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In this work, we introduce a diurnal rodent, the Mongolian gerbil ( Meriones unguiculatus ) (MG) as an alternative to study retinal cone system physiology and pathophysiology in mice. The cone system is of particular importance, as it provides high-acuity and color vision and its impairment in retinal disorders is thus especially disabling. Despite their nocturnal lifestyle, mice are currently the most popular animals to study cone-related diseases due to the high availability of genetically modified models. However, the potential for successful translation of any cone-related results is limited due to the substantial differences in retinal organization between mice and humans. Alternatively, there are diurnal rodents such as the MG with a higher retinal proportion of cones and a macula-like specialized region for improved visual resolution, the visual streak. The focus of this work was the evaluation of the MG's cone system functionality using full-field electroretinography (ERG), together with a morphological assessment of its retinal/visual streak organization via angiography, optical coherence tomography (OCT), and photoreceptor immunohistochemistry. We found that rod system responses in MGs were comparable or slightly inferior to mice, while in contrast, cone system responses were much larger, more sensitive, and also faster than those in the murine counterparts, and in addition, it was possible to record sizeable ON and OFF ERG components. Morphologically, MG cone photoreceptor opsins were evenly distributed throughout the retina, while mice show a dorsoventral M- and S-opsin gradient. Additionally, each cone expressed a single opsin, in contrast to the typical co-expression of opsins in mice. Particular attention was given to the visual streak region, featuring a higher density of cones, elongated cone and rod outer segments (OSs), and an increased thickness of the inner and outer retinal layers in comparison to peripheral regions. In summary, our data render the MG a supreme model to investigate cone system physiology, pathophysiology, and to validate potential therapeutic strategies in that context., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Günter, Belhadj, Seeliger and Mühlfriedel.)

Serum response factor (SRF) controls the expression of muscle contraction and motility genes in mural cells (MCs) of the vasculature. In the retina, MC-SRF is important for correct angiogenesis during development and the continuing maintenance of the vascular tone. The purpose of this study was to provide further insights into the effects of MC SRF deficiency on the vasculature and function of the mature retina in Srf iMCKO mice that carry a MC-specific deletion of Srf . Retinal morphology and vascular integrity were analyzed in vivo via scanning laser ophthalmoscopy (SLO), angiography, and optical coherence tomography (OCT). Retinal function was evaluated with full-field electroretinography (ERG). We found that retinal blood vessels of these mutants exhibited different degrees of morphological and functional alterations. With increasing severity, we found vascular bulging, the formation of arteriovenous (AV) anastomoses, and ultimately, a retinal detachment (RD). The associated irregular retinal blood pressure and flow distribution eventually induced hypoxia, indicated by a negative ERG waveform shape. Further, the high frequency of interocular differences in the phenotype of individual Srf iMCKO mice points to a secondary nature of these developments far downstream of the genetic defect and rather dependent on the local retinal context.

Inherited retinal degenerations (IRDs) are a group of blinding diseases, typically involving a progressive loss of photoreceptors. The IRD pathology is often based on an accumulation of cGMP in photoreceptors and associated with the excessive activation of calpain and poly (ADP-ribose) polymerase (PARP). Inhibitors of calpain or PARP have shown promise in preventing photoreceptor cell death, yet the relationship between these enzymes remains unclear. To explore this further, organotypic retinal explant cultures derived from wild-type and IRD-mutant mice were treated with inhibitors specific for calpain, PARP, and voltage-gated Ca 2+ channels (VGCCs). The outcomes were assessed using in situ activity assays for calpain and PARP and immunostaining for activated calpain-2, poly (ADP-ribose), and cGMP, as well as the TUNEL assay for cell death detection. The IRD models included the Pde6b- mutant rd1 mouse and rd1*Cngb1 -/- double-mutant mice, which lack the beta subunit of the rod cyclic nucleotide-gated (CNG) channel and are partially protected from rd1 degeneration. We confirmed that an inhibition of either calpain or PARP reduces photoreceptor cell death in rd1 retina. However, while the activity of calpain was decreased by the inhibition of PARP, calpain inhibition did not alter the PARP activity. A combination treatment with calpain and PARP inhibitors did not synergistically reduce cell death. In the slow degeneration of rd1 * Cngb1 -/- double mutant, VGCC inhibition delayed photoreceptor cell death, while PARP inhibition did not. Our results indicate that PARP acts upstream of calpain and that both are part of the same degenerative pathway in Pde6b -dependent photoreceptor degeneration. While PARP activation may be associated with CNG channel activity, calpain activation is linked to VGCC opening. Overall, our data highlights PARP as a target for therapeutic interventions in IRD-type diseases.

