Expression of TrkA receptor for neurotrophins in trigeminal neurons innervating the rat gingivomucosal tissue

Neuroscience Letters 418 (2007) 253–256

Expression of TrkA receptor for neurotrophins in trigeminal neurons innervating the rat gingivomucosal tissue Rok Gaˇsperˇsiˇc a,∗ , Uroˇs Kovaˇciˇc b , Andrej C¨or c , Uroˇs Skaleriˇc a a

Department of Oral Medicine and Periodontology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia b Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia c Institute of Histology and Embryology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia Received 11 January 2007; received in revised form 2 March 2007; accepted 18 March 2007

Abstract The purpose of this study was to characterize and evaluate the expression of TrkA receptor in trigeminal ganglion (TG) neurons that innervate the rat gingivomucosal tissue. A retrograde nerve tracer Fluorogold (FG) was injected into the gingiva (group 1) or applied into the gingival sulcus (group 2) of the first right maxillary molar to identify the neurons in TG that innervate the gingivomucosa. After 10 days TG were dissected and FG fluorescence in neurons was observed under UV light microscope. To draw a comparison, approximately 1000 neurons per ganglion from the entire TG (group 3) and approximately 350 neurons per ganglion from the maxillary region in TG (group 4), were analyzed. Expression of TrkA receptor in TG neurons was investigated by immunohistochemistry. About 70% of neurons in groups 1 and 2 contained TrkA receptor, which was statistically significantly more than in groups 3 (41%) and 4 (38%). FG-labeled TrkA-immunopositive neurons were predominantly small or medium-sized (less than 1200 ␮m2 ). However, the neurons innervating the rat gingivomucosa were on average larger than the neurons in the entire TG or in the maxillary region. In conclusion, the majority of neurons in TG that innervate the rat gingivomucosa are small or medium-sized, contain TrkA receptor and are most probably nociceptive. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Rat; Trigeminal ganglion; Gingiva; TrkA receptor; Calcitonin gene-related peptide (CGRP)

Sensory neurons of the trigeminal ganglion (TG) that innervate the rat gingiva are predominantly small or medium-sized [7]. The majority of these neurons and their nerve fibers in the rat gingiva contain neuropeptides calcitonin gene-related peptide (CGRP) and substance P (SP) [7,10]. In inflammatory conditions such as periodontitis [1] or arthritis [17] the expression of SP and CGRP in these neurons is up-regulated. It has been suggested that the nerve growth factor (NGF) is responsible for increased expression of both neuropeptides during inflammation [14]. Tropomyosin-related kinase A receptor (TrkA) is the highaffinity receptor for NGF [6]. Binding to TrkA receptor, NGF regulates the development and maturation of sensory neurons during embryogenesis and is also involved in inflammationrelated nociception in adulthood [13,15]. TrkA is expressed on neuronal as well as non-neuronal cells including B lym-

∗ Corresponding author. Department of Oral Medicine and Periodontology, Faculty of Medicine, Hrvatski trg 6, 1000 Ljubljana, Slovenia. Tel.: +386 1 3002110; fax: +386 1 5222494. E-mail address: [email protected] (R. Gaˇsperˇsiˇc).

0304-3940/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2007.03.060

phocytes, T lymphocytes, monocytes, fibroblasts, chondrocytes, osteoblasts and endothelial cells [11]. Non-neuronal cells of periodontium (gingival keratinocytes and fibroblasts, periodontal ligament cells and epithelial cell rest of Malassez) also express TrkA [11,20,22]. It is not known, however, whether the trigeminal neurons, projecting to the rat gingiva, contain TrkA. In accordance with the putative role of NGF during periodontal inflammation [14], we hypothesized that small and medium size trigeminal neurons innervating the rat gingiva contained TrkA. Ten female Wistar rats (200–220 g) were used in experiments. All efforts were made to minimize the number of experimental animals and their suffering. The Veterinary Administration of the Republic of Slovenia approved the experiments (no. of approval 323–02–211/2003). All procedures were performed under deep anesthesia with a mixture of dihydrothiazine (Rompum, Bayer AG, Leverkusen, Germany, 8 mg/kg; i.p.) and ketamine hydrochloride (Ketalar, Parke–Davis GmbH, Berlin, Germany, 60 mg/kg; i.p.). The rats were anaesthetized and positioned with the mouth held open for observation under a surgical microscope. In group 1 (n = 5), 1 ␮l of 2% aqueous solution of Fluorogold (FG) was

