A2 isoform of mammalian translation factor eEF1A displays increased tyrosine phosphorylation and ability to interact with different signalling molecules The eEF1A1 and eEF1A2 isoforms of translation elongation factor 1A have 98 % similarity and perform the same protein synthesis function catalyzing codon-dependent binding of aminoacyl-tRNA to 80S ribosome . However , the isoforms apparently play different non-canonical roles in apoptosis and cancer development which are awaiting further investigations . We hypothesize that the difference in non-translational functions could be caused , in particular , by differential ability of the isoforms to be involved in phosphotyrosine-mediated signalling . 1 Introduction Translation elongation factor 1A -LRB- eEF1A -RRB- is one of the core members of the elongation machinery providing high efficiency and processivity of the protein synthesis process in the eukaryotic cell -LRB- Negrutskii & El ` skaya , 1998 -RRB- . eEF1A forms a ternary complex with aminoacyl-tRNA and GTP and delivers the correct aminoacyl-tRNA to the A site of mRNA programmed ribosome in the GTP hydrolysis-dependent mode . The GDP-bound form of eEF1A has been shown to interact with deacylated tRNA -LRB- Petrushenko et al. , 1997 -RRB- and can transport it to aminoacyl-tRNA synthetase as the tRNA recycling arm of a tRNA channelling cycle -LRB- Petrushenko , Shalak , Budkevich , Negruskii , & El ` skaya , 2002 -RRB- . It is believed that the translation function of eEF1A might be combined with its involvement in cytoskeletal network maintenance and various signalling pathways -LRB- Lamberti et al. , 2004 -RRB- . eEF1A has been shown to interact with phospholipase C-gamma1 -LRB- PLC-gamma1 -RRB- -LRB- Chang et al. , 2002 ; Kim et al. , 1999 -RRB- and non-receptor tyrosine kinase Txk of the Tec family -LRB- Maruyama , Nara , Yoshikawa , & Suzuki , 2007 -RRB- . Importantly , Txk can form a complex with poly -LRB- ADP-ribose -RRB- polymerase 1 and eEF1A to influence interferon-gamma gene transcription in Th1 cells . Recently Lau , Castelli , Lin , and Macaulay -LRB- 2006 -RRB- identified eEF1A as a novel binding partner for Akt2 rho-associated kinase . In most cases it is not known whether interactions of eEF1A with protein kinase might lead to phosphorylation of eEF1A . Neither has the role of eEF1A in signal transduction pathways been identified . In mammalian cells , two tissue - and development-specific isoforms , eEF1A1 and eEF1A2 , are present . The expression of the 98 % similar isoforms is mutually exclusive -LRB- Kahns et al. , 1998 -RRB- . Interestingly , eEF1A2 , which is present normally only in muscles and neurons , was recently associated with tumor development in tissues that normally express only eEF1A1 -LRB- Amiri et al. , 2007 ; Anand et al. , 2002 -RRB- . How eEF1A2 isoform is involved in malignant transformation is not as yet understood . Since tyrosine kinases are prominent players in cancer development , direct comparison of the ability of eEF1A1 and eEF1A2 to be involved in phosphoTyr-specific signalling processes could help to interpret cancer-related properties of eEF1A2 . In this study , we compared for the first time the interactions of eEF1A1 and eEF1A2 with SH2 and SH3 domains of various signalling molecules . Contrary to eEF1A1 , eEF1A2 was able to interact with SH2 domains of Grb2 , RasGAP , Shc and Shp2 as well as with SH3 domains of Crk , Fgr , Fyn and PLC-gamma1 . Both eEF1A1 and eEF1A2 formed complexes with SH2 domain of PLC-gamma1 . In vivo interaction between endogenous eEF1A and tyrosine phosphatase Shp2 was confirmed in HEK293 cell line and tyrosine phosphorylation of eEF1A1 and eEF1A2 was demonstrated by Western blots with anti-phosphotyrosine-specific antibodies . Importantly , the level of phosphorylation of eEF1A2 was higher than eEF1A1 when isolated from tissues , and also when overexpressed in cells . Furthermore , the level of phosphorylation has been shown to be crucial for in vitro complex formation of eEF1A2 and SH2 domain of Shp2 . 2 Materials and methods 2.1 Cell lines and plasmid constructs HEK293 cells were purchased from the American Type Culture Collection -LRB- Manassas , VA -RRB- and grown according to their instructions . To produce eEF1A1 and eEF1A2 stable cell lines pcDNA 3.1 eEF1A1 and eEF1A2 with the C-terminal His-tags were linearized by MunI and transfected in HEK293 cells using ExGene500 -LRB- Fermentas -RRB- according to manufacturer recommendations . G418 was used to select stable cell lines . 2.2 Bioinformatics ScanSite Program was used for bioinformatic identification of possible SH2 and SH3 domains binding sites within eEF1A1 and eEF1A2 . ScanSite employs the matrix of selectivity values for amino acids at each position relative to an orienting residue as determined by the oriented peptide library technique -LRB- Obenauer , Cantley , & Yaffe , 2003 -RRB- . 2.3 Purification of the eEF1A1 and eEF1A2 isoforms eEF1A1 was purified from rabbit liver using a combination of the gel filtration , anion exchange , cation exchange and hydroxyapatite chromatographies as described previously -LRB- Budkevich et al. , 2002 -RRB- . eEF1A2 was isolated from rabbit muscles using the same procedure except the gel-filtration step was omitted in some cases . To confirm the biological activity of the isolated isoforms , the GDP / -LSB- 3H -RSB- GDP exchange was carried out as in -LRB- Carvalho , Carvalho , & Merrick , 1984 -RRB- . 2.4 Pull down assay Purified GST-SH2 and GST-SH3 domains of various signalling molecules were kindly provided by Prof. Ivan Gout -LRB- London , UK -RRB- -LRB- Gout et al. , 1993 -RRB- . In a pull down assay , 1.5 μg of GST , GST-SH2 or GST-SH3 domains fusion protein was incubated with glutathione-Sepharose 4B beads -LRB- Amersham Biosciences -RRB- for 2 h at 4 °C . The beads were then washed to remove unbound proteins and incubated at 4 °C for 3 h with 1.5 μg of purified eEF1A1 or eEF1A2 . Non-specific interactions were removed by extensive washing and bound proteins eluted by boiling in Laemmli sample buffer . Eluted proteins were resolved by SDS-PAGE , transferred to polyvinylidene difluoride membrane , and detected by immunoblotting with specific antibodies . 2.5 Phosphatase treatment Calf intestine alkaline phosphatase -LRB- CIAP -RRB- -LRB- Fermentas -RRB- has been used to dephosphorylate eEF1A1 and eEF1A2 . The proteins were precipitated from the lysates of stable cell lines on NiNTA and extensively washed with 1 × reaction buffer for CIAP -LRB- 10 mM Tris -- HCl -LRB- pH 7.5 -RRB- , 10 mM MgCl2 -RRB- , then 10 U of CIAP was added and reaction mix was incubated at 37 °C for 1 h. Thereafter the beads were washed two times with 1 × reaction buffer and the proteins bound were eluted by boiling in Laemmli sample buffer . For dephosphorylation of total protein fraction HEK293 cells overexpressing eEF1A1 or eEF1A2 were extracted with 1 × CIAP reaction buffer . To facilitate lysis the cells were passed through a 20-gauge needle 20 times and whole cell extracts were centrifuged at 10,000 × g for 15 min at 4 °C . The supernatant was incubated with 10 U of CIAP at 37 °C for 1 h , in control samples sodium orthovanadate was added to final concentration 1 mM . 