Molluscan memory of injury: evolutionary insights into chronic pain and neurological disorders.

Molluscan preparations have yielded seminal discoveries in neuroscience, but the experimental advantages of this group have not, until now, been complemented by adequate molecular or genomic information for comparisons to genetically defined model organisms in other phyla. The recent sequencing of the transcriptome and genome of Aplysia californica, however, will enable extensive comparative studies at the molecular level. Among other benefits, this will bring the power of individually identifiable and manipulable neurons to bear upon questions of cellular function for evolutionarily conserved genes associated with clinically important neural dysfunction. Because of the slower rate of gene evolution in this molluscan lineage, more homologs of genes associated with human disease are present in Aplysia than in leading model organisms from Arthropoda (Drosophila) or Nematoda (Caenorhabditis elegans). Research has hardly begun in molluscs on the cellular functions of gene products that in humans are associated with neurological diseases. On the other hand, much is known about molecular and cellular mechanisms of long-term neuronal plasticity. Persistent nociceptive sensitization of nociceptors in Aplysia displays many functional similarities to alterations in mammalian nociceptors associated with the clinical problem of chronic pain. Moreover, in Aplysia and mammals the same cell signaling pathways trigger persistent enhancement of excitability and synaptic transmission following noxious stimulation, and these highly conserved pathways are also used to induce memory traces in neural circuits of diverse species. This functional and molecular overlap in distantly related lineages and neuronal types supports the proposal that fundamental plasticity mechanisms important for memory, chronic pain, and other lasting alterations evolved from adaptive responses to peripheral injury in the earliest neurons. Molluscan preparations should become increasingly useful for comparative studies across phyla that can provide insight into cellular functions of clinically important genes.

Chronic spontaneous activity generated in the somata of primary nociceptors is associated with pain-related behavior after spinal cord injury.

Mechanisms underlying chronic pain that develops after spinal cord injury (SCI) are incompletely understood. Most research on SCI pain mechanisms has focused on neuronal alterations within pain pathways at spinal and supraspinal levels associated with inflammation and glial activation. These events might also impact central processes of primary sensory neurons, triggering in nociceptors a hyperexcitable state and spontaneous activity (SA) that drive behavioral hypersensitivity and pain. SCI can sensitize peripheral fibers of nociceptors and promote peripheral SA, but whether these effects are driven by extrinsic alterations in surrounding tissue or are intrinsic to the nociceptor, and whether similar SA occurs in nociceptors in vivo are unknown. We show that small DRG neurons from rats (Rattus norvegicus) receiving thoracic spinal injury 3 d to 8 months earlier and recorded 1 d after dissociation exhibit an elevated incidence of SA coupled with soma hyperexcitability compared with untreated and sham-treated groups. SA incidence was greatest in lumbar DRG neurons (57%) and least in cervical neurons (28%), and failed to decline over 8 months. Many sampled SA neurons were capsaicin sensitive and/or bound the nociceptive marker, isolectin B4. This intrinsic SA state was correlated with increased behavioral responsiveness to mechanical and thermal stimulation of sites below and above the injury level. Recordings from C- and Aδ-fibers revealed SCI-induced SA generated in or near the somata of the neurons in vivo. SCI promotes the entry of primary nociceptors into a chronic hyperexcitable-SA state that may provide a useful therapeutic target in some forms of persistent pain.

