Yung-Jen Huang

Metaplasticity and behavior: how training and inflammation affect plastic potential within the spinal cord and recovery after injury.

Research has shown that spinal circuits have the capacity to adapt in response to training, nociceptive stimulation and peripheral inflammation. These changes in neural function are mediated by physiological and neurochemical systems analogous to those that support plasticity within the hippocampus (e.g., long-term potentiation and the NMDA receptor). As observed in the hippocampus, engaging spinal circuits can have a lasting impact on plastic potential, enabling or inhibiting the capacity to learn. These effects are related to the concept of metaplasticity. Behavioral paradigms are described that induce metaplastic effects within the spinal cord. Uncontrollable/unpredictable stimulation, and peripheral inflammation, induce a form of maladaptive plasticity that inhibits spinal learning. Conversely, exposure to controllable or predictable stimulation engages a form of adaptive plasticity that counters these maladaptive effects and enables learning. Adaptive plasticity is tied to an up-regulation of brain derived neurotrophic factor (BDNF). Maladaptive plasticity is linked to processes that involve kappa opioids, the metabotropic glutamate (mGlu) receptor, glia, and the cytokine tumor necrosis factor (TNF). Uncontrollable nociceptive stimulation also impairs recovery after a spinal contusion injury and fosters the development of pain (allodynia). These adverse effects are related to an up-regulation of TNF and a down-regulation of BDNF and its receptor (TrkB). In the absence of injury, brain systems quell the sensitization of spinal circuits through descending serotonergic fibers and the serotonin 1A (5HT 1A) receptor. This protective effect is blocked by surgical anesthesia. Disconnected from the brain, intracellular Cl- concentrations increase (due to a down-regulation of the cotransporter KCC2), which causes GABA to have an excitatory effect. It is suggested that BDNF has a restorative effect because it up-regulates KCC2 and re-establishes GABA-mediated inhibition.

Peripheral noxious stimulation reduces withdrawal threshold to mechanical stimuli after spinal cord injury: Role of tumor necrosis factor alpha and apoptosis

&NA; Concomitant peripheral nociceptive stimulation after spinal cord injury produces maintained mechanical sensitivity, possibly indicative of persistent pain. Behavioral changes are in parallel with increased TNF&agr;‐induced apoptosis. &NA; We previously showed that peripheral noxious input after spinal cord injury (SCI) inhibits beneficial spinal plasticity and impairs recovery of locomotor and bladder functions. These observations suggest that noxious input may similarly affect the development and maintenance of chronic neuropathic pain, an important consequence of SCI. In adult rats with a moderate contusion SCI, we investigated the effect of noxious tail stimulation, administered 1 day after SCI on mechanical withdrawal responses to von Frey stimuli from 1 to 28 days after treatment. In addition, because the proinflammatory cytokine tumor necrosis factor alpha (TNF&agr;) is implicated in numerous injury‐induced processes including pain hypersensitivity, we assessed the temporal and spatial expression of TNF&agr;, TNF receptors, and several downstream signaling targets after stimulation. Our results showed that unlike sham surgery or SCI only, nociceptive stimulation after SCI induced mechanical sensitivity by 24 h. These behavioral changes were accompanied by increased expression of TNF&agr;. Cellular assessments of downstream targets of TNF&agr; revealed that nociceptive stimulation increased the expression of caspase 8 and the active subunit (12 kDa) of caspase 3, indicative of active apoptosis at a time point consistent with the onset of mechanical allodynia. In addition, immunohistochemical analysis revealed distinct morphological signs of apoptosis in neurons and microglia at 24 h after stimulation. Interestingly, expression of the inflammatory mediator NF&kgr;B was unaltered by nociceptive stimulation. These results suggest that noxious input caudal to the level of SCI can increase the onset and expression of behavioral responses indicative of pain, potentially involving TNF&agr; signaling.

Acute spinal cord injury (SCI) transforms how GABA affects nociceptive sensitization.

