Central canal ependymal cells proliferate extensively in response to traumatic spinal cord injury but not demyelinating lesions.

The adult mammalian spinal cord has limited regenerative capacity in settings such as spinal cord injury (SCI) and multiple sclerosis (MS). Recent studies have revealed that ependymal cells lining the central canal possess latent neural stem cell potential, undergoing proliferation and multi-lineage differentiation following experimental SCI. To determine whether reactive ependymal cells are a realistic endogenous cell population to target in order to promote spinal cord repair, we assessed the spatiotemporal dynamics of ependymal cell proliferation for up to 35 days in three models of spinal pathologies: contusion SCI using the Infinite Horizon impactor, focal demyelination by intraspinal injection of lysophosphatidylcholine (LPC), and autoimmune-mediated multi-focal demyelination using the active experimental autoimmune encephalomyelitis (EAE) model of MS. Contusion SCI at the T9-10 thoracic level stimulated a robust, long-lasting and long-distance wave of ependymal proliferation that peaked at 3 days in the lesion segment, 14 days in the rostral segment, and was still detectable at the cervical level, where it peaked at 21 days. This proliferative wave was suppressed distal to the contusion. Unlike SCI, neither chemical- nor autoimmune-mediated demyelination triggered ependymal cell proliferation at any time point, despite the occurrence of demyelination (LPC and EAE), remyelination (LPC) and significant locomotor defects (EAE). Thus, traumatic SCI induces widespread and enduring activation of reactive ependymal cells, identifying them as a robust cell population to target for therapeutic manipulation after contusion; conversely, neither demyelination, remyelination nor autoimmunity appears sufficient to trigger proliferation of quiescent ependymal cells in models of MS-like demyelinating diseases.

P2X4 receptors influence inflammasome activation after spinal cord injury.

P2X(4) and P2X(7) are the predominant purinergic P2X receptor subtypes expressed on immune and neural cells. These receptor subtypes traffic between intracellular compartments and the plasma membrane and form protein interactions with each other to regulate ATP-dependent signaling. Our recent studies have shown that P2X(7) receptors in neurons and astrocytes activate NLRP1 inflammasomes, but whether P2X(4) receptors regulate inflammasome signaling is essentially unknown. Here, we demonstrate that P2X(4) receptors are expressed in neurons of the spinal cord. We provide direct evidence that spinal cord injury (SCI) induces an innate inflammatory response that leads to increased caspase-l cleavage and production of IL-1ß but not IL-18. Consistent with these findings, P2X(4) knock-out mice showed impaired inflammasome signaling in the cord, resulting in decreased levels of IL-1ß and reduced infiltration of neutrophils and monocyte-derived M1 macrophages, resulting in significant tissue sparing and improvement in functional outcomes. These results indicate that P2X(4) receptors influence inflammasome signaling involving caspase-1 activation and IL-1ß processing in neurons after SCI. P2X(4) might thus represent a potential therapeutic target to limit inflammatory responses associated with SCI and neurodegenerative disorders.

Astrocytes initiate inflammation in the injured mouse spinal cord by promoting the entry of neutrophils and inflammatory monocytes in an IL-1 receptor/MyD88-dependent fashion.

CNS injury stimulates the expression of several proinflammatory cytokines and chemokines, some of which including MCP-1 (also known as CCL2), KC (CXCL1), and MIP-2 (CXCL2) act to recruit Gr-1(+) leukocytes at lesion sites. While earlier studies have reported that neutrophils and monocytes/macrophages contribute to secondary tissue loss after spinal cord injury (SCI), recent work has shown that depletion of Gr-1(+) leukocytes compromised tissue healing and worsened functional recovery. Here, we demonstrate that astrocytes distributed throughout the spinal cord initially contribute to early neuroinflammation by rapidly synthesizing MCP-1, KC, and MIP-2, from 3 up to 12h post-SCI. Chemokine expression by astrocytes was followed by the infiltration of blood-derived immune cells, such as type I "inflammatory" monocytes and neutrophils, into the lesion site and nearby damaged areas. Interestingly, astrocytes from mice deficient in MyD88 signaling produced significantly less MCP-1 and MIP-2 and were unable to synthesize KC. Analysis of the contribution of MyD88-dependent receptors revealed that the astrocytic expression of MCP-1, KC, and MIP-2 was mediated by the IL-1 receptor (IL-1R1), and not by TLR2 or TLR4. Flow cytometry analysis of cells recovered from the spinal cord of MyD88- and IL-1R1-knockout mice confirmed the presence of significantly fewer type I "inflammatory" monocytes and the almost complete absence of neutrophils at 12h and 4days post-SCI. Together, these results indicate that MyD88/IL-1R1 signals regulate the entry of neutrophils and, to a lesser extent, type I "inflammatory" monocytes at sites of SCI.

