Upregulation of cyclooxygenase-2 and thromboxane A2 production mediate the action of tumor necrosis factor-
in isolated rat myenteric ganglia
Matthias Rehn,
Daniela Hild, and
Martin Diener
Institute for Veterinary Physiology, University Giessen, Giessen, Germany
Submitted 18 January 2005
; accepted in final form 2 May 2005
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ABSTRACT
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Intact myenteric ganglia from 4- to 10-day-old rats were isolated from the small intestine. The preparations were cultured overnight, and drugs were applied within this time frame (20 h). Whole cell patch-clamp technique was used to measure basal membrane potential and carbachol-induced depolarization at neurons within these ganglia. Pretreatment with TNF-
(100 ng/ml) hyperpolarized the membrane (from 31.0 ± 2.7 mV under control conditions to 61.2 ± 3.2 mV in the presence of the cytokine) and potentiated the depolarization induced by carbachol (from 5.2 ± 0.7 mV under control conditions to 27.5 ± 2.0 mV in the presence of the cytokine). These effects were mimicked by carbocyclic thromboxane A2 (106 mol/l), a stable thromboxane A2 agonist. The TNF-
action was inhibited by 1-benzylimidazole (2 x 104 mol/l), a thromboxane synthase inhibitor, and BAY U 3405 (5 x 104 mol/l), an inhibitor of thromboxane receptors. Measurements of thromboxane production in the supernatant of the culture revealed an increased concentration of thromboxane B2, the stable metabolite of thromboxane A2, after exposure to TNF-
. Immuncytochemical staining for cyclooxygenase-2 (COX-2) and the neuronal marker microtubule-associating protein-2 revealed an upregulation of COX-2 in myenteric neurons after exposure to the cytokine. These results demonstrate the involvement of COX-2 and the subsequent production of thromboxane A2 in the presence of TNF-
.
COX-2; membrane potential
THE GASTROINTESTINAL IMMUNE system is physiologically involved in the defense against infectious diseases; under pathophysiological conditions, this system plays a central role during intestinal inflammation and immunological disorders (10). Clinical symptoms such as diarrhea induced by stimulation of immune cells within the gut wall are caused by their interaction with the epithelium or the smooth muscle layers (for a review, see Ref. 32).
During inflammation, proinflammatory cytokines such as TNF-
or IFN-
are released from immunoactive cells like T cells or macrophages, which alter enteric nerve function (15, 17) and the motility of the intestine (11, 18). In many cases, these cytokines act via the stimulation of the production of downstream paracrine substances such as prostaglandins (31) or nitric oxide (30) to induce inflammation.
In a recent study, our group (23) observed that incubation of isolated rat myenteric ganglia with TNF-
resulted in a hyperpolarization of the membrane together with a potentiation of the depolarization induced by stimulation of nicotinic receptors with the stable acetylcholine derivative carbachol. This response was paralleled by the nuclear translocation of signal transducer and activator of transcription (STAT5), a transcription factor that often is involved in the alterations in gene transcription evoked by this cytokine (24). The changes in basal membrane potential and in the nicotinic receptor response were suppressed by indomethacin, a nonselective cyclooxygenase (COX) inhibitor, and the COX-2-selective inhibitor nimesulide, whereas the COX-1-selective inhibitor SC-560 only partially inhibited the action of TNF-
(23), suggesting a role of eicosanoids in the signal cascade. The nature of the COX metabolite responsible for this action is unknown. Therefore, in the present study, we tried to answer the question regarding whether TNF-
exposure leads to an upregulation of the inducible form of COX and to identify the arachidonic acid metabolite responsible for the observed actions using electrophysiological (whole cell patch-clamp recording), biochemical (ELISA), and immunocytochemical methods.
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MATERIALS AND METHODS
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Isolation and incubation procedure.
Myenteric ganglia were isolated from the small intestine of 4- to 10-day-old rats. Animals were killed by exsanguination. Animal studies were approved by Regierungspräsidium Giessen (Giessen, Germany).
