The Specific FKBP38 Inhibitor N-(N′,N′-Dimethylcarboxamidomethyl)cycloheximide Has Potent Neuroprotective and Neurotrophic Properties in Brain Ischemia
2006; Elsevier BV; Volume: 281; Issue: 21 Linguagem: Inglês
10.1074/jbc.m600452200
ISSN1083-351X
AutoresFrank Edlich, Matthias Weiwad, Dirk Wildemann, Franziska Jarczowski, Susann Kilka, Marie-Christine Moutty, Günther Jahreis, Christian Lücke, Werner Schmidt, Frank Striggow, Gunter Fischer,
Tópico(s)Peptidase Inhibition and Analysis
ResumoFK506 and FK506-derived inhibitors of the FK506-binding protein (FKBP)-type peptidylprolyl cis/trans-isomerases (PPIase) display potent neuroprotective and neuroregenerative properties in various neurodegeneration models, showing the importance of neuroimmunophilins as targets for the treatment of acute and chronic neurodegenerative diseases. However, the PPIase activity targeted by active site-directed ligands remainsed unknown so far. Here we show that neurotrophic FKBP ligands, such as GPI1046 and N-[methyl(ethoxycarbonyl)]cycloheximide, inhibit the calmodulin/Ca2+ (CaM/Ca2+)-regulated FKBP38 with up to 80-fold higher affinity than FKBP12. In contrast, the non-neurotrophic rapamycin inhibits FKBP38·CaM/Ca2+ 500-fold less affine than other neuroimmunophillins. In the context of the high expression of FKBP38 in neuroblastoma cells, these data suggest that FKBP38·CaM/Ca2+ inhibition can mediate neurotrophic properties of FKBP ligands. The FKBP38-specific cycloheximide derivative, N-(N′,N′-dimethylcarboxamidomethyl)cycloheximide (DM-CHX) was synthesized and used in a rat model of transient focal cerebral ischemia. Accordingly, DM-CHX caused neuronal protection as well as neural stem cell proliferation and neuronal differentiation at a dosage of 27.2 μg/kg. These effects were still dominant, if DM-CHX was applied 2-6 h post-insult. In parallel, sustained motor behavior deficits of diseased animals were improved by drug administration, revealing a potential therapeutic relevance. Thus, our results demonstrate that FKBP38 inhibition by DM-CHX regulates neuronal cell death and proliferation, providing a promising strategy for the treatment of acute and/or chronic neurodegenerative diseases. FK506 and FK506-derived inhibitors of the FK506-binding protein (FKBP)-type peptidylprolyl cis/trans-isomerases (PPIase) display potent neuroprotective and neuroregenerative properties in various neurodegeneration models, showing the importance of neuroimmunophilins as targets for the treatment of acute and chronic neurodegenerative diseases. However, the PPIase activity targeted by active site-directed ligands remainsed unknown so far. Here we show that neurotrophic FKBP ligands, such as GPI1046 and N-[methyl(ethoxycarbonyl)]cycloheximide, inhibit the calmodulin/Ca2+ (CaM/Ca2+)-regulated FKBP38 with up to 80-fold higher affinity than FKBP12. In contrast, the non-neurotrophic rapamycin inhibits FKBP38·CaM/Ca2+ 500-fold less affine than other neuroimmunophillins. In the context of the high expression of FKBP38 in neuroblastoma cells, these data suggest that FKBP38·CaM/Ca2+ inhibition can mediate neurotrophic properties of FKBP ligands. The FKBP38-specific cycloheximide derivative, N-(N′,N′-dimethylcarboxamidomethyl)cycloheximide (DM-CHX) was synthesized and used in a rat model of transient focal cerebral ischemia. Accordingly, DM-CHX caused neuronal protection as well as neural stem cell proliferation and neuronal differentiation