Intrathecal minocycline does not block the adverse effects of repeated, intravenous morphine administration on recovery of function after SCI.

Opioids are among the most effective analgesics for the management of pain in the acute phase of a spinal cord injury (SCI), and approximately 80% of patients are treated with morphine in the first 24 h following SCI. We have found that morphine treatment in the first 7 days after SCI increases symptoms of pain at 42 days post-injury and undermines the recovery of locomotor function in a rodent model. Prior research has implicated microglia/macrophages in opioid-induced hyperalgesia and the development of neuropathic pain. We hypothesized that glial activation may also underlie the development of morphine-induced pain and cell death after SCI. Supporting this hypothesis, our previous studies found that intrathecal and intravenous morphine increase the number of activated microglia and macrophages present at the spinal lesion site, and that the adverse effects of intrathecal morphine can be blocked with intrathecal minocycline. Recognizing that the cellular expression of opioid receptors, and the intracellular signaling pathways engaged, can change with repeated administration of opioids, the current study tested whether minocycline was also protective with repeated intravenous morphine administration, more closely simulating clinical treatment. Using a rat model of SCI, we co-administered intravenous morphine and intrathecal minocycline for the first 7 days post injury and monitored sensory and locomotor recovery. Contrary to our hypothesis and previous findings with intrathecal morphine, we found that minocycline did not prevent the negative effects of morphine. Surprisingly, we also found that intrathecal minocycline alone is detrimental for locomotor recovery after SCI. Using ex vivo cell cultures, we investigated how minocycline and morphine altered microglia/macrophage function. Commensurate with published studies, we found that minocycline blocked the effects of morphine on the release of pro-inflammatory cytokines but, like morphine, it increased glial phagocytosis. While phagocytosis is critical for the removal of cellular and extracellular debris at the spinal injury site, increased phagocytosis after injury has been linked to the clearance of stressed but viable neurons and protracted inflammation. In sum, our data suggest that both morphine and minocycline alter the acute immune response, and reduce locomotor recovery after SCI.

Inflammation increases the development of depression behaviors in male rats after spinal cord injury.

Following spinal cord injury, 18-26% of patients are diagnosed with depressive disorders, compared to 8-12% in the general population. As increased inflammation strongly correlates with depression in both animal and human studies, we hypothesized that the immune activation inherent to SCI could increase depression-like behavior. Thus, we proposed that reducing immune activation with minocycline, a microglial inhibitor, would decrease depression-like behavior following injury. Male Sprague-Dawley rats were given minocycline in their drinking water for 14 days following a moderate, mid-thoracic (T12) spinal contusion. An array of depression-like behaviors (social activity, sucrose preference, forced swim, open field activity) were examined prior to injury as well as on days 9-10, 19-20, and 29-30 post-injury. Peripheral cytokine levels were analyzed in serum collected prior to injury and 10 days post-injury. Hierarchical cluster analysis divided subjects into two groups based on behavior: depressed and not-depressed. Depressed subjects displayed lower levels of open field activity and social interaction relative to their not-depressed counterparts. Depressed subjects also showed significantly greater expression of pro-inflammatory cytokines both before and after injury and displayed lower levels of hippocampal neurogenesis than not-depressed subjects. Intriguingly, subjects who later showed depressive behaviors had higher baseline levels of the pro-inflammatory cytokine IL-6, which persisted throughout the duration of the experiment. Minocycline, however, did not affect serum cytokine levels and did not block the development of depression; equal numbers of minocycline versus vehicle-treated subjects appeared in both phenotypic groups. Despite this, these data overall suggest that molecular correlates of inflammation prior to injury could predict the development of depression after a physical stressor.

Fully Implantable Plantar Cutaneous Augmentation System for Rats Using Closed-loop Electrical Nerve Stimulation.

Plantar cutaneous feedback plays an important role in stable and efficient gait, by modulating the activity of ankle dorsi- and plantar-flexor muscles. However, central and peripheral nervous system trauma often decrease plantar cutaneous feedback and/or interneuronal excitability in processing the plantar cutaneous feedback. In this study, we tested a fully implantable neural recording and stimulation system augmenting plantar cutaneous feedback. Electromyograms were recorded from the medial gastrocnemius muscle for stance phase detection, while biphasic stimulation pulses were applied to the distal-tibial nerve during the stance phase to augment plantar cutaneous feedback. A Bluetooth low energy and a Qi-standard inductive link were adopted for wireless communication and wireless charging, respectively. To test the operation of the system, one intact rat walked on a treadmill with the electrical system implanted into its back. Leg kinematics were recorded to identify the stance phase. Stimulation was applied, with a 250-ms onset delay from stance onset and 200-ms duration, resulting in the onset at 47.58 ± 2.82% of stance phase and the offset at 83.49 ± 4.26% of stance phase (Mean ± SEM). The conduction velocity of the compound action potential (31.2 m/s and 41.6 m/s at 1·T and 2·T, respectively) suggests that the evoked action potential was characteristic of an afferent volley for cutaneous feedback. We also demonstrated successful wireless charging and system reset functions. The experimental results suggest that the presented implantable system can be a valuable neural interface tool to investigate the effect of plantar cutaneous augmentation on gait in a rat model.

Closed-Loop Plantar Cutaneous Augmentation by Electrical Nerve Stimulation Increases Ankle Plantarflexion During Treadmill Walking.

