How I do it: Endoscope-assisted in situ arterial reconstruction of the lower limb.

Arterial reconstruction with the great saphenous vein is a frequently performed vascular surgery technique for revascularization of chronic limb threatening ischemia. Surgeon variations of the procedure are common and aim to balance patency, limb salvage, complications, hospital resources, and technical feasibility. We describe a minimally invasive revascularization option using endoscope assistance for in situ great saphenous vein-arterial bypass to treat infrainguinal occlusive disease. We highlight patient selection, operating room setup, instrument details, and procedure strategies that facilitate the use of this technique. The development and refinement of minimally invasive techniques for lower extremity arterial bypass are critical to reduce wound complications and improve limb salvage outcomes in patients.

Novel oxygen sensing mechanism in the spinal cord involved in cardiorespiratory responses to hypoxia.

As blood oxygenation decreases (hypoxemia), mammals mount cardiorespiratory responses, increasing oxygen to vital organs. The carotid bodies are the primary oxygen chemoreceptors for breathing, but sympathetic-mediated cardiovascular responses to hypoxia persist in their absence, suggesting additional high-fidelity oxygen sensors. We show that spinal thoracic sympathetic preganglionic neurons are excited by hypoxia and silenced by hyperoxia, independent of surrounding astrocytes. These spinal oxygen sensors (SOS) enhance sympatho-respiratory activity induced by CNS asphyxia-like stimuli, suggesting they bestow a life-or-death advantage. Our data suggest the SOS use a mechanism involving neuronal nitric oxide synthase 1 (NOS1) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX). We propose NOS1 serves as an oxygen-dependent sink for NADPH in hyperoxia. In hypoxia, NADPH catabolism by NOS1 decreases, increasing availability of NADPH to NOX and launching reactive oxygen species-dependent processes, including transient receptor potential channel activation. Equipped with this mechanism, SOS are likely broadly important for physiological regulation in chronic disease, spinal cord injury, and cardiorespiratory crisis.

Developmental Maturation of Functional Coupling Between Ventilatory Oscillators in the American Bullfrog.

Many vital motor behaviors - including locomotion, swallowing, and breathing - appear to be dependent upon the activity of and coordination between multiple endogenously rhythmogenic nuclei, or neural oscillators. Much as the functional development of sensory circuits is shaped during maturation, we hypothesized that coordination of oscillators involved in motor control may likewise be maturation-dependent, i.e., coupling and coordination between oscillators change over development. We tested this hypothesis using the bullfrog isolated brainstem preparation to study the metamorphic transition of ventilatory motor patterns from early rhythmic buccal (water) ventilation in the tadpole to the mature pattern of rhythmic buccal and lung (air) ventilation in the adult. Spatially distinct oscillators drive buccal and lung bursts in the isolated brainstem; we found these oscillators to be active but functionally uncoupled in the tadpole. Over the course of metamorphosis, the rhythms produced by the buccal and lung oscillators become increasingly tightly coordinated. These changes parallel the progression of structural and behavioral changes in the animal, with adult levels of coupling arising by the metamorphic stage (forelimb eruption). These findings suggest that oscillator coupling undergoes a maturation process similar to the refinement of sensory circuits over development.

Diving into the mammalian swamp of respiratory rhythm generation with the bullfrog.

All vertebrates produce some form of respiratory rhythm, whether to pump water over gills or ventilate lungs. Yet despite the critical importance of ventilation for survival, the architecture of the respiratory central pattern generator has not been resolved. In frogs and mammals, there is increasing evidence for multiple burst-generating regions in the ventral respiratory group. These regions work together to produce the respiratory rhythm. However, each region appears to be pivotally important to a different phase of the motor act. Regions also exhibit differing rhythmogenic capabilities when isolated and have different CO2 sensitivity and pharmacological profiles. Interestingly, in both frogs and rats the regions with the most robust rhythmogenic capabilities when isolated are located in rhombomeres 7/8. In addition, rhombomeres 4/5 in both clades are critical for controlling phases of the motor pattern most strongly modulated by CO2 (expiration in mammals, and recruitment of lung bursts in frogs). These key signatures may indicate that these cell clusters arose in a common ancestor at least 400 million years ago.

Three brainstem areas involved in respiratory rhythm generation in bullfrogs.

