Effect of exogenous retinoic acid on tooth movement and periodontium healing following tooth extraction in a rat model.

OBJECTIVES: To evaluate the effect of isotretinoin on orthodontic tooth movement (OTM) and wound healing following exodontia. SETTING AND SAMPLE POPULATION: Sixteen 40-day-old male Wistar rats were divided into two groups: (a) OTM and (b) tooth extraction (TE) of the upper 1st molar and OTM. The experimental animals were treated with isotretinoin (7.5 mg/kg) and the control animals with oil solution for 37 days. MATERIALS AND METHODS: The OTM and bone volume were evaluated by the micro-CT and the periodontium healing was assessed by immunohistochemistry for VEGF-C, COX-2 and IL-1ß. RESULTS: The animals of both groups submitted to the TE showed a statistically significant decrease in the bone volume percentage and increase in OTM. No significant difference of OTM and bone volume was observed between the control and experimental group. However, the alveolar bone of the isotretinoin group revealed more medullary spaces with inflammatory, hematopoietic cells, blood vessels and intense immunolabeling for VEGF-C. This group also showed faster gingival regeneration. No significant difference was observed in the COX-2 and IL-1ß labelings following TE between both groups. CONCLUSION: The isotretinoin did not affect the OTM nor did it cause an alteration in maxillary bone volume. This exogenous acid may contribute to the acceleration of gingival healing.

Inactivation of the Odontogenic ameloblast-associated gene affects the integrity of the junctional epithelium and gingival healing.

Odontogenic ameloblast-associated (ODAM) belongs to the secretory calcium-binding phosphoprotein (SCPP) gene cluster. It is expressed by the epithelial ameloblasts during the accrued mineralisation of enamel and by cells of the junctional epithelium (JE), a specialised portion of the gingiva that plays a critical role in periodontal health. In both cases, ODAM localises at the interface between the cells and the tooth surface. It is also present among the cells of the JE, and is distinctively highly expressed in many epithelial tumours. ODAM has been proposed to be a matricellular protein implicated in the adhesion of epithelial cells to tooth surfaces, and possibly in mediating cell status. To gain further understanding of the role of ODAM, we have created an Odam knockout (KO) mouse by deleting coding exons 2-6. Inactivation of the gene was verified by Southern blot, PCR, real-time qPCR and loss of immunostaining for the protein. Young Odam KO mice showed no readily apparent phenotype. No significant differences were observed in enamel volume and density, rod-interrod organisation, and its attrition. However, in older animals, the JE presented some detachment, an increase in inflammatory infiltrate, and apical down-growth. In addition, its regeneration was delayed following a gingivectomy challenge. Our results indicate that inactivation of Odam expression has no dramatic consequence on enamel but the phenotype in older animals replicates some JE changes seen during human periodontal disease. Altogether, our results suggest that ODAM plays a role in maintaining integrity of the JE.

Wells syndrome associated with Churg­Strauss syndrome.

Churg­Strauss syndrome (CSS) is a systemic vasculitis occurring in patients with a history of asthma. Wells' syndrome (WS) is a rare inflammatory dermatosis that clinically resembles cellulitis, and is histologically characterized by eosinophilic infiltration and flame figures. We report a case of WS associated with CSS. There have been three previous reports of WS associated with CSS; ours is the fourth. All cases had bullous lesions, and three cases were positive for antineutrophil cytoplasmic antibodies.

Disruption of periodontal integrity induces expression of apin by epithelial cell rests of Malassez.

