Myeloid Cell Association with Spinal Cord Injury-Induced Neuropathic Pain and Depressive-like Behaviors in LysM-eGFP Mice.

Spinal cord injury (SCI) affects ∼500,000 people worldwide annually, with the majority developing chronic neuropathic pain. Following SCI, approximately 60% of these individuals are diagnosed with comorbid mood disorders, while only ∼21% of the general population will experience a mood disorder in their lifetime. We hypothesize that nociceptive and depressive-like dysregulation occurs after SCI and is associated with aberrant macrophage infiltration in segmental pain centers. We completed moderate unilateral C5 spinal cord contusion on LysM-eGFP reporter mice to visualize infiltrating macrophages. At 6-weeks post-SCI, mice exhibit nociceptive and depressive-like dysfunction compared to naïve and sham groups. There were no differences between the sexes, indicating that sex is not a contributing factor driving nociceptive or depressive-like behaviors after SCI. Utilizing hierarchical cluster analysis, we classified mice based on endpoint nociceptive and depressive-like behavior scores. Approximately 59.3% of the SCI mice clustered based on increased paw withdrawal threshold to mechanical stimuli and immobility time in the forced swim test. SCI mice displayed increased myeloid cell presence in the lesion epicenter, ipsilateral C7-8 dorsal horn, and C7-8 DRGs as evidenced by eGFP, CD68, and Iba1 immunostaining when compared to naïve and sham mice. This was further confirmed by SCI-induced alterations in the expression of genes indicative of myeloid cell activation states and their associated secretome in the dorsal horn and dorsal root ganglia. In conclusion, moderate unilateral cervical SCI caused the development of pain-related and depressive-like behaviors in a subset of mice and these behavioral changes are consistent with immune system activation in the segmental pain pathway. PERSPECTIVE: These experiments characterized pain-related and depressive-like behaviors and correlated these changes with the immune response post-SCI. While humanizing the rodent is impossible, the results from this study inform clinical literature to closely examine sex differences reported in humans to better understand the underlying shared etiologies of pain and depressive-like behaviors following central nervous system trauma.

Rolipram-loaded PgP nanoparticle reduces secondary injury and enhances motor function recovery in a rat moderate contusion SCI model.

Spinal cord injury (SCI) results in immediate axonal damage and cell death, as well as a prolonged secondary injury consist of a cascade of pathophysiological processes. One important aspect of secondary injury is activation of phosphodiesterase 4 (PDE4) that leads to reduce cAMP levels in the injured spinal cord. We have developed an amphiphilic copolymer, poly (lactide-co-glycolide)-graft-polyethylenimine (PgP) that can deliver Rolipram, the PDE4 inhibitor. The objective of this work was to investigate the effect of rolipram loaded PgP (Rm-PgP) on secondary injury and motor functional recovery in a rat moderate contusion SCI model. We observed that Rm-PgP can increase cAMP level at the lesion site, and reduce secondary injury such as the inflammatory response by macrophages/microglia, astrogliosis by activated astrocytes and apoptosis as well as improve neuronal survival at 4 weeks post-injury (WPI). We also observed that Rm-PgP can improve motor functional recovery after SCI over 4 WPI.

Plasticity in Cervical Motor Circuits following Spinal Cord Injury and Rehabilitation.

Neuroplasticity is a robust mechanism by which the central nervous system attempts to adapt to a structural or chemical disruption of functional connections between neurons. Mechanical damage from spinal cord injury potentiates via neuroinflammation and can cause aberrant changes in neural circuitry known as maladaptive plasticity. Together, these alterations greatly diminish function and quality of life. This review discusses contemporary efforts to harness neuroplasticity through rehabilitation and neuromodulation to restore function with a focus on motor recovery following cervical spinal cord injury. Background information on the general mechanisms of plasticity and long-term potentiation of the nervous system, most well studied in the learning and memory fields, will be reviewed. Spontaneous plasticity of the nervous system, both maladaptive and during natural recovery following spinal cord injury is outlined to provide a baseline from which rehabilitation builds. Previous research has focused on the impact of descending motor commands in driving spinal plasticity. However, this review focuses on the influence of physical therapy and primary afferent input and interneuron modulation in driving plasticity within the spinal cord. Finally, future directions into previously untargeted primary afferent populations are presented.

