Lack of evidence for reduced prefrontal cortical serotonin and dopamine efflux after acute tryptophan depletion

Rationale Acute tryptophan depletion (ATD) is a widely used method to study the role of serotonin (5-HT) in affect and cognition. ATD results in a strong but transient decrease in plasma tryptophan and central 5-HT synthesis and availability. Although its use is widespread, the evidence that the numerous functional effects of ATD are caused by actual changes in 5-HT neuronal release is not very strong. Thus far, decreases in 5-HT efflux (thought to reflect synaptic release) were only reported after chronic tryptophan depletion or when ATD was combined with blockade of 5-HT reuptake.
Introduction
A widely applied method for the study of the role of serotonin (5-HT) in cognition and affective processing is acute tryptophan depletion (ATD; Biggio et al. 1974; Fadda et al. 2000b; Fadda 2000). Through dietary depletion of the essential amino acid tryptophan, peripheral and central availability of this precursor of serotonin can be rapidly lowered (Biggio et al. 1974; Moja et al. 1989). This, in turn, leads to reduced synthesis of the monoamine 5-HT (Gessa et al. 1974) and reduced central availability of this neurotransmitter (Fadda et al. 2000b; Lieben et al. 2004a).
Since the introduction of this method, ATD has been successfully applied in both human and rodent research and has implicated the serotonergic system in cognition (e.g. memory impairments, reduced attention and increased impulsivity) and affective processes (e.g. relapse of depressed patients, mild depressive state, aggression and irritability; Fadda 2000; Riedel et al. 2002). Various studies have, furthermore, reported ATD-induced effects on typical ‘prefrontal’ tasks, like reversal learning (Rogers et al. 1999; Finger et al. 2007) and intra- and extradimensional set shifting (Park et al. 1994), but also ATD-induced changes in prefrontal cortex (PFC) activity as measured with functional magnetic resonance imaging (Rubia et al. 2005; Allen et al. 2006; Evers et al. 2005).
Recent literature, however, shows that the reported cognitive and behavioural effects of ATD are not always reproducible and suggests that ATD as a method for studying 5-HT depletion is particularly suitable for subpopulations that show a vulnerability to 5-HT dysregulation (Fadda 2000; Booij et al. 2003; Fusar-Poli et al. 2006; Jans et al. 2007). These subgroups include remitted depressed patients (Bremner et al. 1997; Young and Leyton 2002; Neumeister et al. 2004), females (Nishizawa et al. 1997; Schmitt et al. 2000; Booij et al. 2002; Ellenbogen et al. 1996) and subjects with certain genetic variations in the 5-HT transporter gene (Roiser et al. 2006; Finger et al. 2007). Fusar-Poli et al. (2006) furthermore suggest that the effects observed after ATD might not be mediated solely through a 5-HT-related mechanism, but rather indirectly via compensatory mechanisms of other transmitter systems (see also Praschak-Rieder et al. 2004). One candidate for this is dopamine (DA). Although direct evidence for ATD-induced effects on other transmitter systems, including DA, is lacking, there is compelling evidence that 5-HT can directly regulate dopaminergic activity and, consequently, function (for a current review, see Alex and Pehek 2007).
On the other hand, evidence for the effect of ATD on the actual neuronal release of 5-HT in the brain is sparse and, in most cases, indirect. Human studies are limited in their measurement to imaging studies of central 5-HT synthesis (Nishizawa et al. 1997), 5-HT receptor occupation (Udo de Haes et al. 2002; Praschak-Rieder et al. 2004) and cerebrovascular fluid measurement of tryptophan and 5-HT metabolism (Carpenter et al. 1998). Although these studies partially confirm reduced synthesis of 5-HT and do report lowered levels of the metabolite of 5-HT in cerebrospinal fluid, evidence for reduced release of 5-HT is absent.
A number of rodent studies, in general employing the same method of ATD, suggest that in addition to reduced tryptophan availability and reduced 5-HT synthesis, the actual synaptic release of 5-HT is lowered. Evidence for the assumption that ATD does in fact lower 5-HT release is, however, inconclusive. Microdialysis studies have been able to show reduced extracellular 5-HT after ATD only in the presence of a selective serotonin reuptake inhibitor (SSRI; Fadda et al. 2000b; Bel and Artigas 1996; Stancampiano et al. 1997), while there are no reports that show decreased or unaffected 5-HT efflux in animals that are not treated with SSRIs. This is an important observation, as non-physiological blockade of serotonin reuptake can interfere with normal transmitter release, making a clear-cut interpretation of the data difficult. Furthermore, Marco and Meek (1979) show that SSRI treatment in rats can reduce synthesis, which, in turn, could augment the depletion of 5-HT pools induced by ATD (see also Carlsson and Lindqvist 1978; Moret and Briley 1997; Fuxe et al. 1982 and Yamane et al. 2001).
In line with the evidence for prefrontal mediated effects of ATD mentioned earlier, we examined the effect of ATD on extracellular concentrations of both 5-HT and DA in the PFC in spontaneously active, and behaviourally stimulated, animals. We aimed to investigate whether successful lowering of blood plasma tryptophan concentration with ATD leads to a reduction of medial prefrontal 5-HT efflux in the absence of a serotonergic reuptake blockade.
Materials and methods
All experiments were approved by the Animal Experimentation Committee of the Royal Netherlands Academy of Arts and Sciences and were carried out in agreement with Dutch Laws (Wet op de Dierproeven, 1996) and European regulations (Guideline 86/609/EEC).
Subjects
Subjects were 20 male outbred Wistar rats (Harlan/CPB, Horst, The Netherlands) weighing 175–200 g at arrival. Upon arrival, the animals were socially housed in groups of four in standard type IV macrolon cages where they were kept under a reversed day/night cycle (dimmed red light from 7 a.m. until 7 p.m., white light from 7 p.m. until 7 a.m.) for the duration of the experiment. After surgery and during experimental procedures, the animals were kept in individual cages (25 × 25 × 35 cm).
Surgery
Two weeks after arrival, the animals were subjected to surgery for placement of a jugular vein catheter and implantation of bilateral microdialysis probes. The weight of the animals at time of the surgery was 297–332 g. Rats were anesthetised with intramuscular Hypnorm (0.24 mg/kg fentanyl citrate and 7.5 mg/kg fluanisone, Janssen); subcutaneous Dormicum (0.75 mg/kg midazolam, Roche) was given for muscle relaxation. A silicon catheter was placed in the right jugular vein to allow for blood sampling, as described by Steffens (1969). For the subsequent implantation of the microdialysis probes, the animals were mounted in a stereotactic frame with the toothbar set at −2.5 mm. Bilateral probes were then placed in the medial PFC at an angle of 12° (A + 30 L ± 18; V − 55). Both microdialysis probes and jugular vein cannula were secured to the scull with dental cement and two cranial screws. Postoperative pain reduction was achieved with Temgesic (0.10 mg/kg buprenorphine, Schering-Plough), 0.03 ml/100 g b.w. s.c approximately 2 h after surgery. After surgery, the animals returned to individual cages for the remainder of the experiments.
Experimental procedure
Approximately 1 week after surgery, the animals were assigned to either the experimental or control condition, with the body weight of the animals matched between groups. Fourteen hours before the experiment, food was removed from the home cages. On the day of the measurement, the animals were transferred to the experimentation room where they were kept in cages that contained their own bedding material. For the duration of the experiment, the animals were connected to the microdialysis tubing and blood sampling lines, which were attached to a swivel and counterbalanced beam out of reach of the animals. In this way, the animals were unrestricted in their movement, and the collection of blood samples could be achieved without handling the animals.
The collection of hourly blood samples started as soon as stable efflux of DA and 5-HT was observed (see below), which was approximately 2 h after connection. A first administration of the diet (see below) followed the initial blood sampling; a second administration was given 90 min after the first. Two and a half hours after the first diet administration, the rats were subjected to 20 min of ‘novelty’ stimulation.
During this period, the animals were given access to an identically shaped cage, filled with new bedding material, by means of an opaque sliding door connecting the measurement cage with the ‘novelty’ cage.
Optical movement detectors, connected to a PC running locally written software, were placed on top of the cages to detect possible changes in locomotor behaviour due to diet administration.
All animals were tested individually on separate days. A radio played to mask background noise and minimise interference from experimental procedures.
Treatment
The experimental rats (TRP minus group) were treated orally with a protein–carbohydrate mixture lacking tryptophan (TRP; 4.0 g/kg Solugel P™ and 2.0 g/kg Maltodextrine) in a volume of 10 ml/kg (see Table 1). Two diet administrations were given, spaced 90 min apart. Control animals received the same mixture with added TRP (0.28% TRP of total protein). The exact composition of the mixture and the procedure for administration is identical to that described in Lieben et al. (2004a). To minimise stress, the rats were daily handled and habituated to oral injections with normal tap water (10 ml/kg). To minimise TRP intake through the normal rat chow, food was removed from the home cages 14 h before the start of the experiment. Water, however, remained freely available during this period.