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injected into the palatinal and buccal gingivomucosal connective tissue around the first right maxillary molar. In group 2 (n = 5), 1 ␮l of 2% FG was carefully applied into the palatinal and buccal gingival sulcus of the first right maxillary molar using a Hamilton microsyringe. The surrounding palatinal and buccal mucosa was covered by Vaseline to avoid the spillage of the tracer into the oral cavity. The immunohistochemical and histomorphometrical procedures have been described elsewhere [7] and therefore will only be briefly mentioned here. Animals were sacrificed 10 days after FG application. TGs were dissected, immersion fixed in 10% formalin and embedded in paraffin. Tissues were sectioned and every fourth section (6 ␮m thickness) was collected for a further analysis of TrkA immunoreactivity. Before deparaffinization, FG fluorescence in TG was photographed under a fluorescence microscope (Olympus, IX81, U-MNU2 UV filter, Olympus Optical Company Co., Tokyo, Japan) and these photos were used later to be compared with images obtained with a light microscope. Subsequently, sections were used for TrkA immunohistochemistry using polyclonal anti-rat TrkA antibody (Chemicon, United Kingdom) diluted 1:75. Biotinylated secondary antibody for streptavidin–biotin complex (ABC kit, Dako, Denmark), followed by 3,3diaminobenzidin (DAB, Sigma Chemical Co., Germany) in 0.1% H2 O2 PBS solution were used for antigen visualization. After immunohistochemical procedures, the regions containing FG-labeled neurons, which have been previously identified and photographed under the fluorescence microscope, were observed and photographed under a light microscope (Zeiss-Opton, Opton Feintechnik GmbH, Germany). The photomicrographs were scanned into a computer-based image analysis system with the Microcomputer Imaging Device program (MCID, Imaging Research Inc., Ontario, Canada). Approximately 40–50 sections from each TG were evaluated to estimate the total number of neurons containing FG. To avoid double counting, only cells with visible nuclei were counted. The number of FG-labeled neurons with TrkA immunoreactivity was evaluated in both experimental groups (group 1: injection of FG

into the gingiva, group 2: application of FG into the gingival sulcus). In addition, the cross-sectional areas (CSAs) of FGlabeled neuron cell bodies were measured. To draw a comparison, approximately 1000 neurons per ganglion, representing the entire TG neuronal population (group 3, n = 5), and approximately 350 neurons per ganglion in the maxillary region of TG (group 4, n = 5) were evaluated under the light microscope. These neurons were analyzed in one randomly selected visual field (500 ␮m × 750 ␮m, at 10-fold objective magnification) of every second tissue section. Five TGs for each group (3 and 4) were randomly selected from groups 1 and 2. The data from five TG were pooled for each group separately. The frequencies of immunopositive neurons and small-medium sized (CSA < 1200 ␮m2 ) neurons were calculated for each group. The differences in frequencies of neurons between groups were tested using chi-square test. Overall differences in frequencies between four groups were tested first, followed by pairwise multiple comparison between different pairs of groups. Bonferroni’s correction was used to adjust the p values after multiple comparisons. For each group, mean cross sectional areas (CSAs) and S.D. were calculated separately for immunopositive and immunonegative neurons. The data from the CSAs analysis were first analyzed by means of two-way analysis of variance (2WANOVA). The immunireactivity and four groups were set as independent variables (factors). Further analysis of differences in mean CSAs was carried out by the post-hoc Tukey test. p-value of less than 0.05 was considered to be significant. TrkA immunoreactivity (Fig. 1) was more frequently observed in FG-labeled trigeminal neurons (groups 1 and 2) than in the entire TG (group 3) or in the maxillary region of TG (group 4). The frequencies of TrkA-positive neurons projecting to gingivomucosa (group 1: 67% and group 2: 72%) were statistically significantly higher than the frequencies of TrkA-positive neurons in the entire TG (group 3: 41%) and in the maxillary region (group 4: 38%) (p < 0.001, Table 1). However, there were no statistically significant differences between the groups 1 and 2 and between the groups 3 and 4 regarding the frequencies of TrkA-positive neurons (Table 1).

Fig. 1. Fluorogold-labeled neurons in the right trigeminal ganglion 10 days after tracer application into the gingival sulcus. (a) Fluorescence microscope, objective magnification 40×. Fluorogold-labeled neurons are indicated by the white arrows. (b) Light microscope, objective magnification 40×. The same cells showing TrkA-immunoreactivity. The cells indicated by the black arrows are immunopositive, and the ones indicated by the open arrows are immunonegative.

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Table 1 Total number of neurons, cross-sectional areas (CSAs) and number of neurons smaller than 1200 ␮m2 in the right trigeminal ganglion after immunohistochemical reaction to TrkA CSAs (mean ± S.D.) ␮m2

No. of neuronsa

Group/immunoreactivity

No. of neurons <1200 ␮m2 (%)b

1 (FG injection)

TrkA-positive TrkA-negative

192 93

*

910 ± 380*,+ 1229 ± 464#

142 (74%)* 47 (51%)

2 (FG sulcus)

TrkA-positive TrkA-negative

239 93

*

930 ± 416*,+ 1431 ± 368#

143 (60%)* 22 (24%)

3 (entire TG)

TrkA-positive TrkA-negative

1728 2473

688 ± 334* 987 ± 425

1537 (89%) 1731 (70%)

4 (maxillary region)

TrkA-positive TrkA-negative

737 1184

690 ± 342* 979 ± 438

670 (91%) 864 (73%)

Statistically significant differences in: no. of neurons (frequency): * compared to groups 3 and 4, p < 0.001. CSAs: * compared to immunonegative neurons in each group, p < 0.001; + compared to immunopositive neurons in groups 3 and 4, p < 0.001; # compared to immunonegative neurons in other three groups, p < 0.001. No. of neurons < 1200 ␮m2 (frequency): * compared to immunopositive neurons in other three groups, p < 0.05. a Total number of neurons was pooled from five TG in each subgroup. b Statistical analysis was performed only for immunopositive neurons.

