Evaluation of Buprenorphine in a Postoperative Pain Model in Rats

We evaluated the commonly prescribed analgesic buprenorphine in a postoperative pain model in rats, assessing acute postoperative pain relief, rebound hyperalgesia, and the long-term effects of postoperative opioid treatment on subsequent opioid exposure. Rats received surgery (paw incision under isoflurane anesthesia), sham surgery (anesthesia only), or neither and were treated postoperatively with 1 of several doses of subcutaneous buprenorphine. Pain sensitivity to noxious and nonnoxious mechanical stimuli at the site of injury (primary pain) was assessed at 1, 4, 24, and 72 h after surgery. Pain sensitivity at a site distal to the injury (secondary pain) was assessed at 24 and 72 h after surgery. Rats were tested for their sensitivity to the analgesic and locomotor effects of morphine 9 to 10 d after surgery. Buprenorphine at 0.05 mg/kg SC was determined to be the most effective; this dose induced isoalgesia during the acute postoperative period and the longest period of pain relief, and it did not induce long-term changes in opioid sensitivity in 2 functional measures of the opioid system. A lower dose of buprenorphine (0.01 mg/kg SC) did not meet the criterion for isoalgesia, and a higher dose (0.1 mg/kg SC) was less effective in pain relief at later recovery periods and induced a long-lasting opioid tolerance, indicating greater neural adaptations. These results support the use of 0.05 mg/kg SC buprenorphine as the upper dose limit for effective treatment of postoperative pain in rats and suggest that higher doses produce long-term effects on opioid sensitivity.Relief of postoperative pain is mandated in the Guide for the Care and Use of Animals¹⁸ and the Public Health Service Policy¹⁷ and is a major objective of laboratory animal medicine. Buprenorphine is one of the most commonly used opioid analgesics for postoperative pain in laboratory animals, mainly because of its long duration of action.¹⁰ The typical recommended dose range of buprenorphine in rats is 0.02 to 0.05 mg/kg SC.¹⁰ The upper end of this range, although effective at relieving acute postoperative pain in rats, is associated with side effects such as enhanced postoperative pain after the drug has worn off (rebound hyperalgesia),²³ respiratory depression,²¹ nausea or gastrointestinal distress and pica,²⁵ and neural adaptations (for example, sensitization) that may lead to long-term changes in neural function in the central nervous system and consequent changes in behavior.¹⁴ Central sensitization is a well-studied neural adaptation expressed in the brain and spinal cord and induced by nociceptive stimulation (that is, pain-induced by surgical manipulation) that manifests as hyperalgesia (decreased pain threshold to noxious stimuli) and allodynia (appearance of pain-like responses to nonnoxious tactile stimuli) during the recovery period.^16,29 Central sensitization contributes to persistent pain during the postoperative recovery period (that is, maintenance of increased pain sensitivity during tissue recovery) and chronic pain in some pathologic conditions (that is, persistent pain sensitivity after full tissue recovery). Central sensitization also accounts for the spread of hyperalgesia and allodynia to noninjured areas of the body distal to the injury.³¹ This phenomenon is referred to as ‘secondary pain’ (secondary hyperalgesia and allodynia), because it is not directly associated with the primary injury site.Opioid analgesics inhibit pain by acting on the nervous system to block transduction of pain signals traveling in sensory neurons toward the central nervous system and by facilitating activity of the descending pain inhibition neural pathway.¹⁶ Opioid analgesics also induce neural adaptations in the nervous system, phenomena that underlie the pronounced changes in behavior associated with addiction to narcotics.² Notably, opioid analgesics have been shown to enhance central sensitization initiated by pain transmission.^6,8,14,20 This property means that opiate analgesics facilitate both the inhibition of pain and central sensitization that leads to the enhancement of pain. Because central sensitization is a neural adaptation, the interaction of opiates on this pain mechanism outlasts the presence of the drug; in contrast, opiate effects on pain inhibition are limited to the presence of the drug. This arrangement is thought to account for rebound pain, that is, increased pain sensitivity after the opiate analgesic has worn off. Opiate side effects can compromise the success of recovery by increasing the level of distress experienced during recovery (for example, inducing nausea) and possibly increasing the duration of distress during recovery (for example, allowing for rebound pain). Moreover, and of importance specifically to laboratory animal medicine, the general neural adaptations induced by even a single dose of an opiate analgesic²⁶ may induce changes in the nervous system that alter and therefore compromise the validity of the animal model under study (for example, opioid mechanisms involved in behavioral control).We previously evaluated the feasibility of oral administration of buprenorphine.^15,25 As a basis for comparison, we used the ‘gold-standard’ postoperative buprenorphine dose of 0.05 mg/kg SC. The results of those studies showed that oral administration of buprenorphine was not feasible because the dose necessary to produce analgesia comparable to the standard dose of 0.05 mg/kg SC was 10 times the oral dose recommended in the literature and because the resulting concentration of oral buprenorphine was too bitter for rats to ingest voluntarily in a volume of flavored foodstuff that they could eat in a single meal.^15,25 We also observed that both subcutaneous and oral buprenorphine caused conditioned aversion to flavors,²⁵ suggestive of gastrointestinal distress⁵, with a greater effect for the oral route. Our conclusions and the associated clinical recommendation were limited by our presumption that buprenorphine at 0.05 mg/kg SC was the ideal postsurgical dose.An assessment of the literature that established this dose identified 2 problems. First, little or no research had directly assessed the effect of buprenorphine on pain sensitivity in animals in the hyperalgesic state that characterized the postoperative period,²³ and to our knowledge, no study has directly assessed the dose–response function of postsurgical buprenorphine on hyperalgesia. We hypothesized that endogenous opioids activated during the postoperative period²⁴ might act synergistically with buprenorphine to allow adequate relief of postoperative pain with a lower dose of buprenorphine than is necessary in an algesiometric test, thereby making predictions and extrapolations from algesiometric tests inaccurate. Second, we found that little consideration had been given to the consequences of other physiologic effects of buprenorphine on the recovery process (for example, gastrointestinal distress⁵, rebound hyperalgesia, and allodynia). As stated earlier, recent research on central sensitization has determined that although opioid analgesics inhibit pain sensation acutely, they also enhance neural adaptations that account for rebound pain and other long-term chronic pain conditions.^16,28,29,31 We hypothesized secondarily that a lower dose of buprenorphine, if effective acutely, would result in reduced side effects and be less likely to initiate or enhance neural adaptations, such as rebound hyperalgesia and allodynia.The current study had 2 goals. The first was to establish the minimum dose of buprenorphine needed to relieve acute postoperative pain effectively in rats. As a starting point, we defined effective relief of acute pain as the induction of isoalgesia during the postoperative period; isoalgesia is the normal level of pain sensation, in contrast to analgesia (absence of pain sensation) or hypoalgesia (lower-than-normal pain sensation). The second goal was to evaluate the effect of postoperative buprenorphine on factors that slow recovery (that is, rebound hyperalgesia and allodynia) or create long-term changes (that is, sensitization or tolerance to opiates). We tested our hypothesis by using various doses of buprenorphine in a rat model of incisional pain.^3,4,31 This model was selected because it induces cutaneous and muscular pain common to most surgery and generates mild to moderate persistent pain so that both the acute inhibitory effects of the buprenorphine (that is, pain relief) and the lasting effects of buprenorphine (that is, rebound hyperalgesia) could be studied.     

