Estradiol elicits distinct firing patterns in arcuate nucleus kisspeptin neurons of females through altering ion channel conductances.

Hypothalamic kisspeptin (Kiss1) neurons are vital for pubertal development and reproduction. Arcuate nucleus Kiss1 (Kiss1ARH) neurons are responsible for the pulsatile release of Gonadotropin-releasing Hormone (GnRH). In females, the behavior of Kiss1ARH neurons, expressing Kiss1, Neurokinin B (NKB), and Dynorphin (Dyn), varies throughout the ovarian cycle. Studies indicate that 17ß-estradiol (E2) reduces peptide expression but increases Vglut2 mRNA and glutamate neurotransmission in these neurons, suggesting a shift from peptidergic to glutamatergic signaling. To investigate this shift, we combined transcriptomics, electrophysiology, and mathematical modeling. Our results demonstrate that E2 treatment upregulates the mRNA expression of voltage-activated calcium channels, elevating the whole-cell calcium current and that contribute to high-frequency burst firing. Additionally, E2 treatment decreased the mRNA levels of Canonical Transient Receptor Potential (TPRC) 5 and G protein-coupled K+ (GIRK) channels. When TRPC5 channels in Kiss1ARH neurons were deleted using CRISPR, the slow excitatory postsynaptic potential (sEPSP) was eliminated. Our data enabled us to formulate a biophysically realistic mathematical model of the Kiss1ARH neuron, suggesting that E2 modifies ionic conductances in Kiss1ARH neurons, enabling the transition from high frequency synchronous firing through NKB-driven activation of TRPC5 channels to a short bursting mode facilitating glutamate release. In a low E2 milieu, synchronous firing of Kiss1ARH neurons drives pulsatile release of GnRH, while the transition to burst firing with high, preovulatory levels of E2 would facilitate the GnRH surge through its glutamatergic synaptic connection to preoptic Kiss1 neurons.

A high-throughput focused collimator for OAR-sparing preclinical proton FLASH studies: commissioning and validation.

Objective. To fabricate and validate a novel focused collimator designed to spare normal tissue in a murine hemithoracic irradiation model using 250 MeV protons delivered at ultra-high dose rates (UHDRs) for preclinical FLASH radiation therapy (FLASH-RT) studies.Approach. A brass collimator was developed to shape 250 MeV UHDR protons from our Varian ProBeam. Six 13 mm apertures, of equivalent size to kV x-ray fields historically used to perform hemithorax irradiations, were precisely machined to match beam divergence, allowing concurrent hemithoracic irradiation of six mice while sparing the contralateral lung and abdominal organs. The collimated field profiles were characterized by film dosimetry, and a radiation survey of neutron activation was performed to ensure the safety of staff positioning animals.Main results. The brass collimator produced 1.2 mm penumbrae radiation fields comparable to kV x-rays used in preclinical studies. The penumbrae in the six apertures are similar, with full-width half-maxima of 13.3 mm and 13.5 mm for the central and peripheral apertures, respectively. The collimator delivered a similar dose at an average rate of 52 Gy s-1for all apertures. While neutron activation produces a high (0.2 mSv h-1) initial ambient equivalent dose rate, a parallel work-flow in which imaging and setup are performed without the collimator ensures safety to staff.Significance. Scanned protons have the greatest potential for future translation of FLASH-RT in clinical treatments due to their ability to treat deep-seated tumors with high conformality. However, the Gaussian distribution of dose in proton spots produces wider lateral penumbrae compared to other modalities. This presents a challenge in small animal pre-clinical studies, where millimeter-scale penumbrae are required to precisely target the intended volume. Offering high-throughput irradiation of mice with sharp penumbrae, our novel collimator-based platform serves as an important benchmark for enabling large-scale, cost-effective radiobiological studies of the FLASH effect in murine models.

NK3R signalling in the posterodorsal medial amygdala is involved in stress-induced suppression of pulsatile LH secretion in female mice.

