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

<p>Abstract</p> <p>Background</p> <p>The 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).</p> <p>Method</p> <p>We investigated the expression of complement mRNAs using rt-PCR and the presence of complement proteins associated with PMNs using immunofluroescence and quantitative flow cytometry.</p> <p>Results</p> <p>Stimulated 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.</p> <p>Conclusion</p> <p>Data 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.</p

Postnatal Proteasome Inhibition Induces Neurodegeneration and Cognitive Deficiencies in Adult Mice: A New Model of Neurodevelopment Syndrome

Defects in the ubiquitin-proteasome system have been related to aging and the development of neurodegenerative disease, although the effects of deficient proteasome activity during early postnatal development are poorly understood. Accordingly, we have assessed how proteasome dysfunction during early postnatal development, induced by administering proteasome inhibitors daily during the first 10 days of life, affects the behaviour of adult mice. We found that this regime of exposure to the proteasome inhibitors MG132 or lactacystin did not produce significant behavioural or morphological changes in the first 15 days of life. However, towards the end of the treatment with proteasome inhibitors, there was a loss of mitochondrial markers and activity, and an increase in DNA oxidation. On reaching adulthood, the memory of mice that were injected with proteasome inhibitors postnatally was impaired in hippocampal and amygdala-dependent tasks, and they suffered motor dysfunction and imbalance. These behavioural deficiencies were correlated with neuronal loss in the hippocampus, amygdala and brainstem, and with diminished adult neurogenesis. Accordingly, impairing proteasome activity at early postnatal ages appears to cause morphological and behavioural alterations in adult mice that resemble those associated with certain neurodegenerative diseases and/or syndromes of mental retardation

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

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

Background: The 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). Method: We investigated the expression of complement mRNAs using rt-PCR and the presence of complement proteins associated with PMNs using immunofluroescence and quantitative flow cytometry. Results: Stimulated 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. Conclusion: Data 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

Characterization of recovery, repair, and inflammatory processes following contusion spinal cord injury in old female rats: is age a limitation?

BackgroundAlthough the incidence of spinal cord injury (SCI) is steadily rising in the elderly human population, few studies have investigated the effect of age in rodent models. Here, we investigated the effect of age in female rats on spontaneous recovery and repair after SCI. Young (3&nbsp;months) and aged (18&nbsp;months) female rats received a moderate contusion SCI at T9. Behavioral recovery was assessed, and immunohistocemical and stereological analyses performed.ResultsAged rats demonstrated greater locomotor deficits compared to young, beginning at 7&nbsp;days post-injury (dpi) and lasting through at least 28 dpi. Unbiased stereological analyses revealed a selective increase in percent lesion area and early (2 dpi) apoptotic cell death caudal to the injury epicenter in aged versus young rats. One potential mechanism for these differences in lesion pathogenesis is the inflammatory response; we therefore assessed humoral and cellular innate immune responses. No differences in either acute or chronic serum complement activity, or acute neutrophil infiltration, were observed between age groups. However, the number of microglia/macrophages present at the injury epicenter was increased by 50% in aged animals versus young.ConclusionsThese data suggest that age affects recovery of locomotor function, lesion pathology, and microglia/macrophage response following SCI

Characterization of recovery, repair, and inflammatory processes following contusion spinal cord injury in old female rats: is age a limitation?

BackgroundAlthough the incidence of spinal cord injury (SCI) is steadily rising in the elderly human population, few studies have investigated the effect of age in rodent models. Here, we investigated the effect of age in female rats on spontaneous recovery and repair after SCI. Young (3 months) and aged (18 months) female rats received a moderate contusion SCI at T9. Behavioral recovery was assessed, and immunohistocemical and stereological analyses performed.ResultsAged rats demonstrated greater locomotor deficits compared to young, beginning at 7 days post-injury (dpi) and lasting through at least 28 dpi. Unbiased stereological analyses revealed a selective increase in percent lesion area and early (2 dpi) apoptotic cell death caudal to the injury epicenter in aged versus young rats. One potential mechanism for these differences in lesion pathogenesis is the inflammatory response; we therefore assessed humoral and cellular innate immune responses. No differences in either acute or chronic serum complement activity, or acute neutrophil infiltration, were observed between age groups. However, the number of microglia/macrophages present at the injury epicenter was increased by 50% in aged animals versus young.ConclusionsThese data suggest that age affects recovery of locomotor function, lesion pathology, and microglia/macrophage response following SCI

