Studies of pathogens in small animal models, most often in mice, usually depend on observations of clinical symptoms, as well as sacrifice and harvesting of tissues for use in histopathology or in assays for the acutely replicating pathogen. Such methods have been standard in the field for many years and have yielded a great deal of useful information. The major drawback of such approaches, however, is that the sequential sacrifice of mice precludes the subsequent observation of microbiological, clinical, behavioral, and other out-

From: Methods in Molecular Biology, vol. 292: DNA Viruses: Methods and Protocols Edited by: P. M. Lieberman © Humana Press Inc., Totowa, NJ

comes in the mice being sampled. Significant insights, therefore, from animal-to-animal variations in host-pathogen interactions and therapeutics are therefore likely to be missed. Furthermore, the ability to perform serial studies on the same animal would allow the experimenter to better interpret such animal-to-animal variation.

New imaging technology now allows real-time in vivo imaging of luciferase reporter genes in living mice using a cooled charge-coupled device (CCD) camera (1,2). Because bioluminescence imaging (BLI) has minimal background, the technique is very sensitive for detecting sites of luciferase activity (3). The substrate for firefly luciferase, d-luciferin, crosses cell membranes and the intact blood-brain barrier after intraperitoneal injection, thereby enabling luciferase activity to be detected in any anatomic site in the living mouse. In addition, the low immunogenicity and negligible toxicity of luciferin allows repetitive imaging of the same mouse. Relative amounts of bioluminescence produced in vivo can be quantified by region-of-interest (ROI) analysis, which facilitates comparisons among mice in various experimental groups (2,4). The sensitivity for detecting bioluminescence with BLI is determined by the combination of signal strength and anatomic location. Because hair and overlying tissues scatter and absorb light, luciferase activity is detected more readily in superficial than internal sites in an animal. Darkly pigmented organs and tissues, such as liver, spleen, and skin of some mouse strains, also attenuate light transmission. Nevertheless, BLI can readily detect luciferase activity in internal organs such as brain, liver, lung, and spleen (5,6). Other limitations of the technique include the 2-3-mm spatial resolution of BLI and the 2D imaging capability of first-generation instruments. Nevertheless, BLI provides certain advantages over use of position emission tomography (PET) or magnetic resonance imaging (MRI) in terms of cost, feasibility, throughput, and simplicity for realtime imaging of infectious diseases.

A large number of bacteria and viruses have now been studied using BLI (7-11). Any recombinant microbe to be used for in vivo pathogenesis studies with this methodology ideally should contain a luciferase transgene that is expressed at the highest possible levels in all infected cells. Importantly, the transgene should have minimal, if any, effects on natural pathogenesis of the microbe. We describe here the use of a herpes simplex virus type 1 (HSV-1) recombinant that has been used very successfully in combination with BLI to elucidate the spread and tropism of HSV-1 in vivo in both wild-type and knockout mice (10-12). The method described here elucidates the background information of the generation of the particular virus we have used in our study. Obviously such a description will need to be adapted to the particular needs of the investigator, but it should give some useful general points for the design and generation of other viruses. We anticipate that this methodology will prove use-

ful to many investigators for the study of the pathogenesis of HSV-1 and other viruses.

2. Materials

2.1. Virus Infection Materials

1. High titer (> 4 x 108 PFU/mL) stock of KOSDlux/oriL (see Note 1).

2. Specific pathogen-free 6-8-wk-old mice (see Note 2).

3. Ketamine (Ketaset, Fort Dodge Animal Health).

4. Xylazine (Boehringer Ingelheim Vetmedica).

5. 25-Gage 5/8-inch needles (Becton Dickinson).

6. 28-Gage 1/2-inch U-100 tuberculin syringes (Becton Dickinson).

7. Eppendorf P10 pipetor and tips.

1. CCD camera and computer analysis software (IVIS, Xenogen) (see Note 3).

2. D-Luciferin (Xenogen, Alameda, CA), prepared as a 15 mg/mL stock solution in phosphate-buffered saline (PBS). The solution is sterile-filtered and stored in aliquots at -20°C. The compound is light-sensitive, so the reagent and solution should be protected from light as much as possible.

3. Isoflurane anesthesia delivery system (Harvard) with nose cones and tank.

4. Black paper to use on the imaging shelf of the CCD camera. We use Art Again black paper from Strathmore, which does not produce any background light.

6. Balance.

7. 70% Ethanol solution for decontaminating imaging equipment.

3. Methods

3.1. Infection of Mice

1. Anesthetize mice with 1.75 mg of ketamine and 0.26 mg of xylazine by intraperitoneal (ip) injection. The anesthesia will begin after approx 5 min and last for 15 min.

