Background: Exposure to ethanol in utero leads to several brain development disorders including retinal abnormalities whose underlying cellular pathogenesis remains elusive. We have previously reported changes in electroretinogram recordings in moderate fetal alcohol exposure (MFAE) vervet monkeys. The goal of this study is to characterize the anatomical effects of moderate MFAE during the third trimester in the vervet monkey retina.

Methods: Using immunohistochemistry and Western blots, we analyzed changes in the expression of cell-type specific proteins that may occur in the MFAE retina compared to the normal retina. We also compared the basic retinal anatomy across groups by examining retinal layering and thickness.

Results: Our main result indicates that GFAP (a potent marker of astrocytes) immunoreactivity was increased in the MFAE retina indicating strong astrogliosis. There was no obvious change in the overall anatomy in the MFAE retina and no significant differences in the mean thickness of each retinal layer. Furthermore, no significant changes in the morphology of the photoreceptors, horizontal cells, bipolar cells, and amacrines cells was observed.

Conclusions: These data indicate that astrogliosis is a consequence of prenatal alcohol exposure and might explain the reported changes in the electroretinographic responses.

Background: Exposure to ethanol in utero leads to several brain development disorders including retinal abnormalities whose underlying cellular pathogenesis remains elusive. We have previously reported changes in electroretinogram recordings in moderate fetal alcohol exposure (MFAE) vervet monkeys. The goal of this study is to characterize the anatomical effects of moderate MFAE during the third trimester in the vervet monkey retina.

Methods: Using immunohistochemistry and Western blots, we analyzed changes in the expression of cell-type specific proteins that may occur in the MFAE retina compared to the normal retina. We also compared the basic retinal anatomy across groups by examining retinal layering and thickness.

Results: Our main result indicates that GFAP (a potent marker of astrocytes) immunoreactivity was increased in the MFAE retina indicating strong astrogliosis. There was no obvious change in the overall anatomy in the MFAE retina and no significant differences in the mean thickness of each retinal layer. Furthermore, no significant changes in the morphology of the photoreceptors, horizontal cells, bipolar cells, and amacrines cells was observed.

Conclusions: These data indicate that astrogliosis is a consequence of prenatal alcohol exposure and might explain the reported changes in the electroretinographic responses.

Background: Our national collaborative research initiative is proposing to develop a common infrastructure for Rb research. We are proposing a novel in vivo Rb model using human Rb cells line.

Methods: The rabbit model has advantages over the mouse models: (I) the larger eye size of rabbits, similar to the human infant eye, permits a more accurate injection of the drugs and evaluation of methods of targeted intraocular drug delivery; (II) the rabbit model demonstrated similar fundus appearance and pathologic features to human Rb, including vitreous seeds of viable tumor when the retinal tumor is mid-sized, which are usually found in the late stage in mouse models. The lack of ability to eliminate vitreous seeds is a major reason of current treatment failures in Group C and D tumors; therefore, the rabbit model of Rb may be used as a model to evaluate the effectiveness and various routes of drug delivery.

Results: This is an implementation of an infrastructure for evaluating therapeutic targets. In addition, this finding enables a variety of pharmacokinetic studies, pharmacodynamic and toxicology studies for new therapeutic agents.

Conclusions: This infrastructure meets the growing concern of practitioners and researchers in the field. The common facility is easily accessible to all VHRN members on request, including requests from other sectors.

Background: Our national collaborative research initiative is proposing to develop a common infrastructure for Rb research. We are proposing a novel in vivo Rb model using human Rb cells line.

Methods: The rabbit model has advantages over the mouse models: (I) the larger eye size of rabbits, similar to the human infant eye, permits a more accurate injection of the drugs and evaluation of methods of targeted intraocular drug delivery; (II) the rabbit model demonstrated similar fundus appearance and pathologic features to human Rb, including vitreous seeds of viable tumor when the retinal tumor is mid-sized, which are usually found in the late stage in mouse models. The lack of ability to eliminate vitreous seeds is a major reason of current treatment failures in Group C and D tumors; therefore, the rabbit model of Rb may be used as a model to evaluate the effectiveness and various routes of drug delivery.

Results: This is an implementation of an infrastructure for evaluating therapeutic targets. In addition, this finding enables a variety of pharmacokinetic studies, pharmacodynamic and toxicology studies for new therapeutic agents.

