Annex Found documentation for A124 Investigation into the research of Milena Penkowa

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1 Annex Found documentation for A124 Investigation into the research of Milena Penkowa Overview of paper A124. Role of metallothionein-iii following central nervous system damage. Carrasco J, Penkowa M, Giralt M, Camats J, Molinero A, Campbell IL, Palmiter RD, Hidalgo J. Neurobiol Dis Jun;13(1): Similarities with A143. Lab of the live animal exps and type of exps Hidalgo. Cryo-lesions of brains. Brains were shipped to MP No. & Types of animals used in the animal exps MT-I+II KO mice, MT-III KO mice and wild type controls. 129/Sv genetic background. Number of mice is unclear. MP s exps and time of these exps acc. to MP. Stainings, Counts, microscopy, photos/-data, presentation at Panum Documentation and information requested by Panel, and further keywords and comments for identifying the wanted documentation: -In fig. 11, a northern blot, one wonders whether all 4 gel segments come from same exp or whether they were puzzled together from different origins. All the animal work as well as the northern blots (figure 11) were performed in Hidalgo s lab, according to Hidalgo. The northern blots were done by Carrasco, with contributions from Molinero and Giralt, according to the questionnaires. MP therefore seems to have had a very small role, if any, in relation to fig.11. Paraffin embedded brains The documentation and information found, including the animal key, if found: Keys: D. Lesions. MT-1 transgenic. MERCÉ (i.e. Mercedes Giralt). Several D-Boxes with sections and some slide trays. DD. Mail from Merce Giralt, ,..the relation of the animals and the treatments from the experiment of the Atrian recombinant MT, incl. Handwritten note of Mercé on lesions and MT injection.. All are MTKOs. List of Lesioned and MT-treated mice. Stained sections on slide trays and some DD-boxes. EM. E.M. experiment : Lesions in mice, strains 129 and MT-I+II KO. (And, not relevant for A124 or A143): - Experiment IL6 mice injected with KA : mice, strains 129 and IL6 KO.) Page 1: Mail from X.Carrasco, Identification numbers of brains/animals. Scannings-EM. Page 2: handwritten list For scanning- EM. No EM-samples found. F. Exp. Lesion in MT-3KO and 129Sv (02/2001) Animals no (Mice). Page 2: F-animals, number Several F-Boxes with sections were found. SC. Mail from Juan Hidalgo, 6 June 2003 and two hand-written lists. Lesioned MTKO and 129Sv mice. The A124 is published June 2003, i.e. same time as JH s mail, but probably the mice keyed SC still may be the ones used for the A124. Several SC-boxes. -In JH s mail JH also mentions the J NeuroSci paper animals. However there are no J NeuroSci papers on lesioned animals among the selected papers, so this, JH s mentioning, is probably not of relevance for the Panel s investigation. - On top of one of the hand-written lists are mentioned some slides from GFAP-IL3 mice. These cannot be associated with paper A124. A file concerning instruction for Northern is found. Its origin is Rigshospitalet, though, i.e. the origin is not of MP. -A record on counts of GFAP, a.o.t., is found, The files found for A143 may also be relevant for A124. 1

2 Written documentation found: 2 files found on MP s Hard-disc are copied in the following. (Followed by scanned copies of the found keys). The files found for A143 may also be relevant see document on found documentation for A143. 2

