What is the C2 pedicle, pars interarticularis, and isthmus? Anatomical study and review of the literature regarding these confusing terms with proposal of new nomenclature.

INTRODUCTION: The atypical anatomy of the C2 vertebra has led to terminological discrepancies within reports and studies in the literature regarding the location of its pedicle, pars interarticularis, and isthmus. These discrepancies not only limit the power of morphometric analyses, but they also confuse technical reports regarding operations involving C2, and thus confuse our ability to properly communicate this anatomy. Herein, we examine the variations in nomenclature regarding the pedicle, pars interarticularis, and isthmus of C2, and via an anatomical study, propose new terminology. METHODS: The articular surface and underlying superior and inferior articular processes and adjacent transverse processes were removed from 15 C2 vertebrae (30 sides). Specifically, the areas regarded as the pedicle, pars interarticularis, and isthmus were evaluated. Morphometrics were performed. RESULTS: Our results indicate that, anatomically, C2 has no "isthmus" and that a pars interarticularis for C2, when present, is very short. Deconstruction of the attached parts allowed for visualization of a bony arch extending from the anterior most aspect of the lamina to the body of C2. The arch is composed almost entirely of trabecular bone and without its attached parts, e.g., transverse process, really has no cortical bone laterally. CONCLUSIONS: We propose a more accurate terminology, the pedicle, for pars/pedicle screw placement of C2. Such a term more accurately describes this unique structure of the C2 vertebra and would alleviate terminological confusion in the future literature on this topic.

From OCR and ECAR to energy: Perspectives on the design and interpretation of bioenergetics studies.

Biological energy transduction underlies all physiological phenomena in cells. The metabolic systems that support energy transduction have been of great interest due to their association with numerous pathologies including diabetes, cancer, rare genetic diseases, and aberrant cell death. Commercially available bioenergetics technologies (e.g., extracellular flux analysis, high-resolution respirometry, fluorescent dye kits, etc.) have made practical assessment of metabolic parameters widely accessible. This has facilitated an explosion in the number of studies exploring, in particular, the biological implications of oxygen consumption rate (OCR) and substrate level phosphorylation via glycolysis (i.e., via extracellular acidification rate (ECAR)). Though these technologies have demonstrated substantial utility and broad applicability to cell biology research, they are also susceptible to historical assumptions, experimental limitations, and other caveats that have led to premature and/or erroneous interpretations. This review enumerates various important considerations for designing and interpreting cellular and mitochondrial bioenergetics experiments, some common challenges and pitfalls in data interpretation, and some potential "next steps" to be taken that can address these highlighted challenges.

Flux through mitochondrial redox circuits linked to nicotinamide nucleotide transhydrogenase generates counterbalance changes in energy expenditure.

Compensatory changes in energy expenditure occur in response to positive and negative energy balance, but the underlying mechanism remains unclear. Under low energy demand, the mitochondrial electron transport system is particularly sensitive to added energy supply (i.e. reductive stress), which exponentially increases the rate of H2O2 (JH2O2) production. H2O2 is reduced to H2O by electrons supplied by NADPH. NADP+ is reduced back to NADPH by activation of mitochondrial membrane potential-dependent nicotinamide nucleotide transhydrogenase (NNT). The coupling of reductive stress-induced JH2O2 production to NNT-linked redox buffering circuits provides a potential means of integrating energy balance with energy expenditure. To test this hypothesis, energy supply was manipulated by varying flux rate through ß-oxidation in muscle mitochondria minus/plus pharmacological or genetic inhibition of redox buffering circuits. Here we show during both non-ADP- and low-ADP-stimulated respiration that accelerating flux through ß-oxidation generates a corresponding increase in mitochondrial JH2O2 production, that the majority (∼70-80%) of H2O2 produced is reduced to H2O by electrons drawn from redox buffering circuits supplied by NADPH, and that the rate of electron flux through redox buffering circuits is directly linked to changes in oxygen consumption mediated by NNT. These findings provide evidence that redox reactions within ß-oxidation and the electron transport system serve as a barometer of substrate flux relative to demand, continuously adjusting JH2O2 production and, in turn, the rate at which energy is expended via NNT-mediated proton conductance. This variable flux through redox circuits provides a potential compensatory mechanism for fine-tuning energy expenditure to energy balance in real time.

