Prior studies have shown that removal of vestibular inputs produces lability in blood pressure during orthostatic challenges (Holmes MJ, Cotter LA, Arendt HE, Cass SP, and Yates BJ. Brain Res 938: 62–72, 2002; Jian BJ, Cotter LA, Emanuel BA, Cass SP, and Yates BJ. J Appl Physiol 86: 1552–1560, 1999). Furthermore, these studies led to the prediction that the blood pressure instability results in susceptibility for orthostatic intolerance. The present experiments tested this hypothesis by recording common carotid blood flow (CCBF) in conscious cats during head-up tilts of 20, 40, and 60° amplitudes, before and after the surgical elimination of labyrinthine inputs through a bilateral vestibular neurectomy. Before vestibular lesions in most animals, CCBF remained stable during head-up rotations. Unexpectedly, in five of six animals, the vestibular neurectomy resulted in a significant increase in baseline CCBF, particularly when the laboratory was illuminated; on average, basal blood flow measured when the animals were in the prone position was 41 ± 17 (SE) % higher after the first week after the lesions. As a result, even when posturally related lability in CCBF occurred after removal of vestibular inputs, blood supply to the head was not lower than when labyrinthine inputs were present. These data suggest that vestibular influences on cardiovascular regulation are more complex than previously appreciated, because labyrinthine signals appear to participate in setting basal rates of blood flow to the head in addition to triggering dynamic changes in the circulation to compensate for orthostatic challenges.
- vestibular system
- orthostatic tolerance
- carotid artery
- cerebral blood flow
assumption of an upright posture in humans or a vertical position in quadrupeds can severely challenge normal cardiovascular function. To maintain adequate cerebral perfusion during head-up orientations of the body, peripheral vasoconstriction occurs that restricts blood volume distribution to the lower extremities, thereby maintaining right atrial filling pressure (for review, see Ref. 18). There is evidence to suggest that the vestibular system participates in eliciting alterations in sympathetic outflow during postural changes that serve to maintain stable perfusion of the head. In animals, electrical or natural stimulation of vestibular receptors elicits changes in the activity of sympathetic efferents (23, 25), particularly vasoconstrictor fibers (14, 16). In human subjects, modulation of vestibular nerve activity through head-down neck flexion (7, 20), caloric stimulation of the ear (4), or off-vertical axis rotation (13) also elicits alterations in the firing of vasoconstrictor efferents.
Accordingly, removal of vestibular inputs in animal models was shown to affect the regulation of blood pressure during gravitational challenges. Conscious rats lacking both vestibular and baroreceptor inputs were less able to appropriately adjust blood pressure during gravitational stress produced by centrifugation than vestibular-intact animals with denervated baroreceptors (9). In anesthetized cats, bilateral transection of the eighth cranial nerves resulted in orthostatic intolerance during head-up tilts of the animal's body (5). However, a long-term study of conscious cats revealed that removal of vestibular inputs produces complex effects on the regulation of blood pressure (11). Acutely after bilateral eighth nerve transections, all animals showed blood pressure instability during 60° head-up tilts, but this occurred only when tested under conditions where they were also deprived of visual cues that might indicate body position in space (11). Nonetheless, after 1 wk the animals regained the ability to accurately adjust blood pressure during postural alterations, even when tested in the dark. Subsequent studies have suggested that the recovery of the ability to rapidly adjust blood pressure during postural alterations subsequent to vestibular lesions may be due to the fact that regions of the caudal vestibular nuclei that mediate vestibulosympathetic responses process both labyrinthine and nonlabyrinthine signals reflecting body position in space (12, 24). After vestibular inputs are removed and plastic changes occur in the vestibular nuclei, nonlabyrinthine signals may be sufficient to elicit responses that were previously triggered by alterations in eighth nerve activity (22).
Although previous studies have surmised that loss of vestibular inputs could lead to inadequate perfusion of the head when the body is reoriented from a horizontal to a vertical position (11), this assumption has not been tested directly, which was the major objective of the present study. For this purpose, blood flow through a common carotid artery of conscious felines was recorded as the animals were rotated head up from the horizontal plane by 20, 40, or 60°, before and after the surgical elimination of vestibular inputs. In particular, these experiments examined two hypotheses. The first hypothesis is that common carotid artery blood flow (CCBF) remains stable during head-up rotations of labyrinth-intact animals. The second hypothesis is that eighth nerve transections result in diminished CCBF during orthostatic challenges produced by head-up rotations of the body.
