Parenchymal Markers Research Articles

Abstract WP417: Relevance of Lobar Cerebral Microbleeds in Patients With Deep Intracerebral Hemorrhage

Background: Markers of cerebral small vessel disease (cSVD) severity and prognosis in strictly lobar or strictly deep intracerebral hemorrhages (ICH)/cerebral microbleeds (CMB) are well defined but data are scarce for mixed-location ICH/CMB. We aimed to compare neuroimaging features and outcomes between deep ICH patients with and without lobar CMBs. Methods: Patients with first-ever deep ICH from a prospectively maintained single-center registry were analyzed. Demographics, risk factors and neuroimaging markers of cSVD including CMBs, cortical superficial siderosis, enlarged perivascular spaces (EPVS), white matter hyperintensity volumes (WMH) and brain atrophy scores (range 0-15) were compared between deep ICH patients with lobar CMBs and those without lobar CMBs. The follow-up data were analyzed for ICH recurrence, ischemic stroke (IS) and all-cause mortality. Results: Of 208 patients, 98 (47.1%) had lobar CMBs. Patients with L-CMBs were older (65.3±12.1 vs. 57.4±12.5, p<0.001) and more likely to be hypertensive (98% vs. 90.9%, p=0.037) than those without L-CMBs. Deep CMBs (83.7% vs 56.4%, p<0.001), lacunes (53.1% vs 31.8%, p=0.003) and severe basal ganglia EPVS (40.8% vs 17.3%, p<0.001) were more common in patients with L-CMBs. Patients with L-CMBs had higher volumes of WMH (17.4 ml [IQR 9-38] vs 4.8 ml [IQR 1-13], p<0.001) and atrophy scores (4 [IQR 2-7] vs 3 [IQR 0-6], p=0.007) as compared to patients without L-CMBs. During a median follow-up of 21 months, the ICH recurrence rate was similar between groups (p=0.2), but higher incident IS (3.0 vs 0.0 per 1000-person-months, p=0.016) and all-cause mortality (4.9 vs 1.5 per 1000-person-months, p=0.026) were observed in patients with L-CMBs. In a cox regression model, the presence of lobar CMBs was independently associated with a composite outcome measure (ICH/IS/mortality) after adjusting for age, hypertension, WMH volume, lacune, BG-EPVS and atrophy scores (odds ratio: 2.9, 95%CI:1.01-8.4 p=0.046). Conclusions: Presence of lobar CMBs in patients with deep ICH is associated with parenchymal markers of more severe cSVD and poor prognosis, suggesting a more advanced vasculopathy compared to patients with strictly deep ICH.

Intracranial atherosclerosis and cerebral small vessel disease in intracerebral hemorrhage patients

BackgroundThe association between cerebral small vessel diseases (cSVD) and intracranial atherosclerosis is debated and conflicting results have been reported. We sought to investigate this association in patients with intracerebral hemorrhage (ICH), due to severe cSVD. MethodsConsecutive ICH patients were divided into those meeting criteria for cerebral amyloid angiopathy (CAA) and those with deep hypertensive ICH consistent with hypertensive cSVD (HTN-SVD). White matter hyperintensity volumes (WMH) and microbleed counts (MB) were measured on MRI. CTA was rated for severity of intracranial carotid calcifications and for presence of >50% intracranial stenosis (ICS). Associations of intracranial atherosclerosis severity with type of SVD (CAA vs HTN-cSVD) and with imaging and clinical markers of cSVD burden were analyzed. ResultsThe cohort included 253 CAA and 90 HTN-SVD patients. In multivariable models, the type of cSVD (CAA vs. HTN-cSVD) was not associated with calcification severity (OR=1.04, 95% CI [0.62–3.5], p=0.37) or presence of ICS (OR=0.84, 95% CI [0.21–2.74], p=0.78).We found no association between intracranial atherosclerosis (calcifications and stenoses) and parenchymal markers of cSVD severity (WMH and MB, adjusted p≥0.2 for all comparisons) and no association with presence of dementia before ICH (adjusted p≥0.2 for both comparisons). ConclusionsWe found no association between intracranial atherosclerosis and parenchymal or clinical consequences of cSVD, suggesting that cSVDs while sharing some risk factors are not influenced by upstream larger vessel pathologies.

