ABSTRACT
Parkinson's disease (PD) is characterized by multiple symptoms including olfactory dysfunction, whose underlying mechanisms remain unclear. Here, we explored pathologic changes in the olfactory pathway of transgenic (Tg) mice of both sexes expressing the human A30P mutant α-synuclein (α-syn; α-syn-Tg mice) at 6-7 and 12-14 months of age, representing early and late-stages of motor progression, respectively. α-Syn-Tg mice at late stages exhibited olfactory behavioral deficits, which correlated with severe α-syn pathology in projection neurons (PNs) of the olfactory pathway. In parallel, olfactory bulb (OB) neurogenesis in α-syn-Tg mice was reduced in the OB granule cells at six to seven months and OB periglomerular cells at 12-14 months, respectively, both of which could contribute to olfactory dysfunction. Proteomic analyses showed a disruption in endocytic and exocytic pathways in the OB during the early stages which appeared exacerbated at the synaptic terminals when the mice developed olfactory deficits at 12-14 months. Our data suggest that (1) the α-syn-Tg mice recapitulate the olfactory functional deficits seen in PD; (2) olfactory structures exhibit spatiotemporal disparities for vulnerability to α-syn pathology; (3) α-syn pathology is restricted to projection neurons in the olfactory pathway; (4) neurogenesis in adult α-syn-Tg mice is reduced in the OB; and (5) synaptic endocytosis and exocytosis defects in the OB may further explain olfactory deficits.SIGNIFICANCE STATEMENT Olfactory dysfunction is a characteristic symptom of Parkinson's disease (PD). Using the human A30P mutant α-synuclein (α-syn)-expressing mouse model, we demonstrated the appearance of olfactory deficits at late stages of the disease, which was accompanied by the accumulation of α-syn pathology in projection neurons (PNs) of the olfactory system. This dysfunction included a reduction in olfactory bulb (OB) neurogenesis as well as changes in synaptic vesicular transport affecting synaptic function, both of which are likely contributing to olfactory behavioral deficits.
Subject(s)
Olfaction Disorders , Parkinson Disease , Male , Female , Mice , Humans , Animals , Parkinson Disease/genetics , alpha-Synuclein/genetics , alpha-Synuclein/metabolism , Smell , Proteomics , Mice, Transgenic , Neurogenesis , Olfaction Disorders/genetics , Disease Models, AnimalABSTRACT
Brain damage triggers diverse cellular and molecular events, with astrocytes playing a crucial role in activating local neuroprotective and reparative signaling within damaged neuronal circuits. Here, we investigated reactive astrocytes using a multidimensional approach to categorize their responses into different subtypes based on morphology. This approach utilized the StarTrack lineage tracer, single-cell imaging reconstruction and multivariate data analysis. Our findings identified three profiles of reactive astrocyte responses, categorized by their effects on cell size- and shape- related morphological parameters: "moderate", "strong," and "very strong". We also examined the heterogeneity of astrocyte reactivity, focusing on spatial and clonal distribution. Our research revealed a notable enrichment of protoplasmic and fibrous astrocytes within the "strong" and "very strong" response subtypes. Overall, our study contributes to a better understanding of astrocyte heterogeneity in response to an injury. By characterizing the diverse reactive responses among astrocyte subpopulations, we provide insights that could guide future research aimed at identifying novel therapeutic targets to mitigate brain damage and promote neural repair.
Subject(s)
Astrocytes , Astrocytes/physiology , Animals , Mice , Cell Lineage/physiology , Cluster Analysis , Single-Cell AnalysisABSTRACT
Piriform cortex (PC) is a 3-layer paleocortex receiving primary afferent input from the olfactory bulb. The past decade has seen significant progress in understanding the synaptic, cellular and functional organization of PC, but PC embryogenesis continues to be enigmatic. Here, using birthdating strategies and clonal analyses, we probed the early development and laminar specificity of neurogenesis/gliogenesis as it relates to the organization of the PC. Our data demonstrate a temporal sequence of laminar-specific neurogenesis following the canonical "inside-out" pattern, with the notable exception of PC Layer II which exhibited an inverse "outside-in" temporal neurogenic pattern. Of interest, we found no evidence of a neurogenic gradient along the anterior to posterior axis, although the timing of neuronal migration and laminar development was delayed rostrally by approximately 24 h. To begin probing if lineage affected cell fate in the PC, we labeled PC neuroblasts using a multicolor technique and analyzed their laminar organization. Our results suggested that PC progenitors were phenotypically committed to reach specific layers early in the development. Collectively, these studies shed new light on the determinants of the laminar specificity of neuronal/glial organization in PC and the likely role of subpopulations of committed progenitors in regulating PC embryogenesis.
