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Scientific Reports
Apr 2016

 

Abstract

The Smurf Assay (SA) was initially developed in the model organism Drosophila melanogaster where a dramatic increase of intestinal permeability has been shown to occur during aging (Rera et al., 2011). We have since validated the protocol in multiple other model organisms (Dambroise et al., 2016) and have utilized the assay to further our understanding of aging (Tricoire and Rera, 2015; Rera et al., 2018). The SA has now also been used by other labs to assess intestinal barrier permeability (Clark et al., 2015; Katzenberger et al., 2015; Barekat et al., 2016; Chakrabarti et al., 2016; Gelino et al., 2016). The SA in itself is simple; however, numerous small details can have a considerable impact on its experimental validity and subsequent interpretation. Here, we provide a detailed update on the SA technique and explain how to catch a Smurf while avoiding the most common experimental fallacies.

Keywords: Smurf Assay (蓝精灵分析法), Digestive tract permeability (消化道通透性), Blue dye #1 (蓝色染料#1), Ageing (衰老)

Background

The Smurf Assay (SA) is based on the Drosophila feeding assay described in (Wong et al., 2009). The assay assesses food intake by the co-ingestion of a blue dye, which is not absorbed by the digestive tract, thereby allowing direct quantification. For the SA it is essential that this specific blue dye does not pass an intact intestinal barrier since its readout is a whole body coloration (here blue). This property allows direct in vivo assessment of gut permeability, which has been shown to increase with age (Rera et al., 2011 and 2012).

As recently discussed in Rera et al. (2018), the Smurf Assay is not only a simple way to assess intestinal permeability in vivo, but also an elegant way to assess the physiological age of individuals in a broad range of organisms. As such, it allows novel approaches to study the various events occurring in aging individuals (Tricoire and Rera, 2015; Rera et al., 2018).

In recent years, we have received numerous comments and questions about the initial protocol, leading us to develop the present extended protocol.

Specifics of the dye used: The dye typically used is FD&C blue dye #1, but we have also validated the use of red #40 and fluorescein (Rera et al., 2011 and 2012). We adapted the use of the very same blue #1 to zebrafish (Dambroise et al., 2016) and killifish (Rera et al., 2018) but found it is easier to use fluorescein with nematodes although the same blue #1 can also be used as demonstrated in (Gelino et al., 2016). The dye is non-toxic and does not decrease the lifespan of individuals when exposed during their whole life (Figure 1A). Moreover, no reduction in longevity is detected even when the gut becomes permeable and the dye diffuses into the body, contrary to what was recently suggested in Clark et al. (2015). We confirmed this by placing newly identified Smurfs on normal non-dyed media, and this did not lead to a longer lifespan (Figure 1B).


Figure 1. The blue dye #1 is not toxic neither for non-Smurfs or Smurfs. A. The longevity curve of 1,146 individual female flies maintained on blue medium overlaps the longevity curves of 295 female flies maintained on standard medium by groups of 28-32 individuals (longevity data from Tricoire and Rera, 2015). B. The longevity curve of 173 individual female flies maintained on blue dye for their whole lives overlaps the longevity curve of 172 individual female flies transferred back to standard medium when they became Smurf. This confirms that Smurfs do not die prematurely because the dye gains toxic properties when it diffuses through the gut.

Catching Smurfs
Although we initially described Smurfness as a well-marked, almost binary phenotype (Rera et al., 2011 and 2012; Tricoire and Rera, 2015), Smurfness is, as most phenotypes are, continuous (Figures 2A-2D, Clark et al., 2015). Thus, it is important to understand that the lighter the Smurf is, the greater the chance of misidentifying Smurf individuals. Indeed, the major part of uncertainly identified Smurfs appears in the few days preceding clear mortality acceleration in the population (Figure 3). The continuous nature of the Smurf phenotype can have two main causes. First, the dye might take some time to diffuse through limited gut permeability, thus generating a determined relationship between Smurfness and the level of gut permeability. Second, there can be biological (environmental and/or genetic factors) that can cause variation in the phenotype observed.

Moreover, there can be observer bias, attributable to the experimenter who is sorting and classifying individuals. We noticed that the earlier in the lifespan, and the fewer Smurfs are present in the group, the more likely an observer is to classify individuals as Smurfs, despite subsequently being scored as non-Smurf. The latter is probably inherent in the way we distinguish individuals based on their surrounding individuals, and hence the more Smurf individuals are present, the more stringent we are on their identification. To circumvent this, single individuals could be photographed for independent verification. In practice, however, it is difficult to both sort large numbers of flies and photograph every individual for subsequent blue hue quantification. This problem is less relevant to larger organisms.


Figure 2. The Smurf phenotype is not binary but rather continuous. A. The continuous aspect of the Smurf phenotype was previously described in Clark et al. (2015), but we noticed much more subtle shades of blue in our experimental conditions. B. Nevertheless, only the two categories of Smurfs and non-Smurfs showed significant blue hue difference on all body parts (n = 31 female from the drsGFP genotype, nns = 16, n? = 4, nls = 4 and ns = 7)–a subsequent experiment with larger n was conducted and showed significant differences only between Smurfs and non-Smurfs (not shown). C. The continuous Smurfness distribution is not just due to the Smurf (blue dye based) assay but is also observable in the drsGFP individuals by D. measuring GFP intensity in Smurfs (n = 33) and non-Smurfs (n = 130). The drosomycin promoter-driven GFP expression has been shown to be a surrogate of Smurfness in Rera et al. (2012). Mated 35-40 days old female Drosophila.


