Peripheral nerve injury leads to working memory deficits and dysfunction of the
hippocampus by up-regulation of TNF-α in rodents
Wen-Jie Rena§, Yong Liua§, Li-Jun Zhoua, Wei Lib, Yi Zhonga, Rui-Ping Panga,
Wen-Jun Xina, Xu-Hong Weia, Jun Wanga, He-Quan Zhua, Chang-You Wuc, Zhi-Hai
Qind Guo-Song Liub* and Xian-Guo Liua*
SUPPLEMENTARY FIGURES (1-4)
SUPPLEMENTARY FIGURE LEGENDS
Supplementary Figure 1. No significant difference in locomotor activity was
detected in the animals with SNI, intracerebroventricular injection of drugs and
deletion of TNFR1
(a-c): There was no significant difference in locomotor activity between SNI and
sham groups, used in eight-arm radial maze test (shown in Fig. 1a-i). (d-g): The
locomotor activities in the rats with i.c.v. injection of rrTNF and of anti-TNF,
intra-hippocampal injection of rrTNF or intraperitoneal injection of thalidomide was
not different from vehicle injection groups. (h-i): SNI did not affect the locomotor
activity of WT mice and KO mice. (j-o): There was no significant difference in the
total number of touching objects in NORT tests among groups. n = 5-20 in each group,
Data are presented as means ± s.e.m.
Supplementary Figure 2. SNI has no acute effect on synaptic transmission and
plasticity in contralateral hippocampus, but produces a delayed LTP inhibition
(a): The recordings were made in contralateral side to SNI and sham operation. SNI
did not affect the baseline of fEPSPs and LTP induction by HFS (100 Hz, 50 pulses, 4
trains with 15s intervals), which was delivered 1h after nerve injury (n = 5 in each
group). (b): Similar with ipsilateral hippocampus, the LTP induction in contralateral
hippocampus was also inhibited 23d after SNI (n = 5 in each group). Data are
presented as means ± s.e.m. (c-d): The position of recording electrode in the CA1
region and stimulation electrode in the CA3 region are shown. Data are presented as
means ± s.e.m.
Supplementary Figure 3. TNF-α in plasma increases following SNI
The concentrations of TNF-α in plasma 3d and 7d after SNI were significant higher,
compared with control (0h) (n = 5 at each time point). ** P<0.01 versus control (0h).
Data are presented as means ± s.e.m.
Supplementary Figure 4. Intracerebroventricular injection of rrTNF inhibits
LTP in CA3-CA1 synapses.
(a): HFS was able to induce LTP at CA3-CA1 synapses in aCSF group but not in
rrTNF group. The data were recorded in the rats that had been used for eight-arm
radial maze test following i.c.v. injection rrTNF (shown in Fig. 5 a and b, n = 8 in
each group). (b): Injection of rrTNF (i.c.v., 1 μg/ml, 5 µl) in intact rats did not affect
the baseline of fEPSPs but blocked LTP by HFS, which was delivered 1h after rrTNF
injection (n = 5 in each group). Data are presented as means ± s.e.m.
Supplementary
Figure
5.
Intracerebroventricular
injection
but
not
intra-hippocampal injection of rrTNF induces mechanical allodynia.
(a): Paw withdrawal thresholds decreased significantly in the rats with
intracerebroventricular injection of rrTNF, compared with those before injection in the
same group of rats and with those in aCSF treated group (n=8, P < 0.01,
Mann-Whitney U test). (b): Paw withdrawal thresholds were not affected by
intra-hippocampal injection of rrTNF or aCSF. The tests were performed on 1d before
injection and 3d after last injection.
