We used adult mice (2–4 months of age) of either sex from transgenic strains thy1
-YFP-H and thy1
-YFP16, which express yellow fluorescent protein (YFP) under the control of the neuron-specific Thy-1
promoter (Feng et al., 2000
). The original breeding pairs were purchased from The Jackson Laboratory (Bar Harbor, ME); subsequent stocks of mice used in these experiments were reared in the animal facilities at Drexel University College of Medicine. All experiments were performed in accordance with DUCOM’s Institutional Animal Care and Use Committee and National Institutes of Health guidelines.
Surgical and postoperative procedures
Thy1-YFPH mice were anesthetized with an intraperitoneal injection of xylazine (8 mg/kg) and ketamine (120 mg/kg). Supplements were given during the procedure as needed. A 2- to 3-cm long incision was made in the skin of the back; the spinal musculature was reflected; and the L3-S1 spinal cord segments were exposed by hemi-laminectomies. The cavity made by the laminectomies was perfused with warm sterile Ringer’s solution or artificial cerebrospinal fluid. A small incision was made in the dura overlying the L5 dorsal root near the L3 DRG; a fine forceps (Dumont #5) was introduced subdurally and the L5 dorsal root was crushed for 10 seconds. After images were collected (see below), we attempted to minimize scar formation by tightly applying a piece of thin synthetic matrix membrane (Biobrane, Bertek Pharmaceuticals, Morgantown, WV) over the exposed cord and dura, so that scarring accumulated on the membrane rather than on the dura surface. The matrix membrane was removed and replaced at each imaging session. This membrane was stabilized with a layer of much thicker artificial dura (Gore Preclude MVP Dura Substitute, W.L. Gore and Associates, Flagstaff, AZ) that covered the laminectomy site. The musculature was then closed with sterile 5-0 sutures, and the skin with wound clips. Animals were given subcutaneous injections of lactated Ringer's solution to prevent dehydration and kept on a heating pad until fully recovered from anesthesia. Buprenorphine was given as postoperative analgesia (0.05 mg/kg subcutaneously every 12 h for 2 days). For each imaging session, we reanesthetized and surgically re-exposed the area of interest and repeated the procedures. For conditioning lesions, the sciatic nerve was crushed in the lateral thigh of the ipsilateral hind leg 10 days before the root was crushed. Animals were anesthetized as described above; the skin and superficial muscle layer of the mid thigh were opened; and the sciatic nerve was crushed for 10 s with fine forceps (Dumont #5). The muscle and skin were then closed in layers and the animals were allowed to recover on a heating pad until fully awake.
In vivo imaging and image acquisition
We used a Leica MZ16 fluorescent stereomicroscope or an Olympus BX51 microscope equipped with a fast shutter and a highly sensitive cooled CCD camera (ORCA-Rx2, Hamamatsu, Bridgewater, NJ) controlled by Metamorph software (Molecular Devices, Sunnyvale, CA). Body temperature was maintained by placing the animal on a thermostatically controlled heating pad. Warmed lactated Ringer’s solution was used to superfuse the exposed portion of spinal cord. Images were acquired either as single snapshots or as multiple streams of 10 to 20 frames acquired within 30- to 40-ms exposure time. In-focus images were then selected, and an overview montage was created using Photoshop (Adobe Systems, San Jose, CA). High-resolution confocal images were obtained with a Leica TCS 4D confocal microscope (Heidelberg, Germany). Z stacks were obtained at 0.3-µm step size for 20- to 40-µm depths. Leica TCS-NT acquisition software and Imaris image software (Bitplane AG, Zurich, Switzerland) were used to reconstruct z-series images into maximum intensity projections.