at a dosage of 27.2 μg/kg. These effects were still dominant, if DM-CHX was applied 2-6 h post-insult. In parallel, sustained motor behavior deficits of diseased animals were improved by drug administration, revealing a potential therapeutic relevance. Thus, our results demonstrate that FKBP38 inhibition by DM-CHX regulates neuronal cell death and proliferation, providing a promising strategy for the treatment of acute and/or chronic neurodegenerative diseases. Members of the enzyme class of peptidyl prolyl cis/trans-isomerases (PPIases 2The abbreviations used are: PPIase, peptidyl prolyl cis/trans-isomerase; FKBP, FK506-binding protein; CaM, calmodulin; CaN, calcineurin; RP-HPLC, reverse phase-high performance liquid chromatography; eMCAO, transient focal ischemia induced by injection of endothelin-1; BrdUrd, 5-bromo-2′-deoxyuridine; NeuN, neuronal nuclei; PBS, phosphate-buffered saline; DM-CHX, N-(N′,N′-dimethylcarboxamidomethyl)cycloheximide; ME-CHX, N-[methyl(ethoxycarbonyl)]cycloheximide; Biot-CHX, N-{methylcarboxamido-[N,N′-α,ω-bis(ethylene)decaethyleneglycol]-ω-(N′-biotinamidyl)}-cycloheximide. ; EC 5.2.1.8) are the cytosolic receptors of the immunosuppressive drugs FK506 (tacrolimus), rapamycin (sirolimus), and cyclosporine A (1Takahashi N. Hayano T. Suzuki M. Nature. 1989; 337: 473-475Crossref PubMed Scopus (944) Google Scholar, 2Fischer G. Wittmann-Liebold B. Lang K. Kiefhaber T. Schmid F.X. Nature. 1989; 337: 476-478Crossref PubMed Scopus (1221) Google Scholar). With the exception of cyclosporine A these drugs reversibly inhibit the PPIase activity of members of the FK506-binding protein (FKBP) family and therefore account for numerous physiological effects. Recently, we have shown that FKBP38 is special among the PPIases, because it displays enzymatic activity exclusively in complex with calmodulin/Ca2+ (CaM/Ca2+), thus defining the protein as a CaM/Ca2+-dependent PPIase (3Edlich F. Weiwad M. Erdmann F. Fanghanel J. Jarczowski F. Rahfeld J.U. Fischer G. EMBO J. 2005; 24: 2688-2699Crossref PubMed Scopus (116) Google Scholar). Only the FKBP38·CaM/Ca2+ has affinity for FK506. Interestingly, FK506 and its open chain derivatives were shown to display neuroprotective and neuroregenerative effects in a wide range of animal models mimicking Parkinson disease, dementia, stroke, and nerve damage (3Edlich F. Weiwad M. Erdmann F. Fanghanel J. Jarczowski F. Rahfeld J.U. Fischer G. 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In general, the interpretation of effects caused by FK506 in cells is difficult, because FK506 inhibits not only the enzymatic activity of FKBPs, but also the protein phosphatase activity of calcineurin (CaN, PP2B). CaN inhibition is mediated by complex formation with FK506·FKBP complexes and is thought to be the initial process leading to immunosuppression (20Friedman J. Weissman I. Cell. 1991; 66: 799-806Abstract Full Text PDF PubMed Scopus (363) Google Scholar, 21Liu J. Farmer Jr., J.D. Lane W.S. Friedman J. Weissman I. Schreiber S.L. Cell. 1991; 66: 807-815Abstract Full Text PDF PubMed Scopus (3673) Google Scholar, 22Griffith J.P. Kim J.L. Kim E.E. Sintchak M.D. Thomson J.A. Fitzgibbon M.J. Fleming M.A. Caron P.R. Hsiao K. Navia M.A. Cell. 1995; 82: 507-522Abstract Full Text PDF PubMed Scopus (779) Google Scholar). CaN inhibition by immunophilin-immunosuppressant complexes is used to prevent allograft rejection in transplantation medicine, to treat autoimmune diseases and to circumvent graft-versus-host diseases. Additionally, inhibition of the protein phosphatase was the proposed basis of FK506-mediated neuroprotection, because the FKBP ligand rapamycin, which has no effects on CaN activity, did not exhibit neuroprotective properties (12Sharkey J. Butcher S.P. Nature. 