Brain Research, 596 (1992) 259-268 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
259
BRES 18281
Autoradiographic analysis of 1251-substance P binding in rat spinal cord following chronic constriction injury of the sciatic nerve L.M. Aanonsen a,,, K.C. Kajander b,**, G.J. Bennett b and V.S. Seybold a "Department of Cell Biology and Neuroanatomy, Unicersity of Minnesota, Minneapolis, MN (USA) and I, Neurobiology and Anesthesiology Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, MD (USA) (Accepted 23 June 1992)
Key words: Chronic constriction injury; Pain; Hyperalgesia; Receptor binding; Autoradiography; Substance P; Spinal cord; NK I receptor
Using receptor binding and autoradiographic techniques, changes in Bolton-Hunter labeled t251-substance P (t251-BH-SP) binding were determined in laminae I/II, V and X of rat lumbar spinal cord after chronic constriction injury (CCI) of the sciatic nerve. When compared to the sham-operated side of the control group, SP binding significantly increased ipsilatera! to the CCI in laminae I / ! I at 5, 10 and 20 days after injury and in lamina V at 5 days after injury. Scatchard analysis was performed on the 1251-BH-SP binding to the NK t receptor in laminae I / ! I of rats 5 days after generation of the CCI. A significant decrease in the K a of t251-BH-SP binding was obser,led in laminae I / i l ipsilateral to CCI when compared with the control side (ipsilateral to sham surgery). There was no significant change in the Bmax in laminae I / I I ipsilateral to CCI. The changes in 1251-BH-SP binding in the rat spinal cord that occurred after CC! were found in areas of the spinal cord that receive terminations of nociceptive primary afferent fibers. The increased affinity of the NK~ binding site that we report could result in an increase in SP receptor activation in laminae !/II. Such central changes in SP binding may contribute to the neuropathic pain syndrome observed in rats with the CCI.
INTRODUCTION
The undecapeptide, substance P (SP) is a member of a family of related peptides, the tachykinins, and is considered to be an excitatory transmitter released by primary afferent neurons terminating in the dorsal horn of the spinal cord 47. Numerous line,,; of evidence support the hypothesis that SP is a primary afferent transmitter of nociceptive information. SP-immunoreactive neurons in the dorsal root ganglia have been shown to give rise to AS- and C-fibers35, fibers associated with the transmission of nociceptive information to the spinal cord 31'53. Furthermore, the anatomical distribution of substance P-like immunoreactivity (SPJr)3'24 in the spinal cord parallels the distribution of the dorsal horn terminations of unmyelinated C-fibers and thinly myelinated A/S-fibers. In addition, activation of these fibers results in increased release of SP in the spinal cord 61. Other studies have demonstrated that intrathecal SP alters nociceptive thresholds. For exam-
pie, a decreased tail-flick latency is induced by intrathecally administered S P 16'63, while intrathecal SP antagonists or SP antibodies produce analgesia in the hot-plate and tail-pinch tests 45. In eleetrophysiological studies, iontophoretic application of SP has been shown to excite spinal nociceptive neurons and to facilitate the activation of these tteurons by noxious peripheral stimuli23.2s.49. There is convincing evidence that SP content is increased in the spinal ¢Jorsal horn and its release is increased in response to noxious stimuli. An increase in extraceilular ~;P-ir in the mammalian dorsal horn has been measured in response to noxious mechanical stimuli 29 and noxious cold stimuli 57 using push-pull cannulae. In the case of noxious heating of the skin, an increase in SP-ir is found in the substantia gelatinosa ~7. Formalin injected into the rat hind paw results in an increase in SP-ir in the dorsal h o r n 27'34 and an increase in the expression of the preprotachykinin-A (PPT-A) gene in dorsal root ganglia42. In addition, the early
Correspondence: L. Aanonsen, Biology Department, Macalester College, 1600 Grand Ave., St. Paul, MN 55105, USA. * Present address: Biology Department, Macalester College, 1600 Grand Ave., St. Paul, MN 55105, USA. * * Present address: Department of Oral Science, School of Dentistry, University of Minnesota, Minneapolis, MN 55455, USA.
260 phase of the formalin-induced behavioral response in mice correlates with the time-course for the increase in dorsal horn levels of SPZS; the early behavioral response can be blocked with SP antagonists or antibodies to SP 45. Additional evidence suggests that conditions of chronic pain also engage SP systems. Chronic inflammation, induced by the systemic injection of Freund's adjuvant, results in an increased content of SP-ir in the dorsal spinal cord 5~ and dorsal root ganglia 4a of rats, and an increased release of SP in the dorsal horn of the spinal cord due to movement of the inflamed joint 44,5°. An increased expression of the PPT-A gene has also been demonstrated in the rat spinal cord and dorsal root ganglia after injection of Freund's adjuvant in the hind paw 37. Thus, acute noxious stimuli and the chronic noxious stimulation produced in models of inflammation all appear to result in an increased synthesis, content and release of SP in primary afferent neurons and the spinal dorsal horn. This increased release of SP may contribute to the hyperalgesia seen with inflammation. Chronic pain has been induced by a chronic constriction injury (CCI) of the sciatic nerve of the rat 7. In this case, a neuropathy is induced by placing loose ligatures around the sciatic nerve. This procedure resuits in symptoms that closely resemble the disorders of pain sensation observed in humans with peripheral nerve damage 2'~. Chronic constriction injury in rats produces (1) hyperalgesic responses to noxious thermal, mechanical and chemical stimuli, (2) allodynia and (3) perhaps, dysesthesia or spontaneous pain. It has been shown that the CCI produces a partial and differential deafferentation cf the nerve's peripheral territory. Almost all of the large myelinated axons are interrupted, as are a ~ r y large mai~rity of the small myelinated axons. However, a variable but large percentage of the unmyelinated axons are intact sa2'a°'ag. Thus, the distal nerve is characterized by marked degeneration, whereas the nerve proximal to the injured region appears normal (with possible minor exceptions), and there is no evidence to suggest that any of the nerve's primary afferent neurons die. Consequently, one would expect the terminal arbors of primary afferent neurons to be structurally intact in the spinal cord. The neurochemical pathology of the spinal cord in the CCI model primarily involves the projection of the sciatic nerve, which has been ~hown to terminate largely in the L4 and 1.5 spinal segments 56. In rats with the CCI, SP-ir is significantly reduced ipsilateral to the nerve injury at 10 and 20 days postsurgery TM. In the present study, receptor binding and autoradiographic techniques were used to assess the effects of CCI on
