglandins and leukotrienes, as well as metabolites capable of inducing demyelination (David, Greenhalgh, & Lopez-Vales, 2012). We will discuss the identification and characterization of their roles in SCI.

4.1. MAPK-activated protein kinase 2 This molecular target was identified from our Affymetrix microarray screen of the contused spinal cord 7 days after injury, which corresponds to the peak of the macrophage response (Sroga et al., 2003). This screen identified almost 4500 differentially regulated genes, out of which 2300 were upregulated and 2098 downregulated by 1.5-fold (p < 0.05) in the contused spinal cord relative to laminectomized controls (Ghasemlou, Lopez-Vales, et al., 2010). Genes of interest were identified by categorizing the upand downregulated genes according to function using Gene Ontology, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. We found that the KEGG Pathways with the greatest numbers of genes upregulated are primarily those involved in mediating the inflammatory response (see Table 5.1). The MAPK signaling pathway was of

Table 5.1 KEGG pathways with the highest number of genes upregulated by at least 1.5-fold and p < 0.05, relative to laminectomized controls, 7 days after spinal cord contusion injury KEGG pathway No. of genes

Cytokine–cytokine receptor interaction

46

Focal adhesion

42

MAPK-signaling pathway

36

Regulation of actin cytoskeleton

29

ECM–receptor interaction

28

Leukocyte transendothelial migration

27

Cell adhesion molecules

25

Hematopoietic cell lineage

23

Toll-like receptor signaling pathway

23

Jak–STAT signaling pathway

22

B-cell receptor signaling pathway

22

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interest, as injury and disease often elicit stress-related and inflammatory stimuli that can activate the p38 mitogen-activated protein kinase (MAPK). However, studies inhibiting p38 MAPK after SCI have shown differing results, from slightly improved outcome (i.e., increased standing frequency) and reduced apoptosis (Horiuchi, Ogata, Morino, Chuai, & Yamamoto, 2003; Xu, Wang, et al., 2006) to no improvements in locomotor recovery (Stirling, Liu, Plunet, Steeves, & Tetzlaff, 2008). The inhibitors used to block p38 MAPK, however, block both its beneficial and detrimental effects, which may account for these results. One of the genes further downstream in the p38 MAPK pathway, and so likely to have more selective biological effects, is MK2. MK2 is activated by phosphorylation (Gaestel, 2006), which results in the increased production and activation of matrix metalloproteinases (MMPs) (Xu, Chen, & Bergan, 2006), proinflammatory cytokines (Kotlyarov et al., 1999; Winzen et al., 1999), and nitric oxide (Thuraisingam et al., 2007), factors that have been shown to have detrimental effects after SCI. Moreover, studies using MK2 knockout mice have shown reduced neurotoxicity and neuroinflammation in models of Parkinson’s disease (Gomez-Nicola, Valle-Argos, Pita-Thomas, & Nieto-Sampedro, 2008) and cerebral ischemia (Wang et al., 2002). MK2 can modulate inflammation via binding to AU-rich elements in the 30 untranslated region of cytokine mRNAs such as TNF-a, and help stabilize mRNA transcripts allowing for efficient translation of proinflammatory cytokines (Mahtani et al., 2001; Neininger et al., 2002; Winzen et al., 1999). It can also phosphorylate tristetraprolin (Chrestensen et al., 2004; Mahtani et al., 2001) and Hsp27, and the former can also affect cytokine production (Hitti et al., 2006). We observed that the phosphorylated, active form of MK2 (pMK2) reaches peak expression at 7 days after spinal cord contusion injury in mice, and is expressed in microglia/macrophages, astrocytes, and some neurons (Fig. 5.1). At 12 h after injury, over 60% of GFAPþ astrocytes at the lesion site express pMK2 suggesting that astrocytes at this early phase may contribute to cytotoxic effects possibly via expression of proinflammatory cytokines such as TNF-a (Pineau & Lacroix, 2007). By 7 days after SCI when only 10% of astrocytes express MK2, the astroglial scar may have a beneficial effect by limiting the infiltration of immune cells into the spinal cord (Karimi-Abdolrezaee & Billakanti, 2012). Interestingly, we found a significant improvement in locomotor recovery in MK2/ mice as compared to wild-type littermates after a moderate contusion injury, starting 5 days after injury until the end of the study at day 28. Using the Basso Mouse Scale