Peripheral nerve injury leads to working memory deficits and dysfunction of the
hippocampus by up-regulation of TNF-α in rodents
Wen-Jie Rena§, Yong Liua§, Li-Jun Zhoua, Wei Lib, Yi Zhonga, Rui-Ping Panga,
Wen-Jun Xina, Xu-Hong Weia, Jun Wanga, He-Quan Zhua, Chang-You Wuc, Zhi-Hai
Qin Guo-Song Liub* and Xian-Guo Liua*
SUPPLEMENTARY INFORMATION
SUPPLEMENTARY MATERIALS AND METHODS
Immunohistochemistry
Animals were terminally anesthetized with urethane and perfused through the
ascending aorta with PBS followed by 4% paraformaldehyde in 0.1 M phosphate
buffer. After perfusion, brains were dissected from the skulls and post-fixed in the
same fixative for 24 h and then replaced with 30% sucrose overnight. Frozen brains
were cut in 10 μm for synaptophysin staining and 18 μm for TNF-α staining on
cryostat (LEICA CM3050 S) and sections were immediately mounted on superfrost
slides and left to dry overnight and were blocked with 3% rabbit serum in 0.2% Triton
X-100 in PBS for 2 h at 4°C. For synaptophysin staining the sections were incubated
overnight at 4°C with mouse anti-synaptophysin (Chemicon, for rat) or goat
anti-synaptophysin (Chemicon, for mouse) in blocking solution. On the second day,
the sections were rinsed with PBS and incubated with Alexa 488-coupled rabbit
anti-mouse or anti-goat IgG in PBS (Invitrogen) for 2 h at room temperature. For
TNF-α staining the sections were incubated with goat anti-TNF-α (cell signaling) and
treated with Cy3-conjugated secondary antibody. The slices were then coverslipped
with fluorescent mounting medium (Vector Laboratories) and left for 48 h at 4°C. To
test the specificity of anti-TNF-α used in the present study, a preincubation method
was used. In the experiment the anti-TNF-α was preincubated with TNF-α (R&D
Systems, Inc) at a concentration of 10 µg/ml, which is 5-fold higher than that of the
anti-TNF-α, and then the preincubated antibody was used to detect the
immunoreactivity (IR) of TNF-α in brain sections. The results showed that the
TNF-α-IR detected by preincubated antibody (Fig. 4f) was significantly lower than
that detected by non-preincubated antibody (Fig. 4d).
Novel object recognition test (NORT)
The test was carried out to access the short-term memory and long-term memory of
animals with SNI and sham-operation. The apparatus consisted of a round arena
(diameter: 50cm for mouse and 80cm for rat) with black (for rat) or white (for mouse)
walls and floor. The box and objects were cleaned between trials to stop the build-up
of olfactory cues. Animals received two sessions of 10 minutes each in the empty box
to habituate them to the apparatus and test room. Twenty-four hours later, each rat was
first placed in the box and exposed to two identical objects for 10 min (sample phase).
And then one object was replaced by a new one and the rat was placed back in the box
and exposed to the familiar object and to a novel test object for a further 10-min
(acquisition phase). The short-term memory was tested 10min after “sample phase”
(10-min retention) and long-term memory 24h after sample phase (24h retention) on
the same cohorts of animals. The experimenters measured the time spent exploring
each object. The recognition index was calculated as the percentage ratio of time
spent exploring the novel object over total exploration time during acquisition phase.
In mice experiments, we fine-tuned the level of difficulty of the memory test by
increasing the number of objects during sample phase to three different objects
instead of two identical ones. During the retention interval the most biased object,
usually the least explored one, was replaced by a new novel object. The recognition
index was calculated as the ratio of time spent exploring the novel object over time
spent exploring the most explored object between the two familiar ones. The time
spent exploring the least explored familiar object was not considered, to avoid
potential bias. All behavioral assays were performed by the experimenters who were
blind to the design of the study.
Following the NORT test, animals were perfused for the detection of presynaptic
terminal puncta or TNF-α staining
Measurement of locomotor activity in behavioral test
In eight-arm radial maze test, animal activity (locomotion) was determined by a
simple calculation based on the amount of time spent in the maze and the number of
arms crossed: (no. of arms × 160 cm)/(time (in s) spent in maze), where 160 equals
the length from one arm tip to opposite arm tip. In NORT test, the total number of
touch all objects were accounted. There was no significant difference in locomotion
between SNI and sham groups. The similar results were also observed between drug
injection group and vehicle injection group (Supplementary Figure 1).
