Melanie B. Elliott

Serum and Tissue Cytokines and Chemokines Increase with Repetitive Upper Extremity Tasks

We investigated inflammation in rats performing a low repetition, negligible force (LRNF) or high repetition, negligible force (HRNF) task of reaching and retrieving food pellets at target rates of two or four reaches/min for 2 h/day, for 6–8 weeks. Serum was assayed for 11 cytokines and chemokines; forelimb tissues for four cytokines. Macrophages were counted in forelimb tissues of LRNF rats to add to results from our previous studies of HRNF rats. In HRNF rats, serum IL‐1α, IL‐1β, TNFα, MIP2, MIP3a, and RANTES were elevated in weeks 6 and 8. In contrast, only MIP2 and MIP3a increased in serum of LRNF rats. In 8 week HRNF reach limb tissues, IL‐1α, IL‐1β, TNFα, and IL‐10 increased in distal bones, IL‐1α and ‐β in muscles, and TNFα in tendons. Only IL‐10 increased in LRNF reach limb muscles in week 8. Serum IL‐1α and MIP2 correlated with macrophages in LRNF loose connective tissues, serum MIP3a and MIP2 correlated negatively with grip strength, while serum TNFα, MIP3a, and MIP2 correlated positively with total number of reaches. Thus, several tissue and circulating cytokines/chemokines increase in an exposure dependent manner following short‐term performance of repetitive reaching tasks and correlate with macrophage infiltration and decreasing grip strength.

High force reaching task induces widespread inflammation, increased spinal cord neurochemicals and neuropathic pain.

Repetitive strain injuries (RSI), which include several musculoskeletal disorders and nerve compression injuries, are associated with performance of repetitive and forceful tasks. In this study, we examined in young, adult Sprague-Dawley rats, the effects of performing a voluntary, moderate repetition, high force (MRHF; nine reaches/min; 60% maximum pulling force) task for 12 weeks on motor behavior and nerve function, inflammatory responses in forearm musculoskeletal and nerve tissues and serum, and neurochemical immunoexpression in cervical spinal cord dorsal horns. We observed no change in reach rate, but reduced voluntary participation and grip strength in week 12, and increased cutaneous sensitivity in weeks 6 and 12, the latter indicative of mechanical allodynia. Nerve conduction velocity (NCV) decreased 15% in the median nerve in week 12, indicative of low-grade nerve compression. ED-1 cells increased in distal radius and ulna in week 12, and in the median nerve and forearm muscles and tendons in weeks 6 and 12. Cytokines IL-1alpha, IL-1beta, TNF-alpha, and IL-10 increased in distal forearm bones in week 12, while IL-6 increased in tendon in week 12. However, serum analysis revealed only increased TNF-alpha in week 6 and macrophage inflammatory protein 3a (MIP3a) in weeks 6 and 12. Lastly, Substance P and neurokinin-1 were both increased in weeks 6 and 12 in the dorsal horns of cervical spinal cord segments. These results show that a high force, but moderate repetition task, induced declines in motor and nerve function as well as peripheral and systemic inflammatory responses (albeit the latter was mild). The peripheral inflammatory responses were associated with signs of central sensitization (mechanical allodynia and increased neurochemicals in spinal cord dorsal horns).

A cannabinoid type 2 receptor agonist attenuates blood-brain barrier damage and neurodegeneration in a murine model of traumatic brain injury.

