Kinetic analysis of novel mono- and multivalent VHH-fragments and their application for molecular imaging of brain tumours.

BACKGROUND AND PURPOSE: The overexpression of epidermal growth factor receptor (EGFR) and its mutated variant EGFRvIII occurs in 50% of glioblastoma multiforme. We developed antibody fragments against EGFR/EGFRvIII for molecular imaging and/or therapeutic targeting applications. EXPERIMENTAL APPROACH: An anti-EGFR/EGFRvIII llama single-domain antibody (EG(2)) and two higher valency format constructs, bivalent EG(2)-hFc and pentavalent V2C-EG(2) sdAbs, were analysed in vitro for their binding affinities using surface plasmon resonance and cell binding studies, and in vivo using pharmacokinetic, biodistribution, optical imaging and fluorescent microscopy studies. KEY RESULTS: Kinetic binding analyses by surface plasmon resonance revealed intrinsic affinities of 55 nM and 97 nM for the monovalent EG(2) to immobilized extracellular domains of EGFR and EGFRvIII, respectively, and a 10- to 600-fold increases in apparent affinities for the multivalent binders, V2C-EG(2) and EG(2)-hFc, respectively. In vivo pharmacokinetic and biodistribution studies in mice revealed plasma half-lives for EG(2), V2C-EG(2) and EG(2)-hFc of 41 min, 80 min and 12.5 h, respectively, as well as a significantly higher retention of EG(2)-hFc compared to the other two constructs in EGFR/EGFRvIII-expressing orthotopic brain tumours, resulting in the highest signal in the tumour region in optical imaging studies. Time domain volumetric optical imaging fusion with high-resolution micro-computed tomography of microvascular brain network confirmed EG(2)-hFc selective accumulation/retention in anatomically defined tumour regions. CONCLUSIONS: Single domain antibodies can be optimized for molecular imaging applications by methods that improve their apparent affinity and prolong plasma half-life and, at the same time, preserve their ability to penetrate tumour parenchyma.

Metabolic changes in rat brain following intracerebroventricular injections of streptozotocin: a model of sporadic Alzheimer's disease.

A decrease in cerebral glucose metabolic uptake is an early and characteristic sign of Alzheimer's disease (AD). Streptozotocin (STZ) is a bacterial toxin which damages insulin-producing cells and insulin receptors. Intracerebroventricular (icv) application of STZ in rats has been found to chronically decrease cerebral glucose uptake and produce other effects that bear a resemblance to several other molecular and pathological features of AD. In the present experiments in vivo (1)H MR Spectroscopy with short echo time (3 ms) was used to non-invasively obtain a neurochemical profile of rat brains, 3 weeks and 2 months after double icv injections of STZ or vehicle. Seventeen metabolites were quantified from 27 microL tissue volume which included hippocampus and a part of cerebral cortex, using the LCModel and unsuppressed water signal as an internal reference. Three weeks after icv STZ several metabolites were significantly decreased, the most prominent changes noted in glycerophosphocholine and phosphocholine (-38 +/- 5%), glutathione (-37 +/- 4%), taurine (-30 +/- 19%), glutamate (-26 +/- 14%), phosphocreatine (-23 +/- 15%) and N-acetylaspartate (-16 +/- 6%). On the contrary, the concentration of N-acetylaspartylglutamate was found significantly increased (+38 +/- 18%). After 2 months some of these changes were even more pronounced. We conclude that in vivo (1)H MRS of rat brain following icv STZ injections provides a new input into a better understanding of the critical dependency of neural function and structure on brain glucose consumption, and may be of relevance in further studies of AD pathomechanism.

Magnetic resonance molecular imaging of post-stroke neuroinflammation with a P-selectin targeted iron oxide nanoparticle.

