DeepQuantify: deep learning and quantification system of white blood cells in light microscopy images of injured skeletal muscles.

White blood cells (WBCs) are the most diverse types of cells observed in the healing process of injured skeletal muscles. In the recovery process, WBCs exhibit a dynamic cellular response and undergo multiple changes of the protein expression. The progress of healing can be analyzed by the number of WBCs or by the number of specific proteins observed in light microscopy images obtained at different time points after injury. We propose a deep learning quantification and analysis system called DeepQuantify to analyze WBCs in light microscopy images of uninjured and injured muscles of female mice. The DeepQuantify system features in segmentation using the localized iterative Otsu's thresholding method, masking postprocessing, and classification of WBCs with a convolutional neural network (CNN) classifier to achieve a high accuracy and a low manpower cost. The proposed two-layer CNN classifier designed based on the optimization hypothesis is evaluated and compared with other CNN classifiers. The DeepQuantify system adopting these CNN classifiers is evaluated for quantifying CD68-positive macrophages and 7/4-positive neutrophils and compared with the state-of-the-art deep learning segmentation architectures. DeepQuantify achieves an accuracy of 90.64% and 89.31% for CD68-positive macrophages and 7/4-positive neutrophils, respectively. The DeepQuantify system employing the proposed two-layer CNN architecture achieves better performance than those deep segmentation architectures. The quantitative analysis of two protein dynamics during muscle recovery is also presented.

Characteristics of Tables for Disseminating Biobehavioral Results.

BACKGROUND: To report the complexity and richness of study variables within biological nursing research, authors often use tables; however, the ease with which consumers understand, synthesize, evaluate, and build upon findings depends partly upon table design. OBJECTIVES: To assess and compare table characteristics within research and review articles published in Biological Research for Nursing and Nursing Research. METHOD: A total of 10 elements in tables from 48 biobehavioral or biological research or review articles were analyzed. To test six hypotheses, a two-level hierarchical linear model was used for each of the continuous table elements, and a two-level hierarchical generalized linear model was used for each of the categorical table elements. Additionally, the inclusion of probability values in statistical tables was examined. RESULTS: The mean number of tables per article was 3. Tables in research articles were more likely to contain quantitative content, while tables in review articles were more likely to contain both quantitative and qualitative content. Tables in research articles had a greater number of rows, columns, and column-heading levels than tables in review articles. More than one half of statistical tables in research articles had a separate probability column or had probability values within the table, whereas approximately one fourth had probability notes. CONCLUSIONS: Authors and journal editorial staff may be generating tables that better depict biobehavioral content than those identified in specific style guidelines. However, authors and journal editorial staff may want to consider table design in terms of audience, including alternative visual displays.

Balance Between the Proinflammatory and Anti-Inflammatory Immune Responses with Blood Transfusion in Sepsis.

Blood product transfusion may exacerbate the initial immunosuppressive response of sepsis. Nurses and other patient care providers must be diligent in recognizing and managing a worsening immune status, using flow cytometry to monitor patients' immune status. This type of monitoring may be instrumental in reducing morbidity and mortality in persons with sepsis. This article discusses the recent literature on the associated inflammatory responses that occur with blood transfusion and provides an analysis of alterations in key inflammatory pathways in response to transfusion in a sepsis population.

Time course of chemokine expression and leukocyte infiltration after acute skeletal muscle injury in mice.

Innate pro-inflammatory processes, such as chemokine signaling and leukocyte infiltration, predominate during the first 48 h after an acute skeletal muscle injury. However, the time course of chemokine expression and its relationship to leukocyte infiltration after acute muscle injury within this early post-injury time period has not been investigated. In this study, 46 anesthetized female C57BL/6NHsd mice underwent a closed crush injury of the gastrocnemius muscle and were euthanized 4, 8, 24 and 48 h post-injury. Microarray analysis found 14 chemokine genes to be up-regulated during this period, 12 of which are involved in macrophage or neutrophil chemotaxis, with up-regulation peaking at either 8 or 48 h. RT-PCR analysis on select chemokines confirmed the microarray activation pattern. Neutrophil infiltration patterns mirrored the time course of neutrophil-related chemokines with Gr-1-, 1A8- and 7/4-positive neutrophils infiltrating the muscle 4 h after injury, decreasing at 48 h. Conversely, gene expression and relative quantification levels of macrophage-related chemokines Ccl2 and Ccl7 peaked at 8 h, preceding the infiltration of CD68- and F4/80-positive macrophages, and protein expression of Ccl2 in the muscle. The up-regulation of other macrophage-related chemokines and their receptors peaked at 48 h post-injury.