Abstract TP458: Is There “Crosstalk” Between Intracranial Arterial Pathologies and Small Vessel Disease??

Background: Interactions between intracranial arterial pathologies (IAP) and cerebral small vessel disease (SVD) are an increasingly debated topic. Hypothesis: We analyzed associations between the type/severity of SVD and two IAPs, the intracranial arterial calcifications (ICAC) and intracranial stenosis (ICS) in intracerebral hemorrhage (ICH) patients. Methods: Consecutive ICH patients from a prospective cohort were included. Patients were divided into those meeting Boston criteria for cerebral amyloid angiopathy (CAA) and those with strictly deep hypertensive ICH consistent with hypertensive SVD (HTN-SVD). White matter hyperintensity volume (WMH) and microbleed count (MB) were quantitatively measured on MRI as markers of SVD severity. Head CT angiography was rated for presence of ICAC and for presence of >50% intracranial arterial stenosis (ICS). Associations of IAPs with the type of SVD (CAA vs HTN) as well as imaging markers of SVD severity were analyzed in multivariate models. We also explored the association between IAPs and presence of pre-ICH dementia. Results: The cohort included 253 CAA patients and 90 HTN-SVD. CAA patients were older (73.5 vs 64.8, p<0.001), demonstrating higher WMH (25ml vs 16ml, p<0.001) but lower prevalence of hypertension than HTN-ICH. In univariate comparisons between CAA and HTN-SVD, the presence of ICACs (74% vs 72%, p=0.7) and ICS (7% vs 7.8%, p=0.8) were not different. ICS was not related to the type of SVD in multivariate models either. Using multivariate logistic regression, HTN-SVD was independently associated with presence of ICAC (adjusted OR = 2.56 [95% CI 1.1-6.2, p=0.002), as well as older age, male gender and hypercholesterolemia. We found no association between IAPs and parenchymal markers of SVD severity (WMH and MB) (all p>0.2) and no association with presence of dementia before ICH (p>0.2). Conclusions: HTN-SVD is associated with increased ICAC in multivariate models, suggesting either shared risk factors or direct interactions between SVD and IAP. There is no association of intracranial large artery pathologies (ICAC, ICS) with parenchymal (WMH, MB) or clinical (dementia) consequences of cerebral small vessel diseases.

7 Tesla MRI in Cerebral Small Vessel Disease

Cerebral small vessel disease (SVD) is a major cause of stroke and cognitive decline. Magnetic resonance imaging (MRI) currently plays a central role in diagnosis, and advanced MRI techniques are widely used in research but are limited by spatial resolution. Human 7 Tesla (7T) MRI has recently become available offering the ability to image at higher spatial resolution. This may provide additional insights into both the vascular pathology itself as well as parenchymal markers which could only previously be examined post mortem. In this review we cover the advantages and limitations of 7T MRI, review studies in SVD performed to date, and discuss potential future insights into SVD which 7T MRI may provide.

Localization of bone marrow stromal cells to the injury site after intracerebral hemorrhage in rats

Previous studies demonstrated that intravascular injection of bone marrow stromal cells (BMSCs) significantly improved neurological functional recovery in a rat model of intracerebral hemorrhage (ICH). To further investigate the fate of transplanted cells, we examined the effect of male rat BMSCs administered to female rats after ICH. Twenty-seven female Wistar rats were subjected to ICH surgery. At 24 hours after ICH, these rats were randomly divided into 3 groups and injected intravenously with 1 ml phosphate-buffered saline or 0.5 million or 1 million male rat BMSCs in phosphate-buffered saline. To evaluate the neurological functional outcome, each rat was subjected to a series of behavioral tests (modified neurological severity score and corner turn test) at 1, 7, and 14 days after ICH. The rats were anesthetized intraperitoneally and killed, and the brain tissues were processed at Day 14 after ICH. Immunohistochemistry and in situ hybridization were used to identify cell-specific markers. The male rat BMSCs significantly improved the neurological functional outcome and also significantly diminished tissue loss when intravenously transplanted into the rats after ICH. Immunoassay for bromodeoxyuridine (BrdU) and neuronal markers demonstrated a significant increase in the number of BrdU-positive cells, which indicated endogenous neurogenesis, and a significant increase in the number of cells positive for immature neuronal markers. In situ hybridization showed that more BMSCs resided around the hematoma of the rats treated with the 1-million-cell dose compared with the 0.5-million-cell-dose group. In addition, a subfraction of Y chromosome-positive cells were co-immunostained with the neuronal marker microtubule-associated protein-2 or the astrocytic marker glial fibrillary acidic protein. Male rat BMSCs improve neurological outcome and increase histochemical parameters of neurogenesis when administered to female rats after ICH. This study has shown that the intravenously administered male rat BMSCs enter the brain, migrate to the perihematomal area, and express parenchymal markers.

