The effect of exercise on the cerebral vasculature of healthy aged subjects as visualized by MR angiography.

BACKGROUND AND PURPOSE: Prior studies suggest that aerobic exercise may reduce both the brain atrophy and the decline in fractional anisotropy observed with advancing age. It is reasonable to hypothesize that exercise-induced changes to the vasculature may underlie these anatomic differences. The purpose of this blinded study was to compare high-activity and low-activity healthy elderly volunteers for differences in the cerebrovasculature as calculated from vessels extracted from noninvasive MR angiograms (MRAs). MATERIALS AND METHODS: Fourteen healthy elderly subjects underwent MRA. Seven subjects reported a high level of aerobic activity (64 +/- 5 years of age; 5 men, 2 women) and 7, a low activity level (68 +/- 6 years of age; 5 women, 2 men). Following vessel segmentation from MRA by an individual blinded to subject activity level, quantitative measures of vessel number, radius, and tortuosity were calculated and histogram analysis of vessel number and radius was performed. RESULTS: Aerobically active subjects exhibited statistically significant reductions in vessel tortuosity and an increased number of small vessels compared with less active subjects. CONCLUSIONS: Aerobic activity in elderly subjects is associated with lower vessel tortuosity values and an increase in the number of small-caliber vessels. It is possible that an aerobic exercise program may contribute to healthy brain aging. MRA offers a noninvasive approach to visualizing the cerebral vasculature and may prove useful in future longitudinal investigations.

Computer-assisted measurement of vessel shape from 3T magnetic resonance angiography of mouse brain.

Blood vessel morphology (vessel radius, branching pattern, and tortuosity) is altered by a multitude of diseases. Although murine models of human pathology are important to the investigation of many diseases, there are few publications that address quantitative measurements of murine vascular morphology. This report outlines methods of imaging mice in vivo using magnetic resonance angiograms obtained on a clinical 3T unit, of defining mouse vasculature from these images, and of quantifying measures of vessel shape. We provide examples of both healthy and diseased vasculature and illustrate how the approach can be used to assess pathology both visually and quantitatively. The method is amenable to the assessment of many diseases in both human beings and mice.

Malignancy-associated vessel tortuosity: a computer-assisted, MR angiographic study of choroid plexus carcinoma in genetically engineered mice.

BACKGROUND AND PURPOSE: The ability to assess tumor malignancy and monitor treatment response noninvasively would be of value to both clinicians and animal investigators. This report describes the MR imaging characteristics of a genetically engineered mouse model of choroid plexus carcinoma (CPC) during tumor growth and progression to malignancy. We assess the ability of vessel tortuosity measurements, as calculated from high-resolution MR angiographic (MRA) images, to detect emerging CPC cancers. METHODS: MR images of 9 healthy mice and of 20 CPC mice with precancerous choroid dysplasia or with cancer over a wide range of sizes were analyzed. Two vessel tortuosity measures and a measure of vessel attenuation (vessel count) were calculated from MRA images. Malignancy assessment was based upon a statistical analysis of vessel tortuosity, by using an equation derived from an earlier study of human brain tumor patients. RESULTS: Choroid dysplasia was correctly judged nonmalignant. On the basis of vessel count, neoangiogenesis could not be detected until cancers were full-blown and had reached a volume of approximately 80 mm3. Vessel tortuosity measurements, however, correctly identified emerging malignancy in lesions larger than 0.3 mm3. CONCLUSION: To the best of our knowledge, this report provides the first description of in vivo, MR imaging characteristics of genetically engineered CPC mice during the progression from dysplasia to cancer. Vessel tortuosity measurements offer promise of correctly defining even tiny tumors as malignant.

Symbolic description of intracerebral vessels segmented from magnetic resonance angiograms and evaluation by comparison with X-ray angiograms.