Malaria is a causative factor in about 500.000 deaths each year world-wide. Cerebral malaria is a particularly severe complication of this disease and thus associated with an exceedingly high mortality. Malaria retinopathy is an ocular manifestation often associated with cerebral malaria, and presumably shares a substantial part of its pathophysiology. Here, we describe that indeed murine malaria retinopathy reproduced the main hallmarks of the corresponding human disease. In the living animal, we were able to follow the circulation and cellular localization of malaria parasites transgenically labelled with GFP via non-invasive in vivo retinal imaging. We found that malaria parasites cross the blood-retinal-barrier and infiltrate the neuroretina, concomitant with an extensive, irreversible, and long-lasting retinal neurodegeneration. Furthermore, anti-malarial treatment with dihydroartemisinin strongly diminished the load of circulating parasites but resolved the symptoms of the retinopathy only in part. In summary, we introduce here a novel preclinical model for human cerebral malaria that is much more directly accessible for studies into disease pathophysiology and development of novel treatment approaches. In vivo retinal imaging may furthermore serve as a valuable tool for the early diagnosis of the human disease.

Gene therapy for inherited eye diseases requires local viral vector delivery by intraocular injection. Since large animal models are lacking for most of these diseases, genetically modified mouse models are commonly used in preclinical proof-of-concept studies. However, because of the relatively small mouse eye, adverse effects of the subretinal delivery procedure itself may interfere with the therapeutic outcome. The method described here aims to provide the details relevant to perform a transscleral pars plana virus-mediated gene transfer to achieve an optimized therapeutic effect in the small mouse eye.

Mutations in the Norrin (NDP) gene cause severe developmental blood vessel defects in the retina leading to congenital blindness. In the retina of Ndph-knockout mice only the superficial capillary network develops. Here, a detailed characterization of this mouse model at late stages of the disease using in vivo retinal imaging revealed cystoid structures that closely resemble the ovoid cysts in the inner nuclear layer of the human retina with cystoid macular edema (CME). In human CME an involvement of Müller glia cells is hypothesized. In Ndph-knockout retinae we could demonstrate that activated Müller cells were located around and within these cystoid spaces. In addition, we observed extensive activation of retinal microglia and development of neovascularization. Furthermore, ex vivo analyses detected extravasation of monocytic cells suggesting a breakdown of the blood retina barrier. Thus, we could demonstrate that also in the developmental retinal vascular pathology present in the Ndph-knockout mouse inflammatory processes are active and may contribute to further retinal degeneration. This observation delivers a new perspective for curative treatments of retinal vasculopathies. Modulation of inflammatory responses might reduce the symptoms and improve visual acuity in these diseases.

Full-field electroretinography (ERG) belongs to the gold-standard of electrophysiological test systems in ophthalmology and reflects the sum response of the entire retina to light stimulation. The assessment of the retinal function is a fundamental diagnostic technique not only in the clinical ophthalmology it is also indispensable in the ophthalmic research, in particular, in therapeutic approaches where the in vivo follow up of the benefit after treatment is absolutely necessary. Several current therapeutic approaches have demonstrated long-lasting amelioration in respective disease models and show promise for a successful translation to human patients. In this chapter we provide electroretinography protocols of experimental data which may serve as informative features for upcoming gene therapeutic approaches and clinical trials.

Treatment approaches for inherited eye diseases require local therapeutic molecule delivery by intraocular injection. One important factor that can influence the study outcome is the quality of intraocular administration. The intracompartmental structure (e.g., vitreous) of the eye allows a sustainable release of therapeutic biologicals using an intravitreal delivery. The protocol described here aims at providing the details relevant to perform a transscleral pars plana intravitreal transfer in small eyes using a genetically modified stem cell system. The fact that cells and therewith visually distinct particles are implanted, allows for the assessment of the implantation site and the distribution, and possibilities for temporal follow up studies-hence, valuable information becomes available which can be used to fine-tune the intravitreal delivery technique.

