0306-4522/92 $5.00 4 0.00 Pergamon Press Ltd 4 1992 IBRO
Neuroscience Vol. 50, No. 4, pp. 953-963, 1992
Printed in Great Britain
R A P I D N E U R A L GROWTH: C A L C I T O N I N G E N E - R E L A T E D PEPTIDE A N D SUBSTANCE P-CONTAINING NERVES ATTAIN E X C E P T I O N A L G R O W T H RATES IN REGENERATING DEER ANTLER C. GRAY, e+ M. HUKKANEN,++ Y. T. KONTTINEN,§ G. TERENGHI,~ T. R. ARNETT,* S. J. JONES,* G. BURNSTOCK* a n d J. M. POLAK~ *Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, U.K. :~Oepartment of Histochemistry, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12 ONN, U.K. §Department of Anatomy, University of Helsinki, Siltavuorenpenger, Helsinki 17, Finland A~tract--Deer antler is a unique mineralized tissue which can produce very high growth rates of > 1 cm/day in large species. On completion of antler growth, the dermal tissues which cover the antler are shed and the underlying calcified tissue dies. After several months the old antler is discarded and growth of a new one begins. It is known that deer antlers are sensitive to touch and are innervated. The major aims of this study were to identify and localize by immunohistochemical techniques the type of innervation present, and to find out whether nerve fibres could exhibit growth rates comparable to those of antler. We have taken tissue sections from the tip and shaft of growing Red deer (Cervus elaphus) antlers at three stages of development; shortly after the initiation of regrowth, the rapid growth phase, and near the end of growth. Incubation of tissue sections with antisera to protein gene product 9.5 (a neural cytoplasmic protein), neurofilament triplet proteins (a neural cytoskeletal protein), substance P and calcitonin gene-related peptide (both of which are present in and synthesized by sensory neurons) showed the presence of immunoreactive nerve fibres in dermal, deep connective and perichondrial/periosteal tissues at all stages of antler growth. The sparse distribution of vasoactive intestinal polypeptide-like immunoreactivity was found in dermal tissue only at the earliest stage of antler development. Nerve fibres immunoreactive to neuropeptide Y, C-flanking peptide of neuropeptide Y and tyrosine hydroxylase, all present in postganglionic sympathetic nerves, were not observed at any stage of antler growth. Nerves expressing immunoreactivity for any of the neural markers or peptides employed could not be found in cartilage, osteoid or bone. These results show that antlers are innervated mainly by sensory nerves and that nerves can attain the exceptionally high growth rates found in regenerating antler.
Deer antlers are secondary sexual characters, which mineralize, exfoliate the outer soft tissues (velvet), and, after the rut, are discarded (cast) annually. Developing antler can exhibit growth rates of up to 1.8 cm/day in large deer species. 2° F o r m i n g antler consists of growing tips m a d e up of proliferating undifferentiated m e s e n c h y m a l cells (reserve mesenchyme) which lie b e n e a t h a fibrous p e r i c h o n d r i u m . F u r t h e r d o w n the central core of the (growing) antler is a zone of forming cartilage which becomes hypertrophic, calcifies, a n d is modified first into p r i m a r y a n d then, secondary, spongiosa. 32° This central cartilagenous region is s u r r o u n d e d initially by a collar of de novo woven bone. Overlying the
mineralizing woven b o n e is a layer of periosteum-like tissue which merges imperceptibly with the peric h o n d r i u m . 3 The entire antler is covered by epidermis with a b u n d a n t hair follicles and sebaceous glands but no erector pili muscles. O b s e r v a t i o n s o f deer b e h a v i o u r provides clear evidence t h a t forming antlers are innervated. Deer avoid contact with their soft developing antlers, a n d d e m o n s t r a t e an acute spatial awareness a n d m e m o r y of antler points which are outside their visual range as s h o w n by their skill in sparring with h a r d antlers during the rut. Direct evidence for the i n n e r v a t i o n of antler tissue was reported by Berthold in 1831. 6 Very few experiments involving the d e n e r v a t i o n of antler have been reported. Wislocki a n d Singer 48 found that when one antler was denervated, that antler did not grow in the typical shape or attain the dimensions of the other. They also provided a histological d e m o n stration of i n n e r v a t i o n in the developing antler a n d f o u n d that this was derived from the supraorbital a n d t e m p o r a l b r a n c h e s of the trigeminal nerve. 4~ Since then b o t h myelinated a n d unmyelinated nerves
tTo whom correspondence should be addressed. Abbreviations: ABC, avidin-biotin-peroxidase complex; BSA, bovine serum albumin; CGRP, calcitonin generelated peptide; CPON, C-flanking peptide of neuropeptide Y; DAB, 3,3'diaminobenzidine tetrahydrochloride; LI, like immunoreactivity; NF, neurofilament triplet protein; PBS, phosphate-buffered saline; PGP 9.5, protein gene product 9.5; VIP, vasoactive intestinal peptide. 953
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C. GRAY et al.
have been described in the epidermis and in the vascularized dermal tissue. 46 Although early studies have confirmed the presence of nerves in antler, the methods used do not indicate the type or possible function of the innervation. In this study, we have applied immunocytochemical techniques for the visualization of several neural proteins and peptides in order to localize and to characterize different neural subpopulations in developing antlers. Antisera to protein gene product 9.5 (PGP 9.5) and neurofilament triplet protein (NF) were used to assess the overall innervation. P G P 9.5 is a cytoplasmic ubiquitin C-terminal hydrolase and is known to be present in all neural elements) 4'~6'47 In contrast, N F is a cytoskeletal neural protein ~3 and therefore could be useful as a marker for persistent nerve fibres in rapidly growing tissues. Substance P, one of the major neuropeptides involved in nociception, is localized in primary afferent fibres 3~'38 and is often co-stored with calcitonin gene-related peptide (CGRP), 35 a peptide found to be abundant in sensory nerves in bone tissues. 8'25 Antisera to neuropeptide Y, the C-flanking peptide of neuropeptide Y (CPON), tyrosine hydroxylase and vasoactive intestinal polypeptide (VIP) were used as markers for autonomic nerves, although VIP is also present in some sensory neurons. 41 The main aim of this study was to examine the growth rate of neural elements in growing antler and in particular to look for the presence of peptidelike immunoreactivity (LI) at the rapidly growing antler tip. EXPERIMENTAL PROCEDURES
Tissues and fixation Commercially farmed red deer (Cervus elaphus) antler tissue was purchased from Mr R. Hayward, Slopers Pond Farm, Barnet, Hertfordshire, U.K. Antlers were obtained at two-weekly intervals during mid June and July, a total of three antler specimens. Deer were culled by a high-velocity rifle shot to the head and the antlers removed within 5 min before fixation. Tissue was sliced into pieces of 0.5 x 3.0 cm and fixed by immediate immersion in Zamboni's fluid 4~ for 20 h at +4°C. Tissues were then washed in 15% w/v sucrose and 0.001% w/v sodium azide in 0.01 M phosphate-buffered saline (PBS) at pH 7.2 for two days at +4°C. Antler lengths varied between 10 and 40 cm during the collection period and represented developmental stages from early growth
through the period of rapid elongation to the etad ol extension. The first specimen collected still carried the scab left by casting the previous year's antler and the second was collected during the rapid growth phase. Antlers from these first two deer had substantial amounts of reserve mesenchyme present and a distance between the tip and mineralizing front of over 2.5 cm. In contrast to this, the third specimen was nearing the end of its growth and had minimal amounts of reserve mesenchyme and was clearly mineralizing at 1 cm below the tip. Sections of antler were taken from the tip and approximately 8 cm below the tip. Both longitudinal and transverse sections were studied. Samples of small intestine and nasal skin were used as controls for antibodies and methodology and were treated in the same way. Antisera The antisera used in this study, dilutions and characteristics are shown in Table 1. Omission of primary antiserum or one of the subsequent steps in the avidin-biotinperoxidase complex (ABC) method was included as a control for method specificity. Preabsorption of antisera with an excess of corresponding peptide antigen was used as a control for antibody specificity. Immunohistochemistry Frozen sections (15 pm) cut on a cryostat were mounted on poly-L-lysine-coated slides)° Endogenous peroxidase was inhibited by soaking the sections in 0.3% hydrogen peroxide in methanol for 20 min. Following rinses in PBS, the slides were then placed in a humid chamber and the sections incubated (i) overnight at +4°C with primary antibodies, diluted in PBS containing 1/30 normal goat serum and 0.1% w/v bovine serum albumin (BSA) (Sigma Chemical Co., St Louis, U.S.A.) (Table 1); (ii) with biotinylated goat anti-rabbit IgG, diluted 1:100 in PBS with 0.1% w/v BSA, for 60 rain at room temperature; and (iii) with ABC (avidin and biotinylated horseradish peroxidase both diluted 1/200 in PBS) for 60 min at room temperature, z9 The sections were then incubated for 5min in a solution of 3,Ydiaminobenzidine (DAB) and nickel ammonium sulphate containing glucose and glucose oxidase (EC 1.1.3.4) (Sigma Chemical Co., St Louis, U.S.A.).42 After each step, the slides were rinsed three times in PBS for 5 min. Finally the slides were dehydrated in ethanol, cleared in Inhibisol (Kalon Chemicals, Cramlington, U.K.) and mounted in Pertex (Histolab Products Ab, Gothenburg, Sweden). Assessment of immunoreactive-fibre distribution Results were assessed semi-quantitatively for two reasons: firstly, the number of immunoreactive fibres was low; secondly many fibres, especially those expressing substance P- and VIP-LI, were extremely thin and only visible at high magnification. Immunostained antler sections of approximately 1 cm2 were microscopically monitored for the
Table 1. Characteristics of the antibodies used in the study Dilution
Peptide absorption
PGP 9.5 NF (68,000; 150,000; 200,000 mol.wt)
1/6000 1/2000
N/A N/A
Substance P (synthetic) ~-CGRP (rat, synthetic) Neuropeptide Y CPON (synthetic) Tyrosine hydroxylase
1/4000 1/4000 1/4000 1/4000 1/2000
1.0 nM/ml 0.1 nM/ml N/A N/A N/A
VIP
1/10 000
0.1 nM/ml
Antigen
N/A, not applicable.
