PRODUCT MONOGRAPH Pr TRENTAL® (pentoxifylline sustained release tablets, 400 mg Mfr. Std.) Vasoactive agent ATC Code : C04AD03 2150 St. Elzear Blvd. West Laval, Quebec H7L 4A8 Submission Control No.: 142355 s-a 4.0 Version dated March 30, 2011 Page 1 of 21 Table of Contents PART I: HEALTH PROFESSIONAL INFORMATION. 3
Ejn_6201 2466.2472European Journal of Neuroscience, Vol. 27, pp. 2466–2472, 2008 Augmented brain 5-HT crosses the blood–brain barrierthrough the 5-HT transporter in rat Yasushi Nakatani, Ikuko Sato-Suzuki, Naohisa Tsujino, Akane Nakasato, Yoshinari Seki, Masaki Fumotoand Hideho AritaDepartment of Physiology, Toho University School of Medicine, 5-21-16, Omori-nishi, Ota-ku, Tokyo 143–8540, Japan Keywords: blood–brain barrier, microdialysis, serotonin, SSRI The present study re-evaluated an existing notion that serotonin (5-hydroxytryptamine; 5-HT) could not cross the brain to thecirculating blood via the blood–brain barrier (BBB). To elevate brain 5-HT alone, 5-hydroxytryptophan (5-HTP; 30–75 mg ⁄ kg) wasadministrated intravenously to anaesthetized rats that had undergone gastrointestinal and kidney resections along with liverinactivation (organs contributing to increasing blood 5-HT after 5-HTP administration). A microdialysis method and HPLC systemwere used to determine the brain 5-HT levels in samples collected from the frontal cortex. Blood 5-HT levels were determined fromwhole blood, not platelet-poor plasma, collected from the central vein. We found that blood 5-HT levels showed a significantaugmentation whenever brain 5-HT levels were significantly elevated after the administration of 5-HTP in those rats with theabdominal surgical procedures. This elevation was abolished after pretreatment with a selective serotonin reuptake inhibitor(fluoxetine; 10 mg ⁄ kg i.v.), although brain 5-HT levels remained augmented. These results indicate that augmented brain 5-HT cancross the BBB through the 5-HT transporter from the brain to the circulating blood.
It was previously believed that serotonin (5-hydroxytryptamine; brain ECF 5-HT levels in this study. As 5-HTP decarboxylase, which 5-HT) could not cross from the brain to the periphery via the metabolizes 5-HTP to 5-HT, is found not only in the brain but also blood–brain barrier (BBB). However, recent in vitro studies (Brust in the gut, liver and kidneys (West, 1958), a rise in blood 5-HT et al., 2000; Wakayama et al., 2002) have revealed the presence of levels after 5-HTP administration may not be due to the brain alone 5-HT transporter mRNA in vascular endothelial cells, indicating but also to these other organs. Therefore, to exclude this possibility that the BBB may act as an efﬂux transport system for 5-HT. Based we surgically removed the potentially complicating organs. We on this information, we conducted the present study to re-evaluate administered 5-HTP to those rats whose gastrointestinal tracts and the above concept. In other words, we examined the possibility that kidneys had been completely resected and whose livers had been augmented brain 5-HT may cross the BBB through the 5-HT To conﬁrm the role of the BBB 5-HT transporter in the transport of 5-HT neurons, which are located in the raphe nuclei of the 5-HT from the brain to the periphery, we also examined the effect of brainstem, are known to project to broad areas of the whole brain. In selective serotonin reuptake inhibitors (SSRIs) in those rats. Brain addition, 5-HT neuronal activity exhibits a state-dependent change ECF 5-HT and blood 5-HT levels were measured after 5-HTP (Jacobs & Azmitia, 1992). These neurons regularly ﬁre during waking, administration in rats with and without SSRI pretreatment.
although the ﬁring rate is irregular or silenced during sleep. Therefore, However, it has been established that blood 5-HT is mainly it can be anticipated that there is a steady synaptic release of 5-HT that distributed in platelets and to a lesser degree in the plasma (Artigas occurs over a broad area of the brain during waking. In fact, rat brain et al., 1985; Ortiz et al., 1988). If brain ECF 5-HT can be transported 5-HT levels revealed state-dependent alterations, i.e. high levels via the BBB, it would be expected that the augmented 5-HT in the during waking and low levels during sleep (Penalva et al., 2003). In plasma would move quickly into the platelets. Therefore, in this study the present study, we evaluated the change in extracellular ﬂuid (ECF) we evaluated changes in whole blood, not in platelet-poor plasma, to 5-HT levels in the brain using a microdialysis method and a high- evaluate the BBB efﬂux transport system for 5-HT from the brain to performance liquid chromatography (HPLC) system.
the circulating blood.
