Optimization of a rat lumbar IVD degeneration model for low back pain

Abstract Introduction Intervertebral disc (IVD) degeneration is often associated with low back pain and radiating leg pain. The purpose of this study is to develop a reproducible and standardized preclinical model of painful lumbar IVD degeneration by evaluation of structural and behavioral changes in response to IVD injury with increasing needle sizes. This model can be used to develop new therapies for IVD degeneration. Methods Forty‐five female Sprague Dawley rats underwent anterior lumbar disc needle puncture at levels L4‐5 and L5‐6 under fluoroscopic guidance. Animals were randomly assigned to four different experimental groups: needle sizes of 18 Gauge (G), 21G, 23G, and sham control. To monitor the progression of IVD degeneration and pain, the following methods were employed: μMRI, qRT‐PCR, histology, and biobehavioral analysis. Results T1‐ and T2‐weighted μMRI analysis showed a correlation between the degree of IVD degeneration and needle diameter, with the most severe degeneration in the 18G group. mRNA expression of markers for IVD degeneration markers were dysregulated in the 18G and 21G groups, while pro‐nociceptive markers were increased in the 18G group only. Hematoxylin and Eosin (H&E) and Alcian Blue/Picrosirius Red staining confirmed the most pronounced IVD degeneration in the 18G group. Randall‐Selitto and von Frey tests showed increased hindpaw sensitivity in the 18G group. Conclusion Our findings demonstrate that anterior disc injury with an 18G needle creates severe IVD degeneration and mechanical hypersensitivity, while the 21G needle results in moderate degeneration with no increased pain sensitivity. Therefore, needle sizes should be selected depending on the desired phenotype for the pre‐clinical model.


