Background and Purpose Increases in sympathetic activity and frequency of myocardial damage occur after middle cerebral artery occlusion (MCAO) in Wistar rats, while MCAO in the spontaneously hypertensive rat (SHR) decreases sympathoadrenal activity. Autonomic changes have been suggested to result from damage to the insular cortex (IC).
Methods A lesion of the IC was made using the excitotoxin d,l-homocysteic acid (DLH; 1 mol/L), in urethane-anesthetized Wistar rats and SHRs. Mean arterial pressure (MAP), heart rate, renal sympathetic nerve discharge (SND), ECG, and plasma catecholamines were measured in 14 SHRs and 14 Wistar male rats after a 500-nL injection of DLH or phosphate-buffered saline (PBS) into the IC.
Results Histological examination showed that DLH resulted in neuronal damage throughout the IC. DLH injection initially elevated MAP (at approximately 10 minutes after injection) in Wistar rats but not in SHRs. At 4 hours after the DLH injection, there was a secondary, longer-term increase in MAP in the Wistar rats. MAP decreased in the SHRs after IC lesion such that at 6 hours, lesioned SHRs had a MAP that was significantly lower than that of sham-lesioned SHRs. SND initially increased (at 10 minutes) after DLH injection in Wistar rats. In the SHRs, SND decreased significantly from the initial values, by 3 hours after DLH injection. Plasma catecholamine levels were not significantly changed as a result of IC lesion in the Wistar rats or the SHRs. Heart rates increased in all animals, with no differences between groups. There were no changes in the ECG or in the frequency of cardiac myocytolysis in either strain (sham or lesioned animals).
Conclusions IC lesion in the SHR and Wistar rat therefore appears to result in autonomic changes similar to that seen after MCAO. Unlike MCAO, however, the autonomic changes do not appear to be sufficient to produce myocardial damage.
Considerable evidence has linked hemispheric stroke to cardiac and other autonomic abnormalities.1 2 3 4 5 Stroke models involving middle cerebral artery occlusion (MCAO) in the cat and Wistar rat have demonstrated elevated plasma levels of epinephrine and norepinephrine and sympathetic nerve activity as well as acute myocardial damage.6 7 8 9 The increase in sympathetic activity may have mediated the observed cardiac changes. The autonomic consequences of MCAO also were examined in the spontaneously hypertensive rat (SHR), since hypertension is a major risk factor for stroke.10 11 12 The pattern of autonomic changes was quite different from those observed in Wistar rats. MCAO in the SHR resulted in a decrease in mean arterial pressure and sympathoadrenal activity, with no adverse cardiac effects.13
The insular cortex (IC) may mediate the autonomic changes resulting from acute stroke. While the infarcts in the previous stroke studies in the cat and rat included the insula, they also extensively involved other areas of cortex. A previous investigation in the cat suggested that increases in plasma catecholamines are observed only if the IC is included in the ischemic zone.7 In addition, the IC is a site of limbic and autonomic integration.14 15 16 Autonomic and cardiac effects can be elicited by both electrical and chemical stimulation of the insula.17 18 19 20 21 22
Previous investigations have indicated that exogenous excitatory amino acids will result in a selective lesion of cell bodies in the central nervous system.23 Homocysteate also has been shown to be toxic to cortical neurons.24 In the present study, unilateral lesions of the IC were made using injections of the excitatory amino acid d,l-homocysteate (DLH) to determine if damage to the IC is responsible for the observed autonomic effects of MCAO in the SHR and Wistar rat. A preliminary report of this investigation has been presented in abstract form.25
Materials and Methods
Fourteen SHRs and 14 Wistar rats between 18 and 22 weeks old were used. Food and tap water were provided ad libitum, except that food was removed approximately 12 hours before surgery.
General Surgical Methods
The rats were anesthetized with urethane (1.5 g/kg IP), and core temperature was maintained at 37°C using a rectal thermometer, temperature controller, and heating pad. The right femoral artery was cannulated with PE-50 tubing, filled with heparinized saline. Pulsatile arterial pressure was measured using a Statham P23D transducer and was continuously monitored on a Grass model 7 polygraph. Mean arterial pressure (MAP) was recorded on the polygraph by filtering the pulse pressure with a 0.5-Hz, high-frequency filter. Heart rate was determined using a Grass 7P44 tachograph and was monitored on the polygraph. The right femoral vein was catheterized with PE-10 tubing for the administration of drugs.
