Continuous acceleration of neural activity of the GnRH pulse generator during chronic peripheral infusion of neurokinin 3 receptor agonist in goats.

Secretion of pulsatile gonadotropin-releasing hormone (GnRH) is essential for reproduction. Kisspeptin neurons in the arcuate nucleus (ARC), which coexpress neurokinin B (NKB) and its receptor (NK3R), are believed to be components of the GnRH pulse generator that regulates pulsatile GnRH secretion. We examined the effects of peripheral infusion of senktide, an NK3R selective agonist, on GnRH pulse generator activity by monitoring multiple unit activity (MUA) in the goat ARC. Previous studies have shown that characteristic increases in MUA (MUA volleys) reflect GnRH pulse generator activity. Senktide was infused intravenously or intravaginally for 2 h while recording MUA. Both infusions significantly increased the MUA volley frequency compared with the control. These results demonstrate that peripherally administered senktide acts centrally to sustainably accelerate the neural activity of the GnRH pulse generator throughout the infusion period. This suggests the possibility of practical applications of NK3R agonists for improving reproductive activity in farm animals.

Vitreous metabolomics profiling of proliferative diabetic retinopathy.

AIMS/HYPOTHESIS: Proliferative diabetic retinopathy (PDR) with retinal neovascularisation (NV) is a leading cause of vision loss. This study identified a set of metabolites that were altered in the vitreous humour of PDR patients compared with non-diabetic control participants. We corroborated changes in vitreous metabolites identified in prior studies and identified novel dysregulated metabolites that may lead to treatment strategies for PDR. METHODS: We analysed metabolites in vitreous samples from 43 PDR patients and 21 non-diabetic epiretinal membrane control patients from Japan (age 27-80 years) via ultra-high-performance liquid chromatography-mass spectrometry. We then investigated the association of a novel metabolite (creatine) with retinal NV in mouse oxygen-induced retinopathy (OIR). Creatine or vehicle was administered from postnatal day (P)12 to P16 (during induced NV) via oral gavage. P17 retinas were quantified for NV and vaso-obliteration. RESULTS: We identified 158 metabolites in vitreous samples that were altered in PDR patients vs control participants. We corroborated increases in pyruvate, lactate, proline and allantoin in PDR, which were identified in prior studies. We also found changes in metabolites not previously identified, including creatine. In human vitreous humour, creatine levels were decreased in PDR patients compared with epiretinal membrane control participants (false-discovery rate <0.001). We validated that lower creatine levels were associated with vascular proliferation in mouse retina in the OIR model (p = 0.027) using retinal metabolomics. Oral creatine supplementation reduced NV compared with vehicle (P12 to P16) in OIR (p = 0.0024). CONCLUSIONS/INTERPRETATION: These results suggest that metabolites from vitreous humour may reflect changes in metabolism that can be used to find pathways influencing retinopathy. Creatine supplementation could be useful to suppress NV in PDR. Graphical abstract.

Machine Learning Approach for Intraocular Disease Prediction Based on Aqueous Humor Immune Mediator Profiles.

