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Nerve growth factor facilitates redistribution of adrenergic and non-adrenergic non-cholinergic perivascular nerves injured by phenol in rat mesenteric resistance arteries
Author’s Accepted Manuscript Nerve growth factor facilitates redistribution of adrenergic and non-adrenergic non-cholinergic perivascular nerves injured by phenol in rat mesenteric resistance arteries Ayako Yokomizo, Shingo Takatori, Narumi Hashikawa-Hobara, Mitsuhiro Goda, Hiromu Kawasaki
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S0014-2999(15)30403-9 http://dx.doi.org/10.1016/j.ejphar.2015.12.007 EJP70379
To appear in: European Journal of Pharmacology Received date: 6 August 2015 Revised date: 4 December 2015 Accepted date: 4 December 2015 Cite this article as: Ayako Yokomizo, Shingo Takatori, Narumi HashikawaHobara, Mitsuhiro Goda and Hiromu Kawasaki, Nerve growth factor facilitates redistribution of adrenergic and non-adrenergic non-cholinergic perivascular nerves injured by phenol in rat mesenteric resistance arteries, European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2015.12.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Nerve growth factor facilitates redistribution of adrenergic and non-adrenergic non-cholinergic perivascular nerves injured by phenol in rat mesenteric resistance arteries
Ayako Yokomizo a, Shingo Takatori a, b*, Narumi Hashikawa-Hobara c, Mitsuhiro Goda a, and Hiromu Kawasaki a, b
a
Department of Clinical Pharmaceutical Science, Graduate School of
Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima-naka, Okayama 700-8530, Japan. b
Department of Clinical Pharmacy, College of Pharmaceutical Sciences,
Matsuyama University, 4-2 Bunkyo-cho, Matsuyama, Ehime 790-8578, Japan. c
Department of Life Science, Okayama University of Science, 1-1
Ridai-cho, Okayama 700-0005, Japan.
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Corresponding Author Shingo Takatori, Ph.D. Department of Clinical Pharmacy, College of Pharmaceutical Sciences, Matsuyama University, 4-2 Bunkyo-cho, Matsuyama, Ehime 790-8578, Japan. Tel: +81-89-926-7238 Fax: +81-89-926-7162 E-mail: [email protected]
Abstract We previously reported that nerve growth factor (NGF) facilitated perivascular sympathetic neuropeptide Y (NPY)- and calcitonin gene-related peptide (CGRP)-containing nerves injured by the topical application of phenol in the rat mesenteric artery. We also demonstrated that mesenteric arterial nerves were distributed into tyrosine hydroxylase (TH)-, substance P (SP)-, and neuronal nitric oxide synthase (nNOS)-containing nerves, which had axo-axonal interactions. In the
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present study, we examined the effects of NGF on phenol-injured perivascular nerves, including TH-, NPY-, nNOS-, CGRP-, and SP-containing nerves, in rat mesenteric arteries in more detail. Wistar rats underwent the in vivo topical application of 10% phenol to the superior mesenteric artery, proximal to the abdominal aorta, under pentobarbital-Na anesthesia. The distribution of perivascular nerves in the mesenteric arteries of the 2nd to 3rd-order branches isolated from 8-week-old Wistar rats was investigated immunohistochemically using antibodies against TH-, NPY-, nNOS-, CGRP-, and SP-containing nerves. The topical phenol treatment markedly reduced the density of all nerves in these arteries. The administration of NGF at a dose of 20 µg/kg/day with an osmotic pump for 7 days significantly increased the density of all perivascular nerves over that of sham control levels. These results suggest that NGF facilitates the reinnervation of all perivascular nerves injured by phenol in small resistance arteries.
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Key words Nerve growth factor; Neurotrophic effect; Perivascular nerves; Reinnervation; Phenol-induced nerve injury, Rat mesenteric artery
Chemical compounds
Glycerol (PubChem CID: 753); NGF (PubChem CID: 60160600); Paraformaldehyde (PubChem CID: 712); Penicillin G sodium salt (PubChem CID: 54607813); Phenol (PubChem CID: 996); Picric acid (PubChem CID: 6954); Triton X-100 (PubChem CID: 5590).
List of abbreviations
CGRP, calcitonin gene-related peptide; DRG, dorsal root ganglia; LI, like immunoreactive; NANC, non-adrenergic non-cholinergic nerves; NGF, nerve growth factor; nNOS, neuronal nitric oxide synthase; NPY, neuropeptide Y; PBS, phosphate-buffered saline; p75NTR, neurotrophin
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receptor; SP, substance P; TH, tyrosine hydroxylase; TrkA, tropomyosin receptor kinase A.
1. Introduction Perivascular adrenergic nerves innervating resistance arteries play an important role in maintaining vascular tone and regulating organ and tissue blood flow. Resistance vessels, such as mesenteric arteries, are also innervated by non-adrenergic non-cholinergic (NANC) nerves including calcitonin gene-related peptide (CGRP)-containing (CGRPergic) nerves (Kawasaki et al., 1988) and nitric oxide (NO)-containing nerves (Hatanaka et al., 2006). We previously reported that adrenergic and CGRPergic nerves interacted reciprocally in order to regulate vascular tone (Kawasaki et al., 1990). We also demonstrated that adrenergic and NO-containing nerves innervating rat mesenteric arteries were involved in the modulation of adrenergic neurotransmission (Hatanaka et al., 2006). These findings propose that perivascular adrenergic and NANC nerves
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innervating mesenteric arteries play an important role in regulating vascular tone via axo-axonal interactions. CGRP and substance P (SP) as neuropeptides have been shown to colocalize in the same sensory neuron. However, CGRP-like immunoreactive (LI) and SP-LI nerves have been shown to have different distribution patterns and neuromodulatory roles in nociception and peripheral cardiovascular responses in several species (Skofitsch and Jacobowitz, 1985; Kawasaki et al., 1988; Tsuda and Matsuyama, 1991; Henderson et al., 2006). Therefore, the function of SP-LI nerves in mesenteric arteries has not yet been elucidated. Tyrosine hydroxylase (TH) is a rate-limiting enzyme for noradrenaline synthase and is contained in sympathetic adrenergic nerves, which act as vasoconstrictor nerves. TH-LI nerves have been shown to coexist with neuropeptide Y (NPY)-LI nerves in rat mesenteric arteries and skeletal muscle arteries of various kinds of animals, including rabbits, dogs, cats, and guinea pigs (Pernow et al., 1987; Smyth et al., 2000; Gradin et al., 2003).
