Longitudinal functional imaging studies of stroke are key in identifying the disease progression and possible therapeutic interventions. Here we investigate the applicability of real-time functional optoacoustic imaging for monitoring of stroke progression in the whole brain of living animals.
The mammalian brain depends upon glucose as its main source of energy, and tight regulation of glucose metabolism is critical for brain physiology. Consistent with its critical role for physiological brain function, disruption of normal glucose metabolism as well as its interdependence with cell death pathways forms the pathophysiological basis for many brain disorders. Here, we review recent advances in understanding how glucose metabolism sustains basic brain physiology. We synthesize these findings to form a comprehensive picture of the cooperation required between different systems and cell types, and the specific breakdowns in this cooperation that lead to disease.
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-9?,11?-methanoepoxy Prostaglandin F2? (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.
Cerebral blood flow (CBF) quantification is a valuable tool in stroke research. Mice are of special interest because of the potential of genetic engineering. Magnetic resonance imaging (MRI) provides repetitive, noninvasive CBF quantification. Many MRI techniques require the knowledge of the brain-blood partition coefficient (BBPC) for water. Adopting an MRI protocol described by Roberts et al (1996) in humans, we determined the BBPC for water in 129S6/SvEv mice from proton density measurements of brain and blood, calibrated with deuterium oxide/water phantoms. The average BBPC for water was 0.89 ± 0.03 mL/g, with little regional variation within the mouse brain.
Non-invasive near-infrared fluorescence (NIRF) imaging is a powerful tool to study pathophysiology in a wide variety of animal disease models including brain diseases. However, especially in NIRF imaging of the brain or other deeper laying target sites, background fluorescence emitted from the scalp or superficial blood vessels can impede the detection of fluorescence in deeper tissue. Here, we introduce an effective method to reduce the impact of fluorescence from superficial layers. The approach uses excitation light at two different wavelengths generating two images with different depth sensitivities followed by an adapted subtraction algorithm. This technique leads to significant enhancement of the contrast and the detectability of fluorochromes located in deep tissue layers in tissue simulating phantoms and murine models with stroke.
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.
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.
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.
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.
Impairment of the blood-brain barrier (BBB) after cerebral ischemia leads to extravasation of plasma constituents into the brain parenchyma. We describe a novel method using non-invasive near-infrared fluorescence (NIRF) imaging and bovine serum albumin labeled with a NIRF dye (NIRF-BSA) to detect BBB impairment after middle cerebral artery occlusion (MCAO) in mice. We first explored the time course of BBB impairment after transient MCAO using Evans blue (EB), which binds to plasma albumin in vivo. An initial BBB impairment was observed at 4-8h and a second impairment at 12-16h after reperfusion. No EB extravasation was detected at 8-12h. Non-invasive NIRF imaging with NIRF-BSA confirmed biphasic BBB impairment. Upon co-injection of NIRF-BSA with EB we found a strong correlation between the detected NIRF signal and the amount of extravasated EB (r=0.857, P=0.00178). When MCAO mice received NIRF-BSA together with gadolinium-diethylene triamine penta-acetic acid (Gd-DTPA), T1-weighted images showed Gd-DTPA enhancement at all times while NIRF imaging showed biphasic BBB impairment. In conclusion, NIRF-BSA is a suitable marker of plasma albumin extravasation in the mouse brain. Non-invasive NIRF imaging with NIRF-BSA is a useful tool to study BBB integrity in preclinical models of central nervous system pathology.
Recent experimental studies in rodents suggest that treatment with inhibitors of phosphodiesterase type 5 (PDE5) (tadalafil, sildenafil, zaprinast) not only increases cerebral blood flow but also improves functional recovery after stroke. Here, we investigated in a mouse model of stroke the effects of vardenafil on survival, functional outcome and lesion size after experimental stroke. Mice were subjected to experimental stroke by occlusion of the middle cerebral artery (MCAO) for 45 min. A group of mice received vardenafil (twice 10 mg/kg body weight per day orally over 14 days) starting 3 h after MCAO. Control animals received the vehicle only. Survival, body weight, and behavior were monitored over 4 weeks and brain lesions were measured by T2-weighted MRI, hematoxylin/eosin -- as well as GFAP-staining of cryostat sections, subsequently. The mortality in MCAO-operated animals amounted to 45% until day 10 after stroke and no significant difference in survival between the vardenafil- and vehicle-treatment groups was observed. Compared to sham-operated animals, MCAO-operated mice from both treatment groups demonstrated a significant weight loss until day 5 and regained their body weight by day 14 after ischemia. There was no significant difference between the vardenafil and vehicle-treated MCAO groups. In behavioral studies (sucrose consumption and pole test), analyzing sensorimotor functions as well as a parameter of depression-like symptoms, we observed no significant effect of vardenafil treatment on functional recovery in our model of stroke. Although we observed a trend towards less hemispherical atrophy in the vardenafil compared to the vehicle-treated group four weeks after MCAO our data do not suggest a functionally relevant CNS-tissue protective or regenerative effect in murine stroke.
Ambivalent effects of interleukin-6 on the pathogenesis of ischaemic stroke have been reported. However, to date, the long-term actions of interleukin-6 after stroke have not been investigated. Here, we subjected interleukin-6 knockout (IL-6(-/-)) and wild-type control mice to mild brain ischaemia by 30-min filamentous middle cerebral artery occlusion/reperfusion. While ischaemic tissue damage was comparable at early time points, IL-6(-/-) mice showed significantly increased chronic lesion volumes as well as worse long-term functional outcome. In particular, IL-6(-/-) mice displayed an impaired angiogenic response to brain ischaemia with reduced numbers of newly generated endothelial cells and decreased density of perfused microvessels along with lower absolute regional cerebral blood flow and reduced vessel responsivity in ischaemic striatum at 4 weeks. Similarly, the early genomic activation of angiogenesis-related gene networks was strongly reduced and the ischaemia-induced signal transducer and activator of transcription 3 activation observed in wild-type mice was almost absent in IL-6(-/-) mice. In addition, systemic neoangiogenesis was impaired in IL-6(-/-) mice. Transplantation of interleukin-6 competent bone marrow into IL-6(-/-) mice (IL-6(chi)) did not rescue interleukin-6 messenger RNA expression or the early transcriptional activation of angiogenesis after stroke. Accordingly, chronic stroke outcome in IL-6(chi) mice recapitulated the major effects of interleukin-6 deficiency on post-stroke regeneration with significantly enhanced lesion volumes and reduced vessel densities. Additional in vitro experiments yielded complementary evidence, which showed that after stroke resident brain cells serve as the major source of interleukin-6 in a self-amplifying network. Treatment of primary cortical neurons, mixed glial cultures or immortalized brain endothelia with interleukin 6-induced robust interleukin-6 messenger RNA transcription in each case, whereas oxygen-glucose deprivation did not. However, oxygen-glucose deprivation of organotypic brain slices resulted in strong upregulation of interleukin-6 messenger RNA along with increased transcription of key angiogenesis-associated genes. In conclusion, interleukin-6 produced locally by resident brain cells promotes post-stroke angiogenesis and thereby affords long-term histological and functional protection.
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