Acta Histochem. Cytochem. 38 (3): 229–235, 2005 Molecular Changes in Neurons of Rat Nucleus Ambiguus after Axotomy, As Revealed by a Novel Method of In Vivo Fluorescence Neuronal Labeling Combined with Single-cell RT-PCR Ryuichi Hirota1,2, Kyoko Itoh1, Takeshi Yaoi1, Hitoshi Bamba2, Toshiyuki Uno2, Yasuo Hisa2 and Shinji Fushiki1 1Department of Pathology and Applied Neurobiology and 2Otolaryngology-Head and Neck Surgery, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kawaramachi Hirokoji, Kamigyo-ku, Kyoto 602–8566, Japan Received February 24, 2005; accepted March 7, 2005 To elucidate the molecular changes of were mounted onto membrane slide, and motoneurons which innervate laryngeal DiI-labeled single neurons in NA were muscles after transection of rat recurrent microdissected under a fluorescence laryngeal nerve, we have devised a new microscope. By real-time quantitative RT- method of DiI labeling combined with PCR for single motoneurons, the expres- single-cell RT-PCR, by which nucleus sion levels of GAP-43 and nNOS genes ambiguus (NA) neurons projecting to the were upregulated at 7 days and 1 day intrinsic laryngeal muscles were labeled after axotomy, respectively. The expres- retrogradely by injecting the fluorescent sion levels of Stat3, Reg-2, and Bcl-2 dye DiI into bilateral thyroarytenoid mus- genes were upregulated at 7 days, cles; then either one of the recurrent whereas that of Bax was downregulated laryngeal nerves was transected, and the at 1 and 7 days after axotomy as com- fluorescence-bright single neurons were pared with control levels. With this novel microdissected, followed by single-cell method in which single-cell RT-PCR was reverse transcription (RT)-PCR. Immuno- combined with neurotracer DiI labeling histochemically, many DiI-labeled moto- we could demonstrate a part of the neurons in NA were immunoreactive to molecular cascades involving Stat3 in phosphorylated Stat3 in the nucleus on neurons projecting to laryngeal muscles 1, 7, 10 and 14 days after axotomy. Thirty after axotomy. m-thick frozen sections of brain stem Key words: recurrent laryngeal nerve, nucleus ambiguus, axotomy, single-cell RT-PCR, Stat3 I. Introduction muscles, although it is suggested that immobilization of vocal cord reflects failure in correct innervation [9]. Traumatic injuries to recurrent laryngeal nerve during Molecular mechanisms by which peripheral nerves surgical operation often produce irreversible vocal cord im- regenerate have so far been studied using facial nerve, mobilization, even though end-to-end anastomoses between hypoglossal nerve, and sciatic nerve [12, 14, 17]. On the both transected ends of the nerve are successfully performed contrary, there are few studies on regeneration of vagus [5]. It remains to be clarified, especially from the molecular nerve, particularly the recurrent laryngeal nerve [10]. One viewpoint, whether such vocal cord dysfunction is primarily of the reasons would be that the localization, size and due to impairment of muscles or of neurons that innervate shape of nucleus ambiguus (NA) is not as distinct as those of facial nucleus and hypoglossal nucleus [3]. In addition, Correspondence to: Shinji Fushiki, M.D., Ph.D., Department of neurons in NA project not only to laryngeal muscles but also Pathology and Applied Neurobiology, Graduate School of Medical to muscles localized in pharynx, esophagus, and so on [3]. Science, Kyoto Prefectural University of Medicine, Kawaramachi Conventional histology per se does not help in discrimi- Hirokoji, Kamigyo-ku, Kyoto 602–8566, Japan. nating neurons that project to laryngeal muscles from E-mail: [email protected] other types of neurons located in NA. Therefore, refined 229 230 Hirota et al. techniques are needed to elucidate post-traumatic molecular (KPUM) and approved by the KPUM Animal Care and Use changes of NA neurons which innervate laryngeal muscles. Committee. Here we have attempted to devise a method by which NA motoneurons projecting to laryngeal muscles can be 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine identified by retrograde labeling with a fluorescent dye, perchlorate (DiI) labeling and axotomy DiI, so that fluorescence-bright cells are able to be micro- The animals were deeply anesthetized by intraperito- dissected as a single labeled neuron under fluorescence neal injection of sodium pentobarbital (45 mg/kg) and a 2 l microscope. This novel sampling method has enabled us to of DiI solution diluted in ethanol (1 mg/ml) was injected collect neurons of interest, followed by a single-cell RT- into bilateral thyroarytenoid muscle by Hamilton syringe PCR analysis, whereby molecular changes appearing in NA (Fig. 1). Two weeks after DiI injection, the right recurrent neurons after transection