Noxious input can sensitize pain (nociceptive) circuits within the spinal cord, inducing a lasting increase in spinal cord neural excitability (central sensitization) that is thought to contribute to chronic pain. The development of spinally-mediated central sensitization is regulated by descending fibers and GABAergic interneurons. The current study provides evidence that spinal cord injury (SCI) transforms how GABA affects nociceptive transmission within the spinal cord, recapitulating an earlier developmental state wherein GABA has an excitatory effect. In spinally transected rats, noxious electrical stimulation and inflammation induce enhanced mechanical reactivity (EMR), a behavioral index of nociceptive sensitization. Pretreatment with the GABAA receptor antagonist bicuculline blocked these effects. Peripheral application of an irritant (capsaicin) also induced EMR. Both the induction and maintenance of this effect were blocked by bicuculline. Cellular indices of central sensitization [c-fos expression and ERK phosphorylation (pERK)] were also attenuated. In intact (sham operated) rats, bicuculline had the opposite effect. Pretreatment with a GABA agonist (muscimol) attenuated nociceptive sensitization in intact, but not spinally injured, rats. The effect of SCI on GABA function was linked to a reduction in the Cl- transporter, KCC2, leading to a reduction in intracellular Cl- that would attenuate GABA-mediated inhibition. Pharmacologically blocking the KCC2 channel (with i.t. DIOA) in intact rats mimicked the effect of SCI. Conversely, a pharmacological treatment (bumetanide) that should increase intracellular Cl- levels blocked the effect of SCI. The results suggest that GABAergic neurons drive, rather than inhibit, the development of nociceptive sensitization after spinal injury.

Complete spinal cord injury (SCI) transforms how brain derived neurotrophic factor (BDNF) affects nociceptive sensitization

ABSTRACT Noxious stimulation can induce a lasting increase in neural excitability within the spinal cord (central sensitization) that can promote pain and disrupt adaptive function (maladaptive plasticity). Brain‐derived neurotrophic factor (BDNF) is known to regulate the development of plasticity and has been shown to impact the development of spinally‐mediated central sensitization. The latter effect has been linked to an alteration in GABA‐dependent inhibition. Prior studies have shown that, in spinally transected rats, exposure to regular (fixed spaced) stimulation can counter the development of maladaptive plasticity and have linked this effect to an up‐regulation of BDNF. Here it is shown that application of the irritant capsaicin to one hind paw induces enhanced mechanical reactivity (EMR) after spinal cord injury (SCI) and that the induction of this effect is blocked by pretreatment with fixed spaced shock. This protective effect was eliminated if rats were pretreated with the BDNF sequestering antibody TrkB‐IgG. Intrathecal (i.t.) application of BDNF prevented, but did not reverse, capsaicin‐induced EMR. BDNF also attenuated cellular indices (ERK and pERK expression) of central sensitization after SCI. In uninjured rats, i.t. BDNF enhanced, rather than attenuated, capsaicin‐induced EMR and ERK/pERK expression. These opposing effects were related to a transformation in GABA function. In uninjured rats, BDNF reduced membrane‐bound KCC2 and the inhibitory effect of the GABAA agonist muscimol. After SCI, BDNF increased KCC2 expression, which would help restore GABAergic inhibition. The results suggest that SCI transforms how BDNF affects GABA function and imply that the clinical usefulness of BDNF will depend upon the extent of fiber sparing. HighlightsFixed spaced shock blocks spinally‐mediated central sensitization through BDNF.BDNF inhibits the development of spinally‐mediated central sensitization after SCI.Acute spinal cord injury induces a qualitative shift in how BDNF affects nociceptive transmission.SCI modifies how BDNF affects the Cl− co‐transporter KCC2 and GABAergic inhibition.

Learning about time within the spinal cord: evidence that spinal neurons can abstract and store an index of regularity

Prior studies have shown that intermittent noxious stimulation has divergent effects on spinal cord plasticity depending upon whether it occurs in a regular (fixed time, FT) or irregular (variable time, VT) manner: In spinally transected animals, VT stimulation to the tail or hind leg impaired spinal learning whereas an extended exposure to FT stimulation had a restorative/protective effect. These observations imply that lower level systems are sensitive to temporal relations. Using spinally transected rats, it is shown that the restorative effect of FT stimulation emerges after 540 shocks; fewer shocks generate a learning impairment. The transformative effect of FT stimulation is related to the number of shocks administered, not the duration of exposure. Administration of 360 FT shocks induces a learning deficit that lasts 24 h. If a second bout of FT stimulation is given a day after the first, it restores the capacity to learn. This savings effect implies that the initial training episode had a lasting (memory-like) effect. Two bouts of shock have a transformative effect when applied at different locations or at difference frequencies, implying spinal systems abstract and store an index of regularity (rather than a specific interval). Implications of the results for step training and rehabilitation after injury are discussed.

