Amanda Ellis

Morphine paradoxically prolongs neuropathic pain in rats by amplifying spinal NLRP3 inflammasome activation

Significance Pain after disease/damage of the nervous system is predominantly treated with opioids, but without exploration of the long-term consequences. We demonstrate that a short course of morphine after nerve injury doubles the duration of neuropathic pain. Using genetic and pharmacological interventions, and innovative Designer Receptor Exclusively Activated by Designer Drugs disruption of microglia reactivity, we demonstrate that opioid-prolonged neuropathic pain arises from spinal microglia and NOD-like receptor protein 3 inflammasome formation/activation. Inhibiting these processes permanently resets amplified pain to basal levels, an effect not previously reported. These data support the “two-hit hypothesis” of amplification of microglial activation—nerve injury being the first “hit,” morphine the second. The implications of such potent microglial “priming” has fundamental clinical implications for pain and may extend to many chronic neurological disorders. Opioid use for pain management has dramatically increased, with little assessment of potential pathophysiological consequences for the primary pain condition. Here, a short course of morphine, starting 10 d after injury in male rats, paradoxically and remarkably doubled the duration of chronic constriction injury (CCI)-allodynia, months after morphine ceased. No such effect of opioids on neuropathic pain has previously been reported. Using pharmacologic and genetic approaches, we discovered that the initiation and maintenance of this multimonth prolongation of neuropathic pain was mediated by a previously unidentified mechanism for spinal cord and pain—namely, morphine-induced spinal NOD-like receptor protein 3 (NLRP3) inflammasomes and associated release of interleukin-1β (IL-1β). As spinal dorsal horn microglia expressed this signaling platform, these cells were selectively inhibited in vivo after transfection with a novel Designer Receptor Exclusively Activated by Designer Drugs (DREADD). Multiday treatment with the DREADD-specific ligand clozapine-N-oxide prevented and enduringly reversed morphine-induced persistent sensitization for weeks to months after cessation of clozapine-N-oxide. These data demonstrate both the critical importance of microglia and that maintenance of chronic pain created by early exposure to opioids can be disrupted, resetting pain to normal. These data also provide strong support for the recent “two-hit hypothesis” of microglial priming, leading to exaggerated reactivity after the second challenge, documented here in the context of nerve injury followed by morphine. This study predicts that prolonged pain is an unrealized and clinically concerning consequence of the abundant use of opioids in chronic pain.

Prior exposure to repeated morphine potentiates mechanical allodynia induced by peripheral inflammation and neuropathy

Opioids, such as morphine, induce potent analgesia and are the gold standard for the treatment of acute pain. However, opioids also activate glia, inducing pro-inflammatory cytokine and chemokine production, which counter-regulates the analgesic properties of classical opioid receptor activation. It is not known how long these adverse pro-inflammatory effects last or whether prior morphine could sensitize the central nervous system (CNS) such that responses to a subsequent injury/inflammation would be exacerbated. Here, multiple models of inflammation or injury were induced two days after morphine (5mg/kg b.i.d., five days , s.c.) to test the generality of morphine sensitization of later pain. Prior repeated morphine potentiated the duration of allodynia from peripheral inflammatory challenges (complete Freunds adjuvant (CFA) into either hind paw skin or masseter muscle) and from peripheral neuropathy (mild chronic constriction injury (CCI) of the sciatic nerve). Spinal cord and trigeminal nucleus caudalis mRNAs were analyzed to identify whether repeated morphine was sufficient to alter CNS expression of pro-inflammatory response genes, measured two days after cessation of treatment. Prior morphine elevated IL-1β mRNA at both sites, MHC-II and TLR4 in the trigeminal nucleus caudalis but not spinal cord, but not glial activation markers at either site. Finally, in order to identify whether morphine sensitized pro-inflammatory cytokine release, spinal cord was isolated two days after morphine dosing for five days , and slices stimulated ex vivo with lipopolysaccharide. The morphine significantly induced TNFα protein release. Therefore, repeated morphine is able to sensitize subsequent CNS responses to immune challenges.

Below Level Central Pain Induced by Discrete Dorsal Spinal Cord Injury

Central neuropathic pain occurs with multiple sclerosis, stroke, and spinal cord injury (SCI). Models of SCI are commonly used to study central neuropathic pain and are excellent at modeling gross physiological changes. Our goal was to develop a rat model of central neuropathic pain by traumatizing a discrete region of the dorsal spinal cord, thereby avoiding issues including paralysis, urinary tract infection, and autotomy. To this end, dorsal root avulsion was pursued. The model was developed by first determining the number of avulsed dorsal roots sufficient to induce below-level hindpaw mechanical allodynia. This was optimally achieved by unilateral T13 and L1 avulsion, which resulted in tissue damage confined to Lissauers tract, dorsal horn, and dorsal columns, at the site of avulsion, with no gross physical changes at other spinal levels. Behavior following avulsion was compared to that following rhizotomy of the T13 and L1 dorsal roots, a commonly used model of neuropathic pain. Avulsion induced below-level allodynia that was more robust and enduring than that seen after rhizotomy. This, plus the lack of direct spinal cord damage associated with rhizotomy, suggests that avulsion is not synonymous with rhizotomy, and that avulsion (but not rhizotomy) is a model of central neuropathic pain. The new model described here is the first to use discrete dorsal horn damage by dorsal root avulsion to create below-level bilateral central neuropathic pain.

Morphine amplifies mechanical allodynia via TLR4 in a rat model of spinal cord injury.

