Application Brief: Cellvizio LAB Fibered Fluorescence Microscope and Microdialysis Research

Executive Summary Microdialysis is an integral part of preclinical research to determine extracellular fluid and blood concentrations of metabolites, hormones, drugs, etc, and is often used in quantifying the biochemistry of brain and peripheral tissues. However, it is a molecular-only technique and other imaging modalities are needed to provide the researcher with functional and anatomical information of the animal in vivo. Because of the similarity of fibered fluorescence microscopy (FFM) to microdialysis in terms of probe size and insertion, it is the perfect complement as an imaging modality. The Cellvizio LAB system has been demonstrated to be a highly accurate way of visualizing peripheral nerves, deep brain, individual nerve regeneration, calcium dynamics etc in real-time, with minimal invasiveness, and longitudinally in the same animal. Furthermore, a stereotaxic imaging platform setup can guide the insertion of microdialysis and FFM probes to the exact location of interest. The combination of these systems provide a powerful method of studying brain and nerve dynamics in real-time, in vivo and longitudinally.

Calcium monitoring in the brain at the level of a single neuron

Nerve root from the spinal cord in a Thy1-YFP mouse after surgery to expose the nervous tissue

Application Brief: Cellvizio LAB and Microdialysis Research ver1.0

Background on Microdialysis Microdialysis is an in vivo, minimally invasive procedure used in preclinical research to measure the concentration of unbound markers in fluids of tissues and organs in animals. A thin dialysis tube is implanted in to the tissue of interest, with the semi-permeable membrane of the tube in contact with bodily fluid. The tube is perfused with a physiological liquid and molecular markers in the bodily fluid are exchanged across the membrane. This allows the perfusate to be chemically analyzed and reflects the composition of the fluid in contact.1 Microdialysis allows sampling of extracellular fluid and it simplifies chemical analysis by excluding large molecules from the perfusate.1 For this reason, it is extremely important in observing drug pharmacokinetics and pharmacodynamics. Some of the most commonly detect markers include:1,2 Glucose Lactate Pyruvate Glycerol Glutamate Dopamine Peroxynitrite Hormones Small molecule drugs, etc.

Microdialysis can be performed in peripheral tissues such as muscle, lung, kidney, liver, etc.1 but is most often used in neurobiology research.2,3 However, there are some problems associated with microdialysis, which can be ameliorated by use in combination with imaging modalities.

In vivo neurons in the cortex of a Thy1 YFP mouse

CA1 layer of a Thy1-YFP mouse hippocampus

Application Brief: Cellvizio LAB and Microdialysis Research ver1.0

Applicability of the Cellvizio LAB and Vevo Imaging System One significant drawback of microdialysis is that although it provides a plethora of molecular information, it does not detect any functional information at the tissue of interest. Furthermore, the information captured by microdialysis is always an estimate of the actual concentration of the fluid in question because equilibrium across the membrane is not complete, depending on the membrane pore size, area, rate of flow, diffusion speed, etc.2 Thus other imaging modalities should be used to validate the data. This can be complemented by the Cellvizio LAB system, a high speed fluorescence microscope with miniaturized microprobes capable of high-resolution, real-time endoscopic imaging of fluorescent tissues, cells and markers at a micron level. The system is composed of 300 m probes attached to optical fibers, which are inserted into the tissue of interest, thus operational-wise is very similarly to a microdialysis device. Minimally invasive because of their diameter size, the microprobes have applications for surface, internal and deep imaging of any organ, including brain. Furthermore, image acquisition happens in real-time, making it a perfect complement to be used in conjunction with microdialysis. For example, Vincent et al. used the Cellvizio LAB system to image the peripheral nerves and deep brain of a Thy-1 eYFP (enhanced yellow fluorescent protein) mouse.4 Specifically, axonal changes and nerve regeneration was studied longitudinally following crush injury of the saphenous nerve.4 The non-invasive Cellvizio LAB system allowed the degeneration-regeneration sequence to be recorded daily for a total period of 15 days. The authors then used a 300 m probe to image the striatum and ventral tegmental area (VTA) of a mouse brain.4 Finally, the authors used bicuculline and electrical stimulation to stimulate the mouse brain, to visualize and quantify calcium changes over time.4 The authors concluded that fibered fluorescence microscopy (FFM) system proved to be minimally-invasive, direct, rapid and accurate in measurement of neuronal activity in vivo. Since the Cellvizio LAB provided real-time information, it was correlated in parallel to EEG and could also be complemented with microdialysis to provide the researchers with a more comprehensive data set. The accuracy of results from microdialysis research is integrally linked to the location of the probe placement. Microdialysis reflects the tissue biochemistry and metabolic dynamics only at the exact location where the probe is placed.2,5 It has been suggested that tip position after placement of the probe be checked with micro-CT,2 but this exposes the animal to radiation which can compromise results (especially in tumor models) and is more of a confirmation of location rather than a guidance system to place the probe. On the other hand, the stereotaxic guidance system used in conjugation with the Cellvizio LAB imaging system makes use of accurate 3D coordinate systems to
Application Brief: Cellvizio LAB and Microdialysis Research ver1.0 3

automatically place the microscopy probe at specific brain locations. This was demonstrated by Davenne et al, who used the stereotaxic apparatus to achieve accurate positioning of the Cellvizio LAB probe within the mouse brain.6 This allowed the authors to visualize neurons in different locations of the rostral migratory stream (RMS), and also inside the olfactory bulb (OB). Furthermore, the authors tracked migration of the GFP-labeled cell bodies over a period, with migration speeds ranging from 40-80 m/h.6 The authors concluded that the Cellvizio LAB system was uniquely suited for imaging dynamic processes in the brain, because of its real-time image acquisition, and that it could be used in the brain for 6 hours or more with minimal bleaching and phototoxicity.6 Overall, FFM fibred fluorescence microscopy provides neurobiologists with a powerful tool of realtime, in vivo, in situ, minimally invasive functional imaging of the brain.

Neuromuscular junction of a Thy1-YFP mouse with skeletal muscle fibers marked by topical application of Sytol3

Motor nerve terminals at the neuromuscular junction of Thy1-YFP mouse

Application Brief: Cellvizio LAB and Microdialysis Research ver1.0

References 1. Pea A, Liu P, Hartmut D. Microdialysis in peripheral tissues. Adv Drug Deliv Rev 2000;45:189-216. 2. Smith M, Tisdall MM. Cerebral microdialysis: research technique or clinical tool. Br J Anaesth 2006;97(1):18-25. 3. Zielke HR, Zielke CL, Baab PJ. Direct measurement of oxidative metabolism in the living brain by microdialysis: a review. J Neurochem 2009;109(Suppl. 1):24-29. 4. Vincent P, Maskos U, Charvet I, Bourgeais L, Stoppini L, Leresche N, et al. Live imaging of neural structure and function by fibred fluorescence microscopy. EMBO Rep 2006;7(11):1154-61. 5. Engstrm M, Polito A, Reinstrup P, Romner B, Ryding E, Ungerstedt U, et al. Intracerebral microdialysis in severe brain trauma: the importance of catheter location. J Neurosurg 2005;102:460-9. 6. Davenne M, Custody C, Charneau P, Lledo PM. In vivo imaging of migrating neurons in the mammalian forebrain. Chem Senses 2005 Jan;30 Suppl 1:i115-6.

Application Brief: Cellvizio LAB and Microdialysis Research ver1.0