Developing Compatible Techniques for Magnetic Resonance Imaging of Stroke Pathophysiology

Stroke is the most prevalent neurological disease facing our nation today. Treatments, however, are few and insufficient at reducing the impact of this neurological condition. Experimental animal models are important to improving our understanding of stroke, and for developing new therapies to counter the pathology of stroke. Magnetic Resonance Imaging is the leading tool for the non-invasive investigation of stroke pathophysiology. While most MRI work in animals is conducted under anesthesia, anesthesia has profound effects on cerebral circulation and metabolism, and can affect stroke outcome. Several novel methods were combined with MRI compatible physiologic monitoring equipment to conduct stroke studies in conscious animals. Stress was studied as a factor in these studies and conditioning was utilized to reduce the impact of stress on the animals' physiology. Models of both occlusive and hemorrhagic strokes were successfully implemented within the MRI apparatus. Lastly, experiments using a macrosphere model showed evidence of a pathophysiologic difference between awake and anesthetized animals that undergo stroke

Magnetic resonance imaging of hemorrhagic stroke

Stroke is the third leading killer of Americans and the major cause of disability in our population. It is pathologically obvious yet mechanistically very complex and perplexing. Many individual facets of stroke have been studied independently under very aberrant conditions with attention focused on occlusive stroke. The actual onset of hemorrhagic stroke has never been witnessed though. To achieve this ambitious goal we had to generate most of the procedures we used. With our central theory being that the event of stroke in Stroke Prone Spontaneously Hypertensive Rats (SPSHR) is closely tied to blood pressure, we studied the special physiology of these animals, and used a hypertensive dose of i.v. norepinephrine to induce stroke. We then utilized Magnetic Resonance Imaging (MRI) techniques for imaging conscious animals to image SPSHRs as they underwent hemorrhagic stroke. With a 4.7T MR scanner (Biospec, Bruker Medical) we used diffusion, perfusion, T1 and T2* weighted pulse sequences. In the most robust case, the hemorrhagic insult began at the head of the caudate putamen and was followed by a rapid filling of the ventricular system with blood. The drastic change in cerebral hemodynamics was confirmed with MR angiography. Behavioral tests were run prior to and following stroke, but only proved the ambiguity of such methods in comparison to the certainty of MRI. After imaging, animals were sacrificed and immunocytochemically stained for fibronectin and albumin. All animals were acquired and cared for in accordance with the guidelines published in the Guide for the care and use of laboratory animals (National Institute of Health Publications No. 85-23, Revised 1985)

Imaging the neural substrates involved in the genesis of pentylenetetrazol-induced seizures

Summary: Purpose: Functional imaging of animal models makes it possible to map the functional neuroanatomy contributing to the genesis of seizures. Pentylenetetrazol (PTZ)-induced seizure in rats, a relevant model of human absence and of generalized tonic\u96clonic epilepsy, was used to stimulate seizure activity within 30 s of administration while collecting continuous, high-resolution, multislice images at subsecond intervals. Methods: Pilot studies were conducted to establish a quick and effective PTZ model for the imaging experiments. PTZ was then used to stimulate seizure activity in rats while collecting multislice functional MRI (fMRI) images from the entire forebrain at 4.7 Tesla. Ethosuximide (ESM) also was used to block seizure activity. Results: Within 2-4 s of PTZ administration, a rapid increase in blood oxygen level-dependent (BOLD) signal intensity was noted in the thalamus, especially the anterior thalamic nuclei. Activity in the anterior thalamus peaked 15 s before seizure onset and was more than twofold greater than that in all other thalamic areas. The retrosplenial cortex showed a twofold greater increase in activity as compared with other cortical areas, also peaking at 15 s. The dentate gyrus was twice as active as other hippocampal areas but peaked just before seizure onset. Treatment with ESM blocked seizures, decreasing PTZ-induced activation in most forebrain areas. The anterior thalamus and retrosplenial cortex were essentially blocked by pretreatment with ESM. Conclusions: The anterior thalamus, retrosplenial cortex, and dentate gyrus show the greatest increases in BOLD signal activity before seizure onset. Neurons in these areas may contribute to the neural network controlling the initiation of generalized tonic-clonic seizur

Changes in MRI signal intensity during hypercapnic challenge under conscious and anesthetized conditions

Most functional magnetic resonance imaging (fMRI) studies in animals are conducted under anesthesia to minimize motion artifacts. However, methods and techniques have been developed recently for imaging fully conscious rats. Functional MRI studies on conscious animals report enhanced BOLD signal changes as compared to the anesthetized condition. In this study, rats were exposed to different concentrations of carbon dioxide (CO(2)) while conscious and anesthetized to test whether cerebrovascular reactivity may be contributing to these enhanced BOLD signal changes. Hypercapnia produced significantly greater increases in MRI signal intensity in fully conscious animals (6.7-13.3% changes) as when anesthetized with 1% isoflurane (3.2-4.9% changes). In addition, the response to hypercapnia was more immediate in the conscious condition (< 30s) with signal risetimes twice as fast as in the anesthetized state (60s). Both cortical and subcortical brain regions showed a robust, dose- dependent increase in MRI signal intensity with hypercapnic challenge while the animals were conscious but little or no change when anesthetized. Baseline variations in MRI signal were higher while animals were conscious but this was off set by greater signal intensity changes leading to a greater contrast-to-noise ratio, 13.1 in conscious animals, as compared to 8.0 in the anesthetized condition. In summary, cerebral vasculature appears to be more sensitive to hypercapnic challenge in the conscious condition resulting in enhanced T2* MRI signal intensity and the potential for better BOLD signal changes during functional imaging

