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Sensory innervation of the lateral portion of the lumbar intervertebral disc in rats
The Spine Journal 4 (2004) 275–280
Sensory innervation of the lateral portion of the lumbar intervertebral disc in rats Yasuchika Aoki, MDa,*, Yuzuru Takahashi, MD, PhDa, Kazuhisa Takahashi, MD, PhDa, Tanemichi Chiba, MD, PhDb, Masahiro Kurokawa, MDa, Tomoyuki Ozawa, MDa, Hideshige Moriya, MD, PhDa a
Department of Orthopedic Surgery, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-Ku, Chiba City 260-8670, Japan b Department of Neurobiology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-Ku, Chiba City 260-8670, Japan Received 21 March 2003; accepted 2 October 2003
Abstract
BACKGROUND CONTEXT: An annular tear extending to the outer one-third of the annulus is thought to be one of the causes of low back pain. However, some patients have bilateral low back symptoms, even if the annular tear is localized in the lateral disc. Because nociceptive information from the lateral disc is transmitted by the dorsal root ganglion (DRG) neurons innervating the lateral disc, we investigated the distribution of the DRG neurons innervating the lateral portion of the disc. PURPOSE: To clarify the distribution and pathway of the DRG neurons innervating the lateral portion of the L5–L6 disc in rats. STUDY DESIGN/SETTING: Using the retrograde tracing method, we studied the innervation pattern of the lateral portion of the L5–L6 intervertebral disc in rats. METHODS: The retrograde transport of Fluoro-Gold (F-G; Fluorochrome, Denver, CO) was used in 22 rats. Subjects included a nontreated group (n⫽16) and a sympathectomized group (n⫽6). Seven days after the application of F-G crystals to the left lateral portion of the L5–L6 disc, bilateral T12–L6 DRGs were observed by fluorescent microscopy. RESULTS: In the nontreated group, of all the F-G–labeled neurons, 93.1% were present in the left DRGs and 6.9% were in the right DRGs. The number of labeled neurons was largest in the left L2 DRGs. In the sympathectomized group, the numbers of labeled neurons in the T13, L1 and L2 DRGs were significantly lower than the numbers in the nontreated group. CONCLUSION: Results of this study indicate that DRG neurons innervating the lateral portion of the disc are distributed mainly in the ipsilateral side but also in the contralateral side. The DRG neurons in T13, L1 and L2 innervate the lateral portion of the L5–L6 disc through the paravertebral sympathetic trunks. 쑖 2004 Elsevier Inc. All rights reserved.
Keywords:
Intervertebral disc; Dorsal root ganglion (DRG); Laterality; Discogenic pain; Low back pain; Retrograde tracing; Sympathetic trunks; Sensory fibers
Introduction Some investigators have demonstrated the presence of sensory fibers in the lumbar intervertebral disc in humans
FDA device/drug status: not applicable. This work was supported in whole or in part by the Japanese Society for the Promotion of Science under Grant 1457 1358. Nothing of value received from a commercial entity related to this research. * Corresponding author. Department of Orthopedic Surgery, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba City 260-8670, Japan. Tel.: ⫹81-43-226-2117; fax: ⫹81-43-226-2116. E-mail address: [email protected] (Y. Aoki) 1529-9430/04/$ – see front matter doi:10.1016/j.spinee.2003.10.005
쑖 2004 Elsevier Inc. All rights reserved.
[1–5], rats [6–8] and other animals [9,10]. Recent studies addressing the function of the nerve fibers using immunohistochemical techniques demonstrated that substance P (SP) or calcitonin gene-related peptide (CGRP) immunoreactive nerve fibers were present in the lumbar intervertebral disc and posterior longitudinal ligament [2,4,8,11–13]. These results suggest that the intervertebral discs can be a source of low back pain, because SP and CGRP are believed to be the sensory transmitters of nociceptive information [14–18]. Classically, the nerve fibers in the intervertebral disc have been believed to be supplied segmentally by the corresponding dorsal root ganglia (DRG) [3,6]. However, recent studies have demonstrated nonsegmental innervation patterns in the
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rat lumbar spine [19–22]. Morinaga et al. [19] reported that the ventral portion of the L5–L6 disc was innervated by DRG neurons in L1 and L2. Ohtori et al. [21] reported the DRG neurons innervating the dorsal portion of L5–L6 disc were distributed to multilevel (T13–L6) DRGs. However, the innervation patterns in the lateral portion of the lumbar intervertebral disc have not been clarified. Usually, disc hernia is preceded by one or more attacks of low back pain [23]. Even if the disc hernia is localized in the lateral portion of the disc, the low back pain sometimes appears bilaterally. Furthermore, Slipman et al. [24] reported a random correlation between the side of the patient’s concordantly painful annular tear and the side of the patient’s pain. We hypothesized that nociceptive information from the lateral disc is transmitted to both ipsilateral and contralateral DRGs. It is generally recognized that DRG neurons are divided into two main subpopulations, the small and large neurons [25]. The large neurons are thought to be involved mainly in proprioception. In contrast, most small DRG neurons are thought to be involved in nociception. To examine whether there are some small, nociceptive DRG neurons innervating the contralateral side of the disc, we investigated the distribution and cross-sectional areas of DRG neurons innervating the lateral disc in rats, using a retrograde tracing method. We also hypothesized that the mechanism of innervation was through paravertebral sympathetic trunks. If that hypothesis is correct, it would explain why the blockade of paravertebral sympathetic trunks was effective for some patients with discogenic disorders [26]. To test the hypothesis, we examined the effect of sympathectomy on the distribution of the DRG neurons. In the sympathectomized rats, the DRG neurons innervating the disc through paravertebral sympathetic trunks would not be labeled by the neurotracer. This approach, therefore, elucidates the pathway of axons of the DRG neurons innervating the lateral disc.
