Ying Lu

Selective Inflammatory Pain Insensitivity in the African Naked Mole-Rat (Heterocephalus glaber)

In all mammals, tissue inflammation leads to pain and behavioral sensitization to thermal and mechanical stimuli called hyperalgesia. We studied pain mechanisms in the African naked mole-rat, an unusual rodent species that lacks pain-related neuropeptides (e.g., substance P) in cutaneous sensory fibers. Naked mole-rats show a unique and remarkable lack of pain-related behaviors to two potent algogens, acid and capsaicin. Furthermore, when exposed to inflammatory insults or known mediators, naked mole-rats do not display thermal hyperalgesia. In contrast, naked mole-rats do display nocifensive behaviors in the formalin test and show mechanical hyperalgesia after inflammation. Using electrophysiology, we showed that primary afferent nociceptors in naked mole-rats are insensitive to acid stimuli, consistent with the animals lack of acid-induced behavior. Acid transduction by sensory neurons is observed in birds, amphibians, and fish, which suggests that this tranduction mechanism has been selectively disabled in the naked mole-rat in the course of its evolution. In contrast, nociceptors do respond vigorously to capsaicin, and we also show that sensory neurons express a transient receptor potential vanilloid channel-1 ion channel that is capsaicin sensitive. Nevertheless, the activation of capsaicin-sensitive sensory neurons in naked mole-rats does not produce pain-related behavior. We show that capsaicin-sensitive nociceptors in the naked mole-rat are functionally connected to superficial dorsal horn neurons as in mice. However, the same nociceptors are also functionally connected to deep dorsal horn neurons, a connectivity that is rare in mice. The pain biology of the naked mole-rat is unique among mammals, thus the study of pain mechanisms in this unusual species can provide major insights into what constitutes “normal” mammalian nociception.

Changes in nitric oxide synthase isoforms in the spinal cord of rat following induction of chronic arthritis

Abstract Nitric oxide (NO) possibly plays an important role in the events resulting in hyperalgesia. Nitric oxide synthase (NOS) is a key enzyme in the production of NO. In this study, the relationship between NOS and hyperalgesia in a rat chronic arthritis model was tested. Chronic arthritis was induced by injection of incomplete Freund’s adjuvant into the knee joint cavity unilaterally. The paw withdrawal latency (PWL) to radiant heat was used to detect secondary thermal hyperalgesia induced by the arthritis. After 1 day the PWL of the arthritic hindpaw decreased and it reached its nadir at 3 days after induction of arthritis. The lumbar and cervical enlargement of the spinal cord were removed in different groups of animals 3, 7, 14, or 21 days after induction of arthritis, and frozen tissue sections were cut. Two series of sections were incubated with polyclonal antibodies to neuronal NOS (nNOS) or to inducible NOS (iNOS). nNOS was found to increase gradually in laminae I–III in the lumber but not in the cervical enlargement. The change became most obvious 14 days after induction of arthritis as compared to the control animals. Ependymal cells around the central canal of the lumbar enlargement were more densely stained by anti-iNOS after arthritis. A corresponding change was also found in the cervical enlargement. Computer-assisted image analysis revealed that the mean density of the affected areas in the treated group increased significantly compared with the control animals. This study suggests that the expression of both nNOS and iNOS increase following induction of chronic arthritis, which in turn would presumably lead to an increase in the production of NO. This process could be involved in mediation of the secondary thermal hyperalgesia induced by chronic arthritis.

Nociception in Persistent Pancreatitis in Rats: Effects of Morphine and Neuropeptide Alterations

Background Most animal models of pancreatitis are short-lived or very invasive. A noninvasive animal model of pancreatitis developed in highly inbred rats by Merkord et al. with symptoms persisting for 3 weeks was adopted in the current study to test its validity as a model of visceral pain in commercially available rats. Methods The persistent pancreatitis model was established by tail vein injection of dibutyltin dichloride. Animals were given 10% alcohol in their drinking water to enhance the pancreatitis attack. Blood serum pancreatic enzymes and nociceptive state were monitored for 3 weeks after dibutyltin dichloride or vehicle. Behavioral testing included reflexive withdrawal to mechanical and thermal stimulation of the abdominal area. The effect of morphine on nociceptive behaviors was tested. Histologic analysis of the pancreas and immunohistochemical analysis of substance P and calcitonin gene-related peptide in the spinal cord are included in the study. Results Compared with naïve and vehicle-only injected control groups, rats receiving dibutyltin dichloride demonstrated an increase in withdrawal events after von Frey stimulation and decreased withdrawal latency after thermal stimulation, signaling a sensitized nociceptive state through 7 days. These pain-related measures were abrogated by morphine. Blood serum concentrations of amylase and lipase as well as tissue inflammatory changes and substance P were also significantly elevated during this same time period. Conclusions These results indicate that animals with the dibutyltin dichloride-induced experimental pancreatitis expressed serum, histologic, and behavioral characteristics similar in duration to those present during acute attacks experienced by patients with chronic pancreatitis. These findings and responsivity to morphine suggest the utility of this model developed in a commercially available strain of rats for study of persistent visceral pain.

