Dawn Conklin

Cyclooxygenase-1 in the spinal cord plays an important role in postoperative pain

&NA; Cyclooxygenase‐2 (COX‐2) activity in the spinal cord plays a key role in sensitization to sensory stimuli during acute inflammation. In contrast, intrathecal administration of COX‐2 specific inhibitors has minimal analgesic effects in an incisional model of postoperative pain. We investigated the role of COX isoforms in this model by examining the expression of COX‐1 and the effect of intrathecal COX inhibitors. A 1 cm longitudinal incision was made through skin, fascia and muscles of the plantar aspect of the left paw in male rats, and withdrawal threshold to von Frey filaments measured. Rats were perfused at 1, 2, 3, 5, and 7 days after incision, and COX‐1 immunohistochemistry was performed on L3 to S2 spinal cord and gracile nucleus sections. Other rats received intrathecally the COX‐1 preferring inhibitor, ketorolac, the specific COX‐1 inhibitor, SC‐560, the COX‐2 inhibitor, NS‐398 or vehicle 1 day after surgery. Withdrawal threshold was measured at intervals up to 5 days later. COX‐1 immunoreactivity increased in glia in the ipsilateral L4–L6 spinal dorsal horn and ipsilateral gracile nucleus after incision. Mechanical allodynia peaked on postoperative day 1, and COX‐1 immunoreactivity increased on day 1, peaked on day 2, and declined thereafter. Ketorolac and SC‐560 dose‐dependently increased withdrawal threshold in this model, but NS‐398 had no effect. These results suggest that COX‐1 plays an important role in spinal cord pain processing and sensitization after surgery. Increased COX‐1 activity could precede the up‐regulation of COX‐1 protein, and spinally administered specific COX‐1 inhibitors may be useful to treat postoperative pain.

Induction of astrocyte metallothioneins (MTs) by zinc confers resistance against the acute cytotoxic effects of methylmercury on cell swelling, Na+ uptake, and K+ release

Metallothionein (MT) proteins play an important role in the detoxification of heavy metals. Since methylmercury (MeHg) preferentially accumulates in astrocytes, we investigated the ability of the astrocyte-specific MT isoform, MT-I, to attenuate MeHg-induced cytotoxicity. Increased astrocytic MT expression was achieved by 24-h pretreatment of neonatal rat primary astrocyte cultures with 100 microM zinc (ZnSO4). Subsequently, the astrocytes were treated with MeHg (10 microM), and its toxic effects on cell volume, Na+ uptake, and K+ release were investigated and compared to cells treated with or without MeHg, but in the absence of Zn pretreatment. Pretreatment of astrocytes with Zn was associated with a 2.9-fold increase in MT protein levels (P<0.02), and a 5.6-fold increase in MT mRNA levels (p<0.002) compared to control astrocytes. Astrocytes expressing increased MT protein levels were resistant to MeHg-induced swelling. In isotonic buffer the effect of MeHg on swelling was abolished (p<0.01) by 24-h Zn pretreatment, in such a way that volume profiles in these cells did not differ from controls. Zn-induced increased expression of MTs was also associated with significant attenuation of astrocytic Na+ uptake (p<0.01) and Rb+ (a marker for K+) release (p<0.001) in response to treatment with MeHg. These results demonstrate (1) that astrocytes can be induced to express high levels of MT proteins by pretreatment with Zn, and (2) that Zn confers resistance against the acute effect of MeHg on astrocytic swelling and the associated changes in ion (Na+ and K+) transport. Taken together, the data suggest that astrocytic MT induction offers effective cellular adaptation to MeHg cytotoxicity.