Source
References
Ultraclone, Cambridge, U.K. D. Dahl, Veterans Admin., Boston, MA, U.S.A. Hammersmith Hospital Hammersmith Hospital Hammersmith Hospital Hammersmith Hospital D. Kuhn, Lafayette Clinic, Detroit, MI, U.S.A. Hammersmith Hospital
t6, 23, 47 13 35 19, 35 22 I, 22 33 7
Innervation of antlers
955
presence of immunoreactive fibres and allocated into one of seven tissue types for each deer, namely: epidermis; subepidermis containing hair follicles and sebaceous glands; deep connective tissue containing the major blood vessels; periosteum/perichondrium (tip/shaft); cartilage; osteoid; bone (Figs 1, 2). Nerves observed were allocated one of four categories for each tissue type and deer according to the frequency with which they occurred; I, no immunoreactive nerves detected; II, sparse, 1-10 fibres found in the total sample; III, moderate, 10 20 fibres found in the total sample; IV, abundant, >20 fibres in the total sample. The total sample for each antiserum represents approximately 10 tissue sections/antler region per deer. This represents a total sample in excess of 400 tissue sections. RESULTS I m m u n o r e a c t i v i t y to P G P 9.5, N F , s u b s t a n c e P a n d C G R P was o b s e r v e d in tissue t a k e n f r o m all stages o f antler g r o w t h (Table 2). W h e n c o m p a r e d to the c o n t r o l tissues antlers were relatively sparsely i n n e r v a t e d . P G P 9.5-LI c o u l d only be o b s e r v e d n e a r to the e p i d e r m i s ; in o t h e r r e g i o n s a high b a c k g r o u n d s t a i n i n g was c r e a t e d b y unspecific b i n d i n g o f the a n t i b o d i e s to o s t e o - a n d c h o n d r o b l a s t i c cells in the d e e p e r tissue. N F - L I nerves were o b s e r v e d in s u b e p i d e r m i s , d e e p c o n n e c t i v e tissue a n d p e r i c h o n d r a l / p e r i o s t e a l tissues b o t h as nerves b u n d l e s a n d as i n d i v i d u a l nerve fibres. N F - L I nerves were also p r e s e n t in soft tissues n e a r h y p e r t r o p h i c / c a l c i f y i n g cartilage in t h e central, c a r t i l a g e n o u s , region o f the antler (Figs l, 3), b u t only as sparse a n d s h o r t single fibres (Fig. 4). N F - L I c o u l d n o t be f o u n d in the s p o n g i o s a o r a m o n g the w o v e n b o n e s u r r o u n d i n g it. S u b s t a n c e P- a n d C G R P - L I nerve fibres were f o u n d s u b j a c e n t to the e p i d e r m i s (Figs 1, 2 a n d 5) a n d in the d e e p e r c o n n e c t i v e tissues (Figs 1, 2, 6 - 8 ) . F i b r e s i m m u n o r e a c t i v e to b o t h a n t i s e r a were m o s t
Fig. 1. Diagram showing a growing antler tip during a rapid growth phase. The boxes (2) and (3) mark the location of Figs 2 and 3. C, chondroblastic zone; DC, deep connective tissue; ED, epidermis; HC, hypertrophic/calcifying cartilage; P, periosteum; PC, perichondrium; PS, primary spongiosa; RM, reserve mesenchyme (proliferating chondroblastic region); SED, subepidermis; SS, secondary spongiosa; WB, woven bone. (Diagram not to scale).
a b u n d a n t in the d e e p e r c o n n e c t i v e tissue s u r r o u n d i n g the p e r i c h o n d r i u m / p e r i o s t e u m - l i k e tissue, p a r t i c u larly in the v a s c u l a r region o f the tip. T h e s e i m m u n o reactive fibres were f o u n d b o t h perivascularly a r o u n d small b l o o d vessels a n d as free fibres (Figs 6, 7).
Table 2. Summary of semi-quantitative assessment of fibre distribution Deer
I
I1
III
Length of antler
Tissue
10 cm
Epidermis Subepidermis Deep connective tissue Perichondrium/periosteum Cartilage Osteoid Woven bone
20 cm
Epidermis Subepidermis Deep connective tissue Perichondrium/periosteum Cartilage Osteoid Woven bone
+ + + + + + N/A N/A N/A N/A
Epidermis Subepidermis Deep connective tissue Perichondrium/periosteum Cartilage Osteoid Woven bone
. + + + + + N/A N/A N/A N/A
40 cm
Immunoreactivity NF SP CGRP
PGP 9.5 + + + + + + + N/A N/A N/A N/A
-+ + + + + --. . --+ + + + -. . .
. -+ + + + ----
+ + + + + --. .
+ + + + + + + +
.
-+ + + + + + + --
-. .
+ + --
.
-+ + + . .
VIP
---
. .
. -+ ----
+ + + + + +
--
--
--
N/A, not applicable (see text);--immunoreactive fibres absent; + sparse; + + moderate; + + + abundant.
' i~'i~i~ S ! ¸
i
Innervation of antlers
957
Fig. 3. The central core of developing antler showing hypertrophic cartilage (HC), and vascular lumen (VL). Neurofilament-LI fibres were found in similar tissues to those arrowed. Hematoxylin and eosin counterstaining. (Longitudinal section.) Magnification × 150. Substance P-LI nerve fibres were much more sparse than C G R P - L I fibres, although tissue distribution and localization was similar as evidenced by immunostaining of consecutive tissue sections. Although some P G P 9.5-, N F - , substance P- and C G R P - L I could be detected in most dermal and perichondrial/ periosteal tissue, only deep connective tissue showed
a relatively abundant innervation (Table 2). Nerve fibres containing P G P 9.5-, NF-, substance P- or C G R P - L I were not found within forming cartilage, osteoid or any mineralized antler tissue. Sparse VIP-LI fibres were present in deep connective tissues (Figs l, 2, 9) and occasionally in subepidermal tissue in close proximity to sebaceous
Fig. 4. A neurofilament-Ll fibre (arrowed) within the central cartilaginous region of antler (tor morphology and localization see Figs 1, 3). The fibre is situated in soft tissues adjoining a blood vessel (VL) and hypertrophic cartilage (HC). No immunoreactive fibres for other neural constituents could be found in this or a similar location. (Longitudinal section.) Magnification is × 350.
958
C. GRAYet aL
glands but only at the earliest stage of antler growth. No immunoreactivity could be observed for NPY, CPON or tyrosine hydroxylase in any antler tissue. However, immunoreactivity for these neuropeptide and enzyme antisera were abundant in both intestinal and nasal control tissues. Preabsorptions of peptide antisera with an excess of antigen abolished all immunoreactivity in both antler and control tissues (Table 1). DISCUSSION This study primarily demonstrates that neural elements are able to attain the same exceptionally high growth rates as are found in regenerating antler. Antlers grow by a rapid cellular proliferation, differentiation, hypertrophy, and endochondral ossification from the growing tip 3'4and by the de novo formation of a collar of initially woven bone around the central cartilaginous core? '9 Most of the cellular constituents of antler develop in situ, notable exceptions being the nervous and vascular tissues which must invade forming tissue. It follows that since antler tips are innervated these nerves are able to attain the high growth rates observed in red deer. Using antibodies to PGP 9.5 and NF we have located nerves in antler subepidermis, deep connective tissue, and in periosteum/perichondrium.
