a stimulatory effect by nerve growth factor on the regrowth of adrenergic nerve fibres in the mouse...

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Z. Zellforsch. 146, 15--43 (1973) © by Springer-Verlag 1973 A Stimulatory Effect by Nerve Growth Factor on the Regrowth of Adrenergic Nerve Fibres in the Mouse Peripheral Tissues after Chemical Sympathectomy with 6-Hydroxydopamine Bo Bjerre, Anders BjSrklund and William Mobley Institute of Anatomy and Histology, University of Lund, Lund, Sweden, and Department of Genetics, Stanford University, California, USA I~eceived July 30, 1973 Sumq~/try. Systemically administered Nerve Growth Factor (NGF) had a strong stimulating effect on the regeneration of fully developed adrenergic neurons in the peripheral tissues of the mouse after axotomy induced by 6-hydroxydopamine. The NGF stimulation was investigated at 9 and 21 days after the 6-hydroxydopamine injection, and was observed fluorescence histoehemically as an increase in number, length, and thickness of the outgrowing adrenergic fibre bundles, in the extent and abundance of the terminal ramifications of the regrowing fibres, and in their fluorescence intensity. This increase in the regrowth of the lesioned adre- nergic axons was paralleled by strong and significant increases in the recovery of endogenous noradrenMine in several peripheral tissues. The present findings demonstrate a sensitivity of fully developed adrenergic neurons to NGF during axonal regeneration, and it is suggested that NGF might play a normal physiological role in this process. Key words: Nerve Growth Factor -- Regeneration -- Adrenergic nerves -- Chemical sympathectomy with 6-hydroxydopamine. Introduction One of the most striking features of Nerve Growth Factor (NGF) is its ability to support and stimulate axonal outgrowth, in vivo and in vitro, from developing sensory and sympathetic neurons (for reviews, see Levi-Montalcini and Angeletti, 1968; Levi-MontMeini et al., 1972). However, little is known about its effects on axonal regeneration of these peripheral neuron types in the adult animal. On the other hand, NGF proteins were recently shown to stimulate the growth of new axonal sprouts from lesioned monoamine neurons in the adult rat brain (Bjerre et al., 1973a). Scott and Liu (1964) reported stimnlatory effects by NGF on the regeneration of the central processes of the dorsal root ganglion cells within the dorsal funiele in the piromen-pretreated kitten. Saunders (1972), however, failed to demonstrate any effect by NGF on the regeneration of the peripheral processes of the dorsal root ganglion cells in the adult rat. Moreover, Saunders (1972) has raised criticism against the experiments of Scott and Liu (1964); for this reason it seems that the question whether NGF has any affects on the regenerative growth of adult dorsal root ganglion cells remains open. As far as we know, there has been no studies of the effects by NGF on axonM regeneration of adult sympathetic ganglion cells in vivo. But Silberstein et al. (1971) and Johnson et al. (1972) have pre- sented results suggesting that adult sympathetic neurons in vitro are sensitive to NGF during axonal regeneration; i.e. in the process of "reinnervation" of a de- nervated tissue (iris) in organ culture.

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Page 1: A stimulatory effect by nerve growth factor on the regrowth of adrenergic nerve fibres in the mouse peripheral tissues after chemical sympathectomy with 6-hydroxydopamine

Z. Zellforsch. 146, 15--43 (1973) © by Springer-Verlag 1973

A Stimulatory Effect by Nerve Growth Factor on the Regrowth of Adrenergic Nerve Fibres

in the Mouse Peripheral Tissues after Chemical Sympathectomy with 6-Hydroxydopamine

Bo Bjerre , Anders B jSrk lund and Wi l l i am Mobley

Institute of Anatomy and Histology, University of Lund, Lund, Sweden, and Department of Genetics, Stanford University, California, USA

I~eceived July 30, 1973

Sumq~/try. Systemically administered Nerve Growth Factor (NGF) had a strong stimulating effect on the regeneration of fully developed adrenergic neurons in the peripheral tissues of the mouse after axotomy induced by 6-hydroxydopamine. The NGF stimulation was investigated at 9 and 21 days after the 6-hydroxydopamine injection, and was observed fluorescence histoehemically as an increase in number, length, and thickness of the outgrowing adrenergic fibre bundles, in the extent and abundance of the terminal ramifications of the regrowing fibres, and in their fluorescence intensity. This increase in the regrowth of the lesioned adre- nergic axons was paralleled by strong and significant increases in the recovery of endogenous noradrenMine in several peripheral tissues. The present findings demonstrate a sensitivity of fully developed adrenergic neurons to NGF during axonal regeneration, and it is suggested that NGF might play a normal physiological role in this process.

Key words: Nerve Growth Factor - - Regeneration - - Adrenergic nerves - - Chemical sympathectomy with 6-hydroxydopamine.

Introduction

One of the most s t r ik ing features of Nerve Growth F a c t o r (NGF) is i ts ab i l i ty to suppor t and s t imula te axonal outgrowth , in vivo and in vitro, f rom developing sensory and s y m p a t h e t i c neurons (for reviews, see Levi -Monta lc in i and Angele t t i , 1968; Levi-MontMeini et al., 1972). However , l i t t le is known abou t i ts effects on axonal regenera t ion of these per iphera l neuron types in the adu l t animal . On the other hand, N G F prote ins were recen t ly shown to s t imula te the growth of new axonal sprouts f rom lesioned monoamine neurons in the adu l t r a t b ra in (Bjerre et al., 1973a). Scot t and Liu (1964) r epor ted s t imn la to ry effects b y N G F on the regenera t ion of the central processes of the dorsa l root gangl ion cells wi th in the dorsa l funiele in the p i romen-p re t r ea t ed k i t ten . Saunders (1972), however, fai led to demons t r a t e any effect b y N G F on the regenera t ion of the peripheral processes of the dorsal root gangl ion cells in the adu l t ra t . Moreover, Saunders (1972) has ra ised cr i t ic ism agains t the exper iments of Scot t and Liu (1964); for th is reason i t seems t h a t the quest ion whether N G F has any affects on the regenera t ive growth of adu l t dorsal root gangl ion cells remains open. As far as we know, there has been no s tudies of the effects b y N G F on axonM regenera t ion of adu l t s y m p a t h e t i c gangl ion cells in vivo. But Si lberstein et al. (1971) and Johnson et al. (1972) have pre- sented results suggest ing t h a t adu l t s y m p a t h e t i c neurons in vitro are sensit ive to N G F dur ing axonal regenera t ion ; i.e. in the process of " r e inne rva t ion" of a de- ne rva t ed t issue (iris) in organ culture.

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16 B. Bjerre et al.

Our observat ions of p ronounced effects b y N G F and i ts an t i se rum on the re- genera t ive growth of lesioned catecholamine and indolamine neurons in the adu l t r a t b ra in (Bjerreetal . , 1973, 1974; Stenevi etal . , 1974) posed the quest ion of whether N G F also s t imula tes the process of regenera t ion in the ma tu re per iphera l sympa the t i c nervous system. I n the present work, we chose to s t u d y this p rob lem after chemical a x o t o m y induced b y 6 -hyd roxydopamine (6-OtI-DA). I t is well known t h a t the admin i s t r a t ion of a large dose of 6 -OH-DA to the adu l t an imal causes an a lmost complete and selective degenera t ion of the t e rmina l and para- t e rmina l par t s of the adrenergic axons in most sympa the t i c a l l y i nne rva ted organs wi thout apprec iab ly affecting the ganglionic cell bodies of the adrenergic neuron (Tranzer and Thoenen, 1967, 1968; Malmfors and Sachs, 1968; Thoenen and Tranzer , 1968; Johnson and Sachs, 1970, 1972; for reviews, see Tranzer and Riehards , 1971; Malmfors, 1971; Thoenen, 1971). The regenera t ion process af ter a 6-OI-I-DA-indueed s y m p a t h e e t o m y s tar t s ve ry early, and the adrenergie nerve plexus takes about 2-3 months for a complete regrowth (Haeusler et al., 1969; Jonsson and Sachs, 1970; de Champlain, 1971). The grea t advan tages of the chemical lesioning for the present inves t iga t ion are t h a t tire 6 -OH-DA-induced a x o t o m y and the subsequent regenera t ion process are t h a t reproducible , and several pa r t s of the s y m p a t h e t i c nervous sys tem can be s tud ied in the same animal . Thus the use of 6 -OH-DA offers unique possibil i t ies for s tudy ing the regenera t ion of adrenergie neurons under fa i r ly control led condit ions, and this regenera t ion process should therefore be ideal for the s tudies of possible effects b y N G F on the regenera t ion process of these neurons.

