Visualization 2: Whole-body live mouse imaging by hybrid reflection-mode ultrasound and optoacoustic tomography

Imaging of longitudinal uptake of IRDye-800CW by the bladder. Originally published in Optics Letters on 15 October 2015 (ol-40-20-4643

Visualization 1: Whole-body live mouse imaging by hybrid reflection-mode ultrasound and optoacoustic tomography

Video shows the fly-through the stack of ultrasound and optoacoustic single-wavelength images of a mouse acquired in-vivo. Originally published in Optics Letters on 15 October 2015 (ol-40-20-4643

Liver and gallbladder uptake of ICG.

<p>a) Optoacoustic images through the liver. Grayscale image (left) showing anatomy and ROIs for liver (red) and gallbladder (yellow) analysis. b) FCSI image: fluorescence from ICG overlaid in green on color photograph of cryosection of a mouse sacrificed 10 minutes after injection, showing signal in the liver and gallbladder c) Plot of the signal increase in the liver ROI at 800 nm during single wavelength imaging of the ICG injection. Oscillations are mainly due to breathing motion. The scale is normalized to the maximum value. d) Specific (unmixed) signal from ICG after injection in the liver (black) and gallbladder (blue) ROIs. Each curve is normalized to its own maximum value.</p

Increased mRNA expression of <i>Nrf2</i> and ARE-driven genes in cortical neurons increases glutathione content and resistance to oxidative stress.

<p>A) Total RNA was extracted from pure neuronal cultures and <i>Nrf2</i>, <i>Nqo1</i>, <i>Ho-1</i>, <i>Gclm</i>, <i>Gclc</i> and <i>Keap1</i> mRNA levels were determined by real-time PCR and corrected by <i>β-actin</i> mRNA levels. mRNA levels are expressed as percentage of Thy1-Cre(-)/caNrf2(-) neurons. B) Increased total glutathione (GSH+GSSG) content in Thy1-Cre(+)/ca-Nrf2(+) cortical neurons. C) Neuronal cultures were treated with the indicated concentrations of H₂O₂ and toxicity was determined 24hs later by LDH release assay. Data are expressed as percentage of their respective control. A, B and C, Data are expressed as mean±SD (n≥3). *Significantly different from Thy1-Cre(-)/caNrf2(-) (p≤0.05).</p

Overexpression of ARE-driven genes in Thy1-Cre(+)/caNrf2(+) mice.

<p>Increased mRNA expression levels of Nrf2 and ARE-driven genes in the cortex (A) and spinal cord (B) from 30 days old animals of the indicated genotypes. <i>Nrf2</i>, <i>Nqo1</i>, <i>Ho-1</i>, <i>Gclm</i>, <i>Gclc</i> and <i>Keap1</i> mRNA levels were determined by real-time PCR and corrected by <i>β-actin</i> mRNA levels. No difference in <i>Keap1</i> mRNA levels was observed. Data are expressed as mean±SD (n≥3). *Significantly different from Th1-Cre(-)/caNrf2(-) (p≤0.05). C) hPAP histochemistry in lumbar spinal cord sections from 30 days old mice of the indicate genotypes. Scale bar: 50 µM.</p

Selective overexpression of Nrf2 in type II muscle fibers delays onset but does not extend survival in hSOD1^G93A mice.

<p>A) Increased mRNA expression of Nrf2 and ARE-driven genes in the gastrocnemius muscle from 30 days old non-transgenic (NonTG) and MLC-Nrf2 mice. <i>Nrf2</i>, <i>Nqo1</i>, <i>Ho-1</i>, <i>Gclm</i> and <i>Gclc</i> mRNA levels were determined by real-time PCR and corrected by <i>β-actin</i> mRNA levels. Data are expressed as mean±SD (n≥3). *Significantly different from NonTG (p≤0.05). B) Increased total glutathione (GSH+GSSG) content in the gastrocnemius muscle from 30 days old NonTG and MLC-Nrf2 mice. Data are expressed as mean±SD (n≥3). *Significantly different from NonTG (p≤0.05). C) hPAP histochemistry in muscle sections from 30 days old mice of the indicate genotypes. Scale bar: 50 µM. D) Median onset in MLC-Nrf2(-)/hSOD1^G93A (107 days, n = 17) and MLC-Nrf2(+)/hSOD1^G93A (115.5 days, n = 12) mice. Onset curves are significantly different (p≤0.01, χ² = 6.7). E) Median survival in MLC-Nrf2(-)/hSOD1^G93A (154 days, n = 17) and MLC-Nrf2(+)/hSOD1^G93A (150.5 days, n = 12) mice. Survival curves are not significantly different. F) Disease duration (time from onset to death). All animals are hSOD1^G93A positive. Data are expressed as mean±SD (n as for D and E).</p

Selective overexpression of Nrf2 in neurons delays onset but does not extend survival in hSOD1^G93A mice.

