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A modified rat model of middle cerebral artery thread occlusion under electrophysiological control for magnetic resonance investigations
Abstract
Previous magnetic resonance (MR) investigations of middle cerebral artery (MCA) occlusion in rats were limited by the lack of early post-occlusion MR measurements and/or electrophysiological monitoring. Therefore, we have developed a technique which allows to perform MCA occlusion inside the magnet under simultaneous recording of EEG and direct current (DC) potentials for monitoring the ischemic insult. Rats underwent intraluminal thread occlusion of the right MCA inside the MR tomograph via a catheter extension device, while EEG and DC potentials were recorded by non-magnetic graphite electrodes. The thread was slowly advanced until electrophysiological changes appeared. Diffusion-weighted MR images (DWI) were obtained before and repeatedly after MCA occlusion for up to 7 h. Thereafter, rat brains were frozen in situ or fixed by transcardiac perfusion and investigated by biochemical and histological techniques. In 15 of 18 animals (83%), MCA thread insertion caused immediate EEG changes and a negative DC potential shift at 4.4 +/- 1.8 min (mean +/- SD) after occlusion. In all animals with electrophysiological changes, signal intensity of DWI began to increase within the MCA territory at 12-14 min post-occlusion (the end of the first measurement), and continued to rise throughout the observation period. Ischemia was confirmed by demonstrating focal areas of energy depletion on ATP images. In the animals without electrophysiological changes, DWI or biochemical alterations were absent or confined to the central part of caudate-putamen. The histological lesion area of successfully occluded animals amounted to 70.1 +/- 5.8% of the ipsilateral hemisphere at the level of caudate-putamen.(ABSTRACT TRUNCATED AT 250 WORDS)
... However, other investigators have not supported these conclusions and the discussions for these variations were focused on degree of silicone coating of the thread, 3-0 or 4-0 filament size, length of the filament, and body weight of animals (Garcia, 1993;Holland et a l 1993). It is interesting to note that when comparing two uncoated 4-0 nylon monofilaments from different manufacturers, that subtle differences in material Kohno et al., 1995) occlusion during MRI (see section 1.3.4), achieved successful occlusions in 88% of animals without subarachnoid haemorrhage (Li et ah 1998). ...
... These data indicate that DW I monitors very early disturbances of ion pumps, at times when such changes are thought to be potentially reversible. Autoradiographic techniques for measuring CBF, ATP, glucose, lactate and pH have been used to study the spatial relationship between DW I changes and metabolism following MCAO in the rat (Kohno et al. 1995). In contrast to diffusion-weighted changes observed in global ischaemia (Busza et al. 1992;Verheul et al. 1992), Kohno et al. reported that during the early phase of cerebral ischaemia (30 minutes) the area of hyperintensity seen on DWI was significantly larger than the region of ATP depletion, although it matched the area exhibiting tissue acidosis (Kohno et al. 1995). ...
... Autoradiographic techniques for measuring CBF, ATP, glucose, lactate and pH have been used to study the spatial relationship between DW I changes and metabolism following MCAO in the rat (Kohno et al. 1995). In contrast to diffusion-weighted changes observed in global ischaemia (Busza et al. 1992;Verheul et al. 1992), Kohno et al. reported that during the early phase of cerebral ischaemia (30 minutes) the area of hyperintensity seen on DWI was significantly larger than the region of ATP depletion, although it matched the area exhibiting tissue acidosis (Kohno et al. 1995). This difference became progressively smaller with the evolution of the lesion, such that by 7 hours the area of tissue damage, as indicated by DWI, was identical to the region of ATP depletion and histological infarction. ...
Following a stroke, the ability to discriminate between areas of non-recoverable tissue and potentially salvageable tissue remains a goal of diagnostic imaging. There have been a number of recent developments both in nuclear medicine and in magnetic resonance imaging (MRI) techniques, which may have potential to achieve this goal. This thesis describes the use of these techniques for the evaluation of focal pathophysiology in a rat model of model cerebral artery occlusion (MCAO). Methods were described to use autoradiographic markers to study cerebral blood flow (99mTc-hexamethylpropylene amine oxime) and cerebral hypoxia (125I-iodoazomycin arabinoside) simultaneously following MCAO in the rat. The uptake of these tracers was compared to MRI measurements of the apparent diffusion coefficient of water (ADC) and to histology. It was shown that the combined use of 125I-IAZA or ADC imaging with perfusion imaging may allow differentiation between areas of oligaemic misery perfusion, ischaemic misery perfusion and ischaemic lesion core. MRI was used to examine the effects of cerebral tissue anisotropy on calculation of the spatial distribution of ischaemia in the rat using ADC measurements. Demonstration of grey matter anisotropy led to the use of rotationally invariant ADC measurements which were shown to improve stroke lesion delineation. To investigate the acute changes in NMR parameters of diffusion, perfusion, T1 and T2 a remote controlled MCAO model in the rat was refined for a vertical 8.5T high field magnet. Combined perfusion and diffusion data distinguish between a "moderately affected area", with reduced perfusion but normal diffusion; and a "severely affected area", in which both perfusion and diffusion were significantly reduced. Two novel MRI observations were reported, namely a decrease in T2 and an increase in T1, both within the first few minutes of ischaemia. A rat model of oligaemic misery perfusion was developed in which a large region of homogeneously reduced blood flow was produced without cytotoxic oedema forming. Early NMR changes in CBF, T1 and T2 were noted without reduction in the ADC value. This approach may provide a model of penumbral flow, which may be of value in studying and evaluating neuroprotection.
... DieKohno et al., 1995a;Gyngell et al., 1994a). ...
... Ratten im Magneten durchzuführen (Kohno et al., 1995aGewebeareale signalangehoben bereits wenige Minuten nach Ischämiebeginn (Mintorovitch et al., 1991;Moseley et al., 1990) (s. Abbildung 22). ...
ZUSAMMENFASSUNG
Es ist bekannt, daß ischämische Hirninfarkte über die ersten Tage häufig ein Wachstum zeigen, das mit einer klinischen Verschlechterung einhergehen kann. Diese Schrift beschäftigt sich mit den pathophysiologischen Vorgängen, die für die Entwicklung und Ausbreitung von Hirninfarkten bedeutsam sind. Die Untersuchungen wurden an einem Rattenmodell mit Verschluß der mittleren Hirnarterie durchgeführt. Zur Anwendung kamen u.a. neue Methoden der Kernspintomographie und -spektroskopie, das biochemische Imaging zerebraler Metabolite, neue Verfahren der Bildanalyse, autoradiographische Messungen von Blutfluß und Glukoseumsatz, die in vivo- Chemilumineszenz zur Messung freier Sauerstoffradikale sowie elektrophysiologische Methoden.
In der Umgebung einer fokalen zerebralen Ischämie sind vorübergehende synchronisierte Zelldepolarisationen nachweisbar, die elektrophysiologisch Ähnlichkeit mit Spreading depression haben und zu einer Negativierung des Gleichspannungs- (DC)-Potentials führen. Diese Peri-Infarkt-Depolarisationen werden durch eine fokal erhöhte extrazelluläre Kaliumkonzentration oder exzessive Glutamatausschüttung ausgelöst, treten spontan und irregulär in der Infarktrandzone auf und wandern mit einer Geschwindigkeit von 3-4 mm/min über den Kortex. Sie sind – im Gegensatz zu Spreading depression – charakterisiert durch eine verzögerte Repolarisation und eine fehlende Blutflußantwort bei erhöhtem Stoffwechselbedarf. Peri-Infarkt- Depolarisationen sind deshalb von Episoden mit Gewebehypoxie begleitet. Serielle Messungen von diffusionsgewichteten MR-Bildern ergeben, daß mit jedem Durchlaufen von negativen DC-Potentialshifts eine Zunahme der Läsionsgröße sichtbar wird, die sich nur unvollständig zurückbildet. Es kommt also zu einem treppenförmigen Wachstum der ischämischen Läsion.
Peri-Infarkt-Depolarisationen sind assoziiert mit einem steilen Anstieg der Laktatkonzentration als Hinweis auf die Stimulation der anaeroben Glykolyse bei vermehrtem Glukoseverbrauch, wie anhand von MR-Protonenspektren gezeigt werden konnte. Die Frequenz ihres Auftretens korreliert mit der individuellen Infarktgröße. Werden zusätzliche Depolarisationen in der Umgebung eines Infarktes ausgelöst, vergrößert sich der ischämische Gewebeschaden. Dabei nimmt insbesondere der selektive Neuronenverlust in der Infarktrandzone zu. Proportional zu der Größenzunahme der Infarkte verschlechtert sich das funktionelle Outcome der Tiere. Eine frühe Reperfusion verhindert das Auftreten weiterer Depolarisationen und begrenzt den Gewebeschaden. Die Messung freier Radikale in der Infarktrandzone zeigt jedoch, daß die Reperfusion von einer fünffach erhöhten Radikalproduktion begleitet ist, die als Ursache des sogenannten Reperfusionsschadens angesehen werden kann.
Die dreidimensionale Rekonstruktion von Blutfluß (CBF) und Glukoseumsatz (CMRglc) ergibt, daß die Penumbra nach 1,5-stündiger Ischämie ein größeres Volumen umfaßt als der irreversibel geschädigte Infarktkern. In der Randzone der Infarkte kommt es kumulativ zu einer ausgeprägten Entkopplung von Blutfluß und Glukoseumsatz, die zum Teil auf die Stimulation der anaeroben Glykolyse durch die Depolarisationen zurückzuführen ist. Die Entkopplungsratio CMRglc/CBF ist auf das 5 bis 10fache der Norm gesteigert, so daß ein fokales Energieversagen den selektiven Neuronenverlust in der Randzone von Infarkten erklärt.
Die Korrelation von MR-Bildern mit der Verteilung zerebraler Metabolite und dem lokalen Blutfluß ergibt, daß in den ersten Stunden um den Infarktkern herum eine Penumbrazone existiert, die charakterisiert ist durch einen intakten Energie-stoffwechsel, einen fast normalen Glukosegehalt und eine Laktazidose. In diffusionsgewichteten Bildern kommt die Penumbra ebenso wie der Infarktkern signalangehoben zur Darstellung, wobei die Gesamtgröße der Läsion in den ersten Stunden zunimmt. Die Blutfluß-Schwellenwerte, die nötig sind, um ein normales MR-Signal im Diffusionsbild, einen normalen Gewebe-pH bzw. einen intakten Energiestoffwechsel aufrechtzuerhalten, nehmen mit steigender Ischämiedauer zu. Die Penumbra ist also eine dynamisch sich verändernde Hirnregion in der Umgebung eines wachsenden Infarktkerns und wandert innerhalb von 7-8 Stunden in die Peripherie des Gefäßterritoriums. Zu diesem Zeitpunkt ist die Ausdehnung der ischämischen Läsion im Diffusionsbild identisch mit dem histologischen Infarktareal.
Peri-Infarkt-Depolarisationen können als Schlüsselphänomen der Infarktentwicklung und -ausbreitung angesehen werden (siehe Übersicht S. 120). Derzeit steht der methodisch einwandfreie Nachweis dieses Phänomens beim Menschen noch aus und sollte erbracht werden, bevor die (pharmakologische) Hemmung von Peri-Infarkt- Depolarisationen als ein vielversprechender therapeutischer Ansatz der Schlaganfall- behandlung klinisch erprobt werden kann.