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pMK2/NeuN

pMK2/GFAP

pMK2/Mac-1

A

B

C

D

E

F

Figure 5.1 Immunofluorescence labeling of spinal cord sections 12 h (A and B) and 7 days (C–F) after spinal cord contusion injury, labeled for pMK2, cell type-specific antibodies, and DAPI staining (merged images). Double labeling for pMK2 and NeuN for neurons in the region of the ventral horn (A and D) and pMK2 and GFAP for astrocytes at 500 mm caudal to epicenter (B and E). Double labeling for pMK2 and anti-Mac-1 for macrophages at epicenter of injury (F) and process-bearing microglia at 500 mm caudal to epicenter (C). Arrowheads point to pMK2-immunopositive cells and insets in panels show high-magnification images of pMK2þ cells from the boxed areas. Note that pMK2 labeling is localized primarily to the nuclei of macrophages/microglia, which are double labeled with anti-Mac-1, a cell surface label; as a result, the two labels are not superimposed but the staining nevertheless shows colabeling of the same cells. Scale bar ¼ 100 mm. With permission from Ghasemlou, Lopez-Vales, et al. (2010).

(BMS) to assess locomotor recovery (Basso et al., 2006), MK2-null mice were able to walk almost normally with minor deficits in frequency of plantar stepping and trunk stability (average BMS score of 7.9/9 and subscore of 8.3/11), while the wild-type mice had extensive deficits in locomotion showing either no coordination while stepping or some coordination with severe trunk stability and rotated paws and lift-off and placement (average BMS score of 5.25/9 and subscore of 2.9/11). At 12 h after SCI, there was a 97% reduction in the expression of TNF-a in the spinal cord of MK2 knockout mice. Neutrophil infiltration into the CNS was also reduced by more than 90% at this time-point. At 7 day, which corresponds to peak macrophage numbers and differences in locomotor outcome, the activity of MMP-9 was reduced by about 50% while MMP-2 activity increased by about 50% as determined by gelatin zymography. Previous studies have

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shown that MMP-9, which is produced by macrophages, blood vessels, and astrocytes, mediates breakdown of the blood–spinal cord barrier, resulting in an increased inflammatory response and scar formation after SCI (Noble, Donovan, Igarashi, Goussev, & Werb, 2002); MMP-9/ mice show improved histological and locomotor outcomes after SCI (Noble et al., 2002). In contrast, MMP-2 activity reduces glial scar formation and chondroitin sulfate proteoglycan (CSPG) deposition (Hsu et al., 2006); recovery after SCI is worse in MMP-2 null mice as compared to wild-type mice (Hsu et al., 2006). The changes we observed in cytotoxic and beneficial mediators in MK2 knockout mice after SCI were correlated with reduced secondary damage assessed at 28 day after SCI. These include several histological measures such as reduced ventral horn neuron loss, reduced myelin loss, and increased serotonergic innervation of the ventral horn. In addition, there is a 40% reduction in 3-nitrotyrosine. Our work therefore suggests that MK2 plays multiple roles in different cell types that contribute to inflammation and secondary tissue damage after SCI. This work also suggests that selective pharmacological inhibitors of MK2, would be useful to treat spinal cord injury.

4.2. KCNN4/KCa3.1 This intermediate conductance Ca2þ-activated Kþ channel also known as IK1, KCa3.1, or SK4 (KCNN4 for the gene) is expressed in many types of nonexcitable cells (T cells, red blood cells, and microglia) (Gardos, 1958; Kaushal et al., 2007; Khanna, Chang, Joiner, Kaczmarek, & Schlichter, 1999). Small increases in intracellular Ca2þ (Kd 300 nM) opens the channel leading to Kþ efflux and subsequent increases in Ca2þ influx that can regulate a wide variety of Ca2þ mediated changes. Microglia in vitro express KCa3.1 and experiments using a selective inhibitor (triarylmethane-34 [TRAM-34]) showed that it mediates LPS-induced increase in iNOS, NO and peroxynitrite production, and neurotoxicity (Kaushal et al., 2007). Blocking KCa3.1 channels in vitro with TRAM-34 inhibits p38 MAPK activation (Kaushal et al., 2007). Further, intraocular injection of TRAM-34 reduced retinal ganglion cell loss after optic nerve transection (Kaushal et al., 2007). Other work has also shown that TRAM-34 reduces clinical disability and proinflammatory cytokine production in experimental autoimmune encephalomyelitis (EAE; Reich et al., 2005). KCa3.1 channel blockers were also reported to reduce brain edema, intracranial pressure, and infarct volume in animal model of acute subdural hematoma (Mauler et al., 2004),

