Inhibition of the regenerative growth of central noradrenergic neurons by intracerebrally administered anti-NGF serum

Brain Research, 74 (1974) 1-18 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

Research Reports

I N H I B I T I O N OF T H E R E G E N E R A T I V E G R O W T H OF C E N T R A L NORA D R E N E R G I C N E U R O N S BY I N T R A C E R E B R A L L Y A D M I N I S T E R E D A N T I - N G F SERUM

BO BJERRE, A N D E R S B J O R K L U N D AND U L F STENEVI

Institute of Anatomy and Histology, University of Lund, Lund (Sweden) (Accepted January 24th, 1974)

SUMMARY

The regenerative growth of central monoamine neurons in the adult rat brain, into an autologous iris transplant placed in the caudal diencephalon, was studied. One injection of 2 #1 o f anti-NGF serum, given intracerebrally close to the ascending noradrenergic axons from the locus coeruleus, caused a marked reduction in the regrowth of new axonal sprouts into the transplant, as observed 2 weeks after operation. The inhibitory effect was pronounced when the injection was given at the time of transplantation (which was also the time of axonal damage), and it was considerably less effective when given 4 days after the transplantation, i.e. at a time when the sprouting from the lesioned axons had already started. A similar inhibition of the regrowth of the lesioned noradrenergic locus coeruleus axons was also obtained after preincubation o f the iris transplant in the anti-NGF serum. In contrast, the regrowth from the lesioned dopamine and indolamine fibers in the medial forebrain bundle system seemed unaffected. None of the inhibitory effects was seen in the control specimens treated with different types o f control sera. This indicates that the observed impairment of the formation and regrowth of new axon sprouts from lesioned central noradrenergic neurons was the result of a specific action of the NGF-antibodies by an interference with N G F (or an NGF-like substance) in the brain tissue and/or in the transplanted iris tissue. These observations point to a possible role of endogenous N G F , or an NGF-like substance, in adult central noradrenergic neurons during regeneration.

INTRODUCTION

Central monoamine neurons in the adult rat brain exhibit a remarkable capacity for regenerative growth following axonal damage 6,7,13 (for reviews, see refs. 20 and

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22). The regeneration of such neurons into an autologous iris transplant has been shown to be stimulated by Nerve Growth Factor (NGF)3,s, 21, a protein - - or class of proteins - - that is otherwise known to stimulate the growth of mediodorsal cells of embryonic sensory ganglia, and to be of critical importance during ontogenetic growth and differentiation of peripheral sympathetic noradrenaline neurons (for reviews, see refs. 15 and 16). The stimulatory effect on regenerating central neurons was observed after intraventricular as well as after local intracerebral injections of N G F , and the possibility arose that this sensitivity to exogenous N G F might reflect a general dependence of the central monoamine neurons on endogenous N G F during regeneration, and, perhaps, also during ontogenesis3,2L The probably critical role of endogenous N G F in developing peripheral sympathetic neurons is revealed by the deleterious effects of the antiserum to N G F on the sympathetic neurons in newborn animals. Thus, the anti-NGF serum, given systemically to newborn mice or rats, causes a permanent and almost complete destruction of the peripheral sympathetic nervous system 1°,17 (for review, see ref. 14). Recent results obtained in our laboratory have demonstrated that anti-NGF serum also strongly inhibits the regeneration of fully developed sympathetic neurons after axonal damage 2, suggesting a role for N G F in the mature peripheral nervous system during repair. The present study in the adult rat was to investigate whether anti-NGF serum would interfere with the regenerative growth of lesioned central noradrenaline neurons into an iris transplant implanted in the caudal diencephalon. The study was begun with a series of experiments in which the anti-NGF serum was administered intraventricularly, into the cerebrospinal fluid, but by this route of administration no marked effects were obtained on the regenerative growth from the neuron systems studied. A possible explanation for the lack of response after intraventricular injections of the a n t i - N G F serum was the failure of the antibodies to reach the target cells from the cerebrospinal fluid. Therefore, intracerebral injections of small volumes (12/A) of the anti-NGF serum, placed close to any of the lesioned neuron systems growing into the transplant, seemed to offer more favorable conditions for studying possible effects of the antiserum. The study was thus continued with injections made close to the ascending axons of the locus coeruleus neuron system - - a major noradrenergic system lesioned in the transplanted animals - - and the process of regenerative growth into the denervated iris transplant was studied with the fluorescence histochemical technique. MATERIAL AND METHODS

