In vitro and in vivo DFO-chelatable labile iron release profiles among commercially available intravenous iron nanoparticle formulations.

Intravenous (IV) iron formulations are complex colloidal suspensions of iron oxide nanoparticles. Small changes in formulation can allow more labile iron to be released after injection causing toxicity. Thus, bioequivalence (BE) evaluation of generic IV iron formulations remains challenging. We evaluated labile iron release in vitro and in vivo using a high performance liquid chromatography chelatable iron assay to develop a relational model to support BE. In vitro labile iron release and in vivo labile iron pharmacokinetics were evaluated for Venofer®, Ferrlecit®, generic sodium ferric gluconate complex, InFeD®, Feraheme® and a pre-clinical formulation GE121333. Labile iron release profiles were studied in vitro in 150 mM saline and a biorelevant matrix (rat serum) at 0.952 mgFe/mL. In vivo plasma labile iron concentration-time profiles (t0-240 min) were studied in rats after a 40 mgFe/kg IV dose. In vitro labile iron release in saline was significantly higher compared to rat serum, especially with InFeD®. An in vitro release constant (iKr) was calculated which correlated well with maximal plasma concentrations in the in vivo rat PK model (R2 = 0.711). These data suggest an in vitro to in vivo correlation model of labile iron release kinetics could be applied to BE. Other generic IV iron formulations need to be studied to validate this model.

Improved Intraoperative Visualization of Nerves through a Myelin-Binding Fluorophore and Dual-Mode Laparoscopic Imaging.

The ability to visualize and spare nerves during surgery is critical for avoiding chronic morbidity, pain, and loss of function. Visualization of such critical anatomic structures is even more challenging during minimal access procedures because the small incisions limit visibility. In this study, we focus on improving imaging of nerves through the use of a new small molecule fluorophore, GE3126, used in conjunction with our dual-mode (color and fluorescence) laparoscopic imaging instrument. GE3126 has higher aqueous solubility, improved pharmacokinetics, and reduced non-specific adipose tissue fluorescence compared to previous myelin-binding fluorophores. Dosing and kinetics were initially optimized in mice. A non-clinical modified Irwin study in rats, performed to assess the potential of GE3126 to induce nervous system injuries, showed the absence of major adverse reactions. Real-time intraoperative imaging was performed in a porcine model. Compared to white light imaging, nerve visibility was enhanced under fluorescence guidance, especially for small diameter nerves obscured by fascia, blood vessels, or adipose tissue. In the porcine model, nerve visualization was observed rapidly, within 5 to 10 minutes post-intravenous injection and the nerve fluorescence signal was maintained for up to 80 minutes. The use of GE3126, coupled with practical implementation of an imaging instrument may be an important step forward in preventing nerve damage in the operating room.

Identification of the protein target of myelin-binding ligands by immunohistochemistry and biochemical analyses.

The ability to visualize myelin is important in the diagnosis of demyelinating disorders and the detection of myelin-containing nerves during surgery. The development of myelin-selective imaging agents requires that a defined target for these agents be identified and that a robust assay against the target be developed to allow for assessment of structure-activity relationships. We describe an immunohistochemical analysis and a fluorescence polarization binding assay using purified myelin basic protein (MBP) that provides quantitative evidence that MBP is the molecular binding partner of previously described myelin-selective fluorescent dyes such as BMB, GE3082, and GE3111.

Dual-mode laparoscopic fluorescence image-guided surgery using a single camera.

Iatrogenic nerve damage is a leading cause of morbidity associated with many common surgical procedures. Complications arising from these injuries may result in loss of function and/or sensation, muscle atrophy, and chronic neuropathy. Fluorescence image-guided surgery offers a potential solution for avoiding intraoperative nerve damage by highlighting nerves that are otherwise difficult to visualize. In this work we present the development of a single camera, dual-mode laparoscope that provides near simultaneous display of white-light and fluorescence images of nerves. The capability of the instrumentation is demonstrated through imaging several types of in situ rat nerves via a nerve specific contrast agent. Full color white light and high brightness fluorescence images and video of nerves as small as 100 µm in diameter are presented.

Intraoperative fluorescence imaging of peripheral and central nerves through a myelin-selective contrast agent.

