Brain Research, 578 (1992) 17-25 O 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
17
BRES 17612
Fos-like immunoreactivity in the rat superficial dorsal horn induced by formalin injection in the forepaw: effects of dorsal rhizotomies Catherine Abbadie, Marie-Christine Lombard, Franqoise Morain and Jean-Marie Besson Unit4 de Recherches de Physiopharmacologie du Systdme Nerveux, INSERM U.161 and EPHE. Paris (France) (Accepted 26 November 1991 ) Key words: Immunohistochemistry; Proto-oncogene; Pain; Deafferentation; Spinal cord As previously described at the lumbar spinal level, we found that 2 h after subcutaneous formalin injection in the distal part of the forelimb, Fos-like immunoreactivity (FLI) was induced in the ipsilateral cervical enlargement. Not surprisingly, as the injection site corresponds to the distal part of the C6-C8 dorsal root dermatomes, maximal labelling which predominated in the superficial laminae, was observed in the C6-C8 segments and to a lesser extent in C5. Similar experiments were performed on rats which underwent various types of unilateral dorsal rhizotomies (DRh) 7 days before formalin injection. In animals with C4, C5, T1 and T2 DRh sparing C6-C8 the rostrocaudal distribution was similar to the intact one. But, in animals having C4-T2 DRh sparing one single root, C7, the segmental FLI distribution was modified: it was slightly increased in C7, decreased in C6 and significantly decreased in C8. As expected, no FLI was found in animals with C4 to T2 DRh. The spared root model provides information about the segmental distribution in the cervical spinal cord of the input brought by a single root following stimulation of the distal forelimb, i.e., maximal distribution in the entry segment, but also in the two rostral and one caudal segments. INTRODUCTION The formalin test is a classical standard m o d e l used to study pain mechanisms and to test the effects of analgesic drugs s'12. It consists of a subcutaneous formalin injection into the paw, which induces a stereotypical measurable behavior. Electrophysiological studies showed that dorsal horn nociceptive neurons are activated for at least 1 h following formalin injection 9"1°. Recently, using the Fos immunocytochemical m e t h o d , initially described by H u n t et al. 19, Presley et al. 32 rep o r t e d the expression of the c-fos p r o t o - o n c o g e n e in l u m b a r spinal cord following formalin injection into the hindpaw. M o r e o v e r , Fos-like immunoreactivity ( F L I ) was significantly reduced by systemic m o r p h i n e in a d o s e - r e s p o n s e relationship and these effects were antagonised by naloxone. The Fos immunocytochemistry technique appears to be promising since the p a t t e r n o f c-fos expression is reminiscent of electrophysiological, anatomical and behavioral pain studies and could be useful to test pharmacological agents, even if caution is n e e d e d in the interpretation of Fos-related data. The aim of this study was to quantitatively assess, at the cervical enlargement: (1) the F L I rostrocaudal distribution induced by formalin injection into the rat forepaw, (2) the contribution of the three roots C6, C7 and C8 corresponding to the d e r m a t o m e s where the formalin injection was m a d e 24, and of the single root, C7. F o r
this purpose, we l o o k e d for F L I in intact animals and in animals that underwent various types of unilateral dorsal rhizotomies. We focussed our study on the superficial dorsal horn (laminae I and II) of the spinal cord where small d i a m e t e r primary afferent fibers mainly project (see references in Ref. 13), and where electrophysiological studies show that most of the neurons receive noxious input (see references in Ref. 2). In addition, in o r d e r to visualize the loss of fine afferent fibers in the superficial layers, we examined the histochemistry of fluoride-resistant acid phosphatase ( F R A P ) which is used as a classical m a r k e r of fine (nociceptive) primary afferent fibers 7'21'22. We also considered the effects of various types of rhizotomies on the licking behavior ind u c e d by the formalin injection. MATERIALS AND METHODS Experimental animals Experiments were performed on 35 male albino Sprague-Dawley rats (Charles River, France), weighing 225-250 g. The surgical procedures were performed under general anesthesia (ketamine i.p., 100 mg/kg) supplemented with local anesthesia (2% xylocaine). A dorsal hemilaminectomy from cervical 4 (C4) to thoracic 2 (T2) vertebrae was made on the right side, and various combinations of dorsal root section were carried out, proximal to the ganglia, through small individual openings of the dura mater under a dissection microscope. For each root section, a portion of 1-2 mm long was excised. Care was taken to cause no damage to the blood vessels running along the dorsal roots. The wound was washed with saline solution and muscles and skin sutured layer by layer.
