A Simulation Modelling Study of Referral Distribution Policies in a Centralized Intake System for Surgical Consultation.

Delays beyond recommended wait times, especially for specialist services, are associated with adverse health outcomes. The Alberta Surgical Initiative aims to improve the referral wait time-the time between a referral is received at the central intake to the time a specialist sees the patient. Using the discrete event simulation modelling approach, we evaluated and compared the impact of four referral distribution policies in a central intake system on three system performance measures (number of consultations, referral wait time and surgeon utilization). The model was co-designed with clinicians and clinic staff to represent the flow of patients through the system. We used data from the Facilitated Access to Surgical Treatment (FAST) centralized intake referral program for General Surgery to parameterize the model. Four distribution policies were evaluated - next-available-surgeon, sequential, "blackjack," and "kanban." A sequential distribution of referrals for surgical consultation among the surgeons resulted in the worst performance in terms of the number of consultations, referral wait time and surgeon utilization. The three other distribution policies are comparable in performance. The "next available surgeon" model provided the most efficient and robust model, with approximately 1,000 more consultations, 100 days shorter referral time and a 14% increase in surgeon utilization. Discrete event simulation (DES) modelling can be an effective tool to illustrate and communicate the impact of the referral distribution policy on system performance in terms of the number of consultations, referral wait time and surgeon utilization.

Excess Protein O-GlcNAcylation Links Metabolic Derangements to Right Ventricular Dysfunction in Pulmonary Arterial Hypertension.

The hexosamine biosynthetic pathway (HBP) converts glucose to uridine-diphosphate-N-acetylglucosamine, which, when added to serines or threonines, modulates protein function through protein O-GlcNAcylation. Glutamine-fructose-6-phosphate amidotransferase (GFAT) regulates HBP flux, and AMP-kinase phosphorylation of GFAT blunts GFAT activity and O-GlcNAcylation. While numerous studies demonstrate increased right ventricle (RV) glucose uptake in pulmonary arterial hypertension (PAH), the relationship between O-GlcNAcylation and RV function in PAH is unexplored. Therefore, we examined how colchicine-mediated AMP-kinase activation altered HBP intermediates, O-GlcNAcylation, mitochondrial function, and RV function in pulmonary artery-banded (PAB) and monocrotaline (MCT) rats. AMPK activation induced GFAT phosphorylation and reduced HBP intermediates and O-GlcNAcylation in MCT but not PAB rats. Reduced O-GlcNAcylation partially restored the RV metabolic signature and improved RV function in MCT rats. Proteomics revealed elevated expression of O-GlcNAcylated mitochondrial proteins in MCT RVs, which fractionation studies corroborated. Seahorse micropolarimetry analysis of H9c2 cardiomyocytes demonstrated colchicine improved mitochondrial function and reduced O-GlcNAcylation. Presence of diabetes in PAH, a condition of excess O-GlcNAcylation, reduced RV contractility when compared to nondiabetics. Furthermore, there was an inverse relationship between RV contractility and HgbA1C. Finally, RV biopsy specimens from PAH patients displayed increased O-GlcNAcylation. Thus, excess O-GlcNAcylation may contribute to metabolic derangements and RV dysfunction in PAH.

Identification of novel dynamin-related protein 1 (Drp1) GTPase inhibitors: Therapeutic potential of Drpitor1 and Drpitor1a in cancer and cardiac ischemia-reperfusion injury.

