A longitudinal MRI and TSPO PET-based investigation of brain region-specific neuroprotection by diazepam versus midazolam following organophosphate-induced seizures.

Acute poisoning with organophosphorus cholinesterase inhibitors (OPs), such as OP nerve agents and pesticides, can cause life threatening cholinergic crisis and status epilepticus (SE). Survivors often experience significant morbidity, including brain injury, acquired epilepsy, and cognitive deficits. Current medical countermeasures for acute OP poisoning include a benzodiazepine to mitigate seizures. Diazepam was long the benzodiazepine included in autoinjectors used to treat OP-induced seizures, but it is now being replaced in many guidelines by midazolam, which terminates seizures more quickly, particularly when administered intramuscularly. While a direct correlation between seizure duration and the extent of brain injury has been widely reported, there are limited data comparing the neuroprotective efficacy of diazepam versus midazolam following acute OP intoxication. To address this data gap, we used non-invasive imaging techniques to longitudinally quantify neuropathology in a rat model of acute intoxication with the OP diisopropylfluorophosphate (DFP) with and without post-exposure intervention with diazepam or midazolam. Magnetic resonance imaging (MRI) was used to monitor neuropathology and brain atrophy, while positron emission tomography (PET) with a radiotracer targeting translocator protein (TSPO) was utilized to assess neuroinflammation. Animals were scanned at 3, 7, 28, 65, 91, and 168 days post-DFP and imaging metrics were quantitated for the hippocampus, amygdala, piriform cortex, thalamus, cerebral cortex and lateral ventricles. In the DFP-intoxicated rat, neuroinflammation persisted for the duration of the study coincident with progressive atrophy and ongoing tissue remodeling. Benzodiazepines attenuated neuropathology in a region-dependent manner, but neither benzodiazepine was effective in attenuating long-term neuroinflammation as detected by TSPO PET. Diffusion MRI and TSPO PET metrics were highly correlated with seizure severity, and early MRI and PET metrics were positively correlated with long-term brain atrophy. Collectively, these results suggest that anti-seizure therapy alone is insufficient to prevent long-lasting neuroinflammation and tissue remodeling.

Increased Striatal Presynaptic Dopamine in a Nonhuman Primate Model of Maternal Immune Activation: A Longitudinal Neurodevelopmental Positron Emission Tomography Study With Implications for Schizophrenia.

BACKGROUND: Epidemiological studies suggest that maternal immune activation (MIA) is a significant risk factor for future neurodevelopmental disorders, including schizophrenia (SZ), in offspring. Consistent with findings in SZ research and work in rodent systems, preliminary cross-sectional findings in nonhuman primates suggest that MIA is associated with dopaminergic hyperfunction in young adult offspring. METHODS: In this unique prospective longitudinal study, we used [18F]fluoro-l-m-tyrosine positron emission tomography to examine the developmental time course of striatal presynaptic dopamine synthesis in male rhesus monkeys born to dams (n = 13) injected with a modified form of the inflammatory viral mimic, polyinosinic:polycytidylic acid [poly(I:C)], in the late first trimester. Striatal (caudate, putamen, and nucleus accumbens) dopamine from these animals was compared with that of control offspring born to dams that received saline (n = 10) or no injection (n = 4). Dopamine was measured at 15, 26, 38, and 48 months of age. Prior work with this cohort found decreased prefrontal gray matter volume in MIA offspring versus controls between 6 and 45 months of age. Based on theories of the etiology and development of SZ-related pathology, we hypothesized that there would be a delayed (relative to the gray matter decrease) increase in striatal fluoro-l-m-tyrosine signal in the MIA group versus controls. RESULTS: [18F]fluoro-l-m-tyrosine signal showed developmental increases in both groups in the caudate and putamen. Group comparisons revealed significantly greater caudate dopaminergic signal in the MIA group at 26 months. CONCLUSIONS: These findings are highly relevant to the known pathophysiology of SZ and highlight the translational relevance of the MIA model in understanding mechanisms by which MIA during pregnancy increases risk for later illness in offspring.

Regulation of beige adipocyte thermogenesis by the cold-repressed ER protein NNAT.

