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MicroRNAs (miRNAs) are endogenous, short RNA oligonucleotides that regulate the expression of hundreds of proteins to control cells’ function in physiological and pathological conditions. miRNA therapeutics are highly specific, reducing the toxicity associated with off-target effects, and require low doses to achieve therapeutic effects. Despite their potential, applying miRNA-based therapies is limited by difficulties in delivery due to their poor stability, fast clearance, poor efficiency, and off-target effects. To overcome these challenges, polymeric vehicles have attracted a lot of attention due to their ease of production with low costs, large payload, safety profiles, and minimal induction of the immune response. Poly(N-ethyl pyrrolidine methacrylamide) (EPA) copolymers have shown optimal DNA transfection efficiencies in fibroblasts. The present study aims to evaluate the potential of EPA polymers as miRNA carriers for neural cell lines and primary neuron cultures when they are copolymerized with different compounds. To achieve this aim, we synthesized and characterized different copolymers and evaluated their miRNA condensation ability, size, charge, cytotoxicity, cell binding and internalization ability, and endosomal escape capacity. Finally, we evaluated their miRNA transfection capability and efficacy in Neuro-2a cells and rat primary hippocampal neurons. The results indicate that EPA and its copolymers, incorporating β-cyclodextrins with or without polyethylene glycol acrylate derivatives, can be promising vehicles for miRNA administration to neural cells when all experiments on Neuro-2a cells and primary hippocampal neurons are considered together.

The central nervous system microRNA miR-138-5p has attracted much attention in cancer research because it inhibits pro-apoptotic genes including CASP3. We hypothesize that miR-138-5p downregulation after SCI leads to overexpression of pro-apoptotic genes, sensitizing neural cells to noxious stimuli. This study aimed to identify miR-138-5p targets among pro-apoptotic genes overexpressed following SCI and to confirm that miR-138-5p modulates cell death in neural cells. Gene expression and histological analyses revealed that the drop in miR-138-5p expression after SCI is due to the massive loss of neurons and oligodendrocytes and its downregulation in neurons. Computational analyses identified 176 potential targets of miR-138-5p becoming dysregulated after SCI, including apoptotic proteins CASP-3 and CASP-7, and BAK. Reporter, RT-qPCR, and immunoblot assays in neural cell cultures confirmed that miR-138-5p targets their 3′UTRs, reduces their expression and the enzymatic activity of CASP-3 and CASP-7, and protects cells from apoptotic stimuli. Subsequent RT-qPCR and histological analyses in a rat model of SCI revealed that miR138-5p downregulation correlates with the overexpression of its pro-apoptotic targets. Our results suggest that the downregulation of miR-138-5p after SCI may have deleterious effects on neural cells, particularly on spinal neurons.

Alginate (ALG) is a lineal hydrophilic polysaccharide present in brown algae cell walls, which turns into a gel state when hydrated. Gelation readily produces a series of three dimensional (3D) architectures like fibers, capillaries, and microspheres, used as biosensors and bio‐actuators in a plethora of biomedical applications like drug delivery and wound healing. Hydrogels have made a great impact on regenerative medicine and tissue engineering because they are able to mimic the mechanical properties of natural tissues due to their high water content. Recent advances in neurosciences have led to promising strategies for repairing and/or regenerating the damaged nervous system. Spinal cord injury (SCI) is particularly challenging, owing to its devastating medical, human, and social consequences. Although effective therapies to repair the damaged spinal cord (SC) are still lacking, multiple pharmacological, genetic, and cell‐based therapies are currently under study. In this framework, ALG hydrogels constitute a source of potential tools for the development of implants capable of promoting axonal growth and/or delivering cells or drugs at specific damaged sites, which may result in therapeutic strategies for SCI. In this mini‐review, the current state of the art of ALG applications in neural tissues for repairing the damaged spinal cord is discussed.

