HGF) on Reinnervation of Perivascular Calcitonin Gene-Related Peptide (CGRP)-Containing Nerves Following Phenol-Induced Nerve Injury in the Rat Mesenteric Artery

Neurotrophic Effect of Hepatic Growth Factor (HGF) on Reinnervation of Perivascular Calcitonin Gene-Related Peptide (CGRP)-Containing Nerves Following Phenol-Induced Nerve Injury in the Rat Mesenteric Artery

Narumi Hobara1, Namika Yoshida2, Mitsuhiro Goda2, Ayako Yokomizo2, Yoshihisa Kitamura3, Toshiaki Sendou4, and Hiromu Kawasaki2,* 1Department of Life Science, Okayama University of Science, 1-1 Ridai-cho, Okayama 700-0005, Japan 2Department of Clinical Pharmaceutical Science, 3Department of Pharmaceutical Care and Health Sciences, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima-naka, Okayama 700-8530, Japan 4Department of Hospital Pharmacy, Okayama University Medical School, Okayama University, 2-5-1 Shikata-cho, Okayama 700-8558, Japan

Received August 27, 2008; Accepted October 27, 2008

Abstract. Hepatic growth factor (HGF) has neurotrophic effects in the motor neurons and central nervous system. However, there has been no report about the neurotrophic action on perivascular nerves innervating the resistance artery. We investigated whether HGF can restore innervation or function of perivascular nerves, including neuropeptide Y (NPY)-containing sympathetic adrenergic nerves and calcitonin gene-related peptide (CGRP)-containing nerves, in rat mesenteric artery. To investigate HGF-mediated neurotrophic effects, Wistar rats under pentobarbital-Na anesthesia underwent in vivo perivascular denervation by topical application of phenol on the superior mesenteric artery, and then HGF or nerve growth factor (NGF) was administered for 7 days using an osmotic mini-pump after phenol-treatment. HGF significantly increased the density and number of CGRP-like immunoreactivity (LI)–containing nerve fibers compared with saline administration, while HGF did not affect the density of NPY-containing adrenergic nerve fibers. After 7-day treatment with HGF and phenol, the vascular response of vasodilation was recovered from nerve injury by phenol treatment, but vasoconstriction was not. HGF and NGF induced neurite outgrowth in rat cultured dorsal root ganglia (DRG). These results suggest that HGF has a specific neurotrophic action on reinnervation of vascular CGRP-LI– containing nerve fibers in the rat mesenteric artery and DRG.

Keywords: hepatic growth factor (HGF), neurotrophic effect, calcitonin gene-related peptide (CGRP)-containing nerve, neuropeptide Y (NPY)-containing nerve, phenol-induced perivascular nerve injury

Introduction associated with endothelial function. HGF is well known as an endothelium-specific growth factor that plays a Human hepatic growth factor (HGF) was first purified role as a basic fibroblast factor and survival factor in from the plasma of patients with fulminant hepatic endothelial cells (3). Another report suggested that failure (1, 2). There are many reports that HGF is HGF prevents endothelial cell death under high glucose conditions (4). Although endothelial function of HGF *Correspondence author. has been demonstrated, the effect of perivascular nerve [email protected] function is less well understood. Published online in J-STAGE on December 10, 2008 (in advance) HGF has been shown to have neurotrophic activities doi: 10.1254/jphs.08225FP in the cerebral cortex, sensory neurons, and spinal cord

