Department of Cell Biology
Education and Training
B.A., Queens College, New York
Ph.D., New York University, New York
263 Farmington Avenue
Farmington, CT 06030
The major focus in this laboratory is to elucidate the mechanisms by which leukocytes and pathogens invade the central nervous system (CNS). Movement of cellular elements into the CNS is typically limited by brain microvascular endothelial cells (BMEC) comprising the blood-brain barrier (BBB). It is thus believed that alterations in the BBB contribute to the pathogenesis of various neuroinflammatory, neuroinfectious and neurodegenerative diseases such as multiple sclerosis, AIDS dementia complex and Alzheimer disease. To evaluate how the BBB may be modified in these conditions, we are using both in vitro and in vivo approaches. The in vitro approach involves transwell assays on primary BMECs, isolated and cultured in our lab [Fig. 1]. Such “BBB models” are routinely constructed in the laboratory from mouse or human BMECs, and enable direct measurement of leukocyte transendothelial migration (TEM) and permeability to a wide host of substrates [Fig. 2]. The mechanisms by which cytokines and chemokines influence TEM and permeability at the BBB are currently being investigated with these culture models.
Figure 1. TEM image of a monocyte undergoing transendothelial migration (TEM). In a cell culture model of the BBB, human brain microvascular endothelial cells (BMECs) were cultured atop a hydrated collagen gel coating a Transwell® filter (In Vitro Cell Devel Biol, 2001). Freshly isolated human monocytes were introduced to the upper Transwell® chamber, and the chemokine CCL2 (monocyte chemoattractant protein/MCP-1) (100ng/ml) placed in the bottom chamber. After about 6 hr, the BBB model was fixed in glutaraldehyde and processed for transmission electron microscopy. Two monocytes (arrows) can be seen crossing the BBB model and invading the collagen gel – penetrating between the overhanging processes of two BMECs, to which the infiltrating monocytes still seem to be just barely attached.
Figure 2. Monocyte migration in a 3D endothelial culture. Representative z-stack confocal images, volume rendered with VoxelView software, showing temporal transmigration of CellTracker Green labeled monocytes across endothelial cells labeled with CellTracker Orange, under influence of chemokine CCL2 (monocyte chemoattractant protein/MCP-1) (100ng/ml) placed in the bottom chamber. The endothelial cells were cultured atop hydrated collagen gels supported on Transwell® filters. (In Vitro Cell Devel Biol Anim., 2001)
The in vivo approach uses experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, as a paradigm to study neuroinflammation. Specifically, high resolution, 3D fluorescence microscopy enables the interactions of leukocytes and BMEC to be viewed holistically, highlighting features of the TEM process not discernible with conventional, 2D microscopic methods. Leukocyte interactions with endothelial junctional components are currently a topic of investigation [Fig. 3].
Figure 3. BBB damage in inflamed microvessels during EAE. Representative z-stack confocal images of inflamed spinal cord vessels in mice inflicted with EAE showing three seminal signs of inflammation- (a) Leakage of serum proteins, (b) parenchymal leukocyte infiltration, and (c) separation of parenchymal and endothelial basement membranes to accommodate the extravasated leukocytes. The sections were stained for basement membrane protein Laminin1 (Lam1) (Red) and tight-junction protein Claudin-5 (CLN-5) (Green). DRAQ5 nuclear stain (Blue) reveals the perivascular cellularity surrounding inflamed microvessels. (Microvasc. Res., 2013)
As part of the in vivo approach, this laboratory is also exploiting cell-targeted knockouts of CCL2, a chemokine indispensable for pathogenesis of numerous neuroinflammatory, neuroinfectious and neurodegenerative conditions. Current investigations include determining how elimination of CCL2 from selected cell depots influences different steps in disease pathogenesis [Fig. 4].
Figure 4. CCL2 expression in spinal cord of Astrocyte CCL2 (Astro KO) and Endothelial (Endo KO) CCL2 knockout (KO) mice during EAE. Representative z-stack confocal images from spinal cord cryosections from KO mice at d16 EAE immunostained for chemokine CCL2 (Red) and endothelial marker, CD31 or astrocyte marker, GFAP (Green) are depicted. Cell-specific CCL2-KO mice display loss of CCL2 staining in respective targeted cell types. (a) Astro KO mice show venule-associated CCL2 staining, but lack staining in the parenchymal astrocytes. (b) In contrast, Endo KO mice are deficient in vessel-associated CCL2 staining, but maintain astrocyte staining (right). Boundary of the endothelium is marked with yellow lines. Insets show co-localization of CCL2 with CD31 or GFAP (yellow) in a single z-slice from the respective regions marked by the hatched white boxes, or CCL2 (red) channel alone. (J. Neuroinflammation., 2013, in press)
This laboratory is also investigating how “sentinel” leukocytes initially invade the non-inflamed CNS by crossing the choroid plexus into the cerebrospinal fluid (CSF), and thereafter migrate in the CSF to designated sites within meninges to provoke inflammatory impulses that propagate into the parenchyma and along the CNS axis [Fig. 5].
Figure 5: Leukocytes within the Choroid Plexus (CP) stromal compartment. CP epithelium of a naïve mouse was stained with polyclonal antibody to pan-cytokeratin (FITC, green), and CP stromal capillaries were immunostained with monoclonal anti-CD31 antibody (red). A CD45 antibody was used to stain for any leukocytes present within the CP (blue). Confocal microscopy z-stack images of thick (20 μm) frozen sections of the CP were acquired, and three dimensional rendering was performed using Imaris® image analysis software. Inset showing leukocytes within the stromal compartment between the capillaries and choroidal epithelial cells. (Fluids Barriers CNS, 2012).
