Carboxymethylated and Sulfated Furcellaran from Furcellaria lumbricalis and Its Immobilization on PLA Scaffolds.

This study involved the creation of highly porous PLA scaffolds through the porogen/leaching method, utilizing polyethylene glycol as a porogen with a 75% mass ratio. The outcome achieved a highly interconnected porous structure with a thickness of 25 µm. To activate the scaffold's surface and improve its hydrophilicity, radiofrequency (RF) air plasma treatment was employed. Subsequently, furcellaran subjected to sulfation or carboxymethylation was deposited onto the RF plasma treated surfaces with the intention of improving bioactivity. Surface roughness and water wettability experienced enhancement following the surface modification. The incorporation of sulfate/carboxymethyl group (DS = 0.8; 0.3, respectively) is confirmed by elemental analysis and FT-IR. Successful functionalization of PLA scaffolds was validated by SEM and XPS analysis, showing changes in topography and increases in characteristic elements (N, S, Na) for sulfated (SF) and carboxymethylated (CMF). Cytocompatibility was evaluated by using mouse embryonic fibroblast cells (NIH/3T3).

Sulfation of furcellaran and its effect on hemocompatibility in vitro.

In this study, furcellaran (FUR) obtained from Furcellaria lumbricalis was firstly employed for sulfation via various methods, including SO3-pyridine (SO3∙Py) complex in different aprotic solvents, chlorosulfonic acid and sulfuric acid with a "coupling" reagent N,N'-Dicyclohexylcarbodiimide. Structural characterization through FT-IR, GPC, XPS and elemental analyses confirmed the successful synthesis of 6-O-sulfated FUR derivates characterized by varying degrees of sulfation (DS) ranging from 0.15 to 0.91 and molecular weight (Mw) spanning from12.5 kDa to 2.7 kDa. In vitro clotting assays, partial thromboplastin time (aPTT), thrombin time (TT), and prothrombin time (PT) underscored the essential role of sulfate esters in conferring anticoagulant activity whereas FUR prepared via chlorosulfonic acid with DS of 0.91 reached 311.4 s in aPPT showing almost 4-fold higher anticoagulant activity than native FUR at the concentration 2 mg/mL. MTT test showed all tested samples decreased cell viability in a dose dependent manner while all of them are non-cytotoxic up to the concentration of 0.1 mg/mL. Furthermore, sulfated derivates deposited onto polyethylene terephthalate surface presented substantial decrease in platelet adhesion, as well as absence of the most activated platelet stages. These findings support the pivotal role of O-6 FUR sulfates in enhancing hemocompatibility and provide valuable insights for a comparative assessment of effective sulfating approaches.

Low oral dose of 4-methylumbelliferone reduces glial scar but is insufficient to induce functional recovery after spinal cord injury.

Spinal cord injury (SCI) induces the upregulation of chondroitin sulfate proteoglycans (CSPGs) at the glial scar and inhibits neuroregeneration. Under normal physiological condition, CSPGs interact with hyaluronan (HA) and other extracellular matrix on the neuronal surface forming a macromolecular structure called perineuronal nets (PNNs) which regulate neuroplasticity. 4-methylumbelliferone (4-MU) is a known inhibitor for HA synthesis but has not been tested in SCI. We first tested the effect of 4-MU in HA reduction in uninjured rats. After 8 weeks of 4-MU administration at a dose of 1.2 g/kg/day, we have not only observed a reduction of HA in the uninjured spinal cords but also a down-regulation of CS glycosaminoglycans (CS-GAGs). In order to assess the effect of 4-MU in chronic SCI, six weeks after Th8 spinal contusion injury, rats were fed with 4-MU or placebo for 8 weeks in combination with daily treadmill rehabilitation for 16 weeks to promote neuroplasticity. 4-MU treatment reduced the HA synthesis by astrocytes around the lesion site and increased sprouting of 5-hydroxytryptamine fibres into ventral horns. However, the current dose was not sufficient to suppress CS-GAG up-regulation induced by SCI. Further adjustment on the dosage will be required to benefit functional recovery after SCI.

Spatiotemporal microvascular changes following contusive spinal cord injury.