Imaging cocaine-induced changes in the mesocorticolimbic dopaminergic system of conscious rats

Functional magnetic resonance imaging (fMRI) was used to assess the effects of cocaine on brain activation in fully conscious rats. Methods were developed to image cocaine-induced changes in blood-oxygen-level-dependent (BOLD) signal without the peripheral cardiac and respiratory complications associated with psychostimulant administration. Using spin echo planar imaging (EPI), conscious rats were imaged in a 4.7 T spectrometer prior to and following the intracerebroventricular injection of cocaine (20 microg) in artificial cerebrospinal fluid (10 uL). Within 5 min of injection, there was a significant increase in BOLD signal intensity in the substantia nigra, ventral tegmental area, nucleus accumbens, dorsal striatum and prefrontal cortex, as compared to vehicle controls. Minimal negative BOLD signal changes were observed in response to cocaine and no significant perturbations in normal cardiovascular and respiratory function. These findings demonstrate the technical feasibility of studying psychostimulant-induced brain activity using functional MRI in conscious rats

Imaging cocaine-induced changes in the mesocorticolimbic dopaminergic system of conscious rats

Functional magnetic resonance imaging (fMRI) was used to assess the effects of cocaine on brain activation in fully conscious rats. Methods were developed to image cocaine-induced changes in blood-oxygen-level-dependent (BOLD) signal without the peripheral cardiac and respiratory complications associated with psychostimulant administration. Using spin echo planar imaging (EPI), conscious rats were imaged in a 4.7 T spectrometer prior to and following the intracerebroventricular injection of cocaine (20 microg) in artificial cerebrospinal fluid (10 uL). Within 5 min of injection, there was a significant increase in BOLD signal intensity in the substantia nigra, ventral tegmental area, nucleus accumbens, dorsal striatum and prefrontal cortex, as compared to vehicle controls. Minimal negative BOLD signal changes were observed in response to cocaine and no significant perturbations in normal cardiovascular and respiratory function. These findings demonstrate the technical feasibility of studying psychostimulant-induced brain activity using functional MRI in conscious rats

The macrosphere model: evaluation of a new stroke model for permanent middle cerebral artery occlusion in rats

BACKGROUND AND PURPOSE: The suture middle cerebral artery occlusion (MCAO) model is widely used for the simulation of focal cerebral ischemia in rats. This technique causes hypothalamic injury resulting in hyperthermia, which can worsen outcome and obscure neuroprotective effects. Herein, we introduce a new MCAO model that avoids these disadvantages. METHODS: Permanent MCAO was performed by intraarterial embolization using six TiO(2) macrospheres (0.3-0.4 mm in diameter) or by the suture occlusion technique. Body temperature was monitored, functional and histologic outcome was assessed after 24 h. Additional 16 rats were subjected to macrosphere or suture MCAO. Lesion progression was evaluated using magnetic resonance imaging (MRI). RESULTS: The animals subjected to suture MCAO developed hyperthermia (>39 degrees C), while the temperature remained normal in the macrosphere MCAO group. Infarct size, functional outcome and model failure rate were not significantly different between the groups. Lesion size on MRI increased within the first 90 min and remained unchanged thereafter in both groups. CONCLUSIONS: The macrosphere MCAO model provides reproducible focal cerebral ischemia, similar to the established suture technique, but avoids hypothalamic damage and hyperthermia. This model, therefore, may be more appropriate for the preclinical evaluation of neuroprotective therapies and can also be used for stroke studies under difficult conditions, e.g., in awake animals or inside the MRI scanner

Regional cerebral blood flow and BOLD responses in conscious and anesthetized rats under basal and hypercapnic conditions: implications for functional MRI studies

Anesthetics, widely used in magnetic resonance imaging (MRI) studies to avoid movement artifacts, could have profound effects on cerebral blood flow (CBF) and cerebrovascular coupling relative to the awake condition. Quantitative CBF and tissue oxygenation (blood oxygen level-dependent [BOLD]) were measured, using the continuous arterial-spin-labeling technique with echo-planar-imaging acquisition, in awake and anesthetized (2% isoflurane) rats under basal and hypercapnic conditions. All basal blood gases were within physiologic ranges. Blood pressure, respiration, and heart rates were within physiologic ranges in the awake condition but were depressed under anesthesia (P < 0.05). Regional CBF was heterogeneous with whole-brain CBF values of 0.86 +/- 0.25 and 1.27 +/- 0.29 mL. g-1. min-1 under awake and anesthetized conditions, respectively. Surprisingly, CBF was markedly higher (20% to 70% across different brain conditions) under isoflurane-anesthetized condition compared with the awake state (P < 0.01). Hypercapnia decreased pH, and increased Pco(2) and Po(2). During 5% CO(2) challenge, under awake and anesthetized conditions, respectively, CBF increased 51 +/- 11% and 25 +/- 4%, and BOLD increased 7.3 +/- 0.7% and 5.4 +/- 0.4%. During 10% CO(2) challenge, CBF increased 158 +/- 28% and 47 +/- 11%, and BOLD increased 12.5 +/- 0.9% and 7.2 +/- 0.5%. Since CBF and BOLD responses were substantially higher under awake condition whereas blood gases were not statistically different, it was concluded that cerebrovascular reactivity was suppressed by anesthetics. This study also shows that perfusion and perfusion-based functional MRI can be performed in awake animals.MD/Ph