In addition to this, a few NF-LI nerve fibres were observed in tissues adjacent to hypertrophic cartilage within the central core of the antler. These may represent a population of nerve fibres which are not functionally active. As no neuropeptide-LI could be observed at this location these possibly represent a population of nerve fibres which have been left behind by the growing tip, since cytoskeletal proteins are not likely to undergo as rapid degradation and removal as intravesicular peptides. The principal neuropeptide-like immunoreactivities found were substance P and CGRP which are the major peptide mediators of nociception.49 Localization of substance P and CGRP in antler/bone tissue may also have roles other than that of pain perception. For example, substance P and CGRP act as potent transmitters following antidromic impulse generation in sensorimotor nerves during "axon reflex" action, t2,34In experiments where rat bones had been surgically denervated, varying effects have been observed. Metatarsals and humeri were reduced in size ~7'~8 and femoral length increased. ~8 In a recent study, Hill and co-workers found no change in bone growth although after sympathectomy or capsaicininduced sensory denervation osteoclast distribution on the bone surface was altered. 27 It is not clear how physical stress generated by muscular activity and locomotion have affected the results obtained in these studies. However, antler tissue does not contain
Fig. 5. A strongly stained nerve fibre expressing CGRP-LI (for morphology and localization see Figs l, 2). The apparent staining of the epidermis (ED) is in fact natural pigmentation. The nerve fibre is in close proximity to a hair follicle(HF) and sebaceous gland (SG). (Longitudinal section.) Magnification is × 350.
959
Innervation of antlers
Fig. 6. A substance P-LI nerve fibre (arrowed) within the deep connective tissue of antler (for morphology and localization see Figs 1, 2.) Nerves expressing substance P-LI were all exceptionally thin fibres. These fibres had a similar distribution to those expressing CGRP-LI but they were generally less abundant. The feature marked VL is a vascular lumen of a small blood vessel. (Longitudinal section.) Magnification is ×420. skeletal muscle n o r are antler growing tips load bearing. Antler m a y therefore provide a useful experimental model in which to study the effects of neural influences o n b o n e growth free from physical stresses. Early experiments in which one antler was denervated
reported changes in shape a n d a reduction in size when c o m p a r e d to the other. However, the investigators believed this could have been caused by injury due to the loss of sensation. 48 A subsequent a n d more extensive study showed t h a t denervated antlers were
t
t
1L
Fig. 7. Perivascular CGRP-LI fibres (arrowed) within the deep connective tissue region of antler (for morphology and localization see Figs 1, 2). Similar fibres were also present in periosteal/perichondrial tissue although they were less common. VL, vascular lumen. (Longitudinal section.) Magnification is × 420.
960
C. GgAV et al.
Fig. 8. A transverse section showing a CGRP-LI nerve bundle (arrowed) within the deep connective tissue (for morphology and localization see Figs 1, 2). Fibres can clearly be seen branching out from the main bundle which has been transected. Similar nerve fibre bundles were not found in periosteum/perichondrium or subepidermal tissue. (Transverse section.) Magnification is × 420.
smaller a n d o f altered shape a n d t h a t this occurred m the absence o f any o t h e r external injury. 44 Interestingly, in experiments where trigeminal nerves were electrically stimulated, antlers h a d increased lengths ( > 70%), weights ( > 4 0 % ) a n d altered shapes. ~ Electrical stimulation will o f course induce a release o f
n e u r o p e p t i d e content from terminal vesicles in target tissues. C G R P is a p o t e n t m o d u l a t o r o f bone m e t a b olism. In vivo C G R P a d m i n i s t r a t i o n has been f o u n d to induce hypocalcaemia 4° a n d p r o m o t e calcium u p t a k e by bone. 2 In vitro, C G R P has been s h o w n to increase c A M P , 36 to stimulate osteogenesis, 5 a n d
Fig. 9. VIP-LI nerve fibres (arrowed) within the deep connective tissue of antler (for morphology and localization see Figs 1, 2). VIP-LI nerves were only found in early growing antler (deer I) and were both small and sparse. (Longitudinal section.) Magnification is x 560.
lnnervation of antlers to inhibit bone resorption. ~5'5° Antler osteoclasts are known to be responsive to calcitonin, 2~ and although C G R P is much less potent than calcitonin its local release from nerve terminals may modulate antler bone resorption (and formation) via a calcitonin-like inhibition of osteoclastic resorption. Growing antler is highly vascularized as might be expected in such a metabolically active tissue with a high demand for oxygen and nutrients. A striking feature of antler is that it has vascular cartilage not normally found in postnatal mammals. Substance P and C G R P are both potent vasodilators. ~° Substance P has been shown to stimulate endothelial cell proliferation, migration and neovascularization. 5~'52 Similarly C G R P is a mitogen for endothelial cells. 24 These findings indicate that substance P- and C G R P containing fibres may play an important role in the rapid growth of antler by controlling vascular tone and neovascularization. We found no immunoreactivity in antler tissue for NPY, C P O N or tyrosine hydroxylase. This suggests the lack of sympathetic vasomotor innervation which is surprising in view of the vascular nature of antler tissue. This could be due to a lack of cross-reactivity to these peptides/enzyme in deer, but this is unlikely since fibres immunoreactive to these antisera were observed in the control tissues. In the present study we did not examine any tissue from the proximal, highly mineralized, antler shaft. Neural elements expressing these peptides and catecholamine synthesizing enzyme may exhibit a slower growth rate and be unable to invade the rapidly growing antler tip. However, our findings support the study by Rayner and Ewen who could not demonstrate any evidence of the adrenergic innervation of antlers using a formaldehyde vapour technique. 39
961
In the present study, sparse VIP-LI nerve fibres were observed in deep connective tissue and occasionally near sebaceous glands in the subepidermis of early developing antler, but not in later stages of growth. VIP, which has been shown to be present in both autonomic (sympathetic 26 and parasympathetic ~2) and sensory neurons, 37"41 stimulates smooth muscle relaxation and vasodilation. In bone, it is a potent stimulator of osteoclastic resorption. 2s The absence of VIP-LI from tissues taken at later stages of antler growth suggests that this subpopulation of nerves does not keep pace with antler growth and this finding could support its localization in antler autonomic nerves. CONCLUSION We have conclusively shown that substance Pand C G R P - L I nerve fibres, most probably of sensory origin, can exhibit the exaggerated growth rates found in antler. In addition, this study confirms and extends earlier studies which demonstrated the presence of nerves but the absence of adrenergic innervation in the developing antlers. The localization of substance P- and C G R P - L I , in the dermal, deep connective and perichondrial/periosteal tissues of developing antler supports an important role for sensory nerves in antlerogenesis. Acknowledgements--The authors would like to thank the
BUPA Medical Foundation, the Grand Charity of Freemasons, the Nuffield Foundation and the Science and Engineering Research Council, U.K. (C.G.). the Emil Aaltonen Research Foundation (M.H.) and the Finnish Academy, Finland (Y.T.K.) the Research into Ageing (T.R.A.) and the British Heart Foundation U.K. (G.B.). The authors would also like to express their thanks to Mr P. Bose for the translation of the article by Berthold)
REFERENCES
1. Allen J. M., Potak J. M. and Bloom S. R. (1985) Presence of the predicted C-flanking peptide of neuropeptide Y (CPON) in tissue extracts. Neuropeptides 6, 95-100. 2. Ancill A. K., Bascal Z. A., Whitaker G. and Dacke C. G. (1991) Calcitonin gene-related peptide promotes transient radiocalcium uptake into chick bone in vivo. Expl Physiol. 76, 143 146. 3. Banks W. J. and Newbrey J. W. (1983) Light microscopic studies of the ossification process in developing antlers. In Antler Development in Cervidae (ed. Brown R. D.). Vol. 1, pp. 231 260. Caesar Kleberg Wildlife Research Institute, Kingsville, Texas. 4. Banks W. J. and Newbrey J. W. (1983) Antler development as a unique modification of mammalian endochondral ossification. In Antler Development in Cervidae (ed. Brown R. D.), Vol. 1, pp. 279 306. Caesar Kleberg Wildlife Research Institute, Kingsville, Texas. 5. Bernard G. W. and Shih C. (1990) The osteogenic effect of neuroactive calcitonin gene-related peptide. Peptides !1, 625 ~32. 6. Berthold A. A. (1831) 1Dber das Wachstum, den Abfall und die Wiedererzeugung der Hirschgeweihe. Beitrag z Anat. Zool. u Physiol., pp. 39-96. G6ttingen, Germany. 7. Bishop A. E., Polak J. M., Yiangou Y., Christofides N. D. and Bloom S. R. (1984) The distributions of PHI and VIP in porcine gut and their co-localisation to a proportion of intrinsic ganglion cells. Peptides 5, 255-259. 8. Bjurholm A., Kreicbergs A., Brodin E. and Schultzberg M. (1988) Substance P- and CGRP-immunoreactive nerves in bone. Peptides 9, 165 171. 9. Boyde A., Arnett T. R., Gray C., Loudon A. S. 1. and Maconnachie E. (1989) Scanning electron microscopy of vascular mineralising cartilage in developing antler. Bone 11, 228. 10. Brain S. D. and Williams T. J. (1988) Substance P regulates the vasodilator activity of calcitonin gene-related peptide. Nature 335, 73 75. 11. Bubenik G. A., Bubenik A. B., Stevens E. D. and Binnington A. G. (1982) The effect of neurogenic stimulation on the development and growth of bony tissues. J. exp. Zool. 219, 205 216. 12. Burnstock G. (1990) Co-transmission: the fifth Heymans memorial lecture. Archs int. Pharmacodyn. ThOr. 304, 7 23.