495 496 N Hobara et al motor neurons (5 – 7). In the peripheral nervous system, effort was made to minimize the number of animals HGF has also been reported to have a neurotrophic effect used and their suffering. on adult motor neurons after axotomy of the hypoglossal nerves in vivo (8). However, it remains unclear whether Animal treatments and experimental protocols HGF has trophic effects on peripheral perivascular After anesthesia with sodium pentobarbital (50 mg/kg, nerves. intraperitoneally), an abdominal midline incision was Perivascular nerves play an important role in made in the animal, and the superior mesenteric artery maintenance of vascular tone along with endothelial proximal to the bifurcation from the abdominal aorta cells. The mesenteric artery, which comprises the largest was exposed and topically swabbed with 10% phenol vascular bed in the body, is innervated by many solution (in 90% alcohol–saline) using a cotton bud, perivascular nerves, such as sympathetic adrenergic as described previously (11 – 13). Sham-operated rats nerves, and non-adrenergic non-cholinergic nerves, underwent the same surgical procedures except they were which include calcitonin gene-related peptide (CGRP)- swabbed with the vehicle (saline or 90% alcohol without containing nerves (CGRPergic nerves) (9). Recently, phenol). After swabbing, an antibiotic (penicillin G; we demonstrated that innervation of CGRPergic nerves Sigma Aldrich Japan, Tokyo) was infused around the and neuropeptide Y (NPY)-containing sympathetic surgical area and the incision was closed. After the adrenergic nerves in rat mesenteric resistance arteries operation, the animals were transferred into their indi- was markedly reduced by topical application of phenol vidual cages in a temperature-controlled room and in vivo and that nerve growth factor (NGF) facilitates received intramuscular injection of penicillin G (3.1 mg reinnervation of both types of nerves (10). Furthermore, /kg) for 3 consecutive days. After phenol treatment we recently demonstrated that adrenomedullin, a vaso- and a sham operation, the animals were killed by deep active peptide, has the ability to facilitate reinnervation anesthesia on Day 7 for use in the experiments described of mesenteric perivascular nerves lesioned by topical below. treatment with phenol (11, 12). After the swabbing described above, a mini-osmotic Therefore, the present study was designed to investi- pump (model 2001; Alzet, Alza, Palp Alto, CA, USA) gate whether HGF has neurotrophic activities on the was implanted in the abdominal area to administer reinnervation of mesenteric perivascular nerves after in recombinant human HGF (Mitsubishi Pharmaceutical vivo denervation by using topical treatment with phenol. Co., Tokyo) at a rate of 10 or 30 μg/kg per day and To further investigate the neurotrophic effect of HGF, NGF (Sigma Aldrich Japan) at a rate of 20 μg/kg the density of innervation of perivascular nerves, per day for a period of 7 days. HGF was dissolved in neurogenic vascular responses of the rat mesenteric PBS containing 0.005% Triton-X and NGF was resistance artery and in vitro neurite outgrowth in dissolved in sterile saline and injected into Alzet- primary cultures of dorsal root ganglia (DRG) were osmotic min-pumps. Control animals were implanted examined. with mini-pumps containing saline. The animals were randomly divided into five groups: 1) a normal control Materials and Methods Sham group (Sham); 2) a phenol-saline group (Ph + Saline), animals receiving saline after phenol treatment; Experimental animals 3) a phenol-HGF10 group (Ph + HGF10), animals Eight-week-old Wistar rats (purchased from Shimizu receiving HGF 10 μg/kg per day after phenol treatment; Experimental Animals, Shizuoka) were used in this 4) a phenol-HGF30 group (Ph + HGF30), animals study. The animals were given food and water ad receiving 30 μg/kg per day after phenol treatment; 5) a libitum. They were housed in the Animal Research phenol-NGF group (Ph + NGF), animals receiving NGF Center of Okayama University at a controlled ambient after phenol treatment. temperature of 22°C with 50 ± 10% relative humidity and with a 12-h light/12-h dark cycle (lights on at Systolic blood pressure measurement 8:00 AM). All animal experimental procedures were The systolic blood pressure of each animal was approved by the Committee on Animal Experiments at measured daily by tail-cuff plethysmography (model Okayama