of Effect parasympathetic system stimulation on infarct size in rat focal cerebral ischemia
I. AY*, J. LIU, G. DAI, T. BENNER, H. AY, A.G. SORENSEN.
Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, 02129.
BACKGROUND
Parasympathetic system:
Extensively innervates cerebral vascular system and increases cerebral blood flow (CBF): Fibers originate from the superior salivatory nucleus in the pons, Run along the facial nerve until they synapse in the sphenopalatine ganglion, Innervate blood vessels via the greater superficial petrosal nerve. Regulates several pathways that are involved in pathophysiology of ischemic brain injury other than blood flow: Inhibits cytokine release and thereby prevents cytokine-mediated tissue injury, Reduces neuronal excitability.
149.15
METHODS cont'd
Determination of infarct size:
2 mm thick coronal brain sections were incubated with 2,3,5-triphenyltetrazolium chloride Infarct, ipsilateral non-infarct, and controlateral hemispheric areas were manually outlined by an investigator blinded to the treatment groups using Image J Infarct volume was calculated by multiplying infarct area (contralateral hemispheric area subtracted by ipsilateral non-infarct area) by slice thickness Infarct volume was expressed as a percentage of contralateral hemispheric volume
3
VNS causes an immediate and transient decrease in heart rate in rats.
Chart Window
6 VNS treatment decreases infarct volume in rat transient MCA occlusion.
60
Data analysis:
Data were expressed as mean S.D. Analyzed by repeated measures ANOVA or unpaired t-test P <0.05 were considered statistically significant
The decrease was observed in all the VNS-treated animals. The duration of this effect was around 30 seconds and the amplitude of decrease in heart rate was 22.82 5.13 beats/min (n=60). Representative heart rate tracing from a VNS-treated animal. Arrow shows the time of electrical stimulation.
500
% Infarct Volume
50 40 30 20 10 0
Control VNS
400
*
He a rt R ate (B PM )
300
200
Hypothesis: Modulation of brain's endogenous parasympathetic system may
provide the opportunity to gain access to the region of ischemic but still viable tissue with critically impaired blood flow. Electrical stimulation of unilateral sphenopalatine ganglion (SPG) in rat permanent middle cerebral artery (MCA) occlusion is associated with reduced infarct size (Henninger and Fisher, 2007). SPG's intracranial location may limit the feasibility of its stimulation in humans, whereas vagus nerve is easily accessible in the neck.
100
Experimental Design
Arterial blood pressure and heart rate CBF (control n=4) (VNS n=4) Arterial blood gases Blood glucose Neurological assessment
1:04:00
1:04:10
1:04:20 10
vn s
0
1:04:30
1:04:40
1:04:50
1:05:00
1:05:10
1:05:20
The mean infarct volume and SD was 33.30 9.27% of the contralateral hemispheric volume in control animals (n=10). This was significantly larger than the mean infarct volume in VNS-treated animals (18.22 7.29%, n=10; unpaired t-test: t=4.044, p=0.0008)
4
7 There was no difference between mean arterial blood pressure and heart rate of control and VNS-treated animals before and after the MCA occlusion in rats.
MABP (mmHg) Control Before MCA occlusion 127.96 7.92 129.46 4.85 128.71 8.69 131.12 6.71 P=0.1921 HR (beats/min)
VNS has no effect on rCBF in rat transient MCA occlusion.
Pre-MCA 20 min after After 1st occlusion MCA occlusion stimulation (occlusion) After 3rd stimulation (occlusion) After 5th stimulation (reperfusion)
rCBF (%)
Vagus nerve stimulation (VNS):
Stimulates superior salivatory nucleus, the key structure for cerebrovascular parasympathetic innervation. Safe and effective treatment for refractory partial onset seizures and treatmentresistant depression (George et al., 2002). Under study as a potential therapy for migraine, Alzheimer's disease, traumatic brain injury, and neuropathic pain (George et al., 2002; Smith et al., 2005). Decreases neuronal cell death in gerbil forebrain ischemia (Masada et al., 1996).
