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The artemin-GFRα3 signaling pathway has been implicated in various painful conditions including migraine, cold allodynia, hyperalgesia, inflammatory bone pain, and mouse knees contain GFRα3-immunoreactive nerve endings. We developed high affinity mouse (REGN1967) and human (REGN5069) GFRα3-blocking monoclonal antibodies and, following in vivo evaluations in mouse models of chronic joint pain (osteoarthritic-like and inflammatory), conducted a first-in-human phase 1 pharmacokinetics (PK) and safety trial of REGN5069 (NCT03645746) in healthy volunteers, and a phase 2 randomized placebo-controlled efficacy and safety trial of REGN5069 (NCT03956550) in patients with knee osteoarthritis (OA) pain. In three commonly used mouse models of chronic joint pain (destabilization of the medial meniscus, intra-articular monoiodoacetate, or Complete Freund's Adjuvant), REGN1967 and REGN5069 attenuated evoked behaviors including tactile allodynia and thermal hyperalgesia without discernably impacting joint pathology or inflammation, prompting us to further evaluate REGN5069 in humans. In the phase 1 study in healthy subjects, the safety profiles of single doses of REGN5069 up to 3000 mg (intravenous) or 600 mg (subcutaneous) were comparable to placebo; PK were consistent with a monoclonal antibody exhibiting target-mediated disposition. In the phase 2 study in patients with OA knee pain, two doses of REGN5069 (100 mg or 1000 mg intravenous every 4 weeks) for 8 weeks failed to achieve the 12-week primary and secondary efficacy endpoints relative to placebo. In addition to possible differences in GFRα3 biology between mice and humans, we highlight here differences in experimental parameters that could have contributed to a different profile of efficacy in mouse models versus human OA pain. Additional research is required to more fully evaluate any potential role of GFRα3 in human pain., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: All authors apart from Sylvie Rottey are employees or former employees of Regeneron Pharmaceuticals, Inc. Sylvie Rottey is an employee of Ghent University Hospital, Ghent, Belgium, which was contracted by Regeneron Pharmaceuticals, Inc. to conduct the study., (© 2023 Regeneron Pharamaceuticals, Inc.)

Background and Purpose: Stroke is a major cause of chronic neurological disability. There is considerable interest in understanding how acute transcriptome changes evolve into subacute and chronic patterns that facilitate or limit spontaneous recovery. Here we mapped longitudinal changes in gene expression at multiple time points after stroke in mice out to 6 months., Methods: Adult C57BL/6 mice were subjected to transient middle cerebral artery occlusion. Longitudinal transcriptome levels were measured at 10 time points after stroke from acute to recovery phases of ischemic stroke. Localization and the number of mononuclear phagocytes were determined in the postischemic brain. Whole-mount brain imaging was performed in asplenic mice receiving GFP + (green fluorescent protein)-tagged splenocytes., Results: Sustained stroke-induced mRNA abundance changes were observed in both hemispheres with 2989 ipsilateral and 822 contralateral genes significantly perturbed. In the hemisphere ipsilateral to the infarct, genes associated with immune functions were strongly affected, including temporally overlapping innate and adaptive immunity and macrophage M1 and M2 phenotype-related genes. The strong immune gene activation was accompanied by the sustained infiltration of peripheral immune cells at acute, subacute, and recovery stages of stroke. The infiltrated immune cells were found in the infarcted area but also in remote regions at 2 months after stroke., Conclusions: The study identifies that immune components are the predominant molecular signatures and they may propagate or continuously respond to brain injury in the subacute to chronic phase after central nervous system injury. The study suggests a potential immune-based strategy to modify injury progression and tissue remodeling in ischemic stroke, even months after the initiating event.

Administration of dextran sodium sulfate (DSS) to rodents at varying concentrations and exposure times is commonly used to model human inflammatory bowel disease (IBD). Currently, the criteria used to assess IBD-like pathology seldom include surrogate measures of visceral pain. Thus, we sought to standardize the model and then identify surrogate measures to assess effects on visceral pain. We used various 4% DSS protocols and evaluated effects on weight loss, colon pathology, biochemistry, RNA signature, and open field behavior. We then tested the therapeutic potential of NPY Y1 and/or Y2 receptor inhibition for the treatment of IBD pathology using this expanded panel of outcome measures. DSS caused weight loss and colon shrinkage, increased colon NPY and inflammatory cytokine expression, altered behaviors in the open field and induced a distinct gene metasignature that significantly overlapped with that of human IBD patients. Inhibition of Y1 and/or Y2 receptors failed to improve gross colon pathology. Y1 antagonism significantly attenuated colon inflammatory cytokine expression without altering pain-associated behaviors while Y2 antagonism significantly inhibited pain-associated behaviors in spite of a limited effect on inflammatory markers. A protocol using 7 days of 4% DSS most closely modeled human IBD pathology. In this model, rearing behavior potentially represents a tool for evaluating visceral pain/discomfort that may be pharmacologically dissociable from other features of pathology. The finding that two different NPY receptor antagonists exhibited different efficacy profiles highlights the benefit of including a variety of outcome measures in IBD efficacy studies to most fully evaluate the therapeutic potential of experimental treatments., Competing Interests: All work was funded by and conducted at Regeneron Pharmaceuticals where all authors were employed during the time that these experiments were conducted. This does not alter our adherence to PLOS ONE policies on sharing data and materials related to the work presented here.

