Conditional disruption of Nr5a1 directed by Sox9-Cre impairs adrenal development.

The current study aimed to investigate the effect of Sox9-Cre-directed Nr5a1-conditional knockout (Sox9-Cre;Nr5a1flox/flox) on adrenal development. We showed that SOX9 is expressed by adrenocortical cells at E10.5-E11.5 but is extinguished no later than E12.5. The number of adrenocortical cells significantly reduced in Sox9-Cre;Nr5a1flox/flox mice while the number of cleaved caspase 3-positive cells increased compared to that in the controls at E11.5-E12.5, when the adrenal primordium (AP) is about to expand. This indicated that fetal adrenocortical cells are lost via apoptosis due to Nr5a1 ablation by E12.5. Both medulla formation and encapsulation were perturbed, accompanied by a smaller AP size, in Sox9-Cre;Nr5a1flox/flox mice during embryonic development. Adult Sox9-Cre;Nr5a1flox/flox adrenals were hypoplastic and exhibited irregular organization of the medulla with aberrant sex differentiation in the X zone. Additionally, there were histologically eosin-negative vacuolated cells, which were negative for both the X-zone marker 20αHSD and the steroidogenesis marker 3ßHSD at the innermost cortex of Sox9-Cre;Nr5a1flox/flox adrenals. Although Nr5a1+/- adrenals were hypoplastic, a small number of chromaffin cells were properly located in the center, having normal sex differences in the X-zone. The results collectively provided in-vivo evidence that Nr5a1 plays a critical role in AP expansion and subsequent adrenal development.

Suppression of tooth movement-induced sclerostin expression using ß-adrenergic receptor blockers.

OBJECTIVE: Regulation of bone metabolism by the sympathetic nervous system has recently been clarified. Tooth movement is increased by increased bone metabolic turnover due to sympathetic activation. This study aimed to compare the effects of the ß-adrenergic receptor (ß-AR) blockers atenolol (ß1-AR blocker), butoxamine (ß2-AR blocker) and propranolol (non-selective ß-AR blocker) on tooth movement in spontaneously hypertensive rats (SHR) with sympathicotonia. MATERIALS AND METHODS: Spontaneously hypertensive rats were divided into the following four groups: an SHR control group and groups treated with 0.1 mg/kg atenolol, 1 mg/kg butoxamine or 1 mg/kg propranolol (n = 6 rats/group). Atenolol, butoxamine or propranolol was administered daily to each treatment group, and orthodontic force was applied using a closed-coil spring. Finally, immunohistochemical analysis was performed for receptor activator of nuclear factor kappa-B ligand (RANKL) and sclerostin (SOST). RESULTS: Atenolol, butoxamine and propranolol inhibited tooth movement and increased maxillary alveolar bone volume. Histological analysis revealed that these ß-AR blockers decreased osteoclast activity on the compression side. Furthermore, immunohistochemical analysis revealed that atenolol, butoxamine and propranolol decreased the number of RANKL- and SOST-positive osteocytes on the compression side. CONCLUSIONS: ß-AR blockers decreased tooth movement and downregulated SOST in osteocytes, accompanied by increasing alveolar bone resorption.

Conditioned medium from rat dental pulp reduces the number of osteoclasts via attenuation of adhesiveness in osteoclast precursors.

Dental pulp is known to play crucial roles in homeostasis of teeth and periodontal tissue. Although resorption of bone around the roots of nonvital teeth is occasionally observed in clinical practice, little is known about the role of dental pulp in osteoclastogenesis. Here we evaluated the effects of conditioned medium (CM) from rat dental pulp on osteoclastogenesis. It was found that the CM reduced the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts, but did not alter the mRNA levels of nuclear factor of activated T-cells, cytoplasmic 1 and TRAP. To further understand the mechanism behind these results, we evaluated the effects of CM on osteoclast precursors and found that the CM removed cell processes, resulting in a significant reduction in the number of attached cells and an increase in the number of freely floating cells. Furthermore, the CM suppressed the mRNA levels of focal adhesion kinase and paxillin, which are involved in cell adhesiveness and spreading. Collectively, the present results show that CM from dental pulp serves as an inhibitor of osteoclastogenesis by reducing the number and adhesiveness of osteoclast precursors, suggesting novel therapeutic applicability for osteoporosis.

Suppression of osteoclastogenesis via α2-adrenergic receptors.