was immediately sealed by cyanoacrylate to prevent leakage of F-G. Sympathectomized group (n⫽6) F-G was applied to the same site as in the nontreated group just after the removal of the lumbar paravertebral sympathetic trunks bilaterally from the L3 to L5 levels. Microscopic observation and analysis Seven days after the F-G application, the rats were reanesthetized with sodium pentobarbital and perfused transcardially with 0.9% saline, followed by 500 ml of 4% paraformaldehyde in phosphate buffer (0.1 M; pH 7.4). After laminectomy, bilateral DRGs from T12 to L6 were resected. The specimens were immersed in the same fixative overnight at 4 C, after which they were transferred to 20% sucrose overnight. Each DRG was serially sectioned at 40-µm thickness on cryostat. All the sections were mounted on slides and then coverslipped using Permafluor (Shandon, Inc., Pittsburgh, PA). The sections were observed with a Nikon Eclipse fluorescent microscope (Tokyo, Japan). Only F-G– labeled DRG neurons that had a clear visible nucleus were counted using filter UV-1A (wavelengths of 365 nm for excitation and 420 nm for emission; Fig. 1). To determine
Materials and methods We used 22 male Sprague-Dawley rats weighing 250 to 300 g. The rats were anesthetized with sodium pentobarbital (40 mg/kg, intraperitoneally) and treated aseptically in all surgical procedures. This study was conducted with the permission of the Ethical Committee for Animal Experiments of Chiba University School of Medicine. Nontreated group (n⫽16) A midline abdominal incision was made, and the retroperitoneum was incised. The left iliopsoas muscle was dissected to expose the left lateral aspect of the L5–L6 intervertebral disc. A perforation approximately 0.5 mm in depth was made on the surface of the disc by a 23-gauge needle, and crystals of Fluoro-Gold (F-G; Fluorochrome, Denver, CO) were inserted. The application site was the most lateral point of the left side of the disc. The application site
Fig. 1. Photomicrographs of Fluoro-Gold–labeled neurons in the nontreated group. (a) A small-size neuron (525 µm2) observed in the right L1 dorsal root ganglion (DRG). Scale bar⫽20 µm. (b) An intermediate-size neuron (1,115 µm2) observed in the left L5 DRG. Scale bar⫽20 µm.
Y. Aoki et al. / The Spine Journal 4 (2004) 275–280
whether the F-G–labeled neurons are large, proprioceptive neurons or small, nociceptive neurons, we measured the cross-sectional areas of the labeled neurons in seven rats of the nontreated group and in six rats of the sympathectomized group using a computer-assisted imaging analysis system (NIH Image software, Springfield, VA).
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The segmental distribution of the F-G–labeled neurons is shown in Fig. 2. Sympathectomized group F-G–labeled neurons were observed in the left DRGs from L1 to L5 and in the right DRGs from L3 to L5 (Table 2). No labeled neurons were observed in the left T12, T13 and L6 DRGs. Compared with the nontreated group, the numbers of labeled neurons in the left L1 and L2 DRGs were significantly lower than those in the nontreated group (Fig. 2a), and no labeled neurons were observed in the right T13, L1 and L2 DRGs (Fig. 2b). The segmental distribution of the labeled neurons is shown in Fig. 2.
Statistical analysis We compared the numbers of the F-G–labeled neurons in the DRGs of the same level between the nontreated and sympathectomized groups using nonpaired Welch’s t test. We also compared the mean cross-sectional area of the FG–labeled neurons in the left DRGs and the right DRGs using Welch’s t test. A p value of less than 0.05 was considered to be significant.
Cross-sectional area of cell profiles We measured the cross-sectional area of the cell profiles in seven rats of the nontreated group. The labeled neurons were round or oval (Fig. 1), and their cross-sectional area was within the range of 200 to 2,000 µm2 (Fig. 3). The mean cross-sectional area of the cell profile (mean⫾SD) was 766⫾317 µm2 in the left T13–L5 DRGs and 729⫾193 µm2 in the right T13, L1, L2 and L4 DRGs. No significant differences were found between that of the left DRGs and the right DRGs. If neurons smaller than 800 µm2 were defined as small-size neurons, the ratio of the small-size neurons was 62.3% in the left DRGs and 50% in the right DRGs.