We describe and evaluate methods that create detailed vessel trees by linking vessels that have been segmented from magnetic resonance angiograms (MRA). The tree-definition process can automatically exclude erroneous vessel segmentations. The parent-child connectivity information provided by our vessel trees is important to both surgical planning and to guidance of endovascular procedures. We evaluated the branch connection accuracy of our 3D vessel trees by asking two neuroradiologists to evaluate 140 parent-child connections comprising seven vascular trees against 17 digital subtraction angiography (DSA) views. Each reviewer rated each connection as (1) Correct, (2) Incorrect, (3) Partially correct (a minor error without clinical significance), or (4) Indeterminate. Analysis was summarized for each evaluator by calculating 95% confidence intervals for both the proportion completely correct and the proportion clinically acceptable (completely or partially correct). In order to protect the overall Type I error rate, alpha-splitting was done using a top down strategy. We additionally evaluated segmentation completeness by examining each slice in 11 MRA datasets in order to determine unlabeled vessels identifiable in cross-section following segmentation. Results indicate that only one vascular parent-child connection was judged incorrect by both reviewers. MRA segmentations appeared complete within MRA resolution limits. We conclude that our methods permit creation of detailed vascular trees from segmented 3D image data. We review the literature and compare other approaches to our own. We provide examples of clinically useful visualizations enabled by our methodology and taken from a visualization program now in clinical use.

Computer-assisted visualization of arteriovenous malformations on the home personal computer.

OBJECTIVE: Arteriovenous malformations (AVMs) are difficult lesions to treat, partly because it is difficult to formulate a three-dimensional mental image of the nidus and its supplying arteries, draining veins, and arteries of passage. Our purpose is to develop personal computer software that allows better visualization of complex, three-dimensional, connected vascular anatomy for surgical planning. METHODS: Vessels are defined from magnetic resonance angiograms and are symbolically linked to form vascular trees. The nidus of the AVM is also defined by magnetic resonance angiography. These representations of the nidus and vasculature can be viewed together in a software program that allows the user to color-code groups of vessels or to selectively turn connected groups of vessels "off" to avoid obscuring the part of the image that the user wants to observe. Structures can be viewed from any angle. The vessels can also be shown intersecting any magnetic resonance angiogram slice or superimposed upon digital subtraction angiograms obtained from the same patient. RESULTS: We report results from two patients with AVMs in which our representations were compared with the findings during surgery. Our three-dimensional vascular trees correctly depicted the relationship of the nidus to feeding vessels in three dimensions. We show findings in an additional, unoperated patient for whom vessel trees were created from three-dimensional digital subtraction angiography data and compared with a volume rendering of the original data set. CONCLUSION: Computer-assisted, three-dimensional visualizations of complex vascular anatomy can be helpful in planning the surgical excision of AVMs. Software programs that produce these images can provide important information that is difficult to obtain by traditional techniques. This imaging method is also applicable to guidance of endovascular procedures and removal of complex tumors.

Snapshots of usher-mediated protein secretion and ordered pilus assembly.

Type 1 pilus biogenesis was used as a paradigm to investigate ordered macromolecular assembly at the outer cell membrane. The ability of Gram-negative bacteria to secrete proteins across their outer membrane and to assemble adhesive macromolecular structures on their surface is a defining event in pathogenesis. We elucidated genetic, biochemical, and biophysical requirements for assembly of functional type 1 pili. We discovered that the minor pilus protein FimG plays a critical role in nucleating the formation of the adhesive tip fibrillum. Genetic methods were used to trap pilus subunits during their translocation through the outer membrane usher protein, providing data on the structural interactions that occur between subunit components during type 1 pilus formation. Electron microscopic and biochemical analyses of these stepwise assembly intermediates demonstrated that translocation of pilus subunits occurs linearly through the usher's central channel, with formation of the pilus helix occurring extracellularly. Specialized pilin subunits play unique roles both in this multimerization and in the final ultrastructure of the adhesive pilus.

Registration of 3D cerebral vessels with 2D digital angiograms: clinical evaluation.

