Debayon PaulPostdoctoral Fellow
I’m interested in investigating the association of blood-brain barrier (BBB) damage and leukocyte infiltration into the central nervous system (CNS) parenchyma that underlies neuroinflammation in an animal model of multiple sclerosis (MS), termed experimental autoimmune encephalomyelitis (EAE). Understanding these key events will help in the broader understanding of the mechanisms underlying early stages of MS pathogenesis and emergence of treatment options.
Figure 1. Perivascular leukocyte infiltration in inflamed spinal venules. z-stack images of naïve and d24 EAE spinal cord venules, stained for leukocyte marker CD4, endothelial marker CD31, and nuclear stain DRAQ5. (a) Naïve venule shows intact CD31 staining along the intercellular junctions but absence of CD4 staining or perivascular cellularity. (b) Inflamed d24 EAE venule reveals CD4 staining is largely coincident with perivascular cellularity. (Microvasc. Res., 2013)
To address the question, I’ve recently developed a novel three-dimensional (3D) image visualization and analysis approach on high-resolution confocal images acquired from thick spinal cord cryosections (Paul et al., 2013). Using this, our lab was able to demonstrate a significant heterogeneity in a predominant CNS tight-junction (TJ) protein, Claudin-5 (CLN-5), among the various microvessels (venules, post-capillary venules and capillaries) in a naïve/normal milieu and its focal loss in specific microvascular compartments in EAE.
Figure 2. 3D contour-based quantification of junctional CLN-5 in spinal cord microvessels. CNS microvascular tributaries (e.g. capillaries, post-capillary venules) emerging from a venule of a naïve mouse detailing CLN-5 (green) staining at intercellular junctions. To quantify CLN-5 staining associated with a microvessel in 3D, an individual contour for each confocal z-slice was created by cursoring out the vessel of interest (white arrows) and the individual z-slice contours merged into a 3D contour surface. The contour surface was utilized to isolate the microvessel of interest by masking, and its area approximated the microvascular surface area. An isosurface for the CLN-5 channel was then created from the selected vessel for statistical analysis. (Microvasc. Res., 2013)
Our lab is also interested in exploring the role of CNS cell source-specific CCL2, a chemokine known to surge during neuroinflammation, and contribute to the disease pathology. My subsequent studies, using astrocyte (Astro KO) and endothelial (Endo KO) - specific CCL2 knockout mice generated previously (Ge et al., 2009) in our lab and 3D image analysis, demonstrated that CCL2 released from endothelium and astrocytes in the CNS during disease, uniquely guides leukocytes to cross the respective basement membranes (BMs) (Paul et al., 2014).
Figure 3. Astro KO and Endo KO mice display differences in perivascular cellularity associated with CNS venules in EAE. z-stack images of inflamed venules at d16 EAE stained for BM protein Laminin 1 (Lam 1) (red), CLN-5 (green), and nuclei/DRAQ5 (blue). Longitudinal sections reveal the extent of vessel-associated leukocytes. CLN-5 staining is presented to highlight the endothelial boundary. Insets represent enlarged view of areas highlighted in white hatched boxes, while double-headed arrows denote the ‘perivascular’ space between the endothelial and parenchymal BMs. (a) In wild type (WT) mice, a dense accumulation of DRAQ5+ perivascular cells (representing leukocytes) is seen, with a visibly fragmented pattern of CLN-5 staining. A few leukocytes apparently penetrating the fragmented parenchymal BM are highlighted with a white arrowhead. (b) In Astro KO mice, a similar dense perivascular cellularity and loss of CLN-5 staining is observed, with visibly intact parenchymal BM and lack of parenchymal leukocyte migration. (c) In Endo KO mice, both BM and CLN-5 staining is apparently intact, with minimal perivascular cellularity. (J. Neuroinflammation, 2014)
Alterations in tight junction protein and IgG permeability accompany leukocyte extravasation across the choroid plexus during neuroinflammation. J Neuropathol Exp Neurol. 2014 Nov;73(11):1047-61.
Human ESC-derived MSCs outperform bone marrow MSCs in the treatment of an EAE model of multiple sclerosis. Stem Cell Reports. 2014 Jun 6;3(1):115-30.
Cell-selective knockout and 3D confocal image analysis reveals separate roles for astrocyte-and endothelial-derived CCL2 in neuroinflammation. J Neuroinflammation. 2014 Jan 21;11(1):10. Paul D, Ge S, Lemire Y, Jellison ER, Serwanski DR, Ruddle NH, Pachter JS.
P-glycoprotein regulates trafficking of CD8 T cells to the brain parenchyma. Acta Neuropathol. 2014 Jan 16. Kooij G, Kroon J, Paul D, Reijerkerk A, Geerts D, van der Pol SM, van Het Hof B, Drexhage JA, van Vliet SJ, Hekking LH, van Buul JD, Pachter JS, de Vries HE.
Novel 3D analysis of Claudin-5 reveals significant endothelial heterogeneity among CNS microvessels. Microvasc Res. 2013 Mar;86:1-10. Paul D, Cowan AE, Ge S, Pachter JS.
Active induction of experimental autoimmune encephalomyelitis by MOG35-55 peptide immunization is associated with differential responses in separate compartments of the choroid plexus. Fluids Barriers CNS. 2012 Aug 7;9(1):15. Murugesan N, Paul D, Lemire Y, Shrestha B, Ge S, Pachter JS.
The CCL2 synthesis inhibitor bindarit targets cells of the neurovascular unit, and suppresses experimental autoimmune encephalomyelitis. J Neuroinflammation. 2012 Jul 12;9:171. Ge S, Shrestha B, Paul D, Keating C, Cone R, Guglielmotti A, Pachter JS. [PMID: 22788993]
Coadministration of adenoviral vascular endothelial growth factor and angiopoietin-1 enhances vascularization and reduces ventricular remodeling in the infarcted myocardium of type 1 diabetic rats. Diabetes. 2010 Jan;59(1):51-60. Akita Y, Samuel SM, Paul D, Thirunavukkarasu M, Zhan L, Li C, Maulik N.
Mesenchymal stem cell: present challenges and prospective cellular cardiomyoplasty approaches for myocardial regeneration. Antioxid Redox Signal, 2009 Aug;11(8):1841-55. Paul D, Samuel SM, Maulik N.
Akt/FOXO3a/SIRT1-mediated cardioprotection by n-tyrosol against ischemic stress in rat in vivo model of myocardial infarction: switching gears toward survival and longevity. J Agric Food Chem, 2008 Oct 22;56(20):9692-8.. Samuel SM, Thirunavukkarasu M, Penumathsa SV, Paul D, Maulik N.