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Measuring Rat Serum Osmolality by Freezing Point Osmometry
冰点渗透压测定法测量大鼠的血清渗透压   

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Abstract

Blood serum or plasma osmolality is the measure of electrolyte to water balance in the body’s circulation, and is tightly regulated in physiological states in order to maintain normal levels of serum solute (Bourque, 2008). Osmolality is defined as the number of osmoles of solute per kg of water (mOsm/kg) (Dufour, 1993) and can be measured using different techniques that rely on the colligative properties of the solution. The most commonly used in lab settings are vapour pressure and freezing point osmometry, which are relatively quick and easy to perform. Freezing point osmometry is preferred because it is insensitive to volatile compounds, such as alcohol, that may be present in the solution. Measurement of serum or plasma osmolality is clinically relevant for a number of conditions and diseases, including hypernatremia, diabetic ketoacidosis, and the syndrome of inappropriate antidiuresis (Ellison, 2013; Lupsa and Inzucchi, 2013; Reddi, 2013). In this protocol, we describe the measurement of serum osmolality in rats using the freezing point osmometry technique as originally outlined in our previous study of osmoregulatory perturbations in sepsis (Stare et al., 2015).

Keywords: Osmolality(渗透压), Osmolarity(渗透压), Serum osmolality(血清渗透压), Serum osmolarity(血清渗透压), Osmoregulation(渗透调节)

Materials and Reagents

  1. Underpad (AMG Medical, catalog number: 760-372 )
  2. 21 G x 1 ½ in. needle (BD, catalog number: 305167 )
  3. 5 ml syringe (BD, catalog number: 309646 )
  4. 1.5 ml microcentrifuge tube (Diamed, catalog number: DIATEC610-3169 )
  5. 1,000 μl pipette tips (Diamed, catalog number: DIATEC520-6753 )
  6. Kimwipes (Thermo Fisher Scientific, Fisher Scientific, catalog number: 06-666A )
  7. 1 rat (Rattus rattus; 100-300 g)
  8. Isoflurane (CDMV, catalog number: 19417 )
  9. 100% oxygen
  10. 290 mOsm/kg control solution (ClinitrolTM 290 Reference Solution) (Advanced Instruments, catalog number: 3MA029 )

Equipment

  1. Forceps (Dumont #7b forceps) (Fine Science Tools, catalog number: 11270-20 )
  2. Veterinary gas anesthesia machine (Dispomed, model: 975-3300-000MRI )
  3. Induction chamber (VetEquip, model: 941444 )
  4. 1,000 μl pipette (Gilson, PipetmanTM, model: F123602 )
  5. Nosecone connected to Bain circuit (VetEquip, model: 921463 )
  6. Centrifuge (Spectrafuge 24D) (Labnet International, model: C2400 )
  7. Vortex (Vortex Genie 2) (Scientific Industries, model: SI-0236 )
  8. Freezing point osmometer (Advanced Instruments, model: 3320 Micro-Osmometer )
  9. Ease-Eject 20-Microliter sampler (Advanced Instruments, model: 3M0825 )
  10. Sampler plunger wire (Advanced Instruments, catalog number: 3M0828 )
  11. Sampler tips and chamber cleaners (Advanced Instruments, model: 3MA800 )

