Real-Time Assessment of Ultrasound-Mediated Drug Delivery Using Fibered Confocal Fluorescence Microscopy

Purpose

Transport across the plasma membrane is a critical step of drug delivery for weakly permeable compounds with intracellular mode of action. The purpose of this study is to demonstrate real-time monitoring of ultrasound (US)-mediated cell-impermeable model drug uptake with fibered confocal fluorescence microscopy (FCFM).

Procedures

An in vitro setup was designed to combine a mono-element US transducer, a cell chamber with a monolayer of tumor cells together with SonoVue microbubbles, and a FCFM system. The cell-impermeable intercalating dye, SYTOX Green, was used to monitor US-mediated uptake.

Results

The majority of the cell population showed fluorescence signal enhancement 10 s after US onset. The mean rate constant k of signal enhancement was calculated to be 0.23?±?0.04 min^?1.

Conclusions

Feasibility of real-time monitoring of US-mediated intracellular delivery by FCFM has been demonstrated. The method allowed quantitative assessment of model drug uptake, holding great promise for further local drug delivery studies.     

Transforming growth factor β as a neuronoglial signal during peripheral nervous system response to injury

In contrast to the central nervous system (CNS), the peripheral nervous system (PNS) displays an important regenerative ability which is dependent, at least in part, on Schwann cell properties. The mechanisms which stimulate Schwann cells to adapt their behavior after a lesion to generate adequate conditions for PNS regeneration remain unknown. In this work, we report that adult rat dorsal root ganglion (DRG) neurons are able, after a lesion performed in vivo or when they are dissociated and cultured in vitro, to synthesize transforming growth factor β (TGFβ), a pleiotropic growth factor implicated in wound healing processes and in carcinogenesis. This TGFβ is tentatively identified as the β-1 isoform. Adult rat DRG neurons release a biologically active form of TGFβ which is able to elicit multiple Schwann cell responses including a stimulation to proliferate. Moreover, purified TGFβ-1 produces a Schwann cell morphology alteration and decreases the secretion of tissue-type plasminogen activator (tPA) and enhances the secretion of plasminogen activator inhibitor (PAI) by Schwann cells. This generates conditions which are thought to favor a successful neuritic regrowth. Furthermore, purified TGFβ-1 stimulates type IV collagen mRNA expression in Schwann cells. This subtype of collagen is associated with the process of myelinization. Finally, TGFβ-1 decreases nerve growth factor (NGF) mRNA expression by Schwann cells, an effect which could participate in the maintenance of a distoproximal NGF gradient during nerve regeneration. We propose that neuronal TGFβ plays an essential role as a neuronoglial signal that modulates the response of Schwann cells to injury and participates in the successful regeneration processes observed in the PNS. © 1993 Wiley-Liss, Inc.     

Double-quantum surface-coil NMR studies of sodium and potassium in the rat brain

Double-quantum filtered and conventional single-pulse sodium and potassium NMR spectra were obtained from in situ rat brain at 7.0 T. using a surface coil. In contrast to the ca. 14% decrease observed in single-pulse sodium NMR spectra upon death, increases as large as ca. 800% were observed in double-quantum filtered sodium spectra. Conversely, a ca. 26% increase was observed in single-pulse potassium spectra upon death, while double-quantum filtered potassium spectra decreased below the noise level, for the shortest preparation time used. The decay rate of double-quantum sodium coherence in the in situ rat brain after death was dependent upon the double-quantum preparation time: this behavior results from the nonuniformity of the brain, and may be related to physiological compartmentalization. The potential application to sodium NMR imaging of cerebral functionality is briefly discussed.     

Invited. On the feasibility of MRI-guided focused ultrasound for local induction of gene expression

Gene therapy is a promising approach to the treatment of many forms of disease, including cancer. Of critical concern in its implementation is the ability to control the location, duration, and level of expression of the therapeutic gene. Here, we propose the use of local heat in combination with a heat-sensitive promoter to help accomplish this. Certain members of the family of heat shock protein (hsp) promoters display a regulation that depends strongly on temperature. We present a study of natural hsp70 induction in rat leg by MRI-guided focused ultrasound to investigate the hsp70 promoter as a possible candidate for use in control of gene expression with local heat. A temperature increase of 5–8°C in the focal region for 45 minutes led to a differential expression of the hsp70 mRNA between the focal region and the surrounding tissue ranging from a factor of 3 to 67.     

