Manuel Nieto-Díaz

MicroRNA Dysregulation in the Spinal Cord following Traumatic Injury

Spinal cord injury (SCI) triggers a multitude of pathophysiological events that are tightly regulated by the expression levels of specific genes. Recent studies suggest that changes in gene expression following neural injury can result from the dysregulation of microRNAs, short non-coding RNA molecules that repress the translation of target mRNA. To understand the mechanisms underlying gene alterations following SCI, we analyzed the microRNA expression patterns at different time points following rat spinal cord injury. The microarray data reveal the induction of a specific microRNA expression pattern following moderate contusive SCI that is characterized by a marked increase in the number of down-regulated microRNAs, especially at 7 days after injury. MicroRNA downregulation is paralleled by mRNA upregulation, strongly suggesting that microRNAs regulate transcriptional changes following injury. Bioinformatic analyses indicate that changes in microRNA expression affect key processes in SCI physiopathology, including inflammation and apoptosis. MicroRNA expression changes appear to be influenced by an invasion of immune cells at the injury area and, more importantly, by changes in microRNA expression specific to spinal cord cells. Comparisons with previous data suggest that although microRNA expression patterns in the spinal cord are broadly similar among vertebrates, the results of studies assessing SCI are much less congruent and may depend on injury severity. The results of the present study demonstrate that moderate spinal cord injury induces an extended microRNA downregulation paralleled by an increase in mRNA expression that affects key processes in the pathophysiology of this injury.

MicroRNA dysregulation in spinal cord injury: causes, consequences and therapeutics

Trauma to the spinal cord causes permanent disability to more than 180,000 people every year worldwide. The initial mechanical damage triggers a complex set of secondary events involving the neural, vascular, and immune systems that largely determine the functional outcome of the spinal cord injury (SCI). Cellular and biochemical mechanisms responsible for this secondary injury largely depend on activation and inactivation of specific gene programs. Recent studies indicate that microRNAs function as gene expression switches in key processes of the SCI. Microarray data from rodent contusion models reveal that SCI induces changes in the global microRNA expression patterns. Variations in microRNA abundance largely result from alterations in the expression of the cells at the damaged spinal cord. However, microRNA expression levels after SCI are also influenced by the infiltration of immune cells to the injury site and the death and migration of specific neural cells after injury. Evidences on the role of microRNAs in the SCI pathophysiology have come from different sources. Bioinformatic analysis of microarray data has been used to identify specific variations in microRNA expression underlying transcriptional changes in target genes, which are involved in key processes in the SCI. Direct evidences on the role of microRNAs in SCI are scarcer, although recent studies have identified several microRNAs (miR-21, miR-486, miR-20) involved in key mechanisms of the SCI such as cell death or astrogliosis, among others. From a clinical perspective, different evidences make clear that microRNAs can be potent therapeutic tools to manipulate cell state and molecular processes in order to enhance functional recovery. The present article reviews the actual knowledge on how injury affects microRNA expression and the meaning of these changes in the SCI pathophysiology, to finally explore the clinical potential of microRNAs in the SCI.

Inhibitors of Glioma Growth that Reveal the Tumour to the Immune System

Treated glioblastoma patients survive from 6 to 14 months. In the first part of this review, we describe glioma origins, cancer stem cells and the genomic alterations that generate dysregulated cell division, with enhanced proliferation and diverse response to radiation and chemotherapy. We review the pathways that mediate tumour cell proliferation, neo-angiogenesis, tumor cell invasion, as well as necrotic and apoptotic cell death. Then, we examine the ability of gliomas to evade and suppress the host immune system, exhibited at the levels of antigen recognition and immune activation, limiting the effective signaling between glioma and host immune cells. The second part of the review presents current therapies and their drawbacks. This is followed by a summary of the work of our laboratory during the past 20 years, on oligosaccharide and glycosphingolipid inhibitors of astroblast and astrocytoma division. Neurostatins, the O-acetylated forms of gangliosides GD1b and GT1b naturally present in mammalian brain, are cytostatic for normal astroblasts, but cytotoxic for rat C6 glioma cells and human astrocytoma grades III and I V, with ID50 values ranging from 200 to 450 nM. The inhibitors do not affect neurons or fibroblasts up to concentrations of 4 μM or higher. At least four different neurostatin-activated, cell-mediated antitumoral processes, lead to tumor destruction: (i) inhibition of tumor neo-vascularization; (ii) activation of microglia; (iii) activation of natural killer (NK) cells; (iv) activation of cytotoxic lymphocytes (CTL). The enhanced antigenicity of neurostatin-treated glioma cells, could be related to their increased expression of connexin 43. Because neurostatins and their analogues show specific activity and no toxicity for normal cells, a clinical trial would be the logical next step.