Table 1Composition of the nutritional mixture and determination of the amino acids content of the gelatin-based proteinMixtureValuesProtein (Solugel P®) in 100 ml tap water100Aspartic acid + asparagine5.2Glutamic acid + glutamine9.3Hydroxyproline12.1Serine3.1Glycine22.5Histidine0.5Arginine8.8Threonine1.1Alanine9.3Proline13.3Tyrosine0.4Valine2.1Methionine0.6Cysteine0.2Isoleucine1.4Leucine3.0Hydroxylysine1.4Phenylalanine1.9Tryptophan0.1Lysine3.6Carbohydrate (Malthodextrine) in 80 ml tap water50KCl0.094CaCl2·2H2O2.32l-Tryptophan (Tryp minus group)0l-Tryptophan (control group)0.28The composition of the administered mixture used in the current experiment is depicted in bold. The amino acid content (%) of the protein was obtained from PB Gelatins, Tessenderlo, Belgium.
Blood sampling and quantification of TRP content
On the experimental day, hourly blood samples were taken via the jugular vein catheter and analysed for TRP content. We collected six consecutive blood samples during the experiment. The first sample was taken after a stable microdialysis baseline was established and just before the first diet administration was given. Before the start of the experiment, the rats were habituated to blood sampling to minimise stress (i.e. a syringe was attached to the catheter and blood was withdrawn and immediately returned).
Blood samples (200 μl) were collected in heparin-filled tubes and deproteinised with 5-sulfosalicylic acid (5%). After centrifuging (15 min at 3,220×g), the samples were stored at −80°C until analysis.
After completion of the experiment, the plasma samples were thawed at 4°C, and 20 μl supernatants were transferred to a high-performance liquid chromatograph (HPLC) for analysis (Waters 600E pump and Waters 717plus autosampler, Waters Chromatography b.v., Netherlands; Jasco FP-920 intelligent fluorescence detector, Jasco, Benelux B.V., Netherlands and Shimadzu Class-vp™ software v.5.03 Shimadzu Duisburg, Germany).
The mobile phase consisted of 0.06 mol/l sodium acetate, 9 mmol/l citric acid, 0.37 mmol/l sodium 1-heptane sulfonate (HSA) and 12.5% methanol in milliQ water. The flow rate was kept constant at 1.0 ml/min. Separation of TRP from other components was achieved with a Supelcosil column (LC-18-DB 25 cm × 4.6 mm × 5 μm), with a 2-cm guard-column of the same material (Supelco Superguard™, Supelco USA), at a constant temperature of 40°C. Quantification was achieved by means of fluorescence detection, with the excitation wavelength set at 335 nm and an emission wavelength of 440 nm. TRP content was measured against a calibration curve of an external TRP standard.
Microdialysis probes and measurements
Concentric dialysis probes, manufactured and routinely used in this laboratory (Feenstra and Botterblom 1996), were constructed from fused silica (i.d. 0.075 mm) protected by 25-G needles as inlet and outlet. A Hospal membrane (o.d. 0.32 mm, i.d. 0.24 mm) with 3-mm exposed length was fitted for these experiments.
A week after surgery (Feenstra et al. 2000), the animals were transferred to the test room where the dialysis probes were connected to PEEK (polyetheretherketone)-tubing (o.d. 0.51 mm; i.d. 0.13 mm; Aurora Borealis, Netherlands) attached to a dual-channel swivel (Pronexus, Skärholmen, Sweden). A Univentor 801 microinfusion syringe pump delivered a ringer solution (145 mmol/l NaCl, 1.2 mmol/l CaCl2, 2.7 mmol/l KCl, 1.0 mmol/l MgCl2) at a flow rate of 1.3 μl/min. Dialysate was directly introduced into the electrically actuated HPLC injector (Valco, VICI, C6W), and 50 μl was injected every 15 min. DA and 5-HT were separated on a Supelcosil column (LC-18-DB 25 cm × 4.6 mm × 5 μm) and detected by means of an ANTEC VT-03 electrochemical flowcell (50-μm spacer) set at +0.65 V against a Ag/AgCl reference electrode.
The mobile phase consisted of 0.06 mol/l sodium acetate, 9 mmol/l citric acid, 0.37 mmol/l HSA and 12.5% methanol in milliQ water. The flow rate was kept constant at 1.0 ml/min; a pulse damper (ANTEC, Leyden) was placed with the column inside the DECADE detector unit (ANTEC) and kept at a constant temperature of 40°C.
The DECADE unit was programmed to automatically make an injection every 15 min and simultaneously start registration of the detector output. 5-HT and DA content was measured against a calibration curve of an external standard using Shimadzu Class-VP™ software (Shimadzu, v.5.03). The detection limit (ratio signal to noise = 2) was 0.15 pg of 5-HT and DA injected onto the column.
Drugs and chemicals
The Gelatine hydrolysate (Solugel P™) was obtained from PB Gelatins (Tessenderlo, Belgium). Glucodry 200 was obtained from the Amylumgroup (Koog aan de Zaan, the Netherlands). Potassium chloride (KCl), calciumchloride–dihydrate (CaCl2·2H2O), sodium chloride (NaCl), sodium 1-heptane sulfonate (HSA), methanol and tryptophan were obtained from Sigma-Aldrich Chemie, Germany. 5-Sulfosalicyclic acid dehydrate and magnesium chloride hexahydrate (MgCl2·6H2O) were obtained from Merck Schuchhardt. Sodium acetate and citric acid were purchased at Janssen Chimica, Geel, Belgium
Histological examination
After completion of the experiments, the rats were humanely killed by inhalation of a mixture of CO2/O2 (70/30) followed by 100% CO2. The brains were rapidly taken out of the skull and frozen at −20°C. Coronal (20 μm) sections were cut on a cryostat and stained with thionine for examination with the microscope for the precise location of the microdialysis probes.
Data analysis
Changes in extracellular concentration of DA and 5-HT were calculated as deviations from the baseline defined as the last two samples preceding the first diet administration. Changes in plasma levels of TRP were compared to the first sample preceding diet administration. ‘Movement-counts’, in arbitrary units, registered by the optical movement detector were taken as measure for possible changes in activity. All data were analysed using a repeated measures analysis of variance (ANOVA) with time as within-factor and group/treatment as between-factor. If indicated by the Mauchly’s test of sphericity, the number of degrees of freedom was adjusted by a Huynh–Feldt correction.
If indicated by an ANOVA group or interaction effect, subsequent evaluation of the effects of the treatment on individual samples was done by the Student Newman–Keuls test with repeated measures in which all samples are mutually compared. The level of significance was p < 0.05.
The effect of novelty exposure on 5-HT and DA release and general activity during the measurement was assessed by recalculation of a baseline from the two samples before novelty exposure. The data were then analysed as described above. All data are presented as means±SEM.
Results
Histological examination and animal exclusion Histological sections were examined for placement of the microdialysis probes. Data from rats with incorrectly placed probes were not included in the analysis. After exclusion, both the control group and the TRP minus group included eight animals.
Plasma concentrations Figure 1 shows the change of plasma tryptophan concentrations for both the control and treated group. Due to blockade of several jugular vein cannulas, the control group included seven and the TRP minus group, five animals. Average plasma concentrations (μg/ml) of tryptophan for the initial baseline samples were 18.80 ± 11.92 for the control group and 21.51 ± 15.64 for the TRP minus group.
Fig. 1Plasma tryptophan concentration. Relative concentration (mean±SEM) of tryptophan in plasma compared to baseline. Open triangles correspond to the TRP minus group; closed squares correspond to the control group. The vertical grey bars correspond to, respectively, first diet administration, second diet administration and novelty exposure. Asterisk indicates a significant difference between the two groups, p < 0.05; sigma indicates a significant difference compared to the baseline value, p < 0.05A repeated measures ANOVA with group (TRP minus/control) as between-subjects factor and time as within-subjects factor resulted in a main effect of time [F(4.45,44.50) = 3.07 p < 0.03] and group [F(1,10) = 38.76, p < 0.0005] as well as a group/time interaction [F(4.45,10) = 8.09, p < 0.0005]. An additional t test revealed a significant reduction of the TRP minus group compared to the control group (time point 3: t = 6.00, p < 0.0005; time point 4: t = 7.66, p < 0.0005; time point 5: t = 5.76, p < 0.0005; time point 6: t = 3.15, p = 0.01; Fig. 1). In contrast to the control group that showed an initial increase in plasma tryptophan (up to 167%) and returned to baseline values [F(5,30) = 3.50, p < 0.02], the TRP minus group showed a persistent decrease in plasma tryptophan after the second administration, with a minimum of 29% of baseline 2 h after the first diet administration [F(2.51,10.06) = 15.26, p = 0.001]. A simple contrast ANOVA for the control group revealed that time points 3 and 4 were significantly different from baseline; for the TRP minus, time points 3, 4, 5 and 6 were different from baseline.
Microdialysis
Microdialysis of 5-HT Figure 2a shows the relative change, compared to two baseline samples, in 5-HT efflux over the entire day. An ANOVA with repeated measures revealed no significant group [F(1,14) = 0.57, p > 0.4], time [F(13.49,188.78) = 1.19, p > 0.2] or interaction effect [F(13.49,14) = 0.97, p > 0.4]. Average transmitter concentrations (pg/50 μl) of serotonin for the initial baseline samples were 1.41 ± 0.27 (mean ± SEM) for the control group and 1.48 ± 0.23 (mean ± SEM) for the TRP minus group.
Fig. 2Microdialysis of 5-HT and DA and general activity counts. Relative change in extracellular 5-HT and DA (mean±SEM) and movement compared to baseline. Open triangles correspond to the TRP minus group; closed squares correspond to the control group. a 5-HT, b DA and c movement counts. The vertical grey bars correspond to, respectively, first diet administration, second diet administration and novelty exposure. No significant changes were observedNo significant differences between treated and control group were observed during novelty exposure when values were expressed relative to the original baseline or to a recalculated baseline just before the exposure.