Material and Methods

The N G F Preparat ion

7 S NGF was purified from male mouse submaxillary glands according to Varon et al. (1967). Four separate batches were employed in this study, all having a purity of approximately 95 %, as determined by discontinuous electrophoresis in polyacrylamid gels at p ~ 7.9. When tested for the biological activity of this preparation by the tissue culture method according to Levi-~ontalcini et al. (1954), all four batches of 7 S NGF gave optimal (4-~) fibre responses at a protein concentration of about 10 ng/ml; this concentration thus corresponds to one biological unit (BU) (Varon et al., 1967). In the following, 1 BU is equivalent to 10 ng of the NGF protein.

6-Hydroxydopamine and NGF-Trea tmen t s

154 albino mice (N.M.I~.I.), comprising 4 different experimental groups, were used in the present investigation:

Group 1.22 4-week old female mice received one intravenous injection of 60 mg/kg body weight of 6-hydroxydopamine (6-OI~-DA, AB Hiissle, G6teborg, Sweden) dissolved in saline containing 0.2 mg/ml ascorbic acid. On the next day, 11 of them were given one dose of 4.000 BU/g of NGF (s.c. or i.v.) ; the other 11 received saline similarly. All animals killed 8 days later, i.e. 9 days after the 6-Og-I)A-treatment.

Group 2. 36 4-week old female mice received one intravenous injection of 60 mg/kg of 6-OH-DA, as above. 12 of them were then given 1.000 BU/g, and 6 3.000 BU/g, of NGF (s.c.) daily for the next 6 days, starting on the day after the 6-OI-I-DA-treatment. Control animals received saline similarly. All animals were killed 3 days after the last injection, i.e. 9 days after the 6-OII-DA-treatment.

Group 3. 20 5-week old male mice received one intravenous injection of 220 mg/kg of 6-OH-DA. 12 of them were given 1.000 BU/g of NGF (s. c.) daily for the next 6 days. Control

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NGF and Nerve Regeneration 17

animals received saline similarly. All animals were killed 3 days after the last injection, i.e. 9 days after the 6-OH-DA-treatment.

Group g. 16 4-week old femMe mice received one intravenous injection of 60 mg/kg of 6-0H-DA. 8 of them were then given 1.000 BU/g of NGF (s.c.) daily for the next 5 days (starting on the day after the 6-OH-DA-treatment). After a 2-day interval, the treatment was repeated for another 5 days, and, finally, after another 2-day intervM, it was again repeated for a further 5 days. Thus, the experimentM animals were given a total of 15 daily injections of NGF. Control animals were given saline according to the same schedule. All animals were killed 2 days after the last injection, i.e. 21 days after the 6-OH-DA-treatment.

20 4-week old female and 20 5-week old rome mice given 60 and 220 mg/kg (i.v.) 6-OH-DA, respectively, were investigated one day after injection. A control group consisting of 10 male and 10 female untreated mice of the same age as the experimental animals was included.

Tissue Analyses The animals were killed by exsanguination under light ether anaesthesia. The desired

tissues were rapidly dissected out and anatomically well-defined pieces of tissue were taken both for fluorescence microscopy and for assay of endogenous noradrenaline (NA).

Fluorescence Histochemistry The pieces taken for fluorescence histochemistry of intraneuronal NA consisted of the

entire submaxillary-sublingual gland complex on one side, the whole pancreas, pieces of duodenum and proximal colon, and the two irises. From the heart and spleen small pieces were taken for the fluorescence histoehemistry from the apex of the heart ventricles (and in some sets of experiments also the atria) and from the ventral pole of the spleen, whereas remaining parts of the organs were used for NA determinations.

The irises and the atria were prepared as whole mounts on glass slides and dried in a desic- cator over phosphorous pentoxide in vacuo. Remaining tissues processed for fluorescence histochemistry were frozen in liquid propane, cooled with liquid nitrogen, and freeze-dried. All tissues were treated with formaldehyde gas of optimum humidity according to the Falck- Hillarp method and prepared for fluorescence microscopy (for technical details, see BjSrklund, et al., 1972).

N A Assay Endogenous NA was determined fluorometrically according to Bertler et al. (1958) and

H~ggendal (1963) in whole heart, or in the heart ventricles (atria removed), the submaxillary- sublingual gland complex on one side, spleen, small intestine, and whole brain. For each determination, tissues were pooled from 1-4 animals. Values are expressed as ,ag/g wet weight, except for spleen where the values are expressed as ng/organ (ventral pole removed, see above) to eliminate the error introduced by the marked variation in the blood content of the organ.

Results

E[]ects by N G F on the Regeneration o[ Adrenergie Aocons after 6-OH-DA-Treatment A. His tochemical Findings

When observed one day after t r e a t m e n t with 60 mg/kg of 6 -OH-DA (4-week

old female mice), the number of f luorescent adrenergic fibres in the peripheral

tissues was drast ical ly reduced compared with the un t rea ted controls. Thus,

pract ical ly no fluorescent fibres remained in the iris or the ventr ic le of the heart.

I n other tissues, such as the atria, sa l ivary glands, intestine, spleen, and pancreas,

the varicose, te rminal arborizat ions of the adrenergic axons had disappeared, but

a few non- te rminal axon bundles of vary ing sizes were usual ly left. These bundles

had, typ ica l ly , an increased fluorescence in tens i ty when compared with the in tac t

control animals, and they ran close to the larger arteries entering the i nne rva t ed

2 Z. Zellforseh., Bd. 146

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18 B. Bjerre et al.

tissues; otherwise, the blood vessels were totally devoid of their normal adrenergic innervation.

One day after t reatment with 220 mg/kg of 6-OIt-DA (5-week old male mice), no fluorescent adrenergic terminals were visible in any of the studied peripheral tissues. On the other hand, bundles of preterminal fibres were observed in all peripheral tissues, except the iris and the cardiac ventricles, but their size and fluorescence intensity were generally less than in the animal given 60 mg/kg of 6-OH-DA and observed after the same postinjection time.

Female 4-week old mice, given a single dose of about 4.000 BU/g of NGF s.c. or i.v. one day after 6-OH-DA-treatment (60 mg/kg), were investigated 9 days after the injection of 6-OH-DA (Group 1, see Material and Methods). Such a single injection of a high dose of NGF did not cause any marked increase in the regrowth of the adrenergic fibres in the peripheral tissues, and bioehemicMly there was a significant increase in NA content only in spleen. In some organs, e.g., iris, spleen, and intestine, a slightly more advanced regrowth was observed histochemieally in the NGF-animals when compared with the 6-OH-DA-treated controls. On the other hand, when giving repeated injections of NGF in lower doses (about 1.000 or 3.000 BU/g; Groups 2, 3, and 4) a marked stimulatory effect on the regeneration process was revealed both at 9 and at 21 days after the 6-OH-DA-treatment (see below). No clear-cut difference was found between the two groups of animals given 6 repeated injections of 1.000 or 3.000 BU/g of NGF, with regard to the extent of the adrenergic regrowth observed at 9 days. Therefore these two groups are described together.

In the following, the fluorescence microscopical observations on the effects of repeated daily injections of NGF (6 injections, Groups 2 and 3; 15 injections Group 4) are described in more detail for each of the organs studied. The animals given 60 mg/kg of 6-OH-DA (4-week old female mice) will be referred to as 60 mg- mice and those given 220 mg/kg (5-week old male mice) as 220 mg-miec. The vas deferens and the accessory male genital organs are not included. These tissues are innervated by a special type of sympathetic neurons, the so-cMled short adrenergie neurons (Owman and Sj6strand, 1965; Sj6strand, 1965); the effect by NGF on the regeneration of this neuron type will be published separately.

Iris

9 Days alter Treatment with 6-OH-DA. In the control 60 rag-mice, a few thin bundles of adrenergic fibres had grown into the external edge of the iris at the level of the insertion with the ciliary body, and the bundles in some of the specimens could be seen to arborize into fibres over the external 1/4-1/3 of the iris dilator. No, or occasionally only few, adrenergic fibres were observed in the irises of the control 220 rag-mice at this time.

In the irises of the NGF-treated 60 rag-mice, usually a greater number of thin bundles of preterminal fibres were formed than in the control irises; and in smaller areas within the external 1/3-1/2 of the iris dilator, these bundles had branched to form a sparse elementary ground plexus of varicose fibres. In the NGF-treated 220 rag-mice, a few thin bundles of fibres sometimes had begun growing into the external edge of the irises, but practically no fibres had extended into the iris dilator.

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NGF and Nerve Regeneration 19

21 Days after Treatment with 6-OH-DA (60 mg/kg). In the control irises, a sparse elementary ground plexus of fibres covered about one-third of the circum- ference of the iris dilator (Fig. 1 a). Fibres originating from this plexus reached the sphincter of the iris, but only few of them ran perpendicularly a short distance into the sphincter (Fig. 1 a). In the remaining two-thirds of the circumference of the iris dilator, scattered single fibres, branching from strands or thin bundles of fibres, entered the external 2/3 of the dilator. Although the regenerating fibre bundles often ran along vessels, it was rare to observe any formation of new plexuses in the walls of the small arteries within the irises.

