Functional Brain Imaging Study
of the Central Post-Stroke Pain in Rats
Presenter: Guan-Ying, Chiou (邱冠穎)
School of Medicine
Chung Shan Medical University
Advisors: Bai-Chuang, Shyu PhD (徐百川), Hsiang-Chin, Lu (呂享晉)
IBMS
Academia Sinica
Outline
• Introduction
CPSP: Clinical manifestations and the pathophysiology
14C-iodoantipyrine:
the tracer for blood flow analysis
• Specific aim
• Materials and methods
• Results
• Discussion
• Acknowledgement
Central Post-Stroke Pain (CPSP)
• Central pain: pain initiated or caused by a primary lesion or
dysfunction in the central nervous system
H. M. Merskey & N. Bogduk. IASP Press. (1994)
• First description of CPSP: a form of CPSP is characterized
by spontaneous pain, attacks of allodynia, and dysesthesia
J. Déjerine & G. Roussy. Rev. Neurol. 14 (1906)
Déjerine-Roussy syndrome = Le syndrome thalamique
• Prevalence: 8 ~ 46 % in stroke patients
G. Kumar & C. R. Soni. J. Neurol. Sci. 284 (2009)
• Clinical manifestations: contralateral somatosensory
abnormalities
A. Hyperalgesia: an increased response to a stimulus that is normally
painful
B. Allodynia: pain evoked by stimulus that is usually not painful
C. Paresthesia, dysesthesia, hyperpathia, aftersensation
Central Post-Stroke Pain (CPSP)
• Pathophysiology: the hypotheses
A. Central imbalance
B. Central disinhibition (thermosensory disinhibition)
C. Cerebral sensitization
(→ hyperactivity/hyperexcitability of spinal/supraspinal
nociceptive neurons)
D. Grill illusion theory
G. Kumar & C. R. Soni. J. Neurol. Sci. 284 (2009)
CPSP: Central / Thermosensory disinhibition
Central disinhibition therory
Thermosensory disinhibition therory
Hemorrhagic
lesion (stroke)
Hemorrhagic
lesion (stroke)
•
•
Increased activity: anterior cingulate cortex (ACC), medial thalamus, insula, and periaqueductal
gray (PAG)
Reduced activity: lateral thalamus and ventral posteromedial thalamus (VPM)
H. Klit et al. Lancet Neurol. 8 (2009)
Diagnostic Images of the CPSP Patients
(Left)
Cranial MRI T2WI:
hyperintensity in right parietal area
(Right)
99Tc-ECD single photon emission
computed tomography (SPECT):
hypoperfusion of the corresponding
parietal region
(Left)
CT scan: left putaminal
hemorrhage
(Right)
99Tc-ECD SPECT:
hypoperfusion of frontoparietal area
both sides
J. Kalita et al. Pain Medicine 12 (2011)
14C-Iodoantipyrine ( 14C-IAP)
• Introduced by Sakurada and co-workers in 1979
• Non-volatile, highly lipophilic
• With improved ability to diffuse through the blood-brain barrier
• Degraded at a slow rate
• More accurate tracer for
regional cerebral blood flow (rCBF)
• Acceptable resolution in autoradiography
• Inexpensive
• For CPSP animal model:
rCBF indicates the neuronal activation pattern
Iodoantipyrine
L. C. Glazer. Yale J Biol Med. 61 (1988)
Comparable resolution
High correlation of regional cerebral blood flow (rCBF)
Specific Aim
• To establish the autoradiographic analysis approach of the rat
brain in vitro.
• To investigate the different brain functional images between
control and CPSP rats
Materials and Methods
• Spraque-Dawley adult rats, 250 – 300 g in weight
• Behavior assessment: von Frey test and radioheat test
• Induction of hemorrhage in the right ventral posteromedial (VPM)
/ ventral posterolateral (VPL) thalamic nuclei with collagenase
IV injection
• Connection of the polyethylene 50 (PE 50) tube to the distal end
of the jugular vein: access for intravenous injection of 14Ciodoantipyrine
• Autoradiographic study of the rat brain slices:
Region-of-interest (ROI) analysis
Sham control vs.
