Magnetic resonance imaging of ischemic injury produced by varying severities of photothrombosis differs in neonatal and adult brain.

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.

Cellular correlates of longitudinal diffusion tensor imaging of axonal degeneration following hypoxic-ischemic cerebral infarction in neonatal rats.

Ischemically damaged brain can be accompanied by secondary degeneration of associated axonal connections e.g. Wallerian degeneration. Diffusion tensor imaging (DTI) is widely used to investigate axonal injury but the cellular correlates of many of the degenerative changes remain speculative. We investigated the relationship of DTI of directly damaged cerebral cortex and secondary axonal degeneration in the cerebral peduncle with cellular alterations in pan-axonal neurofilament staining, myelination, reactive astrocytes, activation of microglia/macrophages and neuronal cell death. DTI measures (axial, radial and mean diffusivity, and fractional anisotropy (FA)) were acquired at hyperacute (3 h), acute (1 and 2 d) and chronic (1 and 4 week) times after transient cerebral hypoxia with unilateral ischemia in neonatal rats. The tissue pathology underlying ischemic and degenerative responses had a complex relationship with DTI parameters. DTI changes at hyperacute and subacute times were smaller in magnitude and tended to be transient and/or delayed in cerebral peduncle compared to cerebral cortex. In cerebral peduncle by 1 d post-insult, there were reductions in neurofilament staining corresponding with decreases in parallel diffusivity which were more sensitive than mean diffusivity in detecting axonal changes. Ipsilesional reductions in FA within cerebral peduncle were robust in detecting both early and chronic degenerative responses. At one or four weeks post-insult, radial diffusivity was increased ipsilaterally in the cerebral peduncle corresponding to pathological evidence of a lack of ontogenic myelination in this region. The detailed differences in progression and magnitude of DTI and histological changes reported provide a reference for identifying the potential contribution of various cellular responses to FA, and, parallel, radial, and mean diffusivity.

Comparison of T2 and T2*-weighted MR molecular imaging of a mouse model of glioma.

BACKGROUND: Standard MRI has been used for high-grade gliomas detection, albeit with limited success as it does not provide sufficient specificity and sensitivity to detect complex tumor structure. Therefore targeted contrast agents based on iron oxide, that shorten mostly T2 relaxation time, have been recently applied. However pulse sequences for molecular imaging in animal models of gliomas have not been yet fully studied. The aim of this study was therefore to compare contrast-to-noise ratio (CNR) and explain its origin using spin-echo (SE), gradient echo (GE), GE with flow compensation (GEFC) as well as susceptibility weighted imaging (SWI) in T2 and T2* contrast-enhanced molecular MRI of glioma. METHODS: A mouse model was used. U87MGdEGFRvIII cells (U87MG), derived from a human tumor, were injected intracerebrally. A 9.4 T MRI system was used and MR imaging was performed on the 10 day after the inoculation of the tumor. The CNR was measured prior, 20 min, 2 hrs and 24 hrs post intravenous tail administration of glioma targeted paramagnetic nanoparticles (NPs) using SE, SWI, GE and GEFC pulse sequences. RESULTS: The results showed significant differences in CNR among all pulse sequences prior injection. GEFC provided higher CNR post contrast agent injection when compared to GE and SE. Post injection CNR was the highest with SWI and significantly different from any other pulse sequence. CONCLUSIONS: Molecular MR imaging using targeted contrast agents can enhance the detection of glioma cells at 9.4 T if the optimal pulse sequence is used. Hence, the use of flow compensated pulse sequences, beside SWI, should to be considered in the molecular imaging studies.

Magnetization transfer and diffusion imaging of acute axonal damage in the cerebral peduncle following hypoxia-ischemia in neonatal rats.

BACKGROUND: Magnetic resonance imaging (MRI) of axonal degenerative changes in the cerebral peduncle of the corticospinal tract following cerebral hypoxic-ischemic damage might distinguish infants most appropriate for receiving prompt treatment. The optimal MRI sequence for very early diagnosis of axonal degenerative changes is unknown. We hypothesized that magnetization transfer ratio (MTR) imaging would be more sensitive than traditional MRI, e.g., T(2) or diffusion weighted imaging. METHODS: Transient unilateral cerebral hypoxia-ischemia was produced in the neonatal rat followed by MRI of changes in T(2), the apparent diffusion coefficient (ADC) of water, and MTR, with a focus on the parietal cortex (an ischemic damaged region) and the cerebral peduncle (remote within the corticospinal tract). Rats were imaged at 2 h, 1 d, or 1 wk postinsult. RESULTS: In the cerebral peduncle, MTR and T(2) responded similarly, with alterations occurring ipsilaterally at 1 d postinsult. ADC was most sensitive for detecting changes as early as 2 h postinsult, and this corresponded to a reduced staining of axonal filaments ipsilaterally. CONCLUSION: MTR and T(2) imaging have comparable sensitivity for distinguishing early axonal damage in the cerebral peduncle. ADC imaging is highly sensitive for detecting early disruption of corticospinal axons, supporting its potential hyperacute diagnostic use clinically.

