Intact Rodent Research Articles

Mapping the blood vasculature in an intact human kidney using hierarchical phase-contrast tomography.

The architecture of the kidney vasculature is essential for its function. Although structural profiling of the intact rodent kidney vasculature has been performed, it is challenging to map vascular architecture of larger human organs. We hypothesised that hierarchical phase-contrast tomography (HiP-CT) would enable quantitative analysis of the entire human kidney vasculature. Combining label-free HiP-CT imaging of an intact kidney from a 63-year-old male with topology network analysis, we quantitated vasculature architecture in the human kidney down to the scale of arterioles. Although human and rat kidney vascular topologies are comparable, vascular radius decreases at a significantly faster rate in humans as vessels branch from artery towards the cortex. At branching points of large vessels, radii are theoretically optimised to minimise flow resistance, an observation not found for smaller arterioles. Structural differences in the vasculature were found in different spatial zones of the kidney reflecting their unique functional roles. Overall, this represents the first time the entire arterial vasculature of a human kidney has been mapped providing essential inputs for computational models of kidney vascular flow and synthetic vascular architectures, with implications for understanding how the structure of individual blood vessels collectively scales to facilitate organ function.

Immunofluorescent Verification of Silencing Estrogen Receptor α with siRNA in the Intact Rodent Brain.

The ability to silence the expression of gene products in a chemically, spatially, and temporally specific manner in the brains of animals has enabled key breakthroughs in the field of behavioral neuroscience. Using this technique, estrogen receptor alpha (ERα) has been specifically implicated in a multitude of behaviors in mice, including sexual, aggressive, locomotor, and maternal behaviors, in a variety of brain regions, including the medial preoptic area, ventromedial hypothalamus, and amygdala. In this chapter, we describe the techniques involved in the generation of the small hairpin RNAs (shRNAs) specifically designed to silence ERα, the construction of the adeno-associated viral (AAV) vector for delivery of the shRNA, the procedures to confirm the silencing of ERα (in vitro and in vivo) and in vivo delivery of the shRNAs to the brains of animals.

InVision: An optimized tissue clearing approach for three-dimensional imaging and analysis of intact rodent eyes

SummaryThe mouse eye is used to model central nervous system development, pathology, angiogenesis, tumorigenesis, and regenerative therapies. To facilitate the analysis of these processes, we developed an optimized tissue clearing and depigmentation protocol, termed InVision, that permits whole-eye fluorescent marker tissue imaging. We validated this method for the analysis of normal and degenerative retinal architecture, transgenic fluorescent reporter expression, immunostaining and three-dimensional volumetric (3DV) analysis of retinoblastoma and angiogenesis. We also used this method to characterize material transfer (MT), a recently described phenomenon of horizontal protein exchange that occurs between transplanted and recipient photoreceptors. 3D spatial distribution analysis of MT in transplanted retinas suggests that MT of cytoplasmic GFP between photoreceptors is mediated by short-range, proximity-dependent cellular interactions. The InVision protocol will allow investigators working across multiple cell biological disciplines to generate novel insights into the local cellular networks involved in cell biological processes in the eye.

Engineering an endothelialized, endocrine Neo-Pancreas: Evaluation of islet functionality in an ex vivo model

Islet-based recellularization of decellularized, repurposed rat livers may form a transplantable Neo-Pancreas. The aim of this study is the establishment of the necessary protocols, the evaluation of the organ structure and the analysis of the islet functionality ex vivo.After perfusion-based decellularization of rat livers, matrices were repopulated with endothelial cells and mesenchymal stromal cells, incubated for 8 days in a perfusion chamber, and finally repopulated on day 9 with intact rodent islets. Integrity and quality of re-endothelialization was assessed by histology and FITC-dextran perfusion assay. Functionality of the islets of Langerhans was determined on day 10 and day 12 via glucose stimulated insulin secretion.Blood gas analysis variables confirmed the stability of the perfusion cultivation. Histological staining showed that cells formed a monolayer inside the intact vascular structure. These findings were confirmed by electron microscopy. Islets infused via the bile duct could histologically be found in the parenchymal space. Adequate insulin secretion after glucose stimulation after 1-day and 3-day cultivation verified islet viability and functionality after the repopulation process.We provide the first proof-of-concept for the functionality of islets of Langerhans engrafted in a decellularized rat liver. Furthermore, a re-endothelialization step was implemented to provide implantability. This technique can serve as a bioengineered platform to generate implantable and functional endocrine Neo-Pancreases.