Results Nontreated group F-G applied to the lumbar disc was retrogradely transported to the DRG neurons innervating the disc (Fig. 1). We found 321 F-G–labeled neurons in the 16 rats examined (Table 1). The minimum number was 8, and the maximum number was 50 within a rat. Considering 2,000 to 3,500 neurons are present in a DRG [27], the number of the DRG neurons innervating the lumbar disc was relatively small. F-G–labeled neurons were present in the bilateral DRGs from T13 to L5. No labeled neurons were observed in T12 and L6 DRGs (Table 1). Of the labeled neurons, 93.1% were present in the left (ipsilateral) DRGs, and 6.9% were present in the right (contralateral) DRGs, with the largest number of labeled neurons found in the left L2 DRG.
Discussion There are several reports about the segmental distribution of DRG neurons innervating the lumbar intervertebral disc [10,19–22,28]. Morinaga et al. [19] reported on the innervation pattern of the ventral portion of the L5–L6 disc using
Table 1 Number of Fluoro-Gold–labeled neurons in dorsal root ganglia of the nontreated group (n⫽16) Rat
T12
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Total
0 (0, 0)
(0, (0, (0, (0, (0, (0, (0, (0, (0, (0, (0, (0, (0, (0, (0, (0,
T13 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0)
4 1 0 1 0 0 0 1 2 0 2 1 2 1 1 5
(3, (0, (0, (1, (0, (0, (0, (1, (2, (0, (2, (1, (1, (1, (1, (4,
L1 1) 1) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 1) 0) 0) 1)
21 (17, 4)
10 3 2 1 3 0 1 3 1 2 2 5 5 0 3 8
L2 (10, 0) (3, 0) (2, 0) (1, 0) (2, 1) (0, 0) (1, 0) (3, 0) (1, 0) (2, 0) (2, 0) (4, 1) (4, 1) (0, 0) (3, 0) (7, 1)
49 (45, 4)
4 1 9 5 9 8 9 5 2 13 10 15 5 7 3 19
L3 (4, 0) (1, 0) (9, 0) (5, 0) (6, 3) (8, 0) (8, 1) (5, 0) (2, 0) (12, 1) (10, 0) (14, 1) (5, 0) (7, 0) (3, 0) (18, 1)
124 (117, 7)
2 1 7 0 1 4 6 7 4 4 13 5 3 8 0 4
L4 (2, 0) (1, 0) (7, 0) (0, 0) (1, 0) (4, 0) (5, 1) (7, 0) (3, 1) (4, 0) (13, 0) (5, 0) (3, 0) (8, 0) (0, 0) (4, 0)
69 (67, 2)
1 0 2 1 4 11 0 0 0 0 0 1 1 0 2 8
L5 (1, 0) (0, 0) (1, 1) (1, 0) (3, 1) (11, 0) (0, 0) (0, 0) (0, 0) (0, 0) (0, 0) (1, 0) (1, 0) (0, 0) (2, 0) (6, 2)
31 (27, 4)
0 2 0 1 2 5 1 2 0 0 1 2 3 1 1 6
L6 (0, (2, (0, (1, (1, (5, (1, (2, (0, (0, (1, (2, (3, (1, (1, (6,
0) 0) 0) 0) 1) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0)
27 (26, 1)
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
(0, (0, (0, (0, (0, (0, (0, (0, (0, (0, (0, (0, (0, (0, (0, (0,
Total 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0)
0 (0, 0)
3 (2, 1) indicates a total of 3 Fluoro-Gold–labeled neurons, 2 of which were observed on the left side and 1 on the right side.
21 8 20 9 19 28 17 18 9 19 28 29 19 17 10 50
(20, 1) (7, 1) (19, 1) (9, 0) (13, 6) (28, 0) (15, 2) (18, 0) (8, 1) (18, 1) (28, 0) (27, 2) (17, 2) (17, 0) (10, 0) (45, 5)
321 (299, 22)
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innervated mainly by the L1, L2 and L3 DRG neurons in rats revealed that nociceptive information from any part of the L5–L6 disc is transmitted mainly by the L1 and L2 DRG neurons. The fact that disc lesions most frequently occur at the L4–L5 level in humans, and the L5–L6 level in rats corresponds to the L4–L5 level in humans anatomically, may be one of the explanations why L2 spinal nerve infiltration was effective in treating a patient with discogenic low back pain [29]. Using horseradish peroxidase, Forsythe and Ghoshal [10] have reported the innervation patterns of the dorsolateral portion of the thoracolumbar disc in dogs. In their study, the neurotracer application site was just lateral to the edge of the posterior longitudinal ligament. They reported that some labeled neurons were present in contralateral DRGs. However, they did not describe the ratio of the labeled neurons in the contralateral DRGs. In the present study, we chose the most lateral point of the disc as the application site to clarify the laterality of the innervation patterns in rats. Our test revealed that 6.9% of the sensory fibers in the lateral portion of the disc were derived from DRGs on the contralateral side. Additionally, we measured the cross-sectional areas of the labeled neurons and discovered that some of the labeled neurons observed in DRGs on the contralateral side to the F-G application belonged to a subgroup of small, nociceptive neurons. Although we have not performed immunostaining of the neurotransmitters of nociceptive information, such as SP and CGRP, these results indicate that nociceptive information from the lateral disc can be partially transmitted to contralateral DRGs. It has been reported that some sensory fibers in the lumbar dura mater and the posterior longitudinal ligament are derived by means of the sympathetic trunks in rats [20,26,30– 32]. Suseki et al. [33] have demonstrated the presence of the sensory fibers in lumbar intervertebral discs passing through rami communicantes. Ohtori et al. [21] reported that there were two distinctive pathways from the dorsal portion of the L5–L6 disc to DRGs. One was the pathway through the sinuvertebral nerves, and the other was through the paravertebral sympathetic trunks. In the present study, the number of the F-G–labeled neurons in the ipsilateral T13–L2 DRGs in the sympathectomized group was significantly less than that of the nontreated group. These results
Fig. 2. (a) The segmental distribution of Fluoro-Gold–labeled neurons in the left (ipsilateral) dorsal root ganglia (DRGs) from T12 to L6. (b) The distribution of the mean number of Fluoro-Gold–labeled neurons in the right (contralateral) DRGs from T12 to L6. Mean⫾SE, *p⬍0.01 (unpaired Welch’s t test).
