C. elegans extends lifespan in part because signals from the somatic reproductive tissues activate the nuclear hormone receptor DAF-12.Removal of the germ cells of  Caenorhabditis elegans and Drosophila melanogaster, removing the germline precursor cells increases lifespan. In worms, and possibly also in flies, this lifespan extension requires the presence of somatic reproductive tissues. How the somatic gonad signals other tissues to increase lifespan is not known. The lifespan increase triggered by loss of the germ cells is known to require sterol hormone signaling, as reducing the activity of the nuclear hormone receptor DAF-12, or genes required for synthesis of the DAF-12 ligand dafachronic acid, prevents germline loss from extending lifespan. In addition to sterol signaling, the FOXO transcription factor DAF-16 is required to extend lifespan in animals that lack germ cells. DAF-12/NHR is known to assist with the nuclear accumulation of DAF-16/FOXO in these animals, yet we find that loss of DAF-12/NHR has little or no effect on the expression of at least some DAF-16/FOXO target genes. In this study, we show that the DAF-12-sterol signaling pathway has a second function to activate a distinct set of genes and extend lifespan in response to the somatic reproductive tissues. When germline-deficient animals lacking somatic reproductive tissues are given dafachronic acid, their expression of DAF-12/NHR-dependent target genes is restored and their lifespan is increased. Together, our findings indicate that in C. elegans lacking germ cells, the somatic reproductive tissues promote longevity via steroid hormone signaling to DAF-12.In  C. elegans and other organisms have shown that the reproductive system can also affect the rate at which an animal ages. Removal of C. elegans' germ cells extends lifespan but this effect is not simply due to sterility, as removal of both the somatic reproductive tissues and the germ cells does not extend lifespan. Instead, loss of the germ cells extends lifespan by activating a pathway that requires input from the somatic gonad. In this study, we demonstrate that the somatic reproductive tissues promote longevity by controlling the activity of a steroid signaling pathway that regulates the DAF-12 nuclear hormone receptor.Reproductive tissues are known to generate important intercellular signals. For example, in mammals, the reproductive tissues produce steroid hormones such as estrogen and testosterone that have profound effects on development and physiology. Studies of the nematode  Aging and reproduction are two central aspects of an animal's life history. Although evolutionary theorists have long hypothesized that an intrinsic cost to reproduction may shorten lifespan C. elegans, the somatic reproductive tissues  can have a dramatic lifespan-extending effect. When the germ cells of the worm are removed, the resulting sterile adult animals live 60% longer than they would with an intact germline. Removal of the somatic reproductive tissues along with the germ cells suppresses this lifespan extension, suggesting that the somatic gonad dispatches lifespan-extending signals to other tissues when the germline is gone In daf-9How does the reproductive system influence other tissues? In order for animals lacking the germline to live long, they require a signaling pathway involving the nuclear hormone receptor DAF-12, as well as genes that produce DAF-12 ligands , such as the cytochrome P450 gene C. elegans, as it also performs functions characteristic of the adipose tissue, the liver, and the pancreas.] DAF-16/FOXO is completely required for loss of the germ cells to increase lifespan, and expression of daf-16/FOXO exclusively in the intestine completely rescues the longevity of daf-16(−) mutants lacking a germline The analysis of this longevity pathway has been facilitated by the identification of molecular events that occur in the body when the germ cells are removed. A particularly important event caused by loss of the germ cells is the nuclear accumulation of the evolutionarily conserved lifespan-extending transcription factor DAF-16/FOXO in intestinal cells daf-12/NHR and daf-9/CYP450 are partially required for DAF-16/FOXO to accumulate in intestinal nuclei when the germ cells are removed daf-9/CYP450 mutants lacking germ cells with the DAF-12/NHR ligand Δ4-dafachronic acid rescues DAF-16/FOXO nuclear localization daf-12/NHR is still required for lifespan extension in animals carrying a mutant DAF-16/FOXO protein that localizes constitutively to nuclei daf-12/NHR has another function, apart from regulation of DAF-16/FOXO nuclear localization, in the regulation of longevity by the reproductive system.Previously, we demonstrated that daf-12/NHR might function in the signaling that takes place between the somatic reproductive tissues and the rest of the animal. Like DAF-12/NHR, the somatic gonad has a lifespan-extending function that does not involve DAF-16/FOXO nuclear localization. Germline-deficient animals that lack the somatic gonad do not live long even though DAF-16/FOXO accumulates in nuclei What is the other function of DAF-12/NHR? In this study we have asked whether germ cell (−)] have an extended lifespan that requires both the DAF-12 sterol-signaling pathway and the somatic gonad Worms that lack germ cells  with Δ4-dafachronic acid, one of several isoforms of dafachronic acid hormones shown to bind DAF-12/NHR 4-dafachronic acid was able to increase the lifespan of germline-deficient animals that also lacked the somatic gonad (daf-12(rh61rh411) null mutants that lack the somatic gonad and germ cells were treated with Δ4-dafachronic acid  animals that contained the somatic reproductive tissues. In two out of three experiments, we saw no effect of Δ4-dafachronic acid supplementation (germ cell (−); somatic gonad (−) animals treated with dafachronic acid was as long as that of germ cell (−) animals treated with dafachronic acid  mutant. We found that this construct was able to extend the lifespan of germ cell (−); somatic gonad (−) animals ; somatic gonad (−) animals.Besides being expressed in parts of the somatic gonad,  animals . Likewise system . Thus, Ddaf-9/CYP450. We found that this was the case: expression of DAF-9/CYP450 in ciliated sensory neurons using the cilium-specific che-2 promoter also extended the lifespan of germ cell (−); somatic gonad (−) animals (germ cell (−) animals.We also examined whether DAF-9/CYP450 could extend lifespan when expressed in cells that do not normally express  animals . This fi4-dafachronic acid, increasing dafachronic acid levels by overexpression of DAF-9/CYP450 had a greater effect on the lifespan of germ cell (−); somatic gonad (−) animals than it had on animals with intact gonads: germ cell (−); somatic gonad (−) animals lived longer than did intact animals in several strains carrying multi-copy daf-9/CYP450 transgene arrays. These findings, too, imply that dafachronic acid extends lifespan specifically in germ cell (−); somatic gonad (−) animals.Just as with addition of exogenous Δdaf-9 alleles and the canonical daf-12(m20) allele both completely prevent loss of the germ cells from extending lifespan m20 allele is predicted to eliminate the function of two isoforms of DAF-12/NHR while leaving a third isoform intact daf-12/NHR mutant rh61rh411 is predicted to inactivate all DAF-12/NHR isoforms daf-12(rh61rh411) mutants and found, to our surprise, that they lived slightly longer than intact controls (daf-12(m20) animals was removed rh61rh411 allele. Together, these findings necessitate a revised model for the role of DAF-12/NHR in animals that lack germ cells . We found that expression of GFP under the control of the cdr-6 promoter (Pcdr-6::GFP) was up-regulated in the animal by germ cell ablation  mutant (daf-12/NHR dependent increase in cdr-6 expression in germ cell (−) animals using quantitative RT-PCR (qPCR) (ablation . This cd) mutant . We veriR (qPCR) .cdr-6 expression upon germline removal, then (i) the presence of the somatic gonad should be required for loss of the germline to increase Pcdr-6::GFP expression and (ii) dafachronic acid should be able to substitute for the presence of the somatic gonad in the regulation of this gene. We found that both predictions were met. When we removed the somatic gonad as well as the germ cells, Pcdr-6::GFP expression was no longer elevated; instead, it was similar to the level observed in animals with an intact gonad (germ cell (−); somatic-gonad (−) animals were grown on plates containing Δ4-dafachronic acid, expression of Pcdr-6::GFP was restored to levels that were similar to those of Pcdr-6::GFP-transgenic animals lacking only the germline. As predicted, the increase in Pcdr-6::GFP expression caused by addition of dafachronic acid required a functional DAF-12/NHR protein. In a daf-12(rh61rh411) mutant, we observed no increase in cdr-6 expression upon Δ4-dafachronic acid treatment . The expression of a transcriptional Pdod-24::GFP gene fusion was variable; however, we found that dod-24 expression depended on the presence of the somatic gonad in a daf-12/NHR dependent fashion  mutant background, expression of a Psod-3::GFP fusion still increased upon germ cell removal. Furthermore, this increase in expression still required the somatic gonad (sod-3 by quantitative RT-PCR (qPCR) in glp-1(e2141) mutants, which lack germ cells and are long-lived daf-16/FOXO resulted in a significant drop in sod-3 expression, mutation of daf-12/NHR did not have a statistically significant effect  null mutant and observed that expression of Pcdr-6::GFP still increased relative to intact animals in response to loss of the germline (germ cell (−) animals lowered the expression of Pcdr-6::GFP. Thus, the somatic gonad was still able to modulate expression of cdr-6 when DAF-16/FOXO was not functioning. Notably, the magnitude of up-regulation of Pcdr-6::GFP in the daf-16(mu86) mutant was lower than in a wild-type background. We observed similar expression patterns when we measured cdr-6 mRNA levels by qPCR in glp-1(e2141) mutants, which lack germ cells. Mutation of daf-12/NHR resulted in a significant drop in the level of cdr-6 mRNA, whereas mutation of daf-16/FOXO did not affect cdr-6 mRNA levels , background, Δ4-dafachronic acid extended the lifespan of germ-cell (−); somatic gonad (−) animals, in a daf-16(mu86) background, there was no change in lifespan ; somatic gonad (−) animals fail to live long because they have insufficient dafachronic acid levels. We asked whether the somatic gonad might influence the level of daf-9/CYP450 gene expression, but this was not the case, as levels of DAF-9::GFP were not overtly different (unpublished data). However, the somatic gonad could potentially affect the level of dafachronic acid by alternate mechanisms; for example, by increasing the level of a biosynthetic precursor of dafachronic acid. It is also possible that the somatic gonad regulates the activity of DAF-12/NHR without affecting the total level of dafachronic acid. For example, the somatic gonad could influence the proportion of dafachronic acid in the animal that is available to bind to DAF-12/NHR. Another possibility is that the somatic gonad influences the levels or activities of DAF-12/NHR inhibitors or co-activators, though in this scenario, it is necessary to postulate that increased levels of dafachronic acid can overcome the effects of these co-regulators. It would be interesting to directly measure levels of dafachronic acids in animals that lack the germ cells or the entire reproductive system.One simple model to explain the longevity-promoting activity of the