Learning about Time within the Spinal Cord II: Evidence that Temporal Regularity Is Encoded by a Spinal Oscillator

How a stimulus impacts spinal cord function depends upon temporal relations. When intermittent noxious stimulation (shock) is applied and the interval between shock pulses is varied (unpredictable), it induces a lasting alteration that inhibits adaptive learning. If the same stimulus is applied in a temporally regular (predictable) manner, the capacity to learn is preserved and a protective/restorative effect is engaged that counters the adverse effect of variable stimulation. Sensitivity to temporal relations implies a capacity to encode time. This study explores how spinal neurons discriminate variable and fixed spaced stimulation. Communication with the brain was blocked by means of a spinal transection and adaptive capacity was tested using an instrumental learning task. In this task, subjects must learn to maintain a hind limb in a flexed position to minimize shock exposure. To evaluate the possibility that a distinct class of afferent fibers provide a sensory cue for regularity, we manipulated the temporal relation between shocks given to two dermatomes (leg and tail). Evidence for timing emerged when the stimuli were applied in a coherent manner across dermatomes, implying that a central (spinal) process detects regularity. Next, we show that fixed spaced stimulation has a restorative effect when half the physical stimuli are randomly omitted, as long as the stimuli remain in phase, suggesting that stimulus regularity is encoded by an internal oscillator Research suggests that the oscillator that drives the tempo of stepping depends upon neurons within the rostral lumbar (L1-L2) region. Disrupting communication with the L1-L2 tissue by means of a L3 transection eliminated the restorative effect of fixed spaced stimulation. Implications of the results for step training and rehabilitation after injury are discussed.

Metaplasticity within the spinal cord: Evidence brain-derived neurotrophic factor (BDNF), tumor necrosis factor (TNF), and alterations in GABA function (ionic plasticity) modulate pain and the capacity to learn

Evidence is reviewed that behavioral training and neural injury can engage metaplastic processes that regulate adaptive potential. This issue is explored within a model system that examines how training affects the capacity to learn within the lower (lumbosacral) spinal cord. Response-contingent (controllable) stimulation applied caudal to a spinal transection induces a behavioral modification indicative of learning. This behavioral change is not observed in animals that receive stimulation in an uncontrollable manner. Exposure to uncontrollable stimulation also engages a process that disables spinal learning for 24-48 h. Controllable stimulation has the opposite effect; it engages a process that enables learning and prevents/reverses the learning deficit induced by uncontrollable stimulation. These observations suggest that a learning episode can impact the capacity to learn in future situations, providing an example of behavioral metaplasticity. The protective/restorative effect of controllable stimulation has been linked to an up-regulation of brain-derived neurotrophic factor (BDNF). The disruption of learning has been linked to the sensitization of pain (nociceptive) circuits, which is enabled by a reduction in GABA-dependent inhibition. After spinal cord injury (SCI), the co-transporter (KCC2) that regulates the outward flow of Cl- is down-regulated. This causes the intracellular concentration of Cl- to increase, reducing (and potentially reversing) the inward flow of Cl- through the GABA-A receptor. The shift in GABA function (ionic plasticity) increases neural excitability caudal to injury and sets the stage for nociceptive sensitization. The injury-induced shift in KCC2 is related to the loss of descending serotonergic (5HT) fibers that regulate plasticity within the spinal cord dorsal horn through the 5HT-1A receptor. Evidence is presented that these alterations in spinal plasticity impact pain in a brain-dependent task (place conditioning). The findings suggest that ionic plasticity can affect learning potential, shifting a neural circuit from dampened/hard-wired to excitable/plastic.

Localized and sustained release of brain-derived neurotrophic factor from injectable hydrogel/microparticle composites fosters spinal learning after spinal cord injury

