Dissection of Human Vitreous Body Elements for Proteomic Analysis

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. ^1,2 Abnormal interactions between vitreous substructures and the retina underlie several vitreoretinal diseases, including retinal tear and detachment, macular pucker, macular hole, age-related macular degeneration, vitreomacular traction, proliferative vitreoretinopathy, proliferative diabetic retinopathy, and inherited vitreoretinopathies. ^1,2 The molecular composition of the vitreous substructures is not known. Since the vitreous body is transparent with limited surgical access, it has been difficult to study its substructures at the molecular level. We developed a method to separate and preserve these tissues for proteomic and biochemical analysis. The dissection technique in this experimental video shows how to isolate vitreous base, anterior hyaloid, vitreous core, and vitreous cortex from postmortem human eyes. One-dimensional SDS-PAGE analyses of each vitreous component showed that our dissection technique resulted in four unique protein profiles corresponding to each substructure of the human vitreous body. Identification of differentially compartmentalized proteins will reveal candidate molecules underlying various vitreoretinal diseases.     

Lama1 mutations lead to vitreoretinal blood vessel formation, persistence of fetal vasculature, and epiretinal membrane formation in mice

Background

Valuable insights into the complex process of retinal vascular development can be gained using models with abnormal retinal vasculature. Two such models are the recently described mouse lines with mutations in Lama1, an important component of the retinal internal limiting membrane (ILM). These mutants have a persistence of the fetal vasculature of vitreous (FVV) but lack a primary retinal vascular plexus. The present study provides a detailed analysis of astrocyte and vascular development in these Lama1 mutants.

Results

Although astrocytes and blood vessels initially migrate into Lama1 mutant retinas, both traverse the peripapillary ILM into the vitreous by P3. Once in the vitreous, blood vessels anastomose with vessels of the vasa hyaloidea propria, part of the FVV, and eventually re-enter the retina where they dive to form the inner and outer retinal capillary networks. Astrocytes continue proliferating within the vitreous to form a dense mesh that resembles epiretinal membranes associated with persistent fetal vasculature and proliferative vitreoretinopathy.

Conclusions

Lama1 and a fully intact ILM are required for normal retinal vascular development. Mutations in Lama1 allow developing retinal vessels to enter the vitreous where they anastomose with vessels of the hyaloid system which persist and expand. Together, these vessels branch into the retina to form fairly normal inner retinal vascular capillary plexi. The Lama1 mutants described in this report are potential models for studying the human conditions persistent fetal vasculature and proliferative vitreoretinopathy.     

Proteomic Interactions in the Mouse Vitreous-Retina Complex

Purpose

Human vitreoretinal diseases are due to presumed abnormal mechanical interactions between the vitreous and retina, and translational models are limited. This study determined whether nonstructural proteins and potential retinal biomarkers were expressed by the normal mouse vitreous and retina.

Methods

Vitreous and retina samples from mice were collected by evisceration and analyzed by liquid chromatography-tandem mass spectrometry. Identified proteins were further analyzed for differential expression and functional interactions using bioinformatic software.

Results

We identified 1,680 unique proteins in the retina and 675 unique proteins in the vitreous. Unbiased clustering identified protein pathways that distinguish retina from vitreous including oxidative phosphorylation and neurofilament cytoskeletal remodeling, whereas the vitreous expressed oxidative stress and innate immunology pathways. Some intracellular protein pathways were found in both retina and vitreous, such as glycolysis and gluconeogenesis and neuronal signaling, suggesting proteins might be shuttled between the retina and vitreous. We also identified human disease biomarkers represented in the mouse vitreous and retina, including carbonic anhydrase-2 and 3, crystallins, macrophage inhibitory factor, glutathione peroxidase, peroxiredoxins, S100 precursors, and von Willebrand factor.

Conclusions

Our analysis suggests the vitreous expresses nonstructural proteins that functionally interact with the retina to manage oxidative stress, immune reactions, and intracellular proteins may be exchanged between the retina and vitreous. This novel proteomic dataset can be used for investigating human vitreoretinopathies in mouse models. Validation of vitreoretinal biomarkers for human ocular diseases will provide a critical tool for diagnostics and an avenue for therapeutics.     

