NeuN GFAP CD11b C Involvement of BDNF in the thalamic hypersensitivity in CPSP. Hsi-chien Shih and...
1
NeuN GFAP CD11b C Involvement of BDNF in the thalamic hypersensitivity in CPSP. Hsi-chien Shih and Bai-chuang Shyu , Institute of BioMedical Science, Academia Sinica, Taipei, Taiwan. Abstract After stroke, about 7~10% patient will develop chronic pain syndrome after 6 month later. This chronic pain condition is called central post stroke pain syndrome (CPSP). Lenz supported a hypothesis CPSP is due to the unbalanced oscillation of thalamocortical circuit. Recent research results also indicated that abnormal increased secretion of brain derived neurotrophy factor (BDNF) in spinal cord tissue after spinal cord or peripheral neural injury. Expression of NKCC1 and NCC2, Cl - related channel, were influenced by the over expression of BDNF that the balance and functional role of Cl - in mature neuron were also re-modified. CPSP animal model of brain hemorrhage in ventral-posterior area of thalamus (VP) induced by collagenase injection was well established but the pathological mechanism of this model is not well studied. In the present study, 0.125U/0.5μl type 4 collagense was injected into SD rats’ VP area and nociceptive responses were tested with Von Frey and Plantar test. After 4 weeks of injection, the number of neuron was decreased and the cell number of astroglia, microglia and the mRNA level of BDNF were increased in lesion brain area. The decreasing of neuron was positively correlated with the degree of pain hypersensitivity of CPSP. In the electrophysiology recording, activity of medial- dorsal thalamus nucleus (MD) was enhanced after repeated noxious stimuli in CPSP animal and this enhancement could be blocked by acute TrKB-FC (an extracellular scavenger of BDNF) injection. Instead of inhibition by GABA system in normal rat, MD multiunit activity was enhanced after microinjection of muscimol in CPSP animal. The change of cell composition in VP area was a key factor of CPSP syndrome and over expression of BDNF probably induced by the proliferation of glia or microglia cell. MD neurons plasticity changes after stroke may be due to the composition changes of Cl - related channels that caused by BDNF re-modulation. VPL VPM aterial and Method Preparation of animals Male Sprague–Dawley rats (300–400 g) were housed in an air- conditioned room (21–23°C, humidity 50%, 12-h light/dark cycle starting at 08:00 h) with free access to food and water. All experiments were carried out in accordance with the guidelines of the Academia Sinica Institutional Animal Care and Utilization Committee. Recording of evoked multichannel field potentials and unit activities 16 channels Michigan probes were used to record the extracellular field potentials in the left anterior cingulate cortex (ACC) (about 2.5 mm anterior and 1 mm lateral to bregma; probe inserted 40° from the vertical line) and MD (about 2.2~3.5 mm posterior and 0.5~1.0 mm lateral to bregma; probe inserted perpendicular to the cortical surface). An Ag–AgCl reference electrode was placed in the nasal cavity. The sampling rates of recorded analog signals was 6 kHz in field potentials data and 24KHz in unit data. All data were processed in a multichannel data acquisition system (TDT, Alachua, FL) for a PC and analyzed with MatLab programs. Surgery SD rats were initially anesthetized with 4% isoflurane mixed oxygen air and then placed in a stereotaxic apparatus. Animals were maintained under anesthesia with 1% isoflurane in 30%/70% nitrous oxide/oxygen during the surgery. Rats' body temperatures were maintained at 37.5~36.5°C with a homeothermic blanket system (Model 50–7079, Harvard Apparatus, Holliston, MA). CPSP animal induction and Behavior testing Animals were injected with 0.125U type 4 collagenase in 0.5 μl saline into ventral posteromedial and ventral posteromedial thalamus nuclear, the coordinate is 3.0~3.5 mm posterior and 3.0~3.4 mm lateral to bregma, depth is 5700~6000 μm. Control animals were only injected with 0.5 μl saline. To measure the mechanical and thermal noxious response, Von Frey and Plantar test were applied on bilateral hind limbs before lesion and at 1, 3, 7, 14and 28 day after lesion. Sailine, TrKB FC (1 μ g/1 μl, 0.1 μl/ min) into and muscimol was direction injected with thalamus close to recording site. Immunostain Animal scarified with PBS/paraformadehyde perfusion, rat brain was removed. After 10% to 30% sucrose solution immersion, rat brain was sliced to 30~40μm with frozen section method and the slices included ACC, S1, MD and VPL/VPM was stained with 1’ anti body, Neu-N/Gila or OX42/Glia, and 2’ 488/594nm fluoresce antibody. electrophysiology & immunostain base line 1 3 7 14 21 28 day 1 week habituation lesion lesion behavior test Results Figure 1. A. An example of collagenase lesion in VPL & VPM nuclear. After 1 month of injection. B. and C. Von Fray test and Plantar test of CPSP animal on bilateral hind limbs for 28 days. D correlation between lesion area and behavior testing result. E Open field result of two groups. ☆ Two-way Avova, post hoc p<0.05. 