Noninvasive fluorescence imaging in animal models of stroke.

Noninvasive fluorescence imaging (NFI) is a powerful tool to study physiology and pathophysiology in animal disease models. NFI has been successfully applied in a number of animal disease models including cancer, arthritis, and stroke. Furthermore, several applications in humans have been described. NFI is widely available in research laboratories because it has a number of advantages: It uses non-ionizing radiation and requires comparably simple, inexpensive instrumentation, and easy to handle. Fluorochromes can be detected with high sensitivity, and image acquisition time is relatively short. Furthermore, a plethora of fluorescent imaging agents is available including unspecific, target-specific, and activatable imaging probes. With these probes, biological processes such as inflammation, cell death or enzyme activity, and many others can be visualized in living animals. This review offers an overview of current approaches in NFI of stroke pathophysiology in animal models of cerebral ischemia. First, the instrumentation and the different types of imaging agents for NFI are described. Second, a short introduction to animal models of stroke is provided. Third, examples for NFI in animal models of stroke are given. Finally, the use of NFI in human stroke is critically discussed.

Ultrasound perfusion imaging of small stroke involving the thalamus.

PURPOSE: Ultrasound (US) perfusion imaging of ischemic stroke has mainly been applied to large middle cerebral artery infarction. We investigated whether small stroke involving the thalamus can also be detected. MATERIALS AND METHODS: Phase inversion harmonic imaging (PIHI) was applied to patients with small infarctions involving the thalamus (maximal longitudinal infarct diameter less than 3 cm). PIHI was performed from both the left and right side in axial diencephalic planes. Infarct size and location as well as perfusion properties (MTT maps) were known from MRI. US perfusion parameters were derived from the signal enhancement time course (bolus kinetics, SonoVue for peak-signal increase and time-to-peak. RESULTS: Seventeen patients (52 +/- 11 years, 24% female) with 18 strokes (16 unilateral, 1 bilateral) were included. Six US examinations (18%) were inadequate for analysis due to an insufficient transtemporal bone window. US perfusion depicted 90 % of infarcts with a longitudinal diameter of more than 2 cm. Infarcts with a longitudinal diameter of less than 2 cm were hardly identified. CONCLUSION: PIHI allows identification of a small infarction involving the thalamus subject to infarct size.

Towards noninvasive molecular fluorescence imaging of the human brain.

Fluorescence molecular brain imaging is a new modality allowing the detection of specific contrast agents down to very low concentration ranges (picomolar) in disease models. Here we demonstrate a first noninvasive application of fluorescence imaging in the human brain, where concentrations down to about 100 nM of a nonspecific dye were detected. We argue that due to its high sensitivity, optical molecular imaging of the brain is feasible, which - together with its bedside applicability - makes it a promising technique for use in patients.

A flow sensitive alternating inversion recovery (FAIR)-MRI protocol to measure hemispheric cerebral blood flow in a mouse stroke model.

Blood flow imaging is an important tool in cerebrovascular research. Mice are of special interest because of the potential of genetic engineering. Magnetic resonance imaging (MRI) provides three-dimensional noninvasive quantitative methods of cerebral blood flow (CBF) imaging, but these MRI techniques have not yet been validated for mice. The authors compared CBF imaging using flow sensitive alternating inversion recovery (FAIR)-MRI and (14)C-Iodoantipyrine (IAP)-autoradiography in a mouse model of acute stroke. Twenty-nine male 129S6/SvEv mice were subjected to filamentous left middle cerebral artery occlusion (MCAo). CBF imaging was performed with (14)C-IAP autoradiography and FAIR-MRI using two different anesthesia protocols, namely intravenous infusion of etomidate or inhalation of isoflurane, which differentially affect perfusion. Using (14)C-IAP autoradiography, the average CBF in ml/(100 g*min) was 160+/-34 (isoflurane, n=5) vs. and 59+/-21 (etomidate, n=7) in the intact hemisphere and 43+/-12 (isoflurane, n=5) vs. 36+/-12 (etomidate, n=7) in the MCAo hemisphere. Using FAIR-MRI, the corresponding average CBFs were 208+/-56 (isoflurane, intact hemisphere, n=7), 84+/-9 (etomidate, intact hemisphere, n=7), 72+/-22 (isoflurane, MCAo hemisphere, n=7) and 48+/-13 (etomidate, MCAo hemisphere, n=7). Regression analysis showed a strong linear correlation between CBF measured with FAIR-MRI and (14)C-IAP autoradiography, and FAIR-MRI overestimated CBF compared to autoradiography. FAIR-MRI provides repetitive quantitative measurements of hemispheric CBF in a mouse model of stroke.