It has been well established that brain 5-HT is elevated by administration of 5-hydroxytryptophan (5-HTP), i.e. the 5-HTprecursor (Okada et al., 1972; Lo¨scher et al., 1984; Gartside Materials and methods et al., 1992). We injected 5-HTP intravenously to rats to elevate Animal preparation All procedures involving animals were carried out in accordance Correspondence: Dr Hideho Arita, as above.
with the European Communities Council Directive of 24 November 1986 (86 ⁄ 609 ⁄ EEC) and were approved by the Animal Experimen- Received 26 November 2007, revised 28 February 2008, accepted 11 March 2008 tation Ethics Committee of the Toho University School of Medicine.
ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd Blood–brain barrier and 5-HT Experiments were performed on 25 male Wistar rats weighing 310– at a ﬂow rate of 0.50 mL ⁄ min. The column temperature was 375 (327.6 ± 4.2) g. The rats were adapted to the standard 12 : 12 h maintained at 25 C.
light : dark conditions (lights on at 08.00 h) for > 1 week before the After the stabilization period, three consecutive measurements of experiments. Experiments were then performed during the light 5-HT, which were carried out every 10 min, were done to conﬁrm cycle. The rats were anaesthetized with 50 mg ⁄ kg pentobarbital that the microdialysis sampling from the FC was exhibiting a sodium intraperitoneally. The depth of anaesthesia was continuously steady-state baseline. Thereafter, the three different experimental controlled by maintaining the absence of nociceptive ﬂexion and procedures using 5-HTP and ⁄ or SSRI were carried out (described corneal reﬂexes. Supplementary doses (20% of the original dose) were given intravenously when necessary. The anaesthesia wasmaintained until the end of the experiment, when the animal waskilled with an overdose of anaesthetic. All efforts were made to Analysis of whole-blood 5-HT levels minimize the number of animals used.
Blood (0.5 mL) was obtained in a plastic tube and then 0.5 mL of Catheters were placed into the central vein near the right atrium for saline solution was injected back into the animal in order to maintain a collection of blood samples and for drug injection. To monitor vital stable and constant total blood amount. As per the method of Kremer signs, we measured the arterial blood pressure by placing a catheter et al. (1990), 0.5 mL of blood was suspended in 2.2 mL of water.
into the femoral artery. Heart rate was calculated from the blood Then, 300 lL of the internal standard and 10 lL of a 10% (weight per pressure pulse with a tachometer (AT-601G; Nihon Kohden). The volume) solution of ascorbic acid in water was added to the suspended animals were ﬁxed in a prone position in a stereotaxic frame. Rectal blood sample. The sample was then frozen at )20 C and stored until temperature was maintained at 37 C with a heating lamp.
further analysis of 5-HT.
Rats used in this study had undergone total resection of the 5-HT analyses of the blood samples were conducted within gastrointestinal tract and kidneys, as well as liver inactivation. In 1 week after the experiment. Blood samples were thawed, and then these rats, the operative area was shaved and the abdomen opened by 167 lL of methanol was added to 1 mL of blood sample in order a long midline incision with aseptic precautions. Ligatures were to remove the proteins. Blood samples were centrifuged at 4670 g placed round the celiac and the superior mesenteric arteries as near for 10 min at 4 C. Five hundred microlitres of the supernatant of to the aorta as possible. To inactive the liver, the portal vein was the blood sample was suspended in 4.5 mL of the mobile buffer.
ligated along with the bile duct. The whole intestine was freed from Twenty microlitres of the blood sample was then injected into the its ligaments and all vascular attachments were removed. Total HPLC system. Although we used the same HPLC system to resection of the gastroduodenum and the small and large intestines determine blood 5-HT levels, we applied the following different was performed as quickly and carefully as possible to minimize method for blood 5-HT analysis from that for FC 5-HT analysis.
bleeding. After ligation, the kidneys were also removed. The rectum with its vessels was clamped, ligated distally to the clamp, and then CA-5ODS; 2.1 mm diameter · 150 mm; Eicom). The mobile phase cut between the clamp and the point of ligation. Abdominal muscles consisted of a 0.1-m phosphate acid buffer containing 50 mg ⁄ L and skin were then sutured. After the operation, we conﬁrmed that EDTAÆ2Na and 300 mg ⁄ L sodium 1-octanesulphonate (Nacalai the animal was breathing spontaneously and that the blood pressure Tesque, Japan) as the ion-pair and 20% methanol at pH 6.0. The was maintained within the normal range (mean arterial pressure ﬂow rate was set at 0.22 mL ⁄ min and the column temperature was 121.45 ± 4.11 mmHg; n ¼ 20). The rats were ﬁxed in a prone maintained at 35 C.
position in a stereotaxic frame.