2.6 Immunoprecipitation HEK293 cells were treated under indicated conditions , then washed with ice-cold phosphate-buffered saline -LRB- PBS -RRB- and extracted with the lysis buffer containing 10 mM Tris -- HCl -LRB- pH 7.5 -RRB- , 150 mM NaCl , 5 mM EDTA , 50 mM NaF , 1 % Triton X-100 , 1 mM sodium orthovanadate and a mixture of protease inhibitors -LRB- Roche Applied Science -RRB- . Whole cell extracts were centrifuged at 10,000 × g for 15 min at 4 °C . Endogenous protein complexes were immunoprecipitated from 0.5 mg of total cell lysates with corresponding antibodies -LRB- 1 μg/probe -RRB- and immobilized on protein A-Sepharose beads -LRB- Amersham Biosciences -RRB- . Immune complexes were washed three times with the lysis buffer and used for immunoblotting . As a loading control Western blotting -LRB- WB -RRB- of total cell lysate with anti-β-actin has been used . The effective serum starvation -LRB- 24 h in medium without serum -RRB- and stimulation -LRB- 10 % FBS , 30 min -RRB- has been confirmed in WB of total cell lysates with antibodies against phosphorylated residues S240/S244 in ribosomal protein S6 , which is phosphorylated by S6K1/2 -LRB- effector kinases in PI3K/mTOR pathway -RRB- in response to growth factors stimulation . 2.7 Immunoblot analysis Proteins were separated by SDS-PAGE , transferred onto polyvinylidene difluoride membrane , and incubated for 1 h with blocking solution -LRB- 2 % BSA , Tris-buffered saline , 0.1 % Tween-40 -RRB- . After blocking the membranes were probed 2 h at 4 °C with anti-GST , anti-eEF1A -LRB- Upstate Biotechnology -RRB- , anti-His tag -LRB- Cell Signalling -RRB- , anti-Shp2 -LRB- Cell Signalling -RRB- or anti-pTyr 4G10 -LRB- Upstate Biotechnology -RRB- antibodies . After extensive washing with the solution of Tris-buffered saline and 0.1 % Tween-40 , the membranes were incubated with secondary horseradish peroxidase-conjugated antibody for 1 h at room temperature . The antigen -- antibody complexes were detected using the ECL system -LRB- Millipore -RRB- . When immunoblots had to be re-probed , the membranes were initially stripped and re-blocked prior to incubation with another type of primary antibody . 3 Results 3.1 Existence of SH2/SH3 domain binding sites in the eEF1A1 and eEF1A2 molecules are predicted by bioinformatic analysis First we examined the possibility if the 98 % similar eEF1A1 and eEF1A2 might have distinct binding motifs for signalling molecules . A low stringency Scansite -LRB- Obenauer et al. , 2003 -RRB- search revealed potential SH2/SH3 domain containing protein partners and predicted the existence of several phosphorylation sites for tyrosine kinases in both isoforms -LRB- Fig. 1 -RRB- . However , the SH3 recognition motif specific for Src kinase and adaptor protein Crk was found only in the eEF1A2 molecule . This is an important indication of the possibility of differential involvement of eEF1A2 in the formation of signalling complexes . 3.2 eEF1A1 and eEF1A2 interact in vitro with SH2/SH3 domains of different proteins The ability of eEF1A1 and eEF1A2 to form complexes with SH2 and SH3 domains of different signalling molecules was directly analysed in pull down experiments . The preparative amounts of native eEF1A1 and eEF1A2 were purified from rabbit tissues as described in Section 2 . Importantly , the rabbit and human proteins are 100 % identical . SH2 domains of signalling proteins Grb2 , RasGAP , PLC-gamma1 , Shc , Shp2 and SH3 domains of Crk , Csk , Fgr , Fyn , Src were expressed as recombinant proteins fused with GST , purified from bacteria , immobilized on glutathione sepharose and mixed with eEF1A1 or eEF1A2 . Complexes of eEF1A1 and eEF1A2 with SH2 and SH3 domains of different proteins were detected -LRB- Fig. 2A and B -RRB- . Both eEF1A1 and eEF1A2 interacted with SH2 domain of PLC-gamma1 , while eEF1A2 exclusively formed complexes with SH2 domains of Grb2 , RasGAP , Shc , Shp2 and SH3 domains of Crk , Fgr , Fyn , PLC-gamma1 . It should be noted that principal possibility of the interaction of PLC-gamma1 and eEF1A was shown earlier . The interactions of SH2 and SH3 domains -LRB- Kim et al. , 1999 -RRB- or PH domain -LRB- Chang et al. , 2002 -RRB- of PLC-gamma1 with eEF1A were reported . Our results confirmed the interaction of eEF1A with SH2 and SH3 domains of PLC-gamma1 and were used as internal positive control of interaction with known eEF1A binding partner . Contrary to eEF1A1 , eEF1A2 was found to interact with SH2/SH3 domains of several adaptor proteins . Grb2 and Shc provide a critical link between cell surface growth factor receptors and the Ras and Myc signalling pathways via interactions with cytoplasmic Tyr kinases , G-proteins and other cellular Tyr-phosphorylated proteins . Interestingly , Shc is constitutively activated -LRB- tyrosine-phosphorylated -RRB- in most breast cancer cells where non-typical expression of eEF1A2 was reported -LRB- Frackelton et al. , 2006 ; Tomlinson et al. , 2005 -RRB- . Identification of RasGAP protein as a direct partner of G-protein eEF1A2 may be interesting in two ways . On the one hand , RasGAP may play GTPase activating role for eEF1A2 , because the initial rate of GTP hydrolysis by the intrinsic eEF1A2 GTPase is slightly lower when compared with eEF1A1 -LRB- Kahns et al. , 1998 and our unpublished observation -RRB- . On the other hand , eEF1A2 may influence RasGAP activity and consequently modulate Ras . eEF1A2 interacts also with SH3 domain of Crk which is involved in MAPK signalling , actin cytoskeleton reorganization and insulin receptor signalling -LRB- Feller , 2001 -RRB- . It should be noted that eEF1A is involved in actin cytoskeleton regulation as well -LRB- Gross & Kinzy , 2005 ; Shiina , Gotoh , Kubomura , Iwamatsu , & Nishida , 1994 -RRB- . Fgr and Fyn are Tyr kinases that participate , in particular , in the MAPK/ERK and insulin receptor signalling pathways involved in the control of initiation of translation -LRB- Alper & Bowden , 2005 -RRB- . The interaction of eEF1A2 with SH3 domains of Fgr and Fyn suggests eEF1A2 as a prospective substrate for these enzymes . Thus , eEF1A2 is capable of forming complexes with SH2 and SH3 domains of several signalling molecules . The potentially increased role of eEF1A2 , as compared to eEF1A1 , in phosphoTyr-mediated signalling pathways lends support for the putative oncogenic role assigned to eEF1A2 earlier -LRB- Amiri et al. , 2007 ; Lamberti et al. , 2004 ; Lee , 2003 -RRB- . 3.3 eEF1A1 and eEF1A2 are in complex with Shp2 in vivo Verification of the in vitro formed complexes was done in vivo taking as an example interaction of eEF1A with Shp2 . First , HEK 293 stable cell lines overexpressing eEF1A1-His , eEF1A2-His or GFP protein were used to confirm that endogenous Shp2 protein interacts with eEF1A2 . Interestingly , specific binding of endogenous Shp2 to both isoforms of eEF1A was detected -LRB- Fig. 3A -RRB- . Since only the C-terminal SH2 domain of Shp2 was used as a bait in the experiments in vitro , we suggest eEF1A1 and eEF1A2 bind to different domains of Shp2 . Also , a complex of endogenous eEF1A and Shp2 was immunoprecipitated from HEK293 cells using anti-eEF1A antibodies -LRB- Fig. 3B -RRB- . The complex eEF1A/Shp2 was detected in growing cells . Interestingly , complex formation was more effective in cells synchronized by growth factors starvation and less efficient in cells stimulated with serum , indicating the regulatory nature of this interaction -LRB- Fig. 3B -RRB- . Importantly , eEF1A2 was shown earlier to accumulate in G0 rather than in G1 and S phase cells -LRB- Ann et al. , 1991 -RRB- . However , since anti-eEF1A monoclonal antibodies are specific both for eEF1A1 and eEF1A2 , and the presence of both isoforms in embryonic cells is possible , it is difficult to determine precisely which isoform of eEF1A interacts with Shp2 in vivo . 