Publications by authors named "Alison L Huckenpahler"

8 Publications

  • Page 1 of 1

Noninvasive Imaging of Cone Ablation and Regeneration in Zebrafish.

Transl Vis Sci Technol 2020 09 16;9(10):18. Epub 2020 Sep 16.

Cell Biology, Neurobiology & Anatomy, Medical College of Wisconsin, Milwaukee, WI, USA.

Purpose: To observe and characterize cone degeneration and regeneration in a selective metronidazole-mediated ablation model of ultraviolet-sensitive (UV) cones in zebrafish using in vivo optical coherence tomography (OCT) imaging.

Methods: Twenty-six zebrafish were imaged with OCT, treated with metronidazole to selectively kill UV cones, and imaged at 1, 3, 7, 14, 28, or 56 days after ablation. Regions 200 × 200 µm were cropped from volume OCT scans to count individual UV cones before and after ablation. Fish eyes were fixed, and immunofluorescence staining was used to corroborate cone density measured from OCT and to track monocyte response.

Results: Histology shows significant loss of UV cones after metronidazole treatment with a slight increase in observable blue cone density one day after treatment (Kruskal, Wallis, = 0.0061) and no significant change in blue cones at all other timepoints. Regenerated UV cones measured from OCT show significantly lower density than pre-cone-ablation at 14, 28, and 56 days after ablation (analysis of variance, < 0.01, < 0.0001, < 0.0001, respectively, 15.9% of expected nonablated levels). Histology shows significant changes to monocyte morphology (mixed-effects analysis, < 0.0001) and retinal position (mixed-effects analysis, < 0.0001).

Conclusions: OCT can be used to observe loss of individual cones selectively ablated by metronidazole prodrug activation and to quantify UV cone loss and regeneration in zebrafish. OCT images also show transient changes to the blue cone mosaic and inner retinal layers that occur concomitantly with selective UV cone ablation.

Translational Relevance: Profiling cone degeneration and regeneration using in vivo imaging enables experiments that may lead to a better understanding of cone regeneration in vertebrates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1167/tvst.9.10.18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7500127PMC
September 2020

Noninvasive Imaging and Correlative Histology of Cone Photoreceptor Structure in the Pig Retina.

Transl Vis Sci Technol 2019 Nov 18;8(6):38. Epub 2019 Dec 18.

Department of Ophthalmology and Visual Sciences, University of Louisville, Louisville, KY, USA.

Purpose: To evaluate different methods of studying cone photoreceptor structure in wild-type (WT) and transgenic pigs carrying the human rhodopsin P23H mutant gene (TgP23H).

Methods: For in vivo imaging, pigs were anesthetized with tiletamine-zolazepam and isoflurane and given lidocaine-bupivacaine retrobulbar injections. Stay sutures and a custom head mount were used to hold and steer the head for adaptive optics scanning light ophthalmoscopy (AOSLO). Six WT and TgP23H littermates were imaged at postnatal day 30 (P30), P90, and P180 with AOSLO and optical coherence tomography (OCT), and two additional sets of littermates were imaged at P3 and P15 with OCT only. AOSLO imaging and correlative differential interference contrast microscopy were performed on a P240 WT pig and on WT and TgP23H littermates at P30 and P180.

Results: AOSLO cone density generally underestimates histology density (mean difference ± SD = 24.8% ± 21.4%). The intensity of the outer retinal hyperreflective OCT band attributed to photoreceptors is attenuated in TgP23H pigs at all ages. In contrast, AOSLO images show cones that retain inner and outer segments through P180. At retinal locations outside the visual streak, TgP23H pigs show a heterogeneous degenerating cone mosaic by using two criteria: variable contrast on a split detector AOSLO and high reflectivity on a confocal AOSLO.

Conclusions: AOSLO reveals that the cone mosaic is similar to ex vivo histology. Its use as a noninvasive tool will enable observation of morphologic changes that arise in the cone mosaic of TgP23H pigs over time.

Translational Relevance: Pigs are widely used for translational studies, and the ability to noninvasively assess cellular changes in the cone mosaic will facilitate more detailed investigations of new retinal disease models as well as outcomes of potential therapies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1167/tvst.8.6.38DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6922271PMC
November 2019

RAC-CNN: multimodal deep learning based automatic detection and classification of rod and cone photoreceptors in adaptive optics scanning light ophthalmoscope images.

