Florian C. Roessler

The platelet-rich plasma clot: a standardized in-vitro clot formation protocol for investigations of sonothrombolysis under physiological flows.

No agreement exists about which protocol for in-vitro clot formation is suitable for sonothrombolysis investigations. Lysis rates vary considerably because of different clotting processes and cannot be compared. We aim to establish a new protocol for in-vitro coagulation to permit standardized sonothrombolysis investigations. The proposed procedure is based upon clots prepared from platelet-rich plasma (PRP). This clot material (group A) was compared with the two most commonly used procedures, namely, recalcification of citrate-anticoagulated whole venous blood (group B) and spontaneous clotting of nonanticoagulated venous blood (group C). Histological examinations were performed and clot stability was tested under physiological flow conditions in vitro for all groups (each n = 10). Lysis rates measured by mass loss were compared using buffered plasma and recombinant tissue plasminogen activator (60 kU/ml), or buffered plasma alone. PRP clots displayed a high degree of similarity to emboli specimens in histological examinations and remained stable under pulsatile flow conditions. B and C clots were mechanically unstable and did not resist physiological flow and pressure. Measuring the lysis rate by weighing seems to be inaccurate, with lowest variability in PRP clots. PRP clots appeared more resistant to lysis. PRP clots should be used for standardized sonothrombolysis investigations.

Development of a new clot formation protocol for standardized in vitro investigations of sonothrombolysis.

BACKGROUND Agreement about the most suitable clot formation protocol for sonothrombolysis investigations is lacking. Lysis rates vary strongly owing to different test conditions and, thus, cannot be compared. We aim to establish a simple but physiologically grounded protocol for in vitro coagulation to enable standardized sonothrombolysis investigations. METHOD Clots were generated from platelet-rich plasma (PRP) obtained by centrifugation (10 min, 180 × g) of human venous blood (VB). PRP was mixed with the boundary layer formed between the supernatant and the erythrocyte layer. To achieve clots with different platelet counts, PRP was gradually substituted with platelet-free plasma (PFP), harvested from the supernatant of VB after centrifugation (10 min, 2570 × g). Clot types were examined for histological appearance, hydrodynamic resistance under physiological flows, and lysis rate measured by weight loss after a 2-h treatment with recombinant tissue plasminogen activator (rt-PA) (60 kU/ml). Lysis rates of the most suitable clot were measured after a 1-h treatment with rt-PA (60 kU/ml), and combined treatment with rt-PA and 2-MHz transcranial color-coded sonography (TCCS) (0.179 W/cm(2)) or 2-MHz transcranial Doppler (TCD) (0.457 W/cm(2)). RESULTS With increased platelet count, the hydrodynamic resistance of the artificial clots increased, their histological appearance became more physiological, and lysis rates decreased. The most suitable clots consisted of 1.5-ml PRP, 2.0-ml PFP, and 0.5-ml boundary layer. Their lysis rates were 36.7 ± 7.8% (rt-PA), 40.8 ± 8.6% (rt-PA+TCCS), and 40.4 ± 8.3% (rt-PA+TCD). COMPARISON WITH EXISTING METHODS These systemic investigations were conducted for the first time. CONCLUSION This protocol should be used for standardized sonothrombolysis investigations.

Introduction of a new model for time-continuous and non-contact investigations of in-vitro thrombolysis under physiological flow conditions

BackgroundThrombolysis is a dynamic and time-dependent process influenced by the haemodynamic conditions. Currently there is no model that allows for time-continuous, non-contact measurements under physiological flow conditions. The aim of this work was to introduce such a model.MethodsThe model is based on a computer-controlled pump providing variable constant or pulsatile flows in a tube system filled with blood substitute. Clots can be fixed in a custom-built clot carrier within the tube system. The pressure decline at the clot carrier is measured as a novel way to measure lysis of the clot. With different experiments the hydrodynamic properties and reliability of the model were analyzed. Finally, the lysis rate of clots generated from human platelet rich plasma (PRP) was measured during a one hour combined application of diagnostic ultrasound (2 MHz, 0.179 W/cm2) and a thrombolytic agent (rt-PA) as it is commonly used for clinical sonothrombolysis treatments.ResultsAll hydrodynamic parameters can be adjusted and measured with high accuracy. First experiments with sonothrombolysis demonstrated the feasibility of the model despite low lysis rates.ConclusionsThe model allows to adjust accurately all hydrodynamic parameters affecting thrombolysis under physiological flow conditions and for non-contact, time-continuous measurements. Low lysis rates of first sonothrombolysis experiments are primarily attributable to the high stability of the used PRP-clots.