PURPOSE: Various immune mediators have crucial roles in the pathogenesis of intraocular diseases. Machine learning can be used to automatically select and weigh various predictors to develop models maximizing predictive power. However, these techniques have not yet been applied extensively in studies focused on intraocular diseases. We evaluated whether 5 machine learning algorithms applied to the data of immune-mediator levels in aqueous humor can predict the actual diagnoses of 17 selected intraocular diseases and identified which immune mediators drive the predictive power of a machine learning model. DESIGN: Cross-sectional study. PARTICIPANTS: Five hundred twelve eyes with diagnoses from among 17 intraocular diseases. METHODS: Aqueous humor samples were collected, and the concentrations of 28 immune mediators were determined using a cytometric bead array. Each immune mediator was ranked according to its importance using 5 machine learning algorithms. Stratified k-fold cross-validation was used in evaluation of algorithms with the dataset divided into training and test datasets. MAIN OUTCOME MEASURES: The algorithms were evaluated in terms of precision, recall, accuracy, F-score, area under the receiver operating characteristic curve, area under the precision-recall curve, and mean decrease in Gini index. RESULTS: Among the 5 machine learning models, random forest (RF) yielded the highest classification accuracy in multiclass differentiation of 17 intraocular diseases. The RF prediction models for vitreoretinal lymphoma, acute retinal necrosis, endophthalmitis, rhegmatogenous retinal detachment, and primary open-angle glaucoma achieved the highest classification accuracy, precision, and recall. Random forest recognized vitreoretinal lymphoma, acute retinal necrosis, endophthalmitis, rhegmatogenous retinal detachment, and primary open-angle glaucoma with the top 5 F-scores. The 3 highest-ranking relevant immune mediators were interleukin (IL)-10, interferon-γ-inducible protein (IP)-10, and angiogenin for prediction of vitreoretinal lymphoma; monokine induced by interferon γ, interferon γ, and IP-10 for acute retinal necrosis; and IL-6, granulocyte colony-stimulating factor, and IL-8 for endophthalmitis. CONCLUSIONS: Random forest algorithms based on 28 immune mediators in aqueous humor successfully predicted the diagnosis of vitreoretinal lymphoma, acute retinal necrosis, and endophthalmitis. Overall, the findings of the present study contribute to increased knowledge on new biomarkers that potentially can facilitate diagnosis of intraocular diseases in the future.

Local administration of Neurokinin B in the arcuate nucleus accelerates the neural activity of the GnRH pulse generator in goats.

Kisspeptin neurons in the arcuate nucleus (ARC), which co-express neurokinin B (NKB) and dynorphin A, are termed KNDy neurons. These neurons are candidates for the intrinsic gonadotropin-releasing hormone (GnRH) pulse generator. The central and peripheral administration of NKB or its receptor (NK3R) agonist evokes GnRH pulse generator activity and the subsequent pulsatile GnRH/luteinizing hormone (LH) secretion. However, the mechanism responsible for neural activation of the GnRH pulse generator in goats is unclear. We conducted electrophysiological and histochemical experiments to test the hypothesis that KNDy neurons receive NKB and that the signal is transmitted bilaterally to a population of KNDy neurons. Bilateral electrodes aimed at a cluster of KNDy neurons were inserted into the ovariectomized goat ARC. We observed the GnRH pulse generator activity, represented by characteristic increases in multiple-unit activity (MUA volleys). The unilateral administration of NKB or vehicle in the close vicinity of KNDy neurons under simultaneous MUA recording from both sides revealed that only NKB evoked MUA volley(s) immediately after administration. The timing of the MUA volley(s) evoked on the ipsilateral side was synchronized to that on the contralateral side. The double-labeled ISH for KISS1 and TACR3, which encode kisspeptin and NK3R, respectively, revealed that most KNDy neurons co-expressed TACR3. Therefore, NKB could directly stimulate KNDy neurons, following which the stimulatory signal is immediately transmitted to the entire population of KNDy neurons via connection with their fibers. This mechanism helps synchronize burst activity among KNDy neurons, thereby generating neural signals that govern pulsatile GnRH secretion.

Silkworm recombinant bovine zona pellucida protein 4 (bZP4) as a potential female immunocontraceptive antigen; impaired sperm-oocyte interaction and ovarian dysfunction.

Porcine zona pellucida proteins (ZPs) have been utilized as female immunocontraceptive antigens. The purpose of this study was to explore the potential use of silkworm recombinant bovine ZP4 as an alternative. When the protein was injected with monophosphoryl lipid A (MPL) - an immuno-stimulative agent - into two female goats, marked elevation of the anti-ZP4 titer was detected. Application of the purified specific IgG to a porcine in vitro fertilization system reduced the sperm penetration rate. In one goat, the cyclic profile of serum progesterone disappeared as the anti-ZP4 titer increased. Histological examination of the ovaries revealed degeneration of antral follicles with sparse infiltration of inflammatory cells in the theca, indicating that autoimmune oophoritis had been induced. Together, the present results suggest that recombinant ZP4 disturbs fertilization and exerts a pathogenic effect on follicle development in goats, thus indicating its potential as a female immunocontraceptive antigen.

miR-30a-5p inhibition promotes interaction of Fas+ endothelial cells and FasL+ microglia to decrease pathological neovascularization and promote physiological angiogenesis.