somatic gonad is that the somatic gonad stimulates dafachronic acid production when the germ cells are removed, which in turn affects DAF-12/NHR activity. Indeed, this model is not without precedent, as in humans the somatic reproductive tissues secrete steroid hormones such as androgens, which influence other tissues. In this model, daf-9/CYP450 is expressed in the somatic gonad  animals, daf-9/CYP450, dafachronic acid, and daf-12/NHR play a second role in facilitating the nuclear localization of DAF-16/FOXO germ cell (−) animals Besides modulating the lifespan of daf-12/NHR-dependent up-regulation of cdr-6 and a daf-12/NHR-dependent down-regulation of dod-24 in intact animals. Since activation of DAF-12/NHR is not sufficient to extend lifespan, other lifespan-promoting factors turned on in germline-deficient animals must be necessary for an increased lifespan. Because dafachronic acid extends lifespan in the absence of the somatic gonad, the somatic gonad itself is unlikely to provide these other lifespan-promoting factors. Instead, DAF-16/FOXO is the most likely candidate for the factor activated by loss of the germ cells that is necessary, along with DAF-12/NHR and the somatic gonad, to increase lifespan. Consistent with this idea, genetic inactivation of daf-16/FOXO, like the presence of the germ cells, prevents dafachronic acid from extending lifespan.Giving dafachronic acids to animals with an intact gonad does not extend lifespan . Howeverdaf-12/NHR null mutation, lifespan is extended slightly. This daf-12-independent lifespan-extension pathway (referred to here as the “underlying pathway”) does not require the somatic gonad and is not affected by dafachronic acid. When daf-12(+) activity is present but the somatic gonad is absent or daf-9/CYP450 is mutated, then germ-cell loss does not extend lifespan. This suggests that unliganded DAF-12/NHR prevents germ-cell loss from activating the underlying pathway. In contrast, liganded DAF-12/NHR extends lifespan in response to germ-cell loss, as daf-12(+) animals that have a daf-9(+) genotype plus the somatic gonad live long when the germ cells are removed. In the future, it will be interesting to explore the nature of the underlying daf-12-independent pathway and to learn how daf-12/NHR(+) can affect its activity. Finally, we note that a dual ability of DAF-12/NHR to extend and shorten lifespan is not without precedent. In intact animals, DAF-12/NHR extends lifespan in daf-9/CYP450 reduction-of-function mutants when animals are cultured at 15°C daf-9/CYP450 in the absence of thermosensory neurons An unexpected finding from this study was that DAF-12/NHR has a more complex role in this longevity pathway than previously appreciated. We found that when the germ cells are removed in animals containing a daf-12/NHR mutants, the DAF-16/FOXO target sod-3 is still up-regulated (though perhaps to a lesser extent). Likewise, Wang and Ruvkun showed that the lipase gene K04A8.5 is up-regulated by germline removal in a daf-16/FOXO-dependent but daf-12/NHR-independent fashion daf-16/FOXO is mutated, DAF-12/NHR still retains the ability to affect transcription of genes such as cdr-6 and dod-24. However, DAF-16/FOXO affects the magnitude of this regulation, suggesting that DAF-16/FOXO could have a partial effect on the activity of DAF-12/NHR. In summary, based on the several genes we examined, it appears that DAF-16/FOXO and DAF-12/NHR have distinct effects on the transcriptome of germ-cell deficient animals, although each has minor effects on the activity of the other. This interpretation is supported by a genome-wide microarray analysis of germline-defective daf-16/FOXO and daf-12/NHR mutants .Both sterol signaling and DAF-16/FOXO are required for the long lifespan of germline-deficient animals. The relationship between DAF-12/NHR and DAF-16/FOXO in animals that lack the germ cells is not well understood. Previous work has demonstrated that DAF-12/NHR is partially (but not completely) required for the nuclear accumulation of DAF-16/FOXO in animals that lack the germ cells Although dafachronic-acid signaling and DAF-16/FOXO have distinct effects on gene transcription in animals that lack germ cells, both are required to extend lifespan. Furthermore, dafachronic acid does not override the requirement for DAF-16/FOXO to extend longevity, and rendering DAF-16/FOXO constitutively nuclear does not override the requirement for DAF-12/NHR. These two pieces of data make it unlikely that DAF-12/NHR and DAF-16/FOXO operate in a simple linear pathway, where the transcriptional effects of mutation of one gene would be completely mimicked by the mutation of the other. Instead, the simplest interpretation is that DAF-12/NHR and DAF-16/FOXO function in parallel to promote longevity in animals without germ cells.Therefore, we propose the following model : germ-ceAll strains used in this study were maintained under standard conditions N2daf-12(rh61rh411)CF2479 daf-9(e1406); mgEx662[daf-9p::daf-9 genomic::GFP]daf-9(e1406); mgEx670daf-9(e1406); mgEx663daf-9(e1406); mgEx666daf-9(e1406); mgEx668dpy-5(e907); sEx15369[Pcdr-6::GFP + pCeh361]BC15369 sEx15369[Pcdr-6::GFP + pCeh361] obtained by outcrossing BC15369 3 times to the laboratory N2CF3595 daf-12(rh61rh411); sEx15369[Pcdr-6::GFP + pCeh361]CF3596 agIs6[Pdod-24::GFP]AU68 agIs6[Pdod-24::GFP] obtained by outcrossing AU86 3 times to the laboratory N2CF3556 daf-12(rh61rh411); agIs6[Pdod-24::GFP]CF3600 daf-16(mu86); agIs6[Pdod-24::GFP]CF3601 muIs84[Psod-3::GFP]CF1553 daf-12(rh61rh411); muIs84[Psod-3::GFP]CF3604 daf-16(mu86); sEx15369[Pcdr-6::GFP + pCeh361]CF3597 glp-1(e2141)CF1903 daf-16(mu86); glp-1(e2141)CF1880 glp-1(e2141); daf-12(rh61rh411)CF1658 daf-16(mu86)CF1037 Psod-3::GFP was described previously in daf-9::GFP strains were provided by the Ruvkun Lab and were described previously in Pdod-24::GFP strain was kindly provided by D. Kim. Strains containing Pcdr-6::GFP were obtained from the Genome British Columbia C. elegans Gene Expression Consortium Some nematode strains used in this study were provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources (NCRR). Construction of 3 agarose pads as experimental animals.Germ-cell  or somatic-gonad  precursor cells of newly hatched L1 larvae were killed by laser ablation as described previously p values were determined using the log-rank (Mantel-Cox) method.Lifespan analysis was performed at 20°C as described previously 3. Images were taken using a Retiga EXi Fast1394 CCD digital camera (QImaging) using the 10× objective on a Zeiss Axioplan 2 compound microscope (Zeiss Corporation). Each image was taken with the intestine in focus, since expression of the various transgenes was primarily in the intestine. For each trial, exposure time was calibrated to minimize the number of saturated pixels for that set of animals. Openlab 4.0.2 software (Improvision) was used to quantify the total intensity of fluorescence per worm as measured by intensity of each pixel in the selected area of a frame (i.e. the worm). Vulval expression of Psod-3::GFP, which was very bright, was excluded from quantification, since this structure is not present in animals lacking the gonad. Fluorescence of the entire animal was measured for all other GFP constructs. No expression of any of the constructs was visible in embryos prior to egg laying. Image processing for figures was performed using Adobe Photoshop 7.0 (Adobe).On day 2 of adulthood, animals were anaesthetized on agarose pads containing 0.15 M NaNglp-1(e2141ts) and wild-type N2 animals were raised at 25°C from L1 to day 1 of adulthood, then shifted to 20°C. On day 2 of adulthood, animals were collected for RNA extraction. RNA extraction, purification, and reverse transcription were carried out as described in Sterile http://docs.appliedbiosystems.com/pebiodocs/04303859.pdf). Data were generated from at least two biological repeats. mRNA levels of ama-1, nhr-23, and Y45F10D.4 were used for normalization p values were determined using one-way ANOVA. Primer sequences are available upon request.qPCR was performed using the 7300 Real Time PCR System (Applied Biosystems) and data were analyzed using the Ct method  mutant slightly extends lifespan in a somatic gonad-independent fashion. Removal of the germ cells and the somatic gonad in animals carrying the putative null daf-12(rh61rh411) allele extends lifespan. However, daf-12(rh61rh411) germ cell (−) animals do not live as long as germ cell (−) animals that carry the wild-type allele of daf-12/NHR. Means and p values are listed in (0.07 MB TIF)Click here for additional data file.Figure S2The somatic gonad represses the expression of dod-24. (A) dod-24 requires the somatic gonad for proper expression. The expression of the Pdod-24::GFP transgene was variable; however, across multiple experiments, we observed a consistent trend towards decreased intestinal Pdod-24::GFP expression when the germ cells were removed.  In germ cell (−); somatic gonad (−) animals, Pdod-24::GFP expression levels increased relative to those of intact-gonad and germ cell (−) animals.  Thus, the presence of the somatic gonad suppresses dod-24 expression. (B) GFP driven by the dod-24 promoter was observed throughout the intestine of wild-type animals. Arrowheads indicate position of the head. Original images were rotated and placed on a flat black background. (C) The increase in Pdod-24::GFP caused by removal of the somatic gonad required daf-12/NHR. Expression of Pdod-24::GFP did not increase in daf-12(rh61rh411) mutants in 4 out of 5 trials when the somatic gonad and germ cells were removed. Highly variable changes in overall expression of Pdod-24::GFP were observed when daf-12/NHR was mutated, even in intact animals. (D) daf-16/FOXO had little effect on the ability of the somatic gonad to inhibit dod-24 expression. In daf-16(mu86) mutants lacking the germ cells, removal of the somatic gonad still resulted in increased expression of Pdod-24::GFP. p values for pair-wise comparisons to intact-gonad animals are indicated by: *** p<0.0001, ** p<0.001, * p<0.05, ns p>0.05. Means and p values are listed in (0.73 MB TIF)Click here for additional data file.Table S1Dafachronic acid extends the lifespan of animals that lack both the somatic gonad and germ cells.(0.03 MB XLS)Click here for additional data file.Table S2Overexpression of DAF-9/CYP450 extends the lifespan of animals that lack both the somatic gonad and germ cells.(0.02 MB XLS)Click here for additional data file.Table S3The daf-12(rh61rh411) allele uncovers a lifespan shortening function of DAF-12/NHR in addition to its lifespan promoting activity in germ cell (−) animals.(0.02 MB XLS)Click here for additional data file.Table S4Pcdr-6::GFP up-regulation in animals that lack germ cells requires the somatic gonad and daf-12/NHR.(0.03 MB XLS)Click here for additional data file.Tabe S5Pdod-24::GFP expression changes in a daf-12/NHR-dependent fashion when the germ cells and somatic gonad are removed.(0.03 MB XLS)Click here for additional data file.Table S6Psod-3::GFP up-regulation in animals that lack germ cells is largely independent of daf-12/NHR.(0.03 MB XLS)Click here for additional data file.Table S7Pcdr-6::GFP up-regulation in animals that lack germ cells is largely independent of daf-16/FOXO.(0.03 MB XLS)Click here for additional data file.Table S8Changes in Pdod-24::GFP expression in the absence of germ cells and somatic gonad do not require daf-16/FOXO.(0.03 MB XLS)Click here for additional data file.Table S9DAF-16/FOXO and DAF-12/NHR have distinct effects on gene expression in animals that lack germ cells.(0.03 MB XLS)Click here for additional data file.


