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Evolution of Photochemically Induced Focal Cerebral Ischemia in the Rat: Magnetic Resonance Imaging and Histology
Abstract
Magnetic resonance imaging (MRI) is increasingly used to study the pathophysiological evolution of cerebral ischemia in humans and animals. We have investigated photochemically induced (rose bengal) focal cerebral ischemia, a relatively noninvasive, reproducible model for stroke, and compared the evolution of the ischemic response in vivo and postmortem with MRI and histology, respectively.
MR images weighted for T2, diffusion, and T2* and parallel histological sections stained with cresyl fast violet (CFV) and for glial fibrillary acid protein were obtained from 34 adult male Hooded Lister rats at seven time points (3.75 to 196 hours) after bilateral ischemia induction. From CFV histology, lesion volumes and cell counts were calculated; from diffusion-weighted and T2-weighted images, apparent diffusion coefficients and lesion volumes were determined.
Both MRI and histology revealed a well-defined lesion at 3.75 hours after irradiation and a consistent pattern of temporal evolution; lesion apparent diffusion coefficients decreased significantly by 3.75 hours, increased significantly by day 2, and correlated strikingly with the decline in lesion CFV-positive cell numbers. After day 2, astrocytes and connective tissue cells invaded the infarct. Throughout the time course, lesion volumes determined in vivo and postmortem (after shrinkage correction) agreed well.
MRI changes quantitatively reflect histopathology, revealing reproducible primary and secondary damage characteristics noninvasively. These changes essentially replicate those reported for other animal stroke models and clinically, emphasizing the value both of MRI and the photochemically induced focal cerebral ischemia model in stroke research.
... A range of studies have consistently demonstrated significant decreases in NAA levels in ischemic brain tissue. Studies on animal stroke models have shown that, like in our study, NAA is significantly reduced in the acute phases, only to recover over a time course of 14-28 days [22][23][24]. The reason behind this is mostly due to glial cell proliferation and tissue reorganisation, but also other acetylated compounds may contribute to the signal [17]. ...
... This makes the use of NAA measurements at late stages during infarct maturation, as a marker for treatment responses and infarct development, highly questionable. In the acute and early stages after infarction, NAA seems to reflect the amount of brain damage and neuronal death quite well, as also shown by Lee et al., who found that reduced NAA levels in the infarct correlate with neuronal eosinophilia and pyknosis [24]. This is in agreement with our data and suggests that NAA levels in the early infarct are a useful surrogate marker for neuronal death. ...
... Human studies have likewise demonstrated reductions in NAA several hours after the insult, which seem to stay low for weeks thereafter, although with some variation [10,[25][26][27]. This differs from rodent models where tissue reorganisation and NAA recovery seem to occur at a faster rate [22][23][24]. This is reproduced in our data, but since the purpose of this study is to evaluate whether NAA levels early after infarction (24 h) can be used as a surrogate marker for end-stage tissue damage, this should not affect the translational value of our data. ...
Background
Estimation of brain damage following an ischemic stroke is most often performed within the first few days after the insult, where large amounts of oedematous fluid have accumulated. This can potentially hamper correct measurement of infarcted area, since oedema formation poorly reflects infarct size. This study presents a non-invasive, easily applicable and reliable method to accurately predict long-term evolution and late-stage infarction.
Objective
We performed a longitudinal analysis of brain infarct evolution after MCAO in mice, in order to determine whether water-compensated N-Acetylaspartate (NAA) levels in the infarct area, measured 24 h after the insult, is a suitable marker for late-stage infarction and thereby prognosis.
Methods
Twenty mice were divided into 4 groups and scanned longitudinally at different time-points after MCAO, followed by euthanisation for histology: Group 1) MRI/MRS at day 1 after MCAO (n = 4), Group 2) MRI/MRS at days 1 and 7 after MCAO (n = 5), Group 3) MRI/MRS at days 1, 7, and 14 after MCAO (n = 3), and Group 4) MRI/MRS at days 1, 7, 14, and 28 after MCAO (n = 4). At days 1, 7, 14, and 28, NAA levels were correlated with histological determination of neuronal death based on Nissl and H&E stainings.
Results
Twenty-four hours after the insult, NAA levels in the infarcted area decreased by 35 %, but steadily returned to normal after 28 days. In the acute phases, NAA levels strongly correlated with loss of Nissl substance (r² = −0.874, p = 0.002), whereas NAA levels in later stages reflect glial metabolism and tissue reorganisation. Most importantly, NAA levels 24 h after MCAO was highly correlated with late stage infarction at days 14 and 28 (r² = 0.73, p = 0.01), in contrast to T2 (r² = 0.06, p = 0.59).
Conclusions
By using a fixed voxel, which is easily positioned in the affected area, it is possible to obtain reliable measures of the extent of neuronal loss at early time points independent of oedema and brain deformation. Importantly, NAA levels 24 h after MCAO accurately reflects late-stage infarction, suggesting that NAA is a useful prognostic biomarker early after an ischemic stroke.
... However, PTS does not mimic well all aspects of acute human stroke. PTS simultaneously induces cytotoxic (intracellular) and vasogenic (extracellular) edema with rapid breakdown of the blood-brain barrier [15,19,[45][46][47]; whereas, acute human stroke is characterized by a primary cytotoxic edema, followed by vasogenic edema in several hours [15,19,45]. Another disadvantage of PTS as compared with other focal stroke models is rather narrow or absent of the salvageable ischemic penumbra due to intense development of tissue necrosis and edema. ...
... In general, severe oxidative stress causes necrosis, whereas moderate stress can elicit apoptosis that predominate in the ischemic core and penumbra, respectively. The simultaneous degeneration of neurons, glial cells, and capillaries, and development of vasogenic and intracellular edema in the infarction core were observed at 4 h after PTS in the rat cerebral cortex at the histological and ultrastructural levels [40,47]. Photothrombotic infarct was suggested to be the result of occlusive vascular thrombosis due to platelet aggregation and platelet-endothelium interaction [22,34,35]. ...
... The role of platelet aggregation in PTS is debated. On one hand, platelet aggregation and platelet-containing thrombi were observed in many cases of cerebral photothrombosis [34,35,47]. Different anti-platelet drugs reduced the photothrombotic brain damage. ...
The search of effective anti-stroke neuroprotectors requires various stroke models adequate for different aspects of the ischemic processes. The photothrombotic stroke model is particularly suitable for the study of cellular and molecular mechanisms underlying neurodegeneration, neuroprotection, and neuroregeneration. It is a model of occlusion of small cerebral vessels, which provides detailed study of molecular mechanisms of ischemic cell death and useful for search of potential anti-stroke agents. Its advantages include well-defined location and size of ischemic lesion that are determined by the aiming of the laser beam at the predetermined brain region; easy impact dosing by changing light intensity and duration; low invasiveness and minimal surgical intervention without craniotomy and mechanical manipulations with blood vessel, which carry the risk of brain trauma; low animal mortality and prolonged sensorimotor impairment that provide long-term study of stroke consequences including behavior impairment and recovery; independence on genetic variations of blood pressure and vascular architecture; and high reproducibility. This review describes the current application of the photothrombotic stroke model for the study of cellular and molecular mechanisms of stroke development and ischemic penumbra formation, as well as for the search of anti-stroke drugs.
... Photothrombosis is a relatively simple method technically for producing rather non-invasively a focal infarct in experimental animals (Watson et al., 1985). However, the majority of previous studies using photothrombosis have produced rather large focal infarcts throughout the entire cortex that often include subcortical white matter (Dietrich et al., 1987a;Lee et al., 1996;Pierpaoli et al., 1993;Watson et al., 1985). A few studies have produced smaller infarcts or single vessel infarcts using photothrombosis (e.g. ...
... The photothrombotic stroke method uses a photo-activatable dye, Rose Bengal, to generate coagulation within vessels using experimental conditions that have generally resulted in a relatively large ischemic infarct within the entire depth of the cortex and often also white matter and subcortical structures (Dietrich et al., 1987a;Lee et al., 1996;Pierpaoli et al., 1993). Injection and illumination conditions were chosen to potentially produce minor strokes by selecting conditions that could enhance early platelet disaggregation and thrombus clearance as has been reported to be visualized directly using confocal microscopy (Zhang et al., 2005). ...
... 30-60 mW/cm 2 at 555 nm. Direct comparisons of intensities used between studies are difficult; intensities reported have ranged from 3 to 30 mW or 285 to 580 mW/cm2 for green laser/light (Dietrich et al., 1987b;Harrison et al., 2013;Kao et al., 2014;Zhang et al., 2005) and 100 to 580 mW/cm 2 for white light (Lee et al., 1996;Pevsner et al., 2001). The method involved using a halogen light source, heat filter and fibre optic cable with foil collimator positioned perpendicular to the thinned skull that was covered with an opaque mask with a 3 mm × 3 mm rectangular opening. ...
Background:
Transient ischemic attack, minor stroke and stroke recurrence need improved treatment but lack animal models for research. The aim was to modify photothrombosis methods thereby producing both a minor stroke (with adjacent mild damage) or a minor recurrent stroke.
New method:
A minor stroke, as detected using magnetic resonance imaging and histology, was produced using a low intensity beam of white light with a bright centre, a low dose of Rose Bengal and a short 5min illumination of thinned skull. A recurrent minor stroke was produced by repeating the procedure two days later except the cortical mask was positioned 1.5mm posteriorly.
Results:
The minor photothrombosis procedure produced a small superficial infarct surrounded by a region of scattered necrosis detected histologically. Marked hyperintensities in Diffusion weighted and T2 images identified the infarct. Peri-infarct regions with modest T2 increases corresponded to regions of scattered cell death. A recurrent minor photothrombosis produced additional damage in regions with overlapping mild injury.
Comparison with existing methods:
Previous photothrombosis methods usually produce large cortical infarcts with little penumbra. The current method produces small infarcts with diffuse mild peri-infarct ischemic injury that can be diagnosed using T2 imaging.
Conclusions:
The modified photothrombotic procedure will produce a minor stroke consisting of a small infarct in a region with marked diffusion and T2 hyperintensities and a peri-infarct region of selective necrosis with modest T2 changes. Minor recurrent stroke is readily produced but imaging is key for assessing size and location of each insult.
... The high metabolic demands of neurons make them particularly vulnerable to ischemic insult. In addition, cells killed or irreversibly damaged by the ischemic insult can release factors, including water (i.e., edema), which may spread into and harm nearby tissue that was not initially damaged by the stroke (Lipton, 1999;Witte and Stoll, 1997;van Bruggen et al., 1992;Lee et al., 1996). This process can be observed as delayed or ongoing cell death following the ischemic insult (Lipton, 1999;Witte and Stoll, 1997;Lee et al., 1996). ...
... In addition, cells killed or irreversibly damaged by the ischemic insult can release factors, including water (i.e., edema), which may spread into and harm nearby tissue that was not initially damaged by the stroke (Lipton, 1999;Witte and Stoll, 1997;van Bruggen et al., 1992;Lee et al., 1996). This process can be observed as delayed or ongoing cell death following the ischemic insult (Lipton, 1999;Witte and Stoll, 1997;Lee et al., 1996). Tissue regions that surround the ischemic infarct, which are at risk of ongoing tissue damage are often termed the peri-infarct zone (PIZ) (Carmichael, 2005;Katsman et al., 2003). ...
... Nonetheless, this is only one cellular marker and provides no information about the neurons in the PIZ. In addition to GFAP staining, H&E histology revealed a staining pattern characteristic of edema (diffuse and patchy eosin staining) immediately adjacent to the infarct as has been observed by others using this model (Lee et al., 1996). Nevertheless, the true extent to which edema and altered tissue biochemistry extends from the infarct is difficult to gauge with histology alone. ...
... The highest level of platelet aggregation occurs at the pial surface since it is exposed to the most direct light. It is possible to monitor the progression of the lesion via nuclear magnetic resonance imaging, and multiple MRI studies have plotted the time course of photothrombotic lesions under different parameters [68,70,88,113,123]. injected into the medial saphenous vein or the tail vein. ...
... In all of the above cases, the investigators exposed the brain to unnecessary wavelengths of light, which are not absorbed by the dye molecules, used an unnecessary period of illumination to induce infarcts, and did not perform controls to ensure there were no burns produced by the light source. There is no such thing as a "cold light source" [70]. Excess light energy does not simply disappear, in accordance with the law of conservation of energy. ...
... Excess light energy does not simply disappear, in accordance with the law of conservation of energy. In this case, unabsorbed light is converted to heat energy, which does an unknown amount of tissue damage separate from that caused by the photoexcited dye molecules [70]. ...
... 25 This is contrasted to simultaneous changes in MD and T2W that occur when photothrombosis directly occludes underlying capillary beds, initiating more rapid vascular leakage. 26,27 Finally, to ensure that T2W signal increase involved tissue infarction, we performed NeuN immunohistology on mice receiving occlusions to penetrating arterioles of similar flux, but sacrificed at varying post-occlusion times (Figure 3f). In agreement with MRI data, a gradual increase of infarct size was observed between 2 to 24 h (Figure 3d and e). ...
... This is contrasted to the simultaneous increase in T2 and MD contrast observed when direct capillary thrombosis is induced, i.e. earlier vascular leakage, and indicates a more gradual response to ischemia is occurring after penetrating arteriole occlusion. 26,27 Collectively, this suggests that ischemia caused by loss of flow to the targeted arteriole is the principal factor eliciting the observed microvascular responses. However, we cannot rule out the possibility that there was sub-threshold endothelial damage caused by photosensitization that augmented capillary responses to ischemia. ...