962
C. GRAY et al.
13. Dahl D. and Bignam A. (1977) Preparation of antisera to neurofilament protein from chicken brain and human sciatic nerve. J. comp. Neurol. 176, 645-658. 14. Day I. N., Hinks L. J. and Thompson R. J. (1990) The structure of the human gene encoding protein gene product 9.5 (PGP 9.5), a neuron-specific ubiquitin C-terminal hydrolase. Biochem. J. 268, 52t-524. 15. D'Souza S. M., MacIntyre I., Girgis S. I. and Mundy G. R. (1986) Human synthetic calcitonin gene-related peptide inhibits bone resorption in vitro. Endocrinology 119, 58-61. 16. Doran J. F., Jackson P., Kynoch P. A. and Thompson R. J. (1983) Isolation of PGP 9.5, a new human neurone-specific protein detected by high-resolution two-dimensional electrophoresis. J. Neurochem. 40, 1542-1547. 17. Dysart P. S., Harkness E. M. and Herbison G. P. (1989) Growth of the humerus after denervation. An experimental study in the rat. J. Anat. 167, 147-159. 18. Garcrs G. L. and Santandreu M. E. (1988) Longitudinal bone growth after sciatic denervation in rats. J. Bone Joint Surg. 70B, 315-318. 19. Gibson S. J., Polak J. M., Bloom S. R., Sabate I. M., Mulderry P. M., Ghatei M. A., McGregor G. P., Morrison J. F. B., Kelly J. S., Evans R. M. and Rosenfeld M. G. (1984) Calcitonin gene-related peptide immunoreactivity in the spinal cord of man and eight other species. J. Neurosci. 4, 3101-3111. 20. Goss R. J. (1983) Deer Antlers: Regeneration, Function, and Evolution, Vol. 1, pp. 133-171. Academic Press, New York. 21. Gray C., Taylor M. L., Horton M. A., Loudon A. S. I. and Arnett T. R. (1992) Studies on cells derived from growing deer antler. In The Biology o f Deer (ed. Brown R. D.), Vot 1, pp. 511-519. Springer, New York. 22. Gulbenkian S., Wharton J., Hacker G. W., Varndell I. M., Bloom S. R. and Polak J. M. (1985) Co-localization of neuropeptide Y (NPY) and its C-terminal flanking peptide (C-PON). Peptides 6, 1237-1243. 23. Gulbenkian S., Wharton J. and Polak J. M. (1987) The visualization of cardiovascular innervation in the guinea pig using an antiserum to protein gene product 9.5 (PGP 9.5). J. auton, nerv. Syst. 18, 235-247. 24. Haegerstrand A., Dalsgaard C.-J., Jonzon B., Larsson O. and Nilsson J. (1990) Calcitonin gene-related peptide stimulates proliferation of human endothelial cells. Proc. natn. Acad. Sci. U.S.A. 87, 3299-3303. 25. Hill E. L. and Elde R. (1988) Calcitonin gene-related peptide-immunoreactive nerve fibres in mandibular periosteum of rat: evidence of a primary efferent origin. Neurosci. Lett. 85, 172-178. 26. Hill E. L. and Elde R. (1989) Vasoactive intestinal peptide distribution and colocalization with dopamine-/~hydroxylase in sympathetic chain ganglia of pig. J. auton, nerv. Syst. 27, 229-239. 27. Hill E. L., Turner R. and Elde R. (1991) Effects of neonatal sympathectomy and capsaicin treatment on bone remodeling in rats. Neuroseience 44, 747-755. 28. Hohmann E. L., Levine L. and Tashjian A. H. (1983) Vasoactive intestinal peptide stimulates bone resorption via a cyclic adenosine 3',5'-monophosphate dependant mechanism. Endocrinology 112, 1233-1239. 29. Hsu S. M., Raine L. and Fanger H. (1981) Use of avidin-biotin peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem. 29, 577 -580. 30. Huang W.-M., Gibson S. J., Facer P., Gu J. and Polak J. M. (1983) Improved section adhesion for immunocytochemistry using high molecular weight polymers of L-lysine as a slide coating. Histochemistry 77, 275-279. 31. H6kfelt T., Kellerth J. O., Nilsson G. and Pernow B. (t975) Morphological report for a transmitter role of substance P, immunohistochemical localization in the central nervous system and in primary sensory neurones. Science 190, 889-890. 32. Larsson L.-I., Fahrenkrug J., Schafflitzky De Muckadell O., Sundler F., H~kanson R. and Rehfeld J. F. (1976) Localization of vasoactive intestinal polypeptide (VIP) to central and peripheral neurons. Proc. nam. Acad. Sci. U.S.A. 73, 3197-3200. 33. Lundberg J. M., Terenius L., Hrkfelt T., Martling C. R., Tatemoto K., Mutt V., Polak J. M., Bloom S. R. and Goldstein M. (1982) Neuropeptide Y (NPY)-like immunoreactivity in peripheral noradrenergic neurons and effects of NPY on sympathetic function. Acta physiol, scand. 116, 477-480. 34. Maggi C. A. (1991) The pharmacology of the efferent function of sensory nerves. J. auton. Pharmac. il, 173-208. 35. Merighi A., Gulbenkian S., Gibson S. J. and Potak J. M. (1988) Ultrastructural study of calcitonin gene-related peptide-, neurokinins- and somatostatin-immunoreactive neurones in rat dorsal root ganglia. Evidence of the colocalisation of different peptides in single secretory granules. Cell Tiss. Res. 254, 101-109. 36. Michelangeli V. P., Fletcher A. E., Allan E. H., Nicholson G. C. and Martin T. J. (1989) Effects of calcitonin gene-related peptide on cyclic AMP formation in chicken, rat, and mouse bone cells. J. Bone Miner. Res. 4, 269-272. 37. Morris J. L. and Gibbins I. L. (1992) Co-transmission and neuromodulation. In Autonomic Neuroeffector Mechanisms (eds Burnstock G. and Hoyle H. V.), Vol. 1, pp. 33-119. Harwood, Chur, Switzerland. 38. Pernow B. (1983) Substance P. Pharmac. Rev. 35, 85-141. 39. Rayner V. and Ewen S. W. B. (1981) Do the blood vessels of the antler velvet of the red deer have an adrenergic innervation. Q. Jl exp. Physiol. 66, 81-86. 40. Roos B. A., Fischer J. A., Pignat W., Alander C. B. and Raisz L. G. (1986) Evaluation of the in vivo and in vitro calcium-regulating actions of noncalcitonin peptides proceeded via calcitonin gene expression. Endocrinology llg, 4(~51. 41. Shehab S. A. S., Atkinson M. E. and Payne J. N. (1986) The origins of the sciatic nerve and changes in neuropeptides after axotomy: a double labelling study using retrograde transport of true blue and vasoactive intestinal polypeptide immunohistochemistry. Brain Res. 376, 180--185. 42. Shu S., Ju G. and Fan L. (1988) The glucose oxidase-DAB-nickel method in peroxidase histochemistry of the nervous system. Neurosci. Lett. 85, 169-171. 43. Stefanini M., De Martino C. and Zamboni L. (1967) Fixation of ejaculated spermatozoa for electron microscopy. Nature 216, 173 174. 44. Suttie J. M. and Fennessy P. F. (1985) Regrowth of amputated antlers with and without innervation. J. exp. ZooL 234, 359 366. 45. Tatemoto K., Carlquist M. and Mutt V. (t982) Neuropeptide Y - - a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide. Nature 296, 659-660. 46. Vacek Z. (1955) Innervace lyci rostoucia parohu u cervidu. Cslkgl M o r f 3, 249-264,
Innervation of antlers
963
47. Wilkinson K. D., Lee K. M., Deshpande S., Duerksen-Hughes P., Boss J. M. and Pohl J. (1989) The neuron-specific protein PGP 9.5 is a ubiquitin carboxyl-terminal hydrolase. Science 246, 67ff673. 48. Wislocki G. B. and Singer M. (1946) The occurrence and function of nerves in the growing antler of deer. J. comp. Neurol. 85, 1-19. 49. Woolf C. and Wiensenfeld-Hallin Z. (1986) Substance P and calcitonin gene-related peptide synergistically modulate the gain of the nociceptive flexor withdrawal reflex in the rat. Neurosci. Lett. 66, 226-230. 50. Zaidi M., Chambers T. J., Bevis P. J. R., Beacham J. L., Gaines Das R. E. and MacIntyre I. (1988) Effects of peptides from the calcitonin genes on bone and bone cells. Q. Jl exp. Physiol. 73, 471-485. 51. Ziche M., Morbidelli L., Pacini M., Geppetti P., Alessandrini G. and Maggi C. A. (1990) Substance P stimulates neovascularization in vivo and proliferation of cultured endothelial cells. Microvasc. Res. 40, 264-278. 52. Ziche M., Morbidelli L., Geppetti P., Maggi C. A, and Dolara P. (1991) Substance P induces migration of capillary endothelial cells: a novel NK-1 selective mediated activity. Life Sci. 48, PL7 Pll.
