搜索

An ex vivo Perifusion Method for Quantitative Determination of Neuropeptide Release from Mouse Hypothalamic Explants
用于定量测定小鼠下丘脑外植体神经肽释放的离体灌流方法   

评审
匿名评审
下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

The hypothalamus is a primary brain area which, in mammals, regulates several physiological functions that are all related to maintaining general homeostasis, by linking the central nervous system (CNS) and the periphery. The hypothalamus itself can be considered an endocrine brain region of some sort as it hosts in its different nuclei several kinds of neuropeptide-producing and -secreting neurons. These neuropeptides have specific roles and participate in the regulation of homeostasis in general, which includes the regulation of energy metabolism, feeding behavior, water intake and body core temperature for example.

As previously mentioned, in order to exert their effects, these peptides have to be produced but also, and mostly, to be secreted. In this context, it is of great importance to be able to assess how certain conditions, diseases, or treatments can actually influence the secretion of neuropeptides, thus the function of the different neuropeptidergic circuits.

One method to assess this is the perifusion of hypothalamic explants followed by quantification of peptides within the collected fractions.

Here, we explain step-by-step how to collect fractions during ex vivo perifusion of hypothalamic explants in which one can determine quantitatively neuropeptide/neurohormone release from these viable isolated tissues. Hypothalami perifusion has two great advantages over other existing assays: (1) it allows pharmacological manipulation to dissect out signaling mechanisms underlying release of different neuropeptides/neurohormones in the hypothalamic explants and, (2) it allows simultaneous experiments with different conditions on multiple hypothalami preparations, (3) it is, to our knowledge, the only method that permits the study of neuropeptide secretion in basal conditions and under repeated stimulations with the same hypothalami explants.

Keywords: Perifusion(灌流), Hypothalamus(下丘脑), Hypothalamic explants(下丘脑外植体), Peptide(肽), Neuropeptide(神经肽), Secretion(分泌), Release(释放), Neuroendocrinology(神经内分泌学), Endocrinology(内分泌学)

Background

Perifusion has been regularly used to study the pancreatic islets function. Yet, this assay is on principle adaptable to any endocrine tissue and any peptide or protein secretion.

Indeed, different perifusion systems have already been used in the past and remain a valid procedure used by different research laboratories to study the neuropeptide release from hypothalami in various conditions. For example, Callewaere and colleagues published in 2006 a study in which they analyzed the effect of the chemokine SDF-1 (stromal cell-derived factor-1) on vasotocin-induced AVP (arginine-vasopressin) release. In other studies, perifusion of hypothalamic explants has also been used to analyze SRIH (somatotropin release inhibiting hormone, a.k.a. somatostatin) release from perifused hypothalami (stimulation with an extracellular perifusion with 25 mM KCl) (Tolle et al., 2001; Zizzari et al., 2007).

Yet, currently, regarding hypothalamic explants perifusion, no standardized apparatus or protocol is available. We, ourselves, adapted a protocol from the publication by Callewaere et al., 2006.

With this method, we have recently shown ex vivo how, in mouse, the chemokine CCL2 (CC-chemokine ligand 2) is able to reduce the secretion of the orexinergic hypothalamic neuropeptide Melanin-Concentrating Hormone (MCH) and thus participate in loss of both appetite and weight in a context of high-grade inflammation (Le Thuc et al., 2016).

Materials and Reagents

  1. 15 ml and/or 50 ml centrifuge tubes (Corning, Falcon®, catalog numbers: 352096 and 352070 )
  2. Petri dishes Ø 100 mm (Corning, catalog number: 3262 )
  3. 5 ml pipettes (Corning, Falcon®, catalog number: 357543 )
  4. 10 ml pipettes (Corning, Falcon®, catalog number: 357551 )
  5. 1.5 ml microtubes (SARSTEDT, catalog number: 72.706 )
  6. 500 ml Nalgene Filtration Units–PES FASTER membranes–0.2 µm porosity (Dutscher, catalog number: 029667 )
  7. 0.22 µm filter
  8. 8-Week old mice
  9. Minimum Essential Medium (MEM)–no glutamine, no phenol red, no HEPES (Thermo Fischer Scientific, InvitrogenTM, catalog number: 51200046 )
  10. 2 x 10-3 M Bacitracin (from Bacillus licheniformis) (Sigma-Aldrich, catalog number: 31626 )
  11. L-Glutamine 100x (Thermo Fischer Scientific, InvitrogenTM, catalog number: 25030024 )
  12. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153 )
  13. Protease Inhibitor cocktail (CompleteTM, EDTA-free Protease Inhibitor Cocktail) (Roche Diagnostics)
  14. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
  15. Glucose (Sigma-Aldrich, catalog number: G8270 )
  16. Ultrapure water
  17. ELISA (Phoenix Pharmaceuticals, catalog number: EK-070-47 )
  18. Hypothalamic explant perifusion medium (see Recipes)
  19. 1 M KCl (see Recipes)
  20. Stimulation medium with 60 mM KCl (Iso-osmotic 60 mM KCl, 50 ml) (see Recipes)

