Visualization of perfusion changes with laser speckle contrast imaging using the method of motion history image.

Laser speckle contrast imaging (LSCI) is a real-time imaging modality reflecting microvascular perfusion. We report on the application of the motion history image (MHI) method on LSCI data obtained from the two hemispheres of a mouse. Through the generation of a single image, MHI stresses the microvascular perfusion changes. Our experimental results performed during a pinprick-triggered spreading depolarization demonstrate the effectiveness of MHI: MHI allows the visualization of perfusion changes without loss of resolution and definition. Moreover, MHI provides close results to the ones given by the generalized differences (GD) algorithm. However, MHI has the advantage of giving information on the temporal evolution of the perfusion variations, which GD does not.

Spreading ischemia after aneurysmal subarachnoid hemorrhage.

Spreading depolarization (SD) is a wave of mass neuronal and glial depolarization associated with net influx of cations and water. Prolonged SDs facilitate neuronal death. SD induces tone alterations in cerebral resistance arterioles, leading to either transient hyperperfusion (physiological neurovascular coupling) in healthy tissue or hypoperfusion (inverse neurovascular coupling = spreading ischemia) in tissue at risk for progressive damage. Spreading ischemia has been shown experimentally in an animal model replicating the conditions present following aneurysmal subarachnoid hemorrhage (aSAH), in animal models of the ischemic core and penumbra following middle cerebral artery occlusion, and in patients with aSAH. In animals, spreading ischemia produced widespread cortical necrosis. In patients, spreading ischemia occurred in temporal correlation with ischemic lesion development early and late after aSAH. We briefly review important features of SD and spreading ischemia following aSAH.

Is spreading depolarization characterized by an abrupt, massive release of gibbs free energy from the human brain cortex?

In the evolution of the cerebral cortex, the sophisticated organization in a steady state far away from thermodynamic equilibrium has produced the side effect of two fundamental pathological network events: ictal epileptic activity and spreading depolarization. Ictal epileptic activity describes the partial disruption, and spreading depolarization describes the near-complete disruption of the physiological double Gibbs-Donnan steady state. The occurrence of ictal epileptic activity in patients has been known for decades. Recently, unequivocal electrophysiological evidence has been found in patients that spreading depolarizations occur abundantly in stroke and brain trauma. The authors propose that the ion changes can be taken to estimate relative changes in Gibbs free energy from state to state. The calculations suggest that in transitions from the physiological state to ictal epileptic activity to spreading depolarization to death, the cortex releases Gibbs free energy in a stepwise fashion. Spreading depolarization thus appears as a twilight state close to death. Consistently, electrocorticographic recordings in the core of focal ischemia or after cardiac arrest display a smooth transition from the initial spreading depolarization component to the later ultraslow negative potential, which is assumed to reflect processes in cellular death.

Pericytes in capillaries are contractile in vivo, but arterioles mediate functional hyperemia in the mouse brain.

Modern functional imaging techniques of the brain measure local hemodynamic responses evoked by neuronal activity. Capillary pericytes recently were suggested to mediate neurovascular coupling in brain slices, but their role in vivo remains unexplored. We used two-photon microscopy to study in real time pericytes and the dynamic changes of capillary diameter and blood flow in the cortex of anesthetized mice, as well as in brain slices. The thromboxane A(2) analog, 9,11-dideoxy-9α,11α-methanoepoxy Prostaglandin F2α (U46619), induced constrictions in the vicinity of pericytes in a fraction of capillaries, whereas others dilated. The changes in vessel diameter resulted in changes in capillary red blood cell (RBC) flow. In contrast, during brief epochs of seizure activity elicited by local administration of the GABA(A) receptor antagonist, bicuculline, capillary RBC flow increased without pericyte-induced capillary diameter changes. Precapillary arterioles were the smallest vessels to dilate, together with penetrating and pial arterioles. Our results provide in vivo evidence that pericytes can modulate capillary blood flow in the brain, which may be important under pathological conditions. However, our data suggest that precapillary and penetrating arterioles, rather than pericytes in capillaries, are responsible for the blood flow increase induced by neural activity.

Pathophysiological interference with neurovascular coupling - when imaging based on hemoglobin might go blind.

