Comparing the ocular surface temperature and dry eye condition of keratoconus with normal eyes using infrared thermal imaging.

PURPOSE: This study was conducted to compare the ocular surface temperature in keratoconus eyes with that in normal eyes. METHODS: A total of 27 participants were enrolled, with 10 and 17 participants in the keratoconus and control groups, respectively. Participants in the control group underwent an ophthalmic slit lamp examination and ocular thermography, while an additional corneal tomography was performed for those in the keratoconus group. RESULTS: For patients with keratoconus, the mean upper eyelid temperature (UET) was 32.36 ± 1.02 °C, inner canthus temperature (ICT) was 34.25 ± 0.83 °C, outer canthus temperature (OCT) was 33.62 ± 0.96 °C, initial central corneal temperature (initial CCT) was 33.04 ± 1.03 °C, sixth-second CCT (6 s-CCT) was 32.67 ± 1.19 °C, and the mean change in CCT measured within 6 s (change in CCT within 6 s) was 0.36 ± 0.26 °C. For controls, the values for UET, ICT, OCT, initial CCT, 6 s-CCT, and change in CCT within 6 s were 32.35 ± 1.13 °C, 34.14 ± 0.91 °C, 33.51 ± 1.02 °C, 33.22 ± 1.01 °C, 32.99 ± 1.01 °C, and 0.22 ± 0.17 °C, respectively. Except for the change in CCT within 6 s (p = 0.022), no significant differences were observed in UET (p = 0.973), ICT (p = 0.659), OCT (p = 0.697), initial CCT (p = 0.556) or 6 s-CCT (p = 0.310) between the two groups. CONCLUSION: The keratoconus eyes showed faster changes in CCT and evaporation of tear film after opening the eyes. Therefore, the keratoconus eyes had a higher incidence of dry eye conditions.

The application of artificial intelligence in glaucoma diagnosis and prediction.

Artificial intelligence is a multidisciplinary and collaborative science, the ability of deep learning for image feature extraction and processing gives it a unique advantage in dealing with problems in ophthalmology. The deep learning system can assist ophthalmologists in diagnosing characteristic fundus lesions in glaucoma, such as retinal nerve fiber layer defects, optic nerve head damage, optic disc hemorrhage, etc. Early detection of these lesions can help delay structural damage, protect visual function, and reduce visual field damage. The development of deep learning led to the emergence of deep convolutional neural networks, which are pushing the integration of artificial intelligence with testing devices such as visual field meters, fundus imaging and optical coherence tomography to drive more rapid advances in clinical glaucoma diagnosis and prediction techniques. This article details advances in artificial intelligence combined with visual field, fundus photography, and optical coherence tomography in the field of glaucoma diagnosis and prediction, some of which are familiar and some not widely known. Then it further explores the challenges at this stage and the prospects for future clinical applications. In the future, the deep cooperation between artificial intelligence and medical technology will make the datasets and clinical application rules more standardized, and glaucoma diagnosis and prediction tools will be simplified in a single direction, which will benefit multiple ethnic groups.

UBE2T is a prognostic biomarker and correlated with Th2 cell infiltrates in retinoblastoma.

OBJECTIVE: This study aimed to screen anaplasia-related genes that influence the progression of retinoblastoma (RB) and to identify immune cells associated with the poor prognosis. METHODS: Differentially expressed genes (DEGs) between retina and RB samples were acquired from gene expression omnibus (GEO) database. Candidate hub genes were screened by taking intersections among the co-expressed genes, the hub nodes, and DEGs of the validation set. The hub genes were identified by receiver operating characteristic (ROC) and quantitative real-time PCR (qPCR). Immune infiltration levels of RB tissues were estimated using single-sample gene set enrichment analysis (ssGSEA). The functions of RB cells were detected by CCK8, EDU and flow cytometry assays. RESULTS: 665 DEGs involved in the genesis and progression of RB were acquired from GEO database. 29 candidate hub genes were screened by examining 43 co-expressed genes and 63 hub nodes. 9 hub genes (CHEK1, EXO1, FANCI, GTSE1, MELK, MKI67, NCAPH, PRC1, and UBE2T) strongly related to the anaplastic grades were validated by ROC curve analysis (AUC >0.8). Based on the ssGSEA scores, the immune infiltration levels of Th2 cells were positively associated with anaplastic grade. qPCR assay showed that 9 hub genes were upregulated in RB cells, and UBE2T expressed remarkably high. CCK 8, EDU, and flow cytometry assays revealed that UBE2T silencing inhibited the proliferation of RB cells and incited apoptosis. CONCLUSIONS: The increased infiltration of Th2 cells and upregulated expression of 9 hub genes predict a poor prognosis of RB. UBE2T can be a therapeutic target for RB treatment.