Procedure

  1. Cardiac puncture
    This section describes the cardiac puncture method for acquiring and handling a blood sample in preparation for measuring serum osmolality (see Note A1).
    1. Deep anesthesia will take approximately 10 min overall. Place the rat into the induction chamber. Adjust the oxygen flow rate to 1.5-2 L/min, and the isoflurane vapourizer to 5%.
    2. Once the rat has stopped moving for several minutes (2-4 min), it can be transferred onto the working surface and placed on the mask attached to a Bain circuit.
    3. Place the rat parallel to self in the supine position, with the head towards your non-dominant hand, and tail towards your dominant hand. Adjust the oxygen flow rate to 0.4-0.8 L/min and continue at 3-5% isoflurane.
    4. Verify that it is deeply anesthetized by using the tweezers to pinch the toes. The rat should not respond. Maintain the isoflurane at 3-5% (see Note A2).
    5. Test the plunger on the 5 ml tube, making sure that it moves smoothly. Leave the plunger slightly retracted. Attach the 21 G 1 ½ in. needle.
    6. Using your non-dominant hand, gently press the forelimbs down on the table and inwards into the body so that the claws face towards its hind legs and the forelimbs are parallel to the chest. This 'squeezing' of the forelimbs into the body will lift up the rib cage providing easier access to the heart.
    7. With your dominant hand, insert the needle to the anatomical left of the xiphoid process at a 30° angle (upwards from the chest) and slowly push it into the chest while maintaining a gentle negative pressure with the plunger (see Note A3). You may feel a resistance when you reach the heart, but this is not always the case.
    8. Once you have reached the heart, a small amount of blood will enter the syringe. Release the negative pressure. Let go of the forelimbs, and use your non-dominant hand to stabilize the syringe. Use your dominant hand to slowly pull back on the syringe.
    9. Once you have collected your desired amount of blood (see Note A4), gently apply a small positive pressure by pushing the plunger in, and pull the needle out. A bit of positive pressure will prevent hemolysis during retraction of the needle (see Note A5).
    10. Immediately transfer the blood in the syringe into a 1.5 ml microcentrifuge tube. To do so, remove the needle from the syringe and gently expel the blood into the tube. The blood will have begun to coagulate and if this step is done too quickly, it will cause hemolysis.
    11. Leave at room temperature (RT, ~22 °C) for 30-60 min, or place the tube on ice in an insulated foam container with the lid closed for a minimum of 30 min and up to 9 h (see Note A6 and Representative data).
    12. Follow up with a recommended physical method of euthanization (e.g., pneumothorax, cervical dislocation) as per your institution’s animal use guidelines.

  2. Serum separation
    This section describes the blood processing necessary to prepare the serum sample for osmolality measurement.
    1. Centrifuge the blood sample at 6,000 x g for 5 min. The resultant thick and dark red pellet will contain the haemocytes. The clear, off-white-to-yellow supernatant layer will contain the serum. 1.5 ml of blood provides approximately 0.5-0.7 ml of serum (see Notes B1 and B2).
    2. Pipette the supernatant into a fresh microcentrifuge tube. Be sure not to disturb the pellet when collecting the serum.
    3. Store at 4 °C until ready for further processing, making sure that the lid is completely closed to prevent evaporation.

  3. Osmolality measurement
    This section starts by verifying the reliability of the osmometer by measuring a solution with a known osmolality value (290 mOsm/kg control solution), followed by the method of measuring serum osmolality.
    1. Make sure that your osmometer has been recently calibrated following the manufacturer’s guidelines, and is in good working order.
    2. This is a quality control step to test the osmometer (see Notes C1 and C2).
      1. Attach a sampler tip to the sampler. Use the sampler to draw 20 μl of the 290 mOsm/kg control solution.
      2. Wipe the outside of the sampler tip dry with a Kimwipe, including the tip perimeter. Blot excess sample so that the meniscus is in line with the tip or slightly concave (see Note C3).
      3. Insert the sampler into the cooling chamber. The measurement will take approximately 1 min.
      4. When the measurement has been completed, pull out the sampler, and remove and discard the sampler tip (see Note C4).
      5. Dry the sampler plunger wire with a Kimwipe.
      6. Clean the cooling chamber with a chamber cleaner as per the manufacturer’s instructions
      7. Repeat steps C2 a-f two more times.
    3. Check for any residue at the bottom of your serum sample. Should residue be present, centrifuge the sample again at 6,000 x g for 2.5 min, and pipette the serum into a fresh microcentrifuge tube.
    4. Vortex the sample for 5 sec.
    5. Measure the serum osmolality using the same technique described in step C2, briefly vortexing (~3 sec) the serum in between each measurement (see Notes C1 and C2).
      1. Attach a sampler tip to the sampler. Use the sampler to draw 20 μl of the serum sample.
      2. Wipe the outside of the sampler tip dry with a Kimwipe, including the tip perimeter. Blot excess sample so that the meniscus is in line with the tip or slightly concave (see Note C3).
      3. Insert the sampler into the cooling chamber. The measurement will take approximately 1 min.
      4. When the measurement has been completed, pull out the sampler, and remove and discard the sampler tip.
      5. Dry the sampler plunger wire with a Kimwipe.
      6. Clean the cooling chamber with a chamber cleaner as per the manufacturer’s instructions.
      7. Repeat steps C5 a-f two more times (see Note C5).
    6. When all measurements have been completed, clean the cooling chamber as per the manufacturer’s guidelines.
    7. Report the final value as an average of all serum osmolality measurements.

