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Parasympathetic nerves from the pterygopalatine ganglia may participate in development of cluster headaches, in vascular responses to hypertension and in modulation of damage due to stroke. Stimulation of the nerves elicits cerebral vasodilatation, but it is not known if the nerves tonically influence cerebrovascular tone. We hypothesized that parasympathetics provide a tonic vasodilator influence and tested that hypothesis by measuring cerebral blood flow in anesthetized rats before and after removal of a pterygopalatine ganglion. Ganglion removal led to reduced cerebral blood flow without changing blood pressure. Thus, parasympathetic nerves provide tonic vasodilatory input to cerebral blood vessels.
Cerebral blood vessels are richly innervated both by central pathways (Reis, 1984;Vaucher & Hamel, 1995) and by sympathetic and parasympathetic nerves (Wahl & Schilling, 1993) and are further influenced by metabolic activity of local CNS neurons (Dirnagl et al., 1994;Harder et al., 2002). We have focused on the parasympathetic innervation derived from neurons of the pterygopalatine ganglion (PPG), also referred to as the “sphenopalatine ganglion”. We, like others (Morita-Tsuzuki et al., 1993), have shown that electrical stimulation of the PPG causes cerebral vasodilatation (Talman et al., 2007). Furthermore, we have shown that the PPG is critical for expression of cerebral vasodilatation mediated by hypertension (Talman & Nitschke Dragon, 2000), that the dilatation is dependent on arterial baroreflexes (Talman et al., 1994), and that those dilatory influences are themselves mediated by nitric oxide synthesized by local neurons (Talman & Nitschke Dragon, 2004;Talman & Nitschke Dragon, 2007). One published study (Toda et al., 2000) directly tested whether parasympathetic input to cerebral arteries may contribute tonic vasodilatory influences to the tone in those vessels and another tested that possibility in ophthalmic arteries (Ayajiki et al., 2000). In both studies, however, cerebral blood flow (CBF) and cerebral vascular resistance were assessed by measuring pial arterial diameter and concomitant blood pressure and not by a direct measurement of flow. Furthermore, the study of cerebral vessels after removal of the PPG was performed in dogs (Toda et al., 2000). In contrast, some studies in which influences on cerebrovascular tone were assessed after interruption of parasympathetic input to the vessels reported no change in CBF. (Tanaka et al., 1995b;Branston et al., 1995). In that varying influences on CBF also have been appreciated in different species with stimulation of parasympathetic nerves (Busija & Heistad, 1981;Morita-Tsuzuki et al., 1993;Talman et al., 2007), in that inhibition of nitric oxide synthase attenuates the cerebral blood flow response to stimulation of postganglionic parasympathetic nerves in the rat (Morita-Tsuzuki et al., 1993) and in that much of our own work focuses on rat models of cerebrovascular disease, here we tested the hypothesis that parasympathetic innervation provides a tonic vasodilatory influence on cerebral blood vessels in rat. We used laser flowmetry to assess changes in CBF in the parietal lobe of the brain in rats after acute removal of the ipsilateral pterygopalatine ganglion.