30' 15' pre pre
0
30'
60' VNS
90' VNS
120' VNS
150' VNS
180' VNS
24 h
VNS (n=10) VNS electrode implantation (n=20) MCA occlusion Laser Doppler probe implantation (Control n=4; VNS n=4) Anesthesia & Femoral artery catheterization MCA reperfusion Euthanasia Histology
Control 365.98 11.84 397.34 12.01 371.42 12.74 386.83 21.85 P=0.2644 Control VNS
100
20.81 17.42 21.63 18.01 43.10 15.27 103.26 24.46
3 hours after MCA occlusion VNS Before MCA occlusion 3 hours after MCA occlusion Repeated measures ANOVA
100
23.68 14.65 22.63 18.33 45.26 13.07 94.73 21.05
Aim: investigate To the effect of VNS on infarct size after transient focal cerebral
ischemia in rats.
1 VNS has no effect on arterial blood gases and blood glucose before and after the MCA occlusion in rats.
Before the 60' after the After the MCA occlusion MCA occlusion reperfusion ABGs pH Control VNS pCO2 Control (mmHg) VNS pO2 Control (mmHg) VNS Blood glucose Control (mg/dL) VNS 7.39 0.01 7.39 0.01 43.5 2.58 45.7 1.10 87.6 5.71 81.5 4.01 123 4.65 121 6.23 7.41 0.02 7.40 0.01 41.1 5.78 41.8 2.20 89.2 4.32 84.7 2.87 122 8.56 121 2.85 7.40 0.01 7.40 0.01 40.0 6.78 38.8 5.65 96.1 4.65 89.5 7.43 120 5.98 119 2.43 180' after the MCA occlusion 7.42 0.01 7.42 0.01 36.4 2.13 35.8 2.01 102.6 3.06 101.1 3.92 119 4.86 116 5.42
8 VNS-treated animals have better neurological scores after cerebral ischemia/reperfusion.
4 h after MCA occlusion 2.17 0.18 2.00 0.0 24 h after MCA occlusion 2.8 0.21 1.2 0.10 *
METHODS
Surgical procedures:
Adult male Wistar rats (350-400 g, n=12; Charles River Laboratories) were anesthetized by isoflurane. Rectal temperature was maintained at 37.5 C . Arterial blood pressure and heart rate were continuously monitored. Arterial blood gases and blood glucose were measured intermittently. rCBF was continuously monitored via laser Doppler flowmeter in a subgroup of animals (n=4) from each experimental group. Cerebral ischemia was produced by intraarterial filament occlusion of the right MCA for two hours (Longa et al., 1989). Stimulating electrodes were self-constructed by the method of Smith et al. (2005). Electrodes were implanted on the right vagus nerve, following ischemia.
5 VNS treatment decreases cell death in the penumbra in rat transient MCA occlusion.
Transient occlusion of the right MCA for 2 hours resulted in infarct in the ipsilateral cerebral cortex and underlying striatum (A). In VNS-treated animals, the infarct area was limited to striatum and involved a smaller area of cerebral cortex as compared with untreated animals (B). Images of TTC-stained sections at the level of bregma 0.4 mm from control (A) and VNS-treated (B) animals. A B
VNS
CONCLUSIONS In rat transient MCA occlusion, stimulation of right cervical vagus nerve:
1. has no effect on arterial blood gases and blood glucose, 2. causes transient decrease in arterial blood pressure and heart rate, 3. has no effect on cerebral blood flow, 4. decreases infarct size, 5. improves motor function of animals.
References: 1. Henninger N, Fisher M. (2007) Stimulating circle of willis nerve fibers preserves the diffusion-perfusion mismatch in experimental stroke. Stroke, 38:2779-2786. 2. George MS, Nahas Z, Bohning DE, Kozel FA, Anderson B, Chae JH, Lomarev M, Denslow S, Li X, Mu C. (2002) Vagus nerve stimulation therapy: A research update. Neurology, 59:S56-61. 3. Smith DC, Modgl...