3 1. A file (of ) concerning instruction for Northern: Følgende informationer vil blive brugt til at afgøre hvorvidt eksperimentielt design og prøvesammensætning er optimal med henblik på ekspressionsprofilering med Affymetrix GenChip teknologi. Vi beder venligst om at følgende skema udfyldes som første trin i indlevering af prøver til GenChip analyse. Vi vil gennemgå ansøgningen og give feedback, samt oplysninger omkring pakning og forsendelse af prøver hurtigst muligt. Skema eller tilsvarende oplysninger bedes sendes til Rehannah Borup, Diagnostisk Center, Klinisk Biokemisk Afd. KB-3014, Blegdamsvej 9, 2100 KBH Ø. Rborup@rh.dk Ved indsendelse af prøver til MicroArray Centret skal skemaet vedlægges i udfyldt stand. Oplysninger til brug ved Affymetrix DNA-chip ekspressionsanalyser. Navn: Milena Penkowa Telefon: Afd./Institut: Medicinsk Anatomisk Afd., Panum Inst. M.Penkowa@mai.ku.dk Adresse: Medicinsk Anatomisk Inst., Panum Inst., Blegdamsvej 3, 2100 Kbh Ø Projekt baggrund Metallothionein (MT) er et protein (6-7 kda), som forekommer i lave koncentrationer i det normale centralnervesystem (CNS). Ekspressionen af MT øges signifikant i forbindelse med CNS inflammation og ved patologiske forhold. MT er et neuroprotektivt protein, der forekommer i flere isoformer. MT isoform I og II (MT-I+II) er de bedst kendte og beskrevne MT former, og det er disse isoformer der er signifikant neuroprotektive. MT-I+II reguleres og produceres koordineret og simultant, og kan derfor funktionelt betragtes som éet molekyle. Ved at anvende MT-I+II knock-out mus (MT-I+IIKO mus) og MT-I transgene mus (MTTg mus) samt wildtype mus og rotter, der fik medicinsk MT-II behandling har vi demonstreret betydningen af MT- I+II for neuroprotektion. I korthed virker MT-I+II signifikant antiinflammatorisk, anti-oxiderende og anti-apoptotisk i det patologiske CNS. Kort beskrivelse af eksperimentel procedure og design Vi ønsker at undersøge forskellen i gen-expression (indenfor inflammation, oxidativt stress, apoptose etc) på wildtype mus og MT-I+IIKO efter at musene har fået en hjernelæsion. Desuden undersøges IL-6 KO mus og deres kontroller (wildtypes, der er forskellige fra de wildtypes der er kontroller til MT-I+IIKO s). Vi vil lave hjerneskaden på 3 mus i hver gruppe (ialt 12 mus), og sende hjernerne til jer. Det bedste ville være hvis i selv kunne extrahere RNA et, idet vi så ikke skaber fejlkilder, da vi ikke har stor erfaring i dette. Musehjernerne kan være klar medio september Alternativt giver i os en opskrift og derefter extraherer vi RNA et... Ideen er at se en evt forskel i gen-expression af vigtige gener imellem muse-fænotyperne. Både MT-I+II og IL-6 har kardinalt vigtige roller i det patologiske CNS. Hvis der er mulighed for det, vil vi lave forsøget og tage hjernerne ud ved forskellige tidspunkter, hvis det er OK med jer, fx kunne vi tage hjerner ud ved 8 timer efter skaden, og igen efter 24 timer osv...men dette er op til jer. I så fald, så er det 12 hjerner ved hvert tidspunkt! 3

4 Uanset hvad, så udtages også hjerner fra 2 helt uskadede musehjerner fra hver gruppe/genotype, dvs 8 hjerner ialt Materiale Antal prøver: Hvis hjerner: 12 stk med læsion + 8 stk uden læsion (minimum), ellers 8 pools RNA. Hvis det er OK med prøvevæv fra forskellige tidspunkter efter læsionen, så er det yderligere 12, 24, 36 eller flere hjerner... Navn på prøver: Type: Total RNA Andet (f.eks. uoprenset væv eller celler): RNA-oprensningsmetode: Total RNA koncentration: 1 µg/µl 5 µg/µl 9 µg/µl 2 µg/µl 6 µg/µl 10 µg/µl 3 µg/µl 7 µg/µl 11 µg/µl 4 µg/µl 8 µg/µl 12 µg/µl Udgangsmateriale: Vævstype: Celletype: Chip Mus U74Av2 Mus U74Bv2 Mus U74Cv2 Human U133A Human U133B Human Focus Rotte U34B Rotte U34A Rotte U34C Andet: 4