After the NGF-treatment, the amount of regenerating adrenergic fibres found in the irises was usually markedly increased. Thus, in about 1/22/3 of the circum- ference of the iris dilator, a ground plexus of adrenergic fibres was formed, in some areas appearing even denser than tha t of the normal, intact innervation (Fig. t c). The structural organization of the plexus appeared less regular than in the intact iris, probably because fluorescent fibres, more often than normal, ran in strands of two or more (Fig. 1 c). A fairly large number of fibres ran radially from this ground plexus through the sphincter of the irises (Fig. 1 c), and sometimes a few fibres had started to run a short distance circularly within the sphincter. In the remaining 1/3-1/2 of the iris dilator, the extension and the appearance of the regrowing fibres were similar to those in the areas of the control irises having the most advanced ingrowth. The process of reinnervation of small arteries within the irises was more advanced in the NGF-treated than in the controls. Thus a beginning formation of new plexuses of fibres in the walls of small arteries was observed in the external edge of the irises (Fig. lb) as well as in the external part of the iris dilator. Generally, the fluorescence intensity dis- played by the adrenergic fibres was increased in the NGF-treated animals com- pared with the controls (cf. Figs. 1 a and c).

Salivary Glands

9 Days alter Treatment with 6-OH-DA. The submaxillary and the sublingual glands were investigated. In several irregularly distributed areas of the sub- maxillary gland of the control 60 mgl-miee, an elementary ground plexus had been formed by adrenergic fibres surrounding a fairly large number of the serous acini, whereas other areas, particularly in the most peripheral parts of the gland, were more or less completely devoid of such fibres. The fibres of the elementary ground plexus, however, generally were not completely developed around the whole circumference of the acini as in the untreated animal. Moreover, it was consistently observed that a large number of the regenerated nerve terminals appeared thinner and displayed a lower fluorescence intensity than those in the gland of untreated mice, The adrenergic nerves were seen to enter the gland at the hilum in a few bundles, each displaying a moderate fluorescence intensity. A small number of bundles of fibres were observed continuing from the hilum area out into the gland along larger excretory ducts and arteries in the interlobular septa (Fig. 2 a). The arteries were usually surrounded by very sparse plexuses of fibres. In the sublingual gland, no or very few adrenergic fibres were found among the mucos acini and only occasionally branched fibres were seen close to blood vessels and excretory ducts within the gland.

2*

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Fig. i a--c. Stretch preparations of irises from 4-week old female mice treated with 60 mg/kg of 6-OI-I-I)A and examined 21 days later (Group 4, see Material and Methods). In a, almost the entire width of the iris is covered; in c, the sphincter and the inner part of the dilator (corresponding to the left half of Fig. i a) is shown in the same magnification. The adrenergic supply of the sphincter is seen in the left part of the pictures, a ( × 140). Control mouse treated with saline. Sparse elementary ground plexus of adrenergic fibres of the dilator muscle. A few fibres extend a short distance radially into the sphincter (left). b ( × 220), and e ( × 14~0). NGF animals treated with 15 daily injections of 1.000 BU/g of I~GF beginning on the day after the 6-OH-DA-treatment. Note in c, the dense but irregular ground plexus, the increased fluorescence intensity compared with the control (Fig. la), the frequent strands of two or more fibres in the plexus, and the frequent fibres extending into the sphincter. In b, a thin bundle, of prctermina] adrenergie axons is shown on the external edge of the iris at the level of insertion with the ciliary body. Note the beginning formation of an adrenergic fibre plexus

in the walls of an artery (arrows)

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NGF and Nerve Regeneration 21

Fig. 2 a and b (× 140). Bundles or preterminal adrenergie axons demonstrated in correspon- ding areas of the submaxillary glands from 4-week old female mice treated with 60 mg/kg of 6-OIt-DA and killed 9 days later (Group 2). The bundles run together with arteries and close to excretory ducts in the interlobular septu, a. Control animal, b. NGF animM treated with daily injections of NGF (1.000 BU/g) for 6 consecutive days beginning the day after the 6-OH-DA-treatment. Note the increased number and size of bundles and the increased

fluorescence intensity in the NGF animal

The process of re innerva t ion of the submaxi l la ry gland of the control 220 mr- mice was generally markedly less advanced than in the 60 mg control mice. Thus, as i l lustrated schematically in Fig. 3, a sparse e lementary ground plexus of adrenergic fibres had developed in those parts of the parenchyma localized close

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22 B. Bjerre et al.

9 DAYS AFTER 6-OH-DA TREATMENT

SUBMAXIL, LARY GLAN

HILUM REGIDN

SUBLINGUA GLAND

6-OH- DA- 6-OH-DA+ CONTROL NGF

Fig. 3. Schematic representation of the principal regenerative events (interpreted from tile fluorescence histochemiea] findings) in the salivary (submaxillary and sublingual) glands of 5-week old male mice treated with 6-OH-DA (220 mg/kg) and examined 9 days later (Group 3). The drawings represent longitudinal sections through the glands, including the hilum region of the submaxillary-sublingua] gland complex. Main bundles of preterminal NA-containing axons entering at the hilum together with the vessels branch together with arteries in the interlobular septa (mainly in the submaxillary gland) and give rise to paraterminal and ter- minal ramifications of the axons in the parenehyma of the glands. The diagrams do not claim to be accurate in all details (e.g., exact number and positions of bundles and their branches) Left: the morphological picture of the adrenergic supply as observed in a control animal 9 days after 6-OK-DA-treatment. Right: NG~ animal given daily injections of NGF (1.000 BU/g) for 6 consecutive days beginning on the @y after the 6-OH-DA-treatment. Note the increased number, thickness, and length of bundles of fibres and also the larger areas covered by the

paraterminal and terminal ramifications in the NGF animal

to the hilum area, whereas the regrowing fibres gradually became more sparse at a short distance away from the hilum. Within the outer 1/2-1/3 of the submaxil lary gland parenehyma, only few scattered fibres were visible; this indicates tha t the regenerating adrenergic fibres had grown out from the hilum area to reach approxi- mate ly 1/2-2/3 of the distance to the periphery of the gland (see Fig. 3). No such clear- cut gradient of the regenerating adrenergie fibres was observed in the 9-day female control mice tha t received a lower dose of 6-OH-DA (60 mg/kg) ; in these animals, the regrowing fibres were found with an irregular distribution over most parts of the parenehyma of the gland (see above). Also in the 220 mg-controls, bundles of fibres were seen entering at the hilum, but they often appeared fewer and smaller than those observed in the 60 rag-controls. A few weakly-fluorescent bundles of preterminal fibres followed the interlobular septa within the parenehyma and such bundles sometimes ended in areas almost devoid of fluorescent varicose

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NGF and Nerve Regeneration 23

terminals. Very few fluorescent fibres were found associated with blood vessels and excretory duets in the sublingual gland of the 220 mg-mice, and no fibres had grown in among the aeini of the gland.

The NGF-treated 60 rag-mice showed a striking increase of the adrenergie regrowth into the salivary glands, especially in the submaxillary gland, where the following effects of the NGF-treatment were registered: 1) The fluorescence intensity of the individual adrenergic terminals surrounding the aeini was generally higher than in the controls. 2) In certain areas, irregularly distributed within the parenehyma, the increase in the fluorescence intensity was more accentuated, and here, the individual fibres also appeared markedly thicker than they did in the glands of the controls. Moreover, in these areas, the fibres often ran in strands of two or three around the acini, a phenomenon that was rare in the controls. 3) The adrenergic ground plexus that had developed in the NGF-treated mice gener- ally surrounded the individual aeini more completely than in the control glands. 4) The bundles of fibres--entering the gland along excretory duets and arteries in the interlobular septa from the hilum region--usually were more numerous, were thicker, and displayed a stronger fluorescence intensity in the NGF-treated mice than in the controls (Fig. 2 b). Sometimes, thin bundles of fibres also extended a short distance out among the acini away from the interlobular septa.

The NGF-treatment caused an increased adrenergic regrowth also in the sub- lingual gland of the 60 rag-mice, but the effect was less pronounced than in the submaxillary gland. Thus, in contrast to the control specimens, the NGF-treated specimens had sparse plexuses of adrenergic fibres close to arteries running to- gether with excretory ducts; also a few weakly fluorescent adrenergie terminals surrounded the mucous aeini of the gland (Fig. 4b).

In both the submaxillary and the sublingual glands the extent of the regrowth of the adrenergic fibres was about the same in the NGF-treated 60 rag-mice killed 9 days after the injection of 6-OI-I-DA as in the corresponding controls killed 21 days after the same dose of 6-OH-DA (cf. below and Fig. 4~a and b).