Conclusions: This infrastructure meets the growing concern of practitioners and researchers in the field. The common facility is easily accessible to all VHRN members on request, including requests from other sectors.

Background: Pericytes are contractile cells that wrap along the walls of capillaries. In the brain, pericytes play a crucial role in the regulation of capillary diameter and vascular blood flow in response to metabolic demand. During ischemia, it has been suggested that pericytes may constrict capillaries, and that pericytes remain constricted after reperfusion thus resulting in impaired blood flow.

Methods: Here, we used a mouse model of retinal ischemia based on ligation of the central retinal artery to characterize the role of pericytes on capillary constriction. Ischemia was induced in transgenic mice carrying the NG2 promoter driving red fluorescent protein expression to selectively visualize pericytes (line NG2:DsRed).Changes in retinal capillary diameter at 1 hr after ischemia were measured ex vivo in whole-mounted retinas from ischemic and control eyes (n=4–6/group) using a stereological approach. Vessels and pericytes were three-dimensionally reconstructed using IMARIS (Bitplane). Furthermore, we used a novel and minimally invasive two-photon microscopy approach that allowed live imaging of microvasculature changes in the retina.

Results: Our data show a generalized reduction in capillary diameter in ischemic retinas relative to sham-operated controls in all vascular plexus (ischemia: 4.7±0.2 μm, control: 5.2±0.2 μm, student’s t-test, P<0.001). Analysis of the number of capillary constrictions at pericyte locations, visualized in NG2:DsRed mice, demonstrated a substantial increase in ischemic retinas relative to the physiological capillary diameter reductions observed in controls (ischemia: 1,038±277 constrictions at pericyte locations, control: 60±36 constrictions at pericyte locations, student’s t-test, P<0.01). Live imaging using two-photon microscopy confirmed robust capillary constriction at the level of pericytes on retinal capillaries during ischemia (n=6–8/group).

Conclusions: Collectively, our data demonstrate that ischemia promotes rapid pericyte constriction on retinal capillaries causing major microvascular dysfunction in this tissue. To identify the molecular mechanisms underlying the pathological response of pericytes during ischemia, we are currently carrying out experiments in mice and zebrafish to modulate signaling pathways involved in calcium dynamics leading to contractility in these cells.

Background: Pericytes are contractile cells that wrap along the walls of capillaries. In the brain, pericytes play a crucial role in the regulation of capillary diameter and vascular blood flow in response to metabolic demand. During ischemia, it has been suggested that pericytes may constrict capillaries, and that pericytes remain constricted after reperfusion thus resulting in impaired blood flow.

Methods: Here, we used a mouse model of retinal ischemia based on ligation of the central retinal artery to characterize the role of pericytes on capillary constriction. Ischemia was induced in transgenic mice carrying the NG2 promoter driving red fluorescent protein expression to selectively visualize pericytes (line NG2:DsRed).Changes in retinal capillary diameter at 1 hr after ischemia were measured ex vivo in whole-mounted retinas from ischemic and control eyes (n=4–6/group) using a stereological approach. Vessels and pericytes were three-dimensionally reconstructed using IMARIS (Bitplane). Furthermore, we used a novel and minimally invasive two-photon microscopy approach that allowed live imaging of microvasculature changes in the retina.

Results: Our data show a generalized reduction in capillary diameter in ischemic retinas relative to sham-operated controls in all vascular plexus (ischemia: 4.7±0.2 μm, control: 5.2±0.2 μm, student’s t-test, P<0.001). Analysis of the number of capillary constrictions at pericyte locations, visualized in NG2:DsRed mice, demonstrated a substantial increase in ischemic retinas relative to the physiological capillary diameter reductions observed in controls (ischemia: 1,038±277 constrictions at pericyte locations, control: 60±36 constrictions at pericyte locations, student’s t-test, P<0.01). Live imaging using two-photon microscopy confirmed robust capillary constriction at the level of pericytes on retinal capillaries during ischemia (n=6–8/group).

Conclusions: Collectively, our data demonstrate that ischemia promotes rapid pericyte constriction on retinal capillaries causing major microvascular dysfunction in this tissue. To identify the molecular mechanisms underlying the pathological response of pericytes during ischemia, we are currently carrying out experiments in mice and zebrafish to modulate signaling pathways involved in calcium dynamics leading to contractility in these cells.