Vascular endothelial growth factor (VEGF) treatment during pilocarpine-induced status epilepticus (SE) causes sustained preservation of behavioral function in rats in the absence of enduring neuroprotection (Nicoletti et al., 2010), suggesting the possibility that other cells or mechanisms could be involved in the beneficial effects of VEGF during SE. Astrocytes have been reported to contribute to epileptiform discharges in the hippocampus (Tian et al., 2005; Kang et al., 1998) and to express VEGF receptors (Krum & Rosenstein, 2002). We report here that VEGF treatment significantly alters multiple astrocyte parameters. This study investigated astrocyte morphology one month after SE in animals treated with VEGF or inactivated VEGF control protein during SE. Individual GFAP-immunostained astrocytes from CA1 and dentate gyrus hilus were traced and morphologically quantified, and both somatic and process structures were analyzed. VEGF treatment during SE significantly prevented post-SE increases in number of branch intersections, process length, and node count. Furthermore, analysis of distance to nearest neighboring astrocytes revealed that VEGF treatment significantly increased inter-astrocyte distance. Overall, VEGF treatment during SE did not significantly alter the shape of the astrocytes, but did prevent SE-induced changes in branching complexity, size, and spatial patterning. Because astrocyte morphology may be related to astrocyte function, it is possible that VEGF's enduring effects on astrocyte morphology may impact the functioning of the post-seizure hippocampus., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Pleiotrophin (PTN) is an extracellular matrix-associated protein with neurotrophic and neuroprotective effects that is involved in a variety of neurodevelopmental processes. Data regarding the cognitive-behavioral and neuroanatomical phenotype of pleiotrophin knockout (KO) mice is limited. The purpose of this study was to more fully characterize this phenotype, with emphasis on the domains of learning and memory, cognitive-behavioral flexibility, exploratory behavior and anxiety, social behavior, and the neuronal and vascular microstructure of the lateral entorhinal cortex (EC). PTN KOs exhibited cognitive rigidity, heightened anxiety, behavioral reticence in novel contexts and novel social interactions suggestive of neophobia, and lamina-specific decreases in neuronal area and increases in neuronal density in the lateral EC. Initial learning of spatial and other associative tasks, as well as vascular density in the lateral EC, was normal in the KOs. These data suggest that the absence of PTN in vivo is associated with disruption of specific cognitive and affective processes, raising the possibility that further study of PTN KOs might have implications for the study of human disorders with similar features.

Conditional mutagenesis is becoming a method of choice for studying gene function, but constructing conditional alleles is often laborious, limited by target gene structure, and at times, prone to incomplete conditional ablation. To address these issues, we developed a technology termed conditionals by inversion (COIN). Before activation, COINs contain an inverted module (COIN module) that lies inertly within the antisense strand of a resident gene. When inverted into the sense strand by a site-specific recombinase, the COIN module causes termination of the target gene's transcription and simultaneously provides a reporter for tracking this event. COIN modules can be inserted into natural introns (intronic COINs) or directly into coding exons as part of an artificial intron (exonic COINs), greatly simplifying allele design and increasing flexibility over previous conditional KO approaches. Detailed analysis of over 20 COIN alleles establishes the reliability of the method and its broad applicability to any gene, regardless of exon-intron structure. Our extensive testing provides rules that help ensure success of this approach and also explains why other currently available conditional approaches often fail to function optimally. Finally, the ability to split exons using the COIN's artificial intron opens up engineering modalities for the generation of multifunctional alleles., Competing Interests: All authors of this manuscript are employed by Regeneron Pharmaceuticals, Inc., and some of the authors own a substantial amount of Regeneron stock.

This study investigated hyperreligiosity in persons with partial epilepsy by exploring a relationship between aura symptoms and spirituality. It was reasoned that patients with high frequencies of auras that are suggestive of metaphysical phenomena, termed numinous-like auras, would report increased spirituality of an unconventional form, both during their seizures and generally. Numinous-like auras included: dreaminess/feeling of detachment, autoscopy, derealization, depersonalization, time speed alterations, bodily distortions, and pleasure. A high-frequency aura group, low-frequency aura group, and nonseizure reference group were compared on the Expressions of Spirituality-Revised. The High group had significantly greater Experiential/Phenomenological Dimension and Paranormal Beliefs factor scores than the Low group, and significantly greater Experiential/Phenomenological Dimension factor scores than the reference group. There were no differences between the Low group and the reference group. In addition, there were no differences among the three groups on traditional measures of religiosity. The results provide preliminary evidence that epilepsy patients with frequent numinous-like auras have greater ictal and interictal spirituality of an experiential, personalized, and atypical form, which may be distinct from traditional, culturally based religiosity. This form of spirituality may be better described by the term cosmic spirituality than hyperreligiosity. It is speculated that this spirituality is due to an overactivation and subsequent potentiation of the limbic system, with frequent numinous-like auras indicating sufficient activation for this process to occur. It is likely that numinous-like experiences foster cosmic spirituality in a number of circumstances, including seizures, psychosis, near-death experiences, psychedelic drug use, high-elevation exposure, and also normal conditions., (Wiley Periodicals, Inc. © 2011 International League Against Epilepsy.)

Vascular endothelial growth factor (VEGF) is a vascular growth factor more recently recognized as a neurotrophic factor (for review, see Storkebaum E, Lambrechts D, Carmeliet P. BioEssays 2004;26:943-54). We previously reported that endogenous VEGF protein is dramatically upregulated after pilocarpine-induced status epilepticus in the rat, and that intra-hippocampal infusions of recombinant human VEGF significantly protected against the loss of hippocampal CA1 neurons in this model (Nicoletti JN, Shah SK, McCloskey DP, et al. Neuroscience 2008;151:232-41). We hypothesized that we would see a preservation of cognitive and emotional functioning with VEGF treatment accompanying the neuroprotection previously observed in this paradigm. Using the Morris water maze to evaluate learning and memory, and the light-dark task to assess anxiety, we found a selective profile of preservation. Specifically, VEGF completely preserved normal anxiety functioning and partially but significantly protected learning and memory after status epilepticus. To determine whether the ability of VEGF to attenuate behavioral deficits was accompanied by sustained preservation of hippocampal neurons, we stereologically estimated CA1 pyramidal neuron densities 4 weeks after status epilepticus. At this time point, we found no significant difference in neuronal densities between VEGF- and control-treated status epilepticus animals, suggesting that VEGF could have protected hippocampal functioning independent of its neuroprotective effect., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: Monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD) crystal-induced interleukin 1 beta (IL1beta) release contributes to inflammation in subcutaneous air pouch and peritoneal models of acute gout and pseudogout. However, consequences of IL1 inhibition have not been explored in more clinically relevant models of crystal-induced arthritis., Objective: To develop a novel mouse model of acute gouty ankle arthritis and use it to assess the effects of genetic deletion of IL1 receptor type (IL1R1) and of exogenous mIL1 Trap (a high-affinity blocker of mouse IL1alpha and IL1beta) on pain, synovitis and systemic inflammatory biomarkers., Methods: MSU crystals were injected into the mouse ankle joint and pain and ankle swelling were measured over 4 days. The effects of IL1 inhibition were determined in this model, and in the comparator models of crystal-induced peritonitis and subcutaneous air pouch inflammation., Results: Both IL1R1-null mice and mice pretreated with mIL1 Trap showed reduced neutrophil influx in MSU and CPPD crystal-induced peritonitis and air pouch models (p<0.05). In the ankle joint model, both IL1R1 knockout mice and pretreatment with mIL1 Trap were associated with significant reductions in MSU crystal-induced elevations in hyperalgesia, inflammation, serum amyloid A and the levels of multiple inflammatory cytokines and chemokines (p<0.05). Additionally, it was found that administration of mIL1 Trap after MSU crystal injection reduced established hyperalgesia and ankle swelling., Conclusions: IL1 inhibition both prevented and relieved pain and ankle joint inflammation in response to intra-articular MSU crystals in mice. Results suggested that IL1 Trap has the potential to both prevent and treat gouty arthritis.

