Yearbook 06-07


Matthias Reinhardt
Home Institution Charité - Universitätsmedizin Berlin Host Institution Department of Oncology, Mount Sinai School of Medicine, New York City Mentor: Andreas S. Beutler, M.D. E-Mail: rein.matt@gmail.com
Research Topic see Abstract
Personal Reactions to the U.S. Experience Living and working in New York City! Wow! What should I say? It is impressive, not only by the size of its buildings nor by the number of people who live here, but New York is also amazing because you can see it any way you want to: New York is rich. When you walk down Fifth Avenue and pass the Prada, Armani and Gucci stores, you will see the bright and shiny side of New York with all its jewelery and designer clothes, where any single item can easily cost hundreds of dollars. New York is poor. On almost every corner in Manhattan there are homeless people, begging for food and money. New York is international. Wherever you go in the city you will hear at least two foreign languages spoken, from Italian, Spanish, French, or German to Russian, Chinese, Korean or Indian. New York is American. In Queens or Staten Island you see these typical American houses with a kind of architecture that you won't find anywhere in Europe. New York is very fast paced. 24 hours a day, 7 days a week, people are riding in subways or taxis, or walking through New York's streets trying to get to their next destination as quickly as possible. New York is calm. There is no better place to relax, than lying in the sun in Central Park on a warm spring day or casually strolling through one of the many museums throughout the city.
Greatest Difficulties Encountered Hopefully I don't disappoint the reader by repeating what is written in the yearbook so many times: The “Greatest Difficulty Encountered” for me, as is probably for most of the BMEP students, was to receive the DS-2019 and the visa. But it was worth the hours of standing in line and filing out the paper work to come work in New York. The “Second Greatest Difficulty Encountered” was finding an affordable place to live. Or I should say, a place to sleep, since most of my days are spent in the lab (or downtown Manhattan), not in my room. In New York, housing is a particular problem, due to the through-the-roof prices for apartments. Fortunately, I was able to get a reasonably priced room in the student housing right across the street from the Mount Sinai Medical Center.
Most humorous incident When I began working here in New York, my job consisted mainly of performing surgeries and behavioral testing on rats. For both, I wore scrubs, so that the smell of the rats would not stick to my clothes. One morning, by boss's wife - who also works at Mt Sinai - entered the lab, saw me wearing scrubs and said: “Oh, you are looking surgical today”. But on that day, I was not doing surgery, just testing, so I answered: “No, no. I am looking testicle!” I took me a while until I realized what I just had said.
Helpful Hints for Future Students www.spiegel.de (don't forget Germany!) www.mssm.edu (you want to do research and live in New York City? You should check out this link!) www.protocol-online.org (if your experiments don't work, you might get some useful hints here.) www.kolpingny.org (look here for affordable short time housing in New York City)

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Abstract on Research Topic
Gene transfer of Enhanced Green Fluorescent Protein (EGFP) through intrathecal delivery of self-complementary Adeno-associated Virus 8 (scAAV8)

Authors:
Matthias Reinhardt, Benjamin Storek and Andreas S. Beutler

Background/Introduction:
Chronic pain is being considered as the most live-affecting side-effect of many diseases. According to recent estimations 10-15% of the world population suffer from chronic pain [1]. In severe instances, as in cases of intractable cancer pain, the current treatment of choice consists of high doses of opioid administration. But complete pain relief cannot be always achieved, especially when opioid mediated side effects, as tiredness ,mental clouding or hallucinations, limit the dosage. Previously, a new approach of pain treatment was presented [2]: scAAV8 vector, carrying genes of analgetic proteins, is injected into the cerebrospinal fluid (CSF) at a spinal level. The vector infects intrathecal (IT) cells and causes expression of analgetic proteins. These proteins diffuse autocrine or paracrine to receptors/targets, through which they impede spinal pain transmission. To localize vector mediated protein expression in the IT space, scAAV8 vector, carrying the gene for EGFP, was intrathecally administered. EGFP expression can be easily seen by confocal microscopy; therefore, allowing identification of the target cells of intrathecal gene transfer by scAAV8.

Methods:
Six male Sprague-Dawley rats (weight 350g) were assigned into three groups (each n=2). All rats were IT catheterized by the method developed by Yaksh et al [3]. The tip of the catheter was placed at the lumbar region inside the CSF space. Drug administration was performed through the catheter intrathecally. Group 1 (control): IT administration of 15 µl PBS. Group 2: IT administration of 15 µl scAAV8/EGFP (2x10 9 /particles per µl) Group 3: IT administration of 45 µl scAAV8/EGFP (2x10 9 /particles per µl). The catheter was withdrawn and the animals were closed by wound clips.
Rats were perfused using paraformaldehyde (4%) 16 weeks post IT catheterisation. Dorsal root ganglia (DRG), brain and spinal cord (SC) were immediately excised after perfusion. After being exposed to increasing concentrations of sucrose (10%, 20%, 30%), DRGs (L5, T5, C5), attached SC and occipital cortex were cut on a cryostat to slices of 20µm. They were then attached to subbed slides and coverslipped (Vectashield Hard Set, Vector Labs, Burlingame, CA). Slides were examined under a Zeiss 510 confocal microscope, using Argon Laser (wavelength 488nm) and bandpass filter (505 – 530 nm).

Results:
In rats injected with 15 µl scAAV8/EGFP, EGFP expression was found only in cell bodies of L5 DRGs and the outgoing and incoming dentritical axons. No proof of EGFP expression was found in the SC or brain. The L5, T5, C5 DRGs of rats injected with 45µl scAAV8/EGFP did express EGFP. Intensity of fluorescence decreased from caudal to cranial. At the L5 level, single cells inside the SC were found to be expressing EGFP. No expression was seen in the brain. In all cases of EGFP expression, only large-size neurons could be proven as targets of scAAV8 infection. PBS injected rats showed no fluorescence in comparison to EGFP positive rats.

Conclusion:
Located in the DRGs are the perikarya of all first sensory neurons, including the pain transmitting neurons; therefore, DRGs are an interesting target for any kind of pain therapy. Our pain therapy approach showed that DRGs can be targeted effectively by scAAV8. This suggests that positive results of pain therapy with IT administered scAAV8, carrying genes of analgetic proteins, might be due to the analgetic effect of these proteins inside the DRGs. Further, no central nervous side effects by scAAV8 mediated protein expression in the brain are expected due to this treatment.

References:
[1] Breivik et al.; Prevalence and impact of chronic pain: a systematic review; 11th World Congress on Pain; Sydney, Australia
[2] Storek et al.; Intrathecal long-term gene expression by self-complementary adeno-associated virus type 1 suitable for chronic pain studies in rats; Molecular Pain 2:4; 2006,
[3] Yaksh TL ,Malkmus SA;(1999) Animal models of intrathecal and epidural drug delivery; Spinal Drug Delivery; 1999, pp. 317–344