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We previously reported that the in vivo topical application of phenol, which has been used to block peripheral nerve activity (Wang and Bukoski, 1999), on superior mesenteric arteries markedly reduced the distribution of sympathetic adrenergic NPY-containing nerves and CGRPergic nerves in rat mesenteric resistance arteries. Furthermore, we showed that nerve growth factor (NGF) facilitated the reinnervation of both types of nerves injured by phenol (Hobara et al., 2006). Previous studies found that adrenomedullin (a vasodilator peptide), angiotensin II (a vasoconstrictor peptide), and hepatic growth factor facilitated the reinnervation of mesenteric perivascular nerves injured by topical treatments with phenol (Hobara et al., 2005, 2007a, 2007b, 2008). These findings revealed that facilitatory effects of angiotensin II via the stimulation of angiotensin II type 2 receptors and hepatic growth factor on the reinnervation of mesenteric perivascular nerves were selective to CGRPergic nerves (Hobara et al., 2007b, 2008), thereby implying that the phenol-induced lesion of perivascular nerves is useful for identifying substances with
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neurotrophic effects. However, it currently remains unknown whether NGF facilities the reinnervation of TH-, neuronal nitric oxide synthase (nNOS)-, and SP-containing nerves injured by the application of phenol. Therefore, we herein investigated facilitatory effects of NGF on the reinnervation of phenol-injured perivascular nerves including adrenergic NPY- and TH-containing nerves, as well as CGRPergic nerves, nNOS-, and SP-containing nerves innervating rat mesenteric resistance arteries.
2. Materials and methods 2.1. Experimental animals Eight-week-old Wistar rats were used in this study and purchased from Shimizu Laboratory Supplies Co, Ltd. (Kyoto, Japan). Animals were given food and water ad libitum. They were housed in the Animal Research Center of Okayama University at a controlled ambient temperature of 22°C with 50 ± 10% relative humidity and a 12-h light/12-h dark cycle (lights on at 8:00 AM). This study was carried out to
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minimize the number of animals used and suffering, and was performed in accordance with the Guidelines for Animal Experiments at Okayama University Advanced Science Research Center, Japanese Government Animal Protection and Management Law (No. 115) and Japanese Government Notification on Feeding and Safekeeping of Animals (No. 6).
2.2. In vivo phenol treatment The in vivo phenol treatment used to injure perivascular nerves innervating the mesenteric arteries of the rat was performed according to the method reported by Hobara et al. (2006). Under anesthesia with sodium pentobarbital (50 mg/kg, intraperitoneally), an abdominal midline incision was made in the rat, and the superior mesenteric artery proximal to bifurcation from the abdominal aorta was carefully exposed and topically swabbed with 10% phenol solution (in 90% alcohol-saline) using a cotton bud. After swabbing, an antibiotic (penicillin G; Sigma-Aldrich Japan, Tokyo, Japan) was infused around the surgical area, and the incision was
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then closed. In order to examine the influence of this surgery, sham-operated rats underwent same procedures, but were swabbed with a vehicle (saline or 90% alcohol without including phenol) instead of the phenol solution. After surgery, animals were moved into individual cages in a temperature-controlled room, and received intramuscular injections of penicillin G (3.1 mg/kg) for 3 consecutive days. Seven days after the phenol treatment and sham operation, animals were killed by deep anesthesia for use in experiments described below.
2.3. Administration of NGF NGF was administered intraperitoneally by a continuous infusion through a mini-osmotic pump (model 1007D, Alzet; Alza, Palp Alto, CA, USA) for 7 days. A mini-pump was implanted into the abdominal area immediately after the phenol-swabbing surgery, and NGF was administered at a rate of 20 µg/kg/day according to Hobara et al. (2006). NGF was dissolved in sterile saline and injected into the osmotic
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mini-pump.
2.4. Immunohistochemical study A polyethylene tubing cannula was inserted into the superior mesenteric artery under anesthesia with pentobarbital-Na (50 mg/kg, intraperitoneally), phosphate-buffered saline (PBS) was perfused to remove blood in the vascular bed, and Zamboni solution was then infused. The mesenteric vascular bed was removed together with the intestine as described previously (Hobara et al., 2005, 2006). The 2nd (250-300 µm in diameter) and 3rd branches (200-250 µm in diameter) of the mesenteric artery proximal to the intestine were removed and immersion-fixed in Zamboni solution (2% paraformaldehyde and 15% picric acid in 0.15 M phosphate buffer) for 48 h. After fixation, each artery was repeatedly rinsed in PBS, immersed in PBS containing 0.5% Triton X-100 overnight, and incubated with PBS containing normal goat serum (1: 100) for 60 min. The tissue was then incubated with rabbit polyclonal anti-TH serum (1:
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500) (Chemicon International, Inc., Temecula, CA, USA), rabbit antiNPY serum (1: 300) (Phoenix Pharmaceuticals Inc., Belmont, CA, USA), rabbit polyclonal anti-CGRP serum (Biomol GmbH, Hamburg, Germany) (1: 500), anti-SP serum (1: 200) (Biomol GmbH), or anti-nNOS serum (1: 500) (Zymed Laboratories, South San Francisco, CA, USA) at 4ºC for 72 h. After being incubated, arteries were washed in PBS and sites of the antigen-antibody reaction were revealed by incubation with fluorescein-5-isothiocyanate-labeled goat anti-rabbit IgG (diluted 1: 100) (ICN Pharmaceuticals, Inc., Orange, CA, USA) for 60 min. Thereafter, the artery was thoroughly washed in PBS, mounted on slides, cover-slipped with glycerol/PBS (2: 1 v/v), and observed under a confocal laser scanning microscope (CLSM510, Carl Zeiss GmbH, Jena, Germany) in the Okayama University Medical School Central Research Laboratory.
2.5. Immunohistochemical analysis The immunostaining density of TH- LI, NPY-LI, nNOS-LI, CGRP-LI,
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and SP-LI nerves was analyzed with the method described by Hobara et al. (2005, 2006). Since the fluorescence intensity differed depending on the day of the experiment, the 2nd and 3rd branches of mesenteric arteries were isolated, fixed, and immunostained at the same time on the same day, and were mounted on the same slide glass. Mesenteric arteries isolated from sham-operated rats were used as a control for intensity in each experiment. Confocal projection images of TH, NPY, nNOS, CGRP, and SP immunostaining, which were patched together with 8-10 overlapping images (0.1 µm scanning), were magnified at 20x, digitized as TIF images using a digital camera system (Olympus SP-1000, Olympus, Tokyo, Japan), and imported into a Windows XP computer (Toshiba, Tokyo, Japan) for the quantitative evaluation of TH-LI, NPY-LI, nNOS-LI, CGRP-LI, and SP-LI nerves. The stored digital images were analyzed using image-processing software (Simple PCI; Compix Inc., Imaging Systems, Cranberry Township, PA, USA). The extraction of a specific color and measured field commands were used to extract TH-LI, NPY-LI,
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nNOS-LI, CGRP-LI, and SP-LI areas (which were stained green). Extraction of the signal was carried out using specific protocols based on hue, lightness, and saturation color parameters. A measured field of 100 μm x 100 μm (10,000 μm2, which contained the adventitia layer including immunostained perivascular nerve fibers) was randomly selected on magnified images of the whole mounted artery. The objective areas command was used to calculate the percentage of TH-LI-, NPY-LI-, nNOS-LI-, CGRP-LI-, and SP-LI- positive areas. The intensity of staining was estimated using a point-counting computer program, and the background level was subtracted from the experimental value to yield the corrected intensity. The average density in 3 arteries was taken as the nerve density per animal. In order to determine the number of TH-LI, NPY-LI, nNOS-LI, CGRP-LI, and SP-LI fibers, 5 horizontal lines were drawn on the image of blood vessels in the same region in which the density was estimated by a computer analysis. The number of fibers that crossed each line was then
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counted, and the average number in 3 arteries was taken as the total number of fibers per animal.