Intrathecal gabapentin enhances the analgesic effects of subtherapeutic dose morphine in a rat experimental pancreatitis model.

Background: Morphine sulfate has long been used for analgesia, but clinical applications can be limited by side effects, tolerance, and potential for addiction at therapeutic doses. An agent that produces therapeutic analgesia when coadministered with low-dose morphine could have important clinical uses. The anticonvulsant agent gabapentin has been identified as having antihyperalgesic properties acting on the &agr;2&dgr;1 subunit of N-type voltage-activated calcium channels on dorsal root ganglia neurons. In this study, intrathecal gabapentin, which by itself is ineffective when administered spinally, was combined with low-dose morphine and tested in an acute bradykinin-induced pancreatitis model in rats. Methods: An intrathecal catheter was surgically inserted into the subarachnoid space of male Sprague-Dawley rats. A laparotomy was performed for ligation and cannulation of the bile-pancreatic duct. Rats were pretreated intrathecally with artificial cerebrospinal fluid, gabapentin, morphine, or combined gabapentin and morphine 30 min before bradykinin injection into the bile-pancreatic duct. Spontaneous behavioral activity (cage crossing, rearing, and hind limb extension) was monitored before drug injection (baseline) and after bradykinin injection into the bile-pancreatic duct to assess visceral pain. Results: Spinal pretreatment with up to 300 &mgr;g gabapentin alone was not effective in reducing hind limb extension in this model, but did restore some cage crossing and rearing behaviors. Spinal treatment with low-dose morphine reduced hind limb extension only. Spinal pretreatment with combined gabapentin and subtherapeutic doses of morphine sulfate resulted in restoration of all spontaneous behaviors to surgical baseline levels including elimination of hind limb extension. Conclusion: Combined spinal administration of gabapentin and low doses of morphine significantly reduces pain-related behaviors in this acute rat pancreatitis model, whereas these agents were ineffective when used alone in this dose range. These data suggest that the &agr;2&dgr;1 subunit of the N-type voltage-activated Ca2+ channels is involved in transmission of this visceral pain, likely through effects on primary afferent endings in the spinal cord. Thus, gabapentin may be an effective adjuvant to initial low dose spinal opioid therapy for clinical management of visceral pain.

The Combined Effects of N-type Calcium Channel Blockers and Morphine on Aδ Versus C Fiber Mediated Nociception

Intrathecal &mgr; opiates produce analgesia presynaptically by inhibiting calcium ion influx and postsynaptically by increasing potassium flux. Mu receptors are expressed on presynaptic terminals of unmyelinated (C), but not myelinated (A&dgr;) nociceptors. Thus, &mgr;-opioids such as morphine may act presynaptically to inhibit C, but not A&dgr;, neurotransmission, and postsynaptically on dorsal horn cells that receive input from A&dgr; and/or C fiber nociceptors. N-type calcium ion channel blockers, such as &ohgr;-conotoxin GVIA (&ohgr;-CTX), produce analgesia by impeding flux of calcium ions into A&dgr; and C fiber nociceptor terminals. Thus, morphine and &ohgr;-CTX attenuated C fiber nociception additively, possibly indicating the same presynaptic site of action. Conversely, morphine and &ohgr;- CTX were supraadditively analgesic on an A&dgr; test, indicating that these agents probably have different sites of action. We conclude that although intrathecal application of either morphine or &ohgr;-CTX attenuates both A&dgr; and C fiber mediated nociception in rats, the combined effects are quite different for the two fiber types. Specifically, although coadministration of morphine with &ohgr;-CTX produces an additive, apparently presynaptic antinociception for C fiber-mediated responses, the combination produces a clearly supraadditive, and likely synergistic effect on A&dgr; mediated nociception, probably by acting at pre and postsynaptic sites, respectively. Implications This study demonstrates that combined spinal administration of mu opioids and N-type calcium channel blockers may be useful in providing analgesia for A&dgr; mediated (first, sharp) pain while minimizing the side effects of both drugs.

Mitochondrial Ca(2+) uptake is essential for synaptic plasticity in pain.

The increase of cytosolic free Ca2+ ([Ca2+]c) due to NMDA receptor activation is a key step for spinal cord synaptic plasticity by altering cellular signal transduction pathways. We focus on this plasticity as a cause of persistent pain. To provide a mechanism for these classic findings, we report that [Ca2+]c does not trigger synaptic plasticity directly but must first enter into mitochondria. Interfering with mitochondrial Ca2+ uptake during a [Ca2+]c increase blocks induction of behavioral hyperalgesia and accompanying downstream cell signaling, with reduction of spinal long-term potentiation (LTP). Furthermore, reducing the accompanying mitochondrial superoxide levels lessens hyperalgesia and LTP induction. These results indicate that [Ca2+]c requires downstream mitochondrial Ca2+ uptake with consequent production of reactive oxygen species (ROS) for synaptic plasticity underlying chronic pain. These results suggest modifying mitochondrial Ca2+ uptake and thus ROS as a type of chronic pain therapy that should also have broader biologic significance.

