Inguinal and scrotal hernias are of great concern to pig producers, and lead to poor animal welfare and severe economic loss. Selection against these conditions is highly preferable, but at this time no gene, Quantitative Trait Loci (QTL), or mode of inheritance has been identified in pigs or in any other species. Therefore, a complete genome scan was performed in order to identify genomic regions affecting inguinal and scrotal hernias in pigs. Records from seedstock breeding farms were collected. No clinical examinations were executed on the pigs and there was therefore no distinction between inguinal and scrotal hernias. The genome scan utilised affected sib pairs (ASP), and the data was analysed using both an ASP test based on Non-parametric Linkage (NPL) analysis, and a Transmission Disequilibrium Test (TDT).Significant QTLs (p < 0.01) were detected on 8 out of 19 porcine chromosomes. The most promising QTLs, however, were detected in SSC1, SSC2, SSC5, SSC6, SSC15, SSC17 and SSCX; all of these regions showed either statistical significance with both statistical methods, or convincing significance with one of the methods. Haplotypes from these suggestive QTL regions were constructed and analysed with TDT. Of these, six different haplotypes were found to be differently transmitted (p < 0.01) to healthy and affected pigs. The most interesting result was one haplotype on SSC5 that was found to be transmitted to hernia pigs with four times higher frequency than to healthy pigs (p < 0.00005).For the first time in any species, a genome scan has revealed suggestive QTLs for inguinal and scrotal hernias. While this study permitted the detection of chromosomal regions only, it is interesting to note that several promising candidate genes, including INSL3, MIS, and CGRP, are located within the highly significant QTL regions. Further studies are required in order to narrow down the suggestive QTL regions, investigate the candidate genes, and to confirm the suggestive QTLs in other populations. The haplotype associated with inguinal and scrotal hernias may help in achieving selection against the disorder. Development of inguinal and scrotal hernias usually results from failed obliteration of the processus vaginalis after descent of the testis [processus vaginalis because the processus vaginalis does not obliterate unless the testis reach the scrotum [The occurrence of hernias is a significant problem facing pig producers, and leads to poor animal welfare and severe economic loss. The most commonly occurring hernias are e testis , or frome testis . Complice testis . Further scrotum . This pa2 for these types of hernia range from 0.20 [processus vaginalis and in the closing of the inguinal ring; by association, these genes may also be involved in the occurrence of inguinal and scrotal hernias. Specifically, Insulin-like receptor 3 (INSL3), Müllerian inhibiting substance (MIS), and relaxin are all involved in gubernacular growth [processus vaginalis [Several studies have shown that genetic factors are involved in the development of inguinal and scrotal hernias ,7, howevrom 0.20  to 0.86 rom 0.20 . Severalr growth , while taginalis ,12.The aim of this study was to identify genomic regions affecting the frequency of inguinal and scrotal hernias in pigs. Records from seedstock breeding farms were used in this study. No clinical examinations were executed on the pigs and therefore, there was no distinction between inguinal and scrotal hernias. Examination of these regions may reveal causative genes responsible for the trait, which would be valuable information for use in Marker-Assisted Selection (MAS) schemes directed at decreasing hernia prevalence. Previously, parametric linkage analysis applied to large pedigrees containing many affected individuals has helped in the identification of genes with high penetrance . HoweverThe linkage analysis was performed using an affected sib pair (ASP) test. Figure TDT analysis was applied to all loci in the data set. The significant loci are presented in Table The loci showing significance for both ASP and TDT, and the regions showing high significance (p < 0.005) with only ASP or TDT, were considered to be the most interesting for further analyses. Haplotypes from these regions were constructed and TDT analysis was used to identify those haplotypes most strongly associated with hernia. Six different haplotypes were found to be differently transmitted (p < 0.01). One haplotype on SSC5 was transmitted four times more frequently to pigs with hernias than to healthy pigs (p < 0.00005). Other haplotypes, showing an increased frequency of transmission to affected pigs (p < 0.01) were found on SSC6, SSC15 and SSC17. Table diabetes mellitus and a marker in the 5' region of the insulin gene [Our genomewide scan for QTLs affecting incidence of hernia revealed 9 genomic regions on 8 chromosomes using ASP test p < 0.01), and 6 genomic regions on 6 chromosomes using TDT (p < 0.01). Two of these regions, on SSC5 and SSC17, were significant (p < 0.01) when analysed by both statistical methods  deteriorates over successive generation because of recombination, but the mutation will remain in LD with closely linked markers. It is therefore, possible to define a risk modifying haplotype in a region of the genome surrounding the disease mutation. Once multiple single-point associations within a region have been identified, haplotype based tests for disease associations may be performed. In this investigation, haplotypes consisting of marker alleles in the putative QTL regions were investigated using TDT analysis. Several haplotypes occurred with a higher frequency in the affected pigs compared to the unaffected pigs, indicating that these haplotypes are associated with the occurrence of inguinal and/or scrotal hernias in pigs.processus vaginalis, and a failure of involution at the internal inguinal ring [processus vaginalis. Finally, regulation of collagen metabolism has been shown to play an important role in the development of inguinal and scrotal hernias [processus vaginalis fusion, a process involving substantial tissue remodelling and the characteristic transformation of processus vaginalis epithelium [While most porcine genes are still not mapped, the determination of homologous synteny blocks between human and porcine chromosomes allows us to propose some candidate genes for inguinal and scrotal hernias using comparative genomics -27. The nal ring . In addi hernias ,29. Usin hernias -32. From hernias  and huma hernias , INSL3 i hernias . The CGRithelium ,12. Finaithelium . Moreoveithelium . Additioithelium . Estrogeithelium  demonstrithelium . Patholoithelium . It is tithelium . InteresTo our knowledge, this is the first report of a whole genome scan for markers associated with inguinal and scrotal hernias, and one of the first genetic studies using ASP in livestock. The results reported in this study will encourage us to perform follow-up studies using finemapping approaches in an extended genetic material. Our goal is to discover the causal mutations underlying inguinal and/or scrotal hernias, and to look for shared ancestral haplotypes among pigs with these types of hernias. Ultimately we wish to understand the epistatic interactions between inguinal/scrotal hernia genes, and the interactions between genes and their environment, and their impact on a variety of relevant cofactors associated with this defect. We believe the data contained in the present study represents an important and useful basis to further search for candidate genes causing inguinal and scrotal hernias in pigs. The outcome from these inquiries may also be of relevance to human hernia conditions.For the first time in any species, a genome scan has revealed suggestive QTLs for inguinal and scrotal hernias. Significant QTLs were detected in 8 out of 19 porcine chromosomes (p < 0.01). However, the most promising QTLs were found on SSC1, SSC2, SSC5, SSC6, SSC15, SSC17 and SSCX. These regions were found to be significant when using both statistical methods (ASP and TDT), or with a convincing high significance for either of the methods. Haplotypes from the suggestive QTL regions were constructed and used in TDT analysis. Six different haplotypes were found to be differently transmitted when contrasting hernia pigs with healthy pigs (p < 0.01). One haplotype on SSC5 was transmitted four times more frequently to hernia pigs than to healthy pigs (p < 0.00005). Only chromosomal regions could be detected in this study, but it is noteworthy that several promising candidate genes, including INSL3, MIS, and CGRP, are all located in a highly significant QTL region. Further studies must be performed to narrow down the suggestive QTL regions, investigate the candidate genes, and finally to confirm the suggestive QTLs in other populations.hernia inguinalis or hernia scrotalis, it was necessary to utilise a collection system involving all the breeding farms in Norsvin  in order to access a sufficient number of affected sib pairs (ASP). The power calculations and minimum number of affected sib pairs required for this study was evaluated based on a method suggested by Simianer and Stricker [Due to a generally low incidence of Stricker . Blood sWhen a farmer reported an affected animal, diagnostic procedures were performed by a breeding consultant from Norsvin. Without clinical examination, it is impossible to distinguish direct hernia from indirect hernia, or inguinal hernia from scrotal hernia. No clinical examinations were executed, so the diagnosis of hernia includes both inguinal and scrotal hernias, as well as both indirect and direct hernias. In human and horses, inguinal and scrotal hernias are almost exclusively of the indirect type , therefo2, 2 mM dNTP, 0.01 μm of each primer and 5U Gold Taq polymerase . The PCR program included an initial denaturation step of 10 sec at 95°C, followed by 40 cycles of 30 sec at 94°C, and 30 sec at 55°C-62°C, with a final extension step of 5 min at 73°C. Fragment lengths were determined either by 1) electrophoresing PCR product through a 6% denaturating polyacrylamide gel in an ABI-377 DNA sequencer , where each forward primer was 5'-labeled with one of three fluorophores , or 2) with capillary electrophoresis on an ABI 3730 DNA sequencer , each forward primer 5'-labeled with one of four fluorophores . Genescan and Genotyper version 3.7 software or GeneMapper 3.0  was used for genotype scoring. A computer program was written in house to control for Mendelian inconsistencies in the marker data.Genomic DNA was extracted from a total of 282 pigs (parents and offspring). 130 microsatellite markers from the autosomal chromosomes and 7 microsatellites from the X/Y chromosome were selected from the USMARC Genome Database (2000), based on position, ease of scoring and number of alleles . The marThe family material used in the study was not suitable for constructing genetic maps. Therefore the order of the 137 microsatellites chosen for the genome scan, and the genetic distances between them, were taken from the USMARC Genome Database 2000). The markers were evenly distributed across the 18 autosomal chromosomes and the X-chromosome, including one marker in the pseudoautosomal region. The total length of the map spanned by the marker set was 2335 cM. Average marker heterozygosity was 0.57 and the mean intermarker spacing was 17 cM. The locations of markers are shown in Figure 000. The A non-parametric linkage analysis using the software ALLEGRO 1.0 , requireTwo different test statistics were applied for linkage analysis using the software package ALLEGRO 1.0 . The firTransmission/Disequilibrium Tests  were carj has been passed on from an heterozygous parent to an affected or unaffected offspring, respectively, j has not been passed on from an heterozygous parent  to an affected or unaffected offspring, respectively, and l is the number of alleles at the locus. The TDT test thus took both affected and non-affected siblings into account. A permutation test was carried out according to Lazzeroni and Lange [m permutations, TDT statistics ti for all loci i  were converted to p-values using the empirical estimate;where nd Lange , to corraki = 1 if Tki ≥ ti and aki = 0 otherwise (meaning that pi(ti) is the rate at which the permuted test statistic was larger than the true test statistic). Finally, adjusted p-values (accounting for multiple loci) for the real data were calculated as:where bki = 1 if pi(ti) ≥ minm, with minm being the smallest p-value obtained across loci for permutation m. A Visual Basic program was written to perform this analysis, since, to our knowledge, the permutation option and the option of using both affected and unaffected offspring is not available in any publicly available programs.where 2 with one degree of freedom. For haplotype TDT, only those haplotypes falling in the significant marker regions from the TDT or ASP tests were considered. Haplotypes were retrieved from ALLEGRO output.In addition to the locus-level TDT described above, TDT was also performed on individual alleles, at single loci or haplotypes. The TDT statistic (T) was calculated using equation 1, and nominal significance levels were used, T being distributed approximately as χThe information content (Info) supplied by ALLEGRO is displayed in Figure ASP, affected sib pair; LD, linkage disequilibrium; NPL, non-parametric linkage score; TDT, transmission/disequilibrium test; NSA, numbers of shared alleles; info, information content; INSL3, Insulin like receptor 3; MIS, Müllerian inhibiting substance; CGRP, Calcitonin gene-related peptide; ESR1, estrogen receptor 1; COL9A1, collagen typeIXα; COL2A1, collagen type II; INSL5, Insulin-like hormone 5; CYP19A1, cytochrome P450 family19A1; MAS, master assisted selection.EG coordinated the study, organised and carried out molecular genetic work, performed the statistical analysis of QTL mapping and drafted the paper. MM carried out molecular genetic work. HT was involved in the statistical analysis. HS was involved in planning the project, power calculations, supervising the statistical analysis and writing the paper. SL was involved in planning the project and writing the paper. TM performed the TDT statistical analysis and contributed in writing the paper.