Complement Protein C1q and Adiponectin Stimulate Mer Tyrosine Kinase-Dependent Engulfment of Apoptotic Cells through a Shared Pathway

The failure to clear apoptotic cells is linked to defects in development and autoimmunity. Complement component C1q is required for efficient engulfment of apoptotic cells (efferocytosis), and C1q deficiency leads to development of lupus. We recently identified a novel molecular mechanism for C1q-dependent efferocytosis in murine macrophages. C1q elicited the expression of Mer tyrosine kinase; a receptor that regulates efficient efferocytosis and prevention of autoimmunity. To characterize the C1q–dependent signal transduction mechanism, pathway analysis of the transcriptome from C1q–activated macrophages was performed, and it identified the adiponectin signaling pathway as significantly upregulated with C1q. Adiponectin is structurally homologous to C1q and regulates cellular metabolism via downstream activation of AMP-activated protein kinase (AMPK). Macrophage stimulation with C1q resulted in activation of AMPK, and silencing of AMPK expression using siRNA inhibited C1q–dependent efferocytosis. Adiponectin signaling also stimulates activation of nuclear receptors, and inhibition of the nuclear receptor retinoid x receptor abrogated C1q–dependent Mer expression and efferocytosis. Furthermore, adiponectin elicited Mer expression and Mer-dependent efferocytosis in macrophages similar to cells stimulated with C1q. Collectively, our results suggest that C1q and adiponectin share a common signal transduction cascade to promote clearance of apoptotic cells, and identify a novel molecular pathway required for efficient efferocytosis

Characterization of early and terminal complement proteins associated with polymorphonuclear leukocytes and after spinal cord injury-8

Ate contusion injury to the spinal cord (T9). Lower panels are higher magnifications of selected areas taken from upper panels.<p><b>Copyright information:</b></p><p>Taken from "Characterization of early and terminal complement proteins associated with polymorphonuclear leukocytes and after spinal cord injury"</p><p>http://www.jneuroinflammation.com/content/5/1/26</p><p>Journal of Neuroinflammation 2008;5():26-26.</p><p>Published online 25 Jun 2008</p><p>PMCID:PMC2443364.</p><p></p

Characterization of early and terminal complement proteins associated with polymorphonuclear leukocytes and after spinal cord injury-5

Ytometer. Spinal cord tissues were collected from uninjured control or injured animal 30 minutes, 2 hours, 24 hours, 3 days, or 7 days after SCI. The number of PMNs detected in the injured or uninjured spinal cord was visualized by dot plots (A), and represented as the percent of cells in the spinal cord relative to uninjured control (B). All flow cytometric gates were set using control IgG isotype labeled cells. # = significantly (P < 0.05) different from uninjured control. n = 5. Number of experiment replication = 2.<p><b>Copyright information:</b></p><p>Taken from "Characterization of early and terminal complement proteins associated with polymorphonuclear leukocytes and after spinal cord injury"</p><p>http://www.jneuroinflammation.com/content/5/1/26</p><p>Journal of Neuroinflammation 2008;5():26-26.</p><p>Published online 25 Jun 2008</p><p>PMCID:PMC2443364.</p><p></p

Characterization of early and terminal complement proteins associated with polymorphonuclear leukocytes and after spinal cord injury-2

2000 U/ml), IFN-γ (2000 U/ml), or a combination of TNF-α and IFN-γ. Positive control mRNAs for all complement proteins and inhibitors (CD59 and Crry) of complement activation were isolated from rat liver, while negative controls were absent of mRNAs.<p><b>Copyright information:</b></p><p>Taken from "Characterization of early and terminal complement proteins associated with polymorphonuclear leukocytes and after spinal cord injury"</p><p>http://www.jneuroinflammation.com/content/5/1/26</p><p>Journal of Neuroinflammation 2008;5():26-26.</p><p>Published online 25 Jun 2008</p><p>PMCID:PMC2443364.</p><p></p