Acta Physiol Scand 1979. 106: 271-279

Structural and functional ontogenetic development of the rat portal vein after neonatal 6-hydroxydopaminetreatment BENGT LJUNG, JAN M. LUNDBERG, ANNICA DAHLSTROM and ANN KJELLSTEDT Department of Physiology and Institute of Neurobiology, University of Goteborg, Sweden

LJUNG, B., LUNDBERG, J. M., DAHLSTROM, A. & KJELLSTEDT, A.: Structural and functional ontogenetic development of the rat portal vein after neonatal 6-hydroxydopamine treatment. Acru Physiol Scand. 1979, 106: 271-279. Received 18 Dec. 1978. ISSN 0001-6772. Department of Physiology and Institute of Neurobiology, University of Goteborg, Sweden. The importance of the adrenergic vasomotor nerve supply for the vascular ontogenetic development has been studied in the isolated portal vein preparation from rats, at 5-6 weeks of age, who had either been chemically sympathectomized by a series of postnatal 6-hydroxydopamine (6-OHDA) injections or been receiving the solvent alone. It was found that 6-OHDA treatment largely, but not completely, prevented the outgrowth of the terminal NA fluorescent ground plexus. Nevertheless, the media underwent a seemingly normal differentiation into two layers. Functionally, the portal vein from the 6-OHDA treated animals displayed weak and non-persistent myogenic spontaneous activity; sensitivity to exogenous noradrenaline (NA) was increased 3-fold and maximum stress was increased by 25 % as compared to control. Responses to transmural field stimulation were only obtained at high impulse rates and the maximum response was attenuated. Considering the very sparse adrenergic innervation following 6-OHDA they seemed surprisingly large, however, but since they were abolished by tetrodotoxin and by phenoxybenzamine responses are concluded to be neurogenic and adrenergic in origin. A singular attenuation of neurogenic responses by atropine was found in 6-OHDA treated vessels but not in controls. It is concluded that the adrenergic vasomotor nerve supply seems to exert some trophic influence during ontogenetic development but that the morphologic vascular development is largely governed by other, non-neurogenic mechanisms. As to functional development, 6-OHDA induced sympathectomy causes impaired development of phasic myogenic activity whereas maximum stress is augmented as is the tissue sensitivity to exogenous NA. Key words: Adrenergic vasomotor nerves, rats, ontogenesis, portal vein, trophic effects, vascular neuroeffector, 6-OHDA

In most blood vessels the terminal adrenergic vaso- indicating a generalized P-adrenoceptor distribution motor nerve supply does not reach the individual (Belfrage 1978). smooth muscle cells but is confined to a two-dimenThe media of the adult rat portal vein is divided sional plexus located at the adventitiomedial junc- into an outer longitudinal and an inner circular layer tion. Furthermore there is indirect evidence that in of smooth muscle. The adrenergic ground plexus is the isolated rat portal vein (see Ljung 1976), and in located between these layers (Johansson et al. at least some arterial resistance vessels (see Bel- 1970). At birth, the vessel wall consists of undiffrage 19781, the a-receptor sensitivity also to exo- ferentiated cells and the adrenergic nerves are regenous noradrenaline (NA) is primarily located to stricted to bundles of nonterminal axons in the adthe smooth muscle cells in close contact with the ventitia. During the first few weeks of life the media adrenergic nerve terminals. The P-adrenoceptor becomes differentiated and oriented into the two mediated, inhibitory, vasodilating influence, how- muscle layers. The nerve terminals penetrate the ever, seems to be elicited throughout the media outer, longitudinal layer and ramify on its inside to Acto Plrysiol Scond 106

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B. Ljiing et

LII. of interest to explore the role of the adrenergic vasomotor nerve supply in the ontogenetic development of the vascular effector system. In the present experiments the portal vein of rats chemi-

cally sympathectomized by intense postnatal 6-hydroxydopamine (6-OHDA) treatment (cf. Provost, Bohus & d e Jong 1974) has been studied and compared to control. METHODS

Fig, 1 . Cross-sections of portal veins from (A) a control

rat and (B) a 6-OHDA treated rat. Both rats were 5 weeks of age and pretreated with NA ( 5 pglkg i.v.) 30 min before sacrifice to increase the fluorescense of adrenergic terminals. Note that division of the media into a heavy outer (longitudinal) muscle layer and an inner (circular) layer. The most pronounced terminal plexus can be seen on the inner aspect of the terminal layer in A after 6-OHDA treatment very few adrenergic fibres ( A ) are seen between the muscle layers. Fluorescence micrograph, bar indicates 40 pm.

