Morphology of elastase-induced cerebral aneurysm model in rabbit and rapid prototyping of elastomeric transparent replicas

In this work, we describe a methodology to fabricate transparent elastomeric vascular replicas using rapid prototyping techniques. First, the three-dimensional morphology of an elastase-induced aneurysm model in rabbit is acquired. The morphology is reconstructed from in vivo rotational angiography and it is compared with three-dimensional reconstructions obtained by computerized tomography and magnetic resonance imaging of an intraluminal arterial cast that was obtained from the same animal at sacrifice. Results show that resolution of the imaging modality strongly influences the level of detail, such as small side branches, in the final reconstruction. We developed an average morphology model for elastase-induced aneurysms in rabbits including the surrounding vasculature and describe a method for rapid prototyping of vascular models from the three-dimensional morphology. Our replicas can be manufactured in a short period of time and the final product is optically clear. In addition, the elasticity of the models can be controlled to represent arterial elasticity, which makes them ideal for optical investigations of detailed flow dynamics using measurement tools such as particle image velocimetry.     

Blood flow dynamics in saccular aneurysm models of the basilar artery

Blood flow dynamics under physiologically realistic pulsatile conditions plays an important role in the growth, rupture, and surgical treatment of intracranial aneurysms. The temporal and spatial variations of wall pressure and wall shear stress in the aneurysm are hypothesized to be correlated with its continuous expansion and eventual rupture. In addition, the assessment of the velocity field in the aneurysm dome and neck is important for the correct placement of endovascular coils. This paper describes the flow dynamics in two representative models of a terminal aneurysm of the basilar artery under Newtonian and non-Newtonian fluid assumptions, and compares their hemodynamics with that of a healthy basilar artery. Virtual aneurysm models are investigated numerically, with geometric features defined by beta = 0 deg and beta = 23.2 deg, where beta is the tilt angle of the aneurysm dome with respect to the basilar artery. The intra-aneurysmal pulsatile flow shows complex ring vortex structures for beta = 0 deg and single recirculation regions for beta = 23.2 deg during both systole and diastole. The pressure and shear stress on the aneurysm wall exhibit large temporal and spatial variations for both models. When compared to a non-Newtonian fluid, the symmetric aneurysm model (beta = 0 deg) exhibits a more unstable Newtonian flow dynamics, although with a lower peak wall shear stress than the asymmetric model (beta = 23.2 deg). The non-Newtonian fluid assumption yields more stable flows than a Newtonian fluid, for the same inlet flow rate. Both fluid modeling assumptions, however, lead to asymmetric oscillatory flows inside the aneurysm dome.     

A model for saccular cerebral aneurysm growth by collagen fibre remodelling

The first structural model for saccular cerebral aneurysm growth is proposed. It is assumed that the development of the aneurysm is accompanied by a loss of the media, and that only collagen fibres provide load-bearing capacity to the aneurysm wall. The aneurysm is modelled as an axisymmetric multi-layered membrane, exposed to an inflation pressure. Each layer is characterized by an orientation angle, which changes between different layers. The collagen fibres and fibroblasts within a specific layer are perfectly aligned. The growth and the morphological changes of the aneurysm are accomplished by the turnover of collagen. Fibroblasts are responsible for collagen production, and the related deformations are assumed to govern the collagen production rate. There are four key parameters in the model: a normalized pressure, the number of layers in the wall, an exponent in the collagen mass production rate law, and the pre-stretch under which the collagen is deposited. The influence of the model parameters on the aneurysmal response is investigated, and a stability analysis is performed. The model is able to predict clinical observations and mechanical test results, for example, in terms of predicted aneurysm size, shape, wall stress and wall thickness.     

