Transcriptomic (DNA Microarray) and Metabolome (LC-TOF-MS) Analyses of the Liver in High-Fat Diet Mice after Intranasal Administration of GALP (Galanin-like Peptide).

The aim of this research was to test the efficacy and potential clinical application of intranasal administration of galanin-like peptide (GALP) as an anti-obesity treatment under the hypothesis that GALP prevents obesity in mice fed a high-fat diet (HFD). Focusing on the mechanism of regulation of lipid metabolism in peripheral tissues via the autonomic nervous system, we confirmed that, compared with a control (saline), intranasally administered GALP prevented further body weight gain in diet-induced obesity (DIO) mice with continued access to an HFD. Using an omics-based approach, we identified several genes and metabolites in the liver tissue of DIO mice that were altered by the administration of intranasal GALP. We used whole-genome DNA microarray and metabolomics analyses to determine the anti-obesity effects of intranasal GALP in DIO mice fed an HFD. Transcriptomic profiling revealed the upregulation of flavin-containing dimethylaniline monooxygenase 3 (Fmo3), metallothionein 1 and 2 (Mt1 and Mt2, respectively), and the Aldh1a3, Defa3, and Defa20 genes. Analysis using the DAVID tool showed that intranasal GALP enhanced gene expression related to fatty acid elongation and unsaturated fatty acid synthesis and downregulated gene expression related to lipid and cholesterol synthesis, fat absorption, bile uptake, and excretion. Metabolite analysis revealed increased levels of coenzyme Q10 and oleoylethanolamide in the liver tissue, increased levels of deoxycholic acid (DCA) and taurocholic acid (TCA) in the bile acids, increased levels of taurochenodeoxycholic acid (TCDCA), and decreased levels of ursodeoxycholic acid (UDCA). In conclusion, intranasal GALP administration alleviated weight gain in obese mice fed an HFD via mechanisms involving antioxidant, anti-inflammatory, and fatty acid metabolism effects and genetic alterations. The gene expression data are publicly available at NCBI GSE243376.

Histamine deficiency inhibits lymphocyte infiltration in the submandibular gland of aged mice via increased anti-aging factor Klotho.

OBJECTIVES: Histidine decarboxylase (HDC), a histamine synthase, is expressed in various tissues and is induced by proinflammatory cytokines such as TNFα. As they age, C57BL/6 mice show auto-antibody deposition and lymphocyte infiltration into various tissues, including salivary glands. However, the mechanism underlying cell infiltration and the change in HDC expression in salivary glands with aging remain unclear. Thus, we aimed to elucidate the relationship between histamine and inflammaging. METHODS: We investigated the change in histology and HDC expression in the major salivary glands (parotid, submandibular, and sublingual) of 6-week- and 9-month-old wild-type mice. We also determined the histological changes, cytokine expression, and anti-aging factor Klotho in the salivary glands of 9-month-old wild-type and HDC-deficient (HDC-KO) mice. RESULTS: Cell infiltration was observed in the submandibular gland of 9-month-old wild-type mice. Although most cells infiltrating the submandibular glands were CD3-positive and B220-positive lymphocytes, CD11c-positive and F4/80-positive monocyte lineages were also detected. HDC, TNFα, and IL-1ß mRNA expression increased in the submandibular gland of 9-month-old wild-type mice. The expression of PPARγ, an anti-inflammatory protein, declined in 9-month-old wild-type mice, and Klotho expression increased in 9-month-old HDC-KO mice. Immunohistochemistry showed that Klotho-positive cells disappeared in the submandibular gland of 9-month-old wild-type mice, while Klotho was detected in all salivary glands in HDC-KO mice of the same age. CONCLUSION: Our findings demonstrate the multifunctionality of histamine and can aid in the development of novel therapeutic methods for inflammatory diseases such as Sjogren's syndrome and age-related dysfunctions.

Molecular and Physiological Functions of PACAP in Sweat Secretion.

