healthmedicinet

Animals

Male CD-1 mice (30–35 gr) were housed 3 per cage under controlled conditions (12:12 h
light/dark cycle; room temperature 20–22 °C, humidity 55–60 %) with chow and tap water
were available ad libitum. The experimental procedures were permitted by the Animal
Ethics Committee of the Second University of Naples. Animal care was in observance
with the IASP and European Community (E.C. L358/1 18/ 12/86) guidelines on the use
and protection of animals in experimental research. All efforts were made to reduce
animal suffering and the number of animals used.

Spared Nerve Injury (SNI)

Neuropathy condition was induced according to Decostered and Woolf method 30]. Mice were anesthetized [ketamine (60 mg/kg)?+?xylazine (10 mg/kg)] and the sciatic
nerve was exposed. The tibial and common peroneal nerves were ligated and then transected,
leaving the sural nerve intact. Sham animals were anaesthetized and the sciatic nerve
was exposed, but not transected.

Pain behaviour

Plantar test

Thermal hyperalgesia was evaluated using the plantar test (Ugo Basile). Mice were
placed in clear Plexiglas chambers; a mobile infrared radiant heat source was focused
under the plantar surface of the hind paws. Latency to paw withdrawal was determined
by a timer. The stimulus intensity was preset to obtain a paw withdrawal latency ranging
from 9 to 11 s. Withdrawal threshold of the hind paws was defined as the mean of three
measurements. Dynamic Aesthesiometer. Mechanical allodynia was evaluated using the Dynamic Plantar Aesthesiometer (Ugo
Basile). This equipment employs a single non-flexible filament (0.5-mm diameter) to
apply an increasing force to the plantar surface of the mouse hind paw. Animals were
placed in a cage with a wire mesh floor and allowed to acclimatize before testing.
The filament was applied to the plantar area (plantar territory of the sural nerve)
of the hind paw and it began to exert an increasing upward force, reaching a maximum
of 30 g in 10 s, until the paw was withdrawn. The withdrawal threshold was defined
as the force, in grams, at which the mouse withdrew its paw. Withdrawal thresholds
of ipsilateral and contralateral paws were determined three times, and the reported
value is the mean of the three evaluations. Pain behaviors were evaluated following
PEA or OEA (10 mg/kg, i.p.) repeated treatment (15 days) in sham or SNI mice (up to
30 days). In a separate set of experiments, sham and SNI mice were used for the assessment
of mechanical allodynia 30 days after surgery before and after a single intra-PL-IL
cortex microinjection of 200 nL of vehicle (0.5 % DMSO in artificial cerebrospinal
fluid, ACSF, in mM: 125.0 NaCl, 2.6 KCl, 2.5 NaH2PO4, 1.3 CaCl2, 0.9 MgCl2, 21.0 NaHCO3,
and 3.5 glucose, oxygenated and equilibrated to pH 7.4), or PEA or OEA (6 nmol/mouse).
The surgical preparation for intra-PL-IL cortex administration was performed the day
before the experiment through a 20-gauge stainless steel guide cannula stereotaxically
lowered, by applying coordinates from the atlas of Paxinos and Watson (1986) (AP:
1.4 mm, L: 0.5 mm from bregma, V: 3 mm below the dura) 31]. The drugs doses were chosen based on our previous studies 2], 25].

Tail suspension

Animals were suspended by the tail on a horizontal bar (50 cm from the floor) using
adhesive tape placed 4 cm from the tip of the tail. The duration of immobility was
recorded in seconds over a period of 6 min of habituation. Immobility time was defined
as the absence of escape-directed behaviour.

Object recognition

The experiment started with the habituation period, during which mice were allowed
to freely explore for 1 h the apparatus which consists of a rectangular open box (40?×?30?×?30 cm
width?×?length?×?height) made of grey polyvinyl chloride (PVC) illuminated by a dim
light. The day after each mouse was allowed to explore two identical objects positioned
in the back left and right corners for 5 min (acquisition). A camera recorded the
time spent on exploration of each object. In the test trial, which was carried out
for 2 h after the acquisition, one of the two objects was replaced with a new different
object. The time spent exploring the object was the time the mouse spent with its
nose directed, and within 1 cm, from the object. The behaviour of mice was analyzed
by an observer blind to the treatment. Data were expressed as percentage of recognition
index (RI %), which was calculated as the percentage of the time spent exploring the
novel object / time spent exploring the novel object?+?time spent exploring the familiar
object × 100.