A nonlinear dynamical mechanism for bruit generation by an intracranial saccular aneurysm

 Intracranial saccular aneurysms have been clinically observed to emit a transient sound, a bruit, on each heartbeat. The mechanism causing the bruits has been a matter of contention. A qualitative analysis of the nonlinear dynamical properties of the Shah-Humphrey model for periodic pressure forcing of a thin-necked saccular aneurysm, using the Fung nonlinear constitutive model for the aneurysm material, shows that a small blood pressure jump on each beat, whether the pressure is weakly aperiodic or periodic, induces transients in the radial deformation response of the aneurysmal wall on each heartbeat. These transient vibrations, which have a component with frequency near the natural frequency of the system but are not resonant phenomena and which decay rapidly to a limit cycle during each distinct forcing pressure cycle, can generate the bruits. Received: 21 November 2000 / Revised version: 9 August 2001 / Published online: 23 August 2002 Mathematics Subject Classification (2000): 92B99, 70K40, 70K05 Key words or phrases: Intracranial saccular aneurysm – Bruit – Spectrum – Nonlinear dynamics – Transients – Vortex shedding – Fung model     

In vitro analysis of localized aneurysm rupture

In this study, bulge inflation tests were used to characterize the failure response of 15 layers of human ascending thoracic aortic aneurysms (ATAA). Full field displacement data were collected during each of the mechanical tests using a digital image stereo-correlation (DIS-C) system. Using the collected displacement data, the local stress fields at burst were derived and the thickness evolution was estimated during the inflation tests. It was shown that rupture of the ATAA does not systematically occur at the location of maximum stress, but in a weakened zone of the tissue where the measured fields show strain localization and localized thinning of the wall. Our results are the first to show the existence of weakened zones in the aneurysmal tissue when rupture is imminent. An understanding these local rupture mechanics is necessary to improve clinical assessments of aneurysm rupture risk. Further studies must be performed to determine if these weakened zones can be detected in vivo using non-invasive techniques.     

In vitro development of rabbit pronuclear embryos in rabbit peritoneal fluid

The use of rabbit peritoneal fluid (PF) for the culture of rabbit embryos in vitro was evaluated. Development of zygotes cultured in PF and Earle's balanced salts solution (EBSS) + 10% fetal calf serum (EBSS/FCS) was compared. The effects of increasing the concentration of PF in EBSS and of culturing embryos in fractionated PF were also investigated. In addition, embryonic development in PF was compared to that in vivo. Development to hatching blastocysts was enhanced with PF (73%) compared to EBSS/FCS (3%, p less than 0.001). PF manifested greater mitogenic activity than EBSS/FCS, as indicated by higher cell number in embryos at 48, 72, and 96 h post-mating/hCG (p less than 0.001). PF also promoted blastocyst cell proliferation in a dose-dependent manner (r = 0.98, p less than 0.01); however, embryo growth remained slower than in vivo. Culture in the high (greater than 30,000 Da) molecular mass fraction of PF reduced incidence of hatching (56% vs. 92%, p less than 0.001) and mean cell number in Day 4 blastocysts (151 +/- 4 vs. 243 +/- 5, p less than 0.001). Rates of blastocyst hatching (10%) and cell number (110 +/- 3) were further reduced in the low (less than 30,000 Da) molecular mass fraction. When the high molecular mass fraction was dialyzed, embryos did not develop beyond the early morula stage. This suggests that the interaction or the synergy of high and low molecular mass components of PF is necessary for optimum development of rabbit embryos.     

In vitro neurotransmitter release in an animal model of depression.

Sprague-Dawley rats exposed to uncontrollable shock can be separated by a subsequent shock escape test into two groups: a "helpless" (LH) group which demonstrates a deficit in escape behavior, and a "nonlearned helpless" (NLH) group which shows no escape deficit and acquires the escape response as readily as naive control rats (NC) do. The present studies were designed to examine the correlations between the behavioral differences and the changes of in vitro neurotransmitter release seen in these three groups of rats. The major finding concerned a significant increase in endogenous and K(+)-stimulated serotonin (5-HT) release in the hippocampal slices of LH rats. There were no apparent differences in acetylcholine, dopamine and noradrenaline release in the hippocampus of LH rats as compared to NLH and NC rats. These results add further support to previous studies in our laboratory which implicate presynaptic 5-HT mechanisms in the behavioral deficit caused by uncontrollable shock.     