The animal received a tracheostomy and was placed in a Kopf stereotaxic apparatus. The ECG was monitored using the lead II configuration. The ECG signal was continuously displayed on a Hameg model 205 oscilloscope and stored on a microcomputer for measurement of segments, using an analysis program for electrophysiological experiments (Conrad Yim Software).
The right kidney was exposed using a retroperitoneal approach. With the aid of a Zeiss operating stereomicroscope, the renal nerve branches were isolated from the surrounding tissue, and a loose ligature was placed around one of them. A bipolar silver electrode was used to record nerve activity. The electrode was secured to the nerve, using a dental impression material (Perfourm, Miles Laboratories, Inc). The multi-unit nerve activity was amplified and filtered (100 Hz to 3 kHz; Grass model P15 preamplifier; Neurolog NL100 amplifier). The signal was then fed into the oscilloscope and an audio monitor (Grass, model AM8C). In addition, the rectified and integrated nerve activity was continuously monitored on the polygraph (Grass, model 7P10). Before proceeding, the nerve was tested for a reflex decrease in sympathetic activity with an infusion of phenylephrine (40 to 60 μg/kg IV). The background noise levels were determined with hexamethonium (2 mg/kg IV) at the conclusion of the experimental period.
The cortex dorsal to the right insula was exposed through a small burr hole to allow insertion of a single glass capillary micropipette into the insular cortex.
After completion of surgery, the rat was allowed to stabilize for 30 minutes. The right insular cortex was stimulated electrically (2 milliseconds, 500-μA pulses at 50 Hz for 10 seconds) using a micropipette filled with 3.0 mol/L NaCl. The insula was stimulated at 500-μm increments as the pipette was stereotaxically lowered, until a cardiovascular pressor or depressor site was found. In 8 of the rats from each strain, the micropipette was then raised and filled with 1 mol/L d,l-homocysteic acid (DLH). The micropipette then was lowered to the active site, and 500 nL of DLH was slowly injected into the insula over a 45-second period. This relatively large volume was used in order to obtain as complete a lesion of the IC as possible. In the remaining animals, 500 nL of phosphate-buffered saline (PBS) was injected into the insular cortex. Cardiovascular and autonomic variables were continuously monitored for the following 6 hours.
Arterial blood samples (1.0 mL) were taken 30 minutes before the DLH or PBS injection and at the completion of the 6-hour experimental period. Blood was collected in tubes containing 10 μL of EGTA. The plasma was separated from the cells by centrifugation and transferred into microtubes, sealed, and frozen at −70°C. Plasma norepinephrine and epinephrine levels were determined by reverse-phase high performance liquid chromatography with electrochemical detection, as described previously.26
The animals were perfused via the aorta with 0.9% saline and fixed with formalin. The hearts and brains then were removed. The brains were sectioned (50 μm) with a freezing microtome. Sections were thionin stained and examined with a microscope (Leitz Diaplan), and all pipette tracks/regions of cell damage were drawn using a camera lucida attachment. The heart from each animal was sectioned transversely across the ventricles and embedded in paraffin blocks, which were cut with a microtome (30 μm) and stained with hematoxylin and eosin. A cardiac pathologist, blind to the strain of the rats, examined the hearts for any histological abnormalities.
Statistical comparisons were made using repeated-measures ANOVA and either Tukey’s post hoc test or a paired t test. Comparisons between strains were made using an unpaired t test. A χ2 test was used to compare the frequency of myocardial damage between groups. For all tests, a P value of less than .05 was considered to indicate significance. Values in the results are expressed as mean±SEM.
Changes in Arterial Pressure and Heart Rate
Injection of DLH resulted in an early, significant increase in MAP that peaked after approximately 10 minutes in the Wistar rats (Fig 1A⇓). MAP returned to preinjection levels by 30 minutes. At 4 and 6 hours after the DLH injection, MAP was again significantly higher than its initial value (Fig 1A⇓). In addition, MAP of IC-lesioned Wistar rats was significantly higher than that of sham rats by 4 hours after injection. MAP did not change significantly in the Wistar sham group throughout the experiment (Fig 1A⇓).
