109Use of EPR Spectroscopy to Measure Tissue Oxygen in an Ischemic Flap Model

Methods to reliably measure tissue oxygenation in situ are currently lacking. We have developed a vertically oriented, dorsal, bipedicle flap model that is easy to perform, reliably reproduces tissue ischemia, eliminates craniocaudal variation, and is amenable to studying therapeutic modalities. The effect of narrowing this flap on tissue oxygenation measured with Licox electrodes has previously been presented. In this study we utilize in situ EPR spectroscopy to demonstrate the oxygen gradient in the flap as a function of flap width and placement of a silicone sheet directly under the flap. The effect of wound healing over a 2 week period is demonstrated. Twenty four, 300 gm male Sprague‐Dawley rats underwent creation of the bipedicle flap according to the following groups: 2.5 cm flap with silicone, 2.0 cm flap without silicone, 2.0 cm flap with silicone. Each group of 6 animals was injected with EMS char at 2 cm intervals along the flap and one injection in the control, non‐ischemic tissue. A 4^th group underwent 2.0 cm flaps with silicone and use of lithium phthalocyanin as the paramagnetic material. Wound measurements and EPR spectroscopy were performed on days 3, 7, 10 and 14. On day 14, after EPR measurements, the animals were sacrificed and their wounds excised. One flap and one control wound were preserved for histologic analysis, the other flap and control wounds were prepared for lactate measurements. EPR spectroscopy demonstrated a gradient of oxygen that was lowest in the center of the flap and greatest at either end. Changes in the oxygen gradient correlated with narrowing and placement of the intervening silicone sheet. This new technology has never been utilized in an animal model of impaired wound healing. Comparison of recently developed paramagnetic materials for optimal tissue oxygen and free radical measurements will be presented.     

Rejuvenescence and extension of an urochordate life span following a single,acute administration of an anti-oxidant,butylated hydroxytoluene

Two commonly accepted metabolic theories of aging interpret senescence either in terms of the rate of living, where a fixed total metabolic potential is consumed over an expected lifetime (after which the organism wears out and dies) or, in terms of accumulative oxidative damage resulting in progressive and irreversible changes in metabolic pathways. Protocols based on restricted diets, chronically administered anti-oxidants and the use of established lines of organisms resistant to free radical damage support the metabolic theories of aging by revealing, in many cases, significant extensions of life spans or dramatic anti-aging effects. To test the universality of these metabolic hypotheses of aging, we acutely treated ramets (clonal replicates) from old, long-lived colonies of the urochordate Botryllus schlosseri with lethal doses of the anti-oxidant butylated hydroxytoluene (BHT). This group of organisms has a weekly cyclical and highly synchronized developmental process (blastogenesis), during which all existing zooids are removed by massive apoptosis and phagocytosis processes. In animals treated with BHT, blastogenesis was completely arrested and colonies deteriorated to a morphologically chaotic state. Rescued ramets resorbed BHT treated zooids, regenerated entirely new sets of zooids and then revealed: (1) rejuvenescence and enhanced growth rates and in many cases, (2) up to 4.6 times extension of post-treatment life expectancy. Both metabolic theories for senescence were therefore falsified in B. schlosseri. The possible existence of an aging clock that can be set by the environment is suggested.     

ED point-of-care ultrasound in the diagnosis of ankle fractures in children

In pediatric ankle injury, radiography is the current standard used to differentiate fracture from ligamentous injury; however, the associated cost, increased time, and radiation exposure pose a significant downside to this imaging modality. Point-of-care ultrasound may be an attractive alternative in this setting, as illustrated by this patient case. A 14-year-old boy presented to the emergency department with a left ankle inversion injury sustained while playing soccer. An emergency physician performed ultrasound examination that revealed findings consistent with a nondisplaced Salter-Harris I fracture of the distal fibula. The results of a formal radiograph confirmed this diagnosis. This case report presents the successful use of point-of-care ultrasound for detection of a Salter-Harris I ankle fracture, describes a stepwise approach for this new diagnostic technique in detail, and discusses its value in the setting of pediatric ankle injury.     