One challenge of neuroscience educators is to make accessible to students as many aspects of brain structure and function as possible. The anatomy and function of the cerebrovasculature is among many topics of neuroscience that are underrepresented in undergraduate neuroscience education. Recognizing this deficit, we evaluated methods to produce archival tissue specimens of the cerebrovasculature and the "neurovascular unit" for instruction and demonstration in the teaching lab. An additional goal of this project was to identify the costs of each method as well as to determine which method(s) could be adapted into lab exercises, where students participate in the tissue preparation, staining, etc. In the present report, we detail several methods for demonstrating the cerebrovasculature and suggest that including this material can be a valuable addition to more traditional anatomy/physiology demonstrations and exercises.

Vascular endothelial growth factor (VEGF) is a protein factor which has been found to play a significant role in both normal and pathological states. Its role as an angiogenic factor is well-established. More recently, VEGF has been shown to protect neurons from cell death both in vivo and in vitro. While VEGF's potential as a protective factor has been demonstrated in hypoxia-ischemia, in vitro excitotoxicity, and motor neuron degeneration, its role in seizure-induced cell loss has received little attention. A potential role in seizures is suggested by Newton et al.'s [Newton SS, Collier EF, Hunsberger J, Adams D, Terwilliger R, Selvanayagam E, Duman RS (2003) Gene profile of electroconvulsive seizures: Induction of neurotrophic and angiogenic factors. J Neurosci 23:10841-10851] finding that VEGF mRNA increases in areas of the brain that are susceptible to cell loss after electroconvulsive-shock induced seizures. Because a linear relationship does not always exist between expression of mRNA and protein, we investigated whether VEGF protein expression increased after pilocarpine-induced status epilepticus. In addition, we administered exogenous VEGF in one experiment and blocked endogenous VEGF in another to determine whether VEGF exerts a neuroprotective effect against status epilepticus-induced cell loss in one vulnerable brain region, the rat hippocampus. Our data revealed that VEGF is dramatically up-regulated in neurons and glia in hippocampus, thalamus, amygdala, and neocortex 24 h after status epilepticus. VEGF induced significant preservation of hippocampal neurons, suggesting that VEGF may play a neuroprotective role following status epilepticus.

Neuromedin U (NMU) has recently been reported to have a role in nociception and inflammation. To clarify the function of the two known NMU receptors, NMU receptor 1 (NMUR1) and NMU receptor 2 (NMUR2), during nociception and inflammation in vivo, we generated mice in which the genes for each receptor were independently deleted. Compared to wild type littermates, mice deficient in NMUR2 showed a reduced thermal nociceptive response in the hot plate, but not in the tail flick, test. In addition, the NMUR2 mutant mice showed a reduced behavioral response and a marked reduction in thermal hyperalgesia following capsaicin injection. NMUR2-deficient mice also showed an impaired pain response during the chronic, but not acute, phase of the formalin test. In contrast, NMUR1-deficient mice did not show any nociceptive differences compared to their wild type littermates in any of the behavioral tests used. We observed the same magnitude of inflammation in both lines of NMU receptor mutant mice compared to their wild type littermates after injection with complete Freund's adjuvant (CFA), suggesting no requirement for either receptor in this response. Thus, the pro-nociceptive effects of NMU in mice appear to be mediated through NMUR2, not NMUR1.

Background: Infusion of exogenous vascular endothelial growth factor (VEGF) into adult brain at doses above 60 ng/day induces dramatic angiogenesis accompanied by vascular leak and inflammation. Blood vessels formed by this treatment are dilated and tortuous, exhibiting a pathological morphology. Pathological VEGF-induced angiogenesis is preceded by vascular leak and inflammation, which have been proposed to mediate subsequent angiogenesis., Methods: To test this hypothesis, we infused VEGF into the brains of adult rats to induce pathological angiogenesis. Some of these rats were treated with dexamethasone, a potent anti-inflammatory glucocorticoid, to inhibit inflammation and edema., Results: We demonstrate that inhibition of inflammation by treatment with dexamethasone significantly attenuated VEGF-induced pathological angiogenesis. To present converging evidence that inflammation may be important in this angiogenic process, we also demonstrate that mice genetically deficient in the inflammatory mediator intercellular adhesion molecule-1 have attenuated VEGF-induced angiogenesis. These same mice showed normal amounts of physiological angiogenesis in response to enriched environments, however, suggesting that a generalized reduction in vascular plasticity could not account for their poor angiogenic response to VEGF., Conclusions: Taken together, the data from these experiments suggest that the inflammation which occurs before or during VEGF-induced pathological brain angiogenesis plays a contributory role in the pathological angiogenic process., (Copyright (c) 2007 S. Karger AG, Basel.)