The NGF-treated 220 rag-mice also differed markedly from the corresponding controls with respect to the extent of the adrenergie regrowth into the salivary glands (Fig. 3). Between the NGF-treated and the control 220 rag-mice, the most striking difference was observed in the submaxillary gland, where an elementary ground plexus of adrenergie fibres generally covered larger areas of the parenehyma in the NGF-speeimens than in the controls, as illustrated sehematicMly in Fig. 3. Similar to the controls (left in Fig. 3), there was a clear-cut gradient in the density of the regrowing adrenergic fibres within the parenehyma in the NGF-treated 220 rag-mice (right in Fig. 3), but the NGF treatment had caused the fibres to grow further out into the parenehyma. However, in the most peripheral parts of the gland, i.e., in the regions most distant from the hilum area, few fibres occurred also in the NGF-treated specimens. The number, thickness, length, and fluores- cenee intensity of the bundles demonstrable in the hilum or in the interlobular septa were increased by the NGF-treatment in the 220 rag-mice (el. Fig. 3).

21 Days a[ter Treatment with 6-OH-DA (60 mg/kg). The regeneration process had now progressed in the submaxillary gland of the control mice compared with the corresponding controls investigated 12 days earlier. Thus, a ground plexus of thin, mostly singly running, adrenergie fibres covered practically the whole

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24 B. Bjerre et al.

Fig. 4a - -d ( × 95). Sublingual glands of 4-week old female mice treated with 60 mg/kg of 6-OH-DA. a. Control animal, 2.l days survival (Group 4). A small number of adrenergic termi- nals (arrows) occur scattered among the mucous aeini, b. NGF animal, 9 days survival, given 6 daily injections of 1.000 BU/g of NGF beginning on the day after the 6-OH-DA-treatment (Group 2). Note that the comparable number of adrenergic terminal fibres in this animal and in the control survived for 21 days (Fig. 4a). c. NGF animal, 21 days survival, treated with 15 daily injections of 1.000 BU/g of NGF beginning on the day after the 6-OI-I-DA-treatment (Group 4). The density of fibres and their fluorescence intensity are clearly increased compared with the corresponding control shown in a, but rather similar to the normal picture of the

intact 7-week old female mouse (Fig. 4d)

p a r e n c h y m a of the submax i l l a ry gland. The fibres of the adrenergic ground plexus genera l ly sur rounded the serous acini more comple te ly t han t hey d id a t 9 days af ter the 6-OH-DA-injec t ion , bu t t h e y stil l had a lower fluorescence in t ens i ty t han the t e rmina l s of the u n t r e a t e d mice and the corresponding 6-OH-DA- t r ea t ed mice given N G F (cf. below). A t the hi lum of the submax i l l a ry gland, a few bundles of fibres were observed (Fig. 5 c) ; t hey were of about the same size and abundance as in the corresponding 9-day controls and had a s imilar fluorcs- cence in tens i ty . I n accordance with the observat ions made in the corresponding 9-day controls, a small number of p r edominan t l y th in bundles could be followed also along the excre to ry ducts and arter ies in the in te r lobular septa wi th in the paren- chyma (Fig. 5~). The adrenergic plexuses surrounding the ar ter ies wi th in the

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NGF and Nerve Regeneration 25

parenehyma of the gland now looked more developed than at 9 days. In the sub- lingual gland of the controls, a few branched fibres were found around blood vessels, only rarely forming sparse plexuses. Also, only a few regenerated adrenergic varieose fibres, displaying a low fluoreseenee intensity, were found among the mucous acini in this gland (Fig. 4a).

Also at this more advanced stage of regeneration the NGF-treatment (15 daily injections) caused an increase in the regrowth of the adrenergic fibres into the salivary glands. The quantitative and qualitative differences observed between the NGF-treated and the controls at 21 days after the 6-H-DA-injection re- sembled those described above for the 9-day animals (see above). At 21 days after the 6-OH-DA-treatment, the most obvious effect of the NGF-stimulation was observed to be the development of plexuses of adrenergie varicose fibres surrounding the aeini and the arteries of the submaxillary gland, which differed markedly from that of the corresponding controls. In the NGF-treated mice, the re-formation of the adrenergie innervation seemed complete, but in some respects, the regenerated ground plexus differed from that of normal untreated animals. Thus, in the NGF-treated animals, the fluorescence intensity of the adrenergie fibres was not only restored to normal, but were even somewhat elevated above that observed in the parallely processed untreated specimens. Moreover, in the glands of the NGF-treated animals, the adrenergie fibres often ran two or more together - -somet imes in strands or thin bundles--surrounding the serous aeini, and, in part, the regenerated ground plexus in the NGF-treated animals appeared denser than normal. This impression was derived from obser- vations of aeini whose surface had been cut tangentially, making the adrenergic fibres appear superposed on the aeinar surface. These observations indicated that, in the NGF-treated animals, the adrenergie fibres formed dense basket-like arrangements around considerably more aeini than in the normal untreated animal, and in the 6-OH-DA-treated controls such basket-like arrangements were almost never seen. Both in the hilum of the gland (Fig. 5d) and Mong excretory duets and arteries within the gland (Fig. 5b), bundles of fibres were seen tha t were markedly increased in number, size, and fluorescence intensity compared with the controls (compare Fig. 5 a and b, and e and d, respectively). However, these fibre bundles were not so prominent in the 21-day as in the 9-day NGF-treated mice. In the 21-day NGF-treated animals, more regenerated adrener- gie fibres than in the corresponding 6-OH-DA-treated controls were observed around blood vessels and mucous aeini in the sublingual gland (Fig. 4e). In fact, the regeneration process had now progressed so far in the NGF-treated animals that the extent of the adrenergie fibre supply in the sublinguM gland was com- parable with that of the untreated mice (Fig. 4 d).

In the hilar area of the submaxillary-sublinguM gland complex, small, para- sympathetic ganglia are located. In the normal, untreated mice, a few adrenergie fibres were seen surrounding the ganglia, but only few fibres were found within them. At 9 days after t reatment with 6-OH-DA (60 mg/kg), a few adrenergic fibres, not observed one day after the same treatment, had regenerated and surrounded the ganglia in both the NGF-treated and the control animals. Through each ganglion, a thin bundle of presumably preterminal adrenergie fibres (less clearly observable in the untreated mice) was consistently found running within

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26 B. Bjerre et al.

Fig. 5a--d ( X 105). Submaxillary glands of 4-week old female mice treated with 60 mg/kg of 6-OI-I-DA and killed 21 days later (Group 4). a and b. Pictures showing almost identical areas within the parenchyma with excretory ducts partly running in interlobular septa. a. Control animal, b. NGF animal treated with 15 daily injections of 1.000 BU/g of NGI~ beginning on the day after the 6-OI-I-I)A-~reatment. Compare a and b concerning the size and fluorescence intensity of the preterminM bundles (indicated by arrows) running along the excretory ducts, and concerning the density and the fluorescence intensity of the adrenergic

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NGF and Nerve Regeneration 27

a septum of the ganglion. In the control animals investigated at 9 days, the bundle displayed a low fluorescence intensity and very few fibres arborized from it to penetrate in among the ganglion cell bodies. In the NGF-treated specimens, on the other hand, the bundle had an increased fluorescence intensity, and thin varicose fibres branched from the bundle into the ganglion to partly surround the neuronal perikarya. This was most prominent in the NGF-treated mice examined 21 days after the 6-OH-DA-treatment, where the number of adrenergie varicose fibres was higher than in the normal, untreated animals.

Heart 9 Days a[ter Treatment with 6-OH-DA. Similar to the observation made one

day after treatment with 6-OH-DA, only a few fairly thin bundles of fibres were found running in the endocardium of the atria of both the 60 mg and the 220 mg control mice (Fig. 6 d). At 9 days, these bundles had begun to give off a few single fibres that ran a short distance within the endoeardinm. Practically no muscular innervation was found in the ventricles; but in the 9-day controls, a few thin bundles of fibres were oecassionally seen to run close to arteries devoid of adrener- gie innervation and localized just underneath the epicardium.

Within the atria, a striking effect of the NGF-treatment was observed on the regeneration process of the adrenergic fibres both in the 60 rag- and the 220 mg- mice. Thus, in the NGF-treated animals, bundles of fibres, markedly increased in number, size, and fluorescence intensity compared with those of the corres- ponding control mice, had grown into the endocardinm (compare Fig. 6d and e). A number of these fibres, either singly or as a few bunched together, branched off from the bundles, and - -mos t prominently in the 60 mg-miee--they had begun to form sparse elementary ground plexuses. The effect of NGF was clearly less in the ventricles than in the atria of the heart. Even though fluorescent fibres seemed to occur more often among the muscle cells in the ventricles of the NGF- treated animals than in the corresponding controls, the difference was not marked.

21 Days after Treatment with 6-OH-DA (60 mg/kg). In the atria of the control mice, thin fibre bundles occurred within the endocardinm. In small areas, single fibres or strands of fibres were seen to arborize from the bundles and sometimes to begin to form sparse elementary ground plexuses (Fig. 6b) in a manner similar to that seen in the atria of the NGF-treated 60 rag-mice 9 days after the 6-OH-DA- treatment (cf. above). The supply of adrenergic fibres in the ventricle walls was at this time distinctly more advanced than in the control mice investigated at 9 days, i.e. fairly large numbers of fibres were found also among the muscle cells, especially in the external part of the ventricle wall. Around arteries in the myo- cardium, sparse plexuses of varicose fibres were demonstrated, sometimes in association with thin bundles of probably preterminal fibres.

