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Complete Freund’s Adjuvant-Induced Intervertebral Discitis as an Animal Model for Discogenic Low Back Pain

Lee, Min, MSc*; Kim, Byung-Jo, MD, PhD; Lim, Eun Jeong, MSc; Back, Seung Keun, PhD; Lee, Ju-Han, MD, PhD§; Yu, Sung-Wook, MD, PhD; Hong, Sung-Ha, MSc; Kim, Joo Han, MD, PhD|; Lee, Sang-Heon, MD, PhD; Jung, Woon-Won, PhD#; Sul, Donggeun, PhD*; Na, Heung Sik, MD, PhD

Section Editor(s): Yaksh, Tony L.; Hogan, Quinn H.

doi: 10.1213/ane.0b013e3181b31f39
Analgesia: Pain Mechanisms: Research Reports
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BACKGROUND: Although numerous animal models for low back pain associated with intervertebral disk (IVD) degeneration have been proposed, insufficient data have been provided to make any conclusions regarding pain. Our aim in this study was to determine the reliability of complete Freund’s adjuvant (CFA) injection into the rat spine as an animal model representing human discogenic pain.

METHODS: We studied IVD degenerative changes with pain development after a 10-μL CFA injection into the L5-6 IVD of adult rats using behavioral, histologic, and biochemical studies. Serial histologic changes were analyzed to detect degenerative changes. Expression of calcitonin gene-related peptide (CGRP), prostaglandin E (PGE), and inducible nitric oxide synthase (iNOS) were determined using immunohistochemistry or real-time polymerase chain reaction as support data for pain development. In addition, CGRP immunoreactivity (ir) at the IVD was considered indirect evidence of neural ingrowth into the IVD.

RESULTS: There was a significant increase of the hindpaw withdrawal response in the CFA group until 7 wk postoperatively (P < 0.05). Histologic analyses revealed progressive degenerative changes of the disks without any damage in adjacent structures, including nerve roots. In the CGRP-ir staining study, the bilateral dorsal horns and IVD had positive ir after intradiscal CFA injection. CGRP mRNA expression was increased in the dorsal root ganglion (DRG) at 2 and 4 wk, whereas PGE and iNOS mRNAs were markedly increased at 2 wk. The increment of CGRP expression was higher in allodynic rats compared with nonallodynic rats.

CONCLUSION: Intradiscal CFA injection led to chronic disk degeneration with allodynia, which was suggested by pain behavior and expression of pain-related mediators. The increment of CGRP, PGE, and iNOS also suggest pain-related signal processing between the IVD and the neural pathway in this animal model. This animal model may be useful for future research related to the pathophysiology and development of novel treatment for spine-related pain.

From the *Department of Biomedical Science, Korea University Graduate School; Departments of †Neurology, ‡Physiology, §Pathology, |Neurosurgery, and ¶Rehabilitation Medicine, Korea University College of Medicine; and #Clinical Genomic Institute, Korea University College of Health Science, Seoul, Korea.

Accepted for publication June 4, 2009.

The first two authors (ML and B-JK) contributed equally to this study as co-first authors.

Supported by the Korea Research Foundation Grant, funded by the Korean Government (MOEHRD, Basic Research Promotion Fund; KRF-2006-311-E00080).

Address correspondence and reprint requests to Heung Sik Na, MD, PhD, Department of Physiology, Korea University College of Medicine, 126-1, Anam-dong 5 ga, Seongbuk-gu, Seoul 136-701, Korea. Address e-mail to hsna@korea.ac.kr.

Low back pain (LBP) is one of the most common reasons for physician office visits, with a lifetime prevalence of 60%–90%.1,2 Although any of the spinal structures can be a source of LBP, one likely cause of nonspecific LBP is lumbar intervertebral disk (IVD) disease. IVD degeneration is asymptomatic in most individuals; however, pathologic degeneration with the premature loss of architectural, biological, and biomechanical properties of the IVD can lead to chronic LBP. Despite the various treatment modalities that have been developed for chronic discogenic pain over the last 40 yr, clinical efficacy remains controversial. Furthermore, the pathophysiologic mechanism underlying symptomatic degeneration of the IVD is obscure, although nociceptive neural fiber ingrowth into deeper parts of the IVD after injury, combined with degeneration, is one of the most widely accepted pathomechanisms for chronic discogenic pain.3,4