RATIONALE AND OBJECTIVES: The purpose of this study was to evaluate the accuracy and speed of a new, semiautomatic method of three-dimensional (3D)-two-dimensional (2D) vascular registration. This method should help guide endovascular procedures by allowing interpretation of each digital subtraction angiographic (DSA) image in terms of precreated, 3D vessel trees that contain "parent-child" connectivity information. MATERIALS AND METHODS: Connected, 3D vessel trees were created from segmented magnetic resonance (MR) angiograms. Eleven total DSA images were registered with such trees by using both our method and the current standard (manual registration). The accuracy of each method was compared by using repeated-measures analysis of variance with correction for heterogeneity of variance to evaluate separation of curve pairs on the view plane. Subjective clinical comparisons of the two registration methods were evaluated with the sign test. Registration times were evaluated for both methods and also as a function of the error in the initial estimate of MR angiographic position. RESULTS: The new registration method produced results that were numerically superior to those of manual registration (P < .001) and was subjectively judged to be as good as or better by clinical reviewers. Registration time with the new method was faster (P < .001). If the rotational error in the initial estimate of MR angiographic position is less than 10 degrees around each axis, the registration itself took only 1-2 minutes. CONCLUSION: This method is quicker than and produces results as good as or better than those of manual registration. This method should be able to calculate an initial registration matrix during endovascular embolization and adjust that matrix intermittently with registration updates provided by automatic tracking systems.

Electron microscopy and x-ray diffraction studies of Lotus tetragonolobus A isolectin cross-linked with a divalent Lewisx oligosaccharide, an oncofetal antigen.

The interactions of lectins with multivalent carbohydrates often leads to the formation of highly ordered cross-linked lattices that are amenable to structural studies. A particularly well ordered, two-dimensional lattice is formed from fucose-specific isolectin A from Lotus tetragonolobus cross-linked with difucosyllacto-N-neohexaose, an oligosaccharide possessing the Lewisx determinant, which is an oncofetal antigen. A combination of electron microscopy, x-ray diffraction, simulation of electron micrographs, and molecular model building was used to determine the relative positions of the tetrameric lectin and bivalent carbohydrate within the lattice. X-ray diffraction from unoriented pellets was used to determine the lattice dimensions and analysis of electron micrographs was used to determine the lattice symmetry. Molecular models of the lattice were constructed based on the known structure of the jack bean lectin concanavalin A and the high degree of sequence homology between the two lectins. Using the symmetry and dimensions of the lattice and its appearance in filtered electron micrographs, molecular models were used to determine the orientation of the lectin in the lattice, and to define the range of lectin-oligosaccharide interactions consistent with the structural data. The present study provides the first description of a highly ordered, two-dimensional, cross-linked lattice between a tetravalent lectin and a bivalent carbohydrate.

Bacterial adhesion pili are heterologous assemblies of similar subunits.

P-pili on uropathogenic bacteria are 68-A-diameter rods typically 1 microm in length. These structures project from the outer membrane of Escherichia coli, and contain on their distal tip a thin fibrillum, 25 A in diameter and 150 A long, displaying an adhesin protein responsible for the binding of the bacterium to the surface of epithelial cells lining the urinary tract. Operationally, it is possible to identify three morphologically distinct states of the 68-A-diameter P-pili rods, based on the degree of curvature each can adopt. These states are designated "straight," "curved," and "highly curved." The rods can also be unwound to form thin "threads" that are very similar to the tip fibrillae. Electron microscope data are used to distinguish among these four morphological states and to define limits on the shapes of the pilus proteins. The mechanical properties of the PapA polymers are assessed, and implications of rod polymorphism for pilus function are discussed. A wide variety of data are considered in light of the possibility that all pilins are similar in molecular architecture, with specific differences designed to optimize their specialized functions in the pilus assembly.

Three-dimensional reconstruction of curves from pairs of projection views in the presence of error. I. Algorithms.