After traumatic brain injury (TBI), inflammation participates in both the secondary injury cascades and the repair of the CNS, both of which are influenced by the endocannabinoid system. This study determined the effects of repeated treatment with a cannabinoid type 2 receptor (CB2R) agonist on blood–brain barrier integrity, neuronal degeneration, and behavioral outcome in mice with TBI. We also looked for the presence of a prolonged treatment effect on the macrophage/microglial response to injury. C57BL/6 mice underwent controlled cortical impact (CCI) and received repeated treatments with a CB2R agonist, 0‐1966, or vehicle. After euthanasia at 6 hr or 1, 2, 3, or 7 days postinjury, brains were removed for histochemical analysis. Blood–brain barrier permeability changes were evaluated by using sodium fluorescein (NaF). Perilesional degenerating neurons, injury volumes, and macrophage/microglia cells were quantified by stereological methods. Rota‐rod and open‐field testing were performed to evaluate motor function and natural exploratory behavior in mice. 0‐1966 Treatment resulted in a significant reduction in NaF uptake and number of degenerating neurons compared with the vehicle‐treated group. 0‐1966‐Treated mice demonstrated improvement on rota‐rod and open‐field testing compared with vehicle‐treated mice. These changes in CCI mice treated with 0‐1966 were associated with a prolonged reduction in macrophage/microglia cell counts. In conclusion, repeated treatments with a CB2R agonist, 0‐1966, result in attenuated blood–brain barrier disruption and neuronal degeneration. In addition, repeated treatment with 0‐1966 shows prolonged treatment effects on behavior and the macrophage/microglia cell response over several days.

Acute Effects of a Selective Cannabinoid-2 Receptor Agonist on Neuroinflammation in a Model of Traumatic Brain Injury

Proposed therapeutic strategies for attenuating secondary traumatic brain injury (TBI) include modulation of acute neuroimmune responses. The goal of this study was to examine the acute effects of cannabinoid-2 receptor (CB(2)R) modulation on behavioral deficits, cerebral edema, perivascular substance P, and macrophage/microglial activation in a murine model of TBI. Thirty male C57BL/6 mice underwent sham surgery, or cortical contusion impact injury (CCI). CCI mice received vehicle or the CB(2)R agonist 0-1966 at 1 and 24 h after injury. Performance on the rotarod, forelimb cylinder, and open-field tests were evaluated before and at 48 h after sham or CCI surgery. Cerebral edema was evaluated using the wet-dry weight technique. Immunohistochemical analysis was used to examine changes in substance P and macrophage/microglia-specific Iba1 protein immunoreactivity. Locomotor performance and exploratory behavior were significantly improved in mice receiving 0-1966 (CB(2)R agonist) compared to vehicle-treated mice. Significant reductions were found for cerebral edema, number of perivascular areas of substance P immunoreactivity, and number of activated macrophages/microglial cells in the injured brains of 0-1966-treated mice compared to vehicle-treated mice. The findings show that the effects of the CB(2)R agonist 0-1966 on edema, substance P immunoreactivity, and macrophage/microglial activation, were associated with recovery of acute motor and exploratory deficits. This study provides evidence of acute neuroprotective effects derived from selective CB(2)R activation that may represent an avenue for further development of novel therapeutic agents in the treatment of TBI.

Nociceptive Neuropeptide Increases and Periorbital Allodynia in a Model of Traumatic Brain Injury

Objective.— This study tests the hypothesis that injury to the somatosensory cortex is associated with periorbital allodynia and increases in nociceptive neuropeptides in the brainstem in a mouse model of controlled cortical impact (CCI) injury.

Peripheral neuritis and increased spinal cord neurochemicals are induced in a model of repetitive motion injury with low force and repetition exposure

Performance of high repetition tasks with or without force is associated with peripheral tissue inflammation, decreased nerve function and motor dysfunction. Here, we examined whether a low repetition task with negligible force (LRNF) produces fewer tissue and behavioral pathologies than previously observed with high repetition tasks using our rat model of repetitive motion injury (RMI). Thirty-seven rats were randomized into control or LRNF groups, the latter reaching and grasping a 45 mg food pellet at a rate of 3 reaches/min. This task was performed in 4, 0.5 5 h sessions with 1.5 5 h rest periods for 3 days/week for up to 12 weeks. Examination of distal median nerve, forelimb flexor tendons and bones for ED1-positive cells (macrophages and osteoclasts) revealed increases in nerve and bone in week 12. The nerve also contained increased TNF-alpha expressing cells in week 12. Examination of spinal cord dorsal horns revealed increased immunoexpression of Substance P in week 8 and neurokinin-1 in weeks 8 and 12 in the superficial lamina. Motor behavioral analyses showed no changes in reach rate across weeks, slightly reduced task duration (a measurement of voluntary task participation) in week 12, but significantly increased extra arm movement reversals during reaching in week 8. These extra movement reversals were corrections for missed food pellets during a reach. Thus, performance of even a low repetition, negligible force upper extremity task for 3 months can induce mild peripheral tissue inflammation, neurochemical increases in spinal cord dorsal horns, and declines in fine motor control.