Optimized-Unet: Novel Algorithm for Parapapillary Atrophy Segmentation.

In recent years, an increasing number of people have myopia in China, especially the younger generation. Common myopia may develop into high myopia. High myopia causes visual impairment and blindness. Parapapillary atrophy (PPA) is a typical retinal pathology related to high myopia, which is also a basic clue for diagnosing high myopia. Therefore, accurate segmentation of the PPA is essential for high myopia diagnosis and treatment. In this study, we propose an optimized Unet (OT-Unet) to solve this important task. OT-Unet uses one of the pre-trained models: Visual Geometry Group (VGG), ResNet, and Res2Net, as a backbone and is combined with edge attention, parallel partial decoder, and reverse attention modules to improve the segmentation accuracy. In general, using the pre-trained models can improve the accuracy with fewer samples. The edge attention module extracts contour information, the parallel partial decoder module combines the multi-scale features, and the reverse attention module integrates high- and low-level features. We also propose an augmented loss function to increase the weight of complex pixels to enable the network to segment more complex lesion areas. Based on a dataset containing 360 images (Including 26 pictures provided by PALM), the proposed OT-Unet achieves a high AUC (Area Under Curve) of 0.9235, indicating a significant improvement over the original Unet (0.7917).

Outcomes of combined treatments in patients with retinal arterial macroaneurysm.

PURPOSE: To evaluate the outcome of the combined approach between intravitreal ranibizumab (IVR) and focal laser photocoagulation (FLP) in the treatment of symptomatic retinal arterial macroaneurysm (RAM). METHODS: A total of 10 patients were included in this clinical case series report. They were diagnosed with symptomatic RAM (one eye in each) and assessed by a comprehensive ophthalmologic examination, including fluorescein angiography (FA), optical coherence tomography angiography (OCT-A), and indocyanine green angiography (ICGA). All patients were treated with an IVR followed by an FLP 2 weeks later. If necessary, a second IVR was given 1 month after the first one (or 2 weeks after the first FLP), which was followed by a second FLP treatment 2 weeks later in the needed cases. All cases were followed up for 6 months after the last treatment. RESULTS: Both the retina hemorrhage and edema were resolved by the treatment. No ocular and/or systemic side effects were evident, and no recrudescence of RAM was seen within the 6 months of follow-up. CONCLUSION: The combined treatment of IVRs and FLPs was successful in the management of symptomatic RAM.

An Artificial Intelligent Risk Classification Method of High Myopia Based on Fundus Images.

High myopia is a global ocular disease and one of the most common causes of blindness. Fundus images can be obtained in a noninvasive manner and can be used to monitor and follow up on many fundus diseases, such as high myopia. In this paper, we proposed a computer-aided diagnosis algorithm using deep convolutional neural networks (DCNNs) to grade the risk of high myopia. The input images were automatically classified into three categories: normal fundus images were labeled class 0, low-risk high-myopia images were labeled class 1, and high-risk high-myopia images were labeled class 2. We conducted model training on 758 clinical fundus images collected locally, and the average accuracy reached 98.15% according to the results of fivefold cross-validation. An additional 100 fundus images were used to evaluate the performance of DCNNs, with ophthalmologists performing external validation. The experimental results showed that DCNNs outperformed human experts with an area under the curve (AUC) of 0.9968 for the recognition of low-risk high myopia and 0.9964 for the recognition of high-risk high myopia. In this study, we were able to accurately and automatically perform high myopia classification solely using fundus images. This has great practical significance in terms of improving early diagnosis, prevention, and treatment in clinical practice.

