Ultrasound-Enhanced Transcorneal Drug Delivery for Treatment of Fungal Keratitis.

PURPOSE: Transcorneal drug delivery is hindered by ocular physical and biochemical properties, such as tear production, the epithelial layer of the cornea, and blinking. The aim of this study was to determine whether ultrasound can be applied to increase the transcorneal drug delivery of natamycin used in the treatment of fungal keratitis without dangerously overheating the surrounding ocular tissues. METHODS: To verify the safety of various sets of ultrasound parameters, modeling studies were conducted using OnScale, an ultrasonic wave modeling software. Ultrasound parameters determined optimal for ocular tissue safety were used in a laboratory setting in a jacketed Franz diffusion cell setup. Histological images of the cross-section of the corneas used in experiments were examined for cell damage under a microscope. RESULTS: Increases in transcorneal drug delivery were seen in every treatment parameter combination when compared with the sham treatment. The highest increase was 4.0 times for 5 minutes of pulsed ultrasound at a 25% duty cycle and a frequency of 400 kHz and an intensity of 0.5 W/cm 2 with statistical significance ( P < 0.001). Histological analysis revealed structural damage only in the corneal epithelium, with most damage being at the epithelial surface. CONCLUSIONS: This study suggests that ultrasound is a safe, effective, and minimally invasive treatment method for enhancing the transcorneal drug delivery of natamycin. Further research is needed into the long-term effects of ultrasound parameters used in this study on human ocular tissues.

Feasibility of Therapeutic Ultrasound Application in Topical Scleral Delivery of Avastin.

Purpose: Macromolecules have been shown to be effective in vision-saving treatments for various ocular diseases, such as age-related macular degeneration and diabetic retinopathy. The current delivery of macromolecules requires frequent intraocular injections and carries a risk of serious adverse effects. Methods: We tested the application of therapeutic ultrasound as a minimally invasive approach for the delivery of Avastin into the diseased regions of the eye. Avastin (bevacizumab) is an anti-vascular endothelial growth factor (VEGF) antibody with a molecular weight of 149 kDa. We tested the effectiveness and safety of Avastin delivery through rabbit sclera in vitro using a standard diffusion cell model. Ultrasound at frequencies of 400 kHz or 3 MHz with an intensity of 1 W/cm2 was applied for the first 5 minutes of 1-hour drug exposure. Sham treatments mimicked the ultrasound treatments, but ultrasound was not turned on. Absorbance measurements of the receiver compartment solution were performed at 280 nm using a spectrophotometer. Results: Absorbance measurements indicated no statistical difference between the sham (n = 13) and 400 kHz ultrasound group (n = 15) in the delivery of Avastin through the sclera. However, the absorbance values were statistically different (P < 0.01) between the 3 MHz ultrasound group (0.004, n = 8) and the matched sham group (0.002, n = 7). There was 2.3 times increase in drug delivery in the 3 MHz ultrasound when compared to the corresponding sham group. Histological studies indicated no significant damage in the ultrasound-treated sclera due to ultrasound application. Conclusions: Our preliminary results provided support that therapeutic ultrasound may be effective in the delivery of Avastin through the sclera. Translational Relevance: Our study offers clinical potential for a minimally invasive retinopathy treatment.

Rat cardiopulmonary bypass model: application of a miniature extracorporeal circuit composed of asanguinous prime.

A clinically relevant rat cardiopulmonary bypass (CPB) model would be a valuable tool for investigating pathophysiological and therapeutic strategies on bypass. Previous rat CPB models have been described in the literature; however, they have many limitations, including large circuit surface area, the inability to achieve full bypass, and donor blood requirements for prime. Therefore, we have established a rat CPB model designed to overcome these limitations. The miniature circuit consisted of a filtered reservoir, heat exchanger, membrane oxygenator (surface area = 0.02 m2) with a static priming volume of 2.8 mL, and an inline blood gas monitor. The circuit was primed with 9.5+/-0.5 mL of crystalloid solution and CPB was established on male Sprague-Dawley rats (430-475 g, n = 5) by cannulating the left common carotid artery and the right external jugular vein. The animals were placed on CPB at full flow (111+/-13 mL/kg/ min) for 1 hour and were monitored for and additional 2 hours after the CPB procedure. Hemodynamics, hemoglobin concentration (Hb), and blood gases were analyzed at three time intervals: before, during, and after CPB. The circuit performance was evaluated according to prime volume, compliance, hemodynamic parameters, and gas and heat exchange as described by modified AMMI standards. Data are expressed as mean+/-SD and a repeated-measures analysis of variance with post-Hoc test was used for data comparison between the three time intervals. The ratio of oxygenator surface area to subject body weight for this model is comparable with that of current human adult CPB practice (0.05 m2/kg vs 0.057 m2/kg) Full CPB was achieved and we observed clinically acceptable PaO2, PaCO2, and SvO2 values (209+/-86 mmHg, 25+/-2 mmHg, 78+/-8%, respectively) while on CPB. The use of asanguinous prime did produce statistically significant Hg reduction (15.7+/-0.76 vs. 9.2+/-0.59 g/dL) comparable with clinical practice. No statistically significant differences between pre- and post-CPB hemodynamics and blood gases were found in our study. We have established a miniature circuit consisting of asanquineous prime for a rat CPB model that maintains clinically acceptable results regarding hemodynamic parameters, blood gases, and hemodilution. This model would be valuable for further use in clinically relevant research studies.