2.6. Statistical Analysis All data were expressed as the mean ± S.E.M. An analysis of variance (ANOVA) followed by Tukey’s test was used to determine significance where appropriate. A correlation analysis was carried out using Spearman’s correlation test. P < 0.05 was considered significant.
3. Results 3.1. Distribution of perivascular nerves in the 2nd-3rd branches of mesenteric arteries As shown in Fig. 1, the dense distributions of TH-LI (Fig. 1A), NPY-LI (Fig. 1D), nNOS-LI (Fig. 1G), CGRP-LI (Fig. 1J), and SP-LI (Fig. 1M) nerves in sham control rats were observed outside of the 2nd-3rd branches of mesenteric arteries, and appeared to form a network.
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The densities of TH-LI and CGRP-LI nerves were similar to those of NPY-LI and SP-LI nerves, separately. The densities of adrenergic TH-LI and NPY-LI nerves were greater than those of non-adrenergic nNOS-LI, CGRP-LI, and SP-LI nerves. nNOS-LI nerves exhibited the smallest distribution of density among the immunostained perivascular nerves examined (Fig. 2).
3.2. Effects of NGF on changes in distribution of adrenergic TH-LI and NPY-LI nerves in the mesenteric artery following the topical phenol treatment The topical application of phenol on the superior mesenteric artery markedly reduced the distribution of adrenergic TH-LI (Fig. 1B) and NPY-LI (Fig. 1E) nerves in the 2nd-3rd branches of the distal small artery. The density and number of adrenergic TH-LI (Figs. 3A and 3B) and NPY-LI (Figs. 3C and 3D) nerves were significantly decreased to 40 to 50%, compared to those of sham-treated rats.
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As shown in Figs. 1C and 3, the administration of NGF for 7 days after the topical application of phenol led to the density (Fig. 3A) and number (Fig. 3B) of TH-LI fibers being significantly higher than those in phenol-treated rats. NGF also significantly increased the density (Fig. 3C) and number (Fig. 3D) of NPY-LI nerves (Fig. 1F). Positive correlations were detected between the densities and numbers of TH-LI and NPY-LI nerves in the sham control (TH-LI, P < 0.01, r = 0.9138; NPY-LI, P < 0.01, r = 0.9243), phenol-saline (TH-LI, P < 0.01, r = 0.9583; NPY-LI, P < 0.01, r = 0.9404), and NGF 20 µg/kg/day (TH-LI, P < 0.05, r = 0.9374; NPY-LI, P < 0.05, r = 0.9679).
3.3. Effects of NGF on changes in distribution of nNOS-LI, CGRP-LI, and SP-LI nerves in the mesenteric artery following the topical phenol treatment The densities and numbers of nNOS-LI (Figs. 1H, 4A and 4B), CGRP-LI (Figs. 1K, 4C and 4D), and SP-LI (Figs. 1N, 4E and 4F) were
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significantly lower in phenol-treated rats than in sham control rats. The administration of NGF for 7 days after the phenol treatment led to the densities and numbers of nNOS-LI (Figs. 1I, 4A and 4B), CGRP-LI (Figs. 1L, 4C and 4D) and SP-LI (Figs. 1O, 4E and 4F) nerves being markedly higher than those in phenol-treated rats. Positive correlations were found between the densities and numbers of nNOS-LI, CGRP-LI, and SP-LI nerves in the sham control (nNOS-LI, P < 0.01, r = 0.9489; CGRP-LI, P < 0.01, r = 0.9055; SP-LI, P < 0.05, r = 0.8430), phenol-saline (nNOS-LI, P < 0.01, r = 0.9725; CGRP-LI, P < 0.01, r = 0.9926; SP-LI, P < 0.01, r = 0.8679), and NGF 20 µg/kg/day (nNOS-LI, P < 0.01, r = 0.9693; CGRP-LI, P=0.0639, r = 0.8829; SP-LI, P < 0.05, r = 0.9786). The recovery rate of TH-LI nerves following the treatment with NGF was almost 60%, which was the highest among the five nerves tested (Fig. 5). However, no marked differences were observed in recovery rates among NPY-LI, nNOS-LI, CGRP-LI, and SP-LI nerves.
