Comparative gender differences in local and systemic concentrations of pro-inflammatory cytokines in rats with experimental periodontitis.

BACKGROUND AND OBJECTIVE: There have been few studies of gender differences in response to periodontitis. Thus, we compared gender-specific differences in systemic cytokine concentrations in rats with and without ligature-induced periodontitis. MATERIAL AND METHODS: Experimental periodontal disease was initiated in Sprague-Dawley rats by placing a ligature around the crowns of the second right maxillary molar tooth. Sham-operated control groups were also created. Two weeks later, the right and left maxillary quadrants of teeth, liver and serum were collected from all the rats, and uterine horns were collected from the female rats. Liver and uterine samples were ground in phosphate-buffered saline (10 mg of tissue/mL of phosphate-buffered saline + protease inhibitor) containing a protease inhibitor, and cytokine concentrations were determined by enzyme-linked immunosorbent assay. Digital radiographs were made of maxillary quadrants, and the distance from cemento-enamel junction to alveolar crest was measured using image analysis software. Data were compared by factorial analysis of variance and a post-hoc Tukey test. RESULTS: Female rats with ligatures had greater, but not significantly different, alveolar bone loss than males with ligatures. However, they had higher serum concentrations of interleukin-6, tumor necrosis factor-alpha and C-reactive protein, and liver C-reactive protein (p < 0.05). These females also had higher interleukin-6, tumor necrosis factor-alpha and vascular endothelial growth factor concentrations within the uterine horn, compared to female controls (p < 0.05). Male animals with ligatures had lower serum concentrations of C-reactive protein and higher interleukin-6 and tumor necrosis factor-alpha concentrations within serum, compared to male controls (p < 0.05). CONCLUSION: Our study suggests that females with periodontal disease have a greater risk for inflammatory-based systemic diseases than males.

Effects of induced periapical abscesses on rat pregnancy outcomes.

OBJECTIVE: The objective of this study was to determine the effects of induced periapical abscesses on pregnant rats. DESIGN: In 1/2 of the animals (n=16), the pulps of the maxillary right molars were exposed to the oral environment, which resulted in a periapical abscess. The other 1/2 (n=16) were sham-operated. 1/2 of the animals of both groups became pregnant 2 weeks later. The pregnancy duration, and weight and number of pups were assessed at delivery. Serum, liver and uterine horn samples were taken from all animals at euthanasia and serum IL-6, endothelin-1, TNF-alpha, IL-10, cortisol and insulin were determined by ELISA. Liver concentrations of IL-6, CRP and IL-6 and uterine horn concentrations of IL-6, vascular endothelial growth factor (VEGF), TNF-alpha, IL-10 and IL-1-beta were assessed by ELISA. Blood glucose concentrations were determined using a glucometer. Outcome variables were compared by factorial ANOVA, a post hoc Tukey test, and Pearson's correlation test. RESULTS: Pregnant rats with periapical abscesses had a significantly longer pregnancy and delivered pups with a significantly higher birthweight (p<0.05). They had significantly higher concentrations of IL-6, VEGF, IL-1-beta, and IL-10 within the uterine horn and IL-6, CRP and TNF-alpha within the liver (p<0.01). Blood glucose and serum TNF-alpha, IL-6, endothelin-1, IL-10, and insulin concentrations were significantly higher in the pregnant animals with pulpal abscesses (p<0.01). CONCLUSION: The significant increase in serum TNF-alpha, taken together with significant increases in blood glucose and serum insulin concentrations, suggest that animals with induced periapical abscesses developed insulin resistance, which significantly affected their pregnancy outcomes.

Heterogeneity of channel catfish CTL with respect to target recognition and cytotoxic mechanisms employed.