After the gut was removed from the rat, the serosa was stripped away under optical control and the muscle layer was separated from the mucosa using fine forceps. Then, the muscle was dissociated by incubation at 37°C in DMEM containing 1 mg/ml collagenase type II (Life Technologies, Eggenstein, Germany). The ganglia, forming netlike structures, were collected with a micropipette and placed on ice. This was followed by a wash with DMEM, centrifugation for 10 min (40,000 g), and transfer into Start-V medium (Biochrom, Berlin, Germany) containing penicillin (10,000 U/ml), streptomycin (10 mg/ml), and 10% (vol/vol) FCS (PAA Laboratories, Cölbe, Germany). The ganglia were plated on coverslides (diameter 13 mm) coated with poly-L-lysine (molecular mass >300 kDa; Biochrom) in conventional four-well dishes. The coverslides were placed in the incubator for 45 min to let the ganglionic nets settle down. Then, each well was filled with Start-V medium to a final volume of 500 µl. The four-well chambers were kept in the incubator at 37°C with continuous carbogen [5% CO2 in O2 (vol/vol)] supply. The slides were used in electrophysiological experiments the next day.
Solutions.
The solution for superfusion of the myenteric ganglia during the patch-clamp experiments was a HEPES-buffered Tyrode solution containing (in mmol/l) 140 NaCl, 5.4 KCl, 10 HEPES, 1.25 CaCl2, 1 MgCl2, and 5 glucose. The pH value of this solution was adjusted to 7.4 with NaOH-HCl. The pipettes for the whole cell recordings were filled with a solution containing (in mmol/l) 100 potassium gluconate, 40 KCl, 0.1 EGTA, 10 Tris, 5 ATP, and 2 MgCl2. The pH was adjusted with Tris·HCl to 7.2.
For immunocytochemical experiments, the following solutions were used: a phosphate buffer (100 mmol/l sodium phosphate, pH 7.2), PBS [containing (in mmol/l) 130 NaCl, 8 Na2HPO4, and 1.2 NaH2PO4], and PBS with 0.05% (vol/vol) Triton X-100 (PBS-T).
Patch-clamp experiments.
The myenteric ganglia grown on glass slides were transferred into the experimental chamber (volume 0.5 ml), which was superfused hydrostatically (perfusion rate
1 ml/min). The chamber was mounted on an inverted microscope (Olympus IX-70; Olympus Optical, Hamburg, Germany). All experiments were carried out at room temperature. The patch pipettes had resistances of 510 M
when filled with the standard pipette solution. To obtain a whole cell recording, a suction pulse was used to break the membrane patch under the tip of the pipette after seal formation. Seal resistances were 510 G
. Membrane capacitance was corrected for by cancellation of the capacitance transient (subtraction) using a 10-mV pulse. To distinguish between neurons and nonneuronal cells, a pulse of 50-mV amplitude (starting from a holding potential of 80 mV) and 30-ms duration was used. In this voltage range, only neurons exhibit a fast inward current in the ganglionic preparation after formation of the whole cell configuration (9). This inward current, therefore, was used as a parameter to distinguish the myenteric neurons from enteric glia (23).
Immunocytochemical detection of COX-2 after TNF-
stimulation.
To test the hypothesis that TNF-
could induce the COX-2 isoform, we immunocytochemical investigated the COX-2 signal of TNF-
-stimulated and nonstimulated neurons. To avoid a multilayer of different cells when using the preparation of intact ganglia, this experimental series was performed with dissociated myenteric cells. Cell dissociation was accomplished by mechanical forces acting at the ganglionic nets, which were repeatedly sucked through a 0.4-mm-diameter needle into a syringe and released again. Then, the cell suspension was seeded onto poly-L-lysine-coated coverslips and incubated for 2 days as described above. This incubation allowed neurons to recover from the dissociation protocol and to form new dendritic and axonal processes (9, 27). After 2 days, the medium was exchanged against a medium with or without TNF-
(100 ng/ml). Twenty-hours later, the medium was removed, and the cells were fixed for 15 min at 4°C with paraformaldehyde [4% (wt/vol)] diluted in phosphate buffer. The paraformaldehyde was removed by washing the preparations with phosphate buffer.