University and carried out in accordance TK-370C; UNICOM, Tokyo) throughout the treatment with the Guidelines for Animal Experiments at Okayama period. University Advanced Science Research Center, Japanese Government Animal Protection and Management Law Immunohistochemical study (No. 105) and the Japanese Government Notification The animals treated topically with phenol or vehicle on Feeding and Safekeeping of Animals (No. 6). Every were anesthetized with a large dose of sodium Neurotrophic Effect of Hepatic Growth Factor 497 pentobarbital (50 mg/kg, intraperitoneally). The superior color parameters. A measured field of 100 μm × 100 μm mesenteric artery was cannulated with polyethylene (10000 μm2, which contained the adventitia layer tubing and Zamboni solution (2% paraformaldehyde including immunostained perivascular nerve fibers), was and 15% picric acid in 0.15 M phosphate buffer) was randomly selected on magnified images of the whole infused. Then, the mesenteric artery was removed mount artery. The objective areas command was used to together with the intestine as described previously calculate the percentage of the CGRP-LI– and NPY-LI– (11 – 13). The third branch of the mesenteric artery positive area. The intensity of staining was estimated proximal to the intestine was immersion-fixed in the using a point-counting computer program and the Zamboni solution for 48 h, immersed in PBS containing background level was subtracted from the experimental 0.5% TritonX-100 overnight, and incubated with PBS value to yield the corrected intensity. The average containing normal goat serum (1:100) for 60 min. The density in three arteries was taken as the nerve density tissue was then incubated with rabbit polyclonal anti- per animal. CGRP (Biogenesis, Ltd., Oxford, UK) antiserum at a To determine the number of CGRP-LI and NPY-LI dilution of 1:300 or rabbit polyclonal anti-NPY (Phoenix fibers, five horizontal lines were drawn on the image of Pharmaceuticals, Inc., Belmont, CA, USA) antiserum at the blood vessel in the same region where the density a dilution of 1:300 for 72 h at 4°C. Thereafter, to detect was estimated by computer analysis. Then, the number the sites of the antigen-antibody reaction, the artery was of fibers that crossed each line was counted and the incubated with fluorescein-5-isothiocyanate (FITC)- average of the number in 3 arteries was taken as the labeled goat anti-rabbit IgG (diluted 1:100) (ICN total number of fibers per animal. Pharmaceuticals Inc., Aurora, OH, USA) for 60 min, mounted on a slide, cover-slipped with glycerol/PBS Perfusion of mesenteric vascular beds (2:1 v/v), and observed under a confocal laser scanning The animals were anesthetized with sodium pento- microscope (CLSM510; Carl Zeiss, Tokyo) at the barbital (50 mg/kg, intraperitoneally) and the mesen- Okayama University Medical School Central Research teric vascular bed was isolated and prepared for Laboratory. perfusion as described previously (9, 14). The isolated mesenteric vascular bed was separated from the intestine Immunohistochemical analysis by cutting close to the intestinal wall. Only four main The immunostaining density of CGRP-like immuno- arterial branches from the superior mesenteric trunk reactive (CGRP-LI) and NPY-like immunoreactive running to the terminal ileum were perfused with a (NPY-LI) nerve fibers was analyzed using the method modified (see below) Krebs-Ringer bicarbonate solution described by Hobara et al. (11 – 13). Since the fluores- (Krebs solution). The isolated mesenteric vascular bed cence intensity differed depending on the day of the was then placed on a water-jacketed organ bath experiment, the mesenteric arteries from rats treated maintained at 37°C and perfused with Krebs solution at with each drug after topical phenol treatment on Day 7 a constant flow rate of 5 ml/min with a peristaltic pump and vehicle-treated control rats were isolated, fixed, (model AC-2120; ATTO