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4. Discussion The present immunohistochemical study demonstrated that, in addition to NPY-LI and CGRP-LI nerves reported previously (Kawasaki et al., 1988; Hobara et al., 2006), TH-LI, nNOS-LI, and SP-LI nerves were densely distributed outside rat mesenteric arteries and formed a network. TH, a rate-limiting enzyme for noradrenaline synthase, is contained in sympathetic adrenergic nerves, which act as vasoconstrictor nerves. The adrenergic neuropeptide NPY is known to colocalize with noradrenaline (Torres et al., 1992) and induces peripheral vasoconstriction (McDermott and Bell, 2007). Moreover, previous studies reported that TH-LI nerves coexisted with NPY-LI nerves in arteries of various animals including rats, rabbits, dogs, cats, and guinea pigs (Pernow et al., 1987; Smyth et al., 2000; Gradin et al., 2003). Thus, these findings indicated that TH-LI and NPY-LI nerves are sympathetic adrenergic nerves. We previously reported that rat mesenteric arteries were densely
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innervated by NANC vasodilator CGRP-LI nerves, which contribute to the regulation of vascular tone (Kawasaki et al., 1988; Hobara et al., 2006). CGRP and SP as neuropeptides have been shown to colocalize in the same sensory neuron. However, the distribution of CGRP-LI nerves was shown to be different from that of SP-LI nerves, and both nerves could play different neuromodulatory roles in nociception and peripheral cardiovascular responses in several species (Skofitsch and Jacobowitz, 1985; Kawasaki et al., 1988; Tsuda and Matsuyama, 1991; Henderson et al., 2006). Therefore, the function of SP-LI nerves in rat mesenteric arteries has not yet been elucidated in detail. The present immunohistochemical study revealed the dense distribution of nNOS-LI nerves in the rat mesenteric arteries. This result is supported by our previous findings in which nNOS-LI nerves that use NO as a neurotransmitter were distributed in rat mesenteric arteries, and have inhibitory interactions with adrenergic nerves in rat mesenteric arteries (Hatanaka et al., 2006). The present results suggest that the rat mesenteric
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small resistance artery is densely innervated by various perivascular nerves, which contribute to the maintenance of vascular tone. In the present study, the topical application of phenol on the superior mesenteric artery markedly reduced the distribution of adrenergic TH-LI and NPY-LI nerves as well as NANC nNOS-LI, CGRP-LI, and SP-LI nerves in distal arteries. We previously demonstrated that the topical treatment of rat superior mesenteric arteries with phenol significantly decreased densities of perivascular adrenergic NPY-LI and CGRPergic nerve in the distal small artery (Hobara et al., 2006). Hobara et al. (2006) also showed that NGF treatment for 7 days immediately after the application of phenol restored the innervation of perivascular adrenergic and CGRPergic nerves to control levels. The present results are in consistent with these findings. Furthermore, we herein demonstrated that the topical application of phenol significantly reduced the innervation of adrenergic TH-LI nerves and NANC nNOS-LI and SP-LI nerves, similar to those of NPY-LI and CGRP-LI nerves, and that the administration of
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NGF restored significantly-reduced innervations of these perivascular nerves with the treatment of phenol. We previously reported that nicotine (a nicotinic acetylcholine receptor agonist) markedly restored phenol-injured adrenergic TH-LI nerves (Takatori et al, 2015), while hepatic growth factor preferentially facilitated the reinnervation of perivascular CGRP-LI nerves (Hobara et al., 2008). Thus, NGF appears to exert nonselective neurotrophic effects on perivascular nerves. These results indicated that effects of nicotine and hepatic growth factor differed from those of NGF. NGF is produced at target cells of neurons including Schwann cells, fibroblasts, epidermal cells, and smooth muscle cells, and neurons extend their axons toward higher concentrations of NGF (Richner et al., 2014). NGF acts on two receptors, tropomyosin receptor kinase A (TrkA) receptor and neurotrophin receptor (p75NTR) (Huang and Reichardt, 2001). Mearow et al. (1994) reported that the production of NGF and expression of its receptor were increased at the site of injured and/or
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lesioned peripheral axon to restore damaged neurons. Thus, systemic administration of NGF may facilitate the redistribution of perivascular nerves by upregulating the expression of NGF receptors at the site of phenol-injured axons. In DRG sensory neurons, the binding of NGF to p75NTR has been shown to promote in signaling pathways, leading to apoptosis, and NGF-activated TrkA receptors may stimulate cell survival and neurite extension through the RAS/ mitogen-activated protein kinase and phosphoinositide 3-kinase pathways in sensory neurons (Frade and Barde, 1998; Huang and Reichardt, 2001). Webber et al. (2013) found that the activation of TrkA receptor-signaling cascades protected sensory neurons. However, relationship between these findings and present results needs to be elucidated in more detail. In conclusion, present results suggest that NGF facilitates the reinnervation of phenol-injured perivascular nerves in small resistance arteries and normalizes the perivascular innervation.
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Conflict of interests
The authors declare no competing financial interests.
Acknowledgments
A.Y. and H.K. designed and conducted experiments and analyzed data. S.T. and H.K. contributed to discussions and wrote and reviewed the manuscript. N.H-H and M.G. performed animal treatments and provided technical assistance. S.T. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and accuracy of the data analysis.
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Figure Legends Fig. 1. Typical images showing the effect of a nerve growth factor (NGF) treatment for 7 days on the densities of tyrosine hydroxylase (TH)- (A-C), neuropeptide Y (NPY)- (D-F), neuronal nitric oxide synthase (nNOS)(G-I), calcitonin gene-related peptide (CGRP)- (J-L), and substance P (SP)- (M-O) like immunoreactive (LI)-containing nerves in the 2nd-3rd branches of distal mesenteric arteries after a topical phenol treatment. The horizontal bar in each image indicates 100 mm.
Fig. 2. A bar graph showing the density of various immunopositive nerves in the 2nd-3rd branches of distal mesenteric arteries after a topical phenol treatment in sham rats. The ordinate indicates fold changes over NPY values. **P < 0.01 and ++P < 0.01 vs. NPY and TH, respectively. Each bar indicates the mean ± S.E.M. of 4-18 experiments.
Fig. 3. A bar graph showing the effect of the administration of nerve
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growth factor (NGF) for 7 days on densities and numbers of tyrosine hydroxylase (TH; A and B)-like immunoreactive (LI)- and neuropeptide Y (NPY; C and D)-LI nerves in the 2nd-3rd branches of distal mesenteric arteries after a topical phenol treatment. The ordinate indicates fold changes over the Sham value. **P < 0.01 vs. Sham. Each bar indicates the mean ± S.E.M.
Fig. 4. A bar graph showing the effect of the administration of nerve growth factor (NGF) for 7 days on densities and numbers of neuronal nitric oxide synthase (nNOS; A and B)-, calcitonin gene-related peptide (CGRP; C and D)- and substance P (SP; E and F)-like immunoreactive (LI) nerves in the 2nd-3rd branches of distal mesenteric arteries after a topical phenol treatment. The ordinate indicates fold changes over the Sham value. **P < 0.01 vs. Sham. Each bar indicates the mean ± S.E.M.
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Fig. 5. A bar graph showing the recovery rate following the administration of nerve growth factor (NGF) for 7 days of the density of various perivascular nerves in the 2nd-3rd branches of mesenteric arteries after a topical phenol treatment.
Figure 1
Figure 1
Sham
Phenol
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Phenol + NGF
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TH-LI 100 μm
100 μm
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NPY-LI 100 μm
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SP-LI 100 μm
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Figure 2
Fig. 2.
Density (Ratio) 0.0 0
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CGRP
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Figure 3
Fig. 3.
0 (n)
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Figure 4
Fig. 4.
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A
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Figure 5
Figure 5
Recovery rate (%) 0
20
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TH
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PII: DOI: Reference:
www.elsevier.com/locate/ejphar
S0014-2999(15)30403-9 http://dx.doi.org/10.1016/j.ejphar.2015.12.007 EJP70379
To appear in: European Journal of Pharmacology Received date: 6 August 2015 Revised date: 4 December 2015 Accepted date: 4 December 2015 Cite this article as: Ayako Yokomizo, Shingo Takatori, Narumi HashikawaHobara, Mitsuhiro Goda and Hiromu Kawasaki, Nerve growth factor facilitates redistribution of adrenergic and non-adrenergic non-cholinergic perivascular nerves injured by phenol in rat mesenteric resistance arteries, European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2015.12.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Nerve growth factor facilitates redistribution of adrenergic and non-adrenergic non-cholinergic perivascular nerves injured by phenol in rat mesenteric resistance arteries
Ayako Yokomizo a, Shingo Takatori a, b*, Narumi Hashikawa-Hobara c, Mitsuhiro Goda a, and Hiromu Kawasaki a, b
a
Department of Clinical Pharmaceutical Science, Graduate School of
Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima-naka, Okayama 700-8530, Japan. b
Department of Clinical Pharmacy, College of Pharmaceutical Sciences,
Matsuyama University, 4-2 Bunkyo-cho, Matsuyama, Ehime 790-8578, Japan. c
Department of Life Science, Okayama University of Science, 1-1
Ridai-cho, Okayama 700-0005, Japan.