All experimental procedures conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the University of Pittsburgh's Institutional Animal Care and Use Committee. Data were collected from six purpose-bred adult female cats obtained from Liberty Research (Waverly, NY). Animals were spayed before being included in this study to eliminate cyclic changes in hormonal levels.
Overview of data collection procedures.
The animals were trained over a period of 1–2 mo to remain sedentary, with hindlimbs fully extended, on a tilt table during head-up whole body rotations of 20, 40, or 60° amplitudes. A jacket with attached Velcro straps was placed around the animal's torso; the straps were secured to the sides of the tilt table to prevent the animal's position from shifting during tilting. The animal's head was immobilized by inserting a screw into a bolt mounted on the skull. Blood pressure was recorded during whole body tilts by using a telemetry system, including a transducer and attached transmitter (Data Sciences International, St. Paul, MN) implanted in the animal. The transducer tip was placed into the abdominal aorta, and the transmitter unit was secured in the abdomen. The radio signal generated by this transmitter was detected with the use of a receiver mounted under the tilt table. Blood flow through the left common carotid artery was also recorded during whole body tilts using a perivascular probe (Transonic Systems, Ithaca, NY).
Blood pressure and CCBF were monitored during body tilts under two conditions. During some trials, the lights in the laboratory were illuminated, such that an animal could potentially employ visual cues to determine its body position in space. During other trials, the laboratory was darkened and black cardboard was mounted to the front and sides of the tilt table so that the animal's visual field rotated with its body. Under this condition, an animal was unable to use visual information to determine the orientation of its body.
Data were collected during recording sessions lasting 30 min. Head-up 20, 40, and 60° tilts were randomly disbursed throughout the recording sessions so that animals could not anticipate the amplitude of the next rotation. Tilts persisted for 45 s and were separated by at least 60 s. The tilt table was rotated manually and was secured in the tilted position using a locking device that permitted movement to one of the three predetermined tilt positions. Rotations from the Earth-horizontal to the head-up position were performed rapidly, at a velocity of ∼30°/s at all three amplitudes, to produce a sudden orthostatic challenge. Data collected during trials in which animals vocalized or failed to remain stationary were discarded. Blood pressure and CCBF responses to tilt were recorded in animals with intact eighth cranial nerves over a period of 51–71 days (median of 58 days). Experimental sessions were typically conducted 5 or more days per week, and two recording sessions were usually performed each day. Subsequently, vestibular inputs were surgically eliminated; data recording sessions resumed the day after the surgery and continued for 30 days (although data collection was discontinued in one cat at 10 days after the lesions, due to an injury sustained by the animal that required veterinary intervention including the use of analgesics that could have affected cardiovascular regulation).
Two recovery surgeries were required for each animal. Both surgeries were performed in a dedicated operating suite with the use of sterile procedures. Animals were initially anesthetized by an intramuscular injection of ketamine (20 mg/kg) and acepromazine (0.2 mg/kg). Subsequently, an endotracheal tube and intravenous catheter were inserted. Anesthesia was supplemented as necessary by using 1–1.5% isoflurane vaporized in O2 to maintain areflexia and a stable heart rate. Ringer lactate solution was infused intravenously to replace fluid loss during the surgery. A heating pad and heat lamp were used to maintain core temperature near 38°C.
