Nociceptive Tibialis Anterior reflex activity and phasic descending inhibition Four weeks after spinal cord injury, the rats were rapidly anesthetised with isoflurane (2%) in medicinal air (17% oxygen, at 2 l/min, Synthetic medical air, Ref: X505, Carburos Metallicos) for neurophysiological analysis of nociceptive Tibialis Anterior spinal reflex activity. The nose was then inserted into a plexiglass adapter (Cibertec, S.A.) so that the isoflurane-air mixture (2.0%, 1 l/min) could be administered directly, followed by the subcutaneous injection of atropine. The animal was placed in a supine position on an electric blanket maintained at 37uC (RTC1 Thermal Regulator, Cibertec S.A.). Hair over the left TA muscle and at the mid-thoracic level was removed and both the trunk and the hindlimbs were extended and fixed into a neutral position with adhesive tape during the entire experiment. Bipolar electromyographic responses were recorded by subcutaneaously inserting two multi-stranded TeflonH- coated steel electrodes (Cooner Wire, USA) 0.5 cm into the belly of the Tibialis anterior (TA) muscle of the left limb. In addition, two platinum subdermal electrodes (Ref. E2, Astro-Med Inc., Grass Instruments, USA) were inserted into the tip of the fourth toe and secured with adhesive tape. Lastly, an earth electrode was inserted subcutaneously between the stimulation electrode and the recording electrode at the level of the left ankle. Prior to beginning reflex EMG measurements, the isoflurane anaesthesia level was lowered to 1.2% MAC in medicinal air (1 l/ min). The evoked electromyographic responses of the TA muscle were recorded at a gain of 2K, with a bandwidth filter of between 30 Hz – 10 KHz using a differential AC amplifier (AE3, Cibertec S.A.). The electrical stimulus was applied via a stimulation isolation unit (ISU 165, Cibertec S.A.) from an analogue stimulator (CS20BP, Cibertec S.A.), presented as pulses of 2.0 ms duration. Reflex threshold was identified by characterising the minimal current intensity (mA) required to evoke a clear nociceptive TA reflex EMG response between 0.2 and 1.0 s after stimulation, in over half of ten stimuli. Nociceptive TA reflex activity and temporal summation was evoked during a train of 16 stimuli applied at 1 Hz. The role of descending inhibitory pathways across the T9 spinal cord injury upon nociceptive TA reflex activity was assessed indirectly by applying a percutaneous high frequency electrical conditioning stimulus across the T6-7 vertebral level, by applying an electrical stimulation protocol that has previously been employed to modulate C-fibre evoked potentials [47]. Two platinum subdermal needle electrodes (Ref: E2, Astro-Med INC. Grass, USA) were inserted into each side of the trunk at the T6-7 vertebral level, in order to deliver a stimulus just above the level of the T9 experimental contusion. The high frequency conditioning protocol consisted of a 1 s 100 Hz train, with pulse duration of 0.1 ms, applied every 10 s during a total period of 15 minutes. One minute immediately following the mid-thoracic electrical conditioning stimulation, nociceptive TA reflex activity was recorded using the protocol described above. Similar conditioning electrical stimulation applied to forelimb nerves have been shown to mediate long-lasting inhibition of nociceptive cutaneous spinal reflex activity via a noradrenergic sensitive mechanism [34]. Data were collected with an analog-digital converter (CED Micro 1401 MkII A/D, CED, UK) with data collected at 20 KHz sampling rate. Electromyographic data were integrated using the modulus function of the analysis software (Spike 2, CED, UK) between 0.2 and 0.6 s after the stimulus. Integrated reflex EMG data were analyzed after each stimulus and also grouped for the first and second phase of temporal summation in light of published evidence suggesting that modulation of spinal excitability via descending pathways is evident after a few stimuli [31]. Albumin-Oleic Acid Treatment of Spinal Cord Injury T9 SCI contusion volume analysis and lumbar L4–L5 immunohistochemistry At 30 days after SCI, animals were euthanized with sodium pentobarbital followed by intracardiac perfusion with saline and 4% paraformaldehyde in 0.1 M phosphate buffer (PB). The extracted spinal tissue was transferred to 30% sucrose in 0.1 M phosphate buffer and kept at 4uC for at least two days to provide cryoprotection. Both T6-T13 and lumbar L4–L5 tissue were cut at 30 mm in a Leica CM1900 cryostat and preserved at 230uC until use. Serial sections were collected on slides at a distance of 210 mm between consecutive sections. Thoracic tissue were prepared with Van Gieson’s stain and the volume of the spared tissue was calculated with Cavalieri’s method for volume estimation (see below). For immunohistochemical analysis sections were preincubated for 1 h in a blocking solution composed of 5% normal goat serum (NGS, Vector) and 0.2% Triton X-100 (Merck) diluted in 0.1 M PB in saline. After the general blocking steps, sections were incubated overnight at 4uC with rabbit primary antibodies against serotonin (rabbit anti-5HT, 1:4000, Ref: S5545, Sigma-Aldrich) or microglia (Mouse, anti-CD11b clone OX-42, 1:5000, Ref: Ab1211-100, Abcam). Blocking for nonspecific background staining for pNR1 expression analysis was performed by incubating with a protein block serum (Ultra-V-Block, Ref. TA-125-UV, Thermo Scientific) for 5 minutes at room temperature, prior to overnight incubation with the primary antibody at 4uC (rabbit anti-pNR1, Ser 897 phosphorylated, 1:1000 Ref: DAM1475052; Millipore). Following primary antibody incubation, the tissue was washed three times in 1% NGS (except for pNR1 where PBS was used) and then incubated with the appropriate secondary antibody conjugated with Alexa Fluor 594 (1:1000; 1 hour 4uC; anti-rabbit, Ref: A11012, Invitrogen) or Alexa Fluor 488 (1:1000; 1 hour 4uC; anti-mouse, Ref: A11012, Invitrogen). The sections were then mounted with Fluoromount (Ref F4680, Sigma-Aldrich) and coverslips for the preservation of fluorescence-stained slide tissue (Thermo Scientific Superfrost Ultra Plus, Ref: J3800AMNZ) and analyzed with a fluorescent microscopy. Image acquisition and analysis Spinal cord volume of the injured region was calculated by Cavallieri’s method for volume estimation [48] using Newcast image analysis software (Visiopharm Integrator Software, Visio- pharm). Images of injured and uninjured spinal cords were visualized with VIS in an Olympus BX61 microscope with a DP71 digital camera. Data are expressed as mm3 of tissue referred to a 1 mm slice of spinal cord. For immunohistochemical spinal cord tissue analysis, digital images of the tissue were obtained with a confocal microscope (Leica SP5, Leica) and processed with NIH Image J software (Image J 1.38x, NIH). For each antibody, measurements were carried out over 10–20 images (minimum of 3 images/section/animal; n = 6–7 rats per group) which were sampled from digital images of the tissue obtained with a Leica DFC350 FX camera attached to a DM5000 B Leica epifluorescence microscope. Microscope illumination and data acquisition settings were fixed throughout the entire analysis procedure for each specific analysis. To maintain a constant detection threshold for each image and to compensate for subtle variability of the immunostaining procedure between sections, only immunoreactive 5HT innervation density and number of cells that expressed pNR1 were analysed if the image was at least 50% darker than the average grey level after background subtraction, and shading correction were performed in the areas of grey matter. General 5-HT analysis was performed for the dorsal and ventral horn, while OX-42 area and number of pNR1-positive cells were analyzed specifically within laminae I-II and the IV–VI (with PLoS ONE | www.plosone.org 3 October 2011 | Volume 6 | Issue 10 | e26107