A phase Ib/II study of olutasidenib in patients with relapsed/refractory IDH1 mutant gliomas: Safety and efficacy as single agent and in combination with azacitidine.

2505 Background: Isocitrate dehydrogenase 1 mutations (mIDH1) are present in >70% of patients with Grade II/III gliomas resulting in production and accumulation of (R)-2-hydroxyglutarate causing DNA hypermethylation and promoting tumorigenesis. Olutasidenib is an oral, potent, brain penetrant (Kpuu=0.4 in intact rodent), and selective inhibitor of mutated IDH1 protein. Methods: Patients (pts) with relapsed/refractory (R/R) mIDH1 gliomas received olutasidenib 150 mg BID, orally either as single agent (SA) or in combination (CO) with azacitidine in a dose confirmation phase Ib followed by efficacy evaluation phase II study (NCT: 03684811). Results: As of 31-Oct-2019, 29 pts with R/R mIDH1 glioma were treated with olutasidenib as SA (n=24) or CO (n=5). The median age was 45 yrs (range: 23-64) & 62% were male. WHO Glioma Grade (Gr) at study entry was: II (17%), III (52%) & IV (31%). Median number of prior treatments was 2 (1-5); 86% had received prior temozolomide. mIDH1 status was locally determined (IHC, NGS or PCR): R132H (86%), R132L (7%), R132C (3.5%) & unspecified (3.5%). The median duration of olutasidenib treatment for SA & CO was 4.8 (1-11.4) & 1 (0.2-2.3) months, respectively. Fifteen pts discontinued (disease progression [n=12], AE [n=1], withdrew consent [n=1], other [n=1]). For SA, the most common (>25%) TEAEs (all grades, regardless of attribution) were: fatigue (50%), nausea (50%), diarrhea (33%), ALT increase (29%) & headache (29%). For CO, TEAEs that occurred in ≥ 2 pts were: nausea (n=4), fatigue (n=2), neutropenia (n=2), ALT increase (n=2) & AST increase (n=2). There were 2 protocol defined DLTs in the CO cohort, 1 pt with Gr 4 ALT, Gr 3 AST & Gr 3 GGT elevations & 1 pt with Gr 3 ALT elevation. No pts experienced a TEAE of QTcF prolongation. SA best responses are shown in Table; CO pts are too early for response assessment. The median PFS for SA was 8.3 months. Twenty (87%) and 11 (48%) pts were alive and progression-free at 6 & 12 months, respectively. Conclusions: SA olutasidenib at 150 mg BID demonstrates acceptable safety and tolerability with preliminary clinical activity in glioma pts. Evaluation of CO is ongoing. Updated safety and clinical activity, as well as evaluations of serum/CSF PK/PD will be provided. Clinical trial information: NCT03684811 . [Table: see text]

Perivascular spaces in the brain: anatomy, physiology and pathology.

Perivascular spaces include a variety of passageways around arterioles, capillaries and venules in the brain, along which a range of substances can move. Although perivascular spaces were first identified over 150 years ago, they have come to prominence recently owing to advances in knowledge of their roles in clearance of interstitial fluid and waste from the brain, particularly during sleep, and in the pathogenesis of small vessel disease, Alzheimer disease and other neurodegenerative and inflammatory disorders. Experimental advances have facilitated in vivo studies of perivascular space function in intact rodent models during wakefulness and sleep, and MRI in humans has enabled perivascular space morphology to be related to cognitive function, vascular risk factors, vascular and neurodegenerative brain lesions, sleep patterns and cerebral haemodynamics. Many questions about perivascular spaces remain, but what is now clear is that normal perivascular space function is important for maintaining brain health. Here, we review perivascular space anatomy, physiology and pathology, particularly as seen with MRI in humans, and consider translation from models to humans to highlight knowns, unknowns, controversies and clinical relevance.