Cannabinoid receptor type-2 stimulation, blockade, and deletion alter the vascular inflammatory responses to traumatic brain injury

BackgroundImmunomodulatory therapies have been identified as interventions for secondary injury after traumatic brain injury (TBI). The cannabinoid receptor type-2 (CB2R) is proposed to play an important, endogenous role in regulating inflammation. The effects of CB2R stimulation, blockade, and deletion on the neurovascular inflammatory responses to TBI were assessed.MethodsWild-type C57BL/6 or CB2R knockout mice were randomly assigned to controlled cortical impact (CCI) injury or to craniotomy control groups. The effects of treatment with synthetic, selective CB2R agonists (0-1966 and JWH-133), a selective CB2R antagonist, or vehicle solution administered to CCI groups were assessed at 1-day after injury. Changes in TNF-α, intracellular adhesion molecule (ICAM-1), inducible nitric oxide synthase (iNOS), macrophage/microglial ionized calcium-binding adaptor molecule, and blood-brain-barrier (BBB) permeability were assessed using ELISA, quantitative RT-PCR, immunohistochemistry, and fluorometric analysis of sodium fluorescein uptake. CB2R knockouts and wild-type mice with CCI injury were treated with a CB2R agonist or vehicle treatment.ResultsTNF-α mRNA increased at 6 hours and 1 to 3 days after CCI; a CB2R antagonist and genetic knockout of the CB2R exacerbated TNF-α mRNA expression. Treatment with a CB2R agonist attenuated TNF-α protein levels indicating post-transcriptional mechanisms. Intracellular adhesion molecule (ICAM-1) mRNA was increased at 6 hours, and at 1 to 2 days after CCI, reduced in mice treated with a CB2R agonist, and increased in CB2R knockout mice with CCI. Sodium fluorescein uptake was increased in CB2R knockouts after CCI, with and without a CB2R agonist. iNOS mRNA expression peaked early (6 hours) and remained increased from 1 to 3 days after injury. Treatment with a CB2R agonist attenuated increases in iNOS mRNA expression, while genetic deletion of the CB2R resulted in substantial increases in iNOS expression. Double label immunohistochemistry confirmed that iNOS was expressed by macrophage/microglia in the injured cortex.ConclusionFindings demonstrate that the endogenous cannabinoid system and CB2R play an important role in regulating inflammation and neurovascular responses in the traumatically injured brain. CB2R stimulation with two agonists (0-1966 and JWH-133) dampened post-traumatic inflammation, while blockade or deletion of the CB2R worsened inflammation. Findings support previous evidence that modulating the CB2R alters infiltrating macrophages and activated resident microglia. Further investigation into the role of the CB2R on specific immune cell populations in the injured brain is warranted.

Performance of a repetitive task by aged rats leads to median neuropathy and spinal cord inflammation with associated sensorimotor declines

