Differential induction of c-Fos and phosphorylated ERK by a noxious stimulus after peripheral nerve injury.

PURPOSE: In this study, we compared induction of c-Fos and phosphorylated extracellular signal-regulated kinase (p-ERK) in the spinal dorsal horn after peripheral nerve injury. MATERIALS AND METHODS: We examined the spinal dorsal horn for noxious heat-induced c-Fos and p-ERK protein-like immunoreactive (c-Fos- and p-ERK-IR) neuron profiles after tibial nerve injury. The effect of administration of a MEK 1/2 inhibitor (PD98059) on noxious heat-induced c-Fos expression was also examined after tibial nerve injury. RESULTS: A large number of c-Fos- and p-ERK-IR neuron profiles were induced by noxious heat stimulation to the hindpaw in sham-operated animals. A marked reduction in the number of c-Fos- and p-ERK-IR neuron profiles was observed in the medial 1/3 (tibial territory) of the dorsal horn at 3 and 7 days after nerve injury. Although c-Fos-IR neuron profiles had reappeared by 14 days after injury, the number of p-ERK-IR neuron profiles remained decreased in the tibial territory of the superficial dorsal horn. Double immunofluorescence labeling for c-Fos and p-ERK induced by noxious heat stimulation to the hindpaw at different time points revealed that a large number of c-Fos-IR, but not p-ERK-IR, neuron profiles were distributed in the tibial territory after injury. Although administration of a MEK 1/2 inhibitor to the spinal cord suppressed noxious heat-induced c-Fos expression in the peroneal territory, this treatment did not alter c-Fos induction in the tibial territory after nerve injury. CONCLUSIONS: ERK phosphorylation may be involved in c-Fos induction in normal nociceptive responses, but not in exaggerated c-Fos induction after nerve injury.

Differential Changes in Neuronal Excitability in the Spinal Dorsal Horn After Spinal Nerve Ligation in Rats.

Previous studies demonstrated that peripheral nerve injury induced excessive neuronal response and glial activation in the spinal cord dorsal horn, and such change has been proposed to reflect the development and maintenance of neuropathic pain states. The aim of this study was to examine neuronal excitability and glial activation in the spinal dorsal horn after peripheral nerve injury. We examined noxious heat stimulation-induced c-Fos protein-like immunoreactivity (Fos-LI) neuron profiles in fourth-to-sixth lumbar (L4-L6) level spinal dorsal horn neurons after fifth lumbar spinal nerve ligation (L5 SNL). Immunofluorescence labeling of OX-42 and GFAP was also performed in histological sections of the spinal cord. A significant increase in the number of Fos-LI neuron profiles in the spinal dorsal horn at the L4 level was found at 3 days after SNL, but returned to a level similar to that in sham-operated controls by 14 days after injury. As expected, a decrease in the number of Fos-LI neuron profiles in the spinal dorsal horn at the L5 level was found at 3 days after SNL. However, these profiles had reappeared in large numbers by 14 and 21 days after injury. Immunofluorescence labeling of OX-42 and GFAP indicated sequential activation of microglia and astrocytes in the spinal dorsal horn. We conclude that nerve injury causes differential changes in neuronal excitability in the spinal dorsal horn, which may coincide with glial activation. These changes may play a substantial role in the pathogenesis of neuropathic pain after peripheral nerve injury.

Cross-Excitation in Peripheral Sensory Ganglia Associated with Pain Transmission.

Despite the absence of synaptic contacts, cross-excitation of neurons in sensory ganglia during signal transmission is considered to be chemically mediated and appears increased in chronic pain states. In this study, we modulated neurotransmitter release in sensory neurons by direct application of type A botulinum neurotoxin (BoNT/A) to sensory ganglia in an animal model of neuropathic pain and evaluated the effect of this treatment on nocifensive. Unilateral sciatic nerve entrapment (SNE) reduced the ipsilateral hindpaw withdrawal threshold to mechanical stimulation and reduced hindpaw withdrawal latency to thermal stimulation. Direct application of BoNT/A to the ipsilateral L4 dorsal root ganglion (DRG) was localized in the cell bodies of the DRG and reversed the SNE-induced decreases in withdrawal thresholds within 2 days of BoNT/A administration. Results from this study suggest that neurotransmitter release within sensory ganglia is involved in the regulation of pain-related signal transmission.