Present study was designed to evaluate Jigrine was evaluated in a number of pharmacological test paradigms, viz. open field arena, actophotometer, hole board, rotarod, traction test, grip strength test, spontaneous alternation behavior, passive avoidance task, and phenobarbital sleeping time.Jigrine pretreatment  did not produce any significant effect as compared to normal saline treated animals and was found to be free from any acute undesirable central effects at these two dose levels. Herbal drugs are widely used for the treatment of various diseases. Although herbal drugs often contain highly active pharmacological compounds but much importance is not given to their safety evaluation, may be due to a popular notion “anything herbal is safe.” Lately, with recent increasing interest in traditional or herbal drugs for the prevention and treatment of various disorders, there is also increasing concern about the safety of traditional, herbal product based medicines.per se neuropharmacological effects of jigrine in mice.Jigrine is a polypharmaceutical herbal formulation containing aqueous extracts of 14 medicinal plants used for liver ailments in traditional Indian system of medicine (Unani) . Few stuad libitum. All the procedures carried out on animals were approved by institutional animal ethics committee (JHAEC).Albino mice weighing 20–30 g were used for the study. Animals were supplied by Central Animal House Facility of Jamia Hamdard (Hamdard University) and kept under standard laboratory conditions in 12 h light/dark cycle at 25±2°C. Animals were provided with pellet diet  and water For each of the following tests, mice were divided into three groups of six animals each. Group I served as normal control and received normal saline . Groups II and III received jigrine 1 and 2 ml/kg, p.o., respectively.Mice were observed for their spontaneous motor activity by placing them individually in the actophotometer and number of beam breaks was recorded for 6 min. This apparatus consisted of a square chamber and the activity of the animals was recorded by light beams passing through the chamber and connected to photoelectric cells. The activity was recorded at 0 time and 30, 60, 90, and 120 min after their respective treatments. The actoet al.[Mice were observed for open field activity as per the method of Tricklebank et al. for 6 miMice were observed on the wooden hole board at 0 time (before treatment) and 60 and 120 min after their respective treatments.The wooden hole board apparatus consisted of 60 ×30 cm dimensions with 16 evenly placed holes and painted gray. The number of times the mice dipped their heads in holes during a 3-min trial was counted.Mice were trained to stay on the rotating rod of the rotarod (20 rpm) for 3 min. Trained mice were randomly divided into three groups as described above. All the animals were placed on the rotating rod  60 and 120 min after their respective treatments and the length of time they remained on the rotating rod was recorded.et al.[This test was carried out as per the procedure reported by Anca et al. Animals et al.12Grip strength of animals in all the 3 groups was measured as an indicator of neuromuscular function after 1hour of their respective treatments. The grip strength meter was positioned horizontally. Mice were held by the tail and lowered toward the apparatus. Mice were allowed to grasp smooth metal wire mesh (fore limbs only) and were then pulled backward in the horizontal plane. The force applied to the bar at the moment the grasp was released was recorded as the peak tension (kg). The test was repeated five times within the same session and the highest value from the five trials was recorded as grip strength for that animal.Spontaneous alternation behavior in mice was assessed by using a cross-maze. The maze was made up of plywood (painted gray) and consisted of symmetrical arms (23.5 cm long and 8 cm wide) with 10 cm high sidewalls. The arms extended from a central platform (8 × 8 cm) at a height of 50 cm above the floor and were labeled A, B, C, D. Mice were allowed to traverse the maze freely for 6 min after being placed on the central platform. The number and sequence of arm entries were recorded for calculation of a “percent alternation score.” An alternation was defined as entry into four consecutive arms on overlapping quintuple sets. Five consecutive arm choices within the set of arm choices made up quintuple sets. A quintuple set consisting of arm choices, ABDAC, was considered an alternation. A quintuple set consisting of arm choices, ABDAB, was not considered an alternation. Using this procedure, possible alternation sequences are equal to the number of arm entries minus 4. The percent alternation score is equal to the ratio of  × 100.et al.[The method of Papazova et al. was usedet al.One hour after their respective treatments, all the animals received pentobarbitobne . Time interval between administration of pentobarbital until the loss of righting reflex was recorded as the onset of sleep while the time from the loss of righting reflex to recovery was recorded as the sleeping time.P< 0.05 was considered significant.Results are expressed as mean±SEM. Total variation present in a set of data was estimated by ANOVA followed by Dunnet’s post hoc test. Jigrine was evaluated for its per se effects on locomotor activity, open field activity, hole board activity, rotarod performance, muscle relaxation, muscle grip strength, spontaneous alternation behavior, and sodium pentobarbital induced sleeping time in mice to assess its neuropharmacological safety profile. Locomotor and open


We aimed to characterize the lasting effect of moderate caloric restriction during early pregnancy on offspring energy homeostasis, by focusing on the effects on food intake and body weight as well as on the insulin and leptin systems.Male and female offspring of 20% caloric restricted dams (from 1 to 12 days of pregnancy) (CR) and from control dams were studied. These animals were fed after weaning with a normal-fat (NF) diet until the age of 4 months, and then moved to a high-fat (HF) diet. Blood parameters were measured under fed and 14-h fasting conditions at different ages . Food preferences were also assessed in adult animals.Accumulated caloric intake from weaning to the age of 5 months was higher in CR animals compared with their controls, and this resulted in higher body weight in adulthood in males, but not in females. Both male and female CR animals already showed higher insulin levels at the age of 2 months, under fed conditions, and higher HOMA-IR from the age of 4 months, compared with their controls. CR male animals, but not females, displayed higher preference for fat-rich food than their controls in adulthood and higher circulating leptin levels when they were under HF diet.It is suggested that hyperinsulinemia may play a role in the etiology of hyperphagia in the offspring of caloric restricted animals during gestation, with different outcomes on body weight depending on the gender, which could be associated with different programming effects on later leptin resistance. There is a growing body of evidence showing that the nutritional environment during early life may have later effects on the propensity to suffer from obesity and its related metabolic pathologies ,2. In thHowever, most evidence showing a direct association between gestational malnutrition and a higher propensity to obesity in later life comes from studies in rats, based on severe caloric restriction, generally between 30 to 50%, or severe protein restriction. For instance, Jones and Friedman  showed tOn another hand, little is known about the underlying mechanisms involved in the developmental programming of energy balance under conditions of undernourishment during gestation. The involvement of specific areas of the hypothalamus in the regulation of food intake and energy homeostasis has been clearly established . Hence, The purpose of the present study was to examine the lasting effects of moderate caloric restriction of 20% during the first 12 days of gestation upon offspring energy homeostasis, by focusing on the effects on food intake, body weight and fat accumulation, and on the insulin and leptin systems as possible determinants of potential disorders. In addition, the study was designed to investigate differences between male and female rats and in the effects of a dietary stressor in adult life such as high fat (HF) diet exposure.ad libitum feeding conditions. After the caloric restriction period, rats were allowed to eat ad libitum, and food intake was measured. At day 1 after delivery, excess pups in each litter were removed to keep 10 pups per dam . Weaning was conducted at 21 days of life.The study was performed in male and female rats from 12 different litters, following the protocol below. All rats were housed under controlled temperature (22°C) and a 12 h light-dark cycle (light on from 0800 to 2000), and had unlimited access to tap water and standard chow diet  unless mentioned otherwise. Briefly, virgin female Wistar rats weighing between 200 g and 225 g were mated with male rats . Day of conception (day 0 of pregnancy) was determined by examination of vaginal smears for the presence of sperm, and then female rats were single caged. Pregnant rats were divided into two groups : one with free access to standard chow diet, and the other one underwent a 20% restriction of caloric intake from day 1 to day 12 of pregnancy. Caloric restriction was performed by offering each dam a daily amount of food corresponding to 80% of the calories should be eaten according to body weight. This amount was calculated considering the calories daily consumed by their control animals under At weaning, 24 animals from control dams (controls)  and 24 from caloric restricted dams (CR)  were placed two per cage, paired with another animal of the same group, and fed with standard diet until the age of 4 months; then they were exposed to a high fat (HF) diet . HF diet contained 5.5% calories from soybean oil and 39.5% from lard.Body weight and food intake of the offspring were followed until the age of 5 months. In addition, body fat content  was measured without anesthesia at 2 different ages, when animals were 2 and 5 months old. Body length (from the tip of the nose to the anus) was also measured at the end of the follow up, when animals were 5 months old.ad libitum - animals provided with free access to chow diet - and fasting - animals deprived of food for 14 h. In both conditions, samples were obtained without anesthesia and during the first 2 h of the beginning of the light cycle. Plasma was obtained by centrifugation of blood at 700 g for 10 min, and stored at -20°C until analysis.At 3 different ages , blood samples  were collected (in heparinized containers) from the saphenous vein of control and CR animals, under different feeding conditions: The animal protocol followed in this study was reviewed and approved by the Bioethical Committee of our University and guidelines for the use and care of laboratory animals of the University were followed.Blood glucose concentration was measured by Accu-Chek Glucometer . Plasma insulin concentration was determined using a rat insulin enzyme-linked immunosorbent assay (ELISA) kit  following standard procedures. Plasma leptin concentration was measured using a mouse leptin ELISA kit . Commercial enzymatic colorimetric kits were used for the determination of plasma triglyceride (TG) levels (Triglyceride (INT) 20, Sigma Diagnostics, St Louis, MO, USA) and non-esterified fatty acid (NEFA) .The homeostatic model assessment for insulin resistance (HOMA-IR) was used to assess insulin resistance. It was calculated from fasting insulin and glucose concentration using the formula of Matthews et al : HOMA-IRFood preferences were assessed by a two-bottle preference test as previously described . BrieflyGiven that the animals studied were from six different litters in each treatment group, the effect of litter was simultaneously factored with all data by repeated measures ANOVA. No interactions between the litter and treatment were found across all the data, thus, data were expressed as mean ± S.E.M of animals from the six different litters. Multiple comparisons were assessed by repeated measures ANOVA and two-way ANOVA to determine the effects of