Exercise-Induced Changes to the Macrophage Response in the Dorsal Root Ganglia Prevent Neuropathic Pain after Spinal Cord Injury.

Spinal cord injury (SCI) induces neuropathic pain that is refractory to treatment. Central and peripheral immune responses to SCI play critical roles in pain development. Although immune responses in the dorsal horn have been implicated in SCI-pain, immune mechanisms in the periphery, especially in the dorsal root ganglia (DRG), where nociceptor cell bodies reside, have not been well studied. Exercise is an immunomodulator, and we showed previously that early exercise after SCI reduces pain development. However, the mechanisms of exercise-mediated pain reduction are not understood. Therefore, we examined the 1) underlying immune differences in the spinal cord and DRG between rats with and without pain and 2) immunomodulatory effects of exercise in pain reduction. Rats were subjected to a unilateral contusion at C5 and tested for pain development using von Frey and mechanical conflict-avoidance paradigms. A subgroup of rats was exercised on forced running wheels starting at 5 days post-injury for 4 weeks. We observed greater microglial activation in the C7-C8 dorsal horn of rats with SCI-induced pain compared to rats with normal sensation, and early exercise reduced this activation independently of pain behavior. Further, abnormal pain sensation strongly correlated with an increased number of DRG macrophages. Importantly, exercise-treated rats that maintain normal sensation also have a lower number of macrophages in the DRG. Our data suggest that macrophage presence in the DRG may be an important effector of pain development, and early wheel walking exercise may mediate pain prevention by modulating the injury-induced macrophage response in the DRG. Further supportive evidence demonstrated that rats that developed pain despite exercise intervention still displayed a significantly elevated number of macrophages in the DRG. Collectively, these data suggest that macrophage presence in the DRG may be an amenable cellular target for future therapies.

Translational Challenges of Rat Models of Upper Extremity Dysfunction After Spinal Cord Injury.

There are approximately 17,500 new spinal cord injury (SCI) cases each year in the United States, with the majority of cases resulting from a traumatic injury. Damage to the spinal cord causes either temporary or permanent changes in sensorimotor function. Given that the majority of human SCIs occur in the cervical spinal level, the experimental animal models of forelimb dysfunction play a large role in the ability to translate basic science research to clinical application. However, the variation in the design of clinical and basic science studies of forelimb/upper extremity (UE) function prevents the ease of translation. This review provides an overview of experimental models of forelimb dysfunction used in SCI research with special emphasis on the rat model of SCI. The anatomical location and types of experimental cervical lesions, functional assessments, and rehabilitation strategies used in the basic science laboratory are reviewed. Finally, we discuss the challenges of translating animal models of forelimb dysfunction to the clinical SCI human population.

Delayed Exercise Is Ineffective at Reversing Aberrant Nociceptive Afferent Plasticity or Neuropathic Pain After Spinal Cord Injury in Rats.

Neuropathic pain is a debilitating consequence of spinal cord injury (SCI) that correlates with sensory fiber sprouting. Recent data indicate that exercise initiated early after SCI prevents the development of allodynia and modulated nociceptive afferent plasticity. This study determined if delaying exercise intervention until pain is detected would similarly ameliorate established SCI-induced pain. Adult, female Sprague-Dawley rats with a C5 unilateral contusion were separated into SCI allodynic and SCI non-allodynic cohorts at 14 or 28 days postinjury when half of each group began exercising on automated running wheels. Allodynia, assessed by von Frey testing, was not ameliorated by exercise. Furthermore, rats that began exercise with no allodynia developed paw hypersensitivity within 2 weeks. At the initiation of exercise, the SCI Allodynia group displayed marked overlap of peptidergic and non-peptidergic nociceptive afferents in the C7 and L5 dorsal horn, while the SCI No Allodynia group had scant overlap. At the end of 5 weeks of exercise both the SCI Allodynia and SCI No Allodynia groups had extensive overlap of the 2 c-fiber types. Our findings show that exercise therapy initiated at early stages of allodynia is ineffective at attenuating neuropathic pain, but rather that it induces allodynia-aberrant afferent plasticity in previously pain-free rats. These data, combined with our previous results, suggest that there is a critical therapeutic window when exercise therapy may be effective at treating SCI-induced allodynia and that there are postinjury periods when exercise can be deleterious.

