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 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 , 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 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 , mobilization, even though end-to-end anastomoses between , 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 [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 , , and so on [3]. Science, Kyoto Prefectural University of Medicine, Kawaramachi Conventional 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 . 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 were probe for amplification of each gene of interest except for immediately removed, followed by rapid freezing and kept nNOS. The assay ID for each gene was as follows: at 80LC until use. Thereafter, 30 m-thick horizontal sec- Rn00567901_m1 for GAP-43, Rn00562562_m1 for Stat3, tions were prepared using a freezing microtome throughout Rn00583920_m1 for Reg-2, Rn99999125_m1 for Bcl-2 and the entire rostrocaudal length of NA. The sections were Rn01480158_g1 for Bax. The primers and probe for nNOS mounted onto membrane slides for laser microdissection were designed as follows: 5'-CATCATTCTCGCAGTCA- (Part No. 50103, Molecular Machines & Industries AG, Ger- ACGA-3' (forward), 5'-AGGCAATGCCCCTGAGAAC-3' many) and were dried. Under inverted epifluorescence mi- (reverse), and 5'-CGGCCCTTGGTAGACCTCAGCTAT- croscope, the area of NA was trimmed with micromanipula- GAC-3' (probe). For GAPDH, TaqMan rodent GAPDH tor-driven RNase-free blade and individual neurons labeled control reagent was used (Applied Biosystems). The probe by DiI as shown in Figure 2 were carefully microdissected for each gene of interest was fluorescent-labeled with FAM, from the surrounding neuropil using RNase-free glass and the one for GAPDH with VIC. The cDNA fragments micropipettes, and subsequently transferred into small tubes corresponding to each gene were co-amplified in the same containing cell lysis buffer (Strataprep total RNA microprep PCR reaction tube. PCR reaction was carried out as the kit, Stratagene, USA). manufacturer’s instruction. Multiple comparisons among means for individual gene expressions at distinct post- axotomy days (POD) were performed with Kruskal-Wallis test followed by Scheffé test (StatView, version 5.0).

III. Results Immunohistochemistry The characteristics of histological and immunohisto- chemical analyses for anti-Stat3 and anti-pStat3 are summa- rized in Figure 3 and Table 1. The total number of DiI- labeled NA motoneurons was almost the same at both transected and non-transected sides on all examined PODs (Table 1). Immunohistochemistry showed intense Stat3-immuno- reactivity (IR) localized in cytoplasm or nuclei of NA neurons. The anti-Stat3-antibody that we used reacts with both phosphorylated and non-phosphorylated epitopes of Stat3 peptide. Among DiI-labeled neurons in NA, 80%– Fig. 2. DiI-labeled NA motoneurons identified in 30 m-thick sec- 100% of motoneurons showed strong Stat3-immunoreactiv- tions under inverted epifluorescence microscope. DiI fluorescence ity at transected side on 10 days after axotomy (Fig. 3B, D), present in neurons of NA was clearly observed and single moto- whereas 0–10% of those showed Stat3-IR at non-transected neuron was dissected out using a micromanipulator-driven blade. side (Fig. 3A, C). The number of NA neurons immunoreac- Bar50 m. tive for Stat3 was significantly increased at transected side 232 Hirota et al.

Fig. 3. Immunofluorescence for Stat3 of DiI-labeled neurons in NA at non-transected (A, C) and transected sides (B, D) on 10 days after axotomy (10POD). Immunofluorescence showed intense Stat3-IR localized in cytoplasm (arrow) and/or nuclei (arrowhead) of NA neurons. Among DiI-labeled neurons in NA, most neurons showed intense Stat3-IR at transected side (D), whereas a few neurons did at non-transected side (C). Immunofluorescence for pStat3 of DiI-labeled neurons in NA at non-transected (E, G) and transected sides (F, H) on 10POD. Immunofluorescence showed intense pStat3-IR localized in nuclei (arrowhead) of NA neurons. Among DiI-labeled neurons in NA, most neurons showed intense pStat3-IR in the nucleus at transected side (H), whereas very few neurons did at non-transected side (G). Bar50 m. Molecular Changes in Neurons after Axotomy 233

