cerebrospinal fluid of the rat: decrease after mediobasal hypothalamic lesion and hypophysectomy, increase after adrenalectomy Immunoreactive ACTH in the

Istv\l=a'\nBarna Institute

of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary

cerebrospinal fluid of the rat: decrease after mediobasal hypothalamic lesion and hypophysectomy. increase after adrenalectomy. Acta Endocrinol 1992:126: Barna I. Immunoreactive ACTH in the

350-6. ISSN 0001-5598

The effect of various anaesthetics and of the manipulations of the hypothalamo-pituitary-adrenocortical system (hypophysectomy, adrenalectomy and lesion of the mediobasal hypothalamus) was studied on immunoreactive-ACTH levels in the plasma and in the cerebrospinal fluid in the rat. The anaesthetics used (Hypnorm. pentobarbital, urethan and Ketanest) were without effect on immunoreactive-ACTH concentration in the cerebrospinal fluid. Immunoreactive-ACTH was significantly decreased after hypophysectomy and elevated after adrenalectomy in both cerebrospinal fluid and plasma. Destruction of the mediobasal hypothalamus resulted in reduced immunoreactive-ACTH content in the cerebrospinal fluid (about 20% of control) in both experiments, whereas immunoreactive-ACTH levels in the plasma of the lesioned rats were lower only in one of the two experiments performed. These data suggest that the main source of the immunoreactive-ACTH in the cerebrospinal fluid of the rat is the hypothalamus: the contribution of the pituitary gland being less than 50% of the radioimmunoassayable ACTH. István Barna, Institute PO Box 67, Hungary

of Experimental Medicine. Hungarian Academy of

Adrenocorticotropin (ACTH), ß-endorphin (ßE) and a-melanocyte-stimulating hormone (a-MSH) are syn¬ thesized as parts of the proopiomelanocortin (POMC) precursor molecule (1, 2). The behavioural and neurochemical effects of neuropeptides belonging to the ACTH family following central administration are well docu¬ mented (3, 4). The richest source of POMC-derived peptides in the rat is the pituitary gland, but they were found in significant quantities in the central nervous system (5,6) and in trace amounts in several peripheral tissues (7) as well. In addition, radioimmunoassay (RIA) or combined chromatographic-RIA methods revealed the presence of a number of POMC-derived peptides in the cerebrospinal fluid (CSF) of the rat (8-11). The major part of the immunoreactive-ACTH (ir-ACTH) content in the rat CSF seems to be the 14 kD glycosylated form of ACTH (8). The origin of the ACTH-related peptides in the brain and CSF is quite unclear. Anatomical and physiological data suggest the existence of a retrograde transport of hormones from the pituitary gland to the central nervous system via the portal vessels (12-14). On the other hand, POMC-containing cell bodies have been identified immunocytochemically in the arcuate nucleus of the hypothalamus and in the nucleus tractus solitarius (15, 16). In the hypothalamus, biosynthesis of the

Sciences. H-1450

Budapest,

POMC molecule has also been demonstrated (17). Data suggest that POMC-derived peptides in the CSF and in the plasma form two separate pools representing various sources under different regulation (9, 18-20); further¬ more, that ßE and a-MSH in the CSF are of central origin and their concentrations reflect brain activity (9, 21-

25).

To get information

on

the site of origin and

on

regulation of ir-ACTH in the CSF we tested the effects of hypophysectomy (HYPOX), adrenalectomy (ADX) and mediobasal hypothalamic lesion (MBHX) on the irACTH concentration in the CSF and in the plasma of the rat.

Materials and methods Anaesthetics Aether: Chinoin, Budapest, Hungary, Hypnorm: Janssen Pharmaceuticals Ltd, Oxford, UK; 1.0 ml/kg, intramus¬ cularly. Pentobarbital (Pentobarbitone Sodium): May and Baker Ltd, Dagenham, UK; 40 mg/kg, intraperitoneally (ip). Urethane: Reanal, Budapest, Hungary, 1.4 g/kg, ip. Ketanest: Parke Davis Co., Munich, Germany, 120

mg/kg, ip.