Psychosocial stress negatively impacts reproductive function by inhibiting pulsatile luteinizing hormone (LH) secretion. The posterodorsal medial amygdala (MePD) is responsible in part for processing stress and modulating the reproductive axis. Activation of the neurokinin 3 receptor (NK3R) suppresses the gonadotropin-releasing hormone (GnRH) pulse generator, under hypoestrogenic conditions, and NK3R activity in the amygdala has been documented to play a role in stress and anxiety. We investigate whether NK3R activation in the MePD is involved in mediating the inhibitory effect of psychosocial stress on LH pulsatility in ovariectomised female mice. First, we administered senktide, an NK3R agonist, into the MePD and monitored the effect on pulsatile LH secretion. We then delivered SB222200, a selective NK3R antagonist, intra-MePD in the presence of predator odour, 2,4,5-trimethylthiazole (TMT) and examined the effect on LH pulses. Senktide administration into the MePD dose-dependently suppresses pulsatile LH secretion. Moreover, NK3R signalling in the MePD mediates TMT-induced suppression of the GnRH pulse generator, which we verified using a mathematical model. The model verifies our experimental findings: (i) predator odour exposure inhibits LH pulses, (ii) activation of NK3R in the MePD inhibits LH pulses and (iii) NK3R antagonism in the MePD blocks stressor-induced inhibition of LH pulse frequency in the absence of ovarian steroids. These results demonstrate for the first time that NK3R neurons in the MePD mediate psychosocial stress-induced suppression of the GnRH pulse generator.

Technical note: A small animal irradiation platform for investigating the dependence of the FLASH effect on electron beam parameters.

BACKGROUND: The recent rediscovery of the FLASH effect, a normal tissue sparing phenomenon observed in ultra-high dose rate (UHDR) irradiations, has instigated a surge of research endeavors aiming to close the gap between experimental observation and clinical treatment. However, the dependences of the FLASH effect and its underpinning mechanisms on beam parameters are not well known, and large-scale in vivo studies using murine models of human cancer are needed for these investigations. PURPOSE: To commission a high-throughput, variable dose rate platform providing uniform electron fields (≥15 cm diameter) at conventional (CONV) and UHDRs for in vivo investigations of the FLASH effect and its dependences on pulsed electron beam parameters. METHODS: A murine whole-thoracic lung irradiation (WTLI) platform was constructed using a 1.3 cm thick Cerrobend collimator forming a 15 × 1.6 cm2 slit. Control of dose and dose rate were realized by adjusting the number of monitor units and couch vertical position, respectively. Achievable doses and dose rates were investigated using Gafchromic EBT-XD film at 1 cm depth in solid water and lung-density phantoms. Percent depth dose (PDD) and dose profiles at CONV and various UHDRs were also measured at depths from 0 to 2 cm. A radiation survey was performed to assess radioactivation of the Cerrobend collimator by the UHDR electron beam in comparison to a precision-machined copper alternative. RESULTS: This platform allows for the simultaneous thoracic irradiation of at least three mice. A linear relationship between dose and number of monitor units at a given UHDR was established to guide the selection of dose, and an inverse-square relationship between dose rate and source distance was established to guide the selection of dose rate between 20 and 120 Gy·s-1 . At depths of 0.5 to 1.5 cm, the depth range relevant to murine lung irradiation, measured PDDs varied within ±1.5%. Similar lateral dose profiles were observed at CONV and UHDRs with the dose penumbrae widening from 0.3 mm at 0 cm depth to 5.1 mm at 2.0 cm. The presence of lung-density plastic slabs had minimal effect on dose distributions as compared to measurements made with only solid water slabs. Instantaneous dose rate measurements of the activated copper collimator were up to two orders of magnitude higher than that of the Cerrobend collimator. CONCLUSIONS: A high-throughput, variable dose rate platform has been developed and commissioned for murine WTLI electron FLASH radiotherapy. The wide field of our UHDR-enabled linac allows for the simultaneous WTLI of at least three mice, and for the average dose rate to be modified by changing the source distance, without affecting dose distribution. The platform exhibits uniform, and comparable dose distributions at CONV and UHDRs up to 120 Gy·s-1 , owing to matched and flattened 16 MeV CONV and UHDR electron beams. Considering radioactivation and exposure to staff, Cerrobend collimators are recommended above copper alternatives for electron FLASH research. This platform enables high-throughput animal irradiation, which is preferred for experiments using a large number of animals, which are required to effectively determine UHDR treatment efficacies.