Spinal noradrenergic activation mediates allodynia reduction from an allosteric adenosine modulator in a rat model of neuropathic pain

&NA; Activation of adenosine A1 receptors by endogenous adenosine or synthetic agonists produces anti‐nociception in animal models of acute pain and also reduces hypersensitivity in models of inflammatory and nerve‐injury pain. Allosteric adenosine modulators facilitate adenosine agonist binding to the A1 receptor. The purpose of the current study was to examine the effect, mechanisms of action, and interaction with noradrenergic systems of intrathecal (i.t.) or oral administration of the allosteric adenosine modulator T62 in a rat model of neuropathic pain. A spinal nerve ligation rat model (SNL; ligation of left L5 and L6 spinal nerve roots) was used. One week after SNL surgery, an i.t. catheter was inserted. Withdrawal threshold to mechanical stimulation of the left hind paw was determined before and after surgery, confirming mechanical hypersensitivity. Oral or i.t. T62 reduced hypersensitivity induced by SNL. The effects of i.t. T62 were inhibited by i.t. injection of an A1 receptor antagonist and by an &agr;2‐adrenergic antagonist but not by an A2 adenosine receptor antagonist. Anti‐dopamine &bgr; hydroxylase (D&bgr;H)‐saporin treatment reduce spinal norepinephrine content by 97%, accompanied by an almost complete loss of D&bgr;H immunoreactive axons in the spinal dorsal horn and neurons in the locus coeruleus. The effect of T62 was completely lost in animals treated with anti‐D&bgr;H‐saporin. These data support the hypothesis that activation of the A1 receptor by the allosteric modulator, T62, produces anti‐nociception via spinal noradrenergic activation.

Plasticity in Action of Intrathecal Clonidine to Mechanical but Not Thermal Nociception after Peripheral Nerve Injury

Background Intrathecal clonidine reduces tactile allodynia in animal models of neuropathic pain, and this effect is blocked by atropine. However, the role of tonic spinal cholinergic activity and its interaction with &agr;2-adrenergic systems in normal and neuropathic conditions and to different sensory methods has not been systematically examined. The authors examined cholinergic receptor involvement in thermal and mechanical sensitivity in normal and neuropathic animals and its interaction with intrathecal clonidine. Methods Normal rats and rats that received L5/L6 spinal nerve ligation were tested with acute radiant heat, paw pressure, and punctate mechanical stimulation before and after the intrathecal administration of saline, the muscarinic receptor antagonist, atropine, or a toxin to destroy cholinergic neurons, and then after intrathecal clonidine. Results Atropine, the cholinergic neuronal toxin, and saline did not alter baseline withdrawal thresholds. In nerve-injured rats, neither saline nor atropine altered antinociception from clonidine to a thermal stimulus, but atropine reduced the effect of clonidine to von Frey filament withdrawal threshold (34 ± 5.6 vs. 14 ± 5.8 g [mean ± SEM], saline vs. atropine;P < 0.05) and to withdrawal threshold to paw pressure after clonidine (174 ± 18 g vs. 137 ± 16 g, saline vs. atropine;P < 0.05). Conclusions These data suggest that after nerve injury, mechanical but not thermal antinociception from intrathecal clonidine relies on a muscarinic interaction, because only mechanical antinociception was antagonized by atropine. These results do not favor a regulation of nociceptive transmission by a tonic release of acetylcholine in nerve-injured rats.

Spinal noradrenaline transporter inhibition by reboxetine and Xen2174 reduces tactile hypersensitivity after surgery in rats

Spinal noradrenaline (NA) released in response to noxious stimuli may play an important role in suppression of nociceptive transmission. Here, we investigated the efficacy of a competitive NA transporter inhibitor (reboxetine) and a noncompetitive NA transporter inhibitor peptide, Xen2174, isolated from the Pacific cone snail, to treat tactile hypersensitivity following paw incisional surgery. Male Sprague–Dawley rats were anesthetized, an incision of the plantar aspect of the hind paw was performed, and withdrawal threshold to von Frey filaments near the surgical site determined. Reboxetine (0.5–5 μg) and Xen2174 (0.3–100 μg) increased withdrawal threshold when injected 24 h after paw incision, with a peak effect at 15–60 min, for Xen2174, an ED50 value of 0.64 μg. Administration of Xen2174 (3–30 μg) 15 min before incision also reduced hypersensitivity in a dose‐dependent manner. Withdrawal threshold after the single 30 μg dose was greater than vehicle control even at 2, 3, and 5 days after incision. Doses ≤30 μg did not alter spontaneous behavior. The anti‐hypersensitivity effect of 10 μg of Xen2174 was totally blocked by the α2‐adrenoceptor antagonist, idazoxan, and partially blocked by the muscarinic antagonist, atropine. These data suggest that selective NA transporter inhibition suppresses post‐incisional hypersensitivity through a different mechanism from that of neuropathic pain, since we previously reported that reversal of hypersensitivity by intrathecal clonidine, an α2‐adrenoceptor agonist, following spinal nerve ligation is completely blocked by intrathecal atropine. Finally, these data suggest that intrathecal administration of Xen2174 at the time of spinal anesthesia might produce postoperative analgesia in humans.