All protocols were approved by the institutional animal care and use committees of the University of Iowa and the Department of Veterans Affairs Medical Center, Iowa City and adhered to the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). Adult male Sprague Dawley rats (N= 10) were anesthetized with isoflurane (5% induction and 2-2.5% maintenance) delivered in 100% O2 by nasal cannula to the freely breathing animals. A temperature probe connected to a heating pad was used to maintain body temperature (37°) throughout surgery by means of a temperature controller (YSI Model 73A, Yellow Springs, OH). A cannula (polyethylene, PE-50 tubing) filled with heparinized saline and attached to a transducer and data acquisition system (Power Lab 8/SP; ADInstruments, Colorado Springs) was inserted into a femoral artery for recording arterial blood pressure (AP). The left PPG was exposed with a modification of the method described by Rosen et al (Rosen et al., 1940). The rat’s mouth was kept open with a 1 cm roll of damp cotton gauze inserted between the upper and lower incisors. Doing so moved the temporal muscle posteriorly to aid in exposing the ganglion. The rat’s eye was covered with Triple Antibiotic Ointment (Phoenix Pharmaceutical, St. Joseph, MO). After shaving and preparing the skin with iodine and alcohol we made a 1-1.5 cm incision immediately inferior to the zygomatic arch, exposed the zygomatic bone through blunt dissection, and removed a 1-1.5 cm length of the zygomatic bone with rongeurs. The masseter muscle was then cut and retracted ventrally, to expose the lacrimal gland, which was then retracted dorsally. The maxillary nerve was identified through a microscope, dissected free and retracted dorsally. Using the bony ridge along which the maxillary nerve runs as a guide, the vidian nerve was exposed and the PPG which the nerve enters was isolated. The animal was then placed prone in a stereotaxic frame (DKI Model 1404, David Kopf Instruments, Tujunga, CA). After preparation of the scalp and exposure of the skull a burr hole was made over the parietal lobe ipsilateral to the exposed PPG. The dura was left intact and mineral oil was dripped on it to prevent drying. A laser probe (0.8 mm) mounted in a stereotaxic instrument holder was carefully placed into the mineral oil pool for continuous monitoring of cerebral blood flow (CBF) by a Laserflo Blood Perfusion Monitor (TSI, Model 403A, St. Paul) laser flow meter. Throughout recording of CBF ambient lighting was kept constant. We optimized visualizing the PPG by angling the stereotaxic frame so that the contralateral eye was dependent. As a result, CBF could only be recorded from the cortex ipsilateral to the previously isolated ganglion. The ganglion was again exposed after retraction of surrounding soft tissues. Baseline CBF was established for at least 5 minutes before proceeding with removal of the PPG. Thus, each animal served as its own control. Because we found that touching the ganglion and bony tissues surrounding it often led to artifactual transient increases in CBF with prompt return to basal values, data were collected at baseline before transection and again immediately after transection. Immediately before the resection and during a stable baseline CBF, a sample (0.2-0.25 ml) of arterial blood was removed for blood-gas analysis. All animals freely breathed without ventilatory support throughout the experiments. In that we anticipated that removal of the PPG would lead to a reduction in CBF we chose to accept data if the arterial pCO2 increased after removal of the ganglion (thus attenuating any decrease in flow that might be observed and biasing against our hypothesis). In that pCO2 never fell as a result of ganglionectomy, no parameters were set for such relative hypocarbia. After establishing basal CBF and blood gas values we quickly removed the PPG after cutting its efferent fibers and the vidian nerve as it entered the ganglion. After CBF had achieved a new baseline (typically within 10 minutes of resection) arterial blood was again removed for blood gas analysis.
Data are expressed as mean +/- SEM. CBF measurements are expressed in Laser Doppler (LD) units, a relative quantification of flow. Because of differing basal LD values, changes in CBF were expressed as a percent change from baseline. Data were analyzed by Wilcoxan’s sign test and significance was accepted at a p value ≤ 0.05.
Removal of the PPG caused a gradual 27.9 +/- 7.8 % decrease in CBF (decrease of 13.5 +/- 6.5 laser units; p = 0.047) without changing mean arterial pressure (Fig. 1 and Table 1). Of the 10 animals studied, 9 demonstrated decreased CBF with decreases ranging from 4 LD units (13% fall) to 55 LD units (55% fall). One animal demonstrated a 14 LD unit (18%) increase in CBF after resection. Cerebrovascular resistance, calculated as (mean arterial pressure) ÷ (CBF in LD units), increased (Table 1) after removal of the ganglion from 1.8 +/- 0.3 to 2.9 +/- 0.5 resistance units (p = 0.007). After removal of the PPG, arterial pCO2 increased from 26.7 +/- 3.2 Torr to 40.1 +/- 3.7 Torr (p<0.005) though there was no appreciable change in any animal’s breathing. Throughout the study, arterial pO2 remained greater than 200 Torr in all animals.
