Role of the Peripheral Sympathetic Innervations in Controlling Cerebral Blood Flow after the Transection of Bilateral Superior Cervical Sympathetic Ganglia Two Weeks Later

Background: Cerebral blood vessels are mainly supplied by sympathetic nerves arising from the superior cervical ganglia and cerebral blood volume may be influenced by bilateral superior cervical ganglionectomy (SCG). Various stages of cerebral blood volume changes depended on the time following bilateral excision of SCG. In this study, we emphasize the subacute effect (two weeks) on the local cerebral blood flow (LCBF). Material and Methods: Sprague-Dawley rats weighing 250-400 gm (n = 20) were selected into two groups. Under the ambient temperature 20˚C, the first group animals (n = 10) received sham operation and the other group animals (n = 10) underwent bilateral SCG. The LCBF and O 2 delivery of 14 brain structures were measured for each animal by the use of 14 C-iodoantipyrine technique two weeks after the operation. Results: The average of LCBF was decreased from 150 ml/100 gm/min to 129 ml/100 gm/min after bilateral SCG. Only the LCBF at basal ganglia was increased from 108 ml/min/100 g in the sham-operated group to 118 ml/min/100g in the SCG group. A mean of 14% reduction of LCBF was estimated. In 14 brain structures, the delivery amount of O 2 was all decreased, except in basal ganglia. However, these changes of LCBF and the delivery amount of O 2 at these 14 brain structures did not reach the significant differences. Conclusions: The present results show that chronic effect (two weeks) of bilateral SCG on LCBF was not only in a decrease of the LCBF, but also a decrease of local cerebral O 2 delivery. However, the changes didn't show the significant differences.


Introduction
The relationship between autonomic regulation and cerebral blood flow (CBF) has long been a subject of considerable interest in studying the mechanisms of neurovascular disorders such as hypertensive encephalopathy, migraine and syncope [1].Cerebral blood vessels are mainly supplied by sympathetic nerves arising from the superior cervical ganglia, and the sympathetic nerves, heterogeneously distributed throughout the cere-brocortex [2,3].Although nervous stimulation or adrenergic agonists lead to vasoconstriction, superior cervical ganglionectomy (SCG) or adrenergic antagonists do not usually affect regional CBF [4,5]."Microregional" differences in the cerebrocortex without decreasing global and regional blood flow, resulted from limit flow and/or decrease red cell velocity, has been hypothesized [6].The subacute effect of SCG on cerebral microcirculation remains obscure.In this study, we evaluate local cerebral blood flow (LCBF) and the delivery amount of O 2 at 14 brain structures in experimental rats underwent bilateral SCG.

Material and Methods
Male Sprague-Dawley rats weighing 250 to 400 gm were used for these experiments.These animals were fasted for approximately 12 hours before surgery.

Surgical Techniques
The rats were anesthetized with 3% halothane for 2 -4 minutes until they were calm down.The anesthesia was continued with 1.5% halothane in nitrous oxide (70%) and oxygen (30%) for the period of surgery.The femoral artery and femoral vein were catheterized on both sides of the animals with polyethylene tube (PE-50).The surgical wounds were covered with xylocaine jelly.A plaster cast was fitted from midthorax to midthigh to immobilize the hind limbs and protect the catheters.Then the anesthesia was stopped and the animals were allowed to recover the consciousness.

Experimental Design
The rats were exposed in normal condition (ambient temperature 20˚C) and their physiological condition was assessed by measurement of hematocrit (Hct), hemoglobin (HgB), pH, pCO 2 , pO 2 , volume and saturation of O 2 , systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial blood pressure (MBP), pulse rate (PR), weight, and plasma glucose level.Total 20 male Sprague-Dawley rats were divided into two groups: Group A (n = 10) as the control group underwent sham operation.Group B (n = 10) received bilateral superior cervical ganglionectomy.Local cerebral blood flow and O 2 delivery were measured post each operation.Superior cervical ganglionectomy was performed as: the salivary glands were exposed through sublingular incision in the neck and each superior cervical ganglion was identified at the bifurcation of the common carotid artery beside the trachea.The ganglia were totally removed en bloc from both sides.Sham operation received the same procedure as above except the removal of SCG.After recovering from the anesthesia, the rats were put in a cage with food as usual.Two weeks later, the rats were re-anesthetized for measurement of LCBF.

