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Thyrotropin-releasing hormone in the dorsal vagal complex stimulates hepatic blood flow in rats
Thyrotropin-Releasing Hormone in the Dorsal Vagal Complex Stimulates Hepatic Blood Flow in Rats Masashi Yoneda,1 Takashi Hashimoto,1 Kimihide Nakamura,2 Keisuke Tamori,2 Shiro Yokohama,2 Toru Kono,2 Hajime Watanobe,3 and Akira Terano1 Central administration of thyrotropin-releasing hormone (TRH) enhances hepatic blood flow in animal models. TRH nerve fibers and receptors are localized in the dorsal vagal complex (DVC), and retrograde tracing techniques have shown that hepatic vagal nerves arise mainly from the left DVC. However, nothing is known about the central sites of action for TRH to elicit the stimulation of hepatic blood flow. The effect of microinjection of a TRH analogue into the DVC on hepatic blood flow was investigated in urethane-anesthetized rats. After measuring basal flow, a stable TRH analogue (RX-77368) was microinjected into the DVC and hepatic blood flow response was observed for 120 minutes by laser Doppler flowmetry. Either left or right cervical vagotomy or hepatic branch vagotomy was performed 2 hours before the peptide. Microinjection of RX-77368 (0.5-5 ng) into the left DVC dose-dependently increased hepatic blood flow. The stimulation of hepatic blood flow by RX-77368 microinjection into the left DVC was eliminated by left cervical and hepatic branch vagotomy but not by right cervical vagotomy. By contrast, microinjection of RX77368 into the right DVC did not significantly alter hepatic blood flow. These results suggest that TRH acts in the left DVC to stimulate hepatic blood flow through the left cervical and hepatic vagus, indicating that neuropeptides may act in the specific brain nuclei to regulate hepatic function. (HEPATOLOGY 2003;38:1500-1507.)
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everal lines of anatomic and physiologic evidence suggest the role of the central and autonomic nervous systems in regulating hepatic functions1,2; however, the role of neurotransmitters in the central nervous system to mediate these effects has been poorly understood. In addition to acetylcholine and noradrenaline, neuropeptides have been recognized as novel neurotransmitters in the central and the peripheral nervous system.3 These neuropeptides are reported to act in the brain to induce alterations of physiologic function, including respiratory, cardiovascular, and gastric functions.4-6 In parAbbreviations: TRH, thyrotropin-releasing hormone; DVC, dorsal vagal complex; DMN, dorsal motor nucleus of the vagus; NST, nucleus of the solitary tract. From the 1Department of Gastroenterology, Dokkyo University School of Medicine, Mibu; 2Second Department of Medicine and Surgery, Asahikawa Medical College, Asahikawa; and 3Clinical Research Center, International University of Health and Welfare, Otawara, Japan. Received July 1, 2003; accepted September 8, 2003. Supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (15590681) and the Japan Smoking Health Foundation. Address reprint requests to: Masashi Yoneda, M.D., Department of Gastroenterology, Dokkyo University School of Medicine, Kitakobayashi 880, Mibu, Tochigi 321-0293, Japan. E-mail: [email protected]; fax: (81) 282-86-7761. Copyright © 2003 by the American Association for the Study of Liver Diseases. 0270-9139/03/3806-0023$30.00/0 doi:10.1016/j.hep.2003.09.008 1500
ticular, the effect of central thyrotropin-releasing hormone (TRH) on physiologic, pharmacologic, and pathophysiologic regulation of the gastrointestinal tract has been reported.7 With respect to the stomach, central administration of TRH or a TRH analogue stimulates gastric motility, secretions, and mucosal blood flow through vagal-muscarinic pathways.7-11 We have found that intracisternal injection of a TRH analogue stimulates hepatic blood flow mediated through vagal-muscarinic and nitric oxide– dependent pathways,12 suggesting that hepatic blood flow can be influenced by brain neuropeptides through modulation of the autonomic nervous system. However, nothing is known about the central sites of action for TRH to elicit hepatic blood flow. In the brain, TRH-immunoreactive nerve fibers and terminals and TRH receptors are localized in the dorsal vagal complex (DVC), which includes the dorsal motor nucleus of the vagus (DMN) and the nucleus of the solitary tract (NST),13,14 which are important sites for vagal nerve regulation.15,16 Furthermore, hepatic vagal nerves have been shown to originate mainly from the left DVC by retrograde tracing techniques.16-18 These lines of evidence led us to speculate that TRH acts in the DVC to enhance hepatic blood flow. The present study addressed this
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question by examining the effect of microinjection of TRH into the DVC on hepatic blood flow in rats.
Materials and Methods Animals. Male Wistar rats weighing 250 to 280 g (SLC Co., Shizuoka, Japan) were housed in group cages under conditions of controlled temperature (22-24°C) and illumination (12-hour light cycle starting at 6 AM) for at least 7 days before experiments. Animals were maintained on laboratory chow and tap water. Experiments were performed in rats deprived of food for 24 hours but given free access to water up to the beginning of the study. Protocols describing the use of rats were approved by the animal care committees of Dokkyo University School of Medicine and Asahikawa Medical College and were in accordance with the Ministry of Education, Culture, Sports, Science and Technology of Japan “Guide for the Care and Use of Laboratory Animals.” Chemicals. The following substances were used: a stable TRH analogue, RX-77368, p-Glu-His-(3,3⬘-dimethyl)-Pro-NH2 (Reckitt & Colman, Kingdom-uponHill, England), and urethane (Sigma Chemical Co., St Louis, MO). RX-77368 has similar binding characteristics as authentic TRH on rat brain membranes but is more stable compared with authentic TRH.19,20 RX-77368 was aliquoted in 0.5% bovine serum albumin (Sigma Chemical Co.) and 0.9% saline at a concentration of 1.5 nmol/L and kept frozen at ⫺20°C. The stock solution was diluted in 0.9% saline (pH 7.4) before the experiment. Measurement of Hepatic Blood Flow. All experiments were performed in rats anesthetized with urethane (1.5 g/kg intraperitoneally). Rats underwent a tracheotomy, and PE-260 tubing (Clay Adams, B.D., Parsippany, NJ) was inserted into the trachea to ensure an airway. The right carotid artery was cannulated with PE-50 tubing (Clay Adams, B.D.), and blood pressure was continuously monitored and recorded using a pressure transducer (Uniflow; Baxter, Valencia, CA), a pressure amplifier (PA-001; Star Medical Co., Tokyo, Japan), and a computer (Macintosh G4; Apple Computer, Inc., Cupertino, CA) equipped with a data recording and analysis system (MacLab; AD Instruments Pty Ltd., Castle Hill, Australia). Rats were mounted on ear bars of a stereotaxic apparatus (Kopf model 900; David Kopf Instruments, Tujunga, CA) and positioned to expose the abdomen. A probe (diameter 6 mm, type H; Advance Co. Ltd., Tokyo, Japan) of a laser Doppler flowmeter (ALF 21; Advance Co. Ltd.) was placed on the surface of the lateral left lobe of the liver. The flow signal was averaged with a 3-second time constant and recorded using a computer
