Traumatic Human Spinal Cord Injury: Are Single Treatments Enough to Solve the Problem?

Traumatic spinal cord injury (SCI) results in partial or complete motor deficits, such as paraplegia, tetraplegia, and sphincter control, as well as sensory disturbances and autonomic dysregulation such as arterial hypotension, lack of sweating, and alterations in skin lability. All this has a strong psychological impact on the affected person and his/her family, as well as costs to healthcare institutions with an economic burden in the short, medium, and long terms. Despite at least forty years of experimental animal studies and several clinical trials with different therapeutic strategies, effective therapy is not universally accepted. Most of the published works on acute and chronic injury use a single treatment, such as medication, trophic factor, transplant of a cell type, and so on, to block some secondary injury mechanisms or promote some mechanisms of structural/functional restoration. However, despite significant results in experimental models, the outcome is a moderate improvement in muscle strength, sensation, or eventually in sphincter control, which has been considered non-significant in human clinical trials. Here we present a brief compilation of successful individual treatments that have been applied to secondary mechanisms of action. These studies show limited neuroprotective or neurorestorative approaches in animal models and clinical trials. Thus, the few benefits achieved so far represent a rationale to further explore other strategies that seek better structural and functional restoration of the injured spinal cord.

Use of Cells, Supplements, and Peptides as Therapeutic Strategies for Modulating Inflammation after Spinal Cord Injury: An Update.

Spinal cord injury is a traumatic lesion that causes a catastrophic condition in patients, resulting in neuronal deficit and loss of motor and sensory function. That loss is caused by secondary injury events following mechanical damage, which results in cell death. One of the most important events is inflammation, which activates molecules like proinflammatory cytokines (IL-1ß, IFN-γ, and TNF-α) that provoke a toxic environment, inhibiting axonal growth and exacerbating CNS damage. As there is no effective treatment, one of the developed therapies is neuroprotection of the tissue to preserve healthy tissue. Among the strategies that have been developed are the use of cell therapy, the use of peptides, and molecules or supplements that have been shown to favor an anti-inflammatory environment that helps to preserve tissue and cells at the site of injury, thus favoring axonal growth and improved locomotor function. In this review, we will explain some of these strategies used in different animal models of spinal cord injury, their activity as modulators of the immune system, and the benefits they have shown.

Fast and Sustained Axonal Growth by BDNF Released from Chitosan Microspheres.

Brain-derived neurotrophic factor (BDNF) regulates dendritic branching and dendritic spine morphology, as well as synaptic plasticity and long-term potentiation. Consequently, BDNF deficiency has been associated with some neurological disorders such as Alzheimer's, Parkinson's or Huntington's diseases. In contrast, elevated BDNF levels correlate with recovery after traumatic central nervous system (CNS) injuries. The utility of BDNF as a therapeutic agent is limited by its short half-life in a pathological microenvironment and its low efficacy caused by unwanted consumption of non-neuronal cells or inappropriate dosing. Here, we tested the activity of chitosan microsphere-encapsulated BDNF to prevent clearance and prolong the efficacy of this neurotrophin. Neuritic growth activity of BDNF release from chitosan microspheres was observed in the PC12 rat pheochromocytoma cell line, which is dependent on neurotrophins to differentiate via the neurotrophin receptor (NTR). We obtained a rapid and sustained increase in neuritic out-growth of cells treated with BDNF-loaded chitosan microspheres over control cells (p < 0.001). The average of neuritic out-growth velocity was three times higher in the BDNF-loaded chitosan microspheres than in the free BDNF. We conclude that the slow release of BDNF from chitosan microspheres enhances signaling through NTR and promotes axonal growth in neurons, which could constitute an important therapeutic agent in neurodegenerative diseases and CNS lesions.

Role of Aldynoglia Cells in Neuroinflammatory and Neuroimmune Responses after Spinal Cord Injury.