SP binding sites in the spinal cord at various times after the nerve injury. Increases in SP binding could contribute to the neuropathic pain syndrome observed in rats with CCI. MATERIALS and METHODS 125I-Bolton-Hunter substance P binding in rat spinal cord Atdmals. Male, Sprague-Dawley rats (200-250 g) were used in competition studies to characterize 12SI-Bolton-Hunter substance P (1251-BH-SP) binding to NK l receptors. Each animal was anesthetized with Equithesin 18 (0.3 ml/100 g body weight) and then transcardially perfused with 200 ml of 0.32 M sucrose mixed 1 : 1 with phosphate-buffered saline. Following perfusion, the spinal cord was rapidly removed and minced in a plastic embedding mold in order to obtain a homogeneous sampling of spinal cord tissue. The tissue was frozen on dry ice, sectioned (10 /~m) using a cryostat, and the sections were thaw-mounted on to gelatin-coated slides. Ligand binding protocol. Biochemical and kinetic parameters for 1251-BH-SP binding in rat spinal cord have been reported previously in spinal cord homogenates 13 and in slide-mounted tissue sections 14. The same binding conditions were used in the present study. Briefly, tissue sections were preincubated for 10 rain (room temperature) in 50 mM Tris buffer (pH 7.4) containing 0.02% bovine serum albumin, bacitracin (40 ~g/ml), ethylenediaminetetra-acetate (EDTA; 2.5 mM), leupeptin (4 /~g/ml) and chymostatin (2 ~g/ml). This was followed by two 10-rain washes in the buffer referred to above except that MnC! (3 raM) was substituted for EDTA. The slides were transferred to containers with the same buffer containing 50 pM 12SI-BH-SP ( ~ 2,000 Ci/mmol) or to containers with I~I-BH-SP plus 1 ,¢M unlabeled SP for determination of non-specific binding. The tissue was incubated for 90 rain at room temperature followed by two, 10 rain washes (4°C) in 50 mM Tris buffer (pH 7.4) containing 0.02% BSA. Competition studies. Using the binding protocol described above, competition studies were performed on slide-mounted sections of minced spinal cord tissue from normal, untreated rats in order to confirm the binding specificity of t2Sl-BH-SP. Six different concentrations of each of the competitors were incubated with 50 pM 12"~I-BH-SP. Substance P (0.001-10 nM), neurokinin A (10-1,000 nM) and neurokinin B (0.083-830 nM) were used as the competitors because they have been previously reported to be the most potent ligands fi)r NK t, NK 2 and NKa binding sites, respectively~'4a. Nonspecific binding for the competition studies was determined in the presence of I/~M SP. Slides were incubated in triplicate for determination of total and non-specific binding. The competition studies were repeated three times, each replication with tissue from a different animal. After the final washes, the spinal cord tissue was wiped off of the slides with glass microfiber filters (Whatman G F / A ) and placed in a test tube. Radioactivity was measured by a gamma counter. The 50% inhibitory concentrations (ICs0) were determined from EBDA (Biosoft, Miltown, NJ). The data for SP competition were also analyzed by Scatchard analysis using non-linear regression analysis of the data from LIGAND 36. Chronic constriction injury model Animals. Male, Sprague-Dawley rats (250-300 g) were used in these experiments. This study was performed following the IASP Ethical Guidelines 64. Chronic constriction injury. A unilateral mononeuropathy was produced according to the method described previously7. Briefly, Tats were anesthetized with sodium pentobarbital (40 mg/kg, i.p.) and the common sciatic nerve on the left side of the animal was exposed and consecutive, loosely constrictive ligatures (4) were tied around the nerve. Sham-surgery was performed on the right side of each of these rats. The sham surgery consisted of an identical dissection except the sciatic nerve was not iigated. The animals (7-10 rats/group) were allowed to survive for 2, 5, 10 and 20 days after surgery. The sham-surgery side of the spinal cord served as a control
261 for comparisons of 125I-BH-SP binding between sides of the spinal cord, however, a second control was also included. This second control group included rats that were sham-operated on the left side only (no surgery on the right side) and allowed to survive for 10 (4 rats) and 20 (6 rats) days. These animals were pooled for statistical comparisons since there were no significant differences in ~251-BH-SP binding in any laminae between these two groups. Behavioral assay. Following nerve injury, the animals limped and held the hind paw of the nerve-injured side in a guarded position. Previous studies on the CCI model have shown that the animals also develop significant hyperalgesia in thermal, mechanical and chemical nociceptive tests 2,7. In the present study, each of the animals in the 5, 10 and 20 day survival groups were tested for hyperalgesia using the thermal stimulus paradigm of Hargreaves and coworkers 22. This procedure measures the withdrawal latency from a radiant heat source directed at the proximal half of the plantar surface of each hind paw. Animals were not tested for hyperalgesic responses at 2 days after CCI because it has been reported that exposure to ~oxious heat at this early time evokes edema in the hind paw 7. We wished to avoid the possibility that this peripheral effect of SP h ~ a central counterpart that might influence the binding of 12SI-BH-SP. The behavioral assay for hyperalgesia was conducted just prior to sacrifice of the animals. Four latency measurements for each hind paw were averaged, and a difference score was determined by subtracting the average latency of the control paw from that of the paw ipsilateral to the CCI. Negative scores thus indicate a decrease in threshold on the side ipsilateral to CCI. Statistical analyses were made among groups using one-way ANOVA with Dunnett's post-hoc test. This post-hoc test was chosen since difference scores of the experimental groups were compared only to the difference scores of the control group. The level of significance was set at P < 0.05. Results of the behavioral tests on the animals used in this study were included in a previous report 19. Tissue preparation. At the end of the survival periods and after the be'aavioral tests were performed, the rats were anesthetized with sodium pentobarbital (as described above) and perfused through the aorta with approximately 200 ml of ice-cold phosphate buffered saline mixed 1 : 1 with 0.32 M sucrose. A laminectomy was performed while the animal was embedded in ice, and the L4 segment was identified and removed. The spinal cord segment was then placed in a plastic tissue-embedding mold and frozen on powdered dry ice. The tissue blocks were stored for no more than 2 weeks at -70°C prior to sectioning, The blocks were then mounted on microtome chucks, sectioned (10/zm) on a cryostat and thaw-mounted on to gelatin-coated slides. The tissue was stored at -20°C until receptor binding studies were performed. Receptor binding and autoradiography. For 125I-BH-SP binding on the CCI tissue, slide-mounted tissue sections were processed following the protocol outhned above. The slides were incubated in 50 pM 1251-BH-SP for determination of total binding or in 50 pM 1251-BH-SP plus 1/zM unlabeled SP for determination of non-specific binding. After the final rinses in buffer, the slides were dipped in distilled water (4°C) to remove salts and dried, first under a stream of cool, desiccated air and finally on a slide warmer (30 min at ~ 37°C). The slides were stored desiccated, overnight at 4°C. Autoradiograms were prepared using the technique described by Young and Kuhar 62. Emulsion-coated coverslips (NTB-3 nuclear emulsion, Kodak, Rochester, NY) were apposed to the slide-mounted tissue by applying a drop of Super Glue (Locktite) to the frosted end of the slide. Coverslips were kept in close apposition throughout the exposure period by clipping the slides between Plexiglass sheets. Autoradiograms of 125I microscaies (40 /zCi multilevel reference strips cut to 10/zm; Amersham, Arlingt~a Heights, IL) were prepared in parallel. The slides were stored desiccated in light-tight boxes for exposure periods of 6 and 9 days, and then the emulsion was developed (Kodak Developer D-~9) and fixed (Rapid Fix, Kodak). Following development of the autoradiograms, the tissue was fixed in Carnoy's fixative, stained wi:,,h thionin, dehydrated, and the coverslips were permanently affixed with mounting medium (Entellan, EM Science, Cherry Hill, N J). Scatchard analysis by autoradiography. Scatchard analysis was carried out on autoradiographic data obtained from spinal cords of experi-
Lam 1/11
LamV
D
regionof analysis (SO,O00sq. pm)
1 mm
Fig. 1. Determination of coordinates for autoradiographic analysis. Lain l / I I : at one-half the length of a line from the medial curve of the superficial dorsal horn to the dorsal root entry zone (*). This coordinate is consistent with the termination of small diameter primary afferent neurons of the sciatic nerve 51. Lam V: at threequarters the length of the line from the ventral aspect of the dorsal columns to the lateral curve of the superficial dorsal horn. This coordinate is consistent with the reticulated area in lateral lamina V. Lain X: at the intersection of 2 lines: 1 line extending ventrally from the medial curve of the dorsal horn; 1 line extending laterally from the midpoint between the central canal and the ventral aspect of the dorsal columns.