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

Müller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling Müller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5 has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label Müller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.     

Nrf2 has a protective role against neuronal and capillary degeneration in retinal ischemia-reperfusion injury

Retinal ischemia-reperfusion (I/R) involves an extensive increase in reactive oxygen species as well as proinflammatory changes that result in significant histopathologic damage, including neuronal and vascular degeneration. Nrf2 has a well-known cytoprotective role in many tissues, but its protective function in the retina is unclear. We investigated the possible role of Nrf2 as a protective mechanism in retinal ischemia-reperfusion injury using Nrf2^−/− mice. I/R resulted in an increase in retinal levels of superoxide and proinflammatory mediators, as well as leukocyte infiltration of the retina and vitreous, in Nrf2^+/+ mice. These effects were greatly accentuated in Nrf2^−/− mice. With regard to histopathologic damage, Nrf2^−/− mice exhibited loss of cells in the ganglion cell layer and markedly accentuated retinal capillary degeneration, as compared to wild-type. Treatment with the Nrf2 activator CDDO-Me increased antioxidant gene expression and normalized I/R-induced superoxide in the retina in wild-type but not Nrf2^−/− mice. CDDO-Me treatment abrogated retinal capillary degeneration induced by I/R in wild-type but not Nrf2^−/− mice. These studies indicate that Nrf2 is an important cytoprotective mechanism in the retina in response to ischemia-reperfusion injury and suggest that pharmacologic induction of Nrf2 could be a new therapeutic strategy for retinal ischemia-reperfusion and other retinal diseases.     

The cell cycle and retinal histogenesis fidget mutant mice.

The autoradiographic method using [³H]thymidine has shown that the autosomal recessive mutant gene fidget (gene symbol fi) prolonging the presynthetic period of the cell cycle in the retinal anlage in homozygotes retards the transition of retinal cells to the differentiated state. Some retinal cells of normal embryos (+/+) start their transition to the differentiated state on the 11th day of embryogenesis, while in $\text{fi}\text{fi}$ embryos this process starts only on the 12th day. An active transition of retinal cells to the differentiated state especially in the peripheral zone of the mutant retina takes place 2 days later as compared to normal embryos. The number of differentiating cells in the retina of mutants at the stages of development studied is considerably lower as compared to the norm. The analysis of the cell cycle parameters in 15-day embryos has shown that in the mutants the retina is less mature as compared to +/+ embryos. The sequence of transition of various cell types to the differentiated state in the retina of $\text{fi}\text{fi}$ embryos is the same as in the norm. Gene fidget seems to interfere with proliferative rather than critical (quantal) cell cycles in the developing mouse retina.     

Regulation of the cholesterol efflux transporters ABCA1 and ABCG1 in retina in hemochromatosis and by the endogenous siderophore 2,5-dihydroxybenzoic acid

Hypercholesterolemia and polymorphisms in the cholesterol exporter ABCA1 are linked to age-related macular degeneration (AMD). Excessive iron in retina also has a link to AMD pathogenesis. Whether these findings mean a biological/molecular connection between iron and cholesterol is not known. Here we examined the relationship between retinal iron and cholesterol using a mouse model (Hfe^−/−) of hemochromatosis, a genetic disorder of iron overload. We compared the expression of the cholesterol efflux transporters ABCA1 and ABCG1 and cholesterol content in wild type and Hfe^−/− mouse retinas. We also investigated the expression of Bdh2, the rate-limiting enzyme in the synthesis of the endogenous siderophore 2,5-dihydroxybenzoic acid (2,5-DHBA) in wild type and Hfe^−/− mouse retinas, and the influence of this siderophore on ABCA1/ABCG1 expression in retinal pigment epithelium. We found that ABCA1 and ABCG1 were expressed in all retinal cell types, and that their expression was decreased in Hfe^−/− retina. This was accompanied with an increase in retinal cholesterol content. Bdh2 was also expressed in all retinal cell types, and its expression was decreased in hemochromatosis. In ARPE-19 cells, 2,5-DHBA increased ABCA1/ABCG1 expression and decreased cholesterol content. This was not due to depletion of free iron because 2,5-DHBA (a siderophore) and deferiprone (an iron chelator) had opposite effects on transferrin receptor expression and ferritin levels. We conclude that iron is a regulator of cholesterol homeostasis in retina and that removal of cholesterol from retinal cells is impaired in hemochromatosis. Since excessive cholesterol is pro-inflammatory, hemochromatosis might promote retinal inflammation via cholesterol in AMD.     