Figure 3. All individuals eventually become Smurf prior to death. Estimating Smurf survival time requires taking into account individuals from different moments of the survival experiment to prevent misestimation. A. All 1,146 individual female flies became Smurf prior to death and survived for various duration in that state. B. The uncertainty on the Smurf status has the strongest effect on the youngest identified Smurfs. Restricting Smurf studies to that period is thus at high risk of misestimating their remaining lifespan. We recommend to study them close to the T50 of the population. Original data from (Tricoire and Rera, 2015). C. Proportion of the three different types of living Smurfs at various percent survival in the population. The largest population of uncertain Smurf individuals is restricted to the first few days of Smurf apparition in the population.

Other experimental considerations
The duration of exposure to the dye does not affect survival nor the Smurf Increase Rate (SIR). For ease we now use overnight feeding on the blue dye. The fly population density is of critical importance: we observed that at a too high density, individuals tend to get covered with blue faeces. Although easy to rinse with water (the addition of some ethanol can help immersion) to discern ‘false’ Smurfs from legitimate ones, we do not recommend more than 30 individuals per vial for overnight exposure. For continued exposure to the dye, lower numbers should be considered. Moreover, when learning how to distinguish smurfs we recommend washing flies to confirm the phenotype. These considerations could be especially important as behaviour and the quantity of feaces produced can differ between genetic backgrounds and experimental conditions.

We received a significant number of questions regarding ‘the number of Smurfs with time’. As previously stated in (Rera et al., 2011 and 2012; Tricoire and Rera, 2015; Dambroise et al., 2016), it is the Smurf proportion calculated as at a specific age that increases as a function of age, rather than the absolute number of Smurf individuals. The interpretation of this number is similar to that of mortality risk, as it is related to the age-specific risk of an individual in the population to become a Smurf. Note that Smurfs remain in the population for a short time until they die and thus remain in the numerator of the formula above. The Smurf proportion is thus not equal to the risk of becoming a Smurf, but could be calculated as such (Promislow et al., 1999).

Most of the Smurf-related studies we conduct are based on female flies because, as we described in (Rera et al., 2012), they are easier for Smurf identification, principally since their abdomen is larger. In addition, the age-dependent SIR is weaker in males (see Figure S1A in Rera et al., 2012). This might be due to a much shorter remaining lifespan of males when they are in the Smurf state, as we recently observed (unpublished) and/or their smaller body. It is interesting to notice that in zebrafish the sex-specific SIR intensity was inverted (see Figure S1B in Dambroise et al., 2016). Regardless, male Drosophila do undergo the Smurf transition prior to death (Figure 4), contrary to what was recently suggested in Regan et al., 2016.


Figure 4. The Smurf phenotype occurs in male Drosophila melanogaster. Two examples are pictured: A. A smurf male; B. A non-smurf male. 35 days old males.

Materials and Reagents

  1. Parafilm
  2. 0.22 µm sterile vacuum filter (Corning® bottle-top vacuum filter, Corning, catalog number: 430015 )
  3. 0.45 µm filter (VWR, catalog number: 514-4127 )
    Note: The 0.45 µm filter is for fish.
  4. 30 G syringe (BD, catalog number: 324826 )
  5. Narrow plastic vials
  6. 22-G Micro-Renathane Implantation tubing (Braintree Scientific, catalog number: MRE025 )
  7. Disposable syringe 0.3 ml BD needle 30 G (Insuline) (BD, catalog numbers: 324826 , 320837 )
  8. Soft sponge of approximately 20 mm in height (such as Jaece Industries, catalog number: L800-D )
  9. Drosophila and fish (any source)
  10. FD&C blue dye #1 (Sigma-Aldrich, catalog number: 861146 , SPS Alfachem Ref: 101-2912 and A & Z Food Additives Brilliant Blue FCF(CAS No. 3844-45-9), FD&C Blue 1, E133)
  11. Fluorescein sodium salt (Sigma-Aldrich, catalog number: F6377-500g )
  12. FD&C Red #40, Allura Red (SPS Alfachem)
  13. Bi-distilled deionized water (ddH2O)
  14. Hanks’ balanced salt solution (HBSS, Thermo Fisher Scientific, GibcoTM, catalog number: 14175053 )
  15. Buffered tricaine methanesulfonate (Sigma-Aldrich, catalog number: A5040 ) 164 mg/L in fish tank water
  16. Moldex (VWR, catalog number: 25605.293 )
  17. Blue #1 stock solution (12.8x) (see Recipes)
  18. Dyed media (see Recipes)

Equipment

  1. Magnetic stirrer
  2. 5 L glass beaker
  3. 2 L glass bottle
  4. LED cool white lighting
  5. White background
  6. Epi-fluorescence microscope Nikon Eclipse 80i for nematode Smurfs (Nikon, model: Eclipse 80i )

Software

  1. ImageJ 1.46j and above
  2. GraphPad Prism 6.01

Procedure

  1. Prepare solutions
    See ‘Recipes’ section.

  2. Smurf assay
    Notes:
    1. All fish work was performed following Animals (Scientific Procedures) Act 1986 and UK Home Office approval (PPL 70/8681).
    2. If the blue dye is used at the correct concentration, well dissolved and filtered, no adverse effects are expected from the 30 min incubation period, except some temporary stress and discomfort. After blue-dye incubation, fish should be kept in separate tanks for at least 4 h before joining the main aquarium tanks where water is shared, to ensure all dye is excreted.
    3. Any signs of potential adverse effects such as impaired prolonged swimming and feeding should be brought to the attention of the NACWO and/or NVS and any definite or probably adverse effects should lead culling of the animal using an approved procedure performed by a person registered as competent by the certificate/licensed holder (10.1089/zeb.2016.1248).