Microdialysis of DA Figure 2b shows the relative change, compared to two baseline samples, in DA efflux over the entire day. An ANOVA with repeated measures revealed a significant effect of time [F(6.95,60.58) = 2.76, p < 0.02], but not of group [F(1,14) = 1.62, p > 0.2] or of interaction [F(8.14,14) = 0.98, p > 0.4]. Subsequent analysis of individual groups showed no time effect for either group.Average extracellular transmitter concentrations (pg/50 μl) of dopamine for the initial baseline samples were 0.64 ± 0.15 (mean±SEM) for the control group and 0.72 ± 0.16 (mean±SEM) for the TRP minus group.No significant differences between treated and control group were observed during novelty exposure when values were expressed relative to the original baseline or to a recalculated baseline just before the exposure.
Movement detection
Figure 2c shows the average number of activity counts for both the control group and the TRP minus group. Average movement counts for the initial baseline samples were 123.59 ± 17.71 (mean±SEM) for the control group and 110.38 ± 14.22 (mean±SEM) for the TRP minus group. These data support the behavioural observation of sustained general activity throughout the measurements (e.g. grooming and explorative movement).
A repeated measures ANOVA revealed a significant effect of time over the entire day [F(10.27,143.72) = 2.69, p < 0.005]. No group [F(1,14) = 0.42, p > 0.5] or interaction [F(10.27,14) = 0.55, p > 0.8] effect was observed.
The novelty stimulation did not cause any differential effect on overall activity.
Discussion
To gain insight into the relation between ATD and the release of serotonin (5-HT) in the prefrontal cortex, we measured plasma levels of tryptophan and, simultaneously, PFC efflux of 5-HT and DA in animals that underwent ATD. In line with current literature (Lieben et al. 2004a), ATD induced a rapid decline of free plasma tryptophan within 2 h of the initial administration that lasted throughout the experimental procedure. However, despite this reduction, efflux of prefrontal serotonin (5-HT), as measured in the medial PFC with microdialysis, was unaffected in these animals. In contrast to previous studies, these measurements, in behaviourally active animals, were performed in the absence of a serotonergic reuptake blocker. These results indicate that successful lowering of plasma tryptophan, to levels associated with behavioural effects, does not necessarily induce lowering of 5-HT efflux. Considering the widespread use of ATD as a method for studying the role of serotonin in behavioural and cognitive research, these findings are especially relevant.
The current experiment combined blood sampling through a chronic jugular vein catheter with online microdialysis measurements of 5-HT and DA. This combination of techniques was used to ensure that blood sampling did not hinder the animals in their behaviour or cause unwanted effects on transmitter release.
In line with current literature, we measured a rapid decrease of plasma tryptophan of more than 70%. Although the possibility exists that a greater reduction of plasma tryptophan, which can be achieved with chronic tryptophan depletion (Fadda et al. 2000a), could have induced a reduction of 5-HT efflux, it would not explain the effects on behaviour and affect observed after tryptophan depletions of a magnitude similar to those observed in the current experiment (e.g. Riedel 2004; Lieben et al. 2004b; Delgado 2006). The 4.5-h time-window in which we measured is well within the period for which behavioural effects have been reported, reducing the likelihood that a decrease in 5-HT efflux would have been observed had we measured for a longer period. Lack of sensitivity of the measurement equipment does not seem likely either; with the current detection limit, reductions of either transmitter would have been measurable.
The absence of a clear effect of novelty stimulation on either general activity or transmitter efflux might suggest that the animals were inactive. However, behavioural observation, as well as quantitative movement detection, indicates otherwise, showing that the animals were in fact active throughout the measurements (e.g. grooming, and explorative behaviour), although the active novelty exposure did not induce the expected increase in locomotor activity.
Literature data from both human and rodent studies show that ATD leads to lowering of plasma tryptophan (Biggio et al. 1974; Moja et al. 1989) and decreased synthesis (Gessa et al. 1974) and availability of central 5-HT (Ashley and Curzon 1981; Fadda et al. 2000b; Lieben et al. 2004a), but, to date, it has not been convincingly shown that the ATD lowers efflux of 5-HT in the brain. The few rodent studies that do show reduced efflux after ATD either used chronic treatment with a tryptophan-deficient diet (Fadda et al. 2000b; Van der Stelt et al. 2004) or combined the measurements with drugs that prevent reuptake of 5-HT (Fadda et al. 2000b; Bel and Artigas 1996; Stancampiano et al. 1997; but see Bel and Artigas (1996) who observe an increase, rather than a decrease, of 5-HT efflux in the PFC after ATD in rats on free food). To the best of our knowledge, the current experiment is the first to report on the effect of ATD on prefrontal 5-HT efflux after overnight fasting in the absence of a reuptake blocker, an experimental design that parallels the behavioural studies in both rodents and human subjects. As pointed out in the “Introduction”, this is especially important, as the addition of reuptake inhibition can lead to decreased 5-HT synthesis, and thus, augment the effect of ATD. In light of the behavioural effects observed after acute tryptophan depletion and the general notion that a reduction of synaptic 5-HT release underlies these effects, understanding the effect of ATD on 5-HT efflux, which is thought to reflect release, is essential.
Our data seem to suggest that successful tryptophan depletion does not alter 5-HT release. ATD, however, has convincingly shown to be an effective method to manipulate mood and induce cognitive/behavioural effects in both humans (Fusar-Poli et al. 2006; Riedel et al. 2002) and rodents (Fadda 2000; Riedel 2004). This apparent discrepancy will be discussed in the next section.
Although mood effects after ATD have been reported across populations of subjects, groups with vulnerability for 5-HT dysregulation (e.g. remitted depressed patients, women and people that show a genetic variation in the 5-HT transporter gene) seem particularly sensitive to ATD (Delgado 2006; Booij et al. 2002; Moore et al. 2000; Neumeister 2003; Riedel et al. 2002; Roiser et al. 2006; Jans et al., 2007). It is argued that especially these groups are sensitive to ATD because they already show a reduced availability of central 5-HT that is further reduced by tryptophan depletion. In contrast to non-vulnerable subjects, these subpopulations will be less able to compensate for a temporary reduction in 5-HT synthesis and will experience a greater impact of ATD. Likewise, SSRI-treated animals will experience depletion of 5-HT stores (Marco and Meek 1979) and more readily show decreased 5-HT efflux after ATD than unmedicated rats. Measurements of indices of 5-HT release in response to ATD in ‘vulnerable’ rodents, like serotonin transporter knockout animals (Holmes et al. 2003), or females have not been performed.
In contrast to effects on mood, behavioural and cognitive effects are generally observed in a broader population that also includes healthy subjects (for a review, see Riedel et al. 2002) that do not show a particular sensitivity to 5-HT dysregulation.
The most parsimonious explanation that can unify those observations with the current data is that brain areas outside the medial PFC, which have not been measured in the present experiment, might mediate the behavioural effects. As the innervation of 5-HT in the brain is not uniform (McQuade and Sharp 1997; see for an overview Frazer and Hensler 1999) and some areas appear to be more sensitive to ATD than others (Lieben et al. 2004a; Fusar-Poli et al. 2006), the absence of 5-HT efflux reductions in the medial PFC might not reflect ATD-induced changes in other (prefrontal) areas.
However, this explanation seems to hold only partially at best. As mentioned in the “Introduction”, ATD studies have implicated prefrontal 5-HT in various ‘prefrontal’ tasks, like reversal learning (Rogers et al. 1999; Finger et al. 2007) and intra-, extradimensional set shifting (Park et al. 1994), and shown ATD-induced changes in PFC activity (Rubia et al. 2005; Allen et al. 2006; Evers et al. 2005).
The possibility that other transmitter systems might mediate ATD effects (Fusar-Poli et al. 2006) was investigated by simultaneous measurements of DA. As we reported that DA efflux did not respond to the ATD, the current data cannot confirm this hypothesis, although other possible transmitters remain to be investigated.
While it is not possible to extrapolate these findings to brain areas outside the medial PFC, our present data show that an effective reduction of plasma tryptophan with ATD does not necessarily imply a similar reduction in 5-HT efflux. As ATD continues to be an important and widely used paradigm to study specific roles of 5-HT, it is important to clarify through which mechanism ATD exerts its effects and to what extent changes in 5-HT contribute to the functional effects of ATD.

tryptophan
serotonin (5-ht)
dopamine (da)
microdialysis
medial prefrontal cortex


Bioactive Hydrogel Substrates: Probing Leukocyte Receptor–Ligand Interactions in Parallel Plate Flow Chamber Studies

The binding of activated integrins on the surface of leukocytes facilitates the adhesion of leukocytes to vascular endothelium during inflammation. Interactions between selectins and their ligands mediate rolling, and are believed to play an important role in leukocyte adhesion, though the minimal recognition motif required for physiologic interactions is not known. We have developed a novel system using poly(ethylene glycol) (PEG) hydrogels modified with either integrin-binding peptide sequences or the selectin ligand sialyl Lewis X (SLeX) within a parallel plate flow chamber to examine the dynamics of leukocyte adhesion to specific ligands. The adhesive peptide sequences arginine–glycine–aspartic acid–serine (RGDS) and leucine–aspartic acid–valine (LDV) as well as sialyl Lewis X were bound to the surface of photopolymerized PEG diacrylate hydrogels. Leukocytes perfused over these gels in a parallel plate flow chamber at physiological shear rates demonstrate both rolling and firm adhesion, depending on the identity and concentration of ligand bound to the hydrogel substrate. This new system provides a unique polymer-based model for the study of interactions between leukocytes and endothelium as well as a platform to develop improved scaffolds for cardiovascular tissue engineering.