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(Macintosh G4) equipped with a data recording and analysis system (MacLab). Hepatic blood flow was expressed relative to the basal level. The hydrogen gas clearance technique was used for quantitative measurement of hepatic blood flow. A platinum needle-type electrode (diameter 0.3 mm, MH-50N; MT Giken, Tokyo, Japan) was inserted into the hepatic left lateral lobe, and an AgAgCl reference electrode (MH-10; MT Giken) was placed into the lower peritoneal cavity against the lateral abdominal wall. The platinum contact electrode and a reference electrode were connected to a polarographic and amplifying unit (model DHM-3001; MT Giken) and a computer equipped with a data recording and analysis system (MacLab). Hepatic blood flow was determined by administering hydrogen gas in air through the tracheotomy tube to achieve tissue saturation. The saturation of hydrogen gas required 7 to 8 minutes. The desaturation curves, after removing the source of hydrogen, were recorded and analyzed by a computer equipped with an analogue-to-digital conversion program (Chart V 3.0; AD Instruments). Absolute values of hepatic blood flow were calculated from the desaturation curve and expressed in milliliters per minute per 100 g. Body temperature was kept at 37°C by external heating, and the hepatic surface was continuously rinsed with 0.9% saline (37°C, pH 7.4) to keep moist. Measurement of Portal Blood Flow and Portal Pressure. To measure portal blood flow, we used a transit-time ultrasonic volume flowmeter (model SFA211; Advance Co., Ltd., Tokyo, Japan) connected to a 2-mm specific perivascular flow probe (type 2S; Advance Co., Ltd.) suitable for the rat portal vein. The probe was placed around the portal vein, and the flow signal was averaged with a 3-second time constant and recorded as described above. The superior mesenteric vein was cannulated with PE-50 tubing, and the tip of the cannula was advanced into the portal trunk. Portal venous pressure was monitored as described above. Microinjection and Experimental Protocol. After the surgical preparation was complete, the animals were left undisturbed for 60 minutes for the stabilization of temperature, heart rate, and arterial blood pressure; in addition, basal hepatic blood flow was observed for 30 minutes. The rat head was then fixed in a nose-down position (incisor bar set at ⫺15 mm). The obex region of the dorsal medulla was exposed by resection of the dorsal cervical musculature and removal of the occipital skull plate and small pieces of the cerebellum. Then a glass micropipette (50-70 m diameter) was positioned unilaterally (right or left side) into the DVC according to the following coordinates: 0.6 mm dorsoventral from surface, 0.3 mm rostrocaudal from obex, and 0.5 mm lateral from
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Fig. 1. Effect of microinjection of the TRH analogue, RX-77368, into the left DVC on hepatic blood flow in urethane-anesthetized rats. After a 30-minute basal observation, 0.9% saline vehicle or RX-77368 (5 ng) was microinjected into the left DVC and changes of hepatic blood flow were monitored for 120 minutes by laser Doppler flowmetry. Each point represents the mean ⫾ SE of change in hepatic blood flow compared with the basal period. *P ⬍ .05, **P ⬍ .01 compared with the respective basal period.
obex. RX-77368 (0.1, 0.3, 0.5, 1, 5, and 10 ng) or 0.9% saline vehicle was delivered in a 50-nL volume by pressure ejection over 1 minute using a 1-L Hamilton syringe. The micropipettes were left in place for 3 minutes and then withdrawn. After microinjection, changes of hepatic blood flow were monitored for 120 minutes thereafter. At the end of the experiments, rats were killed by decapitation. Brains were removed and fixed in 10% formalin and 20% sucrose solution for at least 2 days. Frozen sections were sliced at 30 m, mounted, and stained with toluidine blue. Histologic sections were examined microscopically. The location of microinjection sites was identified by visualization of the tip of the micropipette track and marked on plates reproduced from the atlas of Paxinos and Watson.21 In another experiment, either left or right cervical vagotomy, hepatic branch vagotomy, or sham operation was performed 2 hours before microinjection of the peptide. Statistical Analysis. Results are expressed as means ⫾ SE. Comparison of hepatic blood flow after peptide microinjection with basal blood flow was calculated by ANOVA-repeated measurement. Multiple group comparisons were performed by ANOVA and subsequent Dunnett’s test. A P value less than .05 was considered statistically significant.
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Fig. 2. Effect of microinjection of the TRH analogue, RX-77368, into the right DVC on hepatic blood flow in urethane-anesthetized rats. After a 30-minute basal observation, 0.9% saline vehicle or RX-77368 (5 ng) was microinjected into the right DVC and changes in hepatic blood flow were monitored for 120 minutes by laser Doppler flowmetry. Each point represents the mean ⫾ SE of change in hepatic blood flow compared with the basal period.
by laser Doppler flowmetry (Figs. 1 and 2). Microinjection of RX-77368 at 5 ng into the left DVC increased hepatic blood flow from the first 15-minute observation period after the microinjection by 72% ⫾ 16% and returned to the basal line at 75 minutes (Fig. 1). Microinjection of RX-77368 at the same dose into the right DVC slightly increased hepatic blood flow, but this change did not reach a statistically significant level (Fig. 2). The stimulatory action of the TRH analogue into the left DVC on hepatic blood flow was dose related in doses ranging from 0.5 to 5 ng as assessed by change in hepatic blood flow 15 minutes after the microinjection (Fig. 3). Basal hepatic blood flow measured by the hydrogen gas clearance technique was 56 ⫾ 4.8 mL/min/100 g (n ⫽ 10), and microinjection of RX-77368 (5 ng) increased hepatic blood flow with peak response 15 minutes (by 55% ⫾ 13%)
Results Effect of Microinjection of RX-77368 Into the DVC on Hepatic Blood Flow. Microinjection of 0.9% saline vehicle into either the right or left DVC did not influence hepatic blood flow in urethane-anesthetized rats assessed
Fig. 3. Dose-related stimulating effect of microinjection of the TRH analogue, RX-77368, into the left DVC on hepatic blood flow in urethaneanesthetized rats. For other details, see the legend to Fig. 1. Each column represents the mean ⫾ SE of percent change of hepatic blood flow 15 minutes after microinjection. *P ⬍ .05, **P ⬍ .01 compared with the saline vehicle injection group.
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7). Left cervical vagotomy or hepatic branch vagotomy performed 120 minutes before the peptide completely abolished the hepatic circulatory response to microinjection of the TRH analogue (5 ng) into the left DVC (Figs. 8 and 9). Effect of Microinjection of RX-77368 Into the DVC on Mean Arterial Blood Pressure. Microinjection of RX-77368 at a dose of 5 ng into the left DVC did not alter mean arterial blood pressure. Right or left cervical or hepatic branch vagotomy did not influence basal mean arterial blood pressure (Table 1).
Discussion Fig. 4. Effect of microinjection of the TRH analogue, RX-77368, into the left DVC on hepatic blood flow measured by the hydrogen gas clearance technique in urethane-anesthetized rats. After a 30-minute basal observation period, 0.9% saline vehicle or RX-77368 (5 ng) was microinjected into the left DVC and changes in hepatic blood flow were observed for 120 minutes by the hydrogen gas clearance technique. Each point represents the mean ⫾ SE of change in hepatic blood flow compared with the basal period. *P ⬍ .05, **P ⬍ .01 compared with the respective basal period.
after microinjection (Fig. 4); however, percent increase of maximal hepatic blood flow measured by the hydrogen gas clearance technique was lower than that by laser Doppler flowmetry (by 72% ⫾ 16%). The schematic representation of the localization of medullary sites responsive and unresponsive to RX-77368 (5 and 10 ng) is shown in Fig. 5A. Unresponsive sites after microinjection of the TRH analogue included the right DVC and hypoglossal nucleus. In the left DVC, hepatic circulatory response of the TRH analogue microinjection was not significantly different between the NST and the DMN (percent change of hepatic blood flow at 15 minutes: NST, 75% ⫾ 27% [n ⫽ 7]; DMN, 68% ⫾ 21% [n ⫽ 6]; Fig. 5B). Effect of Microinjection of RX-77368 Into the DVC on Portal Blood Flow and Portal Pressure. Basal portal pressure and portal blood flow was 9.9 ⫾ 0.54 cm H2O (n ⫽ 10) and 14.2 ⫾ 2.4 mL/min (n ⫽ 10), respectively. Although microinjection of 0.9% saline vehicle into the left DVC did not modify either portal blood flow or portal pressure, RX-77368 (5 ng) microinjected into the left DVC increased portal blood flow and decreased portal pressure, respectively (Fig. 6A and B). Effect of Vagotomy on Hepatic Blood Flow in Response to Microinjection of RX-77368 Into the Left DVC. Right cervical vagotomy performed 120 minutes before the peptide microinjection did not modify the stimulation of hepatic blood flow induced by microinjection of the TRH analogue (5 ng) into the left DVC (Fig.