Mitochondrial fission is important in physiological processes, including coordination of mitochondrial and nuclear division during mitosis, and pathologic processes, such as the production of reactive oxygen species (ROS) during cardiac ischemia-reperfusion injury (IR). Mitochondrial fission is mainly mediated by dynamin-related protein 1 (Drp1), a large GTPase. The GTPase activity of Drp1 is essential for its fissogenic activity. Therefore, we aimed to identify Drp1 inhibitors and evaluate their anti-neoplastic and cardioprotective properties in five cancer cell lines (A549, SK-MES-1, SK-LU-1, SW 900, and MCF7) and an experimental cardiac IR injury model. Virtual screening of a chemical library revealed 17 compounds with high predicted affinity to the GTPase domain of Drp1. In silico screening identified an ellipticine compound, Drpitor1, as a putative, potent Drp1 inhibitor. We also synthesized a congener of Drpitor1 to remove the methoxymethyl group and reduce hydrolytic lability (Drpitor1a). Drpitor1 and Drpitor1a inhibited the GTPase activity of Drp1 without inhibiting the GTPase of dynamin 1. Drpitor1 and Drpitor1a have greater potency than the current standard Drp1 GTPase inhibitor, mdivi-1, (IC50 for mitochondrial fragmentation are 0.09, 0.06, and 10 µM, respectively). Both Drpitors reduced proliferation and induced apoptosis in cancer cells. Drpitor1a suppressed lung cancer tumor growth in a mouse xenograft model. Drpitor1a also inhibited mitochondrial ROS production, prevented mitochondrial fission, and improved right ventricular diastolic dysfunction during IR injury. In conclusion, Drpitors are useful tools for understanding mitochondrial dynamics and have therapeutic potential in treating cancer and cardiac IR injury.

Biventricular Increases in Mitochondrial Fission Mediator (MiD51) and Proglycolytic Pyruvate Kinase (PKM2) Isoform in Experimental Group 2 Pulmonary Hypertension-Novel Mitochondrial Abnormalities.

Introduction: Group 2 pulmonary hypertension (PH), defined as a mean pulmonary arterial pressure ≥25 mmHg with elevated pulmonary capillary wedge pressure >15 mmHg, has no approved therapy and patients often die from right ventricular failure (RVF). Alterations in mitochondrial metabolism, notably impaired glucose oxidation, and increased mitochondrial fission, contribute to right ventricle (RV) dysfunction in PH. We hypothesized that the impairment of RV and left ventricular (LV) function in group 2 PH results in part from a proglycolytic isoform switch from pyruvate kinase muscle (PKM) isoform 1 to 2 and from increased mitochondrial fission, due either to upregulation of expression of dynamin-related protein 1 (Drp1) or its binding partners, mitochondrial dynamics protein of 49 or 51 kDa (MiD49 or 51). Methods and Results: Group 2 PH was induced by supra-coronary aortic banding (SAB) in 5-week old male Sprague Dawley rats. Four weeks post SAB, echocardiography showed marked reduction of tricuspid annular plane systolic excursion (2.9 ± 0.1 vs. 4.0 ± 0.1 mm) and pulmonary artery acceleration time (24.3 ± 0.9 vs. 35.4 ± 1.8 ms) in SAB vs. sham rats. Nine weeks post SAB, left and right heart catheterization showed significant biventricular increases in end systolic and diastolic pressure in SAB vs. sham rats (LV: 226 ± 15 vs. 103 ± 5 mmHg, 34 ± 5 vs. 7 ± 1 mmHg; RV: 40 ± 4 vs. 22 ± 1 mmHg, and 4.7 ± 1.5 vs. 0.9 ± 0.5 mmHg, respectively). Picrosirius red staining showed marked biventricular fibrosis in SAB rats. There was increased muscularization of small pulmonary arteries in SAB rats. Confocal microscopy showed biventricular mitochondrial depolarization and fragmentation in SAB vs. sham cardiomyocytes. Transmission electron microscopy confirmed a marked biventricular reduction in mitochondria size in SAB hearts. Immunoblot showed marked biventricular increase in PKM2/PKM1 and MiD51 expression. Mitofusin 2 and mitochondrial pyruvate carrier 1 were increased in SAB LVs. Conclusions: SAB caused group 2 PH. Impaired RV function and RV fibrosis were associated with increases in mitochondrial fission and expression of MiD51 and PKM2. While these changes would be expected to promote increased production of reactive oxygen species and a glycolytic shift in metabolism, further study is required to determine the functional consequences of these newly described mitochondrial abnormalities.

Ischemia-induced Drp1 and Fis1-mediated mitochondrial fission and right ventricular dysfunction in pulmonary hypertension.