A n i m a l s a n d sera

Eighty-three female Sprague-Dawley rats (180-200 g body weight) were used. All animals were subjected to cervical sympathectomy by bilateral extirpation o f the superior cervical ganglia prior to the transplantation. A n t i - N G F serum. N G F , highly purified from male mouse submaxillary glands, according to the method described by Bocchini and Angeletti 9, was used to produce the antiserum in rabbits. The rabbit anti-NGF serum thus obtained was a generous

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gift from Dr. Rita Levi-Montalcini (Rome, Italy). When tested for specificity, the antiserum gave only a single precipitation line when reacted with a crude salivary gland extract. Control sera. As a control for non-specific effects o f the anti-NGF serum, another hyperimmune serum - - rabbit anti-mouse thymocyte serum - - was tested as well as normal rabbit serum. The rabbit anti-mouse thymocyte serum was prepared by injecting rabbits intravenously with 10s mouse thymoctes at day 0, day 14, and day 21. Sera were collected 14 days after the last injection.

Operative procedures Iris transplantations were performed, under general barbiturate anesthesia (Brie-

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Fig. 1. Position of the iris transplant in the caudal diencephalon. The lesion produced by the transplantation (cross hatched area) transects two major ascending monoamine fiber systems - - the dorsal catecholamine bundle (DCB), containing predominantly noradrenaline axons, and the medial forebrain bundle (MFB), containing indolamine axons (not represented in the diagram), dopamine axons (originating, i.a., in the substantia nigra, SN), and also some noradrenaline axons (originating in the ventral catecholamine bundle, VCB). In the specimens used for the present study, the transplant is in direct contact with the growing monoamine fibers in these two bundles. The magnitude of ingrowth into the transplant illustrated in the diagram is that observed in control specimens 2 weeks after the transplantation. Filled circles: dopamine-containing cell bodies; open circles: noradrenaline-containing cell bodies. Arrow indicates the site of injection of the anti-NGF serum close to the noradrenaline axons in the DCB, about 1 m m rostral to the locus coeruleus and 3 mm caudal to the site of the lesion. (Schematic drawing after Ungerstedt2a.)

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tal, Lilly, 40 mg/kg), as autologous transplantations according to the technique described by Stenevi et al. 21. In this procedure a thin, fiat, glass rod (about 1.0 mm wide and 0.3 mm thick) is used for lowering the iris by free hand into the caudal diencephalon. The transplant is placed so that the major ascending catecholamine and indolamine pathways in the medial forebrain bundle (MFB), and the dorsal catecholamine bundle (DCB), are transected (see Fig. 1). Only successful transplantations were included in the material (see Results, below). Administration of the sera. The intraventricular and intracerebral injections were made stereotaxically. The intraventricular injections were made into the lateral ventricle and each injection consisted of 25 #1 of undiluted serum. In one set of experiments a single injection of anti-NGF serum was given 7 days after the transplantation (5 animals). In another set of experiments the injections were repeated daily for 7 days, starting on the day of transplantation (7 animals). Six iris-transplanted animals served as controls, and were given normal rabbit serum according to the same schedules. All animals were killed 2 weeks after transplantation. The intraeerebral injections were made ipsilateral to the iris transplant close to the ascending locus coeruleus axons in the DCB, approximately 1 mm rostral to the locus coeruleus (Fig. 1). Two microliters of undiluted serum was given in each injection. Ten animals received a single injection of anti-NGF serum close to the axons in the DCB at the time of transplantation, and 6 animals received a single injection of antiN G F serum close to the DCB axons 4 days after transplantation. Fifteen iris-transplanted animals were used as controls, and were given normal rabbit serum or rabbit anti-mouse thymocyte serum according to the same schedules. All animals were killed 2 weeks after transplantation. In one set of experiments the iris transplant was preincubated (see below) in anti-NGF serum (6 animals) or normal rabbit serum (4 animals) and, in addition, 2 injections (each consisting of 2 #1 of anti-NGF serum and control serum, respectively) were given close to the axons in the DCB. One injection was given at the time of transplantation, and the other 2 weeks later. These animals were killed 4 weeks after transplantation. Preineubations. In this series of experiments the irises were preincubated in undiluted anti-NGF serum (12 animals), and in normal rabbit serum or rabbit antimouse thymocyte serum (12 animals), at room temperature for 15 rain. After this, the irises were immediately placed as transplants in the caudal diencephalon, according to the procedure described above. The animals were killed 2 weeks after transplantation. Histochemical procedure The animals were decapitated under light ether anesthesia. The pons, mesencephalon, and posterior diencephalon were dissected out in one piece (containing the transplant and the injection site(s)), immediately frozen in a liquid propane-propylene mixture at the temperature of liquid nitrogen, and processed for fluorescence microscopy according to the method of Falck and Hillarp (for technical details, see Bj6rklund et al.5). All specimens were serially sectioned in the sagittal plane and evaluated in the fluorescence microscope. The fluorescence colors refer to those observed in the

