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Jun 2017

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Studying the Role of Microglia in Neurodegeneration and Axonal Regeneration in the Murine Visual System
小胶质细胞在小鼠视觉系统神经退变和轴突再生中的作用研究   

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Abstract

Microglia reside in the central nervous system (CNS) and are involved in the maintenance of the physiologic state. They constantly survey their environment for pathologic alterations associated with injury or diseases. For decades, researchers have investigated the role of microglia under different pathologic conditions, using approaches aiming to inhibit or eliminate these phagocytic cells. However, until recently, methods have failed to achieve complete depletion. Moreover, treatments often affected other cells, making unequivocal conclusions from these studies difficult. Recently, we have shown that inhibition of colony stimulating factor 1 receptor (CSF1R) by oral treatment with PLX5622 containing chow enables complete depletion of retinal microglia and almost complete microglia depletion in the optic nerve without affecting peripheral macrophages or other cells. Using this approach, we investigated the role of microglia in neuroprotection in the retina and axon regeneration in the injured optic nerve under different conditions. Thus, this efficient, reliable and easy to use protocol presented here will enable researchers to unequivocally study the contribution of microglia on neurodegeneration and axon regeneration. This protocol can be also easily expanded to other paradigms of acute and chronic injury or diseases in the visual system.

Keywords: Axon regeneration (轴突再生), Neurodegeneration (神经退变), Microglia (小胶质细胞), Microglia depletion (小胶质细胞去除), PLX5622 (PLX5622), CSF1R (CSF1R)

Background

Microglia are resident immune cells of the central nervous system (CNS), which continuously scan their environment for pathologic alterations (Nimmerjahn et al., 2005). Upon detection of such conditions, microglia transform into an activated state and migrate towards the source where they perform different tasks (Kreutzberg, 1996; Davalos et al., 2005). Pathologic alterations, such as neurodegenerative diseases or acute injuries, are associated with neuronal loss and phagocytosis of these dying cells by activated microglia. Therefore, the overall assumption is that microglia are involved in this process (Thanos, 1991; Davalos et al., 2005; Hanisch and Kettenmann, 2007). Besides a possible effect of microglia on dying neurons, microglia are also part of the glial scar, which is formed at the lesion site and impairs axon regeneration in the CNS (Silver, 2004; Kitayama et al., 2011). However, as a clear discrimination of infiltrated blood born macrophages and microglia in the injured nerve was not feasible, the specific role of microglia in glial scar formation and axonal regeneration remained elusive (Silver, 2004; Hanisch and Kettenmann, 2007; Prinz and Priller, 2014; Hilla et al., 2017).

A suitable model to address the role of microglia in CNS degeneration and axonal regeneration is the visual pathway with its retinal ganglion cells (RGCs), whose axons project through the optic nerve (Leibinger et al., 2009; Fischer and Leibinger, 2012). Upon axonal damage, axons normally fail to regenerate (Thanos, 1991; Fischer et al., 2000). The degenerative and regenerative processes, following acute injury, can be easily visualized in retinal wholemounts and optic nerves, respectively as presented in the current protocol. Furthermore, the visual system and particularly RGCs are a suitable model to study neurodegenerative processes in chronic diseases such as multiple sclerosis or glaucoma (Kerrison et al., 1994; Howell et al., 2007; Diekmann and Fischer, 2013; Dietrich et al., 2018). In addition, the optic nerve is widely used as a classical model to study general regenerative failure in the CNS and strategies to facilitate axon growth (Fischer and Leibinger, 2012).

Several studies have been published, suggesting detrimental effects of microglia on RGC survival and axon regeneration whereas other studies indicate a beneficial, neuroprotective role (Thanos et al., 1993; Levkovitch-Verbin et al., 2006; Bosco et al., 2008; Chen et al., 2012; Rice et al., 2015). However, due to a paucity of depletion methods, many studies initially analyzed the contribution of microglia by altering their activation state with pharmacologic approaches (Thanos et al., 1993; Levkovitch-Verbin et al., 2006; Bosco et al., 2008; Jiao et al., 2014). In recent years, genetic methods were developed to achieve microglia depletion (Heppner et al., 2005; Bruttger et al., 2015; Waisman et al., 2015). However, apart from the requirement of transgenic animals, these methods have potential drawbacks such as the development of astrogliosis, secretion of pro-inflammatory cytokines and blood-brain-barrier damage, which complicate data interpretation. Only recently, pharmacologic colony stimulating factor 1 receptor (CSF1R) inhibitors PLX3397 and PLX5622 have been developed, which allow near complete depletion of microglia in the CNS independent of the animals genetic background (Elmore et al., 2014; Spangenberg et al., 2016; Hilla et al., 2017). In fact, we have recently demonstrated that an oral PLX5622 treatment for 21 days leads to a complete depletion of microglia in the retina and almost a total elimination in the optic nerve (Hilla et al., 2017). In accordance with previous studies, this approach selectively induces apoptosis of microglia by CSF1R inhibition, while other phagocytic cells such as peripheral macrophages are not affected (Elmore et al., 2014; Hilla et al., 2017). Although some studies suggested that this treatment causes minor astrocyte activation in the brain, we did not find any in the optic nerve or retina (Elmore et al., 2014; Hilla et al., 2017). Furthermore, as the inhibitor is orally bioavailable and capable of passing the blood brain barrier, PLX5622 can be formulated in standard rodent chow preventing the necessity of regular injections. Thus, this approach allows scientists to unequivocally address the role of microglia for different pathologic CNS paradigms, such as injury or chronic diseases, not only in the visual system, but also in other prominent CNS tissues such as brain and spinal cord. Using this approach in the visual pathway, we found that microglia neither affect the degeneration process of RGCs nor their capability to regenerate injured axons into the optic nerve upon an acute injury (Hilla et al., 2017). The current protocol describes the method for depleting microglia in the visual system and how effects on acute degeneration of RGCs and axon regeneration in the optic nerve can be quantified to identify the contribution of microglia in this context. This protocol can be easily expanded to investigate the involvement of microglia in other paradigms of acute and chronic injury or diseases in the visual system.