Genetically increasing flux through ß-oxidation in skeletal muscle increases mitochondrial reductive stress and glucose intolerance.

Elevated mitochondrial hydrogen peroxide (H2O2) emission and an oxidative shift in cytosolic redox environment have been linked to high-fat-diet-induced insulin resistance in skeletal muscle. To test specifically whether increased flux through mitochondrial fatty acid oxidation, in the absence of elevated energy demand, directly alters mitochondrial function and redox state in muscle, two genetic models characterized by increased muscle ß-oxidation flux were studied. In mice overexpressing peroxisome proliferator-activated receptor-α in muscle (MCK-PPARα), lipid-supported mitochondrial respiration, membrane potential (ΔΨm), and H2O2 production rate (JH2O2) were increased, which coincided with a more oxidized cytosolic redox environment, reduced muscle glucose uptake, and whole body glucose intolerance despite an increased rate of energy expenditure. Similar results were observed in lipin-1-deficient, fatty-liver dystrophic mice, another model characterized by increased ß-oxidation flux and glucose intolerance. Crossing MCAT (mitochondria-targeted catalase) with MCK-PPARα mice normalized JH2O2 production, redox environment, and glucose tolerance, but surprisingly, both basal and absolute insulin-stimulated rates of glucose uptake in muscle remained depressed. Also surprising, when placed on a high-fat diet, MCK-PPARα mice were characterized by much lower whole body, fat, and lean mass as well as improved glucose tolerance relative to wild-type mice, providing additional evidence that overexpression of PPARα in muscle imposes more extensive metabolic stress than experienced by wild-type mice on a high-fat diet. Overall, the findings suggest that driving an increase in skeletal muscle fatty acid oxidation in the absence of metabolic demand imposes mitochondrial reductive stress and elicits multiple counterbalance metabolic responses in an attempt to restore bioenergetic homeostasis.NEW & NOTEWORTHY Prior work has suggested that mitochondrial dysfunction is an underlying cause of insulin resistance in muscle because it limits fatty acid oxidation and therefore leads to the accumulation of cytotoxic lipid intermediates. The implication has been that therapeutic strategies to accelerate ß-oxidation will be protective. The current study provides evidence that genetically increasing flux through ß-oxidation in muscle imposes reductive stress that is not beneficial but rather detrimental to metabolic regulation.

Racial differences in the limb skeletal muscle transcriptional programs of patients with critical limb ischemia.

Critical limb ischemia (CLI) is the most severe manifestation of peripheral artery disease (PAD) and is characterized by high rates of morbidity and mortality. As with most severe cardiovascular disease manifestations, Black individuals disproportionately present with CLI. Accordingly, there remains a clear need to better understand the reasons for this discrepancy and to facilitate personalized therapeutic options specific for this population. Gastrocnemius muscle was obtained from White and Black healthy adult volunteers and patients with CLI for whole transcriptome shotgun sequencing (WTSS) and enrichment analysis was performed to identify alterations in specific Reactome pathways. When compared to their race-matched healthy controls, both White and Black patients with CLI demonstrated similar reductions in nuclear and mitochondrial encoded genes and mitochondrial oxygen consumption across multiple substrates, indicating a common bioenergetic paradigm associated with amputation outcomes regardless of race. Direct comparisons between tissues of White and Black patients with CLI revealed hemostasis, extracellular matrix organization, platelet regulation, and vascular wall interactions to be uniquely altered in limb muscles of Black individuals. Among traditional vascular growth factor signaling targets, WTSS revealed only Tie1 to be significantly altered from White levels in Black limb muscle tissues. Quantitative reverse transcription polymerase chain reaction validation of select identified targets verified WTSS directional changes and supports reductions in MMP9 and increases in NUDT4P1 and GRIK2 as unique to limb muscles of Black patients with CLI. This represents a critical first step in better understanding the transcriptional program similarities and differences between Black and White patients in the setting of amputations related to CLI and provides a promising start for therapeutic development in this population.

Strain-Dependent Variation in Acute Ischemic Muscle Injury.

