30
Neuroscience Letters, 109 (1990) 30 35 Elsevier Scientific Publishers Ireland Ltd.
NSL 06616
Origin of sympathetic and sensory innervation of the temporo-mandibular joint. A retrograde axonal tracing study in the rat Bertil Widenfalk and Mikael Wiberg Hand Surgery Unit, Department of Plastic Surgery, University Hospital, and Department o[' Anatomv Uppsala University, Uppsala (Sweden)
(Received 16 June 1989; Revised version received 25 September 1989: Accepted 27 September 1989) Key word~." Joints innervation: Sympathetic ganglion; Spinal ganglion: Horseradish peroxidase; Rheumatoid arthritis
The cells of origin of sensory and sympathetic innervation of the temporo-mandibularjoint were studied by the intraaxonal transport method. Horseradish peroxidase or lectin-conjugated horseradish peroxidase was injected into the temporo-mandibular joint unilaterally in adult rats. Labelled cells were observed ipsilaterally in the superior cervical and stellate sympathetic ganglia, in the sensory trigeminal ganglion and in the second to fifth dorsal root ganglia: none were found contralaterally. The results are discussed in relation to the hypothesis that a nervous mechanism might be involved in the pathogenesis of joint inflammation.
R h e u m a t o i d arthritis ( R A ) affects not only j o i n t s o f extremities but also others, including the t e m p o r o - m a n d i b u l a r (t-m) j o i n t [2]. Several clinical o b s e r v a t i o n s have indicated that a nervous c o m p o n e n t is involved in the p a t h o g e n e t i c mechanism. F o r instance it has been o b s e r v e d that central or p e r i p h e r a l nerve lesions, affecting one or several limbs, m a y protect the affected limbs from R A (see e.g. ref. 6). The m e c h a nism o f i n v o l v e m e n t o f this nervous c o m p o n e n t is still n o t c o m p l e t e l y u n d e r s t o o d , but it has been p r o p o s e d that an interaction between sensory afferents a n d s y m p a thetic efferents m a y be r e a s o n a b l e , since a p p l i c a t i o n o f substance P into the j o i n t cavity a g g r a v a t e s [12] a n d i m m u n o s y m p a t h e c t o m y a t t e n u a t e s [7], a d j u v a n t - i n d u c e d arthritis. In o r d e r to study the cells o f origin in s y m p a t h e t i c and d o r s a l r o o t ganglia innervating the t-m j o i n t , 25 a d u l t rats weighing a b o u t 300 g were anesthetized with p e n t o b a r b i t a l s o d i u m ( M e b u m a l - V e t ) a n d h o r s e r a d i s h p e r o x i d a s e ( H R P ) o r lectin-conju-
Correspondence." B. Widenfalk, Dept. of Plastic and Hand Surgery, Akademiska sjukhuset, S-751 85 Uppsala, Sweden.
0304-3940/90/$ 03.50 ~:~:,1990 Elsevier Scientific Publishers Ireland Ltd.