Cloning, characterization and expression analysis of a sucrose synthase gene from tropical epiphytic orchid Oncidium Goldiana

A full-length cDNA encoding sucrose synthase was isolated from the tropical epiphytic orchid Oncidium Goldiana. The cDNA is 2829 bp in length containing an open reading frame of 2447 bp encoding 816 amino acids with a predicted molecular mass of 93.1 kDa. The deduced amino acid sequence of O . Goldiana sucrose synthase ( Osus ) shares more than 80% identity with those from other monocotyledonous plants. The sucrose synthase gene was demonstrated to encode a functional sucrose synthase protein by expression as recombinant protein in Escherichia coli . The Osus mRNA is present in all the tissues analysed, with the highest levels in strong sinks such as developing inflorescence and root tips. Incubation with sucrose or glucose resulted in a significant increase in the steady-state Osus mRNA levels in root tips and mature leaves in a similar pattern to maize Sus1 . Expression of the Osus mRNA in mature leaves was markedly enhanced by anaerobic conditions and elevated CO₂. The expression pattern and regulation of the gene suggest that the sucrose synthase plays an important role in the growth and development of the tropical epiphytic orchid O . Goldiana.     

Isolation and characterization of skin-type, type I antifreeze polypeptides from the longhorn sculpin, Myoxocephalus octodecemspinosus

The antifreeze polypeptides (AFPs) are found in several marine fish and have been grouped into four distinct biochemical classes (type I-IV). Recently, the new subclass of skin-type, type I AFPs that are produced intracellularly as mature polypeptides have been identified in the winter flounder (Pleuronectes americanus) and the shorthorn sculpin (Myoxocephalus scorpius). This study demonstrates the presence of skin-type AFPs in the longhorn sculpin (Myoxocephalus octodecemspinosus), which produces type IV serum AFPs. Using polymerase chain reaction-based methods, a clone that encoded for a type I AFP was identified. The clone lacked a signal sequence, indicating that the mature polypeptide is produced in the cytosol. A recombinant protein was produced in Escherichia coli and antifreeze activity was characterized. Four individual Ala-rich polypeptides with antifreeze activity were isolated from the skin tissue. One polypeptide was completely sequenced by tandem MS. This study provides the first evidence of a fish species that produces two different biochemical classes of antifreeze proteins (type I and type IV), and enforces the notion that skin-type AFPs are a widespread biological phenomenon in fish.     