2. For corneal infection, lightly abrade the cornea with eight interlocking strokes of a 25-gage needle. Drop HSV-1 in a 5 |L vol onto both eyes at a maximum concentration of 2 x 106 PFU per eye. Gently massage the eyes with the lids closed for 10 s.

3. For footpad injection, using a 0.5-mL tuberculin syringe, inject up to 20 |L of virus subcutaneously into the footpad.

3.2. Animal Preparation for Imaging

1. To shave mice, anesthetize animals with 2% isoflurane or other approved anesthetic, and use electric clippers to remove hair from areas of interest. Shaving is repeated as needed over the course of an experiment, typically every 4-5 d (see Note 4).

2. Label individual mice with a permanent identification marker, such as an ear punch. If mice were anesthetized for shaving, this is a convenient time to perform the ear punch. To allow rapid identification of mice, use a permanent marker to label the tail of each mouse with the appropriate number.

3. Weigh each mouse in a tared plastic beaker. Anesthesia is not necessary to obtain reliable weights.

3.3. CCD Camera Setup

1. Select the smallest field of view (FOV) that will accommodate the desired number of mice to be imaged at one time (see Note 5). For all our pathogenesis studies, we use the smallest FOV (10 cm) that allows two normal-sized adult mice to be imaged simultaneously.

2. Select the size of image matrix to optimize sensitivity vs spatial resolution. We use a 128 x 128 matrix to enhance camera sensitivity for studies with KOSDlux/oriL.

3. Define image acquisition time. We begin with a 1-min image and subsequently adjust acquisition time based on amounts of bioluminescence.

3.4. Imaging

1. Inject D-luciferin ip at a dose of 15 mg/kg from the 15 mg/mL stock solution, using a 28-gage, 0.3- or 0.5-mL syringe (see note 6).

2. Return animals to cages for 5 min to allow distribution of luciferin throughout tissues.

3. Anesthetize mice with 2% isoflurane, which typically requires 2-3 min.

4. Transfer mice to the stage of the CCD camera, placing anatomic sites of interest closer to the camera. Anesthesia is maintained with 2% isoflurane delivered via nose cones.

5. Begin imaging 10 min after injection of luciferin. Initially, the system acquires a gray-scale photograph of mice. Check this gray-scale image to determine that animals are centered properly under the camera. If animals need to be repositioned, stop the image acquisition and move mice as needed.

6. The system proceeds automatically to the bioluminescent photograph, using the selected imaging parameters.

7. Based on the initial image, adjust matrix size and/or acquisition time as needed to detect bioluminescence without saturating the CCD camera.

8. Obtain images from as many positions as desired. Bioluminescence is automatically presented as a pseudocolor representation superimposed on the gray-scale photograph of each mouse (Fig. 1).

9. Remove mice from the CCD camera and return to cages. Animals typically recover within 5 min.

3.5. Image Analysis

1. Define a uniform minimum threshold for pseudocolor display of relative amounts of light on images (see Note 7).

2. Manually define ROIs for all desired anatomic sites for each mouse (Fig. 1). Use the saturation map feature of the processing software to avoid quantifying light emission from pixels that saturate the CCD camera.

Fig. 1. Bioluminescence imaging 4 d after corneal infection with KOSDlux/oriL. (A) Gray-scale photograph of mouse. (B) Bioluminescence superimposed on gray-scale photograph of mouse. Relative levels of bioluminescence are depicted as a pseudocolor display, with red and blue representing the highest and lowest amounts of photon flux. (C) After applying a minimum threshold value to the image, a ROI is manually defined around the light projected from the eye and periocular tissues.

Fig. 1. Bioluminescence imaging 4 d after corneal infection with KOSDlux/oriL. (A) Gray-scale photograph of mouse. (B) Bioluminescence superimposed on gray-scale photograph of mouse. Relative levels of bioluminescence are depicted as a pseudocolor display, with red and blue representing the highest and lowest amounts of photon flux. (C) After applying a minimum threshold value to the image, a ROI is manually defined around the light projected from the eye and periocular tissues.

3. Determine background bioluminescence from an ROI of the same size. This may be obtained from a mock-infected mouse that was injected with luciferin.

4. Measure bioluminescence units of photon flux (photons/s) in each ROI to correct for differences in image acquisition time.

5. Subtract background bioluminescence from photon flux in each ROI of interest to quantify relative luciferase activity as a measure of amounts of virus.

6. If desired, summarize ROI data over the entire experiment by area-under-the-curve (AUC) analysis, using Kaleidograph (Synergy Software) or other appropriate data analysis software.

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