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fluorescence microscope, equipped with BG 12 (Schott) as mercury lamp filter, and Zeiss 47 + 50 as secondary filters. With this filter setting, the color o f the catecholamine fluorescence will be green to yellow-green, and that o f indolamines yellow. RESULTS

The experimental model, illustrated schematically in Fig. 1, was identical to that employed previously in the study of the stimulatory effects of N G F on the regrowth of lesioned central monoamine neurons3,S, 21. In its position in the caudal diencephalon, the iris transplant will be invaded by sprouting, noradrenaline-containing fibers growing out from the lesioned dorsal catecholamine bundle (DCB), and by (primarily) dopamine-containing fibers from the lesioned medial forebrain bundle (MFB). In control animals observed 7 days after transplantation, the first few noradrenaline and dopamine fibers have grown into the caudal margin o f the transplant bordering on the transected bundlesa,7,2L One week later, the ingrowth of catecholamine fibers from the DCB into the transplant tissue is considerably more advanced, as described below for the control specimens. Therefore, 2 weeks survival after transplantation (and axonal damage) was found suitable for the evaluation of possible inhibitory effects of anti-NGF serum on ingrowth of catecholamine fibers into the transplant, particularly from the DCB. Except in one series of experiments, where the animals were killed 4 weeks after transplantation, all animals observed in the present study were thus sacrificed 2 weeks after transplantation. As pointed out previously 8,2~ the process of 'reinnervation' of the transplanted tissue by the growing central fibers is reproducible when the transplant occurs in the desired position, and will thus allow reliable estimates of the distance o f growth and the magnitude of the ingrowing fibers in the transplant. In order to obtain such a reproducibility only successful transplantations were included in the material. Moreover, to exclude interference by peripheral sympathetic fibers in the transplants, cervical sympathectomy was performed on all experimental and control animals (see Material and Methods section).

(1) Control specimens, 2 weeks survival The control specimens, examined 2 weeks after transplantation, were similar in all control groups. Thus, the various modes of administration of the control sera - single or repeated intraventricular injections, intracerebral injections close to the axons in the DCB, or preincubations of the iris transplant in control sera - - did not markedly influence the extent o f growth, or the magnitude o f ingrowth into the transplant, of sprouting catecholamine fibers originating from the lesioned axon bundles. This was the case for both types o f control sera, normal rabbit serum and rabbit anti-mouse thymocyte serum, and in fact the regrowth into the transplant in the control serum-treated specimens was quite comparable to that in transplants of untreated animals. In the typical control situation, as observed 2 weeks after transplantation, the

Fig. 2. A and B: photomontages illustrating the growth of catecholamine fibers from the transected DCB into the iris transplant (TR) 2 weeks after transplantation. A: control specimen given 2 #1 of normal rabbit serum close to the noradrenaline axons in the DCB (Fig. I) at the time of transplantation. Thin bundles and networks of green-fluorescent fibers have grown into the transplant and extend mainly within the superficial, caudal and rostral, 1/'3 of the transplant; ventrally (down) towards the

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MFB region, and dorsally (up) towards the habenular region. B: specimen given 2 ffl of anti-NFG serum as above. Only very few, delicate, catecholamine fibers (arrows) have grown into the transplant. Note the less abundant 'wild' sprouting caudal to the transplant as compared with the control (Fig. 2A). Dashed lines indicate the caudal border of the transplants ( × 140). The picture is from a specimen with a maximal anti-NGF serum-induced inhibition of the ingrowth from the DCB.