In Vivo Selection and Validation of Liver-Specific Ligands Using a New T7 Phage Peptide Display System

In vivo phage display is a powerful source of new peptide ligands for specific organ targeting by drugs and gene therapy vectors. Since the introduction of this methodology a decade ago, a number of peptides that preferentially react with organ-specific endothelium and parenchymal markers have been selected. One organ that has been conspicuously missing from these selection studies is the liver, which possesses a multitude of acquired and hereditary disorders and represents a highly important therapeutic target. Herein, we set out to fill this gap by introducing a novel peptide display system containing cloned sequences in the tail fiber protein (p17) of phage T7. The p17 display effectively avoids the innate immune system and is well suited both for selection of new liver-specific ligands and for validation of protein sequences that have been implicated in liver targeting by the use of conventional biochemical methods.

Mechanics of edematous lungs.

Using the parenchymal marker technique, we measured pressure (P)-volume (P-V) curves of regions with volumes of approximately 1 cm3 in the dependent caudal lobes of oleic acid-injured dog lungs, during a very slow inflation from P = 0 to P = 30 cmH2O. The regional P-V curves are strongly sigmoidal. Regional volume, as a fraction of volume at total lung capacity, remains constant at 0.4-0.5 for airway P values from 0 to approximately 20 cmH2O and then increases rapidly, but continuously, to 1 at P = approximately 25 cmH2O. A model of parenchymal mechanics was modified to include the effects of elevated surface tension and fluid in the alveolar spaces. P-V curves calculated from the model are similar to the measured P-V curves. At lower lung volumes, P increases rapidly with lung volume as the air-fluid interface penetrates the mouth of the alveolus. At a value of P = approximately 20 cmH2O, the air-fluid interface is inside the alveolus and the lung is compliant, like an air-filled lung with constant surface tension. We conclude that the properties of the P-V curve of edematous lungs, particularly the knee in the P-V curve, are the result of the mechanics of parenchyma with constant surface tension and partially fluid-filled alveoli, not the result of abrupt opening of airways or atelectatic parenchyma.

Mechanisms of recruitment in oleic acid-injured lungs.

Lung recruitment strategies, such as the application of positive end-expiratory pressure (PEEP), are thought to protect the lungs from ventilator-associated injury by reducing the shear stress associated with the repeated opening of collapsed peripheral units. Using the parenchymal marker technique, we measured regional lung deformations in 13 oleic acid (OA)-injured dogs during mechanical ventilation in different postures. Whereas OA injury caused a marked decrease in the oscillation amplitude of dependent lung regions, even the most dependent regions maintained normal end-expiratory dimensions. This is because dependent lung is flooded as opposed to collapsed. PEEP restored oscillation amplitudes only at pressures that raised regional volumes above preinjury levels. Because the amount of PEEP necessary to promote dependent lung recruitment increased the end-expiratory dimensions of all lung regions (nondependent AND dependent ones) compared with their preinjury baseline, the "price" for recruitment is a universal increase in parenchymal stress. We conclude that the mechanics of the OA-injured lung might be more appropriately viewed as a partial liquid ventilation problem and not a shear stress and airway collapse problem and that the mechanisms of PEEP-related lung protection might have to be rethought.

Regional expansion of oleic acid-injured lungs.