Studies on behavioral regulations of morphine tolerance in rats

Opioids remain the drugs of choice in chronic pain treatment, but opioid tolerance, defined as a decrease in analgesic effect after prolonged or repeated use, dramatically limits their clinical utility. Opioid tolerance has classically been studied by implanting spinal catheters in animals for drug administration. This procedure has significant morbidity and mortality, as well as causing an inflammatory response which decreases the potency of opioid analgesia and possibly affects tolerance development. Therefore, we developed and validated a new method, intermittent lumbar puncture (Dautzenberg et al.), for the study of opioid analgesia and tolerance. Using this method, opioid tolerance was reliably induced without detectable morbidity. The dose of morphine needed to induce analgesia and tolerance using this method was about 100-fold lower than that required when using an intrathecal catheter. Only slight inflammation was found at the injection site, dissipated within seven mm. ^ DAMGO, an opioid μ receptor agonist, has been reported to inhibit morphine tolerance, but results from different studies are inconclusive. We evaluated the effect of DAMGO on morphine tolerance using our newly-developed ILP method, as well as other intrathecal catheter paradigms. We found that co-administration of sub-analgesic DAMGO with morphine using ILP did not inhibit morphine tolerance, but instead blocked the analgesic effects of morphine. Tolerance to morphine still developed. Tolerance to morphine can only be blocked by sub-analgesic dose of DAMGO when administered in a lumbar catheter, but not in cervical catheter settings. ^ Finally, we evaluated the effects of Gabapentin (GBP) on analgesia and morphine tolerance. We demonstrated that GBP enhanced analgesia mediated by both subanalgesic and analgesic doses of morphine although GBP itself was not analgesic. GBP increased potency and efficacy of morphine. GBP inhibited the expression, but not the development, of morphine tolerance. GBP blocked tolerance to analgesic morphine but not to subanalgesic morphine. GBP reversed the expression of morphine tolerance even after tolerance was established. These studies may begin to provide new insights into mechanisms of morphine tolerance development and improve clinical chronic pain management. ^

Long-term hyperexcitability of sensory and motor axons in Aplysia californica: Induction by axotomy, serotonin, and transient depolarization

Neuropathic pain is a debilitating neurological disorder that may appear after peripheral nerve trauma and is characterized by persistent, intractable pain. The well-studied phenomenon of long-term hyperexcitability (LTH), in which sensory somata become hyperexcitable following peripheral nerve injury may be important for both chronic pain and long-lasting memory formation, since similar cellular alterations take place after both injury and learning. Though axons have previously been considered simple conducting cables, spontaneous afferent signals develop from some neuromas that form at severed nerve tips, indicating intrinsic changes in sensory axonal excitability may contribute to this intractable pain. Here we show that nerve transection, exposure to serotonin, and transient depolarization induce long-lasting sensory axonal hyperexcitability that is localized to the treated nerve segment and requires local translation of new proteins. Long-lasting functional plasticity may be a general property of axons, since both injured and transiently depolarized motor axons display LTH as well. Axonal hyperexcitability may represent an adaptive mechanism to overcome conduction failure after peripheral injury, but also displays key features shared with cellular analogues of memory including: site-specific changes in neuronal function, dependence on transient, focal depolarization for induction, and requirement for synthesis of new proteins for expression of long-lasting effects. The finding of axonal hyperexcitability after nerve injury sheds new light on the clinical problem of chronic neuropathic pain, and provides more support for the hypothesis that mechanisms of long-term memory storage evolved from primitive adaptive responses to injury. ^

TRPV1 Channels Contribute to Behavioral Hypersensitivity and Spontaneous Activity in Nociceptors After Spinal Cord Injury