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transected monoamine axons in the MFB and the DCB showed a slightly increased fluorescence (though less prominent than 1 week after transplantation), due to a persisting piling up of the catecholamines and indolamines proximal to the lesion. At the lesioned MFB, numerous delicate, fine varicose green-fluorescent (catecholaminecontaining) and yellow-fluorescent (indolamine-containing) fibers were seen to penetrate the narrow zone of necrosis - - surrounding the transplant - - and to enter the transplant. Within the transplant the green-fluorescent catecholamine fibers formed dense bundles and/or dense networks that covered mainly the caudal 1/3-1/2 of the ventral part of the transplant tissue, and they circumscribed the transplant within the surface zone, to reach the rostral side of the transplant (el Fig. 1). The fiber formations could sometimes be followed dorsally in the transplant up towards the region of the DCB (see Fig. 1). Delicate, yellow-fluorescent indolamine fibers, occurring in thin bundles or densely packed patterns, were demonstrated predominantly in the caudal surface zone of the most ventral part of the transplant, but also to some extent intermingled with green-fluorescent fibers in the interior of the transplant. Generally, the yellow-fluorescent fibers were markedly less abundant within the transplant than the green-fluorescent ones. At the lesioned DCB, sprouting, green-fluorescent fibers were demonstrated in the narrow necrotic zone separating the DCB from the transplant (Fig. 3A). Bundles and plexuses of smooth or varicose fibers had grown into the transplant, and covered almost the whole width of the part of transplant localized at the level of the lesioned DCB (el Fig. 1). The green-fluorescent fiber formations extended (mainly within the superficial 1/3 of the transplant) ventrally down to the growing fibers in the MFB region, and dorsally all the way up to the dorsal end of the transplant, situated in the habenular region (Fig. 2A). Thus, with the exception of the central core of the transplant, approximately the dorsal 2/3 of the transplant was invaded by fibers originating from the DCB (Figs. 2A and 4A). The plexuses formed by the arborizations of the growing DCB fibers sometimes had a pattern much resembling the autonomic ground plexus characteristic for the normal sympathetic innervation of the intact iris.

(2) Effects of intraventricularly administered anti-NGF serum, 2 weeks survival In this set of experiments anti-NGF serum was given either as a single injection (25 #1) 7 days after the transplantation, or as 7 repeated daily injections (each of 25/A) from the day of transplantation and onwards. Controls received normal rabbit serum in the same way. In these series of transplantations most of the transplants fell too medial in the MFB region (cf. Fig. 1) to allow any safe conclusion about the growth process from the MFB fibers. With respect to the growth into the transplant from the lesioned DCB fibers, however, it was clear that neither the single nor the repeated anti-NGF serum injections had had any marked effects as observed 2 weeks after the transplantation.

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(3) Effects of intracerebrally administered anti-NGF serum, 2 weeks survival Two microliters of the anti-NGF serum were injected stereotaxically into the brain tissue, close to the axons in the DCB. Practically all axons in this bundle originate in the locus coeruleus (see refs. 19 and 23). The position of the injection (Fig. 1) was approximately 1 mm rostral to the locus coeruleus and 3 mm caudal to the transplant (i.e. the site of axonal damage). In all specimens the site of the injection was determined in the fluorescence microscope. On the basis of previous observations on the area o f diffusion of a fluorescein-labeled immunoglobulin 21, only such specimens were included in the material in which the tip of the injection needle had fallen within about 1 mm from the DCB, without causing any direct damage to the catecholamine axons in the bundle. The area of obvious necrosis caused by the injection usually had a diameter o f about 0.5 mm. The anti-NGF serum was given as a single injection either at the time of transplantation, or 4 days after transplantation. The control animals were injected with the two types o f control sera - - normal rabbit serum and rabbit anti-mouse thymocyte serum - - in the same way.

