Chronic nerve growth factor treatment of normotensive rats

Brain Research, 538 (1991) 251-262 Elsevier

251

BRES 16228

Chronic nerve growth factor treatment of normotensive rats C. Zettler 1, R.J.

Head 2 and R.A. Rush 1

1Departrnent of Physiology, Flinders University of South Australia, Adelaide, S.A. (Australia)and 2CSIRO, Division of Human Nutrition, Adelaide, S.A. (Australia) (Accepted 7 August 1990) Key words: Sympathetic; Hypertension; Nerve growth factor; Vascular; Smooth muscle; Hyperplasia; Hypertroph3,; Catecholamine; Wistar-Kyoto rat

The objectives of this study were to examine the effects of chronic nerve growth factor (NGF) administration on vascular innervation and blood pressure in neonatal rats. Newborn Wistar-Kyoto (WKY) rats bred from normotensive parents were chronically treated with NGF for 8 weeks. Littermate controls received saline. Sympathetic ganglia of treated animals were greatly enlarged and in the superior cervical ganglion neuronal numbers were increased 200% and nuclear areas by 46%. The catecholamine contents of several sympathetically innervated tissues were determined biochemically and found to be significantly elevated in mesenteric arteries, aorta, ileum, adrenal and salivary glands from treated compared to control animals. The catecholamine concentrations were similar to, or exceeded those of the spontaneously hypertensive rat. Histochemically, an aberrant nerve supply was evident occupying a greater volume of the adventitia of the caudal artery and mesenteric arteries. In addition, nerve fibres could be seen penetrating the vessel wall to emerge within the lumen of mesenteric blood vessels. Analysis of the smooth muscle wall of the caudal artery revealed that a small but significant hyperplastic response had been induced. Systolic blood pressures of NGF-treated and control animals were taken at one week intervals from 5 to 8 weeks of age utilizing the tail cuff method. The blood pressures of treated animals were within the normotensive range. It is concluded that chronic NGF treatment leads to changes in vascular innervation and muscle thickness that are similar to those seen in hypertensive animals. Furthermore, the results suggest the elevated levels of NGF seen in peripheral tissues of the spontaneously hypertensive rat are likely to be responsible for the hyperinnervation and resulting hyperplastic responses within vascular tissues, but not exclusively responsible for the elevated blood pressure.

INTRODUCTION

after birth and prior to the d e v e l o p m e n t of hypertension16,31, 45.

The possible central role of the sympathetic nervous system in the d e v e l o p m e n t of hypertension in the spontaneously hypertensive rat ( S H R ) has been recognized by a n u m b e r investigators. Stimulation of the sympathetic outflow in pithed S H R results in a greater elevation of b l o o d pressure than W i s t a r - K y o t o ( W K Y ) controls 51. Differences in tissue concentrations of noradrenaline ( N A ) have been clearly established for a variety of b l o o d vessels of the S H R when c o m p a r e d to their W K Y controls 9'14. F u r t h e r m o r e , several histological studies have shown enhanced sympathetic innervation of the vasculature of the S H R , particularly within the mesenteric artery 31'32'33"45. In addition, Cassis et al. 9 has

Folkow et al. 17'18 suggested, on the basis of hemodynamic evidence, that the e n c r o a c h m e n t of medial smooth muscle cell layers in resistance arteries into a decreasing lumen volume is a primary characteristic of hypertension in the S H R . Sympathetic activity has been shown to exert a t r o p h i c effect on the d e v e l o p m e n t of b l o o d vessels 5, as well as the increased synthesis of vascular protein during the early hypertensive phase in the S H R 52. A causal relationship between an e n h a n c e d sympathetic innervation and vascular s m o o t h muscle cell hyperplasia has recently been established. Lee et al. 34, in a c o m p r e h e n sive study, d e m o n s t r a t e d that medial smooth muscle cell hyperplasia in muscular mesenteric resistance arteries of the S H R is absent after s y m p a t h e c t o m y p r o d u c e d by a c o m b i n e d t r e a t m e n t of antiserum to nerve growth factor ( N G F ) and guanethidine during the first 4 weeks after birth. These results suggest that an e n h a n c e d sympathetic innervation is involved in the induction of the hyperplastic response and altered reactivity o b s e r v e d within the vasculature of the S H R .

