In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

Summary

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.

Abstract

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.

Protocol

1. Building the spinal stabilization device

1. Order the Narishige STS-A Compact Spinal Cord Clamps and the Narishige MA-6N head holding adaptor.
2. Custom design and make a stainless steel base plate to hold the two Narishige parts in alignment so that the animal's head is supported while its spinal column and tail are clamped. Keep in mind that the entire device should fit under the microscope lens usually on a lowered microscope stage.

2. Animal surgery

1. Pre-heat the microscope chamber with an air heating device and maintain it at 37 °C.
2. Weigh the animal and make the anesthetic mix using 100 mg....

Discussion

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 pie.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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, 20mg/ml
Acepromazine	Vedco, Inc.	NADA No: 117-531	Injectable,10mg/ml
Artificial tears ointment	Phoenix Pharmaceuticals, Inc.	NDC No: 57319-760- 25	Lubricant
Betadine	Fisher Scientific	19-061617
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
Gelseal	Amersham	80-6421-43
Lactated Ringers	Baxter Internationl Inc.	2B8609
Buprenex	Reckitt Benckiser	NDC No: 12496- 6757-1	Buprenorphine,injectable
Baytril	Bayer AG	NADA 140-913	Enrofloxacin, antibacterial injectable 2.27% (20ml)
Heating pad - Large	Fine Science Tools	21060-10