Equipment

  1. Racks for microtubes and 15 ml/50 ml conical tubes (Dutscher)
  2. P10, P200, P1000 pipetmen (Gilson)
  3. -20 °C and -80 °C freezer (Sanyo, VWR)
  4. Hole puncher (Maped–any regular hole punch from any stationary brand should do)
  5. Carbogen (5% CO2/95% O2) (Linde France, UN 3156)
  6. Standard Pattern Scissors, Large Loops Sharp/Blunt 14.5 cm (Fine Science Tools, catalog number: 14101-14 )
  7. Extra Thin Iris Scissors, 10.5 cm (Fine Science Tools, catalog number: 14088-10 )
  8. Standard Pattern Forceps with serrated tip (Fine Science Tools, catalog number: 11000-13 )
  9. Dumont #7 forceps with curved tip (Fine Science Tools, catalog numbers: 11271-30 and 11272-30 )
  10. 2 Thermostatic baths (such as JULABO, model: CORIO CD-B27 )
  11. Perifusion chambers (BIOREP TECHNOLOGIES, catalog number: PERI-CHAMBER )
  12. Perifusion chamber filters (BIOREP TECHNOLOGIES, catalog number: PERI-FILTER )
  13. Perifusion tubing set (BIOREP TECHNOLOGIES, catalog number: PERI-TUBSET )
  14. Peristaltic pump (high precision multichannel pump, Ismatec)
  15. Osmometer (Löser Messtechnik, model: Micro-Osmometer Type 6 )

Software

  1. GraphPad Prism (GraphPad software) or any software for statistical data analysis

Procedure

We describe here how to perform manually the hypothalami perifusion assay following these steps (see Figure 1):


Figure 1. Perifusion flow chart. After a 2-h perifusion to reach equilibrium, the sampling procedure consists of a 30-min control basal period, a 30-min period during which drugs are added independently or in combination and a 60-min wash with the medium. After each experiment, an appropriate molecule (e.g., KCl) is applied to control the responsiveness of hypothalamic explants and test tissue viability.

  1. Prepare sufficient hypothalamic explants perifusion medium for the whole experiment (see Recipes; prepare always fresh and no earlier than one day before, stored at 4 °C).
  2. Prepare the stimulation solutions. For example, we have used iso-osmotic 60 mM KCl (see Recipes).
  3. Pre-gas some hypothalamic explant perifusion medium with carbogen (5% CO2/95% O2; all pre-gassing and gassing steps use carbogen) in a 50-ml conical tube for at least 15 min. About 10 ml of the pre-gassed medium will be used ice-cold for brain dissection and hypothalamic collection and the other part, about 40 ml, will be used warmed (34-37 °C) in a thermostatic bath for perifusion.
  4. Prepare perifusion chamber filters (Biorep) for each chamber (2 per chamber) with a hole puncher. Place one filter at each open end of the chamber (see Figures 2F-2H).


    Figure 2. Representative images of the different steps of perifusion experiment. A. Place the mouse brain upside down in a Petri dish containing ice-cold basic medium with the hypothalamus in front of the experimenter (A, B, C and D refer to step 8); B. Remove the cerebellum with a scalpel; C. Recover the hypothalamus with curved Dumont #7 forceps; D. Collect the hypothalamus (shown by the arrow and dashed circle) in a new Petri dish containing ice-cold basic medium; E. To avoid damage to hypothalamus, it can be handled with a large plastic/glass pipette; F. Prior to dissection, prepare perifusion chamber filters; G. Sagittal view of the brain showing the hypothalamus position (a circle in dotted-line is drawn around the hypothalamus); H. Put one filter at each end of the perifusion chamber (step 4);  Prefill the perifusion chambers with warmed pre-gassed perifusion medium (as described in step 6); I. Position the hypothalamus in the center of the perifusion chamber (step 9). Close the chamber and put it back in the thermostatic bath at 37 °C (step 10); J. Complete assembly of the experimental set-up with, in line from right to left, a thermostatic bath at 37 °C which contains tubes filled with perifusion medium (tubes are connected one to one with silicon tubing to perifusion chambers), a peristaltic pump flows the perifusion medium, a second thermostatic bath at 37 °C which receives the perifusion chambers (a perifusion chamber is marked by the red dashed line circle) and, left, the outlet of the perifusion chambers is connected with silicon tubing to low protein absorption 1.5 ml microtubes placed on ice to collect fractions. The perifusion medium flows from right to left.

