Lack of Allodynia and Thalamic Hyper-excitatory in the P2X 7 Knockout Mice after Thalamic Hemorrhage...
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Lack of Allodynia and Thalamic Hyper-excitatory in the P2X 7 Knockout Mice after Thalamic Hemorrhage Lesion Hsi-Chien Shih, Tweety Kuan. Bai-Chung Shyu. Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan Introduction Chronic pain is a serious health challenge in aged society. About 7~15% patients with hemorrhage stroke in lateral thalamus would suffer central post stroke pain (CPSP). But mechanism of CPSP is not clear. In this study, we tested the hypothesis that CPSP is caused by P2X 7 receptor activation after thalamus hemorrhage damage. Wild type mice and P2X 7 knockout mice were injected with type4- collagenase into ventrobasal complex of thalamus. After lesion, the mechanical and thermal allodynia appeared in wild lesion mice group. Multiunit activities in medial dorsal nucleus of thalamus were enhanced and lengthened after noxious electrical stimuli on sciatic. GABAergic inhibitory effect was translated into enhancement. But those abnormal occurrence was not appear in P2X 7 knockout mice lesion group significantly. Preliminary results suggest that the activation of P2X 7 after thalamus hemorrhage damage is an important factor of CPSP. Material and Method Preparation of animals B6 and P2X7 knockout mice (28–32g) 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. Surgery Mice 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 recording. Mice body temperatures were maintained at 38.5~37.5°C with a homeothermic blanket system (Model 50– 7079, Harvard Apparatus, Holliston, MA). CPSP animal induction and Behavior testing Animals were injected with 10mU type 4 collagenase in 0.2 μl saline into ventral posteromedial and ventral posteromedial thalamus nuclear, the coordinate is 1.4~1.6 mm posterior and 1.3~1.5 mm lateral to bregma, depth is 3200~3500 μm. Control animals were only injected with 0.2 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 7, 14, 28 and 35 day after lesion. Saline and muscimol was direction injected with thalamus close to recording site. Recording of evoked multichannel field potentials and unit activities 16 channels Michigan probes were used to record the extracellular field potentials in the right MD (about 1.4~1.6 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. Brain slice preparation and MEQ induction Mice brain was sliced to 250 μm thick fresh brain slices and incubated in aCSF with 95%O 2 /5%CO 2 . After recovery stage, slices was incubated with di-H-MEQ. The fluorescence was measured by AxioImager Z1 system with 20X objective lens, excitation and emission wave length were 360nm and 460nm. To observe cytoplasmic chloride changing, 100uM muscimol were apply in aCSF for 5min. electrophysiology , MEQ and……. base line 7 14 21 28 35 day 1 week habituation lesion lesion behavior test Figure 3. A. Summation numbers per minute of spontaneous multiunit activities in MD before and after muscimol injection in control , B6-lesion and konckout lesion mice. B. Averaged MD unit activity before and after muscimol injection. # two-way Avova, post hoc p<0.05. Enhanced nociceptive response in CPSP mice MD was not occurred in P2X7 knockout mice Figure 2. A. Multiunit responses in MD after repeat noxious electric stimuli on sciatic nerve in control , B6-lesion and konckout lesion mice. B. Relationship between stimuli strength and unit activities within 100ms after stimuli. C. Relationship between stimuli strength and unit activities within 1000ms after stimuli. # two-way Avova, post hoc p<0.05. A B GABAergic inhibition remained in P2X7 knockout mice after thalamus lesion A B Results Development of mechanical and thermal allodynia after lesion Figure 1. A. An example of collagenase lesion in VPL & VPM nuclear after 7 days. B. An example of lesion area changing after 1 month of lesion. C. Von Fray test result. D. Plantar test result. # two-way Avova, post hoc p<0.05. A B C D 7d 30d gW von Frey Plantar day day Summary and Conclusion 1.Mechanical and thermo allodynia were developed quickly after 1~3 weeks lesion in VB complex, to establish a mice hemorrhage CPSP model is possible. 2.Hyperactivity of nociceptive MD response was observed. According to GABA A modulatory results we presented, the inhibitortory role of GABA A system may be altered to excitation after CPSP. 3.Accumulation of cytoplasmic chloride was occurred after CPSP. It indicated that the compositions of chloride-related channels were changed after CPSP, preliminary data of WB supported our hypothesis. 4.In P2X 7 knockout mice lesion group, patterns of allodynia in hind limbs, enhanced and lengthened noxious activities and muscimol enhanced response in MD neuron were not appeared. It Figure 4. A. An example of di-MEQ incubated control MD neuron and the fluorescence change treated with 10uM muscimol. B. An example of di-MEQ incubated CPSP MD neuron and the fluorescence change treated with 10uM muscimol. Flow of cytoplasmic chloride evoked by muscimol treatmant was reversed after CPSP 0 95 96 97 98 99 100 1 2 3 0 1 2 3 4 5 6 7 8 9 10 11 12 13 85 90 95 100 A wt CPSP B mus mus Fluorensence % Fluorensence % N N 3 CO diffusion oxidation N 3 CO + N 360nm 460nm diH-MEQ MEQ + C h l o r i d e - +fluorescence- Composition of channel changed after CPSP C 0 7 14 21 28 35 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 wt-sham wt-lesion ko-lesion 0 7 14 21 28 35 40 60 80 100 120 140 wt-sham wt-lesion ko-lesion Figure 4. Western blotting result of BDNF, KCC2, NKCC1, Trk recptor and GABA A in control , wt-lesion and konckout lesion mice. # t-test p<0.05. numbers/min # # # # # # # # # # # wt wt-lesion ko-lesion Muscimol Muscimol Muscimol
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Transcript of Lack of Allodynia and Thalamic Hyper-excitatory in the P2X 7 Knockout Mice after Thalamic Hemorrhage...
