Sheep as a large animal model for hearing research: comparison to common laboratory animals and humans.

Sensorineural hearing loss (SNHL), caused by pathology in the cochlea, is the most common type of hearing loss in humans. It is generally irreversible with very few effective pharmacological treatments available to prevent the degenerative changes or minimise the impact. Part of this has been attributed to difficulty of translating "proof-of-concept" for novel treatments established in small animal models to human therapies. There is an increasing interest in the use of sheep as a large animal model. In this article, we review the small and large animal models used in pre-clinical hearing research such as mice, rats, chinchilla, guinea pig, rabbit, cat, monkey, dog, pig, and sheep to humans, and compare the physiology, inner ear anatomy, and some of their use as model systems for SNHL, including cochlear implantation surgeries. Sheep have similar cochlear anatomy, auditory threshold, neonatal auditory system development, adult and infant body size, and number of birth as humans. Based on these comparisons, we suggest that sheep are well-suited as a potential translational animal model that bridges the gap between rodent model research to the clinical use in humans. This is especially in areas looking at changes across the life-course or in specific areas of experimental investigation such as cochlear implantation and other surgical procedures, biomedical device development and age-related sensorineural hearing loss research. Combined use of small animals for research that require higher throughput and genetic modification and large animals for medical translation could greatly accelerate the overall translation of basic research in the field of auditory neuroscience from bench to clinic.

Hyposmotic stress causes ATP release in a discrete zone within the outer cortex of rat lens.

Purpose: Purinergic signaling pathways activated by extracellular ATP have been implicated in the regulation of lens volume and transparency. In this study, we investigated the location of ATP release from whole rat lenses and the mechanism by which osmotic challenge alters such ATP release. Methods: Three-week-old rat lenses were cultured for 1 h in isotonic artificial aqueous humor (AAH) with no extracellular Ca2+, hypotonic AAH, or hypertonic AAH. The hypotonic AAH-treated lenses were also cultured in the absence or presence of connexin hemichannels and the pannexin channel blockers carbenoxolone, probenecid, and flufenamic acid. The ATP concentration in the AAH was determined using a Luciferin/luciferase bioluminescence assay. To visualize sites of ATP release induced by hemichannel and/or pannexin opening, the lenses were cultured in different AAH solutions, as described above, and incubated in the presence of Lucifer yellow (MW = 456 Da) and Texas red-dextran (MW = 10 kDa) for 1 h. Then the lenses were fixed, cryosectioned, and imaged using confocal microscopy to visualize areas of dye uptake from the extracellular space. Results: The incubation of the rat lenses in the AAH that lacked Ca2+ induced a significant increase in the extracellular ATP concentration. This was associated with an increased uptake of Lucifer yellow but not of Texas red-dextran in a discrete region of the outer cortex of the lens. Hypotonic stress caused a similar increase in ATP release and an increase in the uptake of Lucifer yellow in the outer cortex, which was significantly reduced by probenecid but not by carbenoxolone or flufenamic acid. Conclusions: Our data suggest that in response to hypotonic stress, the intact rat lens is capable of releasing ATP. This seems to be mediated via the opening of pannexin channels in a specific zone of the outer cortex of the lens. Our results support the growing evidence that the lens actively regulates its volume and therefore, its optical properties, via puerinergic signaling pathways.

The developmental journey of therapies targeting purine receptors: from basic science to clinical trials.

Since the discovery of ATP as an extracellular signalling molecule in 1972, purinergic signalling, mediated by extracellular purines and pyrimidines has been identified in virtually all mammalian tissues and is implicated in regulating fundamental cellular processes. In recent years, there has been an increasing focus on the pathophysiology and potential therapeutic interventions based on purinergic signalling. A vast range of compounds targeting purine receptors are in clinical development, and many more are in preclinical studies, which highlights the fast growth in this research field. As a tribute to Professor Geoffrey Burnstock's legacy in purinergic signalling, we present here a brief review of compounds targeting purine receptors that are in different stages of clinical trials. The review highlights the 50-year journey from basic research on purinergic receptors to clinical applications of therapies targeting purine receptors.

Redox Homeostasis in Ocular Tissues: Circadian Regulation of Glutathione in the Lens?

Accumulating evidence in tissues suggests an interconnection between circadian clocks and redox regulation. Diurnal variations in antioxidant levels, circadian rhythms of antioxidant enzyme activity, and differences in oxidative stress markers at different times of the day all indicate that oxidative stress responses follow a circadian rhythm. Disruptions of circadian rhythms are linked to a number of age-related diseases, including those in the eye. Typically, ocular tissues contain a robust antioxidant defence system to maintain redox balance and minimise oxidative stress and damage. The lens, in particular, contains remarkably high levels of the antioxidant glutathione (GSH). However, with advancing age, GSH levels deplete, initiating a chain of biochemical events that ultimately result in protein aggregation, light scattering, and age-related cataracts. While there is evidence that the lens exhibits circadian rhythms in the synthesis and release of melatonin, little is known about the regulation or function of timekeeping mechanisms in the lens. Since circadian rhythms are disrupted with age, and the depletion of GSH in the lens is a known initiating factor in the development of age-related cataracts, understanding the mechanisms involved in regulating GSH levels may lead to the future development of approaches to manipulate the clock to restore GSH levels and redox balance in the lens, and protect the lens from cataracts.