Co., Tokyo) and superfused and immunostained at the same time on the same day with the same solution at a rate of 0.5 ml/min to prevent and mounted on the same slide glass, while the vehicle- drying. The Krebs solution was bubbled with a mixture treated rats at Day 7 were used as a control for the of 95% O2 plus 5% CO2 before passage through a intensity in each experiment. For quantitative evaluation warming coil maintained at 37°C. The modified Krebs of CGRP-LI and NPY-LI, confocal projection images solution had the following composition: 119.0 mM of CGRP and NPY immunostaining, which consisted of NaCl, 4.7 mM KCl, 2.4 mM CaCl2, 1.2 mM MgSO4, 8 – 10 overlapping images (0.1 μm scanning) patched 25.0 mM NaHCO3, 1.2 mM KH2PO4, 0.03 mM EDTA- together, were magnified 20× and digitized as TIF 2Na, and 11.1 mM glucose (pH 7.4). Changes in the images using a digital camera system (Olympus SP-1000; perfusion pressure were measured with a pressure Olympus, Tokyo) and imported into a Windows XP transducer (model TP-400T; Nihon Kohden, Tokyo) and computer (Toshiba, Tokyo). The stored digital images recorded using a pen recorder (model U-228; Nippon were analyzed using image-processing software (Simple Denshi Kagaku, Tokyo). PCI; Compix, Inc., Imaging Systems, Cranberry Town- ship, PA, USA). The extraction of specific color and Perivascular nerve stimulation (PNS) and bolus injec- measured field commands were used to extract the tion of norepinephrine or CGRP CGRP-LI and NPY-LI areas (which were stained green). After allowing the basal perfusion pressure to stabilize, Extraction of the signal was carried out using specific to observe adrenergic nerve-mediated vasoconstriction, protocols based on the hue, lightness, and saturation the preparation was initially subjected to PNS at 8 and 498 N Hobara et al

12 Hz and bolus injections of norepinephrine (5 and Cell treatments 10 nmol). Thereafter, to observe CGRPergic nerve- To examine whether the HGF induce neurite out- mediated vasodilation, the preparation was contracted growth on DRG cells, cells were incubated without with an α1-adrenoceptor agonist, methoxamine (7 μM), NGF or HGF (control cells), with NGF (100 ng/mL) or in the presence of an adrenergic neuron blocker, HGF (25 or 50 ng/mL). The substances were added to guanethidine (5 μM), which was added to block the the neurons from the second day for 3 days and the adrenergic neurotransmission. The increased perfusion medium was replaced every day. pressure was allowed to stabilize, and the preparation was again subjected to PNS at 2 and 4 Hz and a bolus Immunocytochemical experiments injection of CGRP (50 and 100 pmol). PNS was applied For immunocytochemistry, cells were fixed with 10% by using bipolar platinum ring electrodes placed around formalin for 20 min following incubation with 0.5% the superior mesenteric artery. Rectangular pulses of TritonX-100 for 10 min. After washing with PBS, cells 1 ms and supramaximal voltage (50 V) were given for were incubated with 5% goat serum containing 1% BSA 30 s using an electronic stimulator (model SEN 3301, and NaN3 for 30 min at room temperature. Thereafter, Nihon Kohden). Norepinephrine and CGRP were injected the cells were incubated with 1:500 diluted anti CGRP directly into the perfusate proximal to the arterial rabbit (Biomol International LP, CA, USA) for 60 min at cannula with an infusion pump. A volume of 100 μL room temperature. After washing cells were incubated was injected for 12 s. for 40 min at room temperature with a (FITC)-labeled At the end of each experiment, preparations were goat anti-rabbit IgG (MP Biomedicals, LLC, Tokyo), perfused with 100 μM papaverine to cause complete diluted 1:100 in PBS. After washing with PBS and relaxation. Vasodilator activity is expressed as the distilled water, 2 – 3 drops of Fluoromount (Cosmo Bio percentage of the perfusion pressure at the maximum Co., Ltd., Tokyo), which prevents fading of fluorescent