Retinitis pigmentosa type 43 (RP43) is a blinding disease caused by mutations in the gene for rod phosphodiesterase 6 alpha ( PDE6A ). The disease process begins with a dysfunction of rod photoreceptors, subsequently followed by a currently untreatable progressive degeneration of the entire outer retina. Aiming at a curative approach via PDE6A gene supplementation, a novel adeno-associated viral (AAV) vector was developed for expression of the human PDE6A cDNA under control of the human rhodopsin promotor (rAAV8.PDE6A). This study assessed the therapeutic efficacy of rAAV8.PDE6A in the Pde6a nmf363/nmf363 -mutant mouse model of RP43. All mice included in this study were treated with sub-retinal injections of the vector at 2 weeks after birth. The therapeutic effect was monitored at 1 month and 6 months post injection. Biological function of the transgene was assessed in vivo by means of electroretinography. The degree of morphological rescue was investigated both in vivo using optical coherence tomography and ex vivo by immunohistological staining. It was found that the novel rAAV8.PDE6A vector resulted in a stable and efficient expression of PDE6A protein in rod photoreceptors of Pde6a nmf363/nmf363 mice following treatment at both the short- and long-term time points. The treatment led to a substantial morphological preservation of outer nuclear layer thickness, rod outer segment structure, and prolonged survival of cone photoreceptors for at least 6 months. Additionally, the ERG analysis confirmed a restoration of retinal function in a group of treated mice. Taken together, this study provides successful proof-of-concept for the cross-species efficacy of the rAAV8.PDE6A vector developed for use in human patients. Importantly, the data show stable expression and rescue effects for a prolonged period of time, raising hope for future translational studies based on this approach.

Achromatopsia type 2 (ACHM2) is a severe, inherited eye disease caused by mutations in the CNGA3 gene encoding the α subunit of the cone photoreceptor cyclic nucleotide-gated (CNG) channel. Patients suffer from strongly impaired daylight vision, photophobia, nystagmus, and lack of color discrimination. We have previously shown in the Cnga 3 knockout (KO) mouse model of ACHM2 that gene supplementation therapy is effective in rescuing cone function and morphology and delaying cone degeneration. In our preclinical approach, we use recombinant adeno-associated virus (AAV) vector-mediated gene transfer to express the murine Cnga3 gene under control of the mouse blue opsin promoter. Here, we provide novel data on the efficiency and permanence of such gene supplementation therapy in Cnga3 KO mice. Specifically, we compare the influence of two different AAV vector capsids, AAV2/5 (Y719F) and AAV2/8 (Y733F), on restoration of cone function, and assess the effect of age at time of treatment on the long-term outcome. The evaluation included in vivo analysis of retinal function using electroretinography (ERG) and immunohistochemical analysis of vector-driven Cnga3 transgene expression. We found that both vector capsid serotypes led to a comparable rescue of cone function over the observation period between 4 weeks and 3 months post treatment. In addition, a clear therapeutic effect was present in mice treated at 2 weeks of age as well as in mice treated at 3 months of age at the first assessment at 4 weeks after treatment. Importantly, the effect extended in both cases over the entire observation period of 12 months post treatment. However, the average ERG amplitude levels differed between the two groups, suggesting a role of the absolute age, or possibly, the associated state of the degeneration, on the achievable outcome. In summary, we found that the therapeutic time window of opportunity for AAV-mediated Cnga3 gene supplementation therapy in the Cnga3 KO mouse model extends at least to an age of 3 months, but is presumably limited by the condition, number and topographical distribution of remaining cones at the time of treatment. No impact of the choice of capsid on the therapeutic success was detected.

Characteristics of inherited retinal dystrophies include deficiencies in light perception and nervous conduction within the retina, leading to reduced vision or even blindness. In this context, the loss of function of photoreceptor-specific genes causes a variety of clinically and aetiologically distinct syndromes - each of them belonging to the group of rare diseases. With a prevalence of 1 in 2500, however, inherited retinal diseases are clinically significant and important - especially since these diseases lead to restrictions of a patient's fitness for work and overall quality of life. More than 250 genetic mutations causing the various types of inherited retinal dystrophies have been identified by now (https://sph.uth.tmc.edu/Retnet). In recent years, preclinical research on suitable animal models has yielded important progress in the understanding of the mutations underlying the pathological and molecular biological processes of these diseases. These findings have led to the development of novel and innovative therapeutic strategies for the treatment of inherited retinal dysfunctions, which are still incurable. Meanwhile, many of the successful preclinical studies have led to translational research projects aiming to find treatment options for human patients. However, some preliminary results of these human translational studies indicate the need to optimise and refine the underlying therapeutic concepts., (Georg Thieme Verlag KG Stuttgart · New York.)

