Untargeted blood serum proteomics identifies novel proteins related to neurological recovery after human spinal cord injury.

BACKGROUND: The discovery of new prognostic biomarkers following spinal cord injury (SCI) is a rapidly growing field that could help uncover the underlying pathological mechanisms of SCI and aid in the development of new therapies. To date, this search has largely focused on the initial days after the lesion. However, during the subacute stage of SCI (weeks to months after the injury), there remains potential for sensorimotor recovery, and numerous secondary events develop in various organs. Additionally, the confounding effects of early interventions after the injury are less likely to interfere with the results. METHODS: In this study, we conducted an untargeted proteomics analysis to identify biomarkers of recovery in blood serum samples during the subacute phase of SCI patients, comparing those with strong recovery to those with no recovery between 30 and 120 days. We analyzed the fraction of serum that is depleted of the most abundant proteins to unmask proteins that would otherwise go undetected. Linear models were used to identify peptides and proteins related to neurological recovery and we validated changes in some of these proteins using Enzyme-linked Immunosorbent Assay (ELISA). RESULTS: Our findings reveal that differences in subacute recovery after SCI (from 30 to 120 days) are associated with an enrichment in proteins involved in inflammation, coagulation, and lipid metabolism. Technical validation using commercial ELISAs further confirms that high levels of SERPINE1 and ARHGAP35 are associated with strong neurological recovery, while high levels of CD300a and DEFA1 are associated with a lack of recovery. CONCLUSIONS: Our study identifies new candidates for biomarkers of neurological recovery and for novel therapeutic targets after SCI.

Endocannabinoid signaling in oligodendroglia.

In the central nervous system, oligodendrocytes synthesize the myelin, a specialized membrane to wrap axons in a discontinuous way allowing a rapid saltatory nerve impulse conduction. Oligodendrocytes express a number of growth factors and neurotransmitters receptors that allow them to sense the environment and interact with neurons and other glial cells. Depending on the cell cycle stage, oligodendrocytes may respond to these signals by regulating their survival, proliferation, migration, and differentiation. Among these signals are the endocannabinoids, lipidic molecules synthesized from phospholipids in the plasma membrane in response to cell activation. Here, we discuss the evidence showing that oligodendrocytes express a full endocannabinoid signaling machinery involved in physiological oligodendrocyte functions that can be therapeutically exploited to promote remyelination in central nervous system pathologies.

Trajectory of Serum Levels of Glial Fibrillary Acidic Protein Within Four Weeks Post-Injury Is Related to Neurological Recovery During the Transition from Acute to Chronic Spinal Cord Injury.

The use of biomarkers in spinal cord injury (SCI) research has evolved rapidly in recent years whereby most studies focused on the acute post-injury phase. Since SCI is characterized by persisting neurological impairments, the question arises whether blood biomarkers remain altered during the subacute post-injury time. Sample collection in the subacute phase might provide a better insight in the ongoing SCI specific molecular mechanism with fewer confounding factors compared with the acute phase where, amongst other complications, individuals receive a substantial amount of medication. This study aimed to determine if the temporal dynamics of serum biomarkers of neurodegeneration differ between individuals depending on their extent of neurological recovery in the transition phase between acute and chronic SCI. We performed a secondary analysis of biomarkers in patients with SCI (n = 41) who were treated at a level I trauma center in Germany. Patients with cervical or thoracic SCI regardless of injury severity were included. Blood samples were collected in the acute phase (1-4 days post-injury), and after 30 and 120 days post-injury. Serum protein levels of glial fibrillary acidic protein (GFAP) and neurofilament light protein (NfL) were determined for each time-point of sample collection using R-Plex Assays (Meso Scale Discovery). Linear mixed models were used to evaluate the trajectory of GFAP and NfL over time. Fixed effects of time, neurological recovery, and injury severity, along with the recovery-by-time interaction, were included in models with random slopes and intercepts. GFAP levels increase during the first days after SCI and decrease in subacute to chronic stages. Notably, the trajectory of GFAP over time is significantly associated with the extent of neurological recovery during the transition from acute to chronic SCI with a steeper decline in individuals who recovered better. Serum levels of NfL continue to rise significantly until Day 30 followed by a decrease afterwards, independent of neurological recovery. The trajectory of serum GFAP levels qualifies as a prognostic biomarker for neurological recovery, and facilitates monitoring of disease progression in the sub-acute post-injury phase.

