Inhibiting Inducible Nitric Oxide Synthase with 1400W Reduces Soman (GD)-Induced Ferroptosis in Long-Term Epilepsy-Associated Neuropathology: Structural and Functional Magnetic Resonance Imaging Correlations with Neurobehavior and Brain Pathology.

Organophosphate (OP) nerve agent (OPNA) intoxication leads to long-term brain dysfunctions. The ineffectiveness of current treatments for OPNA intoxication prompts a quest for the investigation of the mechanism and an alternative effective therapeutic approach. Our previous studies on 1400W, a highly selective inducible nitric oxide synthase (iNOS) inhibitor, showed improvement in epilepsy and seizure-induced brain pathology in rat models of kainate and OP intoxication. In this study, magnetic resonance imaging (MRI) modalities, behavioral outcomes, and biomarkers were comprehensively investigated for brain abnormalities following soman (GD) intoxication in a rat model. T1 and T2 MRI robustly identified pathologic microchanges in brain structures associated with GD toxicity, and 1400W suppressed those aberrant alterations. Moreover, functional network reduction was evident in the cortex, hippocampus, and thalamus after GD exposure, and 1400W rescued the losses except in the thalamus. Behavioral tests showed protection by 1400W against GD-induced memory dysfunction, which also correlated with the extent of brain pathology observed in structural and functional MRIs. GD exposure upregulated iron-laden glial cells and ferritin levels in the brain and serum, 1400W decreased ferritin levels in the epileptic foci in the brain but not in the serum. The levels of brain ferritin also correlated with MRI parameters. Further, 1400W mitigated the overproduction of nitroxidative markers after GD exposure. Overall, this study provides direct evidence for the relationships of structural and functional MRI modalities with behavioral and molecular abnormalities following GD exposure and the neuroprotective effect of an iNOS inhibitor, 1400W. SIGNIFICANT STATEMENT: Our studies demonstrate the MRI microchanges in the brain following GD toxicity, which strongly correlate with neurobehavioral performances and iron homeostasis. The inhibition of iNOS with 1400W mitigates GD-induced cognitive decline, iron dysregulation, and aberrant brain MRI findings.

FoxO6 regulates Hippo signaling and growth of the craniofacial complex.

The mechanisms that regulate post-natal growth of the craniofacial complex and that ultimately determine the size and shape of our faces are not well understood. Hippo signaling is a general mechanism to control tissue growth and organ size, and although it is known that Hippo signaling functions in neural crest specification and patterning during embryogenesis and before birth, its specific role in postnatal craniofacial growth remains elusive. We have identified the transcription factor FoxO6 as an activator of Hippo signaling regulating neonatal growth of the face. During late stages of mouse development, FoxO6 is expressed specifically in craniofacial tissues and FoxO6-/- mice undergo expansion of the face, frontal cortex, olfactory component and skull. Enlargement of the mandible and maxilla and lengthening of the incisors in FoxO6-/- mice are associated with increases in cell proliferation. In vitro and in vivo studies demonstrated that FoxO6 activates Lats1 expression, thereby increasing Yap phosphorylation and activation of Hippo signaling. FoxO6-/- mice have significantly reduced Hippo Signaling caused by a decrease in Lats1 expression and decreases in Shh and Runx2 expression, suggesting that Shh and Runx2 are also linked to Hippo signaling. In vitro, FoxO6 activates Hippo reporter constructs and regulates cell proliferation. Furthermore PITX2, a regulator of Hippo signaling is associated with Axenfeld-Rieger Syndrome causing a flattened midface and we show that PITX2 activates FoxO6 expression. Craniofacial specific expression of FoxO6 postnatally regulates Hippo signaling and cell proliferation. Together, these results identify a FoxO6-Hippo regulatory pathway that controls skull growth, odontogenesis and face morphology.

Three-Dimensional GRE T1ρ mapping of the brain using tailored variable flip-angle scheduling.