Materials and Reagents

  1. Glass capillary (World Precision Instruments, catalog number: 1B100F-6 )
  2. Glass slide (VWR, catalog number: 631-0108 )
  3. Surgical filament (Ethicon, catalog number: EH7790 )
  4. Scalpel blades ( B. Braun Melsungen, catalog number: BB511 )
  5. Swabs (Lohmann & Rauscher, catalog number: 13356 )
  6. Nitrocellulose membrane (GE Healthcare, catalog number: RPN303E )
  7. Male and female mice aged between 6-10 weeks
  8. CSF1R-inhibitor PLX5622 (PLX, Plexxikon, commercially not available)
  9. AIN-76A standard rodent chow (Research Diets)
  10. Ketamine (Grovet, Alfasan, Medistar, catalog number: 10002 )
  11. Xylazine (Bayer, Rompun® 2% Injection 25 ml) 
  12. Cholera toxin subunit B, Alexa FluorTM 555 (CTB) (Thermo Fisher Scientific, InvitrogenTM, catalog number: C22843 )
  13. Phosphate buffered saline (PBS) (Thermo Fisher Scientific, catalog number: 14190-094 )
  14. Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: 252549 )
  15. Sucrose (Sigma-Aldrich, catalog number: S9378 )
  16. KP-CryoCompound (KLINIPATH, catalog number: 1620-C )
  17. Double distilled H2O (Carl Roth, catalog number: 3478.2 )
  18. Eye ointment (DR. WINZER, Gent-Ophtal®)
  19. Methanol (Sigma-Aldrich, catalog number: 179957 )
  20. Triton X-100 (Sigma-Aldrich, catalog number: X100
  21. Iba1 antibody (Wako Pure Chemical Industries, catalog number: 019-19741 , RRID: AB_839504, LOT-specific concentration: 1 mg/ml)
  22. βIII-tubulin antibody (BioLegend, catalog number: 801202 , RRID: AB_2313773, LOT-specific concentration: 1 mg/ml)
  23. RNA-binding protein with multiple splicing (RBPMS) antibody (Abcam, catalog number: ab194213 , LOT-specific concentration: 0.25 mg/ml)
  24. Secondary antibodies
    1. Donkey anti mouse 488 (Thermo Fisher Scientific, catalog number: A-21202 , RRID: AB_141607, LOT-specific concentration: 2 mg/ml)
    2. Donkey anti rabbit 594 (Thermo Fisher Scientific, catalog number: A-21207 , RRID: AB_141637, LOT-specific concentration: 2 mg/ml)
  25. Donkey serum (Bio-Rad Laboratories, catalog number: C06SB )
  26. Bovine serum albumin (Sigma-Aldrich, catalog number: A3294 )
  27. Tween 20 (Sigma-Aldrich, catalog number: P1379 )
  28. Paraformaldehyde (PFA) solution (see Recipes)
  29. Sucrose solution (see Recipes)
  30. Blocking solution (sections) (see Recipes)
  31. Blocking solution (wholemount) (see Recipes)

Equipment

  1. Jeweler's forceps (Fine Science Tools, catalog number: 11254-20 )
  2. Capsulotomy scissor (Hermle, catalog number: 564 )
  3. Mouse head holder (KOPF INSTRUMENTS, catalog number: 921-E )
  4. Cryostat (Leica Biosystems, model: CM3050 S )
  5. Fluorescence microscope (ZEISS, model: Axio Observer.D1 )
  6. Confocal laser scanning microscope (Leica Microsystems, model: Leica TCS SP8 )
  7. Binocular (ZEISS, model: Stemi DV4 SPOT )
  8. Cold light source (SCHOTT, model: KL 1600 LED )

Software

  1. Excel 2016 (Microsoft)
  2. SigmaStat 3.1 (Systat)
  3. Photoshop CS6 (Adobe)

Procedure

  1. Microglia depletion
    Notes: 
    1. The CSF1R-inhibitor PLX5622 was kindly provided by Plexxikon Inc. formulated in standard AIN-76A rodent chow at 1,200 mg/kg.
    2. All experimental procedures should be approved by the local animal care committee.
    1. Keep male and female mice aged between 6-10 weeks on a 12 h light/dark cycle with ad libitum access to food and water. 
    2. Exchange rodent chow with PLX5622-containing chow and provide mice ad libitum access for at least 21 days to assure maximum depletion efficiency (Figures 1A, 2A and 3D). Respective control mice receive standard AIN-76A rodent chow without the CSF1R-inhibitor PLX5622.
    3. After 21 days, respective tissue can be harvested to analyze depletion efficiency (Figures 3A and 3B). Otherwise, keep mice on PLX5622-chow for the duration of the experiment to assure continuous microglia depletion. Mice do not show any observable phenotypes when treated with the inhibitor for at least up to 8 weeks.


      Figure 1. Experimental setup for optic nerve injury and axonal regeneration. A. Timeline of the experiment starting by administration of PLX5622 (CSF1R-inhibitor). Afterwards mice were subjected to surgery, and two days prior to sacrifice retinal ganglion cell axons were anterogradely labeled by intravitreal cholera toxin subunit B (CTB) injection. B. Setup for the optic nerve crush surgery. The workplace, as well as the instruments should be sterile. C. Injection scheme for intravitreal CTB injections. A pulled glass capillary containing CTB is inserted retrolentally at an approximately 45°-50° angle into the vitreous body without touching or damaging the ocular lens. D. Confocal image of an optic nerve 21 d after injury showing some CTB-positive regenerating axons crossing the lesion site (red dotted line). The scale above shows exemplary distances where regenerated axons can be counted and divided by the width of the optic nerve at this distance to extrapolate the number of regenerating axons per width of the optic nerve. Scale bar = 100 μm.

  2. Intraorbital optic nerve crush
    1. Make sure that the workplace and the instruments are kept clean and sterile throughout the surgical procedures to minimize the risk of infection (Figure 1B).
    2. Anesthetize mice by intraperitoneal injections of xylazine (16 mg/kg) and ketamine (120 mg/kg).
    3. Check for reflexes (paw withdrawal and corneal reflex) to assure full anesthesia before proceeding with the surgery.
    4. Shave the head using e.g., an electric shaver and perform a sagittal midline incision on the mouse head before fixing it in a mouse head holder.
    5. Fix the left side of the skin with surgical suture to allow undisturbed access to the orbit throughout the surgery.
    6. Use a #11 scalpel to cut along the orbit cavity to get access to the intraorbital space.
    7. In case of excessive bleeding use swabs to absorb the blood, which otherwise would impair your sight.
    8. Gently move the lacrimal gland aside using jeweler’s forceps leaving the tissue intact to get better access to the eye.
    9. Transect the superior oblique, superior rectus and the retractor bulbi using a capsulotomy scissor to gain access to the optic nerve. Take care to avoid accidental optic nerve injury.
    10. Once the optic nerve is accessible, use jeweler’s forceps and crush the optic nerve approximately 1 mm behind the eyeball with consistent pressure for 10 sec. Take care to crush over the entire width as otherwise the injury might remain incomplete (Fischer et al., 2017).
    11. Position the lacrimal gland back and suture the wound using sterile surgical sutures.
    12. Apply eye ointment to prevent infection and desiccation of the eyes.
    13. Afterwards proceed either with the surgery to study axonal regeneration (variant 1) or prepare retinal wholemounts for the analysis of RGC survival (variant 2).