三氧化二砷对视网膜色素上皮细胞增殖的影响

视网膜色素上皮细胞(retinal pigment epithelial cell,RPE)在维护视网膜正常生理功能方面具有极其重要的作用。研究发现,视网膜色素上皮细胞是增殖性玻璃体视网膜疾病(proliferative vitreous retinopathy,PVR)发生发展的主要细胞,而其增殖与细胞内调控信息失调密切相关。多项研究成果表明,三氧化二砷(As2O3)已经被用于医药几千年。其在白血病治疗的使用早在一个世纪以前就有所描述。As2O3在医学上的作用有着悠久的历史。然而,在最近的几个世纪它几乎被遗忘在西方医学。三氧化二砷在白血病、肿瘤的基础研究与临床治疗中已取得较大进展,引起广泛关注,但在眼科领域的研究才刚刚起步.增殖性视网膜疾病的发病日趋严重,已经成为全球性的重大负担,此病所导致的眼部并发症严重影响患者视功能及生活质量,因此,有必要就三氧化二砷对视网膜色素上皮细胞增殖的作用进行综述,以期为眼科疾病的防治研工作提供新的思路和策略。     

Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl

Retinal detachment (RD) describes a separation of the neurosensory retina from the retinal pigmented epithelium (RPE). The RPE is essential for normal function of the light sensitive neurons, the photoreceptors. Detachment of the retina from the RPE creates a physical gap that is filled with extracellular fluid. RD initiates cellular and molecular adverse events that affect both the neurosensory retina and the RPE since the physiological exchange of ions and metabolites is severely perturbed. The consequence for vision is related to the duration of the detachment since a rapid reapposition of the two tissues results in the restoration of vision ¹. The treatment of RD is exclusively surgical. Removal of vitreous gel (vitrectomy) is followed by the removal non essential part of the retina around the detached area to favor retinal detachment. The removed retinal specimens are res nullius (nothing) and consequently normally discarded. To recover RNA from these surgical specimens, we developed the procedure jouRNAl that allows RNA conservation during the transfer from the surgical block to the laboratory. We also standardized a protocol to purify RNA by cesium chloride ultracentrifugation to assure that the purified RNAs are suitable for global gene expression analysis. The quality of the RNA was validated both by RT-PCR and microarray analysis. Analysis of the data shows a simultaneous involvement of inflammation and photoreceptor degeneration during RD.     

Integrated Photoacoustic Ophthalmoscopy and Spectral-domain Optical Coherence Tomography

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging technologies. Existing retinal imaging modalities, such as fundus photography¹, confocal scanning laser ophthalmoscopy (cSLO)², and optical coherence tomography (OCT)³, have significant contributions in monitoring disease onsets and progressions, and developing new therapeutic strategies. However, they predominantly rely on the back-reflected photons from the retina. As a consequence, the optical absorption properties of the retina, which are usually strongly associated with retinal pathophysiology status, are inaccessible by the traditional imaging technologies.Photoacoustic ophthalmoscopy (PAOM) is an emerging retinal imaging modality that permits the detection of the optical absorption contrasts in the eye with a high sensitivity^4-7 . In PAOM nanosecond laser pulses are delivered through the pupil and scanned across the posterior eye to induce photoacoustic (PA) signals, which are detected by an unfocused ultrasonic transducer attached to the eyelid. Because of the strong optical absorption of hemoglobin and melanin, PAOM is capable of non-invasively imaging the retinal and choroidal vasculatures, and the retinal pigment epithelium (RPE) melanin at high contrasts ^6,7. More importantly, based on the well-developed spectroscopic photoacoustic imaging^5,8 , PAOM has the potential to map the hemoglobin oxygen saturation in retinal vessels, which can be critical in studying the physiology and pathology of several blinding diseases ⁹ such as diabetic retinopathy and neovascular age-related macular degeneration.Moreover, being the only existing optical-absorption-based ophthalmic imaging modality, PAOM can be integrated with well-established clinical ophthalmic imaging techniques to achieve more comprehensive anatomic and functional evaluations of the eye based on multiple optical contrasts^6,10 . In this work, we integrate PAOM and spectral-domain OCT (SD-OCT) for simultaneously in vivo retinal imaging of rat, where both optical absorption and scattering properties of the retina are revealed. The system configuration, system alignment and imaging acquisition are presented.     