Measurement of Local Cerebral Blood Flow
The LCBF was measured by the 14 C-iodoantipyrine (IAP) technique developed by Sakurada et al. [7], as modified by Otsuka et al. [8].Before beginning IAP infusion, a single extracorporeal arteriovenous (AV) shunt was formed by shortening and connecting the femoral artery and vein catheters on one side with a 3-cm length of silicone rubber tubing.At the beginning of the experimental period (t = 0), an intravenous infusion of approximately 50uCi of 14 C -IAP in 1ml saline was started using a variable speed infusion pump.The rate of infusion was increased during the experiment according to a schedule that yielded a linearly rising concentration of IAP in the blood.Series arterial blood sampling was performed (every 5 sec starting at t = 0) by puncturing the extracorporeal AV loop with a 22-gauge needle on a plungerless syringe and collecting 60 ~ 80 µl blood over the next 2 -4 sec.The rats were decapitated at t = 30 sec, and their brains were quickly removed., frozen in 2-methylbutane cooled to -45˚C, and stored in a plastic bag at -80˚C until the time of sectioning.
Plasma 14 C-radioactivity was assayed by beta counting.The frozen brain was cut into a series of 20 µm thick sections in a cryostat at -20˚C starting at the area postrema, which lies at the caudal end of the fourth ventricle.Selected brain sections from inferior collicullus, superior collicullus, midbrain, occipital cortex, pons, medulla oblongata, hippocampus, hypothalamus, thalamus, corpus callosum, temporal cortex, frontal cortex, basal ganglia, sensorimotor cortex, cerebellar vermis were dried and placed in X-ray cassettes along with sets of previously calibrated 14 C standards.Many of the areas were selected because they had been included in the reports of Kadekaro et al. [9] and our previous study [10].Un-exposed X-ray film (Kodak SB-5) was placed over the brain section and standards.After approximately 12 days of exposure, the films were developed.The entire autoradiograms were analyzed for radioactivity using an image processing system (MCID, Imaging Research, St. Catharings, Canada).The optical density values were converted to tissue reactivity using the Kety-Sokoloff equation [7].

Measurement of O 2 Delivery
Adjacent frozen samples from fourteen brain regions were immediately used to determinate arterial and venous O 2 saturation.Detail techniques have been published previously [11].Briefly, 20 μm sections were obtained on themicrotome-cryostat at -35˚C under a N 2 atmosphere.The sections were transferred to precooled glass slides and covered with degassed silicone oil and a cover slip.The slides were placed on a microspectrophotometer fitted with an N 2 -flushed cold stage to obtain readings of optical densities at 568, 560, and 506 nm.This three-wavelength method corrects for the light scattering in the frozen blood.Only vessels in the transverse section were studied so that the path of light traversed only the blood, Readings were obtained to determine O 2 saturation in these regions.The O 2 content of blood was determined by multiplying the percent O 2 saturation by the hemoglobin concentration times 1.36.

Statistics
All data was presented with means ± SEM and statistically calculated by unpaired, two-tails t test.

Results
Hemodynamic and physiological data including plasma glucose level, HCT, pH, pulse rate, weight, systolic blood pressure, diastolic blood pressure, mean blood pressure in two groups revealed no significant difference (Table 1).According to the relevant data of blood gas, a significant hyperoxemia (pO 2 from 87.1 to 97 torrs, p < 0.05) was demonstrated, but the pCO 2 , the volume of O 2 and the saturation of O 2 did not reach the significant differences.
LCBF at 14 brain structures was presented in Table 2.Only the LCBF at basal ganglia was increased from 108 ml/min/100 g in the SCG group to 118 ml/min/100 g in the sham-operated group.The others 13 brain structures showed a decreased level of LCBF.The average of LCBF was 150 ml/min/100 g in the sham-operated group and 129 ml/min/100 g in the SCG group.A mean of 14% reduction of LCBF was found at 14 brain structures.However, these changes of LCBF at these 14 brain structures did not reach the significant differences.
The delivery amount of O 2 was also measured at 14 brain structures and summarized in Table 3.In 14 brain structures, the delivery amount of O 2 was all decreased, except in basal ganglia.The differences in the delivery amount of O 2 between sham-operated and symphathectomized group was not significantly meaningful in these 14 brain structures.