Patients suffering from Intermittent Claudication (IC) experience repeated periods of muscle contraction with low blood flow, throughout the day and this may contribute to the hypothesised skeletal muscle abnormalities. However, no study has evaluated the consequences of intermittent contraction with low blood flow on skeletal muscle tissue. Our aim was to generate this basic physiological data, determining the 'normal' response of healthy skeletal muscle tissue. We specifically proposed that the metabolic responses to contraction would be modified under such circumstances, revealing endogenous strategies engaged to protect the muscle adenine nucleotide pool. Utilizing a canine gracilis model (n = 9), the muscle was stimulated to contract (5 Hz) for three 10 min periods (separated by 10 min rest) under low blood flow conditions (80% reduced), followed by 1 hr recovery and then a fourth period of 10 min stimulation. Muscle biopsies were obtained prior to and following the first and fourth contraction periods. Direct arterio-venous sampling allowed for the calculation of muscle metabolite efflux and oxygen consumption.During the first period of contraction, [ATP] was reduced by ~30%. During this period there was also a 10 fold increase in muscle lactate concentration and a substantial increase in muscle lactate and ammonia efflux. Subsequently, lactate efflux was similar during the first three periods, while ammonia efflux was reduced by the third period. Following 1 hr recovery, muscle lactate and phosphocreatine concentrations had returned to resting values, while muscle [ATP] remained 20% lower. During the fourth contraction period no ammonia efflux or change in muscle ATP content occured. Despite such contrasting metabolic responses, muscle tension and oxygen consumption were identical during all contraction periods from 3 to 10 min.repeated periods of muscle contraction, with low blood flow, results in cessation of muscle ammonia production which is suggestive of a dramatic reduction in flux through AMP deaminase. Intermittent Claudication (IC) is the symptomatic manifestation of peripheral vascular disease, suffered by millions of patients worldwide. IC is thought to occur when skeletal muscle blood flow fails to increase adequately during walking ,2 severecomplete ischemia are known to induce a protective response to subsequent prolonged ischemia (preconditioning)  is likely to have been lowered prior to contraction period 2, yet ammonia efflux was extremely high. This indicates that the 20% reduction in resting [ATP], prior to the fourth period of contraction, cannot explain the complete lack of ammonia production. PCr utilization was modestly reduced during the fourth period of contraction, when compared with period 1 (p = 0.051). Although not substantial, it is suggestive of a more rapid increase in ATP derived from oxidative phosphorylation -11,26. AAlterations in substrate utilization can influence muscle pH during contraction, and hence the activation of AMP deaminase, See . There wet al [-1 dry muscle tissue, under all conditions. It is therefore conceivable that the metabolic response to walking, in IC patients, could be greatly altered by earlier walking activity, impacting on the outcome of any clinical assessment and almost certainly complicating the 'disease' phenotype when studied in a laboratory setting.Although there are no previous studies addressing the metabolic response of skeletal muscle to repeated contractions with low blood flow, there are a limited number of intermittent exercise studies (with high blood flow) which can be compared with the present data ,28. Duriet al ,25 proviet al . This efATP loss can exceed 40% even during a single period of contraction with low blood flow  and thusWe can hypothesize that following 3 periods of contractions with low blood flow, adenine nucleotides loss is prevented by an endogenous mechanism, involving modification of AMP deaminase activity. The present study also provides support for the idea that flux through AMP deaminase is not essential for continued muscle contraction while inhibition of AMP deaminase may represent a potential strategy for protecting muscle metabolism in peripheral vascular disease patients -35.2 and PO2) using the Radiometer ABL 625GL (Copenhagen) and venous infusion of heparin, bicarbonate or saline. A gracilis muscle was vascularly isolated, leaving only the main arterial and venous blood flows intact. The distal tendon of the muscle was attached to an isometric force transducer . The popliteal artery was catheterized for recording gracilis muscle perfusion pressure. Heparin was infused following surgery,  for the duration of the experiment. The femoral artery supplying each gracilis muscle was cannulated proximally and distally and attached sequentially to a perfusion pump . The muscle blood flow was fixed by setting the perfusion pump at 6.1 ± 0.1 ml/min. We have previously established this as being similar to the resting flow rate in the gracilis muscle [After an overnight fast, each female dog  was anaesthetized with sodium pentobarbitone (45 ± 1 mg/kg body mass (bm) followed by continuous infusion at 0.10 ± 0.01 mg/kg/min i.v. . The trachea was intubated and the dogs were artificially ventilated . The right brachial artery was cannulated and systemic blood pressure was recorded using a pressure transducer and chart recorder. The left brachial artery and vein were cannulated for collection of arterial blood samples for monitoring of blood parameters , PCOs muscle . This flThe resting length of the muscle was altered to obtain a standard tension of ~400 g/100 g muscle. After a 20 min equilibrium period, baseline values were collected (blood samples and muscle biopsies) and then muscle contraction was induced via electrical stimulation of the obturator nerve . Square-wave impulses of 0.1 msec duration, 5 Hz frequency and a supramaximal voltage (10 V), resulting in complete muscle fiber recruitment and peak twitch tension, were applied for 3 periods of 10 min (with 10 min recovery) followed by 1 hour recovery and then a fourth 10 min period of contraction , creatine and lactate as previously described [Blood gases, whole blood lactate and glucose were measured using the Radiometer ABL 625GL (Copenhagen). The analyzer was calibrated on a daily basis according to the manufactures instructions. Plasma ammonia was determined in 1-ml blood samples treated according to the standard instructions , and indophenol was measured using a spectrophotometer at a wavelength of 630 nm . All biopsy samples were stored in liquid nitrogen. Subsequently, a portion was freeze dried, dissected free from visible connective tissue and blood, powdered and extracted in 0.5 M perchloric acid containing 1 mM EDTA. Following centrifugation, the supernatant was neutralized with 2.2 M KHCOescribed  with theATP adenosine triphosphateAMP adenosine monophosphateIMP inosine monophosphatePCr phosphocreatineIC Intermittent ClaudicationGS glutamine synthetaseJAT originated the study idea and JAT and AM designed and carried out the study. The article was written by AM and JAT.


Lepus timidus) exists in Great Britain, Norway, Sweden, Finland, parts of the Alps and in Eastern Europe, but not in Denmark. Interspecific hybridization has been demonstrated between native Swedish mountain hares and introduced brown hares (Lepus europaeus). During the data collection in a study concerning Danish brown hares we identified 16 hares with a single very divergent haplotype.In Europe the mountain hare (Lepus timidus mtDNA haplotype in the Eastern Danish hare populations is estimated to 6%.Phylogenetic analysis shows that the divergent Danish haplotype is most closely related to the Swedish mountain hare. The frequency of L. europaeus populations but include introgressed brown hares with Swedish L. timidus mtDNA. The most probable explanation of this is natural migration or translocation of introgressed brown hares from Sweden. The impurity of hare populations has implications for conservation and population genetics.In contrast to what is known, the Danish hare populations are not pure  Lepus europaeus Pallas, 1778) is widely distributed throughout Europe, up to 60°N, in Asia Minor and probably south to Israel. It is a popular game animal and its range has expanded to the east both by natural dispersion and by translocations to central and far-east Siberia [The brown hare  covers much of the Palaearctic. In Europe it exists in Great Britain, Norway, Sweden, Finland, parts of the Alps and in Eastern Europe but it does not exist in Denmark [The general range of the mountain hare  were clearly different from the rest and collapsed into a single haplotype. The 16 individuals were obtained from five of the eight populations sampled, and these five populations are all located on islands in the eastern part of Denmark , but not a single L. europaeus sequence in the first 50 hits. The subsequent phylogenetic analysis  and most closely related to the Swedish L. timidus haplotypes  , b) that L. timidus is not reported to be a native breeding species in Denmark [The results show the presence of mountain hare mtDNA in Danish brown hare populations. From mtDNA alone we cannot decide whether these 16 individuals are true mountain hares or introgressed brown hares. This would require further analysis using nuclear gene sequences (e.g.  species ). Howeve Denmark , and c)  Denmark , these iThe most likely origins of introgression are; 1) natural migration of mountain hares from Sweden followed by introgression, or 2) natural migration or translocation of introgressed brown hares from Sweden. However, since the documented distribution of mountain hares in Sweden does not include southern Sweden ,10, natuThe fact that The Baltic Sea occasionally freezes over in very severe winters facilitates natural migration of introgressed brown hares from southern Sweden across the sea barrier to the Eastern Danish islands Figure . This isL. europaeus specimens from sympatric areas carried transmitted L. timidus mtDNA [L. timidus mtDNA and later escaped to the natural hare population.During the mid 1980'ies a network of hare breeding facilities was established in Denmark, and it was initiated with imported hares primarily from Italy, France, Hungary and Sweden . No combus mtDNA , it is pThe detection of only one haplotype in the 16 individuals shows low mtDNA diversity in the total population of hybrids in Denmark. The geographical distribution of the 16 intogressed brown hares , 1.6 μl dNTP , 0.5 μl of each primer (10 pmol/μl), 0.1 μl Taq polymerase (Amersham Pharmacia Biotech), topped up with distilled water to 9 μl and 1 μl template DNA (40–60 ng/μl) was added. Cycling conditions were 94°C for 3 min, and 35 cycles of 94°C for 30 s, 50°C for 40 s and 72°C for 60 s and a final extension step of 72°C for 7 min. Sequencing was conducted under BigDye™ terminator cycling conditions, the reacted products purified using ethanol precipitation and run using an Automatic Sequencer ABI 3730xl. Both strands were sequenced in all samples.A 494 bp fragment of the mtDNA D-loop (control region) was amplified via PCR, using the mammalian control region primers L15997 5'-CACCATTAGCACCCAAAGCT-3' located in the tRNA gene and H16498 5'-CCTGAAGTAGGAACCAGATG-3' . PCR wasSequences were edited using Bioedit version 7.0.0 . IdenticL. europaeus and 20 L. timidus sequences were downloaded from GenBank. Sequences with geographical information were selected to represent haplotypes from all over Europe and Russia. Together with the 19 Danish L. europaeus and the divergent haplotype, these 49 sequences (table 6 generations, and the Markov chain was sampled every 1000 generations. Model parameters were estimated directly from the data and three independent replicates were conducted to avoid entrapment in local optima [Nine l optima . The iniTF carried out all the lab-work, edited and analyzed the sequence data and drafted the manuscript. PV carried out the phylogenetic analysis, prepared the figures and drafted the manuscript. TW organized the samples and drafted the manuscript. All authors have read and approved the final manuscript.