It has been suggested that dependent regions of an injured lung are collapsed and subject to cyclic reopening and collapse during mechanical ventilation. To test this hypothesis, we measured both temporal and spatial heterogeneity of lobar expansion in oleic acid (OA)-injured dogs. Regional volumes were measured in nine dogs (seven supine and two prone) during closed loop sinusoidal oscillations of the lungs before and after OA injury using the parenchymal marker technique. In contrast to computer tomography, the parenchymal marker technique provides absolute measures of regional tissue dimensions as opposed to relative measures of regional air to liquid content. The experiments generated three major findings: (1) OA injury did not lead to the collapse of dependent lung units at FRC, (2) OA injury did not steepen the vertical gradient in regional lung volumes at FRC, and (3) during sinusoidal oscillation of the OA-injured lungs from FRC, dependent regions did not undergo cyclic reopening and collapse. On the basis of these results, we propose an alternative mechanism for the topographic variability in regional impedances and lung expansion after injury, namely liquid and foam in conducting airways.

Regional ventilation in statically and dynamically hyperinflated dogs.

Using the parenchymal marker technique in normal anesthetized dogs, we compared the dynamics of regional lung expansion between two ventilation strategies designed to increase mean thoracic volume. Dynamic hyperinflation (DH was produced by ventilating the lungs at a rate of 50 breaths/min and with a duty cycle of 0.5. Static hyperinflation (SH) was produced through the application of extrinsic positive end-expiratory pressure while the lungs were ventilated at a rate of 15 breaths/min and with a duty cycle of 0.15. Regional tidal volume (VT,r), regional functional residual volume, and the time delay between regional expansion and the flow signal at the common airway were computed for up to 100 regions/lobe in 5 animals. Ventilation strategy had no effect on the overall variance of VT,r within lobes. Although the VT,r measured during SH correlated with VT,r measured during DH, the average correlation coefficient was only 0.69. Ventilation rate-related differences in VT,r and regional functional residual capacity varied with the regional time delay in ways qualitatively consistent with parallel inhomogeneity of unit time constants. However, a large component of frequency-dependent behavior remains unexplained by established mechanisms. We conclude that DH and SH should not be considered equivalent lung unit recruitment strategies.

Difference in Onset of Warm Ischemia and Reperfusion Injury between Parenchymal and Endothelial Cells of the Liver. Evaluation by Purine Nucleoside Phosphorylase and Hyaluronic Acid

The onset of warm ischemia and reperfusion injury in the liver was investigated in a canine model through changes in parenchymal markers [isozyme class V of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT)], endothelial markers [purine nucleoside phosphorylase (PNP) and hyaluronic acid clearance], and the liver metabolism (ketone body ratio) in warm ischemia induced by inflow occlusion using Pringle's maneuver and subsequent reperfusion. In thisin vivomodel, a PNP assay system and a model were designed so as to exclude the influence of wide localization of PNP possibly originating in erythrocytes or the intestine, and to discriminate between PNP of endothelial cells and that of parenchymal cells in the liver. After 45 min of warm ischemia, reperfusion resulted in damage only to endothelial cells, as seen by significant increase in PNP alone (3.6 ± 0.1 U/liter at the end of warm ischemia to 6.8 ± 0.5 U/liter at 5 min after reperfusion,P< 0.01) and significant decrease in hyaluronic acid clearance compared to the 30-min warm ischemia group in which no increase in either marker for parenchymal and endothelial cells was noted. By contrast, after 60 min of warm ischemia, reperfusion resulted in damage to parenchymal cells along with damage to endothelial cells, as seen by significant increases in LDH(V) and ALT (93 ± 4 U/liter and 32 ± 2 IU/liter at the end of warm ischemia to 239 ± 17 U/liter and 165 ± 27 IU/liter at 5 min after reperfusion, respectively), as well as a marked increase in PNP and deterioration of hyaluronic acid clearance compared to the 45-min warm ischemia group. Reperfusion after 120 min of warm ischemia did not show recovery of metabolic function of the liver as evaluated by hepatic mitochondrial redox state. It is suggested that a time lag occurs in the onset of injury between parenchymal cells and endothelial cells and that endothelial cells are temporally earlier in failing than parenchymal cells when the liver is exposed to short-term warm ischemia and subsequent reperfusion.