Systemic Immune Profile Predicts the Development of Infections in Patients with Spinal Cord Injuries.

Patients with spinal cord injury (SCI) frequently develop infections that may affect quality of life, be life-threatening, and impair their neurological recovery in the acute and subacute injury phases. Therefore, identifying patients with SCI at risk for developing infections in this stage is of utmost importance. We determined the systemic levels of immune cell populations, cytokines, chemokines, and growth factors in 81 patients with traumatic SCI at 4 weeks after injury and compared them with those of 26 age-matched healthy control subjects. Patients who developed infections between 4 and 16 weeks after injury exhibited higher numbers of neutrophils and eosinophils, as well as lower numbers of lymphocytes and eotaxin-1 (CCL11) levels. Accordingly, lasso logistic regression showed that incomplete lesions (American Spinal Injury Association Impairment Scale [AIS] C and D grades), the levels of eotaxin-1, and the number of lymphocytes, basophils, and monocytes are predictive of lower odds for infections. On the other hand, the number of neutrophils and eosinophils as well as, in a lesser extent, the levels of IP-10 (CXCL10), MCP-1 (CCL2), BDNF [brain-derived neurotrophic factor], and vascular endothelial growth factor [VEGF]-A, are predictors of increased susceptibility for developing infections. Overall, our results point to systemic immune disbalance after SCI as predictors of infection in a period when infections may greatly interfere with neurological and functional recovery and suggest new pathways and players to further explore novel therapeutic strategies.

Post-COVID Complications after Pressure Ulcer Surgery in Patients with Spinal Cord Injury Associate with Creatine Kinase Upregulation in Adipose Tissue.

The risk of complications following surgical procedures is significantly increased in patients with SARS-CoV-2 infection. However, the mechanisms underlying these correlations are not fully known. Spinal cord injury (SCI) patients who underwent reconstructive surgery for pressure ulcers (PUs) before and during the COVID-19 pandemic were included in this study. The patient's postoperative progression was registered, and the subcutaneous white adipose tissue (s-WAT) surrounding the ulcers was analyzed by proteomic and immunohistochemical assays to identify the molecular/cellular signatures of impaired recovery. Patients with SCI and a COVID-19-positive diagnosis showed worse recovery and severe postoperative complications, requiring reintervention. Several proteins were upregulated in the adipose tissue of these patients. Among them, CKMT2 and CKM stood out, and CKM increased for up to 60 days after the COVID-19 diagnosis. Moreover, CKMT2 and CKM were largely found in MGCs within the s-WAT of COVID patients. Some of these proteins presented post-translational modifications and were targeted by autoantibodies in the serum of COVID patients. Overall, our results indicate that CKMT2, CKM, and the presence of MGCs in the adipose tissue surrounding PUs in post-COVID patients could be predictive biomarkers of postsurgical complications. These results suggest that the inflammatory response in adipose tissue may underlie the defective repair seen after surgery.

A New Score Based on the International Standards for Neurological Classification of Spinal Cord Injury for Integrative Evaluation of Changes in Sensorimotor Functions.

Sensorimotor function of patients with spinal cord injury (SCI) is commonly assessed according to the International Standards for Neurological Classification of Spinal Cord Injury (ISNCSCI). From the ISNCSCI segmental motor and sensory assessments, upper and lower extremity motor scores (UEMS and LEMS), sum scores of pinprick (PP) and light touch (LT) sensation, the neurological level of injury (NLI) and the classification of lesion severity according to the American Spinal Injury Association Impairment Scale (AIS) grade are derived. Changes of these parameters over time are used widely to evaluate neurological recovery. Evaluating recovery based on a single ISNCSCI scoring or classification variable, however, may misestimate overall recovery. Here, we propose an Integrated Neurological Change Score (INCS) based on the combination of normalized changes between two time points of UEMS, LEMS, and total PP and LT scores. To assess the agreement of INCS with clinical judgment of meaningfulness of neurological changes, changes of ISNCSCI variables between two time points of 88 patients from an independent cohort were rated by 20 clinical experts according to a five-categories Likert Scale. As for individual ISNCSCI variables, neurological change measured by INCS is associated with severity (AIS grade), age, and time since injury, but INCS better reflects clinical judgment about meaningfulness of neurological changes than individual ISNCSCI variables. In addition, INCS is related to changes in functional independence measured by the Spinal Cord Independence Measure (SCIM) in patients with tetraplegia. The INCS may be a useful measure of overall neurological change in clinical studies.