Cerebrospinal ACTH in the rat

ACTA endocrinología 1992. 126

Fig. 1. Photomicrograph of the mediobasal hypothalamic lesion (MBHX) on the eighth day after surgery ( x 22). Horizontal bar represents 1 Notice that the lesion destroyed the arcuate region of the hypothalamus but left a small piece of fairly normal median eminence tissue.

Animals and surgery Male Wistar rats ( 2 2 0-2 5 0 g ) of our inbred colony were used. The animals

were

maintained four per cage in

a

temperature (22-24°C) and humidity (55-75%) con¬ trolled room with a 12:12 h light-dark cycle (lights on at 07.00). Standard rat chow and tap water was available ad libitum. Hypophysectomy was performed under aether anaesthesia by the transauricular

approach. Sham operated (SHAM) rats were subjected to

the

same

surgery, but the

pituitary

was

not removed.

Adrenalectomy was performed under pentobarbital an¬ aesthesia. Destruction of the mediobasal hypothalamus was carried out under pentobarbital anaesthesia using stereotactic apparatus and a triangular shaped knife that

3.0 mm wide (26). Sham lesioned animals were prepared for surgery and the knife was towered to the brain in the saggital plane only for 4 mm depth without rotation. The completeness of HYPOX and MBHX were verified on serial sections and that of ADX by macro¬ scopic investigation. For checking HYPOX and MBHX (Fig. 1 ) the sellae and the brains were kept for histological investigation, respectively. Animals with incomplete was

HYPOX, ADX

or

MBHX were excluded from the

experi¬

ments.

Cannulation and

sample collection

Sampling of CSF and trunk blood from HYPOX, ADX and MBHX and from SHAM rats was performed 8-9 days after operations under pentobarbital anaesthesia between 10.00 and 12.00. CSF was obtained by cannu-

351,

mm.

lation of the cisterna magna: a hole was drilled in the midline immediately rostral to the interparietal-occipital bone suture (2 7). Cannulae, 20 mm long, were prepared from stainless steel hypodermic needles (o.d.: 1.1 and i.d.: 0.8 mm). Stylets in cannulae were made from stainless steel wire (d.: 0.75 mm). To keep the appro¬ priate 8.5 mm depth ofcannulation, pieces of 5 mm long silicone tubing were fitted onto the cannulae. These were cut off at an approximately 60° angle. Cannulae were fixed to the skull with dental cement. The stylets were removed 30-3 5 min after cannulation, an appro¬ priate length of polyethylene tubing was fixed onto the cannula and 80-100 yl clear CSF was withdrawn into the tubing with slight negative pressure. Duration of the CSF withdrawal was about 20-30 sec. CSF samples were collected on ice, then centrifuged and stored at 20°C until assayed. After CSF withdrawal rats were decapi¬ tated and trunk blood was collected on ice in polypropy¬ lene tubes containing 100 yl of 20% EDTA. Aliquots of plasma were stored at 20°C until assay of ACTH. —

—

ACTH

radioimmunoassay and statistical evaluation

Plasma and CSF ir-ACTH were determined by direct radioimmunoassay (28). The antibody we used (no. 8514) is directed against the mid-portion of the h-ACTHi_j9 molecule and shows 0.2% cross-reaction with a-MSH. The antiserum does not significantly crossreact with y-MSH, CLIP, ACTH, ,_24, ACTH25-39. ACTHj_ 14 and ACTHi_i9. The sensitivity of the assay was 0.2

fmol/tube (0.9 pg/tube) using h-ACTHi_39 (NIDDK) as labelled hormone iodinated by the chloramine-T meth-

352

acta endocrinologica 1992. 126

lstván Barna

Aliquots

of

pooled CSF (jj\)

12.5 25.0 50.0 100 100

ba

Hy fmol ACTH/tube Fig. 2. Parallelism of serial dilutions of pooled rat cerebrospinal fluid (CSF) with the standard curve in the ACTH radioimmunoassay. Two separate pools of CSF were collected from pentobarbital anaesthetized rats and serial dilutions were made with radioimmunoassay buffer. Each point represents the mean of triplicate (standard curve, open circles) or duplicate (Pool 1, open triangles: and Pool 2, full triangles) determinations.

ur

Fig. 3. Immunoreactive ACTH (ir-ACTH) levels in the cerebrospinal (luid (CSF) of the rat during different anaesthesiae. (Hy: Hypnorm; ba: pentobarbital; ur: urethane: Ke: Ketamine.) Rats were anaesthetized and cannulated between 10.00 and 12.00. Data are presented as mean±SEM (N). No significant differences were observed.