    It is important to standardize the conditions under which Smurfs are examined.
    1. Transfer individuals onto SA medium
      1. Drosophila and nematodes: overnight.
      2. Fish: 30 min.
    2. Transfer individuals onto fresh normal medium for immediate scoring
      1. Drosophila: if you have to process large numbers of vials, you might want to transfer vials one at a time to prevent individuals from clearing the blue dye before scoring.
      2. Nematodes can be examined directly on the fluorescein medium using an epi-fluorescence microscope.
      3. Fish: rinse them until no blue comes out of the individuals in successive and independent fish tanks (Figure 5).


        Figure 5. Identifying Smurfs in zebrafish using the ‘bathing protocol’. A. Successive fish tanks are used to rinse individuals until no further dye is emitted. B. Representative images of Smurf and non-Smurf individuals in WT AB zebrafish.

    3. Count individuals that are positive for Smurfness (SA+) as well as those that are negative (SA-).
      The proportion of Smurfs in the population is then 
    4. Smurf and non-Smurf individuals can then be monitored separately for further analysis.
      An alternative protocol for fish (Raquel Martins and Catarina Henriques): Since penetration of the blue dye through the skin of fish will always be a confounding factor with a dipping protocol, we tested oral administration of the blue dye by following the adult zebrafish gavage protocol published in Collymore et al. (2013). 5 µl of a 5% (m/v) filtered blue dye dissolved in Hanks’ balanced salt solution (HBSS) was administered through oral gavage. Older individuals show the Smurf phenotype whilst we did not observe this in younger individuals (Figure 6C). This method requires training to prevent the experimenter from injuring the gut. It is nevertheless non-lethal, thus allowing further monitoring of individuals post assay.


      Figure 6. Gavage allows to identify zebrafish Smurfs. Smurf zebrafish obtained using the ‘gavage protocol’ (A). All six Zebrafish WT AB individuals of 6 and 29 months of age used (B) and respective quantification of the blue hue that leaked through the gut and is now visible on the skin. (C) Quantifications were performed from digital pictures in ImageJ selecting three different parts of the body/fish, avoiding the blue stripes and quantifying the median of pixels per area. All 3 older fish turned bluer than the younger fish (t-test assuming normality and equal variances: P < 0.05 or more conservative: unequal variance t-test or non-parametric Wilcoxon rank test P ≤ 0.10). See Video 1.

      Video 1. Detailed zebrafish Smurf gavage protocol

      Pre-injection preparation:
      1. Before the gavage procedure, the fish must be fasted for 24 h or at least 4 h prior to gavage, which will empty the intestinal bulb (stomach) contents.
      2. On the flat face, make a cut (10-15 mm deep) in the soft sponge, which will hold the fish for injection.
      3. Then, set the sponge into a 60 mm Petri dish. This will hold water to help maintain sponge moist.

      Anesthesia, Injection and Recovery:
      1. Anaesthetize fish with diluted Tricaine and monitor fish behavior. When fish is quiet, transfer it to the wet sponge (soaked in Tricaine) and move the sponge into a vertical position, as illustrated in the video.
      2. Then, open the zebrafish mouth using the 22-G catheter tubing adjusted to the size of the fish (approximately 2.5 cm), and gently insert the tubing until the tip is past the gills (approximately 1 cm or the length of the tubing). The implantation tubing should not need to be forced. Resistance suggests the tube may be hitting the gill arch or heart. Thus, if there is resistance, gently withdraw, reposition and try again.
      3. When the tube is completely inserted (reaching the middle intestine), inject the material slowly (5 μ of filtered (0.45 μm syringe filter) 5% (w/v) blue #1 diluted in HBSS). To accurately measure 5 μl, pipette 5 μl of the solution onto Parafilm and aspirate with a syringe, avoiding air bubbles. While injecting, make sure that the solution does not exit via the gills or the mouth.
      4. After injecting all the content, remove the fish from the sponge and place into the recovery tank. Fish should be monitored for regurgitation as shown by visualizing the fish actively expelling material from its mouth, or no opercular movement. Fish can be returned to their regular tank, or kept separately in a mating box, once they have recovered.

  3. Blue hue measurements
    Using ImageJ freehand selection tool, ROIs are selected on a picture converted to HSB space (command Image → Type → HSB Stack). Then, the hue is measured by the mean gray value (Analyze → Set measurements… → Mean gray value) in the Hue dimension of the picture.

Data analysis

It is possible to consider two distinct approaches for comparing the Smurf proportion in condition A versus condition B.

  1. At one given time-point, comparing the proportion of Smurfs between conditions A and B:
    1. Use a two-sided binomial test to estimate the statistical significance of the difference between distribution of SA+ individuals amongst the total population, in condition B compared to the distribution in control condition A. We used this approach in (Rera et al., 2011). Since GraphPad Prism Chi-square is just an approximation, one can use the exact test implemented in Excel or R.
    2. Use a Mann-Whitney test on the average proportion of SA+ individuals per vial in condition A and B.
  2. Across multiple time-points, comparing the slope and y-intercept of age-dependent proportion of Smurfs between conditions A and B. We initially described this approach in (Rera et al., 2012) using GraphPad Prism. The calculations follow a method spelled in Chapter 18 of J Zar, Biostatistical Analysis, 2nd edition, Prentice-Hall, 1984. It is equivalent to analysis of covariance (ANCOVA). Similar approaches are available in mixed models, in a binomial model context and should be considered if one wants to correct for experimental confounds, such as batch or other random terms (Gelman et al., 2004).

We recommend the use of method 2 since the time trend ensures a smaller risk of false positives.