mental animals (5 day survival group). Spinal cord sections were incubated in 50 pM 125I-BH-SP with 5 different cor~centrations of unlabeled SP (0.01-1.0 nM). After completion of the binding protocol, the slides were dried and emulsion-coated coverslips were apposed (see above) for a period of 9 days. This tissue ;:~d emulsioncoated coverslips were processed according to the method described above. The densities of autoradiographic grains overlying medial laminae I / I I ~.ere determined on each side of the spinal cord. Control values reflect data ipsilateral to the sham surgery; experimental values reflect data ipsilateral to the CCI. Scatchard analysis was performed with non-linear regression analysis of the data using the McPherson version of LIGAND 36. Ligand binding data used for statistical comparison (n = 3) were selected based on their low variability as determined in Scatchard analysis (correlation coefficient > 0.85).
Computerized image analysis Bilateral measurements of grain densities were taken at 100 x in laminae i / I I (medial), V and X. The medial region of laminae I / l l was chosen for analysis since it has been shown to receive a rich projection of fibers from the sciati~ nerve s'3s'56, particularly nociceptive fibers 31'53'54. Laminae V and X were chosen for analysis since these areas receive terminations of C-fibers 53'54 and thinly myelinated, high-threshold mechanoreceptors 31. Using electrophysiological studies in the rat, laminae I/II, V and X have also been shown to be associated with nociceptive processing 21'4°'6° and contain high densities of spinothalamic tract neurons (see review)TM. Furthermore, all of these regions have been shown to contain moderate to high levels of SP-like immunoreactivity3'24 and SP binding sites 14. The coordinates for the regions of densitometric analysis were determined according to the criteria outlined in Fig. 1. Quantitative measurements of grain densities in these areas were determined using a digital image processing and analysis program for the Macintosh II, Image 1.13b (Dr. W. Rasband, NIH). A video camera attached to the microscope delivered a live image to the digitizer
262 attached to the computer. A threshold function was then used to sum the number of pixels in the images of the grains. Grains densities were determined in the selected areas of the spinal cord in control rats (n = 10) and rats who survived for 2 (n = 7), 5 (n - 8), 10 ( n - - 8 ) and 20 (n = 7) days after CCI. Non-specific binding was found to be < 5% of the total grain density in the areas analyzed and, thus, was not subtracted from total binding values in the analysis of the data. The area of the measurements was approximately 50,000 ~m z. The apposition of the autorad~ograms to the stained tissue sections allowed accurate quantification of the grain densities with respect to laminae identified using a rat stereotaxie atlas 46. Data were converted to fmol/mg tissue by linear transforma-
r'l 5ham (oxpenmental glaup)
T
Laminae I II
•
40e°
GCI (exper,mentaJ g,aup)
O Sham (conlzol gloup) O UntleatlKI (conltolgroup)
e,
36,
3.2
2.8
I
2.3
.!
m ~t C/)
l
Lemtna V
i
•
ID,
2.!
tion of the autoradiographic grain densities according to the standard curve generated from the 125I-microscale standards. For clarity, 'density of silvel" grains' will be referred to as 'tZSl-BH-SP binding' throughout the report.
Statistical analysis The autoradiographic data were analyzed using a three-way (time vs. side of spinal cord vs. laminae) analysis of variance with Duncan's multiple comparison test. The level of significance was set at P < 0.05. There were no significant differences between the two control groups (unilateral sham-surgery) sacrificed at 10 and 20 days postsurgery, so these data were merged for the analysis. There were no side-to-side differences in the merged control group (or in either separate group); nevertheless, we used only the data from the sham-operated side for comparisons with the CCI animals. Post-hoe pair-wise tests included within-group comparisons of the nerve-injured vs. sham-operated sides of the CCI groups. Three sets of comparisons were made at each survival time: (1) the injured side of the experimental group was compared with the sham-operated side of the control grovp, (2) the sham-operated side of the control group was compared with the sham-operated side of experimental group and (3) the injured side of the experimental group was compared to the shamoperated side of the same group. Each of these comparisons were made in each of the laminae where measurements were taken. Note that the within-group comparisons of the nerve-injured vs. sham-operated sides of the CC! animals are not necessarily comparisons of treatment effect vs. a control (the sham-operated side) because of the possibility of a nerve injury-evoked contralateral change. Correlations between changes in 1251-BH-SP binding and hyperalgesia were tested using linear regression analysis.
Materials
t
The following substances were obtained from Sigma Chemical Co. (St. Louis, MO): Tris buffer (Trizma, pH 7.4), bovine serum albumin, bacitracin, EDTA, leupeptin and chymostatin. Substance P, neurokinin A and neurokinin B were obtained from Peninsula (Belmont, CA). t2Sl-Bolton-Hunter labeled substance P ( ~ 2,000 Ci/mmol) was obtained from Amersham (Arlington Heights, IL).
1.5
RESULTS I
i,
I
i
.f:
'
Lamln,, X
Specificity of 12Sl.BH-SPbinding
3,0
3.6
I T
':.4
3.2
3,0 ,
i
t
2
S
, 10
..i 2O
¢.1
I C
Time (days after Iigatlon)
Fig. 2. SP binding in laminae I/!I, V and X in CCI and control rats. SP binding in three regions of the spinal cord in control (n = 10; open circle: sham-operated side; hatched circle: untreated) and after CCI (boxes) at 2 (n = 7), 5 (n --- 8), 10 (n = 8) and 20 (n -- 7) days. The open boxes represent values ipsilateral to sham surgery and the filled boxes represent values ipsilateral to CC1. * significant difference in binding ipsilateral to CCI when compared to the sham-operated side of the control group (P < 0.05, three-way ANOVA with Duncan's post-hoc test); t significant difference in binding between the left (ipsilateral t~ CCI) and right (ipsilateral to sham surgery) sides of the spinal cord (P < 0.05, three-way ANOVA with Duncan's post-hoc test).