PURPOSE: To introduce a new approach called tailored variable flip-angle (VFA) scheduling for SNR-efficient 3D T1ρ mapping of the brain using a magnetization-prepared gradient-echo sequence. METHODS: Simulations were used to assess the relative SNR efficiency, quantitative accuracy, and spatial blurring of tailored VFA scheduling for T1ρ mapping of brain tissue compared with magnetization-prepared angle-modulated partitioned k-space spoiled gradient-echo snapshots (MAPSS), a state-of-the-art technique for accurate 3D gradient-echo T1ρ mapping. Simulations were also used to calculate optimal imaging parameters for tailored VFA scheduling versus MAPSS, without and with nulling of CSF. Four participants were imaged at 3T MRI to demonstrate the feasibility of tailored VFA scheduling for T1ρ mapping of the brain. Using MAPSS as a reference standard, in vivo data were used to validate the relative SNR efficiency and quantitative accuracy of the new approach. RESULTS: Tailored VFA scheduling can provide a 2-fold to 4-fold gain in the SNR of the resulting T1ρ map as compared with MAPSS when using identical sequence parameters while limiting T1ρ quantification errors to 2% or less. In vivo whole-brain 3D T1ρ maps acquired with tailored VFA scheduling had superior SNR efficiency than is achievable with MAPSS, and the SNR efficiency improved with a greater number of views per segment. CONCLUSIONS: Tailored VFA scheduling is an SNR-efficient GRE technique for 3D T1ρ mapping of the brain that provides increased flexibility in choice of imaging parameters compared with MAPSS, which may benefit a variety of applications.

The BBSome Controls Energy Homeostasis by Mediating the Transport of the Leptin Receptor to the Plasma Membrane.

Bardet-Biedl syndrome (BBS) is a highly pleiotropic autosomal recessive disorder associated with a wide range of phenotypes including obesity. However, the underlying mechanism remains unclear. Here, we show that neuronal BBSome is a critical determinant of energy balance through its role in the regulation of the trafficking of the long signaling form of the leptin receptor (LRb). Targeted disruption of the BBSome by deleting the Bbs1 gene from the nervous system causes obesity in mice, and this phenotype is reproduced by ablation of the Bbs1 gene selectively in the LRb-expressing cells, but not from adipocytes. Obesity developed as a consequence of both increased food intake and decreased energy expenditure in mice lacking the Bbs1 gene in LRb-expressing cells. Strikingly, the well-known role of BBS proteins in the regulation of ciliary formation and function is unlikely to account for the obesogenic effect of BBS1 loss as disruption of the intraflagellar transport (IFT) machinery required for ciliogenesis by deleting the Ift88 gene in LRb-expressing cells caused a marginal increase in body weight and adiposity. Instead, we demonstrate that silencing BBS proteins, but not IFT88, impair the trafficking of the LRb to the plasma membrane leading to central leptin resistance in a manner independent of obesity. Our data also demonstrate that postnatal deletion of the Bbs1 gene in the mediobasal hypothalamus can cause obesity in mice, arguing against an early neurodevelopmental origin of obesity in BBS. Our results depict a novel mechanism underlying energy imbalance and obesity in BBS with potential implications in common forms of human obesity.

Computed Tomography and Magnetic Resonance Imaging for Longitudinal Characterization of Lung Structure Changes in a Yucatan Miniature Pig Silicosis Model.

Medical imaging is a rapidly advancing field enabling the repeated, noninvasive assessment of physiological structure and function. These beneficial characteristics can supplement studies in swine by mirroring the clinical functions of detection, diagnosis, and monitoring in humans. In addition, swine may serve as a human surrogate, facilitating the development and comparison of new imaging protocols for translation to humans. This study presents methods for pulmonary imaging developed for monitoring pulmonary disease initiation and progression in a pig exposure model with computed tomography and magnetic resonance imaging. In particular, a focus was placed on systematic processes, including positioning, image acquisition, and structured reporting to monitor longitudinal change. The image-based monitoring procedure was applied to 6 Yucatan miniature pigs. A subset of animals (n= 3) were injected with crystalline silica into the apical bronchial tree to induce silicosis. The methodology provided longitudinal monitoring and evidence of progressive lung disease while simultaneously allowing for a cross-modality comparative study highlighting the practical application of medical image data collection in swine. The integration of multimodality imaging with structured reporting allows for cross comparison of modalities, refinement of CT and MRI protocols, and consistently monitors potential areas of interest for guided biopsy and/or necropsy.

Precision-guided sampling schedules for efficient T1ρ mapping.

