Method Article

Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord

DOI:

10.3791/59084

⸱

February 26th, 2019

 ,  ,  ,  ,  ,  , 
1Department of Neuroanatomy and Molecular Brain Research, Ruhr University Bochum, 2Central Animal Facility of the Medical Faculty, Ruhr University Bochum

* These authors contributed equally

In This Article

Summary

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The combination of transmission electron microscopy and in utero transduction is a powerful approach for studying morphological changes in the fine ultrastructure of the nervous system during development. This combined method allows deep insights into changes in structural details underlying neuroplasticity with respect to their topographical representation.

Abstract

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The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ultrastructural parameters in unambiguously identified topographical structures, affected by a protein of interest that is introduced into the organism via viral transfer. This combined approach allows for a smooth transition from macrostructural to ultrastructural identification by following topographical navigation maps in a tissue atlas. High-resolution electron microscopy of the in-utero-transduced tissue reveals the fine ultrastructure of the neuropil and its plasticity parameters, such as cross-sectioned synaptic bouton areas, the number of synaptic vesicles and mitochondria within a bouton profile, the length of synaptic contacts, cross-sectioned axonal areas, the thickness of myelin sheaths, the number of myelin lamellae, and cross-sectioned areas of mitochondria profiles. The analysis of these parameters reveals essential insights into changes of ultrastructural plasticity in the areas of the nervous system that are affected by the viral transfer of the genetic construct. This combined method can not only be used for studying the direct effect of genetically engineered biomolecules and/or drugs on neuronal plasticity but also opens the possibility to study the in utero rescue of neuronal plasticity (e.g., in the context of neurodegenerative diseases).

Introduction

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No photon can penetrate an ultrathin tissue specimen in the depth grade of an electron. This attributes invaluable advantages to TEM in capturing nanometer resolution images of fine structures when compared to light microscopy techniques. For example, TEM allows for the visualization of intracellular organelles such as mitochondria, melanosomes, and various types of secretory granules, microtubules, microfilaments, cilia, microvilli, and intercellular junctions (cell surface specializations), in particular synapses in the nervous system1,2,3,4. The overall goal of the present methodological study is the ultrastructural recognition of changes in neural plasticity during development upon prenatal interference by combining the state-of-the-art techniques of in utero transduction and TEM. Virally encoded proteins of interest have been transduced in utero into the central nervous system5,6,7, including the spinal cord6. For instance, in utero transduction in combination with TEM has been used for studying the effect of the cell adhesion molecule L1 on motor learning plasticity in L1-deficient mice, in particular with regard to the interplay between L1 and nuclear receptor proteins in cerebellar neurons7.

The analysis of neuroplasticity parameters requires precise information about the localization of the smallest areas within the nervous system. Therefore, it is adequate to describe ultrastructural details and their exact topographical orientation with respect to other structures. In the present study, a specific preparatory method aiming at the detailed investigation of distinct morphological areas based on both light and electron microscopy is presented. This approach combines several techniques of tissue manipulation, starting with in utero transduction of the mouse brain and spinal cord and followed by perfusion fixation, mold-embedding, and processing the tissue for TEM. An essential step included between the embedding and the processing of the tissue for TEM is the documentation of the tissue, using the interference light reflection technique that allows for the precise microphotographic and low-magnification documentation of tissue specimens8,9,10. Incorporated into the present approach, this technique enables researchers to examine topographical and structural details of nervous tissue surfaces and of specimen slice profiles prior to their preparation for TEM.

A special frame for sectioning whole brains corresponds to stereotaxic coordinates. This frame benefits the morphological three-dimensional (3D) reconstruction of areas in nervous tissue and can be used for morphometric analysis. The macrographs of the visualized sections are assigned topographical coordinates, and the serially numbered sections build maps in a tissue atlas.

After resin processing, the embedded tissue is sectioned into ultrathin sections (<70 nm) containing selected areas, according to the maps of the above-mentioned tissue atlas. The ultrathin sections are subjected to TEM to obtain high-resolution images of plasticity parameters (e.g., cross-section profile areas of synaptic boutons or axonal fibers) of their contents and of contacts to neighboring structures within the complex neuropil.

With the method described herein, the smooth transition from visualized macrostructures to micro- and nanostructures permits comparative in-depth studies of morphological neuronal plasticity after in utero transduction of the developing nervous system.