| INTRODUCTION
Low back pain is a leading cause of disability and morbidity in the adult population, affecting 80% of adults within their lifetime. 1,2 Up to 40% of all low back pain is attributed to discogenic pain from intervertebral disc (IVD) degeneration. 3,4 While the pathophysiology of IVD degeneration is not fully understood, changes in the biological and morphological features as well as biomechanical function are known to be involved in this progressive disease. 5,6 The IVD consists of a central nucleus pulposus (NP), an outer annulus fibrosus (AF), and two vertebral endplates that connect the soft tissue with the vertebral body. 7 The NP plays a crucial role in the initiation and progression of IVD degeneration. Various studies demonstrated an increase in inflammatory and catabolic factors and a reduction in anabolic factors in degenerated IVDs in humans, 8,9 and animal models. 6,10 For example, an upregulation of matrix metalloprotease-(MMP)-3 and a reduction of Collagen II in NP tissue obtained from degenerated discs has been shown. 11,12 Moreover, reduction of NP hydration as a result of IVD degeneration can be shown using quantification of T2-weighted images obtained via magnetic resonance imaging (MRI). 13 Degenerative changes in the IVD are often associated with nerve ingrowth and hyper-innervation, whereas in normal IVDs only the periphery of the AF is innervated by nerve fibers. 14 In painful IVD degeneration, higher concentrations of nerve growth and innervationrelated peptides compared to painless degenerative IVDs were shown. 15,16 For example, IVDs obtained from patients with severe back pain and reduced disc height showed an increased amount of nerve fibers in the endplate region, which were immunoreactive for neuropeptides, such as Substance P. 15 This suggests that discogenic pain may be a result of changes in innervation. These changes have been shown to be induced by pro-inflammatory cytokines, including TNFα. 17,18 The importance of TNF-α in the induction of painful IVD degeneration has been recently shown in a study demonstrating that inhibition of TNF-α at the time of induced IVD injury limits long-term pain and degeneration in a rat model. 18 While imaging-based diagnosis including MRI provides detailed images of the IVD tissue, it fails to clearly differentiate between a pathologically painful disc and a disc that does not generate pain. 19,20 Due to the shortcomings of these imaging techniques, IVD degeneration models need to be additionally characterized by assessment of pro-nociceptive markers and painrelated behavior.
To date, there are no clinically validated methods for preventing or reversing IVD degeneration and associated discogenic pain. Current nonsurgical treatments include physical therapy and spinal injections with steroids for pain reduction. 21 Surgical treatments include spinal fusion or arthroplasty procedures to improve stabilization and to reduce pain. 22 Recently, injections of stem cells, growth factors and anti-inflammatory factors are being investigated as potential therapies to attenuate the progression of IVD degeneration. 18,23,24 Prior to testing these treatment modalities in the clinic, small rodent and large animal preclinical models are essential for proof-of-concept testing. 25 Mouse models provide the advantage of using transgenic animals as a powerful tool for mechanistic questions that can isolate parameters like single gene knockout [26][27][28] or aging. 29,30 Large animal models provide the size relevant to human tissues for more translational studies. [31][32][33][34] The rat model, due to its relatively small size, be used in large cohort studies, has relatively large discs compared with the mouse model that allows for injection of therapeutic agents, and supports biobehavioral testing which is a crucial component in studying discogenic pain. 18,25 To assess the progression of IVD degeneration and to test new treatments in rat models, disc needle puncture is frequently used to induce IVD degeneration, and has been previously performed in tail and lumbar discs. 35,36 The tail IVD puncture has the advantage that no spinal surgery is needed and that the needle can be inserted into the tail discs percutaneously. However, it does not take into account the anatomical and mechanical conditions of lumbar spine, the main site of IVD degeneration. 37 On the other hand, injuring the rat lumbar IVD requires an open surgical approach and can be more variable due to harder access. To ensure accurate and reproducible needle insertion into the targeted disc during lumbar spine surgery, use of fluoroscopy has been shown to be a valuable tool to capture of interoperative radiographic images in real time. 38 In addition to the differences in the spinal locations chosen for needle injury, several other factors largely vary between studies, including the needle diameter used, needle insertion site, path of insertion (eg, ventral vs lateral), depth of needle insertion and number of punctures per disc. 39 Controlling multiple factors contributing to the degeneration process is crucial to achieve consistent results and to allow for comparison between different studies.
There is multiple evidence of the efficacy of needle injury in inducing IVD degeneration and discogenic pain in a rat model, as shown by MRI, gene expression analysis, histology, and biobehavioral testing. 18,36,40,41 However, to our knowledge no comprehensive qualitative and quantitative assessment of the changes in the rat lumbar IVD structure, function and pain behavior in response to injury with different needle diameters has been published to date.
In this study, we hypothesize that there is a correlation between needle diameter used for rat lumbar IVD injury, IVD degeneration, and pain-related behavioral measures. Using a highly controlled surgical approach, we aim to demonstrate how lumbar IVD injury with increasing needle diameters affects disc dehydration, catabolic and anabolic factor regulation, disc morphology, as well as structural and functional pain in a rat model. The results of this study will not only help to create a reliable model to mimic moderate and severe disc degeneration but will also highlight differences across outcome measures related to the severity of IVD degeneration. As an additive value, the needle size that will not induce noticeable degeneration, may be marked as the maximal size that can be used for injection therapy to the disc.

| Study design
Forty-five healthy, female CD Sprague Dawley IGS rats (Charles River, Massachusetts), 10 to 12 weeks of age underwent anterior lumbar disc surgery in accordance to the institutional IACUC protocol (IACUC008089). Animals were randomly assigned to four different experimental groups: rats injured with needle sizes of 18 gauge (G) (n = 10), 21G (n = 11), 23G (n = 12), and rats that had sham surgery only (n = 12) ( Figure 1A). 18G and 21G needle sizes were chosen, since they were used in the past to induce IVD degeneration in tail and lumbar rat IVD without the requirement of additional intradiscal injections. 36,40,42 We added the 23G needle group to see if a smaller needle size would be sufficient to induce IVD degeneration in the lumbar spine as shown in rat caudal discs. 36 Under anesthesia and after incision, the anterior lumbar spine was exposed through a transperitoneal anterior approach ( Figure 1B). Under fluoroscopic guidance, each needle was inserted into the middle of the nucleus pulposus (NP, total depth of 2 mm, Figure 1C) of lumbar discs L4-L5 and L5-L6, held for 5 seconds and removed. Afterwards, the incision was closed, and warm fluids, and pain medication were administered. L4-5 and L5-6 were chosen for injury, since we aimed to investigate disc levels that best reflect the situation in humans: L4-5 and L5-S1 (L6 does not exist) are the discs that are most often degenerated and painful in humans. 43 Each of those discs has a unique size, and is exposed to different loading conditions that in turn affect the biological function of each IVD, as seen by a different frequency of degeneration per disc in patients. 43 To monitor the progression of degeneration of the matching spinal segments in the lumbar spine of the different animals, the following methods were employed: T1-and T2-weighted μMRI analysis performed presurgery and at 4-and 8-weeks postsurgery, biobehavioral analysis done presurgery and at 3-and 6-weeks postsurgery, qPCR to detect IVD degeneration and pain (MMP3 [catabolic marker], aggrecan, collagen II [anabolic makers], collagen I and CCN2 [fibrosis markers], TAC1 and TNFα [pro-nociceptive markers]), as well as histological stains (H&E and Alcian blue/Picrosirius Red) performed at week 8 postsurgery ( Figure 1A).