Aldynoglia are growth-promoting cells with a morphology similar to radial glia and share properties and markers with astrocytes and Schwann cells. They are distributed in several locations throughout the adult central nervous system, where the cells of the aldynoglia interact and respond to the signals of the immune cells. After spinal cord injury (SCI), the functions of resident aldynoglia, identified as ependymocytes, tanycytes, and ependymal stem cells (EpSCs) of the spinal cord are crucial for the regeneration of spinal neural tissue. These glial cells facilitate axonal regrowth and remyelination of injured axons. Here, we review the influence of M1 or M2 macrophage/microglia subpopulations on the fate of EpSCs during neuroinflammation and immune responses in the acute, subacute, and chronic phases after SCI.

Characterization of the anti-α-Gal antibody profile in association with Guillain-Barré syndrome, implications for tick-related allergic reactions.

Humans evolved by losing the capacity to synthesize the glycan Galα1-3Galß1-(3)4GlcNAc-R (α-Gal), which resulted in the capacity to develop a protective response mediated by anti-α-Gal IgM/IgG antibodies against pathogens containing this modification on membrane proteins. As an evolutionary trade-off, humans can develop the alpha-Gal syndrome (AGS), a recently diagnosed disease mainly associated with allergic reactions to mammalian meat consumption. The etiology of the AGS is the exposure to tick bites and the IgE antibody response against α-Gal-containing glycoproteins and glycolipids. The objective of this study was to characterize the anti-α-Gal antibody response in association with the immune-mediated peripheral neuropathy, Guillain-Barré syndrome (GBS), and compare it with different factors known to modulate the antibody response to α-Gal such as exposure to tick bites and development of allergic reactions in response to tick bites. The results showed a significant decrease in the IgM/IgG response to α-Gal in GBS patients when compared to healthy individuals. In contrast, the IgM/IgG levels to α-Gal did not change in patients with allergic reactions to tick bites. The IgE response was not affected in GBS patients, but as expected, the IgE levels significantly increased in individuals exposed to tick bites and patients with tick-associated allergies. These results suggest that the immune pathways of anti-α-Gal IgM/IgG and IgE production are independent. Further studies should consider the susceptibility to allergic reactions to tick bites in GBS patients.

Unraveling the Structural Landscape of Chitosan-Based Heparan Sulfate Mimics Binding to Growth Factors: Deciphering Structural Determinants for Optimal Activity.

Chitosan sulfates have demonstrated the ability to mimic heparan sulfate (HS) function. In this context, it is crucial to understand how the specific structural properties of HS domains determine their functionalities and biological activities. In this study, several HS-mimicking chitosans have been prepared to mimic the structure of HS domains that have proved to be functionally significant in cell processes. The results presented herein are in concordance with the hypothesis that sulfated chitosan-growth factor (GF) interactions are controlled by a combination of two effects: the electrostatic interactions and the conformational adaptation of the polysaccharide. Thus, we found that highly charged O-sulfated S-CS and S-DCS polysaccharides with a low degree of contraction interacted more strongly with GFs than N-sulfated N-DCS, with a higher degree of contraction and a low charge. Finally, the evidence gathered suggests that N-DCS would be able to bind to an allosteric zone and is likely to enhance GF signaling activity. This is because the bound protein remains able to bind to its cognate receptor, promoting an effect on cell proliferation as has been shown for PC12 cells. However, S-CS and S-DCS would sequester the protein, decreasing the GF signaling activity by depleting the protein or locally blocking its active site.

A dataset for the analysis of antibody response to glycan alpha-Gal in individuals with immune-mediated disorders.

Humans evolved by losing the capacity to synthesize the glycan Galα1-3Galß1-(3)4GlcNAc-R (α-Gal), which resulted in the development of a protective response mediated by anti-α-Gal IgM/IgG/IgA antibodies against pathogens containing this modification on membrane proteins. As an evolutionary trade-off, humans can develop the alpha-Gal syndrome (AGS), a recently diagnosed disease mediated by anti-α-Gal IgE antibodies and associated with allergic reactions to mammalian meat consumption and tick bites. However, the anti-α-Gal antibody response may be associated with other immune-mediated disorders such as those occurring in patients with COVID-19 and Guillain-Barré syndrome (GBS). Here, we provide a dataset (209 entries) on the IgE/IgM/IgG/IgA anti-α-Gal antibody response in healthy individuals and patients diagnosed with AGS, tick-borne allergies, GBS and COVID-19. The data allows correlative analyses of the anti-α-Gal antibody response with factors such as patient and clinical characteristics, record of tick bites, blood group, age and sex. These analyses could provide insights into the role of anti-α-Gal antibody response in disease symptomatology and possible protective mechanisms.