Sequence effect in the treatment of proliferative diabetic retinopathy with intravitreal ranibizumab and panretinal photocoagulation.

PURPOSE: To compare the outcome of the sequence in the two treatments (intravitreal ranibizumab and panretinal photocoagulation) in high-risk proliferative diabetic retinopathy. METHODS: This retrospective study included 35 patients with newly diagnosed high-risk proliferative diabetic retinopathy in 43 eyes; 18 (22 eyes) received intravitreal ranibizumab before panretinal photocoagulation (intravitreal ranibizumab+ group), while the other 17 (21 eyes) received panretinal photocoagulation before intravitreal ranibizumab (panretinal photocoagulation+ group). Each subject received three intravitreal ranibizumabs that were interleaved with three panretinal photocoagulations. The first treatment (either intravitreal ranibizumab or panretinal photocoagulation) was done 1 week before the second one. The interval between intravitreal ranibizumabs was 4 weeks, panretinal photocoagulation was 2 weeks. The power and pulse duration were determined based upon the status of each retinal spot before each panretinal photocoagulation. The retinal non-perfusion region was measured with fundus fluorescein angiography before and 1 month after the final treatment. The central macular thickness was measured with optical coherence tomography within 1 week before the first treatment, before each panretinal photocoagulation, and 1 month after the final intravitreal ranibizumab. RESULTS: The panretinal photocoagulation energy required for effective treatment was lower in intravitreal ranibizumab+ group in the first and second sessions and in total energy (p < 0.05). Central macular thickness reduction before the second panretinal photocoagulation session was significant in the intravitreal ranibizumab+ group (p < 0.05). CONCLUSION: The sequence used in intravitreal ranibizumab+ group showed clear advantages over that in panretinal photocoagulation+ group in the treatment of proliferative diabetic retinopathy, not only in the use of lower energy for panretinal photocoagulation but also in the more rapid regression of neovascularization and less need of additional treatment.

SC79 protects retinal pigment epithelium cells from UV radiation via activating Akt-Nrf2 signaling.

Excessive Ultra-violet (UV) radiation causes oxidative damages and apoptosis in retinal pigment epithelium (RPE) cells. Here we tested the potential activity of SC79, a novel small molecule activator of Akt, against the process. We showed that SC79 activated Akt in primary and established (ARPE-19 line) RPE cells. It protected RPE cells from UV damages possibly via inhibiting cell apoptosis. Akt inhibition, via an Akt specific inhibitor (MK-2206) or Akt1 shRNA silence, almost abolished SC79-induced RPE cytoprotection. Further studies showed that SC79 activated Akt-dependent NF-E2-related factor 2 (Nrf2) signaling and inhibited UV-induced oxidative stress in RPE cells. Reversely, Nrf2 shRNA knockdown or S40T mutation attenuated SC79-induced anti-UV activity. For the in vivo studies, we showed that intravitreal injection of SC79 significantly protected mouse retina from light damages. Based on these results, we suggest that SC79 protects RPE cells from UV damages possibly via activating Akt-Nrf2 signaling axis.

Repertoires of autophagy in the pathogenesis of ocular diseases.

Autophagy is an important intracellular degradative process that delivers cytoplasmic proteins to lysosome for degradation. Dysfunction of autophagy is implicated in several human diseases, such as neurodegenerative diseases, infectious diseases, and cancers. Autophagy-related proteins are constitutively expressed in the eye. Increasing studies have revealed that abnormal autophagy is an important pathological feature of several ocular diseases. Pharmacological manipulation of autophagy may provide an alternative therapeutic target for some ocular diseases. In this manuscript, we reviewed the relevant progress about the role of autophagy in the pathogenesis of ocular diseases.

Regulation of autophagy by high glucose in human retinal pigment epithelium.