Correspondence: C. Abbadie, INSERM U161, 2 rue d'Alrsia, 75014 Paris, France. Fax: (33) 1 45 88 13 04.
18 Five experimental situations were considered (see Fig. 5): (1) control rats having intact dorsal roots (control; n = 8); (2) sham rats submitted to the surgical procedures except for the lesion of the dorsal roots (sham; n = 2); (3) animals with dorsal rhizotomy of the seven roots C4-T2 (C4-T2 cut; n = 4); (4) animals with dorsal rhizotomy of the four roots C4, C5, T1 and T2, sparing the three roots C6-C8 (C6-C8 spared; n = 5); (5) animals with dorsal rhizotomy of the six roots C4-C8, T1 and T2, sparing the root C7 (C7 spared; n = 6). The animals were allowed to recover in individual cages for a postoperative period of 7 days. On day 7, they were submitted to the formalin test, slightly modified from the test performed by Dubuisson and Dennis 12. The rats were put in a Plexiglas box and deeply anesthetized with 4% halothane for 4 min and then, given 2 x 50 pl subcutaneous (ventrally and dorsally) injections of a 10% formalin solution, in the right forepaw, more specifically between the two middle digits. This area corresponds to the distal portion of the dermatomes of the C6-C8 dorsal roots (Fig. 1). Control experiments consisted of (1) two additional intact rats injected with the same volume of saline in the forepaw, and (2) two non-injected rhizotomized rats in order to detect any Fos labelling not due to the formalin injection.
short PB rinses to take off the excess stain, sections were differentiated in 70% alcohol and the differentiation time was evaluated under the microscope. After being air dried, the slides were coverslipped. To test the specificity of the primary antibody, controls were performed; preabsorption with the corresponding synthetic peptide or omission of any stage in the protocol abolished the staining. For the FRAP reaction, according to the method of G~m6ri 14"15, modified by Silverman and Kruger 3s, the sections were mounted on gelatin-subbed slides and air dried. The slides were rinsed in 0.2 M Tris-maleate buffer (TMB) during 30 s, and incubated for 3
A
ventral
dorsal
ventral
dorsal
C4
C7
C5
C8
Behavioral observations As already described by Dubuisson and Dennis 12, subcutaneous injection of formalin in the forepaw of the rat induces stereotyped behavioral reactions. These reactions consist of: (1) different positions of the injected limb, indicating graded intensities of nociceptive manifestations and arbitrary scaled in degrees from 0 to 2; and (2) intense licking of the injected site scored as degree 3 and considered as the reaction of the animal to the most intense nociceptive manifestation. In the present study, we only looked for the licking behavior. The quantification was done by measuring the duration (in seconds) of this behavior and totalling it up to 50 min after injection.