In the present study, microinjection of a stable TRH analogue, RX-77368, into the left DVC resulted in a dose-related stimulation of hepatic blood flow from the
Fig. 5. (A) Diagram of the coronal section of rat medulla showing responsive and unresponsive sites to unilateral microinjection of the TRH analogue, RX-77368 (5 ng). F, change in blood flow more than 40% over basal; E, change in blood flow less than 40% over basal. Plates are adapted from Paxinos and Watson.21 (B) Effect of microinjection of RX-77368 (5 and 10 ng) into the left NST or DMN on hepatic blood flow in urethane-anesthetized rats. N.S., not significantly different.
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Fig. 7. Effect of right cervical vagotomy on hepatic blood flow in response to microinjection of the TRH analogue, RX-77368, into the left DVC in urethane-anesthetized rats. Right cervical vagotomy was performed 120 minutes before microinjection of RX-77368 (5 ng). For other details, see the legend to Fig. 1.
Fig. 6. Effect of microinjection of the TRH analogue, RX-77368, into the left DVC on portal blood flow and portal pressure in urethaneanesthetized rats. After a 30-minute basal observation, 0.9% saline vehicle or RX-77368 (5 ng) was microinjected into the left DVC and changes in hepatic blood flow were observed for 120 minutes by the hydrogen gas clearance technique. Each point represents the mean ⫾ SE of change in hepatic blood flow compared with the basal period. *P ⬍ .05, **P ⬍ .01 compared with the respective basal period.
first 15-minute observation period in urethane-anesthetized rats determined by laser Doppler flowmetry, a device capable of detecting a relative change of regional tissue blood flow.22 Microinjection of RX-77368 (5 ng) into the left DVC significantly increased hepatic blood flow with a peak response at 15 minutes; thereafter, the enhanced hepatic blood flow returned to baseline at 75 minutes. Doses ranging from 0.5 to 10 ng induced a relative increase in hepatic blood flow assessed 15 minutes after the peptide injection. Microinjection up to 10 ng of the TRH analogue did not further enhance hepatic blood flow, indicating that maximal effect was achieved with the 5-ng (13-pmol) dose. These data indicate that the TRH ana-
logue induces a potent and somewhat long-lasting stimulation of hepatic blood flow when microinjected at a dose in the picomolar range into the left DVC. Likewise, we have reported that intracisternal injection of RX-77368 induces a potent stimulation of hepatic blood flow through vagal pathways in rats.12 In the rat, the DVC seems more responsive to the central effect of TRH on hepatic blood flow than the cerebrospinal fluid. Intracisternal injection requires about a 20 times larger dose of the TRH analogue compared with microinjection into the left DVC to induce the same maximal effect on hepatic blood flow under otherwise identical experimental conditions.12 In the present study, 2 different techniques were used for measurement of hepatic blood flow: laser Doppler flowmetry and the hydrogen gas clearance method. Laser Doppler flowmetry allows real-time but not quantitative measurement. By contrast, the hydrogen
Fig. 8. Effect of left cervical vagotomy on hepatic blood flow in response to microinjection of the TRH analogue, RX-77368, into the left DVC in urethane-anesthetized rats. Left cervical vagotomy was performed 120 minutes before microinjection of RX-77368 (5 ng). For other details, see the legend to Fig. 1. **P ⬍ .01 compared with the sham-operated group.
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branch vagotomy suggests that the peptide may primarily or secondarily activate preganglionic neurons contributing to vagal innervation of the liver.15 Retrograde tracing techniques have shown that most of the hepatic vagal preganglionic efferent neurons (more than 90%-99%) are originating from the left DMN, and orthograde tracing has shown a predominant pattern of hepatic vagal afferent terminations within the left NST.16-18,23 Furthermore, interconnections between the NST and the DMN allow cross-talk between the afferent and efferent loops of vagal nerve.24,25 TRH immunoreactive fibers and terminals are densely localized in the DVC, including the DMN and NST. Quantitative evaluation of TRH immunoreactivity by radioimmunoassay has shown that higher content of TRH is found in the DMN compared with that in the NTS.26 TRH receptors are richly distributed in both the DMN and NST.12 Moreover, TRH cell bodies are localized in raphe nuclei, and TRH terminals in the DVC have been shown to arise from cell bodies in the raphe pallidus and obscurus.27 These lines of anatomic evidence suggest that the left DVC is the more likely site of action for TRH to elicit stimulation of hepatic blood flow. In the present study, an attempt to determine the loci within the DVC (DMN and NST) that are stimulated by TRH was performed by indicating the magnitude of the response in relation to the site of injection (Fig. 4B). This further investigation of the responsive sites in the DVC cannot show the specific site in the left DVC because the hepatic circulatory response of the TRH analogue microinjection was not significantly different between the NST and the DMN (change of hepatic blood flow at 15 minutes: NST, 75% ⫾ 27%; DMN,
Fig. 9. Effect of hepatic branch vagotomy on hepatic blood flow in response to microinjection of the TRH analogue, RX-77368, into the left DVC in urethane-anesthetized rats. Hepatic branch vagotomy was performed 120 minutes before microinjection of RX-77368 (5 ng). For other details, see the legend to Fig. 1. *P ⬍ .05, **P ⬍ .01 compared with the sham-operated group.
gas clearance technique can quantitatively measure blood flow but requires about 15 minutes for one measurement and obtained data are an average value of measuring time. For these reasons, real time points for maximal effect on hepatic blood flow are correctly evaluated by laser Doppler flowmetry but not by the hydrogen gas clearance technique. Thus, we used laser Doppler flowmetry for the further investigations. The left DVC represents a specific site of action for TRH to elicit central stimulation of hepatic blood flow, because the peptide was ineffective when delivered to sites close to but outside the left DVC and also delivered to the right DVC. In addition, the complete suppression of the hepatic microcirculatory response to the TRH analogue microinjected into the left DVC by left cervical or hepatic
Table 1. Effect of Microinjection of TRH Analog, RX 77368, Into the Left Dorsal Vagal Complex on Mean Arterial Pressure in Urethane-Anesthetized Rats Mean Arterial Pressure (mm Hg) Time After Intracisternal Injection (min)
No treatment Saline RX 77368 Left cervical vagotomy Saline RX 77368 Right cervical vagotomy Saline RX 77368 Hepatic branch vagotomy Saline RX 77368
0
15
30
45
60
75
90
105
120
103 ⫾ 4 99 ⫾ 6
98 ⫾ 7 102 ⫾ 4
101 ⫾ 6 99 ⫾ 4
101 ⫾ 4 102 ⫾ 5
99 ⫾ 4 99 ⫾ 6
101 ⫾ 5 102 ⫾ 4
99 ⫾ 4 101 ⫾ 4
102 ⫾ 5 99 ⫾ 5
101 ⫾ 3 102 ⫾ 6
101 ⫾ 6 100 ⫾ 5
99 ⫾ 5 99 ⫾ 7
101 ⫾ 5 101 ⫾ 4
102 ⫾ 4 100 ⫾ 5
100 ⫾ 5 99 ⫾ 6
99 ⫾ 7 100 ⫾ 7
100 ⫾ 4 98 ⫾ 5
99 ⫾ 6 100 ⫾ 5
101 ⫾ 7 100 ⫾ 3
98 ⫾ 5 101 ⫾ 4
100 ⫾ 4 99 ⫾ 5
103 ⫾ 6 100 ⫾ 6
101 ⫾ 5 103 ⫾ 6
100 ⫾ 6 101 ⫾ 4
100 ⫾ 5 101 ⫾ 4
99 ⫾ 5 99 ⫾ 5
100 ⫾ 5 101 ⫾ 6
101 ⫾ 7 100 ⫾ 4
100 ⫾ 5 102 ⫾ 4
99 ⫾ 5 100 ⫾ 5
102 ⫾ 6 99 ⫾ 5
103 ⫾ 4 101 ⫾ 6
100 ⫾ 4 101 ⫾ 6
103 ⫾ 5 102 ⫾ 6
100 ⫾ 4 101 ⫾ 5
101 ⫾ 5 99 ⫾ 6
102 ⫾ 6 100 ⫾ 4
NOTE. Values are means ⫾ SE of 5 rats per group. After basal observation, saline vehicle or RX-77368 (5 ng) was microinjected into the left dorsal vagal complex. Systemic blood pressure was monitored thereafter for 120 minutes. Right or left cervical, or hepatic branch vagotomy was performed 120 minutes before the microinjection of RX-77368 (5 ng).