Limited efficacy of clinical interventions for peripheral arterial disease necessitates a better understanding of the environmental and genetic determinants of tissue pathology. Existing research has largely ignored the early skeletal muscle injury response during hind limb ischemia (HLI). We compared the hind limb muscle response, after 6 hours of ischemia, in two mouse strains that differ dramatically in their postischemic extended recovery: C57BL/6J and BALB/cJ. Perfusion, measured by laser Doppler and normalized to the control limb, differed only slightly between strains after HLI (<12% across all measures). Similar (<10%) effect sizes in lectin-perfused vessel area and no differences in tissue oxygen saturation measured by reflectance spectroscopy were also found. Muscles from both strains were functionally impaired after HLI, but greater muscle necrosis and loss of dystrophin-positive immunostaining were observed in BALB/cJ muscle compared with C57BL/6J. Muscle cell-specific dystrophin loss and reduced viability were also detected in additional models of ischemia that were independent of residual perfusion differences. Our results indicate that factors other than the completeness of ischemia alone (ie, background genetics) influence the magnitude of acute ischemic muscle injury. These findings may have implications for future development of therapeutic interventions for limb ischemia and for understanding the phasic etiology of chronic and acute ischemic muscle pathophysiology.

Our current understanding of the lymphatics of the brain and spinal cord.

The lymphatic system, segregated from the blood vascular system, is an essential anatomical route along which interstitial fluid, solutes, lipids, immune cells, and cellular debris, are conveyed. However, the way these mechanisms operate within the cranial compartment is mostly unknown. Herein, we review current understanding of the meningeal lymphatics, described anatomically over a century ago yet still poorly understood from a functional standpoint. We will delineate the cellular mechanisms by which the meningeal lymphatics are formed and discuss their unique anatomy. Furthermore, this review will discuss the recently-coined "glymphatic system" and the manner by which cerebrospinal fluid (CSF) and interstitial fluid (ISF) are exchanged and thus drained by the meningeal lymphatic vasculature as a key route for conveying cellular waste, solutes, and immune traffic to the deep cervical lymph nodes. The clinical relevance of the meningeal lymphatics will also be described, as they are relevant to various common defects of the lymphatic system. Clin. Anat. 32:117-121, 2019. © 2018 Wiley Periodicals, Inc.

BAG3 (Bcl-2-Associated Athanogene-3) Coding Variant in Mice Determines Susceptibility to Ischemic Limb Muscle Myopathy by Directing Autophagy.

BACKGROUND: Critical limb ischemia is a manifestation of peripheral artery disease that carries significant mortality and morbidity risk in humans, although its genetic determinants remain largely unknown. We previously discovered 2 overlapping quantitative trait loci in mice, Lsq-1 and Civq-1, that affected limb muscle survival and stroke volume after femoral artery or middle cerebral artery ligation, respectively. Here, we report that a Bag3 variant (Ile81Met) segregates with tissue protection from hind-limb ischemia. METHODS: We treated mice with either adeno-associated viruses encoding a control (green fluorescent protein) or 2 BAG3 (Bcl-2-associated athanogene-3) variants, namely Met81 or Ile81, and subjected the mice to hind-limb ischemia. RESULTS: We found that the BAG3 Ile81Met variant in the C57BL/6 (BL6) mouse background segregates with protection from tissue necrosis in a shorter congenic fragment of Lsq-1 (C.B6-Lsq1-3). BALB/c mice treated with adeno-associated virus encoding the BL6 BAG3 variant (Ile81; n=25) displayed reduced limb-tissue necrosis and increased limb tissue perfusion compared with Met81- (n=25) or green fluorescent protein- (n=29) expressing animals. BAG3Ile81, but not BAG3Met81, improved ischemic muscle myopathy and muscle precursor cell differentiation and improved muscle regeneration in a separate, toxin-induced model of injury. Systemic injection of adeno-associated virus-BAG3Ile81 (n=9), but not BAG3Met81 (n=10) or green fluorescent protein (n=5), improved ischemic limb blood flow and limb muscle histology and restored muscle function (force production). Compared with BAG3Met81, BAG3Ile81 displayed improved binding to the small heat shock protein (HspB8) in ischemic skeletal muscle cells and enhanced ischemic muscle autophagic flux. CONCLUSIONS: Taken together, our data demonstrate that genetic variation in BAG3 plays an important role in the prevention of ischemic tissue necrosis. These results highlight a pathway that preserves tissue survival and muscle function in the setting of ischemia.

Diminished force production and mitochondrial respiratory deficits are strain-dependent myopathies of subacute limb ischemia.