In an attempt to elucidate the mechanisms underlying discogenic pain related to IVD degeneration, numerous animal models have been proposed.5 However, the studies using these models have provided insufficient data to make any conclusions regarding pain, even though the animal models have shown significant degeneration of IVD. The relationship between disk morphology and clinically significant discogenic pain remains controversial.6–8 Both disk degeneration and nociceptive neural fiber ingrowth are secondary changes after disk injury; however, disk degeneration and nociceptive neural fiber ingrowth are not always combined. The finding of degenerative disks on magnetic resonance images of asymptomatic subjects does not predict subsequent development of pain, even after several years. Therefore, the previous disk degenerative animal models may not accurately simulate discogenic pain in humans.

Herein, we present a method for producing chronic discogenic pain in rats. Complete Freund’s adjuvant (CFA) was injected into the 5th/6th lumbar (L5-6) IVD to induce chronic discogenic pain associated with degenerative change. CFA induces massive tissue destruction by inflammation, even in small amounts. Several studies have used the method of CFA injection into the IVD for inducing disk degeneration; however, these studies have not included pain measurement, which is the only tool with which to confirm whether pain has developed in the animal model.9,10

The purpose of this study was to determine the reliability of CFA injection into the rat spine as an animal model representing human discogenic pain. To assess its relevance, we performed behavioral, histologic, and biochemical studies.

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METHODS

Animals

Experiments were performed using male Sprague-Dawley rats weighing 200–250 g. The rats were housed in suspended wire mesh cages in a room maintained on a 12-h light/dark cycle (lights on at 07:00 h) and maintained at 22°C–25°C with free access to food and water. All experiments were approved and guided by the Animal Research Policies Committee in our institute.

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CFA Injection

While under general anesthesia (inhaled anesthesia with 0.5%–2% enflurane), the rats were treated aseptically during all surgical procedures. After a midline abdominal incision, a left transperitoneal approach to the spine was used to expose the left ventrolateral side of the L5-6 lumbar disk. Care was taken not to damage major blood vessels near the lumbar spine. The disk levels were identified using the pelvic rim as an anatomic landmark. The L5-6 IVD was then punctured with a 26-gauge needle immediately to the left of the anterior longitudinal ligament. Ten microliters of CFA (Sigma, St. Louis, MO) was injected for 10 min using an infusion pump to the disk (CFA group), and the operative field was then irrigated with normal saline to washout any possible remnants of CFA external to the IVD. The sham surgical procedure group was identical to the CFA group, except that CFA was not administered (sham group).

To investigate any possible CFA leakage that may influence the dorsal root ganglion (DRG), 10 μL of CFA mixed with 1% methylene blue was injected into the L4-S1 IVDs of three rats using the same methods. Twenty-four hours after injection, staining with methylene blue in adjacent structures was examined.

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Behavioral Test

To examine pain development by CFA injection, various pain behavioral tests were performed by a researcher who was blinded to group assignment.

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Mechanical Allodynia in the Hindpaw

Behavioral testing for mechanical allodynia in the CFA (n = 30) and sham (n = 23) groups was performed 1 day before surgery, 1 and 7 days postoperatively (PO), weekly until 8 wk PO, and then 10 wk PO. Mechanical allodynia was assessed by the hindpaw withdrawal threshold in response to probing with a series of calibrated von Frey filaments (3.92, 5.88, 9.80, 19.60, 39.20, 58.80, 78.40, and 147.00 mN [equivalent in grams to 0.4, 0.6, 1.0, 2.0, 4.0, 6.0, 8.0, and 15.0, respectively]; Stoelting, Wood Dale, IL). The 50% withdrawal threshold was determined using the up-down method.11

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Weight Loading Measurement

Based on an idea that LBP affects gait in patients, a special test device12 was used to measure the weight bearing of the rat paws in the CFA (n = 5) and sham (n = 5) groups. This apparatus was composed of a starting box, an arrival box, and a path between both boxes. The floor of the path consisted of eight acrylic plates (5 × 10 cm in width and length, respectively), which are lined up in two rows of four, attached to load cells (Dana Load Cell LTD, Seoul, Korea) that were connected to a computer through an amplifier. This test was initiated by placing the rat in the starting box. While the animal was walking through the path, the investigator identified the plates on which the rat stepped with both hindpaws. Output signals from the load cells attached to these plates were selected for plotting of time-weight curves. The signals generated by improper stepping were repeated two or three times until five time-weight curves were obtained for both hindpaws. The test was performed 1 day before surgery and 7 and 14 days PO. The result was used for statistical analysis after transforming the ratio of the weight-bearing value to body weight in each subject.