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68% ⫾ 21%). However, because these microinjection studies have a problem with the spread of injection material, careful evaluation of further dissection of an effective site in the DVC is needed. In fact, microinjected peptide in a 50-nL volume, which was introduced in the present study, spreads about 600 m in diameter.28 Doses of the TRH analogue (RX-77368), which was microinjected into the brain sites, were varied in the previous study.28-30 Because it is difficult to say whether the microinjection dose of the TRH analogue in the present study was physiologic or pharmacologic, it is very important to further investigate the role of endogenous TRH in hepatic blood flow using different animal models. Hepatic blood flow is regulated by multiple factors.31 The liver is perfused by 2 main blood supplies: the portal vein and the hepatic artery. The liver sinusoid is also considered to regulate hepatic blood flow. In the present study, mean systemic arterial blood pressure was not modified by microinjection of the TRH analogue into the left DVC at a dose that induced the stimulatory effect of hepatic blood flow. Although injection of TRH or a TRH analogue into the cerebrospinal fluid is shown to elevate systemic blood pressure,6,12 this stimulatory effect on systemic blood pressure is mediated through sympathetic pathways.6 Our previous study has also showed that the intracisternal TRH analogue-stimulated hepatic blood flow was abolished by vagotomy, atropine, indomethacin, and L-NAME, but these treatments had no effect on the intracisternal TRH analogue-induced stimulation of blood pressure.12 Furthermore, stimulation of hepatic blood flow induced by microinjection of the TRH analogue into the left DVC was blocked by hepatic branch vagotomy. Taken together, it is not likely that central TRH-induced stimulation of hepatic blood flow is indirectly through possible modulation of systemic arterial blood pressure. In the present study, portal blood flow and portal pressure were also measured after microinjection of the TRH analogue into the left DVC. As a consequence, microinjection of the TRH analogue decreased portal pressure and increased portal blood flow, indicating that central TRH may reduce portal resistance resulting in an increase of portal blood flow. From these results, a change in portal resistance and portal blood flow is speculated to play an important role in the central TRHinduced hepatic hyperemia. This is consistent with previous studies showing that electrical vagal stimulation and administration of acetylcholine produce dilatation of the sinusoids as assessed by in vivo transillumination technique.32,33 The liver is known to be richly innervated,16,34-36 and there has been abundant evidence indicating important roles of the central and the autonomic nervous system in
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hepatic function.1,37,38 Although some information is available on the involvement of central neuropeptides in the modulation of hepatic function,12,39-45 the information on the site of action for these neuropeptides to elicit the regulation of hepatic function is limited. In the present study, we show that a TRH analogue acts in the specific medullary brain site to induce alteration of hepatic function. In conclusion, TRH acts in the left DVC to stimulate hepatic blood flow through the left cervical and hepatic vagus. These functional observations, added to the presence of TRH-immunoreactive fibers and binding sites at this nucleus, suggest a possible stimulatory role of endogenous medullary TRH in the vagal regulation of hepatic function.
References 1. Lautt WW. Hepatic nerves: a review of their functions and effects. Can J Pharmacol Physiol 1980;58:105-123. 2. Shimazu T, Matsushita H, Ishikawa K. Cholinergic stimulation of the rat hypothalamus: effects of liver glycogen synthesis. Science 1976;194:535536. 3. Tache´ Y. CNS peptides and regulation of gastric acid secretion. Ann Rev Physiol 1988;50:19-39. 4. Brown M. Thyrotropin releasing factor: a putative CNS regulator of the autonomic nervous system. Life Sci 1981;28:1789-1795. 5. Lenz HJ, Druge G. Neurohumoral pathways mediating stress-induced inhibition of gastric acid secretion in rats. Gastroenterology 1990;98: 1490-1492. 6. Siren A, Lake C, Feuerstein G. Hemodynamic and neural mechanisms of action of thyrotropin-releasing hormone. Circ Res 1988;62:139-154. 7. Tache´ Y, Yang H, Yoneda M. Vagal regulation of gastric function involves thyrotropin-releasing hormone in the medullary raphe nuclei and dorsal vagal complex. Digestion 1993;54:65-72. 8. Stephens RL, Ishikawa T, Weiner H, Novin D, Tache´ Y. TRH analogue, RX 77368, injected into dorsal vagal complex stimulates gastric secretion in rats. Am J Physiol 1988;254:G639-G643. 9. Yoneda M, Tache´ Y. Central thyrotoropin-releasing factor analog prevents ethanol-induced gastric damage through prostaglandins in rats. Gastroenterology 1992;102:1568-1574. 10. Tanaka T, Guth P, Tache´ Y. Role of nitric oxide in gastric hyperemia induced by central vagal stimulation. Am J Physiol 1993;264:G280-G283. 11. Thiefin G, Tache´ Y, Leung FW, Guth PH. Central nervous system action of thyrotropin-releasing hormone to increase gastric mucosal blood flow in the rat. Gastroenterology 1989;97:405-411. 12. Tamori K, Yoneda M, Nakamura K, Makino I. Effect of intracisternal thyrotropin-releasing hormone on hepatic blood flow in rats. Am J Physiol 1998;274:G277-G282. 13. Manaker S, Rizio G. Autoradiographic localization of thyrotropin-releasing hormone and substance P receptors in the rat dorsal vagal complex. J Comp Neurol 1989;290:516-526. 14. Rinaman L, Miselis RR. Thyrotropin-releasing hormone-immunoreactive nerve terminals synapse on the dentrites of gastric vagal motoneurons in the rat. J Comp Neurol 1990;294:235-251. 15. Gillis RA, Quest JA, Pagani FD, Norman WP. Central centers in the central nervous system for regulating gastrointestinal motility. In: Wood JD, ed. Handbook of Physiology. Bethesda: American Physiological Society, 1989:621-683. 16. Rogers RC, Hermann GE. Central connections of the hepatic branch of the vagus nerve: a horseradish peroxidase histochemical study. J Auton Nerv Syst 1983;7:165-174.