To block unspecific binding sites, the neurons were incubated with a blocking solution prepared in PBS-T with 10% (vol/vol) FCS (PAA Laboratories) at room temperature for 1 h in a moist chamber.
Incubation with the primary anti-rat COX-2 antibody (SC-1747; Santa Cruz, Heidelberg, Germany; polyclonal antibody from the goat) was performed over 36 h at 4°C at a dilution of 1:2,000 in PBS-T with 10% (vol/vol) FCS. To remove the primary antibody, the preparations were washed with PBS-T. Before COX-2 detection, the binding sites of avidin and biotin were blocked by a specific kit (Vector, Linaris, Wertheim-Bettingen, Germany). The primary COX-2 antibody was visualized using an anti-goat biotinylated antibody (1:200 in PBS-T; BA-1000 Vector, Linaris) followed by Cy3-conjugated streptavidin (1:600 in PBS-T; Dianova, Hamburg, Germany) detection. In control experiments, the primary COX-2 antibody was omitted to check for antibody specificity.
COX-2 immunocytochemistry was combined with the immunocytochemical detection of the neuronal marker, microtubule-associating protein-2 [MAP2; Sigma; monoclonal anti-MAP2a/MAP2b produced in mouse against bovine MAP2; used at 1:600 dilution in PBS-T with 10% (vol/vol) FCS], and nuclear staining with 4',6-diamidino-2-phenylindole dilactate (DAPI, used at 1:800 dilution in PBS; MoBiTec, Göttingen, Germany). Fluorescent detection of MAP2 was accomplished with an anti-mouse antibody conjugated to Alexa-Fluor 488 [1:500 in PBS-T with 10% (vol/vol) FCS; MoBiTec].
Coverslipping was performed with an anti-fadant glycerol-phosphate buffer solution (Citifluor AF1; Citifluor, London, UK). Slides were stored at 4°C until they were microscopically analyzed. We used an Olympus BX50 fluorescent microscope (Olympus Optical) for microscopic analyses. Digital images were taken with a black and white camera (Diagnostic Instruments, Sterling Heights, MI), using the Metamorph software package (Visitron Systems, Puchheim, Germany). Image editing software (Adobe Photoshop; Adobe, San Jose, CA) was also used to adjust brightness and contrast.
To quantify the COX-2 signal, photographs were taken with a standardized exposure time (300 ms). COX-2-positive neurons were identified, the number of pixels covered by them was counted, and their mean gray scale value (8-bit resolution) was determined using a conventional image-analysis software.
Immunoassays.
Short-lived thromboxane A2 (TxA2) is rapidly catabolized to the stable B2 (TxB2) form. This derivative was measured in the supernatant of the myenteric cultures by an ELISA kit (Cayman Chemical, Ann Harbor, MI). To standardize the total amount of TxB2, these experiments were performed at dissociated myenteric cells as described for the immunocytochemical experiments. A small aliquot of cell suspension was counted in a Neubauer chamber. Then, about equal amounts of cell suspension were seeded on poly-L-lysine coated coverslips. The preparations were kept in cell culture for 20 h with or without TNF-
. The supernatant was kept at 75°C until TxB2 measurements were performed according to the reference manual of the manufacturer.
Drugs.
Murine TNF-
was dissolved in distilled water containing 1 mg/ml BSA; aliquots were stored at 20°C. Carbocyclic TxA2 (cTxA2; Alexis, Grünberg, Germany) and 1-benzylimidazole (Aldrich, Steinheim, Germany) were dissolved in a stock solution of ethanol and Start-V medium [final maximal content of ethanol in the culture: 0.04% (vol/vol)]. BAY U 3405 (Bayer, Leverkusen, Germany) was dissolved in a stock solution of DMSO and Start-V medium [final content of DMSO in the culture: 0.5% (vol/vol)]. If not indicated otherwise, chemicals were obtained from Sigma (Taufkirchen, Germany).
