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1Gladstone Institute of Neurological Disease, University of California, San Francisco, 2Department of Neurology, University of California, San Francisco
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A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.
Davalos, D., Akassoglou, K. In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy. J. Vis. Exp. (59), e2760, doi:10.3791/2760 (2012).
In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.
1. Building the spinal stabilization device
2. Animal surgery
3. Stabilization of the spinal column and preparation for in vivo imaging
4. In vivo imaging of the mouse spinal cord with two-photon microscopy
5. Repetitive imaging and post-operative care
6. Representative results
All animal procedures were performed under the guidelines set by institutional Animal Care and Use Committees at the University of California, San Francisco and are in accordance with Federal regulations. A picture of the spinal stabilization device and a schematic showing the positioning of a mouse on the device under a microscope lens is shown in Figure 1. Allowing adequate room for breathing movements underneath the animal's body ensures stable in vivo imaging in the spinal cord. Figure 2 shows the close relationship between microglia and the vasculature as it was imaged in vivo in the spinal cord of Cx3cr1GFP/+ transgenic mice18, in which microglia are endogenously labeled with GFP Figure 3 shows examples of repetitive in vivo imaging as it was performed in the same spinal cord areas in mice expressing a fluorescent protein in spinal axons (YFP-H line 3) and microglia (in the Cx3cr1GFP/+ mice).
Figure 1. Stabilization of the mouse spinal column for in vivo imaging using two-photon microscopy. (A) A custom-made steel base plate is used to support and align the STS-A Narishige compact spinal cord clamps and the MA-6N Narishige head holding adaptor as shown here. (B) Proper positioning of adult transgenic mice anesthetized with a KXA anesthetic on the spinal stabilization device. The insert shows the placement of the spinal clamps immediately rostrally and caudally to the laminectomy and the exposed spinal cord tissue.
Figure 2. In vivo imaging of high density microglial cells and blood vessels in the spinal cord of anesthetized mice. Projected but non-aligned z-stacks of (A) highly dense GFP-positive microglia (green) in the spinal cord of a CX3CR1GFP/+ mouse in close proximity with blood vessels (red, labeled with i.v. injection of rhodamine dextran). (B) High magnification image labeled as in (A) of a single microglial cell attached to the wall of a blood vessel with processes extended around the vessel and towards the spinal cord parenchyma. Scale bars, 10μm.
Figure 3. Repetitive in vivo imaging of the same axonal segments and microglia as they were relocated and reimaged in the spinal cord of anesthetized mice on different days. (A) YFP-labeled axons on days 0 and 5. (B). The same vascular structures and microglial cells around them imaged in vivo in the spinal cord of Cx3cr1GFP/+ mice on days 0 and 1. Scale bars, 10μm.
Movie 1. Representative timelapse sequence acquired in vivo from the mouse spinal cord. This sequence shows in detail fine microglial process dynamics (green) and their interactions with the vasculature (red, labeled with i.v. injection of rhodamine dextran) over time within a tissue densely populated with fluorescent structures. The raw images were only corrected for background noise, brightness and contrast and the timelapse movie was constructed by z-projecting sequentially acquired image stacks, without image alignment, averaging or z-selection of individual planes. Blood vessels go through a similar range of z planes as the images of microglia. Z plane depth: 38 μm. Click here to watch the movie.
Movie 2. Timelapse sequence demonstrating the raw stability of the imaging method at the level of a single z-plane. Fast acquisition of the same single z-plane in the spinal cord of CX3CR1GFP/+ mice placed on the spinal stabilization device under KXA anesthesia, demonstrates minimal image displacement between consecutive frames at a scanning rate of 1 frame/s that is faster than the breathing rate of the mouse. The minor residual displacement is probably due to heartbeat. Click here to watch the movie.