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca²⁺), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca²⁺ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca²⁺ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (“slices”) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca²⁺ level. The protocol also allows “in-slice measurement” of absolute Ca²⁺ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca²⁺ signaling as well as the potential involvement of Ca²⁺ in photoreceptor death and retinal degeneration.     

Mutations in Lama1 Disrupt Retinal Vascular Development and Inner Limiting Membrane Formation

The Neuromutagenesis Facility at the Jackson Laboratory generated a mouse model of retinal vasculopathy, nmf223, which is characterized clinically by vitreal fibroplasia and vessel tortuosity. nmf223 homozygotes also have reduced electroretinogram responses, which are coupled histologically with a thinning of the inner nuclear layer. The nmf223 locus was mapped to chromosome 17, and a missense mutation was identified in Lama1 that leads to the substitution of cysteine for a tyrosine at amino acid 265 of laminin α1, a basement membrane protein. Despite normal localization of laminin α1 and other components of the inner limiting membrane, a reduced integrity of this structure was suggested by ectopic cells and blood vessels within the vitreous. Immunohistochemical characterization of nmf223 homozygous retinas demonstrated the abnormal migration of retinal astrocytes into the vitreous along with the persistence of hyaloid vasculature. The Y265C mutation significantly reduced laminin N-terminal domain (LN) interactions in a bacterial two-hybrid system. Therefore, this mutation could affect interactions between laminin α1 and other laminin chains. To expand upon these findings, a Lama1 null mutant, Lama1^tm1.1Olf, was generated that exhibits a similar but more severe retinal phenotype than that seen in nmf223 homozygotes. The increased severity of the Lama1 null mutant phenotype is probably due to the complete loss of the inner limiting membrane in these mice. This first report of viable Lama1 mouse mutants emphasizes the importance of this gene in retinal development. The data presented herein suggest that hypomorphic mutations in human LAMA1 could lead to retinal disease.     

Pigment Epithelium-Derived Factor (PEDF) Peptide Eye Drops Reduce Inflammation,Cell Death and Vascular Leakage in Diabetic Retinopathy in Ins2Akita Mice

Inflammation, neurodegeneration and microvascular irregularities are included in the spectrum of defects associated with diabetic retinopathy. Here, we evaluated intraocular deliverability features of two pigment epithelium-derived factor (PEDF) derivatives given as eye drops and their efficacy in modulating diabetes-induced retinal complications. The antiangiogenic PEDF60–77 (P60) and neuroprotective PEDF78–121 (P78) derivatives were applied to Ins2^Akita mouse eyes once a week for 15 wks at the onset of hyperglycemia. Peptides, labeled with Alexa Fluor 488, were observed penetrating the cornea by 1–4 h and gained access to the ciliary body, retinal pigment epithelium (RPE)-choroid complex, retina microvasculature and vitreous. Peak vitreous levels were 0.2 μg/mL for P60 and 0.9 μg/mL for P78 after 0.5 and 4 h, respectively. Both peptides reduced vascular leakage by ~60% and increased zona occludens 1 (ZO1) and occludin expression in the microvasculature to nondiabetic levels. P60 induced pERK1/2 and P78 promoted pAKT in Muller glia, two signals that were dampened in diabetic conditions. Pharmacologically inhibiting AKT signaling in the retina blocked effects of the peptides on ZO1 and occludin expression. P78 reduced levels of 9/20 cytokines in diabetic vitreous including interferon (IFN)-γ, interleukin (IL)-6, IL-3 and tumor necrosis factor (TNF)-α. P60 lowered levels of 6/20 cytokines but was less effective than P78. Neuroprotective P78 prevented diabetes-induced microglia activation by ~60%, retinal ganglion cell (RGC) death by ~22% and inner plexiform layer thinning by ~13%. In summary, we provide evidence that PEDF bioactive derivatives gained access to the retina by topical delivery and validated their efficacy in reducing diabetic retinopathy complications. Our findings argue for glia regulation of microvascular leakage and an early root cause for RGC degeneration embedded in microglia activation.     

