Gene Expression Analysis of Resident Macrophages in Lipopolysaccharide-stimulated Rat Molar Pulps

Toll-like Receptor 2 Knockout Mice Showed Increased Periapical Lesion Size and Osteoclast Number

“…This receptor is largely expressed in blood leukocytes and different inflammatory cells, participating in the immune response to stimuli by increasing nuclear factor kappa B expression (15,16). It has been shown that TLR2 is expressed by neutrophils (3,4), mast cells (17), monocytes and macrophages (4,18), T cells (19), regulatory T cells (20), and B cells (21). Additionally, studies in murine pulp cells showed TLR2 expression in fibroblasts, odontoblasts, and dendritic cells (22,23).…”

A novel whole tooth‐in‐jaw‐bone culture of rat molars: Morphological, immunohistochemical, and laser capture microdissection analysis

“…The whole tooth‐in‐jaw‐bone culture system presented here may also be useful in analyzing resident type of immunocompetent cells, since the effects of blood‐borne cells can be eliminated. In this regard, we have already demonstrated, using a short‐term whole‐tooth culture for 3 days that lipopolysaccharide (LPS) treatment of cultured rat molars caused the accumulation of resident macrophages and enhanced the expression of Toll‐like receptor 4, CD14, colony stimulating factor‐1, and CX3C chemokine receptor 1 mRNAs in these cells (Chokechanachaisakul et al,2011). Upregulation of these molecules may be involved in the differentiation and subsequent migration of resident macrophages of the pulp (Chokechanachaisakul et al,2011).…”

“…In this regard, we have already demonstrated, using a short‐term whole‐tooth culture for 3 days that lipopolysaccharide (LPS) treatment of cultured rat molars caused the accumulation of resident macrophages and enhanced the expression of Toll‐like receptor 4, CD14, colony stimulating factor‐1, and CX3C chemokine receptor 1 mRNAs in these cells (Chokechanachaisakul et al,2011). Upregulation of these molecules may be involved in the differentiation and subsequent migration of resident macrophages of the pulp (Chokechanachaisakul et al,2011). Moreover, this model may also lay the foundation for defining the molecular mechanism of pulp tissue pathology and thus may contribute greatly to various research fields involving the dentin/pulp complex, including basic aspects of cell biology, mechanobiology, biomaterial sciences, and engineering of the dental pulp tissue.…”

“…The frozen samples ( n = 6, each group) were cut mesio‐distally in a cryostat at 8‐μm thick. Immunostaining was performed using a monoclonal antibody ED2 (anti‐rat resident macrophage; AbD Serotec (MCA342R), Oxford, UK; diluted 1:200) and the avidin–biotin–peroxidase complex method as previously described (Chokechanachaisakul et al,2010a, 2011; Kaneko et al,2008). Following incubation in 3% H 2 O 2 for 5 min, the sections were sequentially incubated with the ED2 antibody diluted with PBS for 24 h at 4°C, biotinylated horse anti‐mouse IgG (rat adsorbed; Vector, Burlingame, CA) for 2 hrs, and avidin–biotin–peroxidase complex (Elite ABC kit; Vector) for 1 h. The sections were then developed with a 3,3′‐diaminobenzidine (DAB) substrate kit (Vector), counterstained with methyl green (Sigma‐Aldrich, St. Louis, MO), and examined under a light microscope (Nikon, Tokyo, Japan).…”

“…Approximately 50 ED2‐positive cells were retrieved from the coronal third of the root pulp of each section into individual tubes filled with a storing medium (RNAlater®, Ambion, Austin, TX), and placed immediately on ice. Finally, a total of ∼200 ED2 positive cells were collected from four serial sections of each sample, as we previously reported (Chokechanachaisakul et al,2011). The average total RNA yield was 1.2 μg per sample with an average corrected A260/A280 ratio of 1.9.…”