Small cerebral infarcts, i.e. microinfarcts, are common in the aging brain and linked to vascular cognitive impairment. However, little is known about the acute growth of these minute lesions and their effect on blood flow in surrounding tissues. We modeled microinfarcts in the mouse cortex by inducing photothrombotic clots in single penetrating arterioles. The resultant hemodynamic changes in tissues surrounding the occluded vessel were then studied using in vivo two-photon microscopy. We were able to generate a spectrum of infarct volumes by occluding arterioles that carried a range of blood fluxes. Those resulting from occlusion of high-flux penetrating arterioles (flux of 2 nL/s or higher) exhibited a radial outgrowth that encompassed unusually large tissue volumes. The gradual expansion of these infarcts was propagated by an evolving insufficiency in capillary flow that encroached on territories of neighboring penetrating arterioles, leading to the stagnation and recruitment of their perfusion domains into the final infarct volume. Our results suggest that local collapse of microvascular function contributes to tissue damage incurred by single penetrating arteriole occlusions in mice, and that a similar mechanism may add to pathophysiology induced by microinfarcts of the human brain.
... Commonly employed techniques for LV measurement include adapted use of imaging software (i.e. Adobe Photoshop CS ©, ImageJ) to manually outline the lesion with a tracing mechanism, such as the free-form or edge-detection selector tools (Di Stefano et al., 2000;Lee et al., 1996;Zivadinov et al., 2012). The metric area is then calculated based on the resolution of the image. ...
... Methods employed once areas of cross-sections are measured must represent the actual volume. Although Simpson's rule (Lee et al., 1996) is frequently used to estimate the volume size from slices in parallel planes, it does not provide a method for visual reconstruction of three dimensional volumes. Moreover, the volume reconstruction by cylindrical approximation (Goldberg et al., 1995) is crude and non-continuous. ...
Accurately measuring the volume of tissue damage in experimental lesion models is crucial to adequately control for the extent and location of the lesion, variables that can dramatically bias the outcome of preclinical studies. Many of the current commonly used techniques for this assessment, such as measuring the lesion volume with primitive software macros and plotting the lesion location manually using atlases, are time-consuming and offer limited precision. Here we present an easy to use semi-automated computational method for determining lesion volume and location, designed to increase precision and reduce the manual labor required. We compared this novel method to currently used methods and demonstrate that this tool is comparable or superior to current techniques in terms of precision and has distinct advantages with respect to user interface, labor intensiveness and quality of data presentation.
... Furthermore, the severity of the ischemic damage was quantified by computation of regional changes in Apparent Diffusion Coefficient (ADC). We compared our findings with those from earlier MRI experiments on the photochemical model (Van Bruggen et al., 1991; Pierpaoli et al., 1993; Lee et al., 1996), including our own (Lanens et al. 1993Lanens et al. , 1995). Part of this work was presented as an abstract and poster at the Fourth Scientific Meeting and Exhibition of the International Society for Magnetic Resonance in Medicine, New York 27/04–3/05/1996, p. 503. A. Van der Linden, M. Verhoye, M. de Ryck. ...
... In this scheme of CBF and ADC events, the ischemic penumbra in grey matter may lie between reductions of ADC by 10 and 25% (0.63–0.52), whereas ADC reductions of 40% and more (0.40 and less) correspond to irreversible damage in the ischemic core (the latter values are also obtained from brain tissue of dead rats (Hoehn-Berlage et al., 1995a,b)). Throughout our experiments, and in contrast with earlier studies (Van Bruggen et al., 1991; Pierpaoli et al., 1993; Lee et al., 1996 ), the ADC values for two contralateral (intact) cortical regions, homologous to the infarct core and medial infarct rim, were 0.46–0.60, representing reductions from the normal 0.70 value by 14 to 34%. ...
We studied the neuroprotective effect of lubeluzole, a NOS (nitric oxide synthase) pathway modulator, on the development of ischemic damage within the first six hours after a photochemically induced neocortical infarct in rats using diffusion-weighted MRI and Apparent Diffusion Coefficient (ADC) maps. A unilateral photochemical infarct was induced in the hindlimb sensorimotor neocortex of Wistar rats. One hour after infarction, rats received either vehicle (n=10) or lubeluzole (n=11; a 0.31 mg/kg i.v. bolus followed by a one-hour 0.31 mg/kg i.v. infusion). During the first six hours after infarct induction, multislice T2- and Diffusion-Weighted magnetic resonance images (MRI) were obtained to measure percent change of volume of ischemic damage, whereas regional ADC maps were used to measure time-dependent density of ischemic damage. Lubeluzole reduced the percent increase of volume of ischemic damage relative to baseline (at 1 h after infarct induction just before drug treatment), by 18% at 5 and 6 hrs after infarct induction. Lubeluzole attenuated the ADC decreases in the peripheral rim of the infarct, but left the ADC values in the core unaffected. In conclusion, the neuroprotectant lubeluzole attenuates growth of ischemic damage as well as its density in the periphery of a photochemically induced neocortical infarct in rats.
... Consiste en la inyección de un colorante fotoactivo (rosa de bengala), por vía intraperitoneal en ratones o por vía intravenosa en ratas, que, tras irradiar el cráneo intacto con un haz de luz a una longitud de onda específica, genera especies reactivas de oxígeno que producen la activación plaquetaria y la formación de un coágulo rico en plaquetas (Fig. 2d). Esto produce un infarto de pequeño tamaño en la región cerebral seleccionada, normalmente en el territorio de la arteria cerebral media, caracterizado por una rápida evolución de la lesión y escasa penumbra [56]. De entre sus ventajas cabe destacar la mínima invasividad de la cirugía, la elevada reproducibilidad de la lesión y una baja mortalidad. ...
Introduction:
Cerebrovascular disease is one of the leading causes of death, disability and dementia around the world. For the most common form of the disease, ischaemic stroke, there is only one drug available, tissue plasminogen activator, and few patients can benefit from this therapy because of the strict inclusion criteria established for its use. This circumstance makes it crucial to search for new forms of treatment to combat the sequelae of the disease, and this requires the development of new biomimetic models that allow for a better understanding of its evolution.
Development:
In this review, we update the platforms and models most widely used in recent years to study the pathophysiology of ischaemic stroke. On the one hand, we review the two- and three-dimensional platforms on which in vitro assays are carried out and, on the other, we describe the most commonly used in vivo experimental models and techniques for assessing ischaemic damage.
Conclusions:
The ultimate aim of developing good experimental models is to find new forms of treatment and thus improve patients' prognosis and quality of life. It is therefore important to generate new in vitro devices and to further refine in vivo models to enable a good clinical translation.
... Because of this intra-arterial occlusion, ischemic cell death progresses rapidly (Dietrich et al., 1986). Therefore, penumbra and collateral channel formation and reperfusion rarely occur (Lee et al., 1996). Research on changes in sensory-motor cortex mapping in response to stroke has exploited this feature (Harrison et al., 2013). ...
Background
Recently, the common marmoset (Callithrix jacchus) has attracted significant interest as a non-human primate stroke model. Functional impairment in non-human primate stroke models should be evaluated quantitatively and successively after stroke, but conventional observational assessments of behavior cannot fully fit this purpose. In this paper, we report a behavioral analysis using MarmoDetector, a three-dimensional motion analysis, in an ischemic stroke model using photosensitive dye, along with an observational behavioral assessment and imaging examination.
Methods
Ischemic stroke was induced in the left hemisphere of three marmosets. Cerebral infarction was induced by intravenous injection of rose bengal and irradiation with green light. The following day, the success of the procedure was confirmed by magnetic resonance imaging (MRI). The distance traveled, speed, activity time, and jumps/climbs were observed for 28 days after stroke using MarmoDetector. We also assessed the marmosets’ specific movements and postural abnormalities using conventional neurological scores.
Results
Magnetic resonance imaging diffusion-weighted and T2-weighted images showed hyperintense signals, indicating cerebral infarction in all three marmosets. MarmoDetector data showed that the both indices immediately after stroke onset and gradually improved over weeks. Neurological scores were the worst immediately after stroke and did not recover to pre-infarction levels during the observation period (28 days). A significant correlation was observed between MarmoDetector data and conventional neurological scores.
Conclusion
In this study, we showed that MarmoDetector can quantitatively evaluate behavioral changes in the acute to subacute phases stroke models. This technique can be practical for research on the pathophysiology of ischemic stroke and for the development of new therapeutic methods.
... Néanmoins, l'inconvénient majeur du modèle est la différence avec la pathologie de l'AVC. La lésion causée par la photothrombose présente simultanément un oedème cytotoxique et vasogénique, des lésions microvasculaires et une rupture de la BHE (Lee et al., 1996). Par ailleurs, ce modèle provoque une lésion ischémique très localisée, sans la zone de pénombre ischémique péri-infarct généralement présente dans la pathologie de l'AVC. ...
L'AVC ischémique est l'une des principales causes de décès et d'invalidité permanente dans le monde. Les processus inflammatoires induits par l'AVC ont été proposés comme des contributeurs clés de la physiopathologie de l'AVC ischémique. Alors que le rôle de la microglie dans l'AVC ischémique a été largement étudié, celles des macrophages associés aux bordures du SNC (BAMs) restent largement inconnues. Notre hypothèse, basée sur les études décrites précédemment et sur la localisation privilégiée des BAMs à l’interface entre le compartiment vasculaire et le parenchyme cérébral, était que les BAMs pouvaient jouer un rôle majeur dans la réponse inflammatoire déclenchée par l’AVC, via la médiation du recrutement et l’infiltration des leucocytes. De plus, sur la base d’études précédentes, nous avons fait l’hypothèse que les BAMs pourraient moduler les réponses inflammatoires induites par l'AVC de manière différente en fonction de l'état inflammatoire basal du cerveau, en particulier au cours du vieillissement.Pour tester cette hypothèse, nous avons étudié l'AVC chez des souris jeunes et âgées avec ou sans déplétion antérieur des BAMs. Nos résultats ont montré que le déficit fonctionnel de l'AVC était aggravé chez les souris âgées déplétées en BAMs. Cette aggravation du résultat fonctionnel s'accompagnait (i) d'une augmentation de l'expression de la P-sélectine endothéliale, (ii) d'une augmentation du roulement et de l'adhésion des leucocytes à la paroi vasculaire, et (iii) d’une infiltration accrue de leucocytes dans l'hémisphère lésé. Ces réponses immunitaires exacerbées étaient présentes à la fois dans la phase aiguë et subaiguë après le début de l'AVC, suggérant ainsi que la présence de BAMs assure un contrôle à long terme de la réponse immunitaire après un AVC. En utilisant le séquençage ARN à partir de BAMs isolés, nous montrons que les BAMs modifient leur phénotype transcriptomique au cours du vieillissement pour surexprimer des gènes impliqués dans la régulation de la réponse immunitaire innée et adaptative et de la présentation des antigènes.Nos résultats montrent que les BAMs acquièrent au cours du vieillissement un rôle central dans l’orchestration de la réponse neuroinflammatoire déclenchées par un AVC, et que leur présence garantit une bonne régulation de la réponse immunitaire.
... Rose Bengal solution (20 mg/kg) was administered through inguinal access of the left femoral vein. A longitudinal incision in the scalp of the animal was performed to expose the bregma and to allow a 15-min application of halogen cold light (diameter of 4.5 mm) on a spot on the left parietal bone 1 mm posteriorly and 1 mm laterally to the Bregma to induce an ischemic vascular lesion 49,50 . After the procedure the animal was sutured, left to recover from anesthesia and returned to the animals' facility. ...
There are no clinical interventions to prevent post-injury epilepsy, a common and devastating outcome after brain insults. Epileptogenic events that run from brain injury to epilepsy are poorly understood. Previous studies in our laboratory suggested Proechimys, an exotic Amazonian rodent, as resistant to acquired epilepsy development in post-status epilepticus models. The present comparative study was conducted to assess (1) stroke-related brain responses 24-h and 30 days after cortical photothrombosis and (2) post-stroke epilepsy between Proechimys rodents and Wistar rats, a traditional animal used for laboratory research. Proechimys group showed smaller volume of ischemic infarction and lesser glial activation than Wistar group. In contrast to Wistar rats, post-stroke decreased levels of pro-inflammatory cytokines and increased levels of anti-inflammatory mediators and growth factors were found in Proechimys. Electrophysiological signaling changes assessed by cortical spreading depression, in vitro and in vivo, showed that Wistar’s brain is most severely affected by stroke. Chronic electrocorticographic recordings showed that injury did not lead to epilepsy in Proechimys whereas 88% of the Wistar rats developed post-stroke epilepsy. Science gains insights from comparative studies on diverse species. Proechimys rodents proved to be a useful animal model to study antiepileptogenic mechanisms after brain insults and complement conventional animal models.
... Intravenous injection of a photosensitive dye (e.g., Rose Bengal or erythrosine B) and light illumination to the cortical cerebral artery induces in situ thrombosis, CBF reduction, and cerebral infarction. [248][249][250][251][252][253][254][255][256][257][258] Light can be illuminated through the thinned skull of the animal brain without craniotomy. Dye-light interaction generates reactive oxygen species that causes endothelial damage and platelet aggregation, leading to the formation of a platelet-rich thrombus at the site of the photochemical reaction. ...
Despite recent advances in recanalization therapy, mechanical thrombectomy will never be a treatment for every ischemic stroke because access to mechanical thrombectomy is still limited in many countries. Moreover, many ischemic strokes are caused by occlusion of cerebral arteries that cannot be reached by intra-arterial catheters. Reperfusion using thrombolytic agents will therefore remain an important therapy for hyperacute ischemic stroke. However, thrombolytic drugs have shown limited efficacy and notable hemorrhagic complication rates, leaving room for improvement. A comprehensive understanding of basic and clinical research pipelines as well as the current status of thrombolytic therapy will help facilitate the development of new thrombolytics. Compared with alteplase, an ideal thrombolytic agent is expected to provide faster reperfusion in more patients; prevent re-occlusions; have higher fibrin specificity for selective activation of clot-bound plasminogen to decrease bleeding complications; be retained in the blood for a longer time to minimize dosage and allow administration as a single bolus; be more resistant to inhibitors; and be less antigenic for repetitive usage. Here, we review the currently available thrombolytics, strategies for the development of new clot-dissolving substances, and the assessment of thrombolytic efficacies in vitro and in vivo .