Microvascular integrity is disrupted following spinal cord injury (SCI) by both primary and secondary insults. Changes to neuronal structures are well documented, but little is known about how the capillaries change and recover following injury. Spatiotemporal morphological information is required to explore potential treatments targeting the microvasculature post-SCI to improve functional recovery. Sprague-Dawley rats were given a T10 moderate/severe (200 kDyn) contusion injury and were perfuse-fixed at days 2, 5, 15, and 45 post-injury. Unbiased stereology following immunohistochemistry in four areas (ventral and dorsal grey and white matter) across seven spinal segments (n = 4 for each group) was used to calculate microvessel density, surface area, and areal density. In intact sham spinal cords, average microvessel density across the thoracic spinal cord was: ventral grey matter: 571 ± 45 mm-2, dorsal grey matter: 484 ± 33 mm-2, ventral white matter: 90 ± 8 mm-2, dorsal white matter: 88 ± 7 mm-2. Post-SCI, acute microvascular disruption was evident, particularly at the injury epicentre, and spreading three spinal segments rostrally and caudally. Damage was most severe in grey matter at the injury epicentre (T10) and T11. Reductions in all morphological parameters (95-99% at day 2 post-SCI) implied vessel regression and/or collapse acutely. Transmission electron microscopy (TEM) revealed disturbed aspects of neurovascular unit fine structure at day 2 post-SCI (n = 2 per group) at T10 and T11. TEM demonstrated a more diffuse and disrupted basement membrane and wider intercellular clefts at day 2, suggesting a more permeable blood spinal cord barrier and microvessel remodelling. Some evidence of angiogenesis was seen during recovery from days 2 to 45, indicated by increased vessel density, surface area, and areal density at day 45. These novel results show that the spinal cord microvasculature is highly adaptive following SCI, even at chronic stages and up to three spinal segments from the injury epicentre. Multiple measures of gross and fine capillary structure from acute to chronic time points provide insight into microvascular remodelling post-SCI. We have identified key vascular treatment targets, namely stabilising damaged capillaries and replacing destroyed vessels, which may be used to improve functional outcomes following SCI in the future.

The Role of Green Tea Catechin Epigallocatechin Gallate (EGCG) and Mammalian Target of Rapamycin (mTOR) Inhibitor PP242 (Torkinib) in the Treatment of Spinal Cord Injury.

Spinal cord injury (SCI) is a devastating condition that has physical and psychological consequences for patients. SCI is accompanied by scar formation and systemic inflammatory response leading to an intense degree of functional loss. The catechin, epigallocatechin gallate (EGCG), an active compound found in green tea, holds neuroprotective features and is known for its anti-inflammatory potential. The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that exists in two functionally distinct complexes termed mTOR complex 1 and 2 (mTORC1; mTORC2). Inhibition of mTORC1 by rapamycin causes neuroprotection, leading to partial recovery from SCI. In this study the effects of EGCG, PP242 (an inhibitor of both complexes of mTOR), and a combination of EGCG and PP242 in SCI have been examined. It has been found that both EGCG and PP242 significantly improved sensory/motor functions following SCI. However, EGCG appeared to be more effective (BBB motor test, from 2 to 8 weeks after SCI, p = 0.019, p = 0.007, p = 0.006, p = 0.006, p = 0.05, p = 0.006, and p = 0.003, respectively). The only exception was the Von Frey test, where EGCG was ineffective, while mTOR inhibition by PP242, as well as PP242 in combination with EGCG, significantly reduced withdrawal latency starting from week three (combinatorial therapy (EGCG + PP242) vs. control at 3, 5, and 7 weeks, p = 0.011, p = 0.007, and p = 0.05, respectively). It has been found that EGCG was as effective as PP242 in suppressing mTOR signaling pathways, as evidenced by a reduction in phosphorylated S6 expression (PP242 (t-test, p < 0.0001) or EGCG (t-test, p = 0.0002)). These results demonstrate that EGCG and PP242 effectively suppress mTOR pathways, resulting in recovery from SCI in rats, and that EGCG acts via suppressing mTOR pathways.