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and reduced infarct volume and improved neurological symptoms after brain ischemia/reperfusion in rats (Chen, Raman, Bodendiek, O’Donnell, & Wulff, 2011). We found that spinal cord contusion injury induced a significant upregulation of KCa3.1 mRNA between 3 and 28 days and protein between 5 and 28 days (Bouhy et al., 2011). We were surprised to find that after SCI, the expression of KCa3.1 was upregulated in reactive astrocytes but not microglia/macrophages in tissue sections (Fig. 5.2). We found, however, that microglia/macrophages acutely isolated from the contused spinal cord 7 days after injury and plated in vitro for 2 h expressed KCa3.1, as did microglia purified from the neonatal CNS. The reasons for the lack of expression in microglia and macrophages in the injured spinal cord in vivo are not clear at present (see Bouhy et al., 2011 for further discussion). Interestingly, however, treatment with TRAM-34 reduced secondary tissue damage including ventral horn neuron loss, tissue loss estimated by GFAP labeling, and axonal loss estimated by antineurofilament labeling. TRAM-34 treatment also reduced expression of IL-1b, TNF-a, and iNOS. Importantly, blocking KCa3.1 with TRAM-34 after spinal cord contusion injury improved locomotor recovery in a dose-dependent manner, being most effective at a dose of 120 mg/kg/days. These findings suggest that KCa3.1 could be a therapeutic target for treatment of SCI. Reactive astrocytes in the injured spinal cord are generally thought of as preventing axon regeneration by forming the glial scar and expressing CSPG that inhibits axon growth (Silver & Miller, 2004). Our findings show that reactive astrocytes might also contribute to the inflammatory response via KCa3.1 A

KCa3.1 B

GFAP C

Merge D

Figure 5.2 (A) Low-magnification image of the contused spinal cord at 7 dpi immunostained for KCa3.1. The area in the box is shown at higher magnification in (B–D). The high-magnification images show double labeling for KCa3.1 (B) and GFAP (C) (arrows) and the merged images with DAPI-stained nuclei (D). Note the KCa3.1 labeling of GFAPþ-reactive astrocytes (arrows); the inset in (D) shows double-labeled profiles at higher magnification (arrow). In the inset, note that the strongest labeling for KCa3.1 is on the membrane while the GFAP labeling is intracellular. Scale bars: A ¼ 500 mm, D ¼ 50 mm, inset ¼ 30 mm. With permission from Bouhy et al. (2011).

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KCa3.1-mediated effects on iNOS and proinflammatory cytokine production and other Ca2þ-mediated effects.