form a plexus between the two media layers (Lundberg et al. 1976). Correlated to this morphologic development a dramatic functional development occurs (Ljung & Stage 1975). The corresponding stages of ontogenesis of the portal vein occur prenatally in species more mature at birth than the rat (Stage & Ljung 1978). Considering the asymmetrical vasomotor nerve supply and the differentiated adrenoceptor distribution in at least some sections of the vasculature it is

Rats of the Sprague-Dawley strain were mated in the laboratory in sufficient number to ascertain that at least two litters would later be delivered on the same day. Each mother rat nursed eight baby rats. Alternate litters received a series of subcutaneous 6-OHDA injections on the 1st. 2nd (100 pglg). 7th, 14th. 20th and 26th (250 pglg) postnatal day. Control litters received S.C. injections of vehicle alone (saline and ascorbic acid 200 pglml). At the time of the expt., during the fifth or sixth weeks of life, one control rat and one 6-OHDA treated rat, born on the same day ("matched") were sacrificed by cervical fracture and the isolated portal veins were either studied directly by histological or histochemical methods (see below) or mounted in an organ bath for isometric recording of contractile activity and thereafter examined morphologically. Orgun burh experiments. The two portal veins from the matched animals were mounted in the same organ bath containing 30 ml of Krebs' solution. One end of each tissue was tied to a tissue holder and the other end to a force transducer (Grass FT 0.03) under 2 m N passive force which was obtained by stretching the vessel to its approximate in vivo length. The tissues were allowed to accommodate in the bath for 1 h before the experiment was started. The active force was recorded on a Grass polygraph and the mean active force of the phasic activity was obtained by electronic integration of the force signal over I min periods. The integrator output was recorded on a separate channel of the polygraph. Responses to exogenous agents were induced by injections directly into the bath fluid in volumes of 0.3-0.9 ml. Concentration-effect relationships for exogenously administered noradrenaline (NA) were obtained by sequential NA administrations at 2 min intervals. The cumulated NA concentrations were: 3, 10, 30 nM . . . 3, 10, 30 pM.Neurogenic responses were elicited

Table I . Efiects of postnatal 6-OHDA treatment on body weight, left ventricular weight and cross sectional urea c?fportul vein measured at 6 weeks of age (mean k S.E.)( n =5) Control Body weight (g) Left ventricular weight (g) Cross sectional area, longitudinal smooth muscle of portal vein (mmz)

6-OHDA

P

0.35+0.016

90f4 0.29f0.013

0.001 0.020

0.14+0.003

0.10+0.007

0.003

121+5

Vuscrrlur ontogenesis after postnutul symputhectomy

Fig. 2 . Stretch preparations from (A) a control rat and (B) a 6-OHDA treated rat. Both rats were 5 weeks of age and the portal veins were mounted at the end of an organ bath experiment. Note the striking paucity of adrenergic nerve fibres after 6-OHDA treatment. The single remaining fibre, however, shows clear varicosities. Mast cells indicated by D. The symbol DD indicates a cluster of small strongly fluorescent cells sometimes observed in the adventitia of the portal vein. Fluorescent micrograph, bar indicates 40 p m .

by transmural field stimulation over 1 rnin periods by means of square wave pulses (0.8 ms; 15 V) at variable frequency. The impulses were delivered between platinum electrodes on either side of each preparation by a Grass stimulator (model S4B) and the voltage was monitored on an oscilloscope. Spontaneous myogenic contraction amplitude was determined as the maximum peak force recorded during a 3 min period prior to the first (‘‘early’’ phase of expt.) and the last (“late”) response which was induced in a particular expt. The amplitude was expressed in absolute (mN) as well as in relative terms (percentage of max force induced by NA). The latter, in turn, was determined as the highest deflection of the force recording obtained during the cumulative NA exposure. In all other respects the magnitude of induced responses were quantitated as the mean force recorded during the period of exposure minus the 18-795877