Introduction
Leukocyte adhesion to sites of inflammation is crucial to eliminate the cause of irritation and repair the surrounding tissue. After the initiation of inflammation by cytokines such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), lipopolysaccharide, and interleukin-3, (IL-3), leukocyte velocity slows dramatically due to leukocyte contact with the vascular wall.9,27,34  Rolling, the initial transient interaction, is mediated by selectin molecules present on the activated endothelial cell surface. E-selectin is an inducible surface glycoprotein that is known to bind the carbohydrate ligand sialyl Lewis X (SLeX), which is present on various leukocytes10,11,13,35,41,43 and mediates stronger adhesions and slower rolling than P- or L-selectin.27 Following this primary contact, firm adhesion takes place via activated β2-integrins binding intracellular adhesion molecule-1 (ICAM-1) expressed on the endothelium, and through the most important member of the β1 integrin subfamily, very late antigen-4 (VLA-4; α4β1). VLA-4 binds vascular cell adhesion molecule-1 (VCAM-1), and is responsible for lymphocyte adhesion to vascular endothelium and leukocyte recruitment to the inflamed area.1,10,11,27 Though well studied, the exact mechanism of adhesion to the vascular endothelium is not known. There are two suggested mechanisms of integrin activation; one proposing that activation is a result of chemokine stimulation of leukocytes2,43 and another suggesting that selectin binding leads to integrin activation.12,42
Several systems have been developed to examine the dynamics of rolling and firm adhesion and to elucidate the exact mechanism of the binding cascade. Leukocytes isolated from human blood have been studied under static and flow conditions on activated endothelial cell monolayers as well as on cells engineered to express endothelial cell adhesion molecules.3,28,37,40 Polystyrene microspheres coated with adhesion ligands have also been shown to interact with stimulated cell monolayers17 and on substrates coated with selectins and other cell adhesion molecules.11 Yeast engineered to display E-selectin has been observed rolling on substrates coated with SLeX. 2 Recently, poly(ethylene glycol) (PEG) has been tethered to gold surfaces to create cell resistant surfaces and spatial gradients of PEG on gold surfaces have been used to study the kinetics of static cell adhesion.32,33 While each of these systems has revealed new insight into the dynamics of leukocyte interactions with endothelial cell adhesion molecules, the ultimate goal must be to establish a simple, cell-free system that more closely mimics the in vivo environment.
PEG hydrogels are crosslinked hydrophilic networks that demonstrate excellent biocompatibility, being highly resistant to protein adsorption and cell adhesion, and causing minimal inflammatory responses.20,21,22,36 These highly swollen networks have similar water content and mechanical properties to soft tissues and may be engineered to contain cell adhesion peptides, growth factors, and therapeutics for localized drug delivery.5,6,26,29,30,39,44 PEG diacrylate hydrogels may be covalently modified with cell adhesive peptide sequences to encourage cell adhesion, spreading, and migration and the interactions of cells with these hydrogels has been extensively studied under static conditions.15,16,24,25,29,31,38 A key benefit of such materials is that the base material, PEG, is intrinsically resistant to protein adsorption, so the adhesive interactions with cells are limited to the factors that are specifically engineered into the hydrogel network. In this work we propose a novel system to study leukocyte adhesion under shear using photopolymerized PEG copolymer hydrogels. After forming thin flat PEG diacrylate base hydrogels, we are able to polymerize a layer of monoacrylate PEG-peptide (or SLeX) to the surface, and using a parallel plate flow chamber, we can observe cell adhesion using video microscopy.
Materials And Methods
All chemicals were purchased from Sigma–Aldrich (St. Louis, MO) unless otherwise stated.
Synthesis of Polyethylene Glycol Diacrylate
Polyethylene glycol diacrylate (PEG-DA) was synthesized by dissolving 12 g dry PEG (MW: 6000; Fluka, Milwaukee, WI) in 16 ml anhydrous dichloromethane (DCM) with an equimolar amount of triethylamine and 0.72 g acryloyl chloride (Lancaster Synthesis, Windham, NH) added dropwise. The mixture was stirred under argon for 24 h, washed with 2 M K2CO3, and separated into aqueous and DCM phases to remove HCl. The DCM phase was dried with anhydrous MgSO4 (Fisher Scientific, Pittsburgh, PA), and the PEG diacrylate was then precipitated in diethyl ether, filtered, and dried under vacuum at room temperature overnight. The resulting polymer was dissolved in N,N-dimethylformamide-d7 and characterized via proton NMR (Avance 400 MHz; Bruker, Billerica, MA) to determine the extent of acrylation.
Synthesis of PEG Derivatives Containing Cell Adhesion Molecules
The cell adhesive peptide sequence used in this study include arginine–glycine–asparagines–serine (RGDS; American Peptide Company, Inc., Sunnyvale, CA), and a peptide containing the cell adhesive leucine–asparagines–valine sequence (glycine–proline–glutamic acid–isoleucine–leucine–asparagines–valine–serine–threonine, GPEILDVST), which was synthesized using standard fluorenylmethoxycarbonyl (Fmoc) chemistry on an Applied Biosystems 431A peptide synthesizer (Foster, CA). The high affinity 4-((N′-2-methylphenyl)ureido)-phenylacetyl-leucine-aspartic acid-valine-proline (Bio1211; Commonwealth Biotechnologies, Inc., Richmond, VA)5,6 was also used as an alternate LDV-containing compound. The non-adhesive sequences used as negative controls were arginine–glycine–glutamic acid–serine (RGES; American Peptide Company, Inc., Sunnyvale, CA) and glycine–proline–glutamic acid–isoleucine–leucine–glutamic acid–valine–serine–threonine (GPEILEVST), also synthesized using Fmoc chemistry on a peptide synthesizer.
Peptides were conjugated to PEG monoacrylate by reaction with acryloyl-PEG-N-hydroxysuccinimide (PEG-NHS; MW 3400; Nektar Therapeutics, Huntsville, AL) in 50 mM sodium bicarbonate (pH 8.5) at a 1:1 molar ratio for 2 h. The mixture was then dialyzed (MWCO 1000), lyophilized, and stored at −20°C. Gel permeation chromatography with UV and evaporative light scattering detectors (Polymer Laboratories, Amherst, MA) was used to determine the coupling efficiency.
The selectin ligand sialyl Lewis X (SLeX) was conjugated to PEG using an avidin–biotin bridge. PEG-NHS (25 mg) was reacted with a lysine–biotin conjugate (biocytin) at a 1:2 molar ratio in 50 mM sodium bicarbonate (pH 8.5) for 2 h. SLeX-biotin (500 μg; Glycotech, Gaithersburg, Maryland) was reacted with avidin (10–15 units mg−1) at a ratio of 2 units avidin per mole of SLeX-biotin in 50 mM sodium bicarbonate (pH 8.5) for 2 h. The reaction mixtures were then combined to allow further conjugation of avidin and biotin, thereby linking acryloyl–PEG–NHS–lysine–biotin to avidin–biotin–SLeX.
Synthesis of Bilayered PEG Copolymer Hydrogels
Hydrogels were formed by first dissolving 0.2 g ml−1 PEG diacrylate in 10 mM HEPES buffered saline (HBS, pH 7.4); the polymer solution was then filter sterilized using a 0.22 μm filter (Gelman Sciences, Ann Arbor, MI). The photoinitiator 2,2-dimethoxy-2-phenyl acetophenone in N-vinylpyrrolidinone (300 mg ml−1) was added at 10 μl ml−1 polymer solution. This mixture was injected between rectangular glass plates separated by 0.5 mm spacers and polymerized under UV light (365 nm, 10 mW cm−2) for 30 s. The top plate was removed and the hydrogel surface rinsed with sterile PBS. A second layer, consisting of 5 μmol ml−1 of either acryloyl-PEG-peptide or acryloyl-PEG-SLeX in HBS and 10 μl ml−1 2,2-dimethoxy-2-phenyl acetophenone in N-vinylpyrrolidinone was then layered on top of the PEG diacrylate base gel, the upper glass plate replaced, and the second layer photopolymerized by exposure to UV light (365 nm, 10 mW cm−2) for 1 min.
Cell Maintenance
JURKAT cells (human T-lymphocytes; ATCC, Manassas, VA) were maintained in RPMI-1640 prepared with 10% fetal bovine serum (FBS; BioWhitaker, Walkersville, MD), 2 mM l-glutamine, 1 unit ml−1 penicillin, and 100 mg l−1 streptomycin (GPS). 300.19/E cells (mouse pre-B lymphoblast; ATCC, Manassas, VA) were sustained in RPMI-1640 prepared with 10% FBS, 1% GPS, and 0.1 mM 2-mercaptoethanol. Cells were maintained at 37°C in a 5% CO2 environment.