... However, disadvantages of this stroke model are major differences from the ischemic stroke pathology. The injury caused by photothrombosis presents simultaneously early acute cytotoxic and vasogenic edema, microvascular injury and blood-brain barrier breakdown (Lee et al., 1996). Additionally, this model causes well-defined ischemic lesion without the penumbra area typically present in stroke pathology. ...
For the last two decades, researchers have placed hopes in a new era in which a combination of reperfusion and neuroprotection would revolutionize the treatment of stroke. Nevertheless, despite the thousands of papers available in the literature showing positive results in preclinical stroke models, randomized clinical trials have failed to show efficacy. It seems clear now that the existing data obtained in preclinical research have depicted an incomplete picture of stroke pathophysiology. In order to ameliorate bench-to-bed translation, in this review we first describe the main actors on stroke inflammatory and immune responses based on the available pre-clinical data, highlighting the fact that the link between leukocyte infiltration, lesion volume and neurological outcome remains unclear. We then describe what is known on neuroinflammation and immune responses in stroke patients, and summarize the results of the clinical trials on immunomodulatory drugs. In order to understand the gap between clinical trials and preclinical results on stroke, we discuss in detail the experimental results that served as the basis for the summarized clinical trials on immunomodulatory drugs, focusing on (i) experimental stroke models, (ii) the timing and selection of outcome measuring, (iii) alternative entry routes for leukocytes into the ischemic region, and (iv) factors affecting stroke outcome such as gender differences, ageing, comorbidities like hypertension and diabetes, obesity, tobacco, alcohol consumption and previous infections like Covid-19. We can do better for stroke treatment, especially when targeting inflammation following stroke. We need to rethink the design of stroke experimental setups, notably by (i) using clinically relevant models of stroke, (ii) including both radiological and neurological outcomes, (iii) performing long-term follow-up studies, (iv) conducting large-scale preclinical stroke trials, and (v) including stroke comorbidities in preclinical research.
... A further advantage is that it decreases the invasion and mortality rates. A significant disadvantage is that the ischemic injury is associated with early intracellular and extracellular edema formation (80), which differ from those observed in human stroke, as intracellular edema is the main indication of acute cerebral ischemia in humans (81). ...
Cerebral ischemia is a leading cause of mortality worldwide. Available treatments are mainly thrombolytic agents for restoring blood flow to the brain. However, this approach has a very narrow treatment window. Despite extensive research, there is still a need for further investigations to identify and develop novel treatment approaches. The present review aimed to summarize and discuss evidence from the literature regarding the best models with which to study cerebral ischemia and the available herbal sources that may provide potential treatment strategies for cerebral ischemia. The present review was based on research published between 1990 and 2020. Herbal remedies provide a promising research area that warrants further attention from researchers in the field. Different models have been used to investigate the pathophysiology of cerebral ischemia/reperfu-sion, and to examine various treatment approaches. The plant kingdom is rich in various phytochemicals with neuroprotective functions. From the literature search performed herein, it can be concluded that middle cerebral and bilateral common carotid artery occlusion models are the most convenient, cost-effective and easily reproducible models. A number of plants, particularly those from Southeast Asia, have used for cerebral ischemia research; however, many more need to be investigated, particularly plants from Africa.
... However, there are some discrepancies when compared to acute human stroke. While the latter is characterized primarily by cytotoxic edema in the earliest stages of ischemia [40], the injury caused by PTS presents simultaneously acute cytotoxic and vasogenic edema due to microvascular injury and blood-brain barrier (BBB) breakdown [41]. Additionally, considering this model causes ischemic lesion with well-defined boundaries, it consequently generates little or no penumbra and collateral blood flow, which is typically present in human ischemic stroke. ...
Stroke is the second-leading cause of death globally and the leading cause of disability in adults. Medical complications after stroke, especially infections such as pneumonia, are the leading cause of death in stroke survivors. Systemic immunodepression is considered to contribute to increased susceptibility to infections after stroke. Different experimental models have contributed significantly to the current knowledge of stroke pathophysiology and its consequences. Each model causes different changes in the cerebral microcirculation and local inflammatory responses after ischemia. The vast majority of studies which focused on the peripheral immune response to stroke employed the middle cerebral artery occlusion method. We review various experimental stroke models with regard to microcirculatory changes and discuss the impact on local and peripheral immune response for studies of CNS-injury (central nervous system injury) induced immunodepression.
... Among the advantages of this model is the possibility of creating small size infarcts to target specific regions [6]. However, there are intrinsic disadvantages of this model since, in contrast with human stroke pathophysiology, its nature is only occlusive, and no growth and maturation of the ischemic penumbra and local collateral flow/reperfusion can take place [101]. ...
Cerebral ischemia can occur at any stage in life, but clinical consequences greatly differ depending on the developmental stage of the affected brain structures. Timing of the lesion occurrence seems to be critical, as it strongly interferes with neuronal circuit development and determines the way spontaneous plasticity takes place. Translational stroke research requires the use of animal models as they represent a reliable tool to understand the pathogenic mechanisms underlying the generation, progression, and pathological consequences of a stroke. Moreover, in vivo experiments are instrumental to investigate new therapeutic strategies and the best temporal window of intervention. Differently from adults, very few models of the human developmental stroke have been characterized, and most of them have been established in rodents. The models currently used provide a better understanding of the molecular factors involved in the effects of ischemia; however, they still hold many limitations due to matching developmental stages across different species and the complexity of the human disorder that hardly can be described by segregated variables. In this review, we summarize the key factors contributing to neonatal brain vulnerability to ischemic strokes and we provide an overview of the advantages and limitations of the currently available models to recapitulate different aspects of the human developmental stroke.
... This hyperintense halo seen on DWI indicated the zone with cytotoxic edema and began to gradually reduce since day 3 post-stroke. MR images of photochemical stroke in a previous report also showed early increase in T 2 signal and decreased diffusion of water, indicating the simultaneous development of substantial vasogenic edema and ischemic infarction [39]. This pattern is different from that seen in human stroke, where infarcts develop with cytotoxic edema, followed by a vasogenic edema which is delayed by several hours [3]. ...
Photochemically induced cerebral ischemia is an easy-manipulated, reproducible, relatively noninvasive, and lesion controllable model for translational study of ischemic stroke. In order to longitudinally investigate the characterization of the model, magnetic resonance imaging, 18F-2-deoxy-glucose positron emission tomography, fluorescence, and bioluminescence imaging system were performed in correlation with triphenyl tetrazolium chloride (TTC), hematoxylin-eosin staining, and immunohistochemistry examinations of glial fibrillary acidic protein, CD68, NeuN, von willebrand factor, and α-smooth muscle actin in the infarct zone. The results suggested that the number of inflammatory cells, astrocytes, and neovascularization significantly elevated in peri-infarct region from day 7 and a belt of macrophage/microglial and astrocytes was formed surrounding infarct lesion at day 14. Both vasogenic and cytotoxic edema, as well as blood brain-barrier leakage, occurred since day 1 after stroke induction and gradually attenuated with time. Numerous cells other than neuronal cells infiltrated into infarct lesion, which resulted in no visible TTC negative regional existence at day 14. Furthermore, recovery of cerebral blood flow and glucose utilization in peri-infarct zone were noted and more remarkably than that in infarct core following the stroke progression. In conclusion, these characterizations may be highly beneficial to the development of therapeutic strategies for ischemic stroke.
... 62 In addition, edema occurs to a substantially greater extent in photothrombotic animal models than in human stroke. 63,64 As it is known that edema can have substantial effects on astrocytes (i.e, end-feet and mitochondrial swelling), metabolic processes, such as glucose uptake from the bloodstream and glycogen accumulation, may occur differently in this model. 65 To the best of our knowledge, this is the first study to demonstrate glycogen accumulation within the PIZ, concomitant with the glial response in the photothrombotic model. ...
Imaging energy metabolites as markers of the energy shuttle between glia and neurons following ischemia is an ongoing challenge. Traditional microscopies in combination with histochemistry reveal glycogen accumulation within glia following ischemia, indicating an altered metabolic profile. Although semi-quantitative histochemical glycogen analysis is possible, the method suffers from typical confounding factors common to histochemistry, such as variation in reagent penetration and binding. In addition, histochemical detection of glycogen does not reveal information on the metabolic fate of glycogen (i.e., lactate production). Therefore, validation of a direct semi-quantitative method to simultaneously image both brain glycogen and lactate in the same tissue section would benefit this research field. In this study, we demonstrate the first application of FTIR spectroscopy for simultaneous direct spectroscopic imaging of brain glycogen and lactate, in situ within ex vivo tissue sections. Serial tissue sections were analysed with GFAP immuno-histochemistry to provide a comparison between the glycogen and lactate distribution revealed by FTIR and the glial distribution revealed by GFAP immuno-histochemistry. The distribution of glycogen revealed by FTIR spectroscopic imaging has been further compared with histochemical detection of glycogen on the adjacent tissue sections. This approach was then applied to study spatio-temporal disturbances in metabolism, relative to glia and neuronal populations, following cerebral ischemia in a murine model of stroke.
... Studies of certain neurological conditions, i.e., brain ischemia, traumatic brain injury, multiple sclerosis, Alzheimer disease, have identified lower levels of NAA likely indicating neuronal death and/or neuronal metabolic down-regulation. For example, in the acute phase of experimental ischemic injury [9], lower NAA in the infarct core parallels the reduction of neuronal number and cell size, nuclear pyknosis and infiltration of polymorphonuclear and mononuclear cells. A similar decrease in NAA levels was found in the ischemic core in patients and the outcome predictions based upon the residual NAA in the infarct core have proved accurate [10,11]. ...
Background and purpose:
We investigated the effects of an intensive impairment-oriented training on neuronal state (assessed by proton MR spectroscopy, 1H-MRS) of the spared motor and premotor cortices in the injured (ipsilesional) hemisphere and clinical impairment in a patient with chronic subcortical stroke.
Methods:
One survivor of a single ischemic stroke located outside of the motor and premotor cortices (assessed on T1-weighted MRI) was studied at six months after stroke. We used functional MRI-guided 1H-MRS to quantify the levels of N-acetylaspartate (NAA - a putative neuronal marker) in the hand representation within ipsilesional primary motor cortex (M1), dorsal premotor cortex (dPM) and supplementary motor area (SMA), and Fugl-Meyer (normal=66 points) test to assess the arm motor impairment immediately before and after a motor training paradigm. Training comprised intensive variable practice (1080 repetitions over 12 day-period) of a reach-to-grasp task with the impaired hand while focusing the learner's attention on an altered movement component, i.e., decreased elbow extension.
Results:
At baseline, the patient was severely impaired (Fugl-Meyer score=25 points) and exhibited lower level of NAA in all areas (M1, 9.2 mM vs. 11.6 ± 2.0 mM in healthy controls; dPM, 8.9 mM vs. 12.2 ± 1.9 mM; SMA, 7.4 mM vs. 11.0 ± 2.3 mM). After training, the patient improved clinically (by 6 points) and displayed higher levels of NAA across all areas (by 0.6-3.3 mM).
Conclusions:
Our data demonstrated that the radiologically normal-appearing ipsilesional motor and premotor areas have the resources to boost behavioral output in response to an intervention. We hope that these data will act as a starting point for further research to test the potential of 1H-MRS measures to provide a biomarker of neuroplasticity in response to restorative therapies in chronic stroke.
... Disadvantages of this model are due to its end-arterial occlusive nature. The rapidly evolving ischemic damage and the endothelial injury at the same time are associated with early cytotoxic (intracellular) and simultaneous vasogenic (extracellular) edema formation; 68 in addition, only a little or no ischemic penumbra and local collateral flow/reperfusion occur in this model. These pathomechanisms are different from those seen in human stroke, in which the cytotoxic edema is a hallmark of acute cerebral ischemia. ...
This review outlines the most frequently used rodent stroke models and discusses their strengths and shortcomings. Mimicking all aspects of human stroke in one animal model is not feasible because ischemic stroke in humans is a heterogeneous disorder with a complex pathophysiology. The transient or permanent middle cerebral artery occlusion (MCAo) model is one of the models that most closely simulate human ischemic stroke. Furthermore, this model is characterized by reliable and well-reproducible infarcts. Therefore, the MCAo model has been involved in the majority of studies that address pathophysiological processes or neuroprotective agents. Another model uses thromboembolic clots and thus is more convenient for investigating thrombolytic agents and pathophysiological processes after thrombolysis. However, for many reasons, preclinical stroke research has a low translational success rate. One factor might be the choice of stroke model. Whereas the therapeutic responsiveness of permanent focal stroke in humans declines significantly within 3 hours after stroke onset, the therapeutic window in animal models with prompt reperfusion is up to 12 hours, resulting in a much longer action time of the investigated agent. Another major problem of animal stroke models is that studies are mostly conducted in young animals without any comorbidity. These models differ from human stroke, which particularly affects elderly people who have various cerebrovascular risk factors. Choosing the most appropriate stroke model and optimizing the study design of preclinical trials might increase the translational potential of animal stroke models.
... Parmi les autres facteurs connus pour jouer un rôle dans ce processus et qui voient leur synthèse augmentée suite à une ischémie, on peut citer : le PDGF qui favoriserait les interactions entre les cellules endothéliales et les péricytes (Renner et al., 2003), l'EPO (Bernaudin et al., 1999), la eNOS qui produit du NO intervenant dans la régulation de la pression sanguine et dans l'angiogenèse (Veltkamp et al., 2002), le TGF-β qui joue un rôle dans la prolifération et la différenciation de nombreux types cellulaires (Massague, 1990 ;Haqqani et al., 2005) et le FGF qui favoriserait la prolifération des cellules endothéliales et l'élongation des vaisseaux Lin et al., 1997). Finalement, il est important de souligner que les EPC ("endothelial progenitor cell") circulantes sont capables de rejoindre les sites de néovascularisation et de s'y différencier en cellules endothéliales, participant ainsi à l'angiogenèse (Asahara & Kawamoto, 2004 (Grome et al., 1988 ;Norris et al., 1994 ;Lee et al., 1996 ;Demougeot et al., 2003). La figure 19 schématise le principe de ce modèle. ...
