David F. Cechetto

Cardiac chronotropic organization of the rat insular cortex.

Clinical evidence implicates the cerebral cortex in the genesis of ECG changes and cardiac arrhythmias. Such findings are not infrequent following acute cortical stroke and during partial seizures. Electrical stimulation of the cerebral cortex, however, only rarely and inconsistently results in cardiac changes. When encountered, attendant alterations in blood pressure and respiration occur; consequently, it is unclear whether the cardiac effects are primary or secondary to these. Phasic insular cortex microstimulation linked to the ECG cycle, a new technique, elicits only heart rate effects, eliminating confounding variables. The insular cortex was chosen for study because of its profuse autonomic and limbic connectivity. Cardiac chronotropic sites were demonstrated in 37 chloralose-anesthetized rats, with tachycardia represented in the rostral posterior insula, and bradycardia in the caudal posterior insula. Both effects were abolished by atenolol but not by atropine, implying their mediation by respective increases or decreases in sympathetic activity. This is the first report of the demonstration of a cortical region wherein stimulation affects heart rate and no other parameter.

Functional and anatomical organization of cardiovascular pressor and depressor sites in the lateral hypothalamic area: I. descending projections

The present study describes the anatomical organization of projections from functionally defined cell groups of the lateral hypothalamic area. Cardiovascular pressor and depressor sites were identified following microinjection (5–50 nl) of 0.01–1.0 M L‐glutamate or D, L‐homocysteate into the anesthetized rat. Subsequent injections of Phaseolus vulgaris‐leucoagglutinin (PHA‐L) or wheat germ agglutinin‐horseradish peroxidase (WGA‐HRP) were made into pressor or depressor sites and their connections with the brainstem and spinal cord were traced.

Human forebrain activation by visceral stimuli.

Visceral function is essential for survival. Discreet regions of the human brain controlling visceral function have been postulated from animal studies (Cechetto and Saper [1987] J. Comp. Neurol. 262:27–45) and suspected from lethal cardiac arrythmias (Cechetto [1994] Integr. Physiol. Behv. Sci. 29:362–373). However, these visceral sites remain uncharted in the normal human brain. We used 4‐Tesla functional magnetic resonance imaging (fMRI) to identify changes in activity in discrete regions of the human brain previously identified in animal studies to be involved in visceral control. Five male subjects underwent heart rate (HR) and/or blood pressure (BP) altering tests: maximal inspiration (MX), Valsalvas maneuver (VM), and isometric handgrip (HG). Increased neuronal activity was observed during MX, VM, and HG, localized in the insular cortex, in the posterior regions of the thalamus, and in the medial prefrontal cortex. To differentiate special visceral (taste) regions from general visceral (HR, BP) regions in these areas, response to gustatory stimulation was also examined; subjects were administered saline (SAL) and sucrose (SUC) solutions as gustatory stimuli. Gustatory stimulation increased activity in the ventral insular cortex at a more inferior level than the cardiopulmonary stimuli. The observed neural activation is the first demonstration of human brain activity in response to visceral stimulation as measured by fMRI. J. Comp. Neurol. 413:572–582, 1999.

Connexin43 null mutation increases infarct size after stroke

Glial‐neuronal interactions have been implicated in both normal information processing and neuroprotection. One pathway of cellular interactions involves gap junctional intercellular communication (GJIC). In astrocytes, gap junctions are composed primarily of the channel protein connexin43 (Cx43) and provide a substrate for formation of a functional syncytium implicated in the spatial buffering capacity of astrocytes. To study the function of gap junctions in the brain, we used heterozygous Cx43 null mice, which exhibit reduced Cx43 expression. Western blot analysis showed a reduction in the level of Cx43 protein and GJIC in astrocytes cultured from heterozygote mice. The level of Cx43 is reduced in the adult heterozygote cerebrum to 40% of that present in the wild‐type. To assess the effect of reduced Cx43 and GJIC on neuroprotection, we examined brain infarct volume in wild‐type and heterozygote mice after focal ischemia. In our model of focal stroke, the middle cerebral artery was occluded at two points, above and below the rhinal fissure. Four days after surgery, mice were killed, the brains were sectioned and analyzed. Cx43 heterozygous null mice exhibited a significantly larger infarct volume compared with wild‐type (14.4 ± 1.4 mm3 vs. 7.7 ± 0.82 mm3, P < 0.002). These results suggest that augmentation of GJIC in astrocytes may contribute to neuroprotection after ischemic injury. J. Comp. Neurol. 440:387–394, 2001.

