Differential regulation of immune responses by odontoblasts.

Odontoblasts (OBs) are cells lining the inner surface of the tooth. Their potential role in host defenses within the tooth is suggested by their production of antimicrobial beta-defensins, but their role needs confirmation. The present study sought to define the roles of human OBs in microbial recognition and innate host responses. Toll-like receptor 2 (TLR2) and TLR4, as well as CCR6, were immunolocalized in human OBs and their dentinal processes in situ. To examine OB function we used organotypic tooth crown cultures to maintain human OBs within their dentin scaffold. Cells in the OB layer of cultured and non-cultured crown preparations expressed mRNA for several markers of innate immunity including chemokine CCL20, chemokine receptor CCR6, TLR2, TLR4 and the OB marker dentin sialophosphoprotein (DSPP). Expression of human beta-defensin 1 (hBD1), hBD2, hBD3, interleukin-8 (IL-8), and CCL20 increased with time in culture. Tooth crown odontoblast (TcOB) cultures were stimulated with agonist that was specific for TLR2 (Pam3CSK4) or TLR4 [Escherichia coli lipopolysaccharide (LPS)]. Nuclear factor-kappaB assays confirmed the TLR2 activity of Pam3CSK4 and the TLR4 activity of LPS. LPS up-regulated IL-1beta, tumor necrosis factor-alpha (TNF-alpha), CCL20, hBD2, IL-8, TLR2 and TLR4; however, Pam3CSK4 down-regulated these mRNAs. IL-1beta, TNF-alpha, CCL20 were also up-regulated from six-fold to 30-fold in TcOB preparations from decayed teeth. Our results show for the first time that OBs express microbial pattern recognition receptors in situ, thus allowing differential responses to gram-positive and gram-negative bacteria, and suggest that pro-inflammatory cytokines and innate immune responses in decayed teeth may result from TLR4 signaling.

Altered localization of Cav1.2 (L-type) calcium channels in nerve fibers, Schwann cells, odontoblasts, and fibroblasts of tooth pulp after tooth injury.

We have determined the localization of Cav1.2 (L-Type) Ca2+ channels in the cells and nerve fibers in molars of normal or injured rats. We observed high levels of immunostaining of L-type Ca2+ channels in odontoblast cell bodies and their processes, in fibroblast cell bodies and in Schwann cells. Many Cav1.2-containing unmyelinated and myelinated axons were also present in root nerves and proximal branches in coronal pulp, but were usually missing from nerve fibers in dentin. Labeling in the larger fibers was present along the axonal membrane, localized in axonal vesicles, and in nodal regions. After focal tooth injury, there is a marked loss of Cav1.2 channels in injured teeth. Immunostaining of Cav1.2 channels was lost selectively in nerve fibers and local cells of the tooth pulp within 10 min of the lesion, without loss of other Cav channel or pulpal labels. By 60 min, Cav1.2 channels in odontoblasts were detected again but at levels below controls, whereas fibroblasts were labeled well above control levels, similar to upregulation of Cav1.2 channels in astrocytes after injury. By 3 days after the injury, Cav1.2 channels were again detected in nerve fibers and immunostaining of fibroblasts and odontoblasts had returned to control levels. These findings provide new insight into the localization of Cav1.2 channels in dental pulp and sensory fibers, and demonstrate unexpected plasticity of channel distribution in response to nerve injury.

Patterns of fluoro-gold entry into rat molar enamel, dentin, and pulp.

Permeabilities of enamel and dentin are not fully understood despite their importance for caries, restorative materials, and pulp-dentin-enamel interactions. We have found that Fluoro-Gold is useful for examining tooth permeability, and we designed studies to test the effects of aging, injury, neural function, and dentinal repair on its influx into vital rat teeth. We used fluorescence microscopy and immunocytochemistry to show that Fluoro-Gold rapidly penetrates enamel, the dentin-enamel junction, and outer dentinal acellular tubules, and then concentrates in odontoblasts, where it remains for weeks. As predicted, influx was greatest in immature teeth, and formation of reparative dentin impeded it. We expected that denervation would disrupt influx, because of neural regulation of dentinal fluid movement, but it did not. Damage to odontoblasts under injured dentin caused increased influx and efflux of Fluoro-Gold. Analysis of our data suggests that permeabilities of enamel and dentin to Fluoro-Gold are age-related, inter-dependent, and regulated by odontoblasts.