31
gated horseradish peroxidase (WGA-HRP) was injected into the joint unilaterally, which was exposed through an incision in the joint capsule. With a Hamilton syringe, the tracer was injected under visual control into the joint cavity. After the injection the needle was left in situ for 5 min before withdrawal to diminish the risk of leakage of tracer into surrounding tissues. Finally the joint capsule, soft tissue and skin was sutured. After varying survival periods the animals were reanesthetized and fixed transcardially with an aldehyde mixture. The superior cervical and stellate sympathetic ganglia and the trigeminal and cervical dorsal root ganglia were removed and rinsed overnight in phosphate buffer containing 30 %w/v sucrose. All ganglia were then cut Ion-
TABLE I SURVEY
OF THE DIFFERENT
DISTRIBUTION GEMINAL
OF LABELLED
RETROGRADE NEURONS
(TRIG) AND DORSAL
EXPERIMENTS,
AND THE NUMBER
IN THE SYMPATHETIC
R O O T (C2 C5) G A N G L I A
AND
(GCS, GS), SENSORY TRI-
IPSILATERALLY
AFTER
INJEC-
T I O N O F 1/zl 10% W G A - H R P O R 1/A 30% H R P I N T O T H E R I G H T (R) O R L E F T (L) T E M P O R O MANDIBULAR JOINT OF THE RAT
Case
Side
Tracer
Survival Number of retrogradely labelled neurons in G C S , G S , T R I G period (h) and C2~Cs ipsilaterally GCS
1
R
2
L
WGA-HRP
12
5
12
64
GS
TRIG
C2
C3
159
12
2
-
224
I
27
C4
C5
-
-
3
L
18
435
106
646
34
124
27
4
L
18
501
136
25l
15
315
75
5
L
24
303
2
459
1
10
-
6
R
24
593
5
543
119
10
7
L
48
186
3
276
30
25
I
8
R
48
305
274
19
6
-
-
9
L
60
421
12
309
18
84
6
-
10
R
60
339
12
326
2
23
2
-
11
R
60
387
19
34
8
-
-
12
R
72
1562
Ill
1024
662
178
23
-
13
R
72
1069
79
1112
315
215
32
1
14
L
96
998
2
218
12
6
2
-
15
L
96
502
7
325
2
35
2
-
16
L
120
550
24
173
2
38
4
17 18
R R
120 12
95 12
15 -
134 34
-
16 -
6 -
-
HRP
19
R
12
150
8
369
-
20 21
R L
24 24
157 191
7
146 63
-
-
-
4
-
-
-
22
L
48
81
4
91
2
-
-
23 24
R R
48 72
8 103
1 2
30 103
5
1 -
-
25
L
72
88
1
41
.
.
.
.
32 g i t u d i n a l l y i n t o 4 0 / t m thick sections o n a f r e e z i n g m i c r o t o m e a n d i n c u b a t e d for H R P h i s t o c h e m i s t r y w i t h t e t r a m e t h y l b e n z i d i n e ( T M B ) [14]. In 4 cases ( N o s . 14, 16, 24, 25) the l o w e r b r a i n s t e m , c o n t a i n i n g the m o t o r t r i g e m i n a l n u c l e u s , was r e m o v e d , rinsed, s e c t i o n e d a n d i n c u b a t e d in the s a m e w a y as d e s c r i b e d a b o v e .
I V
Fig. 1. Peroxidase-positive neurons in the sensory trigeminal ganglion (b 3), dorsal root ganglia (4) and sympathetic ganglia (5, 6) after injection of WGA HRP into the temporo-mandibular joint ipsilaterally. 1 ,: Labelled cells in the mandibular portion of the sensory trigeminal ganglion. 190 x. 1:: small peroxidasepositive neurons in the sensory trigeminal ganglion; survival period 12 hours. 190 x. I~: one large- and two medium-sized labelled cells in the sensory trigeminal ganglion; survival period 96 h. 225 x. 14: round peroxidase-positive cells of varying sizes in the second cervical dorsal root ganglia. 375 x. I ~, 1~: multipolar and fusiform labelled cells in the superior cervical sympathetic ganglia. 475 ×.