4-Methylumbeliferone Treatment at a Dose of 1.2 g/kg/Day Is Safe for Long-Term Usage in Rats.

4-methylumbelliferone (4MU) has been suggested as a potential therapeutic agent for a wide range of neurological diseases. The current study aimed to evaluate the physiological changes and potential side effects after 10 weeks of 4MU treatment at a dose of 1.2 g/kg/day in healthy rats, and after 2 months of a wash-out period. Our findings revealed downregulation of hyaluronan (HA) and chondroitin sulphate proteoglycans throughout the body, significantly increased bile acids in blood samples in weeks 4 and 7 of the 4MU treatment, as well as increased blood sugars and proteins a few weeks after 4MU administration, and significantly increased interleukins IL10, IL12p70 and IFN gamma after 10 weeks of 4MU treatment. These effects, however, were reversed and no significant difference was observed between control treated and 4MU-treated animals after a 9-week wash-out period.

Furcellaran Surface Deposition and Its Potential in Biomedical Applications.

Surface coatings of materials by polysaccharide polymers are an acknowledged strategy to modulate interfacial biocompatibility. Polysaccharides from various algal species represent an attractive source of structurally diverse compounds that have found application in the biomedical field. Furcellaran obtained from the red algae Furcellaria lumbricalis is a potential candidate for biomedical applications due to its gelation properties and mechanical strength. In the present study, immobilization of furcellaran onto polyethylene terephthalate surfaces by a multistep approach was studied. In this approach, N-allylmethylamine was grafted onto a functionalized polyethylene terephthalate (PET) surface via air plasma treatment. Furcellaran, as a bioactive agent, was anchored on such substrates. Surface characteristics were measured by means of contact angle measurements, X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM). Subsequently, samples were subjected to selected cell interaction assays, such as antibacterial activity, anticoagulant activity, fibroblasts and stem cell cytocompatibility, to investigate the Furcellaran potential in biomedical applications. Based on these results, furcellaran-coated PET films showed significantly improved embryonic stem cell (ESC) proliferation compared to the initial untreated material.

Planet of the AAVs: The Spinal Cord Injury Episode.

The spinal cord injury (SCI) is a medical and life-disrupting condition with devastating consequences for the physical, social, and professional welfare of patients, and there is no adequate treatment for it. At the same time, gene therapy has been studied as a promising approach for the treatment of neurological and neurodegenerative disorders by delivering remedial genes to the central nervous system (CNS), of which the spinal cord is a part. For gene therapy, multiple vectors have been introduced, including integrating lentiviral vectors and non-integrating adeno-associated virus (AAV) vectors. AAV vectors are a promising system for transgene delivery into the CNS due to their safety profile as well as long-term gene expression. Gene therapy mediated by AAV vectors shows potential for treating SCI by delivering certain genetic information to specific cell types. This review has focused on a potential treatment of SCI by gene therapy using AAV vectors.

Controlled release of enrofloxacin by vanillin-crosslinked chitosan-polyvinyl alcohol blends.

In transdermal drug delivery applications uniform drug distribution and sustained release are of great importance to decrease the side effects. In this direction in the present research, vanillin crosslinked chitosan (CS) and polyvinyl alcohol (PVA) blend based matrix-type transdermal system was prepared by casting and drying of aqueous solutions for local delivery of enrofloxacin (ENR) drug. Subsequently, the properties including the morphology, chemical structure, thermal behavior, tensile strength, crosslinking degree, weight uniformity, thickness, swelling and drug release of the CS-PVA blend films before and after crosslinking were characterized. In vitro drug release profiles showed the sustained release of ENR by the incorporation of vanillin as a crosslinker into the CS-PVA polymer matrix. Furthermore, the release kinetic profiles revealed that the followed mechanism for all samples was Higuchi and the increase of vanillin concentration in the blend films resulted in the change of diffusion mechanism from anomalous transport to Fickian diffusion. Overall, the obtained results suggest that the investigated vanillin crosslinked CS-PVA matrix-type films are potential candidates for transdermal drug delivery system.

New Model of Ventral Spinal Cord Lesion Induced by Balloon Compression in Rats.