The in vitro effects of aprotinin on twelve different ACT tests.

Aprotinin is frequently used during CPB to reduce post-operative bleeding and attenuate the inflammatory response. The level of anticoagulation in these patients is monitored by using various activated clotting time (ACT) tests, which are generally accepted as being altered by the presence of aprotinin in blood. Therefore, we have investigated the effect of aprotinin on several ACT tests using whole blood from CPB patients. With IRB approval, blood samples were collected from patients undergoing CPB before and after full heparinization (300 u/kg). Each blood sample was divided into two aliquots, and aprotinin was added to one of them to yield a final calculated concentration of 300 KIU/mL. Both aliquots were used simultaneously to perform the 12 ACT tests. A paired Student's t-test was performed on the data. Overall, test results from 9 of 12 devices were significantly increased by aprotinin. Of these, four were increased only when the sample was heparinized, three were elevated by both heparinized and unheparinized blood, and two were elevated only when the sample was unheparinized. Each affected test responded uniquely to aprotinin, producing ACT test results ranging from 12 to 51% above nonaprotinized values. Several tests that were affected by aprotinin using heparinized blood samples were unaffected using unheparinized blood samples. These data emphasizes the unique manner in which individual ACT tests respond to aprotinized blood samples and should be considered when developing institutional policy for anticoagulation of aprotinized patients.

Intrathecal clonidine and severe hypotension after cardiopulmonary bypass.

The use of intrathecal clonidine as an adjunct for the management of chronic pain, intra- and postoperative analgesia is gaining an increase in popularity. However, antinociceptive doses of intrathecal clonidine may produce pronounced hemodynamic side effects, including hypotension and bradycardia. In this report, we present a case of severe hypotension after cardiopulmonary bypass in a patient with intrathecal clonidine infusion. We postulate that the intrathecally administered alpha 2-agonist clonidine reduced our patient's ability to tolerate the hemodynamic lability that is present during the separation from cardiopulmonary bypass by potentially inhibiting sympathetic nervous system activity, renin-angiotensin system, or vasopressin release. The authors report a case of severe hypotension after cardiopulmonary bypass in a patient receiving intrathecal clonidine infusion for chronic neuropathic pain.

How does the age of a blood sample affect it's activated clotting time? Comparison of eight different devices.

Monitoring activated clotting time (ACT) during extracorporeal procedures is virtually universal. The ACT test is usually performed immediately following blood collection. However, certain situations may occur that delay rapid measurement. It is unknown how an aged blood sample affects the ACT measurement. It is hypothesized that the ACT will be affected as a blood sample ages. Multiple blood samples were taken from six patients undergoing cardiopulmonary bypass (CPB). Samples were divided into two groups, heparinized (H) and unheparinized (UH). ACT/HMT tests were performed with each sample on eight devices (Array Actalyke, Gem PCL, Hemochron Jr. Signature, Hemochron Response, Hemochron 801, Hemotec HMS, Rapidpoint Coag, and Sonoclot II) at three different sample ages [< 60 s (fresh blood), 10 min, and 15 min after sample collection. ACT/HMT results of aged samples (10 min and 15 min after sample collection] were compared to ACT/HMT results for fresh blood using a repeated measures analyses of variance (ANOVA) with Student's-Newman-Keuls post hoc test. In the unheparinized group, no device produced an ACT significantly different from the fresh sample counterpart at the 10 min time point. At the 15 min time point, the Hemochron 801 produced a significantly lower average ACT when compared to the fresh sample. (120 +/- 25 vs. 135 +/- 5 s). In the heparinized group, the Actalyke device produced results with 10 and 15 min aged blood that were significantly longer than fresh blood sample results (ACT < 60 s = 426 +/- 66, 10 min = 457 +/- 82, 15 min = 450 +/- 68 s, p < .05). No other device produced significant differences for either time period. Based on this limited sample population, it seems that accurate ACT may be performed on blood samples up to 15 min old in many devices.

Electronic data management for the Hemochron Jr. Signature coagulation analyzer.