Lack of Allodynia and Thalamic Hyper-excitatory in the P2X7 Knockout Mice after Thalamic Hemorrhage Lesion
Hsi-Chien Shih, Tweety Kuan. Bai-Chung Shyu. Institute of Biomedical Sciences, Academia Sinica, Taipei, TaiwanIntroduction Chronic pain is a serious health challenge in aged society. About 7~15% patients with hemorrhage stroke in lateral thalamus would suffer central post stroke pain (CPSP). But mechanism of CPSP is not clear. In this study, we tested the hypothesis that CPSP is caused by P2X7 receptor activation after thalamus hemorrhage damage. Wild type mice and P2X7 knockout mice were injected with type4-collagenase into ventrobasal complex of thalamus. After lesion, the mechanical and thermal allodynia appeared in wild lesion mice group. Multiunit activities in medial dorsal nucleus of thalamus were enhanced and lengthened after noxious electrical stimuli on sciatic. GABAergic inhibitory effect was translated into enhancement. But those abnormal occurrence was not appear in P2X7 knockout mice lesion group significantly. Preliminary results suggest that the activation of P2X7 after thalamus hemorrhage damage is an important factor of CPSP.
Material and MethodPreparation of animalsB6 and P2X7 knockout mice (28–32g) 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.SurgeryMice 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 recording. Mice body temperatures were maintained at 38.5~37.5°C with a homeothermic blanket system (Model 50–7079, Harvard Apparatus, Holliston, MA).CPSP animal induction and Behavior testingAnimals were injected with 10mU type 4 collagenase in 0.2 μl saline into ventral posteromedial and ventral posteromedial thalamus nuclear, the coordinate is 1.4~1.6 mm posterior and 1.3~1.5 mm lateral to bregma, depth is 3200~3500 μm. Control animals were only injected with 0.2 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 7, 14, 28 and 35 day after lesion. Saline and muscimol was direction injected with thalamus close to recording site.
Recording of evoked multichannel field potentials and unit activities16 channels Michigan probes were used to record the extracellular field potentials in the right MD (about 1.4~1.6 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.Brain slice preparation and MEQ inductionMice brain was sliced to 250 μm thick fresh brain slices and incubated in aCSF with 95%O2/5%CO2. After recovery stage, slices was incubated with di-H-MEQ. The fluorescence was measured by AxioImager Z1 system with 20X objective lens, excitation and emission wave length were 360nm and 460nm. To observe cytoplasmic chloride changing, 100uM muscimol were apply in aCSF for 5min.
electrophysiology , MEQ and…….
base line 7 14 21 28 35 day
1 week habituationlesion
lesion behavior test
Figure 3. A. Summation numbers per minute of spontaneous multiunit activities in MD before and after muscimol injection in control , B6-lesion and konckout lesion mice. B. Averaged MD unit activity before and after muscimol injection. # two-way Avova, post hoc p<0.05.
Enhanced nociceptive response in CPSP mice MD was not occurred in P2X7 knockout mice
Figure 2. A. Multiunit responses in MD after repeat noxious electric stimuli on sciatic nerve in control , B6-lesion and konckout lesion mice. B. Relationship between stimuli strength and unit activities within 100ms after stimuli. C. Relationship between stimuli strength and unit activities within 1000ms after stimuli. # two-way Avova, post hoc p<0.05.
A B
GABAergic inhibition remained in P2X7 knockout mice after thalamus lesion A B
Results Development of mechanical and thermal allodynia after lesion
Figure 1. A. An example of collagenase lesion in VPL & VPM nuclear after 7 days. B. An example of lesion area changing after 1 month of lesion. C. Von Fray test result. D. Plantar test result. # two-way Avova, post hoc p<0.05.
A
B
C D7d
30d gW
von Frey Plantar
day day
Summary and Conclusion 1.Mechanical and thermo allodynia were developed quickly after 1~3 weeks lesion in VB
complex, to establish a mice hemorrhage CPSP model is possible.2.Hyperactivity of nociceptive MD response was observed. According to GABAA modulatory
results we presented, the inhibitortory role of GABAA system may be altered to excitation after CPSP.
3.Accumulation of cytoplasmic chloride was occurred after CPSP. It indicated that the compositions of chloride-related channels were changed after CPSP, preliminary data of WB supported our hypothesis.
4. In P2X7 knockout mice lesion group, patterns of allodynia in hind limbs, enhanced and lengthened noxious activities and muscimol enhanced response in MD neuron were not appeared. It suggest that the activation of P2X7 after thalamus hemorrhage damage is an important factor of CPSP.
Figure 4. A. An example of di-MEQ incubated control MD neuron and the fluorescence change treated with 10uM muscimol. B. An example of di-MEQ incubated CPSP MD neuron and the fluorescence change treated with 10uM muscimol.
Flow of cytoplasmic chloride evoked by muscimol treatmant was reversed after CPSP
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+ Chloride -+fluo
resc
ence
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Composition of channel changed after CPSP
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Figure 4. Western blotting result of BDNF, KCC2, NKCC1, Trk recptor and GABAA in control , wt-lesion and konckout lesion mice. # t-test p<0.05.
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