New methods to investigate the GnRH pulse generator.

The exact neural construct underlying the dynamic secretion of gonadotrophin-releasing hormone (GnRH) has only recently been identified despite the detection of multiunit electrical activity volleys associated with pulsatile luteinising hormone (LH) secretion four decades ago. Since the discovery of kisspeptin/neurokinin B/dynorphin neurons in the mammalian hypothalamus, there has been much research into the role of this neuronal network in controlling the oscillatory secretion of gonadotrophin hormones. In this review, we provide an update of the progressive application of cutting-edge techniques combined with mathematical modelling by the neuroendocrine community, which are transforming the functional investigation of the GnRH pulse generator. Understanding the nature and function of the GnRH pulse generator can greatly inform a wide range of clinical studies investigating infertility treatments.

Posterior cruciate ligament rupture and all-epiphyseal repair with suture tape augmentation in a 5-year-old girl: a case report and review of the literature.

INTRODUCTION: Only a few case reports regarding pediatric posterior cruciate ligament (PCL) ruptures without bone avulsion exist in the literature. The present study aims to share our experience in the diagnosis, treatment, and prognosis of a child with a proximal PCL tear. MATERIALS AND METHODS: This article reports a 5-year-old female diagnosed with a proximal PCL tear. The ruptured PCL was repaired with an all-epiphyseal suture tape augmentation (STA) without evidence of growth plate violation. RESULTS: The suture tape was removed under arthroscopy and revealed the PCL was re-attached at 12 months after the first surgery. And at the time of this report, 36 months after surgery, she was doing well without any problems and with negative posterior drawer test. CONCLUSIONS: Pediatric PCL tear without bone avulsion is rare. However, the torn PCL was noticed healed based on an arthroscopic second-look.

HOPE: A Pilot Study of Psilocybin Enhanced Group Psychotherapy in Patients With Cancer.

CONTEXT/OBJECTIVES: Psilocybin-assisted psychotherapy shows promise in treating depression and existential distress in people with serious medical illness. However, its individual-based methodology poses challenges for scaling and resource availability. The HOPE trial (A Pilot Study of Psilocybin Enhanced Group Psychotherapy in Patients with Cancer) is an Institutional Review Boards-approved open-label feasibility and safety pilot study examining psilocybin-assisted group therapy in cancer patients with a DSM-5 depressive disorder (including major depressive disorder as well as adjustment disorder with depressed mood). We report here the safety and clinical outcome measures including six-months follow up data. METHODS: Outcome measures were collected at baseline, two-weeks and 26-weeks postintervention. The study involved three group preparatory sessions, one high-dose (25 mg) group psilocybin session, and three group integration sessions with cohorts of four participants over a three-week intervention. RESULTS: Twelve participants completed the trial. no serious adverse events attributed to psilocybin occurred. The primary clinical outcome measures of change in symptoms of depression on the clinician administered 17-item-HAM-D showed clinically substantial decrease in HAM-D scores from baseline to the two-week timepoint (21.5-10.09, P < 0.001) and the 26-week timepoint (21.5-14.83, P = 0.006). Six out of 12 participants met criteria for remission at two weeks, as defined by HAM-D < 7, three out 12 demonstrated a clinically significant change (4-6 points), and eight out of twelve demonstrated a clinically substantial change (7-12 points). CONCLUSION: This pilot study demonstrated the safety, feasibility, and possible efficacy of psilocybin-assisted group therapy for cancer patients dealing with depressive symptoms. Based on demonstrated efficacy and significant reductions in therapist time, future investigations with the group therapy model are warranted.

Hypothalamic PVN CRH Neurons Signal Through PVN GABA Neurons to Suppress GnRH Pulse Generator Frequency in Female Mice.