shown that caudal arteries from S H R have a greater n u m b e r of nerve axon bundles and a greater area of adventitia occupied by nerve bundles. Similarly, Lee and Saito 35 have d e m o n s t r a t e d that cerebral vessels from S H R are also m o r e densely innervated. M o r e o v e r , several of these studies suggest the increased sympathetic innervation of mesenteric arteries occurs immediately

Correspondence: R.A. Rush, Department of Physiology, Flinders University of South Australia, GPO Box 2100, Adelaide, S.A., 5001 Australia. 0006-8993/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)

252 Endogenous NGF concentrations and NGF gene expression in peripheral tissues correlate with the density of sympathetic innervation 29'46. In a recent study investigating the level of NGF gene expression in cardiac and vascular tissues, Curto et al. 12 demonstrated a 5-fold increased N G F - m R N A concentration in mesenteric arteries from 10-day-old SHR compared to age-matched WKY rats. This increased N G F - m R N A concentration is accompanied by elevated NGF concentrations 14. Since NGF is the only agent known which can induce a sympathetic hyperinnervation, it is possible that the elevated levels of N G F within the SHR is the primary cause of the aberrant innervation and, indirectly, the hyperplastic response of smooth muscle fibres in resistance vessels. If this hypothesis is correct, then the hypertension seen in the SHR may be the direct consequence of elevated N G F levels. The same effect might therefore be produced by direct NGF administration to young normotensive animals. This possibility has been examined by the chronic administration of NGF to newborn WKY rats for a period of 8 weeks.

MATERIALS AND METHODS

Animals WKY and SHR animals were obtained from the Ftinders Medical Centre (Adelaide) colony. Newborn WKY rats from a single litter were separated into 2 groups. The groups were treated every 2nd day from 48h after birth with 3 mg/ kg NGF (n = 6) in phosphate-buffered saline (PBS), pH 7.2 or PBS only (n = 3). A 2nd litter derived from the same sire provided a further 3 animals for the control group and one animal for the NGF-treated group. Animals were sacrificed at 8 weeks of age, 1-2 days after the last injection. NGF administration to one animal was continued to 11 weeks of age. SHR animals were untreated.

NGF purification and characterization Male mice (strain CBA inbred blacks) were obtained from the Waite Institute and the Institute for Medical and Veterinary Sciences, Adelaide. fl-NGF was isolated from submaxillary gland homogenates by a two-stage ion exchange chromatographic procedure according to the method of Mobley et al. 41. The purified fl-NGF was concentrated to 1 mg/ml (determined using the optical density of the solution and the known extinction coefficient for fl-NGF) in PBS, pH 7.2, by positive pressure ultrafiltration (Amicon, YM-10 membrane). To determine purity, a 5/~g sample of the pooled concentrate was loaded onto a 12% sodium dodecyl sulfate (SDS) discontinuous polyacrylamide gel (Mini Protean, Bio-Rad), prepared according to the method of Laemmli3°. No contaminants were detected. A modification of the classical dorsal root ganglion bioassay developed by Cohen et al) l was routinely employed to confirm the purified fl-NGF was biologically active in the ng/ml range.

Blood pressure recordings Indirect systolic blood pressure measurements were taken from conscious rats using a tail-cuff compression method. Rats were placed in a dark, quiet warming box (39 °C) prior to blood pressure determination. Systolic blood pressures were measured by tail cuff plethysmography with an electro-sphygmomanometer and a pneumatic pulse transducer (Narco Biosystems) connected to a Grass polygraph. The average of 6 consecutive measurements per animal

pcr session was taken as a measure of indirect systolic blood pressure. Blood pressure measurements were performed on all adult rats used for breeding, to ensure that each animal had been correctly classified as normotensive. Blood pressures of progeny were taken from 5 weeks of age at weekly intervals (to 8 weeks) and immediately before sacrifice. Similarly, systolic blood pressures of untreated age and weight matched SHR were monitored as a positive control, to demonstrate a rise in blood pressure could be detected in young spontaneously hypertensive animals by the tail-cuff method.