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17. Fox EA, Powley TL. Longitudinal columnar organization within the dorsal motor nucleus represents separate branches of the abdominal vagus. Brain Res 1985;341:269-282. 18. Kohno T, Mori S, Mito M. Cells of origin innervating the liver and their axonal projections with synaptic terminals into the liver parenchyma in rats. Hokkaido J Med Sci 1987;62:933-946. 19. Griffiths EC, McDermott JR, Smith AI. Mechanisms of brain inactivation of centrally-acting thyrotrophin-releasing hormone (TRH) analogues: a high-performance liquid chromatography study. Regul Pept 1982;5:1-11. 20. Hawkins EF, Engel WK. Analog specificity of the thyrotropin-releasing hormone receptor in the central nervous system: possible clinical implication. Life Sci 1985;36:601-611. 21. Paxinos G, Watson C. The rat brain in stereotaxic coordinates. 2nd ed. Sidney: Academic Press, 1986. 22. Almond NE, Wheatley AM. Measurement of hepatic perfusion in rats by laser Doppler flowmetry. Am J Physiol 1992;262:G203-G209. 23. Norgren R, Smith G. Central distribution of subdiaphragmatic vagal branches in the rat. J Comp Neurol 1988;273:207-223. 24. Rogers RC, Kita H, Butcher LL, Novin D. Afferent projections to the dorsal motor nucleus of the vagus. Brain Res Bull 1980;5:365-373. 25. Rogers RC, McTigue DM, Hermann GE. Vagovagal reflex control of digestion: afferent modulation by neural and “endoneurocrine” factors. Am J Physiol 1995;268:G1-G10. 26. Kubek MJ, Rea MA, Hodes ZI, Aprison MH. Quantitation and characterization of thyrotropin-releasing hormone in vagal nuclei and other regions of the medulla oblongata of the rat. J Neurochem 1983;40:13071313. 27. Palkovits M, Mezey E, Eskay RL, Brownstein MJ. Innervation of the nucleus of the solitary tract and the dorsal vagal nucleus by thyrotropinreleasing hormone-containing raphe neurons. Brain Res 1986;373:246251. 28. Siren A, Vonhof S, Feuerstein G. Hemodynamic defense response to thyrotropin-releasing hormone injected into medial preoptic nucleus in rats. Am J Physiol 1991;261:R305-R312. 29. Ishikawa T, Yang H, Tache´ Y. Medullary sites of action of the TRH analogue, RX 77368, to stimulate gastric acid secretion in the rat. Gastroenterology 1989;95:1470-1476. 30. Yang H, Stephens RL, Tache´ Y. TRH analogue microinjected into specific medullary nuclei stimulates gastric serotonin secretion in rats. Am J Physiol 1992;262:G216-G222.
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31. Lautt W, Greenway C. Conceptual review of the hepatic vascular bed. HEPATOLOGY 1987;5:952-963. 32. Koo A, Liang I. Stimulation and blockade of cholinergic receptors in terminal liver microcirculation in rats. Am J Physiol 1979;236:E728E732. 33. Koo A, Liang I. Microvascular filling pattern in rat liver sinusoids during vagal stimulation. J Physiol (Lond) 1979;295:191-199. 34. Skaaring P, Bierring F. On the intrinsic innervation of normal rat liver. Histochemical and scanning electron microscopical studies. Cell Tissue Res 1976;171:141-155. 35. Hermann GE, Kohlerman NJ, Rogers RC. Hepatic-vagal and gustatory afferent interactions in the brainstem of the rat. J Auton Nerv Syst 1983; 9:477-495. 36. Burt AD, Tiniakos D, MacSween RN, Griffiths MR, Wisse E, Polak JM. Localization of adrenergic and neuropeptide tyrosine-containing nerves in the mammalian liver. HEPATOLOGY 1989;9:839-845. 37. Shimazu T. Neuronal regulation of hepatic glucose metabolism in mammals. Diabetes Metab Rev 1987;3:185-206. 38. Jungermann K, Gardemann A, Beuers U, Balle C, Sannemann J, Beckh K, Hartmann H. Regulation of liver metabolism by the hepatic nerves. Adv Enzyme Regul 1987;26:63-88. 39. Farouk M, Geoghegan JG, Pruthi RS, Thomson HJ, Pappas TN, Meyers WC. Intracerebroventricular neuropeptide Y stimulates bile secretion via a vagal mechanism. Gut 1992;33:1562-1565. 40. Nakade Y, Yoneda M, Nakamura K, Makino I, Terano A. Involvement of endogenous CRF in carbon tetrachloride-induced acute liver injury in rats. Am J Physiol 2002;282:R1782-R1788. 41. Yokohama S, Yoneda M, Nakamura K, Makino I. Effect of central corticotropin-releasing factor on carbon tetrachloride-induced acute liver injury in rats. Am J Physiol 1999;276:G622-G628. 42. Yoneda M, Yokohama S, Tamori K, Sato Y, Nakamura K, Makino I. Neuropeptide Y in the dorsal vagal complex stimulates bicarbonate-dependent bile secretion in rats. Gastroenterology 1997;112:1673-1680. 43. Yoneda M, Tamori K, Sato Y, Yokohama S, Nakamura K, Makino I. Central thyrotropin-releasing hormone stimulates hepatic DNA synthesis in rats. HEPATOLOGY 1997;26:1203-1208. 44. Yoneda M, Nakamura K, Yokohama S, Tamori K, Sato Y, Aso K, Aoshima M, et al. Neuropeptide Y stimulates bile secretion via Y1 receptor in the left dorsal vagal complex in rats. HEPATOLOGY 1998;28:670-676. 45. Yoneda M, Watanobe H, Terano A. Central regulation of hepatic function by neuropeptides. J Gastroenterol 2001;36:361-367.
S
everal lines of anatomic and physiologic evidence suggest the role of the central and autonomic nervous systems in regulating hepatic functions1,2; however, the role of neurotransmitters in the central nervous system to mediate these effects has been poorly understood. In addition to acetylcholine and noradrenaline, neuropeptides have been recognized as novel neurotransmitters in the central and the peripheral nervous system.3 These neuropeptides are reported to act in the brain to induce alterations of physiologic function, including respiratory, cardiovascular, and gastric functions.4-6 In parAbbreviations: TRH, thyrotropin-releasing hormone; DVC, dorsal vagal complex; DMN, dorsal motor nucleus of the vagus; NST, nucleus of the solitary tract. From the 1Department of Gastroenterology, Dokkyo University School of Medicine, Mibu; 2Second Department of Medicine and Surgery, Asahikawa Medical College, Asahikawa; and 3Clinical Research Center, International University of Health and Welfare, Otawara, Japan. Received July 1, 2003; accepted September 8, 2003. Supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (15590681) and the Japan Smoking Health Foundation. Address reprint requests to: Masashi Yoneda, M.D., Department of Gastroenterology, Dokkyo University School of Medicine, Kitakobayashi 880, Mibu, Tochigi 321-0293, Japan. E-mail: [email protected]; fax: (81) 282-86-7761. Copyright © 2003 by the American Association for the Study of Liver Diseases. 0270-9139/03/3806-0023$30.00/0 doi:10.1016/j.hep.2003.09.008 1500
ticular, the effect of central thyrotropin-releasing hormone (TRH) on physiologic, pharmacologic, and pathophysiologic regulation of the gastrointestinal tract has been reported.7 With respect to the stomach, central administration of TRH or a TRH analogue stimulates gastric motility, secretions, and mucosal blood flow through vagal-muscarinic pathways.7-11 We have found that intracisternal injection of a TRH analogue stimulates hepatic blood flow mediated through vagal-muscarinic and nitric oxide– dependent pathways,12 suggesting that hepatic blood flow can be influenced by brain neuropeptides through modulation of the autonomic nervous system. However, nothing is known about the central sites of action for TRH to elicit hepatic blood flow. In the brain, TRH-immunoreactive nerve fibers and terminals and TRH receptors are localized in the dorsal vagal complex (DVC), which includes the dorsal motor nucleus of the vagus (DMN) and the nucleus of the solitary tract (NST),13,14 which are important sites for vagal nerve regulation.15,16 Furthermore, hepatic vagal nerves have been shown to originate mainly from the left DVC by retrograde tracing techniques.16-18 These lines of evidence led us to speculate that TRH acts in the DVC to enhance hepatic blood flow. The present study addressed this
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question by examining the effect of microinjection of TRH into the DVC on hepatic blood flow in rats.