Autonomic and myocardial changes in middle cerebral artery occlusion: stroke models in the rat

Stroke models in larger animals such as the cat, dog and monkey are becoming increasingly more expensive and less readily available. However, the rat is an excellent model for focal cerebral ischemia. Rats are readily available, inexpensive and their neuroanatomy and brain function have been studied extensively. Increases in plasma catecholamines and myocardial damage have been observed in clinical stroke. We examined autonomic and myocardial changes in two rat stroke models. In one model only the middle cerebral artery was occluded (MCAO) while the other model involved occlusion of both the MCA and the common carotid artery (MCAO/CCAO). Arterial blood pressure and heart rate were monitored continuously in 25 male rats (326-430 g) that underwent one of the following procedures: (1) MCAO only; (2) MCAO/CCAO; (3) CCAO only; and (4) sham occlusions (SHAM). Arterial blood samples (0.5 ml) for radioenzymatic assay of norepinephrine (NE) and epinephrine (E) were taken twice before the occlusions and at 90 and 180 min after the occlusions. The animals were perfused at the end of the experiment and the heart removed and examined histologically. Tetrazolium salts were reacted with oxidative enzymes to delineate the region of inadequate perfusion. The mean blood pressure and pulse pressure of the SHAM, MCAO/CCAO and CCAO groups significantly declined from initial values (from an average of 78 to 53 mm Hg) during the course of the experiment. However, the mean blood pressure and pulse pressure of the MCAO rats did not change during the experiment, so that the final mean blood pressure and pulse pressure were significantly higher than in the other 3 groups. The levels of both NE and E increased significantly (NE, 1443 +/- 285.9 to 4095 +/- 929 pg/ml; E, 2402 +/- 623 to 3741 +/- 1166 pg/ml) following occlusion in the MCAO group only while the other 3 groups did not change. Four of 6 hearts in the MCAO group were abnormal, showing evidence of subendocardial hemorrhage, ischemic damage or subendocardial congestion. MCAO also resulted in a consistent region of the brain with inadequate perfusion including the insular cortex. These autonomic and myocardial changes appear to mimic some of the changes seen clinically in stroke patients and provide the first acute stroke model for studying autonomic dysfunction in the rat.

Vascular risk factors and Alzheimer’s disease

Vascular cognitive impairment risk factors include stroke, hypertension, diabetes and atherosclerosis. In the elderly, vascular risk factors occur in the presence of high levels of amyloid in the aging brain. Stroke alters the clinical expression of a given load of Alzheimer’s disease (AD) pathology. Experimentally, large vessel infarcts or small striatal infarcts are larger in the presence of amyloid. Patients with minor cerebral infarcts and moderate AD lesions will develop the clinical manifestations of dementia. Moreover, there is also an association between other vascular risk factors and the clinical expression of cognitive decline and dementia. The risk of AD is increased in subjects with adult-onset diabetes mellitus, hypertension, atherosclerotic disease and atrial fibrillation. Experimentally, small striatal infarcts in the presence of high levels of amyloid in the brain exhibit a progression in infarct size over time with enhanced degree of cognitive impairment, AD-type pathology and neuroinflammation compared with striatal infarcts or high amyloid levels alone.

Propofol neuroprotection in cerebral ischemia and its effects on low-molecular-weight antioxidants and skilled motor tasks.

Background: Propofol is neuroprotective when administered immediately after stroke. The therapeutic window, duration of administration, and antioxidant mechanisms of propofol in neuroprotection are not known. The effects of propofol after stroke were examined in the conscious animal. The authors have previously shown that light propofol anesthesia (25 mg · kg−1 · h−1) for a period of 4 h, even if delayed 1 h after the onset of ischemia, decreases infarct volume 3 days after the stroke. Methods: Cerebral ischemia was induced in awake Wistar rats by a local intracerebral injection of the potent vasoconstrictor, endothelin (6 pmol in 3 μl) into the striatum. Propofol treatment after ischemia was delayed up to 4 h, and the infusion period shortened from 4 h to 1 h. Infarct volume was assessed 3 or 21 days after the stroke. Neurologic outcome was evaluated on days 14–21 after ischemia. Tissue ascorbate and glutathione concentrations were evaluated at 4 h and 3 days after ischemia. Results: Infarct volumes were reduced 3 days after ischemia when propofol treatment (25 mg · kg−1 · h−1) was delayed for 2 h (0.5 ± 0.3 mm3) but not 4 h (2.0 ± 0.9 mm3), compared with intralipid controls (2.4 ± 0.7 mm3). The propofol infusion period of 3 h but not 1 h reduced infarct volume. Propofol treatment did not reduce infarct volume 21 days after the stroke, although motor function improvements (Montoya staircase test) were observed 14–21 days after the stroke. Propofol neuroprotection was independent of tissue ascorbate and glutathione concentrations. Conclusions: Concurrent or delayed administration of propofol is neuroprotective 3 days after ischemia. Although there were no differences in infarct volume 21 days after ischemia, propofol-treated animals had functional improvements at this time.