PURPOSE: Patients suffer from complications as a result of unintentional nerve damage during surgery. We focus on improving intraoperative visualization of nerves through the use of a targeted fluorophore and optical imaging instrumentation. PROCEDURE: A myelin-targeting fluorophore, GE3111, was synthesized, characterized for its optical and myelin-binding properties using purified myelin basic protein, and evaluated in mice. Additionally, a compact instrument was adapted to visualize nerves. RESULTS: GE3111 was synthesized using a versatile methodology. Its optical properties were sensitive to the local environment both in vitro and in vivo. Following intravenous injection, central and peripheral nerves were visualized, with the kinetics of nerve uptake modifiable depending on the formulation. Fluorescence polarization showed specific and strong binding to purified myelin basic protein. Nerves were visualized in vivo using a dedicated compact imaging device requiring less than 2.5 mW/cm(2) of illumination at 405 nm. CONCLUSIONS: Fluorescence imaging of nerves through myelin showed a potential for use in image-guided surgery. Intraoperative nerve imaging is an example where contrast agent and instrument development come together as a result of clinical need.

Moderate Recurrent Hypoglycemia Markedly Impairs Set-Shifting Ability in a Rodent Model: Cognitive and Neurochemical Effects.

Recurrent hypoglycemia (RH) is the major complication of intensive insulin treatment for diabetes mellitus. Of particular concern is the perceived potential for long-term impact of RH on cognition. Because diabetic patients have been reported to have deficits in mental flexibility and judgment, both generally considered to be mediated predominantly by the prefrontal cortex, the purpose of the present study was to determine whether RH would affect prefrontal cortex function. Medial prefrontal cortex (mPFC)-mediated set-shifting ability was tested in male Sprague-Dawley rats using a maze-based, food-reward Set-Shift task analogous to the Wisconsin card-sorting task. The performance measure was the number of trials to criterion on both day 1 (initial rule-learning) and day 2 (set-shifting in response to a changed contingency). In vivo microdialysis was used to measure mPFC extracellular glucose, lactate, pyruvate, glutamate, and dopamine. Post-mortem measures within the mPFC included glucose transporter 3 (GluT3) and c-Fos. RH animals had enhanced performance on day 1, consistent with previous work that showed RH to improve subsequent hippocampal function when euglycemic. The key finding of the present work is that RH led to impaired set-shifting performance on day 2, suggesting impairment in e.g. mental flexibility. Consistent with this finding, RH animals show decreased mPFC glycolysis on day 2 compared to controls. Our data show that RH can lead to subsequent impaired judgment, accompanied by reduced prefrontal cortex function. The findings suggest a potential underlying mechanism for the impaired judgment seen in diabetic patients.

Mini-review: impact of recurrent hypoglycemia on cognitive and brain function.

Recurrent hypoglycemia (RH), the most common side-effect of intensive insulin therapy for diabetes, is well established to diminish counter-regulatory responses to further hypoglycemia. However, despite significant patient concern, the impact of RH on cognitive and neural function remains controversial. Here we review the data from both human studies and recent animal studies regarding the impact of RH on cognitive, metabolic, and neural processes. Overall, RH appears to cause brain adaptations which may enhance cognitive performance and fuel supply when euglycemic but which pose significant threats during future hypoglycemic episodes.

Insulin blunts the response of glucose-excited neurons in the ventrolateral-ventromedial hypothalamic nucleus to decreased glucose.

Insulin signaling is dysfunctional in obesity and diabetes. Moreover, central glucose-sensing mechanisms are impaired in these diseases. This is associated with abnormalities in hypothalamic glucose-sensing neurons. Glucose-sensing neurons reside in key areas of the brain involved in glucose and energy homeostasis, such as the ventromedial hypothalamus (VMH). Our results indicate that insulin opens the K(ATP) channel on VMH GE neurons in 5, 2.5, and 0.1 mM glucose. Furthermore, insulin reduced the sensitivity of VMH GE neurons to a decrease in extracellular glucose level from 2.5 to 0.1 mM. This change in the glucose sensitivity in the presence of insulin was reversed by the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin (10 nM) but not by the mitogen-activated kinase (MAPK) inhibitor PD-98059 (PD; 50 microM). Finally, neither the AMPK inhibitor compound C nor the AMPK activator AICAR altered the activity of VMH GE neurons. These data suggest that insulin attenuates the ability of VMH GE neurons to sense decreased glucose via the PI3K signaling pathway. Furthermore, these data are consistent with the role of insulin as a satiety factor. That is, in the presence of insulin, glucose levels must decline further before GE neurons respond. Thus, the set point for detection of glucose deficit and initiation of compensatory mechanisms would be lowered.