We have developed a magnetic resonance molecular imaging method using a novel iron-oxide contrast agent targeted towards P-selectin - MNP-PBP (magnetic nanoparticle-P-selectin binding peptide) - to image endothelial activation following cerebral ischemia/reperfusion. MNP-PBP consists of approximately 1000 PBP ligands (primary sequence: GSIQPRPQIHNDGDFEEIPEEYLQ GGSSLVSVLDLEPLDAAWL) conjugated to a 50 nm diameter aminated dextran iron oxide particle. In vitro P- and E-selectin binding was assessed by competition ELISA. Transient focal cerebral ischemia was induced in male C57/BL 6 mice followed by contrast injection (MNP-PBP; MNP-NH2; Feridex; MNP-PBP-FITC) at 24 h after reperfusion and T(2) magnetic resonance imaging at 9.4 T was performed. Infarction and microvasculature accumulation of contrast agent was assessed in coronal brain sections. MNP-PBP attenuated antibody binding to P-selectin by 34.8 +/- 1.7%. P-selectin was preferentially increased in the infarct hemisphere and MNP-PBP-FITC accumulation in the infarct hemisphere microvasculature was observed. Compared with the nontargeted iron oxide agents MNP-NH2 and Feridex, MNP-PBP showed a significantly greater T(2) effect within the infarction. MR imaging of P-selectin expression with a targeted iron oxide nanoparticle contrast agent may reveal early endothelial activation in stroke and other neuroinflammatory states.

The aging hippocampus: a multi-level analysis in the rat.

In the current experiment we conducted a multi-level analysis of age-related characteristics in the hippocampus of young adult (3 months), middle-aged (12 months), and old (24 months) Fisher 344xBrown Norway hybrid (FBNF1) rats. We examined the relationships between aging, hippocampus, and memory using a combination of behavioral, non-invasive magnetic resonance imaging and spectroscopy, and postmortem neuroanatomical measures in the same rats. Aging was associated with functional deficits on hippocampus-dependent memory tasks, accompanied by structural alterations observed both in vivo (magnetic resonance imaging-hippocampal volume) and postmortem (dentate gyrus neuronal density and neurogenesis). Neuronal metabolic integrity, assessed by levels of N-acetylaspartate with magnetic resonance spectroscopy, was however, preserved. Further, our results suggest that neurogenesis (doublecortin) seems to be related to both performance deficits on hippocampus-dependent tasks and hippocampal volume reduction. The observed pattern of age-related alterations closely resembles that previously reported in humans and suggests FBNF1 rats to be a useful model of normal human aging.

Simultaneous functional magnetic resonance imaging in the rat spinal cord and brain.

Functional magnetic resonance imaging (fMRI) method was developed to investigate the pattern and temporal relationship in neuronal pathways of brain and spinal cord. Signal intensity changes correlating with stimulation patterns were observed simultaneously in the rat spinal cord and brain using fMRI at 9.4 T. Electrical stimulation of the forepaw was used to elicit activity. A quadrature volume RF coil covering both brain and the cervical spinal cord was used. Sets of fast spin echo (FSE) images were acquire simultaneously for both brain and spinal cord fMRI. Experiments were repeated in single animal and across animals. Activities within the dorsal horn of the spinal cord and within the somatosensory cortex were observed consistently within each animal as well as across animals.

Cerebral blood flow response to a hypoxic-ischemic insult differs in neonatal and juvenile rats.

To compare the cerebral blood flow (CBF) response to a transient episode of hypoxia-ischemia producing damage in neonatal and juvenile rats. One- and four-week-old rats were subjected to unilateral carotid artery occlusion plus hypoxia (8% oxygen). Perfusion MR images were acquired either in sham controls or in hypoxic-ischemic rats before, during, 1 h and 24 h after hypoxia-ischemia. At 24 h post hypoxia-ischemia, T2 maps and histology were used to assess damage. In sham controls, CBF increased twofold between the age of one and four weeks. Reductions in CBF ipsilateral to the occlusion occurred during hypoxia-ischemia followed by a substantial recovery at 1 h post in both age groups. However, contralaterally, hyperemia occurred during hypoxia-ischemia in four-week but not one-week-old rats. Similarly, hyperemia occurred ipsilaterally at 24 h post hypoxia-ischemia in four-week but not one-week-olds, corresponding to the distribution of elevations in T2. Despite CBF differences, extensive cell death occurred ipsilaterally in both age groups. The CBF responses to hypoxia-ischemia and reperfusion differ depending on postnatal age, with hyperemia occurring in juvenile but not neonatal rats. The results suggest a greater CBF responsiveness and differential relationship between post-ischemic vascular perfusion and tissue injury in older compared with immature animals.