Two distinct evolutionary conserved neural degeneration pathways characterized in a colonial chordate

Colonial tunicates are marine organisms that possess multiple brains simultaneously during their colonial phase. While the cyclical processes of neurogenesis and neurodegeneration characterizing their life cycle have been documented previously, the cellular and molecular changes associated with such processes and their relationship with variation in brain morphology and individual (zooid) behavior throughout adult life remains unknown. Here, we introduce Botryllus schlosseri as an invertebrate model for neurogenesis, neural degeneration, and evolutionary neuroscience. Our analysis reveals that during the weekly colony budding (i.e., asexual reproduction), prior to programmed cell death and removal by phagocytes, decreases in the number of neurons in the adult brain are associated with reduced behavioral response and significant change in the expression of 73 mammalian homologous genes associated with neurodegenerative disease. Similarly, when comparing young colonies (1 to 2 y of age) to those reared in a laboratory for ∼20 y, we found that older colonies contained significantly fewer neurons and exhibited reduced behavioral response alongside changes in the expression of 148 such genes (35 of which were differentially expressed across both timescales). The existence of two distinct yet apparently related neurodegenerative pathways represents a novel platform to study the gene products governing the relationship between aging, neural regeneration and degeneration, and loss of nervous system function. Indeed, as a member of an evolutionary clade considered to be a sister group of vertebrates, this organism may be a fundamental resource in understanding how evolution has shaped these processes across phylogeny and obtaining mechanistic insight.

Botryllus schlosseri, a marine colonial chordate, has produced valuable insight in the study of tissue regeneration (1, 2), allorecognition (3–6), and stem cells (7, 8). B. schlosseri can be developed as a chordate following sexual reproduction, which gives rise to a tadpole that hatches and finds a subtidal surface to attach to, before metamorphosis into a sessile invertebrate. The metamorphosed individual develops 1 to 3 buds (covered by a common gelatinous tunic) that develop the invertebrate body plan, and several individuals anastomose extracorporeal blood vessels in the tunic to form a colony. Every week this colonial organism undergoes a de novo robust regeneration mediated by adult stem cells that participate in the formation of all body organs, including the central nervous system. The lifespan of B. schlosseri is plastic and amenable to change. Wild colonies are characterized by short lifespans ranging from several months to a few years (9–12) whereas colonies grown in the laboratory can reach 20 y of age (12–17). Systemic changes occur over time in older colonies, including a slower heartbeat, reduced zooid size, decreased regenerative capacity, a shift in cellular composition (e.g., higher level of engulfing phagocytic, cytotoxic, and pigment cells), and shifting patterns of cyclic gene expression associated with aging (17). Despite the key evolutionary position of B. schlosseri as a member of a clade considered to be the sister group of vertebrates (the body plan of the sexually reproduced larval tadpole is along the architecture of most chordate fetuses) (18), and previous literature describing its central nervous system (19–22), little is known about changes in brain morphology, colony behavior, and gene expression associated with the regular, cyclical processes of neural generation and degeneration and how they vary with colony age.Here we introduce B. schlosseri as a model for evolutionary neuroscience. The B. schlosseri genome has been sequenced (5) and an atlas of the molecular and morphological signatures of each developmental stage has been previously generated using microscopy and RNA sequencing (RNA-seq) (22). B. schlosseri can reproduce either sexually through embryogenesis or asexually through blastogenesis. The sexually produced larvae develop two brains (a functional larval brain and the rudiment of the adult brain), a sensory organ detecting light and gravity, a notochord, and a dorsal nerve cord. The larval brain (along with the notochord, segmented musculature, and tail) is absorbed during the settlement and metamorphosis which precedes colony formation. As a sedentary organism, B. schlosseri buds new individuals that together live as a colony composed of individual zooids. Each zooid reproduces asexually in weekly, stem cell-mediated budding cycles (7). Budding occurs during this cycle when, simultaneously throughout the colony, a new generation of buds begin to develop into adult individuals. During budding, all organs including the brain, are created anew without passing through a larval (embryonic and fetal) stage. The life of adult zooids lasts ∼1 wk after which their bodies undergo a synchronized wave of programmed cell death and phagocytic removal called takeover (i.e., TO) (16, 23) in which the nervous systems of old zooids degenerate in tangent to brain formation in the young buds.In this study, we describe 2 different pathways of neurodegeneration that occur during the life cycle of the colonial chordate B. schlosseri. The first occurs every week as a part of a regenerative developmental cycle that occurs independent of age; the second is associated with colony aging. We integrate neural imaging (transmission electron microscopy [TEM] and confocal), three-dimensional (3D) reconstructions, behavioral assay, and RNA-seq of enriched brains (Fig. 1) to study morphological and molecular changes in brains of adult B. schlosseri across developmental stages and ages as associated with changes in zooid behavior. In providing evidence of the cellular and molecular linkages between each neurodegenerative process, we document an unanticipated degree of similarity between the 2 that may be useful in identifying the evolutionary basis of the pathological mechanisms responsible for age-induced loss of neuron structure and function in other phylogeny. Indeed, we argue that given 1) a unique and assayable life cycle, 2) real-time monitoring enabled by transparent body and vasculature, 3) adult tissue specific stem cells that can be enriched and transplanted, 4) morphological differences associated with colony age, and 5) a previously sequenced genome; B. schlosseri provides a novel and valuable platform for the study of neurogenesis, neurodegeneration, and evolutionary neuroscience.Open in a separate windowFig. 1.Sampling and methods used to characterize weekly cycles of neurodegeneration in B. schlosseri colonies. Schematic depicting the various methods employed over the course of the investigation including neural complex dissection, confocal staining, histology, TEM, behavioral experiments and the intervals over which neural complex sampling was conducted (over the weekly cycle and with age in adult individuals). Genetically identical subclones prepared via incision and separation were used in diverse experimental groups.     