Balance between autophagic pathways preserves retinal homeostasis

Aging contributes to the appearance of several retinopathies and is the largest risk factor for aged‐related macular degeneration, major cause of blindness in the elderly population. Accumulation of undegraded material as lipofuscin represents a hallmark in many pathologies of the aged eye. Autophagy is a highly conserved intracellular degradative pathway that plays a critical role in the removal of damaged cell components to maintain the cellular homeostasis. A decrease in autophagic activity with age observed in many tissues has been proposed to contribute to the aggravation of age‐related diseases. However, the participation of different autophagic pathways to the retina physiopathology remains unknown. Here, we describe a marked reduction in macroautophagic activity in the retina with age, which coincides with an increase in chaperone‐mediated autophagy (CMA). This increase in CMA is also observed during retinal neurodegeneration in the Atg5^flox/flox; nestin‐Cre mice, a mouse model with downregulation of macroautophagy in neuronal precursors. In contrast to other cell types, this autophagic cross talk in retinal cells is not bi‐directional and CMA inhibition renders cone photoreceptor very sensitive to stress. Temporal and cell‐type‐specific differences in the balance between autophagic pathways may be responsible for the specific pattern of visual loss that occurs with aging. Our results show for the first time a cross talk of different lysosomal proteolytic systems in the retina during normal aging and may help the development of new therapeutic intervention for age‐dependent retinal diseases.     

Aquaporin 1 Expression in Retinal Neovascularization in a Mouse Model of Retinopathy of Prematurity

Abstract

Recently, it was found that Aquaporin 1 (AQP1) is expressed strongly in proliferating microvessels, but the role of AQP1 in retinal neovascularization remains unknown. Here, we report the distribution of AQP1 expression during neovascularization of the retina in a mouse model of retinopathy of prematurity. AQP1 was expressed in all of the samples examined in P15 mouse and P17 mouse, including experimental and control groups. Immunostaining results showed that AQP1 is located in microvessel endothelia in retinas with proliferative retinopathy and prominently in the outer retina. Expression of AQP1 was significantly increased in experimental animals at P17, compared with control mice. No significant difference was seen in the levels of AQP1 on P12 or P15, compared with control mice. These results suggest that AQP1 may play an important role in retinal neovascularization.     

Lgr5+ amacrine cells possess regenerative potential in the retina of adult mice

Current knowledge indicates that the adult mammalian retina lacks regenerative capacity. Here, we show that the adult stem cell marker, leucine‐rich repeat‐containing G‐protein‐coupled receptor 5 (Lgr5), is expressed in the retina of adult mice. Lgr5⁺ cells are generated at late stages of retinal development and exhibit properties of differentiated amacrine interneurons (amacrine cells). Nevertheless, Lgr5⁺ amacrine cells contribute to regeneration of new retinal cells in the adult stage. The generation of new retinal cells, including retinal neurons and Müller glia from Lgr5⁺ amacrine cells, begins in early adulthood and continues as the animal ages. Together, these findings suggest that the mammalian retina is not devoid of regeneration as previously thought. It is rather dynamic, and Lgr5⁺ amacrine cells function as an endogenous regenerative source. The identification of such cells in the mammalian retina may provide new insights into neuronal regeneration and point to therapeutic opportunities for age‐related retinal degenerative diseases.     