NGF antibody quantification by immunoassay An immunoassay was performed on the sera of treated and control rats to determine whether chronic administration of NGF resulted in the production of antibodies to the factor. Disposable 96 well Costar vinyl plates were coated with fl-NGF (I/~g/ml) in 0.1 M carbonate/bicarbonate buffer, pH 9.6, by incubating at 37 °C for 2 h. Wells were then washed (0.05% Triton X-100 detergent in PBS, pH 7.2) and blocked by incubation with 1% bovine serum albumin (BSA) in 0.05% Triton/PBS for 30 min. Sera from both the NGF-treated and control animals was serially diluted in 0.05% Triton/PBS (pH 7.2) and added to the wells and incubated at 37 °C for 2 h and then at 4 °C overnight. The plates were then washed, as described above, and 100 ~1 of goat alkaline phosphatase conjugated antibodies raised against rat IgG added to each well and incubated for 1 h at 37 °C. The goat alkaline phosphatase conjugated antibodies were prepared from a l mg/ml stock solution (Sigma), diluted 1/500 in 0.05% Triton/PBS with 1% fetal calf serum. Unbound goat antibodies were removed by washing and the alkaline phosphatase visualized by the application of a paranitrophenyl substrate solution (Sigma) at 1 mg/ml in prewarmed diethanolamine buffer (0.1 g/ml; in carbonate/bicarbonate buffer, pH 9.8 at 37 °C). After 15 min the optical density of each well was read on a ELISA microplate reader (Bio-Rad.) at a wavelength of 410 nm. No antibodies raised against the exogenous NGF were detected in the sera from either control or NGF-treated animals.

Histology A. Preparation of tissues. The superior cervical ganglia (SCG) were removed under microscopic control for morphological examination. The ganglia were immediately placed into modified Zamboni's fixative (2% formaldehyde in 0.1 M phosphate buffer with 15% saturated picric acid, pH 7.4) and left overnight. Ganglia were cleared of fixative in dimethylsulfoxide and PBS. The tissues were then dehydrated through increasing strength ethanol, cleared for 1 h in chloroform and embedded in paraffin. Serial 10/~m sections were cut and every 5th section taken, dewaxed and stained for 3 min with 0.025% Thionin blue in 0.05 M sodium acetate buffer, pH 7. Sections were mounted in Eukit (Zeiss). B. Counting procedure. In every section collected, all neurons containing a clear nucleus with at least one visible nucleotus, were counted. The total neuron number per ganglia was obtained by multiplying the summed samples by 5 and corrected for split nuclei by multiplying by t/(t+D) where t is the thickness of the section and D is the true mean nuclear diameter, according to Abercrombie 1. C. Determination of neuronal soma size. Nuclear area measurements within in the SCG from one control and one NGF-treated ganglia were taken by tracing neuronal nuclear circumference at 400x magnification with the aid of a digitizer. Area measurements were performed on 200 neurons from each specimen, selected at random from every 5th section collected. Size frequency histograms were generated for each ganglia using a computer-assisted program and the results were summed and reported as the mean percent of the total number of neurons for each size group. D. Haematoxylin and eosin stain. Caudal artery sections, prepared as described below, were stained with haematoxylin and eosin Y to determine their medial smooth muscle composition2. Smooth muscle layers were counted at a magnification of 1000x using an oil immersion objective, in several sections along the length of the artery. The averaged counts were used for statistical analysis.

253

GROWTH RESPONSE CURVE FOR WKY RATS 200 --o--

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20

30

40

50

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AGE (days)

Fig. 1. Weight gain of neonatal rats treated chronically with NGF compared with controls. There was no statistical difference in weights was observed at any age.

Immunohistochemistry To examine the distribution of perivascular nerve fibres, mesenteric arteries and lengths of caudal arteries (10 mm) immediately adjacent to the tail root were excised and immersed in Zamboni's fixative, as described above. Cryostat sections (10/~m) were cut and processed for indirect immunofluorescence. The primary antiserum, diluted 1:800 in PBS, was raised in rabbit against the general neuronal marker, Protein Gene Product 9.5 (PGP). The specificity of the PGP 9.5 antibody has been documented previously5°. The secondary antibody consisted of fluorescein isothiocyanate (FITC) conjugated to goat anti-rabbit IgG diluted 1:200 in PBS.