CPSP group (R’t VPM/VPL hemorrhage)
Post-lesion behavioral assessment
Connect PE50 tube and start injection
Post-lesion behavioral assessment
Connect PE50 tube distally
to the jugular vein
Euthanasia
5 min
W1
W2
Pre-lesion behavioral
assessment
W3 W4
Heparin (20 U/ml)
0.1 mL/day
Behavioral assessment
10 sec
Decapitate
OCT embedding
Brain slice
1 min
W5
Injection of 14C-IAP
125 μCi/kg of 14C-IAP in 300 μL of 0.9% saline
Right Thalamic Hemorrhage Induced by Injection
of Collagenase
Localization of the lesion:
~ 3.3 mm posterior to the bregma
3.0 mm lateral to the midline
5.6 mm inferior to the surface of the cortex
Jugular Vein Catheterization for 14C-IAP Perfusion
Pain Behavioral Assessment
Mechanical pain (von Frey test)
Thermal pain (radioheat test)
Clip: Radioheat test
Region-of-Interest (ROI) Analysis
• A manual technique
• The boundaries of ROI are based on definitions available in
anatomic atlases.
Relative intensity
Ratio 
A  BA   R  BR 
R  BR 
A: Mean of the ROI
B: Mean of the selected
background region
R: Mean of the reference ROI
(Cerebellum)
Region-of-Interest (ROI) Analysis: Anatomical Maps
ACC
MD
ACC = Anterior cingulate cortex
MD = Medial dorsal thalamic nucleus
VB = Ventrobasal complex
PAG = Periaqueductal gray
Lesion site: VB
Cerebellum as the reference
VB
PAG
Cerebellum
Adapted from G. Paxinos & C. Watson. The Rat Brain: in Stereotaxic Coordinates. 4th ed. (1998)
Results
• Standardization of the autoradiographic signals
• The images of coronal-section brain slices that indicate the
lesion site in VPM/VPL (combined with the map)
• Behavioral assessment: pre-/post-lesion
A. von Frey test for mechanical pain
B. Radioheat test for thermal pain
• Autoradiographic analysis of the control and CPSP rats
•
ROI: relative ratio of radioactivity
The Standard Curves of Autoradiography
Radioactivity: count per minute
Resolution: pixels per mm2
The resolution highly correlates
with radioactivity
Radioactivity = 219.6 X Resolution – 25.2
Radioactivity: μCi
Resolution: pixels per mm2
R2 = 0.9973
Resolution: pixels per mm2
Exposure time: days
4-day exposure as the choice
Histological View of the Lesion Site in VPM/VPL
Right
The lesion site of the CPSP group locates in
the right ventrobasal complex (VPM/VPL)
Behavioral assessment
The Comparison of Relative Ratio in Bilateral ROIs
Control
CPSP
Left brain
Right brain
Linear Regression and Correlation of the Brain Area
PAG
Control (L)
CPSP (L)
ACC
Insula
LH
VMH
PAG
Hypothalamus
Thalamus
Striatum
Cortex
PAG
Hypothalamus
Thalamus
Striatum
Cortex
Control (R)
CPSP (R)
Discussion
• Histological evidence of the lesion site: Precise localization of the
lesion is helpful to clarify the damage range of the ventrobasal
complex and its effects on other brain regions.
• Behavioral assessment: The nociception and thermal sensation
became more significantly sensitive in the side contralateral to the
hemorrhagic lesion as the observation period prolonged, though
subjective judgments could not be ruled out.
Discussion (continued)
• Radioactivity of ROI: significant difference in the bilateral primary
somatosensory cortices, agranular insular cortices (dorsal and
ventral), granular cortices, cingulate cortices (area 1), secondary
motor cortices, prelimbic cortices, and striata, as well as in the
periaqueductal gray.
• Central/Thermosensory disinhibition theory:
• Increased activity: ACC, medial thalamus, insula, and PAG
• Reduced activity: lateral thalamus and VPM
• Correlation of activity: the ventromedial thalamic nucleus, lateral
hypothalamic area, ventromedial hypothalamic center, and the
periaqueductal gray are moderately correlated with the selected
cortical and subcortical regions in the CPSP rats.
Acknowledgement
• N327
• 徐百川 老師
• 黃智偉 老師、管永惠、張維邦、施希建、
吳俊賢、張瑋仁、呂享晉
• Coffee machinery
Thank you for your attention.