| Animal surgery
Animal experiments were performed in accordance to Cedars-Sinai's Institutional Animal Care and Use Committee-approved protocol (IACUC008089). Briefly, under inhalation anesthesia and after incision, an anterior transperitoneal approach to the lumbar spine was uti- lized. An abdominal vertical incision (5 cm) was made at the level of the lumbar spine and blunt dissection surgical techniques were used F I G U R E 1 Study design and surgical approach. A, In an anterior surgical approach, lumbar discs L4-5 and L5-6 were injured with different needle sizes (18G, 21G, and 23G). Per rat, only one needle size was inserted at each of two levels. Sham operated animals with no IVD injury served as controls. Prior to surgery and at 4-and 8-weeks post-surgery μMRI images were taken. Biobehavioral tests were performed prior to surgery and at 3-and 6-weeks postsurgery. After sacrifice at 8 weeks, spines were explanted and the spinal segments injured and uninjured L4-5, L5-6 were analyzed via qPCR and histology. Sac, day of sacrifice; BB, biobehavioral testing. B, Images of the anterior transperitoneal approach to the lumbar spine. C, A needle is inserted into the exposed lumbar disc L5-6. Radiographic image of a rat lumbar spine obtained during surgery to extend the exposure into the abdominal cavity ( Figure 1B,C). The intestines were deflected to the right to expose the abdominal aorta and the left kidney. Anatomical landmarks were then palpated to determine the spinal region to be exposed in upper caudal vertebrae.
The anterior edges of the spinal column were isolated from connective tissue and muscle. Prior to puncture, a mini C-arm was used to clearly identify the level of each IVD ( Figure 1C). Using one Gauge (G) of sterile needle per animal (18G, 21G, or 23G), a disc puncture of 2 mm in depth (to the middle of the IVD) was created in two levels L4-L5 and L5-L6. The desired insertion depth of 2 mm was ensured by surface marking of the needle tip. After the puncture was completed, homeostasis was assured, the peritoneal contents were replaced, and the peritoneum, rectus fascia, and skin were closed in layers. Sham animals underwent the same surgery, but without IVD needle injury. After surgery, the incision was closed, and warm fluids and pain medication (0.05 mg/kg buprenorphine, SC) were administered. Twelve hours after surgery, application of pain medication (0.05 mg/kg buprenorphine, SC) was repeated. Rats were single housed after surgery to minimize risk of injury through companions.
The rats' welfare was assessed daily. No infections, poor conditions, or drug related adverse events were detected.

| Micro-magnetic resonance imaging
To visualize the IVD structure and level of hydration, μMRI imaging, a small animal magnetic resonance imaging scanner, Bruker BioSpec

| Gene expression analysis
Total RNA was isolated from IVDs using TRIzolReagent (Thermo Fisher Scientific) and subsequent homogenization. RNA extraction was done from whole IVD, including the NP and AF, but excluding endplates. Six independent samples (3 × L4-L5 and 3 × L5-L6) were used for RNA extraction and analysis. IVDs were explanted right after rats' euthanasia, shock frozen in liquid nitrogen and stored at −80 C until processing. RNA was isolated via chloroform extraction and RNA cleanup was performed with Qiagen RNeasy mini kit (Qiagen, Germantown Maryland). RNA quality and quantity was determined via spectrophotometry. RNA was transcribed in cDNA using the High-Capacity cDNA Reverse Transcription Kit

| Histology
After sacrifice, whole spines were explanted and fixed in 10% neutral-

