Brain Research 887 (2000) 53±62 www.elsevier.com/locate/bres Research report Nerve growth factor applied onto the olfactory epithelium alleviates degenerative changes of the olfactory receptor neurons following axotomy Hiroki Yasunoa,b , Keijiro Fukazawa b , Tetsuo Fukuoka a , Eiji Kondo a , Masafumi Sakagami b , Koichi Noguchia,* aDepartment of Anatomy and Neuroscience, Hyogo College of Medicine, Hyogo 663-8501, Japan bDepartment of Otolaryngology, Hyogo College of Medicine, Hyogo 663-8501, Japan Accepted 12 September 2000 Abstract The olfactory neuroepithelium of the mammalian nervous system manifests continuous neurogenesis throughout life. Recent studies suggest that neurotrophic factors and their receptors may play a role in the regulation of development and regeneration in the olfactory system. However, there have been very few in vivo studies investigating the effect of exogenous neurotrophic factors in the olfactory system. In the present study, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) were administered into the rat olfactory mucosa for 5 days just after the transection of the olfactory nerve. We then examined the effect of exogenous neurotrophic factors on the degenerative changes in axotomized olfactory receptor neurons (ORNs). Further, we examined the location of their receptors, Trk A and Trk B. We found that both mature and immature ORNs expressed more intense signals for olfactory marker protein and beta-tubulin mRNAs, respectively, when NGF was applied to the axotomized olfactory neuroepithelium for 5 days, compared to the ORNs of saline-treated controls. BDNF at a 10 mg total dose did not show this effect. The effect of NGF applied onto the olfactory epithelium is consistent with the immunohistochemical ®nding that Trk A was present in the dendrites and axon bundles in normal and axotomized ORNs. These results suggest that NGF may protect the degenerative changes in mature and immature ORNs following axotomy through the binding to the Trk A receptor located on the surface of the olfactory epithelium. 2000 Elsevier Science B.V. All rights reserved. Theme: Sensory systems Topic: Olfactory senses Keywords: Olfactory nerve; Olfactory marker protein; Beta-tubulin; Protein gene product 9.5; NGF; BDNF 1. Introduction receptor neurons (ORNs) derived from the observation that a developmentally heterogenous population of receptor In mammals, the olfactory neuroepithelium (OE), neurons is present in adult animals, and autoradiographic situated within the nasal cavity, has the ability to regener- studies have shown that immature neurons in the basal cell ate new neurons continually throughout life from a popula- layer of the OE of adult animals incorporate3 H-thymidine tion of precursor cells present in the basal cell layer [5,34]. These immature neurons progressively move to [15,16]. The concept of continued turnover of olfactory more super®cial layers of the epithelium to become mature ORNs. The process of turnover of ORNs can be modi®ed *Corresponding author. Tel.: 181-798-45-6415; fax: 181-798-45- following the injury of mature ORNs, such as physical or 6417. chemical damage to the OE proper, the axons of the ORNs E-mail address: [email protected] (K. Noguchi). (olfactory nerve), or the olfactory bulb [8,40]. For exam- 0006-8993/00/$ ± see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993(00)02966-8 54 H. Yasuno et al. / Brain Research 887 (2000) 53 ±62 ple, changes in ORNs following the transection of the 2. Materials and method olfactory nerve have been extensively examined. A few days after transection, massive degeneration and neuronal 2.1. Animal procedures programmed cell death of the ORNs occurred and new ORNs, derived from a stem cell population in the OE, All animal experiments conformed to the regulations of generate axons which grow along the olfactory nerve to the the Hyogo College of Medicine Committee on Animal olfactory bulb. Research and were carried out in accordance with the The normal OE is a ciliated pseudostrati®ed columnar guidelines of the NIH on animal care. A total of 24 male epithelium. The basal cells are located at the base of the Sprague-Dawley rats weighing 200±250 g were used. All epithelium and immature ORNs are situated in the lower experimental procedures were performed on rats which layer of the epithelium. The immature ORNs express were deeply anesthetized with sodium pentobarbital (50 growth associated protein 43 (GAP-43/B50) [39,40], mg/kg body weight, i.p.). Additional doses of anesthetics which is expressed during axonal growth and neural were given as needed. development, and tubulin [25,32], which participates in Rats received local anesthetic (1% carbocaine, 200 m1) spindle formation in dividing cells. A third type of cell, the to the site of the skin incision on the middle of the head. mature ORNs, are localized in the middle layer of