Pilot study of dermal and subcutaneous fat structures by MRI in individuals who differ in gender, BMI, and cellulite grading.

BACKGROUND/AIMS: Puckered, dimply skin on the thighs, hips, and buttocks is known as cellulite. The cause of cellulite is not known, although there are a number of different hypotheses. In this study, we use magnetic resonance (MR) micro-imaging to study cellulite skin. To the best of our knowledge, this is the first reported MR study of cellulite. METHODS: High-resolution in vivo MR images of the postlateral thigh skin of two male groups and four female groups were obtained. Subjects were grouped according to their body mass index (BMI) and cellulite grade. A qualitative assessment of how MRI can be used to differentiate skin tissue at different levels of cellulite grading was performed. RESULTS: We found that changes in skin architecture with cellulite can be visualized by in vivo MR micro-imaging. The skin fat layers beneath the dermis and down to the level of muscles are well visualized in the images. Also, the diffuse pattern of extrusion of underlying adipose tissue into dermis is clearly imaged, and was found to correlate with cellulite grading. We also show that other skin tissue parameters such as (a) the percentile of adipose vs. connective tissue in a given volume of hypodermis and (b) the percentile of hypodermic invaginations inside the dermis are correlated with cellulite grade. CONCLUSION: MR images can be interpreted to measure tissue parameters correlated with cellulite. Considering that we had only three subjects in each group, the achievements of this pilot study were highly satisfactory. We have shown that the in vivo micro-MR is a technique able to detect the effects of cellulite and gender. This study can be extended for further investigations of drugs and/or medical devices for cellulite treatment.

Noninvasive assessment of the injured human spinal cord by means of functional magnetic resonance imaging.

STUDY DESIGN: A magnetic resonance imaging technique that enables indirect detection of neuronal activity has been developed for the spinal cord. In the present study, this method, spinal functional magnetic resonance imaging (fMRI), is applied to the first study of the injured spinal cord, with the goal of better clinical assessment of the entire cord. OBJECTIVES: The objectives of this project are: (1) to investigate the neuronal activity that can be detected in the spinal cord caudal to a chronic injury by means of spinal fMRI, and (2) to develop spinal fMRI as a clinical diagnostic tool. SETTING: Institute for Biodiagnostics, National Research Council of Canada, Winnipeg, Manitoba, Canada. METHODS: fMRI of the spinal cord was carried out in 27 volunteers with cervical or thoracic spinal cord injuries (SCIs). Of these volunteers, 18 had complete injuries, and nine had incomplete injuries. Spinal fMRI was carried out in a 1.5 T clinical MR system, using established methods. Thermal stimulation at 10 degrees C was applied to the fourth lumbar dermatome on each leg, and images were obtained of the entire lumbar spinal cord. RESULTS: Areas of neuronal activity were consistently observed in the lumbar spinal cord in response to the thermal stimulation, even when the subjects had no awareness of the sensation. The pattern of activity was notably different compared with noninjured subjects. In general, subjects with complete SCI showed absent or diminished dorsal gray matter activity, but had enhanced ventral activity, particularly contralateral to the stimulation. CONCLUSIONS: Spinal fMRI is able to provide a noninvasive assessment of the injured spinal cord that does not depend on the patient's perception of the stimulus being applied. This work was carried out on a standard clinical MRI system without modification, and so is readily applicable in most MR units. SPONSORSHIP: This work was funded by a grant from the Canadian Institutes of Health Research (CIHR).

Prevalence of infectious diseases in ring-necked pheasant flocks in Poland.