3.4 eEF1A2 and Shp2 interaction is sensitive to dephosphorylation To test if the interaction of eEF1A2 with C-terminal SH2 domain of phosphatase Shp2 is dependent on phosphorylation status of eEF1A , equal amounts of total cell lysates of the stable cell lines expressing eEF1A1 or eEF1A2 were treated with calf intestine alkaline phosphatase , or , in control samples , incubated with phosphatase inhibitor -LRB- sodium orthovanadate -RRB- . Specific interaction of SH2-Shp2 and eEF1A2 was detected only in the samples not treated with CIAP indicating that phosphorylation of eEF1A2 is a necessary condition for eEF1A2/Shp2 complex formation -LRB- Fig. 3C -RRB- . 3.5 eEF1A1 and eEF1A2 are phosphorylated at Tyr in vivo The interaction of eEF1A with SH2 domains of different proteins in vitro as well as the eEF1A -- Shp2 complexes formation in vivo indicate the possibility of tyrosine phosphorylation in the eEF1A molecules . To test if native eEF1A1 and eEF1A2 contain such modifications we probed the isolated eEF1A proteins with monoclonal antibodies specific to phosphorylated tyrosines -LRB- 4G10 -RRB- . As a result , tyrosine phosphorylation of both isoforms was detected -LRB- Fig. 4A -RRB- . Moreover , the phosphorylation of eEF1A1 and eEF1A2 at tyrosine residues was confirmed using the His-tagged eEF1A isoforms precipitated on Ni-NTA sepharose -LRB- Fig. 4B -RRB- . The eEF1A1 and eEF1A2 samples dephosphorylated with calf intestine alcaline phosphatase were used to control specificity of 4G10 antibodies . Notably , eEF1A2 was characterized by greater phosphorylation level as compared to eEF1A1 in both experiments . 4 Discussion Besides its function in translation , eEF1A is thought to be involved in several cellular processes , including embryogenesis , senescence , oncogenic transformation , cell proliferation and organization of cytoskeleton -LRB- reviewed in Gross & Kinzy , 2005 ; Lamberti et al. , 2004 ; Lee , 2003 -RRB- . There are two tissue-specific isoforms of eEF1A . eEF1A1 functions in all mammalian tissues except neurons , cardiomyocytes and myotubes , where eEF1A2 is taking role and is expressed . Recently eEF1A2 was also found to express in human pancreatic islets -LRB- Ahmed , Forsberg , & Bergsten , 2005 -RRB- . Few attempts to differentiate the specific functions of the isoforms have been carried out so far . In vitro , eEF1A2 has slightly decreased translation functions -LSB- our unpublished observation -RSB- . Discovery of putative oncogenic role of eEF1A2 in ovarian cancer -LRB- Lee , 2003 -RRB- as well as opposite apoptosis-related functions of the isoforms -LRB- Chang & Wang , 2007 -RRB- indicated a possibility of distinct participation of eEF1A1 and eEF1A2 in cellular signalling processes . The mechanisms of cancer-related functions of eEF1A2 are not known . We suggested that difference of the isoforms could be related to their distinct ability to be involved into phosphoTyr-mediated signalling pathways . To examine such a possibility we used the panel of cloned SH2 and SH3 domains of various signalling molecules . SH2 domains are known to associate with specific phosphoTyr-containing sites -LRB- Schlessinger & Lemmon , 2003 -RRB- . SH3 domains of adaptor proteins can support the association with a number of signalling proteins and upon cell stimulation can engage adaptor proteins with Tyr-phosphorylated cytoplasmic or membrane-attached partners -LRB- Li , 2005 -RRB- . As a result , we detected the interaction of eEF1A1 and eEF1A2 with SH2 domain of PLC-gamma1 indicating that both isoforms could contain phosphoTyr residues . In addition , eEF1A2 was found associated with SH2 domains of Grb2 , RasGAP , Shc and Shp2 -LRB- Fig. 2A -RRB- . This suggests eEF1A2 plays a more universal role in signal pathways . The finding that eEF1A2 rather than eEF1A1 can interact with SH3 domains of Crk , Fgr and Fyn supports this notion -LRB- Fig. 2B -RRB- . The association of eEF1A and Shp2 is very appealing in terms of the role Shp2 is playing in the cell-activation of Ras , with subsequent activation of PI3K and MAPK signalling pathways -LRB- Mohi & Neel , 2007 -RRB- . In addition , Shp2 regulates actin cytoskeleton reorganization by modulating the activity of small GTP-binding protein Rho -LRB- Burks & Agazie , 2006 ; Harada , Katoh , & Negishi , 2005 -RRB- . It is interesting that eEF1A is an actin-binding protein which can regulate actin bundling as well , but its target substrates have not been identified so far -LRB- Murray , Edmonds , Liu , & Condeelis , 1996 -RRB- . Since direct binding of eEF1A to Shp1 was recently found to have some regulatory consequence , namely eEF1A2-dependent increase of Shp1 phosphatase activity -LRB- Nandan , Yi , Lopez , Lai , & Reiner , 2002 -RRB- the examination of possible effect of eEF1A2 on the Shp2 activity is a tempting task for future investigations . We have observed direct interaction of eEF1A2 with SH2 domain of RasGAP protein . Here we can only speculate about possible involvement of eEF1A2 in the activation of PI3K and MAPK pathways by Ras through RasGAP inactivation -LRB- Yarwood , Bouyoucef-Cherchalli , Cullen , & Kupzig , 2006 -RRB- . Otherwise , RasGAP may play a role of GAP for eEF1A2 . The last notion could be supported by the observation that the subunits of eEF1B , regular exchange factor for eEF1A1 , were not identified as interacting partners for eEF1A2 -LRB- Mansilla et al. , 2002 -RRB- . The phosphorylation of native and overexpressed eEF1A1 and eEF1A2 at tyrosine residues was confirmed by WB with phosphotyrosine-specific antibodies -LRB- Fig. 4A and B -RRB- . Several preps of eEF1A1 and eEF1A2 were analysed and eEF1A2 always demonstrated higher phosphorylation level than eEF1A1 -LRB- data not shown -RRB- . Recently , phosphorylation of Tyr 29 and Tyr 141 of eEF1A was found during an extensive search for all proteins containing phosphotyrosine in human cancer cells -LRB- Rush et al. , 2005 -RRB- . However , since both identified tryptic peptides contained phosphoTyr29 or phosphoTyr141 -LRB- which are identical in eEF1A1 and eEF1A2 -RRB- and the overall search was limited by the peptides containing phosphotyrosine , the unambiguous identification of the phosphorylated isoform in that case was hardly possible . Thus , we have identified several binding partners of eEF1A2 previously unknown . Novel interaction of eEF1A with tyrosine phosphatase Shp2 has been confirmed also in vivo . Potentially augmented ability of eEF1A2 as compared to eEF1A1 to be involved in key signal transduction complexes could be linked to the putative oncogenic role of the former found recently in ovarian cancer -LRB- Anand et al. , 2002 -RRB- . The definite role of eEF1A2 in activation of cell proliferation awaits further investigations .