Notes

  1. Cardiac puncture
    1. Any blood collection method is sufficient for the purpose of measuring serum osmolality. We outline the cardiac puncture method as described in the original article (Stare et al., 2015).
    2. Since the cardiac puncture is a terminal exsanguination procedure, maintaining body heat is unnecessary. Isoflurane can be maintained at maximum levels (5%) throughout the entire procedure.
    3. Approximately 40-70% of the needle will need to be inserted, depending on the size of the rat. If the needle is completely inserted and there is no indication of blood in the syringe, then it is likely that the heart has been missed. In this case, withdraw the needle without removing it from the chest cavity, and try again. If an additional attempt is unsuccessful, replace the needle with a new one. Sometimes an attempt is unsuccessful because the needle becomes blocked.
    4. A 100-300 g rat will provide > 5 ml of blood, however only ~0.15 ml of blood is required to produce the minimum amount of serum (3 x 20 μl, see Note C1) necessary for osmolality measurement. This protocol describes the collection of 1.5 ml of blood because it is the amount that we usually collect for our own experiments (Stare et al., 2015).
    5. Hemolysis is the rupture of erythrocytes and can be visually detected in the serum by a change of colour from yellow/white to red. Hemolysis can alter the composition of the serum and thus potentially alter serum osmolality measurements (Lippi et al., 2012).
    6. A difference between serum and plasma is that serum does not contain coagulants. Therefore it is important to allow the blood to coagulate fully prior to centrifuging the sample. Storage time and temperature recommendations prior to centrifugation of the whole blood sample vary and is to this day debated (Abbadi et al., 2014; Curia et al., 2009; Seifarth et al., 2004), however we routinely place the sample on ice in an insulated foam container with the lid closed for up to 9 h without any adverse effects (see Representative data). Once the serum is isolated, it can be stored at 4 °C for up to 24 to 48 h (see Representative data; Curia et al., 2009).

  2. Serum separation
    1. If the serum layer contains particles that are visible to the eye, re-centrifuge the sample at 6,000 x g for 2-5 min, and collect the serum into a fresh microcentrifuge tube.
    2. Occasionally a thickened, off-white substance appears in the serum layer after the blood has been centrifuged. There are two options to consider: either re-centrifuge at 6,000 x g for 2.5-5 min, or collect the serum around it if possible.

  3. Osmolality measurement
    1. The listed osmometer uses 20 μl per measurement, and has a resolution of ± 1 mOsm/kg. However, we keep a minimum of 200 μl of serum sample.
    2. Keep the lid of the microcentrifuge tube closed in between sampling to prevent evaporation.
    3. When using the sampler, make sure to closely follow the manufacturer’s guidelines to acquiring a reliable and measurable 20 μl sample. This will include ensuring that there are no bubbles in the sample, and keeping a consistent meniscus level. Consistency is very important as these subtle differences can cause inconsistencies with the measurements that may mimic calibration problems with the osmometer or sampler plunger wire deterioration.
    4. Should the measurements of the control solution be outside of the stated value of the standard, re-calibrate the osmometer.
    5. If the measured serum osmolality values are consistently varying by more than 3 mOsm/kg per test, clean the freezing chamber with alcohol as per the manufacturer’s guidelines. If values are still highly variable, replace the plunger wire in the sampler following the manufacturer’s guidelines.

Representative data

We investigated the effects of different coagulation times and temperatures on serum osmolality measurements determined immediately and after one day of cold storage. We found that there was no appreciable impact of various coagulation times for blood samples left at room temperature (RT, ~22 °C; 30 min and 60 min) or ice (~0 °C; 30 min, 60 min, 90 min, and 9 h; Figure 2A and 2B). We also found that serum samples were stable for a day at 4 °C (Figure 2C).