Nerve growth factor (NGF) plays a role in sympathetic neuron integrity and survival. Brain-derived neurotrophic factor (BDNF) also has trophic effects on sympathetic neurons. We report here the serendipitous finding that co-treatment of hippocampus with BDNF and the NGF antagonist TrkA-Fc leads to perivascular inflammation and marked vasoconstriction. This effect is not observed with either reagent alone or in combination with other control proteins. Because NGF supports sympathetic neuron health, we tested the hypothesis that BDNF combined with sympathetic compromise caused this effect. Superior cervical ganglia were removed bilaterally with concurrent BDNF infusion into hippocampus. Perivascular inflammation was observed at 3 days, but not 12 days post treatment, when sympathetic terminals had receded, suggesting that the presence of these terminals was necessary for inflammation. Since sympathetic dysfunction may lead to compensatory overactivity of norepinephrine (NE) signaling, we co-infused BDNF with NE in the hippocampus and observed perivascular inflammation. In humans, sympathetic overactivity has been reported in a variety of vascular diseases. Some of these diseases, e.g. primary Raynaud's, are not accompanied by serious inflammatory disease whereas others, such as scleroderma and systemic lupus, are. We speculate that BDNF may contribute to the transformation of sympathetic dysfunction to inflammatory disease.

In addition to its potent effects on vasculature, it has become clear that vascular endothelial growth factor (VEGF) has effects on both neurons and glia, and recent studies suggest that it can be neuroprotective. To determine potential mechanisms underlying this neuroprotection, recombinant human VEGF was bath applied to adult rat hippocampal slices, and both extracellular and intracellular recordings were used to examine intrinsic properties and synaptic responses of hippocampal principal neurons. Initial studies in area CA1 showed that VEGF significantly reduced the amplitude of responses elicited by Schaffer collateral stimulation, without influencing membrane properties. Similar effects occurred in CA3 pyramidal cells and dentate gyrus granule cells when their major glutamatergic afferents were stimulated. Because VEGF expression is increased after seizures, effects of VEGF were also examined in rats with recurrent spontaneous seizures. VEGF reduced spontaneous discharges in slices from these rats but had surprisingly little effect on epileptiform discharges produced by disinhibition of slices from control rats. These results demonstrate a previously unknown effect of VEGF on neuronal activity and also demonstrate a remarkable potency in the epileptic brain. Based on this, we suggest that VEGF or VEGF-related targets could provide useful endpoints to direct novel therapeutic strategies for epilepsy.

We have previously demonstrated that brain-derived neurotrophic factor (BDNF) induces persistent neuropeptide Y (NPY) production in cortical cultures in an ERK1/2-dependent manner. In some studies, it was shown that BDNF leads to the downregulation of TrkB receptor and some of its downstream responses, whereas in others it does not. We examined whether the BDNF requirement for induction of persistent NPY production correlates with that for induction of phosphorylation of TrkB and ERK1/2. Continuous 24-h exposure to BDNF led to a 2- to 3-fold increase in NPY production (maximal level). While 1 h of BDNF exposure induced NPY production at a half maximal level, 8 h was required for induction of a maximal level. BDNF-induced NPY production was completely inhibited by co-exposure to TrkB-Fc fusion protein (TrkB extracellular domain fused to Fc) and partially inhibited by TrkB-Fc added 1 h after BDNF; TrkC-Fc did not do so. Activation of TrkB receptor was analyzed at two potential tyrosine phosphorylated sites, the activation loop and the Shc binding. BDNF led to coordinated phosphorylation of the two sites that persisted for 6-8 h, and this was not associated with changes in the content of TrkB protein. The presence of BDNF throughout the 6- to 8-h period was required for the persistent phosphorylation of TrkB and ERK1/2. Thus, continuous BDNF activation of TrkB is required for persistent activation of the ERK1/2 pathway and induction of NPY production. We propose that, within the time frame analyzed in this study, BDNF does not lead to the downregulation of TrkB receptor or of the biological responses leading to NPY production.

Vascular endothelial growth factor (VEGF) has been shown to induce angiogenesis when infused continuously into adult rat brain tissue. In addition, VEGF has been shown to enhance permeability in brain vasculature. Adult rats were continuously infused with mouse VEGF into neocortex for up to 7 days. We studied the development of VEGF-induced vasculature in rat neocortex and evaluated the temporal expression of a wide variety of markers for inflammation and vascular leak in relation to the angiogenic response using immunohistochemistry and Western blot analysis. We report here that VEGF-mediated inflammation in brain is characterized by upregulation of ICAM-1 and the chemokine MIP-1alpha, as well as a preferential extravasation of monocytes. VEGF causes a dramatic breakdown of the blood-brain barrier, which is characterized by decreased investment of the vasculature with astroglial endfeet. Perivascular cells, in contrast, increase around the newly formed cerebrovasculature. In addition, breakdown of the blood-brain barrier, leukocyte extravasation, and extracellular matrix deposition occur before vascular proliferation. Furthermore, administration of low doses of VEGF induces permeability and inflammation without appreciable vascular proliferation.

Vascular endothelial growth factor (VEGF) is a vascular growth factor which induces angiogenesis (the development of new blood vessels), vascular permeability, and inflammation. In brain, receptors for VEGF have been localized to vascular endothelium, neurons, and glia. VEGF is upregulated after hypoxic injury to the brain, which can occur during cerebral ischemia or high-altitude edema, and has been implicated in the blood-brain barrier breakdown associated with these conditions. Given its recently-described role as an inflammatory mediator, VEGF could also contribute to the inflammatory responses observed in cerebral ischemia. After seizures, blood-brain barrier breakdown and inflammation is also observed in brain, albeit on a lower scale than that observed after stroke. Recent evidence has suggested a role for inflammation in seizure disorders. We have described striking increases in VEGF protein in both neurons and glia after pilocarpine-induced status epilepticus in the brain. Increases in VEGF could contribute to the blood-brain barrier breakdown and inflammation observed after seizures. However, VEGF has also been shown to be neuroprotective across several experimental paradigms, and hence could potentially protect vulnerable cells from damage associated with seizures. Therefore, the role of VEGF after seizures could be either protective or destructive. Although only further research will determine the exact nature of VEGF's role after seizures, preliminary data indicate that VEGF plays a protective role after seizures.