0 20 40 60 Von Frey exp (26~37, n=8) Day GW 6 8 10 12 Radio Reat (17~37, n=14) Second Mechanical and thermo allodynia occurred after VPL&VPM lesion A 01 3 7 14 21 28 gW 0 20 40 60 80 0 1 3 7 14 21 28 Second 6 8 10 12 0 40 80 120 R² = 0.611933663568679 R² = 0.737710883803482 NeuN NeuN... Von Frey % NeuN % 0 400 800 1200 1600 R² = 0.371538857842569 R² = 0.395009158189154 GFAP GFAP- Astrocyte vs VF:HR Von Frey % GFAP % Con Les 0 50 100 150 200 250 ACC number Con Les 0 50 100 150 200 250 MD number Con Les 0 50 100 150 200 250 VPL, VPM number ACC MD VPM & VPL Neu-N GFAP CD11b Figure 2. A. Immunostaining result of Neu-N (neuron), GFAP (astroglia) and CD11b (microglia) in 4 different rat brain area, included ACC, S1, MD and VPM/VPL area. B. Cell number counting of 500μm 2 in ACC, S1, MD and VPM/VPL collagenase lesion area (right brain, Les) and un-lesion area (left brain, Con). C. Correlation between the immunostaining results and Von Frey test results. Astroglia and microglia cell scars still existed 30 days after collagenase lesion A B C 0 50 100 150 200 250 300 0 200 400 600 800 R² = 0.10724730255045 R² = 0.224197528062334 HL-Von Frey % CD11b % # # # Summary and Conclusion 1.Mechanical and thermo allodynia were developed quickly after 1~3 weeks VP area lesion with collagenase, it is different between long developing duration in human CPSP. 2.The increasing of astroglia and microglia cell number indicated that repair and inflammation still coexisted 30 days after lesion. 3.Hyperactivity of noxious MD response was due to the over- expresses of BDNF, it may been induced by hyper-activation and over-increasing of glia and microglia after hemorrhage. GABA A inhibition system also been altered by the over expression of BDNF C Figure 3. A. Multiunit responses in MD after repeat noxious electric stimuli on sciatic nerve in control and CPSP rat. B. Relationship between stimuli strength and unit activities after stimuli. B. BDNF RT PCR result in normal and collagenase lesion thalamus. D. Effect of TrKB-FC in decreasing of hyperactivities after noxious stimulation. E. Time plot of TrkB-FC effect on unit activity after noxious stimulation. Each result was the summation of 20 sweeps. ☆ Two-way Avova, post hoc p<0.05. # Pair T test, p<0.05. Enhancement of noxious response in MD was widespread in CPSP animal and it could been suppressed by TrKB FC apply A 1s t 3rd TrKB FC 60~120min TrKB FC 240min control rat CPSP rat 1s t 3rd CPSP rat D enhance d 1 ms Stimulate time number 1 0 20 40 60 80 100 # related intensity Con Le s spo n Mus MD spontaneous oscillation activity could been enhanced by GABA A receptor agonist B A Control CPSP Control CPSP Figure 4. A. Spontaneous MD multiunit activities sweeps before and after muscimol injection in control animal. B. Spontaneous MD multiunit activities sweeps before and after muscimol injection in CPSP animal. C. Summation numbers per minute of multiunit activities in control and CPSP animal before and after muscimol injection. D. Averaged MD unit activity of con and CPSP group before and after muscimol treatment. ☆ Two-way Avova, post hoc p<0.05. Muscimol injection Muscimol injection numbe r stren gth B E Spon MuscimolRecovery 0 200 400 600 800 Con CPSP D number Day B C Day Plantar Test Von Frey Test 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 0 50 100 150 R² = 0.231122753643147 R² = 0.0862088176457107 All Lesion area X noxious Response VonFrey% D %(d28/d0) mm 2 Average Max 0 100 200 300 400 500 600 700 Openfield Con CPSP cm/min CD11b 0 20 40 60 80 100 120 0min 60min 120min 0 5 10 15 20 0 200 400 600 800 R² = 0.952957302404163 R² = 0.978396119477836 Control E
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Transcript of NeuN GFAP CD11b C Involvement of BDNF in the thalamic hypersensitivity in CPSP. Hsi-chien Shih and...
NeuN GFAP CD11b
C
Involvement of BDNF in the thalamic hypersensitivity in CPSP.Hsi-chien Shih and Bai-chuang Shyu , Institute of BioMedical Science, Academia Sinica, Taipei, Taiwan.