Evidences showing that under certain circumstances, inflammatory response could be neuroprotective and could also promote adult neurogenesis are growing. In this context, the objective of this work was to investigate the impact of microglial cells in the neuroplastic events. Rats were subjected to photothrombotic ischemia and microglial cells activation was blocked by the mean of poly(ADP-ribose)polymérase-1 (PARP-1) inhibition using 3- aminobenzamide (3-AB) since this protein has been shown to play a major role in this activation. Our results show that PARP-1 activity reduction was associated with a strong repression of the acute microglial activation. Beside, 3-AB treated animals exhibited a decrease in synaptophysin (synaptogenesis) and GAP-43 (axonal growth) expressions. Taken together, our data argue for a supportive role of microglial in adaptive brain plasticity events. According to the preponderant contribution of BDNF in these events, assessment of its cellular localization was performed, and confirmed that these cells represent a significant source. Beside, BDNF immunoreactivity (IR) in microglial cells and BDNF levels in the lesioned and surrounding lesioned areas were found decreased in 3-AB treated animals. However, since this neurotrophin can exert ambivalent biological actions through pro- versus mature forms, we investigate the proper effect of cerebral ischemia on total (Elisa), pro- and mature (Western blotting) expressions. Our results show that total, pro- and mature BDNF expressions are augmented in the early times (4-24h) of ischemia within the lesioned, the surrounding non lesioned and the contralateral cortical areas. At longer time points, total BDNF was still increased at 8d in regions distant from the lesion (hippocampi and contralateral cortex) while pro- and mature forms rise between 8d to 30d in hippocampic territories only. In term of cellular distribution, BDNF-IR was found in neurons but also in non neuronal cells ipsilaterally whereas in the opposite side BDNF staining was restricted to neurons. Our data while raising the question of the pertinence of total BDNF expression in a context of studying its supportive potential action indicate that such assessment has to be coupled with the discrimination of both forms. In addition, our data confirm the important role of BDNF in post-stroke adaptive mechanisms and argue in favour of an important contribution of the hippocampal territory and of the contralateral hemisphere in BDNF related post-stroke neuronal circuit remodelling suggesting that strategies targeting this hemisphere are likely to mediate functional compensation.
... use of platelet activators such as rose bengal administered intravenously which is then photo-activated through the skull) have also been employed. [30] To better model human stroke, thrombi have been used to produce the occlusion. However, despite interest in this kind of model and its arguably greater relevance to the clinical condition, the difficulty of reliably inducing the occlusion has limited its use in drug discovery. ...
This review discusses the reasons for both the difficulty in making therapeutic progress in stroke studies, and the insights that neuroimaging, especially magnetic resonance imaging (MRI) and perfusion computed tomography (CT), are bringing to this difficult area of research. While many advances have been made in the treatment of coronary heart disease and acute coronary syndrome, acute cerebral neuroprotection has almost completely failed to have an impact on stroke (a major unmet clinical need), despite the introduction of tissue plasminogen activator (tPA).
This paper discusses the reasons behind this lack of progress, including the problems posed by animal models of stroke. However, major scientific advances using neuroimaging, both in animals and humans, now permit the rational design of clinical trials and of patient selection to improve the chances of a positive therapeutic outcome. In the future, quantitative neuroimaging will provide surrogates that augment the existing, relatively insensitive, clinical endpoints, thereby reducing both patient numbers, and likely follow-up time, required to power a significant endpoint.
An important therapeutic limitation is currently the difficulty of including MRI early after stroke onset because of the lack of clinics focused on acute cerebral injury. Recent developments in perfusion CT may help alleviate this important bottleneck.
... 106 MRI of photothrombotic stroke shows early increases in T2 signal at the same time as decreased apparent diffusion of water, indicating the simultaneous development of substantial vasogenic edema at the same time as ischemic infarction. 116,117 In acute brain lesions, T2 signal is a function of extracellular water. This means that photothrombosis induces simultaneous vasogenic (extracellular) edema and cytotoxic (intracellular) edema, i.e., leaky vessels and swollen cells at the same time. ...
Rodent stroke models provide the experimental backbone for the in vivo determination of the mechanisms of cell death and neural repair, and for the initial testing of neu-roprotective compounds. Less than 10 rodent models of focal stroke are routinely used in experimental study. These vary widely in their ability to model the human disease, and in their application to the study of cell death or neural repair. Many rodent focal stroke models produce large infarcts that more closely resemble malignant and fatal human infarction than the average sized human stroke. This review focuses on the mechanisms of ischemic damage in rat and mouse stroke models, the relative size of stroke generated in each model, and the purpose with which focal stroke models are applied to the study of ischemic cell death and to neural repair after stroke.
... Decreased T 2 in animal models of forebrain ischemia has not been described before. Chronic extensive gliosis following forebrain ischemia may contribute to this decrease in T 2 by decreasing the amount of free extracellular water in the tissue [6]; hypointense patches on T 2 weighted MR images correspond to regions of dense gliosis following focal ischemia in rat [12]. However, by 7 days post-ischemia in the gerbil ferritin-positive microglia are found in the hippocampus [9], and increased iron concentration in the brain can also cause decreased T 2 [4,7]. ...
Quantitative T2 magnetic resonance (MR) imaging was used to examine gerbil brains 1, 3, 10, and 30 days after 5 min forebrain ischemia. T2 was increased in the dorsal–lateral striatum 1 and 3 days post-ischemia, and in the hippocampus 3 days post-ischemia. T2 was normal 10 days post-ischemia, and decreased in the hippocampus and dorsal-lateral striatum 30 days post-ischemia. Neuronal counts in the dorsal-lateral striatum and CA1 hippocampal region were uniformly decreased 30 days post-ischemia. The increase in T2 shortly after ischemia is attributed to brain edema localized to regions where neuronal injury developed. The late decrease in T2 may be due to decreased water in gliotic tissue, or to ferritin-positive microglia, following forebrain ischemia. Tissue atrophy at later times gave enlarged ventricles on MR images.
... Remote molecular alterations encompass upregulation of proinflammatory cytokines and growth factors (Jander et al, 2000; Kleinschnitz et al, 2004), and rearrangement of neurotransmitter receptors (Redecker et al, 2002). Magnetic resonance imaging studies using the contrast agent gadolinium diethylenetriamine-pentaacetic acid (Gd-DTPA) as the conventional in vivo marker for the BBB showed early and long-lasting breakdown of the BBB within the PT lesion (Lee et al, 1996; Schroeter et al, 2001; Kleinschnitz et al, 2003). However, there were no remote contrast enhancement or signal alterations on T2-weighted (T2-w) images. ...
Magnetic resonance imaging (MRI) is a powerful tool to assess brain lesions, but currently available contrast agents are limited in the assessment of cellular and functional alterations. By use of the novel MRI contrast agent gadofluorine M (Gf) we report on imaging of transient and widespread changes of blood-brain barrier (BBB) properties as a consequence of focal photothrombotic brain lesions in rats. After i.v. application, Gf led to bright contrast in the lesions, but also the entire ipsilateral cortex on T1-weighted MRI. In contrast, enhancement after application of gadolinium diethylenetriamine-pentaacetic acid (Gd-DTPA), a common clinical indicator of BBB leakage was restricted to the lesions. Remote Gf enhancement was restricted in time to the first 24 h after photothrombosis and corresponded to a transient breakdown of the BBB as revealed by extravasation of the dye Evans blue. In conclusion, our study shows that Gf can visualize subtle disturbances of the BBB in three dimensions not detectable by Gd-DTPA. Upon entry into the central nervous system Gf most likely is locally trapped by interactions with extracellular matrix proteins. The unique properties of Gf hold promise as a more sensitive contrast agent for monitoring BBB disturbances in neurologic disorders, which appear more widespread than anticipated previously.
... Photothrombotic ischemia by the rose-bengal method produces a highly consistent focal infarct. Diffusionweighted imaging lesion develops primarily during the first 24 hours, with an expanding volume of DWI deficit continuing over a subsequent 3 to 7 days (van Bruggen et al., 1992; De Ryck et al., 2000; Lee et al., 1996). The extensive thrombosis produced in this model in association with profound blood–brain barrier breakdown may limit the application of identified gene expression in this model to the clinic (that is, lesion development is rapid with little penumbra area to impact upon). ...
Sequencing of the human genome is nearing completion and biologists, molecular biologists, and bioinformatics specialists have teamed up to develop global genomic technologies to help decipher the complex nature of pathophysiologic gene function. This review will focus on differential gene expression in ischemic stroke. It will discuss inheritance in the broader stroke population, how experimental models of spontaneous stroke might be applied to humans to identify chromosomal loci of increased risk and ischemic sensitivity, and also how the gene expression induced by stroke is related to the poststroke processes of brain injury, repair, and recovery. In addition, we discuss and summarise the literature of experimental stroke genomics and compare several approaches of differential gene expression analyzes. These include a comparison of representational difference analysis we have provided using an experimental stroke model that is representative of stroke evolution observed most often in man, and a summary of available data on stroke differential gene expression. Issues regarding validation of potential genes as stroke targets, the verification of message translation to protein products, the relevance of the expression of neuroprotective and neurodestructive genes and their specific timings, and the emerging problems of handling novel genes that may be discovered during differential gene expression analyses will also be addressed.
Cerebral ischemia–reperfusion injury (CI/RI) injury is a common feature of ischemic stroke which occurs when the blood supply is restored after a period of ischemia in the brain. Reduced blood-flow to the brain during CI/RI compromises neuronal cell health as a result of mitochondrial dysfunction, oxidative stress, cytokine production, inflammation and tissue damage. Reperfusion therapy during CI/RI can restore the blood flow to ischemic regions of brain which are not yet infarcted. The long-term goal of CI/RI therapy is to reduce stroke-related neuronal cell death, disability and mortality. A range of drug and interventional therapies have emerged that can alleviate CI/RI mediated oxidative stress, inflammation and apoptosis in the brain. Herein, we review recent studies on CI/RI interventions for which a mechanism of action has been described and the potential of these therapeutic modalities for future use in the clinic.
Injectable hydrogels can generate and support pro-repair environments in injured tissue. Here we used a slow-releasing drug carrying in situ-forming hydrogel to promote post-stroke recovery in a rat model. Release kinetics were measured in vitro and in vivo with MRI, using gadolinium-labeled albumin (Galbumin), which demonstrated prolonged release over multiple weeks. Subsequently, this hydrogel was used for long-term delivery of vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang1) (Gel VEGF + Ang1, n = 14), in a photothrombotically induced cortical stroke lesion in rats. Control stroke animals were intralesionally injected with saline (Saline, n = 10), non-loaded gel (Gel, n = 10), or a single bolus of VEGF + Ang1 in saline (Saline VEGF + Ang1, n = 10). MRI was executed to guide hydrogel injection. Functional recovery was assessed with sensorimotor function tests, while tissue status and vascularization were monitored by serial in vivo MRI. Significant recovery from sensorimotor deficits from day 28 onwards was only measured in the Gel VEGF + Ang1 group. This was accompanied by significantly increased vascularization in the perilesional cortex. Histology confirmed (re)vascularization and neuronal sparing in perilesional areas. In conclusion, intralesional injection of in situ-forming hydrogel loaded with pro-angiogenic factors can support prolonged brain tissue regeneration and promote functional recovery in the chronic phase post-stroke.
Stroke is the second leading cause of death worldwide. Up to 80% of strokes are ischemic and take place due to occlusion of major cerebral arteries or its branches. The pathophysiology of stroke is multifaceted, involving excitotoxicity and activation of inflammatory pathways leading to disturbances in ion channels, oxidative damage, and apoptosis. Thrombolytics are the only FDA-approved drug for ischemic stroke. In order to study the pathophysiology, development of a reliable and reproducible model of ischemic stroke is of great importance. The ideal animal model is the one which can mimic the features of the pathology. This chapter summarizes the models of ischemic stroke with its advantages and limitations.
Stroke is a major cause of disability in adults and children. Recently, we have developed an adult rat model of minor stroke containing a peri-infarct region with a modest T2 increase and mild ischemic damage. We hypothesized that a neonatal minor stroke with mild peri-ischemic changes could also be produced, but with potential ontogenic differences. Using our minor photothrombosis method, we produced a range of severities of ischemic lesions (mini, minor, moderate and severe) within magnetic resonance imaging (MRI) slices of adult and neonatal rats. In both age groups, the lesion region showed a marked increase in T2 and diffusion-weighted intensity and decrease in apparent diffusion coefficient (ADC), corresponding to a cortical infarct detected using fluorojade and hematoxylin and eosin staining. Perilesional regions showed modest increases in T2 and ADC in adults, but not neonates, and this corresponded to scattered cell death, but not necessarily extravasation of plasma protein, i.e. blood-brain barrier disruption. Mini and minor insults in neonates generally showed homogeneous and rather modest changes in T2 and ADC. MR perfusion maps demonstrated a penumbral area of greater hypoperfusion in adults compared with neonates. Together, the results indicate that, in neonatal cortex, a similar severity of photothrombosis occurs throughout the area of photoactivation, whereas, in adult brain, spontaneous clot lysis and/or partial thrombosis occurs adjacent to permanently occluded vessels. Thus, by comparing differing severities of photothrombotic ischemia in neonates and adults, ontogenic differences were detectable using MRI, with mature brain having a greater penumbral region. Mild ischemic injury and scattered cell death in both neonates and adults could be identified by a modest increase in T2 and decrease in ADC. A better understanding of the effects of development on ischemic responses and associated MRI changes will provide a basis for the improved diagnosis of mild or minor ischemic insults relevant to pediatric and adult stroke.