Immunohistochemistry Two hours after the formalin injection, the animals were deeply anesthetized with pentobarbital (55 mg/kg, i.p.) and perfused intracardially with 200 ml of phosphate-buffered saline 0.1 M (PBS) followed by 500 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB). The cervical spinal cord was then removed and postfixed for 4 h in the same fixative and cryoprotected overnight in 30% sucrose in PB. Frontal frozen sections of 40 ~m were cut and collected in PB. The serial sections were divided into two sets, one set was im° munostained for Fos-like protein and the second set was histostained for fluoride-resistant acid phosphatase (FRAP). For Fos immunostaining, the tissue sections were incubated for 30 rain at room temperature in a blocking solution of 3% normal goat serum in PBS with 0.3% Triton-X (NGST). Then the sections were incubated overnight at 4°C in the primary antiserum directed against the c-los protein (kindly provided by Dr. D. Slamon, Departments of Haematology and Oncology at UCLA). The Fos antibody was used at 1:5,000 (for description of the antibody characteristics, see Ref. 32). The incubated sections were washed three times in 1% NGST and incubated in biotinylated goat anti-rabbit IgG for 1 h at room temperature, then washed twice in 1% NGST and incubated for 1 h in Avidin-Biotin-Peroxidase complex (Vectastain, Vector Laboratories), according to the avidin-biotin-peroxydase method of Hsu et al. 17. Finally, according to Mauro et al. 25 and modified by Men6trey et al. 28, the sections were washed three times in PBS and developed in 1-naphthol ammonium carbonate solution (89.5 ml 0.1 M PB, 10 ml 10% 1-naphthol in absolute alcohol and 0.1 ml hydrogen peroxide) for 15 rain and were washed three times in PB to stop the staining reaction. The sections were mounted on gelatin-subbed slides, air dried for the stain to be intensified, and made alcohol-resistant through basic dye enhancement in 0.025 crystal violet solution in PB for 3 rain. After two
C6
B
ventral
5 0 ul, 1 0 % f o r m a l i n
T1
dorsal
5 0 ul, 1 0 % f o r m a l i n
Fig. 1. Dermatomes of the cervicothoraeic roots of the rat brachial plexus and stimulus sites. A: ventral and dorsal aspects of the extremity of rat forelimb showing the extent of the dermatomes corresponding to the C4-T1 dorsal roots (DR). The dermatomes w e r e mapped using electrophysiological recording of the D R fibers activation following cutaneous tactile stimulation. Black areas represent the region from which maximal responses w e r e recorded. Stippled areas r e p r e s e n t zones yielding smaller responses. Note that the C4 D R does not contribute to the inncrvation of the distal forelimb, and the overlapping of two or three adjacent roots (adapted from Lombard et a1.24). B: ventral and dorsal sites of the subcutaneous formalin injection which was done specifically between the two middle digits. Note that these sites correspond to the C6-C8 dermatomes.
19 h at 37°C in substrate solution (0.1 M TMB, 0.1% lead nitrate, 8% sucrose and 5 mM thiamine monophosphate). They were then rinsed in 0.1 M TMB, immersed in 1% ammonium sulfide solution for 20 s until a blackish precipitate developed and washed three times in distilled water. The slides were dehydrated and coverslipped.
Counting of Fos-labelled cells FLI was studied through the C4-T1 spinal segments. Tissue sections were first examined using darkfield microscopy to determine the segmental level according to Molander et al. 29, and the gray matter landmarks. The sections were then examined under lightfield microscopy to localize Fos-positive cells. Labelled nuclei were counted using a camera lucida attachment. All results are expressed as the sum of FLI neurons in three sections per segmental level. Statistical analysis was made to compare the total number of labelled cells, using 1-way analysis of variance for the different groups of animals, and 2-way analysis of variance for the different groups of animals and the spinal level. For multiple comparisons, the Fisher's PLSD test was used. The investigator responsible for plotting and counting the FLI neurons was blind to the experimental situation of each animal. FRAP labelling was observed in adjacent sections to the Fos-labelled ones.
RESULTS
Behavioral observations It is worth mentioning that none of our rhizotomized animals d e v e l o p e d a u t o t o m y of the forelimb within the delay chosen for the e x p e r i m e n t (7 days); only a few (n = 3) exhibited some light scratching of the anterior or posterior face of the shoulder. Several studies have d e m o n s t r a t e d that after formalin injection, animals show two distinct phase in the behavioral response: (1) an early (0-10 min post-injection), and a late (15-50 min post-injection) phases 12'1s. In our study, whatever the group of rats considered, only the late phase was present, certainly due to the initial d e e p halothane anesthesia (see Materials and Methods). Thus, within the first 5 min after the formalin injection (immediately following the 4 min anesthesia), the animals were generally lying or creeping on the floor of the observation cage, showing no c o m p u t a b l e behavioral alterations. F o r the whole p e r i o d of observation (50 rain following formalin injection), the m e a n durations ( S . E . M . ) of the licking behavior were: 299 + 44, 452 +_ 117 and 350 +_ 97 s for the control, C 6 - C 8 spared and C7 spared, respectively. A l t h o u g h there was a tendency for an increase in the C 6 - C 8 spared group, as c o m p a r e d to the control, no significant difference was found between these three groups. F o r the intact animals receiving saline injection and animals having the C 4 - T 2 dorsal rhizotomy, the licking score was below 50 s.