Mutations in the PDE6A gene can cause rod photoreceptors degeneration and the blinding disease retinitis pigmentosa (RP). While a number of pathogenic PDE6A mutations have been described, little is known about their impact on compound heterozygous situations and potential interactions of different disease-causing alleles. Here, we used a novel mouse model for the Pde6a R562W mutation in combination with an existing line carrying the V685M mutation to generate compound heterozygous Pde6a V685M/R562W animals, exactly homologous to a case of human RP. We compared the progression of photoreceptor degeneration in these compound heterozygous mice with the homozygous V685M and R562W mutants, and additionally with the D670G line that is known for a relatively mild phenotype. We investigated PDE6A expression, cyclic guanosine mono-phosphate accumulation, calpain and caspase activity, in vivo retinal function and morphology, as well as photoreceptor cell death and survival. This analysis confirms the severity of different Pde6a mutations and indicates that compound heterozygous mutants behave like intermediates of the respective homozygous situations. Specifically, the severity of the four different Pde6a situations may be categorized by the pace of photoreceptor degeneration: V685M (fastest) > V685M/R562W > R562W > D670G (slowest). While calpain activity was strongly increased in all four mutants, caspase activity was not. This points to the execution of non-apoptotic cell death and may lead to the identification of new targets for therapeutic interventions. For individual RP patients, our study may help to predict time-courses for Pde6a-related retinal degeneration and thereby facilitate the definition of a window-of-opportunity for clinical interventions., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Optical coherence tomography (OCT) is an invaluable diagnostic tool for the detection and follow-up of retinal pathology in patients and experimental disease models. However, as morphological structures and layering in health as well as their alterations in disease are complex, segmentation procedures have not yet reached a satisfactory level of performance. Therefore, raw images and qualitative data are commonly used in clinical and scientific reports. Here, we assess the value of OCT reflectivity profiles as a basis for a quantitative characterization of the retinal status in a cross-species comparative study., Methods: Spectral-Domain Optical Coherence Tomography (OCT), confocal Scanning-Laser Ophthalmoscopy (SLO), and Fluorescein Angiography (FA) were performed in mice (Mus musculus), gerbils (Gerbillus perpadillus), and cynomolgus monkeys (Macaca fascicularis) using the Heidelberg Engineering Spectralis system, and additional SLOs and FAs were obtained with the HRA I (same manufacturer). Reflectivity profiles were extracted from 8-bit greyscale OCT images using the ImageJ software package (http://rsb.info.nih.gov/ij/)., Results: Reflectivity profiles obtained from OCT scans of all three animal species correlated well with ex vivo histomorphometric data. Each of the retinal layers showed a typical pattern that varied in relative size and degree of reflectivity across species. In general, plexiform layers showed a higher level of reflectivity than nuclear layers. A comparison of reflectivity profiles from specialized retinal regions (e.g. visual streak in gerbils, fovea in non-human primates) with respective regions of human retina revealed multiple similarities. In a model of Retinitis Pigmentosa (RP), the value of reflectivity profiles for the follow-up of therapeutic interventions was demonstrated., Conclusions: OCT reflectivity profiles provide a detailed, quantitative description of retinal layers and structures including specialized retinal regions. Our results highlight the potential of this approach in the long-term follow-up of therapeutic strategies.