relaxation induced by papaverine. Vasoconstrictor dyes, were put on the glass cover-slips, which were then activity is expressed as a percent of the perfusion pres- gently applied to the slide glass. CGRP-immunopositive sure. cells were approximately 40% of DRG neurons as reported by De Jonge et al. (15). Primary cultures of DRG cells To quantify neurite outgrowth in neurons, images of Rat DRG neurons were isolated from adult male randomly chosen fields of neuron cultures were Wistar rats (age: 6 – 12 weeks), using procedures that obtained with a Zeiss Axioplan (ZEISS, Germany) and have been reported previously (15, 16). First, animals OLYMPUS DP-50 camera units (Olympus Co., Japan); were anesthetized with a large dose of sodium pento- experiments were performed at the Central Research barbital, intraperitoneally. Then, the spinal cord was Laboratory, Okayama University Medical School. isolated and the ganglia were dissected using micro- Quantification of neurite length was done on exported forceps. After removal of connective tissue, the spinal TIFF files using FluorchemTM8900 (Alpha Innotech, roots were cut close to the ganglia, rapidly replaced in San Leandro, CA, USA). Briefly, the longest neurite collagenase (5 mg/mL for 30 min at room temperature) length was measured from the center of the cell body (Sigma Aldrich Japan) followed by incubation for and counted as neurite outgrowth. 30 min at 37°C with trypsin (Invitrogen Japan, Tokyo) and collagenase 1.25 and 2.5 mg/mL, respectively. The Statistical analyses ganglia suspended with PBS was collected by centrifu- All data are expressed as the mean ± S.E.M. Analysis gation and mechanically dissociated by pipetting with of variance (ANOVA) followed by Tukey’s test was used Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO to determine statistical significance where appropriate. Invitrogen Japan, Tokyo) containing 10% fetal bovine Correlation analysis was carried out using Pearson’s serum (GIBCO Invitrogen Japan), 100 U/mL penicillin, correlation test. P<0.05 was considered statistically and 100 μg/mL streptomycin. The resulting cell suspen- significant. sions were plated on serum-coated Petri dishes to allow the non-neuronal cells to adhere. After 90 min, the Results neurons in suspension were collected and plated on poly-L-lysine (10 μg/mL, Sigma Aldrich Japan)-coated Changes in innervation of CGRP-LI- or NPY-LI nerve 18-mm glass coverslips and maintained for 4 days at fibers in mesenteric arteries following phenol treatment 37°C in a humidified incubator gassed with 5% CO2 with or without HGF or NGF administration and air. As shown in Fig. 1A, the distal mesenteric artery was densely innervated by CGRP-LI nerve fibers, and the Neurotrophic Effect of Hepatic Growth Factor 499

Fig. 1. Representative confocal laser images (A – E) showing changes in the innervation of calcitonin gene-related peptide (CGRP)- like immunoreactivity (LI)–containing nerve fibers in mesenteric arteries after phenol treatment and bar graphs showing changes in the density (F) and number (G) of CGRP-LI–containing nerve fibers in the mesenteric arteries 7 days after topical phenol application and administration of hepatic growth factor (HGF) or nerve growth factor (NGF) treatment. A horizontal bar in the right lower corner of each panel indicates 100 μm. Each bar indicates the mean ± S.E.M. of 5 – 8 experiments. *P<0.05 vs Sham. †P<0.05 vs NGF. density and number of CGRP-LI nerves were lower than densely innervated by NPY-LI nerve fibers, and the that of NPY-LI nerves (Fig. 2A). The density of CGRP- density or number of NPY-LI nerves was much higher LI nerve fibers in the distal small artery was markedly than that of CGRP-LI nerves (Fig. 1A). The density and decreased by approximately 80% after topical phenol number of NPY-LI nerve fibers in the distal small treatment of the superior mesenteric artery (Fig. 1B), arteries were significantly decreased at 7 days