Molecular substrates of potassium spatial buffering in glial cells

It is generally accepted that the foremost mechanism for the buffering of K⁺ from the extracellular space ([K⁺]_o) in the brain is “K⁺ spatial buffering.” This is the process by which glial cells dissipate local K⁺ gradients by transferring K⁺ ions from areas of high to low [K⁺]_o. These glial K⁺ fluxes are mediated mainly by inwardly rectifying K⁺ (Kir) channels. The K⁺ spatial buffering hypothesis has been tested and confirmed in the retina, in which is has been termed as “K⁺ siphoning”. In Müller cells, the primary glial cells of the retina, Kir channels are distributed in a highly non-uniform manner, exhibiting high concentrations in membrane domains facing the vitreous humor (endfeet) and in proximity to the blood vessels (perivascular processes). Such non-uniform distribution of Kir channels facilitates directed K⁺ fluxes in the retina from the synaptic plexiform layers to the vitreous humor and blood vessels. Recent molecular and electrophysiological studies in Müller cells have revealed a high degree of complexity in terms of Kir channel subunit composition, mechanisms of subcellular localization, and regulation. How such complexity fits into their proposed role in buffering [K⁺]_o in retina is the main topic of this article.     

Absence of MMACHC in peripheral retinal cells does not lead to an ocular phenotype in mice

Combined methylmalonic aciduria with homocystinuria (cblC type) is a rare disease caused by mutations in the MMACHC gene. MMACHC encodes an enzyme crucial for intracellular vitamin B₁₂ metabolism, leading to the accumulation of toxic metabolites e.g. methylmalonic acid (MMA) and homocysteine (Hcy), and secondary disturbances in folate and one-carbon metabolism when not fully functional. Patients with cblC deficiency often present in the neonatal or early childhood period with a severe multisystem pathology, which comprises a broad spectrum of treatment-resistant ophthalmological phenotypes, including retinal degeneration, impaired vision, and vascular changes. To examine the potential function of MMACHC in the retina and how its loss may impact disease, we performed gene expression studies in human and mouse, which showed that local expression of MMACHC in the retina and retinal pigment epithelium is relatively stable over time. To study whether functional MMACHC is required for retinal function and tissue integrity, we generated a transgenic mouse lacking Mmachc expression in cells of the peripheral retina. Characterization of this mouse revealed accumulation of cblC disease related metabolites, including MMA and the folate-dependent purine synthesis intermediates AICA-riboside and SAICA-riboside in the retina. Nevertheless, fundus appearance, morphology, vasculature, and cellular composition of the retina, as well as ocular function, remained normal in mice up to 6 or 12 months of age. Our data indicates that peripheral retinal neurons do not require intrinsic expression of Mmachc for survival and function and questions whether a local MMACHC deficiency is responsible for the retinal phenotypes in patients.     

The Role of Histamine in the Retina: Studies on the Hdc Knockout Mouse

The role of histamine in the retina is not well understood, despite it regulating a number of functions within the brain, including sleep, feeding, energy balance, and anxiety. In this study we characterized the structure and function of the retina in mice that lacked expression of the rate limiting enzyme in the formation of histamine, histidine decarboxylase (Hdc^−/− mouse). Using laser capture microdissection, Hdc mRNA expression was assessed in the inner and outer nuclear layers of adult C57Bl6J wildtype (WT) and Hdc^−/−-retinae. In adult WT and Hdc^−/−-mice, retinal fundi were imaged, retinal structure was assessed using immunocytochemistry and function was probed by electroretinography. Blood flow velocity was assessed by quantifying temporal changes in the dynamic fluorescein angiography in arterioles and venules. In WT retinae, Hdc gene expression was detected in the outer nuclear layer, but not the inner nuclear layer, while the lack of Hdc expression was confirmed in the Hdc^−/− retina. Preliminary examination of the fundus and retinal structure of the widely used Hdc^−/−mouse strain revealed discrete lesions across the retina that corresponded to areas of photoreceptor abnormality reminiscent of the rd8 (Crb1) mutation. This was confirmed after genotyping and the strain designated Hdc^rd8/rd8. In order to determine the effect of the lack of Hdc-alone on the retina, Hdc^−/− mice free of the Crb1 mutation were bred. Retinal fundi appeared normal in these animals and there was no difference in retinal structure, macrogliosis, nor any change in microglial characteristics in Hdc^−/− compared to wildtype retinae. In addition, retinal function and retinal blood flow dynamics showed no alterations in the Hdc^−/− retina. Overall, these results suggest that histamine plays little role in modulating retinal structure and function.