Purification and culture of adult rat dorsal root ganglia neurons

To study the trophic requirements of adult rat dorsal root ganglia neurons (DRG) in vitro, we developed a purification procedure that yields highly enriched neuronal cultures. Forty to fifty ganglia are dissected from the spinal column of an adult rat. After enzymatic and mechanical dissociation of the ganglia, myelin debris are eliminated by centrifugation on a Percoll gradient. The resulting cell suspension is layered onto a nylon mesh with a pore size of 10 microns. Most of the neurons, the diameter of which ranged from 17 microns to greater than 100 microns, are retained on the upper surface of the sieve; most of the non-neuronal cells with a caliber of less than 10 microns after trypsinization go through it. Recovery of neurons is achieved by reversing the mesh onto a Petri dish containing culture medium. Neurons to non-neurons ratio is 1 to 10 in the initial cell suspension and 1 to 1 after separation. When these purified neurons are seeded at a density of 3,000 neurons/cm2 in 6 mm polyornithine-laminin (PORN-LAM) coated wells, neuronal survival (assessed by the ability to extend neurites), measured after 48 hr of culture, is very low (from 0 to 16%). Addition of nerve growth factor (NGF) does not improve neuronal survival. However, when neurons are cultured in the presence of medium conditioned (CM) by astrocytes or Schwann cells, 60-80% of the seeded, dye-excluding neurons survive. So, purified adult DRG neurons require for their short-term survival and regeneration in culture, a trophic support that is present in conditioned medium from PNS or CNS glia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multislice proton magnetic resonance spectroscopic imaging in X-linked adrenoleukodystrophy

Multislice proton magnetic resonance spectroscopic imaging permits metabolic analysis of brain tissue in vivo by data acquisition in four oblique axial slices, each 15-mm thick and divided into 0.8-ml single-volume elements. We applied this technique to the systematic study of 25 patients with adrenoleukodystrophy: 3 with the severe childhood or adult cerebral form of the disease, 5 with adrenomyeloneuropathy, 12 with no demonstrable neurological involvement, and 5 women heterozygous for adrenoleukodystrophy who had some degree of neurological disability. Abnormalities on magnetic resonance spectroscopic imaging included a reduction in N-acetyl aspartate, an increase in cholin-containing compounds, and at times, an increase in lactate. Five patients showed abnormalities in the presence of normal-appearing magnetic resonance images, and in 8 other patients the alterations on spectroscopic images were more severe than those demonstrable by magnetic resonance imaging. Correlation with clinical course suggests that an increase in the choline-containing compounds is associated with an active demyelinative process, whereas such compounds are not elevated in lesions that are stable. We conclude that magnetic resonance spectroscopic imaging is a more sensitive indicator of early neurological involvement thatn is magnetic resonance imaging, and that the character of abnormalities detected by the former technique may serve as a gauge of the degree of activity of the demyelinating process and as a guide to the selection and evaluation of therapeutic approaches.     

Study of the interaction of antiplasmodial strychnine derivatives with the glycine receptor

Strychnos icaja Baill. (Loganiaceae) is a liana found in Central Africa known to be an arrow and ordeal poison but also used by traditional medicine to treat malaria. Recently, many dimeric or trimeric indolomonoterpenic alkaloids with antiplasmodial properties have been isolated from its rootbark. Since these alkaloids are derivatives of strychnine, it was important, in view of their potential use as antimalarial drugs, to assess their possible convulsant strychnine-like properties. In that regard, their interaction with the strychnine-sensitive glycine receptor was investigated by whole-cell patch-clamp recordings on glycine-gated currents in mouse spinal cord neurons in culture and by [(3)H]strychnine competition assays on membranes from adult rat spinal cord. These experiments were carried out on sungucine (leading compound of the chemical class) and on the antiplasmodial strychnogucine B (dimeric) and strychnohexamine (trimeric). In comparison with strychnine, all compounds interact with a very poor efficacy and only at concentrations >1 microM with both [(3)H]strychnine binding and glycine-gated currents. Furthermore, the effects of strychnine and protostrychnine, a monomeric alkaloid (without antiplasmodial activity) also isolated from S. icaja and differing from strychnine only by a cycle opening, were compared in the same way. The weak interaction of protostrychnine confirms the importance of the G cycle ring structure in strychnine for its binding to the glycine receptor and its antagonist properties.     

Quantitative cytology on bladder wash versus voided urine: a comparison of results

OBJECTIVES: Quantitative cytology (Quanticyt) is a valuable marker for the identification of high-risk superficial bladder cancer (SBC) patients and can be used to individualize surveillance of patients. A disadvantage is the necessity to perform an invasive procedure to obtain the required bladder wash sample. This study investigated whether quantitative cytology can be performed on voided urine with reliable results, consistent with the quantitative cytology performed on bladder wash samples. METHODS: Between June 2003 and May 2005, 288 voided urine samples in combination with bladder wash samples were obtained from patients with SBC who visited our urologic outpatient department. Quantitative cytology was performed on all samples. Corresponding clinical pathologic features and washed cytopathology results were collected. Linear regression analyses were performed for comparison of results from both types of samples. RESULTS: Ninety-one percent of the samples fell into the low or intermediate region on bladder wash. A clear deviation in the nuclear shape (MPASS) was seen in the voided urine samples, which led to more low-risk results. The clinical characteristics show that this shift is not the result of under-staging. The nuclear content (2c deviation index [DI]) did not change by performing the analysis on urine. CONCLUSION: When urine is correctly processed after voiding, quantitative cytology can be done on these samples. Voided urine-based quantitative cytology can be implemented in daily practice.