Notes

  1. It is crucial to ensure that the bathing solution for fish does not contain clumps of the blue dye powder as they tend to stick to scales, making it harder to distinguish Smurf individuals.
  2. Standardize your Smurf scoring conditions.
  3. Train multiple people to recognize Smurf individuals and cross-check your own scoring with them.
  4. Keep in mind that the genetic background of the individuals you assess for Smurfness can influence its result, so adapt your scoring to it.

Recipes

The FD&C blue dye #1 comes as an extremely volatile dark purple powder. The first step is to put it in an aqueous solution. The maximum concentration we could reach at room temperature is 320 g L-1. This corresponds to a 12.8x stock solution as the final concentration in the medium is 2.5 g per 100 ml (2.5% w/v).

  1. Blue #1 stock solution (12.8x)
    1. Pour 700 ml dH2O into a 5 L glass beaker
    2. Weigh 400 g of the blue #1 powder
    3. Gently pour the powder into the water containing beaker
    4. Add the magnetic stirring bar
    5. Cover the beaker with Parafilm
    6. Stir until the powder is totally dissolved (typically a couple of hours)
    7. Slowly rinse the walls of the beaker with the remaining 300 ml of dH2O
    8. Stir for 30 min
    9. Vacuum filter the blue solution into a sterile bottle
    Notes:
    1. For fish: prepare the 1x solution directly in water extracted from the fish tank and filter it.
    2. For fluorescein: use the same procedure as for the blue #1.
    3. The solution is preferentially used immediately or frozen and not stored at room temperature since its dark color does not allow the observation of any potential contamination.
  2. Dyed medium
    1. Prepare your standard Drosophila food recipe (Rera et al., 2011; Katzenberger et al., 2015; Tricoire and Rera, 2015; Barekat et al., 2016; Regan et al., 2016) with 7.8% less water
    2. After boiling the medium, add the Moldex (VWR)
    3. Add 7.8 ml of the Blue #1 stock solution per 100 ml (final) of food
    4. Stir thoroughly until the coloration is homogenous
    5. Dispense in the narrow vials (minimum 1.25 ml for overnight SA)
    Notes:
    1. For nematodes: use the same procedure as for the blue #1. Although the molar mass of fluorescein is 2.9 times less than that of blue #1 at that concentration the fluorescein does not fluoresce. This allows an easy identification of nematode fluorescent Smurfs directly on the medium.
    2. The fluorescein solution is added directly to the agar plate or mixed with the bacteria.

Acknowledgments

This work was supported by the CNRS to M.R. This protocol is adapted from (Rera et al., 2012; Dambroise et al., 2016). M.J.P.S. is supported by a Sir Henry Wellcome and a Sheffield University Vice Chancellor’s Fellowship and the Natural Environment Research Council (N013832). A.W.McC. is supported by the NERC ACCE Doctoral training program. C.M.H is supported by a Sir. Henry Dale Fellowship by the Wellcome Trust & Royal Society and a Sheffield University Vice Chancellor’s Fellowship. R.R.M is supported by a Sheffield University Doctoral Academy PhD Fellowship. The authors declare no conflict of interest nor competing interests.

References

  1. Barekat, A, Gonzalez, A., Mauntz, R. E., Kotzebue, R. W., Molina, B., El-Mecharrafie, N., Conner, C. J., Garza, S., Melkani, G. C., Joiner, W. J., Lipinski, M. M., Finley, K. D. and Ratliff, E. P. (2016). Using Drosophila as an integrated model to study mild repetitive traumatic brain injury. Sci Rep 6: srep25252.
  2. Chakrabarti, S., Dudzic, J. P., Li, X., Collas, E. J., Boquete, J. P. and Lemaitre, B. (2016). Remote control of intestinal stem cell activity by haemocytes in Drosophila. PLoS Genet 12(5): e1006089.
  3. Clark, R. I., Salazar, A., Yamada, R., Fitz-Gibbon, S., Morselli, M., Alcaraz, J., Rana, A., Rera, M., Pellegrini, M., Ja, W. W. and Walker, D. W. (2015). Distinct shifts in microbiota composition during Drosophila aging impair intestinal function and drive mortality. Cell Rep 12(10): 1656-1667.
  4. Collymore, C., Rasmussen, S. and Tolwani, R. J. (2013). Gavaging adult zebrafish. J Vis Exp (78).
  5. Dambroise, E., Monnier, L., Ruisheng, L., Aguilaniu, H., Joly, J. S., Tricoire, H. and Rera, M. (2016). Two phases of aging separated by the Smurf transition as a public path to death. Sci Rep 6: 23523.
  6. Gelino, S., Chang, J. T., Kumsta, C., She, X., Davis, A., Nguyen, C., Panowski, S. and Hansen, M. (2016). Intestinal autophagy improves healthspan and longevity in C. elegans during dietary restriction. PLoS Genet 12(7): e1006135.
  7. Gelman, A., Trevisani, M., Lu, H. and van Geen, A. (2004). Direct data manipulation for local decision analysis as applied to the problem of arsenic in drinking water from tube wells in Bangladesh. Risk Anal 24(6): 1597-1612.
  8. Katzenberger, R. J., Chtarbanova, S., Rimkus, S. A., Fischer, J. A., Kaur, G., Seppala, J. M., Swanson, L. C., Zajac, J. E., Ganetzky, B. and Wassarman, D. A. (2015). Death following traumatic brain injury in Drosophila is associated with intestinal barrier dysfunction. Elife 4.
  9. Promislow, Tatar, Pletcher and Carey (1999). Below-threshold mortality: implications for studies in evolution, ecology and demography. J Evol Biol 12: 314-328.
  10. Regan, J. C., Khericha, M., Dobson, A. J., Bolukbasi, E., Rattanavirotkul, N. and Partridge, L. (2016). Sex difference in pathology of the ageing gut mediates the greater response of female lifespan to dietary restriction. Elife 5: e10956.
  11. Rera, M., Bahadorani, S., Cho, J., Koehler, C. L., Ulgherait, M., Hur, J. H., Ansari, W. S., Lo, T., Jr., Jones, D. L. and Walker, D. W. (2011). Modulation of longevity and tissue homeostasis by the Drosophila PGC-1 homolog. Cell Metab 14(5): 623-634.
  12. Rera, M., Clark, R. I. and Walker, D. W. (2012). Intestinal barrier dysfunction links metabolic and inflammatory markers of aging to death in Drosophila. Proc Natl Acad Sci U S A 109(52): 21528-21533.
  13. Rera, M., Vallot, C. and Lefrancois, C. (2018). The Smurf transition: new insights on ageing from end-of-life studies in animal models. Curr Opin Oncol 30(1): 38-44.
  14. Tricoire, H. and Rera, M. (2015). A new, discontinuous 2 phases of aging model: Lessons from Drosophila melanogaster. PLoS One 10(11): e0141920.
  15. Wong, R., Piper, M. D., Wertheim, B. and Partridge, L. (2009). Quantification of food intake in Drosophila. PLoS One 4(6): e6063.