different factors . Single comparisons between groups were assessed by Student's t test or Paired t test. The analyses were performed with SPSS for Windows . P < 0.05 was always the threshold of significance.As previously described in the same cohort of dams , 20% matAs shown in Figure The higher body weight of CR male animals compared with their controls can be explained by higher food intake Figure . Daily fUnlike males, no statistically significant differences were found in body weight of CR female animals compared with their controls at any age Figure . NeverthDifferences in body weight between control and CR adult male animals can be mainly attributed to differences in body fat content. At the age of 5 months, CR male animals presented higher percentage of body fat than controls (Student's t test) Figure . HoweverBody length of animals was not different between control and CR animals, either in males or females (data not shown).Circulating glucose, insulin, leptin, triglycerides and NEFA levels were measured in control and CR male and female animals under fed and 14 h fasting conditions at the ages of 2, 4 and 5 months  Figure .Circulating leptin levels of control and CR animals under fed and fasting conditions at the ages of 2, 4 and 5 months are also shown in Table Blood lipid profile was significantly altered in CR animals as of the early stages of life Table . In factFood preferences were measured in adult animals at the age of 3 months with the two-bottle preference test Figure . Both coIt is known that severe caloric restriction during gestation in rats increases the propensity of adverse health outcomes in adulthood, including obesity, although differences according to the magnitude of restriction has been described ,8,9. HerCentral resistance to insulin and/or leptin has been proposed as important mechanisms responsible for the dysregulation of energy homeostasis, which may lead to obesity -17. HereConcerning leptin, moderate caloric restriction led to gender related differences on later leptinemia. Adult CR male animals under NF diet showed a tendency to higher circulating leptin levels under ad libitum feeding conditions compared with their controls, in accordance with their higher body weight. This hyperleptinemia was further increased later on, when animals were 5 months old and under HF diet. In fact, it has been described an increase in leptin levels with age, which seems to be associated to the increased body weigh and fat content . This maImpaired fetal growth by 30% caloric restriction throughout gestation has also been described to be associated with hyperinsulinemia and hyperleptinemia in adult male offspring . These dCirculating lipid profile in animals that underwent caloric restriction during gestation, particularly the elevations of plasma NEFA and TG levels already occurring at the age of 2 months, could also give some clues to explain the apparent insulin resistance in both male and female CR animals. Elevated circulating NEFA levels have been described to play an important role in early molecular events involved in the development of insulin resistance . They diMetabolic programming of hyperphagia due to maternal caloric restriction during gestation could be associated with an alteration in the neuronal development of the central nervous system under these environmental conditions . In factThe reasons for the differences between males and females regarding circulating leptin levels and fat accumulation in adult life due to these particular prenatal conditions are not clearly elucidated yet. Of interest, differences between genders concerning the sensitivity to central leptin and insulin have been previously described ,29. The In addition to the amount of food intake, programming food preferences, as a part of the feeding behavior, may contribute to body weight control and to the development of obesity. This is particularly relevant in humans where obesity development is generally associated with increased appetite and preference for highly caloric food ,34. HereIn summary, it is concluded that a moderate caloric restriction of 20% during the first part of gestation has lasting, gender-dependent effects in the offspring. In particular, these animals are programmed for higher food intake, which seems to be related with central insulin resistance and with higher blood TG and NEFA levels, already present at a juvenile age, and this concludes in higher body weight in adult male rats but not in females. Why programming animals for higher food intake has different outcomes depending on the gender of animals needs further investigation; it is hypothesised that hyperleptinemia, which seems to occur, at least to some extent, in CR males but not in females under these conditions, may play a determinant role in the adult onset of obesity.The authors declare that they have no competing interests.MP participated in the experimental design of the study, carried out the animal procedure and the analysis, and participated in the discussion of the results. TP participated in the animal procedure and the discussion of the results. JS participated in the animal procedure and in the discussion of the results. AP participated in the design and coordination of the study and revised the final version of the manuscript. And CP conceived of the study, and participated in its design and coordination, carried out the discussion of the results and wrote the article. All authors read and approved the final manuscript.


NFKB1 gene was identified which seems to be the first potential functional NFKB1 genetic variation. The main goal of the present investigation was to investigate the NFKB1 -94 insertion/deletion ATTG polymorphism in relation to risk of dilated cardiomyopathy (DCM).Previous studies in experimental and human heart failure showed that nuclear factor kappa B (NF-κB) is chronically activated in cardiac myocytes, suggesting an important involvement of NF-κB in the cardiac remodeling process. A common insertion/deletion  located between two putative key promoter regulatory elements in the NFKB1 -94 insertion/deletion ATTG polymorphism was genotyped by using PCR-PAGE.A total of 177 DCM patients and 203 control subjects were successfully investigated. The NFKB1 -94 insertion/deletion ATTG polymorphism in DCM patients was significantly different from that in control subjects (P = 0.015) and the ATTG2 carrier (ATTG1/ATTG2 + ATTG2/ATTG2) was susceptible to DCM.Genotype frequency of NFKB1 -94 insertion/deletion ATTG polymorphism is associated with DCM.Our data suggested that  Dilated cardiomyopathy (DCM), characterized by left ventricular dilation and systolic dysfunction, is the most common form of heart muscle disease, comprising 60% of the cases of identified cardiomyopathy . This diClinical observations and experimental studies have demonstrated that left ventricular (LV) remodeling and dilation occurs with the progression of end-stage LV failure. It has been reported that in experimental and human heart failure, nuclear factor kappa B (NF-κB) is chronically activated in cardiac myocytes, suggesting an important involvement of NF-κB in the cardiac remodeling process . NF-κB iNFKB1 gene was identified which seems to be the first potential functional NFKB1 genetic variation. The presence of a 4 base pair (bp) deletion resulted in the loss of binding to nuclear proteins, leading to reduced promoter activity [NFKB1 -94 insertion/deletion ATTG polymorphism on the occurrence of DCM.A common insertion/deletion polymorphism  located between two putative key promoter regulatory elements in the activity . A reseaactivity -19. Furtactivity -23. HoweThis study was approved by the hospital ethics committee and all subjects gave written informed consent to participate. One hundred and seventy seven unrelated DCM patients between September 2002 and March 2008 were enrolled in this study. The diagnosis of DCM was made in accordance with the revised criteria established by the 1995 World Health Organization/International Society and Federation of Cardiology Task Force on the Classification of Cardiomyopathy (DCM group) [NFKB1. DNA fragments containing the polymorphism were amplified and the primer sequences were: F 5'-tggaccgcatgactctatca-3', R 5'-ggctctggcttcctagcag-3'. PCR reaction was performed in a total volume of 25 μl, including 2.5 μl 10× PCR buffer, 1.5 mmol/L MgCl2, 0.15 mmol/L dNTPs, 0.5 μmol/L each primer, 100 ng of genomic DNA and 1 U of Tag DNA polymerase. The PCR conditions were 94°C for 4 min, followed by 32 cycles of 30 s at 94°C, 30 s at 64°C and 30 s at 72°C, with a final elongation at 72°C for 10 min. 3 μl PCR products were separated by a 6% polyacrylamide gel and staining with 1.5 g/L argent nitrate. Allele (ATTG)1 yield a 154 bp band and allele (ATTG)2 yield a 158 bp band. About 20% of the samples were randomly selected to perform the repeated assays and the results were 100% concordant.Genomic DNA of each individual was extracted from 200 μl EDTA-anticoagulated peripheral blood samples by a DNA isolation kit from Bioteke  and the procedure was performed according to instruction manual. The polymerase chain reaction (PCR)-polyacrylamide gel electrophoresis (PAGE) method was used to genotype the -94 insertion/deletion ATTG polymorphisms of NFKB1 gene -94 insertion/deletion ATTG polymorphism were obtained by directed counting and Hardy-Weinberg equilibrium were evaluated by chi-square test. Odds ratio (OR) and respective 95% confidence intervals were reported to evaluate the effects of any difference between allelic and genotypes distribution. Probability values of 0.05 or less were regarded as statistically significant in DCM patients compared to healthy controls.All data analyses were carried out using SPSS 13.0 statistical software. Allelic and genotype frequencies of NFKB1 gene -94 insertion/deletion ATTG polymorphism were tested between DCM patients and controls, and observed differences are presented in Table Genotype distributions had no deviation from Hardy-Weinberg equilibrium both in patients and controls. Differences in allelic and genotype distribution of 1/ATTG1 genotype was slightly overrepresented in controls . Furthermore, the P value and OR were 0.005 and 2.304, respectively, ATTG1/ATTG2 + ATTG2/ATTG2 vs. ATTG1/ATTG1 comparison, indicating that ATTG2 carrier (ATTG1/ATTG2 + ATTG2/ATTG2) was susceptible to DCM. The frequency of allele ATTG2 in DCM patients was higher than that in control subjects (62.7% vs. 57.1%), but is not statistically significant (P = 0.118).The overall genotype frequency of DCM patients was significantly different from that of controls. The frequency for ATTGGenetic factors are known to play an important role in the etiology of DCM. The first DCM-associated mutation, in the dystrophin gene, was described in 1993 ,25. The factor in the nucleus of B cells that binds to the enhancer of the kappa light chain of immunoglobulin [NF-κB was discovered by Baltimore and coworkers in 1986 as a globulin ,26. The globulin . Inapproglobulin . NF-κB iglobulin ,23,27. Hglobulin . FurtherNFKB1 promoter in 10 inflammatory bowel disease and 2 controls, and the ATTG1 allele was more frequent in ulcerative colitis than that in controls in the following study. The in vitro promoter expression studies suggest that the ATTG1 allele may result in relatively decreased NFKB1 message and hence decreased p50/p105 NF-κB protein production [The -94 insertion/deletion ATTG polymorphism was identified in a study sequenced the oduction . The assoduction ,17,29.NFKB1 gene -94 insertion/deletion ATTG polymorphism in DCM patients is not significantly different from that of controls. However, the genotype frequency distribution in DCM patients was significantly different from that of controls. The frequency of the ATTG1/ATTG1 genotype of NFKB1 gene -94 insertion/deletion ATTG polymorphism in controls was significantly higher that in DCM patients. We conducted comparison between ATTG1/ATTG1 and (ATTG1/ATTG2+ATTG2/ATTG2) in DCM patients and controls, and further significant difference was observed (p = 0.005). These results indicated that ATTG2 carrier (ATTG1/ATTG2 + ATTG2/ATTG2) was susceptible to DCM. The allelic distribution between DCM patients and controls was different, although not statistically significant (p = 0.118), and the frequency for ATTG2 allele in DCM patients is higher than