Biomed Opt Express 2019 Aug 8;10(8):3815-3832. Epub 2019 Jul 8.

Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA.

Quantification of the human rod and cone photoreceptor mosaic in adaptive optics scanning light ophthalmoscope (AOSLO) images is useful for the study of various retinal pathologies. Subjective and time-consuming manual grading has remained the gold standard for evaluating these images, with no well validated automatic methods for detecting individual rods having been developed. We present a novel deep learning based automatic method, called the rod and cone CNN (RAC-CNN), for detecting and classifying rods and cones in multimodal AOSLO images. We test our method on images from healthy subjects as well as subjects with achromatopsia over a range of retinal eccentricities. We show that our method is on par with human grading for detecting rods and cones.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1364/BOE.10.003815DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6701534PMC
August 2019

Imaging Melanin Distribution in the Zebrafish Retina Using Photothermal Optical Coherence Tomography.

Transl Vis Sci Technol 2018 4;7(5). Epub 2018 Sep 4.

Morgridge Institute for Research, Madison, WI, USA.

Purpose: To demonstrate and validate that photothermal optical coherence tomography (PT-OCT) can image melanin in the retinal pigment epithelium (RPE) and can observe light-driven melanosome translocation in the zebrafish retina.

Methods: A commercial spectral domain OCT system was modified to perform both OCT and PT-OCT. Four adult tyrosinase-mosaic zebrafish with varying levels of melanin expression across their retinas were imaged, and the PT-OCT signal for pigmented and nonpigmented regions were compared. Wild-type dark-adapted ( = 11 fish) and light-adapted ( = 10 fish) zebrafish were also imaged with OCT and PT-OCT. Longitudinal reflectivity and absorption profiles were generated from B-scans to compare the melanin distribution between the two groups.

Results: A significant increase in PT-OCT signal ( < 0.0001, Student's -test) was observed in pigmented regions of interest (ROI) compared to nonpigmented ROIs in the tyrosinase-mosaic zebrafish, which confirms the PT-OCT signal is specific to melanin in the eye. A significant increase in PT-OCT signal intensity ( < 0.0001, Student's -test) was also detected in the light-adapted wild-type zebrafish group compared to the dark-adapted group. Additionally, light-adapted zebrafish display more distinct melanin banding patterns than do dark-adapted zebrafish in PT-OCT B-scans.

Conclusions: PT-OCT can detect different levels of melanin absorption and characterize pigment distribution in the zebrafish retina, including intracellular changes due to light-driven melanosome translocation within the RPE.

Translational Relevance: PT-OCT could quantify changes in pigmentation that occur in retinal diseases. The functional information provided by PT-OCT may also enable a better understanding of the anatomical features within conventional OCT images.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1167/tvst.7.5.4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6126953PMC
September 2018

Intraobserver Repeatability and Interobserver Reproducibility of Ellipsoid Zone Measurements in Retinitis Pigmentosa.

Transl Vis Sci Technol 2018 May 4;7(3):13. Epub 2018 Jun 4.

Ophthalmology & Visual Sciences, Medical College of Wisconsin, Milwaukee, WI, USA.

Purpose: To examine repeatability and reproducibility of ellipsoid zone (EZ) width measurements in patients with retinitis pigmentosa (RP) using a longitudinal reflectivity profile (LRP) analysis.

Methods: We examined Bioptigen optical coherence tomography (OCT) scans from 48 subjects with RP or Usher syndrome. Nominal scan lengths were 6, 7, or 10 mm, and the lateral scale of each scan was calculated using axial length measurements. LRPs were generated from OCT line scans, and the peak corresponding to EZ was manually identified using ImageJ. The locations at which the EZ peak disappeared were used to calculate EZ width. Each scan was analyzed twice by each of two observers, who were masked to their previous measurements and those of the other observer.

Results: On average, horizontal width (HW) was significantly greater than vertical width (VW), and there was high interocular symmetry for both HW and VW. We observed excellent intraobserver repeatability with intraclass correlation coefficients (ICCs) ranging from 0.996 to 0.998 for HW and VW measurements. Interobserver reproducibility was also excellent for both HW (ICC = 0.989; 95% confidence interval [CI] = 0.983-0.995) and VW (ICC = 0.991; 95% CI = 0.985-0.996), with no significant bias observed between observers.