The oxygenation response to functional stimulation: is there a physiological meaning to the lag between parameters?

To investigate the regulation of the hemodynamic response to functional stimulation, functional near-infrared spectroscopy (fNIRS) has been used, due to its ability to assess the dynamics of oxygenated, deoxygenated and total hemoglobin concentration ([oxy-Hb], [deoxy-Hb] and [tot-Hb]). Concerning the latency of these parameters, recent studies have returned a consistent picture when comparing the oxygenation response in the sensorimotor to the visual system: changes in [oxy-Hb] lead those in [deoxy-Hb] by 1.6+/-0.2 s (mean+/-SD) for the sensorimotor system but not for the visual system (0.1+/-0.3 s). A number of physiological differences between these cortical areas may account for such a discrepancy, however, the methodological properties of transcranial NIRS also have a relevant influence. Here we show that for the motor system the latency between changes in oxy- compared to deoxy-Hb vanishes once efforts are made to reduce the effects of a systemic response accompanying sensorimotor activity. We apply two independent approaches to reduce the systemic response and find a simultaneous change in [oxy-Hb] and [deoxy-Hb] even in response to a motor paradigm. The two approaches are: (i) an experimental paradigm with alternating contralateral and ipsilateral motor performance without interspersed rest periods designed to minimize systemic changes and (ii) a global correction scheme in an experiment, comparing a unilateral motor performance to rest. These data shed some doubt on the alleged fundamental physiological difference between cortical hemodynamic regulation in motor and visual cortex and highlight the relevance to respect contributions of the systemic hemodynamics.

Non-invasive detection of fluorescence from exogenous chromophores in the adult human brain.

This is the first report on results proving that fluorescence of exogenous dyes inside the human brain can be excited and detected non-invasively at the surface of the adult head. Boli of indocyanine green (ICG) were intravenously applied to healthy volunteers, and the passage of the contrast agent in the brain was monitored by detecting the corresponding fluorescence signal following pulsed laser excitation at 780 nm. Our hypothesis that the observed fluorescence signal contains a considerable cortical fraction was corroborated by performing measurements with picosecond temporal resolution and analyzing distributions of times of arrival of photons, hence taking advantage of the well-known depth selectivity of that method. Our experimental findings are explained by Monte Carlo simulations modeling the head as a layered medium and taking into account realistic bolus kinetics within the extra- and intracerebral compartment. Although a particular non-specific dye (ICG) was used, the results clearly demonstrate that fluorescence-mediated imaging of the adult human brain is generally feasible. In particular, we will discuss how these results serve as proof of concept for non-invasive fluorescence brain imaging and may thus open the door towards optical molecular imaging of the human brain.

Bed-side assessment of cerebral perfusion in stroke patients based on optical monitoring of a dye bolus by time-resolved diffuse reflectance.

We present a minimally invasive optical method, that is, multi-channel time-domain diffuse near-infrared reflectometry of the head to assess cerebral blood perfusion that is applicable at the bed-side and repetitively at short intervals. Following intravenous injection of an ICG bolus, its transit through intra- and extracerebral tissue is monitored based on changes in moments of distributions of times of flight of photons, recorded with a 4-channel instrument simultaneously on both hemispheres. In healthy volunteers, we found that variance of distributions of times of flight of photons is well suited to assess latency and initial slope of the increase in absorption of intracerebral tissue due to the bolus. We successfully applied our method in two patients demonstrating a reversible cerebral perfusion deficit in an ischemic stroke patient who was treated by thrombolysis and in another patient with a permanent impaired unilateral perfusion due to ipsilateral internal carotid artery occlusion. In either case, we observed a difference in bolus transit time between the hemispheres. In the stroke patient, this difference resolved when re-evaluated 1 day after thrombolysis. The study demonstrates the necessity of a technique with sub-nanosecond time resolution to allow for depth discrimination if clinical perfusion monitoring of cerebrovascular diseases is addressed by optical methods.