BACKGROUND: Autophagy is a self-degradative process that is important for balancing sources of energy at critical times in development and in response to nutrient stress. Retinal pigment epithelium (RPE) works as the outer blood retina barrier and is vulnerable to energy stress-induced injury. However, the effect of high glucose treatment on autophagy is still unclear in RPE. METHODS: Transmission electron microscopy was used to detect the generation of autophagosome. Small interfering RNA (siRNA) and MTT was used to determine the effect of autophagy on cell viability. Western blots and immunohistochemistry were used to detect the expression pattern of autophagic markers, including LC3 and p62. RESULTS: High glucose treatment results in a significant increase in the generation of autophagosome and altered expression of LC3 and p62. High glucose-induced autophagy is independent of mTOR signaling, but is mainly regulated via ROS-mediated ER stress signaling. CONCLUSION: In the scenario of high glucose-induced oxidative stress, autophagy may be required for the removal of damaged proteins, and provide a default mechanism to prevent high glucose-induced injury in RPE.

Abnormal glutamate metabolism in the retina of aquaporin 4 (AQP4) knockout mice upon light damage.

Glutamate is a major excitatory neurotransmitter in the retina. Glutamate neurotoxicity has been implicated in the pathogenesis of several ocular diseases. Aquaporin 4 (AQP4) is a water-selective membrane transport protein, and its knockout could alter retinal neuron excitability. However, the effect of AQP4 knockout on glutamate metabolism is still unclear in the retina. Here, we reported that the retinas in AQP4 knockout mice showed higher glutamate levels than that in wild-type mice upon light damage. AQP4 knockout could result in accelerated apoptosis of retinal cells, increased reactive gliosis, and attenuated survival of RGCs in response to light damage. Moreover, AQP4 knockout could affect the expression pattern of glutamate metabolism-related proteins such as GLAST and GS. Taken together, this study revealed a novel role of AQP4 in regulating glutamate metabolism. Pharmacological manipulation of AQP4 function may represent as a potent therapeutic target in the treatment of neurological ocular disorders.

Ultraviolet (UV) and hydrogen peroxide activate ceramide-ER stress-AMPK signaling axis to promote retinal pigment epithelium (RPE) cell apoptosis.

Ultraviolet (UV) radiation and reactive oxygen species (ROS) impair the physiological functions of retinal pigment epithelium (RPE) cells by inducing cell apoptosis, which is the main cause of age-related macular degeneration (AMD). The mechanism by which UV/ROS induces RPE cell death is not fully addressed. Here, we observed the activation of a ceramide-endoplasmic reticulum (ER) stress-AMP activated protein kinase (AMPK) signaling axis in UV and hydrogen peroxide (H2O2)-treated RPE cells. UV and H2O2 induced an early ceramide production, profound ER stress and AMPK activation. Pharmacological inhibitors against ER stress (salubrinal), ceramide production (fumonisin B1) and AMPK activation (compound C) suppressed UV- and H2O2-induced RPE cell apoptosis. Conversely, cell permeable short-chain C6 ceramide and AMPK activator AICAR (5-amino-1-ß-D-ribofuranosyl-imidazole-4-carboxamide) mimicked UV and H2O2's effects and promoted RPE cell apoptosis. Together, these results suggest that UV/H2O2 activates the ceramide-ER stress-AMPK signaling axis to promote RPE cell apoptosis.

Protective role of Wallerian degeneration slow (Wld(s)) gene against retinal ganglion cell body damage in a Wallerian degeneration model.

Nerve distal axon injury-induced Wallerian degeneration is significantly delayed in Wallerian degeneration slow (Wld(s)) mutant mice, although the associated mechanisms are not completely clear and the role of Wld(s) in retinal ganglion cell (RGC) body damage is not fully understood. In the present study, a Wallerian degeneration model was established in wild-type (WT) and Wld(s) mutant mice by creating mechanical injury in the optic nerves. Wallerian degeneration and RGC body collapse were observed to be significantly delayed in the Wld(s) mice. Electroretinograms (ERG) and visual evoked potentials (VEPs) in Wld(s) mice were also significantly improved at the earlier stages (one week) following injury. The retina immunohistochemistry results showed that Wld(s) mice had more ordered cells and improved inner granular cell layer arrangement compared with the WT mice. Optic nerve Luxol Fast Blue (LFB) staining showed greater axon demyelination in WT mice than in Wld(s) mice. A large number of apoptotic cells were also observed in the WT mice. The present results suggest that the Wld(s) gene may also protect the RGC body following nerve injury.