Unlike Wistar rats, SHRs did not show a significant change in MAP immediately after DLH injection (Fig 1B⇑). MAP of IC-lesioned rats did, however, steadily decline for the duration of the experiment, such that it was significantly lower than the MAP of sham SHR rats, by 4 hours after injection (Fig 1B⇑). The MAP of sham-lesioned SHRs was significantly higher than it was initially, by 4 hours.
Initial heart rates were significantly higher in Wistar rats (375±11 beats per minute [bpm] than in SHRs (319±10 bpm). Heart rates rose significantly in all four groups of animals, beginning at 1 to 3 hours after injection into the IC. At 6 hours, the increases in lesioned and sham Wistar rats were 26±9 and 47±14 bpm, respectively. Similarly, HR increased in lesioned SHRs and sham rats by 29±8 and 60±10 bpm, respectively. There were no significant differences in HR between sham and experimental animals in either strain at any point during the experimental period.
Changes in Sympathetic Nerve Discharge
DLH injection in Wistar rats resulted in a significant increase in renal sympathetic nerve discharge compared with rats injected with PBS 10 minutes after DLH administration (Fig 2A⇓). The level of sympathetic nerve discharge was not significantly different from that of sham rats for the duration of the experimental period (Fig 2A⇓).
There was no significant early (10 minutes) change in renal sympathetic nerve activity in the SHRs after DLH injection (Fig 2B⇑). At 4 hours after injection, sympathetic activity levels were significantly lower (20±6 μv.s) than initial values (42±12 μv.s) and remained significantly lower for the duration of the experiment (16±5 μv.s at 6 hours). In addition, the change in nerve activity was significantly different from that in spontaneously hypertensive, sham-lesioned rats at 1 and 2 hours after DLH injection (Fig 2B⇑).
Renal nerve discharge showed no changes after PBS injection and was stable for the entire 6-hour experimental period in both sham groups of Wistar rats and SHRs (Fig 2⇑).
Changes in Plasma Catecholamines
Initial plasma norepinephrine and epinephrine levels were found to be 2427±493 pg/mL and 2328±344 pg/mL, respectively, in the Wistar rats (combined sham and IC-lesioned animals). SHRs (combined sham and IC-lesioned animals) had initial plasma norepinephrine and epinephrine levels of 1744±553 pg/mL and 1888±194 pg/mL, respectively. Plasma levels of norepinephrine and epinephrine were not significantly changed at 6 hours in SHRs or Wistar rats as a result of IC lesion (Fig 3⇓). Plasma norepinephrine was greater than the initial value in 4 of the 6 IC-lesioned Wistar rats (50% to 400% increases). One IC-lesioned Wistar rat showed no change in plasma norepinephrine levels, and a 30% decrease was observed in the remaining animal. The changes in norepinephrine levels showed a wide variance (Fig 3A⇓). IC-lesioned SHRs appeared to exhibit a slight decrease in plasma epinephrine relative to SHR shams, although this was not a significant change (Fig 3B⇓).
There were no significant changes in PR or QRS intervals in any group. QT intervals were also unaffected by the lesion of the IC in SHRs or Wistar rats (Table⇓).
Occasional myocytolytic fibers were seen in the hearts of some animals in all four groups (Wistar lesion, 2; Wistar sham, 1; SHR lesion, 4; SHR sham, 3). These acute changes were very slight and infrequent. χ2 analysis showed that the frequency of myocardial damage did not differ between animals with IC lesions and sham rats in either strain. In addition, 4 SHRs (2 sham and 2 IC-lesioned rats) showed evidence of mild myocarditis. This damage was considered to be of a long duration and not related to the experimental protocol.
Insular Cortex Damage
DLH injection resulted in extensive neuronal damage throughout the IC of SHRs and Wistar rats. Lesions were evident with thionin staining (Fig 4⇓). All six layers of the IC were included in the lesion (Figs 4⇓, 5⇓, and 6⇓). Portions of the dorsal granular IC were left intact in some animals (Figs 5⇓ and 6⇓). There were no differences between the two strains (Figs 5⇓ and 6⇓). The 500-nL DLH injections also routinely resulted in damage to the dorsal aspect of the piriform cortex, dorsal endopiriform nucleus, and claustrum. In 3 SHRs and 2 Wistar rats, portions of the striatum and basolateral amygdala also were damaged. In the sham rats, PBS injections had no apparent effect on the cells of the cortex.