Activated microglia contribute to convergent nociceptive inputs to spinal dorsal horn neurons and the development of neuropathic pain.

The activation of microglia in the spinal dorsal horn following peripheral nerve injury has been reported previously, and this change has been proposed to contribute to the development of a neuropathic pain state. We recently demonstrated that peripheral nerve injury activated convergent nociceptive inputs to spinal dorsal horn neurons. The present study was designed to further examine the role of microglia in the activation of convergent nociceptive inputs as well as development of a neuropathic pain state after peripheral nerve injury. Tibial nerve injury initially induced hyposensitivity at 3 days post-injury, and this was followed by hypersensitivity to tactile and thermal stimuli at 14 days. The intraperitoneal administration of minocycline (30 mg/kg), an inhibitor of microglial activation, for 8 days starting on the day of surgery prevented increases in OX-42 immunofluorescence labeling in the spinal dorsal horn and the development of tactile and thermal hypersensitivity at 14 days post-injury. The same minocycline treatment (day 0-7) also reduced the nerve injury-induced convergence of nociceptive inputs to spinal dorsal horn neurons, as revealed by double immunofluorescence labeling for c-Fos induced by noxious heat stimulation of the hindpaw and phosphorylated extracellular signal-regulated kinase induced by electrical stimulation of the injured tibial nerve. However, the administration of minocycline for 8 days starting 7 days after surgery did not prevent nerve injury-induced microglial activation, convergent nociceptive inputs, or tactile and thermal hypersensitivity. These results suggest that microglial activation in the early stage following peripheral nerve injury plays an important role in the anomalous convergence of nociceptive signals to spinal dorsal horn neurons and the development of neuropathic pain.

Peripheral nerve injury activates convergent nociceptive input to dorsal horn neurons from neighboring intact nerve.

Previous studies demonstrated that peripheral nerve injury induced excessive nociceptive response of spinal cord dorsal horn neurons and such change has been proposed to reflect the development of neuropathic pain state. The aim of this study was to examine the spinal dorsal horn for convergence of nociceptive input to second-order neurons deafferented by peripheral nerve injury. Double immunofluorescence labeling for c-Fos and phosphorylated extracellular signal-regulated kinase (p-ERK) was performed to detect convergent synaptic input to spinal dorsal horn neurons after the saphenous nerve injury. c-Fos expression and the phosphorylation of ERK were induced by noxious heat stimulation of the hindpaw and by electrical stimulation of the injured or uninjured saphenous nerve, respectively. Within the central terminal field of the saphenous nerve, the number of c-Fos protein-like immunoreactive (c-Fos-IR) cell profiles was significantly decreased at 3 days and returned to the control level by 14 days after the injury. p-ERK immunoreactive (p-ERK-IR) cell profiles were distributed in the central terminal field of the saphenous nerve, and the topographic distribution pattern and number of such p-ERK-IR cell profiles remained unchanged after the nerve injury. The time course of changes in the number of double-labeled cell profiles was similar to that of c-Fos-IR cell profiles after the injury. These results indicate that convergent primary nociceptive input through neighboring intact nerves contributes to increased responsiveness of spinal dorsal horn nociceptive neurons.

Possible involvement of convergent nociceptive input to medullary dorsal horn neurons in intraoral hyperalgesia following peripheral nerve injury.

Previous studies demonstrated that the number of c-Fos protein-like immunoreactive (c-Fos-IR) neurons in the medullary dorsal horn (MDH) evoked by noxious stimulation was increased after peripheral nerve injury, and such increase has been proposed to reflect the development of neuropathic pain state. The aim of this study was to examine the MDH for convergent collateral primary afferent input to second order neurons deafferented by peripheral nerve injury, and to explore a possibility of its contribution to the c-Fos hyperinducibility. Double immunofluorescence labeling for c-Fos and phosphorylated extracellular signal-regulated kinase (p-ERK) was performed to detect convergent synaptic input. c-Fos expression and the phosphorylation of ERK were induced by the intraoral application of capsaicin and by electrical stimulation of the inferior alveolar nerve (IAN), respectively. The number of c-Fos-IR neurons in the MDH induced by the intraoral application of capsaicin was increased after IAN injury, whereas the number of p-ERK immunoreactive neurons remained unchanged. The number of double-labeled neurons, that presumably received convergent primary afferent input from the lingual nerve and the IAN, was significantly increased after IAN injury. These results indicated that convergent primary nociceptive input through neighboring intact nerves may contribute to the c-Fos hyperinducibility in the MDH and the pathogenesis of neuropathic pain following trigeminal nerve injury.