  1. Experimental design
    Seven adult male Westar rats were subjected to the cardiac puncture procedure outlined above. Collected blood was divided into six 1.5 ml microcentrifuge tubes, and left to coagulate at RT or on ice in an insulated foam container with the lid closed for various lengths of time. Each serum sample was separated as described above, and aliquoted into 2 microcentrifuge tubes: one tube was used for immediate osmolality measurement (time point '0 h', and the other was kept at 4 °C for one day (time point '24 h'). Reported values are means of 3-7 measures per sample with ± SEM, and standard deviation (SD) where specified. Statistical analysis and graphing was performed using SigmaPlot 12 and Prism 6 to compare means by One Way ANOVA and paired t-test. A P value of < 0.05 was considered to be significant.
  2. Results

    Table 1. Sample data used for graphing the standard curve



    Figure 1. Standard curve used to test the calibration and performance of the osmometer used in this study

    Table 2. Sample data



    Figure 2. Effects of storage temperature and duration on serum osmolality. A. Serum osmolality values of blood samples (n = 7 each) allowed to coagulate for 30 min to 9 h at RT (~22 °C) or on ice (~0 °C). There was no statistical difference between categories (One Way ANOVA, F(5, 36) = 0.472, P = 0.795); B. Osmolality values normalized to RT-30min; C. Effect of one day of cold storage (4 °C) on serum osmolality values. There was no statistical difference (paired t-test, P = 0.241) between samples that were measured immediately and samples that were stored at 4 °C for one day (0 h, 303.72 ± 0.44 mOsm/kg, n = 26; 24 h, 303.146 ± 0.60 mOsm/kg, n = 26).

Acknowledgments

This protocol is an expanded version of the Materials and Methods – Serum Osmolality protocol appearing in Stare et al. (2015). It has been adapted from McGill Standard Operating Procedures #111-Rat Anesthesia, #301-Rodent Euthanasia and #403-Guidelines for Blood Collection Volume and Frequency, and is approved by the Facility Animal Care Committee of McGill University. Steps C2 and C5 are based on the Ease-Eject® sampler (20 μl) user guide provided by Advanced Instruments, Inc. This work was funded by the Canadian Institutes of Health Research (CIHR) Foundation Grant FDN-143337 and James McGill Research Chair to C.W.B., and a CIHR Frederick Banting and Charles Best Canada Graduate Scholarship – Doctoral Award to J.S. The Quebec Research Fund – Health supports the Research Institute of the McGill University Health Center. The authors thank Dr. M. Prager-Khoutorsky and Dr. M. Verway for providing constructive comments on this manuscript.

References

  1. Abbadi, A., El-Khoury, J. M. and Wang, S. (2014). Stability of serum and plasma osmolality in common clinical laboratory storage conditions. Clin Biochem 47(7-8): 686-687.
  2. Bourque, C. W. (2008). Central mechanisms of osmosensation and systemic osmoregulation. Nat Rev Neurosci 9(7): 519-531.
  3. Curria, A., Pesta, M., Garry, E., Zampa, N., Rosenman, J. and Sacks, J. (2009). Refrigerated and room temperature storage stability of serum osmolality measurements. Clin Chem 55(6): A17-A18.
  4. Dufour D. R. (1993) Osmometry: The rational basis for use of an underappreciated diagnostic tool. American Association for Clinical Chemistry Meeting, Advanced Instruments, Inc. NY. July 13 1993.
  5. Ellison D. H. (2013). Hyponatremia: SIADH. In: Loriaux L. (Ed.). Endocrine Emergencies: Recognition and Treatment. Springer, 74: 115-126.
  6. Lippi, G., Cervellin, G., Favaloro, E. J. and Plebani, M. (2012). Effects of in vitro hemolysis on laboratory testing. In: Sonnta, O. and Plebani, M. (Eds.). In vitro and in vivo hemolysis: An unresolved dispute in laboratory medicine: 39-44.
  7. Lupsa B. C. and Inzucchi S. E. (2013). Diabetic ketoacidosis and hyperosmolar hyperglycemic syndrome. In: Loriaux L. (Ed.). Endocrine Emergencies: Recognition and Treatment. Springer, 115-126.
  8. Reddi A. S. (2013). Disorders of water balance: Hypernatremia. In: Reddi A. S. (Ed.). Fluid, Electrolyte and Acid-Base Disorders. Springer, 133-150.
  9. Seifarth, C. C., Miertschischk, J., Hahn, E. G. and Hensen, J. (2004). Measurement of serum and plasma osmolality in healthy young humans--influence of time and storage conditions. Clin Chem Lab Med 42(8): 927-932.
  10. Stare, J., Siami, S., Trudel, E., Prager-Khoutorsky, M., Sharshar, T. and Bourque, C. W. (2015). Effects of peritoneal sepsis on rat central osmoregulatory neurons mediating thirst and vasopressin release. J Neurosci 35(35): 12188-12197.

