Vascular imprints of neuronal activity: Relationships between the dynamics of cortical blood flow, oxygenation, and volume changes following sensory stimulation

Modern functional neuroimaging methods, such as positron-emission tomography (PET), optical imaging of intrinsic signals, and functional MRI (fMRI) utilize activity-dependent hemodynamic changes to obtain indirect maps of the evoked electrical activity in the brain. Whereas PET and flow-sensitive MRI map cerebral blood flow (CBF) changes, optical imaging and blood oxygenation level-dependent MRI map areas with changes in the concentration of deoxygenated hemoglobin (HbR). However, the relationship between CBF and HbR during functional activation has never been tested experimentally. Therefore, we investigated this relationship by using imaging spectroscopy and laser-Doppler flowmetry techniques, simultaneously, in the visual cortex of anesthetized cats during sensory stimulation. We found that the earliest microcirculatory change was indeed an increase in HbR, whereas the CBF increase lagged by more than a second after the increase in HbR. The increased HbR was accompanied by a simultaneous increase in total hemoglobin concentration (Hbt), presumably reflecting an early blood volume increase. We found that the CBF changes lagged after Hbt changes by 1 to 2 sec throughout the response. These results support the notion of active neurovascular regulation of blood volume in the capillary bed and the existence of a delayed, passive process of capillary filling.

Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex

Cortical blood flow at the level of individual capillaries and the coupling of neuronal activity to flow in capillaries are fundamental aspects of homeostasis in the normal and the diseased brain. To probe the dynamics of blood flow at this level, we used two-photon laser scanning microscopy to image the motion of red blood cells (RBCs) in individual capillaries that lie as far as 600 μm below the pia mater of primary somatosensory cortex in rat; this depth encompassed the cortical layers with the highest density of neurons and capillaries. We observed that the flow was quite variable and exhibited temporal fluctuations around 0.1 Hz, as well as prolonged stalls and occasional reversals of direction. On average, the speed and flux (cells per unit time) of RBCs covaried linearly at low values of flux, with a linear density of ≈70 cells per mm, followed by a tendency for the speed to plateau at high values of flux. Thus, both the average velocity and density of RBCs are greater at high values of flux than at low values. Time-locked changes in flow, localized to the appropriate anatomical region of somatosensory cortex, were observed in response to stimulation of either multiple vibrissae or the hindlimb. Although we were able to detect stimulus-induced changes in the flux and speed of RBCs in some single trials, the amplitude of the stimulus-evoked changes in flow were largely masked by basal fluctuations. On average, the flux and the speed of RBCs increased transiently on stimulation, although the linear density of RBCs decreased slightly. These findings are consistent with a stimulus-induced decrease in capillary resistance to flow.

Nitric oxide mediates the increase in local cerebral blood flow during focal seizures.

The role of nitric oxide (NO) in the increase in local cerebral blood flow (LCBF) elicited by focal cortical epileptic seizures was investigated in anesthetized adult rats. Seizures were induced by topical bicuculline methiodide applied through two cranial windows drilled over homotopic sites of the frontal cortex, and LCBF was measured by quantitative autoradiography by using 4-iodo[N-methyl-14C]antipyrine. Superfusion of an inhibitor of NO synthase, N omega-nitro-L-arginine (NA; 1 mM), for 45 min abolished the increase of LCBF induced by topical bicuculline methiodide (10 mM) [164 +/- 18 ml/100 g per min in the artificial cerebrospinal fluid (aCSF)-superfused side and 104 +/- 12 ml/100 g per ml in the NA-superfused side; P < 0.005]. This effect was reversed by coapplication of an excess of L-arginine substrate (10 mM) (218 +/- 22 ml/100 g per min in the aCSF-superfused side and 183 +/- 31 ml/100 g per min in the NA + L-Arg-superfused side) but not by 10 mM D-arginine, a stereoisomer with poor affinity for NO synthase (193 +/- 17 ml/100 g per min in the aCSF-superfused side and 139 +/- 21 ml/100 g per min in the NA + D-Arg-superfused side; P < 0.005). Superfusion of the guanylyl cyclase inhibitor methylene blue attenuated the LCBF increase elicited by topical bicuculline methiodide by 25% +/- 16% (P < 0.05). The present findings suggest that NO is the mediator of the vasodilation in response to focal epileptic seizures.