Acute intrahippocampal infusion of brain-derived neurotrophic factor (BDNF) leads to long-term potentiation (BDNF-LTP) of synaptic transmission at medial perforant path-->granule cell synapses in the rat dentate gyrus. Endogenous BDNF is implicated in the maintenance of high-frequency stimulation-induced LTP (HFS-LTP). However, the relationship between exogenous BDNF-LTP and HFS-LTP is unclear. First, we found that BDNF-LTP, like HFS-LTP, is associated with enhancement in both synaptic strength and granule cell excitability (EPSP-spike coupling). Second, treatment with a competitive NMDA receptor (NMDAR) antagonist blocked HFS-LTP but had no effect on the development or magnitude of BDNF-LTP. Thus, NMDAR activation is not required for the induction or expression of BDNF-LTP. Formation of stable, late phase HFS-LTP requires mRNA synthesis and is coupled to upregulation of the immediate early gene activity-regulated cytoskeleton-associated protein (Arc). Local infusion of the transcription inhibitor actinomycin D (ACD) 1 hr before or immediately before BDNF infusion inhibited BDNF-LTP and upregulation of Arc protein expression. ACD applied 2 hr after BDNF infusion had no effect, defining a critical time window of transcription-dependent synaptic strengthening. Finally, the functional role of BDNF-LTP was assessed in occlusion experiments with HFS-LTP. HFS-LTP was induced, and BDNF was infused at time points corresponding to early phase (1 hr) or late phase (4 hr) HFS-LTP. BDNF applied during the early phase led to normal BDNF-LTP. In contrast, BDNF-LTP was completely occluded during the late phase. The results strongly support a role for BDNF in triggering transcription-dependent, late phase LTP in the intact adult brain.

Vascular endothelial growth factor (VEGF) plays a critical role during normal embryonic angiogenesis and also in the pathological angiogenesis that occurs in a number of diseases, including cancer. Initial attempts to block VEGF by using a humanized monoclonal antibody are beginning to show promise in human cancer patients, underscoring the importance of optimizing VEGF blockade. Previous studies have found that one of the most effective ways to block the VEGF-signaling pathway is to prevent VEGF from binding to its normal receptors by administering decoy-soluble receptors. The highest-affinity VEGF blocker described to date is a soluble decoy receptor created by fusing the first three Ig domains of VEGF receptor 1 to an Ig constant region; however, this fusion protein has very poor in vivo pharmacokinetic properties. By determining the requirements to maintain high affinity while extending in vivo half life, we were able to engineer a very potent high-affinity VEGF blocker that has markedly enhanced pharmacokinetic properties. This VEGF-Trap effectively suppresses tumor growth and vascularization in vivo, resulting in stunted and almost completely avascular tumors. VEGF-Trap-mediated blockade may be superior to that achieved by other agents, such as monoclonal antibodies targeted against the VEGF receptor.

The results of several studies have contributed to the hypothesis that BDNF promotes seizure activity, particularly in adult hippocampus. To test this hypothesis, BDNF, vehicle (phosphate-buffered saline, PBS), or albumin was infused directly into the hippocampus for 2 weeks using osmotic minipumps. Rats were examined behaviorally, electrophysiologically, and anatomically. An additional group was tested for sensitivity to the convulsant pilocarpine. Spontaneous behavioral seizures were observed in BDNF-infused rats (8/32; 25%) but not in controls (0/20; 0%). In a subset of six animals (three BDNF, three albumin), blind electrophysiological analysis of scalp recordings contralateral to the infused hippocampus demonstrated abnormalities in all BDNF rats; but not controls. Neuronal loss in BDNF-treated rats was not detected relative to PBS- or albumin-treated animals, but immunocytochemical markers showed a pattern of expression in BDNF-treated rats that was similar to rats with experimentally induced seizures. Thus, BDNF-infused rats had increased expression of NPY in hilar neurons of the dentate gyrus relative to control rats. NPY and BDNF expression was increased in the mossy fiber axons of dentate gyrus granule cells relative to controls. The increase in NPY and BDNF expression in BDNF-treated rats was bilateral and occurred throughout the septotemporal axis of the hippocampus. Mossy fiber sprouting occurred in five BDNF-treated rats but no controls. In another group of infused rats that was tested for seizure sensitivity to the convulsant pilocarpine, BDNF-infused rats had a shorter latency to status epilepticus than PBS-infused rats. In addition, the progression from normal behavior to severe seizures was faster in BDNF-treated rats. These data support the hypothesis that intrahippocampal BDNF infusion can facilitate, and potentially initiate, seizure activity in adult hippocampus.

