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Biomedical subjects

W Timmerman

Publications and source records attributed to W Timmerman.

At least 37 records · Page 2Linked to original sources

The significance of extracellular GABA in the substantia nigra of the rat during seizures and anticonvulsant treatments.

The effects of the anti-epileptic drugs valproic acid and gamma-vinyl-GABA (vigabatrin) on the extracellular content of GABA was determined by microdialysis. Probes were implanted in the substantia nigra reticulata (SNR) of rats. It was found that gamma-vinyl-GABA (1000 mg/kg) induced a 4-6-fold increase in the extracellular content of GABA. This increase lasted for at least 72 h. PTZ-induced convulsions were partly antagonized by the GVG treatment. The increase of extracellular GABA after gamma-vinyl-GABA was not affected by infusion of tetrodotoxin. In contrast valproic acid (200 mg/kg), although effective in preventing pentylenetetrazol (PTZ)-induced convulsions, did not affect extracellular GABA in the SNR. PTZ-induced convulsions did not modify extracellular GABA, neither in control rats nor in valproic acid or gamma-vinyl-GABA pretreated animals. The results do not support the idea that extracellular GABA in the SNR plays a significant role in anti-convulsive treatment. However, the present data can also be interpreted that extracellular GABA, as sampled by microdialysis, is not a reliable marker for GABA release.

Animals↗

Semicircadian rhythm of dopamine release in the mediobasal hypothalamus in awake rats during pseudopregnancy: evidence that a thyrotropin-releasing hormone analogue stimulates dopamine release and thereby inhibits prolactin secretion.

The release of dopamine (DA) from tuberoinfundibular (TIDA) neurons during prolactin (PRL) surge and nonsurge periods and the effects of the thyrotropin-releasing hormone (TRH) analogue CG 3703 on DA and PRL secretion were studied in awake pseudopregnant (PSP) rats by simultaneous measurement of extracellular DA levels in the mediobasal hypothalamus (MBH) by means of microdialysis and of plasma PRL concentrations by radioimmunoassay of PRL in blood sampled from a permanent heart cannula. Since basal DA levels were low, the experiments were performed under local infusion of the reuptake inhibitor nomifensine (5 mumol/l), which increased extracellular DA levels 5-fold around the probe, but did not affect the occurrence of the nocturnal PRL surge on day 7-8 of PSP. Under nomifensine conditions, during the interphase (18.00-1.00 h) plasma PRL levels were low (< 50 ng/ml), while the DA release from TIDA neurons was elevated (approximately 250%) relative to the DA values measured during the phase when the nocturnal PRL surge occurred (100%; 1.00-12.00 h; plasma PRL levels were elevated to approximately 300 ng/ml). Thus, semicircadian rhythms were detected both for DA and PRL, which were broadly reciprocal in timing. The TRH analogue CG 3703 (100 and 500 micrograms/animal i.v.), when administered at 00.00 h, dose-dependently increased extracellular DA levels (to 300 and 500% of pretreatment values, respectively) and postponed the PRL surge in a corresponding manner (for approximately 2 and 3 h, respectively). Thus, in addition to its PRL-releasing effects, TRH can also suppress the PRL secretion by stimulating the release of DA from TIDA neurons, thereby increasing the inhibitor DAergic tone on lactotrophs. During the interphase, when DA levels were relatively elevated, administration of the DA blocking agent HA 966 (100 mg/kg body weight i.p.) at 20.00 h resulted in a short-lasting decrease in extracellular DA levels (to approximately 60% of pretreatment values for 30 min) accompanied by an immediate, short-lasting increase in PRL levels (to 400 ng/ml for approximately 1 h). Administration of CG 3703 (500 micrograms/animal i.v.) in combination with HA 966 (100 mg/kg body weight i.p.) at 20.00 h prevented the decrease in DA levels and the rise in plasma PRL values. Thus, the TRH analogue and HA 966 counteracted each other regarding the DAergic system, which again illustrates the stimulatory effect of the TRH analogue on the release of DA from TIDA neurons. Moreover, our results suggest that the interphase and the phase during which the nocturnal PRL surge occurs under the condition of PSP differ with regard to the DAergic inhibition of the PRL secretion; during the interphase the DAergic inhibitory tone prevents the rise in PRL secretion, while during the nocturnal PRL surge period another factor in addition to a decrease in DA levels appears necessary to increase plasma PRL levels.