Assessing neuronal activity by non-invasive functional brain imaging techniques which are based on the hemodynamic response depends totally on the physiological cascade of metabolism and blood flow. At present, functional brain imaging with near infrared spectroscopy (NIRS) or BOLD-fMRI is widely used in cognitive neuroscience in healthy subjects where neurovascular coupling and cerebrovascular reactivity can be assumed to be intact. Local activation studies as well as studies investigating functional connectivity between brain regions of the resting brain provide a rapidly increasing body of knowledge on brain function in humans and animals. Furthermore, functional NIRS and MRI techniques are increasingly being used in patients with severe brain diseases and this use might gain more and more importance for establishing their use in the clinical routine. However, more and more experimental evidence shows that changes in baseline physiological parameters, pharmacological interventions, or disease-related vascular changes may significantly alter the normal response of blood flow and blood oxygenation and thus may lead to misinterpretation of neuronal activity. In this article we present examples of recent experimental findings on pathophysiological changes of neurovascular coupling parameters in animals and discuss their potential implications for functional imaging based on hemodynamic signals such as fNIRS or BOLD-fMRI. To enable correct interpretation of neuronal activity by vascular signals, future research needs to deepen our understanding of the basic mechanisms of neurovascular coupling and the specific characteristics of disturbed neurovascular coupling in the diseased brain.

Elevating intracranial pressure reverses the decrease in deoxygenated hemoglobin and abolishes the post-stimulus overshoot upon somatosensory activation in rats.

BOLD fMRI localizes activated brain areas by measuring decreases of deoxygenated hemoglobin (deoxy-Hb) caused by neurovascular coupling. To date, it is unclear whether intracranial pressure (ICP) modifies deoxy-Hb signaling for brain mapping. In addition, ICP elevation can test whether the BOLD post-stimulus undershoot, a transient hypo-oxygenation following functional activation, is due to vascular compliance rather than elevated cerebral metabolic rate of oxygen (CMRO(2)). We addressed these questions by studying the effect of ICP elevation on neurovascular coupling. In anesthetized rats, a cranial window was implanted over the somatosensory cortex. Using laser Doppler flowmetry and optical spectroscopy, changes in cerebral blood flow (CBF), cerebral blood volume (CBV) and deoxy-Hb were measured during electrical forepaw stimulation. Neuronal activity was monitored by somatosensory evoked potentials. ICP was elevated by subarachnoideal and intracisternal infusion of artificial cerebrospinal fluid. ICP elevation did not abrogate neurovascular coupling. However, the concomitant deoxy-Hb decrease was reduced (ICP=14mmHg) and reversed (ICP=28mmHg). Therefore, the validity of BOLD fMRI has to be questioned during increased ICP. Moreover, the amplitude of the deoxy-Hb post-stimulus overshoot was reduced with ICP elevation. CMRO(2) was not elevated during the post-stimulus response. Therefore, these data provide experimental evidence that the BOLD post-stimulus undershoot is a passive vascular phenomenon.

Pharmacological uncoupling of activation induced increases in CBF and CMRO2.

Neurovascular coupling provides the basis for many functional neuroimaging techniques. Nitric oxide (NO), adenosine, cyclooxygenase, CYP450 epoxygenase, and potassium are involved in dilating arterioles during neuronal activation. We combined inhibition of NO synthase, cyclooxygenase, adenosine receptors, CYP450 epoxygenase, and inward rectifier potassium (Kir) channels to test whether these pathways could explain the blood flow response to neuronal activation. Cerebral blood flow (CBF) and cerebral metabolic rate of oxygen (CMRO(2)) of the somatosensory cortex were measured during forepaw stimulation in 24 rats using a laser Doppler/spectroscopy probe through a cranial window. Combined inhibition reduced CBF responses by two-thirds, somatosensory evoked potentials and activation-induced CMRO(2) increases remained unchanged, and deoxy-hemoglobin (deoxy-Hb) response was abrogated. This shows that in the rat somatosensory cortex, one-third of the physiological blood flow increase is sufficient to prevent microcirculatory increase of deoxy-Hb concentration during neuronal activity. The large physiological CBF response is not necessary to support small changes in CMRO(2). We speculate that the CBF response safeguards substrate delivery during functional activation with a considerable 'safety factor'. Reduction of the CBF response in pathological states may abolish the BOLD-fMRI signal, without affecting underlying neuronal activity.