Corticotropin-releasing hormone (CRH) neurons in the paraventricular nucleus of the hypothalamus (PVN) are central to the stress response. Chemogenetic activation of PVN CRH neurons decreases LH pulse frequency but the mechanism is unknown. In the present study, optogenetic stimulation of PVN CRH neurons suppressed LH pulse frequency in estradiol-replaced ovariectomized CRH-cre mice, and this effect was augmented or attenuated by intra-PVN GABAA or GABAB receptor antagonism, respectively. PVN CRH neurons signal to local GABA neurons, which may provide a possible indirect mechanism by which PVN CRH neurons suppress LH pulse frequency. Optogenetic stimulation of potential PVN GABAergic projection terminals in the hypothalamic arcuate nucleus in ovariectomized estradiol-replaced Vgat-cre-tdTomato mice via an optic fiber implanted in the arcuate nucleus suppressed LH pulse frequency. To further determine whether PVN CRH neurons signal through PVN GABA neurons to suppress LH pulsatility, we combined recombinase mice with intersectional vectors to selectively target these neurons. CRH-cre::Vgat-FlpO mice expressing the stimulatory opsin ChRmine in non-GABAergic CRH neurons alone or in combination with the inhibitory opsin NpHR3.3 in non-CRH-expressing GABA neurons in the PVN were used. Optogenetic stimulation of non-GABAergic CRH neurons suppressed pulsatile LH secretion; however, LH pulse frequency was not affected when CRH neurons were stimulated and PVN GABA neurons were simultaneously inhibited. Together, these studies demonstrate that suppression of LH pulse frequency in response to PVN CRH neuronal activation is mediated by GABAergic signalling intrinsic to the PVN and may incorporate PVN GABAergic projection to the hypothalamic GnRH pulse generator.

Optogenetics studies of kisspeptin neurons.

Optical systems and genetic engineering technologies have made it possible to control neurons and unravel neuronal circuit behavior with high temporal and spatial resolution. The application of optogenetic strategies to understand the physiology of kisspeptin neuronal circuits has evolved in recent years among the neuroendocrine community. Kisspeptin neurons are fundamentally involved in controlling mammalian reproduction but also are implicated in numerous other physiological processes, including but not limited to feeding, energy expenditure, core body temperature and behavior. We conducted a review aiming to shed light on the novel findings obtained from in vitro and in vivo optogenetic studies interrogating kisspeptin neuronal circuits to date. Understanding the function of kisspeptin networks in the brain can greatly inform a wide range of clinical studies investigating infertility treatments, gender identity, metabolic disorders, hot flushes and psychosexual disorders.

GABA Signaling in the Posterodorsal Medial Amygdala Mediates Stress-induced Suppression of LH Pulsatility in Female Mice.

Psychological stress is linked to infertility by suppressing the hypothalamic GnRH pulse generator. The posterodorsal subnucleus of the medial amygdala (MePD) is an upstream regulator of GnRH pulse generator activity and displays increased neuronal activation during psychological stress. The MePD is primarily a GABAergic nucleus with a strong GABAergic projection to hypothalamic reproductive centers; however, their functional significance has not been determined. We hypothesize that MePD GABAergic signalling mediates psychological stress-induced suppression of pulsatile LH secretion. We selectively inhibited MePD GABA neurons during psychological stress in ovariectomized (OVX) Vgat-cre-tdTomato mice to determine the effect on stress-induced suppression of pulsatile LH secretion. MePD GABA neurons were virally infected with inhibitory hM4DGi-designer receptor exclusively activated by designer drugs (DREADDs) to selectively inhibit MePD GABA neurons. Furthermore, we optogenetically stimulated potential MePD GABAergic projection terminals in the hypothalamic arcuate nucleus (ARC) and determined the effect on pulsatile LH secretion. MePD GABA neurons in OVX female Vgat-cre-tdTomato mice were virally infected to express channelrhodopsin-2 and MePD GABAergic terminals in the ARC were selectively stimulated by blue light via an optic fiber implanted in the ARC. DREADD-mediated inhibition of MePD GABA neurons blocked predator odor and restraint stress-induced suppression of LH pulse frequency. Furthermore, sustained optogenetic stimulation at 10 and 20 Hz of MePD GABAergic terminals in the ARC suppressed pulsatile LH secretion. These results show for the first time that GABAergic signalling in the MePD mediates psychological stress-induced suppression of pulsatile LH secretion and suggest a functionally significant MePD GABAergic projection to the hypothalamic GnRH pulse generator.