PURPOSE: To describe, assess, and implement a simple precision estimation framework for optimization of spin-lock time (TSL) sampling schedules for quantitative T1ρ mapping using a mono-exponential signal model. MATERIALS AND METHODS: A method is described for estimating T1ρ precision, and a cost function based on the precision estimates is evaluated to determine efficient TSL sampling schedules. The validity of the framework was tested by imaging a phantom with various sampling schedules and comparing theoretical and experimental precision values. The method utility was demonstrated with in vivo T1ρ mapping of brain tissue using a similar procedure as the phantom experiment. To assist investigators, optimal sampling schedules are tabulated for various tissue types and an online calculator is implemented. RESULTS: Theoretical and experimental precision values followed similar trends for both the phantom and in vivo experiments. The mean absolute percentage error (MAPE) of theoretical estimates of T1ρ map signal-to-noise ratio (SNR) was typically 5% in the phantom experiment and 33% in the in vivo demonstration. In both experiments, optimal TSL schedules yielded greater T1ρ map SNR efficiency than typical schedules. CONCLUSION: The framework can be used to improve the imaging efficiency of T1ρ mapping protocols and to guide selection of imaging parameters.

Detecting activity-evoked pH changes in human brain.

Localized pH changes have been suggested to occur in the brain during normal function. However, the existence of such pH changes has also been questioned. Lack of methods for noninvasively measuring pH with high spatial and temporal resolution has limited insight into this issue. Here we report that a magnetic resonance imaging (MRI) strategy, T(1) relaxation in the rotating frame (T(1)ρ), is sufficiently sensitive to detect widespread pH changes in the mouse and human brain evoked by systemically manipulating carbon dioxide or bicarbonate. Moreover, T(1)ρ detected a localized acidosis in the human visual cortex induced by a flashing checkerboard. Lactate measurements and pH-sensitive (31)P spectroscopy at the same site also identified a localized acidosis. Consistent with the established role for pH in blood flow recruitment, T(1)ρ correlated with blood oxygenation level-dependent contrast commonly used in functional MRI. However, T(1)ρ was not directly sensitive to blood oxygen content. These observations indicate that localized pH fluctuations occur in the human brain during normal function. Furthermore, they suggest a unique functional imaging strategy based on pH that is independent of traditional functional MRI contrast mechanisms.

Sarcolemma-localized nNOS is required to maintain activity after mild exercise.

Many neuromuscular conditions are characterized by an exaggerated exercise-induced fatigue response that is disproportionate to activity level. This fatigue is not necessarily correlated with greater central or peripheral fatigue in patients, and some patients experience severe fatigue without any demonstrable somatic disease. Except in myopathies that are due to specific metabolic defects, the mechanism underlying this type of fatigue remains unknown. With no treatment available, this form of inactivity is a major determinant of disability. Here we show, using mouse models, that this exaggerated fatigue response is distinct from a loss in specific force production by muscle, and that sarcolemma-localized signalling by neuronal nitric oxide synthase (nNOS) in skeletal muscle is required to maintain activity after mild exercise. We show that nNOS-null mice do not have muscle pathology and have no loss of muscle-specific force after exercise but do display this exaggerated fatigue response to mild exercise. In mouse models of nNOS mislocalization from the sarcolemma, prolonged inactivity was only relieved by pharmacologically enhancing the cGMP signal that results from muscle nNOS activation during the nitric oxide signalling response to mild exercise. Our findings suggest that the mechanism underlying the exaggerated fatigue response to mild exercise is a lack of contraction-induced signalling from sarcolemma-localized nNOS, which decreases cGMP-mediated vasomodulation in the vessels that supply active muscle after mild exercise. Sarcolemmal nNOS staining was decreased in patient biopsies from a large number of distinct myopathies, suggesting a common mechanism of fatigue. Our results suggest that patients with an exaggerated fatigue response to mild exercise would show clinical improvement in response to treatment strategies aimed at improving exercise-induced signalling.

Effect of lung inflation level on hyperpolarized 3He apparent diffusion coefficient measurements in never-smokers.