OBJECTIVE: Cold stimuli trigger the conversion of white adipose tissue into beige adipose tissue, which is capable of non-shivering thermogenesis. However, what process drives this activation of thermogenesis in beige fat is not well understood. Here, we examine the ER protein NNAT as a regulator of thermogenesis in adipose tissue. METHODS: We investigated the regulation of adipose tissue NNAT expression in response to changes in ambient temperature. We also evaluated the functional role of NNAT in thermogenic regulation using Nnat null mice and primary adipocytes that lack or overexpress NNAT. RESULTS: Cold exposure or treatment with a ß3-adrenergic agonist reduces the expression of adipose tissue NNAT in mice. Genetic disruption of Nnat in mice enhances inguinal adipose tissue thermogenesis. Nnat null mice exhibit improved cold tolerance both in the presence and absence of UCP1. Gain-of-function studies indicate that ectopic expression of Nnat abolishes adrenergic receptor-mediated respiration in beige adipocytes. NNAT physically interacts with the ER Ca2+-ATPase (SERCA) in adipocytes and inhibits its activity, impairing Ca2+ transport and heat dissipation. We further demonstrate that NHLRC1, an E3 ubiquitin protein ligase implicated in proteasomal degradation of NNAT, is induced by cold exposure or ß3-adrenergic stimulation, thus providing regulatory control at the protein level. This serves to link cold stimuli to NNAT degradation in adipose tissue, which in turn leads to enhanced SERCA activity. CONCLUSIONS: Our study implicates NNAT in the regulation of adipocyte thermogenesis.

Maternal Immune Activation during Pregnancy Alters Postnatal Brain Growth and Cognitive Development in Nonhuman Primate Offspring.

Human epidemiological studies implicate exposure to infection during gestation in the etiology of neurodevelopmental disorders. Animal models of maternal immune activation (MIA) have identified the maternal immune response as the critical link between maternal infection and aberrant offspring brain and behavior development. Here we evaluate neurodevelopment of male rhesus monkeys (Macaca mulatta) born to MIA-treated dams (n = 14) injected with a modified form of the viral mimic polyinosinic:polycytidylic acid at the end of the first trimester. Control dams received saline injections at the same gestational time points (n = 10) or were untreated (n = 4). MIA-treated dams exhibited a strong immune response as indexed by transient increases in sickness behavior, temperature, and inflammatory cytokines. Although offspring born to control or MIA-treated dams did not differ on measures of physical growth and early developmental milestones, the MIA-treated animals exhibited subtle changes in cognitive development and deviated from species-typical brain growth trajectories. Longitudinal MRI revealed significant gray matter volume reductions in the prefrontal and frontal cortices of MIA-treated offspring at 6 months that persisted through the final time point at 45 months along with smaller frontal white matter volumes in MIA-treated animals at 36 and 45 months. These findings provide the first evidence of early postnatal changes in brain development in MIA-exposed nonhuman primates and establish a translationally relevant model system to explore the neurodevelopmental trajectory of risk associated with prenatal immune challenge from birth through late adolescence.SIGNIFICANCE STATEMENT Women exposed to infection during pregnancy have an increased risk of giving birth to a child who will later be diagnosed with a neurodevelopmental disorder. Preclinical maternal immune activation (MIA) models have demonstrated that the effects of maternal infection on fetal brain development are mediated by maternal immune response. Since the majority of MIA models are conducted in rodents, the nonhuman primate provides a unique system to evaluate the MIA hypothesis in a species closely related to humans. Here we report the first longitudinal study conducted in a nonhuman primate MIA model. MIA-exposed offspring demonstrate subtle changes in cognitive development paired with marked reductions in frontal gray and white matter, further supporting the association between prenatal immune challenge and alterations in offspring neurodevelopment.

Strain differences in the extent of brain injury in mice after tetramethylenedisulfotetramine-induced status epilepticus.