Materials and Methods Animals. Male Wistar rats weighing 250 to 280 g (SLC Co., Shizuoka, Japan) were housed in group cages under conditions of controlled temperature (22-24°C) and illumination (12-hour light cycle starting at 6 AM) for at least 7 days before experiments. Animals were maintained on laboratory chow and tap water. Experiments were performed in rats deprived of food for 24 hours but given free access to water up to the beginning of the study. Protocols describing the use of rats were approved by the animal care committees of Dokkyo University School of Medicine and Asahikawa Medical College and were in accordance with the Ministry of Education, Culture, Sports, Science and Technology of Japan “Guide for the Care and Use of Laboratory Animals.” Chemicals. The following substances were used: a stable TRH analogue, RX-77368, p-Glu-His-(3,3⬘-dimethyl)-Pro-NH2 (Reckitt & Colman, Kingdom-uponHill, England), and urethane (Sigma Chemical Co., St Louis, MO). RX-77368 has similar binding characteristics as authentic TRH on rat brain membranes but is more stable compared with authentic TRH.19,20 RX-77368 was aliquoted in 0.5% bovine serum albumin (Sigma Chemical Co.) and 0.9% saline at a concentration of 1.5 nmol/L and kept frozen at ⫺20°C. The stock solution was diluted in 0.9% saline (pH 7.4) before the experiment. Measurement of Hepatic Blood Flow. All experiments were performed in rats anesthetized with urethane (1.5 g/kg intraperitoneally). Rats underwent a tracheotomy, and PE-260 tubing (Clay Adams, B.D., Parsippany, NJ) was inserted into the trachea to ensure an airway. The right carotid artery was cannulated with PE-50 tubing (Clay Adams, B.D.), and blood pressure was continuously monitored and recorded using a pressure transducer (Uniflow; Baxter, Valencia, CA), a pressure amplifier (PA-001; Star Medical Co., Tokyo, Japan), and a computer (Macintosh G4; Apple Computer, Inc., Cupertino, CA) equipped with a data recording and analysis system (MacLab; AD Instruments Pty Ltd., Castle Hill, Australia). Rats were mounted on ear bars of a stereotaxic apparatus (Kopf model 900; David Kopf Instruments, Tujunga, CA) and positioned to expose the abdomen. A probe (diameter 6 mm, type H; Advance Co. Ltd., Tokyo, Japan) of a laser Doppler flowmeter (ALF 21; Advance Co. Ltd.) was placed on the surface of the lateral left lobe of the liver. The flow signal was averaged with a 3-second time constant and recorded using a computer
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(Macintosh G4) equipped with a data recording and analysis system (MacLab). Hepatic blood flow was expressed relative to the basal level. The hydrogen gas clearance technique was used for quantitative measurement of hepatic blood flow. A platinum needle-type electrode (diameter 0.3 mm, MH-50N; MT Giken, Tokyo, Japan) was inserted into the hepatic left lateral lobe, and an AgAgCl reference electrode (MH-10; MT Giken) was placed into the lower peritoneal cavity against the lateral abdominal wall. The platinum contact electrode and a reference electrode were connected to a polarographic and amplifying unit (model DHM-3001; MT Giken) and a computer equipped with a data recording and analysis system (MacLab). Hepatic blood flow was determined by administering hydrogen gas in air through the tracheotomy tube to achieve tissue saturation. The saturation of hydrogen gas required 7 to 8 minutes. The desaturation curves, after removing the source of hydrogen, were recorded and analyzed by a computer equipped with an analogue-to-digital conversion program (Chart V 3.0; AD Instruments). Absolute values of hepatic blood flow were calculated from the desaturation curve and expressed in milliliters per minute per 100 g. Body temperature was kept at 37°C by external heating, and the hepatic surface was continuously rinsed with 0.9% saline (37°C, pH 7.4) to keep moist. Measurement of Portal Blood Flow and Portal Pressure. To measure portal blood flow, we used a transit-time ultrasonic volume flowmeter (model SFA211; Advance Co., Ltd., Tokyo, Japan) connected to a 2-mm specific perivascular flow probe (type 2S; Advance Co., Ltd.) suitable for the rat portal vein. The probe was placed around the portal vein, and the flow signal was averaged with a 3-second time constant and recorded as described above. The superior mesenteric vein was cannulated with PE-50 tubing, and the tip of the cannula was advanced into the portal trunk. Portal venous pressure was monitored as described above. Microinjection and Experimental Protocol. After the surgical preparation was complete, the animals were left undisturbed for 60 minutes for the stabilization of temperature, heart rate, and arterial blood pressure; in addition, basal hepatic blood flow was observed for 30 minutes. The rat head was then fixed in a nose-down position (incisor bar set at ⫺15 mm). The obex region of the dorsal medulla was exposed by resection of the dorsal cervical musculature and removal of the occipital skull plate and small pieces of the cerebellum. Then a glass micropipette (50-70 m diameter) was positioned unilaterally (right or left side) into the DVC according to the following coordinates: 0.6 mm dorsoventral from surface, 0.3 mm rostrocaudal from obex, and 0.5 mm lateral from
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Fig. 1. Effect of microinjection of the TRH analogue, RX-77368, into the left DVC on hepatic blood flow in urethane-anesthetized rats. After a 30-minute basal observation, 0.9% saline vehicle or RX-77368 (5 ng) was microinjected into the left DVC and changes of hepatic blood flow were monitored for 120 minutes by laser Doppler flowmetry. Each point represents the mean ⫾ SE of change in hepatic blood flow compared with the basal period. *P ⬍ .05, **P ⬍ .01 compared with the respective basal period.
obex. RX-77368 (0.1, 0.3, 0.5, 1, 5, and 10 ng) or 0.9% saline vehicle was delivered in a 50-nL volume by pressure ejection over 1 minute using a 1-L Hamilton syringe. The micropipettes were left in place for 3 minutes and then withdrawn. After microinjection, changes of hepatic blood flow were monitored for 120 minutes thereafter. At the end of the experiments, rats were killed by decapitation. Brains were removed and fixed in 10% formalin and 20% sucrose solution for at least 2 days. Frozen sections were sliced at 30 m, mounted, and stained with toluidine blue. Histologic sections were examined microscopically. The location of microinjection sites was identified by visualization of the tip of the micropipette track and marked on plates reproduced from the atlas of Paxinos and Watson.21 In another experiment, either left or right cervical vagotomy, hepatic branch vagotomy, or sham operation was performed 2 hours before microinjection of the peptide. Statistical Analysis. Results are expressed as means ⫾ SE. Comparison of hepatic blood flow after peptide microinjection with basal blood flow was calculated by ANOVA-repeated measurement. Multiple group comparisons were performed by ANOVA and subsequent Dunnett’s test. A P value less than .05 was considered statistically significant.