  3. Variant 1: Anterograde tracing for the analysis of neuronal regeneration
    1. Nineteen days after optic nerve crush repeat Steps B1-B3 to assure optimal surgical conditions before intravitreally injecting 2 μl of the anterograde tracer CTB (5 μg/μl).
    2. Fix the eye with jeweler’s forceps and gently pinch through the cornea with a pulled glass capillary to drain approximately 2 μl of aqueous humor from the anterior chamber.
    3. Fill a pulled glass capillary with 2 μl of the concentrated CTB-solution.
    4. Gently pull out the eye to gain better access to the retrobulbar surface using a pair of jeweler’s forceps. Access the eye retrolentally at a 45°-50° angle to avoid accidental lens injury and insert the glass capillary into the vitreous body (Figure 1C). After injection, quickly remove the needle from the eyeball.
    5. Two days later, anesthetize the mouse as described before and intracardially perfuse the mouse with ice-cold PFA solution (Hilla et al., 2017; Leibinger et al., 2017).
    6. Prepare the optic nerve, embed it in embedding medium for frozen tissue and perform 14 μm cryosections as described previously (Hilla et al., 2017; Leibinger et al., 2017).
    7. These sections can now be directly analyzed under a fluorescence microscope (Figure 1D).

  4. Variant 2: Preparing retinal wholemount for the analysis of neurodegeneration
    1. To analyze RGC-degeneration at a given time point after optic nerve crush, euthanize the mouse according to local animal care guidelines. Perfusion with PFA solution is not required.
    2. Enucleate the eye and place it in a Petri dish filled with PBS. Remove the cornea by cutting along the ora serrata before removing the lens.
    3. Make four radial incisions in the eye cup approximately 90° apart from each other (Figure 2B) leaving the optic disk intact.
    4. Detach the retina from the sclera by cutting the connection between optic disc and sclera and transfer the free-floating retina with vitreous body onto a nitrocellulose membrane with the ganglion cell layer facing up.
    5. Transfer the membrane onto a dry tissue to let the retina suck onto the membrane. Return into PBS filled Petri dish and repeat this step for at least four times.
    6. Carefully detach the vitreous body from the retina using jeweler’s forceps while trying to avoid injury to the retina.
    7. Incubate for 30 min in PFA solution before proceeding to the staining protocol.


      Figure 2. Retinal wholemount preparation and analysis of neuronal survival upon optic nerve injury. A. Timeline of the experiment starting by administration of PLX5622 (CSF1R-inhibitor). Afterwards, mice were subjected to surgery and 21 days later sacrificed to quantify retinal ganglion cell survival after optic nerve injury. B. Schematic drawing of an enucleated eye without the cornea and lens shows the positions of the radial incisions in the retina to achieve a cloverleaf-shape. C. Magnification of a single retinal quadrant explains image acquisition to quantify neuronal survival. Image recording starts approximately one millimeter away from the papilla (semicircle) and progresses to the outer retina without overlap in between the pictures. D. Exemplary confocal image of the ganglion cell layer of a retina 21 days after optic nerve injury. RBPMS-staining (green) shows RGC somata, whereas βIII-tubulin (magenta) additionally highlights RGC axons. The merged picture at the bottom indicates a high overlap between the two RGC markers. Scale bar = 50 μm.

  5. Retinal wholemount staining for the quantification of RGC survival
    1. To analyze RGC survival, retinal wholemounts need to be stained against a suitable neuronal marker.
    2. Permeabilize the retina in 2% Triton X-100 in PBS in a humified chamber for 1 h at room temperature.
    3. Block the tissue using the blocking solution for wholemounts (see Recipes) in a humified chamber for 1 h at room temperature.
    4. Incubate the retinae with the primary antibody (either βIII-tubulin [1:1,000] or RNA-binding protein with multiple splicing [RBPMS, 1:500]) diluted in blocking solution in a humified chamber overnight at 4 °C.
    5. Wash off the nonspecifically bound primary antibodies with PBS three times each for 10 min.
    6. Incubate retinae with secondary antibodies (diluted 1:1,000 in blocking solution) in a humified chamber for 1 h at room temperature.
    7. Wash off the nonspecifically bound secondary antibody with PBS three times each for 10 min.
    8. To visualize Iba1-positive microglia and depletion efficiency one may co-stain the retinae with 1:1,000 Iba1 primary antibody diluted in blocking solution according to the protocol described (Video 1 and Video 2).

      Video 1. Preparation of a retinal wholemount. This video shows the preparation of a retinal wholemount starting with the already enucleated eye in a PBS filled Petri dish. The detailed protocol is described in Procedure D. (All experimental procedures have been approved by the local animal care committee (LANUV Recklinghausen)).

      Video 2. Depletion efficiency in the retina. This video shows a z-scan performed on a confocal laser scanning microscope through representative retinae, which were either treated with the CSF1R-inhibitor PLX5622 (right panel) or control chow (left panel) for 21 d. Microglia (Iba1, red) are only detectable in the control retina. There, they are evenly distributed throughout the retina and mainly localized in the ganglion cell layer (GCL) as well as inner (IPL) and outer plexiform layer (OPL). PLX5622-treated retinae show no microglia in any layer. Staining with the neuronal marker βIII-tubulin (green) shows retinal ganglion cells in the GCL and binding of the secondary antibody to IgG in the non-perfused retina shows positively stained blood vessels, thereby indicating the IPL and OPL.

  6. Optic nerve staining to analyze depletion efficiency
    1. To analyze depletion efficiency, respective tissue may be immunohistochemically stained against microglia/macrophage markers such as Iba1.
    2. Fix respective sections with 100% methanol for 10 min at room temperature.
    3. Block epitopes using the blocking solution for sections (see Recipes) in a humidified chamber for 30 min at room temperature.
    4. Incubate the sections with the Iba1 primary antibody (diluted 1:1,000 in blocking solution) in a humidified chamber overnight at 4 °C.
    5. Wash off the nonspecifically bound primary antibody three times each for 10 min with PBS.
    6. Incubate sections with the secondary antibody (diluted 1:1,000 in blocking solution) in a humidified chamber for 1 h at room temperature.
    7. Wash off the nonspecifically bound secondary antibody three times each for 10 min with PBS.
    8. Note that an optic nerve crush leads to an infiltration of blood-borne macrophages at the lesion site (Figure 3C), which can also be detected by most antibodies for microglia.