This study demonstrates acute vasoconstriction after removal of parasympathetic input to forebrain cerebral arteries and supports a tonic vasodilatory influence by that neural input to the cerebral vasculature. We acknowledge that our study, which did not prohibit an increase in arterial pCO2, likely underestimated that tonic influence. The data are consistent with studies of cerebrovascular regulation in dogs (Toda et al., 2000) but conflict with other studies in rats (Tanaka et al., 1995a). However, in contrast to the latter study that evaluated CBF two weeks after removal of the parasympathetic innervation, the current study evaluated CBF on-line prior to and immediately after transection. Furthermore, the earlier study utilized autoradiographic analysis of CBF and, thus, was limited to one evaluation of CBF. Therefore, it compared CBF in control animals with that in different animals that had lesions. In contrast, the current study utilized intra-animal controls. We cannot say from our work whether vasconstriction was transient and would have cleared within two weeks or if the differences in the two studies reflected use of those different methods for CBF analysis. In that parasympathetic innervation of cerebral vessels is predominantly ipsilateral it is unlikely that any changes in CBF would have been found in the contralateral hemisphere had our methods allowed simultaneous bilateral evaluation of flow. However, again studies vary in their reports of contralateral effects of parasympathetic stimulation with some showing such effects (Seylaz et al., 1988) and others finding no such effects (Toda et al., 2000).
At the moment of ganglionectomy in the current study no nerve fibers other than parasympathetic fibers were transected. It then is unlikely that vasoconstriction seen in this study resulted from an influence of damage to any other nerve inputs to the cerebral vasculature. Vasodilatory trigeminal nerve fibers are quite removed from those of the pterygopalatine ganglion and sympathetic fibers, also distant from the parasympathetic fibers, have further been shown to have little or no influence on CBF in the normotensive range of blood pressures seen in this study (Kelly et al., 1994;Sadoshima et al., 1985;Edvinsson et al., 1993;Heistad et al., 1980).
In addition to vasoconstriction, removal of the PPG led to relative hypercarbia, which would attenuate the vasoconstriction seen here. We have no explanation for the increases in pCO2 that we found. While one study (Hadziefendic & Haxhiu, 1999) suggests that parasympathetic pathways may project to central sites, such as the retrotrapezoid nucleus, (Guyenet, 2008) that are critical for modulating CO2, we know of no study that directly demonstrates such a link.
The current study supports a tonic dilatory influence by parasympathetic nerves on cerebral vessels, but the physiological relevance of that influence remains to be determined. Other studies have suggested physiological implications of the innervation. For example, projections from PPG may participate in linking flow with local neuronal activity (Umemura & Branston, 1995). Other studies have suggested that parasympathetic nerves may provide protection to the brain when exposed to ischemia (Kano et al., 1991;Koketsu et al., 1992;Henninger & Fisher, 2007;Solberg et al., 2008) and we have shown a contribution to vasodilatation during acute hypertension (Agassandian et al., 2003;Talman & Nitschke Dragon, 2000). We have conjectured that parasympathetics to cerebral vessels may also contribute to thermoregulation (Agassandian et al., 2003) by diverting blood flow to the brain while, at the same time, promoting salivation and nasal secretion (Loewy & Spyer, 1990), each a contributor to thermoregulation, especially in rodent species (Stricker, 1970). Parasympathetic influences on cerebral blood vessels may not only play a role in normal physiological function but also may contribute to pathological states. For example, parasympathetic nerves from the PPG may participate in development of cluster headaches, which may respond to blocking function of those same nerves (Kittrelle et al., 1985;Torelli & Manzoni, 2004). The current study suggests that the beneficial effects from blocking those nerves may be mediated through removal of their tonic vasodilatory influences.
This work was supported by VA Merit Review (WTT PI) and in part by NIH R01 HL59593 (WTT PI).
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