PURPOSE: To evaluate the effects of lung volume differences on apparent diffusion coefficient (ADC) measurements on a regional basis, with breath holds at volumes adjusted for differences in lung size across individuals according to the subject's vital capacity (VC). MATERIALS AND METHODS: This study was approved by the local institutional review board and was compliant with HIPAA. Informed consent was obtained from all subjects. Imaging was performed under a physician's Investigational New Drug application from the Food and Drug Administration. ADC changes as a function of inflation levels were evaluated in 24 healthy never-smokers across three lung volumes (20%, 60%, and 100% VC) on the basis of the spirometric data collected from each subject. Response variables based on lung volume and anatomic position were assessed with multifactorial analysis of variance followed by posthoc pair-wise testing. Imaging was performed with a 1.5-T magnetic resonance (MR) unit with use of a two-dimensional gradient-echo fast low-angle shot sequence. RESULTS: Significant differences in ADCs between lung volumes were observed for all inflation levels (20%, 60%, and 100% VC; P < .001), along with significant dependent-nondependent vertical gradients at 20% VC (P < .0001) and 60% VC (P < .0001, left lung only). In addition, significant differences between mean values in the left and right lungs with respect to those in the whole lung were observed at the lower lung inflation levels (20% and 60% VC, P < .01), reaching more uniform expansion at 100% VC. CONCLUSION: The results confirm known anatomic differences in patterns of regional inflation and ventilation with corresponding lung volume changes, emphasizing the need for tight control over lung volume when performing hyperpolarized helium 3 ((3)He) lung studies if (3)He MR imaging is to be used to follow up small longitudinal changes in lung abnormalities.

Acute hydrogen sulfide-induced neurochemical and morphological changes in the brainstem.

Hydrogen sulfide (H2S) is a toxin affecting the cardiovascular, respiratory, and central nervous systems. Acute H2S exposure is associated with a high rate of mortality and morbidity. The precise pathophysiology of H2S-induced death is a controversial topic; however, inhibition of the respiratory center in the brainstem is commonly cited as a cause of death. There is a knowledge gap on toxicity and toxic mechanisms of acute H2S poisoning on the brainstem, a brain region responsible for regulating many reflective and vital functions. Serotonin (5-HT), dopamine (DA), and γ-aminobutyric acid (GABA) play a role in maintaining a normal stable respiratory rhythmicity. We hypothesized that the inhibitory respiratory effects of H2S poisoning are mediated by 5-HT in the respiratory center of the brainstem. Male C57BL/6 mice were exposed once to an LCt50 concentration of H2S (1000 ppm). Batches of surviving mice were euthanized at 5 min, 2 h, 12 h, 24 h, 72 h, and on day 7 post-exposure. Pulmonary function, vigilance state, and mortality were monitored during exposure. The brainstem was analyzed for DA, 3,4-dehydroxyphenyl acetic acid (DOPAC), 5-HT, 5-hydroxyindoleatic acid (5-HIAA), norepinephrine (NE), GABA, glutamate, and glycine using HPLC. Enzymatic activities of monoamine oxidases (MAO) were also measured in the brainstem using commercial kits. Neurodegeneration was assessed using immunohistochemistry and magnetic resonance imaging. Results showed that DA and DOPAC were significantly increased at 5 min post H2S exposure. However, by 2 h DA returned to normal. Activities of MAO were significantly increased at 5 min and 2 h post-exposure. In contrast, NE was significantly decreased at 5 min and 2 h post-exposure. Glutamate was overly sensitive to H2S-induced toxicity manifesting a time-dependent concentration reduction throughout the 7 day duration of the study. Remarkably, there were no changes in 5-HT, 5-HIAA, glycine, or GABA concentrations. Cytochrome c oxidase activity was inhibited but recovered by 24 h. Neurodegeneration was observed starting at 72 h post H2S exposure in select brainstem regions. We conclude that acute H2S exposure causes differential effects on brainstem neurotransmitters. H2S also induces neurodegeneration and biochemical changes in the brainstem. Additional work is needed to fully understand the implications of both the short- and long-term effects of acute H2S poisoning on vital functions regulated by the brainstem.

Sex-based structural and functional MRI outcomes in the rat brain after soman (GD) exposure-induced status epilepticus.