Las lesiones traumáticas de la médula espinal son causa de discapacidad en más de cien mil personasen todo el mundo cada año. La lesión medular (LME) tiene una compleja fisiopatología que incluyemúltiples eventos, respuestas y procesos interrelacionados que afectan a los sistemas nervioso,vascular e inmune y que cambian a lo largo del tiempo. Al inicio del curso temporal de la LME, eltrauma inicial desencadena una serie de alteraciones vasculares, cambios bioquímicos y respuestascelulares secundarias que exacerban el daño inicial. El pronóstico de la LME depende estrechamentede las características de esta fase secundaria, incluyendo el incremento de la apoptosis, la infiltraciónde células inmunes y la respuesta inflamatoria, las alteraciones en la permeabilidad de la barrerahemato-medular, los procesos de degeneración Walleriana y desmielinización, la reactividad glial yla cicatriz glial, así como las respuestas neuroregenerativas. La caracterización de las vías deseñalización y los mecanismos subyacentes a todos estos procesos implicados en el daño secundarioresulta fundamental para identificar nuevas dianas sobre las cuales desarrollar estrategiasterapéuticas efectivas enfocadas a reducir los déficits funcionales inducidos por la lesión LME.Recientes evidencias sugieren que los esfingolípidos juegan un papel activo tras el daño en el SistemaNervioso Central (SNC) así como en diferentes patologías neurodegenerativas. Los esfingolípidos sonuna clase de lípidos basados en un esqueleto de esfingosina a partir del cual se generan una granvariedad de familias de esfingolípidos mediante fosforilación (v.g.: esfingosina-1-fosfato), acilación(v.g.: ceramidas), así como por adición de fosfocolina (esfingomielinas) o de residuos de azúcar (v.g.:cerebrósidos y gangliósidos). Aunque conocidos principalmente por papel como lípidos estructuralesen la biología de membranas, algunos esfingolípidos actúan como moléculas de señalización enprocesos de proliferación, supervivencia, ap, Trauma to the spinal cord causes permanent and incurable disabilities in more than a hundredthousand people every year worldwide. The pathophysiology of the spinal cord injury (SCI) is acomplex intermingled set of events, responses, and processes that affect the nervous, vascular andimmune systems during the days and months that follow the damage. The initial trauma triggers aseries of secondary vascular alterations, biochemical disturbances and cellular responses thatexacerbate the initial damage. The functional outcome from SCI largely depends on prominentfeatures of this secondary phase, including increased apoptosis, immune cell infiltration andinflammatory responses, alterations in blood-spinal cord barrier permeability, Walleriandegeneration and demyelination, glial reactivity and glial scar, as well as neuro-regenerativeresponses. Characterizing the pathways and the mechanisms underlying the secondary damage isbasic to identify targets on which to develop effective therapeutic strategies aimed to restrict thefunctional deficits induced by the SCI.Recent evidences suggest that sphingolipids play an active role following injury to the Central NervousSystem (CNS) as well as in different neurodegenerative diseases. Sphingolipids are a class of bioactivelipids characterized by a sphingosine backbone upon which a large and complex network of familiesis built up by phosphorylation (vg. Sphingosine-1-phosphate), acylation (vg. ceramides), addition ofphosphocoline headgroup (sphingomyelins) or sugar residues (cerebrosides and gangliosides, i.e.glycosphingolipids). Best known for their crucial roles in membrane biology, several sphingolipidmetabolites function as signalling molecules in proliferation, survival, apoptosis, motility, anddifferentiation pathways. The few existing studies indicate that sphingolipid metabolites can playactive roles in different processes of the SCI. Studies in SCI models have established that sphingosine-1-phosphate (S1P), a sphingolipid me

Las lesiones traumáticas de la médula espinal son causa de discapacidad en más de cien mil personasen todo el mundo cada año. La lesión medular (LME) tiene una compleja fisiopatología que incluyemúltiples eventos, respuestas y procesos interrelacionados que afectan a los sistemas nervioso,vascular e inmune y que cambian a lo largo del tiempo. Al inicio del curso temporal de la LME, eltrauma inicial desencadena una serie de alteraciones vasculares, cambios bioquímicos y respuestascelulares secundarias que exacerban el daño inicial. El pronóstico de la LME depende estrechamentede las características de esta fase secundaria, incluyendo el incremento de la apoptosis, la infiltraciónde células inmunes y la respuesta inflamatoria, las alteraciones en la permeabilidad de la barrerahemato-medular, los procesos de degeneración Walleriana y desmielinización, la reactividad glial yla cicatriz glial, así como las respuestas neuroregenerativas. La caracterización de las vías deseñalización y los mecanismos subyacentes a todos estos procesos implicados en el daño secundarioresulta fundamental para identificar nuevas dianas sobre las cuales desarrollar estrategiasterapéuticas efectivas enfocadas a reducir los déficits funcionales inducidos por la lesión LME.Recientes evidencias sugieren que los esfingolípidos juegan un papel activo tras el daño en el SistemaNervioso Central (SNC) así como en diferentes patologías neurodegenerativas. Los esfingolípidos sonuna clase de lípidos basados en un esqueleto de esfingosina a partir del cual se generan una granvariedad de familias de esfingolípidos mediante fosforilación (v.g.: esfingosina-1-fosfato), acilación(v.g.: ceramidas), así como por adición de fosfocolina (esfingomielinas) o de residuos de azúcar (v.g.:cerebrósidos y gangliósidos). Aunque conocidos principalmente por papel como lípidos estructuralesen la biología de membranas, algunos esfingolípidos actúan como moléculas de señalización enprocesos de proliferación, supervivencia, ap