Cell Adhesion and Rolling on Adhesive PEG Gels
Flow assays were performed using a circular parallel plate flow chamber (Glycotech, Gaithersburg, MD) mounted on the stage of a Zeiss Axiovert 135 microscope (Carl Zeiss Inc., Thornwood, NY). The chamber was placed on top of photopolymerized PEG copolymer hydrogels and vacuum sealed to the surface using a portable vacuum pump (Fisher Scientific, Pittsburgh, PA) as shown in Fig. 1. Cell suspensions were drawn through the flow field (1 cm path width, 0.01 in thickness) using a programmable syringe pump (BS-8000 Multi-Phaser™ Programmable Syringe Pump, Braintree Scientific Inc., Braintree, MA) at varying flow rates corresponding to a shear stress range of 3.5–35 dynes cm−2, which is comparable to in vivo shear rates. Cellular interactions with the hydrogels were monitored using a Nikon CoolPix 5000 camera (Nikon Inc., Melville, NY) and transferred to videotape for further analysis.
Figure 1.Schematic of parallel plate flow chamber system. A small pump is used to create a vacuum, sealing the chamber on the surface of the gel. Cells are then perfused over the gel surface using a programmable syringe pump.
Cation Dependent Binding
JURKAT cells were treated with 2 mM magnesium (Mg2+), calcium (Ca2+), or manganese (Mn2+) or with 10 mM EDTA and then perfused through the flow chamber and allowed to settle on the gel for 5 min. Controls were exposed to standard formulations of RPMI-1640 containing 0.4 mM Ca2+ and 0.4 mM Mg2+. Ten fields of view were scanned to get an average number of cells per field of view. Flow rates corresponding to shear stresses of 0.5, 1.0, and 10 dynes cm−2 were used to wash away unbound cells. The number of cells remaining for each shear stress was counted and averaged over several fields of view.
LDV Specificity
JURKAT cells treated with 2 mM Mg2+ were allowed to settle on the LDV gel for 5 min and an average number of cells per field of view was determined. Specificity was demonstrated by the addition 7 μg ml−1 of either a mouse anti-human monoclonal antibody that blocks VLA-4 binding to VCAM-1 (anti-CD49d, clone BU49; Ancell Corporation, Bayport, MN) or an IgG1 isotype control (purified mouse myeloma IgG1; Invitrogen Corporation, Carlsbad, CA) at a shear stress of 0.5 dynes cm−2. The average number of cells bound per field of view was again counted to determine the amount of cells remaining bound to the surface.
Specificity was also demonstrated by the addition of a solution of either 10 mM EDTA or 150 μM Bio1211 introduced into the flow chamber under a shear stress of 0.5 dynes cm−2. An average number of cells bound to the gel under flow was determined every minute for 13 min.
Video Analysis
Cells were allowed to settle on each gel for 5 min. An average of 10 fields of view was scanned and the number of cells settled on the peptide gel was counted. After flow began, fields of view were scanned again and the number of cells remaining (bound to the gel) was counted. After flow was established on the SLeX gels, video was paused and the number of interacting cells was counted. The numbers were averaged over 10 fields of view for each shear stress.
Statistical Analysis
Data were compared with two-tailed, unpaired t-tests; p-values less than 0.05 were considered to be significant.
Results
Synthesis of PEG Hydrogels
PEG hydrogels were formed under UV light in the presence of a photoinitiator between two glass plates. Hydrogels 0.5 mm thick were formed after 30 s of exposure. The addition of an acryloyl-PEG-peptide derivative mixed with the photoinitiator to the surface of the hydrogels resulted in a covalently bound layer of cell adhesive moiety on the surface (Fig. 1).
Quantification of Cell Adhesion and Rolling
Cell rolling on SLeX hydrogels was quantified over a range of shear stresses, and 86.8 ± 11.6 cells per field of view rolled on the gel surface at 3.5 dynes cm−2 with an average rolling velocity of 141.3 μm s−1 (Fig. 2). Rolling decreased with increasing shear stress (7.0 dynes cm−2: 15.2 ± 3.8 cells, average rolling velocity 232.7 μm s−1; 14 dynes cm−2: 8.2 ± 1.9 cells, average rolling velocity 486.8 μm s−1; 21 dynes cm−2: no rolling observed).
Figure 2.300.19/E cells rolling on SLeX modified gel. 300.19 cells transfected with human E-selectin was perfused over a SLeX modified gel. The number of rolling cells were counted per field of view. SLeX ability to support rolling decreased as shear stress increased (*p < 0.02 as compared to shear rate of 3.5 dyne cm−2).
Approximately 98.5 ± 18.6% of cells contacting hydrogel surfaces modified with Bio1211 adhered to the gel surface. In comparison, 41.5 ± 13.4% of cells in contact with the surface of PEG-LDV, an interaction specific for the α4β1 integrin expressed on JURKAT cells, adhered to the hydrogels and 23 ± 8.5% of cells contacting PEG-RGDS hydrogels adhered to the gel surface (Fig. 3). No cells adhesion to PEG-DA or PEG-RGES hydrogels was observed; however the control LEV peptide-modified gels had 1.6 ± 1.2% of cells contacting the surface adhere.
Figure 3.Peptide ability to bind VLA-4. JURKAT cells were allowed to settle on PEG-DA gels conjugated with different peptides at similar densities. After 5 min, cells were subjected to a shear stress of 0.5 dynes cm−2 to remove unbound cells and the remaining cells were counted (*p < 0.02 compared to Bio1211).
Cation Dependant Binding
Cation sensitivity was assessed by the addition of cations or EDTA to cell cultures prior to their exposure to PEG-LDV hydrogels. The presence of 10 mM EDTA had the greatest inhibitory effect on binding, followed by 2 mM Ca2+. Mg2+ and Mn2+ had little effect on the ability of JURKAT cells to bind LDV, and a higher number of cells exposed to both calcium and magnesium were able to bind to the gel surface than those exposed to calcium alone (Fig. 4).
Figure 4.Cation dependent binding of VLA-4 to LDV peptide. JURKAT cells were incubated with 2 mM cations or 10 mM EDTA prior to settling on the LDV gel; controls were exposed to standard formulations of RPMI-1640 containing 0.4 mM Ca2+ and 0.4 mM Mg2+. The percentage of cells on the gel was determined based on the total number of cells before initiating a shear stress of 0.5 dynes cm−2 (*p < 0.05 compared to Control).
LDV Specificity
JURKAT cell binding to PEG-LDV hydrogels was reversed upon the addition of a monoclonal anti-VLA-4 (3.4 ± 2.1% bound). Cells exposed to an isotype-matched control antibody were still able to adhere to the hydrogel surface (88.8 ± 33.4% bound), demonstrating specificity of LDV for the VLA-4 integrin. The higher affinity of Bio1211 compared to the LDV peptide was also observed by the addition of unbound Bio1211 after cells were allowed to settle on PEG-LDV hydrogels (Fig. 5). Bio1211 was able to remove cells from the gel surface better than EDTA through competitive binding of VLA-4.
Figure 5.LDV Specificity for VLA-4. 10 mM EDTA or 1.5 uM Bio1211 was introduced into the flow chamber at a wall shear stress of 0.5 dynes cm−2 after JURKAT cells were allowed to settle on the LDV gel for 5 min. Bio1211, which has a higher affinity for VLA-4 was able to compete the cells off the LDV modified gel surface. EDTA was not as effective as Bio1211 as cells detached more slowly and not completely. The dashed line represents the uninhibited controls, in which 40 ± 15.2% of the cells initially exposed to the hydrogel remained bound through the duration of the experiment. Each data point represents the average percent of bound cells per 10 fields of view and error bars represent the standard deviation of the percent of bound cells within those 10 fields of view.
The influence of peptide concentration on cell adhesion was determined by varying the amounts of LDV bound to the hydrogel surface (Fig. 6). The number of cells bound to the gel increased with LDV concentration, and LDV was better able to support adhesion at lower shear stresses for all concentrations when compared to higher rates of shear.
Figure 6.Dilution effect of LDV. The number of JURKAT cells bound to the gel decreased as the LDV peptide concentration was lowered. LDV was better able to support adhesion at lower shear stresses for all concentrations (*p < 0.05 comparing shear rates at each concentration).
Discussion
The system of hydrophilic hydrogels modified with cell adhesion peptides implemented in this study exhibits the ability to mimic the cell adhesion cascade that occurs during the onset of inflammation in the vascular system. The capacity to modify hydrogels with cell adhesion molecules for the study of cellular interactions with biomaterials for drug delivery and tissue engineering is well documented.4,7,15,16,18,24–26,29 –31,38,39,44 This study implements the use of these materials under physiological flow conditions to examine the mechanisms of leukocyte rolling and firm adhesion.
The formation of thin flat PEG diacrylate hydrogels for use with a parallel plate flow chamber improves upon earlier systems that utilize hydrophobic surfaces with adsorbed cell adhesion molecules. The highly crosslinked structure of swollen PEG networks have water content similar to native vessels, and covalent incorporation of cell adhesive peptides guarantees control of concentration and allows for patterning of one or more adhesion sequences on the gel surface.23 This system is based on a flexible substrate with tunable stiffness, the properties of which can be exploited to examine the responses of different cell types in microenvironments that mimic native tissues in various states of development, remodeling, regeneration, and disease. It has been suggested that the differentiation of cellular function and response could depend significantly on matrix elasticity,8,14,45 and varying the polymer composition or concentration in the hydrogel can alter the permeability and mechanical properties,36 simulating a range of biologically relevant conditions. In addition, the local mobility of adhesive ligands in the solvated hydrogel system should be considerably better than on solid substrates, allowing more variable orientations which could contribute to a greater fraction of accessible ligands available to receptors on the cell surface.