Pivotal roles of Fezf2 in differentiation of cone OFF bipolar cells and functional maturation of cone ON bipolar cells in retina.

During development of the retina, common retinal progenitor cells give rise to six classes of neurons that subsequently further diversify into more than 55 subtypes of neuronal subtypes. Here, we have investigated the expression and function of Fezf2, Fez zinc finger family of protein, in the developing mouse retina. Expression of Fezf2 transcripts was strongly observed in the embryonic retinal progenitors at E14.5 and declined quickly in subsequent development of retina. Then, in postnatal stage at around day 8, Fezf2 was transiently expressed then declined again. Loss-of-function analysis using retinas from mice in which Fezf2 coding region was substituted with ß-galactosidase showed that Fezf2 is expressed in a subset of cone OFF bipolar cells and required for their differentiation. Using electroretinogram, we found that Fezf2 knockout retina exhibited significantly reduced photopic b-wave, suggesting functional abnormality of cone ON bipolar cells. Furthermore, reduced expression of synaptic protein Trpm1 and structural alteration of ON bipolar cell invagination, both of which affected cone photoreceptor terminal synaptic activity, was identified by transmission electron microscopy and immunohistochemistry, respectively. Taken together, our results show that Fezf2 is indispensable in differentiation of bipolar precursors into cone OFF bipolar cells and in functional maturation of cone ON bipolar cells during development of mouse retina. These results contribute to our understanding of how diversity of neuronal subtypes and hence specificity of neuronal connections are established in the retina by intrinsic cues.

Forkhead Box Protein P1 Is Dispensable for Retina but Essential for Lens Development.

Purpose: Forkhead box protein P1 (Foxp1) is a transcriptional repressor expressed in many tissues. We identified Foxp1 as a highly expressed gene in retinal progenitor cells and investigated its roles during eye development. Methods: Mouse eyes with Foxp1 gain- or loss-of-function were established in vitro and in vivo. Results: Foxp1 overexpression in retinal progenitor cells resulted in reduced rod and increased cone photoreceptors. However, retina-specific knockout of Foxp1 was not associated with retinal differentiation abnormalities. Foxp1 was highly expressed in the lens during early development, and continued to be expressed in epithelial and cortical fiber cells until adulthood. At birth, analyses of Foxp1 lens-specific knockout (Foxp1-L-CKO) mice showed no gross morphologic changes in germinal or central epithelial cell compared to the controls. However, the numbers of proliferating and apoptotic cells were significantly increased in Foxp1-L-CKO mice. In addition, clear Y-structures were not observed in either the posterior or anterior sutures of the Foxp1-L-CKO lenses. Mature lenses of Foxp1-L-CKO mice were small and opaque. The fiber cell structure in the core and the cortical fiber cell columns were disturbed in Foxp1-L-CKO mice at postnatal day 14, potentially accounting for the opacity. In addition, epithelial cells were not aligned into columns along the transition zone in Foxp1-L-CKO mice. Taken together, these results suggest that Foxp1 has a role during lens growth in epithelial and differentiating fiber cells. Conclusions: Loss of Foxp1 results in loss of suture and fiber cell alignment, which eventually causes lens opacity, suggesting that Foxp1 has a key role in establishing cortical lens architecture.

Purinergic receptors in the rat lens: activation of P2X receptors following hyperosmotic stress.

PURPOSE: A range of P2Y and P2X receptors is expressed in the rat lens. Because most P2X receptors are located in the cytoplasm, the authors sought to determine whether P2X receptors are functionally active. METHODS: Rat lenses were cultured under either isotonic or hypertonic conditions in the presence of P2 receptor agonists or antagonists. Lenses were fixed and cryosectioned, cell membranes were labeled, and confocal microscopy was used to determine whether the different reagents affected cell morphology. RESULTS: Application of the P2 receptor inhibitor PPADS to lenses cultured under isotonic conditions induced extracellular space dilations between fiber cells in a distinct zone in the outer cortex. This damage was not caused by the inhibition of P2X(1) or P2Y(1) because more selective antagonists for P2X(1) (MRS2159) and P2Y(1) (MRS2179) either did not cause any damage or induced extracellular dilations located between superficial fiber cells at the lens modiolus, respectively. Although the P2 agonists ATPgammaS and ADPbetaS both induced a distinctive disruption to cell morphology in the same localized zone as PPADS, the P2X-specific agonist alpha,beta-methyl-ATP induced no change to cell morphology. However, under hypertonic conditions that cause the insertion of P2X(1,4) into the membranes, alpha,beta-methyl-ATP induced a localized zone of damage that was associated with changes in actin distribution. CONCLUSIONS: The results show that P2X receptors may play a minimal role in mediating ion fluxes in the rat lens under steady state conditions. In contrast, hypertonic cell shrinkage activates previously inactive P2X receptors in the lens, suggesting P2X receptors may play a role in the lens in response to osmotic stress.