The specificity of 1251-BH-SP binding in rat spinal cord was tested in competition studies. These studies revealed that the rank order of potency of related tachykinins for competition of :25I-BH-SP binding was substance P >> neurokinin A > neurokinin B. SP, the most potent competitor, had an ICs0 of 121.2 + 32.6 pM. Neurokinin A was nearly 250 times less potent than SP (ICs0 - 28.1 5:5.9 nM), and neurokinin B was 1,700 X less potent than SP (ICs0 -- 212 5:86•3 nM). Scatchard analysis of the competition data for SP revealed that the Kd of :251-BH-SP binding in rat spinal cord was 397.4 5:32.9 pM.
SP binding in sham-operated controls Sham surgery was performed on only one side of the rat in the control group so that the effects of the surgery alone on ;251-BH-SP binding could be tested• The distribution of ;251-BH-SP binding sites in the rat spinal cord segment L4 was similar to the distribution reported previously :4,33. The greatest :25I-BH-SP bind-
203 ing occurred in laminae I and in the area around the central canal. Moderate levels of ~251-BH-SP binding were observed in laminae III and V and in discrete regions of the ventral horn. Statistical analysis revealed that there were no significant differences ~.n laminae I/II, V and X between the sham-operated side and the unoperated side of the spinal cord when animals were sacrificed 10 or 20 days after surgery.,The data were pooled for 10 and 20 days; these control values appear on the right side of each graph in Fig. 2.
Changes in SP binding in laminae I/I1, V and X following CC! In most cases the within- and b-_.tween-group comparisons were consistent with a simple, unilateral change on the side of the nerve injury. A significant increase in ~251-BH-SP binding ipsilateral to CCI was observed in laminae I / I I at 5, 10 and 20 days after sciatic ligation when these values were compared to the sham-operated side of the control group, and at 2, 5, 10 and 20 days when compared to the contralateral side of the spinal cord (sham-operated) (Fig. 2, top graph). The percent increases in ~251-BH-SP binding over the sham-operated side of the control group at days 5, 10 and 20 were 15.9%, 26.5% and 13.2%, respectively. Fig. 3 is a darkfield photomicrograph of the total (A) and non-specific (B) 125I-BH-SP binding in spinal segment L 4 at 5 days postsurgery. A greater density of silver grains in laminae I/II, especially in the medial aspect of these laminae, was observed on the left side of the spinal cord (ipsilateral to the sciatic
nerve injury) in cemparison with the right side (ipsilateral to sham sl~rgery). The only significant change in ~251-BH-SP binding in lamina V was observed at day 5 after the CCI (Fig. 2, middle graph). This value was found to be significantly different when compared to the sham-operated side of the control group and when compared to the contralateral side (sham-operated). This change represented a 13.6% increase in binding from the sham-operated side of the control group. ~251-BH-SP binding in lamina V returned to the control value by 20 days after sciatic nerve injury. A moderate increase in binding can be seen in the left (ipsilateral to the sciatic nerve injury), deep dorsal horn (region of lamina V) of the darkfield photomicrograph when compared to the right side (ipsilaterai to sham surgery) (Fig. 3A). In lamina X, a significant decrease in 1251-BH-SP binding was observed at 2 days after CCI when compared to the contralateral side of the spinal cord (sham-operated) (Fig. 2, lower graph). No other significant changes in ~251-BH-SP binding occurred in lamina X. It is interesting to note, however, that the pattern of increased binding at day 10 and decreased binding at day 20 was observed both ipsilateral and contralateral to CCI.
Scatchard analysis by autoradiography The dissociation c o n s t a n t ( K d) and Bmax values for ~251-BH-SP binding in laminae I/II ipsilateral to sham surgery (control) and ipsilateral to the CCI (experimental) at 5 days postsurgery were obtained by Scatchard
Fig. 3. Photomicrograph of autoradiograms of 1251-BH-SP binding 5 days after CCI. Darkfield photomicrograph of adjacent sections of L 4 spinal cord incubated under conditions for t25I-BH-SP binding (A) and nonspecific binding (B). The left side of the spinal cord is ipsilateral to the CCI. This section is from a rat sacrificed at 5 days after CCI. Bar -- 400/tm.
264 TABLE I
0.5
Dissociation constant (K d) and Bmax values for 1251-BH-SPin laminae l / ll in control and experimental sides of the spinal cord
g, e-
Grain densities for Scatchard analysis were quantified in laminae I / l l on control and experimental sides of the spinal cord. Scatchard analyses were performed using non-linear regression analysis of the data (McPherson version of LIGAND). The spinal cords used for statistical analysis (n = 3) were selected based on their lack of variability as determined in Scatchard analysis (correlation coefficient > 0.85). Values represent the m e a n + S . E . M . (n = 3). * Student's unpaired t-test, P < 0.05.
