Annulus Fibrosus Injury Induces Acute Neuroinflammation and Chronic Glial Response in Dorsal Root Ganglion and Spinal Cord-An In Vivo Rat Discogenic Pain Model.

Chronic painful intervertebral disc (IVD) degeneration (i.e., discogenic pain) is a major source of global disability needing improved knowledge on multiple-tissue interactions and how they progress in order improve treatment strategies. This study used an in vivo rat annulus fibrosus (AF) injury-driven discogenic pain model to investigate the acute and chronic changes in IVD degeneration and spinal inflammation, as well as sensitization, inflammation, and remodeling in dorsal root ganglion (DRG) and spinal cord (SC) dorsal horn. AF injury induced moderate IVD degeneration with acute and broad spinal inflammation that progressed to DRG to SC changes within days and weeks, respectively. Specifically, AF injury elevated macrophages in the spine (CD68) and DRGs (Iba1) that peaked at 3 days post-injury, and increased microglia (Iba1) in SC that peaked at 2 weeks post-injury. AF injury also triggered glial responses with elevated GFAP in DRGs and SC at least 8 weeks post-injury. Spinal CD68 and SC neuropeptide Substance P both remained elevated at 8 weeks, suggesting that slow and incomplete IVD healing provides a chronic source of inflammation with continued SC sensitization. We conclude that AF injury-driven IVD degeneration induces acute spinal, DRG, and SC inflammatory crosstalk with sustained glial responses in both DRGs and SC, leading to chronic SC sensitization and neural plasticity. The known association of these markers with neuropathic pain suggests that therapeutic strategies for discogenic pain need to target both spinal and nervous systems, with early strategies managing acute inflammatory processes, and late strategies targeting chronic IVD inflammation, SC sensitization, and remodeling.

Lumbar endplate microfracture injury induces Modic-like changes, intervertebral disc degeneration and spinal cord sensitization - an in vivo rat model.

BACKGROUND CONTEXT: Endplate (EP) injury plays critical roles in painful IVD degeneration since Modic changes (MCs) are highly associated with pain. Models of EP microfracture that progress to painful conditions are needed to better understand pathophysiological mechanisms and screen therapeutics. PURPOSE: Establish in vivo rat lumbar EP microfracture model and assess crosstalk between IVD, vertebra and spinal cord. STUDY DESIGN/SETTING: In vivo rat EP microfracture injury model with characterization of IVD degeneration, vertebral remodeling, spinal cord substance P (SubP), and pain-related behaviors. METHODS: EP-injury was induced in 5 month-old male Sprague-Dawley rats L4-5 and L5-6 IVDs by puncturing through the cephalad vertebral body and EP into the NP of the IVDs followed by intradiscal injections of TNFα (n=7) or PBS (n=6), compared with Sham (surgery without EP-injury, n=6). The EP-injury model was assessed for IVD height, histological degeneration, pain-like behaviors (hindpaw von Frey and forepaw grip test), lumbar spine MRI and µCT, and spinal cord SubP. RESULTS: Surgically-induced EP microfracture with PBS and TNFα injection induced IVD degeneration with decreased IVD height and MRI T2 signal, vertebral remodeling, and secondary damage to cartilage EP adjacent to the injury. Both EP injury groups showed MC-like changes around defects with hypointensity on T1-weighted and hyperintensity on T2-weighted MRI, suggestive of MC type 1. EP injuries caused significantly decreased paw withdrawal threshold, reduced axial grip, and increased spinal cord SubP, suggesting axial spinal discomfort and mechanical hypersensitivity and with spinal cord sensitization. CONCLUSIONS: Surgically-induced EP microfracture can cause crosstalk between IVD, vertebra, and spinal cord with chronic pain-like conditions. CLINICAL SIGNIFICANCE: This rat EP microfracture model was validated to induce broad spinal degenerative changes that may be useful to improve understanding of MC-like changes and for therapeutic screening.

Lumbar endplate microfracture injury induces Modic-like changes, intervertebral disc degeneration and spinal cord sensitization - An In Vivo Rat Model.