BACKGROUND AND OBJECTIVE: It has been suggested that epithelial cell rests of Malassez (ERM) may express enamel matrix proteins and play an important role in periodontal regeneration. Two novel proteins, apin (APIN) and amelotin (AMTN), produced by maturation-stage ameloblasts and junctional epithelium, have recently been identified. The objective of this study was to evaluate whether the ERM express APIN and AMTN under normal conditions and after periodontal challenge. MATERIAL AND METHODS: Gingivectomy and orthodontic tooth movement were carried out on the left side of the maxillae of rats. The control group included the untreated contralateral side of these animals and the maxillae of normal, untreated rats. Animals were sacrificed by intracardiac perfusion on days 3 and 5 after the experimental procedures and maxillary molars were decalcified and processed for paraffin embedding. Immunohistochemistry was used to evaluate the expression of various ameloblast products, including APIN, AMTN, ameloblastin (AMBN) and amelogenin (AMEL). RESULTS: At 3 and 5 days after periodontal challenge, ERM were more evident in the periodontal ligament along the root surface and in the root furcations. Immunodetection of APIN, but not of the other three proteins, was observed in the ERM following the disruption of periodontal integrity. No immunolabeling for APIN, AMTN, AMBN and AMEL was detected in the ERM under normal conditions. CONCLUSION: The expression of APIN at an early time-point following disruption of periodontal integrity suggests that this protein may be part of the cascade of events leading to the activation of ERM during periodontal healing and regeneration.

Stress analysis in the mandibular condyle during prolonged clenching: a theoretical approach with the finite element method.

Parafunctional habits, such as bruxism and prolonged clenching, have been associated with functional overloading in the temporomandibular joint (TMJ), which may result in internal derangement and osteoarthrosis of the TMJ. In this study, the distributions of stress on the mandibular condylar surface during prolonged clenching were examined with TMJ mathematical models. Finite element models were developed on the basis of magnetic resonance images from two subjects with or without anterior disc displacement of the TMJ. Masticatory muscle forces were used as a loading condition for stress analysis during a 10 min clenching. In the asymptomatic model, the stress values in the anterior area (0.100 MPa) and lateral area (0.074 MPa) were relatively high among the five areas at 10 min. In the middle and posterior areas, stress relaxation occurred during the first 2 min. In contrast, the stress value in the lateral area was markedly lower (0.020 MPa) than in other areas in the symptomatic model at 10 min. The largest stress (0.050 MPa) was located in the posterior area. All except the anterior area revealed an increase in stress during the first 2 min. The present result indicates that the displacement of the disc could affect the stress distribution on the condylar articular surface during prolonged clenching, especially in the posterior area, probably leading to the cartilage breakdown on the condylar articular surface.

Immunocytochemistry of matrix proteins in calcified tissues: functional biochemistry on section.

The organic matrix of calcified tissues comprises collagenous and/or noncollagenous matrix proteins (NCPs). Identification and precise mapping of these matrix components is essential for determining their function, formulating coherent hypotheses on their mechanism(s) of action, and developing novel therapeutic approaches based on biologics. Fibrillar collagen can be readily identified by its conspicuous structure, however, NCPs, in general, do not individually exhibit characteristic structural features that permit to identify them and morphologically determine their localization. To address this limitation, we have used immunocytochemistry, a form of "biochemistry on section", to correlate composition with structure. For cytochemical characterizations, including immunolabeling, our laboratory has opted for colloidal gold labelings and pioneered their application to calcified tissues because they yield high spatial resolution and are quantitative. Over the years, this approach has been applied to identify and map various NCPs in bone and teeth and, in this review of our work, we will emphasize some selected studies that highlight it application to also achieve functional information.

Nerve-growth-factor-dependent and cell-density-independent survival of septal cholinergic neurons in culture from postnatal rats.

We have established a primary neuronal cell culture technique from the postnatal (P11 to P15) rat CNS to study the nerve growth factor (NGF) response to basal forebrain cholinergic neurons. The survival of septal cholinergic neurons in culture was monitored both by the determination of choline acetyltransferase activity and by counting acetylcholinesterase-positive cells. Cells obtained from postnatal septal regions were found to require a plentiful oxygen supply during the dissociation of the cells. NGF-mediated survival of the septal cholinergic neurons was similarly observed in the cultures by using different plating cell densities up to 12.5 X 10(5) cells/cm2. These results suggest that the promotion by NGF of cell survival in culture is independent of plating cell density.

Effects of nerve growth factor and basic fibroblast growth factor on survival of cultured septal cholinergic neurons from adult rats.