that in controls, although not statistically significant, also indicating that ATTG2 allele might be a risk factor for the susceptibility to DCM.Given the considerable important role of NF-κB pathway involved in initiation and progression of pathogenesis in disease, we investigated the association between -94 insertion/deletion ATTG polymorphism and susceptibility to DCM. The present study shows that the allelic frequency for NFKB1 gene -94 insertion/deletion ATTG polymorphism in DCM patients is significantly different from that in control subjects, and (ATTG1/ATTG2+ATTG2/ATTG2) genotype frequency is higher in DCM patients than that in control subjects. Considering that the ATTG1 allele may result in decreased NFKB1 message and p50/p105 NF-κB protein production, a detrimental role of NF-κB-activation in the initiation and progression of pathogenesis in DCM can be presumed and much further work will be needed for a complete understanding of its mechanism. The main limitation of the present study is the relatively small size of the study population and the lack of replication of the significant association in a second independent cohort of DCM patients.The role of NF-κB in heart has been extensively studied by many authors . Its rolNFKB1 -94 insertion/deletion ATTG polymorphism is significantly associated with DCM. The genotype frequency of (ATTG1/ATTG2+ATTG2/ATTG2) is significantly overrepresented in DCM patients, indicating that ATTG2 carriers are associated with the increased risk of DCM. However, additional studies in a larger number of DCM patients and in different populations could help to establish the true significance of this association.From this study we conclude that the The authors declare that they have no competing interests.BZ, LR and LZ conceived of the study, participated in its design, carried out most of the experiments and drafted the manuscript. YL, LG and YW participated in design of study and helped to draft the manuscript. YC, HX and YS performed sample collection and DNA extraction. YP and ML participated in genotyping. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Detailed human studies of the mechanisms and development of shunt infection in real time are not possible, and we have developed a canine hydrocephalus model to overcome this. The intention of this pilot study was to show that the canine hydrocephalus model could be shunted using conventional "human" shunts, and that a shunt infection could be established so that further studies could then be planned.Canis familiaris) by fourth ventricle obstruction. Four weeks later they were shunted using a Hakim Precision valve. Four of the dogs received shunts whose ventricular catheter had been inoculated with Staphylococcus epidermidis, and three were uninoculated controls. Four weeks after shunting the dogs were sacrificed and necropsy was performed. Removed shunts and tissue samples were examined microbiologically and isolates were subjected to detailed identification and genomic comparison.Hydrocephalus was induced in seven dogs (S. epidermidis in the brain and throughout the shunt system in the four inoculated animals, but in two of these Staphylococcus intermedius was also found. S. intermedius was also isolated from all three "negative" controls. There were slight differences between S. intermedius strains suggesting endogenous infection rather than cross- infection from a point source.All the dogs remained well after shunting. Examination of removed shunt components revealed Shunt infection was established in the canine model, and had the experiment been extended beyond four weeks the typical microbiological, pathological and clinical features might have appeared. The occurrence of unplanned shunt infections in control animals due to canine normal skin flora reflects human clinical experience and underlines the usual source of bacteria causing shunt infection. Staphylococcus epidermidis [S. epidermidis infections is typically low-grade. A shunt-related infection of the central nervous system (CNS) is of particular concern because of the threat to cerebral functions. Treatment may be prolonged and relapses may occur, complicating control of intracranial pressure and giving rise to life-threatening risks such as cognitive and neurological deficits [The introduction of cerebrospinal fluid (CSF) shunts revolutionized the treatment of hydrocephalus and greatly decreased mortality rate. However, over subsequent decades, difficulties in shunt maintenance and problems resulting from infection or occlusion have persisted . Today, dermidis ,10. Thesdermidis . The sevdeficits ,6,12,13.Although many studies have investigated the rate, aetiology and epidemiology of shunt infections, preventive measures are difficult to study, and their pathophysiology is largely unknown due to the difficulties inherent in human studies. Animal models of infection in other implantable devices have not hitherto been suitable for these purposes. We have developed a canine model of chronic obstructive hydrocephalus  which prCanis familiaris) weighing 25–30 kg were used for this study. Animals were obtained from licensed suppliers and quarantined for a minimum of seven days before entering the study. They were maintained in the Cleveland Clinic's fully accredited Animal Care Facility and their maintenance and all experimental procedures were conducted in accordance with the National Institutes of Health (NIH) and The Cleveland Clinic Foundation Animal Research Committee (ARC) guidelines for the use and care of laboratory animals. Each experiment began with the induction of obstructive hydrocephalus (time zero), the details of which have been previously described [Seven young adult (8–9 months) male purpose – bred mongrel dogs . Prior to placement, the ventricular catheter in four of the animals was inoculated with S. epidermidis sensu stricto, F22, a clinical isolate from a proven VP shunt infection. A suspension of the bacteria was prepared in 2 mL of 0.85% saline to give an absorbance at a wavelength of 490 nm of 0.6 equivalent to a bacterial count of 1.0 × 108 colony forming units (cfu) per mL and the catheter was filled with this suspension. The remaining three animals were not inoculated. Post-operative monitoring and care was similar to that described for the post-induction recovery period except that no antibiotics were given. The animals were checked daily and data on temperature, heart rate, food and water intake, and incision site were obtained. The animals were weighed weekly, and closely monitored for clinical signs of infection for the four weeks following the shunt inoculation and implantation procedure. These signs included systemic variables such as sepsis and a persistent fever greater than 39.1°C, neurological variables such as lethargy, coma, seizures, and focal deficits, and local variables such as swelling, redness, soreness, and discharge. If no signs of infection occurred during the 4 weeks of observation, the animals were anaesthetized and a dissection was performed under aseptic conditions.Approximately four weeks after induction, the hydrocephalic animals underwent a standard shunt implantation surgery. The preoperative method of sedation and positioning with the stereotaxic head frame was the same as described previously ,17. The The distal end of the VP shunt was carefully exposed by gross dissection and ligated with silk sutures at the ventricular catheter, proximal and distal to the valve, and at the most distal end of the shunt tubing. Biopsies of the brain parenchyma and meninges adjacent to the ventricular catheter track were obtained via a 2 cm bilateral cranial burr hole. A biopsy of the peripheral tissue adjacent to the valve was obtained and the valve removed. The distal catheter tip was cut and removed along with the remaining peritoneal shunt tubing. The three tissue samples , catheter tips , ligated shunt, and the CSF samples Fig  were sen2 at 35°C, bacterial growth and identification for all samples were determined. The isolates were then sent to the University of Nottingham (R.B.) for further examination. After confirmation of identity using basic tests, each isolate was examined by APIStaph , numerical antibiogram [The tissue samples, catheter tips and lumbar CSF samples underwent routine microbiological processing. The ligated shunt tubing was sonicated for one minute, and fluid withdrawn from the lumen for culture. After incubation overnight in COibiogram  and pulsEach brain and its corresponding meninges was examined for gross signs of encephalitis, meningitis, and ventriculitis (GP). Tissue from the right cerebral hemisphere, which included the ventricular catheter track from each animal was embedded in paraffin wax, sectioned at 8 μm, and stained with haematoxylin and eosin, and the Brown-Brenn tissue Gram stain.All seven animals that underwent the induction of chronic hydrocephalus procedure recovered from surgery. One animal developed symptoms of acute hydrocephalus and required two days of observation in the Critical Care Unit and treatment of raised ICP before resolution. In the remaining animals, vital signs monitored  during the three to four-week period following the induction surgery remained in the normal range. At the time of sacrifice, Evans ratio of all animals was >0.3 confirming ventriculomegaly. There was no incidence of mortality or morbidity in any animal for either the experimental or control groups. Most hydrocephalic animals exhibited signs of lethargy, motor weakness, anorexia, and ataxia in the first seven days immediately following surgery. Vomiting often accompanied neurological signs  reflecting increased ICP. These neurological signs were observed to be transient and resolved within the first one to two days. No animal was observed to be febrile. Autopsy performed in each animal revealed no evidence of intracerebral or intraventricular bleeding, which corroborated with MRI data. In addition, the surgical incision site in all animals healed well and did not show any signs of infection.Six of the seven dogs lost weight during the first week following induction of hydrocephalus. The one animal that did not lose weight following surgery and one other animal whose weight returned to normal following recovery from induction, had shown a weight loss two weeks prior to the shunt implantation surgery. Food intake decreased by almost 50% eight days prior to the shunting procedure. In the four-week period following shunting all animals showed a steady weight gain for the remainder of the study. The animals recovered from the shunt-inoculation procedure very quickly. They had normal consumption of food and fluid the first day post-operatively. Gait and vital signs were normal for the four-week period following the inoculation-shunt procedure. None was febrile and there were no signs of infection of the abdominal or head wounds.At dissection, the animals were deeply anaesthetized, facilitating close examination of the wounds. In five of the seven animals, upon opening the healed sutured skin on the head, fluid collection was observed. In one animal this fluid was thick and slightly discoloured and followed the shunt catheter distally to the valve (neck region). On removal of the ventricular catheters, a plug appeared to block the end of the catheter in five animals. In addition, there was protein matter (non-brain) infiltrating some of the valve systems. No visual signs of infection were observed at the abdominal insertion incision. At autopsy no peritoneal cysts or other abnormalities were observed.S. epidermidis grew this organism from most of the collected samples . In two of these four "S. epi" animals, a second bacterium was also cultured. This was identified (APIStaph) as Staphylococcus intermedius. S. intermedius was grown in broth culture only from the brain tissue and proximal catheter samples of one animal and from the peritoneal tissue sample from the second animal. In the remaining three uninoculated control animals , all samples were positive for S. intermedius. The S. intermedius isolates were compared by APIStaph, numerical antibiogram and PFGE  was achieved and verified by gross examination and culture of hardware and tissue. In addition, the important role of skin flora in seeding of a shunt system during insertion was demonstrated fortuitously by the high rate of