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mean force developed during the 3 min period of spontaneous activity immediately prior to stimulation. NA concentration-effect relationships were obtained by determining the least squares fit of such values from each expt. to a true hyperbolic function. ED, values were determined from the these functions and utilized in logarithmic form (-log ED,,(M)) in statistical calculations. Fluorescence hisrochemistry. Portal veins for histochemistry were dissected from 5-6 weeks old rats, killed by decapitation. The vessels were either ( a ) opened and stretched on a glass slide, as described previously (Lundberg et al. 1976) or ( b ) frozen (together with some splenic tissue for support) in liquid propane, cooled by liquid nitrogen. One series of tissues were mounted according to ( a ) at the end of organ bath expts. After drying over Siccapent” ( u ) or freezedrying in a Hetosic” (model Thieme) freeze dryer ( b ) , the specimens were reacted with paraformaldehyde vapour for 1 h at 80°C. as described previously (e.g. Corrodi & Johnson 1%7). After embedding in paratline the freezedried specimens were sectioned in 10 pm thick sections and mounted in Entellane@with some xylene. All specimens were examined and photographed in a Zeiss Junior fluorescence microscope for transillumination. Some vessels were-prior to freezing-incubated in a Krebs’ solution with Evans blue (10 pglml) in order to decrease the disturbing autofluorescence of elastin in the vessels (De la Lande & Waterson 1%8). By this treatment the green autofluorescence of the elastin fibres is changed into a bright red fluorescence, leaving the CA fibres green fluorescent as usual. To improve the visibility of possibly weakly fluorescent CA fibres in 6-OHDA treated vessels, some animals were given NA i.v. (50 pg/kg) 30 min before killing. This treatment did not alter the fluorescence microscopical picture of these vessels. Effector tissue cross sectional urea. In 5 expts. the tissues were repeatedly rinsed in Krebs’ solution over a 30 min period after the last exposure to NA to allow for return of control activity. The organ bath was then lowered and a beaker filled with Boin’s solution was brought into position for over night fixation at room temperature. The tissues were then dehydrated, embedded, sectioned at 4 pm and stained according to routine procedures. Micrographs (18x24 cm) of the total vascular circumference were then obtained of the 10 vessels and of a calibration scale. After coding, the examiner identified the longitudinal smooth muscle media layer, cut it out from the photograph, weighed it and calculated the cross sectional area by making reference to the weight of the micrograph of a I mm2square obtained in the same way. The code was then deciphered and the average cross-sectional area of the effector tissues and the average maximum stress induced by NA (mN/mm2) was determined for the two groups of tissues. Experimental protocol. Two separate sets of experiments involving functional studies were performed. Following the 1 h incubation period in the first set, where 6 pairs of preparations were studied, the vessels were exposed to transmural field stimulation at I , 4, 16 and 64 Hz at 15 min intervals. A cumulative NA dose-response curve was then determined. After a 30 min rinse Aclu Physbl Scund 106

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B . Ljrrng et (11.

-L

64Hz

81

Control

-

0 4 Hz

-

16Hz

64Hz

lowing composition in mM: NaCl 122, KCI 4.73. NaHCO, 15.5, KH,P04 1.19, MgClz 1.19, glucose 11.5, CaCI, and CaNa, versenate 0.026. The solution was continuously bubbled with 4% CO, in 0, and kept at 38°C. The drugs used were I-noradrenaline bitartrate ( I arterenol, Sigma Chemical Co) acetylcholine chloride (Merck); phenoxybenzamine HCI (Dibenzyline", SK & F); Tetrodotoxine (Sigma); 6-hydroxydopamine (H 88/32, AB Hassle). Statistics. Results are presented as mean f. S.E. Statistical significance has been tested by Student's t-test (pairing design where appropriate) and has been considered probable for P ~ 0 . 0 5 .

2 min

Fig. 3 . Tracings of spontaneous activity and responses to

transmural nerve stimulation at graded frequencies of portal veins from rats, 5 weeks of age, either treated during the neonatal period with 6-OHDA (top) or with vehicle alone (bottom).

RESULTS

development. The intense postnatal 6OHDA treatment seemed to retard the general development of the young rats as evidenced by a few days' delay of the appearance of fur and of the opening of the eyes. At the time of the expt., i.e. at period transmural nerve stimulation (NS) at 16 Hz was the age of 5-6 weeks, the 6-OHDA treated animals applied. The tissues were then exposed to the following displayed ptosis. Their body weight (83k4 g, mean sequence at I5 min intervals: Acetylcholine (ACh) 10 pM, 1 min; Atropine 1 pM, 30 min; ACh 10 pM, 1 min; NS 16 k S.E.. n=6) was 21+4% less than that of their Hz, I min; Tetrodotoxin I pM, 15 min; NS 16 Hz, I min. matched controls (104k6 g. P

Structural and functional ontogenetic development of the rat portal vein after neonatal 6-hydroxydopamine treatment.

Acta Physiol Scand 1979. 106: 271-279 Structural and functional ontogenetic development of the rat portal vein after neonatal 6-hydroxydopaminetreatm...
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