Ankle plantarflexion plays an important role in forward propulsion and anterior-posterior balance during locomotion. This component of gait is often critically impacted by neurotraumas and neurological diseases. We hypothesized that augmenting plantar cutaneous feedback, via closed-loop distal-tibial nerve stimulation, could increase ankle plantarflexion during walking. To test the hypothesis, one intact rat walked on a motorized treadmill with implanted electronic device and electrodes for closed-loop neural recording and stimulation. Constant-current biphasic electrical pulse train was applied to distal-tibial nerve, based on electromyogram recorded from the medial gastrocnemius muscle, to be timed with the stance phase. The stimulation current threshold to evoke plantar cutaneous feedback was set at 30 µA (1·T), based on compound action potential evoked by stimulation. The maximum ankle joint angle at plantarflexion, during the application of stimulation currents of 3.3·T and 6.6·T, respectively, was increased from 149.4° (baseline) to 165.4° and 161.6°. The minimum ankle joint angle at dorsiflexion was decreased from 59.4° (baseline) to 53.1°, during the application of stimulation currents of 3.3·T, but not changed by 6.6·T. Plantar cutaneous augmentation also changed other gait kinematic parameters. Stance duty factor was increased from 51.9% (baseline) to 65.7% and 64.0%, respectively, by 3.3·T and 6.6·T, primarily due to a decrease in swing duration. Cycle duration was consistently decreased by the stimulation. In the control trial after two stimulation trials, a strong after-effect was detected in overall gait kinematics as well as ankle plantarflexion, suggesting that this stimulation has the potential for producing long-term changes in gait kinematics.

Variability in Open-Field Locomotor Scoring Following Force-Defined Spinal Cord Injury in Rats: Quantification and Implications.

Spinal cord injury research in experimental animals aims to define mechanisms of tissue damage and identify interventions that can be translated into effective clinical therapies. Highly reliable models of injury and outcome measurement are essential to achieve these aims and avoid problems with reproducibility. Functional scoring is a critical component of outcome assessment and is currently commonly focused on open field locomotion (the "BBB score"). Here we analyze variability of observed locomotor outcome after a highly regulated spinal cord contusion in a large group of rats that had not received any therapeutic intervention. Our data indicate that, despite tight regulation of the injury severity, there is considerable variability in open-field score of individual rats at 21 days after injury, when the group as a whole reaches a functional plateau. The bootstrapped reference interval (that defines boundaries that contain 95% scores in the population without regard for data distributional character) for the score at 21 days was calculated to range from 2.3 to 15.9 on the 22-point scale. Further analysis indicated that the mean day 21 score of random groups of 10 individuals drawn by bootstrap sampling from the whole study population varies between 9.5 and 13.5. Wide variability between individuals implies that detection of small magnitude group-level treatment effects will likely be unreliable, especially if using small experimental group sizes. To minimize this problem in intervention studies, consideration should be given to assessing treatment effects by comparing proportions of animals in comparator groups that attain pre-specified criterion scores.

SCI and depression: Does inflammation commandeer the brain?

The incidence of depression is almost twice as high in the spinally injured population compared to the general population. While this incidence has long been attributed to the psychological, economic, and social burdens that accompany spinal cord injury (SCI), data from animal studies indicate that the biology of SCI may play an important role in the development of depression. Inflammation has been shown to impact stress response in rodents and humans, and inflammatory cytokines have been associated with depression for decades. The inflammation inherent to SCI may disrupt necessary mechanisms of mental homeostasis, such as serotonin production, dopamine production, and the hypothalamic pituitary adrenal axis. Additionally, gut dysbiosis that occurs after SCI can exacerbate inflammation and may cause further mood and behavior changes. These mediators combined may significantly contribute to the rise in depression seen after SCI. Currently, there are no therapies specific to depression after SCI. Elucidation of the molecular pathways that contribute to SCI-specific depression is crucial for the understanding of this disease and its potential treatments.

Depression-like behavior corresponds with cardiac changes in a rodent model of spinal cord injury.

In previous studies we have shown that approximately 1/3 of male Sprague Dawley rats develop symptoms of depression following a spinal cord injury (SCI). Using established behavioral tests to measure depression in rodents, we found that after SCI, subjects characterized as depressed had decreased sucrose preference, open field activity, social exploration, and burrowing behavior. As some of these tests of depression could be affected by the compromised motor function inherent to the SCI condition, the current study examined whether non-subjective, physiological differences in heart rate and heart rate variability were also associated with depression, as seen in humans. Male Sprague Dawley rats were implanted with radiotelemetry devices and either received a moderate contusion injury or remained intact. The implanted telemetry devices recorded home cage activity, body temperature, heart rate, and heart rate variability for 5 min/h throughout a 30-day post-injury assessment period. Depression behavior was evaluated using a battery of tests conducted on days 9-10 and 19-20 post-injury. Locomotor recovery and pain reactivity were also examined. Hierarchical clustering, based on the behavioral scores collected on the tests of depression, revealed that 28% of the SCI subjects displayed symptoms of depression, relative to the remaining 72% of SCI subjects. The subjects characterized as depressed had significantly lower social interaction and burrowing activity than the group that was not depressed. Interestingly, the subjects behaviorally characterized as depressed also had significantly lower heart rate variability than the not-depressed intact group. There was no difference between not-depressed SCI and intact rats on this measure. Therefore, in addition to behavior, depressed and not-depressed rats differ on measures of physiological function that are associated with depression in humans. These physiological differences further validate the rodent model of depression after SCI.