GSTT1, GSTM1 and GSTP1 impact detoxification of carcinogens by GSTs and have been reported to increase susceptibility to environmentally related health outcomes. Individual factors in arsenic biotransformation may influence disease susceptibility. GST activity is involved in the metabolism of endogenous and exogenous compounds, including catalyzing the formation of arsenic-GSH conjugates.Polymorphisms in GSTT1, GSTP1 and GSTM1 were associated with risk of skin lesions and whether these polymorphisms modify the relationship between drinking water arsenic exposure and skin lesions in a case control study of 1200 subjects frequency matched on age and gender in community clinics in Pabna, Bangladesh in 2001–2002.We investigated whether polymorphisms in GSTT1 homozygous wildtype status was associated with increased odds of skin lesions compared to the null status (OR1.56 95% CI 1.10–2.19). The GSTP1 GG polymorphism was associated with greater odds of skin lesions compared to GSTP1 AA, (OR 1.86 (95%CI 1.15–3.00). No evidence of effect modification by GSTT1, GSTM1 or GSTP1 polymorphisms on the association between arsenic exposure and skin lesions was detected.GSTT1 wildtype and GSTP1 GG are associated with increased risk of skin lesions. Arsenic exposure through drinking water is a global problem, and has reached crisis status in Bangladesh -5. A welGST genes have been associated with susceptibility to a range of diseases, and GST polymorphisms alone and in concert with environmental exposures are associated with disease outcomes and behavior of several enzymes [In vivo studies have shown that GSH serves as a reducing agent required for the reduction of arsenate to arsenite[It has been proposed that there are differences in susceptibility to arsenic due to individual genetic variability in biotransformation of the metal. Polymor enzymes -12. Glut enzymes -12. In v arsenite. GSH als arsenite. GST act arsenite,14. Anim arsenite. Glutath arsenite,12. Chro arsenite,19.GSTM1, GSTT1 and GSTP1 are members of the Mu (μ), Theta (θ), and Pi (π) classes respectively[GSTT1, GSTM1 and GSTP1 alone or in concert with environmental exposures may be associated with increased susceptibility to environmentally related diseases such as cancer and other clinical outcomes[GSTT1 null and GSTM1 null genotypes are deletion polymorphisms and have no θ or μ-glutathione S transferase activity respectively. The GSTP1 polymorphism is a single base pair substitution where adenine is replaced by guanine resulting in an amino acid change in which isoleucine (I105) is replaced by valine (V105), possibly resulting in lower enzyme activity[The biologic control of GST enzymes is multifaceted in that they demonstrate specific patterns of expression that depend on sex, age, tissue, and species and vary between individuals ,20. GSTMpectively. Polymor outcomes,12. The  activity,22. At h activity. We hypoGSTT1, GSTM1, and GSTP1 polymorphisms and skin lesions. In addition, we assessed possible effect-modification by GST genotypes in modifying the risk of arsenic related skin lesions using well water arsenic concentration to estimate exposure.We investigated the relationship between This study was conducted in the Pabna district of Bangladesh, located north of Dhaka on the Pabna (Ganges) River. Pabna was chosen for the following reasons: elevated arsenic was suspected in some of the region's villages due to proximity to the River; Dhaka Community Hospital (DCH) has a well established clinic network in the area; and Pabna is representative of socioeconomic status of much of non-urban Bangladesh. Eligible cases were Pabna residents, at least 16 years of age, with one or more type of skin lesion: diffuse/spotted melanosis, diffuse/spotted keratosis, hyperkeratosis, or leukomelanosis. One physician made the diagnosis, and treatment was provided at DCH when necessary. Controls were healthy individuals diagnosed as free of skin lesions and arsenic related disease randomly selected in a 1:1 ratio from Pabna, age of at least 16 years, living in the same village as cases but not sharing a tube well. Controls were also frequency matched to cases based on gender and age (+/- 3 years). To ensure heterogeneity of exposure and to prevent overmatching on exposure, controls were further selected so as to ensure that 80% were in "low-exposure" arsenic (<50 μg/l) communities and 20% were from suspected "high exposure" (≥50 μg/l) areas. This last ensured that the exposure distribution among controls matched that which has been reported for the Pabna region as a whole.Initial measurements of well arsenic levels were made with Merck field test kits. IndividIn 2001–2002, 1200 subjects were recruited. Physicians, blinded to exposure status, examined potential cases and controls. Trained interviewers administered the questionnaire regarding exposure, lifestyle factors, and collected individual well water samples. Data were collected on liters of water/liquid ingested per day, disease history, residential history including identification of the primary water source (tube well), years of use, and use of a previous tube well.Collection of well water samples was designed to minimize bias. Due to the fact that "high" exposure (≥50 μgAs/l) wells were often painted red and "low" exposure wells (<50 μgAs/l) were often painted green, the field team would have known if they had some indication as to whether the well was above or below 50 μg As/l. However, the field team did not know the arsenic concentration of the well at the time the subject was examined and interviewed, a procedure similar to a study in West Bengal .Additionally, it has been documented that wells are often mislabeled. Thus, tUpon collection of each 100 ml water sample two drops (0.2 ml) of pure nitric acid was added. The samples were stored in a cooler before storage in a 4°C refrigerated room. Analysis of each sample for arsenic concentration was completed using Environmental Protection Agency (EPA) method 200.8 with Inductively Coupled Plasma Mass Spectroscopy (ICP-MS) . The metTwo 10 ml EDTA tubes were used to collect blood and were stored in a cooler on ice until processed with cell lysis solution. Samples were sent to the Molecular Epidemiology Laboratory at Harvard School of Public Health for DNA extraction and genotyping.GSTM1 and GSTT1 genetic polymorphisms were evaluated using a previously described multiplex PCR technique[GSTP1 polymorphism was genotyped by the 5' nuclease assay (TaqMan) using the ABI Prism 7900HT Sequence Detection System . The primers, probes, and reaction conditions are available upon request. Genotyping for GSTT1 and GSTM1 was completed for 1062 subjects, and genotyping for GSTP1 was completed for 1101 subjects. Laboratory personnel were blinded to case status, and a random 5% of the samples were repeated to validate genotyping procedures. Two authors independently reviewed all results with 100% concordance.DNA samples were stored at -80°C. The technique. GSTP1 pAnalysis was restricted to subjects who reported using the same well greater than 6 months to minimize potential for temporal variability in well arsenic concentration. Since subjects were frequency matched by age and sex, these variables were included in all regression models,30. ArseGSTT1, and GSTM1 polymorphisms were tested among controls to ensure Hardy-Weinberg equilibrium. The heterozygote and homozygote variant were not combined for GSTP1.Univariate analyses were performed to describe population characteristics and to identify possible data errors and/or outliers. Continuous variables were summarized using means, medians, standard deviations and ranges, while categorical variables were described using percentages. Bivariate analyses  were conducted explore differences between cases and controls prior to multivariate modeling. The frequency distribution of the Multiple unconditional logistic regression was used to evaluate the associations between arsenic exposure in drinking water on case status. Odds Ratios were obtained from the regression models, as were their 95% confidence intervals. Regression models were fit using Statistical Analysis Systems  version 8.2.GST polymorphisms with skin lesions, adjusting for well arsenic concentration and 2) the modification of the relationship between well arsenic concentration and skin lesions by GST polymorphisms. The joint effects (interaction) of arsenic exposure and each of the genes were evaluated in additive and multiplicative models. Categorical variables were created for measures of arsenic exposure: drinking water arsenic concentration <50 μg/l and ≥50 μg/l. The deletion polymorphisms GSTT1 and GSTM1, were modeled as homozygous wildtype and heterozygotes compared to homozygote null.GSTP1 was modeled to determine whether the heterozygote variant and the homozygote variant might be associated with risk of developing skin lesions compared to wildtype. Variables were created for each combination of arsenic exposure and genotype, with the reference category being low arsenic exposure and either GSTT1 null, GSTM1 null or GSTP1 wildtype AA. Adjusted ORs and 95% CIs were evaluated for deviation from the expected null value on the additive or multiplicative scale. Interaction Contrast Ratios (ICR) and Bootstrap Percentile Method 1 (BP1) 95% CIs were calculated to quantify departure from additivity[GSTT1, GSTM1 or GSTP1 and arsenic exposure and an interaction term compared to the same model excluding the interaction term.Two different analyses were done to investigate: 1) the associations of dditivity. Synergydditivity. To estiSensitivity analysis was conducted by varying weights of controls selected having a well concentration less than 50 μg/l in a weighted logistic regression analysis as described previously. BrieflyGSTM1 and GSTT1 and allele frequencies for GSTP1 among cases and control subjects. The crude frequencies of the GST SNPs were not significantly different between cases and controls.Demographic data are presented in Table GSTT1 wildtype gene carried a higher risk of skin lesions compared to the null polymorphism (OR = 1.56 (95%CI 1.10–2.19). Compared to the reference category GSTP1 AA, the GG genotype had a 86% increase odds of skin lesions (OR = 1.86 95%CI 1.15–3.00). We were not able to detect a significant association with GSTM1 genotype and skin lesions. All models were adjusted for well As level, L/day of total liquid, previous well use, age, sex, education, BMI, chewing tobacco, betel nut use, and smoking status.The main effects for each genotype on skin lesions adjusted for well arsenic concentration was shown in Table GST polymorphisms on skin lesions are shown in Table GSTM1 null, GSTT1 null or GSTP1 AA, respectively. When the results were stratified on exposure, individuals with the GSTT1 wildtype had a higher risk of lesions in the low  exposure group as well as the high exposure group, though the relationship was not significant at the high arsenic exposure level (OR = 1.15 95%CI 0.68–1.94). There is little evidence of effect modification by the GSTM1 genotype. In the low exposure strata, individuals with the GSTP1 GG genotype had a higher risk of skin lesions  than those individuals with the AA genotype. In the high exposure strata there was no significant effect of the GSTP1 genotype. When the analysis is stratified by genotype for GSTP1, results were difficult to interpret due to the insufficient numbers in high exposure and GSTP1 GG category. Likelihood Ratio Tests (LRT) were not statistically significant .Adjusted ORs for the joint effects of well arsenic exposure and each GST polymorphisms are shown in Tables Interaction Contrast Ratios (ICR)s for the joint effects of arsenic exposure and the GSTM1, GSTT1, or GSTP1 for risk of skin lesions  to Valine (105) polymorphism remains to be explained [GSTT1 wildtype expression appears to be associated