Acute exercise prevents the development of neuropathic pain and the sprouting of non-peptidergic (GDNF- and artemin-responsive) c-fibers after spinal cord injury.

Spinal cord injury (SCI) impaired sensory fiber transmission leads to chronic, debilitating neuropathic pain. Sensory afferents are responsive to neurotrophic factors, molecules that are known to promote survival and maintenance of neurons, and regulate sensory neuron transduction of peripheral stimuli. A subset of primary afferent fibers responds only to the glial cell-line derived neurotrophic factor (GDNF) family of ligands (GFLs) and is non-peptidergic. In peripheral nerve injury models, restoration of GDNF or artemin (another GFL) to pre-injury levels within the spinal cord attenuates neuropathic pain. One non-invasive approach to increase the levels of GFLs in the spinal cord is through exercise (Ex), and to date exercise training is the only ameliorative, non-pharmacological treatment for SCI-induced neuropathic pain. The purpose of this study was 3-fold: 1) to determine whether exercise affects the onset of SCI-induced neuropathic pain; 2) to examine the temporal profile of GDNF and artemin in the dorsal root ganglia and spinal cord dorsal horn regions associated with forepaw dermatomes after SCI and Ex; and 3) to characterize GFL-responsive sensory fiber plasticity after SCI and Ex. Adult, female, Sprague-Dawley rats received a moderate, unilateral spinal cord contusion at C5. A subset of rats was exercised (SCI+Ex) on automated running wheels for 20min, 5days/week starting at 5days post-injury (dpi), continuing until 9 or 37dpi. Hargreaves' and von Frey testing was performed preoperatively and weekly post-SCI. Forty-two percent of rats in the unexercised group exhibited tactile allodynia of the forepaws while the other 58% retained normal sensation. The development of SCI-induced neuropathic pain correlated with a marked decrease in the levels of GDNF and artemin in the spinal cord and DRGs. Additionally, a dramatic increase in the density and the distribution throughout the dorsal horn of GFL-responsive afferents was observed in rats with SCI-induced allodynia. Importantly, in SCI rats that received Ex, the incidence of tactile allodynia decreased to 7% (1/17) and there was maintenance of GDNF and artemin at normal levels, with a normal distribution of GFL-responsive fibers. These data suggest that GFLs and/or their downstream effectors may be important modulators of pain fiber plasticity, representing effective targets for anti-allodynic therapeutics. Furthermore, we highlight the potent beneficial effects of acute exercise after SCI.

Chronic at- and below-level pain after moderate unilateral cervical spinal cord contusion in rats.

Chronic neuropathic pain is a significant consequence of spinal cord injury (SCI) that is associated with evoked pain, including allodynia and/or hyperalgesia. Allodynia is defined as a painful response to normally innocuous stimuli, and hyperalgesia occurs when there is an amplified pain response to normally noxious stimuli. We describe a model of a unilateral cervical level (C5) contusion injury where sensory recovery was assessed weekly for 6 weeks in 32 adult, female, Sprague-Dawley rats. Bilateral thermal hyperalgesia and tactile allodynia are detectable in the fore- and hindpaws as early as 7 days post-injury (dpi) and persist for at least 42 days. Paw withdrawal latency in response to a noxious thermal stimulus significantly intra-animal pre-operative values. Change in paw withdrawal latency plateaued at 21 dpi. Interestingly, bilateral forepaw allodynia develops in fewer than 40% of rats as measured by von Frey monofilament testing. Similar results occur in the hindpaws, where bilateral allodynia occurs in 46% of rats with SCI. The contralesional forepaw and both hindpaws of rats showed a slight increase in paw withdrawal threshold to tactile stimuli acutely after SCI, corresponding to ipsilesional forelimb motor deficits that resolve over time. That there is no difference among allodynic and non-allodynic groups in overall spared tissue or specifically of the dorsal column or ventrolateral white matter where ascending sensory tracts reside suggests that SCI-induced pain does not depend solely on the size or extent of the lesion, but that other mechanisms are in play. These observations provide a valid model system for future testing of therapeutic interventions to prevent the onset or to reduce the debilitating effects of chronic neuropathic pain after SCI.

Acute and chronic tactile sensory testing after spinal cord injury in rats.

Spinal cord injury (SCI) impairs sensory systems causing allodynia. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia(3). Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.

Exercise modulates microRNAs that affect the PTEN/mTOR pathway in rats after spinal cord injury.