horseradish peroxidase and choleratoxin B subunit as tracers and concluded that the ventral portion of the lumbar disc was innervated by the L1 and L2 DRGs. Using F-G, Ohtori et al. [21] demonstrated that the dorsal portion of the L5– L6 disc was innervated by the T13–L6 DRGs multisegmentally and also mainly by the L1 and L2 DRGs. Our demonstration that the lateral portion of the L5–L6 disc was
Table 2 Number of Fluoro-Gold–labeled neurons in dorsal root ganglia of the sympathectomized group (n⫽6) Rat
T12
1 2 3 4 5 6 Total
0 0 0 0 0 0 0
(0, (0, (0, (0, (0, (0, (0,
T13 0) 0) 0) 0) 0) 0) 0)
0 0 0 0 0 0 0
(0, (0, (0, (0, (0, (0, (0,
L1 0) 0) 0) 0) 0) 0) 0)
0 1 0 0 0 0 1
(0, (1, (0, (0, (0, (0, (1,
L2 0) 0) 0) 0) 0) 0) 0)
1 2 1 1 2 5 12
L3 (1, 0) (2, 0) (1, 0) (1, 0) (2, 0) (5, 0) (12, 0)
0 3 1 4 7 3 18
L4 (0, 0) (3, 0) (1, 0) (4, 0) (7, 0) (2, 1) (17, 1)
3 0 2 0 9 9 23
L5 (3, 0) (0, 0) (2, 0) (0, 0) (8, 1) (8, 1) (21, 2)
1 3 1 0 6 6 17
L6 (1, 0) (3, 0) (1, 0) (0, 0) (6, 0) (5, 1) (16, 1)
0 0 0 0 0 0 0
3 (2, 1) indicates a total of 3 Fluoro-Gold–labeled neurons, 2 of which were observed on the left side and 1 on the right side.
(0, (0, (0, (0, (0, (0, (0,
Total 0) 0) 0) 0) 0) 0) 0)
5 9 5 5 24 23 71
(5, 0) (9, 0) (5, 0) (5, 0) (23, 1) (20, 3) (67, 4)
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L5–L6 disc to the contralateral DRGs is present in paravertebral sympathetic trunks for the T13 to L2 DRGs and in the ventral primary rami or sinuvertebral nerves for the L3–L5 DRGs. These findings may have shown one of the reasons why lumbar sympathetic blocks sometimes relieve a patient’s low back pain [26]. It has been reported that the nerve fibers in the intervertebral disc are very sparse [3,9]. In the present study we demonstrated the presence of small nociceptive DRG neurons innervating the contralateral side of the disc in rats. There remains the possibility that there are differences in innervation patterns between humans and rats. However, if a similar innervation pattern is present in humans, the patient with a lateral annular tear may feel a pain in the bilateral low back.
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Fig. 3. (a) The size (cross-sectional area) of the Fluoro-Gold–labeled neurons observed in the left (ipsilateral) dorsal root ganglia (DRGs; 162 neurons in the nontreated group). (b) The size (cross-sectional area) of the FluoroGold–labeled neurons observed in the right (contralateral) DRGs (10 neurons in the nontreated group). The cross-sectional area of the neurons was in the range of 201 to 1,000 µm2. Most of them belonged to the subgroup of small- to intermediate-size neurons.