3,4-Dihydroxyphenylacetic Acid↗

Characterisation of the DA-ergic system in the mediobasal hypothalamus: a new approach to simultaneously monitor the release of DA from the TIDA neurons and the PRL secretion from the adenohypophysis in awake rats.

The TIDA neurons, which constitute part of the arcuate nucleus-ME complex, play an important inhibitory role in the regulation of the PRL secretion from the adenohypophysis. To simultaneously study the release of DA from the TIDA neurons and the PRL secretion from the adenohypophysis in awake rats, a microdialysis probe was implanted into the MBH together with a permanent heartcannula in male rats. The extracellular levels of DA in the MBH as measured by microdialysis decreased to 25% of basal values after local infusion of TTX (1 mumol/l), indicating that the released DA was directly derived from neuronal activity. DOPAC levels were not affected. This local infusion of TTX into the MBH induced parallel to the immediate decrease in DA levels, a profound increase in PRL concentration in the blood (from 10 to 55 ng/PRL-RP-2/ml) directly after infusion. Thus, the area in which the dialysis probe was inserted indeed included the DA-ergic neurons that regulate the PRL secretion. Evidence for a functional re-uptake system in the MBH was obtained by local infusion of the re-uptake inhibitor nomifensine (5 mumol/l) which induced an increase in DA release to 350% of basal values, without affecting the DOPAC levels. In spite of this increase in DA levels, the PRL concentration in the blood was not affected. In pseudopregnant female rats, relatively high levels of extracellular DA in the MBH were obtained during the interphase during which the PRL levels are low, while lower DA levels were apparent during the phase the spontaneous nocturnal PRL surge normally appears. Taken together, the approach presented in this study, i.e. the simultaneous measurements of DA in the MBH and PRL in the blood, establishes an advanced method enabling studies on the DA-PRL interactions in awake animals.

3,4-Dihydroxyphenylacetic Acid↗

Characterization of extracellular GABA in the substantia nigra reticulata by means of brain microdialysis.

Brain microdialysis was used to characterize extracellular gamma-aminobutyric acid (GABA) in the substantia nigra reticulata (SNR) of freely moving rats. The extracellular GABA in the SNR was characterized using acutely implanted probes (4-8 h after surgery; day 1) and chronically implanted probes (24 h after surgery; day 2). 3-Mercaptopropionic acid, a glutamic acid decarboxylase inhibitor, was used to identify GABA. This drug induced an immediate decrease in the extracellular GABA levels to 40% of basal values, suggesting that the detected GABA is, at least in part, newly synthesized. The basal levels of extracellular GABA measured either on day 1 or day 2 were not affected by infusion of micromolar amounts of tetrodotoxin. Therefore, a direct coupling between GABA dialysate concentrations and nerve-impulse flow does not seem to exist. Infusion of the GABA uptake inhibitor nipecotic acid (0.5 mmol/l) resulted in a 4-fold increase in the dialysate levels of GABA lasting at least for 3 h on both days. K+ stimulation (60 mmol/l) increased extracellular GABA levels in the SNR to 450% of basal values. This effect again did not differ significantly on day 1 and day 2. The origin of the extracellular GABA in the SNR, as recorded by microdialysis under the two experimental conditions, is discussed.

3-Mercaptopropionic Acid↗

Behavioral and cardiac responses after intracerebroventricular corticotropin-releasing hormone (CRH) administration: role of adrenal cortical hormones.

Intracerebroventricularly (icv) administered corticotropin-releasing hormone (CRH) produces a dose-dependent increase in heart rate in association with behavioral activation. The present study was designed to investigate whether these CRH-induced responses are dependent on adrenal function. The effects of adrenalectomy (ADX) and subsequent corticosterone replacement were studied. Administration icv of 300 ng of CRH failed to produce behavioral activation and tachycardia in ADX rats. Corticosterone replacement restored the CRH-induced behavioral response to preoperative levels, whereas the CRH-induced tachycardia was partially restored. This latter result may be related to the fact that the baseline heart rate of ADX animals appeared to be significantly higher than that of corticosterone-treated ADX animals. It is concluded that circulating adrenal corticosterone in ADX rats is involved in the expression of the behavioral and cardiac effect of central CRH.