Marble-burying

Mice were individually placed in a cage (21?×?38?×?14 cm length x width x height)
containing 5 cm layer of sawdust bedding and fifteen glass marbles (1.5 cm in diameter)
arranged in three rows. Mice were left undisturbed for 15 min under dim light. An
observer blind to the treatment counted the time spent in digging behaviour and the
number of marbles buried (at least two or third buried in the sawdust). At the end
of the test the animal was removed to its own cage.

Western blotting synaptosome

Synaptic protein expression in prefrontal cortex was assessed by western blotting
analysis. The dissected prefrontal cortex tissue samples were lysed in Syn-PER Synaptic
Protein Extraction Reagent (Thermoscientific, USA) (10 ml of Syn-PER Reagent per gram
of tissue), in order to isolate synaptosomes. Lysis buffer contained a cocktail of
phosphatase and proteinase inhibitors, Halt Protease Inhibitor Cocktail (Thermoscientific,
USA), added immediately before use to avoid proteins degradation. Homogenization was
carried out by mean dounce tissue grinder on ice and homogenate was centrifuged at
1200×g for 10 min at 4 °C. The pellet was removed and supernatant was centrifuged at 15000×g for 20 min at 4 °C. After removing supernatant containing cytosolic fraction, synaptosome
pellet was suspended adding 1–2 ml of Syn-PER Reagent per gram of sample. Protein
concentration in each sample was determined using Bradford assay and equal amounts
of total proteins were loaded onto 10 % polyacrylamide gel for Psd-95, Shank1, mGluR5,
mGluR7, NR2a and b, Trkb, 15 % polyacrylamide gel for Homer1a and Homer1b. Proteins
were separated by SDS-PAGE and transferred to nitrocellulose blotting membranes (GE
Healthcare, UK). Following blocking in buffer solution including 5 % milk and 0.1 %
Tween-20 in TBS (Tris HCl 25 mM, NaCl 137 mM), the membranes were immunoblotted with
the following primary antibodies: Homer1a (1:200; Santa Cruz Biotechnology), Homer1b
(1:1000, Santa Cruz Biotechnology), Psd95 (1:1000, Santa Cruz Biotechnology), Shank1
(1:1000, Santa Cruz Biotechnology), mGluR5 (1:1000, Santa Cruz Biotechnology), NR2a
(1:1000, Santa Cruz Biotechnology), NR2b (1:1000, Santa Cruz Biotechnology), Trkb
(1:1000, Santa Cruz Biotechnology). Then blots were incubated in species-appropriate
horseradish peroxidase-conjugated secondary antibodies (1:2000, Santa Cruz Biotechnology)
and proteins of interest visualized by ECL detection (LiteAblot extend, Euroclone)
at molecular weight target. Quantification of the bands intensity on scanned filters
was achieved by Quantity one analysis software (Bio-Rad, USA). Background value was
subtracted to minimize variability across membranes and each lane was normalized for
the corresponding actin value for variation in loading and transfer. Data were analyzed
by one-way ANOVA followed by Turkey’s post-hoc test (level of significance p??0.05). Data were expressed as percentage of the mean of controls. Statistical analyses
were performed with StatView software (version 5.0.1.0; SAS Institute).

Western blotting tissue

After protein determination by Bio-Rad Protein Assay kit (Bio-Rad), equal amounts
of total proteins (30 ?g) for each prefrontal cortex sample were loaded onto 10 %
polyacrylamide gels. Proteins were separated by SDS-PAGE and transferred overnight
to membranes (Immobilon PVDF Membrane, Millipore). The membranes were immunoblotted
overnight using selective antibodies against BDNF (diluted 1:500, Santa Cruz Biotechnology,
Dallas, Texas), pSer473-Akt1, pSer235/236-S6, pSer-240/244-S6 (each diluted 1:500,
Cell Signaling Technology, Beverly, MA). Blots were then incubated with appropriate
horseradish peroxidase-conjugated secondary antibody and target proteins were visualized
by ECL detection (GE Healthcare), followed by quantification through Quantity One
software (Bio-Rad). Antibodies against Akt1 and S6 (both diluted 1:500, Cell Signaling
Technology, Beverly, MA), that are not phosphorylation state-specific were used to
estimate the total amount of proteins. All optical density values were normalized
using an antibody against GAPDH (1:5000, Santa Cruz Biotechnology, Dallas, Texas).
Blots were then incubated in horseradish peroxidase-conjugated secondary antibodies
and target proteins visualized by ECL detection (Pierce, Rockford, IL), followed by
quantification by Quantity One software (Biorad). Normalized values were averaged
and used for statistical comparisons performed by one-way ANOVA, followed by Turkey’s
post-hoc test .