Right ventricular (RV) function determines prognosis in pulmonary arterial hypertension (PAH). We hypothesize that ischemia causes RV dysfunction in PAH by triggering dynamin-related protein 1 (Drp1)-mediated mitochondrial fission. RV function was compared in control rats (n = 50) versus rats with monocrotaline-induced PAH (MCT-PAH; n = 60) both in vivo (echocardiography) and ex vivo (RV Langendorff). Mitochondrial membrane potential and morphology and RV function were assessed before or after 2 cycles of ischemia-reperfusion injury challenge (RV-IR). The effects of Mdivi-1 (25 µM), a Drp1 GTPase inhibitor, and P110 (1 µM), a peptide inhibitor of Drp1-Fis1 interaction, were studied. We found that MCT caused RV hypertrophy, RV vascular rarefaction, and RV dysfunction. Prior to IR, the mitochondria in MCT-PAH RV were depolarized and swollen with increased Drp1 content and reduced aconitase activity. RV-IR increased RV end diastolic pressure (RVEDP) and mitochondrial Drp1 expression in both control and MCT-PAH RVs. IR depolarized mitochondria in control RV but did not exacerbate the basally depolarized MCT-PAH RV mitochondria. During RV IR mdivi-1 and P110 reduced Drp1 translocation to mitochondria, improved mitochondrial structure and function, and reduced RVEDP. In conclusion, RV ischemia occurs in PAH and causes Drp1-Fis1-mediated fission leading to diastolic dysfunction. Inhibition of mitochondrial fission preserves RV function in RV-IR. KEY MESSAGES: Right ventricular ischemia reperfusion (RV-IR) causes RV diastolic dysfunction. IR-induced mitochondrial fission causes RV diastolic dysfunction. In RV-IR Drp1 translocates to mitochondria, binds Fis1 and causes fission and injury. A baseline RV mitochondriopathy in MCT PAH resembles IR-induced mitochondrial injury. Drp1 inhibitors (Mdivi-1 and P110) preserve RV diastolic function post RV-IR.

Distribution and density of contacts from noradrenergic and serotonergic boutons on the dendrites of neck flexor motoneurons in the adult cat.

Serotonergic (5-HT) and noradrenergic (NA) input to spinal motoneurons is essential for generating plateau potentials and self-sustained discharges. Extensor motoneurons are densely innervated by 5-HT and NA synapses and have robust plateau potentials and self-sustained discharges. Conversely, plateau potentials and self-sustained discharges are very rare in flexor motoneurons. The most likely reasons for this difference are that flexor motoneurons have few 5-HT and NA synapses and/or they are distributed distant to the channels responsible for plateau potentials and self-sustained discharges. However, the distribution of 5-HT and NA synapses on flexor motoneurons is unknown. Here we describe the distribution and density of 5-HT and NA synapses on motoneurons that innervate the flexor neck muscle, rectus capitis anterior (RCA), in the adult cat. Using a combination of intracellular staining, fluorescent immunohistochemistry, and 3D reconstruction techniques, we found that 5-HT and NA synapses are widely distributed throughout the dendritic trees of RCA motoneurons, albeit with a strong bias to small-diameter dendrites and to medial dendrites in the case of NA contacts. The number of 5-HT and NA contacts per motoneuron ranged, respectively, from 381 to 1,430 and from 642 to 1,382, which is 2.3- and 1.4-fold less than neck extensor motoneurons (Montague et al., J Comp Neurol 2013;521:638-656). These results suggest that 5-HT and NA synapses on flexor motoneurons may provide a powerful means of amplifying synaptic currents without incurring plateau potentials or self-sustained discharges. This feature is well suited to meet the biomechanical demands imposed on flexor muscles during different motor tasks.

Nonuniform distribution of contacts from noradrenergic and serotonergic boutons on the dendrites of cat splenius motoneurons.