We have previously described an approach to 3D intracerebral vascular reconstruction that uses an MRA as a reconstruction base. Additional vessels seen only by angiography are added by segmenting 2D curves from projection angiograms and reconstructing these curves into 3D, building upon the MRA. Intracerebral vascular reconstruction is difficult for at least two reasons. First, 2D curves must be associated on projection images even when the human eye cannot do so. Second, 3D curves must be reconstructed in the presence of errors such as misregistration, image distortion, and misdefinition of 2D curves. This paper is the first of two that address the specific issue of reconstruction of a 3D curve from a given pair of 2D curves in the presence of error. The method explicitly separates what can and cannot be determined from a pair of projection views. It is also capable of recognizing interruptions produced by viewplane errors, of continuing reconstruction beyond such interruptions, and of localizing and estimating the magnitude of the interruptions. These measurements can also be used to estimate the lengths of regional disparities between a pair of 2D curves, leading to a quantitative estimate of the capacity of a pair of 2D curves to combine to create a 3D object (match value). Match values can be used, in turn, as part of the strategy for automatically associating pairs of 2D curves. This paper provides methods for reconstructing a given pair of 2D curves into 3D in the presence of error and for calculating match values. Error analysis is given in the companion report.

Three-dimensional reconstruction of curves from pairs of projection views in the presence of error. II. Analysis of error.

We have previously described an approach to 3D intracerebral vascular reconstruction that uses an MRA as a reconstruction base. Additional vessels seen only by angiography are added by segmenting 2D curves from projection angiograms and reconstructing these curves into 3D, building upon the MRA. This paper is the second of two that discuss the specific problem of reconstructing a 3D curve from a given pair of 2D curves in the presence of error. The method presented is capable of detecting and handling many errors produced by misregistration, image distortion, or misdefinition of 2D curves. The first paper gives an algorithm. The current paper discusses factors affecting the accuracy of a reconstructed curve, with emphasis upon registration error. We analyze the spatial accuracy of a reconstructed point in terms of the relationships between pixel size, relative viewing angle, 3D point location, and registration error. We provide a theoretical framework that, given the known error properties of a registration algorithm, allows optimization of the viewing geometry so as to produce the highest precision of point reconstruction. A major focus is the effect of registration error upon the reconstruction of a curve. We subdivide registration error into two types, one of which produces smoothly continuous point placement errors and the other of which produces pixel pairing errors. We test our ability to reconstruct a 3D curve in the presence of both. Finally, we summarize approaches to other sources of error. We conclude with a list of recommendations to optimize reconstruction accuracy. When projection points are associated by the rules of epipolar geometry, viewplane point displacements should not exceed 1.5-2 mm along the axis perpendicular to epipolar planes.

The yeast spindle pole body is assembled around a central crystal of Spc42p.

The spindle pole body (SPB) is the microtubule organizing center (MTOC) in the yeast Saccharomyces that plays a pivotal role in such diverse processes as mitosis, budding, and mating. We have used cryoelectron microscopy and image processing to study the structure of isolated diploid SPBs. We show that SPBs are present in two lateral-size classes, sharing a similar vertical architecture comprised of six major layers. Tomographic reconstructions of heparin-stripped SPBs reveal a central hexagonally packed layer. Overexpression of Spc42p results in the growth of a similar layer, forming a crystal that encircles the SPB. Hence, the SPB is an MTOC that utilizes crystallographic packing of subunits in its construction.

Methods for displaying intracerebral vascular anatomy.

We are developing three-dimensional imaging methods to portray vascular anatomy better, including noise-free display of vessels extracted from 3-D data sets, tree-based display, and reconstruction of angiographic data (preliminary work has resulted in the successful reconstruction of aneurysms from angiographic data). Fast, interactive display permits real-time manipulation of viewing orientation.

Development of pilus organelle subassemblies in vitro depends on chaperone uncapping of a beta zipper.