Acute intoxication with tetramethylenedisulfotetramine (TETS) can trigger status epilepticus (SE) in humans. Survivors often exhibit long-term neurological effects, including electrographic abnormalities and cognitive deficits, but the pathogenic mechanisms linking the acute toxic effects of TETS to chronic outcomes are not known. Here, we use advanced in vivo imaging techniques to longitudinally monitor the neuropathological consequences of TETS-induced SE in two different mouse strains. Adult male NIH Swiss and C57BL/6J mice were injected with riluzole (10 mg/kg, i.p.), followed 10 min later by an acute dose of TETS (0.2 mg/kg in NIH Swiss; 0.3 mg/kg, i.p. in C57BL/6J) or an equal volume of vehicle (10% DMSO in 0.9% sterile saline). Different TETS doses were administered to trigger comparable seizure behavior between strains. Seizure behavior began within minutes of TETS exposure and rapidly progressed to SE that was terminated after 40 min by administration of midazolam (1.8 mg/kg, i.m.). The brains of vehicle and TETS-exposed mice were imaged using in vivo magnetic resonance (MR) and translocator protein (TSPO) positron emission tomography (PET) at 1, 3, 7, and 14 days post-exposure to monitor brain injury and neuroinflammation, respectively. When the brain scans of TETS mice were compared to those of vehicle controls, subtle and transient neuropathology was observed in both mouse strains, but more extensive and persistent TETS-induced neuropathology was observed in C57BL/6J mice. In addition, one NIH Swiss TETS mouse that did not respond to the midazolam therapy, but remained in SE for more than 2 h, displayed robust neuropathology as determined by in vivo imaging and confirmed by FluoroJade C staining and IBA-1 immunohistochemistry as readouts of neurodegeneration and neuroinflammation, respectively. These findings demonstrate that the extent of injury observed in the mouse brain after TETS-induced SE varied according to strain, dose of TETS and/or the duration of SE. These observations suggest that TETS-intoxicated humans who do not respond to antiseizure medication are at increased risk for brain injury.

Targeting the CCR6/CCL20 Axis in Entheseal and Cutaneous Inflammation.

OBJECTIVE: To assess the involvement of the CCR6/CCL20 axis in psoriatic arthritis (PsA) and psoriasis (PsO) and to evaluate its potential as a therapeutic target. METHODS: First, we quantified CCL20 levels in peripheral blood and synovial fluid from PsA patients and examined the presence of CCR6+ cells in synovial and tendon tissue. Utilizing an interleukin-23 minicircle DNA (IL-23 MC) mouse model exhibiting key features of both PsO and PsA, we investigated CCR6 and CCL20 expression as well as the preventive and therapeutic effect of CCL20 blockade. Healthy tendon stromal cells were stimulated in vitro with IL-1ß to assess the production of CCL20 by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. The effect of conditioned media from stimulated tenocytes in inducing T cell migration was interrogated using a Transwell system. RESULTS: We observed an up-regulation of both CCR6 and CCL20 in the enthesis of IL-23 MC-treated mice, which was confirmed in human biopsy specimens. Specific targeting of the CCR6/CCL20 axis with a CCL20 locked dimer (CCL20LD) blocked entheseal inflammation, leading to profound reductions in clinical and proinflammatory markers in the joints and skin of IL-23 MC-treated mice. The stromal compartment in the tendon was the main source of CCL20 in this model and, accordingly, in vitro activated human tendon cells were able to produce this chemokine and to induce CCR6+ T cell migration, the latter of which could be blocked by CCL20LD. CONCLUSION: Our study highlights the pathogenic role of the CCR6/CCL20 axis in enthesitis and introduces the prospect of a novel therapeutic approach for treating patients with PsO and PsA.

Acute administration of diazepam or midazolam minimally alters long-term neuropathological effects in the rat brain following acute intoxication with diisopropylfluorophosphate.