suggest that there were also two distinctive pathways of the sensory nerve fibers in the lateral portion of the disc. The sensory fibers originating in the T13–L2 DRGs were thought to pass mainly through the paravertebral sympathetic trunks, and those originating in the L3–L5 DRGs pass mainly through the ventral primary rami of the spinal nerves or sinuvertebral nerves. In the nontreated group, F-G–labeled neurons were present in the contralateral T13–L5 DRGs. In contrast, in the sympathectomized group, the F-G–labeled neurons were present in the contralateral L3–L5 DRGs but not in the contralateral T13–L2 DRGs. These results suggest that the afferent pathway from the lateral portion of the
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Sensory innervation of the lateral portion of the lumbar intervertebral disc in rats Yasuchika Aoki, MDa,*, Yuzuru Takahashi, MD, PhDa, Kazuhisa Takahashi, MD, PhDa, Tanemichi Chiba, MD, PhDb, Masahiro Kurokawa, MDa, Tomoyuki Ozawa, MDa, Hideshige Moriya, MD, PhDa a
Department of Orthopedic Surgery, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-Ku, Chiba City 260-8670, Japan b Department of Neurobiology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-Ku, Chiba City 260-8670, Japan Received 21 March 2003; accepted 2 October 2003
Abstract
BACKGROUND CONTEXT: An annular tear extending to the outer one-third of the annulus is thought to be one of the causes of low back pain. However, some patients have bilateral low back symptoms, even if the annular tear is localized in the lateral disc. Because nociceptive information from the lateral disc is transmitted by the dorsal root ganglion (DRG) neurons innervating the lateral disc, we investigated the distribution of the DRG neurons innervating the lateral portion of the disc. PURPOSE: To clarify the distribution and pathway of the DRG neurons innervating the lateral portion of the L5–L6 disc in rats. STUDY DESIGN/SETTING: Using the retrograde tracing method, we studied the innervation pattern of the lateral portion of the L5–L6 intervertebral disc in rats. METHODS: The retrograde transport of Fluoro-Gold (F-G; Fluorochrome, Denver, CO) was used in 22 rats. Subjects included a nontreated group (n⫽16) and a sympathectomized group (n⫽6). Seven days after the application of F-G crystals to the left lateral portion of the L5–L6 disc, bilateral T12–L6 DRGs were observed by fluorescent microscopy. RESULTS: In the nontreated group, of all the F-G–labeled neurons, 93.1% were present in the left DRGs and 6.9% were in the right DRGs. The number of labeled neurons was largest in the left L2 DRGs. In the sympathectomized group, the numbers of labeled neurons in the T13, L1 and L2 DRGs were significantly lower than the numbers in the nontreated group. CONCLUSION: Results of this study indicate that DRG neurons innervating the lateral portion of the disc are distributed mainly in the ipsilateral side but also in the contralateral side. The DRG neurons in T13, L1 and L2 innervate the lateral portion of the L5–L6 disc through the paravertebral sympathetic trunks. 쑖 2004 Elsevier Inc. All rights reserved.
Keywords:
Intervertebral disc; Dorsal root ganglion (DRG); Laterality; Discogenic pain; Low back pain; Retrograde tracing; Sympathetic trunks; Sensory fibers
Introduction Some investigators have demonstrated the presence of sensory fibers in the lumbar intervertebral disc in humans
FDA device/drug status: not applicable. This work was supported in whole or in part by the Japanese Society for the Promotion of Science under Grant 1457 1358. Nothing of value received from a commercial entity related to this research. * Corresponding author. Department of Orthopedic Surgery, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba City 260-8670, Japan. Tel.: ⫹81-43-226-2117; fax: ⫹81-43-226-2116. E-mail address: [email protected] (Y. Aoki) 1529-9430/04/$ – see front matter doi:10.1016/j.spinee.2003.10.005
쑖 2004 Elsevier Inc. All rights reserved.
[1–5], rats [6–8] and other animals [9,10]. Recent studies addressing the function of the nerve fibers using immunohistochemical techniques demonstrated that substance P (SP) or calcitonin gene-related peptide (CGRP) immunoreactive nerve fibers were present in the lumbar intervertebral disc and posterior longitudinal ligament [2,4,8,11–13]. These results suggest that the intervertebral discs can be a source of low back pain, because SP and CGRP are believed to be the sensory transmitters of nociceptive information [14–18]. Classically, the nerve fibers in the intervertebral disc have been believed to be supplied segmentally by the corresponding dorsal root ganglia (DRG) [3,6]. However, recent studies have demonstrated nonsegmental innervation patterns in the
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rat lumbar spine [19–22]. Morinaga et al. [19] reported that the ventral portion of the L5–L6 disc was innervated by DRG neurons in L1 and L2. Ohtori et al. [21] reported the DRG neurons innervating the dorsal portion of L5–L6 disc were distributed to multilevel (T13–L6) DRGs. However, the innervation patterns in the lateral portion of the lumbar intervertebral disc have not been clarified. Usually, disc hernia is preceded by one or more attacks of low back pain [23]. Even if the disc hernia is localized in the lateral portion of the disc, the low back pain sometimes appears bilaterally. Furthermore, Slipman et al. [24] reported a random correlation between the side of the patient’s concordantly painful annular tear and the side of the patient’s pain. We hypothesized that nociceptive information from the lateral disc is transmitted to both ipsilateral and contralateral DRGs. It is generally recognized that DRG neurons are divided into two main subpopulations, the small and large neurons [25]. The large neurons are thought to be involved mainly in proprioception. In contrast, most small DRG neurons are thought to be involved in nociception. To examine whether there are some small, nociceptive DRG neurons innervating the contralateral side of the disc, we investigated the distribution and cross-sectional areas of DRG neurons innervating the lateral disc in rats, using a retrograde tracing method. We also hypothesized that the mechanism of innervation was through paravertebral sympathetic trunks. If that hypothesis is correct, it would explain why the blockade of paravertebral sympathetic trunks was effective for some patients with discogenic disorders [26]. To test the hypothesis, we examined the effect of sympathectomy on the distribution of the DRG neurons. In the sympathectomized rats, the DRG neurons innervating the disc through paravertebral sympathetic trunks would not be labeled by the neurotracer. This approach, therefore, elucidates the pathway of axons of the DRG neurons innervating the lateral disc.