简介

Smurf分析(SA)最初是在模型生物黑腹果蝇中发现的,其中在老化过程中肠道通透性显着增加(Rera等人,2011年)。 我们已经验证了多种其他模式生物体的协议(Dambroise等人,2016),并利用该分析来进一步了解老化(Tricoire和Rera,2015; Rera等人 。,2018年)。 SA现在也被其他实验室用于评估肠道屏障通透性(Clark等人,2015; Katzenberger等人,2015; Barekat等人 。2016; Chakrabarti等人,2016; Gelino等人,2016)。 SA本身很简单, 然而,许多小细节可能会对其实验的有效性和随后的解释产生相当大的影响。 在这里,我们提供SA技术的详细更新,并解释如何捕捉Smurf,同时避免最常见的实验谬误。

【背景】Smurf分析(SA)基于(Wong等人,2009)中描述的果蝇饲养分析。该测定法通过摄入未被消化道吸收的蓝色染料来评估食物摄入量,从而允许直接定量。对于SA来说,这个特定的蓝色染料不能通过一个完整的肠屏障,因为它的读数是全身着色(这里是蓝色)。这种性质允许直接评估肠道通透性,已经显示其随着年龄的增长而增加(Rera等人,2011和2012)。

正如最近在Rera等人(2018)中所讨论的那样,Smurf分析不仅是评估体内肠渗透性的简单方法,而且也是评估广泛生物体中个体的生理年龄。因此,它允许以新颖的方法来研究在老化个体中发生的各种事件(Tricoire和Rera,2015; Rera等人,2018)。

近几年来,我们收到了很多关于初始协议的意见和问题,导致我们开发了现在的扩展协议。

使用的染料的具体情况:通常使用的染料是FD&C蓝染料#1,但是我们也验证了使用红#40和荧光素(Rera等人,2011和2012)。我们对斑马鱼(Dambroise等人,2016)和kill鱼(Rera 等人,,2018年)进行了相同的蓝色#1调整,但发现它更容易使用荧光素与线虫,尽管如(Gelino等人,2016)中所证实的,也可使用相同的蓝色#1。这种染料是无毒的,在整个生命过程中不会降低个体的寿命(图1A)。此外,与Clark等人(2015)最近提出的建议相反,甚至当肠道变得可渗透并且染料扩散到体内时,也没有检测到寿命的减少。我们通过将新鉴定的Smurfs置于正常的非染色介质上来证实这一点,但这并没有导致寿命延长(图1B)。

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图1.蓝色染料#1对于非蓝精灵或蓝精灵都是无毒的。A.在蓝色培养基上保持的1,146只雌性苍蝇的寿命曲线覆盖了保持的295只雌性苍蝇的寿命曲线标准培养基由28-32人组成(2015年Tricoire和Rera的长寿数据)。 B,在蓝色染料上维持一生的173只雌性苍蝇的寿命曲线与172只雌性苍蝇成为蓝精灵时转移回标准培养基的寿命曲线重叠。这证实蓝精灵不会过早死亡,因为染料通过肠道扩散时会获得毒性。

捕捉蓝精灵
尽管我们最初将Smurfness描述为明显的,几乎是二元的表型(Rera等人,2011和2012; Tricoire和Rera,2015),但Smurfness与大多数表型是连续的(图2A -2D,Clark等人,2015)。因此,理解Smurf越轻,错误识别Smurf个体的机会就越大,这一点很重要。事实上,不确定性蓝精灵的主要部分出现在人口明显加速死亡的前几天(图3)。 Smurf表型的连续性可能有两个主要原因。首先,染料可能需要一段时间才能通过有限的肠道渗透性扩散,从而在Smurfness和肠道通透性水平之间产生确定的关系。其次,可能会导致观察到的表型变异的生物学(环境和/或遗传因素)。