Acute intoxication with organophosphorus cholinesterase inhibitors (OPs) can trigger seizures that rapidly progress to life-threatening status epilepticus. Diazepam, long considered the standard of care for treating OP-induced seizures, is being replaced by midazolam. Whether midazolam is more effective than diazepam in mitigating the persistent effects of acute OP intoxication has not been rigorously evaluated. We compared the efficacy of diazepam vs. midazolam in preventing persistent neuropathology in adult male Sprague-Dawley rats acutely intoxicated with the OP diisopropylfluorophosphate (DFP). Subjects were administered pyridostigmine bromide (0.1 mg/kg, i.p.) 30 min prior to injection with DFP (4 mg/kg, s.c.) or vehicle (saline) followed 1 min later by atropine sulfate (2 mg/kg, i.m.) and pralidoxime (25 mg/kg, i.m.), and 40 min later by diazepam (5 mg/kg, i.p.), midazolam (0.73 mg/kg, i.m.), or vehicle. At 3 and 6 months post-exposure, neurodegeneration, reactive astrogliosis, microglial activation, and oxidative stress were assessed in multiple brain regions using quantitative immunohistochemistry. Brain mineralization was evaluated by in vivo micro-computed tomography (micro-CT). Acute DFP intoxication caused persistent neurodegeneration, neuroinflammation, and brain mineralization. Midazolam transiently mitigated neurodegeneration, and both benzodiazepines partially protected against reactive astrogliosis in a brain region-specific manner. Neither benzodiazepine attenuated microglial activation or brain mineralization. These findings indicate that neither benzodiazepine effectively protects against persistent neuropathological changes, and suggest that midazolam is not significantly better than diazepam. Overall, this study highlights the need for improved neuroprotective strategies for treating humans in the event of a chemical emergency involving OPs.

Suppression of inflammation and fibrosis using soluble epoxide hydrolase inhibitors enhances cardiac stem cell-based therapy.

Stem cell replacement offers a great potential for cardiac regenerative therapy. However, one of the critical barriers to stem cell therapy is a significant loss of transplanted stem cells from ischemia and inflammation in the host environment. Here, we tested the hypothesis that inhibition of the soluble epoxide hydrolase (sEH) enzyme using sEH inhibitors (sEHIs) to decrease inflammation and fibrosis in the host myocardium may increase the survival of the transplanted human induced pluripotent stem cell derived-cardiomyocytes (hiPSC-CMs) in a murine postmyocardial infarction model. A specific sEHI (1-trifluoromethoxyphenyl-3-(1-propionylpiperidine-4-yl)urea [TPPU]) and CRISPR/Cas9 gene editing were used to test the hypothesis. TPPU results in a significant increase in the retention of transplanted cells compared with cell treatment alone. The increase in the retention of hiPSC-CMs translates into an improvement in the fractional shortening and a decrease in adverse remodeling. Mechanistically, we demonstrate a significant decrease in oxidative stress and apoptosis not only in transplanted hiPSC-CMs but also in the host environment. CRISPR/Cas9-mediated gene silencing of the sEH enzyme reduces cleaved caspase-3 in hiPSC-CMs challenged with angiotensin II, suggesting that knockdown of the sEH enzyme protects the hiPSC-CMs from undergoing apoptosis. Our findings demonstrate that suppression of inflammation and fibrosis using an sEHI represents a promising adjuvant to cardiac stem cell-based therapy. Very little is known regarding the role of this class of compounds in stem cell-based therapy. There is consequently an enormous opportunity to uncover a potentially powerful class of compounds, which may be used effectively in the clinical setting.

The chemical convulsant diisopropylfluorophosphate (DFP) causes persistent neuropathology in adult male rats independent of seizure activity.

Organophosphate (OP) threat agents can trigger seizures that progress to status epilepticus, resulting in persistent neuropathology and cognitive deficits in humans and preclinical models. However, it remains unclear whether patients who do not show overt seizure behavior develop neurological consequences. Therefore, this study compared two subpopulations of rats with a low versus high seizure response to diisopropylfluorophosphate (DFP) to evaluate whether acute OP intoxication causes persistent neuropathology in non-seizing individuals. Adult male Sprague Dawley rats administered DFP (4 mg/kg, sc), atropine sulfate (2 mg/kg, im), and pralidoxime (25 mg/kg, im) were monitored for seizure activity for 4 h post-exposure. Animals were separated into groups with low versus high seizure response based on behavioral criteria and electroencephalogram (EEG) recordings. Cholinesterase activity was evaluated by Ellman assay, and neuropathology was evaluated at 1, 2, 4, and 60 days post-exposure by Fluoro-Jade C (FJC) staining and micro-CT imaging. DFP significantly inhibited cholinesterase activity in the cortex, hippocampus, and amygdala to the same extent in low and high responders. FJC staining revealed significant neurodegeneration in DFP low responders albeit this response was delayed, less persistent, and decreased in magnitude compared to DFP high responders. Micro-CT scans at 60 days revealed extensive mineralization that was not significantly different between low versus high DFP responders. These findings highlight the importance of considering non-seizing patients for medical care in the event of acute OP intoxication. They also suggest that OP intoxication may induce neurological damage via seizure-independent mechanisms, which if identified, might provide insight into novel therapeutic targets.