      Figure 3. Depletion efficiency of PLX on Iba1-positive microglia in CNS tissue. A. Maximum intensity projected confocal image of naïve retina wholemount from a control mouse shows Iba1-positive microglia throughout the retina and its different layers. Magnification at the bottom left displays microglia in the ganglion cell layer. In contrast, PLX-treated mice lack microglia throughout all retinal layers resulting in a 100% depletion efficiency (also see Video 2). Scale bar = 1 mm. B. Iba1-positive microglia are equally distributed throughout the naïve optic nerve whereas PLX-application caused massive microglia depletion. In contrast to the retina, some microglia were occasionally visible in the optic nerves (Hilla et al., 2017). Scale bar = 100 μm. C. Twenty-one days after optic nerve crush Iba1-positive cells accumulate at the lesion site in control mice as well as microglia-depleted mice (PLX) whereas the rest of PLX optic nerves remain devoid of signal. These remaining cells are most likely infiltrated hematopoietic macrophages as analyzed elsewhere (Hilla et al., 2017). Scale bar = 100 μm. D. Confocal images show that deprivation of PLX caused Iba1-positive microglia to repopulate the retina underlining the necessity of continuous PLX-treatment for efficient microglia depletion. The box in the bottom left shows a magnification of the ganglion cell layer of the indicated area in the overview. Scale bar = 1 mm.

Data analysis

  1. Variant 1: RGC axon regeneration
    1. Take images of the entire optic nerve section. Start at the lesion site and progress towards the chiasm.
    2. Arrange and merge single images to a complete optic nerve using a suitable image processing program (e.g., Photoshop).
    3. Create a ruler marking the distances to the lesion site you wish to analyze. Overlay it with the optic nerve and use the lesion site as a starting point (Figure 1D).
    4. Quantify axons that cross the defined distances and measure the width of the optic nerve at this distance.
    5. Divide number of regenerating axons crossing the indicated distance by the width of the optic nerve:



    6. Only include sections from complete optic nerves where the tissue between the lesion site and 1 mm after the longest regenerated axon is not disrupted.
    7. To analyze data between two different groups, use a two-way ANOVA with the individual group as the first variable and the distance to the lesion site as the second variable.
    8. A suitable post-hoc test provides information about significant variations between groups in general and, additionally, significant differences for single distances to the lesion site.

  2. Variant 2: RGC survival
    1.  Acquire images of the surviving RGCs by starting approximately 1 mm distal to the papilla with a 400x objective (Figure 2C).
    2. Proceed towards the outer retina and avoid taking images with overlapping areas. Depending on the preparation, four to six images can be recorded (Figures 2C and 2D).
    3. Repeat the above steps (Data analysis, Steps B1-B2) with the other three quadrants.
    4. Using suitable software, quantify RGC numbers per image and divide them by the area of the recorded field to obtain RGC density:



    5. As the RGC density decreases towards the outer retina, calculate the mean density value for one quadrant and then the mean over all four quadrants.
    6. To analyze data between two groups, use Student’s t-test or one-way ANOVA with a suitable post-hoc test for more than two groups.

Notes

  1. To avoid degradation of PLX5622, it is essential to keep chow dry and add only small portions (ca. 20 g/animal) in the cage.
  2. Depletion efficiency in the retina is 100% after a diet on PLX5622 for 14-21 days and highly reproducible. However, in other CNS tissues, some microglia may remain. If also other CNS tissues are analyzed for depletion efficiency, the retina may be used as a positive control.
  3. In case of incomplete anesthesia, mice should receive additional isoflurane anesthesia at 1% isoflurane/99% O2 for the duration of the surgery.
  4. RGC axons normally display only a very limited capacity of regeneration, however it is possible to improve the regenerative outcome by manipulating the regenerative state of RGCs for example by inflammatory stimulation or providing cytokines (Fischer et al., 2000; Leibinger et al., 2016).
  5. In general, high exposure times might be needed to clearly visualize single regenerating CTB-positive axons in the distal part of the optic nerve. As the number of axons is considerably higher at the lesion site, high exposure times in a widefield microscope might cause the fluorescent signal to scatter into the distal part of the optic nerve. This might thereby shift the assumed location of the lesion site into the distal part of the optic nerve. As this would result in incorrect quantification of regenerated axons at different distances to the lesion site, an image of the lesion site at a lower exposure time might be needed to clearly identify lesion site location.
  6. More detailed insights into intraorbital optic nerve crush and intravitreal injections can be found elsewhere (Chiu et al., 2007; Magharious et al., 2011).

Recipes

  1. Paraformaldehyde (PFA) solution
    108 ml Paraformaldehyde (37%)
    100 ml 10x PBS
    ad 1,000 ml ddH2O
  2. Sucrose solution
    30 g Sucrose
    ad 100 ml ddH2O
  3. Blocking solution (sections)
    0.2 g BSA
    0.5 ml serum from secondary antibody host (e.g., donkey serum)
    5 μl Tween 20
    ad 10 ml PBS
  4. Blocking solution (wholemount)
    0.2 g BSA
    1 ml serum from secondary antibody host (e.g., donkey serum)
    5 μl Tween 20
    ad 10 ml PBS

Acknowledgments

This work was supported by the German Research Foundation. We thank Plexxikon Inc. for providing PLX5622 chow. Furthermore, we thank Anastasia Andreadaki for technical support. This protocol was adapted from previous work (Elmore et al., 2014; Hilla et al., 2017).

Competing interests

The authors declare no conflict of interest.

Ethics

All experimental procedures have been approved by the local animal care committee (LANUV Recklinghausen).