(a) Injection given at the time of transplantation As observed in specimens taken 2 weeks after transplantation, this anti-NGF serum treatment had resulted in a markedly reduced ingrowth of fibers from the lesioned DCB into the transplant tissue (Figs. 2 and 3). While in the control specimens large parts of the dorsal 2/3 of the transplant were invaded by green-fluorescent fibers from the DCB (Fig. 2A; see above), the magnitude of ingrowth was very restricted in the anti-NGF serum treated specimens (Fig. 2B), and in the majority of the specimens it was similar to that observed in control specimens taken 1 week earlier. Thus, the sprouting fibers from the DCB generally covered only the caudal 1/4 of the part of the transplant which was in close contact with the lesioned bundle. In a restricted narrow zone at the caudal surface o f the transplant, fibers had also extended ventrally about half-way down to the MFB region, and dorsally up to the habenular region. There was some variation in the magnitude o f the anti-NGF serum effect, so that in 3 of the 10 specimens practically no green-fluorescent fibers were found within the transplant at the DCB (Figs. 2B and 3B), and in one of the 10 the inhibitory effect was uncertain. Although the magnitude of ingrowth into the transplant was much reduced in the experimental animals, the microscopical picture and the growth pattern o f those fibers occurring in the transplant tissue appeared similar in the control and experimental animals. Observations were also made on the axons running up to the transplant within the lesioned DCB (i.e. the preterminal axons ascending close to the site of injection), and on the seemingly random, 'wild' axonal sprouting occurring in the necrosis and the brain tissue caudal to the transplant, around the lesioned axon stumps. In some anti-NGF serum treated animals, particularly in those where very few fibers had grown into the transplant from the DCB, the number of ascending axons fluorescing in the DCB was clearly reduced, and those observed appeared more delicate than in

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b

Fig. 3. A and B: details of the 'wild' sprouting occurring around the lesioned axon stumps of the DCB, and the growth of fibers into the transplant (TR), close to the bundle. Animals killed 2 weeks after transplantation. A : control specimen given 2/~l of rabbit anti-mouse thymocyte serum close to the noradrenaline axons in the DCB (Fig. 1) at the time of transplantation. An abundant, seemingly random axonal sprouting occurs in the partly necrotic brain tissue caudal to the transplant, and the sprouts extend further into the transplant tissue. B: specimen given anti-NGF serum as above. Note the DCB axons ending at the border of the transplant without much sprouting from the lesioned axon stumps. It is also observable that the number of axons fluorescing in the DCB is reduced compared with that of the control (Fig. 3A), and that many of those that are visible appear more delicate and have a lower fluorescence intensity. Dashed lines indicate the caudal border of the transplants ( x 140).

t h e p a r a l l e l l y p r o c e s s e d c o n t r o l s (Fig. 3). I n a d d i t i o n , t h e p a t t e r n o f t h e ' w i l d ' s p r o u t i n g , c a u d a l t o t h e t r a n s p l a n t , w a s in t h e s e s p e c i m e n s less a b u n d a n t t h a n i n t h e c o n -

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trois (Figs. 2 and 3). Due to the marked variability in the fluorescence picture of the ascending axons within the DCB (resulting from technical factors, for instance variation in background fluorescence and variation in fluorescence yield), these effects could not be established with certainty in every single anti-NGF serum treated specimen; it seems quite possible, however, that such effects were consistent phenomena in the anti-NGF serum treated animals. But in no case were there any obvious changes in the fluorescence microscopical appearance of the cell bodies in the locus coeruleus. At the MFB, the growth of green-fluorescent and yellow-fluorescent sprouts into the ventral transplant was generally similar in the experimental and control specimens, and thus the difference in effect of the anti-NGF serum treatment on the growth from the two bundles was very pronounced. Only in a few of the anti-NGF serum treated animals was there a tendency to a slight reduction in the ingrowth from the MFB.

(b) Injection given 4 days after transplantation It has previously been found that the regenerating locus coeruleus neurons in the DCB are most sensitive to the action of exogenous N G F during the very early stages o f the regeneration process3, 21. For this reason, a series of experiments was made to test whether anti-NGF serum would also exert an inhibitory effect on the regrowth of the central noradrenergic neurons if administered a few days after the axonal damage, i.e. at a time when the sprouting process has already started. Anti-NGF serum was injected, 4 days after transplantation, close to the proximal portion o f the DCB, as above (Fig. 1). The results strongly suggest that, when administered at this stage of the regeneration process, the anti-NGF serum no longer has any clear-cut effect on the formation or growth of new axonal sprouts from the lesioned DCB. Only in 1 out of the 6 experimental animals in this group was the magnitude of ingrowth into the transplant from the DCB clearly reduced. In this specimen there was abundant sprouting from the DCB up to the transplant, but the sprouting fibers extended into the transplant to only partly cover the dorsal third o f the transplant. Moreover, the fluorescent axons ascending in the DCB had a more delicate appearance than those in the remaining experimental animals, or those in the controls. Neither the abundance nor the appearance o f the regenerating fibers in the transplants of the remaining anti-NGF serum treated specimens differed from that observed in the control specimens.