O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O B O B O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O B O B O O O O O O O O O O O O O O O O O O B O B O O B I O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O B O O O O O O O O O O O B O O B I O O B O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O 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Effects of temperature and doxorubicin exposure on keratinocyte damage in vitro Cancer chemotherapy treatment often leads to hair loss , which may be prevented by cooling the scalp during drug administration . The current hypothesis for the hair preservative effect of scalp cooling is that cooling of the scalp skin reduces blood flow -LRB- perfusion -RRB- and chemical reaction rates . Reduced perfusion leads to less drugs available for uptake , whereas the reduced temperature decreases uptake of and damage by chemotherapy . Altogether , less damage is exerted to the hair cells , and the hair is preserved . However , the two mechanisms in the hypothesis have not been quantified yet . To quantify the effect of reduced drug damage caused by falling temperatures , we investigated the effect of local drug concentration and local tissue temperature on hair cell damage using in vitro experiments on keratinocytes . Cells were exposed for 4 h to a wide range of doxorubicin concentrations . During exposure , cells were kept at different temperatures . Cell viability was determined after 3 d using a viability test . Control samples were used to establish a concentration -- viability curve . Results show that cell survival is significantly higher in cooled cells -LRB- T < 22 ° C -RRB- than in non-cooled cells -LRB- T = 37 ° C -RRB- , but no significant differences are visible between T = 10 ° C and T = 22 ° C. Based on this result and previous work , we can conclude that there is an optimal temperature in scalp cooling . Further cooling will only result in unnecessary discomfort for the patient and should therefore be avoided . Introduction Chemotherapy-induced hair loss is a feared side effect of cancer treatment -LRB- Katsimbri et al. 2000 -RRB- . Scalp cooling during administration of chemotherapy prevents hair loss -LRB- e.g. , Ridderheim et al. 2003 -RRB- . Cooling can be achieved by means of a cap that is pre-cooled in a freezer or that exchanges coolant with a reservoir . The hair preservative effect of scalp cooling is attributed both to reduced blood flow by vasoconstriction and to reduced reaction rates in the body at the level of the hair follicle . When the scalp is cooled , vasoconstriction is induced , and through this , blood flow to the hair follicle is decreased . This reduced blood flow results in a decrease of the total amount of cytotoxic drug available for uptake in the hair follicle . The effect of reduced perfusion was studied by Janssen et al. -LRB- 2007 -RRB- , and it was found that cooling the scalp to 20 ° C reduces local blood flow down to 20 % of normal . A further decrease in temperature did not result in a further decrease in local blood flow . Based on this , it might be expected that there is a limit in temperature below which the effectiveness of scalp cooling will not increase anymore . However , the hair preservative effect of scalp cooling is also attributed to reduced cell metabolism . Because of reduced temperatures during scalp cooling , cellular drug uptake and damage are assumed to be lower , and with this , hair follicles are thought to be less susceptible to cytotoxic drugs . Decorti et al. -LRB- 2003 -RRB- showed that in vitro doxorubicin uptake in kidney cells at 37 ° C was 4.5 times higher than drug uptake at 4 ° C . In addition , Decorti also showed that doxorubicin uptake is dependent on extracellular concentration . Unfortunately , no studies have been performed to quantify these processes in the human hair follicle . For a better understanding of the functioning of scalp cooling , it is important to quantify the contribution of reduced drug uptake and drug damage , if any , on the hair preservative effect of scalp cooling . To this end , we investigated experimentally the relationship between doxorubicin exposure and cell damage at different temperatures using normal human epidermal keratinocytes . Chemotherapy disrupts the rapidly dividing keratinocytes -LRB- the cells that actually produce the hair shaft -RRB- in the hair follicle -LRB- Cotsarelis and Millar 2001 -RRB- . Therefore , the use of keratinocytes as an in vitro model is indicative for chemotherapy-induced damage to the hair follicle . Moreover , human keratinocytes are commercially available and they are easy to cultivate , which means that with this model , a wide range of boundary conditions can quickly be investigated . The goal of the experiments was to assess the effects of temperature and chemotherapy on keratinocyte survival . Materials and Methods Pooled neonatal normal human epidermal keratinocytes -LRB- NHEK -RRB- were obtained from Cambrex Bio Science Verviers , Belgium -LRB- catalog number CC-2507 -RRB- . These cells have a doubling time of approximately 24 h. Cells were cultured in T-75 flasks at a seeding density of 3,500 cells cm2 under an atmosphere of 95 % air and 5 % CO2 at 37 ° C using 15 ml of keratinocyte general medium -LRB- KGM2 ; Cambrex Bio Science Verviers , catalog number CC-3107 -RRB- . Medium was refreshed the day after plating , and afterwards every other day until cells reached 70 -- 80 % confluence . Then , cells were harvested using 6 ml of trypsin solution and subsequently cryopreserved in liquid nitrogen at a density of 1.2 × 106 cells per milliliter using 80 % KGM2 , 10 % fetal bovine serum , and 10 % dimethyl sulfoxide -LRB- DMSO -RRB- . For the experiments on the effect of temperature and chemotherapy on keratinocyte survival , third passage cells were plated in 24-well plates at a cell density of 6,000 cells per square centimeter . Cells were incubated at 37 ° C for a period of 24 h to allow the cells to recover from handling . Concentrations of doxorubicin in KGM2 -LRB- 0.01 , 0.04 , 0.1 , 0.5 , 1.0 , 3.0 , or 10.0 μg ml − 1 -RRB- were prepared . Cells were then exposed to 250 μl of a specific doxorubicin concentration . Plates were incubated for 4 h at either a low temperature -LRB- TL = 10 ° C -RRB- , a medium temperature -LRB- TM = 22 ° C -RRB- , or a high temperature -LRB- TH = 37 ° C -RRB- . For each combination of temperature and doxorubicin concentration , a sample size of eight was used . Cells receiving medium without any doxorubicin were used as a control group for each specific temperature . At the end of the exposure time , doxorubicin was removed , and cells were washed with 500 μl phosphate-buffered saline -LRB- PBS -RRB- . Fresh medium -LRB- 250 μl per well -RRB- was added to each well , and plates were then incubated at 37 ° C for a post-exposure time of 72 h , after which , a viability assay was performed as described below . The damage to the NHEK cells exposed to different temperatures and doxorubicin concentrations was determined by a colorimetric MTT -LRB- tetrazolium -RRB- viability assay . The assay is based on the observation that viable cells have the ability to metabolize a water-soluble tetrazolium dye 3 - -LRB- 4,5-dimethylthiazol-2-yl -RRB- -2,5-diphenyl tetrazolium bromide -LRB- MTT -RRB- into a product termed purple formazan -LRB- Mosmann 1983 ; Edmondson et al. , 1988 ; Sgouras and Duncan , 1990 -RRB- . The purple formazan can be solubilized , and the optical density of the solute can be determined using a spectrophotometric technique . The resulting absorbance is directly proportional to the number of cells , and this linearity extends over a wide range of cell numbers -LRB- Mosmann 1983 -RRB- . A standard MTT solution was prepared by dissolving 5 mg ml − 1 of MTT -LRB- MTT formazan , Sigma Aldrich , Zwijndrecht , The Netherlands -RRB- in PBS . The standard MTT solution was added to complete medium -LRB- KGM2 -RRB- in a ratio of 1 to 10 to obtain a final solution of 10 % MTT solution and 90 % KGM2 . A volume of 220 μl of this solution was added to all test wells , and the plates were left to incubate at 37 ° C and 5 % CO2 for a period of 45 min . After this period , the MTT/medium solution was removed , and 200 μl of a solution of 90 % DMSO and 10 % Triton X was added to extract the purple formazan salt from the cells . Plates were sealed and kept in dark for 30 min , after which , 100 μl of each well was transferred to a new well of a 96-well plate . The optical density -LRB- OD -RRB- was measured using an automated spectrophotometric plate reader -LRB- Synergy HT , BioTek Instruments Inc. , Winooski , VT , USA -RRB- set to 570 nm and using a reference wavelength of 650 nm . Blanks -LRB- 100 μl of DMSO/Triton X solution only -RRB- were used as an extra control . For each well , the corrected optical density OD -LRB- 570 -- 650 nm -RRB- was provided by the plate reader . Mean optical density and standard error of each temperature and concentration group -LSB- ODT , C ± SE -LRB- ODT , C -RRB- -RSB- and their respective control group -LSB- ODT ,0 ± SE -LRB- ODT ,0 -RRB- -RSB- were calculated . Based on these values , the viability -LRB- S -RRB- for each temperature and concentration group can be calculated as : An analysis of variance -LRB- ANOVA -RRB- was performed using statistical software -LRB- SPSS version 15.0 , SPSS Inc. , Chicago , IL -RRB- . The ANOVA test is used to investigate whether significant differences in mean values exist between two or more groups . In this study , we want to investigate what the effect of temperature on the one hand , and the effect of doxorubicin concentration on the other hand , is on cell survival . Using a general linear model , the viability is statistically modeled as : This means that we investigated whether temperature , concentration , and an interaction term are significantly relevant for describing our data . Where appropriate , a post hoc analysis was used using Tamhane 's T2 criterium -LRB- based on unequal variances -RRB- to test for differences between various doxorubicin concentrations and various exposure temperatures . A significance level of p < 0.05 was used in these statistical tests . Results Microscopic photographs of cells at selected concentrations are shown in Fig. 1 . Here , we can see that the control groups of all temperatures show similar cell counts . With increasing concentration , the number of cells decreases . At 3 μg ml − 1 -LRB- C4 -RRB- , a clear