Propofol Anesthesia Compared to Awake Reduces Infarct Size in Rats

Background Propofol has not been studied directly in animals subject to cerebral ischemia in the conscious state. Strokes are usually induced in animals while they are anesthetized, making it difficult to eliminate anesthetic interactions as a complicating factor. Therefore, to compare the neuroprotective effects of propofol to the unanesthetized state, experiments were performed using a model that induces a stroke in the conscious rat. Methods Cerebral ischemia was induced in awake Wistar rats by a local intracerebral injection of the potent vasoconstrictor endothelin. Four days before the strokes were induced, a guide cannula was implanted for the injection of endothelin. On the day of the experiment, endothelin (6.0 pmol in 3 &mgr;l) was injected into the striatum. Propofol (25 or 15 mg · kg−1 · h−1) or intralipid (vehicle) were infused for 4 h starting immediately after the endothelin injection. In another series, the propofol infusion was begun 1 h after the endothelin injection and continued for 4 h. Three days later, the animals were killed, and the brains were sectioned and stained. Results The propofol group (25 mg · kg−1 · h−1) had a significantly reduced infarct size (0.7 ± 0.21 mm3, first 4 h; 0.27 ± 0.07 mm3, started 1 h after initiation of infarct) compared with the intralipid controls (3.40 ± 0.53 mm3). To exclude a direct interaction between propofol and endothelin, in thiobutabarbital anesthetized rats, endothelin-induced cerebral vasoconstriction was examined using videomicroscopy, with or without propofol. Propofol had no effect on the magnitude or time course of the endothelin-induced vasoconstriction. Conclusions The results show that concurrent or delayed administration of propofol is neuroprotective.

The effects of propofol in the area postrema of rats.

Propofol has an antiemetic effect that may be mediated by &ggr;-aminobutyric acid (GABA) influences on the serotonin system, the mechanism of which is not known. We used three techniques, immunohistochemistry, High Performance Liquid Chromatography, and electrophysiology, to define propofol’s effects on the rat’s brainstem. Paired male Wistar rats received propofol, 20 mg/kg/hr, or Intralipid® for 6 h. The brains were then subjected to immunohistochemical analysis of serotonin. In a separate experiment after a propofol or Intralipid® infusion, cerebrospinal fluid (CSF) was extracted from the fourth ventricle and analyzed for the amount of serotonin and 5-hydroxyindoleacetic acid. Electrophysiological neuronal recordings were made in the area postrema (AP) in response to propofol with and without a GABA or serotonin antagonist. Results showed that immunohistochemical staining for serotonin in the propofol rats was significantly increased (28 ± 12%) in the dorsal raphe and decreased in the AP (17 ± 6%) compared with control. There were no significant changes in the isoflurane-anesthetized animals. Both serotonin and 5-hydroxyindoleacetic acid in the CSF of the fourth ventricle at the level of the AP were significantly reduced by 63% and 36%, respectively. Both propofol and pentobarbital injections reduce AP neuronal activity, but only the propofol response was blocked by bicuculline, a GABA antagonist. We conclude that the reduced levels of serotonin in the AP and the CSF may explain the antiemetic property of propofol. Propofol may also directly act on AP neurons via a GABAA receptor to reduce their activity.

Progressive Increase in Infarct Size, Neuroinflammation, and Cognitive Deficits in the Presence of High Levels of Amyloid

Background and Purpose— In the elderly, cerebral ischemia (CI) occurs in the presence of high levels of amyloid. Neuroinflammation plays a critical role in the pathophysiology of Alzheimer’s disease and CI. This study examined infarct size, neuroinflammation, and cognitive deficits over time in rat models of Alzheimer’s disease and CI. Methods— &bgr;-amyloid toxicity was modeled using bilateral intracerebroventricular injections of &bgr;-amyloid 25 to 35 peptides. CI was modeled using unilateral injections of the potent vasoconstrictor, endothelin-1, into the striatum. Results— Infarct volumes were higher in the presence of amyloid and compared with the CI model alone. In the CI model alone, the infarct volume was significantly smaller 28 days after surgery compared with 7 days after surgery. However, when Alzheimer’s disease and CI models were combined, the infarct volume was significantly larger 28 days after surgery compared with 7 days after surgery. The neuroinflammation in the region of the infarct was also significantly increased. The Barnes circular platform test showed time-dependent increases in memory and learning deficits in the &bgr;-amyloid-treated rats that were even greater when &bgr;-amyloid treatment was combined with CI. Conclusions— CI in the presence of high levels of amyloid results in progressive increases in infarct size, neuroinflammation, and cognitive deficits.