Sweat plays a critical role in human body, including thermoregulation and the maintenance of the skin environment and health. Hyperhidrosis and anhidrosis are caused by abnormalities in sweat secretion, resulting in severe skin conditions (pruritus and erythema). Bioactive peptide and pituitary adenylate cyclase-activating polypeptide (PACAP) was isolated and identified to activate adenylate cyclase in pituitary cells. Recently, it was reported that PACAP increases sweat secretion via PAC1R in mice and promotes the translocation of AQP5 to the cell membrane through increasing intracellular [Ca2+] via PAC1R in NCL-SG3 cells. However, intracellular signaling mechanisms by PACAP are poorly clarified. Here, we used PAC1R knockout (KO) mice and wild-type (WT) mice to observe changes in AQP5 localization and gene expression in sweat glands by PACAP treatment. Immunohistochemistry revealed that PACAP promoted the translocation of AQP5 to the lumen side in the eccrine gland via PAC1R. Furthermore, PACAP up-regulated the expression of genes (Ptgs2, Kcnn2, Cacna1s) involved in sweat secretion in WT mice. Moreover, PACAP treatment was found to down-regulate the Chrna1 gene expression in PAC1R KO mice. These genes were found to be involved in multiple pathways related to sweating. Our data provide a solid basis for future research initiatives in order to develop new therapies to treat sweating disorders.

Ontogenetic development of the water channel protein AQP5 in mouse salivary gland tissue.

Aquaporins (AQP) are a family of channel proteins expressed in the cell membranes of many tissue types. As water channels, they enable the selective permeation of water molecules and thus play an important role in water transport through the plasma membrane. There are numerous AQP sub-types, among which AQP5 is expressed in the salivary glands. The expression and localization of AQP5 in different salivary gland cells of animal models during fetal development and after birth have enabled the physiological functions of AQP5 to be elucidated, but subsequent changes in the adult phase are unknown. It is known that saliva production tends to decrease with age, but it is unclear how AQP5 activity and function changes developmentally, from young to old including gender differences. In the present study, we sampled the parotid, submandibular, and sublingual glands from young (8 weeks old) and aged (12 months old) mice of both sexes to study the effects of age- and sex-related differences in AQP5 expression. Positive fluorescence immunostaining was detected in the membranes of cells from all gland types, and this was enhanced in juvenile mice from both sexes. Western blot analyses revealed that AQP5 expression levels tended to decrease with age in both male and female animals. Conversely, AQP5 gene expression levels did not change significantly with aging, but were found to be high in submandibular gland cells of both sexes, in parotid gland cells of older female mice, and in the sublingual gland cells of young male mice.

An in situ hybridization study of syndecan family during the late stages of developing mouse molar tooth germ.

Expression of syndecan-1, 2, 3, and 4 mRNAs during the late stages of tooth germ formation was investigated by in situ hybridization, using [35S]-UTP-labeled cRNA probes. Syndecan-1 mRNA was mainly expressed in the stellate reticulum and stratum intermedium as well as at the cervical region of dental papilla/dental follicle during E18.5-P3.0. Expression in the dental epithelium was enhanced during the postnatal periods, which was supported by real-time RT-PCR analysis. These spatiotemporal expression patterns may suggest specific roles of syndecan-1 in tooth formation such as tooth eruption or root formation. Syndecan-3 mRNA expression became evident in odontoblasts at E18.5, but compared to collagen type I mRNA, which was strongly expressed at this stage, syndecan-3 expression in odontoblast was restricted in mature odontoblasts beneath the cusps during the postnatal periods. This result was also supported by real-time RT-PCR analysis, and indicated that syndecan-3 may be involved in the progress of dentinogenesis rather than in the initiation of it. Syndecan-4 mRNA roughly showed comparable expression patterns to those of syndecan-3. Syndecan-2 mRNA did not show significant expression during the experimental period, but real-time RT-PCR analysis suggested that syndecan-2 expression might be enhanced with hard tissue formation.

Pituitary adenylate cyclase-activating polypeptide: Protective effects in stroke and dementia.

Evidence shows that pituitary adenylate cyclase-activating polypeptide (PACAP) improves stroke outcomes and dementia. The blood-brain barrier (BBB) controls the peptide and regulatory protein exchange between the central nervous system and the blood; the transport of these regulatory substances across the BBB has been altered in animal models of stroke and Alzheimer's disease (AD). PACAP is a powerful neurotrophin that can cross the BBB, which may aid in the therapy of neurodegenerative diseases, including stroke and AD. PACAP may function as a potential drug in the treatment, prevention, or management of stroke and AD and other neurodegenerative conditions. Here, we review the effects of PACAP in studies on stroke and dementias.