Adult craniofacial morphology results from complex processes that involve growth by bone modelling and interactions of skeletal components to keep a functional and structural balance. Previous analyses of growth dynamics in humans revealed critical changes during late ontogeny explaining particular morphological features in our species. Data on bone modelling patterns from other primate species could help us to determine whether postnatal changes in the growth dynamics of the craniofacial complex are human specific or are shared with other primates. However, characterizations of bone modelling patterns through ontogeny in non-human hominids are scarce and restricted to isolated data on facial and mandibular regions. In the present study, we analyse the bone modelling patterns in an ontogenetic series of Pan and Gorilla to infer the growth dynamics of their craniofacial complex during postnatal development. Our results show that both Pan troglodytes and Gorilla gorilla are characterized by species-specific bone modelling patterns indicative of a mainly forward growth direction during postnatal development. Both species show minor but consistent ontogenetic changes in the distribution of bone modelling fields in specific regions of the face and mandible, in contrast to other regions which show more constant bone modelling patterns. In addition, we carry out a preliminary integrative study merging histological and geometric morphometric data. Both approaches yield highly complementary data, each analysis providing details on specific growth dynamics unavailable to the other. Moreover, geometric morphometric data show that ontogenetic variation in the modelling pattern of the mandibular ramus may be linked to sexual dimorphism.

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 Fuentiduen˜ a (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 Fuentiduen˜ a 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 attrition

Human skull morphology results from complex processes that involve the coordinated growth and interaction of its skeletal components to keep a functional and structural balance. Previous histological works have studied the growth of different craniofacial regions and their relationship to functional spaces in humans up to 14 years old. Nevertheless, how the growth dynamics of the facial skeleton and the mandible are related and how this relationship changes through the late ontogeny remain poorly understood. To approach these two questions, we have compared the bone modelling activities of the craniofacial skeleton from a sample of subadult and adult humans. In this study, we have established for the first time the bone modelling pattern of the face and the mandible from adult humans. Our analyses reveal a patchy distribution of the bone modelling fields (overemphasized by the presence of surface islands with no histological information) reflecting the complex growth dynamics associated to the individual morphology. Subadult and adult specimens show important differences in the bone modelling patterns of the anterior region of the facial skeleton and the posterior region of the mandible. These differences indicate developmental changes in the growth directions of the whole craniofacial complex, from a predominantly downward growth in subadults that turns to a forward growth observed in the adult craniofacial skeleton. We hypothesize that these ontogenetic changes would respond to the physiological and physical requirements to enlarge the oral and nasal cavities once maturation of the brain and the closure of the cranial sutures have taken place during craniofacial development.

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. © The Author(s), 2010.

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.

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. © 2012 VSP.

Azokh Cave is located near the village of the same name in the Nagorno-Karabagh region of the south-eastern part of the Lesser Caucasus (3937.09’ N and 4659.19’ E, 962 metres –a.s.l.). Azokh Cave and other relevant Acheulian sites in the Caucasus (Fig. 1) were described by Lioubine (2002). Together with Mousterian sites (Klein, 1969, 1999; Hoffecker and Cleghorn, 2000; Hoffecker, 2002; Stringer and Andrews, 2005) and sites producing evidence of the Middle-Late Palaeolithic transition (Joris and Adler 2008), the Caucasus region has provided evidence of continuous human settlement of the area throughout the Pleistocene. The geographical location of these sites indicates the persistence of a natural corridor that Lioubine (2002) named the ‘Caucasus isthmus’ and which we describe as the Trans-Caucasian corridor. Based on a geological survey of Quaternary deposits in collaboration with the Armenian Academy of Sciences (Ferna´ndez-Jalvo et al., 2004; King et al., 2003), we observe that the topography of the area has changed considerably due to tectonic compression and periglacial isostasy. This is in agreement with estimations by GPS studies (Mosar, 2006, Mosar et al., 2007) and ESR (Gru¨n et al., 1999) that establishedan uplift rate of12 to14 mm/year or 0.8–1.0 cm/year, respectively. The corridor has changed greatly since the middle Pleistocene, with uplift and erosion altering the landscape, but it is likely that passage through the Caucasian mountains has always been possible. The Trans-Caucasian corridor and other routes via Turkey and towards Asia (Bar-Yosef and Belfer-Cohen, 2001) were migration pathways during the Pleistocene. Fossil humans in the Caucasian area are scarce. The site of Dmanisi in Georgia yielded the earliest known Eurasian hominins (1.7 Ma, Gabunia et al., 2000; Rightmire et al., 2006; Martino´ n- Torres et al., 2008). Late surviving Neanderthals are present at several sites: Mezmaiskaya Cave, in the Northern Caucasus of Russia (30 ka, Skinner et a, Depto. de Geodinámica, Estratigrafía y Paleontología, Fac. de Ciencias Geológicas, TRUE, pub