Adrenal Cortex Hormones↗

Importance of the calcium content infused during microdialysis for the effects induced by D2 agonists on the release of dopamine in the striatum of the rat.

Brain microdialysis was used to investigate whether different calcium concentrations (1.2 and 3.4 mmol/l) of the perfusion fluid influenced the effects of D2 agonists on the release of dopamine in the striatum. We used the D2 agonists (-)N-0437 and (+)PHNO. After both local and systemic administration of (-)N-0437 and (+)PHNO, differences were apparent between their effects at 1.2 mmol/l calcium and 3.4 mmol/l calcium Ringer's solution. Although the drugs induced a similar maximal decrease in the release of dopamine with both calcium concentrations, the potency of the effect was significantly greater at 1.2 mmol/l when compared to 3.4 mmol/l calcium Ringer's solution. Thus, when measuring pharmacological effects of dopaminergic agents, it seems essential to use a Ringer's solution containing the physiological calcium concentration in brain microdialysis.

Animals↗

Enantiomers of monohydroxy-2-aminotetralin derivatives and their activity at dopamine autoreceptors as studied by brain dialysis.

The enantiomers of a series of dopamine (DA) agonists, monohydroxy-2-aminotetralin derivatives, were investigated using brain microdialysis. We used a 5-OH-substituted derivative, N-0437 (2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin), and three 7-OH-substituted derivatives, N-0438 (2-(N-propyl-N-2-thienylethylamino)-7-hydroxytetralin), 7-OH-DPAT (7-hydroxy-2-(N,N-di-n-propylamino)tetralin) and PHNO (4-propyl-9-hydroxynaphthoxazine; position 9 of the naphtoxazines corresponds to position 7 of the aminotetralins). We studied the activity of the enantiomers at autoreceptors regulating the release of DA following their local infusion into the striatum of the rat. We were particularly interested in the activity of R(+)-N-0437, S(-)-N-0438, S(-)-7-OH-DPAT and S(-)-PHNO, which are enantiomers that have been classified as less potent or inactive in previous studies. S(-)-N-0437, R(+)-N-0438, R(+)-7-OH-DPAT and R(+)-PHNO decreased DA release by 45-60%. Thus, these enantiomers are potent agonists at autoreceptors regulating the release of DA. The R(+) enantiomer of the 5-OH-substituted derivative N-0437 possessed antagonistic activity at autoreceptors controlling DA release, increasing DA release by 100%. This finding is consistent with reports showing that one enantiomer of other 5-OH DA agonists displays agonistic activity, while the other has antagonistic properties at DA autoreceptors. The less potent enantiomers of the 7-OH-substituted derivatives S(-)-N-0438, S(-)-7-OH-DPAT and S(-)-PHNO, however, all showed weak agonistic activity at DA autoreceptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

The potential antipsychotic activity of the partial dopamine receptor agonist (+)N-0437.

The (+) enantiomer of the very potent and selective dopamine D-2 agonist, 2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin (N-0437), displays partial agonistic activity at dopamine D-2 receptors. In this study (+)N-0437 was investigated for its antagonistic activity at postsynaptic DA receptors in four behavioural tests which are commonly used to evaluate potential neuroleptic activity, i.e. d-amphetamine-induced stereotypy, passive avoidance responding, intracranial self-stimulation behaviour, and catalepsy. (+)N-0437 (25-50 mumol/kg) was active in the first three models, but did not cause catalepsy. Haloperidol, which was used as a reference compound for classical DA antagonists, showed clear activity in all four models at low doses (0.5-1.0 mumol/kg). (-)N-0437, a full D-2 agonist, displayed no activity in these behavioural models. These results suggest that (+)N-0437 could be used to examine the hypothesis that the use of partial agonists could provide a new treatment for schizophrenia.

Animals↗

Dopaminergic-cholinergic interactions in the striatum: the critical significance of calcium concentrations in brain microdialysis.