Effects of PACAP on Dry Eye Symptoms, and Possible Use for Therapeutic Application.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 27- or 38-amino acid neuropeptide, which belongs to the vasoactive intestinal polypeptide/glucagon/secretin family of peptides. PACAP and its three receptor subtypes are expressed in neural tissues and in the eye, including the retina, cornea, and lacrimal gland. PACAP is known to exert pleiotropic effects on the central nervous system and in eye tissues where it plays important roles in protecting against dry eye. This review provides an overview of current knowledge regarding dry eye symptoms in aged animals and humans and the protective effects, mechanisms of action. In addition, we also refer to the development of a new preventive/therapeutic method by PACAP of dry eye patients.

Regulation of Feeding Behavior and Energy Metabolism by Galanin-like Peptide (GALP): A Novel Strategy to Fight Against Obesity.

Galanin-like peptide (GALP) is composed of 60 amino acid residues and its sequence is highly homologous across species. GALP is produced in the hypothalamic arcuate nucleus and has diverse physiological effects such as the regulation of feeding, energy metabolism, and reproductive behavior. GALP-containing neurons express leptin receptors and these neurons form networks in the hypothalamus that contain various peptides that regulate feeding behavior. Recent studies have revealed that GALP has a central anti-obesity action in addition to its role in food intake regulation. Furthermore, we have found that the respiratory quotient declines shortly after administration of GALP into the lateral ventricle. This suggests that lipid metabolism is accelerated by GALP administration, and identifies a new physiological action for this peptide. In this review article, we summarize our recent research focusing on the mechanism whereby GALP regulates feeding and energy metabolism. We concentrate on the mechanism of regulation of lipid metabolism in peripheral tissues via the autonomic nervous system and outline the effectiveness of the nasal administration of GALP and basic research towards its clinical application.

Comprehensive genetic exploration of selective tooth agenesis of mandibular incisors by exome sequencing.

Tooth agenesis is described as the absence of one or more teeth. It is caused by a failure in tooth development and is one of the most common human developmental anomalies. We herein report genomic analyses of selective mandibular incisor agenesis (SMIA) using exome sequencing. Two Japanese families with SMIA were subjected to exome sequencing, and family with sequence similarity 65 member A (FAM65), nuclear factor of activated T-cells 3 (NFATC3) and cadherin-related 23 gene (CDH23) were detected. In the follow-up study, 51 Japanese and 32 Korean sporadic patients with SMIA were subjected to exome analyses, and 18 reported variants in PAX9, AXIN2, EDA, EDAR, WNT10A, BMP2 and GREM2 and 27 variants of FAM65, NFATC3 and CDH23 were found in 38 patients. Our comprehensive genetic study of SMIA will pave the way for a full understanding of the genetic etiology of SMIA and provide targets for treatment.

Nitrogen-containing bisphosphonate induces a newly discovered hematopoietic structure in the omentum of an anemic mouse model by stimulating G-CSF production.

We previously reported that the injection of nitrogen-containing bisphosphonate (NBP) induced the site of erythropoiesis to shift from the bone marrow (BM) to the spleen. Our previous study established a severely anemic mouse model that was treated with a combination of NBP with phenylhydrazine (PHZ), which induced newly discovered hematopoietic organs in the omentum. No reports have shown that new hematopoietic organs form under any condition. We characterized the structures and factors related to the formation of these new organs. Splenectomized mice were treated with NBP to inhibit erythropoiesis in the BM and then injected with PHZ to induce hemolytic anemia. The mice showed severe anemia and wine-colored structures appeared in the omentum. Some hematopoietic cells, including megakaryocytes, and well-developed sinuses were observed in these structures. Numerous TER119-positive erythroblasts were located with cells positive for PCNA, a cell proliferation marker. C-kit-positive cells were detected and mRNAs related to hematopoiesis were expressed in these structures. Moreover, TER119-positive erythroblasts emerged and formed clusters and hematopoiesis-related factors were detected in the omentum of mice treated with NBP and PHZ. The levels of G-CSF in the serum and hematopoietic progenitor cells (HPCs) in the peripheral blood were increased upon treatment with both NBP and PHZ. These results suggest that the induced hematopoietic structures act as the sites of erythropoiesis and that NBP-induced G-CSF production causes HPC mobilization, homing and colonization in the omentum because they constitutively express some factors, including SDF-1; thus, the newly discovered hematopoietic structure in this study might be formed.

Anti-obesity effect of intranasal administration of galanin-like peptide (GALP) in obese mice.