  5. Connect with appropriate tubing (low protein absorption tubing such as Teflon tubing) the inlet of the perifusion chambers that will contain the hypothalamic explants to a peristaltic pump on one side and the outlet of the chambers to the tubing for fraction collection on the other side.
  6. Prefill the perifusion chambers with warmed pre-gassed perifusion medium. Chambers and tubing will be themselves kept at 34-37 °C in a thermostatic bath.
  7. For every mouse previously euthanized (we use cervical dislocation–chosen method should be fast in order to preserve tissue as much as possible), cut the head and collect the mouse brain in a Petri dish filled with pre-gassed perifusion medium, cooled on ice. To collect the brain, insert the bottom blade of extra thin iris scissors into the foramen magnum, and begin to cut directly up and through the midline of the skull, being careful to keep scissor tips pointed upwards. Then, open the skull using forceps pushing outwards both halves of the skull, thus exposing the brain. Carefully invert the skull: gravity will help the brain to detach from the skull. Using Dumont #7 forceps, carefully slide the forceps along the outer edge of the brain and under the brain from the olfactory lobes, towards the cerebellum. Gently detach the brain from any connective tissue or nerves that prevent it from falling from the skull.
    Note: We used 8-week old mice. Users will determine this regarding each scientific project. A limitation is of course the volume of the perifusion chamber that receives the hypothalamus.
  8. Extract the hypothalami (see Figure 2A). Place the brain upside down in a Petri dish (i.e., the ventral side up and visible and the dorsal side of the brain on the Petri dish bottom) filled with ice-cold perifusion medium (see Figure 2A), cut out the cerebellum and spinal cord parts with a scalpel (see Figure 2B) and isolate the hypothalamus using curved Dumont #7 forceps (see Figure 2C): go carefully as deep as 2 mm around the hypothalamic and circle around them to separate and collect them from the brain. More than one brain can be processed in the same dish. Collect all hypothalamic explants in another Petri dish (see Figure 2D), filled with pre-gassed perifusion medium cooled on ice.
  9. Once all hypothalami are collected, place them immediately in perifusion chambers previously filled, thanks to the peristaltic pump, with gassed and warmed perifusion medium (step 6). Open one end of the chamber and gently deposit the hypothalami in the chamber. Be careful not to block the perifusion flow by having tissues sticking to one end of the perifusion chamber (i.e., don’t push the tissues to close to one end of the chamber or carefully reposition the tissue in the center of the chamber). Close the perifusion chamber (see Figure 2I).
    Note: We used 3 hypothalami per chamber to increase the quantity of peptide release per chamber in order to be able to quantify the release of MCH with ELISA. The number of hypothalami should be determined depending on the experimental setup and conditions (i.e., the neuropeptide of interest, the stimulation of the hypothalami used to release this peptide, and the peptide quantification method).
  10. Place the perifusion chambers containing the hypothalamic explants to the thermostatic bath (34-37 °C) where they will remain during the whole experiment.
  11. Perifuse hypothalamic explants with gassed and warm (34-37 °C) perifusion medium for 2 h prior to any stimulation of the hypothalami in order to establish a baseline level of peptide release by hypothalami. The inlet tubing for the chambers is placed in a bottle containing the perifusion medium. The flow of perifusion medium that goes in the chambers is set with a peristaltic pump at the rate of 0.1 ml/1 min. This rate will be used for the whole experiment.
  12. Collect 3 fractions of the baseline perifusate (1 fraction per 10 min perifusion, and fractions are collected 10 min apart from each other). Each fraction from each chamber will be collected in a separate 1.5 ml microtube in which the outlet tubing will have been previously placed. Aliquot the collected 1-ml perifusate and freeze at -80 °C as quickly as possible to avoid peptide degradation.
  13. Once the baseline fractions are collected, pause the peristaltic pump to eliminate the risk of introducing air bubble in the superfusion tubing and prepare the tubes with the stimulation medium: the inlet tubing for each chamber should be appropriately placed in the corresponding pre-gassed and warmed stimulation solution before starting the peristaltic pump again.
  14. Stimulation will last no longer than 30 min to avoid toxicity and/or desensitization of the tissue. During this step, 3 fractions will be collected (1 fraction = 10 min, fractions are collected 10 min apart from each other). The collected fractions of 1-ml perifusate are aliquoted and freezed at -80 °C rapidly to avoid degradation.
  15. Once the stimulation fractions are collected, pause the peristaltic pump and start a 60-min washout period: place the inlet tubing for each chamber into the bottle containing the gassed and warmed (34-37 °C) hypothalamic explant perifusion medium. During this step, 6 fractions will be collected (1 fraction = 10 min, fractions are collected 10 min apart from each other). Aliquot the collected 1-ml perifusate and freeze at -80 °C as soon as possible to avoid degradation.
  16. After the washout, you can stimulate again all your hypothalamic explants with the appropriate stimulus to induce the release of your neuropeptide of interest in order to control for the viability of the hypothalamic explants–30 min maximum. During this step, 3 fractions will be collected (1 fraction = 10 min, fractions are collected 10 min apart from each other). Aliquot the collected 1-ml perifusate and freeze at -80 °C as soon as possible to avoid degradation.
  17. Turn off the pump. Hypothalamic explants can be saved for further analysis. Explants are collected either with forceps, delicately, or by centrifugation of the chamber in a microtube at 4 °C (see details on Biorep’s website). Freeze at -80 °C.
  18. Wash thoroughly the whole perifusion system (tubing and perifusion chambers) with at least 200 ml of hot autoclaved ultrapure water (pump tubing should be reusable up to 20 times depending on the tubing material).
  19. Quantify the amount/concentration of secreted neuropeptides by ELISA, RIA, flow cytometry, mass spectrometry or other suitable method.