We have established a primary culture technique for neuronal cells from rat basal forebrain from postnatal day 58 (P58) to study the effects of neurotrophic factors on the neurons. The survival of acetylcholinesterase (AChE)-positive neurons of 2-week-old rat septum has already been reported to be strongly supported by nerve growth factor (NGF) in culture. In this culture study of neurons from adult rat brains, the survival of AChE-positive neurons from P58 rat septum was slightly improved by NGF, although low affinity NGF receptor expression was also observed on cultured P58 rat septum neurons as well as on those from 2-week-old rats. The addition of basic fibroblast growth factor (bFGF) improved the survival of AChE-positive neurons cultured from P58 rat septum, but did not promote the survival of neurons from P12 rat septum. These results suggest that NGF changes to a maintenance factor in adult rat brain from a survival factor in postnatal 2-week-old rats. The survival of cholinergic neurons in culture of adult rat septum might be supported by factor(s) other than NGF, such as bFGF.

Involvement of cystatin C in oxidative stress-induced apoptosis of cultured rat CNS neurons.

Oxidative stress is involved in neuronal degeneration in cerebrovascular injury, neuropathology and aging. When rat CNS neurons were cultured in a high (50%) oxygen atmosphere, the neurons died. This high oxygen-induced cell death showed features of apoptotic cell death, characterized by DNA fragmentation, and was blocked by inhibitor of protein synthesis. We found that cystatin C and HuC mRNA, the products of which are an inhibitor of cysteine proteases and an RNA binding protein, respectively, were up-regulated in neurons cultured in the high oxygen atmosphere. In the present study, we focused on cystatin C. Cystatin C protein levels were also increased in neurons cultured in the high oxygen atmosphere. In situ hybridization with an RNA probe for rat cystatin C and immunocytochemistry with anti-human cystatin C antibody showed that microtubule-associated protein 2 (MAP2)-positive neurons expressed cystatin C mRNA and protein, respectively, in the high oxygen atmosphere. These results indicated that oxidative stress stimulates an increase in cystatin C expression in cultured neurons, and that cystatin C might have important roles in regulation of apoptosis elicited by oxidative stress.

SNRK, a member of the SNF1 family, is related to low K(+)-induced apoptosis of cultured rat cerebellar granule neurons.

When cerebellar granule neurons obtained from 11-day-old rats were cultured first in high K(+) medium for 4 days, followed by culture in low K(+) medium, the neurons underwent apoptosis and died. This cell death was prevented by actinomycin D, an inhibitor of RNA synthesis. Commitment time of the protective effect of RNA synthesis inhibition on the cell death was examined by adding actinomycin D at various time points after the switch to the low K(+) medium. More than 50% of the cells died when actinomycin D was added 3 h after changing to the low K(+) medium. To identify what kinds of newly synthesized genes are involved in regulation of the low K(+)-induced death, we performed PCR-based differential subtraction analysis using RNA prepared from the cultured neurons 0 and 3 h after changing to low K(+) medium. We isolated a clone that showed an increase in its mRNA level after changing to the low K(+) medium. This clone encoded the 3' untranslated region of SNRK, a serine/threonine kinase. Tissue distribution analysis showed that the mRNA was expressed mainly in the brain and testis. Developmental analysis in the brain showed that the mRNA expression increased in an age-dependent manner until P28, and was slightly decreased in adults. In situ hybridization analysis showed that the mRNA was expressed throughout the brain. The mRNA was shown to be expressed in neurons by double staining with anti-MAP2 antibody. In addition, anti-N-terminal SNRK antibody stained the nuclei of cultured rat cerebellar granule neurons. These results suggested that SNRK may be involved in regulation of low K(+)-induced apoptosis of cultured cerebellar granule neurons.

Cultured basal forebrain cholinergic neurons from postnatal rats show both overlapping and non-overlapping responses to the neurotrophins.