BACKGROUND CONTEXT : Endplate (EP) injury plays critical roles in painful IVD degeneration since Modic changes (MCs) are highly associated with pain. Models of EP microfracture that progress to painful conditions are needed to better understand pathophysiological mechanisms and screen therapeutics. PURPOSE : Establish in vivo rat lumbar EP microfracture model with painful phenotype. STUDY DESIGN/SETTING : In vivo rat study to characterize EP-injury model with characterization of IVD degeneration, vertebral bone marrow remodeling, spinal cord sensitization, and pain-related behaviors. METHODS : EP-driven degeneration was induced in 5-month-old male Sprague-Dawley rats L4-5 and L5-6 IVDs through the proximal vertebral body injury with intradiscal injections of TNFα (n=7) or PBS (n=6), compared to Sham (surgery without EP-injury, n=6). The EP-driven model was assessed for IVD height, histological degeneration, pain-like behaviors (hindpaw von Frey and forepaw grip test), lumbar spine MRI and µCT analyses, and spinal cord substance P (SubP). RESULTS : EP injuries induced IVD degeneration with decreased IVD height and MRI T2 values. EP injury with PBS and TNFα both showed MC type1-like changes on T1 and T2-weighted MRI, trabecular bone remodeling on µCT, and damage in cartilage EP adjacent to the injury. EP injuries caused significantly decreased paw withdrawal threshold and reduced grip forces, suggesting increased pain sensitivity and axial spinal discomfort. Spinal cord dorsal horn SubP was significantly increased, indicating spinal cord sensitization. CONCLUSIONS : EP microfracture can induce crosstalk between vertebral bone marrow, IVD and spinal cord with chronic pain-like conditions. CLINICAL SIGNIFICANCE : This rat EP microfracture model of IVD degeneration was validated to induce MC-like changes and pain-like behaviors that we hope will be useful to screen therapies and improve treatment for EP-drive pain.

Spinal Cord Sensitization and Spinal Inflammation from an In Vivo Rat Endplate Injury Associated with Painful Intervertebral Disc Degeneration.

Intervertebral disc (IVD) degeneration with Modic-like changes is strongly associated with pain. Lack of effective disease-modifying treatments for IVDs with endplate (EP) defects means there is a need for an animal model to improve understanding of how EP-driven IVD degeneration can lead to spinal cord sensitization. This rat in vivo study determined whether EP injury results in spinal dorsal horn sensitization (substance P, SubP), microglia (Iba1) and astrocytes (GFAP), and evaluated their relationship with pain-related behaviors, IVD degeneration, and spinal macrophages (CD68). Fifteen male Sprague Dawley rats were assigned into sham or EP injury groups. At chronic time points, 8 weeks after injury, lumbar spines and spinal cords were isolated for immunohistochemical analyses of SubP, Iba1, GFAP, and CD68. EP injury most significantly increased SubP, demonstrating spinal cord sensitization. Spinal cord SubP-, Iba1- and GFAP-immunoreactivity were positively correlated with pain-related behaviors, indicating spinal cord sensitization and neuroinflammation play roles in pain responses. EP injury increased CD68 macrophages in the EP and vertebrae, and spinal cord SubP-, Iba1- and GFAP-ir were positively correlated with IVD degeneration and CD68-ir EP and vertebrae. We conclude that EP injuries result in broad spinal inflammation with crosstalk between spinal cord, vertebrae and IVD, suggesting that therapies must address neural pathologies, IVD degeneration, and chronic spinal inflammation.

Ex vivo biomechanical evaluation of Acute lumbar endplate injury and comparison to annulus fibrosus injury in a rat model.

Back pain is often associated with intervertebral disc (IVD) degeneration, and IVD degeneration phenotypes are commonly characterized by annulus fibrosus (AF)-driven and endplate (EP)-driven phenotypes. Few studies of EP injury exist in animal models, even though clinical studies show EP lesions are strongly associated with IVD pathology and pain. This project established an ex-vivo rat lumbar EP injury model and characterized effects of EP injury on motion segment biomechanical properties, as compared to AF injury, a common way of inducing IVD degeneration. Lumbar motion segments (39 total vertebra-IVD-vertebra sections) assigned to Intact (L1/L2), AF injury and EP injury (L3/L4 and L5/L6 randomly selected), and biomechanically tested in axial tension-compression, stress-relaxation and torsional testing in pre-injury and post-injury conditions using a repeated-measures design. EP injury involved superior vertebra endplate puncture transcorporeally and obliquely. AF injury involved mid-line punctures anterior and bilaterally. Axial ROM, tensile stiffness, hysteresis, and neutral zone stiffness were significantly affected by EP injury but not AF injury. Torque range, torsional stiffness and torsional neutral zone stiffness were significantly affected by AF injury but not EP injury. Stress-relaxation fast time constant was decreased for EP injury. EP and AF injuries induced distinct biomechanical changes in lumbar motion segments with EP injury having the largest impact on axial biomechanical properties and AF injury most prominently affecting torsional properties. This study deepens the understanding of biomechanical mechanism of EP-driven low back pain and provides methods and biomechanical characterization for future in vivo studies.