Achromatopsia is an autosomal recessive inherited retinal disease caused by a complete loss of cone photoreceptor function. About 80 % of achromatopsia patients show mutations in the alpha or beta subunit (A3 and B3) of the cGMP controlled cation channel CNG (cyclic nucleotide-gated channel) of cone photoreceptors. Homologous to the human disease, CNGA3 deficient mice reveal a loss of cone specific functionality leading to degeneration of affected cone photoreceptors. The Institute for Ophthalmic Research in Tübingen has now succeeded in curing achromatopsia ACHM2 in an animal model. In this article, we explain the recombinant adeno-associated virus-based approach in detail. Furthermore, applied non-invasive diagnostic techniques for quality and success control, ERG, SLO and OCT, are described. The success of the therapy is indicated by a restored cone photoreceptor function as well as the neuronal processing of retinal signals resulting in a specific, cone-mediated behaviour. The outstanding results derived from the animal model are the starting point for the first human translation of a gene therapy for achromatopsia in Germany., (Georg Thieme Verlag KG Stuttgart · New York.)

Retinitis pigmentosa (RP) is a severe retinal disease characterized by a progressive degeneration of rod photoreceptors and a secondary loss of cone function. Here, we used CNGB1-deficient (CNGB1(-/-)) mice, a mouse model for autosomal recessive RP, to evaluate the efficacy of adeno-associated virus (AAV) vector-mediated gene therapy for the treatment of RP. The treatment restored normal expression of rod CNG channels and rod-driven light responses in the CNGB1(-/-) retina. This led to a substantial delay of retinal degeneration and long-term preservation of retinal morphology. Finally, treated CNGB1(-/-) mice performed significantly better than untreated mice in a rod-dependent vision-guided behavior test. In summary, this study holds promise for the treatment of rod channelopathy-associated retinitis pigmentosa by AAV-mediated gene replacement.

Background: To monitor viability of implanted genetically engineered and microencapsulated human stem cells (MicroBeads) in the mouse eye, and to study the impact of the beads and/or xenogenic cells on retinal integrity., Methodology/principal Findings: MicroBeads were implanted into the subretinal space of SV126 wild type mice using an ab externo approach. Viability of microencapsulated cells was monitored by noninvasive retinal imaging (Spectralis™ HRA+OCT). Retinal integrity was also assessed with retinal imaging and upon the end of the study by light and electron microscopy. The implanted GFP-marked cells encapsulated in subretinal MicroBeads remained viable over a period of up to 4 months. Retinal integrity and viability appeared unaltered apart from the focal damage due to the surgical implantation, GFAP upregulation, and opsin mistargeting in the immediate surrounding tissue., Conclusions/significance: The accessibility for routine surgery and its immune privileged state make the eye an ideal target for release system implants for therapeutic substances, including neurotrophic and anti-angiogenic compounds or protein based biosimilars. Microencapsulated human stem cells (MicroBeads) promise to overcome limitations inherent with single factor release systems, as they are able to produce physiologic combinations of bioactive compounds.

Subretinal injections in mice become increasingly important. Currently, the most prominent application is in gene therapy of inherited eye diseases by means of viral vector delivery to photoreceptors or the retinal pigment epithelium (RPE). Since there are no large animal models for most of these diseases, genetically modified mouse models are commonly used in preclinical proof-of-concept studies. However, because of the relatively small mouse eye, adverse effects of the subretinal delivery procedure itself may interfere with the therapeutic outcome. The protocol described here concerns a transscleral pars plana subretinal injection in small eyes, and may be used for but not limited to virus-mediated gene transfer.

Retinitis pigmentosa (RP) is a group of genetically heterogeneous, severe retinal diseases commonly leading to legal blindness. Mutations in the CNGB1a subunit of the rod cyclic nucleotide-gated (CNG) channel have been found to cause RP in patients. Here, we demonstrate the efficacy of gene therapy as a potential treatment for RP by means of recombinant adeno-associated viral (AAV) vectors in the CNGB1 knockout (CNGB1(-/-)) mouse model. To enable efficient packaging and rod-specific expression of the relatively large CNGB1a cDNA (~4 kb), we used an AAV expression cassette with a short rod-specific promoter and short regulatory elements. After injection of therapeutic AAVs into the subretinal space of 2-week-old CNGB1(-/-) mice, we assessed the restoration of the visual system by analyzing (i) CNG channel expression and localization, (ii) retinal function and morphology and (iii) vision-guided behavior. We found that the treatment not only led to expression of full-length CNGB1a, but also restored normal levels of the previously degraded CNGA1 subunit of the rod CNG channel. Both proteins co-localized in rod outer segments and formed regular CNG channel complexes within the treated area of the CNGB1(-/-) retina, leading to significant morphological preservation and a delay of retinal degeneration. In the electroretinographic analysis, we also observed restoration of rod-driven light responses. Finally, treated CNGB1(-/-) mice performed significantly better than untreated mice in a rod-dependent vision-guided behavior test. In summary, this work provides a proof-of-concept for the treatment of rod channelopathy-associated RP by AAV-mediated gene replacement.