Neurovascular coupling in rat brain operates independent of hemoglobin deoxygenation.

Recently, a universal, simple, and fail-safe mechanism has been proposed by which cerebral blood flow (CBF) might be coupled to oxygen metabolism during neuronal activation without the need for any tissue-based mechanism. According to this concept, vasodilation occurs by local erythrocytic release of nitric oxide or ATP wherever and whenever hemoglobin is deoxygenated, directly matching oxygen demand and supply in every tissue. For neurovascular coupling in the brain, we present experimental evidence challenging this view by applying an experimental regime operating without deoxy-hemoglobin. Hyperbaric hyperoxygenation (HBO) allowed us to prevent hemoglobin deoxygenation, as the oxygen that was physically dissolved in the tissue was sufficient to support oxidative metabolism. Regional CBF and regional cerebral blood oxygenation were measured using a cranial window preparation in anesthetized rats. Hemodynamic and neuronal responses to electrical forepaw stimulation or cortical spreading depression (CSD) were analyzed under normobaric normoxia and during HBO up to 4 ATA (standard atmospheres absolute). Inconsistent with the proposed mechanism, during HBO, CBF responses to functional activation or CSD were unchanged. Our results show that activation-induced CBF regulation in the brain does not operate through the release of vasoactive mediators on hemoglobin deoxygenation or through a tissue-based oxygen-sensing mechanism.

Pathway-specific variations in neurovascular and neurometabolic coupling in rat primary somatosensory cortex.

Functional neuroimaging signals are generated, in part, by increases in cerebral blood flow (CBF) evoked by mediators, such as nitric oxide and arachidonic acid derivatives that are released in response to increased neurotransmission. However, it is unknown whether the vascular and metabolic responses within a given brain area differ when local neuronal activity is evoked by an activity in the distinct neuronal networks. In this study we assessed, for the first time, the differences in neuronal responses and changes in CBF and oxygen consumption that are evoked after the activation of two different inputs to a single cortical area. We show that, for a given level of glutamatergic synaptic activity, corticocortical and thalamocortical inputs evoked activity in pyramidal cells and different classes of interneurons, and produced different changes in oxygen consumption and CBF. Furthermore, increases in stimulation intensities either turned off or activated additional classes of inhibitory interneurons immunoreactive for different vasoactive molecules, which may contribute to increases in CBF. Our data imply that for a given cortical area, the amplitude of vascular signals will depend critically on the type of input, and that a positive blood oxygen level-dependent (BOLD) signal may be a consequence of the activation of both pyramidal cells and inhibitory interneurons.

Hypothermia effects on neurovascular coupling and cerebral metabolic rate of oxygen.

Neuronal activation is accompanied by a local increase in cerebral blood flow (CBF) and in cerebral metabolic rate of oxygen (CMRO(2)), caused by neurovascular and neurometabolic coupling. Hypothermia is used as a neuroprotective approach in surgical patients and therapeutically after cardiac arrest or stroke. The effect of hypothermia on neurovascular coupling is of interest for evaluating brain function in these patients, but has not been determined so far. It is not clear whether functional hyperaemia actually operates at subnormal temperatures. In addition, decreasing brain temperature reduces spontaneous CMRO(2) following a known quantitative relationship (Q(10)). Q(10) determination may serve to validate a recently introduced CMRO(2) measurement approach relying on optical measurements of CBF and hemoglobin concentration. We applied this method to investigate hypothermia in a functional study of the somatosensory cortex. Anesthetized Wistar rats underwent surgical implantation of a closed cranial window. Using laser Doppler flowmetry and optical spectroscopy, relative changes in CBF and hemoglobin concentration were measured continuously. At the same time, an electroencephalogram (EEG) was recorded from the measurement site. By the application of ice packs, whole-body hypothermia was induced, followed by rewarming. Spontaneous EEG, CBF and CMRO(2) were measured, interleaved by blocks of electrical forepaw stimulation. The Q(10) obtained from spontaneous CMRO(2) changes of 4.4 (95% confidence interval 3.7-5.1) was close to published values, indicating the reliability of the CMRO(2) measurement. Lowering brain temperature decreased functional changes of CBF and CMRO(2) as well as amplitudes of somatosensory evoked potentials (SEP) to the same degree. In conclusion, neurovascular and neurometabolic coupling is preserved during hypothermia.