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Protocol

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All procedures on animal subjects have been approved by the institutional animal ethics committees of the federal states of Hamburg and Nordrhein-Westfalen, Germany.  Use sterile instruments, protective gloves and aseptic coats throughout the entire surgical procedure.

1. In Utero Transduction

  1. Prepare adeno-associated virus type 1 (AAV1) coding for the desired target (4 x 1011 viral particles/µL of AAV1) in phosphate-buffered saline (PBS) at pH 7.4. Add 0.1 mg/µL Fast Green and keep the AAV1-Fast-Green mixture at 37 °C.
  2. Prepare a thin capillary tip with the desired shape (8 mm in length, with an outside diameter of 80 µm and an inside diameter of 50 µm), using a micropipette puller (settings: pressure = 500, heat = 700, pull = 0, velocity = 80, time = 200, see the Table of Materials). Break the tip of the capillary so that it is 4-5 mm.
  3. Assemble an aspirator tube (44 cm x 0.7 cm) with the capillary tip and aspirate 15 µL of the AAV1-Fast-Green mixture into the capillary.
  4. Keep the animal subjects at a constant physiological body temperature of 37 °C throughout the entire procedure.
  5. Place a pregnant C57Bl/6 mouse (embryonic day 14.5) into the preincubation chamber and anesthetize the mouse with gaseous 4% isoflurane (with a volumetric airflow rate of 0.6-0.8 L/min).
  6. Subcutaneously, inject buprenorphine (0.1 mg/kg of body weight).
  7. Place the anesthetized mouse on the prewarmed surgical plate (37 °C).
  8. Cover the eyes with a lubricant.
  9. Fit the mouse with the anesthesia mask (gaseous 1.5 % isoflurane at a volumetric airflow rate of 0.6-0.8 L/min)on the surgical plate and shave the abdominal skin region. Wipe the shaved region with 3X 75% ethanol and then with betadine solution.
    NOTE: Monitor the breathing behavior of the anesthetized mouse continuously. Adjust the concentration of the isoflurane gas according to the inhalation-exhalation pattern of the mouse.
  10. Check for the absence of the plantar reflex by squeezing the hind paw phalanges of the mouse.
  11. Open the abdominal cavity by gripping the skin with curved serrated iris forceps (10 cm) and cutting the skin along the linea mediana with straight tungsten carbide scissors (10 cm), and then, by gripping the peritoneal wall with straight Dumont tweezers (12 cm, 0.2 mm x 0.12 mm) and cutting the wall along the linea alba with straight Vanna's scissors (8 cm).
  12. Place a piece of fenestrated paraffin film on the abdominal opening and fix the film on both ends with micro-mosquito hemostatic forceps (12.5 cm, curved).
  13. Expose the uterine horns with a spoon-like device to avoid damage to the embryos inside the uterine horns. Drip a few drops of PBS (37 °C) on the uterine horns and inspect the embryos for damages or malformations inside the uterine sac.
  14. Document the order and position of the embryos in the uterine horns. Turn the embryos carefully inside the uterine sac until the desired position for injection is reached.
  15. Inject 1-2 µL of the AAV1-Fast-Green mixture by visually inspecting the injection site (e.g., brain ventricles) and the dye penetration under a stereomicroscope.
  16. Document the injected embryos and place the uterine horns with the injected embryos back into the abdominal cavity.
  17. Drip a few drops of PBS (37 °C) into the abdominal cavity. Close the cavity by suturing the peritoneal wall (use polyamide 6-0-sized sutures) and the skin (use polyamide 3-0-sized sutures), using Halsted's mosquito hemostatic forceps (12.5 cm, curved). Alternatively, use a simple interrupted pattern or stainless-steel suture/staples to close the abdominal cavity and the skin.