after whereas the vehicle (saline or 90% alcohol) treatment topical phenol application in the superior mesenteric (Sham control) did not reduce the innervation of CGRP- artery (Fig. 2: B, F, and G). Administration of HGF at LI nerve fibers (Fig. 1A). doses of 10 and 30 μg/kg per day caused no significance As shown in Fig. 1 (C, F, and G), administration of change in the density or number of NPY-LI fibers HGF at a dose of 10 μg/kg per day had no effect on the compared with the Phenol-Saline group (Fig. 2: C, D, F, density or number of CGRP-LI nerve fibers, whereas and G). However, the decreased level of NPY-LI fiber administration of HGF at a dose of 30 μg/kg per day density and number following phenol treatment were increased the density and number compared with the restored by the application of NGF (20 μg/kg per day) to Phenol-Saline group (Fig. 1: D, F, and G). NGF admin- the level in the Sham control group (Fig. 2: E, F, and G). istration after topical application of phenol significantly There was a significant difference between the density increased the density and number compared with the and number in the Phenol-Saline group and Phenol-NGF Phenol-Saline group. NGF restored the level of innerva- group (Fig. 2: E, F, and G). tion of CGRP-LI nerve fibers to the level observed in the The relationships between the numbers of CGRP-LI Sham control group (Fig. 1: E – G). and NPY-LI nerve fibers, which were visually counted, As shown in Fig. 2A, the distal mesenteric artery was and the densities of CGRP-LI and NPY-LI nerves (%), 500 N Hobara et al

Fig. 2. Representative confocal laser images (A – E) showing changes in innervation of neuropeptide Y (NPY)-like immunoreactivity (LI)–containing nerve fibers in mesenteric arteries and bar graphs showing changes in the density (F) and number (G) of NPY- LI–containing nerve fibers in the mesenteric arteries 7 days after topical phenol application and administration of hepatic growth factor (HGF) or nerve growth factor (NGF) treatment. A horizontal bar in the right lower corner of each panel indicates 100 μm. Each bar indicates the mean ± S.E.M. of 5 – 8 experiments. *P<0.05 vs Sham. †P<0.05 vs NGF. which were quantified by computer-assisted image Table 1. Systolic blood pressure on day 7 following topical phenol processing, were assessed in mesenteric arteries of all application in rats treated with saline, hepatic growth factor (HGF), or nerve growth factor (NGF) groups. There were significant positive correlations Systolic blood pressure between the density and the numbers of CGRP-LI and Treatments n NPY-LI nerve fibers in the Sham group (CGRP-LI, (mmHg) P<0.05, r = 0.585; NPY-LI, P<0.01, r = 0.762), Phenol- Sham 12 119.4 ± 1.9 Saline group (CGRP-LI, P<0.01, r = 0.822; NPY-LI, Phenol-Saline 18 111.1 ± 2.3 P<0.01, r = 0.740), Phenol-HGF10 group (CGRP-LI, Phenol-HGF (10 μg/kg per day) 6 108.5 ± 2.6 P<0.01, r = 0.927; NPY-LI, P<0.05, r = 0.574), Phenol- Phenol-HGF (30 μg/kg per day) 6 98.8 ± 6.1* HGF30 group (CGRP-LI, P<0.01, r = 0.799; NPY-LI, Phenol-NGF (10 μg/kg per day) 4 101 ± 4.8* P<0.01, r = 0.914), and Phenol-NGF group (CGRP-LI, Each value is a mean ± S.E.M. *P<0.01 vs Sham. P<0.01, r = 0.715; NPY-LI, P<0.05, r = 0.632) (data not shown). dose of 30 μg/kg per day or Phenol-NGF treatment and Changes in systolic blood pressure (SBP) after phenol Phenol-Saline treatment. treatment As shown in Table 1, HGF dose-dependently lowered Effects of HGF or NGF treatment on vasoconstrictor SBP on Day 7 after phenol treatment. NGF also responses to PNS and bolus injection of norepinephrine decreased SBP on Day 7. Significant differences were to phenol treatment rats found in SBP between Phenol-HGF treatment at a As shown in Fig. 3, PNS at 8 and 12 Hz of the Neurotrophic Effect of Hepatic Growth Factor 501