S. intermedius isolation, the three dogs that were not inoculated with S. epidermidis all contracting shunt infections due to S. intermedius. The evidence of infection decreased with samples taken downstream of the catheter in the peritoneal cavity, and none was found in the lumbar fluid.Using the model a 100% infection rate (four out of four animals inoculated with 8cfu/mL bacteria into the ventricular catheter in four animals, in two cases the animals' own S. intermedius was also isolated, as well as from the three uninoculated controls . S. intermedius comprises 90% of all the staphylococci isolated from canine skin [S. intermedius and S. epidermidis were found in the same shunt system the S. intermedius was found in the more proximal portion, and S. epidermidis in the more distal part. Given the flow of CSF through the shunt system this suggests the possibility of bacterial contamination from the proximal end down to the distal end. Comparison of the S. intermedius isolates by three methods (Table In spite of the inoculation of 10ine skin . In two ds Table  showed tS. intermedius has been isolated from a majority of healthy canines [S. intermedius can be confused with Staphylococcus aureus. Both are coagulase positive, and produce a heat – stable nuclease, but S. intermedius does not produce Protein A and most isolates are clumping factor negative. Some variation can occur. Our isolates were clumping factor positive but coagulase negative using human plasma, and they produced a heat – stable nuclease. Their identification was kindly confirmed by Professor François Vandenesch of the University of Lyon, France. canines  although canines -27. S. iThis paper characterizes a canine model of chronic adult obstructive hydrocephalus. The animals survived well with no mortality and low morbidity, providing the opportunity for physiological studies, pathophysiological analyses and diagnostic studies.S. intermedius infections, which once appreciated should be avoidable in future studies, we suggest that the model described here, perhaps with extended post – infection monitoring, is particularly suitable for the study of the pathogenesis, prevention and treatment of experimental shunt infections.In spite of the fortuitous endogenous The authors declare that they have no competing interests.RB contributed to the inception and design of the study, carried out the detailed microbiological investigations, and wrote the manuscript. CB and SMD assisted in the surgery and postoperative management and drafted data. MT and GH carried out the initial laboratory examination of removed shunts and tissues. GP carried out the necropsies and the histopathological investigations. MGL contributed to the inception and design of the study, carried out the hydrocephalus induction, the shunt surgery and the clinical examinations at termination, and oversaw the project in USA. All authors have read and approved the final version of the manuscript.


After 18 weeks under EOD, animals were also trained by using a treadmill for another 6 weeks and then analyzed for physical activity. Both, EOD and endurance exercise increased the resistance of animals to extenuating activity and improved motor coordination. Among the groups that showed the highest performance, AL and EOD trained animals, ALT and EODT respectively, only the EODT group was able to increase glucose and triglycerides levels in plasma after extenuating exercise. No high effects on mitochondrial respiratory chain activities or protein levels neither on coenzyme Q levels were found in gastrocnemius muscle. However, exercise and EOD did increase β-oxidation activity in this muscle accompanied by increased CD36 levels in animals fed under EOD and by changes in shape and localization of mitochondria in muscle fibers. Furthermore, EOD and training decreased muscle damage after strenuous exercise. EOD also reduced the levels of lipid peroxidation in muscle. Our results indicate that EOD improves muscle performance and resistance by increasing lipid catabolism in muscle mitochondria at the same time that prevents lipid peroxidation and muscle damage.Every other day feeding (EOD) and exercise induce changes in cell metabolism. The aim of the present work was to know if both EOD and exercise produce similar effects on physical capacity, studying their physiological, biochemical and metabolic effects on muscle. Male OF-1 mice were fed either  Every other day feeding (EOD) is a procedure that resembles many of the effects of caloric restriction (CR). EOD increases life-span, decrease cancer incidence and protects against age-associated diseases and neurodegeneration or endogenous damage of DNA Ex vivo experiments on rat gastrocnemius muscle exposed to electrical stimulation have suggested synergy between CR and aerobic exercise in muscle bioenergetics The similar effects induced by CR and exercise suggest that both interventions share some mechanisms that modify cell physiology. Both, CR and exercise modify significantly the bioenergetics of muscle. Long-term CR delays the decline of the skeletal muscle aerobic capacity that occurs during aging Most of the studies reported about dietary restriction and exercise have been performed by comparing the effect of CR versus exercise The scope of our research includes the study of different physical activities such as: spontaneous locomotion, grip strength, startle response, motor coordination and resistance to strenuous exercise in mice submitted EOD and/or endurance exercise. All these parameters might be influenced by difference in mice's weight since they were fed in different conditions. However along our experiment we did not find any difference in weight between groups under either AL or EOD . Furthervs. 66.05±10.41% in AL+EOD animals, n = 32, P = 0.002). Further, EOD also produced significant improvement in this test . When we compared the four groups, the higher and more significant improvement was found in the EODT group (P = 0.001), , .vs. 840±108 s, P = 0.015) and covered more distance . Remarkably, EOD group showed almost the same capacity than trained groups . In contrast, plasma glucose levels decreased in the ALT group after exercise although showing similar performance in the extenuating test  (P = 0.012).The two EOD groups were able to maintain or even increase (in the case of EODT) glucose levels after extenuating exercise . A mean ing test . In the In the case of lactate, levels of witness animals showed a tendency to decrease in trained animals  and espevs. AL animals  independently of training (P = 0.001). After extenuating exercise, EOD and EODT groups were able to maintain or even increase TGs levels while they decreased in AL group . After extenuating exercise, both AL and ALT groups showed increases in plasmatic cholesterol while EOD and EODT groups did not present any changes respecting witness animals (P = 0.017) when all AL fed animals  were compared with all animals fed under EOD conditions .In the case of lipids, levels of circulating triglycerides (TGs) in witness animals were significantly lower in animals fed under EOD conditions  AL group . As in t animals . This inP = 0.011). The effect of exercise on initial levels of urea was higher in EOD groups being significant between EOD and EODT groups (P = 0.042). Extenuating exercise did not produce any remarkable change in the levels of plasmatic urea except in the case of EODT group  respecting to sedentary animals  . On the other hand, training slightly but not significantly increased activity in AL fed animals . However, in EODT animals a decrease of activity vs. EOD animals was found being significant in the case of complex II (P = 0.017) but not in the case of complex III (P = 0.379) (P = 0.183). Training induced a slight decreased of this activity in AL fed animals (P = 0.402) being higher and significant in EOD fed animals (P = 0.035). We also determined the coupled activity of complexes I+III and II+III that depend on coenzyme Q (Q) amount in mitochondrial membrane. No changes among groups were found in both activities  . No chan= 0.379) . In the = 0.379)  we foundtivities .9 is the predominant form in mice being around 10 times more abundant than Q10. No significant differences among the different mice groups were found , whereas EOD only induced a non-significant increase (P = 0.305). However, when combined, EOD and exercise highly increased β-oxidation in comparison with the levels found in the AL group (P = 0.027). Further, we determined the level of the fatty acid translocase (CD36), a protein involved in the incorporation of fatty acids from plasma to muscle and in mitochondrial fatty acid oxidation. Surprisingly, EOD significantly increased CD36 levels in comparison with AL animals (P = 0.0009) whereas in EODT animals the levels of this protein decreased to the levels of AL group , which is considered to produce only a mean of 15% deficit in the calorie input due to the fact that animals eat more when they have access to food Edge exploration and elevated plus maze tests indicate that the differences in physical activity were not due to a different degree of anxiety between groups. However, an interesting finding was that, with independence of the feeding procedure, trained animals decreased their total activity in an open field after few minutes probably indicating a higher capacity to recognize new environments. Our outcomes have also shown that EOD and aerobic training increase motor coordination and resistance to exhausting exercise. In the case of running on rolling carpet, EOD slightly increased physical resistance in trained animals whereas it did significantly increase resistance in the sedentary group when compared with the AL group. Interestingly, one of the documented changes in rodents fed under CR is the increase of non-forced physical activity when compared with AL fed animals Another interesting issue was the effect of EOD on the capacity of mobilization of nutrients such as sugars and fats. Our study differs from others mainly in the fact that metabolite analysis of blood was performed in animals that were not fasted overnight. Our aim was to determine the physical capacity and associated parameters, thus, we could not submit the animals to strenuous exercise after fasting. Our data indicate that EOD improves the capacity of mobilization of glucose and lipids maintaining the levels of these metabolites during a strenuous exercise. These results suggest that animals under EOD were more efficient in mobilizing nutrients necessary for physical activity. In agreement with our results, a study made on body builders has proved that restriction in calories during a brief period of time (7 days) increases the capacity to mobilize lipids during running Induction of mitochondrial biogenesis by CR or exercise has been shown in different organisms and tissues The increase in mitochondrial content is considered a well-established adaptation of muscle to exercise, and is mainly referred as mitochondrial biogenesis. In our hands, neither EOD nor exercise induced changes in the activity and amount of proteins involved in respiratory chain. Similar results have been recently shown in other models of CR and exercise where no changes in porin, a mitochondrial mass indicator, have been reported Our results also indicate that both EOD and exercise increase β-oxidation in muscle. Electron microscopy analysis of the mitochondria in gastrocnemius muscle demonstrated that the size, localization and morphology of mitochondria were different in EOD animals. EOD and EODT animals showed more mitochondrial structures in the intermyofibrillar area and around the myofibers. Intermyofibrillar location of mitochondria has been associated with lipid droplets. Thus, these changes in the mitochondria in an intermyofibrillar location in muscle suggest a more efficient activity producing ATP near the sarcomera by using lipid catabolism 9 and Q10 in a different way. Exercise decreased the ratio whereas EOD increased it. These changes probably respond to a more bioenergetic function of Q9 whereas Q10 mainly plays an antioxidant role in mice. CR decreases the levels of reactive oxygen species (ROS) in cells 9/Q10 ratio increases In agreement with previous results, levels of Q were also slightly affected by EOD and exercise EOD protected muscle fibers against oxidative