AbstractAfter stroke, about 7~10% patient will develop chronic pain syndrome after 6 month later. This chronic pain condition is called central post stroke pain syndrome (CPSP). Lenz supported a hypothesis CPSP is due to the unbalanced oscillation of thalamocortical circuit. Recent research results also indicated that abnormal increased secretion of brain derived neurotrophy factor (BDNF) in spinal cord tissue after spinal cord or peripheral neural injury. Expression of NKCC1 and NCC2, Cl - related channel, were influenced by the over expression of BDNF that the balance and functional role of Cl- in mature neuron were also re-modified. CPSP animal model of brain hemorrhage in ventral-posterior area of thalamus (VP) induced by collagenase injection was well established but the pathological mechanism of this model is not well studied. In the present study, 0.125U/0.5μl type 4 collagense was injected into SD rats’ VP area and nociceptive responses were tested with Von Frey and Plantar test. After 4 weeks of injection, the number of neuron was decreased and the cell number of astroglia, microglia and the mRNA level of BDNF were increased in lesion brain area. The decreasing of neuron was positively correlated with the degree of pain hypersensitivity of CPSP. In the electrophysiology recording, activity of medial-dorsal thalamus nucleus (MD) was enhanced after repeated noxious stimuli in CPSP animal and this enhancement could be blocked by acute TrKB-FC (an extracellular scavenger of BDNF) injection. Instead of inhibition by GABA system in normal rat, MD multiunit activity was enhanced after microinjection of muscimol in CPSP animal. The change of cell composition in VP area was a key factor of CPSP syndrome and over expression of BDNF probably induced by the proliferation of glia or microglia cell. MD neurons plasticity changes after stroke may be due to the composition changes of Cl- related channels that caused by BDNF re-modulation.
VPL
VPM
Material and Method
Preparation of animalsMale Sprague–Dawley rats (300–400 g) were housed in an air-conditioned room (21–23°C, humidity 50%, 12-h light/dark cycle starting at 08:00 h) with free access to food and water. All experiments were carried out in accordance with the guidelines of the Academia Sinica Institutional Animal Care and Utilization Committee.
Recording of evoked multichannel field potentials and unit activities
16 channels Michigan probes were used to record the extracellular field potentials in the left anterior cingulate cortex (ACC) (about 2.5 mm anterior and 1 mm lateral to bregma; probe inserted 40° from the vertical line) and MD (about 2.2~3.5 mm posterior and 0.5~1.0 mm lateral to bregma; probe inserted perpendicular to the cortical surface). An Ag–AgCl reference electrode was placed in the nasal cavity. The sampling rates of recorded analog signals was 6 kHz in field potentials data and 24KHz in unit data. All data were processed in a multichannel data acquisition system (TDT, Alachua, FL) for a PC and analyzed with MatLab programs.
SurgerySD rats were initially anesthetized with 4% isoflurane mixed oxygen air and then placed in a stereotaxic apparatus. Animals were maintained under anesthesia with 1% isoflurane in 30%/70% nitrous oxide/oxygen during the surgery. Rats' body temperatures were maintained at 37.5~36.5°C with a homeothermic blanket system (Model 50–7079, Harvard Apparatus, Holliston, MA).
CPSP animal induction and Behavior testingAnimals were injected with 0.125U type 4 collagenase in 0.5 μl saline into ventral posteromedial and ventral posteromedial thalamus nuclear, the coordinate is 3.0~3.5 mm posterior and 3.0~3.4 mm lateral to bregma, depth is 5700~6000 μm. Control animals were only injected with 0.5 μl saline. To measure the mechanical and thermal noxious response, Von Frey and Plantar test were applied on bilateral hind limbs before lesion and at 1, 3, 7, 14and 28 day after lesion. Sailine, TrKB FC (1 μ g/1 μl, 0.1 μl/ min) into and muscimol was direction injected with thalamus close to recording site.
ImmunostainAnimal scarified with PBS/paraformadehyde perfusion, rat brain was removed. After 10% to 30% sucrose solution immersion, rat brain was sliced to 30~40μm with frozen section method and the slices included ACC, S1, MD and VPL/VPM was stained with 1’ anti body, Neu-N/Gila or OX42/Glia, and 2’ 488/594nm fluoresce antibody.
electrophysiology & immunostain
base line 1 3 7 14 21 28 day
1 week habituationlesion
lesion behavior test
Results
Figure 1. A. An example of collagenase lesion in VPL & VPM nuclear. After 1 month of injection. B. and C. Von Fray test and Plantar test of CPSP animal on bilateral hind limbs for 28 days. D correlation between lesion area and behavior testing result. E Open field result of two groups. ☆ Two-way Avova, post hoc p<0.05.