1994; 371: 336-339Crossref PubMed Scopus (441) Google Scholar, 17Dawson T.M. Steiner J.P. Dawson V.L. Dinerman J.L. Uhl G.R. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9808-9812Crossref PubMed Scopus (513) Google Scholar, 23Costantini L.C. Isacson O. Exp. Neurol. 2000; 164: 60-70Crossref PubMed Scopus (75) Google Scholar). In contrast, monofunctional inhibitors of FKBPs, such as GPI1046, GPI1048, GPI1485 (Guilford Pharmaceuticals and Amgen), and V10,367 (Vertex Pharmaceuticals) have been developed, that have no influence on CaN activity, while neuroprotective and neuroregenerative effects of FK506 remain conserved. In the central nervous system, GPI1046 promotes protection and sprouting of serotonin-containing nerve fibers in the somatosensory cortex following parachloramphetamine treatment, induces regenerative sprouting from spared nigro-striatal dopaminergic neurons following MPTP toxicity in mice or 6-hydroxydopamine toxicity in rats, and alleviates the rotational abnormality in 6-hydroxydopamine-treated rats (8Steiner J.P. Connolly M.A. Valentine H.I. Hamilton G.S. Dawson T.M. Hester L. Snyder S.H. Nat. Med. 1997; 3: 421-428Crossref PubMed Scopus (326) Google Scholar, 19Hamilton G.S. Steiner J.P. J. Med. Chem. 1998; 41: 5119-5143Crossref PubMed Scopus (164) Google Scholar). Other monofunctional FK506 derivatives increase branching from developing dopamine neurons in culture, enhance neurite outgrowth of fetal dopamine transplants, increase nerve regeneration, and accelerate functional recovery following peripheral nerve injury (24Gold B.G. Zeleny-Pooley M. Chaturvedi P. Wang M.S. Neuroreport. 1998; 9: 553-558Crossref PubMed Scopus (58) Google Scholar, 25Costantini L.C. Chaturvedi P. Armistead D.M. McCaffrey P.G. Deacon T.W. Isacson O. Neurobiol. Dis. 1998; 5: 97-106Crossref PubMed Scopus (96) Google Scholar). Although the dramatic neuroprotective and neurotrophic effects of FKBP inhibitors imply an involvement of FKBP activity in neuronal cell death, the nature of the FKBP representing the primary target of these drugs remained enigmatic. Until now, 16 FKBPs have been identified in the human genome, two of which, FKBP12 and FKBP52, have been proposed to mediate neurotrophic actions of neuroimmunophilin ligands (12Sharkey J. Butcher S.P. Nature. 1994; 371: 336-339Crossref PubMed Scopus (441) Google Scholar, 26Gold B.G. Densmore V. Shou W. Matzuk M.M. Gordon H.S. J. Pharmacol. Exp. Ther. 1999; 289: 1202-1210PubMed Google Scholar). However, there is accumulating evidence that FKBP12 inhibition does not affect neuronal protection and regeneration. Accordingly, it was shown that FK506 retained its neurite outgrowth-promoting properties in hippocampal cultures from FKBP12 knock-out mice (26Gold B.G. Densmore V. Shou W. Matzuk M.M. Gordon H.S. J. Pharmacol. Exp. Ther. 1999; 289: 1202-1210PubMed Google Scholar). In this report, using (i) comparative PPIase inhibition studies, (ii) expression analysis of FKBPs in neuroblastoma cells, and (iii) application of a novel, highly specific FKBP38 inhibitor in the rat focal cerebral ischemia model, we show that FKBP38·CaM/Ca2+ inhibition constitutes the molecular basis of FKBP ligand-mediated neuroprotective and neurotrophic function. To obtain recombinant FKBP13 the nucleotide sequence encoding the amino acids 27-142 (lacking endoplasmic reticulum transport and retention signals) was amplified by PCR with the following primers: 5′-AATTTCATGAAAAGGAAGATGCAGATCGGGGTC-3′ and 5′-GCTAAAGCTTACAGCTCAGTTCGTCGCTCTATT-3′. The PCR product was subcloned into a pSTBlue-1 vector (Novagen), digested with BspHI/HindIII (New England Biolabs), ligated with a pET28a vector, and transformed into BL21(DE3) Rosetta cells (Novagen). To express FKBP38 in Escherichia coli, the corresponding sequence of the amino acids 1-336 (lacking the membrane anchor) was amplified by PCR using the following primers: 5′-AGTAAGTCATGAGACAACCCCCGGCGG-3′ and 5′-ACGTAAGCTTAAAACAGCCA CTTCCATGG-3′. The PCR product was subcloned into a pSTBlue-1 vector, digested with BspHI/HindIII, and cloned into a pET28a vector and transformed into BL21(DE3) Rosetta cells. The resulting constructs were verified by restriction analysis and DNA sequencing. The expression clones of human FKBP51 and FKBP52 were kindly provided by J. Buchner (Technical University of Munich). Recombinant human FKBP12 and FKBP12.6 were produced using the plasmid pQE60 (Qiagen) in E. coli strain K12 M15/pREP4, as described previously (27Tradler T. Stoller G. Rucknagel K.P. Schierhorn A. Rahfeld J.U. Fischer G. FEBS Lett. 1997; 407: 184-190Crossref PubMed Scopus (60) Google Scholar). Protein expression of the FKBPs was induced by addition of isopropyl β-d-thiogalactopyranoside to a final concentration of 1 mm and incubation for 4 h. Subsequently, cells were harvested by centrifugation at 4 °C for 15 min at 5000 × g. The bacterial pellet from a 6 liters of culture was resuspended in 200 ml of lysis buffer (10 mm Hepes, pH 7.5, 150 mm NaCl) and French pressed. Next the supernatant was centrifuged at 4 °C for 45 min at 35,000 × g. Supernatants containing FKBP51 or FKBP52, respectively, were applied to a nickel-nitrilotriacetic acid column (His Trap HP, 1 ml; Amersham Biosciences) equilibrated with 20 mm Tris/HCl, pH 8.0, using the N-terminal His6-fusion of the two proteins. The separation was performed according to the manufacturer's instructions. Fractions were analyzed by 12.5% (w/v) SDS-PAGE and staining with Coomassie blue. FKBP-containing fractions were dialyzed against 10 mm Hepes buffer, pH 7.8, 1.5 mm MgCl2, 150 mm KCl and loaded on a HiLoad 16/60 Superdex 200 pg (Amersham Biosciences) according to the manufacturer's instructions. The protein fractions were analyzed by 12.5% (w/v) SDS-PAGE and staining with Coomassie Blue. Cell lysates containing FKBP12, FKBP12.6, or FKBP13, respectively, were applied to a Fractogel EMD DEAE-650 (Merck) column and a Reactive Blue 2-Cl 6B (Merck) column equilibrated with 10 mm Tris buffer, pH 8.0. Protein was eluted from the Reactive Blue 2-Cl 6B columnby1 m NaCl. Fractions were analyzed by 15% (w/v) SDS-PAGE and staining with Coomassie Blue. FKBP-containing fractions were dialyzed against 10 mm Hepes buffer, pH 7.5, and applied to a Fractogel EMD (SO3−)-650 column. Protein was eluted by 1 m NaCl and analyzed by 15% (w/v) SDS-PAGE and staining with Coomassie Blue. To purify FKBP38, cell lysate was dialyzed in 10 mm Hepes, pH 7.5, 2 mm CaCl2 and applied to a CaM-Sepharose (Amersham Biosciences) column. Protein was eluted by 5 mm EGTA and analyzed by 12.5% (w/v) SDS-PAGE