Data analysis

All details about data processing and analysis, such as the statistical tests applied, criteria for data inclusion or exclusion have been published in Le Thuc et al., 2016.

Notes

  1. MEM with phenol red can be used when phenol red does not interfere with the peptide quantification method.
  2. For brain and hypothalamus collection, proceed as fast as possible to avoid tissue damage.
  3. Care must be taken not to damage hypothalami, once they have been isolated. We found convenient to transfer hypothalami in the medium with large stem diameter pipette (COPAN, Extra Large Pipette, 207C). As previously mentioned in step 9 of the protocol, we used 3 hypothalami per chamber to increase the quantity of peptide release per chamber in order to be able to quantify the release of MCH with ELISA. The number of hypothalami should be determined depending on the experimental setup and conditions (i.e., the neuropeptide of interest, the stimulation of the hypothalami used to release this peptide, and the peptide quantification method).
  4. Stimulation solutions should be previously gassed with carbogen and warmed in a thermostatic bath (34-37 °C). Gassing should be at saturation in solution and a minimum time of 15 min for pre-gassing is recommended. We found Micro filter candles (VWR, ref 511-0311) convenient to bubble solutions with fine diameter bubbles. The time at which the gassing and warming of these solutions start will depend on the nature of the tested compounds. For example, a stimulation solution containing a stimulating peptide should most likely not be warmed too long before use, while a stimulation solution containing KCl for example can be pre-warmed at convenience. Furthermore, stimuli should be selected in respect to the neuropeptide(s) secreted and the type of neurons investigated.
  5. When collecting fractions during stimulation, care should be taken not to collect the dead volume of the perifusion tubing. The dead volume corresponds to the volume of regular perifusion medium in the perfusion chamber and connecting tubes prior to perifusion of the stimulating medium and therefore corresponds to basal conditions medium. This should be measured for each set-up since it depends on the length of the connecting tubes. In our experimental conditions, this was measured as 1.2 ml and since the speed of the peristaltic pump is set to 0.1 ml/min, the fraction collection was delayed by 12 min.
  6. In some cases, depending on the chosen method for quantification, and on the concentration of the peptide(s) in the collected fractions, some additional steps of to improve peptide extraction or lyophilization to concentrate the sample could be necessary.

Recipes

  1. Hypothalamic explant perifusion medium (400 ml)
    400 ml Minimum Essential Medium–no glutamine, no phenol red, no HEPES
    4 ml of 2 x 10-3 M Bacitracin (final concentration: 20 μM)
    400 mg BSA
    L-Glutamine (final concentration: 2 µM)
    Protease Inhibitor cocktail (cOmpleteTM, EDTA-free Protease Inhibitor Cocktail, Roche) 1x (1 tablet for 50 ml → 8 tablets)
    Filter the medium in a Nalgene Filtration Unit–PES FASTER membranes–0.2 µm porosity
  2. 1 M KCl (100 ml)
    7.455 g KCl (MW = 74.55 g/mol)
    Ultrapure water to 100 ml
    Filter in a Nalgene Filtration Unit–PES FASTER membranes–0.2 µm porosity
  3. Stimulation medium with 60 mM KCl (Iso-osmotic 60 mM KCl, 50 ml)
    47 ml of the hypothalamic explant perifusion medium
    3 ml of 1 M KCl
    Adjust osmolarity to 310-320 mOsm with 0.22 µm filtered Milli-Q water ( Micro-Osmometer Type 6 , Löser)

Acknowledgments

This work was supported by the CNRS, the Fondation pour la Recherche Médicale (DEQ20150331738 to J.L.N. and DRM20101220421 to N.B. and C.R.) and by the French Government (National Research Agency, ANR) through the ‘Investments for the Future’ LABEX SIGNALIFE: program reference #ANR-11-LABX-0028-01. This protocol has been adapted from the previous publication Callewaere et al. (2006) with the precious help of Dr William Rostene (Institut de la Vision, Paris, France).