-0.5
-1.5
-2.5
Control Experimental (CCI)
Kd
Bmax
(pM)
(fmol / mg tissue)
=_"
122.7 + 20.1
23.4 + 6.14
C3
-3.5 45.3 + 3.4 *
26.4 + 2.8
analysis of autoradiographic data (Table I). A significant decrease in the K,~ was observed in laminae I/II ipsilateral to CCI when compared with the control side (ipsilateral to sham surgery). There was no significant change in the Bmax in laminae I/II ipsilateral to the CCI. Fig. 4 represents data from one animal and illustrates Scatchard plots of SP binding in laminae I/II ipsilateral to the CCI (experimental) and ipsilateral to sham surgery (control). Withdrawal latencies and their correlation with changes in 1251-BH.SP binding The latency of the nocifensive withdrawal response was reduced ipsilateral to the nerve injury compared to the contralaterai, sham-operated side (Fig. 5), and, as noted previously 2, the lowered thresholds were accompanied by responses of greater magnitude and dura-
D Control Kd , 141 pM B max = 28.6 fmol/mg
0.8'
• Experimental (CCI) Kd = 39 pM Bmax ==23.7 fmol/mg
0.6'i o m
0.4
I
0.2'
sham
5
10
20
Days after sciatic Iigation Fig. 5. Behavioral test. Animals were tested for their response to a radiant heat stimulus on the day of sacrifice (see text for details). The latency score was computed by subtracting the average latency of the paw ipsilateral to sham surgery from the average latency of the paw ipsilateral to the CCI. Negative scores indicate a shorter latency ipsilateral to the CCI; i.e., hyperalgesia. * significant difference when compared to the sham-operated group ( P
259
BRES 18281
Autoradiographic analysis of 1251-substance P binding in rat spinal cord following chronic constriction injury of the sciatic nerve L.M. Aanonsen a,,, K.C. Kajander b,**, G.J. Bennett b and V.S. Seybold a "Department of Cell Biology and Neuroanatomy, Unicersity of Minnesota, Minneapolis, MN (USA) and I, Neurobiology and Anesthesiology Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, MD (USA) (Accepted 23 June 1992)
Key words: Chronic constriction injury; Pain; Hyperalgesia; Receptor binding; Autoradiography; Substance P; Spinal cord; NK I receptor
Using receptor binding and autoradiographic techniques, changes in Bolton-Hunter labeled t251-substance P (t251-BH-SP) binding were determined in laminae I/II, V and X of rat lumbar spinal cord after chronic constriction injury (CCI) of the sciatic nerve. When compared to the sham-operated side of the control group, SP binding significantly increased ipsilatera! to the CCI in laminae I / ! I at 5, 10 and 20 days after injury and in lamina V at 5 days after injury. Scatchard analysis was performed on the 1251-BH-SP binding to the NK t receptor in laminae I / ! I of rats 5 days after generation of the CCI. A significant decrease in the K a of t251-BH-SP binding was obser,led in laminae I / i l ipsilateral to CCI when compared with the control side (ipsilateral to sham surgery). There was no significant change in the Bmax in laminae I / I I ipsilateral to CCI. The changes in 1251-BH-SP binding in the rat spinal cord that occurred after CC! were found in areas of the spinal cord that receive terminations of nociceptive primary afferent fibers. The increased affinity of the NK~ binding site that we report could result in an increase in SP receptor activation in laminae !/II. Such central changes in SP binding may contribute to the neuropathic pain syndrome observed in rats with the CCI.
INTRODUCTION
The undecapeptide, substance P (SP) is a member of a family of related peptides, the tachykinins, and is considered to be an excitatory transmitter released by primary afferent neurons terminating in the dorsal horn of the spinal cord 47. Numerous line,,; of evidence support the hypothesis that SP is a primary afferent transmitter of nociceptive information. SP-immunoreactive neurons in the dorsal root ganglia have been shown to give rise to AS- and C-fibers35, fibers associated with the transmission of nociceptive information to the spinal cord 31'53. Furthermore, the anatomical distribution of substance P-like immunoreactivity (SPJr)3'24 in the spinal cord parallels the distribution of the dorsal horn terminations of unmyelinated C-fibers and thinly myelinated A/S-fibers. In addition, activation of these fibers results in increased release of SP in the spinal cord 61. Other studies have demonstrated that intrathecal SP alters nociceptive thresholds. For exam-
pie, a decreased tail-flick latency is induced by intrathecally administered S P 16'63, while intrathecal SP antagonists or SP antibodies produce analgesia in the hot-plate and tail-pinch tests 45. In eleetrophysiological studies, iontophoretic application of SP has been shown to excite spinal nociceptive neurons and to facilitate the activation of these tteurons by noxious peripheral stimuli23.2s.49. There is convincing evidence that SP content is increased in the spinal ¢Jorsal horn and its release is increased in response to noxious stimuli. An increase in extraceilular ~;P-ir in the mammalian dorsal horn has been measured in response to noxious mechanical stimuli 29 and noxious cold stimuli 57 using push-pull cannulae. In the case of noxious heating of the skin, an increase in SP-ir is found in the substantia gelatinosa ~7. Formalin injected into the rat hind paw results in an increase in SP-ir in the dorsal h o r n 27'34 and an increase in the expression of the preprotachykinin-A (PPT-A) gene in dorsal root ganglia42. In addition, the early
Correspondence: L. Aanonsen, Biology Department, Macalester College, 1600 Grand Ave., St. Paul, MN 55105, USA. * Present address: Biology Department, Macalester College, 1600 Grand Ave., St. Paul, MN 55105, USA. * * Present address: Department of Oral Science, School of Dentistry, University of Minnesota, Minneapolis, MN 55455, USA.
260 phase of the formalin-induced behavioral response in mice correlates with the time-course for the increase in dorsal horn levels of SPZS; the early behavioral response can be blocked with SP antagonists or antibodies to SP 45. Additional evidence suggests that conditions of chronic pain also engage SP systems. Chronic inflammation, induced by the systemic injection of Freund's adjuvant, results in an increased content of SP-ir in the dorsal spinal cord 5~ and dorsal root ganglia 4a of rats, and an increased release of SP in the dorsal horn of the spinal cord due to movement of the inflamed joint 44,5°. An increased expression of the PPT-A gene has also been demonstrated in the rat spinal cord and dorsal root ganglia after injection of Freund's adjuvant in the hind paw 37. Thus, acute noxious stimuli and the chronic noxious stimulation produced in models of inflammation all appear to result in an increased synthesis, content and release of SP in primary afferent neurons and the spinal dorsal horn. This increased release of SP may contribute to the hyperalgesia seen with inflammation. Chronic pain has been induced by a chronic constriction injury (CCI) of the sciatic nerve of the rat 7. In this case, a neuropathy is induced by placing loose ligatures around the sciatic nerve. This procedure resuits in symptoms that closely resemble the disorders of pain sensation observed in humans with peripheral nerve damage 2'~. Chronic constriction injury in rats produces (1) hyperalgesic responses to noxious thermal, mechanical and chemical stimuli, (2) allodynia and (3) perhaps, dysesthesia or spontaneous pain. It has been shown that the CCI produces a partial and differential deafferentation cf the nerve's peripheral territory. Almost all of the large myelinated axons are interrupted, as are a ~ r y large mai~rity of the small myelinated axons. However, a variable but large percentage of the unmyelinated axons are intact sa2'a°'ag. Thus, the distal nerve is characterized by marked degeneration, whereas the nerve proximal to the injured region appears normal (with possible minor exceptions), and there is no evidence to suggest that any of the nerve's primary afferent neurons die. Consequently, one would expect the terminal arbors of primary afferent neurons to be structurally intact in the spinal cord. The neurochemical pathology of the spinal cord in the CCI model primarily involves the projection of the sciatic nerve, which has been ~hown to terminate largely in the L4 and 1.5 spinal segments 56. In rats with the CCI, SP-ir is significantly reduced ipsilateral to the nerve injury at 10 and 20 days postsurgery TM. In the present study, receptor binding and autoradiographic techniques were used to assess the effects of CCI on