4.3. Secretory leukocyte protease inhibitor The classic model of wound healing in the skin has many of the hallmarks as that observed after SCI (Stadelmann, Digenis, & Tobin, 1998): an inflammatory phase where the sequential infiltration of immune cells contributes to the clearance of tissue debris; the proliferative phase where new blood vessels are formed; and the remodeling phase where cells no longer needed are removed by phagocytosis and collagen/epithelia are laid down forming the scar tissue. This complex process is controlled by various mediators, both pro- and anti-inflammatory as well as those involved in the resolution of inflammation. These phases normally progress in a timely manner but if they do not, the result is a chronic wound (Nwomeh, Yager, & Cohen, 1998), which might also contribute to the ongoing secondary damage in the injured spinal cord. This analogy brought our attention to SLPI, a 12-kD serine protease inhibitor involved in skin wound healing that is produced by activated neutrophils and macrophages, and exerts an anti-inflammatory/resolving effect (Sallenave, 2010). SLPI was also found to suppress the production of proinflammatory mediators in LPS-stimulated macrophages (Jin, Nathan, Radzioch, & Ding, 1997; Zhang, DeWitt, McNeely, Wahl, & Wahl, 1997). Mice deficient in SLPI show impaired skin wound healing while exogenous application of recombinant SLPI (rSLPI) enhanced repair (Ashcroft et al., 2000). Further, treatment with exogenous rSLPI reduced inflammation and tissue damage in models of arthritis (Song et al., 1999) and lung inflammation (Lentsch et al., 1999; Stolk, Rudolphus, & Kramps, 1991), and adenoviral expression in the CNS reduced lesion size after cerebral ischemia (Wang et al., 2003). We therefore studied whether SLPI could have a beneficial role in SCI (Ghasemlou, Bouhy, et al., 2010). SLPI expression in the injured spinal cord mirrored closely the changes seen in the skin after injury: protein levels peak early (3–7 days) after injury and are found primarily in cells that will form the scar tissue (suprabasal epidermal cells in the skin and astrocytes in the spinal cord). Some inflammatory cells are also immunopositive for SLPI, although we observed little if any staining in macrophages in the lesion core. Astrocytes surrounding the lesion core had the most intense immunoreactivity (Fig. 5.3), suggesting that they might play a role in dampening proinflammatory cytokine expression. We then used two different methods to assess the role of SLPI in SCI. Transgenic

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A

B

C

D

E

F

G

H

Figure 5.3 (A) Low-magnification image of spinal cord cross-section 14 days after spinal cord contusion injury double labeled for GFAP and SLPI (L ¼ lesion). Note the increased expression of SLPI (green) in areas bordering the lesion and in neurons in the ventral horn. The area outlined in the box is shown in higher magnification in panels (B–D). (B–D) Higher magnification shows strong SLPI immunoreactivity (C) in reactive astrocytes (B) along the border of the lesion; the merged image with DAPI labeling is shown in panel (D) (L ¼ lesion). (E) Low-magnification image of spinal cord cross-section 1 day postinjury double labeled for neutrophils (antibody 7/4) (red) and SLPI (green). Note also the SLPI labeling in the ventral horn, which appears to be neurons. (F–H) Higher magnification of the area outlined in the box in panel (E) is shown in (F–H). Some neutrophils (F) (arrows) are also labeled for SLPI (G); the merged image is shown in panel (H). The inset in (H) shows higher magnification of the area outlined with the dashed lines showing SLPI labeling in neutrophils. Scale bar in (A and E) ¼ 500 mm, in (D and H) ¼ 100 mm, inset in H ¼ 25 mm. With permission from Ghasemlou, Bouhy, et al. (2010).

mice overexpressing SLPI showed improved locomotor recovery between 3 and 10 days postinjury, however, this effect was lost by day 14. Based on this result, we hypothesized that SLPI may be beneficial in the early phase of SCI when the expression of proinflammatory mediators (cytokines, proteases, etc.) are high, while its anti-inflammatory effects may be detrimental to recovery at later time-points. We therefore treated wild-type mice daily with 1 mg/g rSLPI by intraperitoneal injection for the first 7 days after SCI and found improved locomotor recovery starting 3 days after injury and continuing until the end of the experiment at 28 days (p < 0.05). Histological analysis of the spinal cords from rSLPI-treated animals at 28 days revealed significantly reduced lesion size measured using GFAP-immunoreactivity,