... The macrospheres are then inserted in the ICA by a slow injection of 0.2 mL of saline blocking the blood flow to the MCA (32). Using other experimental stroke models, only few studies performing remote MCAo and monitoring the hyperacute phase of stroke in rodents have been published to date (46)(47)(48)(49)(50)(51)(52)(53)(54). In all those studies, remote occlusion of the MCA was performed using the permanent or temporal suture model, making the macrosphere model the first embolic stroke model to allow for remote occlusion. ...
... In all those studies, remote occlusion of the MCA was performed using the permanent or temporal suture model, making the macrosphere model the first embolic stroke model to allow for remote occlusion. Several modifications for optimizing this technique regarding an improvement of the success rate have been reported (46)(47)(48)(49)(50)(51)(52)(53)(54). However, compared to the macrosphere model, remote occlusion in the suture model is technically more difficult, thus requiring more training for the surgeon. ...
The main challenge of stroke research is to translate promising experimental findings from the bench to the bedside. Many suggestions have been made how to achieve this goal, identifying the need for appropriate experimental animal models as one key issue. We here discuss the macrosphere model of focal cerebral ischemia in the rat, which closely resembles the pathophysiology of human stroke both in its acute and chronic phase. Key pathophysiological processes such as brain edema, cortical spreading depolarizations (CSD), neuroinflammation, and stem cell-mediated regeneration are observed in this stroke model, following characteristic temporo-spatial patterns. Non-invasive in vivo imaging allows studying the macrosphere model from the very onset of ischemia up to late remodeling processes in an intraindividual and longitudinal fashion. Such a design of pre-clinical stroke studies provides the basis for a successful translation into the clinic.
... Owing to the heterogeneous nature of stroke and additional features such as age, sex, race and comorbidities that vary among patients, there is no ideal animal model of human stroke (Mergenthaler and Meisel, 2012); however, the developed models have tried to mimic as much as possible the human condition. Because the middle cerebral artery (MCA) is the most commonly affected artery among stroke patients (Rordorf et al., 1998), one of the most common models for stroke research is MCA occlusion (MCAO) in rodents (Tamura et al., 1981;Kohno et al., 1995). Although these models have helped to unveil the effects of cortical ischemia (Astrup et al., 1981;Neumann-Haefelin et al., 2000;Dijkhuizen et al., 2001), they mislead the researchers' attention to gray matter (GM) injury. ...
... Third, we found neurologic impairment only in 60% of operated marmosets, which is a low rate of success in comparison with the established stroke models for rodents (Tamura et al., 1981;Kohno et al., 1995) and marmosets (Marshall and Ridley, 2003;Freret et al., 2008). This lower success rate could be a disadvantage for applying this model for translational research in the development of neuroprotective drugs as well as cell therapies due to the requirement of a large number of animals. ...
White matter (WM) impairment and motor deficit after stroke are directly related. However, WM injury mechanisms and their relation to motor disturbances are still poorly understood. In humans, the anterior choroidal artery (AChA) irrigates the internal capsule (IC), and stroke to this region can induce isolated motor impairment. The goal of this study was to analyze whether AChA occlusion can injure the IC in the marmoset monkey. The vascular distribution of the marmoset brain was examined by colored latex perfusion and revealed high resemblance to the human brain anatomy. Next, a new approach to electrocoagulate the AChA was developed and chronic experiments showed infarction compromising the IC on magnetic resonance imaging (MRI) scanning (day 4) and histology (day 11). Behavioral analysis was performed using a neurologic score previously developed and our own scoring method. Marmosets showed a decreased score that was still evident at day 10 after AChA electrocoagulation. We developed a new approach able to induce damage to the marmoset IC that may be useful for the detailed study of WM impairment and behavioral changes after stroke in the nonhuman primate.
... The size of the infarct core during the initial 2 h of vascular occlusion increased more rapidly than that of the penumbra. Kohno et al., 1995a,b pMCAO filament Rats (W) 6, 18, 28, 39, 61, 83 and 105 min after MCAO. ...
... Until now, however, there are only a few papers that have reported on simultaneous use of MRI and EEG in animal models of cerebral ischemia. Early studies, in which EEG served to directly ascertain successful MCAO, have demonstrated that early (6–14 min after MCAO) EEG slowing and flattening coincided with increases of the DWI signal (Kohno et al., 1995a,b; Hoehn-Berlage et al., 1995). We speculate that incorporation of EEG recordings into MRI-based assessment of the diffusion–perfusion mismatch model, could contribute to improved differentiation of the penumbra from oligemia or the ischemic core. ...
... Body temperature was kept constant (37 ± 1 °C) by using a warm-water circuit integrated into the animal bed and a feedback-controlled heating pad for MRI acquisition and surgery, respectively. Focal brain ischemia was induced by transient occlusion of the middle cerebral artery (MCA) according to a previously described method 31,32 with an intraluminal monofilament (0.37 mm diameter filament, Doccol Corporation, Redlands, CA, USA). After 60 min of occlusion, the filament was withdrawn and the wound was sutured under local anesthesia. ...
Using superparamagnetic iron oxide nanoparticles (SPION) as a single contrast agent, we investigated dual contrast cerebrovascular magnetic resonance imaging (MRI) for simultaneously monitoring macro- and microvasculature and their association with ischemic edema status (via apparent diffusion coefficient [ADC]) in transient middle cerebral artery occlusion (tMCAO) rat models. High-resolution T1-contrast based ultra-short echo time MR angiography (UTE-MRA) visualized size remodeling of pial arteries and veins whose mutual association with cortical ischemic edema status is rarely reported. ΔR2–ΔR2*-MRI-derived vessel size index (VSI) and density indices (Q and MVD) mapped morphological changes of microvessels occurring in subcortical ischemic edema lesions. In cortical ischemic edema lesions, significantly dilated pial veins (p = 0.0051) and thinned pial arteries (p = 0.0096) of ipsilateral brains compared to those of contralateral brains were observed from UTE-MRAs. In subcortical regions, ischemic edema lesions had a significantly decreased Q and MVD values (p < 0.001), as well as increased VSI values (p < 0.001) than normal subcortical tissues in contralateral brains. This pilot study suggests that MR-based morphological vessel changes, including but not limited to venous blood vessels, are directly related to corresponding tissue edema status in ischemic stroke rat models.
... It may also allow correction for individual differences and facilitate quantification of changes. Methods have nonetheless been developed for performing ischemia inside the magnet (289)(290). ...
Acute brain damage after stroke produces remarkable changes in the brain that can be visualized with a variety of neuroimaging techniques. Some of these techniques are used in patients for diagnostic purposes and are now available to image the rodent brain. However, non-invasive imaging of the brain of rodents is challenging because of the size of the animals and the fact that their handling normally requires sedation or deep anesthesia to avoid stress and movement during image acquisition. In this chapter we will discuss the purpose, advantages and difficulties of applying Magnetic Resonance Imaging (MRI) (part A), Positron Emission Tomography (PET) (part B) and Single Photon Emission Computed Tomography (SPECT) (part C) to image the ischemic brain in rodents.
... The well-known character of DC shifts and polarization changes in nervous tissue during ischemia [38,39,41], reward [9,10] and effect of neuroprotective drugs [21,42,43] may serve as further evidence for neural rather than any other origin of the DC potential. ...
A pilot study has been made of the simultaneous DC
potential and total slow electrical activity changes dur-
ing modeling various metabolic and functional states
of the human brain. The multi-electrode DCEEG re-
cordings have been performed during the hyperven-
tilation (frequent deep one-minute long breathing
motions) and the hypoventilation (voluntary breath
holding). It has been shown that the ischemic state
occurring in hyperventilation is accompanied by the
negative shift of DC potential and increase in the
EEG rhythms amplitude. A distention of brain vessels
during hypoventilation (voluntary breath-hold) and
an improvement of blood supply and thus improve-
ment of vital and functional state of neurons gave rise
to an increase in the EEG rhythm amplitude too,
though against a background of a positive DC-poten-
tial shift. Obtained results are considered with con-
text the generation of the qualitatively different func-
tional states of brain cells during hyper- and hypo-
ventilation which is reflected in their resting potential
and activity. The conducted study show the prospects
for DCEEG and the method we used for DCEEG
data processing to understand the character of func-
tional and metabolic changes in the nervous tissue.
... Another major limitation of current experimental stroke models is the difficulty to combine them with in-bore occlusion of cerebral vessels. In-bore MCAO has been described, [37][38][39] although methods for this are technically very challenging, thus reducing reproducibility. The technique in the current study proved highly advantageous for performing in-bore occlusion. ...
Experimental models are essential for research on ischemic stroke, the second most common cause of death worldwide. The failure of clinical trials on neuroprotective treatment may be due in part to poor animal models. To push the translation of new therapies, we describe a new rat model that captures key elements of human brain ischemia. The model includes imaging and neurointerventional tools that represent the near future of clinical diagnosis and treatment of stroke.
Using Sprague-Dawley rats (n=26), we navigated a microwire with fluoroscopy and MRI guidance from the ventral tail artery to 2 different positions in the middle cerebral artery to establish local occlusion. Animals were scanned with 9.4-T MRI before occlusion, during ischemia, and after reperfusion.
We detected stroke lesions, corresponding to the level of occlusion, in all animals by diffusion-weighted and T2 images. We measured lesion volume (mm(3)±SD) on T2 scans at 24 hours to be 23.2±29.8 in the somatosensory cortex group and 107.9±80 in the striatum group.
We present a new rat model for focal stroke with the possibility to cause lesions in different regions of the brain under fluoroscopic and MRI control. The model will be highly useful for extended studies on the ischemic penumbra, alterations in neural connectivity, and for investigating neurotransmitter-mediated events and biochemical changes in the hyperacute phase of brain ischemia. Also, the model uses clinical routine microcatheters facilitating superselective administration of therapeutics directly to the cerebral circulation.
... If early reperfusion does not explain our findings, this implies that in the negative mismatch zone, cerebral areas have become severely damaged (as indicated by the ADC decrease and later infarction) without an underlying perfusion drop (normal MTT). At first sight this might seem surprising as it is has been shown in territorial ischaemia that ADC drops occur at a brain perfusion level below 34 ml/100 g/min in rats [33] and below 15–20 ml/ 100 g/min in gerbils [34]. On the other hand, ischaemia is obviously not the only " path " to cell damage. ...
Infarction typically develops within the borders of an initial hypoperfused tissue. We prospectively investigated whether in small subcortical stroke patients infarct growth can occur beyond the margins of the affected vascular territories.
In 19 consecutive patients, stroke MRI was performed within 14 h after ictus, and at days 2 and 6 (± 1). Size of diffusion and perfusion disturbances were determined. Infarct volume measured on T2-weighted images on day 6 was considered as imaging endpoint.