Immunohistochemistry of DREZ in whole mounts
Following in vivo
imaging, we harvested tissues and processed them in whole mounts to immunolabel astrocytes, oligodendrocytes, or Schwann cells to locate the CNS/PNS interface. The immunostaining procedure was standard (Wright et al., 2009
), except for the permeabilization steps in which chilled MeOH and 1% sodium borohydride were also used. Mice were perfused transcardially with 0.9% heparinized saline solution followed by 4% paraformaldehyde in phosphate buffered saline (PBS). After 3 hours in situ
postfixation at 4°C, the spinal cord segment (L3–L6) with attached dorsal roots was removed and rinsed in PBS. The tissue was then washed for 30 minutes in a blocking solution containing 0.1 M glycine and 2% bovine serum albumin (BSA) in PBS and treated in cold MeOH for 10 minutes and then 1% sodium borohydride for 5 to 10 minutes. After thorough and extensive rinsing in PBS, the spinal cord was further permeabilized with 0.2% Triton X-100 with 2% BSA in PBS (TBP) for 1 hour and then incubated with primary antibody diluted in TBP overnight. The next day the spinal cord was rinsed thoroughly in TBP and then incubated with appropriate fluorescently conjugated secondary antibodies diluted in the TBP for 1 hour at room temperature. The tissue was then rinsed in PBS, and a thin sheet of dorsal spinal cord was prepared from the DREZ and rootlet, mounted in Vectashield (Vector Laboratories, Burlingame, CA), and stored at 4°C.
Immunohistochemistry of DREZ on cryostat sections
To immunolabel axons at the axotomized DREZ with synaptic vesicle markers, we used the transgenic strain, thy1-YFP16, in which the entire population of large-diameter axons expresses YFP (data not shown). To analyze more axons than superficially located ones, we prepared cryostat sections, rather than whole mounts, of the DREZ after crushing dorsal roots of cervical spinal cord. Using the surgical procedures described earlier, C3–C5 roots were crushed, and the animals were allowed to recover. At 20 days post injury, the C3–C5 spinal cord and roots were harvested, postfixed overnight at 4°C, cryoprotected in 30% sucrose in PBS, and rapidly frozen in Shandon M1 embedding matrix (Thermo Electron Corporation, Pittsburgh, PA). Serial transverse sections were cut on a cryostat at 10 µm (CM3000, Leica) and collected on Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA). For immunostaining, sections were postfixed in 4% paraformaldehyde in PBS for 20 min, rinsed in PBS, and blocked for 1 hour in TBP. The sections were then incubated overnight at 4°C in a cocktail of primary antibodies diluted in TBP. Sections were then rinsed in PBS and incubated with secondary antibodies in TBP for 1hour at room temperature and processed as described above.
Analysis of thy1-YFPH DRGs
L5 DRGs were dissected from unoperated thy1-YFPH mice and processed to obtain serial cryostat sections using the methods described above. Selected sections were stained with a fluorescent Nissl stain (Neurotrace 530/615 red fluorescent Nissl stain; Invitrogen) according to the manufacturer's instructions, washed extensively with PBS, and coverslipped using Vectashield (Vector Laboratories, Burlingame, CA). Neuron counts were made on 5 L5 DRGs from 3 animals. For each DRG at least three randomly selected sections were analyzed, taking care not to use sections that were poorly mounted or stained. Each section selected was at least 30 µm away (three sections) from either of the other selected sections. Using a Retiga EXi (Qimaging) digital camera, the entire section was photographed in segments using the 20x objective on a Leica DMRBE fluorescent microscope. The same section was photographed using both red (Nissl stained cells) and green (YFP+ cells) fluorescent filter cubes to identify neurons containing a nucleus with a visible nucleolus and to determine whether such neurons were YFP-positive. Image segments were collected at 200 magnification and combined to form a montage. Using ImageJ software (National Institutes of Health, Maryland), the cell area of all neurons containing a nucleus with a visible nucleolus from each chosen section was measured. We counted a minimum of 200 neurons per ganglion. If this number was not reached in the three sections chosen, a fourth section was counted, also in its entirety. However, because identification and counting continued even after the minimum of 200 neurons were obtained, we always counted more than 200 Nissl-stained neurons per DRG (mean 218±6 Nissl cells measured/DRG, 4.3% of the measured were YFP+). Histograms representing the cross-sectional area of all Nissl- and YFP-labeled DRG neurons measured were compiled in order to compare the distribution of the YFP-labeled cells with the total cell populations.