简介

血清或血浆渗透压是体内循环中电解质与水平衡的量度,并且在生理状态下受到严格的调节,以维持血清溶质的正常水平(Bourque,2008)。重量摩尔渗透压浓度定义为每公斤水溶质渗透摩尔数(mOsm / kg)(Dufour,1993),并且可以使用依赖于溶液的碰撞性质的不同技术来测量。实验室设置中最常用的是蒸气压和凝固点渗透压测量法,它们相对快速且易于执行。凝固点渗透压是优选的,因为它对溶解中可能存在的挥发性化合物(例如醇)不敏感。血清或血浆渗透压的测量在许多病症和疾病方面具有临床意义,包括高钠血症,糖尿病酮症酸中毒和不良抗生素综合征(Ellison,2013; Lupsa和Inzucchi,2013; Reddi,2013)。在本协议中,我们描述了使用冰点渗透压测定技术测量大鼠血清渗透压的方法,原先在我们以前在败血症中的渗透调节扰动研究中概述(Stare等,2015)。

关键字:渗透压, 渗透压, 血清渗透压, 血清渗透压, 渗透调节

材料和试剂

  1. Underpad(AMG Medical,目录号:760-372)
  2. 21 G×1/2英寸针(BD,目录号:305167)
  3. 5ml注射器(BD,目录号:309646)
  4. 1.5ml微量离心管(Diamed,目录号:DIATEC610-3169)
  5. 1000μl移液管吸头(Diamed,目录号:DIATEC520-6753)
  6. Kimwipes(Thermo Fisher Scientific,Fisher Scientific,目录号:06-666A)
  7. 1大鼠( Rattus rattus; 100-300g)
  8. 异氟烷(CDMV,目录号:19417)
  9. 100%氧
  10. 290mOsm/kg对照溶液(Clinitrol TM sup/290参考溶液)(Advanced Instruments,目录号:3MA029)

设备

  1. 镊子(Dumont#7b镊子)(Fine Science Tools,目录号:11270-20)
  2. 兽用气体麻醉机(Dispomed,型号:975-3300-000MRI)
  3. 感应室(VetEquip,型号:941444)
  4. 1000μl移液管(Gilson,Pipetman TM ,型号:F123602)
  5. Nosecone连接到Bain电路(VetEquip,型号:921463)
  6. 离心机(Spectrafuge 24D)(Labnet International,型号:C2400)
  7. 涡旋(Vortex Genie 2)(Scientific Industries,型号:SI-0236)
  8. 凝固点渗透压计(Advanced Instruments,型号:3320 Micro-Osmometer)
  9. Ease-Eject 20-Microliter sampler(Advanced Instruments,型号:3M0825)
  10. 采样柱塞丝(Advanced Instruments,目录号:3M0828)
  11. 取样器吸头和室内清洁器??(Advanced Instruments,型号:3MA800)