Convergent nociceptive input to spinal dorsal horn neurons after peripheral nerve injury.

The number of c-Fos protein-like immunoreactive (c-Fos-IR) neurons in the spinal dorsal horn evoked by noxious stimulation was previously shown to be increased following peripheral nerve injury, and this increase was proposed to reflect the neuropathic pain state. The aim of this study was to investigate whether anomalous convergent primary afferent input to spinal dorsal horn neurons contributed to nerve injury-induced c-Fos hyperinducibility. Double immunofluorescence labeling for c-Fos and phosphorylated extracellular signal-regulated kinase (p-ERK) was performed to detect convergent synaptic input from different branches of the sciatic nerve after injury to the tibial nerve. c-Fos expression and the phosphorylation of ERK were induced by noxious heat stimulation of the hindpaw and also by electrical stimulation (ES) of the injured tibial nerve, respectively. The number of c-Fos-IR neurons was significantly decreased 3 days after the injury. However, the number of c-Fos-IR neurons returned to the control level 14 days after the injury. P-ERK immunoreactive (p-ERK-IR) neurons were induced in the central terminal field of the tibial nerve by ES of the tibial nerve. The topographic distribution pattern and number of such p-ERK-IR neurons remained unchanged after the nerve injury. The time course of changes in the number of double-labeled neurons, that presumably received convergent primary afferent input, showed a pattern similar to that of c-Fos-IR neurons after the injury. These results indicate that convergent primary nociceptive input through neighboring intact nerves may contribute to c-Fos hyperinducibility in the spinal dorsal horn.

Assessment of intraoral mucosal pain induced by the application of capsaicin.

OBJECTIVE: To develop an objective method for assessing nociceptive behaviour in an animal model of capsaicin-induced intraoral pain. Changes in nociceptive responses were also examined after injury to the inferior alveolar nerve (IAN). DESIGN: Nociceptive responses evoked by the intraoral application of various doses of capsaicin were analyzed in lightly anaesthetized rats. The number of c-Fos protein-like immunoreactive (Fos-LI) neurons in the medullary dorsal horn (MDH) induced by the intraoral application of capsaicin was measured. Behavioural and c-Fos responses were also examined 14 days after injury to the IAN. RESULTS: Larger doses of intraoral capsaicin (1, 10 and 100µg) induced vigorous licking behaviour and c-Fos response in the MDH in a reproducible manner. The magnitudes of both behavioural activity and the c-Fos response from the 10 and 100µg doses of capsaicin were significantly greater than that by the 1µg dose. Injury to the IAN exaggerated the behavioural and c-Fos responses evoked by intraoral capsaicin. CONCLUSIONS: The intraoral application of capsaicin is a valid and reliable method for studying intraoral pain and hyperalgesia following nerve injury.

Effect of adenosine A1 receptor agonist on the enhanced excitability of spinal dorsal horn neurons after peripheral nerve injury.