Experimental protocols Microdialysis procedures and 5-HT measurements A parietal craniotomy was performed, and the dura was opened toadvance the microdialysis probe (0.22 mm diameter; 2 mm exposed The ﬁrst experiment was performed on ﬁve intact rats that did not membrane; A-I-4–02; Eicom, Japan). Using the atlas of the rat brain undergo the abdominal operation. After the stabilization period of (Paxinos & Watson, 1986) as a guide, a probe was slowly and gently 1–2 h, we determined the steady-state level of brain 5-HT by inserted into the left frontal cortex (FC), an area 0.5 mm lateral to the measuring three consecutive microdialysis perfusate samples from midline, 3.2 mm anterior to the bregma and 2.5 mm vertically below the FC at 10-min intervals. Thereafter, 1 mL of 5-HTP (a dose of the dura. The probe was ﬁxed with dental cement, connected to a 75 mg ⁄ kg in saline solution) was administered through the venous microinfusion pump (EP-50; Eicom) and then perfused with Ringer's catheter of the rat.
solution (NaCl, 147 m Microdialysis sampling from the FC was carried out every 10 min m; KCl, 4 mm; CaCl2, 1.9 mm) at a ﬂow rate of 1 lL ⁄ min.
throughout the entire experiment. Blood sampling was performed four A stabilization period of at least 1 h was allowed following probe times as follows. The ﬁrst blood sample was taken prior to the 5-HTP implantation. After the stabilization period, microdialysis sampling administration. Following the 5-HTP administration three further from the FC was carried out every 10 min. The perfusate from the FC blood samples were drawn at 1-h intervals. Note that same amount of was injected into a HPLC system (DAM-300 system; Eicom) using an saline solution was injected back into the animals in order to maintain automatic injector (AS-10; Eicom) and immediately analysed for a stable and constant total blood amount.
5-HT. A reverse-phase column (Eicompak PP-ODS, 4.6 mm diame-ter · 30 mm; Eicom) was used for 5-HT separation. The working electrode was a graphite electrode set at a detector potential of The second experiment was performed on 10 rats that underwent +0.40 V against an Ag ⁄ AgCl2 reference electrode. The composition of complete resection of their gastrointestinal tracts and kidneys, along the mobile phase was 0.1 m phosphoric acid buffer at pH 6.0, with liver inactivation. One millilitre of 5-HTP (a dose of 75 mg ⁄ kg in containing 1% methanol, 2 mm sodium 1-decanesulphonate (as the saline solution) was administered through a venous catheter in six rats, ion-pair) and 0.13 mm ethylenediaminetetraacetic acid (EDTAÆ2Na) with the same amount of a saline solution injected in four rats that ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing LtdEuropean Journal of Neuroscience, 27, 2466–2472 Y. Nakatani et al.
served as the control animals. Following the injection of 5-HTP or saline solution, samples were obtained from the FC every 10 min for Experiment 1: effect of 5-HTP administration on the frontal cortex (FC) and blood 5-HT levels in intact rats Blood sampling was performed three times in the six rats given 5-HTP. The ﬁrst blood sample was taken prior to the injection of The ﬁrst experiment was performed in intact rats that did not undergo 5-HTP (preinjection sample). The second blood sample was obtained the abdominal operation. Figure 1A shows the statistical data for the when the brain 5-HT level had increased to more than two times time course of the changes in FC 5-HT levels before and after 5-HTP higher than the preinjection level, which occurred at 50–70 min after administration. The mean preinjection 5-HT level in FC was 5-HTP administration. The third blood sample was taken when the 0.39 ± 0.04 pg ⁄ 10 lL (n ¼ 5). When 5-HTP was administered brain 5-HT level further increased, which occurred at 10–30 min after intravenously, there was a gradual increase in FC 5-HT levels.
the second blood sample was drawn.
A one-way anova revealed signiﬁcant changes for the time course of In the control experiment, three consecutive blood samples were the mean FC 5-HT level after 5-HTP administration (F4,19 ¼ 9.29, drawn before the saline solution administration and then every 1 h P < 0.0001). There was a signiﬁcant post hoc difference after 5-HTP after saline administration in the four rats that were not given 5-HTP.
administration between before and 40 min (P < 0.01), 50 min(P < 0.01), 60 min (P < 0.001) and 70 min (P < 0.01) after 5-HTP administration. The maximum FC 5-HT level observed at 60 minafter the 5-HTP injection was 1.45 ± 0.44 pg ⁄ 10 lL (n ¼ 5), which The third experiment was conducted to evaluate the effect of SSRI was approximately three times higher than the mean preinjection (ﬂuoxetine hydrochloride; Sigma, USA). The experiment was per- level. FC 5-HT levels gradually decreased thereafter. The mean FC formed on 10 rats that underwent resection of their gastrointestinal tracts and kidneys along with liver inactivation. Prior to SSRI 0.70 ± 0.12 pg ⁄ 10 lL.
pretreatment, we determined the steady-state level of the brain 5-HTby measuring three consecutive microdialysis perfusate samples fromthe FC every 10 min (pretreatment period). Thereafter, 1 mL of SSRI(a dose of 10 mg ⁄ kg in saline solution) was administered to ratsthrough a venous catheter. One millilitre of 5-HTP (a dose of30 mg ⁄ kg in saline solution) was administered through the venouscatheter at 40 min after the SSRI pretreatment. The dose of 5-HTPgiven was reduced to approximately half of that given in Experi-ments 1 and 2. The reason for the reduction in 5-HTP loading was thatextremely high levels of brain 5-HT were noted in the ﬁrstmicrodialysis perfusate sample from the FC when we administeredthe same 5-HTP dose (75 mg ⁄ kg in saline solution) with SSRIpretreatment in the animals.