A majority of persons who have sustained spinal cord injury (SCI) develop chronic pain. While most investigators have assumed that the critical mechanisms underlying neuropathic pain after SCI are restricted to the central nervous system (CNS), recent studies showed that contusive SCI results in a large increase in spontaneous activity in primary nociceptors, which is correlated significantly with mechanical allodynia and thermal hyperalgesia. Upregulation of ion channel transient receptor vanilloid 1 (TRPV1) has been observed in the dorsal horn of the spinal cord after SCI, and reduction of SCI-induced hyperalgesia by a TRPV1 antagonist has been claimed. However, the possibility that SCI enhances TRPV1 expression and function in nociceptors has not been tested. I produced contusive SCI at thoracic level T10 in adult, male rats and harvested lumbar (L4/L5) dorsal root ganglia (DRG) from sham-treated and SCI rats 3 days and 1 month after injury, as well as from age-matched naive control rats. Whole-cell patch clamp recordings were made from small (soma diameter <30 >μm) DRG neurons 18 hours after dissociation. Capsaicin-induced currents were significantly increased 1 month, but not 3 days, after SCI compared to neurons from control animals. In addition, Ca2+ transients imaged during capsaicin application were significantly greater 1 month after SCI. Western blot experiments indicated that expression of TRPV1 protein in DRG is also increased 1 month after SCI. A major role for TRPV1 channels in pain-related behavior was indicated by the ability of a specific TRPV1 antagonist, AMG9810, to reverse SCI-induced hypersensitivity of hindlimb withdrawal responses to heat and mechanical stimuli. Similar reversal of behavioral hypersensitivity was induced by intrathecal delivery of oligodeoxynucleotides antisense to TRPV1, which knocked down TRPV1 protein and reduced capsaicin-evoked currents. TRPV1 knockdown also decreased the incidence of spontaneous activity in dissociated nociceptors after SCI. Limited activation of TRPV1 was found to induce prolonged repetitive firing without accommodation or desensitization, and this effect was enhanced by SCI. These data suggest that SCI enhances TRPV1 expression and function in primary nociceptors, increasing the excitability and spontaneous activity of these neurons, thus contributing to chronic pain after SCI.

A Pre-Clinical Assessment of Minocycline for Treatment of Chronic Neuropathic Pain after Spinal Cord Injury

Patients living with a spinal cord injury (SCI) often develop chronic neuropathic pain (CNP). Unfortunately, the clinically approved, current standard of treatment, gabapentin, only provides temporary pain relief. This treatment can cause numerous adverse side effects that negatively affect the daily lives of SCI patients. There is a great need for alternative, effective treatments for SCI-dependent CNP. Minocycline, an FDA-approved antibiotic, has been widely prescribed for the treatment of acne for several decades. However, recent studies demonstrate that minocycline has neuroprotective properties in several pre-clinical rodent models of CNS trauma and disease. Pre-clinical studies also show that short-term minocycline treatment can prevent the onset of CNP when delivered during the acute stage of SCI and can also transiently attenuate established CNP when delivered briefly during the chronic stage of SCI. However, the potential to abolish or attenuate CNP via long-term administration of minocycline after SCI is unknown. The purpose of this study was to investigate the potential efficacy and safety of long-term administration of minocycline to abolish or attenuate CNP following SCI. A severe spinal contusion injury was administered on adult, male, Sprague-Dawley rats. At day 29 post-injury, I initiated a three-week treatment regimen of daily administration with minocycline (50 mg/kg), gabapentin (50 mg/kg) or saline. The minocycline treatment group demonstrated a significant reduction in below-level mechanical allodynia and above- level hyperalgesia while on their treatment regimen. After a ten-day washout period of minocycline, the animals continued to demonstrate a significant reduction in below-level mechanical allodynia and above-level hyperalgesia. However, minocycline-treated animals exhibited abnormal weight gain and hepatotoxicity compared to gapabentin-treated or vehicle-treated subjects.The results support previous findings that minocycline can attenuate CNP after SCI and suggested that minocycline can also attenuate CNP via long-term delivery of minocycline after SCI (36). The data also suggested that minocycline had a lasting effect at reducing pain symptoms. However, the adverse side effects of long-term use of minocycline should not be ignored in the rodent model. Gabapentin treatment caused a significant decrease in below-level mechanical allodynia and below-level hyperalgesia during the treatment regimen. Because gabapentin treatment has an analgesic effect at the concentration I administered, the results were expected. However, I also found that gabapentin-treated animals demonstrated a sustained reduction in pain ten days after treatment withdrawal. This result was unexpected because gabapentin has a short half-life of 1.7 hours in rodents and previous studies have demonstrated that pre-drug pain levels return shortly after withdrawal of treatment. Additionally, the gabapentin-treated animals demonstrated a significant and sustained increase in rearing events compared with all other treatment groups which suggested that gabapentin treatment was not only capable of reducing pain long-term but may also significantly improve trunk stability or improve motor function recovery.