33 Details of the different retrograde tracing experiments and quantitative values for the distribution of peroxidase-positive cells are given in Table I. General labelling. Labelled neurons were observed in the superior cervical and stellate sympathetic ganglia, the sensory trigeminal ganglion and the second to fifth dorsal root ganglia. All cells were found ipsilateral to the injection side. Different survival periods were used in order to find the optimal one. The maximum number of peroxidase-positive neurons was observed after 72 h when W G A - H R P was used as the tracer, while the optimal survival period was between 12 and 24 h after injection of native HRP, which also yielded a total number of labelled cells that was smaller than when W G A H R P was used. The reasons for these differences are discussed below. Sympathetic ganglia. A maximum of about 1500 peroxidase-positive neurons were found ipsilaterally in the superior cervical and stellate ganglia; about 95% of these were situated in the superior cervical ganglion (Table I). The labelled cells were equally distributed over the ganglia, but differed in size and shape, being multipolar with a diameter of 20-25/tm (Fig. 15) or fusiform, measuring 10-20 ktm (Fig. 16). The multipolar cells were mainly concentrated to the center of the ganglia, while the fusiform ones were most numerous in the outer margin. Sensory trigeminal and dorsal root ganglia. A maximum of 1000 labelled cells were found ipsilaterally in the trigeminal ganglion. (Table I), where they were observed in the mandibular portion [1] (Fig. 10. The dorsal root ganglia were labelled at the level of C2 C5 where a total of 500-800 labelled neurons were observed ipsilaterally, evenly distributed over the ganglia. The overwhelming majority, about 90-95 % were equally distributed between the second and third cervical ganglia, while only some scattered neurons were seen in the fourth and fifth ones (Table I). In both types of sensory ganglia the labelled neurons were round and of varying sizes (Fig. 14). Most frequent (65 %) were the small ones (diameter 15-20/zm), while the medium-sized (25-30/~m) constituted 25% and the large ones (40-55/tm) 10% of the total number of labelled neurons. Technical comments. It has been claimed that W G A - H R P is a superior tracer for retrograde tracing studies [4]. This was also observed in the present study, where the use of W G A - H R P yielded a maximum of up to seven times more labelled neurons than native H R P (Table I). Another difference between the two tracers was that the survival period, when the maximum of labelled cells was observed, was shorter for native H R P than for WGAH R P (Table I). This observation might possibly be explained by the different uptake mechanisms by which the tracer enters the neuron (see e.g. ref. 4), but also by the observation that to reach the nerve endings the tracer has to diffuse through the superficial part of the synovial membrane [17, 19], and this diffusion is supposed to be faster for a smaller molecule such as H R P [10]. The different survival periods also yielded a different pattern of sizes of the labelled cells. Thus, in the sensory trigeminal and dorsal root ganglia, after the shortest survival periods virtually only small labelled neurons were seen (Fig. 12), while after the longest survival periods large ones predominated (Fig. 13), a pattern which has been described previously [5]. The last technical issue to be commented upon is the possible leakage of tracer
34 into the surrounding tissues. The fact that no labelling was observed contralaterally indicated that there was no leakage of tracer into the systemic circulation. To rule out the possibility of spread to the surrounding muscle, the lower brainstem was removed in four cases (Nos. 14, 16, 24, 25), and no retrograde labelling of motor neurons in the m o t o r trigeminal nucleus, innervating the masticatory muscles proper [16], was observed. I n earlier studies [18, 19] investigations have been made of the anatomical and physiological characteristics of the different types of receptors and nerve fibers associated with the t-m joint, but in none of these were the cells of origin of the nerve fibers investigated. The origin of the sensory fibers was studied by Romfh et al. [15] in the cat and they demonstrated that the cells of origin were situated in the mandibular portion of the sensory trigeminal ganglion. The neurons constituted a heterogeneous population with sizes varying between 20 and 60 /lm, but nothing was discussed about any spinal sensory origin. The results of the present study concerning the sensory trigeminal ganglion were in agreement with those obtained by Romfh et al. irrespective of the sizes of the labelled neurons. The most numerous labelled cells in the present study were small. A possible reason why Romfh et al. did not report predominance of that population might have been that their survival periods varied between 24 and 72 h, and in the present study it was clear that with native H R P as a tracer which was also used in the former study the optimal survival time for small neurons was shorter than 24 h. A novel finding in the present study was the impressive sympathetic efferents to t-m joint. This is of special interest in view of the recent proposal that the nervous system might be involved in the pathophysiology of RA [13], and that the sympathetic nervous system is part of this nervous component (see e.g. ref. 3). It seems reasonable to assume that there is some interaction between sensory afferents and sympathetic efferents, a theory considered by others [13]. The majority of labelled cells in the trigeminal and dorsal root ganglia were small, a cell population which has been found to contain substance P [8] and to send terminal collaterals to the superficial laminae of the dorsal horn, observations supporting the idea that these cells are involved in nociceptive transmission [11]. A nociceptive input from joints could therefore modulate the activity in sympathetic efferents, an idea supported by observations of substance P-containing fibers in sympathetic ganglia [9], and this modulation might be of importance for the induction and activity of RA [13]. This study was supported by grants from the Swedish Medical Research Council (project 12X2710) and the Mac Rudberg Foundation.