Subject(s)
Cell Lineage/physiology , Cell Movement/physiology , Neurogenesis/physiology , Neuroglia/physiology , Piriform Cortex/cytology , Piriform Cortex/growth & development , Animals , Female , HEK293 Cells , Humans , Male , Mice , PregnancyABSTRACT
The olfactory tubercle (OT) is located in the ventral-medial region of the brain where it receives primary input from olfactory bulb (OB) projection neurons and processes olfactory behaviors related to motivation, hedonics of smell and sexual encounters. The OT is part of the dopamine reward system that shares characteristics with the striatum. Together with the nucleus accumbens, the OT has been referred to as the "ventral striatum". However, despite its functional importance little is known about the embryonic development of the OT and the phenotypic properties of the OT cells. Here, using thymidine analogs, we establish that mouse OT neurogenesis occurs predominantly between E11-E15 in a lateral-to-medial gradient. Then, using a piggyBac multicolor technique we characterized the migratory route of OT neuroblasts from their embryonic point of origin. Following neurogenesis in the ventral lateral ganglionic eminence (vLGE), neuroblasts destined for the OT followed a dorsal-ventral pathway we named "ventral migratory course" (VMC). Upon reaching the nascent OT, neurons established a prototypical laminar distribution that was determined, in part, by the progenitor cell of origin. A phenotypic analysis of OT neuroblasts using a single-color piggyBac technique, showed that OT shared the molecular specification of striatal neurons. In addition to primary afferent input from the OB, the OT also receives a robust dopaminergic input from ventral tegmentum (Ikemoto, 2007). We used tyrosine hydroxylase (TH) expression as a proxy for dopaminergic innervation and showed that TH onset occurs at E13 and progressively increased until postnatal stages following an 'inside-out' pattern. Postnatally, we established the myelination in the OT occurring between P7 and P14, as shown with CNPase staining, and we characterized the cellular phenotypes populating the OT by immunohistochemistry. Collectively, this work provides the first detailed analysis of the developmental and maturation processes occurring in mouse OT, and demonstrates the striatal nature of the OT as part of the ventral striatum (vST).
Subject(s)
Neurogenesis , Olfactory Tubercle/embryology , Animals , Dopaminergic Neurons/cytology , Dopaminergic Neurons/metabolism , Female , Male , Mice , Myelin Sheath/metabolism , Olfactory Tubercle/cytology , Olfactory Tubercle/growth & developmentABSTRACT
Viruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), use respiratory epithelial cells as an entry point for infection. Within the nasal cavity, the olfactory epithelium (OE) is particularly sensitive to infections which may lead to olfactory dysfunction. In patients suffering from coronavirus disease 2019, deficits in olfaction have been characterized as a distinctive symptom. Here, we used the K18hACE2 mice to study the spread of SARS-CoV-2 infection and inflammation in the olfactory system (OS) after 7â d of infection. In the OE, we found that SARS-CoV-2 selectively targeted the supporting/sustentacular cells (SCs) and macrophages from the lamina propria. In the brain, SARS-CoV-2 infected some microglial cells in the olfactory bulb (OB), and there was a widespread infection of projection neurons in the OB, piriform cortex (PC), and tubular striatum (TuS). Inflammation, indicated by both elevated numbers and morphologically activated IBA1+ cells (monocyte/macrophage lineages), was preferentially increased in the OE septum, while it was homogeneously distributed throughout the layers of the OB, PC, and TuS. Myelinated OS axonal tracts, the lateral olfactory tract, and the anterior commissure, exhibited decreased levels of 2',3'-cyclic-nucleotide 3'-phosphodiesterase, indicative of myelin defects. Collectively, our work supports the hypothesis that SARS-CoV-2 infected SC and macrophages in the OE and, centrally, microglia and subpopulations of OS neurons. The observed inflammation throughout the OS areas and central myelin defects may account for the long-lasting olfactory deficit.