Conclusions: EZ width can be measured using LRPs with excellent repeatability and reproducibility. Our observation of greater HW than VW is consistent with previous observations in RP, though the reason for this anisotropy remains unclear.

Translational Relevance: We describe repeatability and reproducibility of a method for measuring EZ width in patients with RP or Usher syndrome. This approach could facilitate measurement of retinal band thickness and/or intensity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1167/tvst.7.3.13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5989764PMC
May 2018

The Effect of Retinal Melanin on Optical Coherence Tomography Images.

Transl Vis Sci Technol 2017 Apr 3;6(2). Epub 2017 Apr 3.

Department of Cell Biology, Neurobiology, & Anatomy, Medical College of Wisconsin, Milwaukee, WI, USA ; Department of Ophthalmology, Medical College of Wisconsin, Milwaukee, WI, USA ; Department of Biophysics, Medical College of Wisconsin, Milwaukee, WI, USA.

Purpose: We assessed the effect of melanin on the appearance of hyperreflective outer retinal bands in optical coherence tomography (OCT) images.

Methods: A total of 23 normal subjects and 51 patients with albinism were imaged using the Bioptigen high-resolution spectral-domain OCT. In addition, three wild type, three albino ( ), and eight mosaic zebrafish were imaged with the hand-held Bioptigen Envisu R2200 OCT. To identify pigmented versus nonpigmented regions in the mosaic zebrafish, en face summed volume projections of the retinal pigment epithelium (RPE) were created from volume scans. Longitudinal reflectivity profiles were generated from B-scans to assess the width and maximum intensity of the RPE band in fish, or the presence of one or two RPE/Bruch's membrane (BrM) bands in humans.

Results: The foveal RPE/BrM appeared as two bands in 71% of locations in patients with albinism and 45% of locations in normal subjects ( = 0.0003). Pigmented zebrafish retinas had significantly greater RPE reflectance, and pigmented regions of mosaic zebrafish also had significantly broader RPE bands than all other groups

Conclusions: The hyperreflective outer retinal bands in OCT images are highly variable in appearance. We showed that melanin is a major contributor to the intensity and width of the RPE band on OCT. One should use caution in extrapolating findings from OCT images of one or even a few individuals to define the absolute anatomic correlates of the hyperreflective outer retinal bands in OCT images.

Translational Relevance: Melanin affects the appearance of the outer retinal bands in OCT images. Use of animal models may help dissect the anatomic correlates of the complex reflective signals in OCT retinal images.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1167/tvst.6.2.8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5381330PMC
April 2017

Imaging the adult zebrafish cone mosaic using optical coherence tomography.

Vis Neurosci 2016 01;33:E011

Department of Cell Biology, Neurobiology, and Anatomy,Medical College of Wisconsin,Milwaukee,Wisconsin.

Zebrafish (Danio rerio) provide many advantages as a model organism for studying ocular disease and development, and there is great interest in the ability to non-invasively assess their photoreceptor mosaic. Despite recent applications of scanning light ophthalmoscopy, fundus photography, and gonioscopy to in vivo imaging of the adult zebrafish eye, current techniques either lack accurate scaling information (limiting quantitative analyses) or require euthanizing the fish (precluding longitudinal analyses). Here we describe improved methods for imaging the adult zebrafish retina using spectral domain optical coherence tomography (OCT). Transgenic fli1:eGFP zebrafish were imaged using the Bioptigen Envisu R2200 broadband source OCT with a 12-mm telecentric probe to measure axial length and a mouse retina probe to acquire retinal volume scans subtending 1.2 × 1.2 mm nominally. En face summed volume projections were generated from the volume scans using custom software that allows the user to create contours tailored to specific retinal layer(s) of interest. Following imaging, the eyes were dissected for ex vivo fluorescence microscopy, and measurements of blood vessel branch points were compared to those made from the en face OCT images to determine the OCT lateral scale as a function of axial length. Using this scaling model, we imaged the photoreceptor layer of five wild-type zebrafish and quantified the density and packing geometry of the UV cone submosaic. Our in vivo cone density measurements agreed with measurements from previously published histology values. The method presented here allows accurate, quantitative assessment of cone structure in vivo and will be useful for longitudinal studies of the zebrafish cone mosaics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1017/S0952523816000092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5659228PMC
January 2016
-->