Central neuropathic pain (CNP) is a pervasive, debilitating problem that impacts thousands of people living with central nervous system disorders, including spinal cord injury (SCI). Current therapies for treating this type of pain are ineffective and often have dose-limiting side effects. Although opioids are one of the most commonly used CNP treatments, recent animal literature has indicated that administering opioids shortly after a traumatic injury can actually have deleterious effects on long-term health and recovery. In order to study the deleterious effects of administering morphine shortly after trauma, we employed our low thoracic (T13) dorsal root avulsion model (Spinal Neuropathic Avulsion Pain, SNAP). Administering a weeklong course of 10mg/kg/day morphine beginning 24h after SNAP resulted in amplified mechanical allodynia. Co-administering the non-opioid toll-like receptor 4 (TLR4) antagonist (+)-naltrexone throughout the morphine regimen prevented morphine-induced amplification of SNAP. Exploration of changes induced by early post-trauma morphine revealed that this elevated gene expression of TLR4, TNF, IL-1β, and NLRP3, as well as IL-1β protein at the site of spinal cord injury. These data suggest that a short course of morphine administered early after spinal trauma can exacerbate CNP in the long term. TLR4 initiates this phenomenon and, as such, may be potential therapeutic targets for preventing the deleterious effects of administering opioids after traumatic injury.

Unilateral T13 and L1 dorsal root avulsion: methods for a novel model of central neuropathic pain.

Central neuropathic pain is associated with many disease states including multiple sclerosis, stroke, and spinal cord injury, and is poorly managed. One type of central neuropathic pain that is particularly debilitating and challenging to treat is pain that occurs below the level of injury (below-level pain). The study of central neuropathic pain is commonly performed using animal models of stroke and spinal cord injury. Most of the spinal cord injury models currently being used were originally developed to model the gross physiological impact of clinical spinal cord injury. In contrast, the T13/L1 dorsal root avulsion model of spinal cord injury described here was developed specifically for the study of central pain, and as such, was developed to minimize confounding complications, such as paralysis, urinary tract infections, and autotomy. As such, this model induces robust and reliable hindpaw mechanical allodynia. Two versions of the model are described. The first is optimal for testing systemically administered pharmacological manipulations. The second was developed to accommodate intrathecal application of pharmacological manipulations. This model provides an additional means by which to investigate central pain states associated with spinal cord injury, including below-level pain. Finally, a brief discussion of at-level pain measurement is described as it has been suggested in the literature that the mechanisms underlying below- and at-level pain are different.

Indwelling supradural catheters for induction of facial allodynia: surgical procedures, application of inflammatory stimuli, and behavioral testing.

Migraine headaches are debilitatingly painful and poorly managed. Facial allodynia is often associated with migraine, and clinical evidence indicates that it is a critical point in migraine progression. That is, if the migraine can be treated prior to the onset of facial allodynia, the migraine can be halted using triptans, whereas if treatment is administered after facial allodynia has begun, the treatment is ineffective. The meninges and the immune cells therein have been implicated in migraine facial pain. Indeed, application of inflammatory mediators over the meninges has been used to study changes in pain responsive neurons in trigeminal complex, and changes in their receptive fields. Much of this research has been carried out in anesthetized rats, which limits the clinical application. Our indwelling supradural catheter model, in which inflammatory mediators can be administered to the meninges in awake and freely moving rats, allows for the assessment of behavioral changes shortly after injection. Following administration of inflammatory soup (histamine, serotonin, bradykinin, and prostaglandin E2) or the immunogenic HIV-1 coat protein gp120 results in reliable periorbital mechanical allodynia. This model provides an additional means to study the neurocircuitry and neuropharmacology of facial allodynia. Here, we describe detailed methods for the placement of the catheter, injection procedures, and assessment of facial allodynia.

Supradural inflammatory soup in awake and freely moving rats induces facial allodynia that is blocked by putative immune modulators

Facial allodynia is a migraine symptom that is generally considered to represent a pivotal point in migraine progression. Treatment before development of facial allodynia tends to be more successful than treatment afterwards. As such, understanding the underlying mechanisms of facial allodynia may lead to a better understanding of the mechanisms underlying migraine. Migraine facial allodynia is modeled by applying inflammatory soup (histamine, bradykinin, serotonin, prostaglandin E2) over the dura. Whether glial and/or immune activation contributes to such pain is unknown. Here we tested if trigeminal nucleus caudalis (Sp5C) glial and/or immune cells are activated following supradural inflammatory soup, and if putative glial/immune inhibitors suppress the consequent facial allodynia. Inflammatory soup was administered via bilateral indwelling supradural catheters in freely moving rats, inducing robust and reliable facial allodynia. Gene expression for microglial/macrophage activation markers, interleukin-1β, and tumor necrosis factor-α increased following inflammatory soup along with robust expression of facial allodynia. This provided the basis for pursuing studies of the behavioral effects of 3 diverse immunomodulatory drugs on facial allodynia. Pretreatment with either of two compounds broadly used as putative glial/immune inhibitors (minocycline, ibudilast) prevented the development of facial allodynia, as did treatment after supradural inflammatory soup but prior to the expression of facial allodynia. Lastly, the toll-like receptor 4 (TLR4) antagonist (+)-naltrexone likewise blocked development of facial allodynia after supradural inflammatory soup. Taken together, these exploratory data support that activated glia and/or immune cells may drive the development of facial allodynia in response to supradural inflammatory soup in unanesthetized male rats.