and staining with Coomassie Blue. FKBP38-containing fractions were dialyzed against 10 mm Hepes buffer, pH 7.8, 1.5 mm MgCl2, 150 mm KCl and loaded on a HiLoad 16/60 Superdex, 200 pg (Amersham Biosciences), according to the manufacturer's instructions. The protein fractions were analyzed by 12.5% (w/v) SDS-PAGE and staining with Coomassie Blue. The purified FKBPs were subsequently analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and N-terminal protein sequencing, confirming the identity of the proteins. Peptide substrates used were obtained from Bachem (Heidelberg, Germany). FK506 was purchased from Calbiochem. PPIase activity was measured using protease-coupled assays, as described previously (28Kofron J.L. Kuzmic P. Kishore V. Colon-Bonilla E. Rich D.H. Biochemistry. 1991; 30: 6127-6134Crossref PubMed Scopus (488) Google Scholar). FKBP381-336·CaM/Ca2+ PPIase activity was assayed in a reaction mixture containing 1 μm FKBP381-336, 5 μm recombinant human CaM, and 2 mm CaCl2. Inhibition constants for the PPIase activity of the hFKBP381-336·CaM/Ca2+ complex by low molecular weight inhibitors were determined by a competition assay using recombinant FKBP12 (3Edlich F. Weiwad M. Erdmann F. Fanghanel J. Jarczowski F. Rahfeld J.U. Fischer G. EMBO J. 2005; 24: 2688-2699Crossref PubMed Scopus (116) Google Scholar). PPIase activity of 12 nm FKBP12 was inhibited either by 20 nm FK506, 23 nm rapamycin, 1140 nm GPI1046, or 2400 nm N-[methyl(ethoxycarbonyl)]cycloheximide and subsequently recovered by addition of FKBP381-336·CaM/Ca2+. Due to the competition of both FKBPs for inhibitor binding, inhibition constants were determined using the Dynafit software (29Kuzmic P. Anal. Biochem. 1999; 267: 17-23Crossref PubMed Scopus (13) Google Scholar). 4-[2-(3,5-Dimethyl-2-oxocyclohexyl)-2-hydroxyethyl]-2,6-piperidinedione(cycloheximide) and all chemicals were purchased from Sigma (Taufkirchen, Germany) or Fluka (Buchs SG, Switzerland). Analytical and preparative RP-HPLC was performed on Sykam HPLC systems (Germany) with linear gradients of solvent B (0.1% trifluoroacetic acid in acetonitrile) in solvent A (0.1% trifluoroacetic acid in H2O) using HPLC columns of Merck (Germany). Analytical and preparative RP-HPLC were done using a LiChroCART® 125 × 4 column (LiChrospher 100 RP-8, 5 μm) and a Hibar® 250 × 25 column (LiChrosorb® RP-select B, 7 μm), respectively. Product identity was determined by ESI mass spectrometry. 500 mg (1.78 mmol) cycloheximide (C15H23NO4, 281.35 g/mol), 488 mg (2.5 mmol) bromo-acetic acid tert-butylester (C6H11BrO2, 195.1 g/mol), and 442 mg (3.2 mmol) anhydrous K2CO3 were dissolved in 10 ml of N,N-dimethylformamide and stirred for 12 h at room temperature. After evaporation of the solvent, the resulting residue was dissolved in ethyl acetate and filtrated. The crude product was dried over P2O5 following solvent evaporation. The entire amount of the crude product N-[methyl-(tert-butyloxycarbonyl)]cycloheximide was dissolved in 5 ml of a 2.2 m solution of the zinc chloride diethyl ether complex in methylene chloride (Fluka) and stirred for 2 h at room temperature. After evaporation of the solvent and dissolving of the resulting residue in 5 ml of tetrahydrofuran, the product was precipitated by addition of diethyl ether. The crude product was further purified by preparative HPLC to yield 479 mg of compound 2 (79% relating to the applied amount of cycloheximide). 