Leukocyte cell adhesion to the surfaces of modified PEG-DA hydrogels was highly specific, reversible, and sensitive to ligand site density and affinity, demonstrating the efficacy of the system to mimic the events leading to the firm adhesion of immune cells on activated endothelium. The slow rolling of 300.19/E cells on the surfaces of PEG-SLeX hydrogels confirms the integration of SLeX into the system through the use of an avidin–biotin linkage, which did not disrupt the active binding site of the carbohydrate. Incorporation of the RGD and LDV peptides encouraged firm cell adhesion to the materials surface under shear in a concentration dependant manner, and cells remained adherent throughout the duration of the flow experiment. These results illustrate that this method of studying leukocyte adhesion succeeds in mimicking the interactions seen on the in vivo vascular wall under shear. With further optimization, such as incorporation of signaling molecules, this system using of PEG gels can improve insight into the mechanisms of rolling and firm cell adhesion at sites of active vascular disease.
Conclusions
The covalent modification of PEG hydrogel surfaces with cell adhesive peptides was accomplished in continuous layers of adhesive regions. These materials encouraged vascular cell adhesion through both transient interactions with selectin molecules and firm binding via integrins. The system presented here to expose adhesive hydrogels to cells under flow conditions represents a method to study cell–material interactions in environments that closely mimic in vivo environments.

hydrogel
flow chamber
leukocyte adhesion
poly(ethylene glycol)


A refined radio-telemetry technique to monitor right ventricle or pulmonary artery pressures in rats: a useful tool in pulmonary hypertension research

Implantable radio-telemetry methodology, allowing for continuous recording of pulmonary haemodynamics, has previously been used to assess effects of therapy on development and treatment of pulmonary hypertension. In the original procedure, rats were subjected to invasive thoracic surgery, which imposes significant stress that may disturb critical aspects of the cardiovascular system and delay recovery. In the present study, we describe and compare the original trans-thoracic approach with a new, simpler trans-diaphragm approach for catheter placement, which avoids the need for surgical invasion of the thorax. Satisfactory overall success rates up to 75% were achieved in both approaches, and right ventricular pressures and heart and respiratory rates normalised within 2 weeks. However, recovery was significantly faster in trans-diaphragm than in trans-thoracic operated animals (6.4 ± 0.5 vs 9.5 ± 1.1 days, respectively; p < 0.05). Stable right ventricular pressures were recorded for more than 4 months, and pressure changes, induced by monocrotaline or pulmonary embolisms, were readily detected. The data demonstrate that right ventricular telemetry is a practicable procedure and a useful tool in pulmonary hypertension research in rats, especially when used in combination with echocardiography. We conclude that the described trans-diaphragm approach should be considered as the method of choice, for it is less invasive and simpler to perform.
Introduction
Over the last decade, new medical treatments became available for the treatment of pulmonary hypertension (PH) patients, and this development set off a renewed interest in the pathophysiology and haemodynamics of the pulmonary circulation [6, 12, 22]. Experimental PH studies are still largely confined to single measurements of pulmonary arterial (PA) or right ventricular (RV) pressure in anaesthetised, open-chest animals undergoing artificial respiration, which does not allow determining changes of pressures in time. Measuring pulmonary haemodynamics have improved considerably since RV telemetry was firstly introduced by Hess et al. [4], as it allows for continuous monitoring of RV or PA pressures, without introducing artefacts caused by stress or anaesthetics [9]. Moreover, animals fitted with telemetry can serve as their own controls, thus permitting the use of smaller groups of animals than is possible with the single measurement approach in anaesthetised animals [9]. Although many investigators have embraced this new technology with enthusiasm and despite its proven potential, studies actually using this methodology in rats are scarce [4, 13, 16, 18, 19].
One possible explanation for this discrepancy is that the original procedure was only described briefly before [4], complicating its implementation for potential new users. Secondly, the described trans-thoracic approach involves RV catheterisation through complex and highly invasive thoracotomy [4]. In the present study, we hypothesised that RV catheterisation performed via an opening in the diaphragm, thereby omitting a thoracotomy, may be a simpler, less invasive alternative.
In the present report, we provide a detailed description of the alternative trans-diaphragm approach together with a comparison with the original trans-thoracic approach. Furthermore, we illustrate its usefulness when studying the pulmonary circulation, by presenting novel comprehensive pulmonary and cardiac haemodynamic data during the development of monocrotaline-induced pulmonary hypertension.
Materials and methods
All experiments were approved by the Institutional Animal Care and Use Committee of the VU University and were conducted in accordance with the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes, and the Dutch Animal Experimentation Act.
Animals
Thirty-eight male Wistar rats were used (250–300 g; Harlan, Horst, The Netherlands), of which 33 were operated and 5 served as controls (no surgery). The animals were allowed to adapt to their new environment for at least 1 week before surgery. The animals were conventionally housed in pairs under controlled conditions (temperature 21–22°C; humidity 60–65%; 12:12 h light–dark cycle) and had free access to filtered water and standard rat chow (Global 2016, Harlan Teklad, Blackthorn-Bicester, England).
Telemetry system
An implantable radio-telemetry system for blood pressure measurements for small laboratory animals was used, comprising a radio-transmitter (TA11PA-C40) fitted with a 10-cm-long catheter (Data Science International [DSI], St. Paul, MN). Transmitters were turned on a day before implantation and stored in sterilised saline, following the manufacturer’s instructions (http://www.datasci.com/information/index.asp).
The pressure was monitored continuously during surgery to ensure proper position of the tip of the pressure catheter. After successful implantation of the transmitter, RV or PA pressures and locomotor activity were recorded for 10 s every 5 min. From the pressure recordings mean, systolic and diastolic pressure, heart rate, respiratory rate and circadian rhythm were subsequently derived (Dataquest A.R.T. software 4.0, DSI).
Pre-operative care and anaesthesia
All animals received pre-surgical intramuscular buprenorphine analgesia (0.10 mg/kg; Schering-Plough, Maarssen, The Netherlands). For general anaesthesia, isoflurane (2.0% in 1:1 O2/air mix; Pharmachemie, Haarlem, The Netherlands) was used via induction chamber and tracheal intubation (16 G × 51-mm Teflon tube; ventilator settings: breathing frequency 80/min, pressures: 9/0 cmH2O, inspiratory/expiratory ratio 1:1). For extra local analgesia, lidocaine spray (100 mg/ml; AstraZeneca, Zoetermeer, The Netherlands) was applied at the surgical region. Animals were maintained under anaesthesia for an average time of 80 min.
Animals were placed on a heating pad to maintain body temperature and positioned in dorsal recumbency. Hydromellose drops (0.3%; Ratiopharm, Zaandam, The Netherlands) were applied to prevent drying of the eyes. After shaving and disinfection with 70% ethanol of the chest and abdomen, animals were covered with sterile incision foil and the animals were then covered with sterile incision foil (Opraflex, Lohmann & Rauscher, Almere, The Netherlands). A surgical microscope was used (magnification ×16–64; Carl Zeiss, Sliedrecht, The Netherlands) for optimal view.
Surgery: trans-thoracic approach
Implantation of the telemetry transmitter in the abdomen
The abdominal cavity was accessed via a 5-cm midline laparotomy, starting just below the xiphoid process. The transmitter was placed in the peritoneal cavity, with the catheter pointing caudally to prevent liver injury and tissue reaction. The abdominal cavity was covered with gauzes soaked in warm saline and left open until the end of the procedure.
Routing of the pressure catheter to the right ventricle
The heart was exposed by means of a left thoracotomy performed at the sixth intercostal space and mid-clavicular line. The thorax was opened with blunt scissors, respecting the anatomy of the overlying muscle layers and cautiously avoiding injury to the lungs. The pressure catheter was then tunnelled subcutaneously from the peritoneal cavity to the opening in the thorax and temporarily laid aside. Four individual small hooks were used to retract the ribs and expose the heart through a wider, 2 × 2-cm window. The pericardium was then opened with two dressing forceps.
Catheterisation of the right ventricle
A superficial purse string (6–0 Prolene, Ethicon, St-Stevens-Woluwe, Belgium) was placed on the right ventricle free wall, near the apex. Through the purse string, the right ventricle was punctured with a 19 G syringe needle in the direction of the RV outflow tract, avoiding any coronary vessel. After removing the needle, the site was wiped with a sterile cotton stick. The tip of the catheter was then carefully inserted into the right ventricle, using a vessel canulation forceps (0.5–1.0 mm outer diameter, Fine Science Tools, Heidelberg, Germany), avoiding loss of gel from the tip at all times. Live pressure waveform trace confirmed proper RV catheterisation (RV systolic/diastolic pressures are typically ∼25/1 mmHg). Depending on the protocol, the pressure catheter was subsequently advanced for about 2 cm more, positioning the tip of the catheter beyond the pulmonary valves. Again, live pressure waveform trace confirmed correct positioning of the pressure catheter in the pulmonary artery (PA systolic/diastolic pressures are typically ∼25/10 mmHg; Fig. 1, left panel). Finally, the catheter was fixed by the purse string and a small drop of tissue adhesive at the site of insertion (10 μl dispensed by a pipette; Vetbond, 3M, St. Paul MN; Electronic Supplementary Material (ESM) Fig. E-1a).
