mdx Cv3 mouse is a model for electroretinography of Duchenne/Becker muscular dystrophy

Purpose. To identify an animal model for the abnormal scotopic electroretinogram found in a majority of Duchenne and Becker muscular dystrophy patients. Methods. Ganzfeld electroretinograms were recorded in dark-adapted normal C57BL/6 mice, and two strains of mice with different X-linked muscular dystrophy mutations {mdx and mdx Cv}). Responses for the right eye were averaged and the amplitudes and implicit times of the a-wave and b-wave were measured. The electroretinogram was digitally filtered to extract the oscillatory potentials. Statistical analyses included one-way analysis of variance and the Scheffe S test. Results. While the electroretinogram in mdx was normal, in mdx Cv3 the scotopic b-wave was markedly reduced and the oscillatory potentials were delayed, similar to changes observed in Duchenne and Becker muscular dystrophy patients. Some of the mdx <J " 3 animals demonstrated negative configuration electroretinograms, with the b-wave amplitude reduced compared to that of the a-wave. Conclusions. Abnormalities found in the electroretinograms of Duchenne and Becker muscular dystrophy patients led to the identification of dystrophin in human retina and the discover

Development and characterization of transfontanelle photoacoustic imaging system for detection of intracranial hemorrhages and measurement of brain oxygenation: Ex-vivo

We have developed and optimized an imaging system to study and improve the detection of brain hemorrhage and to quantify oxygenation. Since this system is intended to be used for brain imaging in neonates through the skull opening, i.e., fontanelle, we called it, Transfontanelle Photoacoustic Imaging (TFPAI) system. The system is optimized in terms of optical and acoustic designs, thermal safety, and mechanical stability. The lower limit of quantification of TFPAI to detect the location of hemorrhage and its size is evaluated using in-vitro and ex-vivo experiments. The capability of TFPAI in measuring the tissue oxygenation and detection of vasogenic edema due to brain blood barrier disruption are demonstrated. The results obtained from our experimental evaluations strongly suggest the potential utility of TFPAI, as a portable imaging modality in the neonatal intensive care unit. Confirmation of these findings in-vivo could facilitate the translation of this promising technology to the clinic

Snowflake Vitreoretinal Degeneration (SVD) Mutation R162W Provides New Insights into Kir7.1 Ion Channel Structure and Function

<div><p>Snowflake Vitreoretinal Degeneration (SVD) is associated with the R162W mutation of the Kir7.1 inwardly-rectifying potassium channel. Kir7.1 is found at the apical membrane of Retinal Pigment Epithelial (RPE) cells, adjacent to the photoreceptor neurons. The SVD phenotype ranges from RPE degeneration to an abnormal b-wave to a liquid vitreous. We sought to determine how this mutation alters the structure and function of the human Kir7.1 channel. In this study, we expressed a Kir7.1 construct with the R162W mutation in CHO cells to evaluate function of the ion channel. Compared to the wild-type protein, the mutant protein exhibited a non-functional Kir channel that resulted in depolarization of the resting membrane potential. Upon co-expression with wild-type Kir7.1, R162W mutant showed a reduction of I_Kir7.1 and positive shift in ‘0’ current potential. Homology modeling based on the structure of a bacterial Kir channel protein suggested that the effect of R162W mutation is a result of loss of hydrogen bonding by the regulatory lipid binding domain of the cytoplasmic structure.</p></div

Rb⁺ has no effect on Kir7.1 R162W.

<p>Average I–V plot of pEGFP-hKir7.1 (<b>A</b>) and pmCherry-R162W (<b>B</b>) transfected cells. The recordings were obtained in HEPES Ringer’s (Ctrl: open square), 135 mM extracellular K⁺ (closed triangle), or 135 mM extracellular Rb⁺ (open circle). Each data point is the mean ± the SEM of at least 5 experiments. (<b>C</b>) Comparison of the mean fold-increase in the current amplitude due to the exposure of cells to either 135 mM external Rb⁺ (gray bar) or 135 mM external K⁺ (white bar) measured at −140 mV. Error bars are ± SEM.</p

Cellular localization of the Kir7.1 channel.

<p><b>CHO</b> cells expressing the pEGFP-hKir7.1, pEGFP-R162W (<b>A</b>) or both pEGFP-hKir7.1+ pmCherry-R162W (<b>C</b>, <b>D</b>, <b>E</b>) were studied by live cell fluorescence microscopy using a 60X water immersion objective. Kir7.1 localized mainly to the plasma membrane (<b>A</b>. upper panel green: hKir7.1, red: ER and blue: nucleus) in the pEGFP-hKir7.1 transfected cells. pEGFP-R162W expression co-localized with ER labeling (<b>A</b>. middle panel). Control pEGFP expressing cells are shown in the lower panel (<b>A</b>). (<b>B</b>) Line scans (white arrow) of fluorescence intensity distribution of pEGFP-hKir7.1 (<b>A</b>. black trace upper panel), pEGFP-R162W (<b>A</b>. green trace middle panel), and pEGFP (<b>A</b>. dark green trace lower panel) transfected cells. Red and blue traces (<b>B</b>. upper panel and middle panel) represent ER labeling and Hoechst nucleus staining, respectively. In co-transfection experiments, the GFP fluorescence localized to the cellular membrane (<b>C</b>), whereas mCherry fluorescence shows an intracellular aggregated localization (<b>D</b>). Superposition of both red and green fluorescence (<b>E</b>) further illustrates that there is very little co-localization of the wild-type and mutant channel signals. (<b>F</b>) Fluorescence quantification of membrane vs. cytoplasmic expression from five independent co-transfections with pEGFP-hKir7.1 and pmCherry-R162W plasmids is shown in <b>F</b>, p<0.01.</p

Human Kir7.1 model and Kir channel family homology within the C-linker domain.

<p>(<b>A</b>) Tetrameric structural model of Kir7.1 protein and four interacting PIP₂ molecules. The highlighted structure is enlarged for clarity of the interactions between the C-terminal hotspot and the PIP₂ head group. (<b>B</b>) R162 interacts with PIP₂ through 3 hydrogen bonds as shown by the green dotted lines. (<b>C</b>) R162W structure showing the tryptophan residue and its side chain orientation with respect to PIP₂. (<b>D</b>) Comparison of the interaction of both R and W at position 162 with PIP₂ (green dotted line), along with the adjacent K-sharing hydrogen bond (purple dotted line). (<b>E</b>) Topology of the Kir7.1 subunit showing the relative position of the C-linker and Arg (R) 162 residue located adjacent to 2nd trans-membrane domain. (<b>F)</b> The conserved basic residues amongst Kir channels are indicated by upper-case letters. Disease mutations are highlighted by bold-face letters. Residues in the C-linker region are shaded. Numbers represent the first and last residues in the corresponding sequence. The species, name and accession numbers for proteins used for this comparison were as follows: hKir1.1 NM_000220, hKir2.1 NM_010603, hKir2.2 GI: 23110982, hKir3.1 NM_002239, hKir4.1 NM_002241, hKir5.1 NM_018658, hKir7.1 NM_002242, and cKir2.2 GI: 118097849.</p