with a higher risk of disease. The impact of GSTT1, and GSTP1 polymorphisms, arsenic exposure and skin lesions and molecular mechanisms require further study.It has been suggested that GST substrates and glutathione conjugates have the ability to induce a variety of Phase II enzymes, so that polymorphisms in echanisms. The rolxplained , as the GSTM1 wildtype had significantly higher risk of arsenic-induced skin lesions  [GSTT1 or GSTP1 and skin lesions in an arsenic exposed population. Frequencies of GST polymorphisms were similar between the West Bengal study and our results. Due to a much smaller sample size Ghosh et al may have had limited power to detect associations with GSTT1 and GSTP1. Additionally the West Bengal study had only 22 controls and 33 cases who were homozygote null (-/-), and 156 controls and 211 cases who were (+/-) or . They may not have had an adequate sample size to detect a significant association for GSTT1. For GSTP1, they had even fewer subjects which would not have allowed the ability to detect an association. In the Pabna population, we reported that individuals with the GG genotype  had a 1.86 increased odds of skin lesions compared to those individuals with the GSTP1 AA genotype (OR 1.86 (95%CI 1.15–3.00). Ghosh et al reported having 3 cases and 3 controls with the  or GG genotype so they lacked the power to detect the association[GSTP1 activity can compensate for the absence of GSTM1 activity[GSTP1 GG due too few subjects in the high exposure category. However, GSTP1 GG was statistically significant overall for skin lesions, as well as in the low exposure drinking water strata. Despite these limitations, significant differences in risk of skin lesions associated with main effects of GSTT1 and GSTP1 genotypes.Limitations of our study include the possibility of recall bias with regard to reported Liters of liquid consumed per day. Moreover, this measure included water as well as other beverages, resulting in possible exposure misclassification for that variable. As with all sample analysis there is a potential for measurement error. However, rigorous quality control procedures were in place for the analysis of water, as well as for DNA extraction and genotyping. In a previous study in West Bengal India, it was reported by Ghosh et al. that individuals with 24–2.22) . This stsociation. Our res activity. Our stuGSTT1, GSTM1 and GSTP1 polymorphisms, we report that the main effects of GSTT1 wildtype and GSTP1 GG appear to be associated with increased risk of skin lesions. A larger sample size would allow a better investigation of the effect of the GSTP1 polymorphism at higher arsenic exposure. There was no evidence of effect modification of GST polymorphisms and arsenic concentration of drinking water on risk of skin lesions. Further work is required to characterize the potential mechanisms related to arsenic metabolism and GSTT1 and GSTP1 polymorphisms.While we did not detect statistically significant interactions on the multiplicative or additive scale between arsenic level and 2-terminal kinase.GST Glutathione S-transferase, GSH Glutathione, As Arsenic, Monomethylarsonic acid MMA (V), Monomethylarsonous Acid MMA (III), Dimethylarsinic acid DMA (V), multidrug resistant protein transporters (MRP), reactive oxygen species (ROS), LRT Likelihood Ratio Test, OR Odds Ratio, Interaction Contrast Ratios (ICR), Body Mass Index (BMI), Deoxyribonucleic acid (DNA), JNK, c-Jun NHThe author(s) declare that they have no competing interests.KMM was responsible for preparation of the manuscript, analysis of the data, and was involved in lab work and sample preparation.LR was responsible for study design, statistical consultation and manuscript editing.AH was involved in statistical consultation.PW was involved in statistical consultation and manuscript editing.DPM was involved in statistical consultation and manuscript editing.QQ, MR, and GM were responsible for study coordination in Bangladesh, questionnaire data collection, and collection of samples and data entry.TS was responsible for exposure methods and editing of manuscript.EG was responsible for dermatologic consultation regarding skin lesions.LS was responsible for overseeing and conducting genotyping and ensuring quality of data.DCC was the study PI, responsible for study design and management, manuscript editing.All authors approve of the manuscript.


Mice were instilled with 5, 20, and 50 μg of each particle type, and bronchoalveolar lavage was analyzed 24 hr after instillation for inflammatory cells and the level of proinflammatory cytokines. At respective mass-doses, particle-caused detrimental effects ranked in the following order: ufCP > SootL ≥ SootH > Printex90 > PrintexG > DEP. Relating the inflammatory effects to the particle characteristics—organic content, primary particle size, or specific surface area—demonstrates the most obvious dose response for particle surface area. Our study suggests that the surface area measurement developed by Brunauer, Emmett, and Teller is a valuable reference unit for the assessment of causative health effects for carbonaceous UFPs. Additionally, we demonstrated the existence of a threshold for the particle surface area at an instilled dose of approximately 20 cm2, below which no acute proinflammatory responses could be detected in mice.Increased levels of particulate air pollution are associated with increased respiratory and cardiovascular mortality and morbidity. Some epidemiologic and toxicologic research suggests ultrafine particles (UFPs) (< 100 nm) to be more harmful per unit mass than larger particles. Our study was aimed at a quantitative comparison of acute adverse effects of different types of carbonaceous UFPs at a dose range that causes a moderate inflammatory response in lungs. We used six different particle types (primary particle size 10–50 nm, specific surface area 30–800 m In urban air, fine and ultrafine particles (< 100 nm) are most numerous among all particles and represent the highest surface area per mass. This surface can carry large amounts of adsorbed or condensed toxic air pollutants such as organic compounds and transition metals  seem to be especially harmful to susceptible sub-populations such as the elderly, patients with preexisting cardiopulmonary diseases, and diabetics . Ambientn metals . Althougetals , translocate efficiently into the interstitium, and largely evade macrophage clearance . Accordi10 (PM < 10 μm in diameter) and PM2.5 and the number of deaths down to lowest observed exposure concentrations of 2 μg/m3, which gives rise to the no-threshold hypothesis. However, some animal studies suggest a threshold level for PM and also point out that upon transition from the fine to ultrafine particle size range, particle number or surface area, not particle mass, may be more appropriate to characterize adverse effects of PM (a) which of the easily accessible parameters often applied in epidemiologic and toxicologic studies—particle size, surface area, or organic content (OC)—is most appropriate to characterize the inflammatory potency within the ultrafine size range; and b) whether we would be able to detect a threshold level for these particles under controlled experimental settings.A key issue concerning the toxicology of PM is the question whether threshold concentrations exist, below which air pollution has no effect on human health. Exposure–response curves from multiple locations in the United States and Spain have been analyzed . The rests of PM . However2/g, and the OC is between 1 and 20%. To characterize the acute inflammatory events in the lung caused by the particles, mice were killed 24 hr after instillation, and bronchoalveolar lavage (BAL) was performed immediately postmortem.We investigated acute adverse effects of six types of carbonaceous UFPs by intratracheal instillation in healthy mice: two commercially available pigments (PrintexG and Printex90); two laboratory-made flame soot particles with different OC [SootL (low) and SootH (high)]; spark-generated ultrafine carbon particles (ufCP); the standardized reference material (SRM) diesel exhaust particles (DEP) SRM1650a. These particles cover a size range of 10–50 nm. The specific surface area varies between 30 and 800 mRelating the inflammatory dose effect in the lungs to particle characteristics  revealed an obvious correlation for particle surface area in particular. This suggests that specific particle surface area may be a valuable reference parameter for the characterization of detrimental health effects caused by inhaled carbonaceous UFPs. Additionally, our data point to the existence of a threshold below which no acute proinflammatory response in the lung could be found to low doses of instilled particles.We obtained standard reference material DEP from the National Institute of Standards and Technology . Pigment blacks PrintexG and Printex90 were obtained from Degussa . SootH, SootL, and ufCP were generated and filter-sampled in our laboratory in an lipopolysaccharide-free environment. SootH and SootL aerosols were produced by means of a diffusion flame from a mixture of propane and air  at equal settings to ensure particle agglomerates with high (SootH) or low OC (SootL) at the same size distribution . The ufCWe assessed primary particle size by transmission electron microscopy (TEM) of particles resuspended in water. The six particle suspensions were prepared in the same way as for the respective instillation procedures and were immediately mounted on the TEM-grids as described by ad libitum. Animals were 10–12 weeks of age with body weights between 19.6 and 23.1 g during the study. Each of the 22 experimental groups consisted of eight animals. Twenty groups were exposed to particles, and two groups served as control and sham exposed. Mice were anesthetized by intraperitoneal injection of a mixture of xylazine (4.1 mg/kg body weight) and ketamine (188.3 mg/kg body weight). The animals were then intubated by a nonsurgical technique  at 8 weeks of age. The animals were kept in isolated ventilated cages  supplied with filtered air, in a 12-hr light/12-hr dark cycle. Specific pathogen-free hygienic status was approved by a health certificate according to the Federation of European Laboratory Animal Science Associations guidelines . Food anechnique . Using aechnique  and thusg, 20 min at room temperature). The cell-free supernatant from lavages 1 and 2 were pooled and used for biochemical measurements such as lactate dehydrogenase (LDH), total protein, and cytokine concentration. The cell pellet was resuspended in 1 mL RPMI 1640 medium  and supplemented with 10% fetal calf serum ; the number of living cells was determined by the trypan blue exclusion method. We performed cell differentials on the cytocentrifuge preparations . We used the number of polymorphonuclear leukocytes (PMNs) as a marker of inflammation. LDH activity was assayed spectrophotometrically by monitoring the reduction of NAD+ at 366 nm in the presence of lactate. Total protein content was determined spectrophotometrically at 620 nm, applying the Bio-Rad Protein Assay Dye Reagent . We analyzed 50 μl BALF/mouse to assess lavage cytokine concentrations for tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), macrophage inflammatory protein 2 (MIP2), and cytokine-induced neutrophil chemoattractant (KC), using commercial enzyme-linked immunosorbent assays .Twenty-four hours after instillation, mice were anesthetized by intraperitoneal injection of a mixture of xylazine and ketamine and killed by exsanguination. We performed BAL 24 hr after instillation. BAL was performed by cannulating the trachea and infusing the lungs 10 times with 1.0 mL PBS without calcium and magnesium, as described previously . The BALp < 0.05. Single regression models were applied to relate particle properties to the measured effect