Two types of catfish alloantigen-dependent cytotoxic T cells were cloned from PBL from a fish immunized in vivo and stimulated in vitro with the allogeneic B cell line 3B11. Because these are the first clonal cytotoxic T cell lines derived from an ectothermic vertebrate, studies were undertaken to characterize their recognition and cytotoxic mechanisms. The first type of CTL (group I) shows strict alloantigen specificity, i.e., they specifically kill and proliferate only in response to 3B11 cells. The second type (group II) shows broad allogeneic specificity, i.e., they kill and proliferate in response to several different allogeneic cells in addition to 3B11. "Cold" target-inhibition studies suggest that group II CTL recognize their targets via a single receptor, because the killing of one allotarget can be inhibited by a different allotarget. Both types of catfish CTL form conjugates with and kill targets by apoptosis. Killing by Ag-specific cytotoxic T cells (group I) was completely inhibited by treatment with EGTA or concanamycin A, and this killing is sensitive to PMSF inhibition, suggesting that killing was mediated exclusively by the secretory perforin/granzyme mechanism. In contrast, killing by the broadly specific T cytotoxic cells (group II) was only partially inhibited by either EGTA or concanamycin A, suggesting that these cells use a cytotoxic mechanism in addition to that involving perforin/granzyme. Consistent with the presumed use of a secretory pathway, both groups of CTL possess putative lytic granules. These results suggest that catfish CTL show heterogeneity with respect to target recognition and cytotoxic mechanisms.

Effects of deprivation of neonatal nerve growth factor on the expression of neurotrophin receptors and brain-derived neurotrophic factor by dental pulp afferents of the adult rat.

The dental pulp is richly innervated by peptidergic nociceptive neurones that are of special interest because of their central role in dental pain and because they have some features that are not typical of other somatic nociceptors. Here, (35)S-riboprobes were used to determine whether pulpal afferents of adult (2-month-old) rats express the nerve growth-factor (NGF) receptors, p75(NTR) and trkA, which are characteristic of peptidergic nociceptors, and additionally, whether these cells express receptors (trkB and trkC) for other members of the neurotrophin family. In order to begin characterizing the postnatal role of NGF in regulating these neurones, the susceptibility of pulpal afferents to antiserum-mediated early postnatal NGF depletion spanning the period of pulpal innervation development was also examined. In control animals, about 200 trigeminal ganglion cells were labelled after application of the retrograde tracer Fluoro-gold to the first maxillary molar. Among the labelled cells, 79% had positive hybridization signals for p75(NTR), 72% for trkA, 34% for trkB, 1% for trkC, and 77% for BDNF. Neonatal NGF depletion reduced the number of retrogradely labelled pulpal afferents by 33%, with numbers of smaller neurones being most strikingly subnormal. This reduction could be attributed to a partial depletion of the neurone population that expressed p75(NTR) and trkA. Consistent with reports that NGF-responsive neurones also express BDNF, NGF deprivation resulted in a reduction in the number of pulpal afferents that expressed BDNF to an extent similar to that seen for trkA. In contrast, anti-NGF exposure had little effect on the number of pulpal afferents that expressed trkB. These findings indicate that most pulpal afferents in the adult express the NGF receptors p75(NTR) and trkA, and thus have a continuing potential susceptibility to NGF-mediated regulation of functions such as neuropeptide and BDNF synthesis. However, only a subpopulation of this group of neurones requires NGF in order to develop connections to the pulp during the neonatal period. Few, if any, pulpal afferents express the high-affinity neurotrophin-3 (NT3) receptor trkC, although many have large cell bodies typical of NT3-responsive sensory neurones. A small subpopulation of pulpal afferents seems to express no neurotrophin receptors, yet it is unlikely that these cells belong to the class of small sensory cells known to bind isolectin IB4.

Course and composition of the nerves that supply the mandibular teeth of the rat.