Basal forebrain cholinergic neurons respond in vitro and in vivo to nerve growth factor (NGF) and to brain-derived neurotrophic factor (BDNF). It is not clear to what extent the neurons that respond to these two factors, or to neurotrophin-3 or -4/5 (NT-3; NT-4/5) are identical or only partially overlapping populations. We have addressed this issue in cultures of basal forebrain neurons derived from 2-week-old postnatal rats, using choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) as cholinergic markers. Cholinergic neuron survival was enhanced in the presence of NGF, BDNF and NT-4/5. NT-4/5 was as effective as BDNF. NT-3 was without effect at this age, although in cultures derived from embryonic forebrain, cholinergic differentiation was induced by NT-3. Cotreatment with NGF and BDNF resulted in small, but consistent increases in the number of ChAT-positive neurons, compared with either factor alone. NT-4/5 was also found to be additive with NGF, whereas cotreatment with BDNF and NT-4/5 showed no additivity. NT-3 had no additive effects with any other neurotrophin on any cholinergic parameters in postnatal cultures. Taken together, the results indicate the existence in postnatal rat brain of a large overlapping population of cholinergic neurons that are responsive to ligands for the neurotrophin receptors TrkA (NGF) and TrkB (BDNF and NT-4/5), but not TrkC (NT-3), and small distinct populations that show specificity for NGF or BDNF but not both. We hypothesize that cholinergic neurons projecting into different regions of the hippocampus may derive trophic support from distinct neurotrophins.

High potassium enhances secretion of neurotrophic factors from cultured astrocytes.

Elevation of extracellular potassium concentration ([K+]o) in the central nervous system (CNS), which is observed such after physiological stimuli and during ischemia, is known to be regulated by astrocytes. We suspected that in response to increased [K+]o, astrocytes might secrete some neurotrophic factor(s) to promote the survival of active and/or ischemically damaged neurons. In the present study, we examined neurotrophic activity contained in HK-ACM, i.e., astrocyte-conditioned medium (ACM) obtained after culturing astrocytes in 40 mM potassium-containing medium (HK medium). Addition of HK-ACM to basal forebrain cultures from postnatal 2-week-old (P2w) rats increased both the choline acetyltransferase (ChAT) activity (4.40-fold) and the number of ChAT-positive neurons (2.01-fold) as compared with non-conditioned HK medium. On the other hand, the neurotrophic effects of LK-ACM, i.e., ACM collected after culturing astrocytes in 4 mM potassium-containing medium (LK medium), were much weaker (2.85- and 1.41-fold for ChAT activity and number of ChAT-positive neurons, respectively) than those of HK-ACM. The neurotrophic effects of ACMs increased in a manner dependent on potassium concentration and on astrocyte culture time. Addition of an antibody against nerve growth factor (NGF) neutralized the neurotrophic effects of HK- and LK-ACMs. Direct quantification of NGF protein in ACMs by the two-site ELISA method demonstrated that a high concentration of potassium enhanced NGF secretion from cultured astrocytes. These results suggested that astrocytes secrete NGF in response to [K+]o elevation in the CNS.

High oxygen atmosphere for neuronal cell culture with nerve growth factor. I. Primary culture of basal forebrain cholinergic neurons from fetal and postnatal rats.

Cholinergic neurons cultured from postnatal days 11-13 (P11-P13) rat basal forebrain showed better survival in the culture condition using a 50% O2 atmosphere with and without nerve growth factor (NGF) than in a low (10 or 20%) O2 atmosphere. Except for the culture at a low cell density, the beneficial effect of the highly oxidized culture condition was found in the culture from P3 neurons, but not from embryonic day 18 neurons. The survival of microtubule-associated protein 2 (MAP2)-positive neurons in culture from P3 basal forebrain regions was more enhanced in a 50% O2 atmosphere than in 20% and also 10% O2 atmosphere. The viable number of the MAP2-positive neurons in a 10% O2 condition was about half of that in a 20% condition. These results suggest that the response of the cultured neurons to an incubator O2 concentration changes during the neuronal development in CNS from fetal to postnatal stages.

Generation of reactive oxygen species, release of L-glutamate and activation of caspases are required for oxygen-induced apoptosis of embryonic hippocampal neurons in culture.

