NEUROINFLAMMATION

Inflammation is a complex process, mediated by cellular and molecular mechanisms caused by a response to a tissue damage from an aggressive agent, whether if biological, chemical or physical origin. This process occurs, ultimately, with the purpose of promoting defense, repair and tissue regeneration. The inflammatory process leads to changes in blood vessels that have their diameter and flow altered, with the objective of leading to increased vascular permeability and consequent leakage of fluids and cells into the extracellular space of the affected tissue. This sequence of events generates the cardinal signs of inflammation, which are: pain, heat, redness, edema, with loss or alteration of function. The process occurs through mechanisms induced by cytokines and that despite having local manifestation, it can lead to systemic responses involving the whole organism with fever, chills, tremors, tachycardia, leukocytosis, sweating, diuresis and blood dyscrasias. In the cascade of events related to inflammation, there is initially a local stimulus that promotes morphological and functional changes in the attacked tissue that trigger the release of signaling molecules, the defensins that have a chemotactic effect on monocytes, neutrophils and lymphocytes, and pro-inflammatory mediators. that are directly involved in the next inflammatory phases. There is, then, the recognition of aggression and the aggressor agent by the receptors of cells of the immune system and release of inflammatory mediators, of the cyclooxygenase pathway that will release prostaglandin, prostacyclins and thromboxanes and by the lipooxygenase pathway that will produce leukotrienes (Figure 1). Then, there is a modification of the local microcirculation promoting vasodilation, initially arteriolar and subsequently of the venules by the action of histamine release by mast cells, and associated with an increase in local blood flow, generate the cardinal flushing and heat signals.

Selection of Boar Sperm by Reproductive Biofluids as Chemoattractants

Chemotaxis is a spermatozoa guidance mechanism demonstrated in vitro in several mammalian species including porcine. This work focused on follicular fluid (FF), periovulatory oviductal fluid (pOF), the medium surrounding oocytes during in vitro maturation (conditioned medium; CM), progesterone (P4), and the combination of those biofluids (Σ) as chemotactic agents and modulators of spermatozoa fertility in vitro. A chemotaxis chamber was designed consisting of two independent wells, A and B, connected by a tube. The spermatozoa are deposited in well A, and the chemoattractants in well B. The concentrations of biofluids that attracted a higher proportion of spermatozoa to well B were 0.25% FF, 0.25% OF, 0.06% CM, 10 pM P4 and 0.25% of a combination of biofluids (Σ2), which attracted between 3.3 and 12.3% of spermatozoa (p < 0.05). The motility of spermatozoa recovered in well B was determined and the chemotactic potential when the sperm calcium channel CatSper was inhibited, which significantly reduced the % of spermatozoa attracted (p < 0.05). Regarding the in vitro fertility, the spermatozoa attracted by FF produced higher rates of penetration of oocytes and development of expanded blastocysts. In conclusion, porcine reproductive biofluids show an in vitro chemotactic effect on spermatozoa and modulate their fertilizing potential.

Recellularization of bronchial extracellular matrix with primary bronchial smooth muscle cells

Severe asthma is associated with an increased airway smooth muscle (ASM) mass and an altered composition of the extracellular matrix (ECM). Studies have indicated that ECM-ASM cell interactions contribute to this remodeling and its limited reversibility with current therapy. Three-dimensional matrices allow the study of complex cellular responses to different stimuli in an almost natural environment. Our goal was to obtain acellular bronchial matrices and then develop a recellularization protocol with ASM cells. We studied equine bronchi as horses spontaneously develop a human asthma-like disease. The bronchi were decellularized using Triton/Sodium Deoxycholate. The obtained scaffolds retained their anatomical and histological properties. Using immunohistochemistry and a semi-quantitative score to compare native bronchi to scaffolds revealed no significant variation for matrixial proteins. A DNA quantification and electrophoresis indicated that most of DNA was 29.6 ng/mg of tissue ± 5.6 with remaining fragments of less than 100 bp. Primary ASM cells were seeded on the scaffolds. Histological analysis after recellularization showed that ASM cells migrated and proliferated primarily in the decellularized smooth muscle matrix, suggesting a chemotactic effect of the scaffolds. This is the first report of primary ASM cells preferentially repopulating the smooth muscle matrix layer in bronchial matrices. This protocol is now being used to study the molecular interactions occurring between the asthmatic ECMs and ASM to identify effectors of asthmatic bronchial remodeling.

Sequential Delivery of Dual Growth Factors from Injectable Chitosan-Based Composite Hydrogels

Local administration of platelet-derived growth factor-BB (PGDF-BB) and bone morphogenetic protein-2 (BMP-2) in a sequential release manner could substantially promote bone healing. To achieve this goal, a delivery system that could sustain the release of PGDF-BB and BMP-2 by way of temporal separation was developed. One type of PGDF-BB-encapsulated alginate microsphere and another type of BMP-2-encapsulated microsphere with a core-shell structure were respectively produced using emulsification methods. These two types of microspheres were then embedded into chitosan/glycerophosphate hydrogel for constructing composite gels. Some of them were found to be injectable at ambient temperature and had thermo-sensitive features near physiological temperature and pH. The optimally formulated composite gels showed the ability to control the release of PGDF-BB and BMP-2 in a sequential fashion in which PDGF-BB was released earlier than BMP-2. In vitro release patterns indicated that the release rates could be significantly regulated by varying the embedded amount of the factor-encapsulated microspheres, which can in turn mediate the temporal separation release interval between PGDF-BB and BMP-2. The released PDGF-BB and BMP-2 were detected to be bioactive based on their respective effects on Balb/c 3T3 and C2C12 cells. These results suggest that the presently developed composite gels have the potential for bone repair by synergistically utilizing the early chemotactic effect of PDGF-BB and the subsequent osteogenic and angiogenic functions of PDGF-BB and BMP-2.

Effect of the CCL5-Releasing Fibrin Gel for Intervertebral Disc Regeneration

Objective To explore if chemokine (C-C motif) ligand 5 (CCL5) delivery could recruit annulus fibrosus (AF) cells to the injury sites and facilitate the repair of ruptured AF. Design The effects of CCL5 on bovine AF cells in vitro were tested by transwell assay and quantitative real-time polymerase chain reaction. Fibrin gel containing CCL5 was used to treat annulotomized bovine caudal discs cultured under dynamic loading conditions. After 14 days of loading, the samples were collected for histological examination. A pilot animal study was performed using sheep cervical discs to investigate the effect of fibrin gel encapsulated with CCL5 for the treatment of ruptured AF. After 14 weeks, the animals were sacrificed, and the discs were scanned with magnetic resonance imaging before histopathological examination. Results CCL5 showed a chemotactic effect on AF cells in a dose-dependent manner. AF cells cultured with CCL5 in vitro did not show any change of the gene expression of CCL5 receptors, catabolic and proinflammatory markers. In vitro release study showed that CCL5 exhibited sustained release from the fibrin gel into the culture media; however, in the organ culture study CCL5 did not stimulate homing of AF cells toward the defect sites. The pilot animal study did not show any repair effect of CCL5. Conclusions CCL5 has a chemotactic effect on AF cells in vitro, but no ex vivo or in vivo regenerative effect when delivered within fibrin gel. Further study with a stronger chemotactic agent and/or an alternate biomaterial that is more conductive of cell migration is warranted.