The health status of ring-necked pheasants in view of the prevalence of infectious diseases was estimated in Polish pheasantries in the years 1997-2000. Anatomicopathological, microbiological and serological examinations were carried out on birds derived from 26 pheasantries, including birds randomly selected from 18 flocks and sick or dead birds sent from 8 pheasantries. Antibodies specific to the following viruses were detected in serum blood samples: HE, AE, AP, REO, AI, Adeno group 1, MD, ND, as well as Mycoplasma gallisepticum specific antibodies. However, in none of the examined flocks was the presence of antibodies against reticuloendoteliosis virus found. Marble spleen disease and salmonellosis proved to be the most frequent cause of death during the growing period.

Functional magnetic resonance imaging of the human brain based on signal enhancement by extravascular protons (SEEP fMRI).

Functional magnetic resonance imaging (fMRI) studies of the human brain were carried out at 3 Tesla to investigate an fMRI contrast mechanism that does not arise from the blood oxygen-level dependent (BOLD) effect. This contrast mechanism, signal enhancement by extravascular protons (SEEP), involves only proton-density changes and was recently demonstrated to contribute to fMRI signal changes in the spinal cord. In the present study it is hypothesized that SEEP fMRI can be used to identify areas of neuronal activity in the brain with as much sensitivity and precision as can be achieved with BOLD fMRI. A detailed analysis of the areas of activity, signal intensity time courses, and the contrast-to-noise ratio (CNR), is also presented and compared with the BOLD fMRI results. Experiments were carried out with subjects performing a simple finger-touching task, or observing an alternating checkerboard pattern. Data were acquired using a conventional BOLD fMRI method (gradient-echo (GE) EPI, TE = 30 ms), a conventional method with reduced BOLD sensitivity (GE-EPI, TE = 12 ms), and SEEP fMRI (spin-echo (SE) EPI, TE = 22 ms). The results of this study demonstrate that SEEP fMRI may provide better spatial localization of areas of neuronal activity, and a higher CNR than conventional BOLD fMRI, and has the added benefit of lower sensitivity to field inhomogeneities.

Mapping of neuronal function in the healthy and injured human spinal cord with spinal fMRI.

Functional magnetic resonance imaging of the human spinal cord is carried out with a graded thermal stimulus in order to establish the relationship between signal changes and neural activity. Studies of the lumbar spinal cord in 15 healthy subjects with 10 degrees C stimulation of the skin overlying the calf demonstrate a pattern of activity that matches the neuronal anatomy of the spinal cord. This pattern shows primarily dorsal horn activity, with expected components of motor reflex activity as well. Moreover, a later response shifting to noxious cold over time is also demonstrated with a shift to more dorsal horn activity. Signal intensity changes detected at different degrees of thermal stimulation have a biphasic nature, with much larger signal changes below 15 degrees C as the stimulus becomes noxious, and agree well with electrophysiological results reported in the literature. These findings demonstrate a strong correspondence between Spinal fMRI results and neural activity in the human spinal cord. Spinal fMRI is also applied to studies of the injured spinal cord, below the site of injury. Results consistently demonstrate activity in the spinal cord even when the subjects cannot feel the stimulus being applied. Signal intensity changes demonstrate the same stimulus-response pattern as that in noninjured subjects, but the areas of activity in the spinal gray matter are notably altered. In subjects with complete injuries, activity is absent ipsilateral to the thermal stimulation, but appears to be enhanced on the contralateral side. These findings demonstrate the reliability of Spinal fMRI and its clinical potential.

Extravascular proton-density changes as a non-BOLD component of contrast in fMRI of the human spinal cord.

The fractional signal intensity change (Delta S/S) observed during activation in T(2)-weighted fMRI of the spinal cord has previously been shown to depend linearly on the echo time (TE) but to have a positive value of roughly 2.5% extrapolated to zero TE. In this study we investigated the origin of this finding by measuring the Delta S/S in spinal fMRI with very short TEs. Our results demonstrate that the Delta S/S does not approach zero, but has a value as high as 3.3% at TE = 11 ms. At TEs > 33 ms we observed the linear relationship between Delta S/S and TE as in previous studies. These data demonstrate that there is a non-BOLD contribution to signal changes observed in spinal fMRI. We hypothesize that this contribution is a local proton density increase due to increased water exudation from capillaries with increased blood flow during neuronal activation, and term this effect "signal enhancement by extravascular protons" (SEEP).

