Samuel Adamson

Evidence for Lymphatics in the Developing and Adult Human Choroid.

PURPOSE Lymphatics subserve many important functions in the human body including maintenance of fluid homeostasis, immune surveillance, and tumor metastasis. Our aim was to provide structural and phenotypic evidence of lymphatic-like structures in the human choroid, including details of its development. METHODS Using multiple-marker immunohistochemistry (IHC), choroids from human fetal eyes (8-26 weeks gestation) and adults (17-74 years) were examined with lymphatic- and vascular-specific markers: prospero homeobox-1 (PROX-1), lymphatic vascular endothelium receptor-1 (LYVE-1), podoplanin, D2-40, endomucin, VEGF-C, vascular endothelial growth factor receptor-3 (VEGFR-3 or Flt4), UEA lectin, platelet endothelial cell adhesion molecule-1 (PECAM-1), CD34, and CD39. Transmission electron microscopy (TEM) was used to establish evidence for choroidal lymphatics, and to provide details of stratification and relative frequency of lymphatics compared to choroidal blood vessels. RESULTS Immunohistochemistry and TEM indicated a central-to-peripheral topography of lymphatic formation, with numerous blind-ended lymph sacs just external to the choriocapillaris, as well as the presence of infrequent precollector and collector lymphatic channels. Characteristic ultrastructural features of lymphatics in adult human choroid included anchoring filaments, luminal flocculent protein but absence of erythrocytes, fragmented and/or absent basal lamina, absence of intracellular Weibel-Palade bodies, infrequent pericyte ensheathment, and lack of fenestrae. CONCLUSIONS The system of blind-ended initial lymphatic segments seen just external to the fenestrated vessels of the choriocapillaris is ideally placed for recirculating extracellular fluid and strategically placed for immune surveillance. The presence of a system of lymphatic-like channels in the human choroid provides an anatomical basis for antigen presentation in the posterior eye, with a possible route from the eye to the sentinel lymph nodes, similar to that already described for anterior eye lymphatics.

Loss of Survival Factors and Activation of Inflammatory Cascades in Brain Sympathetic Centers in Type 1 Diabetic Mice

Neuroinflammation and neurodegeneration have been observed in the brain in type 1 diabetes (T1D). However, little is known about the mediators of these effects. In T1D mice with 12- and 35-wk duration of diabetes we examined two mechanisms of neurodegeneration, loss of the neuroprotective factors insulin-like growth factor I (IGF-I) and IGF-binding protein-3 (IGFBP-3) and changes in indoleamine 2,3-dioxygenase (IDO) expression in the brain, and compared the response to age-matched controls. Furthermore, levels of matrix metalloproteinase-2 (MMP-2), nucleoside triphosphate diphosphohydrolase-1 (CD39), and ionized calcium-binding adaptor molecule 1 (Iba-1) were utilized to assess inflammatory changes in astrocytes, microglia, and blood vessels. In the diabetic hypothalamus (HYPO), we observed 20% reduction in neuronal soma diameter (P<0.05) and reduced neuronal expression of IGFBP-3 (-32%, P<0.05) and IGF-I (-15%, P<0.05) compared with controls at 35 wk. In diabetic HYPO, MMP-2 expression was increased in astrocytes (46%, P<0.01), and IDO⁺ cell density rose by (62%, P<0.05). CD39 expression dropped by 30% (P<0.05) in microglia and blood vessels. With 10 wk of systemic treatment using minocycline, an anti-inflammatory agent that crosses the blood-brain barrier, MMP-2, IDO, and CD39 levels normalized (P<0.05). Our results suggest that increased IDO and early loss of CD39⁺ protective cells lead to activation of inflammation in sympathetic centers of the CNS. As a downstream effect, the loss of the neuronal survival factors IGFBP-3 and IGF-I and the neurotoxic products of the kynurenine pathway contribute to the loss of neuronal density observed in the HYPO in T1D.

Increased indoleamine 2,3-dioxygenase and quinolinic acid expression in microglia and müller cells of diabetic human and rodent retina

Purpose We investigated the relationship between inflammation, neuronal loss, and expression of indoleamine 2, 3-dioxygenase (IDO) and quinolinic acid (QUIN) in the retina of subjects with type 1 diabetes (T1D) and type 2 diabetes (T2D) and in the retina of rats with T1D. Methods Retinas from T1D (n = 7), T2D (n = 13), and 20 age-matched nondiabetic human donors and from T1D (n = 3) and control rats (n = 3) were examined using immunohistochemistry for IDO, QUIN, cluster of differentiation 39 (CD39), ionized calcium-binding adaptor molecule (Iba-1, for macrophages and microglia), Vimentin (VIM; for Müller cells), neuronal nuclei (NeuN; for neurons), and UEA1 lectin (for blood vessels). Results Based on morphologic criteria, CD39+/ionized calcium binding adaptor molecule 1(Iba-1+) resident microglia and CD39−/Iba-1+ bone marrow–derived macrophages were present at higher density in T1D (13% increase) and T2D (26% increase) human retinas when compared with controls. The density and brightness of IDO+ microglia were increased in both T1D and T2D human retinas. The intensity of QUIN+ expression on CD39+ microglia and VIM+ Müller cells was greatly increased in both human T1D and T2D retinas. T1D retinas showed a 63% loss of NeuN+ neurons and T2D retinas lost approximately 43% when compared with nondiabetic human retinas. Few QUIN+ microglia-like cells were seen in nondiabetic retinas, but the numbers increased 18-fold in T1D and 7-fold in T2D in the central retina. In T1D rat retinas, the density of IDO+ microglia increased 2.8-fold and brightness increased 2.1-fold when compared with controls. Conclusions Our findings suggest that IDO and QUIN expression in the retinas of diabetic rats and humans could contribute to the neuronal degeneration that is characteristic of diabetic retinopathy.