Fig. 1left panel: Live pressure recordings while advancing the pressurecatheter into the pulmonary artery. Notice the stepwise increase in diastolic pressures when manoeuvring the catheter beyond the pulmonary valves (arrow), whereas the systolic pressures remained unchanged. right panel: Live pressure recordings while injecting a bolus injection of microspheres (arrow; 19 μm, 1.5 million/100 g i.v.). Upon embolisation, a significant increase in systolic, diastolic and developed RV pressures was observed. Furthermore, in both recordings, the effect of breathing is noticeable
Closing of the chest and abdomen
After the catheterisation, blood clots if any were removed from the pleural cavity with a moist cotton stick. The ribs were approximated with two individual sutures (5–0 Vicryl, Ethicon). To promote full expansion of the lungs, a small burst of positive pressure was applied (maximally 10 cmH2O for 1 s). A chest tube (18 G), used as air outlet, was removed after proper thorax excursions were observed. The chest muscles were laid back in layers (no suturing necessary). Then, the transmitter was sutured to the abdominal muscle (5–0 Perma-Hand, Ethicon) incorporating the suture rib of the device. The abdominal wall was sutured in a running subcuticular pattern (5–0 Vicryl). Finally, the skin of the chest and abdomen was closed with a running suture (5–0 Vicryl).
Surgery: trans-diaphragm approach
Opening of abdomen and the diaphragm
The abdominal cavity was accessed via a 5-cm midline laparotomy, with the abdominal wall retracted by two individual hooks. To expose the diaphragm, a retraction suture was used to lift the xiphoid process during the whole procedure. The visceral organs were covered temporarily with wet gazes, and the liver was gently pushed down by the weight of a blade holder. A small midline incision in the diaphragm was then made, with small sharp scissors, from the xiphoid process until the tendinous part of the diaphragmatic membrane. The two sides of the diaphragm were held aside with two retraction sutures, providing optimal exposure of the heart (ESM Fig. E-1b).
Catheterisation of the right ventricle
RV catheterisation was performed similarly, as described above. In short, the pericardium was opened by two dressing forceps, and a purse string was placed just right of the apex. The pressure catheter was then inserted into the right ventricle through a small puncture made with a syringe needle and fixed in place.
Closing the diaphragm and abdomen
After catheterisation, blood clots in the thorax, if any, were removed. The diaphragm was closed with a running suture (5–0 Vicryl), starting from the ventral side, with the pressure catheter sticking out the diaphragm at the dorsal end of the incision. After that, the retraction suture through the xiphoid process was removed. To promote full expansion of the lungs, a small burst of positive pressure was applied, as described above, with the chest tube between the sutures of the diaphragm, until proper thorax excursions were visually confirmed. To further secure closure of the diaphragm, a small amount of tissue adhesive (Vetbond, 3M) was applied. Finally, the transmitter was implanted in the peritoneal cavity, fixed to the abdominal wall with the catheter facing caudally, and the abdomen was closed, as described above.
Post-operative care
To compensate for loss of fluids, all animals received warm sterile saline at the end of the procedure (5 ml intraperitoneal). After final skin closure, the animals were allowed to regain consciousness.
In the first 24 h of recovery, the animals were housed in individual cages. Each cage was placed halfway on a heating pad, in such a way that half of the cage was maintained above it and the other at room temperature. During this period, the animals were monitored three times, received post-surgical analgesia when clinically indicated (buprenorphine 0.10 mg/kg subcutaneous) and were provided with drinking gel pads and softened rat chow. After 24 h, the animals were conventionally housed in pairs for a full recovery, and they were inspected and weighted daily. Animals were considered fully recovered from surgery when their appearance and behaviour were normal, their surgical wounds healed, their pre-surgical body weight regained, when the pressures normalised and circadian rhythm was restored. The study period was ended after 4 months.
Experimental protocol
To induce acute and chronic pulmonary hypertension, four animal received an intravenous bolus injection of microspheres [25] (tail; 1.5 million /100 g body mass in sterile saline; polystyrene, mean diameter 19 μm (#7520), Duke Scientific, Fremont, CA) and three animals monocrotaline subcutaneous [24] (MCT; 80 mg/kg in sterile saline; Sigma-Aldrich, Zwijndrecht, The Netherlands), respectively. Experimental interventions were performed no earlier then 2 weeks after the surgery.
RV echocardiography
After a 2-week recovery, trans-thoracic echocardiographic measurements (ProSound SSD-4000 system equipped with a 13-Mhz linear transducer [UST-5542], Aloka, Tokyo, Japan) were performed on anaesthetised but spontaneously breathing rats (isoflurane 2.0% in 1:1 O2/air mix), according to the standards of the American Society of Echocardiography [10, 15]. To minimise the effects of isoflurane on cardiac function, the time under anaesthesia was set to a maximum of 15 min [14].
The measurements were compared with untreated rats to check for possible adverse effects related to the procedure. Cardiac output, tricuspid annular plane systolic excursion (TAPSE), RV wall thickness (RVWT) and RV end diastolic diameter (RVEDD) were measured, as described before by Hardziyenka et al. [3]. TAPSE is a parameter for RV systolic function, which is measured in the apical four-chamber view of the heart and expresses the displacement of the lateral tricuspid annulus towards the apex during systole [10].
Estimation of pulmonary vascular resistance and RV power output
Additional echocardiographic measurements were performed in MCT-treated rats and untreated but operated controls, just before the injection, 2 weeks after injection and when the first clinical signs of RV heart failure developed (as indicated by weight loss, dyspnoea and lethargy, after which the rats were euthanised). Pulmonary vascular resistance (PVR) and RV power output (RV-power) were estimated, by combining telemetric pressure data and echocardiographic flow data.
PVR was estimated by Poiseuille’s law: [PVR] ≈ [mean PAP]/[cardiac output] ≈ (0.61 × [systolic RV pressure] + 2 mmHg)/[cardiac output] [1, 23].
RV-power was estimated using a simplified pressure–volume analysis, assuming rectangular pressure–volume loops: [RV-power] ≈ [systolic RV pressure] × [stroke volume] × [heart rate] = [systolic RV pressure] × [cardiac output] [21, 23].
Autopsy
To investigate potential effects of chronic implantation of the telemetry transmitters, the animals were euthanised at the end of the study protocol by exsanguination under isoflurane (4.0% in 1:1 O2/air mix). Macroscopic examination of right ventricle, lungs, diaphragm and intercostal muscles were performed, and organ weights (heart, lungs, liver, spleen, kidneys) were measured and compared with controls. Animals treated with MCT or microspheres were excluded from these analyses.
Statistical analyses
All measurements, unless otherwise stated, are presented as mean ± standard error of the mean. Normal distribution was verified, and Student’s t test, analysis of variance or Fisher Exact test were used, when appropriate (SPSS 13.0, SPSS, Chicago IL). A p value less than 0.05 was considered statistically significant.
Results
Success rate
Seventeen animals were operated using the trans-thoracic approach, and 16 animals were operated using the trans-diaphragm approach.
In the trans-thoracic group, 8 animals showed stable pressure signals for the whole study period of 4 months. Five animals did not recover form surgery, and 4 animals developed instable pressure signals, caused by clot formation inside the pressure catheter within 2 weeks of recovery. Of the 9 failed procedures, 4 occured in the first four attempts.
In the trans-diaphragm group, 9 animals showed stable pressure signal for the whole study period of 4 months. Six animals did not recover form surgery, and one animal developed instable pressure signals, caused by clot formation inside the pressure catheter within 2 weeks of recovery. As in the trans-thoracic group, 4 of the failures occurred in the first four attempts.
By discarding the first 4 animals in each procedure, as we consider this the learning curve, the overall success rate was 8 of 13 (62%) for the trans-thoracic approach and 9 of 12 (75%) for the trans-diaphragm approach.
Recovery from surgery
Within a week post-surgery, heart and respiratory rates and circadian rhythm normalised in all rats, and no differences were observed between the two approaches (Table 1). Although all animals regained their pre-surgical body weight within 2 weeks, full recovery was significantly faster in the trans-diaphragm- than the trans-thoracic-operated animals (6.4 ± 0.5 vs 9.5 ± 1.1 days, respectively; p < 0.05).
Table 1Main results of this study, all values in mean ± SEM Trans-thoracic approach (n = 17)Trans-diaphragm approach (n = 16)Controls (n = 5)Success ratea8/13 (62%)7/9 (79%)n.r.Recovery BW to pre-surgical level (days)d9.5 ± 1.16.4 ± 0.5*n.r. Heart rate (bpm)e362 ± 4.4359 ± 5.7350–400b [8, 17] Respiratory rate (rpm)e90 ± 2.491 ± 3.880–100b [11, 17, 20] Circadian rhythm (days)e3.4 ± 0.43.7 ± 0.6n.r.Pressurese RVSP (mmHg)25 ± 1.124 ± 0.921–26b [16] RVDP (mmHg)1.7 ± 0.32.2 ± 0.21–5c [5]RV echocardiographyd Cardiac output (ml/min)107 ± 4.9116 ± 6.9110 ± 4.9 TAPSE (mm)3.6 ± 0.13.7 ± 0.23.4 ± 0.1 RVWT (mm)1.0 ± 0.11.0 ± 0.10.9 ± 0.1 RVEDD (mm)3.6 ± 0.13.7 ± 0.13.6 ± 0.1Autopsyf, organ weights (g) Heart1.4 ± 0.11.4 ± 0.21.3 ± 0.1 Lungs1.5 ± 0.11.5 ± 0.21.5 ± 0.2 Liver15.4 ± 0.515.0 ± 0.615.1 ± 0.5 Spleen0.65 ± 0.030.68 ± 0.040.64 ± 0.04 Kidneys2.4 ± 0.22.3 ± 0.22.3 ± 0.1n Number of animals, n.r. not relevant, BM body mass, RVSP RV systolic pressure, RVDP RV diastolic pressure, TAPSE tricuspid annular plane systolic excursion, RVWT RV wall thickness, RVEDD RV end diastolic diameter*p < 0.05aFirst four attempts were discarded.bNormal values for Wistar rats, awake and at rest, measured by radio-telemetrycNormal values for Wistar rats, measured during acute pressure measurements under anaesthesiadAnalyses on surviving animals onlyeAnalyses on successfully operated animals only (stable signal)fAnalyses on surviving and untreated animals only (no microspheres or monocrotaline)
RV pressures normalised in all animals within 2 weeks after surgery (RV systolic/diastolic pressures ∼25/1 mmHg), irrespective of the method used (Table 1). PA systolic/diastolic pressures were estimated to be ∼25/10 mmHg (Fig. 1, left panel). Activity had no significant effect on the stability of the pressure signals.