ACTR-52. PHASE 1 STUDY OF FT-2102, AN INHIBITOR OF MUTANT IDH1, IN PATIENTS WITH RELAPSED/REFRACTORY IDH1 MUTANT GLIOMAS: PRELIMINARY SAFETY AND CLINICAL ACTIVITY

Abstract BACKGROUND Isocitrate dehydrogenase mutations (mIDH1) are present in > 70% of patients with Grade II/III gliomas resulting in production and accumulation of (R)-2-hydroxyglutarate causing DNA hypermethylation and promoting tumorigenesis. FT-2102 is a potent, brain penetrant (Kpuu=0.4 in intact rodent) and selective inhibitor of mIDH1. METHODS Patients with advanced relapsed/refractory mIDH1 gliomas received FT-2102 150mg BID, orally. Following a dose confirmation 3 + 3 Phase 1b, 20 pts enrolled in a Simon 2 stage Phase 2 study (NCT: 03684811). RESULTS As of 01-May-2019, 23 with glioma (Grade at enrollment: II/III/IV; n=4, n=12 & n=7 respectively) were treated with FT-2102 monotherapy. The patient’s median age was 46 years (range: 23–64) & 61% were male. Median number of prior treatments was 2 (range 1–5) and 78% had received prior temozolomide. mIDH1 status was locally determined (IHC, NGS or PCR): R132H, R132L, R132C & unspecified (n=15, n=2, n=1 & n=5). Median duration of FT-2102 treatment to date was 40 days (range: 26–177), with 1 patient discontinuing (disease progression). Treatment emergent adverse events (all grades, regardless of attribution) that occurred in >10% of were: fatigue (22%), nausea (17%), diarrhea (17%), ALT increase (13%), headache (13%) and AST increase (13%), none leading to treatment discontinuation. None experienced an adverse event of QTcF prolongation or skin hyperpigmentation. There were no protocol-defined DLT’s. To date, six had at least 1 evaluable post-baseline response assessment, with best response of 1 PR (unconfirmed as of data cutoff), 3 SD and 2 PD. Responses of patients remaining on FT-2102 will be updated as available. Evaluations of serum/CSF pharmacokinetics and selected pharmacodynamics will be provided. CONCLUSION FT-2102 at 150 mg BID demonstrates acceptable safety and tolerability with potential clinical activity in with gliomas. The Phase 2 study is ongoing, evaluating both single agent, FT-2102 and in combination with azacitidine.

A newly developed anesthetic based on a unique chemical core

Intravenous anesthetic agents are associated with cardiovascular instability and poorly tolerated in patients with cardiovascular disease, trauma, or acute systemic illness. We hypothesized that a new class of intravenous (IV) anesthetic molecules that is highly selective for the slow type of γ-aminobutyric acid type A receptor (GABAAR) could have potent anesthetic efficacy with limited cardiovascular effects. Through in silico screening using our GABAAR model, we identified a class of lead compounds that are N-arylpyrrole derivatives. Electrophysiological analyses using both an in vitro expression system and intact rodent hippocampal brain slice recordings demonstrate a GABAAR-mediated mechanism. In vivo experiments also demonstrate overt anesthetic activity in both tadpoles and rats with a potency slightly greater than that of propofol. Unlike the clinically approved GABAergic anesthetic etomidate, the chemical structure of our N-arylpyrrole derivative is devoid of the chemical moieties producing adrenal suppression. Our class of compounds also shows minimal to no suppression of blood pressure, in marked contrast to the hemodynamic effects of propofol. These compounds are derived from chemical structures not previously associated with anesthesia and demonstrate that selective targeting of GABAAR-slow subtypes may eliminate the hemodynamic side effects associated with conventional IV anesthetics.