| Biobehavioral testing
The Randall-Selitto and von Frey tests were was used as complimentary measures of mechanical sensitivity. The von Frey test is frequently used in similar studies that test pain behavior in rat models for IVD degeneration. 18,40,41 While von Frey filaments detect tactile sensitivity, the Randall-Selitto test allows to assess response thresholds to deep mechanical pressure. 44 The Rotarod assay was included as a measure of motor capacity. Behavioral testing was conducted by a treatment-blind experimenter between 3:00 PM and 7:00 PM in the Biobehavioral Research Core at Cedars-Sinai. On each testing day, rats were brought into the Biobehavioral Research Core at least 30 minutes prior to the test session in order to habituate them to the environment. For Rotarod testing, movement-evoked pain was assessed during ambulation on a Rotorod (San Diego Instruments).
The rats were placed on the rotating rod for a 210-seconds trial repeated three times at 30 minutes interval. For each trial, the rod was set at a start speed of 3 rpm that remained constant for 30 seconds, then, the rod gradually accelerated from 3 rpm to 30 rpm over a 3 minutes period. The latency to fall off the rod was averaged across the trials. For the Randall-Selitto test, The Ugo Basile Analgesy-Meter (www.ugobasile.com) was used to measure mechanical hyperalgesia, 45 as described previously. 46 The experimenter gently restrained the rat in one hand for testing on the paw pinch apparatus and with the other hand guided the hind paw to be tested on the plinth under the coneshaped pusher. A weight operated by the experimenter pressing a pedal-switch exerted a force at a constant rate of 16 g per second.
When the rat elicited paw withdrawal or showed nocifensive behavior, the experimenter released the pedal and recorded the applied force. Three measures for each paw were collected then averaged.
For von Frey testing, an electronic von Frey (www.iitcinc.com) was used to assess mechanical/tactile allodynia as previously described. 47 The animals were placed in a plexiglas testing chamber

| Statistics
Prior to study start, the sample size was determined using G* Power software. The results indicated the following: to observe a 1-fold difference at a p value of 0.05 with a power set to 80%, our needed sample size was 16. To account for any possible variation, we increased our sample size by 50%. The unit of observation for gene expression and MRI analysis was disc and for biobehavioral tests rat. Both L4-5 and L5-6 discs were kept in the same analysis. Variation due to disc level (L4-L5 vs L5-6) was evenly distributed across the testing groups to account for differences in the different spinal disc levels. All statistical analyses were performed using Prism 7 (GraphPad Software, Inc., La Jolla, California); P < .05 was considered to be statistically F I G U R E 2 Injury with a 18G needle results in the most severe disc degeneration: μMRI analysis. A, Representative images of 18G needle injured IVDs L4-5 and L5-6 over time. IVD degeneration is evidenced by the reduced signal of T1 and T2 area. Left and middle images show lumbar spine segments L2-S1. The right images show a magnification of L4-5 and L5-6. B, Quantitative analysis results of T1-weighted area and high signal NP area (T2-weighted) analysis at 4-and 8-weeks postinjury relative to presurgery. Results are displayed for levels L4-5 and L5-6 combined (sham, 18G, 21G, and 23G injured groups). *Indicates statistical differences between different needle sizes. # Indicates statistical differences between different time points. Results are presented as mean ± SD; *( # ) P < .05, **( ## ) P < .01, ***( ### ) P < .001, ****( #### ) P < .0001. C, Regression of disc degeneration parameters (μMRI) on needle diameters (mm). Shown is the reduction of T1 area and NP high intensity area (T2) of discs injured with different needle diameters (0.64 mm/23G, 0.82 mm/21G and 1.27 mm/18G) at 4-and 8-weeks postsurgery. Needle diameter (mm) explained 44% to 47% of the variability in the T1 area and 17% to 19% of the variability in NP hydration. μMRI data were normalized to uninjured discs of the same level from rats undergoing sham surgery   Figure 2C).