In vitro characterization of anti-glucosylceramide rabbit antisera

Glucosylceramides (GlcCer) are biosynthetic precursors of glycosphingolipids. They are widely distributed in biological systems where they exhibit numerous biological functions. Studies on the localization of glucosylceramides in different tissues have used biochemical methods only since specific antibodies against GlcCer were not previously available. We have characterized two commercially available rabbit antisera which were prepared against GlcCer of plant origin (1-O-(beta-D-glucopyranosyl)-N-acyl-4-hydroxysphinganine; GlcCer-3) or human origin (1-O-(beta-D-glucopyranosyl)-N-acyl-sphingosine; GlcCer-2) and claimed to be specific for GlcCer. The antisera were also able to detect specifically GlcCer species in crude lipid extracts from human epidermis after separation by thin-layer chromatography. The reagents are sensitive since both antisera reacted at dilutions higher than 1:500 with their homologous antigen in the nanogram range in thin layer immunostaining or dot-blot assays. The antisera are specific for GlcCer although they did not differentiate between GlcCer-2 and GlcCer-3 containing sphingosine or 4-hydroxysphinganine. The antisera also reacted with N-stearoyl-DL-dihydroglucocere-broside indicating that the naturally occurring structural variations in the amino alcohol moiety are not determining the specificity. No crossreactivity was observed with other mono- or diglycosylceramides (galactosylceramides, lactosyl-ceramide), free ceramides or structurally unrelated lipids (cholesterol, sphingomyelin, or phospholipids). Therefore, the glycosylmoiety seems to represent the major antigenic determinant. Finally, the antisera also proved to be useful for the immunohistochemical localization of GlcCer in human epidermis by which earlier biochemical data on the distribution of GlcCer in the various epidermal layers were confirmed.     

In vitro evaluation of sperm quality

This paper highlights selected laboratory analyses that are currently used to evaluate sperm, and describes why results from these assays do not consistently correlate with sperm fertility. Reasons for the disconnect between the two are due in part to the definition and reliability of the fertility data collected, to the complexity of the spermatozoon itself, to imprecision of some measurements, and to uncontrollable factors not associated to either the laboratory analysis or the sperm sample. Each sperm must possess a number of different attributes to fertilize an oocyte, and individual laboratory assays measure only one or a few of these attributes. Current and past data, correlating laboratory assay data with sperm fertility are presented in an effort to determine which types of assays are important to conduct and when to conduct them. Even though laboratory assay results do not allow accurate evaluation of the fertilizing potential of a semen sample, these assays are important to enable culling of poor quality samples.     

In vitro and in vivo evaluation of intravesical docetaxel loaded hydrophobically derivatized hyperbranched polyglycerols in an orthotopic model of bladder cancer

The objective of this study was to evaluate the tolerability, to establish a dosing regimen, and to evaluate the efficacy of intravesical docetaxel (DTX) formulations in a mouse model of bladder cancer. DTX in commercial formulation (Taxotere, DTX in Tween 80) or loaded in hyperbranched polyglycerols (HPGs) was evaluated. The synthesis and characterization of HPGs with hydrophobic cores and derivatized with methoxy poly(ethylene glycol) in the shell and further functionalized with amine groups (HPG-C(8/10)-MePEG and HPG-C(8/10)-MePEG-NH(2)) is described. Intravesical DTX in either commercial or HPGs formulations (up to 1.0 mg/mL) was instilled in mice with orthotopic bladder cancer xenografts and was well tolerated with no apparent signs of local or systemic toxicities. Furthermore, a single dose of intravesical DTX (0.5 mg/mL) loaded in HPGs was significantly more effective in reducing the tumor growth in an orthotopic model of bladder cancer than the commercial formulation of Taxotere. In addition, DTX-loaded HPG-C(8/10)-MePEG-NH(2) was found to be more effective at lower instillation dose than DTX (0.2 mg/mL)-loaded HPG-C(8/10)-MePEG. Overall, our data show promising antitumor efficacy and safety in a recently validated orthotopic model of bladder cancer. Further research is warranted to evaluate its safety and efficacy in early phase clinical trials in patients refractory to standard intravesical therapy.     