Cutaneous lycopene and beta-carotene levels measured by resonance Raman spectroscopy: high reliability and sensitivity to oral lactolycopene deprivation and supplementation.

Carotenoids, naturally occurring lipophilic micronutrients, possess an antioxidant activity associated with protection from damage induced by free radicals. The present study investigated an innovative non-invasive method to measure cutaneous levels of lycopene and beta-carotene and to monitor the distribution of orally administered lactolycopene in human skin and plasma. A double-blind placebo-controlled randomized study was performed in 25 volunteers, who were under a lycopene-deprived diet (4 weeks prior to study until end of the study) and orally received either lactolycopene or placebo for 12 weeks. Skin and plasma levels of lycopene and beta-carotene were monitored monthly using Raman spectroscopy and HPLC, respectively. Cutaneous levels of lycopene and beta-carotene monitored by resonance Raman spectroscopy showed high reliability. Irrespective of the investigated area, cutaneous levels were sensitive to lycopene deprivation and to oral supplementation; the forehead showed the closest correlation to lycopene variation in plasma. Plasma and skin levels of lycopene were both sensitive to oral intake of lactolycopene and, interestingly, also skin levels of beta-carotene. Thus, oral supplementation with lycopene led to an enrichment of beta-carotene in human skin, possibly due to the fact that carotenoids act in the skin as protection chains, with a natural protection against free radicals.

Endogenous signals initiating inflammation in the injured nervous system.

Glial cells are known to respond to a variety of neural injuries and play an important role in tissue damage and repair in the injured nervous system. This glial response, which is initially characterized by the expression of proinflammatory cytokines and chemokines and the attraction of microglial cells toward sites of injury, literally occurs within seconds to minutes of the injury. This suggests that signals that are endogenous to the nervous system are responsible for initiating neuroinflammation. In this review, we summarize the most recent advances made in the identification of these endogenous signals and describe the receptors and signaling pathways by which these ligands stimulate the production of cytokines and chemokines. Among these endogenous damage signals are ligands for toll-like receptors, including several heat shock proteins and extracellular matrix components, as well as self-derived RNA and DNA and associated proteins. Growing evidence also suggests that nucleotides released upon injury and acting through P2 receptors, such as ATP and UTP or their analogues, could serve as endogenous signals for the rapid response of glial cells.

Toll-like receptor signaling is critical for Wallerian degeneration and functional recovery after peripheral nerve injury.

Toll-like receptors (TLRs) bind specific components conserved among microorganisms as well as endogenous ligands produced by necrotic cells, injured axons, and the extracellular matrix. Here, we investigated whether TLRs are involved in regulating the immune response, Wallerian degeneration (WD), and nerve regeneration after sciatic nerve lesion. Early expression of interleukin-1beta and monocyte chemoattractant protein-1 was compromised in the sciatic nerve distal stump of mice deficient in TLR signaling. In addition, significantly fewer macrophages were recruited and/or activated in the sciatic nerve distal stump of TLR2-, TLR4-, and MyD88-deficient mice compared with wild-type littermates, whereas WD, axonal regeneration, and recovery of locomotor function were impaired. In contrast, animals that received a single microinjection of TLR2 and TLR4 ligands at the site of sciatic nerve lesion had faster clearance of the degenerating myelin and recovered earlier than saline-injected control rats. Finally, rats that had altered innate immune response through dexamethasone treatment exhibited three times more myelin debris in their sciatic nerve distal stump and a significant delay in recovery of locomotor function. Our results provide strong evidence that TLR signaling plays a critical role in orchestrating the innate immune response leading to efficient and rapid clearance of inhibitory myelin debris and nerve regeneration.

