Benjamin B. Guarino

Metabotropic Glutamate Receptor-5 and Protein Kinase C-Epsilon Increase in Dorsal Root Ganglion Neurons and Spinal Glial Activation in an Adolescent Rat Model of Painful Neck Injury

There is growing evidence that neck pain is common in adolescence and is a risk factor for the development of chronic neck pain in adulthood. The cervical facet joint and its capsular ligament is a common source of pain in the neck in adults, but its role in adolescent pain remains unknown. The aim of this study was to define the biomechanics, behavioral sensitivity, and indicators of neuronal and glial activation in an adolescent model of mechanical facet joint injury. A bilateral C6-C7 facet joint distraction was imposed in an adolescent rat and biomechanical metrics were measured during injury. Following injury, forepaw mechanical hyperalgesia was measured, and protein kinase C-epsilon (PKCɛ) and metabotropic glutamate receptor-5 (mGluR5) expression in the dorsal root ganglion and markers of spinal glial activation were assessed. Joint distraction induced significant mechanical hyperalgesia during the 7 days post-injury (p < 0.001). Painful injury significantly increased PKCɛ expression in small- and medium-diameter neurons compared to sham (p < 0.05) and naïve tissue (p < 0.001). Similarly, mGluR5 expression was significantly elevated in small-diameter neurons after injury (p < 0.05). Spinal astrocytic activation after injury was also elevated over sham (p < 0.035) and naïve (p < 0.0001) levels; microglial activation was only greater than naïve levels (p < 0.006). Mean strains in the facet capsule during injury were 32.8 ± 12.9%, which were consistent with the strains associated with comparable degrees of hypersensitivity in the adult rat. These results suggest that adolescents may have a lower tissue tolerance to induce pain and associated nociceptive response than do adults.

Activating transcription factor 4, a mediator of the integrated stress response, is increased in the dorsal root ganglia following painful facet joint distraction

Chronic neck pain is one of the most common musculoskeletal disorders in the US. Although biomechanical and clinical studies have implicated the facet joint as a primary source of neck pain, specific cellular mechanisms still remain speculative. The purpose of this study was to investigate whether a mediator (activating transcription factor; 4ATF4) of the integrated stress response (ISR) is involved in facet-mediated pain. Holtzman rats underwent C6/C7 facet joint loading that produces either painful (n=16) or nonpainful (n=8) responses. A sham group (n=9) was also included as surgical controls. Behavioral sensitivity was measured and the C6 dorsal root ganglia (DRGs) were harvested on day 7 to evaluate the total and neuronal ATF4 expression. In separate groups, an intra-articular ketorolac injection was administered either immediately (D0 ketorolac) or 1 day (D1 ketorolac) after painful facet joint loading. Allodynia was measured at days 1 and 7 after injury to assess the effects on behavioral responses. ATF4 and BiP (an indicator of ISR activation) were separately quantified at day 7. Facet joint loading sufficient to elicit behavioral hypersensitivity produced a threefold increase in total and neuronal ATF4 expression in the DRG. After ketorolac treatment at the time of injury, ATF4 expression was significantly (P<0.01) reduced despite not producing any attenuation of behavioral responses. Interestingly, ketorolac treatment at day 1 significantly (P<0.001) alleviated behavioral sensitivity at day 7, but did not modify ATF4 expression. BiP expression was unchanged after either intervention time. Results suggest that ATF4-dependent activation of the ISR does not directly contribute to persistent pain, but it may sensitize neurons responsible for pain initiation. These behavioral and immunohistochemical findings imply that facet-mediated pain may be sustained through other pathways of the ISR.

TRANSIENT NERVE ROOT COMPRESSION LOAD AND DURATION DIFFERENTIALLY MEDIATE BEHAVIORAL SENSITIVITY AND ASSOCIATED SPINAL ASTROCYTE ACTIVATION AND MGLUR5 EXPRESSION