OBJECTIVE: Reduced skeletal muscle mitochondrial function might be a contributing mechanism to the myopathy and activity based limitations that typically plague patients with peripheral arterial disease (PAD). We hypothesized that mitochondrial dysfunction, myofiber atrophy, and muscle contractile deficits are inherently determined by the genetic background of regenerating ischemic mouse skeletal muscle, similar to how patient genetics affect the distribution of disease severity with clinical PAD. METHODS: Genetically ischemia protected (C57BL/6) and susceptible (BALB/c) mice underwent either unilateral subacute hind limb ischemia (SLI) or myotoxic injury (cardiotoxin) for 28 days. Limbs were monitored for blood flow and tissue oxygen saturation and tissue was collected for the assessment of histology, muscle contractile force, gene expression, mitochondrial content, and respiratory function. RESULTS: Despite similar tissue O2 saturation and mitochondrial content between strains, BALB/c mice suffered persistent ischemic myofiber atrophy (55.3% of C57BL/6) and muscle contractile deficits (approximately 25% of C57BL/6 across multiple stimulation frequencies). SLI also reduced BALB/c mitochondrial respiratory capacity, assessed in either isolated mitochondria (58.3% of C57BL/6 at SLI on day (d)7, 59.1% of C57BL/6 at SLI d28 across multiple conditions) or permeabilized myofibers (38.9% of C57BL/6 at SLI d7; 76.2% of C57BL/6 at SLI d28 across multiple conditions). SLI also resulted in decreased calcium retention capacity (56.0% of C57BL/6) in BALB/c mitochondria. Nonischemic cardiotoxin injury revealed similar recovery of myofiber area, contractile force, mitochondrial respiratory capacity, and calcium retention between strains. CONCLUSIONS: Ischemia-susceptible BALB/c mice suffered persistent muscle atrophy, impaired muscle function, and mitochondrial respiratory deficits during SLI. Interestingly, parental strain susceptibility to myopathy appears specific to regenerative insults including an ischemic component. Our findings indicate that the functional deficits that plague PAD patients could include mitochondrial respiratory deficits genetically inherent to the regenerating muscle myofibers.

Meningocele manqué: a comprehensive review of this enigmatic finding in occult spinal dysraphism.

The term meningocele manqué (MM) was coined in 1972 to describe a broad range of surgical findings characterized by intradural bands tethering neural structures to the dorsal dura. Over the following decades, reports continued to lump intradural tethering bands associated with a variety of comorbidities under the umbrella term MM. In more recent years, disorders previously called MM have been identified as embryologically distinct and were reclassified. While this sectioning continues, there remains a set of intradural tethering disorders for which no better term than MM exists. Herein, we comprehensively review the literature surrounding MM, including embryological disorders, clinical, radiographic, and surgical presentation, as well as alternative classification methods and MM treatment.

Nerve root anomalies: making sense of a complicated literature.

INTRODUCTION: Nerve root anomalies (NRAs) are a set of well-described congenital irregularities for which several classification systems have been devised over the years. CONCLUSION: This comprehensive review examines the anatomy and characteristics of NRAs; their surgical, radiographic, and cadaveric prevalence rates; clinical and radiographic presentations; and surgical management. In addition, the top 5 NRA classification systems are presented and related.

Junctional neural tube defect in a newborn: report of a fourth case.

INTRODUCTION: A discontinuous, functionally disconnected spinal cord is an extremely rare finding, with only three known reports in the literature. Titled junctional neural tube defect (JNTD), this newly reported dysraphism is believed to arise from a developmental error occurring during junctional neurulation, a transitory stage of development marked by the end of primary neurulation and the beginning of secondary neurulation. Herein, we report a newborn case of JNTD. CASE REPORT: We report a newborn boy born with anorectal atresia. Physical examination revealed normal movement in the upper and lower extremities. Imaging revealed distal sacral agenesis and a spinal cord that was discontinuous at the thoracolumbar junction. Three vertebral segments inferiorly, at the L2 vertebral level, the distal end of the spinal cord (i.e., conus medullaris) were visualized. No signs of a tethered cord were identified. CONCLUSIONS: Characterized by an error in junctional neurulation in which the primary and secondary NT fail to integrate appropriately, JNTD has been recently classified. We believe the current patient to represent only the fourth reported case of JNTD in the literature.