TNF-α promotes human retinal pigment epithelial (RPE) cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression through activation of Akt/mTORC1 signaling.

Tumor necrosis factor-alpha (TNF-α) promotes in vitro retinal pigment epithelial (RPE) cell migration to initiate proliferative vitreoretinopathy (PVR). Here we report that TNF-α promotes human RPE cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression. Inhibition of MMP-9 by its inhibitor or its neutralizing antibody inhibited TNF-α-induced in vitro RPE cell migration. Reversely, exogenously-added active MMP-9 promoted RPE cell migration. Suppression Akt/mTOR complex 1(mTORC1) activation by LY 294002 and rapamycin inhibited TNF-α-mediated MMP-9 expression. To introduce a constitutively active Akt (CA-Akt) in cultured RPE cells increased MMP-9 expression, and to block mTORC1 activation by rapamycin inhibited its effect. RNA interference (RNAi)-mediated silencing of SIN1, a key component of mTOR complex 2 (mTORC2), had no effect on MMP-9 expression or secretion. In conclusion, this study suggest that TNF-α promotes RPE cell migration by inducing MMP-9 expression through activation of Akt/ mTORC1, but not mTORC2 signaling.

Tumor necrosis factor-alpha (TNF-α)-mediated in vitro human retinal pigment epithelial (RPE) cell migration mainly requires Akt/mTOR complex 1 (mTORC1), but not mTOR complex 2 (mTORC2) signaling.

When rhegmatogenous retinal detachment occurs, tumor necrosis factor-alpha (TNF-α) among other cytokines leaks into the subretinal space, induces resident retinal pigment epithelial (RPE) cells to migrate, which is the initial step of proliferative vitreoretinopathy (PVR). In the current study, we aim to understand how this is regulated by focusing the cellular mechanisms involved. Here we identified an Akt/Tuberous sclerosis protein 2 (TSC2)/mTOR complex1 (mTORC1) signaling pathway after TNF-α treatment to mediate RPE cell migration. Suppression of mTORC1 activation, either by its inhibitor rapamycin, or by activation of its suppressor AMP activated protein kinase (AMPK), inhibited TNF-α-mediated RPE cell migration, while RNA interference (RNAi)-mediated knocking-down of SIN1 or Rictor, two key components of mTOR complex 2 (mTORC2), had no significant effect on TNF-α-induced RPE cell migration. Our data provide initial evidence that TNF-α-mediated in vitro RPE cell migration mainly requires Akt/mTORC1, but not mTORC2 signaling. The results of this study may lead to indentify novel signaling targets against PVR.

EGCG protects against UVB-induced apoptosis via oxidative stress and the JNK1/c-Jun pathway in ARPE19 cells.

Ultraviolet B (UVB) radiation is part of the spectrum of light produced by the sun. This form of radiation has been implicated as one of the potential etiological factors causing age-related macular degeneration (AMD). Oxidative injury to the retinal pigment epithelium (RPE) has also been thought to play a key role in AMD. The aim of the present study was to determine the mechanism by which UVB causes damage to the RPE cells, whether it occurs through oxidative stress and the mitogen-activated protein kinase (MAPK) pathway and whether the green tea extract, (-)-epigallocatechin gallate (EGCG), has a protective role. Cell viability assays were used to determine the viability of the cells under different conditions. Cell death caused by apoptosis was determined using fluorescein isothiocyanate conjugated-annexin V/PI labeling, followed by flow cytometry. Intracellular reactive oxygen species (ROS) levels were measured by flow cytometry. Western blot analysis was used to detect UVB-induced MAPK signaling pathways. The findings showed that UVB induced apoptosis, which increased intracellular ROS in ARPE19 cells. Inhibition of c-Jun NH2-terminal kinase (JNK) with a specific inhibitor augmented this apoptosis, and anisomycin (an activator of JNK) attenuated this apoptosis. In addition, UVB decreased the phosphorylation of JNK1 and c-Jun. Finally, EGCG reduced the ROS generation and apoptosis, and also partially blocked the decreased phosphorylation of JNK1 and c-Jun by UVB irradiation. The findings show that UVB irradiation is able to induce apoptosis in ARPE19 cells through oxidative stress, but EGCG treatment attenuates this damage. In this situation, the JNK pathway plays an anti-apoptotic role. The use of selective activators or antioxidants may be useful in reducing the oxidative damage occurring in AMD.