References

  1. Callewaere, C., Banisadr, G., Desarmenien, M. G., Mechighel, P., Kitabgi, P., Rostene, W. H. and Melik Parsadaniantz, S. (2006). The chemokine SDF-1/CXCL12 modulates the firing pattern of vasopressin neurons and counteracts induced vasopressin release through CXCR4. Proc Natl Acad Sci U S A 103(21): 8221-8226.
  2. Le Thuc, O., Cansell, C., Bourourou, M., Denis, R. G., Stobbe, K., Devaux, N., Guyon, A., Cazareth, J., Heurteaux, C., Rostene, W., Luquet, S., Blondeau, N., Nahon, J. L. and Rovere, C. (2016). Central CCL2 signaling onto MCH neurons mediates metabolic and behavioral adaptation to inflammation. EMBO Rep 17(12): 1738-1752.
  3. Tolle, V., Zizzari, P., Tomasetto, C., Rio, M. C., Epelbaum, J. and Bluet-Pajot, M. T. (2001). In vivo and in vitro effects of ghrelin/motilin-related peptide on growth hormone secretion in the rat. Neuroendocrinology 73(1): 54-61.
  4. Zizzari, P., Longchamps, R., Epelbaum, J. and Bluet-Pajot, M. T. (2007). Obestatin partially affects ghrelin stimulation of food intake and growth hormone secretion in rodents. Endocrinology 148(4): 1648-1653.

简介

下丘脑是一个主要的脑区域,在哺乳动物中,通过连接中枢神经系统(CNS)和周围环境来调节与维持一般体内平衡有关的几种生理功能。下丘脑本身可以被认为是某种类型的内分泌大脑区域,因为它在其不同的核中存在几种神经肽产生和分泌神经元。这些神经肽具有特定的作用并参与一般的体内平衡调节,其中包括调节能量代谢,摄食行为,进水量和身体核心温度。
   如前所述,为了发挥其效果,必须产生这些肽,而且大部分是分泌的。在这种情况下,能够评估某些病症,疾病或治疗如何能够真正影响神经肽的分泌以及不同的神经肽能电路的功能是非常重要的。
   评估这一点的一种方法是下丘脑外植体的渗透,然后定量收集的部分中的肽。
   在这里,我们逐步解释如何在体外渗出下丘脑外植体中收集分数,其中可以从这些活的分离组织定量测定神经肽/神经激素释放。下丘脑渗出与其他现有测定相比有两大优点:(1)允许药理学操作来解剖下丘脑外植体中不同神经肽/神经激素释放的信号传导机制,以及(2)允许在多种下丘脑制剂上同时进行不同条件的实验,(3)据我们所知,这是允许研究神经肽分泌在基础条件下和用同样的下丘脑外植体反复刺激的唯一方法。
【背景】已经经常使用渗透研究胰岛功能。然而,该测定原则上适用于任何内分泌组织和任何肽或蛋白质分泌。
   事实上,过去已经使用了不同的渗透系统,并且仍然是不同研究实验室在各种条件下研究下丘脑神经肽释放的有效程序。例如,Callewaere及其同事在2006年发表了一项研究,分析了趋化因子SDF-1(基质细胞衍生因子-1)对血管舒张素诱导的AVP(精氨酸 - 加压素)释放的影响。在其他研究中,下丘脑外植体的渗透也被用于分析从浸润性下丘脑中释放的生长激素释放抑制激素(又称生长抑素)(用25mM KCl进行细胞外渗透的刺激)(Tolle等人,2001; Zizzari等人,,2007)。
   然而,目前,关于下丘脑外植体渗出,没有标准化的装置或方案可用。我们自己修改了Callewaere等人,2006年出版的协议。
   通过这种方法,我们最近已经显示,在小鼠中,趋化因子CCL2(CC-趋化因子配体2)能够减少orexin能下丘脑神经肽黑色素浓度激素(MCH)的分泌,从而参与丧失两者食欲和体重在高度炎症的背景下(Le Thuc等,2016)。

关键字:灌流, 下丘脑, 下丘脑外植体, 肽, 神经肽, 分泌, 释放, 神经内分泌学, 内分泌学

材料和试剂

  1. 15 ml和/或50 ml离心管(Corning,Falcon ®,目录号:352096和352070)
  2. 培养皿Ø100毫米(康宁,目录号:3262)
  3. 5ml移液器(Corning,Falcon ®,目录号:357543)
  4. 10 ml移液器(Corning,Falcon ®,目录号:357551)
  5. 1.5ml微管(SARSTEDT,目录号:72.706)
  6. 500毫升Nalgene过滤装置-PES FASTER膜 - 0.2微米孔隙度(Dutscher,目录号:029667)
  7. 0.22μm过滤器
  8. 8周龄老鼠
  9. 最低必需培养基(MEM) - 无谷氨酰胺,无酚红,无HEPES(Thermo Fischer Scientific,Invitrogen TM,目录号:51200046)
  10. (来自地衣芽孢杆菌)(Sigma-Aldrich,目录号:31626)
  11. L-谷氨酰胺100x(Thermo Fischer Scientific,Invitrogen TM,目录号:25030024)
  12. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A2153)
  13. 蛋白酶抑制剂混合物(Complete TM,不含EDTA的蛋白酶抑制剂混合物)(Roche Diagnostics)
  14. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
  15. 葡萄糖(Sigma-Aldrich,目录号:G8270)
  16. 超纯水
  17. ELISA(Phoenix Pharmaceuticals,目录号:EK-070-47)
  18. 下丘脑外植体渗透介质(见食谱)
  19. 1 M KCl(见配方)
  20. 具有60mM KCl(等渗60mM KCl,50ml)的刺激培养基(参见食谱)