Fig. 3. Changes in vasoconstrictor responses to perivascular nerve stimulation (PNS) and to bolus infusion of norepinephrine (NE) in perfused mesenteric vascular beds 7 days after topical phenol application and administration of hepatic growth factor (HGF) or nerve growth factor (NGF) treatment. *P<0.05 vs Sham control. perfused mesenteric vascular beds with resting tone receptors (19). produced a frequency-dependent increase in perfusion Vasodilatations in responses to PNS (2 and 4 Hz) in pressure due to vasoconstriction. The vasoconstrictor Phenol-Saline-treated rat preparations were significantly responses to PNS at 8 and 12 Hz in the Phenol-Saline smaller than those in Sham preparations (Fig. 4). As group were smaller than those in the Sham group shown in Figs. 4, the PNS-induced (1 Hz) vasodilator (Fig. 3). Furthermore, in the Phenol-Saline and Phenol- responses of preparations from Phenol-Saline, Phenol- HGF (10 or 30 μg/kg per day) groups, the vaso- HGF (10 or 30 μg/kg per day), and Phenol-NGF groups constrictor response to PNS at 12 Hz, but not 8 Hz, was were similar in magnitude to Sham preparations. The also significantly smaller than the Sham group (Fig. 3). vasodilator responses to PNS at 2 and 4 Hz in the In the Phenol-NGF group, the vasoconstrictor response Phenol-HGF (30 μg/kg per day but not 10 μg/kg per to PNS at 12 Hz was smaller than that in the Sham day) group were greater than those in the Phenol-Saline group, but there was no significant difference between group and no significant difference was found between the Phenol-NGF and Sham group. As shown in Fig. 3, the Sham and Phenol-HGF groups (Fig. 4). The Phenol- vasoconstrictor responses to bolus injections of nor- NGF group showed PNS-induced vasodilator responses epinephrine were not significantly affected by treatment similar to those in the Phenol-HGF group. Bolus with Phenol-Saline, Phenol-HGF, or Phenol-NGF. injection of CGRP at concentrations of 50 and 100 pmol produced long-lasting depressor responses, which Effects of treatment with HGF or NGF on vasodilator mimicked PNS-induced vasodilation and was blocked responses to PNS or bolus injection of CGRP to phenol by CGRP(8 – 37) (data not shown). As shown in treatment rats Fig. 4, vasodilator responses to injected CGRP at any To maintain active tone of the mesenteric artery, each concentration in the Phenol-Saline group were of a preparation was contracted by continuous perfusion of similar degree to those in the Sham group. However, 7 μM methoxamine (α1-adrenergic receptor agonist) in in the Phenol-HGF (10 or 30 μg/kg per day) group, the presence of 5 μM guanethidine (adrenergic neuron 50 pmol CGRP–induced vasodilation was greater than blocker), which was added to block adrenergic neuro- that in the Phenol-Saline group, while the level of transmission. In this preparation, PNS at 2 and 4 Hz 100 pmol CGRP–induced vasodilation was the same as caused a frequency-dependent decrease in perfusion those in the Sham and Phenol-Saline groups. The pressure due to vasodilation. The vasodilator response to vasodilation in response to CGRP (50 or 100 pmol) in PNS has been shown to be mediated by CGRPergic the Phenol-NGF group was significantly smaller than nerves since the response was blocked by CGRP(8 – 37) that in the Sham group and Phenol-Saline group. (CGRP-receptor antagonist) and capsaicin (CGRP depletor) (17, 18). Bolus injections of CGRP also Effect of treatment with HGF or NGF on neurite induced a concentration-dependent vasodilation, which outgrowth of DRG neurons has been shown to be mediated by postsynaptic CGRP In cultured DRG cells, sizes of the CGRP-LI positive 502 N Hobara et al

Fig. 4. Changes in vasodilator responses to perivascular nerve stimulation (PNS) and to bolus infusion of calcitonin gene-related peptide (CGRP) in perfused mesenteric vascular beds 7 days after topical phenol application and administration of hepatic growth factor (HGF) or nerve growth factor (NGF) treatment. Active tone was produced by 7 μM methoxamine in the presence of 5 μM guanethidine. *P<0.05 vs Sham. †P<0.05 vs NGF.

