Human dental pulp stromal cell conditioned medium alters endothelial cell behavior.

BACKGROUND: Angiogenesis is of utmost importance for tissue regeneration and repair. Human dental pulp stromal cells (hDPSCs) possess angiogenic potential, as they secrete paracrine factors that may alter the host microenvironment. However, more insight into how hDPSCs guide endothelial cells (ECs) in a paracrine fashion is yet to be obtained. Therefore, the current study aimed to investigate the effect(s) of conditioned medium derived from hDPSCs (hDPSC-CM) on EC behavior in vitro. METHODS: hDPSCs were harvested from third molars scheduled for surgical removal under informed consent. The angiogenic profile of hDPSC-CM was identified using human angiogenesis antibody array and enzyme-linked immunosorbent assay (ELISA). Using real-time reverse transcription-polymerase chain reaction (RT-PCR) and ELISA, the mRNA and protein expression level of specific angiogenic biomarkers was determined in human umbilical vein endothelial cells (HUVECs) exposed to hDPSC-CM. The effect of hDPSC-CM on HUVEC attachment, proliferation and migration was evaluated by crystal violet staining, MTT, transwell migration along with real-time cell monitoring assays (xCELLigence; ACEA Biosciences, Inc.). A Matrigel assay was included to examine the influence of hDPSC-CM on HUVEC network formation. Endothelial growth medium (EGM-2) and EGM-2 supplemented with hDPSC-CM served as experimental groups, whereas endothelial basal medium (EBM-2) was set as negative control. RESULTS: A wide range of proangiogenic and antiangiogenic factors, including vascular endothelial growth factor, tissue inhibitor of metalloproteinase protein 1, plasminogen activator inhibitor (serpin E1), urokinase plasminogen activator and stromal cell-derived factor 1, was abundantly detected in hDPSC-CM by protein profiling array and ELISA. hDPSC-CM significantly accelerated the adhesion phases, from sedimentation to attachment and spreading, the proliferation rate and migration of HUVECs as shown in both endpoint assays and real-time cell analysis recordings. Furthermore, Matrigel assay demonstrated that hDPSC-CM stimulated tubulogenesis, affecting angiogenic parameters such as the number of nodes, meshes and total tube length. CONCLUSIONS: The sustained proangiogenic and promaturation effects of hDPSC-CM shown in this in vitro study strongly suggest that the trophic factors released by hDPSCs are able to trigger pronounced angiogenic responses, even beyond EGM-2 considered as an optimal culture condition for ECs.

Instrument fracture in root canals - where, why, when and what? A study from a student clinic.

AIM: To investigate the incidence of instrument fracture during nonsurgical root canal treatment performed by undergraduate dental students and to assess the outcome of the treatment if the remaining fragment was left or removed from the root canal. METHODOLOGY: A retrospective analysis was performed by reviewing assessment forms (n = 3854) filled out for each root canal treatment over a 10-year period (1997-2006) at the University of Bergen, Norway. Based on the assessment forms, all instrument fractures and data concerning type and dimension of the fractured instruments were identified. Moreover, the affected tooth, root, root curvature and the vertical level of the fragment in the root canal were recorded. To assess the outcome of the root canal treatment, radiographs taken at follow-ups were evaluated by two individual examiners. RESULTS: The overall incidence of instrument fracture during the period was 1.0% on a tooth level. Instrument fracture occurred significantly more often in molars and in teeth rated as difficult preoperatively. Of all instrument fractures, 39.5% were located in the mesio-buccal canals of molars, and 76.5% of the fragments were located apically. A significant high percentage of instruments of small apical diameter (sizes 006-015) fractured in relative straight root canals. The treatment was successful in 71.4% of the cases where the instrument fragment was removed, but only in 56.5% of the cases where the fragment remained in the root canal. Significantly more instruments were removed from teeth with a primary infection (P ≤ 0.05). The success rate for teeth with instrument fracture was 72.7% for vital teeth, 58.3% for primary infected teeth and 42.9% in retreatment cases. CONCLUSIONS: Within the limits of the present study, the results indicate that the preoperative diagnosis is important for the final treatment outcome and that removal of the fragment is important in cases with primary infection.

