The effect of unilateral disruption of the centrifugal visual system on normal eye development in chicks raised under constant light conditions.

The centrifugal visual system (CVS) comprises a visually driven isthmic feedback projection to the retina. While its function has remained elusive, we have previously shown that, under otherwise normal conditions, unilateral disconnection of centrifugal neurons in the chick affected eye development, inducing a reduced rate of axial elongation that resulted in a unilateral hyperopia in the eye contralateral to the lesion. Here, we further investigate the role of centrifugal neurons in ocular development in chicks reared in an abnormal visual environment, namely constant light. The baseline ocular phenotype of constant light-reared chicks (n = 8) with intact centrifugal neurons was assessed over a 3-week post-hatch time period and, subsequently, compared to chicks raised in normal diurnal lighting (n = 8). Lesions of the isthmo-optic tract or sham surgeries were performed in another seventeen chicks, all raised under constant light. Ocular phenotyping was performed over a 21-day postoperative period to assess changes in refractive state (streak retinoscopy) and ocular component dimensions (A-scan ultrasonography). A pathway-tracing paradigm was employed to quantify lesion success. Chicks raised in constant light conditions with an intact CVS developed shallower anterior chambers combined with elongated vitreous chambers relative to chicks raised in normal diurnal lighting. Seven days following surgery to disrupt centrifugal neurons, a significant positive correlation between refractive error asymmetry between the eyes and lesion success was evident, characterized by hyperopia in the eye contralateral to the lesion. By 21 days post-surgery, these contralateral eyes had become emmetropic, while ipsilateral eyes had developed relative axial hyperopia. Our results provide further support for the hypothesis that the centrifugal visual system can modulate eye development.

A new method to investigate how mechanical loading of osteocytes controls osteoblasts.

Mechanical loading, a potent stimulator of bone formation, is governed by osteocyte regulation of osteoblasts. We developed a three-dimensional (3D) in vitro co-culture system to investigate the effect of loading on osteocyte-osteoblast interactions. MLO-Y4 cells were embedded in type I collagen gels and MC3T3-E1(14) or MG63 cells layered on top. Ethidium homodimer staining of 3D co-cultures showed 100% osteoblasts and 86% osteocytes were viable after 7 days. Microscopy revealed osteoblasts and osteocytes maintain their respective ovoid/pyriform and dendritic morphologies in 3D co-cultures. Reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) of messenger ribonucleic acid (mRNA) extracted separately from osteoblasts and osteocytes, showed that podoplanin (E11), osteocalcin, and runt-related transcription factor 2 mRNAs were expressed in both cell types. Type I collagen (Col1a1) mRNA expression was higher in osteoblasts (P < 0.001), whereas, alkaline phosphatase mRNA was higher in osteocytes (P = 0.001). Immunohistochemistry revealed osteoblasts and osteocytes express E11, type I pro-collagen, and connexin 43 proteins. In preliminary experiments to assess osteogenic responses, co-cultures were treated with human recombinant bone morphogenetic protein 2 (BMP-2) or mechanical loading using a custom built loading device. BMP-2 treatment significantly increased osteoblast Col1a1 mRNA synthesis (P = 0.031) in MLO-Y4/MG63 co-cultures after 5 days treatment. A 16-well silicone plate, loaded (5 min, 10 Hz, 2.5 N) to induce 4000-4500 µÎµ cyclic compression within gels increased prostaglandin E2 (PGE2) release 0.5 h post-load in MLO-Y4 cells pre-cultured in 3D collagen gels for 48, 72 h, or 7 days. Mechanical loading of 3D co-cultures increased type I pro-collagen release 1 and 5 days later. These methods reveal a new osteocyte-osteoblast co-culture model that may be useful for investigating mechanically induced osteocyte control of osteoblast bone formation.

Disruption of the centrifugal visual system inhibits early eye growth in chicks.

PURPOSE: Emmetropization, the process by which neonatal refractive errors are reduced toward zero, is partially dependent on brain-retina connectivity. Here, we investigated the role of the centrifugal visual system, a visually driven retinal feedback projection, as one potential influence on this complex mechanism. METHODS: Lesions of the isthmo-optic nucleus/tract or sham surgeries were performed in fifty-four 4- to 5-day-old chicks to disrupt centrifugal efferents to the contralateral retina. Prior to surgery, baseline refractive error measurements were made using streak retinoscopy. Postoperative ocular phenotyping, which (in addition to retinoscopy) comprised A-scan ultrasonography and infrared keratometry, was performed 7 days and 21 days postsurgery. A pathway-tracing paradigm was used to determine lesion success, whereby an injection of wheat-germ agglutinin was made into the vitreous chamber contralateral to the lesion. Postmortem, tissue processing, immunohistochemistry, and stereological analysis of intact centrifugal neurons were performed. Subsequently, chicks were divided into quartile groups based on percentage lesion success. RESULTS: Seven days postsurgery, chicks in the quartile of highest percentage lesion success exhibited significant axial hyperopia in the "treated eye" (contralateral to the lesion) relative to the "control eye" (ipsilateral to the lesion) eye, when compared with subjects within quartile groups of lower percentage lesion success (P = 0.004). However, by 21 days postsurgery, the induced hyperopia was no longer evident. CONCLUSIONS: Unilateral disruption of centrifugal efferents to the retina of the contralateral eye induces an initial axial hyperopia, which is subsequently reversed through increased vitreous elongation in the affected eyes.