Azokh Cave is located near the village of the same name in the Nagorno-Karabagh region of the south-eastern part of the Lesser Caucasus (3937.09’ N and 4659.19’ E, 962 metres –a.s.l.). Azokh Cave and other relevant Acheulian sites in the Caucasus (Fig. 1) were described by Lioubine (2002). Together with Mousterian sites (Klein, 1969, 1999; Hoffecker and Cleghorn, 2000; Hoffecker, 2002; Stringer and Andrews, 2005) and sites producing evidence of the Middle-Late Palaeolithic transition (Joris and Adler 2008), the Caucasus region has provided evidence of continuous human settlement of the area throughout the Pleistocene. The geographical location of these sites indicates the persistence of a natural corridor that Lioubine (2002) named the ‘Caucasus isthmus’ and which we describe as the Trans-Caucasian corridor. Based on a geological survey of Quaternary deposits in collaboration with the Armenian Academy of Sciences (Ferna´ndez-Jalvo et al., 2004; King et al., 2003), we observe that the topography of the area has changed considerably due to tectonic compression and periglacial isostasy. This is in agreement with estimations by GPS studies (Mosar, 2006, Mosar et al., 2007) and ESR (Gru¨n et al., 1999) that establishedan uplift rate of12 to14 mm/year or 0.8–1.0 cm/year, respectively. The corridor has changed greatly since the middle Pleistocene, with uplift and erosion altering the landscape, but it is likely that passage through the Caucasian mountains has always been possible. The Trans-Caucasian corridor and other routes via Turkey and towards Asia (Bar-Yosef and Belfer-Cohen, 2001) were migration pathways during the Pleistocene. Fossil humans in the Caucasian area are scarce. The site of Dmanisi in Georgia yielded the earliest known Eurasian hominins (1.7 Ma, Gabunia et al., 2000; Rightmire et al., 2006; Martino´ n- Torres et al., 2008). Late surviving Neanderthals are present at several sites: Mezmaiskaya Cave, in the Northern Caucasus of Russia (30 ka, Skinner et a

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 deer's normal and pedicle skin. © 2010 Pita-Thomas et al.

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 antler's innervation, the effects of the antler's 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. © 2010 Wiley-Liss, Inc.

[Background] Gene expression profiles of non-model mammals may provide valuable data for biomedical and evolutionary studies. However, due to lack of sequences compared. The results show that crossspecies hybridization affected neither the distribution of the hybridization reproducibility information of other species, DNA microarrays are currently restricted to humans and a few model species. This limitation may be overcome by using arrays developed for a given species to analyse gene expression in a related one, an approach known as "cross-species analysis". In spite of its potential usefulness, the accuracy and reproducibility of the gene expression measures obtained in this way are still open to doubt. The present study examines whether or not hybridization values from cross-species analyses are as reproducible as those from samespecies analyses when using Affymetrix oligonucleotide microarrays., [Results] The reproducibility of the probe data obtained hybridizing deer, Old-World primates, and human RNA samples to Affymetrix human GeneChip® U133 Plus 2.0 was compared. The results show that crossspecies hybridization affected neither the distribution of the hybridization reproducibility among different categories, nor the reproducibility values of the individual probes. Our analyses also show that a 0.5% of the probes analysed in the U133 plus 2.0 GeneChip are significantly associated to un-reproducible hybridizations. Such probes-called in the text un-reproducible probe sequences- do not increase in number in cross-species analyses., [Conclusion] Our study demonstrates that cross-species analyses do not significantly affect hybridization reproducibility of GeneChips, at least within the range of the mammal species analysed here. The differences in reproducibility between same-species and cross-species analyses observed in previous studies were probably caused by the analytical methods used to calculate the gene expression measures. Together with previous observations on the accuracy of GeneChips for cross-species analysis, our analyses demonstrate that cross-species hybridizations may provide useful gene expression data. However, the reproducibility and accuracy of these measures largely depends on the use of appropriated algorithms to derive the gene expression data from the probe data. Also, the identification of probes associated to un-reproducible hybridizations-useless for gene expression analyses- in the studied GeneChip, stress the need of a re-evaluation of the probes' performance.