The method described here allows for stable and repetitive in vivo imaging of densely populated fluorescent cellular structures in the spinal cord of anesthetized mice using two-photon microscopy. The achieved stability is a result of a custom-made spinal stabilization device and an anesthetic regimen that reduces respiratory-induced movement artifact. The spinal stabilization device allows breathing space underneath the mouse body and can be built using commercially available spinal clamps and head mounting piece (Fig. 1). The method is highly reproducible, minimally invasive and consistently generates raw data that can be used for experimental analyses without extensive image post-processing (Fig.2 and Video 1). This technique can therefore be used for studies of cell-cell interactions in the abundance of commercially available fluorescent mouse lines (Figs. 1-3 and Video 1). This method can resolve not only densely populated cellular structures but also rapidly occurring cellular functions or responses (Videos 1, 2). For example, it can be used to quantitatively measure microglial process dynamics over time as well as distance travelled during chemotactic behavior. Quantification can be performed as we previously described for imaging microglial responses in the living brain8. Moreover, it can be used in combination with other experimental approaches such as the use of fluorescently labeled microspheres to measure microglial phagocytosis in the spinal cord in vivo.
The ability to perform repetitive in vivo imaging of the exact same area in the spinal cord of the same animals on different days allows studying the progression of biological phenomena and the dissection of the sequence of events that could for example be involved with the development of disease. Successful application of this method for longitudinal studies relies on the availability of tissue landmarks for reliably relocating the areas that were previously imaged. This needs to be taken into account in particular for spinal cord injury studies. For example in some of the commonly used spinal cord injury models such as spinal cord contusion or dorsal hemisection the massive necrotic lesion that is generated at the epicenter of the injury causes significant axonal and vascular rearrangement that might hinder relocation of the previously imaged area. This can be overcome by either performing repetitive in vivo imaging at the lesion edge or by selecting an injury model that causes a targeted, smaller lesion, such as the thin needle transection model19.
The method described here exposes a small segment of the spinal cord, by performing a single laminectomy over the target imaging area. The underlying dura matter has been left intact where possible. This ensures minimal pertubation of the imaged tissue underneath the meninges and minimizes the injury incurred to the animal's spine. The overall stabilization scheme can easily be adapted to image either through the interlaminar space without performing a laminectomy 20 or through multiple serial laminectomies if a smaller or a larger imaging window is preferred for a given study respectively.
As is common with most in vivo techniques, the results obtained by using this in vivo imaging method may greatly depend on the use of proper anesthesia. The KXA anesthetic mix that is recommended here has also been used in imaging studies of different tissues before 21-24 and was selected exclusively based on its ability to significantly reduce breathing movements. Other anesthetic approaches may produce comparable results to this KXA mix.
The spinal cord imaging technique described in this protocol provides a powerful tool for spinal cord research, since the ability to record cell-cell interactions in real time can greatly facilitate in vivo studies of the spinal cord in physiology and pathology.
We have nothing to disclose.
This work was supported by the National Multiple Sclerosis Society grant RG4595A1/T to DD and the NIH/NINDS grants NS051470, NS052189 and NS066361 to K.A. Figures and movies adapted and/or reprinted from Davalos et al., J Neurosci Methods. 2008 Mar 30;169(1):1-7 Copyright 2008, with permission from Elsevier.
|Rhodamine B dextran||Invitrogen||D1841||70 kDa, diluted in
ACSF (3% w/v)
|Ketamine HCl||Bioniche Pharma||NDC No: 67457-001-10||Injectable, 50mg/ml|
|Anased||Lloyd, Inc.||NADA No: 139-236||Xylazine injectable,
|Acepromazine||Vedco, Inc.||NADA No: 117-531||Injectable,10mg/ml|
|Artificial tears ointment||Phoenix Pharmaceuticals, Inc.||NDC No: 57319-760- 25||Lubricant|
|McPherson-Westcott Scissors||World Precision Instruments, Inc.||555500S||Curved, blunt-tipscissors|
|Straight Forceps||World Precision Instruments, Inc.||555047FT||Toothed tip forceps|
|Small vessel cauterize||Fine Science Tools||18000-00|
|Gelfoam||Pharmacia Corporation (Pfizer)||Mixer Mill MM400|
|Compact spinal cord clamps||Narishige International||STS-A|
|Head holding adaptor||Narishige International||MA-6N|
|Lactated Ringers||Baxter Internationl Inc.||2B8609|
|Buprenex||Reckitt Benckiser||NDC No: 12496- 6757-1||Buprenorphine,injectable|
|Baytril||Bayer AG||NADA 140-913||Enrofloxacin,
|Heating pad - Large||Fine Science Tools||21060-10|
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