-1 2 5 8 11 14 17 20 23 26 290
20
40
60
Von Frey exp (26~37, n=8)
HR HL
Day
GW
-1 2 5 8 11 14 17 20 23 26 296
8
10
12
Radio Reat (17~37, n=14)
HR
Seco
nd
Mechanical and thermo allodynia occurred after VPL&VPM lesionA
0 1 3 7 14 21 28
gW
0
20
40
60
80
0 1 3 7 14 21 28
Seco
nd
6
8
10
12
0 20 40 60 80100
120140
160180
2000
40
80
120R² = 0.611933663568679R² = 0.737710883803482
NeuN
NeuN vs VF:HR
Von Frey %
Neu
N %
0 20 40 60 80 1001201401601802000
400
800
1200
1600
R² = 0.371538857842569R² = 0.395009158189154
GFAPGFAP-Astro-cyte vs VF:HRPower (GFAP-Astrocyte vs VF:HR)
Von Frey %
GFA
P %
Con Les0
50
100
150
200
250ACC
num
ber
Con Les0
50
100
150
200
250MD
num
ber
Con Les0
50
100
150
200
250VPL, VPM
num
ber
ACC
MD
VPM
& V
PL
Neu-N GFAP CD11b
Figure 2. A. Immunostaining result of Neu-N (neuron), GFAP (astroglia) and CD11b (microglia) in 4 different rat brain area, included ACC, S1, MD and VPM/VPL area. B. Cell number counting of 500μm2 in ACC, S1, MD and VPM/VPL collagenase lesion area (right brain, Les) and un-lesion area (left brain, Con). C. Correlation between the immunostaining results and Von Frey test results. # Pair T test, p<0.05.
Astroglia and microglia cell scars still existed 30 days after collagenase lesionA B
C
0 50 100 150 200 250 3000
200
400
600
800
R² = 0.10724730255045R² = 0.224197528062334
HL-Von Frey %
CD11
b %
#
##
Summary and Conclusion 1. Mechanical and thermo allodynia were developed quickly after 1~3 weeks VP area lesion with
collagenase, it is different between long developing duration in human CPSP.2. The increasing of astroglia and microglia cell number indicated that repair and inflammation still
coexisted 30 days after lesion. 3. Hyperactivity of noxious MD response was due to the over-expresses of BDNF, it may been
induced by hyper-activation and over-increasing of glia and microglia after hemorrhage. GABA A inhibition system also been altered by the over expression of BDNF that maybe the reason why MD neuron expressed hyperactivation after noxious stimuli.
C
Figure 3. A. Multiunit responses in MD after repeat noxious electric stimuli on sciatic nerve in control and CPSP rat. B. Relationship between stimuli strength and unit activities after stimuli. B. BDNF RT PCR result in normal and collagenase lesion thalamus. D. Effect of TrKB-FC in decreasing of hyperactivities after noxious stimulation. E. Time plot of TrkB-FC effect on unit activity after noxious stimulation. Each result was the summation of 20 sweeps. ☆ Two-way Avova, post hoc p<0.05.# Pair T test, p<0.05.
Enhancement of noxious response in MD was widespread in CPSP animal and it could been suppressed by TrKB FC apply A 1st 3rd
TrKB FC 60~120min TrKB FC 240min
cont
rol r
at
CPSP
rat
1st 3rd
CPSP
rat
D enhanced
1
ms
Stimulate time
num
ber
10
20
40
60
80
100#
rela
ted
inte
nsity
Con Les
spon
Mus
MD spontaneous oscillation activity could been enhanced by GABAA
receptor agonistBA Control CPSP
Control CPSP
Figure 4. A. Spontaneous MD multiunit activities sweeps before and after muscimol injection in control animal. B. Spontaneous MD multiunit activities sweeps before and after muscimol injection in CPSP animal. C. Summation numbers per minute of multiunit activities in control and CPSP animal before and after muscimol injection. D. Averaged MD unit activity of con and CPSP group before and after muscimol treatment. ☆ Two-way Avova, post hoc p<0.05.
Muscimol injection Muscimol
injection
num
ber
strength
B
E
Spon Muscimol Recovery0
200
400
600
800Con CPSP
D
num
ber
Day
B C
Day
Plantar TestVon Frey Test
0.5 1 1.5 2 2.5 3 3.5 4 4.5 50
50
100
150
R² = 0.231122753643147
R² = 0.0862088176457101
All Lesion area X noxious ResponseVonFrey%Linear (VonFrey%)
D
%(d
28/d
0)
mm2Average Max
0
100
200
300
400
500
600
700
Openfield
Con CPSP
cm/m
in
CD11b
0
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0min 60min 120min180min 240min
0 5 10 15 200
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R² = 0.952957302404163
R² = 0
Control
E
