Laszlo Miskolczi

Saccular aneurysm induction by elastase digestion of the arterial wall: A new animal model

OBJECTIVE To develop a rabbit aneurysm model that is more realistic in gross appearance and histological features than previous models and to enable the development of a larger animal model. METHODS Ten rabbits received porcine pancreatic elastase, five at the right common carotid artery bifurcation and five others at the right superior thyroid artery origin. One control animal received collagenase and another received papaverine, each at the right superior thyroid artery origin. The agents were topically delivered to the arterial adventitia with a microsyringe after surgical exposure of the targeted arteries. The arteries were monitored for aneurysm growth with a video camera for up to 3 hours and were then removed and processed for histology. RESULTS Saccular aneurysms developed in one of five animals after elastase application at the carotid bifurcation and in all five animals receiving elastase at the superior thyroid artery origin. Among the six aneurysms, recurrent minor hemorrhages occurred in four, thrombosis of the aneurysm sac in three, and rupture causing severe bleeding in one. Histological sections revealed thin-walled aneurysms composed only of collagen fibers and some cellular elements. No saccular dilation resulted from papaverine application. Collagenase application resulted in a hemorrhagic-thrombotic lesion in the arterial wall but no aneurysm formation. CONCLUSION Arterial saccular aneurysms were induced in rabbits by topical application of elastase with an easy and efficient method. These aneurysms are histologically similar to natural aneurysms, and their arterial nature renders them more authentic than those of surgical models. This aneurysm model may serve as a foundation for further aneurysm research.

Quantitative analysis of injured, necrotic, and apoptotic cells in a new experimental model of intracerebral hemorrhage

ObjectiveTo develop a new survival model of intracerebral hemorrhage (ICH) in rabbits and study the patterns of cellular injury in different regions 24 hrs after introduction of hematoma. Quantitation and characterization of injured cells in regions adjacent and distant to the hematoma have not been performed. DesignProspective case-control study. SubjectsTen New Zealand rabbits. InterventionWe introduced ICH in six anesthetized New Zealand rabbits by autologous blood injection under arterial pressure in the deep white matter in the frontal lobe. Measurements and Main ResultsHematoxylin and eosin staining was performed in six animals with ICH after 24 hrs to quantify intact, injured, and necrotic cells in regions proximal and distant to the hematoma, and the results were compared with four control animals. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining was performed to quantify apoptotic cells in specified regions in five animals with ICH, and the results were compared with four control animals. All cell counts were performed by one investigator who used 100× oil emersion microscopy. The presence of localized hematoma was confirmed in all six animals with blood infusion. Compared with controls, animals with ICH had a significantly higher proportion of swollen cells in both the inner (55.9% ± 3.0% vs. 26.8% ± 1.7%;p < .05) and the outer (59.8% ± 4.6% vs. 27.7% ± 4.5%;p < .05) rim of the perihematoma region. A small proportion of shrunken dark cells were observed in both the inner (4.0% ± 1.5%) and the outer (3.6% ± 1.0%) rim of the perihematoma region. The remaining cells were considered morphologically intact. A large proportion of cells trapped within the matrix of the hematoma were either shrunken dark cells (48.8% ± 16.4%) or swollen (38.8% ± 15.1%). In the TUNEL-stained sections, a high burden of apoptotic cells was observed in the matrix of the hematoma (17.5 ± 6.3 cells per high power field) but not in the perihematoma regions (less than two cells per high power field). ConclusionsA reproducible model of ICH in rabbits is described. At 24 hrs, the perihematoma region contains relatively large proportions of morphologically intact or reversibly injured (swollen) cells, suggesting the possibility of an extended window for therapeutic intervention.

Hemodynamics of Carotid Artery Atherosclerotic Occlusive Disease

Hemodynamic mechanisms for the initiation and progression of carotid bifurcation atherosclerotic occlusive disease have been extensively researched during the past few decades. Attention has focused on the carotid bulb, or sinus, where most atherosclerotic plaques are found. Herein, the authors review the seminal works that have led to an understanding of not only complex local hemodynamics but also the elicited specific biologic response. In addition, new analysis of the age-dependent morphologic maturation of the human carotid bifurcation is unveiled. Understanding the role of hemodynamics in atherogenesis may lead to the improvement of minimally invasive endovascular therapy and noninvasive strategies.