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Thermal Allodynia in the Rat Tail

The tail-flick response to hot (40°C) and cold (4°C) water that was developed as a behavioral test in a neuropathic pain rat model13 was performed in the CFA (n = 5) and sham (n = 5) groups. The test was performed 1 day before surgery and 7 and 14 days PO. The latency to a tail-flick response after dipping the tail into water was measured with a cutoff time of 15 s. Each test was repeated five times, and the averaged results were used for analysis.

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Mechanical Allodynia in Low Back

The responses to probing with a von Frey filament (2.0 g) were evaluated in the CFA (n = 5) and sham (n = 5) groups. We applied the filament on the back of the spinous process of the L5 or L6 vertebra. After 10 applications, the number of withdrawal responses or screaming sounds was measured 1 day before surgery and 7 and 14 days PO. The occurrence of responses was expressed as a percentage.

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Histology

Two, 4, and 8 wk after the CFA injection, each rat (n = 3 for each time period) was killed to observe the pathologic changes in the IVD. The sham animals (n = 3) were killed 8 wk after the sham operation. The lumbar spines were removed from the rats and the disks, together with intact adjacent vertebral body bone, were fixed in 10% neutral buffered formalin for 1 wk, decalcified in EDTA, and processed for paraffin sectioning. Blocks of tissue were embedded in paraffin and cut into sagittal and horizontal sections (5 μm in thickness) using a microtome. The sections were stained with hematoxylin and eosin and analyzed qualitatively under a light microscope (B × 50; Olympus) at magnifications ranging from 12.5 to 100× for evidence of changes in the nucleus pulposus (NP), annulus fibrosus (AF), and adjacent structures, including the nerve roots and vertebral body bone.

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Immunohistochemistry

Calcitonin Gene-Related Peptide (CGRP)-Immunoreactivity (ir) in the Superficial Dorsal Horn

Four weeks after CFA injection and the sham operation, rats (n = 6 for each group) were perfused transcardially with normal saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS [pH 7.4]). Spinal cord segments at the level of L5 were removed and postfixed for 6–8 h in the same fixative. The spinal cord segments were cryoprotected with 30% sucrose and horizontally cut with a freezing microtome (HM 550; MICROM; Thermo Fisher Scientific Inc., Waltham, MA) into 15-μm sections. Immunohistochemistry was performed on free-floating sections with anti-CGRP (1:80,000; Peninsula Lab, Belmont, CA) according to the avidin biotin complex method (Vector Elite Kit; Vector, Burlingame, CA). Before staining, sections were rinsed with 1% bovine serum albumin and 10% normal goat serum (NGS) for 1 h and incubated in primary antibodies for 48 h at 4°C.

Sections were rinsed in 0.05 M PBS and 3% NGS. The secondary antibody was biotinylated goat anti-rabbit IgG. The avidin-biotinylated horseradish peroxidase complex was detected using 0.05% diaminobenzidine containing 0.01% hydrogen peroxide. Sections were dehydrated with graded alcohol, cleared in xylene, and coverslipped. The tissues from the CFA and sham groups were processed in parallel.

We measured the density of CGRP-ir in two spinal cord sections from each rat using a computer-assisted image analysis system (NIH Image Software, Bethesda, MD). Images of the spinal cord sections were captured with a 34× objective and a charged coupled device camera, and converted to digital images with a gray value ranging from 0 to 255. For each spinal cord section, we calculated the mean density of the superficial dorsal horn (laminae I–IV; gray areas in Fig. 4D) by subtracting the background density (areas of laminae VI and VII; Fig. 4D) from the mean density of laminae I–IV.

Figure 4.

Figure 4.