A holistic approach to unravelling chondroitin sulfation: Correlations between surface charge, structure and binding to growth factors.

Chondroitin sulfate (CS) is a relevant family of polysaccharides that participates in a large variety of biological events that are related to neural processes by regulating various growth factors through the pattern and degree of sulfation of the polysaccharide. However, their own complexity makes their optimization for biomedical applications a difficult undertaking. Thus, a different perspective has to be taken. Herein, we show that the particular sulfate distribution within the disaccharide repeating-unit plays a key role in the binding of growth factors (GFs). In particular, this disposition modulates the surface charge of the helical structure that, interestingly, has a significant influence on the binding capacity of CSs with several GFs. This fact should be carefully considered in the design of new ligands with improved activity as GFs ligands.

Biochemical profiling of rat embryonic stem cells grown on electrospun polyester fibers using synchrotron infrared microspectroscopy.

Therapeutic options for spinal cord injuries are severely limited; current treatments only offer symptomatic relief and rehabilitation focused on educating the individual on how to adapt to their new situation to make best possible use of their remaining function. Thus, new approaches are needed, and interest in the development of effective strategies to promote the repair of neural tracts in the central nervous system inspired us to prepare functional and highly anisotropic polymer scaffolds. In this work, an initial assessment of the behavior of rat neural progenitor cells (NPCs) seeded on poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) fiber scaffolds using synchrotron-based infrared microspectroscopy (SIRMS) is described. Combined with a modified touch imprint cytology sample preparation method, this application of SIRMS enabled the biochemical profiles of NPCs on the coated polymer fibers to be determined. The results showed that changes in the lipid and amide I-II spectral regions are modulated by the type and coating of the substrate used and the culture time. SIRMS studies can provide valuable insight into the early-stage response of NPCs to the morphology and surface chemistry of a biomaterial, and could therefore be a useful tool in the preparation and optimization of cellular scaffolds. Graphical abstract Synchrotron IR microspectroscopy can provide insight into the response of neural progenitor cells to synthetic scaffolds.

Synthesis, physicochemical characterization and biological evaluation of chitosan sulfate as heparan sulfate mimics.

Despite the relevant biological functions of heparan sulfate (HS) glycosaminoglycans, their limited availability and the chemical heterogeneity from natural sources hamper their use for biomedical applications. Chitosan sulfates (ChS) exhibit structural similarity to HSs and may mimic their biological functions. We prepared a variety of ChS with different degree of sulfation to evaluate their ability to mimic HS in protein binding and to promote neural cell division and differentiation. The structure of the products was characterized using various spectroscopic and analytical methods. The study of their interaction with different growth factors showed that ChS bound to the proteins similarly or even better than heparin. In cell cultures, a transition effect on cell number was observed as a function of ChS concentration. Differences in promoting the expression of the differentiation markers were also found depending on the degree of sulfation and modification in the chitosan.

Quantitative proteomics reveals Piccolo as a candidate serological correlate of recovery from Guillain-Barré syndrome.

Guillain-Barré syndrome (GBS) is an autoimmune-mediated peripheral neuropathy of unknown cause. However, about a quarter of GBS patients have suffered a recent bacterial or viral infection, and axonal forms of the disease are especially common in these patients. Proteomics is a good methodological approach for the discovery of disease biomarkers. Until recently, most proteomics studies of GBS and other neurodegenerative diseases have focused on the analysis of the cerebrospinal fluid (CSF). However, serum represents an attractive alternative to CSF because it is easier to sample and has potential for biomarker discovery. The goal of this research was the identification of serum biomarkers associated with recovery from GBS. To address this objective, a quantitative proteomics approach was used to characterize differences in the serum proteome between a GBS patient and her healthy identical twin in order to lessen variations due to differences in genetic background, and with additional serum samples collected from unrelated GBS (N = 3) and Spinal Cord Injury (SCI) (N = 3) patients with similar medications. Proteomics results were then validated by ELISA using sera from additional GBS patients (N = 5) and healthy individuals (N = 3). All GBS and SCI patients were recovering from the acute phase of the disease. The results showed that Piccolo, a protein that is essential in the maintenance of active zone structure, constitutes a potential serological correlate of recovery from GBS. These results provided the first evidence for the Piccolo´s putative role in GBS, suggesting a candidate target for developing a serological marker of disease recovery.