levels, and a multiple linear regression model was used to assesss simultaneously the effect of particle surface area and OC in relation to the inflammatory response. We used response values of individual animals for correlation analysis.All values are reported as the mean ± SE of eight animals. We used analysis of variance (ANOVA), as calculated by the commercial statistical package Statgraphics , to establish the statistical significance of differences between the experimental groups. We applied the Tukey honestly significant difference procedure to evaluate the significant differences between the 18 particle groups  and the sham control group. Differences were considered significant at In all but two experimental groups, protein concentration in BALF remained unchanged. Only SootL and ufCP at 50 μg significantly elevated total protein levels > 300 μg/mL, compared with a level of about 200 μg/mL in control and water-instilled animals (data not shown). Even at the highest doses, none of the six investigated particle types significantly altered LDH levels within 24 hr after instillation (data not shown). Similarly, the number of nonvital leukocytes retrieved by BAL remained unaffected in all groups (data not shown). Taken together, the latter findings point to the absence of cytotoxic short-term effects of the analyzed particles up to the dose of 50 μg/mouse.The total number of retrieved BAL leukocytes remained unchanged across the different doses and particles studied. Thus, we took the percentage of BAL neutrophils as a cellular marker to quantify early lung inflammation. As shown in To characterize the degree of inflammatory changes in the lung at a molecular level, we analyzed the amount of various pro-inflammatory cytokines released into the alveolar space: IL1-β and TNF-α, two cytokines commonly produced by activated monocytes, and MIP2 and KC, two potent neutrophil attractants that represent the murine functional homologs to human IL-8. Comparable with the conditions for PMN influx, the instillation of water did not alter cytokine concentrations in BALF, and the instillation of ufCP particles generated the highest cytokine levels at each dose. BALF concentrations of IL-1β are shown in r2 = 0.38. In contrast, a relatively strong correlation (r2 = 0.65) became evident when the inflammation response was related to particle specific surface area for all six types of instilled particles (p < 0.001).Primary particle size, OC, and particle surface area at a given dose were related to end points of lung inflammation and inflammatory cell activation. In articles . The cor2 shown in 2, and for the lowest observed adverse effect level (LOAEL) to a surface area of 22.1 cm2, represented by the 5-μg SootL dose. Consistently, the dose–effect curves for all inflammatory end points, most evidently for BALF cytokine levels, feature a point that marks the onset of a measurable inflammation response. Below this threshold, no particle-related increase in BAL PMN counts or cytokine concentrations was measurable. To confirm this threshold phenomenon, we instilled two additional doses of our most potent particle specimen, the ufCPs, at quantities < 5 μg. In concordance with comparatively low surface burdens of the other particles, BET surface doses of 4 cm2 and 16 cm2, representing ufCP particle quantities of 0.5 and 2 μg, did not induce a significant PMN influx or alter basal cytokine concentrations in BALF within 24 hr  to investigate adverse effects of ultrafine laboratory-generated surrogates and a standardized exhaust PM at a dose range that causes moderate inflammatory responses in the lung, and b) to assess which of the easily accessible parameters often applied in epidemiologic and toxicologic studies—particle size, surface area, or OC—is most suitable to characterize the inflammatory potency within the UFP size range. None of the instilled materials at any dose caused an increase in LDH concentration or the number of dead cells in BALF. This finding indicates the absence of acute tissue damage within 24 hr, even at the highest doses for all six types of carbonaceous nanoparticles, and is an indication for moderate doses of particles. Nevertheless, neutrophil recruitment of up to 60% was induced at the highest doses. This response is within the range of those described by others using various types of particles  UfCP particles did not cause a significant inflammatory response, whereas 40 cm2 (5 μg) was effective. On the basis of these results, we estimate a threshold of about 20 cm2 BET surface area. A threshold of 20 cm2 in mice was also suggested by the instillation of relatively high mass doses of quite different particle species by 2  vs. 3.0 (± 0.3) pg/mL, p < 0.05) and were within the range between 2- and 5-μg ufCP instillations. Additionally, it is notable that for subchronic particle inhalation in rats, a 200- to 300-cm2 threshold level for particle-induced lung effects has been described , rest ventilating of 15 m3/day, and deposition efficiency of 70%] suggests that lung burdens of urban residents may exceed 150 cm2/day, which is two orders of magnitude lower than the critical surface dose extrapolated from our data. Individuals suffering from respiratory or cardiovascular diseases might show significant lower effect thresholds and therefore be more susceptible to particulate air pollution. Assuming that deposited particles accumulate in the lungs (It would certainly be most interesting to extrapolate our experimental findings to man and environmental settings. Converting the experimental threshold of 20 cmhe lungs , the surhe lungs . Overall10 and daily cardiorespiratory mortality (Epidemiologic studies show no indication of a threshold below which no adverse effects can be found. For example, analysis of data from the 20 largest U.S. cities between 1987 and 1994 provided no evidence for a threshold between PMortality . As revi2 for particle exposure in healthy mice, below which no inflammatory response to the instilled particles could be demonstrated in the lung. Despite the well-known limitations of animal-to-human extrapolations, this threshold for acute pulmonary inflammatory reactions might be in relevant proximity to lung burdens of susceptible individuals chronically exposed to highly polluted air.In summary, this dose–response assessment of six different types of carbonaceous UFPs confirms the surface area concept within the nanosize range. Therefore, particle surface area may be the most appropriate parameter to evaluate the inflammatory potential and thus predict possible adverse health effects of ultrafine ambient particles. We identified a threshold surface area of approximately 20 cm


Habitual background loading was removed from the tibiae of 18 female adult mice by hindlimb-unloading. For 20 min/d, 5 d/wk, the left tibia of each mouse was subjected to oscillatory 0.6 g accelerations at 45 Hz while the right tibia served as control. Sham-loaded (n = 9) and normal age-matched control (n = 18) mice provided additional comparisons. Oscillatory accelerations, applied in the absence of weight bearing, resulted in 70% greater bone formation rates in the trabeculae of the metaphysis, but similar levels of bone resorption, when compared to contralateral controls. Quantity and quality of trabecular bone also improved as a result of the acceleration stimulus, as evidenced by a significantly greater bone volume fraction (17%) and connectivity density (33%), and significantly smaller trabecular spacing (−6%) and structural model index (−11%). These in vivo data indicate that mechanosensory elements of resident bone cell populations can perceive and respond to acceleratory signals, and point to an efficient means of introducing intense physical signals into a biologic system without putting the matrix at risk of overloading. In retrospect, acceleration, as opposed to direct mechanical distortion, represents a more generic and safe, and perhaps more fundamental means of transducing physical challenges to the cells and tissues of an organism.A range of tissues have the capacity to adapt to mechanical challenges, an attribute presumed to be regulated through deformation of the cell and/or surrounding matrix. In contrast, it is shown here that extremely small oscillatory accelerations, applied as unconstrained motion and inducing negligible deformation, serve as an anabolic stimulus to osteoblasts  This is particularly true in bone tissue where the mineralized matrix seemingly protects the resident cell population from high levels of deformation, and thus higher loads are considered necessary to transduce load information to osteoblasts and osteocytes. The mechano-responsiveness of bone was recognized as early as the 16more is better principle, recent work suggests that matrix strains two orders of magnitude below this threshold can be anabolic to bone Contrasting with this In contrast to a matrix deformation dependent pathway for mechanotransduction, the frequency sensitivity of the adaptive system points towards a more fundamental, perhaps unrecognized, pathway by which physical signals interact with the tissues and cells. Indeed, a mechanism that would allow a cell to sense mechanical signals directly without reliance on matrix strain would obviate the need for compensatory tissue-level amplification mechanisms in vivo, inducing motion but no direct deformation. It was hypothesized that the resident cell population would be able to sense and respond to these acceleratory signals as anabolic and that the product of the cell response would culminate in a morphology with enhanced tissue quantity and architecture.In the study reported here, bone's habitual loading environment was removed, and very small-amplitude oscillatory accelerations were applied All procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). Adult (19 wk) female BALB/cByJ mice were used for all experiments. To suppress the potential confounding interference of the experimental physical stimulus with that induced by normal functional load bearing, the first group of 18 mice were tail-suspended for 21 d such that their hindlimbs failed to make contact with the ground In vivo micro-computed tomography  was used to quantify bone morphology and micro-architecture in left and right tibiae of all experimental-, sham-, and age-matched control mice. Baseline scans were performed one day prior to commencing the respective protocol (day −1) and on the day of sacrifice (day 21). To aid in the determination of indices of bone formation, calcein injections were administered to all mice on days 9 and 19 . To minimize the technical complications inherent in determining bone formation and bone resorption activity on sections of the same bone, these analyses were performed on different subsets of mice. Nine tibiae of the experimental mice and eight tibiae of the age-matched control mice were prepared to allow for the analysis of bone formation , while the remaining tibiae were used to quantify bone resorption .An acceleration device comprising a transducer and function generator delivered high-frequency sinusoidal accelerations to the tibia along its longitudinal axis. Similar to previous experiments using whole body vibration in mice in vivo microCT scans using an isometric voxel size of 11.5 µm for trabecular bone and 21 µm for cortical bone. Metaphyseal trabecular bone of the proximal tibia was quantified in a region located between 300 µm and 600 µm distal from the growth plate. For cortical bone, a mid-diaphyseal region spanning 300 µm centered on the midsection of the tibia. Noise in the reconstructed images was minimized using a 3D Gaussian filter for which “sigma” and “support” were set at 0.5 and 1, respectively. Bone was segregated via thresholding routines as described previously Trabecular and cortical bone morphology of the tibia