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Fig. 2. Effect of microinjection of the TRH analogue, RX-77368, into the right DVC on hepatic blood flow in urethane-anesthetized rats. After a 30-minute basal observation, 0.9% saline vehicle or RX-77368 (5 ng) was microinjected into the right DVC and changes in hepatic blood flow were monitored for 120 minutes by laser Doppler flowmetry. Each point represents the mean ⫾ SE of change in hepatic blood flow compared with the basal period.
by laser Doppler flowmetry (Figs. 1 and 2). Microinjection of RX-77368 at 5 ng into the left DVC increased hepatic blood flow from the first 15-minute observation period after the microinjection by 72% ⫾ 16% and returned to the basal line at 75 minutes (Fig. 1). Microinjection of RX-77368 at the same dose into the right DVC slightly increased hepatic blood flow, but this change did not reach a statistically significant level (Fig. 2). The stimulatory action of the TRH analogue into the left DVC on hepatic blood flow was dose related in doses ranging from 0.5 to 5 ng as assessed by change in hepatic blood flow 15 minutes after the microinjection (Fig. 3). Basal hepatic blood flow measured by the hydrogen gas clearance technique was 56 ⫾ 4.8 mL/min/100 g (n ⫽ 10), and microinjection of RX-77368 (5 ng) increased hepatic blood flow with peak response 15 minutes (by 55% ⫾ 13%)
Results Effect of Microinjection of RX-77368 Into the DVC on Hepatic Blood Flow. Microinjection of 0.9% saline vehicle into either the right or left DVC did not influence hepatic blood flow in urethane-anesthetized rats assessed
Fig. 3. Dose-related stimulating effect of microinjection of the TRH analogue, RX-77368, into the left DVC on hepatic blood flow in urethaneanesthetized rats. For other details, see the legend to Fig. 1. Each column represents the mean ⫾ SE of percent change of hepatic blood flow 15 minutes after microinjection. *P ⬍ .05, **P ⬍ .01 compared with the saline vehicle injection group.
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7). Left cervical vagotomy or hepatic branch vagotomy performed 120 minutes before the peptide completely abolished the hepatic circulatory response to microinjection of the TRH analogue (5 ng) into the left DVC (Figs. 8 and 9). Effect of Microinjection of RX-77368 Into the DVC on Mean Arterial Blood Pressure. Microinjection of RX-77368 at a dose of 5 ng into the left DVC did not alter mean arterial blood pressure. Right or left cervical or hepatic branch vagotomy did not influence basal mean arterial blood pressure (Table 1).
Discussion Fig. 4. Effect of microinjection of the TRH analogue, RX-77368, into the left DVC on hepatic blood flow measured by the hydrogen gas clearance technique in urethane-anesthetized rats. After a 30-minute basal observation period, 0.9% saline vehicle or RX-77368 (5 ng) was microinjected into the left DVC and changes in hepatic blood flow were observed for 120 minutes by the hydrogen gas clearance technique. Each point represents the mean ⫾ SE of change in hepatic blood flow compared with the basal period. *P ⬍ .05, **P ⬍ .01 compared with the respective basal period.
after microinjection (Fig. 4); however, percent increase of maximal hepatic blood flow measured by the hydrogen gas clearance technique was lower than that by laser Doppler flowmetry (by 72% ⫾ 16%). The schematic representation of the localization of medullary sites responsive and unresponsive to RX-77368 (5 and 10 ng) is shown in Fig. 5A. Unresponsive sites after microinjection of the TRH analogue included the right DVC and hypoglossal nucleus. In the left DVC, hepatic circulatory response of the TRH analogue microinjection was not significantly different between the NST and the DMN (percent change of hepatic blood flow at 15 minutes: NST, 75% ⫾ 27% [n ⫽ 7]; DMN, 68% ⫾ 21% [n ⫽ 6]; Fig. 5B). Effect of Microinjection of RX-77368 Into the DVC on Portal Blood Flow and Portal Pressure. Basal portal pressure and portal blood flow was 9.9 ⫾ 0.54 cm H2O (n ⫽ 10) and 14.2 ⫾ 2.4 mL/min (n ⫽ 10), respectively. Although microinjection of 0.9% saline vehicle into the left DVC did not modify either portal blood flow or portal pressure, RX-77368 (5 ng) microinjected into the left DVC increased portal blood flow and decreased portal pressure, respectively (Fig. 6A and B). Effect of Vagotomy on Hepatic Blood Flow in Response to Microinjection of RX-77368 Into the Left DVC. Right cervical vagotomy performed 120 minutes before the peptide microinjection did not modify the stimulation of hepatic blood flow induced by microinjection of the TRH analogue (5 ng) into the left DVC (Fig.
In the present study, microinjection of a stable TRH analogue, RX-77368, into the left DVC resulted in a dose-related stimulation of hepatic blood flow from the
Fig. 5. (A) Diagram of the coronal section of rat medulla showing responsive and unresponsive sites to unilateral microinjection of the TRH analogue, RX-77368 (5 ng). F, change in blood flow more than 40% over basal; E, change in blood flow less than 40% over basal. Plates are adapted from Paxinos and Watson.21 (B) Effect of microinjection of RX-77368 (5 and 10 ng) into the left NST or DMN on hepatic blood flow in urethane-anesthetized rats. N.S., not significantly different.
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Fig. 7. Effect of right cervical vagotomy on hepatic blood flow in response to microinjection of the TRH analogue, RX-77368, into the left DVC in urethane-anesthetized rats. Right cervical vagotomy was performed 120 minutes before microinjection of RX-77368 (5 ng). For other details, see the legend to Fig. 1.
Fig. 6. Effect of microinjection of the TRH analogue, RX-77368, into the left DVC on portal blood flow and portal pressure in urethaneanesthetized rats. After a 30-minute basal observation, 0.9% saline vehicle or RX-77368 (5 ng) was microinjected into the left DVC and changes in hepatic blood flow were observed for 120 minutes by the hydrogen gas clearance technique. Each point represents the mean ⫾ SE of change in hepatic blood flow compared with the basal period. *P ⬍ .05, **P ⬍ .01 compared with the respective basal period.
first 15-minute observation period in urethane-anesthetized rats determined by laser Doppler flowmetry, a device capable of detecting a relative change of regional tissue blood flow.22 Microinjection of RX-77368 (5 ng) into the left DVC significantly increased hepatic blood flow with a peak response at 15 minutes; thereafter, the enhanced hepatic blood flow returned to baseline at 75 minutes. Doses ranging from 0.5 to 10 ng induced a relative increase in hepatic blood flow assessed 15 minutes after the peptide injection. Microinjection up to 10 ng of the TRH analogue did not further enhance hepatic blood flow, indicating that maximal effect was achieved with the 5-ng (13-pmol) dose. These data indicate that the TRH ana-
logue induces a potent and somewhat long-lasting stimulation of hepatic blood flow when microinjected at a dose in the picomolar range into the left DVC. Likewise, we have reported that intracisternal injection of RX-77368 induces a potent stimulation of hepatic blood flow through vagal pathways in rats.12 In the rat, the DVC seems more responsive to the central effect of TRH on hepatic blood flow than the cerebrospinal fluid. Intracisternal injection requires about a 20 times larger dose of the TRH analogue compared with microinjection into the left DVC to induce the same maximal effect on hepatic blood flow under otherwise identical experimental conditions.12 In the present study, 2 different techniques were used for measurement of hepatic blood flow: laser Doppler flowmetry and the hydrogen gas clearance method. Laser Doppler flowmetry allows real-time but not quantitative measurement. By contrast, the hydrogen
Fig. 8. Effect of left cervical vagotomy on hepatic blood flow in response to microinjection of the TRH analogue, RX-77368, into the left DVC in urethane-anesthetized rats. Left cervical vagotomy was performed 120 minutes before microinjection of RX-77368 (5 ng). For other details, see the legend to Fig. 1. **P ⬍ .01 compared with the sham-operated group.