Decreased blood pressure response in mice deficient of the α1b-adrenergic receptor

To investigate the functional role of different α1-adrenergic receptor (α1-AR) subtypes in vivo, we have applied a gene targeting approach to create a mouse model lacking the α1b-AR (α1b−/−). Reverse transcription–PCR and ligand binding studies were combined to elucidate the expression of the α1-AR subtypes in various tissues of α1b +/+ and −/− mice. Total α1-AR sites were decreased by 98% in liver, 74% in heart, and 42% in cerebral cortex of the α1b −/− as compared with +/+ mice. Because of the large decrease of α1-AR in the heart and the loss of the α1b-AR mRNA in the aorta of the α1b−/− mice, the in vivo blood pressure and in vitro aorta contractile responses to α1-agonists were investigated in α1b +/+ and −/− mice. Our findings provide strong evidence that the α1b-AR is a mediator of the blood pressure and the aorta contractile responses induced by α1 agonists. This was demonstrated by the finding that the mean arterial blood pressure response to phenylephrine was decreased by 45% in α1b −/− as compared with +/+ mice. In addition, phenylephrine-induced contractions of aortic rings also were decreased by 25% in α1b−/− mice. The α1b-AR knockout mouse model provides a potentially useful tool to elucidate the functional specificity of different α1-AR subtypes, to better understand the effects of adrenergic drugs, and to investigate the multiple mechanisms involved in the control of blood pressure.

Correlation of regional cerebral blood flow and change of plasma sodium concentration during genesis and satiation of thirst

Positron emission tomography studies were conducted during genesis of moderate thirst by rapid i.v. infusion of hypertonic saline (0.51 M) and after satiation of thirst by drinking water. The correlation of regional cerebral blood flow with the change in the plasma Na concentration showed a significant group of cerebral activations in the anterior cingulate region and also a site in the middle temporal gyrus and in the periaqueductal gray. Strongest deactivations occurred in the parahippocampal and frontal gyri. The data are consistent with an important role of the anterior cingulate in the genesis of thirst.

Blood flow and oxygen delivery to human brain during functional activity: Theoretical modeling and experimental data

Coupling of cerebral blood flow (CBF) and cerebral metabolic rate for oxygen (CMRO2) in physiologically activated brain states remains the subject of debates. Recently it was suggested that CBF is tightly coupled to oxidative metabolism in a nonlinear fashion. As part of this hypothesis, mathematical models of oxygen delivery to the brain have been described in which disproportionately large increases in CBF are necessary to sustain even small increases in CMRO2 during activation. We have explored the coupling of CBF and oxygen delivery by using two complementary methods. First, a more complex mathematical model was tested that differs from those recently described in that no assumptions were made regarding tissue oxygen level. Second, [15O] water CBF positron emission tomography (PET) studies in nine healthy subjects were conducted during states of visual activation and hypoxia to examine the relationship of CBF and oxygen delivery. In contrast to previous reports, our model showed adequate tissue levels of oxygen could be maintained without the need for increased CBF or oxygen delivery. Similarly, the PET studies demonstrated that the regional increase in CBF during visual activation was not affected by hypoxia. These findings strongly indicate that the increase in CBF associated with physiological activation is regulated by factors other than local requirements in oxygen.

Effects of anesthesia on functional activation of cerebral blood flow and metabolism

Functional brain mapping based on changes in local cerebral blood flow (lCBF) or glucose utilization (lCMRglc) induced by functional activation is generally carried out in animals under anesthesia, usually α-chloralose because of its lesser effects on cardiovascular, respiratory, and reflex functions. Results of studies on the role of nitric oxide (NO) in the mechanism of functional activation of lCBF have differed in unanesthetized and anesthetized animals. NO synthase inhibition markedly attenuates or eliminates the lCBF responses in anesthetized animals but not in unanesthetized animals. The present study examines in conscious rats and rats anesthetized with α-chloralose the effects of vibrissal stimulation on lCMRglc and lCBF in the whisker-to-barrel cortex pathway and on the effects of NO synthase inhibition with NG-nitro-l-arginine methyl ester (l-NAME) on the magnitude of the responses. Anesthesia markedly reduced the lCBF and lCMRglc responses in the ventral posteromedial thalamic nucleus and barrel cortex but not in the spinal and principal trigeminal nuclei. l-NAME did not alter the lCBF responses in any of the structures of the pathway in the unanesthetized rats and also not in the trigeminal nuclei of the anesthetized rats. In the thalamus and sensory cortex of the anesthetized rats, where the lCBF responses to stimulation had already been drastically diminished by the anesthesia, l-NAME treatment resulted in loss of statistically significant activation of lCBF by vibrissal stimulation. These results indicate that NO does not mediate functional activation of lCBF under physiological conditions.