Subject(s)
COVID-19 , Myelin Sheath , Olfactory Bulb , Olfactory Mucosa , SARS-CoV-2 , Animals , COVID-19/pathology , COVID-19/complications , Mice , Olfactory Mucosa/pathology , Olfactory Mucosa/virology , Olfactory Bulb/pathology , Olfactory Bulb/virology , Myelin Sheath/pathology , Myelin Sheath/metabolism , Microglia/pathology , Microglia/metabolism , Microglia/virology , Mice, Transgenic , Angiotensin-Converting Enzyme 2/metabolism , Olfaction Disorders/pathology , Olfaction Disorders/virology , Disease Models, Animal , Male , Inflammation/pathology , Inflammation/virology , Macrophages/pathology , FemaleABSTRACT
Microglia invade the neuroblast migratory corridor of the rostral migratory stream (RMS) early in development. The early postnatal RMS does not yet have the dense astrocyte and vascular scaffold that helps propel forward migrating neuroblasts, which led us to consider whether microglia help regulate conditions permissive to neuroblast migration in the RMS. GFP-labeled microglia in CX3CR-1GFP/+ mice assemble primarily along the outer borders of the RMS during the first postnatal week, where they exhibit predominantly an ameboid morphology and associate with migrating neuroblasts. Microglia ablation for 3 d postnatally does not impact the density of pulse labeled BrdU+ neuroblasts nor the distance migrated by tdTomato electroporated neuroblasts in the RMS. However, microglia wrap DsRed-labeled neuroblasts in the RMS of P7 CX3CR-1GFP/+;DCXDsRed/+ mice and express the markers CD68, CLEC7A, MERTK, and IGF-1, suggesting active regulation in the developing RMS. Microglia depletion for 14 d postnatally further induced an accumulation of CC3+ DCX+ apoptotic neuroblasts in the RMS, a wider RMS and extended patency of the lateral ventricle extension in the olfactory bulb. These findings illustrate the importance of microglia in maintaining a healthy neuroblast population and an environment permissive to neuroblast migration in the early postnatal RMS.
Subject(s)
Microglia , Neural Stem Cells , Mice , Animals , Neural Stem Cells/physiology , Lateral Ventricles , Cell Movement/physiology , Olfactory Bulb/physiologyABSTRACT
The formation of the olfactory nerve and olfactory bulb (OB) glomeruli begins embryonically in mice. However, the development of the olfactory system continues throughout life with the addition of new olfactory sensory neurons (OSNs) in the olfactory epithelium (OE). Much attention has been given to the perinatal innervation of the OB by OSN axons, but in the young adult the process of OSN maturation and axon targeting to the OB remains controversial. To address this gap in understanding, we used BrdU to label late-born OSNs in young adult mice at postnatal day 25 (P25-born OSNs) and timed their molecular maturation following basal cell division. We show that OSNs in young adults undergo a sequential molecular development with the expression of GAP 43 (growth-associated protein 43) > AC3 (adenylyl cyclase 3) > OMP (olfactory marker protein), consecutively, in a time frame of â¼8 d. To assess OSN axon development, we implemented an in vivo fate-mapping strategy to label P25-born OSNs with ZsGreen. Using sampling intervals of 24 h, we demonstrate the progressive extension of OSN axons in the OE, through the foramen of the cribriform plate, and onto the surface of the OB. OSN axons reached the OB and began to target and robustly innervate specific glomeruli â¼10 d following basal cell division, a time point at which OMP expression becomes evident. Our data demonstrate a sequential process of correlated axon extension and molecular maturation that is similar to that seen in the neonate, but on a slightly longer timescale and with regional differences in the OE.