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Corresponding Author Shingo Takatori, Ph.D. Department of Clinical Pharmacy, College of Pharmaceutical Sciences, Matsuyama University, 4-2 Bunkyo-cho, Matsuyama, Ehime 790-8578, Japan. Tel: +81-89-926-7238 Fax: +81-89-926-7162 E-mail: [email protected]
Abstract We previously reported that nerve growth factor (NGF) facilitated perivascular sympathetic neuropeptide Y (NPY)- and calcitonin gene-related peptide (CGRP)-containing nerves injured by the topical application of phenol in the rat mesenteric artery. We also demonstrated that mesenteric arterial nerves were distributed into tyrosine hydroxylase (TH)-, substance P (SP)-, and neuronal nitric oxide synthase (nNOS)-containing nerves, which had axo-axonal interactions. In the
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present study, we examined the effects of NGF on phenol-injured perivascular nerves, including TH-, NPY-, nNOS-, CGRP-, and SP-containing nerves, in rat mesenteric arteries in more detail. Wistar rats underwent the in vivo topical application of 10% phenol to the superior mesenteric artery, proximal to the abdominal aorta, under pentobarbital-Na anesthesia. The distribution of perivascular nerves in the mesenteric arteries of the 2nd to 3rd-order branches isolated from 8-week-old Wistar rats was investigated immunohistochemically using antibodies against TH-, NPY-, nNOS-, CGRP-, and SP-containing nerves. The topical phenol treatment markedly reduced the density of all nerves in these arteries. The administration of NGF at a dose of 20 µg/kg/day with an osmotic pump for 7 days significantly increased the density of all perivascular nerves over that of sham control levels. These results suggest that NGF facilitates the reinnervation of all perivascular nerves injured by phenol in small resistance arteries.
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Key words Nerve growth factor; Neurotrophic effect; Perivascular nerves; Reinnervation; Phenol-induced nerve injury, Rat mesenteric artery
Chemical compounds
Glycerol (PubChem CID: 753); NGF (PubChem CID: 60160600); Paraformaldehyde (PubChem CID: 712); Penicillin G sodium salt (PubChem CID: 54607813); Phenol (PubChem CID: 996); Picric acid (PubChem CID: 6954); Triton X-100 (PubChem CID: 5590).
List of abbreviations
CGRP, calcitonin gene-related peptide; DRG, dorsal root ganglia; LI, like immunoreactive; NANC, non-adrenergic non-cholinergic nerves; NGF, nerve growth factor; nNOS, neuronal nitric oxide synthase; NPY, neuropeptide Y; PBS, phosphate-buffered saline; p75NTR, neurotrophin
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receptor; SP, substance P; TH, tyrosine hydroxylase; TrkA, tropomyosin receptor kinase A.
1. Introduction Perivascular adrenergic nerves innervating resistance arteries play an important role in maintaining vascular tone and regulating organ and tissue blood flow. Resistance vessels, such as mesenteric arteries, are also innervated by non-adrenergic non-cholinergic (NANC) nerves including calcitonin gene-related peptide (CGRP)-containing (CGRPergic) nerves (Kawasaki et al., 1988) and nitric oxide (NO)-containing nerves (Hatanaka et al., 2006). We previously reported that adrenergic and CGRPergic nerves interacted reciprocally in order to regulate vascular tone (Kawasaki et al., 1990). We also demonstrated that adrenergic and NO-containing nerves innervating rat mesenteric arteries were involved in the modulation of adrenergic neurotransmission (Hatanaka et al., 2006). These findings propose that perivascular adrenergic and NANC nerves
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innervating mesenteric arteries play an important role in regulating vascular tone via axo-axonal interactions. CGRP and substance P (SP) as neuropeptides have been shown to colocalize in the same sensory neuron. However, CGRP-like immunoreactive (LI) and SP-LI nerves have been shown to have different distribution patterns and neuromodulatory roles in nociception and peripheral cardiovascular responses in several species (Skofitsch and Jacobowitz, 1985; Kawasaki et al., 1988; Tsuda and Matsuyama, 1991; Henderson et al., 2006). Therefore, the function of SP-LI nerves in mesenteric arteries has not yet been elucidated. Tyrosine hydroxylase (TH) is a rate-limiting enzyme for noradrenaline synthase and is contained in sympathetic adrenergic nerves, which act as vasoconstrictor nerves. TH-LI nerves have been shown to coexist with neuropeptide Y (NPY)-LI nerves in rat mesenteric arteries and skeletal muscle arteries of various kinds of animals, including rabbits, dogs, cats, and guinea pigs (Pernow et al., 1987; Smyth et al., 2000; Gradin et al., 2003).
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We previously reported that the in vivo topical application of phenol, which has been used to block peripheral nerve activity (Wang and Bukoski, 1999), on superior mesenteric arteries markedly reduced the distribution of sympathetic adrenergic NPY-containing nerves and CGRPergic nerves in rat mesenteric resistance arteries. Furthermore, we showed that nerve growth factor (NGF) facilitated the reinnervation of both types of nerves injured by phenol (Hobara et al., 2006). Previous studies found that adrenomedullin (a vasodilator peptide), angiotensin II (a vasoconstrictor peptide), and hepatic growth factor facilitated the reinnervation of mesenteric perivascular nerves injured by topical treatments with phenol (Hobara et al., 2005, 2007a, 2007b, 2008). These findings revealed that facilitatory effects of angiotensin II via the stimulation of angiotensin II type 2 receptors and hepatic growth factor on the reinnervation of mesenteric perivascular nerves were selective to CGRPergic nerves (Hobara et al., 2007b, 2008), thereby implying that the phenol-induced lesion of perivascular nerves is useful for identifying substances with
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neurotrophic effects. However, it currently remains unknown whether NGF facilities the reinnervation of TH-, neuronal nitric oxide synthase (nNOS)-, and SP-containing nerves injured by the application of phenol. Therefore, we herein investigated facilitatory effects of NGF on the reinnervation of phenol-injured perivascular nerves including adrenergic NPY- and TH-containing nerves, as well as CGRPergic nerves, nNOS-, and SP-containing nerves innervating rat mesenteric resistance arteries.