At the initial examination, the mean diffusion lesion [apparent diffusion coefficient (ADC) lesion size, 1.82 ± 1.2 ml] was larger (p = 0.0002) than the perfusion lesion [mean transit time (MTT) lesion size, 0.72 ± 0.69 ml]. Such an "inverse mismatch" (ADC lesion > MTT lesion) was present in 14/19 patients at baseline and in all patients on day 2. Final lesion volume at day 6 was 3.2 ± 1.6 ml which was larger than the initial perfusion deficit (p = 0.02).
In small subcortical ischaemic stroke "inverse mismatch" is frequent and infarction develops beyond the initial perfusion disturbance. This indicates that cytotoxic processes probably triggered by the infarct core are a dominant mechanism for lesion growth. Areas with normal perfusion but which are threatened by cytotoxic damage developing over several days seem prime targets for neuroprotective therapy.
... We used Eppendorf microloaders (Eppendorf AG, Hamburg , Germany) and a controlled heating procedure involving a MF-830 micro forge (Narishige Scientific Instrument Laboratory, Tokyo, Japan) for the manufacture of the filaments as presented in detail inFigure 1. We defined that the tip of the occluding filaments should have a diameter between 275 and 300 μm according to Kohno et al. [16]. The diameter of all microloaders was measured at the tip, behind the tip, 1 cm and 2 cm from the tip, using CAST ® software (Visiopharm A/S, Hørsholm , Denmark) (Additional file 1). the manufacturing of the occluding filaments. ...
We are currently investigating microglial activation and neuronal precursor cell (NPC) proliferation after transient middle cerebral artery occlusion (tMCAo) in rats. This study aimed: (1) to investigate differences in hippocampal NPC proliferation in outbred male spontaneously hypertensive rats (SHRs) and Sprague-Dawley rats (SDs) one week after tMCAo; (2) to present the practical use of the optical fractionator and 2D nucleator in stereological brain tissue analyses; and (3) to report our experiences with an intraluminal tMCAo model where the occluding filament is advanced 22 mm beyond the carotid bifurcation and the common carotid artery is clamped during tMCAo.
Twenty-three SDs and twenty SHRs were randomized into four groups subjected to 90 minutes tMCAo or sham. BrdU (50 mg/kg) was administered intraperitoneally twice daily on Day 4 to 7 after surgery. On Day 8 all animals were euthanized. NeuN-stained tissue sections were used for brain and infarct volume estimation with the 2D nucleator and Cavalieri principle. Brains were studied for the presence of activated microglia (ED-1) and hippocampal BrdU incorporation using the optical fractionator.
We found no significant difference or increase in post-ischemic NPC proliferation between the two strains. However, the response to remote ischemia may differ between SDs and SHRs. In three animals increased post-stroke NPC proliferation was associated with hippocampal ischemic injury. The mean infarct volume was 89.2 +/- 76.1 mm3 in SHRs and 16.9 +/- 22.7 mm3 in SDs (p < 0.005). Eight out of eleven SHRs had ischemic neocortical damage in contrast to only one out of 12 SDs. We observed involvement of the anterior choroidal and hypothalamic arteries in several animals from both strains and the anterior cerebral artery in two SHRs.
We found no evidence of an early hippocampal NPC proliferation one week after tMCAo in both strains. Infarction within the anterior choroidal artery could induce hippocampal ischemia and increase NPC proliferation profoundly. NPC proliferation was not aggravated by the presence of activated microglia. Intraluminal tMCAo in SHRs gave a more reliable infarct with neocortical involvement, but affected territories supplied by the anterior cerebral, anterior choroidal and hypothalamic arteries.
... Following the initial reports of a dramatic decrease in ADC following cerebral ischemia 5 considerable effort was invested in developing methods whereby occlusion could take place within the magnet. 104 It was thus possible to follow the time course in detail starting at the time of ischemia both for focal and global ischemia. It was found that the ADC can start to fall dramatically within 2 min of global ischemia being induced via cardiac arrest, 105 and that the decline is biphasic, with an initial rapid fall of about 40% within minutes, followed by a continuous gradual decline attributed to a drop in temperature. ...
This review examines the way in which microscopic tissue parameters can affect MR experiments which are sensitive to diffusion. The interaction between the intra‐ and extravascular as well as that between the intra‐ and extracellular spaces is examined. Susceptibility gradients due to the presence of deoxyhemoglobin can cause diffusion‐induced signal losses which are significant in functional magnetic resonance experiments, particularly at higher main magnetic field strengths. This is also true of the fast response that manifests itself as an early negative signal change in functional magnetic resonance experiments. The fields surrounding paramagnetic vessels are described and the way in which diffusion in these fields contributes to functional signal changes is examined. Flow in the capillary bed can be a confounding factor in experiments which aim to examine the diffusion characteristics of extravascular water. It is potentially also a method for assessing capillary perfusion. The intravoxel incoherent motion experiment is described in terms of how significantly this effect can influence diffusion attenuation curves from water. The major models for describing water diffusion in tissue are presented, as are the main experimental results that have contributed to an understanding of the mechanisms of diffusion contrast. The widely accepted view that changes in the diffusion characteristics are caused by a shift of water to the intracellular space and a concomitant change in extracellular tortuosity is examined critically. More recent experiments that indicate that a reduction in the intracellular diffusion may occur simultaneously with the cell swelling are described and their compatibility with existing models discussed. Copyright © 2001 John Wiley & Sons, Ltd.
Abbreviations used
2FDG‐6P fluor‐2‐deoxyglucose‐6‐phosphate
ADC apparent diffusion coefficient
BOLD blood oxygen level dependent
CNS central nervous system
CSF cerebro spinal fluid
DTI diffusion tensor imaging
DWI diffusion weighted imaging
fMR functional magnetic resonance
FR fast response
IVCM intravoxel coherent motion
IVIM intravoxel incoherent motion
MCAO middle cerebral artery occlusion
MRI magnetic resonance imaging
NMDA N ‐methyl‐ D ‐aspartate
NMR nuclear magnetic resonance
PFG pulsed field gradient
rBF regional blood flow
SGP short gradient pulse
TE echo time.
... MR detection of the earliest episodes of periinfarct CSD after acute ischemia is handicapped because of the lack of an appropriate model to produce ischemia within the magnet of the MR scanner. We have developed a modification of the suture model for MCAO in rats (Longa et aI., 1990) comparable to recently described models (Kohno et aI., 1995), which enables us to visualize CSD in the very beginning of acute ischemia using high-speed ADC measurements made during and af ter the MCA occlusion. ...
The suture model for middle cerebral artery occlusion (MCAO) was used to induce acute ischemia in rats remotely within a magnetic resonance (MRI) scanner. Serial MR diffusion weighted imaging (DWI) was performed during remote MCAO using an echo planar imaging technique. MR perfusion imaging was performed before and after occlusion using the bolus tracking technique. Transient apparent diffusion coefficient (ADC) changes were detected in six of seven rats as early as 2.7 +/- 1.5 min post MCAO. ADC values declined transiently to 70.1 +/- 6.0% of control and recovered to 95.5 +/- 6.8% of control within 3.3 +/- 2.9 min. These ADC changes propagated bidirectionally away from the ischemic core with a speed of 3.0 +/- 1.1 mm/min. Transient ADC decreases only occurred in ischemic areas characterized by moderately decreased tissue perfusion. Propagation toward cortical regions with severe tissue perfusion deficits was not detected. DWI can detect the earliest dynamic, reversible ADC changes in the ischemic tissue. The speed of propagation of the decreasing ADC wave, the waveform characteristics, and the occurrence in moderately perturbated tissue are compatible with cortical spreading depression.
Basic relevant information on methodologies used in neurological disease models can be extremely hard to find. Originally published in 2006, this important reference work contains 30 chapters from over 60 internationally recognized scientists and covers every major methodology and disease model used in neuroscience research. Divided into two major sections, the first deals with general methodologies in neuroscience research covering topics from animal welfare and ethical issues to surgical procedures, post-operative care and behavioral testing. Section two covers every major disease model including traumatic brain injury, ischemia and stroke, to Parkinson's, motor neurone disease, epilepsy and sleep disorders. Delivering critical methodological information and describing small animal models for almost all major neurological diseases, this book forms an essential reference for anyone working in neuroscience, from beginning students to experienced researchers and medical professionals.
Basic relevant information on methodologies used in neurological disease models can be extremely hard to find. Originally published in 2006, this important reference work contains 30 chapters from over 60 internationally recognized scientists and covers every major methodology and disease model used in neuroscience research. Divided into two major sections, the first deals with general methodologies in neuroscience research covering topics from animal welfare and ethical issues to surgical procedures, post-operative care and behavioral testing. Section two covers every major disease model including traumatic brain injury, ischemia and stroke, to Parkinson's, motor neurone disease, epilepsy and sleep disorders. Delivering critical methodological information and describing small animal models for almost all major neurological diseases, this book forms an essential reference for anyone working in neuroscience, from beginning students to experienced researchers and medical professionals.
Basic relevant information on methodologies used in neurological disease models can be extremely hard to find. Originally published in 2006, this important reference work contains 30 chapters from over 60 internationally recognized scientists and covers every major methodology and disease model used in neuroscience research. Divided into two major sections, the first deals with general methodologies in neuroscience research covering topics from animal welfare and ethical issues to surgical procedures, post-operative care and behavioral testing. Section two covers every major disease model including traumatic brain injury, ischemia and stroke, to Parkinson's, motor neurone disease, epilepsy and sleep disorders. Delivering critical methodological information and describing small animal models for almost all major neurological diseases, this book forms an essential reference for anyone working in neuroscience, from beginning students to experienced researchers and medical professionals.
Basic relevant information on methodologies used in neurological disease models can be extremely hard to find. Originally published in 2006, this important reference work contains 30 chapters from over 60 internationally recognized scientists and covers every major methodology and disease model used in neuroscience research. Divided into two major sections, the first deals with general methodologies in neuroscience research covering topics from animal welfare and ethical issues to surgical procedures, post-operative care and behavioral testing. Section two covers every major disease model including traumatic brain injury, ischemia and stroke, to Parkinson's, motor neurone disease, epilepsy and sleep disorders. Delivering critical methodological information and describing small animal models for almost all major neurological diseases, this book forms an essential reference for anyone working in neuroscience, from beginning students to experienced researchers and medical professionals.
Basic relevant information on methodologies used in neurological disease models can be extremely hard to find. Originally published in 2006, this important reference work contains 30 chapters from over 60 internationally recognized scientists and covers every major methodology and disease model used in neuroscience research. Divided into two major sections, the first deals with general methodologies in neuroscience research covering topics from animal welfare and ethical issues to surgical procedures, post-operative care and behavioral testing. Section two covers every major disease model including traumatic brain injury, ischemia and stroke, to Parkinson's, motor neurone disease, epilepsy and sleep disorders. Delivering critical methodological information and describing small animal models for almost all major neurological diseases, this book forms an essential reference for anyone working in neuroscience, from beginning students to experienced researchers and medical professionals.