2. Telemacrophotography of Isolated Tissues

  1. Preparation of buffers
    1. Prepare Sörensen's buffer (1 L) by dissolving 14.95 g of Na2HPO4 and 2.18 g of KH2PO4 in 1 L of distilled water under stirring at 200 rpm. For perfusion of late gestation pups and/or pups in the post-natal time periods, use an appropriate size of needles for abdominal or intragastric injection and make sure that the concentration of the terminal sodium pentobarbital anesthesia does not exceed 180 mg/kg of body weigh.
    2. Prepare Mugnaini's fixation solution (5 L) by heating 500 mL of distilled water to 75 °C and adding 50 g of paraformaldehyde powder under stirring at 200 rpm, adding 200 µL of 5 N NaOH, adding 1,500 mL of Sörensen's buffer, 1,750 mL of distilled water, and 500 mL of 25% glutaraldehyde. Fill up to 5,000 mL with distilled water. Use this final buffer for perfusion.
      NOTE: Prepare Mugnaini's fixation solution under the hood, wear protective glasses, and avoid fumes. Add methylene blue (0.05 g/L) for a better visualization of the perfusion.
  2. Mouse perfusion and tissue isolation
    1. Transcardially perfuse the pregnant mice that carry the transduced embryos (in the case of embryo studies) or the born transduced pubs at the desired age (e.g., postnatal day 24) according to standard procedures6,7,11,12,13,14,15,16,17, using intraperitoneal terminal sodium pentobarbital anesthesia (200 mg/kg of body weight).
    2. Inject the mice transcardially with heparin solution (500 U) using a 26 G, 1 in needle and, before fixation, infuse the mice transcardially with 10 mL of PBS to flush out the blood from the body, and perfuse them transcardially with 30 mL of 40 °C prewarmed Mugnaini's fixation solution.
      NOTE: For adult mice, perform an alternative retrograde perfusion via the abdominal aorta14.
    3. Isolate the perfused tissue of interest (e.g., whole brain or spinal cord) and postfix the tissue in at least 10 mL of Mugnaini's fixation solution for another 24 h at 4 °C.
    4. Wash the tissue in 10 mL of PBS for 3 h at room temperature.
  3. Embedding in agarose, plus documentation and sectioning
    1. Adjust the isolated tissue (e.g., whole brain) in a special frame with a reproducible sectioning angle8,9,10. Alternatively, use a vibratome with an adjustable cutting thickness.
    2. Place the nervous tissue in the frame, adjust the tissue for telemacrography, and document the coordinates.
    3. Prepare 3% low-melting agarose-embedding medium: add 3 g of agarose in 100 mL of Sörensen's buffer and heat the mixture in a water bath to 90 °C.
    4. Pour 3% agarose (30 °C) in the frame that contains the tissue. Cover the frame with a warm metal block and wait until it is hardening. During hardening, use telemacrographic devices to image the embedded tissue and its coordinates within the frame.
    5. Transfer the agarose-embedded tissue into a frame with cutting gaps corresponding to the coordinates of the first frame.
    6. Cut the embedded tissue into sections of desired thickness (e.g., 1.5 mm) with a device with a thin and vibrating razor blade (see Table of Materials).
      NOTE: To improve the gliding of the razor blade, drip a few drops of glycerin onto the embedded tissue.
    7. Image each tissue section in PBS and collect the images into a folder.

3. Preparation of the Isolated Tissue for Transmission Electron Microscopy

NOTE: Perform all further steps of incubation in glass dishes with tightly closable lids on a shaking platform under the hood.

  1. Wash the tissue sections for 2x 30 min in PBS. Incubate the sections in 2% aqueous osmium tetroxide solution (OsO4) for 2 h at room temperature.
    CAUTION: Osmium tetroxide is toxic and may be harmful when it comes in contact with skin.
  2. Wash the osmicated sections for 2x 30 min in PBS.
  3. Incubate the sections in 30%, 50%, and 70% ethanol at room temperature for 10-15 min (optional: incubate in 70% ethanol at 4 °C overnight).
  4. Image the osmicated specimens in 70% ethanol under LED RGB light8,9,10 (2x 15 W) applied to the sample from the left and right side at an angle of 45°. Use black dishes and a dull black background to minimize scattering and the reflection of the light during illumination.
    CAUTION: Do not allow the section to dry out during imaging.
  5. Create an atlas of the section images with coordinates by collecting images in series in a folder.
  6. Incubate the specimens in 100% ethanol (2x for 30 min) and 100% propylene oxide (2x for 30 min) at room temperature.
    CAUTION: Do not allow the sections to dry out while changing solutions.
  7. Mix 260 mL of resin with 240 mL of dodecenylsuccinic anhydride in a glass vessel while gently stirring with a glass bar. Periodically check for inhomogeneity, bubbles, and smears. Very gently, stir by hand for at least 45 min.
  8. Prepare resin/propylene oxide in a ratio of 1:2 and 1:1 and add 3% accelerator (2,4,6-Tris(dimethylaminomethyl)phenol).
  9. Incubate the tissue in the 1:2 embedding solution from step 3.8 for 2 h and then in the 1:1 embedding solution from step 3.8 for 2 h at room temperature on a rotating wheel.
  10. Place the tissue in flat polypropylene dishes, cover the tissue with fresh resin containing 3% accelerator, and cure the embedded tissue at 65-85 °C for 12-24 h.
  11. Cool down the embedded tissue to room temperature and remove the resin-embedded specimens from the polypropylene dishes.

