Neuropeptides (1991) l&69-73 @ Longman Group UK Ltd 1991

Ventricular Fluid Neuropeptides in Parkinson’s Disease. II. Levels of Substance P-Like lmmunoreactivity

H. CRAMER*, S. JOST”, C. REUNER”, E. MILIOSt, J. GEIGER* and F. MUNDlNGERt

*Department of Neurology and tDepartment of Stereotactic Neurosurgery, University of Freiburg, 7800 Freiburg, FRG

Abstract-Substance P-like immunoreactivity (SPLI) was determined in cerebroventricular fluid of patients with extrapyramidal motor diseases. Patients with Parkinson’s disease (PD) showed a SPLI concentration decreased by 30% compared with patients without extrapyramidal disease. No differences were apparent for patients with dystonia. Fluid obtained from the foramen Monro showed higher SPLI concentrations than fluid from a lateral ventricle, indicating that hypothalamic sources are important for ventricular substance P. Lateral ventricular SPLI was particularly low in parkinsonian patients which raises the possibility of a decreased SPergic activity in basal ganglia occurring in PD.

Introduction

Anatomic, biochemical and pharmacological evid- ence has associated the undecapeptide substance P (SP) with dopaminergic neurons in the striatum and substantia nigra (1-5). Post mortem studies of patients with Parkinson’s disease (PD) showed decreased cerebral concentrations of substance P-like immunoreactivity (SPLI) (6, 7). Nigro- striatal SPLI was found decreased also in patients with Huntington’s disease (8). Moreover, the issue of an involvement of SP in the pathophysiology of nigrostriatal dysfunction was raised when increased levels of SPLI were observed in lumbar

Date received 6 July 1990 Date accepted 6 August 1990

cerebrospinal fluid (CSF) of patients with PD (9). These authors concluded hypothetically that failing dopaminergic transmission might induce a compensatory increase in SPergic neuronal act- ivity, reflected by increased CSF SPLI. Among various brain regions studied the striatum, substantia nigra and the hypothalamus contain the highest concentrations of SPLI (10-12). It appeared, therefore, interesting to us to examine levels of SPLI in ventricular fluid of patients with PD and other movement disorders. Additionally, since CSF SPLI was found to comprise various molecular forms of SP (13)) in contrast to cerebral SPLI which is represented mainly by the authentic unadecapeptide (12), we separated these forms in CSF specimen by high performance liquid chroma- tography (HPLC) followed by radioimmunoassay (RIA).

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70 NHJROPEPTIURS

Patients and Methods

Patients

141 patients admitted for therapeutic stereotactic surgery were examined (Table 1). 68 patients (age ranged 37-80 years) suffered from PD with severe tremor and/or rigidity. The mean duration of disease was 5.1 f 0.4 years. 34 patients presented with extrapyramidal hyperkinesia: 15 of these had torsion dystonia (TD), 19 had spasmodic torti- collis. 13 patients presented with isolated tremor of limbs or head diagnosed as essential tremor which had lasted for 21.5 + 4.0 years until operation. Of the other patients 14 patients had chronic stable multiple sclerosis with myoclonia or intention tremor. Six patients had temporal lobe epilepsy. Six patients were admitted for action myoclonus as a sequela of traumatic cerebral hypoxia 7.3 f 1.5 years earlier.

For comparative purpose we also studied lum- bar CSF of 6 patients who underwent gaz cisterno- graphy for the suspicion of ponto-cerebellar angle tumor and whose CT-findings were negative.

Ventricular puncture targeted to the foramen Monro was performed on one side with a stereotaxic technique (14) under local anaesthesia between 9 and 11 a.m. The exact site of the puncture was ascertained by ventriculography. Fluid obtained before ventriculography and stereotaxic lesioning was immediately frozen and stored at -40°C until analysis.

Methods

Determination of substance P-like immunoreac- tivity

Patients were admitted because of resistance to medical treatment. The majority of patients with PD had been treated with L-dopa, bromocriptin or biperidin. Patients with epilepsy were medicated with phenobarbital (3); phenytoin (3), valproic acid (2)) carbamazepin (1) and clonazepam (1). Of the patients with essential tremor one received metixin chloride, one oxazepam.

SPLI was determined as described (16, 17) using an antiserum raised in rabbits which shows little crossreaction with the closely related undecapep- tide physalemine (15%), with cassinin (<3%), with substance IS (0.1%) and less than 1% crossreaction with eledoisine, bombesin and somatostatin. The antibody has an affinity for SP5-11 at least equal of that for SPl-11 and does not detect SPl-5 and SPl-7 which implicates that it

Table 1 Clinical data and ventricular CSF levels of total protein and Substance P-like Immunoreactivity (SPLI) in patients with Parkinson’s disease, dystonic syndromes, essential tremor, multiple sclerosis, postanoxic myoclonus, epilepsy and healthy controls with lumbar puncture. Results are shown as mean f. S.E.M.