Morphological variation in the tyrosine receptor kinase A-immunoreactive periodontal ligament epithelium of developing and mature rats.

Tyrosine receptor kinase A (trkA) is the high-affinity receptor for nerve growth factor. It has been found in several non-neuronal cell types, indicating biological roles independent of neural function, as well as in the nervous system. An initial study demonstrated that an antibody to the full extracellular domain did not label periodontal ligament epithelium (PLE; also known as epithelial rests of Malassez), but that another antibody which recognises a truncated 41-kDa form of trkA did label PLE. Thus, truncated trkA-immunoreactive (-IR) PLE was further investigated here in developing molars of young rats, and in its mature form in adult rat molars, for its reaction to moderate or deep molar injuries, and for its appearance along the continuously erupting incisors of mature rats. In some of the adult rat molars we also analysed the association of nerve fibres with PLE using antibodies for p75 neurotrophin receptor or peripherin. Rat jaws were fixed with 4% formaldehyde and demineralised, and bound antibody was detected with avidin-biotin-peroxidase and diaminobenzidine or fluorescence procedures. Light microscopy showed great variation in the appearance of trkA-IR PLE and considerable morphological changes during the eruption of molars and incisors. By electron microscopy it was shown that trkA-IR was not uniformly distributed in PLE cells but rather was concentrated in the peripheral zones of each cell cluster. Tooth injury did not influence the form or occurrence of PLE unless there was specific destruction of a ligament region. Qualitative analyses of nerve fibres showed that they only rarely innervated PLE in adult rats, indicating that the truncated receptor has non-neuronal functions in this epithelium. These results suggest that neurotrophin growth factors, acting via truncated trkA receptors, affect the interactions between PLE cells and the periodontal ligament, with fewer PLE interactions with nerves. Furthermore, the expression of these receptors on PLE supports the possibility that these cells are active during tooth development and eruption rather than being merely passive remnants of the degenerating tooth sheath. The similar trkA-IR of PLE and junctional epithelium, as well as their structural association, suggests interactions between these two epithelia.

The exact expression of glial fibrillary acidic protein (GFAP) in trigeminal ganglion and dental pulp.

The expression in various cell types of peripheral tissues of glial fibrillary acidic protein (GFAP), first discovered as an intermediate filament specific for astrocytes, remains controversial owing to numerous reports of a wide distribution for GFAP-immunoreactivity in various cells. The present study employed immunohistochemistry to investigate the precise expression of GFAP in the dental pulp and trigeminal ganglion of adult rats and wild-type mice as well as GFAP-knockout mice. The exhibition of GFAP-immunoreactivity in the trigeminal ganglion was further examined by a reverse transcription polymerase chain reaction (RT-PCR) technique, and in situ hybridization histochemistry using a specific cRNA probe prepared by us. The immunoreaction for GFAP was recognizable in the axons, Schwann cells, and the fibroblasts in the dental pulp of rats and wild-type littermate mice. However, mice with null mutations in the GFAP gene remained immunoreactive for GFAP in all these locations. Intense GFAP-immunoreactivity was found in a small number of satellite cells in the trigeminal ganglion in all animals examined in this study. RT-PCR analysis demonstrated bands for the GFAP gene corresponding to the length expected from the primer design in the samples of trigeminal ganglion and dental pulp. In situ hybridization histochemistry also showed intense signals for GFAP mRNA in some satellite cells of the trigeminal ganglion, but never in the neurons. These data suggest that the GFAP-immunoreactive molecules in the pulpal axons and fibroblasts react non-specifically with the polyclonal antibody and are probably a closely related type of intermediate filament.

Chronic tooth pulp inflammation causes transient and persistent expression of Fos in dynorphin-rich regions of rat brainstem.