2. Materials and methods 2.1. Experimental animals Eight-week-old Wistar rats were used in this study and purchased from Shimizu Laboratory Supplies Co, Ltd. (Kyoto, Japan). Animals were given food and water ad libitum. They were housed in the Animal Research Center of Okayama University at a controlled ambient temperature of 22°C with 50 ± 10% relative humidity and a 12-h light/12-h dark cycle (lights on at 8:00 AM). This study was carried out to
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minimize the number of animals used and suffering, and was performed in accordance with the Guidelines for Animal Experiments at Okayama University Advanced Science Research Center, Japanese Government Animal Protection and Management Law (No. 115) and Japanese Government Notification on Feeding and Safekeeping of Animals (No. 6).
2.2. In vivo phenol treatment The in vivo phenol treatment used to injure perivascular nerves innervating the mesenteric arteries of the rat was performed according to the method reported by Hobara et al. (2006). Under anesthesia with sodium pentobarbital (50 mg/kg, intraperitoneally), an abdominal midline incision was made in the rat, and the superior mesenteric artery proximal to bifurcation from the abdominal aorta was carefully exposed and topically swabbed with 10% phenol solution (in 90% alcohol-saline) using a cotton bud. After swabbing, an antibiotic (penicillin G; Sigma-Aldrich Japan, Tokyo, Japan) was infused around the surgical area, and the incision was
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then closed. In order to examine the influence of this surgery, sham-operated rats underwent same procedures, but were swabbed with a vehicle (saline or 90% alcohol without including phenol) instead of the phenol solution. After surgery, animals were moved into individual cages in a temperature-controlled room, and received intramuscular injections of penicillin G (3.1 mg/kg) for 3 consecutive days. Seven days after the phenol treatment and sham operation, animals were killed by deep anesthesia for use in experiments described below.
2.3. Administration of NGF NGF was administered intraperitoneally by a continuous infusion through a mini-osmotic pump (model 1007D, Alzet; Alza, Palp Alto, CA, USA) for 7 days. A mini-pump was implanted into the abdominal area immediately after the phenol-swabbing surgery, and NGF was administered at a rate of 20 µg/kg/day according to Hobara et al. (2006). NGF was dissolved in sterile saline and injected into the osmotic
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mini-pump.
2.4. Immunohistochemical study A polyethylene tubing cannula was inserted into the superior mesenteric artery under anesthesia with pentobarbital-Na (50 mg/kg, intraperitoneally), phosphate-buffered saline (PBS) was perfused to remove blood in the vascular bed, and Zamboni solution was then infused. The mesenteric vascular bed was removed together with the intestine as described previously (Hobara et al., 2005, 2006). The 2nd (250-300 µm in diameter) and 3rd branches (200-250 µm in diameter) of the mesenteric artery proximal to the intestine were removed and immersion-fixed in Zamboni solution (2% paraformaldehyde and 15% picric acid in 0.15 M phosphate buffer) for 48 h. After fixation, each artery was repeatedly rinsed in PBS, immersed in PBS containing 0.5% Triton X-100 overnight, and incubated with PBS containing normal goat serum (1: 100) for 60 min. The tissue was then incubated with rabbit polyclonal anti-TH serum (1:
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500) (Chemicon International, Inc., Temecula, CA, USA), rabbit antiNPY serum (1: 300) (Phoenix Pharmaceuticals Inc., Belmont, CA, USA), rabbit polyclonal anti-CGRP serum (Biomol GmbH, Hamburg, Germany) (1: 500), anti-SP serum (1: 200) (Biomol GmbH), or anti-nNOS serum (1: 500) (Zymed Laboratories, South San Francisco, CA, USA) at 4ºC for 72 h. After being incubated, arteries were washed in PBS and sites of the antigen-antibody reaction were revealed by incubation with fluorescein-5-isothiocyanate-labeled goat anti-rabbit IgG (diluted 1: 100) (ICN Pharmaceuticals, Inc., Orange, CA, USA) for 60 min. Thereafter, the artery was thoroughly washed in PBS, mounted on slides, cover-slipped with glycerol/PBS (2: 1 v/v), and observed under a confocal laser scanning microscope (CLSM510, Carl Zeiss GmbH, Jena, Germany) in the Okayama University Medical School Central Research Laboratory.
2.5. Immunohistochemical analysis The immunostaining density of TH- LI, NPY-LI, nNOS-LI, CGRP-LI,
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and SP-LI nerves was analyzed with the method described by Hobara et al. (2005, 2006). Since the fluorescence intensity differed depending on the day of the experiment, the 2nd and 3rd branches of mesenteric arteries were isolated, fixed, and immunostained at the same time on the same day, and were mounted on the same slide glass. Mesenteric arteries isolated from sham-operated rats were used as a control for intensity in each experiment. Confocal projection images of TH, NPY, nNOS, CGRP, and SP immunostaining, which were patched together with 8-10 overlapping images (0.1 µm scanning), were magnified at 20x, digitized as TIF images using a digital camera system (Olympus SP-1000, Olympus, Tokyo, Japan), and imported into a Windows XP computer (Toshiba, Tokyo, Japan) for the quantitative evaluation of TH-LI, NPY-LI, nNOS-LI, CGRP-LI, and SP-LI nerves. The stored digital images were analyzed using image-processing software (Simple PCI; Compix Inc., Imaging Systems, Cranberry Township, PA, USA). The extraction of a specific color and measured field commands were used to extract TH-LI, NPY-LI,
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nNOS-LI, CGRP-LI, and SP-LI areas (which were stained green). Extraction of the signal was carried out using specific protocols based on hue, lightness, and saturation color parameters. A measured field of 100 μm x 100 μm (10,000 μm2, which contained the adventitia layer including immunostained perivascular nerve fibers) was randomly selected on magnified images of the whole mounted artery. The objective areas command was used to calculate the percentage of TH-LI-, NPY-LI-, nNOS-LI-, CGRP-LI-, and SP-LI- positive areas. The intensity of staining was estimated using a point-counting computer program, and the background level was subtracted from the experimental value to yield the corrected intensity. The average density in 3 arteries was taken as the nerve density per animal. In order to determine the number of TH-LI, NPY-LI, nNOS-LI, CGRP-LI, and SP-LI fibers, 5 horizontal lines were drawn on the image of blood vessels in the same region in which the density was estimated by a computer analysis. The number of fibers that crossed each line was then
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counted, and the average number in 3 arteries was taken as the total number of fibers per animal.
2.6. Statistical Analysis All data were expressed as the mean ± S.E.M. An analysis of variance (ANOVA) followed by Tukey’s test was used to determine significance where appropriate. A correlation analysis was carried out using Spearman’s correlation test. P < 0.05 was considered significant.