Assessing the immune status of critically ill trauma patients by flow cytometry.

BACKGROUND: Unintentional injury or trauma remains the leading cause of death among young adults. About one fifth of these trauma patients require care in an intensive care unit (ICU) because of severity of injuries and comorbidities. Patients hospitalized in an ICU are at increased risk for nosocomial infections, such as urinary tract infections, pneumonia, bacteremia, and wound infections. Many of these patients will develop sepsis or septic shock, and some will progress to multiple organ failure and death. The balance between the proinflammatory and counterinflammatory immune response appears to be a driving factor in this progression. At present, there is no proposed method for the timely detection of the immune status in trauma patients, making rational decisions to use immune-altering therapies difficult. OBJECTIVE: We demonstrate that flow cytometry, with its capabilities to characterize and/or enumerate (a) leukocyte subtypes, (b) leukocyte activation markers, (c) leukocyte-derived cytokines and microvesicles, and (d) leukocyte function is well suited to monitor the immune status of critically ill trauma patients. METHODS: Information for the review was obtained from the extant literature. DISCUSSION: We suggest that flow cytometry is a research method that might aid nurse scientists in investigating the immune status of critically ill patients, the recovery status of conditions such as hemorrhagic shock and tissue injury and the relationship between cancer disease progression and symptoms. Therefore, flow cytometry has the potential to broaden nursing research priority areas so that a comprehensive approach to understanding the cellular response is attained.

Comparison of methodologic quality and study/report characteristics between quantitative clinical nursing and nursing education research articles.

AIM: To compare the methodologic quality and study/report characteristics between quantitative clinical nursing and nursing education research articles. BACKGROUND: The methodologic quality of quantitative nursing education research needs to advance to a higher level. Clinical research can provide guidance for nursing education to reach this level. METHOD: One hundred quantitative clinical research articles from-high impact journals published in 2007 and 37 education research articles from high impact journals published in 2006 to 2007 were chosen for analysis. RESULTS: Clinical articles had significantly higher quality scores than education articles in three domains: number of institutions studied, type of data, and outcomes. CONCLUSION: The findings indicate three ways in which nursing education researchers can strengthen the methodologic quality of their quantitative research. With this approach, greater funding may be secured for advancing the science of nursing education.

Mouse model of muscle crush injury of the legs.

Because crush injury to skeletal muscle is an important cause of morbidity in natural disaster and battlefield settings, a reproducible and refined animal model of muscle crush injury is needed. Both open and closed small-animal models of skeletal muscle crush injury are available but are limited by their need for surgical isolation of the muscle or by the adverse effect of fibular fracture, respectively. In the current study, we developed and validated a novel, noninvasive mouse model of lower-extremity muscle crush injury. Despite the closed nature of our model, gross evidence of muscle damage was evident in all mice and was verified microscopically through hematoxylin and eosin staining. The injury elicited both neutrophil and macrophage infiltration at 24 and 48 h after injury. The area percentage and mean antigen area of F4/80-positive macrophages were higher at 48 h than at 24 h after injury, and CD68-positive macrophage area percentage and mean antigen area differed significantly between injured and uninjured muscle. In addition, the incidence of fibular fracture was one third lower than that reported for an alternative noninvasive model. In conclusion, our model is a reproducible method for muscle crush injury in the mouse pelvic limb and is a refinement of previous models because of its decreased bone fractures and reduction of animal numbers.

Macrophage colony stimulating factor-induced macrophage differentiation influences myotube elongation.

BACKGROUND: Unaccustomed exercise, high-intensity dynamic sports activities, or the resumption of normal weight-bearing after a period of disuse can induce skeletal muscle injury, which activates an inflammatory response followed by muscle regeneration. Specific subsets of macrophages are involved in muscle regeneration. But the exact role of macrophage differentiation during muscle regeneration remains to be elucidated. OBJECTIVE: The objective of the study was to examine the effect of macrophage colony stimulating factor (M-CSF)-differentiated, lipopolysaccharides (LPS)-stimulated-macrophage-conditioned medium on muscle-cell proliferation, fusion, and elongation, which are key events during muscle regeneration and myogenesis. METHOD: Murine C2C12 myoblasts were cultured in conditioned medium obtained from PU5-1R macrophages that were (a) undifferentiated, unstimulated; (b) M-CSF-differentiated, unstimulated; (c) undifferentiated, LPS-stimulated; or (d) M-CSF-differentiated, LPS-stimulated. Myoblast proliferation ratio, nuclei number, and length were measured. RESULTS: C2C12 cells cultured in conditioned medium from M-CSF-differentiated, LPS-stimulated macrophages had significantly more nuclei and greater length than cells cultured in conditioned medium from undifferentiated, LPS-stimulated macrophages. Dilution and denaturization of the M-CSF-differentiated, LPS-stimulated-macrophage medium prevented a marked increase in C2C12 nuclei number and length. However, the C2C12 myoblast proliferation ratio was significantly greater in conditioned medium from undifferentiated, LPS-stimulated macrophages than in conditioned medium from M-CSF-differentiated, LPS-stimulated macrophages. CONCLUSIONS: M-CSF-differentiated, LPS-stimulated macrophages may influence myogenesis and the early and terminal stages of muscle regeneration. This knowledge may aid in developing therapies that will directly expedite muscle repair and lead to faster rehabilitation and reduced rehabilitation costs.