Differential Adhesiveness and Neurite-promoting Activity for Neural Cells of Chitosan, Gelatin, and Poly-L-Lysine Films

Chitosan (Ch) and some of its derivatives have been proposed as good biomaterials for tissue engineering, to construct scaffolds promoting tissue regeneration. In this work we made composite films from Ch and mixtures of Ch with gelatin (G) and poly-l-lysine (PLL), and evaluated the growth on these films of PC12 and C6 lines as well as neurons and glial cells derived from cerebral tissue and dorsal root ganglia (DRG). C6 glioma cells proliferated on Ch, G, and Ch + G films, although metabolic activity was decreased by the presence of the G in the mixtures. NGF-differentiated PC12 cells, adhered preferentially on Ch and films containing PLL. Unlike NGF-treated PC12 cells, cortical and hippocampal neurons showed good adhesion to Ch and Ch + G films, where they extended neurites. Astrocytes adhered on Ch, Ch + G, and Ch + PLL mixtures, although viability decreased during the culture time. Olfactory ensheathing cells (OEC) adhered and proliferated to confluency on the wells covered with Ch + G films. Neurites from DRGs exhibited high extension on these films. These results demonstrate that Ch + G films have excellent adhesive properties for both neurons and regeneration-promoting glia (OEC). These films also promoted neurite extension from DRG, making them good candidates for tissue engineering of nerve repair.

Gene expression of axon growth promoting factors in the deer antler.

The annual regeneration cycle of deer (Cervidae, Artiodactyla) antlers represents a unique model of epimorphic regeneration and rapid growth in adult mammals. Regenerating antlers are innervated by trigeminal sensory axons growing through the velvet, the modified form of skin that envelopes the antler, at elongation velocities that reach one centimetre per day in the common deer (Cervus elaphus). Several axon growth promoters like NT-3, NGF or IGF-1 have been described in the antler. To increase the knowledge on the axon growth environment, we have combined different gene-expression techniques to identify and characterize the expression of promoting molecules not previously described in the antler velvet. Cross-species microarray analyses of deer samples on human arrays allowed us to build up a list of 90 extracellular or membrane molecules involved in axon growth that were potentially being expressed in the antler. Fifteen of these genes were analysed using PCR and sequencing techniques to confirm their expression in the velvet and to compare it with the expression in other antler and skin samples. Expression of 8 axon growth promoters was confirmed in the velvet, 5 of them not previously described in the antler. In conclusion, our work shows that antler velvet provides growing axons with a variety of promoters of axon growth, sharing many of them with deers normal and pedicle skin.

Factors promoting neurite outgrowth during deer antler regeneration

Every year male deers completely regenerate their antlers. During this process, antlers are reinnervated by sensory fibers, growing at the highest rate recorded for any adult mammal. Despite its clinical potential, only a few studies have dealt with this fascinating phenomenon. Among the possible factors underlying fast growth of the antlers innervation, the effects of the antlers endocrine and paracrine factors were evaluated, using an in vitro assay for sensory neurite growth. We found that soluble molecules secreted by the velvet, the modified skin that covers the antler, strongly promote neurite outgrowth. Using specific blocking antibodies, we demonstrated that nerve growth factor is partially responsible for these effects, although other unidentified molecules are also involved. On the contrary, neither endocrine serum factors nor antler substrates promoted neurite outgrowth, although antler substrata from deep velvet layers cause neurite outgrowth orientation. Taken together, our results point to the existence in the deep velvet of an environment that promotes oriented axon growth, in agreement with the distribution of the antler innervation.

Life-history traits of the Miocene Hipparion concudense (Spain) inferred from bone histological structure.

Histological analyses of fossil bones have provided clues on the growth patterns and life history traits of several extinct vertebrates that would be unavailable for classical morphological studies. We analyzed the bone histology of Hipparion to infer features of its life history traits and growth pattern. Microscope analysis of thin sections of a large sample of humeri, femora, tibiae and metapodials of Hipparion concudense from the upper Miocene site of Los Valles de Fuentidueña (Segovia, Spain) has shown that the number of growth marks is similar among the different limb bones, suggesting that equivalent skeletochronological inferences for this Hipparion population might be achieved by means of any of the elements studied. Considering their abundance, we conducted a skeletechronological study based on the large sample of third metapodials from Los Valles de Fuentidueña together with another large sample from the Upper Miocene locality of Concud (Teruel, Spain). The data obtained enabled us to distinguish four age groups in both samples and to determine that Hipparion concudense tended to reach skeletal maturity during its third year of life. Integration of bone microstructure and skeletochronological data allowed us to identify ontogenetic changes in bone structure and growth rate and to distinguish three histologic ontogenetic stages corresponding to immature, subadult and adult individuals. Data on secondary osteon density revealed an increase in bone remodeling throughout the ontogenetic stages and a lesser degree thereof in the Concud population, which indicates different biomechanical stresses in the two populations, likely due to environmental differences. Several individuals showed atypical growth patterns in the Concud sample, which may also reflect environmental differences between the two localities. Finally, classification of the specimens’ age within groups enabled us to characterize the age structure of both samples, which is typical of attritional assemblages.