Serum Levels of Glial Fibrillary Acidic Protein and Neurofilament Light Protein Are Related to the Neurological Impairment and Spinal Edema after Traumatic Spinal Cord Injury.

Neurological examination in the acute phase after spinal cord injury (SCI) is often impossible and severely confounded by pharmacological sedation or concomitant injuries. Therefore, diagnostic biomarkers that objectively characterize severity or the presence of SCI are urgently needed to facilitate clinical decision-making. This study aimed to determine if serum markers of neural origin are related to: 1) presence and severity of SCI, and 2) magnetic resonance imaging (MRI) parameters in the very acute post-injury phase. We performed a secondary analysis of serological parameters, as well as MRI findings in patients with acute SCI (n = 38). Blood samples were collected between Days 1-4 post-injury. Serum protein levels of glial fibrillary acidic protein (GFAP), neuron-specific enolase (NSE), and neurofilament light protein (NfL) were determined. A group of 41 age- and sex-matched healthy individuals served as control group. In the group of individuals with SCI, pre-operative sagittal and axial T2-weighted and sagittal T1-weighted MRI scans were available for 21 patients. Serum markers of neural origin are different among individuals who sustained traumatic SCI depending on injury severity, and the extent of the lesion according to MRI in the acute injury phase. Unbiased Recursive Partitioning regression with Conditional Inference Trees (URP-CTREE) produced preliminary cut-off values for NfL (75.217 pg/mL) and GFAP (73.121 pg/mL), allowing a differentiation between individuals with SCI and healthy controls within the first 4 days after SCI. Serum proteins NfL and GFAP qualify as diagnostic biomarkers for the presence and severity of SCI in the acute post-injury phase, where the reliability of clinical exams is limited.

The Structure of the Spinal Cord Ependymal Region in Adult Humans Is a Distinctive Trait among Mammals.

In species that regenerate the injured spinal cord, the ependymal region is a source of new cells and a prominent coordinator of regeneration. In mammals, cells at the ependymal region proliferate in normal conditions and react after injury, but in humans, the central canal is lost in the majority of individuals from early childhood. It is replaced by a structure that does not proliferate after damage and is formed by large accumulations of ependymal cells, strong astrogliosis and perivascular pseudo-rosettes. We inform here of two additional mammals that lose the central canal during their lifetime: the Naked Mole-Rat (NMR, Heterocephalus glaber) and the mutant hyh (hydrocephalus with hop gait) mice. The morphological study of their spinal cords shows that the tissue substituting the central canal is not similar to that found in humans. In both NMR and hyh mice, the central canal is replaced by tissue reminiscent of normal lamina X and may include small groups of ependymal cells in the midline, partially resembling specific domains of the former canal. However, no features of the adult human ependymal remnant are found, suggesting that this structure is a specific human trait. In order to shed some more light on the mechanism of human central canal closure, we provide new data suggesting that canal patency is lost by delamination of the ependymal epithelium, in a process that includes apical polarity loss and the expression of signaling mediators involved in epithelial to mesenchymal transitions.

Cannabinoid Receptor 1 associates to different molecular complexes during GABAergic neuron maturation.