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at and near the lesion epicenter. Myelin sparing was improved for a distance of 900 mm rostral and caudal to the lesion epicenter, and motor neuron survival was increased over more than 600 mm rostral and caudal to the lesion epicenter. Serotonergic sprouting 1 mm caudal to the injury was also increased more than 60% in rSLPI-treated animals (p < 0.05) as compared to injured control mice. To identify how systemic delivery of rSLPI could have such beneficial effects in the spinal cord, we examined the localization of SLPI in vivo before and after injury. Because SLPI had previously been reported to localize to leukocyte nuclei where it could bind to NF-kB binding sites to attenuate the expression of proinflammatory immune response genes (Taggart et al., 2005), we focused on leukocytes in the peripheral circulation. We found that after a single intraperitoneal injection of rSLPI in adult mice with SCI, almost 100% of the leukocytes in the peripheral circulation were immunoreactive for SLPI at 3 h after injection as compared to only 30% of cells from vehicle-treated mice. Importantly, based on optical density measurements, we found a fourfold greater SLPI immunoreactivity in rSLPI-treated mice as compared to vehicle-treated controls. This data clearly indicate that rSLPI is likely to be taken up by leukocytes in the circulation. Western blot of the injured spinal cord also showed increased SLPI and Ik-Ba, the inhibitor of NF-kB activity, in rSLPI-treated mice at 12 and 24 h, relative to vehicle-treated controls. Increased SLPI immunoreactivity was found in small, round cells in the injury site that appeared to be infiltrating leukocytes. mRNA analysis of cytokines regulated by SLPI and NF-kB showed a 40% reduction in TNF-a levels (p < 0.05) 12 h after SCI. Our data suggest that SLPI can modulate the inflammatory milieu of the injured spinal cord, resulting in beneficial effects on locomotor recovery and tissue protection during the first week after injury, by exerting antiinflammatory effects. These inflammatory effects are likely due to NF-k B-mediated production of TNF-a and other proinflammatory molecules. While only one or two injections of rSLPI were effective in other models of peripheral inflammation such as arthritis (Song et al., 1999) and skin wound (Ashcroft et al., 2000), daily treatment was required for improved recovery after SCI. The reasons for this are not known at present. Our data provides further evidence that during the first week after injury, the spinal cord tissue environment is one that promotes secondary tissue damage, which can be reduced at least in part by treatment with rSLPI as endogenously produced SLPI is not sufficient to provide effective protection.

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The use of SLPI to treat SCI may be fast-tracked as it has received orphan drug status by the FDA for the treatment of cystic fibrosis and genetic emphysema.

4.4. Phospholipase A2 PLA2 enzymes and downstream pathways play a role in a variety of inflammatory conditions. PLA2s hydrolyze membrane phospholipids at the sn-2 position to release a free fatty acid and generate a lysophospholipid. If the fatty acid released is arachidonic acid, it can produce a variety of prostaglandins and leukotrienes via cyclooxygenase-1 and 2 (COX-1 and 2) and lipooxygenase enzymes, respectively. If the phospholipid is phosphatidylcholine, then the lysophospholipid released by the actions of PLA2 will give rise to LPC (also known as lysolecithin), a potent demyelinating agent. We reported that LPC can induce rapid demyelination in the spinal cord within 4 days (Ousman & David, 2000, 2001). There are about 20 mammalian forms of PLA2, which have different specificities, activation conditions, intracellular or extracellular localization, and tissue distribution. There are also about two dozen downstream bioactive metabolites of PLA2. Therefore, PLA2s, the various downstream enzymes, and the metabolites they produce comprise a major pathway controlling a wide variety of inflammatory responses. In addition to proinflammatory mediators, this pathway can also generate proresolution mediators that play a role in the naturally occurring resolution of inflammation (Serhan et al., 2008). We will focus here on our recent work on the expression and role of different members of the PLA2 superfamily in SCI. There are two classes of PLA2—secreted (sPLA2) and intracellular. The latter include calcium-dependent (cPLA2) and calcium-independent (iPLA2) forms. The former require micromolar concentrations of calcium and are activated upon phosphorylation. These enzymes play a role in membrane turnover and host defense (Brown, Chambers, & Doody, 2003; Murakami, Nakatani, Atsumi, Inoue, & Kudo, 1997). We found increase in expression at the mRNA and protein level after SCI in only 3 of the 14 PLA2s examined (Lopez-Vales et al., 2011). These include sPLA2 group IIA (sPLA2 GIIA), cPLA2 GIVA and iPLA2 GVIA, which showed differences in the time-duration after injury when they are expressed, and differences in the cell types that express them. The expression of sPLA2 GIIA was detected during the first week after spinal cord contusion injury in mice (mainly between 3 and 7 days). The activated phosphorylated form of cPLA2 GIVA,