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CGRP-ir in IVD

To confirm the presence of nerve fiber ingrowth into the CFA-injected IVD (L5-6), we reacted paraffin-embedded sagittal sections from rats (n = 4) that were killed 4 wk after CFA injection, including the CFA-injected disk and adjacent disks (as control disks) of the upper and lower levels with anti-CGRP (1:50). The paraffin blocks were cut into sections, 6-μm thick. After mounting the sections on glass slides, the sections were dewaxed in xylene and rehydrated in descending grades of ethanol. After rinsing in PBS three times, antigen retrieval was achieved using the pressure cooking method in 10 mM citrate buffer. To quench endogenous peroxidase reactivity, the retrieved tissues were treated with 3% hydrogen peroxide and 10% methanol. The sections were pretreated with 1% bovine serum albumin and 10% NGS for 20 min, then incubated in primary antibody for 24 h. The subsequent steps were the same as the immunostaining procedures of the spinal cord, as described earlier. The omission of the primary antibodies was used as a negative control.

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Tissue Collection and Isolation of RNA

Based on the behavioral test results, allodynic rats were selected if they showed robust mechanical allodynia (>50% decrease in withdrawal threshold compared with baseline). Among the allodynic rats of the CFA group, three rats with a greater degree of decrement in the withdrawal threshold at each time period (2, 4, and 8 wk) were killed. The rats in the sham group were also killed at the same time period (n = 3 for each time period). For euthanasia, animals were injected intraperitoneally with an overdose of sodium pentobarbital. Both sides of the L5 DRG were removed aided by a surgical microscope. Tissues were frozen at −80°C until RNA isolation. Total RNA was isolated from rat DRG tissues using TRIzol reagent (Invitrogen Corp., Carlsbad, CA) following the manufacturer’s protocol and stored at −80°C until further processing. The concentration and purity of the total RNA were determined spectrophotometrically at 260 nm and were assessed using an Agilent™ Bioanalyzer (Agilent, Palo Alto, CA).

To investigate the correlation between the pain behavior results and the expression of CGRP in the DRG, we selected the three most allodynic rats and three nonallodynic rats (<20% decrease in withdrawal threshold compared with baseline) in CFA-injected rats, and isolated total RNAs for each time period with the same methods as described earlier.

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Real-Time Polymerase Chain Reaction

Real-time polymerase chain reaction (RT-PCR) was performed to verify the differential expression of CGRP, prostaglandin E (PGE), and inducible nitric oxide synthase (iNOS) using a Roche LightCycler system (Roche Diagnostics, GmbH Mannheim, Germany) and the SYBR Green method. The relative gene expression was determined by using the comparative CT method. All reactions were performed in a total volume of 20 μL of reaction mixture containing 2.0 μL of 10X buffer (50 mM KCl, 10 mM Tris-HCl [pH 8.3], and 0.001% gelatin), 2.0 μL of 2.5 mM MgCl2, 1.6 μL of 2.5 mM dNTP, 1.0 μL of 5′ primer (10 pmol/μL), 1.0 μL of 3′ primer (10 pmol/mL), 0.4 μL of amplitaq (5 U/mL), a 10,000-fold dilution of 1.0 μL of SYBR Green I (molecular probes), 1.0 μL of cDNA, and 10.0 μL of sterile water. The thermal cycling conditions for PCR were an initial denaturation for 3 min at 95°C, followed by 40 cycles of 52°C for 5 s, and 72°C for 12 s. The conditions for β-globin were initial denaturation for 3 min at 95°C, followed by 40 cycles of 54°C for 5 s, and 72°C for 12 s. For the RT-PCR analysis, PCRs were performed for each cDNA sample, and all PCR products were run on 2.0% agarose gel to confirm the presence of a single amplicon. Negative controls (except templates) were included in the PCR reaction to ensure specific amplification. LightCycler software version 4.0 (Roche) was used for the analysis of the quantitative PCR. The authenticity of the PCR products was confirmed by melting curve analysis using LightCycler software. The values obtained from each sample were normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA expression. Levels of each gene expression in all experimental groups were compared with the levels of the expression of the sham group. Those experiments were repeated three times to verify consistent results.

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Statistical Analysis

Repeated one-way analysis of variance, Friedman rank test, Mann–Whitney U-test, and pairwise post hoc comparisons were performed to determine whether there were any significant differences across the behavioral test scores obtained from a given animal group on different experimental days, and the data were expressed as the mean ± sem. For analysis of CGRP expression at the superficial dorsal horn between the CFA injection group and the sham group, the Mann–Whitney U-test was performed, and the data were expressed as the mean ± sd. Time-based differences of CGRP, iNOS, and PGE were analyzed using the Kruskal–Wallis test, and the Tukey honestly significant difference was used for post hoc analysis. A P <0.05 was considered significant.