CB1 cannabinoid receptor is widely expressed in the central nervous system of animals from late prenatal development to adulthood. Appropriate activation and signaling of CB1 cannabinoid receptors in cortical interneurons are crucial during perinatal/postnatal ages and adolescence, when long-lasting changes in brain activity may elicit subsequent appearance of disorders in the adult brain. Here we used an optimized immunoprecipitation protocol based on specific antibodies followed by shot-gun proteomics to find CB1 interacting partners in postnatal rat GABAergic cortical neurons in vitro at two different stages of maturation. Besides describing new proteins associated with CB1 like dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex (DLAT), fatty acid synthase (FASN), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ), voltage-dependent anion channel 1 (VDAC1), myosin phosphatase Rho-interacting protein (MPRIP) or usher syndrome type-1C protein-binding protein 1 (USHBP1), we show that the signaling complex of CB1 is different between maturational stages. Interestingly, the CB1 signaling complex is enriched at the more immature stage in mitochondrial associated proteins and metabolic molecular functions, whereas at more mature stage, CB1 complex is increased in maturation and synaptic-associated proteins. We describe also interacting partners specifically immunoprecipitated with either N-terminal or C-terminal CB1 directed antibodies. Our results highlight new players that may be affected by altered cannabinoid signaling at this critical window of postnatal cortical development.

Revisiting CB1 cannabinoid receptor detection and the exploration of its interacting partners.

BACKGROUND: Cannabinoid receptor 1 (CB1) identification by western blot (WB) has generated a great deal of controversial data making the interpretation of the results difficult. Our purpose is to find the most adequate experimental conditions to detect CB1 by WB and immunoprecipitation (IP) as a first step towards the study of CB1 interactome. NEW METHOD: We use CB1 knockout mice tissue as negative controls and describe appropriate sample handling conditions for CB1 detection by WB and IP from brain and cortical neuron cultures. RESULTS: Sample heating above 65 °C greatly impaired CB1 detection by WB, since it favored the formation of high molecular weight aggregates. We also show the convenience of using n-dodecyl-ß-d-maltoside (DDM) as a detergent for the detection of CB1 by WB and, mostly, for IP. COMPARISON WITH EXISTING METHOD(S): We obtain consistent and specific CB1 detection by WB and IP using four different commercial antibodies and KO tissue for an accurate CB1 identification. We clarify the identification of the receptor in complex samples compared with the diverse and unclear results obtained using standard WB methods. CONCLUSIONS: We establish experimental guidelines for the detection of CB1 by WB and the study of CB1 interacting proteins by IP. We propose a new interpretation of CB1 WB and IP data based on the folding and packing state of the protein and the detergent used. The standardization of the most advantageous conditions for coimmunoprecipitation (CoIP) would be a useful tool for the future study of the interactome of CB1.

The incidence and etiology of sperm DNA fragmentation in the ejaculates of males with spinal cord injuries.

STUDY DESIGN: Retrospective descriptive study. OBJECTIVES: To determine the incidence and probable etiology of sperm DNA fragmentation (SDF) in a sample of males with spinal cord injury (SCI). SETTING: Hospital in Toledo, Spain; University-based Genetics laboratory in Madrid, Spain. METHODS: Semen collected by vibro-stimulation from 27 males with various levels of spinal cord injury. Classical semen parameters, SDF, leukocytospermia and pro-oxidant capacity were assessed and compared with a cohort of normozoospermic fertile donors (n = 10). RESULTS: Males with SCI presented with lower semen quality compared with normozoospermic donors with respect to progressive motility (p = 0.0002), SDF (p < 0.00005), pro-oxidant capacity (p = 0.0191) and leukocytospermia (p < 0.00005). Although there was no significant correlation between semen quality and time since the lesion occurred, the period of abstinence appeared to be positively correlated with SDF (r = 0.486; p = 0.041). When the semen parameters of males with SCI were categorized based on those with cervical and thoracic lesions, sperm concentration was higher for those with cervical damage (p = 0.0257). Males with complete lesions (AIS A) had ejaculates that were lower in progressive motility (p = 0.0040) than those with incomplete injuries (AIS B-D). CONCLUSIONS: Ejaculates of males with SCI have excessively elevated SDF when compared with normozoospermic donors, which is likely to be associated with coincident high levels of leucocytospermia and pro-oxidant capacity. We propose that these phenomena are caused by the accumulation and degeneration of spermatozoa in the cauda epididymidis.

Elevated Autoantibodies in Subacute Human Spinal Cord Injury Are Naturally Occurring Antibodies.