Differential Requirement for CCR6 in IL-23-Mediated Skin and Joint Inflammation.

CCR6 is important for the trafficking of IL-17A-producing γδ T cells and required for the development of psoriasiform dermatitis in an IL-23 intradermal injection model. The role of CCR6, however, in IL-23-mediated joint inflammation is unclear. We herein hydrodynamically delivered IL-23 minicircle DNA into wild-type and CCR6-deficient (CCR6-knockout) mice to induce overexpression of IL-23 systemically. After IL-23 gene transfer, wild-type mice exhibited concurrent skin and joint changes that recapitulate some features found in human psoriatic skin and joints. CCR6-knockout mice were resistant to IL-23-induced skin inflammation but exhibited no changes in joint inflammation compared with wild-type mice. Depletion of neutrophils protected wild-type mice from skin and joint disease without suppressing T helper type 17 cytokine expression. In contrast, mice lacking γδ T cells showed a partial reduction in neutrophilic recruitment and a significant decrease in IL-17A expression in skin and paw tissue. Thus, in an IL-23-mediated model that allows concurrent assessment of both skin and joint disease, we showed that CCR6 is critical for inflammation in the skin but not in the joint. Furthermore, our data suggest that neutrophils and γδ T cells are key effector cells in IL-23-mediated skin and joint inflammation in mice.

TSPO PET Using [18F]PBR111 Reveals Persistent Neuroinflammation Following Acute Diisopropylfluorophosphate Intoxication in the Rat.

Acute intoxication with organophosphates (OPs) can trigger status epilepticus followed by persistent cognitive impairment and/or electroencephalographic abnormalities. Neuroinflammation is widely posited to influence these persistent neurological consequences. However, testing this hypothesis has been challenging, in part because traditional biometrics preclude longitudinal measures of neuroinflammation within the same animal. Therefore, we evaluated the performance of noninvasive positron emission tomography (PET), using the translocator protein (TSPO) radioligand [18F]PBR111 against classic histopathologic measures of neuroinflammation in a preclinical model of acute intoxication with the OP diisopropylfluorophosphate (DFP). Adult male Sprague Dawley rats administered pyridostigmine bromide (0.1 mg/kg, im) 30 min prior to administration of DFP (4 mg/kg, sc), atropine sulfate (2 mg/kg, im) and 2-pralidoxime (25 mg/kg, im) exhibited moderate-to-severe seizure behavior. TSPO PET performed prior to DFP exposure and at 3, 7, 14, 21, and 28 days postexposure revealed distinct lesions, as defined by increased standardized uptake values (SUV). Increased SUV showed high spatial correspondence to immunohistochemical evidence of neuroinflammation, which was corroborated by cytokine gene and protein expression. Regional SUV metrics varied spatiotemporally with days postexposure and correlated with the degree of neuroinflammation detected immunohistochemically. Furthermore, SUV metrics were highly correlated with seizure severity, suggesting that early termination of OP-induced seizures may be critical for attenuating subsequent neuroinflammatory responses. Normalization of SUV values to a cerebellar reference region improved correlations to all outcome measures and seizure severity. Collectively, these results establish TSPO PET using [18F]PBR111 as a robust, noninvasive tool for longitudinal monitoring of neuroinflammation following acute OP intoxication.

Preliminary evidence of increased striatal dopamine in a nonhuman primate model of maternal immune activation.