Catecholamine assay using high-performance fiquid chromatography (HPLC) The following tissues were used for catecholamine estimations after rapid excision; thoracic and abdominal aorta, mesenteric artery, heart, spleen, ileum, salivary and adrenal glands. All tissues were washed in ice-chilled saline, blotted, weighed, and placed in 0.4 M perchloric acid (PCA). An internal standard, dihydroxybenzoic acid (DHBA), was added to each sample to permit correction for loss of amines during isolation. Tissues were snap frozen at

BLOOD PRESSURE MEASUREMENTS SHR vs WKY 180

Fig. 3. Comparison of sizes of superior cervical ganglia from rats treated chronically with NGF (A) and controls (B).

-80 °C prior to assay. Tissues were homogenized with a Polytron tissue disperser and catecholamines isolated from the supernatant according to the method of Head et al. 22 and assayed chemically using HPLC. The noradrenaline (NA), dopamine (DA) and adrenaline (A) content of perchloric acid extracts of tissues was performed with a fully automated HPLC apparatus. The latter comprised solvent delivery systems (ETPOKORTEC, K25D pumps), an auto-injector (ETPOKORTEC, K65 automated sample injector), a 5 #m HPLC-grade alumina column, a reverse phase column (Waters C18), valve switching units (CSIRO Division of Human Nutrition), electrochemical detector (ECD, LKB model 2143) and chart recorder. The perchloric acid extracts were automatically adjusted to pH 8.4, transferred to the alumina column, eluted with low pH buffers and further purified on the reverse phase column. Samples were pumped through a silica column prior to loading onto alumina in 0.05 M Tris-HC1 buffer pH 8, with 100 mg/! EDTA (flow rate 1 ml/min) to isolate the catecholamines. Catecholamines were eluted from the alumina column with 0.05 M NaH2PO 4 and 0.1 M citric acid, pH 3.6, to which 100 mg/l EDTA, 50 mg/l sodium octyl sulfonate and 40 ml/! methanol had been added (flow rate 1 ml/min). Separation was achieved by passage through the C18 u Bondapak analytical column. Eluted catecholamines were detected electrochemically and their concentrations calculated by comparing peak heights with those of standard preparations (100 ng/ml of NA, A, DA and DHBA in 500

TABLE I --

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Comparison of mean organ weights from control and NGF-treated animals

SHR

Weights represent the mean of 6 organs with the exception of control hearts (n = 5). SCG, superior cervical ganglion.

Organ

t

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Weight (rag) NGF

I

5

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6 7 AGE (weeks)

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8

9

Fig. 2. Comparison of indirect systolic blood pressures from chronically NGF-treated, control and age-matched spontaneously hypertensive rats.

Heart Adrenal gland Kidney Salivary gland SCG

611.3 + 29.2 + 677.3 + 170.0 + 6.2 +

Control 51.3 6.7 69.3 23.1 1.5"*

** Significance level o f P < 0.001.

652.8 + 29.4 + 734.0 + 174.2 + 2.2 +

27.6 4.7 87.5 43.7 0.4

254 S.C.G. NEURONAL

NUCLEAR

AREA DISTRIBUTION

TABLE II Comparison of neuronal numbers and neuronal nuclear areas within the SCG from control and NGF-treated animals

50

Raw counts represent the mean 5 SCG. NGF

30

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20

a.

39,161+ 18,692 + 94.2 + 10.9 +

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3,609** 1,723 1.6pm z 1.4/~m

18,167 + 9,517 + 64.4 + 9.1 +

1,898 996 1.1 pm 2 1.2#m

** Significance level of P < 0.005. o

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SIZE (urn sq)

Fig. 4. The effect of NGF treatment on nuclear areas within neurons of the superior cervical ganglia. Values represent the mean percent of neurons within each size group.

animals w e r e o b s e r v e d , with the e x c e p t i o n o f s y m p a t h e t i c ganglia (Table I). Blood pressure

The /~1 0.4 M PCA to which 80 pl 2 M Tris, pH 10.6 had been added). Standards were run before and after every 4th sample. Final tissue catecholamine concentrations were calculated by compensating for internal standard recovery rates which ranged between 50 and 98% depending on the tissue examined. Statistical analysis All analyses were performed using a two-tailed Student's t-test. All data are expressed as the mean + standard error of the mean (S.E.M.).

mean

systolic b l o o d

pressure

of N G F - t r e a t e d

animals at 8 w e e k s of age (125.3 + 1.1 m m H g , n = 7) was not g r e a t e r t h a n saline t r e a t e d c o n t r o l s (131.8 + 1.8, n = 6, Fig. 2). N o r did any a n i m a l e x c e e d p a r e n t a l b l o o d pressure