was assessed from the Indices of bone formation were assessed in metaphyseal trabecular and diaphyseal endocortical bone . Following euthanasia, the left and right tibia were fixated in 10% formalin and dehydrated in isopropyl alcohol. The samples were infiltrated with a series of three solutions comprising methyl methacrylate (85%), n-butyl phthalate (15%), and benzoyl peroxide , and then embedded in polymerized methyl methacrylate. Coronal 7 µm sections were cut from the metaphysis with a rotary microtome  while transverse 40 µm sections were cut from the mid-diaphysis with a diamond wire saw . Calcein labels were traced using standard software  that computed the amount of single labeled surface (sLS/BS), double-labeled surface (dLS/BS), mineralizing surface (MS/BS), mineral apposition rate (MAR), and bone formation rate (BFR/BS) Resorption activity was assessed in trabecular bone of the metaphysis by staining for tartrate resistant acid phosphatase (TRAP). Following euthanasia, each tibia was fixed in 10% formalin, decalcified in 2.5% formic acid, dehydrated in increasing concentrations of ethyl alcohol , and then embedded in glycol methacrylate . Coronal 7 µm sections of the proximal tibia were cut on a rotary microtome  and stained for TRAP activity, as described previously Differences in histologic and morphometric indices between the left and right tibiae within the same group of mice were determined with non-parametric Wilcoxon tests. Wilcoxon tests were also used to compare microCT based bone morphology between baseline (day −1) and completion (day 21). Differences between AGE-CTR, CTR, and ACC tibiae or SHAM, SHAM-CTR, and CTR tibiae were evaluated by Kruskal-Wallis tests with Dunn post-hoc tests. SigmaStat for Windows 3.10  was used for all statistical comparisons. Statistical significance was set at 5%. All data were presented as mean±standard deviation, unless specified otherwise.At baseline, there were no differences in trabecular or cortical bone morphology between tibiae pertaining to the different groups used in this study Table 1.In trabecular bone of the metaphysis, the application of very small amplitude, high frequency accelerations for 20 min/d over a 3 wk period was associated with 70% greater (p<0.05) bone formation rates when compared to contralateral control tibae Fig. 1. Trabecular bone morphology and micro-architecture were also sensitive to the application of low-amplitude, high-frequency oscillatory accelerations Fig. 2. Sham control animals were included in the experimental design to confirm that the attachment of the actuator apparatus itself was not responsible for any measurable effect and that the unilateral application of the stimulus did not produce a systemic effect. To address the former, there were no significant differences in any trabecular or cortical morphological parameter between sham-stimulated limbs (SHAM) and their contralateral controls (SHAM-CTR) Table 3.The primary aim of this study was to determine whether low-level accelerations can be sensed by bone in the absence of habitual functional loading. A secondary aim was to investigate the ability of this stimulus to attenuate unloading induced changes in bone's cellular activity and morphology. To this end, data from CTR and ACC tibiae were compared to normal age-matched controls (AGE-CTR). Compared to trabecular MS/BS (18.2±3.7%), MAR (1.2±0.1 µm/d), and BFR/BS (83.1±17.5 µm/yr) of age-matched controls, the removal of weight bearing (CTR) significantly (p<0.05) reduced indices of bone formation, as indicated by the 45% lower MS/BS, 52% lower MAR, and 74% lower BFR/BS Fig. 1. in vivo microCT scans between baseline and 3wk showed that cortical area in CTR tibiae decreased significantly by 2% (p = 0.009) during the experimental period. In contrast, ACC tibiae did not lose significant amounts of bone. Both groups, however, were significantly different from AGE-CTR which gained 2% (p = 0.003) in cortical area over the 3wk experimental period.Compared to age matched controls at 3wk, trabecular bone of CTR tibae was characterized by significantly lower BV/TV (49%), Conn.D (40%), Tr.N (13%), Tr.Th (20%) while SMI (63%), and Tb.Sp (15%) were significantly greater. With the application of short bouts of accelerations, these relative differences to normal controls decreased to 40% (BV/TV), 20% (Conn.D), 6% (Tr.N), 19% (Tr.Th), 46% (SMI), and 10% (Tb.Sp) Fig. 3. Many biologic systems have the ability to perceive and respond to functional challenges by adapting, but the physical signal which dominates the regulatory input has been difficult to identify. Bone, as an example of an adaptive tissue, can readily adapt to new functional challenges by either increasing bone mass in response to increased demand, or by reducing bone mass in response to disuse. Considering the sensitivity of the skeletal system, however, it is puzzling that such highly orchestrated responses are associated with relatively small deformations. Even during extremely strenuous activities, peak strains in the appendicular skeleton rarely exceed 0.3% strain in vivo sensitivity of bone cells to even extremely small accelerations reported here suggests an alternative and efficient pathway for the transduction of physical signals to the transcriptional machinery of the nucleus (The mechanisms by which physical signals are sensed by a cell have routinely focused on specific components of the mechanical information resulting from load, such as stretch or shear of the cell membrane. The  nucleus Fig. 4. Recent evidence has demonstrated the sensitivity of the musculo-skeletal system to extremely low-level whole body vibrations If acceleration of the nucleus contributes to the recognition of functional load-bearing, it may be surprising that cortical and trabecular bone did not respond similarly. However, this regional specificity has been observed in other studies This mouse model benefits from the uni-lateral application of the acceleration stimulus to a single limb, allowing the use of an internal contra-lateral control limb and thereby reducing the potential of confounding factors and elevating statistical power. Comparisons between the experimental and sham-control mice confirmed that neither the attachment of the tibia to the device, nor the presence of electromagnetic fields from the transducer were confounding variables, and supports the conclusion that altered regulation of cellular activity and bone morphology were due to the accelerations itself. The use of a genetic mouse strain that is exceptionally sensitive to unloading, while its sensitivity to low level, high frequency loading is only average High frequency accelerations, applied in the absence of habitual background loading were readily sensed by bone cells and resulted in trabecular bone formation and morphology distinct from disuse alone. Similar to other physical signals that do not necessarily require matrix deformation to be influential in the adaptive process, such as pulsed electromagnetic fields or ultrasound


As a standard for measuring tissue oxygenation in human tumors, the invasive, computerized polarographic needle electrode system (pO2 histography) was used for comparing the different non invasive measurements.The present study was conducted to analyze the value of  and tumor to blood ratios (FMISOT/B) were calculated. FDG PET of the lymph node metastases was performed 71 ± 17 min after intravenous administration. To visualize as many vessels as possible by CDS, a contrast enhancer  was administered. Color pixel density (CPD) was defined as the ratio of colored to grey pixels in a region of interest. From CDS signals two parameters were extracted: color hue – defining velocity (v) and color area – defining perfused area (A). Signal intensity as a measure of tissue perfusion (TP) was quantified as follows: TP = vmean × Amean.Until now a total of 38 Patients with malignancies of the head and neck were examined. Tumor tissue pO2 polarography , CPD and FMISOT/M. Only a slight correlation between TP and the fraction of pO2 values ≤ 10.0 mmHg, median and mean pO2 could be detected. After exclusion of four outliers the absolute values of the Pearson correlation coefficients increased clearly. There was no relevant association between mean or maximum FDG uptake and the different polarographic- as well as the CDS parameters.In order to investigate the degree of linear association, we calculated the Pearson correlation coefficient. Slight (|r| > 0.4) to moderate (|r| > 0.6) correlation was found between the parameters of pOCDS and FMISO PET represent different approaches for estimation of therapy relevant tumor hypoxia. Each of these approaches is methodologically limited, making evaluation of clinical potential in prospective studies necessary. Tumor hypoxia has been associated with malignant progression, representing an increasing probability of recurrence, loco regional spread and distant metastases. In addition, the hypoxic fraction in solid tumors reduces their sensitivity to conventional treatment modalities, modulating therapeutic response to ionizing radiation or certain chemotherapeutic agents ,8,10,25.2 histography) represents one standard for measuring tissue oxygenation in human malignancies  Fluoromisonidazole (FMISO) and [18F] Fluorodeoxyglucose (FDG) positron-emission-tomography (PET) for determination of tumor oxygenation. In addition two factors related to oxygen delivery, the tumor vascularisation and tumor perfusion were measured by contrast-enhanced color duplex sonography (CDS).We present the results of our analyses of a cohort of patients with head and neck malignancies, being examined with direct, invasive measurements of tissue oxygenation by pO2 measurement (last measurement). None of the patients had previously been treated for their malignancies. Because of different treatment modalities  no clinical analysis was performed. The study was approved by the medical ethical committee of the University Aachen, Germany. After explanation of the rationale, risks, and benefits of the examination, informed consent was obtained from all patients.Lymph node metastases of 38 patients with histologically verified head and neck malignancies  were enrolled in a prospective clinical evaluation between October 2002 and January 2005. Standard sonographic, CT and PET criteria were used for the diagnosis of metastatic lymph nodes. In 20 patients the diagnosis were confirmed histologically. Patients underwent the following measurements within one week: contrast-enhanced color duplex sonography (CDS), FDGPET, FMISOPET and polarographic pOMeasurements were performed in one lymph node metastases in each patient. In order to ensure measurements in the same suspected lymph nodes, sonographically examined lymph nodes were marked on diagnostic CT scans or in the case of lymph node conglomerates, the extension of the scanned node was marked on skin. PET examinations were realized in most of the patients within two days  using skin markers and positioning lasers for reproducible data acquisition resulting in a corresponding slice location. No immobilization device was used. Sonographic studies, selection of representative images and parameter calculation as well as polarographic measurement were performed by one person each respectively, resulting in an objective and independent data acquisition. As detailed information of all used techniques was published by our group ,6,27,34 ® in 11 ml sterile water for injection. 