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on the other hand, was increased between 3 and 28 days; with a threefold increase at day 3 that decreased to about a twofold increase between 14 and 28 days. The active 52-kDa cleaved form of iPLA2 is increased three- to fourfold between 7 and 28 days except at day 14 when it reaches a peak of about sixfold. sPLA2 GIIA and iPLA2 GVIA are widely expressed in most cell types (astrocytes, oligodendrocytes, neurons, and microglia/ macrophages) in the injured spinal cord with some key differences. sPLA2 GIIA is expressed mainly in astrocytes and oligodendrocytes, while iPLA2 GVIA is expressed mainly in oligodendrocytes. cPLA2 GIVA, on the other hand, is expressed mainly by neurons and oligodendrocytes in the injured cord. We dissected out the functional roles of three PLA2s in SCI using a panel of small molecule inhibitors that were selective for each of the three PLA2s or inhibitors that were able to partially or completely block all three. Please see our publication of details of the selectivity and potency of these inhibitors, and additional information on related work on these inhibitors (Lopez-Vales et al., 2011). Blocking sPLA2 GIIA with a highly selective small amide inhibitor (GK115) yielded improved recovery of locomotor control assessed using the BMS analysis. Treatment with this inhibitor also led to reduced myelin loss and tissue damage, and improved serotonergic innervation of the ventral horn. The reduction in myelin loss correlates with the increased expression of sPLA2 GIIA in oligodendrocytes. Other work has also shown that injection of sPLA2 GIII, a bee venom form of PLA2 into the intact spinal cord induced myelin damage and tissue loss (Liu et al., 2006). Our work with a selective inhibitor (GK115) provides direct evidence for a role for sPLA2 GIIA in the pathology and functional loss seen after SCI. Inhibition of sPLA2 has also been shown to be neuroprotective after transient global brain ischemia in gerbils (Wang et al., 2009). In contrast to blocking sPLA2 GIIA, blocking iPLA2 GVIA with a selective pentafluorokeone inhibitor (FKGK11) did not improve locomotor function except for some minimal effects in the BMS subscore at 28 days. The inhibitor treatment, however, reduced myelin loss and some tissue loss but had no effect on survival of ventral motor neurons or on serotonergic innervation of the ventral horns after injury. The lack of effect on ventral horn neuron survival and reinnervation may underlie the poor recovery of locomotor function. As blocking iPLA2 GVIA can significantly reduce myelin loss and tissue loss, the combined blocking of iPLA2 GVIA and sPLA2 GIIA might be expected to provide better recovery as compared to blocking each alone.

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To our surprise, blocking cPLA2 GIVA with a highly selective 2-oxoamide inhibitor (AX059) resulted in worsening of all functional and histological outcome measures. Locomotor function assessed with the BMS analysis, tissue sparing, and ventral motor neuron survival were all significantly worse in mice treated with the cPLA2 GVIA inhibitor (LopezVales et al., 2011). This was an unexpected result as cPLA2 GIVA has been reported to be detrimental in other models of neurological disorders such as cerebral ischemia (Bonventre et al., 1997) and EAE (Kalyvas et al., 2009). In brain ischemia/reperfusion injury, cPLA2 was thought to contribute to blood–brain barrier disruption through the p38 MAPK pathway (Nito et al., 2008). In the EAE study, we reported that the same inhibitor (AX059) used in SCI had beneficial effects. We therefore assessed the effects of spinal cord contusion in cPLA2 GIVA null mice, which also showed significant worsening of various outcome measures after SCI as was seen with the inhibitor treatment. cPLA2 GIVA is expressed in ventral motor neurons in the normal uninjured spinal cord; our findings therefore suggest that loss of this expression may be detrimental to the survival of these neurons. This may have to do with its role in membrane turnover related to synaptic vesicle release from the large motor neurons projecting to neuromuscular junctions. cPLA2 has also been reported to play a role in survival of neurons in hippocampal slice cultures and in preventing calcium-mediated cell death of other nonneural cell types (Casas et al., 2006; Forlenza, Mendes, Marie, & Gattaz, 2007; Oikawa et al., 2005). The importance of cPLA2 was further confirmed using a pan-PLA2 inhibitor (FKGK2) that blocked all three PLA2s to about 90%. Treatment with this inhibitor after SCI also led to worsening of all outcome measures assessed. These findings suggest that the beneficial effects of blocking sPLA2 GIIA and iPLA2 GVIA can be lost if cPLA2 is inhibited. Interestingly, a weak pan-PLA2 inhibitor that partially blocked all three inhibitors to about 50% level (cPLA2 by 62%, iPLA2 by 45%, and sPLA2 by 52%) was the most effective in promoting locomotor recovery and reducing histopathology, including about a threefold increase in serotonergic innervation. We showed that this effect appears to be mediated by upregulation of cPLA2, COX-2, and prostaglandin E synthetic pathway via the EP1 receptor, suggesting potential cross talk between various PLA2s as has been reported in other models (Murakami & Kudo, 2001; Offer et al., 2005). These findings show that different members of the PLA2 superfamily can have either beneficial or detrimental effects in SCI (Table 5.2). Interestingly, we found that the same inhibitors had different effects in EAE, a model of