Point-of-care testing (POC, POCT) laboratory devices are being introduced into operating suites and critical care units in ever increasing numbers. The small, portable devices have gained in popularity because of their ease of use and the rapid availability of test results. POCT is an integral part of extracorporeal technology (ECT). A challenge associated with the growth of POC technology is related to management of the data generated by these devices. In the field of ECT, storing, retrieving, analyzing, viewing and charting quality control (QC) and patient test data generated with POC coagulation instruments is essential. We evaluated a premarket version of data management software developed for the Hemochron Jr. Signature coagulation analyzer, a PC-based software capable of fulfilling our objective. A database comprised of greater than 50 plasma and electronic QC results and greater than 140 patient sample results for ACT, PT, and aPTT tests was transferred from a Hemochron Jr. Signature device to two different PCs, each equipped with Hemochron ReportMaker software supplied by the manufacturer. Data files were transferred directly from the coagulation test unit to the PCs via an RS-232 cable. A variety of charts, reports, and file listings were created from the datasets using the software menus. Transfer of the complete database required less than 5 min. The relative speed and simplicity of the data interface promotes frequent charting of QC data, permitting real-time monitoring and early identification of data trends or values requiring intervention. If a subset of QC data is found to be incomplete, altered, or unacceptable, all patient samples tested during that period can be promptly identified. The software also includes data query tools useful for sorting and selecting specific subsets of patient and QC data. Electronic data management can facilitate compliance with quality control requirements and assist clinicians and laboratory personnel in the collection, storage, and review of quality control and patient test data. In addition, the patient and QC data are readily accessible when necessary for use in risk management assessment, accreditation, or litigation proceedings.

Comparison of five point-of-care prothrombin and activated partial thromboplastin time devices based on age of blood sample.

Delays in processing statium (STAT) blood samples have led to the production of an increasing number of point-of-care tests. Product inserts recommend measuring blood samples immediately after procurement, suggesting that delays may invalidate the test results. We studied the effect of the age of blood samples on point-of-care (POC) prothrombin time (PT) and an activated partial thromboplastin time (aPTT) result. Informed consent was obtained from 11 patients undergoing cardiopulmonary bypass (CPB). Blood samples (40 mL) were taken from each patient. Each blood sample was used to perform five PT tests and six aPTT tests on five POC devices (Gem PCL, Hemochron 801, Hemochron Jr. Signature, Hemochron Response, Rapidpoint Coag) at three different sample ages [< 60 s (fresh blood), 10 and 18 min after sample collection]. Blood samples were procured in a plastic syringe devoid of air bubbles, which was left undisturbed between tests but was gently agitated before initiating the 10- and 18-min tests. For tests requiring citrated whole blood, a fraction of each sample was anticoagulated (3.8% citrate) at each age. Statistical analysis was used for comparison of test results for fresh blood to aged samples (10 and 18 min). Test values were recorded as International Normalized Ratio (INR) and seconds for PT and aPTT, respectively. Two devices, the Hemochron 801 and Hemochron response showed statistically, although not clinically, significant variation in PT test results when the samples were aged to 10 and 18 minutes. As for aPTT results, Hemochron 801, Hemochron response, Hemochron Jr. signature, and Gem PCL showed statistically significant variation at 18 minutes. One device (Hemochron 801) reported results with 10-min aged blood that were statistically different from fresh blood. None of the aPTT tests results from any device produced results with aged blood that were clinically different from fresh blood. This study suggests that, in the tests evaluated, blood samples that have aged 10 or 18 min will produce clinically relevant aPTT and PT results, respectively.

High-volume, zero balanced ultrafiltration improves pulmonary function in a model of post-pump syndrome.

The systemic inflammatory response syndrome (SIRS), which may develop following cardiopulmonary bypass (CPB), can cause postoperative complications that contribute to the morbidity and mortality associated with open-heart surgery. Inflammatory mediators such as cytokines, are thought to play an important role in SIRS. Zero Balance Ultrafiltration (Z-BUF) is thought to reduce the quantity of inflammatory mediators associated with CPB and may attenuate the adverse effects of bypass. Following ethics committee approval, both an unfiltered experimental group and Z-BUF treatment group consisting of Yorkshire pigs (41 +/- 19 kg) were anesthetized, ventilated, instrumented, cannulated and placed on CPB for 60 minutes. Following CPB, an infusion of low-dose endotoxin (1 microg/kg) was administered I.V. and the animals were monitored for 3.5 hours. The Z-BUF treatment group (n = 5) received high-volume Z-BUF (122 ml/kg +/- 41) and the unfiltered experimental group (n = 5) did not. Hemodynamics, blood gases, and pulmonary functions were measured before, during, and after CPB. Following euthanasia, the middle lobe of the lung was prepared for histological analysis. Necropsy of the lung sample was weighed before and after dehydration to evaluate lung water content. During the experimental time course, plasma samples were evaluated for Interleukin-8 (IL-8) concentrations. Arterial PO2's (mmHg) in the unfiltered experimental group showed a significant reduction at 3.5 hours post CPB when compared to baseline while the Z-BUF treatment group PaO2 did not significantly change. There was a significant difference in the PaO2 between the unfiltered experimental and Z-BUF group at the final 3.5 hour time point (78 +/- 32 vs. 188 +/- 92 mmHg respectively). Pulmonary compliance (ml/cmH2O) was significantly reduced in both the unfiltered experimental and Z-BUF treatment groups with the unfiltered experimental group being the most significant. Lung wet/dry ratios were established and results found the unfiltered experimental group ratio significantly greater than that of the Z-BUF treatment group. Morphometric analysis of histologic lung sections confirmed pulmonary injury in the unfiltered experimental group and protection in the Z-BUF treatment group. This study suggests that Z-BUF provides higher arterial PO2's and lung compliances while reducing pulmonary edema and lung injury in a porcine model of PPS.