Rapid saccular aneurysm induction by elastase application in vitro

OBJECTIVE To develop a new saccular aneurysm model in vitro using elastase to study aneurysm initiation, growth, and rupture and to create a new in vivo aneurysm model to test endovascular therapies. METHODS Seventeen common carotid arteries excised from freshly killed pigs and sheep were treated with seven different methods of elastase delivery. The arteries were mounted in a saline-filled flow chamber. They received pulsatile flow for 48 hours, or until the resulting aneurysms ruptured. Changes were continuously monitored with video camera recordings and validated with histological sections. RESULTS All eight arteries treated topically, either on the intimal or on the adventitial surface, with elastase concentrations greater than 1 U/mm2, developed saccular aneurysms; five of them ruptured within 48 hours. All four arteries treated with surface concentrations of 0.1 U/mm2 via microcatheter infusion into the lumen developed fusiform aneurysms. None of the arteries that received surface concentrations less than 0.1 U/mm2 developed aneurysms. Histological sections revealed a reduced number of cellular element in a stretched collagen matrix at the dome of the saccular aneurysms. CONCLUSION After empirically testing several methods of elastase delivery, we were able to induce saccular, bifurcation-type aneurysms in animal arterial specimens. These aneurysms are histologically similar and more authentic than surgical models. The procedure is easy and reproducible. Our results suggest a possible enzymatic role in aneurysm formation and highlight the dramatic effects of selective arterial elastic damage. Also, the rapid growth of our experimental aneurysms may reflect the speed of the natural process.

A Novel Intravascular Drug Delivery Method Using Endothelial Biotinylation and Avidin-Biotin Binding

In this study, a novel intravascular drug delivery system was developed in which a drug injected from a catheter was fixed to the vasculature of the targeted tissue. Cellular proteins of viable endothelial cells were first biotinylated directly by biotinylation reagents, and then bound by an avidinated drug or, using avidin as a linker, a biotinylated drug. In the initial experiments, we studied in vitro the biotinylation of cultured bovine aortic endothelial cells (BAECs) by applying biotinylation reagents (NHS-LC-biotin or sulfo-NHS-LC-biotin) onto the washed intact BAEC monolayers and showed that the amount of biotin bound to the cells depended on the concentration of the biotinylation reagents applied. The cell-bound biotin decreased with time after the biotinylation. When fluorescein-labeled avidin (FITC-avidin) was applied to the biotinylated BAEC monolayers, the FITC-avidin readily bound to the cells. An LDH-release assay showed that sulfo-NHS-LC-biotin was only slightly cytotoxic to the BAECs and a colony formation assay showed only slight adverse effects of the reagent. In vivo studies were carried out on the renal arteries of normal rabbits. A solution of NHS-LC-biotin was injected through a catheter to one kidney to biotinylate its vasculature and the vehicle to the other as control, followed by a perfusion with saline. Finally, a solution of FITC-avidin was injected to both kidneys that were then reperfused with the blood flow following the withdrawal of the catheters. In the histological sections, more than 85% of glomeruli was stained with fluorescein in the biotinylated kidney, whereas no glomeruli were stained in the control. In the kidneys harvested 2 days after the same procedure, most glomeruli were still brightly stained. In the final experiment, biotinylated kidneys were injected with a solution of avidin, followed by a solution of fluorescein-biotin. Control kidneys had no prior biotinylation but received the same injections of avidin and fluorescein-biotin as above. More than 80% of glomeruli were stained in the biotinylated kidneys but none in the controls. This indicated that biotinylated drugs can be anchored to the biotinylated vasculature through avidin without being flushed away by blood flows. No apparent adverse effect was found in the functions of biotinylated kidneys. We propose that this drug delivery system is feasible for the treatment of some pathological conditions of blood vessels such as microvascular proliferation in malignant tumors and for continuous drug delivery in certain target organs.

Angiographic assessment of the performance of flow divertors to treat cerebral aneurysms.