Cerebral cortical hyperactivation in response to mental stress in patients with coronary artery disease

The central nervous system (CNS) effects of mental stress in patients with coronary artery disease (CAD) are unexplored. The present study used positron emission tomography (PET) to measure brain correlates of mental stress induced by an arithmetic serial subtraction task in CAD and healthy subjects. Mental stress resulted in hyperactivation in CAD patients compared with healthy subjects in several brain areas including the left parietal cortex [angular gyrus/parallel sulcus (area 39)], left anterior cingulate (area 32), right visual association cortex (area 18), left fusiform gyrus, and cerebellum. These same regions were activated within the CAD patient group during mental stress versus control conditions. In the group of healthy subjects, activation was significant only in the left inferior frontal gyrus during mental stress compared with counting control. Decreases in blood flow also were produced by mental stress in CAD versus healthy subjects in right thalamus (lateral dorsal, lateral posterior), right superior frontal gyrus (areas 32, 24, and 10), and right middle temporal gyrus (area 21) (in the region of the auditory association cortex). Of particular interest, a subgroup of CAD patients that developed painless myocardial ischemia during mental stress had hyperactivation in the left hippocampus and inferior parietal lobule (area 40), left middle (area 10) and superior frontal gyrus (area 8), temporal pole, and visual association cortex (area 18), and a concomitant decrease in activation observed in the anterior cingulate bilaterally, right middle and superior frontal gyri, and right visual association cortex (area 18) compared with CAD patients without myocardial ischemia. These findings demonstrate an exaggerated cerebral cortical response and exaggerated asymmetry to mental stress in individuals with CAD.

Force-mediated kinetics of single P-selectin/ligand complexes observed by atomic force microscopy

Leukocytes roll along the endothelium of postcapillary venules in response to inflammatory signals. Rolling under the hydrodynamic drag forces of blood flow is mediated by the interaction between selectins and their ligands across the leukocyte and endothelial cell surfaces. Here we present force-spectroscopy experiments on single complexes of P-selectin and P-selectin glycoprotein ligand-1 by atomic force microscopy to determine the intrinsic molecular properties of this dynamic adhesion process. By modeling intermolecular and intramolecular forces as well as the adhesion probability in atomic force microscopy experiments we gain information on rupture forces, elasticity, and kinetics of the P-selectin/P-selectin glycoprotein ligand-1 interaction. The complexes are able to withstand forces up to 165 pN and show a chain-like elasticity with a molecular spring constant of 5.3 pN nm−1 and a persistence length of 0.35 nm. The dissociation constant (off-rate) varies over three orders of magnitude from 0.02 s−1 under zero force up to 15 s−1 under external applied forces. Rupture force and lifetime of the complexes are not constant, but directly depend on the applied force per unit time, which is a product of the intrinsic molecular elasticity and the external pulling velocity. The high strength of binding combined with force-dependent rate constants and high molecular elasticity are tailored to support physiological leukocyte rolling.

Positron-emission tomography imaging of long-term shape recognition challenges

Long-term visual memory performance was impaired by two types of challenges: a diazepam challenge on acquisition and a sensory challenge on recognition. Using positron-emission tomography regional cerebral blood flow imaging, we studied the effect of these challenges on regional brain activation during the delayed recognition of abstract visual shapes as compared with a baseline fixation task. Both challenges induced a significant decrease in differential activation in the left fusiform gyrus, suggesting that this region is involved in the automatic or volitional comparison of incoming and stored stimuli. In contrast, thalamic differential activation increased in response to memory challenges. This increase might reflect enhanced retrieval attempts as a compensatory mechanism for restoring recognition performance.

Forebrain mechanisms of nociception and pain: Analysis through imaging

Pain is a unified experience composed of interacting discriminative, affective-motivational, and cognitive components, each of which is mediated and modulated through forebrain mechanisms acting at spinal, brainstem, and cerebral levels. The size of the human forebrain in relation to the spinal cord gives anatomical emphasis to forebrain control over nociceptive processing. Human forebrain pathology can cause pain without the activation of nociceptors. Functional imaging of the normal human brain with positron emission tomography (PET) shows synaptically induced increases in regional cerebral blood flow (rCBF) in several regions specifically during pain. We have examined the variables of gender, type of noxious stimulus, and the origin of nociceptive input as potential determinants of the pattern and intensity of rCBF responses. The structures most consistently activated across genders and during contact heat pain, cold pain, cutaneous laser pain or intramuscular pain were the contralateral insula and anterior cingulate cortex, the bilateral thalamus and premotor cortex, and the cerebellar vermis. These regions are commonly activated in PET studies of pain conducted by other investigators, and the intensity of the brain rCBF response correlates parametrically with perceived pain intensity. To complement the human studies, we developed an animal model for investigating stimulus-induced rCBF responses in the rat. In accord with behavioral measures and the results of human PET, there is a progressive and selective activation of somatosensory and limbic system structures in the brain and brainstem following the subcutaneous injection of formalin. The animal model and human PET studies should be mutually reinforcing and thus facilitate progress in understanding forebrain mechanisms of normal and pathological pain.