(4) Effects of preincubation of the iris transplant in anti-NGF serum, 2 weeks survival It is known that the rat iris, as well as other peripheral tissues, contains endogenous N G F , or NGF-like proteins, and it has been shown that this N G F is released from the iris when cultured in vitro 12. The present procedure - - incubating the iris transplant in anti-NGF serum prior to its placement in the posterior diencephalon - was used with the intention o f accomplishing two things. Firstly, by a specific antigenantibody reaction at least some of the N G F or NGF-like antigens contained in the iris might be blocked by NGF-antibodies. Secondly, NGF-antibodies could also, more unspecifically, become attached to or accumulated in the transplant tissue and then be

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released, at least to some extent, from the transplant in situ. In the latter case, the NGF-antibodies would have the possibility of exerting an effect on the brain tissue, at the level of the lesioned axon stumps. Preincubation o f the transplant tissue in anti-NGF serum for 15 min at room temperature did indeed result in a reduced ingrowth of new catecholamine fibers into the transplant from the DCB (Fig. 4), but not from the MFB. Within the transplant, in 10 out o f the 12 experimental specimens, there were markedly fewer green-fluorescent catecholamine fibers originating from the DCB than in the corresponding controls, and they covered a much smaller area of the transplant. In 2 of these transplants, practically no fluorescent sprouts had grown into the transplant at all, which indicates an almost total inhibition of the ingrowth. By and large, the number of fibers from the DCB in the transplant, and the area of the transplant covered by them, was comparable to that obtained after a single intracerebral injection of anti-NGF serum close to the DCB axons (see above). However, in the specimens carrying irises preincubated in anti-NGF serum, the fluorescence microscopical picture o f the ascending catecholamine axons in the lesioned DCB did not differ clearly from that of the parallelly processed controls, and there was no certain effect on the 'wild' sprouting around the lesioned axon stumps. With respect to the lack of observable effect on the growth from the MFB, it should be pointed out that this growth is much less extensive than that from the DCB at 14 days after transplantation. For this reason the situation at the MFB is less suitable for the evaluation o f small inhibitory effects.

(5) Effects of preincubation of the iris transplant in anti-NGF serum, combined with intracerebral injections of the antiserum, 4 weeks survival The purpose of this series o f experiments was the study of longer term effects of anti-NGF serum on the growth o f new axonal sprouts from the lesioned, noradrenergic neurons o f the DCB, into the iris transplant. The intention was, further, to administer larger amounts o f antiserum than in the previous experiments. Therefore, besides preincubating the iris transplant in anti-NGF serum, two injections (2 #1 each) of antiserum were made, close to the proximal portion o f the DCB (Fig. 1) - - one at the time of transplantation, another 2 weeks later. The controls received normal rabbit serum similarly.

Fig. 4. A and B : growth of green-fluorescent fibers from the transected DCB into the transplant (TR) 2 weeks after transplantation. A: control specimen having the iris transplant incubated for 15 rain in normal rabbit serum prior to its transplantation. Thick bundles and networks of catecholamine fibers have grown into the transplant to cover large areas, excepting the central area of the transplant tissue. B: specimen with the iris transplant incubated as above but in anti-NGF serum. The ingrowth of catecholamine fibers from the DCB is much reduced. Sprouting fibers from the DCB are found in the caudal 1/3-1/2 of the part of the transplant which is in close contact with the lesioned DCB. They are also found in thin bundles, and extend in the caudal surface zone of transplant, ventrally (down) towards the MFB region and dorsally (up) towards the habenular region. Dashed lines indicate border of the transplants ( x 140).