程序

  1. 心脏穿刺
    本节介绍准备测量血清渗透压浓度时采集和处理血液样本的心脏穿刺方法(参见注释A1)。
    1. 深层麻醉将需要大约10分钟。将大鼠放入诱导室。将氧气流速调节至1.5-2L/min,将异氟烷蒸发器调节至5%。
    2. 一旦大鼠已经停止移动几分钟(2-4分钟),它可以转移到工作表面上并且放置在附接到Bain回路的面罩上。
    3. 将大鼠平行于自己在仰卧位置,头朝向你的非优势手,并尾部对你的优势手。将氧气流速调节至0.4-0.8 L/min,并在3-5%异氟烷下继续
    4. 使用镊子夹住脚趾,确认其深度麻醉。大鼠不应作出反应。保持异氟烷3-5%(见注A2)。
    5. 测试5毫升管上的柱塞,确保它平稳地移动。使柱塞略微缩回。安装21 G 1?英寸针。
    6. 使用非惯用手,轻轻地将前肢向下压在桌子上,并向内进入身体,使爪子朝向它的后腿,前肢平??行于胸部。这种"挤压"前肢进入身体将抬起肋骨,提供更容易接近心脏
    7. 用优势手,将针头以30°角(从胸部向上)插入剑突过程的解剖左侧,并缓慢推入胸部,同时用活塞保持轻微的负压(见注A3)。当你到达心脏时,你可能会感到抵抗,但这并不总是这样
    8. 一旦你到达了心脏,少量的血液将进入注射器。释放负压。放开前肢,用你的非优势手稳定注射器。用你的惯用手慢慢地拉回注射器。
    9. 收集所需量的血液后(参见注释A4),通过推入柱塞轻轻地施加小的正压力,然后将针头拉出。一点正压将防止针头缩回期间溶血(见注释A5)。
    10. 立即将注射器中的血液转移到1.5ml微量离心管中。为此,从注射器中取出针头,轻轻地将血液排入试管。血液将开始凝结,如果这个步骤做得太快,它将引起溶血。
    11. 在室温(RT,?22℃)下放置30-60分钟,或将管置于隔热泡沫容器中的冰上,盖子关闭至少30分钟和至多9小时(见注A6和代表数据)。
    12. 按照您机构的动物使用指南,按照推荐的物理安乐死方法(例如,气胸,颈椎脱臼)。

  2. 血清分离
    本节介绍准备血清样品进行渗透压测量所需的血液处理
    1. 将血样以6000×g离心5分钟。所得的深和深红色沉淀将含有血细胞。透明的,灰白色至黄色的上清液层将含有血清。 1.5ml血液提供约0.5-0.7ml血清(见注释B1和B2)。
    2. 吸取上清液到新鲜的微量离心管中。当收集血清时,不要打扰沉淀。
    3. 储存在4°C,直到准备进一步处理,确保盖子完全关闭,以防止蒸发
  3. 渗透压测量
    本节首先通过测量已知摩尔渗透压浓度值(290 mOsm/kg对照溶液)的溶液,然后通过测量血清渗透压浓度的方法来验证渗透压计的可靠性。
    1. 确保您的渗透压力计最近已按照制造商的指南进行校准,并且运行状况良好。
    2. 这是测试渗透压力计的质量控制步骤(见注释C1和C2)。
      1. 将取样器端头连接到取样器。使用取样器绘制20微升290 mOsm/kg对照溶液。
      2. 用kimwipe擦拭采样器尖端的外部,包括尖端周边。多余的样品,使弯月面与尖端对齐或略凹(见注释C3)
      3. 将采样器插入冷却室。测量将需要大约1分钟。
      4. 当测量完成后,拉出取样器,取出并丢弃取样器尖端(见注C4)。
      5. 用kimwipe干燥取样柱塞线。
      6. 按照制造商的说明
        用腔室清洁剂清洁冷却室
      7. 重复步骤C2 a-f两次以上。
    3. 检查血清样品底部的残留物。如果存在残留物,将样品再次以6000xg离心2.5分钟,并将血清移至新鲜的微量离心管中。
    4. 涡旋样品5秒。
    5. 使用步骤C2中描述的相同技术测量血清渗透压,在每次测量之间短暂涡旋(?3秒)血清。(见注释C1和C2)。
      1. 将取样器端头连接到取样器。使用取样器绘制20μl血清样品。
      2. 用kimwipe擦拭采样器尖端的外部,包括尖端周边。多余的样品,使弯月面与尖端对齐或略凹(见注释C3)
      3. 将采样器插入冷却室。测量将需要大约1分钟。
      4. 测量完成后,拉出取样器,取出并丢弃取样器尖端。
      5. 用kimwipe干燥取样柱塞线。
      6. 按照制造商的说明用腔室清洁剂清洁冷却室。
      7. 重复步骤C5 a-f两次(见注释C5)。
    6. 当所有测量完成后,按照制造商的指南清洁冷却室。
    7. 报告最终值作为所有血清渗透压浓度测量的平均值。