Basic relevant information on methodologies used in neurological disease models can be extremely hard to find. Originally published in 2006, this important reference work contains 30 chapters from over 60 internationally recognized scientists and covers every major methodology and disease model used in neuroscience research. Divided into two major sections, the first deals with general methodologies in neuroscience research covering topics from animal welfare and ethical issues to surgical procedures, post-operative care and behavioral testing. Section two covers every major disease model including traumatic brain injury, ischemia and stroke, to Parkinson's, motor neurone disease, epilepsy and sleep disorders. Delivering critical methodological information and describing small animal models for almost all major neurological diseases, this book forms an essential reference for anyone working in neuroscience, from beginning students to experienced researchers and medical professionals.
ElectroEncephaloGraphy (EEG) may be helpful for studying and monitoring CerebroVascular Diseases (CVD). Transitory or persistent EEG modifications, related to functional and anatomical changes induced by cerebral ischemia, may indeed have both diagnostic and prognostic value in CVD patients. The use of electrophysiology to monitor CVD patients is based on the rationale that cerebral activity is strictly dependent on adequate cerebral blood perfusion. EEG changes can indeed be detected early - already after 10 s - after a significant decrease in cerebral perfusion, before reversible clinical symptoms and long before ischemic damage. A total arrest of cerebral blood flow induces instead—constantly and in few seconds—the suppression of cerebral electrical activity that, if flow is not rapidly restored, becomes permanent and it is followed by brain death. Furthermore, continuous EEG monitoring in acute CVD provides real-time and dynamic data on cerebral dysfunction and it may add useful information to clinical and imaging-based outcome prediction. In this chapter, EEG changes consequent both to cerebral ischemia and to hemorrhages will be described.
Filamentous occlusion of the middle cerebral artery (MCA) is probably the most frequently used model in experimental stroke research. It results in reproducible lesions in the cortex and striatum and can be used to induce both permanent and transient MCA occlusion (MCAo). It allows for the study of both acute stroke treatment as well as interventions that target stroke recovery. This chapter gives a practical “hands-on” approach for performing this model in mice and rats.
The EEG signal is the closest available measure of brain function and is exquisitely sensitive to ischemia. Data from animal models and from intraoperative monitoring suggest that continuous EEG monitoring is able disclose changes suggestive of impeding ischemia before infarction occurs. Several small studies in patients with aneurysmal subarachnoid hemorrhage now provide preliminary evidence that it can also be successfully applied to detection of delayed cerebral ischemia with a good sensitivity. These studies relied on quantitative analysis, underscoring the necessity to introduce such methods in clinical practice. Further improvements in quantitative EEG analysis, integration in a multimodality monitoring framework, and prospective validation studies are now required to confirm the utility of EEG monitoring for delayed cerebral ischemia detection.
This chapter describes several most common research techniques and methods developed by investigators to enhance the usefulness of the laboratory rat. Wild rats are aggressive and much more difficult to handle than domesticated laboratory rats. Investigators have designed several devices to safely handle wild rats in the laboratory. Dim red light can be used to calm wild rats and facilitate their handling and restraint in laboratory environments. Wild rats can be safely removed from cages using heavy cloth bags that the animals readily enter. Identification of individual rats or groups of rats is often required in biomedical research studies. Cage cards may be all that is necessary for many studies but rigorous individual identification is often required for drug safety studies done according to Good Laboratory Practice (GLP) regulations and for other types of experiments. Individual identification may also help prevent problems associated with inadvertent switching of cage cards or accidents such as escape of similar colored animals. Procedures for collection of bodily fluids and principles of drug or test substance administration are also described. Moreover, specialized techniques for cardiovascular and neurophysiology research are illustrated.
Krueger K, Busch E. Protocol of a thromboembolic stroke model in the rat: Review of the experimental procedure and comparison of models. Invest Radiol 2002;37:600–608. Rationale and Objective. Different models to study the pathophysiology of cerebral ischemia and to evaluate therapeutic strategies exist. Described is the detailed procedure of a thromboembolic stroke model in the rat most closely resembling human embolic stroke and compare the model to other equivalent rodent models. Materials and Methods. An evaluation of a new thromboembolic stroke model was performed on 35 male Wistar rats. After preparation of the carotid artery, a catheter was introduced into the external carotid artery. During injection of autologous, fibrin-rich emboli into the internal carotid artery the common carotid artery was temporarily occluded. Regional cerebral blood flow (rCBF) and lesion size were determined. Results. Twelve fibrin-rich blood clots of 1.5 mm in length were necessary in order reliably to occlude the origin of the middle cerebral artery. A stable decrease of rCBF and lesion size was confirmed by autoradiography, diffusion, and perfusion MRI, TTC-staining, biochemical imaging, and histology. Conclusion. In this animal model, the situation of human cerebral ischemia is simulated closely. The model is suitable for investigations of the pathophysiology of stroke and facilitates studies on the effects of thrombolytic therapy.
Perfusion-weighted imaging (PWI), using the method of arterial spin tagging, is strongly T1-dependent. This translates into a high field dependency of the perfusion signal intensity. In order to determine the expected signal improvement at higher magnetic fields we compared perfusion-weighted images in rat brain at 4.7 T and 7 T. Application of PWI to focal ischemia and functional activation of the brain and the use of two different anesthetics allowed the observation of a wide range of flow values. For all these (patho-)physiological conditions switching from 4.7 T to 7 T resulted in a significant increase of mean perfusion signal intensity by a factor of 2.96. The ratio of signal intensities of homotopic regions in the ipsi- and contralateral hemisphere was field-independent. The relative contribution of a) T1 relaxation time, b) net magnetization, c) the Q-value of the receiver coils and d) the degree of adiabatic inversion to the signal improvement at higher field strength were discussed. It was shown that the main parameters contributing to the higher signal intensity are the lengthening of T1 and the higher magnetization at the higher magnetic field.
The 45-T Hybrid Magnet will use NbTi and NbâSn Cable-In-Conduit Conductors (CICC) operating at 10 kA to produce a field of 14-T on axis in a 710-mm diameter magnet bore. A candidate NbâSn wire has been evaluated and selected for this application. However, the copper stabilizer of some wires has been shown to be susceptible to contamination, from unknown sources, which results in low Residual Resistivity Ratios (RRR). Here the authors report a characterization of wire given abbreviated heat treatment schedules designed to improve the RRR. It is shown that the RRR is readily improved by modifying the heat treatment schedule while critical current density remains above specified minimums. RRR and critical current density as a function of heat treatment and critical current density as a function of axial strain are reported.
Diffusions- und perfusionsgewichtete magnetresonanztomographische Techniken werden zunehmend in der Akutdiagnostik zerebraler Ischämien eingesetzt. Die pathophysiologischen Hintergründe von Signaländerungen in der diffusionsgewichteten MR-Bildgebung (DW-MRT) sind komplex, und der klinische Einsatz erfordert eine detaillierte Kenntnis der Ursachen von DW-MRT Signaländerungen.
Untersuchungen an verschiedenen tierexperimentellen Modellen zeigen, dass eine Wasserverschiebung von extra- nach intrazellulär die wesentliche Grundlage von Signalanstiegen in der DW-MRT darstellt. Solche Wasserverschiebungen treten unter verschiedenen pathophysiologischen Bedingungen auf und können sowohl Ausdruck einer reversiblen Membrandepolarisation wie bei der Spreading depression sein oder einen irreversiblen Prozeß einleiten wie bei der anoxischen Depolarisation. Bei der zerebralen Ischämie ist der Signalanstieg in der DW-MRT Ausdruck einer zytotoxischen Zellschwellung, und die DW-MRT ist die einzige nichtinvasive Methode zu deren Nachweis. Da die zytotoxische Zellschwellung am Beginn der ischämischen Kaskade steht, weist die DW-MRT ischämisches Gewebe zu einem Zeitpunkt nach, zu dem konventionelle T2-gewichtete MR-Sequenzen und die zerebrale Computertomographie noch einen unauffälligen Befund ergeben.
Die perfusionsgewichtete MR-Bildgebung (PW-MRT) ist eine Methode zur semiquantitativen Bestimmung der Gewebeperfusion und stellt eine komplementäre Methode bei der Akutdiagnostik der zerebralen Ischämie im tierexperimentellen und humanen Bereich dar.
Die technischen Grundlagen der DW-MRT und PW-MRT werden dargestellt und die Korrelation der MR-Signaländerungen mit metabolischen, hämodynamischen und histologischen Parametern wird diskutiert.
Summary
New MR sequences are an unique tool for the detection of acute and chronic cerebral ischemia and yield information not readily available from other techniques on the tissue status. The background of the signal changes in diffusion-weighted MRI (DWI) is complex. In general, DWI visualises cytotoxic cell swelling. Experimental studies on DWI have shown that DWI is sensitive to water shifts from the extracellular into the intracellular space. Water shifts within these compartments can occur with a large variety of pathophysiological events. They can be due to membrane depolarisations that can be either reversible such as with spreading depression or irreversible as with anoxic depolarisations.
Perfusion-weighted MRI (PWI) allows for the semiquantitative assessment of tissue perfusion and is a complementary technique to DWI.
DWI and PWI have opened new vistas for the evaluation of acute ischaemic injuries under experimental und clinical conditions. The correlation of DWI and PWI changes with metabolic, haemodynamic and histological parameters is discussed.
Oligemic regions, in which the cerebral blood flow is reduced without impaired energy metabolism, have the potential to evolve toward infarction and remain a target for therapy. The aim of this study was to investigate this oligemic region using various MRI parameters in a rat model of focal oligemia. This model has been designed specifically for remote-controlled occlusion from outside an MRI scanner. Wistar rats underwent remote partial MCAO using an undersize 0.2 mm nylon monofilament with a bullet-shaped tip. Cerebral blood flow (CBFASL), using an arterial spin labeling technique, the apparent diffusion coefficient of water (ADC), and the relaxation times T1 and T2 were acquired using an 8.5 T vertical magnet. Following occlusion there was a decrease in CBFASL to 35 ± 5% of baseline throughout the middle cerebral artery territory. During the entire period of the study there were no observed changes in the ADC. On occlusion, T2 rapidly decreased in both cortex and basal ganglia and then normalized to the preocclusion values. T1 values rapidly increased (within approximately 7 min) on occlusion. In conclusion, this study demonstrates the feasibility of partially occluding the middle cerebral artery to produce a large area of oligemia within the MRI scanner. In this region of oligemic flow we detect a rapid increase in T1 and decrease in T2. These changes occur before the onset of vasogenic edema. We attribute the acute change in T2 to increased amounts of deoxyhemoglobin; the mechanisms underlying the change in T1 require further investigation. Magn Reson Med 44:706–712, 2000. © 2000 Wiley-Liss, Inc.
In the last decade, in vivo MR methods have become established tools in the drug discovery and development process. In this review, several successful and potential applications of MRI and MRS in stroke, rheumatoid and osteo-arthritis, oncology and cardiovascular disorders are dealt with in detail. The versatility of the MR approach, allowing the study of various pathophysiological aspects in these disorders, is emphasized. New indication areas, for the characterization of which MR methods have hardly been used up to now, such as respiratory, gastro-intestinal and skin diseases, are outlined in a subsequent section. A strength of MRI, being a non-invasive imaging modality, is the ability to provide functional, i.e. physiological, readouts. Functional MRI examples discussed are the analysis of heart wall motion, perfusion MRI, tracer uptake and clearance studies, and neuronal activation studies. Functional information may also be derived from experiments using target-specific contrast agents, which will become important tools in future MRI applications. Finally the role of MRI and MRS for characterization of transgenic and knock-out animals, which have become a key technology in modern pharmaceutical research, is discussed. The advantages of MRI and MRS are versatility, allowing a comprehensive characterization of a diseased state and of the drug intervention, and non-invasiveness, which is of relevance from a statistical, economical and animal welfare point of view. Successful applications in drug discovery exploit one or several of these aspects. In addition, the link between preclinical and clinical studies makes in vivo MR methods highly attractive methods for pharmaceutical research. Copyright © 1999 John Wiley & Sons, Ltd.