A rodent model to advance the field treatment of crush muscle injury during earthquakes and other natural disasters.

Approximately 170 earthquakes of 6.0 or higher magnitude occur annually worldwide. Victims often suffer crush muscle injuries involving impaired blood flow to the affected muscle and damage to the muscle fiber membrane. Current rescue efforts are directed toward preventing acute kidney injury (AKI), which develops upon extrication and muscle reperfusion. But field-usable, muscle-specific interventions may promote muscle regeneration and prevent or minimize the pathologic changes of reperfusion. Although current rodent crush injury models involve reperfusion upon removal of the crush stimulus, an analysis of their methodological aspects is needed to ensure adequate simulation of the earthquake-related crush injury. The objectives of this systematic review are to (a) describe rodent crush muscle injury models, (b) discuss the benefits and limitations of these models, and (c) offer a recommendation for animal models that would increase our understanding of muscle recovery processes after an earthquake-induced crush muscle injury. The most commonly used rodent model uses a clamping or pressing crush stimulus directly applied to murine hindlimb muscle. This model has increased our understanding of muscle regeneration but its open approach does not adequately represent the earthquake-related crush injury. The model we recommend for developing field-usable, muscle-specific interventions is a closed approach that involves a nonclamping crush stimulus. Findings from studies employing this recommended model may have greater relevance for developing interventions that lessen the earthquake's devastating impact on individual and community health and quality of life, especially in developing countries.

Body weight and leukocyte infiltration after an acute exercise-related muscle injury in ovariectomized mice treated with estrogen and progesterone.

In both rats and mice, an acute skeletal muscle injury leads to leukocyte infiltration in which the leukocytes remove dead fibers and cellular debris, induce a secondary injury, and/or promote myofiber regeneration. Short-term exogenous estrogen treatment attenuates this leukocyte infiltration and prevents body weight gain in rat exercise-induced skeletal muscle injury models. But these estrogen effects may not occur in mice because body weight gain does not consistently occur in ovariectomized mice treated with estrogen. Additionally, progesterone may also attenuate this leukocyte infiltration without affecting body weight. The aim of the current study was to compare body weight and leukocyte infiltration in exercise-injured skeletal muscle of ovariectomized mice treated with exogenous estrogen and progesterone for the short period of 17 days with that of ovariectomized-placebo-treated mice and gonadal-intact male and female mice. There was no significant difference in body weight between the ovariectomized-estrogen-treated and the ovariectomized-placebo-treated mice. The amount of intramuscular leukocyte infiltration of ovariectomized mice treated with estrogen or progesterone was not significantly different from that of ovariectomized-placebo-treated mice. However, in the injured muscle, the mean area of the leukocyte antigen, 7/4, of the ovariectomized-estrogen-treated group was 2-3-fold greater than that of the ovariectomized-placebo-treated, ovariectomized-estrogen-progesterone-treated, and intact male groups (p<.05), suggesting that the 7/4-positive leukocytes of the ovariectomized-estrogen-treated group were larger or had more antigen. In conclusion, ovariectomized mice demonstrate a different body weight and leukocyte response to short-term estrogen treatment than that of ovariectomized rats, and short-term estrogen treatment modulates leukocyte phenotype. These data broaden our understanding of estrogen's effects on body weight and leukocyte infiltration, and may aid in increasing our understanding of how males and females differ in response to acute muscle injury.

Methodological quality and scientific impact of quantitative nursing education research over 18 months.

The methodological quality of nursing education research has not been rigorously studied. The purpose of this study was to evaluate the methodological quality and scientific impact of nursing education research reports. The methodological quality of 133 quantitative nursing education research articles published between July 2006 and December 2007 was evaluated using the Medical Education Research Study Quality Instrument (MERSQI).The mean (+/- SD) MERSQI score was 9.8 +/- 2.2. It correlated (p < .05) with several scientific impact indicators: citation counts from Scopus (r = .223), Google Scholar (r = .224), and journal impact factor (r = .216); it was not associated with Web of Science citation count, funding, or h Index. The similarities between this study's MERSQI ratings for nursing literature and those reported for the medical literature, coupled with the association with citation counts, suggest that the MERSQI is an appropriate instrument to evaluate the quality of nursing education research.

