Development of in situ forming implants for controlled delivery of punicalagin.

Due to efficient drainage of the joint, the development of intra-articular depots for long-lasting drug release is a difficult challenge. Moreover, a disease-modifying osteoarthritis drug (DMOAD) that can effectively manage osteoarthritis has yet to be identified. The current study was undertaken to explore the potential of injectable, in situ forming implants to create depots that support the sustained release of punicalagin, a promising DMOAD. In vitro experiments demonstrated punicalagin's ability to suppress production of interleukin-1ß and prostaglandin E2, confirming its chondroprotective properties. Regarding the entrapment of punicalagin, it was demonstrated by LC-MS/MS to be stable within PLGA in situ forming implants for several weeks and capable of inhibiting collagenase upon release. In vitro punicalagin release kinetics were tunable through variation of solvent, PLGA lactide:glycolide ratio, and polymer concentration, and an optimized formulation supported release for approximately 90 days. The injection force of this formulation steadily increased with plunger advancement and higher rates of advancement were associated with greater forces. Although the optimal formulation was highly cytotoxic to primary chondrocytes if cells were exposed immediately or shortly after implant formation, upwards of 70 % survival was achieved when the implants were first allowed to undergo a 24-72 h period of phase inversion prior to cell exposure. This study demonstrates a PLGA-based in situ forming implant for the controlled release of punicalagin. With modification to address cytotoxicity, such an implant may be suitable as an intra-articular therapy for OA.

Isolation of Transcriptomic-Quality Total RNA from Mouse Spinal Cords.

Assessing cells, proteins, and total RNA in the spinal cord is vital for advancing our understanding of neuroinflammation and neurodegenerative diseases. For instance, immune cells infiltrate the spinal cord in the experimental autoimmune encephalomyelitis (EAE) model, commonly used to study multiple sclerosis. Thus, it is valuable to assess total RNA to determine the neuronal and inflammatory profiles in the spinal cord. Further, RNA profiles are useful for deciphering the effects of drugs or chemicals on neuroinflammation and neurodegenerative diseases such as EAE. The purpose of this protocol and the online video illustrating it is to describe and demonstrate the expulsion of the spinal cord from the mouse spinal column and homogenization of the spinal cord using liquid nitrogen for optimal RNA isolation. Although we present this method with spinal cords from EAE mice, the technique is broadly applicable, including RNA isolation from the spinal cords of healthy mice. Proper performance of these steps is critical to achieving a sufficient yield of transcriptomic-quality spinal cord RNA when combined with final isolation using commercially available kits. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Isolation of the spinal cord from the spinal column Support Protocol: Preparation of blunt-end needle for spinal cord isolation Basic Protocol 2: Spinal cord homogenization using liquid nitrogen Basic Protocol 3: Assessment of RNA purity, quantification, and integrity.

TCDD Inhibition of IgG1 Production in Experimental Autoimmune Encephalomyelitis (EAE) and In Vitro.

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD) is a ligand for the aryl hydrocarbon receptor (AhR). TCDD is well-characterized to produce immunotoxicity, including suppression of antibody production. Previously we showed that TCDD inhibited myelin oligodendrocyte glycoprotein (MOG) peptide-specific IgG and attenuated disease in experimental autoimmune encephalomyelitis (EAE) model in mice. Thus, the purpose of this study was to characterize the effects of TCDD on IgG subclasses in EAE and in vitro and assess effects in B cells derived from various tissues. TCDD modestly suppressed intracellular IgG expression in splenocytes (SPLC), but not bone marrow (BM) or lymph node (LN) cells. To further understand TCDD's effects on IgG, we utilized LPS and LPS + IL-4 in vitro to stimulate IgG3 and IgG1 production, respectively. TCDD preferentially suppressed IgG1+ cell surface expression, especially in SPLC. However, TCDD was able to suppress IgG1 and IgG3 secretion from SPLC and B cells, but not BM cells. Lastly, we revisited the EAE model and determined that TCDD suppressed MOG-specific IgG1 production. Together these data show that the IgG1 subclass of IgG is a sensitive target of suppression by TCDD. Part of the pathophysiology of EAE involves production of pathogenic antibodies that can recruit cytolytic cells to destroy MOG-expressing cells that comprise myelin, so inhibition of IgG1 likely contributes to TCDD's EAE disease attenuation.