Discussion
Sympathetic nerves supply different parts of the cerebrovascular system including the main pial arteries at the base of the brain, the pial arterial and venous systems of the convexities, and parts of the intracerebral (parenchymal) vascular system [12].Using the techniques of immunofluorescence and histofluorescence, cerebral vessels have been proved to be innerved by peripheral sympathetic nervous system, mainly arising from the ipsilateral   superior cervical ganglion [13].These nerve fibers are distributed mainly in rostral areas of the brain and heterogeneously distributed to cortical vessels to the level of arterioles [5].The innervation also provides a protective mechanism for brain to insure an adequate O 2 supply to caudal regions under hypoxic conditions by limiting flow to cortical areas [6].
Several studies about sympathetic influences on cerebral vessels have been discussed, especially in SCG, by measuring the cerebral blood volume (CBV) and regional CBF.Eklof et al. discovered CBF reduced about 30% in monkeys undergo bilateral SCG within 2 weeks but the same results were not observed by Waltz et al in the cat after monolateral SCG [14,15].Several experimental studies revealed the various stages of CBV depended on the time following bilateral SCG [12,16,17].In serial mice studies, Edvinsson et al. found, compared with sham-operated controls, CBV was decreased to a level 15% to 28% below the control values twelve hours after SCG, increased to a level 15% to 34% above the control values one day later, and returned to normal range within only one week [12,16].He hypothesized the vasoconstriction was caused by norepinephrine leakage from the degenerating nerves shortly after operation; vasodilatation resulted from a consequence of disappearance of the transmitter one day later; and normalization of vascular tone was caused by supersensitivity of denervated vascular receptors to circulating catecholamines in chronic phase [12].
However, in the studies emphasized on the long term effects (8 -10 weeks) of unilateral SCG on non-anesthetized rabbits, Aubineau et al. [18], found a mean of 17% reduction of CBF compared to the heterolateral cortex.This phenomenon was observed from 8 to 30 days after the operation.Intravenous infusion of noradrenaline did not significantly modify CBF in both hemispheres.There were no signs of supersensitivity to catecholamines.They concluded that, as in the peripheral circulation, chronic sympathectomy affects the equilibrium of the vascular smooth muscle fibers, but that circulating amines play no compensatory role in the cerebral circulation because of the blood-brain barriers.So the reduction of CBF could be attributed to many factors after sympathectomy: decrease in the thickness of the turnica media of the cerebral vessels [19], a modification of membrane mechanisms of the smooth muscle fibers [20], depolarization without any possible modification in the threshold of excitability, a hypersensitivity to calcium ion, and possible metabolic changes revealed by a decrease in sodium-potassium-adenosine triphosphatase activity and modification in the level of adenosine triphosphate [18].Suppression of the whole cervical sympathetic innervation could also result in a reduction in pineal gland activity [21], alterations in the numerous neuroendocrine systems [22], or a modification in the activity of the carotid sinus [23].Some of these phenomena may modify CBF and CBV in the long term.
In our present study, two weeks postoperatively, the bilateral SCG rats displayed a mean of 14% reductive level of LCBF as compared to those of the sham-operated rats.In the 14 brain structure, the LCBF was all decreased, except in the basal ganglia.It showed the subacute effect of bilateral SCG was not only in the CBF, but also in the LCBF.The delivery amount of O 2 was also decreased in these regions.Our results were compatible with previous reports [11,18].Based on these results, we could conduct the study to investigate the further effect of bilateral SCG in the rats with heat stroke.

Conclusions
Although the change of LCBF and local cerebral O 2 delivery in the 14 brain structure didn't show the significant differences, our present results show that subacute effect (two weeks) of bilateral SCG on CBF was not only in a decrease of LCBF, but also a decrease of local cerebral O 2 delivery.About 14% decrease level of LCBF was