笔记

  1. 心脏穿刺
    1. 任何血液收集方法足以用于测量血清渗透压浓度的目的。我们概述了原始文章(Stare等人。,2015)中所述的心脏穿刺方法。
    2. 由于心脏穿刺是终末放血过程,因此不需要保持体热。在整个过程中异氟醚可以保持在最大水平(5%)。
    3. 根据大鼠的大小,需要插入大约40-70%的针。如果针完全插入并且在注射器中没有血液的指示,则很可能心脏已经丢失。在这种情况下,请取出针头,不要将其从胸腔中取出,然后重试。如果再次尝试不成功,请更换新针。有时尝试不成功,因为针被阻塞。
    4. 100-300g大鼠将提供> 5毫升的血液,但只需要?0.15毫升的血液产生最小量的血清(3×20微升,见注释C1)渗透压测量所必需的。该协议描述了1.5ml血液的收集,因为它是我们通常为我们自己的实验收集的量(Stare等人。,2015)。
    5. 溶血是红细胞的破裂,并且可以通过从黄色/白色到红色的颜色变化在血清中目测检测。溶血可以改变血清的组成,从而潜在地改变血清渗量浓度测量(Lippi等人,2012)。
    6. 血清和血浆之间的差异是血清不含凝血剂。因此,重要的是允许血液在离心样品之前完全凝结。在全血样品离心之前的存储时间和温度推荐是变化的,并且是今天讨论的(Abbadi等人,2014; Curia等人,2009; Seifarth然而,我们通常将样品置于隔热泡沫容器中的冰上,盖子关闭长达9小时,没有任何不良影响(见代表数据)。一旦分离血清,可以在4℃下储存长达24至48小时(参见代表性数据; Curia等人,2009)。

  2. 血清分离
    1. 如果血清层含有眼睛可见的颗粒,则以6,000×g离心样品2-5分钟,并将血清收集到新的微量离心管中。
    2. 偶尔在血液离心后血清层中出现增稠的灰白色物质。有两个选择要考虑:或者在6,000英寸xg 重新离心2.5-5分钟,或者尽可能收集周围的血清。

  3. 渗透压测量
    1. 列出的渗透压计每个测量使用20μl,分辨率为±1 mOsm/kg。然而,我们保留至少200μl的血清样品。
    2. 在取样之间保持微量离心管的盖子关闭,以防止蒸发
    3. 当使用取样器时,确保严格遵循制造商的指南,以获得可靠和可测量的20μl样品。这将包括确保样品中没有气泡,并保持一致的弯月面水平。一致性非常重要,因为这些细微的差异可能导致与可能模拟渗透计或取样器柱塞丝劣化的校准问题的测量不一致。
    4. 如果对照溶液的测量值超出标准的规定值,请重新校准渗透计
    5. 如果测量的血清渗透压值每次测试一致地变化超过3mOsm/kg,根据制造商的指南用酒精清洁冷冻室。如果值仍然高度可变,请按照制造商的指南更换进样器中的柱塞线。

代表数据

我们调查不同的凝血时间和温度对立即和冷藏一天后测定的血清渗透压浓度测量的影响。我们发现,对于在室温(RT,?22℃; 30分钟和60分钟)或冰(?0℃; 30分钟,60分钟,90分钟,和9小时;图2A和2B)。我们还发现血清样品在4℃下稳定一天(图2C)。

  1. 实验设计
    使七只成年雄性Westar大鼠进行上述心脏穿刺程序。将收集的血液分成六个1.5ml微量离心管,并在室温或冰上在隔热泡沫容器中凝结,盖子关闭不同的时间。如上所述分离每个血清样品,并等分到2个微量离心管中:一个管用于立即重量摩尔渗透压浓度测量(时间点'0h',另一个管在4℃保持一天(时间点'24h报告的值是每个样品3-7个测量的平均值,具有±SEM,和指定的标准偏差(SD)。使用SigmaPlot 12和Prism 6进行统计分析和绘图,以通过单因素方差分析(One Way ANOVA) > t -test。 P 值<0.05被认为是重要的。
  2. 结果