The primary, cell-type specific antibodies included anti glial fibrillary acidic protein (GFAP, mouse monoclonal, 1:1000, Chemicon, Millipore, Billerica, MA) to label astrocytes, anti-myelin oligodendrocyte glycoprotein (MOG, goat polyclonal, 1:200, R&D Systems, Minneapolis, MN) for labeling oligodendrocytes and anti-SC/2E (mouse monoclonal, 1:1000, Cosmo Bio USA, Carlsbad, CA) or laminin-1 (rat monoclonal, 1:200, Abcam, Cambridge, MA) to label Schwann cells. Mouse monoclonal antibodies to a synaptic vesicle protein, SV2 (1:10, Developmental Studies Hybridoma Bank, Iowa City, Iowa), or to synaptotagmin 2 (znp-1, 1:2000, Zebrafish International Resource Center, Eugene, OR) were used to label synaptic vesicles. To learn more about the phenotype of YFP+ DRG neurons, selected sections from L5 DRGs were labeled with one or more of the following methods: Neurons containing phosphorylated epitopes of high-molecular-weight neurofilament were identified using the SMI 312 antibody (mouse monoclonal antibody, 1:1000 dilution, Covance Inc., Princeton, NJ). The population of small primary afferent neurons that expresses the trkA neurotrophin receptor was labeled using an antibody to calcitonin gene-related polypeptide (CGRP, rabbit polyclonal antibody to rat CGRP, 1:2000, Bachem Americas, Torrance, CA). The population of small DRG neurons that does not express the trkA neurotrophin receptor was labeled using Griffonia simplicifolia IB4 lectin (biotin conjugate, 5µg/ml, Sigma-Aldrich, St. Louis, MO). Secondary antibodies used were Alexa 647-conjugated donkey anti-mouse 1:200, Invitrogen, Eugene, OR), Alexa-Fluor 568-conjugated goat anti-mouse IgG1 (1:200, Invitrogen, Eugene, OR), Alexa-Fluor 647-conjugated donkey anti-rabbit IgG (1:200, Invitrogen, Carlsbad, CA) and rhodamine-red conjugated rabbit anti-goat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).
Electron microscopy of the DREZ
The mice were perfused transcardially (with heparinized Tyrode’s solution followed by 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M Na-cacodylate buffer. The spinal cord segments L3–L6 were then removed as one piece and rinsed in 0.1M Na-cacodylate buffer, mounted on an agarose support, and placed in the vibratome well containing chilled buffer. The most superficial longitudinal slice containing the DREZ (0< 250µm thickness) was cut and further processed for electron microscopy. To target our electron microscopic analysis to the area where axons had stalled, we applied fiducial markers to the surface of the spinal cord slice. The spinal cord sections were flattened with insect pins in Sylgard silicone elastomer-lined 35-mm petri dishes. A 1.0% solution of 1, 1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine-5,5'-disulfonic acid (DiI, Invitrogen, Carlsbad, CA) was dissolved in dichloromethylene and loaded into a micropipette (resistance of 5–10 MΩ). Crystals of DiI were iontophoretically applied to the surface of the spinal cord slice in an area of the DREZ with bulb-tipped axons (e.g., see ). To render the DiI crystal electron dense, we excited the DiI crystals near their excitation wavelength in the presence of 3, 3’-diaminobenzidine (DAB, 5.0 mg/mL, Sigma-Aldrich) until the DiI crystal was replaced with a dark red/brown DAB precipitate (~20 minutes). After photoconversion, spinal cord slices were trimmed to contain the area of interest using the electron-dense fiducial markers as reference points. Tissue blocks were stained with 1.0% osmium tetroxide reduced in 1.5% potassium ferrocyanide for 45 minutes, then dehydrated in an ascending ethanol series, infiltrated with Araldite 502 Embed 812 resin, and polymerized at 60°C for 48 hours. Polymerized tissue blocks were sectioned (0.5 µm) with a glass knife on a Leica Ultracut R microtome (Leica, Wetzlar, Germany) until the fiducial markers were located. Serial ultrathin sections (60–70 nm) were cut and mounted on pioloform-coated slot grids. Sections were counterstained with 2.0% aqueous uranyl acetate and Reynold’s lead citrate. Sections were viewed at 75 kV on a Hitachi H-600 transmission electron microscope. Serial electron micrographs were captured at 6000× and scanned at a resolution of 1000 dpi.
Figure 11 Ultrastructural analysis of DR axons stopped at the DREZ, revealing presynaptic differentiation. The L5 root of a Thy1-YFPH mouse was crushed 13 days previously. A, Low-magnification, transmitted-light view, superimposed on fluorescence image, of the (more ...)