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  21. Leibinger, M., Andreadaki, A., Golla, R., Levin, E., Hilla, A. M., Diekmann, H. and Fischer, D. (2017). Boosting CNS axon regeneration by harnessing antagonistic effects of GSK3 activity. Proc Natl Acad Sci U S A 114(27): E5454-E5463.
  22. Leibinger, M., Muller, A., Andreadaki, A., Hauk, T. G., Kirsch, M. and Fischer, D. (2009). Neuroprotective and axon growth-promoting effects following inflammatory stimulation on mature retinal ganglion cells in mice depend on ciliary neurotrophic factor and leukemia inhibitory factor. J Neurosci 29(45): 14334-14341.
  23. Levkovitch-Verbin, H., Kalev-Landoy, M., Habot-Wilner, Z. and Melamed, S. (2006). Minocycline delays death of retinal ganglion cells in experimental glaucoma and after optic nerve transection. Arch Ophthalmol 124(4): 520-526.
  24. Magharious, M. M., D'Onofrio, P. M. and Koeberle, P. D. (2011). Optic nerve transection: a model of adult neuron apoptosis in the central nervous system. J Vis Exp(51): 2241.
  25. Nimmerjahn, A., Kirchhoff, F. and Helmchen, F. (2005). Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science 308(5726): 1314-1318.
  26. Prinz, M. and Priller, J. (2014). Microglia and brain macrophages in the molecular age: from origin to neuropsychiatric disease. Nat Rev Neurosci 15(5): 300-312.
  27. Rice, R. A., Spangenberg, E. E., Yamate-Morgan, H., Lee, R. J., Arora, R. P., Hernandez, M. X., Tenner, A. J., West, B. L. and Green, K. N. (2015). Elimination of microglia improves functional outcomes following extensive neuronal loss in the hippocampus. J Neurosci 35(27): 9977-9989.
  28. Silver, J. and Miller, J. H. (2004). Regeneration beyond the glial scar. Nat Rev Neurosci 5(2): 146-156.
  29. Spangenberg, E. E., Lee, R. J., Najafi, A. R., Rice, R. A., Elmore, M. R., Blurton-Jones, M., West, B. L. and Green, K. N. (2016). Eliminating microglia in Alzheimer's mice prevents neuronal loss without modulating amyloid-β pathology. Brain 139(Pt 4): 1265-1281.
  30. Thanos, S. (1991). The relationship of microglial cells to dying neurons during natural neuronal cell death and axotomy-induced degeneration of the rat retina. Eur J Neurosci 3(12): 1189-1207.
  31. Thanos, S., Mey, J. and Wild, M. (1993). Treatment of the adult retina with microglia-suppressing factors retards axotomy-induced neuronal degradation and enhances axonal regeneration in vivo and in vitro. J Neurosci 13(2): 455-466.
  32. Waisman, A., Ginhoux, F., Greter, M. and Bruttger, J. (2015). Homeostasis of microglia in the adult brain: review of novel microglia depletion systems. Trends Immunol 36(10): 625-636.

简介

小胶质细胞存在于中枢神经系统(CNS)中,并参与维持生理状态。他们不断调查他们的环境,以寻找与受伤或疾病相关的病理改变。几十年来,研究人员使用旨在抑制或消除这些吞噬细胞的方法,研究了小胶质细胞在不同病理条件下的作用。然而,直到最近,方法未能实现完全耗尽。此外,治疗通常会影响其他细胞,因此难以从这些研究中得出明确的结论。最近,我们已经表明,通过用含有食物的PLX5622口服治疗抑制集落刺激因子1受体(CSF1R)能够完全消除视网膜小胶质细胞并且几乎完全消除视神经中的小胶质细胞,而不影响外周巨噬细胞或其他细胞。使用这种方法,我们研究了小胶质细胞在视网膜神经保护中的作用以及在不同条件下受损视神经中的轴突再生。因此,这里提出的这种有效,可靠和易于使用的方案将使研究人员能够明确地研究小胶质细胞对神经变性和轴突再生的贡献。该方案还可以容易地扩展到视觉系统中的急性和慢性损伤或疾病的其他范例。

【背景】 小胶质细胞是中枢神经系统(CNS)的常驻免疫细胞,其持续扫描其环境以进行病理改变(Nimmerjahn et al。,2005)。在检测到这种情况后,小胶质细胞转变为活化状态并向其进行不同任务的来源迁移(Kreutzberg,1996; Davalos et al。,2005)。病理改变,例如神经退行性疾病或急性损伤,与活化的小胶质细胞的神经元丢失和这些垂死细胞的吞噬作用有关。因此,总体假设是小胶质细胞参与该过程(Thanos,1991; Davalos et al。,2005; Hanisch和Kettenmann,2007)。除小胶质细胞对垂死神经元的可能影响外,小胶质细胞也是胶质瘢痕的一部分,胶质瘢痕在损伤部位形成并损害CNS中的轴突再生(Silver,2004; Kitayama et al。, 2011)。然而,由于明显区分受损神经中浸润的血液中存在的巨噬细胞和小胶质细胞是不可行的,小胶质细胞在胶质瘢痕形成和轴突再生中的特定作用仍然难以捉摸(Silver,2004; Hanisch和Kettenmann,2007; Prinz和Priller, 2014; Hilla et al。,2017)。

解决小胶质细胞在中枢神经系统退化和轴突再生中作用的合适模型是其视网膜神经节细胞(RGCs)的视觉通路,其轴突通过视神经突出(Leibinger et al。。,2009 ; Fischer和Leibinger,2012年)。轴突损伤时,轴突通常无法再生(Thanos,1991; Fischer et al。,2000)。急性损伤后的退行性和再生性过程可以分别在视网膜全身和视神经中容易地显现,如本方案中所示。此外,视觉系统,特别是RGC是研究慢性疾病(如多发性硬化或青光眼)神经退行性过程的合适模型(Kerrison 等。,1994; Howell 等。,2007; Diekmann和Fischer,2013; Dietrich et al。,2018)。此外,视神经被广泛用作研究CNS中一般再生衰竭的经典模型和促进轴突生长的策略(Fischer和Leibinger,2012)。