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(a) Control specimens The control specimens received iris transplants which had been preincubated in control serum, and were given two injections of control serum close to the axons ofthe DCB. The general fluorescence microscopical appearance, and the magnitude of ingrowth of fibers into the transplant from the DCB and the MFB, did not differ significantly from untreated transplanted specimens which had the same survival time. At 4 weeks after transplantation, the amine accumulations in the transected axons of the DCB and the MFB were weakly fluorescent, and could only be observed close to the site of the lesion. The number of fibers which had grown into the transplant, as well as the area covered by them, had markedly increased as compared with the 2-week specimens. At the MFB, green-fluorescent fibers growing in bundles or densely packed irregular patterns covered almost the whole ventral part of the transplant tissue. Among these fiber formations, several bundles or dense irregular patterns of yellowfluorescent indolamine fibers were sometimes demonstrated. At the DCB, numerous green-fluorescent fibers extended in several bundles into the transplant and invaded almost entirely the surviving parts of the transplant tissue. Ventrally, they intermingled with the growing fibers which originated in the MFB, and dorsally, they reached, in great numbers, the dorsal end of the transplant. The sprouting fibers from the DCB grew in the transplant in bundles of varying size; they branched profusely and arborized into terminal plexuses that had, in part, a clear resemblance to the autonomic ground plexus formed by the normal sympathetic innervation in the intact iris (cf. ref. 7). Four weeks after transplantation almost the entire surviving transplant was covered thus by regenerated catecholamine fibers which had grown out from the two lesioned bundles. (b) Anti-NGF serum treated specimens Preincubation of the iris transplant in anti-NGF serum, in combination with two injections of the anti-NGF serum close to the DCB caused, at 4 weeks after transplantation, a clearly reduced ingrowth from the lesioned DCB in 4 out of the 6 analyzed specimens. In these, bundles and plexus-like formations of fibers covered, approximately, the caudal 1/3-1/2 of the transplant facing the lesioned DCB. Ventrally, down to the MFB region, and dorsally, up to the habenular region, fibers or fiber formations extended mainly in the caudal 113 of the transplant. Sometimes networks of fibers also covered the surface zone of the dorsal end of the transplant. The numbers of regenerated DCB fibers demonstrated within these transplants were in general comparable to those observed 2 weeks after transplantation in control specimens. Microscopically, no certain differences were found between control and experimental animals with respect to the growth pattern of regenerated DCB fibers. The morphology of the ascending axons in the DCB was difficult to observe at this stage, but in at least one of the experimental specimens, fewer fluorescent axons than normal were visible in the DCB at the lesion site. In two of the anti-NGF serum treated specimens both the number of DCB fibers in the transplant and the area of the transplant covered by the ingrowing fibers

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were similar to those of the controls. It is possible, however, that in one of these two specimens one o f the injections of anti-NGF serum had leaked out into the fourth ventricle, which might explain the lack of effect in this case. As in the previous experiments, the ingrowth of green- and yellow-fuorescent fibers from the MFB into the transplant was not clearly influenced by the anti-NGF serum treatment. DISCUSSION

The present observations provide evidence that the antiserum to N G F impairs axonal sprouting from adult central noradrenergic neurons, when administered intracerebrally at the time of axonal damage. Since control sera (normal rabbit serum and another hyperimmune rabbit serum) did not have this effect, it seems likely that the growth impairment is the result of an interference with N G F or an NGF-like substance in the tissue, through a specific action of the NGF-antibodies. There are observations from experiments with fluorescein-labeled immunoglobulin 21 that this type of protein reaches a high concentration only within a limited distance (in the order of 1-1.25 mm) from the injection site. This supports the idea that the action of the anti-NGF serum, when injected close to the locus coeruleus axons in the mesencephalon, is locally restricted, and probably confined to the region of the lesioned axons. Preliminary observations have also indicated that injections placed close to the cell bodies in the locus coeruleus can induce inhibition of the regenerative growth from the lesioned axons, possibly indicating that the inhibitory action of the anti-NGF serum can be elicited at different parts of lesioned neurons. The present observations on the effect of intracerebral injection o f anti-NGF serum, in combination with the findings that exogenous N G F - - injected in the same position in the mesencephalon - - exerts a stimulatory effect on the sprouting process from the locus coeruleus axons 21, suggest that endogenous N G F may play a role in the regenerative growth of adult, central, noradrenergic neurons. This hypothesis would require that N G F or NGF-like proteins can be produced in the brain, since the bloodbrain barrier can be expected largely to prevent such proteins from entering the brain from the periphery via the blood streamL In fact, there is some indication of such a local production of N G F , or NGF-like proteins, from the detection (by radioimmunoassay technique) of small amounts of 7S NGF-antigens in mouse brain 11. Like many other peripheral tissues, the iris contains NGF-antigens, which are released from the iris when cultured in vitro ~2. Thus, in the experimental situation employed, the transplanted iris tissue might be another source of N G F , of importance for the stimulation of the 'reinnervation' process. It seems possible that in the experiments with a preincubation of the iris in anti-NGF serum, the N G F (or NGF-like material) contained in the iris was blocked, either in situ, or after having been released from its storage sites, and this blockade might thus have been the cause of the growth impairment seen in these experiments. In the reverse experiment el, in which the iris transplant was preincubated in N G F with the intention of increasing its N G F content, the sprouting from the lesioned noradrenergic neurons was stimulated - - thus provid-