Microdialysis sampling from the FC continued every 10 min throughout the experiment, with blood samples drawn three timesduring the study period. The ﬁrst blood sample was obtained duringthe control period, i.e. prior to SSRI pretreatment. The second bloodsample was drawn 30 min after SSRI pretreatment, which was 10 minprior to the 5-HTP administration. The third blood sample was drawnat the time when we observed an apparent increase in the FC 5-HTlevel after the 5-HT administration, which was 30–90 min after 5-HTPadministration.
In the control experiment, instead of SSRI pretreatment animals were given 1 mL of saline solution through the venous catheterafter the control period, which was described above. One millilitreof 5-HTP was given through the venous catheter at 40 min afterthe saline pretreatment. Microdialysis sampling from the FCcontinued every 10 min throughout the experiment. Blood sampleswere drawn three times during the control experiment. The ﬁrstsample was drawn during the control period, the second 30 minafter saline pretreatment and the third 0–90 min after 5-HTPadministration.
Fig. 1. The effects of 5-HTP administration on (A) the brain and (B) the blood Statistical analysis 5-HT levels in intact rats. An arrow indicates the timing of the 5-HTPadministration. (A) Time course showing changes in the mean 5-HT level in A one-way anova was used to analyse the data obtained in the FC before and after 5-HTP administration (75 mg ⁄ kg, i.v.) in the intact rats.
Experiment 1 while a two-way anova for repeated measures was The preinjection level of the FC 5-HT consists of the mean of three successive used for data obtained in Experiments 2 and 3. Signiﬁcant main data points obtained at 10, 20 and 30 min before 5-HTP administration.
(B) Time course showing changes in the mean blood 5-HT level before and effects were further analysed with a Scheffe´ post hoc test. Effects were after 5-HTP administration (75 mg ⁄ kg, i.v) in the intact rats. Data are considered to be statistically signiﬁcant when P-values were < 0.05.
expressed as the mean ± SE (n ¼ 5). ##P < 0.01, ###P < 0.001, ####P < 0.0001 All data are expressed as the mean ± SE.
vs. preinjection values.
ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience, 27, 2466–2472 Blood–brain barrier and 5-HT Figure 1B shows the time course for the changes in blood 5-HT at 60 min after 5-HTP administration. On the other hand, rat 4 levels. The mean blood 5-HT level before 5-HTP administration was expressed a slow and small increase in the FC 5-HT level after 5-HTP 1.95 ± 0.10 lg ⁄ mL. A one-way anova revealed signiﬁcant changes administration, with a FC 5-HT level of 14.1 pg ⁄ 10 lL at 60 min and in the time course for the mean blood 5-HT level after 5-HTP a peak FC 5-HT level of 105.8 pg ⁄ 10 lL at 120 min after 5-HTP administration (F4,3 ¼ 111.5, P < 0.0001). There was a signiﬁcant administration. As the time courses for the changes in FC 5-HT levels post hoc difference between before and 60 min (P < 0.0001), differed among the six rats after 5-HTP administration, we obtained 120 min (P < 0.001) and 180 min (P < 0.001) after 5-HTP admin- the ﬁrst postinjection blood sample at a time when the FC 5-HT level istration. The maximum blood 5-HT level observed at 60 min after the was more than two times higher than the preinjection level. Thus, the 5-HTP injection was 4.40 ± 0.14 lg ⁄ mL, which was approximately ﬁrst postinjection blood samples (open symbol for each response two times higher than the preinjection blood 5-HT level. The blood curve) were drawn between 50 and 70 min after 5-HTP administration 5-HT levels continued to remain high until 180 min after 5-HTP in this experiment. The second postinjection samples were taken when administration, although FC 5-HT levels showed an apparent decrease the FC 5-HT level further increased, which occurred at 10–30 min (see Fig. 1A).
after the ﬁrst postinjection blood samples were drawn.