The relation between immunohistochemical product discreteness and leukocyte manual quantification reliability.

The discreteness of immunohistochemical products may interfere with detecting individual cells when obtaining manual quantification even with the application of a nuclear stain. To examine the influence of discreteness, we: (1) determined the relation between manual quantification reliability and discreteness, (2) analyzed the reliability between the manual and computer-assisted approaches, and (3) determined the relation between the reliability of the manual and computer approaches and discreteness. Sixty murine injured skeletal muscle sections were immunolabeled using the antineutrophil antibody, 7/4. Using computer-generated section images, an observer counted the number of cells on 2 different occasions (T1 and T2). Computer cell counts per section and discreteness per section (as indicated by the max pixel area per section) were determined. A moderate correlation was found for the relation between manual reliability and discreteness (P<0.01). The rankings of manual and computer counts were different (P<0.01), suggesting a lack of reliability between these approaches. The relation between the reliability of the manual and computer approaches and discreteness was moderately correlated (P<0.01). Samples with a computer count 200 or more had a greater mean max pixel area than those with a count <200 (P<0.0001). In conclusion, manual quantification may be more appropriate with samples with a leukocyte count <200, because discreteness is higher within this cell count range. Alternatively, when samples consist of 200 or more cells and discreteness is low, then the approaches that account for discreteness should be utilized.

Neutrophil infiltration in exercise-injured skeletal muscle: how do we resolve the controversy?

Neutrophils have not consistently been detected in exercise-injured skeletal muscle and, therefore, neutrophil infiltration in this muscle has become a controversial issue. Thirty-eight animal and human studies that assessed injured muscle for neutrophils and employed acute exercise (e.g. level, uphill or downhill running, eccentric contractions, or swimming) were analysed to help clarify the relationship between neutrophil infiltration and exercise-induced muscle injury. Findings from nearly three-quarters of the reviewed studies suggest that neutrophil accumulation follows exercise-induced muscle injury. Intramuscular neutrophil infiltration was present in 85% and 55% of the animal and human studies, respectively. However, no consistent relationship between the potential damaging effect of the exercise type and neutrophil infiltration can be conclusively established from these studies. Specific animal-related factors that could influence these results include age, animal strain, catecholamines, corticosterone, acute stressors and muscle type, whereas a specific human-related influencing factor is physical activity status. Factors affecting both animal and human studies could include sex hormones, muscle sampling techniques and neutrophil detection approaches. General categories of methods that have been used to detect neutrophil infiltration are microscopy, myeloperoxidase (MPO) biochemical assay, antibody staining and white blood cell radionuclide imaging. Only studies employing white blood cell radionuclide imaging have consistently detected neutrophil infiltration. However, antibody staining with a quantitative analysis is currently the most feasible, valid and sensitive method. Research recommendations, therefore, are warranted to resolve the neutrophil infiltration controversy. We propose two approaches for animal studies. The first approach encompasses (i) studying or measuring factors that could influence neutrophil infiltration; (ii) using quantitative antibody staining analysis (in all studies and employing a panel of anti-neutrophil antibodies); (iii) examining the relationship between fibre morphological changes and neutrophil antigen expression; and (iv) developing a neutrophil antibody-radionuclide imaging technique. The second approach will yield animal findings complementing or addressing the gaps from the human exercise studies. For human studies, we suggest that (i) physical activity status is investigated; (ii) quantitative antibody staining analysis is performed (including staining injured muscle with a panel of antibodies such as anti-elastase, anti-MPO, anti-CD11b and anti-CD15 or assessing injured muscle using both immunohistochemistry and the MPO biochemical assay); and (iii) the relationship between fibre morphological changes and neutrophil antigen expression is examined. Studies that incorporate these recommendations could lead to an increased understanding of whether neutrophils are essential for the recovery from an exercise-induced muscle injury.

Indices of leukocyte infiltration and muscle recovery after eccentric contraction-induced injury in young and adult male mice.

The movement of leukocytes (e.g., neutrophils and macrophages) into skeletal muscle is a critical response after a sports-related injury. Age could influence this response. The major objectives of the study were to determine whether there is a relationship between age and the degree of (1) leukocyte response and (2) muscle recovery. The left plantarflexor muscles of 42 young and adult male mice underwent eccentric contractions, which can cause injury. After 1 or 5 days, leukocyte infiltration and muscle recovery were assessed in the soleus or medial gastrocnemius muscles. No relationship between age and leukocyte infiltration was found (P = .36). Neutrophils, but not macrophages, were detected at 1-day recovery. A significant relationship between age and muscle recovery was detected (P = .0498), suggesting that muscle recovery may be decreased with increased age.

