Comparison of Nociceptive Effects of Buprenorphine,Firocoxib, and Meloxicam in a Plantar Incision Model in Sprague–Dawley Rats

Due to their reduced frequency of dosing and ease of availability, NSAIDs are generally preferred over opioids for rodent analgesia. We evaluated the efficacy of the highly COX2-selective NSAID firocoxib as compared with meloxicam and buprenorphine for reducing allodynia and hyperalgesia in rats in a plantar incision model of surgical pain. After a preliminary pharmacokinetic study using firocoxib, Sprague–Dawley rats (n = 12 per group, 6 of each sex) were divided into 6 groups: no surgery (anesthesia only), saline (surgery but no analgesia), buprenorphine (0.05 mg/kg SC every 8 h), meloxicam (2 mg/kg SC every 24 h), and 2 dosages of firocoxib (10 and 20 mg/kg SC every 24 h). The nociception assays were performed by using von Frey and Hargreaves methodology to test mechanical allodynia and thermal hyperalgesia. These assays were performed at 24 h before and at 20, 28, 44, and 52 h after start of surgery. None of the analgesics used in this study produced significantly different responses in allodynia or hyperalgesia from those of saline-treated rats. In the Hargreaves assay, female saline-treated rats experienced significantly greater hyperalgesia than did males. These findings add to a growing body of literature suggesting that commonly used dosages of analgesics may not provide sufficient analgesia in rats experiencing incisional pain.

Institutions performing studies using animals have both an ethical responsibility and a legal requirement to administer appropriate analgesia to research subjects.²⁵ Controlling postoperative pain can hasten return to normal function, minimize healing time, and prevent the development of chronic pain. In humans, postoperative pain is prevalent, with 80% of patients reporting acute pain, ranging from moderate to extreme, after surgery.⁴ Postoperative pain is not restricted to human medicine but also extends to veterinary analgesia. Therefore, we must continue to scrutinize analgesic protocols and strive to implement novel therapies.During a surgical incision, peripheral tissue injury leads to central sensitization. This process results in hyperalgesia, an increased response to a painful stimulus, and allodynia, a painful response to a normally benign stimulus.⁶⁰ Several models that are used to test nociception in rodents have demonstrated repeatability, including plantar incision, antigen-induced inflammation, and capsaicin injection.^7,8 Among these, the plantar incision model is minimally invasive and allows for objective measurements of acute postoperative pain.^11,12 In addition, this model may be particularly advantageous, because surgical incision pain in humans appears to be better controlled in terms of persistency, duration, and pain management than are other causes of nerve injury and inflammation.⁶⁰ In the plantar incision model, allodynia is assessed by using the von Frey assay of mechanical stimuli,⁴⁷ whereas hyperalgesia is assessed by using the Hargreaves assay of thermal stimuli.²³When making decisions regarding analgesia, choosing an appropriate medication that maximizes pain control and minimizes side effects is important. The 2 most commonly used classes of analgesics in rodents are NSAID and opioids. In human and veterinary medicine, the use of cyclooxygenase (COX) 2-selective NSAID is preferred because of lower potential for the adverse gastrointestinal and kidney side effects that result from COX1 inhibition.²⁹ However, the most commonly used NSAID in laboratory rodents are ketoprofen (COX nonselective), meloxicam (minimally COX2-seletive), and carprofen (variably COX selectivity by species).^31,37,43 Although newer NSAID such as firocoxib have an extremely high selectivity for the COX2 pathway (approximately 400-fold more selective for COX2)³⁷ and are being used more frequently in companion and large animals, little is known about the efficacy of firocoxib in laboratory rodents. A recent study has assessed the use of this medication in mice,⁴⁹ but no published dose determination or efficacy studies are available for rats.Buprenorphine, widely considered the ‘gold standard’ opioid analgesic for rodents,^5,18,52 is a Schedule III controlled substance under the Controlled Substances Act.⁴² Many researchers do not hold a DEA license, thus creating a barrier to use of this medication. Slow-release formulations of buprenorphine cannot be obtained in New York due to state restrictions against compounded controlled drugs for research use in animals. Standard-formulation buprenorphine must be dosed fairly frequently (every 6 to 8 h in mice and rats) to maintain efficacy, thus increasing the need for handling and injections.^5,6 In addition, potentially detrimental side effects of buprenorphine have been reported in rodents and humans, including increased activity, reduced food intake, respiratory depression, and pica.^35,46,56 These considerations must be weighed against the potential analgesic relief that the medication is intended to provide.In the current study, we first conducted a pharmacokinetic analysis of firocoxib in Sprague–Dawley rat plasma to determine an appropriate dosing frequency. We then evaluated the use of firocoxib for reducing allodynia and hyperalgesia in Sprague–Dawley rats by using a plantar incisional model of postoperative pain, comparing the efficacy of firocoxib with those of meloxicam and buprenorphine. In light of previous studies in our lab using firocoxib in a similar mouse model of plantar incisional pain,⁴⁹ we hypothesized that firocoxib and buprenorphine would produce comparable antinociception in rats.     