OBJECTIVE: Exposure to the nerve agent, soman (GD), induces status epilepticus (SE), epileptogenesis, and even death. Although rodent models studying the pathophysiological mechanisms show females to be more reactive to soman, no tangible sex differences in brains postexposure have been reported. In this study, we used multimodal imaging using MRI in adult rats to determine potential sex-based biomarkers of soman effects. METHODS: Male and female Sprague Dawley rats were challenged with 1.2 × LD50 soman followed by medical countermeasures. Ten weeks later, the brains were analyzed via structural and functional MRI. RESULTS: Despite no significant sex differences in the initial SE severity after soman exposure, long-term MRI-based structural and functional differences were evident in the brains of both sexes. While T2 MRI showed lesser soman-induced neurodegeneration, large areas of T1 enhancements occurred in females than in males, indicating a distinct pathophysiology unrelated to neurodegeneration. fMRI-based resting-state functional connectivity (RSFC), indicated greater reductions in soman-exposed females than in males, associating with the T1 enhancements (unrelated to neurodegeneration) rather than T2-hyperintensity or T1-hypointensity (representing neurodegeneration). The wider T1 enhancements associating with the decreased spontaneous neuronal activity in multiple resting-state networks in soman-exposed females than males suggest that neural changes unrelated to cellular atrophy impinge on brain function postexposure. Taken together with lower spontaneous neural activity in soman-exposed females, the results indicate some form of neuroprotective state that was not present in males. SIGNIFICANCE: The results indicate that endpoints other than neurodegeneration may need to be considered to translate sex-based nerve agent effects in humans.

The BBSome regulates mitochondria dynamics and function.

OBJECTIVE: The essential role of mitochondria in regulation of metabolic function and other physiological processes has garnered enormous interest in understanding the mechanisms controlling the function of this organelle. We assessed the role of the BBSome, a protein complex composed of eight Bardet-Biedl syndrome (BBS) proteins, in the control of mitochondria dynamic and function. METHODS: We used a multidisciplinary approach that include CRISPR/Cas9 technology-mediated generation of a stable Bbs1 gene knockout hypothalamic N39 neuronal cell line. We also analyzed the phenotype of BBSome deficient mice in presence or absence of the gene encoding A-kinase anchoring protein 1 (AKAP1). RESULTS: Our data show that the BBSome play an important role in the regulation of mitochondria dynamics and function. Disruption of the BBSome cause mitochondria hyperfusion in cell lines, fibroblasts derived from patients as well as in hypothalamic neurons and brown adipocytes of mice. The morphological changes in mitochondria translate into functional abnormalities as indicated by the reduced oxygen consumption rate and altered mitochondrial distribution and calcium handling. Mechanistically, we demonstrate that the BBSome modulates the activity of dynamin-like protein 1 (DRP1), a key regulator of mitochondrial fission, by regulating its phosphorylation and translocation to the mitochondria. Notably, rescuing the decrease in DRP1 activity through deletion of one copy of the gene encoding AKAP1 was effective to normalize the defects in mitochondrial morphology and activity induced by BBSome deficiency. Importantly, this was associated with improvement in several of the phenotypes caused by loss of the BBSome such as the neuroanatomical abnormalities, metabolic alterations and obesity highlighting the importance of mitochondria defects in the pathophysiology of BBS. CONCLUSIONS: These findings demonstrate a critical role of the BBSome in the modulation of mitochondria function and point to mitochondrial defects as a key disease mechanism in BBS.

Molecular heterogeneity in the choroid plexus epithelium: the 22-member γ-protocadherin family is differentially expressed, apically localized, and implicated in CSF regulation.

The choroid plexus (CP) epithelium develops from the ependyma that lines the ventricular system, and plays a critical role in the development and function of the brain. In addition to being the primary site of CSF production, the CP maintains the blood-CSF barrier via apical tight junctions between epithelial cells. Here we show that the 22-member γ-protocadherin (γ-Pcdh) family of cell adhesion molecules, which we have implicated previously in synaptogenesis and neuronal survival, is highly expressed by both CP epithelial and ependymal cells, in which γ-Pcdh protein localization is, surprisingly, tightly restricted to the apical membrane. Multi-label immunostaining demonstrates that γ-Pcdhs are excluded from tight junctions, basolateral adherens junctions, and apical cilia tufts. RT-PCR analysis indicates that, as a whole, the CP expresses most members of the Pcdh-γ gene family. Immunostaining using novel monoclonal antibodies specific for single γ-Pcdh proteins shows that individual epithelial cells differ in their apically localized γ-Pcdh repertoire. Restricted mutation of the Pcdh-γ locus in the choroid plexus and ependyma leads to significant reductions in ventricular volume, without obvious disruptions of epithelial apical-basal polarity. Together, these results suggest an unsuspected role for the γ-Pcdhs in CSF production and demonstrate a surprising molecular heterogeneity in the CP epithelium.