I Arvidsson, J., Location of cat trigeminal ganglion cells innervating dental pulp of upper and lower canines studied by retrograde transport of horseradish peroxidase, Brain Res., 99 (1975) 135 139. 2 Blackwood,H.J.J., The anatomy and pathology of the mandibular joint, J. Bone Joint Surg., 43 (1961) 396. 3 Christensen, K. and Henriksen, O., The reflexsympatheticdystrophy syndrome, Scand. J. Rheumatol., 12 (1983) 263-267.
35 4 Gonatas, N.K., Harper, C., Mizutani, T. and Gonatas, J.O., Superior sensitivity of conjugates of horseradish peroxidase with wheat germ agglutinin for studies of retrograde axonal transport, J. Histochem. Cytochem., 27 (1979) 728-734. 5 Grant, G., Arvidson, J., Robertson, B. and Ygge, J., Transganglionic transport of horseradish peroxidase in primary neurons, Neurosci. Lett., 12 (1979) 23-28. 6 Hamilton, S., Unilateral rheumatoid arthritis in hemiplegia, J. Can. Assoc. Radiol., 34 (1983) 49-50. 7 Herfort, R.A., Extended Sympathectomy in the treatment of advanced rheumatoid arthritis, New York State J. Med., 56 (1956) 1292-1294. 8 H6kfelt, T., Elde, R., Johansson, O., Luft, R., Nilsson, G. and Arimura, A., Immunohistochemical evidence for separate populations of somatostatin-containing and substance P-containing primary afferent neurons in the rat, Neuroscience, l (1976) 131 136. 9 H6kfelt, T., Elfvin, L.-G., Schultzberg, M., Goldstein, M. and Nilsson, G., On the occurrence of Substance P-containing fibers in sympathetic ganglia: Immunohistochemical evidence, Brain Res., 132 (1977) 2%41. l0 Kushner, I. and Sommerville, J.A., Permeability of human synovial membrane to plasma proteins. Relationship to molecular size and inflammation, Arthritis Rheum., 14 (1971) 560-570. 11 Leeman, S.E. and Gamse, R., Substance P in sensory neurons, Trends Pharmacol. Sci., 21 (1981) 119-121. 12 Levine, J.D., Clark, R., Devor, M., Helms, C., Moskowitz, M.A. and Basbaum, A.J., Intraneuronal substance P contributes to the severity of experimental arthritis, Science, 226 (1984) 547-549. 13 Levine, J.D., Goetzl, E.J. and Basbaum, A.J., Contribution of the nervous system to the pathophysiology of rheumatoid arthritis and other polyarthritides, Rheum. Dis. Clin. North Am., 13 (1987) 369-383. 14 Mesulam, M.M., Tetramethylbenzidine for horseradish peroxidase histochemistry. A non-carcinogenic, blue reaction product for visualizing neuronal afferents and efferents, J. Histochem. Cytochem., 26 (1978) 10t%117. 15 Romfh, J.H., Capra, N.F. and Gatipon, G.B., Trigeminal nerve and temporomandibular joint of the cat: A horseradish peroxidase study, Exp. Neurol., 65 (1979) 99-106. 16 Szentagothai, J., Functional representation in the motor trigeminal nucleus, J. Comp. Neurol., 90 (1949) 111 120. 17 Widenfalk, B., Elfvin, L.-G. and Wiberg, M., Origin of sympathetic and sensory innervation of the elbow joint in the rat: a retrograde axonal tracing study with wheat germ agglutinin conjugated horseradish peroxidase, J. Comp. Neurol., 271 (1988) 313-318. 18 Wyke, B.D., Neurophysiological aspects of joint function with particular reference to the temporomandibular joints, J. Bone Joint Surg., 43 (1961) 39(~397. 19 Wyke, B.D., The neurology of joints, Ann. R. Coll. Surg. Engl., 41 (1967) 25-50.
Neuroscience Letters, 109 (1990) 30 35 Elsevier Scientific Publishers Ireland Ltd.