Rapamycin sensitive mTOR activation mediates nerve growth factor (NGF) induced cell migration and pro-survival effects against hydrogen peroxide in retinal pigment epithelial cells.

Patients with age related macular degeneration (AMD) have a loss of vision in the center of the visual field. Oxidative stress plays an important role in this progress. Nerve growth factor (NGF) is important for the survival and maintenance of sympathetic and sensory neurons and NGF eye drops improve visual acuity and electro-functional activity in patients with AMD. However, the molecular mechanisms and signaling events involved in this have not been fully investigated. Using cultured human retinal pigment epithelial (RPE) cells, we demonstrate here that NGF protects RPE cells against hydrogen peroxide (H(2)O(2))-induced cell apoptosis. NGF also induces RPE cell migration, the latter is important for retinal regeneration and the recovery from AMD. H(2)O(2) decreases S6 phosphorylation and cell viability, which is restored by NGF. Rapamycin, the pharmacologic inhibitor of mammalian target of rapamycin (mTOR), diminished NGF-induced S6 phosphorylation, cell migration and protective effects against oxidative stress. Collectively, we conclude that activation of rapamycin sensitive mTOR signaling mediates NGF induced cell migration and pro-survival effects in H(2)O(2) treated RPE cells.

Xenogeneic immunosuppression of human umbilical cord mesenchymal stem cells in a major histocompatibility complex-mismatched allogeneic acute graft-versus-host disease murine model.

Mesenchymal stem cells (MSCs) hold great promise for treating immune disorders owing to their immunoregulatory capacity, but the mechanism remains controversial. As we show here, the mechanism of human umbilical cord mesenchymal stem cell (HUCMSC)-mediated immunosuppression involves TGF-ß and indoleamine 2,3-dioxygenase (IDO). In this study, we investigated the influence of xenogeneic HUCMSCs on acute graft-versus-host disease (aGVHD) in murine allogeneic bone marrow transplantation (BMT). In the HUCMSC-treated group, lethally irradiated DBA/2(H-2Kd) mice were adoptively transferred with expanded HUCMSCs, bone marrow (BM), and splenocytes (SCs) from C57BL/6 (H-2Kb) mice. Recipients in the control group were transferred only BM and SCs. The two groups were compared in survival, weight, histopathologic specimens, and aGVHD scoring. In the HUCMSC-treated group, 60% of the mice survived past day 30 after BMT, but in the control group, all mice died within 18 d. The mice treated with HUCMSCs exhibited light symptoms of aGVHD after day 30. The results suggest that xenogeneic HUCMSCs could alleviate aGVHD symptoms and prolong survival after allogeneic BMT. Our study suggests that in vitro expanded HUCMSCs might be used to inhibit severe aGVHD effectively in allogeneic hematopoietic cell transplantation clinically.

The calcium-sensing receptor mediates bone turnover induced by dietary calcium and parathyroid hormone in neonates.

