0306-4522/92$5.00+ 0.00 Pergamon Press Ltd 0 1992IBRO
NeuroscienceVol. 48, No. 4, pp. 985-993, 1992 Printed in Great Britain
c-fos EXPRESSION IN RAT LUMBAR SPINAL CORD DURING THE DEVELOPMENT OF ADJUVANT-INDUCED ARTHRITIS C. ABBADIE*
and J.-M. BESSON
Uniti de Recherches de Physiopharmacologie du Systime Nerveux, iNSERM U.161 and EPHE, 2 rue d’Al&a, 75014 Paris, France Abstract-A parallel clinical and behavioral study of adjuvant-induced arthritis in the rat showed four stages in the time-course of the disease: preclinical (first week), acute (weeks 2-4), post-acute (weeks 5-8) and recovery weeks 9-11) [Calvino et nl. (1987) Behau. Brain Res. 24, 1l-291. As several studies have reported the expression of the proto-oncogene c-j& in spinal cord neurons following acute noxious peripheral stimuli, the aim of this study was to quantitatively assess Fos-like immunoreactivity in lumbar spinal cord neurons at various times of adjuvant-induced arthritis development, i.e. one, two, three, 11 and 22 weeks post-inoculation. The total number of Fos-like immunoreactive neurons in the lumbar enlargement correlated with the observed development of adjuvant-induced arthritis, i.e. Fos-like immunoreactivity was absent at one week, moderate at two weeks, greatly increased at three weeks, decreased at 11 weeks and returned to control values at 22 weeks. At three weeks, at the peak of Fos-like immunoreactivity distribution and acute stage of hyperalgesia, maximal labeling was observed in L3 and L4 spinal segments. In these segments, the most densely labeled region was the neck (laminae V and VI) of the dorsal horn (55%) and the ventral horn (35%) as compared to the superficial laminae (laminae I and II; 5%) and the nucleus proprius (laminae III and IV; 5%). These data indicate that c-fos expression induced by chronic inflammation is better expressed in deeper laminae than in the superficial ones, and that the number of Fos-positive cells correlates with behavioral studies. Thus, the use of Fos-like immunoreactivity in the chronic inflammatory pain model seems to be an interesting tool to study possible effects of various pharmacological compounds such as analgesic or anti-inflammatory drugs.
An experimental model to study “chronic” pain is adjuvant-induced arthritis (AIA) in the rat (see references in Ref. 5), which resembles human rheumatoid polyarthritis. It is induced by subcutaneous injection of Freund’s adjuvant (killed Mycobacterium butyricum suspended in mineral oil) into the base
of the rat tai1.35,36This disease. predominantly affects distal hindlimb joints (ankles). Among the observed changes, the most marked are decreases in locomotion and increases in scratching behavior.*s14 An initial inflammatory response develops within hours, but dramatic clinical signs appear from the 10th day post-inoculation and the changes last over several weeks.10,i1,14The model of AIA which induces numerous biochemical changes at both the periphery and the central nervous system has been used for this study of potential analgesic and antiinflammatory drugs (see references in Ref. 5). In addition, from the electrophysiological point of view, the disease has been shown to induce dramatic modifications in the activity of both superficial (I and II) and deeper (V and VI) laminae of the dorsal horn
*To whom correspondence should be addressed. AIA, adjuvant-induced arthritis; -LI, like immunoreactive/immunoreactivity; NRST normal rabbit serum in phosphate-buffered saline with T&on-X; PB, phosphate buffer; PBS, phosphate-buffered saline.
Abbrrviufions:
neurons receiving noxious inputs. These modifications involve (i) the appearance of high spontaneous firing rate, frequently associated with bursting patterns and sometimes sudden dramatic increases in the absence of any intentional stimulation, and (ii) an increase of neuronal responsivity to mechanical stimulation.g26 These data are reminiscent of clinical observations in human rheumatoid disease, where flashes of pain can appear spontaneously, or can be caused by simple touch at the inflamed area. The aim of this study was to quantitatively evaluate the Fos-like immunoreactivity (-LI) in lumbar spinal cord neurons during AIA development. The expression of the proto-oncogene c-fos in spinal cord neurons following various acute noxious peripheral stimuli has been demonstrated in numerous studies.6~‘s~22~27*38~43~44 More interestingly, during acute inflammation, induced, either by plantar injection of complete Freund’s adjuvant*’ or by carageenan,34 an increase in Fos-LI was reported in dorsal horn neurons of the spinal cord; some of these neurons co-localized with dynorphin,34 or are at the origin of ascending pathways. *’ These two latter experimental conditions strongly differ from rheumatoid arthritis induced by injection of Freund’s adjuvant into the tail, which develops over several weeks and which mainly involves distal hindlimb joints.
985
986
C.
