Morphological aspects of the peripheral nervous system.

<p>The protective effect of repeated administrations of PEA was evaluated on oxaliplatin-damaged DRGs on day 21. 5 μm DRG sections were stained by the Azan-Mallory method. Light micrographs (original magnification 20X) were analyzed by measuring the incidence of eccentric nucleoli and multinucleolated neurons. Each value represents the mean of 12 rats per group, performed in two different experimental sets. **P<0.01 versus vehicle + vehicle; ^^P<0.01 versus oxaliplatin + vehicle.</p

Glial activation profile in the spinal cord.

<p><i>Astrocytes</i>. The effect of repeated treatment with PEA (30 mg kg^-1 daily i.p.) was evaluated in oxaliplatin-treated rats on day 21. The number of GFAP-positive cells was measured in the dorsal horn of the spinal cord. Images (original magnification 20X) of sections of lumbar spinal cord of oxaliplatin-treated animals (oxaliplatin + vehicle) are reproduced in comparison with control (vehicle + vehicle). Representative immunohistochemical staining after PEA treatments is shown (20X). Each value represents the mean of 12 rats per group, performed in two different experimental sets. *P<0.05 versus vehicle + vehicle; ^P<0.05 versus oxaliplatin + vehicle.</p

HT-29 cell viability after 24 and 48h incubation.

<p>HT-29 cells were treated with increasing concentrations of oxaliplatin (1–100 μM) in the presence or in the absence of PEA (10 μM). Incubation was allowed for 24h or 48h. Cell viability was measured by MTT assay. Control condition was arbitrarily set as 100% and values are expressed as the mean ± S.E.M. of three experiments.</p><p>*P<0.05</p><p>**P<0.01 in comparison to control (oxaliplatin 0 μM).</p><p>HT-29 cell viability after 24 and 48h incubation.</p

ATF3 expression levels in sciatic nerve and L4-L5 DRGs.

<p>The protective effect of PEA was evaluated on the peripheral nervous tissue of treated animals on day 21. A representative immunohistochemical staining for ATF3 in 10 μm longitudinal sciatic nerve sections is shown (original magnification 20X). Densitometric analysis was performed to obtain a quantitative measurement for sciatic nerve and DRG neurons. Each value represents the mean of 12 rats per group, performed in two different experimental sets. *P<0.05 and **P<0.01 versus vehicle + vehicle; ^P<0.05 and ^^P<0.01 versus oxaliplatin + vehicle.</p

Glial activation profile in the brain cortex.

<p><i>Astrocytes</i>. The effect of repeated treatment with PEA (30 mg kg^-1 daily i.p.) was evaluated in oxaliplatin-treated rats on day 21. The number of GFAP-positive cells were measured in the somatosensory area 1. Representative immunohistochemical staining (20X) and quantitative measurements are shown. Each value represents the mean of 12 rats per group, performed in two different experimental sets. *P<0.05 versus vehicle + vehicle; ^P<0.05 versus oxaliplatin + vehicle.</p

Behavioral measures.

<p><i>Pain</i>: <i>mechanical non-noxious and noxious stimuli</i>. a) The Von Frey test was used to measure the pain threshold as a response evoked by a non-noxious stimulus. b) Paw-pressure test was used to measure sensitivity to a mechanical noxious stimulus. <i>Motor coordination</i>. The integrity of the animals’ motor coordination was assessed using a Rota-rod apparatus measuring c) the time spent to keep the balance and d) the number of falls, in 600 s. Animals were treated daily i.p. with 2.4 mg kg^-1 oxaliplatin or vehicle. PEA (30 mg kg^-1) was administered daily i.p. Behavioral evaluations were performed on day 21, 24h after treatment (pre) and 60 min after a new injection. Control animals were treated with vehicles. Each value represents the mean of 12 rats per group, performed in two different experimental sets. **P<0.01 versus vehicle + vehicle; ^P<0.05 and ^^P<0.01 versus oxaliplatin + vehicle.</p

Morphometric determinations performed on the soma area of DRG Neurons.

<p>**P<0.01 in comparison to vehicle + vehicle treated rats</p><p>^^P<0.01 vs oxaliplatin + vehicle group.</p><p>Morphometric determinations performed on the soma area of DRG Neurons.</p

Glial activation profile in the brain cortex.

<p><i>Microglia</i>. The effect of repeated treatment with PEA (30 mg kg^-1 daily i.p.) was evaluated in oxaliplatin-treated rats on day 21. The number of Iba1-positive cells were measured in the somatosensory area 1. Representative immunohistochemical staining (20X) and quantitative measurements are shown. Each value represents the mean of 12 rats per group, performed in two different experimental sets. *P<0.05 versus vehicle + vehicle; ^P<0.05 versus oxaliplatin + vehicle.</p

Inflammation-related mediators.

<p>On day 21, protein expression levels of IκBα were quantified by immunoblot in a) DRG and b) spinal cord; c) protein expression levels of COX2 were quantified by immunoblot in spinal cord Animals were treated daily i.p. with 2.4 mg kg^-1 oxaliplatin or vehicle for 21 days. PEA (30 mg kg^-1) was repeatedly administered i.p. (daily for 20 days starting from the first day of oxaliplatin administration). Control animals were treated with vehicles. Densitometric analysis is shown. β-actin normalization was performed for each sample. Each value represents the mean of 5 rats per group, performed in two different experimental sets. ^P<0.05 and ^^P<0.01 versus oxaliplatin + vehicle.</p

Electrophysiological recording of <i>NS neuron</i> activity.

<p>Representative peristimulus time histograms (PSTHs) show the responses of a single spinal NS neuron to a mechanical noxious stimulation (von Frey filaments 5.8N/20mm² for 3sec) in vehicle (A), oxaliplatin (B) and oxaliplatin + PEA (C) treated rats on day 21 of treatment. The lower panels show the onset (E), the duration of excitation (F), and the frequency (G) of the evoked activity of NS neurons in the three groups. Each point represents the mean ± S.E.M of 2–3 neurons recorded for each animal of different groups of rats (n = 3–6). ***P<0.001 indicates significant differences versus vehicle + vehicle, ^#P<0.05 and ^##P<0.01 indicate significant differences versus oxaliplatin + PEA.</p