We have analyzed central Fos immunoreactivity (Fos-IR) brainstems of adult rats after three clinically relevant dental injuries: filled dentin (DF) cavities that cause mild pulp injury and heal within 1-2 weeks; open pulp exposures (PX) that cause gradual pulp loss and subsequent periodontal lesions; and filled pulp exposures (PXF). By 1 week after DF cavities, no Fos-IR remained except for sites such as lateral-ventral periolivary nucleus (LVPO) that had Fos-IR in all rats including controls. PX injury induced (1) a delayed transient expression of Fos at 1-2 weeks at three loci (ipsilateral neurons in dorsomedial nucleus oralis, paratrigeminal nucleus, and trigeminal tract), (2) persistent ipsilateral Fos for at least 4 weeks after injury in dynorphin (Dyn)-rich regions (rostral lateral solitary nucleus, periobex dorsal nucleus caudalis), and (3) late Fos-IR at 2-4 weeks (bilateral superficial cervical dorsal horn, contralateral dorsal nucleus caudalis, contralateral rostral lateral solitary nucleus). Rats with PXF injury were examined at 2 weeks, and they had greater numbers and more extensive rostro-caudal distribution of Fos neurons than the PX group. One week after PX injury, Fos-IR neurons were found in regions with strong Dyn-IR central fibers. Co-expression of Dyn and Fos was found in some unusually large neurons of the ipsilateral rostral lateral solitary nucleus, trigeminal tract, and dorsal nucleus caudalis. Immunocytochemistry for the p75 low affinity neurotrophin receptor (p75NTR) or for calcitonin gene-related peptide (CGRP) showed no consistent change in trigeminal central endings in any Fos-reactive brainstem areas, despite the extensive structural and cytochemical reorganization of the peripheral endings of the dental neurons. The Fos responses of central neurons to tooth injury have some unusual temporal and spatial patterns in adult rats compared to other trigeminal injury models.

Neurotrophin receptors and nerve growth factor are differentially expressed in adjacent nonneuronal cells of normal and injured tooth pulp.

High-affinity tyrosine kinase A (trkA) neurotrophin receptors on neurons and nonneuronal cells elicit differentiation or survival functions in response to nerve growth factor (NGF), whereas the low-affinity neurotrophin (p75) receptor modulates trkA activity or can independently cause apoptosis or NFkappaB-mediated survival functions. We examined dental tissues for the presence of trkA-like immunoreactivity (trkA-IR), to determine which nonneuronal cell types express it in normal compared with inflamed teeth and how the trkA-positive cells relate to those expressing the p75 receptor and/or NGF. Normal and injured rat molars (dentin cavity for 4 h, 16-24 h, 3 days, 16 days, or 5 weeks) were immunoreacted using the ABC detection system for two anti-trkA antibodies (sTA, Santa Cruz Biotechnology; rTA, L. Reichardt) and antibodies against p75 and NGF, all of which also stained pulpal nerve fibers. We report that, when using the sTA antibody (recognizing the intracellular carboxy terminal), nonneuronal trkA-IR was found in odontoblasts of normal teeth and also in invading polymorphonuclear leukocytes (PMNs) and reparative odontoblasts after injury. When using rTA (recognizing the extracellular domain of the receptor), nonneuronal trkA-IR was only found in odontoblasts. Odontoblasts also had NGF-IR but did not label for NGF mRNA. The lack of odontoblast NGF mRNA suggests that NGF is passed from fibroblasts to the adjacent odontoblasts, where it is picked up by receptor-mediated mechanisms for regulation of odontoblast function. Tooth injury disrupts this system such that trkA-IR decreases in injured odontoblasts, p75 decreases in fibroblasts, and NGF is upregulated by fibroblasts and accumulates in the injured pulp and surviving odontoblasts. Pulpal NGF may contribute to chemoattraction for the invading leukocytes or their sTA-IR may have been induced in response to pulpal NGF. Thus, tooth pulp has a different distribution of nonneuronal NGF and its paracrine receptors during inflammation compared with normal conditions.

Up-regulation of class A Ca2+ channels in trigeminal ganglion after pulp exposure.

We have studied changes in the level of calcium channel expression in the cell bodies of neurons located in the maxillary division of the trigeminal ganglion following induction of persistent pulpitis by pulp exposure in the right maxillary molars. Using anti-peptide antibodies to the alpha1 subunit of class A (P-/Q-type) voltage-gated calcium channels, we observed slight increases in the expression level three days following surgery and approximately 4 fold increase by eight days following the lesion. These changes in the expression of the alpha1 subunit of class A calcium channels may have functional implications in the responses of nociceptive neurons to chronic inflammation.

Dental injury models: experimental tools for understanding neuroinflammatory interactions and polymodal nociceptor functions.