Women exposed to a variety of viral and bacterial infections during pregnancy have an increased risk of giving birth to a child with autism, schizophrenia or other neurodevelopmental disorders. Preclinical maternal immune activation (MIA) models are powerful translational tools to investigate mechanisms underlying epidemiological links between infection during pregnancy and offspring neurodevelopmental disorders. Our previous studies documenting the emergence of aberrant behavior in rhesus monkey offspring born to MIA-treated dams extends the rodent MIA model into a species more closely related to humans. Here we present novel neuroimaging data from these animals to further explore the translational potential of the nonhuman primate MIA model. Nine male MIA-treated offspring and 4 controls from our original cohort underwent in vivo positron emission tomography (PET) scanning at approximately 3.5-years of age using [18F] fluoro-l-m-tyrosine (FMT) to measure presynaptic dopamine levels in the striatum, which are consistently elevated in individuals with schizophrenia. Analysis of [18F]FMT signal in the striatum of these nonhuman primates showed that MIA animals had significantly higher [18F]FMT index of influx compared to control animals. In spite of the modest sample size, this group difference reflects a large effect size (Cohen's d = 0.998). Nonhuman primates born to MIA-treated dams exhibited increased striatal dopamine in late adolescence-a hallmark molecular biomarker of schizophrenia. These results validate the MIA model in a species more closely related to humans and open up new avenues for understanding the neurodevelopmental biology of schizophrenia and other neurodevelopmental disorders associated with prenatal immune challenge.

A magnetic resonance imaging study of early brain injury in a rat model of acute DFP intoxication.

Current treatments for seizures induced by organophosphates do not protect sufficiently against progressive neurodegeneration or delayed cognitive impairment. Developing more effective therapeutic approaches has been challenging because the pathogenesis of these delayed consequences is poorly defined. Using magnetic resonance imaging (MRI), we previously reported brain lesions that persist for months in a rat model of acute intoxication with the OP, diisopropylfluorophosphate (DFP). However, the early spatiotemporal progression of these lesions remains unknown. To address this data gap, we used in vivo MRI to longitudinally monitor brain lesions during the first 3 d following acute DFP intoxication. Adult male Sprague Dawley rats acutely intoxicated with DFP (4mg/kg, sc) were MR imaged at 6, 12, 18, 24, 48, 72h post-DFP, and their brains then taken for correlative histology to assess neurodegeneration using FluoroJade C (FJC) staining. Acute DFP intoxication elicited moderate-to-severe seizure activity. T2-weighted (T2w) anatomic imaging revealed prominent lesions within the thalamus, piriform cortex, cerebral cortex, hippocampus, corpus striatum, and substantia nigra that corresponded to neurodegeneration, evident as bands of FJC positive cells. Semi-quantitative assessment of lesion severity demonstrated significant regional variation in the onset and progression of injury, and suggested that lesion severity may be modulated by isoflurane anesthesia. These results imply that the timing of therapeutic intervention for attenuating brain injury following OP intoxication may be regionally dependent, and that longitudinal assessment of OP-induced damage by MRI may be a powerful tool for assessing therapeutic response.

Corrigendum: High-throughput discovery of novel developmental phenotypes.

This corrects the article DOI: 10.1038/nature19356.

Architecture of the rat nephron-arterial network: analysis with micro-computed tomography.

Among solid organs, the kidney's vascular network stands out, because each nephron has two distinct capillary structures in series and because tubuloglomerular feedback, one of the mechanisms responsible for blood flow autoregulation, is specific to renal tubules. Tubuloglomerular feedback and the myogenic mechanism, acting jointly, autoregulate single-nephron blood flow. Each generates a self-sustained periodic oscillation and an oscillating electrical signal that propagates upstream along arterioles. Similar electrical signals from other nephrons interact, allowing nephron synchronization. Experimental measurements show synchronization over fields of a few nephrons; simulations based on a simplified network structure that could obscure complex interactions predict more widespread synchronization. To permit more realistic simulations, we made a cast of blood vessels in a rat kidney, performed micro-computed tomography at 2.5-µm resolution, and recorded three-dimensional coordinates of arteries, afferent arterioles, and glomeruli. Nonterminal branches of arcuate arteries form treelike structures requiring two to six bifurcations to reach terminal branches at the tree tops. Terminal arterial structures were either paired branches at the tops of the arterial trees, from which 52.6% of all afferent arterioles originated, or unpaired arteries not at the tree tops, yielding the other 22.9%; the other 24.5% originated directly from nonterminal arteries. Afferent arterioles near the corticomedullary boundary were longer than those farther away, suggesting that juxtamedullary nephrons have longer afferent arterioles. The distance separating origins of pairs of afferent arterioles varied randomly. The results suggest an irregular-network tree structure with vascular nodes, where arteriolar activity and local blood pressure interact.