Ischemia-induced angiogenesis contributes to various neuronal and retinal diseases, and often results in neurodegeneration and visual impairment. Current treatments involve the use of anti-VEGF agents but are not successful in all cases. In this study we determined that miR-30a-5p is another important mediator of retinal angiogenesis. Using a rodent model of ischemic retinopathy, we show that inhibiting miR-30a-5p reduces neovascularization and promotes tissue repair, through modulation of microglial and endothelial cell cross-talk. miR-30a-5p inhibition results in increased expression of the death receptor Fas and CCL2, to decrease endothelial cell survival and promote microglial migration and phagocytic function in focal regions of ischemic injury. Our data suggest that miR-30a-5p inhibition accelerates tissue repair by enhancing FasL-Fas crosstalk between microglia and endothelial cells, to promote endothelial cell apoptosis and removal of dead endothelial cells. Finally, we found that miR-30a levels were increased in the vitreous of patients with proliferative diabetic retinopathy. Our study identifies a role for miR-30a in the pathogenesis of neovascular retinal disease by modulating microglial and endothelial cell function, and suggests it may be a therapeutic target to treat ischemia-mediated conditions.

A Single Pheromone Receptor Gene Conserved across 400 My of Vertebrate Evolution.

Pheromones are crucial for eliciting social and sexual behaviors in diverse animal species. The vomeronasal receptor type-1 (V1R) genes, encoding members of a pheromone receptor family, are highly variable in number and repertoire among mammals due to extensive gene gain and loss. Here, we report a novel pheromone receptor gene belonging to the V1R family, named ancient V1R (ancV1R), which is shared among most Osteichthyes (bony vertebrates) from the basal lineage of ray-finned fishes to mammals. Phylogenetic and syntenic analyses of ancV1R using 115 vertebrate genomes revealed that it represents an orthologous gene conserved for >400 My of vertebrate evolution. Interestingly, the loss of ancV1R in some tetrapods is coincident with the degeneration of the vomeronasal organ in higher primates, cetaceans, and some reptiles including birds and crocodilians. In addition, ancV1R is expressed in most mature vomeronasal sensory neurons in contrast with canonical V1Rs, which are sparsely expressed in a manner that is consistent with the "one neuron-one receptor" rule. Our results imply that a previously undescribed V1R gene inherited from an ancient Silurian ancestor may have played an important functional role in the evolution of vertebrate vomeronasal organ.

A neurokinin 3 receptor-selective agonist accelerates pulsatile luteinizing hormone secretion in lactating cattle.

Pulsatile gonadotropin-releasing hormone (GnRH) secretion, which is indispensable for follicular development, is suppressed in lactating dairy and beef cattle. Neurokinin B (NKB) neurons in the arcuate nucleus of the hypothalamus are considered to play an essential role in generating the pulsatile mode of GnRH/luteinizing hormone (LH) secretion. The present study aimed to clarify the role of NKB-neurokinin 3 receptor (NK3R) signaling in the pulsatile pattern of GnRH/gonadotropin secretion in postpartum lactating cattle. We examined the effects of the administration of an NK3R-selective agonist, senktide, on gonadotropin secretion in lactating cattle. The lactating cattle, at approximately 7 days postpartum, were intravenously infused with senktide (30 or 300 nmol/min) or vehicle for 24 h. The administration of 30 or 300 nmol/min senktide significantly increased LH pulse frequency compared to in the control group during 0-4 or 20-24 h after infusion, respectively. Moreover, LH and follicle-stimulating hormone levels were gradually increased by 300 nmol/min administration of senktide during the 0-4-h sampling period. Ultrasonography of the ovaries was performed to identify the first postpartum ovulation in senktide-administered lactating cattle. The interval from calving to first postpartum ovulation was significantly shorter in the 300 nmol/min senktide-administered group than in the control group. Taken together, these findings suggest that senktide infusion elicits an increase in LH pulse frequency that may stimulate follicular development and, in turn, induce the first postpartum ovulation in lactating cattle.