Lymphangiogenesis is induced during development of periodontal disease.

Lymphangiogenesis, the formation of new lymphatics, is associated with chronic inflammation and tissue injury, and its role is to enhance lymphatic flow, immune cell transport, and antigen clearance. It is unknown if lymphangiogenesis takes place during periodontal disease development, and we hypothesized that growth of lymphatic vessels occurs in gingiva during development of periodontitis in mice. Inflammation was induced in gingiva with Porphyromonas gingivalis gavage, and bone resorption was verified after 42 days. Growth of lymphatic and blood vessels was measured after immunofluorescent staining with LYVE-1 and CD31. Expression of vascular endothelial growth factors and 2 inflammatory cytokines was investigated 10 days post-infection. Gingival lymphangiogenesis was found 10 days and 42 days post-infection, but proliferation of vessels was observed only in the shortest observation period. Epithelial expression of vascular growth factors (VEGF) A, C, and D was observed in gingiva, and increased numbers of immune cells expressing VEGF-C were found after infection, along with up-regulation of IL-1ß and TNF-α at protein levels. We conclude that lymphangiogenesis takes place in gingiva during periodontal disease development, and that up-regulation of vascular growth factor C in recruited immune cells is likely important for the growth of lymphatic vessels.

Differential expression of neuropeptide Y Y1 receptors during pulpal inflammation.

AIM: To study the pattern of neuropeptide Y (NPY) Y1 receptor (Y1R) localization in the normal dental pulp and during different stages of pulpal inflammation. The hypothesis was that the expression of Y1R varies during different stages of pulpitis. METHODOLOGY: Pulp exposure injury was made on first molar teeth of Sprague-Dawley rats. Animals were killed 3, 7, 14, 21, 28 and 46 days after pulp exposure. Jaws were removed, decalcified and processed for immunohistochemistry for identification and localization of Y1R. Double labelling was performed using antibodies for laminin, CD43 and CD4 with Y1R. RT-PCR was performed to verify gene expression of Y1R in dental pulp and trigeminal ganglion. RESULTS: RT-PCR revealed the presence of NPY-Y1R in the dental pulp, trigeminal ganglion and gingiva. With immunohistochemistry, in control rats, Y1R was mainly located in capillaries and small vessels in the dental pulp as well as in the odontoblastic layer. No YIR was noted on immune cells in normal pulp. In pulpitis, immune cells such as CD43+ granulocytes and CD4+ lymphocytes expressed Y1R. As pulpitis progressed, there was a significant decrease in number of blood vessels expressing Y1R in the odontoblast layer when compared to control rat pulp. CONCLUSIONS: This study gives evidence that Y1R is a modulator of pulpal inflammation.

Apexogenesis after initial root canal treatment of an immature maxillary incisor - a case report.

AIM: To present a case where a traumatized, immature tooth still showed capacity for continued root development and apexogenesis after root canal treatment was initiated based on an inaccurate pulpal diagnosis. SUMMARY: Traumatic dental injuries may result in endodontic complications. Treatment strategies for traumatized, immature teeth should aim at preserving pulp vitality to ensure further root development and tooth maturation. A 9-year-old boy, who had suffered a concussion injury to the maxillary anterior teeth, was referred after endodontic treatment was initiated in tooth 21 one week earlier. The tooth had incomplete root length, thin dentinal walls and a wide open apex. The pulp chamber had been accessed, and the pulp canal instrumented to size 100. According to the referral, bleeding from the root made it difficult to fill the root canal with calcium hydroxide. No radiographic signs of apical breakdown were recorded. Based on radiographic and clinical findings, a conservative treatment approach was followed to allow continued root development. Follow-up with radiographic examination every 3rd month was performed for 15 months. Continued root formation with apical closure was recorded. In the cervical area, a hard tissue barrier developed, which was sealed with white mineral trioxide aggregate (MTA). Bonded composite was used to seal the access cavity. At the final 2 years follow-up, the tooth showed further root development and was free from symptoms.

Sensory pulpal nerve fibres and trigeminal ganglion neurons express IL-1RI: a potential mechanism for development of inflammatory hyperalgesia.