Effects of baclofen on colon inflammation-induced Fos, CGRP and SP expression in spinal cord and brainstem.

The present study demonstrates sites of expression for Fos protein in the brainstem and lumbosacral spinal cord of rats subjected to mustard oil irritation of the colon. The protective effect of baclofen, a selective GABA(B) receptor agonist, on the induced Fos protein increases was determined. Mustard oil injected into the lumen of the colon produces an acute site-specific inflammation. Immunocytochemical localization of Fos protein in neuronal nuclei was evident after 1 h, was greatest at 2 h and was still evident but declining at 8 h. In the spinal cord the majority of Fos labeled neurons were localized in the superficial laminae of lumbar (L6) cord with more found in the sacral (S1) cord. Some labeled neurons were also found in the deeper spinal laminae, intermediolateral nucleus and around lamina X. Brainstem sites expressing Fos included the nucleus of the solitary tract in the medulla, parabrachial, locus coeruleus, pontine and caudal dorsal raphe nuclei and periaqueductal gray. Weak Fos protein labeling existed in a few cells in vehicle control animals. Systemic administration of the GABA(B) receptor agonist, baclofen (10 mg/kg, i.p.), significantly reduced Fos expression in the spinal cord after mustard oil treatment but significantly increased the relative number of nuclei labeled in the nucleus of the solitary tract. Baclofen also significantly decreases dorsal horn CGRP immunoreactivity relative to the increased levels seen after inflammation of the colon. The SP content increases observed after inflammation of the colon were not altered by baclofen. These data suggest that: (1) neurons in regions important for nociceptive transmission, descending inhibitory control and autonomic control are activated by noxious stimulation of the colon, and (2) baclofen specifically reduces Fos expression in the superficial dorsal horn of the spinal cord induced by nociceptive afferent input.

Enkephalin-encoding herpes simplex virus-1 decreases inflammation and hotplate sensitivity in a chronic pancreatitis model

BackgroundA chronic pancreatitis model was developed in young male Lewis rats fed a high-fat and alcohol liquid diet beginning at three weeks. The model was used to assess time course and efficacy of a replication defective herpes simplex virus type 1 vector construct delivering human cDNA encoding preproenkephalin (HSV-ENK).ResultsMost surprising was the relative lack of inflammation and tissue disruption after HSV-ENK treatment compared to the histopathology consistent with pancreatitis (inflammatory cell infiltration, edema, acinar cell hypertrophy, fibrosis) present as a result of the high-fat and alcohol diet in controls. The HSV-ENK vector delivered to the pancreatic surface at week 3 reversed pancreatitis-associated hotplate hypersensitive responses for 4–6 weeks, while control virus encoding β-galactosidase cDNA (HSV-β-gal) had no effect. Increased Fos expression seen bilaterally in pain processing regions in control animals with pancreatitis was absent in HSV-ENK-treated animals. Increased met-enkephalin staining was evident in pancreas and lower thoracic spinal cord laminae I–II in the HSV-ENK-treated rats.ConclusionThus, clear evidence is provided that site specific HSV-mediated transgene delivery of human cDNA encoding preproenkephalin ameliorates pancreatic inflammation and significantly reduces hypersensitive hotplate responses for an extended time consistent with HSV mediated overexpression, without tolerance or evidence of other opiate related side effects.

A plethora of painful molecules.

Pain is a fundamental experience with a complex and multi-layered neurobiological basis. In recent years a powerful battery of techniques has been brought to bear to unravel the mechanisms by which painful stimuli are transduced and processed. There have been several recent discoveries regarding the molecular transduction mechanisms in nociceptors and novel molecular and cellular mechanisms underlying the spinal processing of painful stimuli. The mechanisms by which sensory neurons initiate hyperalgesia and touch evoked pain (allodynia) have been addressed particularly successfully in recent studies. The rich variety of key molecular players that have emerged in physiological and pathophysiological pain states reflects the sophistication and uniqueness of this vitally important sense.

Differential antinociceptive effects of spinal opioids on foot withdrawal responses evoked by C fibre or Aδ nociceptor activation

Intrathecal application of μ, δ, and κ opioids attenuate responses on several tests of animal nociception. However, the potency of these opioids differ depending on which tests were used. One factor contributing to these discrepancies is that different types of noxious stimuli activate different sets of nociceptor types, which may be differentially sensitive to opiate inhibition. To examine this hypothesis, we used a recently developed behavioural test which allows for differential assessment of nociception evoked by the activation of myelinated (Aδ) and unmyelinated C thermonociceptors. Administration of a κ‐selective agonist was ineffective on either type of response. δ1 drugs were slightly more potent on C fibre‐mediated responses than on Aδ‐mediated responses. Intrathecal μ and δ2 drugs were antinociceptive on both Aδ and C nociceptor‐mediated responses. However, unlike the δ1 effects, the dose‐response curves for μ and δ2 drugs were significantly more steep for Aδ than for C fibre‐mediated responses, potentially indicating differences in the mechanisms by which the drugs act on these 2 response types.