100 mg (295 μmol) N-(carboxymethyl)cycloheximide, 306 mg (588 μmol) of PyBOP (520 g/mol) and 72 mg (882 μmol) of dimethylamine hydrochloride were dissolved in 20 ml of methylene chloride and cooled to 0 °C. After dropwise addition of 130 μl N-morpholine in 5 ml of methylene chloride, the resulting reaction mixture was stirred for 1 h at 0 °C and subsequently for 12 h at room temperature. Following evaporation of the solvent, the crude product was purified by preparative HPLC to yield 67 mg of N-(N′,N′-dimethylcarboxamidomethyl)cycloheximide (62%). The identity of the synthesized compounds was verified by ESI-MS and NMR. NMR experiments were performed in CdCl3 at 25 °C using a DRX 500 spectrometer (Bruker, Rheinstetten, Germany). 1H NMR data of were acquired with a 5-mm inverse triple-resonance probe with XYZ-gradient capability at 500.13 MHz resonance frequency; 13C NMR data were obtained with a 5-mm broadband probe at 125.76 MHz resonance frequency. Standard one-dimensional and two-dimensional pulse sequences were employed. All spectra were processed with the XWIN-NMR 3.5 software (Bruker) and referenced to chloroform. 1H NMR (500.13 MHz) data are as follows: δ 4.55 (2H, HCH2,NAc), δ 4.18 (1H, HOH, C8), δ 3.03 (3H, HCH3, NAc,trans), δ 2.92 (3H, HCH3,NAc,cis), δ 2.85/2.51 (2H, HCH2,C3), δ 2.85/2.51 (2H, HCH2,C5), δ 2.61 (1H, HCH,C11), δ 2.49 (1H, HCH,C9), δ 2.47 (1H, HCH, C4), δ 2.18 (1H, HCH,C13), δ 1.91/1.83 (2H, HCH2,C14), δ 1.87/1.59 (2H, HCH2,C12), δ 1.60 (1H, HCH,C8), δ 1.60/1.33 (2H, HCH2,C7) δ 1.22 (3H, HCH3,C13), δ 0.97 (3H, HCH3, C11). 13C NMR (125.76 MHz) data are as follows: δ 216.6, 172.2, 172.0, 166.1, 66.5, 50.2, 42.6, 40.5 (2 signals), 39.0, 37.8, 37.7, 33.1, 26.7, 26.4, 18.4, 14.2, 36.2, 35.7. mRNA from SH-SY5Y, PC3, and human Jurkat cells was prepared using the RNeasy mini kit (Qiagen) according to the manufacturer's protocol. Ribonuclease inhibitor (Calbiochem) was added to the mRNA preparations. The prepared mRNA was reverse-transcribed into cDNA using oligo(dT) primers (Novagen) and the Omniscript RT kit (Qiagen). Real-time PCR was performed using a QuantiTect SYBR Green PCR kit (Qiagen) and the Bio-Rad iCycler according to the manufacturer's protocol. To receive amplicons of the various FKBP cDNAs the following primers were used: FKBP12, 5′-GAG TGC AGG TGG AAA CCA TC-3′ (forward) and 5′-GTA GTG CAC CAC GCA GGT CTG-3′ (reverse); FKBP13, 5′-ATG AAA AGG AAG CTG CAG ATC GGG GTC-3′ (forward) and 5′-GCG CGA TTT GAT GGG ACA GTG-3′ (reverse); FKBP25, 5′-ATG GCG GCG GCC GTT CCA C-3′ (forward) and 5′-CTG CAG AAA CTT GAT AAT G-3′ (reverse); FKBP38, 5′-ATG GGA CAA CCC CCG G-3′ (forward) and 5′-CTG CCA TGG AGC CCG AGC C-3′ (reverse); FKBP51, 5′-ATG GGC ATC CGG AGA ACC AAA C-3′ (forward) and 5′-CAG TGA ATG CCA CAT CTC TGC-3′ (reverse); FKBP52, 5′-ATG GGA CAG TCC TCC AAC GAT C-3′ (forward) and 5′-GAT TCC GCC ATC TTC CTC TTC-3′ (reverse); actin, 5′-GAT GAT GAT ATC GCC GCG CTC-3′ (forward) and 5′-CAC GAT GGA GGG GAA GAC G-3′ (reverse). The eukaryotic translation assay was performed for 60 min at 30 °C using the Flexi®Rabbit reticulocyte lysate system (Promega) according to the manufacturer's instructions. To prevent degradation of sample luciferase RNA, 40 units of RNasin ribonuclease inhibitor was added to the reaction. The concentration of synthesized firefly luciferase was determined by luminescence measurement in a scintillation counter (Wallac1450, PerkinElmer Life Sciences) at 25 °C. Animals—Studies