Modulatory effect of pineapple peel extract on lipid peroxidation,catalase activity and hepatic biomarker levels in blood plasma of alcohol-induced oxidative stressed rats

Objective

To investigate the ability of the methanolic extract of pineapple peel to modulate alcohol-induced lipid peroxidation, changes in catalase activities and hepatic biochemical marker levels in blood plasma.

Methods

Oxidative stress was induced by oral administration of ethanol (20% w/v) at a dosage of 5 mL/kg bw in rats. After 28 days of treatment, the rats were fasted overnight and sacrificed by cervical dislocation. Blood was collected with a 2 mL syringe by cardiac puncture and was centrifuged at 3 000 rpm for 10 min. The plasma was analyzed to evaluate malondialdehyde (MDA), catalase activity, aspartate aminotransferase (AST), alkaline phosphatase (ALP) and alanine aminotransferase (ALT) concentrations.

Results

Administration of alcohol caused a drastic increase (87.74%) in MDA level compared with the control. Pineapple peel extract significantly reduced the MDA level by 60.16% at 2.5 mL/kg bw. Rats fed alcohol only had the highest catalase activity, treatment with pineapple peel extract at 2.5 mL/kg bw however, reduced the activity. Increased AST, ALP and ALT activities were observed in rats fed alcohol only respectively, treatment with pineapple peel extract drastically reduced their activities.

Conclusions

The positive modulation of lipid peroxidation, catalase activities as well as hepatic biomarker levels of blood plasma by the methanolic extract of pineapple peels under alcohol-induced oxidative stress is an indication of its protective ability in the management of alcohol-induced toxicity.     

Axillary nodal metastasis and resection margins as predictors of Loco Regional Recurrence in Breast Cancer Patients

BackgroundSurgical resection margins (RM), axillary nodal involvement and lymph node ratio (LNR) determine loco-regional control (LRC) in breast cancer management. Late presentation precludes breast conservation therefore surgical option is usually mastectomy and adjuvant chemoradiation minimize loco-regional recurrence (LRR).ObjectiveWe investigated the prognostic role of lymph nodes positive for malignancy (pN), LNR and RM on LRR of breast cancer in a tertiary hospital in Ibadan, Nigeria.MethodsLongitudinal cohort study of 225 females with breast carcinoma managed and followed up for 5-years with end point of LRR or not. Chi-square test and logistic regression analysis were used to evaluate the interaction of resection margin and proportion of metastatic lymph nodes with LRR. The receiver-operator curve was plotted to determine the proportion of metastatic lymph nodes which predicted LRR.ResultsNinety-nine percent had modified radical mastectomy and 163 (72.4%) had negative resection margins. A mean of 11 axillary lymph nodes were harvested at surgery. The age, positive resection margin and number of harvested nodes with malignant cells are associated with LRR. The overall 5-year LRR rate was 16%.ConclusionLRR is dependent on lymph node involvement as well as and tumor aggressiveness.