Ultrasound perfusion imaging of cysts in the midbrain.

AIM: Cerebral ultrasound perfusion examinations have been focused on the diencephalic plane. We describe perfusion abnormalities in the mesencephalon. METHOD: Phase-Inversion-Harmonic-Imaging (PIHI, Siemens Sonoline Elegra) was performed in a patient with ponto-mesencephalic cysts, that was defined by MRI. The signal increase (bolus kinetics, SonoVue) was quantified for peak-signal-increase (PSI) and time-to-peak (TTP). RESULTS: PSI and TTP could be quantified for the ipsi- and contralateral posterior cerebral arteries, the ipsilateral middle cerebral artery, the mesencephalon and the ipsilateral temporal lobe. The contrast agent influx was diminished or extinct in the area of the cysts. PSI and TTP delineated the area of the cystic structure well. CONCLUSION: PIHI in the mesencephalic plane is feasible and allows identification of pathological structures within the mesencephalon.

Cross talk in the Lambert-Beer calculation for near-infrared wavelengths estimated by Monte Carlo simulations.

Using the modified Lambert-Beer law to analyze attenuation changes measured noninvasively during functional activation of the brain might result in an insufficient separation of chromophore changes ("cross talk") due to the wavelength dependence of the partial path length of photons in the activated volume of the head. The partial path length was estimated by performing Monte Carlo simulations on layered head models. When assuming cortical activation (e.g., in the depth of 8-12 mm), we determine negligible cross talk when considering changes in oxygenated and deoxygenated hemoglobin. But additionally taking changes in the redox state of cytochrome-c-oxidase into account, this analysis results in significant artifacts. An analysis developed for changes in mean time of flight--instead of changes in attenuation--reduces the cross talk for the layers of cortical activation. These results were validated for different oxygen saturations, wavelength combinations and scattering coefficients. For the analysis of changes in oxygenated and deoxygenated hemoglobin only, low cross talk was also found when the activated volume was assumed to be a 4-mm-diam sphere.

Determining changes in NIR absorption using a layered model of the human head.

A theoretical approach is presented to determine absorption changes in different compartments of a layered structure from distributions of times of flight of photons. In addition resulting changes in spatial profiles of time-integrated intensity and mean time of flight are calculated. The capability of a single-distance, time-domain method to determine absorption changes with depth resolution is tested on a layered phantom. We apply this method to in vivo measurements on the human head (motor stimulation, Valsalva manoeuvre) and introduce a small-sized time-domain experimental set-up suitable for bedside monitoring.

Spontaneous low frequency oscillations of cerebral hemodynamics and metabolism in human adults.

UNLABELLED: We investigated slow spontaneous oscillations in cerebral oxygenation in the human adult's visual cortex. The rationale was (1) to demonstrate their detectability by near infrared spectroscopy (NIRS); (2) to analyze the spectral power of as well as the phase relationship between the different NIRS parameters (oxygenated and deoxygenated hemoglobin and cytochrome-oxidase; oxy-Hb/deoxy-Hb/Cyt-ox). Also (3) influences of functional stimulation and hypercapnia on power and phase shifts were investigated. The results show that-in line with the literature-low frequency oscillations (LFO) centred around 0.1 s(-1) and even slower oscillations at about 0.04 s(-1) (very low frequency, VLFO) can be distinguished. Their respective power differs between oxy-Hb, deoxy-Hb, and Cyt-ox. Either frequency (LFO and VLFO) is altered in magnitude by functional stimulation of the cortical area examined. Also we find a change of the phase shift between the vascular parameters (oxy-Hb, tot-Hb) and the metabolic parameter (Cyt-ox) evoked by the stimulation. It is shown that hypercapnia attenuates the LFO in oxy-Hb and deoxy-Hb. CONCLUSIONS: (1) spontaneous vascular and metabolic LFO and VLFO can be reproducibly detected by NIRS in the human adult. (2) Their spectral characteristics and their response to hypercapnia are in line with those described in exposed cortex (for review see (Hudetz et al., 1998)) and correspond to findings with transcranial doppler sonography (TCD) (Diehl et al., 1995) and fMRI (Biswal et al., 1997). (3) The magnitude of and phase relation between NIRS-parameters at the LFO may allow for a local noninvasive assessment of autoregulatory mechanisms in the adult brain.