5.5 ml were given as a bolus whereas 5.5 ml were infused with 300 ml/h. CDS was performed in 32 patients.Technical details and adjustment of the sonography device were used as previously published ,6,27. InThis examination could be realized in 32 patients. All sonographic studies were recorded on digital video. From these video recordings, representative horizontal and longitudinal scans were selected for assessment. Depending on the size of the investigated nodes, this resulted in 5 to 16  = 4.38) images of each lymph node. For visualization of tumor vascularisation, the maximal systolic phases were used. Sonographic studies, selection of representative images and parameter calculation were performed by one person each, respectively. In a region of interest, representing the extent of vascularisation in the investigated slice, CPD was defined as ratio of colored pixels to gray pixels. The mean CPD was calculated in order to find a representative value for imaged vascularisation.®, Chameleon-Software Corp., Leipzig, Germany) was available. In 18 patients this software was used for evaluation of blood flow dynamics in the lymph node metastases until now. Cervical lymph nodes were examined at the largest diameter to record perfusion signals of intranodal vessels. All sonographic studies were recorded and perfusion signals of every single image were read out automatically from the region of interest (ROI). The ROI was defined in advance, encompassing the whole node's area, sparing out surrounding tissue.In the course of the study the commercially software  and pulsatility index (PI) in all vessels of the region of interest (ROI) . Every s® scanner with an axial field of view of 16.2 cm . The spatial resolution in the transaxial and axial orientation of this PET is nearly isotopic. In the reconstructed images, the full width at half maximum (FWHM) is about 6.0 mm measured at the center of the field of view (FOV) using a ramp filter with a 0.5 cut-off frequency.In 24 patients FDG PET as well as FMISO PET examinations could be performed. All PET studies were carried out using an ECAT EXACT 922/47FDG PET of the tumor region was performed 71 ± 17 min after intravenous administration of 264 ± 46 MBq FDG, applying the whole-body tool with 8 min emission scanning and 4 min transmission scanning for each bed position. After correction for attenuation using the transmission scan optimized by a segment μ-map with empirical attenuation coefficients, the data were reconstructed with the OSEM algorithm . All patFMISO PET consisted of one static scan of the relevant region – as defined by sonography or computed tomography – performed 120 min after intravenous administration of 314 ± 42 MBq FMISO. The acquisition time included 15 min emission scanning followed by 4 min transmission scanning. Attenuation correction and reconstruction processing was done according to FDG PET. Three venous blood samples  were taken at each static scan. After correction according to the half-life period the mean values were calculated. The radioactivity concentration was measured in a calibrated well counter . After correction for decay the mean values were calculated.T/M). To calculate tumor to blood ratios (FMISOT/B) at 120 min after administration of FMISO, the average radioactivity concentration of the three blood samples was used.The tumor was defined according to the image data of the FDG-PET and the puncture computer tomography (CT) scans. In cases where the tumour was not clearly visible in the FMISO scan, FDG data were used to delineate the malignant lesion and define a region of interest. Rectangular regions of interest depending to the tumor size were placed over the tumor and ipsilateral nuchal muscles in order to calculate FMISO tumor to muscle ratios  of the tumor were calculated after normalization of the radioactivity concentration to the injected radioactivity and the body weight. Additionally, the mean SUV was approximately corrected for partial volume effects by applying recovery coefficients obtained from phantom studies ...2-histograph . Sterile polarographic needle electrodes with stainless steel shafts and a mean diameter of 300 μm were used. For each patient the needle electrodes were placed and guided CT-controlled in the tumor after visual matching of CT- and PET-scans without general or local anesthesia, avoiding larger necrotic areas by preceding diagnostic CT scans. In this way we realized 95–400 single pO2 measurements per lymph node  resulting in a representative distribution of pO2 values. The relative frequency of pO2 readings with values ≤ 2.5 mmHg, ≤ 5.0 mmHg and ≤ 10.0 mmHg as well as mean and median pO2 were calculated to asses the biological and clinical relevance of oxygenation measurement [Tumor oxygenation was measured in 36 patients with polarographic needle electrodes, using a pOData are summarized by calculating relative frequencies, as well as suitable measurements of location and variation. The degree of linear relationship between different, polarographically measured parameters of tumor hypoxia, CPD, TP and the FDG-, and FMISO-PET parameters investigated by computing Pearson correlation coefficients and displayed graphically using scatter plots.®12.0 statistical analysis software package.All statistical analyses were performed using the SPSSThe interindividual range of mean color pixel density in the investigated lymph nodes was between 2.4% and 16.4% . The average TP was 0.065 cm/s with a range from 0.012 cm/s to 0.173 cm/s and a SD of 0.051 cm/s. A mean number of 8.0  recorded slices per measurement were evaluated.SUVmean = 1.69, SD = 0.40; average FMISOSUVmax = 1.98, SD = 0.50) and FDG  as well as tumor-to-muscle ratios of FMISO  and tumor-to-blood ratios of FMISO  were calculated for the total tumor regions.Mean and maximum SUV of FMISO . The average mean pO2 was 17.6 mmHg . The mean proportion of pO2 values ≤ 2.5 mmHg was 29.3% , of values ≤ 5.0 mmHg 38.4%  and of values ≤ 10.0 mmHg 48.9% .The average median pOT/M, FMISOT/B, CPD and TP, we calculated the Pearson correlation coefficient. Correlations are listed in Table T/B and the hypoxic fraction ≤ 2.5 mmHg could be detected. There was no relevant correlation between FMISOT/B and the other polarographically, as well as sonographically, measured parameters. There was also no relevant association between TP and the hypoxic fractions ≤ 2.5 mmHg as well as ≤ 5.0 mmHg. When graphically analyzing the observed correlations through an evaluation of the corresponding scatter plots  with apparent discrepancy between FMISO uptake and pOT/B and FDGSUVmax with a Pearson correlation coefficient of 0.403. No relevant correlation between FMISOSUVmax/mean and CPD/TP or the polarograhpic parameters could be detected. Figure 2 polarography, CDS, FDG and FMISO PET in a normoxic and a hypoxic tumor.There was no relevant association between mean or maximum FDG uptake and the different polarographic- as well as the CDS parameters with an absolute value of the Pearson correlation coefficient ranging between 0.056 and 0.241. There was only a moderate association between FMISOResults of preclinical and clinical investigations during the last ten years have confirmed that tumor hypoxia precedes malignant progression by several mechanisms, including an increased expression of transcription factors and gene products involved in tumor progression and induction of genomic instability. In those investigations, the transcriptional factor HIF-1 has emerged as a major regulator of adaptive processes (including angiogenesis) that can support tumor cell survival, proliferation, invasion, and tumor spread. It has also been shown that hypoxia can enhance malignant progression and increase aggressiveness through clonal selection . Numerou2 of 30 to 50 cells in front of the probe and not a direct measurement of intracellular oxygenation, it enables an estimation of radio biologically relevant intracellular oxygenation. This could be proved by the use of comet assay and pO2 polarograph in anaplastic R3327-AT Dunning prostate tumors, resulting in high correlations between mean pO2 and mean comet moment as a parameter of overall strand break induction [2 histography, an evaluation of intratumoral oxygenation heterogeneity, but not of spatial information is possible. As an invasive method it is confined to superficial, well accessible tumors or lymph node metastases. Evaluating the different methods for determination of tumor oxygenation, we found only slight to moderate correlations between pO2 histography and most of the non-invasive measurable parameters. When analyzing the differences in correlation through an evaluation of the scatter plots  with low mean and median pO2 values based on inhomogeneous distribution of the different hypoxic fractions. There was a high percentage of readings ≤ 10 mmHg and a low percentage of readings ≤ 2.5 or ≤ 5.0 mmHg. CPD or TP only deliver a mean value of tumor vascularisation or perfusion. The method is therefore unable to reproduce any heterogeneity of tumor oxygenation resulting in a decreased correlation between CPD or TP and the hypoxic fractions ≤ 2.5 and ≤ 5.0 mmHg.Comparing the parameters of pO2 polarography must be expected. Rejecting central necrotic tumor parts from CDS evaluation, we found good correlation between CDS and FMISO parameters. Patient 4 also showed an apparent discrepancy between FMISO uptake and pO2-polarography. This patient had a lymph node metastasis with a necrotic part and a part with vital tumor tissue  and perfusion (TP), an estimation of average tissue oxygenation without quantification of oxygenation heterogeneity was possible. The later could be realized by the FMISO parameters especially by FMISOClinical relevance of polarographic determination of tumor oxygenation was elucidated in several clinical studies. But recently the emphasis has been on the potential use of non-invasive approaches. CDS and FMISO PET for estimation of therapy relevant tumor hypoxia represent different non invasive approaches for analysis of tumor hypoxia. But each of these approaches is methodologically limited. Consequently clinical potential must be substantiated in a prospective study, including uniform treatment modalities in order to be more than only bricks in the puzzle of therapy relevant tumor hypoxia.Financial competing interestsThe pharmaceutical company Hoffmann- La Roche partially financed the costs for FMISO, a substance used to carry out PET scans.The authors declare that there are no other competing interests.BG has made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data; MP has been involved in acquisition of data; MP has been involved in acquisition of data; PR has made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data; MZ has made substantial contributions to conception and design, acquisition of data; HJK has been involved in acquisition of data, analysis and interpretation of data; SS has been involved in statistical analysis and interpretation of data; BA has been involved in acquisition of data; CD has made substantial contributions to conception and design; KH has made contributions to conception and design, GMP production of tracer; HHC has made contributions to conception and design, GMP production of tracer; TS acquisition of data, analysis and interpretation of data; PM acquisition of data, analysis and interpretation of data; ED acquisition of data, analysis and interpretation of data; MJE has been involved in analysis and interpretation of data and has made contributions to conception and design.