Table 1. The ratios of Stat3- and pStat3-immunoreactive neurons divided by total DiI-labeled neurons in NA Stat3 pStat3 meanMSD transected side (n) non-transected side (n) transected side (n) non-transected side (n) 1POD 76.9M4.7%* (86) 5.3M6.1% (95) 82.0M6.6%* (85) 4.7M9.5% (88) 7POD 81.5M14.2%* (87) 9.0M7.0% (83) 87.7M14.7%* (82) 7.1M6.5% (86) 10POD 82.4M11.4%* (81) 6.2M6.4% (82) 91.8M6.0%* (84) 3.2M4.1% (81) 14POD 82.4M15.2%* (77) 11.1M7.4% (75) 81.8M16.0%* (87) 8.2M12.9% (91) The ratios of both Stat3 and pStat3-IR neurons were significantly increased in transected side as compared with those in non-transected side (p0.0001) at every evaluated POD. n: total DiI-labeled neurons in NA. as compared with that at non-transected side on 1, 7, 10 and upregulated at 1POD and returned to control level at 7POD 14 PODs (p0.0001 vs. control side, Table 1). Anti-pStat3 (Fig. 4A, B). The relative expression level of Stat3 mRNA antibody recognized Stat3 only when phosphorylated at was downregulated at 1POD followed by a significant tyrosine 705, and demonstrated that the immunoreactivity upregulation at 7POD (Fig. 5A). The expression of Reg-2 was confined to the nucleus. Among DiI-labeled neurons in mRNA was not detected in control (non-transected side); NA, 80%–100% of neurons showed intense pStat3-IR in however, it was discernible at 1POD and was significantly the nucleus at transected side (Fig. 3F, H), whereas 0–10% upregulated at 7 POD (Fig. 5B). The expression level of Bax of neurons showed pStat3-IR at control side at 10 days mRNA was significantly decreased at 1POD and increased after axotomy (Fig. 3G, H). No pStat3-IR was observed at 7POD, which remained at lower level than in control (Fig. in neuronal cytoplasm on either side or at any time point 5C). The Bcl-2 mRNA was not detected in control and at examined. The number of NA neurons showing pStat3-IR 1POD, whereas it was highly expressed at 7POD (Fig. 5D). was significantly increased at transected side as compared with that at non-transected side on 1, 7, 10 and 14 PODs (p0.0001 vs. control side, Table 1).

Single-cell microdissection for DiI-labeled neurons DiI-labeled NA motoneurons could be identified as flu- orescent bright cells in the 30 m-thick sections that had been put onto the membrane slide under inverted epifluores- cence microscope. Observing the sections under both phase contrast and excitation with epifluorescence apparatus, single motoneurons were trimmed and dissected out with micromanipulator-driven RNase-free blade and transferred into small tubes with glass microcapillaries (Fig. 2).

Real-time quantitative RT-PCR The relative expression level of GAP-43 mRNA was Fig. 4. The relative expression level of GAP-43 (A) and nNOS gene significantly upregulated at 7POD and that of nNOS was (B) by real-time quantitative RT-PCR. *p0.05, **p0.01.

Fig. 5. The relative expression level of Stat3 (A), Reg-2 (B), Bax (C) and Bcl-2 (D) genes by real-time quantitative RT-PCR. The expression levels of Stat3, Reg-2, and Bcl-2 genes were significantly upregulated at 7POD, whereas that of Bax was significantly downregulated at 1 and 7PODs as compared with control levels. *p0.05, **p0.01. 234 Hirota et al.