Lastly, laser capture microdissection (LCM) is being used to evaluate gene expression profiles – in situ – of designated CNS vascular, epithelial, and parenchymal cells at various stages following CNS insult. LCM can be coupled various profiling platforms to detect single genes, larger groups of genes (several hundreds) or whole transcriptomes [Fig. 6].
Figure 6: Immuno-LCM allows retrieval of tissue from specific CP compartments. Evidence of histological purity. Immunofluorescence was performed using FITC-conjugated pan-cytokeratin antibody to highlight the CP epithelium (green), while immunohistochemistry using alkaline phosphatase detection with NBT/BCIP as substrate was carried-out to label the endothelium of CP stromal capillaries (dark brown). LCM was performed on a Pixcell IIe LCM unit. Images both BEFORE and AFTER LCM, as well as LCM retrieved tissue deposited on the cap, are shown to highlight selective retrieval of CP stromal capillary (top row) and CP choroidal epithelial tissues (bottom row). (Fluids Barriers CNS, 2012)
In collaboration with ImStem Biotechnology, current efforts are aimed at evaluating how transplanted human, embryonic stem cell-derived mesenchymal stems cells direct therapeutic and reparative effects in the disease CNS.
- Genetic Profiling for the Blood Brain Barrier Diseases
- 3D Image Visualization and Analysis with Imaris*
- Studying the Choroid Plexus During Neuroinflammation in 3D
- Studying the Blood-Brain Barrier Tight Junction Proteins in 3D.
Lab Members See posters of current work!
Debayon Paul, Valentina Baena, Shujun Ge, Xi Jiang, Evan R. Jellison, Timothy Kiprono, Dritan Agalliu and Joel S. Pachter. Appearance of claudin-5+ leukocytes in the central nervous system during neuroinflammation: a novel role for endothelial-derived extracellular vesicles. Journal of Neuroinflammation (2016) 13:292 DOI 10.1186/s12974-016-0755-8. pdf
Shrestha B, Paul D, Pachter JS. Alterations in Tight Junction Protein and IgG Permeability Accompany Leukocyte Extravasation Across the Choroid Plexus During Neuroinflammation. . J Neuropathol Exp Neurol. 2014 Oct 6.
Wang X, Kimbrel EA, Ijichi K, Paul D, Lazorchak AS, Chu J, Kouris NA, Yavanian GJ, Lu SJ, Pachter JS, Crocker SJ, Lanza R, Xu RH. 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.
Paul D, Ge S, Lemire Y, Jellison ER, Serwanski DR, Ruddle NH, Pachter JS . Cell-selective knockout and 3D confocal image analysis reveals separate roles for astrocyte- and endothelial-derived CCL2 in neuroinflammation. Dec 2013, J. Neuroinflammation, In press.
Kooij G, Reijerkerk A, Kroon J, Paul D, Geerts D, van der Pol SMA, van het Hof B, Drexhage J, van Vliet SJ, Hekking LHP, van Buul JD, Pachter JS, de Vries HE. 2013. P-glycoprotein regulates trafficking of CD8+ T cells to the central nervous system, Dec 2013. Acta Neuropathologica, In press.
Paul D, Cowan AE, Ge S, Pachter JS. Novel 3D analysis of Claudin-5 reveals significant endothelial heterogeneity among CNS microvessels. Microvasc Res. 2013 Mar;86:1-10.
Murugesan N, Paul D, Lemire Y, Shrestha B, 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.
Ge S, Shrestha B, Paul D, Keating C, Cone R, Guglielmotti A, 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
Demarest TG, Murugesan N, Shrestha B, Pachter JS. .Rapid expression profiling of brain microvascular endothelial cells by immuno-laser capture microdissection coupled to TaqMan® low density array. J Neurosci Methods. 2012;206(2):200-4.
Murugesan N, Demarest TG, Madri JA, Pachter JS. Brain regional angiogenic potential at the neurovascular unit during normal aging. Neurobiol Aging. 2012 May;33(5):1004.e1-16.
Murugesan N, Macdonald JA, Lu Q, Wu SL, Hancock WS, Pachter JS. Analysis of mouse brain microvascular endothelium using laser capture microdissection coupled with proteomics. Methods Mol Biol. 2011;686:297-311.
Macdonald JA, Murugesan N, Pachter JS. Endothelial cell heterogeneity of blood-brain barrier gene expression along the cerebral microvasculature. Neurosci Res. 2010 May 15;88(7):1457-74.
Ge S, Murugesan N, Pachter JS. Astrocyte- and endothelial-targeted CCL2 conditional knockout mice: critical tools for studying the pathogenesis of neuroinflammation. J Mol Neurosci. 2009 Sep;39(1-2):269-83.
Ge S, Song L, Serwanski DR, Kuziel WA, Pachter JS. Transcellular transport of CCL2 across brain microvascular endothelial cells. J Neurochem. 2008 Mar;104(5):1219-32.
Song L, Ge S, Pachter JS. Caveolin-1 regulates expression of junction-associated proteins in brain microvascular endothelial cells. Blood. 2007 Feb 15;109(4):1515-23.