The role of N and C termini in the antifreeze activity of winter flounder (Pleuronectes americanus) antifreeze proteins

Antifreeze proteins (AFPs) are found in many marine fish and have been classified into five biochemical classes: AFP types I-IV and the antifreeze glycoproteins. Type I AFPs are alpha-helical, partially amphipathic, Ala-rich polypeptides. The winter flounder (Pleuronectes americanus) produces two type I AFP subclasses, the liver-type AFPs (wflAFPs) and the skin-type AFPs (wfsAFPs), that are encoded by distinct gene families with different tissue-specific expression. wfsAFPs and wflAFPs share a high level of identity even though the wfsAFPs have approximately half the activity of the wflAFPs. Synthetic polypeptides based on two representative wflAFPs and wfsAFPs were generated to examine the role of the termini in antifreeze activity. Through systematic exchange of N and C termini between wflAFP-6 and wfsAFP-2, the termini were determined to be the major causative agents for the variation in activity levels between the two AFPs. Furthermore, the termini of wflAFP-6 possessed greater helix-stabilizing ability compared with their wfsAFP-2 counterparts. The observed 50% difference in activity between wflAFP-6 and wfsAFP-2 can be divided into approximately 20% for differences at each termini and approximately 10% for differences in the core. Furthermore, the N terminus was determined to be the most critical component for antifreeze activity.     

Comparative proteomic analysis of extracellular proteins of enterohemorrhagic and enteropathogenic Escherichia coli strains and their ihf and ler mutants

Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC, respectively) strains are closely related human pathogens that are responsible for food-borne epidemics in many countries. Integration host factor (IHF) and the locus of enterocyte effacement-encoded regulator (Ler) are needed for the expression of virulence genes in EHEC and EPEC, including the elicitation of actin rearrangements for attaching and effacing lesions. We applied a proteomic approach, using two-dimensional polyacrylamide gel electrophoresis in combination with matrix-assisted laser desorption ionization-time of flight mass spectrometry and a protein database search, to analyze the extracellular protein profiles of EHEC EDL933, EPEC E2348/69, and their ihf and ler mutants. Fifty-nine major protein spots from the extracellular proteomes were identified, including six proteins of unknown function. Twenty-six of them were conserved between EHEC EDL933 and EPEC E2348/69, while some of them were strain-specific proteins. Four common extracellular proteins (EspA, EspB, EspD, and Tir) were regulated by both IHF and Ler in EHEC EDL933 and EPEC E2348/69. TagA in EHEC EDL933 and EspC and EspF in EPEC E2348/69 were present in the wild-type strains but absent from their respective ler and ihf mutants, while FliC was overexpressed in the ihf mutant of EPEC E2348/69. Two dominant forms of EspB were found in EHEC EDL933 and EPEC E2348/69, but the significance of this is unknown. These results show that proteomics is a powerful platform technology for accelerating the understanding of EPEC and EHEC pathogenesis and identifying markers for laboratory diagnoses of these pathogens.     

Structure of a Novel Shoulder-to-Shoulder p24 Dimer in Complex with the Broad-Spectrum Antibody A10F9 and Its Implication in Capsid Assembly

Mature HIV-1 viral particles assemble as a fullerene configuration comprising p24 capsid hexamers, pentamers and dimers. In this paper, we report the X-ray crystal structures of the p24 protein from natural HIV-1 strain (BMJ4) in complex with Fab A10F9, which recognizes a conserved epitope in the C-terminal domain of the BMJ4 p24 protein. Our structures reveal a novel shoulder-to-shoulder p24 dimerization mode that is mediated by an S-S bridge at C177. Consistent with these structures, the shoulder-to-shoulder dimer that was obtained from the BMJ4 strain was also observed in p24 proteins from other strains by the introduction of a cysteine residue at position 177. The potential biological significance was further validated by the introduction of a C177A mutation in the BMJ4 strain, which then displays a low infectivity. Our data suggest that this novel shoulder-to-shoulder dimer interface trapped by this unique S-S bridge could represent a physiologically relevant mode of HIV-1 capsid assembly during virus maturation, although Cys residue itself may not be critical for HIV-I replication.