4. Selection of Ultrastructural Neuroplasticity Parameters for Quantitative Analysis

  1. Mapping the area of interest
    1. Choose an area of interest (e.g., hippocampus or cerebellum) and localize the area in the section atlas by choosing the image from the atlas (step 3.5) that contains this area.
    2. Sketch the borders of the area of interest onto the section image and find/superimpose these region borders onto the resin specimen.
    3. Scratch-mark the borders of the area of interest (e.g., hippocampus or cerebellum) on the resin specimen, using a fine needle gauge (26 G, 1 in).
    4. Heat the resin specimen to 85 °C in an oven to soften the resin for trimming or, alternatively, use a trimming device, a thin blade, or sandpaper.
    5. Excise the area of interest from the resin specimen with a razor blade (see Table of Materials). Mount the specimen on holding bars of acrylic glass of the required caliber (e.g., with a diameter of 8 mm and a length of 1 cm) with glue. Trim the mounted specimen for semi- and ultrathin sectioning.
    6. Prepare semithin (0.75 µm) and ultrathin (70 nm) sections of the trimmed area using an ultramicrotome: set it at 1.5 mm/s for 0.75 µm thickness and at 0.7 mm/s for 70 nm thickness.
    7. Collect the semithin sections on glass carriers and stain the sections with 1% toluidine blue in PBS (for 4 min).
    8. Wash the sections several times in deionized water. Examine the stained sections under the light microscope using 4x (NA of 0.1 ∞/-), 10x (NA of 0.22 ∞/0.17), 40x (NA of 0.65 ∞/0.17), and 100x (NA of 1.25 ∞/0.17) objectives.
    9. Collect ultrathin sections on nickel grids. Subject the grids to TEM at 180 kV and at 3,200x, 6,000x, and/or 8,000x magnification.
  2. TEM Analysis
    1. Choose the ultrastructural parameters of interest for quantitative TEM analysis (e.g., boutons with vesicles and mitochondria or myelinated and nonmyelinated axons) and take TEM images of these parameters under 3,500x, 6,000x and/or 8,000x magnification.

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Results

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For reliable and fast anesthesia of mice, numerous safety parameters were considered, and an optimized workspace of the anesthesia unit proved to be adequate (Figure 1A). The unit is designed to control the mixture of liquid isoflurane and ambient air with a precision required for successful surgery on small animals, such as mice and rats. Air and isoflurane are mixed in the vaporizer according to the desired settings and delivered into a box...

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Discussion

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A crucial step of in utero transduction is the injection procedure. The precise injection into brain ventricles or into another area of interest requires experience and hands-on skill. The thinner the microcapillary tip, the less tissue damage may occur; however, this is at the cost of increasing injection pressure. In contrast to in utero electroporation19,20,21,22, the survival rate of the in...

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Disclosures

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The authors have nothing to disclose.