Neonatal brain injury remains a devastating condition, with poor outcomes despite the institution of an effective neuroprotective strategy of therapeutic hypothermia. There is an urgent need to develop additional neuroprotective strategies and to tailor our clinical predictive ability for families and their infants. Such goals could be more readily achieved if reliable early clinical indicators or biomarkers existed. This review will explore the relation between magnetic resonance (MR) imaging biomarkers and the degree of brain pathology observed in our translational piglet model of perinatal asphyxia. We also suggest biomarker relevance at a cellular level. The review will describe the development needed to optimize and simplify the use of biomarkers to speed up future trials of neuroprotection.
This reviewing article is intended to provide an overview on rodent models of stroke and a summary of recent MRI findings from such animal models as well as their potential impacts on experimental and clinical stroke studies.
Background and purpose:
Recovery from stroke is hypothesized to involve the reorganization of surviving cortical areas. To study the functional organization of sensorimotor cortex at multiple time points before and after stroke, we performed longitudinal light-based motor mapping of transgenic mice expressing light-sensitive channelrhodopsin-2 in layer 5 cortical neurons.
Methods:
Pulses of light stimulation were targeted to an array of cortical points, whereas evoked forelimb motor activity was recorded using noninvasive motion sensors. Intrinsic optical signal imaging produced maps of the forelimb somatosensory cortex. The resulting motor and sensory maps were repeatedly generated for weeks before and after small (0.2 mm3) photothrombotic infarcts were targeted to forelimb motor or sensory cortex.
Results:
Infarcts targeted to forelimb sensory or motor areas caused decreased motor output in the infarct area and spatial displacement of sensory and motor maps. Strokes in sensory cortex caused the sensory map to move into motor cortex, which adopted a more diffuse structure. Stroke in motor cortex caused a compensatory increase in peri-infarct motor output, but did not affect the position or excitability of sensory maps.
Conclusions:
After stroke in motor cortex, decreased motor output from the infarcted area was offset by peri-infarct excitability. Sensory stroke caused a new sensory map to form in motor cortex, which maintained its center position, despite becoming more diffuse. These data suggest that surviving regions of cortex are able to assume functions from stroke-damaged areas, although this may come at the cost of alterations in map structure.
The effect of thrombolytic therapy was studied in rats submitted to thromboembolic stroke by intracarotid injection of autologous blood clots. Thrombolysis was initiated after 15 minutes with an intracarotid infusion of recombinant tissuetype activator (10 mg/kg body weight). Reperfusion was monitored for 3 hours using serial perfusion- and diffusion magnetic resonance imaging, and the outcome of treatment was quantified by pictorial measurements of ATP, tissue pH, and blood flow. In untreated animals, clot embolism resulted in an immediate decrease in blood flow and a sharp decrease in the apparent diffusion coefficient (ADC) that persisted throughout the observation period. Thrombolysis successfully recanalized the embolized middle cerebral artery origin and led to gradual improvement of blood flow and a slowly progressing reversal of ADC changes in the periphery of the ischemic territory, but only to transient and partial improvement in the center. Three hours after initiation of thrombolysis, the tissue volume with ADC values less than 80% of control was 39 ± 22% as compared to 61 ± 20% of ipsilateral hemisphere in untreated animals (means ± SD, P=.03) and the volume of ATP-depleted brain tissue was 25 ± 31% as compared to 46 ± 29% in untreated animals. Recovery of ischemic brain injury after thromboembolism is incomplete even when therapy is started as early as 15 minutes after clot embolism. Possible explanations for our findings include downstream displacement of clot material, microembolism of the vascular periphery, and events associated with reperfusion injury.Keywords: Thromboembolic stroke; Thrombolysis; Rat; rt-PA; Diffusion MRI, Perfusion MRI; Biochemical imaging; rCBF
In order to examine the effect of EGF1 peptides in directing nanoparticles to thrombi, the synthesized EGF1 peptide was conjugated
to poly (ethyleneglycol)-poly (lactide) (PEG-PLA) nanoparticles (NP) to form EGF1-NP. A lipophilic fluorescent dye, coumarin-6,
was incorporated into EGF1-NP so as to detect its loading and release capacity. The binding ability of EGF1-NP with TF-expressing
cells was shown to be significantly higher than that of the non-conjugated NP. Following an intravenous administration, fluorescence
was distributed along the vessel wall of the thrombosis regions in the model rats injected with coumarin-6-loaded EGF1-NP.
The in vitro and in vivo results suggest that EGF1-NP is a promising drug delivery system for targeting cerebral thrombi.
Keywordsantithrombotic-targeting drug delivery-epidermal growth factor-like domain-tissue factor-nanoparticle
In a strategy for anti-thrombotic therapy, we have expressed EGFP-EGF1 fusion protein, in which EGF1 can bind with tissue factor (TF). EGFP has previously been widely used as a fluorescent protein marker. EGFP-EGF1 protein was thiolated and conjugated to the malemide covering on the pegylated nanoparticles (NP) to form the EGFP-EGF1-NP. The EGFP-EGF1-NP was characterized in terms of morphology, size and zeta potential. In vitro cell viability experiment confirmed that the biodegradable EGFP-EGF1-NP was safe. To evaluate the delivering ability of EGFP-EGF1-NP, a fluorochrome dye, Dir, was incorporated into the nanoparticle, and the loading capacity and release property of the particle were examined. In vitro results showed that the binding ability of EGFP-EGF1-NP with TF-expressing cells was significantly stronger than that of non-conjugated NP. In vivo multispectral fluorescent imaging demonstrated that EGFP-EGF1-NP had high specificity and sensitivity in targeting thrombi. Our study demonstrated that EGFP-EGF1-NP is a promising TF-targeting drug delivery system for thrombolytic treatment.
Hypertension is more common among African Americans than Americans of European descent. However, the genetic etiology has not been defined. Similarly, lipoprotein (Lp) (a), an independent risk factor for cardiovascular disease, is higher among African Americans. To explore the relationship between Lp (a) and hypertension, we measured the blood pressure of transgenic mice expressing apolipoprotein(a), the unique protein moiety of lipoprotein(a). As controls, we also determined blood pressure for apoE deficient mice, low density lipoprotein-receptor (LDL-R) deficient mice, and wild type C57Bl/6 mice. Apo(a) expression was not associated with hypertension. Surprisingly, LDL-R deficient mice exhibited male-associated hypertension. This observation could explain the higher incidence of atherosclerosis in male LDL-R deficient mice and human familial hypercholesterolemia (FH) patients. LDL-R deficient mice were more sensitive to photochemically induced cerebral stroke. However, this hypersensitivity was only modestly associated with sexual dimorphism. The presented data suggest that LDL-R deficiency results in hitherto unrecognized changes in the vascular tone.
The photothrombotic ring stroke model with sustained underperfusion followed by late spontaneous reperfusion (Gu et al. 1999) was employed to study its morphological consequences. The exposed crania of adult male Wistar rats were subjected to a ring-shaped laser irradiation beam simultaneously with intravenous erythrosin B infusion. The ischemic volume was calculated from serial sections throughout the ischemic lesions at 4, 10, 24, 48, and 72 h and 7 days and 28 days after irradiation. The ischemic volume, expressed as a percentage of the ipsilateral hemispheric volume, increased steadily from 4 to 10 to 24 h to reach its maximum value at 48 h after irradiation; at 3 days, 7 days, and 28 days, the ischemic volume was reduced to 75%, 24%, and 22% of the value at 48 h. Evaluation of ischemic volumes at different anteroposterior levels revealed that the reduced ischemic volume at 72 h and later was mainly due to morphological restoration in the centrally located, nonirradiated region at risk. An initial enlargement and development of cystic coagulation necrosis was observed in the cortical areas corresponding to the ring lesion itself. In the region at risk, a gradually deteriorating neuropil and nerve cell morphology were observed over time, with maximum severity at 48 h postirradiation. At this time, most laminae II and III neurons in the region at risk exhibited eosinophilia and pyknosis but no incrustations, with small islands of less damaged neurons randomly scattered. At 72 h and up to 28 days after irradiation, these cell characteristics were no longer observed and the region at risk was well populated with neurons that had a chiefly unremarkable cytological appearance. Neuronal counts in the central part of the region at risk were performed; no significant difference in neuronal density was observed between sham-operated controls and at 28 days after irradiation. In conclusion, the consistent, late spontaneous reperfusion coincided with remarkable tissue recovery as assessed morphologically in the region at risk. The data suggest that nerve cell repair may occur even after the detection, by conventional morphological methods, of prolonged critical ischemic neuronal damage in the setting of acute ischemic stroke.
A neuroprotective effect can be obtained with N-[(4-cycloheptylaminopyrid-3-yl)sulphonyl]N′-cycloheptyl urea (BM27), a pyrid-3-yl-sulphonylurea structurally related to torasemide, a loop diuretic. We have investigated the neuroprotective effect of BM27 by magnetic resonance imaging and use of the photothrombotic model of cerebral infarction in the rat. This method enables non-invasive quantification of the extent of the cerebral oedema from T2-weighted spin-echo images.
This article reports the evolution of the extent of oedema with time (0.5, 1, 2, 4, 6, 24 and 48 h, 7 and 15 days and 1 month after induction of the lesion) in rats pretreated with 5mg kg−1 BM27 or an appropriate control. At all times, the rats treated with BM27 had, on average, smaller lesions than control rats (30% decrease between 2 h and 6 h).
These results strongly suggest a significant (P < 0.01) but modest neuroprotective effect of BM27 in ischaemic cerebral stroke. Further investigations should be performed to determine if BM27 or its analogues are of clinical interest.
This study aimed at developing a dual setup of the photothrombotic ring stroke model with or without late spontaneous reperfusion in the region at risk and to explore the morphological consequences. The exposed crania of adult male Wistar rats were subjected to a ring-shaped laser-irradiation beam (o.d. 5.0 mm, 0.35 mm thick) for 2 min simultaneously with intravenous erythrosin B (17 mg/kg) infusion. Transcardial carbon-black perfusion revealed that a laser intensity of 0.90 W/cm(2) resulted in late, that is, starting at 72 h, spontaneous reperfusion, whereas the lowest laser intensity that produced lack of reperfusion at 7 days post-irradiation was 1.84 W/cm(2). Laser-Doppler flowmetry showed prompt cortical cerebral blood flow (cCBF) reduction both in the ring lesion and region at risk (12% and 25% of control values) after high-intensity irradiation; these reduced flow values were more rapid and pronounced than in the low-intensity irradiation setup as previously shown. The high- compared with low-intensity irradiation setup produced more frequent occurrence of thrombi in the ring-lesion region and a larger ischemic cortical lesion with a more rapid pace of ischemic cellular changes in the ring-lesion region and the region at risk. The region at risk transformed into pannecrosis in the high-intensity, but recovered morphologically in the low-intensity irradiation setup. This dual photothrombotic setup with or without spontaneous reperfusion enables the study of events related to ischemic cell survival or death in an anatomically predefined region at risk.
Apoptosis-related cell death is linked to oxidative stress and caspases in experimental cerebral ischemia. However, the role of oxidative stress in caspase activation and subsequent apoptotic cell death after cerebral ischemia is unknown. The authors evaluated the role of oxidative stress in ischemic cerebral infarction after photothrombosis and the relation between oxidative stress and caspase-related cell death 6 and 24 hours after ischemia with and without U-74389G, a potent free radical scavenger (10 mg/kg, 30 minutes before and after ischemia induction). Reactive oxygen species, detected by hydroethidine oxidation, and cytosolic cytochrome c were detected in early ischemic lesions. Western blot analysis showed the cleaved form and the increased level of the proform of caspase-3 in the ischemic lesion 24 hours after ischemia. Decreased caspase-3 immunoreactivity was detected in the antioxidant-treated group after ischemia. Decreased DNA fragmentation and laddering were detected and the lesion was smaller in the treated group after ischemia compared with the untreated group. Oxidative stress and cytochrome c release occur in the ischemic lesion after photothrombotic ischemia. The free radical scavenger attenuated caspase-3 up-regulation, DNA fragmentation, and the final lesion. The authors concluded that oxidative stress may mediate caspase-related apoptotic cell death and subsequent cortical infarction after photothrombotic ischemia.
This investigation analyzed the potential of high-resolution magnetic resonance imaging (MRI) at a field strength of 7T to depict leukocyte infiltration and glial responses after focal cerebral ischemia induced by photothrombotic occlusion of cerebral microvessels. For this purpose we superimposed multiparametric MRI (apparent diffusion coefficient, T2, perfusion-weighted, and gadolinium-DTPA-enhanced T1-weighted imaging) on tissue sections stained for phagocytes and astrocytes and, moreover, assessed the regional distribution of tissue pH and ATP content by invasive biochemical methods. Comparing the histological data with the various MRI parameters, high-resolution MRI did not allow a spatial discrimination between distinct areas of phagocyte accumulation or astroglial scar formation, based on image contrast or even quantitative parameter value differences. However, MRI parameters underwent characteristic changes and differentiated distinct stages of tissue remodeling between days 3 and 14 after photothrombosis. Low apparent diffusion coefficient (ADC) and high T2 values indicated an early stage (3 days) with necrosis and beginning glial activation. Normal ADC and reduced T2 elevation characterized an infarct with advanced glial activation and infiltration of hematogenous cells at 7 days after photothrombosis. Heterogeneous ADC together with T2 elevation reflected a late infarct stage (14 days) when pseudocystic degeneration and scar formation had occurred.