High fat diet causes inferior vertebral structure and function without disc degeneration in RAGE-KO mice.

Back pain and spinal pathologies are associated with obesity in juveniles and adults, yet studies identifying causal relationships are lacking and none investigate sex differences. This study determined if high fat (HF) diet causes structural and functional changes to vertebrae and intervertebral discs (IVDs); if these changes are modulated in mice with systematic ablation for the receptor for advanced glycation endproducts (RAGE-KO); and if these changes are sex-dependent. Wild-type (WT) and RAGE-KO mice were fed a low fat (LF) or HF diet for 12 weeks starting at 6 weeks, representing the juvenile population. HF diet led to weight/fat gain, glucose intolerance, and increased cytokine levels (IL-5, MIG, and RANTES); with less fat gain in RAGE-KO females. Most importantly, HF diet reduced vertebral trabecular bone volume fraction and compressive and shear moduli, without a modifying effect of RAGE-KO, but with a more pronounced effect in females. HF diet caused reduced cortical area fraction only in WT males. Neither HF diet nor RAGE-KO affected IVD degeneration grade. Biomechanical properties of coccygeal motion segments were affected by RAGE-KO but not diet, with some interactions identified. In conclusion, HF diet resulted in inferior vertebral structure and function with some sex differences, no IVD degeneration, and few modifying effects of RAGE-KO. These structural and functional deficiencies with HF diet provide further evidence that diet can affect spinal structures and may increase the risk for spinal injury and degeneration with aging and additional stressors. Back pain and spinal pathologies are associated with obesity in juveniles and adults, yet studies identifying causal relationships are lacking and none investigate sex differences.

Development of a standardized histopathology scoring system for intervertebral disc degeneration and regeneration in rabbit models-An initiative of the ORSspine section.

BACKGROUND: The rabbit lumbar spine is a commonly utilized model for studying intervertebral disc degeneration and for the pre-clinical evaluation of regenerative therapies. Histopathology is the foundation for which alterations to disc morphology and cellularity with degeneration, or following repair or treatment are assessed. Despite this, no standardized histology grading scale has yet been established for the spine field for any of the frequently utilized animal models. AIMS: The purpose of this study was to establish a new standardized scoring system to assess disc degeneration and regeneration in the rabbit model. MATERIALS AND METHODS: The scoring system was formulated following a review of the literature and a survey of spine researchers. Validation of the scoring system was carried out using images provided by 4 independent laboratories, which were graded by 12 independent graders of varying experience levels. Reliability testing was performed via the computation of intra-class correlation coefficients (ICC) for each category and the total score. The scoring system was then further refined based on the results of the ICC analysis and discussions amongst the authors. RESULTS: The final general scoring system involves scoring 7 features (nucleus pulposus shape, area, cellularity and matrix condensation, annulus fibrosus/nucleus pulposus border appearance, annulus fibrosus morphology, and endplate sclerosis/thickening) on a 0 (healthy) to 2 (severe degeneration) scale. ICCs demonstrated overall moderate to good agreement across graders. An addendum to the main scoring system is also included for use in studies evaluating regenerative therapeutics, which involves scoring cell cloning and morphology within the nucleus pulposus and inner annulus fibrosus. DISCUSSION: Overall, this new scoring system provides an avenue to improve standardization, allow a more accurate comparison between labs and more robust evaluation of pathophysiology and regenerative treatments across the field. CONCLUSION: This study developed a histopathology scoring system for degeneration and regeneration in the rabbit model based on reported practice in the literature, a survey of spine researchers, and validation testing.

Development of a standardized histopathology scoring system for intervertebral disc degeneration in rat models: An initiative of the ORS spine section.