Male rats treated with subchronic PCP show intact olfaction and enhanced interest for a social odour in the olfactory habituation/dishabituation test

The olfactory system participates in many sensory processes, and olfactory endophenotypes appear in a variety of neurological disorders such as Alzheimer’s and Parkinson’s disease, depression and schizophrenia. Social withdrawal is a core negative symptom of schizophrenia and animal models have proven to be invaluable for studying the neurobiological mechanisms and cognitive processes behind the formation of social relationships. The subchronic phencyclidine (PCP) rat model is a validated model for negative symptoms of schizophrenia, such as impaired sociability. However, the complete range of social behaviour and deficits in the model are still not fully understood. Intact rodent olfaction is essential for a wide range of social behaviour and disrupted olfactory function could have severe effects on social communication and recognition. In order to examine the olfactory ability of male rats treated with subchronic PCP, we conducted an olfactory habituation/dishabituation test including both non-social and social odours. The subchronic PCP-treated rats successfully recognized and discriminated among the odours, indicative of intact olfaction. Interestingly, the subchronic PCP-treated rats showed greater interest for a novel social odour compared to the saline-treated rats and the rationale remains to be elucidated. Our data indicate that subchronic PCP treatment does not disrupt olfactory function in male rats. By ruling out impaired olfaction as cause for the poor social interaction performance in subchronic PCP-treated rats, our data supports the use of NMDA receptor antagonists to model the negative symptoms of schizophrenia.

Epigenetic Regulation of Glutamic Acid Decarboxylase 67 in a Hippocampal Circuit.

GABAergic dysfunction in hippocampus, a key feature of schizophrenia (SZ), may contribute to cognitive impairment in this disorder. In stratum oriens (SO) of sector CA3/2 of the human hippocampus, a network of genes involved in the regulation of glutamic acid decarboxylase GAD67 has been identified. Several of the genes in this network including epigenetic factors histone deacetylase 1 (HDAC1) and death-associated protein 6 (DAXX), the GABAergic enzyme GAD65 as well as the kainate receptor (KAR) subunits GluR6 and7 show significant changes in expression in this area in SZ. We have tested whether HDAC1 and DAXX regulate GAD67, GAD65, or GluR in the intact rodent hippocampus. Stereotaxic injections of lentiviral vectors bearing shRNAi sequences for HDAC1 and DAXX were delivered into the SO of CA3/2, followed by laser microdissection of individual transduced GABA neurons. Quantitative PCR (QPCR) analyses demonstrated that inhibition of HDAC1 and DAXX increased expression of GAD67, GAD65, and GluR6 mRNA. Inhibition of DAXX, but not HDAC1 resulted in a significant increase in GluR7 mRNA. Our data support the hypothesis that HDAC1 and DAXX play a central role in coordinating the expression of genes in the GAD67 regulatory pathway in the SO of CA3/2.

ATPS-61NEW SMALL MOLECULES THAT KILL HYPOXICALLY TRANSFORMED GLIOMA STEM-LIKE CELLS

High grade gliomas (HGGs) are lethal brain cancers, accounting for 78% of adult onset primary CNS malignancies. HGGs are resistant to current conventional therapies, including radiation therapy (RT), chemotherapy, and treatment with anti-angiogenic agents. Populations of cancer cells surviving these treatments regenerate leading to recurrence in more than 95% of patients. Spontaneous tumor necrosis within avascular tumor domains imparts poor clinical prognosis as glioma cells are reprogrammed by hypoxic transcription factors to adopt ‘stem-cell’ like properties; commonly referred to as glioma ‘stem-like’ cells (GSCs). GSCs possess several properties of normal stem cells, including self-aggregation, expression of stem cell markers, ability to survive in hypovascularized tumor regions, ability to survive detachment, and most importantly, the ability of tumor regrowth. GSCs can stop proliferating in hypoxic and acidic perinecrotic regions, which increases their resistance to RT and conventional anti-mitotic therapies. Hypoxically reprogrammed GSCs represent an important drug target to prevent the recurrence of HGGs. Drug targeting of hypovascularized GSCs is challenging as acidic and hypoxic tumor domains promote cell cycle arrest. Thus, therapeutic agents must be cytotoxic, not cytostatic, and need to kill independently of proliferative status. We developed novel small molecules that triggers ‘mis-trafficking’ of endosomes containing urokinase protein cargo in HGGs, and killing non-proliferating and proliferating glioma cells by an irreversible caspase-independent necrotic pathway. We identified that more than 90% of glioma cells within 22 patient derived xenografts (PDX) express intracellular PAI-1 and uPA and that 40-80% of these cells co-express the pluripotent stem cell transcription factors Oct4 and Nanog. Our lead small molecule crosses the intact rodent BBB and selectively kills glioma cells within intracerebral PDX glioma xenografts (PDX). The anti-glioma cytotoxicities of these small molecules is cell cycle independent, unaffected by hypoxia, and could represent an effective adjunctive therapy to be combined with radiotherapy, anti-mitotic, and/or anti-angiogenic therapies.

