NeuN GFAP CD11b

1. Involvement of BDNF in the thalamic hypersensitivity in CPSP. Hsi-chien Shih and Bai-chuangShyu, Institute of BioMedical Science, Academia Sinica, Taipei, Taiwan. Results Abstract Mechanical and thermo allodynia occurred after VPL&VPM lesion Enhancement of noxious response in MD was widespread in CPSP animal and it could been suppressed by TrKB FC apply 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. A B C Plantar Test Von Frey Test 12 A 1st 3rd B 1st 3rd 80 60 10 Second number CPSP rat control rat gW 40 8 20 0 6 E C D strength 0 1 28 1 28 3 7 14 21 0 3 7 14 21 Day Day TrKB FC 240min enhanced TrKB FC 60~120min D E number related intensity CPSP rat %(d28/d0) Stimulate time 1 Con Les ms mm2 Material and Method 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. 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. 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. Astroglia and microglia cell scars still existed 30 days after collagenase lesion MD spontaneous oscillation activity could been enhanced by GABAAreceptor agonist Surgery A B Neu-N GFAP CD11b 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). A B CPSP Control spon ACC 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. VPL VPM Mus MD C D CPSP Control 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 MatLabprograms. number # # VPM & VPL # Muscimol injection Muscimol injection 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. Immunostain NeuN GFAP CD11b C CD11b Animal scarified with PBS/paraformadehydeperfusion, 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. Summary and Conclusion 1 week habituation electrophysiology & immunostain • 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. • The increasing of astrogliaand microglia cell number indicated that repair and inflammation still coexisted 30 days after lesion. • 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. lesion 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 immunostainingresults and Von Frey test results. # Pair T test, p<0.05. 7 14 21 28 day 1 3 base line behavior test lesion