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RESULTS

CFA Leakage

A sparse amount of CFA leakage was observed during needle removal from the IVD; however, no CFA leakage was observed during injection or even after needle removal. On dissection 24 h after injection, IVDs were well stained with methylene blue, whereas adjacent structures including the DRG, nerve root, and spinal cord were not stained at all (Fig. 1).

Figure 1.

Figure 1.

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Behavioral Study

Mechanical Allodynia in the Hindpaw

As shown in Figure 2, the CFA group showed a marked decrease in the withdrawal threshold of the bilateral hindpaws, whereas the sham group did not exhibit such changes. Before injection, the left and right hindpaw withdrawal thresholds were near 15 g in both the CFA and sham groups. However, in the CFA group, the withdrawal thresholds in both hindpaws 1–49 days after CFA injection were significantly lower than not only the withdrawal thresholds before CFA injection (*P < 0.05 versus preinjection value [Friedman repeated analysis of variance followed by the pairwise post hoc test]) but also the withdrawal thresholds of the sham group (#P < 0.05 versus the sham group [Mann–Whitney U-test]). Thus, these results suggest that CFA injection into the L5-6 IVD resulted in mechanical allodynia.

Figure 2.

Figure 2.

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Weight Bearing

Both groups showed no significant differences in the ratio of weight-bearing values to body weight after surgery compared with baseline. The weight load in the CFA group was 40.2% ± 0.8% at baseline, 39.4% ± 0.75% at 7 days, and 40.0% ± 0.71% at 14 days after CFA injection. The sham group had 39.6% ± 1.03% at baseline, 40.0% ± 0.45% at 7 days, and 40.0% ± 0.71% at 14 days after surgery.

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Thermal Allodynia in Rat Tail

The CFA and sham groups did not show any statistical differences in the mean latency (seconds) after surgery (14.4 ± 0.4 and 14.8 ± 0.2 at 7 and 14 days in the CFA group versus 14.6 ± 0.4 and 14.8 ± 0.2 in the sham group, respectively) compared with baseline (14.4 ± 0.4 in the CFA group and 14.6 ± 0.4 in the sham group).

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Mechanical Allodynia in the Low Back

The CFA and sham groups did not show any statistical differences in the mean response frequency after surgery (64% and 48% at 7 and 14 days in the CFA group versus 58% and 52% in the sham group, respectively) compared with baseline (52% in the CFA group and 46% in the sham group).

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Histology

The NP of sham-operated disks, which were obtained 8 wk PO, showed prominent notochordal cells with a smaller portion of chondrocyte-like cells, and the AF was characteristically well organized with lamellar sheets of collagen (Fig. 3A). However, the NP 2 wk after CFA injection exhibited a markedly decreased number of notochordal cells and proliferation of chondrocyte-like cells, whereas the AF was relatively well organized (Fig. 3B). In addition, the NPs 4 and 8 wk after CFA injection showed a loss of notochordal cells and were replaced by chondrocyte-like cells. Furthermore, the collagen layers of the AFs were intermingled with the fibrocartilagenous portions of the NPs (Figs. 3C–E). However, there was no evidence of current or previous inflammatory reactions in adjacent structures, including nerve roots and the epidural space, 2, 4, and 8 wk after the CFA injection. Those findings were observed consistently in every specimen.

Figure 3.

Figure 3.

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Immunohistochemistry

CGRP-ir in the Superficial Dorsal Horn

The CGRP-ir of the superficial dorsal horns of the spinal cord at the L5 level was higher in the CFA group (124.85 ± 11.48) compared with the sham group (111.21 ± 4.98, P = 0.047; Fig. 4). However, there was no difference between the two sides of the dorsal horn (data not shown).

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CGRP-ir in the IVD

CGRP-ir nerve fibers were evident in the CFA-injected disks in three of four rats. CGRP-ir was mainly localized to the ventral parts of the degenerated IVDs. Nerve fibers stained with CGRP were present between adjacent lamellae of the AF (Fig. 5); however, adjacent normal disks did not contain CGRP-ir (data not shown).

Figure 5.

Figure 5.