The major subassemblies of virulence-associated P pili, the pilus rod (comprised of PapA) and tip fibrillum (comprised of PapE), were reconstituted from purified chaperone-subunit complexes in vitro. Subunits are held in assembly-competent conformations in chaperone-subunit complexes prior to their assembly into mature pili. The PapD chaperone binds, in part, to a conserved motif present at the C terminus of the subunits via a beta zippering interaction. Amino acid residues in this conserved motif were also found to be essential for subunit-subunit interactions necessary for the formation of pili, thus revealing a molecular mechanism whereby the PapD chaperone may prevent premature subunit-subunit interactions in the periplasm. Uncapping of the chaperone-protected C terminus of PapA and PapE was mimicked in vitro by freeze-thaw techniques and resulted in the formation of pilus rods and tip fibrillae, respectively. A mutation in the leading edge of the beta zipper of PapA produces pilus rods with an altered helical symmetry and azimuthal disorder. This change in the number of subunits per turn of the helix most likely reflects involvement of the leading edge of the beta zipper in forming a right-handed helical cylinder. Organelle development is a fundamental process in all living cells, and these studies shed new light on how immunoglobulin-like chaperones govern the formation of virulence-associated organelles in pathogenic bacteria.

Haemophilus influenzae pili are composite structures assembled via the HifB chaperone.

Haemophilus influenzae is a Gram-negative bacterium that represents a common cause of human disease. Disease due to this organism begins with colonization of the upper respiratory mucosa, a process facilitated by adhesive fibers called pili. In the present study, we investigated the structure and assembly of H. influenzae pili. Examination of pili by electron microscopy using quick-freeze, deep-etch and immunogold techniques revealed the presence of two distinct subassemblies, including a flexible two-stranded helical rod comprised of HifA and a short, thin, distal tip structure containing HifD. Genetic and biochemical studies demonstrated that the biogenesis of H. influenzae pili is dependent on a periplasmic chaperone called HifB, which belongs to the PapD family of immunoglobulin-like chaperones. HifB bound directly to HifA and HifD, forming HifB-HifA and HifB-HifD complexes, which were purified from periplasmic extracts by ion-exchange chromatography. Continued investigation of the biogenesis of H. influenzae pili should provide general insights into organelle development and may suggest novel strategies for disease prevention.

Three-dimensional reconstruction of intracranial vessels from biplane projection views.

The three-dimensional (3D) reconstruction of intracerebral vessels from two-dimensional (2D) projection views is an important clinical problem that, so far, has eluded solution. This report describes a new approach that uses projection images to build arterial trees progressively from an underlying 3D network, using a new method to pair shadow images on widely separated projection views. As a test of our general methodology, we have reconstructed a middle cerebral arterial tree from two projection views of a magnetic resonance dataset and have tested the accuracy of reconstruction against the original 3D dataset. This report describes the general approach to 3D vascular reconstruction and the computer program used to perform the final reconstruction step. The results suggest that accurate, 3D reconstruction of intracranial vessels is indeed possible from as few as two projection views.

Effects of anterolateral spinal lesions on escape responses of rats to hindpaw stimulation.

In order to determine the effects of spinal cord lesions on nociceptive sensitivity of rodents, methods were developed to assess the speed of operant escape responses to electrocutaneous stimulation (ES). ES was delivered across the dorsal and ventral surfaces of either hindpaw, producing a current path through deep tissues. In order to guide establishment of a range of stimulus intensities for this manner of stimulation, a preliminary human psychophysical experiment was conducted with stimulation between the dorsal and ventral surfaces of a finger. For the human subjects, detection thresholds averaged 0.13 mA, and thresholds for a sharp (but nonpainful) sensation were 0.42 mA. Levels of stimulation between these thresholds for detection and a sharp quality elicited sensations of tingle or itch. Thresholds for reports of pain averaged 0.67 mA. On the basis of these results, intensities of ES ranging from 0.05 to 1.0 mA were presented to the feet of rats that were trained to perform an escape response with one forelimb. Thresholds for escape averaged slightly less than 0.1 mA; responding was consistent at 0.4 mA; and response probability and speed were maximal at approximately 0.8 mA. Thus, the rats responded aversively at intensities below those rated as sharp or painful by the human subjects, but the speed of escape reached a plateau at intensities that were above pain threshold for the human subjects. Unilateral thoracic lesions of the lateral spinal column of rats produced a contralateral hypalgesia. Escape thresholds were elevated, and the speed of escape responses to all intensities was reduced. This effect depended upon interruption of axons in the middle and anterior portions of one lateral column, corresponding to the location of long ascending pathways for nociception, including the spinothalamic tract. The speed of escape responding increased over 20 weeks of postoperative testing of animals with the largest lesions. This confirms results obtained previously from monkeys (by means of a similar paradigm), and corresponds to clinical reports of humans who have received spinal lesions for control of intractable pain. Thus, the location and organization of nociceptive pathways in the spinal cord of rodents appear to be similar to those of primates, and similar adaptations occur following interruption of these pathways.