MicroRNA-208b progressively declines after spinal cord injury in humans and is inversely related to myostatin expression.

The effects of long-term physical inactivity on the expression of microRNAs involved in the regulation of skeletal muscle mass in humans are largely unknown. MicroRNAs are short, noncoding RNAs that fine-tune target expression through mRNA degradation or by inhibiting protein translation. Intronic to the slow, type I, muscle fiber type genes MYH7 and MYH7b, microRNA-208b and microRNA-499-5p are thought to fine-tune the expression of genes important for muscle growth, such as myostatin. Spinal cord injured humans are characterized by both skeletal muscle atrophy and transformation toward fast-twitch, type II fibers. We determined the expression of microRNA-208b, microRNA-499-5p, and myostatin in human skeletal muscle after complete cervical spinal cord injury. We also determined whether these microRNAs altered myostatin expression in rodent skeletal muscle. A progressive decline in skeletal muscle microRNA-208b and microRNA-499-5p expression occurred in humans during the first year after spinal cord injury and with long-standing spinal cord injury. Expression of myostatin was inversely correlated with microRNA-208b and microRNA-499-5p in human skeletal muscle after spinal cord injury. Overexpression of microRNA-208b in intact mouse skeletal muscle decreased myostatin expression, whereas microRNA-499-5p was without effect. In conclusion, we provide evidence for an inverse relationship between expression of microRNA-208b and its previously validated target myostatin in humans with severe skeletal muscle atrophy. Moreover, we provide direct evidence that microRNA-208b overexpression decreases myostatin gene expression in intact rodent muscle. Our results implicate that microRNA-208b modulates myostatin expression and this may play a role in the regulation of skeletal muscle mass following spinal cord injury.

Localization of Metal Electrodes in the Intact Rat Brain Using Registration of 3D Microcomputed Tomography Images to a Magnetic Resonance Histology Atlas.

Simultaneous neural recordings taken from multiple areas of the rodent brain are garnering growing interest because of the insight they can provide about spatially distributed neural circuitry. The promise of such recordings has inspired great progress in methods for surgically implanting large numbers of metal electrodes into intact rodent brains. However, methods for localizing the precise location of these electrodes have remained severely lacking. Traditional histological techniques that require slicing and staining of physical brain tissue are cumbersome and become increasingly impractical as the number of implanted electrodes increases. Here we solve these problems by describing a method that registers 3D computed tomography (CT) images of intact rat brains implanted with metal electrode bundles to a magnetic resonance imaging histology (MRH) atlas. Our method allows accurate visualization of each electrode bundle’s trajectory and location without removing the electrodes from the brain or surgically implanting external markers. In addition, unlike physical brain slices, once the 3D images of the electrode bundles and the MRH atlas are registered, it is possible to verify electrode placements from many angles by “reslicing” the images along different planes of view. Furthermore, our method can be fully automated and easily scaled to applications with large numbers of specimens. Our digital imaging approach to efficiently localizing metal electrodes offers a substantial addition to currently available methods, which, in turn, may help accelerate the rate at which insights are gleaned from rodent network neuroscience.