| Gene expression of IVD degeneration markers is regulated in response to injury with 18G and 21G needles
To investigate changes in gene regulation in IVDs in response to different needle injuries, quantitative RT-PCR of the IVDs of L4-5 and L5-6 combined were evaluated after the animals' sacrifice at week 8. An upregulation of the catabolic marker, MMP3, was detected in the 18G and 21G groups vs sham control. Furthermore, MMP3 levels were elevated in the 21G vs the 23G group ( Figure 3A). Collagen I levels, an indicator of fibrotic activity in the disc were increased in the 21G group vs sham group as well as vs 23G ( Figure 3B). Gene expression of CNN2, another marker for fibrotic activity, was upregulated in 21G group compared to sham control and 18G ( Figure 3C). Expression F I G U R E 4 IVD degeneration and NP/AF lesion in response to needle puncture injury is most pronounced in the 18G group. A, H&E stained lumbar discs from sham and 18G, 21G, and 23G injured groups, explanted at 2 months postsurgery. B, Magnification of the NP site represented by black rectangle on row. C, Alcian blue/Picrosirius Red stained lumbar discs from sham and 18G, 21G, and 23G injured groups, explanted at 2 months postsurgery. D, Magnification of the NP site represented by white rectangle on row. E, Magnification of the AF needle insertion site represented by yellow rectangle on row. NP, nucleus pulposus; AF, annulus fibrosus, EP, end plate, GP, growth plate of the main NP matrix gene, aggrecan, was downregulated in all injury groups compared with sham control ( Figure 3D). Expression of collagen II was downregulated in the 21G group vs sham control only ( Figure 3E). The neuropeptide gene, TAC1, was not significantly regulated ( Figure 3F). TNF-α was only detectable in a subset of the samples analyzed. However, an upregulation in the 18G group compared to sham control and 21G groups was found ( Figure 3G).  Figure 4C,D). At the needle insertion site, the AF was disrupted in all injury groups ( Figure 4E).

| Mechanical sensitivity is increased in response to IVD injury with a 18G needle
Longitudinal analysis with the Randall-Selitto device demonstrated an increased normalized paw withdrawal threshold in the sham group at week 6 vs presurgery ( Figure 5A). Comparison between groups 6 weeks after injury showed a reduced normalized withdrawal threshold in the 18G needle group compared with the sham and 21G groups ( Figure 5B). Regarding the longitudinal von Frey testing, a reduction in the normalized paw withdrawal threshold of 18G needle injured animals was detected at weeks 3 and 6 post-injury compared to pre-surgery. Furthermore, an increase was detected in the 23G group at week 6 vs week 3 ( Figure 5C). In the group comparison, at week 6, the normalized paw withdrawal threshold of the 18G needle group was lower than 21G and 23G group ( Figure 5D). Rotarod evaluation detected no differences in the rats' latency to fall over time or between groups ( Figure 5E,F).