Preoperative inhibition of cyclooxygenase-1 in the spinal cord reduces postoperative pain.

Intrathecal administration of cyclooxygenase (COX)-1, but not COX-2, specific inhibitors given on postoperative day 1 has analgesic effects in an incisional model of postoperative pain. We investigated the effects of preoperative administration of intrathecal COX inhibitors in this model. Fifteen minutes before surgery, rats received intrathecally the COX-1 preferring inhibitor, ketorolac, the specific COX-1 inhibitor, SC-560, the COX-2 inhibitor, NS-398, or vehicle. A 1-cm longitudinal incision was then made through skin, fascia, and muscles of the plantar aspect of a left paw in male rats. Withdrawal threshold to von Frey filaments was measured at 2 h, 4 h, and at intervals up to 5 days later. Ketorolac and SC-560 increased withdrawal threshold to mechanical stimulation, but NS-398 had no significant effect. These results suggest that COX-1 plays an important role in spinal cord pain processing and sensitization after surgery and that preoperative intrathecal administration of specific COX-1 inhibitors may be useful to treat postoperative pain.

Intrathecal Morphine Reduces Allodynia after Peripheral Nerve Injury in Rats via Activation of a Spinal A1 Adenosine Receptor

Background:The degree to which intrathecally administered morphine can alleviate hypersensitivity in animals after peripheral nerve injury is controversial, and the mechanisms by which morphine works in these circumstances are uncertain. In normal animals, morphine induces adenosine release, and in vitro data suggest that this link is disrupted after peripheral nerve injury. Therefore, using a controlled, blinded study design, the authors tested intrathecal morphine efficacy in rats with peripheral nerve injury and the role of spinal A1 adenosine receptors in the action of morphine. Methods:Male rats underwent intrathecal catheter implantation and lumbar spinal nerve ligation, resulting in hypersensitivity to tactile stimulation of the paw. Intrathecal morphine alone or with naloxone or the specific A1 adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentyxanthine (DPCPX), was administered, and withdrawal threshold to von Frey filament application to the hind paw was determined. Results:Intrathecal morphine (0.25–30 &mgr;g) dose-dependently reversed mechanical hypersensitivity after spinal nerve ligation, with an ED50 of 0.79 &mgr;g. The effect of morphine was blocked by intrathecal naloxone. Intrathecal DPCPX alone had no effect on withdrawal threshold after spinal nerve ligation but completely reversed the effect of morphine, with an ID50 of 5.6 &mgr;g. Conclusions:This study is in accord with two recent reports that support short-term efficacy of intrathecal morphine to reverse hypersensitivity to mechanical stimuli in animal models of neuropathic pain. Despite previous studies demonstrating that morphine releases less adenosine after peripheral nerve injury, the current study suggests that the antihypersensitivity effect of morphine in these conditions is totally reliant on A1 adenosine receptor activation.

Transfection and overexpression of metallothionein-I in neonatal rat primary astrocyte cultures and in astrocytoma cells increases their resistance to methylmercury-induced cytotoxicity.