difference is visible between the high temperature group and both the medium and low temperature group . The difference between these groups at other concentrations is less well defined . It can be seen that there is a difference in cell morphology between 0.04 μg ml − 1 -LRB- C2 -RRB- and 0.5 μg ml − 1 -LRB- C3 -RRB- . Cells in the latter group , and in groups with higher doxorubicin concentrations , show enlarged and flattened cell shape and increased granularity . These are characteristics of senescence or aging , which is known to be caused by doxorubicin -LRB- Roninson 2003 -RRB- . The results of the viability determination are shown in Fig. 2 . This figure shows the cell viability as a function of doxorubicin concentration for different doxorubicin exposure temperatures . In this figure , we can see that the viability of keratinocytes as a function of doxorubicin concentration shows a decreasing S-curve with increasing concentrations . Viability levels are slightly above 1 for low concentrations -LRB- 0.01 μg ml − 1 -RRB- , and with increasing concentrations , viability gradually drops towards zero for high concentrations -LRB- 10 μg ml − 1 -RRB- . For different temperature groups , cell viability at a specific concentration is always lowest for the high temperature group . In the mid-section of the concentration range -LRB- i.e. , 0.1 -- 1 μg ml − 1 -RRB- , the differences between the high temperature group -LRB- TH -RRB- and the two lower temperature groups -LRB- TL and TM -RRB- are more pronounced . Cell viability for these lower temperature groups decreases more slowly than for the high temperature group , until at a concentration of 1 μg ml − 1 , a rapid drop in cell viability is visible . The difference between the individual temperature groups at low and high concentrations is therefore small . Figure 1 . Microscopic photographs of cells exposed to selected doxorubicin concentrations . Photographs are taken at a magnification of ten times . C1 = 0.01 μg ml − 1 , C2 = 0.04 μg ml − 1 , C3 = 0.5 μg ml − 1 , C4 = 3.0 μg ml − 1 , C5 = 10 μg ml − 1 . Figure 2 . Cell viability as a function of doxorubicin concentration at different temperatures . Delta : TL -LRB- 10 ° C -RRB- , filled square : TM -LRB- 22 ° C -RRB- , and open circle : TH -LRB- 37 ° C -RRB- . Data points show the mean and standard error of eight replicates . The results of a significance test are shown in Table 1 . From an ANOVA analysis , we found that the effect of doxorubicin concentration on NHEK viability was highly significant -LSB- F -LRB- 6,161 -RRB- = 165.213 , p < 0.001 -RSB- , as was the effect of temperature on NHEK viability -LSB- F -LRB- 6,161 -RRB- = 37.054 , p < 0.001 -RSB- . The effect size of doxorubicin -LRB- ; partial eta squared -RRB- was higher than the effect size of temperature -LRB- = 0.88 compared to = 0.36 -RRB- . For all temperature groups , increased doxorubicin concentration has a significant decreasing effect on cell viability . This effect was highly significant for the high temperature group -LSB- F -LRB- 6,49 -RRB- = 119.400 , p < 0.001 -RSB- , the intermediate temperature group -LSB- F -LRB- 6,49 -RRB- = 33.166 , p < 0.001 -RSB- , and the lowest temperature group -LSB- F -LRB- 6,49 -RRB- = 68.225 , p < 0.001 -RSB- Based on these results , a post hoc analysis was performed for each temperature group to check where a significant transition in cell viability as function of doxorubicin concentration occurs . This transition point may be viewed as the inflection point of the S-curve . For the low and medium temperature group , we found a transition point between 1.0 and 3.0 μg ml − 1 . The highest temperature group has a transition point slightly below 0.5 μg ml − 1 . The ANOVA analysis also revealed a significant interaction between doxorubicin and temperature on cell viability -LSB- F -LRB- 6,161 -RRB- = 2.754 , p = 0.002 -RSB- , although the effect is smaller than that of temperature and doxorubicin -LRB- = 0.199 -RRB- . This significant interaction term shows that the effect of temperature on cell viability is not the same at the levels of different doxorubicin concentrations . For low and high doxorubicin concentrations , there is hardly any influence of a lower temperature on cell survival . In the mid-section of the graph this influence is far more substantial . The results of the ANOVA analysis are summarized in Table 1 . It can be seen that significant differences exist between TL and TH for concentrations of 0.1 μg ml − 1 and up , except for a concentration of 1 μg ml − 1 . At this concentration , the significance level is not high enough , but still , a trend is visible -LRB- p < 0.1 -RRB- . Between TM and TH , significant differences exist for concentrations of 0.5 μg ml − 1 and up , except for a concentration of 1 μg ml − 1 where no significant difference is visible . The only significant difference between TL and TM exists at a concentration of 10 μg ml − 1 , although a trend is visible at a concentration of 0.1 μg ml − 1 . Table 1 . Results of an ANOVA analysis for determining the significance between different temperature groups at a specific doxorubicin concentrationDoxorubicin -LRB- μg ml − 1 -RRB- F valuep valueTL vs THTM vs THTL vs TM0 .01 F -LRB- 2,21 -RRB- = 0.3150.733 0.04 F -LRB- 2,21 -RRB- = 1.9580.166 0.1 F -LRB- 2,21 -RRB- = 15.187 < 0.001 p < 0.01 p < 0.1 * 0.5 F -LRB- 2,21 -RRB- = 31.605 < 0.001 p < 0.001 p < 0.001 1F -LRB- 2,21 -RRB- = 4.380 < 0.05 p < 0.1 * 3F -LRB- 2,21 -RRB- = 12.908 < 0.001 p < 0.01 p < 0.01 10F -LRB- 2,21 -RRB- = 22.587 < 0.001 p < 0.001 p < 0.05 p < 0.05 p Values are shown with their respective significance level . Trends -LRB- p < 0.1 -RRB- are marked with an asterisk . See text for more detail . Discussion We examined the role reduced metabolism has on the hair preservative effect of scalp cooling . For this , a controlled in vitro experiment was conducted to assess the damage exerted by doxorubicin on human epidermal keratinocytes for a range of chemotherapy concentrations and exposure temperatures . The effects of temperature and doxorubicin on cell damage were determined from cell viability measurements -LRB- MTT assay -RRB- . We found that increased doxorubicin concentrations have a significant decreasing effect on keratinocyte cell viability . Viability was close to 1 for low concentrations and close to 0 for high concentrations . In between , the relationship between doxorubicin concentration and cell viability shows an S-curve . We also found that reduced temperature has a significant increasing effect on keratinocyte cell viability . The two cooled temperature groups -LRB- 10 ° C and 22 ° C -RRB- show a higher cell viability for each doxorubicin concentration than the 37 ° C group . At the mid-range of doxorubicin concentrations -LRB- 0.04 -- 1 μg ml − 1 -RRB- , the effect of reduced temperature is more pronounced than in the extreme values of doxorubicin concentration . This observation is confirmed by the fact that an interaction term of doxorubicin concentration and exposure temperature was significant . The increased effect of reduced temperature was significant for doxorubicin concentrations of 0.1 μg ml − 1 and higher . We found no significant differences between the low -LRB- 10 ° C -RRB- and medium -LRB- 22 ° C -RRB- group . The results of the experiment are in line with the hypothesis of scalp cooling . In this hypothesis , the reduced temperature caused by scalp cooling will result in lower perfusion and a lower cellular metabolism . With this , the supply , uptake and damage of a chemotherapeutic agent will be diminished . For low doxorubicin concentrations -LRB- 0.01 and 0.04 μg ml − 1 -RRB- , the MTT assay for each temperature group showed a higher viability than the respective control group . Because of the fact that each specific temperature group has its own control group , it is unlikely that this effect is caused by a measurement error . This means that apparently , cells are metabolically more active when exposed to a small amount of doxorubicin . It is possible that cells in the control group are close to reaching confluence at the time that viability is determined . As cells reach confluence , there is not enough room for new cells . Therefore , the rate at which cells divide will become lower , and hence , their metabolic activity will decrease . Another possibility is that a small amount of doxorubicin will stimulate cells to repair damage exerted to the cells . Hence , it may actually result in larger cell growth compared to a control group receiving no doxorubicin . At this point , however , we have no indication for which explanation may be true . Another remarkable result is that there is no significant difference between the medium and low temperature group . Thus , it seems that there is a limit in effect of reduced temperature . In a pilot study to determine optimal doxorubicin exposure temperature , we found that an exposure temperature of 26 ° C also showed increased cell viability -LRB- data not shown -RRB- , and this increase did not differ from other temperature groups . During scalp cooling , a skin temperature of approximately 20 ° C is reached . Based on the results of our experiment , one might expect that the limit in effect of reduced temperature during scalp cooling may be reached for a temperature as high as 26 ° C . This means that during scalp cooling , the effect of reduced metabolism is already at its maximum . However , care has to be taken when the results of an in vitro experiment are generalized . Therefore , further studies on the effect of reduced temperature on cell damage are needed to exactly define this limit . Ideally , an in vitro hair follicle model is used in these studies . Our current study confines the boundary conditions in doxorubicin concentration and exposure temperature that can be used in these further studies . Recently , it was shown that there is a limit in perfusion during scalp cooling -LRB- Janssen et al. 2007 -RRB- . Cooling the scalp to approximately 18 ° C resulted in a decrease in perfusion to 20 % . Further cooling did not result in any further decrease in perfusion . The findings of the current study indicate that there is no difference in cell survival between 22 ° C and 10 ° C. Based on these findings , we can conclude that there is an optimal temperature in scalp cooling . Further cooling will only result in unnecessary discomfort for the patient . Therefore , strong cooling during scalp cooling should be avoided .