Neuronal hyperactivity has been implicated in abnormal pain sensation following peripheral nerve injuries. Previous studies have indicated that the activation of adenosine A1 receptors (A1R) in the central and peripheral nervous systems produces an antinociceptive effect. However, the mechanisms involved in the peripheral effect are still not fully understood. The effects of the local application of the selective A1R agonist, 2-chloro-N(6)-cyclopentyladenosine (CCPA) on neuronal hyperactivity were examined in this study using a neuropathic pain model induced by a tibial nerve injury. We utilized Fos protein-like immunoreactivity induced by noxious heat stimulation to examine changes in the number of Fos protein like immunoreactive (Fos-LI) neuron profiles in the spinal dorsal horn, and behavioral analysis for mechanical and thermal sensitivities. The nerve injury induced an exaggerated Fos response to noxious heat stimulation. The number of Fos-LI neuron profiles was significantly decreased and their distribution was restricted to the central terminal field of the spared peroneal nerve 3 days after the injury. The number of Fos-LI neuron profiles returned to control levels and a large number of these profiles were observed in the central terminal field of the injured tibial nerve 14 days after the injury. These enhanced Fos responses were attenuated by the local application of CCPA. The nerve injury also resulted in mechanical allodynia and thermal hyperalgesia. The local application of CCPA inhibited thermal hyperalgesia, but was less effective against mechanical allodynia. These results indicated that activation of peripheral A1R plays a role in the regulation of nerve injury-induced hyperalgesia.

Fos protein-like immunoreactive neurons induced by electrical stimulation in the trigeminal sensory nuclear complex of rats with chronically injured peripheral nerve.

The rat trigeminal sensory nuclear complex (TSNC) was examined for Fos protein-like immunoreactive (Fos-LI) neurons induced by electrical stimulation (ES) of the lingual nerve (LN) at 2 weeks after injury to the LN or the inferior alveolar nerve (IAN). Intensity-dependent increase in the number of Fos-LI neurons was observed in the subnucleus oralis (Vo) and caudalis (Vc) of the spinal trigeminal tract nucleus irrespective of nerve injury. The number of Fos-LI neurons induced by ES of the chronically injured LN at A-fiber intensity (0.1 mA) was significantly increased in the Vo but not the Vc. On the other hand, in rats with chronically injured IAN, the number of Fos-LI neurons induced by ES of the LN at C-fiber intensity (10 mA) was significantly increased in the Vc but not the Vo. These results indicated that injury of a nerve innervating intraoral structures increased the c-Fos response of Vo neurons to A-fiber intensity ES of the injured nerve. A similar nerve injury enhanced the c-Fos response of Vc neurons to C-fiber intensity ES of a spared uninjured nerve innervating an intraoral territory neighboring that of the injured nerve. The present result show that nerve injury causes differential effects on c-Fos expression in the Vo and Vc, which may explain complexity of neuropathic pain symptoms in clinical cases.

Muscarinic receptor immunoreactivity in the superior salivatory nucleus neurons innervating the salivary glands of the rat.

The superior salivatory nucleus (SSN) contains preganglionic parasympathetic neurons to the submandibular and sublingual salivary glands. Cevimeline, a muscarinic acetylcholine receptor agonist, stimulates the salivary glands and is presently used as sialogogue in the treatment of dry mouth. Since cevimeline passes through the blood-brain barrier, it is also able to act on muscarinic acetylcholine receptors in the central nervous system. Our preliminary experiment using the whole-cell patch-clamp technique has shown that cevimeline excites SSN neurons in rat brain slices, suggesting that SSN neurons have muscarinic acetylcholine receptors; however, it is unclear which subtypes of muscarinic acetylcholine receptors exist in SSN neurons. In the present study, we investigated immunohistochemically muscarinic acetylcholine receptor subtypes, M1 receptor (M1R), M2R, M3R, M4R, and M5R in SSN neurons. SSN neurons innervating the salivary glands, retrogradely labeled with a fluorescent tracer from the chorda-lingual nerve, mostly expressed M3R immunoreactivity (-ir) (92.3%) but not M1R-ir. About half of such SSN neurons also showed M2R- (40.1%), M4R- (54.0%) and M5R-ir (46.0%); therefore, it is probable that SSN neurons co-express M3R-ir with at least two of the other muscarinic receptor subtypes. This is the first report to show that SSN neurons contain muscarinic acetylcholine receptors.

Persistent hypoglossal artery with hypoplasia of the vertebral and posterior communicating arteries.