Optogenetic stimulation of Kiss1ARC terminals in the AVPV induces surge-like luteinizing hormone secretion via glutamate release in mice.

Kisspeptin neurons are mainly located in the arcuate (Kiss1ARC, vis-à-vis the GnRH pulse generator) and anteroventral periventricular nucleus (Kiss1AVPV, vis-à-vis the GnRH surge generator). Kiss1ARC send fibre projections that connect with Kiss1AVPV somata. However, studies focused on the role of Kiss1ARC neurons in the LH surge are limited, and the role of Kiss1ARC projections to AVPV (Kiss1ARC→AVPV) in the preovulatory LH surge is still unknown. To investigate its function, this study used optogenetics to selectively stimulate Kiss1ARC→AVPV and measured changes in circulating LH levels. Kiss1ARC in Kiss-Cre-tdTomato mice were virally infected to express channelrhodopsin-2 proteins, and optical stimulation was applied selectively via a fibre optic cannula in the AVPV. Sustained 20 Hz optical stimulation of Kiss1ARC→AVPV from 15:30 to 16:30 h on proestrus effectively induced an immediate increase in LH reaching peak surge-like levels of around 8 ng/ml within 10 min, followed by a gradual decline to baseline over about 40 min. Stimulation at 10 Hz resulted in a non-significant increase in LH levels and 5 Hz stimulation had no effect in proestrous animals. The 20 Hz stimulation induced significantly higher circulating LH levels on proestrus compared with diestrus or estrus, which suggested that the effect of terminal stimulation is modulated by the sex steroid milieu. Additionally, intra-AVPV infusion of glutamate antagonists, AP5+CNQX, completely blocked the increase on LH levels induced by Kiss1ARC→AVPV terminal photostimulation in proestrous animals. These results demonstrate for the first time that optical stimulation of Kiss1ARC→AVPV induces an LH surge-like secretion via glutamatergic mechanisms. In conclusion, Kiss1ARC may participate in LH surge generation by glutamate release from terminal projections in the AVPV.

Posterodorsal Medial Amygdala Urocortin-3, GABA, and Glutamate Mediate Suppression of LH Pulsatility in Female Mice.

The posterodorsal subnucleus of the medial amygdala (MePD) is an upstream modulator of the hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary-adrenal (HPA) axes. Inhibition of MePD urocortin-3 (Ucn3) neurons prevents psychological stress-induced suppression of luteinizing hormone (LH) pulsatility while blocking the stress-induced elevations in corticosterone (CORT) secretion in female mice. We explore the neurotransmission and neural circuitry suppressing the gonadotropin-releasing hormone (GnRH) pulse generator by MePD Ucn3 neurons and we further investigate whether MePD Ucn3 efferent projections to the hypothalamic paraventricular nucleus (PVN) control CORT secretion and LH pulsatility. Ucn3-cre-tdTomato female ovariectomized (OVX) mice were unilaterally injected with adeno-associated virus (AAV)-channelrhodopsin 2 (ChR2) and implanted with optofluid cannulae targeting the MePD. We optically activated Ucn3 neurons in the MePD with blue light at 10 Hz and monitored the effect on LH pulses. Next, we combined optogenetic stimulation of MePD Ucn3 neurons with pharmacological antagonism of GABAA or GABAB receptors with bicuculline or CGP-35348, respectively, as well as a combination of NMDA and AMPA receptor antagonists, AP5 and CNQX, respectively, and observed the effect on pulsatile LH secretion. A separate group of Ucn3-cre-tdTomato OVX mice with 17ß-estradiol replacement were unilaterally injected with AAV-ChR2 in the MePD and implanted with fiber-optic cannulae targeting the PVN. We optically stimulated the MePD Ucn3 efferent projections in the PVN with blue light at 20 Hz and monitored the effect on CORT secretion and LH pulses. We reveal for the first time that activation of Ucn3 neurons in the MePD inhibits GnRH pulse generator frequency via GABA and glutamate signaling within the MePD, while MePD Ucn3 projections to the PVN modulate the HPG and HPA axes.