Past clinical and experimental evidence suggests that cerebral aneurysms can be successfully excluded from the circulation solely by the endovascular placement of a flow diverting device across the aneurysm neck. These devices promote intraaneurysmal flow stasis and concomitant thrombosis by redirecting flow away from the aneurysm. To comprehensively test the efficacy of such flow divertors, we are implanting devices with three different porosities in a large cohort of elastase-induced aneurysms in rabbits. Treatment efficacy is quantified by a mathematical model that is fit to aneurysmal angiographic contrast washout curves. Results from three animals implanted with different device porosities are presented here. The model competently captures the behavior of the aneurysmal washout curves and provides reliable indices of device efficacy. Preliminary analysis indicates that immediately after implantation, the device with medium porosity performs better than the devices with lower and higher porosities

Vessel size measurements in angiograms: Manual measurements

Vessel size measurement is perhaps the most often performed quantitative analysis in diagnostic and interventional angiography. Although automated vessel sizing techniques are generally considered to have good accuracy and precision, we have observed that clinicians rarely use these techniques in standard clinical practice, choosing to indicate the edges of vessels and catheters to determine sizes and calibrate magnifications, i.e., manual measurements. Thus, we undertook an investigation of the accuracy and precision of vessel sizes calculated from manually indicated edges of vessels. Manual measurements were performed by three neuroradiologists and three physicists. Vessel sizes ranged from 0.1-3.0 mm in simulation studies and 0.3-6.4 mm in phantom studies. Simulation resolution functions had full-widths-at-half-maximum (FWHM) ranging from 0.0 to 0.5 mm. Phantom studies were performed with 4.5 in., 6 in., 9 in., and 12 in. image intensifier modes, magnification factor = 1, with and without zooming. The accuracy and reproducibility of the measurements ranged from 0.1 to 0.2 mm, depending on vessel size, resolution, and pixel size, and zoom. These results indicate that manual measurements may have accuracies comparable to automated techniques for vessels with sizes greater than 1 mm, but that automated techniques which take into account the resolution function should be used for vessels with sizes smaller than 1 mm.

Neurological deficits associated with the elastase-induced aneurysm model in rabbits

Abstract Objective: Although the rabbit elastase-induced aneurysm model is currently used widely for endovascular research and device testing, procedural causes leading to animal morbidity and mortality have not yet been clearly described. We conducted a retrospective study to analyse factors contributing to neurological deficits in rabbits that underwent the elastase-induced aneurysm creation procedure at our research center from 2002 to 2005 in order to improve the technique and reduce procedure-related morbidity and mortality. Methods: A total sample of 38 animals that underwent the procedure under the same conditions was analysed in two groups: animals that presented neurological deficits (ND, n=15) and animals that were neurological deficit free (NDF, n=23). Data were collected by reviewing the animal records and radiographic images from the procedures. Statistical analyses using the Mann–Whitney test, unpaired t-test with Welch correction and Fishers exact tests were performed to compare the two groups based on variables associated with endothelial injury and activation of the coagulation cascade. Results: The variables of animal weight (signifying state of health of the animal), total procedure time, total balloon occlusion time and clot formation were found to be significantly and/or very significantly correlated to ND presentation. Discussion: Successful creation of the rabbit elastase-induced aneurysm model depends on careful control over several technical details. Important variables governing outcome have been identified here. A specific, improved endovascular arrangement that facilitates maneuvering of the devices and reduces the risk of air emboli is presented.

Improved method of magnification factor calculation for the angiographic measurement of neurovascular lesion dimensions

Accurately evaluating the size of a neurovascular lesion is essential for properly devising treatment strategies. The magnification factor must be considered in order to measure the dimension of a lesion from an angiogram. Although a method to calculate the magnification of the lesion by linear interpolation of the measurable magnification factors of two markers has been in use, this paper shows that it can be inaccurate. By deriving the exact formula for calculating the magnification factor at the level of the lesion, the error generated by the linear interpolation of magnification factor has been evaluated. This error was found to depend on source‐to‐skin distance (SSD), the location of the lesion in the head, and the head size. The closer the head is to the focal spot and the nearer the lesion is to the center of the head, the larger is the error. Since clinicians tend to use high geometric magnification (i.e., small SSD) in interventional procedures, there exists a possible consequential error of more than 3% in lesion sizing if the linear‐interpolation calculation method is used. It is thus recommended that the exact formula derived here be used to calculate the magnification factor to improve accuracy. PACS number(s): 87.57.–s, 87.57.Nk, 87.59.Dj

Geometry of Elastase-Induced Aneurysms in Rabbits

Thirty New Zealand White rabbits were used in the study. The diameter of the origin of the CCA measured immediately after elastase incubation correlated positively to the aneurysm neck at three week follow up. Moreover, the aneurysm neck both after creation and three week follow-up had a positive correlation with the final dome height. Finally, the dome height was found to be related to the angle between the innominate artery and the aneurysm centerline for dome diameter/neck ratio of <1.5. These results indicate that neck width immediately after creation and the curvature of the parent artery are linked to aneurysm dimensions.Copyright