Acknowledgements

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The authors thank the colleagues of the animal facility at the Medical Faculty, Ruhr-University Bochum, for their support and animal care.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
2,4,6-Tris(dimethyl-aminomethyl)phenolServa36975
26 G x 1'' needleHenke-Sass, Wolf GmbH
410 Anaesthesia Unit for air pumpBiomedical Instruments (Univentor)8323102
Adeno-associated virus serotype 1 (AAV1)UKE (Viral Core Facility)-For references and target areas of AAV1 see: https://www.addgene.org/viral-vectors/aav/aav-guide/ and also: Designer gene delivery vectors: molecular engineering and evolution of adeno-associated viral vectors for enhanced gene transfer. Kwon I, Schaffer DV. Pharm Res. 2008 Mar;25(3):489-99. Recombinant AAV viral vectors pseudotyped with viral capsids from serotypes 1, 2, and 5 display differential efficiency and cell tropism after delivery to different regions of the central nervous system. Burger C, Gorbatyuk OS, Velardo MJ, Peden CS, Williams P, Zolotukhin S, Reier PJ, Mandel RJ, Muzyczka N. Mol. Ther. 2004 Aug;10(2):302-17. Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis. McCarty DM, Monahan PE, Samulski RJ. Gene Ther. 2001 Aug;8(16):1248-54.
AgaroseSigma-AldrichA9414low gelling agarose
Air PumpBiomedical Instruments (Univentor)Eheim 100
AralditeCIBA-GEIGY23857.9resin for embedding of tissue
aspirator tune assembliesSigma-AldrichA5177-5EA
Breathing Mask Mouse Anodized AluminiumBiomedical Instruments (Univentor)-
buprenorphineTemgesicampulespainkiller
capillariesScience-ProductsGB100TF-10with fillament
Dodecenylsuccinic anhydrideFluka44160
Dumont tweezers (#3, 12 cm, straight, 0.2 x 0.12 mm)FST11203-23
electric shaverPhillips-
Ethicon sutures (Ethilon, 6-0 and 3-0)Ethicon-polyamide
eye lubricantBepanthene-
Fast GreenSigma-AldrichF7252for visualization of injected liquids
Gas Routing Switch 4/2 connectorsBiomedical Instruments (Univentor)8433020
halsted Mosquito hemostatic forceps (12.5 cm, straight)FST13011-12
Heparin-NatriumRatiopharm25 000 I.E./5 mL
Induction box for mice
with horizontally moving lid.
Inner dimensions: LxBxH: 155 mm x 115 mm x 130 mm.
Wall thickness: 6 mm		Biomedical Instruments (Univentor)-
iris forceps (10 cm, curved, serrated)FST14007-14
iris scissors (11 cm, straight, tungsten carbide)FST14501-14
Isofluran OP Tisch, electrically heated, sm
Outer dimensions: 257mm x110 mm x 18 mm.
Heating area: 190 mm x 90 mm
The removal of the isoflurane escaping
the breathing mask is downwards in compliance with the
regulations		Biomedical Instruments (Univentor)-
isoflurane (Attane)JD medicalinhalation anesthesia
LED RGB lightsCameoCLQS15RGBWLEDs 2 x 15 W
Light microscope Basic DM ELeica-4x (N.A. 0.1 ∞/-), 10x (N.A. 0.22 ∞/0.17), 40x (N.A. 0.65 ∞/0.17), 100x (N.A. 1.25 ∞/0.17) objectives
micropipette pullerScience-ProductsP-97
Mosquito hemostatic forceps (12.5 cm, curved)FST13010-12
Nickel grids, 200 meshTed Pella1GC200
Osmium (VIII)-oxidDegussa73219
Propylene oxideFluka82320
razor bladesSchick87-10489
Sodium pentobarbital (Narcoren)Merial GmbH-
TC01mR 1-Channal temperature controller with feedbackBiomedical Instruments (Univentor)-
Technovit 4004 two components glueKulzer
TelemacrodeviceCanon-Canon Spiegelreflex Kamera EOS2000D, EF-S 18-55 mm f/3.5-5.6 IS STM Objective, Extension below 150 mm, Manual Extension Tube 7 mm ring, 14 mm ring, 28 mm ring, Macro reverse ring (58 mm), Canon copy stand.
Thermopuller P-97Sutter Instruments-
thin vibrating razor blade deviceKrup-with Szabo thin blades
toluidine blueSigma-Aldrich89640
Transmission electron microscope C20Phillips-up to 200 kV
Tygon 6/4 Tubing material for connection of all parts
Outer diameter: 6 mm
Inner diameter: 4 mm
Wa
ll thickness: 1 mm		Biomedical Instruments (Univentor)-
Ultracut EReichert-Jung-ultramicrotome
Univentor ScavengerBiomedical Instruments (Univentor)8338001
Vannas scissors (8 cm, straight)FST15009-08

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Transmission Electron MicroscopySynaptic Bouton AnalysisMyelin Sheath ThicknessTissue Atlas NavigationOsmium Tetroxide StainingUltramicrotome SectioningAdeno Associated Virus InjectionEmbryonic Mouse Brain

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