(paternal

141.2 m m H g ;

maternal

129.1 and

139.5 m m H g ) , which are within t h e n o r m o t e n s i v e r a n g e (100-140 m m H g ) . N G F t r e a t m e n t to o n e a n i m a l for 11 w e e k s did not result in any o b v i o u s b l o o d p r e s s u r e c h a n g e f r o m the level r e c o r d e d at 8 w e e k s of age (124 + 1.9 m m H g ) . D i r e c t c o m p a r i s o n o f N G F - t r e a t e d animals (n = 6) to their l i t t e r m a t e c o n t r o l s (n = 3) r e v e a l e d no

RESULTS

significant d i f f e r e n c e s in their systolic b l o o d p r e s s u r e s T r e a t e d animals s h o w e d no visible d e t r i m e n t a l effects

(125.0 + 1.3 cf. 127.5 + 1.2 m m H g ) .

However the 3

of the N G F a d m i n i s t r a t i o n . B o d y weights of N G F - t r e a t e d

control animals f r o m the 2nd litter d e r i v e d f r o m a m o t h e r

animals i n c r e a s e d with age at the s a m e rate as controls

with a h i g h e r b l o o d p r e s s u r e (139.5 m m H g ) , s h o w e d a

(Fig. 1). Similarly, after sacrifice no significant differ-

slightly e l e v a t e d , t h o u g h still n o r m o t e n s i v e p r e s s u r e of

e n c e s in o r g a n weights b e t w e e n

136.1

control and t r e a t e d

+ 0.2 m m H g .

This v a r i a t i o n in c o n t r o l b l o o d

Fig. 5. Fluorescence micrographs of transverse sections of caudal arteries from control (A) and NGF-treated (B) rats. Immunohistochemistry was performed with PGP 9.5 antibodies to reveal all peripheral nerve fibres. Note the increased area of adventitia occupied by nerve fibre bundles in the tissue from the NGF-treated animal.

255

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Fig. 6. Fluorescence micrographs of transverse sections of mesenteric blood vessels from control (A,C,E) and NGF-treated (B,D,F) rats. Peripheral nerve fibres were immunohistochemically labelled with antibodies raised against PGP 9.5. Note the increased nerve fibre density at the adventitial-medial boundary (D) and within the adventitia (B,F) in blood vessels from treated animals, x 160, bar = 100/~m.

256 46% increase in mean neuronal nuclear area (Table 11). Examination of size frequency histograms showed a 55% shift towards larger nuclear areas ( > 8 # m e) in NGFtreated rats (Fig. 4). These results indicate that the increase in ganglionic volume, in response to N G F treatment, is in part due to the hypertrophy and hyperplasia of sympathetic neurons. However, the total increase in ganglion volume was also due to an obvious increase in the number of nerve fibres originating from within the ganglion, although this was not quantified.

Vascular innervation

Fig. 7. A mesenteric blood vessel from an NGF-treated rat, immunohistochemically labelled with PGP 9.5. Note the aberrant growth of nerve fibres into the lumen of the blood vessel, x 160.

pressures is responsible for the apparent lowering of pressure in the NGF-treated animals. Fig. 2 also shows a significant increase in blood pressure from 6 weeks of age in untreated S H R animals monitored at the same time.

Morphometric analysis of sympathetic ganglia Macroscopic examination revealed N G F had elicited a dramatic and obvious increase in the volume of all sympathetic ganglia from treated animals. These included the sympathetic chain at all levels, and coeliac ganglia. Fig. 3 shows the increased size of a representative SCG from an NGF-treated animal compared to a ganglion from a control animal. The chronic administration of N G F resulted in an 96% increase of total neuron number within the SCG and a

A

In both the mesenteric and caudal arteries, an aberrant nerve plexus was seen, which indicated an increased perivascular fibre density. Within the caudal artery, this abnormal growth was seen as an overabundance of nerve fibre bundles within the adventitia (Fig. 5). Similarly, the mesenteric arteries of treated animals displayed an increased number of nerve fibres within the adventitia, and additionally, an increased number of fibres within the mesentery (Fig. 6). A striking feature of this increased fibre growth within the mesenteric vessels, was the presence of nerve fibre bundles penetrating through the vessel wall into the lumen (Fig. 7).