Fos immunocytochemistry results General observations. A s the sections were d e v e l o p e d
C4
°4°"
C 5 ~ C8 ~e'"
c6~T1~ Fig. 2. Camera lucida drawings showing the rostrocaudal distribution of Fos-like immunoreactive (FLI) neurons, in the spinal cervical enlargement, 2 h after formalin injection into the ipsilateral forepaw of an intact rat. Each schema includes all labelled cells in a single 40-/~m section; each dot represents one labelled cell. The most intense labelling is noted at the C5-C8 segments, which correspond to the spinal cord projection of the distal forepaw afterents. Note that there is a more restricted rostrocaudal distribution of FLI cells in the superficial dorsal horn than in the deeper layers of the spinal gray. The boundaries of the superficial laminae and of the reticular part of the neck of the dorsal horn are outlined for orientation.
in naphthol solution instead of diaminobenzidine, F L I nuclei were identified as d a r k violet round structures, often containing non-stained nucleoli. In our experimental conditions, 7 days after the surgical p r o c e d u r e , no labelling or only a few labelled cells (less than 4 cells/section) were found in sham o r in rhizotomized animals not injected with formalin. Similar observations were m a d e in intact rats receiving saline injection. A f t e r formalin injection, the ipsilateral rostrocaudal and laminar distribution of F L I neurons in the cervical spinal cord of control rats is shown in Fig. 2. L a b e l l e d cells were e n c o u n t e r e d in all the considered spinal segments ( C 4 - T 1 ) , and they were exclusively localized in the dorsal horn. The greater n u m b e r of labelled neurons was o b s e r v e d in the superficial laminae of C 5 - C 8 segments, and m o r e precisely was concentrated in the medial part. M o r e sparse Fos labelling was found all through the d e e p e r layers, laminae V, VI and X, and to a lesser extent in laminae I I I and IV. Contralateral to the injected paw, only a few cells (less than 0/section) were Fos-positive in some animals; they were r a n d o m l y distributed in the whole gray matter. A c c o r d i n g to these observations showing that after
20 formalin injection F L I was best e v o k e d in the superficial laminae, we focussed our study on these laminae in order to gauge the effects of various types of dorsal rhizotomies. Before comparing the results of the different experimental situations, we quantified in each group, the segmental rostrocaudal distribution in the six spinal levels, C 4 - T 1 of F L I neurons. These levels correspond to the segments of the projection of the injected d e r m a t o m e s and to the adjacent segments. Segmental distribution (Figs. 3-5). In control rats, the most intense labelling was found in C 5 - C 8 segments where the respective n u m b e r of F L I neurons was 50 + 12 (mean + S . E . M . ) , 81 + 14, 93 + 12 and 76 + 12. The n u m b e r of cells in these four segments r e p r e s e n t e d
93% of the total n u m b e r of labelled cells in the C 4 - T 1 extent. The distribution was maximal in C7. H o w e v e r there is no significant difference between C7 and the two adjacent C6 and C8 segments. In contrast, c o m p a r e d to C7, the n u m b e r of cells in C5, T1 and C4 were significantly smaller, 50 + 12 ( P < 0.01), 20 + 4 and 4 + 2 ( P < 0.001), respectively. Interestingly, when comparing the n u m b e r of cells in the C5 and T1 segments which are adjacent to the three segments corresponding to the injected d e r m a t o m e s ( C 6 - C 8 ) , there was a significant higher n u m b e r of cells in the rostral C5 than in the caudal T1 segment ( P < 0.05). In C 6 - C 8 spared, the segmental distribution was quite similar to the control one. Intense labelling was also ob-
FRAP
Fos
t
a
¢
t .
'c J
Fig. 3. These photomicrographs illustrate the location of Fos-like immunoreactive (FLI) neurons and of FRAP mapping in the superficial laminae of the dorsal horn in C7 segment, in adjacent sections. Three experimental situations are represented: intact rat (A), C4-T2 dorsal rhizotomy sparing the three C6-C8 roots (B) and C4-T2 dorsal rhizotomy sparing the C7 root (C). Note that the number of FLI neurons, in these three groups is apparently similar, while FRAP labelling slightly decreases when three roots are spared, but dramatically diminishes when only the C7 root is spared.