    表1.用于绘制标准曲线的示例数据



    图1.用于测试本研究中使用的渗透压计的校准和性能的标准曲线

    表2.样本数据



    图2.储存温度和持续时间对血清渗透压的影响 A.血液样品(每种n = 7)的血清渗透压值允许在室温下凝结30分钟至9小时?22℃)或在冰上(?0℃)。在类别之间没有统计学差异(单因素方差分析,单位方差分析(5,36)= 0.472,P = 0.795); B.渗透压值归一化到RT-30min; C.一天冷藏(4℃)对血清渗透压值的影响。在立即测量的样品与在4℃下储存一天(0小时)的样品之间没有统计学差异(配对t检验,P = 0.241) ,303.72±0.44mOsm/kg,n = 26; 24小时,303.146±0.60mOsm/kg,n = 26)。

致谢

该协议是出现在Stare等人中的材料和方法 - 血清渗透压协议的扩展版本。 (2015)。它已经改编自McGill标准操作程序#111-大鼠麻醉,#301-啮齿动物安乐死和#403-血液收集量和频率指南,并且由McGill大学的设施动物护理委员会批准。步骤C2和C5基于Advanced Instruments,Inc。提供的Ease-Eject ?取样器(20μl)用户指南。该工作由加拿大健康研究所(CIHR)Foundation Grant FDN -143337和James McGill研究主席CWB,CIHR Frederick Banting和Charles最佳加拿大研究生奖学金 - 博士学位奖魁北克研究基金 - 健康支持麦吉尔大学健康中心的研究所。作者感谢M. Prager-Khoutorsky博士和M. Verway博士对这份手稿提出建设性意见。

参考文献

  1. Abbadi,A.,El-Khoury,JM和Wang,S.(2014)。 
  2. Bourque,CW(2008)。  中枢机制的渗透和systemic osmogulation。 Nat Rev Neurosci 9(7):519-531。
  3. Curria,A.,Pesta,M.,Garry,E.,Zampa,N.,Rosenman,J。和Sacks,J。(2009)。  Hyponatremia:SIADH。 In:Loriaux L.(Ed。)。 内分泌紧急情况:识别和治疗。 Springer,74:115-126。
  4. Lippi,G.,Cervellin,G.,Favaloro,EJ和Plebani,M.(2012)。  体外溶血对实验室测试的影响。和Plebani,M。(Eds。)。 体外和体内溶血:实验室医学中未解决的争议:39-44。
  5. Lupsa BC and Inzucchi SE(2013)。  糖尿病酮症酸中毒和高渗性高血糖综合征。 In:Loriaux L.(Ed。)。 内分泌紧急情况:识别和治疗。 Springer,115-126。
  6. Reddi AS(2013)。  水平衡障碍:高钠血症。 In:Reddi AS(Ed。)。 流体,电解质和酸碱失调。 Springer,133-150
  7. Seifarth,CC,Miertschischk,J.,Hahn,EG和Hensen,J.(2004)。  测量健康年轻人中血清和血浆渗透压 - 时间和储存条件的影响。临床化学实验室Med(临床化学实验室)42(8):927- 932。
  8. Stare,J.,Siami,S.,Trudel,E.,Prager-Khoutorsky,M.,Sharshar,T.和Bourque,CW(2015)。  腹膜脓毒症对大鼠中枢神经调节神经元介导口渴和加压素释放的影响 J Neurosci 35(35):12188-12197。

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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: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Stare, J. and Bourque, C. W. (2016). Measuring Rat Serum Osmolality by Freezing Point Osmometry. Bio-protocol 6(19): e1950. DOI: 10.21769/BioProtoc.1950.
  2. Stare, J., Siami, S., Trudel, E., Prager-Khoutorsky, M., Sharshar, T. and Bourque, C. W. (2015). Effects of peritoneal sepsis on rat central osmoregulatory neurons mediating thirst and vasopressin release. J Neurosci 35(35): 12188-12197.

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