Regional lung volume trajectories during expiratory flow in dogs.

Parenchymal markers were placed in the left caudal lobes of anesthetized dogs, and the three-dimensional positions of the markers were tracked by biplane videofluoroscopy during expiration from total lung capacity (TLC) to approximately 30% TLC at steady expiratory flows of 0.3-7% TLC/s. Regional volumes of samples of parenchyma with volumes at TLC of 1-5 cm3 were obtained by computing the volumes of tetrahedral defined by taking the markers, four at a time, as apices of the tetrahedra, Regional volume (Vr), as a fraction of volume at TLC, was plotted against average volume (VL), as a fraction of volume at TLC, and Vr was fit by a quadratic function of VL. The initial slopes of the plots, dVr/dVL at VL = 1, varied by +/- 26% from the mean slope of 1. The curvatures were highly correlated with the slopes, and the Vr vs. VL plots formed a nest of "onion skins". The initial slopes were weakly correlated with the vertical position of the centroid of the tetrahedron, with the slope increasing by approximately 20%/cm, on average, in the dorsal direction in the supine dog. The vertical gradient in Vr accounted for approximately 30% of the total variability; small-scale heterogeneity contributed the remaining 70%. These results confirm earlier quasistatic measurements of nonuniform Vr and provide the first data on curvature of Vr trajectories. The mechanisms that cause the nongravitational component of Vr variability and the curvatures of the trajectories are unknown.

Nonuniform expansion of constricted dog lungs.

The parenchymal marker technique was used to measure regional tidal volumes of samples of lung parenchyma in four open-chest supine dogs. Radiopaque markers that had been implanted in the lower lobe were tracked by biplane video fluoroscopy during sinusoidal volume oscillations at tidal volumes of approximately 20% of total lung capacity and frequencies of 1-40 breaths/min before and after methacholine was administered by aerosol. The volumes of tetrahedrons with apexes at four markers were computed, and sine waves were fit to the data for volume vs. time for each tetrahedron. The ratio of mean regional volume to mean airway pressure decreased by 10-45% after exposure to methacholine. Dynamic lung elastance and resistance of the constricted lungs were larger than control, and both were frequency dependent. Regional elastance and resistance varied considerably among tetrahedrons, and these were also frequency dependent. The data were fit by a model in which tissue elastance was uniform and nearly equal to elastance in the control state, but small-airway resistance was high and variable. We conclude that the lung contracts under bronchoconstriction but that the increased dynamic elastance and resistance of the constricted lung may be primarily the result of nonuniform increased airway resistance at the level of the terminal bronchioles.

Characterization of luminal and basal cells flow-sorted from the adult rat mammary parenchyma

Differentially expressed membrane antigens have been used to flow-sort viable luminal epithelial and myoepithelial cells from freshly disaggregated adult virgin rat mammary parenchyma. Resulting cultures and clones have been characterized morphologically and by a panel of antibodies that recognise cell-type-specific cytoskeletal antigens in the intact mammary gland. Five clonal phenotypes were recognisable by morphological criteria, three (types 1-3) exclusively associated with sorted luminal epithelial (25.5-positive) cells, and two (types 4 and 5) generated from the sorted myoepithelial (CALLA/neutral endopeptidase 24.11-positive) cells. All clones derived from myoepithelial cells continued to express a basal parenchymal marker in the form of the rat equivalent of human cytokeratin 14, while smooth muscle alpha-actin was expressed by the small slowly growing type 4 clones but was found in fewer cells in rapidly proliferating type 5 myoepithelially derived clones. Two of the luminal clone types, characterized by an attenuated appearance and slow growth (types 1/2), expressed only luminal-specific markers, including the equivalent of human cytokeratins 7/18/19. Type 3 clones, by contrast, consisted of rapidly proliferating cells, many of which either co-expressed CK14 and CK18 antigens (type 3a) or were composed of a mosaic of CK14+/CK18-, CK14+/CK18+ and CK14-/CK18+ cells (type 3b). The sorted myoepithelially derived clones grew faster than clones from sorted luminal cells as evidenced by the larger fraction of cells synthesizing DNA. All types of clone could be obtained from both isolated ducts and alveoli, when these were cloned separately, although there were some differences in their relative frequency.(ABSTRACT TRUNCATED AT 250 WORDS)

Regional ventilation during spontaneous breathing and mechanical ventilation in dogs.