SP binding sites in the spinal cord at various times after the nerve injury. Increases in SP binding could contribute to the neuropathic pain syndrome observed in rats with CCI. MATERIALS and METHODS 125I-Bolton-Hunter substance P binding in rat spinal cord Atdmals. Male, Sprague-Dawley rats (200-250 g) were used in competition studies to characterize 12SI-Bolton-Hunter substance P (1251-BH-SP) binding to NK l receptors. Each animal was anesthetized with Equithesin 18 (0.3 ml/100 g body weight) and then transcardially perfused with 200 ml of 0.32 M sucrose mixed 1 : 1 with phosphate-buffered saline. Following perfusion, the spinal cord was rapidly removed and minced in a plastic embedding mold in order to obtain a homogeneous sampling of spinal cord tissue. The tissue was frozen on dry ice, sectioned (10 /~m) using a cryostat, and the sections were thaw-mounted on to gelatin-coated slides. Ligand binding protocol. Biochemical and kinetic parameters for 1251-BH-SP binding in rat spinal cord have been reported previously in spinal cord homogenates 13 and in slide-mounted tissue sections 14. The same binding conditions were used in the present study. Briefly, tissue sections were preincubated for 10 rain (room temperature) in 50 mM Tris buffer (pH 7.4) containing 0.02% bovine serum albumin, bacitracin (40 ~g/ml), ethylenediaminetetra-acetate (EDTA; 2.5 mM), leupeptin (4 /~g/ml) and chymostatin (2 ~g/ml). This was followed by two 10-rain washes in the buffer referred to above except that MnC! (3 raM) was substituted for EDTA. The slides were transferred to containers with the same buffer containing 50 pM 12SI-BH-SP ( ~ 2,000 Ci/mmol) or to containers with I~I-BH-SP plus 1 ,¢M unlabeled SP for determination of non-specific binding. The tissue was incubated for 90 rain at room temperature followed by two, 10 rain washes (4°C) in 50 mM Tris buffer (pH 7.4) containing 0.02% BSA. Competition studies. Using the binding protocol described above, competition studies were performed on slide-mounted sections of minced spinal cord tissue from normal, untreated rats in order to confirm the binding specificity of t2Sl-BH-SP. Six different concentrations of each of the competitors were incubated with 50 pM 12"~I-BH-SP. Substance P (0.001-10 nM), neurokinin A (10-1,000 nM) and neurokinin B (0.083-830 nM) were used as the competitors because they have been previously reported to be the most potent ligands fi)r NK t, NK 2 and NKa binding sites, respectively~'4a. Nonspecific binding for the competition studies was determined in the presence of I/~M SP. Slides were incubated in triplicate for determination of total and non-specific binding. The competition studies were repeated three times, each replication with tissue from a different animal. After the final washes, the spinal cord tissue was wiped off of the slides with glass microfiber filters (Whatman G F / A ) and placed in a test tube. Radioactivity was measured by a gamma counter. The 50% inhibitory concentrations (ICs0) were determined from EBDA (Biosoft, Miltown, NJ). The data for SP competition were also analyzed by Scatchard analysis using non-linear regression analysis of the data from LIGAND 36. Chronic constriction injury model Animals. Male, Sprague-Dawley rats (250-300 g) were used in these experiments. This study was performed following the IASP Ethical Guidelines 64. Chronic constriction injury. A unilateral mononeuropathy was produced according to the method described previously7. Briefly, Tats were anesthetized with sodium pentobarbital (40 mg/kg, i.p.) and the common sciatic nerve on the left side of the animal was exposed and consecutive, loosely constrictive ligatures (4) were tied around the nerve. Sham-surgery was performed on the right side of each of these rats. The sham surgery consisted of an identical dissection except the sciatic nerve was not iigated. The animals (7-10 rats/group) were allowed to survive for 2, 5, 10 and 20 days after surgery. The sham-surgery side of the spinal cord served as a control
261 for comparisons of 125I-BH-SP binding between sides of the spinal cord, however, a second control was also included. This second control group included rats that were sham-operated on the left side only (no surgery on the right side) and allowed to survive for 10 (4 rats) and 20 (6 rats) days. These animals were pooled for statistical comparisons since there were no significant differences in ~251-BH-SP binding in any laminae between these two groups. Behavioral assay. Following nerve injury, the animals limped and held the hind paw of the nerve-injured side in a guarded position. Previous studies on the CCI model have shown that the animals also develop significant hyperalgesia in thermal, mechanical and chemical nociceptive tests 2,7. In the present study, each of the animals in the 5, 10 and 20 day survival groups were tested for hyperalgesia using the thermal stimulus paradigm of Hargreaves and coworkers 22. This procedure measures the withdrawal latency from a radiant heat source directed at the proximal half of the plantar surface of each hind paw. Animals were not tested for hyperalgesic responses at 2 days after CCI because it has been reported that exposure to ~oxious heat at this early time evokes edema in the hind paw 7. We wished to avoid the possibility that this peripheral effect of SP h ~ a central counterpart that might influence the binding of 12SI-BH-SP. The behavioral assay for hyperalgesia was conducted just prior to sacrifice of the animals. Four latency measurements for each hind paw were averaged, and a difference score was determined by subtracting the average latency of the control paw from that of the paw ipsilateral to the CCI. Negative scores thus indicate a decrease in threshold on the side ipsilateral to CCI. Statistical analyses were made among groups using one-way ANOVA with Dunnett's post-hoc test. This post-hoc test was chosen since difference scores of the experimental groups were compared only to the difference scores of the control group. The level of significance was set at P < 0.05. Results of the behavioral tests on the animals used in this study were included in a previous report 19. Tissue preparation. At the end of the survival periods and after the be'aavioral tests were performed, the rats were anesthetized with sodium pentobarbital (as described above) and perfused through the aorta with approximately 200 ml of ice-cold phosphate buffered saline mixed 1 : 1 with 0.32 M sucrose. A laminectomy was performed while the animal was embedded in ice, and the L4 segment was identified and removed. The spinal cord segment was then placed in a plastic tissue-embedding mold and frozen on powdered dry ice. The tissue blocks were stored for no more than 2 weeks at -70°C prior to sectioning, The blocks were then mounted on microtome chucks, sectioned (10/zm) on a cryostat and thaw-mounted on to gelatin-coated slides. The tissue was stored at -20°C until receptor binding studies were performed. Receptor binding and autoradiography. For 125I-BH-SP binding on the CCI tissue, slide-mounted tissue sections were processed following the protocol outhned above. The slides were incubated in 50 pM 1251-BH-SP for determination of total binding or in 50 pM 1251-BH-SP plus 1/zM unlabeled SP for determination of non-specific binding. After the final rinses in buffer, the slides were dipped in distilled water (4°C) to remove salts and dried, first under a stream of cool, desiccated air and finally on a slide warmer (30 min at ~ 37°C). The slides were stored desiccated, overnight at 4°C. Autoradiograms were prepared using the technique described by Young and Kuhar 62. Emulsion-coated coverslips (NTB-3 nuclear emulsion, Kodak, Rochester, NY) were apposed to the slide-mounted tissue by applying a drop of Super Glue (Locktite) to the frosted end of the slide. Coverslips were kept in close apposition throughout the exposure period by clipping the slides between Plexiglass sheets. Autoradiograms of 125I microscaies (40 /zCi multilevel reference strips cut to 10/zm; Amersham, Arlingt~a Heights, IL) were prepared in parallel. The slides were stored desiccated in light-tight boxes for exposure periods of 6 and 9 days, and then the emulsion was developed (Kodak Developer D-~9) and fixed (Rapid Fix, Kodak). Following development of the autoradiograms, the tissue was fixed in Carnoy's fixative, stained wi:,,h thionin, dehydrated, and the coverslips were permanently affixed with mounting medium (Entellan, EM Science, Cherry Hill, N J). Scatchard analysis by autoradiography. Scatchard analysis was carried out on autoradiographic data obtained from spinal cords of experi-
Lam 1/11
LamV
D
regionof analysis (SO,O00sq. pm)
1 mm
Fig. 1. Determination of coordinates for autoradiographic analysis. Lain l / I I : at one-half the length of a line from the medial curve of the superficial dorsal horn to the dorsal root entry zone (*). This coordinate is consistent with the termination of small diameter primary afferent neurons of the sciatic nerve 51. Lam V: at threequarters the length of the line from the ventral aspect of the dorsal columns to the lateral curve of the superficial dorsal horn. This coordinate is consistent with the reticulated area in lateral lamina V. Lain X: at the intersection of 2 lines: 1 line extending ventrally from the medial curve of the dorsal horn; 1 line extending laterally from the midpoint between the central canal and the ventral aspect of the dorsal columns.