Proinflammatory cytokine synthesis in the injured mouse spinal cord: multiphasic expression pattern and identification of the cell types involved.

We have studied the spatial and temporal distribution of six proinflammatory cytokines and identified their cellular source in a clinically relevant model of spinal cord injury (SCI). Our findings show that interleukin-1beta (IL-1beta) and tumor necrosis factor (TNF) are rapidly (<5 and 15 minutes, respectively) and transiently expressed in mice following contusion. At 30-45 minutes post SCI, IL-1beta and TNF-positive cells could already be seen over the entire spinal cord segment analyzed. Multilabeling analyses revealed that microglia and astrocytes were the two major sources of IL-1beta and TNF at these times, suggesting a role for these cytokines in gliosis. Results obtained from SCI mice previously transplanted with green fluorescent protein (GFP)-expressing hematopoietic stem cells confirmed that neural cells were responsible for the production of IL-1beta and TNF for time points preceding 3 hours. From 3 hours up to 24 hours, IL-1beta, TNF, IL-6, and leukemia inhibitory factor (LIF) were strongly upregulated within and immediately around the contused area. Colocalization studies revealed that all populations of central nervous system resident cells, including neurons, synthesized cytokines between 3 and 24 hours post SCI. However, work done with SCI-GFP chimeric mice revealed that at least some infiltrating leukocytes were responsible for cytokine production from 12 hours on. By 2 days post-SCI, mRNA signal for all the above cytokines had nearly disappeared. Notably, we also observed another wave of expression for IL-1beta and TNF at 14 days. Overall, these results indicate that following SCI, all classes of neural cells initially contribute to the organization of inflammation, whereas recruited immune cells mostly contribute to its maintenance at later time points.

A novel method for multiple labeling combining in situ hybridization with immunofluorescence.

In situ hybridization (ISH) is a particularly useful method to investigate de novo mRNA expression in tissue sections. High specificity and sensitivity of this technique combined with the great preservation of tissue and cellular morphology conferred by fixatives such as 4% paraformaldehyde, pH 9.5, make ISH a tool of choice for detecting genes of interest in individual cells in the central nervous system (CNS). Here we describe a novel method that combines radioactive ISH with immunofluorescence on the same tissue section to identify cell populations expressing selected mRNA transcripts. This novel method has several major advantages over previously described double-labeling light microscopic methods combining enzymatic immunohistochemistry and ISH including (1) complete protection against loss of hybridization signal that normally occurs during the immunoenzymatic reaction, (2) improved immunolabeling sensitivity due to the proteinase K digestion step during ISH, (3) detection of several proteins specific for different cell populations on the same tissue section, and (4) counterstaining of tissue sections without affecting visualization of immunolabeling. This new method will be particularly useful for investigators looking to identify cell populations producing mRNAs expressed in low abundance such as cytokines, chemokines, and growth factors in the intact and/or injured mammalian CNS.

Prevention of pneumococcal disease in mice immunized with conserved surface-accessible proteins.

The development of a vaccine against Streptococcus pneumoniae has been complicated by the existence of at least 90 antigenically distinct capsular serotypes. Common protein-based vaccines could represent the best strategy to prevent pneumococcal infections, regardless of serotype. In the present study, the immunoscreening of an S. pneumoniae genomic library allowed the identification of a novel immune protein target, BVH-3. We demonstrate that immunization of mice with BVH-3 elicits protective immunity against experimental sepsis and pneumonia. Sequence analysis revealed that the bvh-3 gene is highly conserved within the species. Since the BVH-3 protein shows homology at its amino-terminal end with other pneumococcal proteins, it was of interest to determine if protection was due to the homologous or to the protein-specific regions. Immunoprotection studies using recombinant BVH-3 and BVH-3-related protein fragments as antigens allowed the localization of surface-exposed and protective epitopes at the protein-specific carboxyl termini, thus establishing that BVH-3 is distinct from other previously reported protective protein antigens. Immunization with a chimeric protein comprising the carboxyl-terminal regions of BVH-3 and of a BVH-3-related protein improved the protection by targeting two surface pneumococcal components. Thus, BVH-3 and the chimeric protein hold strong promise as vaccine components to control pneumococcal disease.