was immediately sealed by cyanoacrylate to prevent leakage of F-G. Sympathectomized group (n⫽6) F-G was applied to the same site as in the nontreated group just after the removal of the lumbar paravertebral sympathetic trunks bilaterally from the L3 to L5 levels. Microscopic observation and analysis Seven days after the F-G application, the rats were reanesthetized with sodium pentobarbital and perfused transcardially with 0.9% saline, followed by 500 ml of 4% paraformaldehyde in phosphate buffer (0.1 M; pH 7.4). After laminectomy, bilateral DRGs from T12 to L6 were resected. The specimens were immersed in the same fixative overnight at 4 C, after which they were transferred to 20% sucrose overnight. Each DRG was serially sectioned at 40-µm thickness on cryostat. All the sections were mounted on slides and then coverslipped using Permafluor (Shandon, Inc., Pittsburgh, PA). The sections were observed with a Nikon Eclipse fluorescent microscope (Tokyo, Japan). Only F-G– labeled DRG neurons that had a clear visible nucleus were counted using filter UV-1A (wavelengths of 365 nm for excitation and 420 nm for emission; Fig. 1). To determine
Materials and methods We used 22 male Sprague-Dawley rats weighing 250 to 300 g. The rats were anesthetized with sodium pentobarbital (40 mg/kg, intraperitoneally) and treated aseptically in all surgical procedures. This study was conducted with the permission of the Ethical Committee for Animal Experiments of Chiba University School of Medicine. Nontreated group (n⫽16) A midline abdominal incision was made, and the retroperitoneum was incised. The left iliopsoas muscle was dissected to expose the left lateral aspect of the L5–L6 intervertebral disc. A perforation approximately 0.5 mm in depth was made on the surface of the disc by a 23-gauge needle, and crystals of Fluoro-Gold (F-G; Fluorochrome, Denver, CO) were inserted. The application site was the most lateral point of the left side of the disc. The application site
Fig. 1. Photomicrographs of Fluoro-Gold–labeled neurons in the nontreated group. (a) A small-size neuron (525 µm2) observed in the right L1 dorsal root ganglion (DRG). Scale bar⫽20 µm. (b) An intermediate-size neuron (1,115 µm2) observed in the left L5 DRG. Scale bar⫽20 µm.
Y. Aoki et al. / The Spine Journal 4 (2004) 275–280
whether the F-G–labeled neurons are large, proprioceptive neurons or small, nociceptive neurons, we measured the cross-sectional areas of the labeled neurons in seven rats of the nontreated group and in six rats of the sympathectomized group using a computer-assisted imaging analysis system (NIH Image software, Springfield, VA).
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The segmental distribution of the F-G–labeled neurons is shown in Fig. 2. Sympathectomized group F-G–labeled neurons were observed in the left DRGs from L1 to L5 and in the right DRGs from L3 to L5 (Table 2). No labeled neurons were observed in the left T12, T13 and L6 DRGs. Compared with the nontreated group, the numbers of labeled neurons in the left L1 and L2 DRGs were significantly lower than those in the nontreated group (Fig. 2a), and no labeled neurons were observed in the right T13, L1 and L2 DRGs (Fig. 2b). The segmental distribution of the labeled neurons is shown in Fig. 2.
Statistical analysis We compared the numbers of the F-G–labeled neurons in the DRGs of the same level between the nontreated and sympathectomized groups using nonpaired Welch’s t test. We also compared the mean cross-sectional area of the FG–labeled neurons in the left DRGs and the right DRGs using Welch’s t test. A p value of less than 0.05 was considered to be significant.
Cross-sectional area of cell profiles We measured the cross-sectional area of the cell profiles in seven rats of the nontreated group. The labeled neurons were round or oval (Fig. 1), and their cross-sectional area was within the range of 200 to 2,000 µm2 (Fig. 3). The mean cross-sectional area of the cell profile (mean⫾SD) was 766⫾317 µm2 in the left T13–L5 DRGs and 729⫾193 µm2 in the right T13, L1, L2 and L4 DRGs. No significant differences were found between that of the left DRGs and the right DRGs. If neurons smaller than 800 µm2 were defined as small-size neurons, the ratio of the small-size neurons was 62.3% in the left DRGs and 50% in the right DRGs.