Aldynoglia cells and modulation of RhoGTPase activity as useful tools for spinal cord injury repair.

A combined approach in spinal cord injury (SCI) therapy is the modulation of the cellular and molecular processes involved in glial scarring. Aldaynoglial cells are neural cell precursors with a high capacity to differentiate into neurons, promote axonal growth, wrapping and myelination of resident neurons. These important characteristics of aldaynoglia can be combined with specific inhibition of the RhoGTPase activity in astroglia and microglia that cause reduction of glial proliferation, retraction of glial cell processes and myelin production by oligodendrocytes. Previously we used experimental central nervous system (CNS) injury models, like spinal cord contusion and striatal lacunar infarction and observed that administration of RhoGTPase glycolipid inhibitor or aldaynoglial cells, respectively, produced a significant gain of functional recovery in treated animals. The combined therapy with neuro-regenerative properties strategy is highly desirable to treat SCI for functional potentiation of neurons and oligodendrocytes, resulting in better locomotor recovery. Here we suggest that treatment of spinal lesions with aldaynoglia from neurospheres plus local administration of a RhoGTPase inhibitor could have an additive effect and promote recovery from SCI.

Expression of Early Growth Response Gene-2 and Regulated Cytokines Correlates with Recovery from Guillain-Barré Syndrome.

Guillain-Barré syndrome (GBS) is an immune-mediated peripheral neuropathy. The goal of this research was the identification of biomarkers associated with recovery from GBS. In this study, we compared the transcriptome of PBMCs from a GBS patient and her healthy twin to discover possible correlates of disease progression and recovery. The study was then extended using GBS and spinal cord injury unrelated patients with similar medications and healthy individuals. The early growth response gene-2 (EGR2) was upregulated in GBS patients during disease recovery. The results provided evidence for the implication of EGR2 in GBS and suggested a role for EGR2 in the regulation of IL-17, IL-22, IL-28A, and TNF-ß cytokines in GBS patients. These results identified biomarkers associated with GBS recovery and suggested that EGR2 overexpression has a pivotal role in the downregulation of cytokines implicated in the pathophysiology of this acute neuropathy.

Inhibition of glial proliferation, promotion of axonal growth and myelin production by synthetic glycolipid: A new approach for spinal cord injury treatment.

PURPOSE: After spinal cord injury (SCI) a glial scar is generated in the area affected that forms a barrier for axon growth and myelination, preventing functional recovery. Recently, we have described a synthetic glycolipid (IG20) that inhibited proliferation of human glioma cells. We show now that IG20 inhibited the proliferation of astrocytes and microglial cells, the principal cellular components of the glial scar, and promoting axonal outgrowth and myelin production in vitro. METHODS: Glial cells were inhibited with IG20 (IC50≈10 µM) and studied by RT-PCR, Western Blotting, immunoprecipitation and fluorescence microscopy. Axonal outgrowth in dorsal root ganglia (DRG) and myelin production by oligodendrocytes were analyzed by immunocytochemistry. Adult rats were assayed in spinal cord contusion model and the recovery of treated animals (n = 6) and controls (n = 6) was followed. RESULTS: The IG20 was localized in the cytosol of glial cells, forming a complex with RhoGDIα, a regulator of RhoGTPases. Treatment of astroglial cultures with IG20 increase the expression of BDNF receptor genes (TrkBT1, TrkB Full). IG20 reduced the astroglial marker GFAP, while increasing production of myelin basic protein in oligodendrocytes and promoted axonal outgrowth from DRG neurons. Local injection of IG20, near a spinal cord contusion, promoted the recovery of lesioned animals analyzed by BBB test (P <  0.05). CONCLUSIONS: We propose that inhibition of astrocytes and microglia by IG20 could be diminished the glial scar formation, inducing the re-growth and myelination of axons, these elements constitute a new approach for SCI therapy.