Slow disease progression in a C57BL/6 pten-deficient mouse model of prostate cancer.

Prostate-specific deletion of Pten in mice has been reported to recapitulate histological progression of human prostate cancer. To improve on this model, we introduced the conditional ROSA26 luciferase reporter allele to monitor prostate cancer progression via bioluminescence imaging and extensively backcrossed mice onto the albino C57BL/6 genetic background to address variability in tumor kinetics and to enhance imaging sensitivity. Bioluminescence signal increased rapidly in Pten(p-/-) mice from 3 to 11 weeks, but was much slower from 11 to 52 weeks. Changes in bioluminescence signal were correlated with epithelial proliferation. Magnetic resonance imaging revealed progressive increases in prostate volume, which were attributed to excessive fluid retention in the anterior prostate and to expansion of the stroma. Development of invasive prostate cancer in 52-week-old Pten(p-/-) mice was rare, indicating that disease progression was slowed relative to that in previous reports. Tumors in these mice exhibited a spontaneous inflammatory phenotype and were rapidly infiltrated by myeloid-derived suppressor cells. Although Pten(p-/-) tumors responded to androgen withdrawal, they failed to exhibit relapsed growth for up to 1 year. Taken together, these data identify a mild prostate cancer phenotype in C57BL/6 prostate-specific Pten-deficient mice, reflecting effects of the C57BL/6 genetic background on cancer progression. This model provides a platform for noninvasive assessment of how genetic and environmental risk factors may affect disease progression.

Leptin resistance contributes to obesity and hypertension in mouse models of Bardet-Biedl syndrome.

Bardet-Biedl syndrome (BBS) is a heterogeneous genetic disorder characterized by many features, including obesity and cardiovascular disease. We previously developed knockout mouse models of 3 BBS genes: BBS2, BBS4, and BBS6. To dissect the mechanisms involved in the metabolic disorders associated with BBS, we assessed the development of obesity in these mouse models and found that BBS-null mice were hyperphagic, had low locomotor activity, and had elevated circulating levels of the hormone leptin. The effect of exogenous leptin on body weight and food intake was attenuated in BBS mice, which suggests that leptin resistance may contribute to hyperleptinemia. In other mouse models of obesity, leptin resistance may be selective rather than systemic; although mice became resistant to leptin's anorectic effects, the ability to increase renal sympathetic nerve activity (SNA) was preserved. Although all 3 of the BBS mouse models were similarly resistant to leptin, the sensitivity of renal SNA to leptin was maintained in Bbs4 -/- and Bbs6 -/- mice, but not in Bbs2 -/- mice. Consequently, Bbs4 -/- and Bbs6 -/- mice had higher baseline renal SNA and arterial pressure and a greater reduction in arterial pressure in response to ganglionic blockade. Furthermore, we found that BBS mice had a decreased hypothalamic expression of proopiomelanocortin, which suggests that BBS genes play an important role in maintaining leptin sensitivity in proopiomelanocortin neurons.

A knockin mouse model of the Bardet-Biedl syndrome 1 M390R mutation has cilia defects, ventriculomegaly, retinopathy, and obesity.