NSL 06616
Origin of sympathetic and sensory innervation of the temporo-mandibular joint. A retrograde axonal tracing study in the rat Bertil Widenfalk and Mikael Wiberg Hand Surgery Unit, Department of Plastic Surgery, University Hospital, and Department o[' Anatomv Uppsala University, Uppsala (Sweden)
(Received 16 June 1989; Revised version received 25 September 1989: Accepted 27 September 1989) Key word~." Joints innervation: Sympathetic ganglion; Spinal ganglion: Horseradish peroxidase; Rheumatoid arthritis
The cells of origin of sensory and sympathetic innervation of the temporo-mandibularjoint were studied by the intraaxonal transport method. Horseradish peroxidase or lectin-conjugated horseradish peroxidase was injected into the temporo-mandibular joint unilaterally in adult rats. Labelled cells were observed ipsilaterally in the superior cervical and stellate sympathetic ganglia, in the sensory trigeminal ganglion and in the second to fifth dorsal root ganglia: none were found contralaterally. The results are discussed in relation to the hypothesis that a nervous mechanism might be involved in the pathogenesis of joint inflammation.
R h e u m a t o i d arthritis ( R A ) affects not only j o i n t s o f extremities but also others, including the t e m p o r o - m a n d i b u l a r (t-m) j o i n t [2]. Several clinical o b s e r v a t i o n s have indicated that a nervous c o m p o n e n t is involved in the p a t h o g e n e t i c mechanism. F o r instance it has been o b s e r v e d that central or p e r i p h e r a l nerve lesions, affecting one or several limbs, m a y protect the affected limbs from R A (see e.g. ref. 6). The m e c h a nism o f i n v o l v e m e n t o f this nervous c o m p o n e n t is still n o t c o m p l e t e l y u n d e r s t o o d , but it has been p r o p o s e d that an interaction between sensory afferents a n d s y m p a thetic efferents m a y be r e a s o n a b l e , since a p p l i c a t i o n o f substance P into the j o i n t cavity a g g r a v a t e s [12] a n d i m m u n o s y m p a t h e c t o m y a t t e n u a t e s [7], a d j u v a n t - i n d u c e d arthritis. In o r d e r to study the cells o f origin in s y m p a t h e t i c and d o r s a l r o o t ganglia innervating the t-m j o i n t , 25 a d u l t rats weighing a b o u t 300 g were anesthetized with p e n t o b a r b i t a l s o d i u m ( M e b u m a l - V e t ) a n d h o r s e r a d i s h p e r o x i d a s e ( H R P ) o r lectin-conju-
Correspondence." B. Widenfalk, Dept. of Plastic and Hand Surgery, Akademiska sjukhuset, S-751 85 Uppsala, Sweden.
0304-3940/90/$ 03.50 ~:~:,1990 Elsevier Scientific Publishers Ireland Ltd.
31