Subject(s)
Olfactory Bulb/cytology , Olfactory Bulb/growth & development , Olfactory Mucosa/cytology , Olfactory Mucosa/growth & development , Olfactory Receptor Neurons/cytology , Animals , Mice , Neurogenesis/physiologyABSTRACT
The anterior commissure (AC) is a phylogenetically conserved inter-hemispheric connection found among vertebrates with bilateral symmetry. The AC connects predominantly olfactory areas but many aspects of its development and structure are unknown. To fill this gap, we investigated the embryonic and postnatal development of the AC by tracing axons with DiI and the piggyback transposon multicolor system. With this strategy, we show that axon growth during establishment of the AC follows a strictly regulated timeline of events that include waiting periods ("regressive strategies") as well as periods of active axon outgrowth ("progressive strategies"). We also provide evidence that these processes may be regulated in the midline via overexpression of chondroitin sulfate proteoglycans. Additionally, we demonstrate that the ipsi- and contralateral innervation of piriform cortex occurs simultaneously. Morphologically, we found that 20% of axons were myelinated by postnatal day (P) 22, in a process that occurred fundamentally around P14. By immunohistochemistry, we described the presence of glial cells and two new subtypes of neurons: one expressing a calretinin (CR)-/MAP2+ phenotype, distributed homogeneously inside the AC; and the other expressing a CR+/MAP2+ phenotype that lies beneath the bed nucleus of the stria terminalis. Our results are consistent with the notion that the AC follows a strictly regulated program during the embryonic and postnatal development similarly to other distal targeting axonal tracts.
Subject(s)
Anterior Commissure, Brain/embryology , Piriform Cortex/embryology , Animals , Anterior Commissure, Brain/ultrastructure , Axons/ultrastructure , Female , Male , Mice , Myelin Sheath/ultrastructure , Neuroglia/cytology , Neurons/cytology , Piriform Cortex/cytologyABSTRACT
PURPOSE: Chitosan is a natural polysaccharide which can form gels and scaffolds that support its use as a biomaterial in various tissue engineering applications. A useful feature of chitosan polymer is that you can manipulate its properties easily. Thus, in this work we studied the effect of varying chitosan concentration in the topography and the biological properties of the chitosan films, as well as the effects in the structure of 3D gels in order to be used as nerve bridges. METHODS: Analysis of film topographies were addressed by swelling test and atomic force microscopy (AFM). In vitro biological properties were assessed through MTT viability assays on cultures of blood-brain barrier forming endothelial (bEnd5), glioma (C6) and postmitotic neuron (NGF-differentiated PC12) cell lines. The structure of tridimensional gels was studied by environmental scanning electron microscopy.â© RESULTS: Topography of 1% chitosan films showed a AFM profile with higher nano-roughness profile than that observed in 2% films, which was smoother. Moreover, swelling rate was not affected. Topography changes affected cell viability as shown by the MTT assays. Our results showed that 2% chitosan films promoted higher proliferation and viability of C6 and PC12 respectively than 1% films. Conversely, neither 1% nor 2% films promoted viability of bEnd5 cells. In order to establish theâ©feasibility of both type of chitosan solutions as nerve bridges, we constructed 3D gels by alkaline precipitation. Resulting gels showed that only 2% gels were rigid enough to be effectively used as nerve bridges. CONCLUSIONS: These results establish that changes in chitosan concentration affects the polymer surface topography, which has a direct effect in the growing cell behavior. Additionally, higher concentration of chitosan gels are required to be used in neural tissue engineering.