Oxygen-induced cell death in embryonic neurons is a useful in vitro model of neuronal apoptosis to study the molecular mechanisms underlying the cell death induced by oxidative stress. In the present study, we examined the involvement of reactive oxygen species and glutamate in the high (50%) oxygen-induced death of cultured hippocampal neurons. During the course of cell death, increases in O2- and hydrogen peroxide (H2O2) levels were observed. On the other hand, superoxide dismutase (SOD), catalase and deferoxamine (DFX), which have inhibitory effects on the generation of O2-, H2O2 and hydroxyl radicals, respectively, protected the neurons. These results suggested that both O2- and H2O2 play important roles in this apoptosis. Antagonists of NMDA and AMPA/kinate (AMPA/KA) receptors and an inhibitor of glutamate release partially prevented the apoptosis, suggesting that exposure to high oxygen enhances glutamate release, which results in activation of NMDA receptor and AMPA/KA receptor. In addition, specific nitric oxide (NO) scavenger and NO synthetase inhibitors blocked the apoptosis, indicating that NO and/or peroxynitrite are involved in this mechanism of cell death. Caspase inhibitors also blocked the neuronal apoptosis. These results suggested that multiple effectors including generation of reactive oxygen species, release of L-glutamate and activation of caspases are activated during the death induced by high oxygen.

The survival of striatal cholinergic neurons cultured from postnatal 2-week-old rats is promoted by neurotrophins.

Although the expression of nerve growth factor (NGF) in the rat striatum is the highest at 2 postnatal weeks (P2w), the action of NGF at that age has not been studied in detail. We examined the effects of several neurotrophic factors, including NGF, on striatal cholinergic neurons cultured from P2w rats. We also examined the effects of a cyclic AMP (cAMP) analog and high K(+)-evoked depolarization. NGF specifically promoted the survival of choline acetyltransferase (ChAT)-positive neurons, and consequently increased the ChAT activity per well, whereas it did not induce the ChAT activity per cholinergic neuron. NGF-responsiveness was the highest in striatal cultures from P2w rats, but it was almost lost in cultures from P4w rats. Brain-derived neurotrophic factor (BDNF), neurotrophin-4/5 (NT-4/5), and a cAMP analog had survival-promoting effects on striatal total neurons including cholinergic neurons. On the other hand, high K+ hardly promoted the survival of striatal cholinergic neurons in cultures from P2w rats, although it increased the viable number of total striatal neurons. High K+ did not increase the ChAT activity in any tested cultures from postnatal 3- to 28-day-old rats. These results demonstrated that NGF prevented the death of striatal cholinergic neurons in cultures from P2w rats, but not from P4w rats, and that high K+ could not rescue these deaths. We propose that cholinergic neurons in the striatum are programmed to die at P2w, and that this programmed cell death can be restored by neurotrophins, but not by depolarization.

High potassium and cyclic AMP analog promote neuronal survival of basal forebrain cholinergic neurons in culture from postnatal 2-week-old rats.

We found depolarization-dependent promotion of survival of cultured basal forebrain cholinergic neurons from postnatal 2-week-old rats. Over 30 mM KCl (high K+) as well as nerve growth factor (NGF) induced considerably high choline acetyltransferase (ChAT) activity and the increase was potentiated by the addition of BAY K8644, a Ca2+ channel agonist. The increase in ChAT activity by high K+ was due to the increased number of viable acetylcholinesterase-positive and ChAT-positive neurons. Also, a cyclic AMP analog gave the same effect as high K+, but its ability to induce the ChAT activity was higher than that of high K+. On the other hand, both high K+ and NGF had very little effects on the survival of the cultured cholinergic neurons from 10-week-old rats. Cyclic AMP analog induced considerable increase in ChAT activity and promotion of survival of cholinergic neurons in the 10-week-old culture. These findings showed that the neuronal death occurring just of the end of synapse formation in rat basal forebrain cholinergic neurons could be prevented by NGF and high K+, but the death of older cholinergic neurons could not. We propose the possibility that the death of postnatal 2-week-old basal forebrain cholinergic neurons in culture might be programmed cell death.

Synergistic effects of brain-derived neurotrophic factor and ciliary neurotrophic factor on cultured basal forebrain cholinergic neurons from postnatal 2-week-old rats.

