Hal X. Nguyen

Quantitative analysis of cellular inflammation after traumatic spinal cord injury: evidence for a multiphasic inflammatory response in the acute to chronic environment

Traumatic injury to the central nervous system results in the disruption of the blood brain/spinal barrier, followed by the invasion of cells and other components of the immune system that can aggravate injury and affect subsequent repair and regeneration. Although studies of chronic neuroinflammation in the injured spinal cord of animals are clinically relevant to most patients living with traumatic injury to the brain or spinal cord, very little is known about chronic neuroinflammation, though several studies have tested the role of neuroinflammation in the acute period after injury. The present study characterizes a novel cell preparation method that assesses, quickly and effectively, the changes in the principal immune cell types by flow cytometry in the injured spinal cord, daily for the first 10 days and periodically up to 180 days after spinal cord injury. These data quantitatively demonstrate a novel time-dependent multiphasic response of cellular inflammation in the spinal cord after spinal cord injury and are verified by quantitative stereology of immunolabelled spinal cord sections at selected time points. The early phase of cellular inflammation is comprised principally of neutrophils (peaking 1 day post-injury), macrophages/microglia (peaking 7 days post-injury) and T cells (peaking 9 days post-injury). The late phase of cellular inflammation was detected after 14 days post-injury, peaked after 60 days post-injury and remained detectable throughout 180 days post-injury for all three cell types. Furthermore, the late phase of cellular inflammation (14-180 days post-injury) did not coincide with either further improvements, or new decrements, in open-field locomotor function after spinal cord injury. However, blockade of chemoattractant C5a-mediated inflammation after 14 days post-injury reduced locomotor recovery and myelination in the injured spinal cord, suggesting that the late inflammatory response serves a reparative function. Together, these data provide new insight into cellular inflammation of spinal cord injury and identify a surprising and extended multiphasic response of cellular inflammation. Understanding the role of this multiphasic response in the pathophysiology of spinal cord injury could be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions.

Shifts in macrophage phenotypes and macrophage competition for arginine metabolism affect the severity of muscle pathology in muscular dystrophy

Duchenne muscular dystrophy (DMD) is the most common, lethal, muscle-wasting disease of childhood. Previous investigations have shown that muscle macrophages may play an important role in promoting the pathology in the mdx mouse model of DMD. In the present study, we investigate the mechanism through which macrophages promote mdx dystrophy and assess whether the phenotype of the macrophages changes between the stage of peak muscle necrosis (4 weeks of age) and muscle regeneration (12 weeks). We find that 4-week-old mdx muscles contain a population of pro-inflammatory, classically activated M1 macrophages that lyse muscle in vitro by NO-mediated mechanisms. Genetic ablation of the iNOS gene in mdx mice also significantly reduces muscle membrane lysis in 4-week-old mdx mice in vivo. However, 4-week mdx muscles also contain a population of alternatively activated, M2a macrophages that express arginase. In vitro assays show that M2a macrophages reduce lysis of muscle cells by M1 macrophages through the competition of arginase in M2a cells with iNOS in M1 cells for their common, enzymatic substrate, arginine. During the transition from the acute peak of mdx pathology to the regenerative stage, expression of IL-4 and IL-10 increases, either of which can deactivate the M1 phenotype and promote activation of a CD163+, M2c phenotype that can increase tissue repair. Our findings further show that IL-10 stimulation of macrophages activates their ability to promote satellite cell proliferation. Deactivation of the M1 phenotype is also associated with a reduced expression of iNOS, IL-6, MCP-1 and IP-10. Thus, these results show that distinct subpopulations of macrophages can promote muscle injury or repair in muscular dystrophy, and that therapeutic interventions that affect the balance between M1 and M2 macrophage populations may influence the course of muscular dystrophy.