mental animals (5 day survival group). Spinal cord sections were incubated in 50 pM 125I-BH-SP with 5 different cor~centrations of unlabeled SP (0.01-1.0 nM). After completion of the binding protocol, the slides were dried and emulsion-coated coverslips were apposed (see above) for a period of 9 days. This tissue ;:~d emulsioncoated coverslips were processed according to the method described above. The densities of autoradiographic grains overlying medial laminae I / I I ~.ere determined on each side of the spinal cord. Control values reflect data ipsilateral to the sham surgery; experimental values reflect data ipsilateral to the CCI. Scatchard analysis was performed with non-linear regression analysis of the data using the McPherson version of LIGAND 36. Ligand binding data used for statistical comparison (n = 3) were selected based on their low variability as determined in Scatchard analysis (correlation coefficient > 0.85).
Computerized image analysis Bilateral measurements of grain densities were taken at 100 x in laminae i / I I (medial), V and X. The medial region of laminae I / l l was chosen for analysis since it has been shown to receive a rich projection of fibers from the sciati~ nerve s'3s'56, particularly nociceptive fibers 31'53'54. Laminae V and X were chosen for analysis since these areas receive terminations of C-fibers 53'54 and thinly myelinated, high-threshold mechanoreceptors 31. Using electrophysiological studies in the rat, laminae I/II, V and X have also been shown to be associated with nociceptive processing 21'4°'6° and contain high densities of spinothalamic tract neurons (see review)TM. Furthermore, all of these regions have been shown to contain moderate to high levels of SP-like immunoreactivity3'24 and SP binding sites 14. The coordinates for the regions of densitometric analysis were determined according to the criteria outlined in Fig. 1. Quantitative measurements of grain densities in these areas were determined using a digital image processing and analysis program for the Macintosh II, Image 1.13b (Dr. W. Rasband, NIH). A video camera attached to the microscope delivered a live image to the digitizer
262 attached to the computer. A threshold function was then used to sum the number of pixels in the images of the grains. Grains densities were determined in the selected areas of the spinal cord in control rats (n = 10) and rats who survived for 2 (n = 7), 5 (n - 8), 10 ( n - - 8 ) and 20 (n = 7) days after CCI. Non-specific binding was found to be < 5% of the total grain density in the areas analyzed and, thus, was not subtracted from total binding values in the analysis of the data. The area of the measurements was approximately 50,000 ~m z. The apposition of the autorad~ograms to the stained tissue sections allowed accurate quantification of the grain densities with respect to laminae identified using a rat stereotaxie atlas 46. Data were converted to fmol/mg tissue by linear transforma-
r'l 5ham (oxpenmental glaup)
T
Laminae I II
•
40e°
GCI (exper,mentaJ g,aup)
O Sham (conlzol gloup) O UntleatlKI (conltolgroup)
e,
36,
3.2
2.8
I
2.3
.!
m ~t C/)
l
Lemtna V
i
•
ID,
2.!
tion of the autoradiographic grain densities according to the standard curve generated from the 125I-microscale standards. For clarity, 'density of silvel" grains' will be referred to as 'tZSl-BH-SP binding' throughout the report.
Statistical analysis The autoradiographic data were analyzed using a three-way (time vs. side of spinal cord vs. laminae) analysis of variance with Duncan's multiple comparison test. The level of significance was set at P < 0.05. There were no significant differences between the two control groups (unilateral sham-surgery) sacrificed at 10 and 20 days postsurgery, so these data were merged for the analysis. There were no side-to-side differences in the merged control group (or in either separate group); nevertheless, we used only the data from the sham-operated side for comparisons with the CCI animals. Post-hoe pair-wise tests included within-group comparisons of the nerve-injured vs. sham-operated sides of the CCI groups. Three sets of comparisons were made at each survival time: (1) the injured side of the experimental group was compared with the sham-operated side of the control grovp, (2) the sham-operated side of the control group was compared with the sham-operated side of experimental group and (3) the injured side of the experimental group was compared to the shamoperated side of the same group. Each of these comparisons were made in each of the laminae where measurements were taken. Note that the within-group comparisons of the nerve-injured vs. sham-operated sides of the CC! animals are not necessarily comparisons of treatment effect vs. a control (the sham-operated side) because of the possibility of a nerve injury-evoked contralateral change. Correlations between changes in 1251-BH-SP binding and hyperalgesia were tested using linear regression analysis.
Materials
t
The following substances were obtained from Sigma Chemical Co. (St. Louis, MO): Tris buffer (Trizma, pH 7.4), bovine serum albumin, bacitracin, EDTA, leupeptin and chymostatin. Substance P, neurokinin A and neurokinin B were obtained from Peninsula (Belmont, CA). t2Sl-Bolton-Hunter labeled substance P ( ~ 2,000 Ci/mmol) was obtained from Amersham (Arlington Heights, IL).
1.5
RESULTS I
i,
I
i
.f:
'
Lamln,, X
Specificity of 12Sl.BH-SPbinding
3,0
3.6
I T
':.4
3.2
3,0 ,
i
t
2
S
, 10
..i 2O
¢.1
I C
Time (days after Iigatlon)
Fig. 2. SP binding in laminae I/!I, V and X in CCI and control rats. SP binding in three regions of the spinal cord in control (n = 10; open circle: sham-operated side; hatched circle: untreated) and after CCI (boxes) at 2 (n = 7), 5 (n --- 8), 10 (n = 8) and 20 (n -- 7) days. The open boxes represent values ipsilateral to sham surgery and the filled boxes represent values ipsilateral to CC1. * significant difference in binding ipsilateral to CCI when compared to the sham-operated side of the control group (P < 0.05, three-way ANOVA with Duncan's post-hoc test); t significant difference in binding between the left (ipsilateral t~ CCI) and right (ipsilateral to sham surgery) sides of the spinal cord (P < 0.05, three-way ANOVA with Duncan's post-hoc test).