而且,由于正在对个人进行分类和分类的实验者可能会有观察者的偏见。我们注意到,在寿命越早,组中出现蓝精灵数量越少,观察者就越有可能将个体归类为蓝精灵,尽管随后被评为非Smurf。后者可能是固有的,因为我们根据他们周围的个人来区分个体,因此,Smurf个体越多,我们对他们的鉴定就越严格。为了规避这一点,可以拍摄单个人进行独立验证。然而,在实践中,难以对大量的苍蝇进行分类,并对每个人进行后续的蓝色色调量化。这个问题与较大的生物体不太相关。
“”src
图2. Smurf表型不是二元的,而是连续的。A. Smurf表型的连续方面先前在Clark等人(2015)中有描述,但是我们在我们的实验条件下注意到更多微妙的蓝色阴影。 B.然而,只有两类Smurfs和非Smurfs在所有身体部位(n = 31,女性来自drsGFP基因型,n = ns = 16,n = = 4,n = 4和n = 7) - 进行后续的较大n的实验,仅在Smurfs和非Smurfs之间显示出显着差异,蓝精灵(未显示)。 C.连续的Smurfness分布不仅仅是由于Smurf(基于蓝色染料的)测定,而且在drsGFP个体中也可以通过D.在Smurfs(n = 33)和非Smurfs(n = 130)中测量GFP强度来观察。已经显示,drosomycin启动子驱动的GFP表达已被证明是Rera等人的Smurfness的替代品(2012)。交配35-40天龄的雌性果蝇。

“”src
图3.所有个体在死亡之前最终都会成为Smurf。估计Smurf生存时间需要考虑来自生存实验不同时刻的个体,以防止错误估计。答:在死亡之前,所有1,146只雌性苍蝇都成为蓝精灵,并在该状态下存活了不同的时间。 B. Smurf状态的不确定性对最年轻的Smurfs有最大的影响。因此限制Smurf研究到那个时期是误判他们的剩余寿命的高风险。我们建议研究他们接近人口的50%。原始数据来自(Tricoire和Rera,2015)。 C.人口中不同生存百分比的三种不同类型的活蓝精灵的比例。不确定的Smurf个体的最大的人口被限制在人口中Smurf幻象的头几天。

其他实验考虑因素
暴露于染料的持续时间不影响存活,也不影响Smurf增加率(SIR)。为了方便起见,我们现在使用蓝色染料隔夜喂食。苍蝇种群密度是至关重要的:我们观察到密度过高时,个体倾向于被蓝色的粪便覆盖。虽然容易用水冲洗(加入一些乙醇可以帮助浸泡)从合法的“蓝精灵”中辨别出“虚假”的蓝精灵,但是我们并不建议每瓶超过30人过夜接触。为了继续接触染料,应考虑较少的数字。而且,在学习如何区分蓝精灵时,我们推荐洗苍蝇以确认表型。这些考虑可能是特别重要的,因为行为和产生的粪便的数量可以在遗传背景和实验条件之间不同。

我们收到了很多关于“与时间有关的蓝精灵数量”的问题。如之前在(Rera等人,2011和2012; Tricoire和Rera,2015; Dambroise等人,2016)中所述,Smurf比例计算为< img class =“videopic donotsetwh”width =“150”height =“44”alt =“”src =“/ attached / image / 20180201 / 20180201012723_8764.jpg”/>在一个特定的年龄增加作为一个函数的年龄比绝对数量的Smurf个体。这个数字的解释与死亡率风险相似,因为它与个体在个体中年龄别风险成为Smurf有关。请注意,蓝精灵在人口中保持一段时间,直到死亡,因此保持在上述公式的分子中。 Smurf比例因此不等于成为Smurf的风险,但可以这样计算(Promislow等人,1999)。

我们进行的大多数Smurf相关研究都是以女性苍蝇为基础的,因为正如我们在(Rera等人,2012年)中所描述的那样,他们更容易进行Smurf鉴定,主要是因为他们的腹部较大。另外,年龄依赖性SIR在男性中较弱(参见Rera等人,2012年的图S1A)。这可能是由于我们最近观察到(未发表)和/或他们较小的身体,所以在他们处于Smurf状态时,男性的剩余寿命要短得多。有趣的是,在斑马鱼中,性别特异性SIR强度被颠倒(参见Dambroise等人,2016年的图S1B)。无论如何,雄性果蝇在死亡之前确实经历了Smurf转变(图4),与2016年在Regan等人最近提出的建议相反。

“”src
图4. Smurf表型出现在雄性黑腹果蝇。下面是两个例子:A.一个smurf男性; B.一个非smurf男性。 35日龄的男性。

关键字:蓝精灵分析法, 消化道通透性, 蓝色染料#1, 衰老

材料和试剂

  1. Parafilm
  2. 0.22微米无菌真空过滤器(Corning 瓶顶真空过滤器,康宁,目录号:430015)
  3. 0.45微米过滤器(VWR,目录号:514-4127)
    注:0.45微米过滤器适用于鱼类。
  4. 30G注射器(BD,目录号:324826)
  5. 狭窄的塑料瓶
  6. 22-G微型Renathane植入管(Braintree Scientific,目录号:MRE025)
  7. 一次性注射器0.3毫升BD针30 G(Insuline)(BD,目录号:324826,320837)
  8. 大约20毫米高的软海绵(如Jaece Industries,目录号:L800-D)
  9. 果蝇和鱼(任何来源)
  10. FD&amp; C蓝色染料#1(Sigma-Aldrich,目录号:861146,SPS Alfachem Ref:101-2912和A&amp; Z食品添加剂Brilliant Blue FCF(CAS No.3844-45-9),FD&amp; C Blue 1 ,E133)
  11. 荧光素钠盐(Sigma-Aldrich,目录号:F6377-500g)
  12. FD&amp; C Red#40,Allura Red(SPS Alfachem)
  13. 双蒸去离子水(ddH2O)
  14. Hanks平衡盐溶液(HBSS,Thermo Fisher Scientific,Gibco TM,目录号:14175053)
  15. 缓冲的三卡精甲磺酸盐(西格玛奥德里奇,目录号:A5040)164毫克/升鱼缸水
  16. Moldex(VWR,目录号:25605.293)
  17. 蓝色#1储备溶液(12.8x)(见食谱)
  18. 染色的媒体(见食谱)