(Accepted 18 May 1992)
Neuroscience Vol. 50, No. 4, pp. 953-963, 1992
Printed in Great Britain
R A P I D N E U R A L GROWTH: C A L C I T O N I N G E N E - R E L A T E D PEPTIDE A N D SUBSTANCE P-CONTAINING NERVES ATTAIN E X C E P T I O N A L G R O W T H RATES IN REGENERATING DEER ANTLER C. GRAY, e+ M. HUKKANEN,++ Y. T. KONTTINEN,§ G. TERENGHI,~ T. R. ARNETT,* S. J. JONES,* G. BURNSTOCK* a n d J. M. POLAK~ *Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, U.K. :~Oepartment of Histochemistry, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12 ONN, U.K. §Department of Anatomy, University of Helsinki, Siltavuorenpenger, Helsinki 17, Finland A~tract--Deer antler is a unique mineralized tissue which can produce very high growth rates of > 1 cm/day in large species. On completion of antler growth, the dermal tissues which cover the antler are shed and the underlying calcified tissue dies. After several months the old antler is discarded and growth of a new one begins. It is known that deer antlers are sensitive to touch and are innervated. The major aims of this study were to identify and localize by immunohistochemical techniques the type of innervation present, and to find out whether nerve fibres could exhibit growth rates comparable to those of antler. We have taken tissue sections from the tip and shaft of growing Red deer (Cervus elaphus) antlers at three stages of development; shortly after the initiation of regrowth, the rapid growth phase, and near the end of growth. Incubation of tissue sections with antisera to protein gene product 9.5 (a neural cytoplasmic protein), neurofilament triplet proteins (a neural cytoskeletal protein), substance P and calcitonin gene-related peptide (both of which are present in and synthesized by sensory neurons) showed the presence of immunoreactive nerve fibres in dermal, deep connective and perichondrial/periosteal tissues at all stages of antler growth. The sparse distribution of vasoactive intestinal polypeptide-like immunoreactivity was found in dermal tissue only at the earliest stage of antler development. Nerve fibres immunoreactive to neuropeptide Y, C-flanking peptide of neuropeptide Y and tyrosine hydroxylase, all present in postganglionic sympathetic nerves, were not observed at any stage of antler growth. Nerves expressing immunoreactivity for any of the neural markers or peptides employed could not be found in cartilage, osteoid or bone. These results show that antlers are innervated mainly by sensory nerves and that nerves can attain the exceptionally high growth rates found in regenerating antler.
Deer antlers are secondary sexual characters, which mineralize, exfoliate the outer soft tissues (velvet), and, after the rut, are discarded (cast) annually. Developing antler can exhibit growth rates of up to 1.8 cm/day in large deer species. 2° F o r m i n g antler consists of growing tips m a d e up of proliferating undifferentiated m e s e n c h y m a l cells (reserve mesenchyme) which lie b e n e a t h a fibrous p e r i c h o n d r i u m . F u r t h e r d o w n the central core of the (growing) antler is a zone of forming cartilage which becomes hypertrophic, calcifies, a n d is modified first into p r i m a r y a n d then, secondary, spongiosa. 32° This central cartilagenous region is s u r r o u n d e d initially by a collar of de novo woven bone. Overlying the
mineralizing woven b o n e is a layer of periosteum-like tissue which merges imperceptibly with the peric h o n d r i u m . 3 The entire antler is covered by epidermis with a b u n d a n t hair follicles and sebaceous glands but no erector pili muscles. O b s e r v a t i o n s o f deer b e h a v i o u r provides clear evidence t h a t forming antlers are innervated. Deer avoid contact with their soft developing antlers, a n d d e m o n s t r a t e an acute spatial awareness a n d m e m o r y of antler points which are outside their visual range as s h o w n by their skill in sparring with h a r d antlers during the rut. Direct evidence for the i n n e r v a t i o n of antler tissue was reported by Berthold in 1831. 6 Very few experiments involving the d e n e r v a t i o n of antler have been reported. Wislocki a n d Singer 48 found that when one antler was denervated, that antler did not grow in the typical shape or attain the dimensions of the other. They also provided a histological d e m o n stration of i n n e r v a t i o n in the developing antler a n d f o u n d that this was derived from the supraorbital a n d t e m p o r a l b r a n c h e s of the trigeminal nerve. 4~ Since then b o t h myelinated a n d unmyelinated nerves
tTo whom correspondence should be addressed. Abbreviations: ABC, avidin-biotin-peroxidase complex; BSA, bovine serum albumin; CGRP, calcitonin generelated peptide; CPON, C-flanking peptide of neuropeptide Y; DAB, 3,3'diaminobenzidine tetrahydrochloride; LI, like immunoreactivity; NF, neurofilament triplet protein; PBS, phosphate-buffered saline; PGP 9.5, protein gene product 9.5; VIP, vasoactive intestinal peptide. 953
954
C. GRAY et al.
have been described in the epidermis and in the vascularized dermal tissue. 46 Although early studies have confirmed the presence of nerves in antler, the methods used do not indicate the type or possible function of the innervation. In this study, we have applied immunocytochemical techniques for the visualization of several neural proteins and peptides in order to localize and to characterize different neural subpopulations in developing antlers. Antisera to protein gene product 9.5 (PGP 9.5) and neurofilament triplet protein (NF) were used to assess the overall innervation. P G P 9.5 is a cytoplasmic ubiquitin C-terminal hydrolase and is known to be present in all neural elements) 4'~6'47 In contrast, N F is a cytoskeletal neural protein ~3 and therefore could be useful as a marker for persistent nerve fibres in rapidly growing tissues. Substance P, one of the major neuropeptides involved in nociception, is localized in primary afferent fibres 3~'38 and is often co-stored with calcitonin gene-related peptide (CGRP), 35 a peptide found to be abundant in sensory nerves in bone tissues. 8'25 Antisera to neuropeptide Y, the C-flanking peptide of neuropeptide Y (CPON), tyrosine hydroxylase and vasoactive intestinal polypeptide (VIP) were used as markers for autonomic nerves, although VIP is also present in some sensory neurons. 41 The main aim of this study was to examine the growth rate of neural elements in growing antler and in particular to look for the presence of peptidelike immunoreactivity (LI) at the rapidly growing antler tip. EXPERIMENTAL PROCEDURES
Tissues and fixation Commercially farmed red deer (Cervus elaphus) antler tissue was purchased from Mr R. Hayward, Slopers Pond Farm, Barnet, Hertfordshire, U.K. Antlers were obtained at two-weekly intervals during mid June and July, a total of three antler specimens. Deer were culled by a high-velocity rifle shot to the head and the antlers removed within 5 min before fixation. Tissue was sliced into pieces of 0.5 x 3.0 cm and fixed by immediate immersion in Zamboni's fluid 4~ for 20 h at +4°C. Tissues were then washed in 15% w/v sucrose and 0.001% w/v sodium azide in 0.01 M phosphate-buffered saline (PBS) at pH 7.2 for two days at +4°C. Antler lengths varied between 10 and 40 cm during the collection period and represented developmental stages from early growth
through the period of rapid elongation to the etad ol extension. The first specimen collected still carried the scab left by casting the previous year's antler and the second was collected during the rapid growth phase. Antlers from these first two deer had substantial amounts of reserve mesenchyme present and a distance between the tip and mineralizing front of over 2.5 cm. In contrast to this, the third specimen was nearing the end of its growth and had minimal amounts of reserve mesenchyme and was clearly mineralizing at 1 cm below the tip. Sections of antler were taken from the tip and approximately 8 cm below the tip. Both longitudinal and transverse sections were studied. Samples of small intestine and nasal skin were used as controls for antibodies and methodology and were treated in the same way. Antisera The antisera used in this study, dilutions and characteristics are shown in Table 1. Omission of primary antiserum or one of the subsequent steps in the avidin-biotinperoxidase complex (ABC) method was included as a control for method specificity. Preabsorption of antisera with an excess of corresponding peptide antigen was used as a control for antibody specificity. Immunohistochemistry Frozen sections (15 pm) cut on a cryostat were mounted on poly-L-lysine-coated slides)° Endogenous peroxidase was inhibited by soaking the sections in 0.3% hydrogen peroxide in methanol for 20 min. Following rinses in PBS, the slides were then placed in a humid chamber and the sections incubated (i) overnight at +4°C with primary antibodies, diluted in PBS containing 1/30 normal goat serum and 0.1% w/v bovine serum albumin (BSA) (Sigma Chemical Co., St Louis, U.S.A.) (Table 1); (ii) with biotinylated goat anti-rabbit IgG, diluted 1:100 in PBS with 0.1% w/v BSA, for 60 rain at room temperature; and (iii) with ABC (avidin and biotinylated horseradish peroxidase both diluted 1/200 in PBS) for 60 min at room temperature, z9 The sections were then incubated for 5min in a solution of 3,Ydiaminobenzidine (DAB) and nickel ammonium sulphate containing glucose and glucose oxidase (EC 1.1.3.4) (Sigma Chemical Co., St Louis, U.S.A.).42 After each step, the slides were rinsed three times in PBS for 5 min. Finally the slides were dehydrated in ethanol, cleared in Inhibisol (Kalon Chemicals, Cramlington, U.K.) and mounted in Pertex (Histolab Products Ab, Gothenburg, Sweden). Assessment of immunoreactive-fibre distribution Results were assessed semi-quantitatively for two reasons: firstly, the number of immunoreactive fibres was low; secondly many fibres, especially those expressing substance P- and VIP-LI, were extremely thin and only visible at high magnification. Immunostained antler sections of approximately 1 cm2 were microscopically monitored for the
Table 1. Characteristics of the antibodies used in the study Dilution
Peptide absorption
PGP 9.5 NF (68,000; 150,000; 200,000 mol.wt)
1/6000 1/2000
N/A N/A
Substance P (synthetic) ~-CGRP (rat, synthetic) Neuropeptide Y CPON (synthetic) Tyrosine hydroxylase
1/4000 1/4000 1/4000 1/4000 1/2000
1.0 nM/ml 0.1 nM/ml N/A N/A N/A
VIP
1/10 000
0.1 nM/ml
Antigen
N/A, not applicable.