Recent research has shown that peripheral mechanisms of pain are much more complex than previously thought, and they differ for acutely injured normal tissues compared with chronic inflammation or neuropathic (nerve injury) pain. The purpose of the present review is to describe uses of dental injury models as experimental tools for understanding the normal functions of polymodal nociceptive nerves in healthy tissues, their neuroinflammatory interactions, and their roles in healing. A brief review of normal dental innervation and its interactions with healthy pulp tissue will be presented first, as a framework for understanding the changes that occur after injury. Then, the different types of dental injury that allow gradation of the extent of tissue damage will be described, along with the degree and duration of inflammation, the types of reactions in the trigeminal ganglion and brainstem, and the type of healing. The dental injury models have some unique features compared with neuroinflammation paradigms that affect other peripheral tissues such as skin, viscera, and joints. Peripheral inflammation models can all be contrasted to nerve injury studies that produce a different kind of neuroplasticity and neuropathic pain. Each of these models provides different insights about the normal and pathologic functions of peripheral nerve fibers and their effects on tissue homeostasis, inflammation, and wound healing. The physical confinement of dental pulp and its innervation within the tooth, the high incidence of polymodal A-delta and C-fibers in pulp and dentin, and the somatotopic organization of the trigeminal ganglion provide some special advantages for experimental design when dental injury models are used for the study of neuroinflammatory interactions.

Neurotrophin receptor expression is induced in a subpopulation of trigeminal neurons that label by retrograde transport of NGF or fluoro-gold following tooth injury.

Tissue responses to injury are regulated by neurotrophins and neurotrophin receptor levels and can involve both retrograde and paracrine/autocrine trophic signaling. To determine how neurotrophins may contribute to the injury response, the timing and the extent of the up-regulation of neurotrophins and their receptors was examined in a model system which is particularly well suited for the analysis of trophic signaling pathways in response to injury. Injury to the occlusal surfaces of rat molar cusps induces a localized increase in nerve growth factor (NGF) expression in the dental pulp within 4-6 h. Radiolabeled NGF was transported in a receptor-mediated fashion from the teeth to a subset of neurons in the trigeminal ganglion within 15 h, indicating that these neurons possess NGF receptors (trk A and/or p75NTR). To test for NGF responses in the tooth sensory afferent neurons, levels of expression of neurotrophins and their receptors were examined by in situ hybridization in the trigeminal ganglion at 0, 4, 12, 20, 28 and 52 h post-injury. Within the maxillary division of the trigeminal ganglion, trk A expression was elevated at 4 h post-injury, with a maximum increase (2-fold) after 52 h. p75NTR was increased by 28 h post-injury and was increased 1.35-fold by 52 h. BDNF mRNA was increased 12 h after injury (1.8-fold), and 2.5-3-fold at 52 h post-injury. The trk B expression was increased only late after injury (28 and 52 h). To determine the receptor/neurotrophin phenotype of trigeminal neurons with projections to the molar teeth, these neurons were double-labeled with the retrograde tracer fluoro-gold and probes for either BDNF or trk B. The results show that tooth-innervating trigeminal neurons express BDNF, but not trk B. The timing of mRNA expression after injury and the phenotype of identified trigeminal neurons suggests a complex signaling cascade in which NGF at the injury site regulates NGF receptor expression at the levels of the cell body as well as increases in BDNF expression. Upregulated BDNF may act in a paracrine fashion on neighboring trigeminal cells expressing trk B. This signaling cascade may be a common feature of the response to mild peripheral inflammatory injuries within nociceptive pathways.

NGF depletion reduces ipsilateral and contralateral trigeminal satellite cell reactions after inferior alveolar nerve injury in adult rats.

Following peripheral nerve injury, neuronal cell functions in sensory ganglia shift from normal maintenance and neurotransmission toward survival and regeneration. A rapid modulation of glial cell activity, which is related to changes in neuronal-support cell interaction, also occurs after nerve injury. Nerve growth factor (NGF) is required for the survival and maintenance of specific populations of sensory and sympathetic neurons, and changes in neuronal gene expression after axonal injury are due in part to a loss of NGF retrograde transport from the periphery to the cell body. A similar role for NGF in modulating support cell responses to peripheral nerve injury, however, has not been demonstrated. Using an autoimmune model, we assessed the effects of NGF depletion in adult rats on the injury-induced expression of glial fibrillary acid protein immunoreactivity (GFAP-IR) in the ipsilateral and contralateral trigeminal ganglia (TG). Unilateral inferior alveolar nerve crush resulted in a bilateral, NGF-dependent trigeminal satellite cell response. In control rats there was a widespread induction of GFAP-IR in the ipsilateral as well as the contralateral TG. In contrast, GFAP-IR was reduced to the mandibular division of the ipsilateral TG in NGF-depleted rats, and the contralateral up-regulation of GFAP-IR was entirely abolished. Bilateral sympathectomy failed to mimic the effects of autoimmunization. Our results provide evidence that NGF depletion inhibits injury-induced satellite cell responses, independent of its effects on sympathetic nerve function.