the The left skull, rostral to the bone suture bregma, was epithelium and express olfactory marker protein (OMP) opened using a micro drill and the left olfactory bulb was [4,6,10,27,30,43], a 19 kDa cytoplasmic protein. Immature exposed. A microsurgery scalpel was inserted to a depth of and mature neurons express neural cell adhesion molecules 4 mm against the olfactory bulb side of the cribriform plate [28], which are important during all stages of neurode- and the ®la olfactoria were transected according to the velopment and are especially important for synaptic struc- method reported by Harding et al. [19]. In a preliminary turing and protein gene product 9.5 (PGP 9.5) [23,36], experiment, we con®rmed that our method could success- which is a neurospeci®c peptide. A fourth type of cell in fully transect the olfactory nerves by checking the thick- the OE are supporting cells, which are located in the upper ness of the olfactory epithelium. Next, NGF (20 mg/ml, layer of the epithelium. Injury of ORNs also affects the recombinant rat NGF, R&D Systems, Minneapolis, MN, neurochemical expression pattern and axotomized ORNs USA), or BDNF (20 mg/ml, recombinant human BDNF, lose almost all OMP immunoreactive pro®les after about a Sumitomo Pharmaceutical, Osaka, Japan) was adminis- week [31]. tered twice a day for 5 days. Rats received NGF or BDNF It is well known that several growth factors play a solution using a Hamilton syringe intermittently for 15 min critical role in the regulation of neurogenesis in the until a total volume of 50 ml of solution had been injected developing central nervous system. The typical neuro- into the left nasal cavity. In control rats, saline was trophic factors, nerve growth factor (NGF) and brain- administered. The total dose of 10 mg of NGF or BDNF derived neurotrophic factor (BDNF), are known to have was administered over a 5 day period. This dose was crucial roles in the differentiation, survival, morphogenesis determined according to the doses of neurotrophins, topical and regeneration of sensory neurons [2,20,24,26,37]. Re- injections of which were reported previously (6 mg/day cently, high-af®nity receptors of the neurotrophic factor NGF [11,41], 5 mg/day BDNF [35], 12 mg/day BDNF tyrosine kinases (Trks) have been cloned; NGF is the [12]), and considering the very limited area of the olfac- preferred ligand of Trk A [7] and BDNF is the preferred tory epithelium. ligand of Trk B [13]. A number of studies have suggested that endogenous neurotrophins play a role in the regulation 2.2. In situ hybridization histochemistry of neurogenesis within normal development of the OE and altered turnover of OE following injury to the ORNs. For Five days after surgery, rats were deeply anesthetized example, NGF injected into the olfactory bulb is trans- with sodium pentobarbital and perfused transcardially with ported to the OE for 18 h in the mouse [29]. Antibodies to 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). NGF injected in the olfactory bulb induce degeneration of The left olfactory mucosa was dissected, post®xed over- ORNs [38]. ORNs can be matured in vitro by treatment night, followed by immersion in 20% sucrose in phosphate with neurotrophins, especially treatment with a combina- buffer for cryoprotection. After 1 or 2 days, olfactory tion of NGF and BDNF [33]. However, there has been no mucosae were frozen with powdered dry ice, cut trans- in vivo experiments in which exogenous neurotrophins versely with a cryostat into 10 mm thick sections, thaw- were applied into the nasal cavity. In this study, we mounted onto Vectabond-coated slides (Vector Laborator- administered NGF or BDNF into rats' olfactory mucosa, ies, Burlingame, CA, USA) and stored at 2808C until use. and examined in vivo the effect on axotomized ORNs. The An olfactory marker protein-speci®c probe, a mature ORN dependent measures examined were altered mRNA expres- marker, and a beta-tubulin-speci®c probe, an immature sion of neurochemical markers using in situ hybridization ORN marker, were used. The probes consisted of 48-base histochemistry, and we also examined their receptors, Trk oligonucleotides and the OMP probe was complementary A and Trk B, using immunohistochemistry. to bases 439±486 and beta-tubulin probe to bases 18±65 of H. Yasuno et al. / Brain Research 887 (2000) 53 ±62 55 the respective cDNAs. These probes were labeled with drated in a graded ethanol series, cleared in xylene, and 35S-deoxyadenosine triphosphate (NEN, Boston, MA, coverslipped. USA) and terminal deoxynucleotidyl transferase (Amer- sham, Buckinghamshire, UK), giving a speci®c activity of 2.4. Quantitative analysis 1.031099 ±1.5310 cpm/mg. Tissue sections were hybrid- ized after thawing, without any pretreatment, overnight at Each experimental group consisted of six rats. For the in 428C in humidi®ed boxes with 53105 c.p.m.