Source
References
Ultraclone, Cambridge, U.K. D. Dahl, Veterans Admin., Boston, MA, U.S.A. Hammersmith Hospital Hammersmith Hospital Hammersmith Hospital Hammersmith Hospital D. Kuhn, Lafayette Clinic, Detroit, MI, U.S.A. Hammersmith Hospital
t6, 23, 47 13 35 19, 35 22 I, 22 33 7
Innervation of antlers
955
presence of immunoreactive fibres and allocated into one of seven tissue types for each deer, namely: epidermis; subepidermis containing hair follicles and sebaceous glands; deep connective tissue containing the major blood vessels; periosteum/perichondrium (tip/shaft); cartilage; osteoid; bone (Figs 1, 2). Nerves observed were allocated one of four categories for each tissue type and deer according to the frequency with which they occurred; I, no immunoreactive nerves detected; II, sparse, 1-10 fibres found in the total sample; III, moderate, 10 20 fibres found in the total sample; IV, abundant, >20 fibres in the total sample. The total sample for each antiserum represents approximately 10 tissue sections/antler region per deer. This represents a total sample in excess of 400 tissue sections. RESULTS I m m u n o r e a c t i v i t y to P G P 9.5, N F , s u b s t a n c e P a n d C G R P was o b s e r v e d in tissue t a k e n f r o m all stages o f antler g r o w t h (Table 2). W h e n c o m p a r e d to the c o n t r o l tissues antlers were relatively sparsely i n n e r v a t e d . P G P 9.5-LI c o u l d only be o b s e r v e d n e a r to the e p i d e r m i s ; in o t h e r r e g i o n s a high b a c k g r o u n d s t a i n i n g was c r e a t e d b y unspecific b i n d i n g o f the a n t i b o d i e s to o s t e o - a n d c h o n d r o b l a s t i c cells in the d e e p e r tissue. N F - L I nerves were o b s e r v e d in s u b e p i d e r m i s , d e e p c o n n e c t i v e tissue a n d p e r i c h o n d r a l / p e r i o s t e a l tissues b o t h as nerves b u n d l e s a n d as i n d i v i d u a l nerve fibres. N F - L I nerves were also p r e s e n t in soft tissues n e a r h y p e r t r o p h i c / c a l c i f y i n g cartilage in t h e central, c a r t i l a g e n o u s , region o f the antler (Figs l, 3), b u t only as sparse a n d s h o r t single fibres (Fig. 4). N F - L I c o u l d n o t be f o u n d in the s p o n g i o s a o r a m o n g the w o v e n b o n e s u r r o u n d i n g it. S u b s t a n c e P- a n d C G R P - L I nerve fibres were f o u n d s u b j a c e n t to the e p i d e r m i s (Figs 1, 2 a n d 5) a n d in the d e e p e r c o n n e c t i v e tissues (Figs 1, 2, 6 - 8 ) . F i b r e s i m m u n o r e a c t i v e to b o t h a n t i s e r a were m o s t
Fig. 1. Diagram showing a growing antler tip during a rapid growth phase. The boxes (2) and (3) mark the location of Figs 2 and 3. C, chondroblastic zone; DC, deep connective tissue; ED, epidermis; HC, hypertrophic/calcifying cartilage; P, periosteum; PC, perichondrium; PS, primary spongiosa; RM, reserve mesenchyme (proliferating chondroblastic region); SED, subepidermis; SS, secondary spongiosa; WB, woven bone. (Diagram not to scale).
a b u n d a n t in the d e e p e r c o n n e c t i v e tissue s u r r o u n d i n g the p e r i c h o n d r i u m / p e r i o s t e u m - l i k e tissue, p a r t i c u larly in the v a s c u l a r region o f the tip. T h e s e i m m u n o reactive fibres were f o u n d b o t h perivascularly a r o u n d small b l o o d vessels a n d as free fibres (Figs 6, 7).
Table 2. Summary of semi-quantitative assessment of fibre distribution Deer
I
I1
III
Length of antler
Tissue
10 cm
Epidermis Subepidermis Deep connective tissue Perichondrium/periosteum Cartilage Osteoid Woven bone
20 cm
Epidermis Subepidermis Deep connective tissue Perichondrium/periosteum Cartilage Osteoid Woven bone
+ + + + + + N/A N/A N/A N/A
Epidermis Subepidermis Deep connective tissue Perichondrium/periosteum Cartilage Osteoid Woven bone
. + + + + + N/A N/A N/A N/A
40 cm
Immunoreactivity NF SP CGRP
PGP 9.5 + + + + + + + N/A N/A N/A N/A
-+ + + + + --. . --+ + + + -. . .
. -+ + + + ----
+ + + + + --. .
+ + + + + + + +
.
-+ + + + + + + --
-. .
+ + --
.
-+ + + . .
VIP
---
. .
. -+ ----
+ + + + + +
--
--
--
N/A, not applicable (see text);--immunoreactive fibres absent; + sparse; + + moderate; + + + abundant.
' i~'i~i~ S ! ¸
i
Innervation of antlers
957
Fig. 3. The central core of developing antler showing hypertrophic cartilage (HC), and vascular lumen (VL). Neurofilament-LI fibres were found in similar tissues to those arrowed. Hematoxylin and eosin counterstaining. (Longitudinal section.) Magnification × 150. Substance P-LI nerve fibres were much more sparse than C G R P - L I fibres, although tissue distribution and localization was similar as evidenced by immunostaining of consecutive tissue sections. Although some P G P 9.5-, N F - , substance P- and C G R P - L I could be detected in most dermal and perichondrial/ periosteal tissue, only deep connective tissue showed
a relatively abundant innervation (Table 2). Nerve fibres containing P G P 9.5-, NF-, substance P- or C G R P - L I were not found within forming cartilage, osteoid or any mineralized antler tissue. Sparse VIP-LI fibres were present in deep connective tissues (Figs l, 2, 9) and occasionally in subepidermal tissue in close proximity to sebaceous
Fig. 4. A neurofilament-Ll fibre (arrowed) within the central cartilaginous region of antler (tor morphology and localization see Figs 1, 3). The fibre is situated in soft tissues adjoining a blood vessel (VL) and hypertrophic cartilage (HC). No immunoreactive fibres for other neural constituents could be found in this or a similar location. (Longitudinal section.) Magnification is × 350.
958
C. GRAYet aL
glands but only at the earliest stage of antler growth. No immunoreactivity could be observed for NPY, CPON or tyrosine hydroxylase in any antler tissue. However, immunoreactivity for these neuropeptide and enzyme antisera were abundant in both intestinal and nasal control tissues. Preabsorptions of peptide antisera with an excess of antigen abolished all immunoreactivity in both antler and control tissues (Table 1). DISCUSSION This study primarily demonstrates that neural elements are able to attain the same exceptionally high growth rates as are found in regenerating antler. Antlers grow by a rapid cellular proliferation, differentiation, hypertrophy, and endochondral ossification from the growing tip 3'4and by the de novo formation of a collar of initially woven bone around the central cartilaginous core? '9 Most of the cellular constituents of antler develop in situ, notable exceptions being the nervous and vascular tissues which must invade forming tissue. It follows that since antler tips are innervated these nerves are able to attain the high growth rates observed in red deer. Using antibodies to PGP 9.5 and NF we have located nerves in antler subepidermis, deep connective tissue, and in periosteum/perichondrium.