Also at 21 days, there was a markedly increased adrenergic innervation of the atria in the NGF-treated specimens, compared -with the controls. Under NGF-

terminal ground plexus within the parenehyma, c and d. Hilum region of submaxillary sub- lingual gland complex, c. Control animal, d. NGt0 animal given I5 daily injections of 1.000 BU/g of NGF, as in b. Note the increased number and size of the pretermirtal fibre bundles, and their increased fluorescence intensity compared with the control picture in c, which shows an other-

wise very similar part of the hilum region

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28 B. Bjerre et al.

Fig. 6a--e (× 125). Stretch preparations of atria from female mice treated with 6-OH-DA (60 mg/kg) at age 4 weeks, a. Untreated mouse. The adrenergie nerves iorm a terminal ground plexus in the endoeardium, b. Control animal 21 days after the 6-OH-DA-treatment (Group 4).

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NGF and Nerve Regeneration 29

stimulation, the regeneration process had advanced so far that the innervation in several areas of the atria was similar to that of the untreated mice (Fig. 6a). In some patchy areas, the number of thin bundles or strands of fibres crossing the endocardium was even snpranormal (el. Fig. 6 a and c), while in other areas, the fibres appeared clearly fewer than normal. Plexuses of fibres, more developed than in the control animals, closely surrounded blood vessels in the atria. In the ventricle, the extent of regeneration of adrenergie fibres among the muscle cells was not clearly different in the NGF-treated animals from that in the controls; however, the fluorescence intensity of the fibres was possibly somewhat higher in the NGF-treated animals.

Intestine

Two parts of the intestine, duodenum and proximal colon, were investigated, but the changes in the adrenergic fibre supply induced either by the 6-OH-DA- or by the NGF-treatments were similar in both parts; they will therefore be des- scribed together.

9 Days alter Treatment with 6-OH-DA. There was an almost total disappearance of the adrenergic fibres in the intestine one day after treatment with 6-Ott-DA (60 or 220 mg/kg), but 8 days later, regenerating fibres could be demonstrated. In the 60 mg control mice, a few adi'energic varicose fibres were found in the myent~rie plexus of Anerbaeh (Fig. 7 a). These fibres were very irregularly distri- buted, so that patches of the Auerbaeh's plexus, localized close to thin bundles of preterminal fibres entering the intestine along arteries, had a sparse supply of weakly fluorescent adrenergic fibres, whereas the major part of the plexus had no such fibre supply (Fig. 7 a). Only occasional delicate adrenergie fibres occurred in or close to the submucous plexus of Meissner (Fig. 7 a). Arteries entering the intestine were very sparsely surrounded by adrenergic fibres, but sometimes thin, weakly fluorescent, bundles of fibres followed the arteries a short distance within the muscle or subserosal layers. No fluorescent fibers extended into the mucosal layer.

The situation in the 9-day 220 mg control mice was similar to that in the 60 mg controls, but the number of regenerating fibres in the intestine was even less than in the 60 rag-mice. Thus, some of the 220 mg controls were still practically devoid of adrenergie fibres 9 days after the 6-OH-DA-treatment.

In most of the animals, the NGF-treatment was found to have markedly stinmlated the regeneration process of adrenergic fibres into the intestine, but a few of the NGF-treated mice did not differ clearly from the corresponding controls. The NGF-induced stimulation was observed in both the 60 mg and the 220 mg

Thin bundles of fibres cross through the endocardium, and weakly fluorescent fibres or strands of fibres have begun to form an elementary ground plexus, e. NGF animal 21 days after the 6-OI-I-DA-treatment, followed by 15 daily injections of NGF (1.000 BU/g) (Group 4). Note the increased fluorescence intensity compared with b and the larger number of thin bundles and strands of fibres crossing the endocardium, d. Control animal 9 days after the 6-OH-DA- treatment (Group 2). Bundles of fibres arborizing within the endocardium, e. NGF animal 9 days after the 6-OI-I-DA-treatment, followed by 6 daily injections of NGF (3.000 BU/g) (Group 2). A huge complex of very large bundles is seen arborizing within the endocardium,

much enlarged compared with the control animal in d

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Fig. 7 a - - c ( × 110). Proximal colon from 4-week old fcmMe mice treated with 6-OH-DA (60 mg/kg), a. Control animal 9 days after the 6-OIt-DA-treatment (Group 2). A few fluo- rescent fibres are seen around non-fluorescent cells of Auerbach's plexus (-~). A few thin strands of adz'energic fibres follow small arteries within the submucosM layer (<-). b. NGF animal 9 days after the 6-OH-DA-treatment followed by 6 daily injections of NGF (1.000 BU/g) (Group 2). The regrowth of adrenergic fibres is much increased compared with the controls (Fig. 7a). The normally non-fluorescent parasympathetic nerve cells of Auerbach's plexus appear highly fluorescent (-~). This is partly due to a slight diffusion, but mainly to the very large number of intensively fluorescent adrenergic fibres surrounding the intramural ganglion cell bodies in dense basket-like networks. Strands of fluorescent fibres are located in the muscle layer proper, probably close to smM1 blood vessels. Note also the fibres around ganglion cell bodies in the Meissner's plexus (<-) and in the submucosM layer, c. NGF animal 21 days after the 6-OH-DA-treatment, followed by 15 daily injections of NGF (1.000 BU/g) (Group 4). Oblique section through Auerbach's plexus showing a part of this plexus where an

almost normal adrenergic supply has been re-established

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NGF and Nerve Regeneration 31

animals, although the extent of rcgrowth was less after the higher dose also in the NGF-treated animals. Several differences between the NGF-treated specimens and the controls were noted: 1. The adrenergie fibres had grown further in the NGF-treated mice than in the controls. This was clearly observed when following the branching of fibres from an individual bundle entering the intestine close to an artery. Fibres originating from such a bundle covered a larger area of the intestine in the NGF-treated animals than in the controls, and in the NGF-treated animals fibres were seen to extend a short distance also into the mueosal layer. 2. The density of the regenerated adrenergic fibres was increased by the NGF- t reatment (Fig. 7b). This was noted both in the Auerbaeh's and Meissner's plexuses, as well as in the muscle layer proper, and in Auerbaeh's plexus, parti- cularly in the NGF-treated 60 mg-miee, the regenerated fibres often formed basket-like networks around the intramural ganglion cell bodies, rarely seen in the controls (Fig. 7 b). 3. The fluoreseenee intensity displayed by the adrenergic fibres showed an overall increase after NGF-treatment (Fig. 7 b). As a consequence, the individual, intensely fluorescent fibres surrounding the intramural ganglion cell bodies in the NGF-treated 60 mg-miee were often difficult to distinguish from each other, and these highly fluorescent fibres caused the whole ganglion cell bodies to appear green-fluorescent (Fig. 7b). 4. The reinnervation of the arteries in the intestine was also affected to some extent by the NGF-treatment , and the bundles of preterminal adrenergie axons, running close to the arteries in the walls of the intestine, appeared to be increased in number, size, and fluores- cence intensity after NGF-treatment .

21 Days alter Treatment with 6-OH-DA (60 mg/kg). Compared with the 60 mg control mice investigated 9 days after the 6-OH-DA-treatment, the regeneration of adrenergie fibres had progressed further in the 21-day controls, and this was observed mainly in 4 respects. 1. Generally, larger parts of the Auerbach's plexus were reached by adrenergie fibres, although the regrowth was still very limited, and some parts of this plexus completely lacked adrenergie fibres. 2. A small number of fibres terminated in Meissner's plexus. 3. Adrenergie fibres were found in the muscle layer proper and to a small extent fibres ran a short distance up into the mueosal layer. 4. The fluorescence intensity displayed by the adrenergie fibres was somewhat higher than after 9 days survival.

The regeneration of the adrenergie fibres in the intestine of the NGF-treated mice was much increased and had advanced so that the morphological picture of the adrenergie fibre supply part ly resembled that of the normal, untreated animal (el. Fig. 7e). There were differences from normal animals in two respects: 1. The adrenergic supply of Auerbaeh's plexus was less developed than normally in some areas of the intestine in the NGF-treated animals. 2. The number of adrenergie fibres terminating in Meissner's plexus, and running in the submneosal layer was generally somewhat reduced compared to normal. On the other hand, the number of fibres extending into the mueosal layer, and the distance covered by them within this layer, appeared normal. Also, the adrenergie vascular supply in the intestine was close to the normal.

Irrespective of treatment, no changes were seen in the appearance of the small, intensely yellow-fluorescent, serotonin-containing, enterochromaffin cells in the mueosal epithelium.