stress even in animals that suffered endurance activity. This higher protection may be related to lower muscle damage after extenuating activity in non-trained EOD animals determined by the levels of creatine kinase in plasma. Our results agree with several other reports that suggest that EOD protects muscle against oxidative stress and even avoids muscle loss during aging In summary, our work indicate that a nutritional stress induced by the EOD model of CR together with a moderate increase of energy expenditure through physical exercise produces metabolic changes that increase the efficiency of mitochondrial activity in muscle, reduces oxidative damage and improves physical performance. Subtle modifications at the cellular and biochemical levels in response to dietary stress seem to be the basis for a higher mitochondrial efficiency. Taking into consideration that the decline in physical activity during age is a common factor in many species, the results shown here suggest that the combination of the reduction of calorie intake and the practice of aerobic exercise would also increase physical performance in humans and then, improve their quality of life.A cohort of 64 non-consanguineous swiss-OF1 male mice aged 6 weeks was used . Animals were housed into enriched environmental conditions in groups of 6 animals per polycarbonate cage in a colony room under a 12 h light/dark cycle (8:00 AM–8:00 PM) under temperature (22±3°C) and humidity controlled. Animals were maintained accordingly to a protocol approved by the Pablo de Olavide University Ethical Committee and following the international rules for animal research.ad libitum (AL) and the other half under CR using the every-other-day feeding model (EOD) ad libitum for all the groups. After four months under these conditions, each group was randomly subdivided again in a sedentary group and a trained group following a mild forced aerobic exercise protocol. During the first two weeks, a training protocol was performed by a routine increasing both speed and time on a treadmill  until reaching 20 meters/min and 20 min. This protocol consisted in the 70–80% of average maximum speed reached in the initial tests. After that, animals were trained at this speed for 20 min/5 days a week during the following 6 weeks similarly to already published endurance exercise protocols ad libitum and sedentary group; ALT, ad libitum and trained group; EOD, caloric restricted and sedentary group and EODT, caloric restricted and trained group. Weight was determined every 15 days.Animals were first randomly assigned to two initial groups: half of the animals were fed All physical activity analysis was carried out between 9–11 hours in the morning and just after a feeding day for the EOD groups. Spontaneous activity was determined by using an open field box (26×39 cm)  determined by the number of broken light beams during a period of 10 min per animal. Peripheral exploration was determined directly by eye in sessions of 5 min as anxiety measure. Also, elevated plus maze test was used to quantify anxiety levels during 5 min per animal. Press force tests were performed by using a Grip Strength . For startle response, animals were placed individually inside a startle chamber . The startle response was determined by using a piezoelectric accelerometer controlled by homemade software. Startle stimulus was 100 ms at 125 db and the response is indicated as the average from 20 to 30 recordings. Motor activity and coordination tests were performed on Rotarod . After an adaptation period, a test was performed to combine coordination and resistance by using Rotarod speed acceleration up to 100 rpm and determining the maximum riding time of animals at this speed.In the case of extenuating activity, all groups of animals performed a week of habituation before test. Half of the animals from each group (n = 8) were exposed to extenuating physical exercise on treadmill associated with electric stimuli, without inclination and fastening speed by 5 meters/min every 5 min. We established the end of the experiment at the moment the animal stopped for more than 5 seconds under electric stimuli without trying to move back to the treadmill. Animals were sacrificed by cervical dislocation and dissection was performed just after the test was finished. The rest of the animals of each group remained as witnesses without running and were sacrificed at the same time than the runners. These witness animals were considered as controls of the plasmatic metabolic situation of animals before exercise. To avoid any effect of nutrient uptake on extenuating activity performance, all groups received food during the day before the exercise test.g and stored in small aliquots kept at −80°C until the determination of different blood metabolites. Metabolites were analyzed by using commercial kits for triglycerides (Randox TR210), cholesterol (Randox CH201), urea (Randox UR457), uric acid (Randox UA233), L-lactate , glucose (Randox GL2623), total protein (Randox TP245) and albumin . Creatine kinase (CK) activity was analyzed by the CK-NAC  test. In all cases, samples were always processed in parallel with the respective quality controls provided by the supplier.Blood was collected by cardiac puncture just after cervical dislocation. Plasma was obtained by centrifugation in Vacuette Z serum Sep. Clot activator tubes for 10 min at 3000×g to eliminate debris and nuclei. Protein was determined by Stoschek-modified Bradford's method Gastrocnemius muscles were dissected immediately after sacrifice and frozen in liquid nitrogen. After thawing and clearing from connective tissue, muscle was homogenized by using lysis buffer  supplemented with 1 mM PMSF and protease inhibitor cocktail 1∶50 (Sigma) in a 1∶9 volume-to-weight ratio, followed by centrifugation for 10 min at 700×1 as electron acceptor (ε340: 6.81 mM−1 cm−1). Complex II was measured in 50 mM phosphate buffer pH 7.0 by kinetic quantification of the reduction of 0.1 mM diclorophenol-indophenol (DCPIP) at 600 nm for 2 min at 30°C after adding 32 mM succinic acid to the reaction (ε600: 19 mM−1 cm−1). Complex III was determined in 50 mM phosphate buffer pH 7.5 by kinetic quantification of the reduction of the cytochrome C (0.05 mM) at 550 nm for 2 min at 30°C after adding decylubiquinol (0.05 mM) as electron supplier to the reaction (ε550: 21 mM−1 cm−1). Complex IV was measured in 10 mM phosphate buffer pH 7.0 by kinetic quantification of oxidation of reduced cytochrome C as electron donor at 550 nm for 2 min at 38°C using oxygen as electron acceptor (ε550: 21 mM−1 cm−1). The combined activities of complex I+III and II+III were determined in 50 mM phosphate buffer pH 7.5 by determining the kinetics of the reduction of cytochrome C for 2 min at 30°C by adding NADH (0.1 mM) (complex I+III) or succinic acid (3 mM) (complex II+III) as electrons donors (ε550: 21 mM−1 cm−1). For each determination, specific inhibitors such as rotenone , actimycin A , sodium azide  and ferric cyanide , were used as previously indicated Mitochondria chain complexes activities were measured in a spectrophotometer (Thermo Spectronic Unicam UV 500). Complex I was measured in 20 mM phosphate buffer pH 8.0 by kinetic quantification of 0.2 mM NADH consumption at 340 nm for 2–3 min at 30°C after adding 1 mM CoQ340: 6.22 mM−1 cm−1). Inhibition with oligomycin (0.2 mg/ml) was used to specifically determine mitochondrial ATPase activity.Complex V/ATPase activity was determined by the measurement of its ATP phosphatase activity. Assay was performed in Hepes-Mg pH 8.0 (50 mM) by adding NADH (0.2 mM), phosphoenol pyruvate (2.5 mM), pyruvate kinase (10 mg/ml), lactate dehydrogenase (5 mg/ml) and antimycin A (0.2 mg/ml). After 2 min of incubation, ATP (25 mM) pH 7.0 was added (ε412: 13.6 mM−1 cm−1).Citrate synthase (CS) activity was determined in 75 mM Tris-HCl buffer (pH 8.0) by quantification of ditio-bis-nitrobenzoate (0.1 mM) reduction in the presence of Triton X-100 1%, acetyl CoA (7 mg/ml) and oxalacetate (5 mM) at 412 nm for 2 min at 30°C  for 10 min followed by a vigorous shake with vortex. Q was extracted with hexane as indicated Whole muscle homogenate was obtained after disruption in lysis buffer as above indicated by using a mechanical driven homogenizer and further centrifugation at 700×g to remove debris. Supernatant was mixed with two times concentrated Laemmli Buffer  and proteins separated by SDS-PAGE. Proteins were transferred to Hybond ECL nitrocelullose membranes  and visualized by using the mouse monoclonal antibodies against the 39 kDa subunit of NADH:ubiquinone oxidoreductase , 70 kDa subunit of Succinate:ubiquinone oxidoreductase , core 2 subunit Ubiquinol:cytochrome c oxidoreductase , subunit Vb of the Cytochrome c oxidase  and α-subunit of ATP synthase   at 1∶1000 dilution and HRP-labeled anti-mouse IgG at 1∶1000. Blots were visualized by ECL technique and quantified by using the Quantity One 1-D analysis software (Biorad). Blots were digitalized and quantified by using Quantity One software (Biorad). Protein expression levels were corrected for whole protein loading determined by staining membrane with Red Ponceau.MDA levels were measured according to the method of Gérard-Monnier et al. with some modifications Freshly isolated muscle samples were fixed with 2.5% Glutaraldehyde in PBS and further with 1% Osmium Tetroxide, dehydrated with acetone and embedded in araldite resin. Sections (50 nm) were obtained by using an ultramicrotome (LKB 8800 Ultrotome III). After placing onto a cooper grid, slices were stained for 5 min with uranyl acetate and lead citrate followed by 2 min incubation with lead citrate. Samples were visualized by using a transmission electron microscope (Philips CM10). Ten random images  were analyzed to measure the area occupied by mitochondria, the number of mitochondrial structures per unit of area, the area per mitochondria (at least 10 mitochondria per image) and the roundness of each mitochondrial structure by using the ImageJ software version 1.42i .P<0.05.SigmaStat 3.5 program was used for the statistical analysis and figures were performed by using SigmaPlot 10.0 program (Systat Software Inc). All data are expressed as means ± S.E. For all experiments, 16 mice per group were analyzed. The information obtained from each group was statistically processed pursuant to the most suitable technique for each case. Student t-test or two ways Analysis of Variance (ANOVA) followed by Post-Hoc pairwise multiple comparison procedures (Bonferroni t-test) were performed. The critical significance level α was  = 0.050 and, then, statistical significance was defined as


Mycoplasma haemofelis is the most pathogenic of the species; ‘Candidatus Mycoplasma haemominutum’ and ‘Candidatus Mycoplasma turicensis’ are less pathogenic. The natural route of transmission of feline haemoplasma infection has not been confirmed, but fleas are implicated. When disease results, common clinical signs are pallor, lethargy, anorexia, weight loss, depression, dehydration and pyrexia. Treatment with tetracyclines or fluoroquinolones is usually effective at resolving clinical disease, but clearance of infection may not result.The feline haemotropic mycoplasmas (‘haemoplasmas') are a group of bacteria that can induce haemolytic anaemia in cats. The feline haemoplasmas are found worldwide, although prevalence varies geographically.M haemofelis.Older male non-pedigree cats are believed to be at increased risk of haemoplasma infection, although younger cats are possibly more likely to show clinical disease associated with The significance of feline haemoplasma infection is difficult to determine due to the existence of asymptomatic carrier cats and the variable pathogenicity of the haemoplasma species. Polymerase chain reaction (PCR) results should be interpreted in the light of the patient's clinical signs and haematological findings, infecting haemoplasma species and level of haemoplasma DNA present in the blood. Trial antibiotic treatment for haemoplasmosis may be warranted in suspected cases while awaiting PCR results.Aspects of feline haemoplasmosis, particularly risk factors, pathogenesis, diagnostic methods and treatment, have been the focus of much recent research. This article draws on the current evidence base with a view to helping clinicians diagnose and manage cases more effectively.