一些研究已经发表,表明小胶质细胞对RGC存活和轴突再生的不利影响,而其他研究表明有益的神经保护作用(Thanos et al。,1993; Levkovitch-Verbin et al。 ,2006; Bosco et al。,2008; Chen et al。,2012; Rice et al。,2015)。然而,由于缺乏耗尽方法,许多研究最初通过药理学方法改变了它们的活化状态,分析了小胶质细胞的作用(Thanos et al。,1993; Levkovitch-Verbin et al。 ,2006; Bosco et al。,2008; Jiao et al。,2014)。近年来,开发了遗传方法以实现小胶质细胞耗竭(Heppner et al。,2005; Bruttger et al。,2015; Waisman et al。,2015)。然而,除了转基因动物的需要之外,这些方法具有潜在的缺点,例如星形胶质细胞增生的发展,促炎细胞因子的分泌和血脑屏障损伤,这使数据解释复杂化。直到最近,药物集落刺激因子1受体(CSF1R)抑制剂PLX3397和PLX5622才得以开发,其允许CNS中小胶质细胞几乎完全耗尽,而与动物遗传背景无关(Elmore et al。,2014 ; Spangenberg et al。,2016; Hilla et al。,2017)。事实上,我们最近证明口服PLX5622治疗21天导致视网膜中小胶质细胞完全消耗,几乎完全消除了视神经(Hilla et al。,2017)。根据以前的研究,这种方法通过CSF1R抑制选择性地诱导小胶质细胞凋亡,而其他吞噬细胞如外周巨噬细胞不受影响(Elmore et al。,2014; Hilla et al。 ,2017)。虽然有些研究表明这种治疗会导致大脑中星形胶质细胞的微小活动,但我们在视神经或视网膜中没有发现任何活动(Elmore et al。,2014; Hilla et al。,2017)。此外,由于抑制剂具有口服生物可利用性并且能够通过血脑屏障,因此PLX5622可配制在标准啮齿动物食物中,从而无需定期注射。因此,这种方法使科学家能够明确地解决小胶质细胞对不同病理性CNS范例的作用,例如损伤或慢性疾病,不仅在视觉系统中,而且在其他突出的CNS组织如脑和脊髓中。在视觉通路中使用这种方法,我们发现小胶质细胞既不影响RGC的退化过程,也不影响它们在急性损伤时再生受损轴突进入视神经的能力(Hilla et al。,2017)。目前的方案描述了视觉系统中消除小胶质细胞的方法,以及如何量化对视神经中RGC急性变性和轴突再生的影响,以确定小胶质细胞在这种情况下的贡献。该方案可以很容易地扩展到调查小胶质细胞参与视觉系统中的急性和慢性损伤或疾病的其他范例。

关键字:轴突再生, 神经退变, 小胶质细胞, 小胶质细胞去除, PLX5622, CSF1R

材料和试剂

  1. 玻璃毛细管(World Precision Instruments,目录号:1B100F-6)
  2. 载玻片(VWR,目录号:631-0108)
  3. 手术丝(Ethicon,目录号:EH7790)
  4. 手术刀刀片(B. Braun Melsungen,目录号:BB511)
  5. 棉签(Lohmann& Rauscher,目录号:13356)
  6. 硝酸纤维素膜(GE Healthcare,目录号:RPN303E)
  7. 雄性和雌性小鼠年龄在6-10周之间
  8. CSF1R抑制剂PLX5622(PLX,Plexxikon,商业无法提供)
  9. AIN-76A标准啮齿动物食物(研究饮食)
  10. 氯胺酮(Grovet,Alfasan,Medistar,目录号:10002)
  11. 甲苯噻嗪(拜耳,Rompun ® 2%注射液25 ml) 
  12. 霍乱毒素亚基B,Alexa Fluor TM 555(CTB)(Thermo Fisher Scientific,Invitrogen TM ,目录号:C22843)
  13. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,目录号:14190-094)
  14. 多聚甲醛(PFA)(西格玛奥德里奇,目录号:252549)
  15. 蔗糖(Sigma-Aldrich,目录号:S9378)
  16. KP-CryoCompound(KLINIPATH,目录号:1620-C)
  17. 双蒸H 2 O(Carl Roth,目录号:3478.2)
  18. 眼膏(DR.WINZER,Gent-Ophtal ®)
  19. 甲醇(Sigma-Aldrich,目录号:179957)
  20. Triton X-100(Sigma-Aldrich,目录号:X100) 
  21. Iba1抗体(Wako Pure Chemical Industries,目录号:019-19741,RRID:AB_839504,LOT-特异性浓度:1 mg / ml)
  22. βIII-tubulin抗体(BioLegend,目录编号:801202,RRID:AB_2313773,LOT-特异性浓度:1 mg / ml)
  23. 具有多个剪接的RNA结合蛋白(RBPMS)抗体(Abcam,目录号:ab194213,LOT特异性浓度:0.25 mg / ml)
  24. 二抗
    1. 驴抗小鼠488(Thermo Fisher Scientific,目录号:A-21202,RRID:AB_141607,LOT特异性浓度:2mg / ml)
    2. 驴抗兔594(Thermo Fisher Scientific,目录号:A-21207,RRID:AB_141637,LOT特异性浓度:2 mg / ml)
  25. 驴血清(Bio-Rad Laboratories,目录号:C06SB)
  26. 牛血清白蛋白(Sigma-Aldrich,目录号:A3294)
  27. 吐温20(西格玛奥德里奇,目录号:P1379)
  28. 多聚甲醛(PFA)溶液(见食谱)
  29. 蔗糖溶液(见食谱)
  30. 阻止解决方案(部分)(见食谱)
  31. 阻塞解决方案(wholemount)(参见食谱)

设备

  1. 珠宝商的钳子(精细科学工具,目录号:11254-20)
  2. Capsulotomy剪刀(Hermle,目录号:564)
  3. 鼠标头支架(KOPF INSTRUMENTS,目录号:921-E)
  4. Cryostat(Leica Biosystems,型号:CM3050 S)
  5. 荧光显微镜(蔡司,型号:Axio Observer.D1)
  6. 共聚焦激光扫描显微镜(Leica Microsystems,型号:Leica TCS SP8)
  7. 双目(ZEISS,型号:Stemi DV4 SPOT)
  8. 冷光源(肖特,型号:KL 1600 LED)

软件

  1. Excel 2016(微软)
  2. SigmaStat 3.1(Systat)
  3. Photoshop CS6(Adobe)

程序

  1. 小胶质细胞耗尽
    注意: 
    1. CSF1R抑制剂PLX5622由Plexxikon Inc.以标准AIN-76A啮齿动物饲料配制,浓度为1,200 mg / kg。
    2. 所有实验程序均应得到当地动物护理委员会的批准。
    1. 将年龄在6-10周之间的雄性和雌性小鼠保持在12小时光照/黑暗周期,随意随意获取食物和水。 
    2. 用含有PLX5622的食物交换啮齿动物食物并提供至少21天的小鼠随意访问,以确保最大的消耗效率(图1A,2A和3D)。各对照小鼠接受标准AIN-76A啮齿动物饲料,不含CSF1R抑制剂PLX5622。
    3. 21天后,可以收获各自的组织以分析耗尽效率(图3A和3B)。否则,在实验期间将小鼠放在PLX5622-chow上以确保连续的小胶质细胞耗尽。当用抑制剂治疗至少8周时,小鼠不会出现任何可观察到的表型。


      图1.视神经损伤和轴突再生的实验装置。 :一种。通过施用PLX5622(CSF1R-抑制剂)开始实验的时间表。然后对小鼠进行手术,并且在处死前两天,通过玻璃体内霍乱毒素亚单位B(CTB)注射对视网膜神经节细胞轴突进行顺行标记。 B.视神经挤压手术的设置。工作场所以及仪器应该是无菌的。 C.玻璃体内注射CTB的注射方案。将含有CTB的拉制玻璃毛细管以大约45°-50°的角度重新插入玻璃体中,而不会接触或损坏眼睛晶状体。 D.损伤后21天视神经的共聚焦图像,显示一些CTB阳性再生轴突穿过病变部位(红色虚线)。上面的比例示出了可以计算再生轴突的示例性距离,并且除以该距离处的视神经的宽度,以推断视神经的每个宽度的再生轴突的数量。比例尺=100μm。