Damaged axons in the adult mammalian central nervous system (CNS), including those of the spinal cord, have extremely limited endogenous capacity to regenerate. This is the result of both the intrinsic and extrinsic inhibitory factors that limit the regeneration of adult neurons. Despite attempts to limit or eliminate the extrinsic inhibitory components, regeneration of adult neurons in the CNS is still limited. Therefore, additional factors that can further enhance the intrinsic plasticity of adult neurons need to be considered. Herein, we examine the effects of brain-derived neurotrophic factor (BDNF), a known growth factor for neuronal survival and plasticity, using an in vivo delivery method for a localized and sustained delivery to the spinal cord. A highly versatile injectable biomaterial platform for the sustained delivery of BDNF was developed using a physical blend of hyaluronic acid (HA) and methylcellulose (MC), in combination with poly-lactic-co-glycolic acid (PLGA) microparticles. Contemporary studies examining the plasticity of the CNS suggest that the spinal cord is an important site for activity-dependent learning that can mediate motor function after injury or disease. Here we utilized such a learning paradigm in combination with local and sustained BDNF application (at L3-S2) to foster spinal learning after complete spinal cord injury in rodents. Our data suggest that composite biomaterial systems such as the one described herein can be utilized for the sustained and localized delivery of therapeutics following damage to the spinal cord.

Engaging pain fibers after a spinal cord injury fosters hemorrhage and expands the area of secondary injury

&NA; In humans, spinal cord injury (SCI) is often accompanied by additional tissue damage (polytrauma) that can engage pain (nociceptive) fibers. Prior work has shown that this nociceptive input can expand the area of tissue damage (secondary injury), undermine behavioral recovery, and enhance the development of chronic pain. Here, it is shown that nociceptive input given a day after a lower thoracic contusion injury in rats enhances the infiltration of red blood cells at the site of injury, producing an area of hemorrhage that expands secondary injury. Peripheral nociceptive fibers were engaged 24 h after injury by means of electrical stimulation (shock) applied at an intensity that engages unmyelinated pain (C) fibers or through the application of the irritant capsaicin. Convergent western immunoblot and cyanmethemoglobin colorimetric assays showed that both forms of stimulation increased the concentration of hemoglobin at the site of injury, with a robust effect observed 3–24 h after stimulation. Histopathology confirmed that shock treatment increased the area of hemorrhage and the infiltration of red blood cells. SCI can lead to hemorrhage by engaging the sulfonylurea receptor 1 (SUR1) transient receptor potential melastatin 4 (TRPM4) channel complex in neurovascular endothelial cells, which leads to cell death and capillary fragmentation. Histopathology confirmed that areas of hemorrhage showed capillary fragmentation. Co‐immunoprecipitation of the SUR1‐TRPM4 complex showed that it was up‐regulated by noxious stimulation. Shock‐induced hemorrhage was associated with an acute disruption in locomotor performance. These results imply that noxious stimulation impairs long‐term recovery because it amplifies the breakdown of the blood spinal cord barrier (BSCB) and the infiltration of red blood cells, which expands the area of secondary injury.

Pain Input After Spinal Cord Injury (SCI) Undermines Long-Term Recovery and Engages Signal Pathways That Promote Cell Death

Pain (nociceptive) input caudal to a spinal contusion injury increases tissue loss and impairs long-term recovery. It was hypothesized that noxious stimulation has this effect because it engages unmyelinated pain (C) fibers that produce a state of over-excitation in central pathways. The present article explored this issue by assessing the effect of capsaicin, which activates C-fibers that express the transient receptor potential vanilloid receptor-1 (TRPV1). Rats received a lower thoracic (T11) contusion injury and capsaicin was applied to one hind paw the next day. For comparison, other animals received noxious electrical stimulation at an intensity that engages C fibers. Both forms of stimulation elicited similar levels of c-fos mRNA expression, a cellular marker of nociceptive activation, and impaired long-term behavioral recovery. Cellular assays were then performed to compare the acute effect of shock and capsaicin treatment. Both forms of noxious stimulation increased expression of tumor necrosis factor (TNF) and caspase-3, which promotes apoptotic cell death. Shock, but not capsaicin, enhanced expression of signals related to pyroptotic cell death [caspase-1, inteleukin-1 beta (IL-1ß)]. Pyroptosis has been linked to the activation of the P2X7 receptor and the outward flow of adenosine triphosphate (ATP) through the pannexin-1 channel. Blocking the P2X7 receptor with Brilliant Blue G (BBG) reduced the expression of signals related to pyroptotic cell death in contused rats that had received shock. Blocking the pannexin-1 channel with probenecid paradoxically had the opposite effect. BBG enhanced long-term recovery and lowered reactivity to mechanical stimulation applied to the girdle region (an index of chronic pain), but did not block the adverse effect of nociceptive stimulation. The results suggest that C-fiber input after injury impairs long-term recovery and that this effect may arise because it induces apoptotic cell death.