Ventromedial hypothalamic lesions change the expression of cell proliferation-related genes and morphology-related genes in rat pancreatic islets

Studies in normal rats and ob/ob mice indicated that islet neogenesis does not occur in the intact rodent pancreas. We previously reported that ventromedial hypothalamic (VMH) lesions stimulated cell proliferation of rat pancreatic islet B and acinar cells primarily through a cholinergic receptor mechanism and examined how gene families involved in cell proliferation in total pancreatic tissue are regulated after VMH lesions formation. This study examined how gene families involved in cell proliferation in pancreatic islets alone are regulated after VMH lesions formation. Pancreatic islet RNA was extracted, and differences in gene expression profiles between rats at day 3 after VMH lesioning and sham-VMH-lesioned rats were investigated using DNA microarray and real-time polymerase chain reaction. VMH lesions regulated genes that were involved in functions related to cell cycle and differentiation, growth, binding, apoptosis and morphology in pancreas islets. Real-time polymerase chain reaction also confirmed that gene expression of polo-like kinase 1 (Plk1) and topoisomerase (DNA) II α 170 kDa (Top2a), and stanniocalcin 1 (Stc1) were upregulated at day 3 after the VMH lesions. Ventromedial hypothalamic lesions may change the expression of cell proliferation-related genes and morphology-related genes in rat pancreatic islets.

Feasibility study of hidden flow imaging based on laser speckle technique using multiperspectives contrast images

This paper demonstrates the insertion of lens array in the front of a CCD camera in a laser speckle imaging (LSI) like-technique to acquire multiple speckle reflectance projections for imaging blood flow in an intact biological tissue. In some of LSI applications, flow imaging is obtained by thinning or removing of the upper tissue layers to access blood vessels. In contrast, with the proposed approach flow imaging can be achieved while the tissue is intact. In the system, each lens from an hexagonal lens array observed the sample from slightly different perspectives and captured with a CCD camera. In the computer, these multiview raw images are converted to speckled contrast maps. Then, a self-deconvolution shift-and-add algorithm is employed for processing yields high contrast flow information. The method is experimentally validated first with a plastic tube filled with scattering liquid running at different controlled flow rates hidden in a biological tissue and then extensively tested for imaging of cerebral blood flow in an intact rodent head experience different conditions. A total of fifteen mice were used in the experiments divided randomly into three groups as follows: Group 1 (n=5) consisted of injured mice experience hypoxic ischemic brain injury monitored for ~40min. Group 2 (n=5) injured mice experience anoxic brain injury monitored up to 20min. Group 3 (n=5) experience functional activation monitored up to ~35min. To increase tissue transparency and the penetration depth of photons through head tissue layers, an optical clearing method was employed. To our knowledge, this work presents for the first time the use of lens array in LSI scheme.

In vivo identification of sentinel lymph nodes using MRI and size‐controlled and monodispersed magnetite nanoparticles

To develop a sentinel lymph node (SN) identification method using accurately synthesized magnetic nanoparticles (MNPs), as an enhanced specific SN tracer in combination with magnetic resonance imaging (MRI) in intact rodent and SN metastasis models. Three sizes of MNPs were originally synthesized. We developed an experimental rat SN model, with brachial lymph nodes (Br) as the SN and the axillary lymph node (Ax) as the second lymph node, and injection of MNPs via the front paw. SN detectability was evaluated in vivo using T1 -weighted MR images after injection of the synthesized MNPs, and the amount of iron in the Br and in the Ax was assessed using inductively coupled plasma optical emission spectrometry. The highest ratios of the amount of iron in the Br versus the Ax were 3.1 and 3.3, using 20-nm MNPs after 2- and 24-hour injections. The appropriate dose and particle diameter for MRI detection was optimized, and the SN was optimally distinguished in the normal and metastatic rat models using MRI after a 0.4 mg/kg 20-nm MNP injection. We developed and optimized a useful SN identification method using MRI in rodent models.