Supplementary Materials
Future Work
• Collect more autoradiographic and behavioral data for further
confirmation.
• Statistical Parametric Mapping (SPM): reconstruction of the
whole brain image that localizes the significant nuclei or area
→ still under the troubleshooting of appropriate Matlab® program
• Electrophysiological investigation for the significant brain area:
electric stimulation and the response pattern
Statistical Parametric Mapping (SPM)
P. T. Nguyen et al. Neurolmage 23 (2004)
SPM Images: Comparison of CPSP and Control
Control
CPSP
Pain System of the Brain
• Lateral pain system
A. Primary somatosensory cortex: sensory-discriminative
dimension of pain
B. Secondary somatosensory cortex: pain intensity
C. Insula: thermal and nociceptive information processing
• Medial pain system
A. Medial and intralaminar thalamic nuclei → Anterior
cingulate cortex: affective-emotional aspect of pain
CPSP: Central disinhibition
• Stroke in the lateral thalamus could disinhibit the activity of medial thalamus
and cause pain.
H. Head & G. Holmes. Brain 34 (1911)
• The loss of the GABAergic neurons in ventral posterolateral (VPL) thalamic
nuclei caused by early nonspecific deafferentation, the return of function in
the large afferent system now lacking, results in intrinsic inhibition of VPL
and subsequently activates other cortical areas in an unlearned fashion
which results in a sensation never experienced before.
R. Melzack & J. D. Loeser. Pain 4 (1978)
• An indirect route of disinhibition via thalamic reticular nuclei that contain
inhibitory interneurons
D. Jeanmonod et al. Brain Apr. 119 (1996)
• Central pain results from the loss of descending controls from interoceptive
cortex on brainstem homeostatic sites that drive thermoregulatory behavior
by way of the medial thalamus and anterior cingulate cortex.
A. D. Craig. Trends Neurisci. 26 (2003)
CPSP: the Proposed Mechanisms
A. Central disinhibition
B. The thermosensory
disinhibition therory
C. The intra-thalamic
inhibitory hypothesis
D. The deafferentation
hypothesis
E. The dynamic
reverberation theory
H. Klit et al. Lancet Neurol. 8 (2009)
Laterality of lesions
• Right sided lesions predominate among CPSP patients at both
cortical and thalamic levels.
• Non-dominant hemisphere: specialization in monitoring somatic
state?
S. Canavero & V. Bonicalzi. Cambridge University Press (2007)
Diagnostic Criteria for CPSP
H. Klit et al. Lancet Neurol. 8 (2009)
Iodoantipyrine: Partition Coefficient
Non-polar solvent: water partition coefficient
14C-Iodoantipyrine vs. 14C-antipyrine
Least polar
Most polar
Brain tissue: blood partition coefficient of 14CIodoantipyrine = 0.8
A. Uniform density throughout the rat brain
B. Post-administration of equilibrated blood
Darker circle inside tissue: blood
O. Sakurada et al. Am. J. Physiol. 234 (1978)
Statistical Parametric Mapping (SPM)
P. T. Nguyen et al. Neurolmage 23 (2004)
On-going research
• Early application of P2X7 antagonist could help to reduce the CPSP
development on hyper-pain sensitivity, hyper neuronal excitability, also
reduces the microglia aggregation, P2X7, TNFα, IL-6, IL-1β but not
BDNF elevation. The reduction or less occurrences of CD11b and
P2X7 positive cells indicating an anti-inflammatory effects via
inhibition of P2X7 during post stroke brain traumatic progression,
which possibly preserve spaces for the traumatic tissues undergoes
later recovery processes.
• Hyperactivity of noxious MD response was due to the over-expresses
of BDNF, it may be induced by hyper-activation and over-increasing of
glia and microglia after hemorrhage. GABAA inhibition system of the
reticular nucleus was altered by the over expression of BDNF. And it
maybe the mechanism underlying the enhanced MD neuronal activity
after noxious stimuli in CPSP.
P2X7 involving in a series of sequential signaling
affects microglia and neuronal activity resulting CPSP
ATP activated
microglia
Ca2+
P2X7
ProIL-1β
Glu
IL-1β
Glu
Glu
MD neuron inputs ↑
Pain
stimulation
Modified from Bennett et. al, J of Theoretical Biology, 2009, 261:1-16