* indicates P 0.05, ** indicates P 0.01 compared to untreated BV2 cells. or 3 months. The second is Dihydroergotamine Mesylate an active vaccination study in which we examined 16 month aged APPSw/NOS2-/- mice treated with A vaccination for 4 weeks. Results There is a significant activation of the MMP2 and MMP9 proteinase degradation systems by anti-A immunotherapy, regardless of whether this is delivered through active vaccination or passive immunization. We have characterized this activation by gene expression, protein expression and zymography assessment of MMP activity. Conclusions Since the MMP2 and MMP9 systems are heavily implicated in the pathophysiology of intracerbral hemorrhage, these data may provide a potential mechanism of microhemorrhage due to immunotherapy. Increased activity of the MMP system, therefore, is likely to be a major factor in increased microhemorrhage occurrence. After injection with a lethal dose of ketamine, the mice were perfused intracardially with 25 ml normal saline. Brains were rapidly removed and bisected in the mid-sagittal plane. One Rabbit Polyclonal to ENTPD1 half of each brain was immersion fixed in 4% paraformaldehyde, while the other was snap-frozen in liquid nitrogen and stored at -80C. Frozen sections of the fixed hemibrain were collected following cryoprotection through sucrose. 25 m sections were collected and stored in DPBS+sodium azide at 4C until needed. The frozen hemibrain was pulverized using a mortar and pestle on dry ice. Brain powder was then stored at -80C until needed. em Passive immunization study: /em After injection with a lethal dose of pentobarbital, the mice were perfused intracardially with 25 ml normal saline. Brains were rapidly removed and bisected in the mid-sagittal plane. One half of each brain was immersion fixed in 4% paraformaldehyde, while the other was dissected into frontal cortex, posterior Dihydroergotamine Mesylate cortex, hippocampus, cerebellum and rest of leftover brain tissue. These pieces were expensive frozen and stored at -80C. Eight 25 m sections equally spaced 600 mm apart were selected from our active vaccination study for free floating immunohistochemistry for MMP9 (1: 1000, Rabbit polyclonal, Millipore, Billireca, MA) as explained previously . Quantitative real-time RT-PCR Approximately 40 mg frozen brain powder (for the active vaccination study) or the whole right hippocampus (for the passive immunization study) was used for RNA extraction using the PerfectPure RNA tissue kit (5 Primary Inc., Gaithersburg, MD). RNA concentrations were determined by UV spectrophotometry and cDNA produced using the cDNA archive kit (Applied Biosystems, Foster City, CA). Real-time PCR was performed using the TaqMan Gene Expression assay kit (Applied Dihydroergotamine Mesylate Biosystems, Foster City, CA) according to the manufacturer’s instructions and Dihydroergotamine Mesylate as previously explained . All genes are normalized to 18 S rRNA. Normal non-transgenic mice served as the comparator and fold changes were calculated using the -delta delta Ct) method  The following genes were analyzed: 18 s (Hs99999901_s1), MMP2 (Mm00439498_m1), MMP3 (Mm00440295_m1), MMP9 (Mm00600163_m1), MT1-MMP (Mm00485054_m1), TIMP1 (Mm00441818_m1), TIMP2 (Mm00441825_m1). ELISA measurement Active vaccination study only: Protein was extracted from 4 brains for each genotype using 100 mg pulverized brain powder in PBS with total protease inhibitor (Sigma-Aldrich, St Louis MO) and quantified using the BCA protein assay kit (Pierce Biotechnology Inc. Rockford, IL, performed according to manufacturer’s instructions). We used commercially available packages to assess MMP3, MMP9, TIMP1, MMP2 and TIMP2 and ran the assays according to manufacturer’s recommendations (R&D Systems, Minneapolis, MN). All data were normalized to the total protein to yield ng/mg protein. Zymography Enzymatic activities of tissue MMPs were measured using zymography in brain samples from your active vaccination study only: Protein was extracted using 100 mg pulverized brain powder in PBS and quantified immediately using the BCA protein assay kit (Pierce Biotechnology Inc. Rockford, IL. Performed according to manufacturer’s instructions). Protein samples were immediately separated on a precast 10% gelatin zymogram gel (Invitrogen, Carlsbad, CA). The gel was removed, incubated in zymogram renaturing buffer for 30 minutes, equilibrated for 30 minutes in zymogram developing buffer at room temperature and then incubated immediately at 37C with softly agitation in new zymogram developing buffer (all buffers obtained from Invitrogen, Carlsbad, CA). The next day, the gel was washed gently with water and incubated in Commassie blue (0.1% Commassie blue, 20% methanol, 10% Dihydroergotamine Mesylate acetic acid). The gel was stained.