| DISCUSSION
This is the first study using multiple assessment methods to demonstrate the responses in rat lumbar IVD degeneration, pro-nociceptive marker expression, and pain-related behavior to IVD injury with increasing needle diameters (decreasing gauge). Our results show a correlation between the needle diameter and the hydration of lumbar F I G U R E 5 Biobehavioral tests show increased sensitivity to paw pressure (Randall-Selitto) and mechanical stimuli (von Frey) in rats injured with a 18G needle. The results of, A,B, Randall-Selitto, C,D, von Frey and, E,F, Rotarod biobehavioral tests of rats injured with 18G, 21G, and 23G needles, performed presurgery as well as at weeks 3 and 6 postsurgery are shown. Top, A,C,E, differences between time-points are shown. Bottom, B,D,F, differences between experimental groups are shown. *Indicates significant differences between experimental groups: #a: pre vs 3 weeks, #b: pre vs 6 weeks, #c: 3 vs 6 weeks *P < .05, **P < .01, ***P < .001 IVD. Injury with a 18G needle, the largest diameter, had the strongest IVD degeneration-inducing effect, as demonstrated by the greatest loss of NP water content and most severe lesion of the AF and NP, it had the biggest impact on pro-nociceptive mRNA marker regulation, and it increased sensitivity to mechanical stimuli. Catabolic, anabolic and fibrosis gene marker regulation in the IVD was detected in both the 18G and 21G needle injury group.
Micro-MRI analysis demonstrated a reduction in disc area (T1-weighted) and NP hydration (T2-weighted) values in the 18G needle group compared to sham control and to 23G needle groups at weeks 4 and 8 postsurgery. Furthermore, a correlation between needle diameter (mm) and disc area as well as NP hydration values was detected. Our results are in line with studies in rat caudal spines showing an induction of disc degeneration in response to disc puncture with 18G and 21G needles. 35,36,48 For example, a reduced X-ray-based disc high index and poorer disc hydration in rat caudal spines injured with an 18G compared to 25G needle and uninjured controls was shown. 36 Quian et al. demonstrated an induction of IVD degeneration at 2 weeks postinjury in a rat tail model injured with a 18G, but not a 26G needle, using T2-weighted imaging. 49 Furthermore, a significant reduction in T2 density and MRI index in response to rat caudal spine injury with a 18G or 21G needle compared to intact controls was shown. 35 To our knowledge there is no prior study comparing the effect of different needle diameters on the induction of IVD degeneration in rat lumbar discs, although the degree of lumbar disc degeneration is strongly associated with degenerative disc disease, a common cause of low back pain. 50 To induce IVD degeneration in rat lumbar spine, needles of varying diameters have been used. 18,[40][41][42] For example, a recent study by our group demonstrated the successful induction of IVD degeneration in lumbar rat spine by inserting a 18G needle in a retroperitoneal approach, which resulted in a similar reduction of IVD hydration using μMRI compared to the present study. 42 Also in line with our study, rat lumbar disc injury with a 21G needle resulted in an increase in IVD degeneration compared to pre-surgical controls, as demonstrated via MRI and H&E staining. 40 In contrast, a study by Li et al showed injury of lumber discs with a 27G needle to be sufficient to create a slight lesion of the NP and to induce loss of disc hydration at week 8 postsurgery. 38 While the histological findings are in line with our findings, our study results do not confirm that a single puncture with a needle ≥23G is sufficient to induce loss of IVD hydration in the same model system. When using a small needle diameter, a more efficient induction of IVD degeneration might be achieved in a combinatory approach with intradiscal PBS or TNF-α injections, as demonstrated in prior research. 18,41 It is noteworthy that no clear signs of IVD degeneration were detected in the 23G group. For cell therapeutic injections into the IVD, use of very small needles can create high shear stress and consequent low cell viability or high viscosity. 51 Therefore, the maximal needle size for therapeutic injections is important to know. Based on our data we suggest choosing a maximal needle size of 23G for this purpose.
Our study showed an upregulation of MMP3 mRNA expression in response to both injuries with a 18G and 21G needle, but not 23G. Similar to our findings, MMP-3 has been shown to be increased in severely but not mildly degenerated human NPs. 52 Furthermore, a study by Yang et al. detected a decrease in aggrecan gene expression levels in mouse injured tail discs at 2, 6, and 12 weeks postinjury. 53 In contrast, no downregulation of aggrecan gene expression was detected in response to rat caudal tail injury, using a 18G needle. 36 Our study detected a decrease in collagen II and increase in collagen I and CNN2 gene expression levels in the 21G group only. In human and animal degenerated NP tissues, a reduction in collagen II and abnormal deposition of collagen 1 have been shown. 11,54 Similarly, in rat injured caudal IVDs, an upregulation of collagen I and downregulation of collagen 2 has been detected at 2, 4, and 8 weeks postinjury. 36,55 Furthermore, increased CNN2 expression in degenerated human IVDs has been detected. 56 We observed that fibrosis markers expression (collagen I and CCN2) was clearly enhanced in the 21G vs with IVD degeneration and negatively with normalized paw withdrawal thresholds. 18 In this study, TNFα gene overexpression was only observed in a subset of samples following disc injury. While increased TNFα levels have been frequently shown to be associated with the development and progression of IVD degeneration, and with discogenic pain in rat models, evidence is usually provided in the literature on a protein level. 18,55,57 The observed differences might be therefore due to differences in TNFα gene and protein expression at the time point investigated.