EFFICACY OF THE INVERTED INTERNAL LIMITING MEMBRANE FLAP TECHNIQUE WITH VITRECTOMY FOR RETINAL DETACHMENT ASSOCIATED WITH MYOPIC MACULAR HOLES.

PURPOSE: To evaluate the anatomical and functional outcome of the inverted internal limiting membrane (ILM) flap technique with vitrectomy for retinal detachment associated with macular hole (MHRD) in highly myopic eyes. METHODS: In this retrospective observational study, 21 eyes with MHRD that underwent vitrectomy with conventional ILM peeling (ILM-peeling group, n = 11) or the inverted ILM flap technique (ILM flap group, n = 10) combined with C3F8 tamponade were enrolled in this study. The initial retinal reattachment rate, macular hole closure rate, and postoperative visual acuity at the final visit were compared between the ILM-peeling group and ILM flap group. RESULTS: There was no significant difference in the retinal reattachment rate between ILM-peeling and ILM flap groups (91% and 100%, respectively). The macular hole closure rate in the ILM flap group was 80% (8 of 10 eyes), and was significantly higher than 36% (4 of 11 eyes) in the ILM-peeling group (P = 0.039). Among 12 eyes that achieved macular hole closure, there was no significant difference in median visual acuity after vitrectomy between the ILM flap group and ILM-peeling group (logMAR unit [Snellen acuity]: 1.0 [20/200] and 0.76 [20/125], respectively, P = 0.300). CONCLUSION: Compared with conventional ILM peeling, the inverted ILM flap technique was more effective for macular hole closure after vitrectomy for MHRD in myopic eye but showed no advantage in the postoperative visual outcome in this study.

PERSISTENT OVERPRODUCTION OF INTRAOCULAR VASCULAR ENDOTHELIAL GROWTH FACTOR AS A CAUSE OF LATE VITREOUS HEMORRHAGE AFTER VITRECTOMY FOR PROLIFERATIVE DIABETIC RETINOPATHY.

PURPOSE: The purpose of this study was to investigate whether vitreous levels of vascular endothelial growth factor (VEGF) predict late vitreous hemorrhage (VH) after vitrectomy for proliferative diabetic retinopathy, and how VEGF level changes in patients with postoperative late VH. METHODS: Eighty-five eyes of 68 patients with proliferative diabetic retinopathy who underwent vitrectomy were analyzed retrospectively. Vitreous samples were collected from eyes undergoing primary vitrectomy and from eyes with late VH undergoing second vitrectomy. Vitreous VEGF levels were measured using enzyme-linked immunosorbent assay. The relationship between VEGF level and late VH (>4 weeks) occurring during follow-up as well as clinical findings, and changes in VEGF level in eyes with late VH undergoing second vitrectomy were analyzed. RESULTS: Late VH occurred in 20 (24%) of 85 eyes, and 9 eyes required second vitrectomy. Vitreous levels of VEGF were significantly higher (median: 1,945 pg/mL; P < 0.0001) in eyes with late VH than in those without. Preexisting iris neovascularization (P < 0.0001), hypertension (P = 0.002), and proteinuria (P = 0.040) were also significant risk factors of late VH. Multivariate logistic regression analysis showed that a higher vitreous VEGF level was independently associated with a risk of postoperative late VH in patients with proliferative diabetic retinopathy (odds ratio: 20.8, 95% confidence interval: 2.72-159.47; P = 0.003). Vitreous VEGF level at second vitrectomy in patients with late VH was significantly lower compared with that at primary vitrectomy, but remained elevated (median: 1,610 pg/mL; P = 0.023). CONCLUSION: In patients with proliferative diabetic retinopathy, high intraocular VEGF level at primary vitrectomy was identified as an independent risk factor of postoperative late VH. Persistent overproduction of intraocular VEGF may be associated with postoperative late VH.

