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Characterization of Hippocampal Adult-borne Granule Cells in a Transient Cerebral Ischemia Model

海马成体新生颗粒细胞在短暂性脑缺血模型中的特征

MC Mihai Ceanga
MG Madlen Guenther
II Ina Ingrisch
AK Albrecht Kunze

发布: 2021年01月20日第11卷第2期 DOI: 10.21769/BioProtoc.3890 浏览次数: 3587

评审: Qihui WuAnthony FlamierAnonymous reviewer(s)

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Abstract

Long-term consequences of stroke significantly impair the quality of life in a growing population of stroke survivors. Hippocampal adult neurogenesis has been hypothesized to play a role in the pathophysiology of cognitive and neuropsychiatric long-term sequelae of stroke. Reliable animal models of stroke are paramount to understanding their biomechanisms and to advancing therapeutic strategies. We present a detailed protocol of a transient cerebral ischemia model which does not cause direct ischemic damage in the hippocampus, allowing investigations into the pathophysiology of long-term neurocognitive deficits of stroke. Furthermore, we describe a protocol for obtaining acute hippocampal slices for the purpose of electrophysiological and morphological characterization of adult-borne granule cells. Particularities relating to performing electrophysiological recordings from small cells, such as immature adult-borne granule cells, are also discussed. The present protocol may be complemented by multi-modal investigations (behavioral, morpho-structural, biochemical), to hopefully facilitate research and advances into the long-term sequelae of stroke and the discovery of new therapeutic opportunities.

Keywords: Stroke (中风) search MCAO (大脑中动脉闭塞) search Adult-borne granule cells (成体新生颗粒细胞) search Acute hippocampal slices (急性海马脑片) search Whole cell patch clamp (全细胞膜片钳) search

Background

Stroke is a major cause of mortality and morbidity in the developed world causing both acute as well as delayed deficits. While intervention strategies targeting restoration of blood flow in ischemic stroke have become more efficient at reducing acute morbidity and mortality, long-term consequences of stroke such as post-stroke depression and post-stroke cognitive impairment and dementia presently evade clinical therapy (Wang et al., 2010; Loubinoux et al., 2012; Mijajlovic et al., 2017). The pathophysiology of these delayed neurocognitive deficits is insufficiently understood. Adult-borne granule cells have been hypothesized to play a role in cognitive and neuropsychiatric sequelae of stroke, however, research in this domain has trailed investigations of other diseases involving adult neurogenesis, such as epilepsy (Jakubs et al., 2006; Jessberger et al., 2007). Several animal models of brain ischemia exist, ranging from reversible global ischemia models to permanent focal ischemia models or even more localized cortical infarcts induced by photothrombosis (Sicard and Fisher, 2009). However, global ischemia models may directly affect the viability of (adult-borne) hippocampal granule cells, thus inducing confounding factors regarding the pathophysiology of neurocognitive deficits, while focal infarcts may cause less robust secondary changes in the brain. Indeed, previous research found that the infarct size correlates with the degree of morphological changes observed in adult-borne granule cells, with transient middle cerebral artery occlusion (MCAO) models having a more profound influence than cortical infarcts (e.g., by photothrombosis, Niv et al., 2012). We therefore present a flexible MCAO model which can reliably induce an extensive, cortico-subcortical ischemia pattern, but without direct ischemic damage of the hippocampus (Sicard and Fisher, 2009). The advantage of the present method is a scalable volume of infarction as a function of ischemia time (with larger infarct volumes inducing a more widespread perturbation in the process of adult neurogenesis), whereby a high percentage of animals survive to allow behavioral, electrophysiological, morphological and biochemical characterization at delayed time points after stroke. Adult borne granule cells can be fluorescently marked either using transgenic models (Couillard-Despres et al., 2006) or by using retroviral labeling techniques (Niv et al., 2012). Transgenic DCX/DsRed mice (Couillard-Despres et al., 2006) express the red fluorescent protein DsRed under the control of the doublecortin (DCX) promoter. DCX is a migration protein which is expressed in immature neurons at the time of synaptic integration and intrinsic maturation roughly up to the fourth week post-mitosis. Alternatively, retroviral labeling can be used to follow the maturation of dentate granule cells born at the moment of intrahippocampal virus injection (Niv et al., 2012).

Materials and Reagents

Middle cerebral artery occlusion

  1. Mini Vessel Clip (Aesculap Surgical Instruments, catalog number: FD562R )
  2. Silicone rubber-coated monofilament (Doccol, catalog number: 702056PK5Re )
  3. Silk suture size 7-0, 100 yards/spool (Teleflex, catalog number: 103-S )
  4. Silk suture size 3-0 with needle (Covidien, Sofsilk, catalog number: SS694 )
  5. Surgical tape (3M Transpore, 3M ID 70200739582 )
  6. Softasept N (Braun, catalog number: 3887138 )
  7. Cotton pads and cotton swabs

Acute hippocampal slice electrophysiology
  1. Petri dish
  2. 50 ml Falcon tube (e.g., Falcon, catalog number 10788561 )
  3. 15 ml Falcon tube (e.g., Falcon, catalog number 10773501 )
  4. Razor blades (e.g., Gillette silver blue)
  5. Air stone (Robu Glasfilter, catalog number 18105 )
  6. Filter paper (Sartorius, catalog number: FT-3-303-055 )
  7. Superglue (e.g., UHU, catalog number: 36016 )
  8. Syringe filters, 0.22 µm (Kinesis, catalog number: ESF-MC-13-022 )
  9. Thick walled borosilicate glass capillaries (Science products, catalog number: GB150F-8P )