GnRH pulse generator frequency is modulated by kisspeptin and GABA-glutamate interactions in the posterodorsal medial amygdala in female mice.

Kisspeptin neurons in the arcuate nucleus of the hypothalamus generate gonadotrophin-releasing hormone (GnRH) pulses, and act as critical initiators of functional gonadotrophin secretion and reproductive competency. However, kisspeptin in other brain regions, most notably the posterodorsal subnucleus of the medial amygdala (MePD), plays a significant modulatory role over the hypothalamic kisspeptin population; our recent studies using optogenetics have shown that low-frequency light stimulation of MePD kisspeptin results in increased luteinsing hormone pulse frequency. Nonetheless, the neurochemical pathways that underpin this regulatory function remain unknown. To study this, we have utilised an optofluid technology, precisely combining optogenetic stimulation with intra-nuclear pharmacological receptor antagonism, to investigate the neurotransmission involved in this circuitry. We have shown experimentally and verified using a mathematical model that functional neurotransmission of both GABA and glutamate is a requirement for effective modulation of the GnRH pulse generator by amygdala kisspeptin neurons.

Provocative tests with Kisspeptin-10 and GnRH set the scene for determining social status and environmental impacts on reproductive capacity in male African lions (Panthera leo).

Understanding the hypothalamic factors regulating reproduction facilitates maximising the reproductive success of breeding programmes and in the management and conservation of threatened species, including African lions. To provide insight into the physiology and pathophysiology of the hypothalamic-pituitary-gonadal reproductive axis in lions, we studied the luteinising hormone (LH) and steroid hormone responses to gonadotropin-releasing hormone (GnRH) and its upstream regulator, kisspeptin. Six young (13.3 ± 1.7 months, 56.2 ± 4.3 kg) and four adult (40.2 ± 1.4 months, 174 ± 6 kg) male lions (Ukutula Conservation Centre, South Africa) were used in this study. Lions were immobilised with a combination of medetomidine and ketamine and an intravenous catheter was placed in a jugular, cephalic or medial saphenous vein for blood sampling at 10-min intervals for 220 min. The ten-amino acid kisspeptin which has full intrinsic activity (KP-10, 1 µg/kg) and GnRH (1 µg/kg) were administered intravenously to study their effects on LH and steroid hormone plasma concentrations, measured subsequently by ELISA and liquid chromatography tandem mass spectrometry (LC-MS/MS), respectively. Basal LH levels were similarly low between the age groups, but testosterone and its precursor levels were higher in the adult animals. Adult lions showed a significant LH response to KP-10 (10-fold) and GnRH (11-fold) administration (p < 0.05 and P < 0.001, respectively) whereas in young lions LH increased significantly only in response to GnRH. In adults alone, testosterone and its precursors steadily increased in response to KP-10, with no significant further increase in response to GnRH. Plasma levels of glucocorticoids in response to KP-10 remained unchanged. We suggest that provocative testing of LH and steroid stimulation with kisspeptin provides a new and sensitive tool for determining reproductive status and possibly an index of exposure to stress, environmental insults such as disease, endocrine disruptors and nutritional status. 272 words.

Role of Posterodorsal Medial Amygdala Urocortin-3 in Pubertal Timing in Female Mice.

Post-traumatic stress disorder impedes pubertal development and disrupts pulsatile LH secretion in humans and rodents. The posterodorsal sub-nucleus of the medial amygdala (MePD) is an upstream modulator of the hypothalamic gonadotropin-releasing hormone (GnRH) pulse generator, pubertal timing, as well as emotional processing and anxiety. Psychosocial stress exposure alters neuronal activity within the MePD increasing the expression of Urocortin3 (Ucn3) and its receptor corticotropin-releasing factor type-2 receptor (CRFR2) while enhancing the inhibitory output from the MePD to key hypothalamic reproductive centres. We test the hypothesis that psychosocial stress, processed by the MePD, is relayed to the hypothalamic GnRH pulse generator to delay puberty in female mice. We exposed C57Bl6/J female mice to the predator odor, 2,4,5-Trimethylthiazole (TMT), during pubertal transition and examined the effect on pubertal timing, pre-pubertal LH pulses and anxiety-like behaviour. Subsequently, we virally infected Ucn3-cre-tdTomato female mice with stimulatory DREADDs targeting MePD Ucn3 neurons and determined the effect on pubertal timing and pre-pubertal LH pulse frequency. Exposure to TMT during pubertal development delayed puberty, suppressed pre-pubertal LH pulsatility and enhanced anxiety-like behaviour, while activation of MePD Ucn3 neurons reduced LH pulse frequency and delayed puberty. Early psychosocial stress exposure decreases GnRH pulse generator frequency delaying puberty while inducing anxiety-behaviour in female mice, an effect potentially involving Ucn3 neurons in the MePD.