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branch vagotomy suggests that the peptide may primarily or secondarily activate preganglionic neurons contributing to vagal innervation of the liver.15 Retrograde tracing techniques have shown that most of the hepatic vagal preganglionic efferent neurons (more than 90%-99%) are originating from the left DMN, and orthograde tracing has shown a predominant pattern of hepatic vagal afferent terminations within the left NST.16-18,23 Furthermore, interconnections between the NST and the DMN allow cross-talk between the afferent and efferent loops of vagal nerve.24,25 TRH immunoreactive fibers and terminals are densely localized in the DVC, including the DMN and NST. Quantitative evaluation of TRH immunoreactivity by radioimmunoassay has shown that higher content of TRH is found in the DMN compared with that in the NTS.26 TRH receptors are richly distributed in both the DMN and NST.12 Moreover, TRH cell bodies are localized in raphe nuclei, and TRH terminals in the DVC have been shown to arise from cell bodies in the raphe pallidus and obscurus.27 These lines of anatomic evidence suggest that the left DVC is the more likely site of action for TRH to elicit stimulation of hepatic blood flow. In the present study, an attempt to determine the loci within the DVC (DMN and NST) that are stimulated by TRH was performed by indicating the magnitude of the response in relation to the site of injection (Fig. 4B). This further investigation of the responsive sites in the DVC cannot show the specific site in the left DVC because the hepatic circulatory response of the TRH analogue microinjection was not significantly different between the NST and the DMN (change of hepatic blood flow at 15 minutes: NST, 75% ⫾ 27%; DMN,
Fig. 9. Effect of hepatic branch vagotomy on hepatic blood flow in response to microinjection of the TRH analogue, RX-77368, into the left DVC in urethane-anesthetized rats. Hepatic branch vagotomy was performed 120 minutes before microinjection of RX-77368 (5 ng). For other details, see the legend to Fig. 1. *P ⬍ .05, **P ⬍ .01 compared with the sham-operated group.
gas clearance technique can quantitatively measure blood flow but requires about 15 minutes for one measurement and obtained data are an average value of measuring time. For these reasons, real time points for maximal effect on hepatic blood flow are correctly evaluated by laser Doppler flowmetry but not by the hydrogen gas clearance technique. Thus, we used laser Doppler flowmetry for the further investigations. The left DVC represents a specific site of action for TRH to elicit central stimulation of hepatic blood flow, because the peptide was ineffective when delivered to sites close to but outside the left DVC and also delivered to the right DVC. In addition, the complete suppression of the hepatic microcirculatory response to the TRH analogue microinjected into the left DVC by left cervical or hepatic
Table 1. Effect of Microinjection of TRH Analog, RX 77368, Into the Left Dorsal Vagal Complex on Mean Arterial Pressure in Urethane-Anesthetized Rats Mean Arterial Pressure (mm Hg) Time After Intracisternal Injection (min)
No treatment Saline RX 77368 Left cervical vagotomy Saline RX 77368 Right cervical vagotomy Saline RX 77368 Hepatic branch vagotomy Saline RX 77368
0
15
30
45
60
75
90
105
120
103 ⫾ 4 99 ⫾ 6
98 ⫾ 7 102 ⫾ 4
101 ⫾ 6 99 ⫾ 4
101 ⫾ 4 102 ⫾ 5
99 ⫾ 4 99 ⫾ 6
101 ⫾ 5 102 ⫾ 4
99 ⫾ 4 101 ⫾ 4
102 ⫾ 5 99 ⫾ 5
101 ⫾ 3 102 ⫾ 6
101 ⫾ 6 100 ⫾ 5
99 ⫾ 5 99 ⫾ 7
101 ⫾ 5 101 ⫾ 4
102 ⫾ 4 100 ⫾ 5
100 ⫾ 5 99 ⫾ 6
99 ⫾ 7 100 ⫾ 7
100 ⫾ 4 98 ⫾ 5
99 ⫾ 6 100 ⫾ 5
101 ⫾ 7 100 ⫾ 3
98 ⫾ 5 101 ⫾ 4
100 ⫾ 4 99 ⫾ 5
103 ⫾ 6 100 ⫾ 6
101 ⫾ 5 103 ⫾ 6
100 ⫾ 6 101 ⫾ 4
100 ⫾ 5 101 ⫾ 4
99 ⫾ 5 99 ⫾ 5
100 ⫾ 5 101 ⫾ 6
101 ⫾ 7 100 ⫾ 4
100 ⫾ 5 102 ⫾ 4
99 ⫾ 5 100 ⫾ 5
102 ⫾ 6 99 ⫾ 5
103 ⫾ 4 101 ⫾ 6
100 ⫾ 4 101 ⫾ 6
103 ⫾ 5 102 ⫾ 6
100 ⫾ 4 101 ⫾ 5
101 ⫾ 5 99 ⫾ 6
102 ⫾ 6 100 ⫾ 4
NOTE. Values are means ⫾ SE of 5 rats per group. After basal observation, saline vehicle or RX-77368 (5 ng) was microinjected into the left dorsal vagal complex. Systemic blood pressure was monitored thereafter for 120 minutes. Right or left cervical, or hepatic branch vagotomy was performed 120 minutes before the microinjection of RX-77368 (5 ng).
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68% ⫾ 21%). However, because these microinjection studies have a problem with the spread of injection material, careful evaluation of further dissection of an effective site in the DVC is needed. In fact, microinjected peptide in a 50-nL volume, which was introduced in the present study, spreads about 600 m in diameter.28 Doses of the TRH analogue (RX-77368), which was microinjected into the brain sites, were varied in the previous study.28-30 Because it is difficult to say whether the microinjection dose of the TRH analogue in the present study was physiologic or pharmacologic, it is very important to further investigate the role of endogenous TRH in hepatic blood flow using different animal models. Hepatic blood flow is regulated by multiple factors.31 The liver is perfused by 2 main blood supplies: the portal vein and the hepatic artery. The liver sinusoid is also considered to regulate hepatic blood flow. In the present study, mean systemic arterial blood pressure was not modified by microinjection of the TRH analogue into the left DVC at a dose that induced the stimulatory effect of hepatic blood flow. Although injection of TRH or a TRH analogue into the cerebrospinal fluid is shown to elevate systemic blood pressure,6,12 this stimulatory effect on systemic blood pressure is mediated through sympathetic pathways.6 Our previous study has also showed that the intracisternal TRH analogue-stimulated hepatic blood flow was abolished by vagotomy, atropine, indomethacin, and L-NAME, but these treatments had no effect on the intracisternal TRH analogue-induced stimulation of blood pressure.12 Furthermore, stimulation of hepatic blood flow induced by microinjection of the TRH analogue into the left DVC was blocked by hepatic branch vagotomy. Taken together, it is not likely that central TRH-induced stimulation of hepatic blood flow is indirectly through possible modulation of systemic arterial blood pressure. In the present study, portal blood flow and portal pressure were also measured after microinjection of the TRH analogue into the left DVC. As a consequence, microinjection of the TRH analogue decreased portal pressure and increased portal blood flow, indicating that central TRH may reduce portal resistance resulting in an increase of portal blood flow. From these results, a change in portal resistance and portal blood flow is speculated to play an important role in the central TRHinduced hepatic hyperemia. This is consistent with previous studies showing that electrical vagal stimulation and administration of acetylcholine produce dilatation of the sinusoids as assessed by in vivo transillumination technique.32,33 The liver is known to be richly innervated,16,34-36 and there has been abundant evidence indicating important roles of the central and the autonomic nervous system in
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hepatic function.1,37,38 Although some information is available on the involvement of central neuropeptides in the modulation of hepatic function,12,39-45 the information on the site of action for these neuropeptides to elicit the regulation of hepatic function is limited. In the present study, we show that a TRH analogue acts in the specific medullary brain site to induce alteration of hepatic function. In conclusion, TRH acts in the left DVC to stimulate hepatic blood flow through the left cervical and hepatic vagus. These functional observations, added to the presence of TRH-immunoreactive fibers and binding sites at this nucleus, suggest a possible stimulatory role of endogenous medullary TRH in the vagal regulation of hepatic function.
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