5 Dato 5. aug 2002 Underskrift: Milena Penkowa Spørgsmål vedrørende analysen kan rettes til: Rehannah Borup, KB3014, tlf , Finn Cilius Nielsen, KB 3013, tlf , Susanne Smed, KB 4111, tlf , A file (of ) with a record on counts: New cell counts. Counted different animals in each group. Area is 0,4 mm2. Cell counts in black are of round/amoeboid (not ramified) lectin+ cells. The blue numbers are of ramified microglial cells. Controls The normal mice: 42,30,50 The MTTg* mice: 29,37,27 The IL6Tg* mice: 81,72,61 The crossed mice: 41,48,52 1 dpl The normal mice: 75,73,89. 22,34,40 The MTTg* mice: 25,14,10. 9,11,3 The IL6Tg* mice: 181,174, ,48,54 The crossed mice: 24,19,35. 15,30,19 3 dpl The normal mice: 161,143, ,31,37 The MTTg* mice: 37,44,31. 6,10,2 The IL6Tg* mice: 305,292, ,41,50 The crossed mice: 49,63,62. 12,28,16 6 dpl The normal mice: 165,147, ,30,41 The MTTg* mice: 36,44,31. 8,6,12 The IL6Tg* mice: 273,294, ,38,51 The crossed mice: 42,60,41. 19,30,17 5

6 10 dpl The normal mice: 76,95,83. 34,49,48 The MTTg* mice: 32,36,29. 11,12,7 The IL6Tg* mice: 91,75,58. 51,39,55 The crossed mice: 32,31,41. 23,18,31 20 dpl: The normal mice: 29,26,36. 40,50,51 The MTTg* mice: 11,10,8. 15,13,17 The IL6Tg* mice: 20,21,16. 71,65,50 The crossed mice: 13,3,10. 29,19,37 GFAP+ cells Controls The normal mice: 29,27,19 The MTTg* mice: 22,29,31 The IL6Tg* mice: 54,58,42 The crossed mice: 40,36,38 1 dpl The normal mice: 38,29,43 The MTTg* mice: 65,54,51 The IL6Tg* mice: 63,68,70 The crossed mice: 67,74,79 3 dpl The normal mice: 112,95,96 The MTTg* mice: 162,173,178 The IL6Tg* mice: 170,175,186 The crossed mice: 198,184,167 6 dpl The normal mice: 104,90,93 The MTTg* mice: 186,188,179 The IL6Tg* mice: 189,199,211 The crossed mice: 190,201, dpl The normal mice: 103,91,95 The MTTg* mice: 193,179,198 The IL6Tg* mice: 185,172,187 The crossed mice: 208,197,216 6

7 20 dpl The normal mice: 53,64,56 The MTTg* mice: 158,177,151 The IL6Tg* mice: 135,146,153 The crossed mice: 180,182,151 CD3+ CELLS Controls The normal mice: 0,1,2 The MTTg* mice: 1,1,0 The IL6Tg* mice: 3,1,0 The crossed mice: 0,0,1 1 dpl The normal mice: 27,30,35 The MTTg* mice: 22,19,15 The IL6Tg* mice: 67,74,63 The crossed mice: 26,28,34 3 dpl The normal mice: 44,46,39 The MTTg* mice: 20,25,18 The IL6Tg* mice: 83,92,77 The crossed mice: 41,45,38 6 dpl The normal mice: 43,45,40 The MTTg* mice: 21,28,11 The IL6Tg* mice: 82,90,75 The crossed mice: 41,44,36 10 dpl The normal mice: 45,48,42 The MTTg* mice: 24,21,9 The IL6Tg* mice: 28,33,35 The crossed mice: 20,26,12 20 dpl The normal mice: 35,45,41 The MTTg* mice: 14,10,6 The IL6Tg* mice: 31,30,24 The crossed mice: 15,23, End of files found. Scanned copies of found keys on the following pages: 7

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