  2. 眶内视神经挤压
    1. 确保工作场所和器械在整个手术过程中保持清洁和无菌,以尽量降低感染风险(图1B)。
    2. 通过腹膜内注射甲苯噻嗪(16mg / kg)和氯胺酮(120mg / kg)麻醉小鼠。
    3. 检查反射(缩足和角膜反射),以确保在进行手术前完全麻醉。
    4. 使用电动剃须刀例如剃掉头部,并在将其固定在鼠标头支架上之前,在鼠标头上进行矢状中线切口。
    5. 用手术缝合线固定皮肤左侧,以便在整个手术过程中不受干扰地进入眼眶。
    6. 使用#11手术刀沿着轨道腔切开以进入眶内空间。
    7. 如果出血过多,请使用拭子吸收血液,否则会损害您的视力。
    8. 使用珠宝商的镊子轻轻移动泪腺,使组织保持完整,以便更好地接近眼睛。
    9. 使用囊切开术剪刀切除上斜肌,上直肌和牵开器肺,以进入视神经。注意避免意外的视神经损伤。
    10. 一旦可以接触到视神经,使用珠宝钳的镊子并用一致的压力将视神经压在眼球后面大约1 mm,持续10秒。注意压碎整个宽度,否则伤害可能仍然不完整(Fischer et al。,2017)。
    11. 将泪腺定位回来并使用无菌手术缝合线缝合伤口。
    12. 涂抹眼膏,以防止感染和眼睛干燥。
    13. 然后进行手术以研究轴突再生(变体1)或制备视网膜整体用于分析RGC存活(变体2)。

  3. 变型1:用于分析神经元再生的顺行追踪
    1. 视神经挤压后19天重复步骤B1-B3以确保在玻璃体内注射2μl顺行示踪剂CTB(5μg/μl)之前的最佳手术条件。
    2. 用珠宝钳的镊子固定眼睛,用拉动的玻璃毛细管轻轻夹住角膜,从前房排出大约2μl的房水。
    3. 用2μl浓缩的CTB溶液填充拉出的玻璃毛细管。
    4. 使用一对珠宝商的镊子轻轻拉出眼睛,以更好地进入眼球后表面。以45°-50°的角度进行眼部后入眼,以避免意外的镜片损伤,并将玻璃毛细管插入玻璃体内(图1C)。注射后,迅速将针头从眼球上取下。
    5. 两天后,如前所述麻醉小鼠,并用冰冷的PFA溶液心内灌注小鼠(Hilla et al。,2017; Leibinger et al。,2017) 。
    6. 准备视神经,将其嵌入冷冻组织的包埋介质中,并如前所述进行14μm冷冻切片(Hilla et al。,2017; Leibinger et al。,2017) 。
    7. 现在可以在荧光显微镜下直接分析这些切片(图1D)。

  4. 变型2:准备视网膜整体装置用于分析神经变性
    1. 为了分析视神经挤压后给定时间点的RGC变性,根据当地动物护理指南对小鼠实施安乐死。不需要用PFA溶液灌注。
    2. 摘除眼睛并将其置于装有PBS的培养皿中。在取下镜头之前,沿锯齿锯切开角膜去除角膜。
    3. 在眼杯中做四个径向切口,彼此间隔大约90°(图2B),使光盘保持完整。
    4. 通过切断视神经盘和巩膜之间的连接将视网膜从巩膜上分离,并将具有玻璃体的自由浮动视网膜转移到硝酸纤维素膜上,神经节细胞层朝上。
    5. 将膜转移到干燥的组织上,让视网膜吸到膜上。返回PBS充满培养皿并重复该步骤至少四次。
    6. 使用珠宝商的镊子小心地将玻璃体从视网膜上分离,同时尽量避免损伤视网膜。
    7. 在进行染色方案之前,在PFA溶液中孵育30分钟。


      图2.视网膜整体制备和视神经损伤后神经元存活的分析。 A.通过施用PLX5622(CSF1R抑制剂)开始实验的时间线。然后,对小鼠进行手术,并在21天后处死,以量化视神经损伤后的视网膜神经节细胞存活。 B.没有角膜和晶状体的去核眼的示意图显示了视网膜中的径向切口的位置以实现三叶草形状。 C.单个视网膜象限的放大解释了图像采集以量化神经元存活。图像记录从乳头(半圆)开始大约1毫米处并且前进到视网膜外部,而图像之间没有重叠。 D.视神经损伤后21天视网膜神经节细胞层的示例性共聚焦图像。 RBPMS染色(绿色)显示RGC somata,而βIII-微管蛋白(品红色)另外突出RGC轴突。底部的合并图片表示两个RGC标记之间的高重叠。比例尺=50μm。

  5. 用于定量RGC存活的视网膜wholemount染色
    1. 为了分析RGC存活,需要针对合适的神经元标记物染色视网膜整体。
    2. 在室温下在湿化室中在PBS中的2%Triton X-100中使视网膜透化1小时。
    3. 使用用于整个装置的封闭溶液(参见配方)在室温下在湿化室中封闭组织1小时。
    4. 将视网膜与第一抗体(βIII-微管蛋白[1:1,000]或具有多个剪接的RNA结合蛋白[RBPMS,1:500])一起孵育,在4℃下在湿化室中在封闭溶液中稀释过夜。
    5. 用PBS洗去非特异性结合的一抗三次,每次10分钟。
    6. 将视网膜与第二抗体(在封闭溶液中以1:1,000稀释)在室温下在湿化室中孵育1小时。
    7. 用PBS洗涤非特异性结合的二抗,每次三次,持续10分钟。
    8. 为了可视化Iba1阳性小胶质细胞和耗竭效率,可以根据所述方案(视频1和视频2)用在封闭溶液中稀释的1:1,000 Iba1一抗共同染色视网膜。