The significance of NMR water diffusion measurements performed at short diffusion times (<10 ms) for brain tissue is examined. An apparent restriction to diffusion for both healthy and cytotoxically edematous tissue is shown: cytotoxic edema lengthens the diffusion time at which this phenomenon is visible. The dramatic reduction in apparent diffusion coefficient (ADC) observed in the core of cytotoxic edema is explained in terms of the enclosure of extracellular water in non-contiguous pockets in conjunction with the shift of water from the extra-to the intracellular space. The model presented provides an explanation for the ADC reduction without recourse to changes in the cell membrane permeability to water, or unrealistic values for the extra- and intracellular diffusion coefficients.
The interleaved-spiral magnetic resonance imaging (MRI) technique was implemented and optimized on a Bruker Biospec 47/30
scanner. The method gives rise to high-resolution images with a time saving factor of up to 8, as compared to the conventional
approach. A multifunctional pulse sequence for the fast interleaved-spiral MRI was composed. These functions include spin
intensity imaging, transverse relaxation timeT
2 and apparent diffusion-weighted imaging. The method was used to obtain the dynamic responses of a rat brain during ischemia.
Filamentous occlusion of the middle cerebral artery (MCA) is probably the most frequently used model in experimental stroke
research. It results in reproducible lesions in the cortex and striatum and can be used to induce both permanent and transient
MCAO. It allows for the study of both acute stroke treatment as well as interventions that target stroke recovery. This chapter
gives a practical “hands-on” approach for performing this model in mice and rats.
Key wordsBrain ischemia-Focal ischemia-Middle cerebral artery occlusion-Filament model
Acute brain damage after stroke produces remarkable changes in the brain that can be visualized with a variety of neuroimaging
techniques. Some of these techniques are used in patients for diagnostic purposes and are now available to image the rodent
brain. However, noninvasive imaging of the brain of rodents is challenging because of the size of the animals and the fact
that they normally need to be anesthetized to avoid stress and movement during image acquisition. In this chapter, we will
discuss the purpose, advantages, and difficulties of applying Magnetic Resonance Imaging (MRI) (part A) and Positron Emission
Tomography (PET) (part B) to image the ischemic brain in rodents.
Key wordsADC-DTI-DWI-fMRI-MRI-PET-PWI-Rodents-Stroke
We report the technical details and validation of an improved rat model for thromboembolic stroke and rt-PA induced reperfusion, which closely resembles clinical embolic stroke. The middle cerebral artery (MCA) was proximally occluded by injection of twelve medium sized (1.5×0.35 mm), fibrin-rich autologous blood clots. On inspection, densely packed clot material was found at the ipsilateral MCA origin in all untreated animals. Autoradiographic rCBF measurements showed severe ischemic deficit throughout the ipsilateral MCA territory in untreated animals. The volume in which flow values fell below 30 ml/100 g per min was 54±14% of the hemispheric volume. In all rt-PA treated animals the proximal MCA was recanalised, and the volume with flow values below 30 ml/100 g per min was reduced to 29±17%. Histological findings paralleled the spatial spread of the CBF deficit. The rat model presented is well-suited for investigations of the specific pathophysiology of thromboembolic stroke. Furthermore it allows detailed studies of thrombolytically induced reperfusion, beyond the question of successful recanalisation.
Stroke is a major cause of mortality and disability. The management with thrombolytic therapy has to be initiated within 3-4 h and is associated with limitations like increased risk of intracranial hemorrhage and progression of cerebral injury. Immunophilin inhibitors such as cyclosporine A and tacrolimus have been shown to afford neuroprotection by improving neurological functions and infarct volume in models of ischemic stroke. In the present study, the effect of rapamycin in middle cerebral artery occlusion (MCAo) model of ischemic stroke was evaluated. Ischemic stroke was induced in rats by occluding the MCA using the intraluminal thread. After 1 h of MCAo, animals were administered rapamycin (50, 150, 250 μg/kg, i.p.). After 2 h of occlusion, reperfusion was done. Thirty minutes after reperfusion, animals were subjected to diffusion-weighted magnetic resonance imaging for assessment of protective effect of rapamycin. Twenty-four hours after MCAo, motor performance was assessed, the animals were euthanized and the brains were removed for estimation of malondialdehyde, glutathione, nitric oxide and myeloperoxidase. Significant improvement was observed with rapamycin 150 and 250 μg/kg in percent infarct area, apparent diffusion coefficient and signal intensity as compared to vehicle treated group. Rapamycin treatment ameliorated motor impairment associated with MCAo and significantly reversed the changes in levels of malondialdehyde, glutathione, nitric oxide and myeloperoxidase. The results of the present study indicate neuroprotective effect of rapamycin in MCAo model of stroke. Therefore, rapamycin might be considered as a therapeutic strategy for stroke management.
Original experimental studies in nonhuman primate models of focal ischemia showed flow-related changes in evoked potentials that suggested a circumferential zone of low regional cerebral blood flow with normal K(+) homeostasis, around a core of permanent injury in the striatum or the cortex. This became the basis for the definition of the ischemic penumbra. Imaging techniques of the time suggested a homogeneous core of injury, while positing a surrounding 'penumbral' region that could be salvaged. However, both molecular studies and observations of vascular integrity indicate a more complex and dynamic situation in the ischemic core that also changes with time. The microvascular, cellular, and molecular events in the acute setting are compatible with heterogeneity of the injury within the injury center, which at early time points can be described as multiple 'mini-cores' associated with multiple 'mini-penumbras'. These observations suggest the progression of injury from many small foci to a homogeneous defect over time after the onset of ischemia. Recent observations with updated imaging techniques and data processing support these dynamic changes within the core and the penumbra in humans following focal ischemia.
Because there is no background signal from xenon in biological tissue, and because inhaled xenon is delivered to the brain by blood flow, we would expect a perfusion deficit, such as is seen in stroke, to reduce the xenon concentration in the region of the deficit. Thermal polarization yields negligible xenon signal relative to hyperpolarized xenon; therefore, hyperpolarized xenon can be used as a tracer of cerebral blood flow. Using a rat permanent right middle cerebral artery occlusion model, we demonstrated that hyperpolarized (129)Xe MRI is able to detect, in vivo, the hypoperfused area of focal cerebral ischemia, that is the ischemic core area of stroke. To the best of our knowledge, this is the first time that hyperpolarized (129)Xe MRI has been used to explore normal and abnormal cerebral perfusion. Our study shows a novel application of hyperpolarized (129)Xe MRI for imaging stroke, and further demonstrates its capacity to serve as a complementary tool to proton MRI for the study of the pathophysiology during brain hypoperfusion.
Protein changes induced by traumatic or ischemic brain injury can serve as diagnostic markers as well as therapeutic targets for neuroprotection. The focus of this chapter is to provide a representative overview of preclinical brain injury and proteomics analysis protocols for evaluation and discovery of novel biomarkers. Detailed surgical procedures have been provided for inducing MCAo and implantation of chronic indwelling cannulas for drug delivery. Sample collection and tissue processing techniques for collection of blood, CSF, and brain are also described including standard biochemical methodology for the proteomic analysis of these tissues.
The dynamics of proteomic analysis is a multistep process comprising sample preparation, separation, quantification, and identification of proteins. Our approach is to separate proteins first by two-dimensional gel electrophoresis according to charge and molecular mass. Proteins are then fragmented and analyzed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Identification of proteins can be achieved by comparing the mass-to-charge data to protein sequences in respective databases.
Neue Therapieverfahren beim akuten ischämischen Schlaganfall erfordern zunehmend eine bessere Information über das Ausmaß der instantanen Struktur- und Funktionsstörung, um Therapieziele definieren und die Einschätzung der Prognose verbessern zu können. Die neueren multimodalen Bildgebungsverfahren der Kernspintomographie erlauben eine präzise Beurteilung der aktuellen Schädigung und der Perfusionsläsion des Gehirns mit hoher Ortsauflösung während die zeitliche und funktionelle Störung praktisch nicht kontinuierlich messbar ist. In der vorliegenden Arbeit wurde untersucht, ob durch die Messung der interhemisphärischen Kopplung im EEG ein Meßverfahren zur Erfassung der funktionellen ischämischen Läsion möglich ist. An freiwilligen Probanden und Patienten mit akutem Schlaganfall wurde ein Schlaganfallscore (ESS) erhoben. Mittels Kernspintomographie wurde das Ausmaß der strukturellen ischämischen Läsion und das Volumen der Perfusionsstörung gemessen. Mehrfachableitungen des 2 Kanal-EEGs wurden einer speziellen Analyse der Frequenzkopplung (ISI) und zeitlichen Frequenzpräsentation in Form eines Verlaufsindex (HI) analysiert. Normalpersonen zeigten eine stabile, reproduzierbare Interhemisphärenkopplung im Frequenzbereich von 7-15 (ISI 81 SD 9,2). Mit zunehmendem Schweregrad des ischämischen Insultes nimmt der ISI signifikant ab. Es besteht eine enge Korrelation zwischen Ischämievolumen und ISI. Verlaufsuntersuchungen zeigten, dass die Erholungsphase nach Insult im EEG abgebildet wird. Auch die kontralaterale Hemisphäre zeigt oft mit einer Latenz von Stunden und Tagen eine Funktionsstörung. Die Ergebnisse zeigen, dass mit dem ISI ein einfaches bedside-EEG Monitoring möglich ist und aktuelle ischämische Perfusionsdefizite detektiert werden können. Zusätzlich lassen sich kontralaterale Funktionsstörungen (Diaschisis) nachweisen, welche in der Bildgebung nicht präsent sind. Zusammen mit der Bildgebung bietet das Verfahren die Möglichkeit, das Stadium des Insultes besser und zeitnah abzubilden und damit Therapieeffekte zu erfassen. Die Störungen gehen vermutlich über die Läsionsseite hinaus, welches für die Einschätzung der Rehabilitation der gesunden Hemisphäre Bedeutung gewinnen könnte.