Cell cultures

Cortices of 17 days embryonic (E-17) mice were prelieved, dissected carefully in ice,
typsinized in 0.25 % Trypsin-EDTA for 20 min. Tissues were centerfuged (2000 rpm)
for 1 min and filtered. Cells were seeded in Poly D-Lysine- overnight pre-treated
multiwells in serum free medium (Neurobasal Medium?+?2 % B27 growth factor). Cells
were left to grow by changing half of medium every 3 days and then they were treated
at 14 DIV.

Cultured neurons western blotting

Total lysates from neuron cultures obtained through RIPA buffer lysis were analyzed
by western blot experiment after treatment with PEA (100 nM, 24 h). Denaturated protein
(50 ?g) was loaded on 10 % polyacrilamide gel and transferred on a Polyvinylidene
Fluoride (PVDF) membrane incubated overnight at 4 °C with the horseradish peroxidase
conjugated antibodies: goat polyclonal anti-mGluR5 (1:500; Santa Cruz Biothecnology),
rabbit polyclonal anti-Nr2b (1:500; Abcam), goat polyclonal anti-NR2a (1:1000; Santa
Cruz Biothecnology), and then with the relative secondary antibody for 1 h. Reactive
bands were visualized on a X-ray film. Whereas the same membrane trip was used for
revealing the expression of more than one protein of interest, a mild stripping at
60 °C for 10 min was done. Monoclonal anti-?-tubulin antibody (1:1000; Sigma) was
used as housekeeping protein to check for identical protein loading. Images were captured,
stored, and analyzed with the Quantity One software (BioRad, Hercules, CA).

In vivo single unit extracellular recordings

Mice for electrophysiological recordings were anaesthetized with pentobarbital (50 mg/kg,
i.p.) and placed in a stereotaxic device (David Kopf Instruments, Tujunga, CA). Body
temperature was maintained at 37 ° C with a temperature-controlled heating pad. In
all surgical preparations, the scalp was incised and holes were drilled in the skull
overlying the site of recording, medial prefrontal cortex (AP: +1–2.9, L: 0.2–0.3
from bregma and V: 1.2–3 mm below dura) and contralateral (right) with respect to
the nerve insult (left). Anaesthesia was maintained with a constant continuous infusion
of propofol (5–10 mg/kg/h, i.v.). A glass-insulated tungsten filament electrode (3–5
M?) (FHC Frederick Haer Co., ME) was stereotaxically lowered into the mPFC and the
recorded signals were amplified and displayed on a digital storage oscilloscope to
ensure that the unit under study was unambiguously discriminated throughout the experiment.
Signals were processed by an interface CED 1401 (Cambridge Electronic Design Ltd.,
UK) and analyzed through Spike2 software (CED, version 4) to create peristimulus rate
histograms online and to store and analyze digital records of single-unit activity
off-line. Configuration, shape, and height of the recorded action potentials were
monitored and recorded continuously. Neurons showed a cluster of spikes with an increased
frequency showing typically a Gaussian pattern of distribution after stimulation.
We measured the duration of the excitation (in seconds) as the period of the increased
firing activity which exceeds the average baseline value +2 standard deviations (SDs).
Moreover, we measured the the onset of excitation (in milliseconds) which was considered
as the time from the application of the stimulus (artifact) to the first-evoked spike
which exceeds the average baseline value +2 SD. Moreover, the EAP slope, indicating
rate of activation of synaptic receptors, was used for the evaluation of the efficacy
of synaptic transmission. Electrophysiolgical evaluations were performed in the following
groups of mice (?=?10): 1) sham mice; 2) 15 or 30 days SNI mice.