Nigrosome 1 visibility at susceptibility weighted 7T MRI—A dependable diagnostic marker for Parkinson's disease or merely an inconsistent, age-dependent imaging finding?

Background Visualisation of nigrosome 1, a substructure of the healthy substantia nigra, was restricted in susceptibility weighted MR imaging in almost all patients with Parkinsons disease studied so far. The purpose of this study was to determine the degree of visibility of this substructure in subjects without Parkinson’s disease and to examine the potential link between increasing brain iron accumulation with age and its detectability. Methods In 46 subjects (21 women, 25 men; 19 to 75 y; mean age: 44.5; SD: 15.6) examined with susceptibility weighted MR imaging at 7T visibility of nigrosome 1 was rated and classified. We assessed differences related to age and to signal intensities in the substantia nigra, red nucleus and putamen as correlates of the individual iron concentration. Results In 93% nigrosome 1was at least unilaterally clearly present. In 24% at least one-sided limited visibility was observed. Using predefined classification criteria the specificity of the visibility across all age groups reached approximately 94%. We found no correlation with increasing iron concentrations with age. Conclusion Aging with a related increase in iron concentration probably does not affect the visibility of nigrosome 1 at 7T SWI MRI. Our results support the role of this feature as a future differential diagnostic tool but further large-scale prospective studies are needed to better define the extent of a “limited visibility” to which an individual can be considered healthy.

In Vitro Examination of the Thrombolytic Efficacy of Desmoteplase and Therapeutic Ultrasound Compared with rt-PA

The aim of the study described here was to evaluate the thrombolytic efficacy of combined treatment with the fibrin-selective plasminogen activator desmoteplase (DSPA) and therapeutic ultrasound (sonothrombolysis [STL]) compared with conventional rt-PA (recombinant tissue plasminogen activator) treatment in vitro. Lysis rates were determined by the weight loss of platelet-rich plasma (PRP) clots treated with rt-PA (60 kU/mL) or DSPA (2 μg/mL) combined with pulsed wave ultrasound (2 MHz, 0.179 W/cm(2)). To reveal the individual effects of medication and ultrasound, lysis rates were also determined for DSPA monotherapy and for combined treatment with rt-PA and ultrasound. Clots solely placed in plasma served as the control group. Lysis increased significantly with rt-PA (26.5 ± 7.8%) and DSPA (30.5 ± 6%) compared with the control group (18.2 ± 5.9%) (each p < 0.001). DSPA lysis was more effective than rt-PA lysis (without STL: p = 0.015, with STL: p = 0.01). Combined treatment with DSPA and 2-MHz STL significantly exceeded rt-PA lysis (32.8% vs. 26.5%, p < 0.001).

Microvascular Imaging in Acute Ischemic Stroke

Microvascular imaging (MVI), a new ultrasound technology, is used to analyze brain perfusion at the patients bedside. This study aims to evaluate the diagnostic and prognostic value of MVI in patients with acute ischemic stroke (AIS).

Acute brain infarction detected by CCT and stroke risk in patients with transient ischemic attack lasting <1 hour

Background and purpose: This study aimed to determine the frequency and associated factors of acute brain infarction (ABI) detected by noncontrast cranial computed tomography (CCT) in patients with transient ischemic attack (TIA) of symptom duration <1 h and to investigate the association between evidence of ABI and short-term risk of stroke. Methods: During a 54-month period (starting November 2007), consecutive patients with TIA (symptom duration <1 h) admitted and imaged with CCT were prospectively evaluated. Adjusted logistic regression was used to estimate odds ratios (ORs). Results: Of 1021 patients (mean age, 74.5 ± 11 years; 52% female) with TIA (symptom duration <1 h) imaged with CCT at admission, 68 patients (6.7%; 95% CI, 5.3–8.3%) exhibited TIA-related ABI. Adjusted logistic regression showed that ABI was independently correlated with atrial fibrillation (AF) (OR, 3.3; 95% CI, 1.4–7.9; p = 0.006) and time between onset and CT assessment >6 h (OR, 2.5; 95% CI, 1.1–6.1; p = 0.034). During hospitalization (5 ± 3 d), 22 patients (2.2%; 95% CI, 1.4–3.1%) developed a stroke. Patients with ABI had higher stroke rates than those without (10.3% and 1.6%, respectively; p < 0.001). Adjusted logistic regression revealed that stroke risk was independently correlated with ABI (OR, 5.3; 95% CI, 1.8–15.0; p = 0.002) and AF (OR, 2.6; 95% CI, 1.1–6.4; p = 0.026). Conclusions: Detection of ABI by CCT in TIA patients with symptom duration <1 h may depend on timing of CCT assessment and presence of AF. Evidence of ABI indicates an elevated stroke risk during hospitalization.

