Scoring Central Nervous System Inflammation, Demyelination, and Axon Injury in Experimental Autoimmune Encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a common immune-based model of multiple sclerosis (MS). This disease can be induced in rodents by active immunization with protein components of the myelin sheath and Complete Freund's adjuvant (CFA) or by the transfer of myelin-specific T effector cells from rodents primed with myelin protein/CFA into naïve rodents. The severity of EAE is typically scored on a 5-point clinical scale that measures the degree of ascending paralysis, but this scale is not optimal for assessing the extent of recovery from EAE. For example, clinical scores remain high in some EAE models (e.g., myelin oligodendrocyte glycoprotein [MOG] peptide-induced model of EAE) despite the resolution of inflammation. Thus, it is important to complement clinical scoring with histological scoring of EAE, which also provides a means to study the underlying mechanisms of cellular injury in the central nervous system (CNS). Here, a simple protocol is presented to prepare and stain spinal cord and brain sections from mice and to score inflammation, demyelination, and axonal injury in the spinal cord. The method for scoring leukocyte infiltration in the spinal cord can also be applied to score brain inflammation in EAE. A protocol for measuring soluble neurofilament light (sNF-L) in the serum of mice using a Small Molecule Assay (SIMOA) assay is also described, which provides feedback on the extent of overall CNS injury in live mice.

Peroxisome Proliferator-Activated Receptor-δ Deficiency in Microglia Results in Exacerbated Axonal Injury and Tissue Loss in Experimental Autoimmune Encephalomyelitis.

Peroxisome proliferator-activated receptor (PPAR)-δ is a nuclear receptor that functions to maintain metabolic homeostasis, regulate cell growth, and limit the development of excessive inflammation during immune responses. Previously, we reported that PPAR-δ-deficient mice develop a more severe clinical course of experimental autoimmune encephalomyelitis (EAE); however, it was difficult to delineate the role that microglia played in this disease phenotype since PPAR-δ-deficient mice exhibited a number of immune defects that enhanced CNS inflammation upstream of microglia activation. Here, we specifically investigated the role of PPAR-δ in microglia during EAE by using mice where excision of a floxed Ppard allele was driven by expression of a tamoxifen (TAM)-inducible CX3C chemokine receptor 1 promoter-Cre recombinase transgene (Cx3cr1CreERT2: Ppardfl/fl). We observed that by 30 days of TAM treatment, Cx3cr1CreERT2: Ppardfl/fl mice exhibited Cre-mediated deletion primarily in microglia and this was accompanied by efficient knockdown of Ppard expression in these cells. Upon induction of EAE, TAM-treated Cx3cr1CreERT2: Ppardfl/fl mice presented with an exacerbated course of disease compared to TAM-treated Ppardfl/fl controls. Histopathological and magnetic resonance (MR) studies on the spinal cord and brains of EAE mice revealed increased Iba-1 immunoreactivity, axonal injury and CNS tissue loss in the TAM-treated Cx3cr1CreERT2: Ppardfl/fl group compared to controls. In early EAE, a time when clinical scores and the infiltration of CD45+ leukocytes was equivalent between Cx3cr1CreERT2: Ppardfl/fl and Ppardfl/fl mice, Ppard-deficient microglia exhibited a more reactive phenotype as evidenced by a shorter maximum process length and lower expression of genes associated with a homeostatic microglia gene signature. In addition, Ppard-deficient microglia exhibited increased expression of genes associated with reactive oxygen species generation, phagocytosis and lipid clearance, M2-activation, and promotion of inflammation. Our results therefore suggest that PPAR-δ has an important role in microglia in limiting bystander tissue damage during neuroinflammation.

Pharmacokinetics, tissue distribution and safety of gold nanoparticle/PKC Delta inhibitor peptide hybrid in rats.

Gold nanoparticles (GNPs) are extremely useful for drug delivery, due in part to their highly tunable nature. However, this variability has prevented a clear understanding of the pharmacokinetics and toxicity of GNPs for drug delivery. Here, we present the clearance, organ distribution and acute toxicity testing of our drug delivery system which uses GNPs and two penta-peptides, to deliver a rationally designed peptide drug. We found that with or without our therapeutic, the GNP/peptide hybrid cleared rapidly from the blood in rats and accumulated mostly in the liver and spleen, although it was also detectable in several other organs. There were subtle but detectable differences between the behavior of our GNP hybrids with or without the therapeutic peptide. The GNP/peptide hybrid showed no evidence of toxicity at single doses up to 16 times the therapeutic dose, as measured by a battery of tests including, blood cell makeup, levels of markers of liver, kidney and spleen function, organ mass indexes, and histology. These results underline the importance of testing the pharmacokinetics and toxicity of all GNP preparations, as even minor changes to the surface coatings of GNPs can influence their behavior. On the other hand, the results herein can help guide the design and use of similar GNP/peptide drug delivery systems.

Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles.

Protein kinase C-delta inhibitor (PKCδi) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a cell-penetrating peptide, TAT, for intracellular delivery. However, TAT has shown non-specific biological activities. Gold nanoparticles (GNPs) can be used as drug delivery carriers without recognized toxicity. Therefore, we have used a GNP/peptide hybrid to deliver PKCδi. Two short peptides (P2: CAAAAE and P4: CAAAAW), at a 95:5 ratio, were used to modify the surface properties of GNP. GNPs conjugated with PKCδi (GNP/PKCi) are stable in distilled water, 0.9% NaCl, and phosphate-buffered saline (PBS) containing bovine serum albumin or fetal bovine serum. Intravenous injection of GNP-PKCi was previously shown to prevent ischemia-reperfusion injury of the lung. This article outlines a protocol to formulate GNP/PKCi and assess the physiochemical properties of GNP/PKCi. We have used similar methods to formulate other peptide-based drugs with GNP. This article will hopefully draw more attention to this novel intracellular drug delivery technology and its applications in vivo.

The Intersection of Central Dopamine System and Stroke: Potential Avenues Aiming at Enhancement of Motor Recovery.

Dopamine, a major neurotransmitter, plays a role in a wide range of brain sensorimotor functions. Parkinson's disease and schizophrenia are two major human neuropsychiatric disorders typically associated with dysfunctional dopamine activity levels, which can be alleviated through the druggability of the dopaminergic systems. Meanwhile, several studies suggest that optimal brain dopamine activity levels are also significantly impacted in other serious neurological conditions, notably stroke, but this has yet to be fully appreciated at both basic and clinical research levels. This is of utmost importance as there is a need for better treatments to improve recovery from stroke. Here, we discuss the state of knowledge regarding the modulation of dopaminergic systems following stroke, and the use of dopamine boosting therapies in animal stroke models to improve stroke recovery. Indeed, studies in animals and humans show stroke leads to changes in dopamine functioning. Moreover, evidence from animal stroke models suggests stimulation of dopamine receptors may be a promising therapeutic approach for enhancing motor recovery from stroke. With respect to the latter, we discuss the evidence for several possible receptor-linked mechanisms by which improved motor recovery may be mediated. One avenue of particular promise is the subtype-selective stimulation of dopamine receptors in conjunction with physical therapy. However, results from clinical trials so far have been more mixed due to a number of potential reasons including, targeting of the wrong patient populations and use of drugs which modulate a wide array of receptors. Notwithstanding these issues, it is hoped that future research endeavors will assist in the development of more refined dopaminergic therapeutic approaches to enhance stroke recovery.