Nancy L. Buechler

Effects of laparotomy on spontaneous exploratory activity and conditioned operant responding in the rat: a model for postoperative pain.

Background:Treatment of postsurgical pain is a major use of analgesics, particularly after abdominal surgery. Analgesics display a number of limiting side effects, including sedation, cognitive impairment, and ileus. Although several postoperative rodent models have been developed, these models do not address these concerns. Methods:A model is presented in the rat in which a subcostal incision is performed, penetrating into the peritoneal cavity. The behavioral effects of this surgical procedure are assessed using exploratory locomotor activity and conditioned operant responding. The effects of morphine and ketorolac were assessed in both behavioral paradigms. Results:Laparotomy decreased ambulation and rearing by approximately 50% 24 h after surgery, and stereotypy (small confined movements) was affected to a lesser degree. The effects of laparotomy on conditioned operant responding were more complex. Total number of sucrose pellets earned was decreased for 2–3 days after laparotomy; however, the amount of time required was increased for up to 2 weeks. Morphine reversed the effects of surgery on ambulation and stereotypy but not rearing, and the dose–effect curve for morphine was shifted to the left by 5 mg/kg ketorolac. Ketorolac produced significant improvement in operant responding after laparotomy, and coadministration of ineffective doses of morphine and ketorolac produced a positive response. Conclusion:The current model is consistent with behavioral aspects of postoperative pain seen clinically. The effects of morphine and ketorolac alone and in combination were consistent with the reported analgesic efficacy and occurrence of side effects found with these agents clinically.

Opioid self-administration in the nerve-injured rat : Relevance of antiallodynic effects to drug consumption and effects of intrathecal analgesics

Background:Neuropathic pain is associated with several sensory abnormalities, including allodynia as well as spontaneous pain. Opioid intake in neuropathic pain patients is motivated by alleviation of both pain and allodynia. However, laboratory animal studies rely almost exclusively on reflexive withdrawal measures of allodynia. The authors examined the pharmacology of self-regulated intake of opioids in rats with or without nerve injury and compared the rate of drug intake to reversal of allodynia. Methods:Rats were implanted with intravenous catheters, and the L5 and L6 spinal nerves were ligated in half of these animals. Rats were then trained to self-administer a commonly abused opioid (heroin) and commonly prescribed opioids (morphine, fentanyl, hydromorphone, and methadone). In addition, rats trained to self-administer heroin were given either clonidine or adenosine spinally before self-administration sessions to assess opioid-sparing effects. Results:Nerve injury significantly decreased the reinforcing effects of low doses of opioids, and only doses of each opioid that reduced mechanical hypersensitivity maintained self-administration after spinal nerve ligation. The rate of drug consumption was correlated with the duration of the antiallodynic effect for each dose of opioid. Intrathecal administration of clonidine or adenosine reversed mechanical hypersensitivity, but only clonidine reduced heroin self-administration in rats with spinal nerve ligation. Conclusion:Opioid self-administration is significantly altered by nerve injury, with rate of drug intake being correlated with reversal of allodynia. Intrathecal clonidine, but not adenosine, produces opioid-sparing effects in self-administering rats. The neurobiologic mechanisms that regulate opioid consumption in rats therefore seem to be altered after nerve injury.

SIRT1 inhibition during the hypoinflammatory phenotype of sepsis enhances immunity and improves outcome

Mechanism‐based sepsis treatments are unavailable, and their incidence is rising worldwide. Deaths occur during the early acute phase of hyperinflammation or subsequent postacute hypoinflammatory phase with sustained organ failure. The acute sepsis phase shifts rapidly, and multiple attempts to treat early excessive inflammation have uniformly failed. We reported in a sepsis cell model and human sepsis blood leukocytes that nuclear NAD+ sensor SIRT1 deacetylase remodels chromatin at specific gene sets to switch the acute‐phase proinflammatory response to hypoinflammatory. Importantly, SIRT1 chromatin reprogramming is reversible, suggesting that inhibition of SIRT1 might reverse postacute‐phase hypoinflammation. We tested this concept in septic mice, using the highly specific SIRT1 inhibitor EX‐527, a small molecule that closes the NAD+ binding site of SIRT1. Strikingly, when administered 24 h after sepsis, all treated animals survived, whereas only 40% of untreated mice survived. EX‐527 treatment reversed the inability of leukocytes to adhere at the small intestine MVI, reversed in vivo endotoxin tolerance, increased leukocyte accumulation in peritoneum, and improved peritoneal bacterial clearance. Mechanistically, the SIRT1 inhibitor restored repressed endothelial E‐selectin and ICAM‐1 expression and PSGL‐1 expression on the neutrophils. Systemic benefits of EX‐527 treatment included stabilized blood pressure, improved microvascular blood flow, and a shift toward proimmune macrophages in spleen and bone marrow. Our findings reveal that modifying the SIRT1 NAD+ axis may provide a novel way to treat sepsis in its hypoinflammatory phase.