Structural polymorphism of bacterial adhesion pili.

Bacterial adhesion pili are designed to bind specifically and maintain attachment of bacteria to target cells. Uropathogenic P-pili are sufficiently mechanically resilient to resist the cleansing action of urine flow that removes most other bacteria. P-pili are 68 A in diameter and approximately 1 micron long, and are composed of approximately 1,000 copies of the principal structural protein, PapA. They are attached to the outer membrane by a minor structural protein, PapH and are terminated by an approximately 20 A diameter fibrillus composed of PapK, PapE and PapF, which presents the host-binding adhesin PapG. The amino-acid sequences of PapA, PapE, and PapF are similar, with highly conserved C-termini being responsible for binding to PapD, the periplasmic chaperone. Our three-dimensional reconstruction indicates that pili are formed by the tight winding of a much thinner structure. A structural transition allows the pilus to unravel without depolymerizing, producing a thin, extended structure five times the length of the original pilus.

Construction of a microphage variant of filamentous bacteriophage.

The intergenic region in the genome of the Ff class of filamentous phage (comprising strains fl, fd and M13) genome constitutes 8% of the viral genome, and has essential functions in DNA replication and phage morphogenesis. The functional domains of this region may be inserted into separate sites of a plasmid to function independently. Here, we demonstrate the construction of a plasmid containing, sequentially, the origin of (+)-strand synthesis, the packaging signal and a terminator of (+)-strand synthesis. When host cells harboring this plasmid (pLS7) are infected with helper phage they produce a microphage particle containing all the structural elements of the mature, native phage. The microphage is 65 A in diameter and about 500 A long. It contains a 221-base single-stranded circle of DNA coated by about 95 copies of the major coat protein (gene 8 protein).

The effect of stimulus duration on noxious-stimulus induced c-fos expression in the rodent spinal cord.

C-fos is a proto-oncogene that is expressed within some neurons following depolarization. The protein product, fos, has been proposed as an anatomical marker for neuronal activity following noxious peripheral stimulation. However, the literature on noxious-stimulus induced fos expression contains several puzzling observations on the time course and laminar distribution of neuronal labeling within the spinal cord. This study has analyzed the effect of stimulus duration on the expression of fos-like immunoreactivity (FLI) within the spinal cord of anesthetized rats. In order to examine the time course of fos expression following brief periods of stimulation, we required a type of stimulus that was intense enough to activate nociceptors but that did not produce tissue damage. We have therefore employed pulsed, high intensity electrical stimulation, with stimulus durations ranging from 3 s to 24 h. The results indicate that stimulus duration has a profound effect upon the number of labeled cells, the intensity of neuronal labeling, the laminar pattern of FLI, and the time course of fos expression. Brief stimulation periods induce relatively few and relatively lightly labeled neurons, located predominantly within the most superficial laminae of the dorsal horn. Maximal immunoreactivity appears approximately 2 h after stimulation has ceased, and disappears within hours. Continuous stimulation produces many more labeled cells, darker labeling, and FLI within both dorsal and ventral laminar regions. Maximal FLI is seen after approximately 4.5 h of continuous stimulation, with reduction in the number of labeled cells thereafter. These data indicate that the results of any study employing c-fos as a marker for neuronal activity may be affected by the duration of the exciting stimulus.