Experimental Stroke Disease Phenotyping Using Mrimrs

Due to the vital role of energy metabolism for proper tissue function, noninvasive 31P MRS has been applied to monitor levels of HEP such as ATP and PCr, characterizing the metabolic state of tissue (Figure 8.2). Cerebral HEP synthesis not matching HEP consumption will ultimately lead to energy failure, to brain dysfunction, and ultimately to brain death. This is the case, e.g., for status epilepticus, during which energy consumption is dramatically increased. Prolonged seizures have been shown to compromise tissue levels of PCr and later of ATP with a concomitant increase of intracellular pH reflecting anaerobic glucose metabolism [7]. Brain ischemia has analogous metabolic consequences: during global cerebral ischemia, PCr levels disappear within 2 min following cessation of blood flow, while ATP reservoirs are depleted within typically 4 min following cardiac arrest [9]. Intracellular pH values drop from a normal value of 7.2 to values around 6.5, depending on the resting blood glucose levels [7-9]. Additional :H-MRS investigations demonstrated that energy failure is accompanied by massive build-up of intracellular lactate levels due to anaerobic glycolysis [53,54].

HEP levels as indicator of tissue state have been shown to be of limited value: due to highly efficient regulatory mechanisms only severe metabolic stress will lead to observable changes of HEP steady-state levels. As long as their synthesis rate accounts for the energy consumption, HEP levels will remain unchanged. HEP turnover, on the other hand, has to match the demands of tissue function; thus, a direct assessment of turnover rates provides superior information. This has been demonstrated in normal rat brain exposed to various "workloads." while cerebral steady-state ATP levels were not different for pentobarbital and isoflurane anesthetized rats and animals with mild epileptic seizures following administration of the 7 -butyric amino acid A (GABAa) receptor antagonist bicuculline, there was a high correlation between the forward rate constant, kf, for the creatine kinase reaction

as a measure for ATP turnover, and the cerebral energy consumption as reflected by the integrated electrical activity [55]. A similar correlation has been observed for the glucose perfused rat heart [56]. Kinetic information was derived from so-called magnetization transfer experiments, in which the transfer of a magnetic label between two reaction partners (in the case of the creatine kinase reaction PCr and ATP) was monitored [56-59].

MRS studies, while providing valuable mechanistic information, are limited (1) by the lack of sensitivity; (2) by a small dynamic range (it is difficult to detect drug effects of less than 20 to 30%); and (3) by poor spatial resolution, Therefore, MRS has played a minor role in the management of stroke patients and for the development of novel antiischemic therapies. In contrast, MRI methods emerged as an indispensable tool to identify and stage cerebral ischemic tissue (see also Chapter 9).

The classical MRI method for assessment of cerebral infarction is based on T2 contrast: accumulation of water in the infarcted brain area (vasogenic edema) leads to significantly elevated T2 values providing a good demarcation between ischemic and intact tissue. The contrast-to-noise ratio is high enough to allow for application of tissue segmentation algorithms based on intensity thresholding and the method has been extensively applied to quantitative assessments of drug effects on infarct volume in animal models of focal cerebral ischemia [36,60]. There is an excellent correlation between infarct volumes determined in vivo using T2-weighted MRI and lesion volumes derived from histology [61].

Tissue areas displaying increased T2 values are already irreversibly damaged; thus, earlier indicators that provide a significant contrast for tissue that is still salvageable would be highly relevant. Cerebral ischemia is caused by occlusion of a major brain artery, therefore assessment of CBF should immediately reflect hypoperfused brain regions. A number of MRI methods to assess local CBF has been developed [62-64] and applied to studies of stroke patients [65] or in animal models of stroke. In rat focal cerebral ischemia models, perfusion deficits have been visualized within seconds after occlusion of a major brain artery such as the MCA [42,66]. Relative perfusion values as compared to normally perfused brain regions can be obtained in a straightforward manner, but the determination of absolute values in ml/g tissue/min remains difficult. Yet, assessment of absolute values would be essential as reduced values of tissue perfusion do not necessarily imply tissue necrosis, and there are region-specific perfusion thresholds for neuronal function and neuronal survival [67].