Objective:
Diffusion-weighted magnetic resonance imaging (DWI) detects acute ischemic infarcts with high lesion conspicuity. Determination of infarct age is difficult on DWI alone because infarct signal intensity (SIinfarct) on DWI is influenced by T2 properties ("T2 shine-through"). Maps of the apparent diffusion coefficient (ADC) reflect pure diffusion characteristics without T2 effects but have low lesion conspicuity. Thus, in clinical practice, combined use of DWI and ADC maps is required. Exponential DWI (eDWI) is an innovative means of MRI-diffusion data analysis that merges the advantages of DWI and ADC maps. The authors hypothesized that SIinfarct on eDWI would correlate with infarct age. The authors studied 114 consecutive patients who had 120 ischemic strokes with clearly determined onset times and who underwent echo-planar DWI. The eDWI were generated by dividing the signal intensity on DWI by that on the corresponding T2 image on a pixel-by-pixel basis. SIinfarct on eDWI was measured in the lesion core and expressed as a percentage of contralateral control tissue. On eDWI, relative SIinfarct changed significantly with infarct age (P < .0001). When patients were sorted in infarct-age groups, no significant differences were found within the first 120 hours. However, for patients studied within 5 days, the mean relative SIinfarct was significantly higher compared with patients studied > or = 8 days after stroke (P < .05). For all infarcts up to 5 days old, the eDWI signal intensity was higher than control tissue (hyperintense appearance). All infarcts > 10 days old had an eDWI signal intensity lower than control tissue (hypointense appearance). The authors concluded that the use of eDWI, as a single set of images, reliably differentiates acute infarcts (< or = 5 days old) from infarcts > 10 days old. This feature would be expected to be helpful when the distinction between acute and nonacute infarction cannot be determined on clinical grounds.
Oxidative stress has been associated with the development of blood-brain barrier disruption and cellular injury after ischemia. The cytosolic antioxidant, copper/zinc superoxide dismutase, has been shown to protect against blood-brain barrier disruption and infarction after cerebral ischemia-reperfusion. However, it is not clear whether copper/zinc superoxide dismutase can protect against evolving ischemic lesions after thromboembolic cortical ischemia. In this study, the photothrombotic ischemia model, which is physiologically similar to thromboembolic stroke, was used to develop cortical ischemia. Blood-brain barrier disruption and oxidative cellular damage were investigated in transgenic mice that overexpress copper/zinc superoxide dismutase and in littermate wild-type mice after photothrombotic ischemia, which was induced by both injection of erythrosin B (30 mg/kg) and irradiation using a helium neon laser for 3 min. Free radical production, particularly superoxide, was increased in the lesioned cortex as early as 4 h after ischemia using hydroethidine in situ detection. The transgenic mice showed a prominent decrease in oxidative stress compared with the wild-type mice. Blood-brain barrier disruption, evidenced by quantitation of Evans Blue leakage, occurred 1 h after ischemia and gradually increased up to 24 h. Compared with the wild-type mice, the transgenic mice showed less blood-brain barrier disruption, a decrease in oxidative DNA damage using 8-hydroxyguanosine immunohistochemistry, a subsequent decrease in DNA fragmentation using the in situ nick-end labeling technique, and decreased infarct volume after ischemia. From these results we suggest that superoxide anion radical is an important factor in blood-brain barrier disruption and oxidative cellular injury, and that copper/zinc superoxide dismutase could protect against the evolving infarction after thromboembolic cortical ischemia.
Over the last two decades several studies have suggested the role of photothrombotic occlusion of cerebral microvessels using rose bengal, resulting in small strokes in rodents that resemble those in humans. This paper describes such a photothrombotic method of acute small stroke induction in rats with histopathologic and in vivo magnetic resonance imaging (MRI) observations from 3 to 6 h after irradiation, which is homologous to a human autopsy specimen. Utilizing 30 min of irradiation with minimal beam intensity (0.1 W/cm(2)) cold white light in conjunction with 20 mg of intravenous (iv) rose bengal as a rapid infusion, small infarcts were induced photochemically in the frontal lobes of six rats. The infarcts showed a consistent pattern on histologic and in vivo MR sections when examined within 7 h or less of irradiation. Both MRI and histologic sections were comprised of (a) a superior zone of infarcted neurons, (b) a middle curvilinear transition zone of edema on MRI and histologically vacuolated neuropil, and (c) an inferior zone of normal neurons. Shorter duration water-sensitive (T2)- and postgadolinium longer duration (T1)-weighted signal decay images both showed a curvilinear hyperintense transition zone of edema. The mean infarct and transition zone areas measured from the histologic sections were comparable to those measured on the MRI. The infarct model described above allows in vivo observations using MRI with the potential for use in testing putative neuroprotective agents. As demonstrated by a comparison with the histologic features of such infarcts in surgical and autopsy brain specimens, the model is relevant to acute human ischemic infarcts.
To determine if the phenomenon of transient normalization of T2 relaxation in the subacute stage of ischemic stroke is associated with either magnetic susceptibility effects secondary to hemorrhage or changes in tissue water content. MATERIALS and
We utilized a rat model of transient, focal, cerebral ischemia. The possibility of hemorrhage was evaluated with T2*-weighted (T2*W) imaging and histology. Changes in water content were assessed by brain wet-to-dry weight.
Susceptibility effects were not evident in T2*W images, and neither red blood cells nor unchelated Fe(III) was found in hematoxylin and eosin (H-E)- or Prussian Blue-stained sections, respectively. However, between the peak of T2 contrast and the point of transient T2 normalization, water content consistently decreased by an average of 3%.
Transient T2 normalization is associated with normalization of water content and can occur without evidence of hemorrhage.
To identify the temperature dependent change of the apparent diffusion coefficient (ADC) of water in brain tissue, the ADC values of normal rat brain were measured over a range of body temperatures with monitoring of head temperature using a small water reference implanted under the temporalis muscle. An initial experiment using thermocouples implanted into the cortex, caudate-putamen, temporalis muscle, and rectum demonstrated that temperature in all regions were highly correlated over a temperature range from 33 to 39 degrees C. In another group of normal rats, brain ADC values varied almost uniformly with body temperature over the temperature range 33-39 degrees C, implying that brain ADC values accurately reflect changes in brain temperature. The effects of focal ischemia and administration of the noncompetitive N-methyl-D-aspartate (NMDA) antagonist, CNS-1102, on ADC were also examined, using the suture middle cerebral artery (MCA) occlusion model while maintaining the body temperature at 37 degrees C. ADC values and therefore brain temperature in the nonischemic and ischemic hemispheres were not affected by the drug. These experiments suggest that brain ADC measurement could be useful in animal studies and, potentially, in humans to assess the effects of pharmacologic intervention on brain temperature.
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.
Recent developments in diffusion-weighted imaging (DWI) have enabled the pathological changes that occur during cerebral ischaemia to be studied. The present studies utilised DWI to investigate the development of early ischaemic changes following permanent middle cerebral artery (MCA) occlusion in the rat, which represents a model of stroke. An increased DWI signal was seen in the region of the occluded MCA and this was detectable as early as 1 h postocclusion. DWI images were obtained at nine stereotactic levels throughout the brain, providing a quantifiable measure of the volume of increased signal intensity in each animal. At 1 h post-MCA occlusion the hyperintense areas were seen in the frontoparietal cortex and lateral caudate nucleus; these areas represent the core of the infarct and no protection is seen with any compounds in these areas. There was a progressive increase in the area of hyperintensity up to 4 h post-MCA occlusion, and at this time point the hyper-intensity was seen in the dorsolateral cortex and caudate nucleus. At 4 h post-MCA occlusion there was a significant correlation between the volume of hemispheric and cortical ischaemic damage measured using DWI and histology. Thus, it appears that the increased DWI signal seen during the early time points after MCA occlusion was demarcating tissue that was destined for infarction. The area beyond the hyperintense region at 1 h represents the so-called "penumbral" region, because with increasing time (post-MCA occlusion) this area became incorporated into the infarct. There was also a slight increase in infarct size between 4 and 24 h, when assessed using DWI or histology, although two groups of animals were being compared, as opposed to the time-course study, in which just one group of animals was used. At 24 h post-MCA occlusion there was a good correlation between DWI, histology, and conventional T2 weighted imaging. There was no further increase in size of the infarct between 24 h and 7 days as assessed using histology and T2-weighted imaging. DWI could not be used to quantify infarct volume at 7 days because there was no uniform signal in the damaged area. At 7 days the area of infarction actually appeared to be darker in the diffusion-weighted images. The hyperintensity seen in diffusion-weighted images appears to decrease some time between 24 h and 7 days.(ABSTRACT TRUNCATED AT 400 WORDS)
In the limb plexus, motor axons destined for limb muscles diverge along separate pathways to innervate muscles derived from either the dorsal or ventral premuscle masses. We have examined the axonal guidance cues involved in this initial, specific pathway choice at the plexus by making dorsoventral (D/V) limb bud reversals prior to innervation. Chick/quail chimeras were used to determine the proximodistal level of the reversal in tissue sections. The specificity of the projections to dorsal or ventral nerve trunks was assessed by retrograde HRP labeling at ages prior to motoneuron death. Axons corrected for the reversal when the level of the graft was proximal to the plexus, and when the reversed limb and its gross nerve pattern were normal. If all of these conditions were not satisfied, aberrant innervation patterns were observed. Axonal trajectories were analyzed within the host tissue, at the host-graft border, and within rotated tissue to determine where along the pathway guidance cues might be located. Special attention was given to cases in which axons compensated for the reversal to project in accord with the positions of their soma in the lateral motor column. In these correcting cases, after normal D/V sorting in the spinal nerves of the host, motor axons altered their trajectories upon entering rotated graft tissue as they approached and traversed the plexus. Because corrections were within rotated tissue and not proximal to it, the D/V pathway cues are unlikely to be long-range target-derived signals, but rather appear to be closely associated with positional information in the plexus region and also more proximally in the tissue surrounding the distal spinal nerves.
We used a photochemical technique which induces thrombotic infarction by intravenous injection of the fluorescein derivative Rose Bengal and focal illumination of the intact skull surface. Following such photochemi-cally induced infarcts in the sensorimotor neocortex of rats, posttreatment with flunarizine, a class IV calcium antagonist, within a critical period of the first 6 h after infarction, results in marked sparing of sensorimotor function (tactile/proprioceptive limb placing reactions), while animals remain normoglycemic and are free of drug-induced behavioral toxicity. This could reflect flunarizine-induced coping of neuronal tissue with ischemia-related ionic shifts. It is argued that photochemical thrombosis and middle cerebral artery occlusion can fruitfully complement each other as experimental stroke models.
We have characterised the Ca2+ channel blocking properties of a new non-peptide Ca2+ channel antagonist, SB 201823-A, in cultures of rat sensory neurones. The IC50 for SB 201823-A against total Ca2+ current in sensory neurones was 4.9 μM. SB 201823-A showed little selectivity for sub-types of neuronal Ca2+ channel but was selective for Ca2+ channels over Na+ and K+ channels. Efficacy against other types of cation channel such as agonist gated channels was not assessed. SB 201823-A was neuroprotective in vivo when administered post-ischaemia in one focal and one global model of neuronal ischaemia. In the rat photothrombotic focal lesion model, SB 201823-A administered i.p. 10 min post-ischaemia resulted in a dramatic reduction in lesion volume. In the gerbil bilateral carotid artery occlusion global model, SB 201823-A dosed i.p. 30 min post-occlusion resulted in both histological and functional improvements when compared to vehicle treated animals. These data suggest that such novel neuronal Ca2+ channel antagonists may have potential in ameliorating both the pathological and functional consequences of stroke in man.
A derivation is given of the effect of a time-dependent magnetic field gradient on the spin-echo experiment, particularly in the presence of spin diffusion. There are several reasons for preferring certain kinds of time-dependent magnetic field gradients to the more usual steady gradient. If the gradient is reduced during the rf pulses, H1 need not be particularly large; if the gradient is small at the time of the echo, the echo will be broad and its amplitude easy to measure. Both of these relaxations of restrictions on the measurement of diffusion coefficients by the spin-echo technique serve to extend its range of applicability. Furthermore, a pulsed gradient can be recommended when it is critical to define the precise time period over which diffusion is being measured.
The theoretical expression derived has been verified experimentally for several choices of time dependent magnetic field gradient. An apparatus is described suitable for the production of pulsed gradients with amplitudes as large as 100 G cm−1. The diffusion coefficient of dry glycerol at 26°±1°C has been found to be (2.5±0.2)×10−8 cm2 sec−1, a value smaller than can ordinarily be measured by the steady gradient method.
The development of the Rose Bengal model of cerebral infarction in the rat is monitored using rapid NMR imaging techniques. The lesion volume,T
1,T
2, and the apparent diffusion coefficient (ADC) are measured in the time interval between 24 and 168 h postinduction. VolumetricT
1-weighted 3D imaging of the lesions produced by this model is shown to have a standard error of about 5% over the first 3 days postinduction, rising to about 15% at 1 week. Both theT
1 andT
2 contrasts are found to be more durable than those in ADC and are, hence, more suitable for volumetric monitoring over this time period. An elevation in ADC is also observed in tissue outside the lesion and this is interpreted as being caused by the transport of edema away from the lesion.
Thesis (Ph. D.)--University of Cambridge, 1995.