Subject(s)
Chitosan/chemistry , Animals , Cell Line , Cell Proliferation/drug effects , Cell Survival/drug effects , Chitosan/pharmacology , Microscopy, Atomic Force , PC12 Cells , Rats , Surface Properties , Tissue EngineeringABSTRACT
BACKGROUND: Spinal bridge implants are strategic to provide growth surfaces for axonal regeneration after spinal cord injuries. The design of an appropriate substrate, one that is suitable for implantation, must involve careful testing of the biomaterial properties both in vitro and in vivo. OBJECTIVE: The goal of this work was to test the structure, stability and biological response after spinal bridges implantation of several biopolymers, composed of mixtures of agar (AG), as structural matrix scaffold, with κ-carrageenan (Kc), gelatin (G), xanthan gum (Xn) and polysulfone (PS). METHODS: Biopolymer structures were studied by environmental scanning electron microscopy, whereas the stability of gels was analyzed by in vitro degradation and swelling tests. The biocompatibility of these materials and their ability to promote cell growth and axonal regeneration were studied by implantation of spinal bridges containing empty linear channels in an acute rat spinal cord transection model at thoracic level (T8). RESULTS AND CONCLUSIONS: All gel mixtures gave rise to porous structures and they were stables to degradation, excepting the AG+G mixture. Spinal bridges constructed from all mixtures were implanted during a month in adult rats. After this time a low host reaction occurred to all bridge materials as well as neurite and cell ingrowths through the empty channels. Neurites within the bridges were mostly peripheral sensory fibers such as those positive for CGRP, whereas there was a lack of regeneration of central axons crossing from the spinal tissue to bridges. Many of these neurites established closed contacts with non-myelin Schwann cells. The histological analysis revealed a high accumulation of collagen fibers within the channels. Unexpected was the apparent loss of channels linearity which affected the growth of neurites and cells, indicating the need for additional regeneration strategies and vertebrae bridge fixing.
Subject(s)
Agar/chemistry , Biocompatible Materials/chemistry , Guided Tissue Regeneration/instrumentation , Nerve Regeneration/physiology , Spinal Cord Injuries/physiopathology , Spinal Cord Injuries/surgery , Spinal Cord/physiopathology , Animals , Equipment Failure Analysis , Gels/chemistry , Guided Tissue Regeneration/methods , Male , Materials Testing , Prosthesis Design , Rats , Rats, Wistar , Tissue ScaffoldsABSTRACT
Astrocytes are a heterogeneous population of glial cells with multifaceted roles in the central nervous system. Recently, the new method for the clonal analysis Star Track evidenced the link between astrocyte heterogeneity and lineage. Here, we tested the morphological response to mechanical injury of clonally related astrocytes using the Star Track approach, which labels each cell lineage with a specific code of colors. Histological and immunohistochemical analyses at 7 days post injury revealed a variety of morphological changes that were different among distinct clones. In many cases, cells of the same clone responded equally to the injury, suggesting the dependence on their genetic codification (intrinsic response). However, in other cases cells of the same clone responded differently to the injury, indicating their response to extrinsic factors. Thus, whereas some clones exhibited a strong morphological alteration or a high proliferative response to the injury, other clones located at similar distances to the lesion were apparently unresponsive. Concurrence of different clonal responses to the injury reveals the importance of the development determining the astrocyte features in response to brain injuries. These features should be considered to develop therapies that affect glial function.
Subject(s)
Astrocytes/pathology , Brain Injuries/pathology , Cerebral Cortex/pathology , Animals , Astrocytes/metabolism , Biomarkers/metabolism , Brain Injuries/etiology , Cell Proliferation , Clone Cells/metabolism , Clone Cells/pathology , Disease Models, Animal , Electroporation , Hypertrophy , MiceABSTRACT
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.