We have investigated, in neonates, whether the calcium-sensing receptor (CaR) mediates the effects of dietary calcium on bone turnover and/or modulates parathyroid hormone (PTH)-induced bone turnover. Wild-type (WT) pups and pups with targeted deletion of the Pth (Pth(-/-)) gene or of both Pth and CaR (Pth(-/-)CaR(-/-)) genes were nursed by dams on a normal or high-calcium diet. Pups nursed by dams on a normal diet received daily injections of vehicle or of PTH(1-34) (80 µg/kg) for 2 weeks starting from 1 week of age. In pups receiving vehicle and fed by dams on a normal diet, trabecular bone volume, osteoblast number, type 1 collagen-positive area, and mineral apposition rate, as well as the expression of bone-formation-related genes, all were reduced significantly in Pth(-/-) pups compared with WT pups and were decreased even more dramatically in Pth(-/-)CaR(-/-) pups. These parameters were increased in WT and Pth(-/-) pups but not in Pth(-/-)CaR(-/-) pups fed by dams on a high-calcium diet compared with pups fed by dams on a normal diet. These parameters also were increased in WT, Pth(-/-), and Pth(-/-)CaR(-/-) pups following exogenous PTH treatment; however, the percentage increase was less in Pth(-/-)CaR(-/-) pups than in WT and Pth(-/-) pups. In vehicle-treated pups fed by dams on either the normal or high-calcium diet and in PTH-treated pups fed by dams on a normal diet, the number and surfaces of osteoclasts and the ratio of RANKL/OPG were reduced significantly in Pth(-/-) pups and less significantly in Pth(-/-)CaR(-/-) pups compared with WT pups. These parameters were further reduced significantly in WT and Pth(-/-) pups from dams fed a high-calcium diet but did not decrease significantly in similarly treated Pth(-/-)CaR(-/-) pups, and they increased significantly in PTH-treated pups compared with vehicle-treated, genotype-matched pups fed by dams on the normal diet. These results indicate that in neonates, the CaR mediates alterations in bone turnover in response to changes in dietary calcium and modulates PTH-stimulated bone turnover.

Parathyroid hormone contributes to regulating milk calcium content and modulates neonatal bone formation cooperatively with calcium.

To determine whether PTH and calcium (Ca) interact in neonatal bone formation, female lactating mice either heterozygous (PTH(+/-)) or homozygous (PTH(-/-)) for targeted deletion of the pth gene were fed either a normal (1% Ca, 0.6% phosphate) or high-Ca diet (2% Ca and 0.4% phosphate). Dietary effects on milk Ca content and Ca-regulating hormones were determined in dams, and the effects of milk content were assessed on bone turnover in 3-wk-old pups. On the normal diet, milk Ca and 1,25-dihydroxyvitamin D(3) levels were lower, but milk PTH-related protein levels were higher in the PTH(-/-) dams compared with the PTH(+/-) dams. On the high-Ca diet, milk Ca levels were higher, but milk 1,25-dihydroxyvitamin D(3) and PTH-related protein levels were lower in both PTH(+/-) and PTH(-/-) dams. In pups fed by PTH(-/-) dams compared with pups fed by PTH(+/-) dams on normal diets, bone mineral density, trabecular bone volume relative to tissue volume, and the number of osteoblasts were reduced in both PTH(+/-) (32.5 +/- 1.2 vs. 39.6 +/- 1.5 mg/cm(2), P < 0.05; 23.3 +/- 1.6 vs. 29.2 +/- 2.8%, P < 0.01; and 94.2 +/- 8.2 vs. 123.5 +/- 3.5/mm(2), P < 0.01, respectively) and PTH(-/-) (20.4 +/- 0.9 vs. 27.0 +/- 1.2 mg/mm(2), P < 0.05; 16.8 +/- 1.9 vs. 19.3 +/- 2.1%, P < 0.05; and 48.6 +/- 7.9 vs. 90.5 +/- 8.6/mm(2), P < 0.01, respectively) pups but were lower in the PTH(-/-) pups compared with the PTH(+/-) pups. In contrast, in pups fed by either PTH(+/-) or PTH(-/-) dams on the high-Ca diet, bone mineral density, bone volume/tissue volume, and osteoblast numbers were significantly higher, in both PTH(+/-) (50.5 +/- 1.7 vs. 58.7 +/- 2.0 mg/mm(2), P < 0.05; 37.9 +/- 5.2 vs. 46.1 +/- 5.1, P < 0.05; and 120.5 +/- 9.2 vs. 159.3 +/- 14.7/mm(2), P < 0.01, respectively) and PTH(-/-) (33.0 +/- 1.2 vs. 47.5 +/- 2.2 mg/mm(2), P < 0.001; 23.8 +/- 3.1 vs. 35.9 +/- 2.0, P < 0.05; and 78.7 +/- 10.1 vs. 99.8 +/- 13.6/mm(2), P < 0.05, respectively), and were highest in the PTH(+/-) pups fed by the PTH(+/-) dams on the high-Ca diet. These results indicate that PTH can modulate Ca content of milk, and that PTH and Ca can each exert cooperative roles on osteoblastic bone formation in the neonate.