Injury to the cervical nerve roots is a common source of neck pain. Animal models of nerve root compression have previously established the role of compression magnitude and duration in nerve root-mediated pain and spinal inflammation; yet, the response of the spinal glutamatergic system to transient nerve root compression and its relationship to compression mechanics have not been studied. The glutamate receptor, mGluR5, has a central role in pain, and its expression by neurons and astrocytes in the spinal cord may be pivotal for neuronal-glial signaling. This study quantified spinal GFAP and mGluR5 expression following nerve root compressions of different magnitudes and durations in the rat. Compression to the C7 nerve root was applied for a duration that was either above (10 min) or below (3 min) the critical duration for mediating afferent discharge rates during compression. To also test for the effect of the magnitude of the compression load, either a 10 gf or a 60 gf was applied to the nerve root for each duration. Mechanical allodynia was assessed, and the C7 spinal cord was harvested on day 7 for immunofluorescent analysis. Double labeling was used to localize the expression of mGluR5 on astrocytes (GFAP) and neurons (MAP2). Seven days after injury, 10 min of compression produced significantly greater behavioral sensitivity (P<0.001) and spinal GFAP expression (P=0.002) than 3 min of compression, regardless of the compression magnitude. Nerve root compression at 60 gf produced a significant increase (P<0.001) in spinal mGluR5 for both of the durations studied. There was no difference in the distribution of mGluR5 between astrocytes and neurons following nerve root compression of any type. The glutamatergic and glial systems are differentially modulated by the mechanics of nerve root compression despite the known contribution of glia to pain through glutamatergic signaling.

Spinal microglial proliferation is evident in a rat model of painful disc herniation both in the presence of behavioral hypersensitivity and following minocycline treatment sufficient to attenuate allodynia

Although spinal glia acquire a reactive profile in radiculopathy, glial cell proliferation remains largely unstudied. This study investigated spinal glial proliferation in a model simulating painful disc herniation; the C7 nerve root underwent compression and chromic gut suture exposure or sham procedures. A subset of injured rats received minocycline injections prior to injury. Allodynia was assessed and bromodeoxyuridine (BrdU) was injected 2 hr before tissue harvest on day 1 or 3. Spinal cell proliferation and phenotype identification were assayed by fluorescent colabeling with antibodies to BrdU and either glial fibrillary acidic protein (astrocytes) or Iba1 (microglia). At day 1, ipsilateral allodynia was significantly increased (P < 0.001) for injury over sham. Minocycline treatment significantly decreased ipsilateral allodynia to sham levels at day 1 (P < 0.001). At day 3, ipsilateral allodynia remained and contralateral allodynia was also present for injury (P< 0.003) over sham. The number of BrdU‐positive cells in the ipsilateral spinal dorsal horn at day 1 after injury was significantly elevated (P < 0.001) over sham. Approximately 70% of BrdU‐positive cells labeled positively for Iba1; dividing microglia were significantly increased (P < 0.004) in the ipsilateral dorsal horn at day 1 following injury compared with sham. Spinal cellular proliferation after injury was not changed by minocycline injection. By day 3, the number of BrdU‐positive cells had returned to sham levels bilaterally. Data indicate that spinal microglia proliferate after injury but that proliferation is not abolished by minocycline treatment that attenuates allodynia, indicating that spinal microglial proliferation may be related to injury and may not be linked to changes in sensory perception.

Upregulation of BDNF and NGF in cervical intervertebral discs exposed to painful whole-body vibration.

Study Design. In vivo study defining expression of the neurotrophins, brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), in cervical intervertebral discs after painful whole-body vibration (WBV). Objective. The goal of this study is to determine if BDNF and NGF are expressed in cervical discs after painful WBV in a rat model. Summary of Background Data. WBV is a possible source of neck pain and has been implicated as increasing the risk for disc disorders. Typically, aneural regions of painful human lumbar discs exhibit hyperinnervation, suggesting nerve ingrowth as potentially contributing to disc degeneration and pain. BDNF and NGF are upregulated in painfully degenerate lumbar discs and hypothesized to contribute to this pathology. Methods. Male Holtzman rats underwent 7 days of repeated WBV (15 Hz, 30 min/d) or sham exposures, followed by 7 days of rest. Cervical discs were collected for analysis of BDNF and NGF expression through RT-qPCR and Western blot analysis. Immunohistochemistry also evaluated their regional expression in the disc. Results. Vibration significantly increases BDNF messenger ribonucleic acid (mRNA) levels (P = 0.036), as well as total-NGF mRNA (P = 0.035). Protein expression of both BDNF (P = 0.006) and the 75-kDa NGF (P = 0.045) increase by nearly 4- and 10-fold, respectively. Both BDNF mRNA (R2 = 0.396; P = 0.012) and protein (R2 = 0.280; P = 0.035) levels are significantly correlated with the degree of behavioral sensitivity (i.e., pain) at day 14. Total-NGF mRNA is also significantly correlated with the extent of behavioral sensitivity (R2 = 0.276; P = 0.044). Both neurotrophins are most increased in the inner annulus fibrosus and nucleus pulposus. Conclusion. The increases in BDNF and NGF in the cervical discs after painful vibration are observed in typically aneural regions of the disc, consistent with reports of its hyperinnervation. Yet, the induction of nerve ingrowth into the disc was not explicitly investigated. Neurotrophin expression also correlates with behavioral sensitivity, suggesting a role for both neurotrophins in the development of disc pain. Level of Evidence: N/A