The input-output properties of motoneurons are dynamically regulated. This regulation depends, in part, on the relative location of excitatory and inhibitory synapses, voltage-dependent and -independent channels, and neuromodulatory synapses on the dendritic tree. The goal of the present study was to quantify the number and distribution of synapses from two powerful neuromodulatory systems that originate from noradrenergic (NA) and serotonergic (5-HT) neurons. Here we show that the dendritic trees of motoneurons innervating a dorsal neck extensor muscle, splenius, in the adult cat are densely, but not uniformly innervated by both NA and 5-HT boutons. Identified splenius motoneurons were intracellularly stained with Neurobiotin. Using 3D reconstruction techniques we mapped the distributions of contacts formed by NA and 5-HT boutons on the reconstructed dendritic trees of these motoneurons. Splenius motoneurons received an average of 1,230 NA contacts (range = 647-1,507) and 1,582 5-HT contacts (range = 1,234-2,143). The densities of these contacts were 10 (NA) to 6 (5-HT)-fold higher on small compared to large-diameter dendrites. This relationship largely accounts for the bias of NA and 5-HT contacts on distal dendrites and is partially responsible for the higher density of NA contacts on dendrites located more than 200 µm dorsal to the soma. These results suggest that the neuromodulatory actions of NA and 5-HT are compartmentalized and regulate the input-output properties of motoneurons according to precisely arranged interactions with voltage-dependent and -independent channels that are primarily located on small-diameter dendrites.

Dendrite-derived supernumerary axons on adult axotomized motor neurons possess proteins that are essential for the initiation and propagation of action potentials and synaptic vesicle release.

Axotomy can trigger profound alterations in the neuronal polarity of adult neurons in vivo. This can manifest itself in the development of new axon-like processes emanating from the tips of distal dendrites. Previously, these processes have been defined as axonal based on their axonal morphology. This study extends this definition to determine whether, more importantly, these processes possess the prerequisite molecular machinery to function as axons. Using a combination of intracellular labeling and immunohistochemistry, we demonstrate that the distribution of voltage-gated sodium channels on these processes matches the arrangement of these channels that is necessary for the initiation and conduction of action potentials. At terminal bouton-like structures they possess key proteins necessary for the release of synaptic vesicles (SV2 and synaptophysin). Thus, axon-like processes emanating from the tips of distal dendrites represent a rearrangement of neuronal polarity whereby axotomized neurons can develop additional functional axons in vivo.

Axonal regeneration and development of de novo axons from distal dendrites of adult feline commissural interneurons after a proximal axotomy.

Following proximal axotomy, several types of neurons sprout de novo axons from distal dendrites. These processes may represent a means of forming new circuits following spinal cord injury. However, it is not know whether mammalian spinal interneurons, axotomized as a result of a spinal cord injury, develop de novo axons. Our goal was to determine whether spinal commissural interneurons (CINs), axotomized by 3-4-mm midsagittal transection at C3, form de novo axons from distal dendrites. All experiments were performed on adult cats. CINs in C3 were stained with extracellular injections of Neurobiotin at 4-5 weeks post injury. The somata of axotomized CINs were identified by the presence of immunoreactivity for the axonal growth-associated protein-43 (GAP-43). Nearly half of the CINs had de novo axons that emerged from distal dendrites. These axons lacked immunoreactivity for the dendritic protein, microtubule-associated protein2a/b (MAP2a/b); some had GAP-43-immunoreactive terminals; and nearly all had morphological features typical of axons. Dendrites of other CINs did not give rise to de novo axons. These CINs did, however, each have a long axon-like process (L-ALP) that projected directly from the soma or a very proximal dendrite. L-ALPs were devoid of MAP2a/b immunoreactivity. Some of these L-ALPs projected through the lesion and formed bouton-like swellings. These results suggest that proximally axotomized spinal interneurons have the potential to form new connections via de novo axons that emerge from distal dendrites. Others may be capable of regeneration of their original axon.

Distribution of contacts from vestibulospinal axons on the dendrites of splenius motoneurons.