Somatosensory evoked fast optical intensity changes detected non-invasively in the adult human head.

This work is the first to report optical intensity changes (DeltaI/I approximately 0.05%) with a latency between 60 and 160ms after electrical median nerve stimulation at 5Hz detected non-invasively through the intact adult human skull in volunteers. The signal is localised and reproducible when measuring at the same position on successive examinations. Compared to previous reports of fast optical changes in the human adult by a single group (Psychophysiology, 32 (1995) 505) the here reported changes are much smaller. They are in line with results from a photon transport calculation on a head model employing data from exposed cortical tissue. The origin of the signal found here is still unclear, however, they might be the non-invasive equivalent to the scattering changes found in exposed cortical tissue studies (J. Neurophysiol., 78 (1997) 1707).

Near-infrared spectroscopy: does it function in functional activation studies of the adult brain?

Changes in optical properties of biological tissue can be examined by near-infrared spectroscopy (NIRS). The relative transparency of tissues including the skull to near-infrared light is the prerequisite to apply the method to brain research. We describe the methodology with respect to its applicability in non-invasive functional research of the adult cortex. A summary of studies establishing the 'typical' response in NIRS vascular parameters, i.e. changes in the concentration of oxygenated and deoxygenated haemoglobin, over an activated area is followed by the validation of changes in the cytochrome-oxidase redox state in response to a visual stimulus. Proceeding from these findings a rough mapping of this metabolic response over the motion-sensitive extrastriate visual area is demonstrated. NIRS measures concentration changes in deoxygenated haemoglobin [deoxy-Hb] which are assumed to be the basis of fMRI BOLD contrast (blood oxygenation level-dependent). The method is therefore an excellent tool to validate assumptions on the physiological basis underlying the fMRI signal, due to its high specificity as to the parameters measured. Questions concerning the concept of 'activation'/'deactivation' and that of the linearity of the vascular response are discussed. To challenge the method we finally present results from a complex single-trial motor paradigm study testing the hypothesis, that premotor potentials (contingent negative variation) can be examined by functional techniques relying on the vascular response. Some of the work described here has been published elsewhere.

Estimation of megacity growth: simple rules versus complex phenomena.

"The growth of large urban aggregates (megacities) is analogous to the development of self-organized structures known in physics. Using empirical data about changes in the built-up areas of different cities as input, the self-organizing model employed here suggests that megacities evolve towards a hierarchical form of spatial organization, and provides estimates of the size of subclusters that compose the urban aggregate.... The model has been validated by reproducing the evolution of the Berlin area over a period of 35 years (1910-45). Using the same assumptions, the evolution of the built-up area of Daegu (Korea) is simulated up to the year 2010."

[The effect of vitamin A and beta-carotene on the vitamin E status, ejaculation parameters and health of boar used for insemination]. / Einfluss von Vitamin A und beta-Carotin auf Vitamin E-Haushalt, Ejakulatparameter und Gesundheit beim Besamungseber.

In this study consequences of vitamin A-supplementation to the vitamin E-status was investigated in the boar. Three groups of boars, each with 9 animals were fed over a period of seven month with 30000 I.E. Vit. A/kg concentrate (group A), 90 mg b-carotene + 1000 I.E. Vit. A/kg (group B) and 1000 I.E. Vit. A/kg (group C). Every boar was given 100 mg Vit. E/kg plus 50 ml soybean oil/kg to induce oxidative stress. After four month group C showed a higher amount of tocopherol in serum (p < 0.05). The amount of tocopherol in serum of the group B were exactly between group A and C. The amount of retinol in serum of the group C began to decrease after three month due to the high reserve capacity of the liver (p < 0.01). The retinyl ester in serum reflected the state of supply. 90 mg b-carotene led to an efficiency of 15000 I.E. Vit. A. The vitamin antagonism between Vit. A and Vit. E is not based on an antagonism of the intestinal resorption. There was no influence on the daily sperm production caused by different supplementations. The sperm quality was lowered in group C; the number of defective sperm increased (p < 0.001). The supplementation of soybean oil lead to an increase of the saturated fatty acids in the fatty acid pattern of the sperm cells. The increase of saturated fatty acids was the lowest in group C that showed the highest amount of tocopherol in serum.