<p>A) Median onset in Thy1-Cre(-)/caNrf2(-)/hSOD1^G93A (107 days, n = 10), Thy-1Cre(+)/caNrf2(-)/hSOD1^G93A (109 days, n = 12), Thy1-Cre(-)/caNrf2(+)/hSOD1^G93A (115 days, n = 7) and Thy1-Cre(+)/caNrf2(+)/hSOD1^G93A mice (122.5 days, n = 12). Onset curves are significantly different (p≤0.05, χ² = 10.6). B) Median survival in Thy1-Cre(-)/caNrf2(-)/hSOD1^G93A (155 days, n = 10), Thy-1Cre(+)/caNrf2(-)/hSOD1^G93A (157.5 days, n = 12), Thy1-Cre(-)/caNrf2(+)/hSOD1^G93A (156 days, n = 7) and Thy1-Cre(+)/caNrf2(+)/hSOD1^G93A mice (163.5 days, n = 12). Survival curves are not significantly different. C) Disease duration (time from onset to death). All animals are hSOD1^G93A positive. Data are expressed as mean±SD (n as for A and B).</p

<i>The</i> β<i>-geo</i> gene trap cassette was inserted in <i>Sms1</i> intron 3 and leads to disruption of testes-specific transcripts and protein expression.

<p>(A) Schematic drawing of the β-geo gene trap cassette (yellow bars) and its insertion in intron 3 of the <i>Sms1</i> gene. Coding exons are indicated by brown colour. Genotyping primers (TMEM23_fw, TMEM23_rv and Spli_rv2) are indicated as black arrowheads. The location of the <i>lacZ</i> probe used for Northern blotting is depicted by a grey line. (B) Mice were genotyped by triplet-PCR. The wild-type allele: 999bp, mutant allele 577bp; Ladder (L), heterozygous (+/-), wild-type (+/+), homozygous (-/-). (C) Schematic illustration of the four protein coding splice variants. Red line illustrates the gene trap insertion site. <i>Sms1</i> specific Northern blot probe, (801bp), is indicated by a grey bar. (D) Northern blot of testes RNA revealed a reduction of testes specific (<i>Sms1</i>_001, _002, _004) <i>Sms1</i> splice variants in <i>Sms1</i>^-/- animals. (E) Quantification of the <i>Sms1</i>_001 and <i>Sms1</i>_004 splice variants in testes of <i>Sms1</i>^-/- in comparison to <i>Sms1</i>^<i>WT</i> animals. (F) Immunoblot analysis of testes tissue with HPRT as a loading control. (G) Quantification of testes SMS1 protein levels of <i>Sms1</i>^<i>WT</i> and <i>Sms1</i>^-/-. <i>Sms1</i>^<i>WT</i> (n = 3), <i>Sms1</i>^-/- (n = 3). Data is shown as mean ± SEM, *p≤0.05, **p≤0.01, ***p≤0.001.</p

Histological analysis of testes and epididymides of <i>Sms1</i>^-/- males indicates impaired spermatogenesis.

<p>(A, B) H&E staining of <i>Sms1</i>^<i>WT</i> (A) and <i>Sms1</i>^-/- (B) accumulation of round cells (black arrow heads) in the <i>Sms1</i>^-/- epididymal lumen, instead of Sz (black arrow in A). Scale bar: 50μm. (C–F) IHC for SMS1. (C,E) <i>Sms1</i>^<i>WT</i>; (D,F) <i>Sms1</i>^-/-. (G, H) IHC for SMS2. (G) <i>Sms1</i>^<i>WT</i>, (H) <i>Sms1</i>^-/-. Scale bar: 20μm. (I, J) TUNEL staining of <i>Sms1</i>^<i>WT</i> and <i>Sms1</i>^-/- testes. (I) Apoptosis (black arrows) in <i>Sms1</i>^<i>WT</i> seminiferous tubules (left panel), <i>Sms1</i>^-/- (right panel, scale bar: 200μm). (J) Higher magnification revealed apoptotic clusters near the basal lamina and apoptotic material in the tubule lumen of <i>Sms1</i>^-/- seminiferous tubules. (K) Quantification of TUNEL-positive events per tubule revealed a higher rate of apoptosis in <i>Sms1</i>^-/- testes compared to <i>Sms1</i>^<i>WT</i>. Scale bar: 20μm. Blood-testis-barrier (BTB), blood-epididymis barrier (BEB), spermatocyte (Sc), Sertoli-cell only phenotype (SCO), spermatozoa (Sz), spermatid (Spt), elongating spermatid (elSpt).</p

Lipid profile of <i>Sms1</i> mutant testes PCaa, PCae and lysoPC species, compared to <i>Sms1</i> wild-type levels.

<p>Lipid levels with the highest intensities are shown for diacylphosphatidylcholines (PCaa, A) plasmalogens (PCaes, B) and lyos-phosphatidylcholines (lysoPCs, C). Additionally, predicted major fatty acids are listed. Data is presented as mean percentage ± SEM, with wild-type levels set to 100% (<i>Sms1</i> wild-type, n = 15; <i>Sms1</i> mutant, n = 17); *p≤0.05, **p≤0.01, ***p≤0.001.</p