of recurrent laryngeal nerve can be laryngeal nerve was transected under deep anesthesia. The analyzed in details with a high specificity and precision. We animals were allowed to live for 1, 7, 10 or 14 days after have evaluated expression of Stat3 mRNA in NA neurons axotomy (five animals were allotted to each group). with a single-cell RT-PCR together with immunohisto- chemistry and have demonstrated that time-dependent Immunohistochemistry post-transcriptional modifications, viz., phosphorylation and Under deep anesthesia with sodium pentobarbital, translocation of transcription factor Stat3, ensue in NA animals were perfused transcardially with ice-cold 4% motoneurons after severing recurrent laryngeal nerve. In ad- formaldehyde in 0.1 M phosphate buffer (pH 7.4), and dition, expression patterns of apoptosis-associated genes and dissected brain stems were post-fixed in the same fixa- a gene that is purportedly Stat3-dependent were evaluated tive overnight prior to cryoprotection with 20% sucrose in after axotomy with a single-cell RT-PCR. phosphate buffered saline (PBS). Thereafter, 10 m-thick horizontal sections were prepared using a freezing micro- II. Materials and Methods tome throughout the entire rostrocaudal extent of the NA in the medulla oblongata. Animals All the sections were observed and photographed under Twenty-five seven-week-old male rats (Sprague- an epifluorescence microscope (Olympus BX51, Japan). Dawley) were purchased from a local breeder (Shimizu DiI-labeled neurons in NA were counted and every sixth Laboratory Animals, Kyoto, Japan) and maintained in a section was used for immunohistochemistry. temperature- and humidity-controlled animal facility with a The sections were incubated with blocking buffer (10% 12:12-hr light:dark cycle. Experimental animals used in the goat serum, 1% BSA, 0.3% Triton-X in PBS) at room tem- present study were handled according to the “Guidelines perature for 1 hr, and then reacted with the primary antibody for the Care and Use of Laboratory Animals in Biomedical appropriately diluted at 4LC overnight, followed by incuba- Research” of Kyoto Prefectural University of Medicine tion with the secondary antibody at room temperature for Fig. 1. Diagram showing method for DiI labeling of NA motoneurons projecting to bilateral thyroarytenoid muscles and transection of the right recurrent laryngeal nerve. Two l of DiI solution was injected into bilateral thyroarytenoid muscles for retrograde labeling of neurons. Two weeks after the injection, the right recurrent laryngeal nerve was transected. Molecular Changes in Neurons after Axotomy 231 2 hr. Primary antibodies included a rabbit polyclonal anti- cDNA synthesis and quantitative real-time RT-PCR phospho-Stat3 (pStat3) IgG (1:100) (Cell Signaling, USA) Total cDNAs from each single cells were synthesized and a rabbit polyclonal anti-Stat3 IgG (1:1,000) (Santa Cruz and amplified as described previously in Oda et al. [16]. Biotechnology, USA). Alexa 488-cojugated goat anti-rabbit Briefly, first strand cDNAs were synthesized with SMART IgG (Molecular Probes, 1:250 diluted in blocking solution) oligo II primer and 3'-anchored primer using Superscript II was used as a secondary antibody. After incubation with RNase H reverse transcriptase (Invitrogen, USA) or Power- secondary antibodies, the sections were placed in Slow-Fade script (Clontech, USA). Second strand cDNA synthesis equilibration buffer at room temperature for 10 min, and and amplification of total cDNAs were carried out using then mounted with Slow-Fade/glycerol. They were observed Advantage 2 PCR polymerase mix (Clontech, USA). The under epifluorescence microscope. optimized number of thermal cycling was set at 18 for the The numbers of pStat3- and Stat3-immunoreactive following real-time quantitative RT-PCR. neurons that were positive for DiI-labeling were counted The relative expression levels of genes interest, name- throughout the entire area of NA. ly, GAP-43, nNOS, Stat3, Reg-2, Bcl-2, Bax and glycer- aldehyde-3-phosphate dehydrogenase (GAPDH), were Single-cell microdissection for DiI-labeled neurons measured by real-time quantitative RT-PCR using TaqMan Individual neurons of NA were isolated as follows. Universal PCR Master Mix (Applied Biosystems, USA) on DiI-labeling of NA neurons and nerve injury were done as an ABI PRISM 7000 Sequence Detection System (Applied described above. One and seven days after nerve injury, Biosystems). TaqMan gene expression assays (Applied the animals were sacrificed by intraperitoneal injection Biosystems) were used as a set of primers and TaqMan with excess doses of pentobarbital. The brainstems were probe for amplification of each gene of interest except for immediately removed, followed by rapid freezing