Angiopoietin-1 (Ang1) is a ligand for the endothelial specific receptor tyrosine kinase, Tie2, that protects the adult peripheral vasculature from vascular leakage. We tested the hypothesis that increases in levels of Ang1 reduce blood-brain barrier (BBB) leakage in ischemic brain. Mice were subjected to embolic middle cerebral artery (MCA) occlusion. Recombinant adenoviruses expressing Ang1 (Ad-Ang1) or a control gene encoding green fluorescent protein (Ad-GFP), or recombinant Ang1 protein, BowAng1, was administered to mice before MCA occlusion. Regional cerebral blood flow (rCBF), the brain tissue content of Evans Blue, and ischemic lesion volume were measured. Serum levels of Ang1 (183+/-31.9 microg/ml, n=4) were detected in mice receiving Ad-Ang1 or in mice treated with BowAng1 (262+/-35.4 microg/ml, n=7) but not in the control mice (n=11). Six hours after MCA occlusion, mice receiving Ad-GFP (n=8) or control protein (n=7) showed large Evans Blue leakage in the ipsilateral hemisphere (0.46+/-0.05 or 0.55+/-0.16 ng/mg tissue) whereas mice receiving Ad-Ang1 (n=6) or BowAng1 (n=7) had significantly (P<0.05) less Evans Blue leakage (0.26+/-0.07 or 0.14+/-0.03 ng/mg tissue). Infusion of recombinant human vascular endothelial growth factor (rhVEGF(165)) to ischemic mice resulted in significant (P<0.05) increases in Evans Blue leakage (1.24+/-0.34 ng/mg tissue, n=7) compared with the control mice. In contrast, infusion of rhVEGF(165) in ischemic mice receiving Ad-Ang1 did not significantly increase Evans Blue dye in the ipsilateral hemisphere (0.22+/-0.06 ng/mg tissue, n=6). Moreover, 24 h after ischemia mice receiving Ad-Ang1 had a significantly smaller ischemic lesion volume (22.6+/-2.7%, n=8) than the lesion volume in mice receiving Ad-GFP (44.7+/-3.7%, n=8), although rCBF reduced to approximately 20% of the contralateral levels in both groups of mice 10 min after ischemia. Our data demonstrate that Ang1 reduces BBB leakage in ischemic brain and consequently decreases ischemic lesion volume.

Evidence suggests that brain-derived neurotrophic factor (BDNF) may be important in the pathophysiology of depression, in addition to its role as a neurotrophic factor for sensory neurons. The authors conducted a series of experiments examining the behavioral profile of BDNF heterozygous knockout and wild-type mice. The heterozygous and wild-type mice did not differ on measures of activity, exploration, or hedonic sensitivity, or in the forced swim test. When assessed in the learned helplessness paradigm, heterozygous mice were slower to escape after training than were wild-type mice (p = .02). This effect may be accounted for by the fact that these mice demonstrate a reduced sensitivity to centrally mediated pain, apparent on the hot plate and Formalin injection tests of nociception. Overall, heterozygous mice were not more likely to display anxious or depressive-like behaviors and, consequently, may not constitute a murine model of genetic vulnerability to mood and anxiety disorders.

Heparin-binding growth-associated molecule (HB-GAM) (pleiotrophin) is a highly conserved extracellular matrix-associated protein implicated in a diverse range of developmental processes, including the formation and plasticity of neuronal connections. Using gene targeting, we have in the present study created HB-GAM-deficient mice that are viable and fertile and show no gross anatomical abnormalities. The hippocampal structure as well as basal excitatory synaptic transmission in the area CA1 appear normal in the mice lacking HB-GAM. However, hippocampal slices from HB-GAM-deficient mice display a lowered threshold for induction of long-term potentiation (LTP), which reverts back to the wild-type level by application of HB-GAM. HB-GAM expression in hippocampus is activity-dependent and upregulated in several neuropathological conditions. Thus, we suggest that HB-GAM acts as an inducible signal to inhibit LTP in hippocampus., (Copyright 2001 Academic Press.)

Growing axons follow highly stereotypical pathways, guided by a variety of attractive and repulsive cues, before establishing specific connections with distant targets. A particularly well-known example that illustrates the complexity of axonal migration pathways involves the axonal projections of motor neurons located in the motor cortex. These projections take a complex route during which they first cross the midline, then form the corticospinal tract, and ultimately connect with motor neurons in the contralateral side of the spinal cord. These obligatory contralateral connections account for why one side of the brain controls movement on the opposing side of the body. The netrins and slits provide well-known midline signals that regulate axonal crossings at the midline. Herein we report that a member of the ephrin family, ephrin-B3, also plays a key role at the midline to regulate axonal crossing. In particular, we show that ephrin-B3 acts as the midline barrier that prevents corticospinal tract projections from recrossing when they enter the spinal gray matter. We report that in ephrin-B3(-/-) mice, corticospinal tract projections freely recross in the spinal gray matter, such that the motor cortex on one side of the brain now provides bilateral input to the spinal cord. This neuroanatomical abnormality in ephrin-B3(-/-) mice correlates with loss of unilateral motor control, yielding mice that simultaneously move their right and left limbs and thus have a peculiar hopping gait quite unlike the alternate step gait displayed by normal mice. The corticospinal and walking defects in ephrin-B3(-/-) mice resemble those recently reported for mice lacking the EphA4 receptor, which binds ephrin-B3 as well as other ephrins, suggesting that the binding of EphA4-bearing axonal processes to ephrin-B3 at the midline provides the repulsive signal that prevents corticospinal tract projections from recrossing the midline in the developing spinal cord.

During the past decade, there has been a surge of interest in growth factors (GFs) that act selectively on vascular endothelium and perivascular cells. Studies employing mutant mice or the administration of recombinant proteins have suggested that these factors not only mediate the proliferation of endothelial cells, but also regulate vascular differentiation, regression, and permeability. During and after cerebral ischemia, brain vasculature becomes leaky and unstable, and the normally impermeable blood-brain barrier breaks down. Several days after the ischemic insult, endothelial cells begin to proliferate, and angiogenesis occurs. Expression studies have shown that key vascular GFs are regulated, during these processes, in a complex and coordinated manner. The distinct pattern of regulation exhibited by each vascular GF suggests a unique role for each factor during the initial vascular destabilization and subsequent angiogenesis that occurs after cerebral ischemia. Data from studies in other biological systems support these suggested roles. Thus, manipulation of vascular GFs may prove to be an effective means of stabilizing or enriching brain vasculature after ischemia, and ameliorating the detrimental effects of blood-brain barrier breakdown and vessel regression after stroke.

Various studies have shown that brain-derived neurotrophic factor (BDNF) increases neuronal excitability and is localized and upregulated in areas implicated in epileptogenesis. Seizure activity increases the expression of BDNF mRNA and protein, and recent studies have shown that interfering with BDNF signal transduction inhibits the development of the epileptic state in vivo. These results suggest that BDNF contributes to epileptogenesis. Further analysis of the cellular and molecular mechanisms by which BDNF influences excitability and connectivity in adult brain could provide novel concepts and targets for anticonvulsant or anti-epileptogenic therapy.