The first surgery was performed to mount a fixation plate to the skull and to implant the blood pressure transducer and perivascular probe. To implant the blood pressure transducer, an incision was made in the right hindlimb to expose the femoral artery. A small opening was then made in the arterial wall so that the transducer's tip could be introduced and advanced 3–5 cm rostrally into the abdominal aorta. The incision in the arterial wall was closed using Vetbond tissue adhesive (3M, St. Paul, MN), and a ligature was employed to secure the transducer to the artery. An incision was made through the abdominal wall, and the transmitter unit was passed subcutaneously, deep to the inguinal ligament, into the peritoneal cavity and secured to the abdominal muscles using sutures. The perivascular probe was implanted through a longitudinal incision on the left ventrolateral aspect of the animal's neck. Blunt dissection through the plane of the sternothyroid and sternohyoid muscles provided access to the common carotid artery. Care was taken not to disturb surrounding tissue while the perivascular probe was placed around the artery and secured in place using sutures. The cable from the probe was routed subcutaneously, and the connector was attached using dental cement to the skull behind the fixation bolt. Animals recovered for at least 3 wk after this surgery before data collection was initiated.
A second surgery was performed after initial data collection was complete to eliminate vestibular inputs. For this purpose, the tympanic bulla on each side of the skull was opened using a ventrolateral approach to expose the cochlea. A drill was used to remove temporal bone near the base of the cochlea, thereby producing a labyrinthectomy that rendered the vestibular apparatus dysfunctional. This procedure also provided access to the internal auditory canal. The eighth cranial nerve was transected under microscopic observation within the internal auditory canal. Thus two independent lesions affecting the vestibular system were made on both sides to ensure that vestibular inputs were eliminated. In no case did nystagmus or a tonic deviation in eye position occur after the surgery, suggesting that the peripheral lesions were complete. To ensure that animals received proper hydration and nutrition during the postsurgical period, ∼100 ml of Ringer lactate solution were administered subcutaneously each day and feeding was done by hand until the animal's spontaneous consumption of food and water returned to prelesion levels. After all data recording was completed, animals were euthanized with the use of an overdose of pentobarbital sodium followed by transcardial perfusion with saline.
Data recording and analysis.
During recording sessions, a cable was used to connect the perivascular probe to a Transonics Systems TS420 perivascular flow module, which provided instantaneous volume flow measurements. Both the pulsatile volume flow and mean volume flow outputs from this unit were recorded digitally using a Cambridge Electronic Design (Cambridge, UK) 1401-plus data collection system interfaced with a Macintosh (Apple Computer, Cupertino, CA) G4 computer. A voltage proportional to blood pressure produced by the Data Sciences International telemetric blood pressure system was also digitized. In addition, the voltage supplied by a potentiometer mounted on the tilt table provided for a recording of table position. All signals were sampled at 100 Hz. An example of the data recorded is shown in Fig. 1.
The Spike-2 software package (Cambridge Electronic Design) was used for data analysis. Each data file considered a 60-s interval, consisting of the 15-s period before the onset of each tilt and the 45-s period during which the animal was rotated head-up. For each 1-s bin in this interval, the following parameters were determined: mean systolic blood pressure, mean diastolic blood pressure, and mean CCBF. Mean arterial pressure was calculated using the following formula: [(·diastolic blood pressure) + (·systolic blood pressure)]. Resistance of the head vasculature was calculated by dividing mean arterial pressure by CCBF. Typically, 32 successful trials were conducted per day per animal, with 16 performed in the dark and 16 executed when the laboratory was illuminated. Approximately one-third of the tilts were performed at each of the three amplitudes. The mean values obtained for identical trials performed on a particular day (i.e., at a particular rotation amplitude and laboratory lighting condition) were pooled to generate an average daily curve of mean arterial pressure and CCBF responses to that testing condition. Values obtained from these average daily curves were employed for statistical analyses, which considered the effects of rotation amplitude, the availability of visual information reflecting body position in space, and vestibular lesions on mean arterial pressure and CCBF determined for the following time points subsequent to the onset of tilts: 2, 4, 6, 8, 10, 15, and 40 s. In addition, measurements taken at 14 and 5 s before each tilt were analyzed to ascertain the effects of laboratory lighting conditions and vestibular lesions on baseline mean arterial pressure and CCBF. Table 1 summarizes the number of average daily curves that provided the data for analyses. A prior study reported that compensation for the effects of vestibular lesions on regulation of blood pressure during postural alterations occurred after 1 wk (11). For this reason, data recorded in the first week after the removal of labyrinthine inputs and in subsequent weeks were considered as separate groups during analyses.