Despite the variety of experimental models of spinal cord injury (SCI) currently used, the model of the ventral compression cord injury, which is commonly seen in humans, is very limited. Ventral balloon compression injury reflects the common anatomical mechanism of a human lesion and has the advantage of grading the injury severity by controlling the inflated volume of the balloon. In this study, ventral compression of the SCI was performed by the anterior epidural placement of the balloon of a 2F Fogarty's catheter, via laminectomy, at the level of T10. The balloon was rapidly inflated with 10 or 15 µL of saline and rested in situ for 5 min. The severity of the lesion was assessed by behavioral and immunohistochemical tests. Compression with the volume of 15 µL resulted in severe motor and sensory deficits represented by the complete inability to move across a horizontal ladder, a final Basso, Beattie and Bresnahan (BBB) score of 7.4 and a decreased withdrawal time in the plantar test (11.6 s). Histology and immunohistochemistry revealed a significant loss of white and gray matter with a loss of motoneuron, and an increased size of astrogliosis. An inflation volume of 10 µL resulted in a mild transient deficit. There are no other balloon compression models of ventral spinal cord injury. This study provided and validated a novel, easily replicable model of the ventral compression SCI, introduced by an inflated balloon of Fogarty´s catheter. For a severe incomplete deficit, an inflated volume should be maintained at 15 µL.

Yarrowia lipolytica Adhesion and Immobilization onto Residual Plastics.

Research in cell adhesion has important implications in various areas, such as food processing, medicine, environmental engineering, biotechnological processes. Cell surface characterization and immobilization of microorganisms on solid surfaces can be performed by promoting cell adhesion, in a relatively simple, inexpensive, and quick manner. The adhesion of Yarrowia lipolytica IMUFRJ 50682 to different surfaces, especially potential residual plastics (polystyrene, poly(ethylene terephthalate), and poly(tetrafluoroethylene)), and its use as an immobilized biocatalyst were tested. Y. lipolytica IMUFRJ 50682 presented high adhesion to different surfaces such as poly(tetrafluoroethylene) (Teflon), polystyrene, and glass, independent of pH, and low adhesion to poly(ethylene terephthalate) (PET). The adhesion of the cells to polystyrene was probably due to hydrophobic interactions involving proteins or protein complexes. The adhesion of the cells to Teflon might be the result not only of hydrophobic interactions but also of acid-basic forces. Additionally, the present work shows that Y. lipolytica cell extracts previously treated by ultrasound waves (cell debris) maintained their enzymatic activity (lipase) and could be attached to polystyrene and PET and used successfully as immobilized biocatalysts in hydrolysis reactions.

Age-related changes in the tooth-bone interface area of acrodont dentition in the chameleon.

Chameleon teeth develop as individual structures at a distance from the developing jaw bone during the pre-hatching period and also partially during the post-hatching period. However, in the adult, all teeth are fused together and tightly attached to the jaw bone by mineralized attachment tissue to form one functional unit. Tooth to bone as well as tooth to tooth attachments are so firm that if injury to the oral cavity occurs, several neighbouring teeth and pieces of jaw can be broken off. We analysed age-related changes in chameleon acrodont dentition, where ankylosis represents a physiological condition, whereas in mammals, ankylosis only occurs in a pathological context. The changes in hard-tissue morphology and mineral composition leading to this fusion were analysed. For this purpose, the lower jaws of chameleons were investigated using X-ray micro-computed tomography, laser-induced breakdown spectroscopy and microprobe analysis. For a long time, the dental pulp cavity remained connected with neighbouring teeth and also to the underlying bone marrow cavity. Then, a progressive filling of the dental pulp cavity by a mineralized matrix occurred, and a complex network of non-mineralized channels remained. The size of these unmineralized channels progressively decreased until they completely disappeared, and the dental pulp cavity was filled by a mineralized matrix over time. Moreover, the distribution of calcium, phosphorus and magnesium showed distinct patterns in the different regions of the tooth-bone interface, with a significant progression of mineralization in dentin as well as in the supporting bone. In conclusion, tooth-bone fusion in chameleons results from an enhanced production of mineralized tissue during post-hatching development. Uncovering the developmental processes underlying these outcomes and performing comparative studies is necessary to better understand physiological ankylosis; for that purpose, the chameleon can serve as a useful model species.