21
control
C6,C7,C8 spared
C7 spared
Fig. 4. Camera lucida drawings showing the cervical rostrocaudal distribution of Fos-like immunoreactive (FLI) neurons in the superficial dorsal horn, 2 h after formalin injection into the ipsilateral forepaw. Three experimental situations are represented: intact rat (control), C4-T2 dorsal rhizotomy sparing the three C6-C8 roots (C6-C8 spared), and C4-T2 dorsal rhizotomy sparing the C7 root (C7 spared). Each schema includes all labelled cells in one 40/~m section; each dot represents a single labelled cell. Note that in control and in C6-C8 spared animals the most intense labelling is observed in the C5-C8 segments, while in C7 spared animal, the number of FLI neurons is lower in C6 and even more in C8.
served in C 5 - C 8 (72 + 16, 103 + 16, 101 + 14 and 76 _ 12, respectively). H e r e again, the n u m b e r of cells in C5 was significantly higher than in T1 ( P < 0.05). In C7 spared, the distribution differed from those observed in both control and in C 6 - C 8 spared. A l t h o u g h , the main intense labelling was found in the C 5 - T 1 segments, there was a clear p e a k in C7 (116 + 25), comp a r e d to the adjacent C6 (64 + 23) and C8 (40 + 15) segments. H o w e v e r , this difference was only significant for C8 (P < 0.01). In this case, the n u m b e r of F L I cells was also higher in C5 than in T1, however there was no
significant difference. F o r the animals that were submitted to the C 4 - T 2 dorsal rhizotomy, almost no F L I cells (less than 4) could be observed.
Comparison of the different experimental situations. Considering the total n u m b e r of F L I neurons in the spinal segment from C 4 - T 1 , there was no significant difference (F2.16 = 0.6, P = 0.6) between the three groups of animals (control, C 6 - C 8 spared, and C7 spared). The total n u m b e r of labelled cells were 323 + 47, 390 + 58 and 289 + 87, respectively. We found no correlation
22
A
control 150
c,
~
c5
100
ison segment by segment showed differences between the control and the C7 spared: (1) a small increase in the number of FLI cells in C7, in C7 spared (but nonsignificant); and (2) a decrease in C6 and C8, in C7 spared which was only significant in C8 (P < 0.01).
50
FRAP mapping 0
O9 r"
o G)
Z
C4 C5 C6 C7
As shown in Fig. 3, in the C7 segment for the control group, the FRAP labelling was localized in the whole substantia gelatinosa (laminae II). In C6-C8 spared and in C7 spared, we observed a disappearance of the labelling in the lateral part of this lamina. We noticed a weak decrease in the median part in C6-C8 spared, while there was a dramatic decrease in the whole substantia gelatinosa in the C7 spared. After the C4-T2 dorsal rhizotomy, there was a total disappearance of the F R A P labelling.
C8 T1
C6, C7, C8 spared 150
._.= 100
== O
E
i
50
_E G)
/
0 C4 C5 C6 C7 C8 T1
T
DISCUSSION
O LL
C7 spared 150
100
50
$
0 C4
C5
C6
C7
C8
T1
Spinal Segments
Fig. 5. Quantitative rostrocaudal distributions of Fos-like immunoreactive (FLI) neurons and schemas illustrating the different dorsal rhizotomies. Three experimental situations are represented: intact rat (control; n = 8), C4-T2 dorsal rhizotomy sparing the three C6-C8 roots (C6-C8 spared; n = 5), and C4-T2 dorsal rhizotomy sparing the C7 root (C7 spared; n = 6). A: quantification of the rostrocaudal distributions of FLI cells evoked by formalin injection, in the C4-T1 spinal enlargement: spinal segments vs the number of FLI neurons (mean + S.E.M.). The number of cells is the sum of three sections per segment. Note that in control and in C6-C8 spared animals the most intense labelling is observed in the C5-C8 segments, while in C7 spared animal, the number of FLI neurons is lower in C6 and even more in C8. For each diagram, significance is expressed taking C7 as reference (*P < 0.05; **P < 0.01; ***P < 0.001). B: these schemata illustrate various combinations of unilateral dorsal root sections which are made intradurally, between the ganglia and the spinal cord (see Materials and Methods).