Mapping of KNDy neurons and immunohistochemical analysis of the interaction between KNDy and substance P neural systems in goat.

A population of neurons in the arcuate nucleus (ARC) coexpresses kisspeptin, neurokinin B (NKB), and dynorphin, and therefore they are referred to as KNDy neurons. It has been suggested that KNDy neurons participate in several brain functions, including the control of reproduction. The present study aimed to advance our understanding of the anatomy of the KNDy neural system. We first produced an antiserum against goat kisspeptin. After confirming its specificity, the antiserum was used to histochemically detect kisspeptin-positive signals. Using the colocalization of kisspeptin and NKB immunoreactivity as a marker for KNDy neurons, we mapped distributions of their cell somata and fibers in the whole brain (except the cerebellum) of ovariectomized (OVX) goats. KNDy neuronal somata were distributed throughout the ARC, and were particularly abundant in its caudal aspect. KNDy neuronal fibers projected into several areas within the septo-preoptic-hypothalamic continuum, such as the ARC, median eminence, medial preoptic nucleus, and bed nucleus of the stria terminalis. Kisspeptin immunoreactivity was not found outside of the continuum. We then addressed to the hypothesis that substance P (SP) is also involved in the KNDy neural system. Double-labeling immunohistochemistry for kisspeptin and SP revealed that KNDy neurons did not coexpress SP, but nearly all of the KNDy neuronal somata were surrounded by fibers containing SP in the OVX goats. The present results demonstrate anatomical evidence for a robust association between the KNDy and SP neural systems.

An administration of TAK-683 at a minimally effective dose for luteinizing hormone stimulation under the absence of the ovary induces luteinizing hormone surge in ovary-intact goats.

The present study aimed to evaluate hormonal responses and their association with the TAK-683 blood concentrations in goats administered TAK-683 at a low dose, which had been previously determined as the minimally effective dose for luteinizing hormone (LH) stimulation in ovariectomized goats. In Experiment 1, 5 µg of TAK-683 treatment had no significant stimulatory effect on LH secretion in ovariectomized Shiba goats (n = 4). In Experiment 2, cycling goats received the treatment of prostaglandin F2α and progesterone-releasing controlled internal drug releasing (CIDR) to induce the follicular phase, then they were treated with 5 µg of TAK-683 (hour 0) intravenously (n = 4, IV) or subcutaneously (n = 3, SC) or with vehicle intravenously (n = 4, control) at 12 h after CIDR removal. Blood samples were collected at 10-min (-2-6 h), 2-h (6-24 h), or 6-h (24-48 h) intervals. Ovarian ultrasonographic images were assessed daily to confirm ovulation after the treatment. A surge-like release of LH was immediately observed after injection in all animals in the IV (peak time: 4.2 ± 0.6 h, peak concentration: 73.3 ± 27.5 ng/ml) and SC (peak time: 4.6 ± 0.4 h, peak concentration: 62.6 ± 23.2 ng/ml) groups, but not in the control group. Ovulation was detected within 3 days after TAK-683 injection in all animals in the IV and SC groups, and the interval period from TAK-683 administration to ovulation in the IV group was significantly (P < 0.05) shorter than that of the control group. No significant changes were observed between the IV and SC groups in terms of luteal diameter and blood progesterone levels after ovulation. The present findings suggest that the involvement of one or more ovarian factor(s) is indispensable for a TAK-683-induced LH surge leading to ovulation in goats.

Effects of intravenous administration of neurokinin receptor subtype-selective agonists on gonadotropin-releasing hormone pulse generator activity and luteinizing hormone secretion in goats.