Interactions between neutrophils and macrophages promote macrophage killing of rat muscle cells in vitro

Current evidence indicates that the physiological functions of inflammatory cells are highly sensitive to their microenvironment, which is partially determined by the inflammatory cells and their potential targets. In the present investigation, interactions between neutrophils, macrophages and muscle cells that may influence muscle cell death are examined. Findings show that in the absence of macrophages, neutrophils kill muscle cells in vitro by superoxide‐dependent mechanisms, and that low concentrations of nitric oxide (NO) protect against neutrophil‐mediated killing. In the absence of neutrophils, macrophages kill muscle cells through a NO‐dependent mechanism, and the presence of target muscle cells causes a three‐fold increase in NO production by macrophages, with no change in the concentration of inducible nitric oxide synthase. Muscle cells that are co‐cultured with both neutrophils and macrophages in proportions that are observed in injured muscle show cytotoxicity through a NO‐dependent, superoxide‐independent mechanism. Furthermore, the concentration of myeloid cells that is necessary for muscle killing is greatly reduced in assays that use mixed myeloid cell populations, rather than uniform populations of neutrophils or macrophages. These findings collectively show that the magnitude and mechanism of muscle cell killing by myeloid cells are modified by interactions between muscle cells and neutrophils, between muscle cells and macrophages and between macrophages and neutrophils.

Polymorphonuclear leukocytes promote neurotoxicity through release of matrix metalloproteinases, reactive oxygen species, and TNF-α

As the first immune cells to infiltrate the nervous system after traumatic PNS and CNS injury, neutrophils (polymorphonuclear leukocytes, PMNs) may promote injury by releasing toxic soluble factors that may affect neuronal survival. Direct neurotoxicity of matrix metalloproteinases (MMPs), reactive oxygen species (ROS), and cytokines released by PMNs was investigated by culturing dorsal root ganglion (DRG) cells with PMN‐conditioned media containing MMP inhibitor (GM6001), ROS scavengers, or tumor necrosis factor αR (TNF‐αR) neutralizing antibody. Although DRGs exposed to PMN‐conditioned media had 53% fewer surviving neurons than controls, neuronal cell loss was prevented by GM6001 (20 μmol/L), catalase (1000 U/mL), or TNF‐αR neutralizing antibody (1.5 μg/mL), elevating survival to 77%, 94%, and 95%, respectively. In accordance with protection by GM6001, conditioned media collected from MMP‐9 null PMNs was less neurotoxic than that collected from wild‐type PMNs. Additionally, MMP inhibition reduced PMN‐derived ROS; removal of ROS reduced PMN‐derived MMP‐9 activity; and TNF‐α inhibition reduced both PMN‐derived MMP‐9 activity and ROS in PMN cultures. Our data provide the first direct evidence that PMN‐driven neurotoxicity is dependent on MMPs, ROS, and TNF‐α, and that these factors may regulate PMN release of these soluble factors or interact with one another to mediate PMN‐driven neurotoxicity.

Expression of a muscle‐specific, nitric oxide synthase transgene prevents muscle membrane injury and reduces muscle inflammation during modified muscle use in mice

Nitric oxide (NO) can function as either a pro‐inflammatory or anti‐inflammatory molecule, depending upon its concentration and the microenvironment in which it is produced. We tested whether muscle‐derived NO affects muscle inflammation and membrane lysis that occur in modified muscle use. Transgenic mice with muscle‐specific over‐expression of neuronal NO synthase (nNOS) were generated in which transgene expression was driven by the human skeletal muscle actin promoter. Transgenic mice and non‐transgenic littermates were subjected to hindlimb muscle unloading followed by reloading, which causes muscle inflammation and membrane lysis. NOS expression decreased in transgenic and non‐transgenic mice during muscle unloading. Muscle inflammation was assessed by immunohistochemistry after 24 h of muscle reloading following 10 days of unloading. Soleus muscles of non‐transgenic mice showed significant increases in the concentrations of neutrophils (4.8‐fold) and macrophages (11.3‐fold) during reloading, compared to mice that experienced unloading only. Muscles of transgenic mice showed 51 % fewer neutrophils in reloaded muscles than those of non‐transgenic mice, but macrophage concentrations did not differ from non‐transgenic mice. Muscle membrane damage was determined by measuring influx of an extracellular marker dye. Significantly more membrane damage occurred in muscles of non‐transgenic mice experiencing reloading than in ambulatory controls. However, membrane damage in the reloaded muscles of transgenic mice did not differ from that in ambulatory mice. In vitro cytotoxicity assays confirmed that mouse neutrophils lyse muscle cell membranes, and showed that inhibition of NOS in muscle and neutrophil co‐cultures significantly increased neutrophil‐mediated lysis of muscle cells. Together, these data show that muscle‐derived NO can function as an anti‐inflammatory molecule in muscle that experiences modified loading, and that NO can prevent neutrophil‐mediated damage of muscle cell membranes in vivo and in vitro.