Reagents

  1. NaCl (Carl Roth, catalog number: 9265.1 )

  2. KCl (Carl Roth, catalog number: 6781.1 )

  3. CaCl2 (Carl Roth, catalog number: A119.1 )

  4. MgCl2 (Carl Roth, catalog number: 2189.1 )

  5. MgSO4 (Carl Roth, catalog number: 0261.1)

  6. NaHCO3 (Carl Roth, catalog number: HN01.2 )

  7. NaH2PO4 (Carl Roth, catalog number: T879.2 )

  8. Glucose (Carl Roth, catalog number: HN06.3 )

  9. Sucrose (Carl Roth, catalog number: 9286.2 )

  10. Na-Ascorbate (Sigma-Aldrich, catalog number: A4034 )

  11. K-gluconate (Carl Roth, catalog number: 2621.2 )

  12. MgATP (Sigma-Aldrich, catalog number: A9187 )

  13. NaGTP (Sigma-Aldrich, catalog number: G8877 )

  14. EGTA (Sigma-Aldrich, catalog number: 03777)

  15. HEPES (Carl Roth, catalog number: HN77.2 )

  16. KOH (Carl Roth, catalog number: K017.1 )

  17. Biocytin (Tocris, catalog number: 3349 )

  18. Streptavidin-Cy3 (Sigma-Aldrich, catalog number: S6402-1ML )

  19. TBS (Thermo Fisher, catalog number: 28358 )

  20. Normal Donkey Serum (abcam, catalog number: ab7475 )

  21. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )

  22. 4% PFA (Sigma-Aldrich, catalog number: 1004965000 )

  23. Fluoromount (Sigma-Aldrich, catalog number: F4680 )

  24. Slicing solution (final) (see Recipes)

  25. Artificial cerebrospinal fluid (final) (see Recipes)

  26. Slicing solution 10x stock (see Recipes)

  27. aCSF 10x Stock, 500 ml (see Recipes)

  28. 2 M Glucose stock solution (see Recipes)

  29. 1 M CaCl2 stock solution (see Recipes)

  30. 1 M MgCl2 stock solution (see Recipes)

  31. Slicing solution (see Recipes)

  32. aCSF (see Recipes)

  33. Intracellular solution (see Recipes)

  34. TBS Plus (see Recipes)

Equipment

Middle cerebral artery occlusion

  1. Needle holder (Fine Science Tools, catalog number: 12010-14 )

  2. Blunt surgical scissors (Fine Science Tools, catalog number: 14003-14 )

  3. Standard forceps (Fine Science Tools, catalog number: 11000-13 )

  4. Curved standard forceps (Fine Science Tools, catalog number: 11001-12 )

  5. Hartman hemostat (Fine Science Tools, catalog number: 13002-10 )

  6. Delicate suture tying forceps (Fine Science Tools, catalog number: 11063-07 )

  7. Spring scissors (Fine Science Tools, catalog number: 15018-10 )

  8. Temperature controller (FHC, catalog number: 40-90-8D )

  9. Heating pad (FHC, catalog number: 40-90-2-02 )

  10. Mini Rectal Thermistor Probe (FHC, catalog number: 40-90-5D-02 )

  11. Stereomicroscope with cold-light source (Zeiss, Stemi 508, catalog number: 495009-0016-000 )


Acute hippocampal slice electrophysiology

  1. 200 ml beaker

  2. Laboratory timer

  3. Curved blunt surgical scissors (Fine Science Tools, catalog number: 14003-14 )

  4. Small scissors (Fine Science Tools, catalog number: 14184-09 )

  5. Adson forceps (Fine Science Tools, catalog number: 11006-12 )

  6. Small curved forceps (Fine Science Tools, catalog number: 11152-10 )

  7. Spatula (Fine Science Tools, catalog number: 10099-15 )

  8. Microloader microcapillaries (Eppendorf, catalog number: 5242956003 )

  9. Vibrating blade microtome (Leica VT1200 S, catalog number: 1491200S001 )

  10. Water bath (GFL Water Bath, catalog number: 1108 )

  11. Osmometer (Gonotec Osmomat 010)

  12. Peristaltic pump (Ismatec ISM830, catalog number: GZ-78016-02 )

  13. Upright electrophysiology microscope ( Zeiss Axio Examiner.Z1 )

  14. Patch clamp amplifier (Molecular Devices, catalog number: Axopatch 200B-2 )

  15. Digitizer (Molecular Devices, catalog number: Digidata 1550B4 )

  16. Micropipette puller (Sutter Instruments, P-97 Flaming/Brown, catalog number: P-97 )

  17. Constant current stimulation unit (Digitimer Ltd., DS3)

  18. (Optional) Commercial slice chamber (Science Products, Brain Slice Keeper1, catalog number: BSK1 )

Software

  1. ZEN 2.3 (blue edition) imaging software (ZEISS Germany)

  2. pCLAMP 10.7 (Molecular Devices, Axon Instruments)

Procedure

English
中文翻译

文章信息

版权信息

© 2021 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

  1. Ceanga, M., Guenther, M., Ingrisch, I. and Kunze, A. (2021). Characterization of Hippocampal Adult-borne Granule Cells in a Transient Cerebral Ischemia Model. Bio-protocol 11(2): e3890. DOI: 10.21769/BioProtoc.3890.
  2. Ceanga, M., Keiner, S., Grünewald, B., Haselmann, H., Frahm, C., Couillard-Després, S., Witte, O. W., Redecker, C., Geis, C. and Kunze, A. (2019). Stroke accelerates and uncouples intrinsic and synaptic excitability maturation of mouse Hippocampal DCX+ adult-born granule cells. J Neurosci 39(9): 1755-1766.

    3. ris ris Download Citation in RIS Format

分类

神经科学 > 神经系统疾病

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