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32 B. Bjerre et al.

Spleen

9 Days alter Treatment with 6-OH-DA. In contrast to the observations made one day after t reatment with 6-OH-DA, larger blood vessels entering at the hilum of the spleen were reinnervated, although sparsely, by adrenergic fibres both in the 60 mg and the 220 mg control mice. A few bundles of adrenergic non- terminal axons were seen entering the spleen at the hilum area in these animals. Within the parenchyma, thin fluorescent f ibres--not observed one day after t reatment with 6-OH-DA~were demonstrated around the small arteries, but compared with the normal picture, the number of fibres was quite small, parti- cularly in the animals treated with 220 mg/kg of 6-OtI-DA.

Within the piece of the spleen (the ventral pole) analysed fluorescence histo- chemicMly, the regrowth of adrenergic fibres was not clearly increased after NGF-treatment. However, there was some variation between specimens, and a few of the NGF-treated mice differed slightly from the corresponding controls by having an increased number of sparsely innervated small arteries within the parenchyma. In addition, bundles of fibres, increased in size and displaying a higher fluorescence intensity than in the controls, were occasionally demonstrated along larger arteries penetrating the spleen.

21 Days alter Treatment with 6-OH-DA (60 mg/kg). Compared with the situa- tion 9 days after t reatment with 6-OH-DA, the regeneration process had continued considerably in the 21-day animals. Thus the regenerating fibres had grown around the small arteries within the parenchyma of the spleen, but their density was still not normal. The bundles of fibres entering at the hilum appeared rather similar to those of the 9-day animals in number, size, and fluorescence intensity.

After NGF-treatment most ~nimMs showed no clear-cut increase in the extent of regrowth of the adrenergic fibres. But in a few NGF-treated mice, the fluorescence intensity of the individual adrenergic fibres--also in the preterminM fibre bun- dies--within the spleen seemed to be increased compared with the controls.

Pancreas

9 Days after Treatment with 6-OH-DA. Whereas no adrenergic nerve terminals and only few bundles of non-terminal adrenergic axons close to larger arteries were demonstrable within the pancreas one day after t reatment with 6-OH-DA, 8 days later, some adrenergic fibres were found to have regenerated. Thus, in the 60 and 220 mg controls, besides bundles of non-terminal axons, sparse plexuses of fibres closely surrounding mainly larger and to some minor extent also smaller arteries were found. Usually no, or only very few, adrenergic fibres were observed within the exocrine parenchyma of the gland at this time after the 6-OH-DA- treatment. A few fibres sometimes ran around, but not within, some pancreatic islets localized close to large blood vessels (Fig. 8a); otherwise the islets were totally devoid of adrenergic fibres.

The stimulatory effect of NGF on the process of regeneration of adrenergic fibres in the pancreas was clear-cut, both with respect to arteries and to the exo- crine and the endocrine parts of the gland. In the NGF-treated 60 rag-mice, a larger number of small and large arteries were surrounded by plexuses of adrener- gic fibres, where the individual fibres often appeared thicker and more intensely fluorescent than those observed in the controls. Also, bundles of fibres, increased

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NGF and Nerve t~egeneration 33

Fig. 8a--c. Pancreatic islets of 4-week old female mice treated with 6-OH-DA (60 mg/kg). a. Control animal 9 days after the 6-OI-I-DA-treatment (Group 2). A few adrenergie fibres are demonstrated round a small islet. No fibres are seen among the islet cells. (× 180). b. NGF animal 9 days after the 6-OH-DA-troatment, followed by 6 daily injections of NGF (1.000 BU/g) (Group 2). One large and a few smaller islets surrounded and invaded by adren- ergic fibres and also partly by thin bundles of fibres (× 125). c. NGF animal 21 days after the 6-OH-DA-treatment, followed by 15 daily injections of NGt~ (1.000 BU/g) (Group 4). The regrowing fibres have formed a network, in and round the islet, that is comparab]e ta that

of the normM untreated mice ( × 180)

in number , thickness, length, and fluorescence in tens i ty , were found close to ar ter ies of va ry ing sizes. Sca t te red wi thin the exoerine pa renehyma , delieat, e adrenergie fibres were found more often t han in the controls. However , the most conspicuous effect of the N G F - t r e a t m e n t was observed in the endocrine p a r t of pancreas , where the islets were suppl ied with adrenergie fibres to a cons iderably greater ex ten t t h a n in the controls. Around bo th small and large islets, a vary ing , somet imes large, number of h ighly f luorescent adrenergie fibres, or bundles of fibres, were found (Fig. 8b). W i t h i n some large islets adrenergie fibres were sca t te red among the endocrine cells, and the morphological p ic ture of the adren- ergie fibre supp ly of some of the islets was thus close to t h a t of the normal ani- mal.

3 Z. ZelIforsch., Bd. 146

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34~ B. Bjerre et al.

The regrowth of adrenergie fibres in the NGF-treated 220 rag-mice varied between individual animals. Thus, about half of them did not markedly differ from the corresponding controls, whereas the others showed a clear effect of the NGF-t rea tment and appeared similar to the NGF-treated 60 rag-mice also killed 9 days after the 6~Ott-DA-treatment.

21 days after treatment with 6-OH-DA (60 mg/kg). Also at this time after the 6-OH-DA-treatment, the regeneration of the adrenergie fibres was in most cases clearly more advanced in the NGF-treated specimens than in the controls. In the controls fairly sparse plexuses of adrenergie fibres were common around not only large but also small arteries within the parcnehyma. Moreover, bundles of preterminal fibres of varying size were observed running close to arteries most often the larger ones within the exocrine parenehyma. Round a small number of the islets, a few thin fibres were seen. Scattered, mainly within large islets, two or three thin fibres were usually demonstrated in each section. Thus, 21 days after t reatment with 6-OH-DA, the adrenergic supply of the pancreas was far from re-established in the control mice.

Whereas a few NGF-treated specimens did not clearly differ from the controls most of them had an adrenergic fibre supply similar to that of the untreated mice. In such NGF-treated animals, the plexuses of adrcnergic terminals that had been restored around the small and large arteries were markedly increased and covered most of the arteries found within the pancreas. Also, numerous bundles of pre- terminal fibres ran close to these vessels. Generally, the fibres and the bundles of fibres found around or close to the arteries seemed thicker and displayed a higher fluorescence intensity in the NGF-treated animals. The fluorescent fibres within the exocrine parenehyma of the NGF-treated mice were increased in nmnber and fluorescent intensity compared with the controls. Around most large and small islets, several adrencrgic fibres were seen, and within islets of all sizes networks of delicate adrenergie fibres were formed to such an extent that the islets were in- distinguishable from those of the untreated mouse. (cf. Fig. 8 e).

B. Chemical Findings

The endogenous noradrenaline (NA) levels were measured in brain and several peripheral tissues at 1, 9, and 21 days after 60 or 220 mg/kg of 6-OH-DA, and compared with the levels in normal untreated mice of the same age and sex (Fig. 9a-f). One day after 60 mg/kg of 6-OH-DA (female mice), the endogenous NA was depleted in all peripheral tissues to about 10% or less of the original levels. In brain, the reduction was much smaller (about 20%) but statistically significant (0 .01<p<0.05) . With 6-OH-DA at the higher dose (220 mg/kg, male mice), the NA depletion seemed to be somewhat more accentuated in all investi- gated peripheral tissues, and the return of endogenous NA, as measured 9 days after the 6-OH-DA-treatment, was less after 220 mg/kg than after 60 mg/kg of 6-OH-DA. Irrespective of which dose of 6-OH-DA was administered, t reatment with NGF (i.000 or 3.000 BU/g body weight) for 6 consecutive days resulted in an accelerated recovery of NA in the salivary glands, whole heart and spleen. Thus, 9 days after the 6-OH-DA injection, the NGF-t rea tment had caused an increase in NA to about 120-140% in the salivary glands, 170-250% in whole heart, and 135-180% in spleen, compared with the controls given 6-OtI-DA alone (cf.

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NGF and Nerve l~egeneration 35

Fig. 9a-e). A single injection of a high dose of NGF (4.000 BU/g body weight) given the day after the 6-Ott-DA-treatment (60 mg/kg) caused, at 9 days, a statistically significant increase in the NA levels only in the spleen. In the sali- vary glands, intestine, and spleen--but not in the hear t - - the differences between the NA levels in the control and the NGF-treated mice were further accentuated by a prolonged NGF-treatment (15 daily injections of 1.000 BU/g of NGF), resulting, 21 days after the 6-OI-I-DA-treatment (60 mg/kg), in NA levels that were increased to about 175% of control (given 6-OtI-DA alone) in the salivary gland, 165% in whole heart, 220% in spleen, and 170% in intestine (Fig. 9a-d). The restoration of NA in the NGF-treated animals was about 55, 60, and 90 % of normal in heart, intestine, and spleen, respectively, whereas the corresponding values in the controls (given 6-OIt-DA alone) were only about 30, 35, and 40% respectively. In the salivary glands, the prolonged NGF-treatment caused an increase in endogenous NA to supranormal levels (about 140 % of the concentra- tion in the normal untreated mice) (Fig. 9 a).