In addition to this, a few NF-LI nerve fibres were observed in tissues adjacent to hypertrophic cartilage within the central core of the antler. These may represent a population of nerve fibres which are not functionally active. As no neuropeptide-LI could be observed at this location these possibly represent a population of nerve fibres which have been left behind by the growing tip, since cytoskeletal proteins are not likely to undergo as rapid degradation and removal as intravesicular peptides. The principal neuropeptide-like immunoreactivities found were substance P and CGRP which are the major peptide mediators of nociception.49 Localization of substance P and CGRP in antler/bone tissue may also have roles other than that of pain perception. For example, substance P and CGRP act as potent transmitters following antidromic impulse generation in sensorimotor nerves during "axon reflex" action, t2,34In experiments where rat bones had been surgically denervated, varying effects have been observed. Metatarsals and humeri were reduced in size ~7'~8 and femoral length increased. ~8 In a recent study, Hill and co-workers found no change in bone growth although after sympathectomy or capsaicininduced sensory denervation osteoclast distribution on the bone surface was altered. 27 It is not clear how physical stress generated by muscular activity and locomotion have affected the results obtained in these studies. However, antler tissue does not contain
Fig. 5. A strongly stained nerve fibre expressing CGRP-LI (for morphology and localization see Figs l, 2). The apparent staining of the epidermis (ED) is in fact natural pigmentation. The nerve fibre is in close proximity to a hair follicle(HF) and sebaceous gland (SG). (Longitudinal section.) Magnification is × 350.
959
Innervation of antlers
Fig. 6. A substance P-LI nerve fibre (arrowed) within the deep connective tissue of antler (for morphology and localization see Figs 1, 2.) Nerves expressing substance P-LI were all exceptionally thin fibres. These fibres had a similar distribution to those expressing CGRP-LI but they were generally less abundant. The feature marked VL is a vascular lumen of a small blood vessel. (Longitudinal section.) Magnification is ×420. skeletal muscle n o r are antler growing tips load bearing. Antler m a y therefore provide a useful experimental model in which to study the effects of neural influences o n b o n e growth free from physical stresses. Early experiments in which one antler was denervated
reported changes in shape a n d a reduction in size when c o m p a r e d to the other. However, the investigators believed this could have been caused by injury due to the loss of sensation. 48 A subsequent a n d more extensive study showed t h a t denervated antlers were
t
t
1L
Fig. 7. Perivascular CGRP-LI fibres (arrowed) within the deep connective tissue region of antler (for morphology and localization see Figs 1, 2). Similar fibres were also present in periosteal/perichondrial tissue although they were less common. VL, vascular lumen. (Longitudinal section.) Magnification is × 420.
960
C. GgAV et al.
Fig. 8. A transverse section showing a CGRP-LI nerve bundle (arrowed) within the deep connective tissue (for morphology and localization see Figs 1, 2). Fibres can clearly be seen branching out from the main bundle which has been transected. Similar nerve fibre bundles were not found in periosteum/perichondrium or subepidermal tissue. (Transverse section.) Magnification is × 420.
smaller a n d o f altered shape a n d t h a t this occurred m the absence o f any o t h e r external injury. 44 Interestingly, in experiments where trigeminal nerves were electrically stimulated, antlers h a d increased lengths ( > 70%), weights ( > 4 0 % ) a n d altered shapes. ~ Electrical stimulation will o f course induce a release o f
n e u r o p e p t i d e content from terminal vesicles in target tissues. C G R P is a p o t e n t m o d u l a t o r o f bone m e t a b olism. In vivo C G R P a d m i n i s t r a t i o n has been f o u n d to induce hypocalcaemia 4° a n d p r o m o t e calcium u p t a k e by bone. 2 In vitro, C G R P has been s h o w n to increase c A M P , 36 to stimulate osteogenesis, 5 a n d
Fig. 9. VIP-LI nerve fibres (arrowed) within the deep connective tissue of antler (for morphology and localization see Figs 1, 2). VIP-LI nerves were only found in early growing antler (deer I) and were both small and sparse. (Longitudinal section.) Magnification is x 560.
lnnervation of antlers to inhibit bone resorption. ~5'5° Antler osteoclasts are known to be responsive to calcitonin, 2~ and although C G R P is much less potent than calcitonin its local release from nerve terminals may modulate antler bone resorption (and formation) via a calcitonin-like inhibition of osteoclastic resorption. Growing antler is highly vascularized as might be expected in such a metabolically active tissue with a high demand for oxygen and nutrients. A striking feature of antler is that it has vascular cartilage not normally found in postnatal mammals. Substance P and C G R P are both potent vasodilators. ~° Substance P has been shown to stimulate endothelial cell proliferation, migration and neovascularization. 5~'52 Similarly C G R P is a mitogen for endothelial cells. 24 These findings indicate that substance P- and C G R P containing fibres may play an important role in the rapid growth of antler by controlling vascular tone and neovascularization. We found no immunoreactivity in antler tissue for NPY, C P O N or tyrosine hydroxylase. This suggests the lack of sympathetic vasomotor innervation which is surprising in view of the vascular nature of antler tissue. This could be due to a lack of cross-reactivity to these peptides/enzyme in deer, but this is unlikely since fibres immunoreactive to these antisera were observed in the control tissues. In the present study we did not examine any tissue from the proximal, highly mineralized, antler shaft. Neural elements expressing these peptides and catecholamine synthesizing enzyme may exhibit a slower growth rate and be unable to invade the rapidly growing antler tip. However, our findings support the study by Rayner and Ewen who could not demonstrate any evidence of the adrenergic innervation of antlers using a formaldehyde vapour technique. 39
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In the present study, sparse VIP-LI nerve fibres were observed in deep connective tissue and occasionally near sebaceous glands in the subepidermis of early developing antler, but not in later stages of growth. VIP, which has been shown to be present in both autonomic (sympathetic 26 and parasympathetic ~2) and sensory neurons, 37"41 stimulates smooth muscle relaxation and vasodilation. In bone, it is a potent stimulator of osteoclastic resorption. 2s The absence of VIP-LI from tissues taken at later stages of antler growth suggests that this subpopulation of nerves does not keep pace with antler growth and this finding could support its localization in antler autonomic nerves. CONCLUSION We have conclusively shown that substance Pand C G R P - L I nerve fibres, most probably of sensory origin, can exhibit the exaggerated growth rates found in antler. In addition, this study confirms and extends earlier studies which demonstrated the presence of nerves but the absence of adrenergic innervation in the developing antlers. The localization of substance P- and C G R P - L I , in the dermal, deep connective and perichondrial/periosteal tissues of developing antler supports an important role for sensory nerves in antlerogenesis. Acknowledgements--The authors would like to thank the
BUPA Medical Foundation, the Grand Charity of Freemasons, the Nuffield Foundation and the Science and Engineering Research Council, U.K. (C.G.). the Emil Aaltonen Research Foundation (M.H.) and the Finnish Academy, Finland (Y.T.K.) the Research into Ageing (T.R.A.) and the British Heart Foundation U.K. (G.B.). The authors would also like to express their thanks to Mr P. Bose for the translation of the article by Berthold)
REFERENCES
1. Allen J. M., Potak J. M. and Bloom S. R. (1985) Presence of the predicted C-flanking peptide of neuropeptide Y (CPON) in tissue extracts. Neuropeptides 6, 95-100. 2. Ancill A. K., Bascal Z. A., Whitaker G. and Dacke C. G. (1991) Calcitonin gene-related peptide promotes transient radiocalcium uptake into chick bone in vivo. Expl Physiol. 76, 143 146. 3. Banks W. J. and Newbrey J. W. (1983) Light microscopic studies of the ossification process in developing antlers. In Antler Development in Cervidae (ed. Brown R. D.). Vol. 1, pp. 231 260. Caesar Kleberg Wildlife Research Institute, Kingsville, Texas. 4. Banks W. J. and Newbrey J. W. (1983) Antler development as a unique modification of mammalian endochondral ossification. In Antler Development in Cervidae (ed. Brown R. D.), Vol. 1, pp. 279 306. Caesar Kleberg Wildlife Research Institute, Kingsville, Texas. 5. Bernard G. W. and Shih C. (1990) The osteogenic effect of neuroactive calcitonin gene-related peptide. Peptides !1, 625 ~32. 6. Berthold A. A. (1831) 1Dber das Wachstum, den Abfall und die Wiedererzeugung der Hirschgeweihe. Beitrag z Anat. Zool. u Physiol., pp. 39-96. G6ttingen, Germany. 7. Bishop A. E., Polak J. M., Yiangou Y., Christofides N. D. and Bloom S. R. (1984) The distributions of PHI and VIP in porcine gut and their co-localisation to a proportion of intrinsic ganglion cells. Peptides 5, 255-259. 8. Bjurholm A., Kreicbergs A., Brodin E. and Schultzberg M. (1988) Substance P- and CGRP-immunoreactive nerves in bone. Peptides 9, 165 171. 9. Boyde A., Arnett T. R., Gray C., Loudon A. S. 1. and Maconnachie E. (1989) Scanning electron microscopy of vascular mineralising cartilage in developing antler. Bone 11, 228. 10. Brain S. D. and Williams T. J. (1988) Substance P regulates the vasodilator activity of calcitonin gene-related peptide. Nature 335, 73 75. 11. Bubenik G. A., Bubenik A. B., Stevens E. D. and Binnington A. G. (1982) The effect of neurogenic stimulation on the development and growth of bony tissues. J. exp. Zool. 219, 205 216. 12. Burnstock G. (1990) Co-transmission: the fifth Heymans memorial lecture. Archs int. Pharmacodyn. ThOr. 304, 7 23.