设备

  1. 微管支架和15 ml / 50ml锥形管(Dutscher)
  2. P10,P200,P1000移液器(Gilson)
  3. -20°C和-80°C冰箱(Sanyo,VWR)
  4. 打孔机(任意固定品牌的任何定孔打孔) -
  5. Carbogen(5%CO 2/95%O 2)(Linde France,UN 3156)
  6. 标准图案剪刀,大圈夏普/钝14.5厘米(精细科学工具,目录号:14101-14)
  7. 超薄虹膜剪刀,10.5厘米(精细科学工具,目录号:14088-10)
  8. 标准图案钳具有锯齿尖(Fine Science Tools,目录号:11000-13)
  9. Dumont#7镊子弯曲尖端(精细科学工具,目录号:11271-30和11272-30)
  10. 2恒温浴(如JULABO,型号:CORIO CD-B27)
  11. 渗透室(BIOREP TECHNOLOGIES,目录号:PERI-CHAMBER)
  12. 渗透室过滤器(BIOREP TECHNOLOGIES,目录号:PERI-FILTER)
  13. 渗透管套(BIOREP TECHNOLOGIES,目录号:PERI-TUBSET)
  14. 蠕动泵(高精度多通道泵,Ismatec)
  15. 渗透计(LöserMesstechnik,型号:Micro-Osmometer Type 6)

软件

  1. GraphPad Prism(GraphPad软件)或用于数据统计分析的任何软件

程序

我们在这里描述如何按照这些步骤手动进行下丘脑渗透测定(见图1):


图1.渗透流程图。在2小时的渗透达到平衡后,抽样程序由30分钟的对照基期,30分钟的时间组成,在此期间药物独立地加入或组合并用培养基洗涤60分钟。在每个实验之后,应用适当的分子(例如,KCl)来控制下丘脑外植体的反应性和测试组织活力。

  1. 为整个实验准备足够的下丘脑外植体渗透培养基(参见食谱;准备总是新鲜,不早于前一天,在4℃下储存)。
  2. 准备刺激解决方案。例如,我们使用了等渗60 mM KCl(见配方)。
  3. 将含有碳原子(5%CO 2/95%O 2)的一些下丘脑外植体渗出培养基预先气化,所有预充气和放气步骤使用碳霉素)在50ml锥形管至少15分钟。大约10ml的预先加入的培养基将用于冷冻用于脑解剖和下丘脑收集,另一部分约40ml将在恒温槽中用温热(34-37℃)进行渗透。 />
  4. 使用穿孔机为每个室(每个室2个)准备渗透室过滤器(Biorep)。在室的每个开口端放置一个过滤器(见图2F-2H)。


    图2.渗透实验的不同步骤的代表性图像A.将小鼠脑上皮倒置在含有在实验者前面具有下丘脑的冰冷的基础培养基的培养皿中(A,B ,C和D表示步骤8); B.用手术刀取下小脑; C.用弯曲的Dumont#7钳子恢复下丘脑; D.在含有冰冷的基础培养基的新培养皿中收集下丘脑(箭头和虚线所示) E.为避免损伤下丘脑,可用大型塑料/玻璃移液器处理; F.在解剖之前,准备渗透室过滤器;显示下丘脑位置的大脑的矢状视图(虚线中的一圈被淹死在下丘脑周围); H.将一个过滤器放在渗透室的两端(步骤4);用预热的预先吹入的介质预填充渗透室(如步骤6所述)。 I.将下丘脑置于渗透室的中心(步骤9)。关闭腔室并将其放回37℃的恒温槽中(步骤10); J.实验装置的完全组装,从右到左依次是在37℃下的恒温槽,其中包含充满渗透介质的管(管与硅管连接成一个一个到渗透室),蠕动泵流入渗透介质,37℃的第二个恒温槽,其接收渗透室(一个渗透室由红色虚线圆圈标记),并且左边,渗出室的出口与硅管连接到低蛋白质吸收将1.5ml微管置于冰上收集级分。渗透介质从右向左流动。