Chitosan, Gelatin and Poly(L-Lysine) Polyelectrolyte-Based Scaffolds and Films for Neural Tissue Engineering

Biomaterial implants are a promising strategy to replace neural tissue that is lost after traumatic nerve damage. Chitosan (Ch) is a suitable material for nerve implantation when it is used at a minimum amount of 2% (w/v). The goal of this study was to determine the best mixture of 2% Ch with gelatin (G) and poly(L-lysine) (PLL) for use in neural tissue engineering. Using different physicochemical approaches we showed that all mixtures formed polyelectrolyte complexes with distinct electrostatic interactions between their compounds. This gave rise to different gel morphologies, among which Ch + G exhibited a significantly smaller pore size, unlike Ch + G + PLL. However, thermal resistance to degradation and the wettability of the Ch-based films were not affected. Additionally, these differences affected glial cells growth in long-term (14 days) cultures performed on Ch-based films. Astrocytes and olfactory ensheathing cells proliferated on G and Ch + G films which induced both flattened and spindle cell morphologies. Meanwhile, cortical and hippocampal neurons were similarly viable in all studied films and significantly lower than those observed in controls. Lastly, neurites from dorsal root ganglia extended the most on Ch + G films. These results show that a Ch + G mixture is a promising candidate for use in neural tissue engineering.

Deer antler innervation and regeneration.

Nervous system injuries are a major cause of impairment in the human society. Up to now, clinical approaches have failed to adequately restore function following nervous system damage. The regenerative cycle of deer antlers may provide basic information on mechanisms underlying nervous system regeneration. The present contribution reviews the actual knowledge on the antler innervation and the factors responsible for its regeneration and fast growth. Growing antlers are profusely innervated by sensory fibers from the trigeminal nerve, which regenerate every year reaching elongation rates up to 2 cm a day. Antler nerves grow through the velvet in close association to blood vessels. This environment is rich in growth promoting molecules capable of inducing and guiding neurite outgrowth of rat sensory neurons in vitro. Conversely, endocrine regulation failed to show effects on neurite outgrowth in vitro, in spite of including hormones of known promoting effects on axon growth. Additional studies are needed to analyze unexplored factors promoting on growth in antlers such as electric potentials or mechanical stretch, as well as on the survival of antler innervating neurons.

Diadenosine tetraphosphate (Ap4A) inhibits ATP-induced excitotoxicity: a neuroprotective strategy for traumatic spinal cord injury treatment.

Reducing cell death during the secondary injury is a major priority in the development of a cure for traumatic spinal cord injury (SCI). One of the earliest processes that follow SCI is the excitotoxicity resulting from the massive release of excitotoxicity mediators, including ATP, which induce an excessive and/or prolonged activation of their receptors and a deregulation of the calcium homeostasis. Diadenosine tetraphosphate (Ap4A) is an endogenous purinergic agonist, present in both extracellular and intracellular fluids, with promising cytoprotective effects in different diseases including neurodegenerative processes. In a search for efficient neuroprotective strategies for SCI, we have tested the capability of Ap4A to reduce the excitotoxic death mediated by the ATP-induced deregulation of calcium homeostasis and its consequences on tissue preservation and functional recovery in a mouse model of moderate contusive SCI. Our analyses with the murine neural cell line Neuro2a demonstrate that treatment with Ap4A reduces ATP-dependent excitotoxic death by both lowering the intracellular calcium response and decreasing the expression of specific purinergic receptors. Follow-up analyses in a mouse model of contusive SCI showed that acute administration of Ap4A following SCI reduces tissue damage and improves motor function recovery. These results suggest that Ap4A cytoprotection results from a decrease of the purinergic tone preventing the effects of a massive release of ATP after SCI, probably together with a direct induction of anti-apoptotic and pro-survival pathways via activation of P2Y2 proposed in previous studies. In conclusion, Ap4A may be a good candidate for an SCI therapy, particularly to reduce excitotoxicity in combination with other modulators and/or inhibitors of the excitotoxic process that are being tested.