Recent evidence suggests that neurokinin B (NKB), a member of the neurokinin (tachykinin) peptide family, plays a pivotal role in gonadotropin-releasing hormone (GnRH) pulse generation. Three types of neurokinin receptors (NKRs), NK1R, NK2R and NK3R, are found in the brain. Although NKB preferentially binds to NK3R, other NKRs are possibly also involved in NKB action. The present study examined the effects of intravenous administration of the NKR subtype-selective agonists GR73632 (NK1R), GR64349 (NK2R), and senktide (NK3R) on GnRH pulse generator activity and luteinizing hormone (LH) secretion. Multiple-unit activity (MUA) was monitored in ovariectomized goats (n = 5) implanted with recording electrodes. Characteristic increases in MUA (MUA volleys) were considered GnRH pulse generator activity. Although three NKR agonists dose-dependently induced an MUA volley and an accompanying increase in LH secretion, the efficacy in inducing the volley markedly differed. As little as 10 nmol of senktide induced an MUA volley in all goats, whereas a dose of 1000 nmol was only effective for the NK1R and NK2R agonists in two and four goats, respectively. When the treatment failed to evoke an MUA volley, no apparent change was observed in the MUA or LH secretion. Similar effects of the NK2R and NK3R agonists were observed in the presence of estradiol. The results demonstrated that NK3R plays a predominant role in GnRH pulse generation and suggested that the contributions of NK1R and NK2R to this mechanism may be few, if any, in goats.

The effects of chronic subcutaneous administration of an investigational kisspeptin analog, TAK-683, on gonadotropin-releasing hormone pulse generator activity in goats.

The continuous activation of the kisspeptin receptor by its agonists causes the abrogation of kisspeptin signaling, leading to decreased pulsatile luteinizing hormone (LH) secretion. Employing this phenomenon as a tool for probing kisspeptin action, this study aimed to clarify the role of kisspeptin in gonadotropin-releasing hormone (GnRH) pulse generation in goats. We examined the effects of chronic administration of TAK-683, an investigational kisspeptin analog, on LH secretion, GnRH immunostaining, pituitary responses to exogenous GnRH, and GnRH pulse generator activity, reflected by a characteristic increase in multiple-unit activity (MUA volley). An osmotic pump containing TAK-683 was subcutaneously implanted on day 0. TAK-683 treatment dose-dependently suppressed pulsatile LH secretion on day 1. Higher doses of chronic TAK-683 profoundly suppressed pulsatile LH secretion but had little effect on GnRH immunostaining patterns and pituitary responses to GnRH on day 5. In ovariectomized goats, MUA volleys occurred at approximately every 30 min on day -1. On day 5 of chronic TAK-683 administration, pulsatile LH secretion was markedly suppressed, whereas MUA volleys were similar to those observed on day -1. Male pheromones and senktide (neurokinin B receptor agonist) induced an MUA volley but had no effect on LH secretion during chronic TAK-683 administration. The results indicate that the chronic administration of a kisspeptin analog profoundly suppresses pulsatile LH secretion without affecting GnRH content, pituitary function or GnRH pulse generator activity, and they suggest an indispensable role for kisspeptin signaling in the cascade driving GnRH/LH pulses by the GnRH pulse generator.

Profile of intraocular immune mediators in patients with age-related macular degeneration and the effect of intravitreal bevacizumab injection.

PURPOSE: To measure intraocular cytokine levels in patients with exudative age-related macular degeneration and analyze changes in the cytokine profile 2 days after intravitreal bevacizumab injection. METHODS: This prospective case-control study enrolled 37 patients (37 eyes) with age-related macular degeneration including polypoidal choroidal vasculopathy. Twenty-eight age-matched patients (28 eyes) who underwent cataract surgery were used as controls. Undiluted aqueous humor samples were collected after intravitreal bevacizumab injection. Two days after intravitreal bevacizumab injection, cataract surgery was performed and undiluted aqueous humor samples were collected at the beginning of surgery (10 eyes). Twenty-three cytokines were measured using flow cytometry. P values were corrected in multiple comparisons using the conservative Bonferroni-Holm method. The level of significance was set at 0.0022 (0.05/23). RESULTS: At baseline, aqueous humor levels of vascular endothelial growth factor, angiogenin, interferon gamma-inducible protein (IP)-10, macrophage inflammatory protein (MIP)-1ß, monokine induced by interferon γ (Mig), and monocyte chemotactic protein (MCP)-1 were significantly higher in the age-related macular degeneration group than in the control group (P < 0.0022). The result of exploratory multivariate analysis showed that elevated angiogenin level was an important factor that discriminates the two groups (P = 0.0004). Two days after intravitreal bevacizumab injection, vascular endothelial growth factor levels tended to be reduced (P = 0.049), whereas interleukin (IL)-6 and IL-8 levels increased significantly (P < 0.0022). CONCLUSION: Vascular endothelial growth factor and also angiogenin, IP-10, MCP-1, MIP-1ß, and Mig may be related to the pathogenesis of age-related macular degeneration. Intravitreal bevacizumab injection increases inflammatory cytokine levels, suggesting the induction of an inflammatory process.