  5. 连接适当的管道(低蛋白质吸收管,如特氟龙管),将包含下丘脑外植体的渗出室的入口连接到一侧的蠕动泵,另一侧的腔室出口到管道,用于在另一侧进行部分收集。
  6. 用预热的预充气介质预填充渗透室。房间和管道本身将在34-37°C的恒温槽中保存。
  7. 对于以前安乐死的每只老鼠(我们使用颈椎脱位选择的方法应该是快速的,以尽可能多地保存组织),切开头部,并将小鼠脑收集在填充有预充气的渗透介质的培养皿中,在冰上冷却。要收集大脑,将一个超薄的虹膜剪刀的底部刀片插入孔中,并开始直接切开并穿过颅骨的中线,小心地将剪刀尖端向上指向。然后,用镊子打开颅骨,向外推动颅骨的两半,从而暴露大脑。仔细翻转颅骨:重力将帮助大脑从头骨脱离。使用Dumont#7镊子,小心地将镊子沿着脑的外边缘和大脑从嗅角向小脑滑向小脑。轻轻地从任何结缔组织或神经分离大脑,防止它从头骨掉落。
    注意:我们使用8周龄的老鼠。用户将确定每个科学项目。限制当然是接收下丘脑的渗出室的体积。
  8. 提取下丘脑(见图2A)。将大脑倒置在充满冰冷的浸润培养基的培养皿(即,腹侧朝上,培养皿底部的大脑的可见和背侧)(参见图2A)用手术刀切开小脑和脊髓部分(参见图2B),并使用弯曲的Dumont#7镊子分离下丘脑(见图2C):在下丘脑周围仔细深入2毫米,并围绕它们分开,从大脑收集他们。可以在同一道菜中处理多于一个的大脑。收集另一个培养皿中的所有下丘脑外植体(参见图2D),填充在冰上冷却的预先充气的渗透培养基。
  9. 一旦收集了所有的下丘脑,就可以将它们立即放在先前填充的渗透室中,这要归功于蠕动泵,有气体和温热的渗透介质(步骤6)。打开房间的一端,轻轻地将海马座放在房间中。注意不要通过使组织粘附在渗出室的一端( ,不要将组织靠近腔室的一端或仔细重新定位组织的位置)来阻止渗出物流动房间中心)。关闭渗透室(见图2I)。
    注意:我们每个室使用3个下丘脑来增加每个室的肽释放量,以便能够用ELISA量化MCH的释放。应根据实验设置和条件( ,感兴趣的神经肽,用于释放该肽的下丘脑的刺激,以及肽量化方法)。
  10. 将包含下丘脑外植体的渗出室放入恒温槽(34-37℃),在整个实验过程中保留下来。
  11. 浸润性下丘脑外植体在气胸和温暖(34-37°C)浸润培养基下,任何刺激下丘脑之前2 h,以建立下丘脑肽释放的基线水平。用于腔室的入口管被放置在包含渗透介质的瓶子中。进入腔室的渗出介质的流动以蠕动泵以0.1ml / 1分钟的速度设定。这个速率将用于整个实验。
  12. 收集基线perifusate的3个部分(每10分钟1次,每次10分钟收集分数)。来自每个室的每个部分将被收集在单独的1.5ml微管中,其中出口管将被预先放置在其中。将收集的1毫升perifusate等分,并尽快在-80℃冷冻以避免肽降解。
  13. 一旦收集了基准分数,暂停蠕动泵,以消除在灌注管中引入气泡的风险,并用刺激介质制备管:每个室的入口管应适当放置在相应的预放气和加热刺激溶液再次启动蠕动泵前。
  14. 刺激持续不超过30分钟以避免组织的毒性和/或脱敏。在该步骤中,将收集3个级分(1份= 10分钟,将馏分相互收集10分钟)。将收集的1ml过氧化物的级分等分并快速冷冻至-80℃以避免降解。
  15. 一旦收集刺激部分,暂停蠕动泵并开始60分钟清洗期:将每个腔室的入口管放入含有气体和温热(34-37℃)下丘脑外植体浸润培养基的瓶子中。在该步骤中,将收集6个级分(1份= 10分钟,将馏分相互收集10分钟)。将收集的1毫升perifusate等分,并尽快在-80℃冷冻,以免降解。
  16. 在冲洗后,您可以用适当的刺激再次刺激所有下丘脑外植体,以诱导您的神经肽的释放,以控制下丘脑外植体的活力 - 最大30分钟。在该步骤中,将收集3个级分(1份= 10分钟,将馏分相互收集10分钟)。将收集的1 ml perifusate等分,并在-80°C下冷冻可能避免退化。
  17. 关闭泵。下丘脑外植体可以节省进一步分析。外植体采用镊子精细收集,或通过在4℃的微管中离心室(参见Biorep网站的详细信息)。冻在-80°C。
  18. 用至少200毫升热压灭菌超纯水彻底清洗整个渗透系统(管道和渗透室)(根据管道材料,泵管应可重复使用20次)。
  19. 通过ELISA,RIA,流式细胞仪,质谱法或其他合适的方法定量分泌的神经肽的量/浓度。