Spinal cord injury (SCI) results in long-term neurological and systemic consequences, including antibody-mediated autoimmunity, which has been related to impaired functional recovery. Here we show that autoantibodies that increase at the subacute phase of human SCI, 1 month after lesion, are already present in healthy subjects and directed against non-native proteins rarely present in the normal spinal cord. The increase of these autoantibodies is a fast phenomenon-their levels are already elevated before 5 days after lesion-characteristic of secondary immune responses, further supporting their origin as natural antibodies. By proteomics studies we have identified that the increased autoantibodies are directed against 16 different nervous system and systemic self-antigens related to changes known to occur after SCI, including alterations in neural cell cytoskeleton, metabolism and bone remodeling. Overall, in the context of previous studies, our results offer an explanation to why autoimmunity develops after SCI and identify novel targets involved in SCI pathology that warrant further investigation.

Cells in the adult human spinal cord ependymal region do not proliferate after injury.

In vertebrates that regenerate the injured spinal cord, cells at the ependymal region proliferate and coordinate the formation of bridges between the lesion stumps. In mammals, these cells also proliferate profusely around the central canal after spinal cord injury, although their actual contribution to repair is controversial. In humans, however, the central canal disappears from early childhood in the majority of individuals, being replaced by astrocyte gliosis, ependymocyte clusters, and perivascular pseudo-rosettes. In this human ependymal remnant, cells do not proliferate under normal conditions, but it is not known if they do after a lesion. Here, we studied the human ependymal remnant after traumatic spinal cord injury using samples from 21 individuals with survival times ranging from days to months post-injury. With three different monoclonal antibodies raised against two different proliferation markers (Ki67 and MCM2), we found that the ependymal remnant in adult humans does not proliferate after injury at any time or distance from the lesion. Our results seriously challenge the view of the spinal cord ependymal region as a neurogenic niche in adult humans and suggest that it would not be involved in cell replacement after a lesion. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Laser-Capture Microdissection for the Analysis of Rat and Human Spinal Cord Ependyma by qPCR.

In the last few decades many efforts have been dedicated to decipher the nature and regenerative potential of neurogenic niches and endogenous stem cells after damage of the central nervous system. In the spinal cord, it has been largely focused on the ependymal region, which hosts neural precursors/stem cells (NSC) in rodents but differs between species and ages. In the current chapter, we detail our protocol to study the gene expression profile of this region using fresh frozen blocks of rat and human post-mortem spinal cords. We describe how to prepare and process those tissues, how to identify and dissect the ependymal region using Laser-Capture Microdissection (LCMD), and how to isolate and amplify RNA with different integrity states to finally obtain enough material for performing gene expression assays using Taqman® Low Density Arrays. LCMD technique maintains tissue integrity allowing for subsequent analysis without manipulation steps that may alter molecular properties of cells and the eventual loss of delicate cell types in comparison with other approaches that require previous disaggregation of the tissue and cell manipulation before isolation.

Spicing Up Pharmacology: A Review of Synthetic Cannabinoids From Structure to Adverse Events.

Recreational use of synthetic cannabinoids (SCB), a class of novel psychoactive substances is an increasing public health problem specifically in Western societies, with teenagers, young adults, and the prison population being the most affected. Some of these SCB are analogs of tetrahydrocannabinol, aminoalkylindoles, and other phytocannabinoid analogs have been detected in herbal preparations generically called "Spice." Spice, "K2" or "fake cannabis" is a general term used for variable herbal mixtures of unknown ingredients or chemical composition. SCB are highly potent CB1 cannabinoid receptor agonists falsely marketed and sold as safe and legal drugs. Here, we present an overview of the endocannabinoid system, CB, and SCB chemical structures and activity at CB receptors. Finally, we highlight the psychological effects of SCB, particularly on learning and memory, and adverse clinical effects including on the cardiovascular system, kidneys, and CNS, including psychosis. Taken together, it is clear that many SCB are extremely dangerous and a major public health problem.

Wnts Are Expressed in the Ependymal Region of the Adult Spinal Cord.