Functional magnetic resonance imaging of the human cervical spinal cord with stimulation of different sensory dermatomes.

Functional MR imaging (fMRI) of the cervical spinal cord was carried out in 13 healthy volunteers. A cold stimulus was applied, at different times, to three different sensory dermatome regions overlying the right hand and forearm: the thumb side of the palm, the little finger side of the palm, and the forearm below the elbow. Stimulation of these areas is expected to involve the 6(th), 8(th), and 5(th) cervical spinal cord segments respectively. Whereas true activations are expected to correspond to the region being stimulated, false activations such as arising from noise and motion, are not. The results demonstrate that clustering of active pixels into groups based on their intensity time courses discriminates false activations from true activations. Following clustering, the distribution of activity observed with fMRI matched the expected regions of neuronal activation with the different areas of stimulation on the hand and forearm.

Functional magnetic resonance imaging of tonic pain and vasopressor effects in rats.

In functional magnetic resonance imaging (fMRI) studies, an elevation in blood pressure (BP) in individuals with a poor autoregulatory response may increase cerebral blood flow, potentially enhancing the blood oxygenation level dependent response. To investigate the role of BP changes, the cerebral activation to either tonic pain or the infusion of the vasopressor norepinephrine was correlated with the accompanying BP changes in alpha-chloralose anesthetized rats. Immediately after formalin (2%) injection into the forepaw, fMRI detected an activation that was correlated with the BP increase and additional activations that were independent of blood pressure changes 5-40 minutes later. The activation detected with the administration of the vasopressor norepinephrine, which does not cross the blood-brain barrier was correlated to both the amount and rate of increase in BP. The response ranged from being sparse, localized within cortex or widespread during modest, moderate or severe elevations in BP, respectively. The cerebral circulatory effects of hypertension should be considered as contributing to changes in cerebral blood oxygenation in fMRI studies involving increases in BP.

Spin-echo versus gradient-echo fMRI with short echo times.

Blood-oxygen level dependent signal changes in the visual cortex were investigated as a function of echo time with spin-echo and gradient-echo EPI at 1.5 T and 3 T. The linear relationship between the fractional signal change and the echo time was apparent in all cases. Relaxation rate changes determined from the slope of this linear relation agree with published values, intercept values extrapolated to an echo time of zero, however, were 0.66% to 1.0% with spin-echo EPI, and 0.11% to 0.35% with gradient-echo EPI. Spin-echo and gradient-echo EPI can therefore yield similar signal changes at sufficiently short echo times.

Characterization of contrast changes in functional MRI of the human spinal cord at 1.5 T.

Contrast changes observed in functional magnetic resonance imaging in the human spinal cord were investigated with both motor and sensory tasks over a range of echo times. Data were acquired using a single-shot fast spin-echo sequence at 1.5 Tesla. Data were analyzed with two different correlation thresholds and the effects of altering the order of repeated experiments was also investigated. Plots of the fractional signal change as a function of echo time yielded linear functions with slopes corresponding to relaxation rate changes of -0.30 sec(-1) with sensory stimulation and approximately -0.50 sec(-1) with a motor task. However, the fractional signal change extrapolated to an echo time of zero was significantly greater than zero in each case and was roughly 2.5%. This suggests that in addition to the BOLD effect there is a baseline signal change which occurs concomitant to neuronal activation in the spinal cord.

The engineering of an interventional MRI with a movable 1.5 Tesla magnet.

The engineering of a novel intra-operative MRI system is described. A movable, 1.5 Tesla MRI magnet was placed in a neurosurgical operating room without affecting established neurosurgical procedure. The system allows fast, high-quality MR intra-operative imaging of the brain and spine without the necessity of patient transportation. A neuro-navigational device capable of displaying and updating spatially referenced MR images in the operating room was integrated with the MRI system. Over 100 procedures have been carried out with this system without limiting surgical access and without compromising traditional neurosurgical, nursing or anesthetic techniques. J. Magn. Reson. Imaging 2001;13:78-86.