Normal development of dental innervation and nerve/tissue interactions in the colony-stimulating factor-1 deficient osteopetrotic mouse.

Dental innervation occurs concurrently with tooth development, eruption, and root formation and is suggested to interact with developing tissues. The purpose of the present study was to investigate dental innervation in osteopetrotic (op/op) mice, which carry a mutation of colony-stimulating factor-1 (CSF-1) and demonstrate sparse macrophages and osteoclasts, failure of bone resorption, lack of tooth eruption, and poor root formation. Jaw tissues from 21 mice in different age groups (7 days, 18 days, 26 days, 5 weeks, and 3 months) were prepared for immunocytochemistry and light microscopy. Immunocytochemistry with the neuronal marker protein gene product 9.5 (PGP 9.5), macrophage marker F4/80, double-labeling with F4/80 and PGP 9.5, and histochemical analysis using tartarate-resistant acid phosphatase (TRAPase) were carried out in selected sections. Molar and incisor development were arrested in the op/op mouse, and both types of teeth had bony occlusion of the eruptive pathway and failure of root formation. Third molar development in the normal mouse is delayed until after birth; therefore, it encounters different bone barriers and jaw structures than are present when first and second molars and incisors begin to develop after the second embryonic week. All three molars, however, completed crown formation prior to eruption failure. Partial root formation was seen in several homozygous op/op mice, and, in those cases, there was partial development of the periodontal ligament. Innervation of dental tissues that successfully formed was essentially normal in the mutant mice despite phenotypic deficiencies in macrophages and osteoclasts. The periodontal ligament was innervated with PGP 9.5-immunoreactive Ruffini mechanoreceptive endings in those cases in which the ligament formed, and op/op mice had remarkably normal sensory innervation of molar and incisor pulp despite failure of bone resorption, failure of root development, and arrested eruption. This study shows that op/op mice develop normal innervation in dental tissues and that dental nerve development proceeds independently of bone abnormalities and root failure in this animal.

Dental innervation and CGRP in adult p75-deficient mice.

Adult dental tissues have unusual neurotrophin biology. Pulpal fibroblasts express nerve growth factor (NGF) and the low-affinity p75 neurotrophin receptor, their sensory nerve fibers express p75 and trk A, and pulpal sympathetic fibers lack p75. Following tooth injury, there is increased pulpal NGF, sprouting of sensory nerve endings, and increased immunoreactivity for the sensory neuropeptide calcitonin gene-related peptide (CGRP). In the present study, we have analyzed tooth structure and innervation of pulp and periodontal ligament in young (6-8 weeks, 3 months) and older (5-12 months) adult mice carrying a null mutation in the p75 gene and compared the results with those of age-matched wild-type controls. Our hypotheses were that tooth structure would be abnormal and that pulpal innervation would be greatly reduced because it consists primarily of nociceptive fibers that have been found to be severely depleted in skin of p75(-/-) mice. Tissues were fixed, X-rayed for gross dental morphology, decalcified, and analyzed for immunoreactivity for CGRP and for a general nerve marker, protein gene product 9.5. Radiographs showed worn-down molar crowns in p75-deficient mice. Light microscopy confirmed the accelerated molar wear and showed intense CGRP immunoreactivity in pulp nerve endings of mutant mice, compared with a gradual decrease in CGRP intensity in controls during normal aging. The CGRP intensity in 5-12-month-old pairs of mice was threefold greater in the mutants (P < 0.03), and in younger mice the mutant always had more CGRP than its matched control. The innervation of molar ligament in all p75-deficient mice was similar to that of controls except there was nerve sprouting near bone loss in mutants. The incisors of mutant mice did not have unusual wear and their pulpal CGRP immunoreactivity remained normal, but their periodontal ligament had fewer thin branched nerve endings at all ages. Thus, most innervation of teeth and their supporting tissues developed normally, and the only neural changes in p75(-/-) mutant mice were the reduction of incisor ligament sensory receptors and increased molar CGRP. Sensory nerves in teeth gradually lose neuropeptide intensity during aging, but that did not happen in the mutant mice, suggesting that the accelerated molar wear stimulated persistent high levels of CGRP.