We evaluated the effects of the different patterns of chest wall deformation that occur with different body positions and modes of breathing on regional lung deformation and ventilation. Using the parenchymal marker technique, we determined regional lung behavior during mechanical ventilation and spontaneous breathing in five anesthetized recumbent dogs. Regional lung behavior was related to the patterns of diaphragm motion estimated from X-ray projection images obtained at functional residual capacity (FRC) and end inspiration. Our results indicate that 1) in the prone and supine positions, FRC was larger during mechanical ventilation than during spontaneous breathing; 2) there were significant differences in the patterns of diaphragm motion and regional ventilation between mechanical ventilation and spontaneous breathing in both body positions; 3) in the supine position only, there was a vertical gradient in lung volume at FRC; 4) in both positions and for both modes of breathing, regional ventilation was nonlinearly related to changes in lobar and overall lung volumes; and 5) different patterns of diaphragm motion caused different sliding motions and differential rotations of upper and lower lobes. Our results are inconsistent with the classic model of regional ventilation, and we conclude that the distribution of ventilation is determined by a complex interaction of lung and chest wall shapes and by the motion of the lobes relative to each other, all of which help to minimize distortion of the lung parenchyma.

Cardiogenic motion of right lung parenchyma in anesthetized intact dogs.

Cardiogenic motion of the right lung parenchyma (CGLM) was measured in six morphine-pentobarbital-anesthetized dogs (11-16 kg) under conditions of varying paced and spontaneous sinus heart rates. Motion of 1-mm-diameter percutaneously implanted radiopaque lung parenchymal markers were measured using a computer-based biplane video-roentgenographic assembly. Correlation of the amplitudes and phases of marker motions to the R wave of the electrocardiogram (ECG) were determined utilizing a modified fast Fourier transform algorithm. Of initial importance was the observation that CGLM was significantly greater than the variability of repeated measurements. The total amplitudes of motion of the markers (where AT = square root A2X + A2Y + A2Z and AX, AY, AZ are the amplitudes of marker motion along the orthogonal X, Y, and Z body axes) in the right apical, cardiac, and diaphragmatic lobes were 0.025 +/- 0.005 (SE), 0.046 +/- 0.005, and 0.023 +/- 0.025 cm, respectively, (P less than 0.01, cardiac lobe vs. others). The total amplitude of motion observed in the cardiac lobe was equivalent to earlier observations of total amplitude (0.045 +/- 0.004 cm) of marker motion in response to high frequency airway oscillation at a pump stroke volume of 22-26 ml. An analysis of the higher harmonics of marker oscillation suggested that the second harmonic of CGLM is also larger than the variability of our measurements.

Biplane videoroentgenographic analysis of dynamic regional lung strains in dogs.

A method is described for determining the spatial distribution of pulmonary parenchymal strains in the intact canine thorax, using measurements of displacement of metallic (1-mm-diam)) markers percutaneously implanted throughout the parenchyma of the right lung. Dogs are supported head up or head down in a water-immersion respirator with the animal's airway connected to ambient air. Tracking of the parenchymal markers is accomplished by stereo biplane videoroentgenographic recordings, which allow high temporal (60/S) and spatial (+/- 1.5 mm) resolution measurements of the "tagged" lungs during various respiratory maneuvers. After transferring the video information to a stop-action video disc, an operator-interactive computer program is used to input the geometric coordinates of the markers into the computer. The true spatial coordinates are then determined after correction for pincushion and magnification distortions. Spatial and temporal distributions of regional parenchymal strains are obtained by determining the distance between markers on a frame-by-frame basis over the extent of the respiratory cycle. Data indicate nonuniformity in regional lung parenchymal strains.