Gamma-aminobutyric acid modulates local brain oxygen consumption and blood flow in rat cerebellar cortex.

In the awake brain, the global metabolic rate of oxygen consumption is largely constant, while variations exist between regions dependent on the ongoing activity. This suggests that control mechanisms related to activity, that is, neuronal signaling, may redistribute metabolism in favor of active networks. This study examined the influence of gamma-aminobutyric acid (GABA) tone on local increases in cerebellar metabolic rate of oxygen (CeMR(O(2))) evoked by stimulation of the excitatory, glutamatergic climbing fiber-Purkinje cell synapse in rat cerebellum. In this network, the postsynaptic depolarization produced by synaptic excitation is preserved despite variations in GABAergic tone. Climbing fiber stimulation induced frequency-dependent increases in synaptic activity and CeMR(O(2)) under control conditions. Topical application of the GABA(A) receptor agonist muscimol blocked the increase in CeMR(O(2)) evoked by synaptic excitation concomitant with attenuation of cerebellar blood flow (CeBF) responses. The effect was reversed by the GABA(A) receptor antagonist bicuculline, which also reversed the effect of muscimol on synaptic activity and CeBF. Climbing fiber stimulation during bicuculline application alone produced a delayed undershoot in CeBF concomitant with a prolonged rise in CeMR(O(2)). The findings are consistent with the hypothesis that activity-dependent rises in CeBF and CeMR(O(2)) are controlled by a common feed-forward pathway and provide evidence for modification of cerebral blood flow and CMR(O(2)) by GABA.

Effect of hyperoxia on neurogenic plasma protein extravasation in the rat dura mater.

OBJECTIVE: We aimed to study the effect of normobaric hyperoxia on neurogenic inflammation of the rat dura mater. BACKGROUND: Inhalation of 100% oxygen is a first-line therapy for the treatment of acute cluster headache (CH). However, the mechanisms underlying the antinociceptive effect of oxygen are poorly understood. Sumatriptan, which is also effective in aborting CH attacks, is known to inhibit neurogenic inflammation of the dura mater. We hypothesized that hyperoxia reduces dural plasma protein extravasation in the model of electrically stimulating the rat trigeminal ganglion. METHODS: Unilateral stimulation of the trigeminal ganglion was performed in anesthetized male Sprague-Dawley rats. We assessed plasma protein extravasation (PPE) in the ipsilateral dura mater under normoxic (group 1) and hyperoxic conditions (group 2: pO(2) 200 mmHg; group 3: pO(2) 300 mmHg; group 4: pO(2) 400 mmHg). The study results were compared to the effect of sumatriptan (300 microg/kg) on dural PPE. RESULTS: Under normoxic conditions, the calculated extravasation ratio was 1.72 +/- 0.2. Hyperoxic treatment (groups 2, 3, 4) significantly attenuated dural PPE. At oxygen levels of 400 mmHg, the PPE ratio was 1.14 +/- 0.2 (P < .01). After IV application of sumatriptan (300 microg/kg), PPE was nearly abolished (PPE ratio: 1.06 +/-0.17). CONCLUSION: Our findings demonstrate that hyperoxia is able to inhibit dural PPE. Hyperoxia may play an anti-inflammatory role in neurogenic inflammation, but further studies are needed to clarify whether this effect is either caused by prejunctional mechanisms or by modulation of the vascular permeability at postcapillary venules.

Functional imaging with laser speckle contrast analysis: vascular compartment analysis and correlation with laser Doppler flowmetry and somatosensory evoked potentials.