Results Nontreated group F-G applied to the lumbar disc was retrogradely transported to the DRG neurons innervating the disc (Fig. 1). We found 321 F-G–labeled neurons in the 16 rats examined (Table 1). The minimum number was 8, and the maximum number was 50 within a rat. Considering 2,000 to 3,500 neurons are present in a DRG [27], the number of the DRG neurons innervating the lumbar disc was relatively small. F-G–labeled neurons were present in the bilateral DRGs from T13 to L5. No labeled neurons were observed in T12 and L6 DRGs (Table 1). Of the labeled neurons, 93.1% were present in the left (ipsilateral) DRGs, and 6.9% were present in the right (contralateral) DRGs, with the largest number of labeled neurons found in the left L2 DRG.
Discussion There are several reports about the segmental distribution of DRG neurons innervating the lumbar intervertebral disc [10,19–22,28]. Morinaga et al. [19] reported on the innervation pattern of the ventral portion of the L5–L6 disc using
Table 1 Number of Fluoro-Gold–labeled neurons in dorsal root ganglia of the nontreated group (n⫽16) Rat
T12
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Total
0 (0, 0)
(0, (0, (0, (0, (0, (0, (0, (0, (0, (0, (0, (0, (0, (0, (0, (0,
T13 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0)
4 1 0 1 0 0 0 1 2 0 2 1 2 1 1 5
(3, (0, (0, (1, (0, (0, (0, (1, (2, (0, (2, (1, (1, (1, (1, (4,
L1 1) 1) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 1) 0) 0) 1)
21 (17, 4)
10 3 2 1 3 0 1 3 1 2 2 5 5 0 3 8
L2 (10, 0) (3, 0) (2, 0) (1, 0) (2, 1) (0, 0) (1, 0) (3, 0) (1, 0) (2, 0) (2, 0) (4, 1) (4, 1) (0, 0) (3, 0) (7, 1)
49 (45, 4)
4 1 9 5 9 8 9 5 2 13 10 15 5 7 3 19
L3 (4, 0) (1, 0) (9, 0) (5, 0) (6, 3) (8, 0) (8, 1) (5, 0) (2, 0) (12, 1) (10, 0) (14, 1) (5, 0) (7, 0) (3, 0) (18, 1)
124 (117, 7)
2 1 7 0 1 4 6 7 4 4 13 5 3 8 0 4
L4 (2, 0) (1, 0) (7, 0) (0, 0) (1, 0) (4, 0) (5, 1) (7, 0) (3, 1) (4, 0) (13, 0) (5, 0) (3, 0) (8, 0) (0, 0) (4, 0)
69 (67, 2)
1 0 2 1 4 11 0 0 0 0 0 1 1 0 2 8
L5 (1, 0) (0, 0) (1, 1) (1, 0) (3, 1) (11, 0) (0, 0) (0, 0) (0, 0) (0, 0) (0, 0) (1, 0) (1, 0) (0, 0) (2, 0) (6, 2)
31 (27, 4)
0 2 0 1 2 5 1 2 0 0 1 2 3 1 1 6
L6 (0, (2, (0, (1, (1, (5, (1, (2, (0, (0, (1, (2, (3, (1, (1, (6,
0) 0) 0) 0) 1) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0)
27 (26, 1)
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
(0, (0, (0, (0, (0, (0, (0, (0, (0, (0, (0, (0, (0, (0, (0, (0,
Total 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0)
0 (0, 0)
3 (2, 1) indicates a total of 3 Fluoro-Gold–labeled neurons, 2 of which were observed on the left side and 1 on the right side.
21 8 20 9 19 28 17 18 9 19 28 29 19 17 10 50
(20, 1) (7, 1) (19, 1) (9, 0) (13, 6) (28, 0) (15, 2) (18, 0) (8, 1) (18, 1) (28, 0) (27, 2) (17, 2) (17, 0) (10, 0) (45, 5)
321 (299, 22)
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innervated mainly by the L1, L2 and L3 DRG neurons in rats revealed that nociceptive information from any part of the L5–L6 disc is transmitted mainly by the L1 and L2 DRG neurons. The fact that disc lesions most frequently occur at the L4–L5 level in humans, and the L5–L6 level in rats corresponds to the L4–L5 level in humans anatomically, may be one of the explanations why L2 spinal nerve infiltration was effective in treating a patient with discogenic low back pain [29]. Using horseradish peroxidase, Forsythe and Ghoshal [10] have reported the innervation patterns of the dorsolateral portion of the thoracolumbar disc in dogs. In their study, the neurotracer application site was just lateral to the edge of the posterior longitudinal ligament. They reported that some labeled neurons were present in contralateral DRGs. However, they did not describe the ratio of the labeled neurons in the contralateral DRGs. In the present study, we chose the most lateral point of the disc as the application site to clarify the laterality of the innervation patterns in rats. Our test revealed that 6.9% of the sensory fibers in the lateral portion of the disc were derived from DRGs on the contralateral side. Additionally, we measured the cross-sectional areas of the labeled neurons and discovered that some of the labeled neurons observed in DRGs on the contralateral side to the F-G application belonged to a subgroup of small, nociceptive neurons. Although we have not performed immunostaining of the neurotransmitters of nociceptive information, such as SP and CGRP, these results indicate that nociceptive information from the lateral disc can be partially transmitted to contralateral DRGs. It has been reported that some sensory fibers in the lumbar dura mater and the posterior longitudinal ligament are derived by means of the sympathetic trunks in rats [20,26,30– 32]. Suseki et al. [33] have demonstrated the presence of the sensory fibers in lumbar intervertebral discs passing through rami communicantes. Ohtori et al. [21] reported that there were two distinctive pathways from the dorsal portion of the L5–L6 disc to DRGs. One was the pathway through the sinuvertebral nerves, and the other was through the paravertebral sympathetic trunks. In the present study, the number of the F-G–labeled neurons in the ipsilateral T13–L2 DRGs in the sympathectomized group was significantly less than that of the nontreated group. These results
Fig. 2. (a) The segmental distribution of Fluoro-Gold–labeled neurons in the left (ipsilateral) dorsal root ganglia (DRGs) from T12 to L6. (b) The distribution of the mean number of Fluoro-Gold–labeled neurons in the right (contralateral) DRGs from T12 to L6. Mean⫾SE, *p⬍0.01 (unpaired Welch’s t test).