In vitro model systems for evaluation of smooth muscle cell response to cryoplasty

Restenosis is a major health care problem, with approximately 40% of angioplasties resulting in restenosis. Mechanisms related to elastic recoil, cell proliferation, and extracellular matrix (ECM) synthesis are implicated. In vivo studies have demonstrated the potential for cryotherapy to combat the process of restenosis, but the mechanisms whereby freezing and/or cooling can reduce or eliminate smooth muscle cell (SMC) proliferation and ECM synthesis are not well known. While in vivo testing is ultimately necessary, in vitro models can provide important information on thermal parameters and mechanisms of injury. However, it is important to carefully choose the model system for in vitro work on cryoinjury characterization to adequately reflect the clinical situation. In this study, we examined the differences in response to cryoinjury by SMCs from different species (rat, pig, and human) and in different cellular environments (suspension vs. tissue equivalent). Tissue equivalents, composed of cells embedded in collagen or fibrin gel, provide a 3-D tissue-like environment, while allowing for controlled composition. As reported here, all SMCs showed similar trends, but rat cells appeared less sensitive to cooling at faster cooling rates in suspension, while human SMCs were less sensitive to temperatures just above freezing when embedded in collagen. In addition, the SMCs were less sensitive in suspension than they were in collagen. Cells in suspension exhibited 70% viability at -11 degrees C, whereas cells in the tissue equivalent model showed only 30% survival. Future studies will aim to more adequately represent the conditions in restenosis by providing inflammatory and proliferative cues to the cells.     

Structure of pulsatile flow in a model of elastic cerebral aneurysm

This study examines the effect of aneurysmal wall elasticity on the structure of flow within an elastic aneurysm during pulsatile flow. We visualized flow structure in a model of an elastic saccular aneurysm located at the bifurcation of the anterior cerebral artery and extending to the anterior communicating artery, and measured changes in the diameter of the aneurysm wall during pulsatile flow using particle imaging velocimetry (PIV). We similarly measured these features during steady flow by PIV and found that dilation of the aneurysmal wall absorbed the dynamic energy within the aneurysm. Accordingly, aneurysm wall elasticity functions as a biocompatible reaction that relieves wall shear stress acting on the vascular wall during pulsatile flow, and should thus inhibit the development and rupture of an aneurysm.     

In vitro evaluation of an RGD-functionalized chitosan derivative for enhanced cell adhesion

Tissue repair is a spontaneous process that is initiated on wounding. However, if this complex mechanism is impaired or not sufficient the use of biomaterials might increase the chance of successful healing. In this view, an RGD-functionalized polymer was developed to promote dermal healing. A water-soluble chitosan derivative, carboxymethyl-trimethylchitosan (CM-TM-chitosan) was synthesized and GRGDS-moieties were grafted to the backbone at a concentration of 59nmol/mg polymer to increase cell-biomaterial interaction. Tested in vitro with cultured human dermal fibroblasts, the developed polymer showed good biocompatibility and the initial adhesion was increased by 3-5 times due to the GRGDS-moieties. Moreover, cell spreading was specific to the interaction with GRGDS, giving a 12-fold increase of cells showing a fully spread morphology within 30min. Overall, CM-TM-chitosan conjugated with GRGDS-peptides may prove useful as a biomaterial in wound healing.     

In vitro evaluation of nanoparticles spleen capture

After intravenous injection, the main part of nanoparticles trapped by the spleen are concentrated in the marginal zone. The first step of this capture is the adhesion of the particles to the marginal zone macrophages. As classical techniques of cell suspension preparation did not allow to isolate without damage these actively capturing cells, tightly bound to a well-developed reticular meshwork, we designed a tissue slice incubation method, in order to study in vitro the interaction of nanoparticles with these particular macrophages, in conditions close to in vivo. In a serum supplemented medium, this in vitro model was able to give similar uptake profile than after intravenous injection of nanoparticles thus proving its validity. Surprisingly, no significant decrease of nanoparticles capture was observed when the medium was depleted from complement, immunoglobulins or proteins affine for heparin, while substitution of serum by purified albumin allowed a near optimal uptake. Addition of competitive ligands for lectin-like receptors did not show any clear inhibition of spleen capture. On the other hand, the scavenger receptor blocking agents, such as maleylated albumin or polyinosinic acid, induced a strong reduction of the spleen nanoparticles uptake. Thus, this paper proposes an in vitro binding assay as a reliable method to investigate the spleen capture of a large variety of nanoparticulate drug carriers. It is also a useful methodology to highlight the interactions between spleen cells and nanoparticles. The data obtained suggest that capture of nanoparticles depends on a multifactorial and complex phenomenon involving for a part albumin and the scavenger receptor.