AIM: To localize interleukin-1 receptor type I (IL-1RI) in rat dental pulp and trigeminal ganglion (TG) and to test the hypothesis that pulpal inflammation increases neuronal expression of IL-1RI. METHODOLOGY: Female Wistar rats were subjected to unilateral pulp exposures in the maxillary and mandibular first molars, whereas the contralateral jaws served as untreated controls. Seven days later the animals were transcardiacally perfused and the jaws and the TGs were removed and prepared for immunohistochemistry. Immunoreactivity for IL-1RI was examined alone (DAB) and together with calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), CD31 or CD34 by multiple-labelling immunofluorescence. Quantification of IL-1RI-immunoreactive (-IR) cells in the maxillary and mandibular division of the ganglion was performed in parasagittal immunoreacted sections of the right and left TGs. Data were analysed with Mann-Whitney Rank Sum test (P < 0.05). RESULTS: Interleukin-1 receptor type I was found on sensory (CGRP-IR) and sympathetic (NPY-IR) nerve fibres and on blood vessels (CD31- and CD34-IR) in the dental pulp. It was also localized on sensory neurons and axons in the TG. Pulpal inflammation significantly increased the expression of IL-1RI in the TG (P < 0.001). CONCLUSIONS: The localization of IL-1RI on sensory nerve fibres and its up-regulation in TG neurons during pulpal inflammation may imply a direct effect of IL-1 in pulpal nociception. The presence of IL-1RI on sympathetic nerve fibres and on blood vessels may indicate a vasoactive role of the same cytokine in the pulp.

Dens invaginatus and treatment options based on a classification system: report of a type II invagination.

AIM: To give an overview of treatment options for dens invaginatus based on a classification system. SUMMARY: Dens invaginatus is a dental malformation which may give endodontic complications. Treatment may vary in relation to anatomy, and a classification system for dens invaginatus forms the basis for discussion. A clinical case, classified as a type II invagination (Oehlers' classification), is also presented. Clinical and radiographic examination revealed an invagination penetrating into the apical third of the root canal in tooth number 12. The tooth was immature with an open apex, apical pathosis and a labial fistula. To control the infection, ultrasonic removal of the invagination was necessary, as the invagination prevented complete cleaning and shaping of the root canal. After chemo-mechanical preparation and dressing with calcium hydroxide, an apical plug of MTA was placed, followed by restoration of the tooth with resin-bonded composite. Healing of the lesion with hard tissue formation was confirmed at follow-up. KEY LEARNING POINTS * Knowledge about classification and anatomical variations of teeth with dens invaginatus is important in endodontic decision making. * A classification system may be helpful when treatment options are considered. * Classification of dens invaginatus requires a thorough preoperative radiographic examination.

Delta (delta) opioid receptors in small and medium-sized trigeminal neurons supporting the dental pulp of rats.

The control of pain perception is a challenge in clinical dentistry, most prominent during tooth pulp inflammation. The tooth pulp is a well-defined target, and is densely supplied by a sensory trigeminal innervation. Opioids are signaling molecules that are suggested to participate in pain perception. Here we analysed the presence of delta opioid receptor (DOR) in trigeminal neurons innervating the tooth pulp of rat molars. Immunohistochemical and ultrastructural analysis revealed that DOR was identified in peripheral nerves in the molar dental pulp, both in the root and the coronal pulpal parts, with branching in the highly innervated subodontoblast layer. DOR was localised in about one third of all the trigeminal dental neurons, identified by means of retrograde neuronal transport of fluorogold (FG) from the dental pulp. Of the DOR-labeled neurons, nearly all were small and medium-sized (147.5-1,810.2 microm(2), mean 749.1 +/- 327.3 microm(2)). Confocal microscopy confirmed that DOR-immunoreactivity was distributed as granules in the neuronal cytoplasm. Approximately 70% of the DOR-immunoreactive neurons were also immunopositive for vanilloid receptor 1 (TRPV1). Ultrastructural analysis demonstrated DOR-immunoreactivity in the unmyelinated and in some of the myelinated nerve fibers in the dental pulp. These results indicate that DOR may influence the function in a subset of small and medium-sized trigeminal sensory neurons supporting the tooth, which are mainly known for their ability to mediate nociceptive stimuli. Agonists, acting on DOR, may thus have an influence on a subpopulation of nociceptive neurons supporting the rat tooth.

