Long-Term In Vivo Imaging of Multiple Organs at the Single Cell Level

<div><p>Two-photon microscopy has enabled the study of individual cell behavior in live animals. Many organs and tissues cannot be studied, especially longitudinally, because they are located too deep, behind bony structures or too close to the lung and heart. Here we report a novel mouse model that allows long-term single cell imaging of many organs. A wide variety of live tissues were successfully engrafted in the pinna of the mouse ear. Many of these engrafted tissues maintained the normal tissue histology. Using the heart and thymus as models, we further demonstrated that the engrafted tissues functioned as would be expected. Combining two-photon microscopy with fluorescent tracers, we successfully visualized the engrafted tissues at the single cell level in live mice over several months. Four dimensional (three-dimensional (3D) plus time) information of individual cells was obtained from this imaging. This model makes long-term high resolution 4D imaging of multiple organs possible.</p> </div

Visualizing radiation-induced thymocyte apoptosis in ear-thymus graft.

<p>A nude BALB/c chimera containing DsRed⁺ hematopoietic cells were transplanted with a thymus from a B6 CD45.1 neonatal mouse (<48 hours old) into the ear pinna. Five weeks later, the ear pinna containing thymus graft was irradiated with 8.5 Gy. Cell apoptosis was then followed over time by in vivo two-photon imaging after injection of FAM-FLIVO. The images were taken 113 to 137 µm deep from surface. Representative pictures from each group are shown. The percentages of apoptotic cells are shown. Similar experiments have been repeated for three times. Green = EGFP; red = apoptotic cells. Scale bar = 50 µm.</p

Histological analysis of adult tissues transplanted into ear pinnae (H&E stain, scale bar = 100 µm).

<p>B6 CD45.1 adult tissues or pieces of organs were subcutaneously transplanted into syngeneic mouse ear pinnae. Ear-tissues were harvested at different time points post transplantation. Similar experiments have been repeated for at least 3 times.</p

Ear-tissue can be visualized at the cellular level in living animal.

<p>(a) This picture showing the location of an engrafted heart graft in the ear pinna. (b) Skeletal muscle and small intestine tissues from EGFP mice were subcutaneously transplanted into the ear pinnae of BALB/c nude mice. The images were obtained 9 (muscle) and 22 (intestine) weeks after transplantation. The images were taken 107 µm (muscle) deep from surface. The depth for intestine could not be determined. (c) Heart and kidney tissues from EGFP mice were subcutaneously transplanted into the ear pinnae of BALB/c nude mice. Rhodomin B conjugated dextran was injected i.v. to visualize the blood vessels. The images were obtained 9 (heart) and 2 (kidney) weeks after transplantation. Green = EGFP; red = dextran. (d) EGFP⁺ neonatal thymic tissue was subcutaneously transplanted into the ear pinnae of BALB/c nude mice. Eight weeks later, the mice were irradiated and transplanted with DsRed⁺ T cell depleted bone marrow cells. The image was taken 4 weeks after bone marrow transplantation at the depth of 103 µm. Green = EGFP; red = DsRed. Scale bar = 100 µm.</p

T cell reconstitution in mice engrafted with ear-thymus.

<p>Thymuses from B6 CD45.1 neonatal mice (<48 hours old) were transplanted into the ear pinnae of BALB/c nude mice. T cell reconstitution was monitored phenotypically and functionally. (a) Thymic grafts were harvested 5 months after transplantation. siTREC numbers were determined in thymocytes. This is a representative of three similar experiments. Each group contained 3 animals. P = not significant. (b&c) Phenotypic T cell recovery. Peripheral T cell recovery was monitored in peripheral blood by flow cytometry. This is a representative of two similar assays with similar results. Each group contained more than 16 animals. ^*P<0.05; (d&e) Skin transplantation. More than three months after ear-thymic transplantation, ear-thymus recipients were transplanted with third-party C3H/HeJ skin grafts. Survival of skin grafts was monitored daily after transplantation. Pictures (d) were taken 5 weeks after skin grafting. The combined results from two similar experiments are shown. Each group contained 10–12 animals.</p

Histological analysis of fetal tissues transplanted into ear pinnae (H&E stain, scale bar = 100 µm).

<p>B6 CD45.1 near-term fetal tissues were subcutaneously transplanted into syngeneic mouse ear pinnae. Ear-tissues were harvested at different time points post transplantation. Similar experiments have been repeated for at least 3 times.</p

LOD plot of PR interval (black, left y-axis) and <i>Tnni3k (purple</i> right y-axis) on chromosome 3.

<p>LOD plot of PR interval (black, left y-axis) and <i>Tnni3k (purple</i> right y-axis) on chromosome 3.</p

Genotype effects at rs13477506.

<p>(A) <i>Tnni3k</i> expression (Illumina probe ILMN_3023962) as a function of genotype at rs13477506 in F2 mice; homozygous 129P2: AA, green; 129P2-FVBN/J heterozygous: AB, turquoise; homozygous FVBN/J: BB, blue; the darker shades represent the independent validation of the <i>Tnni3k</i> transcription levels by Q-PCR (right y-axis). (B) PR interval as a function of genotype at rs13477506 in F2 mice. Error bars indicate standard errors.</p

Overview of the locations of the linkage regions on chromosome 3.

<p>(A) Entire mouse chromosome 3 with refseq genes indicated in dark blue, black bar: 1.5 LOD drop of the PR-QTL; dark red bar 20 Mb DBA.AKR congenic region; purple bar 1.5 LOD drop of the Tnni3k eQTL; grey bar minimal region of overlap. (B) 1 Mb close up of the minimal region of overlap; the positions of rs49812611 (associated with nonsense mediated decay in DBA/2J) and rs13477506 (QTLs) are indicated. (C) Haplotypes of a panel of 9 inbred mouse strains as determined by the mouse phylogeny viewer (<a href="http://msub.csbio.unc.edu/" target="_blank">http://msub.csbio.unc.edu/</a>) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003113#pgen.1003113-Yang1" target="_blank">[19]</a>.</p

<i>Tnni3k</i> prolongs the PR interval.

<p>(A) Congenic mice carrying the AKR/J green haplotype in a DBA/2J genetic background display the green haplotype PR interval duration. Overexpression of tagged <i>hTNNI3k</i> in a DBA/2J background significantly prolongs the PR interval. Colors show the haplotype of each strain at the <i>Tnni3k</i> locus, error bars indicate standard deviations. (B–D) Examples of ECG traces of DBA/2J (B), AKR.DBA.<i>hrtfm2</i> congenic (C) and DBA/2J overexpressing h<i>TNNI3K</i> (D).</p