Role of the parahippocampal cortex in memory for the configuration but not the identity of objects: converging evidence from patients with selective thermal lesions and fMRI.

The parahippocampal cortex and hippocampus are brain structures known to be involved in memory. However, the unique contribution of the parahippocampal cortex remains unclear. The current study investigates memory for object identity and memory of the configuration of objects in patients with small thermo-coagulation lesions to the hippocampus or the parahippocampal cortex. Results showed that in contrast to control participants and patients with damage to the hippocampus leaving the parahippocampal cortex intact, patients with lesions that included the right parahippocampal cortex (RPH) were severely impaired on a task that required learning the spatial configuration of objects on a computer screen; these patients, however, were not impaired at learning the identity of objects. Conversely, we found that patients with lesions to the right hippocampus (RH) or left hippocampus (LH), sparing the parahippocampal cortex, performed just as well as the control participants. Furthermore, they were not impaired on the object identity task. In the functional Magnetic Resonance Imaging (fMRI) experiment, healthy young adults performed the same tasks. Consistent with the findings of the lesion study, the fMRI results showed significant activity in the RPH in the memory for the spatial configuration condition, but not memory for object identity. Furthermore, the pattern of fMRI activity measured in the baseline control conditions decreased specifically in the parahippocampal cortex as a result of the experimental task, providing evidence for task specific repetition suppression. In summary, while our previous studies demonstrated that the hippocampus is critical to the construction of a cognitive map, both the lesion and fMRI studies have shown an involvement of the RPH for learning spatial configurations of objects but not object identity, and that this takes place independent of the hippocampus.

Object-location memory impairment in patients with thermal lesions to the right or left hippocampus.

Memory for object-location was investigated by testing subjects with small unilateral thermolesions to the medial temporal lobe using small-scale 2D (Abstract) or large-scale 3D (Real) recall conditions. Four patients with lesions of the left hippocampus (LH), 10 patients with damage to the right hippocampus (RH) and 9 matched normal controls (NC) were tested. Six task levels were presented in a pseudorandom order. During each level, subjects viewed one to six different objects on the floor of a circular curtained arena 2.90 m in diameter for 10 s. Recall was tested by marking the locations of objects on a map of the arena (Abstract recall) and then by replacing the objects in the arena (Real recall). Two component errors were studied by calculating the Location Error (LE), independent of the object identity and the configuration error by finding the best match to the presented configuration. The RH group was impaired relative to the NC for nearly all combinations of recall and error types. An impairment was observed in this group even for one object and it deepened sharply with an increasing object number. Damage to the right perirhinal or parahippocampal cortices did not add to the impairment. Deficits in the LH group were also observed, but less consistently. The data indicate that spatial memory is strongly but not exclusively lateralised to the right medial temporal lobe.

A battery of tests for quantitative examination of idiothetic and allothetic place navigation modes in humans.

Research into the neural mechanisms of place navigation in laboratory animals has led to the definition of allothetic and idiothetic navigation modes that can be examined by quantitative analysis of the generated tracks. In an attempt to use this approach in the study of human navigation behavior, 10 young subjects were examined in an enclosed arena (2.9 m in diameter, 3 m high) equipped with a computerized tracking system. Idiothetic navigation was studied in blindfolded subjects performing the following tasks-Simple Homing, Complex Homing and Idiothesis Supported by Floor-Related Signals. Allothetic navigation was examined in sighted subjects instructed to find in an empty arena the acoustically signaled unmarked goal region and later to retrieve its position using tasks (Natural Navigation, Cue-Controlled Navigation, Snapshot Memory, Map Reading) that evaluated different aspects of allothesis. The results indicate that allothetic navigation is more accurate than idiothetic, that the poor accuracy of idiothesis is due to angular rather than to distance errors, and that navigation performance is best when both allothetic and idiothetic modes contribute to the solution of the task. The proposed test battery may contribute to better understanding of the navigation disturbances accompanying various neurological disorders and to objective evaluation of the results of drug therapy and of rehabilitation procedures.