ProDisc cervical arthroplasty does not alter facet joint contact pressure during lateral bending or axial torsion.

Study Design. A biomechanical study of facet joint pressure after total disc replacement using cadaveric human cervical spines during lateral bending and axial torsion. Objective. The goal was to measure the contact pressure in the facet joint in cadaveric human cervical spines subjected to physiologic lateral bending and axial torsion before and after implantation of a ProDisc-C implant. Summary of Background Data. Changes in facet biomechanics can damage the articular cartilage in the joint, potentially leading to degeneration and painful arthritis. Few cadaveric and computational studies have evaluated the changes in facet joint loading during spinal loading with an artificial disc implanted. Computational models have predicted that the design and placement of the implant influence facet joint loading, but limited cadaveric studies document changes in facet forces and pressures during nonsagittal bending after implantation of a ProDisc. As such, little is known about the local facet joint mechanics for these complicated loading scenarios in the cervical spine. Methods. Seven osteoligamentous C2–T1 cadaveric cervical spines were instrumented with a transducer to measure the C5–C6 facet pressure profiles during physiological lateral bending and axial torsion, before and after implantation of a ProDisc-C at that level. Rotations at that level and global cervical spine motions and loads were also quantified. Results. Global and segmental rotations were not altered by the disc implantation. Facet contact pressure increased after implantation during ipsilateral lateral bending and contralateral torsion, but that increase was not significant compared with the intact condition. Conclusion. Implantation of a ProDisc-C does not significantly modify the kinematics and facet pressure at the index level in cadaveric specimens during lateral bending and axial torsion. However, changes in facet contact pressures after disc arthroplasty may have long-term effects on spinal loading and cartilage degeneration and should be monitored in vivo.

Contact pressure in the facet joint during sagittal bending of the cadaveric cervical spine.

The facet joint contributes to the normal biomechanical function of the spine by transmitting loads and limiting motions via articular contact. However, little is known about the contact pressure response for this joint. Such information can provide a quantitative measure of the facet joints local environment. The objective of this study was to measure facet pressure during physiologic bending in the cervical spine, using a joint capsule-sparing technique. Flexion and extension bending moments were applied to six human cadaveric cervical spines. Global motions (C2-T1) were defined using infra-red cameras to track markers on each vertebra. Contact pressure in the C5-C6 facet was also measured using a tip-mounted pressure transducer inserted into the joint space through a hole in the postero-inferior region of the C5 lateral mass. Facet contact pressure increased by 67.6 ± 26.9 kPa under a 2.4 Nm extension moment and decreased by 10.3 ± 9.7 kPa under a 2.7 Nm flexion moment. The mean rotation of the overall cervical specimen motion segments was 9.6 ± 0.8° and was 1.6 ± 0.7° for the C5-C6 joint, respectively, for extension. The change in pressure during extension was linearly related to both the change in moment (51.4 ± 42.6 kPa/Nm) and the change in C5-C6 angle (18.0 ± 108.9 kPa/deg). Contact pressure in the inferior region of the cervical facet joint increases during extension as the articular surfaces come in contact, and decreases in flexion as the joint opens, similar to reports in the lumbar spine despite the difference in facet orientation in those spinal regions. Joint contact pressure is linearly related to both sagittal moment and spinal rotation. Cartilage degeneration and the presence of meniscoids may account for the variation in the pressure profiles measured during physiologic sagittal bending. This study shows that cervical facet contact pressure can be directly measured with minimal disruption to the joint and is the first to provide local pressure values for the cervical joint in a cadaveric model.