O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O B I O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O B O O O O B O O O B O O O O O O O O O O O O B O B O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O B O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B I O O O O

scalp cooling
doxorubicin
keratinocytes
viability


Impact of the mesh fixation technique on operation time in laparoscopic repair of ventral hernias Background Fixation of the prosthesis is one of the critical components of laparoscopic repair of ventral and incisional hernia -LRB- LRVIH -RRB- . The impact of the fixation technique used on operative time has never been analyzed . We compared the duration of the operation according to the fixation technique used in a series of 138 patients with primary umbilical hernia . Introduction Laparoscopic repair of ventral and incisional hernia -LRB- LRVIH -RRB- is gaining increasing popularity due to its low recurrence rate , short hospital stay , and low complication rate . Fixation of the prosthesis is one of the critical components of LRVIH . Currently , the two most popular methods of mesh fixation are the use of helical tackers either with or without transabdominal sutures -LRB- TAS -RRB- . Overall it appears that the two laparoscopic mesh fixation techniques are similar in outcomes -LSB- 1 -RSB- . However , the impact of the applied fixation technique on operative time has never been analyzed . We compared the duration of the operation according to the fixation technique used in a series of patients with primary umbilical hernia -LRB- PUH -RRB- who underwent a straightforward repair by using completely standardized techniques . Patients and methods Data were collected from operative reports of all 167 patients who underwent laparoscopic repair of PUH up to January 2007 . To make the data more homogenous , we excluded patients who required adhesiolysis -LRB- n = 13 -RRB- or simultaneously underwent another procedure -LRB- n = 12 -RRB- , or with whom a minor complication occurred intraoperatively , i.e. , bleeding or equipment problem -LRB- n = 4 -RRB- . The remaining 138 patients underwent straightforward repair of PUH by using a completely standardized technique , and they represented the study group used to compare operative times between the two mesh fixation techniques . Pneumoperitoneum was established by using a Veress needle . Three trocars -LRB- 10 , 5 , and 5 mm -RRB- were inserted left laterally . A 1-mm-thick expanded polytetrafluoroethylene mesh -LRB- DualMesh ® , WL Gore , Flagstaff , AZ -RRB- of 15 by 10 cm was used to overlap the hernia opening by at least 3 -- 4 cm . The mesh was fixed using one of the following two techniques : either -LRB- 1 -RRB- with tackers -LRB- ProTack ® , TycoUSS , Norwalk , CT -RRB- along the periphery of the mesh at intervals of 1 -- 1.5 cm and eight TAS placed equidistant also along the periphery -LRB- n = 107 , further called the TAS technique -RRB- or -LRB- 2 -RRB- with a double ring of tackers alone without the use of TAS -LRB- n = 31 , further called the DC technique -RRB- . With this technique , also known as a `` double crown technique '' -LSB- 2 -RSB- , the outer ring of tackers is the same as in the TAS technique . The inner ring of tackers is placed around the hernia opening about 1 cm apart . Transabdominal sutures were pulled through the abdominal wall with a suture passer -LRB- Gore Suture Passer Instrument ® , WLGore , Flagstaff , AZ -RRB- . In the first 84 patients , the method of mesh fixation was based on the surgeon 's preference . For the last 54 patients , the mesh fixation technique performed was randomly chosen in conjunction with another study being done at our hospital that compares postoperative pain following these two methods of mesh fixation . Once the fixation was completed , the abdomen was desufflated and the trocar site of 10 mm and skin were closed . The time taken from the stab wound for insertion of a Veress needle to completion of skin closure was recorded to the nearest minute . Statistical analysis was done using the t test . Significance was set at a P < 0.05 . Results Of the 138 patients in our study group , 106 were males and 36 females . Mean age was 55.0 ± 12.3 years . The TAS and DC groups were found to be well matched for sex and age . Mean operating time for the TAS technique was 50.6 min compared to 41.4 min for the DC technique . Mean difference in operating time was 9.2 min . This difference was significant -LRB- P = 0.002 -RRB- . Mean postoperative hospital stay was identical for both groups and equaled 1 day . Postoperative complications included seroma lasting longer than 6 weeks in seven patients and chronic pain at TAS sites in one patient . All seromas resolved without intervention . The patient with persisting pain underwent relaparoscopy and removal of all eight TAS that provided complete pain relief . There were no significant differences in morbidity between the two groups . No recurrences were detected during a mean follow-up of 26.4 ± 25.6 months . Discussion Besides prosthetic material that must sufficiently overlap the hernia defect , a reliable fixation of the prosthesis against the abdominal wall is a crucial component for success of LRVIH . Since the early years of LRVIH , there has been a strong belief among pioneers of this technique that reliable fixation of the mesh can only be achieved with the TAS technique -LSB- 3 , 4 -RSB- and , in addition , that sutured mesh fixation is an imperative in LRVIH -LSB- 5 -RSB- . Another technique of fixation introduced later and consisting of a double crown of tackers only has been gaining increasing popularity due to a few specified advantages : technical simplicity , fewer incisions in the skin and possibly shorter operative time -LSB- 2 , 6 -RSB- . One of the most interesting issues on LRVIH currently being debated is which of these two techniques is better . Prospective randomized studies comparing the two techniques are missing . Meta-analysis of published literature indicates that the two laparoscopic mesh fixation techniques are similar in main outcome parameters such as recurrence and complication rate -LSB- 1 -RSB- . In the same analysis , the use of tackers alone resulted in a slightly shorter operative time than when the TAS technique was used . However , in most of the studies that were included in this meta-analysis , fixation with tackers was performed by applying only a single row of tackers or technical details of the tacker fixation technique were not explained . Reliable data comparing TAS technique and DC technique are definitely missing . We decided to compare the two techniques in a maximally homogenous model of the procedure : same site of hernia , same logistics of the operation , same prosthetic material , same fixation device , and same operation technique until the moment that the outer ring of tackers is completed . The only difference between the two techniques was the completion of the mesh fixation . In one technique eight TAS had to be inserted and in the other technique usually 6 -- 8 tackers of the inner crown . Once the fixation had been completed , whatever the method was , the rest of the procedure was again identical . Obviously , the difference in operative times between the two operative techniques can be entirely accounted to the difference in time needed for insertion of eight TAS compared to the time needed for application of an inner crown of tackers . Since tacking of the inner crown takes definitely not more than 1 min in laparoscopic repair of PUH , insertion of eight TAS required at least 8 min . This strongly indicates that insertion of every single TAS prolongs LRVIH by approximately 1 min . A laparoscopic repair of PUH is definitely the least complex procedure among all LRVIHs . Insertion of TAS is probably easier than in other LRIVHs due to a central location of the hernia , a general absence of adhesions , maximal space between the distended abdominal wall and the bowel underneath , and an excellent view . It may be anticipated that insertion of TAS during more complex laparoscopic repairs of incisional hernias at less suitable sites , in the presence of adhesions and proximity of the bowel can be much more challenging and as a consequence will require more time than during repair of a PUH . Our results indicate that LRVIH by using the DC technique indeed requires less operative time than when the TAS technique is used . As long as no significant differences between the two fixation techniques are demonstrated on issues of recurrence , complications , and postoperative pain , the time difference we have measured might be an argument in favor of the DC technique , especially when mesh fixation would require a large number of TAS .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

laparoscopy
study, time
surgical mesh
surgical fixation devices