1. Studies in animal models of stroke have provided an invaluable contribution to our current understanding of the pathogenesis of cerebral ischaemia. The strengths of stroke research in animals are: 1) the ability to control the severity, duration, location and cause of the ischaemia, variables which confound interpretation of human stroke data; 2) co-existent disease states and variations in cerebrovascular anatomy are avoided; and 3) physiological parameters such as blood pressure, blood gases, temperature and plasma glucose (all of which influence the magnitude of the ischaemic lesion) can be closely monitored and controlled. Taking all these things on board, it is possible to induce a consistent focal ischaemic lesion in animal models of stroke (e.g. the permanent occlusion of the middle cerebral artery (MCA) in the rat). This has resulted in the wide use of animal models for assessment of anti-ischaemic drug efficacy as well as for research into the pathophysiological sequelae of stroke. 2. Traditionally focal ischaemia models involved permanent occlusion of a major cerebral artery such as the MCA. However, since vessel occlusion is seldom permanent in human stroke more recent developments have incorporated reperfusion (following ischaemia) into the design of the animal model. This has been achieved by reversible occlusion of cerebral vessels using 1) intraluminal filaments; 2) microclips; 3) the abluminal application of potent and prolonged vasoconstrictors; or 4) the introduction of emboli into the cerebral circulation.(ABSTRACT TRUNCATED AT 250 WORDS)
The mechanisms that give rise to ischemic brain damage have not been definitively determined, but considerable evidence exists that three major factors are involved: increases in the intercellular cytosolic calcium concentration (Ca++i), acidosis, and production of free radicals. A nonphysiological rise in Ca++i due to a disturbed pump/leak relationship for calcium is believed to cause cell damage by overactivation of lipases and proteases and possibly also of endonucleases, and by alterations of protein phosphorylation, which secondarily affects protein synthesis and genome expression. The severity of this disturbance depends on the density of ischemia. In complete or near-complete ischemia of the cardiac arrest type, pump activity has ceased and the calcium leak is enhanced by the massive release of excitatory amino acids. As a result, multiple calcium channels are opened. This is probably the scenario in the focus of an ischemic lesion due to middle cerebral artery occlusion. Such ischemic tissues can be salvaged only by recirculation, and any brain damage incurred is delayed, suggesting that the calcium transient gives rise to sustained changes in membrane function and metabolism. If the ischemia is less dense, as in the penumbral zone of a focal ischemic lesion, pump failure may be moderate and the leak may be only slightly or intermittently enhanced. These differences in the pump/leak relationship for calcium explain why calcium and glutamate antagonists may lack effect on the cardiac arrest type of ischemia, while decreasing infarct size in focal ischemia. The adverse effects of acidosis may be exerted by several mechanisms. When the ischemia is sustained, acidosis may promote edema formation by inducing Na+ and Cl- accumulation via coupled Na+/H+ and Cl-/HCO3- exchange; however, it may also prevent recovery of mitochondrial metabolism and resumption of H+ extrusion. If the ischemia is transient, pronounced intraischemic acidosis triggers delayed damage characterized by gross edema and seizures. Possibly, this is a result of free-radical formation. If the ischemia is moderate, as in the penumbral zone of a focal ischemic lesion, the effect of acidosis is controversial. In fact, enhanced glucolysis may then be beneficial. Although free radicals have long been assumed to be mediators of ischemic cell death, it is only recently that more substantial evidence of their participation has been produced. It now seems likely that one major target of free radicals is the microvasculature, and that free radicals and other mediators of inflammatory reactions (such as platelet-activating factor) aggravate the ischemic lesion by causing microvascular dysfunction and blood-brain barrier disruption.(ABSTRACT TRUNCATED AT 400 WORDS)
The mammalian central nervous system is composed of a large and diverse array of its two principle cell types, neurons and glia. Given this extreme cellular heterogeneity, it is not surprising that targets of neurotoxic insult often are diverse and unpredictable. In devising strategies to assess neurotoxicity, one must overcome the obstacle posed by not knowing where to look for damage. Our approach to the detection and localization of sites of toxicant-induced damage has been to take advantage of two general responses to nervous system insult, astrogliosis and argyrophilia. Assays for GFAP, the principle intermediate filament protein of astrocytes, have been devised to quantify astrogliosis and a modified cupric silver degeneration stain has been employed to examine patterns of argyrophilia. Using these methods, we found that increases in GFAP and the occurrence of argyrophilia are consistent responses to neurotoxic insults. Moreover, increments in these two indicators reveal dose-, time- and region-dependent patterns of neurotoxicity at toxicant dosages below those that cause light microscopic evidence of cell loss or damage. The rapid onset of astrogliosis and argyrophilia following specific toxicant exposures suggests that the signals underlying these events are among the earliest that can be linked to the neurotoxic state.
Rapid MRI of the molecular diffusion of water demonstrated cerebral infarcts in 32 patients. We studied these patients at various times following the onset of ischemic symptoms and found that diffusion-weighted imaging revealed the infarcts sooner than conventional T2-weighted spin-echo imaging did; four hyperacute infarcts were shown only by diffusion-weighted imaging. Acute infarcts had lower apparent diffusion coefficients (ADCs) than noninfarcted regions did. This relative difference in ADC reached a nadir in the first 24 hours and rose progressively thereafter. Chronic infarcts showed a relative increase in diffusion and were readily distinguishable from acute infarcts. The technique takes less than 2 minutes to apply using a standard 1.5-tesla scanner in the clinical setting. Diffusion-weighted imaging has the potential to play a role in improving the early anatomic diagnosis of stroke and therefore in the development and implementation of early stroke interventions.
The distribution of brain cell injury following transient ischemia is remarkable because only certain neurons in distinct brain regions are destroyed (selective neuronal death). Because excitatory neurotransmitters (glutamate and aspartate) cause a similar pattern of selective neuronal death, it seemed only natural to associate these effects with the trauma of ischemia. This led to the formulation of the excitotoxin hypothesis, which explains selective neuronal death as a result of excessive interstitial concentration increases of excitatory amino acids during ischemia, resulting in the opening of receptor-coupled ionophores, of which calcium channels are of particular interest. A large influx of calcium associated with impaired intracellular calcium sequestration mechanisms due to energy failure activates a host of catabolic enzymes that ultimately will cause neuronal death. The purpose of this work was (a) to measure extracellular glutamate concentration increases during ischemia in a selective vulnerable brain region (rat CA1 hippocampus), (b) to evaluate the toxicity of such a concentration increase, and (c) to investigate the relationship between ischemia-induced glutamate accumulation and changes of calcium homeostasis. The execution of these experiments required a method that was able to sample excitatory amino acids in the brain extracellular space for subsequent analysis by high performance liquid chromatography (HPLC). The choice of the microdialysis technique proved most satisfactory and further mathematical analysis made it possible to transform dialysate glutamate concentrations to extracellular concentrations. The study demonstrated that extracellular glutamate in CA1 reached toxic concentrations during ischemia. There appeared to be a clear correlation between ischemia-induced glutamate accumulation and the decrease in extracellular calcium since both changes were prevented in the denervated CA1 (the destruction of glutamatergic innervation from CA3 protects CA1 pyramidal neurons from ischemic damage). By contrast, blockade of N-methyl-D-aspartate (NMDA) receptors with the glutamate antagonist APV was only partially effective in preventing the ischemia-induced calcium changes in CA1. Taken together, these results support the excitotoxin hypothesis but question the rational of treating neuronal injury caused by transient global ischemia exclusively with NMDA antagonists.
At high magnetic fields (7 and 8.4 T), water proton magnetic resonance images of brains of live mice and rats under pentobarbital anesthetization have been measured by a gradient echo pulse sequence with a spatial resolution of 65 x 65-microns pixel size and 700-microns slice thickness. The contrast in these images depicts anatomical details of the brain by numerous dark lines of various sizes. These lines are absent in the image taken by the usual spin echo sequence. They represent the blood vessels in the image slice and appear when the deoxyhemoglobin content in the red cells increases. This contrast is most pronounced in an anoxy brain but not present in a brain with diamagnetic oxy or carbon monoxide hemoglobin. The local field induced by the magnetic susceptibility change in the blood due to the paramagnetic deoxyhemoglobin causes the intra voxel dephasing of the water signals of the blood and the surrounding tissue. This oxygenation-dependent contrast is appreciable in high field images with high spatial resolution.
Diffusion-weighted MR images were compared with T2-weighted MR images and correlated with 1H spin-echo and 31P MR spectroscopy for 6-8 h following a unilateral middle cerebral and bilateral carotid artery occlusion in eight cats. Diffusion-weighted images using strong gradient strengths (b values of 1413 s/mm2) displayed a significant relative hyperintensity in ischemic regions as early as 45 min after onset of ischemia whereas T2-weighted spin-echo images failed to clearly demonstrate brain injury up to 2-3 h postocclusion. Signal intensity ratios (SIR) of ischemic to normal tissues were greater in the diffusion-weighted images at all times than in either TE 80 or TE 160 ms T2-weighted MR images. Diffusion- and T2-weighted SIR did not correlate for the first 1-2 h postocclusion. Good correlation was found between diffusion-weighted SIR and ischemic disturbances of energy metabolism as detected by 31P and 1H MR spectroscopy. Diffusion-weighted hyperintensity in ischemic tissues may be temperature-related, due to rapid accumulation of diffusion-restricted water in the intracellular space (cytotoxic edema) resulting from the breakdown of the transmembrane pump and/or to microscopic brain pulsations.
Recent work has shown that a sustained increase in cytosolic Ca2+ concentration is often linked to the onset of cytotoxicity. Sten Orrenius and colleagues describe several biochemical mechanisms that are stimulated by such a Ca2+ increase and can directly mediate cell death by causing disruption of the cytoskeleton, DNA fragmentation and extensive damage to other cell components.
An increase in cytosolic free calcium concentration ([Ca2+]i) may trigger irreversible cell injury following cerebral ischemia. We have measured changes in [Ca2+]i in cat cortex in vivo during ischemia produced by 1 hour of middle cerebral artery occlusion and during 30 minutes of reperfusion. Indo-1, a fluorescent Ca2+ indicator, was loaded into the exposed cortex by superfusion, and changes in the [Ca2+]i signal (400/506 nm ratio) were measured microfluorometrically during ultraviolet excitation (340 nm). The nicotinamide adenine dinucleotide/reduced nicotinamide adenine dinucleotide (NAD/NADH) redox state and hemodynamic changes were measured simultaneously. The animals showing severe deterioration in their electroencephalograms (EEG) showed a progressive increase in the [Ca2+]i signal during ischemia (baseline: 1.46 +/- 0.05; 60 minutes after occlusion: 2.99 +/- 0.37; n = 7). At 30 minutes following reperfusion, the animals showing little recovery in their EEG exhibited a further increase in [Ca2+]i (4.71 +/- 0.87, n = 3), whereas animals showing significant recovery in their EEG also showed recovery of [Ca2+]i (1.55 +/- 0.09, n = 4). By contrast, the moderate or mild stroke animals with less deterioration in their EEGs showed no increase in [Ca2+]i during either ischemia or reperfusion. These data suggest that the increase in [Ca2+]i might be closely related not only to deterioration of brain function during ischemia but also to poor recovery during the reperfusion period.
This study was carried out with a recently developed model of focal cerebral ischemia in the rat based on the photochemical induction of thrombotic stroke using the dye Rose Bengal. We examined the change in the volume of the lesion and brain water content, in separate groups of rats, at different times (1, 4, 24, 72, and 168 h) after the induction of the ischemic lesion. The volume of ischemic damage increased rapidly between 1 and 24 h after the ischemic insult and decreased between 24 and 168 h. The lesion at 168 h was significantly larger than that following 1 h of ischemia and similar to that obtained at 4 h, suggesting that the maximum extent of tissue damage (without the involvement of significant edema) was reached within the first 4 h in this model. The enlargement of the lesion after 4 h correlated closely with changes in brain water content.
Alterations in the blood-brain barrier to proteins, and regional water and electrolyte content were documented in a rat model of photochemically induced small-vessel thrombosis leading to infarction. Horseradish peroxidase (HRP) or Evans blue was given immediately following a 2-min photochemical sensitization period. At 5 min following irradiation, multifocal sites of peroxidase extravasation were noted within the irradiated area. Ultrastructural examination revealed endothelial cells filled with HRP which in some cases extended into the basal lamina and extracellular spaces. At 15 min, protein leakage was more pronounced within the irradiated zone and reaction product was also apparent within the subarachnoid and perivascular spaces of brain regions remote from the site of irradiation. Widespread staining on the surface of the irradiated hemisphere was apparent in rats perfused 8 h following Evans blue infusion. Water content increased significantly by 15 min within the irradiated zone but not in brain regions remote from this site. Although vasogenic edema is an early event in this stroke model, increases in water content are restricted to the irreversibly damaged site. In contrast, protein tracer escaping from microvessels coursing within the irradiated zone was widely distributed. These findings implicate endothelial barrier dysfunction in the genesis of tissue injury in this model. Morphological evidence for the capability of macromolecules to escape from a site of evolving infarction and to migrate to distances remote from the area of primary microvascular damage is also discussed.
Cerebral ischemia leading to infarction was produced in rats by intravascular thrombosis induced by a photochemical reaction between systemically injected rose bengal and green light (560 nm) transmitted through the intact skull for a 2-min period. At 2 or 15 min following photochemical sensitization, animals were perfusion-fixed for scanning (SEM) and transmission (TEM) electron microscopic analyses of the cerebral vasculature. At 2 minutes, ultrastructural examination of cortical regions destined to undergo infarction revealed numerous platelet aggregates within both pial and intraparenchymal vessels. Platelets close to the endothelial walls were routinely degranulated with pseudopodia. Endothelial cells were frequently swollen and contained dilated mitochondria and granular endoplasmic reticulum. The endothelial luminal membrane structure was shown by high-power TEM to be focally damaged. If brain temperature was reduced by 4 degrees C during the photochemical sensitization period, the platelet response was inhibited without interfering with other ultrastructural changes. These results are consistent with the hypothesis that photochemically induced endothelial alterations stimulate platelet activation and implicate abnormal endothelial function as a primary event in the pathogenesis of photochemically induced cerebral infarction.