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous disorder that results in retinal degeneration, obesity, cognitive impairment, polydactyly, renal abnormalities, and hypogenitalism. Of the 12 known BBS genes, BBS1 is the most commonly mutated, and a single missense mutation (M390R) accounts for approximately 80% of BBS1 cases. To gain insight into the function of BBS1, we generated a Bbs1(M390R/M390R) knockin mouse model. Mice homozygous for the M390R mutation recapitulated aspects of the human phenotype, including retinal degeneration, male infertility, and obesity. The obese mutant mice were hyperphagic and hyperleptinemic and exhibited reduced locomotor activity but no elevation in mean arterial blood pressure. Morphological evaluation of Bbs1 mutant brain neuroanatomy revealed ventriculomegaly of the lateral and third ventricles, thinning of the cerebral cortex, and reduced volume of the corpus striatum and hippocampus. Similar abnormalities were also observed in the brains of Bbs2(-/-), Bbs4(-/-), and Bbs6(-/-) mice, establishing these neuroanatomical defects as a previously undescribed BBS mouse model phenotype. Ultrastructural examination of the ependymal cell cilia that line the enlarged third ventricle of the Bbs1 mutant brains showed that, whereas the 9 + 2 arrangement of axonemal microtubules was intact, elongated cilia and cilia with abnormally swollen distal ends were present. Together with data from transmission electron microscopy analysis of photoreceptor cell connecting cilia, the Bbs1 M390R mutation does not affect axonemal structure, but it may play a role in the regulation of cilia assembly and/or function.

Transcriptomic profile analysis of brain inferior colliculus following acute hydrogen sulfide exposure.

Hydrogen sulfide (H2S) is a gaseous molecule found naturally in the environment, and as an industrial byproduct, and is known to cause acute death and induces long-term neurological disorders following acute high dose exposures. Currently, there is no drug approved for treatment of acute H2S-induced neurotoxicity and/or neurological sequelae. Lack of a deep understanding of pathogenesis of H2S-induced neurotoxicity has delayed the development of appropriate therapeutic drugs that target H2S-induced neuropathology. RNA sequencing analysis was performed to elucidate the cellular and molecular mechanisms of H2S-induced neurodegeneration, and to identify key molecular elements and pathways that contribute to H2S-induced neurotoxicity. C57BL/6J mice were exposed by whole body inhalation to 700 ppm of H2S for either one day, two consecutive days or 4 consecutive days. Magnetic resonance imaging (MRI) scan analyses showed H2S exposure induced lesions in the inferior colliculus (IC) and thalamus (TH). This mechanistic study focused on the IC. RNA Sequencing analysis revealed that mice exposed once, twice, or 4 times had 283, 193 and 296 differentially expressed genes (DEG), respectively (q-value < 0.05, fold-change> 1.5). Hydrogen sulfide exposure modulated multiple biological pathways including unfolded protein response, neurotransmitters, oxidative stress, hypoxia, calcium signaling, and inflammatory response in the IC. Hydrogen sulfide exposure activated PI3K/Akt and MAPK signaling pathways. Pro-inflammatory cytokines were shown to be potential initiators of the modulated signaling pathways following H2S exposure. Furthermore, microglia were shown to release IL-18 and astrocytes released both IL-1ß and IL-18 in response to H2S. This transcriptomic analysis data revealed complex signaling pathways involved in H2S-induced neurotoxicity and may provide important associated mechanistic insights.

The podosomal-adaptor protein SH3PXD2B is essential for normal postnatal development.

Podosome-type adhesions are actin-based membrane protrusions involved in cell-matrix adhesion and extracellular matrix degradation. Despite growing knowledge of many proteins associated with podosome-type adhesions, much remains unknown concerning the function of podosomal proteins at the level of the whole animal. In this study, the spontaneous mouse mutant nee was used to identify a component of podosome-type adhesions that is essential for normal postnatal growth and development. Mice homozygous for the nee allele exhibited runted growth, craniofacial and skeletal abnormalities, ocular anterior segment dysgenesis, and hearing impairment. Adults also exhibited infertility and a form of lipodystrophy. Using genetic mapping and DNA sequencing, the cause of nee phenotypes was identified as a 1-bp deletion within the Sh3pxd2b gene on mouse Chromosome 11. Whereas the wild-type Sh3pxd2b gene is predicted to encode a protein with one PX domain and four SH3 domains, the nee mutation is predicted to cause a frameshift and a protein truncation altering a portion of the third SH3 domain and deleting all of the fourth SH3 domain. The SH3PXD2B protein is believed to be an important component of podosomes likely to mediate protein-protein interactions with membrane-spanning metalloproteinases. Testing this directly, SH3PXD2B localized to podosomes in constitutively active Src-transfected fibroblasts and through its last SH3 domain associated with a transmembrane member of a disintegrin and metalloproteinase family of proteins, ADAM15. These results identify SH3PXD2B as a podosomal-adaptor protein required for postnatal growth and development, particularly within physiologic contexts involving extracellular matrix regulation.