The effects of estradiol and progesterone on plantarflexor muscle fatigue in ovariectomized mice.

The aim of this study was to examine specific and interactional effects of estradiol and progesterone on the time-to-fatigue of eccentrically contracted plantarflexor muscles and on the percent of plantarflexor isometric torque remaining immediately after an eccentric contraction (EC) protocol. Ovariectomized 6- to 8-week-old C57BL/6 mice were implanted with 21-day 0.05 mg-placebo, 0.05 mg-17-beta estradiol (OE), 15 mg-progesterone (OP), or estradiol and progesterone pellets (OEP). On the 16th day of hormone treatment, the isometric torque of the left plantarflexor muscles was measured. The left plantarflexor muscles then underwent 1 set of 150 ECs followed by 2 immediate post-EC isometric torque measurements. A group of ovarian-intact female mice of a similar age underwent the same isometric torque measurements and EC protocol. Plantarflexor muscle fatigue during ECs took 30%-41% longer to occur in the OP group (n = 9) than it did in the intact (n = 8, P = 0.02), OC (n = 11, P = 0.003), and OEP (n = 9, P = 0.007) groups. Peak active isometric torque had decreased immediately after ECs at 2 time points (M1 and M2). The OP group exhibited the greatest percent of isometric torque remaining immediately after ECs (M1, P = 0.03; M2, P = 0.04). These findings suggest that progesterone reduces muscle fatigue in response to ECs and that this progesterone effect is blunted when estradiol also is present. Therefore, ovarian hormone status may need to be considered when evaluating a response to physical activities, especially those activities involving ECs.

Inactivity: symptoms associated with gastrocnemius muscle disuse during pregnancy.

This longitudinal study assessed change in gastrocnemius muscle metabolism across antepartum bed rest and recovery during the first 6 weeks postpartum as well as symptoms during recovery. The convenience sample consisted of 65 pregnant women, hospitalized on bed rest for a mean of 24.8 days (range 5-70 days). A dual wavelength hemoglobin/ myoglobin spectrophotometer and ergometer assessed muscle reoxygenation times after plantar flexion exercise from hospital admission through 6 weeks postpartum. The Postpartum Symptom Checklist assessed symptoms of muscle deconditioning. The time for gastrocnemius muscle reoxygenation after exercise significantly increased across antepartum bed rest (t = -2.1, P <.05) and significantly decreased during the 6-week postpartum period (t = 1.83, P <.05). Women who were on bed rest prior to hospital admission had significantly higher reoxygenation scores upon enrollment (t = -8.6, P <.05). Symptoms of postpartum muscle soreness, deconditioning, and difficulty with mobility were reported. Postpartum assessment and rehabilitation are needed to facilitate recovery and prevent long-term injury.

Desmin characteristics of CD11b-positive fibers after eccentric contractions.

PURPOSE: The purpose of this study was to determine the desmin characteristics of fibers infiltrated by CD11b-leukocytes in fast- and slow-twitch murine muscles after eccentric contractions. METHODS: We developed a computer image analysis procedure to quantify the percentage of intramyofiber CD11b-leukocyte infiltration. Only CD11b-positive fibers that exhibited identifiable borders were examined. Patterns of leukocyte infiltration of these fibers were distinguished and confirmed quantitatively by determining the mean percentage of leukocyte infiltration. We examined the desmin characteristics of these fibers with the same image analysis system and quantified the percentage of the fiber that demonstrated antidesmin staining. The influence of sex and muscle type on desmin characteristics and percentage of CD11b-leukocyte infiltration also was analyzed. RESULTS: The percentage of CD11b-leukocyte infiltration was less than 50% for the majority of fibers. Three intramyofiber leukocyte infiltration patterns were identified that had significantly different mean percentages of leukocyte infiltration: 14%, 27%, and 47% (P < 0.001). Of the three desmin characteristics (normal, increased, and decreased) that were observed, almost three fourths of the fibers exhibited increased desmin. A significant difference in the percentage of increased desmin was found among the CD11b-leukocyte infiltration patterns (P = 0.01). Fibers with the least amount of CD11b-leukocyte infiltration showed the greatest amount of increased desmin. Sex and muscle type did not influence any of these findings. CONCLUSION: Increased desmin appears to be associated with an early stage of intramyofiber leukocyte infiltration after eccentric contractions.