962
C. GRAY et al.
13. Dahl D. and Bignam A. (1977) Preparation of antisera to neurofilament protein from chicken brain and human sciatic nerve. J. comp. Neurol. 176, 645-658. 14. Day I. N., Hinks L. J. and Thompson R. J. (1990) The structure of the human gene encoding protein gene product 9.5 (PGP 9.5), a neuron-specific ubiquitin C-terminal hydrolase. Biochem. J. 268, 52t-524. 15. D'Souza S. M., MacIntyre I., Girgis S. I. and Mundy G. R. (1986) Human synthetic calcitonin gene-related peptide inhibits bone resorption in vitro. Endocrinology 119, 58-61. 16. Doran J. F., Jackson P., Kynoch P. A. and Thompson R. J. (1983) Isolation of PGP 9.5, a new human neurone-specific protein detected by high-resolution two-dimensional electrophoresis. J. Neurochem. 40, 1542-1547. 17. Dysart P. S., Harkness E. M. and Herbison G. P. (1989) Growth of the humerus after denervation. An experimental study in the rat. J. Anat. 167, 147-159. 18. Garcrs G. L. and Santandreu M. E. (1988) Longitudinal bone growth after sciatic denervation in rats. J. Bone Joint Surg. 70B, 315-318. 19. Gibson S. J., Polak J. M., Bloom S. R., Sabate I. M., Mulderry P. M., Ghatei M. A., McGregor G. P., Morrison J. F. B., Kelly J. S., Evans R. M. and Rosenfeld M. G. (1984) Calcitonin gene-related peptide immunoreactivity in the spinal cord of man and eight other species. J. Neurosci. 4, 3101-3111. 20. Goss R. J. (1983) Deer Antlers: Regeneration, Function, and Evolution, Vol. 1, pp. 133-171. Academic Press, New York. 21. Gray C., Taylor M. L., Horton M. A., Loudon A. S. I. and Arnett T. R. (1992) Studies on cells derived from growing deer antler. In The Biology o f Deer (ed. Brown R. D.), Vot 1, pp. 511-519. Springer, New York. 22. Gulbenkian S., Wharton J., Hacker G. W., Varndell I. M., Bloom S. R. and Polak J. M. (1985) Co-localization of neuropeptide Y (NPY) and its C-terminal flanking peptide (C-PON). Peptides 6, 1237-1243. 23. Gulbenkian S., Wharton J. and Polak J. M. (1987) The visualization of cardiovascular innervation in the guinea pig using an antiserum to protein gene product 9.5 (PGP 9.5). J. auton, nerv. Syst. 18, 235-247. 24. Haegerstrand A., Dalsgaard C.-J., Jonzon B., Larsson O. and Nilsson J. (1990) Calcitonin gene-related peptide stimulates proliferation of human endothelial cells. Proc. natn. Acad. Sci. U.S.A. 87, 3299-3303. 25. Hill E. L. and Elde R. (1988) Calcitonin gene-related peptide-immunoreactive nerve fibres in mandibular periosteum of rat: evidence of a primary efferent origin. Neurosci. Lett. 85, 172-178. 26. Hill E. L. and Elde R. (1989) Vasoactive intestinal peptide distribution and colocalization with dopamine-/~hydroxylase in sympathetic chain ganglia of pig. J. auton, nerv. Syst. 27, 229-239. 27. Hill E. L., Turner R. and Elde R. (1991) Effects of neonatal sympathectomy and capsaicin treatment on bone remodeling in rats. Neuroseience 44, 747-755. 28. Hohmann E. L., Levine L. and Tashjian A. H. (1983) Vasoactive intestinal peptide stimulates bone resorption via a cyclic adenosine 3',5'-monophosphate dependant mechanism. Endocrinology 112, 1233-1239. 29. Hsu S. M., Raine L. and Fanger H. (1981) Use of avidin-biotin peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem. 29, 577 -580. 30. Huang W.-M., Gibson S. J., Facer P., Gu J. and Polak J. M. (1983) Improved section adhesion for immunocytochemistry using high molecular weight polymers of L-lysine as a slide coating. Histochemistry 77, 275-279. 31. H6kfelt T., Kellerth J. O., Nilsson G. and Pernow B. (t975) Morphological report for a transmitter role of substance P, immunohistochemical localization in the central nervous system and in primary sensory neurones. Science 190, 889-890. 32. Larsson L.-I., Fahrenkrug J., Schafflitzky De Muckadell O., Sundler F., H~kanson R. and Rehfeld J. F. (1976) Localization of vasoactive intestinal polypeptide (VIP) to central and peripheral neurons. Proc. nam. Acad. Sci. U.S.A. 73, 3197-3200. 33. Lundberg J. M., Terenius L., Hrkfelt T., Martling C. R., Tatemoto K., Mutt V., Polak J. M., Bloom S. R. and Goldstein M. (1982) Neuropeptide Y (NPY)-like immunoreactivity in peripheral noradrenergic neurons and effects of NPY on sympathetic function. Acta physiol, scand. 116, 477-480. 34. Maggi C. A. (1991) The pharmacology of the efferent function of sensory nerves. J. auton. Pharmac. il, 173-208. 35. Merighi A., Gulbenkian S., Gibson S. J. and Potak J. M. (1988) Ultrastructural study of calcitonin gene-related peptide-, neurokinins- and somatostatin-immunoreactive neurones in rat dorsal root ganglia. Evidence of the colocalisation of different peptides in single secretory granules. Cell Tiss. Res. 254, 101-109. 36. Michelangeli V. P., Fletcher A. E., Allan E. H., Nicholson G. C. and Martin T. J. (1989) Effects of calcitonin gene-related peptide on cyclic AMP formation in chicken, rat, and mouse bone cells. J. Bone Miner. Res. 4, 269-272. 37. Morris J. L. and Gibbins I. L. (1992) Co-transmission and neuromodulation. In Autonomic Neuroeffector Mechanisms (eds Burnstock G. and Hoyle H. V.), Vol. 1, pp. 33-119. Harwood, Chur, Switzerland. 38. Pernow B. (1983) Substance P. Pharmac. Rev. 35, 85-141. 39. Rayner V. and Ewen S. W. B. (1981) Do the blood vessels of the antler velvet of the red deer have an adrenergic innervation. Q. Jl exp. Physiol. 66, 81-86. 40. Roos B. A., Fischer J. A., Pignat W., Alander C. B. and Raisz L. G. (1986) Evaluation of the in vivo and in vitro calcium-regulating actions of noncalcitonin peptides proceeded via calcitonin gene expression. Endocrinology llg, 4(~51. 41. Shehab S. A. S., Atkinson M. E. and Payne J. N. (1986) The origins of the sciatic nerve and changes in neuropeptides after axotomy: a double labelling study using retrograde transport of true blue and vasoactive intestinal polypeptide immunohistochemistry. Brain Res. 376, 180--185. 42. Shu S., Ju G. and Fan L. (1988) The glucose oxidase-DAB-nickel method in peroxidase histochemistry of the nervous system. Neurosci. Lett. 85, 169-171. 43. Stefanini M., De Martino C. and Zamboni L. (1967) Fixation of ejaculated spermatozoa for electron microscopy. Nature 216, 173 174. 44. Suttie J. M. and Fennessy P. F. (1985) Regrowth of amputated antlers with and without innervation. J. exp. ZooL 234, 359 366. 45. Tatemoto K., Carlquist M. and Mutt V. (t982) Neuropeptide Y - - a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide. Nature 296, 659-660. 46. Vacek Z. (1955) Innervace lyci rostoucia parohu u cervidu. Cslkgl M o r f 3, 249-264,
Innervation of antlers
963
47. Wilkinson K. D., Lee K. M., Deshpande S., Duerksen-Hughes P., Boss J. M. and Pohl J. (1989) The neuron-specific protein PGP 9.5 is a ubiquitin carboxyl-terminal hydrolase. Science 246, 67ff673. 48. Wislocki G. B. and Singer M. (1946) The occurrence and function of nerves in the growing antler of deer. J. comp. Neurol. 85, 1-19. 49. Woolf C. and Wiensenfeld-Hallin Z. (1986) Substance P and calcitonin gene-related peptide synergistically modulate the gain of the nociceptive flexor withdrawal reflex in the rat. Neurosci. Lett. 66, 226-230. 50. Zaidi M., Chambers T. J., Bevis P. J. R., Beacham J. L., Gaines Das R. E. and MacIntyre I. (1988) Effects of peptides from the calcitonin genes on bone and bone cells. Q. Jl exp. Physiol. 73, 471-485. 51. Ziche M., Morbidelli L., Pacini M., Geppetti P., Alessandrini G. and Maggi C. A. (1990) Substance P stimulates neovascularization in vivo and proliferation of cultured endothelial cells. Microvasc. Res. 40, 264-278. 52. Ziche M., Morbidelli L., Geppetti P., Maggi C. A, and Dolara P. (1991) Substance P induces migration of capillary endothelial cells: a novel NK-1 selective mediated activity. Life Sci. 48, PL7 Pll.
(Accepted 18 May 1992)