Mass Spectrometry in Pharmacokinetic Studies of a Synthetic Compound for Spinal Cord Injury Treatment.

The studies of drugs that could constitute a palliative to spinal cord injury (SCI) are a continuous and increasing demand in biomedicine field from developed societies. Recently we described the chemical synthesis and antiglioma activity of synthetic glycosides. A synthetic sulfated glycolipid (here IG20) has shown chemical stability, solubility in polar solvents, and high inhibitory capacity over glioma growth. We have used mass spectrometry (MS) to monitor IG20 (m/z = 550.3) in cells and tissues of the central nervous system (CNS) that are involved in SCI recovery. IG20 was detected by MS in serum and homogenates from CNS tissue of rats, though in the latter a previous deproteinization step was required. The pharmacokinetic parameters of serum clearance at 24 h and half-life at 4 h were determined for synthetic glycoside in the adult rat using MS. A local administration of the drug near of spinal lesion site is proposed.

NFAT transcription factors regulate survival, proliferation, migration, and differentiation of neural precursor cells.

The study of factors that regulate the survival, proliferation, and differentiation of neural precursor cells (NPCs) is essential to understand neural development as well as brain regeneration. The Nuclear Factor of Activated T Cells (NFAT) is a family of transcription factors that can affect these processes besides playing key roles during development, such as stimulating axonal growth in neurons, maturation of immune system cells, heart valve formation, and differentiation of skeletal muscle and bone. Interestingly, NFAT signaling can also promote cell differentiation in adults, participating in tissue regeneration. The goal of the present study is to evaluate the expression of NFAT isoforms in NPCs, and to investigate its possible role in NPC survival, proliferation, migration, and differentiation. Our findings indicate that NFAT proteins are active not only in neurogenic brain regions such as hippocampus and subventricular zone (SVZ), but also in cultured NPCs. The inhibition of NFAT activation with the peptide VIVIT reduced neurosphere size and cell density in NPC cultures by decreasing proliferation and increasing cell death. VIVIT also decreased NPC migration and differentiation of astrocytes and neurons from NPCs. In addition, we identified NFATc3 as a predominant NFAT isoform in NPC cultures, finding that a constitutively-active form of NFATc3 expressed by adenoviral infection reduces NPC proliferation, stimulates migration, and is a potent inducer of NPC differentiation into astrocytes and neurons. In summary, our work uncovers active roles for NFAT signaling in NPC survival, proliferation and differentiation, and highlights its therapeutic potential for tissue regeneration.

Synthetic glycolipids for glioma growth inhibition developed from neurostatin and NF115 compound.

Neurostatin, a natural glycosphingolipid, and NF115, a synthetic glycolipid, are inhibitors of glioma growth. While neurostatin shows high inhibitory activity on gliomas its abundance is low in mammalian brain. On the contrary NF115 exhibits less inhibitory activity on gliomas, but could be prepared by chemical synthesis. In this study we describe synthetic compounds, structurally related to NF115, capable of inhibiting glioma growth at low micromolar range. We used DNA microarray technology to compare the genes inhibited in U373-MG human glioma cells after treatment with the natural or synthetic inhibitor. New synthetic compounds were developed to interact with the product of Rho GDP dissociation inhibitor alpha gene, which was repressed in both treatments. Compounds that were inhibitors of glioma cell growth in assays for [3H]-thymidine incorporation were then injected in C6 tumor bearing rats and the tumor size in each animal group were measured. The GC-17, GC-4 and IG-5 are new compounds derived from NF115 and showed high antiproliferative activity on tumor cell lines. The GC-17 compound inhibited U373-MG glioblastoma cells (3.2 µM), the effects was fifty times more potent than NF115, and caused a significant reduction of tumor volume (P<0.05) when tested in Wistar rats allotransplanted with C6 glioma cells.

