- emulsion was deposited on either side of the tail base. Pertussis toxin (300 ng/200 µL; 180, List Biological Laboratories) was injected intraperitoneally (ip) on days 0 and 2 after MOG immunization. CHCA (C2020, Sigma) was dissolved in phosphate-buffered
The migration of leukocytes into the CNS drives the neuropathology of multiple sclerosis (MS). This penetration likely utilizes energy resources that remain to be defined. Using the experimental autoimmune encephalomyelitis (EAE) model of MS, we determined that macrophages within the perivascular cuff of post-capillary venules are highly glycolytic as manifested by strong expression of lactate dehydrogenase A (LDHA) that converts pyruvate to lactate. These macrophages expressed prominent levels of monocarboxylate transporter-4 (MCT-4) specialized in secreting lactate from glycolytic cells. The functional relevance of glycolysis was confirmed by siRNA-mediated knockdown of LDHA and MCT-4, which decreased lactate secretion and macrophage transmigration. MCT-4 was in turn regulated by EMMPRIN (CD147) as determined through co-expression/co-immunoprecipitation studies, and siRNA-mediated EMMPRIN silencing. The functional relevance of MCT-4/EMMPRIN interaction was affirmed by lower macrophage transmigration in culture using the MCT-4 inhibitor, α-cyano-4-hydroxy-cinnamic acid (CHCA), a cinnamon derivative. CHCA also reduced leukocyte infiltration and the clinical severity of EAE. Relevance to MS was corroborated by the strong expression of MCT-4, EMMPRIN and LDHA in perivascular macrophages in MS brains. These results detail the metabolism of macrophages for transmigration from perivascular cuffs into the CNS parenchyma and identifies CHCA and diet as potential modulators of neuro-inflammation in MS.
Insulin-like growth factor binding protein-7: A marker of conjunctivalization in an animal model of limbal stem cell deficiency
Park, M;Mazalo, J;Di Girolamo, N;
The Ocular SurfaceMay 21, 2019
Product: Cholera Toxin (AZIDE-FREE) from Vibrio cholerae
Within the basal ganglia circuit, the GPe is critically involved in motor control. Aside from Foxp2+ neurons and ChAT+ neurons that have been established as unique neuron types, there is no consensus on the classification of GPe neurons. Properties of the remaining neuron types are poorly defined. In this study, we leverage new mouse lines, viral tools, and molecular markers to study GPe neurons. By examining multiple modalities, we sought to better define GPe neuron subtypes. We found that Sox6 represents a novel, defining marker for GPe neuron subtypes. Lhx6+ neurons that lack the expression of Sox6 were devoid of both parvalbumin and Npas1. This result confirms previous assertions of the existence of a unique Lhx6+ population. Neurons that arise from the Dbx1+ lineage were similarly abundant in the GPe and displayed a heterogeneous makeup. Tracing experiments revealed that Npas1+-Nkx2.1+ neurons represent the principal, non-cholinergic, cortically-projecting neurons; they project profusely in the cortex and are part of a cortico-pallidal-cortical loop. Lastly, analysis of the spatial distribution and electrophysiological properties of a number of GPe neuron types further confirms the diversification of GPe subtypes. In summary, we provide improved descriptions of GPe neuron subtypes. By delineating different GPe neurons and their synaptic partners, our findings establish the circuit substrates that can be important for motor function and dysfunction. Our findings reconcile some of the discrepancies that arose from differences in techniques or the reliance on pre-existing tools.
Microglia suppress the secondary progression of autoimmune encephalomyelitis
- Difco Laboratories) by subcutaneous injection. Pertussis toxin (200 ng; List Biological Laboratories, Campbell, CA, USA) was intraperitoneally administrated at 0‐ and 48‐hr postimmunization. EAE scores were assessed daily
For active immunization 59 573 mice were immunized subcutaneously into the flanks with 200 µg 574 MOG35-55 peptide (Anawa) emulsified in complete Freund’s adjuvant (CFA; InvivoGen) and 575 200 ng pertussis toxin (List Biological Laboratories Inc.) intraperitoneally on day 0 and day 2 post-immunization.
Brain endothelium possesses several organ-specific features collectively known as the blood-brain barrier (BBB). In addition, trafficking of immune cells in the healthy central nervous system (CNS) is tightly regulated by CNS vasculature. In CNS autoimmune diseases such as multiple sclerosis (MS), these homeostatic mechanisms are overcome by autoreactive lymphocyte entry into the CNS causing inflammatory demyelinating immunopathology. Previous studies have shown that pericytes regulate the development of organ-specific characteristics of brain vasculature such as the BBB and astrocytic end-feet. Whether pericytes are involved in the control of leukocyte trafficking remains elusive. Using adult, pericyte-deficient mice (Pdgfbret/ret), we show here that brain vasculature devoid of pericytes shows increased expression of VCAM-1 and ICAM-1, which is accompanied by increased leukocyte infiltration of dendritic cells, monocytes and T cells into the brain, but not spinal cord parenchyma. Regional differences enabling leukocyte trafficking into the brain as opposed to the spinal cord inversely correlate with the pericyte coverage of blood vessels. Upon induction of experimental autoimmune encephalitomyelitis (EAE), pericyte-deficient mice succumb to severe neurological impairment. Treatment with first line MS therapy - fingolimod significantly reverses EAE, indicating that the observed phenotype is due to the massive influx of immune cells into the brain. Furthermore, pericyte-deficiency in mice that express myelin oligodendrocyte glycoprotein peptide (MOG35-55) specific T cell receptor (Pdgfbret/ret; 2D2Tg) leads to the development of spontaneous neurological symptoms paralleled by massive influx of leukocytes into the brain, suggesting altered brain vascular immune quiescence as a prime cause of exaggerated neuroinflammation. Thus, we show that pericytes indirectly restrict immune cell transmigration into the CNS under homeostatic conditions and during autoimmune-driven neuroinflammation by inducing immune quiescence of brain endothelial cells.