Subject(s)
Chitosan/chemistry , Gelatin/chemistry , Neurites/metabolism , Neurons/cytology , Polylysine/chemistry , Tissue Scaffolds/chemistry , Animals , Cell Adhesion , Cell Line, Tumor , Cell Proliferation , Cells, Cultured , Ganglia, Spinal/cytology , Neuroglia/cytology , Neuroglia/metabolism , Neurons/metabolism , PC12 Cells , Rats , Rats, WistarABSTRACT
Olfaction is the most relevant chemosensory sense of the rodents. General odors are primarily detected by the main olfactory system while most pheromonal signals are received by the accessory olfactory system. The first relay in the brain occurs in the olfactory bulb, which is subdivided in the main and accessory olfactory bulb (MOB/AOB). Given that the cell generation time is different between AOB and MOB, and the cell characterization of AOB remains limited, the goal of this work was first, the definition of the layering of AOB/MOB and second, the determination of cellular phenotypes in the AOB in a time window corresponding to the early postnatal development. Moreover, since reelin (Reln) deficiency has been related to olfactory learning deficits, we analyzed reeler mice. First, we compared the layering between AOB and MOB at early embryonic stages. Then, cell phenotypes were established using specific neuronal and glial markers as well as the Reln adaptor protein Dab1 to analyse differences in both genetic backgrounds. There was no apparent difference in the cell phenotypes among AOB and MOB or between wild type (wt) and reeler animals. However, a disruption in the granular cell layer of reeler with respect to wt mice was observed. In conclusion, the AOB in Reln-deficient mice showed similar neuronal and glial cell types being only affected the organization of granular neurons.
ABSTRACT
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.
Subject(s)
Chitosan/chemistry , Gelatin/chemistry , Nervous System/cytology , Nervous System/drug effects , Polylysine/chemistry , Tissue Engineering/methods , Tissue Scaffolds/chemistry , Animals , Astrocytes/cytology , Astrocytes/drug effects , Astrocytes/metabolism , Cell Adhesion/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Chemical Phenomena , Electrolytes/chemistry , Ganglia, Spinal/cytology , Hippocampus/cytology , Nervous System/metabolism , Neurites/drug effects , Neurites/metabolism , Olfactory Bulb/cytology , Porosity , Rats , Rats, Wistar , Temperature , WettabilityABSTRACT
Dab1 mediates reelin signalling and plays critical roles in early brain development such as the stereotypical positioning of neurons in the brain. The olfactory bulb undergoes a prominent layering reorganization, but shows not apparent differences between wild type and reeler in the layer organization. Therefore, an accurate regional and cellular simultaneous analysis of these molecules becomes essential to clarify the role played by Dab1 upon Reelin effect. The present study reveals a strong and consistent Dab1 mRNA and protein expressions, throughout the olfactory bulb layers in both wild type and reeler mice. In addition, noteworthy is the pattern of Dab1 location within cell nuclei in both strains. Furthermore, a temporal increment of Dab1 expression levels is detected from P0 to P15 in both strains, being the protein quantity higher in reeler than in wild type mice. Altogether, our results revealed that Reln acts directly from projection neurons via the production of different Reln fragments. Changes in the pattern of Dab1 expression could reflect an alternative Reln function in postnatal and adult stages, besides a possible regulation of Dab1 by other molecules distinct to Reln.
Subject(s)
Cell Adhesion Molecules, Neuronal/metabolism , Extracellular Matrix Proteins/metabolism , Gene Expression Regulation, Developmental , Nerve Tissue Proteins/metabolism , Olfactory Bulb/metabolism , Serine Endopeptidases/metabolism , Animals , Animals, Newborn , Gene Expression , Mice , Mice, Neurologic Mutants , Nerve Tissue Proteins/analysis , Nerve Tissue Proteins/genetics , Neurons , RNA, Messenger/analysis , Reelin Protein , Time FactorsABSTRACT
Ordered gelation under spin-coating conditions, as reported here, is a suitable method to order cells in biogels. Cell ordering is of great importance for functional repair of central nervous system (CNS) injuries, because therapies must include strategies to bridge chystic gaps and facilitate axon growth towards its target. Organized biocompatible and biodegradable substrates may be used for this purpose, to supply trophic support and provide directional cues for neuronal process outgrowth. Atomic force microscopy (AFM) and low temperature scanning electron microscopy (LTSEM), confirmed that fibrils in kappa-carrageenan/chitosan and fibrin hydrogels prepared under spin-coating conditions, were longitudinally arranged. The cell model was conveniently tested using rat C6 glioma cells. C6 cells were distributed regularly in fibrin gels formed under centrifugal force. The ability of ordered fibrin scaffolds to promote uniform distribution of transplanted cells, was confirmed by fluorescence microscopy.