We observed a rare case of the right persistent hypoglossal artery (PHA) in the posterior cranial fossa of a deceased 74-year-old Japanese man who did not have any clinical manifestations related to this anomaly when alive. The large-sized PHA originating from the cervical internal carotid artery passed through the hypoglossal canal together with the hypoglossal nerve and reached the posterior cranial fossa to anastomose the basilar artery. In addition, the ipsilateral vertebral artery and bilateral posterior communicating arteries were hypoplastic. Here, we discuss the developmental mechanisms underlying the formation of the PHA and the spectrum of diseases related to its presence.

Differential activation of mitogen-activated protein kinases and glial cells in the trigeminal sensory nuclear complex following lingual nerve injury.

Mitogen-activated protein kinases (MAPKs) play a pivotal role in the mediation of cellular responses to a variety of signaling molecules. The current study demonstrates phosphorylation of extracellular signal-regulated kinase (ERK) and p38 MAPK in each subdivision of the trigeminal sensory nuclear complex (TSNC) following lingual nerve injury. Immunohistochemical labeling for phosphorylated ERK (p-ERK) or phosphorylated p38 (p-p38) MAPK was performed in histological sections of the brainstem. A transient increase in the immunoreactivity for p-ERK was found in each subdivision of the TSNC followed by a prolonged increase in the immunoreactivity for p-p38 MAPK after nerve injury. Double immunofluorescence labeling with cell-specific markers revealed that ERK and p38 MAPK were phosphorylated predominantly by OX-42-positive microglia or GFAP-positive astrocytes. Increased immunofluorescence labeling for OX-42 and GFAP indicated that microglia and astrocytes were activated by nerve injury in the TSNC. Activation of MAPKs and glial cells in the rostral subdivisions of the TSNC was comparable with that in the subnucleus caudalis of the trigeminal spinal tract nucleus (Vc). We conclude that differential activation of MAPKs and glial cells in the rostral subdivisions of the TSNC as well as the Vc may have a substantial role in the pathogenesis of neuropathic pain following trigeminal nerve injury.

Tunicamycin-induced cell death in the trigeminal ganglion is suppressed by nerve growth factor in the mouse embryo.

The effect of nerve growth factor (NGF) on tunicamycin (Tm)-treated neurons in the trigeminal ganglion was investigated by use of caspase-3 immunohistochemistry. In intact embryos at embryonic day 16.5, only a few caspase-3-immunoreactivity were detected in the ganglion neurons. Mean +/- SE of the density of the immunoreactivity was 0.22 +/- 0.03%. In contrast, the number of the immunoreactive neurons was increased at 24 h after injection of 0.5 microg Tm in 1 microl of 0.05 N NaOH solution into mouse embryos at embryonic day 15.5. The density of immunoreactivity was also increased (mean +/- SE = 1.44 +/- 0.11%) compared to intact and 0.05 N NaOH-treated embryos (mean +/- SE = 0.35 +/- 0.03%). The Tm treatment caused increase of the number of trigeminal neurons representing apoptotic profiles (intact, mean +/- SE = 79.3 +/- 8.5; 0.05 N NaOH, mean +/- SE = 132 +/- 11.5; 0.5 microg Tm, mean +/- SE = 370.2 +/- 64.8). In addition, NGF significantly prevented the increase of density of the immunoreactivity (mean +/- SE = 0.54 +/- 0.16%) and the number of apoptotic cells (mean +/- SE = 146.2 +/- 11.3). Saline application (without NGF) had no effect on Tm-induced increase of the immunoreactivity (mean +/- SE = 1.78 +/- 0.23%) or the apoptotic profiles (mean +/- SE = 431.9 +/- 80.5). These results indicate that Tm-induced cell death in the trigeminal ganglion is suppressed by NGF in the mouse embryo.

Brn-3a deficiency transiently increases expression of calbindin D-28 k and calretinin in the trigeminal ganglion during embryonic development.