The state of water in cerebral ischemia was studied by using the proton nuclear magnetic resonance (1H-NMR) method. Cerebral ischemia was induced experimentally in Mongolian gerbils by unilateral ligation of the common carotid artery. Longitudinal (T1) and transverse (T2) relaxation times of the ischemic brain were measured with a pulse FT-NMR spectrometer and the water content was determined by the wet/dry method. Quantitative analysis of the relaxation times was performed sequentially during the initial 7 hours following ligation and the data were compared with those of brain edema previously reported by S. Naruse in the rat. Characteristic findings in brain ischemia include prolongation of the slow component of T2 and increase in the water content. A quantitative comparison of relaxation rate and water content demonstrates that ischemic brain edema in Mongolian gerbils is different from cytotoxic and vasogenic types of brain edema. When R2 (1/T2) was plotted against the water content, the slope value of ischemia in the gerbil was between the slope values of the TET intoxication and cold injury induced edemas reported previously. From these results, it might be said that ischemic brain edema includes both the cytotoxic and vasogenic types of brain edema. Glycerol was demonstrated to affect brain ischemia by decreasing the water content and by shortening the slow component of T2. By analysis of the relaxation times and water content, we examined the pathophysiological characteristics of water molecules in ischemic brain tissue.
At the present time, brain oedema is classified into two types: cytotoxic and vasogenic (Klatzo, 1967) in accordance with two basically different pathological mechanisms involved, each characterized by distinctly different profiles of concurrent changes. In most instances, the labelling of oedema as vasogenic or cytotoxic is only relative, since it is exceptional that only one of the two mechanisms would be operating exclusively. However, an understanding of the nature and dynamics of various brain disorders, associated with oedema, should be enhanced considerably by recognition of relative involvement of these two basic types of oedema in various phases of a pathological process.
We have used a photochemical reaction in vivo to induce reproducible thrombosis leading to cerebral infarction in rats. After the intravenous injection of rose bengal, a potent photosensitizing dye, an ischemic lesion was formed by irradiating the left parietal convexity of the exposed skull for 20 minutes with green light (560 nm) from a filtered xenon arc lamp. Animals were allowed to survive from 30 minutes to 15 days after irradiation. Early microscopic alterations within the irradiated zone included the formation of thrombotic plugs and adjacent red blood cell stasis within pial and parenchymal vessels. Scanning electron microscopy revealed frequent platelet aggregates adhering to the vascular endothelium, often resulting in vascular occlusion. Carbon-black brain perfusion demonstrated that occlusion of vascular channels progressed after irradiation and was complete within 4 hours. Histopathological examination at 1, 5, and 15 days revealed that the associated infarct evolved reproducibly through several characteristic stages, including a phase of massive macrophage infiltration. Although cerebral infarction in this model is initiated by thrombosis of small blood vessels, the fact that the main pathological features of stroke are consistently reproduced should permit its use in assessing treatment regimens. Further, the capability of producing infarction in preselected cortical regions may facilitate the study of behavioral, functional, and structural consequences of acute and chronic stroke.
In the rat middle cerebral artery occlusion (MCAO) model of embolic stroke, calcium antagonists, as well as competitive and noncompetitive N-methyl-D-aspartate (NMDA) antagonists have been studied extensively by various groups. However, there are divergent conclusions concerning their efficiencies. These discrepancies may be due to the different experimental conditions and/or rat strains used. In this study we have assessed in five commonly used rat strains, under identical experimental conditions, the ability of one representative of each of these drug classes to reduce the infarct size in this model. Drugs or corresponding vehicle were injected immediately after permanent unilateral MCAO and the infarct volume was determined 48 hr later by magnetic resonance imaging. In vehicle-treated controls, the total infarct volume was strain-dependent (range, 70-200 microliters), primarily due to variations in the cortex. The extent of total infarct volume reduction (up to 65%) elicited by the three drugs also was strain-dependent, the main effect and variation being observed in the cortex (range, 0-83% reduction). No difference in efficiency was seen between the competitive and noncompetitive NMDA antagonist, whereas the calcium antagonist showed better results in all five strains. The potential to reduce the infarct size in the rat MCAO model with calcium or NMDA antagonists depends upon the strain used. The exact reasons and mechanisms are presently not understood. Such knowledge would certainly add to the confidence in this model and would allow more accurate predictions concerning the clinical efficacy of drugs in stroke trials.
The application of bipolar diffusion sensitizing gradient pulses to significantly reduce the diffusion time is described. This approach is combined with the rapid U-FLARE imaging sequence. Three diffusion-sensitized types of experiments are compared and their suitability for detecting restricted diffusion is discussed. Experiments using a modification of the diffusion weighting by varying the diffusion time between 1.6 and 6.0 ms obtained nonmonoexponential signal attenuation curves from both healthy brains and postmortem. This behavior is indicative of restricted diffusion, but as it is detectable only at short diffusion times, in contrast to a restriction due to impermeable barriers, we have termed this "apparent restriction."
Our purpose was to use whole brain echo planar magnetic resonance imaging (MRI) to identify and characterize diffusion abnormalities in acute cerebral ischemia. We studied 40 patients as early as 3 hours after onset of signs and symptoms of cerebral ischemia. Diffusion-weighted imaging (DWI) of the entire brain could be completed in 3 seconds or, using seven different diffusion sensitivities (maximum b = 1,271 sec/mm2), in 48 seconds. Measurements and synthetic maps were made of apparent diffusion coefficients (ADC), a physiological parameter that characterizes the self-diffusion of water in tissue. Early ischemic lesions were identified with DWI as hyperintense regions of decreased ADC in all patients who subsequently developed infarction, before changes were evident on conventional MRI in cases studied earlier than 6 hours after onset of ischemic symptoms. Lesions as small as 4 mm in diameter were identified. The extent of lesions within white matter was best defined by controlling for the anisotropic effect of axonal orientation. The mean ADC (+/- SD) for control regions in the 36 patients was 9.15 (+/- 2.91) x 10(-4) mm2/sec. Mean ADC of ischemic regions was 56% of control values at 6 hours or less and stayed significantly reduced for 3 to 4 days after onset of ischemia. The relative ADC increased progressively over time to be pseudonormalized at 5 to 10 days and elevated in the chronic state, making the distinction of acute lesions adjacent to chronic infarcts readily apparent. DWI with echo planar imaging measures a unique physiological parameter that is sensitive to ischemic changes before conventional MRI. Its potential role in the quantitative study of human stroke pathophysiology and therapeutics is the subject of further investigation.
In experimental stroke research one of the key questions is whether animals models truly reflect human disease. One important reason for modelling stroke is to produce tissue changes that can be used to validate the correlation between the results of advanced radiological imaging and pathology. The aim of this study was to compare two minimally invasive rat models of focal ischaemic stroke as to their ability to simulate clinical stroke reproducibly. In one model a focal infarct was induced using the photochemical Rose Bengal technique. The second model was based on transvascular occlusion of the middle cerebral artery using a cervical approach. In all animals we performed MRI at different times with two different paramagnetic contrast agents. The time course of blood-brain-barrier disruption in the Rose Bengal model differed entirely from that in human brain ischaemia. The experimental stroke showed marked contrast enhancement in the first hour after the onset of ischaemia. On the other hand, the MRI changes in the suture occlusion model were very similar to the changes observed in human brain ischaemia: no early disruption of the blood-brain-barrier and increased T2-signal 4-6 h after the onset of stroke. We hope that the suture occlusion model will be used for research not only by basic and clinical scientists but also by radiologists, who, using modern imaging methods, may greatly influence the diagnosis as well as the management of this devastating disease.
The occlusion of the middle cerebral artery was used as an experimental acute stroke model in 30 cats. The diffusion of water was followed by diffusion-sensitized MRI between 1 and 15 h after induction of stroke. It is demonstrated that images representing the trace of the diffusion tensor provide a much more accurate delineation of affected area than images representing the diffusion in one direction only. The reason is that the strong contrast caused by the anisotropy and orientation of myelin fibers is completely removed in the trace of the diffusion tensor. The trace images show a small contrast between white and gray matter. The diffusion coefficient of white matter is decreased in acute stroke to approximately the same extent as gray matter. It is further shown that the average lifetime of water in extra and intracellular space is shorter than 20 ms both for healthy and ischemic tissue indicating that myelin fibers are permeable to water. The anisotropy contrast did not change before or after induction of stroke, nor after sacrifice. Together, these observations are consistent with the view that the changes in water diffusion during acute stroke are directly related to cytotoxic oedema, i.e., to the change in relative volume of intra- and extracellular spaces. Changes in membrane permeability do not appear to contribute significantly to the changes in diffusion.
The regional distribution and temporal evolution of the diffusion coefficient (Dw) of water in rat brain was measured during and after transient middle cerebral artery (MCA) occlusion. Male Wistar rats (n = 14) were subjected to 2 h of middle cerebral artery occlusion, induced by intracarotid insertion of a filament. Diffusion (n = 14) and perfusion (n = 7) weighted magnetic resonance imaging were performed before, and at various time points after MCA occlusion, ranging from 30 min up to 7 days. Our data demonstrate that the temporal profiles of Dw differ between the severely and the least damaged regions of tissue. In the core of the lesion, where the tissue evolved to necrosis, Dw declined significantly (P < 0.001) within 0.5 h after onset of ischemia, and remained depressed until 24 h after withdrawal of the suture. However, no statistically significant decline in Dw was found in the perifocal regions containing morphologically intact cells. Perfusion MRI qualitatively exhibited a hypoperfusion and reperfusion during, and after 2 h MCA occlusion, respectively. A significant (r > or = 0.71, P < 0.01) correlation was found between delta Dw (the difference in Dw between the ipsilateral ischemic and homologous contralateral control regions) obtained immediately before withdrawal of the suture (2 h of ischemia) and at specific early time points after withdrawal of the suture, and the degree of ischemic cell damage. No significant (P > 0.01) correlation was detected at an early time points of ischemia or at other time points after withdrawal of the suture.(ABSTRACT TRUNCATED AT 250 WORDS)
To correlate the findings on diffusion-weighted magnetic resonance (MR) images with the cytologic and histologic findings in ischemic tissue.
A photochemical model of cerebral infarction in rats was studied with diffusion- and T2-weighted MR imaging. The development of lesions was followed from 20 minutes to 5 days after the onset of ischemia. Apparent water diffusion coefficient (ADC) maps were calculated and correlated with light and electron microscopic findings.
T2-weighted images clearly showed vasogenic edema but did not enable distinction between areas with cellular damage and the surrounding edematous regions. In contrast, the ADC, which was elevated in nonischemic edematous regions, was diminished in areas with histologic evidence of ischemic damage or necrosis. In the core of the infarct, the ADC became elevated when electron microscopy revealed cellular lysis.
Diffusion-weighted images may help ascertain the extent of cellular damage and death after stroke.
The noninvasive nature of NMR imaging enables serial studies on a single animal. In 12 male Wistar rats, the dynamic progression of a photochemically induced (Rose Bengal) infarct was studied starting immediately after induction and up to 10 days. The results demonstrated that both T2- and postcontrast T1-weighted NMR images are required to discern the time dependent dynamics of the ischemic process. The ischemic lesion was already visible on T2-weighted images within 30 min after the induction. Twenty-four hours after the insult, both area and intensity reached maximum values. Hereafter and up to day 10, both parameters decreased. Postcontrast T1- weighted images revealed a blood-brain barrier (BBB) rupture immediately after the induction which persisted until 10 days after the insult. The application of contrast agents such as Gd-DOTA or Gd-HP-DO3A also allowed the detection of 10-day-old lesions which were not always discernable on T2-weighted images. The penetration of both contrast agents in the affected area proceeded slower the first 5 days after the insult while at day 10, maximum contrast enhancement was reached almost immediately after administration of the contrast agent. At 24 hr after the insult, the discrepancy between the lesion area as determined on T2-weighted images and on postcontrast T1-weighted images was maximal. At this stage, the lesion was characterized by central core with a leaky BBB surrounded by a reversible zone which appeared enhanced on T2-weighted images.
The evolution of a photochemically induced cerebral thrombotic infarction was followed in rats during the first week after the insult by means of NMR imaging and histology. Heavily T2-weighted images provided an excellent lesion detection and a high specificity for the discrimination of different histological abnormalities. The T2-weighted images showed a brain lesion evolving during the first 24 h from a homogeneous hyperintense area, histologically corresponding to diffuse vasogenic and cytotoxic oedema with concomitant neuronal necrosis, to an iso-intense area with a hyperintense seam, which microscopically correlated with increased vascular permeability at the periphery of the lesion. The hyperintense seam was observed up to day 7, but at that time coincided with gliomesodermal repair reaction which could be verified histochemically and ultrastructurally. It may be concluded that NMR-micro-imaging at a moderately high field, enables early detection and adequate follow-up of small cerebral infarctions in rats.
The sensitivity of magnetic resonance image signals of the brain to the change in the cerebral blood oxygenation was measured in gradient echo images of rat brains at a field strength of 7 T. The sensitivity depended on the blood vessel volume relative to the tissue volume within the image voxel, and signal intensities in the cortical area were well correlated with the change in the venous blood de-oxygenation level at the sagittal sinus. Tissue signals in the image (15 ms echo time) showed a sensitivity of 10-20% change for the full range of deoxygenation level from 0-100%. From these observations and image simulations, the extent of the signal response to some neuro-stimulation which induces an increase in regional cerebral blood flow has been estimated for 4 T field strength.
Cerebral circulation: stroke
- Jcm Burst
Mechanisms of lipid peroxidation potentiated by ischemia in the brain
- M D Watson Bd Ginsberg
Influence of tissue-type plasminogen activator on blood flow during evolution of thrombotic stroke
- B D Watson
- Dietrich
- M D Busto R Ginsberg
- C F Hoyng
Concepts and techniques of experimental stroke induced by cerebrovascular photothrombosis
- B D Watson
- Dietrich
- H Prado R Nakayama
- H Kanemitsu
- N N Futrell
- H Yao
- C G Markgraf
- P Wester
Mitigation of evolving cortical infarction in rats by recombinant tissue plasminogen activator following photochemically induced thrombosis
- B D Watson
- W D Prado R Dietrich
- M D Busto R Scheinberg P Ginsberg
Therapeutic modulation of brain temperature: relevance to ischemic brain injury
- M D Ginsberg
- L L Sternau
- My-T Globus
- W D Dietrich
- R Busto