A comparison of MR imaging of a mouse model of glioma at 0.2 T and 9.4 T.

Both 0.2 T and 9.4 T MRI systems were used to image a mouse model of glioma. RF coils were designed for both fields. A spin-echo, multi-echo pulse sequence was used to determine T(2) relaxation times of both brain and tumor tissues. Contrast-to-noise ratio was calculated based on the selected echo time. The results showed that 0.2 T is suitable for mouse model imaging, however total scan time must be long to achieve high enough SNR. T(2) relaxation times of the tumor and brain tissues can be measured at 0.2 T and are 2.1 and 1.8 times respectively longer at 0.2 T than at 9.4 T. Contrast to noise ratio for tumor and brain was better at high field than at the low field. We concluded that 0.2 T may be used to study mouse model of glioma using spin echo pulse sequence, yet the total scan time is long (about 40 min), resolution is lower (∼250 µm × 250 µm) and slice thickness (3mm) must be large enough to obtain sufficient SNR.

Detection of T(2) changes in an early mouse brain tumor.

The aim of the study was to determine the effect of early tumor growth on T(2) relaxation times in an experimental glioma model. A 9.4-T magnetic resonance imaging (MRI) system was used for the investigations. An animal model (n=12) of glioma was established using an intracranial inoculation of U87MGdEGFRvIII cells. The imaging studies were performed from Day 10 through Day 13 following tumor inoculation. Tumor blood vessel density was determined using quantitative immunochemistry. Tumor volume was measured daily using MR images. T(2) values of the tumor were measured in five areas across the tumor and calculated using a single exponential fitting of the echo train. The measurements on Days 10 and 13 after tumor inoculation showed a 20% increase in T(2). The changes in T(2) correlated with the size of the tumor. Statistically significant differences in T(2) values were observed between the edge of the tumor and the brain tissue on Days 11, 12 and 13 (P=.014, .008, .001, respectively), but not on Day 10 (P=.364). The results show that T(2)-weighted MRI may not detect glioma during an early phase of growth. T(2) increases in growing glioma and varies heterogenously across the tumor.

Mild cerebral hypoxia-ischemia produces a sub-acute transient inflammatory response that is less selective and prolonged after a substantial insult.

Cerebral ischemia initiates various injurious processes including neuroinflammatory responses such as activation of microglia and increases in cytokine and nitric oxide release. Evidence primarily from in vitro studies, indicates that neuroinflammatory effects can be either beneficial or harmful, possibly related to stimulus strength. We investigated using in vivo models, the effect of a mild or substantial cerebral hypoxia-ischemia on: cerebral microglial/macrophage activation (ED1), pro-inflammatory cytokines (tumor necrosis factor-alpha), nitrosative stress (nitrotyrosine) and permanent brain damage. A mild insult produced a transient (1-2 days post) increase in activated microglia/macrophages within subcortical white and not gray matter but transiently increased cytokine or nitrotyrosine expression in cortex and not white matter. There was also prolonged scattered cell death in cortex and white matter over weeks along with loss of myelin/axons and cortical atrophy at 4 weeks post-insult. In contrast, a substantial insult produced white and gray matter necrosis, cyst formation and atrophy, along with increases in tumor necrosis factor and nitrotyrosine staining within both white and gray matter starting at 1-2 days post-insult. Microglial/macrophage staining was increased starting at 1-week post a substantial insult and remained elevated for weeks thereafter. Thus, a transient neuroinflammatory response occurs following a mild insult whereas prolonged scattered cell death occurs for weeks, particularly in white matter. Insult severity also affects the progression of the neuroinflammatory response, which is prolonged after a substantial insult. Effective therapy will need to be customized for insult severity and timing; and, monitoring the injury processes with imaging or biomarkers may help guide treatment.

Functional magnetic resonance imaging within the rat spinal cord following peripheral nerve injury.