The potential of multiparametric MRI parameters for differentiating between reversibly and irreversibly damaged brain tissue was investigated in an experimental model of focal brain ischemia in the rat. The middle cerebral artery (MCA) was occluded by intraluminal suture insertion for 60 or 90 min, followed by 4.5 h of reperfusion. The apparent diffusion coefficient (ADC) of brain water, T-1 and T-2 relaxation times, and CBFi, an MR-derived index of cerebral perfusion, were repeatedly measured and correlated with the outcome from the ischemic impact. A novel user-independent approach for segmentation of ADC maps into classes of increasing injury was introduced to define regions of interest (ROIs) in which these parameters were evaluated. MCA occlusion led to a graded decline of ADC, which corresponded with both the severity of flow reduction and an increase in T-1 and T-2 relaxation times. Removal of the suture led to a triphasic restitution of blood flow consisting of a fast initial rise, a secondary decline, and final normalization. Postischemic reperfusion led to a rise of ADC irrespective of the duration of ischemia. However, the quality of recovery declined with increasing severity of the ischemic impact. Throughout the observation time, T-1 and T-2 showed a continuous increase, the intensity of which correlated with the severity of ADC decline during ischemia. Particularly with longer ischemia time, elevated T-2 in combination with reduced ADC yielded a lower probability of recovery during recirculation, while intraischemic perfusion information contributed less to the prediction of outcome. In conclusion, the combination of MR parameters at the end of ischemia correlated with the probability of tissue recovery but did not permit reliable differentiation between reversibly and irreversibly damaged tissue.
Animal stroke models have played a unique role in understanding normal and ischemic brain metabolism and function. Sophisticated methods for the quantitative mapping of local cerebral blood flow, glucose utilization, and protein synthesis were developed and validated in rats. Rat is the commonly used species in experimental stroke studies and is one of the most suitable animals. The use of small animals for stroke research studies provides advantages of lower cost and greater acceptability from an ethical perspective compared to larger animals. In contrast, the disadvantage of using small animals is the lissencephalic morphology of their brains; whereas, large animals have gyrencephalic brains and considerable amount of neocortex like humans. Setting high standards for experimental studies will improve their relevance to human stroke and will help in the development of novel therapies for stroke. Scientists should be cautious not to over-interpret experimental data. The widespread use of novel technologies with stroke models has improved their utility. Many critical discoveries in the stroke field have stemmed from experimental models. Genetically manipulated animals have opened a new approach to stroke pathophysiology. This rapidly developing field holds great potential for improving stroke care, despite its already well recognized limitations.
Middle cerebral artery occlusion was performed in rats while the animals were inside the nuclear magnetic resonance (NMR) tomograph. Successful occlusion was confirmed by the collapse of amplitude on an electrocorticogram. The ultrafast NMR imaging technique UFLARE was used to measure the apparent diffusion coefficient (ADC) immediately after the induction of cerebral ischemia. ADC values of normal cortex and caudate-putamen were 726 +/- 22 x 10(-6) mm2/s and 659 +/- 17 x 10(-6) mm2/s, respectively. Within minutes of occlusion, a large territory with reduced ADC became visible in the ipsilateral hemisphere. Over the 2 h observation period, this area grew continuously. Quantitative analysis of the ADC reduction in this region showed a gradual ADC decrease from the periphery to the core, the lowest ADC value amounting to about 60% of control. Two hours after the onset of occlusion, the regional distribution of ATP and tissue pH were determined with bioluminescence and fluorescence techniques, respectively. There was a depletion of ATP in the core of the ischemic territory (32 +/- 20% of the hemisphere) and an area of tissue acidosis (57 +/- 19% of the hemisphere) spreading beyond that of ATP depletion. Regional CBF (rCBF) was measured autoradiographically with the iodo[14C]antipyrine method. CBF gradually decreased from the periphery to the ischemic core, where it declined to values as low as 5 ml 100 g-1. When reductions in CBF and in ADC were matched to the corresponding areas of energy breakdown and of tissue acidosis, the region of energy depletion corresponded to a threshold in rCBF of 18 +/- 14 ml 100 g-1 min-1 and to an ADC reduction to 77 +/- 3% of control. Tissue acidosis corresponded to a flow value below 31 +/- 11 ml 100 g-1 min-1 and to an ADC value below 90 +/- 4% of control. Thus, the quantification of ADC in the ischemic territory allows the distinction between a core region with total breakdown of energy metabolism and a corona with normal energy balance but severe tissue acidosis.
NMR bolus track measurements were correlated with autoradiographically determined regional cerebral blood flow (rCBF). The NMR method is based on bolus infusion of the contrast agent gadolinium diethylenetriaminepentaacetate and high-speed T*2-sensitive NMR imaging. The first pass of the contrast agent through the image plane causes a transient decrease of the signal intensity. This time course of the signal intensity is transformed into relative concentrations of the contrast agent in each pixel. The mean transit time and relative blood flow and volume are calculated from such indicator dilution curves. We investigated whether this NMR technique correctly expresses the relative rCBF. The relative blood flow data, calculated from NMR bolus track experiments, and the absolute values of iodo[14C]antipyrine autoradiography were compared. A linear relationship was observed, indicating the proportionality of the transient NMR signal change with CBF. Excellent interindividual reproducibility of calibration constants is observed (r = 0.963). For a given NMR protocol, bolus track measurements calibrated with autoradiography after the experiment allow determination of absolute values for rCBF and regional blood volume.
The regional evolution of brain infarction was studied in Wistar rats submitted to remotely controlled thread occlusion of the middle cerebral artery. Occlusion was performed in the magnet of an NMR tomography system to allow continuous recording of diffusion-weighted images. After 30 min (n = 6) or 2 h (n = 9), cerebral blood flow was measured by [14C] iodoantipyrine autoradiography while the regional distribution of ATP, glucose, lactate, and pH was imaged using pictorial bioluminescence and fluoroscopic methods. In diffusion-weighted images, the hemispheric lesion area (HLA) at the level of caudate-putamen amounted to 54.2 +/- 10.9% after 30 min and to 67.0 +/- 5.9% after 2 h vascular occlusion. These areas corresponded to the regions exhibiting tissue acidosis (60.8 +/- 9.3% and 70.4 +/- 4.5%), but were clearly larger than those in which ATP was depleted (22.3 +/- 20.8% and 49.6 +/- 12.9% after 30 min and 2 h, respectively). The threshold of blood flow for the increase of signal intensity in diffusion-weighted images increased between 30 min and 2 h occlusion from 34 to 41 ml/100 g per minute, the threshold of acidosis from 40 to 47 ml/100 g per minute, and the threshold for ATP depletion from 13 to 19 ml/100 g per minute. Our study demonstrates that diffusion-weighted imaging detects both the core and the penumbra of the evolving infarction but is not able to differentiate between the two parts. It further shows that the ischemic lesion grows during the initial 2 h of vascular occlusion, and that the size of the infarct core increases more rapidly than that of the penumbra.
The classic concept of the viability thresholds of ischemia differentiates between two critical flow rates, the threshold of electrical failure and the threshold of membrane failure. These thresholds mark the upper and lower flow limits of the ischemic penumbra which is thought to suffer only functional but not structural injury. Recent studies of the functional and metabolic disturbances suggest a more complex pattern of thresholds. At declining flow rates, protein synthesis is inhibited at first (at a threshold of about 0.55 ml/gm/min), followed by a stimulation of anaerobic glycolysis (at 0.35 ml/gm/min), the release of neurotransmitters and the beginning disturbance of energy metabolism (at about 0.20 ml/min), and finally the anoxic depolarization (< 0.15 ml/gm/min). The penumbra, as defined by the classic flow thresholds, does not remain viable for extended periods. Since viability of the tissue requires maintenance of energy-dependent metabolic processes, penumbra is redefined as a region of constrained blood supply in which the energy metabolism is preserved. Imaging of the penumbra by combining autoradiographic cerebral blood flow measurements with bioluminescent images of adenosine triphosphate (ATP) demonstrates a gradual expansion of the infarct core (in which ATP is depleted) into the penumbra until, after a few hours, the penumbra has disappeared. It is suggested that the limited survival of the penumbra is due to periinfarct depolarizations, which result in repeated episodes of tissue hypoxia, because the increased metabolic workload is not coupled to an adequate increase of collateral blood supply. This explains pharmacological suppression of periinfarct depolarizations lowering the threshold of metabolic disturbances and reducing the volume of the ischemic infarct.
The quantitative NMR parameters T1, T2, rho, and apparent diffusion coefficient (ADC) were determined during the 7 h after middle cerebral artery occlusion in rats. In the normal caudate-putamen (CP), 869 +/- 145 ms and 72 +/- 2 ms for T1 and for T2, respectively, were found; the corresponding values for cortex were 928 +/- 117 ms and 73 +/- 2 ms. The ADC showed significant dependence on gradient direction: diffusion along x resulted in 534 +/- 53 microns 2/s (CP) and 554 +/- 62 microns 2/s (cortex), and along y in 697 +/- 58 microns 2/s (CP) and 675 +/- 53 microns 2/s (cortex). In the ischemic territory, a continuous increase over time of both relaxation times was observed in the CP, leading to an increase of 29 +/- 20% (T1) and 51 +/- 41% (T2) above control level. ADC dropped to 63 +/- 15% of control in the CP and to 74 +/- 4% of control in the temporal cortex. No significant change was noted in proton density during the observation period. Strongest ADC reduction was in the center of the ischemic territory (< or = 60% of control) surrounded by a region of lesser reduction (< or = 80% of control). During the early part of the study, the area of reduced ADC was larger than that of elevated relaxation times. Toward the end of the experiment, the area of increased relaxation times approached that of decreased ADC at < or = 80% of control. Good agreement of histological presentation of infarct with the total area of decreased ADC (< or = 80%) was demonstrated.
Following permanent occlusion of the left middle cerebral artery (MCA) in rats, electrophysiological and hemodynamic characteristics of the periinfarct border zone were investigated in sham-operated (n = 6), untreated (n = 6), and MK-801-treated (3.0 mg/kg; n = 6) animals. For this purpose, direct current potential (DC), EEG, and blood flow (laser-Doppler flowmetry) were recorded from the cortex in the periphery of the MCA territory. In sham-operated rats, a single negative cortical DC deflection was observed after electrocoagulation of the cortex, whereas in untreated MCA-occluded animals, three to eight transient DC deflections were monitored during the initial 3 h of ischemia. The duration of these cortical DC shifts gradually increased from 1.2 +/- 0.3 to 3.7 +/- 2.7 min (mean +/- SD; p less than 0.05) during this time. In animals treated intraperitoneally with MK-801 (3.0 mg/kg) immediately after MCA occlusion, the number of cortical DC shifts significantly declined to one to three deflections (p less than 0.005). The EEG of the treated animals revealed low-amplitude burst-suppression activity. In the untreated and treated experimental group, the reduction of cortical blood flow amounted to 69 +/- 25 and 49 +/- 13% of control, respectively. Despite the more pronounced cortical oligemia, MK-801 treatment resulted in a significant decrease of the volume of the ischemically injured tissue from 108 +/- 38.5 (untreated group) to 58 +/- 11.5 (p less than 0.05) mm3. Our results suggest that repetitive cortical DC deflections in the periinfarct border zone contribute to the expansion of ischemic brain infarcts.