Although urethane decreased MAP, it did provide stable long-term anesthesia. In addition, urethane was used previously to develop an effective stroke model in the Wistar and SHR.8 13 To compare the results of the previous stroke studies with those of the present experiment, urethane was chosen as the most appropriate anesthetic agent. HR increased significantly in all four groups of animals. This therefore appears to be an additional effect of urethane anesthesia itself and is consistent with previous observations in our laboratory.13
Initial Effects of DLH Injection
The initial response to DLH injection in the Wistar rats was an increase in MAP and renal sympathetic nerve activity. Previously, it has been shown there are pressor/sympathoexcitatory and depressor/sympathoinhibitory regions within the insular cortex of the Wistar rat.18 20 In this investigation, the effect of a 500-nL DLH injection, which would probably stimulate both regions, was always a pressor response, accompanied by sympathoexcitation in the Wistar rats. It therefore appears that the effect of stimulation of neurons inducing pressor/sympathoexcitatory responses dominates that of the region containing depressor/sympathoinhibitory neurons. This has been confirmed in a separate study in our laboratory.27 The initial response to DLH stimulation in the SHR was nonsignificant and more variable than that of the Wistar rats (Fig 1B⇑). This inability to obtain significant pressor/sympathoexcitatory responses in the IC of the SHR also has been confirmed in our laboratory.27 In that study, smaller injections (200 nL) of DLH into the IC of SHRs anesthetized with propofol (2,6 isopropylphenol) resulted in similar responses to those seen in the present investigation.27 These initial effects of DLH injection into the IC of Wistar rats and SHRs are presumably due to an excitatory action at glutamate receptors.28
Long-Latency Autonomic and Cardiovascular Changes
In addition to excitation, injection of excitatory amino acids such as DLH has been shown to result in cellular damage in neurons.23 24 The resulting lesion of the IC in this study also induced autonomic changes. Similarly, MCAO in the Wistar rat as well as in the SHR has been shown to result in an infarct that includes the IC.8 9 13 Furthermore, MCAO has been shown to result in autonomic and cardiac changes similar to those seen in the clinical stroke population.8 9 In the present experiments, the autonomic changes appeared to correlate with those seen after MCAO in Wistar rats and SHRs.
MCAO in the Wistar rat results in a significant increase in renal sympathetic nerve activity.8 In addition, MAP was maintained at a level that was higher than that of sham MCAO Wistar rats.8 In the present experiment, a lesion of the IC, using DLH, in Wistar rats resulted in an increase in MAP. While renal sympathetic nerve discharge was not significantly increased at the end of the experiment, it did appear to remain higher than that of shams. The pattern of sympathetic nerve discharge and MAP changes therefore appears to be similar for IC lesion and MCAO.
MCAO in Wistar rats also has been shown to result in an increase in plasma norepinephrine.8 It appears, however, that an IC lesion alone is insufficient to cause a significant change in plasma catecholamine levels in the Wistar rat. MCAO results in more extensive damage to the IC and regions of the cortex beyond the insula.8 13 Thus, the catecholaminergic changes resulting from MCAO may be due to more extensive damage of the insula and/or inclusion of other cortical areas within the ischemic zone.
A lesion of the IC in the SHR appears to result in a reduction in renal sympathetic drive in the SHR. Renal sympathetic tone decreased significantly as a result of IC lesion in the SHR. This indicates that IC damage in the SHR results in changes in tone that are specific to the renal and possibly other sympathetic nerves. This fall in sympathetic activity may be related to the observed moderate fall in blood pressure. While MAP was not significantly lower than its initial value in the IC-lesioned SHR, it did decline and was significantly lower than that of the sham animals at 4 hours after DLH injection. The moderate fall in MAP that was observed in the present study may be related to renal factors. The influence of the renal sympathetic nerves on the renin-angiotensin system has been well documented and is summarized in a recent review.29 The decrease in renal sympathetic activity seen in the present investigation may have resulted in the decrease in MAP.
MAP rose toward the end of the experimental period in the SHR shams. This may be due to a gradual decrease in the depressant effect of the anesthesia. This effect is likely more obvious in the SHR, since the present study and our previous results indicate that, unlike the Wistar rat, the intact contralateral insular cortex does not have a tonic inhibitory effect on sympathetic tone in this strain.13 27 Thus, subcortical and peripheral mechanisms compensating for the depressant effect of anesthesia might be more effective in the SHR.