Sex ratio Mean age Total protein Patient group Number f/m + S.E.M. (gW

I Parkinson’s 68 33135 60.1 + 1.1 0.15 + 0.01 disease

II Torsion 15 5/10 32.0 + 4.2 0.14 f 0.02 dystonia

III Torticohis 19 4115 42.2 + 2.7 0.17 + 0.02 IV Essential 13 5/s 56.8 rt 4.4 0.12 f 0.01

tremor V Multiple 14 915 38.4 + 2.3 0.20 + 0.02

sclerosis VI Epilepsy 6 115 35.3 + 4.6 0.18 f 0.05 VII Postanoxic 6 l/5 21.2 f 6.0 0.19 + 0.04

myoclonus VIII Controls with 6 l/5 43.8 + 5.3 0.41 f 0.06

lumbar puncture Total V-VII 26 11/15 33.7 f 2.5 0.19 + 0.02

* Significantly different from Total V-VII (P C 0.01) and Group V (P < 0.05). Student’s two-sided t-test.

SPLI (fmollml)

7.8 f 0.5 *

9.3 f 1.4

9.5 k 1.3 8.2 f 1.1

10.7 + 1.5

12.2 f 3.8 11.6 f 2.2

9.3 k 1.6

11.3 + 1.2

VJZNTRICULAR FLUID NEUROPEPTIDES IN PARKINSON’S DISEASE 71

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Figure High performance liquid chromatographic separation of substance P-like immunoreactivity in ventricular CSFof patients with PD. Horizontal bars indicate the elution volumes of authentic SPI-11, SP2-11, SP4-11, SPS-11, pyroglutamyl-SPS-11 and SPl-11-m. (A) CSF from lateral ventricle, means of nine patients, calculated for each fraction. (B) CSF from foramen Monro, means of four patients.

is directed exclusively against the C-terminal portion of the SP molecule. As a tracer we used 8-tyrosyl-SP (Peninsula Labs, Belmont, USA) which was iodinated by the cloramin-T-method and purified by ion-exchange chromatography on a 6 x lcm carboxymethyl-cellulose column (Whatman Co) using gradient elution starting with lOm1 of 0.002 molar ammonium acetate solution (pH 4.6) followed by 0.2 molar ammonium acetate solution (pH 4.6). The immunological identity between synthetic SP and SPLI from CSF was ascertained by preparing dilution curves of con- centrated CSF and synthetic SP, both curves showing parallelism. The detection limit of the assay was 15 pg/ml. Intra-assay and inter-assay variations coefficient were 5% and 11% respectively. The recovery of synthetic substance P added to CSF was 98%.

The chemical nature of SPLI was examined by reverse phase high performance liquid-chromato- graphy (HPLC) on a Spherisorb-ODS column with a gradient elution system consisting of 0.1 M triethylammoniumformiate (TEAF)/acetonitrile 75:25 and 0.14M TEAF/acetonitrile 50:50 of pH 2.5. As statistical tests for significance analysis of variance (ANOVA) and students two-sided t-test were used.

Results

Ventricular SPLI levels of patients from the com- parison groups were in the range of levels in lumbar CSF, obtained from neurologically intact

control patients (Tabie 1). For all patients cor- relations between sex or age and ventricular SPLI levels showed no influence of these variables (e.g. r - 0.128 for age and SPLI).

Analysis of variance (ANOVA) did not reveal any statistically significant differences between patient groups. However, patients with PD showed a SPLI level of 7.8 f 0.5 fmoYm1 which was 30% lower than the mean level of the group of patients with MS or other non-extrapyramidal diseases suggestive for a trend towards lower levels in.PD as also shown by t-test. Patients with other extrapyramidal disorders (torsion dystonia, torti- collis) also showed somewhat low levels of the SPLI with, however, no significant differences between groups.

Analysis of SPLI as to the exact site of puncture according to ventriculography revealed slightly higher mean levels for foraminal samples com- pared to supraforaminal samples (Table 2a & b). However, a statistically significant difference was only observed for the large patient group with PD.

Combined analysis with high performance liquid chromatography and RIA of CSF specimen from PD patients (n = 13) revealed marked heterogenity of SPLI (Figure). Major peaks co- eluted with SPl-11 (about 22%), X2-11 (13%), SP4-11 (10%) and SP5-11 (18%). Further peaks on the chromatograms were identified as pyroglu- tamyl-SPS-11 (11%) and sulfoxidated species of SP (12%). There were no differences between samples from the two compartments studied.