(3.6±0.42(N=8)pmol/lvs6.1±0.33(N=7)pmol/lin controls). Ir-ACTH levels were high in the CSF and in the plasma of ADX rats (Fig. 5), but the increase in CSF (22.3 ± 1.17 (N 9) pmol/1 vs 12.9±0.68 (N 9) pmol/1 in sham operated rats) was smaller than in plasma (176.4± 22.65 (N 9) pmol/1 vs 77.3±15.59 (N 9) pmol/1). MBHX resulted in a significant decrease of ir-ACTH =

od. Intra- and inter-assay coefficients were 4.7 and 7%, respectively. In this study 50 yl aliquots of plasma and 40 yl aliquots of the individual CSF samples (plus 10 yl RIA buffer) were assayed except the displacement experi¬ ment (Fig. 2). Ir-ACTH levels (pmol/1) are given as mean ± sem with numbers of determinations in paren¬ theses. Analysis of variance was followed by Dunn's or Dunnett's test (29, 30) as appropriate.

Results

Ke

=

=

=

concentrations in the CSF in both

experiments (MBHX Exp. 1: 1.1±0.39 (N 8) pmol/1 vs 5.8±1.26 (N 8) pmol/I, data are not shown in the figure; MBHX Exp. 2: 5.3 ±0.91 (N=9) pmol/1 vs 22.6±1.31 (N 9) pmol/1, Fig. 6). Plasma ACTH levels of the lesioned rats did not =

=

=

decrease significantly in the first MBHX experiment, but we observed a significant difference in the second MBHX experiment (MBHX Exp. 1: 29.1 ± 3.50 (N 8) pmol/1 vs 51.5 ±10.90 (N 8) pmol/1, data are not shown in the figure; MBHX Exp. 2: 33.4±7.33 (N 8) pmol/1 vs 155.9±43.65 (N 8) pmol/1 Fig. 6). =

In our ACTH-RIA system serial dilutions of CSF pooled from several animals paralleled the calibration curve (Fig. 2) validating the assay for ir-ACTH measurement in the CSF.

The effect of four different anaesthetics: Hypnorm, urethane and Ketanest on ir-ACTH con¬ centration in the CSF is shown in Fig. 3. There were no significant differences in the mean ir-ACTH levels of the four groups (Hypnorm: 12.9±0.88 pmol/1 (N=7); pentobarbital: 9.9Ü.52 pmol/1 (N=7); urethan: 11.7±0.59 pmol/1 (N 9); Ketanest: 13.9± 1.51 pmol/

=

=

=

pentobarbital,

Discussion

KN=7)).

The present results confirm that rat CSF contains significant amounts of ir-ACTH. Under the conditions used in this study, we found levels ranging from 5 to 25 fmol/ml which is in agreement with the data reported by Carnes et al. (10) and Kiser et al. (11). These experiments were performed over a period of about one year and considerable variation of CSF ir-ACTH levels between the different control groups was seen (sham operation to HYPOX, to ADX and to MBHX Exp. 1 and 2;

=

Both CSF and plasma ir-ACTH levels were signifi¬ cantly reduced after HYPOX (Fig. 4), but the change was more pronounced in plasma (1.1 ±0.08 (N 8) pmol/1 vs 94.9 ± 10.40 (N= 7) pmol/1 in controls) than in CSF =

Cerebrospinal ACTH in

ACTA ENDOCRINOLOGÍA 1992. 126

the rat

353

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CO

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