Immunohistochemistry for neuron-specific nuclear protein (NeuN), caspase-3, calcitonin gene-related peptide (CGRP), and calcium-binding proteins was performed on the trigeminal ganglion (TG) in wild type and Brn-3a knockout mice at embryonic days 12.5-16.5 (E12.5-E16.5). In Brn-3a knockout mice, the number of NeuN-immunoreactive (ir) neuron profiles increased at E14.5 (40.0% increase) and decreased at E16.5 (28.3% reduction) compared to wild type mice. Caspase-3-ir neuron profiles were abundant in the TG of wild type mice at E12.5-E16.5. However, the loss of Brn-3a decreased the number of caspase-3-ir neuron profiles at E12.5 (69.7% reduction) and E14.5 (51.7% reduction). At E16.5, the distribution of caspase-3-ir neuron profiles was barely affected by the deficiency. CGRP-ir neuron profiles were observed in the TG of wild type mice but not knockout mice at E12.5. At E14.5 and E16.5, CGRP-ir neuron profiles were abundant in both wild type and knockout mice. Calbindin D-28 k (CB)-ir neuron profiles decreased in the TG of mutant mice at E12.5 compared to wild type mice (56.4% reduction). At E14.5, however, Brn-3a deficiency transiently increased CB-ir neuron profiles (169.4% increase as compared to wild type mice). Calretinin (CR)-ir neuron profiles could not be detected in the TG of wild type mice at E12.5-16.5. However, numerous CR-ir neuron profiles transiently appeared in the knockout mouse at E14.5. Parvalbumin (PV)-ir neurons appeared in wild type and knockout mice at E14.5. At this stage, the number of large (>50 mum(2)) PV-ir neuron profiles in knockout mice was fewer than that in wild type mice. The number and cell size of PV-ir neuron profiles were barely affected by the deficiency at E16.5. The present study indicates that the loss of Brn-3a causes increase of TG neurons at E14.5 and decrease of TG neurons at E16.5. It is also suggested that Brn-3a deficiency affects the number and cell size of CGRP- and calcium-binding protein-containing neurons at E12.5 and E14.5. Caspase-3-dependent cell death of CB- and CR-ir neurons may be suppressed by the deficiency at E14.5.

Effect of connective tissue growth factor (CCN2/CTGF) on proliferation and differentiation of mouse periodontal ligament-derived cells.

BACKGROUND: CCN2/CTGF is known to be involved in tooth germ development and periodontal tissue remodeling, as well as in mesenchymal tissue development and regeneration. In this present study, we investigated the roles of CCN2/CTGF in the proliferation and differentiation of periodontal ligament cells (murine periodontal ligament-derived cell line: MPL) in vitro. RESULTS: In cell cultures of MPL, the mRNA expression of the CCN2/CTGF gene was stronger in sparse cultures than in confluent ones and was significantly enhanced by TGF-beta. The addition of recombinant CCN2/CTGF (rCCN2) to MPL cultures stimulated DNA synthesis and cell growth in a dose-dependent manner. Moreover, rCCN2 addition also enhanced the mRNA expression of alkaline phosphatase (ALPase), type I collagen, and periostin, the latter of which is considered to be a specific marker of the periosteum and periodontium; whereas it showed little effect on the mRNA expression of typical osteoblastic markers, e.g., osteopontin and osteocalcin. Finally, rCCN2/CTGF also stimulated ALPase activity and collagen synthesis. CONCLUSION: These results taken together suggest important roles of CCN2/CTGF in the development and regeneration of periodontal tissue including the periodontal ligament.

Involvement of caspase cascade in capsaicin-induced apoptosis of dorsal root ganglion neurons.

Capsaicin induces apoptosis in some types of neurons, but the exact molecular mechanism remains unclear. In this study, capsaicin was systemically administrated in newborn rats and the dorsal root ganglion (DRG) neurons were examined for caspase-immunoreactivity. Capsaicin-induced neuronal apoptosis was revealed by TUNEL. TUNEL-positive neurons rapidly increased, reaching the peak at 24 h post-injection when 10.6% of DRG neurons were apoptotic. Neurons expressing immunoreactivity for activated caspases-9 and -3 concomitantly increased. At 24 h, 15.9% and 17.7% of DRG neurons exhibited immunoreactivity for caspase-9 and caspase-3, respectively. DNA fragmentation signal and caspase-immunoreactivity were detected in less than 0.5% of DRG neurons of vehicle control rats. The immunoreactivity and TUNEL-positivity returned to the vehicle control level by 120 h. Double label immunohistochemistry revealed co-expression of caspase-9 and DNA fragmentation or caspase-3 and DNA fragmentation. These results suggest that the caspase cascade is involved in the primary neuronal apoptosis induced by neurotoxin capsaicin.