horseradish peroxidase and choleratoxin B subunit as tracers and concluded that the ventral portion of the lumbar disc was innervated by the L1 and L2 DRGs. Using F-G, Ohtori et al. [21] demonstrated that the dorsal portion of the L5– L6 disc was innervated by the T13–L6 DRGs multisegmentally and also mainly by the L1 and L2 DRGs. Our demonstration that the lateral portion of the L5–L6 disc was
Table 2 Number of Fluoro-Gold–labeled neurons in dorsal root ganglia of the sympathectomized group (n⫽6) Rat
T12
1 2 3 4 5 6 Total
0 0 0 0 0 0 0
(0, (0, (0, (0, (0, (0, (0,
T13 0) 0) 0) 0) 0) 0) 0)
0 0 0 0 0 0 0
(0, (0, (0, (0, (0, (0, (0,
L1 0) 0) 0) 0) 0) 0) 0)
0 1 0 0 0 0 1
(0, (1, (0, (0, (0, (0, (1,
L2 0) 0) 0) 0) 0) 0) 0)
1 2 1 1 2 5 12
L3 (1, 0) (2, 0) (1, 0) (1, 0) (2, 0) (5, 0) (12, 0)
0 3 1 4 7 3 18
L4 (0, 0) (3, 0) (1, 0) (4, 0) (7, 0) (2, 1) (17, 1)
3 0 2 0 9 9 23
L5 (3, 0) (0, 0) (2, 0) (0, 0) (8, 1) (8, 1) (21, 2)
1 3 1 0 6 6 17
L6 (1, 0) (3, 0) (1, 0) (0, 0) (6, 0) (5, 1) (16, 1)
0 0 0 0 0 0 0
3 (2, 1) indicates a total of 3 Fluoro-Gold–labeled neurons, 2 of which were observed on the left side and 1 on the right side.
(0, (0, (0, (0, (0, (0, (0,
Total 0) 0) 0) 0) 0) 0) 0)
5 9 5 5 24 23 71
(5, 0) (9, 0) (5, 0) (5, 0) (23, 1) (20, 3) (67, 4)
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L5–L6 disc to the contralateral DRGs is present in paravertebral sympathetic trunks for the T13 to L2 DRGs and in the ventral primary rami or sinuvertebral nerves for the L3–L5 DRGs. These findings may have shown one of the reasons why lumbar sympathetic blocks sometimes relieve a patient’s low back pain [26]. It has been reported that the nerve fibers in the intervertebral disc are very sparse [3,9]. In the present study we demonstrated the presence of small nociceptive DRG neurons innervating the contralateral side of the disc in rats. There remains the possibility that there are differences in innervation patterns between humans and rats. However, if a similar innervation pattern is present in humans, the patient with a lateral annular tear may feel a pain in the bilateral low back.
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Fig. 3. (a) The size (cross-sectional area) of the Fluoro-Gold–labeled neurons observed in the left (ipsilateral) dorsal root ganglia (DRGs; 162 neurons in the nontreated group). (b) The size (cross-sectional area) of the FluoroGold–labeled neurons observed in the right (contralateral) DRGs (10 neurons in the nontreated group). The cross-sectional area of the neurons was in the range of 201 to 1,000 µm2. Most of them belonged to the subgroup of small- to intermediate-size neurons.
suggest that there were also two distinctive pathways of the sensory nerve fibers in the lateral portion of the disc. The sensory fibers originating in the T13–L2 DRGs were thought to pass mainly through the paravertebral sympathetic trunks, and those originating in the L3–L5 DRGs pass mainly through the ventral primary rami of the spinal nerves or sinuvertebral nerves. In the nontreated group, F-G–labeled neurons were present in the contralateral T13–L5 DRGs. In contrast, in the sympathectomized group, the F-G–labeled neurons were present in the contralateral L3–L5 DRGs but not in the contralateral T13–L2 DRGs. These results suggest that the afferent pathway from the lateral portion of the
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Naylor, 5 years later, described the dynamic role of weight-bearing influence on the deformed disc in the foramen of the stenotic spine [3].
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