were performed on male Sprague-Dawley rats (250-280 g) obtained from Harlan Winkelmann (Borchen, Germany). The animals were maintained under constant environmental conditions with ambient temperature of 21 ± 2 °C and relative humidity of 40%. They were housed with a 12-h light-dark cycle and given food and water ad libitum. Induction of Focal Cerebral Ischemia—The procedure for the induction of focal cerebral ischemia by occlusion of the middle cerebral artery via intracerebral microinjection of endothelin 1 was modified from that published previously by Sharkey and Butcher (30Sharkey J. Butcher S.P. J. Neurosci. Methods. 1995; 60: 125-131Crossref PubMed Scopus (86) Google Scholar). Anesthesia was induced with halothane in a mixture of nitrous oxide/oxygen (70:30) and maintained with 2-3% halothane during the following procedures: rats were placed in a Kopf stereotaxic frame and further anesthetized via nose cone. For the induction of focal cerebral ischemia, a burr hole was drilled (1 mm diameter) into the skull (coordinates: anterior 0.90 mm from bregma, lateral 5.2 mm to satura sagittalis) and a 29-gauge cannula was lowered 7.5 mm below the dura according to the rat brain atlas of Paxinos and Watson (31Paxinos G. Watson C. The Rat Brain in Stereotoxic Coordinates, 5th Ed. Elsevier Academic Press, San Diego, CA2005Google Scholar). To induce occlusion of the middle cerebral artery, rats received an injection of 60 pmol of endothelin 1 (ED-1, Sigma) in 3 μlof0.1 m phosphate-buffered saline over a time period of 5 min. After a further 5 min, the cannula was slowly withdrawn. For the intracerebroventricular application of DM-CHX, a second burr hole was drilled into the skull (coordinates: posterior 0.80 mm from bregma, lateral 1.5 mm to satura sagittalis) and a 29-gauge cannula was lowered 4.5 mm below the dura. Intracerebroventricular applications of DM-CHX were performed either during (i) ischemia, 6 and 24 h after reperfusion; (ii) 2, 6, and 24 h; or (iii) 6 and 24 h after reperfusion. Rats were maintained at 37-38 °C throughout the operation procedure. For controls, following a middle incision in sham operated animals, a burr hole was drilled into the skull, but no endothelin 1 was injected. Determination of Infarct Volume—After a survival time of 7 days after eMCAO, animals were anesthetized by an intraperitonial injection of pentobarbital and perfusion-fixed transcardially with saline followed by 4% paraformaldehyde in 0.1 m phosphate buffer. Brains were then removed carefully, post-fixed in the same fixative for 2 h, and placed in a rodent brain matrix (rat, Activational Systems Inc., Scientific Instrumentation). 1-mm coronal brain slices were cut with a razor blade at 14 predetermined anterior-posterior levels. After cryoprotection in 30% sucrose, slices were rapidly frozen in isopentane and stored at -80 °C. Four to five cryostat sections (30 μm) from each brain slice were cut in acryo-microtome and stained with toluidine blue. The extent of cortical and striatal damage following ischemic injury was documented with microphotographic images from the Nissl stained slices showing the anterior-posterior level according to the brain atlas of Paxinos and Watson (31Paxinos G. Watson C. The Rat Brain in Stereotoxic Coordinates, 5th Ed. Elsevier Academic Press, San Diego, CA2005Google Scholar). The extent of the infarct area at each level was calculated by integrating the area of damage at