Pathological increases in vascular leakage lead to edema and swelling, causing serious problems in brain tumors, in diabetic retinopathy, after strokes, during sepsis and also in inflammatory conditions such as rheumatoid arthritis and asthma. Although many agents and disease processes increase vascular leakage, no known agent specifically makes vessels resistant to leaking. Vascular endothelial growth factor (VEGF) and the angiopoietins function together during vascular development, with VEGF acting early during vessel formation, and angiopoietin-1 acting later during vessel remodeling, maturation and stabilization. Although VEGF was initially called vascular permeability factor, there has been less focus on its permeability actions and more effort devoted to its involvement in vessel growth and applications in ischemia and cancer. Recent transgenic approaches have confirmed the profound permeability effects of VEGF (refs. 12-14), and have shown that transgenic angiopoietin-1 acts reciprocally as an anti-permeability factor when provided chronically during vessel formation, although it also profoundly affects vascular morphology when thus delivered. To be useful clinically, angiopoietin-1 would have to inhibit leakage when acutely administered to adult vessels, and this action would have to be uncoupled from its profound angiogenic capabilities. Here we show that acute administration of angiopoietin-1 does indeed protect adult vasculature from leaking, countering the potentially lethal actions of VEGF and inflammatory agents.

Brain-derived neurotrophic factor (BDNF) modulates neuropeptide levels in hippocampus and cortex of young adult rats. Neuropeptide levels are altered in some age-related disorders, such as Alzheimer's and Parkinson's Disease. BDNF may be able to rectify peptide abnormalities but, because plasticity decreases with age, BDNF may not alter peptide levels as readily in aged animals. To determine if BDNF would regulate peptide levels in aged rats, young, aged memory-impaired, and unimpaired rats were infused with BDNF or vehicle into hippocampus and cortex. Cell profile counts, cell profile areas, fiber counts, and/or fiber terminal densities were measured for sections immunostained for neuropeptide Y (NPY), somatostatin (SOM), cholecystokinin-8 (CCK), and dynorphin A(1-8) (DYN). Results showed that BDNF upregulated cortical NPY-immunoreactivity (ir) and SOM-ir, upregulated hippocampal NPY-ir, and downregulated hippocampal DYN-ir in both aged and young rats. In addition, BDNF significantly and selectively normalized the areas of atrophied deep cortical CCK-ir cell profiles in aged-impaired rats. Finally, decreased CCK-ir fiber density was found in the hippocampal formation of aged memory-impaired rats.

Transgenic mice overexpressing brain-derived neurotrophic factor from the beta-actin promoter were tested for behavioral, gross anatomical and physiological abnormalities. Brain-derived neurotrophic factor messenger RNA overexpression was widespread throughout brain. Overexpression declined with age, such that levels of overexpression decreased sharply by nine months. Brain-derived neurotrophic factor transgenic mice had no gross deformities or behavioral abnormalities. However, they showed a significant passive avoidance deficit. This deficit was dependent on continued overexpression, and resolved with age as brain-derived neurotrophic factor transcripts decreased. In addition, the brain-derived neurotrophic factor transgenic mice showed increased seizure severity in response to kainic acid. Hippocampal slices from brain-derived neurotrophic factor transgenic mice showed hyperexcitability in area CA3 and entorhinal cortex, but not in dentate gyrus. Finally, area CA1 long-term potentiation was disrupted, indicating abnormal plasticity. Our data suggest that overexpression of brain-derived neurotrophic factor in the brain can interfere with normal brain function by causing learning impairments and increased excitability. The results also support the hypothesis that excess brain-derived neurotrophic factor could be pro-convulsant in the limbic system.

The expression of the neurotrophic factor BDNF increases during learning-related events and is decreased in the hippocampus of Alzheimer's Disease patients, suggesting that it plays a role in learning, memory, and/or age-related memory deficits. We examined the expression of BDNF and its high affinity receptor, trkB, in young and aged Sprague-Dawley rats. BDNF and trkB mRNA were measured by semi-quantitative in situ hybridization and BDNF protein was measured by ELISA. Significant decreases with age were detected for BDNF mRNA in the pons, BDNF protein in the midbrain, and trkB mRNA in many areas of the brain. Rats were evaluated on the Morris water maze before sacrifice so that BDNF and trkB levels could be related to cognitive status. Regression analyses revealed that decreased trkB mRNA in the pons significantly predicted impaired memory performance in aged rats. These results suggest that decreases in trkB mRNA with age are more widespread than decreases in BDNF, and that BDNF decreases are restricted to more caudal brain regions., (Copyright 1998 Elsevier Science B.V.)

Fusion proteins comprising the Fc domain of human IgG and extracellular domains of receptor tyrosine kinases can neutralize the activity of their cognate ligands when administered in molar excess. We have generated a fusion protein using the ectodomain of TrkB (TrkB-Fc). Although the ability of TrkB-Fc to neutralize the activity of brain-derived neurotrophic factor (BDNF) in vitro has been demonstrated, there have been no conclusive demonstrations of its ability to neutralize the activity of BDNF in vivo. We co-infused TrkB-Fc with BDNF into the cortex and hippocampus of adult rats to determine whether TrkB-Fc would interfere with the ability of BDNF to upregulate neuropeptide Y (NPY). We report here that rather than neutralizing the activity of exogenous BDNF, co-infusion with the TrkB-Fc fusion protein greatly increased the volume of tissue in which neuropeptide Y immunostaining was upregulated. In addition, TrkB-Fc greatly enhanced BDNF's distribution through adult brain parenchyma. TrkB-Fc also markedly increased the otherwise limited diffusion of BDNF into brain parenchyma following intraventricular infusion. These results show that rather than neutralizing or sequestering BDNF, the TrkB-Fc, at close to molar equivalence to BDNF, can function as a carrier for BDNF and thus enhance the delivery or penetration of this polypeptide into the brain., (Copyright 1998 Academic Press.)