(Pearson's r = 0.4, P = 0.53) between the total number of FLI cells and the behavioral scores expressed by licking scores. If we compared the segmental distribution in the cervical spinal enlargement, there was no significant difference between the control, C6-C8 spared and C7 spared groups (F2,10 = 1.9, P = 0.15). In contrast, the compar-
In the present study, we found comparable results at the cervical spinal level in non-lesioned animals injected with formalin in the forepaw, to those described by Presley et al. 32 at the lumbar spinal level following formalin injection in the hindpaw. These authors described maximal labelling along three lumbar segments (L3-L5), we observed maximal labelling also along three cervical segments (C6-C8), with a decrease in the Fos labelling rostrally and caudally to these segments. In both studies, there was a similar laminar distribution, i.e., intense staining in laminae I and II, less dense in laminae V, VI and X, and a few FLI neurons in laminae III, IV and in the ventral horn. On the other hand, the laminae labelling differed according to the segmental level: in superficial laminae, FLI neurons distribution was less extensive than in the deeper ones. Despite the fact that we both used the same antibody, Presley et al. 32 found a higher number of FLI cells; this could be due to the fact that our animals were anesthetized for the formalin injection, as evidenced by the abolition of the first behavioral phase. This is reminiscent of recent results from Porro et al. 3~ who observed that in the rat spinal cord, 2-deoxyglucose uptake was less pronounced in pentobarbital-anesthetized animals than in non-anesthetized. Since the present study was concerned with the effects of dorsal rhizotomy, and as both anatomical and electrophysiological methods show that small diameter fibers mainly terminate in these laminae (see references in Ref. 13), we decided to focus our study on the superficial layers of the dorsal horn. Moreover, Fos staining in laminae I and II represented the region with the greater number of Fos positive cells in the gray matter. The most intensive staining was observed in the medial part of
23 laminae I and 11, as also reported by Men6trey et al. 27 and Presley et al. 32 at the lumbar level. With the 2-deoxyglucose technique, and with a similar formalin injection in the forepaw, the highest increase in activity was also found in the medial part of the superficial region of the ipsilateral dorsal horn 31. Thus, the segmental labelling follows the somatotopic organization of the cutaneous afferent fibers corresponding to the stimulus site (distal part of the limb), that is consistent with electrophysiological studies reporting a medial location of neurons driven by stimulation applied in distal peripheral fields4. We found no FLI neurons in laminae I and II, in non-stimulated (no formalin injection) rhizotomized rats. This result contrasts with those of Sharp et al. 34, reporting that trigeminal nerve section provoked FLI in laminae I and II of the spinal trigeminal nucleus pars caudalis and those of Chi et al. 6, relating that sciatic nerve section induced FLI in lumbar spinal cord. These discrepant results could be explained by the difference in the lesions: in our study, we made root sections whereas the others carried out a nerve section. These two lesions mainly differ by the fact that: (1) dorsal root section resuits in rapid degeneration of afferent fibers 16, which is almost complete by 7 days postlesion, whereas nerve section results in the survival of still functionally excitable afferent fibers; and (2) neuroma formation was absent after dorsal root section whereas it was facilitated following nerve section, according to Chi et al. 6. Although spontaneous neuronal hyperactivity has been reported after dorsal rhizotomyz3, it develops only in those animals showing scratching and/or autotomy. In the present study, no rhizotomized animal presented autotomy. On the other hand, we observed no FLI cells in sham rats, so no immunoreactivity could be due to the surgical procedure. The analysis of the number of Fos-positive cells along the cervical enlargement provides information about the segmental distribution of the input brought by dorsal roots following stimulation of the distal forelimb. In intact animals, we observed maximal Fos labelling in C6-C8 segments, thus, consequently with Lombard et al. 24, we confirmed that the formalin injection site mainly corresponds to the dermatome of the C7 root, but also to the dermatomes of C6 and C8 roots which overlap with C7. In addition, the number of FLI neurons was significantly higher in C5 than in T1 (which are localized two segments rostraUy and caudally to C7). Two different explanations could explain this: (1) formalin injection can spread out to the C5 dermatome, since it covers the most medial digit, but this is unlikely, since the T1 dermatome also covers one digit (the most lateral) and could also be affected by formalin; and (2) afferent fibers when entering the spinal cord project more in the