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ing evidence that N G F contained in, or associated with, the transplant can exert stimulatory effects. An alternative or contributory explanation for the effect of preincubation in anti-NGF serum on the regenerative growth could be that the NGF-antibodies were released from the transplant in its position in the brain, and reached the region of the lesioned noradrenergic axons. Here, the antibodies would have the possibility of affecting the lesioned axon stumps, and perhaps also - - at a later stage - - the regrowing axon sprouts. In this case, the inhibitory effect might - - at least partly - - reflect a blocking of N G F (or NGF-like material) within the axons and/or the axon sprouts, or in elements associated with them. In a parallel study on the mouse z, it has been demonstrated that anti-NGF serum also causes a strong inhibition of the regrowth of peripheral noradrenergic axons after axonal damage induced by the neurotoxic drug 6-hydroxydopamine. Thus, following one subcutaneous injection of 0.1 ml/g of anti-NGF serum (given the day after the 6-hydroxydopamine treatment) there was a strong reduction in the reformation of the terminal plexuses and in the recovery of endogenous NA in the peripheral organs. The anti-NGF serum treatment caused a marked atrophy of the cell bodies, and a reduction in the noradrenaline content of the cell bodies and their surviving axon parts. However, the neurons - - including the axon stumps - - seemed to survive, suggesting that the inhibited regeneration was not due to a degeneration of the sympathetic neurons. Rather, the impairment of the sprouting process and the regrowth of the sprouting fibers could be regarded as a consequence of an inhibitory effect of the anti-NGF serum on the general performance of the regenerating neurons, including also the production of the noradrenaline transmitter. In the present experiments on the locus coeruleus noradrenergic neurons, fluorescence microscopical changes were sometimes observed in the lesioned axon bundle, i.e. in the surviving portions of the ascending locus coeruleus axons, but there was no direct evidence for an actual degeneration of axons in the bundle. Thus, as in the case of the lesioned peripheral noradrenergic neurons, it seems probable that the observed changes in the proximal stumps reflect a reduced accumulation of noradrenaline in the axons. This could signify that, in addition to the effects on the formation and regrowth of the sprouting fibres, the administration of anti-NGF serum close to the regenerating axons in the DCB causes a reduction in the production or the down-transport of noradrenaline in the lesioned locus coeruleus neurons. At any rate, the effects observed fluorescence histochemically in regenerating central and peripheral noradrenergic neurons after a n t i - N G F serum treatment are qualitatively quite similar, suggesting that the mode of action of the NGF-antibodies in the two systems is in principle the same. The hypothesis that endogenous N G F or NGF-like proteins participate in the physiological regulation or stimulation of the sprouting process which takes place in lesioned noradrenergic neurons has much support in studies on both developing and fully developed neurons in vivo and in vitro (for discussion, see ref. 4). There are interesting similarities between the NGF-induced effect on the metabolism and ultrastructure of the intact nerve cells (as observed in developing sympathetic and sensory ganglia in vivo and in vitro; see refs. 15 and 16) and the characteristic features of the

REGENERATION OF CENTRAL N A NEURONS

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retrograde perikaryal response of nerve cells to axon damage (see ref. 18). This generally holds true for, e.g., the increases observed in protein and RNA synthesis and in the accumulation of neurofilaments and neurotubules. Therefore, the effects induced by NGF and the anti-NGF serum on the regenerating neuron could, perhaps, be viewed as an augmentation and a diminution, respectively, of the retrograde, regenerative response of the nerve cells to the axon damage. Interestingly, in the present experimental model, the anti-NGF serum and the NGF injections were considerably more effective when given at the time of axonal damage than when given 4 days later, i.e. when the sprouting had already begun. If regarded in relation to the retrograde perikaryal response, this could signify that the role of endogenous NGF or NGF-like proteins in axonal regeneration might be of special significance during the very early stages, or in the triggering, of the retrograde response in the locus coeruleus neurons. ACKNOWLEDGEMENTS

The authors wish to thank Annika Borgelin, Britt Nilsson, and Gertrude Stridsberg for their skilful technical assistance. The study was supported by grants from the Faculty of Medicine, University of Lurid, the Magnus Bergvall Foundation, National Institutes of Health, USPHS (NS 06701-07), and from the Swedish Medical Research Council (04X-3874).

REFERENCES

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