Mean and standard deviation plotted in black

iled in each experiment. Stimuli were applied to the partially innervated medial aspect of the plantar surface of the hind paw, an area innervated by the saphenous nerve. Mechanical withdrawal thresholds were calculated from von Frey hair force response curves. Animals were housed in Perspex holding chambers with metal mesh floors and allowed to habituate for 10 min. A range of calibrated von Frey hairs were applied to the plantar surface of the hind paw, with a total of five applications per weighted hair. From these data, force response curves were generated and withdrawal values were calculated as the weight at which withdrawal frequency = 50%. Tactile allodynia was assessed in the metal mesh floored enclosures using a brush moved across the plantar surface of the hind paw where a withdrawal scored one, with no response zero. This was repeated a total of five times giving a maximum score of five per session. Cold allodynia: a single drop of acetone was applied to the plantar surface of the hind paw using a 1 ml syringe a maximum of five times giving a maximum score of five if the animal exhibited licking/shaking behavior in response to each application. Thermal hyperalgesia: animals were held in Perspex enclosures with a glass floor. A radiant heat source was positioned under the hind paw, and the latency was recorded for the time taken for the animal to move the hind paw away from the stimulus. This was repeated three times and a mean value calculated for each test. Formalin Testing: animals were habituated to glass floored testing enclosures as above. A single 50 l injection of 5% formalin was administered to the plantar surface of the right hind paw by intradermal injection. Immediately following formalin injection, animals were placed into the testing enclosures. Time spent exhibiting pain-like behaviors and the total number of pain-like behaviors was recorded in five MedChemExpress CSP-1103 minute bins for sixty minutes. Data are shown as the classical biphasic response with behavioral responses pooled for the first phase 015 min and second phase 2060 min. Blinding of nociceptive behavioral studies are routine in the laboratory however where animal welfare/experimental design prohibits this, it cannot be implemented. For instance, in nerve-injured animals blinding is not possible as controls are nave. The lack of blinding may have introduced some subjective bias into these experiments, which is in part mitigated by behavioral data is supported by the inclusion of experiments in which measurements are not subjective. 2.3. Electromyographic experiments A well-defined method for minimally invasive preferential selection of either C- or A- fiber mediated nociceptive pathways was used. Noxious withdrawal responses to A- and C-nociceptor selective stimulation were carried out as previously described, by measurement of electromyographic activity in biceps femoris. Animals were anesthetized using isoflurane induction, and the external jugular vein and trachea were cannulated to allow maintenance of airway and anesthesia. Following surgery, anesthesia was switched to alfaxalone, and animals were maintained at a steady level of anesthesia by continuous pump PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19840865 perfusion via the jugular vein for the remainder of the experiment. Bipolar electrodes were made with Teflon coated stainless steel wire implanted into the bicep femoris. EMG recordings were amplified and filtered by a combination of in-house built and Neurolog preamplifier and band pass filters. Animals were