Mathematical models in GnRH research.

Mathematical modelling is an indispensable tool in modern biosciences, enabling quantitative analysis and integration of biological data, transparent formulation of our understanding of complex biological systems, and efficient experimental design based on model predictions. This review article provides an overview of the impact that mathematical models had on GnRH research. Indeed, over the last 20 years mathematical modelling has been used to describe and explore the physiology of the GnRH neuron, the mechanisms underlying GnRH pulsatile secretion, and GnRH signalling to the pituitary. Importantly, these models have contributed to GnRH research via novel hypotheses and predictions regarding the bursting behaviour of the GnRH neuron, the role of kisspeptin neurons in the emergence of pulsatile GnRH dynamics, and the decoding of GnRH signals by biochemical signalling networks. We envisage that with the advent of novel experimental technologies, mathematical modelling will have an even greater role to play in our endeavour to understand the complex spatiotemporal dynamics underlying the reproductive neuroendocrine system.

Two-fragment Segond fracture validates historical descriptions of independent soft tissue attachments.

This is a case report of a 26-year-old male who sustained a Segond fracture in the context of an acute anterior cruciate ligament (ACL) rupture incurred while downhill skiing. Further work-up revealed that the Segond fracture consisted of two distinct fragments with separate soft tissue attachments, including the capsule-osseous layer of the iliotibial band and the short arm of the biceps femoris. Imaging showed interval healing of the Segond fracture between initial presentation and the performance of arthroscopic ACL reconstruction approximately 4 months later. As intraoperative evaluation demonstrated that anatomic ACL reconstruction restored translational and rotatory knee stability, surgical repair of the Segond fracture, or the anterolateral complex of the knee more broadly, was not required. Maintenance of translational and rotatory knee stability was confirmed at serial post-operative appointments up through final follow-up.Level of evidence Level V.

Non-anatomic tunnel position increases the risk of revision anterior cruciate ligament reconstruction.

PURPOSE: Anterior cruciate ligament (ACL) graft failure is a complication that may require revision ACL reconstruction (ACL-R). Non-anatomic placement of the femoral tunnel is thought to be a frequent cause of graft failure; however, there is a lack of evidence to support this belief. The purpose of this study was to determine if non-anatomic femoral tunnel placement is associated with increased risk of revision ACL-R. METHODS: After screening all 315 consecutive patients who underwent primary single-bundle ACL-R by a single senior orthopedic surgeon between January 2012 and January 2017, 58 patients were found to have both strict lateral radiographs and a minimum of 24 months follow-up without revision. From a group of 456 consecutive revision ACL-R, patients were screened for strictly lateral radiographs and 59 patients were included in the revision group. Femoral tunnel placement for each patient was determined using a strict lateral radiograph taken after the primary ACL-R using the quadrant method. The center of the femoral tunnel was measured in both the posterior-anterior (PA) and proximal-distal (PD) dimensions and represented as a percentage of the total distance (normal center of anatomic footprint: PA 25% and PD 29%). RESULTS: In the PA dimension, the revision group had significantly more anterior femoral tunnel placement compared with the primary group (38% ± 11% vs. 28% ± 6%, p < 0.01). Among patients who underwent revision; those with non-traumatic chronic failure had statistically significant more anterior femoral tunnel placement than those who experienced traumatic failure (41% ± 13% vs. 35% ± 8%, p < 0.03). In the PD dimension, the revision group had significantly more proximal femoral tunnel placement compared with the primary group (30% ± 9% vs 38% ± 9%, p < 0.01). CONCLUSION: In this retrospective study of 58 patients with successful primary ACL-R compared with 59 patients with failed ACL-R, anterior and proximal (high) femoral tunnels for ACL-R were shown to be independent risk factors for ACL revision surgery. As revision ACL-R is associated with patient- and economic burden, particular attention should be given to achieving an individualized, anatomic primary ACL-R. Surgeons may reduce the risk of revision ACL-R by placing the center of the femoral tunnel within the anatomic ACL footprint. LEVEL OF EVIDENCE: Level III.