As Fig. 9 b and e show, the effect of NGF on heart NA seemed to be confined mainly to the atria. Thus, when the ventricles of heart were assayed separately (at 9 days after 60 mg/kg of 6-OH-DA), the NGF-treatment did not cause any significant increase in the return of endogenous NA (Fig. 9e). This parallels the fluorescence microscopical findings (see above) which demonstrated a much stronger effect of NGF on the regrowth of adrenergic nerves in the atria than in the ventricles of the heart.

In brain, six consecutive injections of 1.000 BU/g of NGF caused a slight, but statistically not significant, increase in the NA concentration 9 days after the 6-OIt-DA-treatment (60 mg/kg) (Fig. 9f).

Discussion

The effects of NGF on the regrowth of lesioned adrenergic nerves was studied in mice subjected to chemical sympathectomy with 6-OIt-DA. To keep the amount of injected NGF protein within reasonable limits, the experiments were carried out in 4-5-week old mice. At this age, the adrenergic innervation of the peripheral organs is fully established (de Champlain et al., 1970; Owman et al., 1971; Mirkin, 1972) but the body weight is only 15-20 g. Two different doses of 6-Ott-DA, 60 and 220 mg/kg, were given in a single intravenous in- jection. One day after injection, both doses resulted in a complete disappearance of the fluorescent terminal and paraterminal ramifications of the adrenergic axons in all peripheral tissues investigated, i.e. iris, salivary glands, atria and ventricles of the heart, intestine, spleen, and pancreas. The difference between the two doses was seen in the extent of fluorescent preterminal fibre bundles that remained in the organs. In all tissues, except the iris and the ventricles of the heart, bundles of preterminal adrenergic fibres persisted, but after the higher dose of 6-OIt-DA, the bundles were generally reduced in size and fluorescence intensity compared with those observed in the animals given the lower dose. This probably indicated a more extensive degeneration of preterminal fibres after the higher dose of 6-OtI-DA, which agrees with the previous findings of Mahnfors (1971).

3*

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36 B. Bjerre et al.

150-

100.

50

SALIVARY GLANDS NA CONTENT (100°/o:1.46 pg/g NA) £ (100%=l.83pglg NA) O '

[ ] NORMAL

[ ] 6-OH-DA CONTROL

• 6-OH-DA+NGF (SINGLE INJ.)

[ ] 6-OH-DA+NGF (REPEATED INJ.)

9O` 9O` 9 o" £ Norma{ 1day 9days 9 days 21 days

TIME AFTER 6-OH-DA TREATMENT

150-

100-

o z

50-

HEART NA CONTENT (100%=0.72 pg/g NA] 9 (100%= 0.52 Pg/g NA) O"

[ ] NORMAL

[ ] 6-OH-DA CONTROL

• 6-OH-DA+NGF (SINGLE INJ.)

[ ] 6-OH-DA+NGF (REPEATED INJ.)

FT-IN.D. 9O` 9o"

Normal 1day £ O £

9 days 9 days 21 days TIME AFTER 6-OH-DA TREATMENT

150-

100

50.

SPLEEN NA CONTENT (100 %= 23.7 ng/organ NA) £ (100 °/o = 23.4 ng/organ NA) O

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NGF and Nerve Regeneration 37

HEART VENTRICLES NA CONTENT (100 °/o= 0.55 pg/g NA) £

[] NORMAL

[] 6-OH-BA CONTROL

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BRAIN NA CONTENT (100 °/o = 0.32 pg/g NA) £

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TIME AFTER 6-OH-DA TREATMENT

f Fig. 9 e and f

Fig. 9a--f. NA content in various organs of mice treated with 6-OH-DA (4-week old female mice, 60 mg/kg; 5-week old male mice, 220 mg/kg). Effect by NGF given either as a single injection (4.000 BU/g; Group 1, see Material and Methods) one day after the 6-OH-DA- treatment or as repeated daily injections of 1.000 or 3.000 BU/g (female mice; Group 2) and 1.000 BU/g (male mice; Group 3) for 6 days following the 6-OH-DA-treatment. Controls were given saline similarly, and the animals were killed 9 days after the 6-OH-DA-treatment. The female mice, killed 21 days after the 6-OH-DA-tre~tment, were given 1.000 BU/g of NGF or saline daily, for 15 days (Group 4). The results are expressed in per cent of normal untreated controls of the same sex and age. Each bar is the mean -~ S.E.M. of 4-12 determinations. Differences between NGF-treated and control animals (given 6-Ott-DA alone): * = 0.05 >

p > 0.01; ** = 0.01 > p > 0.001; *** = p < 0.001. Student's t-test

The extensive t e rmina l degenera t ion induced b y the 6 -O H -D A - t r e a tme n t s was ref lected in a s t rong d e p l e t i o n - - b y more t han 9 0 % - - o f the endogenous N A in all t issues except the brain. Despi te the more pronounced affect b y the higher dose (220 mg/kg) on the p re te rmina l axons, as revealed his tochemical ly , this t r e a t m e n t only s l ight ly accen tua t ed the N A deplet ion.

Combined evidence f rom electron-microscopical (Cobb and Bennet t , 1971; Tranzer and Richards , 1971), b iochemical (Jonsson, 1971; Thoenen, 1971), and fluorescence h is toehemieal s tudies (Jonsson and Sachs, 1970, 1972; de Champlain , 1971; Malmfors, 1971; Sachs, 1971) indica te t h a t the 6 -OH-DA- indueed axonal degenera t ion is a ve ry r a p i d proeess in the per iphera l nervous sys tem. The f irst signs of axona l degenera t ion are revea led wi th the e lect ron-microscope 1-2 hrs af ter in jec t ion of a large dose of 6 -OI t -DA (Cobb and Bennet t , 1971 ; Tranzer and Riehards , 1971); a t 24 hrs, the adrenergic axon profiles are e i ther comple te ly absen t or represen ted b y an i rregular , e lectron-dense body (Cobb and Benne t t ,

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38 B. Bjerre st al.

1971). Thus, at the t ime when the NGF-t rea tment was begun in the present study, i.e. at one day after the 6-OH-DA-treatment, the degeneration process was prob- ably very advanced or even completed. There are thus good reasons for believing that the effects induced by the NGF-t rea tment were due to an interference with regeneration rather than degeneration in the present experiments.

In the mouse (Malmfors, 1971), the first fluorescence histoehemical signs of sprouting from the lesioned adrenergic axons are observed 2-4 days after the 6- OI-I-DA-injection. Within the next 2-3 months, adrenergic terminal networks closely resembling those of the normal intact animal are regenerated (Haeusler et al., 1969; Jonsson and Sachs, 1970; de Champlain, 197t). The effects of NGF on this regeneration process were evaluated at an early stage (9 days after 6-Ott-DA- injection) and at a more advanced stage (21 days), during which time the animals were treated with 6 or 15 daily subcutaneous injections, respectively, of 7S NGF. The doses of the 7S NGF preparation given (10-30 ~g/g, equivalent to about 1.000-3.000 BU/g, in each injection) are, on a weight basis, equivalent to those necessary to produce a near maximum response on the sympathetic ganglia in the newborn mouse (Levi-Montalcini and Angeletti, 1968 ; Edwards et al., 1972).

A stimulatory effect by the NGF-treatments on the regeneration of the adrenergie nerves was demonstrated both in the fluorescence histochemistry of the regrowing fibres and in the fluorimetric determinations of the recovery of the endogenous NA levels. The magnitude of the NGF-stimulation varied in the sense tha t there was a clear variation in the extent to which the fibres had regener- ated in the NGF-treated animals. This is not surprising in view of the variations in the regrowth process that occurred already in the corresponding control animals, given 6-OH-DA alone. These variations were observed between different animals, but, more important, also between different organs in the same animal, differences tha t might be due to, e.g., a variation in the extent to which the injected 6-OH-DA damaged the adrenergic axons in different tissues and in different animals. The most obvious example of this variation in the regrowth of the chemically lesioned adrenergic nerves was seen in the two irises of the same animal. In this paired organ, the extent of regrowth was seldom the same on the two sides, and sometimes the difference between the sides was pronounced. Similar differences were seen in all tissues at 9 or 21 days after the 6-Oft-DA-treat- ment, and in each set of experiments there were usually a few specimens from the NGF-treated animals that had a fluorescence histochemical picture that did not clearly differ from those control animals that had a more advanced,regrowth of the lesioned axons. Since clear st imnlatory effects most often were observed in other tissues of the same animal, it seems highly probable that the NGF-treat- ment was effective in these cases, but that the effect was obscured by the variance in the experimental system.