Current descriptions of the organization of synapses on the dendritic trees of spinal motoneurons indicate that the inputs are arranged in several patterns: some are widely distributed; some are distributed to proximal dendrites; others are distributed based on the trajectory of the dendrites. However, the principles governing the organization of synapses on spinal motoneurons remain poorly defined. Our goal was to extend the descriptions of the distribution of synapses, identified by their source, on the dendritic trees of spinal motoneurons. We combined anterograde and intracellular staining techniques in cats to determine the distribution of contacts between excitatory axons from the rostral aspect of the descending vestibular nucleus and the dendrites of motoneurons supplying a dorsal neck muscle, splenius. In five of five motoneurons, the contacts were preferentially distributed on dendrites medial to the soma. This qualitative observation was confirmed by using Monte Carlo methods. The results from this analysis showed that the distribution of contacts can be explained not by the overall distribution of the dendritic membrane area but rather by a systematic innervation of the medial regions of the dendritic trees (P < 0.02). Despite this selectivity, there was no additional bias in the distribution of contacts to proximal vs. distal dendrites. By concentrating excitatory synapses in a restricted region of the dendritic tree, the actions of vestibulospinal connections on neck motoneurons may be increased as a result of a greater probability of activating persistent inward currents on the dendrites.

The temporal sequence of morphological and molecular changes in axotomized feline motoneurons leading to the formation of axons from the ends of dendrites.

At 8-12 weeks post axotomy, unusual distal processes (UDPs) with axon-like structural (uniform diameter, tortuous) and molecular (growth-associated protein [GAP]43, absence of microtubule-associated protein [MAP]2a/b immunoreactivity) features emerge from distal motoneuron dendrites (Rose et al. [2001] Eur J Neurosci 13:1166-1176). In this study, we determine the time course of molecular and morphological changes associated with the formation of axons from dendrites. Motoneurons innervating neck muscles in the adult cat were permanently axotomized for 2, 4, 20, or 35 weeks and intracellularly stained with Neurobiotin. Computer-assisted reconstructions were used to map the location of MAP2a/b and GAP-43 immunoreactivity. At 2 and 4 weeks post axotomy, all UDPs had short appendages, giving them an arboreal appearance. They were immunoreactive for GAP-43 and lacked immunostaining for MAP2a/b. Axon-like UDPs were not seen until 8-12 weeks post axotomy. By 20 and 35 weeks post axotomy, some axon-like UDPs acquired morphological features of axons with synaptic connections (right-angled branching, bouton-like specializations). GAP-43 immunoreactivity was not detected in any axotomized motoneurons by 20 weeks post axotomy, whereas all UDPs remained devoid of MAP2a/b immunoreactivity even at 35 weeks post axotomy. These molecular changes accompanied structural modifications to proximal regions of "dendrites" giving rise to UDPs. The distance from the ends of the UDPs to the soma did not change. Thus, all UDPs begin as simple, arboreal structures with molecular features of growing axons, but over a period of 35 weeks, some UDPs slowly acquire morphological and molecular features of motoneuron axons with synaptic connections. These results suggest a new modus operandi for axonal growth and the establishment of new synaptic connections after injury.

Alterations to neuronal polarity following permanent axotomy: a quantitative analysis of changes to MAP2a/b and GAP-43 distributions in axotomized motoneurons in the adult cat.

Following axotomy, morphologically unusual, distal processes (UDPs) emerge from motoneuron dendrites. These processes contain an axonal protein, growth-associated protein 43 (GAP-43) but lack immunostaining for the dendritic protein microtubule-associated protein 2a/b (MAP2a/b). Thus, it appears that neuronal polarity alters following axotomy. Our goal was to describe this change in neuronal polarity on a more detailed and quantitative level. We asked two questions: Following axotomy, where in the entire neuron does the immunoreactivity for MAP2a/b and GAP-43 change and do these changes reflect a transformation of dendrite to axon or growth from terminal dendrites? Using intracellular labeling and immunocytochemistry, changes in MAP2a/b and GAP-43 immunoreactivity were also found in processes with a morphology typical of terminal branches of intact motoneurons (called simple distal processes [SDPs]), as well as UDPs. Trajectories (the path from the soma to a single terminus) with UDPs and SDPs were longer than trajectories without these processes, and trajectories with UDPs were the longest. Trajectories without UDPs or SDPs were similar in length to trajectories from intact motoneurons. The distance from the soma to the point where MAP2a/b immunoreactivity became absent in trajectories with UDPs or SDPs was similar to the length of trajectories from intact motoneurons. Thus, following axotomy, two morphologically distinct types of axon-like processes emerge from dendrites. The formation of these processes does not involve a transformation of the original dendrite, but rather growth at the ends of dendrites.