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The index of myocardial performance has prognostic power in patients with cardiomyopathy and following myocardial infarction. As the index of myocardial performance has been shown to be preload and afterload dependent, the effect of altering contractility on IMP and its components with left ventricular dysfunction has been incompletely delineated.Chronic left ventricular dysfunction was induced in 10 canines using coronary microsphere embolization. Each dog was instrumented and imaged with 2D echo and Doppler. At the same atrially paced rate, contractility was increased with a dobutamine infusion and then following 4 weeks of oral digoxin.With chronic left ventricular dysfunction, a reduced left ventricular ejection fraction  and increased index of myocardial performance  due to isovolumic contraction time lengthening and shortened left ventricular ejection time were noted. Dobutamine increased ejection fraction (p < 0.001), reduced left ventricular end diastolic pressure (p < 0.01), and reduced the index of myocardial performance  due to isovolumic contraction time, isovolumic relaxation time, and left ventricular ejection time shortening. Digoxin increased ejection fraction (p < 0.05), reduced left ventricular end diastolic pressure (p < 0.05), and reduced the index of myocardial performance  due to isovolumic contraction time shortening (p < 0.001). Both dobutamine and digoxin lengthened the diastolic filling period (p < 0.01).Increased inotropy with digoxin and dobutamine reduced the index of myocardial performance in dogs with left ventricular dysfunction. Shortened isovolumic contraction time, increased diastolic filling period, and reduced left ventricular end diastolic pressure with digoxin may provide insight into its efficacy in heart failure. The index of myocardial performance (IMP), calculated as the sum of the isovolumic relaxation time (IRT) and isovolumic contraction time (ICT) divided by the left ventricular (LV) ejection time (LVET), has been utilized as a combined systolic-diastolic index that is prognostic in patients with dilated cardiomyopathy  and postSystematic evaluations of IMP in the normal and abnormal left ventricle have recently been performed using a canine model of chronic ischemic LV dysfunction. IMP was demonstrated to be preload and afterload dependent but not heart rate dependent ,10. In tDigoxin, an oral inotropic agent, also has beneficial symptomatic effects in heart failure patients with either reduced or preserved LV function . HoweverThe goals of this study were:  to deterThe animals used in this study were maintained in accordance with the guidelines of the Committee on Animal Studies at Wayne State University School of Medicine and with the position of the American Heart Association on research animal use. The protocol was approved by the Animal Investigation Committee of Wayne State University. Anesthesia was induced in 10 conditioned mongrel dogs (16–24 kg) with intramuscular morphine sulfate (1.5 mg/kg) and acepromazine (1.1 mg/kg) followed in 15 minutes by 30 mg/kg of intravenous ketamine hydrochloride. Maintenance anesthesia was produced by intravenous morphine sulfate (1.5 mg/kg/hr) and pentobarbital (3 mg/kg/hr). The dogs were intubated and artificially ventilated with a Harvard respirator using room air. Using fluoroscopic guidance, two 7F high fidelity catheters (Millar Instruments) were introduced via the right carotid artery and advanced to the left ventricle and ascending aorta. A #7 multipurpose Judkins catheter was introduced through a sheath (Cordis) into the right femoral artery and advanced into the left coronary ostium. Continuous electrocardiographic monitoring was performed using lead II. At held end expiration, ECG, LV pressures, dP/dT, and central aortic pressures were obtained at 100 mm/s using an 8 channel physiologic recorder (Gould). Simultaneous 2 dimensional echocardiograms and Doppler were obtained from with the use of a phased array echocardiograph (Aloka 880). Transesophageal 4 chamber view was obtained from a 5 MHz probe placed in the mid-esophagus. Both transaortic and transmitral pulsed Doppler recordings were obtained from the LV outflow tract and from beyond the tips of the mitral leaflets in the left ventricle at 100 mm/s. Mitral regurgitation was assessed from the 4-chamber view as the size of the maximal color flow jet divided by the left atrial area.Ischemic LV dysfunction was induced by left main coronary artery plastic microsphere injections (58 ± 2 microns) (3M) injected in boluses of 17,500 microspheres every 5–10 minutes until the peak positive dP/dT was reduced by 25% and the LV end diastolic pressure was >12 mm Hg. LV dysfunction  was produced in 45–60 minutes with only mild mitral regurgitation . This approach ultimately leads to a model of chronic LV dysfunction where the extent of LV dysfunction may be titrated [At 8 weeks post coronary microsphere embolization, the animals were anaesthetized, intubated, instrumented, and ventilated as above. Using fluoroscopy, a #7 thermodilution catheter and a #5 bipolar pacing wire were advanced from the left internal jugular vein to the pulmonary artery and right atrium respectively. Atrial pacing commenced 10 beats above baseline rate with a PR interval <160 msec. Transthoracic and transesophageal echocardiography and Doppler were obtained as above. Continuous electrocardiographic monitoring was performed using lead II. The above hemodynamic, echocardiographic, and Doppler parameters were obtained after 10 minutes of steady state atrial pacing. Dobutamine was infused at a rate of 2–2.5 micrograms/kg/minute for 15 minutes. Care was taken to adjust the rate of infusion to ensure that atrial pacing was maintained. The above parameters were then obtained. The infusion was discontinued and the dogs were observed until hemodynamics and echocardiographic assessment of LV systolic function returned to the pre-dobutamine LV dysfunction baseline. The above parameters were then obtained as a baseline for digoxin therapy. The vessels were repaired, and the animals were recovered.On the following day, each dog received 0.02 mg/kg/digoxin in their food daily to a maximal dose of 0.25 mg digoxin for 4 weeks ,14. At 1LV dysfunction was induced in 8 additional dogs as described above. At 8 and 12 weeks post embolization, the dogs were instrumented as described above. Hemodynamics, echocardiography, spectral, and color Doppler were obtained at baseline and with atrial pacing 10 beats above baseline. The same atrial rate was used for each dog at 8 and 12 weeks post embolization.For all stages and time periods, LV pressures, dP/dT, cardiac outputs, and aortic pressures were measured from the average of 3 consecutive cycles at held end expiration. The time constant of LV pressure decline was calculated using the Weiss method . LV end For all stages and time periods, all Doppler indices were measured from the average of 3 consecutive cycles at held end expiration. From transmitral Doppler indices, peak rapid filling velocity (E) and peak atrial filling velocity (A) were measured. The E/A ratio was calculated. The rapid filling deceleration time was calculated as the time interval from the peak rapid filling velocity to the time mitral flow decelerated to the zero baseline. The tracing was extrapolated to the zero baseline if atrial filling commenced prior to mitral flow fully decelerating to zero. Diastolic, rapid filling, and atrial filling velocity integrals were determined. Mitral regurgitation was assessed using the ratio of the maximal color flow jet area in the left atrium during systole divided by the left atrial area.From the transmitral pulsed Doppler, the time interval between the end of mitral filling and the onset of mitral filling was determined ("a"). From transaortic pulsed Doppler, the time interval from the beginning of aortic flow to the end of aortic flow was determined ("b" or LVET). The IMP can be calculated asIMP = (a-b)/bTo determine the IRT, the time from the R wave to the end of aortic flow was subtracted from the R wave to the onset of mitral flow interval. As "a-b" represents total isovolumic time, the ICT is the difference between IRT and "a-b" . The ICTIntraobserver variability for calculation of IMP was determined from 25 randomly selected stages. Each determination was performed 4 weeks apart. The average difference between determinations was 0.008 ± 0.002. Intraobserver and interobserver variability for LV echocardiographic volume was determined by selecting end diastolic and end systolic frames from the echocardiogram of 10 previously studied dogs. Each frame was analyzed 3 weeks apart by 2 observers (see acknowledgement). The average difference for end diastolic or end systolic volume was 2.2 ± 0.9 cc for intraobserver variability and 3.4 ± 1.7 cc for interobserver variability.All data was expressed as mean ± standard deviation. Differences between a variable among stages was assessed using paired T testing. A p < 0.05 was considered to be significant.th chronic LV dysfunction, LV volumes, LV mass, heart rate, and LV end diastolic pressure were increased. Ejection fraction and peak +dP/dT declined. Parameters of relaxation (Tau and E/A) were also abnormal as compared to baseline. The deceleration time was also shortened suggesting reduced LV chamber compliance. This finding may be secondary to the interstitial and replacement fibrosis seen in the myocardium with this model of chronic LV dysfunction [Table function . IMP wasTable Table IMP has been identified as a useful prognostic parameter following acute myocardial ,17,18 inBoth inotropic agents significantly increased peak +dP/dT and LV ejection fraction though the effect of digoxin was smaller. IMP was markedly reduced with dobutamine infusion with reductions in both ICT and IRT despite a reduction in LVET. The reduction in ICT was >70% and due to a combination of prolongation of time to mitral closure (12 msec) and shortening of time to onset of aortic flow (18 msec). Similarly oral digoxin for 1 month resulted in a reduction in the IMP due to a 61% reduction in ICT. Shortening of the ICT was likely related to the prolongation of the time to mitral closure (24 msec). With both agents, LV filling pressures declined and the diastolic filling period increased suggesting that reductions in total isovolumic time resulted in greater time for diastolic filling. With dobutamine, stroke volume increased and relaxation improved allowing a greater filling volume to enter a more relaxed left ventricle with lower resultant filling pressures. With digoxin, stroke volume also increased and was associated with a longer diastolic filling period resulting in a lower LV filling pressure. This finding with digoxin is intriguing as its use in patients with preserved LV function (ejection fraction >40%) has been both advocated and questioned ,21.a model of LV dysfunction in which a positive inotrope is administered or a negative inotrope administered in a model with normal LV function.Using a swine model, LaCorte  demonstrHowever, the relationship between contractile alterations and IMP is less well delineated in the left ventricle with reduced LV function. Most information regarding the effects of contractile alterations are based on an acute ischemic episode from a normal resting LV function . There aAs this is an experimental study, the applicability of these findings and relation to intact humans is always a limitation. The general anesthesia utilized may influence the results. However, this anesthesia regimen has been utilized in multiple experimental studies ,10,12 anThough IMP has been demonstrated to be load dependent ,10, it cIn conclusion, the effect of increasing inotropic stimulation in a model of mild chronic canine LV dysfunction was determined. Increasing inotropy with digoxin and dobutamine reduced IMP primarily due to ICT shortening. Dobutamine had the additional effects of IRT and LVET shortening. Of note, a shortened ICT, increased diastolic filling period, and reduced LV end diastolic pressure with digoxin may provide insight into its efficacy in heart failure.ICT = isovolumic contraction timeIMP = Index of myocardial performanceIRT = isovolumic relaxationLV = left ventricularLVET = left ventricular ejection timeThe author(s) declare that they have no competing interests.As this is a single author paper, the author designed, acquired and analyzed the data. The author drafted and wrote the manuscript.