Microdialysis

Microdialysis experiments were performed in awake and freely moving mice. In brief,
mice were anaesthetized and stereotaxically implanted with concentric microdialysis
probes into the mPFC using coordinates: AP: 1.4–1.8 mm, L: 0.3–05 mm from bregma and
V: 3.0 mm below the dura. Microdialysis probes were constructed with 22G (0.41 mm
I.D., 0.7 mm O.D.) stainless steel tubing: inlet and outlet cannulae (0.04 mm I.D.,
0.14 mm O.D.) consisted of fused silica tubing. The probe had a tubular dialysis membrane
(Enka AG, Wuppertal, Germany) 1.3 mm in length. Following a recovery period of 24 h,
dialysis was commenced with ACSF (NaCl 147 mM, CaCl
₂
2.2, KCl 4 mM; pH 7.2) perfused at a rate of 1 ?L/min by a Harvard Apparatus infusion
pump. Following a 60-min equilibration period, 12 consecutive 30-min dialysate samples
were collected. At the end of experiments, mice were anaesthetized and their brains
perfused fixed via the left cardiac ventricle with heparinised paraformaldehyde saline
(4 %). Brains were dissected out and fixed in a 10 % formaldehyde solution for 2 days.
The brain were cut in 40-?m thick slices and observed under a light microscope to
identify the probe locations. Dialysates were analyzed through an high performance
liquid chromatography method. The system comprised a Varian ternary pump (mod. 9010),
a C18 reverse-phase column, a Varian refrigerated autoinjector (mod. 9100), a Varian
fluorimetric detector. Dialysates were precolumn derivatized with Opthaldialdehyde
(10 ?l dialysate?+?10 ?l o-pthaldialdehyde) and amino acid conjugates resolved using
a gradient separation. The mobile phase consisted of 2 components: 1) 0.2 M sodium
phosphates buffer (pH 5.8), 0.1 M citric acid (pH 5.8) and 2) 90 % acetonitrile, 10 %
distilled water. Additionally, to determine the amount of area peak due to D-Aspartate,
a parallel sample was incubated with 2 ml of purified D-Asp Oxidase for 15 min at
37 °C and chromatographed as above. The total disappearance or the reduction of area
peak corresponding to D-Asp elution peak confirmed the presence of D-Asp and gave
the exact amount of the content of D-Asp. A typical analysis is represented in Fig. 5. Data were collected by a Dell Corporation PC system 310 interfaced by Varian Star
6.2 control data and acquisition software. The data were expressed as mean of five
samples for each mice.

Measurement of N-acylethanolamines levels (PEA and OEA)

The extraction, purification and quantification of N-acylethanolamines from tissues
has been performed as previously described 32]. Briefly, tissues were dounce-homogenized and extracted with chloroform/methanol/Tris–HCl
50 mmol/l pH 7.5 (2:1:1, vol/vol) containing internal standards [2H]4 PEA and [2H]2
OEA 50 pmol each). The lipid-containing organic phase was dried down, weighed, and
pre-purified by open-bed chromatography on silica gel. Fractions were obtained by
eluting the column with 99:1, 90:10 and 50:50 (v/v) chloroform/methanol. The 90:10
fraction was used for PEA and OEA quantification by liquid chromatography—atmospheric
pressure chemical ionization—mass spectrometry by using a Shimadzu high-performance
liquid chromatography apparatus (LC-10ADVP) coupled to a Shimadzu (LCMS-2020) quadrupole
mass spectrometry via a Shimadzu atmospheric pressure chemical ionization interface
as previously described [32[. LC analysis was performed in the isocratic mode using
a Discovery C18 column (15 cm?×?4.6 mm, 5 ?m) and methanol/water/acetic acid (85:15:1
by vol.) as mobile phase with a flow rate of 1 ml/min. The amounts of componds in
tissues, quantified by isotope dilution with the abovementioned deuterated standards,
are expressed as pmol/mg.

Immunohistochemistry and immunofluorescence

Under pentobarbital anesthesia (50 mg/kg, i.p.), animals were transcardially perfused
with saline solution followed by 4 % paraformaldehyde in 0.1 M phosphate buffer. The
brains were excised, post fixed for 3 h in the perfusion fixative, cryoprotected for
72 h in 30 % sucrose in 0.1 M phosphate buffer, and frozen in Optimal cutting temperature
(O.C.T.) embedding compound. Transverse sections (20 ?m) were cut using a cryostat
and thaw-mounted onto glass slides. Slides were incubated overnight with primary antibody
solutions for the microglial cell marker Iba-1 (rabbit anti-ionized calcium binding
adapter molecule-1; 1:1000; Wako Chemicals, Germany). Possible non-specific labeling
of mouse secondary antibody was detected by using secondary antibody alone. Following
incubation, sections were washed and incubated for 2 h with secondary antibody solution
(donkey anti-rabbit Alexa FluorTM 488; 1:1000; Molecular Probes, USA). Slides were
washed, coverslipped with Vectashield mounting medium (Vector Laboratories, USA),
and visualized under a Leica fluorescence microscope. The number of cells positive
for Iba-1 was determined within a box measuring 2×10
⁴
?m
²
that was placed in the m-PFC. Six sections were assessed from one animal and three
animals were used for each group. To avoid cell over-counting, only DAPI-counterstained
cells were considered as positive profiles. Iba-1 -positive cells were identified
as resting (with small somata bearing long, thin and ramified processes), activated
microglia (with hypertrophy together with retraction of processes to a length shorter
than the diameter of the somata) or hyper-branched microglia (with increased number
of branches).