Effects of low-frequency stimulation of the subthalamic nucleus on movement in Parkinson 's disease Excessive synchronization of basal ganglia neural activity at low frequencies is considered a hallmark of Parkinson 's disease -LRB- PD -RRB- . However , few studies have unambiguously linked this activity to movement impairment through direct stimulation of basal ganglia targets at low frequency . Furthermore , these studies have varied in their methodology and findings , so it remains unclear whether stimulation at any or all frequencies ≤ 20 Hz impairs movement and if so , whether effects are identical across this broad frequency band . To address these issues , 18 PD patients chronically implanted with deep brain stimulation -LRB- DBS -RRB- electrodes in both subthalamic nuclei were stimulated bilaterally at 5 , 10 and 20 Hz after overnight withdrawal of their medication and the effects of the DBS on a finger tapping task were compared to performance without DBS -LRB- 0 Hz -RRB- . Tapping rate decreased at 5 and 20 Hz compared to 0 Hz -LRB- by 11.8 ± 4.9 % , p = 0.022 and 7.4 ± 2.6 % , p = 0.009 , respectively -RRB- on those sides with relatively preserved baseline task performance . Moreover , the coefficient of variation of tap intervals increased at 5 and 10 Hz compared to 0 Hz -LRB- by 70.4 ± 35.8 % , p = 0.038 and 81.5 ± 48.2 % , p = 0.043 , respectively -RRB- . These data suggest that the susceptibility of basal ganglia networks to the effects of excessive synchronization may be elevated across a broad low-frequency band in parkinsonian patients , although the nature of the consequent motor impairment may depend on the precise frequencies at which synchronization occurs . Introduction There is extensive evidence that neuronal activity is abnormally synchronized at low frequencies in Parkinson 's disease -LRB- PD -RRB- and in animal models of parkinsonism -LRB- reviewed in Gatev et al. , 2006 ; Hammond et al. , 2007 ; Uhlhaas and Singer , 2006 -RRB- . However , this does not , by itself , prove that pathological synchrony is mechanistically important in parkinsonism . More persuasive evidence would be the impairment of voluntary movement by the artificial synchronization of neural activity in the basal ganglia . Such synchronization is possible by stimulating deep brain electrodes implanted for the treatment of PD at low frequencies , rather than at those frequencies above 100 Hz used for therapeutic benefit . Electrical stimulation of surgical targets like the subthalamic nucleus -LRB- STN -RRB- simultaneously activates neural elements in the vicinity of the electrode and this synchronous activity is then propagated onwards , as evinced by evoked pallidal -LRB- Brown et al. , 2004 ; Hashimoto et al. , 2003 -RRB- , cortical -LRB- MacKinnon et al. , 2005 -RRB- and muscular activity -LRB- Ashby et al. , 1999 , 2001 -RRB- . So far there have been several reports of the impairment of movement by stimulation of the STN at frequencies ≤ 20 Hz in patients with PD . Moro et al. -LRB- 2002 -RRB- and Chen et al. -LRB- 2007 -RRB- studied finger tapping during DBS at 5 Hz and 20 Hz , respectively , and found this to be slowed . However , Timmermann et al. -LRB- 2004 -RRB- , using the motor United Parkinson 's Disease rating scale -LRB- UPDRS -RRB- , failed to confirm a worsening during DBS at 5 and 20 Hz , but did find increased bradykinesia with stimulation at 10 Hz . Another study evaluated tapping performance over several frequencies within the same patients , but only found weak effects that involved relative rather than absolute impairments in motor performance , superimposed upon an overall tendency for movement to improve with increasing stimulation frequency -LRB- Fogelson et al. , 2005 -RRB- . Accordingly , it is unclear whether stimulation at any or all frequencies ≤ 20 Hz impairs movement and if so , whether effects are identical across this broad frequency band . The issue is an important one , as although spontaneous synchrony tends to occur at frequencies centered around 20 Hz in PD -LRB- Hammond et al. , 2007 -RRB- , it occurs at rather lower frequencies in the 1-methyl-4-phenyl-1,2,3,6 , - tetrahydropyridine -LRB- MPTP -RRB- primate model of PD -LRB- Goldberg et al. , 2004 ; Raz et al. , 1996 , 2000 -RRB- . Here we contrast the effects of STN stimulation and thereby extrinsically imposed synchronization at a number of frequencies ≤ 20 Hz , to establish whether all such frequencies impair movement and , if so , whether they impair movement in the same way . To this end we studied performance in a simple finger tapping task , as this is objective and correlates with motor impairment -LRB- Giovannoni et al. , 1999 ; Rabey et al. , 2002 -RRB- , and considered changes in task execution according to baseline performance -LRB- Chen et al. , 2006a -RRB- . Materials and methods Patients and surgery Twenty patients participated with informed consent and the permission of the local ethics committees -LRB- 5 females , mean age 59.5 ± 1.4 years ; mean disease duration 13.5 ± 1.0 years -RRB- . Their clinical details are summarized in Table 1 . Fourteen of these patients had also been recorded at least 6 months previously in a different paradigm involving stimulation at 20 Hz , 50 Hz and 130 Hz -LRB- Chen et al. , 2007 -RRB- . Implantation of bilateral STN DBS electrodes was performed in all subjects for treatment of Parkinson 's disease at least 6 months prior to study -LRB- mean 34.7 ± 5.9 months -RRB- . The DBS electrode used was model 3389 -LRB- Medtronic Neurological Division , Minneapolis , USA -RRB- with four platinum -- iridium cylindrical surfaces -LRB- 1.27 mm diameter and 1.5 mm length -RRB- and a centre-to-centre separation of 2 mm . Contact 0 was the most caudal and contact 3 was the most rostral . The intended coordinates at the tip of contact 0 were 10 -- 12 mm from the midline , 0 -- 2 mm behind the midcommissural point and 3 -- 5 mm below the anterior commissural -- posterior commissural line . Adjustments to the intended coordinates were made in accordance with the direct visualization of STN in individual stereotactic MRI -LRB- Hariz et al. , 2003 -RRB- and , in the patients operated in Taiwan -LRB- n = 7 -RRB- , the results of microelectrode recordings . Correct placement of the DBS electrodes in the region of the STN was further supported by : -LSB- 1 -RSB- effective intra-operative macrostimulation ; -LSB- 2 -RSB- post-operative T2-weighted MRI compatible with the placement of at least one electrode contact in the STN region ; -LSB- 3 -RSB- significant improvement in UPDRS motor score during chronic DBS off medication -LRB- 22.7 ± 3.0 -RRB- compared to UPDRS off medication with stimulator switched off -LRB- 52.6 ± 4.8 ; p < 0.00001 , paired t-test -RRB- . One patient was excluded due to the absence of significant improvement in UPDRS motor score during chronic DBS and another one due to missing clinical data . Protocol All patients were assessed after overnight withdrawal of antiparkinsonian medication , although the long action of many of the drugs used to treat PD meant that patients may still have been partially treated when assessed . They were studied when the stimulator was switched off and during bilateral STN stimulation at 5 Hz , 10 Hz and 20 Hz . The stimulation types were assessed in pseudo-randomized order across patients , as was the presentation order of trials within a stimulation type . Stimulation contacts , amplitude and pulse duration were the same as utilized for therapeutic high frequency stimulation in each subject -LRB- see Table 1 -RRB- . There was no evidence of capsular spread during stimulation , as determined by clinical examination . Patients were not informed of the stimulation type . We did not stimulate one side at a time to avoid possible functional compensation by the non-stimulated side . We waited 20 min after changing between conditions before testing . This is sufficient time to elicit about 75 % of DBS effects -LRB- Temperli et al. , 2003 -RRB- . Task The task was repetitive depression of a keyboard key as fast as possible by rapid alternating flexion and extension of the index finger at the level of the metacarpophalangeal joint -LRB- Chen et al. , 2006a , 2007 -RRB- . Tapping was performed in two runs of 30 s , separated by ∼ 30-s rest and each hand tested separately -LRB- giving four runs per condition -RRB- . Data from one side were rejected as these were collected contralateral to a previous unilateral pallidotomy -LRB- case 18 in Table 1 -RRB- . The number of taps made with the index finger in 30 s was recorded and the run from each pair with the best performance selected for analysis , as this was less likely to be affected by fatigue , or the effects of impaired arousal/concentration . Statistics The results of the tapping task in patients were analyzed according to their baseline performance -LRB- e.g. without stimulation -RRB- . The lower limit of normal baseline performance was obtained by testing ten healthy age matched control subjects -LRB- 20 sides , 4 males , mean age 57 years , range 52 -- 64 years -RRB- using the same tapping task . The mean tapping rate in this control group was 162 taps/30 s . The lower limit of the normal range -LRB- e.g. mean − -LSB- 2 × standard deviation -RSB- -RRB- in this control group was 127 taps per 30 s . The 35 tapping sides studied in the 18 patients were accordingly divided into those with baseline performance within normal limits -LRB- n = 17 ; the mean tapping performance across this group , 157 taps/30 s , was still lower than the mean tapping performance in healthy subjects -RRB- and those with baseline tapping rates lower than normal limits -LRB- n = 18 ; mean tapping performance 58 taps/30 s -RRB- . The rationale behind this approach was to select those sides -LRB- with baseline performance within normal limits -RRB- in which any deleterious effects of DBS would not be overshadowed by the beneficial effects of DBS-induced suppression of spontaneous pathological activity or limited by floor