Immune Mediators Profiles in the Aqueous Humor of Patients with Simple Diabetic Retinopathy.

Various immune mediators identified to date are associated with the development of advanced forms of diabetic retinopathy (DR), such as proliferative DR and diabetic macular edema, although the exact pathophysiological mechanisms of early stages of DR such as simple DR remain unclear. We determined the immune mediator profile in the aqueous humor of eyes with simple DR. Fifteen eyes of fifteen patients with simple DR were studied. Twenty-two eyes of twenty-two patients with cataracts and no DR served as controls. Undiluted aqueous humor samples were collected, and a cytometric bead array was used to determine the aqueous humor concentrations of 32 immune mediators comprising 13 interleukins (IL), interferon-γ, interferon-γ-inducible protein-10 (IP-10), monocyte chemoattractant protein-1, macrophage inflammatory protein (MIP)-1α, MIP-1ß, regulated on activation, normal T cell expressed and secreted (RANTES), monokine induced by interferon-γ, basic fibroblast growth factor (bFGF), Fas ligand, granzyme A, granzyme B, interferon-inducible T-cell alpha chemoattractant (ITAC), fractalkine, granulocyte macrophage colony-stimulating factor, granulocyte colony-stimulating factor (G-CSF), vascular endothelial growth factor (VEGF), angiogenin, tumor necrosis factor-α, and CD40 ligand. Among the 32 immune mediators, 10 immune mediators, including bFGF, CD40 ligand, fractalkine, G-CSF, IL-6, IL-8, MIP-α, MIP-1ß, and VEGF, showed significantly higher aqueous humor concentrations and the Fas ligand had significantly lower concentration (p < 0.05) in eyes with simple DR compared with control eyes. Of these 10 cytokines with significant concentration alteration, protein-protein interaction analysis revealed that 8 established an intricate interaction network. Various immune mediators may contribute to the pathogenesis of simple DR. Attention should be given to the concentrations of immune mediators in ocular fluids even in simple DR. Large-scale studies are warranted to assess whether altered aqueous humor concentrations of these 10 immune mediators are associated with an increased risk of progression to advanced stages of DR.

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.

Healing rate of macular edema secondary to branch retinal vein occlusion in two years after initiation of intravitreal ranibizumab later combined with other treatment as needed and characteristics of refractory cases.

PURPOSE: To investigate the 2-year healing rate of macular edema (ME) secondary to branch retinal vein occlusion (BRVO) treated initially with intravitreal ranibizumab (IVR) and later combined with other treatment as needed, and the characteristics of refractory cases. METHODS: 130 patients (130 eyes) with BRVO-ME who received IVR initially were studied. Anti-vascular endothelial growth factor drug was additionally administered when ME relapsed or persisted. Photocoagulation was performed when the non-perfusion area (NPA) was ≥5 disc diameter (DD), and/or when ME relapsed due to microaneurysm. Patients were classified into a healed group [ME resolved in <2 years or mild ME remained without best-corrected visual acuity (BCVA) loss for ≥6 months] or refractory group (ME persisted for ≥2 years). RESULTS: 110 eyes were classified into the healed group, and 20 eyes into the refractory group. The healed group and refractory group had, respectively, mean follow-up periods of 21.2 and 37.4 months, and frequencies of NPA ≥5 DD of 55.5 and 25.0% (p = 0.015). In the healed group, mean BCVA (logMAR) improved significantly compared to baseline in all the periods until 24 months after treatment initiation and at the last visit (p<0.001). In the refractory group, mean BCVA improved significantly compared to baseline until 12 months after treatment initiation (p<0.05 for all periods), but was not significantly different at 18 or 24 months or at the last visit. CONCLUSION: In patients with BRVO-ME treated initially with IVR and later given additional treatments as needed, the healing rate was 84.6%. In eyes that healed within 2 years, BCVA improved relative to baseline throughout 24 months and at the last visit. In refractory eyes, BCVA improved only until 12 months, and thereafter deteriorated to baseline level at the last examination.

Ectopic inner foveal layer as a factor associated with metamorphopsia after vitrectomy for epiretinal membrane.