As pointed out in Section 8.2, a consequence of cellular energy failure is the loss of ion homeostasis; the altered cellular osmolality leads to a massive influx of water and, hence, to cell swelling. This cytotoxic edema is reflected by a decrease of the water ADC value. The ADC measured in a voxel is the weighted average of the intra- and extracellular contributions. Since the extracellular diffusion coefficient is larger than the intracellular one, an increase in the intracellular volume fraction will lead to a decrease of the ADC. The correlation of cerebral ADC values with measurements of electrical conductivity in brain parenchyma in a neurotoxic lesion model supported this interpretation [68]. Nevertheless, this view is an oversimplification; additional factors to be considered are changes of diffusion constants within the individual tissue compartments [69,70]. Indeed, ADC reduction of the intracellular metabolites after global ischemia or cytotoxic lesions were observed [71].

Decreases in cerebral ADC values occur within minutes after onset of ischemia [72], i.e., diffusion-weighted MRI detects early microstructural changes following an ischemic insult. More importantly even, it has been demonstrated at least in animal models of global [73] and focal ischemia [74] that ADC changes are reversible: in both cases tissue reperfusion led to normalization of ADC values that were decreased during the ischemic episode. Similarly, it has been demonstrated that pharmacological interventions in NMDA-induced neurotoxicity normalized ADC values in newborn rats [68]. The evolution of the water ADC in ischemic tissue shows a characteristic profile both in stroke patients [75] and in animals models of focal cerebral ischemia [76]. The initial drop to 60% of the baseline values is followed by a pseudonormalization, which is commonly observed between 4 to 8 days after the insult.

The combination of the various MR readouts (T2, Tj, CBF, CBV, ADC) provides a comprehensive characterization of the ischemic brain parenchyma (see also Chapter 9, Section 9.2.3). As the temporal evolution following infarction is different for the various parameters, a combination of them may provide a fingerprint regarding the state of the infarcted tissue (Figure 8.4). For instance, decreased CBF and ADC but still normal T2 values indicate brain regions with reversible tissue damage that might be potentially saved by pharmacological treatment, while areas with decreased CBF, ADC and increased T2 values are destined to becoming necrotic. Hence, the tissue stroke signature [76,77] might be used to predict the eventual final outcome of the infarction and to identify the brain region that might be responsive to a therapeutic intervention. Unfortunately, applications of such concepts were of limited success up to now [78].

Pathological brain regions are characterized by deviations of MRI parameters from "normality." Needless to say, that this procedure is to some extent arbitrary. How much must T2 values deviate from normality to be identified as abnormal? This depends on a variety of factors such as the general signal-to-noise ratio of the image and the morphological heterogeneity of the corresponding brain area. Moreover, clinical stroke is highly heterogeneous, spontaneous recanalization may occur with profound influence on the evolution of the MRI parameters and thus to their prognostic quality. In animal models, rather homogeneous conditions can be achieved. For instance, in the rat unilateral permanent MCAO model, it has been found that the extent of the lesion derived from CBF maps recorded 1 h postinfarction (based on relative CBF values in the ischemic region that were significantly smaller than contralateral values) predicted the outcome at 48 h with good accuracy in untreated animals [76]. In contrast, the ischemic regions as derived from ADC and T2 maps at 1 h were significantly smaller than those determined at 48 h, i.e., there was significant growth of the lesion area both in ADC- and T2-weighted images. Hence, the prognostic

FIGURE 8.4 Characteristic tissue signatures based on MRI parameters for rat permanent MCA occlusion. At 2 h after occlusion CBF, CBV, and ADC are decreased in the infarcted area (left side) as compared to the healthy control side, while T2 maps display no left/right asymmetry yet. At 24 h, CBF, CBV, and ADC remain decreased in the lesion area, while T2 now is clearly elevated due to the formation of a vasogenic edema. The temporal evolution of the various MRI parameters allows staging of tissue and potentially the prediction whether a specific region might by salvagable by a therapeutic intervention. (Adapted from Rudin, M. et al., Exp. Neurol., 169, 56, 2001. With permission.)