Galanin-like peptide (GALP) has an anti-obesity effect in rats and mice. It has been reported that the uptake of GALP by the brain is higher after intranasal administration than with intravenous injection. This study therefore aimed to clarify the effect of intranasal administration of GALP on the feeding behavior of lean and obese mice. Autoradiography revealed the presence of (125)I-GALP in the olfactory bulb and the brain microcirculation. The body weights of ob/ob mice gradually increased during vehicle treatment, but remained unchanged in response to repeated intranasal administration of GALP, with both ob/ob and diet-induced obese mice displaying significantly decreased food intake, water intake and locomotor activity when treated with GALP. These results suggest that intranasal administration is an effective route whereby GALP can exert its effect as an anti-obesity drug.

Pituitary adenylate cyclase-activating polypeptide enhances saliva secretion via direct binding to PACAP receptors of major salivary glands in mice.

Xerostomia, or dry mouth, is a common syndrome that is generally treated with artificial saliva; however, no other effective methods have yet been established. Saliva secretion is mainly under the control of the autonomic nervous system. Pituitary adenylate cyclase-activating polypeptide (PACAP) is recognized as a multifunctional neuropeptide in various organs. In this study, we examined the effect of PACAP on saliva secretion, and detected the distribution of the PACAP type 1 receptor (PAC1R) in major salivary glands, including the parotid, submandibular, and sublingual glands, in 9-week-old male C57BL/6 mice. Intranasal administration of PACAP 38 increased the amount of saliva secreted, which was not inhibited by atropine pretreatment. Immunohistochemical analysis showed that PAC1R was distributed in the three major salivary glands. In the parotid and sublingual glands, PAC1R was detected in striated duct cells, whereas in the submandibular gland, a strong PAC1R immunoreaction was detected in tall columnar epithelial cells in the granular ducts (i.e., pillar cells), as well as in some striated duct cells. PACAP significantly increased the concentration of epidermal growth factor in saliva. These results suggest that PACAP directly regulates saliva secretion by controlling the absorption activity in the ducts, and that pillar cells regulate the function of granular epithelial cells in the granular duct, such as the secretion of growth factors into the saliva. Collectively, these results suggest the possibility of PACAP as a new effective treatment of xerostomia. Anat Rec, 299:1293-1299, 2016. © 2016 Wiley Periodicals, Inc.

The expression of embryonic globin mRNA in a severely anemic mouse model induced by treatment with nitrogen-containing bisphosphonate.

BACKGROUND: Mammalian erythropoiesis can be divided into two distinct types, primitive and definitive, in which new cells are derived from the yolk sac and hematopoietic stem cells, respectively. Primitive erythropoiesis occurs within a restricted period during embryogenesis. Primitive erythrocytes remain nucleated, and their hemoglobins are different from those in definitive erythrocytes. Embryonic type hemoglobin is expressed in adult animals under genetically abnormal condition, but its later expression has not been reported in genetically normal adult animals, even under anemic conditions. We previously reported that injecting animals with nitrogen-containing bisphosphonate (NBP) decreased erythropoiesis in bone marrow (BM). Here, we induced severe anemia in a mouse model by injecting NBP injection in combination with phenylhydrazine (PHZ), and then we analyzed erythropoiesis and the levels of different types of hemoglobin. METHODS: Splenectomized mice were treated with NBP to inhibit erythropoiesis in BM, and with PHZ to induce hemolytic anemia. We analyzed hematopoietic sites and peripheral blood using morphological and molecular biological methods. RESULTS: Combined treatment of splenectomized mice with NBP and PHZ induced critical anemia compared to treatment with PHZ alone, and numerous nucleated erythrocytes appeared in the peripheral blood. In the BM, immature CD71-positive erythroblasts were increased, and extramedullary erythropoiesis occurred in the liver. Furthermore, embryonic type globin mRNA was detected in both the BM and the liver. In peripheral blood, spots that did not correspond to control hemoglobin were observed in 2D electrophoresis. ChIP analyses showed that KLF1 and KLF2 bind to the promoter regions of ß-like globin. Wine-colored capsuled structures were unexpectedly observed in the abdominal cavity, and active erythropoiesis was also observed in these structures. CONCLUSION: These results indicate that primitive erythropoiesis occurs in adult mice to rescue critical anemia because primitive erythropoiesis does not require macrophages as stroma whereas macrophages play a pivotal role in definitive erythropoiesis even outside the medulla. The cells expressing embryonic hemoglobin in this study were similar to primitive erythrocytes, indicating the possibility that yolk sac-derived primitive erythroid cells may persist into adulthood in mice.