Nonsurgically retreated root filled teeth--radiographic findings after 20-27 years.

AIM: To identify periapical changes in nonsurgically retreated root-filled teeth 20-27 years after root canal treatment. METHODOLOGY: From an original material of 429 roots, retreated by undergraduate students in a teaching clinic, 112 roots in 70 individuals could be evaluated radiographically 20-27 years after treatment. The same roots had been studied 10-17 years earlier. The periapical condition was registered and compared by three observers in two series of intraoral radiographs taken 10-17 and 20-27 years after treatment. A retrospective analysis was performed to gain information about probable endodontic and nonendodontic reasons for extractions of lost roots, by evaluating their periapical status immediately after retreatment and at the 10-17-year follow-up. RESULTS: Favourable outcomes were observed in 11 roots that had radiolucencies at the 10-17-year follow-up. Eight of these roots had periapical pathosis preoperatively, five of them filled with surplus root filling material. The percentage of cases recorded as normal condition at the final follow-up was 95.5%, including five cases initially recorded with increased width of the apical periodontal space. Delayed healing as a result of surplus root filling material explained most of the cases with favourable outcome assessed many years after treatment. Twenty-eight roots were lost because of extraction during the observation period, 17 during the last 10 years. Based on status at previous follow-ups, endodontic failure seems to represent a minor reason for extraction in the material. CONCLUSION: Late periapical changes, with more successful cases, were recorded when a 10-17-year follow-up after root canal treatment was extended for another 10 years. Persistent asymptomatic periapical radiolucencies, especially those with overfill, should generally not be classified as failures, as many of them will heal after an extended observation period.

NK1, NK2, NK3 and CGRP1 receptors identified in rat oral soft tissues, and in bone and dental hard tissue cells.

The distribution of the tachykinin receptors neurokinin-1 (NK1), neurokinin-2 (NK2) and neurokinin-3 (NK3), and the calcitonin gene-related peptide-1 (CGRP1) receptor were examined in rat teeth and tooth-supporting tissues by immunohistochemical methods and light and confocal microscopy. Western blot analysis was performed to identify the NK1- and the CGRP1-receptor proteins in the dental pulp. The results showed that odontoblasts and ameloblasts, cementoblasts and cementocytes, osteoblasts and osteocytes are all supported with the tachykinin receptors NK1 and NK2, but a distinct, graded cellular labeling pattern was demonstrated. The ameloblasts were also positive for CGRP1 receptor. Blood vessels in oral tissues expressed the tachykinin receptors NK1, NK2 and NK3, and the CGRP1 receptor. Both gingival and Malassez epithelium were abundantly supplied by NK2 receptor. Pulpal and periodontal fibroblasts demonstrated NK1 and NK2 receptors. Western blot analysis identified both the NK1- and the CGRP1-receptor proteins in the dental pulp. These results clearly indicate that the neuropeptides substance P, neurokinin A, neurokinin B and CGRP, released from sensory axons upon stimulation, directly modulate the function of the different types of bone and dental hard tissue cells, and regulate functions of blood vessels, fibroblasts and epithelial cells in oral tissues.

Periapical changes following root-canal treatment observed 20-27 years postoperatively.

AIM: The aim of the present study was to identify periapical changes 20-27 years after root-canal treatment. METHODOLOGY: The periapical condition of 265 roots filled by undergraduate students was evaluated in two series of intraoral radiographs taken 10-17 and 20-27 years after treatment. Roots (72) not recorded with a normal periapical situation on both occasions by two observers, were re-evaluated by other two examiners, separately and jointly. Final decisions about diagnoses were made by all four examiners. A strict definition was used for the identification of cases with an unfavourable outcome. RESULTS: Favourable outcomes were observed in 6.4% of the roots that had radiolucencies at the 10-17-year follow-up. Periapical radiolucencies after 20-27 years appeared in 1.5% of all other roots. The radiographic failure frequency for the total material was 4.9%. The percentage of cases with normal periapical findings at the final follow-up was 86.4%, whilst 8.7% were recorded with increased width of the apical periodontal space. Delayed healing owing to surplus root-filling material explained nearly all of the cases with favourable outcome assessed many years after treatment. CONCLUSIONS: Late periapical changes, with more successes than failures, were recorded when a 10-17-year follow-up period after root-canal treatment was extended for another 10 years.