Cortical spreading depression (CSD) protects hippocampal and cortical neurons from an otherwise lethal ischemic insult delivered days later. The present study was undertaken to evaluate changes in the expression of BDNF following CSD, distinct from lesion effects and its possible involvement in delayed ischemic tolerance. CSD was elicited by KCl application and a cortical lesion was made by hyperosmolar NaCl application. BDNF mRNA was examined by in situ hybridization and Northern blot up to 7 days post-CSD. BDNF protein content was measured by ELISA. In the cortex, BDNF protein was mildly elevated despite minimal increases of mRNA in the NaCl lesion group. CSD specifically up-regulated BDNF mRNA at 4 h, followed by a delayed secondary increase at 2-3 days. BDNF protein exhibited smaller biphasic increases at 24 h and 3-7 days post-CSD which were significantly higher than the NaCl lesion group. In the hippocampus, BDNF protein levels showed a delayed decrease in both groups independent of mRNA changes, but CSD specifically delayed this decrease. Thus, CSD can alter BDNF levels independent of lesion effects. The increased BDNF following CSD in the cortex is consistent with the involvement of BDNF in cortical ischemic tolerance. BDNF could not, however, be directly related to ischemic tolerance in the hippocampus.

Purpose: The role of magnetic resonance imaging (MRI) in the diagnosis of osteomyelitis in foot infections in diabetics was investigated. The accuracy, sensitivity, and specificity of MRI, plain radiography, and nuclear scanning were determined for diagnosing osteomyelitis, and a cost comparison was made., Methods: Twenty-seven patients with diabetic foot infections were studied prospectively. All patients underwent MRI and plain radiography. Twenty-two patients had technetium bone scans, and 19 patients had Indium scans. Nineteen patients had all four tests performed. Patients with obvious gangrene or a fetid foot were excluded., Results: The diagnosis of osteomyelitis was established by pathologic specimen (n = 18), bone culture (n = 3), or successful response to medical management (n = 6). Osteomyelitis was confirmed in nine of the pathologic specimens. The diagnostic sensitivity, specificity, and accuracy for MRI was 88%, 100%, and 95%, respectively, for plain radiography it was 22%, 94%, and 70%, respectively, for technetium bone scanning it was 50%, 50%, and 50%, respectively, and for Indium leukocyte scanning it was 33%, 69%, and 58%, respectively. The data were analyzed statistically with the two-tailed Fisher's exact test. MRI was the only test that was statistically significant (p < 0.01)., Conclusions: MRI appeared to be the single best test for the diagnosis of osteomyelitis associated with diabetic foot infections. It had a better diagnostic accuracy than conventional modalities and appeared to be more cost-effective than the frequently used Indium scan.

The expression of neuropeptides and neurotrophic factors is altered in the hippocampus after seizure induction in rats. Because the increase in brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) mRNAs precede changes in neuropeptide expression after seizure, it is possible that BDNF and NGF mediate subsequent alterations in peptide expression. To test this hypothesis directly, BDNF or NGF was infused into the hippocampus and cortex of adult rats. To ascertain the regional specificity of any observed effects of neurotrophin administration on neuropeptide expression, infusions into the striatum were also studied. To control for specificity, vehicle was also infused into the same sites. Peptide and mRNA alterations were assessed by Northern analysis, immunohistochemistry and radioimmunoassay. BDNF produced elevations of peptide and mRNA for neuropeptide Y and cholecystokinin in hippocampus and cortex, and somatostatin in cortex. BDNF increased mRNAs for neuropeptide Y, cholecystokinin, substance P and dynorphin in striatum. In contrast, BDNF decreased dynorphin peptide and mRNA in hippocampus. NGF's effects were limited to small mRNA increases, without corresponding changes in peptide levels, for neuropeptide Y in hippocampus and striatum, substance P in cortex and cholecystokinin in striatum. The distinct and limited effects of NGF infusion on neuropeptide expression demonstrate that BDNF's effects are not non-specific results of protein infusion into the brain. These findings indicate that BDNF may play a regionally specific role in modulating neuropeptide expression in the normal brain as well as in various pathophysiological states.

Research has demonstrated environmentally induced plasticity of hippocampal dentate gyrus-evoked potentials. Other research has shown a role of the NMDA receptor in dentate gyrus long-term potentiation (LTP). The authors tested the role of the NMDA receptor in one form of environmentally induced plasticity, in which transferring animals from their home cages to another environment results in significant excitatory postsynaptic potential (EPSP) enhancement and concomitant depression of the population spike. Rats were chronically implanted with stimulating electrodes in the perforant path and recording electrodes in the dentate gyrus bilaterally. Evoked potentials were recorded from freely behaving rats for four 20-minute sessions (1/wk), which took place immediately following an environmental transfer. Rats received 0.00, 0.05, 0.08, or 0.10 mg/kg MK-801 s.c. 30 minutes prior to recording sessions in either an ascending- or descending-dose series. Results showed that MK-801 produced a reduction of the EPSP enhancement, which takes place over the 20-minute session. The effects of MK-801 on spike depression varied as a function of dose series and time within a session, suggesting a long-term effect of MK-801 on spike depression. There was no detected effect of MK-801 on behavior. Results suggest a role of the NMDA receptor in this form of environmentally induced plasticity with different effects of NMDA receptor antagonism on EPSP enhancement and spike depression.

Two cases of corneal hematomas initially manifested as corneal blood staining, but were further complicated by the presence of persistent epithelial defects and stromal thinning. Both cases occurred following cataract surgery. Scanning electron and light microscopic examination in the second case demonstrated a loss of keratocytes, stromal collagen breakdown, and epithelial cell degeneration over the center of the hematoma. Large corneal hematomas may cause epithelial degeneration by functioning as barriers to nutrients and metabolic factors from the anterior chamber. Unlike corneal blood staining, conservative treatment is often insufficient therapy for corneal hematomas with associated persistent epithelial defects. Complications from epithelial defects, including corneal thinning, descemetocele, and perforation, may necessitate more aggressive treatment, even surgery. An epithelial defect overlying blood staining should suggest the presence of a corneal hematoma.