Acute progressive paravascular placoid neuroretinopathy with negative-type electroretinography in paraneoplastic retinopathy

A 59-year-old Caucasian male presented in May 2015 and underwent full ophthalmic examination and automated visual field test. Informed consent was obtained for serial multimodal imaging and serum anti-retinal antibody analysis. The study protocol adhered to the tenets of the Declaration of Helsinki and was approved by the Human Research Ethics Office of the University of Western Australia (RA/4/1/7916).

Imaging modalities included near-infrared reflectance (NIR), blue-light autofluorescence (AF), fluorescein and indocyanine green angiographies, spectral domain optical coherence tomography (SD-OCT, Spectralis HRA + OCT, Heidelberg Engineering, Heidelberg, Germany), ultra-widefield colour and green-light AF imaging (P200Tx, Optos plc, Dunfermline, UK), microperimetry (MAIA, CenterVue, Padova, Italy) and adaptive optics (AO) imaging (rtx1 camera, Imagine Eyes, Orsay, France). Electrophysiology was performed according to the International Society for Clinical Electrophysiology of Vision (ISCEV) standards (RETIport 3.2, Roland Consult, Brandenburg, Germany).

Human posterior eye cups were obtained from the Lions Eye Bank, Western Australia. The use of human tissue for research was approved by the University of Western Australia Human Research Ethics Office (Trial number: 2012-090).

The tissue was fixed in formalin and embedded in paraffin, cut at 4 microns and submitted for routine hematoxylin and eosin staining. Immunohistochemistry was performed including appropriate positive and negative controls.

Paraffin-embedded human retinal or prostate tumour sections were heated at 50 °C for 1 h. The sections were then deparaffinized in xylene and rehydrated in graded series of ethanol. Heat epitope retrieval was performed in a rice cooker by boiling the deparaffinized sections for 20 min in antigen retrieval buffer (10 mM Sodium Citrate, 0.05% Tween20, pH 6). The sections in the buffer were then cooled on ice. After washing with TBST (50 mM Tris–HCl, 150 mM NaCl, 0.05% Tween20), sections were blocked for 1–2 h at room temperature with tris-buffered saline (TBS, 50 mM Tris–HCl, 150 mM NaCl) containing 10% normal goat serum and 1% BSA. The sections were then probed at 4 °C overnight with human serum (1:50), rabbit anti-recoverin (1:50), rabbit anti-synaptophysin (1:200), rabbit anti-Ki67 (1:200), mouse anti-GAPDH (1:100) or mouse anti-alpha-enolase (1:100) in TBS/1% BSA. After five TBS washes, sections were incubated with DAPI (1ug/ml) and goat antihuman AlexaFluor 488 (1:250), goat anti-rabbit AlexaFluor 488 or AlexaFluor 546 (1:250), anti-mouse AlexaFluor 546 (1:250) (Invitrogen), correspondingly. Negative controls were performed by omitting the primary antibody.

Human retinal tissue was lysed in RIPA lysis buffer (Sigma-Aldrich, St. Louis, MO, USA) with 1% protein inhibitor cocktail (Sigma) followed by shaking at 4 °C for 1 h. The lysate was then centrifuged at 13,000 rpm for 30 min at 4 °C and the supernatant collected and stored at −20 °C. Protein concentration of the lysate was determined by Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA). Human liver lysates were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Purified recombinant proteins were obtained from Abcam (Cambridge, England, UK, GAPDH and alpha-enolase) and Abnova (Taipei, Taiwan, PNMA2). Twenty micrograms of total protein from tissue lysates or 1 μg of a purified recombinant protein was mixed with Sample Buffer (Invitrogen, Thermo Fisher, Scientific, Waltham MA, USA) and heated at 70 °C for 10 min before loading onto a NuPAGE 4–12% Bis–Tris gel (Invitrogen). Protein samples were separated by gel electrophoresis in 1× MES buffer (Invitrogen) and then transferred onto a PVDF membrane (Immobilon-FL, Merck Millipore, Billerica, MA, USA) at 80 V for 90 min in transfer buffer (25 mM Tris, 190 mM glycine, 20% methanol, pH 8.3). After transfer, the membrane was incubated with Odyssey^® Blocking Buffer (LI-COR, Lincoln, NE, USA) at room temperature for 1 h. The membrane then was incubated with human serum (1:200) or rabbit anti-recoverin antibody (1:1000, ab85292, Abcam) in Odyssey^® Blocking Buffer plus 0.1% Tween20 at 4 °C overnight. After washing in TBST 3 times, the membrane was incubated with IRDye^® 800CW goat antihuman IgG (1:15,000; LI-COR) in Odyssey^® Blocking Buffer plus 0.1% Tween20 and 0.01% SDS for 1 h at room temperature. After 3 TBST washes, the membrane was washed once with TBS without Tween20. The membrane was then imaged at 800 nm wavelength by the Odyssey Infrared Imager (Model 9120, LI-COR).