BACKGROUND: Rats are a widely accepted preclinical model for evaluating intervertebral disc (IVD) degeneration and regeneration. IVD morphology is commonly assessed using histology, which forms the foundation for quantifying the state of IVD degeneration. IVD degeneration severity is evaluated using different grading systems that focus on distinct degenerative features. A standard grading system would facilitate more accurate comparison across laboratories and more robust comparisons of different models and interventions. AIMS: This study aimed to develop a histology grading system to quantify IVD degeneration for different rat models. MATERIALS & METHODS: This study involved a literature review, a survey of experts in the field, and a validation study using 25 slides that were scored by 15 graders from different international institutes to determine inter- and intra-rater reliability. RESULTS: A new IVD degeneration grading system was established and it consists of eight significant degenerative features, including nucleus pulposus (NP) shape, NP area, NP cell number, NP cell morphology, annulus fibrosus (AF) lamellar organization, AF tears/fissures/disruptions, NP-AF border appearance, as well as endplate disruptions/microfractures and osteophyte/ossification. The validation study indicated this system was easily adopted, and able to discern different severities of degenerative changes from different rat IVD degeneration models with high reproducibility for both experienced and inexperienced graders. In addition, a widely-accepted protocol for histological preparation of rat IVD samples based on the survey findings include paraffin embedding, sagittal orientation, section thickness < 10 µm, and staining using H&E and/or SO/FG to facilitate comparison across laboratories. CONCLUSION: The proposed histological preparation protocol and grading system provide a platform for more precise comparisons and more robust evaluation of rat IVD degeneration models and interventions across laboratories.

A perspective on the ORS Spine Section initiative to develop a multi-species JOR Spine histopathology series.

This perspective summarizes the genesis, development, and potential future directions of the multispecies JOR Spine histopathology series.

Ex-vivo biomechanics of repaired rat intervertebral discs using genipin crosslinked fibrin adhesive hydrogel.

Microdiscectomy is the current standard surgical treatment for intervertebral disc (IVD) herniation, however annulus fibrosus (AF) defects remain unrepaired which can alter IVD biomechanical properties and lead to reherniation, IVD degeneration and recurrent back pain. Genipin-crosslinked fibrin (FibGen) hydrogel is an injectable AF sealant previously shown to partially restore IVD motion segment biomechanical properties. A small animal model of herniation and repair is needed to evaluate repair potential for early-stage screening of IVD repair strategies prior to more costly large animal and eventual human studies. This study developed an ex-vivo rat caudal IVD herniation model and characterized torsional, axial tension-compression and stress relaxation biomechanical properties before and after herniation injury with or without repair using FibGen. Injury group involved an annular defect followed by removal of nucleus pulposus tissue to simulate a severe herniation while Repaired group involved FibGen injection. Injury significantly altered axial range of motion, neutral zone, torsional stiffness, torque range and stress-relaxation biomechanical parameters compared to Intact. FibGen repair restored the stress-relaxation parameters including effective hydraulic permeability indicating it effectively sealed the IVD defect, and there was a trend for improved tensile stiffness and axial neutral zone length. This study demonstrated a model for studying IVD herniation injury and repair strategies using rat caudal IVDs ex-vivo and demonstrated FibGen sealed IVDs to restore water retention and IVD pressurization. This ex-vivo small animal model may be modified for future in-vivo studies to screen IVD repair strategies using FibGen and other IVD repair biomaterials as an augment to additional large animal and human IVD testing.

Males and females exhibit distinct relationships between intervertebral disc degeneration and pain in a rat model.

Back pain is linked to intervertebral disc (IVD) degeneration, but clinical studies show the relationship is complex. This study assessed whether males and females have distinct relationships between IVD degeneration and pain using an in vivo rat model. Forty-eight male and female Sprague-Dawley rats had lumbar IVD puncture or sham surgery. Six weeks after surgery, IVDs were evaluated by radiologic IVD height, histological grading, and biomechanical testing. Pain was assessed by von Frey assay and dorsal root ganglia (DRG) expression of Calca and Tac1 genes. Network analysis visualized which measures of IVD degeneration most related to pain by sex. In both females and males, annular puncture induced structural IVD degeneration, but functional biomechanical properties were similar to sham. Females and males had distinct differences in mechanical allodynia and DRG gene expression, even though sex differences in IVD measurements were limited. Network analysis also differed by sex, with more associations between annular puncture injury and pain in the male network. Sex differences exist in the interactions between IVD degeneration and pain. Limited correlation between measures of pain and IVD degeneration highlights the need to evaluate pain or nociception in IVD degeneration models to better understand nervous system involvement in discogenic pain.