Biventricular adaptation to volume overload in mice with aortic regurgitation.

BACKGROUND: Aortic valve regurgitation is usually caused by impaired coaptation of the aortic valve cusps during diastole. Hypercholesterolemia produces aortic valve lipid deposition, fibrosis, and calcification in both mice and humans, which could impair coaptation of cusps. However, a link between hypercholesterolemia and aortic regurgitation has not been established in either species. The purpose of this study was to ascertain the prevalence of aortic regurgitation in hypercholesterolemic mice and to determine its impact on the left and right ventricles. METHODS AND RESULTS: Eighty Ldlr-/-/Apob100/100/Mttpfl/fl/Mx1Cre+/+ ("Reversa") hypercholesterolemic mice and 40 control mice were screened for aortic regurgitation (AR) with magnetic resonance imaging at age 7.5 months. The prevalence of AR was 40% in Reversa mice, with moderate or severe regurgitation (AR+) in 19% of mice. In control mice, AR prevalence was 13% (p = 0.004 vs. Reversa), and was invariably trace or mild in severity. In-depth evaluation of cardiac response to volume overload was performed in 12 AR-positive and 12 AR-negative Reversa mice. Regurgitant fraction was 0.34 +/- 0.04 in AR-positive vs. 0.02 +/- 0.01 in AR-negative (mean +/- SE; p < 0.001). AR-positive mice had significantly increased left ventricular end-diastolic volume and mass and reduced ejection fraction in both ventricles. When left ventricular ejection fraction fell below 0.60 in AR-positive (n = 7) mice, remodeling occurred and right ventricular systolic function progressively worsened. CONCLUSION: Hypercholesterolemia causes aortic valve regurgitation with moderate prevalence in mice. When present, aortic valve regurgitation causes volume overload and pathological remodeling of both ventricles.

Effects of deep sedation or general anesthesia on cardiac function in mice undergoing cardiovascular magnetic resonance.

BACKGROUND: Genetically engineered mouse models of human cardiovascular disease provide an opportunity to understand critical pathophysiological mechanisms. Cardiovascular magnetic resonance (CMR) provides precise reproducible assessment of cardiac structure and function, but, in contrast to echocardiography, requires that the animal be immobilized during image acquisition. General anesthetic regimens yield satisfactory images, but have the potential to significantly perturb cardiac function. The purpose of this study was to assess the effects of general anesthesia and a new deep sedation regimen, respectively, on cardiac function in mice as determined by CMR, and to compare them to results obtained in mildly sedated conscious mice by echocardiography. RESULTS: In 6 mildly sedated normal conscious mice assessed by echo, heart rate was 615 +/- 25 min-1 (mean +/- SE) and left ventricular ejection fraction (LVEF) was 0.94 +/- 0.01. In the CMR studies of normal mice, heart rate was slightly lower during deep sedation with morphine/midazolam (583 +/- 30 min-1), but the difference was not statistically significant. General anesthesia with 1% inhaled isoflurane significantly depressed heart rate (468 +/- 7 min-1, p < 0.05 vs. conscious sedation). In 6 additional mice with ischemic LV failure, trends in heart rate were similar, but not statistically significant. In normal mice, deep sedation depressed LVEF (0.79 +/- 0.04, p < 0.05 compared to light sedation), but to a significantly lesser extent than general anesthesia (0.60 +/- 0.04, p < 0.05 vs. deep sedation). In mice with ischemic LV failure, ejection fraction measurements were comparable when performed during light sedation, deep sedation, and general anesthesia, respectively. Contrast-to-noise ratios were similar during deep sedation and during general anesthesia, indicating comparable image quality. Left ventricular mass measurements made by CMR during deep sedation were nearly identical to those made during general anesthesia (r2 = 0.99, mean absolute difference < 4%), indicating equivalent quantitative accuracy obtained with the two methods. The imaging procedures were well-tolerated in all mice. CONCLUSION: In mice with normal cardiac function, CMR during deep sedation causes significantly less depression of heart rate and ejection fraction than imaging during general anesthesia with isoflurane. In mice with heart failure, the sedation/anesthesia regimen had no clear impact on cardiac function. Deep sedation and general anesthesia produced CMR with comparable image quality and quantitative accuracy.