effects due to major baseline impairment -LRB- Chen et al. , 2006a , 2007 -RRB- . Four subjects had sides distributed across the two groups of differing baseline tapping performance . Tapping rates and coefficients of variation were normally distributed -LRB- one-sample Kolmogorov -- Smirnov tests , p > 0.05 -RRB- . Repeated-measures ANOVAs with within-subjects simple contrasts -LRB- comparing different frequencies of stimulation to no stimulation -RRB- were performed in SPSS -LRB- SPSS for Windows version 11 , SPSS Inc. , Chicago , IL , USA -RRB- . Mauchly 's test was used to determine the sphericity of the data entered in the ANOVAs , and where data were non-spherical Greenhouse -- Geisser corrections applied . Means ± standard error of the means are presented in the text . Results Low-frequency stimulation had no reliable clinical effect and did not consistently induce tremor , mobile dyskinesia , or dystonic posturing . Four patients experienced dystonic postures during the experiment and one had increased tremor . However , these effects were seen for overlapping stimulation frequencies . We divided the tapping sides into two groups according to whether or not tapping performance off DBS was within normal limits established on 20 sides in 10 healthy age-matched subjects -LRB- see Materials and methods -RRB- . ANOVA of tapping scores with factors FREQUENCY -LRB- four levels : 0 , 5 , 10 and 20 Hz -RRB- and BASELINE TAPPING PERFORMANCE -LRB- two levels : within normal limits and less than normal limits -RRB- demonstrated a within-subjects interaction between FREQUENCY and BASELINE TAPPING PERFORMANCE -LRB- F -LSB- 3,48 -RSB- = 4.224 , p = 0.01 -RRB- . Data were further analyzed with separate ANOVAs in each baseline tapping performance group . In those patients with baseline tapping performance within normal limits , ANOVA with the factor FREQUENCY confirmed that the latter was a significant main effect -LRB- F -LSB- 3,48 -RSB- = 3.777 , p = 0.016 -RRB- . Within-subjects contrasts indicated that tapping during 5 and 20 Hz stimulation was worse than during no stimulation -LRB- F -LSB- 1,16 -RSB- = 6.385 , p = 0.022 and F -LSB- 1,16 -RSB- = 8.793 , p = 0.009 , respectively -RRB- . The average deterioration in tapping rate during 5 and 20 Hz stimulation compared to no stimulation -LRB- 0 Hz -RRB- in this group was 11.8 ± 4.9 % and 7.4 ± 2.6 % , respectively -LRB- Fig. 1 -RRB- . There was a trend towards a decreased tapping performance at 10 Hz compared to 0 Hz -LRB- F -LSB- 1,16 -RSB- = 3.578 , p = 0.077 -RRB- . There was no effect of FREQUENCY in patients with baseline tapping performance below normal limits -LRB- ANOVA , F -LSB- 1.9,32.8 -RSB- = 2.202 , p = 0.128 -RRB- . We also analyzed the variability in tapping as measured by the coefficient of variation -LRB- CV -RRB- of the time intervals between successive taps on those sides with baseline tapping performance within normal limits . ANOVA with the factor FREQUENCY -LRB- four levels : 0 , 5 , 10 and 20 Hz -RRB- revealed a significant effect of FREQUENCY -LRB- F -LSB- 3,48 -RSB- = 3.408 , p = 0.025 -RRB- . Within-subjects contrasts indicated that the CV increased during 5 and 10 Hz stimulation compared with no stimulation -LRB- F -LSB- 1,16 -RSB- = 5.144 , p = 0.038 and F -LSB- 1,16 -RSB- = 4.852 , p = 0.043 , respectively -RRB- . The average increase of the CV during 5 and 10 Hz stimulation compared to 0 Hz in this group was 70.4 ± 35.8 % and 81.5 ± 48.2 % , respectively -LRB- Fig. 2 -RRB- . There was no difference between the CV at 20 Hz compared to 0 Hz -LRB- F -LSB- 1,16 -RSB- = 0.871 , p = 0.365 -RRB- . There was , however , a trend for the CV with 5 Hz stimulation to exceed that with 20 Hz stimulation -LRB- t-test ; p = 0.059 -RRB- . Discussion We have shown that STN DBS at a variety of low frequencies can slow distal upper limb movements in PD patients with relatively preserved baseline tapping function at the time of study . The effect was present with DBS at 5 Hz and 20 Hz in line with previous studies -LRB- Chen et al. , 2007 ; Fogelson et al. , 2005 ; Moro et al. , 2002 -RRB- , and there was a trend towards a similar effect with stimulation at 10 Hz -LRB- Timmermann et al. , 2004 -RRB- . These effects were apparent when tapping sides were separately analyzed according to whether the level of baseline performance was within or outside of normal limits , in line with previous studies suggesting that deleterious effects of DBS are more evident on those sides with relatively preserved baseline performance -LRB- Chen et al. , 2006a , 2007 -RRB- . The effect was not apparent during stimulation on those sides with impaired baseline performance , either because of confounding , albeit mild , suppressive effects of low-frequency DBS on spontaneous pathological oscillations or because of floor effects -LRB- Chen et al. , 2007 ; Fogelson et al. , 2005 -RRB- . In principle , then , the susceptibility of basal ganglia -- cortical loops to the effects of excessive synchronization may be elevated across a broad low-frequency band in parkinsonian patients . Accordingly , the relatively different frequency ranges of pathological synchronization in patients and MPTP-treated primates -LRB- Hammond et al. , 2007 -RRB- may be more indicative of the resonance properties of basal ganglia networks in the different situations , rather than any fundamental differences in the mechanism of bradykinesia . However , it must be stressed that this is a generalization , and although synchronization at different frequencies may conspire to disturb movement , there may still be subtle differences in the way movement is impaired . This is brought out by the differential effects of low-frequency stimulation on the variation in tapping intervals , evident in differences in the coefficient of variation and hence independent of any differences in tapping rate . Only DBS at 5 and 10 Hz increased temporal variability , whereas DBS at 20 Hz selectively decreased tapping rates without changing tapping variability . The implication is that basal ganglia networks are involved in processing related to the temporal patterning and regularity of movement and that these circuits may be particularly susceptible to disruption by pathological synchronization at frequencies ≤ 10 Hz . In support of basal ganglia involvement in the temporal patterning of movement , PD patients have increased temporal variability in finger tapping -LRB- Giovannoni et al. , 1999 ; Shimoyama et al. , 1990 -RRB- , and temporal variability in motor performance is a very early feature of Huntington 's disease -LRB- Hinton et al. , 2007 -RRB- . Indeed , Flowers considered increased variability of movement in both time and space to be one of the core components of motor dysfunction in PD , along with a basic slowness of movement , and a difficulty in initiating and maintaining movement -LRB- Sheridan et al. , 1987 -RRB- . This variability in motor performance may also relate to the phenomenon of freezing . No overt freezing episodes were observed during tapping in our patients , but an increased variability of stride has been shown in PD patients experiencing freezing of gait independent of frank freezing episodes -LRB- Hausdorff et al. , 2003 -RRB- . However , a primary disturbance of temporal patterning is not the only potential interpretation for the increased variability seen during stimulation at 5 Hz and 10 Hz . Tremor was not seen during low-frequency stimulation -LRB- except in one patient -RRB- , in agreement with Timmermann et al. -LRB- 2004 -RRB- , nor were there any obvious and consistent dyskinesias . Nevertheless , it is possible that synchronization at frequencies ≤ 10 Hz induced subtle hyperkinesias that led to increased temporal variability across taps . A previous case report describes dyskinetic movements induced by STN DBS at 5 Hz -LRB- Liu et al. , 2002a -RRB- , and there is increasing evidence that excessive synchronization over 4 -- 10 Hz within basal ganglia circuits may be related to both levodopa-induced dyskinesias in PD -LRB- Alonso-Frech et al. , 2006 -RRB- and mobile elements of dystonia -LRB- Chen et al. , 2006b ; Liu et al. , 2002b ; Silberstein et al. , 2003 -RRB- . Relevant in this regard , a recent study demonstrated an increased variability of speech rate in patients treated with l-DOPA and suggested that this effect was related to dyskinesia -LRB- De Letter et al. , 2006 -RRB- . Variability of swing movement was also observed in the gait of dyskinetic CP children -LRB- Abel et al. , 2003 -RRB- . In summary , our results provide further evidence that DBS of the STN over a relatively broad band of low frequencies can impair movement , in line with other more circumstantial evidence of an association between low-frequency synchrony in basal ganglia -- cortical loops and altered movement -LRB- see recent reviews by Gatev et al. , 2006 ; Uhlhaas and Singer , 2006 ; Hammond et al. , 2007 -RRB- . The present results also raise the important possibility that the detailed profile of motor abnormalities evident in extrapyramidal diseases depends to some extent on the precise frequencies at which pathological synchronization occurs . Indeed , some differences in the details of the effects of pathological synchrony at different frequencies might be anticipated , given the evidence for selective tuning of basal ganglia -- cortical sub-circuits -LRB- Fogelson et al. , 2006 -RRB- .

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