Diagnosing periapical lesions--disagreement and borderline cases.

AIM: The aim of this paper is to present a selection of disagreement and borderline cases from a methodological study on the radiographic diagnosis of periapical disease. METHODOLOGY: Thirty-two roots, 12% of the material in an earlier study, were subjected to joint discussion because of diagnostic problems. The aim was consensus. Six cases representing different teeth/roots illustrating typical problems are presented. RESULTS: The varying morphology of apical areas and varying density of surrounding bone present a number of challenges. Even so, the joint discussion resulted in an agreement for all the cases. Twenty-five diagnoses were established and seven cases were not diagnosed owing to substandard radiographs. CONCLUSION: A detailed analysis of the periodontal ligament space, the lamina dura, trabecular pattern and bone marrow spaces is mandatory for the radiographic diagnosis of periapical disease

Incompletely fractured teeth associated with diffuse longstanding orofacial pain: diagnosis and treatment outcome.

AIM: To evaluate the diagnostic value of different clinical findings, and analyse the symptoms experienced from teeth with incomplete dentinal fractures in patients with diffuse orofacial pain. Secondly, to present the outcome of different treatment modalities in these patients. METHODOLOGY: Thirty-two patients, referred to the teaching clinic at the Faculty of Dentistry, University of Bergen, with poorly localized orofacial pain, were finally diagnosed with 46 incompletely fractured teeth. The distribution and pattern of pain was thoroughly recorded for each patient. The final diagnosis, incompletely fractured tooth, was obtained after removal of fillings and direct inspection of possible fracture lines. RESULTS: Many of the patients had suffered diffuse orofacial pain for more than 1 year, and had earlier consulted dental and medical expertise. Direct inspection of fracture lines, combined with staining solution and transillumination proved to be the best diagnostic tools. The longer the duration of pain before the diagnosis of an incompletely fractured tooth was established, the more diffuse was the distribution of pain. Endodontic or restorative treatment relieved the symptoms in 90% of the patients, whilst persisting symptoms in 10% were considered part of an orofacial pain complex of obscure aetiology. CONCLUSION: This study shows that the diagnosis of the incompletely fractured teeth in patients with longstanding diffuse orofacial pain symptoms are time consuming and represent a diagnostic problem. With appropriate endodontic and/or restorative treatment, symptoms were relieved in the majority of cases.

Prodromal herpes zoster--a diagnostic challenge in endodontics.

This case report highlights the diagnostic challenge that herpes zoster represents if pain develops in the prodromal stage. A 58-year-old male presented with pain in the left maxilla. The symptoms had lasted for 7 months. The first premolar had been extracted soon after the onset, and the second premolar root filled shortly thereafter. Symptoms were experienced as sudden pain attacks lasting for several hours and analgesics gave some pain relief. Clinical examination showed that the second premolar was tender to percussion. No sinus tract or swelling were present. Radiographic examination showed previously root-filled second premolar and first molar teeth, and no evidence of apical pathosis. Due to the uncertainty about the quality of the root filling in the second premolar and incomplete root filling in the first molar, retreatment was started prior to prosthetic treatment in the region. The pain continued and became more intense during the treatment. A diagnosis of herpes zoster was determined, when an acute attack with oedema and vesicles occurred, 2 months after retreatment was started. In the present case, therefore, the primary attack presented itself after months and the herpes zoster diagnosis could not be made until then. points. Key learning points. * A long lasting prodromal stage is an unusual event. * Symptoms combined with inadequate technical standard of root fillings may confuse the diagnostics. * No available data support or suggest the use of antiviral treatment as a diagnostic tool.