Vascular smooth muscle High power microscopical analysis of haematoxylin and eosin stained caudal arteries revealed that within the smooth muscle wall, exogenous N G F treatment had induced a hyperplastic response (Fig. 8). Caudal arteries from control animals contained 7.5 + 0.3 (n = 6) smooth muscle cell layers whereas those from NGF-treated animals contained 8.8 + 0.2 (n = 6) layers. This

f l

Fig. 8. Photomicrographs of haematoxylin and eosin-stained transverse sections caudal arteries. Smooth muscle cell layers were increased from 7.5 in control tissues (A) to 8.8 in NGF-treated tissues (B).

257 difference was statistically significant at the level of P < 0.001. Catecholamine determinations In a number of tissues from chronically NGF-treated

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VASCULAR CATECHOLAMINE LEVELS IN WKY RATS

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animals, the NA, A and DA content was significantly greater when compared to corresponding tissues from untreated WKY controls (Figs. 9-11). This became strikingly apparent in the mesenteric arteries and aorta from chronically treated animals. Comparisons between

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Fig. 9. Catecholamine concentrations of vascular tissues (MES, mesenteric vessels; A O R . T H , thoracic aorta; A O R . A B , abdominal aorta) from control and NGF-treated rats. **Indicates significance at the P < 0.005 level.

-r Fig. 10. Catecholamine concentrations of non-vascular tissues (SAL: salivary gland) from control and N G F - t r e a t e d rats. *Indicates significance at the P < 0.05 level. **Indicates significance at the P < 0.005 level.

258 ADRENAL z 0 II,Z m

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Thoracic aorta. The NA content of the thoracic aorta from untreated animals was relatively low (0.17 + 0.04 /~g/g) and other amines, if present, were below the level of detection (< 10 ng/g). The NA content of treated animals however was elevated 1600% (P < 0.005) to that found in control tissues (Fig. 9). In addition DA concentrations were also significantly elevated (P < 0.005) to 117 + 50 ng/g. Abdominal aorta. The NA content per gram of tissue was greater in all treated animals and corresponded to a 420% increase (P < 0.005). A could not be detected in either control or treated animals. However, treated animals displayed significantly elevated levels of DA (55.5 ng/g, P < 0.005) whereas untreated animals were again characterized by non-detectable amounts of DA in the in the tissue preparation (Fig. 9). Heart. The NA content of hearts per gram of tissue was not elevated in treated animals (1.60 _+ 0.3/~g/g) when compared to untreated controls (1.22 + 0.13/ag/g). No significant change was seen for either adrenaline concentrations (109 + 25 vs 61.4 + 11 ng/g) or dopamine concentrations (41.5 + 5.0 vs 39.0 _+ 7.0 ng/g; Fig. 10). Spleen. NGF-treated animals showed no significant differences in catecholamine concentration per gram of tissue with respect to untreated controls (Fig. 10). Salivary gland. NA and DA contents of whole salivary glands were significantly greater (P < 0.005) in NGFtreated animals, corresponding to a 100% increase. In contrast no significant differences in A content were found between NGF-treated and untreated controls (Fig. 10). Ileum. The NA content measured per gram of tissue was significantly greater in NGF-treated animals, corresponding to a 170% increase over controls (P < 0.005). There was no corresponding increase in either A and DA levels when compared to controls (Fig. 10). Adrenalgland. A comparison of catecholamine content of adrenal glands from NGF-treated and untreated animals showed that within treated animals, tissue A and DA levels were elevated 84% (P < 0.005) and 149% (P < 0.005), respectively. NA levels were also increased by 40% (P < 0.005) over controls (Fig. 11). DISCUSSION

the catecholamine content of individual tissues are described below. The detection limit of the assay has been described previously as 10 ng/g of tissue 22. Mesenteric arteries. The catecholamine content was significantly greater in mesenteric arteries from treated animals (Fig. 9). The increase in NA and A content within this tissue was approximately 350% (P < 0.005). A 100% increase in DA content was also evident (P < 0.005).