Gene expression of connective tissue growth factor (CTGF/CCN2) in calcifying tissues of normal and cbfa1-null mutant mice in late stage of embryonic development.

Connective tissue growth factor (CTGF/CCN2), one of the most recently described growth factors, is produced by chondrocytes, vascular endothelial cells, and transforming growth factor (TGF)-beta-stimulated fibroblasts. CTGF was isolated from a chondrosarcoma-derived chondrocytic cell line, HCS-2/8, and found to be normally expressed in cartilage tissues, especially in hypertrophic chondrocytes, and also to stimulate both the proliferation and the differentiation of chondrocytes in vitro. Therefore, CTGF is thought to be one of the most important regulators of endochondral ossification in vivo. Herein we describe the expression pattern of the ctgf gene in the calcifying tissues of normal developing mouse embryos in comparison with that in core binding factor a1 (Cbfa1)-targeted mutant (cbfa1-null) mouse embryos, in which impaired development and growth were characteristically observed in the skeletal system. After 15 days of development (E15), the expression of ctgf was detected in the zone of hypertrophy and provisional calcification, in which ossification proceeds toward the epiphysis during the skeletal development of the mouse embryo. Furthermore, ctgf was expressed in developing molar and incisal tooth germs around the perinatal stage. However, no expression of the gene was found in the cbfa1-null mouse embryos. These results indicate that CTGF may have certain important roles in the development of the calcifying tissues in the mouse embryo.

Brn-3a deficiency increases tyrosine hydroxylase-immunoreactive neurons in the dorsal root ganglion.

Immunohistochemistry for tyrosine hydroxylase (TH) was performed on the dorsal root ganglia (DRG) in wild-type, heterozygous and Brn-3a knockout mice at embryonic day 18.5. TH-immunoreactive (-IR) neurons were detected in the DRG of wild-type and heterozygous mice, but their proportion was greatly increased by the loss of Brn-3a function (wild-type and heterozygot, 8.4%; knockout, 20.9%). IR neurons were of various sizes in wild-type (mean+/-S.D.=118.1+/-55.4 microm2, range=26.6-306.3 microm2) and heterozygous mice. In the knockout mice, however, TH-IR neurons were mostly small (mean+/-S.D.=68.2+/-34.3 microm2, range=11.8-166.8 microm2). The present study suggests that Brn-3a may normally suppress TH expression in many small DRG neurons but activate TH expression in large DRG neurons.

Activation of the caspase cascade underlies the rat trigeminal primary neuronal apoptosis induced by neonatal capsaicin administration.

The systemic administration of capsaicin is known to cause a massive loss of sensory primary neurons in newborn rats. Here we examined the trigeminal ganglion neurons immunohistochemically for the possible induction of activated forms of caspases-9 and -3 following a subcutaneous injection of capsaicin in newborn rats. The DNA fragmentation signal was labeled by a TUNEL method. TUNEL-positive neurons were rare (< 0.5%) at 24 h after injection of the vehicle without capsaicin. After the capsaicin injection, TUNEL-positive neurons began to increase by 12 h, reached a peak at 24 h (11.4%), and returned to the control level by 120 h. Vehicle control levels of caspase- 9-immunoreactive (ir) and caspase-3-ir neurons were low (< 0.5%). Neonatal capsaicin administration induced caspase-9-immunoreactivity (ir) and -3-ir. The temporal distributions of caspase-9-ir and caspase-3-ir neurons were similar to those of TUNEL-positive neurons with peak expressions at 24 h of 13.2 and 11.1%, respectively. A double-stain analysis at 24 h post-injection indicated 72% of TUNEL-positive neurons were caspase-9-ir, and 70% caspase-3-ir. Conversely, 78 and 68% of caspase-9-ir and caspase-3-ir neurons, respectively, were TUNEL-positive. Comparison of two adjacent sections immunostained for the two different antigens revealed the co-expression of the two caspases. These results suggest that neonatal capsaicin triggers the caspase cascade and, thereby, induces trigeminal primary neuronal apoptosis.