Groups of 4 guinea-pigs were immunized with acid extracts prepared from bovine myelin (EF), normal human liver tissue and malignant or benign neoplastic tissues in Freund's complete adjuvant . All the animals immunized with EF developed clinical symptoms of EAE within 21 days of the initial immunization, whilst some of the animals immunized with certain tumour extracts developed symptoms which closely resembled those of EAE. Control animals immunized with FCA only remained asymptomatic. Cellular immunity to the various extracts in immunized animals was assessed 20 days after immunization by i.d. skin testing, and upon killing at Day 21 with the direct peritoneal-exudate macrophage migration inhibition (MMI) test. Brains and spinal cords were removed at killing, fixed in formalin and processed for histological examination. I.d. skin testing was shown to be most consistent in demonstrating positive delayed hypersensitivity, whilst the MMI test frequently gave negative results in the presence of pronounced skin responses to specific extracts. Thus it was shown that 3/4 animals immunized with basic proteins extracted from an adenocarcinoma of the lung or related hepatic metastases, and 1/2 animals immunized with an extract of a carcinoma of the breast, gave intense erythema and induration responses 5 mm in diameter 24 h after i.d. challenge with EF. No such response was obtained in animals immunized with basic proteins extracted from normal human liver, any of the other neoplastic tissues, or in control animals immunized with FCA only. Examination of brains and spinal cords from animals immunized with EF revealed dense infiltration by mononuclear cells in the ependyma and choroid plexus of levels in the spinal cord. Examination of brains and spinal cords from animals immunized with the lung-tumour extract or related hepatic metastases which showed demonstrable immunological cross-reactivity with EF in immunized animals, revealed a number of inflammatory changes characterized by dense infiltrates of mononuclear cells sub-ependymally, and perivascular cuffing in the cortex. However, no significant lesions were seen in the spinal cords of these animals. Polyacrylamide-gel electrophoresis of the 2 tumour extracts exerting this apparent encephalitogenic effect did not reveal proteins within the mol. wt range of EF. Thus the observed pathological effects and cross-reactivity with EF were probably not due to contamination with nervous-tissue components. It is suggested that these tumour extracts may have contained a component or components other than EF, immunologically cross-reactive with EF, and capable of inducing the observed encephalitis.


A young male patient was undergoing myringoplasty for right ear chronic suppurative otitis media. While drilling in the middle ear cavity, duramater was breached accidentally. Surgeons were, however, allowed to complete the procedure. Keeping the seriousness of such a complication in mind, an urgent neurosurgical intervention was sought and noncontrast computed tomography head scan was done to analyse the extent of the injury. Osteoplastic craniotomy had to be performed subsequently to evacuate the contusional haematoma over the right temporoparietal region. Throughout the procedure, patient's vitals were monitored vigilantly to prevent any further deterioration of his condition. All the available resources were tapped judiciously to maintain intracranial pressure within normal limits. With a quick responsiveness on the part of the anaesthesia team, an active decision making, appropriate and remarkable anaesthetic management both intra and postoperatively, and good ICU care, a young patient could be salvaged and discharged successfully within a week with no immediate or residual complications related to myringoplasty or any neurological deficit. The complication rate of myringoplasty is low in all series. Kotecha A 16-year-old, 70-kg ASA grade I male was undergoing myringoplasty for right ear CSOM in routine elective operation theatre under general anaesthesia. Unexpectedly, the duramater was breached over the aditus after 2 hours of initiating the surgery, which was recognised as a sudden give way by the otolaryngiologists. Keeping the impending cerebral compromise in mind, we sought an emergency neurosurgical consultation and decided for an urgent noncontrast computed tomography (NCCT) head scan. Meanwhile, temporalis muscle flap was placed over the breached dura by the operating surgeon. The wound was closed in two layers over gel foam and a drain. The patient was electively ventilated in ICU while awaiting NCCT head scan report with Assist Control Ventilation (volume cycled) and was maintained on continuous intravenous infusions of inj. Midazolam 0.25 mcg/kg/minute and inj. Vecuronium 1 mcg/kg/minute. Vitals were maintained within normal limits. The patient was catheterised, a nasogastric tube inserted and fundoscopy done to rule out papilloedema.NCCT head scan revealed intraparenchymal haematoma of size 4.5 × 2 × 3 cm with minimal mass effect and peri-lesional oedema with few foci of pneumocephalus in right temporal region,extra-axial bleed  in right tentorium cerebelli andbony defect in right petrous temporal bone with fluid in middle and external ear cavity.In ICU, complete haemogram, prothombin time (PT), activated partial thromboplastin time (aPTT) and international normalised ratio (INR) were done. Patient was given inj. Phenytoin 800 mg i.v. (loading dose) and inj. Vit. K one amp. i.m. along with the antibiotic cover.2O, urine output was adequate. The above regimen was implemented to maintain the vitals and cerebral haemodynamics within physiological limits. Inj. Ondansetron 6.0 mg i.v. (for postoperative nausea and vomiting) and inj. Diclofenac sodium 75 mg i.m.  were administered. Inj. Vecuronium and inj. Midazolam infusions were stopped. When patient was fully conscious with good cognition, adequate protective airway reflexes and motor power, he was extubated after giving inj. Glycopyrrolate 5 mcg/kg i.v. and inj. Neostigmine 50 mcg/kg i.v. There were no signs of any residual neurological deficit. The patient was observed in ICU. Subsequent NCCT head scan was normal. Treatment regimen consisted of inj. Amikacin, inj. Augmentin, inj. Phenytoin, inj. Mannitol and syp. Citicoline. The patient was discharged on the seventh postoperative day.An urgent right temporo-parietal osteoplastic craniotomy was planned for the drainage of contusional haematoma in 15° head up position . High riVariations in the anatomy of the temporal bone, such as anteriorly placed lateral venous sinus, low middle cranial fossa dura or a prominent Korner's septum create additional difficulties for the inexperienced surgeon, leading to various complications. The effe2), cerebral blood flow (CBF) and ICP, since N2O is 35 times more diffusible than nitrogen, pneumocephalus and VAE (if it occurs) can expand dangerously and, therefore, was avoided. Oxygen–air mixture is preferable but we used 100% oxygen along with isoflurane 0.4–0.6 vol.% as air was not available at our institution. Isoflurane produces a reduction in the CMRO2 which is greater than that produced by equivalent Minimum Alveolar Concentration (MAC) values of halothane, but the CBF increase with isoflurane is minimal below 1.1 MAC.[Major goals during anaesthesia include (1) maintenance of adequate perfusion and oxygenation of normal brain, (2) optimising operative conditions to facilitate resection, (3) ensuring rapid emergence of anaesthesia at the conclusion of the procedure to facilitate neurological assessment, and when appropriate, 4) accommodating intraoperative electrophysiological monitoring.[ accommod2 responsiveness is preserved.[2 and inj. Vecuronium, as such, has no cerebral and few systemic side effects.Veselis and colleagues, using PET (Positron Emission Tomography) observed a global reduction in CBF of 12% after 0.15 mg/kg of inj. Midazolam to awake healthy human volunteers and noted that the decrease occurred preferentially in brain regions associated with arousal, attention and memory. Further,reserved. Inj. FenThough we used the above technique, target controlled infusion (TCI) or total intravenous anaesthesia (TIVA) using inj. Propofol are being used at some centres, more so during awake craniotomies. At the end of surgery, a rapid recovery of consciousness is essential so that the level of response can be assessed along with Glasgow Coma Scale and to rule out any neurological deficits. Use of a good antibiotic regimen, phenytoin (antiepileptic), mannitol (osmolar diuretic), dihyropyridine calcium channel blocker like nimodipine  and a vitamin B choline like citicoline (neuroprotective agent), helps in rapid postoperative recovery.Thus, a seemingly normal procedure can pose a challenge to an anaesthesiologist's competence. When such complications occur, he should be able to take prompt decisions, act swiftly and use all the available resources optimally to affect a good patient outcome.