Lateral breakdown of nonendodontic origin adjacent to maxillary left incisors.

CASE REPORT: A 16-year-old female presented with a labial fistula located between the central and lateral left maxillary incisors. The teeth had normal colour, responded positively to pulp testing, demonstrated negative percussion tests and had no evidence of periodontal pockets. The patient reported no history of trauma, but mentioned that she had received orthodontic treatment. Radiographic examination showed bone loss between these two teeth. Explorative surgery followed by antibiotic treatment was performed, but a fistula reappeared after 22 months. Surgical retreatment combined with antibiotic treatment resulted in gradual healing over a three-year period. No root canal treatment was performed. Aetiological considerations connected to tissue injury and inflammation are discussed. Inflammation induced disturbances in local homeostasis may possibly explain the lateral breakdown of bone. Such areas of reduced resistance may, under unfavourable conditions, be infected by blood-born pathogens. Information about such aberrant cases is important in endodontic decision making.

Neurokinin-1 receptor expression in the mature dental pulp of rats.

Substance P induces inflammatory reactions in peripheral tissues including the dental pulp, but its regulatory effects in target tissues are dependent on receptor signalling. Here the expression of the substance-P receptor neurokinin-1 (NK1) in the mature molar pulp of the rat was examined in order to localize the main target areas for substance P. A polyclonal antibody directed against the C-terminal of the receptor was used, and immunohistochemistry was performed by the avidin-biotin peroxidase complex method. The results showed that the NK1 receptor was intensely expressed along vessel-like structures in the odontoblast and subodontoblast layer. A granulated and diffusely distributed NK1-receptor labelling was found along larger blood vessels in the root pulp and pulp proper. NK1 receptor-positive cells were frequently observed in the cell-rich zone beneath the odontoblast layer. The results indicate that, in the mature rat molar pulp, the main targets for substance P acting through the NK1 receptors are tissues related to blood vessels in the odontoblast and subodontoblast area. Furthermore, the expression of NK1 receptors on cells located in the subodontoblast area could indicate that substance P also affects cell functions in this area.

Root resorption and apical breakdown during orthodontic treatment of a maxillary lateral incisor with dens invaginatus.

A 13-year-old girl was referred for endodontic treatment of a maxillary right lateral incisor with root resorption and apical radiolucency after orthodontic treatment. Radiographically the tooth had an invagination canal (Oehlers' Type III) and responded positively to pulp testing. The invagination canal extending to the lateral resorption was opened. Vital bleeding tissue was found, and the canal was cleaned and obturated. The pulp in the main root canal remained vital. The radiolucent lesion gradually decreased during the 3-year follow-up. The bone resorption seen here, despite vital tissue in the invagination canal, was probably due to osteoclastic activity caused by a low grade chronic infection, influenced by an injury-induced inflammation in the periodontium during orthodontic treatment.

Nerve fibers immunoreactive to calcitonin gene-related peptide, substance P, neuropeptide Y, and dopamine beta-hydroxylase in innervated and denervated oral tissues in ferrets.

The effect of sensory and sympathetic denervation on the localization and distribution of nerve fibers immunoreactive (IR) to calcitonin gene-related peptide (CGRP), substance P (SP), neuropeptide Y (NPY), and dopamine beta-hydroxylase (DBH) was studied in the dental pulp, periodontal ligament (PDL), and gingiva in ferrets. Unilateral axotomy was performed by resection of the inferior alveolar nerve (IAN) 10 days before the experiment (Group 1); sympathectomy, by unilateral removal of the cervical ganglion 5 days before the experiments (Group 2). Immunohistochemistry was performed on free-floating sections by the avidin-biotin-peroxidase technique. A considerably higher density of sensory fibers IR to CGRP and SP was found in the dental pulp than in PDL and gingiva. The majority of pulpal fibers were located in the walls of blood vessels. A subodontoblastic network of fibers IR to CGRP and SP was lacking in incisors and canines and was found only in the coronal pulp in premolars and molars. Sympathetic fibers were sparsely distributed in the pulp, and they were mainly confined to large vessels running centrally in the root pulp as well as the larger vessels in apical PDL and alveolar bone. Gingiva was well supplied with CGRP- and SP-IR nerves, and some NPY and DBH fibers were located in association with larger vessels. Round cell-like structures within the basal part of the epithelium were CGRP-IR. Axotomy induced a complete loss of CGRP- and SP-IR fibers in the anterior part of the jaws, whereas sympathectomy caused a reduction, but not a total loss, of NPY- and DBH-IR nerves. It is concluded that, except for some distributional differences, the oral tissues in the ferret have an abundant sensory innervation similar to that found in other species.