Laser Speckle Contrast Analysis (LASCA), a novel, high-resolution blood flow imaging method, was performed on rat somatosensory cortex during functional activation. In the same animals, cerebral blood flow (CBF) was measured with Laser Doppler Flowmetry. To obtain a quantitative estimate of the underlying neuronal activity, somatosensory evoked potentials were recorded simultaneously with an epidural EEG. Our results show that: 1. CBF changes measured by LASCA or LDF are nonlinearly dependent on the magnitude of electrical neural activity revealed by somatosensory evoked potentials. 2. The magnitude of relative CBF changes measured by LASCA and LDF shows a strong correlation. 3. LASCA imaging localizes the highest relative changes of CBF in microcirculatory areas, with a smaller contribution by larger vessels. This study demonstrates that LASCA is a reliable method that provides 2D-imaging of CBF changes that are comparable to LDF measurements. It further suggests that functional neuroimaging methods based on CBF enhance areas of microcirculation and thus might prove more accurate in localizing neural activity than oxygenation related methods like BOLD-fMRI.

Activity-induced tissue oxygenation changes in rat cerebellar cortex: interplay of postsynaptic activation and blood flow.

Functional neuroimaging relies on the robust coupling between neuronal activity, metabolism and cerebral blood flow (CBF), but the physiological basis of the neuroimaging signals is still poorly understood. We examined the mechanisms of activity-dependent changes in tissue oxygenation in relation to variations in CBF responses and postsynaptic activity in rat cerebellar cortex. To increase synaptic activity we stimulated the monosynaptic, glutamatergic climbing fibres that excite Purkinje cells via AMPA receptors. We used local field potentials to indicate synaptic activity, and recorded tissue oxygen partial pressure (P(tiss,O2)) by polarographic microelectrodes, and CBF using laser-Doppler flowmetry. The disappearance rate of oxygen in the tissue increased linearly with synaptic activity. This indicated that, without a threshold, oxygen consumption increased as a linear function of synaptic activity. The reduction in P(tiss,O2) preceded the rise in CBF. The time integral (area) of the negative P(tiss,O2) response increased non-linearly showing saturation at high levels of synaptic activity, concomitant with a steep rise in CBF. This was accompanied by a positive change in P(tiss,O2). Neuronal nitric oxide synthase inhibition enhanced the initial negative P(tiss,O2) response ('dip'), while attenuating the evoked CBF increase and positive P(tiss,O2) response equally. This indicates that increases in CBF counteract activity-induced reductions in P(tiss,O2), and suggests the presence of a tissue oxygen reserve. The changes in P(tiss,O2) and CBF were strongly attenuated by AMPA receptor blockade. Our findings suggest an inverse relationship between negative P(tiss,O2) and CBF responses, and provide direct in vivo evidence for a tight coupling between activity in postsynaptic AMPA receptors and cerebellar oxygen consumption.

Principal neuron spiking: neither necessary nor sufficient for cerebral blood flow in rat cerebellum.

Neuronal activity, cerebral blood flow, and metabolic responses are all strongly coupled, although the mechanisms behind the coupling remain unclear. One of the key questions is whether or not increases in spiking activity in the stimulated neurons are sufficient to drive the activity-dependent rises in cerebral blood flow (CBF) that form the basis of the signals used in functional neuroimaging such as the blood oxygen level-dependent (BOLD) signal. To this end the present study examined the effect of enhanced spike activity per se on CBF in rat cerebellar cortex under conditions of disinhibition, achieved by blocking GABA(A) receptors using either bicuculline or picrotoxin. Purkinje cell spiking activity and local field potentials were recorded by glass microelectrodes, and laser Doppler flowmetry was used to monitor CBF. Disinhibition increased Purkinje cell spiking rate to 200-300% of control without incurring any increase in basal CBF. This demonstrates that increased spike activity per se is not sufficient to affect basal CBF. The neurovascular coupling between excitatory synaptic activity and CBF responses evoked by inferior olive (climbing fibre) stimulation was preserved during disinhibition. Thus, the unchanged basal CBF in the presence of the dramatic rise in Purkinje cell spiking rate was not explained by impaired synaptic activity-CBF coupling. On the basis of our previous and the present studies, we conclude that increased spiking activity of principal neurons is neither sufficient nor necessary to elicit CBF responses and in turn BOLD signals, and that activation-dependent vascular signals reflect excitatory synaptic activity.