Two-way ANOVA revealed statistically significant main effects of immunoreactivity (F = 337, p < 0.001) and groups (F = 84, p < 0.001) on CSAs. The same analysis also showed a statistically significant (F = 4.4, p = 0.004) interaction effect of both independent variables. Post hoc tests revealed that CSAs of TrkA-positive neurons were statistically significantly (p < 0.001) smaller than CSAs of TrkA-negative neurons in each group. CSAs of TrkA-positive neurons and of TrkA-negative neurons, respectively, were statistically significantly (p < 0.001) larger in groups 1 and 2 than in groups 3 and 4. However, the differences in the CSAs of TrkA-positive neurons between groups 1 and 2 and between groups 3 and 4 were not statistically significant. CSAs of TrkA-negative neurons in group 2 were statistically significantly (p < 0.001) larger than in group 1. There was no significant difference in CSAs of TrkA-negative neurons between groups 3 and 4. More than 60% of TrkA-positive neurons in each group were smaller than 1200 ␮m2 (Table 1). Among TrkA-positive neuronal cell bodies, the frequencies of small and medium-sized neurons (CSA < 1200 ␮m2 ) in group 1 (74%) and group 2 (60%) were statistically significantly smaller (p < 0.05) than in group 3 (89%) and in group 4 (91%). There was no statistically significant difference between group 3 (the entire TG) and group 4 (the maxillary region) in this regard (Table 1). Our results showed that approximately 40% of the neurons in TG of adult rat contain TrkA receptor. Similar percentages of TrkA-positive neurons in TG and in the dorsal root ganglion (DRG) were found in adult mice [4] and rats [3,16], respectively. The rat TG neurons are of various sizes: small (<500 ␮m2 ), medium (500–1200 ␮m2 ) and large (1200 and up to 4000 ␮m2 ) [9,12]. In the present study, approximately 90% TrkA-positive neurons in TG were small and medium-sized. Accordingly, the diameter of TrkA expressing DRG neurons similarly corresponds well to a range typical for small and medium-sized nociceptive neurons [3,5,8]. We demonstrated that approximately 70% of TG neurons innervating the rat gigivomucosa (FG-labeled neurons in groups 1 and 2) con-

tained TrkA, which was significantly more than the frequencies of TrkA-positive neurons in the entire TG and in the maxillary region. This data correspond well with the CGRP and SP immunoreactivity that is similarly more frequently observed in these neurons than in the entire TG [7]. Neurons innervating gingival connective tissue (group 1) were smaller compared to neurons that innervate sulcular epithelium (group 2), which corresponds to our previous observations [7] and confirms that the gingivomucosal tissue of the rat is innervated by two different sensory neuron populations, identified by the two different retrograde labeling procedures used in our study (see also [7]). Furthermore, the mean CSAs of the sensory neurons innervating the rat gingivomucosa were larger than the sensory neurons representing the entire TG and the maxillary region in TG. Nevertheless, our results suggest that about 70% of the neurons innervating the rat gingivomucosa are nociceptive neurons, because it has recently been shown that TrkA is selectively expressed in nococeptive DRG neurons regardless of their sizes [5]. TrkA is a high-affinity receptor for NGF that was first studied for its essential role in neuronal growth and survival [15]. In addition, several reports also indicate that NGF, whose level is elevated in the inflamed tissue, modulates the experimentally induced inflammatory processes in the rat skin [19,21]. It has been suggested that the alteration in neuropeptides expression in sensory neurons may contribute to neurogenic inflammation in periodontium [14]. Such changes could be a consequence of increased production of NGF during periodontal inflammation, because the synthesis of SP and CGRP in the TG is increased after experimentally induced periodontal inflammation [1] and after treatment of TG neurons with NGF in vitro [18] and in vivo [2]. Notably, systemic administration of anti-NGF neutralizing antibodies prevented the up-regulation of neuropeptides in the primary sensory neurons innervating the inflamed skin [21]. Further studies are required to examine the proposed mechanisms using experimental periodontitis model in rats.

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Acknowledgements The authors thank Prof. Dr. J. Sketelj for helpful advices during the preparation of this manuscript. We thank Mrs. N. Godniˇc for expert technical assistance with immunohistochemical proˇ cedures, Mr. T. Zele for preparation of the figures and Mrs. M. Perpar for reviewing the grammatical issues of the manuscript.

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