PURPOSE: To analyse the relationship between the extent of metamorphopsia and optical coherence tomography (OCT) parameters including the ectopic inner foveal layer (EIFL) in patients with epiretinal membrane (ERM) who underwent pars plana vitrectomy (PPV). METHODS: This study included 63 eyes of 62 patients who underwent PPV for ERM. Metamorphopsia was assessed by measuring the metamorphopsia score for vertical lines (MV) and metamorphopsia score for horizontal lines (MH) using M-CHARTS. Central retinal thickness (CRT), EIFL thickness and disruption of outer retinal layers were measured before and after surgery, and the relationship of these parameters with metamorphopsia scores at 3 months after vitrectomy was analysed. RESULTS: Preoperative MV correlated with preoperative CRT and EIFL (p = 0.005 and 0.045 respectively). Postoperative MH correlated with preoperative CRT (p = 0.048), while postoperative MV correlated with preoperative CRT and EIFL (p = 0.002 and 0.0006 respectively). Postoperative MH correlated with postoperative CRT and EIFL (p = 0.001 and 0.002 respectively), and postoperative MV also correlated with postoperative CRT and EIFL (p = 0.0008 and < 0.0001 respectively). MH and MV did not correlate with outer retinal layer disruptions, both before and after vitrectomy. When multiple regression analysis was performed on preoperative and postoperative OCT parameters that correlated with postoperative metamorphopsia scores, only postoperative EIFL was identified as an independent factor associated with postoperative MV (p = 0.042). CONCLUSION: When PPV was conducted for ERM, postoperative EIFL was found to be a significant factor associated with postoperative metamorphopsia score.

Long-Term Outcome of Eyes with Vitrectomy for Submacular and/or Vitreous Hemorrhage in Neovascular Age-Related Macular Degeneration.

PURPOSE: To study long-term clinical outcomes in patients with submacular hemorrhage (SMH) and/or vitreous hemorrhage (VH) associated with neovascular age-related macular degeneration (nAMD) and the real-world clinical situation of adding anti-VEGF therapy after pars plana vitrectomy (PPV). METHODS: In this retrospective case series, 25 eyes with SMH and/or VH associated with nAMD were treated by PPV and followed up for at least 24 months. When exudative changes were unresolved or recurred after PPV, additional intravitreal anti-VEGF therapy was given. RESULTS: The reasons for performing PPV were SMH (8 eyes) and VH (17 eyes) associated with nAMD. Mean best-corrected visual acuity (BCVA) of eyes with SMH improved significantly at 6 months (P < 0.01) and 12 months (P < 0.05) after PPV. Mean BCVA of eyes with VH improved at 1, 3, 6, 12, 18, and 24 months (P < 0.01) and at the final visit (P < 0.05). Post-PPV anti-VEGF therapy was initiated in 6 of 8 (75.0%) eyes with SMH and 7 of 17 (47.1%) eyes with VH. Of the 13 eyes given anti-VEGF therapy after PPV, 11 eyes had anti-VEGF therapy initiated within 10 months after surgery. Dry macula rate after PPV was 50.0% in SMH and 70.6% in VH. CONCLUSIONS: BCVA improved in eyes with SMH at 6 and 12 months after PPV, and the BCVA was maintained until the end of the study. BCVA improved significantly in eyes with VH at all time points after PPV. In eyes undergoing PPV for nAMD, recurrence of exudative changes after 11 months from the initial PPV was rare.

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.

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.

Case of peripheral fibrovascular proliferative retinopathy associated with morning glory disc anomaly.

It is known that the retinal detachments (RDs) associated with a morning glory disc anomaly (MGDA) usually appear from around the disc anomaly and complications by peripheral fibrovascular proliferation and tractional RD are very rare. We report our findings in an eye with MGDA that had a tractional RD, massive exudation from a peripheral temporal fibrovascular proliferation and vasculatures stretched by this proliferation and by a contraction of the hyaloidal membrane. The RD was successfully treated by vitrectomy and encircling buckling surgery.

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.

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.

Effectiveness of prophylactic intravitreal bevacizumab injection to proliferative diabetic retinopathy patients with elevated preoperative intraocular VEGF in preventing complications after vitrectomy.

PURPOSE: This study aimed to elucidate the effects of intravitreal bevacizumab (IVB) injections for the prevention of post-vitrectomy complications in proliferative diabetic retinopathy (PDR) patients with elevated vitreous vascular endothelial growth factor (VEGF) concentration. DESIGN: Prospective case series. METHODS: Thirty-three patients (42 eyes) with PDR who underwent primary vitrectomy in the Department of Ophthalmology, Tokyo Medical University Hospital were studied. We measured VEGF concentrations in vitreous humor collected at the time of vitrectomy using ELISA. IVB injections were performed after vitrectomy in patients with vitreous VEGF levels exceeding 1,000 pg/mL. New bleeding occurring within 1 month of vitrectomy was defined as early vitreous hemorrhage (VH). MAIN OUTCOME MEASURE: The incidence of complications after vitrectomy including postoperative early VH. RESULTS: IVB injections were administered to 11 eyes (26%) with vitreous VEGF concentrations exceeding 1,000 pg/mL. None of the 11 eyes that received an IVB injection developed early VH. Among 31 eyes (74%) with vitreous VEGF concentrations lower than 1,000 pg/mL, two eyes (6%) developed early VH after vitrectomy. CONCLUSIONS: Prophylactic IVB injections administered to patients with elevated preoperative intraocular VEGF concentrations were effective in preventing post-vitrectomy early VH.

Recent trends of ocular complications in patients with atopic dermatitis.