Nerve growth factor depletion by autoimmunization produces thermal hypoalgesia in adult rats.

Nerve growth factor (NGF) plays a role in mechanisms of inflammation and hyperalgesia in adult animals. We sought to determine if NGF depletion produced by autoimmunization of adult rats altered their thermal sensitivity to an acute noxious thermal stimulus. Anti-NGF IgG was not detected in the cerebrospinal fluid of any tested samples. Only those rats with the highest anti-NGF serum titers showed significant (P < 0.05) thermal hypoalgesia measured using the hot plate test (52 degrees C): the mean (+/-S.D.) hind paw lick latency of rats in the high anti-NGF titer group was 18.0 +/- 4.6 s compared to means of 10.8 +/- 4.3 s, 9.2 +/- 2.6 s and 10.1 +/- 3.0 s in the medium, low and control groups, respectively. Thus, NGF depletion by autoimmunization is a useful model for investigating the role of NGF in behavioral responses of adult rats to noxious stimuli, providing high titers of antibody are present.

Periapical replacement resorption of permanent, vital, endodontically treated incisors after orthodontic movement: report of two cases.

Radiographic observations show that periapical replacement resorption (PARR) is a frequent and unpredictable sequella of orthodontic movement of permanent maxillary incisors. Only the apical root portion undergoes resorption, and it is subsequently replaced with normal bone. PARR also occurs without orthodontic tooth movement, but these resorptions differ as to type and location. PARR clearly occurs in teeth with vital pulps after orthodontic movement; however, our two cases and a review of 43 other cases indicate that endodontically treated incisors show a statistically significant lesser frequency and severity of apical resorption than untreated teeth. The role the vital pulp plays in this apical resorption is yet to be determined. A hypothetical explanation is offered suggesting that pulpal neuropeptides may be involved in PARR in both vital and endodontically treated incisors. The role calcium hydroxide plays in endodontically treated teeth is also considered.

Different localizations of growth-associated protein (GAP-43) in mechanoreceptors and free nerve endings of adult rat periodontal ligament, dental pulp and skin.

Distributions of growth-associated protein-43 (GAP-43) in the periodontal ligament and dental pulp of adult rats were studied by light and electron microscopy. The mature periodontal ligament and dental pulp contained numerous GAP-43-positive neural elements, comprising periodontal Ruffini endings and thin nerve fibers, but expression patterns differed among the kinds of nerves. In the periodontal ligament of rat molars, immunoelectron microscopy revealed that GAP-43 like immunoreactivity in the Ruffini ending, an essential mechanoreceptor, was confined to the Schwann sheaths around the axon terminals and was not in the axon terminals themselves, unlike free endings that revealed axonal GAP-43. However, the lamellar Schwann cells associated with the cutaneous receptors did not exhibit any GAP-43 like immunoreactivity though they were intensely reactive for low affinity nerve growth factor receptor (p75-NGFR), a marker for lamellar Schwann cells in mechanoreceptors. The characteristically uniform expression of GAP-43 in the Schwann lamellae that surround the Ruffini mechanoreceptors of rat molar ligament suggests that Schwann cells are involved in the GAP-43 mediated plasticity of these receptors. On the other hand, the pulpal nerves were filled with the reaction products in their axonal spaces, suggesting the potential for neuronal plasticity during normal function and after tooth injury.

Chronic dexamethasone treatment and its effects on sensory neuropeptides, pulpal injury reactions and reparative dentin.