We investigated microRNAs (miRs) associated with PTEN/mTOR signaling after spinal cord injury (SCI) and after hind limb exercise (Ex), a therapy implicated in promoting spinal cord plasticity. After spinalization, rats received cycling Ex 5 days/week. The expression of miRs, their target genes and downstream effectors were probed in spinal cord tissue at 10 and 31 days post injury. Ex elevated expression of miR21 and decreased expression of miR 199a-3p correlating with significant change in the expression of their respective target genes: PTEN mRNA decreased and mTOR mRNA increased. Western blotting confirmed comparable changes in protein levels. An increase in phosphorylated-S6 (a downstream effector of mTOR) within intermediate grey neurons in Ex rats was blocked by Rapamycin treatment. It thus appears possible that activity-dependent plasticity in the injured spinal cord is modulated in part through miRs that regulate PTEN and mTOR signaling and may indicate an increase in the regenerative potential of neurons affected by a SCI.

Validity of acute and chronic tactile sensory testing after spinal cord injury in rats.

Spinal cord injury (SCI) impairs sensory systems causing allodynia. Measuring the development of allodynia in rodent models of SCI is challenging due to spinal shock and marked motor impairments. Assessment of SCI-induced allodynia is not standardized across labs, making interpretation of results difficult. Therefore, we validated sensory threshold assessment after SCI and developed a novel assessment of allodynia prior to motor recovery in a rat SCI model. One hundred fifty-six Sprague-Dawley rats received T8 laminectomy or mild to moderate SCI using the OSU SCI device (0.3 mm to 1.3 mm cord displacement). To determine tactile thresholds, von Frey hairs (VFH) were applied in Up-Down or ascending order to the dorsal or plantar hindpaw. The most efficient and valid procedures that maintain high sensitivity and specificity were identified. Ten Up-Down VFH applications yielded stable thresholds; reducing the risk of threshold decay and unnecessary exposure to painful stimuli. Importantly, distraction of SCI-rats with food revealed differential decay of thresholds than when distraction is not provided. The new test uses dorsal VFH stimulation and is independent of trunk or hindlimb control. Acute dorsal VFH thresholds collected before recovery of hindlimb weight support accurately predicted plantar VFH thresholds measured at late timepoints (chi(2)=8.479; p<0.05). Thus, standardized testing early after SCI using the dorsal VFH test or later using 10 stimuli in the Up-Down test produces valid measures of tactile sensation across many SCI severities. Early detection of allodynia in experimental SCI will allow identification of mechanisms responsible for pain development and determine targets for therapeutic interventions.

Remote activation of microglia and pro-inflammatory cytokines predict the onset and severity of below-level neuropathic pain after spinal cord injury in rats.

Spinal cord injury (SCI) impairs sensory systems causing chronic allodynia. Mechanisms underlying neuropathic pain have been more extensively studied following peripheral nerve injury (PNI) than after central trauma. Microglial activation, pro-inflammatory cytokine production and activation of p38 MAP kinase pathways may induce at-level allodynia following PNI. We investigated whether midthoracic SCI elicits similar behavioral and cellular responses below the level of injury (lumbar spinal cord; L5). Importantly, we show that anatomical connections between L5 and supraspinal centers remain intact after moderate SCI allowing direct comparison to a well-established model of peripheral nerve injury. We found that SCI elicits below-level allodynia of similar magnitude to at-level pain caused by a peripheral nerve injury. Moreover, the presence of robust microglial activation in L5 cord predicted allodynia in 86% of rats. Also increased phosphorylation of p38 MAP kinase occurred in the L5 dorsal horn of allodynic rats. For below-level allodynia after SCI, TNF-alpha and IL-1beta increased in the L5 dorsal horn by 7 dpo and returned to baseline by 35 dpo. Interestingly, IL-6 remains at normal levels early after SCI and increases at chronic time points. Increased levels of pro-inflammatory cytokines also occurred in the thalamus after SCI-induced allodynia. These data suggest that remote microglial activation is pivotal in the development and maintenance of below-level allodynia after SCI. Fractalkine, a known activator of microglia, and astrocytes were not primary modulators of below-level pain. Although the mechanisms of remote microglial activation are unknown, this response may be a viable target for limiting or preventing neuropathic pain after SCI in humans.