Epidemiological studies have demonstrated a relationship between advancing age and susceptibility to risk factors for median neuropathies and musculoskeletal disorders. In this study, we determined if performance of a voluntary reaching task by aged rats induced sensorimotor declines, median nerve dysfunction and increased inflammatory cytokines in peripheral nerves, muscle and spinal cord neurons. Aged (14 mon) rats were trained for 15 min/day for 4 weeks to learn a high repetition, low force (HRLF) task (19 reaches/min; 15% maximum pulling force). Aged task rats performed the task for 2 h/day, 3 days/wk, for 12 weeks (until they were 18 mon of age). No behavioral changes were detected in normal controls (NC) or food-restricted controls (FR C) as they aged. However, grip strength declined in HRLF rats in weeks 6-12 (P<0.01 each) and 12-week trained-only rats (TR; P<0.05), compared to NC. Mechanical hypersensitivity was present in weeks 9 and 12 HRLF reach limb forepaws (P<0.01 and P<0.05, respectively), and 12-week HRLF support limb forepaws (P<0.01) and hindpaws (P=0.03), compared to NC. By week 12, median nerve conduction velocity declined 23%, bilaterally, in HRLF (P<0.001 each), and 13% in TR (P<0.05), compared to NC. Tumor necrosis factor alpha (TNFα) increased in 12-week HRLF muscle (P=0.005), median nerve (P<0.01), and neurons in superficial lamina of HRLF cervical spinal cords (P<0.01), compared to NC. interleukin 1 beta (IL1β) also increased in superficial lamina neurons (P<0.01). Loss of grip strength was correlated with median nerve conduction slowing (r=0.70) as well as increased nerve and muscle TNFα (r=-0.38 and r=-0.41, respectively); decrease in forepaw withdrawal thresholds was correlated with median nerve conduction slowing (r=0.81), increased nerve TNFα (r=-0.59), and increased TNFα and IL1β in neurons in spinal cord dorsal horns (r=-0.52 and r=-0.47, respectively). Thus, aged rats performing a repetitive task exhibited sensorimotor declines that were associated with decreased median nerve conduction, and increased pro-inflammatory cytokines in the median nerve and cervical spinal cord neurons.

An investigation of cerebral edema and injury volume assessments for controlled cortical impact injury.

UNLABELLED Using the controlled cortical impact (CCI) model, our laboratory compared edema in contralateral and ipsilateral regions to help clarify conflicting reports of contralateral edema and for enhanced assessment and interpretation of CCI injury pathophysiology. This investigation examined regional edema in response to graded injury severities over time with regards to tissue damage. Prior to injury rats were anesthetized with ketamine and xylazine (1:1). CCI injury parameters were set at 4.0m/s and 120 to 130 ms. Rats were randomized to receive moderate or severe injuries set at 2.0 and 3.0mm depths, respectively. Cerebral edema and injury volume were examined separately following euthanasia with pentobarbital. Cerebral edema was measured using the wet-dry weight technique at 24 or 48 h after injury. Sham animals underwent all surgical procedures except the impact injury. Injury volume was quantified using 2,3,5-triphenyltetrazolium chloride staining at 24h or 7 days after injury. The results of this investigation confirm that cerebral edema is absent in the uninjured, contralateral hemisphere after moderate and severe CCI injury. There were regional differences in cerebral edema formation in the hemisphere ipsilateral to injury that were dependent on injury severity and the length of time after injury. Tissue damage was reduced over 7 days following moderate CCI injury. CONCLUSIONS (1) the absence of edema in the contralateral hemisphere allows it to serve as a valid control for edema formation, (2) misrepresenting injury volume because of edema continues to be a problem for evaluating CCI injury and treatment efficacy, and (3) reduced injury volume over 7 days following CCI injury suggests tissue recovery after initial dysfunction.

Delayed thalamic astrocytosis and disrupted sleep-wake patterns in a preclinical model of traumatic brain injury

Traumatic brain injury (TBI) involves diffuse axonal injury and induces subtle but persistent changes in brain tissue and function and poses challenges for early detection of neurological injury. The present study uses an automated behavioral analysis system to assess alterations in rodent behavior in the subacute phase in a preclinical mouse model of TBI, controlled cortical impact (CCI) injury. In the first few weeks following CCI, mice demonstrated normal exploratory behaviors and other typical home‐cage behaviors. However, beginning 4 weeks post‐injury, CCI mice developed disruptions in sleep–wake patterns, including an increased number of awakenings from sleep. Such impaired sleep maintenance was accompanied by an increased latency to reach peak sleep in CCI mice. These sleep disruptions implicate involvement of the thalamocortical network, the activity of which must be tightly regulated to control sleep maintenance. After injury, there was an increase in reactive microglia in thalamic regions as well as delayed reactive astrocytosis that was evident in the thalamic reticular nucleus, which preceded the development of sleep disruptions. These data suggest that cortical injury may trigger inflammatory responses in deeper neuroanatomical structures, including the thalamic reticular nucleus. Such engagement of the thalamus may perturb the thalamocortical network that regulates sleep/awake patterns and contribute to sleep disruptions observed in this model as well as those documented in patients with TBI.