Leptin signaling and the intervertebral disc: Sex dependent effects of leptin receptor deficiency and Western diet on the spine in a type 2 diabetes mouse model.

Type 2 diabetes and obesity are associated with back pain in juveniles and adults and are implicated in intervertebral disc (IVD) degeneration. Hypercaloric Western diets are associated with both obesity and type 2 diabetes. The objective of this study was to determine if obesity and type 2 diabetes result in spinal pathology in a sex-specific manner using in vivo diabetic and dietary mouse models. Leptin is an appetite-regulating hormone, and its deficiency leads to polyphagia, resulting in obesity and diabetes. Leptin is also associated with IVD degeneration, and increased expression of its receptor was identified in degenerated IVDs. We used young, leptin receptor deficient (Db/Db) mice to mimic the effect of diet and diabetes on adolescents. Db/Db and Control mice were fed either Western or Control diets, and were sacrificed at 3 months of age. Db/Db mice were obese, while only female mice developed diabetes. Female Db/Db mice displayed altered IVD morphology, with increased intradiscal notochordal band area, suggesting delayed IVD cell proliferation and differentiation, rather than IVD degeneration. Motion segments from Db/Db mice exhibited increased failure risk with decreased torsional failure strength. Db/Db mice also had inferior bone quality, which was most prominent in females. We conclude that obesity and diabetes due to impaired leptin signaling contribute to pathological changes in vertebrae, as well as an immature IVD phenotype, particularly of females, suggesting a sex-dependent role of leptin in the spine.

Correction: Dietary polyphenols as a safe and novel intervention for modulating pain associated with intervertebral disc degeneration in an in-vivo rat model.

[This corrects the article DOI: 10.1371/journal.pone.0223435.].

Phlpp1 is associated with human intervertebral disc degeneration and its deficiency promotes healing after needle puncture injury in mice.

Back pain is a leading cause of global disability and is strongly associated with intervertebral disc (IVD) degeneration (IDD). Hallmarks of IDD include progressive cell loss and matrix degradation. The Akt signaling pathway regulates cellularity and matrix production in IVDs and its inactivation is known to contribute to a catabolic shift and increased cell loss via apoptosis. The PH domain leucine-rich repeat protein phosphatase (Phlpp1) directly regulates Akt signaling and therefore may play a role in regulating IDD, yet this has not been investigated. The aim of this study was to investigate if Phlpp1 has a role in Akt dysregulation during IDD. In human IVDs, Phlpp1 expression was positively correlated with IDD and the apoptosis marker cleaved Caspase-3, suggesting a key role of Phlpp1 in the progression of IDD. In mice, 3 days after IVD needle puncture injury, Phlpp1 knockout (KO) promoted Akt phosphorylation and cell proliferation, with less apoptosis. At 2 and 8 months after injury, Phlpp1 deficiency also had protective effects on IVD cellularity, matrix production, and collagen structure as measured with histological and immunohistochemical analyses. Specifically, Phlpp1-deletion resulted in enhanced nucleus pulposus matrix production and more chondrocytic cells at 2 months, and increased IVD height, nucleus pulposus cellularity, and extracellular matrix deposition 8 months after injury. In conclusion, Phlpp1 has a role in limiting cell survival and matrix degradation in IDD and research targeting its suppression could identify a potential therapeutic target for IDD.

Dietary polyphenols as a safe and novel intervention for modulating pain associated with intervertebral disc degeneration in an in-vivo rat model.

Developing effective therapies for back pain associated with intervertebral disc (IVD) degeneration is a research priority since it is a major socioeconomic burden and current conservative and surgical treatments have limited success. Polyphenols are naturally occurring compounds in plant-derived foods and beverages, and evidence suggests dietary supplementation with select polyphenol preparations can modulate diverse neurological and painful disorders. This study tested whether supplementation with a select standardized Bioactive-Dietary-Polyphenol-Preparation (BDPP) may alleviate pain symptoms associated with IVD degeneration. Painful IVD degeneration was surgically induced in skeletally-mature rats by intradiscal saline injection into three consecutive lumbar IVDs. Injured rats were given normal or BDPP-supplemented drinking water. In-vivo hindpaw mechanical allodynia and IVD height were assessed weekly for 6 weeks following injury. Spinal column, dorsal-root-ganglion (DRG) and serum were collected at 1 and 6 weeks post-operative (post-op) for analyses of IVD-related mechanical and biological pathogenic processes. Dietary BDPP significantly alleviated the typical behavioral sensitivity associated with surgical procedures and IVD degeneration, but did not modulate IVD degeneration nor changes of pro-inflammatory cytokine levels in IVD. Gene expression analyses suggested BDPP might have an immunomodulatory effect in attenuating the expression of pro-inflammatory cytokines in DRGs. This study supports the idea that dietary supplementation with BDPP has potential to alleviate IVD degeneration-related pain, and further investigations are warranted to identify the mechanisms of action of dietary BDPP.