gated horseradish peroxidase (WGA-HRP) was injected into the joint unilaterally, which was exposed through an incision in the joint capsule. With a Hamilton syringe, the tracer was injected under visual control into the joint cavity. After the injection the needle was left in situ for 5 min before withdrawal to diminish the risk of leakage of tracer into surrounding tissues. Finally the joint capsule, soft tissue and skin was sutured. After varying survival periods the animals were reanesthetized and fixed transcardially with an aldehyde mixture. The superior cervical and stellate sympathetic ganglia and the trigeminal and cervical dorsal root ganglia were removed and rinsed overnight in phosphate buffer containing 30 %w/v sucrose. All ganglia were then cut Ion-
TABLE I SURVEY
OF THE DIFFERENT
DISTRIBUTION GEMINAL
OF LABELLED
RETROGRADE NEURONS
(TRIG) AND DORSAL
EXPERIMENTS,
AND THE NUMBER
IN THE SYMPATHETIC
R O O T (C2 C5) G A N G L I A
AND
(GCS, GS), SENSORY TRI-
IPSILATERALLY
AFTER
INJEC-
T I O N O F 1/zl 10% W G A - H R P O R 1/A 30% H R P I N T O T H E R I G H T (R) O R L E F T (L) T E M P O R O MANDIBULAR JOINT OF THE RAT
Case
Side
Tracer
Survival Number of retrogradely labelled neurons in G C S , G S , T R I G period (h) and C2~Cs ipsilaterally GCS
1
R
2
L
WGA-HRP
12
5
12
64
GS
TRIG
C2
C3
159
12
2
-
224
I
27
C4
C5
-
-
3
L
18
435
106
646
34
124
27
4
L
18
501
136
25l
15
315
75
5
L
24
303
2
459
1
10
-
6
R
24
593
5
543
119
10
7
L
48
186
3
276
30
25
I
8
R
48
305
274
19
6
-
-
9
L
60
421
12
309
18
84
6
-
10
R
60
339
12
326
2
23
2
-
11
R
60
387
19
34
8
-
-
12
R
72
1562
Ill
1024
662
178
23
-
13
R
72
1069
79
1112
315
215
32
1
14
L
96
998
2
218
12
6
2
-
15
L
96
502
7
325
2
35
2
-
16
L
120
550
24
173
2
38
4
17 18
R R
120 12
95 12
15 -
134 34
-
16 -
6 -
-
HRP
19
R
12
150
8
369
-
20 21
R L
24 24
157 191
7
146 63
-
-
-
4
-
-
-
22
L
48
81
4
91
2
-
-
23 24
R R
48 72
8 103
1 2
30 103
5
1 -
-
25
L
72
88
1
41
.
.
.
.
32 g i t u d i n a l l y i n t o 4 0 / t m thick sections o n a f r e e z i n g m i c r o t o m e a n d i n c u b a t e d for H R P h i s t o c h e m i s t r y w i t h t e t r a m e t h y l b e n z i d i n e ( T M B ) [14]. In 4 cases ( N o s . 14, 16, 24, 25) the l o w e r b r a i n s t e m , c o n t a i n i n g the m o t o r t r i g e m i n a l n u c l e u s , was r e m o v e d , rinsed, s e c t i o n e d a n d i n c u b a t e d in the s a m e w a y as d e s c r i b e d a b o v e .
I V
Fig. 1. Peroxidase-positive neurons in the sensory trigeminal ganglion (b 3), dorsal root ganglia (4) and sympathetic ganglia (5, 6) after injection of WGA HRP into the temporo-mandibular joint ipsilaterally. 1 ,: Labelled cells in the mandibular portion of the sensory trigeminal ganglion. 190 x. 1:: small peroxidasepositive neurons in the sensory trigeminal ganglion; survival period 12 hours. 190 x. I~: one large- and two medium-sized labelled cells in the sensory trigeminal ganglion; survival period 96 h. 225 x. 14: round peroxidase-positive cells of varying sizes in the second cervical dorsal root ganglia. 375 x. I ~, 1~: multipolar and fusiform labelled cells in the superior cervical sympathetic ganglia. 475 ×.