Speckle Tracking in Transcranial Ultrasound Allows Noninvasive Analysis of Pulsation Patterns of the Third Ventricle

Cerebrospinal fluid (CSF) flow is sensitive to many cerebral disorders. We aimed to develop a noninvasive bedside method to detect physiological and pathological CSF phenomena by measuring pulsation patterns of the third ventricle. By transcranial B-mode ultrasound, electrocardiography (ECG)-gated video loops of the third ventricle were acquired. “Speckle tracking” software was used to quantify the relative change of its width. We conducted measurements of nine cardiac cycles in 11 healthy subjects in sitting and in supine position during Valsalva maneuver to investigate the influence of an increased intracranial pressure on the relative deformation of the third ventricle. In one patient with occlusive hydrocephalus, 19 cardiac cycles were measured in sitting position before and after removal of a tumorous obstruction of the aqueduct of Sylvius. Healthy subjects expressed a pulse-related increased width of the third ventricle ( max ¯ : +5.69, 95% confidence interval [CI] = [4.38, 7.00]). No significant difference was found between the sitting and the supine position in healthy adults. In the preoperative state of occlusive hydrocephalus, we found a negative, pulse-related deformation ( min ¯ : −1.86, 95% CI = [−2.15, −1.58]) with delayed onset. After surgery, the deformation pattern resembled that of our healthy controls. The difference between pre- and postoperative condition was significant (p < 0.001). Transcranial B-mode sonography can be used to record small movements of the sidewalls of the third ventricle. This noninvasive bedside method is suitable to assess CSF pulsatility within the third ventricle and might be able to distinguish between physiological and pathological flows.

Comparing outcome and recanalization results in patients with anterior circulation stroke following endovascular treatment with and without a treatment with rt-PA: A single-center study

Endovascular treatment (ET), in addition to a therapy with intravenous recombinant tissue plasminogen activator IV rt‐PA in patients with acute ischemic stroke, has been found to improve outcome. However, data about ET in patients who have not received therapy with rt‐PA due to contraindications for IV rt‐PA are sparse. Comparison of ET with IV rt‐PA versus ET alone in patients with stroke is done using a proximal intracranial arterial occlusion (internal carotid artery, middle cerebral artery (M1‐Segment)).

Clot Formation in the Presence of Acetylsalicylic Acid Leads to Increased Lysis Rates Regardless of the Chosen Thrombolysis Strategy.

Background: Patients with acute ischemic strokes frequently take an acetylsalicylic acid (ASA) premedication. We determined the impact of ASA on different thrombolysis strategies in vitro. Methods: For two clot types made from platelet-rich plasma (one with and one without ASA) lysis rates were measured by weight loss after 1 h for five different groups: in control group A clots were solely placed in plasma; in groups B and C clots were treated with rt-PA (60 kU/ml), and in groups D and E clots were treated with desmoteplase (DSPA; 2 µg/ml). Ultrasound (2 MHz, 0.179 W/cm2) was included in groups C and E. The fibrin mesh structures of the clots were investigated by electron microscopy. Results: For both clot types lysis rates increased significantly for all treatment strategies compared to their control group (each p < 0.001). The addition of ASA significantly increased the lysis rate in all 5 groups (each p < 0.001) and led to a ceiling effect concerning the treatment. A semiquantitative analysis of transmission electron micrographs revealed a decreased fibrin density for clots with ASA. For both clot types DSPA and ultrasound led to a significant dissolution of the fibrin mesh (both p = 0.029). Conclusions: In vitro ASA pretreatment leads to significantly increased lysis rates due to a weaker fibrin mesh in platelet-rich plasma clots.