Functional magnetic resonance imaging (fMRI) was used to detect the effects of graded peripheral nerve injury at the spinal level. Graded peripheral nerve injury in rats was accomplished by transection of nerves entering the spinal cord at the L3 and L4 levels of the spinal cord segments. Electrical stimulation of the hindpaw was used to elicit activity within the spinal cord. The stimulation experimental paradigm consisted of 62 functional images, 5 slices each, with a total of 3 rest and 2 stimulation periods. A 9.4 T MRI system and a quadrature volume rf coil covering the lumbar spinal cord were used for the fMRI study. Sets of fast spin echo images were acquired repeatedly following sham preparatory surgery under control conditions and in rats following sham surgery (pre nerve cut), followed by L3 nerve and then L4 nerve section. In rats with sham surgery, there was a significant activation within the dorsal horn of slices corresponding to L3 and L4 spinal cord segments. Following section of the L3 nerve, there was a reduction in the number of active voxels in the L3 and L4 spinal cord segments. The activation was reduced further by sectioning of the L4 nerve. Thus, following an increasing loss of axonal connections to the spinal cord, there was a decreasing number of active voxels within the spinal cord. The results demonstrate that spinal fMRI in the rat has sufficient sensitivity to detect within the spinal cord the effects of a graded reduction in peripheral connectivity.

Transient hypertension concurrent with forepaw stimulation enhances functional MRI responsiveness in infarct and peri-infarct regions.

Although functional magnetic resonance imaging (fMRI) is gaining use as a tool to assess cerebral recovery following various insults, the effects of potential confounders such as hypertension are poorly defined. We hypothesized that after stroke, transient hypertension during an fMRI study could produce a detected activation unrelated to neuronal activity within the infarct. Thus, the effect of norepinephrine induced increases in blood pressure (BP) on the fMRI response to forepaw stimulation were investigated in controls or 1 week after transient middle cerebral artery occlusion in rats. Images were smoothed spatially and voxels correlating to either forepaw stimulation or the change in BP time courses were analyzed. Transient hypertension increased the signal intensity and numbers of voxels correlating to the BP time courses within and adjacent to the ischemic infarct and these exceeded the response in the contralateral hemisphere or in controls. With left paw stimulation at normotension, there was a loss of activation in right sensory-motor cortex -- a region with necrosis and disruption of cerebral vessels. As BP increased left paw stimulation also resulted in the detection of activation in the infarcted sensory-motor cortex and peri-infarct regions. Thus, BP changes synchronous with tasks in fMRI studies can result in MR signal changes consistent with a loss of cerebral blood flow (CBF) autoregulation rather than neuronal activation in necrotic brain. After stroke, the use of stressful tasks associated with BP changes in fMRI studies should be limited or the BP change should be considered as a potential source of MR signal changes.

An integrated radio frequency probe and cranial clamp for intraoperative magnetic resonance imaging: technical note.

OBJECTIVE: To design an integrated radio frequency (RF) head probe and cranial clamp for intraoperative magnetic resonance imaging (MRI) that do not interfere with the operating procedures. METHODS: A concept based on four inductively coupled rings was developed and applied for an intraoperative RF probe. The probe was integrated with a specially designed cranial clamp and incorporated into the intraoperative MRI system. RESULTS: The design of the RF probe allows splitting the probe into two separate parts; the lower two rings and matching ring are permanently incorporated into the patient table, and the two upper rings can be removed to expose the patient's head during neurosurgery. The probe produces a homogeneous B1 field over the entire region of interest with sufficient sensitivity to obtain high quality images. The cranial clamp, made of MRI compatible materials, is asymmetrical to allow variable head positioning. CONCLUSION: The described RF head probe and cranial clamp have been used successfully in more than 400 brain surgeries without compromising sterility of the operating area. Pre-, intra-, and postsurgical MRI scans have been obtained without a need to move a patient or reposition the head for imaging sessions. The images were of high quality and free of susceptibility or eddy currents artifacts. With minor modifications, the integrated RF probe and cranial clamp can be used successfully in other intraoperative MRI systems.

Diabetes, leukoencephalopathy and rage.

Longstanding diabetes mellitus damages kidney, retina, peripheral nerve and blood vessels, but brain is not usually considered a primary target. We describe direct involvement of the brain, particularly white matter, in long-term (9 months) experimental diabetes of mice, not previously modeled, correlating magnetic resonance (MR) imaging with quantitative histological assessment. Leukoencephalopathy and cerebral atrophy, resembling that encountered in diabetic humans, developed in diabetic mice and was accompanied by time-related development of cognitive changes in behavioural testing. Increased RAGE (receptor for advanced glycation end products) expression, a mediator of widespread diabetic complications, increased dramatically at sites of white matter damage in regions of myelination. RAGE expression was also elevated within neurons, astrocytes and microglia in grey matter and within oligodendrocytes in white matter. RAGE null diabetic mice had significantly less neurodegenerative changes when compared to wild-type diabetic mice. Our findings identify a robust and novel model of cerebral, particularly white matter, involvement with diabetes associated with abnormal RAGE signaling.