A procedure for occluding the stem of the proximal middle cerebral artery of the rat is described. The operation is performed under anaesthesia through a small subtemporal craniectomy. After occlusion, 3 animals were perfused with carbon block and 8 with a FAM fixative (40% formaldehyde, glacial acetic acid, and methanol). The findings were compared with sham-operated animals. Carbon black studies demonstrated an area of impaired perfusion corresponding to the territory of the occluded artery in each animal. Neuropathological studies invariably showed that there was ischaemic brain damage in the cortex and basal ganglia. The frontal cortex was involved in every animal, as was the lateral part of the neostriatum; the sensorimotor and auditory cortex were involved in most animals, whereas the occipital cortex and medial striatum were involved only infrequently. The damage produced by ischaemia could be readily distinguished from the small local lesion seen at the surgical site in sham-operated animals. The ability to produce a consistent focal ischaemic lesion in the rodent brain provides a technical approach that is sufficiently reproducible to enable investigation of the pathophysiology of ischaemia using recently developed autoradiographic and neurochemical methods.
In the periphery of ischemic brain lesions, transient spreading depression-like direct current (DC) deflections occur that may be of pathophysiological importance for determining the volume of the ischemic infarct. The effect of these deflections on cerebral blood flow, tissue oxygen tension, and electrophysiology was studied in rats submitted to intraluminal thread occlusion of the middle cerebral artery (MCA) and compared with the changes following potassium chloride (KCl)-induced spreading depression of intact animals. Immediately after MCA occlusion, cortical laser-Doppler flow (LDF) in the periphery of the MCA territory sharply decreased to 35 +/- 14% of control (mean +/- SD; p < 0.05), tissue PO2 declined from 28 +/- 4 to 21 +/- 3 mm Hg (p < 0.05), and EEG power fell to approximately 80% of control. During 7-h occlusion, 3-11 DC deflections with a mean duration of 5.2 +/- 4.8 min occurred at irregular intervals, and EEG power gradually declined to 66 +/- 16% of control (p < 0.05). During the passage of DC deflections, LDF did not change, but PO2 further declined to 19 +/- 4 mm Hg (p < 0.05). KCl-induced depolarizations of intact rats were significantly shorter (1.4 +/- 0.5 min; p < 0.05) and were accompanied by a 43% increase in LDF (p < 0.05) and a slight but significant increase in tissue PO2 from 22 +/- 4 to 25 +/- 4 mm Hg (p < 0.05). The comparison of periinfarct and KCl-induced depolarizations demonstrates that oxygen requirements are not coupled to an appropriate flow response in the periinfarct zone with severely reduced blood flow.(ABSTRACT TRUNCATED AT 250 WORDS)
We have examined the incidence and size of infarction after occlusion of different portions of the rat middle cerebral artery (MCA) in order to define the reliability and predictability of this model of brain ischemia. We developed a neurologic examination and have correlated changes in neurologic status with the size and location of areas of infarction. The MCA was surgically occluded at different sites in six groups of normal rats. After 24 hr, rats were evaluated for the extent of neurologic deficits and graded as having severe, moderate, or no deficit using a new examination developed for this model. After rats were sacrificed the incidence of infarction was determined at histologic examination. In a subset of rats, the size of the area of infarction was measured as a percent of the area of a standard coronal section. Focal (1-2 mm) occlusion of the MCA at its origin, at the olfactory tract, or lateral to the inferior cerebral vein produced infarction in 13%, 67%, and 0% of rats, respectively (N = 38) and produced variable neurologic deficits. However, more extensive (3 or 6 mm) occlusion of the MCA beginning proximal to the olfactory tract--thus isolating lenticulostriate end-arteries from the proximal and distal supply--produced infarctions of uniform size, location, and with severe neurologic deficit (Grade 2) in 100% of rats (N = 17). Neurologic deficit correlated significantly with the size of the infarcted area (Grade 2, N = 17, 28 +/- 5% infarction; Grade 1, N = 5, 19 +/- 5%; Grade 0, N = 3, 10 +/- 2%; p less than 0.05). We have characterized precise anatomical sites of the MCA that when surgically occluded reliably produce uniform cerebral infarction in rats, and have developed a neurologic grading system that can be used to evaluate the effects of cerebral ischemia rapidly and accurately. The model will be useful for experimental assessment of new therapies for irreversible cerebral ischemia.
To develop a simple, relatively noninvasive small-animal model of reversible regional cerebral ischemia, we tested various methods of inducing infarction in the territory of the right middle cerebral artery (MCA) by extracranial vascular occlusion in rats. In preliminary studies, 60 rats were anesthetized with ketamine and different combinations of vessels were occluded; blood pressure and arterial blood gases were monitored. Neurologic deficit, mortality rate, gross pathology, and in some instances, electroencephalogram and histochemical staining results were evaluated in all surviving rats. The principal procedure consisted of introducing a 4-0 nylon intraluminal suture into the cervical internal carotid artery (ICA) and advancing it intracranially to block blood flow into the MCA; collateral blood flow was reduced by interrupting all branches of the external carotid artery (ECA) and all extracranial branches of the ICA. In some groups of rats, bilateral vertebral or contralateral carotid artery occlusion was also performed. India ink perfusion studies in 20 rats documented blockage of MCA blood flow in 14 rats subjected to permanent occlusion and the restoration of blood flow to the MCA territory in six rats after withdrawal of the suture from the ICA. The best method of MCA occlusion was then selected for further confirmatory studies, including histologic examination, in five additional groups of rats anesthetized with halothane. Seven of eight rats that underwent permanent occlusion of the MCA had resolving moderately severe neurologic deficits (Grade 2 of 4) and unilateral infarcts averaging 37.6 +/- 5.5% of the coronal sectional area at 72 hours after the onset of occlusion.(ABSTRACT TRUNCATED AT 250 WORDS)
A summary of animal experimental models for the production and treatment of global and regional cerebral ischemia is presented. The methodological peculiarities of the various procedures are reviewed and discussed with respect to the theoretical and clinical relevance.Copyright © 1991 S. Karger AG, Basel
The layering of a luciferin-luciferase solution on brain slices makes endogenous ATP visible. Rat brains, frozen in situ and sliced at 16 micrometer thickness at a temperature of--15 degrees C, were fixed by a ternary mixture of ethanol, formalin and dioxane at--20 degrees C for 15 min, and dried at 40 degrees C for 12 h. Luciferin, luciferase and a small quantity of magnesium sulfate were dissolved into an arsenate buffer solution containing 1% polyvinylpyrrolidone, 2% gelatin and 1% glycerol. The solution was then frozen into a column, sliced at 40 micrometer thickness at--15 degrees C and placed on the precooled brain slice. A luminiferous luciferin-ATP reaction begins when the brain slice reaches room temperature and persists for more than 10 min. This technique therefore makes possible the optical and/or photographic determination of the endogenous concentration of brain ATP. Capability of the technique is also demonstrated.
Focal brain ischemia was induced by middle cerebral artery occlusion in the rat. The volume of cerebral damage was determined 2 days later by MRI in vivo and in the same animals histologically. The edema volume as measured by MRI and the histologically determined infarction was highly correlated. As a consequence, the neuroprotective effect of the N-methyl-D-aspartate (NMDA) receptor antagonists CGP 40116 and MK 801 were similar with both methods. Excitotoxic neurodegeneration in the rat striatum was induced by direct injection of quinolinic acid. The degree of damage was evaluated in vivo 1 day later by quantitative MRI, and 7 days later by measuring the activities of neuronal marker enzymes choline acetyltransferase and glutamic acid decarboxylase. Striatal damage assessed using the three approaches was highly correlated. Cerebroprotective efficacy of the NMDA receptor antagonist CGP 40116 was indistinguishable based on all methods. MRI was more reproducible than the enzymatic methods and was faster and simpler than histologic examination for routine analysis of excitotoxic damage and cerebroprotection in vivo in a pharmaceutical research environment.
We examined serial changes of diffusion- (DWI) and T2-weighted (T2WI) magnetic resonance images 30 minutes to 3 hours after intraluminal suture occlusion of the middle cerebral artery (MCA) in eight rats and after sham occlusion in four. We correlated the abnormal areas on DWI and T2WI with postmortem areas of infarction determined by 2,3,5-triphenyltetrazolium chloride (TTC), 24 hours after the operation. The 30-minute DWI in each MCA-occluded rat demonstrated increased signal intensity in the ipsilateral MCA territory, while T2WI showed no changes. At 3 hours, the ipsilateral DWI signal intensity increased further and the area of abnormality slightly increased. In some animals, the 3-hour T2WI disclosed an area of hyperintensity significantly smaller than that seen on the 30-minute DWI. TTC staining demonstrated an extensive MCA infarction in all rats with permanent MCA occlusion, confirmed by hematoxylin and eosin staining. The percent infarcted area of coronal brain sections, as determined by TTC staining, correlated significantly with areas on similar DWI sections at both 30 minutes and 3 hours. Sham-occluded control animals did not display any changes on DWI, T2WI, or TTC staining. The present study suggests that DWI is a very sensitive modality for detecting early ischemic brain injury, being highly correlated with post-mortem area of infarction, and may be useful to assess pharmacologic intervention.
Gradient-recalled echo-planar (T2*-weighted) imaging was used to noninvasively monitor regional blood oxygenation state changes in real time during transient episodes of focal ischemia in cat brain. Varying ischemic intervals (12 s to 30 min) were caused by middle cerebral artery occlusion. A rapid signal drop was noted upon occlusion, due to deoxygenation of static blood in the ischemic tissues. Upon successful reperfusion, the signal intensity recovered immediately and increased above (overshot) the baseline level before slowly returning to normal. The "overshoot" response was strongly dependent on the duration of the ischemic interval and is thought to reflect reactive hyperemia.
It has become increasingly clear that a stroke lesion usually consists of a densely ischemic focus and of perifocal areas with better upheld flow rates. At least in rats and cats, some of these perifocal ("penumbral") areas subsequently become recruited in the infarction process. The mechanisms may involve an aberrant cellular calcium metabolism and enhanced production of free radicals. In general, though, the metabolic perturbation in the penumbra requires better characterization. The objective of this article was to define flow distribution in a rat model of reversible middle cerebral artery (MCA) occlusion, so as to allow delineation of the metabolic aberrations responsible for the subsequent infarction. We modified the intraluminal filament occlusion model recently developed by Koizumi et al. (1986), and described in more detail by Nagasawa and Kogure (1989), adopting it for use in both spontaneously breathing and artificially ventilated rats. Successful occlusion of the MCA (achieved in about 9/10 rats) was judged by unilateral EEG depression in ventilated rats, and neurological deficits, such as circling, in spontaneously breathing ones. CBF in the ipsilateral hemisphere was reduced to nearly constant values after 20, 60, and 120 min of occlusion, flow rates in the focus being about 10% and in the perifocal ipsilateral areas about 15-20% of control (contralateral side). When the filament was left in place (permanent occlusion) 2,3,5-triphenyl tetrazolium chloride (TTC) staining and histopathology after 24 h showed a massive infarct on the occluded side, extending from caudoputamen and overlaying cortex to the occipital striate cortex. Animals recirculated after 60 min of MCA occlusion, and allowed to survive 7 days for histopathology, showed infarction of the caudoputamen (lateral part or whole nucleus) in 5/6 animals and selective neuronal necrosis in one animal. The neocortex showed either infarcts, selective neuronal necrosis, or no damage. There was some overlap between neocortical areas which were infarcted and those which were salvaged by reperfusion. In general, though, both the CBF data and the recovery studies with a histopathological endpoint define large parts of the neocortex as perifocal (penumbral) areas which lend themselves to studies of metabolic events leading to infarction.