Event-related functional MRI: Past, present, and future

The past two decades have seen an enormous growth in the field of human brain mapping. Investigators have extensively exploited techniques such as positron emission tomography and MRI to map patterns of brain activity based on changes in cerebral hemodynamics. However, until recently, most studies have investigated equilibrium changes in blood flow measured over time periods upward of 1 min. The advent of high-speed MRI methods, capable of imaging the entire brain with a temporal resolution of a few seconds, allows for brain mapping based on more transient aspects of the hemodynamic response. Today it is now possible to map changes in cerebrovascular parameters essentially in real time, conferring the ability to observe changes in brain state that occur over time periods of seconds. Furthermore, because robust hemodynamic alterations are detectable after neuronal stimuli lasting only a few tens of milliseconds, a new class of task paradigms designed to measure regional responses to single sensory or cognitive events can now be studied. Such “event related” functional MRI should provide for fundamentally new ways to interrogate brain function, and allow for the direct comparison and ultimately integration of data acquired by using more traditional behavioral and electrophysiological methods.

Conscious recollection and the human hippocampal formation: evidence from positron emission tomography.

We used positron emission tomography (PET) to examine the role of the hippocampal formation in implicit and explicit memory. Human volunteers studied a list of familiar words, and then they either provided the first word that came to mind in response to three-letter cues (implicit memory) or tried to recall studied words in response to the same cues (explicit memory). There was no evidence of hippocampal activation in association with implicit memory. However, priming effects on the implicit memory test were associated with decreased activity in extrastriate visual cortex. On the explicit memory test, subjects recalled many target words in one condition and recalled few words in a second condition, despite trying to remember them. Comparisons between the two conditions showed that blood-flow increases in the hippocampal formation are specifically associated with the conscious recollection of studied words, whereas blood-flow increases in frontal regions are associated with efforts to retrieve target words. Our results help to clarify some puzzles concerning the role of the hippocampal formation in human memory.

Cerebrovascular alterations in mice lacking neuronal nitric oxide synthase gene expression.

Nitric oxide (NO) is known to mediate increases in regional cerebral blood flow elicited by CO2 inhalation. In mice with deletion of the gene for neuronal NO synthase (NOS), CO2 inhalation augments cerebral blood flow to the same extent as in wild-type mice. However, unlike wild-type mice, the increased flow in mutants is not blocked by the NOS inhibition, N omega-nitro-L-arginine, and CO2 exposure fails to increase brain levels of cGMP. Topical acetylcholine elicits vasodilation in the mutants which is blocked by N omega-nitro-L-arginine, indicating normal functioning of endothelial NOS. Moreover, immunohistochemical staining for endothelial NOS is normal in the mutants. Thus, following loss of neuronal NOS, the cerebral circulatory response is maintained by a compensatory system not involving NO.

Platelet-derived growth factor BB induces functional vascular anastomoses in vivo.

Neovascularization that generates collateral blood flow can limit the extent of tissue damage after acute ischemia caused by occlusion of the primary blood supply. The neovascular response stimulated by the BB homodimeric form of recombinant platelet-derived growth factor (PDGF-BB) was evaluated for its capacity to protect tissue from necrosis in a rat skin flap model of acutely induced ischemia. Complete survival of the tissue ensued, when the original nutritive blood supply was occluded, as early as 5 days after local PDGF-BB application, and the presence of a patent vasculature was evident compared to control flaps. To further evaluate the vascular regenerative response, PDGF-BB was injected into the muscle/connective tissue bed between the separated ends of a divided femoral artery in rats. A patent new vessel that functionally reconnected the ends of the divided artery within the original 3- to 4-mm gap was regenerated 3 weeks later in all PDGF-BB-treated limbs. In contrast, none of the paired control limbs, which received vehicle with an inactive variant of PDGF-BB, had vessel regrowth (P < 0.001). The absence of a sustained inflammatory response and granulation tissue suggests locally delivered PDGF-BB may directly stimulate the angiogenic phenotype in endothelial cells. These findings indicate that PDGF-BB can generate functional new blood vessels and nonsurgically anastomose severed vessels in vivo. This study supports the possibility of a therapeutic modality for the salvage of ischemic tissue through exogenous cytokine-induced vascular reconnection.