rostral direction than caudally. The fact that the distribution of C6-C8 spared was the same as the control one, reinforces the assertion that the information coming from the distal part of the forelimb arrives through three main roots: C6-C8 and also confirms the second hypothesis, i.e., fiber projection is more rostral than caudal, since in this case the C5 root was cut, but the labelling in the corresponding segment was still higher than in T1. Surprisingly, when only the C7 root was spared at the center of six cut roots, the total number of FLI neurons in the cervical enlargement slightly (but not significantly) diminished in contrast to FRAP labelling which dramatically decreased. Although the total number of FLI cells in this case, was close to the control one, the segmental distribution changed. Not surprisingly, in this case, the number of FLI neurons peaked in C7 and diminished in adjacent segments: decreased in C6 (not significantly) and more particularly in C8 (P < 0.01). Thus, these data show and also confirm that primary afferent fibers from a single dorsal root mainly project in their entry segment, and in one caudal and two rostral segments. Several studies on afferent fiber projections have been reported, with different data depending on fiber diameter, spinal level, species, and technique used. It has generally been shown that A6-fibers project over three or four segments 3°'39, whereas C-fibers project only over one or two segments 1'26'3°'36'38'39. The fact that in the C7 spared, the number of FLI neurons, in the C7 segment was similar to the control tends to suggest that the input arising from this remaining C7 root is sufficient to produce an activation equivalent to that seen in intact animals. This observation is surprising, since although the entry segment receives the majority of the fine diameter primary afferent fibers arising from a dorsal r o o t 36'38 there is a well-known overlap of adjacent root projection in the spinal gray matter. This overlap of projection areas from adjacent peripheral territories has been clearly demonstrated by Williams et al. 42. However, in our experimental conditions, it could be that sections of adjacent roots can suppress segmental inhibitory control mechanisms normally acting on nociceptive afferent input (see references in Ref. 40). This hypothesis seems to be sustained by our behavioral observations, since the licking score (although representing only one aspect of the nociceptive behavioral reactions, i.e., supraspinal- integrated behavior) exhibited by the C7 spared animals was as intense as that of the control animals. Additional experiments using the spared root paradigm but focussing on C6 or C8 would be necessary to confirm our hypothesis. From a functional point of view, the interpretation of Fos labelling should be made with caution, for the following reasons: the expression of c-fos does not provide information on the function of the cell, and it is not
24 possible to differ between the cellular activation resulting from nociceptive input and a more general activation following a stimulation; on the other hand, all activated cells do not compulsorily express FOS5'19'41. Nevertheless, in some studies, Fos labelling seems to correlate with animal nociceptive reactions 27"32 and in fact, our data are reminiscent of other studies, i.e., distribution of Fos-positive n e u r o n s correlates with the known distribution of nociceptive cells in the spinal dorsal horn (see references in Ref. 2). More precisely, a good correlation
ported by the behavioral observations of Kirk and Denny-Brown2° describing in the m o n k e y an enlargement, within the first week post-lesion, of an isolated dorsal root dermatome after "intradural section of the three neighboring dorsal root above and below the dermatome area." These authors evaluated this enlargement by measuring the monkey's reactions to pinprick stimulation. They attributed these modifications of behavioral reactions to central disinhibitory mechanisms, since administration of subconvulsive doses of strychnine induced the same effect. Finally, our results together with the above data have to be considered in parallel with clini-
has been shown between the time-course of excitatory effects induced in dorsal horn neurons following formalin injection and behavioral manifestations9'1°'32.
cal investigations reporting sensory disturbances mainly
Finally, concerning the pathophysiological aspects of deafferentation-induced sensory disturbances, our results
hyperaesthesia, hyperalgesia and/or allodynia in partially deafferented zones of the body after dorsal rhizotomies
are in good agreement with experimental and clinical investigations. Electrophysiological studies performed in the dorsal horn of cats with partial deafferentation 3 or spared root lesion 33 indicated that the n u m b e r of neu-
(see references in Ref. 11) or avulsion of part of the brachial plexus dorsal roots in humans 37 (and see references in Ref. 43).
rons responding to noxious stimulation did not significantly change in the partially deafferented spinal segments. Concerning a possible suppression or weakening of segmental inhibitory controls, this hypothesis is sup-
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