设备

  1. 磁力搅拌器
  2. 5升玻璃烧杯
  3. 2升玻璃瓶
  4. LED冷白色照明
  5. 白色背景
  6. 外荧光显微镜尼康Eclipse 80i线虫蓝精灵(尼康,型号:Eclipse 80i)

软件

  1. ImageJ 1.46j及以上
  2. GraphPad棱镜6.01

程序

  1. 准备解决方案
    见“食谱”部分。

  2. 蓝精灵分析
    注意:
    1. 所有的鱼类工作都是在1986年动物(科学程序)法案和英国内政部批准(PPL 70/8681)之后进行的。
    2. 如果蓝色染料以正确的浓度使用,溶解和过滤良好,30分钟潜伏期内不会产生副作用,除了一些暂时的压力和不适。在蓝色孵化后,鱼在分开水的主要水族箱之前应该保持在独立的水槽中至少4小时,以确保所有的染料都被排出体外。
    3. 任何潜在不利影响的迹象,例如长时间游泳和喂养受损,应引起NACWO和/或NVS的注意,并且任何明确的或可能的不利影响应该导致使用由个人执行的批准程序扑杀动物注册为证书/持证持有人(10.1089 / zeb.2016.1248)的主管。

    标准化审查蓝精灵的条件非常重要。
    1. 将个人转移到SA媒体上
      1. 果蝇和线虫:过夜。
      2. 鱼:30分钟
    2. 将个体转移到新鲜的正常培养基上以便立即打分
      1. 果蝇:如果您必须处理大量的小瓶,您可能需要一次转移一个小瓶以防止个体在计分前清除蓝色染料。
      2. 线虫可以用荧光显微镜直接在荧光素培养基上检测。
      3. 鱼:冲洗它们,直到连续和独立鱼缸中的个体没有蓝色出现(图5)。


        图5.使用“沐浴协议”识别斑马鱼中的蓝精灵。 :一种。连续的鱼缸被用来冲洗个人,直到没有进一步的染料排放。 B.WT AB斑马鱼中Smurf和非Smurf个体的代表性图像。

    3. 计算对Smurfness(SA +)以及负面(SA-)为阳性的个体。
      然后,人口中Smurfs的比例为 />
    4. 然后可以分别监视Smurf和非Smurf个人的进一步分析。
      对于鱼的替代方案(Raquel Martins和Catarina Henriques):由于蓝色染料通过鱼的皮肤渗透将始终是浸渍方案的混杂因素,因此我们测试了口服蓝色染料遵循Collymore等人发表的成年斑马鱼灌胃方案(2013年)。通过口服灌胃施用5μl溶于Hanks平衡盐溶液(HBSS)中的5%(m / v)过滤的蓝色染料。老年人表现出Smurf表型,而我们没有在年轻人中观察到这一点(图6C)。这种方法需要培训,以防止实验者伤害肠道。然而它是非致死性的,因此可以进一步监测检测后的个体。


      图6. Gavage可以识别斑马鱼Smurfs。使用“灌胃方案”(A)获得的Smurf斑马鱼。 (B)和分别量化通过肠道泄漏的蓝色色调,并且现在在皮肤上可见,所有六个斑马鱼WT AB个体的6个月和29个月龄使用。 (C)从ImageJ中的数字图像中进行量化,选择身体/鱼的三个不同部分,避免蓝色条纹并量化每个区域的像素的中值。所有3种较老的鱼变得比年轻的鱼更蓝(假设正态性和等方差:<0.05或更保守:不等方差t 测试或非参数Wilcoxon秩和检验P ≤0.10)。见视频1.

      s视频1

      注射前准备:
      1. 在灌胃程序之前,鱼在管饲之前必须禁食24小时或至少4小时,这将清空肠球(胃)内容物。
      2. 在平坦的表面上,在软海绵上做一个切口(10-15毫米深),这将保持注射鱼。
      3. 然后,将海绵放入60毫米的培养皿中。这将持有水,以帮助保持海绵湿润。

      <麻醉,注射和恢复:
      1. 用稀释的Tricaine麻醉鱼并监测鱼的行为。当鱼安静时,将其转移到湿海绵(用三文鱼浸泡),并将海绵移动到垂直位置,如视频所示。
      2. 然后,使用调整到鱼的大小(约2.5厘米)的22-G导管管打开斑马鱼嘴,轻轻插入管,直到尖端超过鳃(约1厘米或管的长度)。植入管不应该被迫。阻力表明管可能击中鳃弓或心脏。因此,如果有阻力,轻轻撤回,重新定位,再试一次。
      3. 当试管完全插入(到达中肠)时,缓慢注入(HBSS稀释5%过滤(0.45μm注射器过滤器)5%(w / v)蓝色1号)。为了精确测量5μl,吸取5μl的溶液到Parafilm上并用注射器抽吸,避免气泡。在注射的同时,确保溶液不会通过鳃或嘴出口。
      4. 在注入所有内容后,将海绵中的鱼取出并放入回收罐中。如图所示,通过观察鱼从嘴里积极排出物质,或者没有运动,可以监测鱼的反流情况。
        一旦鱼类恢复正常,鱼可以放回正常水箱,或者分开存放在配套箱内
  3. 蓝色色调测量
    使用ImageJ手绘选择工具,在转换为HSB空间的图像(命令图像→类型→HSB堆栈)上选择ROI。然后,通过图片的色相维度中的平均灰度值(分析→设置测量...→平均灰度值)来测量色调。

数据分析

有可能考虑两种不同的方法来比较条件A中的Smurf比例与条件B.