For most multiphasic motor patterns, rhythm and pattern are produced by the same circuit elements. For respiration, however, these functions have long been assumed to occur separately. In frogs, the ventilatory motor pattern produced by the isolated brainstem consists of buccal and biphasic lung bursts. Previously, two discrete necessary and sufficient sites for lung and buccal bursts were identified. Here we identify a third site, the Priming Area, important for and having neuronal activity correlated with the first phase of biphasic lung bursts. As each site is important for burst generation of a separate phase, we suggest each major phase of ventilation is produced by an anatomically distinct part of an extensive brainstem network. Embedding of discrete circuit elements producing major phases of respiration within an extensive rhythmogenic brainstem network may be a shared architectural characteristic of vertebrates. ABSTRACT: Ventilation in mammals consists of at least three distinct phases: inspiration, post-inspiration and late-expiration. While distinct brainstem rhythm generating and pattern forming networks have long been assumed, recent data suggest the mammalian brainstem contains two coupled neuronal oscillators: one for inspiration and the other for active expiration. However, whether additional burst generating ability is required for generating other phases of ventilation in mammals is controversial. To investigate brainstem circuit architectures capable of producing multiphasic ventilatory rhythms, we utilized the isolated frog brainstem. This preparation produces two types of ventilatory motor patterns, buccal and lung bursts. Lung bursts can be divided into two phases, priming and powerstroke. Previously we identified two putative oscillators, the Buccal and Lung Areas. The Lung Area produces the lung powerstroke and the Buccal Area produces buccal bursts and - we assumed - the priming phase of lung bursts. However, here we identify an additional brainstem region that generates the priming phase. This Priming Area extends rostral and caudal of the Lung Area and is distinct from the Buccal Area. Using AMPA microinjections and reversible synaptic blockade, we demonstrate selective excitation and ablation (respectively) of priming phase activity. We also demonstrate that the Priming Area contains neurons active selectively during the priming phase. Thus, we propose that three distinct neuronal components generate the multiphase respiratory motor pattern produced by the frog brainstem: the buccal, priming and powerstroke burst generators. This raises the possibility that a similar multi-burst generator architecture mediates the three distinct phases of ventilation in mammals.

Localization of essential rhombomeres for respiratory rhythm generation in bullfrog tadpoles using a binary search algorithm: Rhombomere 7 is essential for the gill rhythm and suppresses lung bursts before metamorphosis.

Frog metamorphosis includes transition from water breathing to air breathing but the extent to which such a momentous change in behavior requires fundamental changes in the organization of the brainstem respiratory circuit is unknown. Here, we combine a vertically mounted isolated brainstem preparation, "the Sheep Dip," with a search algorithm used in computer science, to identify essential rhombomeres for generation of ventilatory motor bursts in metamorphosing bullfrog tadpoles. Our data suggest that rhombomere 7, which in mammals hosts the PreBötC (PreBötzinger Complex; the likely inspiratory oscillator), is essential for gill and buccal bursts. Whereas rhombomere 5, in close proximity to a brainstem region associated with the mammalian expiratory oscillator, is essential for lung bursts at both stages. Therefore, we conclude there is no rhombomeric translocation of respiratory oscillators in bullfrogs as previously suggested. In premetamorphic tadpoles, functional ablation of rhombomere 7 caused ectopic expression of precocious lung bursts, suggesting the gill oscillator suppresses an otherwise functional lung oscillator in early development.

Transmission of the respiratory rhythm to trigeminal and hypoglossal motor neurons in the American Bullfrog (Lithobates catesbeiana).

Spatially distinct, interacting oscillators in the bullfrog medulla generate and coordinated buccal and lung ventilatory rhythms, but how these rhythms are transmitted onto trigeminal and hypoglossal motor neurons is unknown. Using a vertically-mounted isolated brainstem preparation, the Sheep Dip, we identified the regions of the brainstem containing motor nuclei using a solution capable of blocking synaptic release and, following washout, locally exposed these regions to 5 µM NBQX and/or 50 µM AP5. Local application of NBQX significantly reduced the amplitude of buccal and lung bursts on the trigeminal nerve, and lung bursts on the hypoglossal nerve. Local AP5 caused a significant reduction in lung burst amplitude on both nerves, but for buccal bursts, hypoglossal amplitude increased and trigeminal amplitude was unchanged. Local co-application of NBQX and AP5 eliminated fictive respiratory motor output completely in both nerves. These results are consistent with mammalian data, suggesting a critical role for glutamate in transmission of respiratory activity from oscillators to motor neurons.