From the Cover: MagneticResonance Imaging Reveals Progressive Brain Injury in Rats Acutely Intoxicated With Diisopropylfluorophosphate.

Acute intoxication with organophosphates (OPs) can trigger seizures that progress to status epilepticus, and survivors often exhibit chronic neuropathology, cognitive impairment, affective disorders, and/or electroencephalographic abnormalities. Understanding how acute injury transitions to persistent neurological sequelae is critical to developing medical countermeasures for mitigating damage following OP-induced seizures. Here, we used in vivo magnetic resonance imaging (MRI) to monitor the spatiotemporal patterns of neuropathology for 1 month after acute intoxication with diisopropylfluorophosphate (DFP). Adult male Sprague Dawley rats administered pyridostigmine bromide (0.1 mg/kg, im) 30 min prior to successive administration of DFP (4 mg/kg, sc), atropine sulfate (2 mg/kg, im), and 2-pralidoxime (25 mg/kg, im) exhibited moderate-to-severe seizure behavior. T2-weighted and diffusion-weighted MR imaging prior to DFP exposure and at 3, 7, 14, 21, or 28 days postexposure revealed prominent lesions, tissue atrophy, and ventricular enlargement in discrete brain regions. Lesions varied in intensity and/or extent over time, with the overall magnitude of injury strongly influenced by seizure severity. Importantly, lesions detected by MRI correlated spatially and temporally with histological evidence of brain pathology. Analysis of histogram parameters extracted from frequency distributions of regional apparent diffusion coefficient (ADC) values identified the standard deviation and 90th percentile of the ADC as robust metrics for quantifying persistent and progressive neuropathological changes. The interanimal and interregional variations observed in lesion severity and progression, coupled with potential reinjury following spontaneous recurrent seizures, underscore the advantages of using in vivo imaging to longitudinally monitor neuropathology and, ultimately, therapeutic response, following acute OP intoxication.

Editor's Highlight: Spatiotemporal Progression and Remission of Lesions in the Rat Brain Following Acute Intoxication With Diisopropylfluorophosphate.

Similar to organophosphate (OP) nerve agents, diisopropylfluorophosphate (DFP) rapidly and irreversibly inhibits acetylcholinesterase, leading to convulsions that can progress to status epilepticus (SE). However, in contrast to the OP nerve agents, the long-term consequences of DFP-induced SE are not well known. Thus, we characterized the spatiotemporal profile of neuropathology during the first 2 months following acute DFP intoxication. Adult, male Sprague Dawley rats administered pyridostigmine bromide (0.1 mg/kg, im) 30 min prior to successive administration of DFP (4 mg/kg, sc), atropine sulfate (2 mg/kg, im), and 2-pralidoxime (25 mg/kg, im), exhibited moderate-to-severe seizure behavior, yet survived until euthanized at 0.5 to 60 days post exposure. Analyses of brains and hearts stained with hematoxylin-eosin, or of brains immunostained for neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP), or ionized binding adapter molecule 1 (IBA1), revealed progressive neuronal cell death, neuroinflammation, and tissue remodeling across limbic brain regions and the cerebral cortex, with no detectable pathology in the cerebellum or the heart. The lesion type and progression varied according to brain region and time after exposure. Across multiple brain regions, neuronal necrosis peaked after the first week, and neuroinflammation persisted at least 2 months after intoxication. Notably, mineralization was observed at later times in the thalamus, and to a more limited extent, in the hippocampus. Lesion severity was influenced by the initial seizure severity, and spontaneous recurrent seizures were associated with more severe brain damage. These findings parallel descriptions of neuropathology in preclinical models of acute intoxication with OP nerve agents, and other seizurogenic chemicals, suggesting conserved mechanisms of pathology downstream of chemical-induced SE.

Pair bond formation leads to a sustained increase in global cerebral glucose metabolism in monogamous male titi monkeys (Callicebus cupreus).