      视频1.准备视网膜整体装置。

      视频2.视网膜的耗尽效率。

  6. 光学神经染色分析耗竭效率
    1. 为了分析耗竭效率,可以对小胶质细胞/巨噬细胞标记物如Iba1进行免疫组织化学染色。
    2. 在室温下用100%甲醇将各个部分固定10分钟。
    3. 使用阻断溶液在室温下在加湿室中对切片(参见配方)封闭表位30分钟。
    4. 将切片与Iba1一抗(在封闭溶液中1:1,000稀释)在加湿室中于4℃孵育过夜。
    5. 用PBS洗涤非特异性结合的一抗三次,每次10分钟。
    6. 将第二抗体(在封闭溶液中以1:1,000稀释)在加湿室中在室温下孵育1小时。
    7. 用PBS洗掉非特异性结合的二抗,每次3次,每次10分钟。
    8. 请注意,视神经挤压导致病变部位的血源性巨噬细胞浸润(图3C),大多数小胶质细胞抗体也可检测到这种情况。


      图3. PLX对CNS组织中Iba1阳性小胶质细胞的消耗效率 A.来自对照小鼠的幼稚视网膜整体上的最大强度投影共聚焦图像显示整个视网膜及其不同层中的Iba1阳性小胶质细胞。左下方的放大显示神经节细胞层中的小胶质细胞。相反,PLX处理的小鼠在所有视网膜层中缺乏小胶质细胞,导致100%的消耗效率(也参见视频2)。比例尺= 1毫米。 B.Iba1阳性小胶质细胞在幼稚视神经中均匀分布,而PLX应用导致大量小胶质细胞耗竭。与视网膜相反,视神经中偶尔会出现一些小胶质细胞(Hilla et al。,2017)。比例尺=100μm。 C.视神经挤压后21天,Iba1阳性细胞在对照小鼠以及小胶质细胞耗尽小鼠(PLX)的损伤部位积聚,而PLX视神经的其余部分保持缺乏信号。如其他地方所分析的,这些剩余的细胞很可能是浸润的造血巨噬细胞(Hilla et al。,2017)。比例尺=100μm。 D.共聚焦图像显示,PLX的剥夺导致Iba1阳性小胶质细胞重新填充视网膜,强调了连续PLX治疗有效小胶质细胞耗竭的必要性。左下方的框显示了概览中指示区域的神经节细胞层的放大倍数。比例尺= 1毫米。

数据分析

  1. 变体1:RGC轴突再生
    1. 拍摄整个视神经节的图像。从病变部位开始,朝着交叉的方向前进。
    2. 使用合适的图像处理程序(例如,Photoshop)将单个图像排列并合并到完整的视神经。
    3. 创建标尺,标记您要分析的病变部位的距离。用视神经覆盖它并以病变部位为起点(图1D)。
    4. 量化穿过限定距离的轴突并测量此距离处视神经的宽度。
    5. 将超过指示距离的再生轴突的数量除以视神经的宽度:



    6. 仅包括来自完整视神经的切片,其中损伤部位之间的组织和最长再生轴突之后1mm的组织未被破坏。
    7. 要分析两个不同组之间的数据,请使用双向ANOVA,其中单个组作为第一个变量,距离病变部位的距离作为第二个变量。
    8. 一个合适的事后检验提供了有关群体之间的重大差异的信息,另外,还提供了到病变部位的单一距离的显着差异。

  2. 变式2:RGC生存
    1.  通过400x物镜从乳头远端开始约1 mm处获取幸存的RGC图像(图2C)。
    2. 向外视网膜前进,避免拍摄重叠区域的图像。根据准备,可以记录四到六个图像(图2C和2D)。
    3. 用其他三个象限重复上述步骤(数据分析,步骤B1-B2)。
    4. 使用合适的软件,量化每个图像的RGC数量,并将它们除以记录区域的面积,以获得RGC密度:



    5. 随着RGC密度朝向外视网膜减小,计算一个象限的平均密度值,然后计算所有四个象限的平均密度值。
    6. 要分析两组之间的数据,请使用学生的 t - 测试或单向ANOVA,并对两组以上进行适当的事后检验。

笔记

  1. 为避免PLX5622降解,必须保持食物干燥,并在笼子中加入少量( ca。 20克/动物)。
  2. 在PLX5622上饮食14-21天后,视网膜的消耗效率为100%,并且具有高度可重复性。然而,在其他CNS组织中,可能残留一些小胶质细胞。如果还分析其他CNS组织的耗竭效率,则视网膜可以用作阳性对照。
  3. 在麻醉不完全的情况下,在手术期间,小鼠应在1%异氟烷/ 99%O 2 下接受额外的异氟醚麻醉。
  4. RGC轴突通常仅显示非常有限的再生能力,但是可以通过操纵RGC的再生状态来改善再生结果,例如通过炎性刺激或提供细胞因子(Fischer 等人,,2000)。 ; Leibinger et al。,2016)。
  5. 通常,可能需要高暴露时间以清楚地显现视神经远端部分中的单个再生CTB阳性轴突。由于在损伤部位的轴突数量相当高,在宽视场显微镜中的高曝光时间可能导致荧光信号散射到视神经的远端部分。这可能因此将病变部位的假定位置移动到视神经的远端部分。由于这将导致在到病变部位的不同距离处的再生轴突的不正确量化,可能需要在较低暴露时间的病变部位的图像以清楚地识别病变部位位置。
  6. 有关眶内视神经挤压和玻璃体内注射的更详细见解可以在其他地方找到(Chiu et al。,2007; Magharious et al。,2011)。

食谱

  1. 多聚甲醛(PFA)溶液
    108毫升多聚甲醛(37%)
    100毫升10倍PBS
    ad 1,000 ml ddH2O
  2. 蔗糖溶液
    30克蔗糖
    ad 100 ml ddH 2 O.
  3. 阻止解决方案(部分)
    0.2克BSA
    来自二抗宿主的0.5ml血清(例如,驴血清)
    5μlTween20
    ad 10毫升PBS
  4. 阻塞解决方案(wholemount)
    0.2克BSA
    来自二抗宿主的1ml血清(例如,驴血清)
    5μlTween20
    广告10毫升PBS

致谢

这项工作得到了德国研究基金会的支持。我们感谢Plexxikon Inc.提供PLX5622食品。此外,我们感谢Anastasia Andreadaki的技术支持。作者宣称没有利益冲突。该协议改编自先前的工作(Elmore et al。,2014; Hilla et al。,2017)。

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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Hilla, A. M. and Fischer, D. (2018). Studying the Role of Microglia in Neurodegeneration and Axonal Regeneration in the Murine Visual System. Bio-protocol 8(16): e2979. DOI: 10.21769/BioProtoc.2979.
  2. Hilla, A. M., Diekmann, H. and Fischer, D. (2017). Microglia are irrelevant for neuronal degeneration and axon regeneration after acute injury. J Neurosci 37(25): 6113-6124.
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