Figure 2B shows the changes in FC 5-HT levels before and after saline administration in four rats that had total removal of their Experiment 2: effect of 5-HTP administration on FC 5-HT levels gastrointestinal tracts and kidneys, along with liver inactivation. The and blood 5-HT levels in rats that had undergone the abdominal 0.53 ± 0.07 pg ⁄ 10 lL. Little or no change in the FC 5-HT levels The second experiment was performed in 10 rats that had undergone was observed throughout the experiment and up to 120 min after total removal of their gastrointestinal tracts and kidneys, along with saline administration. Thus for these rats, sequential blood samplings liver inactivation. Figure 2 shows the individual time course for the were obtained at 60 and 120 min after saline administration.
changes in the FC 5-HT levels before and after 5-HTP or saline Figure 3 shows the statistical data for the time course changes in the administration. The mean FC 5-HT level during the preinjection blood 5-HT levels after 5-HTP and saline administrations. The mean period was 0.89 ± 0.19 pg ⁄ 10 lL (n ¼ 6). After 5-HTP was admin- blood 5-HT level before 5-HTP administration was 2.14 ± 0.11 istered intravenously, there was a gradual increase observed in the FC lg ⁄ mL (n ¼ 6). There was a gradual increase in the mean blood 5-HT 5-HT levels, although there were marked differences for the time level after 5-HTP administration, whereas there was little change course and magnitude of the changes in the FC 5-HT level among the noted for the mean blood 5-HT level after saline administration.
six rats examined (Fig. 2A). For example, rat 2 exhibited a relatively A two-way anova revealed signiﬁcant changes in the time course for rapid and large increase in the FC 5-HT level after 5-HTP the mean blood 5-HT level after 5-HTP administration. The interaction administration, with a peak FC 5-HT level of 186.3 pg ⁄ 10 lL seen effect on the time course of the mean blood 5-HT level was signiﬁcantfor 5-HTP administration · saline administration (F1,2 ¼ 43.65,P < 0.001). In addition, individual anova revealed a signiﬁcantchange in the time course for the mean blood 5-HT level after 5-HTPadministration (F3,2 ¼ 50.41, P < 0.001), which was not seen aftersaline administration (F3,2 ¼ 0.77, P ¼ 0.50). With regard to themean blood 5-HT level after 5-HTP administration, there was a Fig. 2. The effects of (A) 5-HTP and (B) saline administration on FC 5-HTlevel in rats that had undergone total removal of their gastrointestinal tracts andkidneys, along with liver inactivation. (A) Time courses showing changes inindividual FC 5-HT levels before and after 5-HTP administration in six rats that Fig. 3. Time courses showing changes in the mean blood 5-HT levels before had undergone the abdominal operation. The arrow indicates the timing of and after 5-HTP (s, n ¼ 6) and saline (d, n ¼ 4) administrations in rats that 5-HTP administration (75 mg ⁄ kg, i.v). (B) Time courses showing changes in had undergone total removal of their gastrointestinal tracts and kidneys, along individual FC 5-HT levels before and after saline administration (arrow) in four with liver inactivation. The arrow indicates the time of the 5-HTP or saline rats that had undergone the abdominal operation. Open symbols indicate the administration. Data are expressed as the mean ± SE. ###P < 0.001 vs.
times when blood samples were drawn.
preinjection values. *P < 0.05, **P < 0.01, 5-HTP vs. Saline.
ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing LtdEuropean Journal of Neuroscience, 27, 2466–2472 Y. Nakatani et al.
signiﬁcant post hoc difference observed between the preinjectionsample and the ﬁrst (P < 0.001) and second (P < 0.001) postinjectionsamples. There was also a signiﬁcant post hoc difference between5-HTP administration and saline administration for both the ﬁrst(P < 0.05) and second (P < 0.01) postinjection samples.
Experiment 3: combined effects of SSRI and 5-HTPadministrations on FC 5-HT levels and blood 5-HT levelsin rats that had undergone the abdominal operation The third experiment was also performed in 10 rats that had undergonetotal removal of their gastrointestinal tracts and kidneys, along withliver inactivation. Figure 4A shows individual time courses for thechanges in FC 5-HT levels before and after 5-HTP administration inrats pretreated with SSRI. SSRI administration was performed afterthe control period and at the point where steady-state FC 5-HT levelscould be obtained (Fig. 4B). There was a small nonsigniﬁcant increasein FC 5-HT levels after SSRI administration. The mean FC 5-HT levelat 30 min after SSRI administration was 1.03 ± 0.20 pg ⁄ 10 lL, whilethe 0.46 ± 0.11 pg ⁄ 10 lL. The ﬁrst blood sample was obtained duringthe control period while the second blood sample was drawn at 30 minafter SSRI pretreatment. When 5-HTP (30 mg ⁄ kg in saline solution)was given intravenously to SSRI-pretreated rats, a more rapid increasein FC 5-HT levels was observed when compared to the correspondingdata for rats without SSRI pretreatment (Fig. 2A). The third bloodsample was obtained at 30–60 min after 5-HTP administration.