Whole body vibration induces forepaw and hind paw behavioral sensitivity in the rat

Whole body vibration (WBV) has been linked to neck and back pain, but the biomechanical and physiological mechanisms responsible for its development and maintenance are unknown. A rodent model of WBV was developed in which rats were exposed to different WBV paradigms, either daily for 7 consecutive days (repeated WBV) or two single exposures at Day 0 and 7 (intermittent WBV). Each WBV session lasted for 30 min and was imposed at a frequency of 15 Hz and RMS platform acceleration of 0.56 ± 0.07 g. Changes in the withdrawal response of the forepaw and hind paw were measured, and were used to characterize the onset and maintenance of behavioral sensitivity. Accelerations and displacements of the rat and deformations in the cervical and lumbar spines were measured during WBV to provide mechanical context for the exposures. A decrease in withdrawal threshold was induced at 1 day after the first exposure in both the hind paw and forepaw. Repeated WBV exhibited a sustained reduction in withdrawal threshold in both paws and intermittent WBV induced a sustained response only in the forepaw. Cervical deformations were significantly elevated which may explain the more robust forepaw response. Findings suggest that a WBV exposure leads to behavioral sensitivity.

Facet joint contact pressure is not significantly affected by ProDisc cervical disc arthroplasty in sagittal bending: a single-level cadaveric study

BACKGROUND CONTEXT Total disc arthroplasty is a motion-preserving spinal procedure that has been investigated for its impact on spinal motions and adjacent-level degeneration. However, the effects of disc arthroplasty on facet joint biomechanics remain undefined despite the critical role of these posterior elements on guiding and limiting spinal motion. PURPOSE The goal was to measure the pressure in the facet joint in cadaveric human cervical spines subjected to sagittal bending before and after implantation of the ProDisc-C (Synthes Spine Company, L.P, West Chester, PA, USA). STUDY DESIGN A biomechanical study was performed using cadaveric human cervical spines during sagittal bending in the intact and implanted conditions. METHODS Seven C2-T1 osteoligamentous cadaveric cervical spines were instrumented with a transducer to measure the C5-C6 facet pressure profiles during physiological sagittal bending, before and after implantation of a ProDisc-C at that level. Rotations of the index segment and global cervical spine were also quantified. RESULTS The mean C5-C6 range of motion significantly increased (p=.009) from 9.6°±5.1° in the intact condition to 16.2°±3.6° after implantation. However, despite such changes in rotation, there was no significant difference in the facet contact pressure during extension between the intact (64±30 kPa) and implanted (44±55 kPa) conditions. Similarly, there was no difference in facet pressure developed during flexion. CONCLUSIONS Although implantation of a ProDisc-C arthroplasty device at the C5-C6 level increases angular rotations, it does not significantly alter the local facet pressure at the index level in flexion or extension. Using a technique that preserves the capsular ligament, this study provides the first direct measurement of cervical facet pressure in a disc arthroplasty condition.

The potential for salmon fibrin and thrombin to mitigate pain subsequent to cervical nerve root injury

Nerve root compression is a common cause of radiculopathy and induces persistent pain. Mammalian fibrin is used clinically as a coagulant but presents a variety of risks. Fish fibrin is a potential biomaterial for neural injury treatment because it promotes neurite outgrowth, is non-toxic, and clots readily at lower temperatures. This study administered salmon fibrin and thrombin following nerve root compression and measured behavioral sensitivity and glial activation in a rat pain model. Fibrin and thrombin each significantly reduced mechanical allodynia compared to injury alone (p < 0.02). Painful compression with fibrin exhibited allodynia that was not different from sham for any day using stimulation by a 2 g filament; allodynia was only significantly different (p < 0.043) from sham using the 4 g filament on days 1 and 3. By day 5, responses for fibrin treatment decreased to sham levels. Allodynia following compression with thrombin treatment were unchanged from sham at any time point. Macrophage infiltration at the nerve root and spinal microglial activation were only mildly modified by salmon treatments. Spinal astrocytic expression decreased significantly with fibrin (p < 0.0001) but was unchanged from injury responses for thrombin treatment. Results suggest that salmon fibrin and thrombin may be suitable biomaterials to mitigate pain.