Neuropeptide W-Induced Hypophagia is Mediated Through Corticotropin-Releasing Hormone-Containing Neurons.

Neuropeptide W (NPW), which was originally isolated from the porcine hypothalamus, has been identified as the endogenous ligand for both the NPBWR1 (GPR7) and NPBWR2 (GPR8) receptors. These receptors, which belong to the orphan G protein-coupled receptor (GPCR) family, share a high sequence homology with the opioid and somatostatin receptor families. NPW and NPBWR1 are widely distributed in the rat central nervous system (CNS). While the intracerebroventricular (i.c.v.) injection of NPW elevates plasma corticosterone levels, the intravenous administration of NPW in conjunction with a corticotropin-releasing hormone (CRH) antagonist blocks NPW-induced corticosterone secretion. It has been reported that NPW is involved in regulating the hypothalamus-pituitary-adrenal cortex (HPA) axis and that i.c.v. administration of NPW decreases feeding behavior. The aim of the present study was to ascertain if NPW's role in feeding regulation is mediated (or not) through corticotropin-releasing hormone (CRH)-containing neurons. We found that NPW-containing axon terminals make synapses with CRH-immunoreactive cell bodies and dendritic processes in the hypothalamic paraventricular nucleus (PVN). The central infusion of NPW significantly induced c-Fos expression in CRH-immunoreactive neurons in the mouse PVN, but not in vasopressin- or oxytocin-immunoreactive neurons. To determine if NPW regulates feeding behavior through CRH neurons, the feeding behavior of mice was studied following the i.c.v. administration NPW in the presence or absence of pretreatment with a CRH antagonist. While NPW administration decreased feeding activity, the CRH antagonist inhibited this effect. These results strongly suggest that NPW regulates feeding behavior through CRH neurons in the mouse brain.

Regulation of Osteoclast Multinucleation by the Actin Cytoskeleton Signaling Network.

Although it is known that osteoclasts are multinucleated cells that are responsible for bone resorption, the mechanism by which their size is regulated is unclear. We previously reported that an actin-rich superstructure, termed the zipper-like structure, specifically appears during the fusion of large osteoclast-like cells (OCLs). Actin cytoskeleton reorganization in osteoclasts is regulated by a signaling network that includes the macrophage colony-stimulating factor (M-CSF) receptor, a proto-oncogene, Src, and small GTPases. Here, we examined the role of actin reorganization in the multinucleation of OCLs differentiated from RAW 264.7 cells using various pharmacological agents. Jasplakinolide, which stabilizes actin stress fibers, induced the development of small OCLs, and the Src inhibitor SU6656 and the dynamin inhibitor dynasore impaired the maintenance of the podosome belt and the zipper-like structure. These inhibitors decreased the formation of large OCLs but increased the number of small OCLs. M-CSF is known to stimulate osteoclast fusion. M-CSF signaling via Src up-regulated Rac1 activity but down-regulated Rho activity. Rac1 and Rho localized to the center of the zipper-like structure. Rho activator II promoted the formation of small OCLs, whereas the Rho inhibitor Y27632 promoted the generation of large OCLs. These results suggest that the status of the actin cytoskeleton signaling network determines the size of OCLs during cell fusion.

Nuclei of the human raphe.

Along the raphe of the brain stem, a series of small neuronal groups can be observed in the medulla oblongata, the pons and the mesencephalon. The neurons located in and adjacent to the raphe are considered to produce mainly serotonin (5-HT). The groups of nuclei containing 5-HT were first reported in experimental animals in the early 1960s. The presence of such nuclei, however, has not yet been brought to light in the human brainstem except the few atlases, although in several neuroanatomy textbooks, extrapolated data are shown in the form of drawings as if they were the data from the human brain. The aim of this study is to present microscopic photos of such raphe nuclei made from serial sections of the human brainstem, and to clarify the differences between findings in human and textbook drawings from animal data.

Evaluation of the spinal cord white matter.

Morphological findings or evaluations of the nervous system have traditionally concentrated on cell somata; evaluations of the white matter have not been put forward up to now. This study was conducted to evaluate the white matter in the spinal cord with the LPH discriminative staining method which was proposed by Goto. Thanks to the minimum shrinkage ratio (10 ± 0% in length) which this technique allows, it is possible to evaluate the sizes of nerve axons, and to compare the arrangement of nerve fibers in various parts of the spinal white matter. As the axonal sizes reflect nerve conduction velocities, we would like to emphasize that these sizes or the differences in the arrangement of axons may be important for a better understanding of neurosymptomatology.