Transient blood pressure changes affect the functional magnetic resonance imaging detection of cerebral activation.

Functional magnetic resonance imaging (fMRI) provides an indirect measure of cerebral activation that could be altered by factors directly affecting cerebral blood flow independent of changes in neuronal activation. Presently, we investigate how changes in blood pressure (BP) affect the activation detected with fMRI. fMRI scans were acquired in 33 rats under control conditions and following transient BP increases (norepinephrine, IV) or decreases (arfonad, IV) with and without electrical stimulation of the forepaw. Voxels correlating to either the stimulation or the change in BP time courses were identified. During transient hypertension, irrespective of forepaw stimulation, BP increases (i.e., >10 mm Hg) produced a transient increase in the blood oxygen level-dependent (BOLD) intensity resulting in a significant numbers of voxels correlating to the BP time courses (P < 0.05), and the number of these voxels increased as BP increased, becoming substantial at BP > 30 mm Hg. The activation patterns with BP increases and stimulation overlapped spatially resulting in an enhanced cerebral activation to simultaneous forepaw stimulation (P < 0.05). BP decreases (>10 mm Hg) produced corresponding decreases in BOLD intensity, causing significant numbers of voxels correlating to the BP decreases (P < 0.005), and these numbers increased as BP decreased (P < 0.001). The BP decreases and stimulation time courses and responses were distinct, and hypotension did not affect the detection of the activation response to forepaw stimulation. The results indicate that substantial hypertension accompanying a stimulation paradigm produces a BOLD response that enhances the cerebral activation detected, whereas hypotension does not affect the detection of neuronal activation but does produce responses that could be interpreted as a 'deactivation'.

White matter damage precedes that in gray matter despite similar magnetic resonance imaging changes following cerebral hypoxia-ischemia in neonatal rats.

We hypothesized that the cerebral injury produced by hypoxia-ischemia (HI) in neonatal rats would differ in white compared with gray matter as detected histologically or with magnetic resonance (MR) imaging methods. Maps of T2 and the apparent diffusion coefficient (ADC) of water were acquired in 1-week-old rats at times prior to cerebral HI (right carotid artery occlusion plus 1.5 h of hypoxia), within the last 5-10 min of HI, and 1 h or 24 h after HI. Near the end of HI, ADC decreased and T2 increased in both cortical gray and subcortical white matter within the cingulum of the HI hemisphere. One hour after HI, ADC partially recovered, but T2 remained increased and then increased further by 24 h post-HI. In contrast to the similar MR responses in white and gray matter, histological evidence for irreversible cell damage occurred in white matter earlier than in gray matter within the HI hemisphere. At 1 h post-HI, rarefied or disrupted nerve fibers and an increase in TUNEL-positive cells were observed within white matter in the cingulum, whereas neurons within the cortical gray matter appeared normal. By 24 h post-HI, damage was apparent in both white and gray matter. Thus, MR imaging detected acute tissue edema following cerebral HI in both gray and white matter but did not distinguish between the early irreversible tissue injury detected histologically in white but not gray matter in this rather severe model of neonatal encephalopathy.

Magnetic resonance imaging of differential gray versus white matter injury following a mild or moderate hypoxic-ischemic insult in neonatal rats.

Selective white matter injury in the pre-mature infants suggests it has a greater susceptibility to hypoxia-ischemia. To investigate whether white matter injury would predominate following a mild hypoxic-ischemic insult, 7-day-old rats underwent either mild or moderate hypoxia-ischemia and magnetic resonance imaging 24 h later. Mild and moderate hypoxia-ischemia were produced by unilateral carotid artery occlusion plus exposure to hypoxia for either 45-50 or 90 min at ambient temperatures of 34.5 or 35.5 degrees C, respectively. Following mild hypoxia-ischemia, there was a significant increase in T(1) and T(2) within periventricular white matter (e.g. corpus callosum) in the hemisphere ipsilateral to the occlusion compared to that contralaterally and less of an increase within gray matter (e.g. cortex and striatum). This corresponded to relatively selective white matter injury detected histologically. Following a moderate hypoxia-ischemia, both gray and white matter was severely injured with marked increases in T(1) and T(2) occurring in both white and gray matter regions ipsilateral to the hypoxia-ischemia. We conclude that a mild insult, consisting of a short duration of hypoxia-ischemia at a slightly lower body temperature than a moderate hypoxic-ischemic insult, produces enhanced injury in white matter and a relative sparing of gray matter.