Figure 4B shows individual time courses for the changes in FC 5-HT levels after 5-HTP administration in rats without SSRIpretreatment. Saline solution was administered in the same manner Fig. 4. The effects of 5-HTP administration on individual FC 5-HT levels in as for the SSRI-pretreated rats. When 5-HTP (30 mg ⁄ kg in saline rats (A) with or (B) without SSRI pretreatment. Note that this experiment was solution) was administered intravenously, we observed differences for performed in rats that had undergone total removal of their gastrointestinal tracts both the time course and the magnitude of the changes of the FC 5-HT and kidneys, along with liver inactivation. (A) Time courses showing changes levels among the ﬁve rats studied, which was similar to our ﬁndings in for the individual FC 5-HT levels before and after 5-HTP administration in ﬁveSSRI pretreated rats. The arrow labelled SSRI indicates the time of pretreatment Experiment 2. For example, rats 6 and 7 showed rapid and large with SSRI (10 mg ⁄ kg i.v.). The arrow labelled 5-HTP indicates the time of increases in the FC 5-HT levels after 5-HTP administration. Thus, in 5-HTP administration (30 mg ⁄ kg, i.v.). Open symbols indicate the time when these cases blood sampling was performed at 40 min after 5-HTP blood samples for 5-HT analysis were drawn. Note that blood samples obtained administration. In the remaining three rats, blood sampling was not during the control period (ﬁrst blood sample) were drawn 20 min before SSRI performed until FC 5-HT levels clearly increased. In rat 10, a blood pretreatment. The second blood sample was obtained at 30 min after SSRIpretreatment, corresponding to 10 min before 5-HTP administration in all ﬁve sample was obtained at 60 min after 5-HTP administration, and in rats rats. The third blood sample was drawn at the point where we observed an 8 and 9 samples were drawn at 90 min after 5-HTP administration.
apparent increase in the FC 5-HT level after 5-HTP administration in each rat.
Figure 5A shows statistical data for changes in the FC 5-HT levels See text for details. (B) Time courses showing changes in the individual FC after 5-HTP administration in rats with and without SSRI pretreat- 5-HT levels before and after 5-HTP administration in the ﬁve rats that did notreceive any SSRI pretreatment (saline administration). The arrows labelled with ment. The mean FC 5-HT level at the 5-HTP postinjection (Fig. 5A) saline or 5-HTP indicate the times when saline or 5-HTP were administered.
represents the FC 5-HT level obtained from the last microdialysis Open symbols indicate the times when blood samples for 5-HT analysis were sampling point after 5-HTP administration. The mean FC 5-HT levels obtained. The times when the ﬁrst, second and third blood samples were drawn after 5-HTP administration in rats with and without SSRI pretreatment are the same as those described in A.
were 45.40 ± 12.75 pg ⁄ 10 lL (n ¼ 5) and 42.35 ± 12.55 pg ⁄ 10 lL(n ¼ 5), respectively. One-way anova revealed signiﬁcant changes in administration, which was in contrast to the very small change noted the mean FC 5-HT levels after 5-HTP administration in both the rats after the saline pretreatment (saline pretreatment, Fig. 5B). Two-way with SSRI pretreatment (F4,2 ¼ 12.34, P < 0.01) and those without anova revealed signiﬁcant changes in the time course after 5-HTP SSRI pretreatment (F4,2 ¼ 10.93, P < 0.01).
administration for both SSRI and saline pretreatment. As illustrated in Figure 5B shows the statistical data for the time courses of the Fig. 5B, the interaction effect for (SSRI + 5-HTP) administra- changes in blood 5-HT levels after 5-HTP administration in rats with tion · (saline + 5-HTP) administration was found to be signiﬁcant and without SSRI pretreatment. The mean blood 5-HT level before (F1,2 ¼ 55.12, P < 0.01). Individual anova revealed a signiﬁcant SSRI pretreatment (control, Fig. 5B) was 2.02 ± 0.07 lg ⁄ mL change in the time courses of the mean blood 5-HT level after (n ¼ 5). There was a small decrease in the mean blood 5-HT level (SSRI + 5-HTP) administration (F4,2 ¼ 8.34, P < 0.05) and (sal- after SSRI pretreatment (SSRI pretreatment; Fig. 5B), whereas there ine + 5-HTP) administration (F4,2 ¼ 69.38, P < 0.0001). With regard was little change observed for the mean blood 5-HT level after 5-HTP to the mean blood 5-HT level after SSRI or saline pretreatment, there administration (5-HTP postinjection; Fig. 5B). On the other hand, the was a signiﬁcant post hoc difference noted between the control and the mean blood 5-HT level after 5-HTP administration in rats without SSRI ⁄ saline pretreatments (P < 0.05) only in the SSRI-pretreated SSRI pretreatment (Fig. 5B) showed a marked increase after 5-HTP rats. As for the mean blood 5-HT level after 5-HTP administration, ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience, 27, 2466–2472 Blood–brain barrier and 5-HT that all neurotransmitters are retained in the brain. However, recentin vitro studies (Brust et al., 2000; Wakayama et al., 2002) haverevealed the presence of 5-HT transporter mRNA in vascularendothelial cells. This indicates that the BBB may act as an efﬂuxtransport system for 5-HT. Based on this new data, we conducted thepresent in vivo functional study. As a result, we found that indeed5-HT can cross from the brain into the circulating blood via the BBB.