Low to moderate risk of nerve damage during peroneus longus tendon autograft harvest.

PURPOSE: This study aims to evaluate the proximity of the tendon stripper to both the peroneal and sural nerves during peroneus longus tendon (PLT) autograft harvesting. METHODS: Ten fresh-frozen human cadaveric lower extremities were used to harvest a full-thickness PLT autograft using a standard closed blunt-ended tendon stripper. The distance to the sural nerve from the PLT (at 0, 1, 2 and 3 cm proximal to lateral malleolus (LM), and the distance to the peroneal nerve and its branches from the end of the tendon stripper were measured by two separate observers using ImageJ software. RESULTS: The average distance from the PLT to the sural nerve increased significantly from 0 to 2 cm proximal to LM. The average distance to the sural nerve at the LM was 4.9 ± 1.5 mm and increased to 10.8 ± 2.4 mm (2 cm proximal to LM). The average distance from the tendon stripper to the deep peroneal nerve was 52.9 ± 11.4 mm. The average distance to the PLT branch of peroneal nerve was 29.3 ± 4.2 mm. The superficial peroneal nerve, which coursed parallel and deep to the tendon stripper, was on average 5.2 ± 0.7 mm from the end of the stripper. No transection injuries of the nerves were observed in any of the ten legs after harvesting. CONCLUSION: This cadaver study found during a full-thickness PLT harvest, the distances between the tendon stripper and the nerves were greater than 5 mm with an initial incision at 2 cm proximal to LM which is recommended.

Knees with straight Blumensaat's line have small volume of femoral intercondylar notch.

PURPOSE: Smaller femoral intercondylar notch volume has been identified as a risk factor for anterior cruciate ligament injury. The present study aims to investigate differences in the intercondylar notch volume based on differences in the morphology of Blumensaat's line. METHODS: Eighty-eight (88) subjects (42 male and 46 female: median age 27: range 15-49), were included in this study. Using 3-dimensional computed tomography (3D-CT), the volume of the intercondylar notch was calculated using a truncated-pyramid shape simulation with the formula: [Formula: see text]. Femoral condyle height (h) was measured in the sagittal plane of the knee in 3D-CT. The area of the intercondylar notch was measured in the axial slice containing the most proximal level (S1) and most distal level (S2) of Blumensaat's line. In the sagittal view of the knee, Blumensaat's line morphology was classified into either straight or hill type. Statistical analysis was performed to compare h, S1, S2, and notch volume between the straight and hill type groups. RESULTS: Thirty-six subjects were classified as having straight type morphology and 52 subjects were classified as having hill type morphology. The measured h, S1, and S2, of the straight and hill types were 29 ± 4 and 31 ± 4 mm, 213 ± 72 and 205 ± 51 mm2, 375 ± 114 and 430 ± 94 mm2, respectively. The calculated femoral intercondylar notch volume of the straight and hill types was 8.1 ± 2 and 9.5 ± 2 cm3, respectively. Straight type knees showed significantly smaller S2 (p = 0.04), and notch volume (p = 0.01) when compared with hill type knees. CONCLUSION: Intercondylar notch volume was significantly smaller in knees with straight type Blumensaat's line morphology. Considering that Blumensaat's line represents the roof of the femoral notch, morphological variations in Blumensaat's line are likely to reflect variation in notch volume. For clinical relevance, as a smaller notch volume is a risk factor for ACL injury, straight type Blumensaat's line may also be considered a potential risk factor for ACL injury. LEVEL OF EVIDENCE: Level III.