33 Details of the different retrograde tracing experiments and quantitative values for the distribution of peroxidase-positive cells are given in Table I. General labelling. Labelled neurons were observed in the superior cervical and stellate sympathetic ganglia, the sensory trigeminal ganglion and the second to fifth dorsal root ganglia. All cells were found ipsilateral to the injection side. Different survival periods were used in order to find the optimal one. The maximum number of peroxidase-positive neurons was observed after 72 h when W G A - H R P was used as the tracer, while the optimal survival period was between 12 and 24 h after injection of native HRP, which also yielded a total number of labelled cells that was smaller than when W G A H R P was used. The reasons for these differences are discussed below. Sympathetic ganglia. A maximum of about 1500 peroxidase-positive neurons were found ipsilaterally in the superior cervical and stellate ganglia; about 95% of these were situated in the superior cervical ganglion (Table I). The labelled cells were equally distributed over the ganglia, but differed in size and shape, being multipolar with a diameter of 20-25/tm (Fig. 15) or fusiform, measuring 10-20 ktm (Fig. 16). The multipolar cells were mainly concentrated to the center of the ganglia, while the fusiform ones were most numerous in the outer margin. Sensory trigeminal and dorsal root ganglia. A maximum of 1000 labelled cells were found ipsilaterally in the trigeminal ganglion. (Table I), where they were observed in the mandibular portion [1] (Fig. 10. The dorsal root ganglia were labelled at the level of C2 C5 where a total of 500-800 labelled neurons were observed ipsilaterally, evenly distributed over the ganglia. The overwhelming majority, about 90-95 % were equally distributed between the second and third cervical ganglia, while only some scattered neurons were seen in the fourth and fifth ones (Table I). In both types of sensory ganglia the labelled neurons were round and of varying sizes (Fig. 14). Most frequent (65 %) were the small ones (diameter 15-20/zm), while the medium-sized (25-30/~m) constituted 25% and the large ones (40-55/tm) 10% of the total number of labelled neurons. Technical comments. It has been claimed that W G A - H R P is a superior tracer for retrograde tracing studies [4]. This was also observed in the present study, where the use of W G A - H R P yielded a maximum of up to seven times more labelled neurons than native H R P (Table I). Another difference between the two tracers was that the survival period, when the maximum of labelled cells was observed, was shorter for native H R P than for WGAH R P (Table I). This observation might possibly be explained by the different uptake mechanisms by which the tracer enters the neuron (see e.g. ref. 4), but also by the observation that to reach the nerve endings the tracer has to diffuse through the superficial part of the synovial membrane [17, 19], and this diffusion is supposed to be faster for a smaller molecule such as H R P [10]. The different survival periods also yielded a different pattern of sizes of the labelled cells. Thus, in the sensory trigeminal and dorsal root ganglia, after the shortest survival periods virtually only small labelled neurons were seen (Fig. 12), while after the longest survival periods large ones predominated (Fig. 13), a pattern which has been described previously [5]. The last technical issue to be commented upon is the possible leakage of tracer
34 into the surrounding tissues. The fact that no labelling was observed contralaterally indicated that there was no leakage of tracer into the systemic circulation. To rule out the possibility of spread to the surrounding muscle, the lower brainstem was removed in four cases (Nos. 14, 16, 24, 25), and no retrograde labelling of motor neurons in the m o t o r trigeminal nucleus, innervating the masticatory muscles proper [16], was observed. I n earlier studies [18, 19] investigations have been made of the anatomical and physiological characteristics of the different types of receptors and nerve fibers associated with the t-m joint, but in none of these were the cells of origin of the nerve fibers investigated. The origin of the sensory fibers was studied by Romfh et al. [15] in the cat and they demonstrated that the cells of origin were situated in the mandibular portion of the sensory trigeminal ganglion. The neurons constituted a heterogeneous population with sizes varying between 20 and 60 /lm, but nothing was discussed about any spinal sensory origin. The results of the present study concerning the sensory trigeminal ganglion were in agreement with those obtained by Romfh et al. irrespective of the sizes of the labelled neurons. The most numerous labelled cells in the present study were small. A possible reason why Romfh et al. did not report predominance of that population might have been that their survival periods varied between 24 and 72 h, and in the present study it was clear that with native H R P as a tracer which was also used in the former study the optimal survival time for small neurons was shorter than 24 h. A novel finding in the present study was the impressive sympathetic efferents to t-m joint. This is of special interest in view of the recent proposal that the nervous system might be involved in the pathophysiology of RA [13], and that the sympathetic nervous system is part of this nervous component (see e.g. ref. 3). It seems reasonable to assume that there is some interaction between sensory afferents and sympathetic efferents, a theory considered by others [13]. The majority of labelled cells in the trigeminal and dorsal root ganglia were small, a cell population which has been found to contain substance P [8] and to send terminal collaterals to the superficial laminae of the dorsal horn, observations supporting the idea that these cells are involved in nociceptive transmission [11]. A nociceptive input from joints could therefore modulate the activity in sympathetic efferents, an idea supported by observations of substance P-containing fibers in sympathetic ganglia [9], and this modulation might be of importance for the induction and activity of RA [13]. This study was supported by grants from the Swedish Medical Research Council (project 12X2710) and the Mac Rudberg Foundation.
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