Sex Differences in Rat Intervertebral Disc Structure and Function Following Annular Puncture Injury.

STUDY DESIGN: A rat puncture injury intervertebral disc (IVD) degeneration model with structural, biomechanical, and histological analyses. OBJECTIVE: To determine if males and females have distinct responses in the IVD after injury. SUMMARY OF BACKGROUND DATA: Low back pain (LBP) and spinal impairments are more common in women than men. However, sex differences in IVD response to injury have been underexplored, particularly in animal models where sex differences can be measured without gender confounds. METHODS: Forty-eight male and female Sprague Dawley rats underwent sham, single annular puncture with tumor necrosis factor α (TNFα) injection (1×), or triple annular puncture with TNFα injection (3×) surgery. Six weeks after surgery, lumbar IVDs were assessed by radiologic IVD height, spinal motion segment biomechanical testing, histological degeneration grading, second harmonic generation (SHG) imaging, and immunofluorescence for fibronectin and α-smooth muscle actin. RESULTS: Annular puncture injuries significantly increased degenerative grade and IVD height loss for males and females, but females had increased degeneration grade particularly in the annulus fibrosus (AF). Despite IVD height loss, biomechanical properties were largely unaffected by injury at 6 weeks. However, biomechanical measures sensitive to outer AF differed by sex after 3× injury-male IVDs had greater torsional stiffness, torque range, and viscoelastic creep responses. SHG intensity of outer AF was reduced after injury only in female IVDs, suggesting sex differences in collagen remodeling. Both males and females exhibited decreased cellularity and increased fibronectin expression at injury sites. CONCLUSION: IVD injury results in distinct degeneration and functional healing responses between males and females. The subtle sex differences identified in this animal model suggest differences in response to IVD injury that might explain some of the variance observed in human LBP, and demonstrate the need to better understand differences in male and female IVD degeneration patterns and pain pathogenesis. LEVEL OF EVIDENCE: N/A.

Inhibiting tumor necrosis factor-alpha at time of induced intervertebral disc injury limits long-term pain and degeneration in a rat model.

BACKGROUND: Painful intervertebral disc (IVD) degeneration has tremendous societal costs and few effective therapies. Intradiscal tumor necrosis factor-alpha (TNFα) is commonly associated with low back pain, but the direct relationship remains unclear. PURPOSE: Treatment strategies for low back pain require improved understanding of the complex relationships between pain, intradiscal pro-inflammatory cytokines, and structural IVD degeneration. A rat in vivo lumbar IVD puncture model was used to 1) determine the role of TNFα in initiating painful IVD degeneration, and 2) identify statistical relationships between painful behavior, IVD degeneration, and intradiscal pro-inflammatory cytokine expression. METHODS: Lumbar IVDs were punctured anteriorly and injected with TNFα, anti-TNFα, or saline and compared with sham and naive controls. Hindpaw mechanical hyperalgesia was assayed weekly to determine pain over time. 6-weeks post-surgery, animals were sacrificed, and IVD degeneration, IVD height, and intradiscal TNFα and interleukin-1 beta (IL-1ß) expressions were assayed. RESULTS: Intradiscal TNFα injection increased pain and IVD degeneration whereas anti-TNFα alleviated pain to sham level. Multivariate step-wise linear regression identified pain threshold was predicted by IVD degeneration and intradiscal TNFα expression. Pain threshold was also linearly associated with IVD height loss and IL-1ß. DISCUSSION: The significant associations between IVD degeneration, height loss, inflammation, and painful behavior highlight the multifactorial nature of painful IVD degeneration and the challenges to diagnose and treat a specific underlying factor. We concluded that TNFα is an initiator of painful IVD degeneration and its early inhibition can mitigate pain and degeneration. Intradiscal TNFα inhibition following IVD injury may warrant investigation for its potential to alter downstream painful IVD degeneration processes.