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, and ciliary neurotrophic factor (CNTF), a member of the neurocytokine family, are known to have synergistic effects on motoneurons, but such synergistic effect has not been studied in detail especially in the brain. In the present study, we examined the synergistic effects of BDNF and CNTF on the survival of basal forebrain cholinergic neurons cultured from postnatal 2-week-old (P2w) rats. Although BDNF is well-known to promote the survival of basal forebrain cholinergic neurons in P2w culture, CNTF had little effect on the survival of choline acetyltransferase (ChAT)-positive neurons and did not increase ChAT activity in the culture. However, CNTF enhanced BDNF-mediated promotion of cell survival of cholinergic neurons when added concomitantly. BDNF alone induced only a three-fold increase in ChAT activity in control cultures, but the concomitant addition of CNTF resulted in an eight-fold increase. CNTF did not enhance BDNF-mediated cell survival of total neurons from the basal forebrain, hippocampus or cerebellum, suggesting that the synergistic effects of CNTF on the BDNF-mediated increase of viability might be strong in basal forebrain cholinergic neurons. CNTF also enhanced the neurotrophin-4/5-mediated increase of ChAT activity, but not the nerve growth factor (NGF)-mediated one. Furthermore, the BDNF-mediated increase was also enhanced by leukemia inhibitory factor but not by interleukin-6. Similar synergistic pattern between neurotrophins and cytokines were also observed in the induction of ChAT activity in embryonic basal forebrain culture. These results suggest that TrkB, a functional high-affinity receptor of BDNF and NT-4/5, and LIFR beta, a receptor component contained in CNTF and LIF receptor complex, might be involved in the observed synergistic effects.

PACAP has a neurotrophic effect on cultured basal forebrain cholinergic neurons from adult rats.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a member of the vasoactive intestinal polypeptide gene family (VIP) that was originally isolated from rat hypothalamus. The high affinity PACAP receptor, PAC1, is expressed in the basal forebrain area of adult, as well as developing rat brain. Hippocampus, a targeting area of basal forebrain cholinergic neurons, contains PACAP. Thus, hippocampal-derived PACAP may have an effect on basal forebrain cholinergic neurons. Indeed, we have reported that PACAP showed neurotrophic effects on these neurons in embryonic and early postnatal culture. Here we report that PACAP has a neurotrophic effect on adult cholinergic neurons in culture. PACAP increases the number of choline acetyltransferase immunoreactive neurons about 2-fold. A similar effect was observed on treatment with cAMP analogue but not nerve growth factor. PACAP also improved the survival and neurite outgrowth of total neurons. These results indicate that PACAP acts as a neurotrophic factor even on adult neurons in vitro.

Interleukin-6 as a neurotrophic factor for promoting the survival of cultured basal forebrain cholinergic neurons from postnatal rats.

Human recombinant interleukin-6 (IL-6, B-cell stimulating factor-2) was capable of supporting neuronal survival in cholinergic neuron culture, prepared from 10-day-old rat brain septal region. Cell survival of the cultured cholinergic neurons was estimated by measuring the remaining choline acetyltransferase (ChAT) activities after 6 days of culture. IL-6 at a concentration of 5 ng/ml maintained a more than 3-fold higher ChAT activity in the culture as compared with that in cultures without IL-6. The maximal dose of IL-6 was near 50 ng/ml. The concomitant addition of mouse nerve growth factor (NGF) and IL-6, both at maximal doses, had a synergistic effect on cholinergic cell survival. These results indicate that IL-6 can act as a neurotrophic agent, independent of the action of NGF, supporting neuronal survival of cultured postnatal rat septal cholinergic neurons. On the other hand, IL-6 did not affect the differentiation of the cultured embryonic rat septal cholinergic neurons, differently from the differentiation action by NGF.

Increase in choline acetyltransferase activity in septum of rats after transient forebrain ischemia: a possible role of factors released in the hippocampus.

Choline acetyltransferase (ChAT) activity increased in rat septum 2 weeks after a transient forebrain ischemia. Extracts were prepared from hippocampus in which CA1 pyramidal neurons had been selectively destroyed by the ischemic insult. ChAT activity in septal neuronal cultures treated with these extracts for 6 days was significantly higher than that in control cultures.