Brain microdialysis experiments were performed to assess the effects of calcium (1.2 mmol/l and 3.4 mmol/l) in the perfusion solution on a variety of pharmacological treatments known to affect the release of dopamine (DA) and/or acetylcholine (ACh). Intrastriatal infusion of the muscarinic receptor agonist oxotremorine (100 microM), the selective dopamine D-2 receptor agonist (-)-N-0437 (1 microM), and the indirect DA agonists (+)amphetamine (10 microM) and nomifensine (1 microM) via the dialysis probe did not affect the overflow of ACh when the perfusion fluid contained 3.4 mmol/l calcium. In contrast, these compounds produced pronounced decreases in the overflow of ACh at 1.2 mmol/l calcium. Intrastriatal infusion of the muscarinic receptor antagonist atropine (1 microM) increased the output of ACh both at 1.2 mmol/l and 3.4 mmol/l calcium. The selective DA D-2 receptor antagonist (-)-sulpiride (1 microM) did not affect the overflow of ACh at either calcium concentration. Infusion of oxotremorine and atropine had no effect on the overflow of DA at either 1.2 mmol/l at 3.4 mmol/l calcium. (-)-N-0437 decreased and (-)-sulpiride increased DA overflow, both effects being independent of the calcium concentration in the perfusion fluid. Nomifensine and (+)amphetamine caused relatively (but not absolutely) larger increases in the overflow of DA at 1.2 mmol/l calcium. These findings emphasize the critical importance of the calcium concentration of the perfusion fluid in determining the nature of pharmacological responses in microdialysis experiments, and demonstrate that locally applied dopaminergic drugs can modulate striatal cholinergic function.

Acetylcholine↗

Effects of D-2 agonists on the release of dopamine: localization of the mechanism of action.

Brain microdialysis was used to localize the mechanism of action of the effect induced by the D-2 agonists (-)-2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin [(-)-N-0437] and (+)-4-propyl-9-hydroxynaphtoxazine [(+)-PHNO], on the release of DA in the striatum. Both agonists induced a stronger decrease in the release of DA when administered systemically in comparison to local administration. This suggests that the action of D-2 agonists is not exclusively mediated by autoreceptors regulating the release of DA at the level of the nerve terminals. By co-infusing nomifensine (10 microM) the effect of intrastriatally administered D-2 agonists on the release of DA could be completely abolished. As both agonists were effective when administered systemically in normal rats and in rats with kainic acid lesions performed in the striatum during nomifensine infusion, the effects induced by D-2 agonists seem to be partly mediated by autoreceptors situated on cell bodies, regulating the impulse flow of the neuron. In addition, D-2 receptors located on postsynaptic structures participating in the striatonigral feedback loops were suggested to be involved. (-)-N-0437 and (+)-PHNO were less effective after systemic administration when kainic acid lesioned rats were used in comparison with normal rats. Thus, D-2 agonists interact in a complex way with D-2 receptors for displaying their effect on the release of DA: autoreceptors situated on nerve terminals and on cell bodies as well as D-2 receptors located on postsynaptic structures participating in the striatonigral neuronal loops may all be involved to a certain extent in the mechanism of action of D-2 agonists.

Animals↗

In vivo evidence for the existence of autoreceptors on dopaminergic, serotonergic, and cholinergic neurons in the brain.

Intrastriatal infusions as well as systemic administration of the selective D-2 antagonist (-)-sulpiride caused similar increases in the dialysate levels of dopamine (DA) to about 180% of controls. A similar conclusion was drawn when the selective D-2 agonist (-)-N-0437 was infused intrastriatally or administered systemically: both routes of administration caused a decrease in the release of DA to about 40-50% of controls. In order to evaluate the properties of synthesis-modulating autoreceptors on dopaminergic and serotonergic neurons we have estimated the synthesis rate of serotonin (5-HT) or DA by monitoring the 5-HTP or DOPA formation in the dialysates during infusion of a decarboxylase inhibitor. Infusion of (-)-N-0437 decreased the DOPA formation, whereas infusion of (-)-sulpiride increased the dialysate levels of DOPA; these results indicate that the D-2 receptors controlling the synthesis of DA are localized on nerve terminals. Administration of the selective 5-HT-IA agonist 8-hydroxy-dipropyl-aminotetraline (8-OH-DPAT) resulted in a decrease in the synthesis rate of 5-HT. When 8-OH-DPAT was infused via the dialysis membrane, the agonist was unable to modify the release of 5-HT. The effects of infusion of the muscarinic agonist oxotremorine and the muscarinic antagonist atropine were dependent on the presence of the esterase inhibitor neostigmine in the perfusion fluid. In the absence of neostigmine, oxotremorine caused a pronounced decrease in the output of acetylcholine (ACh), whereas atropine was without effect. In the presence of neostigmine oxotremorine was without effect but infusion of atropine or other anticholinergics caused a pronounced increase in the dialysate levels of ACh. It is concluded that the autoreceptor controlling the release of ACh is of the M3-type and that the receptor is not fully occupied during normal conditions. In conclusion, microdialysis of neurotransmitters is a valuable tool for the study of autoreceptors in vivo. The presented studies provided evidence for the existence of autoreceptors controlling the synthesis and/or release of DA, 5-HT, as well as ACh in the striatum.