The effects of body temperature and mass on the postprandial metabolic responses of the African egg-eating snakes Dasypeltis scabra and Dasypeltis inornata

African egg-eating snakes (Dasypeltis) feed only on freshly laid bird eggs which they perforate within their esophagus before swallowing the liquid contents and regurgitating the empty shell. Compared to a snake’s typical intact meal, the liquid diet of Dasypeltis would expectedly generate a more moderate postprandial metabolic response and specific dynamic action (SDA). Free-ranging Dasypeltis feed over a range of ambient temperatures and thereby experience predicted temperature-dependent shifts in the duration and magnitude of their postprandial metabolic response. Such shifts would undoubtedly be shared among different species and age classes of Dasypeltis. To examine these expectations, we measured pre- and postprandial metabolic rates of adult Dasypeltis inornata and adult and neonate Dasypeltis scabra in response to liquid egg meals weighing 20% of snake body mass at 20, 25, 27, 30, and 32°C. With an increase in body temperature, postprandial metabolic profiles of neonate and adult snakes became narrower and shorter in duration. Specific dynamic action varied among temperature treatments, increasing from 20 to 32°C. Standard metabolic rate, postprandial peak metabolic rate, and SDA scaled with mass exponents that typically did not differ from 1.0. As expected, Dasypeltis digesting a liquid egg diet experienced a more modest postprandial response and SDA, expending on average only 10.6% of the meal’s energy on the breakdown, absorption, and assimilation of the egg meal, whereas other colubrids consuming intact rodent or fish meals expend on average 16.3% of the meal’s energy on digestion and assimilation. Actively foraging and feeding throughout the avian egg laying season enable Dasypeltis to survive when eggs are not available. The adaptive suite of traits that enable Dasypeltis to consume eggs of large relative size and ingest only the liquid contents may also be joined by physiological adaptations specific to their liquid diet and extended bouts of fasting.

Abstract P285: Impaired Recovery of Left Ventricular Function After Acute Myocardial Reperfusion Injury Is Rescued by the Anti-inflammatory Peptide Annexin-A1 ex Vivo

The glucocorticoid-regulated protein annexin-A1 (ANX-A1) is a key effector molecule of anti-inflammatory glucocorticoid actions. We have shown that the N-terminal derived peptide ANX-A1(2–26) preserves cardiomyocyte viability after metabolic inhibition in vitro. Our hypothesis was that ANX-A1 preserves the myocardium post-ischemic injury. ANX-A1(2–26)(0.3μM) prevented adult rat cardiomyocyte injury, whether present for hypoxia-reoxygenation (LDH release reduced from 4.1±0.7- to 1.1±0.2-fold n=11 P<0.001) or only on reoxygenation (LDH release reduced to 1.4±0.4-fold n=11, P<0.001). Similar protection at both time points was also evident on cardiomyocyte viability (trypan blue exclusion). The recovery of post-ischemic left ventricular (LV) function in intact rodent hearts was also protected. After 20mins reperfusion, the recovery of LV developed pressure (LVDP) remained significantly impaired in untreated rat hearts (47±5% baseline, n=11) whereas addition of ANX-A1(2–26)(0.3μM) on reperfusion improved LVDP recovery (to 66±7% baseline, n=13, p<0.05). Phosphorylation of both Akt (1.9±0.2-fold, P<0.05) and phospholamban (3.4±0.4-fold, P<0.05) was also enhanced by ANX-A1(2–26). Similar ANX-A1(2–26)-induced rescue of LV function was observed in mouse hearts after acute myocardial reperfusion injury (57±7% of baseline, n=11 untreated hearts vs 78±8% baseline, n=6 ANX-A1(2–26)-treated hearts, p<0.05). ANX-A1(2–26) cardioprotection was associated with attenuated cardiac enzyme release (LDH, CK) and Akt phosphorylation (3.1±0.8-fold, P<0.005). In contrast, deficiency of endogenous ANX-A1 further exacerbated recovery of post-ischemic LV function, across LVDP (to 29±4% baseline, P<0.001), LV end-diastolic pressure (to 5.5±1.0-fold baseline, P<0.001), LV+dP/dt (to 33±4% baseline, P<0.001), LV-dP/dt (to 36±4% baseline, P<0.001) and rate-pressure product (to 24±4% baseline, P<0.001). This is the first evidence that ANX-A1 or its mimetic ANX-A1(2-26) preserve post-ischemic recovery of LV function. ANX-A1 is thus an endogenous regulator of LV function. Furthermore, ANX-A1-based therapies may thus represent a novel clinical approach for the prevention and treatment of reperfusion injury.