3. Results 3.1. Distribution of perivascular nerves in the 2nd-3rd branches of mesenteric arteries As shown in Fig. 1, the dense distributions of TH-LI (Fig. 1A), NPY-LI (Fig. 1D), nNOS-LI (Fig. 1G), CGRP-LI (Fig. 1J), and SP-LI (Fig. 1M) nerves in sham control rats were observed outside of the 2nd-3rd branches of mesenteric arteries, and appeared to form a network.
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The densities of TH-LI and CGRP-LI nerves were similar to those of NPY-LI and SP-LI nerves, separately. The densities of adrenergic TH-LI and NPY-LI nerves were greater than those of non-adrenergic nNOS-LI, CGRP-LI, and SP-LI nerves. nNOS-LI nerves exhibited the smallest distribution of density among the immunostained perivascular nerves examined (Fig. 2).
3.2. Effects of NGF on changes in distribution of adrenergic TH-LI and NPY-LI nerves in the mesenteric artery following the topical phenol treatment The topical application of phenol on the superior mesenteric artery markedly reduced the distribution of adrenergic TH-LI (Fig. 1B) and NPY-LI (Fig. 1E) nerves in the 2nd-3rd branches of the distal small artery. The density and number of adrenergic TH-LI (Figs. 3A and 3B) and NPY-LI (Figs. 3C and 3D) nerves were significantly decreased to 40 to 50%, compared to those of sham-treated rats.
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As shown in Figs. 1C and 3, the administration of NGF for 7 days after the topical application of phenol led to the density (Fig. 3A) and number (Fig. 3B) of TH-LI fibers being significantly higher than those in phenol-treated rats. NGF also significantly increased the density (Fig. 3C) and number (Fig. 3D) of NPY-LI nerves (Fig. 1F). Positive correlations were detected between the densities and numbers of TH-LI and NPY-LI nerves in the sham control (TH-LI, P < 0.01, r = 0.9138; NPY-LI, P < 0.01, r = 0.9243), phenol-saline (TH-LI, P < 0.01, r = 0.9583; NPY-LI, P < 0.01, r = 0.9404), and NGF 20 µg/kg/day (TH-LI, P < 0.05, r = 0.9374; NPY-LI, P < 0.05, r = 0.9679).
3.3. Effects of NGF on changes in distribution of nNOS-LI, CGRP-LI, and SP-LI nerves in the mesenteric artery following the topical phenol treatment The densities and numbers of nNOS-LI (Figs. 1H, 4A and 4B), CGRP-LI (Figs. 1K, 4C and 4D), and SP-LI (Figs. 1N, 4E and 4F) were
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significantly lower in phenol-treated rats than in sham control rats. The administration of NGF for 7 days after the phenol treatment led to the densities and numbers of nNOS-LI (Figs. 1I, 4A and 4B), CGRP-LI (Figs. 1L, 4C and 4D) and SP-LI (Figs. 1O, 4E and 4F) nerves being markedly higher than those in phenol-treated rats. Positive correlations were found between the densities and numbers of nNOS-LI, CGRP-LI, and SP-LI nerves in the sham control (nNOS-LI, P < 0.01, r = 0.9489; CGRP-LI, P < 0.01, r = 0.9055; SP-LI, P < 0.05, r = 0.8430), phenol-saline (nNOS-LI, P < 0.01, r = 0.9725; CGRP-LI, P < 0.01, r = 0.9926; SP-LI, P < 0.01, r = 0.8679), and NGF 20 µg/kg/day (nNOS-LI, P < 0.01, r = 0.9693; CGRP-LI, P=0.0639, r = 0.8829; SP-LI, P < 0.05, r = 0.9786). The recovery rate of TH-LI nerves following the treatment with NGF was almost 60%, which was the highest among the five nerves tested (Fig. 5). However, no marked differences were observed in recovery rates among NPY-LI, nNOS-LI, CGRP-LI, and SP-LI nerves.
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4. Discussion The present immunohistochemical study demonstrated that, in addition to NPY-LI and CGRP-LI nerves reported previously (Kawasaki et al., 1988; Hobara et al., 2006), TH-LI, nNOS-LI, and SP-LI nerves were densely distributed outside rat mesenteric arteries and formed a network. TH, a rate-limiting enzyme for noradrenaline synthase, is contained in sympathetic adrenergic nerves, which act as vasoconstrictor nerves. The adrenergic neuropeptide NPY is known to colocalize with noradrenaline (Torres et al., 1992) and induces peripheral vasoconstriction (McDermott and Bell, 2007). Moreover, previous studies reported that TH-LI nerves coexisted with NPY-LI nerves in arteries of various animals including rats, rabbits, dogs, cats, and guinea pigs (Pernow et al., 1987; Smyth et al., 2000; Gradin et al., 2003). Thus, these findings indicated that TH-LI and NPY-LI nerves are sympathetic adrenergic nerves. We previously reported that rat mesenteric arteries were densely
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innervated by NANC vasodilator CGRP-LI nerves, which contribute to the regulation of vascular tone (Kawasaki et al., 1988; Hobara et al., 2006). CGRP and SP as neuropeptides have been shown to colocalize in the same sensory neuron. However, the distribution of CGRP-LI nerves was shown to be different from that of SP-LI nerves, and both nerves could play different neuromodulatory roles in nociception and peripheral cardiovascular responses in several species (Skofitsch and Jacobowitz, 1985; Kawasaki et al., 1988; Tsuda and Matsuyama, 1991; Henderson et al., 2006). Therefore, the function of SP-LI nerves in rat mesenteric arteries has not yet been elucidated in detail. The present immunohistochemical study revealed the dense distribution of nNOS-LI nerves in the rat mesenteric arteries. This result is supported by our previous findings in which nNOS-LI nerves that use NO as a neurotransmitter were distributed in rat mesenteric arteries, and have inhibitory interactions with adrenergic nerves in rat mesenteric arteries (Hatanaka et al., 2006). The present results suggest that the rat mesenteric
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small resistance artery is densely innervated by various perivascular nerves, which contribute to the maintenance of vascular tone. In the present study, the topical application of phenol on the superior mesenteric artery markedly reduced the distribution of adrenergic TH-LI and NPY-LI nerves as well as NANC nNOS-LI, CGRP-LI, and SP-LI nerves in distal arteries. We previously demonstrated that the topical treatment of rat superior mesenteric arteries with phenol significantly decreased densities of perivascular adrenergic NPY-LI and CGRPergic nerve in the distal small artery (Hobara et al., 2006). Hobara et al. (2006) also showed that NGF treatment for 7 days immediately after the application of phenol restored the innervation of perivascular adrenergic and CGRPergic nerves to control levels. The present results are in consistent with these findings. Furthermore, we herein demonstrated that the topical application of phenol significantly reduced the innervation of adrenergic TH-LI nerves and NANC nNOS-LI and SP-LI nerves, similar to those of NPY-LI and CGRP-LI nerves, and that the administration of
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NGF restored significantly-reduced innervations of these perivascular nerves with the treatment of phenol. We previously reported that nicotine (a nicotinic acetylcholine receptor agonist) markedly restored phenol-injured adrenergic TH-LI nerves (Takatori et al, 2015), while hepatic growth factor preferentially facilitated the reinnervation of perivascular CGRP-LI nerves (Hobara et al., 2008). Thus, NGF appears to exert nonselective neurotrophic effects on perivascular nerves. These results indicated that effects of nicotine and hepatic growth factor differed from those of NGF. NGF is produced at target cells of neurons including Schwann cells, fibroblasts, epidermal cells, and smooth muscle cells, and neurons extend their axons toward higher concentrations of NGF (Richner et al., 2014). NGF acts on two receptors, tropomyosin receptor kinase A (TrkA) receptor and neurotrophin receptor (p75NTR) (Huang and Reichardt, 2001). Mearow et al. (1994) reported that the production of NGF and expression of its receptor were increased at the site of injured and/or
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lesioned peripheral axon to restore damaged neurons. Thus, systemic administration of NGF may facilitate the redistribution of perivascular nerves by upregulating the expression of NGF receptors at the site of phenol-injured axons. In DRG sensory neurons, the binding of NGF to p75NTR has been shown to promote in signaling pathways, leading to apoptosis, and NGF-activated TrkA receptors may stimulate cell survival and neurite extension through the RAS/ mitogen-activated protein kinase and phosphoinositide 3-kinase pathways in sensory neurons (Frade and Barde, 1998; Huang and Reichardt, 2001). Webber et al. (2013) found that the activation of TrkA receptor-signaling cascades protected sensory neurons. However, relationship between these findings and present results needs to be elucidated in more detail. In conclusion, present results suggest that NGF facilitates the reinnervation of phenol-injured perivascular nerves in small resistance arteries and normalizes the perivascular innervation.