Statistical analyses were performed using SPSS Version 11 software (SPSS, Chicago, IL). Data from each animal were considered separately. Statistical significance was set at P < 0.05, and pooled data are presented as means ± SE. To identify the effects of tilt amplitude on CCBF in labyrinth-intact animals, multivariate analyses of variance were performed, which considered blood flow at each time point vs. tilt amplitude; post hoc tests [least significant difference (LSD)] established whether CCBF differed during 40 and 60° tilts from that during 20° tilts. To determine the effects of visual information and vestibular lesions on baseline CCBF, univariate analyses of variance were performed, with the presence of vestibular lesions (prelesion, first week postlesion, subsequent postlesion period) serving as the dependent variable. Separate analyses were executed for data recorded when the laboratory was illuminated, and those recorded when the laboratory was dark; post hoc tests (LSD) were performed to elucidate the effects of vestibular lesions on baseline CCBF. Similar analyses were executed to indicate the effects of vestibular lesions on baseline mean arterial pressure and resistance of the head vasculature. To ascertain whether vestibular lesions resulted in lability in blood flow to the head during postural alterations, the percent change in CCBF from baseline resulting from 60° head-up tilts was considered. Specifically, a multivariate analyses of variance combined with LSD post hoc tests compared the percent change in CCBF from baseline at each time point, for trials conducted before removal of vestibular inputs, trials performed in the first week after the vestibular neurectomy, and trials carried out subsequently.
Effects of postural alterations on CCBF in animals with an intact vestibular apparatus.
Figure 2 depicts the effects of head-up tilts on CCBF in each animal before removal of vestibular inputs. Separate columns illustrate data collected when the laboratory was dark and data gathered when the laboratory was illuminated. In five of the six animals, CCBF decreased slightly (<5%) during the first few seconds after the animal was rotated head up, but then it rebounded to a slightly higher level than baseline. Similar changes in CCBF occurred when animals were tested in the dark as when recordings were performed in an illuminated laboratory, and the alterations in blood flow during 40 and 60° tilts were not significantly different from those resulting from 20° tilts. Only in animal 2 did a modest perturbation in CCBF (∼10% decrease) occur at the onset of 60° head-up rotations, although blood flow recovered to baseline levels within 8 s after the onset of the rotation. Figure 3 shows the changes in CCBF from pretilt levels for all animals combined, and it indicates that, on average, blood flow to the head decreased <5% at the onset of 60° head-up tilts when vestibular inputs were present.
Effects of removal of vestibular inputs on baseline CCBF.
Baseline CCBF was determined from measurements taken at 5 s before each tilt. To demonstrate that baseline blood flow was sufficiently stable to employ a single time point for analyses, we also compared measurements taken at 14 and 5 s before each tilt. As shown in Table 2, blood flows ascertained at the two times preceding tilts were always nearly identical, suggesting that baseline CCBF ascertained at a single time point was adequate for our purposes.
The effects of removal of vestibular inputs on baseline CCBF measured at 5 s before each rotation of the animals are shown in Fig. 4. Removal of vestibular inputs caused baseline CCBF to increase significantly in five of the six animals (see Fig. 4). In four of the five cases where baseline CCBF increased after the vestibular neurectomy, blood flow remained elevated throughout the survival period; in the remaining animal, recordings were discontinued after 10 days such that the long-term consequences of the lesions on CCBF were not ascertained. The same trends are evident in Fig. 5A, which shows the effects of vestibular lesions on average baseline CCBF of all animals combined. Figure 6 provides further details regarding the effects of vestibular lesions on baseline CCBF in two animals. Each symbol on this figure depicts the average baseline blood flow ascertained 5 s before tilts were delivered at each amplitude on every day recordings were performed; filled circles depict baseline blood flows measured in the dark, whereas open squares depict baseline blood flows measured when the laboratory was illuminated. Thus six symbols are typically plotted for every day that recordings were conducted, as three tilt amplitudes were delivered in the two laboratory lighting conditions. These examples illustrate that an abrupt change in baseline CCBF resulted from the removal of vestibular inputs. Animal 6 is the only case where baseline CCBF did not increase after the elimination of labyrinthine signals; in this cat, blood flow to the head decreased slightly from prelesion values, but this occurred only when the animal was in the dark.