Null mutation of myeloperoxidase in mice prevents mechanical activation of neutrophil lysis of muscle cell membranes in vitro and in vivo.

Membrane lysis is a common and early defect in muscles experiencing acute injuries or inflammation. Although increased mechanical loading of muscles can induce inflammation and membrane lysis, whether mechanical loads applied to muscle can promote the activation and cytolytic capacity of inflammatory cells and thereby increase muscle damage is unknown. We tested whether mechanical loads applied to mouse muscle cells in vitro can increase membrane lysis, and whether neutrophil‐mediated lysis of muscle cells is promoted by mechanical loads applied in vitro and in vivo. Cyclic loads applied to muscle cells for 24 h in vitro produced little muscle cell lysis. Similarly, the addition of neutrophils to muscle cell cultures in the presence of superoxide dismutase (SOD) produced little muscle cell lysis. However, when cyclic mechanical loads were applied to neutrophil–muscle co‐cultures in the presence of SOD, there was a synergistic effect on muscle cell lysis, suggesting that mechanical loading activates neutrophil cytotoxicity. However, application of mechanical loads to co‐cultures of muscle cells and neutrophils that are null mutants for myeloperoxidase (MPO) showed no mechanical activation of neutrophil cytotoxicity. This indicates that loading promotes neutrophil cytotoxicity via MPO. Activity assays confirmed that mechanical loading of neutrophil–muscle co‐cultures significantly increased MPO activity. We further tested whether muscle membrane lysis in vivo was mediated by neutrophils when muscle was subjected to modified loading by using a mouse model of muscle reloading following a period of unloading. We observed that MPO −/− soleus muscles showed a significant 52% reduction in membrane lysis compared to wild‐type mice, although the mutation did not decrease inflammatory cell extravasation. Together, these in vitro and in vivo findings show that mechanical loading activates neutrophil‐mediated lysis of muscle cells through an MPO‐dependent pathway.

Deficiency in complement C1q improves histological and functional locomotor outcome after spinal cord injury

Although studies have suggested a role for the complement system in the pathophysiology of spinal cord injury (SCI), that role remains poorly defined. Additionally, the relative contribution of individual complement pathways in SCI is unknown. Our initial studies revealed that systemic complement activation was strongly influenced by genetic background and gender. Thus, to investigate the role of the classical complement pathway in contusion-induced SCI, male C1q knock-out (KO) and wild-type (WT) mice on a complement sufficient background (BUB) received a mild-moderate T9 contusion injury with the Infinite Horizon impactor. BUB C1q KO mice exhibited greater locomotor recovery compared with BUB WT mice (p < 0.05). Improved recovery observed in BUB C1q KO mice was also associated with decreased threshold for withdrawal from a mild stimulus using von Frey filament testing. Surprisingly, quantification of microglia/macrophages (F4/80) by FACS analysis showed that BUB C1q KO mice exhibited a significantly greater percentage of macrophages in the spinal cord compared with BUB WT mice 3 d post-injury (p < 0.05). However, this increased macrophage response appeared to be transient as stereological assessment of spinal cord tissue obtained 28 d post-injury revealed no difference in F4/80-positive cells between groups. Stereological assessment of spinal cord tissue showed that BUB C1q KO mice had reduced lesion volume and an increase in tissue sparing compared with BUB WT mice (p < 0.05). Together, these data suggest that initiation of the classical complement pathway via C1q is detrimental to recovery after SCI.