Intranasal administration of PACAP: uptake by brain and regional brain targeting with cyclodextrins.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a potent neurotrophic and neuroprotectant that is transported across the blood-brain barrier in amounts sufficient to affect brain function. However, its short half-life in blood makes it difficult to administer peripherally. Here, we determined whether the radioactively labeled 38 amino acid form of PACAP can enter the brain after intranasal (i.n.) administration. Occipital cortex and striatum were the regions with the highest uptake, peaking at levels of about 2-4% of the injected dose per gram of brain region. Inclusion of unlabeled PACAP greatly increased retention of I-PACAP by brain probably because of inhibition of the brain-to-blood efflux transporter for PACAP located at the blood-brain barrier. Sufficient amounts of PACAP could be delivered to the brain to affect function as shown by improvement of memory in aged SAMP8 mice, a model of Alzheimer's disease. We found that each of three cyclodextrins when included in the i.n. injection produced a unique distribution pattern of I-PACAP among brain regions. As examples, ß-cyclodextrin greatly increased uptake by the occipital cortex and hypothalamus, α-cyclodextrin increased uptake by the olfactory bulb and decreased uptake by the occipital cortex and striatum, and (2-hydropropyl)-ß-cyclodextrin increased uptake by the thalamus and decreased uptake by the striatum. These results show that therapeutic amounts of PACAP can be delivered to the brain by intranasal administration and that cyclodextrins may be useful in the therapeutic targeting of peptides to specific brain regions.

Calcified tissue formation of subcutaneously transplanted mouse dental pulp.

It is well established that dental pulp has the ability to form calcified tissue, however, the precise process of calcified tissue formation and its characteristics are still undetermined. In this study we examined the process and the matrix components of the calcified tissues by means of subcutaneously transplanted dental pulp tissue. The mid-third of the mouse incisor pulp was transplanted into abdominal subcutaneous tissue. Two calcified tissues were independently formed within the implanted pulp at 7 days after the implantation, one developed in the peripheral region and the other was formed in the middle region of the pulp. Histological investigation indicated the existence of hypertrophic chondrocytes in the peripheral calcified tissue. Immunohistochemical study indicated the colocalization of types I and II collagen surrounding these cells. RT-PCR analysis indicated the transient expression of type II collagen at 7 days and the constant expression of type I collagen, osteonectin, osteocalcin and dentin matrix protein-1 and 2 at all examined times. Dentin sialophosphoprotein was only detected at 28 days after the transplantation. These results indicated that dental pulp cells might have the capacity to form calcified tissue by implanted dental pulp and it is possible that the difference of local environments induced the cells to form different types of calcified tissues within the implanted pulp.

In situ proliferation and differentiation of macrophages in dental pulp.

The presence of macrophages in dental pulp is well known. However, whether these macrophages proliferate and differentiate in the dental pulp in situ, or whether they constantly migrate from the blood stream into the dental pulp remains unknown. We have examined and compared the development of dental pulp macrophages in an organ culture system with in vivo tooth organs to clarify the developmental mechanism of these macrophages. The first mandibular molar tooth organs from ICR mice aged between 16 days of gestation (E16) to 5 days postnatally were used for in vivo experiments. Those from E16 were cultured for up to 14 days with or without 10% fetal bovine serum. Dental pulp tissues were analyzed with immunohistochemistry to detect the macrophages and with reverse transcription and the polymerase chain reaction (RT-PCR) for the detection of factors related to macrophage development. The growth curves for the in vivo and in vitro cultured cells revealed similar numbers of F4/80-positive macrophages in the dental pulp. RT-PCR analysis indicated the constant expression of myeloid colony-stimulating factor (M-CSF) in both in-vivo- and in-vitro-cultured dental pulp tissues. Anti-M-CSF antibodies significantly inhibited the increase in the number of macrophages in the dental pulp. These results suggest that (1) most of the dental pulp macrophages proliferate and differentiate in the dental pulp without a supply of precursor cells from the blood stream, (2) M-CSF might be a candidate molecule for dental pulp macrophage development, and (3) serum factors might not directly affect the development of macrophages.