Acetylcholine↗

The effects of the enantiomers of the dopamine agonist N-0437 on food consumption and yawning behaviour in rats.

The enantiomers of the potent and selective dopamine (DA) D-2 receptor agonist 2-(N-propyl-N-thienylethyl-amino)-5-hydroxytetralin, N-0437, were tested for their effects on palatable food consumption and yawning behaviour in rats. (-)-N-0437 (1.0 and 5.0 mumols/kg). This confirms the agonistic action of (-)-N-0437 on postsynaptic receptors as food consumption is considered to be related to stimulation of postsynaptic DA receptors. Yawning behaviour was stimulated by (-)-N-0437 (0.5 mumol/kg) and could be antagonized by the autoreceptor-selective antagonist (+)-UH 232 (25 mumols/kg), which suggests an agonistic action on DA autorecptors. (+)-N-0437 (5.0 and 10.0 mumols/kg) also reduced food consumption and the effect could be antagonized by YM 09151-2 (0.03 mumol/kg). The weaker effect of (+)-N-0437 on food intake in comparison to that induced by (-)-N-0437 can be explained if it assumed that (+)-N-0437 is a partial agonist. (+)-N-0437 did not induce yawning behaviour in rats, suggesting that autoreceptors mediating the release of DA may be involved in stimulating yawning by DA agonists.

Animals↗

The enantiomers of the dopamine agonist N-0437: in vivo and in vitro effects on the release of striatal dopamine.

The enantiomers of the potent and selective dopamine (DA) D-2 receptor agonist 2-(N-propyl-N-2-thienylethyl-amino)-5-hydroxytetralin, N-0437, were tested for their pharmacological actions on DA D-2 autoreceptors in vivo, by measuring DA release by microdialysis during local administration of both drugs and in vitro, by measuring their effects on the electrically stimulated release of [3H]DA from striatal slices. In both experimental situations (-)-N-0437, at low doses, acted as an agonist on receptors controlling DA release. However, in vivo at a concentration of 10 microM (-)-N-0437 induced a short-lasting increase in DA release and in vitro the inhibitory effect of (-)-N-0437 was significantly less pronounced at higher concentrations (1-10 microM). (+)-N-0437 (0.1-10 microM) showed an antagonistic action both in vivo and in vitro. Following inhibition of the neuronal impulse flow with tetrodotoxin, (+)-N-0437 failed to increase DA release suggesting the effect of this enantiomer is not associated with an amphetamine-like action on the nerve terminal. The use of the DA re-uptake inhibitor GBR 12909 confirmed the antagonism of (+)-N-0437 towards DA receptors. Since it is known that (+)-N-0437 acts as an agonist on DA D-2 autoreceptors controlling the synthesis of DA, these results provide additional evidence for the existence of distinct DA D-2 autoreceptor populations involved in the release and synthesis of DA. The differential actions of (-)- and (+)-N-0437 gave rise to mutual antagonism of the actions of the enantiomers both in vivo and in vitro, thus providing a strong argument for using the enantiomers instead of the racemate in clinical situations.

Animals↗

Microdialysis and striatal dopamine release: stereoselective actions of the enantiomers of N-0437.