ABBADIEand
A detailed parallel clinical and behavioral study of AIA in the rat showed four stages in the time-course of the disease: preclinical (first week), acute (weeks 2-4), post-acute (weeks 5-8) and recovery (weeks 9-l l).’ Thus, our study took into consideration these four stages. For this purpose, at various times of AIA, we made an analysis of the segmental distribution of Fos-LI neurons in five spinal segments (L2-L6), and in the laminar distribution over four regions. Preliminary reports have partly described these data.‘,2 EXPERIMENTAL PROCEDURES
Experimental animals
Experiments were performed on 42 male Sprague-Dawley rats (Charles River, France), weighing 200-225g at the beginning of the experiment. Polyarthritis was induced by intradennal injection of Fnund’s adjuvant into the base of the tail (killed Mycobacteriwn butyricum suspended in mineral oil);% the inoculation was made at the breeding center (Charles River, France). Control animals received vehicle alone. Guidelines on ethical standards for investigations of experimental pain in animals were followed.13 The number of experimental arthritic animals was kept to a minimum. They were housed three to a large cage, the floor of which was covered with sawdust to minimize the possibility of interactions between rats placed in close contact. They were kept in an animal room at a constant temperature of 22”C, with a 12-h alternating light-dark cycle. Food and water were available ad libitum; the food was directly available on the sawdust in the cages to minimize the need for the animals to make potentially painful movements to obtain food. Rats were k&d at various post-inoculation periods: one, two, three, 11 and 22 weeks after Freund’s adjuvant injection. Every experimental group included seven animals. Immunohistochemistry
Animals were deeply anesthetized with pentobarbital (55 mg/kg, i.p.) and perfused intracardially with 200 ml of 0.1 M phosphate-buffered saline (PBS) followed by 500 ml of 4% paraformaldehyde in 0.1 M phosphate buEer (PB). The lumbar spinal cord was then removed and postfued for 4 h in the same fixative and cryoprotected overnight in 30% sucrose in PB. Frontal frozen sections of 40 pm were cut and collected in PB. The serial sections were immunostained for c-&-like protein according to the avidin-biotin-peroxidase method of Hsu et aLz’ The tissue sections were incubated for 30 min at room temperature in a blocking solution of 3% normal rabbit serum in PBS with 0.3% T&on-X NNRST). The sections were then incubated overnight at 4°C in the phmary ant&rum directed against the c-10s protein (OA 11-823, Cambridge Research Biochemicals). The Fos antibody was used at 1: 2000. The incubated sections were washed three times in 1% NRST and incubated in biotinylated rabbit anti-sheep IgG for 1 h at room temperature, then washed twice in 1% NRST and incubated for 1 h in avidin-biotin-peroxidase complex (Vectastain, Vector Laboratories). FinalIy, according to Mauro et al.,25modified by Menktrey et al.,m the sections were washed three times in PBS and developed in I-naphtol ammonium carbonate solution (89.5 ml 0.1 M PB, 10 ml 10% I-naphtol in absolute alcohol and 0.1 ml hydrogen peroxide) for 15min, and were washed three times in PB to stop the staining reaction. The sections were mounted on gelatin-subbed slides and air-dried for the stain to be intensified and made alcoholresistant through basic dye enhancement in 0.025% crystal
J.-M. BESON violet solution in PB for 3 min. After two short PB I inses to take off the excess of 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 (OP-1 I-3210, CRB) or omission of any stage in the protocol abolished the staining Counting of Fos -labeled cells Fos-LX was studied through five lumbar spinal levels, L2-L6. Tissue sections were first examined using dark-field microscopy to determine the segmental level according to Molander et al.,3z and the gray matter landmarks. The sections were then examined under light-field microscopy to localize Fos-positive cells. Labeled nuclei were counted using a camera lucida attachment. For each rat, three measures were made: (i) the total number of Fos-LI neurons for the five lumbar spinal segments, (ii) the number of Fos-LI neurons per lumbar level and (iii) the number of Fos-LI neurons per region in the spinal gray matter. According to Presley et al., 38 four regions were defined: superficial dorsal horn (laminae I and II; superficial), nucleus proprius (laminae III and IV; nucleus proprius), neck of ihe horsal.hom (laminae V and VI; neck) and the ventral erav flaminae VII. VIII. IX and X: ventral). All results a;e kxpressed as the sum’of the number of F&-L1 neurons in three sections per one segmental level. Statistical analysis was made to compare the numbers of labeled cells, using one-way analysis of variance for the total number in the whole lumbar enlargement in the different groups of animals, two-way analysis of variance for the different groups of animals and the spinal level, and three-way analysis of variance for the different groups of animals, the spinal level, and the region. For multiple comparisons, the PLSD Fisher’s test was used. The investigator responsible for plotting and counting the Fos-LI neurons was unaware of the treatment of each animal. RESULTS
General observations
With our procedures, Fos-LI nuclei appeared as dark-violet round structures, often containing nonstained nucleoli. No labeling or only a few labeled cells (