Differential membrane redistribution of P2X receptor isoforms in response to osmotic and hyperglycemic stress in the rat lens.

P2X(1, 2, 3, 4, 6 and 7) are all expressed in a differentiation-dependent manner in the rat lens. However, in the lens outer cortex the subcellular distribution of all P2X isoforms is predominantly associated with a pool of receptors located in cytoplasmic vesicles. Here we investigate whether osmotic and hyperglycemic stress can alter the subcellular distribution of this cytoplasmic pool of P2X receptors. We show that in a discrete zone of the deeper outer cortex an isoform and stimulus-specific shift in the subcellular distribution of P2X receptors occurs from the cytoplasm to defined membrane domains. In response to hypertonic stress P2X(1) and P2X(4) isoforms became more closely associated with the broad sides of fiber cells, while under hypotonic conditions P2X(4) and P2X(6) isoforms associate with the narrow side membranes. No such changes in subcellular distribution were observed for P2X(2,3 and 7) isoforms. Lens cultured in 50 mM glucose exhibited cell swelling in this zone but only P2X(4) associated with narrow side membranes. Our results indicate P2X receptors can be differentially recruited to specific membrane domains of lens fiber cells by osmotic and hyperglycemic stress. Furthermore they suggest the involvement of specific P2X isoforms in the regulation of fiber cell volume and the initiation of diabetic cataract.

Molecular and functional mapping of regional differences in P2Y receptor expression in the rat lens.

Extracellular ATP has been shown to mobilize intracellular Ca(2+) in cultured ovine lens epithelial cells and in human lens epithelium, suggesting a role for purines in the modulation of lens transparency. In this study, we characterized the expression profiles of P2Y receptor isoforms throughout the rat lens at both the molecular and the functional levels. RT-PCR indicated that P2Y(1), P2Y(2), P2Y(4) and P2Y(6) are expressed in the lens, while P2Y(12), P2Y(13) and P2Y(14) are not. Immunohistochemistry, using isoform specific antibodies, indicated that the epithelium does not express P2Y(1) and P2Y(2), but that the underlying fiber cells, which differentiate from the epithelial cells, exhibit strong membranous labeling. Although co-expressed in fiber cells, differences in P2Y(1) and P2Y(2) expression were apparent. P2Y(1) expression extended deeper into the lens than P2Y(2), and its expression co-localized with Cx50 gap junction plaques, while P2Y(2) did not. Labeling for P2Y(4) and P2Y(6) receptors were observed in both epithelial cells and fiber cells, but the labeling was predominantly cytoplasmic in nature. While purine agonist (ATP, ADP, UTP and UDP) application to the lens induced mobilization of intracellular Ca(2+) in cortical fiber cells, little to no effect was observed in the anterior and equatorial epithelium. Thus the inability of UTP and UDP to mobilize intracellular Ca(2+) in the epithelium and the predominately cytoplasmic location of P2Y(4) and P2Y(6) suggests that these receptors may represent an inactive pool of receptors that may be activated under non-physiological conditions. In contrast, our results indicated that P2Y(1) and P2Y(2) are functionally active in fiber cells and their differential subcellular expression patterns suggest they may regulate distinct processes in the lens under steady state conditions.

Molecular identification and localization of P2X receptors in the rat lens.

There is growing evidence suggesting that purine-mediated signaling pathways play important roles in the ocular lens. We have previously reported the expression of P2Y receptors in the lens; and in this present study, we show that P2X receptors are also expressed. Transcripts for P2X1-7 receptors were detected in lens fiber cells using the reverse-transcription polymerase chain reaction (RT-PCR). Western blot analysis confirmed the expression of all P2X receptor subtypes at the protein level. Immunohistochemistry revealed P2X1 and P2X7 expression in the cytoplasm of cortical fiber cells. P2X2 expression was confined to the apical-apical interface between epithelial and fiber cells with little expression in cortical fiber cells. In contrast, P2X3, P2X4, P2X5 and P2X6 were expressed throughout the lens extending from the outer cortex through to the core. In the outer cortex, immunolocalisation of these P2X receptors was predominantly cytoplasmic. However, deeper into the lens, P2X receptor immunolabeling became more membranous, indicating the recruitment of P2X receptors from cytoplasmic vesicles into the membranes of mature fiber cells in the lens core. Western blotting confirmed regional differences in P2X receptor expression. The differential expression of P2X receptors in the ocular lens indicates that the purinergic signaling pathways may be involved in the maintenance of lens homeostasis.