Neural differentiation of transplanted neural stem cells in a rat model of striatal lacunar infarction: light and electron microscopic observations.

The increased risk and prevalence of lacunar stroke and Parkinson's disease (PD) makes the search for better experimental models an important requirement for translational research. In this study we assess ischemic damage of the nigrostriatal pathway in a model of lacunar stroke evoked by damaging the perforating arteries in the territory of the substantia nigra (SN) of the rat after stereotaxic administration of endothelin-1 (ET-1), a potent vasoconstrictor peptide. We hypothesized that transplantation of neural stem cells (NSCs) with the capacity of differentiating into diverse cell types such as neurons and glia, but with limited proliferation potential, would constitute an alternative and/or adjuvant therapy for lacunar stroke. These cells showed neuritogenic activity in vitro and a high potential for neural differentiation. Light and electron microscopy immunocytochemistry was used to characterize GFP-positive neurons derived from the transplants. 48 h after ET-1 injection, we characterized an area of selective degeneration of dopaminergic neurons within the nigrostriatal pathway characterized with tissue necrosis and glial scar formation, with subsequent behavioral signs of Parkinsonism. Light microscopy showed that grafted cells within the striatal infarction zone differentiated with a high yield into mature glial cells (GFAP-positive) and neuron types present in the normal striatum. Electron microscopy revealed that NSCs-derived neurons integrated into the host circuitry establishing synaptic contacts, mostly of the asymmetric type. Astrocytes were closely associated with normal small-sized blood vessels in the area of infarct, suggesting a possible role in the regulation of the blood brain barrier and angiogenesis. Our results encourage the use of NSCs as a cell-replacement therapy for the treatment of human vascular Parkinsonism.

Lipid and ganglioside alterations in tumor cells treated with antimitotic oleyl glycoside.

Oleyl 2-acetamido-2-deoxy-α-D-glucopyranoside (1) was previously shown to exhibit antimitotic activity on glioma (C6) and melanoma (A375) cell lines. Preliminary studies about its mechanism of action using (1)H MAS NMR suggested that 1 may be altering the metabolism of lipids. We have now studied the effect of 1 on the fatty acid, sphingolipid and ganglioside content in a line of carcinomic human alveolar epithelial cells (A549) using UPLC-MS. Oleic acid and NB-DNJ were used as positive controls for inhibition of fatty acid and ganglioside synthesis, respectively. Compound 1 (10 µM) was more efficient than oleic acid in reducing fatty acid levels of A549 cells, producing a decrease in the range of 40-15%, depending on the acyl chain length and the number of insaturations. In addition, glycoside 1 caused a reduction on ganglioside content of A549 tumor cell line and accumulation of lactosylceramide, the common metabolic precursor for ganglioside biosynthesis. Alteration of ganglioside metabolism was also observed with two galactosylated derivatives of 1, which caused a more pronounced increase in lactosylceramide levels. Compound 1 at higher concentrations (above 30 µM) produced drastic alterations in glycosphingolipid metabolism, leading to cell metabolic profiles very different from those obtained at 10 µM. These biochemical changes were ascribed to activation of endoplasmic reticulum stress pathways.

Detection of metabolite changes in C6 glioma cells cultured with antimitotic oleyl glycoside by 1H MAS NMR.

The synthetic glycoside, oleyl N-acetyl-alpha-D-glucosaminide (1), was previously shown to exhibit antimitotic activity on rat (C6) and human (U-373) glioma lines. To obtain information about its mechanism of action, metabolite changes in C6 glioma cells were analyzed after treatment with 1 using high-resolution magic angle spinning (1)H NMR. Compound 1 caused either a decrease or an increase in the intensity of the signal assigned to coenzyme A (CoA) metabolites depending on the concentration used. The data obtained from the (1)H NMR spectra of cells cultured with 1, combined with those obtained after treatment with oleic acid (an inhibitor of acetyl-CoA carboxylase) and phenyl butyrate (a known antineoplastic agent), suggest that 1 may be altering the metabolism of fatty acids and induce apoptosis of C6 glioma cells. These results point to NMR spectroscopy as an efficient technique for monitoring the response of the cells to therapeutic agents.