Initial sensory nerve reactions to dental injuries include terminal sprouting and intensified immunoreactivity for calcitonin gene-related peptide (CGRP) and substance P (SP); those reactions are reduced at 4 days after injury when rats are treated daily with dexamethasone (DEX) [17]. Here we have analyzed long-term effects of DEX (daily, 0.2 mg/kg) on wound healing, sensory nerve sprouting, and CGRP/SP intensity at 7-14 days after cavity preparation. All DEX treated rats had loss of appetite and stopped growing during the postoperative periods while controls had normal postoperative growth. After 7-14 days, CGRP immunoreactivity (IR) was decreased to one-third of normal (P < 0.05) compared to vehicle in both the intact and injured molar pulp, and SP also decreased, but the neuropeptide intensity in adjacent periodontal innervation was not changed. Pulpal injury and inflammation were reduced by DEX treatment, but reparative dentin was formed just as well in the DEX rats as in the vehicle group. When the injured teeth formed fibrous dentin, there was sprouting of nerves towards that matrix, and DEX did not inhibit that reaction. The sprouts could contain intense neuropeptide immunoreactivity in DEX rats even though the CGRP/SP intensity in uninjured pulp was reduced. We conclude that (1) chronic DEX treatment causes a generalized decrease in CGRP and SP neuropeptides in pulpal nerves but not in periodontal ligament; (2) it reduces abscess formation in injured teeth; (3) it does not block reparative dentin formation; and (4) it does not block sprouting of pulpal nerves towards fibrous dentin. The selective loss of pulpal neuropeptides CGRP and SP during dexamethasone treatment may be caused by reduced dental function since there was substantial loss of appetite and chronic weight loss during the 1-2 week treatment periods.

Anterograde transport of neurotrophins and axodendritic transfer in the developing visual system.

Neurotrophic factors support the differentiation and survival of neurons and influence properties of synaptic transmission. The neurotrophic hypothesis postulates a retrograde action of trophic factors: their production and release by target cells and their uptake by innervating axons. Besides the retrograde route of trophic messengers, the survival of neurons and the development of synapses is thought to be also regulated by anterograde, afferent trophic signals. We now show that exogenous neurotrophins are transported in the anterograde direction, from cell bodies to the axon terminals, and that the intact neurotrophin is released after anterograde transport, taken up and utilized by second-order visual neurons in the developing chick brain. These results suggest that anterogradely transported neurotrophins may play a role in synaptic plasticity and may have effects at more than one synapse beyond the initial release site.

Axonal transport of fluorescent carbocyanine dyes allows mapping of peripheral nerve territories in gingiva.

Sensory innervation of gingival tissue can cause neurogenic inflammation that depends on the extent of the branching area of the peripheral nerve fibers. We designed the present study to determine whether single trigeminal axons branch to both the buccal and palatal gingiva of maxillary molars of adult rats. Accumulation via retrograde transport of DiI (red) or DiA (green) fluorescent carbocyanine dyes in neurons of trigeminal ganglia was evaluated 7 days after applying one dye to the buccal sulcus and the other to the palatal sulcus. Both dyes were absorbed through the junctional epithelium, and the two sites each labeled similar numbers and sizes of neurons in the lateral zone of the maxillary division (44% from buccal and 46% from palatal gingiva). Double-labeled neurons had the same size (32.5 +/- 6.70 microns, mean circumference +/- S.D.) and location as single-labeled neurons, and they were 9% of the total. This study shows that exogenous dyes can diffuse into mucosa and thereby allow in vivo mapping of sensory nerve branching patterns to several intact tissues per animal. We found that 9% of the labeled cells extended to both the buccal and palatal gingiva. Thus, inflammation that spreads from one gingival region to the other could have a neurogenic mechanism involving trigeminal sensory neurons that extend their peripheral branches to innervate both buccal and palatal gingiva of adult rat molars.

Odontoblast processes in dentin revealed by fluorescent Di-I.

There has been controversy about the length and structure of the odontoblast process within dentin since the earliest histologic studies of teeth. Our objective was to use the fluorescent carbocyanine dye Di-I combined with a new gelatin embedment procedure and confocal microscopy to determine the structure and extent of odontoblast processes in developing and mature rat teeth, injured rat molars, reparative dentin, and adult monkey teeth. We found that odontoblast processes do not extend into outer dentin or to the dentin-enamel junction except during early stages of development. Those in innervated regions of crown are long and straight, whereas those in roots are extensively branched and shorter. Cavity injury to crown dentin caused odontoblast fragments to be aspirated into outer dentin. In reparative dentin the odontoblast processes were branched and similar to those in roots. We used photoconversion and electron microscopy to show that Di-I fills the entire odontoblast after gelatin embedment, including the cytoplasm. This is a different type of carbocyanine staining from any previously reported, and it also stains other cells in adjacent hard tissues such as bone and cementum. The Di-I-gelatin method is a new way to use carbocyanine dyes. It has enabled us to solve a long-standing controversy about the histology of teeth, and it should be useful for many other studies of cell structure.