PURPOSE: To elucidate the recent trends in prevalence and characteristics of ocular complications of atopic dermatitis (AD). STUDY DESIGN: Cross-sectional observational study. METHODS: Among AD patients who visited our department between 2012 and 2015, 70 patients (140 eyes; recent AD group) who gave informed consent to participate in the study were analyzed. Following a medical interview, ophthalmological examinations were conducted for ocular complications related to AD. The data were compared to those of 280 AD patients (560 eyes) analyzed in a similar study conducted at our department in 1991-1993 (previous AD group). RESULTS: Blepharitis was found in 58 eyes (41%) in the recent AD group, and the frequency was significantly lower compared to the previous AD group (294 eyes, 53%) (p < 0.05). Tears in retina or pars plana ciliaris occurred in 22 eyes (4%) in the previous AD group, compared with none in the recent AD group (p < 0.01). Retinal detachment was observed in 12 eyes (2%) in the previous AD group, and none in the recent AD group (p < 0.01). Atopic keratoconjunctivitis (AKC) increased significantly in the recent AD group compared to the previous AD group (74.3% vs. 39.5%) (p < 0.001). Patients with a habit of slapping around the eye decreased significantly from 32.5% in the previous AD group to 12.1% in the recent AD group (p < 0.001). CONCLUSIONS: Ocular complications in AD patients show a trend of decrease in recent years, which presumably is attributed to educational activities to increase patient awareness and advances in therapeutic strategy.

Absence of Posterior Vitreous Detachment Is a Risk Factor of Severe Bleb-Related Endophthalmitis.

PURPOSE: Bleb-related endophthalmitis (BRE) after glaucoma surgery is an infection caused by bacteria in the avascular bleb gaining access into the eye. We report the clinical features and outcome of 10 consecutive eyes with severe BRE treated at our hospital. METHODS: Ten patients (10 eyes) with stage IIIb BRE after trabeculectomy diagnosed and treated at the Department of Ophthalmology, Tokyo Medical University, between April 2013 and May 2015, were studied. Patient background, type of glaucoma, interval from the first trabeculectomy, pretreatment bleb findings, causative microorganisms, surgical methods, status of posterior vitreous detachment (PVD), and postoperative visual acuity were examined. RESULTS: The 10 patients comprised 8 males and 2 females, with mean age of 70.6 years at BRE onset. The types of glaucoma were open-angle glaucoma in 7 patients, neovascular glaucoma in 2, and secondary glaucoma in 1. All eyes underwent trabeculectomy combined with mitomycin C prior to the development of BRE. The interval from the first glaucoma surgery to onset of endophthalmitis was 8.5 ± 4.1 years. Examination of the bleb revealed leakage of aqueous humor from the avascular bleb in all eyes. Bacteria were isolated from intraocular samples of 8 eyes; namely, Viridans streptococci in 5 eyes, Staphylococcus epidermidis in 1, Branhamella catarrhalis in 1, and coagulase-negative Staphylococci in 1. BRE was treated by vitrectomy in 9 eyes and enucleation in 1 eye. PVD was produced intentionally during vitrectomy in 6 eyes. Histopathological examination of the enucleated eye showed no PVD. Visual acuity improved by 3 lines or more in 6 patients, while decimal visual acuity remained lower than 0.1 in 4 patients. CONCLUSION: BRE developed frequently in eyes with no PVD. The absence of PVD may be a risk factor of severe BRE.

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.

Structure and zonal expression of olfactory receptors in the olfactory epithelium of the goat, Capra hircus.

The mammalian olfactory system employs sophisticated mechanisms to detect and recognize an extensive range of smells. In rodents, the olfactory epithelium (OE), situated within the nasal cavity, mainly comprises four defined endoturbinates and several ectoturbinates. Olfactory receptors (ORs) belong to a large family, comprising over 1,000 genes in rodents, which are expressed in olfactory sensory neurons in the OE that detect odor molecules. The rodent OE is divided into four topographically distinct zones, defined by individual OR distribution. However, although the structural complexity and the zonal organization of mammalian OE may contribute to successfully interpreting olfactory information, it remains poorly understood. In this study, we investigated the nasal cavity structure and zonal organization of the OE in goats. Morphological observations revealed that the goat nasal cavity possessed well-developed endoturbinates and ectoturbinates and had a structure similar to that of rodents and sheep, previously reported in other studies. In situ hybridization was used to analyze the expression pattern of ORs, NADPH:quinone oxidoreductase 1, and olfactory cell adhesion molecules as markers of zonal organization in the goat OE. Based on the expression patterns of these genes, we concluded that the goat OE was divided into four zones. The well-developed structure of the nasal cavity and distribution of each OR in the OE were similar to those found in rodents, suggesting that these features were highly conserved between mammals and may have fundamental roles in discriminating among numerous odor molecules in the environment.

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.

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.

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.