Intracerebroventricular injection of rrTNF or TNF antibody
The rat anesthetized with chloral hydrate was placed in a stereotaxic frame and a
cannula was implanted in the lateral cerebral ventricle according to the following
co-ordinates: 1.2 mm caudal to the bregma, 1.8 mm lateral to the sagittal suture and
3.7-4.3 mm below the surface of the skull. Three days after operation, recombinant rat
TNF (rrTNF, R&D Systems, 1 µg/ml) or artificial cerebrospinal fluid (aCSF) was
injected intracerebroventricularly in a volume of 5 µl in 10 min for 3 times (daily).
Three days after last injection the memory tests started.
To test the effect of intracerebroventricular injection of TNF-α antibody on memory
impairment produced by SNI, cannula was implanted 7 days before SNI and TNF-α
antibody (anti-rat TNF-α antibody, R&D Systems, 250 µg/ml) or aCSF (in a volume
of 5 µl in 10 min) was injected into lateral ventricle 2h before SNI and in 7
consecutive days after SNI (daily). Three days after cessation of injection, the
memory function was first evaluated with NORT, and then with eight-arm radial maze
test. The levels of TNF-α in CSF and in hippocampus were accessed following the
behavioral tests.
In the preliminary experiment, we found that 3h after i.c.v. injection of rrTNF at 1
µg/ml (5 µl in volume in 10min), the concentrations of TNF-α in CSF was 231 ±
10pg/ml. Previous study reported the volume of rat’s CSF is about 2 ml (Grant et al,
1991; Kim et al, 1996). Considering the increase in TNF-α in CSF persisted for at
least 20d after SNI and the peak concentration of TNF-α in CSF following SNI was
156.3 ± 2.8 pg/ml (Fig. 4a), the rrTNF at a concentration of 1 µg/ml and the TNF
antibody at 250 µg/ml were used for intracerebroventricular injection.
Intra-hippocampal injection of rrTNF
The cannulae were implanted in bilateral CA1 region of the rostral hippocampus in
rats under chloral hydrate anesthesia. The co-ordinates of implantation were following:
3.4 mm caudal to the bregma, 2.5 mm lateral to the sagittal suture and 3.5 mm below
the surface of the skull. Seven days after operation rrTNF (200 ng/ml) was injected
for 3 times (daily) bilaterally in a volume of 0.5 μl over 1 min period. The injection
needles were left in the cannula for 1 min after injection. Three days after cessation of
injection, the NORT and eight-arm radial maze tests were successively performed.
The levels of TNF-α in CSF and in hippocampus were measured after memory tests.
As shown in Fig. 4b, the increase in TNF-α in hippocampus persisted for at least
40d following SNI and the peak concentration reached to 74.8 ± 1.8 pg/100 mg tissue.
As hippocampus of is about 60 mg, rrTNF at a concentration of 200ng/ml (0.5 µl in
each side) was injected. In control group the same volume of aCSF was injected.
TNF-α bioassay
To harvest rat cerebrospinal fluid (CSF), the anesthetized animal was placed in the
stereotaxic frame and foramen magnum was exposed but dura kept intact. CSF was
collected with a tiny needle connected to a syringe (1ml). Animals were sacrificed by
decapitation after collecting CSF and the brains were rapidly removed at 4 °C ice-cold
PBS. Hippocampus was weighed and homogenized in ice-cold PBS using a Potter
homogenizer (1000 rpm, 10 strokes). The homogenates and the CSF were centrifuged
for 15min (12000 rpm, 4 °C). Blood was collected from left ventricle of the animals
to abstract plasma. After sample standing for 1h in room temperature, raw plasma was
drawn off and then centrifuged for 10min (1000rpm, 4 °C). The supernatant was
abstracted and stored in -20℃ for TNF-α bioassay. The amount of TNF-α was
determined using anti-rat TNF-α ELISA Kits (R&D Systems, Minneapolis, MN, USA)
according to the manufacturer’s protocol.
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