Echocardiography revealed normal cardiac output (∼110 ml/min), TAPSE (∼3.5 mm), RVWT (∼1.0 mm) and RVEDD (∼3.6 mm) in all rats, and no differences were seen between the differently operated animals and controls (Table 1). Furthermore, other signs of RV dysfunction, like pericardial effusion and tricuspid regurgitation, were not present in any animal.
Acute pressure effects of pulmonary embolisation by microspheres
RV telemetry could readily detect acute changes in RV pressure, induced by a bolus injection of microspheres (Fig. 1, right panel). Upon embolisation, systolic RV pressures increased from 21 ± 1 to 38 ± 3 mmHg (p < 0.01) and diastolic RV pressures increased from 2 ± 1 to 7 ± 2 mmHg (p < 0.05).
Haemodynamic changes during the development of MCT-induced pulmonary hypertension
Changes in haemodynamic parameters during the development of MCT-induced PH are shown in Fig. 2 (also in ESM Fig. E-2). Four weeks after MCT injection, the first clinical signs of RV heart failure developed. During this period, there was a gradual rise in RV systolic pressure, from 22 ± 2 mmHg at baseline to 38 ± 4 mmHg at 2 weeks and 72 ± 5 mmHg at 4 weeks (p < 0.01 vs control). At 4 weeks, cardiac output dropped dramatically, from 100 ± 7 ml/min at baseline to 107 ± 8 at 2 weeks and 52 ± 4 ml/min at 4 weeks (p < 0.01 vs control). In addition, the poor haemodynamic condition of the MCT-treated rats at 4 weeks was confirmed by a simultaneous decline in TAPSE from 3.6 ± 0.1 mm during the first 2 weeks to 1.8 ± 0.2 mm at 4 weeks (p < 0.01 vs control). During the development of PH, the right ventricle remodelled initially by increasing RVWT (1.0 ± 0.1 mm at baseline, 1.1 ± 0.1 mm at 2 weeks and 1.3 ± 0.1 mm at 4 weeks, respectively; p < 0.01 vs control) but subsequently by increasing RVEDDs as well (3.5 ± 0.1 mm at baseline, 3.6 ± 0.1 mm at 2 weeks and 7.0 ± 0.5 mm at 4 weeks, respectively; p < 0.01 vs control).
Fig. 2Haemodynamic changes during the development of MCT-induced pulmonary hypertension. Four weeks after the rats received MCT, clinical signs of RV heart failure developed. In these 4 weeks, systolic pressures continued to rise, while cardiac output could not be maintained. The presence of RV remodelling was indicated by increased wall thickness and diameter. The pulmonary vascular resistance continued to rise as well, while at 4 weeks, the increase in the RV-power was inadequate to compensate for the dramatically increased afterload. Triangles connected by dashed lines: MCT-treated; squares connected by uninterrupted lines: control group; n = 3 for both groups. All values are in mean ± SEM, Asterisk, p < 0.05; double asterisk, p < 0.01 compared to control (post-hoc analyses). Please note that sometimes the icons conceal the error bars. For explanation of the abbreviations, see corresponding text
During the development of PH, PVR increased continuously, from 1.2 ± 0.2 × 109 N m−5 s at baseline, to 1.9 ± 0.2 × 109 N m−5 s at 2 weeks and 7.9 ± 0.9 × 109 N m−5 s at 4 weeks (p < 0.01 vs control). RV-power was increased at 2 weeks, but no further increase was seen at 4 weeks (RV-power = 4.9 ± 0.7 mW at baseline, 9.1 ± 0.9 mW at 2 weeks, 8.3 ± 0.7 mW at 4 weeks, respectively; p < 0.01 vs controls).
Autopsy
Chronic implantation of the telemetry transmitter in both approaches did not result in any differences in organ weight as compared to control (Table 1), and no apparent pulmonary embolisms were seen.
Autopsy revealed only local fibrosis of the right ventricle near the insertion point of the catheter (ESM Fig. E-3a). In the trans-thoracic approach, animals showed normal healing of the intercostal muscles. In the trans-diaphragm approach, no herniation or dehiscence of the diaphragm were seen in any animal, and often, the liver was partially accreted to the healed wound of the diaphragm (ESM Fig. E-3b).
Discussion
The present study describes in detail two surgical techniques to monitor RV or PA pressures over time by radio-telemetry in rats. To our best knowledge, we are the first research group to describe the trans-diaphragm approach and its comparison with the previously used trans-thoracic approach. We have demonstrated that: (1) in our hands, both the trans-diaphragm as well as the trans-thoracic approach have satisfactory success rates, especially when considering the complexity of the procedures, (2) the time-to-recovery was significantly shorter with the trans-diaphragm approach than the trans-thoracic approach, (3) measured physiological parameters recovered fully in both methods, and there were no permanent detrimental effects on RV, intercostal muscles or diaphragm, (4) using RV-telemetry, acute and chronic pressure changes in the pulmonary circulation can be readily detected and (5) RV-telemetry, in combination with echocardiography, allow thorough monitoring of pulmonary and cardiac haemodynamic changes over time.
Fully recovered animals showed normal RV structure, as expressed by RVWT and RVEDD, and normal function, as expressed by cardiac output and TAPSE. In addition, in both approaches, RV pressure normalised shortly after surgery, and autopsy revealed only minor fibrosis in the direct proximity of the catheter. Therefore, permanent detrimental effects of RV-telemetry on the RV can be excluded for both techniques. In addition, examination of other organs revealed no abnormalities, as was described by others [4]. In animals operated using the trans-diaphragm approach, macroscopic examination revealed normal wound healing of the diaphragm. Although we have not tested the strength of the healed diaphragm, others did not find significant differences between a normal (untouched) and a sutured diaphragm [7]. The natural history of small defects in the diaphragm is relatively benign, as they almost always heal spontaneously [2], especially when the diaphragm is fixated by other organs [26]. Furthermore, the observation of a normal respiratory rate seen in the recovered animals confirms that the trans-diaphragm approach is safe and a good alternative for the trans-thoracic approach.
In our experience, insertion of the pressure catheter into the RV and its advance further into the pulmonary artery when desired is comparatively easy to perform in the trans-diaphragm approach. This relative ease of RV catheterisation is reflected in the higher success rate compared to the trans-thoracic approach and might be well explained by the more caudal and inferior direction by which the catheter enters the heart. Approaching the heart in this manner entails minimal manipulation and manoeuvring of the catheter during the process of RV catheterisation, resulting in a lower incidence of clot formation inside the catheter. This complication occurred more frequently in trans-thoracic-operated animals as a direct consequence of excessive manoeuvring and accidental squeezing of the catheter causing it to lose or displace some of its anti-thrombolitic gel. The strongest advocate of the trans-diaphragm approach over the trans-thoracic approach, however, is the significantly faster recovery of the animals. Prompt recovery from surgery is particularly important in rats with MCT-induced PH because of low survival rates beyond 4 weeks after MCT injection.
In this study, we used echocardiography to validate RV-telemetry in vivo. Additionally, through the combination of pressure data, obtained by RV-telemetry, with echo-Doppler flow data, we were able to study the haemodynamics of the pulmonary circulation in greater detail [23]. MCT induced pulmonary and cardiac remodelling, at the same time as PVR, RVWT and RVEDD increased. Cardiac adaptation to overcome the increasing afterload became inadequate by the fourth week of MCT injection, in such that there was no further increase in RV-power to maintain adequate cardiac output while RV systolic pressure continued to rise. Detailed studies of these observations are planned for the near future.
In the present study, we have focussed on the refinement of the RV-telemetry technique in rats, but this does not exclude its applicability in mice and other rodents. Indeed, in their recent study, Schwenke et al. [18] reported long-term monitoring of PA pressure in mice using radio-telemetry.
In conclusion, we described a new, easier-to-perform, mildly invasive trans-diaphragm-based RV-telemetry approach for long-term monitoring of PA and RV pressures in the rat model of MCT-induced PH. Our findings may be applied to improve our understanding of the disease processes involved in PH and develop better treatment strategies for the disease.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Electronic Supplementary Material
M.L. Handoko et al. A refined radio-telemetry technique to monitor right ventricle or pulmonary artery pressures in rats. Pflugers Arcg – Eur J Physiol 2007. Fig. E-1, E-2, E-3. (PDF 292 kb)

pulmonary artery
diaphragm
heart
respiration
circulation
blood pressure
cardiac output
power output
hypertensive rats
vascular remodelling