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Real-Time Polymerase Chain Reaction

Figure 6 shows the change in expression of each gene in the DRG neurons at each timepoint in the CFA-treated allodynic rat group compared with the sham control group. Both PGE and iNOS mRNA expression had a marked increase at 2 wk (7.16- and 4.41-fold; P = 0.015 and P = 0.031, respectively) after CFA injection compared with the sham control group. However, the increases at 4 wk (1.72- and 1.83-fold, respectively) and 8 wk (1.38- and 2.04-fold, respectively) were not that high. CGRP mRNA expression was increased at 2 wk (2.83-fold; P = 0.049) and 4 wk (2.95-fold; P = 0.042), but decreased near the level of the control group at 8 wk (1.15-fold).

Figure 6.

Figure 6.

Comparing CGRP expression between allodynic and nonallodynic rats, CGRP expression was significantly increased in allodynic rats at 4 wk after CFA injection (P = 0.025; Fig. 7).

Figure 7.

Figure 7.

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DISCUSSION

In this study, we attempted to develop a disk-related pain model with CFA injection into the L5-6 IVD of rats. The model is characterized by the following: 1) disk degenerative changes, 2) an increase in the CGRP-ir of the superficial dorsal horns of the spinal cord at the level of L5, 3) increased expression of CGRP, iNOS, and PGE mRNA in the DRG, 4) CGRP-ir in the injected disk, and 5) indirect evidence of mechanical allodynia in the hindpaw.

We observed the expression of several local mediators and neurotransmitters, which are related to pain to verify development of the pain model. CGRP expression was significantly increased in the entire lamina I and II areas of the bilateral dorsal horns and the DRG in the CFA group. In addition, a significant increase in the expression of CGRP in allodynic rats compared with nonallodynic rats suggested that the increased expression was well correlated to pain behavior study results. Our study also showed a marked increase of PGE and iNOS in DRG 2 wk after CFA injection, which are well-known mediators to sensitize sensory neurons to maintain hyperalgesia. A study suggested that the NO and prostaglandin signaling pathways may interact in some way to regulate the inflammatory process, resulting in development of hyperalgesia.14 Persistent activation of nociceptors activates NO synthases,15 which suggests a link involving nitric oxide synthase and NO with pain. Other researchers have shown a significant increase in nitrate and nitrite levels in the sciatic nerves in the chronic neuropathic rat model and extensive expression of NO correlating with increasing pain intensity in chronic pain patients.15,16 These results support a close relationship between NO elevation and local pain processing, regardless of pain origin and type. Prostaglandins are also involved in inflammation, especially PGE and PGF, which have been recognized to contribute to the initiation and maintenance of primary hyperalgesia.1718 Based on the previous study results, this study results suggest that IVD inflammation and degeneration induced by CFA injection might induce increased release of local inflammatory mediators, resulting in stimulation of free nerve endings in the AF. The signal transmitted through free nerve endings in the IVD may evoke expression of PGE and iNOS in the DRG, resulting in development of allodynia as evidenced by an increased expression of CGRP in the DRG and spinal neurons.

It is generally recognized that there are no nerve fibers located in normal lumbar disks, except at the outermost part of the AF. However, under pathologic inflammatory conditions, some sensory nerve fibers extend into the inner layers of human IVD tissue where free nerve endings are stimulated, leading to pain symptoms.19–22 The nerve ingrowth into the inner layer of the IVD may be caused by some signals that provoke and direct the ingrowth. In one study, it was observed that there were higher levels of interleukin-6 and interleukin-8 in painful disks when compared with asymptomatic disks, regardless of the degree of degeneration, suggesting that production of proinflammatory mediators in the disk may be a major factor for the development of pain symptoms.23 The ir of CGRP observed in the ventral portion of the IVD in our study was consistent with the findings in IVDs in patients with painful degeneration.24 CGRP is found in unmyelinated, slow-conducting sensory nerves, which are believed to be involved in nociceptive transmission or neuromodulation. Although adjacent control disks did not reveal CGRP-ir, the presence of CGRP-ir in the CFA-injected disks indicates that the disks can be a source of pain sensation. Therefore, our study findings, such as histologic degeneration in the IVD, increased pain-related mediators in the DRG and spinal cord, and expression of CGRP in the IVD support the previously suggested pathomechanism that discogenic pain may be developed by stimulation of nociceptive neural fibers that grow into an injured IVD through an inflammatory reaction.