Statistics.
Results are given as means ± SE (with n = no. of investigated neurons). Significance of differences was tested by paired and unpaired two-tailed Student's t-test or one-way ANOVA test when the mean values of more than two groups had to be compared.
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RESULTS
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Effect of inhibitors of the thromboxane pathway.
Control experiments confirmed the previous observation (23) that preincubation with TNF-
(100 ng/ml for 20 h) caused a hyperpolarization of the membrane, i.e., control neurons showed a basal membrane potential of 31.0 ± 2.7 mV (n = 6), whereas neurons pretreated with the cytokine had a basal membrane potential of 61.2 ± 3.2 mV (n = 10, P < 0.05 vs. control). In addition, in TNF-
-pretreated neurons, the depolarization evoked by carbachol (5 x 105 mol/l) was potentiated from a depolarization of 5.2 ± 0.7 mV under control conditions (n = 6) to a depolarization of 27.5 ± 2.0 mV (n = 10, P < 0.05) in the presence of the cytokine (Fig. 1A). The most reasonable explanation for the potentiation of the carbachol response by TNF-
is an increase in the driving force for cation influx due to the hyperpolarized basal membrane potential.
The effects of the cytokine were blocked by COX-2 inhibitors as reported earlier (23). Because TxA2 is one metabolite downstream of the COXs, 1-benzylimidazole (2 x 104 mol/l), a thromboxane synthase inhibitor (26), and BAY U 3405 (5 x 104 mol/l), an inhibitor of thromboxane receptors (19), were tested for their abilities to interfere with the TNF-
response. All inhibitors were administered simultaneously with the cytokine, i.e., 20 h before the patch-clamp experiments. In the presence of 1-benzylimidazole, the hyperpolarization induced by TNF-
was completely suppressed, i.e., in the combined presence of the cytokine and the thromboxane synthase inhibitor, basal membrane potential only amounted to 20.2 ± 3.0 mV (n = 5, Fig. 1, B and D). Also, the potentiation of the carbachol-induced depolarization was abolished, i.e., in the presence of TNF-
combined with 1-benzylimidazole, the cholinergic agonist only induced a depolarization of 4.0 ± 0.5 mV (n = 5). A similar suppression of the TNF-
action was observed with the thromboxane receptor blocker BAY U 3405 (Fig. 1, C and D). The inhibitors alone affected neither the basal membrane potential nor the response to carbachol (Fig. 1D).
Mimicry by a stable thromboxane derivative.
Based on these inhibitor data, cTxA2 (106 mol/l), a stable TxA2 derivative (6), was tested for its ability to mimic the action of TNF-
. Two experimental protocols were used: the thromboxane analog was applied either in preincubation for 20 h (Fig. 2A) or applied during the whole cell recording at neurons not pretreated with any drugs (Fig. 2B). In both cases, the TNF-
effect was mimicked by the eicosanoid. Neurons pretreated with cTxA2 for 20 h showed a hyperpolarization of the membrane, i.e., a basal membrane potential of 41.8 ± 5.4 mV (n = 6) compared with 24.7 ± 7.5 mV (n = 6) in the untreated control. In addition, the depolarization evoked by carbachol was upregulated (Fig. 2, A and C). In contrast to cTxA2, two stable prostaglandin analogs, when applied with the same protocol (20-h pretreatment), were ineffective. In the presence of iloprost (106 mol/l), a stable PGI2 analog (4), basal membrane potential amounted to 31.6 ± 2.4 mV and carbachol evoked a depolarization of only 4.0 ± 1.0 mV (n = 7). In the presence of 16,16-dimethyl-PGE2 (106 mol/l), the corresponding data were 24.1 ± 1.9 and 4.7 ± 0.8 mV (n = 9) for basal membrane potential and carbachol response, respectively.