Probe with chest shielding for improved breast MRI.

The design and construction of an RF coil system for use in MR breast imaging is described. The two-ring, tuned Helmholtz coil, with its axis perpendicular to the chest, surrounds a single pendant breast and is coupled both internally and to the MRI transmitter/receiver by mutual induction. The addition of two symmetrical RF shields minimizes losses in the chest and significantly improves performance. Images obtained from eight healthy volunteers showed that the coil permitted imaging of breasts of diverse size with an in-plane resolution of 0.27 x 0.27 mm and a slice thickness of less than 2 mm at a field strength of 3 T as well as 1.5 T. The use of shields with surface coils in general is advocated as a method for improving signal-to-noise ratio. Magn Reson Med 43:917-920, 2000.

Analysis of ovariectomy and estrogen effects on body composition in rats by X-ray and magnetic resonance imaging techniques.

Resistance of bone to fracture--bone strength--has been shown to depend on both the amount of bone and its architectural spatial organization. In vivo magnetic resonance (MR) techniques have the capability of imaging bone tissue, including the trabecular microarchitecture and the marrow composition. We have applied in vivo and ex vivo MR methods to the tibia in an ovariectomized rat model of osteoporosis. Specifically, in vivo high-resolution three-dimensional MR imaging and localized MRS were facilitated by specialized coils and high field magnets, resulting in enhanced sensitivity of detection. As a result, in vivo and ex vivo differences in marrow composition were found between sham-ovariectomized, ovariectomized, and ovariectomized animals treated with 17-beta-estradiol. Estrogen effects were detected in vivo 7 days after surgery (3 days into treatment) as a decrease in the tibial fat signal level. The in vivo effects of ovariectomy were observed 56 days after surgery as an increase in MR image fat signal level and spectral fat/water ratio in the proximal tibia. Ex vivo measurements of tibial marrow water signal discriminated clearly between the sham and ovariectomized groups and showed increased individual variations in the treatment group. Imaging further showed that the highest fat content is observed in the epiphysis. Computed tomography confirmed ovariectomy-induced loss of bone in the proximal tibial metaphysis compared with the sham group. This loss of cancellous bone with ovariectomy is consistent with the MR observations of increases in both fat and water in the metaphysis. These data showed that MR techniques complement X-ray techniques in the bone, water, and fat compositional analysis of the appendicular skeleton in response to ovariectomy and pharmacological treatment.

A mobile high-field magnetic resonance system for neurosurgery.

OBJECT: The authors' goal was to place a mobile, 1.5-tesla magnetic resonance (MR) imaging system into a neurosurgical operating room without adversely affecting established neurosurgical management. The system would help to plan accurate surgical corridors, confirm the accomplishment of operative objectives, and detect acute complications such as hemorrhage or ischemia. METHODS: The authors used an actively shielded 1.5-tesla magnet, together with 15 mtesla/m gradients, MR console computers, gradient amplifiers, a titanium, hydraulic-controlled operating table, and a radiofrequency coil that can be disassembled. The magnet is moved to and from the surgical field by using overhead crane technology. To date, the system has provided unfettered access in 46 neurosurgical patients. In all patients, high-definition T1- and/or T2-weighted images were rapidly and reproducibly acquired at various stages of the surgical procedures. Eleven patients underwent craniotomy that was optimized after preincision imaging. In four patients who harbored subtotally resected tumor, intraoperative MR imaging aided the surgeon in removing the remaining tumor. Interestingly, the intraoperative administration of gadolinium demonstrated a dynamic expansion of enhancement beyond the preoperative contrast contour in patients with malignant glioma. These zones of new enhancement proved, on examination of biopsy samples, to be tumor. CONCLUSIONS: The authors have demonstrated that high-quality MR images can be obtained in the operating room within reasonable time constraints. Procedures can be conducted without compromising or altering traditional neurosurgical, nursing, or anesthetic techniques. It is feasible that within the next decade intraoperative MR imaging may become the standard of care in neurosurgery.