IV. Discussion and motoneuron survival factor [15], acts on motoneurons in an autocrine and paracrine mode to enhance survival [15]. We have demonstrated that, after axotomy of recurrent Noteworthy is the fact that the Reg-2 promoter contains laryngeal nerve in rat, the number of Stat3-immunoreactive three Stat3 binding sites and is upregulated in response to neurons in the NA was increased, accompanied with phos- Stat3 activation [8]. In our present study, Stat3 phosphoryla- phorylation and nuclear translocation of Stat3. In addition, tion in NA motoneurons started at 1 day after axotomy and we have devised a method to study gene expression of a soon reached a plateau that it maintained for the following 2 neuron of interest whereby retrograde labeling by DiI weeks, and that Reg-2 mRNA was induced significantly in enabled us to identify a specific neuron, followed by single NA motoneurons on the lesioned side at 1 POD and further cell microdissection and single-cell RT-PCR. With this com- upregulated at 7POD, demonstrating that Reg-2 expression bination of methods to focus on a single neuron that inner- is induced by axotomy in NA motoneurons as shown previ- vates the region of interest, we have successfully analyzed ously for facial [19] as well as spinal motoneurons [13]. changes of gene expression after axotomy. Genes that we Therefore, our data on Reg-2 correspond to the report by have studied were GAP-43, nNOS, Stat3, Reg-2, Bax and Schweizer et al. [19] in that Stat3 is a likely regulator of Bcl-2. Our method herein described differs from previously Reg-2 expression in vivo. reported methods for molecular analyses for collected Among the genes that we have studied, Bax showed homogenous or pure cell populations, including a combina- downregulation after axotomy. When considering the fact tion of laser capture microdissection (LCM) and quantita- that apoptosis of facial motoneurons after nerve lesion in tive RT-PCR [21], and immunophenotyping for dissociated neonatal mice is absent in Bax-deficient mice [6], it might be cells followed by LCM and RT-PCR [11]. argued that Bax plays an essential role in lesion-mediated GAP-43 is a protein localized specifically in the growth motoneuronal death. In contrast, an apoptosis-inhibiting fac- cone and plays a role in neurite formation and regeneration tor [7], Bcl-2 showed upregulation at 7POD. It is tempting to [2]. Uno et al. [20] reported that GAP-43 mRNA transiently speculate that upregulation of Bcl-2 could lead to additional increased in the motoneurons in the NA after transection of cellular responses besides preventing apoptosis, since it is recurrent laryngeal nerve in rat by in situ hybridization. known that Bcl-2 overexpression promotes axonal regenera- They showed that signals for GAP-43 mRNA reached a tion of retinal ganglion cells [4]. peak on POD7. In addition, they demonstrated in a similar Our findings that both Bcl-2 and Reg-2 were upregulat- experimental system of recurrent laryngeal nerve transection ed in NA motoneurons after axotomy suggest that they play that neuronal nitric oxide synthase (nNOS) activity was in- an important role in the cellular programs leading to regen- duced in the NA motoneurons on POD7, reaching a peak on eration of motoneurons after lesion, involving JAK-Stat sig- POD14 [1]. Our present observation on the expression of naling pathways. Further studies with a combination of DiI GAP-43 with single-cell RT-PCR is compatible with that in labeling and a single-cell RT-PCR would further contribute previous reports, although that of nNOS seems different in to understanding molecular pathogenesis that underlies terms of its time course. The reason for the difference in recurrent laryngeal nerve transection. results would be due to the difference in methodology; previous reports studied nNOS activity by NADPH diapho- V. Acknowledgments rase histochemistry, whereas we measured nNOS mRNA by single-cell RT-PCR. Supported in part by Grants-in-Aid for Scientific Our observation that Stat3 immunoreactivity increased Research from the Ministry of Education, Culture, Sports, in NA motoneurons after axotomy and its translocation from Science and Technology, Japan. the cytoplasm to the nucleus together with phosphorylation is worth mentioning, since it has been shown that activation of Stat3 by phosphorylation on position Tyr705 and en- VI. References hanced nuclear translocation occurs within 3 hr in rat facial 1. Bamba, H., Uno, T., Koike, S., Shogaki, K. and Hisa, Y. (2000) and hypoglossal neuron following axotomy [18]. Schwaiger Induction of nitric oxide synthase activity in nucleus ambiguus et al. [18] suggested that activation of Stat3, as a transcrip- motoneurons after injury to the rat recurrent laryngeal nerve. Acta tion factor, might be involved in the switch from the physio- Otolaryngol. 120; 327–329. logical gene expression to an alternative program that is 2. Benowitz, L. 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