Evidence for the effectiveness of NGF treatment NGF plays a key role in the development of several components of the sympathetic and sensory nervous systems. Changes in neuronal number and nuclear area within the SCG have been well documented during normal development, and in response to short-term NGF treatment 23,zs. During normal development there is a decrease in the total number of neurons in this ganglion,

259 at a time when peripheral connections are being established 7. The ability of postnatal N G F treatment to rescue neurons from naturally occurring cell death, is thought to reflect the survival of those neurons that would normally die following a competitive interaction for N G F within the target field 24'37. A. Ganglia. Gross dissection of the sympathetic chains of NGF-treated animals revealed enlarged ganglia at all levels as expected from previous findings 37. These changes were quantified in the SCG, where neuronal numbers were elevated more than 2-fold. Studies on the effects of N G F in the SCG of neonatal rats have reported increases in neuronal numbers of an average of 2- to 3-fold as well as increases in nerve fibres and number of non-neuronal cells 23'37. The present results therefore fall within the reported range for increased neuronal numbers. Furthermore, the results indicate that the additional neurons survive for at least 8 weeks after birth. However, whether this is associated with a continued requirement for exogenous NGF is not known. No obvious enlargement was seen in sensory ganglia, but no histological studies were performed. B. Perivascular nerves. Our study has shown not only an increase in the number of perivascular nerve fibres, but also an irregularity in the distribution of these nerves. In some aspects the innervation is similar to that of the SHR. Sympathetic innervation of the SHR has been extensively examined and most workers conclude that resistance vessels such as the caudal and mesenteric arteries receive an increased number of sympathetic nerve fibres 9A6'45. One striking difference between NGFtreated and SHR animals is the abnormal growth of nerve fibres into the wall of the mesenteric blood vessels, penetrating even into the lumen. This presumed chemotactic response of the fibres has been reported in early studies of chicken embryos carrying tumour implants. A sarcoma, later shown to be releasing NGF, was implanted onto the chorio-allantoic membrane. The circulating N G F stimulated the invasion by peripheral nerves of many blood vessels, forming large neuromas within the lumen 36. The neuronal marker, PGP, used in our study, does not allow the differentiation between sensory or sympathetic nerves, so the proportion of each fibre type could not be determined. However, preliminary studies using two specific markers, substance P and tyrosine hydroxylase (T-OH), did show that both fibre types were present in both the mesenteric and caudal arteries (results not shown).

Biochemical assessment of hyperinnervation The comparison of tissue NA levels in the SHR and its normotensive control (WKY) have been made in an attempt to understand the neural mechanisms that

underlie the initiation and/or maintenance of essential hypertension 9'1°'13'14. These studies have conclusively demonstrated elevated N A concentrations in a number of vascular and non-vascular tissues in the adult SHR when compared to their age-matched normotensive controls. Tissues which display elevated catecholamine levels (2- to 5-fold) include the mesenteric artery 3A5'22, renal artery z~, caudal artery 9"15, kidney 15'22, aorta 15'22, adrenal gland a5 and vas deferens 22. In a recent study by Donohue et al. 15, the appearance of elevated levels of NA in the mesenteric arteries was shown to be present from 2 days of age and occurred in other tissues (aorta, kidney, spleen and adrenal glands) before the development of hypertension, between 5 and 10 weeks of age. Both acute and chronic NGF administration to neonatal rats have been shown to induce increased synthesis of enzymes involved in catecholamine production within sympathetic neurons, including the rate-limiting enzyme T-OH 2s'47. Exogenous N G F stimulates T - O H activity in the superior cervical ganglia in a dose-dependent manner 28. This implies the availability of N G F to sympathetic noradrenergic neurons is involved in the regulation of neurotransmitter production via T - O H synthesis, and thus in the maintenance of neurotransmitter levels in the periphery. In our NGF-treated animals, there was a significant elevation of catecholamines in several tissues, but the effect was most pronounced in blood vessels. The most striking effects of NGF treatment were seen in the mesenteric arteries where the noradrenaline level was elevated by approximately 350%, and in the thoracic aorta, which rose from almost undetectable levels in the control animals to levels exceeding those of the abdominal aorta, suggesting a dense innervation had resulted from the treatment. In control animals only the abdominal aorta receives an innervation 8. The magnitude and distribution of catecholamine concentrations in the NGF-treated animals is remarkably similar to that reported for the SHR 9A5'22. The raised NA concentrations in the mesenteric vessels and adrenal glands, and the normal levels in the heart and spleen are almost identical in the NGF-treated and SHR animals. The only exception is the aorta, in which the NA concentration is greater in NGF-treated animals. These findings are in agreement with the hyperinnervation seen morphologically. Moreover, this close correlation between the two animals is supportive of the view that it is an abnormality in the production of NGF alone which is responsible for the hyperinnervation seen in the SHR. That aberrant fibres which we detected in the lumen of mesenteric vessels are not present in the SHR, probably indicates that the delivery of N G F in the SHR is not via the blood, but directly from cells within the particular

260 target tissues. This is consistent with both the observation that N G F is not normally detectable in blood 19 and the presence of N G F m R N A in peripheral tissues 12.