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.

Resveratrol Attenuates Microvascular Inflammation in Sepsis via SIRT-1-induced Modulation of adhesion molecules in ob/ob mice

Obesity, a sirtuin‐1 (SIRT‐1) ‐deficient state, increases morbidity and resource utilization in critically ill patients. SIRT‐1 deficiency increases microvascular inflammation and mortality in early sepsis. The objective of the study was to study the effect of resveratrol (RSV), a SIRT‐1 activator, on microvascular inflammation in obese septic mice.

Sirtuin-2 Regulates Sepsis Inflammation in ob/ob Mice

Objective Obesity increases morbidity and resource utilization in sepsis patients. Sepsis transitions from early/hyper-inflammatory to late/hypo-inflammatory phase. Majority of sepsis-mortality occurs during the late sepsis; no therapies exist to treat late sepsis. In lean mice, we have shown that sirtuins (SIRTs) modulate this transition. Here, we investigated the role of sirtuins, especially the adipose-tissue abundant SIRT-2 on transition from early to late sepsis in obese with sepsis. Methods Sepsis was induced using cecal ligation and puncture (CLP) in ob/ob mice. We measured microvascular inflammation in response to lipopolysaccharide/normal saline re-stimulation as a “second-hit” (marker of immune function) at different time points to track phases of sepsis in ob/ob mice. We determined SIRT-2 expression during different phases of sepsis. We studied the effect of SIRT-2 inhibition during the hypo-inflammatory phase on immune function and 7-day survival. We used a RAW264.7 (RAW) cell model of sepsis for mechanistic studies. We confirmed key findings in diet induced obese (DIO) mice with sepsis. Results We observed that the ob/ob-septic mice showed an enhanced early inflammation and a persistent and prolonged hypo-inflammatory phase when compared to WT mice. Unlike WT mice that showed increased SIRT1 expression, we found that SIRT2 levels were increased in ob/ob mice during hypo-inflammation. SIRT-2 inhibition in ob/ob mice during the hypo-inflammatory phase of sepsis reversed the repressed microvascular inflammation in vivo via activation of endothelial cells and circulating leukocytes and significantly improved survival. We confirmed the key finding of the role of SIRT2 during hypo-inflammatory phase of sepsis in this project in DIO-sepsis mice. Mechanistically, in the sepsis cell model, SIRT-2 expression modulated inflammatory response by deacetylation of NFκBp65. Conclusion SIRT-2 regulates microvascular inflammation in obese mice with sepsis and may provide a novel treatment target for obesity with sepsis.

Involvement of the Lateral Amygdala in the Antiallodynic and Reinforcing Effects of Heroin in Rats after Peripheral Nerve Injury

Background:Neuropathic pain alters opioid self-administration in rats. The brain regions altered in the presence of neuropathic pain mediating these differences have not been identified, but likely involve ascending pain pathways interacting with the limbic system. The amygdala is a brain region that integrates noxious stimulation with limbic activity. Methods:&mgr;-Opioid receptors were blocked in the amygdala using the irreversible antagonist, &bgr;-funaltrexamine, and the antiallodynic and reinforcing effects of heroin were determined in spinal nerve-ligated rats. In addition, the effect of &bgr;-funaltrexamine was determined on heroin self-administration in sham-operated rats. Results:&bgr;-Funaltrexamine decreased functional activity of &mgr;-opioid receptors by 60 ± 5% (mean ± SD). Irreversible inhibition of &mgr;-opioid receptors in the amygdala significantly attenuated the ability of doses of heroin up to 100 &mgr;g/kg to reverse hypersensitivity after spinal nerve ligation. Heroin intake by self-administration in spinal nerve-ligated rats was increased from 5.0 ± 0.3 to 9.9 ± 2.1 infusions/h after administration of 2.5 nmol of &bgr;-funaltrexamine in the lateral amygdala, while having no effect in sham-operated animals (5.8 ± 1.6 before, 6.7 ± 0.9 after). The antiallodynic effects of 60 &mgr;g/kg heroin were decreased up to 4 days, but self-administration was affected for up to 14 days. Conclusions:&mgr;-Opioid receptors in the lateral amygdala partially meditate heroins antiallodynic effects and self-administration after peripheral nerve injury. The lack of effect of &bgr;-funaltrexamine on heroin self-administration in sham-operated subjects suggests that opioids maintain self-administration through a distinct mechanism in the presence of pain.