Figures 4 and 5A also indicate that removal of vestibular inputs altered the effects of laboratory lighting conditions on baseline CCBF. When the vestibular system was intact, baseline blood flow was either slightly higher when the animals were in the dark than when the laboratory was illuminated (n = 3) or CCBF was not statistically different between the two conditions (n = 3). In contrast, subsequent to the bilateral vestibular neurectomies in five of the six cases, baseline CCBF was higher when the animals were in the light than when they were in the dark. In two of the five cases where removal of vestibular inputs resulted in a significantly higher CCBF when the animals were in the light, this effect was evident during the first week after the lesions; in the other three cases, the effect was only significant subsequent to a 1-wk postlesion recovery period.
Figure 7 shows the effects of vestibular neurectomies on mean baseline blood pressure in most of the animals; data are not available for animal 2 or for animal 1 after 1 wk after removal of vestibular inputs, due to failure of the blood pressure transducer. A comparison of Figs. 4 and 7 reveals that in most cases the postlesion increase in blood flow through the carotid artery was not simply due to increased perfusion pressure. For example, in animals 4 and 5, removal of vestibular inputs resulted in a large increase in CCBF when the animals were in an illuminated laboratory, although mean baseline blood pressure was not significantly different from that before removal of vestibular inputs. In animal 3, vestibular lesions produced a large increase in baseline CCBF when the animals were in the dark, which was accompanied by a decrease in mean blood pressure. Because the consequences of removal of vestibular inputs on CCBF cannot be explained simply by a change in perfusion pressure, these data suggest that the lesions affected specific mechanisms that regulate blood flow to the head. This conclusion is further supported by Fig. 5B, which indicates the effects of vestibular lesions on the average resistance of the head vasculature of all animals combined. This figure shows that across the cases, resistance was lower in head blood vessels after removal of vestibular inputs than when labyrinthine signals were present.
Effects of removal of vestibular inputs on CCBF during head-up tilts.
Figure 8 illustrates the combined effects of a bilateral vestibular neurectomy and 60° head-up rotations on blood flow through the common carotid artery. To simplify comparisons, blood flow changes are plotted relative to those that occurred before vestibular lesions; in addition, because removal of vestibular inputs substantially altered baseline CCBF, the data shown in Fig. 8 were normalized to the baseline flow ascertained before each tilt. When most animals were in the dark, the percent changes in CCBF relative to baseline during 60° head-up tilts were not significantly different after removal of vestibular inputs than when the labyrinths were intact. During 60° head-up tilts delivered in an illuminated laboratory subsequent to vestibular lesions, in two animals (animals 3 and 4) CCBF dropped significantly more relative to baseline during than when labyrinthine inputs were present. However, in both of these cases, removal of vestibular inputs also produced an increase in baseline CCBF (see Fig. 4) that was larger in magnitude than the tilt-related lability in blood flow to the head. Figure 9 compares absolute CCBF during 60° head-up tilts before and subsequent to the removal of labyrinthine inputs. Note that blood flow measured after lesions (indicated by dashed lines) was equal to or greater than flow ascertained before lesions (indicated by a solid line). The only exception is animal 6, where CCBF was decreased 10–14% from that when vestibular inputs were intact, but this occurred only during tilts conducted in the dark in the first week after lesions.
The major finding of this study was that baseline CCBF increased significantly after the removal of vestibular inputs, particularly when the laboratory was illuminated. Although prior experiments have established that bilateral damage to the peripheral or central vestibular system produces lability in blood pressure during orthostatic challenges (5, 10, 11, 17), the present findings show that removal of vestibular inputs does not result in diminished blood flow to the head during 60° tilts in the pitch plane. Although the hypothesis that a bilateral vestibular neurectomy produces susceptibility for orthostatic intolerance was not supported, the present findings suggest that labyrinthine signals participate in regulating baseline head blood flow. These data also provide a mechanism to account for the common complaint of patients with vestibular disorders of increased “pressure in the head” (1, 8); this sense of pressure could be the end result of increased blood flow through head blood vessels.