Previously, we have examined the effects of MCAO in the SHR.13 As in the present investigation, renal sympathetic nerve discharge declined significantly after MCAO in the SHR.13 In addition, MAP also declined, and it was to a greater extent than that seen after IC lesion. Plasma epinephrine levels also declined significantly after MCAO in the SHR, while the decrease was not significant after IC lesion.13 The greater fall in MAP after MCAO, relative to IC lesion, may be related to the more significant decrease in plasma epinephrine levels in MCAO animals compared with IC-lesioned rats. Thus, IC lesion was not sufficient to decrease sympathoadrenal activity to the same extent observed after MCAO.
ECG Changes and Myocardial Damage
Lesion of the IC did not appear to have any adverse cardiac effects in either rat strain. This is consistent with the results of MCAO in the SHR.13 MCAO in the Wistar rat, however, did result in a prolonged QT interval and increased incidence of cardiac myocytolysis.8 30 Cardiac deficits in the Wistar rat after experimental stroke have been suggested to result from elevated plasma catecholamine levels.8 This also may be the mechanism of cardiac damage after stroke in humans.4 5 31 In the present investigation, plasma catecholamines were not significantly elevated in the Wistar rat. The lack of any cardiac effects in the present investigation is therefore consistent with the hypothesis that these changes are mediated by elevated levels of plasma catecholamines.
Insular Cortex Lesions
The relatively large volume of DLH resulted in an identifiable lesion of the entire IC by 6 hours after injection. Smaller volumes were not used in order to avoid partial lesions of the insula. The insula previously has been shown to contain both pressor and depressor regions.18 20 The lesions made in the present investigation appear to have encompassed both of these functional regions. This was the intended result, as it would not have been possible to accurately determine the relative damage to the pressor and depressor areas in a partial lesion of the IC. Despite the relatively large volume of DLH, portions of the dorsal granular IC were left intact in some animals. This may have contributed to the lack of catecholaminergic and cardiac deficits.
This study did not examine the role of the surrounding cortical regions in autonomic regulation. It is possible that these areas are involved in the response to MCAO in Wistar rats and SHRs. The present experiments were aimed at determining whether damage to the IC, without interrupting cortical blood flow, could produce changes similar to those after MCAO. This appears to be the case, but a role for the surrounding cortical areas cannot be excluded.
There is substantial evidence that increased glutamate release and overstimulation of cortical cells is involved in the mechanism of neuronal death after cerebral ischemia.32 33 The DLH injections of the present investigation probably had transient excitatory effects followed by neuronal death. In previous experimental stroke studies in the Wistar rat, we have observed initial increases in sympathetic activity and MAP followed by more long-term autonomic changes.8 This pattern is similar to that seen after DLH injection into the IC. Thus, this type of lesion may be appropriate for studying the effects of ischemic cell damage in localized cortical regions.
DLH microinjections produce consistent lesions of the IC in SHRs and Wistar rats. Lesion of the IC results in a pattern of autonomic changes that are similar but not identical to those seen after experimental stroke (MCAO) in SHRs and Wistar rats. IC lesion results in an increase in renal sympathetic nerve activity and MAP in the Wistar rat. The same procedure causes renal sympathetic nerve discharge and MAP decreases in the SHR. A lesion of the IC does not result in the changes in plasma catecholamine levels or cardiac pathology seen after MCAO. This lack of plasma catecholaminergic and cardiac myocytolytic changes may be due to the incomplete destruction of IC neurons. Conversely, cortical areas surrounding the IC, which are damaged after MCAO, may be involved in the cardiac effects of experimental stroke.
This work was supported by the Heart and Stroke Foundation of Ontario. K.S. Butcher is the recipient of a Heart and Stroke Foundation of Canada Traineeship. D.F. Cechetto is a Career Investigator with the Heart and Stroke Foundation of Ontario. We would like to express our appreciation to Dr Collette Guiraudon for her assistance with the analysis of the cardiac pathology.
- Received May 6, 1994.
- Revision received October 14, 1994.
- Accepted November 30, 1994.
- Copyright © 1995 by American Heart Association
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