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Conflict of interests
The authors declare no competing financial interests.
Acknowledgments
A.Y. and H.K. designed and conducted experiments and analyzed data. S.T. and H.K. contributed to discussions and wrote and reviewed the manuscript. N.H-H and M.G. performed animal treatments and provided technical assistance. S.T. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and accuracy of the data analysis.
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Figure Legends Fig. 1. Typical images showing the effect of a nerve growth factor (NGF) treatment for 7 days on the densities of tyrosine hydroxylase (TH)- (A-C), neuropeptide Y (NPY)- (D-F), neuronal nitric oxide synthase (nNOS)(G-I), calcitonin gene-related peptide (CGRP)- (J-L), and substance P (SP)- (M-O) like immunoreactive (LI)-containing nerves in the 2nd-3rd branches of distal mesenteric arteries after a topical phenol treatment. The horizontal bar in each image indicates 100 mm.
Fig. 2. A bar graph showing the density of various immunopositive nerves in the 2nd-3rd branches of distal mesenteric arteries after a topical phenol treatment in sham rats. The ordinate indicates fold changes over NPY values. **P < 0.01 and ++P < 0.01 vs. NPY and TH, respectively. Each bar indicates the mean ± S.E.M. of 4-18 experiments.
Fig. 3. A bar graph showing the effect of the administration of nerve
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growth factor (NGF) for 7 days on densities and numbers of tyrosine hydroxylase (TH; A and B)-like immunoreactive (LI)- and neuropeptide Y (NPY; C and D)-LI nerves in the 2nd-3rd branches of distal mesenteric arteries after a topical phenol treatment. The ordinate indicates fold changes over the Sham value. **P < 0.01 vs. Sham. Each bar indicates the mean ± S.E.M.
Fig. 4. A bar graph showing the effect of the administration of nerve growth factor (NGF) for 7 days on densities and numbers of neuronal nitric oxide synthase (nNOS; A and B)-, calcitonin gene-related peptide (CGRP; C and D)- and substance P (SP; E and F)-like immunoreactive (LI) nerves in the 2nd-3rd branches of distal mesenteric arteries after a topical phenol treatment. The ordinate indicates fold changes over the Sham value. **P < 0.01 vs. Sham. Each bar indicates the mean ± S.E.M.
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Fig. 5. A bar graph showing the recovery rate following the administration of nerve growth factor (NGF) for 7 days of the density of various perivascular nerves in the 2nd-3rd branches of mesenteric arteries after a topical phenol treatment.
Figure 1
Figure 1
Sham
Phenol
A
Phenol + NGF
B
C
TH-LI 100 μm
100 μm
D
E
100 μm
F
NPY-LI 100 μm
G
100 μm
H
100 μm
I
nNOS-LI 100 μm
J
100 μm
100 μm
K
L
100 μm
100 μm
100 μm
M
N
O
CGRP-LI
SP-LI 100 μm
100 μm
100 μm
Figure 2
Fig. 2.
Density (Ratio) 0.0 0
0.2
0.6
1.0
NPY
TH
nNOS
CGRP
P < 0.01
SP
Figure 3
Fig. 3.
0 (n)
0.2
0.6
1.0
1.4
NPY-LI nerves
0.2 0 (n)
0.6
1.0
1.4
TH-LI nerves Density (Ratio) Density (Ratio)
**
**
S
P P+N
(18)
**
P < 0.01
P P+N
(18) (18)
C
S
(10)
P < 0.01
(10) (10)
A Number (Ratio) Number (Ratio)
0
0.2
0.6
1.0
1.4
0.2 0
0.6
1.0
1.4
(n)
S
S
(10)
**
P P+N
(18)
**
P < 0.01
P P+N
(10) (10)
**
P < 0.01
(18) (18)
D
(n)
B
Figure 4
Fig. 4.
nNOS-LI nerves
A
P < 0.01
1.0
** 0.6
0.2 0 (n)
(11) (11)
S
B
1.4
Number (Ratio)
Density (Ratio)
1.4
1.0
** 0.6 0.2 0
(11)
P < 0.01
(n)
P P+N
(11) (11)
S
P
(11)
P+N
CGRP-LI nerves
C P < 0.01
1.0 0.6
**
0.2 0
(n)
(10) (10)
S
D
1.4
Number (Ratio)
Density (Ratio)
1.4
P < 0.01
1.0
**
0.6 0.2 0
(10)
(n)
P P+N
(10) (10)
S
P
(10)
P+N
SP-LI nerves
E P < 0.01
1.0
**
0.6 0.2 0
(n)
(10) (10)
S
F
1.4
(10)
P P+N
Number (Ratio)
Density (Ratio)
1.4
P < 0.01
1.0
**
0.6 0.2 0
(n)
(10) (10)
S
(10)
P P+N
Figure 5
Figure 5
Recovery rate (%) 0
20
40
60
TH
NPY
nNOS
CGRP
SP