Several lines of evidence suggest that the increases in CCBF after a bilateral vestibular neurectomy were the consequence of alterations in the activity of neural efferent pathways innervating blood vessels in the head. First, in most cases, the increases in blood flow through the common carotid artery were not related simply to alterations in blood pressure, as was demonstrated by a decrease in the resistance of the head vasculature. More importantly, the blood flow increases were most prominent when the laboratory was illuminated. If a nonspecific mechanism (e.g., discomfort or inflammation related to the inner ear surgery) was responsible for the alterations in CCBF after vestibular lesions, then similar blood flows would be expected when the animal was in the dark as in the light. In addition, in most cases, the increases in blood flow persisted for many weeks, and it did not rapidly return to baseline as the animals recovered from the bilateral labyrinthectomy. The simplest explanation for these data is the presence of a neural mechanism that integrates vestibular, visual, and perhaps other sensory inputs. This mechanism regulates baseline flow through head blood vessels, such that the neural drive to produce vasoconstriction decreases if labyrinthine signals are eliminated, particularly when visual information regarding the environment is available. The existence of such a mechanism is supported by the fact that the increases in baseline CCBF observed after removal of vestibular inputs (average of 41 ± 17%) is comparable to the change in cerebrovascular resistance resulting from stimulation of the cut end of the cervical sympathetic chain (2). The large effects of vestibular lesions on blood flow through the carotid artery raise the possibility that labyrinthine signals play an appreciable role in regulating sympathetic outflow to head blood vessels, although this notion is yet to be tested experimentally. It is also feasible that removal of vestibular inputs resulted in vasodilatation of cerebral blood vessels by altering tonic activity of perivascular nerve fibers that regulate the resistance of the cerebrovascular bed (6). Further studies will be required to ascertain which particular neural pathways regulating blood flow to the head receive tonic influences from the vestibular system.
Studies on human subjects have provided contradictory evidence as to whether vestibular stimulation affects cerebral blood flow (3, 19, 21), and no study has suggested that the vestibular system plays a prominent role in regulating cerebral vascular resistance. There are several possible explanations for the differences in findings between our study and experiments conducted in humans. First, the studies on human subjects employed head-down neck flexion to stimulate vestibular receptors, which should have elicited a modest change in the activity of a subset of vestibular afferents. In contrast, we performed a vestibular neurectomy, which completely eliminated the firing of labyrinthine afferents. Thus the alterations in vestibular inputs induced during our study were much larger than those produced during prior experiments in humans. Furthermore, head down neck flexion is a complex stimulus, which can activate many different sensory receptors, including neck proprioceptors, baroreceptors, and receptors in the airway. It is feasible that vestibular stimulation resulting from head-down neck flexion does alter cerebral resistance but that this effect is offset by changes in head blood flow due to activation of other sensory receptors. Additional experiments will thus be required to ascertain whether the vestibular system contributes to regulating the resistance of the cerebrovascular bed in humans.
In summary, although several previous studies have suggested that bilateral vestibular lesions can produce orthostatic intolerance (5, 10, 11, 17), the present data demonstrate that removal of vestibular inputs does not compromise CCBF during the orthostatic challenge provided by 60° head-up tilts. Instead, a bilateral vestibular neurectomy typically resulted in tonic increases in blood flow to the head, particularly when animals were also provided visual information regarding their environment. These data show that vestibular influences on cardiovascular regulation are more complex than previously appreciated, in that labyrinthine signals appear to participate in setting basal rates of blood flow to the head in addition to triggering dynamic changes in the circulation to compensate for orthostatic challenges. The functional significance of regulation of baseline blood flow to the head by the vestibular system remains to be elucidated. Even so, these findings support the hypothesis raised in previous studies (14–16) that vestibular influences on the sympathetic nervous system are patterned and can induce distinct effects in different vascular beds.
This work was supported by National Institute of Deafness and Other Communications Disorders (NIDCD) Grant R01 DC-00693 (to B. J. Yates). Core support was provided by NIDCD Grant P30-DC-05205.
The authors thank Katie Wilkinson and Brian Sadacca for valuable technical assistance in the completion of these studies.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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