A comprehensive review of the anterior fontanelle: embryology, anatomy, and clinical considerations.

PURPOSE: Fontanelles are a regular feature of infant development in which two segments of bone remain separated, leaving an area of fibrous membrane or a "soft spot" that acts to accommodate growth of the brain without compression by the skull. Of the six fontanelles in the human skull, the anterior fontanelle, located between the frontal and parietal bones, serves as an important anatomical diagnostic tool in the assessment of impairments of the skull and brain and allows access to the brain and ventricles in the infant. METHODS: Using a standard database search, we conducted a review of the anterior fontanelle, including its embryology, anatomy, pathology, and related surgical implications. CONCLUSIONS: The diagnostic value of the anterior fontanelle, through observation of its shape, size, and palpability, makes the area of significant clinical value. It is important that clinicians are aware of the features and associated pathologies of this area in their everyday practice.

Mitochondrial therapy improves limb perfusion and myopathy following hindlimb ischemia.

Critical limb ischemia is a devastating manifestation of peripheral arterial disease with no effective strategies for improving morbidity and mortality outcomes. We tested the hypothesis that cellular mitochondrial function is a key component of limb pathology and that improving mitochondrial function represents a novel paradigm for therapy. BALB/c mice were treated with a therapeutic mitochondrial-targeting peptide (MTP-131) and subjected to limb ischemia (HLI). Compared to vehicle control, MTP-131 rescued limb muscle capillary density and blood flow (64.7±11% of contralateral vs. 39.9±4%), and improved muscle regeneration. MTP-131 also increased electron transport system flux across all conditions at HLI day-7. In vitro, primary muscle cells exposed to experimental ischemia demonstrated markedly reduced (~75%) cellular respiration, which was rescued by MTP-131 during a recovery period. Compared to muscle cells, endothelial cell (HUVEC) respiration was inherently protected from ischemia (~30% reduction), but was also enhanced by MTP-131. These findings demonstrate an important link between ischemic tissue bioenergetics and limb blood flow and indicate that the mitochondria may be a pharmaceutical target for therapeutic intervention during critical limb ischemia.

Subacute limb ischemia induces skeletal muscle injury in genetically susceptible mice independent of vascular density.

OBJECTIVE: The primary preclinical model of peripheral artery disease, which involves acute limb ischemia (ALI), can result in appreciable muscle injury that is attributed to the acuity of the ischemic injury. A less acute model of murine limb ischemia using ameroid constrictors (ACs) has been developed in an attempt to mimic the chronic nature of human disease. However, there is currently little understanding of how genetics influence muscle injury following subacute arterial occlusion in the mouse. METHODS: We investigated the influence of mouse genetics on skeletal muscle tissue survival, blood flow, and vascular density by subjecting two different mouse strains, C57BL/6 (BL6) and BALB/c, to ALI or subacute limb ischemia using single (1AC) or double (2AC) AC placement on the femoral artery. RESULTS: Similar to ALI, the 2AC model resulted in significant tissue necrosis and limb perfusion deficits in genetically susceptible BALB/c but not BL6 mice. In the 1AC model, no outward evidence of tissue necrosis was observed, and there were no differences in limb blood flow between BL6 and BALB/c. However, BALB/c mice displayed significantly greater muscle injury, as evidenced by increased inflammation and myofiber atrophy, despite having no differences in CD31(+) and SMA(+) vascular density and area. BALB/c mice also displayed significantly greater centralized myonuclei, indicating increased muscle regeneration. CONCLUSIONS: The susceptibility of skeletal muscle to ischemia-induced injury is at least partly independent of muscle blood flow and vascular density, consistent with a muscle cell autonomous response that is genetically determined. Further development of preclinical models of peripheral artery disease that more accurately reflect the nature of the human disease may allow more accurate identification of genetic targets for therapeutic intervention.

"A Low-Cost Microfluidics System for Light Microscopy Experiments".

Microfluidics devices are powerful tools for studying dynamic processes in live cells, especially when used in conjunction with light microscopy. There are many applications of microfluidics devices including recording dynamic cellular responses to small molecules or other chemical conditions in perfused media, monitoring cell migration in constrained spaces, or collecting media perfusate for the study of secreted compounds in response to experimental inputs/manipulations. Here we describe a configurable low-cost (channel-based) microfluidics platform for live-cell microscopy, intended to be useful for experiments that require more precision/flexibility than simple rubber spacers, but less precision than molded elastomer-based platforms. The materials are widely commercially available, low-cost, and device assembly takes only minutes.