FIGURE 8.4 Characteristic tissue signatures based on MRI parameters for rat permanent MCA occlusion. At 2 h after occlusion CBF, CBV, and ADC are decreased in the infarcted area (left side) as compared to the healthy control side, while T2 maps display no left/right asymmetry yet. At 24 h, CBF, CBV, and ADC remain decreased in the lesion area, while T2 now is clearly elevated due to the formation of a vasogenic edema. The temporal evolution of the various MRI parameters allows staging of tissue and potentially the prediction whether a specific region might by salvagable by a therapeutic intervention. (Adapted from Rudin, M. et al., Exp. Neurol., 169, 56, 2001. With permission.)

value of CBF measurements was found to be superior to that of ADC and T2 values. This has also been reported in clinical stroke (see also Chapter 9, Section 9.2.1): a significant perfusion/diffusion mismatch, with the "perfusion lesion" being larger than that derived from ADC maps, implied significant growth of the ADC lesion [79].

Over many years, research in stroke was focused on the acute phase, i.e., the first 48 h following infarction. The therapy objective was to protect tissue from becoming infarcted by intervening at the early steps in the pathophysiological cascade. However, therapy successes achieved in animal models of focal cerebral ischemia could not be translated into the clinics due to a variety of reasons. A critical factor is the short time window available for successful cytoprotective interventions, probably of only a few hours, which is a severe hurdle for clinical drug development. In recent years, focus in stroke research was shifted to later phases of the pathology, in particular the inflammatory component of the disease has been addressed. Correspondingly, MRI methods to assess these aspects of cerebral ischemia have been developed.

Inflammation is characterized by massive infiltration of immune-competent cells, predominantly blood-borne monocytes that infiltrate tissue by transcytosis to become activated macrophages. These cells of the monocyte phagocytotic system (MPS) are often found at the border of ischemic/necrotic tissue. Ultrasmall particles of iron-oxide (USPIO) have been developed as contrast agents for visualization of structures with phagocytotic function, such as the spleen or lymph nodes [80]. These nanoparticles are efficiently internalized by monocytes/macrophages via absorptive endocytosis and, hence, can be used to track such cells. Feasibility of USPIO-based macrophage imaging in the brain has been demonstrated in the rat experimental autoimmune encephalomyelitis model of multiple sclerosis [81,82] (see also Chapter 12). In these studies, focal areas characterized by shortening of T2 and T2 have been observed 24 h following the systemic administration of USPIOs. Histological analysis confirmed the presence of USPIO-labeled macrophages in the inflamed tissue regions. Application of the same approach to models of permanent [83] and transient MCAO in rats [18] displayed massive macrophage infiltration in focal ischemia. During the first 2 days after vessel occlusion USPIO accumulation was predominantly observed in patches in the core lesion comprising the caudate putamen and at the periphery of the infarction. At later time points, maximal label accumulation was observed at the boundary of the lesion. USPIO loaded macrophages could be monitored up to 7 days following systemic administration of the contrast agent.

MRI can be applied to visualize different phases of the pathophysiological cascade of stroke, from the initial vascular occlusion visualized by MR angiography [84,85] to the chronic phase with neuro-inflammatory and apoptotic processes. Application of functional MRI (fMRI) methods allows this comprehensive structural and physiological characterization to be complemented by readouts of CNS function. Obviously, focal cerebral ischemia translates into loss of functionality in the affected brain areas. In the rat permanent MCA occlusion model sensory stimulation of the forepaw contralateral to the infarction side did not evoke a hemodynamic response in the infarcted somatosensory cortex [86,87]. Similarly, global stimulation using bicuculline identified significant functional dropouts in the infarcted territory [86]. Functional deficits could be identified in transient MCA occlusion of short duration, despite apparent structural integrity of the tissue [88]. Functional readouts are particularly attractive when studying functional recovery, e.g., following cytoprotective therapy (see Section 8.6.3).

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