In this study, we applied various pain behavior measurements, including mechanical or thermal allodynia on the low back, tail, or hindpaws and paw pressure on the floor during gait. However, no measurements, except indirect measurement using the hindpaw withdrawal test, revealed reliable changes after CFA injection. Although LBP is a major symptom in patients with lumbar discogenic pain, many patients experience extremity pain. The development of pain along the lower limb has been demonstrated by stimulation of the IVD using intradiscal electrothermal annuloplasty.25 This phenomenon was attributed to referred pain because of the convergence of afferent neurons onto common neurons within the central nervous system. The L5-6 IVD of rats has been shown to be multi-segmentally innervated by the T13-L6 DRG. The sensory fibers from the upper lumbar DRG have been shown to innervate the L5-6 IVD through the paravertebral sympathetic trunks, whereas the sensory fibers from the lower lumbar DRG may innervate the L5-6 disk through the sinovertebral nerves.26 The hindpaw in which mechanical allodynia signs were shown in this study is mainly innervated by sensory fibers from the L4 and L5 DRG.27 Although any dermatomal area from T13-L6 could be used for mechanical stimuli, based on previous reports related to the sensory fiber distribution of the IVD, we applied the hindpaw withdrawal response, which is one of the most commonly used methods for demonstrating pain behavior in various neuropathic animal models.28–30

Despite this physiologic explanation for incremental hindpaw sensitivity in disk inflammation, the possibility of radiculopathic pain caused by direct nerve root inflammation because of leakage of CFA still remains. CFA, a water-in-oil emulsion containing heat-killed mycobacteria or mycobacterial cell wall components, is a very strong adjuvant to induce intense local inflammation at the site of the injection. Because of the massive destruction of tissue by inflammation at the injection site, research guidelines recommend significant caution and restriction in the use of this adjuvant.31 If hindpaw sensitivity was caused by direct inflammation of the nerve root by CFA leakage, histologic evidence would be noted at the nerve root. On histologic evaluation, the degenerative changes progressed slowly, resulting in complete loss of notochordal cells by 4 wk in the CFA injection group. However, adjacent structures, including nerve roots and the psoas muscles that cover the injection site of the IVD, were completely intact. In addition, we could not find any methylene blue stain in adjacent structures, except the IVD 24 h after injection of CFA mixed with methylene blue. This suggests that injection of CFA into the L5–6 IVD led to selectively significant degeneration of the disk, and the hindpaw sensitivity may not have been caused by direct nerve root inflammation. The negative response in various behavioral studies that were mostly used for evaluation of pain in radiculopathy or neuropathy models may also explain that the mechanism of hindpaw sensitivity is different from that of neuropathies or radiculopathies. Abnormal signals (persistent ectopic discharge) from neuromas or the DRGs are important for the central sensitization and the resultant generation of chronic neuropathic pain after peripheral nerve injury. Based on this hypothesis, our study results, which showed nerve fiber ingrowth into the IVD and increases in biochemical makers for pain in the DRG and spinal dorsal horn, may be related to central sensitization. In addition, nonnoxious mechanical stimuli applied to the plantar surface of the hindpaw may be interpreted as painful stimuli.

It has been estimated that each rat month of life is roughly equivalent to 2.5 human years.32 In this animal model, the slowly progressive degenerative changes and prolonged pain for 7 wk after CFA injection, which is roughly equivalent to 4–5 human years, are compatible with human chronic discogenic pain and degenerative morphologic changes. In addition, the decrement in pain behavior without improvement in degenerative changes after 8 wk could explain a spontaneous improvement of pain without morphologic improvement of the disk in patients with chronically degenerated disks. The decrement in pain behavior without improvement of degenerative changes after 8 wk may also explain the discrepancy between morphologic changes and symptoms in human disks. However, because the degeneration in this model was developed by inflammation using CFA, this model has no relevance to the pathophysiology of age-related spontaneous disk degeneration with LBP in humans.

Although we used the withdrawal response using von Frey hair at the hindpaws, which are remote from the CFA injection site of the L5–6 IVD, the response was significantly increased in the CFA injection group compared with the sham group. We cannot conclude that this is a pure discogenic pain model; however, we developed an animal model with disk degeneration induced by disk inflammation and observed that the model had significant pain behavior and expression of specific pain-related transmitters and mediators. A future study is needed to develop a more reliable direct pain measurement method related to discogenic LBP, but we believe our study will promote progress in research related to the pathophysiology and development of novel treatments for spine-related pain.

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ACKNOWLEDGMENTS

The authors thank Seok Joo Hong, MD, PhD for valuable comments in study design.

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