The second reason that is cited as to why 5-HT cannot cross the BBB is related to the 5-HT content that is found within the organs. Ithas been reported that > 90% of the total 5-HT content in the wholebody is distributed within the gastrointestinal tract (West, 1958;Gaginella, 1995), and that only a very small percentage of the total5-HT content is found within the brain. Therefore, it has been believedthat the augmented ECF 5-HT in the brain does not contribute to anysigniﬁcant changes in the 5-HT levels within the circulating blood.
Therefore, we administered 5-HTP intravenously in rats that hadundergone the abdominal operation in an attempt to elevate brain5-HT alone. As a result, we found that whole-blood 5-HT levelssigniﬁcantly increased whenever brain ECF 5-HT levels were elevatedby the 5-HTP administration in the rats undergoing the abdominaloperation. Therefore, it is reasonable to hypothesize that theaugmented brain ECF 5-HT does contribute to a signiﬁcant changein 5-HT levels within the circulating blood. In other words, augmentedbrain ECF 5-HT can translocate from the brain into the blood via theBBB.
This hypothesis may be further supported by the data concerning regional differences of the 5-HTP decarboxylase, which is the enzymeresponsible for metabolizing 5-HTP to 5-HT. A study by West (1958)demonstrated that there was high 5-HTP decarboxylase activity in thekidneys (188 lg ⁄ tissue) and the liver (125 lg ⁄ tissue), with only avery low activity noted in the gastrointestinal tract (1–2 lg ⁄ tissue).
The brain exhibited moderate activity for 5-HTP decarboxylase(32 lg ⁄ tissue). In addition, 5-HT that is produced by the gastrointes- Fig. 5. The effects of 5-HTP administration on (A) FC 5-HT levels and tinal tract is thought to be metabolized by monoamine oxidase in the (B) blood 5-HT levels in rats with SSRI or saline pretreatment. All rats liver, as it circulates through the portal vein (Gillis, 1985). Therefore, underwent a surgical procedure to totally remove their gastrointestinal tractsand kidneys, along with liver inactivation. Arrows labelled SSRI or saline it is unlikely that the gastrointestinal tract would make any signiﬁcant indicate the times of the SSRI (10 mg ⁄ kg i.v) or saline pretreatments. Arrows contribution towards increasing blood 5-HT when 5-HTP is admin- labelled 5-HTP indicate the times of the 5-HTP (30 mg ⁄ kg i.v) administrations.
istered to the intact rat.
s, Data obtained in rats with SSRI pretreatment (n ¼ 5); d, data points from With regard to intrarenal formation of 5-HT by renal decarboxylase, rats without SSRI pretreatment (saline administration, n ¼ 5). Data areexpressed as the mean ± SE. #P < 0.05, ##P < 0.01, ####P < 0.0001 vs.
Stier & Itskovitz, 1985) demonstrated that administration of 5-HTP control. **P < 0.01, SSRI + 5-HTP vs. Saline + 5-HTP.
resulted in an increase in urinary 5-HT without a concomitant increasein plasma 5-HT in the rat. Based on this result, it is reasonable to there was a signiﬁcant post hoc difference observed between the speculate that intrarenal formation of 5-HT may not contribute to an control and the 5-HTP postinjection (P < 0.0001) in the rats that were increase in 5-HT in whole blood after 5-HTP administration.
not pretreated with SSRI.
Based on data by West (1958), the whole organs that exhibit 5-HTP There was also a signiﬁcant post hoc difference noted in the blood decarboxylase activity include the kidneys, the liver, the gastrointes- 5-HT level for the 5-HTP postinjection between the SSRI- and saline- tinal tract, the brain and the skin. After 5-HTP administration in rats pretreated rats (P < 0.01).
that underwent resection of their gastrointestinal tracts and kidneysalong with liver inactivation, the skin in addition to the brain were thetwo organs found to be capable of augmenting the whole-blood 5-HT levels. However, skin has been demonstrated to show the lowest The present study revealed that whole-blood 5-HT levels exhibited amount of enzyme activity (1 lg ⁄ tissue). Thus, it is less likely that signiﬁcant augmentation when brain 5-HT levels were elevated after 5-HT in the skin contributes to any signiﬁcant augmentation of 5-HT 5-HTP administration in rats that had undergone total removal of their in the whole blood after 5-HTP administration, even though we can gastrointestinal tracts and kidneys, along with liver inactivation.
not completely rule out this possibility.
This result implies that 5-HT may cross the brain into the circulating The other new ﬁnding of the present study is that SSRI pretreatment blood via the BBB. However, there are two major reasons that may be in rats lacking functional kidneys, gastrointestinal tract and liver cited as evidence as to why 5-HT cannot possibly cross from the brain abolished elevation of whole-blood 5-HT levels that was induced by into the circulating blood via the BBB.