Pyruvate modulation of redox potential controls mouse sperm motility.

Sperm gain fertilization competence in the female reproductive tract through a series of biochemical changes and a requisite switch from linear progressive to hyperactive motility. Despite being essential for fertilization, regulation of sperm energy transduction is poorly understood. This knowledge gap confounds interpretation of interspecies variation and limits progress in optimizing sperm selection for assisted reproduction. Here, we developed a model of mouse sperm bioenergetics using metabolic phenotyping data, quantitative microscopy, and spectral flow cytometry. The results define a mechanism of motility regulation by microenvironmental pyruvate. Rather than being consumed as a mitochondrial fuel source, pyruvate stimulates hyperactivation by repressing lactate oxidation and activating glycolysis in the flagellum through provision of nicotinamide adenine dinucleotide (NAD)+. These findings provide evidence that the transitions in motility requisite for sperm competence are governed by changes in the metabolic microenvironment, highlighting the unexplored potential of using catabolite combination to optimize sperm selection for fertilization.

"Modeling Diffusive Search by Non-Adaptive Sperm: Empirical and Computational Insights".

During fertilization, mammalian sperm undergo a winnowing selection process that reduces the candidate pool of potential fertilizers from ~106-1011 cells to 101-102 cells (depending on the species). Classical sperm competition theory addresses the positive or 'stabilizing' selection that acts on sperm phenotypes within populations of organisms but does not strictly address the developmental consequences of sperm traits among individual organisms that are under purifying selection during fertilization. It is the latter that is of utmost concern for improving assisted reproductive technologies (ART) because 'low fitness' sperm may be inadvertently used for fertilization during interventions that rely heavily on artificial sperm selection, such as intracytoplasmic sperm injection (ICSI). Importantly, some form of sperm selection is used in nearly all forms of ART (e.g., differential centrifugation, swim-up, or hyaluronan binding assays, etc.). To date, there is no unifying quantitative framework (i.e., theory of sperm selection) that synthesizes causal mechanisms of selection with observed natural variation in individual sperm traits. In this report, we reframe the physiological function of sperm as a collective diffusive search process and develop multi-scale computational models to explore the causal dynamics that constrain sperm 'fitness' during fertilization. Several experimentally useful concepts are developed, including a probabilistic measure of sperm 'fitness' as well as an information theoretic measure of the magnitude of sperm selection, each of which are assessed under systematic increases in microenvironmental selective pressure acting on sperm motility patterns.

Mitochondria inside acute myeloid leukemia cells hydrolyze ATP to resist chemotherapy.

Despite early optimism, therapeutics targeting oxidative phosphorylation (OxPhos) have faced clinical setbacks, stemming from their inability to distinguish healthy from cancerous mitochondria. Herein, we describe an actionable bioenergetic mechanism unique to cancerous mitochondria inside acute myeloid leukemia (AML) cells. Unlike healthy cells which couple respiration to the synthesis of ATP, AML mitochondria were discovered to support inner membrane polarization by consuming ATP. Because matrix ATP consumption allows cells to survive bioenergetic stress, we hypothesized that AML cells may resist cell death induced by OxPhos damaging chemotherapy by reversing the ATP synthase reaction. In support of this, targeted inhibition of BCL-2 with venetoclax abolished OxPhos flux without impacting mitochondrial membrane potential. In surviving AML cells, sustained polarization of the mitochondrial inner membrane was dependent on matrix ATP consumption. Mitochondrial ATP consumption was further enhanced in AML cells made refractory to venetoclax, consequential to downregulations in both the proton-pumping respiratory complexes, as well as the endogenous F1-ATPase inhibitor ATP5IF1. In treatment-naive AML, ATP5IF1 knockdown was sufficient to drive venetoclax resistance, while ATP5IF1 overexpression impaired F1-ATPase activity and heightened sensitivity to venetoclax. Collectively, our data identify matrix ATP consumption as a cancer-cell intrinsic bioenergetic vulnerability actionable in the context of mitochondrial damaging chemotherapy.