Background: Rods and cones are critical for light detection. Although there has been considerable work done in elucidating the molecular mechanisms involved in rod development, not much is known about how the cone cell fate decision is made by the multipotent retinal progenitor cells during development. Analysis of the promoter regions of Nrl and trβ2, rod and cone differentiation factors respectively, revealed DNA binding motifs of two POU-domain containing transcription factors, Pou2f1 and Pou2f2. Preliminary experiments showed that Pou2f1/2 are expressed during the peak of cone genesis in the embryonic retina. Therefore, we hypothesize that Pou2f1/2 specify cone cell fate in the developing retina.

Methods: We used immunofluorescence and in situ hybridization to establish the spatiotemporal expression of Pou2f1/2 during retinogenesis. We performed in vivo electroporation in post-natal mice to misexpress Pou2f1/2 and used antibodies specific to proteins expressed in cones such as Rxrγ and S-opsin to count cones. Using ex vivo electroporation of embryonic retinal explants, we knocked out Pou2f1 and Pou2f2 using CRISPR/Cas9 gRNAs at the peak of cone production window. Finally, we transfected post-natal retinal explants with a combination of regulatory elements of Nrl or thrb with control backbone vector, Pou2f1 or Pou2f2 using electroporation.

Results: We found that Pou2f1/2 are expressed in retinal progenitor cells in the developing retina and subsequently in the differentiated cones. Pou2f1/2 misexpression outside the cone genesis window led to an increase in cones at the expense of rods. Pou2f1/2 indel knockouts generated by CRISPR/Cas9 gRNAs led to a decrease in cones and a converse increase in rods. Finally, we found that Pou2f1/2 activate the cis-regulatory module (CRM) of the thrb gene and repress the activity of the CRM of Nrl.

Conclusions: These results uncover novel players that establish the complex gene regulatory network for cone photoreceptor fate specification in the retinal progenitor cells. We anticipate that this work should help us devise improved replacement therapies in the future utilizing stem cells for retinal degenerative diseases such as aged-related macular degeneration (AMD) and Stargardt’s disease.

Background: Rods and cones are critical for light detection. Although there has been considerable work done in elucidating the molecular mechanisms involved in rod development, not much is known about how the cone cell fate decision is made by the multipotent retinal progenitor cells during development. Analysis of the promoter regions of Nrl and trβ2, rod and cone differentiation factors respectively, revealed DNA binding motifs of two POU-domain containing transcription factors, Pou2f1 and Pou2f2. Preliminary experiments showed that Pou2f1/2 are expressed during the peak of cone genesis in the embryonic retina. Therefore, we hypothesize that Pou2f1/2 specify cone cell fate in the developing retina.

Methods: We used immunofluorescence and in situ hybridization to establish the spatiotemporal expression of Pou2f1/2 during retinogenesis. We performed in vivo electroporation in post-natal mice to misexpress Pou2f1/2 and used antibodies specific to proteins expressed in cones such as Rxrγ and S-opsin to count cones. Using ex vivo electroporation of embryonic retinal explants, we knocked out Pou2f1 and Pou2f2 using CRISPR/Cas9 gRNAs at the peak of cone production window. Finally, we transfected post-natal retinal explants with a combination of regulatory elements of Nrl or thrb with control backbone vector, Pou2f1 or Pou2f2 using electroporation.

Results: We found that Pou2f1/2 are expressed in retinal progenitor cells in the developing retina and subsequently in the differentiated cones. Pou2f1/2 misexpression outside the cone genesis window led to an increase in cones at the expense of rods. Pou2f1/2 indel knockouts generated by CRISPR/Cas9 gRNAs led to a decrease in cones and a converse increase in rods. Finally, we found that Pou2f1/2 activate the cis-regulatory module (CRM) of the thrb gene and repress the activity of the CRM of Nrl.

Conclusions: These results uncover novel players that establish the complex gene regulatory network for cone photoreceptor fate specification in the retinal progenitor cells. We anticipate that this work should help us devise improved replacement therapies in the future utilizing stem cells for retinal degenerative diseases such as aged-related macular degeneration (AMD) and Stargardt’s disease.