Social bonds, especially attachment relationships, are crucial to our health and happiness. However, what we know about the neural substrates of these bonds is almost exclusively limited to rodent models and correlational experiments in humans. Here, we used socially monogamous non-human primates, titi monkeys (Callicebus cupreus) to experimentally examine changes in regional and global cerebral glucose metabolism (GCGM) during the formation and maintenance of pair bonds. Baseline positron emission tomography (PET) scans were taken of thirteen unpaired male titi monkeys. Seven males were then experimentally paired with females, scanned and compared, after one week, to six age-matched control males. Five of the six control males were then also paired and scanned after one week. Scans were repeated on all males after four months of pairing. PET scans were coregistered with structural magnetic resonance imaging (MRI), and region of interest (ROI) analysis was carried out. A primary finding was that paired males showed a significant increase in [18F]-fluorodeoxyglucose (FDG) uptake in whole brain following one week of pairing, which is maintained out to four months. Dopaminergic, "motivational" areas and those involved in social behavior showed the greatest change in glucose uptake. In contrast, control areas changed only marginally more than GCGM. These findings confirm the large effects of social bonds on GCGM. They also suggest that more studies should examine how social manipulations affect whole-brain FDG uptake, as opposed to assuming that it does not change across condition.

Effects of pair bonding on dopamine D1 receptors in monogamous male titi monkeys (Callicebus cupreus).

Pair bonding leads to increases in dopamine D1 receptor (D1R) binding in the nucleus accumbens of monogamous prairie voles. In the current study, we hypothesized that there is similar up-regulation of D1R in a monogamous primate, the titi monkey (Callicebus cupreus). Receptor binding of the D1R antagonist [11 C]-SCH23390 was measured in male titi monkeys using PET scans before and after pairing with a female. We found that within-subject analyses of pairing show significant increases in D1R binding in the lateral septum, but not the nucleus accumbens, caudate, putamen, or ventral pallidum. The lateral septum is involved in a number of processes that may contribute to social behavior, including motivation, affect, reward, and reinforcement. This region also plays a role in pair bonding and paternal behavior in voles. Our observations of changes in D1R in the lateral septum, but not the nucleus accumbens, suggest that there may be broadly similar dopaminergic mechanisms underlying pair bonding across mammalian species, but that the specific changes to neural circuitry differ. This study is the first research to demonstrate neuroplasticity of the dopamine system following pair bonding in a non-human primate; however, substantial variability in the response to pairing suggests the utility of further research on the topic.

Imaging, Behavior and Endocrine Analysis of "Jealousy" in a Monogamous Primate.

Understanding the neurobiology of social bonding in non-human primates is a critical step in understanding the evolution of monogamy, as well as understanding the neural substrates for emotion and behavior. Coppery titi monkeys (Callicebus cupreus) form strong pair bonds, characterized by selective preference for their pair mate, mate-guarding, physiological and behavioral agitation upon separation, and social buffering. Mate-guarding, or the "maintenance" phase of pair bonding, is relatively under-studied in primates. In the current study, we used functional imaging to examine how male titi monkeys viewing their pair mate in close proximity to a stranger male would change regional cerebral glucose metabolism. We predicted that this situation would challenge the pair bond and induce "jealousy" in the males. Animals were injected with [18F]-fluorodeoxyglucose (FDG), returned to their cage for 30 min of conscious uptake, placed under anesthesia, and then scanned for 1 hour on a microPET P4 scanner. During the FDG uptake, males (n=8) had a view of either their female pair mate next to a stranger male ("jealousy" condition) or a stranger female next to a stranger male (control condition). Blood and cerebrospinal fluid samples were collected and assayed for testosterone, cortisol, oxytocin, and vasopressin. Positron emission tomography (PET) was co-registered with structural magnetic resonance imaging (MRI), and region of interest analysis was carried out. Bayesian multivariate multilevel analyses found that the right lateral septum (Pr(b>0)=93%), left posterior cingulate cortex (Pr(b>0)=99%), and left anterior cingulate (Pr(b>0)=96%) showed higher FDG uptake in the jealousy condition compared to the control condition, while the right medial amygdala (Pr(b>0)=85%) showed lower FDG uptake. Plasma testosterone and cortisol concentrations were higher during the jealousy condition. During the jealousy condition, duration of time spent looking across at the pair mate next to a stranger male was associated with higher plasma cortisol concentrations. The lateral septum has been shown to be involved in mate-guarding and mating-induced aggression in monogamous rodents, while the cingulate cortex has been linked to territoriality. These neural and physiological changes may underpin the emotion of jealousy, which can act in a monogamous species to preserve the long-term integrity of the pair.