Mechanisms of apoptosis in chronic lymphocytic leukemia (CLL)

Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by the accumulation of terminally differentiated, mature B cells that do not progress beyond the G1 stage of cell cycle, suggesting that these cells possess intrinsic defects in apoptosis. Treatment relies heavily on chemotherapy (primarily nucleoside analogs and glucocorticoids) that may initially be effective in patients, but ultimately give rise to refractory, untreatable disease. The purpose of this study was to determine whether key components of the apoptotic machinery were intact in CLL lymphocytes, especially in patients refractory to therapy. ^ Activation of proteases has been shown to be at the core of the apoptotic pathway and this work demonstrates that protease activation is required for glucocorticoid and nucleoside analog-induced apoptosis in CLL cells. Inhibitors of serine proteases as well as caspase inhibitors blocked induced DNA fragmentation, and a peptide inhibitor of the nuclear scaffold (NS) protease completely suppressed both induced and spontaneous apoptosis. However, the NS protease inhibitor actually promoted several pro-apoptotic events, such as caspase activation, exposure of surface phosphatidylserine, and loss of mitochondrial membrane potential. These results suggested that the NS protease may interact with the apoptotic program in CLL cells at two separate points. ^ In order to further investigate the role of the NS protease in CLL, patient isolates were treated with proteasome inhibitors because of previous results suggesting that the ISIS protease might be a β subunit of the proteasome. Proteasome inhibitors induced massive DNA fragmentation in every patient tested, even in those resistant to the effects of glucocorticoid and nucleoside analogs in vitro. Several other features of apoptosis were also promoted by the proteasome inhibitor, including mitochondrial alterations such as release of cytochrome c and drops in mitochondrial membrane potential. Proteasome inhibitor-induced apoptosis was associated with inhibition of NFκB, a proteasome-regulated transcription factor that has been implicated in the suppression of apoptosis in a number of systems. The NS protease inhibitor also caused a decrease in active NFκB, suggesting that the proapoptotic effects of this agent might be due to depletion of NFκB. ^ Given these findings, the role of NFκB, in conferring survival in CLL was investigated. Glucocorticoid hormone treatment was shown to cause decreases in the activity of the transcription factor, while phorbol dibutyrate, which blocks glucocorticoid-induced DNA fragmentation, was capable of upregulating NFκB. Compellingly, introduction of an undegradable form of the constitutive NFκB inhibitor, IκB, caused DNA fragmentation in several patient isolates, some of which were resistant to glucocorticoid in vitro. Transcription of anti-apoptotic proteins by NFκB was postulated to be responsible for its effects on survival, but Bcl-2 levels did not fluctuate with glucocorticoid or proteasome inhibitor treatment. ^ The in vitro values generated from these studies were organized into a database containing numbers for over 250 patients. Correlation of relevant clinical parameters revealed that levels of spontaneous apoptosis in vitro differ significantly between Rai stages. Importantly, in vitro resistance to nucleoside analogs or glucocorticoids predicted resistance to chemotherapy in vivo, and inability to achieve remission. ^

The pain experience of elderly hospice patients with cancer

Purpose/objectives. A grounded theory design was used to identify, describe, and generate a theoretical analysis of the pain experience of elderly hospice patients with cancer. ^ Sample. Eleven participants over the age of 65, receiving services from a for-profit hospice were interviewed in their homes. ^ Methods. Broad unstructured face to face audio-taped interviews were transcribed verbatim and analyzed using constant-comparative method of analysis. ^ Findings. Pain was described as a hierarchy of chronic, acute, and psychological pain with psychological pain as the worst. Suffering was the basic social problem of pain. Participants dealt with suffering by the basic social process of enduring. Enduring had two sub-processes, maintaining hope and adjusting. Trusting in a higher being and finding meaning were mechanisms of maintaining hope. Mechanisms of adjusting were dealing with uncertainty, accepting, and minimizing pain. ^ Implications for nursing practice. Nurses need to recognize and value the hard work of enduring to deal with suffering. Assisting elderly hospice patients with cancer to address the sub-processes of enduring and their mechanisms can foster enduring. ^