The rodent dentition has become an important model for investigations of interactions between dental tissues and peripheral neurons. Although experimental nerve injury has been widely used for such studies, there is uncertainty about the courses of nerve fibers supplying the mandibular teeth. In order to clarify this, we used a mixture of monoclonal antibodies against neurofilament proteins to enhance demonstration of nerve fibers so that small nerves could be readily traced in serial frozen sections of mandibles of Sprague Dawley rats ranging in age from embryonic day (E) 18 to postnatal day (P) 90. The 1st molar and anterior portion of the 2nd molar were innervated by small nerves that emerged as distinct branches of the IAN trunk at or near the mandibular foramen. In contrast, the nerve supply to the 3rd molar and posterior part of the 2nd molar was a branch of the lingual nerve that bypassed the mandibular canal altogether. The IAN trunk split into the mental nerve and a large branch to the incisor about 2 mm anterior to the mandibular foramen. Thick branches of the incisor nerve descended into the incisor socket to form a dense plexus of nerve fiber bundles extending along the length of the incisor periodontium. The sparse pulpal innervation of the incisor was provided by a few thin fascicles that emerged from the caudal portion of the periodontal plexus to enter the incisor apex. The dental branches of the IAN and lingual nerve seen in the adult were well established and readily identifiable at age E18 even though their targets were limited to the follicles of the developing teeth. These studies show that the trigeminal branches that supply the mandibular teeth can be identified at a wide range of ages as distinct nerves at a considerable distance proximal to their targets. This detailed information on the courses taken by the dental nerves can provide an anatomical basis for increased precision in characterization and perturbation of neural pathways from the molars and incisor.

Induction of target cell apoptosis by channel catfish cytotoxic cells.

This study examines cytotoxic mechanisms used by channel catfish peripheral blood-derived effector cells. Transmission electron microscopy (TEM), coupled with [(3)H]thymidine DNA fragmentation (JAM) and terminal deoxynucleotidyl nick-end labeling (TUNEL) assays, provided the first evidence that catfish peripheral blood cytotoxic effectors killed allogeneic targets via an apoptotic pathway. TEM demonstrated that the effector cell population present within peripheral blood leukocytes (PBLs) was composed of agranular lymphocytes that formed conjugates with, and induced apoptosis in, allogeneic target cells. Both JAM and TUNEL assays showed that PBLs induced target cell DNA fragmentation within 1 h of coculture. In addition, fixed effectors did not induce target cell necrosis or apoptosis, and target cell lysis was completely inhibited by chelation of free Ca(2+) by EGTA. These results suggest that catfish peripheral blood-derived effector cells utilize a secretory mechanism rather than a ligand-based mechanism to trigger apoptosis.

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.

Effects of neonatal exposure to anti-nerve growth factor on the number and size distribution of trigeminal neurones projecting to the molar dental pulp in rats.

The first aim of the present study was to determine whether depletion of endogenous nerve growth factor (NGF) during early postnatal development results in a long-term deficit in the number of trigeminal ganglion cells and axons projecting to the molar pulp. The second aim was to identify selectivity of the effects of NGF deprivation for any specific size group among pulp neurones. Newborn Sprague-Dawley rats were given subcutaneous injections of either rabbit anti-mouse-NGF serum or non-immune (control) rabbit serum for a period of 1 month. At age 4 months, Fluoro-gold (FG) was applied to the pulp chamber of the right maxillary first molar. One week later the animals were perfusion-fixed, and the trigeminal ganglia were removed and serially sectioned with a cryostat. Labelled neurones were seen only in the trigeminal ganglia ipsilateral to the injected teeth. The area of every labelled cell profile was measured, and from these data, estimates of the true number and size distribution of FG-labelled cells were obtained by recursive translation. Ganglia of control animals had a mean of 197 labelled neurones, all in the maxillary division, and most of the somas were of medium or large diameter. NGF-deprived animals had significantly fewer (mean = 145) FG-labelled cells in the trigeminal ganglion ipsilateral to the injected tooth. Neurones with somas of less than 30 microns dia were most strikingly subnormal in anti-NGF treated animals (64% of controls). In accordance with the greater susceptibility of small neurones to anti-NGF exposure, deficits in apical nerve fibres of the mandibular first molar were greater in degree and duration for unmyelinated axons than for myelinated axons. It is concluded that NGF is an important mediator in regulation of postnatal development of the sensory innervation of the dental pulp. The results also indicate that postnatal development of at least one class of larger pulpal afferent neurones is regulated by factors other than NGF.