First experiences using the new Powerplex® ESX17 and ESI17 kits in casework analysis and allele frequencies for two different regions in Germany

DNA databases are the most efficient tools in criminal investigations with unknown perpetrators. Due to a significant number of random matches in cross-border DNA profile exchanges, the European Network of Forensic Science Institutes (ENFSI) proposed the addition of further short tandem repeats (STRs) to European DNA databases. Therefore, the new Powerplex® ESX17 and Powerplex® ESI17 kits from Promega comprised the 11 established DNA database STRs and additionally the well-known loci D1S1656 and D12S391, as well as D2S441, D10S1248, and D22S1045. The latter three STRs are thereby established as so-called mini-STRs to fulfill the increasing requirements regarding sensitivity and reproducibility for analysis of minute amounts of DNA. Here, we provide allele frequencies for the five additional STRs from two populations from Germany. A test regarding suitability and robustness of the new kits for routine trace analysis showed that it is more likely to obtain a meaningful profile using Powerplex® ESX17 and Powerplex® ESI17 kits compared to the Powerplex® ES kit. However, for both new kits the range of template DNA amount is rather small, e.g., slightly more DNA than recommended resulted in DNA profiles which could not be reliably evaluated due to allelic drop-in or imbalances and overshoots. In our opinion, the new kits are very promising new tools in forensic trace analysis even though handling and evaluation should yet be carried out with great caution.     

Evaluation of different GABA receptor agonists in the kindled amygdala seizure model in rats

The effects of three specific GABA receptor agonists, muscimol, progabide, and gaboxadol, on kindled seizures were evaluated in amygdala-kindled rats. The only compound that exerted significant anticonvulsant effects at nonsedative doses was progabide. Thus, after i.p. administration of 100 mg/kg progabide, a significant increase in seizure latency and significant decreases in duration of motor seizures and amygdala afterdischarges were determined. A decrease in severity of motor seizures was found only after 200 mg/kg progabide which, however, gave rise to marked sedation and muscle relaxation. Muscimol and gaboxadol were almost inactive in attenuating kindled seizures even at doses that produced pronounced side effects. Assuming that the amygdala-kindled rat is a useful model of complex partial seizures with secondary generalization in the human, the data suggest that GABA receptor agonists are not effective against this type of epilepsy (muscimol, gaboxadol) or effective only at large doses (progabide).     

Prevention of amygdala kindling with an inhibitor of gamma-aminobutyric acid uptake

A novel, specific inhibitor of gamma-aminobutyric acid (GABA) uptake, SKF 89976-A (N-[4,4-diphenyl-3-butenyl]-nipecotic acid), was administered daily (15 mg/kg, i.p.) 30 min prior to amygdala stimulation in adult female rats. Whereas control rats developed full kindled seizures after 9.4 +/- 1.2 amygdala stimulations. SKF 89976-A-treated rats had not progressed beyond an early stage of kindled seizures by the termination of drug treatment after 22 episodes of daily amygdala stimulation. Following cessation of SKF 89976-A treatment, full kindled seizures developed after 4.1 +/- 0.9 additional amygdala stimuli. The data suggest that SKF 89976-A can inhibit generalization of amygdala-kindled seizures, possibly by enhancement of central GABAergic activity.     

Comparison of the anticonvulsant effects of two novel GABA uptake inhibitors and diazepam in amygdaloid kindled rats

Summary Two novel, specific inhibitors of GABA uptake, namely SKF 89976-A (N-[4,4-diphenyl-3-butenyl]-nipecotic acid) and SKF 100330-A (N-[4,4-diphenyl-3-butenyl]-guvacine) were tested for anticonvulsant effects in amygdaloid kindled female rats. The anticonvulsant effectiveness of the compounds was compared with that of diazepam. SKF 89976-A and SKF 100330-A produced dosedependent anticonvulsant effects on all seizure parameters measured in fully kindled rats, i.e. they inhibited seizure severity, increased seizure latency, and decreased the duration of motor seizures and EEG afterdischarges. ED 50s for inhibition of seizure severity were 4.6 and 15.1 mg/kg (0.014 and 0.045 mmol/kg) i.p. for SKF 100330-A and SKF 89976-A, respectively. For comparison, the ED 50 of diazepam was 1.9 mg/kg (0.0067 mmol/kg) i.p. Observation of behaviour indicated that the novel GABA uptake blockers exerted no side-effects in anticonvulsant doses, whereas diazepam produced sedative effects at all active dosage levels. The data demonstrate that SKF 100330-A and SKF 89976-A are potent, non-sedative anticonvulsant drugs in the kindling model of epilepsy, and these compounds thus may deserve interest as potential antiepileptic drugs with a very selective mechanism of action.     

Evidence for impaired GABAergic activity in the substantia nigra of amygdaloid kindled rats

The activity of the GABA-synthesizing enzyme glutamate decarboxylase (GAD) was determined in synaptosomal fractions from 12 brain regions of amygdaloid kindled rats. The only significant difference in regional GAD activities between kindled animals and sham-operated controls was a 40% decrease of GAD activity in the substantia nigra. The data suggest that impaired GABAergic function in the nigra may be involved in the initiation and propagation of amygdaloid-kindled seizures.     

The impact of DNA contamination of bone samples in forensic case analysis and anthropological research

Contamination precautions and quality control are great issues when human bones are investigated genetically. This is especially true for historical samples with only minute amounts of usually highly degraded DNA. But also in forensic routine analysis, sometimes DNA has to be isolated from bones in equally bad conditions, e.g. from burned victims. In such cases, there are several eventualities to contaminate the sample with foreign DNA, for example caused by the recovery of the bones, by trace investigation on a crime scene, or - of course - during handling in the lab. We present the investigation of artificially contaminated historical bone samples which contained no original DNA. Three different kind of contamination were studied: (1) touching of the samples, (2) application of saliva, and (3) application of pure DNA. The samples were genetically investigated without and with the employment of a defined cleaning protocol of the bones. The results show that pure DNA can usually not be removed from the bones and that saliva is a similar thread for subsequent DNA analysis. After the cleaning procedure about 70% of saliva contaminated samples still yielded reproducible STR profiles implicating severe problems for the investigation of highly degraded bone fragments. Simple touching of the specimens seems not to be a real problem for genetic investigations since the obtained signals were not reproducible.