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Fig. 2. Typical tracings of basal membrane potential and depolarization evoked by carbachol (5 x 105 mol/l, solid bars) in myenteric neurons pretreated for 20 h with carbocyclic thromboxane A2 (cTxA2; 106 mol/l; A) or in untreated neurons in the absence or presence of cTxA2 (106 mol/l, open bar; B). In this latter group of experiments, carbachol was applied twice, i.e., before (first solid bar) and after (second solid bar) the administration of cTxA2. C: means ± SE of basal membrane potential (bottom bars) and depolarization evoked by carbachol (top bars) in the presence cTxA2; n = 6 in both groups.
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When carbachol was administered to the neurons not pretreated with any eicosanoid, the cholinergic agonist evoked only a depolarization of 7.2 ± 3.1 mV (n = 6; first solid bar in Fig. 2B). cTxA2 induced a prompt hyperpolarization of the membrane from 24.7 ± 7.5 to 37.8 ± 6.2 mV (n = 6), an effect that did not reach statistical significance due to its large variability. However, the depolarization induced by carbachol was significantly enhanced to a value of 17.3 ± 3.2 mV (n = 6, second solid bar in Fig. 2B, P < 0.05 vs. carbachol-induced depolarization in the absence of the eicosanoid).
Effect of TNF-
on thromboxane production.
The amount of TxB2, the stable metabolite of TxA2, in the supernatant of the culture dishes was measured in the presence and absence of TNF-
(100 ng/ml for 20 h) to find out whether the cytokine stimulates the production of this eicosanoid. Indeed, in supernatants from cytokine-treated cultures, the amount of TxB2 per cell increased from 0.524 ± 0.091 fg/cell under control conditions to 0.974 ± 0.194 fg/cell in the presence of the cytokine (n = 10, P < 0.05; Fig. 3).
Upregulation of COX-2 expression after TNF-
stimulation.
Because TNF-
is known from other cells to cause an upregulation of COX-2 (3, 28), which might be responsible for the observed production of thromboxanes in the culture, immunocytochemical staining for COX-2 was performed. Cellular nuclei were labeled simultaneously with DAPI to mark the individual cells (Fig. 4, right). Neurons were stained with the neuronal marker MAP2. Control cells without cytokine treatment did already show COX-2 expression. However, the staining of TNF-
-stimulated cells was more condensed and more intensive (Fig. 4, left). Labeling with MAP2 revealed that the cells expressing COX-2 were indeed neurons (Fig. 4, middle).

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Fig. 4. Immunocytochemical staining against cyclooxygenase-2 (COX-2; left) or microtubule-associating protein 2 (MAP2; middle) and nuclear staining with 4',6-diamidino-2-phenylindole dilactate (right). Top row: primary antibody against COX-2 was omitted to evaluate background fluorescence. Middle row: control cells. Bottom row: cells were pretreated for 20 h with TNF- (100 ng/ml).
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The content of COX-2 was quantified by image analysis, i.e., by counting the number of pixels and their gray tones of individual neurons stained for COX-2. Under control conditions, the mean gray value of COX-2-expressing neurons (n = 314) amounted to 6.7 ± 0.4 gray scale values/pixel, which increased to 15.5 ± 0.7 gray scale values/pixel in the neurons pretreated with TNF-
(n = 357, P < 0.05 vs. untreated cells; Fig. 5).

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Fig. 5. Mean gray scale value/pixel for COX-2 signals in control cells (n = 314) and cells pretreated with 100 ng/ml TNF- (n = 357). Values are means ± SE. *P < 0.05 vs. control.
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DISCUSSION
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The communication between the immune system and the nervous system is in general mediated by soluble messengers (22). An important group of paracrine substances upregulated by cytokines during immunological reactions or inflammation is the metabolites of arachidonic acid such as prostaglandins, thromboxanes, and leukotrienes (14). These compounds can then act at the enteric nervous system (4, 7, 17) to affect intestinal functions.