Looking beyond the intervertebral disc: the need for behavioral assays in models of discogenic pain.

Orthopedic research into chronic discogenic back pain has commonly focused on aging- and degeneration-related changes in intervertebral disc structure, biomechanics, and biology. However, the primary spine-related reason for physician office visits is pain. The ambiguous nature of the human condition of discogenic low back pain motivates the use of animal models to better understand the pathophysiology. Discogenic back pain models must consider both emergent behavioral changes following pain induction and changes in the nervous system that mediate such behavior. Looking beyond the intervertebral disc, we describe the different ways to classify pain in human patients and animal models. We describe several behavioral assays that can be used in rodent models to augment disc degeneration measurements and characterize different types of pain. We review rodent models of discogenic pain that employed behavioral pain assays and highlight a need to better integrate neuroscience and orthopedic science methods to extend current understanding of the complex and multifactorial pathophysiology of discogenic back pain.

Annular puncture with tumor necrosis factor-alpha injection enhances painful behavior with disc degeneration in vivo.

BACKGROUND CONTEXT: Painfulintervertebral disc degeneration is extremely common and costly. Effective treatments are lacking because the nature of discogenic pain is complex with limited capacity to distinguish painful conditions from age-related changes in the spine. Hypothesized sources of discogenic pain include chronic inflammation, neurovascular ingrowth, and structural disruption. PURPOSE: This study aimed to investigate inflammation, pro-neurovascular growth factors, and structural disruption as sources of painful disc degeneration STUDY DESIGN/SETTING: This study used an in vivo study to address these hypothesized mechanisms with anterior intradiscal injections of tumor necrosis factor-alpha (TNFα), pro-neurovascular growth factors: nerve growth factor and vascular endothelial growth factor (NGF and VEGF), and saline with additional sham surgery and naïve controls. Depth of annular puncture was also evaluated for its effects on structural and painful degeneration. METHODS: Rat lumbar discs were punctured (shallow or deeper puncture) and intradiscally injected with saline, TNFα, or NGF and VEGF. Structural disc degeneration was assessed using X-ray, magnetic resonance imaging (MRI), and histology. The rat painful condition was evaluated using Von Frey hyperalgesia measurements, and substance P immunostaining in dorsal root ganglion (DRG) was performed to determine the source of pain. RESULTS: Saline injection increased painful responses with degenerative changes in disc height, MRI intensity, and morphologies of disc structure and cell. TNFα and NGF/VEGF accelerated painful behavior, and TNFα-injected animals had increased substance P in DRGs. Deeper punctures led to more severe disc degeneration. Multiple regression analysis showed that the painful behavior was correlated with disc height loss. CONCLUSIONS: We concluded that rate and severity of structural disc degeneration was associated with the amount of annular disruption and puncture depth. The painful behavior was associated with disc height loss and discal inflammatory state, whereas pro-inflammatory cytokines might play a more important role in the level of pain, which might have resulted from enhanced DRG sensitization. These in vivo painful disc degeneration models with different severities of structural changes may be useful for investigating discogenic pain mechanisms and for screening therapies, although interpretations must note the differences between all surgically induced animal models and the human condition.

Assessment of functional and behavioral changes sensitive to painful disc degeneration.

The development of an in vivo rodent discogenic pain model can provide insight into mechanisms for painful disc degeneration. Painful disc degeneration in rodents can be inferred by examining responses to external stimuli, observing pain-related behaviors, and measuring functional performance. This study compared the sensitivity of multiple pain and functional assessment methods to disc disruption for identifying the parameters sensitive to painful disc degeneration in rats. Disc degeneration was induced in rats by annular injury with saline injection. The severity of disc degeneration, pain sensitivity, and functional performance were compared to sham and naïve control rats. Saline injection induced disc degeneration with decreased disc height and MRI signal intensity as well as more fibrous nucleus pulposus, disorganized annular lamellae and decreased proteoglycan. Rats also demonstrated increased painful behaviors including decreased hindpaw mechanical and thermal sensitivities, increased grooming, and altered gait patterns with hindpaw mechanical hyperalgesia and duration of grooming tests being most sensitive. This is the first study to compare sensitivities of different pain assessment methods in an in vivo rat model of disc degeneration. Hindpaw mechanical sensitivity and duration of grooming were the most sensitive parameters to surgically induced degenerative changes and overall results were suggestive of disc degeneration associated pain.