GSK3β negatively regulates oligodendrocyte differentiation and myelination in vivo

Glycogen synthase kinase 3β (GSK3β) is an essential integrating molecule for multiple proliferation and differentiation signals that regulate cell fate. Here, we have examined the effects of inhibiting GSK3β on the development of oligodendrocytes (OLs) from their oligodendrocyte precursors (OP) in vivo by injection into the lateral ventricle of postnatal mice and ex vivo in organotypic cultures of isolated intact rodent optic nerve. Our results show that a range of GSK3β inhibitors (ARA-014418, lithium, indirubin, and L803-mt) increase OPs and OLs and promote myelination. Inhibition of GSK3β stimulates OP proliferation and is prosurvival and antiapoptotic. The effects of GSK3β inhibition in OPs is via the canonical Wnt signaling pathway by stimulating nuclear translocation of β-catenin. However, direct comparison of the effects of Wnt3a and GSK3β inhibition in optic nerves shows that they have opposing actions on OLs, whereby GSK3β inhibition strikingly increases OL differentiation, whereas Wnt3a inhibits OL differentiation. Notably, GSK3β inhibition overrides the negative effects of Wnt3a on OLs, indicating novel GSK3β signaling mechanisms that negatively regulate OL differentiation. We identify that two mechanisms of GSK3β inhibition are to stimulate cAMP response element binding (CREB) and decrease Notch1 signaling, which positively and negatively regulate OL differentiation and myelination, respectively. A key finding is that GSK3β inhibition has equivalent effects in the adult and stimulates the regeneration of OLs and remyelination following chemically induced demyelination. This study identifies GSK3β as a profound negative regulator of OL differentiation that contributes to inefficient regeneration of OLs and myelin repair in demyelination.

Partial Disinhibition Is Required for Transition of Stimulus-Induced Sharp Wave–Ripple Complexes Into Recurrent Epileptiform Discharges in Rat Hippocampal Slices

Sharp wave-ripple complexes (SPW-Rs) in the intact rodent hippocampus are characterized by slow field potential transients superimposed by close to 200-Hz ripple oscillations. Similar events have been recorded in hippocampal slices where SPW-Rs occur spontaneously or can be induced by repeated application of high-frequency stimulation, a standard protocol for induction of long-lasting long-term potentiation. Such stimulation is reminiscent of protocols used to induce kindling epilepsy and ripple oscillations may be predictive of the epileptogenic zone in temporal lobe epilepsy. In the present study, we investigated the relation between recurrent epileptiform discharges (REDs) and SPW-Rs by studying effects of partial removal of inhibition. In particular, we compared the effects of nicotine, low-dose bicuculline methiodide (BMI), and elevated extracellular potassium concentration ([K(+)](o)) on induced SPW-Rs. We show that nicotine dose-dependently transformed SPW-Rs into REDs. This transition was associated with reduced inhibitory conductance in CA3 pyramidal cells. Similar results were obtained from slices where the GABAergic conductance was reduced by application of low concentrations of BMI (1-2 μM). In contrast, sharp waves were diminished by phenobarbital. Elevating [K(+)](o) from 3 to 8.5 mM did not transform SPW-Rs into REDs but significantly increased their incidence and amplitude. Under these conditions, the equilibrium potential for inhibition was shifted in depolarizing direction, whereas inhibitory conductance was significantly increased. Interestingly, the propensity of elevated [K(+)](o) to induce seizure-like events was reduced in slices where SPW-Rs had been induced. In conclusion, recruitment of inhibitory cells during SPW-Rs may serve as a mechanism by which hyperexcitation and eventually seizure generation might be prevented.