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Table 2a Clinical data and ventricular CSF levels of total protein and Substance P-like Immunoreactivity (SPLI) in patients with Parkinson’s disease, dystonic syndromes, essential tremor, multiple sclerosis, CSF obtained from lateral ventricle. Results are shown as mean + S.E.M.

Sex ratio Mean age Patient group Number f/m + S.E.M.

I Parkinson’s 23 1419 60.0 5 2.3 disease

II Torsion 5 2J3 32.2 + 5.0 dystonia

III Torticollis 6 115 40.8 + 4.7 IV Essential 9 316 61.0 + 5.5

tremor V Multiple 3 3/o 44.7 f 6.2

sclerosis VI Epilepsy 3 112 32.3 + 9.2

Total I-VI 49 24125 52.4 + 2.3

* Significantly different from Group I, Table 2b (P < 0.01). Student’s two-sided t-test.

Total protein

(gl!,

0.13 + 0.01

0.13 + 0.03

0.14 & 0.03 0.13 + 0.02

0.15 + 0.04

0.14 + 0.04 0.14 + 0.01

SPLI (fmollml)

5.8 + 0.6 *

7.1 + 2.0

11.3 f 3.6 7.9 + 1.6

10.4 f 5.3

12.8 + 3.1 7.7 f 0.8

Table 2b Clinical data and ventricular CSF levels of total protein and Substance P-like Immunoreactivity (SPLI) in patients with Parkinson’s disease, dystonic syndromes, essential tremor, epilepsy and multiple sclerosis, CSF obtained from foramen Monro. Results are shown as mean 5 S.E.M.

Patient group

I Parkinson’s disease

II Torsion dystonia

III Torticollis IV Essential

tremor V Multiple

sclerosis VI Epilepsy

Total I-VI

Number

45

10

13 4

11

3 86

Sex ratio Mean age Total protein SPLI

f/m + S.E.M. (gll) (fmollml)

19126 60.1 f 1.2 0.16 + 0.01 8.8 + 0.7

317 31.9 f 6.0 0.15 + 0.02 10.1 f 1.9

3110 42.8 f 3.4 0.18 rt 0.02 8.7 + 1.1 2l2 47.5 f 5.2 0.12 + 0.03 8.9 + 1.3

615 36.7 f 2.3 0.21 + 0.03 10.8 f 1.4

o/3 38.3 f 3.5 0.21 f 0.09 11.6 f 7.9 33153 49.9 * 1.7 0.17 f 0.01 9.3 + 0.5

Discussion

There are, at least to our knowledge, no published studies on cerebroventricular fluid SPLI in patients with basal ganglia disease, in contrast to studies of lumbar CSF SPLI. These reported SPLI levels to be unchanged (15,16) or increased (9) in patients with PD. This study shows that concentra- tions of SPLI in ventricular fluid of PD patients are low as compared to patients without extrapyrami- da1 disorders, arguing against an enhanced SPergic neuronal activity in PD (9).

most abundant in SP among various cerebral nuclei (10, 11). In this study, levels were found higher when fluid was sampled from the foramen Monro than from a supraforaminal level in the lateral ventricle, suggesting an important contri- bution from hypothalamic areas. Moreover, lowest SPLI levels were encountered in the lateral ventricle of parkinsonian patients. Since CSF levels of neuropeptides are believed to reflect the release from adjacent tissues (17, 18), this finding raises the hypothesis of a decreased utilization and release of substance P in basal ganglia in PD.

SPLI levels of striatum and hypothalamus, two The chemical identity of SPLI in CSF is of ventricle-lining structures, were reported to be the particular interest. Toresson et al (13) showed that

VENTRICULAR FLUID NEUROPEFTIDES IN PARKINSON’S DISEASE 73

the bulk of SPLI in lumbar fluid did not coelute with synthetic SPl-11, although producing dilution curves that were parallel to the SP standard curve, using a C-terminally directed antibody. Upon trypsinization most of the SP-like material co- eluted with authentic SP. Those authors concluded that CSF SPLI should exist mainly in an N-termi- nally extended form, possibly related to SP-pre- cursors previously isolated from human brain tissue (19). In our system heterogeneity of SPLI is clearly demonstrated. However, in contrast to the study of Toresson et al we detected a major fraction of SPLI coeluting with the undecapeptide and with C-terminal fragments like SP2-11 and SP5-11. The latter fragment appears to have biological effects similar to the undecapeptide (20). Further studies are necessary to clarify the significance of various forms of SPLI in the CSF and possible differences between CSF compartments.

Acknowledgement

This work was supported by the Deutsche Forschungsgemeins- chaft (contracts CR37/3-1 and CR37/3-2). We are indebted to Mrs U. Piotrowski for valuable technical assistance and to Miss C. Settele for carefully typing the manuscript.

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