Intrathecal Administration of a Cylcooxygenase-1, but Not a Cyclooxygenase-2 Inhibitor, Reverses the Effects of Laparotomy on Exploratory Activity in Rats

Studies of hypersensitivity to mechanical stimuli after incisional surgery suggest that cyclooxygenase (COX)-1, but not COX-2, in the spinal cord participates in postoperative pain. In the current study, we sought to determine the role of COX isoenzymes after laparotomy, examining spontaneous exploratory behavior rather than withdrawal reflexes. Adult male Sprague-Dawley rats underwent subcostal laparotomy surgery under isoflurane anesthesia or received anesthesia without surgery. Exploratory locomotor activity was measured on the first postoperative day after intrathecal injection of dimethyl sulfoxide (vehicle) or COX-1 (SC-560) or COX-2 (NS-398) inhibitors. Laparotomy reduced ambulation, rearing, and rapid small movements (stereotypy) similarly in animals without intrathecal catheters and those receiving intrathecal vehicle control. SC-560 produced a dose-related return to normal exploratory behavior with complete return at doses of 20 &mgr;g and larger. In contrast, NS-398 in doses up to 50 &mgr;g failed to increase exploratory behavior. These data with exploratory behavior and laparotomy agree with studies with reflexive withdrawal responses after incisional surgery and indicate that COX-1 inhibition reduces pain responses after surgery. Spinal COX-1 inhibition completely restores exploratory activity, including rearing behavior that stretches the abdominal muscles. These data suggest that targeting COX-1 in the spinal cord may produce postoperative analgesia.

Potential therapeutic action of nitrite in sickle cell disease

Sickle cell disease is caused by a mutant form of hemoglobin that polymerizes under hypoxic conditions, increasing rigidity, fragility, calcium influx-mediated dehydration, and adhesivity of red blood cells. Increased red cell fragility results in hemolysis, which reduces nitric oxide (NO) bioavailability, and induces platelet activation and inflammation leading to adhesion of circulating blood cells. Nitric Oxide inhibits adhesion and platelet activation. Nitrite has emerged as an attractive therapeutic agent that targets delivery of NO activity to areas of hypoxia through bioactivation by deoxygenated red blood cell hemoglobin. In this study, we demonstrate anti-platelet activity of nitrite at doses achievable through dietary interventions with comparison to similar doses with other NO donating agents. Unlike other NO donating agents, nitrite activity is shown to be potentiated in the presence of red blood cells in hypoxic conditions. We also show that nitrite reduces calcium associated loss of phospholipid asymmetry that is associated with increased red cell adhesion, and that red cell deformability is also improved. We show that nitrite inhibits red cell adhesion in a microfluidic flow-channel assay after endothelial cell activation. In further investigations, we show that leukocyte and platelet adhesion is blunted in nitrite-fed wild type mice compared to control after either lipopolysaccharide- or hemolysis-induced inflammation. Moreover, we demonstrate that nitrite treatment results in a reduction in adhesion of circulating blood cells and reduced red blood cell hemolysis in humanized transgenic sickle cell mice subjected to local hypoxia. These data suggest that nitrite is an effective anti-platelet and anti-adhesion agent that is activated by red blood cells, with enhanced potency under physiological hypoxia and in venous blood that may be useful therapeutically.

Sirtuin 2 Regulates Microvascular Inflammation during Sepsis

Objective. Sepsis and septic shock, the leading causes of death in noncoronary intensive care units, kill more than 200,000/year in the US alone. Circulating cell-endothelial cell interactions are the rate determining factor in sepsis inflammation. Sirtuin, a seven-member family of proteins (SIRT1–7), epigenetically controls inflammation. We have studied the roles of SIRTs 1, 3, and 6 in sepsis previously. In this project, we studied the role of SIRT2 on sepsis-related inflammation. Methods. Sepsis was induced in C57Bl/6 (WT), SIRT2 knockout (SIRT2KO), and SIRT2 overexpressing (SIRT2KI) mice by cecal ligation and puncture (CLP). We studied leukocyte/platelet adhesion using intravital microscopy and E-selectin/ICAM-1 adhesion molecule expression in the small intestine with immunohistochemistry (IHC) six hours post-CLP/sham surgery. We also studied 7-day survival rates in WT, SIRT2KO, and SIRT2KI sepsis mice. Results. Compared to WT mice, SIRT2KO mice show exaggeration while SIRT2KI mice show attenuation of cellular adhesion with sepsis in the small intestine. We also show that the small intestinal E-selectin and ICAM-1 expressions increased in SIRT2KO and decreased in SIRT2KI mice versus those in WT sepsis mice. We show that the 7-day survival rate is decreased in SIRT2KO and increased in SIRT2KI sepsis mice. Conclusion. SIRT2 modulates microvascular inflammation in sepsis and affects survival.