An intracerebral dialysis method was used to test both enantiomers of the very potent and selective dopamine (DA) D-2 agonist 2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin, N-0437, for their actions on DA receptors in the striatum of the rat. (-)N-0437 induced a 60% decrease in DA release, which was independent of the presence or absence of a kainic acid lesion placed unilaterally in the striatum. Stereotyped behaviour was apparent following administration of the (-) enantiomer. Thus, (-)N-0437 displayed an agonistic action on both pre- and postsynaptic D-2 receptors. (+)N-0437 did not induce any effect in the release model after peripheral administration nor did it induce any form of stereotypy. A comparison between the effects of (-)N-0437 after oral (10 mumol/kg) and transdermal (10 mumol/kg) administration showed the advantages of the latter mode of administration. Transdermal application induced a much longer duration of action of the drug (13 h) in comparison with the oral mode (5 h). Thus, transdermal administration may be a very useful method of drug application for therapeutic use.

3,4-Dihydroxyphenylacetic Acid↗

Hemorrhoidal ligation. A review of efficacy.

Two hundred forty-one patients undergoing hemorrhoidal rubber band ligation over a five-year period were reviewed to focus on complications. Three patients (1.2 percent) were hospitalized. Two, on oral anticoagulants, recovered from bleeding complications. One patient developed a band-related abscess that resolved with drainage. Twenty patients (8.3 percent) reported pain associated with the procedure. Three patients went on to subsequent surgical hemorrhoidectomy. There were no deaths in the series. This report agrees with previously published series highlighting the efficacy and cost containment of hemorrhoidal ligation. Continued reliance on this approach to hemorrhoidal disease is justified.

Abscess↗

Experimental endotoxemia in pregnancy: in situ glomerular microthrombus formation associated with impaired glomerular adenosine diphosphatase activity.

The mechanism of increased sensitivity for endotoxin in pregnancy as reflected by the formation of microthrombi in renal glomeruli is unknown. It has been shown that reduced glomerular diphosphatase (ADPase) activity in the rat kidney greatly increases the intraglomerular thrombotic tendency. We now studied experimental intraglomerular thrombosis ex vivo in association with glomerular ADPase activity in pregnant and nonpregnant control rats after infusion of either endotoxin or saline solution. Each animal (Wistar rat) was equipped with a permanent vena jugularis catheter and received either endotoxin (1.0 micrograms/kg body weight) (n = 6) or saline solution (n = 5) 7 days before being killed; nonpregnant rats were also treated with endotoxin (n = 5) or saline solution (n = 4). On day 21, before the animals were to be killed, they were anesthetized and their left kidneys were perfused with adenosine diphosphate solution (10 micrograms/ml) and platelet-rich plasma (1 x 10(9) cells/ml). Perfused kidneys were processed for light microscopy, electron microscopy, enzyme cytochemistry at the ultrastructural level, and immunohistology. The results showed decreased ADPase activity exclusively in the glomerular basement membrane of kidneys of pregnant rats treated with endotoxin in contrast to the findings in control rats. In addition, exclusively in the group of endotoxin-treated pregnant rats, significantly increased intraglomerular platelet aggregation could be detected after alternate perfusion ex vivo. We suggest that, in the present model, enhanced susceptibility of glomerular ADPase for endotoxin is due to pregnancy-associated factors that have yet to be identified. This increased susceptibility may promote in situ formation of intraglomerular microthrombi.

Animals↗

Presacral and postsacral extraspinal ependymoma. Report of a case and review of the literature.

A 29-year-old woman underwent surgery for an expanding subcutaneous postsacral mass. At surgery the mass was found to be an extraspinal ependymoma, completely separated from the central nervous system. The tumor was completely excised, with no evidence of recurrence 12 months postoperatively. Review of the literature reveals that only 17 cases of extraspinal postsacral ependymoma and 28 cases of presacral ependymoma have been reported. Postsacral tumors usually present as subcutaneous masses, but presacral tumors present with constipation, abdominal mass, neurologic deficit, and bony erosion. The treatment of choice for this tumor is wide local excision. Radiation may be of some value for local disease; chemotherapy has not been successful. Local recurrence has occurred in 23.5 per cent of the postsacral cases; 50 per cent of these patients died. The local recurrence rate is 14 per cent for patients with presacral masses, and the subsequent mortality rate has been 75 per cent. Distant metastases have occurred in three patients with postsacral tumors and in two patients with presacral tumors. The greatest chance for cure of this tumor lies with complete excision at the initial operation.

Adult↗