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Table 5.2 The timing of expression of the three PLA2s, after SCI, the cell types they are expressed in and their roles cPLA2 GIVA iPLA2 GVIA sPLA2 GIIA

Increased 3–7 days Time period expressed in SCI

Increased 3–28 days

Increased 7–28 days

Cell types Astrocytes and Neurons, expressing oligodendrocytes; also in oligodendrocytes PLA2s some neurons and microglia/macrophages

Oligodendrocytes; also some astrocytes, neurons, and microglia/ macrophages

Effects in Detrimental SCI

Detrimental

Beneficial

CNS autoimmune disease (Kalyvas et al., 2009). Unlike in SCI, blocking cPLA2 GIVA with AX059 reduced the severity of clinical paralysis during the initial phase of EAE when treatment was started before the onset of symptoms. In contrast, blocking iPLA2 GVIA with FKGK11 gave the most profound improvement in EAE. This is in striking contrast to the minimal effects of blocking iPLA2 in SCI. Further, treatment with the weak panPLA2 inhibitor (AX115) had no effect in EAE when treatment was given prior to onset of symptoms, but eliminated the remission phase (i.e., making EAE worse) when given after the onset of symptoms. Blocking iPLA2 GVIA was most effective in EAE while the weak pan-inhibitor (AX115) and the sPLA2 GIIA inhibitor (GK115) were most effective in SCI. These two studies show that members of the PLA2 superfamily can have very different roles in different neurological conditions and point to the need to determine their roles using selective small molecule inhibitors or gene knockout mice.

5. CONCLUSIONS The inflammation that occurs in the injured spinal cord contributes to secondary tissue damage and functional loss. It involves all cell types in the CNS, as well as immune cells from the periphery. Although this innate immune response mainly involves microglia, peripheral macrophages, and neutrophils, other cell types such as astrocytes may also contribute by producing factors that regulate leukocyte responses. The immune response after SCI is complex and involves many cell types and molecular pathways. We have used different approaches to identify several molecular targets that

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modulate the inflammatory response in SCI. These approaches include: Affymetirx gene array screening that led to the identification of MK2 that helps orchestrate cytotoxic responses; searching for molecules identified in other CNS conditions that led to the identification of KCa3.1 in astrocytes that mediate inflammation in SCI; studying molecules involved in wound healing in tissues such as the skin that led to the identification of the beneficial role of SLPI in SCI; and examining the role of lipolytic enzymes belonging to the PLA2 superfamily that led to the production of various eicosanoids and other lipid mediators of inflammation. Our work using small molecule inhibitors, recombinant proteins, and gene knockout mice has provided direct evidence for the role of these molecules in SCI. In all but one (MK2), small molecule inhibitors (KCa3.1 and PLA2s) or recombinant proteins (SLPI) were shown to be effective in reducing secondary damage and promoting recovery of locomotor function after SCI, indicating that these may be suitable therapeutic candidates to target for SCI in humans. Effective treatment in humans will likely require a combination of neuroprotection strategies such as these, combined with treatments to promote axon sprouting and regeneration.

ACKNOWLEDGMENTS Work presented here was supported by grants from the Canadian Institutes of Health Research (CIHR) to S. D. J. Z. is supported by a postdoctoral fellowship from the Fonds de la recherche en sante´ du Que´bec (FRSQ) and the CIHR Neuroinflammation Training Program; N. G. was supported by Studentship awards from the CIHR and FRSQ.

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