each stereotactic level and the distances between the various levels. Using a light microscope (Nikon, Eclipse TE 3000) equipped with a 4× objective, the image analysis was performed with Lucia software, Version 4.2.1. The data were statistically analyzed by non-paired Student's t tests. Data are represented as mean ± S.E. Statistical significance was accepted at the level of 0.001 or 0.01 of probability. Quantification of Cell Proliferation and Neuronal Differentiation— For labeling mitotic cells, the thymidine analogue 5-bromo-2′-deoxyuridine (BrdUrd, Sigma) was used. Immediately after eMCAO surgery, animals received an intraperitoneal BrdUrd injection of 50 mg per kg body weight. This treatment was repeated during the following 5 days. Preparation of cryosections was performed as described above 7 days after eMCAO. For BrdUrd and NeuN labeling, sections were washed three times with 0.1 m phosphate-buffered saline (PBS) and incubated in 2 n HCL for 60 min at 37 °C. Unspecific binding was blocked by 1.5% goat normal serum (Alexis, Gruenberg, Germany). Slices were then incubated with rat anti-BrdUrd (1:50, Serotec, Oxford, UK) and mouse anti-NeuN (1:500, Chemicon, Hampshire, UK) antibodies overnight at 4 °C. For BrdUrd staining, a fluorescein isothiocyanate-labeled goat anti rat IgG 2a (1:200, Serotec), and for NeuN staining, a Alexa Fluoro 594-labeled goat anti-mouse IgG (H+L, 1:200, MoBiTec, Göttingen, Germany), was used. After final washing in PBS, sections were mounted and coverslipped using Vectashild with 4′,6-diamidino-2-phenylindole (Alexis, Gruenberg, Germany). For quantification of BrdUrd-positive cells and newly differentiated neurons (double-labeled with BrdUrd and NeuN), damaged cortical areas near the core of the injection of endothelin, corresponding to the inner zone of penumbra, were analyzed. Pictures were captured using the LUCIA imaging software package (Version 4.8, Laboratory Imaging, Prague, Czech Republic) and an Eclipse TE2000-S inverted fluorescence microscope with a 20× objective (Nikon, Düsseldorf, Germany). BrdUrd-positive nuclei and double-labeled cells were counted in three to five different cortical areas (each 237 μm2). Results are given as the mean value of all sections per animal. Five to eight animals per treatment were used. Ladder Rung Walking Test—To assess loss and recovery of function after focal cerebral ischemia, a ladder rung walking test, according to Metz et al. (32Metz G.A. Whishaw I.Q. Neuroscience. 2002; 111: 325-336Crossref PubMed Scopus (85) Google Scholar), was performed. A first series of training and test sessions were performed 2 days and 1 day before eMCAO surgery, respectively. Another series of training and test sessions was applied 6 and 7 days after eMCAO. In the training sessions, all animals crossed the ladder five times using a regular rung pattern of 2-cm distance from rung to rung. The total ladder length was 1 m. The following day, the same animals were tested three times on an irregular rung pattern where the distance between rungs varied between 1 and 4 cm. The rung pattern was changed between each test run to avoid memory and learning effects. Each session was monitored. Errors of hind and fore limb placements were counted and scored according to the following scheme (which includes score and type of misplacement): 0, correct placement; 1, limb position on same rung wa
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