Fig. 5. Representative images showing neurite outgrowth of calcitonin gene-related peptide (CGRP)-immunopositive neurons in rat dorsal root ganglia (DRG) cells (A – D) and effect of treatment with hepatic growth factor (HGF) or nerve growth factor (NGF) (E). Arrowheads in images show outgrown neurites. In panel E, a bar graph shows changes in mean neurite length after treatment with HGF or NGF. The ordinate indicates fold increase over the control neurite length. Each bar indicates the mean ± S.E.M. *P<0.05 vs Control. cell bodies were 20.3 ± 6.9 μm (n = 46). As shown Discussion in Fig. 5, treatment with HGF at 25 or 50 ng/mL concentration-dependently increased neurite outgrowth In the present study, topical application of phenol in cultured DRG cells. Treatment with NGF at around the rat superior mesenteric artery in vivo resulted 100 ng/mL also markedly increased neurite outgrowth. in marked reduction of both CGRP-LI and NPY-LI Extended neurite length induced by HGF at 25 and nerves, which innervated in the distal small mesenteric 50 ng/mL and NGF at 100 ng/mL were 82.5 ± 33.1, artery. This finding is in good accordance with our 118.2 ± 59.6, and 113.3 ± 26.5 μm, respectively. There previous reports (10 – 12), supporting the fact that was a significant difference between the control and the method using phenol is effective for inducing HGF at 50 ng/mL, but not 25 ng/mL, or NGF at perivascular denervation in vivo. The present study is 100 ng/mL. The immunostaining assay showed that the first to demonstrate that HGF facilitates reinnerva- outgrown neurites were CGRP-LI–positive (Fig. 5). tion of perivascular CGRP-LI nerves injured by topical phenol treatment in the rat superior mesenteric artery. Our previous studies showed that phenol-induced Neurotrophic Effect of Hepatic Growth Factor 503 reduction of perivascular innervation was prevented by present results that HGF has specific neurotrophic effect NGF treatment (10). Furthermore, adrenomedullin, a on CGRP-LI fibers in the mesenteric artery. However, it potent vasodilator peptide, facilitates the reinnervation is still unclear whether HGF acts directly or indirectly on of perivascular NPY-LI and CGRP-LI nerves injured by DRG neurons. Hashimoto et al. (24) reported that c-Met topical application of phenol in the rat mesenteric artery mRNA was not detected in DRG but was detected in the (11). Taken together, it is likely that HGF, like NGF and spinal dorsal horn. Therefore, it is possible that HGF adrenomedullin, has the ability to enhance reinnervation might act on glia cells, which release trophic factors of the perivascular nerves. However, HGF seems to have such as NGF, neurotrophine-3, and brain-derived a different action from NGF and adrenomedullin since neurotrophic factor. Our findings showed that HGF HGF cancelled only the phenol-induced reduction of alone was effective to promote axonal outgrowth in the innervation of CGRP-LI nerves without affecting adult rat cultured DRG neurons. However, HGF has NPY-LI. been reported to have no outgrowth promoting activity In the present study, a high dose of HGF after phenol in mouse cultured embryonic DRG neurons (22). The treatment, which facilitated reinnervation of CGRP-LI difference between the present observations and other nerves, restored the CGRPergic nerve-mediated vaso- reported results may be the HGF concentrations used. dilator response to PNS of perfused mesenteric vascular In the study on mouse embryonic cells, HGF at a beds, suggesting that the vasodilator function of concentration of 12 ng/mL was used, while the present CGRPergic nerves is recovered by HGF. However, study showed no significant increase in neurite out- adrenergic nerve-mediated vasoconstrictor responses by growth at 25 ng/mL concentration. PNS were not affected by treatment of HGF, which had In conclusion, the present results suggest that HGF no effect on reinnervation of sympathetic NPY-LI facilitates the reinnervation of CGRP-LI nerve fibers nerves. These functional results are well correlated with injured by topical phenol treatment. The results also results obtained by immunohistochemical studies. These suggest that HGF, like NGF, has the ability to regenerate results suggest that HGF accelerates only reinnervation and/or reinnervate perivascular nerves as a neurotrophic and/or regenerations of CGRPergic vasodilator nerves factor. and restores their vasodilator function. Interestingly, the present study showed that the Phenol-HGF–treated group had lower systolic blood pressure compared References with the Phenol-Saline–treated group. Therefore, it is 1 Nakayama H, Tsubouchi H, Gohda E, Koura M, Nagahama J, assumed that the blood pressure lowering effect may Yoshida H, et al. Stimulation of DNA synthesis in adult rat result from restored CGRPergic nerve function by HGF hepatocytes in primary culture by sera from patients with since HGF had no effect on injured NPY-LI adrenergic fulminant hepatic failure. Biomed Res. 1985;6:231–237. vasoconstrictor nerves. 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