The sensitivity of diffusion-weighted MRI was compared to that of T2-weighted MRI following temporary middle cerebral artery occlusion (MCA-O) for 33 min followed by 4 h of reperfusion in rats. Diffusion-weighted spin-echo images using strong gradients (b value of 1413 s/mm2) demonstrated a significant increase in signal intensity in ischemic regions as early as 14 min after onset of ischemia in comparison to the normal, contralateral hemisphere (p less than 0.05). This hyperintensity returned to baseline levels during reperfusion. T2-weighted images showed no evidence of brain injury during the temporary occlusion. In three rats subjected to permanent MCA-O, diffusion-weighted MRI demonstrated an increased signal intensity on the first image following occlusion and continued to increase during the 4-h observation period. T2-weighted images failed to demonstrate significant injury until approximately 2 h after MCA-O. Signal intensity ratios of ischemic to normal tissues were greater in the diffusion-weighted images than in the T2-weighted MR images at all time points (p less than 0.05). Close anatomical correlation was found between the early and sustained increase in diffusion-weighted MRI signal intensity and localization of infarcts seen on post-mortem histopathology.
To develop a simple, relatively noninvasive small-animal model of reversible regional cerebral ischemia, we tested various methods of inducing infarction in the territory of the right middle cerebral artery (MCA) by extracranial vascular occlusion in rats. In preliminary studies, 60 rats were anesthetized with ketamine and different combinations of vessels were occluded; blood pressure and arterial blood gases were monitored. Neurologic deficit, mortality rate, gross pathology, and in some instances, electroencephalogram and histochemical staining results were evaluated in all surviving rats. The principal procedure consisted of introducing a 4-0 nylon intraluminal suture into the cervical internal carotid artery (ICA) and advancing it intracranially to block blood flow into the MCA; collateral blood flow was reduced by interrupting all branches of the external carotid artery (ECA) and all extracranial branches of the ICA. In some groups of rats, bilateral vertebral or contralateral carotid artery occlusion was also performed. India ink perfusion studies in 20 rats documented blockage of MCA blood flow in 14 rats subjected to permanent occlusion and the restoration of blood flow to the MCA territory in six rats after withdrawal of the suture from the ICA. The best method of MCA occlusion was then selected for further confirmatory studies, including histologic examination, in five additional groups of rats anesthetized with halothane. Seven of eight rats that underwent permanent occlusion of the MCA had resolving moderately severe neurologic deficits (Grade 2 of 4) and unilateral infarcts averaging 37.6 +/- 5.5% of the coronal sectional area at 72 hours after the onset of occlusion.(ABSTRACT TRUNCATED AT 250 WORDS)
We describe a new focal ischemia model consisting of unilateral middle cerebral artery occlusion with a silicone rubber cylinder attached to a nylon surgical thread inserted through the internal carotid artery in rats. Recirculation was accomplished by pulling the thread out of the artery. We evaluated the reliability of this model and studied the influence of reperfusion of the brain by measuring regional cerebral blood flow in 30 rats and by using conventional neuropathologic methods after different periods of occlusion in 48 rats. The anterior neocortex and the lateral part of the caudate putamen, which were supplied by the occluded middle cerebral artery, were the regions most frequently damaged. After 1 hour of occlusion in five rats, in the cortex supplied by the occluded artery mean +/- SD blood flow was 0.19 +/- 0.08 ml/g/min (approximately 15% of that in the corresponding region of five sham-operated control rats), and mild scattered ischemic cell change was observed. Three (n = 5) or six (n = 5) hours of occlusion reduced blood flow more severely and caused severe ischemic cell changes in the cortex supplied by the occluded artery in proportion to the duration of ischemia. Characteristically, in five rats subjected to 3 hours of occlusion followed by 3 hours of recirculation, blood flow was restored and spongy edematous change was observed in the cortex supplied by the recirculated artery. This change resulted in hypoperfusion of the neighboring cortical region surrounding the recirculated area. Our model should be useful in various investigations of the influence of reperfusion on focal ischemic brain injury.
Focal cerebral ischemia was induced by occlusion of the middle cerebral artery in rats. The volumetric assessment of infarcted tissue, 2 days following occlusion, was calculated from the examination of eight preselected coronal sections. Five differing rat strains were examined. A small and variable infarcted volume was seen in Wistar-Kyoto rats; Sprague-Dawley rats had a relatively large, but still variable, infarcted volume. Of the normotensive rat strains, the most reproducible volume of infarcted tissue was seen in Fischer-344 rats; also the absolute value of the infarcted volume did not vary from one series to another in this strain. Chronic arterial hypertension, studied in both normal and stroke-prone spontaneously hypertensive rats, was associated with significantly larger infarction volumes. Age does not change the volume of necrosis: Fischer-344 rats were studied at 3, 9, and 20 months of age, and no significant differences were noted between these ages. Experimental diabetes was induced by the administration of streptozotocin 3 days prior to middle cerebral artery occlusion. Severe hyperglycemia (greater than 400 mg/dl) was associated with a considerably increased volume of infarction. The variability of the resultant lesion is high in the most commonly studied strains, but our results suggest that, for studies in normotensive rats, the use of the Fischer-344 strain produces a standardized and repeatable infarction that may be significantly modified by experimental interventions. Age is not a factor that affects the occlusion-induced infarction; in contrast, both chronic arterial hypertension and experimental diabetes aggravate the histological consequences of middle cerebral artery occlusion in the rat. We conclude that quantitative histological evaluation of infarct size allows a meaningful assessment of the gravity of focal cerebral ischemia.
This investigation describes a surgical approach for ligation of the middle cerebral artery (MCA) in the young rat and evaluates consequences of the occlusion with a neurologic exam for motor deficits, Evans blue test for blood-brain barrier leaks, and light microscopy for histologic changes after 3 days. Evans blue extravasation and the lesion were limited to cortex at the burr hole site in occluded and sham operated rats. MCA occlusion beyond the point of origin of the striate branches in the young rat results in neither neurological deficits, dye markings, nor histologic changes in the distal vascular field to indicate an infarct. Apparently, the young rodent collateral supply maintains the tissue in a viable state.
The regional evolution of brain infarction was studied in Wistar rats submitted to remotely controlled thread occlusion of the middle cerebral artery. Occlusion was performed in the magnet of an NMR tomography system to allow continuous recording of diffusion-weighted images. After 30 min (n = 6) or 2 h (n = 9), cerebral blood flow was measured by [14C] iodoantipyrine autoradiography while the regional distribution of ATP, glucose, lactate, and pH was imaged using pictorial bioluminescence and fluoroscopic methods. In diffusion-weighted images, the hemispheric lesion area (HLA) at the level of caudate-putamen amounted to 54.2 +/- 10.9% after 30 min and to 67.0 +/- 5.9% after 2 h vascular occlusion. These areas corresponded to the regions exhibiting tissue acidosis (60.8 +/- 9.3% and 70.4 +/- 4.5%), but were clearly larger than those in which ATP was depleted (22.3 +/- 20.8% and 49.6 +/- 12.9% after 30 min and 2 h, respectively). The threshold of blood flow for the increase of signal intensity in diffusion-weighted images increased between 30 min and 2 h occlusion from 34 to 41 ml/100 g per minute, the threshold of acidosis from 40 to 47 ml/100 g per minute, and the threshold for ATP depletion from 13 to 19 ml/100 g per minute. Our study demonstrates that diffusion-weighted imaging detects both the core and the penumbra of the evolving infarction but is not able to differentiate between the two parts. It further shows that the ischemic lesion grows during the initial 2 h of vascular occlusion, and that the size of the infarct core increases more rapidly than that of the penumbra.
Focal cerebral ischemia causes rapid intensity changes in diffusion-weighted images (DWI) and elevated lactate as detected by localized proton spectroscopy (1H-MRS). To investigate whether such changes can also be evoked by perischemic depolarizations, we combined DWI and 1H-MRS measurements with DC potential recordings. About 40 min after occlusion of the middle cerebral artery in a rat, a negative DC deflection was observed indicating transient cell depolarization. Coincidentally with the depolarization a transient increase of the DWI signal intensity and a partially reversible increase of lactate occurred in the periphery of the ischemic territory. These results show that peri-ischemic depolarization, known to contribute to the evolution of cerebral infarction, evokes disturbances that can be detected by DWI and 1H-MRS.
Diffusion-weighted nuclear magnetic resonance imaging has been shown to detect early ischemia-related alterations in experimental stroke. This raises the question of whether the observed increase in signal intensity is correlated with changes in cerebral metabolism. After middle cerebral artery occlusion, nuclear magnetic resonance diffusion images were recorded and compared with the regional concentration of cerebral metabolites and with histology of identical planes.
Seven anesthetized Fischer rats were subjected to permanent occlusion of the middle cerebral artery. T1, T2, and diffusion images (b factors ranging from 0 to 1500 s/mm2) were measured in three to five planes after 7 hours. Thereafter, brains were frozen in situ for histology and quantitative bioluminescence imaging of ATP, glucose, lactate, and for fluorescence imaging of tissue pH.
Seven hours after middle cerebral artery occlusion, the apparent diffusion coefficient was reduced from 615 +/- 97 x 10(-6).mm2.s-1 (contralateral brain) to 359 +/- 42 x 10(-6).mm2.s-1 (ischemic brain; mean +/- SD, P < .01). A precise topical coincidence was demonstrated between changes in nuclear magnetic resonance diffusion images, pattern of histological damage, ATP-depleted areas, and local tissue acidosis, the lesion area amounting to between 24.1% and 27.6% of the hemisphere at the level of the caudate-putamen. The area of elevated brain lactate clearly exceeded the acidic core of the infarct and included the slightly alkaline border zone.
The data demonstrate that after 7-hour middle cerebral artery occlusion, the reduction of the apparent diffusion coefficient in nuclear magnetic resonance diffusion images reflects precisely the region of histological injury, breakdown of energy metabolism, and tissue acidosis.
Our purpose was to assess the effectiveness of middle cerebral artery occlusion in producing acute focal ischemia in the rat by the use of Koizumi's and Longa's methods, in which occlusion is achieved by passing a nylon thread into the internal carotid artery.
Cerebral blood flow was measured by using the hydrogen clearance method, and the brains were examined histologically to assess ischemic damage.
By Koizumi's method profound reduction in cerebral blood flow was achieved in 28 of 30 rats (93%). The mean cerebral blood flow in the middle cerebral artery territory was 10.7 (95% confidence interval, 9.9-11.5) ml/100 g per minute. By Longa's method reduction in cerebral blood flow was achieved in only 29 of 52 rats (56%), and in these animals mean cerebral blood flow was 33 (95% confidence interval, 28.3-33.7) ml/100 g per minute (p < 0.001 compared with Koizumi's method). Cerebral blood flow was reduced to < 16 ml/100 g per minute in only seven animals (24%).
By Koizumi's method the depth of ischemia is more profound, occlusion is achieved in a much higher proportion of cases, and the incidence of perforation of the intracranial internal carotid is much less frequent than by Longa's method.
Ischemic penumbra in a model of reversible middle cerebral artery occlusion in the rat
- Memezawa