Correlation of complement fragment C5a with inflammatory cytokines in the vitreous of patients with proliferative diabetic retinopathy.

PURPOSE: To determine the vitreous concentration of complement fragment C5a in patients with proliferative diabetic retinopathy (PDR) and the relation between C5a and inflammatory cytokines including vascular endothelial growth factor (VEGF) and monocyte chemotactic protein-1 (MCP-1). METHODS: Vitreous samples were obtained at the time of vitrectomy from 12 eyes of 11 PDR patients and from 11 eyes of 11 patients without diabetes with macular disease (controls). Vitreous and serum concentrations of human C5a, VEGF, and MCP-1 were quantified using FACS Caliber flow cytometer. RESULTS: Vitreous concentration of C5a increased significantly in patients with PDR [median (range): 928.7 (46.6 to 3,319.4) pg/ml] compared with controls [58.7 (22.2 to 1,432.4) pg/ml; p < 0.01]. In PDR patients, vitreous concentration of C5a correlated significantly with those of VEGF (p < 0.05) and MCP-1 (p < 0.05). CONCLUSIONS: Our results suggest that C5a may play an important role in the pathogenesis of PDR and work in concert with inflammatory cytokines such as VEGF and MCP-1 in pathological angiogenesis.

Effects of soluble CD14 and cytokine levels on diabetic macular edema and visual acuity.

PURPOSE: The pathogenesis of diabetic retinopathy has been suggested to be associated with ocular inflammation. Macrophages and monocytes that infiltrate the eye are known to express CD14. After shedding from the membrane-bound CD14, soluble CD14 (sCD14) is released, which could potentially activate inflammatory signaling. In this study, the authors investigated ocular fluid and serum levels of vascular endothelial growth factor (VEGF), sCD14, and other inflammatory cytokines in patients with diabetic macular edema (DME). Furthermore, the authors determined any potential correlation between these factors and visual acuity. METHODS: Vitreous fluid, aqueous humor, and serum samples from 14 eyes with DME and 24 control eyes were investigated. Soluble CD14, interleukin 8, interferon-inducible protein 10, monocyte chemotactic protein 1, monokine induced by interferon γ, and VEGF were measured simultaneously by FACSCalibur flow cytometer. Visual acuity was measured in all patients with DME before surgery, with the assessors being blinded to the patients' diagnoses. RESULTS: All factors were significantly elevated in vitreous fluid of DME eyes. Soluble CD14 and VEGF levels in vitreous fluid and aqueous humor were significantly higher in patients with DME than in nondiabetic controls (P < 0.05). In patients with DME, vitreous and aqueous humor concentrations of sCD14 correlated significantly. In these patients, vitreous fluid concentration of sCD14 correlated significantly with that of VEGF or interleukin 8 or monocyte chemotactic protein 1. In addition, there was a significant positive correlation between preoperative visual acuity and intraocular sCD14 concentrations. CONCLUSION: Soluble CD14 may act as key regulator of VEGF production and contribute to the pathogenesis of diabetic retinopathy.

Electrophysiological and morphological evidence for synchronized GnRH pulse generator activity among Kisspeptin/neurokinin B/dynorphin A (KNDy) neurons in goats.