The specificity of 1251-BH-SP binding in rat spinal cord was tested in competition studies. These studies revealed that the rank order of potency of related tachykinins for competition of :25I-BH-SP binding was substance P >> neurokinin A > neurokinin B. SP, the most potent competitor, had an ICs0 of 121.2 + 32.6 pM. Neurokinin A was nearly 250 times less potent than SP (ICs0 - 28.1 5:5.9 nM), and neurokinin B was 1,700 X less potent than SP (ICs0 -- 212 5:86•3 nM). Scatchard analysis of the competition data for SP revealed that the Kd of :251-BH-SP binding in rat spinal cord was 397.4 5:32.9 pM.
SP binding in sham-operated controls Sham surgery was performed on only one side of the rat in the control group so that the effects of the surgery alone on ;251-BH-SP binding could be tested• The distribution of ;251-BH-SP binding sites in the rat spinal cord segment L4 was similar to the distribution reported previously :4,33. The greatest :25I-BH-SP bind-
203 ing occurred in laminae I and in the area around the central canal. Moderate levels of ~251-BH-SP binding were observed in laminae III and V and in discrete regions of the ventral horn. Statistical analysis revealed that there were no significant differences ~.n laminae I/II, V and X between the sham-operated side and the unoperated side of the spinal cord when animals were sacrificed 10 or 20 days after surgery.,The data were pooled for 10 and 20 days; these control values appear on the right side of each graph in Fig. 2.
Changes in SP binding in laminae I/I1, V and X following CC! In most cases the within- and b-_.tween-group comparisons were consistent with a simple, unilateral change on the side of the nerve injury. A significant increase in ~251-BH-SP binding ipsilateral to CCI was observed in laminae I / I I at 5, 10 and 20 days after sciatic ligation when these values were compared to the sham-operated side of the control group, and at 2, 5, 10 and 20 days when compared to the contralateral side of the spinal cord (sham-operated) (Fig. 2, top graph). The percent increases in ~251-BH-SP binding over the sham-operated side of the control group at days 5, 10 and 20 were 15.9%, 26.5% and 13.2%, respectively. Fig. 3 is a darkfield photomicrograph of the total (A) and non-specific (B) 125I-BH-SP binding in spinal segment L 4 at 5 days postsurgery. A greater density of silver grains in laminae I/II, especially in the medial aspect of these laminae, was observed on the left side of the spinal cord (ipsilateral to the sciatic
nerve injury) in cemparison with the right side (ipsilateral to sham sl~rgery). The only significant change in ~251-BH-SP binding in lamina V was observed at day 5 after the CCI (Fig. 2, middle graph). This value was found to be significantly different when compared to the sham-operated side of the control group and when compared to the contralateral side (sham-operated). This change represented a 13.6% increase in binding from the sham-operated side of the control group. ~251-BH-SP binding in lamina V returned to the control value by 20 days after sciatic nerve injury. A moderate increase in binding can be seen in the left (ipsilateral to the sciatic nerve injury), deep dorsal horn (region of lamina V) of the darkfield photomicrograph when compared to the right side (ipsilaterai to sham surgery) (Fig. 3A). In lamina X, a significant decrease in 1251-BH-SP binding was observed at 2 days after CCI when compared to the contralateral side of the spinal cord (sham-operated) (Fig. 2, lower graph). No other significant changes in ~251-BH-SP binding occurred in lamina X. It is interesting to note, however, that the pattern of increased binding at day 10 and decreased binding at day 20 was observed both ipsilateral and contralateral to CCI.
Scatchard analysis by autoradiography The dissociation c o n s t a n t ( K d) and Bmax values for ~251-BH-SP binding in laminae I/II ipsilateral to sham surgery (control) and ipsilateral to the CCI (experimental) at 5 days postsurgery were obtained by Scatchard
Fig. 3. Photomicrograph of autoradiograms of 1251-BH-SP binding 5 days after CCI. Darkfield photomicrograph of adjacent sections of L 4 spinal cord incubated under conditions for t25I-BH-SP binding (A) and nonspecific binding (B). The left side of the spinal cord is ipsilateral to the CCI. This section is from a rat sacrificed at 5 days after CCI. Bar -- 400/tm.
264 TABLE I
0.5
Dissociation constant (K d) and Bmax values for 1251-BH-SPin laminae l / ll in control and experimental sides of the spinal cord
g, e-
Grain densities for Scatchard analysis were quantified in laminae I / l l on control and experimental sides of the spinal cord. Scatchard analyses were performed using non-linear regression analysis of the data (McPherson version of LIGAND). The spinal cords used for statistical analysis (n = 3) were selected based on their lack of variability as determined in Scatchard analysis (correlation coefficient > 0.85). Values represent the m e a n + S . E . M . (n = 3). * Student's unpaired t-test, P < 0.05.
-0.5
-1.5
-2.5
Control Experimental (CCI)
Kd
Bmax
(pM)
(fmol / mg tissue)
=_"
122.7 + 20.1
23.4 + 6.14
C3
-3.5 45.3 + 3.4 *
26.4 + 2.8
analysis of autoradiographic data (Table I). A significant decrease in the K,~ was observed in laminae I/II ipsilateral to CCI when compared with the control side (ipsilateral to sham surgery). There was no significant change in the Bmax in laminae I/II ipsilateral to the CCI. Fig. 4 represents data from one animal and illustrates Scatchard plots of SP binding in laminae I/II ipsilateral to the CCI (experimental) and ipsilateral to sham surgery (control). Withdrawal latencies and their correlation with changes in 1251-BH.SP binding The latency of the nocifensive withdrawal response was reduced ipsilateral to the nerve injury compared to the contralaterai, sham-operated side (Fig. 5), and, as noted previously 2, the lowered thresholds were accompanied by responses of greater magnitude and dura-
D Control Kd , 141 pM B max = 28.6 fmol/mg
0.8'
• Experimental (CCI) Kd = 39 pM Bmax ==23.7 fmol/mg
0.6'i o m
0.4
I
0.2'
sham
5
10
20
Days after sciatic Iigation Fig. 5. Behavioral test. Animals were tested for their response to a radiant heat stimulus on the day of sacrifice (see text for details). The latency score was computed by subtracting the average latency of the paw ipsilateral to sham surgery from the average latency of the paw ipsilateral to the CCI. Negative scores indicate a shorter latency ipsilateral to the CCI; i.e., hyperalgesia. * significant difference when compared to the sham-operated group ( P