The Wnt family of proteins plays key roles during central nervous system development and in several physiological processes during adulthood. Recently, experimental evidence has linked Wnt-related genes to regulation and maintenance of stem cells in the adult neurogenic niches. In the spinal cord, the ependymal cells surrounding the central canal form one of those niches, but little is known about their Wnt expression patterns. Using microdissection followed by TaqMan® low-density arrays, we show here that the ependymal regions of young, mature rats and adult humans express several Wnt-related genes, including ligands, conventional and non-conventional receptors, co-receptors, and soluble inhibitors. We found 13 genes shared between rats and humans, 4 exclusively expressed in rats and 9 expressed only in humans. Also, we observed a reduction with age on spontaneous proliferation of ependymal cells in rats paralleled by a decrease in the expression of Fzd1, Fzd8, and Fzd9. Our results suggest a role for Wnts in the regulation of the adult spinal cord neurogenic niche and provide new data on the specific differences in this region between humans and rodents.

Cannabinoids to treat spinal cord injury.

Spinal cord injury (SCI) is a devastating condition for which there is no standard treatment beyond rehabilitation strategies. In this review, we discuss the current knowledge on the use of cannabinoids to treat this condition. The endocannabinoid system is expressed in the intact spinal cord, and it is dramatically upregulated after lesion. Endogenous activation of this system counteracts secondary damage following SCI, and treatments with endocannabinoids or synthetic cannabinoid receptor agonists promote a better functional outcome in experimental models. The use of cannabinoids in SCI is a new research field and many questions remain open. Here, we discuss caveats and suggest some future directions that may help to understand the role of cannabinoids in SCI and how to take advantage of this system to regain functions after spinal cord damage.

CB1 cannabinoid receptor enrichment in the ependymal region of the adult human spinal cord.

Cannabinoids are involved in the regulation of neural stem cell biology and their receptors are expressed in the neurogenic niches of adult rodents. In the spinal cord of rats and mice, neural stem cells can be found in the ependymal region, surrounding the central canal, but there is evidence that this region is largely different in adult humans: lacks a patent canal and presents perivascular pseudorosettes, typically found in low grade ependymomas. Using Laser Capture Microdissection, Taqman gene expression assays and immunohistochemistry, we have studied the expression of endocannabinoid system components (receptors and enzymes) at the human spinal cord ependymal region. We observe that ependymal region is enriched in CB1 cannabinoid receptor, due to high CB1 expression in GFAP+ astrocytic domains. However, in human spinal cord levels that retain central canal patency we found ependymal cells with high CB1 expression, equivalent to the CB1(HIGH) cell subpopulation described in rodents. Our results support the existence of ependymal CB1(HIGH) cells across species, and may encourage further studies on this subpopulation, although only in cases when central canal is patent. In the adult human ependyma, which usually shows central canal absence, CB1 may play a different role by modulating astrocyte functions.

The ependymal region of the adult human spinal cord differs from other species and shows ependymoma-like features.

Several laboratories have described the existence of undifferentiated precursor cells that may act like stem cells in the ependyma of the rodent spinal cord. However, there are reports showing that this region is occluded and disassembled in humans after the second decade of life, although this has been largely ignored or interpreted as a post-mortem artefact. To gain insight into the patency, actual structure, and molecular properties of the adult human spinal cord ependymal region, we followed three approaches: (i) with MRI, we estimated the central canal patency in 59 control subjects, 99 patients with traumatic spinal cord injury, and 26 patients with non-traumatic spinal cord injuries. We observed that the central canal is absent from the vast majority of individuals beyond the age of 18 years, gender-independently, throughout the entire length of the spinal cord, both in healthy controls and after injury; (ii) with histology and immunohistochemistry, we describe morphological properties of the non-lesioned ependymal region, which showed the presence of perivascular pseudorosettes, a common feature of ependymoma; and (iii) with laser capture microdissection, followed by TaqMan® low density arrays, we studied the gene expression profile of the ependymal region and found that it is mainly enriched in genes compatible with a low grade or quiescent ependymoma (53 genes); this region is enriched only in 14 genes related to neurogenic niches. In summary, we demonstrate here that the central canal is mainly absent in the adult human spinal cord and is replaced by a structure morphologically and molecularly different from that described for rodents and other primates. The presented data suggest that the ependymal region is more likely to be reminiscent of a low-grade ependymoma. Therefore, a direct translation to adult human patients of an eventual therapeutic potential of this region based on animal models should be approached with caution.