Back pain in migrant farmworker families and farmworker high school students from Starr County, Texas

Back symptoms are a major global public health problem with the lifetime prevalence ranging between 50-80%. Research suggests that work-related factors contribute to the occurrence of back pain in various industries. Despite the hazardous nature, strenuous tasks, and awkward postures associated with farm work, little is known about back injury and symptoms in farmworker adults and children. Research in the United States is particularly limited. This is a concern given the large proportion of migrant farmworkers in the United States without adequate access to healthcare as well as a substantial number of youth working in agriculture. The present study describes back symptoms and identifies work-related factors associated with back pain in migrant farmworker families and farmworker high school students from Starr County, TX. Two separate datasets were used from two cohort studies "Injury and Illness Surveillance in Migrant Farmworkers (MANOS)" (study A: n=267 families) and "South Texas Adolescent Rural Research Study (STARRS)" (study B: n=345). Descriptive and inferential statistics including multivariable logistic regression were used to identify work-related factors associated with back pain in each study. In migrant farmworker families, the prevalence of chronic back pain during the last migration season ranged from 9.5% among youngest children to 33.3% among mothers. Chronic back pain was significantly associated with increasing age; fairly bad/very bad quality of sleep while migrating; fewer than eight hours of sleep at home in Starr County, TX; depressive symptoms while migrating; self-provided water for washing hands/drinking; weeding at work; and exposure to pesticide drift/direct spray. Among farmworker adolescents, the prevalence of severe back symptoms was 15.7%. Severe back symptoms were significantly associated with being female; history of a prior accident/back injury; feeling tense, stressed, or anxious sometimes/often; lifting/carrying heavy objects not at work; current tobacco use; increasing lifetime number of migrant farmworker years; working with/around knives; and working on corn crops. Overall, results support that associations between work-related exposures and chronic back pain and severe back symptoms remain after controlling for the effect of non-work exposures in farmworker populations. ^

Blood-oxygen-level-dependent (BOLD) signal changes in total cortex and subcortex, pain, reward, and vigilance regions during mechanical pressure pain after morphine administration

Morphine is the most common clinical choice in the management of severe pain. Although the molecular mechanisms of morphine have already been characterized, the cerebral circuits by which it attenuates the sensation of pain have not yet been studied in humans. The objective of this two-arm (morphine versus placebo), between-subjects study was to examine whether morphine affects pain via pain-related cortical circuits, but also via reward regions that relate to the motivational state, as well as prefrontal regions that relate to vigilance as a result of morphine's sedative effects. Cortical activity was measured by the blood-oxygen-level-dependent (BOLD) signal changes using functional magnetic resonance imaging (fMRI). ^ The novelty of this study is at three levels: (i) to develop a methodology that will assess the average BOLD signal across subjects for the pain, reward, and vigilance cortical systems; (ii) to examine whether the reward and/or sedative effects of morphine are contributing factors to cortical regions associated with the motivational state and vigilance; and (iii) to propose a neuroanatomical model related to the opioid-sensitive effects of reward and sedation as a function of cortical activity related to pain in an effort to assess future analgesics. ^ Consistent with our hypotheses, our findings showed that the decrease in total pain-related volume activated between the post- and the pre-treatment morphine group was about 78%, while the post-treatment placebo group displayed only a 5% decrease when compared to pre-treatment levels of activation. The volume increase in reward regions was 451% in the post-treatment compared to the pre-treatment morphine condition. Finally, the volumetric decrease in vigilance regions was 63% in the posttreatment compared to the pre-treatment morphine condition. ^ These findings imply that changes in the blood flow of the reward and vigilance regions may be contributing factors in producing the analgesic effect under morphine administration. Future studies need to replicate this study in a higher resolution fMRI environment and to assess the proposed neuroanatomical model in patient populations. The necessity of pain research is apparent, since pain cuts across different diseases especially chronic ones, and thus, is recognized as a vital public health developing area. ^