5-HTP administration, even though the brain 5-HT levels remained First, the BBB is formed by tight junctions of the brain capillary increased. These results suggest that the 5-HT transporters that are endothelial cells. The role of these junctions is to prevent neurotrans- located on the brain endothelial cells play the inevitable role mitters including 5-HT from crossing the junction and thus ensuring associated with crossing from the brain via the BBB into the ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing LtdEuropean Journal of Neuroscience, 27, 2466–2472 Y. Nakatani et al.
circulating blood. As discussed earlier, the in vitro study by Brust Brust, P., Friedrich, A., Krizbai, I.A., Bergmann, R., Roux, F., Ganapathy, V. & et al. (2000) revealed the presence of a 5-HT transporter mRNA in the Johannsen, B. (2000) Functional expression of the serotonin transporter inimmortalized rat brain microvessel endothelial cells. J. Neurochem., 74, brain endothelium, indicating that cerebral endothelial cells are able to actively participate in the removal of the released 5-HT within the Fuxe, K. & Agnati, L.F. (1991) Two principal modes of electrochemical brain. This possibility was functionally proven in the present in vivo communication in the brain: volume versus wiring transmission. In Fuxe, K.
physiological study that used the SSRI. Thus, we hypothesized that & Agnati, L.F. (eds), Volume Transmission in the Brain: Novel Mechanisms the 5-HT transporters located on the brain endothelial cells may act as for Neural Transmission. Raven Press, New York, pp. 1–9.
Fuxe, K., Dahlstro¨m, A., Ho¨istad, M., Marcellino, D., Jansson, A., Rivera, A., the efﬂux transport system for the 5-HT that crosses from the brain Diaz-Cabiale, Z., Jacobsen, K., Tinner-Staines, B., Hagman, B., Leo, G., into the circulating blood.
Staines, W., Guidolin, D., Kehr, J., Genedani, S., Belluardo, N. & Agnati, What physiological role does the 5-HT transporter located on the L.F. (2007) From the Golgi–Cajal mapping to the transmitter-based brain endothelium actually play? It has been established that the 5-HT characterization of the neuronal networks leading to two modes of braincommunication: Wiring and volume transmission. Brain Res. Rev., 55, 17– transporter is present not only in the synaptic terminals of 5-HT neurons but also in the brain endothelial cells (Brust et al., 2000; Gaginella, T.S. (1995) Serotonin in the intestinal tract: a synopsis. In Gaginella, Wakayama et al., 2002). Therefore, we can hypothesize that both 5-HT T.S. & Galligan, J.J. (eds), Serotonin and Gastrointestinal Function. CRC transporters play a signiﬁcant role in 5-HT homeostasis within the Press, Boca Raton, pp. 1–9.
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5-hydroxyindoles in plasma and brain. Neuropharmacology, 23, 1041– In summary, increased brain ECF 5-HT is removed from the brain Okada, F., Saito, Y., Fujieda, T. & Yamashita, I. (1972) Monoamine changes into the circulating blood via the 5-HT transporter system located on in the brain of rats injected with L-5-hydroxytryptophan. Nature, 238, 355– the brain endothelial cells.
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Perosino G.C., 2012. Scienze della Terra (cap. 2 - modulo V). CREST (To). 2 - ELEMENTI DI PALEOECOLOGIA 2.1 - Definizione di ecologia Il termine ecologia deriva dal greco oikos che significa "casa" o "posto per vivere". L'ecologia è lo studio degli organismi nella loro casa, ma viene anche definita come lo studio delle relazioni fra organismi o gruppi di organismi ed il loro ambiente e tenta di definire l'insieme di innumerevoli fenomeni e relazioni che caratterizzano sistemi molto complessi. Nello stagno, per esempio, avvengono processi di sedimentazione che, nel tempo, portano al suo riempimento. I materiali che si depositano sul fondo hanno caratteri che dipendono da quelli fisici e chimici delle acque e soprattutto dagli apporti di detriti minerali ed organici dagli ambienti circostanti. La presenza di organismi, solitamente rigogliosa, costituisce l'aspetto più rilevante ed arricchisce i sedimenti di sostanza organica. Ma la presenza di quegli organismi, sia come quantità, sia come composizione di specie, dipende dai caratteri fisici e chimici delle acque e questi a loro volta dipendono anche dal clima della regione nella quale si trova quello stagno. Il clima a sua volta influenza la composizione della vegetazione che si trova intorno al bacino, nell'ambito della quale trovano rifugio animali che si nutrono di prede che trovano nell'acqua. È un insieme di relazioni il cui elenco sembra senza fine, ma che, man mano che vengono studiate, consentono di ottenere un quadro generale sempre più attendibile di quell'ambiente.