Neurons in the arcuate nucleus (ARC) that concomitantly express kisspeptin, neurokinin B (NKB) and dynorphin A are termed KNDy neurons and are likely candidates for the intrinsic gonadotropin-releasing hormone (GnRH) pulse generator. Our hypothesis is that KNDy neurons are functionally and anatomically interconnected to generate discrete neural signals that govern pulsatile GnRH secretion. Our goal was to address this hypothesis using electrophysiological and anatomical experiments in goats. Bilateral electrodes targeting KNDy neurons were implanted into ovariectomized goats, and GnRH pulse generator activity, represented by characteristic increases in multiple-unit activity (MUA volleys), was measured. Spontaneous and pheromone- or senktide (an NKB receptor agonist)-induced MUA volleys were simultaneously recorded from both sides of the ARC. An anterograde tracer, biotinylated dextran amine (BDA), was also injected unilaterally into the ARC of castrated male goats, and the distribution of fibers containing both BDA and NKB was examined using dual-labeling histochemistry. The results showed that MUA volleys, regardless of origin (spontaneous or experimentally induced), occur simultaneously between the right and left sides of the ARC. Tract tracing indicated that axons projecting from NKB neurons in the ARC were directly apposed to other NKB neuronal cells located bilaterally in the ARC. These results demonstrate that GnRH pulse generator activity occurs synchronously between both sides of the ARC in goats and that KNDy neurons are bilaterally interconnected in the ARC via NKB-containing fibers. Taken together, the results suggest that KNDy neurons form a neuronal circuit to synchronize burst activity among KNDy neurons and thereby generate discrete neural signals that govern pulsatile GnRH secretion.

Differential effects of continuous exposure to the investigational metastin/kisspeptin analog TAK-683 on pulsatile and surge mode secretion of luteinizing hormone in ovariectomized goats.

The aim of the present study was to determine if the estradiol-induced luteinizing hormone (LH) surge is influenced by the constant exposure to TAK-683, an investigational metastin/kisspeptin analog, that had been established to depress the pulsatile gonadotropin-releasing hormone (GnRH) and LH secretion in goats. Ovariectomized goats subcutaneously received TAK-683 (TAK-683 group, n=6) or vehicle (control group, n=6) constantly via subcutaneous implantation of an osmotic pump. Five days after the start of the treatment, estradiol was infused intravenously in both groups to evaluate the effects on the LH surge. Blood samples were collected at 6-min intervals for 4 h prior to the initiation of either the TAK-683 treatment or the estradiol infusion, to determine the profiles of pulsatile LH secretion. They were also collected at 2-h intervals from -4 h to 32 h after the start of estradiol infusion for analysis of LH surges. The frequency and mean concentrations of LH pulses in the TAK-683 group were remarkably suppressed 5 days after the start of TAK-683 treatment compared with those of the control group (P<0.05). On the other hand, a clear LH surge was observed in all animals of both groups. There were no significant differences in the LH concentrations for surge peak and the peak time of the LH surge between the TAK-683 and control groups. These findings suggest that the effects of continuous exposure to kisspeptin or its analog on the mechanism(s) that regulates the pulsatile and surge mode secretion of GnRH/LH are different in goats.

Kisspeptin and GnRH pulse generation.

The reproductive neuropeptide gonadotropin-releasing hormone (GnRH) has two modes of secretion. Besides the surge mode, which induces ovulation in females, the pulse mode of GnRH release is essential to cause various reproductive events in both sexes, such as spermatogenesis, follicular development, and sex steroid synthesis. Some environmental cues control gonadal activities through modulating GnRH pulse frequency. Researchers have looked for the anatomical location of the mechanism generating GnRH pulses, the GnRH pulse generator, in the brain, because an artificial manipulation of GnRH pulse frequency is of therapeutic importance to stimulate or suppress gonadal activity. Discoveries of kisspeptin and, consequently, KNDy (kisspeptin/neurokinin B/dynorphin) neurons in the hypothalamus have provided a clue to the possible location of the GnRH pulse generator. Our analyses of hypothalamic multiple-unit activity revealed that KNDy neurons located in the hypothalamic arcuate nucleus might play a central role in the generation of GnRH pulses in goats, and perhaps other mammalian species. This chapter further discusses the possible mechanisms for GnRH pulse generation.