In the control animals treated with 6-OH-DA alone, there were--irrespective of the above-mentioned variat ions--certain consistent differences in the regrowth process in different tissues. Most conspicuously, the regeneration in the sub- maxillary gland progressed considerably faster than that of the other investigated tissues, and this difference was observed both histoehemiea]ly and biochemically. At 9 days after 60 mg/kg of 6-OH-DA, the NA level was about 60% of normal in the salivary glands, but only about 20-30% in other tissues ; at 21 days, the eorre-

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NGF and Nerve l~egeneration 39

sponding figures were about 80 % and 30-40 % of normal (see Fig. 9). I t is interesting that the tissue where the regeneration of the adrenergic axons proceeded fastest in the controls--the submaxillary gland--also is, by far, richest in endogenous NGF (Levi-Montalcini and Angeletti, 1968).

Fluorescence histochemieally, the stimulatory effect of exogenous NGF on the regenerating adrenergic nerves was observed in one or more of the following respects. 1. The bundles of preterminal fibres growing along blood vessels into the denervated organs were increased in thickness, length, and number. 2. The regrowing fibres arborized more abundantly and covered larger areas within the tissues. The regenerated fibres were restricted to those areas that normally had an adrenergie innervation, but in the animals analysed 21 days after the 6-Ott-DA- treatment, some regions--the submaxillary gland and certain parasympathetic ganglia--had a density of adrenergic fibres that exceeded that of the normal, untreated animals. 3. The fluorescence intensity of the individual terminal or preterminal adrenergic fibres--while reduced in the 6-OtI-DA-treated control animals when compared with normal--was often clearly elevated in the NGF- treated animals.

The NGF-induced increase in preterminal fibre bundles (1) probably signifies an increased outgrowth of new axonal sprouts from the proximal ends of the lesioned axons out to the denervated areas, which would be consistent also with the apparent hyperinnervation obtained, e.g., in the submaxillary gland in the NGF-stimulated animals at 21 days after the 6-OH-DA-treatment. This increased outgrowth could, it seems, be due to an increased sprouting from the lesioned axons, or to a more successful outgrowth or a better survival of the initially formed axon sprouts.

The observation (2) of an increased terminal arborization of the regenerating fibres--best demonstrated in certain tissues such as the submaxillary gland and the intestine of the mice treated with the higher dose of 6-OH-DA (220 mg/kg) ; cf. Fig. 3--suggests that NFG also caused an acceleration of the growth of the sprouting fibres. This acceleration resulted in an advancement of the re-innervation process in the NGF-stimulated animals. Thus, for example, the terminal plexus re- formed in the submaxillary gland at 9 days in the NGF-treated animals (given 60 mg/kg of 6-OH-DA) was similar to, or even more advanced than, that observed at 21 days in the controls, given 6-OH-DA alone. In fact, seemingly normal ter- minal plexuses were partly restored in, for instance, the atria and the intestine al- ready by 21 days, and even earlier in the submaxillary gland and the pancreatic islets, in the NGF-treated animals.

The increased fluorescence intensity of the regenerating axons (3) caused by the NGF-treatment signifies a restoration of the intra-axona] NA concentration towards normal. In the peripheral nervous system, it has been observed that the fluorescence intensity of outgrowing adrenergic axons, during ontogenesis (de Champlain et al., 1970) and during regeneration (Olson and Ma]mfors, 1970) is often lower than in the mature adrenergic axon terminals, but they are still readily demonstrable in the fluorescence microscope. This is generally true also after chemical sympathectomy with 6-OH-DA (cf. Jonsson and Sachs, 1972). In the rat, Brimijoin and Molinoff (1971) and Brimijoin (1972) have observed a marked decrease in NA synthesizing enzyme dopamine-fi-hydroxylase (but not

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40 B. Bjerre et al.

tyrosine hydroxylase) in the sympathetic ganglia and a decreased transport of this enzyme down the sciatic nerve a few days after destruction of the adrenergic terminals with 6-OIt-DA. This suggests that the lower-than-normal content of NA in the regrowing axons could reflect a reduced capacity for synthesis of the t ransmit ter in the regenerating neurons. In the intact newborn animal, NGF- t rea tment has been shown to cause a selective induction of both tyrosine hydroxy- lase and dopamine-/~-hydroxylase in the developing sympathetic ganglia (Hendry and Iversen, 197t; Thoenen et al., 1971; Thoenen, 1972). Thus it seems possible that the increased fluorescence intensity of the regenerating fibres in the NGF- treated mice could be due to a st imulatory effect of NGF on the synthesis of ETA.

As reflected in the NA determinations the magnitude of the NGF-induced response in different organs was faMy similar, especially in those animals sub- jected to a prolonged NGF-treatment . There were, however, some differencies; the most conspicuous one was observed in the response of the adrcnergic fibres re-innervating the atria and the ventricles of the heart. Thus, in contrast to the atria, there was no clear-cut effect in the cardiac ventricles, and this was established both histochemically and biochemically. In the assays of whole heart (atria included), the NA concentration was increased to about 250% of control in the NGF-treated g-day mice treated with 60 mg/kg of 6-OH-DA, but when the ventricles were assayed separately, there was no significant increase in NA after the same t reatment (Fig. 9d and e).

A puzzling discrepancy between the histochemical and the biochemical findings occurred in the spleen: The NA levels were consistently significantly increased in the NGF-treated animals (most prominently in the 21-day group where the NA content of the NGF-treated animals was more than twice that of the controls), but no distinct differences could be observed histoehemieally. The most likely explanation for this would be that the NGF-indueed growth response was--as in the hear t=regional ly restricted. Thus, as the histochemistry was carried out only on a small piece of the spleen (the ventral pole), this might have failed to include the critical region.

The simplest way to explain differences in effects observed in various tissues would be in terms of differences in responsiveness to exogenous NGF between different sympathetic neurons. But it also seems possible that the differences could be related to differences in the growth environment provided by the various tissues. From in vitro studies of dissociated, developing neurons, Burnham et al. (1972) suggested that sympathetic and sensory neurons are sensitive to exogenous NGF only in those situations where the support provided by surrounding tissue elements is inadequate. According to this hypothesis, NGF in the present situation would supplement for deficiencies in the support of the growing fibres. Obviously, such a difference in growth support would readily explain differences in response to exogenous NGF during regeneration.

Conclusions

The results demonstrate that NGF exerts strong stimulatory effects on the regrow~h of adrenergic fibres and the return of endogenous NA in the peripheral tissues of the mouse after axotomy induced by 6-OH-DA. The NGF stimulation was observed both in number, length, and thickness of the outgrowing fibre

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NGF and Nerve Regeneration 41

bundles, in the extent and abundance of the terminal ramifications of the re- growing fibres, and also in their fluorescence intensity. These NGF effects on peripheral NA neurons are comparable to those previously reported by Bjerre et al. (1973I) and Stenevi et al. (1974) on the regenerative sprouting of central catecholamine and indolamine neurons in the adult rat brain, suggesting that these various monoamine neuron types have a similar sensitivity to the NGF proteins, at least during regeneration.

From the extensive and fundamental work of Levi-MontMeini, Angeletti and their co-workers (see Levi-Montalcini and Angeletti, 1968 ; Levi-Montalcini et al., 1972) it seems probable that NGF plays an important role in the development and maintenance of the sympathetic nervous system. The present observations point to a possible role of NGF in the growth of adrenergie neurons also after their development has been completed. After axonal damage, the neuron is placed in a situation in many respects resembling that found during ontogenesis, and it is an attractive idea that NGF eould indeed play a similar physiological role in both these situations. In fact, the effects of NGF on the ontogenetic growth of the peripheral adrenergic neurons in the newborn mouse, as described fluorescence histochemically by Olson (1967) and bioehemically by Crain and Wiegand (1961), are in several respects similar to those obtained on the regenera- tive growth in the present study. The magnitude of the NGF-induced growth response--as judged by the fluorescence histochemieal picture or by the percentage increase in NA indueed by the NGF-treatment-- thus seems to be similar in those different situations, suggesting that, during regeneration, the responsiveness of the fully developed sympathetic neurons is comparable to that during onto- genesis. On these grounds it seems reasonable to assume that the mechanism of action of NGF during development and during regeneration could be similar. In this context, it is also interesting to note that, from preliminary observations (Bjerre, Bj6rklund, Mobley and Rosengren, in preparation), NGF also appears to elicit collateral sprouting from intact adult, sympathetic neurons.

Acknowledgements. The skilful technical assistance of Ingegerd Andersson, Lilian Bengts- son, Anne-Marie 01sson, and Eva Svensson is gratefully aeknoMedged.

The study was supported by grants from the Faculty of Medicine, University ot Lund, from the Swedish Medical Research Council (grants No. 04 × -3874, 04 × -712, and 04 × -56), and from the Magnus Bergvall Foundation.

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Dr. Bo Bjerre Tornblad Institute Biskopsgatan 7 S-223 62 Land Sweden