The rd1 natural mutant is one of the first and probably the most commonly studied mouse model for retinitis pigmentosa (RP), a severe and frequently blinding human retinal degeneration. In several decades of research, the link between the increase in photoreceptor cGMP levels and the extremely rapid cell death gave rise to a number of hypotheses. Here, we provide clear evidence that the presence of cyclic nucleotide gated (CNG) channels in the outer segment membrane is the key to rod photoreceptor loss. In Cngb1(-/-) × rd1 double mutants devoid of regular CNG channels, cGMP levels are still pathologically high, but rod photoreceptor viability and outer segment morphology are greatly improved. Importantly, cone photoreceptors, the basis for high-resolution daylight and colour vision, survived and remained functional for extended periods of time. These findings strongly support the hypothesis of deleterious calcium (Ca(2+))-influx as the cause of rapid rod cell death and highlight the importance of CNG channels in this process. Furthermore, our findings suggest that targeting rod CNG channels, rather than general Ca(2+)-channel blockade, is a most promising symptomatic approach to treat otherwise incurable forms of cGMP-related RP.

Congenital absence of cone photoreceptor function is associated with strongly impaired daylight vision and loss of color discrimination in human achromatopsia. Here, we introduce viral gene replacement therapy as a potential treatment for this disease in the CNGA3(-/-) mouse model. We show that such therapy can restore cone-specific visual processing in the central nervous system even if cone photoreceptors had been nonfunctional from birth. The restoration of cone vision was assessed at different stages along the visual pathway. Treated CNGA3(-/-) mice were able to generate cone photoreceptor responses and to transfer these signals to bipolar cells. In support, we found morphologically that treated cones expressed regular cyclic nucleotide-gated (CNG) channel complexes and opsins in outer segments, which previously they did not. Moreover, expression of CNGA3 normalized cyclic guanosine monophosphate (cGMP) levels in cones, delayed cone cell death and reduced the inflammatory response of Müller glia cells that is typical of retinal degenerations. Furthermore, ganglion cells from treated, but not from untreated, CNGA3(-/-) mice displayed cone-driven, light-evoked, spiking activity, indicating that signals generated in the outer retina are transmitted to the brain. Finally, we demonstrate that this newly acquired sensory information was translated into cone-mediated, vision-guided behavior.

Purpose: Retinal blood vessel diameter and arteriovenous ratio (AVR) are commonly used diagnostic parameters. Because vascular walls are typically not visible in funduscopy, clinical AVR estimation is based on the lumen rather than the entire vessel diameter. Here the authors used a transgenic mouse model to quantify AVR in vivo based on total vessel dimensions (wall and lumen)., Methods: Confocal scanning laser ophthalmoscopy (cSLO) and indocyanine green angiography of the retinal vasculature were performed in wild-type and transgenic mice expressing green fluorescent protein (GFP) under the transcriptional control of the smooth muscle type α-actin (αSMA) promoter. Spectral-domain-OCT and ERG were performed to control for integrity of retinal structure and function in vivo and histology to demonstrate the location of GFP expression., Results: Native cSLO imaging and angiography yielded only inner vessel diameters similar to those observed through clinical funduscopy. In αSMA-GFP mice, autofluorescence imaging of the GFP-marked vascular walls also allowed the determination of outer vessel diameters. The mean AVR based on either inner diameter (AVR(id) = 0.72 ± 0.08) or outer diameter (AVR(od) = 0.97 ± 0.09) measurements were significantly different (P < 0.01)., Conclusions: Transgenic αSMA-GFP expression in murine vessel wall components allowed quantification of retinal vessel outer diameters in vivo. Although arterioles and venules differ in lumen and vessel wall width, they share a common outer diameter, leading to an AVR(od) close to unity. Because vessel walls are primary targets in common hypertensive and metabolic diseases, αSMA-GFP transgenic mice may prove valuable in the detailed assessment of such disorders in vivo.