The sympathetic nervous system is known to regulate osteoclast development. However, the involvement of α2-adrenergic receptors (α2-ARs) in osteoclastogenesis is not well understood. In the present study, their potential role in osteoclastogenesis was investigated. Guanabenz, clonidine and xylazine were used as agonists of α2-ARs, while yohimbine and idazoxan were employed as antagonists. Using RAW264.7 pre-osteoclast and primary bone marrow cells, the mRNA expression of the osteoclast-related genes nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), tartrate-resistant acid phosphatase (TRAP) and cathepsin K was evaluated following induction with receptor activator of nuclear factor κB ligand (RANKL). TRAP staining was also conducted to assess effects on osteoclastogenesis in mouse bone marrow cells in vitro. Administration of 5-20 µM guanabenz (P<0.01, for RANKL-only treatment), 20 µM clonidine (P<0.05, for RANKL-only treatment) and 20 µM xylazine (P<0.05, for RANKL-only treatment) attenuated RANKL-induced upregulation of NFATc1, TRAP and cathepsin K mRNA. Furthermore, the reductions in these mRNAs by 10 µM guanabenz and 20 µM clonidine in the presence of RANKL were attenuated by 20 µM yohimbine or idazoxan (P<0.05). The administration of 5-20 µM guanabenz (P<0.01, for RANKL-only treatment) and 10-20 µM clonidine (P<0.05, for RANKL-only treatment) also decreased the number of TRAP-positive multi-nucleated osteoclasts. Collectively, the present study demonstrates that α2-ARs may be involved in the regulation of osteoclastogenesis.

Topographic anatomy of the posterior ramus of thoracic spinal nerve and surrounding structures.

STUDY DESIGN: Cadaver dissection. OBJECTIVE: To examine the potential points of spinal nerve entrapment and the articular branches in the thoracic spine. SUMMARY OF BACKGROUND DATA: Despite many cadaver studies focused on the cervical and lumbar spinal nerves, detailed anatomy of the thoracic nerve branches is missing from the viewpoint of painful neuropathy on the thoracic region. METHODS: A total of 120 pairs of thoracic spinal nerves out of 10 donated cadavers were dissected. Detailed anatomy of the posterior ramus and medial/lateral branches and their fine branches in the entire thoracic region was investigated by both macroscopic and stereomicroscopic dissections. RESULTS: The posterior ramus of the thoracic nerve passed through the narrow space between the bony structures and adjacent fibrous tissue. It is sent to the first branch, which is called "the descending branch," before bifurcating into medial and lateral branches. The medial branch runs posterolaterally, then turns medially along the edge of multifidus, and passes between that and semispinalis, whereas the lateral branch runs underneath the intertransverse ligament. Both medial and lateral cutaneous branches penetrated the thoracolumbar fascia, and the medial cutaneous branch penetrated the tendinous portion of back muscles. CONCLUSION: Several points might be listed as potential sites of entrapment along the course of the posterior ramus of the thoracic nerve and its branches, leading to the cause of thoracic back pain. In addition, the articular branch entering the facet (zygapophyseal) joint originated from the descending branch, which was the first branch of the posterior ramus.

Inhibitory effect of CGRP on osteoclast formation by mouse bone marrow cells treated with isoproterenol.

The present study was designed to elucidate the mode of action of isoproterenol (Isp; adrenergic beta-agonist) and to characterize the effect of the calcitonin gene-related peptide (CGRP; sensory neuropeptide) on osteoclast formation induced by Isp in a mouse bone marrow culture system. Treatment of mouse bone marrow cells with Isp generated tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells (MNCs) capable of excavating resorptive pits on dentine slices, and caused an increase in receptor activator of NF-kappaB ligand (RANKL) and a decrease in osteoprotegerin (OPG) production by the marrow cells. The osteoclast formation was significantly inhibited by OPG, suggesting the involvement of the RANKL-RANK system. CGRP inhibited the osteoclast formation caused by Isp or soluble RANKL (s-RANKL) but had no influence on RANKL or OPG production by the bone marrow cells treated with Isp, suggesting that CGRP inhibited the osteoclast formation by interfering with the action of RANKL produced by the Isp-treated bone marrow cells without affecting RANKL or OPG production. This in vitro data suggest the physiological interaction of sympathetic and sensory nerves in osteoclastogenesis in vivo.