Coexpression of vasoactive intestinal polypeptide and substance P in reinnervating pulpal nerves and in trigeminal ganglion neurones after axotomy of the inferior alveolar nerve in the rat.

The effect of this axotomy on the expression of vasoactive intestinal polypeptide (VIP) in trigeminal ganglion neurones and nerve fibres in the first molar was examined immunohistochemically 3 weeks postsurgically in rats. A distinct upregulation of VIP-like immunoreactivity was found in 3 to 4% of the neurones (mean dia., 20.9 +/- 0.45 microns; mean cross-sectional area, 367 +/- 13.2 microns 2) in the mandibular region after axotomy. An almost complete coexpression was registered in neurones upregulated for VIP and growth-associated protein 43. Coexpression of VIP and substance P (SP) was found in a small number of the immunoreactive (IR) small-sized neurones, mainly in those in which VIP and SP were both weakly immunoreactive. In the uninjured ganglion, weakly labelled VIP-positive granules were frequently traced at high magnification in the cytoplasm of small neurones. No VIP-IR fibres were found in the control molar pulp, except for a few in the root pulp near the apex. However, 3 weeks after axotomy, a number of VIP-containing nerve fibres were found in the molar pulp and apical periodontium. Coarse VIP-IR fibres directed towards the odontoblast layer were a common finding. In some of these fibres VIP was shown to colocalize with SP. These results indicate that VIP is clearly expressed and transported in regenerating primary afferent neurones after axotomy of the inferior alveolar nerve.

Upregulation of growth associated protein 43 expression and neuronal co-expression with neuropeptide Y following inferior alveolar nerve axotomy in the rat.

Growth associated protein 43 (GAP 43) is an acidic membrane-bound phosphoprotein produced at high levels in developing and regenerating neurons. It is a substrate for protein kinase C and suggested to be involved in calcium-regulated release of axonal vesicular-contained neurotransmitters. Expression of GAP 43 has been demonstrated in the uninjured cat dental pulp which receives its sensory nerve supply from the trigeminal ganglion. The aim of this study was a detailed mapping of the spatial and time-dependent expression of GAP 43 and co-expression of neuropeptide Y (NPY) in dental peripheral target tissues and trigeminal neurons subsequent to inferior alveolar nerve (IAN) axotomy in rats, as background for later low-level laser studies. Unilateral sectioning of IAN, resulting in an almost complete loss of sensory nerve fibers in the ipsilateral dental pulp of the first molar, was performed. The avidin biotin complex (ABC) method was used to evaluate peripheral changes in GAP 43 expression at 4, 7 and 10 days. Ganglionic changes in GAP 43 and co-localization of neuronal NPY expression was examined at 4, 10 and 21 days using either the ABC method or double immunofluorescence labelling techniques and confocal microscopy. Axotomy resulted in an early upregulation and change in the peripheral distribution of GAP 43 in nerve profiles already 4 days post IAN axotomy suggesting a Schwann cell origin. Ten days post axotomy a pronounced upregulation of GAP 43 immunoreactivity could be demonstrated in neurons located in the mandibular region of the trigeminal ganglion, compared to the contralateral uninjured side. The peripheral and ganglionic upregulation of GAP 43 continued to persist at 21 days. A concomitant time-delayed shift and co-expression of NPY was demonstrated throughout in the GAP 43-upregulated ganglion cells 10 days post axotomy. Furthermore, confocal microscopy indicated that the intraneuronal distribution of NPY and upregulated GAP 43 expression showed a similar conformity and distribution in both perinuclear regions and cell periphery.