MR molecular imaging of early endothelial activation in focal ischemia.

Focal ischemia followed by reperfusion initiates a harmful P- and E-selectin-mediated recruitment of leukocytes in brain microvasculature. In this study, we tested whether a novel magnetic resonance (MR) contrast agent (Gd-DTPA-sLe(x) A), which is designed to bind to activated endothelium could be detected by MR imaging (MRI) in a focal stroke mouse model. MRIs (9.4T) of the brain were acquired 24 hours after transient middle cerebral artery occlusion. T1 maps were acquired repeatedly before and up to 1.5 hours after the intravenous injection of either Gd-DTPA or Gd-DTPA-sLe(x) A. Analysis of images included a pixel-by-pixel subtraction of T1 maps from the precontrast T1 maps and quantification of T1 within the ischemic area. After injection of Gd-DTPA-sLe(x) A, T1 decreased compared with precontrast levels, and an interhemispheric difference between the pre-post contrast T1 developed within the stroke lesion at a mean time of 52 minutes after injection (p < 0.05). Animals injected with Gd-DTPA did not exhibit changes in T1 signal intensity between regions of the ipsilateral and contralateral hemispheres, indicating that the reductions in T1 observed with Gd-DTPA-sLe(x) A were unrelated to blood-brain barrier breakdown. Fluorescent-labeled sLe(x) A administered intravenously was observed to bind to the endothelium of injured but not control brain. The study suggests that the contrast agent Gd-DTPA-sLe(x) A can be used to visualize early endothelial activation after transient focal ischemia in vivo with MRI.

Functional MRI of the rat lumbar spinal cord involving painful stimulation and the effect of peripheral joint mobilization.

PURPOSE: To examine neuronal activation in the spinal cord due to secondary hyperalgesia resulting from intrajoint capsaicin injection, and the effect of physiotherapy manipulation, using functional magnetic resonance imaging (fMRI), in alpha-chloralose anesthetized rats. MATERIALS AND METHODS: FMRI of the rat lumbar spinal cord was performed at 9.4 Tesla. Stimuli included injection of 25 microL of capsaicin (128 microg/mL in 7.5% dimethyl sulfoxide [DMSO]) into the right forepaw or 75 microL into the right ankle joint followed by a light touch stimulus, with and without physiotherapy manipulation. RESULTS: Activation of pain areas of the spinal cord (dorsal horn) was found in all animals after injection of capsaicin into the plantar surface of the rat hindpaw and ankle joint. Overlay maps depicting activations and deactivations showed significant reproducibility between experiments. Greater overlay of activations were observed for intrajoint compared to intradermal capsaicin injection. The distribution of activations after stimulation of the hindpaw using a light touch stimulus was somewhat more varied; activation of the dorsal horn was evident, with greater overlap resulting when joint mobilization was not performed. CONCLUSION: Results suggest a trend toward decreased areas of activation in the spinal cord associated with pain, as a result of hyperalgesia, following physiotherapy joint mobilization.

Functional MRI involving painful stimulation of the ankle and the effect of physiotherapy joint mobilization.

We examined whether cerebral activation due to secondary hyperalgesia resulting from intrajoint capsaicin injection could be detected using functional magnetic resonance imaging (fMRI) in alpha-chloralose anesthetized rats. We also examined whether we could detect analgesic changes in the central nervous system response to pain as a result of physiotherapy joint manipulation. Robust activation of areas of the brain known to be associated with the processing of pain, namely the anterior cingulate (bilateral), frontal cortex (bilateral) and sensory motor cortex (contralateral), was found in all animals following injection of 25 microl of capsaicin (128 microg/ml in 7.5% DMSO) into the plantar surface of the rat hindpaw (n = 7) and 75 microL into the ankle joint (n = 13). Significantly greater activation was observed when capsaicin was injected into the plantar surface of the hindpaw compared to the ankle joint. Mechanical allodynia and secondary hyperalgesia following capsaicin injection into the ankle joint also resulted in activation of the same brain regions. Trends toward decreased areas of activation in brain regions associated with pain in animals following physiotherapy joint mobilization were observed.