Characterization of early and terminal complement proteins associated with polymorphonuclear leukocytes in vitro and in vivo after spinal cord injury

BackgroundThe complement system has been suggested to affect injury or disease of the central nervous system (CNS) by regulating numerous physiological events and pathways. The activation of complement following traumatic CNS injury can also result in the formation and deposition of C5b-9 membrane attack complex (C5b-9/MAC), causing cell lysis or sublytic effects on vital CNS cells. Although complement proteins derived from serum/blood-brain barrier breakdown can contribute to injury or disease, infiltrating immune cells may represent an important local source of complement after injury. As the first immune cells to infiltrate the CNS within hours post-injury, polymorphonuclear leukocytes (PMNs) may affect injury through mechanisms associated with complement-mediated events. However, the expression/association of both early and terminal complement proteins by PMNs has not been fully characterized in vitro, and has not observed previously in vivo after traumatic spinal cord injury (SCI).MethodWe investigated the expression of complement mRNAs using rt-PCR and the presence of complement proteins associated with PMNs using immunofluroescence and quantitative flow cytometry.ResultsStimulated or unstimulated PMNs expressed mRNAs encoding for C1q, C3, and C4, but not C5, C6, C7 or C9 in culture. Complement protein C1q or C3 was also detected in less than 30% of cultured PMNs. In contrast, over 70% of PMNs that infiltrated the injured spinal cord were associated with C1q, C3, C7 and C5b-9/MAC 3 days post-SCI. The localization/association of C7 or C5b-9/MAC with infiltrating PMNs in the injured spinal cord suggests the incorporation or internalization of C7 or C5b-9/MAC bound cellular debris by infiltrating PMNs because C7 and C5b-9/MAC were mostly localized to granular vesicles within PMNs at the spinal cord epicenter region. Furthermore, PMN presence in the injured spinal cord was observed for many weeks post-SCI, suggesting that this infiltrating cell population could chronically affect complement-mediated events and SCI pathogenesis after trauma.ConclusionData presented here provide the first characterization of early and terminal complement proteins associated with PMNs in vitro and in vivo after SCI. Data also suggest a role for PMNs in the local internalization or deliverance of complement and complement activation in the post-SCI environment.

Immunosuppressants Affect Human Neural Stem Cells In Vitro but Not in an In Vivo Model of Spinal Cord Injury

Clinical immunosuppression protocols use calcineurin inhibitors, such as cyclosporine A (CsA) or tacrolimus (FK506), or mammalian target of rapamycin (mTOR) inhibitors, such as sirolimus (rapamycin). These compounds alter immunophilin ligand signaling pathways, which are known to interact downstream with mediators for human neural stem cell (hNSC) differentiation and proliferation, suggesting that immunosuppressants may directly alter hNSC properties. We investigated whether immunosuppressants can exert direct effects on the differentiation, proliferation, survival, and migration of human central nervous system‐derived stem cells propagated as neurospheres (hCNS‐SCns) in vitro and in an in vivo model of spinal cord injury. We identified unique, immunosuppressant‐dependent effects on hCNS‐SCns differentiation and proliferation in vitro. All immunosuppressants tested increased neuronal differentiation, and CsA and rapamycin inhibited proliferation in vitro. No immunosuppressant‐mediated effects on hCNS‐SCns survival or migration in vitro were detected. These data suggested that immunosuppressant administration could alter hCNS‐SCns properties in vivo. We tested this hypothesis by administering immunosuppressants to constitutively immunodeficient spinal cord injured mice and assessed survival, proliferation, differentiation, and migration of hCNS‐SCns after 14 weeks. In parallel, we administered immunosuppressants to immunocompetent spinal cord injury (SCI) mice and also evaluated hCNS‐SCns engraftment and fate. We identified no effect of immunosuppressants on the overall hCNS‐SCns fate profile in either xenotransplantation model. Despite a lower level of human cell engraftment in immunocompetent SCI mice, functional locomotor recovery was observed in animals receiving hCNS‐SCns transplantation with no evidence of allodynia. These data suggest that local cues in the microenvironment could exert a stronger influence on hCNS‐SCns than circulating levels of immunosuppressants; however, differences between human and rodent metabolism/pharmokinetics and xenograft versus allograft paradigms could be determining factors.

Quantitative assessment of immune cells in the injured spinal cord tissue by flow cytometry: a novel use for a cell purification method.

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck & Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.