Pain behavior assessments using von Frey showed behavioral signs of mechanical hypersensitivity at 6 weeks postsurgery vs presurgery in rats that were injured with an 18G needle, but not with a 21G needle in our study. The increases in pain sensitivity could either be due to pain primarily from the painful degenerating disc (discogenic pain) but could also be from increased radicular (nerve) pain from compression due to increased spinal central canal and lateral recess stenosis secondary to intervertebral disc collapse. 58 The dependence of pain behavior on the severity of IVD degeneration has been shown in a previous study that detected a more rapid development of behavioral changes (tested via pressure hyperalgesia directly over the punctured discs) in response to IVD injury of L4-5 and L5-6 in rat using a larger drill (0.8 mm, equivalent to 21G) vs a smaller drill (0.5 mm, equivalent to 25G). 59 In line with our study, no reduction in von Frey 50% force withdrawal were detected in this study using those sizes. 59 Furthermore, the lack of significant changes in the 21G group is in line with a study from Liu et al showing no changes in 50% withdrawal thresholds using von Frey in response to anterior lumbar disc puncture with a 21G needle compared with presurgery. 40 In contrast, rat lumbar IVD injury using a 26G needle in combination with PBS and TNF-α injections into the discs demonstrated a significant reduction of paw withdrawal thresholds normalized to presurgery using von Frey. 18,41 Randall-Selitto behavior tests that were performed in our study showed a similar pattern as von Frey testing. Lack of significant differences between the sham and 18G group at 3 and 6 weeks using von Frey and between pre-and postsurgery in the 18G group using Randall-Selitto testing may be a result of a limited sample number and small effect size and should be therefore tested with larger rat cohorts in the future. Furthermore, differences between von Frey and Randall-Selitto may be a result of the mode of stimulation (tactile perception vs mechanical pressure). Different modes of stimulation may activate different nociceptors and/or mechanoreceptors, leading to different responses. 44 The lack of differences in the animals' motor function in our model via Rotarod is in line with the literature showing no differences in motor function in a rat lumbar IVD injury model using a 26G needle in combination with PBS. 41 In this model of IVD degeneration we utilize the rat lumbar intervertebral disc injury, which requires an open anterior surgical approach and is more invasive compared to the rat-tail model. We believe that the lumbar model more accurately represents the human lumbar IVD condition, as rat tails do not have facet joints and have significantly different mechanical and viscoelastic properties. 60 Moreover, the highly gelatinous NP of rat caudal IVDs may limit the translation of these findings to human IVDs, which contain a more fibrous NP tissue. 61 Beckstein et al demonstrated that the disc tissue material properties are similar between rat and human disc after adjusting for geometry, despite differences in GAG and water composition. 62 This study is not without limitations. Firstly, time points for imaging and biobehavioral testing did not match, which makes it difficult to compare these outcome measures. Secondly, the study's  44 Future studies should also look at the effect of the IVD injury on sensory neurons residing in the DRG and the spinal cord plasticity as a result of the injury and/or intervention. Finally, nerve or vascular in-growth into the IVD should be studied. There is no doubt that nervous system plays an active role in backpain generation. Therefore, deeper understanding of the underlying mechanisms that mediate the interaction between the sensory neurons and IVD cells is needed.
In conclusion, we show a method to reliably modulate the degree of lumbar IVD degeneration and associated pain responses by varying the size of the needle puncture. This will allow us to mimic the spectrum of IVD degeneration seen in the human population, which can range from mild to severe. In our study, IVD puncture with a 21G needle clearly demonstrated signs of moderate disc degeneration, but we did not show reliable pain responses when using this needle. This model may be utilized for testing of cell-based therapies and other therapeutic agents in order to prevent discogenic pain, since the ORS Spine Section emphasized the need of selecting moderate severity disc degeneration and avoiding too mild or too advanced degeneration. 23 Injury with a 18G needle induced severe disc degeneration, associated with discogenic pain in our study. Our model may be used to study therapeutic agents for treatment of discogenic pain, which may be applied in combination with stem cell therapies and biomaterials approaches, to repopulate the disc space with viable cells and to restore the structural integrity of the disc. Prior to this several things should be considered: To elucidate the effect of needle injury on IVD function and pain behavior in more depth, more diverse biobehavioral assays (eg, open field, tail suspension or grip force tests) as well as biomechanics assessment of the IVD segments (eg, motion testing) should be included. In addition, detection of quantitative changes in IVD glycosaminoglycans (GAGs) in response to needle injury should be considered, since GAGs are known to be reduced during early disc degeneration. 67 Finally, a combinatory approach of a 21G needle with a pro-inflammatory agent, such as TNF, should be considered to explore the options to achieve a uniform moderate degeneration and consistent behavioral pain responses.

ACKNOWLEDGMENTS
This study was partially supported by NIH/NIAMS K01AR071512 to D. S. The authors wish to acknowledge Cedars-Sinai's Imaging Core facility for performing the μMRI scans, Cedars-Sinai's Biobank and Translational Research Core for performing the histological analysis and slide scanning, and Samuel Fuchs for illustrations in Figure 1A.

CONFLICT OF INTEREST
The authors have no conflict of interest.