Arterial thickening in SHR Accelerated smooth muscle cell growth and increased peripheral resistance are characteristic features of arteries in genetically hypertensive animals 6'42. There is good evidence that the increased smooth muscle cell mass confers a functional predisposition to increased vascular resistance, as vessels have an increased maximal force generating capability and undergo proportionately larger luminal area reductions for a given level of smooth muscle cell activation 43. The observation that these changes occur before elevation in blood pressure, have raised the possibility that medial smooth muscle cell hyperplasia within resistance vessels may initiate the development of hypertension 17'18 (reviewed in ref. 20). In our study, there is a significant, but small, increase in the number of smooth muscle cell layers in the caudal artery. This increased wall thickness in the absence of an increase in blood pressure suggests that the hyperplasia is due to a t r o p h i c response of muscle to its innervation, rather than as a consequence of increased lumenal pressure. Other factors may also be important in this hyperplastic response, such as elevated plasma levels of angiotensin II seen in the S H R 4°, which have the potential to exert a t r o p h i c influence on vascular smooth muscle 39. Implications for the study of hypertension in the SHR Many factors appear to contribute to the increased total peripheral resistance observed in the SHR. These include a rarefication of the vascular bed and a general constriction of the vasculature (reviewed in refs. 20, 42). There is evidence to suggest that the general constriction is due to the combined effect of an increased sympathetic activity in visceral nerve fibres 25 27,38,48 and an enhanced sympathetic innervation of vascular tissues. The latter is thought to induce both structural differences in the resistance vessels i.e. medial smooth muscle cell hyperplasia 34,44 (reviewed in ref. 20) and altered vascular reactivity 9,49. It is not possible to determine from our study whether the enhanced sympathetic innervation is physiologically functional. It would therefore be interesting to examine, in a future study, the contractile

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response of hyperinnervated blood vessels in NGFtreated animals to nerve stimulation and pharmacological agents. The primary objective of this study was to determine whether chronic administration of N G F to neonatal animals would effect blood pressure. Our findings clearly indicate that blood pressure is unaffected by the treatment despite the dramatic effects produced on blood vessel innervation and medial structure. This was not due to technical difficulties of blood pressure measurement in young animals by the tail cuff method, since we were able to readily detect the rising pressure in age-matched S H R monitored at the same time. Since many of the changes observed in our treated animals were similar to those seen in the SHR, and elevated N G F and its m R N A have been seen in peripheral tissues of the SHR, it seems likely that the hyperinnervation of the S H R is due to elevated levels of NGF. However the absence of an elevated blood pressure in our treated animals argues that some other factor(s) is important in the initiation and maintenance of hypertension. The trophic effects of a hyperactive sympathetic nervous system 4 and angiotensin 1139 o n vascular smooth muscle are potential candidates. It is also possible that the level of sympathetic nerve activity in our animals is not identical to that of the SHR; an enhancement of which has been implicated in the initiation phase of the hypertension 25'26'27'48. A n o t h e r possibility is that in the SHR, N G F is elevated not only in peripheral tissues, but also in the central nervous system which would result in alterations to N G F sensitive neurons. It is unlikely that systemic N G F administration would reach the brain or spinal cord after closure of the blood-brain barrier. Whether the systemic N G F treatment had any effect on the central nervous system and sympathetic nerve activity will need to be investigated in future experiments. Finally, the failure of N G F treatment alone to induce hypertension may reflect the importance of other abnormalities in young SHRs, such as the renal reninangiotensin system.

Acknowledgements. We would like to thank Jim Vahaviolos, Phillipa Wilson and Dianna Bridges of the Department of Human Physiology, Hinders Medical Centre, Adelaide, and S. Jefferson of the Department of Human Nutrition, CSIRO, for their expert technical assistance throughout the year. This work was supported by the National Heart Foundation of Australia.

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