  1. 在一个给定的时间点,比较条件A和B之间Smurfs的比例:
    1. 使用双侧二项式检验来估计SA +个体在总体群体中的分布差异的统计学显着性,条件B与对照条件A中的分布相比较。我们在(Rera等人, ,2011)。由于GraphPad Prism卡方只是一个近似值,可以使用在Excel或R中实现的精确测试。
    2. 在条件A和B中,对每个小瓶的SA +个体的平均比例使用Mann-Whitney检验。
  2. 在多个时间点上,比较条件A和B中Smurfs的年龄相关比例的斜率和y轴截距。我们最初使用GraphPad Prism(2012年)描述了这种方法(Rera等人,2012年) 。计算遵循J Zar,Biostatistical Analysis,第2版,Prentice-Hall,1984年第18章中的方法。它相当于协方差分析(ANCOVA)。类似的方法在混合模型中是可用的,在二项模型背景下,并且如果想要校正实验混淆,例如批处理或其它随机项(Gelman等人,2004),应该考虑相似的方法。 br />

我们建议使用方法2,因为时间趋势确保误报的风险较小。

笔记

  1. 确保鱼类沐浴液不含蓝色染料粉末的团块是非常重要的,因为它们往往会粘在鳞片上,使其难以区分Smurf个体。
  2. 规范您的Smurf评分条件。
  3. 培训多个人认识Smurf个人,并与他们交叉检查自己的得分。
  4. 请记住,你所评估的Smurfness个体的遗传背景可能会影响其结果,所以请适应你的评分。

食谱

FD&amp; C蓝色染料#1是非常易挥发的深紫色粉末。第一步是将其放入水溶液中。我们在室温下可以达到的最大浓度是320g L -1。这对应于12.8x储备溶液,因为培养基中的最终浓度是2.5g / 100ml(2.5%w / v)。

  1. 蓝色#1储备溶液(12.8x)
    1. 将700毫升dH 2 O倒入5升玻璃烧杯中
    2. 称重400克蓝#1粉
    3. 轻轻地把粉末倒入装有烧杯的水中
    4. 添加磁力搅拌棒
    5. 用Parafilm
      盖上烧杯
    6. 搅拌,直到粉末完全溶解(通常是几个小时)
    7. 用剩余的300ml dH 2 O缓慢冲洗烧杯的壁。
    8. 搅拌30分钟
    9. 将蓝色溶液真空过滤到无菌瓶中
    注意:
    1. 对于鱼类:直接在从鱼缸中提取的水中准备1x溶液并将其过滤。
    2. 对于荧光素:使用与蓝色#1相同的步骤。
    3. 该溶液优选立即使用或冷冻,并且不在室温下储存,因为其深色不允许观察到任何潜在的污染。
  2. 染色的媒介
    1. 准备你的标准果蝇食谱(Rera et al。,2011; Katzenberger et al。,2015; Tricoire and Rera,2015; Barekat < 2016); Regan et al。,2016),水量减少7.8%
    2. 煮沸介质后,加入Moldex(VWR)

    3. 每100毫升(最后)的食物添加7.8毫升的蓝#1储备液
    4. 彻底搅拌至着色均匀
    5. 分配在狭窄的小瓶(过夜SA最少1.25毫升)
    注意:
    1. 对于线虫:使用与蓝色#1相同的步骤。尽管在该浓度下荧光素的摩尔质量比蓝色#1的摩尔质量小2.9倍,但是荧光素不发荧光。这使得可以直接在培养基上直接识别线虫荧光Smurfs。
    2. 荧光素溶液直接加到琼脂平板上或与细菌混合。

致谢

这项工作得到了CNRS到M.R的支持。该协议改编自(Rera等人,2012年,Dambroise等人,2016年)。 M.J.P.S.由亨利•惠康爵士和谢菲尔德大学副校长研究员和自然环境研究委员会(N013832)支持。 A.W.McC。由NERC ACCE博士培训计划提供支持。 C.M.H得到一位爵士的支持。维尔康姆信托基金会亨利戴尔奖学金皇家学会和谢菲尔德大学校长奖学金。 R.R.M由谢菲尔德大学博士学位博士研究生支持。作者声明不存在利益冲突或利益冲突。

参考

  1. Barekat,A,Gonzalez,A.,Mauntz,RE,Kotzebue,RW,Molina,B.,El-Mecharrafie,N.,Conner,CJ,Garza,S.,Melkani,GC,Joiner,WJ,Lipinski,MM, Finley,KD和Ratliff,EP(2016)。 使用果蝇作为研究轻度重复性创伤性脑损伤的综合模型
    6:srep25252。
  2. Chakrabarti,S.,Dudzic,J.P.,Li,X.,Collas,E.J.,Boquete,J.P.和Lemaitre,B。(2016)。 通过果蝇中的血细胞远程控制肠干细胞活性 a PLoS Genet 12(5):e1006089。
  3. Clark,RI,Salazar,A.,Yamada,R.,Fitz-Gibbon,S.,Morselli,M.,Alcaraz,J.,Rana,A.,Rera,M.,Pellegrini,M.,Ja,WW和Walker,DW(2015)。 果蝇衰老过程中微生物组成的明显变化会损害肠道功能并驱动死亡。 Cell Rep 12(10):1656-1667。
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引用:Martins, R. R., McCracken, A. A., Simons, M. J., Henriques, C. M. and Rera, M. (2018). How to Catch a Smurf? – Ageing and Beyond… In vivo Assessment of Intestinal Permeability in Multiple Model Organisms. Bio-protocol 8(3): e2722. DOI: 10.21769/BioProtoc.2722.
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