数据分析

关于数据处理和分析的所有细节,例如应用的统计测试,数据包含或排除的标准已在2016年的Le Thuc等人公布。

笔记

  1. 当酚红不干扰肽定量方法时,可以使用酚红的MEM
  2. 对于大脑和下丘脑的收集,尽快进行,以避免组织损伤。
  3. 一旦被隔离,必须小心不要损伤下丘脑。我们发现在大直径移液管(COPAN,超大型移液器,207C)的培养基中转运下丘脑是方便的。如方案的步骤9中所述,我们使用每个室3个下丘脑来增加每个室的肽释放量,以便能够用ELISA量化MCH的释放。应根据实验设置和条件(即,感兴趣的神经肽,用于释放该肽的下丘脑的刺激)和肽定量方法来确定下丘脑的数量。
  4. 刺激溶液应先用碳水气体放置,并在恒温槽(34-37℃)中温热。放气应处于饱和状态,推荐使用预充气的最短时间为15分钟。我们发现微过滤蜡烛(VWR,参考511-0311)方便气泡溶液与细直径气泡。这些解决方案的起泡和升温开始的时间将取决于被测化合物的性质。例如,含有刺激肽的刺激溶液在使用前最可能不会被加热太久,而含有KCl的刺激溶液可以在方便的情况下预热。此外,应该对分泌的神经肽和所研究的神经元的类型选择刺激。
  5. 在刺激期间收集分数时,应注意不要收集渗液管的死体积。死体积对应于在刺激介质渗出之前的灌注腔室和连接管中的常规渗透介质的体积,因此对应于基础条件介质。应该对每个设置进行测量,因为它取决于连接管的长度。在我们的实验条件下,测量为1.2毫升,由于蠕动泵的速度设定为0.1毫升/分,收集的分数延迟了12分钟。
  6. 在某些情况下,根据所选择的定量方法和收集的级分中肽的浓度,可能需要一些额外的步骤来改善肽提取或冻干浓缩样品。

食谱

  1. 下丘脑外植体渗透培养基(400毫升)
    400毫升最低必需培养基 - 无谷氨酰胺,无酚红,无HEPES
    4毫升2×10 3 -3杆菌肽(终浓度:20μM)
    400毫克BSA
    L-谷氨酰胺(终浓度:2μM)
    蛋白酶抑制剂混合物(cOmplete TM,不含EDTA的蛋白酶抑制剂鸡尾酒,Roche)1x(1片为50ml→8片)
    过滤Nalgene过滤装置中的介质 - PES FASTER膜 - 0.2μm孔隙度
  2. 1 M KCl(100 ml)
    7.455克KCl(MW = 74.55克/摩尔)
    超纯水至100 ml
    过滤器在Nalgene过滤装置中PES-PES FASTER膜 - 0.2μm孔隙度
  3. 具有60mM KCl(等渗60mM KCl,50ml)的刺激培养基
    47 ml下丘脑外植体渗透培养基
    3毫升1M KCl 将0.22摩尔过滤的Milli-Q水(Micro-Osmometer Type 6,Löser)将渗透压调节至310-320 mosm,

致谢

这项工作得到了国家自然科学基金委,“基金会基金会(JLN,DEQ 20150331738和NB和CR”)以及法国政府(国家研究机构ANR)通过“未来投资”的支持。LABEX SIGNALIFE:计划参考#ANR-11的LabX-0028-01。该协议已经由以前的出版物Callewaere等人(2006)改编自William Rostene博士(法国巴黎研究所)的宝贵帮助。

参考

  1. Callewaere,C.,Banisadr,G.,Desarmenien,MG,Mechighel,P.,Kitabgi,P.,Rostene,WH和Melik Parsadaniantz,S。(2006)。趋化因子SDF-1 / CXCL12通过CXCR4调节血管加压素神经元的发射模式并抵消诱导的加压素释放。 Proc Natl Acad Sci USA 103(21):8221-8226。
  2. Le Thuc,O.,Cansell,C.,Bourourou,M.,Denis,RG,Stobbe,K.,Devaux,N.,Guyon,A.,Cazareth,J.,Heurteaux,C.,Rostene, Luquet,S.,Blondeau,N.,Nahon,JL和Rovere,C.(2016)。  MCH神经元上的中枢CCL2信号介导代谢和行为适应炎症。 17(12):1738-1752。
  3. Tolle,V.,Zizzari,P.,Tomasetto,C.,Rio,MC,Epelbaum,J.and Bluet-Pajot,MT(2001)。< a class =“ke-insertfile”href =“http: /www.ncbi.nlm.nih.gov/pubmed/11174017“target =”_ blank“> 体外和体外生长素释放肽/胃动素相关肽对生长的作用大鼠中的激素分泌。神经内分泌学73(1):54-61。
  4. Zizzari,P.,Longchamps,R.,Epelbaum,J.and Bluet-Pajot,MT(2007)。 Obestatin部分影响生长激素释放激素对啮齿动物食物摄取和生长激素分泌的刺激。内分泌学 148(4):1648-1653。 />
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Le Thuc, O., Noël, J. and Rovère, C. (2017). An ex vivo Perifusion Method for Quantitative Determination of Neuropeptide Release from Mouse Hypothalamic Explants. Bio-protocol 7(16): e2521. DOI: 10.21769/BioProtoc.2521.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。