A major proinflammatory cytokine is TNF-
. TNF-
is upregulated in the muscular layer/myenteric plexus region during inflammation (12). In a recent study, our group (23) observed that the cytokine affects basal membrane potential and the response to nicotinic receptor stimulation at myenteric neurons. Possible paracrine substances mediating this response were supposed by us to be prostaglandins because blockers of COXs inhibited the action of TNF-
. However, when we tested their abilities to mimic the action of TNF-
, none of the tested prostaglandin analogs, i.e., neither iloprost, a stable PGI2 analog (4), nor 16,16-dimethyl-PGE2 mimicked the action of the cytokine. Therefore, we focused on a potential involvement of TxA2 in the mediation of the TNF-
response. Indeed, 1-benzylimidazole, a thromboxane synthase inhibitor (26), and BAY U 3405, a thromboxane receptor antagonist (19), inhibited the action of TNF-
(Fig. 1). In contrast, cTxA2, a stable TxA2 derivative (5), mimicked the TNF-
effect after long- and short-time administration (Fig. 2). This suggests that TNF-
stimulates the production of TxA2, which then affects the myenteric neurons. An interaction between these pathways has already been observed in human monocytes (2), although, in cells, TxA2 stimulates the synthesis of TNF-
.
A prerequisite for the formation of thromboxane is the conversion of arachidonic acid into PGG2 and PGH2 by COXs. Two different isoforms of COX are known: the constitutively expressed COX-1 and an inducible form, COX-2 (31). Increased COX-2 activity can be found during inflammation in nearly every organ or tissue, including the gut (29) and nervous system (16, 20, 25). This upregulation was manifested by PCR (13) and Western and Northern blotting (21) as well as immunolabeling (16) experiments. Our results demonstrate an upregulation of COX-2 after TNF-
treatment at rat myenteric ganglionic cells as shown by immunocytochemistry (Figs. 5 and 6). In this preparation, COX-2 is already constitutively expressed but clearly upregulated after exposure to the cytokine. It is not clear whether the basal expression of COX-2 might be induced by the isolation and culture procedure; however, in human colonic specimens, COX-2 mRNA was found already under control conditions (25). Double labeling with a neuronal marker, MAP2, confirmed that the cells within the ganglionic culture expressing this enzyme were neurons. Consequently, it seems reasonable to conclude that TNF-
stimulates COX-2 expression at enteric neurons followed by the production of TxA2 and a change in the basal electrophysiological properties of the enteric neurons.

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Fig. 6. Schematic drawing summarizing the action of TNF- produced, e.g., by macrophages on enteric neurons.
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A strong stimulation of the content of TxB2, the stable metabolite of TxA2, which even exceeded the production of PGE2, has been measured in rectal dialysis specimens of patients with colitis ulcerosa (14). A stimulation of the production of TxB2 could also be observed in the ganglionic preparation after exposure to TNF-
(Fig. 3), suggesting a role for thromboxanes in the pathogenesis of intestinal inflammatory diseases. In general, the role of this arachidonic acid metabolite in gastrointestinal functions has only been scarcely studied compared with the detailed knowledge accumulated about the gut actions of prostaglandins or leukotrienes (for a review, see Ref. 32). However, because stimulation of thromboxane receptors has been shown to alter intestinal epithelial ion transport (6, 26), to evoke contractions of the longitudinal smooth muscle (5), to mediate antigen-induced muscle contractions (1), and to trigger spontaneous contractions and Ca2+ waves in intestinal smooth muscle reaggregates (8), TxA2 might be an important modulator of gastrointestinal functions acting at the epithelium, the smooth muscle, and the enteric neurons, the key players for the control of autonomous gut functions (Fig. 6).
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GRANTS
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This work was supported by Deutsche Forschungsgemeinschaft Grant Di 388-1.
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ACKNOWLEDGMENTS
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It is a pleasure to acknowledge Melanie Simon (Center for Internal Diseases, Giessen, Germany) for helpful cooperation in the measurements of TxB2 and Anne Siefjediers for expert help in the use of the image-analysis programs.
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FOOTNOTES
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Address for reprint requests and other correspondence: M. Diener, Institut für Veterinär-Physiologie, Universität Gießen, Frankfurter Str. 100, D-35392 Gießen, Germany (e-mail: Martin.Diener{at}vetmed.uni-giessen.de)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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