Metallothionein-I (MT-I) was expressed in neonatal rat primary astrocyte cultures and an astrocytoma cell line by pGFAP-MT-I plasmid transfection under the control of the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter. Following transient transfection of the pGFAP-MT-I plasmid, MT-I mRNA and MT-I protein levels were determined by northern blot and immunoprecipitation analyses, respectively. The ability of cells over-expressing MT-I to withstand acute methylmercury (MeHg) treatment was measured by the release of preloaded Na251CrO4, an indicator of membrane integrity. Transfection with the pGFAP-MT-I plasmid led to increased mRNA (2. 5-fold in astrocytes and 7.4-fold in astrocytomas) and MT-I protein (2.4-fold in astrocytes and 4.0-fold in astrocytomas) levels compared with their respective controls. Increased expression of MT-I was associated with attenuated release of Na251CrO4 upon MeHg (5 microM) treatment. These results demonstrate that MT-I can be highly expressed both in primary astrocyte cultures and astrocytomas by pGFAP-MT-I plasmid transfection, and lend credence to the hypothesis that increased expression of MT-I affords protection against the cytotoxic effects of MeHg. Taken together, the data suggest that MT offer effective cellular adaptation to MeHg cytotoxicity.

Spinal adrenergic and cholinergic receptor interactions activated by clonidine in postincisional pain.

Background Previous pharmacologic and molecular studies suggest that the &agr;2-adrenoceptor subtype A is the target for spinally administered &agr;2-adrenergic agonists, i.e., clonidine, for pain relief. However, intrathecally administered &agr;2 C antisense oligodeoxynucleotide was recently reported to decrease antinociception induced by clonidine in the rat, suggesting non-A sites may be important as well. The current study sought to determine the subtype of &agr;2 adrenoceptors activated by clonidine in a rodent model for human postoperative pain, and to examine its interaction with spinal cholinergic receptors. Methods Postoperative hypersensitivity was produced in rats by plantar incision of the hind paw and punctuate mechanical stimuli were applied around the wound 24 h after surgery. Effects of intrathecal clonidine and 2-(2,6-diethylphenylamino)-2-imidazoline (ST91) on withdrawal thresholds to the stimulus were determined. To examine the adrenoceptor subtype and its interaction with spinal cholinergic receptors, animals were intrathecally pretreated with vehicles BRL44408 (an &agr;2 A subtype–preferring antagonist), ARC239 (an &agr;2 non-A subtype–preferring antagonist), atropine (a muscarinic antagonist), and mecamylamine (a nicotinic antagonist). Results Intrathecal ST91 showed a significantly greater efficacy when compared with clonidine. The analgesic effect of clonidine was diminished by pretreatment with either adrenoceptor antagonist, whereas the effect of ST91 was solely blocked by ARC239 pretreatment. Atropine and mecamylamine abolished the effect of clonidine effect but not the effect of ST91. Conclusions Both &agr;2 A and &agr;2 non-A adrenoceptors, as well as spinal cholinergic activation, are important to the antihypersensitivity effect of clonidine after surgery. ST91 is more efficacious in this model than clonidine and relies entirely on &agr;2 non-A adrenoceptors.

Intrathecal morphine and ketorolac analgesia after surgery: comparison of spontaneous and elicited responses in rats

Pain after surgery results in significant morbidity, and systemic opioids often fail to provide adequate analgesia without marked sedation and respiratory depression. Intrathecal morphine provides better analgesia, but is limited by delayed respiratory depression. Intrathecal injection of the cyclooxygenase inhibitor, ketorolac, has recently entered clinical trials, and the current study examined the interaction between intrathecal morphine and ketorolac to treat postoperative pain. We also sought to compare these treatments on a commonly used assessment of withdrawal threshold and a new assessment of spontaneous behavior after surgery. Male Sprague Dawley rats and underwent hind paw incision or subcostal laparotomy surgery. Intrathecal morphine, ketorolac, or their combination were injected on the first postoperative day, with outcome measure being return to pre‐surgery withdrawal threshold with von Frey filament testing of the paw after paw incision, or return to pre‐surgery exploratory activity after laparotomy. Intrathecal morphine completely reversed the effects of surgery in both models, but intrathecal ketorolac only partially reversed them. Ketorolac enhanced the potency of morphine several fold in both models, and did so synergistically after paw incision. In all cases drug potency was greater for spontaneous than elicited responses. These data confirm that spinal opioid receptor and cyclooxygenase enzyme inhibition diminish elicited tactile hypersensitivity after surgery, and that they similarly return spontaneous behavior to normal. Differences in drug potency could reflect fundamental differences in outcome measures or in the surgical procedures themselves. These data support combination study of intrathecal morphine and ketorolac for postoperative pain.